# Injectable thermoresponsive magnetic hydrogel composite incorporating iron oxide and hydroxyapatite nanoparticles for bone tissue engineering

Wednesday, 28th September - 16:00 - Targeted drug delivery and Nanocarriers - Tissue engineering and regenerative nanomedicine - Tower 24 - Room 107 - Oral presentation - Abstract ID: 505

**Dr. Padmalosini Muthukumaran**¹, **Prof. Seeram Ramakrishna**², **Prof. Balázs Gulyás**¹, **Prof. Raju V. Ramanujan**³, **Prof. Dinesh Kumar Srinivasan**¹

1. Lee Kong Chian School of Medicine, Nanyang Technological University, 2. Centre for Nanofibers and Nanotechnology, National University of Singapore, 3. School of Materials Science and Engineering, Nanyang Technological University

**Introduction:** Injectable bone cements (IBC) are liquid at normal conditions, but when injected into bone defect, they will stabilize and mold to the defect shape and aids in bone regeneration. Commercially available IBC are based on polymethylmethacrylate, calcium phosphate or calcium sulfate. These IBC have several drawbacks such as low radiopacity, lack of bioactivity, heat induced necrosis of surrounding tissue, poor mechanical strength etc. The objective of this study is to fabricate a novel thermosensitive injectable bone cement with incorporation of super paramagnetic iron oxide nanoparticles and nanohydroxyapatite (nHA) and test their suitability of bone healing.

**Methods:** Composite hydrogel was prepared by homogenization of Poly (N-isopropyl acrylamide)-co-acrylic acid (pNIPAM-co-AA) polymer solution with citrate modified magnetic iron oxide nanoparticles (MNP, 15-20 nm), nHA (200 nm) and strontium ranelate (SR). The obtained hydrogel was characterized for its structural, thermal and mechanical properties. Human mesenchymal stem cells (hMSC) were grown on the composite hydrogels in vitro and the structure, proliferation, osteogenic differentiation and mineralization ability of the cells on the hydrogel was analysed using MTS, alkaline phosphatase (ALP) assays, alizarin red staining and immunocytochemistry.

**Results:** The hydrogel composite comprised of a microporous structure with uniform distribution of the nanoparticles. LCST of the composite was observed as 350°C with 80.25% water content. FT-IR analysis showed characteristic peaks for amide, acid, cyanide, phosphate and carbonate groups. The onset of thermal degradation was observed at 1440°C with residual weight of 16% at 900°C. The cells grown on hydrogel composite showed significantly higher proliferation and ALP activity (p<0.01). Further, the quantification of ARS staining revealed significantly higher mineralization (p<0.01) in the composite hydrogel. Immunohistochemistry studies showed that the cells expressed both CD90 and osteocalcin, indicating that the cells are differentiating towards osteogenic lineage.

**Discussion:** The results suggest that the fabricated composite hydrogel could serve as injectable bone cements for non-invasive or minimally invasive defect filling in bone tissue engineering. The hydrogel is also capable of inducing mesenchymal stem cell differentiation towards osteogenic lineage.

**Acknowledgement:** This work was supported by MOE AcRF Tier 1 research grant (1T1-01/15) awarded to Asst. Prof. Dinesh Kumar Srinivasan, LKCMedicine, NTU.
<figure><img src="image_46.png" /><figcaption>Figure structure of hydrogel.jpg</figcaption></figure>
# Biogenic Gold Nanoparticles for Complete Recovery of Dermal Burn Wounds

Wednesday, 28th September - 16:25 - Targeted drug delivery and Nanocarriers - Tissue engineering and regenerative nanomedicine - Tower 24 - Room 107 - Oral presentation - Abstract ID: 277

**Dr. Suvadra Das**<sup>1</sup>, **Mr. Asim Halder**<sup>1</sup>, **Dr. Partha Roy**<sup>2</sup>, **Ms. Anwesha Banerjee**<sup>3</sup>, **Mr. Durbadal Ojha**<sup>3</sup>, **Mr. Saptarshi Mandal**<sup>1</sup>, **Dr. Debprasad Chattopadhyay**<sup>3</sup>, **Prof. Arup Mukherjee**<sup>1</sup>

1. University of Calcutta, 2. ADAMAS University, 3. ICMR Virus Unit, I.D. and B.G. Hospital

**Introduction:** Despite developments in regenerative medicine areas, the skin burn recovery remained a burden-some problem in critical care. The aim of this study was to arrive at safe flavonoid anchored gold nanoparticles and investigating them in dermal burn wound animal models.

**Methods:** Flavonolignan Silymarin is a proclaimed antioxidant and immunostimulant available from Silybum marianum. One pot bio-catalytic technique was applied for silymarin conjugated gold nanoparticles synthesis. Course of the reaction was followed systematically in LCMS and the appearance of gold plasmonic response was recorded in time scale. Silymarin conjugated gold nanoparticles (SmAunp) were characterized in DLS, XRD, FTIR, TEM, FESEM and ICPMS. Burn wound experiments were run in mice as per the guidelines of IAEC, University of Calcutta (Registration No. 506/01/a/CPCSEA, 2009-2010) approval No 506/01/a/CPCSEA/CUTech03. Experimental burn wound was inflicted under ether anaesthesia and the animals in group of 10 were treated with 5 µl SmAunp in water (600 µg/ml), standard silver sulfadiazine or saline control solutions. Dorsal skin burn was inflicted with 10 mm circular brass probe, 20 sec exposure at 100 0C. Recovery or not was recorded over a fourteen days treatment period. Histopathology and tissue biochemistry were recorded in each group intermittently and at the end of the experiment.

**Results:** Molecular bio-organic conjugation was confirmed in final product LCMS and in FTIR studies. Particle plasmon response was recorded at 540 nm and the average size in TEM was observed at 12.07 nm. SAED pattern and XRD observations proved fcc crystalline structure for the metallic gold. SmAunp exhibited excellent healing properties like wound closure, granulation, and vascularisation in comparison to standard drug. Histological analysis showed significant epithelialization in wounds treated with SmAunp.

**Discussion:** Wound healing properties of bio safe gold nanoparticles are reported for the first time. SmAunp exerted appropriate cytokines regulation and it is likely that the stem cells in the remaining hair follicular bulge were activated due to SmAunp. Favourable antioxidant properties of SmAunps were another factor for accelerated recovery.

**Acknowledgement:** Financial support to Dr. Suvadra Das from University Grants Commission (Sanction no. F.15-1/2015-16/PDFWM-2015-17-WES-33815(SA-II)), Government of India is thankfully acknowledged.
<figure><img src="image_47.png" /><figcaption>(A) TEM image of SmAunp (inset XRD spectrum of SmAunp). Histological features of partial thickness burn wounds on day 14 after burn injury (hematoxylin & eosin stain), (B) normal skin, (C) untreated wound, (D) SmAunp treated wound.</figcaption></figure>

Tem xrd studies and histological features.jpg
# WORKSHOP: Different approaches for the formation of synthetic hydrogels based on hybrid physically-chemically cross-linked networks

Wednesday, 28th September - 16:50 - Targeted drug delivery and Nanocarriers - Tissue engineering and regenerative nanomedicine - Tower 24 - Room 107 - Workshop presentation - Abstract ID: 413

Dr. Maxime Grillaud ¹, Mr. Maarten Bakker ¹, Dr. Patricia Dankers ¹

1. Eindhoven University of Technology

Many polymers with different types of cross-linking have been designed to develop various hydrogels for industrial and fundamental applications. In particular, they have attracted significant interest in the field of regenerative medicine, as drug delivery vehicles or scaffolds for tissue engineering. However, the main challenging limitation for their extensive use is their low mechanical stability. Many recent efforts have been made to overcome this burden, in particular by the elaboration of double network (DN) hydrogels. Despite the promising results of the few existing physically-chemically cross-linked DN hydrogels, the preparation procedures require long photopolymerization time and toxic precursor materials. Moreover, only the comparison of the mechanical properties between the hybrid DN hydrogel made in very specific conditions with the corresponding two separate networks is studied. The fundamental understanding of the combination of two different cross-links for a rational design of new hybrid DN hydrogels is a challenge.

Herein, we provide synthetic strategies to tailor in-situ formation of DN hydrogels based on physically-chemically cross-linked networks. Three approaches to combine catalyst-free click reaction and hydrogen bonding for in-situ hydrogel formation will be described (see Figure). More in detail, the covalent cross-linking consists of the click reaction between azides and cyclooctyne derivatives, referred to as strain-promoted azide-alkyne coupling (SPAAC), and the non-covalent cross-linking is based on supramolecular interactions via a four-fold hydrogen bonding motif, the ureido-pyrimidinone (UPy) unit.

The mechanical properties of these new hydrogels are studied by rheology and their morphology are analyzed by confocal microscopy. A detailed comparison of the hydrogel properties is discussed according to the approach selected and the ratio of the two types of cross-linking. In particular, this work shows the benefits to associate the dynamicity afforded by supramolecular interactions for gel recovering with the strength provided by the covalent cross-links for enhanced material stiffness. We propose that this work offers both a novel versatile platform for the easy preparation of DN hydrogels under physiological conditions with tunable properties, and one more step in the fundamental understanding of structure-property relationship of physically-chemically cross-linked hydrogels.
<figure><img src="image_48.png" /><figcaption>Figure abstract.jpg</figcaption></figure>
# The Drive to Master the Foundation Principles of Nanoscale interactions with living Organisms

Thursday, 29th September - 09:00 - Plenary Speeches - Amphitheatre 25 - Oral presentation - Abstract ID:
565

## Prof. Kenneth Dawson¹

1. Centre for BioNano Interactions, School of Chemistry, University College Dublin

The nanoscale is unique in biology, and our capacity to engineer on that scale will be transformative. Thus, the endogenous (intrinsic) machinery of biology is defined and operates on the nanoscale. Typical biomolecules and assemblies that are actively transported around organisms by specific motors and drivers are between 5-80nm. This means that nanoparticles are also actively (using the energy of the cell) transported around cells and biological barriers all unlike small molecules which passively partition into biological compartments (cells, organs etc). Secondly, the power of being able to communicate with, and use those endogenous mechanisms of biology is potentially transformative in practical terms. That is an enduring fact that renders our effort to draw the power of nano to the challenge of medicine. However, these could remain only words, or broad ideas in our generation, if we do not learn how to engage this enormous potential to interact with the machinery of organisms. There are

deep challenges. First of all, the complexity of the interaction is remarkable, much more so than for small cell and molecules, or large particles. Capturing this new capacity for benefit of human society will require dedication and commitment, indeed an exceptional generational effort, rather than peripheral or aspirational research. Both the potential, and the challenge, of this field may have been underestimated, but now now we have faced the need to invest in

guiding principles and laws governing the whole arena, the true picture is unfolding quickly. We are optimistic. We discuss progress being made in understanding how interactions between nanoscale objects and living organisms occur, and their governing principles. [2,3] We argue that the future lies in pressing forward to develop a truly microscopic (molecular scale) understanding between the nanoscale and living organisms. [4]

## References

1. Proceedings of the National Academy of Sciences 104 (7), 2050-2055 (2007).

2. Nature nanotechnology 7 (12), 779-786 (2012)

3. Nature nanotechnology 7 (1), 62-68 (2012)

4. Nature nanotechnology 10 (5), 472-479 (2015)
# Biocompatible Metal Organic Frameworks in Nanomedicine

Thursday, 29th September - 09:40 - Plenary Speeches - Amphitheatre 25 - Oral presentation - Abstract ID:
30

Dr. christian serre¹

1. ENS-ESPCI-PSL-CNRS

Metal Organic Frameworks (MOFs) are a recent class of tuneable porous hybrid solids based on metal sub-units connected to organic complexing molecules (carboxylates, phosphonates, azolates...) delimiting an ordered porous network. Their unprecedented structural and chemical diversity is of potential interest for many applications such as gas storage, separation or catalysis, among others.(1) Recently, some of us demonstrated their high potential in biomedicine for drug delivery, release of biologically active gases or imaging.(2) This led in most cases to very high drug loading capacities and a prolonged release using a wide range of active drugs with different polarities and sizes.(3) We will report here the recent progresses in this field from the synthesis, degradability, toxicity and biodistribution of nanoparticles of biocompatible MOFs,(4) to their encapsulation, release and in vitro or in vivo properties.(5-10)

## References

1-Themed issue: Metal-organic frameworks, Chem. Soc. Rev., 2014

2 M. Gimenez et al., Coord. Chem. Rev., 2015

3- (a) P. Horcajada et al. Angew. Chem., Int. Ed. 2006, 45, 5974

4- Baati, T.; et al., Nat. Mater., 2010, 9, 172

5- T. Chalati, et al., Nanomedicine, 2011, 6(10), 1657

6- J.E. Eubank et al. APL Mater., 2014, 2, 124112

7- Agostoni, V et al., Adv. Healthcare. Mater. 2013, 2(12), 1630-1637.

8- (a) E. Bellido et al. Langmuir, 2014, 30(20), 5911-5920; (b) E. Bellido et al. Adv. Health. Mater., 2015 4(8), 1246-1257

9- V. Agostoni et al. Scientific Reports, 2015, 5, 7925

10- Cooper, L. et al. Chem. Comm. 2015, 51(27), 5848-5851
# Dendrimers as tools towards nanomedicine

Thursday, 29th September - 10:45 - Plenary Speeches - Amphitheatre 25 - Oral presentation - Abstract ID:
250

Dr. Anne-Marie Caminade¹

1. LCC-CNRS Toulouse

Dendrimers [1] are hyperbranched and nanosized macromolecules, synthesized step-by-step in an iterative fashion to ensure a perfect control of all their structural parameters (chemical composition, size, weight, etc.), and which may have outstanding properties. [2] Most of these properties of dendrimers depend on their terminal functions, but surprisingly, the internal structure may also play a key role, especially when considering the biological properties. [3]

We synthesize phosphorus-containing dendrimers (i.e. dendrimers having a phosphorus atom at all the branching points), and we can play with both their internal structure [4] and the type of their terminal functions. These dendrimers, when specifically functionalized (see Figure), display many biological properties ranging from transfection to immuno-stimulation, [5] from antivirals to anti-prion, anti-inflammatory [5, 6, 7] and anticancer [8] agents. These biological properties of phosphorus-containing dendrimers will be emphasized.

## References

[1] Dendrimers. Towards Catalytic, Material and Biomedical Uses. Caminade A.M., Turrin C.O., Laurent R., Ouali A., Delavaux-Nicot B, Eds., John Wiley & Sons, Chichester (UK) 2011.

[2] Caminade A.M., Ouali A., Laurent R., Turrin C.O., Majoral J.P., Chem. Soc. Rev. 2015, 44, 3890.

[3] Caminade A.M., Fruchon S., Turrin C.O., Poupot M., Ouali A., Maraval A., Garzoni M., Maly M., Furer V., Kovalenko V., Majoral J.P., Pavan G.M., Poupot R. Nature Comm. 2015, 6, 7722.

[4] Galliot C., Larré C., Caminade A.M., Majoral J.P., Science 1997, 277, 1981.

[5] Hayder M., Poupot M., Baron M., Nigon D., Turrin C.O., Caminade A.M., Majoral J.P., Eisenberg R.A., Fournié J.J., Cantagrel A., Poupot R., Davignon J.L., Science Transl. Med. 2011, 3, 81ra35.

[6] Blattes E., Vercellone A., Eutamène H., Turrin C.O., Théodorou V., Neyrolles O., Majoral J.P., Caminade A.M., Prandi J., Nigou J., Puzo G., Proc. Nat. Acad. Sci. USA 2013, 110, 8795.

[7] Le Dall J., Fruchon S., Garzoni M., Pavan G.M., Caminade A.M., Turrin C.O., Blanzat M., Poupot R. Nanoscale 2015, 7, 17672.

[8] El Brahmi N., Mignani S.M., Caron J., El Kazzouli S., Bousmina M.M., Caminade A.M., Cresteil T., Majoral J.P. Nanoscale 2015, 7, 3915.
<figure><img src="image_49.png" /></figure>

Examples of biologically active functions linked to the surface of a
phosphorus-containing dendrimer

<caption>Dendrimer bio.png</caption>
# From Nano Shape & Self Recognition to Flexibility in Cancer Treatment and Differentiation

Thursday, 29th September - 11:25 - Plenary Speeches - Amphitheatre 25 - Oral presentation - Abstract ID:
527

Prof. dennis discher ¹

1. University of Pennsylvania

From viruses to tissue matrices, biology is filled with remarkable polymeric structures that motivate mimicry with goals of both clarifying and exploiting biological principles. Filamentous viruses inspired our development and computations of worm-like polymer micelles – ‘filomicelles’ – that persist in the circulation and deliver even better than spheres [1]. However, particles of any type interact with innate immune molecules and also phagocytes while nearby ‘Self’ cells are spared due to a polypeptide that limits phagocytic clearance [2]. The phagocyte’s cytoskeleton forcibly drives the decision downstream of adhesion, proving analogous to how matrix elasticity directs stem cell fate [3, 4].

[1] Y. Geng, P. Dalhaimer, S. Cai, R. Tsai, M. Tewari, T. Minko, and D.E. Discher. Shape effects of filaments versus spherical particles in flow and drug delivery. Nature Nanotechnology (2007) 2: 249-255.

[2] P.L. Rodriguez, T. Harada, D.A. Christian, D.A. Pantano, R.K. Tsai, and D.E. Discher. Minimal ‘Self’ peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles. Science (2013) 339: 971-975.

[3] A. Engler, S. Sen, H.L. Sweeey, and D.E. Discher. Matrix elasticity directs stem cell lineage specification. Cell (2006) 126: 677-689.

[4] J. Swift, I.L. Ivanovska, ... and D.E. Discher. Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-directed Differentiation. Science (2013) 341: 1240104-1 to 15.
# Magnetic force-based skeletal muscle tissue engineering for in vitro drug testing

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 64

Dr. Akira Ito¹, Mr. Kazushi Ikeda¹, Mr. Ryusuke Imada¹, Dr. Masanori Sato¹, Dr. Yoshinori Kawabe¹, Prof. Masamichi Kamihira¹

1. Kyushu University

**Introduction:** Skeletal muscle tissue engineering holds great promise for pharmacological studies. In vitro culture systems are available for drug discovery for patients with injured, diseased and age-related muscle. For drug testing, conventional 2D cell culture systems are based on formation of myotubes on tissue culture dishes. However, the 2D cell culture systems have limitations to mimic in vivo skeletal muscle functions mainly due to lacking the architecture of native muscles. One of the most important characteristics of skeletal muscle is contractile force generation ability, and tissue-engineered skeletal muscle constructs should mimic the architecture of native muscles and reproduce contractile force generation. In the present study, we demonstrate an in vitro system for drug testing using tissue-engineered skeletal muscle constructs. **Methods:** We have developed a fabrication method of functional skeletal muscle tissue constructs by using a magnetic force-based tissue engineering technique, in which myoblasts were labeled with magnetite nanoparticles, and assembled by magnetic field to form a cell-dense and aligned skeletal muscle-like structure. Small-molecular drugs are used as a model drug. **Results and Discussion:** In response to small-molecular drugs, myotube differentiation of myoblasts were promoted in 2D cell culture. However, the levels of contractile force generation of tissue-engineered skeletal muscle constructs treated with small-molecular drugs were not consistent with those of myotube differentiation in 2D cell culture. On the other hand, there was a high correlation between sarcomere formation as well as contractile activity in two-dimensional cell culture and contractile force generation of tissue-engineered skeletal muscle constructs. These observations indicate that the contractility data is indispensable for in vitro drug screening.
Characterization of Noble Metal Nanoparticles
functionalized by molecule-capping method with mixed
organic ligands carried out by SR-XPS and SERS

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 69

Ms. Laura Carlini ¹, Prof. Chiara Battocchio ¹, Prof. Paolo Postorino ², Ms. Claudia Fasolato ², Dr. Ilaria Fratoddi ², Dr. Iole Venditti ², Ms. Giovanna Testa ², Dr. Fabio Sciubba ²

1. Roma Tre University, 2. Sapienza University of Rome

This work is focused on the study of the structural, electronic and morphological properties of Noble Metal Nanoparticles (MNP-s) for innovative applications in nano-medicine and nano-biotechnology.A key point for the technological development of nano-structured materials and practical biomedical devices based on MNP-s is the achievement of a fine control of the stability and toxicity of the system.The chemical functionalization of the MNP-s can be done by means of capping metallic clusters with appropriate organic ligands.The molecule-capping method provide a reliable control of particle composition, shape and size distribution making the MNP-s suitable for active purposes in catalysis, nanoelectronics, sensing and bioanalysis.In this communication, we present MNP-s (Au and Ag) functionalized with mixed organic ligands (DEA and 3MPS) prepared with different metal/thiol stoichiometric ratios.The molecular overlayer has been selected on purpose for the biomedical applications, as well as to stabilize the nanoparticles.The changes in the stoichiometric ratio between metal and different capping agents can influence the chemical properties of ligands functional groups and the dimension of the functionalized MNP-s.Moreover, the biocompatibility of the system depends strictly on the charge and thickness of capping molecules layer.Therefore, the study of the interaction at the interface between the organic ligand and MNP-s at atomic level is the first step for the fabrication and developing of very suitable nanomaterials for diagnostic and therapy.The characterization of the nano-systems was carried out by means of Synchrotron Radiation induced X-ray Photoelectron Spectroscopy (SR-XPS) and Self Enhanced Raman Spectroscopy (SERS).SR-XPS provides information on the local bonding environment of a given species and it has been demonstrated to be a unique tool for investigating the nature of the interaction at the capping agent/metal nanoparticle interface, as well as the chemical structure of MNPs surface.In SERS, the Raman intensity diffused by molecules close to a nano-curved metallic surface is highly enhanced by the localized surface plasmon resonance (LSPR), allowing the spectroscopical investigation of molecular monolayers.In conclusion, we compared the semi-quantitative SR-XPS and SERS analysis to obtain a better understanding of the system exploring the potential synergy between different techniques in order to give new insights in the field of nanomaterials.
<figure><img src="image_50.png" /></figure>

Noble Metal Nanoparticle functionalized by sodium 3-mercapto-1-propansulfonate (3MPS) and 2-diethylaminoethanethiol hydrochloride (DEA).

<figure><img src="image_51.png" /></figure>

SERS spectra of functionalized MNPs.

