Surface modification of PLGA nanoparticles with biotinylated chitosan for the sustained in vitro release and the enhanced cytotoxicity of epirubicin

Colloids and Surfaces B: Biointerfaces 138 (2016) 1–9

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Surface modification of PLGA nanoparticles with biotinylated chitosan for the sustained in vitro release and the enhanced cytotoxicity of epirubicin Hongli Chen a,∗ , Li Qin Xie a , Jingwen Qin a , Yajing Jia a , Xinhua Cai b , WenBin Nan a , Wancai Yang b,∗ , Feng Lv c , Qi Qing Zhang c a

School of Life Science and Technology, The Key Laboratory of Biomedical Material, Xinxiang Medical University, Xinxiang, China School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, China c Institute of Biomedical Engineering, Chinese Academy of Medical Sciences, Tianjin, China b

a r t i c l e

i n f o

Article history: Received 2 September 2015 Received in revised form 20 October 2015 Accepted 16 November 2015 Keywords: PLGA nanoparticle Chitosan Biotin Epbrubicin

a b s t r a c t In this study, poly(d,l-lactide-co-glycolide) nanoparticles (PLGA NPs) with biotinylated chitosan (Bio-CS)surface modification were prepared to be usded as a tumor-targeted and prolonged delivery system for anticancer drugs. Epirubicin (EPB), as a model drug, was encapsulated into Bio-CS surface modified PLGA (Bio-CS-PLGA) NPs with a drug encapsulation efficiency of 84.1 ± 3.4%. EPB-loaded Bio-CS-PLGA NPs were spherical shaped, and had a larger size and higher positive zeta potential compared to the unmodfied EPBloaded PLGA NPs. The in vitro drug releases showed that EPB-loaded Bio-CS-PLGA NPs exhibited relatively constant drug release kinetics during the first 48 h and the drug burst release significantly decreased in comparison to the unmodified PLGA NPs. The results of MTS assays showed that Bio-CS-PLGA NPs markedly increased the cytotoxicity of EPB, compared to both the unmodified PLGA NPs and the CS-PLGA NPs. The uptakes of NPs in human breast cancer MCF-7 cells were evaluated by the flow cytometry and the confocal microscope. The results revealed that Bio-CS-PLGA NPs exhibited a greater extent of cellular uptake than the unmodified PLGA NPs and CS-PLGA NPs. Moreover, the cellular uptake of Bio-CS-PLGA NPs was evidently inhibited by the endocytic inhibitors and the receptor ligand, indicating that biotin receptor-mediated endocytosis was perhaps involved in the cell entry of Bio-CS-PLGA NPs. In MCF-7 tumor-bearing nude mice, EPB-loaded Bio-CS-PLGA NPs were efficiently accumulated in the tumors. In summary, Bio-CS-PLGA NPs displayed great potential for application as the carriers of anticancer drugs. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Nanoparticles (NPs) can be accumulated in tumors after systemic administrations, and their biodistribution is mainly determined by their physical and biochemical properties, such as the particle size and surface property, the nature of the polymer, and the characteristics of the drug [1,2]. Polylactic-co-glycolic acid (PLGA) is FDA-approved biodegradable polymers [3–5]. Various kinds of NPs prepared from PLGA have been investigated and showed great potential as carriers for cancer therapy in patients [4–6]. However, the main drawback of PLGA NPs is their non-specific interaction with cells and proteins

∗ Corresponding authors at: 601 Jinsui Road, Hongqi District, Xinxiang 453003, China. Fax: +86 373 3029887. E-mail addresses: [email protected] (H. Chen), [email protected] (W. Yang). http://dx.doi.org/10.1016/j.colsurfb.2015.11.033 0927-7765/© 2015 Elsevier B.V. All rights reserved.

which lead to drug accumulation in nontarget tissues [5–8]. Their lack of suitable functional groups for efficient covalent conjugation with bioactive ligands constitutes another drawback. Unmodified PLGA NPs can be internalized by many types of non-phagocytic cells in culture. By tuning the size, shape and surface properties of PLGA NPs, the cellular uptakes of encapsulated drugs can be significantly improved [5,7]. Antibody, aptamer and even small molecule surface-modified PLGA NPs exhibit preferential binding to the target cells and/or enhanced cellular internalization, compared to those cells lacking the targeted receptor or ligand [2,9,10]. Active-targeting to cancer cells is a widely acceptable strategy for drug delivery, in which ligand-conjugates can directly interact with complementary moieties on the surface of target cancer cells [1]. In our previous reports, we prepared various chitosan (CS)based surface-modified PLGA NPs to be used as drug carriers [11] and also found that using biotinylated CS (Bio-CS) conjugate as a surface-modified material could realize the distribution of func-

