Drug Delivery: Antibody-Functionalized Porous Silicon Nanoparticles for Vectorization of Hydrophobic...

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Antibody-Functionalized Porous Silicon Nanoparticles for Vectorization of Hydrophobic Drugs

Emilie Secret , Kevin Smith , Valentina Dubljevic , Eli Moore , Peter Macardle , Bahman Delalat , Mary-Louise Rogers , Terrance G. Johns , Jean-Olivier Durand , Frédérique Cunin , * and Nicolas H. Voelcker *

We describe the preparation of biodegradable porous silicon nanoparticles (pSiNP) functionalized with cancer cell targeting antibodies and loaded with the hydrophobic anti-cancer drug camptothecin. Orientated immobilization of the antibody on the pSiNP is achieved using novel semicarbazide based bioconjugate chemistry. To demonstrate the generality of this targeting approach, the three antibodies MLR2, mAb528 and Rituximab are used, which target neuroblastoma, glioblastoma and B lymphoma cells, respec-tively. Successful targeting is demonstrated by means of fl ow cytometry and immunocytochemistry both with cell lines and primary cells. Cell viability assays after incubation with pSiNPs show selective killing of cells expressing the receptor corresponding to the antibody attached on the pSiNP.

1. Introduction

Nanomedicine holds great promise for cancer therapy. [ 1–5 ] In particular, the use of nanomaterials as anticancer drug nanovectors is expected to overcome some of the issues inherent to conventional chemotherapy, including the poor

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DOI: 10.1002/adhm.201200335

E. Secret, Dr. J. O. Durand, Dr. F. CuninInstitut Charles Gerhardt MontpellierUMR 5253 CNRS-ENSCM-UM2-UM1Ecole Nationale Supérieure de Chimie de Montpellier34296 Montpellier, France E-mail: [email protected] K. Smith, Dr M. L. RogersCentre for NeuroscienceDepartment of Human PhysiologySchool of Medicine Flinders UniversityBedford Park, SA 5042, Australia V. Dubljevic, Prof. T. G. JohnsMonash Institute of Medical ResearchClayton, VIC 3168, Australia E. Moore, Dr. B. Delalat, Prof. N. H. VoelckerMawson InstituteUniversity of South AustraliaMawson Lakes, SA 5095, AustraliaE-mail: [email protected] Dr P. MacardleDepartment of Immunology Allergy and ArthritisSchool of MedicineFlinders UniversityBedford Park, SA 5042, Australia

Adv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201200335

pharmacokinetic profi les of the anti-cancer drugs and their lack of tumor specifi city. The propensity of nanoscale materials (compared to single small mol-ecules) to accumulate in a tumor through the enhanced permeation and retention (EPR) effect provides many possibilities to design effective drug delivery nanosys-tems that vectorize poorly water soluble toxic anticancer drug to tumor sites. [ 6 ] Nanoparticles or other delivery systems studied for cancer therapy include lipo-somes, [ 7 , 8 ] polymers spheres and cap-sules, [ 9 , 10 ] semi-conductor quantum dots, [ 11 , 12 ] iron oxide [ 13 , 14 ] and gold nano-particles, [ 15 , 16 ] carbon nanotubes, [ 17 , 18 ]

mesoporous silica nanoparticles [ 19 , 20 ] and metal-organic frame-works (MOFs). [ 21 ] Porous silicon (pSi) nanoparticles (pSiNP) are particularly attractive for nanomedicine because they are biodegradable [ 22–26 ] in vivo and the degradation product, silicic acid, is non-toxic. The few studies using pSiNP for biomed-ical applications published so far have focused on imaging, [ 24 ] non-targeted drug delivery, [ 27 ] photodynamic therapy, [ 28 ] photo-thermal therapy [ 29 ] and immunotherapy. [ 30 ]

While nanoparticles tend to passively accumulate in tumor tissue due to the EPR effect, which is ascribed to enhanced vas-cular permeability in tumors, [ 31 ] active endocytosis of nanopar-ticles targeting cancer cells is equally desired in order to further increase the concentration of the anticancer drug intracellularly, and to limit its toxic effects in normal tissue. This is achiev-able by targeting membrane receptors overexpressed in cancer cells. Different types of targeting agents have been attached to nanoparticles, [ 32 ] including peptides, [ 33 ] antibodies, [ 34 , 35 ] small molecules as folic acid [ 36 ] or mannose, [ 19 ] and RNA aptamers. [ 12 ] Vectorization of drugs using functionalized pSiNP in cancer therapy has not been described yet.

In this work, we pursued the vectorization of the hydrophobic alkaloid camptothecin using pSiNP targeted towards various types of cancer cells. Camptothecin is an anticancer agent that targets a nuclear enzyme: DNA topoisomerase I, which plays a crucial role in the removal of the DNA supercoiling associated with DNA replication and transcription. [ 37 , 38 ] This drug entered in phase I and II clinical trials, but had to be abandoned fol-lowing the onset of toxic manifestations because of overexpo-sure. [ 39 ] Vectorization of camptothecin with nanoparticles is a highly promising route to avoid the drawbacks of this drug, and breakthroughs in this fi eld have recently been demo nstrated

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Scheme 1 . (a) Reaction scheme for drug loading and antibody-grafting on the pSiNP surface. Si-H terminated nanoparticles undergo hydrosilylation with protected semicarbazide, followed by deprotection, drug loading and reaction with periodate-oxidized antibodies; (b–d) schematic showing vectorization of the hydrophobic alkaloid camptothecin using pSiNP to cancer cells.

