PLGA nanoparticles against HER-positive breast cancer cells

Accepted Manuscript Title: Enhanced delivery of Paclitaxel using electrostatically-conjugated Herceptin-bearing PEI/PLGA nanoparticles against HER-positive breast cancer cells Author: Kongtong Yu Jinlong Zhao Zunkai Zhang Yin Gao Yulin Zhou Lesheng Teng Youxin Li PII: DOI: Reference:

S0378-5173(15)30379-3 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.11.033 IJP 15371

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

24-8-2015 29-10-2015 20-11-2015

Please cite this article as: Yu, Kongtong, Zhao, Jinlong, Zhang, Zunkai, Gao, Yin, Zhou, Yulin, Teng, Lesheng, Li, Youxin, Enhanced delivery of Paclitaxel using electrostatically-conjugated Herceptin-bearing PEI/PLGA nanoparticles against HER-positive breast cancer cells.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.11.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Enhanced delivery of Paclitaxel using electrostatically-conjugated Herceptin-bearing PEI/PLGA nanoparticles against HER-positive breast cancer cells Kongtong Yu, Jinlong Zhao, Zunkai Zhang, Yin Gao, Yulin Zhou, Lesheng Teng* [email protected], Youxin Li* [email protected] College of Life Science, Jilin University, Qianjin Street No.2699, Changchun, Jilin Province, 130012, China *

Corresponding authors at: Tel.: +86-431-85155320; Fax: +86-431-85155320.

Graphical Abstract

Abstract We have developed a novel nanoparticle delivery system fabricated from polyethylenimine (PEI) and poly(d,l-lactide-co-glycolide) (PLGA), which were able to deliver the chemotherapeutic agent Paclitaxel, while the biomacromolecule Herceptin acted as a targeting ligand that was conjugated onto the surfaces of the nanoparticles via electrostatic interactions. In this study, these electrostatically-conjugated Herceptin-bearing PEI/PLGA nanoparticles (eHER-PPNs) were optimized and employed as vectors to target HER2-positive breast cancer cells. The eHER-PPNs had an average diameter of ~280 nm and a neutral surface charge (1.00 ± 0.73 mV), which remained stable under physiological conditions. The anticancer effects of eHER-PPNs were investigated in HER2-positive BT474 cells and HER2-negative MCF7 cells. The eHER-PPNs showed enhanced cytotoxicity that was dependent on the receptor expression levels and the incubation time. These conjugated nanoparticles deliver Paclitaxel more efficiently (p < 0.001) than unmodified PPNs, Herceptin and the combined effects of these two monotherapies . Furthermore, the chemically-conjugated Herceptin-bearing PEI/PLGA nanoparticles (cHER-PPNs) were fabricated as a comparison. The eHER-PPNs exhibited lower cell viability (46.7%) than that of cHER-PPNs (65.1%). The targeting ability of eHER-PPNs was demonstrated through confocal microscopy images and flow cytometry, which showed that eHER-PPNs displayed higher cellular uptake efficiency (p < 0.001) in comparison with cHER-PPNs. Therefore, eHER-PPNs could provide promising platforms for the delivery of therapeutic drugs against HER2-positive breast cancers.

Keywords:

Targeted

Polyethylenimine.

delivery;

Electrostatic

attraction;

Herceptin;

Paclitaxel;

1. Introduction Currently, effective drug delivery in cancer treatment remains a challenge. Polymer nanoparticles, as drug delivery vectors, play an essential role in the delivery of anticancer drugs (Mu and Feng, 2003; Zhang et al., 2006). These nanoparticles allow the encapsulation of hydrophobic drugs, while also providing the advantages of efficient drug loading, sustained and controlled release, high stability, prolonged circulation time and facile surface modification (Date et al., 2007; Gómez-Gaete et al., 2007; Palma et al., 2014). They are also designed to achieve active targeted delivery of drugs through the use of pH- or temperature-sensitive materials or through the incorporation of biological molecules such as folic acid, galactose, small nucleic acids, RGD motifs, peptides and antibodies into the structures of the nanoparticles (Hossain et al., 2015; Kang et al., 2003; Na et al., 2006). Among these biological molecules, antibodies are the most promising and have been applied in preclinical and clinical settings as adjuvants or for targeted therapy (Bethge and Sandmaier, 2004; Rao and Schmader, 2007). Herceptin (Trastuzumab), a recombinant humanized monoclonal antibody, targets the extracellular domain of the human epidermal growth factor receptor 2 (HER2), which is overexpressed in 25–30% of invasive breast cancers (Mi et al., 2012). It has been approved by the FDA as a single agent or for use in combination with chemotherapy in the treatment of HER2-positive breast cancers. Herceptin achieves its anticancer effects through multiple mechanisms, which include blocking HER2 extracellular domain cleavage, inhibiting intracellular signaling pathways, promoting antibody-dependent cellular toxicity, reducing

