# Development of novel anti-Kv 11.1 antibody-conjugated PEG-TiO2 nanoparticles for targeting pancreatic ductal adenocarcinoma cells

Angelica Sette, Jolanda Spadavecchia, Jessem Landoulsi, Sandra Casale, Bernard Haye, Olivia Crociani, Annarosa Arcangeli

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## To cite this version:

Angelica Sette, Jolanda Spadavecchia, Jessem Landoulsi, Sandra Casale, Bernard Haye, et al.. Development of novel anti-Kv 11.1 antibody-conjugated PEG-TiO2 nanoparticles for targeting pancreatic ductal adenocarcinoma cells. Journal of Nanoparticle Research, 2013, 15, pp.2111. 10.1007/s11051-013-2111-6 hal-00983778

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# Development of novel anti-Kv 11.1 antibody-conjugated PEG-TiO₂ nanoparticles for targeting pancreatic ductal adenocarcinoma cells

Angelica Sette · Jolanda Spadavecchia · Jessem Landoulsi · Sandra Casale · Bernard Haye · Olivia Crociani · Annarosa Arcangeli

Received: 14 May 2013 / Accepted: 31 October 2013 / Published online: 16 November 2013
© The Author(s) 2013. This article is published with open access at Springerlink.com

**Abstract** Titanium dioxide (TiO₂) has been widely used in many nanotechnology areas including nanomedicine, where it could be proposed for the photodynamic and sonodynamic cancer therapies. However, TiO₂ nanoformulations have been shown to be toxic for living cells. In this article, we report the development of a new delivery system, based on nontoxic TiO₂ nanoparticles, further conjugated with a monoclonal antibody against a

novel and easily accessible tumor marker, e.g., the Kv 11.1 potassium channel. We synthesized, by simple solvothermal method, dicarboxylic acid-terminated PEG TiO₂ nanocrystals (PEG-TiO₂ NPs). Anti-Kv 11.1 monoclonal antibodies (Kv 11.1-Mab) were further linked to the terminal carboxylic acid groups. Proper conjugation was confirmed by X-ray photoelectron spectroscopy analysis. Kv 11.1-Mab-PEG-TiO₂ NPs efficiently recognized the specific Kv 11.1 antigen, both in vitro and in pancreatic ductal adenocarcinoma (PDAC) cells, which express the Kv 11.1 channel onto the plasma membrane. Both PEG TiO₂ and Kv 11.1-Mab-PEG-TiO₂ NPs were not cytotoxic, but only Kv 11.1-Mab-PEG-TiO₂ NPs were efficiently internalized into PDAC cells. Data gathered from this study may have further applications for the chemical design of nanostructures to be applied for therapeutic purposes in pancreatic cancer.

Angelica Sette and Jolanda Spadavecchia have contributed equally to this work.

**Electronic supplementary material** The online version of this article (doi:10.1007/s11051-013-2111-6) contains supplementary material, which is available to authorized users.

A. Sette · O. Crociani · A. Arcangeli (✉)
Section of Internal Medicine, Department of Experimental and Clinical Medicine, University of Florence, Viale GB Morgagni 50, 50134 Florence, Italy
e-mail: annarosa.arcangeli@unifi.it

J. Spadavecchia · J. Landoulsi · S. Casale
Laboratoire de Réactivité de Surface, UMR CNRS 7197, Université Pierre & Marie Curie-Paris VI, Site d'Ivry-Le Raphaël, 94200 Ivry-sur-Seine, France

B. Haye
Chimie de la Matière Condensée de Paris, Collège de France, Université Pierre & Marie Curie – Paris VI, 11 place Marcelin Berthelot, Paris, France