in vitro [ 40 , 41 ] and in vivo [ 42 , 43 ] with mesoporous silica nanoparticles functionalized with folic acid. Here, we show that vectorization of camptothecin with biodegradable pSiNP is an attractive alter-native to mesoporous silica nanoparticles. Furthermore, pSiNP, loaded with camptothecin, were bio-functionalized with a series of cell-targeting antibodies using a novel conjugation strategy ( Scheme 1 a). In contrast to the common non-specifi c antibody immobilization procedures, we used an oriented immobiliza-tion strategy where the active site (Fab fragment) of the antibody remains accessible for biomolecular recognition. The nanoparti-cles were fi rst hydrosilylated to functionalize them with a semi-carbazide group. [ 44 , 45 ] Antibody binding was then performed after periodate oxidation of the Fc region’s carbohydrate side-chain to generate an aldehyde. To demonstrate the generality of the vectorization approach, three antibodies were grafted on the pSiNP: MLR2 (monoclonal antibody to p75NTR), mAb528 (monoclonal antibody to EGFR) and Rituximab (monoclonal antibody to CD20), targeting glioblastoma, neuroblastoma and B cell lymphoma cells, respectively. The loading and release kinetic of camptothecin in the pSiNP were also studied, and the camp-tothecin-loaded nanoparticles grafted with the antibodies were found to be very effi cient to target and kill cancer cells in vitro .

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Figure 1 . (a,b) TEM images of the pSiNP at low (a) and high (b) resolution. (c) SEM image of a100 nm on (b) and (c).

2. Results and Discussion

2.1. Preparation and Functionalization of the pSi Nanoparticles

pSiNP were prepared by electrochemical etching of crystalline silicon, followed by lifting up the porous membrane, fracturing the membrane into particles by ultrasoni-cation and fi ltering the formed particles through a 0.2 μ m membrane. Scanning elec-tron microscopy (SEM) and transmission electron microscopy (TEM) images of the obtained sample show mesoporous nanopar-ticles with sizes in the range of 35–195 nm ( Figure 1 ). Dynamic light scattering meas-urements confi rmed that nanoparticles with dimensions between 40 nm and 200 nm with a mean size of 115 nm were prepared (data not shown). Such a size range is suit-able for targeted (anti-cancer) drug delivery applications where the size of the nanocar-riers should not exceed 400 nm for vascular extravasation (EPR effect), and neither fall

below 10 nm to avoid renal clearance. [ 6 ] SEM and TEM images also revealed that the nanoparticles featured 2D-channels. Nitrogen adsorption-desorption analysis performed on the sam-ples allowed us to calculate the average pore channel diameter, the porous volume, and the specifi c surface area of the nano-particles. The pSiNP had a porous volume of 1.5 mL g − 1 , a BET surface of 502 m 2 g − 1 and mesopores with an average diameter of 23.5 nm.

Immobilization of the antibodies on the surface of the freshly etched pSiNP was realized following the different steps outlined in Scheme 1 . Coupling between the pSiNP and the antibody involved a semicarbazide functional linker, which was chosen since it allows orientated attachment of the anti-body by its carbohydrate side-chain (Fc fragment), thereby keeping its active site accessible for potential biorecognition. [ 45 ] Silicon hydride terminated pSiNP were fi rst hydrosilylated with tert-butyl-2-[(allylamino)carbonyl]hydrazine-carboxylate, a Boc-protected semicarbazide functional alkene. Protection of the semicarbazide was required to avoid interference with the hydrosilylation reaction. Deprotection of the semicarbazide linker was achieved by treatment with trifl uoroacetic acid (TFA). IR spectra of the hydrosilylated nanoparticles before and

heim

n individual pSiNP. Scale bar is 200 nm on (a), and



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Figure 2 . IR spectra of (a) pSiNP hydrosilylated with the protected semicarbazide and (b) pSiNP after deprotection of the semicarbazide group. The insert shows a zoom of the region between 1200 cm − 1 and 3800 cm − 1 .

after deprotection of the semicarbazide linker are presented in Figure 2 . Spectrum a shows bands at 1706 cm − 1 and 1655 cm − 1 , corresponding to the C = O carbamate and to the C = O urea stretching modes, respectively, which demonstrate the presence of the Boc-protected semicarbazide on the pSiNP. Bands at 2971 cm − 1 , 2921 cm − 1 and 2847 cm − 1 were assigned to the C-H stretching vibrations of the aliphatic chains, and of the methyl group in the Boc protected semicarbazide. After the deprotec-tion step, the C = O carbamate band disappeared (spectrum b), indicating that the Boc groups were successfully removed. Removal of the Boc group is also consistent with the decrease of the intensity of the bands in the C-H stretching region. In both spectra, bands observed at 2245 cm − 1 and 2103 cm − 1 attributed to Si-H stretching modes are indicative of incomplete Si site coverage by the semicarbazide ligand, which is expected for this reaction. [ 56 , 57 ]

After the deprotection of the semicarbazide, the nanoparti-cles were loaded with camptothecin by simple impregnation and agitation of the nanoparticles in a 2.5 mg mL − 1 solution of camptothecin in dimethylformamide (DMF). Camptothecin loading was performed following semicarbazide functionaliza-tion but prior to the coupling of the antibody in order to avoid premature drug release during functionalization and subse-quent rinsing steps and avoid exposure of the antibody to a denaturing organic solvent. To determine the amount of camp-tothecin loaded in the porous silicon nanoparticles, we dissolved the particles in a KOH solution, and measured the absorbance of this solution at 432 nm. We determined that approximately

