Biodegradable Nanoparticles for Direct or Two-Step Tumor

with the results presented as relative cell number versus log fluorescence intensity. ...... Wai Hei Tse , Laszlo Gyenis , David W. Litchfield , J...
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Bioconjugate Chem. 2006, 17, 139−145

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Biodegradable Nanoparticles for Direct or Two-Step Tumor Immunotargeting Leila Nobs,† Franz Buchegger,‡ Robert Gurny,*,† and Eric Alle´mann†,§ Department of Pharmaceutical Technology and Biopharmaceutics, School of Pharmaceutical Sciences, Ecole de Pharmacie Gene`ve-Lausanne, University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland, and Service of Nuclear Medicine, University Hospital of Geneva, Rue Micheli-du-Crest 24, 1211 Geneva 14, Switzerland. Received May 11, 2005; Revised Manuscript Received September 9, 2005

In this study, selective cancer cell targeting of biodegradable poly(lactic acid) (PLA) nanoparticles (NPs) has been investigated in vitro. SKOV-3 (HER2 positive) ovarian cancer and Daudi (CD20 positive) lymphoma cell targeting was mediated by anti-HER2 (trastuzumab, Herceptin) and anti-CD20 (rituximab, Mabthera) monoclonal antibodies (mAbs), respectively. The mAb against nonexpressed antigen serving on each cell as isotype matched irrelevant control. Two different targeting approaches have been studied, a direct method using antibody-labeled NPs (mAb-NPs) and a pretargeting method using the avidin-biotin technology. For the direct protocol, fluorescent PLA-NPs were prepared including 10% 1-pyrenebutanol (PB)-labeled PLA in the NP-preparation (PB-NP). Thiol groups were covalently bound to the PB-NP, and the resulting thiolated PB-NP were coupled with the two mAbs using a bifunctional cross-linker. The effective targeting of cells by mAb-PB-NP was shown by flow cytometry analysis. Clearly anti-HER2-PB-NP specifically bound to the SKOV-3 cells and not to the Daudi cells, while anti-CD20-PB-NPs bound to Daudi cells but not to SKOV-3 cells. Specific mAb-PB-NP binding to tumor cells produced a mean 10-fold or higher signal increase compared to irrelevant IgG-PB-NPs. For the pretargeting protocol, plain PLA-NPs were also thiolated and NeutrAvidin-Rhodamine Red-X (NAR) coupled to the functionalized PLA-NPs with sulfo-MBS. The two-step method was evaluated in vitro by incubating SKOV-3 cells first with biotinylated mAbs followed by NAR-NPs. The relative fluorescence associated to the specific binding of NPs produced a 6-fold increase in flow cytometry signal compared to nonspecific binding. In conclusion, these experiments have shown that NPs covalently coupled with antibodies or NAR can specifically and efficiently bind to cancer cells in both a pretargeting and a direct approach, suggesting that functionalized NPs may be a useful drug carrier for tumor targeting.

INTRODUCTION One of the major problems facing cancer chemotherapy is the undesirable side effects. These are mainly of nonspecific nature due to distribution of the chemotherapeutic drugs to tissues other than tumor. To overcome the undesirable side effects and to increase local concentration of the drug at the target site, several drug-delivery systems have been investigated, such as drug-polymer conjugates (1), liposomes (2, 3), micelles (4, 5), and polymeric nanoparticles (NPs)1 (6, 7). Colloidal carrier systems, such as liposomes and NPs, present some relevant advantages over other drug carriers: (a) a high drug loading capacity; (b) the possibility to control size and permeability of the carrier; (c) the protection of the encapsulated drug from metabolism or early excretion. Among the different carriers, biodegradable NPs emerged as suitable means of carrying bioactive molecules to tissues of interest, especially due to their long shelf life, to the potential for control of release rate of the drug, and to the well established methods of preparation (8, 9). Furthermore, long circulating “stealth” * Corresponding author: Robert Gurny, Phone: +41 22 379 61 46. Fax: +41 22 379 65 67. E-mail: [email protected]. † University of Geneva. ‡ University Hospital of Geneva. § Current address: Bracco Research SA, Route de la Galaise 31, 1228 Plan-les-Ouates, Geneva, Switzerland. 1 Abbreviations: PLA, poly(lactic acid); FACS, fluorescenceactivated cell sorting; NAR, NeutrAvidin Rhodamine Red-X.; NP, nanoparticle; mAb, monoclonal antibody; mAb-NP, mAb labeled nanoparticle; NAR-NPs, NeutrAvidin-labeled NP.; PB-NP, 1-pyrenebutanol loaded NP.; mAb-biot, biotinylated mAb; sulfo-MBS, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester.

