Preparation of PGA-PAE-micelles for Enhanced Anti-tumor Efficacy of

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Biological and Medical Applications of Materials and Interfaces

Preparation of PGA-PAE-micelles for Enhanced Anti-tumor Efficacy of Cisplatin Yazhou Chen, Li Zhang, Yingjie Liu, Shiming Tan, Ruidan Qu, Zirong Wu, Yue Zhou, and Jing Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04259 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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ACS Applied Materials & Interfaces

Preparation of PGA-PAE-micelles for Enhanced Anti-tumor Efficacy of Cisplatin Yazhou Chen a, Li Zhang a, Yingjie Liu a, Shiming Tan a, Ruidan Qu a Zirong Wu a, Yue Zhou b,*, Jing Huang a,*

a

School of Life Science, East China Normal University, Shanghai 200241, PR China.

b

Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, PR China.

* Corresponding authors: E-mail: [email protected] (Y. Zhou) E-mail: [email protected] (J. Huang)

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ABSTRACT: Poly-γ-L-glutamic acid (PGA) is an outstanding drug carrier candidate owning to its excellent biodegradability and biocompatibility. PGA carrier may shield toxic drugs from the body and enable the delivery of poorly soluble or unstable drugs thereby minimize the side effects and improve drug efficacy. However, the limitation of PGA as a drug carrier is low drug loading efficiency (DLE), which is usually below 30%. In this study, we reported a chemical modification method using L-phenylalanine ethyl ester (PAE). PGA-PAE construct was amphiphilic, which could form micelles in aqueous solution. CDDP, a commonly used chemotherapy drug whose side-effect is well-known, was used as a model molecule to test the drug-loading efficiency of PGA-PAE. In this paper, two sizes of CDDP-loaded PGA-PAE micelles (M(Pt)-1 and M(Pt)-2) were prepared, the average diameter of M(Pt)-1 was 106 ± 6 nm and M(Pt)-2 was 210 ± 9 nm. The DLE of M(Pt)-1 and M(Pt)-2 were 52.8 ± 2.2% and 55.8 ± 1.2%, respectively. Both exhibited excellent biocompatibility, stability and drug-retaining capability in physiological condition. The in vitro accumulative drug-releasing profile, IC50 for different tumor cell lines Hela, A549, and HCCC9810, and in vivo pharmacokinetics were similar between these two micelles, however, M(Pt)-1 showed higher tumor tissue retention and longer efficient cancer cell internalization time (up to 20 d). Our results suggested PGA-PAE micelle carriers reduced the toxicity of CDDP and its size at around 100 nm was the better for CDDP high-efficacy. KEYWORDS: poly-γ-L-glutamic acid (PGA); Cisplatin; micelles; drug-loading efficiency; EPR effect

INTRODUCTION Cancer is among the major diseases threatening people’s health worldwide1-2. At present, major clinical interventions are surgery, radiotherapy, biotherapy and chemotherapy, among which chemotherapy has been used as a systematic treatment3 apart from its well-known various side effects. Cisplatin (CDDP) is one of the most used chemotherapy drug for the treatment of many malignancies in breast, liver, lung, neck, ovary, testicle and bladder due to its broad spectrum of anti-tumor activity4-5. The molecular mechanism of CDDP treatment is the formation of inter- and intra-strand cross-linked DNA adducts that activate cell apoptosis6-7, thus is toxic to both cancer and normal cells. Therefore, the side effect of CDDP treatment is very severe, especially, its renal toxicity raises a major concern8-10. Recently, nanomedicine has emerged as promising therapeutics for treatment of this intractable problem for the unique properties of the nano-carriers including prolonged circulation in the bloodstream followed by systemic accumulation at the disease site, which is also called the enhanced permeability and retention effect (EPR effect)11-14. Micelles from copolymers are popular therapeutic delivering carriers due to their biodegradable property and ability to assemble and disassemble under certain conditions, these characters allowed loading and release of therapeutic molecules in specific environments15-18. In order to achieve better therapeutic results, the research on micelles have been from focus on developing new methods and applications, to focus on improving the properties of micelles, such as the uniformity, high drug loading efficiency, etc, to achieve better


