Star-Shaped Amphiphilic Hyperbranched Polyglycerol Conjugated

May 6, 2016 - Academy of Military Medical Sciences, Beijing 100850, China. §. The School of Pathology and Laboratory Medicine, University of Western ...
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Star-Shaped Amphiphilic Hyperbranched Polyglycerol Conjugated with Dendritic Poly(L‑lysine) for the Codelivery of Docetaxel and MMP‑9 siRNA in Cancer Therapy Xiaoyan Zhou,† Qianqian Zheng,† Changyong Wang,‡ Jiake Xu,§ Jian-Ping Wu,∥ Thomas Brett Kirk,∥ Dong Ma,*,† and Wei Xue*,† †

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China ‡ Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences, Beijing 100850, China § The School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia ∥ 3D Imaging and Bioengineering Laboratory, Department of Mechanical Engineering, Curtin University, Perth, Australia

ABSTRACT: The drug/gene codelivery is a promising strategy for cancer treatment. Herein, to realize the codelivery of docetaxel and MMP-9 siRNA plasmid efficiently into tumor cells, a star-shaped amphiphilic copolymer consisting of hyperbranched polyglycerol derivative (HPG-C18) and dendritic poly(L-lysine) (PLLD) was synthesized by the click reaction between azido-modified HPG-C18 and propargyl focal point PLLD. The obtained HPG-C18-PLLD could form the nanocomplexes with docetaxel and MMP-9, and the complexes showed good gene delivery ability in vitro by inducing an obvious decrease in MMP-9 protein expression in MCF-7 cells. The apoptosis assay showed that the complex could induce a more significant apoptosis to breast cancer cells than that of docetaxel or MMP-9 used alone. In vivo assay indicated that the codelivery strategy displayed a better effect on tumor inhibition. Moreover, HPG-C18-PLLD displayed lower toxicity as well as better blood compatibility compared to polyethylenimine PEI-25k, which may be the result of that HPG-C18-PLLD showed the comparative MMP-9 delivery ability in vivo compared with PEI-25k even if it showed the slight lower transfection efficiency in vitro. Therefore, HPG-C18-PLLD is a safe and effective carrier for the codelivery of drug/gene, which should be encouraged in tumor therapy. KEYWORDS: tumor therapy, co-delivery, star-shaped copolymer, hyperbranched polyglycerol, poly(L-lysine) dendrimer

1. INTRODUCTION Nowadays, cancer has become the leading reason to death in the world, and millions of people are diagnosed with cancer every year.1−3 At present, chemotherapy, radiation treatment, and surgical treatment are commonly used for cancer therapy in clinic. But surgical therapy is helpless in the face of the transferring tumor, and the chemotherapy and radiation treatment usually induce serious adverse effects and drug resistance.4−6 In recent years, the drug/gene codelivery method has emerged to yield promising results in cancer therapy, because the strategy could enhance synergistic effects, decrease the © XXXX American Chemical Society

occurrence and impact of drug resistance, and also reduce the adverse side effects.7 Yin et al. reported a redox-sensitive micelle based on hyaluronic acid derivative to codeliver paclitaxel and siRNA, which showed a good combined therapy effect.8 Gaspar et al. synthesized the triblock copolymer consisting of poly(2-ethyl-2-oxazoline)-poly(L-lactide) and polyethylenimine (PEI) to codeliver mcDNA and doxorubicin (DOX).9 Xu et al. developed the PEI/cis-aconitic−anhydride Received: February 6, 2016 Accepted: May 6, 2016