© 2012 WILEY-VCH Verlag GmAdv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201200335

315 nmol of camptothecin were loaded per mg of functionalized pSiNP, which is considerable payload. In comparison, on mes-oporous silica nanoparticles, 1.6 to 29 nmol of camptothecin were loaded per mg of nanoparticles. [ 41 , 42 ]

In parallel, chemical modifi cations were performed on the antibodies of interest to prepare them for coupling with the semicarbazide-functionalized pSiNP. The carbohydrate side-chain on the Fc fragment of the antibodies was fi rst oxidized using NaIO 4 to generate aldehyde functional groups. [ 58 ] These react readily with the hydrazine functionality of the deprotected semicarbazide and form a stable covalent bond. [ 45 ] Unlike the commonly used carbodiimide coupling chemistry, our approach allows the site-specifi c immobilization of the antibodies on the pSiNP. We used oxidation procedure to retain antibody activity and observed that short oxidation times of 30 minutes or less and rapid removal of the oxidant during the workup were crucial since long exposure of the antibody to sodium periodate led to non-specifi c oxidation and loss of the antibody’s specifi city (data not shown). Gel fi ltration purifi cation process was therefore preferred to purifi cation by successive dialysis. In a subsequent reaction, the oxidized antibodies were labeled with fl uorescein isothiocyanate (FITC) to facilitate analysis by fl ow cytometry, and fi nally coupled to the semicarbazide-functionalized pSiNP. The antibody grafting time was deliberately kept short to mini-mize camptothecin release from the nanoparticles.

Three antibodies were grafted on the pSiNP via this route, which are MLR2, mAb528 and Rituximab. The MLR2 antibody was developed to target the p75NTR receptor on nerve cells. [ 48 ] p75NTR is overexpressed on some neuroblastoma cell lines including on SH-SY5Y neuroblastoma cells. The BSR fi broblast cell line, which does not express p75NTR receptor, was chosen as a negative control. [ 48 ] Second, the mAb528 antibody targets the epithelial growth factor receptor (EGFR), which is overexpressed in several types of cancer including brain, breast, lung, colon, kidney or prostate cancer. Targeting of EGFR by the mAb528-functionalized nanoparticles was investigated on the two dif-ferent glioblastoma cell lines U87MG and U87MG transfected with the de2-7EGFR gene (U87MG. Δ 2–7). U87MG is a glioma cell line and expresses 5 × 10 4 wild type EGFR per cell. The de2-7EGFR gene is the most common EGFR mutation in glioma. U87MG. Δ 2–7 express both the wild type EGFR but co-express de2-7EGFR at 2 × 10 6 receptors per cell. For mAb528, SH-SY5Y served as a negative control since this cell line does not express EGFR. Finally, Rituximab was used in this study to target the CD20 receptor on B lymphocytes. Rituximab is already in clinical use as a therapeutical antibody in lymphoma therapy. [ 59 ] CD20 is overexpressed on B cell lymphomas including Burkitt’s lym-phoma HRIK cells and DOHH-2 (follicular B cell lymphoma). As a control cell line, an immortalized T lymphocyte cell line (Jurkat) was chosen, which do not express CD20. In all three cases, a signifi cant decrease in the absorption of the antibody solution at 280 nm after coupling to the pSiNP was observed proving successful coupling (data not shown).

The release kinetics of camptothecin from antibody-grafted nanoparticles was studied for the pSi-MLR2 nanoparticles, by monitoring the fl uorescence intensity of camptothecin in cell culture medium ( Figure 3 ). For comparison, the release kinetics of camptothecin from pSiNP, which were functionalized with the deprotected semicarbazide but not yet with the antibody,

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Figure 3 . Release kinetic of camptothecin from (✕) pSiNP without anti-body, and ( � ) pSiNP grafted with MLR2 antibody.

was also investigated. In both cases, about 80% of the loaded camptothecin was released from the nanoparticles within 3 hours, indicating that the presence of the antibody at the sur-face of the pSiNP did not signifi cantly affect the drug release profi le. Admittedly, the total amount of camptothecin released from the antibody-grafted nanoparticles was slightly lower than the amount of camptothecin released from the nanopar-ticles without antibodies. This difference was attributed to the fact that some camptothecin was released during the antibody grafting step.

2.2. Cancer Cell Targeting with Antibody-Functionalized Nanoparticles

Flow cytometry was used to test the ability of FITC labeled anti-body-functionalized nanoparticles to target cancer cells, as cell surface binding is essential for effi cient uptake of the nanopar-ticles and the release of cytotoxic drug either at the cell surface or inside the cells. Note that for this experiment, the nanoparti-cles were not loaded with camptothecin. The FITC fl uorescence histograms are presented ( Figure 4 ) for three cell types SH-SY5Y, U87MG, and HRIK, as well as for the respective control cell types BSR, SH-SY5Y and Jurkat. The fl uorescence signal observed on all the spectra after cells were incubated for 30 min with PBS corresponds to cell autofl uorescence (black curves). When the SH-SY5Y, U87MG, and B lymphocyte cells were exposed to the antibody-functionalized pSiNP - pSi-MLR2, pSi-MAb528 and pSi-Rituximab, respectively - a signifi cant shift of the histograms (Figure 4 a,c,e) to higher fl uorescence intensity was observed (red curve), indicating that the antibody-function-alized nanoparticles effectively bind to the cells within 30 min-utes. In contrast, when the same experiment was performed on the control cells, which do not express the receptor for the anti-body bound to the pSiNP, no shift of the fl uorescence curve was observed (Figure 4 b,d,f), suggesting that the three nanopar-ticle-bound antibodies retain their specifi city. This experiment hence demonstrated the successful targeting of antibody-func-tionalized pSiNP to cells expressing the corresponding receptor within a time frame over which only a small percentage of drug (around 15%) is released.

