# Niosome nanoparticles loaded with essential oils for wound dressing applications

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 328

<u>Mrs. Sara García Salinas</u>¹, <u>Mrs. Hellen Elizondo</u>¹, <u>Dr. Víctor Sebastián</u>¹, <u>Dr. Manuel Arruebo</u>¹, <u>Dr. Silvia Irusta</u>¹, <u>Dr. Gracia Mendoza</u>¹

1. University of Zaragoza

**Introduction:** In nanomedicine the improvement of nanoparticulated systems used in the treatment of different pathologies through local, targeted or controlled release has played a crucial role. Niosomes are one of these nanoparticulated systems, vesicles made by self-assembly of non-ionic surfactants, cholesterol and stabilizers such as dicetyl phosphate (DCP). On the other hand, microorganism resistance is becoming one of the main challenges in medicine research. That is why nowadays plants that were believed to have medicinal properties are studied by its bactericidal, anti-inflammatory and regenerative ability in wound healing, among others. Combination of those materials could be used in topical wound care in order to obtain more targeted and efficient approaches.

**Methods:** The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of essential oils (EO) reported as bactericidal in Escherichia coli and Staphylococcus aureus were evaluated before being encapsulated in niosomes. EO tested were Carvacrol, Eugenol, Cinnamaldehyde, Tymol, Squalene, Tyrosol and Rosmarinic acid. Inhibitory and bactericidal effects were analyzed by the dilution method using agar broth cultures by contacting different concentrations of EO with bacteria for 24 hours.

**Results:** The antibacterial effects of EO tested are shown in Table I. It can be seen that Carvacrol, Cinnamaldehyde and Tymol showed higher antibacterial efficiency in both E. coli and S. aureus, being MIC and MBC between 0.1 and 0.5 mg/mL. On the other hand, Eugenol displayed higher bactericidal effects in E. coli than in S. aureus. In this project, we have also carried out the niosome synthesis through a methodology based in microfluidics, using an interdigitated micromixer. Vesicle sizes are 238 ± 41 nm and their Z potential is -33 ± 3mV.

**Discussion:** In spite of all the efforts that have been made towards the development of artificial wound dressings, none of the currently available options combine all the requirements necessary for quick and optimal cutaneous regeneration. Further studies will be carried out to test the regenerative and anti-inflammatory ability of these EO and to encapsulate them into niosomes, so that the combination may make optimum smart bioactive dressings with a controlled release of the natural substrates.
<table><caption>Table i.png</caption><thead><tr><th rowspan="2"></th><th colspan="2">Escherichia coli</th><th colspan="2">Staphylococcus aureus</th></tr><tr><th>MIC (mg/mL)</th><th>MBC (mg/mL)</th><th>MIC (mg/mL)</th><th>MBC (mg/mL)</th></tr></thead><tbody><tr><td>Carvacrol</td><td>0.2</td><td>0.4</td><td>0.1</td><td>0.2</td></tr><tr><td>Eugenol</td><td>0.4</td><td>0.5</td><td>1.2</td><td>1.3</td></tr><tr><td>Cinnamaldehyde</td><td>0.2</td><td>0.3</td><td>0.4</td><td>0.5</td></tr><tr><td>Tymol</td><td>0.1</td><td>0.3</td><td>0.1</td><td>0.2</td></tr><tr><td>Squalene</td><td>&gt; 4.0</td><td>&gt; 4.0</td><td>&gt; 4.0</td><td>&gt; 4.0</td></tr><tr><td>Tyrosol</td><td>&gt; 4.0</td><td>&gt; 4.0</td><td>&gt; 2.0</td><td>&gt; 2.0</td></tr><tr><td>Rosmarinic acid</td><td>&gt; 4.0</td><td>&gt; 4.0</td><td>&gt; 4.0</td><td>&gt; 4.0</td></tr></tbody></table>
# Simple and Efficient Approach for siRNA Encapsulation into PCLs by Freeze-thawing Method

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 337

Ms. Ayaka Okamoto¹, Dr. Hiroyuki Koide¹, Mr. Hiroki Tsuchida¹, Dr. Hidenori Ando¹,
Ms. Saki Ariizumi¹, Ms. Chiaki Kiyokawa¹, Mr. Masahiro Hashimoto¹, Dr. Tomohiro Asai¹,
Dr. Takehisa Dewa², Prof. Naoto Oku¹

1. University of Shizuoka School of Pharmaceutical Sciences, 2. Graduate School of Engineering, Nagoya Institute of Technology

[Introduction] Small interfering RNA (siRNA) has been widely studied for the treatment of intractable diseases, such as cancer. Since naked siRNA is unstable in plasma and has low transfection efficiency, the siRNA delivery system is indispensable for the establishment of systemic siRNA therapy. In the present study, we report an effective and easy-to-use approach for siRNA encapsulation into liposomes by freeze-thawing. We found that freeze-thawing of single-layer polycation liposomes (PCLs)/siRNA complex forms multi-layer structures, suggesting siRNA was effectively packaged between the lipid layers of multi-layer PCLs.

[Method] Dicetyl phosphate-diethylenetriamine conjugate-based PCLs were incubated with siRNA to form PCLs/siRNA complexes (conventional lipoplex). The lipoplex was frozen in liquid nitrogen and thawed in a water bath with vortexing to prepare freeze-thawed lipoplex. Structure of the freeze-thawed lipoplex was observed by transmission electron microscopy. To confirm stability of the siRNA, conventional or freeze-thawed lipoplexes were incubated with fetal bovine serum for 72 h, and then undegraded siRNA was extracted and detected by electrophoretic assay. Gene silencing effect of the freeze-thawing lipoplex was evaluated by reporter gene assay using B16F10 murine melanoma cells transduced with firefly luciferase gene. In order to determine the biodistribution of siRNA in the freeze-thawed lipoplexes, alexa750-modified siRNA (alexa-siRNA) formulated in PEGylated freeze-thawed lipoplex was administrated intravenously to tumor-bearing mice. Then, accumulation of the alexa-siRNA in each organ and tumor was detected by in vivo imaging system.

[Result and Discussion] The transmission electron microscopic observation showed that the freeze-thawed lipoplex formed a multi-layer structure after the freeze-thawing of single-layered PCLs/siRNA complex. The siRNA formulated in freeze-thawed lipoplexes was not degraded even after incubation with 90% fetal bovine serum for 72 h while siRNA formulated in the conventional lipoplexes was degraded. These results indicate that siRNA was packed between the lipid layers and prevented from exposure to nucleases. The freeze-thawed lipoplex showed significantly higher gene-silencing efficacy compared with the conventional ones. Additionally, more amount of alexa-siRNA formulated in PEGylated freeze-thawed lipoplexes showed long-term blood circulation and accumulated in tumor compared with the PEGylated conventional lipoplexes. Our results provide an effective strategy for systemic siRNA delivery with a quite simple procedure.
# Adsorption of Bilirubin by Chitosan Coated Activated Carbon Prepared from Date Pits

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 345

Ms. Ameera Seyedzadeh ¹, Ms. Asil Mwafy ¹

1. United Arab Emirates University

The aim of this process was to prepare activated carbon (AC) from date pit powder and observe its adsorption efficiency of bilirubin toxin. Adsorption plays a vital role in the removal of toxins from the blood stream of patients with liver failure. It has been acknowledged from preceding literature that AC has a high capacity to adsorb albumin-bound toxins, which is why it is expended for this purpose. In order to increase the adsorption of bilirubin, an increase in the surface area of the AC was necessary. This increase was achieved through pyrolysis. Furthermore, to increase the capacity of absorbance, the AC was coated with chitosan gel, which contains several groups on its chains that act as interaction sites. Results indicated that the presence of the AC lead to a dramatic cut in bilirubin and the more the AC added to a sample, the faster the rate of adsorption as well as a higher capacity of adsorption allowable.
# Using MEMS Resonant Mass Measurement to Characterize Mass, Density and Count of Nano-scale Particles

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 353

Dr. Hanna Jankevics Jones¹, Mrs. Rachel Bott¹, Mr. Stephen Carrington¹, Dr. Matthew Barea¹

1. *Malvern Instruments*

## Introduction

Using the innovative method of resonant mass measurement (RMM), we present data showing how mass, density and concentration from particle count-for particles ranging from nanometers up to micrometers can be determined. The technology utilizes a suspended MEMS microchannel resonator, through which individual particles transit across the resonator altering the resonant frequency, which is then detected using an optical-based method. Changes in frequency observed when a particle enters the microchannel resonator are proportional to the buoyant mass of the particle, and can be translated into mass, size or surface area[1]. RMM therefore allows particle concentration to be determined as well as particle mass in a single measurement. Furthermore, RMM enables differentiation of mixtures of particles types based on density differences. For example, silicone oil droplets found in therapeutic pre-filled syringes can be discriminated from protein aggregates, thus enabling accurate quantification of both silicon oil content and protein aggregate concentration[2]. Folzer et al have also shown differences in aggregate densities for protein particles stemming from different proteins such as BSA and IgG[3]. RMM also has the capability of resolving mass differences between uncoated and coated nano-scale particles, as shown by the binding of proteins to latex beads[4].

## Methods & Results

Exemplifying the technique, we show how size and concentration of among other gold nanoparticles in suspension can be determined, as well as particle density determination of a range of materials including polystyrene latex, mesoporous silica and metal nanoparticles.

## Discussion

Mass is an inherent attribute of all particles, and exploiting this as a detection technique offers a more universal method for characterizing nano-scale particles in a wide variety of dispersants. The ability to measure the particle density is critical for characterization of nano particles, to understand and optimize the production of nano particles for applications such as for example drug delivery, diagnostics or imaging.

## REFERENCES

[1] T.P. Burg, M. Godin, S.M. Knudsen, W. Shen, G. Carlson, J.S. Foster, K. Babcock and S. Manalis, *Nature* 446, 1066-1069, 2007.

[2] A.R. Patel, D. Lau, and J. Liu, *Anal. Chem.* 84, 6833-6840, 2012.

[3] E. Folzer, T. Khan, R. Schmidt, C. Finkler, J. Huwyler, H-C. Mahler, and A.V. Koulov, *J.Pharm.Sci.*, 1-7, 2015.

[4] M.R. Nejadnik and Wim Jiskoot, *J.Pharm.Sci.* 1-7, 2014.
# Polydopamine, a potential mucopenetrative nanomaterial capable of multimodal therapy for bladder cancer

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 384

Ms. Barbara Poinard ¹, Mr. Samuel Neo ², Ms. Angeline Tan ², Mr. Roy Tan ², Prof. Koon Gee Neoh ³, Dr. James Kah ³

1. NUS (National University of Singapore) Graduate School of Science and Engineering, 2. Ngee Ann polytechnic, 3. NUS
(National University of Singapore)

The high rate of treatment failure for superficial Bladder cancer is partly due to the short residence time of drugs and low uptake, both of which can be explained by urine voiding as well as the presence of a thin mucus layer on the surface of the urothelium. Hence, the development of novel therapeutics that would improve drug retention for patients with bladder cancer is significant.

The present study aims to exploit the many properties of polydopamine (PDA) to design a biodegradable muco penetrative polymeric nanocarrier of Ce6, a photosensitizer drug. PDA is hydrophilic and slightly negatively charged which minimizes hydrophobic interactions and electrostatic interactions with the negatively charged mucus mesh. Furthermore, it is a natural mimic of melanin, biocompatible, easily synthesized and capable of photothermal properties thus enabling multimodal therapy (photodynamic (PDT) and photothermal (PTT)) to increase the drug retention and hence cell kill efficacy of non-muscle invasive bladder cancer cells.

To date, we have successfully characterized and optimized PDA loaded with Ce6 in vitro and studied many parameters such as their loading capacity, stability in different media and release profiles. It has been demonstrated that PDA could effectively load up to 3 microM of Ce6 with an initial burst release and a saturation at day 7. The therapeutic effect of PDA-Ce6 has also been compared to free Ce6 and PDA alone in bladder cancer cells with a significantly higher cell kill when subjected to the dual PDT-PTT. Furthermore, preliminary studies have been performed probing the interaction between mucin and PDA. Results with the Transwell system seemed to indicate that PDA were capable of permeating through the mucus mesh.

The development of these mucopenetrative particles could possibly also be applied in other kinds of cancer or drug delivery therapy which requires passage through the mucus barrier.
# Development of a Long-circulating Liposomal Carrier Coated with Serum Albumin via Ligand

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 403

Ms. Hikari Sato¹, Mr. Yuta Nakamura¹, Prof. Akihiro Kishimura², Prof. Takeshi Mori², Prof. Yoshiki Katayama²

1. Graduate school of System Life Science, Kyushu University, 2. Department of Applied Chemistry, Kyushu University

**Introduction:** Polyethylene glycol (PEG)-modified liposome is known as the most succeeded drug carrier because of its long blood half-life. However, it has a problem with its repeated administration because it is rapidly removed from blood after the second administration by produced antibodies against PEG. Here, we focused on serum albumin (SA) for an alternative molecule to provide stealth property to liposome. SA is the most abundant protein in plasma (40 g/L). Since SA can avoid glomerular filtration and degradation in endocytosis, it has a long blood half-life (20 days). Moreover, SA works as an endogenous drug carrier for exogenous drugs and can avoid being caught by immune system. We utilized SA to camouflage the surface of liposome to prolong its blood circulation time. Here, we propose modification of the liposome surface via SA-specific ligands. As such a ligand, we selected octadecanedioic acid, which is reported to bind to albumin. Because of the remaining terminal carboxyl group of octadecanedioic acid after modification of PEG-lipid, insertion of the ligand into hydrophobic liposome's bilayer will be prevented.

**Method:** Octadecanedioic acid-modified lipid (1) was synthesized. Then, a liposome containing lipid 1 (1LL) was prepared by a hydration method. To confirm the stability of 1LL in physiological saline, 1LL was incubated in DPBS containing SA from bovine (BSA) at r.t. Change of liposome's size was monitored for a week.

**Results and Discussion:** In the absence of BSA, 1LL aggregated in DPBS after 2 days. On the other hand, in the presence of BSA, 1LL kept narrow size distribution and dispersed stably for a week. It was speculated that the aggregation of 1LL was caused by hydrophobic interaction between ligands, however, the hydrophobic interaction seems to be suppressed by coating of 1LL with SA. This result indicates that 1LL was successfully coated with SA, contributing significant stabilization of 1LL in physiological saline. 1LL would be applicable to a drug carrier with enhanced blood circulation. Result of blood circulation time of the liposome in mice will be presented.
<figure><img src="image_94.png" /><figcaption>Iconan.jpg</figcaption></figure>
CNTs recruit different serum protein assortments on
the biocorona

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 412

Mrs. ESPERANZA PADÍN GONZÁLEZ ¹, Mrs. Nerea Iturrioz ¹, Mrs. Eloisa González-lavado ¹, Ms.

Carmen Pesquera ², Mr. Fernando González ², Ms. Monica L. Fanarraga ¹

1. Universidad de Cantabria-IDIVAL, 2. Universidad de Cantabria

Carbon nanotubes (CNT) have a highly reactive surface that has the intrinsic ability of recruiting surrounding proteins on their surfaces through non-covalent interactions. The CNT biocorona has a chief role in CNT toxicity and bioaccumulation. This protein coating has many effects in vivo providing the nanotube with different biomimetic identities,1,2 dictating the bio-distribution,3 being critical in cellular recognition and CNT biodegradation.4

The formation of the protein corona has been extensively studied in recent years but the underlying biochemical processes taking place on the proteins that bind the nanotube are not completely understood. The interaction of proteins with the CNTs depends on the actual physicochemical properties of the surface of the nanotube,5,6 and also on the structure of the interacting proteins. Most soluble proteins (such as serum albumin) have hydrophobic cores that are only exposed after pseudo-denaturation.7 The exposure of the polypeptide core by partial unfolding of the protein depends on many factors, among others, (i) temperature, (ii) environmental reducing conditions, (iii) time, (iv) solvents, (v) detergents, etc.8 Accordingly, one single protein can display different interactive behaviors with the same nanomaterial depending on these conditions. Here we show how the protein corona of CNTs functionalized with blood serum, recruits diverse assortments of serum proteins on the nanotube surface when exposed to different conditions during functionalization. Our results reveal that the functionalization of the CNT is qualitative and quantitative dependent on all the above environmental conditions.

(1) Nel, A. E., et al., Nat. Mater. 2009, 8 (7), 543-557.

(2) Monopoli, M. P., et al., Nat. Nanotechnol. 2012, 7 (12), 779-786.

(3) Maruyama, K., et al., Biomed Res. Int. 2015, 2015, 793186.

(4) Gea, C., et al., PNAS. 2011, 101, 16968-16973.

(5) Du, J., et al., JNN. 2011, 11, 10102-10110.

(6) Sopotnik, M., et al., Carbon. 2015, 95, 560-572.

(7) Sengupta, B., et al., RSC Advances. 2015, 5, 82395-82402.

(8) Arakawa, T., et al., Adv. Drug Deliver. Rev. 2001, 46, 306-326.
<figure><img src="image_95.png" /><figcaption>**Figure 1.** Functionalization of Nanocyl® NC3100™ MWCNTs with blood serum under different conditions reveals specific biocorona. SDS-PAGE electrophoresis of CNTs functionalized with serum proteins</figcaption></figure>

<caption>Figure1 abstract.jpg</caption>
# In vitro correction of congenital disorder of glycosylation type Ia (CDG Ia) using PLGA nanoparticles loaded with GDP-Man.