nanoparticles such as poly(ethylene glycol)-coated NP (PEGNP) have proved to greatly enhance the therapeutic index of the drug (10-12). In fact, owing to their prolonged circulation time in blood, they more readily accumulate in areas with leaky vasculature, such as tumors, than plain NPs. However, a longcirculation time of NPs might be insufficient to obtain tumor control since a specific interaction with tumor cells does not occur. A promising strategy to achieve direct drug delivery is the development of active targeting of cancer cells via interactions mediated by ligands such as antibodies, lectins, and peptides, as presented with liposomes (13, 14). Active tumor targeting of NPs may be achieved with either direct targeting or the pretargeting-multistep method. In direct targeting, NPs should be covalently coupled with the ligand and the resulting drug carrier be administered at once. In the pretargeting approach, the therapeutic molecule is not coupled with the ligand and is administered after an appropriate delay time following the targeting ligand. This delay allows time for the antibody to localize and concentrate in the tumor. Commonly, pretargeting protocol involves an avidin-biotin system (15) or bispecific antibodies (16). In this study, we explored the two approaches to target biodegradable poly(lactic acid) (PLA) NPs to tumor cells. AntiHER2 mAbs (trastuzumab, Herceptin) and anti-CD20 mAbs (rituximab, Mabthera) were used as targeting ligands. Two cell lines were used, SKOV-3 human ovarian cancer cells expressing HER2 antigen, and Daudi lymphoma cells, expressing CD20 antigen. On each cell line, the antibody directed against the nonexpressed antigen served as isotype-matched irrelevant control immunoglobulin. In the direct approach, NPs exposing mAbs at their surface were incubated with the two tumor cell

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lines. In the pretargeting protocol, tumor cells were pretargeted with biotinylated mAbs prior to the administration of avidinlabeled NPs. Cell interaction of fluorescence-labeled NPs was measured by flow cytometry.

EXPERIMENTAL PROCEDURES Materials. Poly(DL-lactic acid) (PLA) (100DL 4A, Mw 57 kDa) was a gift from Alkermes (Cincinnati, OH). 4-Hydroxyazobenzene-2-carboxylic acid (HABA) and phosphate-buffered saline (PBS) were from Sigma (Buchs, Switzerland). m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), sulfosuccinimidyl 6-(biotinamido)hexanoate (sulfo-NHS-LCbiotin), BCA protein assay kit, and D-Salt dextran plastic desalting columns were supplied by Pierce (Rockford, IL). NeutrAvidin Rhodamine Red-X (NAR) was from Molecular Probes (Leiden, The Netherlands). Cell Lines. SKOV-3, human ovarian carcinoma cells expressing HER2, and Daudi, human B lymphoma cells expressing CD20 (American Type Culture Collection ATCC, Manassas, VA), were cultured in 75-cm2 culture flasks in RPMI-1640 with Glutamax I (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (Brunschwig, Basle Switzerland), 100 units/mL of penicillin, and 100 µg/mL of streptomycin (Gibco) in an atmosphere of 95% humidified air and 5% CO2 at 37 °C. Cells were maintained in exponential growth using adequate splitting and renewal of medium. SKOV-3 cells were maintained as a monolayer, and for all the experiments, cells were harvested from subconfluent cultures using trypsin-EDTA (Gibco) and were resuspended in fresh complete medium. Unless stated otherwise, all in vitro studies were performed at 37 °C under gentle rotation in tubes containing phosphate-buffered saline (PBS) with 0.1% bovine serum albumin. Biotinylated Antibodies. Antibodies for targeting assay included the anti-HER2 mAb (trastuzumab, Herceptin) and antiCD20 mAb (rituximab, Mabthera) (Roche, Basle, Switzerland). Prior to the coupling reactions, the antibodies were purified by size exclusion chromatography using a D-Salt dextran plastic desalting column. For the pretargeting approach, anti-HER2 and anti-CD20 mAbs were biotinylated as follows: 1 mL of purified mAbs (5 mg/mL) was mixed with sulfo-NHS-LC-biotin in bicarbonate buffer (pH 8.3) at a molar reaction ratio of 5 molecules of biotins per mAb molecule. After incubation for 1 h at room temperature, the unconjugated sulfo-NHS-LC-biotin molecules were removed from the mixture by size exclusion chromatography using a D-Salt dextran plastic desalting column. The fractions containing mAb were pooled, and the mAb concentration was determined by absorbance at 280 nm assuming an extinction coefficient of 1.4 M-1 cm-1 (mg/mL)-1. The molar ratio of mAb-to-biotin was assessed by the colorimetric method using the dye 4-hydroxyazobenzene-2-carboxylic acid (HABA) (17). The biotinylated mAbs (mAb-biot) were stored at 4 °C until use. The capacity of biotinylated mAbs to bind to cells was assessed in vitro on both SKOV-3 and Daudi cells using a twostep targeting protocol. SKOV-3 or Daudi cells in suspension (5 × 105 cells/mL) in PBS were incubated with 100 µg/mL of either biotinylated anti-HER2 or biotinylated anti-CD20 mAbs during 2 h. Unbound antibodies were removed by three successive centrifugations (400g, 5 min). Thereafter, 4 µL of NAR (1 mg/mL) were added to the cells and incubated for 1 h. The cells were rinsed with cold PBS three times before being analyzed by flow cytometry as described below. Nanoparticles Coupled with NeutrAvidin Rhodamine Red-X. Plain NPs were prepared by a salting-out method according to a previously described method (18). Thiol functions were covalently bound to the surface of the plain nanoparticles to allow the subsequent covalent binding of ligands to the NPs.