A

DOI: 10.1021/acsami.6b01611 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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2.2. Synthesis of Azido-Modified Amphiphilic HPGC18 (HPG−C18-N3). According to our previous work,24 the amphiphilic HPG-C18 was first synthesized. Then, 1.0 g of HPG-C18 was dissolved in 50 mL of mixed tetrahydrofuran/ dimethylformamide (v/v = 1:1) solvent, then 8 g of 4-toluene sulfonyl chloride and 30 mL of pyridine were added in ice bath and stirred. After 2 h, the solution was stirred at 25 °C for another 24 h. Then, by removing the solvent under reduced pressure, the remainder was dropped into excess diethyl ether. The precipitate was dried in vacuum drying oven for 24 h to obtain the HPG-C18-sulfanilic acid ester. Then, 1.0 g of HPGC18-sulfanilic acid ester and 0.2 g of NaN3 were dissolved in 10 mL of distilled water. The mixture was stirred under the N2 atmosphere at 60 °C. After 24 h, the mixture was dialyzed for 3 d (molecular weight cutoff (MWCO) = 1000, USA) and then frozen to dry to obtain HPG−C18-N3 (yield = 45%). The content of azido group in HPG−C18-N3 was determined to be 8.3 mmol/g by elemental analysis. 2.3. Synthesis of the Star-Shaped Copolymer (HPGC18-PLLD). The propargyl focal point PLLD (generation = 4) was synthesized according to our previous report.25 For HPG-C18-PLLD synthesis, 0.1 g of HPG−C18-N3, 1.5 g of PLLD, and 0.25 g of CuSO4·5H2O were dissolved in 20 mL of distilled water. Subsequently, 0.99 g of sodium ascorbate was added under the protection of N2. The solution was stirred at 40 °C for 3 d. Then, the product was dialyzed for 3 d (MWCO = 3000, USA) and then lyophilized to obtain HPG-C18-PLLD (yield = 30%). To confirm the chemical structure of HPG-C18-PLLD, 1H NMR and Fourier transform infrared (FT-IR) analyses were performed. The 1H NMR spectrum was recorded in D2O through an NMR spectrometer (Bruker DPX-300, Germany) at 25 °C. The FT-IR spectra were measured on an FT-IR spectrometer (Bruker VERTEX 70, Germany) with the wavenumber ranging from 4000 to 500 cm−1. The molecular weight and its distribution of HPG-C18-PLLD were analyzed using a gel permeation chromatography (GPC, Malvern, U.K). HPG-C18-PLLD was dissolved in 0.8 mol/L aqueous NaNO3 solution at 35 °C and then performed by GPC (poly(ethylene oxide) as the standard). 2.4. Docetaxel Loading. To load the hydrophobic DOC into complex, 20 mg of DOC was dissolved in 2 mL of acetone, added dropwise into 10 mL of distilled water containing 100 mg of HPG-C18-PLLD at 4 °C. This solution was stirred for 24 h in the dark at 25 °C and was then dialyzed for 24 h (MWCO = 500, USA). The DOC-loaded HPG-C18-PLLD complex (HPG-C18-PLLD/DOC) was obtained after filtration and lyophilization. The content of DOC in HPG-C18-PLLD/DOC complex was confirmed by high-performance liquid chromatography (HPLC, Agilent 1200, USA). The HPLC was equipped with a Zorbax Eclipse XDB-C18 column (5 μm, 4.6 × 150 mm). The mixed solution consisting of purity water, methanol, and acetonitrile (25/35/40, v/v/v) was used as mobile phase, and the flow rate was 1.0 mL/min. The effluents were recorded at 230 nm, and the content of DOC was calculated by comparing with the standard curve.26 All samples were filtered (0.45 μm) before the experiment. For DOC release experiment, 1 mL of aqueous HPG-C18PLLD/DOC was enclosed in the dialysis bag (MWCO = 2000) and then immersed in 10 mL of media (phosphate-buffered saline (PBS) with 10% Tween 80) at 37 °C. At predetermined intervals, half of media was taken out, and then an equal volume

nanoparticles and used them for DOX and Bcl-2 siRNA codelivery and displayed the promising therapy to metastatic lung tumor with low side effects.10 Among these, the biggest challenge in tumor combined therapy at present is to synthesize a carrier with safety and high efficiency. Dendritic poly(L-lysine) (PLLD) and its derivatives have attracted more and more attention in drug and gene delivery recently due to their well-defined architecture, biodegradation, and low cytotoxicity.11−13 However, compared with other widely used cationic carriers, such as PEI and PAMAM, PLLD shows a lower gene transfection efficiency, which limits its future applications in clinic.14,15 Therefore, PLLD with higher generations must be synthesized.16,17 In fact, it is difficult to obtain the higher generations PLLDs with accurate chemical structures due to the steric hindrance of the dendrimer and the difficult purification accompanied by the increasing generations. Our previous works have proved that it is a promising strategy to achieve improved gene transfection efficiency of PLLD by conjugating low-generation PLLDs to a molecule with multifunctional groups to form a star-shaped copolymer,18,19 which may be resulted from the improvement of the local cationic charge density and the segment flexibility for binding gene. Moreover, in the consideration of the intravenous drug and gene codelivery, a blood-compatible carrier must be designed. Hyperbranched polyglycerol (HPG) is a widely used polymer with a globular polymeric structure that comprises a polyether backbone with massive hydroxyl groups.20,21 The polyether backbone, which is similar to poly(ethylene glycol) (PEG), gives it good blood compatibility and an attractive application in biomedicine.22 Its abundant hydroxyl groups on spherical surface allow the conjugation as other hyperbranched polymers.23 We have synthesized an amphiphilic HPG derivative that showed good blood compatibility and highefficiency drug delivery into tumor cells.24 In this work, an amphiphilic octadecane (C18)-modified HPG (HPG-C18) was synthesized through the one-pot reaction and then conjugated with poly(L-lysine) dendrons through a click reaction to form the star-shaped copolymer. The hydrophobic C18 segments could encapsulate the hydrophobic docetaxel, and poly(L-lysine) dendrons interact with MMP-9 siRNA plasmid. The obtained coloaded complex was explored for MCF-7 tumor therapy. Moreover, its biocompatibility including toxicity and blood compatibility was also studied.