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tional biotin groups on the surface of PLGA NPs [12]. Biotin, one of the B complex vitamin families, is a growth promoter at cellular level [10,13–18]. Biotin content in cancerous tumors is often higher than that in normal tissue [1,10]. Thus, biotin has been generally used as an effective cancer-targeting ligand to modify the polymeric prodrugs and the nanodrug carriers [19,20]. We therefore deduced that Bio-CS-PLGA NPs maybe used as a novel tumor-targeted carrier for the anticancer drugs. In this study, epirubicin (EPB) was used as a model anticancer drug to be encapsulated into Bio-CS-PLGA NPs, and the EPB loading and in vitro release behaviors were also studied. We further evaluated the cytotoxicity and the cellular uptake of EPB-loaded Bio-CS-PLGA NPs in MCF-7 human breast cancer cells, on which biotin receptor was largely overexpressed. Meanwhile, several endocytosis inhibitors including chlorpromazine, flipin, amiloride and the ligand biotin for biotin receptor, were selected to preliminarily study the potential cell entry mechanism of Bio-CS-PLGA NPs. 2. Materials and methods 2.1. Materials PLGA (MW 10,000–30,000, the ratio of lactic to glycolic acid in the copolymer is 50:50, with uncapped end-group) was purchased from Shandong Institute of Medical Instruments. CS (low molecular weight, >80% deacetylation, Brookfield viscosity 20.000 cps) and Polyvinyl alcohol (PVA, wt 30,000–70,000) was purchased from Sigma–Aldrich (St. Louis, Mo, USA). EPB (purity 99.5%) was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. Biotin, chlorpromazine, filipin, and hydrochloride amiloride were purchased from Sigma–Aldrich. Quant* Tag Biotin Kit (BDK-2000) was purchased from Vector Laboratories, Inc. MCF-7 cell line was obtained from Cancer Institute & Hospital, Chinese Academy of Medical Sciences. All other chemical reagents were of analytical or HPLC grade and obtained from commercial sources. 2.2. Synthesis of biotinylated CS (Bio-CS) Bio-CS was synthesized, as shown in Fig. 1(a). In brief, biotin (0.5 mol), dicyclohexylcarbodiimide (DCC, 0.5 mol) and Nhydroxysuccinimide (NHS, 0.6 mol) were mixed in 20 mL DMF followed by stirring for 18 h. The reaction mixture was filtered and the excess of diethyl ether was then added to get the precipitate of NHS-biotin. The thus-obtained crude product of NHS-biotin was purified by the recrystallization in 2-propanol (70 ◦ C). To conjugate biotin with CS, 10 mL 1% CS in acetic acid solution was added dropwise into certain amount of NHS-Bio in DMF, and the mixture was then continuously stirred for 20 h at room temperature. After that, the mixture was dialyzed against deionized water using dialysis tubing with Mw cut-off of 8000 Da (Spectrum Laboratories, USA) for 48 h with 6-water exchanges, and then freeze dried to obtain Bio-CS. 2.3. Preparation of blank and EPB-loaded NPs PLGA NPs were prepared by the modified solvent displacement method as described previously [11,12]. Typically, 100 mg of PLGA was dissolved in 4 mL methylene chloride (CH2 Cl2 ) and EPB was dissolved in 500 ␮L deionized water with the concentration of 10 mg/mL. The EPB aqueous solution was added drop wise to methylene chloride, and the mixture was then sonicated by an ultrasonic probe (UH-500A, Autoscience Instrument, Co., Ltd.) to form primary emusion (W1 /O). After that, the emulsion was poured into 100 mL deionized water containing 0.5% (W/V) PVA (W2 phase) under stirring and continuously stirred at 400 rpm for

4 h. After the filtration through membrane filter with the pore size of 0.8 ␮m, the NP suspension was centrifuged at 15,000 rpm for 10 min. The sediment product was resuspended in 10 mL water and then freeze–dried (Labconco, USA) to get the powder of EPB-loaded PLGA NPs. In addition, blank PLGA NPs were also prepared by the same method only without the addition of EPB. To activate the carboxylic groups on the surfaces of PLGA NPs, 100 mg of PLGA NP powder was dispersed in 5 mL of PBS (pH 5.0) containing EDC (40 mg) and NHS (20 mg). CS or Bio-CS (60 mg) was then added to this suspension and the resulting mixture was stirred for 24 h at ambient temperature. Upon completion of the reaction, the excess coupling reagent was removed by centrifugation to give the powders of CS-PLGA or Bio-CS-PLGA NPs. Furthermore, blank CS-PLGA and Bio-CS-PLGA NPs were also prepared at the same time.