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 421

Dr. Barbara Bortot¹, Dr. Eleonora De Martino², Dr. Alessandra Tesser¹, Prof. Giovanni Tosi³, Prof. Barbara Ruozi³, Dr. Giovanni Maria Severini¹

1. Institute for Maternal and Child Health IRCCS Burlo Garofolo, 2. University of Trieste, 3. Department of Life Sciences of University of Modena and Reggio Emilia

Congenital disorder of glycosylation syndrome type Ia (CDG Ia) is an autosomal recessive multisystem disorder with neurological involvement due to mutations into gene sequence that codifies phosphomannomutase 2 (PMM2), enzyme involved in N-glycosylation pathway. Mutated PMM2 leads to a missed conversion of mannose-6P to mannose 1P which results in low concentrations of GDP-MAN in cytosol and little branched oligosaccharide chains. Previous studies have been demonstrating a reduced activity of several lysosomal enzymes such as α-fucosidase, β-hexosaminidase, β-glucuronidase and α-mannosidase in leukocytes and sera of CDG-Ia patients. We propose an in vitro treatment of CDG fibroblasts using PLGA nanoparticles loaded with guanosine diphosphate D-mannose (GDP-Man), a nucleotide-activated sugar essential to building olygosaccharide chains by-passing glycosylation pathway reaction catalized by PMM2. PLGA-NPS loaded with the commercial guanosine 5'-diphospho-D-mannose sodium salt were prepared by W/O/W (double emulsion) solvent evaporation method. Human fibroblast controls and CDG 1a fibroblasts were obtained from skin biopsies. Specific activities of β-galactosidase, α-mannosidase, β-glucuronidase were measured using 4-methyl-umbelliferyl β-D-galactopyranoside, 4-methyl-umbelliferyl α-d-Mannopyranoside, 4-methyl-umbelliferyl β-d-glucuronide substrates.

In order to determine in vitro hypoglycosylation correction we assayed specific activity of different lysosomal enzymes including β-galactosidase, α-mannosidase and β-glucoronidase from CDG 1a patient's fibroblast cultures. Three fibroblast cultures derived from three CDG patients, called F1, F2 and F3, were incubated with PLGA nanoparticles loaded with 1.5 ug of GDP-Man, corresponding to 2,3 nmol of GDP-Man for three or six hours at 37°C.

The specific activity of lysosomal enzymes like α-mannosidase and β-glucoronidase were assayed at three different times after treatment: 24 hours, 48 hours and 72 hours. As shown in Figure 1, α-mannosidase attains levels of activity close to fibroblast control activity after 48 hours post-incubation in F1, F2 and F3, demonstrating the efficiency of the proposed treatment. To date there is no therapy for CDG-Ia patients, but PLGA nanoparticles loaded with GDP-Man might provide a suitable treatment to bypass PMM2 deficiency and improve protein glycosylation due to their high biodegradability and toxicity profile.

Keywords: Congenital disorder of glycosylation, poly(lactic-co-glycolic acid)

nanoparticles, Guanosine diphosphate and Mannose, phosphomannomutase 2, lysosomal enzyme, specific activity.
<figure><img src="image_96.png" /><figcaption>Fig. 1: Correction using GDP-Man PLGA nanoparticles of a-mannosidase activity in three cultured fibroblasts obtained from CDG patients. Analysis was repeated three times and the mean ± s.d. is reported.</figcaption></figure>

Gdp-man plga nps correction.jpg
Effect of the nanoparticle shape on the cancer treatment: the case of pluronic F127 stabilized and doxorubicin loaded magnetite nanocubes and nanospheres.

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 444

Dr. Timur Nizamov¹, Dr. Anastasia Garanina¹, Prof. Alexander Savchenko¹, Prof. Alexander Majouga¹

1. NUST MISiS

Magnetic nanoparticles are extensively prepared for the potential use in nanomedicine fields: MRI and drug delivery. Magneto-niosomes – magnetic nanoparticles, stabilized by non-ionic surfactants are the ones of the promising agents in such fields. They are examples of theranostic agents combining therapy (drug delivery) and diagnostics (MRI contrast agents) in one agent. The other rarely studied aspect is the effect of nanoparticle shape on toxicity, drug delivery and relaxivity. The following work is dedicated to synthesizing of magneto-niosomes with different core shapes (spherical and cubic), and investigating the effect of nanoparticles shape on relaxivity and toxicity towards PC3 and LNCaP cancer cell cultures.

Nanospheres and nanocubes were synthesized by thermal decomposition of iron oleate in 1-octadecene. Shape control was reached by different stabilizers molar ratio in reaction medium. According to TEM measurements average size of the nanoparticles is 20±5 nm. The synthesized nanoparticles were transferred into water by non-ionic surfactant (Pluronic F127) solution. DLS data demonstrates that nanoparticles average size has risen up to 90±10 nm due to niosomes formation. The hydrophilized nanoparticles were loaded with doxorubicin by addition of its solution into hydrosol and stirring. Doxorubicin excess was taken away by centrifugation and supernatant removing. Spectrophotometry was used to determine the doxorubicin loading (the average drug loading is 13,5% of drug carrier). Loaded niosomes cytotoxicity was measured on PC3 and LNCaP cultures by MTT assay and compared to cytotoxicity of unloaded nanoparticles and free doxorubicin after 48 hour of incubation.

The obtained data demonstrate lower cytotoxicity of doxorubicin loaded nanoparticles compared to free dox-
orubicin at the same doxorubicin concentrations in cell culture. This happens due to slow doxorubicin releasing
from Pluronic shell of magneto-niosomes which play a role of drug carrier. Also nanocubes based niosomes has
shown a bit higher cytotoxicity compared to analogous nanospheres based agents. This effect is caused by four
times higher uptake of nanocubes versus nanospheres. Both cubic and spherical niosomes demonstrated rela-
tively high T2-relaxivity (240 mM-1*c-1 and 262 mM-1*c-1 respectively).

The authors knowledge financial support from Ministry of Education and Science of the Russian Federation (14.607.21.0132, RFMEFI60715X0132).
# Atomic Force Acoustic Microscopy for the Characterization of Gold Nanoparticles Embedded into a Polymeric Matrix and its Promissory Application in Nanomedicine

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 445

**Dr. Daniela Geraldo**¹, **Mr. Eduardo Jeraldo**¹, **Prof. Ramiro Arratia-perez**¹

1. Universidad Andres Bello

In this work, we report for the first time a morphological study of gold nanoparticles embedded on a polymeric matrix using Atomic Force Acoustic Microscopy (AFAM). The polymer based-gold nanoparticles were prepared by a green approach using biomass from macroseaweed, as a mixture of reducing agents containing not only water soluble polysaccharides but also phytoactive compounds. It was demonstrated that AFM in the acoustic mode is a powerful tool that allows obtain conclusive information about the morphology of a natural polymer acting not only as a reducing agent but also as stabilizing agent for the gold nanoparticles. The formation of gold nanoparticles was monitored by UV-Vis spectroscopy and Transmission Electron Microscopy (TEM) confirming the presence of monodisperse and isotropic nanostructures. Finally, the hemolytic activity of the gold nanoparticles embedded into the polymeric matrix was tested in order to compare their intrinsic cytotoxicity with those ones prepared using the well-known inorganic reducing agent, sodium citrate (SC). The obtained results strongly suggest that these eco-friendly gold nanoparticles could be used on human cells due its less toxicity which expand their use on nanomedicine.

Acknowledgements: Financial support for this work was provided by the Air Force Office of Scientific Research Project FA9550-12-1-0367 and Proyecto Núcleo UNAB DI-622-14N.
<figure><img src="image_97.png" /><figcaption>Gold nps embedded into polymer by afam 10x10.jpg</figcaption></figure>
# Preparation and characterization of multimeric system of RGD-grafted PMMA-nanoparticles as a targeted drug-delivery system for paclitaxel

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 484

Ms. Brenda Vianey Gibbens-Bandala¹, Dr. Enrique Morales-Avila¹, Dr. Blanca Eli Ocampo-García², Dr. Guillermina Ferro-Flores²

1. Universidad Autónoma del Estado de México, 2. Instituto Nacional de Investigaciones Nucleares

In cancer, therapeutic alternatives include the use of antineoplastic drugs; however, current drugs lack selectivity for only to damaged tissue, which produce severe adverse effects. Improvements in the development of polymer-based nanoparticles as drug-delivery systems, offers new therapeutic opportunities. Among them, ligand-mediated targeting, interacts with specific receptors on target cells, increases selectivity and efficacy and allows controllable drug delivery, increasing therapeutic benefits, while minimizing side effects. Additionally, it offers the possibilities of encapsulating poorly-soluble drugs, protecting therapeutic molecules, and modifying the pharmacokinetic profile of nanoparticle-incorporated drugs.

The aim of this research was to prepare and to characterize poly(methyl methacrylate) (PMMA) nanoparticles grafted with the -Arginine, Glycine, Aspartic Acid (RGD)- peptide sequence as a promising smart drug delivery system for Paclitaxel (PTX), directed to sites with integrin receptor overexpression, such as angiogenesis sites in some solid tumors.

Characterization techniques included FT-IR spectroscopy, particle size by dynamic light scattering (DLS), zeta potential, morphology by transmission electron microscopy (TEM), entrapment efficiency (%EE), drug release profile and cytotoxicity in positive integrin receptor C6 cell line. The vibrational spectroscopy characterization indicated the formation of the PMMA polymer, PMMA-PTX or RGD-PMMA-PTX modified polymer. RGD-PMMA-PTX size distribution was found to be 17.58 ± 7.45 nm with a zeta potential of

-38.73 ± 5.62 mV and of spheroidal form. According to the boxLucas Model, PTX was incorporated into polymeric matrix with an entrapment efficiency maximum of 100% (HPLC). These nanoparticles showed sustained in vitro release with maximum release percentages of 55% and 40% after 21 days at pH 5.3 and 7.4, respectively, a relatively fast release of PTX in the first 5 hours at pH 5.3, but not for pH 7.4 attributable to the diffusion and dissolution of PTX that was entrapped superficially in the polymer matrix. RGD-PMMA-PTX nanoparticles were shown to be more cytotoxic to C6 glioblastoma cells than non-targeted nanoparticles and free PTX.

These results indicate that RGD-PMMA-PTX is suitable for specific PTX delivery in cells that over-express αvβ3 receptors in processes of angiogenesis. In this study, the small amount of PTX required to produce an anti-proliferative effect opens the possibility of reducing side effects.
<figure><img src="image_98.png" /></figure>

Preparation of multimeric system of rgd-grafted
pmma-ptx nanoparticles.jpg
# Effect of Ligand Shell Composition on Biodistribution of Ultrasmall Gold Nanoparticles

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 506

Dr. Yao Ding¹, Dr. Tom Coulter¹, Ms. Cristina Espinosa Garcia¹, Dr. Sarah Hale¹, Mr. Alessandro Pace¹, Dr. Ketan Patel¹, Ms. Usoa Aguilera Peral¹, Mrs. Angela Robinson¹, Dr. Dan Palmer¹, Dr. Phil Williams¹, Dr. Meike Roskamp¹

1. Midatech Pharma Plc

Cytotoxic chemotherapy is the standard of care for many types of cancer despite the frequently observed severe side effects. The targeted delivery of chemotherapeutics has great potential to reduce these effects by increasing drug concentrations within the target tissue, thereby reducing the required dose [1]. Targeted delivery can be an extremely useful approach to achieving a therapeutically active concentration whilst ensuring acceptable systemic toxicity [2].

Midatech Pharma Plc uses novel ultrasmall gold nanoparticles (GNPs) with a gold core diameter of 1.6 – 1.8 nm as a functionalizable vehicle for tissue-selective delivery (Figure 1). This platform is highly flexible, allowing incorporation of various components dependent upon the therapeutic application [3]. In the current study we describe GNPs that are coated with thiol-modified carbohydrates and bifunctional polyethylene glycols (PEGs). We investigated the effect of different chemical ligand shell compositions on the biodistribution and pharmacokinetic profile of these particles using an in vivo rat model. By varying the charge and ligand size, we showed that Midatech's novel ultrasmall gold nanoparticles can be customized to vary circulation times and tissue selective accumulation. Also, due to their very small size and carbohydrate based ligand shell these particles are less likely to form a protein corona compared to larger nanoparticle analogues [4], and are therefore especially interesting candidates for active targeting strategies.

In conclusion, Midatech GNPs offer a highly flexible and customizable drug delivery system which may be tailored for a range of cancer types and other diseases.

## References

[1] S. M. Sagnella, J. A. McCarroll, M. Kavallaris, Nanomedicine, 10, 1131 (2014).

[2] M. Shilo, M. Motiei, P. Hana, R. Popovtzer, Nanoscale, 6, 2146 (2014).

[3] midatechpharma.com.

[4] K. Zarschler, L. Rocks, N. Licciardello, L. Boselli, E. Polo, K. P. Garcia, L. De Cola, H. Stephan, K. A. Dawson, Nanomedicine, 12 (6), 1663-1701.
<figure><img src="image_99.png" /><figcaption>Figure 1. Functionalized gold nanoparticles for drug delivery (R = COOH, NH2).</figcaption></figure>

Generic particle.jpg
# Influence of biophysical anomalies on forest tree quality

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 563

Dr. Aigars Indriksons¹, Mr. Maris Eglite¹, Dr. Imants Liepa¹

1. Latvia University of Agriculture, Forest faculty

Biolocation is a method which allows to estimate the biophysical anomalies on the earth surface, underground and air by considering of changes in condition of dowsing rod or other indicator in hands of dowser. The most popular version of biophysical anomalies is connected with flows of underground water. Water has an 81 times higher relative dielectric coefficient as air. However structures of different networks suggest for another nature of radiation which does not connect with water flow.

There is very few research carried out of this theme in Latvia. In 1979 there was the section of Bioelectronics established by the A. Popov's scientific-technically society of Radio engineering, electronics and communication, which in fact worked in Institute of Physics of Latvian Academy of Science.

The research carried out in Liepupe Forestry suggests that the pine (Pinus sylvestris L.) trees growing on biophysical anomalies are more vigorous and those growing on points of intersection are with wider diameter. However, the pine trees measured by City Jelgava gives a converse suggestion – the trees growing on places with biophysical anomalies are thinner and also with lower density.

According to news of Finnish forest scientist V. Altonen, during the 30ties of the 20 century a number of scientists have tried to clarify if there in forest is some influence of biophysical anomalies to the flora. K. Müller (1935; 1936) indicates that trees growing on points of intersection of anomalies are decayed. He has observed that trees growing on the biophysical anomalies are more infected by diseases and more abundant are the agents of diseases – parasitic fungi and bacteria.

Till up to now there are no scientifically secure evidence between any external physical fields and biophysical anomalies obtained. However, if something can not been proven or rejected in scientifically proper way, it does not mean that it is not existing and has not to been explored with increasingly growing technical resources.
Keywords: biophysical anomaly, biolocation methods, tree growth.
Engineered Nanoparticles for Rapid Delivery of Betulinic Acid in MDA MB 231 and HEp-2 Cancer Cell Lines

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 279

<u>Mr. Asim Halder</u>¹, <u>Ms. Pritha Mukherjee</u>¹, <u>Mrs. Subarna Ghosh</u>¹, <u>Dr. Urmi Chatterji</u>¹, <u>Prof. Arup Mukherjee</u>¹

1. University of Calcutta

Introduction: A range of triterpenoids have gained attention in cancer chemotherapy due to promising pre-clinical results. Triterpenoids are however constrained in therapeutics for both the solubility and permeability limitations. Betulinic acid (BA) is a pentacyclic triterpenoid available in Betula alba (white birch) bark. BA is enlisted under the RAID (Rapid Access to Intervention Development) program of NCI, USA. The compound is a strong inhibitor of topoisomerase and induces caspase activation and mitochondrial membrane damage. BA is widely available and economic but suffers seriously in bio-pharmaceutics. Smart nanoparticle drug delivery devices were therefore conceived as one solution for BA delivery. Lactoferrin was used for nanoparticle ligand tethering for successful particle propagation up to the cancer cell nucleus.

Methods: PLGA nanoparticles loaded with BA (BAnps) were prepared in modified solvent diffusion method using PLF 127 as a stabilizer. Lactoferrin conjugation on BAnps (Lf-BAnps) was achieved in EDC/NHS coupling reactions. Nanoparticles were characterized in DLS, AFM, TEM, XRD, FTIR and SDS-PAGE. In-vitro anticancer efficacy was evaluated in two metastatic cancer cell lines: MDA-MB-231 and Hep-2.

Results and Discussion: The average particle size of Lf-BAnps in DLS was 120 nm. The entrapment efficiency of BA (75.42%) and non-fickian release pattern were determined by RP HPLC. The FT-IR and SDS-PAGE analysis confirmed lactoferrin interactions with BAnps. Lf-BAnps showed significantly strong antiproliferative and cytotoxic effects on both the cancer cell lines (IC50 4.0 µg/ml). Time-dependent uptake and quantitative count in flow-cytometry showed early time-point intracellular entry of Lf-BAnps. Specific cellular localization was confirmed by confocal microscopy. Increased sub-G1 population and decreased G2/M population indicating the apoptotic potential and cellular arrest of Lf-BAnps on cancer cells. Rapid localization and lactoferrin bio-functionalization in nanoscale has proved as one successful strategy in chemotherapeutic drug targeting.
<figure><img src="image_100.png" /></figure>

Nanoparticles characterization and anticancer
evaluation study.jpg
# Hybrid biocompatible silica nanoparticles as theranostic agent

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 122

Mr. Ritu Raj¹, Ms. Carina Crucho², Prof. José Paulo Sequeira Farinha², Prof. Carlos Baleizão²

1. CQFM – Centro de Química-Física Molecular and IN – Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Lisboa, Portugal, Department of Life Science, National Institute of Technology, Rourkela, Odisha, India, 2. CQFM – Centro de Química-Física Molecular and IN – Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Lisboa, Portugal

**Introduction:** Hybrid silica nanoparticles have attracted much attention owing to their potential biomedical applications in diagnostic and precision drug delivery for cancer detection and treatment. The inorganic silica core can be used to transport cargo and impart properties such as fluorescence, whereas the polymeric shell can be used to convey water solubility, biocompatibility, long blood circulation times, and bioconjugation. The goal of our work is to develop “smart” hybrid nanoparticles (NPs) with theranostic (therapeutic + diagnostic) functionalities that carry a fluorescent dye for traceability and imaging, feature a mechanism for release control, and are able to accommodate large drug loads and deliver their cargo to the desired location. Here we focus on the preparation of fluorescent silica nanoparticles (SiNPs) with a poly(lactide-co-glycolide) (PLGA) biocompatible and biodegradable copolymer shell.