<caption>Functionalized mnp.jpg</caption>

<figure><img src="image_52.png" /><figcaption>Sr-xps spectra.jpg</figcaption></figure>
# Incorporation of paclitaxel into hollow-p4VP nanoparticles to improve breast cancer chemotherapy

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 74

Ms. Maria del Carmen Leiva Arrabal¹, Ms. Julia Jiménez-López¹, Dr. Rafael Contreras-Caceres², Ms. Laura Cabeza¹, Dr. Gloria Perazzoli¹, Dr. Raúl Ortiz³, Prof. Consolación Melguizo¹, Prof. Juan Manuel López-romero², Prof. Jose Prados¹

1. Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, Spain; Biosanitary Institute of Granada (ibs.GRANADA), Andalusian Health Service (SAS), University of Granada, Spain. 2. Department of Organic Chemistry, University of Málaga, Málaga, Spain. 3. Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, Spain; Department of Health Sciences, University of Jaén, Jaén, Spain.

**Introduction:** Paclitaxel (PTX) is being currently used in the treatment of breast cancer, though it presents some drawbacks as low solubility, no tumor specificity and high toxicity. PTX loading into nanoparticles (NPs) may overcome those limitations, avoiding the use of toxic Cremophor EL in the formulation, and improving treatment efficiency. In this work, we have developed hollow poly(4-vinylpyridine) nanoparticles (p4VP) for PTX delivery, and their biocompatibility and antitumor activity against breast cancer has been in vitro studied.

**Methods:** Firstly, biocompatibility of p4VP was analyzed by a hemolysis assay with human blood. Sulphorodamine B method was used to compare PTX and PTX-loaded p4VP (PTX@p4VP) cytotoxicity on human breast MCF7 cancer cells and MCF-10A normal cell line. Moreover, their activity against multicellular tumor spheroids (MTS) formed from MCF7 cells, was studied by tracking their volume every 2 days, and by immunostaining with TUNEL to compare cell death after different treatments.

**Results:** Hemolysis assay with human blood proved no toxicity of p4VP. In addition, cell death was improved by the use of PTX@p4VP in both cell lines, whereas the effect was more evident on tumor cells (2.9-fold reduction of PTX IC50 vs 1.7-fold). Furthermore, MTS treated with PTX@p4VP showed a significant volume reduction than those treated with PTX. However, both treatments achieved a reduction in MTS volume in comparison to controls and blank p4VP. Immunofluorescence corroborated those data by staining death cells on the surface of MTS treated with PTX and PTX@p4VP.

**Discussion:** According to those results, p4VP present good biocompatibility that make them suitable for in vivo drug delivery. They are also able to enhance PTX antitumor effect against breast cancer cells and breast MTS, which resemble tumor mass. In the light of our findings, PTX@p4VP represent a promising delivery system for the improvement of chemotherapy involved in the treatment of breast tumor.
<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

Figures:

A) Cytotoxicity of PTX@p4VP vs PTX in MCF7 breast tumor and MCF10A normal cell lines.

B) Inmunofluorescence of MTS to stain apoptotic cells after different treatments.

C) Hemolysis percentage in human blood incubated with blank p4VP NPs.

P4vp nps with ptx against breast cancer.jpg
# Tannin-chitosan composite nanoparticles as potencial nanomedicine to prevent urinary tract infections

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 77

Dr. Sergio Madrigal-Carballo ¹, Ms. Emilia Alfaro-Viquez ², Dr. Christian Krueger ², Prof. Jess Reed ²

1. National Center for Biotechnological Innovations (CENIBiot), CeNAT-CONARE, 2. Reed Research Group, University of Wisconsin-Madison

Introduction. Naturally occurring renewable resources that inhibit microbial adhesion are important alternatives to antibiotics and may help stem the alarming increase in drug resistant bacteria. Bacterial invasion of gut epithelial cells (enterocytes) provides a mechanism by which pathogens are protected from complements, antibodies, and other immune defense molecules, which in turn, allows the pathogens to colonize and persist in the gut. We have invented novel composite biomaterial comprising formulations of tannin, extracted from cranberries (fruit, juice or press cake), and chitosan, manufactured from shrimp shells. The composites are versatile and can be formulated into useful biomaterials, such as nanoparticles, 3-dimensional foams, and films. Methods. Chitosan binds to negatively charged tannins by an electrostatic interaction driven by its positively charged amino group. This interaction allows developing stable hybrid nanoparticles via ionotropic gelation with tripolyphosphate (TPP), suitable as a therapeutic controlled release system for urinary tract infections. We study the effect of cranberry tannin-chitosan composite nanoparticles on the invasion of Caco-2 cells by Uro-pathogenic Escherichia coli (UPEC) strain 5011, supplied by the UW-University Hospital (Madison, WI, USA). Results. Results of a dose-response experiment indicate the cranberry tannin-chitosan composite nanoparticles significantly reduced invasion of Caco-2 cells by extra-intestinal pathogenic E. coli (ExPEC). The nanoparticles significantly inhibited the ability of the pathogen to invade the Caco-2 cells by 40%, 80% and 96% at a total polyphenolic concentration of 0.2 µg, 0.5µg and 0.75µg GAE/ml (respectively). Discussion. Figure shows scanning electron micrographs exploring the effect of tannin-chitosan composite nanoparticles on ExPEC surface structures that are involved in cell adhesion and subsequent inhibition of invasion of intestinal epithelial cells in vitro. When the pathogen was exposed to the tannin-chitosan composite nanoparticle material (Fig. C), extensive coating and cross-linking on multiple bacteria was seen (Fig. B), compared to control (Fig. A). Results suggest the cranberry tannin-chitosan composite nanoparticles physically coat the surface virulence factors of ExPEC, which in turn prevents invasion of the intestinal epithelial cell.
<figure><img src="image_55.png" /><figcaption>Tannin-chitosan nanoparticles interactions with
expec.png</figcaption></figure>
# Lung cancer: a new approach to paclitaxel treatment using PLGA nanocarriers

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 90

Ms. Julia Jiménez-López¹, Ms. Maria del Carmen Leiva Arrabal¹, Dr. Mazen El-hammadi², Ms. Laura Cabeza¹, Dr. Gloria Perazzoli¹, Dr. Lucía Martín-Banderas², Dr. Raúl Ortiz³, Prof. Jose Prados¹, Prof. Consolación Melguizo¹

1. Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, Spain; Biosanitary Institute of Granada (ibs.GRANADA), Andalusian Health Service (SAS), University of Granada, Spain. 2. Pharmacy and Pharmaceutical Technology, University of Sevilla, 3. Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, Spain; Department of Health Sciences, University of Jaén, Jaén, Spain.

## Introduction

Lung cancer is the type of cancer with the highest mortality rate worldwide. Paclitaxel (PTX), an antimitotic drug, is widely administered for the treatment of choice for this type of cancer and other solid tumours. However, PTX has many limitations and is associated with many undesirable effects like neutropenia, neurotoxicity or hypersensitivity reactions. In this work a new type of polymeric nanoparticles of lactic and glycolic acid (PLGA) loaded with this drug for the treatment of lung cancer is proposed to solve these limitations.

## Methods

Proliferation assays with sulforhodamine B in two human lung tumor cell lines, A549 and NCI-H460, were performed in order to compare the effect of free PTX and NPs loaded with this drug (NP-PTX). Cytotoxicity studies with blank nanoparticles were performed. The mechanisms of action of PTX and NP-PTX were tested with cell cycle assays by flow cytometry. For cellular uptake studies with fluorescence microscopy, A549 was incubated with Nile red (NR) and NR-loaded NPs at different times. Finally, multicellular tumour spheroids (MTS) assays in A549 were performed and MTS were treated with PTX, NP-PTX and blank NPs at the IC50 dose of free PTX. MTS growth was monitored with imaging microscopy.

## Results

The results obtained in proliferation assays showed significant dose reduction IC50 with NP-PTX up to 3.63 and 3.79 times lower in A549 and NCI-H460, compared to free PTX. No inhibition was observed in the growth of any cell lines with blank nanoparticles. The results showed enhanced cell cycle accumulation in G2 and sub-G1 phase in cells treated with NP-TX facing PTX-treated cells. Intracellular uptake results suggest that NP-PTX facilitate the incorporation of NR into the cell compared with free NR. Finally, the MTS assays showed a significant and greater reduction in the volume of spheroids treated with NP-PTX (73%) than treated with free PTX (46%). No significant difference was observed between MTS untreated and treated with blank NPs.

## Discussion

In short, these promising in vitro results obtained suggest that PLGA NPs provide a potential strategy as a mechanism for PTX encapsulation and could allow increasing the therapeutic index of this drug.
<figure><img src="image_56.png" /></figure>

In vitro results of a polymeric nanoparticle loaded
with paclitaxel for the treatment of lung cancer.png
# Nanofibers Preparation by Free-Liquid Surface Electrospinning for Cartilage Tissue Engineering

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 95

Ms. Parinita Agrawal ¹, Prof. Krishna Pramanik ¹

1. National Institute of Technology, Rourkela, Odisha

Cartilage degeneration due to aging like osteoarthritis, or developmental abnormalities and trauma causes severe damage to cartilage tissue. Adult cartilage tissue has limited self-repair capacity thus the deformity is generally not self-cured. Tissue engineering can provide cure to this problem by designing scaffold of appropriate composition to mimic the extra cellular matrix of the damaged tissue on which the stem cells could grow to form functional cartilage tissue. Electrospinning is the method for formation of nanoscale diameter fibrous matrix, which provides high surface area, beneficial for cell adhesion. Free-liquid surface electrospinning is an advanced method that provides higher productivity. In the present study, we explore electrospinning for fabricating scaffolds from chitosan (CS) blended with regenerated silk fibroin (SF). To improve the processing ability of the blend solution, poly(ethylene oxide) (PEO) was added to the blend which was then successfully electrospun. SF is a naturally occurring biodegradable fibrous protein having good mechanical properties and biocompatibility. CS has intrinsic antibacterial activity and biocompatibility that makes it suitable for the tissue engineering applications.

The morphology of nanofibrous matrix was characterized by scanning electron microscopy (SEM). Crystalline nature and hydrophilicity of the nanofibrous matrix was confirmed by X-ray diffractometry and contact angle analysis, respectively. The matrix was found to possess biodegradation rate and tensile strength adequate for cartilage tissue replacement. The electrospun nanofibers also supported growth and proliferation of mesenchymal stem cells (MSCs) as observed by field emission SEM and MTT studies. Histological and fluorescence microscopic analysis confirmed the differentiation of MSCs to chondrocytes, on the nanofibrous matrix. The confirmatory study by quantitative-PCR validated the potential of the prepared nanofibrous matrix for cartilage tissue engineering applications.

Keywords: Nanofiber, electrospinning, tissue engineering, chitosan, silk fibroin, poly(ethylene oxide)
# Evaluation of chloroaluminium phthalocyanine-loaded magnetic nanoemulsion as drug delivery device to treat glioblastoma using hyperthermia and photodynamic therapy

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 101

Dr. Leonardo Barcelos de Paula ¹, Prof. Fernando Lucas Primo ², Prof. Marcelo Rodrigues Pinto ³,
Prof. Paulo Cesar Morais ⁴, Prof. Antonio Claudio Tedesco ¹

1. Department of Chemistry - University of Sao Paulo - USP - Ribeirao Preto/SP, 2. Department of Bioprocess and Biotechnology - Univ. Estadual Paulista Júlio de Mesquita Filho - UNESP - Araraquara/SP, 3. University of Uberaba - Uberaba/MG, 4. College of Chemistry and Chemical Engineering - Anhui University - Hefei

**Introduction:** Brain tumors known as glioma are the most common neoplasms found in the central nervous system of adults. In a brain cancer tumor cancer stem cells (CSCs) represent a small fraction of the total cancer cell population. Studies have suggested similarities of CD133+ CSC with the proneural subtype and of CD133 CSC with the mesenchymal subtype. **Methods:** We developed two samples magnetic nanoemulsions (MNEs) loaded with magnetic nanoparticles (MNPs) plus photoactivated drugs, chloroaluminium phthalocyanine (ClAlPc), (0.15×10¹⁶ or 1.50×10¹⁶ MNP/mL), were obtained through a spontaneous emulsification process based on oil-in-water mixtures. All the cell lines (human mesenchymal stem cells derived from bone marrow - BM-MSC and the human glioma - U87MG and T98G) were cultivated in alpha-MEM, DMEM-low and 10% fetal bovine serum at 37 °C and 5% CO2, respectively. The AC magnetic field during the hyperthermia (HPT) studies operates at 1 MHz with 40 Oe magnetic field amplitude. Photodynamic therapy (PDT) protocol used a typical diode laser set at 670 nm and operates with 100, 200, and 700 mJ/cm² fluency. For different treatments the cell viability was assessed by the MTT test. **Results and Discussion:** Particle size analysis showed that all colloidal formulations produced in the present study and containing ClAlPc were on the nanoscale size, with typical mean diameter around 200 nm and size distribution value of 0.25. Cytotoxicity assay was conducted with the MNE/ClAlPc samples at the two distinct MNP concentrations while keeping fixed the ClAlPc concentration (0.5 µmol/L). Synergism of the combined HPT and PDT treatments, revealing an increase of up to 60-70% in cell death. **Conclusion:** From the present study we found that the HPT treatment reduces the cell viability in about 15%, regardless the magnetic nanoparticle content within the MNE. However, a superior reduction in cell viability of about 70% was found while combining the HPT and PDT treatments. Moreover, confocal microscopy images clearly indicated the cytoplasm localization and active site of the as-prepared drug delivery device. Therefore, we envisage that the combined treatment of HPT and PDT represents a promising paradigm for brain cancer intervention, such as glioblastoma.
Synthesis, research and functionalization of hybrid
contrast agents based on gadolinium doped magnetite

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 136

Mrs. Iana Tcareva ¹, Prof. Alexander Majouga ², Prof. Alexander Savchenko ¹, Mr. Maksim Abakumov ³, Mr. Igor Shchetinin ¹

1. National University of Science and Technology, MISiS, 2. Lomonosov Moscow State University, 3. The Pirogov Russian
National Research Medical University (RNRMU)

Magnetic Resonance Tomography, MRI, is one of the most used instruments for non-invasive clinical diagnostics.
In contrast to radiological investigations, MRI has no danger of radiation exposure to produce images using radio
frequency electromagnetic radiation with very low energy. The most researches in this area are focused on the
development of contrast agents, that can provide a clearer distinction between healthy and diseased tissue. The
majority of contrast agents are magnetic nanoparticles (MNP).

MNPs of iron oxide are used for the diagnostics of many diseases such as cardiovascular, neurological and cancer tumors. Liver and prostate cancer are the most prevalent of malignant tumors. Currently a priority re-
searches of contrast agents for MRI are hybrid contrast agents, providing comprehensive diagnostic information
about the dynamics of the disease progression. Gadolinium chelates and magnetite are the most appropriate
T1 and T2 contrast agents, respectively, but these compounds can provide toxic effect on healthy cells. One of
the ways to prevent toxicity is the creation of hybrid contrast agents based on gadolinium doped magnetite.

In this work gadolinium doped magnetite nanoparticles were prepared by two techniques: thermal decomposi-
tion of iron and gadolinium acetylacetonates in diphenyl ether and hydrolysis of Fe3+ и Gd3+ salts mixture in the
presence of polyethyleneimine, followed by addition of hydrazine hydrate and microwave irradiation. These
nanoparticles are designed to be used as a hybrid contrast agents for hepatocellular and prostate carcinomas
visualization.

Obtained nanoparticles were investigated by methods of transmission electron microscopy, X-ray diffraction, Mössbauer spectroscopy, VSM magnetometry and thermogravimetric analysis. Also the toxicity of nanoparticles and their T2-relaxation time were measured in vitro. In vivo examinations on mouse model of liver cancer were carried out.

The authors knowledge financial support from Ministry of Education and Science of the Russian Federation (14.607.21.0132, RFMEFI60715X0132).
# In-depth investigation on DNA-AgNCs designs for adenosine detection

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 184

Ms. Shi Ting Lee ¹, Dr. Siu Yee New ¹

1. The University of Nottingham Malaysia Campus

Silver nanocluster (AgNCs) as like other metal nanoclusters has size of approaching Fermi wavelength of electron ( 2 nm). This nanomaterial no longer possesses surface plasmon resonance (SPR). Indeed, its energy structure is in a discrete level that similar to those of molecules, enable it to absorb intense light and emit strong fluorescence colour upon excited with UV light. Among stabilising ligands, DNA template has particularly gained more attention. Apart from its versatility, it may due to the feasibility to integrate DNA aptamer into the template. This aptamer functions exactly like antibody and binds specific to a target analyte. Hitherto, there is no detailed study about the impact of sequence template arrangement (i.e. AgNCs nucleation sequence and target recognizing sequence) on the sensing performance of AgNCs. In this work, we are investigating the effect of different combination between AgNCs nucleation sequence (NC) and adenosine aptamer (Apt A) onto adenosine detection. Three different designs have been formulated in order to systematically position AgNCs nucleation sequences and adenosine aptamer in the same template, namely Apt A 5, Apt A 3 and Apt A M with the last letter implies the position of AgNCs nucleation sequence at 5'-end, 3'-end and in the middle. Among these designs, only Apt A 5 design gives selective result against adenosine, with a 0.5-fold enhancement in the emission intensity observed. The linear range of detection is wide and across four order of magnitude, 1 – 2500 µM. We deduced that the fluorescent increment is correlated to the guanine rich bases in the aptamer sequence.

<figure><img src="image_57.png" /></figure>

Graphic abstract - dna-agncs design to adenosine detection.png
# Noncovalent Assembly of Carbon Nanotubes: Toward the Construction of Nanotube-Based Breast Cancer Therapy Nanovectors

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 204

Dr. Ayhan Unlu¹, Mr. Mehdi Partovi Meran², Ms. Bircan Dinc³, Mr. Yasin Celikok⁴, Prof. Isil Albeniz⁵, Prof. Seniha Guner²

1. Trakya University, 2. Istanbul Technical University, 3. Istanbul Kemerburgaz University, 4. Yeni Yuzyil University, 5. Istanbul University

In recent years a wide range of different nanoscale drug delivery vectors have been evaluated. Among the numerous nanoscale systems, carbon nanotubes (CNTs) hold great potential for diverse applications and are becoming a viable component of biomedical science. Apart from their uses in the cellular imaging with diagnostic effects in nanomedicine, CNTs are promising drug carriers in the drug delivery systems for cancer therapies. One of the key advantages of CNTs in biomedical applications is that they can be easily internalized by cells and, therefore, can act as delivery vehicles for a variety of molecules relevant to therapy and diagnosis. In addition, the high aspect ratio of CNTs offer great advantages over existing delivery vectors, as the high surface area provides multiple attachment sites for drug targeting. The main obstacle for utilization of pristine CNTs are their scarce solubility in any aqueous environment due to the graphitic nature of their sidewalls. To overcome this problem, CNT surfaces should be modified by noncovalent coating or by covalent functionalization approaches. In this study, we describe a non-covalent Single-Walled Carbon Nanotubes (SWCNT)-doxorubicin supramolecular complex that can be developed for cancer therapy. In light of the unique properties of SWCNT and the hydrophilic and flexible ability of PEG chains, we linked PEG chains and pyrene molecules, which can strongly attach to SWCNT surface via π-π stacking. Different monocarboxylic acid-terminated PEGs were synthesized and subsequently PEG-Pyrene was prepared via an esterification reaction of PEG-COOH with pyrene methanol. Finally, SWCNT and PEG-pyrene heterogeneous mixture was sonicated and stirred vigorously in THF to gain PEG modified SWCNT. We initially examined the cytotoxicity of different PEG-modified SWCNT samples in MDA-MB-231 breast cancer cell line and determine IC50 values for this type cells by using MTT cell proliferation assay in our study. The results indicated that PEG-modified SWCNTs have significantly lower cytotoxicity. At the final stage, the interaction between Doxorubicin and functionalized SWCNT was studied by monitoring the emission spectrum of doxorubicin by fluorescence spectrophotometry.
Molecular dynamics simulation of interaction of lysine
dendrimer and Semax peptide

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 214

Mrs. Elena Popova ¹, Prof. Igor Neelov ², Mr. Victor Kuznetsov ¹, Mr. Sergey Petunov ¹, Mr. Angrey Radilov ¹

1. Research Institute of hygiene, occupational pathology and human ecology, 2. ITMO University

Peptide drug delivery systems development is one of the most important tasks in modern pharmacology. Therapeutic peptides are efficient and have minimum side effects. Parenteral route of peptide administration is the most effective. The main problem in oral route is the action of proteolytic enzymes on peptides and their low mucosal permeability. It is proposed to use dendrimers to solve the problem. Dendrimer is a macromolecule with a symmetrical branched structure. It has stable charge and size. Dendrimers reduce toxic effect of peptides and ensure targeted delivery into cells (e.g., cancer). In some cases, dendrimers with drugs complexes have a much more pronounced therapeutic effect. The regulatory peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) as a model one was chosen. It has an antioxidant, antihypoxic and neuroprotective effects. It is used for acute ischemic stroke prevention, traumatic brain injury, diseases of optic nerve and glaucoma optic neuropathy treatment, during rehabilitation after stroke.

Currently, method of molecular dynamics is the main method of polymer complexes and biopolymer systems modelling. The simulation was performed via this method for systems consisting of one lyzine dendrimer molecule (32 NH3+ groups), 8, 16 and 24 Semax molecules, water and chloride counterions in a cubic cell with periodic boundary conditions. Further energy minimizations and simulations using GROMACS 4.5.6 package and AMBER_99SB-ildn force fields were performed. The potential energy of this force field consists of valence bonds and angles deformation energy, angles of internal rotation, Van der Waals and electrostatic interactions. Complexes with peptides were formed in 30-60 ns. The equilibrium size and anisotropy of the complexes were close. Radial distribution of atom number shows that dendrimer was inside, and peptides mainly on the surface of complexes. Hydrogen bonds and ion pairs number of the third and the second complexes were less because of the less close contact between dendrimers and peptides. It is shown that dendrimer of third generation can attach about 22 Semax molecules.

Keywords: dendrimers, peptides, computer simulation, molecular dynamics method.
# Salinomycin nanoparticles induce selective toxicity toward tumor cells rather than stroma in orthotopic model of pancreatic cancer

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 229

Dr. zahra Daman¹, Dr. Yuhua Wang², Prof. Leaf Huang²

1. Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, 2. Laboratory of drug targeting, Eshelman School of Pharmacy, University of North Carolina in Chapel Hill, NC, USA

## Introduction

Salinomycin (SAL), a selective inhibitor of cancer stem cells (CSCs), was recently recognized for its ability to inhibit proliferation and induce apoptosis in various tumors including highly chemo-resistant ones. The aim of this study was to deliver SAL to orthotopic model of pancreatic cancer by the aid of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and explore some mechanistic questions regarding this treatment.

## Methods

SAL-loaded NPs were prepared and investigated in terms of pharmaceutical properties. MTT assay was used to study cell proliferation in AsPC-1-luc cells. In vivo antitumor study was performed in nude mice orthotopically implanted with AsPC-1-luc cells and treated by IV administration of saline and SAL NPs for control and test groups, respectively. Tumor growth was monitored weekly through in vivo imaging of luciferase activity. The collected tumors from SAL-treated and control groups were used for protein extraction and immunofluorescent staining of different markers.