2.4. Characterization of the NPs 2.4.1. Surface morphology Surface morphologies of NPs were visualized by a field emission scanning electron microscopy (SEM, Jeol, JSM-6700F, Akishima Toky, Japan) system. Before imaging, the freeze–dried samples were coated by platinum for 4 min.

2.4.2. Particle size analysis and zeta potential measurement The particle size and size distribution were measured by 90 Plus Particles Size Analyzer (Malvern nanoZS, England) based on the laser light scattering technique. Before measurement, the freshly prepared NPs were appropriately diluted and the final concentrations were about 0.5 mg/mL. The zeta potential was determined by Zeta Potential Analyzer in deionized water.

2.4.3. Drug loading content and encapsulation efficiency determination Briefly, 10 mg of NPs was dissolved in 1 mL of methylene chloride and the loaded EPB was subsequently extracted with 0.05 M HCl solution. The recovery rate was determined from the control experiments involving a mixture of blank NPs and a known amount of EPB. The amount of EPB was determined using high performance liquid chromatography (HPLC) method as described previously [21]. The EPB loading contents (LC) and encapsulation efficiency (EE) values can be calculated as following Eqs. (1) and (2): LC =

The mass of EPB in the NPs × 100% The mass of NPs weight

(1)

EE =

The mass of EPB in the NPs × 100% Total mass of EPB

(2)

2.4.4. In vitro drug release The in vitro releases of EPB from above NPs were studied using dynamic dialysis method in phosphate buffer solution (PBS, pH 6.8) containing 0.01% (w/v) sodium azide. Briefly, 20 mg of EPB-loaded NPs was suspended in 5 mL of PBS and vibrated in a water-bath shaker at 75 rpm at 37 ◦ C. At designated time intervals, all samples were centrifuged at 15,000 rpm for 10 min, the supernatants were taken out for the detection of EPB content and 5 mL of fresh release media were then added at the same time. The EPB release amounts were determined by HPLC analysis as above described. For the evaluation of the release kinetics, the release data were fitted into first order, zero order and Higuchi models. The subsequent selection of the optimum model was based on the comparison of the relevant correlation coefficients.

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Fig. 1. The synthesis route of Bio-CS (a) and the illustration for preparation of Bio-CS-PLGA NPs (b).

2.5. Analysis of the chemical composition and the biotin content on the surface of NPs The chemical composition of the surface layer of NPs was analyzed by X-ray photoelectron spectroscopy (XPS, PHI-1600, PerkinElmer, MN, USA). The binding energy spectrum was recorded from 0 to 1100 eV with pass energy of 80 eV under the fixed transmission mode. For the further quantitative analysis of chemical composition, two samples in each group were detected and the survey scans were performed in two different areas on each sample. The biotin content on the surface of NPs was quantitatively determined using a commercially available Quant* Tag Biotin Kit (BDK-2000, Vectorlabs, CA, USA). The kit reagents can chemically react with free or bound biotin to produce a stable and colored product that can be quantified by a spectrophotometer. The contents of bound biotin on the surface of NPs were determined in accordance with the instructions. 2.6. Cell culture Human breast cancer MCF-7 cells were cultured in Dubelco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 mM sodium pyruvate, 1.5 ␮g/L of sodium bicarbonate, and 1% penicillin–streptomycin, and incubated in a SANYO CO2 incubator (Sanyo, Osaka, Japan) at 37 ◦ C in a humidified-environment of 5% carbon dioxide. 2.7. In vitro cytotoxicity In vitro cytotoxicities of EPB-loaded NPs were evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-