**Method:** We prepared monodisperse fluorescent SiNPs incorporating a perylenediimide (PDI) derivative in the silica network, and from the SiNPs external surface, a dense polymer shell of PLGA was grown by ring-opening polymerization (ROP), with different monomers ratio to modulate the degradation rate of the shell. The biocompatibility of the NPs was tested against MCF-7 human breast carcinoma cells. Cell viability and confocal studies were performed to assess the efficacy of the hybrid NPs.

**Result & Discussion:** The synthesized SiNPs were spherical and homogenous with an average diameter of 31 nm which was confirmed by Transmission electron microscopy (TEM) and Dynamic Light Scattering (DLS). The increased diameter of the Hybrid NPs due to the formation of a PLGA shell on the surface of the silica core has been established by TEM and DLS. The cell viability (MTT assay) showed a high level of biocompatibility, and confocal images confirmed the time-dependent internalization of hybrid NPs into the MCF7 cells.

**Acknowledgments:** This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER), projects RECI/CTM-POL/0342/2012, UID/NAN/50024/2013, and PTDC/CTM-POL/3698/2014. R.R. thanks, Erasmus Mundus Experts Sustain Ph.D. grant.
<figure><img src="image_101.png" /></figure>

Abstract for iconan.jpg
# Comparison of the effect of selenium Nano particles and sodium selenite on the serum and hepatic lipid profiles of rats following experimental exposure to cadmium.

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 472

Dr. Saeid KarimiDehkordi¹, Dr. Abdonnaser Mohebbi¹, Prof. Mohammad Reza Aslani¹, Dr. Afshin

Jafari Dehkordi¹, Dr. Akram Shahabi¹

1. Shahrekord University

**Introduction:**

Cd exposure alters cellular normal process leading to dysfunction in different organs. Several studies have reported the cardiovascular effects of Cd in human and animal models. Some studies have revealed the lipid metabolism alterations in Cd exposed human and experimental animals. Cd causes modification in the blood lipid profile toward a atherogenic condition.

Selenium is known due to its antioxidant function. The reduction of Se salts in presence of proteins forms Se nanoparticles (Nano-Se) which possess greater biological activity than ion form. It is well known that Se alleviates cadmium toxicity by preventing oxidative damage. However the effect of Se on the lipid metabolism in case of Cd toxicity has not been studied.

The aim of present work was to study the probable effect of Se in two different form (sodium selenite, Na-Se, and Nano-Se) on the lipid metabolism in Cd intoxicated rats.

**Methods:**

Male rats were divided in 4 different experimental groups. A control group and other rats were orally received Cd (3mg/kg b.w) alone or with red Nano-Se (20-30 nm) or Na-Se (0.1 mg/kg b.w) for 35 days. Serum and liver lipid profile were determined using standard methods.

**Results:**

Cd exposure increased blood and liver TG, total cholesterol, LDL-C and VLDL-C and decreased HDL-C in affected rats compared to control group. Serum and liver TG concentrations were decreased near control group by receiving both Nano-Se and Na-Se, 29% and 38% respectively. The liver total cholesterol and LDL-C contents were not affected by Na-Se while Nano-Se caused a statistically significant reduction on them. Our data revealed that receiving both forms of Se led to decrease in serum concentrations of total cholesterol and its lipoprotein containers, meanwhile, receiving Nano-Se had a relatively greater effect than Na-Se.

**Discussion:**

Based on our finding, Nano-Se has stronger ameliorative effects on lipid metabolism disorders in Cd intoxicated rats in comparison to Na-Se. The main mechanism of this effects were not clear. However, It is well known that biologic properties of Se Nano-Se specially antioxidant activity, are dependent on the particle size and smaller particles have greater activity.
# Mannose Nanoparticles from Guar Gum: Macrophage Vectorization, Drug Delivery and Leishmanicidal Efficacy against Wild and Drug Resistant Strains

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation -
Abstract ID: 244

Prof. Arup Mukherjee¹, Mr. Asim Halder¹, Mr. Sumanta Kumar Ghosh¹, Ms. Nandita Ghosh¹, Dr. Ena Ray Banerjee¹

1. University of Calcutta

**Introduction:** Guar gum (GG) is obtained in sufficient purity from Cyamopsis tetragonoloba seeds. Unlike cel-lulose, GG presents one β 1-4 mannose chain interposed with α 1-6 galactose substituents. GG hydrates hugely in mannose surfaced coiled assembly formations. We are successful for the first time in extracting mannose nanoparticles (GMn) from GG. GMns observed rapid receptor gated uptake in macrophages. Drug loaded GMns were used against macrophage resident L. donovani strains.

**Methods:** Temperature controlled GG hydrolysis in sulfuric acid (64%) yielded GMns spheroids, TEM size 48.8 nm, ζ potential -20.4mv. Epichlorohydrin in liquor ammonia reactions on GMn provided cationic modifications and fluorescent rhodamin dye was linked following isothiocyanate chemistry. Quercetin (Qr) was loaded in GMns by porous diffusion in phosphate buffer. The loading efficiency in HPLC was 87% w/w and FT-IR studies confirmed nano-chemistry. Qr loaded GMns efficacy against L. donovini AG 83 strains were studied against axenic and macrophage infested amastigotes.

**Results and Discussion:** GMns and Qr loaded GMns were taken up rapidly in mouse peritoneal macrophages. Uptake was complete within 60 min and 2 mM mannose but not galactose could inhibit that significantly. Con-canavallin A agglutination experiments confirmed GMns 42% surface mannose density. Qr loaded GMns were safe and powerful leishmanicidals against wild and drug resistant strains (Table 1).

Table 1. Antileishmanial efficacy of quercetin loaded GMns against Leishmania donovani.
<table><caption>Table 1. Antileishmanial efficacy of quercetin loaded GIMs against *Leishmania donovani* AG 83</caption><thead><tr><th rowspan="2">Doug</th><th colspan="3">Axonic amastigote</th><th colspan="3">IC<sub>50</sub> (from 1:SD 1 replication) µM</th><th rowspan="2">Cytotoxicity Macrophage cells CC<sub>10</sub> µM</th></tr><tr><th>WT</th><th>SSG resistant</th><th>PMM resistant</th><th>WT</th><th>SSG resistant</th><th>PMM resistant</th></tr></thead><tbody><tr><td>Amphotericin B</td><td>0.2±0.05<sup>a</sup></td><td>0.4±0.05<sup>a</sup></td><td>0.35±0.05<sup>a</sup></td><td>0.15±0.05<sup>a</sup></td><td>0.2±0.05<sup>a</sup></td><td>0.18±0.05<sup>a</sup></td><td>14</td></tr><tr><td>Sodium stibogluconate (SSG)</td><td>3.6±0.40</td><td>130±20</td><td>115±16</td><td>1.6±0.30</td><td>18.3±3</td><td>17.3±2.60</td><td>27</td></tr><tr><td>Paromomycin (PMM)</td><td>10±2<sup>b</sup></td><td>380±40<sup>a</sup></td><td>330±30<sup>a</sup></td><td>8±2<sup>a</sup></td><td>125±15<sup>a</sup></td><td>115±13<sup>a</sup></td><td>248</td></tr><tr><td>Qy-loaded GIMs</td><td>25±4<sup>b</sup></td><td>30±5<sup>b</sup></td><td>26±6.3<sup>b</sup></td><td>18±5<sup>a</sup></td><td>23±3<sup>b</sup></td><td>24±4.2<sup>b</sup></td><td>6.4±10<sup>2</sup></td></tr><tr><td>Qy</td><td>31.2±2.1<sup>a</sup></td><td>145.5±24<sup>a</sup></td><td>75±10<sup>a</sup></td><td>33.0±6.5<sup>a</sup></td><td>123±16<sup>a</sup></td><td>64.0±9.5<sup>a</sup></td><td>12±10<sup>2</sup></td></tr></tbody></table>

WT (Wild Type), SSG (sodium stibogluconate) and PMM (paromomycin). * P<0.05 highly significant, **P<0.5 significant and # P<0.1 no significant difference compared with SSG group.

Table 1. antileishmanial efficacy of quercetin loaded gmms against leishmania donovani..png
# PAMAM dendrimer curcumin conjugate as a supramolecular polyphenol; a biomimetic approach to improve anticancer properties of curcumin

Friday, 30th September - 13:30 - Poster Session & Sponsors Exhibition - Patio 25 - Poster presentation - Abstract ID: 316

Dr. Vahid Erfani-Moghadam¹, Prof. Majid Sadeghizadeh², Dr. Alireza Nomani³

1. Department of Biotechnology, Faculty of advanced Medical Technology, Golestan University of Medical sciences, Gorgan, Iran, 2. Department of Genetics, Faculty of biological sciences, Tarbiat Modares University of Tehran, Tehran, 3. Department Of Pharmaceutical Biomaterials, School Of Pharmacy, Zanjan University Of Medical Sciences

**Introduction:** Nanotechnology researchers try to design novel drugs with new properties, such as enhanced bioavailability, biocompatibility and/or biodegradability. Curcumin is a potent natural anticancer agent, but its efficiency is limited by drawbacks, such as very low solubility, high rate of degradation and low rate of absorption of its hydrophobic molecules in vivo. In this study, we investigated the cell uptake and anticancer effects of curcumin on U87MG brain carcinoma cell line and HFSF-PI3, as a normal human fibroblastic cells, by preparing PAMAM (generation 4) dendrimer curcumin conjugates.

**Methods:** The curcumin conjugation to the surface of PAMAM dendrimers and its conjugation quantity were evaluated by FTIR and NMR methods. Then, cytotoxicity, cell cycle (by PI) and apoptosis (by PI/Annexin-V-FLUOS methods) of various PAMAM-based curcumin conjugates were investigated using U87MG cells. Also, real-time PCR method was used to evaluate the relative gene expression of anti-apoptotic genes; Xiap, RB1 and P21.

**Results:** The cytotoxicity studies showed that the IC50 of the free curcumin solution, 20% and 40% curcumin-PAMAM conjugated and PAMAM-curcumin encapsulation, were 48, 0.9, 2.5 and 13.5 µM, respectively, on U87MG cancer cell line. Results of the cell cycle and apoptosis analyses indicated that with the curcumin conjugated or encapsulated to PAMAM dendrimer, sub G1 population and apoptosis were significantly increased compared to that of the free curcumin. The results showed that Xiap and P21 gene expression in U87MG cells significantly decreased.

**Discussion:** Overall, the obtained results showed that dendrimer-based curcumin formulation induces apoptosis in U87MG cancer cells. Results showed that the application of these nanocarriers significantly increased the cell uptake of curcumin in comparison to the free curcumin. At the same time, by conjugating to the PAMAM surface, curcumin obtains a better water stability and perhaps longer therapeutic effect than PAMAM-encapsulated curcumin. In summery, based on the fact that supramolecular polyphenols have strong anticancer effects compared with small molecules, it seems that the synthesis of such big molecules can be considered as an effective biomimetic approach and as an appropriate drug delivery system for curcumin to U87MG cancer cells and probably as a suitable candidate for in vivo researches.
nanoMIL100: a novel efficient tool to target lung cancer

Friday, 30th September - 16:00 - Targeted drug delivery and Nanocarriers - Nanomedicine for cancer diagnosis & therapy - Amphitheatre 25 - Oral presentation - Abstract ID: 48

Dr. Teresa Simon-Yarza¹, Dr. Monica Gimenez-marques¹, Ms. Rhizlaine Mrimi¹, Ms. Angelika Mielcarek², Dr. Ruxandra Gref³, Dr. Patricia Horcajada¹, Dr. christian serre¹, Prof. Patrick Couvreur⁴

1. Institut Lavoisier, UMR8180 CNRS/Université de Versailles St-Quentin en Yvelines, 2. Ecole Normale Superieure, 3. Institut Galien, UMR8612 CNRS/Université Paris-Sud, 4. Institut Galien, UMR8612 CNRS, Université Paris-Sud

Lung cancer (LC) is a major health problem, accounting for near 27% of all cancer deaths. Each year, more people die of LC than colon, breast, and prostate cancers combined (1). This fact evidences the urgent need to improve current available therapy. To increase LC therapy and to reduce its toxicity we propose the use of MOF-based nanoparticles (NP). In particular, we have developed a formulation that passively targets the lungs without any visible signs of toxicity based on MIL-100(Fe), a mesoporous iron(III) trimesate MOF.

NP were prepared and characterized as described elsewhere (2). To evaluate their in vivo biodistribution and toxicity at early times (up to 24 h), a first study was performed in Sprague-Dawley rats. An important accumulation of the NP was observed in the lungs by measuring the iron concentration by atomic absorption spectroscopy. In accordance with our previous data, animals did not present any signs of toxicity. This result encouraged the evaluation of the impact of the loading of Gemcitabine-monophosphate (GMP), a well-known drug used in LC treatment, into MIL100 NP, on drug organ accumulation.

Tritiated-GMP was encapsulated by the impregnation method as previously described (3). C57BL/6JRj mice were treated iv with 10 mg/kg of the free drug and the equivalent dose of MIL100 encapsulated drug. When administered in NP, GMP concentration in lungs was five-fold higher than free GMP after 15 min and remained three-fold larger after 8h (Figure 1).

Thus, MIL100 NP appear as an efficient tool to target the lungs to treat pulmonary diseases.

1. American Cancer Society statistics (last revised 2015).

2. P. Horcajada et al. Chem. Rev., 2012, 112(2) 1232.

3. V. Rodriguez-Ruis et al. J Drug Target. 2015, 23(7-8), 759.

Figure 1 Drug accumulation (measured as disintegrations per second per mg of organ).
<figure><img src="image_102.png" /><figcaption>Figure 1.jpg</figcaption></figure>
# Hyperbranched polyglycerol-docetaxel treatments for bladder cancer and the characterization of treated bladder tissue using MALDI-MS imaging

Friday, 30th September - 16:25 - Targeted drug delivery and Nanocarriers - Nanomedicine for cancer diagnosis & therapy - Amphitheatre 25 - Oral presentation - Abstract ID: 257

Dr. David Plackett¹, Dr. Clement Mugabe², Ms. Shujun Lin³, Dr. Guobin Sun³, Dr. Nancy Ford³, Dr. Richard Liggins², Prof. Helen Burt¹

1. Faculty of Pharmaceutical Sciences, University of British Columbia, 2. The Centre for Drug Research and Development (CDRD), 3. The Centre for High-Throughput Phenogenomics (CHTP), Faculty of Dentistry, University of British Columbia

## Introduction

In North America, bladder cancer is the 4th most common cancer in men and the 9th in women and is reported to have the highest lifetime cost per patient of all cancers. Furthermore, innovations in the treatment of bladder cancer have been slow to develop and there remain significant unmet medical needs. The use of mucoadhesive nanoparticles based on amine-modified hyperbranched polyglycerols (HPGs) as vehicles for delivery of the taxane drug docetaxel (DTX), including drug uptake, efficacy, safety and pharmacokinetics, has been the subject of our recent research. In this context, the aim of the study reported here was to demonstrate that DTX could be visualized and quantified in bladder tissue by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging. The objectives of the research were to compare HPG-DTX formulations with Taxotere®, an existing commercial treatment, when applied to ex vivo pig bladder samples, which would then be sectioned and examined using MALDI-MS imaging. Results in terms of drug quantitation and penetration in the tissue could then be compared with results obtained using alternative analytical methods.

## Methods

Freshly obtained pig bladders were transferred to pH 7.4 buffer and cut into 1 x 1 cm samples. These samples, urothelium side upwards, were placed in Franz diffusion cells and the donor compartments filled with 300 µls of either an HPG-DTX formulation or a Taxotere® formulation. Following incubation at 37oC, the samples were removed from the cells and frozen before cutting into thin sections at 90o to the urothelium surface. The surfaces of these sections were then examined using MALDI-MS imaging and compared with treated and untreated controls.

## Results

The distribution and concentrations of DTX in pig bladder tissue determined using MALDI-MS imaging, as illustrated in the Figure, were comparable with results obtained previously using an established radiolabeled drug method and confirmed the higher loading of DTX in bladder tissue when using an HPG formulation.

## Conclusions

The MALDI-MS imaging method provides a convenient means of visualizing DTX in treated bladder tissue and is a potentially useful alternative to the use of radiolabeled drug or other methods for drug quantitation.
<figure><img src="image_103.png" /></figure>

Visualization and quantitation of docetaxel in an
hpg-dtx-treated pig bladder section using maldi-ms
imaging.jpg
# Albumin as a Nitric Oxide-Traffic Protein : Novel Anticancer Agent

Friday, 30th September - 16:50 - Targeted drug delivery and Nanocarriers - Nanomedicine for cancer diagnosis & therapy - Amphitheatre 25 - Oral presentation - Abstract ID: 180

Prof. MASAKI OTAGIRI¹, Dr. Yu Ishima², Prof. Toru Maruyama³

1. Faculty of Pharm Sci., Sojo University, 2. Faculty of Pharm Sci, Kumamoto University, 3. Faculty of Pharm Sci, Kumamoto University

**Objective** While Nitro Oxide (NO) has a wide range of biological functions, NO therapy still has problems that need to be overcome, such as its short half-life in vivo (0.1 s). To accomplish this, we examined human serum albumin (HSA) because it is the most abundant plasma protein and endogenous S-nitrosothiols in human plasma are largely associated with HSA. S-nitrosated HSA (SNO-HSA) is significantly more stable than low molecular weight S-nitrosothiols. It should also be noted that the cellular uptake of NO from HSA with one NO-moiety results in cytoprotective effects, whereas HSA with more SNO-groups induces apoptosis, an effect that is probably useful in cancer therapy. We introduced two type SNO-HSA, ca 7 conjugated SNO groups using chemical modification (Poly-SNO-HSA) and an S-nitrosated recombinant HSA dimer (SNO-AL-Dimer), as possible cancer therapeutic applications.

