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Anti-Tumor Activity of Docetaxel PLGA-PEG Nanoparticles with a Novel Anti-HER2 scFv | OMICS International
ISSN: 2157-7439
Journal of Nanomedicine & Nanotechnology

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Anti-Tumor Activity of Docetaxel PLGA-PEG Nanoparticles with a Novel Anti-HER2 scFv

Thi Thuy Duong Le1, Thi Minh Lua Dang1, Thi My Nhung Hoang2, Thi Huyen La1, Thi Minh Huyen Nguyen1, Thanh Tam Nguyen3 and Quang Huan Le1*

1Department of Animal Cell Technology, Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay District, Hanoi, Vietnam

2Department of Cell Biology, Faculty of Biology, Hanoi University of Sciences, Viet Nam National University, 334 Nguyen Trai Road, Thanh Xuan District, Ha Noi, Vietnam

3Department of Organic Synthesis, Institute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay District, Hanoi, Vietnam

*Corresponding Author:
Quang Huan Le
Department of Animal Cell Technology
Institute of Biotechnology, Vietnam Academy of Science
and Technology, 18 Hoang Quoc Viet Road
Cau Giay District, Hanoi, Vietnam
Tel: 2-0106-333-091
E-mail: [email protected]

Received Date: October 20, 2014; Accepted Date: January 28, 2015; Published Date: February 08, 2015

Citation: Le TTD, Dang TML, Hoang TMN, La TH, Nguyen TMH, et al. (2015) Anti-Tumor Activity of Docetaxel PLGA-PEG Nanoparticles with a Novel Anti-HER2 scFv 6:267. doi: 10.4172/2157-7439.1000267

Copyright: © 2015 Le TTD, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Background: In this study, we developed pegylated (poly(D,L-lactide-co-glycolide) (PLGA-PEG) nanoparticles for loading docetaxel and improving active target in cancer cells because it’s advantages over other nanocarriers such as excellent biocompatibility, biodegradability and mechanical strength and these nanoparticles were conjugated with molecules of a novel anti-HER2 single chain fragment (scFv) by a simple carbodiimide modified method. ScFvs have potential advantages over whole antibodies such as more rapid tumor penetration and clearance. In addition, to investigate cellular uptake of targeted nanocarriers, many studies had performed by linking with fluorescent factors but in this study 6-histidine-tag fused with novel anti-HER2 scFv antibodies can be used to purify protein and study binding activity and cellular uptake of targeting nanoparticles that it was not changed their characterization in vitro. Furthermore, cytotocixity of these nanoparticles was also investigated in BT474 (HER2 overexpress) and MDA-MB-231 (HER2 underexpress) cells.

Results: Docetaxel loaded nanoparticles (Doc-NPs) with a mean size of 105 nm and zeta potential of -25 mV were prepared by nanoprecipitation method. Conjugation of a novel single chain fragment of antibody against epidermal growth factor receptor 2 to Doc-NPs by covalent coupling via cross-linker EDC and NHS resulted in an increase of mean size and zeta potential of targeted nanoparticles (scFv-Doc-NPs) with 135 nm and -32 mV respectively. The scFv- Doc-NPs bound specifically to BT474 cells but no MDA-MB-231 cells was investigated by flow cytometry. Especially, confocal fluorescence scanning microscopy revealed internalization of the scFv-Doc-NPs by the targeted cancer cells through anti-Histag antibodies with Alexa Fluor 546. Moreover, the scFv-Doc-NPs showed stronger cytotoxicity on BT474 cells than MDA-MB-231 cells with IC50 values of 0.234 and 0.535 μM, respectively.

Conclusion: Here we report anti-HER2 scFv labeled and docetaxel loaded PLGA-PEG nanoparticles in order to broaden the applications of this new targeted drug delivery system in the therapy of HER2 overexpressed cancers. Also, this drug delivery system represents a promising approach to improve the efficacy of nanoparticles in active targeting for HER2-overexpressed cancer therapy.