O. Crociani · A. Arcangeli
DI.V.A.L. Toscana srl, Via Madonna del Piano 6, 50019 Sesto Fiorentino (FI), Italy

**Keywords** Nanoparticles · PEG–TiO₂ · XPS analysis · Kv 11.1 · Pancreatic cancer · Nanomedicine

## Introduction

Nanophase engineering expands nowadays in rapidly growing number of structural and functional materials, both inorganic and organic. This allows the manipulation of nanostructures for different applications from mechanics to electronics (Caricato et al. 2007; Xu et al. 2011). Nanotechnology has recently received
great attention also for application in the biomedical field. In particular, nanoparticles (NPs) can be viewed as synthetic materials with considerable potential applications in biomedicine due to their unique ways to interact with matter (Wagner et al. 2006; Kim et al. 2010; Jokerst et al. 2011). NPs can be used either for diagnosis, as contrast agents in optical, photoacoustic, and magnetic resonance imaging, or for therapy. In the latter case, NPs can either be activated (e.g., by hyperthermia or photodynamic activation) or used as carriers for drug delivery. Nanocarriers are able to increase tumor exposure to therapeutic agents, improving treatment effects either by prolonging circulation times, protecting the carried drug from degradation, or enhancing tumor uptake (Fernandez-Fernandez et al. 2011).

Titanium dioxide (TiO₂) has been widely used in many nanotechnology areas such as paint industry, electronics, gas sensors, and environmental engineering (Song et al. 2008; De Marco et al. 2010). More recently, it has been applied to biomedicine. The production of reactive oxygen species (ROS) under ultraviolet and ultrasound irradiation (Ashikaga et al. 2000; Ninomiya 2012) makes TiO₂ an efficient photocatalyst. For these reasons, TiO₂ can be applied for the photodynamic and sonodynamic cancer therapies (Yamaguchi 2011). While TiO₂ is nontoxic (Fei Yin et al. 2013), the toxicity of nanoformulation of TiO₂ is debatable. This different behavior is related to the small size of NPs accompanied by a high specific surface area of interaction (Huang et al. 2010), as well as to their ability to easily cross biologic barriers such as the skin, lung, and intestine. Indeed, TiO₂ NPs were found to be toxic for mouse fibroblasts (Jin et al. 2008) and even more toxic for human cells (Lai et al. 2008). Consistently, human bronchial epithelial cells lost their viability at very low concentrations of TiO₂ NPs, an effect mediated by ROS production (Park et al. 2008). One strategy to prevent the toxicity of TiO₂ nanoformulations is their coating with polyethylene glycol (PEG). PEG is a hydrophilic, nonionic polymer that exhibits excellent biocompatibility (Jokerst et al. 2011). When used for drug delivery purposes, the addition of PEG to polymer particles produced an increased circulation time, which in turn prevented NPs uptake by the reticuloendothelial system (Jacobson et al. 2010).

Over the last few decades, the application of targeted therapeutic strategies to cancer has improved response and survival. To maximize the therapeutic

activity while minimizing toxic side effects, it is possible to conjugate drugs to a targeted delivery system. Targeted NPs represent good drug delivery systems, to improve specific drug distribution and avoid systemic toxicity. Targeting of NPs can be achieved by either passive or active procedures. Passive targeting simply relies on NPs accumulation inside the tumor mass, an effect mediated by the so-called enhanced permeability and retention effect (Fernandez-Fernandez et al. 2011). However, low tumor specificity and suboptimal drug concentration at the tumor site strongly limit the efficacy of passive targeting approaches. For active targeting, it is necessary to incorporate, on the NPs surface, a targeting moiety (e.g., antibodies, peptides, etc.) able to bind an easily accessible and, hopefully, tumor-specific cell surface receptor. Such binding can facilitate NPs intracellular uptake and in turn, drug delivery. Preclinical studies showed that antibody-targeted NPs have better antitumor activity compared to nontargeted NPs, due to increased tumor localization (Zhang and Sun 2004; Kirpotin et al. 2006).

We report in this article, a new strategy for a targeted-therapeutic approach in cancer, based on actively targeted NPs. We synthesized dicarboxylic acid-terminated PEG TiO₂ nanocrystals by a simple one-step solvothermal method, and further conjugated the PEG–TiO₂ NPs with a monoclonal antibody (Arcangeli 2006), being able to bind a cell surface protein (e.g., the Kv 11.1 potassium channel), which is aberrantly expressed in many types of human cancers (D'Amico et al. 2013).

## Materials and methods

### Materials

Ti(IV) isopropoxide (Ti(OPri)₄ or TTIP 97 %; benzyl alcohol (BzOH ≥ 99 %)), (poly(ethyleneglycol) bis (carboxymethyl)ether) (PEG diacide MW:600), cysteamine (CYS), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), DMF (dimethylformammide for molecule biology 99 %), buffer solution (PBS pH 7.4) were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France). The S5-P peptide was synthesized by PRIMM company. All solvents were reagent grade and used without any further
purification. Experiments were carried out at room temperature if not specified otherwise.