angiogenesis and disturbing DNA repair (Mi et al., 2012; Zhao and Feng, 2014). The synergistic effect has been observed for the co-delivery of Herceptin and conventional cytotoxic drugs such as Paclitaxel, Docetaxel or Carboplatin (Mi et al., 2012; Parveen et al., 2010). Herceptin has also been conjugated onto the surface of nanocarriers for use as a targeting ligand to direct the delivery of Paclitaxel to HER2-positive breast cancer cells (Gilbert et al., 2003; Jung et al., 2007). The combination of Herceptin and Paclitaxel not only endows the nanocarriers with targeting behavior, but also sensitizes cancer cells to Paclitaxel and thus achieves enhanced tumor suppression (Baselga et al., 2004; Ratcliffe et al., 2001). The current conjugation approach was primarily achieved through chemically crosslinking between Herceptin and some functional groups on the surface of the nanocarrier. However, for this monoclonal antibody Herceptin with a complex superior structure and a high molecular weight of 145.5 kDa, each reaction step must be carefully planned in order to avoid loss of activity (Hamilton, 2015; Liu, 2014; Liu et al., 2010). In addition, the closely chemical crosslinking may give rise to steric hindrance between two nearby Herceptin during recognition or hinder the release of the delievered drug after endocytosis. There is a strong need for a better approach that provides milder reaction conditions than are encountered in chemical crosslinking reactions. Polyethylenimine (PEI) has been widely used in gene transfection and drug delivery applications due to the proton sponge effect (Nimesh, 2013; Olbrich et al., 2001). This polymer exhibits a hyper branched structure and bears many primary amine groups, which are protonated under physiological conditions (Dehshahri et al., 2009; Nguyen et al., 2014). The high positive charge of PEI can cause this polymer to exhibit electrostatic interactions

with negatively charged biological molecules (nucleic acids or proteins) and thus facilitate cellular uptake. It has been reported that PEI-coated nanoparticles were used as drug vectors and exhibited enhanced cellular uptake efficiency (Chumakova et al., 2008; Liu et al., 2014). However, very litte attention has focused on the electrostatically-driven combination of cationic PEI and anionic Herceptin macromolecules. In our new approach, PLGA/PEI nanoparticles (PPNs) were first prepared, and Herceptin was subsequently electrostatically conjugated onto the PPNs (eHER-PPNs). Paclitaxel, a commonly chemotherapeutic agent that is used to treat a wide spectrum of cancers such as breast, ovarian cancer, and small and non-small cell lung cancer (Koo et al., 2013; Razi et al., 2015), was encapsulated in the hydrophobic core of these nanoparticles. Electrostatic adsorptions of anionic Herceptin and cationic PPNs were investigated at different PPNs-to-Herceptin weight ratios. The antibody binding efficiency was characterized in terms of the Herceptin loading. In addition, we fabricated

chemically-conjugated

Herceptin-bearing PPNs (cHER-PPNs) for comparison. The cytotoxic effects of the eHER-PPNs were investigated via MTT assays in HER2-positive BT474 cells and HER2-negative MCF7 cells and compared with free Herceptin, Paclitaxel and other formulations. The targeting ability of eHER-PPNs was evaluated through flow cytometry analysis and confocal images in BT474 and MCF7 cells in comparison with cHER-PPNs.

2.1. Materials Paclitaxel was purchased from Huiang Biopharma Co., Ltd. (Guilin, China). Herceptin (Mw, 145.5 kDa) was provided by Lvye Pharma (Yantai, China). Poly(d,l-lactide-co-glycolide)

(PLGA, Mw 5-10 kDa, 50: 50) was purchased from Boehringer Ingelheim Pharma GmbH & Co. KG (Ingelheim, Germany). Polyethylenimine (PEI, Mw 25 kDa, branched), polyvinyl alcohol (PVA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), methylene chloride (DCM) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other chemical reagents and solvents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All of the reagents and solvents were of analytical grade. All cell culture media, supplements and markers were obtained from Thermo Fisher Scientific (Waltham, MA, USA). BCA Protein Assay Kit was obtained from Dingguo Changsheng Biotech Co., Ltd. (Beijing, China). BT474 and MCF7 breast cancer cell lines were obtained from American Type Culture Collection (ATCC, USA). All experiments were performed using deionized water.

2.2. Preparation of the nanoparticles 2.2.1 PEI/PLGA nanoparticles (PPNs) The preparation of PPNs conducted using an oil-in water (O/W) emulsion-solvent evaporation method (Chan et al., 2009; Ravi Kumar et al., 2004). Briefly, 72 mg of PLGA and 8 mg of Paclitaxel were dissolved in 8 mL of DCM as the organic phase. Meanwhile, various weight ratios (0.0, 0.2%, 0.4%, 0.8%, 1.6%, 3.2% and 4.8%, w/v) of PEI dissolved in an aqueous PVA solution (2%, w/v) served as the aqueous phase. The organic phase was then added dropwise into the aqueous phase with an oil-water ratio of 1:1 (v/v) and homogenized at 22000 rpm (T25, IKA, Germany) for 1 min twice at a 30 s interval. Next, 14 mL of deionized water was added and homogenized at 22000 rpm for 1 min twice at a 30 s interval.