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Immunocytochemistry was used to confi rm the targeting of primary cells from brain tissue isolates that express the p75 receptor by the pSi-MLR2 nanoparticles. As demonstrated in Figure 4 , the pSi-MLR2 nanoparticles, labeled with FITC can target cells that express p75NTR, we further wanted to demon-strate this specifi city in mixed primary cultures. After culturing primary motor neurons that express p75NTR [ 54 ] with astrocytes that were all isolated from E12 mice embryonic spinal cords, the cells were fi xed and subjected to immunocytochemistry ( Figure 5 ). p75NTR is present on motor neurons (goat anti-p75NTR, red), and motor neurons were cultured on a bed of astrocytes (identifi ed by GFAP staining; pink) that do not con-tain p75NTR. The nuclei of all cells is seen by DAPI staining (blue). The pSi-MLR2 nanoparticles, labeled with FITC (green), stained a small proportion of cells, and exclusively those that also stained with p75NTR (so co-coloured as yellow). Notably, none of the pSi-MLR2 nanoparticles, labeled with FITC were present on astrocytes (pink cells), thus indicating that pSi-MLR2 targeting also worked for primary cells.

The targeting of lymphocytes was also confi rmed by means of imaging fl ow cytometry ( Figure 6 ). The fl uorescence due to the FITC labeled pSi-Rituximab was observed only on the DOHH-2 cells, which are B cells expressing the CD20 receptor. The absence of fl uorescence on the Jurkat cells indicates that the nanoparticles did not bind to T cells. Hence, binding was specifi c for the anti-CD20 antibody and not due to passive inter-nalization of the nanoparticles.

2.3. Vectorization of Camptothecin to Cancer Cells

The ability of the antibody-functionalized pSiNP loaded with camptothecin to target and kill cancer cells in vitro was inves-tigated in SH-SY5Y, and in U87MG and U87MG. Δ 2–7, which express the p75 and EGFR receptors, respectively.

SH-SY5Y and BSR cells were treated with free MLR2 anti-body, with MLR2-grafted pSiNP (pSi-MLR2), with pSiNP loaded with campthotecin, but not grafted with MLR2 antibody (pSi-CPT), and with pSiNP loaded with camptothecin and grafted with MLR2 antibody (pSi-MLR2-CPT). In addition, NSC-34, which are made through fusion of mouse neuroblastoma and spinal motor neurons and also express the p75NTR receptor, were exposed to the same nanoparticle treatments. Cells were treated with the nanoparticles for 30 minutes at 50 μ g mL − 1 concentration, rinsed and the cell culture medium was changed. The cells were then incubated for 4 days. A Trypan Blue exclu-sion assay was performed in order to count the living cells. In a control experiment, the cells were incubated directly for 4 days with the nanoparticles, without the rinsing step. The results are presented in Figure 7 for the SH-SY5Y (a) the con-trol BSR (b), and the NSC-34 (c). These results clearly indicate that, MLR2-grafted pSiNP (pSi-MLR2) without camptothecin were non-toxic to the cells. 90 to 100% of the SH-SY5Y cells and of the NSC-34 cells remained alive after being exposed to the targeting pSi-MLR2 nanoparticles for 4 days, independently of the rinsing step. About 90% of the BSR cells exposed to the pSi-MLR2 nanoparticles survived after 4 days of incubation as well. When the cells were incubated for 4 days with pSi-CPT (omitting the rinsing step), 100% and 99% of cell death was

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Figure 4 . Flow cytometric analysis of the various types of cells incubated for 30 minutes with PBS (black), and with pSiNP grafted with the anti-body (red). (a) SH-SY5Y/pSi-MLR2, (b) BSR/pSi-MLR2, (c) U87MG/pSi-mAb528, (d) SH-SY5Y/pSi-mAb528, (e) HRIK/pSi-Rituximab, (f) Jurkat/pSi-Rituximab.

observed for SH-SY5Y and NSC-34, respectively, and up to 45% for BSR. In this case, cell death was attributed to the release of camptothecin into the cell culture medium over the course of the experiment. In contrast, if the medium containing pSi-CPT particles was removed after 30 minutes, no signifi cant cell

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death was observed for either the SH-SY5Y and BSR. In con-trast, 43% of dead cells were counted for the NSC-34 cells. This suggests that fusion cell line is more sensitive to camptothecin than SH-SY5Y. When the cells were treated with pSi-MLR2-CPT, 69% of SH-SY5Y cells and 96% of NSC-34 which both express

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Figure 5 . Immunofl uorescence microscopy image of the pSi-MLR2 nano-particles targeting motor neurons cells growing on astrocytes. Pink: glia (GFAP staining), red: p75 expressing motor neurons, blue: nuclei: green: FITC labeled pSi-MLR2 nanoparticles.