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Optimization of the reaction conditions and characterization of the thiolated NPs has been presented elsewhere (19). Briefly, carboxylic acid groups of PLA NPs were activated with 1-Ethyl3-(3-dimethylaminopropyl)carbodiimide (EDAC) to allow the covalent binding of cystamine. Thereafter, disulfide bonds of cystamine were reduced using Tris(2-carboxyethyl)phosphine (TCEP). Thiolated NPs were subsequently used to covalently bind mAbs for the direct approach or NeutrAvidin Rhodamine Red-X (NAR) for the pretargeting protocol. The preparation and characterization of NAR-labeled NPs (NAR-NPs) has been described previously (20). Antibody Coupling to Fluorescent Nanoparticles for Direct Targeting. Fluorescent NPs were prepared by the saltingout process with 10% (w/w) of 1-pyrenebutanol-labeled PLA and 90% of unlabeled PLA (21). The fluorescent polymer, 1-pyrenebutanol-labeled PLA, was used in this experiment as a fluorescent tracer for flow cytometry analysis. The fluorescence excitation and emission spectra of 1-pyrenebutanol contained in the NPs were determined in PBS (FluoroMax spectrofluorometer, Spec Indutries, NJ). The mean Z size of the labeled NPs was determined by dynamic light scattering (Malvern Instruments, Ltd, UK). Thiol functions were covalently bound to fluorescent NPs as described above and the resulting thiolated PB-NP were coupled to purified Herceptin or Mabthera using the bifunctional crosslinker, sulfo-MBS. mAbs (2 mg/mL) were incubated over 45 min at room temperature with sulfo-MBS in PBS (pH 7.4) at four different molar ratios, 1:5; 1:10; 1:20; 1:40 (mAb:sulfoMBS). The excess of sulfo-MBS was removed from the mixture by size exclusion chromatography using the desalting columns. The fractions containing the maleimide-activated mAbs were collected, and their concentration was determined. Thereafter, 500 µL of mAb (1 mg/mL) was incubated with 500 µL of thiolated PB-NP (20 mg/mL) and gently shaken for 60 min at room temperature. Subsequently, the uncoupled mAbs were removed by four cycles of centrifugation (20000g, 10 min). The supernatant of the centrifugation step was used for the determination of uncoupled mAb by a BCA assay (microplate procedure). Finally, mAb-conjugated NPs (mAb- PB-NP) were stored in PBS at 4 °C. Trials were carried out on plain PB-NP (without thiol functions) to determine the fraction of noncovalently attached mAb on the surface of the NPs. Nanoparticle Binding. For the direct targeting, 100 µL of a 1 mg/mL suspension of specific or irrelevant mAb-PB-NP was incubated for 45 min with 5 × 105 SKOV-3 or Daudi cells. Cells were rinsed with three successive centrifugation steps (400g, 5 min) with cold PBS to remove unbound nanoparticles, and the mean relative fluorescence was measured by cytometry analysis. For pretargeting experiments, SKOV-3 cells (5 × 105 cells/ ml) were incubated for 2 h with 100 µg/mL of biotinylated antiHER-2 or anti-CD20 mAbs (negative control). PBS containing unbound mAb was removed, and cells were washed with two cycles of successive centrifugations (400g, 5 min). Subsequently, 100 µL of a 1 mg/mL suspension of NAR-NPs was added, and cells wrer incubated for 1 h. Three successive centrifugation steps (400g, 5 min) with cold PBS were carried out to remove unbound NAR-NPs and resuspended in PBS for flow cytometry analysis. For both targeting approaches, parallel incubations were performed with unlabeled NPs and with irrelevant mAbs to assess the nonspecific interaction of the nanoparticles with cells. Other controls included the incubation during 45 min of free mAb (100 µg) prior to the incubation of mAb-PB-NP or NARNPs. Flow Cytometry Analysis. The capacity of mAb-NP-BPs and of biotinylated mAbs to bind cells was analyzed using flow