The emulsion was stirred overnight in order to evaporate the organic solvent. The obtained PPNs were washed twice in order to remove the unencapsulated Paclitaxel or excess PEI and subsequently collected by centrifugation at 15000 rpm for 15 min at 4 °C (Allegra 64R, Beckman Coulter, USA). The collected PPNs were lyophilized at -40 °C for 24 h using a BenchTop Freeze Dryer (AdVantage 2.0, SP Scientific, USA) and stored at 4 °C until use. The same method was applied to prepare the rhodamine B-loaded PPNs except that extra 1 mg of rhodamine B was added into the organic phase. During the preparation of the blank PPNs (bPPNs), only PLGA was dissolved in DCM.

2.2.2 Electrostatically-conjugated Herceptin-bearing PPNs (eHER-PPNs) eHER-PPNs were prepared according to a previously described method (Lee et al., 2009). Briefly, eHER-PPNs with various PPNs-to-Herceptin weight ratios ranging between 1: 10 and 5: 1 were prepared by adding lyophilized PPNs of various weights to Herceptin solution which Herceptin was in sodium acetate/acetic acid buffer (20 mM, pH 6.0), and mixed for 5 s. The complex solution was kept at room temperature for 30 min. The final concentration of Herceptin was 1 mg/mL.

2.2.3 Chemically-conjugated Herceptin-bearing PPNs (cHER-PPNs) cHER-PPNs were synthesized by the previously mentioned method, but with a slight modification (Arya et al., 2011). Briefly, 10 mg of NHS, 40 mg of EDC were dissolved in 2 mL of PBS (0.1 M, pH 6.0). This was followed by addition of 500 μL of Herceptin solution, in which the Herceptin has been dissolved in PBS (0.1 M, pH 7.4) at a concentration of 10

mg/mL. After 30 min, 5 mg of lyophilized PPNs that had been redispersed into in 2 mL of PBS (0.1 M, pH 6.0) was added to the above solution and left at room temperature for 4 h. The reaction mixture was dialyzed against deionized water to remove excess reagent and unconjugated Herceptin, before it was collected by centrifugation at 12000 rpm for 15 min at 4 °C. The product of cHER-PPNs was obtained after freeze-drying treatment.

2.3. Characterization of the nanoparticles The average particle size, size distribution and zeta potential of the nanoparticles were analyzed by the dynamic light scattering (DLS) technique using a Zetasizer Nano ZS90 (Malvern, UK) at 25 °C. Prior to the measurement, lyophilized nanoparticles were reconstituted in deionized water using ice-cooled sonication. DLS measurements were presented as the z-average diameter, polydispersity index (PDI), and ζ-potential. The data were calculated using the Malvern software package. All samples were measured in triplicate. The drug loading was determined directly by measuring the amount of Paclitaxel that was entrapped within the nanoparticles. Briefly, the lyophilized nanoparticles were redissolved in 2 mL of acetonitrile and sonicated for 5 min, and then 8 mL of methanol was added and mixed for high performance liquid chromatography (HPLC, Waters, USA) characterization with an absorption peak at 228 nm. The total amount of Paclitaxel in the nanoparticles was determined from the peak area, which was correlated with a standard curve. All analysis was performed in triplicate. The drug loading (DL) and encapsulation efficiency (EE) were calculated using the following Eqs. (1) and (2);

 weight of paclitaxel encapsulated in nanoparticles  DL    100% weight of nanoparticles  

(1)

 weight of paclitaxel encapsulated in nanoparticles  EE    100% weight of paclitaxel actually used  

(2)

The amount of Herceptin that was adsorbed onto the surfaces of the nanoparticles was examined indirectly. In particular the supernatant from all of the centrifugation steps of preparation process was collected for quantitative analysis using the bicinchoninic acid (BCA) method. Subsequently, 100 μL of a Herceptin solution and 200 μL of a BCA working solution were added into a 96-well plate and incubated for 30 min at 37 °C, before they were measured at 562 nm using a microplate reader (Multiskan Spectrum, Thermo, USA). The amount of Herceptin on the surfaces of the nanoparticles was calculated using Eq. (3). The Herceptin load (HL) is defined as the percentage of the amount of Herceptin present on all of the nanoparticles in a given sample.

HL  ((initial amount added to formulation  amount of free herceptin determined in supernatant) / amount of nanoparticles) 100%

(3)

The morphology of the nanoparticles was observed by transmission electron microscopy (TEM, H-800, Hitachi, Japan). The eHER-PPNs were resuspended in deionized water and sonicated before they were introduced onto a copper grid and the excess solution was subsequently blotted away with filter paper (Salihov et al., 2015). The thin layer of eHER-PPNs was obtained by evaporating the surface liquid at room temperature. These samples were subsequently examined by TEM using an acceleration voltage of 200 kV.

2.4. Native protein gel shift assay

6% Tris-glycine gel was used to study the interaction between PPNs and Herceptin via native polyacrylamide gel electrophoresis (Native-PAGE). The freshly prepared eHER-PPNs in various PPNs-to-herceptin weight ratios ranging between 1: 10 and 5: 1 were filtered through a 0.45 μm microfiltration membrane prior to sampling. The electrophoresis was initiated by pre-running the solution for 5 min at a voltage of 220 V and subsequently running the solution at a fixed voltage of 120 V for 40 min using the ice bath to cool the solution. The fronts were tracked with Bromophenol blue and the gel were stained with Coomassie brilliant blue R250 (Dingguo, China). Destaining was performed in water containing 10% methanol and 10% acetic acid. The stained gel was visualized under the GelDoc XR System (Bio-rad, USA).