## TiO₂ synthesis and Kv 11.1-Mab immobilization

TiO₂ colloids were prepared according to the following procedure, as previously described by (Spadavecchia et al. 2011): in brief, 1 mL portion of TTIP (3.36 mmol) was added to 0.75 mL of PEG Diacide (MW: 600), along with 5 mL of BzOH (TTIP/PEG-diacid 1/5). The solution was stirred for 30 min at room temperature after which the reaction mixtures were transferred into a steel autoclave equipped with a Teflon cap and kept at 200 °C for 18 h. During this time, the solution turns from clear to yellow milky suspension. The reaction was then stopped, and the suspension was centrifuged two times (9,000 rpm for 30 min). The precipitate becomes white to slightly yellow upon washing with ethanol and dichloromethane two times and was dried under nitrogen. 10 mg of nPEG-TiO₂ was dissolved in 5 mL of DMF containing a solution of NHS (60 mM) and EDC (30 mM) in water for 4 h (activation). After rinsing in DMF and EtOH, the nPEG-TiO₂ was treated and centrifuged two times (9,000 rpm for 30 min). The precipitate becomes white to slightly yellow upon washing with ethanol two times and was dried under nitrogen. The binding of Kv 11.1-Mab was performed at pH 7.4 using PBS buffer solution. 50 µL (1 mg/mL) of Kv 11.1-Mab in PBS solution was added into 2 mL of PEG-TiO₂ NPs and stirred for 2 h at room temperature. After this time, Kv 11.1-Mab-PEG-TiO₂ nanoparticles were centrifuged two times (9,000 rpm for 30 min), dried under nitrogen, and analyzed by X-ray photoelectron spectroscopy (XPS) analysis (see below).

In order to check the specificity of the Kv 11.1-Mab-PEG-TiO₂, anti-IgG was also used and conjugated to PEG-TiO₂ following the same procedure used for the Kv 11.1-Mab antibody.

## XPS analysis

XPS analyses were performed using a SPECS (Phoibos MCD 100), X-ray photoelectron spectrometer (SPECS, Germany) equipped with a monochromatized aluminum X-ray source ($h\nu$ = 1,486.74 eV), and Phoibos 100 hemispherical energy analyzer. A pass energy of 20 eV was used for survey scan, and

10 eV as pass energy for narrow scans. The samples were fixed on the support, and no charge stabilization device was used. The pressure in the analysis chamber during measurement was around $10^{-10}$ Torr. The photoelectron collection angle ($\theta$) between the normal to the sample surface and the analyzer axis was 0. The following sequence of spectra was recorded: survey spectrum, O 1s, N 1s, C 1s and Ti 2p. The data treatment was performed using the Casa XPS software (Casa Software Ltd., UK). The peaks were decomposed using a linear baseline, and a component shape defined by the product of a Gauss and Lorentz function, in 70:30 ratio. Molar concentration ratios were calculated using peak areas normalized according to Scofield factors.

## ELISA assay

The ELISA assay was used to detect and verify the proper orientation of the Kv 11.1-Mab conjugated to PEG-TiO₂ nanoparticles. The S5-P peptide was used to cover a 96-well plate, and incubated overnight at 4 °C. After three washes with 0.05 %, Tween-20 in PBS, PEG-TiO₂, and Kv 11.1-Mab-PEG-TiO₂ NPs were diluted at different concentrations in BSA 3 % plus 0.05 % Tween-20 in PBS and incubated for 2 h at room temperature. 100 µL/well of antimouse secondary antibody (Sigma) (1:500) was dispensed and incubated for 1 h at room temperature. The assay was developed by the addition of 100 µL/well TMB substrate (Sigma), and the reaction was stopped with the addition of 100 µL/well of 0.5 M HCl. Plates were analyzed measuring the absorbance at 450 nm. For the ELISA assay on living cells, PANC-1 and NIH-3T3 were seeded to semiconfluence in 96-well plate in DMEM plus 10 % fetal bovine serum (FBS) and incubated overnight at 37 °C and 5 % CO₂. After three washes with PBS, Kv 11.1-Mab-PEG-TiO₂ and IgG-Mab-PEG-TiO₂ were diluted at different concentrations into culture medium and added to the cells for 2 h at room temperature. The following steps were the same as described above.