## Results

Nano formulation of SAL was prepared in suitable size and loading traits. SAL (3.5 mg/kg every other day) blocked tumor growth by 52% compared to the control group after nine injections. Western blotting of tumor protein extracts indicated that SAL treatment leads to up-regulation of E-cadherin and β-catenin expressions in AsPC-1 orthotopic tumor. Noteworthy, immunofluorescence staining of adjacent tumor sections demonstrated that treatment with SAL NPs leads to considerable apoptosis in the tumor cells rather than the stroma.

## Discussion

Herein, we utilized PEG-PLGA as a biocompatible and biodegradable polymer to formulate SAL into polymeric NPs, so that it could be stably solubilized for further investigations. Regarding in vivo treatments, a 52% reduction in tumor size compared to the control group highlights SAL-loaded PLGA NPs as a promising system for pancreatic cancer treatment. The results of concurrent CD31/α-SMA staining and the TUNEL assay showed that the apoptotic pancreatic cancer cells were mainly distributed in the tumor nest, which confirms efficient intratumoral extravasation and penetration of SAL-loaded NPs. Our data suggested that SAL can harness epithelial-mesenchymal transition through increased co-expression of E-cadherin and its binding protein, β-catenin. However, more mechanistic research is needed before any translation of SAL-based treatments into clinics.
<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /><figcaption>Fig iconan.jpg</figcaption></figure>
Antibacterial effects of gold-chitosan nanocomposites
on human macrophages infected by intracellular
pathogenic bacteria

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 267

Dr. Gracia Mendoza ¹, Ms. Anna Regiel-futyra ², Dr. Vanesa Andreu ¹, Dr. Víctor Sebastián ³, Dr.

Agnieszka Kyziol ², Prof. Grażyna Stochel ², Dr. Manuel Arruebo ³

1. University of Zaragoza, 2. Jagiellonian University, 3. Universidad de Zaragoza

Introduction: Bacteria have developed resistances to antibiotics with the subsequent negative consequences in the treatment of different pathogenic infections. Nanomaterials have emerged as a potential bactericidal approach to avoid multidrug resistances. We previously demonstrated that gold-chitosan nanocomposites show bactericidal but not cytotoxic action at the doses tested (Regiel-Futyra et al., 2015). Here, we evaluated their action in a co-culture model of macrophages infected by different bacteria to closely mimic a real infection scenario.

Methods: Nanocomposites were prepared by a solvent evaporation method by using chitosan with medium average molecular weight and chitosan based gold nanoparticles dispersions with two different initial gold precursor concentrations, 1 and 2 mM. Then, gold nanoparticles and chitosan-gold nanocomposites were characterized by UV-Vis spectroscopy, transmission electron microscopy and Fourier Transform Infrared Spectra analysis. Cytotoxic effects on different cell lines and bacteria were evaluated separately (Regiel-Futyra et al., 2015). Cytotoxic effects of gold chitosan nanocomposites on macrophages infected with Gram positive (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli) were evaluated and the antimicrobial activity was studied by the colony-forming unit assay.

Results: Gold chitosan nanocomposites exhibited potent antibacterial effects at the subcytotoxic concentrations assayed in both infection macrophage models being more accentuated in the Gram positive bacteria model in which one of our experimental groups exerted a complete bactericidal effect obtaining no bacteria colonies after treatment of the infected macrophages with the gold-chitosan based nanocomposites.

Discussion: The development of novel bactericidal strategies is imperative regarding the high prevalence of multidrug resistances arising in bacteria. By 2050 antimicrobial resistance would lead to 10 million people deaths every year and a reduction of 2% to 3.5% in Gross Domestic Product. Our study showed that at non cytotoxic concentrations, gold chitosan nanocomposites were able to kill both Gram positive and Gram negative bacteria in infection models in vitro without damaging host cells and avoiding the well-known cyto- and genotoxic effects of other metal nanomaterials such as silver and copper nanoparticles. This approach represents a potential basis in the design and development of bactericidal materials to efficiently halt the progression of bacteria colonization avoiding the growing problem of multidrug resistances.
# Multifunctional polymer-modified liposomes that capture and neutralize toxic protein, histones

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 361

Mr. Hiroki Tsuchida ¹, Dr. Hiroyuki Koide ¹, Mr. Masahiko Nakamoto ², Ms. Anna Okishima ³, Ms. Saki Ariizumi ³, Ms. Chiaki Kiyokawa ³, Dr. Tomohiro Asai ¹, Prof. Yu Hoshino ², Prof. Naoto Oku ¹

1. Department of Medical Biochemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 2. Department of Chemical Engineering, Kyushu University, 3. Department of Medical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka

Poly-N-isopropylacrylamide (pNIPAm) based polymer nanoparticles (NPs) with the capacity to recognize, capture and neutralize the target toxic peptide melittin in vivo by multiple weak interactions are of significant interest as “plastic antidotes”. The multipoint interactions are generated by N-tert-butylacrylamide (TBAm, a hydrophobic monomer) and acrylic acid (AAc, a negatively charged monomer). However, the NPs have low biocompatibilities and short circulation time. To improve these problems, we focused on modification of linear polymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization of NIPAm, TBAm and AAc onto liposomal surface. Histones, major mediators of sepsis, were chosen as target proteins. The linear polymer library was prepared by changing the AAc ratio and polymer length. Liposomal sizes and zeta-potentials were not significantly different between before and after polymer modification onto the liposomal surface even in the increasing of AAc ratio and polymer length. The modification rates of polymers depend on AAc ratio and polymer length. The circulation times of polymer-modified liposomes were far longer than linear polymers. Interactions between polymer-modified liposomes and histones were monitored by quartz crystal microbalance (QCM). As a result, polymers including higher density of AAc enhanced affinity of liposomes to the histones. In addition, there is an optimal length to induce high affinity to the histones. We next demonstrated toxin neutralization effect of polymer-modified liposomes using mouse endothelial 2H-11 cells. The optimized polymer-modified liposomes inhibited histones cytotoxicity in vitro. These results indicate that the liposomes bound and neutralized target protein, histones. To confirm whether polymer-modified liposomes neutralize histones in vivo, optimized polymer-modified liposomes were intravenously injected into mice after the lethal injection of histones via tail vein. The polymer-modified liposomes improved survival rate of mice exposed to lethal dose of histones. Namely, polymer-modified liposomes neutralized histones in the bloodstream. The functional liposomes capable of capturing and neutralizing the target biomacromolecules can be used as new antidotes.
Hypoxia-directed and activated theranostic agent:
Imaging and treatment of solid tumor

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 366

Ms. Hyun Min Kim ¹, Prof. Eun-joong Kim ², Mr. Hyunseung Lee ¹, Prof. Jong Seung Kim ³, Dr. Kwan Soo Hong ¹

1. Bioimaging Research Team, Korea Basic Science Institute, 2. Department of Biomedical Engineering, School of medicine,

Kyung Hee University, 3. Department of Chemistry, Korea University

Hypoxia, a distinguished feature of various solid tumors, has been considered as a key marker for tumor pro-
gression. Inadequate vasculature and high interstitial pressures result in relatively poor drug delivery to these
tumors. Herein, we developed an antitumor theranostic agent, 4, which is activated in hypoxic conditions and
can be used for the diagnosis and treatment of solid tumors. Compound 4, bearing biotin, a tumor-targeting
unit, and SN38, an anticancer drug, proved to be an effective theranostic agent for solid tumors. SN38 plays
a dual role: as an anticancer drug for therapy and as a fluorophore for diagnosis, thus avoids an extra fluo-
rophore and limits cytotoxicity. Compound 4, activated in the hypoxic environment, showed high therapeutic
activity in A549 and HeLa cells and spheroids. In vivo imaging of solid tumors confirmed the tumor-specific
localization, deep tissue penetration and activation of 4, as well as the production of a strong anticancer effect
through the inhibition of tumor growth in a xenograft mouse model validating it as a promising strategy for the
treatment of solid tumors.

<figure><img src="image_60.png" /><figcaption>Hypoxia figure1.jpg</figcaption></figure>
# Chemically cross-linked silk fibroin hydrogel with enhanced elastic properties, biodegradability, and biocompatibility

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 368

<u>Mr. minhee kim</u>¹, <u>Mr. seung hyun lee</u>¹, <u>Prof. Won Ho Park</u>¹

1. Chungnam national university

## Introduction

In this study, the synthesis of SF hydrogel via a chemical cross-linking reaction using γ-ray irradiation was investigated and the resultant hydrogel was characterized. Two different hydrogels were investigated: SF P-gel induced by β-sheet structure and chemically cross-linked hydrogel prepared by γ-ray irradiation. The secondary structures and strengths of the gels were compared. Furthermore, the swelling behavior, biodegradation, and cell viability of human mesenchymal stem cells (hMSCs) on the SF hydrogel were evaluated.

## Methods

Freshly regenerated SF solutions of various concentrations (2.3%-7.9%) were poured into a petri dish and irradiated with γ-rays from a Co-60 source. The radiation dose varied from 15-60 kGy and the dose rate was 15 kGy/h, at room temperature. The irradiated samples were cut into small pieces to compare with SF P-gel with regard to various properties.

## Result and discussion

The radiation technique seems to be an excellent pathway for the preparation of hydrogels because polymer solutions undergo chemical cross-linking on irradiation to yield a hydrogel. By contrast, most chemical cross-linking reactions of SF can induce a modification of proteins, resulting in structural destabilization and inflammation by exposure to toxic organic solvents and cross-linking agents. In this study, the SF C-gel was prepared by γ-ray irradiation, which induced intermolecular cross-linking reactions. In order to investigate the effect of irradiation on the gelation of the SF solution, the solutions were γ-ray irradiated at various doses of 15, 30, 45, 60, 75, and 90 kGy. Regardless of the SF concentration and irradiation doses, SF hydrogel was formed immediately after irradiation, as shown in Figure 1A. However, as shown in Figure B, cracks occurred in the hydrogel at doses higher than 60 kGy. A suitable absorption dose for an SF solution was 60 kGy. Therefore, the experiment was performed using the dose of 60 kGy.
A

<figure><img src="image_61.png" /></figure>

B

<figure><img src="image_62.png" /><figcaption>Figure.png</figcaption></figure>
# Non-invasive In Vivo Imaging and Tracking of Dendritic Cells Migration using MR/NIR Dual Modal Contrast Agent

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 373

Dr. Jee-Hyun Cho¹, Ms. Hyun Min Kim², Dr. Hye Sun Park², Dr. Kwan Soo Hong²

1. Bioimaging Research Team, Korea Basic Institute, 2. Bioimaging Research Team, Korea Basic Science Institute

Dendritic cells (DCs) play a key role in immune system as potent antigen-presenting cells (APC), which are uniquely capable of initiating a primary immune response. After DCs capture and process antigen in peripheral tissues, they begin to mature and migrate to secondary lymphoid tissues, where they activate naïve T cells. Therefore, DC migration into the draining lymph nodes is critical for T cell priming. Here, we developed a novel magnetic resonance (MR)/near-infrared (NIR) imaging contrast agent, which was synthesized by a facile fluorescent dye coating on magnetic nanoparticles (MNP). The dye-coated MNP was used as a high-performance MR/NIR imaging contrast agent with a high T2 relaxivity (r2) value of approximately 308 mM-1s-1, and NIR fluorescence by biocompatible indocyanine green dye to support non-invasive in vivo tracking of DCs migration into lymph nodes. The impact of the dye-coated MNPs on DC viability and maturation was systematically investigated using MTT assays and FACS analysis. And the labeling effects of DCs with the dye-coated MNPs were investigated in changes on DC phenotype or maturation potential. Importantly, the migration of DCs via lymphatic drainage and homing into the lymph node were monitored through real-time NIR fluorescence and MR imaging. Taken together, this novel MR/NIR contrast agent has potential application as a non-invasive imaging agent for both inducing T cell priming and imaging of DC-based immunotherapy.

<figure><img src="image_63.png" /><figcaption>Abstract2 figure.jpg</figcaption></figure>
# Matryoshka-type enteric microparticles for the treatment of tuberculosis

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 376

Dr. Vanesa Andreu¹, Ms. Ane Larrea¹, Dr. Salvador Alfaro², Ms. Begoña Gracia¹, Dr. Gracia Mendoza¹, Dr. Víctor Sebastián¹, Dr. José Antonio Ainsa¹, Dr. Manuel Arruebo¹

1. University of Zaragoza, 2. National Polytechnic Institute

## Introduction

The World Health Organization reported in 2015 that the tuberculosis still constitutes a major cause of morbidity and mortality worldwide. The treatment used nowadays presents different limitations such as non-localised delivery of drugs, high dose and large treatment duration as well as the side effects that it produces.

The aim of this work was to investigate a new polymeric drug-delivery system that may provide enhanced drug delivery at the site of infection, hence reducing drug associated side-effects and the probability of developing drug resistance. In that sense, matryoshka-type enteric microparticles containing rifampicin-loaded PLGA nanoparticles were developed as a potential effective oral therapeutic alternative to achieve appropriate intracellular concentrations in tuberculosis-infected alveolar macrophages.

## Methods

Rifampicin-loaded PLGA nanoparticles (NPs) were prepared by the oil-in-water single-emulsion solvent-evaporation method and those NPs were encapsulated in turn within enteric monodisperse Eudragit-based-microcapsules using the double emulsification and evaporation method. The morphological characterization of micro- and nanoparticles has been carried out by several techniques (SEM, TEM, DLS, FT-IR).

In vitro antimycobacterial activity of rifampicin, unloaded-PLGA nanoparticles and rifampicin-loaded PLGA nanoparticles were determined on M. tuberculosis H37Rv at intracellular and extracellular pH. Also, in vitro permeability of these NPs through the intestinal epithelium was evaluated using an artificial monolayer of gastrointestinal epithelial cells.

## Results

The colloidal hydrodynamic sizes of rifampicin-PLGA NPs were 145.2 ± 31.2 nm. HPLC analysis indicated that the EE and DL achieved for these NPs were 5.4 ± 0.55 and 1.08 ± 0.11 g/w %, respectively. SEM morphological analysis of enteric Eudragit-microcapsules revealed a bipyramidal structure of micrometric size (6.1 ± 1.9 µm). The enteric coating remains unaltered under simulated gastric conditions and the inner antibiotic-loaded NPs were rapidly released under simulated intestinal conditions.

Antimicrobial activity of the free and the encapsulated rifampicin showed similar levels of bacterial eradication.

## Discussion

An intracellular accumulation and slow antibiotic release can be achieved using orally administered enteric microparticles containing rifampicin-loaded PLGA NPs if they were able to extravasate through the intestinal epithelium into the blood stream with the sufficient blood circulation half-life to reach tuberculosis-infected alveolar macrophages.
Regulation of angiogenesis through the efficient
delivery of microRNAs into endothelial cells using
polyamine-coated carbon nanotubes

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 377

Dr. Andrea Masotti ¹, Dr. Mark Miller ², Ms. Antonella Celluzzi ¹, Dr. Lorrain Rose ², Dr. Federico Micciulla ³, Dr. Patrick Hadoke ², Dr. Stefano Bellucci ³, Dr. Andrea Caporali ²

1. Ospedale Pediatrico Bambino Gesù, 2. University of Edinburgh, 3. INFN-Laboratori Nazionali di Frascati

Introduction

MicroRNAs (miRNAs) directly regulate gene expression at a post-transcriptional level and represent an attrac-
tive therapeutic target for a wide range of diseases. Here, we report a novel strategy for delivering miRNAs to
endothelial cells (ECs) to regulate angiogenesis, using polymer functionalized carbon nanotubes (CNTs).

Methods

CNTs were coated with two different polymers, polyethyleneimine (PEI) or polyamidoamine dendrimer (PA-MAM), followed by conjugation of miR-503 oligonucleotides as recognized regulators of angiogenesis.

Results

We demonstrated a reduced toxicity for both polymer-coated CNTs, compared with pristine CNTs or polymers
alone. Moreover, polymer-coated CNT stabilized miR-503 oligonucleotides and allowed their efficient deliv-
ery to ECs. The functionality of PAMAM-CNT-miR-503 complexes was further demonstrated in ECs through
regulation of target genes, cell proliferation and angiogenic sprouting and furthermore, in a mouse model of
angiogenesis.

Discussion

This comprehensive series of experiments demonstrates that the use of polyamine-functionalized CNTs to de-
liver miRNAs is a novel and effective means to regulate angiogenesis.
# How to enhance the functionality of microencapsulated cells by using graphene oxide nanoparticles

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 383

**Dr. Laura Saenz del Burgo**¹, **Dr. Jesús Ciriza**¹, **Dr. Argia Acarregui**¹, **Mr. Haritz Gurruchaga**¹, **Prof. Rosa María Hernández**¹, **Dr. Gorka Orive**¹, **Prof. Jose Luis Pedraz**¹

1. University of the Basque Country

**Introduction:** Cell microencapsulation consists on the immobilization of cells within an alginate matrix surrounded by a semi-permeable membrane that allows the release of therapeutic drugs. This technology represents an opportunity to develop long-term de novo synthesized drug delivery systems for the treatment of chronic diseases. However, several challenges need to be overcome before it can be translated into the clinic. For instance, a higher viability of the cells would be highly desirable. Graphene oxide (GO) has shown to promote the adhesion and proliferation of several cell types. Thus, we fabricated hybrid alginate-GO capsules in order to improve the viability of erythropoietin (EPO) secreting C2C12 myoblasts.

**Methods:** Myoblasts were encapsulated into alginate or hybrid alginate-GO microcapsules. First, we studied the main physical parameters of the microcapsules. Next, early apoptosis and viability of the cells was quantified by flow cytometry. Also, the CCK8 assay was performed to analyze the metabolic activity and the cell membrane integrity was determined. Finally, EPO production was detected by Elisa. For in vivo studies, syngeneic C3H mice were implanted and blood samples were collected weekly for hematocrit determination.

**Results and Discussion:** We were able to produce microcapsules with GO in their core and proved that their physical characteristics were not substantially modified. Also, we determined that 25-50 µg/ml of GO is the most suitable concentration for enhancing the viability of encapsulated cells, since this rank provided lower cell death and better metabolic activity. However, even at the lowest GO concentration, the quantity of EPO detected on the medium was low, most likely due to its adsorption to the GO surface. Therefore, we tested if a protein pre-coating could attenuate the capacity of GO to adhere the therapeutic protein. Interestingly, serum-coating effectively avoided the adsorption of EPO to the nanoparticles and enhanced, even more, the viability, membrane integrity and drug release of encapsulated cells. Finally, myoblasts encapsulated in hybrid alginate-protein coated GO microcapsules showed to be functional in vivo as they increased hematocrit levels on mice. Therefore, these results provide another step for the future pre-clinical application of GO nanoparticles in combination with cell microencapsulation.
<figure><img src="image_64.png" /><figcaption>Microcapsules composed by 1.5 alginate and 200µg go 20x.jpg</figcaption></figure>
Cellular internalization mechanisms of
polyamine-coated carbon nanotubes

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 386

Ms. Antonella Celluzzi ¹, Dr. Alessandro Paolini ¹, Dr. Andrea Masotti ¹

1. Ospedale Pediatrico Bambino Gesù

Introduction

Carbon nanotubes (CNTs) are emerging as promising systems for transfection of drugs, small molecules and, recently, have been widely studied for the delivery of nucleic acids. In our previous paper, we reported the preparation of efficient delivery systems consisting of polymer-functionalized carbon nanotubes (CNTs). These systems efficiently delivered microRNAs (miRNAs) in endothelial cells (ECs).

Methods

These compounds were obtained through coating of CNTs with two polyamine polymers (i.e., polyethyleneimine, PEI and polyamidoamine dendrimer, PAMAM), followed by complexation with miRNA mimics. In particular, both PEI-coated and PAMAM-coated CNTs were able to stabilize miR-503 oligonucleotides and allowed an efficient delivery of polyplexes to ECs.

We further investigated the cellular internalization mechanism of polymer-coated CNTs compared to polymers
alone. Using inhibitors of clathrin-mediated endocytosis (i.e, chlorpromazine) and caveolae-mediated uptake
(i.e., filipin/genistein) we studied the internalization mechanisms of polymer-coated CNTs by different cell lines.

Results

FACS analysis suggested that these compounds may enter cells following different internalization pathways. In this contribution we will show the obtained preliminary results.

Discussion

Inhibition assays may contribute to understand the internalization processes by polyamines/dendrimers and polyamine-coated CNTs.
# Synthesis of polymer coated Co0.5Zn0.5Fe2O4 nanoparticles and their cytotoxicity on human carcinoma cells

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 395

Dr. Zulqurnain Ali¹, Dr. Rashda Abbasi², Dr. Muhammad Atif¹, Ms. Javeria Arshad¹, Mr. Abdul Jabbar Khan¹, Dr. Nafees Ahmad²

1. Air University, 2. Institute of Biomedical and Genetic Engineering, Islamabad

Over recent decades, the application of magnetic nanoparticles has been broadly investigated with their advance applications in the areas of biotechnology, medicinal drug delivery, magnetic fluid hyperthermia, and magnetic resonance imaging. Spinel ferrite nanoparticles are of importance because of their well-known unique optical, electronic and magnetic properties. Spinel ferrite nanoparticles have no preferred direction of magnetization, good saturation magnetization and high permeability. Being magnetically soft; they can easily magnetized and demagnetized, and electrically insulating. For this reason, in this study, cobalt zinc ferrite (Co0.5 Zn0.5 Fe2O4) magnetic nanoparticles (CZF) were used for further investigation. However, there are no data about biological activity of CZF, including their cellular uptake, toxicity, effects on cell proliferation, phenotype and functional activity. Therefore, these aspects remain a subject of particular interest. Multifunctional CZF were synthesized by sol gel method with high colloidal stability having room temperature ferromagnetism. The synthesis method is cost-effective, easy to scale up and reproducible. For biological applications CZF were polymer coated with amphiphilic polymer. The amphiphilic polymer can be functionalized with organic, organo-metallic or anticancer drug molecules; for this linkage the molecules need to bear a free amino group. Via the amino group these molecules are then directly attached to the maleic anhydride rings of the amphiphilic polymer. In this study 1% anhydride rings of amphiphilic polymer were reacted with fluorescent dye (cresyl violet perchlorate) used as model system for anti-cancer drug loaded polymer shell. The amount of functional molecule can be varied up to 25% of the anhydride rings, which provides greater affinity of drug loading in polymer shell. CFZ were further characterized by XRD, FTIR, UV-Vis spectroscopy and gel electrophoresis. The in vitro cytotoxicity of CZF was examined via the MTT assay on HepG2 (Liver Cancer cells). Concisely, 104 cells per well were seeded in to 96-well plates and exposed to different concentrations of CFZ for 24 h. The CFZ induce significant death of the carcinoma cells (HepG2). Stimulatingly, this seems to be a noteworthy improvement towards the ability of surface functionalized multifunctional CZF as carriers for drugs for anti-cancer therapy.
<figure><img src="image_65.png" /></figure>

Ali abstract.png
# Ratiometric real-time measurement of protein kinase activity with fluorophore labeled polyion complexes

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 402

Mr. Takanobu Nobori¹, Prof. Akihiro Kishimura¹, Prof. Takeshi Mori¹, Prof. Yoshiki Katayama¹

1. Department of Applied Chemistry, Kyushu University

## Introduction

The dysfunction of protein kinase (PK) activity results in various human diseases. Thus, a measurement method for PKs is not only valuable for understanding the molecular mechanism of complicated cellular events but also for diagnosis and drug discovery. Recently, fluorescence probes which get turn on during PK-mediated phosphorylation of substrate peptides are one of the attractive methods due to sharp contrast in simple optical readout systems. However, it is still difficult to realize quantitative real-time analysis and monitoring of PK activity in living cells. Therefore, we demonstrate a novel quantitative real-time detection system of PK activity. Our system is based on a polyion complex (PIC) nanoparticle embedded with a PK-responsive fluorescence signal and a second constant internal standard signal.