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therapeutic results. A lot of biodegradable materials have been shown to be ideal nano-carriers, including chitosan (CS)19-20, polylactic acid (PLA)21-22, and poly-γ-L-glutamic acid (PGA)23, but not all of them can be easily manipulated to desirable molecular weight. PGA is a linear polymer which can be easily hydrolyzed by acid. By adjusting the hydrolysis time, the unified PGA molecular weight could be obtained24. However, the limitation of the PGA as nano drug carrier is the low drug loading efficiency (DLE), which is below 30 %25-27. Therefore, it is of great significance to improve this aspect. On the other hand, L-phenylalanine ethyl ester (PAE), a hydrophobic molecule, can be used to modify a lot of polymers such as alginate28, short peptide29, due to the ease of their synthetic modifications. Also, PAE can modify PGA by ligation to its –COOH side chain. Ideally, the amphiphilic PGA-PAE products are able to self-assemble into micelles with a homogeneous diameter in aqueous solution (Figure 1). The size of the nano-carriers is also a very important feature which can affect their EPR effect30-31. It has been reported that sodium chloride (NaCl) could affect the size of the micelles during the their formation32. Therefore, in the present study, we adjusted the concentration of NaCl to achieve precise control of the size of the PGA-PAE micelles. In addition, the physical and biological properties of the micelles were investigated and compared in this study, including drug loading efficiency, stability and size, in vitro cytotoxicity to different of cancer cells, in vivo biodistribution and antitumor activity.

MATERIALS AND METHODS Materials. Fluorescein isothiocyanate (FITC), L-phenylalanine ethylester (PAE) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Aladdin Reagent (Shanghai, China). Cisplatin (CDDP) and 4, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). For in vitro cell culture, human cervical carcinoma cell line (HeLa), human alveolar adenocarcinoma cell line (A549), and human gallbladder cancer cell line (HCCC 9810) were purchased from Cell Bank of Shanghai, Chinese Academy of Sciences (CAS, Shanghai, China). HeLa, A549, HCCC 981 cells were cultured at 37 ℃ under a humidified 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (Clark Bioscience, Houston USA), 100 units/mL penicillin, and 100 units/mL streptomycin. Preparation of size-regulated PGA-PAE micelles. PGA (poly-γ-L-glutamic acid) was produced and purified in our laboratory using a bacterial strain reported elsewhere33-35. In order to obtain low molecular weight PGA, 1 % PGA in aqueous solution was adjusted the pH to 3 using HCl (1 M) and incubated in a 98 ℃ water bath for 10 min, 20 min, 30 min, respectively. The reactions was halted by cooling down in ice water, and then adjusted the pH to neutral using NaOH (10 M). The solution was purified by dialysis (Biosharp, MW = 3500 Da) in distilled water for 36 h, the distilled water changed per 6 h. Hydrolysis 30 min resulted in PGA of molecular weight around 300 KD, which was used for PAE modification (Figure S1) in the presence of 50 mM EDC (Aladdin, Shanghai, China). The reaction was allowed to proceed for 12 h. After lyophilization, the PGA-PAE powder was stored in -20 ℃ for further experiments. PGA-PAE micelles of different sizes were prepared according to a previous report32.