Keywords

Active targeting; Docetaxel; ScFv; Polymeric nanoparticles; Anti-HER2, Targeted cancer drug

Introduction

Chemotherapy has many side effects because of nonspecific bio distribution of chemotherapeutic agents. Therefore, many nanoparticle drug delivery systems have been designed to improve the efficacy of anticancer agents, minimize side effects and to enhance biocompatibility, serum stability [1]. The surface modification of nanoparticles with peptides, nucleic acids, antibodies, aptamers, or small molecules that bind to antigens on the surface of cancer cells or cancer tissues may be considered as an efficiently targeted delivery of cancer drugs [2]. The HER2 receptor is one of the major targets for the design of targeted anticancer drugs. These receptors are internalized by receptor-mediated endocytosis and are readily accessible to antibodybased therapy. Therefore it promotes intracellular accumulation of anti-HER2-covered immunonanocarriers, including anticancer drugs [3]. Amplification or over-expression of the ERBB2 gene occurs in approximately 15-30% of breast cancers [4,5]. Over- expression is also known to occur in ovarian, stomach, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma [6]. Therefore, among the targeting moieties, monoclonal antibodies specific to certain antigens on the surface of cancer cells have been used most often for the targeting of nanoparticles to tumor sites [7]. Recombinant antibody fragments (single-chain antibodies, scFv) which have been widely used as targeting moieties of nanocarriers both in vitro [8] and in vivo [9] are particularly attractive because their binding properties can readily be engineered using directed evolution [10]. PLGA has generated tremendous interest due to its excellent biocompatibility, biodegradability, and mechanical strength [11]. Moreover, PLGA molecules have hydrophobic nature, therefore hydrophobic drugs, including most anticancer agents, can be easily loaded into PLGA nanoparticles [12]. It has also been used by many researchers for passive and active targeting of anticancer agents [13].

Docetaxel is a member of the taxane drug class, which also includes the chemotherapeutic medication paclitaxel. Although docetaxel remains twice as potent as paclitaxel (due to docetaxel’s effect on the centrosome of the mitotic spindle), the two taxanes have been observed to have comparable efficacy. Several recent articles have found “no evidence those regimens containing docetaxel yield greater benefits than those including paclitaxel.”While efficacy between the two agents has been observed to be equivalent, paclitaxel may cause fewer side effects. Additionally, it has been noted that docetaxel is prone to cellular drug resistance via a variety of different mechanisms (Figure 1).

nanomedicine-nanotechnology-characterization

Figure 1: 1HNMR characterization of PLGA-COOH (A) and PLGA-PEG (B). PLGA: δ 5.2 (m, ((OCH(CH3)C(O)OCH2 C(O))n)); δ 4.8 (m, ((OCH(CH3) C(O)OCH2C(O))n)); δ 1.6 (d, ((OCH(CH3)C(O)OCH2C(O))n). PEG: δ 3.7 (s, (CH2CH2O)m).

Docetaxel is a leading chemotherapeutic drug for breast carcinoma, ovarian cancer, head and neck, and lung cancer, using nanoparticulate systems [13-18]. In this study, we report on the preparation and characterization of anti HER2 scFv-conjugated pegylated PGLA nanoparticles as a targeted delivery system for docetaxel. The quantity of monoclonal antibodies attached to the surface of the nanoparticles was characterized. Their ability to target and enter HER2-overexpressed BT-474 cells was studied and compared with MDA-MB-231 cells as a negative control.

Materials and Methods

Materials

Docetaxel anhydrous (Doc) of purity was purchased from Shanghai Bioman Pharma Co. Ltd, Shanghai, China. Poly(D,L-lactide-co glycolide) with terminal carboxylate groups (PLGA, lactide:glycolide, 50:50, Mw ~ 7000 - 1700), bifunction poly(ethylene glycol 2-aminoethyl ether acetic acid (NH2-PEG-COOH, Mw ~ 3400), 1-Ethyl-3-(3- dimethyllaminopropyl) carbodiimide (EDC) and N-hydroxysuccimide (NHS) were obtained from Sigma-Aldrich (St Louis, MO). Molecular biology buffers were purchased from Sigma. 4-Aminobenzoic acid p-aminobenzoic acid and 3-(4,5- dimethylthiazol-2-yl)-(2,5-diphenyl tetrazolium bromide) (MTT) was purchase from Promega (US). Monoclonal mouse anti-hexahistidine antibody was purchased from Abcam (Cambridge, MA), anti-mouse secondary antibodies conjugated with Alexa 546 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). All other organic solvents were analytical grade from Fisher Scientific.