## Methods

5-(and 6) -carboxytetramethyl-rhodamine (TAMRA) was modified to poly-L-aspartic acid (pAsp-TAMRA) as an- ionic polymer. As for cationic polymer, Cy5 and protein kinase Cα (PKCα) substrate peptide were modified to Dextran (Dex-Cy5-pep). After PICs formation of these polymers through electrostatic interaction, various concentrations of the PKCα were added and changes in the TAMRA and Cy5 fluorescence intensities were mon- itored.

## Results and Discussion

Once pAsp-TAMRA and Dex-Cy5-pep formed the PICs, TAMRA fluorescence was selectively quenched and Cy5 fluorescence tended to be constant. This uneven quenching behavior of the two signals was controlled by adjusting each fluorophore content in each polymer. High content of TAMRA in pAsp-TAMRA (2.1 mol%) in- duced concentration quenching of TAMRA, while low content of Cy5 in Dex-Cy5-pep (0.4 mol%) did not show the quenching. In the monitoring of each fluorescence intensities with phosphorylation reaction of PICs, the recovery of TAMRA fluorescence intensity was observed with increasing reaction time and PKCα concentration. On the other hand, Cy5 fluorescence intensity was still constant. This result indicated that the electrostatic in- teraction between two types of polymers were weaken by phosphorylation and PICs were dissociated, which resulted in TAMRA fluorescence recovery from quenching. In this research, we realized this probe enables quantitative and real-time detection of PKCα activity. In near future, we will apply this PIC type probe to the measurement of PK activity in living cells.
<figure><img src="image_66.png" /><figcaption>Figure for abstract.jpg</figcaption></figure>
Synthetic hydrogels based on hybrid
physically-chemically cross-linked networks

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 422

Dr. Maxime Grillaud ¹, Mr. Maarten Bakker ¹, Dr. Patricia Dankers ¹

1. Eindhoven University of Technology

Many polymers with different types of cross-linking have been designed to develop various hydrogels for indus-
trial and fundamental applications. In particular, they have attracted significant interest in the field of regen-
erative medicine, as drug delivery vehicles or scaffolds for tissue engineering. However, the main challenging
limitation for their extensive use is their low mechanical stability. Many recent efforts have been made to over-
come this burden, in particular by the elaboration of double network (DN) hydrogels. Despite the promising
results of the few existing physically-chemically cross-linked DN hydrogels, the preparation procedures require
long photopolymerization time and toxic precursor materials. Moreover, only the comparison of the mechan-
ical properties between the hybrid DN hydrogel made in very specific conditions with the corresponding two
separate networks is studied. The fundamental understanding of the combination of two different cross-links
for a rational design of new hybrid DN hydrogels is a challenge.

Herein, we provide synthetic strategies to tailor in-situ formation of DN hydrogels based on physically-chemicaly cross-linked networks. Three approaches to combine catalyst-free click reaction and hydrogen bonding for in-situ hydrogel formation will be described (see Figure). More in detail, the covalent cross-linking consists of the click reaction between azides and cyclooctyne derivatives, referred to as strain-promoted azide-alkyne coupling (SPAAC), and the non-covalent cross-linking is based on supramolecular interactions via a four-fold hydrogen bonding motif, the ureido-pyrimidinone (UPy) unit.

The mechanical properties of these new hydrogels are studied by rheology and their morphology are analyzed
by confocal microscopy. A detailed comparison of the hydrogel properties is discussed according to the approach
selected and the ratio of the two types of cross-linking. In particular, this work shows the benefits to associate
the dynamicity afforded by supramolecular interactions for gel recovering with the strength provided by the
covalent cross-links for enhanced material stiffness. We propose that this work offers both a novel versatile
platform for the easy preparation of DN hydrogels under physiological conditions with tunable properties, and
one more step in the fundamental understanding of structure-property relationship of physically-chemically
cross-linked hydrogels.
<figure><img src="image_67.png" /><figcaption>Figure abstract.jpg</figcaption></figure>
# A Novel niosome gene delivery approach for central nervous system disorders

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 438

Mr. Mohamed Mashal¹, Dr. Noha Attia², Prof. Gustavo Puras³, Prof. Jon Zarate⁴, Dr. Cristina Soto Sanchez⁵, Prof. Eduardo Fernandez⁵, Prof. Jose Luis Pedraz³

1. University of the Basque Country, UPV/EHU, Spain, 2. University of the Basque Country, UPV/EHU. University of Alexandria,

3. University of the Basque Country, UPV/EHU, 4. University of the Basque Country UPV/EHU, 5. University of Miguel Hernandez

## Introduction

Glial cells are part of the nervous tissue where they nourish protect and regulate neuronal function. Therefore, they are ideal vehicle for gene therapy of brain diseases. Niosomes represent a recent safe approach for gene delivery purposes. Thus, our aim was to formulate an effective niosome formulation based on the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) to deliver genes to nervous tissue.

## Methods

Modified reverse phase evaporation technique was applied to obtain our niosomes. After their physicochemical characterization, niosomes were complexed with pCMS-EGFP reporter plasmid to form nioplexes. Afterwards, nioplexes were used to transfect NT2 cells as well as primary neuronal cells from rat embryonic cortex in vitro followed by studying their uptake and internalization pathways. Finally, Nioplexes were injected to transfect rat cerebral cortex in vivo.

## Results

Nioplexes depicted appealing physicochemical properties as gene delivery vehicles. Additionally, they could transfect both NT2 and primary glial cells without compromising cell viability as detected by microscopy and flowcytometry. NT2 showed high uptake of nioplexes (59.6±7.01%), possibly due to their endocytosis by all the tested mechanisms; clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME) and macropinocytosis. Nevertheless, their co-localization with both CME and lysosomes could explain the relatively lower transfection (up to 16%). When nioplexes were injected into rat cerebral cortex, they had tropism to transfect glial cells.

## Conclusion

Thanks to its effectiveness to transfect cortical glial cells in rat brain without the known risks of viral vectors, our niosome vector could be an interesting novel non-viral formulation for gene therapy targeting severe diseases of the brain.
# Proteins adsorption upon nanoparticles: from the physicochemical basis to the functional impacts

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 474

Mr. Laurent Marichal¹, Dr. Jean-philippe Renault¹, Dr. Jean-christophe Aude², Dr. Yves Boulard³, Dr. Serge Pin¹, Dr. Jean Labarre²

1. CEA Saclay, NIMBE, 2. CEA Saclay, iBiTec-S, 3. CEA Saclays, iBiTec-S

The production and utilization of nanoparticles has been strongly increasing over the last decades, raising concerns regarding their biological effects and health hazards. Due to their nanometric size, nanoparticles have two major properties, (i) a high specific surface and (ii) the ability to cross biological barriers. Thus, they can interact with a large amount of biological compounds (especially proteins) and impact biological processes.

We study adsorption of proteins on silica nanoparticles (SiNP). We used yeast protein extracts in order to assess which kinds of proteins tend to adsorb on SiNP and which do not. Using proteomics (mass Spectrometry Shotgun analysis), we identified several hundreds of adsorbed and non-adsorbed proteins. The statistical comparative analysis of these two sets of proteins revealed the physicochemical determinants relevant for adsorption: the overrepresentation of arginine residues and of large disordered regions, as well as an impoverishment in secondary structures and hydrophobic aminoacids. These results are consistent with the notion that disorder and flexibility favor protein adsorption.

Using GO term analysis, we also evidenced that RNA binding proteins have a marked affinity for SiNP. We selected one of them, NPL3, a protein carrying poly(A) mRNA from nucleus to cytoplasm. We constructed two truncated mutants fused to GFP (see figure) and found that the C-ter region of NPL3 containing several repetitions of the Arg-Gly-Gly (RGG) motif is responsible for the high affinity of the protein for SiNP.

In vivo, the Arg residue of RGG motif is frequently dimethylated. We synthesized peptides (25-mer) containing RGG repetitions and some versions where Arg residues were dimethylated. The data indicate that Arg dimethylation significantly increases the affinity of the peptides for SiNP. We are currently planning to use calorimetry with these peptides in order to assess if the adsorption process, in these cases, is entropy or enthalpy driven.

Our data opens the way for predicting potential biological impacts of nanoparticles.
<figure><img src="image_68.png" /></figure>

Figure: Representation of the adsorbed and non adsorbed fractions of the fluorescent proteins, namely NPL3 and 2 truncated mutants called RRM (RNA Recognition Motif) and RGG on silica nanoparticle. Total protein extracts were produced from yeast strains expressing the different fusion proteins.

Npl3 mutants affinity towards sinp.jpg
Flexible cortical Multi-electrode array implant for neural
recording in minipig

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 475

Mr. Jean-Marie Mayaudon ¹, Dr. Lionel Rousseau ², Dr. Gaëlle Offranc Piret ¹, Dr. Blaise Yvert ³

1. INSERM, BrainTech Laboratory U1205, Grenoble, 2. ESIEE Paris, ESYCOM Université Paris-Est, 3. Univ. Grenoble Alpes,
BrainTech Laboratory U1205, Grenoble

Multi-Electrode Arrays are key neuronal interfacing systems in neurophysiological and clinical research to bet-
ter understand healthy and pathological neural network. In neuronal implant domain, one of the major chal-
lenge is to propose a biocompatible device that remain stable over a long period of time. Flexible implants are
promising to reduce tissue damage and inflammation on the long term, as they limit the frictions at the implant
tissue interface.

Based on the micro and nano technologies, we fabricate a fully flexible 256 microelectrodes. Several biocom-
patible polymers can be used to achieve the implant that allow to vary and test different implant thicknesses
(range between 5 to 20 µm). The MEA implant is arranged in several independent strips that cover most of the
cortical surface, and shape the brain regions in order to keep a close contact distance from neural tissue, reduce
micromotions, and avoid implant displacement.

Electrochemical characterization of electrodes will be achieved and biocompatibility of the implant will be as-
sessed on rat primary cortical cells cultures.

Performance and stability of this device will be tested for in-vivo cortical recording in the minipig.
# Exudate Triggered Metal Corrosion for Self-Powered Wound Healing Application

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 512

Mr. Sun Woong Han¹, Mr. Keun Ho Lee¹, Mr. Tae Hoon Ki¹, Prof. Hong Koo Baik¹

1. Yonsei University

We developed a galvanic wound healing patch that can promote skin wound healing without the use of any external electric devices. Electrical fields have been proven to modulate skin cell behavior to enhance skin wound healing. Our galvanic wound healing patch generated electrical fields by galvanic corrosion triggered electric stimulation for skin wound healing. The galvanic corrosion properties have been proven to modulate skin cell behavior to enhance skin wound healing. In vivo data demonstrated that the galvanic patch promoted the wound healing process through enhanced cellular metabolism and migration. This wound healing property may lead to a clinically relevant galvanic wound healing patch therapy for self-powered wound healing application.
# Collagen glycation versus chronologically-aged fibroblasts in keratinocyte differentiation of reconstructed human skin

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 556

**Ms. Roberta Rigon**¹, **Mr. Christian Hausmann**², **Mr. Christopher Wolff**², **Dr. Julia Tigges**³, **Prof. Ellen Fritsche**³, **Dr. Marus Chorilli**⁴, **Dr. Christian Zoschke**², **Prof. Monika Schäfer-korting**²

1. Universidade Estadual Paulista "Júlio de Mesquita Filho", Araraquara, Brazil and Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany, 2. Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany, 3. IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany, 4. Department of Drugs and Medicines - Univ. Estadual Paulista Júlio de Mesquita Filho - UNESP - Araraquara/SP

Skin morphology markedly changes over the lifespan, but the effects on the skin barrier function remain to be elucidated for nanocarrier-enhanced and -targeted topical drug delivery [1]. Herein, we investigated the effects of collagen glycation and fibroblast age, respectively, on keratinocyte differentiation in reconstructed human skin (RHS).

For glycation, collagen was incubated with 10 mM D-ribose for 3 weeks, dialyzed in water for 48 h, and analyzed by fluorescence spectroscopy [2]. Glycated collagen was mixed with non-glycated collagen 1:1 and used for RHS. Juvenile keratinocytes were used for the epidermal compartment of all RHS. Fibroblasts for glycated and juvenile (non-glycated) RHS were from juvenile foreskin; too, in addition we used fibroblasts from mammary reduction surgeries of 60- to 70-year-old female donors. RHS were cultured for two weeks after the exposure to the air-liquid-interface and then subjected to reflectance confocal microscopy, immunofluorescence staining or skin surface pH analysis.

We detected 30% of collagen glycation by fluorescence spectroscopy and significant amounts of carboxymethyl lysine staining in glycated models. Stratum corneum thickness increased in glycated and aged RHS 2-fold compared to juvenile RHS with the most dense stratum corneum appearance in glycated RHS. The surface pH of glycated models exceeded the surface pH of RHS with chronologically-aged fibroblasts and juvenile RHS. Expression of keratinocyte terminal differentiation markers like filaggrin and loricrin as well as integrin expression was enhanced in glycated RHS whilst dermal thickness decreased in glycated models compared to juvenile and aged RHS (Figure 1), which is in accordance to literature [3].

Taken together, both approaches mimic hallmarks of cutaneous aging. Collagen glycation affects keratinocyte differentiation more extensively than the replacement of juvenile by chronologically-aged fibroblasts, highlighting the impact of glycation for the skin barrier function. Future studies will evaluate the functionality of the skin barrier.

## References

[1] Biniek et al. (2015). J Dermatol Sci 80: 94-101.

[2] Pennacchi et al. (2015). Tissue Eng Part A 21:2417-25.

[3] Pageon et al. (2015). BioResearch Open Access, 4.1.
<figure><img src="image_69.png" /><figcaption>Figure 1. Effect of collagen glycation and chronologically-aged fibroblasts into RHS on epidermal differentiation.</figcaption></figure>

Figure 1. effect of collagen glycation and chronologically-aged fibroblasts into rhs on epidermal differentiation.jpg
# Nanoformulation of imipramine loaded resealed erythrocytes as potent anti-leishmanial, which targets unique 'prokaryotic TopA' homolog in Leishmania

Thursday, 29th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 97

Ms. Sumedha Mukherjee¹, Ms. Devyani Shukla¹, Dr. Somdeb Bosedasgupta¹

1. IIT BHU, Varanasi

**Introduction:**

Uncontrolled use of traditional anti-leishmanials leading to the emergence of drug resistant parasites has necessitated the search for potent drugs against novel drug targets clear and evident. A serendipitous BLAST search using prokaryotic TopA sequence led us to identify its homolog, LdBPK_210180.1 in Leishmania. The sequence corresponds to a unique Topoisomerase IA (LdTopIA), having prokaryotic homologs and being absent in higher eukaryotes. Previously anti-depressants have been shown to exhibit anti-leishmanial activity and imipramine, which is one such anti-depressant has been reported to inhibit mycobacterial Topoisomerase I (MttopoI), so we thought of testing efficacy of imipramine as an inhibitory agent against LdTopIA.

**Methods:**

Sequence similarity of E.coli TopA and LdTopIA was established using CLUSTAL-W and homology modeling of LdTopIA was performed using SWISS-MODEL using E.coli TopA as template (PDB ID-1CY1). Functional complementation studies were performed in RFM 475 (an E.coli TopoI null-DNA gyrasets mutant strain) in presence and absence of imipramine. In-silico docking was also studied for imipramine and LdTopIA. Nanoformulation of imipramine was carried out for entrapment in resealed erythrocytes for efficient delivery to the effected organs.

**Results:**

Homology modeling and functional characterization shows LdTopIA has active site residues similar to its prokaryotic homologs. Though the enzyme complements E.coli TopA in RFM 475 strain, it fails to do the same in presence of imipramine. In-silico docking studies also show that imipramine binds to the active site of LdTopIA suggesting that imipramine is a potent inhibitor of LdTopIA.

**Discussion:**

The results establish LdTopIA as a unique drug target capable of killing the parasite while respite the host. Since, liver and spleen are the effected organs in visceral leishmaniasis, nanoformulation of imipramine-loaded resealed erythrocytes can be an effective therapy to combat the disease as the reticuloendothelial system readily phagocytoses such RBCs. Such strategy can lead to personalized medicines where one's own red blood cells can be used as drug delivery vessel and thereby prove to be a successful new strategy for treating leishmaniasis.
<figure><img src="image_70.png" /><figcaption>Imipramine binds to ldtopia active site.jpg</figcaption></figure>
# Platinum nanoparticles as multifunctional active nanocarriers integrating the function of high-performance antioxidant drugs

Thursday, 29th September - 16:00 - Targeted drug delivery and Nanocarriers - Nanomedicine for cancer diagnosis & therapy - Amphitheatre 25 - Oral presentation - Abstract ID: 252

Ms. Deborah Pedone¹, Dr. Elisa De Luca², Dr. Mauro Moglianetti², Dr. Roberto Marotta³, Mr. Tiziano Catelani³, Dr. Barbara Sartori⁴, Dr. Heinz Amenitsch⁴, Prof. Saverio Francesco Retta⁵, Dr. Pier Paolo Pompa⁶

1. Istituto Italiano di Tecnologia, Center for Biomolecular Nanotechnologies; University of Salento, Department of Engineering for Innovation, 2. Istituto Italiano di Tecnologia, Center for Biomolecular Nanotechnologies, 3. Electron Microscopy Laboratory, Nanochemistry Department, Istituto Italiano di Tecnologia, 4. Graz University of Technology, Institute of Inorganic Chemistry, 5. Department of Clinical and Biological Sciences, University of Torino, 6. Istituto Italiano di Tecnologia

## Introduction

In recent years, nanomaterials that mimic natural enzymes (nanozymes) have elicited huge interest in nanomedicine (Wei and Wang, Chemical Society Reviews, 2013). In this work, we explored the use of citrate-capped platinum nanoparticles (Pt NPs) as nanozymes that act simultaneously as drugs and carriers in oxidative stress-mediated diseases.

## Methods

We synthesized citrate-capped Pt NPs of 5 and 20 nm of diameter and performed a systematic characterization of their superoxide dismutase (SOD)-, catalase (CAT)- and peroxidase (HRP)- like activities in cell-free environment. As the combined function of drug and carrier requires that Pt NPs are cytocompatibles, we performed a systematic toxicity assessment (WST-1, LDH leakage, TUNEL, and DCF assay), investigating the internalization of Pt NPs and their subcellular localization by TEM and ICP-AES analyses.

Finally, to test the hypothesis that citrate-capped Pt NPs can act as a scavenging material in biological systems, we tested Pt NPs on the cellular model of a cerebrovascular disease, namely Cerebral Cavernous Malformation (CCM), characterized by an abnormal angiogenesis and associated with a significant increase in intracellular reactive oxygen species (ROS) levels.

## Results and Discussion

We showed that pure, monodisperse, and endotoxin-free Pt NPs do not exert any toxicity on several cell lines, independently of the size. We proved that the absence of toxicity of Pt NPs is related to their compartmentalization and stability within the endo/lysosomal vesicles and to the absence of release of Pt ions.

We demonstrated, for the first time, that Pt nanozymes are capable to restore physiological ROS homeostasis in a real experimental model of CCM disease, finding that Pt NPs can completely recover the cellular phenotype, similar to that of wild type cells (Fig.1).

This is possible because of the strong and broad antioxidant nanozyme activity of Pt NPs, which are simultaneously endowed with strong CAT-, HRP-, and SOD-like activities, with superior performance than natural enzymes (Moglianetti et al., Nanoscale, 2016).
These findings are important and of broad interest, and open up novel perspectives in nanomedicine for the development of multifunctional active nanocarriers integrating the function of high-performance antioxidant drugs with strong potential for therapies of complex oxidative stress-related diseases.

<figure><img src="image_71.png" /><figcaption>Pt nps restore ros homeostasis in cellular model of oxidative stress mediated diseases.jpg</figcaption></figure>
# Transport of Liposome Encapsulated Drugs in Voxelized Computational Model of Brain Tumors

Thursday, 29th September - 16:25 - Targeted drug delivery and Nanocarriers - Nanomedicine for cancer diagnosis & therapy - Amphitheatre 25 - Oral presentation - Abstract ID: 239

Mr. Ajay Bhandari¹, Dr. Ankit Bansal², Dr. Anup Singh³, Dr. Niraj Sinha¹

1. Indian Institute of Technology, Kanpur, 2. Indian Institute of Technology, Roorkee, 3. Indian Institute of Technology, Delhi

**Introduction:**

Cancer is one of the leading causes of death all over the world. Various treatment strategies are now a days used for treatment of cancers such as chemotherapy, radiotherapy and surgical removal. Liposome mediated drug delivery has emerged as an excellent anticancer therapy due to its ability to deliver drugs at site of action and reducing the chances of side effects to the healthy tissues. Computational modelling provides us a non-invasive approach to predict the transport mechanism of liposomes to tumor site and their accumulation within tumor tissues. Aim of this study is to perform computer simulations to qualitatively and quantitatively analyze the deposition of liposomes in human brain tumors. Additionally, the size effect is incorporated by considering stealth (PEG encapsulated doxorubicin) and conventional liposomes. Their accumulation results are compared with the free drug (doxorubicin).

**Methods:**

In this study computational fluid dynamics has been used to develop a voxelized model based on DCE-MRI. Complete analogy is maintained between MRI slice and CFD slice by assigning all the variables voxelwise. All the fluid flow and solute transport equations have been simulated to predict the transport of liposomes to tumor site. To the best of our knowledge this is the first study to predict the transport of liposomes in realistic heterogeneous vasculature of tumors computationally.