the p75NTR receptor, died upon 30 minutes of exposure and rinsing, and 96% of SH-SY5Y cells and 96% of NSC-34 cells died when the rinsing step was omitted. The toxicity of the MLR2-grafted pSiNP loaded with camptothecin was therefore signifi cantly increased compared to the pSi-CPT nanoparticles without MLR2. In addition, control experiments on BSR cells with the pSi-MLR2-CPT nanoparticles showed no signifi cant cell death (about 11%) upon 30 minutes of exposure to the nanoparticles. These results demonstrate successful vectoriza-tion of the camptothecin to neuroblastoma and primary neuron cells using MLR2 functionalized pSiNP.

pSi vectorization was also investigated for glioblastoma cell lines U87MG and U87MG. Δ 2–7 which were treated with mAb528-grafted pSiNP (pSi-mAb528), pSiNP loaded with camp-tothecin but not grafted with mAb528 antibody (pSi-CPT), and pSiNP loaded with camptothecin and grafted with the mAb528


Figure 6 . Images taken by an imaging fl ow cytometer. (a) DOHH-2 cells anwere chosen randomly. Ch01 images are brightfi eld images, Ch02 are fl uor

antibody (pSi-mAb528-CPT). In this case, two different concen-trations of nanoparticles were tested on each type of cells. The cells were treated with the nanoparticles for 1 hour, rinsed, the cell culture medium changed, and then incubated for 4 days to determine the effect of the nanoparticle treatment on cell via-bility by means of a MTS assay. In a parallel experiment, the cells were incubated directly for 4 days with the nanoparticles, omitting the rinsing step. The cell viability results are presented in Figure 8 . When not loaded with camptothecin, pSi-mAb528 showed a low level of toxicity on the two types of cells. At the lower concentration of pSiNP tested (10 μ g mL − 1 ), all of the U87MG and of the U87MG. Δ 2–7 cells remained viable after 4 days of incubation with pSi-mAb528. At a higher nanoparticle concentration (50 μ g mL − 1 ), these percentages decreased to 85% of live U87MG and 95% of live U87MG. Δ 2–7 cells. pSi-CPT nanoparticles killed about 90% of the U87MG cells when the rinsing step was omitted. In contrast, only modest cytotoxicity was observed when the medium containing pSi-CPT at both 50 and 10 μ g mL − 1 was rinsed after 1 hour. This residual cyto-toxicity is presumably due to camptothecin which has already been released from the nanoparticles during the initial incu-bation, before the rinsing step. Finally, in both cases (with or without rinsing the cells after 1 hour of incubation), signifi cant cell killing was observed after incubation with the targeting pSi-mAb538-CPT. Similar results were obtained using U87MG. Δ 2–7 cells, where the targeting pSi-mAb528-CPT nanoparticles virtu-ally eliminated all of the cells (Figure 8 b). Therefore, pSiNP deri-vatized with the anti-EGFR antibody mAb528 were rapidly inter-nalized (within 1 hour) and released their camptothecin payload leading to effective death in cells expressing the wild type EGFR or the glioma associated mutant de2-7EGFR.

3. Conclusion

In conclusion, we have successfully demonstrated the potential of pSiNP for the delivery of hydrophobic drugs to kill cancer cells. pSiNP were prepared by electrochemical etching of Si.

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d (b) Jurkat cells, incubated with pSi-Rituximab labeled with FITC. Images escence images (excitation 488 nm, emission 480–560 nm).



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Figure 7 . Trypan Blue exclusion cell viability assays of (a) SH-SY5Y cells, (b) BSR cells and (c) NSC-34 cells incubated with the MLR2 antibody (MLR2), the pSiNP grafted with the MLR2 antibody (pSi-MLR2), the pSiNP loaded with camptothecin (pSi-CPT) and the pSiNP loaded with camptothecin and grafted with the MLR2 antibody (pSi-MLR2-CPT). Black: cells were incubated for 30 minutes with the nanoparticles, rinsed and incubated for 4 days before analysis. White: Cells were incubated for 4 days with the nanoparticles without a rinsing step. For each treatment, we used at least 3 replicates and signifi cance was analyzed by standard deviation.

The average size of pSiNP was 115 nm, with a specifi c surface area (500 m 2 g − 1 ) and an average pore size of 23.5 nm. These nanoparticles were therefore suitable to perform drug delivery. pSiNP were functionalized with antibodies in an original and controlled way to target the corresponding over-expressed anti-gens in cancer cells. Functionalized pSiNP were effi ciently taken up by cancer cells over-expressing the corresponding receptor in less than one hour as shown by fl ow cytometry, whereas control cells without the receptors did not interact with pSiNP. Immunocytochemistry confi rmed the specifi city of the uptake of pSiNP on primary motor neurons. After the demonstration of the functionalized pSiNP specifi city, pSiNP were loaded with camptothecin and cell killing was only observed on three types of cancer cells which over-expressed the corresponding receptor of the antibody-functionalized pSiNP, without affecting cells

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which do not express the receptor, when cells were washed 30 minutes to 1 hour after incubation. Work is in progress to further exploit the high potential of pSiNP for application in oncology, on animal models.