Cell culture

Human breast cancer cell lines BT474 (HER2-positive) and MDA-MB-231 (HER2-negative) (American Type Culture Collection) were obtained from Institute of Biotechnology (VAST). The cell lines were cultivated in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37ºC in a humidified incubator with 5% CO2. The cells were maintained in an exponential growth phase by periodic subcultivation.

Synthesis of PLGA-PEG

Copolymer PLGA-PEG was synthesized by conjugation of NH2- PEG-COOH to PLGA-COOH. Briefly, 150 mg of PLGA-COOH were dissolved in 1 ml DCM. PLGA-carboxylate was converted into PLGA-NHS by adding 1 mg of NHS and 2 mg of EDC in 1 ml DCM with gentle stirring. PLGA-NHS obtained was precipitated with 10 ml ethyl ether/methanol washing solvent by centrifugation at 3000 rpm for 10 min and the washing process was repeated two times to remove residual EDC/NHS. The PLGA-NHS pellet was dried under vacuum for 30 min to remove residual ether and methanol. After drying under vacuum, 100 mg of PLGA-NHS was dissolved in 2 ml DCM followed by addition 10 mg of NH2- PEG-COOH and 7.5 mg of N,Ndiisopropylethylamine. The mixture solution was incubated for 24 h at room temperature under gentle stirring. Then, this mixture washed by ether/methanol washing solvent and centrifuged to remove unreacted PEG. The resulting PLGA-PEG copolymer was dried under vacuum and used for nanoparticle preparation without further treatment. The following are the main nuclear magnetic resonance (NMR) peaks of the sample:

1H-NMR (CDCl 3 at 300 Hz) d 5.2 (m, (OCH(CH3) C(O)OCH2C(O) n-(CH2CH2O)m), 4.8 (m, (OCH(CH3)C(O) OCH2C(O)n-(CH2CH2O) m), 3.7 (s, (OCH(CH3)C(O)OCH2C(O) n-(CH2CH2O)m), 1.6 (d, (OCH(CH3)C(O)OCH2C(O)) n-(CH2CH2O)m.

Preparation of docetaxel-loaded PLGA-PEG nanoparticles

The Doc encapsulated PLGA-PEG copolymer nanoparticles were prepared using a nanoprecipitation method. Briefly, 10 mg of PLGAPEG copolymer and 1 mg of Doc were dissolved in acetone. This organic phase was emulsified with an aqueous solution of polyvinyl alcohol (0.5% w/v) by sonication using a probe sonicator at amplitude 5 for 60 seconds. Nanoparticles were formed immediately, and gently stirred at room temperature for 4 hours to evaporate the organic solvent. The resulting nanoparticle suspension was then collected by ultrafiltration. The washing process was repeated three times in order to remove the adsorbed drugs. The washed nanoparticles were then freeze- dried using the Thermo Electron’s Modulyo freeze dryer (USA) for 8 h.

Characterization of docetaxel-loaded PLGA-PEG nanoparticles

Size and zeta potential measurements: The average size and polydispersity of Doc-loaded PLGA-PEG nanoparticles (Doc-NPs) were analyzed by dynamic light scattering (DLS). Zeta potential of Doc-NPs was evaluated in deionized water ( ? 1 mg/ml) using the electrophoretic mode of Zetasizer 3000 HS (Malvern instruments Ltd., United Kingdom) at 25ºC. Each sample was measured in triplicate.

Surface morphology: Field emission scanning electron microscopy (FESEM) system (Hitachi S-4800 FE-SEM) was used to determine the shape and surface morphology of nanoparticles produced. Particles were coated with gold under vacuum before scanning electron microscopy.

Drug encapsulation efficiency: The docetaxel entrapped in the Doc-NPs and measured by NanoDrop 1000 spectrophotometer (Canada) and quantified by UV-Vis spectrophotometer at 275 nm for Doc was previously described [19]. Briefly, 3 mg nanoparticles were dissolved in 2 ml of DCM (dichloromethane). DCM was evaporated in nitrogen atmosphere and residue was resuspended in DMSO for analysis by UV-Vis spectrophotometer.