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cytometry. The cells were suspended in 500 µL of cold PBS prior the measurements. For the pretargeting protocol, mean fluorescence intensity of 10000 cells was analyzed using a FACSfits cytometer (Becton Dickinson, San Jose, CA) equipped with an argon-ion laser adjusted to 488 nm. For the direct targeting method, the mean fluorescence intensity was measured using a FACSVAntage flow cytometer (Becton Dickinson, San Jose, CA). The excitation of 1-pyrenebutanol (PB-NP) was performed using an UV laser with two wavelengths at 351 and 364 nm (Coherent Laser, Inc. Santa Clara, CA) set at 20 mW. The emission signal was analyzed using the reading channel FL5-H corresponding to wavelengths ranging from 383 to 407 nm. Median fluorescence F was calculated using Cellquest software (Becton Dickinson) with the results presented as relative cell number versus log fluorescence intensity. Cells incubated with only PBS were used for background fluorescence calibration.

RESULTS Fluorescent, Thiolated Nanoparticles. Plain NPs and fluorescent NPs were obtained by a salting-out procedure (18). For the fluorescent labeling of NP, the 1-pyrenebutanol-PLA conjugate was used at 10% of unconjugated PLA. Both plain NPs and fluorescent NPs (PB-NP) showed very similar characteristics in terms of particle size and redispersibility. The mean size of the plain NPs and PB-NP was 265 and 270 nm, respectively. After freeze-drying the NPs or PB-NPs, complete redispersion of all formulations could be achieved in water. Spectofluorimetric assay revealed that the PB-NP were efficiently loaded with 1-pyrenebutanol-PLA (results not shown). Further results suggested that notably no subsequent spontaneous release occurred. The maximum for the excitation and emission wavelengths of 1-pyrenebutanol NPs was found at 375 and 390 nm, respectively. The thiolation reaction was very reproducible on both fluorescent and nonfluorescent NPs with an incorporation of 104 ( 2 mmol of thiol functions per mol of PLA, for nonfluorescent NPs and 89 ( 10 mmol for PB-NP. The average size of plain NPs and PB-NP after the thiolation reaction was 278 and 297 nm, respectively, corresponding to a size increase of 5 to 10% only compared to the original size. Direct Coupling with Antibodies. The coupling reaction between the two different mAbs and the thiolated PB-NP was carried out in the presence of the bifunctional cross-linker sulfoMBS. Four different molar ratios of sulfo-MBS to mAbs were evaluated. An equal concentration of maleimide-mAb was used for all the coupling reactions with thiolated PB-NP. Figure 1 summarizes the results of the coupling reaction between maleimide-mAbs and thiolated PB-NP. For both anti-CD20 and anti-HER2 mAbs the amount of mAbs bound to the surface of the PB-NP increased when an increased molar ratio between mAbs and sulfo-MBS was used. Amounts of 1.5, 2.5, and 5.6 mmol of mAbs per mol of PLA were bound to the PB-NP using a mAbs to sulfo-MBS molar ratio of 1:10, 1:20, and 1:40, respectively. These three formulations were retained for the in vitro studies. At the lowest molar ratio between sulfo-MBS and mAbs (1:5), no coupling of mAbs to the surface of the nanoparticles could be demonstrated. Between 2500 and 2700 mAbs (25 to 27% of total mAb) were coupled to the thiolated PB-NP surface at the highest molar ratio of sulfo-MBS. This corresponds to coupling of approximately 19% of the total measurable thiol functions on the surface of NPs. Control reactions on PB-NP lacking thiol functions showed that only a minor amount of mAbs (2.8%) bound nonspecifically to the particles. The average size of the mAb-PB-NP after the coupling reaction was ranging from 340 to 410 nm, depending on the amount of mAbs bound to the particles.