Briefly, 10 mg/mL PGA-PAE (DMSO solution) were added into the same volume of aqueous solution dropwise under stirring. The size of the micelles was regulated by adding the 0.1 M NaCl or distilled water during the formation of the micelles. The resulting solutions were dialyzed against distilled water for 3 days at room temperature using a dialysis bag (MW = 3500 Da) and intensively dialyzed against. Fluorescent labeling of the PGA-PAE copolymers. The FITC labeled PGA-PAE copolymers were prepared by stirring the PGA-PAE (10 µM) and FITC (20 µM) in DMSO solution for 48 h. After being dialyzed against distilled water for 3 days in dark at room temperature using a dialysis bag (MW = 3500 Da) to remove salts and excess. After that, the resulting solutions were lyophilized to obtain FITC-PGA-PAE powders and kept in dark until in vitro experiment. Characterization. The synthesis of PGA-PAE was confirmed by 1H NMR analysis (Performing on a Varian 699.804 MHz NMR spectrometer, Agilent Technologies, USA) at 298.2 ± 0.1 K. Gel permeation chromatography (GPC) measurements were performed on a DMSO GPC system (Agilent PLgel 5um MIXED-C column, 1525 HPLC pump with 2414 Refractive Index detector) using DMSO solution as eluent (flow rate: 1 ml/min, 35 °C). The transmission electron microscopy (TEM) images were taken to analyze the micelles size and distribution (using a Hitachi microscope at an acceleration voltage of 100 kV, HT7700, Hitachi, Japan). The samples were dispersed onto the ultrathin carbon-coated copper TEM grids using micropipettes and dried under ambient condition. The hydrodynamic diameters, particle dispersion index (PDI) and zeta potentials of the PGA-PAE micelles were measured by Dynamic Light Scattering (DLS, Zeta sizer Nano ZS90, Malvern Instruments, UK). DLS was performed on a DynaPro NanoStar from Wyatt Technology using Dynamics software version 7.1.7. The date acquisition parameters included water solvent, spherical radius of gyration (Rg) model, temperature of 20.0 ℃, and 30 acquisitions. The UV-Vis spectra were recorded to confirm the FITC was successfully labeled on PGA-PAE micelles by using a UV-Vis spectrometer (Cary60, Agilent Technologies, USA). Drug loading efficiency (DLE), drug loading content (DLC) and the stability of M(Pt)s. To measure the DLE of the micelles, 10.0 mg CDDP and 100 mg PGA-PAE were fully dissolved in 10 mL DMSO solution. Thereafter, the mixtures were added to the same volume of aqueous solution under stirring. Another 10.0 mg CDDP was added into the mixture and incubated at room temperature for 3 days. After dialyzing in distilled water for 12 h to remove excess free CDDP, M(Pt)s loaded with CDDP were obtained and stored at 4 ℃. Concentrations of Pt was determined by ICP-MS. DLE % and DLC % were calculated from the formula below: DLE % = loading amount of drug in micelles/initial feeding amount of drug for loading × 100% DLC % = loading amount of drug in micelles/total weight of M(Pt)s × 100% The stability of M(Pt)s were assessed via incubating them with PBS for up to 7 days. For every 24 h, the solutions were observed for any agglomeration or sediment.