The standard curve of Doc was linear in the range of 50-1000 g/ ml in dimethyl sulfoxide (DMSO). The encapsulation efficiency of Doc was measured by the amount of Doc encapsulated comparing with the total amount of drug used in formulation, multiplied by 100.

ScFv expression and purification

The anti-HER2 scFv protein was previously expressed in E. coli strain BL21 [20]. Briefly, the fusion gene of anti-HER2 scFv containing His6-tag on C-terminus was cloned into a T7 promoter-based E. coli expression vector, pET-22a(+). E.coli bacterial cultures were incubated at 37oC in lysogeny broth (LB) growth medium at 28oC. The anti-HER2 scFv expression was induced by addition of 0.5 mM isopropyl-L-thio-β- D-galactopyranoside (IPTG) and grown to log phase (A600 nm = 0.8) for 12 h. The cells were harvested, centrifuged, and the pellet was resuspended in lysis buffer (0.01 M Tris-HCl, pH 8.3, with 0.1 M NaCl and 10 mM EDTA) and sonicated on ice. The lysate was then centrifuged at 22,000 g for 30 min at 4oC. The pellet was used for purification of His6-tagged protein on Ni2+-NTA column (Qiagen) under denaturing conditions according to the manufacturer’s instructions. The protein was denatured with 8 M urea, refolded for 5 h using a linear gradient from 8 to 0 M urea and eluted with 250 mM imidazole. For final purification of anti-HER2 scFv, elution fractions were diluted 20-fold, applied onto Q Sepharose FF 1-ml column (GE Healthcare) and eluted using linear gradient from 0 to 500 mM NaCl.

SDS/PAGE analysis of the proteins was performed according to standard protocols using 12.5% (for the other recombinant proteins) polyacrylamide gels.

Conjugation of scFv to nanoparticles

The conjugation of anti-HER2 scFv to Doc-NPs was accomplished via crosslinking of –COOH and – NH2 using a modification of carbodiimide method (DeNardo et al.). Briefly, 1 ml of Doc-NPs (2 mg/ml) was incubated with 100 l of 4 mM EDC and 100 l of 10 mM NHS for 15 minutes at room temperature with gentle stirring. Then the activated particles were covalently linked to 50 l of scFv (1 mg/ml) for 2 hours at room temperature and gently vortex. The reaction mixture was quenched by adding hydroxylamine (to give a final concentration of 5 to 10 mM). The Doc-NPs conjugated with anti-HER2 scFv (Doc- NPs-scFv) was purified from unconjugated protein and by-products by ultrafiltration. The NPs suspensions were kept at 4oC until use.

Flow cytometry

Flow cytometry (FCM) was used to evaluate binding of Doc-NPsscFv to target (HER2 positive BT474) and nontarget (HER2 negative MDA-MB-231) cells. Cells were collected from culture and centrifuged for 5 min at 4ºC and 1500 rpm. Cells were then incubated with 200 l of Doc-NPs-scFv for 30 min at room temperature (RT). As a control, cells were separately grown in the absence of nanoparticles. Samples were then washed 3 times with PBS 1. Samples were incubated with monoclonal mouse anti-hexahistidine antibody for 30 min at RT. After that, samples were washed by PBS 1 and supernatant was discarded. Samples were incubated with Alexa Fluor 546 goat anti-mouse antibody for 30 min at RT and then centrifuged for 5 min at 4oC; all but 500 l of supernatant was discarded. 500 l of PBS 1 was added to each sample, and flow cytometric analysis was performed on FACSCanto II Cytometer (BD Biosciences) and BD FACSDivaTM software.

Cellular uptake studies

The cellular uptake of Doc-NPs and Doc-NPs- scFv by cells was observed by fluorescence microscope. BT474 and MDA-MB-231 cells were allowed to adhere to a glass coverslips in 12-well plate for 24 h before experiments. The cells were then incubated with 100 g/ ml of Doc-NPs or Doc-NPs-scFv for 1 h at 37oC. After washing twice with PBS, samples were incubated with monoclonal mouse antihexahistidine primary antibody for 1 h at 4ºC and then samples were washed three times by PBS 1. Samples were incubated with Alexa Fluor 546 goat anti-mouse secondary antibody for 1 h at RT and washed by PBS 1 three times. Nucleus was counterstained with Hoechst, the cells were fixed with 4% formaldehyde for 10 min and analyzed by confocal fluorescence scanning microscopy (Zeiss LMS 510 confocal microscopy).