2.5. Stability

To determine the stability of eHER-PPNs in physiological conditions, the lyophilized eHER-PPNs was resuspended in PBS (0.01 M, pH 7.4) with and without 10% fetal bovine serum (FBS, v/v), and subsequently incubated at 37 °C. Size and dispersity measurements were acquired at 0.5 h, 1 h, 2 h, 4 h and 24 h, respectively. Dissociated Herceptin was separated and detected using the BCA Protein Assay Kit at fixed time intervals. The stability of these nanoparticles was evaluated by both the maintenance of both the particle size and the polydispersity. In addition, the content analysis of dissociated Herceptin as also evaluated.

2.6. In vitro release

The release of Paclitaxel from nanoparticles was studied using the dialysis method. Briefly, 10 mg sample of drug-loaded nanoparticles that were dispersed into 1 mL of PBS (0.01 M, pH 7.4) containing 0.5% Tween-80 (v/v) were placed in dialysis bags (MWCO, 8 kDa). These dialysis bags were then immersed into a container containing 50 mL of the above PBS and kept shaking on an orbital shaker at 120 rpm at 37 °C. The measurements were taken at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 1 d, 2 d, 3 d and 4 d, respectively. At designated time intervals, 5 mL of the release medium was collected and replaced with the same volume of fresh medium. The release medium was analyzed for its Paclitaxel content. Methylene chloride was added to the release medium, vortexed in order to dissolve the nanoparticles and then centrifuged. The aqueous layer was discarded and the organic layer was left to settle and the

drug

present

in

this

organic

layer

was

subsequently

redissolved

in

an

acetonitrile/methanol solvent mixture (2: 8, v/v) and characterized by HPLC at 228 nm.

2.7. Cell cultures

Two human breast cancer cell lines, HER2-positive BT474 cells and HER2-negative MCF7 cells, were used in our study. Cells were grown in RPMI-1640 medium that was supplemented with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in the presence of 5% CO2.

2.8. Cellular uptake study

2.8.1. Cytotoxicity

For cytotoxicity measurements, BT474 and MCF7 cells were incubated in 96-well plates at a density of 1 × 104 cells per well. Following the overnight incubation, the old medium was removed and replaced with the same volume of fresh medium containing free Herceptin (2.82 µg), Paclitaxel and dilutions of pre-prepared nanoparticles (at a Paclitaxel concentration of 2 µM) and the cells were subsequently incubated for 4 h. The pre-prepared nanoparticles included PPNs, eHER-PPNs, cHER-PPNs and physical mixtures of Herceptin and PPNs (pHER-PPNs). The culture medium was substituted with fresh complete medium and then incubated for 24 h, 48 h and 72 h of incubation, respectively. Subsequently, 20 μL of MTT solution (5 mg/mL) was added to each well and the cells were incubated for an additional 4 h. The growth medium containing MTT solution was discarded and 150 μL of DMSO was added to each well to dissolve the produced formazan crystals. The absorbance of formazan was measured using a microplate reader at a test wavelength of 570 nm and reference wavelength of 630 nm. The cell viability was defined as the percentage of the absorbance of the experiments over that of the controls. Each condition was tested using 8 repeated measurements.

2.8.2. Confocal microscopy

The cellular uptake of the nanoparticles was visualized using confocal microscopy, in which the nanoparticles were labeled using two fluorescent markers and will thus be denoted as double fluorescence labeled samples.. In particular,

rhodamine B was used to mark PPNs

and FITC was to mark Herceptin. The rhodamine B-loaded PPNs were prepared by the method described in Section 2.2.1. To label herceptin, FITC dissolved in DMSO was added drop wise into pre-perpared Herceptin solution, in which Herceptin had been dissolved in carbonate buffer solution (pH 9.0) in a weight ratio of 1: 7 and reacted at 4 °C for 8 h in the dark. The unreacted FITC was removed through a dialysis process. The degree of cross-linking was determined using a spectrophotometer (721-100, Xinmao, China) and the cross-linking ratio was 40 mole of FITC per mole of Herceptin. For cellular uptake, BT474 and MCF7 cells were seeded into a glass-bottom Petri dish (NEST, China) at a density of 2 × 104 cells per dish and cultured to reach 70% confluency. The growth medium was subsequently removed and replaced with a medium containing eHER-PPNs and cHER-PPNs (that had been labeled with rhodamine B and FITC) at a Paclitaxel concentration of 2 µM, and incubated for 2 h prior to the removal of the medium. Cells were then washed three times with cold PBS (0.01 M, pH 7.4) to remove debris or dye, and fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature. The cells were imaged using a confocal laser scanning microscope (LSM710, Carl Zeiss, Germany) that was equipped with an argon laser using a FITC filter (Ex 488 nm, Em 525 nm) and a rhodamine B filter (Ex 540 nm, Em 625 nm).