4. Experimental Section Cell Lines : BSR cells, a clone of baby hamster kidney fi broblast cells [ 46 ]

were kindly provided by Christine Tuffereau (Laboratorie de Génétique des Virus, CNRS, France). NSC-34 cells, a hybrid of neuroblastoma cells with embryonic spinal cord cells, were kindly supplied by Dr. Neil Cashman (University of Toronto, Canada). The U87MG. Δ 2–7 glioma cell line has been described previously in detail. [ 47 ]

Antibodies : Anti-p75NTR mouse monoclonal antibody (MLR2) was made and purifi ed as previously described. [ 48 ] Affi nity purifi ed rabbit polyclonal GFAP antibody was from Dako (Glostrup, Denmark). Rituximab (MabThera) was purchased form Roche (Basel, Switzerland) in 100 mg vials and stored at 4 ° C prior to use. The anti-EGFR antibody mAb528 was produced and purifi ed in the Biological Production Facility (Ludwig Institute for Cancer Research, Heidelberg, Melbourne, Australia) as previously described. [ 49 ]

Cell Culture : All plasticware was from Greiner Bio-One (Belgium). DMEM, Penicillin/Streptomyocin (PSG), Glutamax I (GX), fetal bovine serum (FBS), Hank’s Buffered Salt Solution (HBSS), mouse laminin, 0.05% Trypsin-EDTA 1X, 0.05% trypsin-EDTA 10X, Neurobasal, B27, horse serum, MEM, HEPES, antibody diluents, DAPI were all from Gibco (Life Technologies, Melbourne, Australia). Trypsin inhibitor, 2-mercaptoethanol, poly-L-ornithine, paraformaldehyde, donkey serum were all from Sigma-Aldrich, Australia. DNAse was from Roche Diagnostics Australia. Recombinant human BDNF was from eBioscience, San Diego, California. Human GDNF was from Gro Pep, Adelaide, Australia. Recombinant Rat CNTF was from R&D Systems, Minneapolis, Minnesota. U87MG and U87MG. Δ EGFR cells were maintained in DMEM/5% FCS; media for U87MG. Δ EGFR also contained G418 (400 mg mL − 1 ). The cell lines Jurkat (acute T cell leukaemia) and DOHH-2 (follicular B cell lymphoma) were stored in liquid nitrogen. Upon thawing, they were maintained in RPMI1640 medium supplemented with 5% fetal calf serum. Prior to use, cells were washed in media and the cell concentration adjusted to 10 7 mL − 1 . Further dilutions were made in media as required.

Preparation of pSiNP : Boron-doped Si p + + -type wafers (0.0008–0.0012 Ω · cm resistivity, < 100 > orientation) from Siltronix, France were electrochemically etched in a 3:1 volume ratio solution of aqueous hydrofl uoric acid (HF) (48%)/ ethanol (obtained from Sigma-Aldrich). Etching was performed in a Tefl on cell with a platinum ring counter electrode. A constant current of 200 mA cm − 2 was applied for 150 seconds, followed by rinsing 3 times with ethanol. The porous layer was then electropolished in a 3.3% HF solution in ethanol with a constant current of 4 mA · cm − 2 for 250 seconds. After 3 rinses with ethanol, the porous layer was placed in ethanol in a glass vial. After degassing the sample for 20 minutes under a nitrogen stream, the pSi fi lm was fractured by ultrasonication over 16 hours. The largest particles were then removed by centrifugation at 2700 g for 2 minutes. The supernatant was then fi ltered through a 0.2 μ m nylon fi lter membrane (Carl Roth). In order to remove the smallest particles, the solution was fi nally centrifuged at 22000 g for 30 minutes. The pellet of pSiNP was then redispersed in absolute ethanol.

Characterization of the pSiNP : Scanning electron microscopy (SEM) images were obtained using a high resolution Field Emission Gun S-4800 Hitachi microscope. To avoid sample charging anomalies, the pSi samples were coated with a thin layer of gold prior to the SEM analysis. Transmission electron microscopy (TEM) images were obtained using a Jeol 1200 EX II microscope. Dynamic light scattering (DLS) measurements were obtained on a Malvern 4800 instrument. Nitrogen adsorption-desorption volumetry experiments were performed at − 196 ° C using an ASAP2020 Micromeretics instrument. Prior to the analysis, 20.5 mg of pSiNP were outgassed overnight in situ at 30 ° C.

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Figure 8 . MTS cell viability assays of (a) U87MG cells incubated with pSiNP at 50 μ g mL − 1 , (b) U87MG cells incubated with pSiNP at 10 μ g mL − 1 , (c) U87MG. Δ 2–7cells incubated with pSiNP at 50 μ g mL − 1 and (d) U87MG. Δ 2–7cells incubated with pSiNP at 10 μ g mL − 1 . Black: cells were incubated for 60 minutes with the nanoparticles, rinsed and incubated further for 4 days before analysis. White: Cells were incubated for 4 days with the nano-particles (signifi cance was analyzed by standard deviation).