In vitro cytotoxicity assays

The in vitro cytotoxicity of the following docetaxel formulations was tested on BT474 and MDA-MB-231 cells using the MTT test: Doc-PLGA-PEG, Doc-PLGA-PEG-scFv, and free docetaxel. Unloaded nanoparticles were used as a control. BT474 and MDA-MB-231 cells were first grown in 96-well plates at the density of 5×102 viable cells/ wells and incubated for 24 hours to allow cell attachment. The medium was replaced by 100 l of the formulation at different concentrations of 30-400 nM followed by incubation for 72 h at 37ºC. For free docetaxel, a stock solution was prepared in DMSO (1M Docetaxel). The DMSO concentration in the medium was lower than 0.5%, at which level it has no effect on cell proliferation. The cell viability was detected by MTT assay (Promega, US) according to manufacturer’s instruction. Each assay was repeated three times. Cell viability was calculated using the following equation:

Cell viability (%) = (Ints/Intcontrol) 100 (1)

Where Ints is the colorimetric intensity of cells incubated with the samples, and Intcontrol is the colorimetric intensity of cells incubated with the DMEM medium only

Statistical analysis

Statistical analysis was performed with the statistical Prim Graph version 5 software. The nonparametric test was used to calculate the probability of significant differences among the groups. Statistical significance was defined as p<0.05.

Characterization techniques

UV-visible (UV-vis) absorption spectroscopic measurements were recorded on a single beam UV-vis spectrometer, Agilent 8453, using quartz cells of 1 cm path length and methanol as the reference solvent at room temperature. Also, Fourier Transform Infra-Red (FTIR) measurements were recorded on a Shimadzu FT-IR 4300 instrument using KBr pellets at room temperature. Transmission electron microscopic (TEM) images of the nanoparticles were taken with a LEO 912AB instrument operated at an accelerating voltage of 120 kV with line resolution of 0.3 nm at room temperature. The samples for TEM measurements were prepared by placing a droplet of the colloidal solution onto a carbon-coated copper grid and allowing it to dry in air naturally. X-ray diffraction (XRD) was carried out with a Bruker D8 ADVANCE X-ray Diffractometer, using the wavelength of 0.15406 nm (CuK------------) radiations at room temperature. Based on the TEM images we determined the size distributions of the final product by counting at least 300 particles. The elemental analyses for carbon, hydrogen, nitrogen, sulfur and oxygen were performed using a Thermo Finnigan Flash EA CHNS-O analyzer. We determined the gold percentage in the Folate-4Atp-AuNP by Shimadzu model AA-670 atomic absorption spectrophotometer.

Results and Discussion

Synthesis of PLGA-PEG polymer

Carboxyl-functionalized PLGA-PEG copolymer was synthesized by direct conjugation of PLGA-COOH with NH2-PEG-COOH, both having fixed block length, to generate PLGA-PEG. The basic chemical structure of PLGA-PEG copolymer was confirmed by 1H-NMR (Figure 2). One of the prominent features is a peak at 3.4 ppm, matching the methylene groups of PEG. Overlapping doublets at 1.6 ppm are attributed to the methyl groups of the D- and L-lactic acid repeat units. The multiples at 5.2 ppm and 4.8 ppm correspond to the lactic acid – CH and the glycolic acid –CH, respectively, with the high complexity of the peaks resulting from different D-lactic, L-lactic glycolic acid sequences in the polymer backbone. The carboxyl group located at the end terminal of the hydrophilic PEG block is available for surface chemistry on the nanoparticle surface.

nanomedicine-nanotechnology-derivetives

Figure 2: TEM images of NPs (A), Doc-NPs (B) and scFv-Doc-NPs derivetives (C).

Preparation and characterization of docetaxel-loaded nanoparticles

Docetaxel was encapsulated in the pegylated PLGA nanoparticles with carboxyl end groups by the nanoprecipitation method. The physicochemical characteristics of the nanoparticles are summarized in Table 1.