## Measurement of cell viability

The viability of two pancreatic ductal adenocarcinoma (PDAC) cell lines, Panc-1 and MIA PaCa-2, in the presence of PEG-TiO₂ and Kv 11.1-Mab-PEG-TiO₂ NPs, was assessed using the WST-1 assay (Roche,
Mannheim, Germany). Cells were seeded into 96-well cell culture plates at a density of 3,000 cells/well in DMEM plus 10 % FBS. On the next day, cell medium was replaced by 100 µL of fresh medium containing PEG–TiO₂ or Kv 11.1-Mab-PEG–TiO₂ at different concentrations. Cells were cultured for 24 h in a humidified incubator at 37 °C and 5 % CO₂. Before the WST-1 assay, the medium in each sample was replaced with fresh medium that did not contain PEG–TiO₂. WST-1 reagent (10 µL) was added into each well, followed by incubation at 37 °C for 1 h. Then, the amount of formazan dye formed after WST-1 cleavage carried out by living cells was measured at 450 nm.

## Immunofluorescence

MIAPaCa-2, Panc-1, and NIH-3T3 were plated onto glass coverslips coated with Fibronectin (Sigma) (20 µg/mL). The day after plating, coverslips were washed twice with PBS and fixed by treating for 1 h with 4 % paraformaldehyde in PBS, at room temperature. After three further washes in PBS, coverslips were incubated for 15 min in PBS plus 1 % SDS at room temperature for antigen retrieval, then treated for 15 min with 100 mM glycine at room temperature (to quench residual crosslinking activity of the formaldehyde) and permeabilized for 4 min with PBS plus 0.01 % Triton X-100. Unspecific binding sites were blocked for 30 min at room temperature with blocking solution (10 % fetal calf serum (FCS) in PBS) was then incubated for 1 h at room temperature with mouse anti-hERG1 monoclonal antibody (Enzo Life Technologies) (1 µg/mL) in blocking solution. After three washes with PBS, cells were incubated for 45 min at room temperature with Alexa-488-labeled secondary antimouse antibody (invitrogen) diluted in blocking solution (1:500). Coverslips were washed three times in PBS and mounted on glass microscope slides in fluorescence ProLong Gold Antifade Reagent with DAPI (invitrogen).

## Transmission electron microscopy (TEM) analysis

TEM images of PEG–TiO₂ NPs were recorded using a JEOL JEM 1011 microscope operating at an accelerating voltage of 100 kV. The samples for TEM analyses were prepared by dropping a dilute solution of PEG–TiO₂ dissolved in dichloromethane onto

carbon-coated copper grid and then allowing the solvent to evaporate.

TEM images of PDAC and NIH-3T3 cells treated with PEG–TiO₂ and Kv 11.1-Mab-PEG–TiO₂ NPs were acquired using the same apparatus, following the procedure described below. Panc-1 and MIA PaCa-2 cells were grown in a six-well plate, and then incubated with 0.01, 0.05, or 0.1 % of PEG–TiO₂ and with 0.01, 0.05, or 0.1 % of Kv 11.1-Mab-PEG–TiO₂ for 48 h in a humidified incubator at 37 °C and 5 % CO₂. Subsequently, cells were detached with 0.05 % trypsin plus 0.02 % EDTA, washed with PBS, and fixed in 2 % glutaraldehyde in phosphate buffer 0.1 M. Then, a procedure was applied, consisting of five steps: dehydration, impregnation, inclusion, ultramicrotomy, and contrasting.

Dehydration: the samples were gradually dehydrated by bath of ethanol 50, 70, and 95 % for 5 min each, and two baths of 10 min in ethanol 100 %. Then, ethanol was replaced by propylene oxide in two baths of 10 min: the first one in a mixing 50/50 ethanol propylene oxide, and the second in propylene oxide pure. Impregnation: the dehydrated samples were impregnated in a solution of Araldite (20 mL Araldite CY212; 22 mL DDSA—dodecenyl succinic anhydride, used as hardener; and 1.1 mL BDMA—benzyl dimethyl amine—used as accelerator). The impregnation has been done in three steps: the first one with a mixing of 2/3 volume of propylene oxide and 1/3 volume of araldite for 1 h, the second with a mixture of 1/3 volume of propylene oxide and 2/3 volume of araldite overnight at 4 °C and, finally, in pure Araldite. Inclusion: the samples impregnated by araldite were incubated at 60 °C for 3 days, to obtain hard blocks. Ultramicrotomy: blocks were cut in ultrathin section of 70 nm with an ultramicrotome (Ultracut Reichert, France). Contrasting: slides obtained were put onto copper grid and contrasted with a 50/50 mix of uranyl acetate 5 % and ethanol 100 % and rinsed with ethanol 50 %.