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Releasing profiles of CDDP from M(Pt)s. The releasing of CDDP from M(Pt)s in PBS containing 10% fresh sera form BABL/c mice at 37 ℃ was evaluated by dialysis method (Biosharp, MW = 3500 Da)27. The released amount of CDDP outside the dialysis bag was sampled and measured at specific time points. In vitro toxicity of M(Pt)s. In vitro toxicity of M(Pt)s were tested against three different tumor cell lines (HeLa, A549, and HCCC9810). 100 µL cells suspensions were seeded in 96-well culture plates (104 cells per well) and cultured for 12 h at 37 ℃ and 5% CO2, the culture supernatant was replaced with 100 µL fresh DMEM containing either M(Pt)s or free CDDP in a CDDP equivalent concentration ranging from 0 to 40 µg/mL. Cell proliferation for 24 h, 48 h and 72 h were evaluated by MTT assay. In brief, 20 µL MTT at the concentration of 5 mg/mL was added into each well, after additional 4 h incubation, the medium in each well was replaced by 200 µL DMSO. The absorbance of each well at 570 nm was measured by a Powerwave microplate reader (XS, Bio-Tek). The IC50 values were calculated using Graphpad prism 5.0. Cellular uptake assay. HeLa cells (2.5 × 104 cells per well) were seeded in 6-well culture plates and cultured in 250 µL cell medium overnight. After cell attachment, FITC-labeled M(Pt)-1 and M(Pt)-2 were added and incubated for additional 1 h at 37 ℃. The cultural medium was removed and the cells were carefully washed with PBS. Then, cells were immersed into 4% (w/v) paraformaldehyde for 30 min at 4 ℃. DAPI staining were performed to visualize cell nuclei. Images were taken by the confocal microscope (CLSM, TCS SP5, Leica, Germany). To qualified cellular uptake, cells were detached by trypsin. After centrifugation for 5 min at 1500 r/min, the cells were re-suspended in PBS. Data for 1 × 104 gated events were collected, and analyzed by flow cytometer (FACS, Beckman, California, USA). Pharmacokinetics. The in vivo experiments were carried out according to the guidelines for care and use of laboratory animals of National Institutes of Health. The experiment protocols were approved by the ethics committee of East China Normal University. BABL/c mice of average weight 20 g were randomly divided into 3 groups (CDDP, M(Pt)-1 and M(Pt)-2). The tail vein were carried out at dosage of 4 mg/kg body weight (on CDDP basis). The blood samples were collected from the orbital cavity at the time points of 5 min, 0.5 h, 1 h, 4 h, 10 h and 24 h. The samples were decomposed by heating in chloroazotic acid and the Pt contents in each samples were measured by ICP-MS (7500A, Thermo, USA). Tissue distribution. The biodistribution of M(Pt)s was investigated in BABL/c nude mice (average weight 20g) bearing transplant tumors. To establish the xenografted tumor models, HeLa cells suspension at a number of 1 × 106 were subcutaneously injected into the right back of mice. The tumor volumes were calculated according to the formula: tumor volume = width2 × length × π/6. When the average volume of the tumors researched 150 mm3, the tumor-bearing mice were randomly divided into 3 groups and intravenously injected with CDDP, M(Pt)-1 and M(Pt)-2 (4 mg/kg, on CDDP basis), respectively. At specific time points after administration, 3 mice were sacrificed and autopsied. Major organs (heart, liver, spleen,



lung and kidney) and tumors were removed and milled. Then, the contents of Pt in these tissues were measured by ICP-MS. In vivo antitumor efficacy. To establish the HeLa tumor xenograft models in BAL/c nude mice, 106 HeLa cells were subcutaneously injected into right back of the mice. When the tumor volume reached 70 - 100 mm3, mice were divided into five groups (n = 5). Each group was intravenously injected with Saline, PGA-PAE, M(Pt)-1, M(Pt)-2, and CDDP, respectively. The injection dosage was 4 mg/kg on CDDP basis. The injection volume was 150 µL. The mice were injected via tail vein at a specific time points (1 d, 3 d, 5 d and 7 d). The tumor volume and body weight of each mouse were recorded every two days. All mice were sacrificed at the end of the experiment period (20 d) and all the tumors from different groups were collected for analysis. In addition, major organs including heart, lung, liver, spleen and kidney were harvested, following with 2 days fixing in 4% formalin solution (w/v) at 25 ℃. The major organs were embedded in paraffin, and sectioned into cross-section slices of a thickness of 4 µm. The cross-sections were stained with hematoxylin and eosin (H & E). Then, the stained cross-sections were observed under an optical microscope (Leica, Wetzlar, Germany).