2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Briefly, MCF-7 cells were seeded into a 96-well plate at a density of 4 × 104 cells/well. After the cells form monolayer, the culture media were replaced by solutions of EPB-loaded NPs or free EPB with drug dose of 10 ␮g/mL. After a further 2 h incubation, NPs or EPB solutions were then replaced with fresh media after several washing steps and the cells were then incubated for 24, 48, 72, 96 and 120 h. At designated time intervals, 20 ␮L MTS solution with concentration of 0.5 mg/mL was added to each well and incubated for a futher 3 h. After that, the culture media were removed and 100 ␮L of isopropanol were then added. Finally, the absorbance of cells were detected at 450 nm using the microplate reader (Thermo Multiscan MK3, Thermo Fisher Scientific, MA, USA) with the plain cell culture media as the control. Cell viability was expressed by the ratio between the absorbance of the cells incubated with EPB-loaded NPs (or free EPB) to that of the cells incubated with blank culture media only. In addition, the inhibitory effects of blank NPs with concentration range of 0.2–5 mg/mL on the proliferation of MCF-7 cells were also assessed using MTS assay to preliminarily evaluate the biosecurity of this novel carrier. 2.8. Cellular uptake of NPs The uptakes and distributions of the NPs in MCF-7 cells were evaluated by the confocal microscope using the red flourescence of EPB. Briefly, the cells were seeded in a chambered coverglass system (LABTEK, Nagle Nunc, Napersville, IL, USA) and incubated with the EPB-loaded NPs at EPB concentration of 10 ␮g/mL at 37 ◦ C for 2 h. After that, the cells were rinsed three times with cold PBS and then fixed with 4% paraformaldehyde. Finally, the fixed cells were counterstained with 4 ,6-diamidino-2-phenylindole (DAPI)

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Fig. 2. SEM images (a) and XPS spectra (b) of EPB-loaded PLGA, CS-PLGA and Bio-CS-PLGA NPs.

(Sigma–Aldrich, St. Louis, MO, USA) to visualize nuclei and were observed under a confocal laser scanning microscope (LSM 410, Zeiss, Jena, Germany). The celluar uptakes of EPB-loaded NPs were further quantificationally determined by the flow cytometry (Beckman–Coulter, Miami, FL, USA). Typically, the MCF-7 cells were seeded in 6-well tissue culture plates at a density of 2 × 105 cells/well and cultured in DMEM media at 37 ◦ C, 5% CO2 for 24 h. The cells were then incubated with EPB-loaded NPs at 37 ◦ C for further 2 h. After removal of the supernatants, the cells were trypsinized and then centrifuged at 1500 rpm for 5 min to obtain cell pellets. After that, the cell pellets were washed twice with PBS and then resuspended in PBS. It was note worthy that the cells were processed in ice bath (4 ◦ C) to inhibit the further cellular uptake. The fluorescence signals of MCF-7 cells were detected using a coulter EPICS/XL MCL Beckman–Coulter and the resulting data were analyzed using the Expo32 software. 2.9. The cell entry mechanism of EPB-loaded Bio-CS-PLGA NPs Various endocytic inhibitors including chlorpromazine, filipin, hydrochloride amiloride and the receptor ligand of biotin were used to further study the potential cell entry mechanism of EPB-loaded Bio-CS-PLGA NPs. MCF-7 cells were pre-incubated with these inhibitors or biotin at their nontoxic concentrations e.g., 10 ␮g/mL for chlorpromazine, 5 ␮g/mL for filipin, 10 ␮g/mL for hydrochloride amiloride, and 10 ␮g/mL for biotin, in serum-free media for 24 h at 37 ◦ C. Afterwards, the culture media were removed and the fresh media containing EPB-loaded NPs were then added. After further 2 h incubation, the cells were washed three times with PBS and analyzed by the flow cytometry method as described above. The cells, only treated with NPs, were used as the control and their uptake was designed as 100%. 2.10. The distribution of NPs in the tumor-bearing mice NPs were firstly labeled with a near-IR dye, Cy5.5 NHS ester. Briefly, NPs and Cy5.5 NHS ester were dissolved in dried dimethyl sulfoxide (DMSO) and reacted for 72 h at room temperature by avoiding light. Excess dye molecules were removed by centrifugation filtration through 3 kDa MWCO Amicon filters (Millipore,