## Results

Dicarboxylic acid-terminated PEG–TiO₂ nanocrystals were prepared according to the procedure previously described (Spadavecchia et al. 2011). In this procedure, PEG diacide plays twin activities: (i) as a relevant reagent precursor in the chemical synthesis,
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Schematic image of the formation of amide links between the COOH groups of the TiO₂ NP and NH₂ groups of the antibody</figcaption></figure>

and (ii) as a stabilizer of the TiO₂ NP. In fact, the adsorbed PEG on the surface of TiO₂ particles enhances stabilization by both exerting steric effects and leading to the formation of a monodisperse phase.

The synthesized NPs were used as a support for peptide conjugation. We conjugated a monoclonal antibody directed against the Kv 11.1 potassium channel protein (Kv 11.1-Mab) which is over expressed in several types of human cancers (D'Amico et al. 2013). PEG–TiO₂ NPs were activated in EDC/NHS solution for 3 h, and then the antibody molecule was immobilized on the surface of activated NPs through the formation of amide links between the COOH groups of the TiO₂ NP and NH₂ groups of the antibody (Fig. 1).

The morphology of Kv 11.1-Mab-PEG–TiO₂ NPs, as revealed by TEM, showed separated and well-defined NPs with a unique cubic shape (Fig. 2b). Both the shape and size of Kv 11.1-Mab-PEG–TiO₂ NPs were identical to those of the PEG–TiO₂ NPs without peptide, as previously reported (Spadavecchia et al. 2011) (see also Fig. 2a). By analyzing several TEM images, Kv 11.1-Mab-PEG–TiO₂ NPs were observed to exhibit a narrow size distribution (near 7 nm, see the histogram in Fig. 2c). Moreover, from TEM pictures, it is possible to note that while PEG–TiO₂ NPs are close to each others, the distance between each Kv 11.1-Mab-PEG–TiO₂ NPs is greater by about 20 nm (compare pictures in Fig. 2a, b). Taking into account that the length of the Kv 11.1 Mab is about 15 nm (Foley et al. 2007), we can estimate that the antibody influence interparticle distance due a double combination of electrostatic and steric repulsion of the antibody linked to the PEG-chain present on the NPs. It is worth noting that all experiments were carried out at pH 7.4, a value at which the amino acids of the Kv 11.1-Mab (pI: 6.2–6.5) is positively charged. This implies that the intracellular uptake of Kv 11.1-Mab (see below) is favored only by the specific recognition

of the antibody to the Kv 11.1 protein expressed on the cancer cells. Finally, TEM images were also recorded on Kv 11.1-Mab-PEG–TiO₂ NPs after incubation in FCS at 37 °C for 24 h: no relevant modification in terms of size and shape was observed (Fig. S1, Supplementary Section).

More direct evidence for the crystallinity of the NPs was confirmed by X-ray diffraction analysis (XRD) performed on Kv 11.1 Mab-PEG–TiO₂ NPs powder. Results showed particularly broad diffraction peaks, which are characteristic of very small crystallite sizes (Fig. 2d). The stability of the carboxylate ligand influences Ti–O–Ti bridge formation, thereby enhancing growth rates of various crystallographic planes. X-ray diffraction spectra also indicated the presence of NPs with a preferred growth orientation along the c-axis of the anatase lattice. As shown in Fig. 2d, all reflections correspond to anatase with a particle size of about 7 nm [calculated from the (101) reflection using Scherrer's equation, as previously performed (Ciccarrella et al. 2009)]. These findings indicate the presence of NPs in anatase form, highly crystalline, and without apparent structural defects. Furthermore, the particle size determined by XRD is in a good agreement to that obtained by TEM.