Sample Mean size ± SD(nm) PDI ± SD Zeta potential ± SD(mV) EE
PLGA-PEG 87 ± 5 0.08 ± 0.02 - 24 ± 0.3  
Doc-PLGA-PEG 105 ± 4 0.1 ± 0.07 - 25 ± 0.5 43%
ScFv-Doc-PLGA- 145 ± 9 0.1 ± 0.07 - 32 ± 1  
PEG        

Table 1: Physicochemical characteristics of PLGA-PEG, Doc-PLGA-PEG and scFv-Doc-PLGA-PEG nanoparticles (n=3).

One of the most important characteristic of nanoparticle systems is their size and size distribution. The biodistribution, toxicity, and targeting ability of these systems is determined by their size. Pore sizes in tumor microvasculature vary between 100 nm and 780 nm [21]. Many studies have recognized the suitable size of these carriers for extravasation and accumulation in solid tumors below 400 nm [22]. As shown in Table 1, the nontargeted nanoparticles had a size of 105 ± 4 nm, whereas the monoclonal antibody-targeted nanoparticles were larger at 145 ± 9 nm. Conjugation may be a reason for the larger size of the targeted nanoparticles. Several steps of the centrifugation and freeze-drying process may be another reason for the larger size of the targeted nanoparticles [23]. Scanning electron microscopy showed that the nanoparticles and targeted nanoparticles were spherical and rather homogeneous in size (Figure 3).

nanomedicine-nanotechnology-restriction

Figure 3: The cloning and expression of pET22b(+) vector containing anti- HER2 scFv gene in E. coli cells. 3A. Lane M: DNA marker 1kb (Fermentas), lane 1: anti-HER2 scFv gene; 3B. E. coli clones; 3C. Lane M: DNA marker 1kb, lanes 1-10: anti-HER2 scFv gene analyzed by restriction enzymes from E. coli clones.

For nanoparticles and monoclonal antibody -targeted nanoparticles, the zeta potential was -24 ± 0.3 mV and – 32 ± 1 mV, respectively. This result shows that the negative value of mean zeta potential of the targeted nanoparticles is increased due to coupling of anti-HER2 scFv to the nanoparticles, containing several ion groups and suggesting that conjugation of monoclonal antibodies to the nanoparticles leads to an increase in the negative surface charge of targeted nanoparticles.

Previous studies have shown that in order to release taxane drugs at a sustainable rate from PLGA NPs, the drug loading concentrations should be limited [24], especially in the case of pegylated NPs [25]. Therefore, NPs containing variable amounts of docetaxel were synthesized by adjusting docetaxel drug loading at 10% by weight of the added polymer and it was satisfactory rate for goog drug encapsulation efficiency [26]. Table 1 also shows the drug loading efficiency and the PLGA-PEG nanoparticles prepared with nanoprecipitation method in this study resulted in a high drug encapsulation efficiency of 43%.

Purification of anti-HER2 scFv

Anti-HER2 scFv antibodies were used as targeting molecules for directed delivery of Doc-loaded PLGA-PEG NPs to tumor cells. Targeting proteins were produced in E. coli and purified as described in Materials and Methods. The proteins obtained were of the expected molecular weight and homogeneity according to SDS-PAGE (Figure 4).

nanomedicine-nanotechnology-Purification

Figure 4: Purification of anti-HER2 scFv. 12.5% SDS-PAGE confiming the purification of anti-HER2 scFv (31 kDa), CoomassieBrillian Blue R-25 stained gel; Lane M: Standard protein marker (Thermo Scientific); Lane 1: total protein of culture extract; Lane 2, 3 and 4: elution fractions ofscFv protein.

Flow cytometric analysis

To test whether Doc-NPs-scFv would differentially bind to HER2- positive and HER2-negative cells, the binding of Doc-NPs-scFv to BT474 and MDA-MB-231 cells evaluated by FCM analysis. Previous studies have well established that BT474 is a human breast cancer cell line that overexpresses HER2 protein on its surface (3.7 106 receptors/ cell) and that MDA-MB-231 cell is a HER2-negative human

breast cancer cell line (7 103 receptors/cell) [27-30]. Consistent with the original report, Doc-NPs-scFv (blue curve) demonstrated a higher binding to the HER2-positive BT474 cells than the HER2- negative MDA-MB-231 cells (Figure 5). The mean fluorescent intensity of Doc-NPs-scFv binding with BT474 cells is about 3 times higher than that with MDA-MB-231 cells. Since scFv may facilitate binding with the target.

nanomedicine-nanotechnology-overexpress

Figure 5: FMC analysis of binding activity of HER2-overexpress BT474 and HER2-underexpress MDA-MB-231 cells treated with Doc-NPs solution as control and scFv-Doc-NPs derivative at RT for 30 min in PBS.