The pore diameter was determined using the Broekhoff and de Boer (BdB) method [ 50 , 51 ] whilst the samples’ surface area was determined from the Brunnauer–Emmett–Teller (BET) theory. [ 52 , 53 ]

Functionalization of the pSiNP with semicarbazide : Nanoparticles dispersed in ethanol were centrifuged at 22000 g for 30 minutes, and rinsed twice with THF. Nanoparticles were then dispersed in a solution of the protected semicarbazide (tert-butyl-2-[(allylamino)carbonyl]hydrazine-carboxylate) at 10 − 1 M in THF (3 mL). The synthesis of the semicarbazide is described elsewhere. [ 45 ] Hydrosilylation reaction was performed at 85 ° C for 3 hours under N 2 refl ux. Nanoparticles were then centrifuged and rinsed twice with THF, once with ethanol and once with dichloromethane (CH 2 Cl 2 ). To remove the Boc-group, nanoparticles functionalized with the protected semicarbazide were centrifuged at 22000 g for 30 minutes and then dispersed in a trifl uoroacetic acid (TFA)/ CH 2 Cl 2 (40% v/v) solution (5 mL). The obtained dispersion was agitated at room temperature for 4 hours. Then nanoparticles were rinsed twice with CH 2 Cl 2 , once with ethanol, once with water, once with ethanol and fi nally once with CH 2 Cl 2 .

Loading of the pSiNP with camptothecin : After the deprotection of the semicarbazide, nanoparticles were dispersed in a 2.5 mg mL − 1 camptothecin in dimethylformamide (DMF) solution (1 mL). The mixture was agitated in the dark for 2 hours at room temperature. The nanoparticles were then centrifuged at 22000 g for 30 minutes and rinsed in water and centrifuged again at 22000 g for 15 minutes.

The release experiments were performed by monitoring the fl uorescence intensity of camptothecin in cell culture medium. The solution was excited at 340 nm, the fl uorescence emission was monitored at 440 nm using a Perkin Elmer LS55 fl uorimeter.

Oxidation of the antibody : Oxidation of mAb528 and Rituximab: A diluted solution of the antibody at 10 mg mL − 1 was prepared with a 0.01 M sodium phosphate, 0.15 M sodium chloride buffer at pH = 7.2. A solution of sodium periodate (NaIO 4 ) at 0.1 M in the same buffer was added to the antibody solution at a ratio 1/10 in volume. The mixture was then allowed to react under agitation for 30 minutes in the dark at room temperature. The antibody was fi nally purifi ed by gel fi ltration with

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a Sephadex G25 column using a 0.1 M sodium phosphate and 0.15 M sodium chloride chromatography buffer at pH = 7.2.

Oxidation of MLR2: The antibody was diluted to be at a concentration of 2 mg mL − 1 with a 20 mM sodium acetate, 0.15 M sodium chloride buffer at pH = 5. A solution of NaIO 4 at 20 mM in the same buffer was added to the antibody solution at a ratio 1/1 in volume. The mixture was then allowed to react under agitation for 45 minutes in the dark at room temperature. Then the antibody was purifi ed by successive dialysis. The fi rst dialysis was performed in 2 L of the 20 mM sodium acetate, 0.15 M sodium chloride buffer at pH = 5 for 2 hours with a dialysis membrane which had a cutoff of 10 kDa. Then 3 successive dialyses were performed each for 2 hours in a 0.01 M potassium phosphate solution with 1% of Triton-X 100 at pH 6.5.

FITC labeling of the antibody : After oxidation, the antibody was dialyzed against Phosphate Buffered Saline (PBS, composed of 137 mM of NaCl, 10 mM of phosphate and 2.7 mM of KCl at pH = 7.4) for 24 hours and then against a 0.1 M solution of sodium bicarbonate NaHCO 3 for 24 hours. The concentration of the antibody was measured by spectrophotometry, using an Agilent HP8453. FITC was dissolved at 2 mg mL − 1 in DMSO just prior to use. For an antibody solution concentration lower than 1 mg mL − 1 , FITC was added at a molar ratio of 1/5 of the antibody. For an antibody concentration greater than 1 mg mL − 1 , the FITC was added at a molar ratio of 1/10 of the antibody. FITC was added to the antibody solution, and the solution was agitated in the dark at room temperature for 4 hours. The antibody was fi nally purifi ed from the unreacted FITC by gel fi ltration with a Sephadex G25 column with a 0.1 M sodium phosphate and 0.15 M sodium chloride chromatography buffer at pH = 7.2.

Coupling of the antibody with the semicarbazide-functionalized pSiNP : The pH of the antibody solution was adjusted to pH = 6.0 with a 2 M HCl solution. Nanoparticles were dispersed in the antibody solution and allowed to react for 30 minutes at room temperature under agitation in the dark. The nanoparticles were then centrifuged at 22000 g for 15 minutes, and rinsed in water 3 times. They were fi nally dispersed in PBS.



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Determination of the concentration of nanoparticles in solution : Inductively coupled plasma–optical emission spectroscopy (ICP-OES) analysis were performed to determine the concentration of nanoparticles in the different solutions. The analyses were performed on a Perkin-Elmer 5300V. Samples were prepared at a concentration range of 1–10 ppm at pH = 2.0. A calibration curve was obtained by analyzing different dilutions from a sample at a stock concentration of 1 mg mL − 1 .

Flow cytometry : BSR and SH-SY5Y cell line stocks were cultured in complete DMEM containing 10% FBS, PSG, GX. Cell lines were trypsinized, centrifuged at 400 g, resuspended in supplement free DMEM and analyzed by fl ow cytometry as previously described. [ 48 ] 1.0 × 10 7 cells mL − 1 were suspended in 50 μ L of PBS and incubated at 4 ° C for 30 minutes with the MLR2 functionalized pSiNP. The cells were then washed with ice-cold PBS (pH 7.2)/1.0% normal bovine serum/0.1% azide (cell wash buffer) and read on an Accuri Cytometer C6 Flow Plus systems (BD Biosciences). At least 30 000 cells were measured by list mode and measurements were performed on a single cell basis, and displayed as frequency histograms. Dead cells and debris were gated out of the analysis on the basis of forward and side scatter, and data analyzed by CFlow Plus Software (BD). The same experiment was performed with U87MG and SH-SY5Y cell lines with the mAb528 functionalized pSiNP, and with HRIK and Jurkat lymphocytes with the Rituximab functionalized pSiNP.