Billerica, MA, USA) and washed with water for over 8 times. In vivo imaging technique was used to evaluate the distribution of NPs in nude mice bearing MCF-7 tumor. Female Balb/c nude mice were inoculated subcutaneously with 200 mL cell suspensions containing 1 × 106 MCF-7 cells. When the tumors grew up to 300–400 mm3 in average, the mice were randomly divided into five groups, respectively treated with free CY5.5, PLGA, PLGA, Bio-CS-PLGA NPs and physiological saline (control). Cy5.5-labeled NPs or free Cy5.5 was injected into the tumor-bearing nude mice via tail vein, and then imaged using Maestro in vivo Imaging System Cri (Woburn, MA, USA) at the predefined times. All mice were sacrificed at 24 h post injection and the major organs were collected for further detection. 2.11. Statistical analysis Data were analyzed using an F-test with subsequent T-tests (equal variance) for the comparison between two different groups. For 3 or more groups, ANOVA test was used followed by a least significant differences method. Results were considered statistically significant at P < 0.05. All data reported were mean value ± S.D., unless otherwise noted. 3. Results and discussions 3.1. The preparation and characterization of NPs with EPB loading Bio-CS was synthesized and the DS of biotin group in its molecule was about 31%, which was determined by the 1 H NMR and ICP methods[12]. We have prepared PLGA NPs using this method and successfully realized the surface coating with Bio-CS by the covalent binding method, shown in Fig. 1(b) [13]. In this current study, an optimum feed ratio of Bio-CS to PLGA NP matrix of 0.6:1 (w/w) was used to prepare Bio-CS surface-modified PLGA NPs with EPB loading. Furthermore, EPB-loaded CS surface-modified PLGA NPs were also prepared as the control by the same method. EPB-loaded NPs were firstly characterized in terms of their morphology, surface chemical composition and biotin content. The SEM images are shown in Fig. 2(a). Like PLGA NPs, CS-PLGA and Bio-CSPLGA NPs had the regularly spherical shapes with the relatively

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Fig. 3. Cumulative release profiles of EPB from PLGA, CS-PLGA and Bio-CS-PLGA NPs.

uniform distributions, indicating that the surface modification methods used in this study could not influence the morphology of PLGA NPs. Next, we assessed the surface chemical composition of PLGA, CS-PLGA and Bio-CS-PLGA NPs using XPS, which can provide the surface chemical composition of the NPs in the depth range of 5–7 nm, to confirm the presence of biotin groups on the surface of NPs. Fig. 2(b) shows the XPS spectra of PLGA, CS-PLGA and Bio-CS-PLGA NPs. Because chitosan contains nitrogen element, we could evaluate the coating efficiencies of CS and Bio-CS on PLGA NPs by the detection of nitrogen content. Compared with PLGA NPs, the characteristic signals of N 1s (atomic orbital 1s of nitrogen) were observed both in the spectra of CS-PLGA and Bio-CA-PLGA NPs, and the weight percentages of nitrogen were about 5.2% and 4.8%, respectively. In a similar way, biotin groups on the surface of NPs could be assessed by the detection of sulphur content. Obviously, no sulphur element was observed both in the XPS spectra of PLGA NPs and CS-PLGA NPs (Fig. 2(a) and (b)). However, the characteristic signal of S 1s (atomic orbital 1s of sulphur) appeared in the XPS spectrum of Bio-CS-PLGA NPs (Fig. 2(c)), confirming the surface distribution of biotin groups on Bio-CS-PLGA NPs. In view of the fact that the surface biotin density of Bio-CS-PLGA NPs was directly related to their tumor-targeting capability, we further determined the surface biotin density of Bio-CS-PLGA NPs using Quant* Tag Biotin Kit. In this study, the biotin density on the surface of NPs was (7.33 ± 0.71) × 10−9 mol/mg NPs when the feed ratio of Bio-CS to PLGA NPs was about 0.6:1 (w/w). Moreover, we measured the size, size distribution and surface charged property of Bio-CS-PLGA NPs. Table 1 shows the mean diameter and zeta potential of various NPs. The mean diameter of EPB-loaded PLGA NPs was 248.4 ± 21.0 nm with a polydispersity index of 0.154 ± 0.079, indicating a relatively narrow size distribution. The zeta potential of EPB-loaded PLGA NPs was −21.21 ± 2.13 mV, but changed to be −31.61 ± 2.37 mV after activation with EDC, which suggested that a large number of COOH gourps were produced on the NPs surface due to the hydrolysis of PLGA. It is widely accepted that zeta potential is an important parameter for the NPs as it can influence both the physical stability and the biological mucoadhesion of the NPs [7,24]. In theory, the more pronounced zeta potentials in the range of 20–40 mV, being positive or negative, is favorable for stabilizing the NPs in colloid solutions. In addition, the NPs with positive surface charges are much easier to be uptaken by the cancer cells due to their interactions with negatively charged cell membranes [7]. Thus, we believed that the positively charged CS-PLGA and Bio-CS-PLGA NPs could preferably carry EPB to enter cancer cells in comparison to the negatively charged PLGA NPs.