2.8.3. Flow cytometry

Cellular uptake of nanoparticles was quantified using flow cytometry. Briefly, BT474 and MCF7 cells were seeded in 24-well plates (Thermo Fisher Scientific, China) at a density of 2 × 105 cells/well and cultivated overnight. Then, the growth medium was replaced with medium containing double fluorescence labeled eHER-PPNs and cHER-PPNs at a Paclitaxel concentration of 2 µM. The cells were then incubated for 3 h, washed three times with PBS (0.01 M, pH 7.4), harvested using trypsin and resuspended in PBS (0.01 M, pH 7.4) for flow cytometry (EPICS XL/XL-MCL, Beckman Coulter, USA) analysis.

2.9. Statistical analysis

Statistical analysis of data was performed using the Student’s t-test and two-tailed. All values are presented as mean ± SD, with p values of < 0.05 indicating statistical significance.

3. Results and Discussion

3.1. Preparation of eHER-PPNs

The core-shell structure of the eHER-PPNs is shown in Fig. 1. PLGA and PEI polymer were choosen as the components that

constituted the polymeric core. The widely-used

chemotherapy agent Paclitaxel was loaded in the polymeric matrix of the PPNs, which were

prepared by an emulsion-solvent evaporation method. The PEI was used to provide cationic group on the surface of PPNs to conjugate with the biological therapy agent Herceptin, which was used as a targeting ligand. The cationic PPNs-based core and the anionic Herceptin-based shell were drawn together by electrostatic interactions. The ratios of PEI and PPNs-to-herceptin weight ratios were optimized to provide the appropriate particle size, surface charge and ligand density. The ratios of PEI were designated to be 0%, 0.2%, 0.4%, 0.8%, 1.6%, 3.2% and 4.8%. The size and Zeta potential of PPNs are listed in Table 1, from which it can be seen that the size of the PPNs of the various PEI ratios varied from 240 to 290 nm, indicating a narrow size distribution. The Zeta potential ranged from 0 to 45 mV, which was determined by the positive charge of the PEI on the PPNs surface. This value influenced the amount of Herceptin conjugated onto the surface of PPNs. The Herceptin loading amount increased as the surface charge increased, was accompanied by an increase of the eHER-PPNs diameter, which making it difficult for cellular uptake. A few relevant studies have shown that smaller particles with a uniform size distribution, higher surface charge, and a moderate surface ligand had a higher cellular uptake (Kratz and Warnecke, 2012). Accordingly, we have choosen a PEI ratio of 1.6% for the fabrication of our

PPNs to obtain

a compromise between the size and the Zeta potential. We then incorporated a moderate targeting ligand, which was important for efficient drug delivery by the eHER-PPNs. Native-PAGE and BCA assays were performed to evaluate the ability of PPNs to bind with Herceptin at different weight ratios and ensure that Herceptin would retain its structural integrity and that it would not denature during the preparation. The ratios of PPNs-to-Herceptin were designated to be 1: 10, 1: 5, 1: 2.5, 1: 1, 2.5: 1 and 5: 1. The

unreacted herceptin was separated and applied to a Native-PAGE and compared with the native herceptin. As shown in Table 2 and Fig.2, at low PPNs-to-Herceptin ratios, PPNs could not effectively bind with Herceptin and the free Herceptin was still visible. However, the amount of free Herceptin began to decrease with increasing of PPNs-to-Herceptin ratios. Also, the binding efficiency was at a high level but the amount of reacted Herceptin was low when the PPNs-to-Herceptin ratio was as high as 5: 1. It could be seen that when the PPNs-to-herceptin ratio was 1: 1, the binding efficiency was the highest and the amount of free herceptin was nearly absent. Therefore, the PPNs-to-Herceptin ratio of 1: 1 was selected to fabricate the eHER-PPNs and was used for all subsequent experiments.

3.2. Characterization of eHER-PPNs The PPNs, eHER-PPNs and cHER-PPNs were prepared. The particle size, PDI, Zeta potential and drug loading were measured by DLS, HPLC and BCA assay, respectively. The results are listed in Table 3. The eHER-PPNs and cHER-PPNs both exhibited certain increases in their diameters and decreases in their Zeta potentials in comparison

with PPNs,

which indicated that Herceptin had been successfully conjugated onto the the surface of PPNs (Lee et al., 2009). EE of Paclitaxel that was encapsulated within eHER-PPNs and cHER-PPNs was determined to be 60.5 ± 1.9% and 63.8 ± 2.1% in comparison with a value of 66.8 ± 3.0%. observed for the Paclitaxel that was encapsulated within the PPNs. The slight decrease of the EE in the former two cases might be due to the surface Herceptin that restricted the amount of Paclitaxel that could be encapsulated near the surface. The TEM images of the PPNs and eHER-PPNs are shown in Fig.3. It can be seen that the eHER-PPNs

were spherical with a core-shell structure and the conjugation of Herceptin onto the surface was visualized. In contrast,, the PPNs exhibiteda homogeneous spherical structure. Fig. 3 also showed that the size had increased after conjugation, which was consistent with the size measured by DLS analyzer.