Fluorescence image analysis was performed on an ImageStream X (ISX) imaging fl ow cytometer (Amnis, Seattle). Jurkat and DOHH-2 cells were washed and adjusted to 5 × 10 6 mL − 1 in RPMI1640 media and then incubated with 30 μ g of pSi-Rituximab conjugated with FITC for 30 min the dark at room temperature. Cells were washed by centrifugation at 400 g for 5 minutes in PBS/1%FCS before being resuspended in 75 μ L of PBS/1%FCS. Cells were excited with a 488 nm laser and emission collected in the 480–560 nm band. The instrument collects images on two CCD cameras, including both brightfi eld and multiple fl uorescence images. Images from 10 000 events were collected and post-acquisition analysis was performed using IDEAS software (Amnis).

Immunocytochemistry : Primary motor neuron extraction and culture: all animal related procedures were approved by the Flinders University Animal Ethics Committee. E12.5 embryonic motor neurons were extracted, purifi ed and cultured as we have previously described. [ 54 ] Wells (or glass cover slips) were coated with poly-L-ornithine (0.01%) overnight, washed with sterile water and then incubated with mouse laminin (2.5 μ g mL − 1 for 2 hours) in HBSS prior to addition of motor neurons. Cells were cultured in neurobasal media supplemented with 1% B27, 1% Glutamax, 10% horse serum, 25 μ M BME, 1 ng mL − 1 BDNF, 1 ng mL − 1 GDNF, 10 ng mL − 1 CNTF. Media was changed 24 hours after plating, then every 2 days. After 9 days, cultures were rinsed in PBS and fi xed in 4% paraformaldehyde for 15 minutes on ice, in preparation for immunocytochemistry. Fixed cultures were blocked overnight in antibody diluent (10% donkey serum) at 4 ° C, shaking. Following blocking, primary antibodies and pSi nanoparticles were added. Primary antibodies were added as follows, in varying combinations; 5 μ g pSi-MLR2 labeled with FITC, 25 μ g pSi-MLR2 labeled with FITC, 1:1000 rabbit anti Glial Fibrillary Acidic Protein (GFAP; Dako), all in antibody diluents (1% donkey serum) for 3 days at 4 ° C under agitation. Afterwards, cells were washed in PBS 3 times for 10 minutes each. Secondary antibodies were added at 1:800, in antibody diluents (1% donkey serum) for 3 hours at room temperature under agitation. After incubation, cells were again washed in PBS 3 times for 10 minutes each. The fi rst wash contained 1.25 μ g mL − 1 DAPI. The cells were imaged with an Olympus IX71 inverted fl uorescent microscope. All images were taken at 20x magnifi cation, with varying exposure times.

Cell viability : SH-SY5Y and BSR cells were trypsinized and then incubated with samples (pSi-MLR2-CPT, pSi-MLR2, pSi-CPT or MLR2) at 50 μ g mL − 1 nanoparticle concentration for 30 minutes under agitation at 37 ° C. Following incubation, half the cells were centrifuged at 400 g and resuspended in cell culture media twice and then placed in wells of a 24-well plate at 5000 cells/well. The other half of the cells was transferred immediately after incubation into wells of a 24-well plate at a concentration of 5000 cells/well. Both washed and unwashed cells

© 2012 WILEY-VCH Verlag GAdv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201200335

were cultured in DMEM (5% FBS, 1% PSG, 1% Glutamax) in 5% CO 2 at 37 ° C for 72 hours. After incubation, a Trypan Blue exclusion assay was performed as previously described to count the live and dead cells. [ 55 ] Briefl y, the cells were washed with PBS 3 times and trypsinized. Trypan Blue (0.4%) was added to 100 μ L of cell suspension in an equal volume and incubated at room temperature for 5 minutes. The viable cells were counted using a haemocytometer (Invitrogen, Ltd., Renfrew, UK). Values of viability of treated cells were expressed as a percentage of that from corresponding control cells. All experiments were repeated at least 3 times.

U87MG and U87MG. Δ 2–7 cells were placed in wells of a 96-well plate at 1000 cells/well. They were incubated with the different samples (pSi-mAb528, pSi-CPT or pSi-mAb528-CPT) at a nanoparticle concentration of 10 and 50 μ g mL − 1 at 37 ° C. Half of them were incubated for 1 hour, then washed, the medium was changed, and fi nally incubated for 4 days. The other half of the cells was incubated directly for 4 days. After incubation, a MTS assay (Promega cell titer 96 aqueous one solution cell proliferation assay) was performed in order to count the live and dead cells. Values of viability of treated cells were expressed as a percentage of that from corresponding control cells. All experiments were repeated at least 3 times.

Acknowledgements This material is based upon work supported by the PHC FAST 2011 Program # 24136Y. We acknowledge D. Cot and T. Cacciaguerra for help with the imaging, M. Alnakhli for providing glioblastoma cells, and M.-N. Labour for helpful discussions. Finally, we thank M. Cicera for his help in preparing graphical illustrations.

Received: September 20, 2012Published online:

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