The powders of Kv 11.1-Mab-PEG–TiO₂ NPs were also characterized by XPS analysis to check the nature of chemical functions present on the NP surface. Representative C 1s peaks recorded on TiO₂ NPs before and after the immobilization of the Kv 11.1-Mab are given in Fig. 3. The interpretation of this peak is based on the methodology developed specifically for biosystems (Rouxhet and Genet 2011) and other complex interfaces with biologic interest (Landoulsi et al. 2008; Spadavecchia et al. 2013). Before the immobilization of Kv 11.1 Mab, the C 1s peak exhibited (i) a component at 284.8 eV due to carbon bound only to carbon and/or hydrogen [C–(C,H)]; (ii)
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Structural analysis of Kv 11.1-Mab-PEG–TiO₂: a TEM pictures of clusters of PEG–TiO₂ NPs in solution; b TEM pictures of clusters of Kv 11.1-Mab-PEG–TiO₂ NPs in solution; c particle size distribution of Kv 11.1-Mab-PEG–TiO₂ NPs; and d XRD Pattern</figcaption></figure>

an intense component at about 286.3 eV due to carbon
making a single bond with oxygen and/or nitrogen [C–
(O,N)], which reveals the dominating presence of PEG
function; and (iii) a component at about 289.0 eV
attributed to carboxyl and ester functions [O–C=O].
These results confirm the expectations regarding the

<figure><img src="image_5.png" /><figcaption>**Fig. 3** XPS analysis. C 1s and N 1s peaks recorded on PEG–TiO₂ NPs a before and b after immobilization of Kv 11.1-Mab</figcaption></figure>

chemical function present on the nPEG–TiO₂ NPs.
After immobilization of the Kv 11.1-Mab, a signifi-
cant decrease of the contribution at 286.3 eV is
observed, and a contribution at about 287.9 eV, due
to carbon making one double bond [C=O] with oxygen
in amide, appeared. This suggests that the protein is
present on the NP surface. Further support to these
findings is given by the analysis of the N 1s peak
(Fig. 3), which shows a noticeable increase after
protein immobilization. The main component in the N
1s peak is situated at about 400.0 eV, attributable to
amide or amine [C–N], with no additional components
near 401.6 eV, due to protonated amine. The presence
of nitrogen before the immobilization of the Kv 11.1-
Mab is due to compounds from adventitious contam-
ination, as broadly observed with XPS on high energy
solid surfaces.

In order to achieve a NP system that is capable of
targeting specific cells, the binding procedure must
preserve the biologic activity, and in particular, the
specificity, of the antibody. Therefore, we first eval-
uated the binding capacity and specificity of the Kv
11.1-Mab-PEG–TiO₂NPs, performing an ELISA
assay against the peptide (S5-P peptide) used for
raising the antibody (Guasti et al. 2008). This assay
can be also used to verify the proper orientation of the
Kv 11.1-Mab conjugated to the PEG–TiO₂ NPs.
Figure 4a shows the 450-nm absorbance values
obtained after incubation of either PEG–TiO₂ or Kv
11.1-Mab-PEG–TiO₂ NPs onto S5-P peptide-coated
<figure><img src="image_6.png" /><figcaption>**Fig. 4** NPs' interaction with PDAC cells: a binding specificity of Kv 11.1-Mab-PEG-TiO₂ NPs determined through an ELISA assay performed on the S5P peptide; b Kv 11.1 expression in NIH-3T3, MIAPaCa-2, and Panc-1 cells. Immunofluorescence was performed on cells seeded onto glass slides coated with 20 µg/mL Fibronectin, by means of Kv 11.1-Mab and Alexa-488-conjugated secondary antibody. Photographs were taken</figcaption></figure>

or BSA-coated wells. PEG-TiO₂ NPs showed no binding to the S5-P peptide, whereas Kv 11.1-Mab-PEG-TiO₂ NPs recognized and bound the S5-P peptide-coated wells in a dose-dependent manner. In particular, Kv 11.1-Mab-PEG-TiO₂ NPs at 0.1 % concentration gave an absorbance value of roughly half of that obtained with 10 µg/mL of the whole antibody molecule, suggesting good binding efficiency of the antibody to the NPs, as well as the maintenance of good antigen recognition. Conversely, no binding of Kv 11.1-Mab-PEG-TiO₂ NPs to BSA-coated wells was detected. We can conclude that the Kv 11.1-Mab, also when bound to PEG-TiO₂ NPs, efficiently and specifically binds its proper antigen.