3.3. Stability The stability of eHER-PPNs was evaluated by particle size, polydispersity and quantitative analysis in which the eHER-PPNs were incubated in PBS (0.01 M, pH 7.4) in the prescence or in the absence of 10% FBS at 37 °C (Fig. 4). The particle sizes of the eHER-PPNs were taken at a predetermined time. During 24 h of incubation, the size of eHER-PPNs reduced by approximately 20 nm in PBS (0.01 M, pH 7.4). This behavior may be attributed to the Herceptin on the surface of eHER-PPNs undergoing partial dissociation at physiological pH, which differed form its incubation conditions. However, the size of eHER-PPNs initially increased and then decreased during the incubation in PBS (0.01 M, pH 7.4) with 10% FBS. The adsorbed serum proteins might lead to an increase in size (Khademi-Azandehi and Moghaddam, 2015). As time passed, eHER-PPNs formed stronger electrostatic interactions with the serum proteins and assumed more compact structures, so that the sizes of the eHER-PPNs decreased. Thus, the size of eHER-PPNs fabricated at PPNs-to-Herceptin ratio of 1: 1 stabilized at around 275 nm and remained unchanged at 24 h, suggesting that they could maintain stable in physiological conditions without undergoing flocculation during their release stage. The quantification of Herceptin in the incubation medium shown in Table 1 and Fig. 4C also supported the results of particle size analysis. Furthermore, the

polydispersity of eHER-PPNs in PBS (0.01 M, pH 7.4) with or without 10% FBS increased during the dissociation process and then decreased to the initial status, indicating that no aggregation occurred between two microparticles during incubation.

3.4. In vitro release The in vitro release behavior of Paclitaxel from PPNs, eHER-PPNs and cHER-PPNs was monitored over 120 h at 37 ºC (Fig. 5). It could be seen that the release profiles of all nanoparticles exhibited a biphasic pattern: an initial burst release that was followed by a slow sustained release, which could be attributed to the paclitaxel near the surface. The percentage of paclitaxel within the initial 6 h was 36.2%, 28.6%, and 25.4% for the PPNs, eHER-PPNs, and the cHER-PPNs, respectively. The release rate of the eHER-PPNs and cHER-PPNs was slower than that of PPNs, which illustrated that the presence of Herceptin on the surface reduced the release rate of Paclitaxel during the release stage. When the profiles of eHER-PPNs and cHER-PPNs were compared, it was apparent that a slow release profile was observed for cHER-PPNs. This result might be due to the fact that the Herceptin did not undergo any significant degree of dissociation from the surface of cHER-PPNs. In the following hours, Paclitaxel was released from the eHER-PPNs/cHER-PPNs in a sustained manner and kept the pace with PPNs, and finally reached a plateau phase. The accumulative release of Paclitaxel within the initial 120 h was 78%, 71.2%, and 65.2% for PPNs, eHER-PPNs, and cHER-PPNs, respectively. The data indicated that the presence of Herceptin on the surface prevented the release of Paclitaxel to some degree. There was an advantage for eHER-PPNs provide a key advantages: the faster Paclitaxel release in tumor site was helpful

to achieve therapeutic window and was benefiticial for tumor treatment.

3.5. Cytotoxicity The biocompatibility of two blank nanoparticles (defined as bPPNs and eHER-bPPNs) was evaluated in BT474 cells by an MTT assay. It could be seen from Fig. 6 there was no significant cytotoxicity encountered when the nanoparticle concentration was at 50 μg/mL (which exceeded the applied concentration of 28.2 μg/mL), explaining their good biocompatibility. The eHER-bPPNs displayed a certain degree of cytotoxicity when nanoparticles concentration was equal or greater than 100 μg/mL. the cytotoxicity of Paclitaxel-loaded eHER-PPNs was evaluated in BT474 cells and MCF7 cells and compared with that of free Paclitaxel, free Herceptin, Paclitaxel-loaded PPNs and Paclitaxel-loaded pHER-PPNs (Diermeier et al., 2005). The Paclitaxel and herceptin concentrations had been optimized to prevent non-selective cytotoxicity. As shown in Fig. 7, the cellular uptake of eHER-PPNs displayed receptor-dependent and time-dependent behavior. Notably, the BT474 cells treated with eHER-PPNs revealed higher cytotoxicity compared with PPNs, suggesting their effective targeting of the HER2 receptors. However, the MCF7 cells did not exhibit notable differences in cytotoxicity of eHER-PPNs and PPNs. The BT474 cells treated with eHER-PPNs revealed poor viability (58.2%, 50.2% and 46.7%) when compared with MCF7 cells (73.6%, 63.1% and 57.7%) after incubation for 24, 48 and 72 h, respectively. The BT474 cells treated with pHER-PPNs showed higher viability than those treated with eHER-PPNs, further explaining that the increased toxicity effects of eHER-PPNs was