RESULTS Synthesis and characteristics of M(Pt)s. PAE was conjugated to PGA according to a well-developed precipitation and dialysis method23, 36. The 1H NMR spectrum confirmed that PAE was successfully conjugated to PGA (Figure 2A). The two types of the micelles was formed either to disperse PGA-PAE DMSO solution in distilled water only or 0.1 M NaCl solution. The CDDP (cisplatin) loaded micelles with different size were prepared (M(Pt)-1 and M(Pt)-2). The drug loading efficiency (DLE %) of M(Pt)-1 and M(Pt)-2 were 52.8 ± 2.2% and 55.8 ± 1.2%, respectively (Table 1.). The drug loading content (DLC %) for M(Pt)-1 was 10.5 ± 0.5, and the DLC % for M(Pt)-2 was 11.1 ± 0.3. The TEM images showed that M(Pt)-1 and M(Pt)-2 had a spherical shape and uniform size (Figure 2B and C). The dynamic light scattering (DLS) analysis demonstrated that M(Pt)-1 had a hydrodynamic diameter of 107 ± 6 nm and M(Pt)-2 had a hydrodynamic diameter of 221 ± 9 nm (Figure 2D and E), respectively. The PDI (Polymer dispersity index) (both 0.09) and zeta potential of two sizes of micelles suggested the two sizes of micelles had a good monodispersity in solution (Figure S4). Furthermore, the time-elapsed size and zeta potential evolution of the two sizes of micelles in PBS solution were recorded, respectively. The result showed that there were no significant size changes for both sizes of micelles (Figure S5B). Zeta potential showed no significant difference between them (Figure S5C), implying that the two types of micelles were stable in PBS conditions. The accumulative drug release from M(Pt)-1 and M(Pt)-2 were tested. Two different conditions including PBS (pH = 7.4) and PBS + 10% serum of BABL/c mice (pH = 7.4) were performed (Figure 3). M(Pt)-1 and M(Pt)-2 in PBS were served as a control, the experimental group were in PBS + 10% serum (pH = 7.4). After 7 days, in the PBS + 10% serum group, an accumulative release for M(Pt)-1 was 52.9 ± 2.3% and M(Pt)-2 was 50.7 ± 2.2%. In contrast, in the PBS control group, the accumulative release were only 25.2 ± 2.4% and 21.8 ± 1.8% for M(Pt)-1 and M(Pt)-2, respectively.