Fig. 4. Cytotoxicity of blank PLGA, CS-PLGA and Bio-CS-PLGA NPs against MCF-7 cells at 24 h (a) and 48 h (b) after administrations. Viabilities of cells incubated with free EPB, EPB-loaded PLGA, CS-PLGA and Bio-CS-PLGA NPs. (c) *P < 0.05, compared with the EPB. **P < 0.01, compared with the EPB. **P < 0.01, compared with the CSPLGA NPs.

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Table 1 Characteristic parameters of EPB-loaded NPs.

PLGA NP CS-PLGA NP Bio-CS-PLGA NP

PLGA:CS/Bio-CS (W/W)a

Yield (%)b

Polydispersity indexc

Particle size (nm)c

Zeta potential (mV)c

EE (%)d

1:0 1:0.6 1:0.6

91.4 ± 2.6 77.7 ± 2.0 74.7 ± 2.6

0.154 ± 0.079 0.268 ± 0.171 0.213 ± 0.064

248.4 ± 21.0 273.3 ± 26.2 268.3 ± 23.4

−21.21 ± 2.13 24.45 ± 1.29 24.21 ± 2.31

84.1 ± 3.4 72.0 ± 3.5 71.4 ± 3.6

Values express as mean ± S.D. (n = 5). a PLGA/CS or PLGA/Bio-CS (mg/mg). b Yield (%) was expressed as the percentage of the NP in the the materials (PLGA, EPB and CS or Bio-CS) (mg/mg). c The size and size distribution (mean value ± S.D.) determined by the dynamic laser light scattering with three times. d Encapsulation efficiency (mean value ± S.D.) the NP (mean value ± S.D.) determined by HPLC with three times.

3.2. The drug loading capability and in vitro release property As shown in Table 1, the EPB loading content and encapsulation efficiency in PLGA NPs were 4.36% and 84.1 ± 3.4%, respectively. In contrast, EPB loading contents of EPB-loaded CS- and Bio-CSPLGA NPs decreased to be 3.48% and 3.46%, respectively, and the encapsulation efficiencies evidently decreased to be 72.0 ± 3.5% and 71.4 ± 3.6% at the same time. Above results implied that a small quantity of EPB leaked from PLGA NPs during the process of surface modifications. However, the EPB leak might be favor to reduce the drug distribution on the surface layer of PLGA NPs [11]. Fig. 3 shows the cumulative release curves of EPB from the NPs. It was clear that 78–84% of the loaded EPB was released after 10 days, indicating a large proportion of EPB could be released from these NPs. All of the profiles were characterized by a rapid procedure of initial drug releases followed by a continuous period of slow drug releases after 48 h. We believed EPB that was adsorbed on the NP surface and loosely encapsulated was firstly released during the initial release phase and that loaded in the inner of the NPs was then slowly released during the later phase. Compared with EPBloaded PLGA NPs, EPB-loaded CS-PLGA NP and Bio-CS-PLGA NP both exhibited obviously sustained releases during the first 24-h period, suggesting that the surface modification effectively reduced EPB distribution on the surface of PLGA NPs, which was manifested by the reduced drug releases from CS- and Bio-CS-PLGA NPs [11]. 3.3. In vitro cytotoxicity of EPB-loaded NPs on human breast cancer MCF-7 cells The inhibitory effect of blank Bio-CS-PLGA NPs was firstly assessed to preliminarily evaluate their biosecurity. From the result data (Fig. 4), it could be seen that all blank NPs, including PLGA NPs, CS- and Bio-CS-PLGA NPs, had no significant influence on the proliferation of MCF-7 cells in NP concentration range of 0.2–5 mg/mL at 24 h (Fig. 4(a)) and 48 h (Fig. 4(b)) post administrations, thus it could be deduced that that all blank NPs had no cytotoxicity in the concentration range used in this study. Next, we measured the cytotoxicities of EPB-loaded NPs with EPB concentration of 10 ␮g/mL on MCF-7 cells at 24, 48, 72, 96, and 120 h after administrations. As shown in Fig. 4(c), all EPB-loaded NPs showed higher cytotoxicity than free EPB at 48, 72, 96 and 120 h, respectively (P < 0.05), indicating that EPB-loaded NPs had a stronger and more durable inhibitory effect on the proliferation of MCF-7 cells. Additionally, EPB-loaded Bio-CS-PLGA NPs exhibited a higher cytotoxicity both than EPB-loaded PLGA NPs and CS-PLGA NPs. For examples, cell viability values of MCF-7 cells with treatments of free EPB, EPB-loaded PLGA, CS-PLGA and Bio-CS-PLGA NPs were respectively 26.3%, 21.2%, 19.4%, and 18.1% at 48 h, and respectively 74.1%, 54.5%, 46.6% and 36.1% at 120 h. We believed that surface-modification of PLGA NPs with CS or Bio-CS could increase the cellular uptake of the loaded drug, thus remarkably enhanced its in vitro anticancer activity. Furthermore, EPB-loaded Bio-CS-PLGA NPs had a significantly higher cytotoxicity than CSPLGA NPs, suggesting that the surface modification of biotin groups