**Results and Discussion** The transfer of NO from Poly-SNO-HSA to cells was faster and more pronounced. Surprisingly, the inflow of NO results in apoptotic cell death by ROS induction and caspase-3 activation and not cytoprotection.

Interestingly, NO donors such as nitroglycerin have been reported to reverse resistant to anticancer agents. Therefore, we evaluated the effect of Poly-SNO-HSA on the resistance of human myelogunous leukemic cells (K562 cells) to doxorubicin (dx). The results showed that treatment with Poly-SNO-HSA increased its accumulation in dx-resistant K562 cells (K562/dx). Furthermore, Poly-SNO-HSA enhanced the anticancer effect of dx in K562/dx mice. Poly-SNO-HSA reversed dx resistance by decreasing the expression of P-gp and HIF-1-α.

The SNO-AL-Dimer was found to specifically deliver large amounts of cytotoxic NO to tumor tissue but not to normal organs in C26 tumor-bearing mice. In this respect, the SNO-AL-Dimer is superior to SNO-HSA and GS-NO. Interestingly, S-nitrosation improved the uptake of the HSA dimer in tumor tissue through augmenting the enhanced permeability and retention (EPR) effect. The data suggest that the SNO-AL-Dimer behaves not only as an anticancer therapeutic drug but also as a potentiator of the EPR effect. Therefore, the SNO-AL-Dimer might be a very appealing carrier for utilizing the EPR effect in cancer therapeutics.
Nanoparticles in proton and heavy ion therapy

Friday, 30th September - 17:15 - Targeted drug delivery and Nanocarriers - Nanomedicine for cancer diagnosis & therapy - Amphitheatre 25 - Oral presentation - Abstract ID: 559

Dr. erika porcel¹, Ms. Marta Bolsa¹, Ms. Daniela Salado¹, Dr. Lenka Stefancikova¹, Mr. Vladimir Ivosev¹, Prof. Sandrine Lacombe¹

1. ISMO Université Paris Sud/CNRS

Introduction :Radiotherapy, one of the main treatments in cancer, can be improved by the use of heavy atoms, as radiation enhancers. Many investigations are conducted in this area. The challenge is to increase the radiation damage on tumor whilst preserving healthy tissue by improving targeting. Recent developments in nanotechnology brought new perspectives by using nanoparticles (NP), which can be specifically functionalized.

Methods :We studied, using plasmid probes, the complex molecular damage radio-enhancement induced by NP. The effect at cellular scale was investigated by clonogenic assay. The internalisation and localisation of NP in cells was performed by complementary methods of microscopy (confocal, electronic...).

Results :We have shown, using plasmid probes, that platinum nanoparticles (PtNP) strongly enhance complex molecular damage induced as well by carbon ions [1], as by protons [2] or gamma rays [3]. This effect is not due to the nature of the incoming radiation but explained by the auto-amplification of electron cascades into the nanoparticles. Similar results were found with gadolinium based nanoparticles (GBN) which give the possibility to associate RMN imaging to radiotherapy. Furthermore, a decrease of mammalian cell survival was also observed when GBN or PtNP are associated to ions radiation [4].

Discussion :These results allow us to measure how the use of heavy nanoparticles could improve treatments by enhancing efficiency and targeting of radiations into the tumor. Further assays are in progress to understand the biological mechanisms underlying these effects [5]. New nanoparticles are considered to find the best candidate.

[1] Porcel et al. Platinum nanoparticles: a promising material for future cancer therapy? Nanotechnology 21, 85103 (2010).

[2] Schlathölter et al. Improving proton therapy by metal-containing nanoparticles: nanoscale insights. International Journal of Nanomedicine 11, 1549-1556 (2016).

[3] Porcel et al. Nano-Sensitization under gamma rays and fast ion radiation. J. Phys.: Conf. Ser. 373 (2012).

[4] Porcel et al. The use of theranostic gadolinium-based nanoprobes to improve radiotherapy performances. Nanomedicine 10 (2014).

[5] Stefancikova et al. Effect of gadolinium-based nanoparticles on nuclear DNA damage and repair in glioblastoma tumor cells. Journal of Nanobiotechnology 14:63 (2016).
Frontier biomedical research: from multi-functional
bio-interfaces to biomaterials and tissue engineering

Friday, 30th September - 16:00 - Tissue engineering and regenerative nanomedicine - Tower 24 - Room 101 - Oral presentation - Abstract ID: 503

Prof. Maria Tomoaia-Cotisel ¹

1. Babes-Bolyai University of Cluj-Napoca, Faculty of Chemistry and Chemical Engineering, 11 Arany Janos Str., 400028
Cluj-Napoca, Romania

The progress over a quarter century on understanding molecular self-assemblies of various biomolecules, like, fatty acids, lipids, proteins and drugs, and colloidal characteristics of inorganic nanoparticles (NPs), such as hydroxyapatite (HapNPs) as well as of AuNPs and AgNPs, has allowed us to develop a practical strategy for syntheses of innovative nanobiomaterials. These nanobiomaterials can be used as scaffolds in cell culture or in biomedical devices with improved anti-microbial properties. Thus, new horizons are opened up from multi-functional biointerfaces and innovative materials to tissue engineering and nanomedicine with vast biomedical applications. The structure and properties of these materials are investigated by cutting-edge experimental tools existing in our Center of Physical Chemistry, in Babes-Bolyai University platform of research (e.g., AFM, STM, fluorescence microscopy, SEM and TEM, Langmuir-Blodgett techniques for self-assembly (LBT), DSC calorimetry, and various spectroscopic techniques: UV-Vis, FTIR, RMN, RAMAN and RES.

Innovative Hap, modified with Si, Mg and Zn: Hap-Si-Mg-Zn, and functional scaffolds of these materials with collagen:COL represent the first report on the effects of Si, Mg and Zn, simultaneously present within the layered ceramic scaffolds in human osteoblasts culture. For this goal, the cellular expression of osteoblasts markers: like collagen, osteopontin and osteocalcin were visualized by fluorescence microscopy and by using immuno-cytochemical staining methods. Results indicate that combined scaffolds made of Hap/COL, Hap-Si/COL and Hap-Si-Mg-Zn/COL layers have an improved stimulating activity to osteoblasts compared with native scaffolds (e.g., made only from pure Hap), particularly in promoting the formation of mineralized bone matrix. Moreover Hap-Si-Mg-Zn/COL combined layered scaffolds substantially enhanced osteoblasts activity and adhesion, as evidenced by cell expression of collagen, osteopontin and osteocalcin as well as of F-actin stress fibers, in vitro. Thus, current study clearly demonstrated that the incorporation of Si, Mg and Zn within Hap could be an active, safe and inexpensive tool for potential clinical applications in orthopedic surgery, bone cancer therapy and nanomedicine. Acknowledgements: This research was supported by the UEFISCDI through the 171, 241 and 257 projects.
# The Sustainable Release of Vancomycin and Its Degradation Products from Collagen/Hydroxyapatite Nano/Micro Structured Layers Prepared using Different Techniques

Friday, 30th September - 16:25 - Tissue engineering and regenerative nanomedicine - Tower 24 - Room 101 - Oral presentation - Abstract ID: 83

Dr. Tomas Suchy¹, Dr. Monika Supova¹, Dr. Eva Klapkova², Dr. Vaclava Adamkova³, Dr. Jan Zavora³, Dr. Margit Zaloudkova⁴, Dr. Martin Braun¹, Dr. Rastislav Ballay⁵, Dr. Frantisek Denk¹, Dr. Jiri Rebicek⁶, Dr. Marek Pokorny⁶, Dr. Pavla Sauerova⁷, Dr. Marie Hubalek Kalbacova⁷, Dr. Lukas Horny⁸, Dr. Jan Vesely⁸, Mr. Tereza Vonavkova⁸

1. Department of Composites and Carbon Materials, Institute of Rock Structure and Mechanics, Academy of Sciences of the Czech Republic, Prague, 2. Department of Medical Chemistry and Clinical Biochemistry, Charles University, 2nd Medical School and University Hospital Motol, Prague, 3. Clinical microbiology and ATB centre, Institute of Medical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, 4. Department of Composites and Carbon Materials, Institute of Rock Structure and Mechanics, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 5. 1st Department of Orthopaedics, 1st Faculty of Medicine, Charles University in Prague and Motol University Hospital, Prague, 6. Contipro Biotech s.r.o., R&D Department, Dolni Dobrouc, 7. Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, Prague, 8. Faculty of Mechanical Engineering, Czech Technical University in Prague, Prague

## Introduction

Infections of the musculoskeletal system present a serious challenge in the field of orthopaedic and trauma medicine. The aim of the experiment was to develop a resorbable layer via the controlled elution of antibiotics to be used as a bone/implant bioactive interface particularly in the case of prosthetic joint infections or as a preventative procedure regarding primary joint replacement in a potentially infected site.

## Methods

Nano- or micro-structured layers based on collagen (type I, VUP Medical, Czech Republic) and 0, 5 or 15wt% of hydroxyapatite nanoparticles (avg. 150nm, Sigma Aldrich, Germany) were prepared employing the lyophilisation or electrospinning of dispersions with or without 10wt% vancomycin hydrochloride (Mylan S.A.S, France) and subsequently cross-linked by EDC/NHS (Sigma Aldrich, Germany). Pure cross-linked collagen/hydroxyapatite electrospun mats were subsequently impregnated with 10wt% vancomycin. The in vitro release rates of vancomycin and its inactive degradation products were characterized by HPLC. The antimicrobial effects of the layers were determined using agar diffusion testing against four different clinical isolates. The in vitro biological evaluation was conducted using SAOS-2 cells in direct contact with the layers or 24h infusions (MTS/LDH tests).

## Results

The maximum concentration of the released active form of vancomycin (700mg/l after 3hours, 150mg/l 21stday) was assessed by means of the vancomycin impregnation of cross-linked electrospun layers. The lowest concen-
tration was determined for those layers electrospun directly from a collagen solution with vancomycin. Agar diffusion tests revealed that the electrospun impregnated layers exhibited the highest activity. Modification using hydroxyapatite exerts no strong effect on vancomycin evolution. All the tested samples showed a sufficient cytocompatibility rate with no indication of cytotoxic effects.

## Discussion

The higher specific surface of nanostructured layers probably plays a negative role in the preparation process due to the higher rate of vancomycin elution to the cross-linking solution. This may be overcome via the subsequent impregnation of the cross-linked layers. Our results suggest that the local application of high-dose vancomycin via drug delivery carriers provides a safe therapeutic osteomyelitis treatment method that prevents the development of bacterial resistance.

## Acknowledgments

This study was supported by the Technology Agency of the Czech Republic (project no. TA04010330).

<figure><img src="image_104.png" /><figcaption>Iconan suchy.jpg</figcaption></figure>
# Gold Nanoshell-Assisted Wireless Activation of Myotube Contraction

Friday, 30th September - 16:50 - Tissue engineering and regenerative nanomedicine - Tower 24 - Room 101 - Oral presentation - Abstract ID: 547

**Mr. Attilio Marino**¹, **Dr. Satoshi Arai**², **Dr. Yanyan Hou**², **Prof. Madoka Suzuki**², **Prof. Gianni Ciofani**¹

1. Istituto Italiano di Tecnologia, 2. Waseda University

## INTRODUCTION

Different approaches have been developed in the recent years for muscle cell stimulation and for myotube contraction, aiming at various applications in tissue engineering, regenerative medicine, and bionics. Mild heat stimulation of muscle cells within physiological range represents an intriguing strategy for the modulation of cell functions. In this context, plasmonic properties of gold nanostructures can be exploited upon near-infrared (NIR) excitation in order to remotely heat cells, also into deep tissues, owing to the low absorption in the NIR (λ 800 nm) [1].

Although the effects of nanoparticle-assisted photo-thermal stimulation have been widely investigated in neurons and nerves, no significant studies on muscle cells can be found in the literature. In this work, for the first time, photo-thermal conversion was exploited to remotely stimulate muscle cells by using gold nanoshells (NS) in combination with NIR radiation.

## MATERIALS AND METHODS

Au@SiO2 NS in water suspension (50 µg/ml, Nano-Composix) were centrifuged and re-dispersed in the cell medium (50 µg/ml). NS were characterized with scanning electron microscopy and UV-Vis absorbance was also investigated (Hitachi F-2700).

Intracellular temperature dynamics in response to a NIR stimulation (0.25 W/cm2) were assessed both in presence and in absence of the NS (Nano-Composix, 50 µg/ml) treatment, after 5 days of differentiation, by using a temperature-sensitive fluorescent thermometer targeting endoplasmic/sarcoplasmic reticulum (ER thermo yellow). [2] Calcium imaging was performed by using Fluo-4 AM fluorescent indicator.

## RESULTS AND DISCUSSIONS

Intracellular temperature increments of about 5°C were induced by the NIR stimulation in the presence of NS. The increments of temperature were demonstrated able to efficiently induce a myotube contraction. Fluorescence Ca2+ imaging analysis demonstrated as no intracellular Ca2+ transients can be detected during the NIR + NS stimulation, so indicating that Ca2+ fluxes are not involved in the described phenomena.

Our results report, for the first time, a "wireless" activation of muscle cells mediated by light and nanoparticles, envisaging applications in tissue engineering as well as in bionics.

## REFERENCES

[1] J. Yong et al. Adv. Healthc. Mater. 2014, 3, 1862–186.

[2] S. Arai et al. Sci. Rep. 2014, 4.
# Complete 3D regeneration of functional organs by manipulation of the pluripotent stem cells

Friday, 30th September - 17:15 - Tissue engineering and regenerative nanomedicine - Tower 24 - Room 101 - Oral presentation - Abstract ID: 47

Prof. Hiroshi Kagami¹

1. Shinshu University

Complete regeneration of the functional organs have been very difficult in many biological species. We have been challenged the regeneration of the functional organs by use of the pluripotent stem cells isolated from early avian embryos. These stem cells were cultured, genetically modified and micro-injected into the other avian embryos. Completely functional legs and hearts could be regenerated and the organ shape in 3D level were almost same as that of the control organs. Moreover, the mice ES cells could be committed into avian embryos and the mice derived neuron or muscle could be regenerated in the avian embryos.

All of the experimental procedures and results of the regeneration of the stem cell-derived organs will be shown as movie.
# Evaluation of theranostic dendrimers for radiotherapy and MRI of gliomas

Friday, 30th September - 16:00 - Nanomedicine for cancer diagnosis & therapy - Tower 24 - Room 103 -
Oral presentation - Abstract ID: 466

Ms. Flonja Liko¹, Prof. Eduardo Fernandez-Megia², Prof. Francois Hindré¹

1. *Micro et Nanomédecines Biomimétiques* (MINT) UMR-S 1066, IBS-CHU, University of Angers, Angers, 2. *Center for Research in Biological Chemistry and Molecular Materials* (CiQUS), University of Santiago de Compostela, Santiago de Compostela.

**Aim:** A new oncologic strategy, based on the integration of nanovectorized radiotherapy and locoregional delivery was evaluated for the treatment of gliomas. Our focus was the synthesis of heterofunctional dendrimers to facilitate personalized management of the most common and lethal type of primary brain tumors. Gallic acid-triethylene glycol (GATG) dendrimers, fully functionalized with DTPA derivatives, were the nanovectors of choice to deliver the radiotherapeutic 188-Re and paramagnetic nuclei Gd(III), targeting the radiotherapeutic dose to the tumor site with a minimally invasive stereotactic surgery, in an F98 rat glioma model. Intravenous injection was used to further investigate the pharmacokinetics, throughout body distribution and clearance profiles of these dendrimers.

**Methods:** F98 tumor cells were implanted intracerebrally in syngeneic Fischer rats (n=15). A comparison of brain retention and tissue biodistribution between 188Re-perrhenate (n=6) and (188Re+Gd)-2[G3]-DTPA dendrimers (n=6) was done following CED (3.7 MBq/injection) at Day 20 post-tumor implantation, using an osmotic pump (0.5µL/min for 20 minutes). Brain retention of Gd-2[G3]-DTPA dendrimers in F98 glioma rats, was also evaluated by MRI, at 24h post-CED (n=3). Intravenous injection was used to mimic the behavior of dendrimers in the bloodstream, following their brain clearance. To investigate if the generation sizes of dendrimers influenced any differences in their pharmacokinetics, throughout body distribution and clearance profiles, three highest generations (2[G2]-2[G4]) of GATG dendrimers were synthesised and radiolabeled with technetium. Rats (n=36) were intravenously injected with 3.7 MBq of 99mTc-2[G2]-DTPA, 99mTc-2[G3]-DTPA, and 99mTc-2[G4]-DTPA dendrimers, and put in individual metabolic cages. They were sacrificed at 1, 6, 24 and 48 hours post-injection (n=9 per each time interval; 3 rats per generation of dendrimers). The content activity of urine, feces and each organ was determined using a gamma counter and expressed as percentage of injected dose/organ (%ID).

**Conclusion:** The use of dendrimers as nanovectors prevented the fast brain clearance of the radionuclide on its own, and prolonged the confinement of the internal radiation at the tumor site. Molecular weight and architecture of dendrimers had an important role on the in vivo behavior of these nanovectors. The easy dual labeling procedure opens the perspective of multimodality in therapy and imaging of gliomas.
<figure><img src="image_105.png" /><figcaption>Gatg dendrimers for integrated radiotherapy and mr imaging of glioblastoma.jpg</figcaption></figure>
# Targeting Hypoxia in Advanced Prostate Cancer Using Tirapazamine-Copper Nanoparticles

Friday, 30th September - 16:25 - Nanomedecine for cancer diagnosis & therapy - Tower 24 - Room 103 - Oral presentation - Abstract ID: 253

Dr. Wafa Al-Jamal¹

1. University of East Anglia/ School of Pharmacy

**Introduction:** Hypoxia is considered a hallmark of cancer and a common characteristic of locally advanced solid cancers, including prostate cancer. Tumor hypoxia plays a key role in promoting angiogenesis, metastasis, and drug resistance. Hypoxia-activated prodrugs have opened the door for specific treatment of the solid and metastatic tumors with lower side effects. Tirapazamine (TPZ) is the most advanced hypoxia-activated prodrug. TPZ has shown great specificity and potency in inhibiting tumor growth at moderate to severe hypoxic conditions. TPZ is currently in phase III clinical trials to treat cervical cancer, however its efficacy in vivo has been limited due to its poor penetration in tumor tissues. The present work aims to develop novel TPZ-copper nanoparticles to target and penetrate hypoxic prostate tumor mass, following systemic administration in vivo.