**Results and Discussions:**

Simulation has been performed for 48 hours. Results indicate that stealth liposomes accumulate more and remain for longer period of time in tumors as compared to conventional liposomes and free drug. Stealth liposome concentration has been estimated approximately 20 fold more than that of free drug and it remains in the tumor tissue for longer period as compared to conventional liposomes and free drug. These values have been validated with those in literature. Reason behind stealth liposome more accumulation in tumors is their slower plasma and higher vascular clearance. This increases the therapeutic effect of drug and also their probability of killing tumor cells due to long drug exposure. This computational study potentially enables medical professionals to forecast the overall effectiveness of the drug and optimize treatment strategy for each patient.
<figure><img src="image_72.png" /><figcaption>Contour plots of concentration.png</figcaption></figure>
# Sensitization of sarcoma tumors with short chain sphingolipid liposomes

Thursday, 29th September - 16:50 - Targeted drug delivery and Nanocarriers - Nanomedicine for cancer diagnosis & therapy - Amphitheatre 25 - Oral presentation - Abstract ID: 510

Dr. Sara Zalba Oteiza¹, Mr. Joost A. P. Rens¹, Dr. Jeroen Rovers², Prof. Marcel Verheij³, Dr. Timo L. M. Ten Hagen¹

1. Erasmus Medical Center, 2. Ceronco Biosciences, 3. Antoni van Leeuwenhoek

**Introduction:** Multidrug resistance (MDR) is one of the main challenges in cancer chemotherapy. The over-expression of multidrug transporters like P glycoprotein (Pgp) is a common cause of resistance1. Liposomes enriched in short chain sphingolipids (SCS) may interact with cancer cell membranes improving permeability to encapsulated drugs2-4. Our aim is to evaluate the effect of SCS liposomes in resistant tumor cells with the aim of improve their chemo-sensitivity.

**Methods:** MES-SA/MX2 is a resistant human utero sarcoma cell line derived from MES-SA, with Pgp over-expression. Liposomes loaded with doxorubicin were enriched (SCS-LP) or not (LP) with SCS, characterized and incubated with both cell lines to evaluate drug internalization and cytotoxic effect. Biodistribution, phar-macokinetics and efficacy was evaluated in xenograft tumor bearing mice with the above mentioned sensitive and resistant cell lines.

**Results:** In vitro assays showed a higher effect of SCS-LP in MES-SA/MX2, which was related with an increase in the sensitivity to doxorubicin. Actually SCS-LP were able to revert their resistance compared to sensitive cell results. On the MES-SA cell line all liposomes had a similar effect under all conditions tested. A higher accu-mulation of doxorubicin in resistant tumors after SCS-LP administration was observed in mice bearing these tumors subcutaneously, compared to the sensitive tumor and LP. Besides, nuclear accumulation of doxorubicin was superior in resistant tumors under SCS-LP treatment. Finally, enriched liposomes showed a faster clear-ance but were able to control the tumor growth more efficiently than non SCS liposomes in resistant tumors, whereas in sensitive tumor no difference was found.

**Discussion:** Based on our in vitro and in vivo results we conclude that the use of doxorubicin-SCS-LP reverts the resistance of MES-SA/MX2. Our data indicates that sensitivity to the drug is improved through bypassing or compensation of Pgp activity. Although the formulation needs to be optimized, e.g. to avoid serum instability, these liposomes represent a very promising tool to treat cancer, especially MDR tumors.

1. Adv Drug Deliv Rev. 2013 Nov;65(13-14):1852-65.

2. J Biomed Nanotechnol. 2016 Apr;12(4):630-44.

3. Pharm Res. 2015 Apr;32(4):1354-67.

4. Pharm Res. 2013 Jul;30(7):1883-95.
# Immune checkpoint blockade in melanoma by new targeted Doxorubicin immunoliposomes

Thursday, 29th September - 17:15 - Targeted drug delivery and Nanocarriers - Nanomedecine for cancer diagnosis & therapy - Amphitheatre 25 - Oral presentation - Abstract ID: 511

Mrs. María Merino Díaz¹, Mrs. Ana Margarita Contreras Sandoval ¹, Dr. Noelia Casares ², Dr. Iñaki Troconiz ¹, Dr. Pedro Berraondo ², Dr. Timo L. M. Ten Hagen ³, Dr. Sara Zalba Oteiza ³, Dr. Maria J Garrido ¹

1. University of Navarra, 2. CIMA, 3. Erasmus Medical Center

**Introduction:** Programmed Cell Death Ligand 1(PD-L1), expressed in most solid tumors, binds to T-cells through its receptor PD-1, leading to the immunoresistance development and tumor progression [1]. Anti-PD-L1 monoclonal antibody (mAb) blocks this ligand promoting antitumor activity of T-cells [2]. Moreover, Doxorubicin (Dox) liposomes improve the therapeutic effect over free drug. Both therapies can be combined in a single formulation of Dox liposomes decorated with anti-PD-L1 Fab' fragments. Thus, the aim of this work is to develop and characterize Dox targeted liposomes and evaluate its antitumor effect in vitro and in vivo.

**Methods:** Dox liposomes (LPDOX) were prepared by film hydration method [3] and drug loaded by sulphate gradient [4]. DSPE-PEG2000-Mal micelles were coupled to anti-PD-L1-Fab' fragments and incorporated in pre-formed LPDOX with post insertion method, obtaining targeted liposomes (LPDOXFab'). After physicochemical characterization, drug release was measured in FBS at 37 °C for 1h. B16 OVA melanoma mouse cell line was used for in vitro/in vivo studies. In vitro cytotoxicity (IC50) was obtained after 24h of drug exposure and Dox cell uptake was measured by FACS and confocal microscopy after 4h of treatment. Finally, in vivo antitumor efficacy was studied in tumour bearing mice.

**Results:** LPDPXFab' and LPDOX were around 130 nm, PDI < 0.1 and 95±7.5% of encapsulation. Ligand conjugation was 40±7.4%. Drug release reached 20% after 1h in FBS. Targeted liposomes were able to bind specifically PD-L1, showing no internalization. IC50 values for both formulations were similar after 24h of exposure. There was a delay in melanoma tumor growth with no significant differences in both liposomes. However, survival for LPDOXFab' was higher, suggesting a benefit that deserves to be explored.

**Conclusion:** LPDPXFab' have been successfully developed using the post-insertion method. In vitro and in vivo studies revealed similar efficacy for both formulations. However, the difference in the survival suggests an improvement of the targeted formulation that will be deeper examined in further studies.

[1] Crit Rev Oncol Hematol. 2014 Jan; 89(1):140-65.

[2] Eur J Cancer. 2013 Sep; 49(14):2968-71.

[3] Eur J Pharm Biopharm. 2012 Jun; 81(2):273-80.

[4] J Control Release. 2012 Jun 10; 160(2): 117-34.
Titanate nanotubes as new preclinical theranostic platform against prostate cancer: vectorization and immobilization of docetaxel or of gold nanoparticles

Thursday, 29th September - 17:40 - Targeted drug delivery and Nanocarriers - Nanomedecine for cancer diagnosis & therapy - Amphitheatre 25 - Oral presentation - Abstract ID: 14

<u>Mr. Alexis Loiseau</u>¹, Dr. Julien Boudon¹, Dr. Céline Mirjolet², Prof. Gilles Créhange², Prof. Stéphane Roux¹, Prof. Nadine Millot¹

1. Université Bourgogne Franche-Comté, 2. CGFL

Titanate nanotubes (TiONts) are synthetized by a hydrothermal process [1]. Their uncommon morphology obtained by controlled parameters allows them to be internalized more easily into cells. This permits their use as novel transfection agents [2] without inducing cytotoxicity while providing a radiosensitization effect [3]. These TiONts are combined to docetaxel (DTX), a molecule widely used for the treatment of cancers. Currently, injected drugs only reach very weakly tumor sites. In the last decade, the development of nanotechnologies has offered a new strategy to vectorize an active substance. In this work, two developments of nanohybrids are presented to fight against prostate cancer [4]: the first approach consists in combining TiONts and DTX and in a second one TiONts and gold nanoparticles (AuNPs) are combined together. This project is based on intraprostatic injection of the nanohybrids.

A particular attention was paid on the elaboration of functionalized-TiONts nanohybrids. On the one hand, docetaxel molecules were grafted by an original pathway onto TiONts. DOTA macrocycles were also grafted on the latter, allowing a radiotracer to be chelated, in order to evaluate the biodistribution of TiONts in mice by nuclear imaging. Biodistribution kinetics showed that more than 70% of nanohybrids were localized into the tumor 96 hours after injection. Mice receiving nanohybrid-RT (Radiation Therapy) exhibited a significant tumor growth delay compared to mice receiving free DXL-RT [5]. On the other hand, AuNPs functionalized by DTDTPA molecules are grafted on TiONts. This combination confers not only the nanovectorization via TiONts but also therapeutic effect via AuNPs [6]. In both cases, the suspension stability of nanohybrids was increased by the addition of biocompatible polyethylene oxide polymers and physico-chemical characterizations were realized to assert each functionalization step of our engineered nanohybrids.

References:

[1] A.-L. Papa et al., J. Phys. Chem. C, 113 (2009)

[2] A.-L. Papa et al., Nanotoxicology, 7, 6 (2013)

[3] C. Mirjolet et al., Radiother. Oncol., 108, 1 (2013)

[4] J. Boudon et al., Nanomedecine. O. C. Press: chapt 16

[5] C. Mirjolet et al., Eur. J. Cancer, 50, 6 (2014)

[6] C. Alric et al., Gold Bulletin, 41, 2 (2008)
# The endothelial glycocalyx controls interactions of nanoparticles with the endothelium and their translocation across the blood-tissue border

Thursday, 29th September - 16:00 - Targeted drug delivery and Nanocarriers - Tower 24 - Room 101 - Oral presentation - Abstract ID: 414

Dr. Bernd Uhl¹, Dr. Stephanie Hirn¹, Mr. Roland Immler¹, Mrs. Karina Mildner², Dr. Leonhard Möckl³, Prof. Markus Sperandio¹, Prof. Christoph Bräuchle³, Dr. Christoph Reichel¹, Dr. Dagmar Zeuschner², Prof. Fritz Krombach¹

1. Walter Brendel Centre of Experimental Medicine, Klinikum der Universität München, Ludwig-Maximilians-Universität München, Munich, Germany, 2. Electron Microscopy Unit, Max Planck Institute for Molecular Biomedicine, Münster, Germany, 3. Department of Chemistry and Center for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Munich, Germany

A prerequisite for the use of nanoparticles (NPs) in diagnostic and therapeutic applications is the targeted delivery to the site of injury. The endothelial glycocalyx (eGCX) is a glycoprotein-polysaccharide meshwork coating the luminal surface of the vasculature. Its role for the regulation of interactions between blood-borne NPs and the microvascular endothelium, however, remains obscure.

Multiphoton in vivo microscopy was employed on the cremasteric microvasculature of wildtype mice to analyze the structure of the eGCX and the deposition of NPs. The eGCX was visualized using labeled wheat germ agglutinin. Carboxyl quantum dots (QDs) were chosen as model NPs. Heparinase, hyaluronidase, and neuraminidase were used to systemically degrade the eGCX. Direct binding of NPs to endothelial molecules was examined in vitro using flow chambers. The uptake and translocation of NPs were addressed in vivo employing transmission electron microscopy (TEM). The effect of ischemia-reperfusion (I/R) injury on the eGCX and the deposition of NPs were examined using an I/R model.

In the microvasculature of control animals, a high eGCX layer was found in all vessel segments, while interactions of QDs with the endothelium were low. Upon enzymatic degradation of the eGCX, however, its size was significantly reduced and NP-endothelium interactions increased considerably. Moreover, endothelial uptake and transendothelial translocation of NPs after enzymatic degradation of the eGCX were demonstrated in vivo using TEM. Binding of QDs to endothelial signaling and adhesion molecules, which were exposed to the blood-stream upon enzymatic degradation of the eGCX, was observed by in vivo microscopy and confirmed in flow chambers. Employing I/R on the cremaster muscle caused a significant reduction of the eGCX and a dramatic increase in NP-endothelium interactions.

Consequently, the eGCX effectively controls NP-endothelium interactions in the microvasculature. It was found to physically cover endothelial adhesion and signaling molecules thereby preventing endothelial attachment, uptake, and translocation of NPs through different layers of the vessel wall. Conversely, degradation of the eGCX under pathologic conditions promoted interactions of NPs with microvascular endothelial cells thus identifying the injured eGCX as an essential element of the blood-tissue border facilitating the targeted delivery of nanomaterials to diseased tissue.
Magnetic hyperthermic response of nanocomposites
based on PEG2000-gallol-coated iron oxide
nanoparticles dispersed in
poly(N-isopropylacrylamide-co-acrylamide) hydrogels

Thursday, 29th September - 16:25 - Targeted drug delivery and Nanocarriers - Tower 24 - Room 101 - Oral presentation - Abstract ID: 463

Mr. Kevin C. Behan ¹, Ms. Sarah M. Martyn ¹, Dr. Aylvin A. Dias ², Prof. Andreas Heise ³, Dr. Dermot F. Brougham ⁴

1. Dublin City University, School of Chemical Sciences, Glasnevin, Dublin 9, 2. DSM Ahead, Urmonderbaan 22, 6167 RD Sittard-Geelen, 3. Royal College of Surgeons, Department of Pharmaceutical and Medicinal Chemistry, 123 St. Stephen's Green, Dublin 2, 4. University College Dublin, School of Chemistry, Stillorgan Road, Belfield, Dublin 4

Nanoparticles can be incorporated into hydrogel matrices by various means. Non-covalent methods such as blending and in situ co-precipitation are straightforward, but can result in uncontrolled diffusion of the nanoparticles out of the gel. Covalent methods graft reactive groups onto the nanoparticle surface, which can then act as nano-crosslinkers, forming stable networks. [1]

Superparamagnetic iron oxide nanoparticles were synthesised step-wise from 6 nm seeds, according to the method by Sun. The hydrophobic oleic acid and oleylamine ligands were exchanged with the cathecol-bearing methoxy-PEG2000-gallol. These modified iron oxide nanoparticles could be transferred almost quantitatively into ethanol.

Subsequently, the methoxy-PEG2000-gallol coated nanoparticles were mixed with a solution containing various amounts of N-isopropylacrylamide, acrylamide, PEG700-diacrylate, ammonium persulfate, and TEMED. The solutions underwent thermal radical polymerisation to obtain the magnetic hydrogel nanocomposite. After purification, the gels were exposed to an AC magnetic field (475 kHz, 12.44 mT). Deswelling with respect to nanoparticle loading and acrylamide concentration was investigated. The methoxy-PEG chains on the nanoparticle surface ensure facile dispersion, a stable nanocomposite, with reproducible heating profile and deswelling behaviour.

Dhyd of the iron oxide nanoparticles was 44 nm and DTEM was 12 nm. For gels containing 200 µL of nanoparticle suspension (22.6 mM Fe, Tf=24.3°C), the deswelling ratio in the AC field was 8.6, 7.3, and 5.1%, respective of increasing acrylamide content. For gels containing 400 µL of nanoparticle suspension (45.1 mM Fe, Tf=32.1°C), the deswelling ratio was 39.7, 26.3, and 19.0%, respectively. For gels containing 800 µL of nanoparticle suspension (90.3 mM Fe, Tf=35.4°C), the deswelling ratio was 45.4, 43.1, and 26.4%, respectively.

The amount of acrylamide influences both the rates of swelling and deswelling in the hydrogel. Increasing iron oxide nanoparticle loading increases the temperature of the gels and accelerates the deswelling further. Both swelling and deswelling are reproducible for a given gel. It is planned to investigate deswelling of the gels at a baseline temperature of 37 °C.

[1] Li, Y., Huang, G., Zhang, X., Li, B., Chen, Y., Lu, T., Lu, T. J. and Xu, F. (2013), Magnetic Hydrogels and Their Potential Biomedical Applications. Adv. Funct. Mater., 23: 660–672. doi:10.1002/adfm.201201708
<figure><img src="image_73.png" /><figcaption>Figure 1: (A) TEM image of 12 wt% iron oxide nanoparticles. (B) Densitometry ratios of magnetic hydrogel. (C) Heating profiles of gels as function of [Fe]. (D) Densitometry state based on scyliamidine contrast (left: AAm120; middle: AAm200; right: AAm400)</figcaption></figure>

Abstract fig.png
# Increasing the stability and antimicrobial activity of antimicrobial peptides after association to Lipid nanocapsules

Thursday, 29th September - 16:50 - Targeted drug delivery and Nanocarriers - Tower 24 - Room 101 - Oral presentation - Abstract ID: 53

Ms. Nada Matougui¹, Dr. Anne Claire Groo², Dr. Anita Umerska¹, Dr. Helena Bysell³, Prof. Patrick Saulnier¹

1. Université Angers, UMR-S1066 Angers, France, 2. Normandie Univ, UNICAEN, Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN), 14000 Caen, France, 3. SP Technical Research Institute of Sweden

**Introduction:**

Resistant infections are estimated to be responsible of 700.000 deaths worldwide. These figures, often underestimated highlight the importance of the antimicrobial resistance (AMR). Education toward the misuse of antibiotics is no more sufficient. Finding new therapies to overcome resistance becomes imperative. Antimicrobial peptides are under the spotlight. Their ability to act rapidly, at low concentrations and in a not specific way makes them interesting antibiotic molecules with less potential to induce resistance. Nevertheless, their development is hampered by physico-chemical and biological instabilities.

The aim of this work is to explore the potential of lipid nanocapsules (LNCs) to deliver AA230, DPK060 and LL37, three cationic, hydrophilic and biosourced antimicrobial peptides.

**Methods:**

Peptide adsorption on the surface of the LNCs (LNC-AMP) and/or encapsulation within their lipidic core (LNC-RM-AMP) was performed. The in vitro antimicrobial activity of the AMPs and nanoformulated AMPs was assessed by the determination of the minimal inhibitory concentrations against different Gram (-) and Gram (+) bacteria and The kinematics of the inhibitory effect was conducted for each formulation against the sensitive strains. Proteolysis assays was performed to inviggate peptides stability after association.

**Results:**

The results showed a good association efficiency after adsorption to the surface of LNCs. The encapsulation in the core succeeded except for the peptide AA230. The antimicrobial activity was preserved in all cases. Moreover, a potentiation of the in vitro anti-bacterial activity, reflected by a lower minimal inhibitory concentrations (MICs) was observed for the adsorbed peptides. The time kill-kinetics studies confirm the bactericidal killing of the nanoformulated peptides at concentrations up to 8 times lower than the MIC of the peptide alone. Moreover, proteolysis assays performed with trypsin as model enzyme, demonstrated a partial protection of adsorbed LL37 against proteolytic degradation compared to the plain peptide over 4hours.

**Discussion:**

Overall, these data indicated that LNCs are promising nanocarriers for the delivery of AMPs. The two strategies studied are to be investigated. The better knowledge of the peptide properties and the exploration of the mechanism of action responsible of the synergistic effect will definitely contribute to consolidate the potential of such nanoformulated peptides as next-generation antibiotics.
<table><thead><tr><th rowspan="2">Bacterial strains</th><th colspan="6">Minimum Inhibitory Concentrations (µg/mL)</th></tr><tr><th>LL37</th><th>LNC-LL37</th><th>DPK060</th><th>LNC-DPK060</th><th>AA230</th><th>LNC-AA230</th></tr></thead><tbody><tr><td>Staphylococcus aureus</td><td>8-16</td><td>8-16</td><td>4</td><td>2-4</td><td></td><td></td></tr><tr><td>Methicillin-resistant Staphylococcus aureus clinical strain (MESA)</td><td>8-16</td><td>4</td><td>4</td><td>1</td><td></td><td></td></tr><tr><td>Pseudomonas aeruginosa</td><td>8-16</td><td>16</td><td>8</td><td>8-16</td><td>2-4</td><td>1</td></tr><tr><td>Pseudomonas aeruginosa clinical strain</td><td>8-16</td><td>4</td><td>16</td><td>4-8</td><td>4-8</td><td>2</td></tr><tr><td>Bactericida coli ATCC 25932</td><td>16</td><td>8</td><td>8</td><td>4</td><td>2-4</td><td>1</td></tr><tr><td>Bactericida coli clinical strain</td><td>16</td><td>8</td><td>4-8</td><td>2-4</td><td>1-2</td><td>0.5</td></tr><tr><td>Acinetobacter baumannii ATCC BAA-1730</td><td>16</td><td>4</td><td>4-8</td><td>4-8</td><td>2-4</td><td>0.25</td></tr></tbody></table>

Minimal inhibitory concentrations of native and associated antimicrobial peptides.png
# Biological Recognition of Biomolecular Corona

Thursday, 29th September - 17:15 - Targeted drug delivery and Nanocarriers - Tower 24 - Room 101 - Oral presentation - Abstract ID: 261

Ms. Sandra Lara¹, Ms. Fatima Alnasser¹, Dr. Ester Polo¹, Dr. David Garry¹, Ms. Maria-cristina

Lo-giudice¹, Dr. Delyan Hristov¹, Dr. Yan Yan¹, Prof. Kenneth A. Dawson¹

1. Centre For BioNano Interactions (CBNI), School of Chemistry and Chemical Biology, University College Dublin

## Introduction

When the nanoparticle surface comes in contact with a biological milieu, adsorption processes may lead to a well-defined surface-assembly of biomolecules derived from the environment. Appropriately presented, and sufficiently long-lived ('hard corona') biomolecular motifs presented at the surface would define how a nanoparticle first interacts with and is recognised by cells. Besides their fundamental importance, such questions likely define many key impacts on efficacy of nanoparticle-based therapies, including their bio-distribution and nanomedicine targeting. For instance, well-defined active processes driven by receptors on the specialized cells of the liver could drive nanoparticle accumulation. However, little is known of the molecular organisation of various proteins in the corona. Hence, the detailed molecular interactions between the presented protein motifs and receptors are yet to be elucidated.

## Methods

Here we present a systematic method to advance such hypotheses, by direct investigation of nanoparticle-receptor interactions in biological milieu. Firstly, we have built a cell receptor library by transfection of interest to host cells. This system allows us to examine the dependence of nanoparticle uptake to a given receptor in appropriate biological milieu. In addition, this approach overcomes the limitations that some receptors cannot be isolated in a functionally relevant form, as their expression levels are interdependent in endogenously expressed cells. In parallel, we also have developed tools by using monoclonal antibodies to detect epitope presentation on the nanoparticle surface. Altogether, we have been able to characterise the biomolecular corona with molecular details and their interactions with different receptors.