attributed to HER2 receptor-mediated endocytosis instead of cytotoxic superposition of PPNs and Herceptin. Further cytotoxicity experiments were conducted to evaluate the immunoreactivity of herceptin on the surfaces of eHER-PPNs that were fabricated through electrostatic interactions and on the surface of the cHER-PPNs that were fabricated by chemical conjugation were used as a comparison (Fig. 8). Results demonstrated that BT474 cells treated with cHER-PPNs showed higher viability (74.3%, 69% and 65.1%) than the cells treated with eHER-PPNs (58.2%, 50.2% and 46.7%) after incubation for 24 h, 48 h and 72 h, respectively. These differences might be due to the activity of Herceptin or incomplete release of PPNs. The crosslinking conditions employed during the preparation of the cHER-PPNs might damage structure and immunoreactivity of the incorporated Herceptin. However, the mild conditions employed to facilitate the electrostatic attractions would not influence the activity of Herceptin incorporated onto

the surfaces of eHER-PPNs. During

recognition process, the strong chemical crosslinking might give birth to inflexible hindrance between two nearby Herceptin units. In addition, it might inhibit the separation of the PPNs from the eHER-PPNs, leading to an incomplete release of PPNs after endocytosis. In contrast, the weaker electrostatic attractions might not influence the separation process of PPNs form the eHER-PPNs. The fact that eHER-PPNs exhibited better cytotoxicity effect in comparison with cHER-PPNs, eventually contributed to the enhanced cytotoxicity.

3.6. Cellular uptake by confocal microscopy

It had been demonstrated in cytotoxicity experiments that Herceptin could be acted as a targeting ligand for HER2 receptor. Here we compared the cellular uptake efficiency between the eHER-PPNs and cHER-PPNs to show that eHER-PPNs could more efficiently play the target and enter into the cancer cells. Fig. 9 shows the confocal images of BT474 cells and MCF7 cells that were incubated with the eHER-PPNs and cHER-PPNs for 2 h, respectively. The red fluorescence represented rhodamine B-loaded PPNs, the green fluorescence represented FITC labeled Herceptin on the surface of eHER-PPNs, while the blue fluorescence represented the nucleus stained by DAPI. It could be seen from the merged images that the nuclei were circumvented by superimposed fluorescence (yellow fluorescence) of eHER-PPNs, indicating that the PPNs and Herceptin had been simultaneously internalized into cells and did not undergo fluorescence intensity

dissociation during endocytosis. In contrast, much stronger

was exhibited by the BT474 cells than by the MCF7 cells. The

eHER-PPNs groups showed higher cellular uptake in accordance with the stronger fluorescence intensity observed in comparison compared with the groups treated with cHER-PPNs. These results supported that Herceptin could function as a targeting ligand to facilitate the uptake of eHER-PPNs into cancer cells by receptor mediated endocytosis. Therefore, the eHER-PPNs fabricated through electrostatic interactions could more efficiently delivered Paclitaxel than was possible with cHER-PPNs that were fabricated by chemical conjugation.

3.7. Cellular uptake by flow cytometry

The internalization of the eHER-PPNs and cHER-PPNs into BT474 cells and MCF7 cells was also quantitatively evaluated by flow cytometry. The results of these experiments are shown in Fig. 10. After 3 h of incubation, the fluorescence intensity exhibited by BT474 cells that had been treated with cHER-PPNs and eHER-PPNs internalized into BT474 cells was 16.4, 36.1 for FITC and 18.7, 49.5 for rhodamine B, respectively. The internalized fluorescence intensity of FITC and rhodamine B showed positive correlation. As a comparison, MCF7 cells that had been treated with cHER-PPNs and eHER-PPNs showed the same level of fluorescence intensity, which indirectly illustrated that enhanced cellular uptake efficiency was due to targeted delivery. These results quantitatively confirmed the observation in confocal microscopy imaging.

4. Conclusion We designed, fabricated and evaluated a novel drug delivery system, denoted as eHER-PPNs, which incorporated Paclitaxel as a chemotherapy agent in the PLGA/PEI matrix, Herceptin as a targeting ligand on the surface of the nanoparticles by electrostatic interactions. It has shown that eHER-PPNs exhibited significantly higher degree of cytotoxicity in HER2-positive cancer cells than that observed in HER2-negative cancer cells. Furthermore, eHER-PPNs showed enhanced cytotoxicity compared with PPNs, free herceptin and pHER-PPNs, suggesting that Herceptin acted as a targeting ligand instead of simply cytotoxic superposition of Herceptin and PPNs. The eHER-PPNs also displayed higher cytotoxicity and cellular uptake efficiency in comparison with cHER-PPNs that were fabricated by chemical conjugation. Therefore, the use of electrostatic interactions provided a suitable method for the

preparation of protein-loaded nanoparticles owing to the mild reaction conditions involved. This work demonstrated that eHER-PPNs could potentially serve as efficient carriers of chemotherapeutic agents for the treatment of breast cancer.

Acknowledgements This work was supported by the Gentral Lab of General Biology, Jilin University, China.

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Figure Captions

Fig. 1. Schematic illustration of the eHER-PPNs.

Fig.2. Native-PAGE of eHER-PPNs. Lane 1: PPNs, Lane 2-7: PPNs-to-Herceptin weight ratios: 5: 1, 2.5: 1, 1: 1, 1: 2.5, 1: 5 and 1: 10, respectively, Lane 8: native Herceptin (10 µg).