**Methods:** To improve the encapsulation of TPZ in nanoparticles, TPZ-copper complexes, Cu(TPZ)2, were prepared and characterized using different analytical techniques (HPLC, IR, UV/Vis, spectrofluorometry and MALDI-TOF). Next, a remote loading method was developed to encapsulate stable Cu(TPZ)2 complexes in different lipid-based nanoparticles. Cu(TPZ)2-nanoparticles were characterized using dynamic light scattering, spectrofluorometry and HPLC. The cytotoxicity of TPZ and Cu(TPZ)2 complexes, and Cu(TPZ)2-nanoparticles was assessed in vitro, using prostate cancer cells cultured under normoxia and hypoxia. The cytotoxicity was evaluated using resurazin cell viability assay.

**Results:** Cu(TPZ)2 complexes were more hydrophobic in nature, which led to an efficient drug loading in the lipid nanoparticles. The drug loading was dependent on the lipid composition, drug: lipid and the hydrating buffers used. Our results showed that the cytotoxicity of the Cu(TPZ)2-nanoparticles was highly selective to hypoxia. It was also dependent on the cell line, drug concentration, and the incubation time.

**Conclusions:** In this work, we showed that the complexation of TPZ with copper was an attractive approach to promote TPZ encapsulation in lipid nanoparticles. This novel nanomedicine could overcome TPZ shortcomings in vitro and in vivo, and offer a promising approach to target advanced prostate cancer in patients.

**Acknowledgements:** This work was supported by Prostate Cancer UK (Grant CDF-12-002), the Engineering and Physical Sciences Research Council (EPSRC) (EP/M008657/1), and University of East Anglia.
# Theoretical Reactivity of Cell-Surface Receptors in Presence of Nanodiamonds as Carriers

Friday, 30th September - 16:50 - Nanomedecine for cancer diagnosis & therapy - Tower 24 - Room 103 -
Oral presentation - Abstract ID: 433

Dr. Norma Flores-Holguín¹, Prof. Linda-Lucila Landeros-Martínez¹, Dr. Erasmo Orrantia-Borunda¹

1. Centro de Investigación en Materiales Avanzados, S.C.

## Introduction

The estrogen receptors (ER) have allowed offering a better prognosis to patients with breast cancer. The most common treatment is Tamoxifen (TAM), a Selective Estrogen Receptor Modulator (SERM), which can inhibit the estrogen effects in the breast cancer neoplastic cells without altering the beneficial effects it has on bone, cardiovascular and nervous system [1]. However, even though TAM has good results, it also has side effects such as blood clots and endometrial cancer.

For this reason a study of the theoretical reactivity parameters of two cell-surface receptors, arginine-glycine-aspartic acid tripeptide (RGD) that is present in the glycoprotein fibronectine and Aspargine-Glycine-Arganine tripeptide (NGR) found in the aminopeptidase, was developed trying to find their interaction with TAM drug.

## Methodology

The computational characterization of the peptides RGD and NGR, and the complex ND-TAM/RGD, ND-TAM/NGR, and ND-TAM complex was made using the Conceptual Density Functional Theory, DFT [2] with M06 functional [3] and 6-31G (d) basis set [4]. The reactivity parameters as ionization potential, electron affinity and chemical hardness were calculated. The cross reactivity was done and the frontier orbitals were obtained for all the studied systems.

## Results and Discussion

According with electronic density distribution, the ND-TAM complex inactivates the drug on its surface. This does not permit to bind it with the cell-surface receptors in healthy cells. Nevertheless, the difference in pH of cancer cells will allow the binding in the active site of the receptor. The analysis of the frontier molecular orbitals confirm the inactivation of TAM when it is carried by the nanodiamond. It can be observed in Figure 1. Also, the cross-reactivity indicated that TAM reacts with greater facility in presence of RGD than NGR.

**Keywords:** breast cancer, Tamoxifen, Estrogen Receptor, DFT

[1] F. Cammarata-Scalisi¹, M. Balza, A. Arenas de, M.M. Sotolongo, F. Stock, J.L. Valderrama-Landaeta, Revista Médica de la Extensión Portuguesa, 2 (2008)

[2]. P. Hohenberg, W. Kohn, Physical Review, 136 (1964) B864-B871.

[3] Y. Zhao, D. Truhlar, Theoretical Chemistry Accounts, 120 (2008) 215-241.

[4] V.A. Rassolov, M.A. Ratner, J.A. Pople, P.C. Redfern, L.A. Curtiss, Journal of Computational Chemistry, 22 (2001) 976-984.
<figure><img src="image_106.png" /><figcaption>Figure 1.jpg</figcaption></figure>
Photocatalytic TiO2 Nanoparticles for Tumor Therapy

Friday, 30th September - 16:00 - Nanomedecine for cancer diagnosis & therapy - Tower 24 - Room 105 -
Oral presentation - Abstract ID: 56

Ms. Susanne Koch ¹, Dr. Sofia Dembski ², Dr. Stephan Hackenberg ³, Dr. Karine Heuzé ⁴

1. University of Bordeaux / Fraunhofer ISC Würzburg, 2. Fraunhofer ISC Würzburg, 3. Department of Oto-Rhino-Laryngology, Plastic, Aesthetic and Reconstructive Head and Neck Surgery, University Clinic Würzburg, 4. University of Bordeaux

Despite advances in treatments for head and neck cancers, patient survival rates have only slightly increased over the last decades [1]. Thus, novel therapy strategies need to be developed to enhance current standard treatments like surgery, radiation or chemotherapy. Photocatalytic nanoparticles (NPs) are a serious candidate as they may combine their function as a therapeutic agent with surfaces that can be functionalized to mediate cellular uptake, carry drugs or molecules for diagnostic.

In this study, TiO2 (anatase) NPs with diameters of 10 nm were synthesized via hydrothermal treatment. The NPs and their in situ grafted organic surface groups were characterized by XRD, TEM, zeta potential, dynamic light scattering (DLS), thermogravimetric analysis, photocatalytic activity measurements and IR-spectroscopy. Stabilization of the synthesized NPs in cell culture media was examined via sedimentation studies (absorption spectroscopy, DLS). Cytotoxicity tests and tumor treatment experiments were carried out in a human tumor cell line (FaDu) and human bone marrow-derived mesenchymal stem cells with the help of the MTT colorimetric staining method.

After complete characterization of the synthesized TiO2 NPs, they were stabilized in cell culture media using a polycarboxylate ether as a novel stabilization agent. A perfect stability over at least one day and agglomerate sizes less than 100 nm were achieved [2]. Within this research the biocompatibility of non-activated NPs was demonstrated. For the treatment of tumor cells a novel concept was developed; the photocatalytic NPs were activated by UV light which modified their surface groups. Hereupon, NPs exhibited dose-dependent cytotoxic properties in FaDu while non-malignant cells were significantly less affected.

In summary, the novel NPs are attractive for the treatment of superficial mucosa malignancies of the head and neck due to their cancer specific toxicity and since they can be activated prior to cell exposure. This allows tumor therapy without UV irradiation of cells avoiding UV-induced DNA damages in non-targeted cells. Furthermore, pre-treatment UV activation allows the NP application in deeper cancer formations.

[1] R. Siegel et al. CA Cancer J. Clin. 65 (2015) 5–29.

[2] S. Koch et al. Colloids Surf. B: Biointerfaces 143 (2016) 7-14.
<figure><img src="image_107.png" /></figure>

Vitality of cancer cells and human stem cells treated
with pre-activated tio2 nanoparticles.png
# Immobilized ERK2 and GSK-3beta magnetic microparticles for targeted protein phosphorylation

Friday, 30th September - 16:25 - Nanomedecine for cancer diagnosis & therapy - Tower 24 - Room 105 -
Oral presentation - Abstract ID: 299

Dr. Marcela Slovakova¹, Ms. Lenka Hromádková², Mr. Rudolf Kupčík¹, Dr. Daniela Řípová³, Prof.

Zuzana Bílková¹

1. University of Pardubice, Faculty of chemical technology, 2. National institute of mental health, Department of Neurobiology and AD center, Klecany. Charles University in Prague, Faculty of Science, Prague, 3. National institute of mental health, Department of Neurobiology and AD center, Klecany

**Introduction:** Micro and nanoparticles with immobilized enzymes as recoverable, stable and specific catalysts are applied in a variety of technologies, in biomedical applications and widely in research. Magnetic microparticles bring the advantage of the non-contaminating and very specific and sensitive reaction on their substrates, peptides and proteins. This work is based on the use of proline-directed protein kinases: extracellular signal-regulated kinase (ERK2) and glycogen synthase kinase 3β (GSK-3β) immobilized to magnetic microparticles for targeted recombinant tau 1-441 phosphorylation.

**Methods:** In order to obtain highly efficient carrier for the targeted phosphorylation of peptides/proteins, two kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, and metal cations Ni2+ or Co3+ functionalities. Relevant methods of covalent immobilization, non-oriented and oriented, were chosen and reaction conditions were optimized. Phosphorylation of low molecular peptides and operational and storage stabilities of kinase-superparamagnetic particles were performed. Soluble and immobilized ERK2 and GSK-3β were applied for recombinant tau 1-441 phosphorylation. Tryptic phosphopeptides enrichment was performed by ion-metal affinity chromatography using TiO2 magnetic nanoparticles. The level and the position of phosphorylation sites were identified by using MALDI-LTQ-Orbitrap MS. Western blot was carried out to confirm phosphorylation of tau 1–441 (ENZO and rPeptide) by soluble or immobilized ERK2 and GSK-3β kinases using specific anti-tau and anti-phospho-tau antibodies.

**Results:** The effect of immobilization was confirmed due to the enzymes ability to phosphorylate low molecular synthetic peptides. The ERK2 and GSK-3β kinases immobilized on SeraMag beads using the carbodiimide chemistry proved the ability to phosphorylate peptides in 10 cycles without significant loss in activity. Using carriers with immobilized ERK2 or GSK-3β we performed the efficient phosphorylation of model recombinant protein tau 1-441. Presence and position of phosphorylation along the peptide chains were detected by MALDI MS and confirmed also by Western blot with anti-tau phospho-specific antibodies.

**Discussion:** Present results document very well the ability of ERK2 and GSK-3β superparamagnetic particles to phosphorylate the target peptides and recombinant tau 1-441 with desired efficiency and specificity. The process of phosphorylation can be better controlled; subsequent purification of phosphorylated tau can be omitted.

**Acknowledgement:** EU project NADINE No. 246513
Evaluation of magnetic nanoparticles coated by Taxol
imprinted polymer for controlled drug delivery in mouse
breast cancer model

Friday, 30th September - 16:50 - Nanomedecine for cancer diagnosis & therapy - Tower 24 - Room 105 -
Oral presentation - Abstract ID: 468

Prof. Hamid Hashemi-Moghaddam ¹, Mrs. Elnaz Mirsaeed-Gazi ¹, Prof. Saeed Zavareh ²

1. Damghan Branch, Islamic Azad University, 2. Damghan University

Nanoparticles (NPs) have been extensively investigated to improve delivery efficiency of therapeutic and di-
agnostic agents. In this study, magnetic molecularly imprinted polymer (MIP) was synthesized by using poly
DOPA. Synthesized MIP was used for controlled Taxol delivery in a spontaneous model of breast adenocarci-
noma in Balb/c mice in the presence of an external magnetic field. Antitumor effectiveness of Taxol imprinted
polymer (Taxol-IP) was evaluated in terms of tumor-growth delay, tumor-doubling time, inhibition ratio, and
histopathology. Results showed higher efficacy of Taxol-IP in the presence of magnetic field upon suppressing
tumor growth than free Taxol and Taxol-IP without magnetic field. The Taxol and Fe distribution among tis-
sues were evaluated by high-performance liquid chromatography and flame atomic absorption spectrometry,
respectively. The obtained results, showed significantly deposition of Taxol in the Taxol-IP treated group with
magnetic field. Thus, magnetic Taxol-IP is promising for breast cancer therapy with high efficacy.
# Enhanced antifungal activity of itraconazole by the supercritical antisolvent technique

Friday, 30th September - 16:00 - Targeted drug delivery and Nanocarriers - Tower 24 - Room 107 - Oral presentation - Abstract ID: 304

Dr. Jayvadan Patel ¹

1. Faculty of Pharmacy, Sankalchand Patel University, Visnagar, Gujarat, India

Itraconazole is broad spectrum antifungal drug that have problems of poor solubility and bioavailability of conventional dosage forms. Attempts to overcome the solubility problems are solubilisation with mixed micelles or forming a complex using cyclodextrins but these approaches are of limited success. One tried to improve the in vivo performances of poorly soluble drug by reducing the particles size using supercritical antisolvent technique (SAA). Micronization of itraconazole dissolved in acetone, dimethyl sulfoxide and ethanol with supercritical carbon dioxide as antisolvent was successfully performed using a supercritical antisolvent technique. The effect of a few process parameters such as precipitation temperature, the pressure and solute concentration in the liquid solution has been studied to evaluate their influence on morphology and size of particles. The micronised itraconazole were evaluated for drug content, particle size analysis and in vitro dissolution profiles. Fourier transform infrared spectroscopy, differential scanning calorimetry and PXRD patterns was used to study the possible changes after micronization of itraconazole. The dissolution rate was increased after micronized compared with pure itraconazole in distilled water, pH 1.2 buffer and pH 7.0 buffer. Micronized and pure itraconazole were evaluated for their therapeutic efficacy in the treatment of experimental oral candidiasis induced by Candida albicans in immunosuppressed rats. This antifungal activity was analyzed by microbiological and histopathological techniques. Microbiologically, micronized itraconazole significantly (p<0.05) reduced the number of colony forming units (CFU) sampled from the oral cavity of rats treated for eight consecutive days, compared to untreated control rats. Histologically, the untreated control animals showed numerous hyphae on the epithelium of the dorsal surface of the tongue. In contrast no hyphal colonization of the epithelium was seen in micronized itraconazole-treated animals indicating improved bioavailability of micronised itraconazole as compared with pure itraconazole. In conclusion, SAA process could be a useful method for the micronization of itraconazole and its solubility, dissolution rate and antifungal activities were significantly increased by micronization. Improve the in vivo performances of poorly soluble drug by reducing the particles size using supercritical antisolvent technique (SAA) is a novel approach.
# WORKSHOP: Synthesis of amphiphilic hyaluronan containing-phenyl fatty acids for the preparation of polymeric micelles for applications in drug delivery

Friday, 30th September - 16:25 - Targeted drug delivery and Nanocarriers - Tower 24 - Room 107 - Workshop presentation - Abstract ID: 68

**Dr. Gloria Huerta-Angeles**¹, **Ms. Alena Matelova**², **Mrs. Zdislava Brunová**¹, **Dr. Daniela Šmejkalová**¹, **Prof. Vladímir Velebný**¹

1. CONTIPRO, 2. Department of Chemistry, Faculty of Technology, Tomas Bata University in Zlín

## Introduction

Polymeric micelles (PM) are particularly interesting for drug delivery applications because of their characteristic features e.g., size, surface, solubility under physiological conditions and enhanced permeability, which help them to penetrate and be taken up by cells. Hyaluronan (HA) is a natural polysaccharide, ubiquitous in the human body, therefore, it is suitable for medical applications. Moreover, HA allows specific targeting.

## Methods

In this work, novel amphiphilic hyaluronan (HA) based polymeric micelles were prepared in base of HA grafted with omega-phenylalkanoic acids (ω-PAA), including 4-phenylbutyric, 6-phenylhexanoic, 8-phenyloctanoic or 11-tolylundecanoic acids, which are aromatic fatty acids normally occurring in fats of natural origin. The prepared derivatives were encapsulated of aromatic hydrophobic drugs in order to evaluate its loading capacity (ie. trans-resveratrol, retinyl palmitate and quercetine).

## Results

The synthesis of HA-grafted with ω-PAA (HA-g-PA) was mediated by mixed anhydrides¹ that allows HA modification without degradation or formation of toxic subproducts. The reactivity of ω-PAA towards esterification has decreased with the increasing length of the aliphatic spacer between the aromatic ring and carboxylic moiety. The novel HA derivatives were found to be not cytotoxic and self-assemble from very low CMC. Furthermore, Polymeric micelles (PM) were characterized by small size (cca 30 nm, PDI; 0.2).

## Discussion

Comparing to our previously reported work², the drug loading capacity of HA-g-PA. The drug loading content increased substantially likely because of pi-pi interactions between the micelle core and loaded aromatic hydrophobic drug. The formation of well-defined hydrophobic nanodomains can be reached by defining the degree of modification of HA. This work describes a facile strategy to achieve well defined HA derivatives possessing an effective drug loading content, which is a critical factor for designing polymeric micelles³.

## References

1. Huerta-Angeles, G., et al., Carbohydrate Polymers, 2014, 111, 883-891.

2. Šmejkalová, D., et al., Biomacromolecules, 2014, 15(11), 4012-4020.

3. Liang, Y et al., Biomaterials, 2015, 71, 1-10.
# Effects of nanoparticles on gastrointestinal disorders and therapy

Friday, 30th September - 18:15 - Video Presentations - premc.org - Video presentation - Abstract ID: 217

Ms. Rabia Riasat¹, Prof. Nie Guangjun¹, Ms. Zertashia Tariq²

1. CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190, China., 2. Department of Biological Sciences, Islamia University, Bahawalpur, Pakistan.