## Results and Discussion

In this presentation, we will illustrate the recognition of biomolecular corona by a couple of key receptors, low-density-lipoprotein receptor (LDLR) and Fc-gamma receptor I (FcγRI), which are abundant in the liver. By using both flow cytometry and microscopy, we have shown that the 'labelling' of nanoparticles by biomolecular adsorption processes allows for ubiquitous nanoparticle multi-pathway involvement in biological processes, which governs the liver accumulation of nanoparticles.
# Synthesis-Dependent Surface Defects and Morphology of Hematite Nanoparticles and Their Effect on Cytotoxicity in Vitro

Thursday, 29th September - 17:40 - Targeted drug delivery and Nanocarriers - Tower 24 - Room 101 - Oral presentation - Abstract ID: 82

**Mr. Dean Cardillo**¹, **Dr. Moeava Tehei**¹, **Dr. Md Shahriar Hossain**¹, **Mr. Md Monirul Islam**¹, **Ms. Kathrin Bogusz**¹, **Dr. Dongqi Shi**¹, **Dr. David Mitchell**¹, **Prof. Michael Lerch**¹, **Prof. Anatoly Rosenfeld**¹, **Dr. Stéphanie Corde**², **Dr. Konstantin Konstantinov**¹

1. University of Wollongong, 2. Prince of Wales Hospital

In this study we investigated the toxicity of hematite nanoparticles on the Madin-Darby Canine Kidney (MDCK) cell line. Hematite precursors were prepared using precipitation and spray precipitation, which were then subsequently annealed to form crystalline iron oxide nanoparticles. The crystalline structure, morphology and magnetic properties of these nanoparticles were investigated using X-ray Diffraction, Scanning Electron Microscopy, Transmission Electron Microscopy, and Physical Properties Measurement System. X-ray Photoelectron and Raman Spectroscopy were also used in order to probe the fine structure and surface characteristics. The biological effect of these nanoparticles was also assessed, using a clonogenic assay for overall cytotoxicity, as well as flow cytometric studies to investigate cellular uptake of the nanoparticles, as well as their effects on the cellular reactive oxygen species (ROS) levels.

While the traditional precipitation method produced roughly spherical hematite particles, the use of spray precipitation resulted in highly porous nanorods. Through XPS and Raman analysis it was found that these nanoparticles feature a small defects of maghemite (γ-Fe2O3) on their surface, in the form of tetrahedrally-coordinated Fe3+. This is due to a thermodynamically-favoured phase transition on their surface due to being formed from amorphous iron oxide precursors. The concentration of these defects were found to decrease when the precursor was annealed at the higher temperature.

It was found that these defects have an effect on the biological activity, with the spherical nanoparticles showing much lowered toxicity and ROS generation than their rod-shaped counterparts. The particles featuring these maghemite defects, which has been shown to scavenge free radical species, even showed to lower the ROS levels in the cytometric assay relevant to the control. The rod shape of the spray precipitation nanoparticles also had preferential uptake into the MDCK cells compared to the spherical nanoparticles.

This work highlights the need for in-depth understanding of the materials properties of nanomaterials in order to understand the factors governing their toxicity, including effects on their fine structure from synthesis parameters.
<figure><img src="image_74.png" /><figcaption>Picture2.png</figcaption></figure>
# Fate of Various Dendrimers with Different Sizes and Surfaces after Subcutaneous and Intradermal Administration

Thursday, 29th September - 16:00 - Nanomedicine for cancer diagnosis & therapy - Tower 24 - Room 103 -
Oral presentation - Abstract ID: 310

Prof. Chie Kojima¹

1. Osaka Prefecture University

Dendrimers are synthetic macromolecules with highly controllable structures, which are potent multifunctional nanoparticles for drug delivery system and imaging. There are many reports on intravenous administration of dendrimer, but few on intradermal and subcutaneous administration. In this study, various dendrimers of different generations (G2, G4, G6, and G8) and different surfaces (amino, carboxyl, acetyl and collagen peptide modification) were prepared and radiolabeled. First, the radiolabeled dendrimers were intradermally administrated to the right footpad of rats. All G2 dendrimers were predominantly accumulated in the kidney. Amino-terminal, acetyl-terminal and carboxy-terminal dendrimers of greater than G4 were mostly located at the injection site, in blood and in the popliteal lymph node, respectively. Interestingly, the carboxy-terminal dendrimers were not largely recognized by macrophages and T cells in the lymph node. Finally, the lymph detection was performed by single photon emission computed tomography (SPECT) imaging using various dendrimers. (Figure 1). Carboxy-terminal dendrimers of greater than G4 and amino-terminal G6 dendrimer (G6-NH2) successfully visualized the popliteal lymph node (indicated by arrows), but acetyl-terminal G6 dendrimer (G6-Ac) and carboxy-terminal G2 dendrimer (G2-COOH) did not. Compared with the COOH-terminal dendrimers of higher than G4, G6-NH2 was largely retained at the injection site (indicated by arrow heads). These results suggest that carboxy-terminal dendrimers of greater than G4 are useful for drug delivery and imaging of sentinel lymph node, which the first lymph node draining tumor cells. Next, the radiolabeled dendrimers were subcutaneously administrated into tumor-bearing mice, and monitored by using SPECT imaging. Acetylated G4 dendrimer (Ac-G4) and collagen peptide-conjugated dendrimer (CP-G4) were largely retained at the injection site for at least 1 day (indicated by arrows). Ac-G4 were partly accumulated in the kidney (indicated by K), but the CP-G4 was not (Figure 2). On the other hand, these dendrimers were accumulated in the liver and the kidney following intravenous administration. These results indicate that the subcutaneously injected dendrimers did not largely gain substantial access to the systemic circulation, which is useful for a depot of drug around the injection site.
<figure><img src="image_75.png" /><figcaption>Fig.1.jpg</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>Fig.2.jpg</figcaption></figure>
# Specific nanoparticle targeting of the EGF-receptor using single-domain antibodies.

Thursday, 29th September - 16:25 - Nanomedicine for cancer diagnosis & therapy - Tower 24 - Room 103 - Oral presentation - Abstract ID: 46

Dr. Kristof Zarschler¹, Dr. Louise Rocks², Dr. Eugene Mahon², Dr. Kanlaya Prapainop², Dr. Holger Stephan¹, Prof. Kenneth A. Dawson²

1. Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden - Rossendorf, 2. Centre For BioNano Interactions (CBNI), School of Chemistry and Chemical Biology, University College Dublin

## Introduction

For effective localization of functionalized nanoparticles at diseased tissues such as solid tumors or metastases through biorecognition, appropriate targeting vectors directed against selected tumor biomarkers are a key prerequisite. The diversity of such oncotropic vector molecules ranges from proteins, including antibodies and fragments thereof, through aptamers and glycans to short peptides and small molecules.

In the presented work we analyze the specific nanoparticle targeting capabilities of a small camelid single-domain antibody (sdAb), representing a potential recognition agent for the epidermal growth factor receptor (EGFR).

## Methods

Bioconjugation of EGFR-specific sdAbs to different nanomaterials and characterization of sdAb-conjugates covering in vitro cancer cell imaging, cell proliferation as well as EGFR phosphorylation and signaling are described. The specificity of the sdAb-conjugates is investigated by way of receptor RNA silencing techniques with increasing complexity in vitro by introducing increasing concentrations of human or bovine serum.

## Results and Discussion

The results show that sdAb-functionalized nanomaterials can effectively target the EGFR, even in more complex bovine and human serum conditions where targeting specificity is largely conserved for increasing serum concentrations. For highly affine targeting ligands such as sdAbs, targeting a receptor such as EGFR with low serum competitor abundance, receptor recognition function can still be partially realized in complex conditions. Moreover, sdAb-mediated biorecognition of EGFR is not restricted to particular nanomaterials, but was observed to work efficiently in combination with a variety of materials.
# Differential Modulation of Biological Properties In Vitro by a Range of cRGDY Peptides on Clinically Translated Dual-Modality Silica Nanoparticles

Thursday, 29th September - 16:50 - Nanomedicine for cancer diagnosis & therapy - Tower 24 - Room 103 - Oral presentation - Abstract ID: 359

Dr. Miriam Benezra¹, Dr. Pauliah Mohan¹, Prof. Ulrich Weisner², Prof. Michelle Bradbury¹

1. Memorial Sloan-Kettering Cancer Center, New York, NY, 2. Cornell University, Ithaca, NY

**Introduction:**

Ultra-small Dual Modality nanoparticles, termed Cornell dots (i.e., C' dots) may provide a disease-directed platform for delivery of small cancer molecules for theranostic purposes. For clinical advancement of such platforms is the need to characterize at the cellular and molecular levels, to light biological properties of these probes. We used silica nanoparticle encapsulate Cy 5 dyes (C' dots), surface functionalized with methoxy-terminated polyethylene glycol (PEG), that bind to it different numbers of integrin-targeting cRGDY ligands (N-6, 14, 18) (i.e., cRGDY-PEG-C' dots). Their activities were compared in vitro on several biological systems using cells that over express the αvβ3 receptor, a human melanoma (M21) and human umbilical vein endothelial (HUVEC) cells.

**Methods :**

Binding and internalization - flow cytometry; Migration performed by time lapse. Adhesion and spreading - SpectraMax5 micro plate reader; Vi-Cell Counter for cell Viability, Effect on signaling - Western blot analysis.

**Results:**

Dose response and time-dependent binding revealed binding saturation at 50 nM with 14 or 18 cRGDY-PEG-C' dots at two hours as opposed to 100 nM with 6 cRGDY-PEG-C' dots at four hours incubation. Internalization studies revealed a higher uptake (15-20%) of the 14 and 18 particles in 370C compared to 6 cRGDY-PEG-C' dots on M21. Binding specificity was demonstrated using antibodies against the αvβ3 receptor. There was no binding of any of the particle on cells that don't express the αv receptor subunit. Minor binding was observed with two different sizes of scramble peptide-cRADY (6 or 15-20). Additionally, a higher migration rate of HUVEC cells, adhesion with spreading on fibronectin coated plates of M21 cells and proliferation of M21 cells were shown with 14 or 18 cRGDY-PEG-C' dots compared to 6 cRGDY-PEG-C' dots. Activation of the FAK pathway by integrin was also several fold (x2) more with 14 cRGDY as opposed to 6 cRGDY on the particle.

**Discussion:**

Changes in vitro on the level of binding and key biological processes-cell migration, adhesion/spreading, proliferation process were greater with 14-18 cRGDY-PEG-C' dots. This paves the way in designing a diagnostic platform for primary tumor and cancer metastasis or therapeutic purpose for processes like angiogenesis and wound healing.
<figure><img src="image_77.png" /><figcaption>Figure 1: Multimodal C⁰ dot design for n,δ,ε integrins and characterization [A] Schematic representation of (c)RGDy-PEGylated core-shell silica nanoparticle with surface-bonding nectins and integrins and core-containing reactive dye molecules (dots) [B] Specific binding of cRGDy-PEG-C⁰ dots (N=6,14,18) to M21-L cells on against M21-L cells lacking surface integrin expression. Detection of binding was done by flow cytometry. Each bar represents mean±SD of 3 replicates.</figcaption></figure>
# Traceable Iron Oxide Based Nanoparticles for Antigen/Adjuvant in vivo Delivery to Lymph Nodes

Thursday, 29th September - 17:15 - Nanomedicine for cancer diagnosis & therapy - Tower 24 - Room 103 -
Oral presentation - Abstract ID: 27

Ms. Ane Ruiz de Angulo¹, Dr. Aintzane Zabaleta¹, Ms. Zuriñe Baz¹, Dr. Jordi Llop¹, Prof. Juan Carlos Mareque Rivas¹

1. CIC biomaGUNE

Most of the currently available vaccines are based on the inactivation of the pathogen, which is not a valid strategy for fighting several chronic infections and, especially, cancer.[1,2] Here we present PEGylated iron oxide based nanoparticles (IONPs) as traceable delivery system for directing tumour antigens and adjuvants to lymph nodes (LNs), leading to improved antitumor immunity and survival outcomes. The nanovehicles allow using lower doses of the immunostimulatory molecules to achieve a given response and thereby enhance their safety profiles. Moreover, other bioactive molecules and drugs can be co-delivered to further improve the efficacy and therapeutic index of the anticancer therapy.

We designed and created PEGylated IONP-filled micelles carrying short oligodeoxynucleotides with CpG motifs as adjuvants and ovalbumin (OVA) as antigen. Doping with rhodamine B-phospholipids[3] was used to monitor in vitro cell uptake, whilst chemistry-free radiolabeling with 67Ga could be applied to study the biodistribution in vivo by SPECT imaging. The immunostimulatory potential of the constructs was tested analyzing cytokine, antibody production and cellular mediated immunity. Immunization against tumour-antigen was performed to demonstrate the anti-tumour immunity.

We obtained water soluble micelles with a diameter below 100nm, which were internalized through the endocytic pathway and co-localized with the toll-like receptor 9 (TLR9). SPECT imaging showed accumulation of these constructs in the LNs and both in vitro and in vivo assays demonstrated enhanced immunostimulatory activity than with the free molecules. The analysis of adaptive immune response demonstrated the production of anti-OVA antibodies and the activation of antigen specific CD8+ T cells, able to significantly slow down the appearance of tumour.

The nanoconstructs were able to deliver the immunostimulatory molecules in the correct intracellular organelle, where the CpGs could reach TLR9 and, therefore, induce cytokine production in vitro. There was no systemic effect in vivo, concluding that the effect was directed to lymphoid organs. The CD8+ T cell subset was successfully activated against the antigen, crucial characteristic for generating anticancer immunity. The anti-tumour studies showed the potential of these systems for developing cancer vaccines.[4]

## References

(1)Landrith, T.A. et al. Semin.Immunopathol.2015,37,261-270.

(2)Schreiber, R.D. et al. Science.2011,331,1565-1570.

(3)Cobaleda-Siles, M. et al. Small.2014,10,5054-5067.

(4)Ruiz-de-Angulo, A. et al. ACS Nano.2016,10,1602-1618.
<figure><img src="image_78.png" /><figcaption>Figure1.png</figcaption></figure>
# Magnetic-nanoparticles as a theranostic tool for liver metastases in a murine model

Thursday, 29th September - 17:40 - Nanomedicine for cancer diagnosis & therapy - Tower 24 - Room 103 -
Oral presentation - Abstract ID: 330

Mr. Borja Herrero De La Parte ¹, Dr. Eneko Garayo Urabayen ¹, Dr. Oihane Kistiñe Arriortua Llarena ², Dr. Jose Javier Echevarria-uraga ³, Prof. Jose Angel Garcia Martinez ¹, Prof. Ignacio García-Alonso Montoya ⁴, Prof. Fernando Plazaola Muguruza ¹, Ms. Irati Rodrigo Arrizabalaga ²

1. University of the Basque Country, UPV/EHU, 2. BCMaterials, 3. Galdakao-Usansolo Hospital, 4. University of the

**Introduction:** The treatment of liver metastases is still a challenge. Magnetic hyperthermia is being explored as a new therapy. Functionalized magnetic nanoparticles (MNP) can be adhered to tumour tissues and then heated selectively by an alternating magnetic field (AMF). The temperature increment due to the MNP heating of MNP might destroy the tumours spearing healthy liver tissue. The aim of our experiment was to develop a reliable and selective magnetic-hyperthermia therapy for diagnosis and treatment of liver metastases in a rat model. RGD-peptides functionalized MNP, intravascular infusion of MNP and an electromagnetic applicator to achieve selective heating have been designed and an "in vivo" experiment was carried out to check the effectivity of the treatment.

**Methods:** Colorectal cancer cells were inoculated into the left lateral liver lobe (LLL) of WAG/RijHsd rats. Fe3O4 nanoparticles covered with PMAO and functionalized with RGD peptides were synthetized in order to adhere them to the tumour tissue. Using a microcannula placed into the hepatic artery, MNP-group was infused with MNP and saline-group was infused with the same volume of saline. After 12 hours, MRI studies were practised by 1,5T MRI machine and magnetic hyperthermia was applied by an alternating magnetic field of 14kA/m intensity and 606kHz frequency, during 21 minutes. The temperature was monitored with 3 probes placed in the tumour, LLL and rectum. 12 hours later, both groups were sacrificed and the liver removed for histopathological analysis.

**Results:** MRI study showed decay of the signal intensity (SI) in the liver and in the periphery of the tumour. Moreover, scattered foci may be observed within the tumours. After thermal exposure, tumour temperature increased 4.66 ± 0.99°C in saline-group and 7.89 ± 1.2°C in MNP group (p<0.001) (Figure 1). The values of the necrotic tissue, relative to the total tumour tissue, ranged between 1-13% and 13-99% for the saline-group and MNP-group (p<0.01), respectively.

**Discussion:** MNP adhered to liver metastases and could be monitored by MRI. A higher thermal increase in liver and tumour tissue was reached in MNP-infused animals, showing that MNP can be used in magnetic hyperthermia therapy, although further experiments are needed.
<figure><img src="image_79.png" /><figcaption>Figure1.jpg</figcaption></figure>
Hybrids of biomolecules and carbon nanotubes:
nanodevices for biosensing

Thursday, 29th September - 16:00 - Biological & medical nanodevices and biosensors - Bionanocatalysis and nanobiosystems - Tower 24 - Room 105 - Oral presentation - Abstract ID: 104

Prof. KAZUO UMEMURA ¹, Mr. Shusuke Oura ¹, Mr. Yu Ishizaka ¹

1. Tokyo University of Science

Carbon nanotubes (CNTs) have unique nanostructures and physicochemical properties. For example, when CNTs are irradiated by visible lights, near-infrared (NIR) photoluminescence is observed. On the other hand, when CNTs are irradiated by NIR lights, significant adsorption of NIR is observed. There is another unique phenomenon in fluorescent spectroscopy. When some fluorescent dyes adsorbed on CNT surfaces, fluorescence from the dyes are almost quenched even when the dyes are irradiated by suitable lights for excitation. Using the physicochemical properties, various new methods for nanobiosensing such as detecting single-mismatch of DNA sequences have been reported by various authors.

We have reported nanoscopic characterization of hybrids of DNA and CNTs (DNA-CNT hybrids) in order to obtain fundamental information of interaction between DNA and CNTs. [1-6] Furthermore, we have investigated reactions of DNA binding proteins with DNA-CNT hybrids to establish biochemical reactions on CNT surfaces. In this paper, we systematically studied optical responses of DNA-CNT hybrids under various conditions. UV-vis, NIR, photoluminescence, and fluorescent spectroscopies were applied to the same samples for comparison. As a result, we found that DNA-CNT hybrids can detect changes of the spectra caused by minute perturbation such as injection of chemicals. Our results will be a basis of application of CNTs to nanobiosensing. In addition, causative proper use of the several optical measurements dependent on research purposes will be discussed.

[1] T Hayashida and K Umemura, Colloids Surfaces B, 143, 526-531 (2016).

[2] K Umemura. Nanomaterials, 2015, 5(1), 321-350(2015).

[3] S Oura, M Ito, D Nii, Y Homma, K Umemura. Colloids Surfaces B, 126, 496-501 (2015).

[4] D Nii, T Hayashida, Y Yamaguchi, S Ikawa, T Shibata, K Umemura, Colloids Surfaces B, 121, 325-330(2014).

[5] D Nii, T Hayashida, K Umemura. Colloids Surfaces B, 106, 234-239(2013).

[6] T Hayashida, K Umemura. Colloids Surfaces B, 101, 49-54(2013).
# Biomimetic compartmentalization approach in designing nanoreactor with organelle-like function

Thursday, 29th September - 16:25 - Biological & medical nanodevices and biosensors - Bionanocatalysis and nanobiosystems - Tower 24 - Room 105 - Oral presentation - Abstract ID: 274

Dr. Vimalkumar Balasubramanian¹, Ms. Alexandra Correia¹, Dr. Hongbo Zhang¹, Ms. Flavia Fontana¹, Mr. Ermei Mäkilä², Prof. Jarno Salonen², Prof. Jouni Hirvonen¹, Dr. Helder A. Santos¹

1. University of Helsinki, 2. University of Turku

## Introduction

In nature, biological cell is regarded as an intricate microenvironment, in which myriad of enzymes work together to catalyze concurrently multiple chemical reactions concurrently that are crucial for metabolism and cell functions. Inspired by nature, scientists manifest an increasing interest to mimic these compartmentalized enzyme regulatory mechanisms, not only to understand the biological process of cellular metabolism and functions, but most importantly to design customized biomimetic nanomachineries with a tremendous potential in a range of applications from biochemicals, pharmaceutics, diagnostics to, biomedicine, smart materials, and synthetic organelles. Therefore, mimicking the chemical transformations exhibited by enzymes in cellular compartments has triggered intensive research interest for creating new dynamic materials with tunable enzyme reactivity. In this work, we aim to develop a compartmentalized cellular nanoreactor consisting of porous silicon nanoparticles (PSi NPs) entrapped with an enzyme, horseradish peroxidase (HRP), and surface coated with a cancer cell membranes to demonstrate the design of biomimetic cellular nanoreactors and their impact on improving cellular functions for biomedical applications.

## Methods

PSi NPs have been used as host material to the entrapment of HRP enzymes within the pores. To construct a compartmentalized cellular nanoreactor, purified cancer cell membranes coated the surface of PSi NPs by an extrusion. The enzyme activity and kinetics of the compartmentalized cellular nanoreactors were evaluated using an enzymatic assay. To demonstrate the function of biomimetic cellular nanoreactors in improving cellular functions, different in-vitro cell experiments were performed.

## Results and Discussion

The results from the physicochemical characterization of nanoreactors indicate the successful loading of HRP in PSi NPs and the cancer cell membrane surface coating. The enzyme activity and kinetics analyses showed an enhanced substrate affinity and reaction rate compared to free HRP (Figure 1B), suggesting a high catalytic activity of nanoreactors. The in-vitro cell experiments demonstrated that nanoreactors can readily integrate with cells and can supplement the cellular functions under oxidative stress conditions. This cellular nanoreactor featuring a biocompartment enclosed by cellular membrane closely resembling nature's biocompartmentalization is an innovative biomimetic approach, which further helps the customized designing of biomimetic nanomachineries that can impact the repairing of cellular functions in pathological conditions.
<figure><img src="image_80.png" /><figcaption>**Figure 1.** A) Schematic representation of the biomimetic compartmentalized nanoreactor based on the cancer cell membrane coated PSi NPs loaded with HRP enzymes. B) Enzyme activity of nanoreactors analyzed using amplex-red fluorescence assay, compared with different controls. C) Transmission electron micrograph of the compartmentalized cellular nanoreactors.</figcaption></figure>

<caption>Figure 1.png</caption>
NBelyax® Nanoparticles for disinfection and sterilization of living and nonliving surfaces tested in vitro and in situ

Thursday, 29th September - 16:50 - Biological & medical nanodevices and biosensors - Bionanocatalysis and nanobiosystems - Tower 24 - Room 105 - Oral presentation - Abstract ID: 426

Dr. Leon Albarran ¹, Mrs. Gabriela León ¹, Mr. Sergio León ¹, Dr. Paola Arteaga ¹

1. Gresmex S.A. de C.V.

Gresmex is a Mexican company with 17 years of experience in manufacturing cleaning products and personal care.