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In vitro toxicity of M(Pt)s and Cellular uptake. For potential drug carriers, good biocompatibility is a prerequisite for its clinical application. The toxicity of PGA-PAE micelles to HeLa cells was evaluated by standard MTT assay. Cells were incubated with the micelles for 72 h. Figure S6 showed that the cell viability was over 90% at a PGA-PAE micelle concentration even up to 250 µg/mL. Therefore, PGA-PAE micelles showed good biocompatibility. This observation is consistent with the previously reports37-38. In order to test their in vitro cytotoxicity, M(Pt)-1 and M(Pt)-2 were incubated with human cancer cells HeLa, A549 and HCCC9810 at different concentrations and times. The concentrations are ranged from 1.25 µg/mL to 40 µg/mL. Three time points were selected, 24 h, 48 h, and 72 h, and MTT assay was used to evaluate the amount of the cells proliferated (Figure 4, Figure S7 and Table S1.). As the results presented, time and concentration dependent cancer cells inhibition effect were observed. The representative results of 72 h incubation with HeLa cells were shown in Figure 4C and Table S1. The IC50 of M(Pt)-1 was 4.9 ± 0.8, M(Pt)-2 was 7.9 ± 0.9 and free CDDP was 3.5 ± 0.3. Apparently, the IC50 of both M(Pt)s were much higher than free CDDP. This might be ascribed to the delayed drug release behavior of M(Pt)-1 and M(Pt)-2, resulting in the significantly decreased toxicity of the CDDP loaded micelles. Moreover, the M(Pt)-1 of size 100 nm showed significantly better cell inhibition effect than M(Pt)-2, which indicated that 100 nm M(Pt)-1 had higher antitumor ability. To clarify the enhanced tumor cell inhibition effect of M(Pt)-1, we compared the cellular uptake behavior of M(Pt)-1 and M(Pt)-2. FITC was used to label the CDDP loaded micelles (as shown in Figure S8). CLSM and FACS were used to measure the endocytosis rate. As shown in Figure 5, M(Pt)-1 and M(Pt)-2 could both be effectively uptaken by HeLa cells as short as 1-hour incubation. The M(Pt)-1 group showed more tumor cell uptaking rate in the form of much stronger fluorescent signal seen in the confocal images (Figure 5A). The enhanced uptake behavior may be caused by two reasons. First, the amount of the M(Pt) uptaken by each cell was more. Second, the amount of the cells which uptake M(Pt) was more. The second reason was confirmed by FACS results (Figure 5 B-E). The above results indicated that endocytosis of M(Pt)-1 was easier to HeLa cells than M(Pt)-2, resulting in an enhanced HeLa cells inhibited behavior. Pharmacokinetics and biodistribution. The metabolic behavior of M(Pt)-1 and M(Pt)-2 in blood were tested in BABL/c mice. The concentration of chelated platinum (Pt) was used as the evaluation criterion and free CDDP as control. CDDP, M(Pt)-1 or M(Pt)-2 was intravenously injected into the mice (4 mg/kg, on CDDP basis). The blood samples were collected and measured at defined time points (5 min, 0.5 h, 1 h, 4 h, 10 h and 24 h). CDDP was quickly cleared out from blood after tail vein injection, dropping to as low as 500 ng pt/mL blood within 10 h. Both M(Pt)-1 and M(Pt)-2 showed significantly prolonged blood circulation time compared to free CDDP (P < 0.01), with concentration remained over 1500 ng pt/mL blood even after 24 h (Figure 6). The positive charge of outer shell of the micelles could protect themselves from the renal and liver clearance. This also in favor of the passive accumulation of drug loaded micelles in tumor and enhance their EPR effect (Figure 7A). To visualize the biodistribution of the two kinds of micelles with different sizes in BABL/c nude mice bearing xenograft HeLa tumors, Indocyanine green (ICG) was used as a replacement of CDDP. ICG is the ideal NIR fluorescent dye approved by the Food and Drug



Administration (FDA) for clinical use39-40. Here, ICG loaded micelles were designated as M(ICG)-1 and M(ICG)-2 in contrast to M(Pt)-1 and M(Pt)-2. Both were prepared according to the previous reports41-42. Briefly, ICG (2 mg/mL in DMSO) and PGA-PAE (10 mg/mL in DMSO) were mixed and then were added into the same volume of distilled water or 0.1 M NaCl solution. The results from TEM and DLS showed that their sizes were remained the same as M(Pt)-1 and M(Pt)-2 (Figure S11). When tumor volume grown to 150 mm3，free ICG, M(ICG)-1 or M(ICG)-2 was intravenously injected into the mice. Imagines were taken by an in vivo imaging system (IVIS) after 10 h. The tumors and main organs including heart, liver and kidney were removed and imaged by IVIS (Figure 7B). In M(ICG)-1 group, the tumors demonstrated a much higher fluorescence intensity than those in the ICG and M(ICG)-2 group (Figure 7C). The fluorescence intensity in liver were lower in the M(ICG)-1 group compared with that in M(ICG)-2 group , although both were higher compared to CDDP group. In kidney，CDDP group showed a much higher accumulation compared to both M(ICG) groups. No detectable fluorescent signal was found in hearts. To quantitatively evaluate the biodistribution of the M(Pt)s micelles, CDDP, M(Pt)-1 or M(Pt)-2 was intravenously injected into mice (4 mg/kg, on CDDP basis). After 10 h, Pt concentrations in tumors, livers and kidneys were determined by ICP-MS. As shown in (Figure 7D), in tumors, M(Pt)-1 demonstrated a much higher distribution than M(Pt)-2 and free CDDP groups (P

Preparation of PGA-PAE-micelles for Enhanced Anti-tumor Efficacy of

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