Gastrointestinal (GI) diseases are the diseases that affect any part of the gastrointestinal tract including acute, chronic, recurrent or functional disorders. There are a numbers of factors affecting the biology of GI tract. Nanoparticles are one of them in causing diseases. As there are many conventional therapeutic strategies for the treatment of GI diseases but these are not very efficient. Nanotechnology is the emerging and rapidly evolving field of the current era with new hopes in the field of nano-medicine for the detection, prevention and the treatment of diseases. Chemotherapeutic drug delivery in the field of nanotechnology has gained much attention and focus recently. Nano materials have wider range of potential applications for the detection and treatment of diseases while toxicological effects cannot be neglected and safe and non-toxic nano drugs should be considered for the treatment of pathological and physiological gastrointestinal diseases to reduce the existing conventional treatments. The parameters such as shape, size, surface chemistry and geometry of nanoparticles are important to consider in the designing of nano carrier. The review aims at integrating toxicological effects of nano materials and their safe and effective role in the treatment of GI disorders. Although there are disorders caused by nanoparticles but counteracting as well in a safe and targeted delivery of the conventional drugs into the GI system.

<figure><img src="image_108.png" /><figcaption>Img1.jpg</figcaption></figure>
# Synthesis and characterization of biocompatible, non-toxic dopamine coated novel flower shapes core (Fe)/ porous hollow shell (Fe3O4) super paramagnetic Fe/Fe3O4 nanoparticles

Friday, 30th September - 18:30 - Video Presentations - premc.org - Video presentation - Abstract ID: 263

Ms. Rabia Riasat¹, Prof. Nie Guangjun¹, Ms. Zertashia Tariq², Ms. Muntaha Sakeena³

1. CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing, 100190, China., 2. Department of Biological Sciences, Islamia University, Bahawalpur, Pakistan., 3. EME, NUST, Islamabad, Pakistan

Superparamagnetic flower shaped Fe/Fe3O4 nanoparticles with iron core and hollow and porous shell were synthesized by thermal decomposition of iron penta carbonyl. Controlled oxidation of these nanoparticles was done by acid etching. Fe/Fe3O4 core shell iron oxide nanoparticles were synthesizing first as the template material at annealing temperature of 120°C and solution based conversion of Fe/PHFe3O4 nanoparticles was done through two way annealing progression of heating time, first at 100°C then at 250°C with constant heating rate of 5°C/min. Phase confirmation of these nanoparticles were done by X-ray diffraction (XRD) and morphological structures were analysed by transmission electron microscopy (TEM) and higher resolution transmission electron microscopy (HRTEM) and showed that nanostructures were approximately in 16 nm to 20 nm in size. While the opening pores in the shell were 1-3nm in size and the hollow cavity between iron core and shell is about 2nm hollow and porous Fe/Fe3O4 nanoparticles were surface functionalized by dopamine polymer. The dopamine coating was confirmed by Fourier transform infrared (FTIR) spectra. Nanoparticles showed magnetic saturation of 38 emu/g analysed by vibrating sample magnetometer (VSM) and showing the magnetic property as well after coating with dopamine. The as prepared nanoparticles are hydrophilic to aqueous media and are not toxic to cells (Caco-2 and Hep G2). These nanoparticles can be exploited for a number of applications including target definite therapeutic applications by encapsulating the required drug according to disease profile and can be used a nanomedicine for the iron deficiency control as by possessing large surface area and nano-sized particle profile.

Key words

Core shell nanoparticles, SPIONS, super paramagnetic iron oxide nanoparticles, dopamine
<figure><img src="image_109.png" /><figcaption>Rabia paper figure.png</figcaption></figure>
# Competitive intelligence study of a non-viral vector for gene therapy of Parkinson's disease
(Cinvestav-PX-001)

Friday, 30th September - 18:45 - Video Presentations - premc.org - Video presentation - Abstract ID: 439

Dr. AMERICA PADILLA¹, Dr. Daniel Martínez-Fong¹, Dr. Victor Manuel Téllez-López¹, Dr. Armando De Jesús Espadas-Álvarez¹

1. Centro de Investigación y de Estudios Avanzados del IPN

## Introduction

Cinvestav-PX-001 is a nanoparticle-based gene delivery system capable of targeting therapeutic genes to dopaminergic neurons in the brain and therefore induces a site-directed therapy for Parkinson's disease (PD). Experiments in rats with PD-like lesions suggest that this treatment helps to restore dopaminergic function and improve motor symptoms. Cinvestav-PX-001 may have significant commercial potential. This new treatment is safer compared to more commonly used viral vectors for therapeutic indication of PD, because they can induce adverse side effects. Competitive advantages include: high neuronal specificity in addressing the long-term transgene expression, safety and a simple, reproducible and cost-effective synthesis. The aim of this study was to obtain the competitive outlook of the available technologies related with the use of a non-viral vector for gene therapy of PD and thus provide support for strategic decisions in our organization based on the position of intellectual property.

## Methods

A prior art analysis based on patents and scientific documents was performed to identify potential competing patents. We applied analysis tools for designing commercialization strategy and these include: a) technological and competitive intelligence, b) market research (size, segments, trends, drivers and growth potential) and c) regulatory environment, in which the technology competes. These aspects were analyzed in order to improve the timeliness and quality of inputs to the decision process.

## Results and Discussion

The invention is directed to the market of supplies for gene therapy, the projected growth for the market is up to 2.8 trillion on 2024, and the main leading companies are located in United States, Japan, United Kingdom, France, Germany, Italy and Spain. As a result of this analysis, the commercialization strategy for Cinvestav-PX-001 considers provide a non-exclusive license to an international licensee in USA and/or Europe with the experience and ability to complete its commercial development (clinical phase and regulatory affairs) and the creation of a spinoff company from Cinvestav with innovative vision in new therapeutic applications of Cinvestav-PX-001. Project supported by Fund of Innovation (Finnova 224222), Ministry of Economy and Conacyt Mexico. We appreciate the support of ISIS Enterprise and Newton Fund in IP analysis.
<figure><img src="image_110.png" /><figcaption>Diapositiva1.jpg</figcaption></figure>
Authors Index

<table><tr><td>Abakumov, M.</td><td>104</td></tr><tr><td>Abbasi, R.</td><td>121</td></tr><tr><td>Abbaspour, H.</td><td>36</td></tr><tr><td>Acarregui, A.</td><td>118</td></tr><tr><td>Adamkova, V.</td><td>229</td></tr><tr><td>Agarwal, M.</td><td>16</td></tr><tr><td>Agrawal, P.</td><td>102</td></tr><tr><td>Aguilera Peral, U.</td><td>211</td></tr><tr><td>Ahmad, N.</td><td>121</td></tr><tr><td>Aid Launais, R.</td><td>73, 77</td></tr><tr><td>Ainsa, J.</td><td>116</td></tr><tr><td>Akhtar, A.</td><td>21</td></tr><tr><td>Al Jamal, W.</td><td>235</td></tr><tr><td>Albarran, L.</td><td>163</td></tr><tr><td>Albeniz, I.</td><td>106</td></tr><tr><td>Alejo, T.</td><td>70</td></tr><tr><td>Alfaro Viquez, E.</td><td>98</td></tr><tr><td>Alfaro, S.</td><td>116</td></tr><tr><td>Ali, Z.</td><td>121</td></tr><tr><td>Alizadeh, E.</td><td>179</td></tr><tr><td>Alnasser, F.</td><td>148</td></tr><tr><td>Amenitsch, H.</td><td>136</td></tr><tr><td>Ammar, S.</td><td>66</td></tr><tr><td>Anand, B.</td><td>13</td></tr><tr><td>Ando, H.</td><td>196</td></tr><tr><td>Andreu, V.</td><td>110, 116</td></tr><tr><td>Antoccia, A.</td><td>68</td></tr><tr><td>Anyfantakis, M.</td><td>39</td></tr><tr><td>Arai, S.</td><td>231</td></tr><tr><td>Ariizumi, S.</td><td>111, 196</td></tr><tr><td>Armengaud, J.</td><td>7</td></tr><tr><td>Arratia Perez, R.</td><td>207</td></tr><tr><td>Arriortua Llarena, O.</td><td>158</td></tr><tr><td>Arruebo, M.</td><td>70, 110, 116, 190, 194</td></tr><tr><td>Arshad, J.</td><td>121</td></tr><tr><td>Arteaga, P.</td><td>163</td></tr><tr><td>Asai, T.</td><td>31, 111, 196</td></tr><tr><td>Ashrafi Parchin, R.</td><td>179</td></tr><tr><td>Aslani, M.</td><td>218</td></tr><tr><td>Atif, M.</td><td>121</td></tr><tr><td>Attia, N.</td><td>127</td></tr><tr><td>Aude, J.</td><td>128</td></tr></table>