In this project the active nanoparticle NBelyax® was developed. The invention of this material began in 2008 as part of the research enterprise environment to a material or substance capable of removing viruses.

In the first stage of innovation we worked on the elimination of rotavirus considering it as the most resistant virus to external factors, and that it could serve as a reference for the removal and inactivation of any other type of virus. The second stage was focused on the evaluation of effectiveness against a wider spectrum of microbiological organisms, including viruses, bacteria, fungi, spores, mycobacteria and trypanosomes. The mechanism of action is theoretically based on the nanoparticle attacking the genetic material of pathogenic microorganisms.

The Bioselectivity of the active ingredient NBelyax® gives us the advantage over other developments or products to eliminate all pathogens without causing any damage to the cells or the body of the user. Moreover, protect against infections supports better healing wounds existing prior to use.

The third stage was the inclusion in the portfolio of finished antiseptics, disinfectants and sterilants, in order to give a high disinfecting power and therefore a high added value to our products. That was the birth of the Eviter® products which include sanitizers, soaps, creams and cold sterilants designed for use in medical, clinical and surgical areas

NBelyax®, being broad spectrum disinfectant and high level is an important tool for prevention of diseases caused by active infections.

It is noteworthy that Eviter® can be used in any environment to create biosecure environments such as schools, museums, offices, transportation and avoid contagion large vectors that trigger pandemics.
# Cloning, expression and purification of Pseudomonas aeruginosa azurin, a small redox protein and its application in molecular electronics

Thursday, 29th September - 17:15 - Biological & medical nanodevices and biosensors - Bionanocatalysis and nanobiosystems - Tower 24 - Room 105 - Oral presentation - Abstract ID: 22

Ms. Neeti Kalyani¹, Prof. Prashant Mishra¹

1. IIT Delhi

## INTRODUCTION

Redox metalloproteins are getting considerable interest nowadays due to their applications in creating hybrid sub micrometre-sized electronic components for biosensors, transistors and memory devices. These bioshanodevices overcome the limitations of silicon-based devices like tunnelling effects, efficiency, and heat. These are operationally simple, cost efficient and suitable for real time detection. Azurin from Pseudomonas aeruginosa is an oxidoreductase, which possess an inherent, efficient electron transfer capability, occurring at single electron level in a very fast and directional way. Also, this has been proved to stabilize p53 tumor suppressor protein and hence, useful in cancer therapy. Azurin has a great potential to be used in the molecular electronics. But the purification of this protein is difficult and require multiple steps which result in low yield. We have cloned the protein and purify it in single step and got better yield of the protein. We have confirmed its electron transfer properties with cyclic voltammetry and conductive AFM.

## METHOD

Azurin gene was cloned and expressed in Escherichia coli and purified by Ni-NTA (Nickel-Nitrilo acetic acid) chromatography. Recombinant azurin was characterized using UV-Visible, FTIR, Circular Dichroism and Raman Spectroscopy. Azurin was immobilized directly on gold via thiol linkage and on silicon and mica using 3-mercaptopropyl trimethoxysilane as linker. Conductive atomic force microscopy was also done to study the morphology and conductivity of azurin layer on different substrates.

## RESULTS AND DISCUSSION

Azurin gene was successfully cloned and expressed in good amount. Pure band of azurin was obtained after single step of Ni-NTA purification. The excellent current density of azurin layer on gold was observed in cyclic voltammetry experiments. The sharp oxidation and reduction potentials peaks were as reported for the wild type azurin. In conductive atomic force microscopy the current observed was higher in the azurin immobilized sample than the blank one.

## CONCLUSION

We were successful in getting the pure active protein in very good quantity which can be used directly for electronic studies. Same protein can be used as the acceptor and donor of electrons. Its small size and efficient electron transfer capability make it a desirable molecule for the development of biosensors.
<figure><img src="image_81.png" /><figcaption>Azurin protein for biosensor.png</figcaption></figure>

<figure><img src="image_82.png" /></figure>
# A modified Hodgkin–Huxley model for nanoelectronics

Thursday, 29th September - 17:40 - Biological & medical nanodevices and biosensors - Bionanocatalysis and nanobiosystems - Tower 24 - Room 105 - Oral presentation - Abstract ID: 392

Prof. Peter Burke¹

1. University of California, Irvine

## A modified Hodgkin–Huxley model for nanoelectronics

In the Hodgkin–Huxley model[1], biological ion pumps push current through a membrane until it is the electrical capacitance of the membrane is charged up, at which point it generates a voltage which turns off the ion channel pumps. When a lipid bilayer is brought into close proximity to a nano-electronic device (such as a 1d carbon nanotube[2], quasi-1d silicon nanowire, 2d graphene sheet[3], or any other of the many classes of nanoelectronics devices), a similar phenomenon occurs: The current through the ion channel charges up the electrical capacitance of the lipid bilayer. However, because the nano-electronic devices have capacitance of their own (including both classical electrical capacitance as well as quantum capacitance[4] due to the Pauli exclusion principle), these also get charged by the ion channels, and ultimately turn off the ion channels, even for simple ion channels that are not voltage dependent. This modified Hodgkin–Huxley model can account for the electrical behavior of nanoelectronic interfaces to a electrically active ion channels, a growing interest with applications from neurons and cadiomyocytes to even smaller, electrophysiologically active organelles such as chloroplasts and even mitochondria[5].

[1]A. L. Hodgkin and A. F. Huxley, "A quantitative description of membrane current and its application to conduction and excitation in nerve," J. Physiol., vol. 117, no. 4, pp. 500–544, Aug. 1952.

[2]W. Zhou, Y. Y. Wang, T.-S. Lim, T. Pham, D. Jain, and P. J. Burke, "Detection of single ion channel activity with carbon nanotubes," Sci. Rep., vol. 5, p. 9208, 2015.

[3]Y. Y. Wang, T. D. Pham, K. Zand, J. Li, and P. J. Burke, "Charging the quantum capacitance of graphene with a single biological ion channel.," ACS Nano, vol. 8, no. 5, pp. 4228–4238, Apr. 2014.

[4]C. Rutherglen and P. Burke, "Nanoelectromagnetics: Circuit and Electromagnetic Properties of Carbon Nanotubes," Small, vol. 5, no. 8, pp. 884–906, 2009.

[5]K. Zand, T. Pham, A. Davila Jr., D. C. Wallace, and P. J. Burke, "Nanofluidic platform for single mitochondria analysis using fluorescence microscopy," Anal Chem, vol. 85, no. 12, pp. 6018–6025, 2013.
<figure><img src="image_83.png" /></figure>

Screenshot from 2016-07-15 13 28 01.png
# WORKSHOP: SEEC Microscopy, a live and label-free analysis technique in the fields of Materials and Life Sciences

Thursday, 29th September - 16:00 - Nano-Imaging for diagnosis, therapy and delivery - Tower 24 - Room 107 - Workshop presentation - Abstract ID: 574

Mr. Nicolas Medard ¹, Mr. Imed Ayadi ¹

1. Nanolane

SEEC Microscopy is a label-free analysis technique offering new characterization capabilities such as the live nanoscale imaging, the multiplex molecular interaction analysis or the real-time quantitative study. The technique implements unique optical sensitive sensors (SEEC sensors) with specific contrast-enhanced properties enabling the live visualization of samples down to nanoscale (0.1nm). In addition, a proprietary algorithm (Q-SEEC) enables quantitative analyses (surface interaction and topography analyses) with an accuracy of 0.3nm. SEEC Microscopy is dedicated to the study of samples in the fields of Materials and Life Sciences. Thanks to its high lateral sensitivity (down to 1 nm), the technique can be applied for the analysis of nanofilms, nanopatterns, nanotubes, nanoparticles or DNA molecules... Successful analyses were recently performed on polyelectrolytes, lipid layers, biofilms, biochips samples.

The N-Lab Station, brand-new SEEC equipment, is commercializedin two versions: the ‘MAT’ version (for analyses in dry) with a precise thermal control stage and the ‘BIO’ version (for analyses in dry and liquid) including complete thermal and fluidic systems (injection pump, fluidic switch, fluidic chamber, flowmeter...).

Both versions are providedwith the ‘LabSoft’ software for sequence planning, live data display and treatment.

More information: http://nlab.nano-lane.com/

SEND AN EMAIL TO nicolas.medard@eolane.com TO SECURE YOUR SEAT FOR THIS WORKSHOP.

<figure><img src="image_84.png" /><figcaption>SEEC analysis of enzymatic reactions
1. Topographic and 3D images (t₀ and t₀+120min) 2. Biochip thickness vs. reaction time</figcaption></figure>

Secc analysis of enzymatic reactions.png

<figure><img src="image_85.png" /></figure>

N-lab station.png
# Functionalization of Emissive Conjugated Polymer Nanoparticles by Coprecipitation: Consequences for Particle Photophysics and Colloidal Properties

Thursday, 29th September - 16:45 - Nano-Imaging for diagnosis, therapy and delivery - Tower 24 - Room 107 - Oral presentation - Abstract ID: 523

**Prof. Gareth Redmond** ¹

1. University College Dublin

Recent developments in materials synthesis, bio-conjugation methods and luminescence techniques have led to a rapid proliferation of novel fluorescence-based approaches to imaging in the life sciences. However, for high-resolution or long-time duration imaging applications, molecular dyes suffer from limitations of low brightness, poor photo-stability and fluorescence intermittency (blinking). Consequently, we are exploring a potentially promising alternative based on highly fluorescent conjugated polymer materials. Here, the functionalization of polyfluorene (PFO) nanoparticles by coprecipitation of the conjugated polymer with an amphiphilic comb polymer, consisting of a hydrophobic polystyrene backbone with hydrophilic, carboxylic acid-terminated polyethylene oxide side-chains (PS-PEG-COOH), is investigated. The comb polymer affects the properties of the formed hybrid nanoparticles. Non-functionalized particles are typically larger (28 nm) than functionalized ones (20 nm); this size difference impacts peak molar extinction coefficients accordingly. Zeta potentials are negative, consistent with negative surface charge on PFO particles due to chemical defect formation, with additional charge on functionalized particles due to the pendant carboxylic acid groups. Emission quantum yields of functionalized particles are typically larger, consistent with lower efficiency of energy transfer to quenchers in smaller particles and weaker PFO interchain interactions due to chain dilution. The trend in per-particle fluorescence brightness values, as confirmed by single particle fluorescence imaging, reflects the size-dependent nanoparticle absorption cross sections. Photostability studies on aqueous dispersions of hybrid particles indicate mild photobrightening under continuous illumination while PFO particles exhibit slow exponential emission decay. Functionalized particles also tend to be more resistant to aggregation during exposure to adenocarcinoma cells; see Figure (polyfluorene nanoparticles (blue) in MCF-7 cells (red / phalloidin-TRITC)). Generally, the hybrid particles exhibit more favourable time-, pH- and medium-dependent stabilities, likely due to steric and electrostatic stabilization by PEG- carboxylic acid functionalities. Overall, the functionalized particles exhibit attractive properties: Reasonably small size, tight size distribution, high absorption cross section, radiative rate and emission quantum yield, excellent brightness and photostability, and good colloidal stability.
<figure><img src="image_86.png" /><figcaption>Mcf-7sample7image3series z07c1 2.jpg</figcaption></figure>
# Innovative SPIONs for multimodal imaging: MRI/PET and MRI/optical imaging

Thursday, 29th September - 17:10 - Nano-Imaging for diagnosis, therapy and delivery - Tower 24 - Room 107 - Oral presentation - Abstract ID: 13

Dr. Julien Boudon¹, Dr. Guillaume Thomas¹, Dr. Lionel Maurizi¹, Prof. Nadine Millot¹

1. Université Bourgogne Franche-Comté

Amongst non-invasive methods, single photon emission computed tomography (SPECT), positron emission tomography (PET) and magnetic resonance imaging (MRI) are routinely used to detect pathological tissues. Each of these imaging modalities has unique advantages along with intrinsic limitations (for example MRI has an excellent spatial resolution but suffers from a lack of sensitivity). To circumvent these hurdles, ongoing efforts are made to develop multimodal methods. Similarly, bimodal medical imaging relying on MRI coupled to Optical Imaging (OI) may improve the accuracy of a diagnosis by combining the sensitivity of OI and the resolution of MRI.

In this field, nanoparticle (NP) technologies represent the most promising strategy to elaborate multimodal contrast agents. Superparamagnetic iron oxide nanoparticles (SPIONs) are currently being developed for several biological applications such as hyperthermia, drug delivery, or MRI [1-2]. For the latter application, SPIONs are usually used as water proton transverse relaxation time (T2) contrast agents.

The SPIONs presented in this talk for multimodal imaging (MRI/PET and MRI/optical imaging) are developed by taking advantage of (i) the originality of their simultaneous synthesis and colloidal stabilization in one step through a continuous hydrothermal synthesis device (synthesis time <10 s, conditions T, P up to the supercritical water field) [3-4] and (ii) the association of these nanoparticles with innovative chelating agents (NODAGA, MANOTA etc.) [5] or innovative fluorophores like phthalocyanine derivatives for near-IR detection and photodynamic therapy [6].

For all the nanohybrids developed, the grafting of the different ligands is confirmed by exhaustive characterizations. The imaging agents are stable under physiological conditions and show no cytotoxicity. Yields of radiolabeling indicate an efficient chelation and subsequent spectroscopic studies address the imaging capabilities of each probe in the well characterized SPIONs. In vitro, in vivo and biodistribution results of the final NPs will be presented.

[1] L. Maurizi et al., Langmuir, 2009, 8857

[2] L. Maurizi et al., J. Biomed. Nanotechnol. 2015, 126

[3] L. Maurizi et al., Chem. Commun., 2011, 11706

[4] G. Thomas et al., RSC Adv., 2015, 78614

[5] G. Thomas et al., Nanotechnology, 2016, 135604

[6] J. Boudon et al., Chem. Commun., 2013, 7394
<figure><img src="image_87.png" /><figcaption>Iconan2016 spions image.png</figcaption></figure>
# Elaboration of a new in vivo imaging system based on multimodal upconversion nanoparticles

Thursday, 29th September - 17:35 - Nano-Imaging for diagnosis, therapy and delivery - Tower 24 - Room 107 - Oral presentation - Abstract ID: 410

Mr. Julien Santelli¹, Dr. Lechevallier Séverine², Dr. Robert Mauricot³, Prof. Daniel Cussac⁴, Prof. Marc Verelst⁵

1. CEMES-CNRS, Université de Toulouse, CNRS, France | I2MC, Université de Toulouse, INSERM, Hôpitaux de Toulouse, France,

2. CHROMALYS SAS, 29 rue Jeanne Marvig BP 94347 31055 TOULOUSE cedex 4, France, 3. CEMES-CNRS, Université de

Toulouse, CNRS, France, 4. I2MC, Université de Toulouse, INSERM, Hôpitaux de Toulouse, France, 5. CEMES-CNRS, Université

de Toulouse, CNRS, France | CHROMALYS SAS, 29 rue Jeanne Marvig BP 94347 31055 TOULOUSE cedex 4, France

## Background

In vivo imaging can be achieved with various techniques (MRI, CT-scan, PET-scan, scintigraphy, fluorescence...) depending on the nature of the target tissue, cell or tracer. Nevertheless, there is actually no reference method that allows for optimal results: each technique has advantages and limitations in depth, resolution, sensitivity, tissue damage or cost.

## Methods

We have developed multimodal nanoparticles for biological imaging: a Gadolinium oxysulfide core allows MRI and CT-scan imaging whereas the addition of other lanthanides as dopants enables the acquisition of fluorescence properties. The combination of the different techniques gives maximal information with only one tracer. This brings much more flexibility and robustness in the results comparatively to other tracers currently available on the market. We have found that Ytterbium/Thulium codoped nanophosphors are particularly suited for in vivo imaging as they emit strong luminescence after upconversion of near infrared photons. Briefly, this enables the imaging of deeper targets whilst minimizing tissue lesions compared to classical fluorescence obtained after visible laser excitation. Indeed, it benefits from tissue transparency and lower excitation energy.

## Findings

Here we report the development of a complete in vivo fluorescence tracking system: Ytterbium/Thulium codoped Gadolinium oxysulfide as tracer and dedicated macroscopic imaging device. Our latest results are very promising: we are able to follow the kidney-injected tracer on the surface of the skin after two months. This let us foresee real applications to achieve our final goal: over-time detection of nanoparticle-labeled mesenchymal/stromal cells after grafting in a solid organ. In the end, it will signify the availability of an innovative and robust tool for cell therapy proof of concept.
# The Curious Case of 1D and 2D Carbon Nanostructure Pharmacology & Toxicology

Friday, 30th September - 09:00 - Plenary Speeches - Amphitheatre 25 - Oral presentation - Abstract ID: 573

Prof. Kostas Kostarelos¹

1. University of Manchester

Carbon nanomaterials may have captured the excitement and interest of myriads of scientists and two Nobel prizes, but have only just entered a phase of maturity in their development and utilization for medical purposes. Graphene-based materials that constitute some of the newest nanocarbon types today, have mainly been explored as components of biosensors and for construction of matrices in tissue engineering. The capacity of graphene to act as a platform for various therapeutic and diagnostic agents has also been reported, however not as coherently. Among the recent advances made with graphene-related materials, their use as components of innovative delivery systems is full of promise, however there are challenges facing these exciting new tools both in terms of biological activity and toxicological profiling. This presentation will attempt to offer a comparative perspective on the pharmacological and toxicological profiles revealed for 1D (carbon nanotubes, CNT) and 2D (graphene oxide sheets, GO) carbon nanostructures. This aims to be an illustration of the importance rational and systematic research work plays to unravel the unparalleled properties of carbon nanomaterials and allow their utilization as clinical tools.
Nucleic acid chemistry for nanomedicine

Friday, 30th September - 09:40 - Plenary Speeches - Amphitheatre 25 - Oral presentation - Abstract ID: 526

Prof. philippe Barthelemy ¹

1. INSERM U1212

The combination of nucleic acids chemistry (e.g., nucleoside, nucleotides, oligonucleotides) with supramolec-
ular principles provides an efficient and powerful approach to prepare well-defined systems with tunable
physico-chemical properties and functions. We develop new nano-systems based on nucleic acids chemistry
for i) drug delivery applications (therapeutic, theranostic), and ii) tissue engineering. This communication will
present novel “smart” nucleic acid derivatives (nucleolipids, lipid-oligonucleotide conjugates) developed in our
lab [1].

References

[1]

- Latxague, L., Ramin, M.A., Appavoo, A., Berto, P., Maisani, M., Ehret, C., Chassande, O., and Barthélémy, P. (2015) Angewandte Chemie, DOI: 10.1002/ange.201409134.

- Oumzil, K. Benizri, S. Tonelli, G. Staedel, C. Appavoo, A. Chaffanet, M. Navailles L. and Barthélémy P. (2015) ChemMedChem DOI: 10.1002/cmdc.201500381

- Luvino, D. ; Khiati, S. ; Oumzil, K. ; Rocchi, P. ; Camplo, M. ; and Barthélémy P. (2013) J. Control. Release, 172, 954-961

- Khiati, S., Luvino, D., Oumzil, K., Chauffert, B., Camplo, M. and Barthélémy P. (2011) ACS Nano, 5 (11), pp 8649-8655.

- Patwa, A., Gissot, A., Bestel, I. and Barthélémy, P. (2011) Chem. Soc. Reviews, 40, 5844–5854.

- Godeau, G., Bernard, J., Staedel, C., Barthélémy, P. (2009) Chem. Commun., 5127-5129.

- Moreau, L., Camplo, M., Wathier, M., Taib, N., Laguerre, M., Bestel, I., Grinstaff, M.W., Barthélémy, P. (2008) J. Am. Chem. Soc. 130, 14454-14455.
<figure><img src="image_88.png" /><figcaption>Diapositive1.jpg</figcaption></figure>
Nanotechnologies for targeted delivery of nucleic acid

Friday, 30th September - 10:45 - Plenary Speeches - Amphitheatre 25 - Oral presentation - Abstract ID: 517

Prof. Elias Fattal¹

1. Université Paris-Sud

Hyaluronic acid (HA) is a glycosaminoglycan, the main constituent of the extracellular matrix and the natural ligand of CD44 receptor. The association of HA with nanotechnology allow to target the cancer stem cells through the CD44 receptor overexpressed on the surface of these cells. Lipoplexes containing a HA-dioleoyl phosphatidylethanolamine conjugate (HA-DOPE) were designed for this purpose. They were prepared from cationic liposomes and used to complex small interfering RNA (siRNAi). Targeting the CD44 receptor on lung cancer cells was shown to improve the inhibition effect of siRNA using the luciferase gene as a target. The internalization mechanism of the lipoplexes was shown to be mediated by both the CD44 receptor and caveolae. This approach has been applied successfully to deliver the same siRNA anti-luciferase in a mouse model of lung metastases demonstrating a higher inhibition than the non-targeted formulation. We have also associated an aptamer antiCD44 to the surface of liposomes. These vectors have also shown great potential for siRNA delivery in vitro and in vivo to cells overexpressing CD44 avoiding the toxicity problems related to lipoplexes. These data validates the relevance of CD44 as a target for the delivery of macromolecular drugs by nanotechnology. Finally, we have designed inhaled formulation for siRNA delivery in the treatment of lung injury disease, to reduce inflammation. Dendriplexes delivery of an antiTNF siRNA have shown, after intratracheal administration, a potent inhibition of this cytokine demonstrating the potential of nanotechnologies to also deliver locally the siRNA.
# Nanostructured Biomaterials for Medical and Biological Applications

Friday, 30th September - 11:25 - Plenary Speeches - Amphitheatre 25 - Oral presentation - Abstract ID: 558

Jackie Ying¹

1. Institute of Bioengineering and Nanotechnology

Nanostructured materials have been developed for various medical and biological applications. They have been designed as stimuli-responsive drug delivery systems and sustained protein delivery systems. Nanocomposite systems have also been derived to provide simultaneous drug delivery and bioimaging functions as theranostic systems. Micellar nanocomplexes have been synthesized with green tea-based ingredients as unique carrier materials that offer synergistic therapeutic effects with the drugs to be delivered.