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Figure 1. Summary of the coupling reaction between thiolated NPs and mAbs (anti-HER2 and anti-CD20 mAbs). Influence of the molar ratio (mAb:sulfo-MBS, values given in parentheses) on the coupling reaction on the ratio of mAbs obtained at the surface of the NPs.

NeutrAvidin-Rhodamine Red-X-Coupled NPs. After activation of NAR with sulfo-MBS, 500 µL of the activated NAR solution (360 µg/mL) was used for the coupling reaction with an equal volume of NPs (20 mg/mL). The quantification of the fluorescent Neutravidin on the NP surface revealed that 5.0 mmol of NAR per mol of PLA was covalently attached to the surface of the NAR-NP, corresponding to approximately 1300 molecules of NAR per NP. In control reactions the concentration of activated NAR nonspecifically bound to nonthiolated NPs was as low as 0.6 mmol of NAR per mol of PLA. The mean particle size increased from 278 nm before coupling to 320 nm after the coupling reaction. Biotinylation of Antibodies. The water-soluble NHS-LCbiotin was chosen due to its extended spacer arm that limits steric hindrance and provides optimal reactivity with avidin and permits the biotin to reach the lysine groups (22). Starting with a NHS-LC-biotin-to-antibody molar ratio of 5:1, an average of three molecules of biotin was introduced per antibody molecule, as determined in the colorimetric assay with HABA reagent. The ability of biotinylated mAbs to bind to specific antigens was assessed in vitro with a two step-targeting protocol using free NAR. Anti-HER2 bound specifically to SKOV-3 cells but not to Daudi cells. On the contrary, anti-CD20 interacted with Daudi cells, but not with SKOV-3 cells (Figures 2A and 2B). Results of a competition assay with radiolabeled Rituximab indicated that the immunoreactivity of Rituximab coupled with 3 to 4 NHS-LC-biotin per antibody molecule as measured with the HABA assay was only slightly impaired with loss of about 25% compared with native Rituximab. Higher biotinylation degrees of the antibody resulted in stronger impairment of the immunoreactivity in this assay (data not shown). Direct Targeting. The three retained PB-NP formulations were tested for each type of mAb. The targeting of SKOV-3 showed that all anti-HER2 formulations bound specifically to the cells. Whereas, when anti-CD20-PB-NP were incubated with SKOV-3 cells only little cell interaction was observed compared with plain NPs and no significant difference was observed among the three anti-CD20-PB-NP formulations (Figures 3A and 4A). The binding efficiency of targeted NP to the cells was dependent on the mAb:sulfo-MBS ratio, since increased fluorescence was observed for the 1:20 molar ratio compared with 1:10. However, the highest ratio of sulfo-MBS (1:40) that resulted in the highest amount of antibody coupled per NP showed the lowest binding among the three NP formulations, suggesting that this formulation resulted in significant inactivation of antibody. Based on these results, the optimum molar

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Figure 2. Flow cytometry analysis of biotinylated mAbs biding to SKOV-3 and Daudi cells. (A) SKOV-3 cells were preincubated with biotinylated anti-HER2 (black peak) or biotinylated anti-CD20 (white peak) followed by NAR. B) Daudi cells were preincubated with biotinylated anti-CD20 (black peak) or biotinylated anti-HER2 (white peak) followed by NAR.