We then assessed whether Kv 11.1-Mab-PEG-TiO₂ NPs properly recognized the whole antigen, e.g., the Kv 11.1 potassium channel, when expressed on the plasma membrane of living cells. To this purpose, we

using a C1 confocal microscope (Nikon); c binding of Kv 11.1-Mab-PEG-TiO₂ NPs on cells: MIAPaCa-2 and NIH-3T3 cells were treated with different concentrations of Kv 11.1-Mab-PEG-TiO₂ and IgG-Mab-PEG-TiO₂; d cell viability (WST-1) assay performed on MIAPaCa-2 and Panc-1 cells in the presence of either PEG-TiO₂ or Kv 11.1-Mab-PEG-TiO₂ NPs, both at 0.1 % concentration

used two human PDAC cell lines (MIAPaCa-2 and Panc-1), which express Kv 11.1 at the plasma membrane level (see immunofluorescence pictures in Fig. 4b). On the other hand, NIH-3T3 cells, which are devoid of Kv 11.1 expression (Fig. 4b) were used as negative control. Figure 4c shows data relative to Panc-1 and NIH-3T3 cells: Kv 11.1-Mab-PEG-TiO₂ NPs efficiently bound Panc-1 cells, in a dose-dependent manner, whereas they did not bind NIH-3T3 cells. On the other hand, NPs conjugated with an irrelevant antibody (IgG-Mab-PEG-TiO₂) did not bind either Panc-1 or NIH-3T3 cells. Hence, Kv 11.1-Mab-PEG-TiO₂ NPs efficiently and specifically bind their antigen, when it is physiologically expressed onto the plasma membrane of tumor cells.

We then assessed the cytotoxicity of PEG-TiO₂ and Kv 11.1-Mab-PEG-TiO₂ NPs, both diluted at 0.1 % concentration into culture medium, through the
**Fig. 5** TEM pictures of PDAC cells trated with PEG–TiO₂ NPs or Kv 11.1-Mab-PEG–TiO₂ NPs: a, c Panc-1 treated with PEG–TiO₂ NPs (0.1 %). b MIAPaCa-2 treated with Kv 11.1-Mab-PEG–TiO₂ NPs (0.1 %); d Panc-1 treated with Kv 11.1-Mab-PEG–TiO₂ NPs (0.1 %); TEM was performed after exposure for 48 h to either PEG–TiO₂ or Kv 11.1-Mab-PEG–TiO₂ NPs. *Scale bars* are reported on the *bottom left* of each panel

nPEG-TiO₂

<figure><img src="image_7.png" /></figure>

Kv11.1-PEG-TiO₂

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

WST1 assay. Both MiaPaCa-2 and Panc-1 cells were analyzed, and the corresponding results are shown in Fig. 4d: neither PEG–TiO₂ nor Kv 11.1-Mab-PEG–TiO₂ NPs affected cell viability, in both types of cells.

Finally, we evaluated by TEM analysis whether Kv 11.1-Mab-PEG–TiO₂ NPs could by internalized into PDAC cells. TEM was performed on 70-nm sections of either MIAPaCa-2 or PANC-1 cells, after exposure for 48 h to either PEG–TiO₂ or Kv 11.1-Mab-PEG–TiO₂ NPs, both at 0.1 % concentration. Pictures are shown in Fig. 5 and in the Supplementary Section, Fig. S2. Kv 11.1-MAb-PEG–TiO₂ NPs were found to

be well internalized (see Fig. 5b), whereas PEG–TiO₂ NPs showed undetectable intracellular localization (Fig. 5a, c). Both panels (a) and (b) show non contrasted cells, and the NPs are clearly evident (see the white box in panel (b) and its higher magnification below). Only Kv 11.1-Mab-TiO₂ NPs are present inside the cell, as evidenced in the picture in panel (b). In particular, a large endocytotic vesicle is present inside the cytoplasm, filled with clusters of NPs. In some cells, we found that Kv 11.1-MAb-PEG–TiO₂ NPs reached the nucleus (Fig. 5d, S2). Note that panels (c) and (d) show contrasted cells, and the NPs
are hence more conspicuous only in the magnifications reported below the corresponding panels. The size and shape of the Kv 11.1-MAb-PEG-TiO₂ NPs internalized into the cells remained unchanged compared with NPs in aqueous solution (compare the magnification with the scale bar at 20 nm in the inset to Figs. 2b, 5d).