<table><tr><td>Avvakumova, S.</td><td>55</td></tr><tr><td>Ayadi, I.</td><td>168</td></tr><tr><td>Baigl, D.</td><td>39</td></tr><tr><td>Baik, H.</td><td>131</td></tr><tr><td>Bakar Bin Abdul Majeed, A.</td><td>17</td></tr><tr><td>Bakker, M.</td><td>86, 125</td></tr><tr><td>Balasubramanian, V.</td><td>161</td></tr><tr><td>Baldassarre, A.</td><td>28</td></tr><tr><td>Baleizão, C.</td><td>216</td></tr><tr><td>Ballay, R.</td><td>229</td></tr><tr><td>Banerjee, A.</td><td>84</td></tr><tr><td>Banerjee, E.</td><td>219</td></tr><tr><td>Bansal, A.</td><td>138</td></tr><tr><td>Baraket, A.</td><td>34, 63</td></tr><tr><td>Barcelos De Paula, L.</td><td>103</td></tr><tr><td>Barea, M.</td><td>198</td></tr><tr><td>Barthelemy, p.</td><td>175</td></tr><tr><td>Battocchio, C.</td><td>68, 94</td></tr><tr><td>Bausells, J.</td><td>34, 63</td></tr><tr><td>Baz, Z.</td><td>156</td></tr><tr><td>Behan, K.</td><td>144</td></tr><tr><td>Belkahla, H.</td><td>66</td></tr><tr><td>Bell, C.</td><td>21</td></tr><tr><td>Bellini, M.</td><td>56</td></tr><tr><td>Bellucci, S.</td><td>117</td></tr><tr><td>Benezra, M.</td><td>154</td></tr><tr><td>Berger, A.</td><td>73</td></tr><tr><td>Bergonzo, P.</td><td>14</td></tr><tr><td>Berrahal, Y.</td><td>59</td></tr><tr><td>Berraondo, P.</td><td>141</td></tr><tr><td>Bhandari, A.</td><td>138</td></tr><tr><td>Bjerkvig, R.</td><td>25</td></tr><tr><td>Bogusz, K.</td><td>149</td></tr><tr><td>Bolsa, M.</td><td>227</td></tr><tr><td>Bordi, F.</td><td>28</td></tr><tr><td>Borrego Dorado, I.</td><td>12, 23</td></tr><tr><td>Bortot, B.</td><td>204</td></tr><tr><td>Bose, B.</td><td>16</td></tr><tr><td>Bosedasgupta, S.</td><td>134</td></tr><tr><td>Bott, R.</td><td>198</td></tr><tr><td>Boudon, J.</td><td>142, 171</td></tr></table>
<table>
  <tr>
    <td>Boukherroub, R.</td>
    <td>3</td>
    <td>Corde, S.</td>
    <td>149</td>
  </tr>
  <tr>
    <td>Boulard, Y.</td>
    <td>128</td>
    <td>Corne, G.</td>
    <td>25</td>
  </tr>
  <tr>
    <td>Bradbury, M.</td>
    <td>154</td>
    <td>Correia, A.</td>
    <td>161</td>
  </tr>
  <tr>
    <td>Braun, M.</td>
    <td>229</td>
    <td>Corsi, F.</td>
    <td>56</td>
  </tr>
  <tr>
    <td>Brito, M.</td>
    <td>42</td>
    <td>Coulter, T.</td>
    <td>211</td>
  </tr>
  <tr>
    <td>Bros, M.</td>
    <td>30</td>
    <td>Couvreur, P.</td>
    <td>222</td>
  </tr>
  <tr>
    <td>Brougham, D.</td>
    <td>144</td>
    <td>Crankin, E.</td>
    <td>81</td>
  </tr>
  <tr>
    <td>Brunová, Z.</td>
    <td>243</td>
    <td>Crespo, H.</td>
    <td>42</td>
  </tr>
  <tr>
    <td>Brust, M.</td>
    <td>51</td>
    <td>Crucho, C.</td>
    <td>216</td>
  </tr>
  <tr>
    <td>Bräuchle, C.</td>
    <td>143</td>
    <td>Créhange, G.</td>
    <td>142</td>
  </tr>
  <tr>
    <td>Budak, H.</td>
    <td>61</td>
    <td>Cussac, D.</td>
    <td>173</td>
  </tr>
  <tr>
    <td>Burke, P.</td>
    <td>166</td>
    <td>Czuba, E.</td>
    <td>81</td>
  </tr>
  <tr>
    <td>Burt, H.</td>
    <td>224</td>
    <td>Daman, Z.</td>
    <td>108</td>
  </tr>
  <tr>
    <td>Bysell, H.</td>
    <td>146</td>
    <td>Dankers, P.</td>
    <td>86, 125</td>
  </tr>
  <tr>
    <td>Bílková, Z.</td>
    <td>240</td>
    <td>Das, S.</td>
    <td>79, 84</td>
  </tr>
  <tr>
    <td>Cabeza, L.</td>
    <td>96, 100</td>
    <td>Davis, T.</td>
    <td>81</td>
  </tr>
  <tr>
    <td>Caminade, A.</td>
    <td>90</td>
    <td>Dawson, K.</td>
    <td>88, 148, 153</td>
  </tr>
  <tr>
    <td>Caporali, A.</td>
    <td>117</td>
    <td>De Azevedo, C.</td>
    <td>62</td>
  </tr>
  <tr>
    <td>Cardillo, D.</td>
    <td>149</td>
    <td>De Luca, E.</td>
    <td>136</td>
  </tr>
  <tr>
    <td>Carlini, L.</td>
    <td>94</td>
    <td>De Martino, E.</td>
    <td>204</td>
  </tr>
  <tr>
    <td>Carregal, S.</td>
    <td>51</td>
    <td>De Rose, R.</td>
    <td>81</td>
  </tr>
  <tr>
    <td>Carrington, S.</td>
    <td>198</td>
    <td>Dembski, S.</td>
    <td>238</td>
  </tr>
  <tr>
    <td>Carvalho, A.</td>
    <td>35</td>
    <td>Denk, F.</td>
    <td>229</td>
  </tr>
  <tr>
    <td>Casares, N.</td>
    <td>141</td>
    <td>Devineau, S.</td>
    <td>39</td>
  </tr>
  <tr>
    <td>Catelani, T.</td>
    <td>136</td>
    <td>Devoisselle, J.</td>
    <td>7</td>
  </tr>
  <tr>
    <td>Cayero Otero, M.</td>
    <td>23</td>
    <td>Dewa, T.</td>
    <td>31, 196</td>
  </tr>
  <tr>
    <td>Cayero, M.</td>
    <td>12</td>
    <td>Dias, A.</td>
    <td>144</td>
  </tr>
  <tr>
    <td>Celichowski, G.</td>
    <td>188</td>
    <td>Dinc, B.</td>
    <td>106</td>
  </tr>
  <tr>
    <td>Celikok, Y.</td>
    <td>106</td>
    <td>Ding, Y.</td>
    <td>211</td>
  </tr>
  <tr>
    <td>Celluzzi, A.</td>
    <td>28, 117, 120</td>
    <td>Discher, d.</td>
    <td>92</td>
  </tr>
  <tr>
    <td>Chaibi, S.</td>
    <td>63</td>
    <td>Domeradzka Gajda, K.</td>
    <td>188</td>
  </tr>
  <tr>
    <td>Charnay, C.</td>
    <td>7</td>
    <td>Dosumu, A.</td>
    <td>75</td>
  </tr>
  <tr>
    <td>Chatterji, U.</td>
    <td>214</td>
    <td>Dubey, K.</td>
    <td>13</td>
  </tr>
  <tr>
    <td>Chattopadhyay, D.</td>
    <td>84</td>
    <td>Echevarria Uraga, J.</td>
    <td>158</td>
  </tr>
  <tr>
    <td>Chauvierre, C.</td>
    <td>73, 77</td>
    <td>Eglite, M.</td>
    <td>213</td>
  </tr>
  <tr>
    <td>Chen, L.</td>
    <td>81</td>
    <td>El Hammadi, M.</td>
    <td>100</td>
  </tr>
  <tr>
    <td>Cho, J.</td>
    <td>27, 115</td>
    <td>Elizondo, H.</td>
    <td>194</td>
  </tr>
  <tr>
    <td>Chopineau, J.</td>
    <td>7</td>
    <td>Emami, J.</td>
    <td>10</td>
  </tr>
  <tr>
    <td>Chorilli, M.</td>
    <td>43, 48, 132</td>
    <td>Erfani Moghadam, V.</td>
    <td>221</td>
  </tr>
  <tr>
    <td>Churyukina, K.</td>
    <td>187</td>
    <td>Errachid, A.</td>
    <td>63</td>
  </tr>
  <tr>
    <td>Cieślak, M.</td>
    <td>188</td>
    <td>Errachid, A.</td>
    <td>34</td>
  </tr>
  <tr>
    <td>Ciofani, G.</td>
    <td>231</td>
    <td>Espadas Álvarez, A.</td>
    <td>247</td>
  </tr>
  <tr>
    <td>Ciriza, J.</td>
    <td>118</td>
    <td>Espinosa García, C.</td>
    <td>211</td>
  </tr>
  <tr>
    <td>Colombo, M.</td>
    <td>55</td>
    <td>Esquivel Alfaro, M.</td>
    <td>5</td>
  </tr>
  <tr>
    <td>Comparetti, E.</td>
    <td>180</td>
    <td>Farinha, J.</td>
    <td>216</td>
  </tr>
  <tr>
    <td>Contreras Caceres, R.</td>
    <td>96</td>
    <td>Fasolato, C.</td>
    <td>94</td>
  </tr>
  <tr>
    <td>Contreras Sandoval, A.</td>
    <td>141</td>
    <td></td>
    <td></td>
  </tr>
</table>
<table>
  <tr>
    <td>Fattal, E.</td>
    <td>177</td>
    <td>Guner, S.</td>
    <td>106</td>
  </tr>
  <tr>
    <td>Fernandez Megia, E.</td>
    <td>233</td>
    <td>Gurruchaga, H.</td>
    <td>118</td>
  </tr>
  <tr>
    <td>Fernandez, E.</td>
    <td>127</td>
    <td>Habchi, M.</td>
    <td>14</td>
  </tr>
  <tr>
    <td>Ferro Flores, G.</td>
    <td>209</td>
    <td>Hackenberg, S.</td>
    <td>238</td>
  </tr>
  <tr>
    <td>Fiandra, L.</td>
    <td>56</td>
    <td>Hadjersi, t.</td>
    <td>63</td>
  </tr>
  <tr>
    <td>Figueiras, E.</td>
    <td>42</td>
    <td>Hadoke, P.</td>
    <td>117</td>
  </tr>
  <tr>
    <td>Flores Holguin, N.</td>
    <td>236</td>
    <td>Haghighipak, z.</td>
    <td>36</td>
  </tr>
  <tr>
    <td>Fontana, F.</td>
    <td>71, 161</td>
    <td>Halder, A.</td>
    <td>79, 84, 214, 219</td>
  </tr>
  <tr>
    <td>Ford, N.</td>
    <td>224</td>
    <td>Hale, S.</td>
    <td>211</td>
  </tr>
  <tr>
    <td>Fracassi, A.</td>
    <td>68</td>
    <td>Han, S.</td>
    <td>131</td>
  </tr>
  <tr>
    <td>Fratoddi, I.</td>
    <td>68, 94</td>
    <td>Hart, L.</td>
    <td>19</td>
  </tr>
  <tr>
    <td>Fritsche, E.</td>
    <td>132</td>
    <td>Hasanzadeh, F.</td>
    <td>10</td>
  </tr>
  <tr>
    <td>Gaillard, J.</td>
    <td>7</td>
    <td>Hashemi Moghaddam, H.</td>
    <td>241</td>
  </tr>
  <tr>
    <td>Gamez Herrera, E.</td>
    <td>190</td>
    <td>Hashimoto, M.</td>
    <td>196</td>
  </tr>
  <tr>
    <td>Garanina, A.</td>
    <td>182, 206</td>
    <td>Hausmann, C.</td>
    <td>132</td>
  </tr>
  <tr>
    <td>Garayo Urabayen, E.</td>
    <td>158</td>
    <td>Heise, A.</td>
    <td>144</td>
  </tr>
  <tr>
    <td>Garcia Martinez, J.</td>
    <td>158</td>
    <td>Hemadi, M.</td>
    <td>66</td>
  </tr>
  <tr>
    <td>García Alonso Montoya, I.</td>
    <td>158</td>
    <td>Henriksen Lacey, M.</td>
    <td>51</td>
  </tr>
  <tr>
    <td>García Hevia, L.</td>
    <td>64</td>
    <td>Herlem, G.</td>
    <td>66</td>
  </tr>
  <tr>
    <td>García Salinas, S.</td>
    <td>194</td>
    <td>Hernández, R.</td>
    <td>118</td>
  </tr>
  <tr>
    <td>Garrido, M.</td>
    <td>141</td>
    <td>Herrero De La Parte, B.</td>
    <td>158</td>
  </tr>
  <tr>
    <td>Garry, D.</td>
    <td>148</td>
    <td>Heuzé, K.</td>
    <td>238</td>
  </tr>
  <tr>
    <td>Geraldo, D.</td>
    <td>207</td>
    <td>Hindré, F.</td>
    <td>233</td>
  </tr>
  <tr>
    <td>Ghali, L.</td>
    <td>21</td>
    <td>Hirn, S.</td>
    <td>143</td>
  </tr>
  <tr>
    <td>Ghanbari, M.</td>
    <td>179</td>
    <td>Hirvonen, J.</td>
    <td>71, 161</td>
  </tr>
  <tr>
    <td>Gharbi, T.</td>
    <td>66</td>
    <td>Hobernik, D.</td>
    <td>30</td>
  </tr>
  <tr>
    <td>Ghosh, N.</td>
    <td>219</td>
    <td>Hodges, N.</td>
    <td>75</td>
  </tr>
  <tr>
    <td>Ghosh, S.</td>
    <td>214, 219</td>
    <td>Hoffmann, C.</td>
    <td>25</td>
  </tr>
  <tr>
    <td>Gibbens Bandala, B.</td>
    <td>209</td>
    <td>Hong, K.</td>
    <td>27, 29, 112, 115</td>
  </tr>
  <tr>
    <td>Gimenez Marques, M.</td>
    <td>222</td>
    <td>Horcajada, P.</td>
    <td>222</td>
  </tr>
  <tr>
    <td>Glass, J.</td>
    <td>81</td>
    <td>Horny, L.</td>
    <td>229</td>
  </tr>
  <tr>
    <td>González Lavado, E.</td>
    <td>64, 202</td>
    <td>Hoshino, Y.</td>
    <td>111</td>
  </tr>
  <tr>
    <td>González, F.</td>
    <td>64, 202</td>
    <td>Hossain, M.</td>
    <td>149</td>
  </tr>
  <tr>
    <td>González, J.</td>
    <td>64</td>
    <td>Hou, Y.</td>
    <td>231</td>
  </tr>
  <tr>
    <td>González, M.</td>
    <td>43, 48</td>
    <td>Hristov, D.</td>
    <td>148</td>
  </tr>
  <tr>
    <td>Govender, T.</td>
    <td>185</td>
    <td>Hromádková, L.</td>
    <td>240</td>
  </tr>
  <tr>
    <td>Grabbe, S.</td>
    <td>30</td>
    <td>Huang, L.</td>
    <td>108</td>
  </tr>
  <tr>
    <td>Gracia, B.</td>
    <td>116</td>
    <td>Hubalek Kalbacova, M.</td>
    <td>229</td>
  </tr>
  <tr>
    <td>Gref, R.</td>
    <td>222</td>
    <td>Huerta Angeles, G.</td>
    <td>243</td>
  </tr>
  <tr>
    <td>Grigorakaki, C.</td>
    <td>25</td>
    <td>Hussain, B.</td>
    <td>61</td>
  </tr>
  <tr>
    <td>Grillaud, M.</td>
    <td>86, 125</td>
    <td>Hussien, s.</td>
    <td>37</td>
  </tr>
  <tr>
    <td>Grobelny, J.</td>
    <td>188</td>
    <td>Iglesias Jerez, R.</td>
    <td>12, 23</td>
  </tr>
  <tr>
    <td>Groo, A.</td>
    <td>146</td>
    <td>Ikeda, K.</td>
    <td>93</td>
  </tr>
  <tr>
    <td>Guangjun, N.</td>
    <td>244, 245</td>
    <td>Imada, R.</td>
    <td>93</td>
  </tr>
  <tr>
    <td>Guari, Y.</td>
    <td>7</td>
    <td>Immler, R.</td>
    <td>143</td>
  </tr>
  <tr>
    <td>Guerreiro, P.</td>
    <td>42</td>
    <td>Indriksons, A.</td>
    <td>213</td>
  </tr>
  <tr>
    <td>Gulyás, B.</td>
    <td>82</td>
    <td></td>
    <td></td>
  </tr>
</table>
<table>
  <tbody>
    <tr>
      <td>Irusta, S.</td>
      <td>70, 190, 194</td>
      <td>Kozajda, A.</td>
      <td>188</td>
    </tr>
    <tr>
      <td>Ishima, Y.</td>
      <td>226</td>
      <td>Krishnamoorthy, S.</td>
      <td>25</td>
    </tr>
    <tr>
      <td>Ishizaka, Y.</td>
      <td>160</td>
      <td>Krombach, F.</td>
      <td>143</td>
    </tr>
    <tr>
      <td>Islam, M.</td>
      <td>149</td>
      <td>Krueger, C.</td>
      <td>98</td>
    </tr>
    <tr>
      <td>Ito, A.</td>
      <td>93</td>
      <td>Kupčík, R.</td>
      <td>240</td>
    </tr>
    <tr>
      <td>Iturrioz, N.</td>
      <td>202</td>
      <td>Kurt, H.</td>
      <td>61</td>
    </tr>
    <tr>
      <td>Ivosev, V.</td>
      <td>227</td>
      <td>Kuznetsov, V.</td>
      <td>107</td>
    </tr>
    <tr>
      <td></td>
      <td></td>
      <td>Kwela, J.</td>
      <td>46</td>
    </tr>
    <tr>
      <td>Jafari Dehkordi, A.</td>
      <td>218</td>
      <td>Kwon, I.</td>
      <td>1</td>
    </tr>
    <tr>
      <td>Jankevics Jones, H.</td>
      <td>198</td>
      <td>Kyzioł, A.</td>
      <td>110</td>
    </tr>
    <tr>
      <td>Jeraldo, E.</td>
      <td>207</td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Jiménez López, J.</td>
      <td>96, 100</td>
      <td>L. Fanarraga, M.</td>
      <td>64, 202</td>
    </tr>
    <tr>
      <td>Johnston, A.</td>
      <td>81</td>
      <td>Labarre, J.</td>
      <td>128</td>
    </tr>
    <tr>
      <td>Juenet, M.</td>
      <td>73, 77</td>
      <td>Lacombe, S.</td>
      <td>227</td>
    </tr>
    <tr>
      <td>Józefowicz, M.</td>
      <td>46</td>
      <td>Landeros Martinez, L.</td>
      <td>236</td>
    </tr>
    <tr>
      <td></td>
      <td></td>
      <td>Lara, S.</td>
      <td>148</td>
    </tr>
    <tr>
      <td>Kagami, H.</td>
      <td>232</td>
      <td>Larrea, A.</td>
      <td>116</td>
    </tr>
    <tr>
      <td>Kah, J.</td>
      <td>199</td>
      <td>Lee, H.</td>
      <td>27, 29, 112</td>
    </tr>
    <tr>
      <td>Kalyani, N.</td>
      <td>164</td>
      <td>Lee, K.</td>
      <td>131</td>
    </tr>
    <tr>
      <td>Kamihira, M.</td>
      <td>93</td>
      <td>Lee, S.</td>
      <td>105</td>
    </tr>
    <tr>
      <td>Kamouni Belghiti, D.</td>
      <td>14</td>
      <td>Lee, s.</td>
      <td>113</td>
    </tr>
    <tr>
      <td>Kaneno, R.</td>
      <td>180</td>
      <td>Leiva Arrabal, M.</td>
      <td>96, 100</td>
    </tr>
    <tr>
      <td>Kang, J.</td>
      <td>27, 29</td>
      <td>Lerch, M.</td>
      <td>149</td>
    </tr>
    <tr>
      <td>Kar, K.</td>
      <td>13</td>
      <td>Letourneur, D.</td>
      <td>73, 77</td>
    </tr>
    <tr>
      <td>Karimi Dehkordi, S.</td>
      <td>218</td>
      <td>León, G.</td>
      <td>163</td>
    </tr>
    <tr>
      <td>Karimi, B.</td>
      <td>179</td>
      <td>León, S.</td>
      <td>163</td>
    </tr>
    <tr>
      <td>Karimi, N.</td>
      <td>36</td>
      <td>Li, B.</td>
      <td>73, 77</td>
    </tr>
    <tr>
      <td>Katayama, Y.</td>
      <td>123, 200</td>
      <td>Li, J.</td>
      <td>31</td>
    </tr>
    <tr>
      <td>Kawabe, Y.</td>
      <td>93</td>
      <td>Liepa, I.</td>
      <td>213</td>
    </tr>
    <tr>
      <td>Kent, S.</td>
      <td>81</td>
      <td>Liggins, R.</td>
      <td>224</td>
    </tr>
    <tr>
      <td>Khan, A.</td>
      <td>121</td>
      <td>Liko, F.</td>
      <td>233</td>
    </tr>
    <tr>
      <td>Khlobystov, A.</td>
      <td>182</td>
      <td>Lin, S.</td>
      <td>224</td>
    </tr>
    <tr>
      <td>Khowessah, o.</td>
      <td>32</td>
      <td>Liu, D.</td>
      <td>71</td>
    </tr>
    <tr>
      <td>Ki, T.</td>
      <td>131</td>
      <td>Liz Marzán, L.</td>
      <td>51</td>
    </tr>
    <tr>
      <td>Kiessling, F.</td>
      <td>4</td>
      <td>Llop, J.</td>
      <td>156</td>
    </tr>
    <tr>
      <td>Kiger, L.</td>
      <td>39</td>
      <td>Lo Giudice, M.</td>
      <td>148</td>
    </tr>
    <tr>
      <td>Kim, E.</td>
      <td>112</td>
      <td>Loiseau, A.</td>
      <td>142</td>
    </tr>
    <tr>
      <td>Kim, H.</td>
      <td>27, 112, 115</td>
      <td>Luisetto, I.</td>
      <td>68</td>
    </tr>
    <tr>
      <td>Kim, J.</td>
      <td>112</td>
      <td>López Romero, J.</td>
      <td>96</td>
    </tr>
    <tr>
      <td>Kim, m.</td>
      <td>113</td>
      <td>Lúcio, M.</td>
      <td>35</td>
    </tr>
    <tr>
      <td>Kishimura, A.</td>
      <td>123, 200</td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Kiyokawa, C.</td>
      <td>111, 196</td>
      <td>Madiehe, A.</td>
      <td>50</td>
    </tr>
    <tr>
      <td>Klapkova, E.</td>
      <td>229</td>
      <td>Madrigal Carballo, S.</td>
      <td>5, 98</td>
    </tr>
    <tr>
      <td>Koch, S.</td>
      <td>238</td>
      <td>Maghzi, P.</td>
      <td>10</td>
    </tr>
    <tr>
      <td>Koide, H.</td>
      <td>111, 196</td>
      <td>Mahon, E.</td>
      <td>153</td>
    </tr>
    <tr>
      <td>Kojima, C.</td>
      <td>151</td>
      <td>Maibohm, C.</td>
      <td>42</td>
    </tr>
    <tr>
      <td>Konstantinov, K.</td>
      <td>149</td>
      <td>Majouga, A.</td>
      <td>104, 182, 206</td>
    </tr>
    <tr>
      <td>Kostarelos, K.</td>
      <td>174</td>
      <td>Manai, R.</td>
      <td>14</td>
    </tr>
  </tbody>
</table>
<table><tr><td>Mandal, S.</td><td>84</td></tr><tr><td>Mani, V.</td><td>17</td></tr><tr><td>Mann, S.</td><td>81</td></tr><tr><td>Mareque Rivas, J.</td><td>156</td></tr><tr><td>Marichal, L.</td><td>39, 128</td></tr><tr><td>Marino, A.</td><td>231</td></tr><tr><td>Marinov, L.</td><td>8</td></tr><tr><td>Marotta, R.</td><td>136</td></tr><tr><td>Martyn, S.</td><td>144</td></tr><tr><td>Martín Banderas, L.</td><td>12, 23, 100</td></tr><tr><td>Martínez Fong, D.</td><td>247</td></tr><tr><td>Maruyama, T.</td><td>226</td></tr><tr><td>Mashal, M.</td><td>127</td></tr><tr><td>Mason, A.</td><td>30</td></tr><tr><td>Masotti, A.</td><td>28, 117, 120</td></tr><tr><td>Matchuk, O.</td><td>186, 187</td></tr><tr><td>Matelova, A.</td><td>243</td></tr><tr><td>Matougui, N.</td><td>146</td></tr><tr><td>Mauricot, R.</td><td>173</td></tr><tr><td>Maurizi, L.</td><td>171</td></tr><tr><td>Mayaudon, J.</td><td>130</td></tr><tr><td>Mazzucchelli, S.</td><td>56</td></tr><tr><td>Medard, N.</td><td>168</td></tr><tr><td>Melguizo, C.</td><td>96, 100</td></tr><tr><td>Mendes, P.</td><td>19</td></tr><tr><td>Mendoza, G.</td><td>70, 110, 116, 194</td></tr><tr><td>Merino Díaz, M.</td><td>141</td></tr><tr><td>Metelkina, O.</td><td>182</td></tr><tr><td>Meyer, M.</td><td>50</td></tr><tr><td>Micciulla, F.</td><td>117</td></tr><tr><td>Micheau, O.</td><td>66</td></tr><tr><td>Mielcarek, A.</td><td>222</td></tr><tr><td>Mihály, J.</td><td>8</td></tr><tr><td>Mildner, K.</td><td>143</td></tr><tr><td>Miller, M.</td><td>117</td></tr><tr><td>Millot, N.</td><td>142, 171</td></tr><tr><td>Ming Tatt, L.</td><td>17</td></tr><tr><td>Miotke, M.</td><td>46</td></tr><tr><td>Mirjolet, C.</td><td>142</td></tr><tr><td>Mirsaeed Gazi, E.</td><td>241</td></tr><tr><td>Mishra, P.</td><td>164</td></tr><tr><td>Mitchell, D.</td><td>149</td></tr><tr><td>Mitchell, P.</td><td>19</td></tr><tr><td>Moglianetti, M.</td><td>136</td></tr><tr><td>Mohan, P.</td><td>154</td></tr><tr><td>Mohebbi, A.</td><td>218</td></tr><tr><td>Momekov, G.</td><td>8</td></tr></table>