Figure 4. Direct targeting protocol. Effect of mAb:sulfo-MBS ratio on the binding capacity of mAbs-NPs to specific antigens expressed on the surface of the cells. (A) Anti-HER2-NPs incubated with SKOV-3 cells using anti-CD20-NPs and unlabeled NPs as negative controls. Data are mean of four independent experiments ( SD. (B) Anti-CD20NPs incubated with Daudi cells using anti-HER2-PB-NP and unlabeled PB-NP as negative controls. Data are mean of three independent experiments ( SD. Values in parentheses indicate initial molar ratios mAb:sulfo-MBS.

Figure 3. Direct targeting protocol. Flow cytometry analysis of targeted PB-NP binding to SKOV-3 and Daudi cells. (A) SKOV-3 cells were incubated with either anti-HER2-PB-NP (black peak) or anti-CD20PB-NP (grey peak) during 45 min. (B) Daudi cells were incubated with either anti-CD20-PB-NP (black peak) or anti-HER2-PB-NP (grey peak) during 45 min. Cells incubated alone were used as background fluorescence (white peak).

ratio used was 1:20 (mAb:sulfo-MBS). This anti-HER2-PBNP formulation showed significantly higher binding activity up to 13-fold than the equivalent anti-CD20-NPs formulation. The targeting ability of the same mAb-PB-NP reagents was also assessed on Daudi cells. A very similar result as on SKOV-3 was again observed on Daudi cells with strong interaction of the specific mAb-coupled NPs and low background activity with irrelevant mAb. At the 1:20 molar ratio of activation with sulfo-MBS, binding impairment was also observed with rituximab (Figures 3B and 4B). The 1:20 antiCD20-NP formulation showed up to 10-fold higher binding activity than anti-HER2-NPs in cells expressing CD20 antigens. No significant difference was observed among the three antiHER2-NP formulations.

Figure 5. Direct targeting protocol. Study of the influence of free mAbs on the interaction between cells and targeted-NPs. Cells were incubated either with targeted-NPs for 45 min (1st and 3rd bar) or with free mAbs for 45 min followed by the incubation of targeted-NPs over 45 min (2nd and 4th bar). Data are mean of four independent experiments ( SD for anti-HER2-NPs on SKOV-3 cells and mean of three independent experiments ( SD for anti-CD20-NPs on Daudi cells.

As a further demonstration of the specificity of mAb-PB-NP interaction with cells, flow cytometry analyses were also carried out on both cells incubated with free mAbs followed by the treatment of mAb-NPs. As illustrated in Figure 5, the association of anti-HER-NPs with SKOV-3 could be competitively inhibited by the preincubation of free anti-HER2, indicating that the cell-

Tumor Targeting with Nanoparticles

Figure 6. Pretargeting protocol. Study of the interaction between SKOV-3 cells prelabeled with biotinylated mAbs anti-HER2-biot and anti-CD20-biot followed by the incubation of NAR-NPs. First step: preincubation with mAbs during 2 h; second step: interaction with NAR-NPs during 60 min. Two control experiments were performed with free anti-HER2 (not biotin conjugated) or NAR-NPs without antibody. Data are mean of three independent experiments ( SD.

association with NPs was mediated through the HER2 antigens on SKOV-3 cells. Similar results were obtained with Daudi cells (Figure 5). Two-Step Targeting. The cell association of NAR-NPs with the two-step approach was evaluated on SKOV-3 cells. Results shown in Figure 6 demonstrated that the cells preincubated with biotinylated anti-HER2 mAb showed significantly higher fluorescence intensity compared with those treated with biotinylated anti-CD20 mAbs. The mean fluorescence of cell-associated NPs measured with biotinylated anti-HER2 was 6-fold higher that obtained with biotinylated anti-CD20 using equivalent concentrations of NARNPs. Cells incubated with NAR-NPs in the absence of mAbs confirmed that the specific binding of the NPs to the cells was mediated by the biotinylated-anti-HER2. When anti-HER2 mAbs without biotin were used to pretarget the cells, only a small amount of NAR-NPs interacted with the cells, demonstrating that the association between NPs and cells is due to the strong affinity between NAR and biotin.