## Discussion

TiO₂ nanoformulations have been widely used in many nanotechnology areas, but their use in nanomedicine for drug delivery purposes is still hampered by the high toxicity that TiO₂ NPs exert on living cells. In this article, we report the development of new, nontoxic TiO₂ nanoparticles, further conjugated with a monoclonal antibody against a novel and easily accessible tumor marker, e.g., the Kv 11.1 potassium channel, proposed for drug delivery purposes.

We synthesized, by simple solvothermal method, dicarboxylic acid-terminated PEG-TiO₂ NPs, further conjugated with the Kv 11.1-Mab, characterized by a high homogenous shape and extremely small size (about 7 nm). Hence, we obtained TiO₂ NPs with a large surface area, and therefore with an ensuing increase of the number of available surface-active sizes (Di Paola et al. 2008). The narrow-size distribution should lead to a high photonic efficiency favoring a higher interfacial charge carrier transfer rate and a better photocatalytic activity of NPs.

PEG-TiO₂ NPs were further conjugated with a monoclonal antibody directed against a novel tumor target, e.g., Kv 11.1 potassium channel. While the relevance of ion channels in tumor cell biology has been increasing in the recent years (Arcangeli and Yuan 2011) this is the first article exploiting voltage-dependent potassium channels as targets for nanodeivery. Only a recent article (Li et al. 2013) showed the development of nanoparticles carrying small interfering RNAs against Kv 11.1 channels, further stressing the relevance of these channel proteins in innovative cancer treatments. We showed that Kv 11.1-Mab-PEG-TiO₂ NPs efficiently and specifically recognized the specific Kv 11.1 antigen, both in vitro and in PDAC cells which express the Kv 11.1 channel onto the plasma membrane. The proper orientation of the targeting moiety promotes the antigen binding and the subsequent spontaneous internalization by the target

cells without using physical methods such as electroporation (Xu et al. 2007). Indeed, we proved this fact by showing that only Kv 11.1-MAb-PEG-TiO₂ NPs are well internalized into PDAC cells. In particular, we observed large endocytotic vesicle inside the cytoplasm, filled with clusters of NPs. Such appearance is consistent with a mechanism of endocytosis underlying Kv 11.1-MAb-PEG-TiO₂ NPs intracellular uptake, similar to that reported for other NPs in other cellular types (Wilhelm et al. 2003; Riviere et al. 2007). It is worth noting that the uptake kinetics of NPs by cells have been only slightly characterized and quantified as a function of their size and shape (Chithrani et al. 2006). Most studies have focused on liposomes (Chenevier et al. 2000) and polymer particles (Jaulin et al. 2000; Alyaudtin et al. 2001) which are generally larger than 100 nm (Alyaudtin et al. 2001). Very recently (Grudzinski et al. 2013), carbon-coated iron NPs were shown to be internalized into melanoma cells, with a pattern of internalization similar to that observed by us.

Finally, we showed that both PEG TiO₂ and Kv 11.1-Mab-PEG-TiO₂ NPs were not cytotoxic to PDAC cells. Since other studies have shown a significant toxicity of TiO₂ NPs (Jin et al. 2008; Okuda-Shimazaki et al. 2010), our data stress the prominent role of the coating (e.g., PEG) in avoiding cytotoxicity (Mano et al. 2012), a fact that could be related to either the nature or the charge of the coating.

The lack of cytotoxicity stresses even more the possible applications of the targeted NPs, which we have developed for cancer therapy. In fact, our NPs could be used for therapeutic purposes by either exploiting the photocatalytic features of TiO₂ with the ensuing ROS production or introducing new moieties (e.g., porphyrins, phthalocyanines) or cytotoxic drugs inside the NPs. Due to the low toxicity of the PEG-TiO₂ NPs that we have developed, both these approaches will be tested in vivo using appropriate PDAC mouse models.

**Acknowledgments** The present study was supported by grants from the Italian Association for Cancer Research (AIRC) to AA. The authors would like to thank Christophe Méthivier (LRS-UPMC-Paris) for technical assistance and discussion about XPS analysis.

**Open Access** This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
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