on NPs was favor to increase the anticancer activity of the loaded drug. We believed it was perhaps due to that Bio-CS-PLGA NPs had a higher affinity for MCF-7 cells, on which biotin receptor was often over-expressed. 3.4. Uptake of NPs in MCF-7 human breast cancer cells The confocal images are shown in Fig. 5(a). Compared with PLGA NPs, the obviously enhanced fluorescence signals were observed both for EPB-loaded CS-PLGA and Bio-CS-PLGA NPs. For three types of EPB-loaded NPs, the fluorescence signals were all mainly located in the cytoplasm of MCF-7 cells. It indicated that EPB could be successfully carried by these NPs into MCF-7 cells after treatments for 2 h, but the large amounts of EPB were not released from these NPs during this period. Next, we quantitatively measured the cellular uptakes of EPB-loaded NPs using the flow cytometry method. As shown in Fig. 5(b), EPB-loaded CS-PLGA and Bio-CS-PLGA NPs both exhibited the higher fluorescence intensities in MCF-7 cells in comparison to EPB-loaded PLGA NPs. This was basically consistent with the results of confocal images. From the above results, it could be deduced that the surface properties of NPs can influence the cellular internalization process. For example, compared with EPB-loaded PLGA NPs, the uptakes of EPB-loaded CS-PLGA and Bio-CS-PLGA NPs in MCF-7 cells were increased by ∼22% (P < 0.05) and ∼41% (P < 0.01), respectively, perhaps due to the following reasons. First, according to the previous reports [22,23], CS-PLGA and Bio-CS-PLGA NPs were much easier to be uptaken by cancer cells via phagocytosis or endocytosis than PLGA NPs due to their more highly hydrophilic surfaces. Second, the surface charge of NPs could also influence their cellular uptake efficiency. NPs with positive charges often exhibit the higher cellular uptakes than the neutral and negatively charged NPs due to their electrostatic interactions with the anionic membrane [24–26]. Besides, the fluorescence intensity of EPB-loaded Bio-CS-PLGA NPs in MCF-7 cells was significantly higher than that of CS-PLGA NPs. It suggested that Bio-CS-PLGA NPs entered cancer cells by the different mechanism of cell entry other than CS-PLGA NPs. Biotin is a growth promoter of cells. Its content in cancerous tumors is significantly higher than that in the normal tissue. Recently, various biotin-conjugated macromolecular carriers were developed and showed the significantly increased uptakes of the loaded drugs in cancer cells through the process of receptor-mediated endocytosis [10,13–18]. Hence, we believed the higher cellular uptake of EPB-loaded Bio-CS-PLGA NPs was possibly because they entered MCF-7 cells via biotin-receptor mediated endocytosis. 3.5. The cell entry mechanism of EPB-loaded Bio-CS-PLGA NPs In order to obtain the information about the mechanism of particle cellular internalization, we further evaluated the uptakes of EPB-loaded Bio-CS-PLGA NPs in MCF-7 cells under the presence of the endocytic inhibitors, including chlorpromazine, filipin and amiloride, and the receptor ligand of biotin using the flow cytometry method. Chlorpromazine can inhibit the clathrin-mediated

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Fig. 5. Confocal image (a) and FACS results (b) of MCF-7 cells after 2 h incubation with EPB-loaded NPs.

slight influence on the cellular uptakes of CS-PLGA and PLGA NPs. According to the previous reports [13,14,30–32], we believed that Bio-CS-PLGA NPs would be able to enter MCF-7 cells, on which biotin receptor over-expressed, by the biotin-receptor mediated endocytosis, thus their cellular uptake was significantly inhibited by the ligand biotin. 3.6. In vivo targeted delivery of Bio-CS-PLGA NPs

Fig. 6. The inhibitory effects of various endocytic inhibitors and biotin on the uptakes of EPB-loaded Bio-CS-PLGA NPs in MCF-7 cells. The control group cells were treated with EPB-loaded Bio-CS-PLGA, CS-PLGA and PLGA NPs, respectively, but without adding inhibitors. The uptake of Bio-CS-PLGA NPs in MCF-7 cells was expressed as 100%. *P < 0.05 and **P < 0.01, compared with the control.