Fig. 3. The TEM images of PPNs (A) and eHER-PPNs (B).

Fig. 4. Stability of eHER-PPNs in PBS (0.01 M, pH 7.4) with or without 10% FBS; particle size (A) and polydispersity (B). Binding efficiency of Herceptin in PBS (0.01 M, pH 7.4) (C). N=3.

Fig. 5. In vitro release profiles of Paclitaxel from PPNs, eHER-PPNs and cHER-PPNs in PBS (0.01 M, pH 7.4) containing 0.5% Tween-80 (v/v). N=3.

Fig. 6. In vitro cell viability of BT474 cells and MCF7 cells treated with bPPNs and eHER-bPPNs at nanoparticle concentrations of 50, 100 and 200 μg/mL, respectively. N=8.

Fig. 7. In vitro cell viability of BT474 cells (A) and MCF7 cells (B) treated with Paclitaxel, Herceptin, PPNs, eHER-PPNs and pHER-PPNs after incubation for 24 h, 48 h and 72 h, respectively. N=8.

Fig. 8. In vitro cell viability of BT474 cells (A) and MCF7 cells (B) treated with eHER-PPNs and cHER-PPNs after incubation for 24 h, 48 h and 72 h, respectively. N=8.

Fig. 9. The CLSM images showed the cellular uptake of double fluorescence labeled eHER-PPNs and cHER-PPNs in BT474 cells and MCF7 cells.

Fig.10. Quantitative study of cellular uptake efficiency of double fluorescence labeled eHER-PPNs and cHER-PPNs after 3 h incubation in BT474 cells and MCF7 cells: the fluorescence intensity of FITC (A) and the fluorescence intensity of rhodamine B (B). N=3.

Tables

Table 1 Optimization of PEI ratio in W phase on eHER-PPNs. Note that eHER-PPNs are fabricated at PPNs-to-Herceptin ratios of 1: 5 (Herceptin is excessive). Data represent mean ± SD, n = 3. PPNs

Ratio of PEI in W

eHER-PPNs

phase (%, w/v)

Zeta potential (mV)

Size (nm)

PDI

0

-0.44 ± 0.40

244.4 ± 3.4

0.191 ± 0.021

1.26 ± 0.38

245.8 ± 6.4

0.222 ± 0.044

0.2%

10.40 ± 1.28

245.6 ± 3.8

0.132 ± 0.039

-0.11 ± 0.49

256.5 ± 5.3

0.235 ± 0.051

0.4%

15.50 ± 1.45

245.1 ± 2.6

0.118 ± 0.011

0.01 ± 0.59

260.2 ± 5.1

0.177 ± 0.029

0.8%

26.90 ± 2.33

248.6 ± 1.8

0.149 ± 0.043

-0.53 ± 0.13

269.7 ± 4.5

0.231 ± 0.065

1.6%

32.40 ± 3.03

251.0 ± 2.2

0.247 ± 0.037

-1.01 ± 0.21

282.4 ± 7.6

0.283 ± 0.057

3.2%

40.80 ± 3.21

272.3 ± 6.4

0.160 ± 0.034

-0.84 ± 0.62

307.3 ± 8.5

0.218 ± 0.049

4.8%

44.60 ± 2.87

290.4 ± 7.6

0.146 ± 0.023

-0.72 ± 0.23

325.4± 10.2

0.195 ± 0.038

a

Zeta potential (mV) Size (nm)

Binding efficiency is defined as percentage of the amount of reacted herceptin on the amount

of herceptin used during the preparation process.

PDI

e

Table 2 Optimization of eHER-PPNs at various PPNs-to-Herceptin ratios for their particle size, PDI and binding efficiency. Note that 1.6% PEI ratio is used to fabricate the PPNs. Data represent mean ± SD, n = 3. PPNs-to-Herceptin

eHER-PPNs

Binding efficiency (%)

(w/w)

Size (nm)

5:1

263.2 ± 8.2

0.230 ± 0.032

91.3 ± 0.8

2.5:1

261.0 ± 8.6

0.179 ± 0.025

55.2 ± 1.2

1:1

282.8 ± 5.8

0.133 ± 0.047

92.7 ± 0.6

1:2.5

284.0 ± 4.1

0.188 ± 0.054

42.3 ± 0.5

1:5

287.4 ± 8.4

0.177 ± 0.028

18.6 ± 0.4

1:10

299.0 ± 7.7

0.181 ± 0.033

16.4 ± 1.3

PDI

Table 3 Characteristics of the optimized formulations: particle size, PDI, Zeta potential and EE. Data represent mean ± SD, n = 3. Formulation

Size (nm)

PDI

Zeta potential (mV)

EE (%)

PPNs

249.1 ± 2.2

0.152 ± 0.053

30.20 ± 3.30

66.8 ± 3.0

eHER-PPNs

280.7 ± 7.8

0.228 ± 0.112

1.00 ± 0.73

60.5 ± 1.9

cHER-PPNs

268.4 ± 6.6

0.271 ± 0.061

11.80 ± 0.75

63.8 ± 2.1