DISCUSSION Nanoparticle Fluorescence Labeling and Activation for Covalent Ligand Coupling. The use of biodegradable NPs for drug delivery of therapeutic agents is now well established (23, 24). Among different carrier systems, and particularly with respect to liposomes, NPs have proved to be suitable candidates for antitumor drug therapy. NPs present notably a high drug loading capacity and a variety of well-defined biodegradable polymers, allow the controlled release rate of the drug, and finally show a high storage and in vivo stability. To further improve the therapeutic index of anticancer agent, NPs must be designed to target cancer cells. The reliable way to achieve this goal is the tumor targeting mediated by ligands such as antibodies. To our knowledge, there are very few reports regarding tumor targeting using mAb-labeled biodegradable NPs (25-27) and no study at all on the multistep targeting approach. Hence, the main goal of the present study was the development of a novel concept of functionalized biodegradable NPs designed to specifically target antigen receptors on tumor cells. In the course of development of the NP-targeting, we made several unexpected observations. We previously described the evaluation and selection, among three different methods, of the NP thiolation used here based on EDAC and cystamine that provided low nonspecific reaction with the NPs (19). An unexpected difficulty was observed with the fluorescence

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labeling of the NPs. Initially, nile red loaded NPs had been selected for flow cytometry studies. Nile red is commercially available highly hydrophobic fluorescent marker. It has been used previously to label NPs for in vitro investigations (28, 29). However, in the setting presented here, nile red staining of NPs was unstable; the dye leaked out and labeled cells even in the absence of NP targeting. As an alternative, we coupled commercially available NeutrAvidin Rhodamine Red-X onto the NPs. In this reagent, biotin binding sites remain available (20), providing double function as fluorescent dye and two-step reagent for binding to biotinylated antibodies. As a drawback, however, the fluorescence labeling is not independent from the NP-targeting function in this approach. Even though we did not observe release of this reagent from the NPs, we cannot exclude that this happened at low level. 1-Pyrenebutanol coupled to PLA chains appeared finally as optimal fluorescence labeling. The use of 10% of 1-pyrenebutanol-PLA for the preparation PLANPs was efficient and gave a stable fluorescent labeling, well adapted for flow cytometry. The other functions of the NPs, such as solubility and particle size, were not altered in the presence of this fluorescent PLA. The coupling reaction carried out on both fluorescent and nonfluorescent NPs were highly efficient leading to the formation of covalent thioether bonds. In this study, it was demonstrated that NPs can be easily labeled by two types of mAbs, anti-HER2 and anti-CD20 mAbs, and that by varying the molar ratio of the cross-linker, the mAb density on the particles be tailored. Cancer Cell Association. The ability of NPs to specifically bind tumor cells was shown with the direct and two-step targeting approaches on two different cancer cell lines, SKOV-3 and Daudi. For direct targeting, mAbs were covalently bound to the surface of the PB-NP, and it was demonstrated that the antigen binding activity of the mAbs was dependent on the density of the mAb on the PB-NP surface. In fact, the amount of total cell-associated PB-NP decreased when a high molar ratio was used (1:40), suggesting that inactivation of the antigen binding domain had occurred. Increased sulfo-MBS substitution on mAbs resulted in a higher cell targeting capacity until a maximum was reached. Nevertheless, further studies must be carried out, particularly in vivo, to determine the most favorable mAb density, taking into account that excessive mAb densities on particles may induce rapid clearance from plasma, as shown with immunoliposomes (30). For the two types of cells, only minor NP-cell interaction was observed in the absence of mAbs, demonstrating that PB-NP bound specifically to the cells via the antigen-antibody interaction. This was also confirmed by the incubation of cells with NPs labeled with irrelevant mAbs. The second approach investigated in this study was the pretargeting approach. Multistep targeting is promising since it has the potential of increasing tumor-to-normal-tissue ratios, and as a result, to enhance the therapeutic index of pretargeted drug compared with the use of direct targeting. Frequently, pretargeting protocols involve the use of the avidin-biotin system. Avidin-biotin presents the advantage of dose amplification, since avidin can bind four molecules of biotin. In this study, NeutrAvidin, a chemically deglycosylated avidin, has been preferred to avidin. NeutrAvidin is similar to avidin with respect to its biotin binding affinity but it present less nonspecific binding to cells and has an increased circulation time in the blood compartment. To allow FACS analysis, a fluorescent labeled NeutrAvidin has been chosen: NeutrAvidin Rhodamine Red-X (NAR). It has been shown in this study that NAR-NPs recognized and bound specifically to cells preincubated with biotinylated specific mAbs, demonstrating that the pretargeting approach is feasible with NPs. NAR-NP can be potentially used