endocytosis [27]. Filipin can inhibit the caveolae-mediated uptake by the interaction with cholesterol in the cell membrane [28]. Amiloride, as a specifc Na+ /H+ exchange inhibitor, can selectively inhibit cell macropinocytosis [29]. Biotin can competitively inhibit the biotin-receptor endocytosis. Thus, we could use these substances to evaluate the cell entry route of Bio-CS-PLGA NPs in this study. Fig. 6 shows the cellular uptakes of EPB-loaded NPs in MCF-7 cells at EPB concentration of 10 ␮g/mL under the different conditions and the cellular uptake of EPB-loaded Bio-CS-PLGA NPs without adding any inhibitors was designed as 100%. Evidently, the cellular uptakes of various EPB-loaded NPs were more or less inhibited by the endocytic inhibitors of chlorpromazine, amiloride and filipin. It could be deduced that more than one mechanism, e.g., the clathrin-mediated endocytosis and macropinocytosis, were involved simultaneously in the cell internalizations of these NPs. Alternatively, the addition of free biotin also significantly decreased the uptake of Bio-CS-PLGA NPs in MCF-7 cells, but exerted only

To further evaluate the in vivo delivery of Bio-CS-PLGA NPs, we labeled PLGA, CS-PLGA and Bio-CS-PLGA NPs with Cy5.5 and then assessed their biodistributions in MCF-7 tumor-bearing mice after intravenous injections. At 12 h after injection, free Cy5.5 could not be detected, indicating that Cy5.5 was rapidly eliminated from the body, whereas Cy5.5 labeled NPs were almost distributed the whole body of tumor-bearing mouse at same time (Fig. 7a), suggesting that these NPs could prolonged the in vivo circulation time of Cy5.5. For example, the distributions of Cy5.5-Bio-CS-PLGA NPs in the liver, kidney and tumor were evidently observed in tumor-bering mouse (Fig. 7a). In addition, compared to the other treatments, only Cy5.5-Bio-CS-PLGA NPs were accumulated in the tumor at 24 h after administration (Fig. 7b), which further confirmed the tumor-targeted delivery of Cy5.5-Bio-CS-PLGA NPs. 4. Conclusion In this study, we successfully prepared EPB-loaded Bio-CS-PLGA NPs with active functional group of biotin on their surface. The in vitro release of EPB from Bio-CS-PLGA NPs exhibited a marked sustained release and a reduced initial burst release compared to EPB release from PLGA NPs. EPB-loaded Bio-CS-PLGA NPs exhibited the increased cytotoxicity in MCF-7 cells both compared to EPB-loaded PLGA and CS-PLGA NPs due to their enhanced cellular uptake. Moreover, the cellular internalization of Bio-CS-PLGA NPs was inhibited by the endocytic inhibitors of chlorpromazine, filipin and amiloride, indicating that more than one mechanism were involved simultaneously in their cell entry. Moreover, the addition of excess biotin significantly reduced the uptake of Bio-CS-PLGA NPs in MCF-7 cells, suggesting that these NPs were also able to enter into cancer cells

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Fig. 7. Tissue distributions of Cy5.5-labeled NPs in tumor-bearing nude mice. (a) Fluorescence images of MCF-7 tumor-bearing nude mice treated with free Cy5.5, Cy5.5-PLGA NPs, Cy5.5-CS-PLGA NPs, and Cy5.5-Bio-CS-PLGA NPs at 12 h after administrations. The control mice were injected with 200 ␮L normal saline. (b) Fluorescence image of major tissues isolated from tumor-bearing nude mice at 24 h after administrations.

via biotin receptor endocytosis. In MCF-7 tumor-bearing nude mice, Bio-CS-PLGA NPs were mainly distributed in the tumor after 24 h post administration. In conclusion, Bio-CS-PLGA NPs displayed a great potential as a novel carrier for anticancer drugs. Acknowledgments This research was supported in part by The Key Technologies R&D Program of Henan Province (No. 122102210148), the grants from the National Natural Sciences Foundation (Nos. U1304819, 81401519), and the grants for the Innovative Team for Science and Technology from the Departments of Education, Sciences and Technologies, Henan, China. References [1] A.A. Alexander-Bryant, W.S. Vanden Berg-Foels, X. Wen, et al., Bioengineering strategies for designing targeted cancer therapies, Adv. Cancer Res. 118 (2013) 1–59.

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