In addition, nanostructure processing has been employed in creating synthetic cell culture substrates for the expansion and controlled differentiation of stem cells. Nanostructured scaffolds have also been obtained for cell and tissue engineering.
# Investigating the Eremostachys laciniata (EL) and Curcumin Longa (CL) encapsulated by Solid Lipid Nanoparticles (SLN) in order to treat the inflammatory diseases

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 76

Mr. akbar vaseghi¹, Ms. Haleh Seyedabasi², Ms. Mina Ghanbari³, Dr. Alireza Panahi⁴, Ms. Elaheh Alizadeh⁵, Mr. Bager Karimi⁶, Mr. Reza Ashrafi Parchin⁷

1. Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran, 2. Department of Agriculture, Khoy Payamnor University, Western Azerbaijan, Iran, 3. Department of Applied Chemistry, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, 4. Department of Biology, Faculty of Basic Sciences, University of Mohaghegh Ardabili, Ardabil, Iran, 5. Department of Biotechnology, Payame Noor University, Tehran, 6. Department of Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran, 7. Department of Biotechnology and Plants Breeding, Gorgan University of Agricultural Sciences and Natural Resources, Iran

Solid Lipid Nanoparticles (SLN) have emerged as a next-generation drug delivery system with potential applications in pharmaceutical field, cosmetics, research, clinical medicine and other allied sciences. Since a decade, trials are being made to utilize SLN as alternative drug delivery system to colloidal drug delivery systems such as lipid emulsions, liposomes and polymeric nanoparticles. Eremostachys laciniata(EL), an Iranian traditional medicinal herb, and Curcumin Longa (CL) are a thick root which are used in local traditional medicine in order to treat the inflammatory diseases. In this project, we are studying nano-encapsulation by using high-pressure homogenizer method with EL and CL effective material mixtures, isolated from rhizomes, and SLN (we call NANO-ChiCo). We compared the NANO-ChiCo with piroxicam ointment in order to treat the inflammatory diseases, e.g., osteoarthritis, rheumatoid arthritis, and reiter's syndrome. After one month of treatment with the SLN-ChiCo and piroxicam ointment, our recent results for all groups showed significant improvement compared to the control groups.
# Effects of paclitaxel delivery by carbon nanotubes on prostate cancer cells and monocytes

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 85

Mr. Edson Comparetti¹, Dr. Valber Pedrosa², Dr. Ramon Kaneno¹

1. Institute of Biosciences of Botucatu, UNESP - Univ Estadual Paulista, Department of Microbiology and Immunology, 2.

Institute of Biosciences of Botucatu, UNESP - Univ Estadual Paulista, Department of Chemistry and Biochemistry

Carbon nanotubes (NT) have properties that make them useful in the biomedical field, for instance for controlled drug delivery. Indeed, NT can be loaded with antitumor drugs, such as paclitaxel (PTX), in order to guide drug release within the tumor cells. Considering the high-energy metabolism of tumor cells, we revested PTX-loaded NT with glucose molecules (GLY) in order to test their toxicity on both human prostate cancer cells and monocytes of health donors. First, we increased the polydispersity of NT in aqueous solution synthesizing them with polyethyleneimine (PEI) and polyethyleneimine combined with glucose (PEI-GLY). Analysis by Fourier transform infrared spectroscopy (FTIR - Nicoletspectrometer Nexus 670) confirmed the incorporation of polymer on NT walls. Transmission electron microscopy was used to study NT structure and morphological changes induced by incorporated material. The amount of PTX incorporated by NT was estimated by UV/visible spectroscopy. Cytotoxic activity on prostate cancer cells (LNCaP) was confirmed by flow cytometry after 48h incubation with nanoparticles. Analysis of cell viability was based on Annexin V- (apoptosis) and 7AAD- (cell death) labeling and analysis by flow cytometry in five experimental conditions: a) NT; b) NT-PTX; c) NT-GLY; d) NT-GLY-PTX and e) equivalent concentration of pure PTX. NT-PTX showed as high cytotoxic activity as pure PTX, that was significantly higher than NT alone or NT-GLY. The most interesting finding was that cytotoxic effects of NT-GLY-PTX had slightly higher than pure PTX and NT-PTX, indicating that glucose favors the interaction of particles with tumor cells (Fig.1). We also analyzed the effect of NT on the viability and phenotype of monocytes of health donors exposed to NT, NT-PTX, NT-GLY and NT-GLY-PX. Our results show that expression of CD80, CD86, HLA-DR and CD83 was not hindered by exposition to nanotubes (any of preparations), while all formulations increased the expression of PD-L1 molecules on monocytes. These results indicate that glucose enhance the drug-carrier property of NT, and has low toxic effect on normal monocytes.
<figure><img src="image_89.png" /><figcaption>Fig. 1 - Effects of modified carbon nanotubes on morphology and viability of tumor cells. Dot plot depicts the effect of NT-PTX, NT-GLY-PTX and pure PTX (C, E and F) on the size and granulocyte of prostate cancer cell line LNCaP. Pseudocolor graphs represent early apoptosis (Annexin V+, Q9), late apoptosis (Annexin V+/AAD⁻ cells, Q10) and dead cells (AAD⁻ cells, Q11).</figcaption></figure>

Cell death.jpg
Nanoscale Engineering of Hybrid Magnetite-Carbon Nanofibre Materials for MRI Contrast Agents

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 111

Ms. Olga Metelkina¹, Dr. Graham Rance², Dr. Galina Pavlovskaya², Prof. Alexander Savchenko¹,
Prof. Andrei Khlobystov², Prof. Alexander Majouga¹, Dr. Anastasia Garanina¹

1. *National University of Science and Technology, MISiS, 2. The University of Nottingham*

Magnetic nanomaterials show significant promise as contrast agent materials for magnetic resonance imaging (MRI). We have developed a new highly efficient one-step synthesis of magnetically-functionalised hollow carbon nanofibres, where the size of magnetite nanoparticles formed on carbon nanofibres is controlled by the mass ratio of the magnetite precursor (iron acetylacetonate) to nanofibres which act as templates and supports for the nucleation and growth of the magnetic nanoparticles. The physicochemical and magnetic properties of the new hybrid nanomaterials are evaluated and optimised with the aim to ultimately produce an efficient “smart” MRI theranostic agent – a material that allows the combined diagnosis (with MRI), treatment (with magnetic targeting) and follow-up of a disease (with MRI) – currently in high demand for various clinical applications. We have shown that our magnetite-nanofibre materials are effectively solubilised in water resulting in a stable suspension that has been employed as a “negative” contrast agent with an excellent transverse relaxivity (R2) of (268 ± 13) mM/s. Moreover, their attractive magnetic properties make them especially suited for magnetic tracking and thus of particular interest for personalised nanomedicine.

The authors are grateful to Ministry of Education and Science of the Russian Federation. Grant number RFMEFI60715X0132.
<figure><img src="image_90.png" /><figcaption>Longitudinal r1 and transverse r2 relaxivity.png</figcaption></figure>
Cytotoxicity of ICD-85 NPs on Human Cervical Carcinoma HeLa Cells through Caspase-8 Mediated Pathway

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 148

Prof. Abbas Zare Mirakabadi¹

1. Razi Vaccine and Serum Research Institute

The biological application of nanoparticles (NPs) is a rapidly developing area of nanotechnology that raises new possibilities in the treatment of human cancers. The cytotoxicity of ICD-85 was evaluated by MTT and LDH assays. The apoptotic effect of free ICD-85 and ICD-85 NPs on HeLa cells was assessed using caspase-8 colorimetric assay. The MTT assay showed that ICD-85 NPs could enhance the in-vitro cytotoxicity against HeLa cells compared to the free ICD-85. The IC50 value at 72 h was reduced from 25 ± 2.9 µg/mL for free ICD-85 to 15.5 ± 2.4 µg/mL for ICD-85 NPs. However, LDH assay demonstrated that ICD-85 has dose-dependent cytotoxicity on HeLa cells while ICD-85 NPs exhibited weaker cytotoxicity on same cells. The results also indicate that ICD-85-induced apoptosis on HeLa cells is associated with the activation of caspase-8. Moreover, caspase-8 assay analysis demonstrated that the ICD-85 NPs induced a higher apoptotic rate in HeLa cells compared to free ICD-85. Our results demonstrated that the encapsulation of ICD-85 enhances its anti-proliferative effects. Taken together, these results suggest that the delivery of ICD-85 in nanoparticles may be a promising approach for low toxicity and high affectivity in the treatment of cancer
# Will a Carbon Nanosheet serve as a replacement to membrane components in a Nanodisc?

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 206

Dr. Suresh Vepuri ¹, Prof. Mahmoud Soliman ¹, Prof. Thirumala Govender ¹

1. Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Westville, Durban, South Africa

Nanodisc is a nano size architecture of an intrinsic biological membrane protein. This in vitro nano biological science process is rapidly evolved into a technology that produce solubilized membrane proteins in their native environment for biomedical and drug discovery applications. The organization of nanodiscs involve a protein enclosed phospholipid bilayer system held together by membrane scaffold proteins (MSPs). MSPs are condensed forms of apolipoprotein (apo) A-I which wrap around a patch of a lipid bilayer to form a disc-like particle or nanodisc. MSPs basically act as a ribbon/sheet to tie the lipid assembly, and simultaneously provide aqueous solubility for the system. Therefore, several engineered MSPs are designed and evaluated by reconstitution. Typically, the MSPs are produced by using recombinant technology and protein purification. To simplify the process, we are interested in designing simple organic structures as substitute for the total membrane components that include both MSPs and lipid layer. As an initiative we attempted a molecular modelling study to assess the propensity of a carbon nanosheet as supporting membrane structure for the protein to form a nanodisc. In this process, we identified a bacterial outer membrane protein porin as example structure to model the carbon nanosheet variant of its nanodisc (Fig 1.). A molecular dynamics study was performed to compare the binding interactions and conformational stability between the native nanodisc model of porin and the nanosheet variant. The nanosheet protein variant was found to be stable and offered a strong hydrophobic interaction network for the protein assembly. Our preliminary study results are interesting and strongly support our idea of nanosheet frame work as substitute for biological membrane system in current nanodisc technology. We anticipate that the carbon nanosheet with its native hydrophobic design and proven flexibility in surface chemical modification, shall be a good alternate for MSPs and could bring new horizons in nanodisc technology.

Acknowledgements: NRF-KIC Travel grant, UKZN-College of Health Sciences, UKZN-Nanotechnology Platform and CHPC Capetwon, South Africa.

<figure><img src="image_91.png" /><figcaption>Graphene variant of nanodisc.jpg</figcaption></figure>
# Accumulation of Doxorubicin Conjugates with Dendritic Polymers and Vector Protein in Normal and Tumor Cells in vitro

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 223

Prof. Irina Zamulaeva¹, Ms. Olga Matchuk¹, Dr. Nikita Yabarov¹, Ms. Elena Nikolskaja¹

1. National Medical Research Radiological Centre of the Ministry of Health of the Russian Federation

## Introduction

Anticancer agents available today on the market cannot guarantee satisfactory results. There are a number of factors that reduce the efficacy of the drugs. On the one hand, one of the ways to overcome these problems is using increased doses of a drug or application of high toxic agents, which often leads to serious side effects and raises the risk of multidrug resistance occurrence. But, on the other hand, one of the most effective ways to enhance the effectiveness of anticancer drugs is development of new methods for target delivery of chemotherapeutic agents into tumor cells. The aim of this study was a comparative analysis of accumulation of dendritic polymers conjugated with anticancer drug and vector protein in breast cancer and normal cells in vitro. In addition, removal of these drugs from cancer cells was also studied.

## Methods

Polyamidoamine dendrimers of the second generation (G2) were used. Accumulation of doxorubicin (Dox), its conjugates with G2-dendrimers (G2-Dox) and recombinant third domain of alpha-fetoprotein as vector protein (3D-G2-Dox) in normal and cancer cells was studied by flow cytometry and laser confocal microscopy. The study objects were cells of peripheral blood mononuclear fraction of healthy donors and cells of breast adenocarcinoma lines MCF-7 and MCF-7/MDR1 differing in chemosensitivity.

## Results

G2-Dox and 3D-G2-Dox accumulated in cancer cells of the both lines better than free Dox (p<0.05). However, removal of these drugs out of MCF-7 and MCF-7/MDR1 cells was significantly different: in the latter case free Dox was completely excluded from the cells in 24 hours, while Dox accumulating in composition with dendrimers still remained in the cells. It is important that 3D-G2-Dox (unlike G2-Dox) accumulated in normal cells worse than free Dox (p<0.01).

## Discussion

The results indicate that using of 3D-G2-Dox is the most promising course because it accumulates in cancer cells better but in normal cells worse than free Dox. Furthermore, it can be assumed that applying of 3D-G2-Dox would be especially useful in cases of multi-drug resistance associated with the high expression of P-glycoprotein.

This work was supported by grant of Russian Scientific Foundation # 15-15-10013.
# Cytotoxic Effects of Ionizing Radiation and Doxorubicin Conjugates with Dendritic Polymer and Vector Protein on Breast Cancer Cells in vitro

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 228

Ms. Olga Matchuk¹, Ms. Kristina Churyukina¹, Dr. Nikita Yabarov¹, Ms. Elena Nikolskaja¹, Prof. Irina Zamulaeva¹

1. *National Medical Research Radiological Centre of the Ministry of Health of the Russian Federation*

## Introduction

Dendrimers are perspective nanocarriers for anticancer drugs, but unfortunately their conjugates and complexes with drugs lack a targeted action that limits their application. Conjugation of dendrimers with ligands for specific receptors on the tumor cells surface seems promising approach for increasing therapeutic efficiency and decreasing systemic toxicity of anticancer agents. The aim of the study was to evaluate cytotoxic effects of dendrimers conjugated with doxorubicin (Dox) and alpha-fetoprotein recombinant third domain (3D) as vector protein on cancer cells after single and combined application in vitro. Combined effects of ionizing radiation and dendrimers are of great interest because radiation therapy is well known to be used in the treatment of cancer patients in about half of all cases.

## Methods

Polyamideamine dendrimers of the second generation (G2) were used after conjugation with 3D and Dox. The cytotoxic effects and intracellular distribution of Dox were studied by MTT-test, light and laser confocal microscopy 24 hours after ionizing radiation exposure of breast adenocarcinoma cells (MCF-7 and MCF-7/MDR1 lines) following by incubation with free Dox, G2-dendrimers loaded with Dox (G2-Dox), or conjugates of G2-dendrimers with 3D and Dox (3D-G2-Dox) during 2 hours.

## Results

G2-Dox and 3D-G2-Dox significantly decreased number of MCF-7 cells by 10% and number of MCF-7/MDR1 cells by 25-30% as compared to control (p<0,05). Thus, free Dox exerted more pronounced cytotoxic effect on MCF-7 cells and the same effect on chemoresistant MCF-7/MDR1 cells as compared to the conjugates. For MCF-7 line using both conjugates in combination to irradiation was not effective because the conjugates did not increase the cytotoxic effects of radiation exposure. For MCF-7/MDR1 line subadditive effects were shown after the combined treatment with each drug and radiation. The highest synergy factor (0.91) was found for 3D-G2-Dox.

## Discussion

In terms of overall cytotoxic effects on stable tumor cell lines, using the studied conjugates is justified only in combination with irradiation and only in the case of high expression of P-glycoprotein, which causes the multidrug resistance of cancer cell line MCF-7/MDR1.

This work was supported by grant of Russian Scientific Foundation # 15-15-10013.
# Mutagenicity testing in non-transformed and transformed human breast cell lines after exposure to silver nanoparticles in combination with aluminum chloride, butylparaben, or di-n-butylphthalate

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 292

Dr. Maciej Stępnik¹, Ms. Katarzyna Domeradzka-Gajda¹, Dr. Joanna Roszak¹, Dr. Anna Kozajda¹, Ms. Anna Smok-Pieniązek¹, Prof. Jarosław Grobelny², Dr. Emilia Tomaszewska², Prof. Grzegorz Celichowski², Dr. Małgorzata Cieślak³, Dr. Dorota Puchowicz³

1. Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, Łódź, 2. Department of Materials Technology and Chemistry, University of Łódź, 3. Scientific Department of Unconventional Technologies and Textiles, Textile Research Institute, Łódź

## Introduction

Application of silver nanoparticles (AgNP) in cosmetic products is growing rapidly, however very little is known about their biological interactions with other cosmetic ingredients. Considering in vitro and in vivo studies demonstrating accumulation of AgNP in tissues and DNA damage in mammalian cells following exposure to AgNP, the safety issue of using different cosmetic formulations containing AgNP becomes an urgent issue. The aim of this study was to assess potential mutagenic effects of AgNP in combination with other ingredients/impurities of cosmetic products such as: aluminum chloride (AlCl3), butylparaben (BPB) and dibutylphthalate (DBPH).

## Methods

AgNP were characterized using the UV-Vis spectroscopy, dynamic light scattering (DLS) and scanning transmission electron microscopy (STEM). The mean size of metallic core of two citrate stabilized AgNP was 15 ± 3 nm and 45 ± 10 nm. The hydrodynamic size of the particles was 19 ± 4 nm and 58 ± 10 nm. Mutagenicity of the AgNP in combination with AlCl3, BPB, or DBPH in three human cell lines, i.e. MCF-7, MDA-MB-231 (both cell types representing breast adenocarcinoma, estrogen receptor(ER)-positive and ER-negative, respectively) and MCF-10A (non-transformed cells), was measured using the in vitro micronucleus test. For comparison, an effect of silver nitrate was also assessed.

## Results

Considerable differences in cytotoxicity of both AgNP on normal and cancer cell lines were seen. For example, the highest acceptable concentration (% cytostasis <55%) of AgNP15 used for a 24 h exposure of MCF-10A cells was 47.1 µg/ml, while for MCF-7 and MDA-MB-321 it was 1 µg/ml or 1.2 µg/ml, respectively. The nanoparticles did not show any mutagenic effect neither in combination with the AlCl3 (≤500 µM), BPB (≤200 µM) nor DBPH (≤35 µM). Silver nitrate did not show any mutagenicity at up to acceptable concentrations of 1.5 µg/ml (MCF-10A) or 1 µg/ml (MCF-7 and MDA-MB-321).

## Discussion

The results indicate that the AgNP do not show mutagenic activity in the breast cells lines and that selected
cosmetic additives/impurities do not modulate the mutagenic/cytotoxic potential of AgNP.

Funded by the Polish National Science Centre (grant 2012/07/B/NZ7/04197). In this study the apparatus purchased in POIG.01.03.01-00-004/08 project were used.
# Electrospun PCL nanofibers loaded with carvacrol for wound dressing applications

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 294

Mr. Enrique Gamez Herrera ¹, Dr. Silvia Irusta ¹, Dr. Manuel Arruebo ¹

1. University of Zaragoza

## Introduction

In chronic wounds, an appropriate protection is necessary for a correct healing and actual passive wound dressings are not suitable in these cases. For acute or chronic wounds properties that reduce healing times and associated pain at the same time that maintain humidity, pH and optimum temperature are required. An important property of dressing materials is the formation of an effective barrier against the entry of pathogens.

Essential oils (of thyme, cinnamon, sage, etc.) have antimicrobial properties and some also have demonstrated anti-inflammatory, analgesic and healing properties. Besides they present lower cytotoxicity against eukaryotic cells, and a reduced tendency to promote bacterial resistance than synthetic antimicrobials (e.g. antibiotics).

Electrospinning is a fast and easy method to obtain nanofibers with high surface area, high surface to volume ratio and good mechanical properties. Biocompatible synthetic and natural polymers as poly-caprolactone (PCL) and chitosan can be used. Furthermore, by this process nanofibers can be loaded with the mentioned natural compounds.

In this work a fast and easy method to produce PCL nanowebs loaded with carvacrol, a natural active compound that have antimicrobial properties, is reported. This compound was selected because minimal bactericidal concentration (MBC) values are below 0.2 mg/mL and 0.4 mg/mL against S. Aureus and E. Coli respectively.

## Methods

For the electrospinning process a 10% w/w PCL solution in DCM:DMF 1:1 has been prepared. Then, the appropriate amount of carvacrol has been added to the solution to obtain a 20% w/w in the fibers. Feeding flowrate was 1 mL/h, nozzle-to-plate-distance was 18 cm and voltage used was the necessary to stabilize the Taylors's cone. Drug loading achieved has been determined by GC-MS and the obtained fibers have been characterized by SEM and FTIR.

## Results and conclusions

The average diameters of the obtained PCL nanofibers were: 240±40 nm. FTIR spectra show bands related to the natural compounds beside those of PCL corroborating the formation of the composite. Essential oil loading measured by GC-MS was 11.15±0.97% w/w.
<figure><img src="image_92.png" /><figcaption>Pcl10car20.jpg</figcaption></figure>
# Evaluation of Epigenetic Changes in Repetitive Sequences of Human DNA Induced by Nanoparticles: A Pilot Study for Nanoparticle-Epigenomics Interaction

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 305

Dr. Amornpun Sereemaspun ¹, Ms. Siwaporn Nilyai ²

1. Department of Anatomy, Faculty of Medicine, Chulalongkorn University, 2. Nanobiomedicine Laboratory, Faculty of Medicine, Chulalongkorn University

BackgroundEngineered nanoparticles (ENPs) are one of the most nanomaterials that wildly used in various fields including biomedical applications. However, the adverse effects of ENPs on health risk still need to be concerned. ENPs have been reported to be matter that cause cellular damage through either direct or indirect, cellular oxidative stress is one of the most nanotoxicity have been found. Reactive oxygen species (ROS) is cause of cellular oxidative stress that leads to intracellular macro molecules damages and may impact on DNA methylation changes. In this study we aim to investigate the effect of ROS induced by ENPs on DNA methylation that is one important of epigenetic mechanisms. Human embryonic kidney (Hek 293) and human keratinocyte (HaCaT) cells were used as model to expose with three different types of ENPs, AuNPs, SiNPs and CSNPs.

MethodsWe evaluated cytotoxicity of cells by measuring viability, morphology and ROS levels. Global DNA methylation levels were measured by 5-methylcytosine immunocytochemistry staining, and we also investigated DNA methylation levels of retrotransposable elements, LINE-1 and Alu by using combined bisulfite restriction analysis technique (COBRA).

ResultsWe found ROS level was increased in SiNPs exposed HaCaT cells only. DNA hypomethylation of global and Alu elements was showed in cells were exposed with SiNPs and CSNPs in HaCaT cells only. LINE-1 did not change in both of Hek 293 and HaCaT cells. Furthermore, the inversion of Alu DNA methylation level in HaCaT cells exposed with SiNPs and CSNPs was found in pretreated with antioxidant.

ConclusionOur study demonstrated the new insight that DNA methylation of Alu elements represents the global DNA methylation of cell exposed with ENPs. In this study, the alteration of DNA methylation level in ENPs exposed cells is ROS independent and specific to cell types.
<figure><img src="image_93.png" /><figcaption>Amornpun abstract.png</figcaption></figure>