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for the conjugation of any biotinylated ligand. Furthermore, it may also be possible to simultaneously target different antigens with the same avidin-labeled formulation. A surprising result, however, was observed in the use of the biotin-avidin reagents in single step targeting. The successful use of these reagents in the two-step NP-targeting had shown that they were perfectly operational, as indicated also by the quality controls. However, when trying to use the same reagents in a one-step targeting whereby the NAR-NPs were first precoated with the biotinylated antibodies, washed, and then incubated with cells, no targeting was observed. When trying to explain the apparent difference between the two- and onestep targeting result, we remained essentially with three hypotheses. First, the immunoreactivity of the antibodies coupled with a mean of three biotins per antibody molecule was conserved when used as soluble agent, but might be strongly reduced when reacted first with the NAR-NPs due to sterical hindrance on the NP surface. Second, when used as two-step reagent, antibodies with conserved immunoreactivity might be positively selected, while, when first reacted with the avidinNPs, selection of antibodies might occur for the number of available biotin sites (from the Gaussian distribution) and therefore a higher probability of reduced immunoreactivity. Finally, in the two-step reaction, the number of reaction sites on the cell surface might become amplified by the three biotins per antibody molecule, while in the NAR-NP precoating, any loss of immunoreactivity due to sterical hindrance would result in a reduced number of potential binding sites for the cells. These different effects might possibly occur in combination and could explain the loss of binding capacity of the two-step reagents in a one-step targeting in combination with NPs. Despite the numerous advantages of the pretargeting approach and the promising results in preclinical (31-33) and clinical studies (34, 35), there are some points that must be considered. Avidin, like streptavidin, is a foreign protein for humans and, therefore, has shown considerable immunogenicity in vivo, thus limiting the number of times that it can be administered. As an alternative to both avidin and streptavidin, chemically modified avidins, such as NeutrAvidin, have been proposed, but their efficacy and their potential low immunogenicity remains to be demonstrated in vivo. Furthermore, it is not yet known to what extent the presence of endogenous biotin or biotinases can interfere in the pretargeting approach (36), even though clinical results have shown the feasibility of this multistep targeting. An important condition for good targeting results in pretargeting systems is the relatively slow internalization or even absence of internalization of the mAbs after binding the antigens. In fact, it has to be kept in mind that the antibodies should remain available for a sufficient period of time after binding to the target to allow the avidin-biotin interaction to take place. On the other hand, it has been proven that internalizing antibodies can be necessary for improved efficacy of certain therapeutic agents (37). In this study internalizing target HER2 and the noninternalizing target CD20 were used as models. Other internalizing mAbs can be coupled to the NPs for the therapy of lymphoma, such as anti-CD22, a humanized mAb (Epratuzumab) that is presently in clinical phase II study (38). At the present stage of the work, it is not possible to define which targeting strategy is preferred to deliver NPs to tumor cells. More work has to be accomplished, especially in vivo studies have to be undertaken using different tumor models (solid and hematopoietic). Nevertheless, the proof-of-concept of cancer cell targeting with NPs has been demonstrated on two cell lines using two mAbs as models. Universal NPs have been designed to target specific antigens, and these targeted PLA NPs can be applied to other target antigens.

Nobs et al.

ACKNOWLEDGMENT This work was supported by a grant from Cancer Research Switzerland (#938-09-1999) and from the Geneva Cancer League. The authors thank D. Wohlwend for technical assistance with regard to cytofluorometric experiments, Mrs. C. Grannavel and C. Paschoud for assistance in the preparation of particular reagents, and C. Nguyen for kindly supplying the 1-pyrenebutanol-labeled poly(lactic acid).

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