Cellular uptake experiment

The results showed that Doc-NPs-scFv generated enhanced fluorescence intensity in HER2-positive BT474 cells, comparing with HER2-negative MDA-MB-231 cells (Figure 6). To further explore the in vitro cancer targeting of Doc-NPs-scFv against the HER2- overexpressing cells, we perfomed in vitro cellular uptake studies with both BT474 and MDA-MB-231 cells using confocal fluorescence microscopy. The cells were incubated with antibody-conjugated or antibody-unconjugated nanoparticles as described and then fixed and detected the bound nanoparticles by indirect immunofluorescence by using anti-histag primary antibody and Alexa 488-conjugated antimouse secondary antibody. Nuclei were counterstained with Hoechst. The images clearly indicated that the Doc-NPs-scFv were mainly accumulated in the cytoplasm around the nucleus of BT474 cells. Accordingly, Doc-NPs-scFv could be internalized into the BT474 cells and carry the anticancer drugs into the cytoplasm. Compared to BT474 cells, fluorescent signal of Doc-NPs-scFv entered MDA-MB-231 cells was much less; suggesting again that the scFv facilitated the uptake of nanoparticles into the cells.

nanomedicine-nanotechnology-Confocal

Figure 6: Confocal microscopy images of HER2-overexpress BT474 and HER2-underexpress MDA-MB-231 cells treated with Doc-NPs or scFv-Doc- NPs. The nuclei were stained with Hoechst. The merged images of Alexa 546 and the Hoechst channels. BT474 cells exposed to scFv-Doc-NPs at 100 μg/ ml for 1 h.

In vitro cellular cytotoxicity assay

A series of in vitro cytotoxicity assays was performed to evaluate the anticancer potential of Doc -NPs, Doc-NP-scFv and using BT-474 and MDA-MB-321 cells after an incubation of 72 h at 37oC (Figure 7). Anti-HER2 scFv-conjugated and unconjugated nanoparticles with no drug loading were also tested to determine the effects of conjugation on cell viability. Statistical analysis showed that the drug-unloaded nanoparticles did not influence on cell viability. Doc-NPs was highly cytotoxic for both BT-474 and HCT116 cells and they were also more toxic than free docetaxel. However, the most cytotoxicity of Doc-NP-scFv was in BT474 cells, while the cell toxicity of MDA-MB-231 cells was lower. The greater efficiency of the Doc- NP-scFv derivative can be explained by their specific interaction with HER2 antigens on the surface of BT-474 cells, indicating the potential of these nanoparticles to treat human HER2-positive cancer cells.

nanomedicine-nanotechnology-Cytotoxic

Figure 7: Cytotoxic effects of free Doc and Doc-NPs with or without anti- HER2 scFv on target BT474 (A) and non-target MDA-MB-231 (B) cells. Cell viability was analyzed after incubation with different NPs and free Doc (equivalent docetaxelconcentrations (0 μM, 0.05 μM, 0.5 μM, 1 μM, 2.5 μM, and 5 μM) of free Doc ( ), Doc-NPs ( ) and scFv-Doc-NPs ( ) using cytotoxicity assay Kit (MTT).

Conclusion

In this study, anti-HER2 scFv-decorated pegylated PLGA nanoparticles with suitable characterization were successfully prepared for targeted delivery of docetaxel. We next demonstrated enhanced NP delivery to HER2-positive cancer cells as compared to HER2- negative cancer cells. The results suggest that Doc-NPs-scFv may have application potentials in targeted therapy against HER2-positive caner cells.

Acknowledgements

This work was supported by State Program ‘Application- oriented basic scientific research’ Project number: 04/2011/HÐ-NCCBUD and Project number VAST-02.02/1314.

References


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