<table><tr><td>Momekova, D.</td><td>8</td></tr><tr><td>Morais, P.</td><td>103</td></tr><tr><td>Morales Avila, E.</td><td>209</td></tr><tr><td>Mori, T.</td><td>123, 200</td></tr><tr><td>Mrimi, R.</td><td>222</td></tr><tr><td>Mugabe, C.</td><td>224</td></tr><tr><td>Mukherjee, A.</td><td>79, 84, 214, 219</td></tr><tr><td>Mukherjee, P.</td><td>214</td></tr><tr><td>Mukherjee, S.</td><td>134</td></tr><tr><td>Mura, F.</td><td>28</td></tr><tr><td>Muthukumaran, P.</td><td>82</td></tr><tr><td>Mwafy, A.</td><td>197</td></tr><tr><td>Mäkilä, E.</td><td>71, 161</td></tr><tr><td>Möckl, L.</td><td>143</td></tr><tr><td>Nakamoto, M.</td><td>111</td></tr><tr><td>Nakamura, Y.</td><td>200</td></tr><tr><td>Neelov, I.</td><td>107</td></tr><tr><td>Neo, S.</td><td>199</td></tr><tr><td>Neoh, K.</td><td>199</td></tr><tr><td>Nessark, B.</td><td>34</td></tr><tr><td>Nessark, F.</td><td>34</td></tr><tr><td>New, S.</td><td>105</td></tr><tr><td>Nicolis, S.</td><td>55</td></tr><tr><td>Nieder, J.</td><td>35, 42</td></tr><tr><td>Nikolova, I.</td><td>8</td></tr><tr><td>Nikolskaja, E.</td><td>186, 187</td></tr><tr><td>Nilyai, S.</td><td>192</td></tr><tr><td>Nizamov, T.</td><td>206</td></tr><tr><td>Nobori, T.</td><td>123</td></tr><tr><td>Nomani, A.</td><td>221</td></tr><tr><td>Nourdin, y.</td><td>63</td></tr><tr><td>Nyalosaso, J.</td><td>7</td></tr><tr><td>Ocampo García, B.</td><td>209</td></tr><tr><td>Odorico, M.</td><td>7</td></tr><tr><td>Ojha, D.</td><td>84</td></tr><tr><td>Okamoto, A.</td><td>31, 196</td></tr><tr><td>Okishima, A.</td><td>111</td></tr><tr><td>Oku, N.</td><td>31, 111, 196</td></tr><tr><td>Oliveira, R.</td><td>62</td></tr><tr><td>Ollivier, V.</td><td>73, 77</td></tr><tr><td>Onani, M.</td><td>50</td></tr><tr><td>Orive, G.</td><td>118</td></tr><tr><td>Orrantia Borunda, E.</td><td>236</td></tr><tr><td>Ortiz De Solorzano, I.</td><td>70</td></tr><tr><td>Ortiz, R.</td><td>96, 100</td></tr><tr><td>Osborne, S.</td><td>75</td></tr></table>
<table>
  <tr>
    <td>Otagiri, M.</td>
    <td>226</td>
    <td>Pramanik, K.</td>
    <td>102</td>
  </tr>
  <tr>
    <td>Oura, S.</td>
    <td>160</td>
    <td>Prapainop, K.</td>
    <td>153</td>
  </tr>
  <tr>
    <td>Ozolina, R.</td>
    <td>35</td>
    <td>Prat, O.</td>
    <td>7</td>
  </tr>
  <tr>
    <td>Pace, A.</td>
    <td>211</td>
    <td>Prieto, M.</td>
    <td>70</td>
  </tr>
  <tr>
    <td>Padilla, A.</td>
    <td>247</td>
    <td>Primo, F.</td>
    <td>103</td>
  </tr>
  <tr>
    <td>Padín González, E.</td>
    <td>202</td>
    <td>Prosperi, D.</td>
    <td>55, 56</td>
  </tr>
  <tr>
    <td>Pajot Augy, E.</td>
    <td>14</td>
    <td>Puchowicz, D.</td>
    <td>188</td>
  </tr>
  <tr>
    <td>Palissot, V.</td>
    <td>25</td>
    <td>Puras, G.</td>
    <td>127</td>
  </tr>
  <tr>
    <td>Palmer, D.</td>
    <td>211</td>
    <td>Quinn, J.</td>
    <td>81</td>
  </tr>
  <tr>
    <td>Panahi, A.</td>
    <td>179</td>
    <td>Radilov, A.</td>
    <td>107</td>
  </tr>
  <tr>
    <td>Pandolfi, L.</td>
    <td>55</td>
    <td>Raj, R.</td>
    <td>216</td>
  </tr>
  <tr>
    <td>Paolini, A.</td>
    <td>28, 120</td>
    <td>Ramakrishna, S.</td>
    <td>82</td>
  </tr>
  <tr>
    <td>Park, H.</td>
    <td>27, 115</td>
    <td>Rance, G.</td>
    <td>182</td>
  </tr>
  <tr>
    <td>Park, W.</td>
    <td>113</td>
    <td>Real Oliveira, M.</td>
    <td>35</td>
  </tr>
  <tr>
    <td>Partovi Meran, M.</td>
    <td>106</td>
    <td>Rebicek, J.</td>
    <td>229</td>
  </tr>
  <tr>
    <td>Pascual Garcia, C.</td>
    <td>25</td>
    <td>Redmond, G.</td>
    <td>169</td>
  </tr>
  <tr>
    <td>Patel, J.</td>
    <td>242</td>
    <td>Reed, J.</td>
    <td>98</td>
  </tr>
  <tr>
    <td>Patel, K.</td>
    <td>211</td>
    <td>Regiel Futyra, A.</td>
    <td>110</td>
  </tr>
  <tr>
    <td>Pavlovskaya, G.</td>
    <td>182</td>
    <td>Reichel, C.</td>
    <td>143</td>
  </tr>
  <tr>
    <td>Pedone, D.</td>
    <td>136</td>
    <td>Renault, J.</td>
    <td>128</td>
  </tr>
  <tr>
    <td>Pedraz, J.</td>
    <td>118, 127</td>
    <td>Rens, J.</td>
    <td>140</td>
  </tr>
  <tr>
    <td>Pedrosa, V.</td>
    <td>180</td>
    <td>Retta, S.</td>
    <td>136</td>
  </tr>
  <tr>
    <td>Perazzoli, G.</td>
    <td>96, 100</td>
    <td>Riasat, R.</td>
    <td>244, 245</td>
  </tr>
  <tr>
    <td>Perera, A.</td>
    <td>23</td>
    <td>Rigon, R.</td>
    <td>43, 48, 132</td>
  </tr>
  <tr>
    <td>Persuy, M.</td>
    <td>14</td>
    <td>Rizzuto, M.</td>
    <td>56</td>
  </tr>
  <tr>
    <td>Pesquera, C.</td>
    <td>64, 202</td>
    <td>Robinson, A.</td>
    <td>211</td>
  </tr>
  <tr>
    <td>Petunov, S.</td>
    <td>107</td>
    <td>Rocks, L.</td>
    <td>153</td>
  </tr>
  <tr>
    <td>Pikramenou, Z.</td>
    <td>75</td>
    <td>Rodero, C.</td>
    <td>43</td>
  </tr>
  <tr>
    <td>Pin, S.</td>
    <td>128</td>
    <td>Rodrigo Arrizabalaga, I.</td>
    <td>158</td>
  </tr>
  <tr>
    <td>Pinto, M.</td>
    <td>103</td>
    <td>Romero, R.</td>
    <td>42</td>
  </tr>
  <tr>
    <td>Piret, G.</td>
    <td>130</td>
    <td>Rose, L.</td>
    <td>117</td>
  </tr>
  <tr>
    <td>Pisani, C.</td>
    <td>7</td>
    <td>Rosenfeld, A.</td>
    <td>149</td>
  </tr>
  <tr>
    <td>Plackett, D.</td>
    <td>224</td>
    <td>Roskamp, M.</td>
    <td>211</td>
  </tr>
  <tr>
    <td>Plazaola Muguruza, F.</td>
    <td>158</td>
    <td>Roszak, J.</td>
    <td>188</td>
  </tr>
  <tr>
    <td>Poinard, B.</td>
    <td>199</td>
    <td>Rousseau, L.</td>
    <td>14, 130</td>
  </tr>
  <tr>
    <td>Pokorny, M.</td>
    <td>229</td>
    <td>Roux, S.</td>
    <td>142</td>
  </tr>
  <tr>
    <td>Polo, E.</td>
    <td>148</td>
    <td>Rovers, J.</td>
    <td>140</td>
  </tr>
  <tr>
    <td>Polzonetti, G.</td>
    <td>68</td>
    <td>Roy, P.</td>
    <td>84</td>
  </tr>
  <tr>
    <td>Pompa, P.</td>
    <td>136</td>
    <td>Ruiz De Angulo, A.</td>
    <td>156</td>
  </tr>
  <tr>
    <td>Popova, E.</td>
    <td>107</td>
    <td>Ruozi, B.</td>
    <td>204</td>
  </tr>
  <tr>
    <td>Popova, M.</td>
    <td>8</td>
    <td>Russo, M.</td>
    <td>68</td>
  </tr>
  <tr>
    <td>Porcaro, F.</td>
    <td>68</td>
    <td>Sadeghi, H.</td>
    <td>10</td>
  </tr>
  <tr>
    <td>Porcel, e.</td>
    <td>227</td>
    <td>Sadeghizadeh, M.</td>
    <td>221</td>
  </tr>
  <tr>
    <td>Possas Abreu, M.</td>
    <td>14</td>
    <td>Saenz Del Burgo, L.</td>
    <td>118</td>
  </tr>
  <tr>
    <td>Postorino, P.</td>
    <td>94</td>
    <td>Sahoo, A.</td>
    <td>16</td>
  </tr>
  <tr>
    <td>Prado, M.</td>
    <td>59</td>
    <td>Sakeena, M.</td>
    <td>245</td>
  </tr>
  <tr>
    <td>Prados, J.</td>
    <td>96, 100</td>
    <td></td>
    <td></td>
  </tr>
</table>
<table><tr><td>Sako, M.</td><td>31</td></tr><tr><td>Salado, D.</td><td>227</td></tr><tr><td>Salazar Cabrera, R.</td><td>12, 23</td></tr><tr><td>Salonen, J.</td><td>71, 161</td></tr><tr><td>Salvioni, L.</td><td>55</td></tr><tr><td>Santelli, J.</td><td>173</td></tr><tr><td>Santini, B.</td><td>55</td></tr><tr><td>Santos, H.</td><td>71, 161</td></tr><tr><td>Sanz, G.</td><td>14</td></tr><tr><td>Sartori, B.</td><td>136</td></tr><tr><td>Sato, H.</td><td>200</td></tr><tr><td>Sato, M.</td><td>93</td></tr><tr><td>Saudagar, P.</td><td>41</td></tr><tr><td>Sauerova, P.</td><td>229</td></tr><tr><td>Saulnier, P.</td><td>146</td></tr><tr><td>Savchenko, A.</td><td>104, 182, 206</td></tr><tr><td>Schlesser, V.</td><td>25</td></tr><tr><td>Schäfer Korting, M.</td><td>132</td></tr><tr><td>Sciubba, F.</td><td>94</td></tr><tr><td>Scorsone, E.</td><td>14</td></tr><tr><td>Sebastián, V.</td><td>70, 110, 116, 194</td></tr><tr><td>Seddik El Hak, A.</td><td>63</td></tr><tr><td>Sennato, S.</td><td>28</td></tr><tr><td>Sereemaspun, A.</td><td>192</td></tr><tr><td>Serre, c.</td><td>89, 222</td></tr><tr><td>Severini, G.</td><td>204</td></tr><tr><td>Seyedabasi, H.</td><td>179</td></tr><tr><td>Seyedzadeh, A.</td><td>197</td></tr><tr><td>Shahabi, A.</td><td>218</td></tr><tr><td>Shahbazi, M.</td><td>71</td></tr><tr><td>Shams, a.</td><td>22</td></tr><tr><td>Shchetinin, I.</td><td>104</td></tr><tr><td>Shi, D.</td><td>149</td></tr><tr><td>Shukla, D.</td><td>134</td></tr><tr><td>Sibuyi, N.</td><td>50</td></tr><tr><td>Siew Wei, Y.</td><td>17</td></tr><tr><td>Silva, F.</td><td>42</td></tr><tr><td>Simon Yarza, T.</td><td>222</td></tr><tr><td>Singh Shekhawat, D.</td><td>13</td></tr><tr><td>Singh, A.</td><td>138</td></tr><tr><td>Sinha, N.</td><td>138</td></tr><tr><td>Skepu, A.</td><td>50</td></tr><tr><td>Slovakova, M.</td><td>240</td></tr><tr><td>Smok Pieniążek, A.</td><td>188</td></tr><tr><td>Soleimannejad, J.</td><td>44</td></tr><tr><td>Soliman, M.</td><td>185</td></tr><tr><td>Sorrentino, L.</td><td>56</td></tr></table>

<table><tr><td>Soto Sanchez, C.</td><td>127</td></tr><tr><td>Sperandio, M.</td><td>143</td></tr><tr><td>Srinivasan, D.</td><td>82</td></tr><tr><td>Stefancikova, L.</td><td>227</td></tr><tr><td>Stephan, H.</td><td>153</td></tr><tr><td>Stochel, G.</td><td>110</td></tr><tr><td>Strankowska, J.</td><td>46</td></tr><tr><td>Strankowski, M.</td><td>46</td></tr><tr><td>Strozyk, M.</td><td>51</td></tr><tr><td>Stępnik, M.</td><td>188</td></tr><tr><td>Such, G.</td><td>81</td></tr><tr><td>Suchy, T.</td><td>229</td></tr><tr><td>Sun, G.</td><td>224</td></tr><tr><td>Supova, M.</td><td>229</td></tr><tr><td>Suzuki, M.</td><td>231</td></tr><tr><td>Szegedi, A.</td><td>8</td></tr><tr><td>Séverine, L.</td><td>173</td></tr><tr><td>Taherzade, S.</td><td>44</td></tr><tr><td>Takahje, M.</td><td>17</td></tr><tr><td>Taki, M.</td><td>57</td></tr><tr><td>Tan, A.</td><td>199</td></tr><tr><td>Tan, R.</td><td>199</td></tr><tr><td>Tariq, Z.</td><td>244, 245</td></tr><tr><td>Tarlani, A.</td><td>44</td></tr><tr><td>Tcareva, I.</td><td>104</td></tr><tr><td>Tedesco, A.</td><td>103</td></tr><tr><td>Tehei, M.</td><td>149</td></tr><tr><td>Ten Hagen, T.</td><td>140, 141</td></tr><tr><td>Tesser, A.</td><td>204</td></tr><tr><td>Testa, G.</td><td>94</td></tr><tr><td>Thomann, J.</td><td>25</td></tr><tr><td>Thomas, G.</td><td>171</td></tr><tr><td>Thovhogi, N.</td><td>50</td></tr><tr><td>Thurecht, K.</td><td>81</td></tr><tr><td>Tigges, J.</td><td>132</td></tr><tr><td>Tomaszewska, E.</td><td>188</td></tr><tr><td>Tomoaia Cotisel, M.</td><td>228</td></tr><tr><td>Tosi, G.</td><td>204</td></tr><tr><td>Tozzi, A.</td><td>28</td></tr><tr><td>Trendafilova, I.</td><td>8</td></tr><tr><td>Troconiz, I.</td><td>141</td></tr><tr><td>Truffi, M.</td><td>56</td></tr><tr><td>Tsuchida, H.</td><td>111, 196</td></tr><tr><td>Téllez López, V.</td><td>247</td></tr><tr><td>Ugolini, A.</td><td>68</td></tr><tr><td>Uhl, B.</td><td>143</td></tr></table>
<table><tr><td>Umemura, K.</td><td>160</td><td>Wilhelm, C.</td><td>2</td></tr><tr><td>Umerska, A.</td><td>146</td><td>Williams, P.</td><td>211</td></tr><tr><td>Unlu, A.</td><td>106</td><td>Wolff, C.</td><td>132</td></tr><tr><td>Urena, H.</td><td>5</td><td>Yabbarov, N.</td><td>186, 187</td></tr><tr><td>V. Ramanujan, R.</td><td>82</td><td>Yagci Acar, h.</td><td>53</td></tr><tr><td>Valiente, R.</td><td>64</td><td>Yan, Y.</td><td>148</td></tr><tr><td>Vaseghi, a.</td><td>179</td><td>Ying, J.</td><td>178</td></tr><tr><td>Velebný, V.</td><td>243</td><td>Yvert, B.</td><td>130</td></tr><tr><td>Venditti, I.</td><td>68, 94</td><td>Yüce, M.</td><td>61</td></tr><tr><td>Vepuri, S.</td><td>185</td><td>Zabaleta, A.</td><td>156</td></tr><tr><td>Verelst, M.</td><td>173</td><td>Zalba Oteiza, S.</td><td>140, 141</td></tr><tr><td>Verheij, M.</td><td>140</td><td>Zaloudkova, M.</td><td>229</td></tr><tr><td>Vesely, J.</td><td>229</td><td>Zamulaeva, I.</td><td>186, 187</td></tr><tr><td>Vial, S.</td><td>59</td><td>Zarate, J.</td><td>127</td></tr><tr><td>Vijayaraj Kumar, P.</td><td>17</td><td>Zare Mirakabadi, A.</td><td>184</td></tr><tr><td>Vilas Boas, V.</td><td>35</td><td>Zarschler, K.</td><td>153</td></tr><tr><td>Villegas, J.</td><td>64</td><td>Zavareh, S.</td><td>241</td></tr><tr><td>Von Stosch, M.</td><td>62</td><td>Zavora, J.</td><td>229</td></tr><tr><td>Vonavkova, T.</td><td>229</td><td>Zeuschner, D.</td><td>143</td></tr><tr><td>Wang, S.</td><td>21</td><td>Zhang, H.</td><td>71, 161</td></tr><tr><td>Wang, Y.</td><td>108</td><td>Zine, N.</td><td>34, 63</td></tr><tr><td>Weisner, U.</td><td>154</td><td>Zoschke, C.</td><td>132</td></tr><tr><td>Wen, X.</td><td>21</td><td>Zouaoui, A.</td><td>34</td></tr><tr><td>Wenger, J.</td><td>59</td><td>Řípová, D.</td><td>240</td></tr><tr><td>Whittaker, M.</td><td>81</td><td>Šmejkalová, D.</td><td>243</td></tr></table>
<figure><img src="image_111.png" /></figure>

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