2. MATERIALS AND METHODS 2.1. Materials. Glycidol (96%) and 1,1,1-tris(hydroxymethyl) propane (TMP, 99%) were obtained from Aladdin Industrial Corporation (Shanghai, China). Potassium methoxide solution (25% in methanol) was obtained from Sigma-Aldrich (USA). 1,2-Epoxyoctadecane was purchased from Tokyo Chemical Industry Corporation (Tokyo, Japan). Docetaxel (DOC, 99%) and PEI (25 kDa, PEI-25k) were obtained from Aladdin (Shanghai, China). A pcDNA3 plasmid was used to construct the vector that expresses small interference RNA (siRNA) for MMP-9 and enhanced green fluorescent protein by Invitrogen Corp (Shanghai). Annexin VPE Apoptosis Detection Kit I was obtained from Becton, Dickinson, and Company (USA). All other chemicals were purchased as AR and used directly. B

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2.8. Apoptosis Assay. For apoptosis assay, 5 × 104 cells were routinely maintained in plates. After 24 h, the media were substituted by 500 μL of DMEM that contained the samples, as follows: blank HPG-C18-PLLD, HPG-C18-PLLD/DOC (DOC: 0.08 μg/well), HPG-C18-PLLD/MMP-9 (w/w = 80, MMP-9:2 μg/well), and HPG-C18-PLLD/DOC/MMP-9 (w/ w = 80, DOC: 0.08 μg/well, MMP-9:2 μg/well). Every sample was set three parallel holes. PBS was used as negative control. The formulations were substituted by fresh DMEM after 6 h. The cells were incubated for another 42 h. Subsequently, all of the trypsinized and washed cells were analyzed for death or apoptosis by staining with 5 μL of Annexin V-PE and 5 μL of 7AAD for 15 min, followed by flow cytometry (BD caliber, U.S.A). Data were analyzed by FlowJo analysis software. 2.9. In Vivo Assay. The nude mice bearing MCF-7 tumors (4−5 weeks old, 18−20 g) were obtained from the Jinan University Center for Animal Experiment. This experimental design has been approved by the Institutional Administration Panel for Laboratory Animal Care. The mice were set randomly as six groups with five mice per group. Subsequently, various samples including blank HPG-C18-PLLD (400 mg/kg HPGC18-PLLD, dissolved in 0.2 mL of PBS), HPG-C18-PLLD/ DOC (0.2 mg/kg DOC, dissolved in 0.2 mL of PBS), HPGC18-PLLD/MMP-9 (5 mg/kg MMP-9, dissolved in 0.2 mL of PBS) and HPG-C18-PLLD/DOC/MMP-9 (0.2 mg/kg DOC and 5 mg/kg MMP-9, dissolved in 0.2 mL of PBS) were injected into the xenografted MCF-7 tumor mice. All groups were injected every day. PBS and PEI-25k/MMP-9 (5 mg/kg MMP-9) were set as the control groups in this study. All animals were sacrificed 21 d later. According to the formula W = L2/2, the tumor volume was calculated. (W and L represent the widest diameter and the longest diameter, respectively) 2.10. Biocompatibility. 2.10.1. Toxicity. Cytotoxicity. The CCK-8 assay was used to assess the cytotoxicity of blank HPGC18-PLLD. Briefly, MCF-7 cells were cultured into a 96-well plate. After 24 h of incubation, blank HPG-C18-PLLD at various concentrations were added and then incubated for another 48 h. PBS and PEI-25k were used as the control groups. Subsequently, the media were removed, and then 100 μL of DMEM (containing 10 μL of CCK-8) was added. The absorbance of supernatant was tested at 450 nm using a microplate reader. In Vivo Toxicity. The blank HPG-C18-PLLD was injected into mice through the tail vein injection (500 mg/kg mouse), and PBS was set as control group. All of the mice were kept in standard housing conditions. After 7 d of feeding, all mice were sacrificed, and their major organs were separated and fixed in 4% (w/v) paraformaldehyde for histological observation. 2.10.2. Blood Compatibility. Hemolysis Assay. The hemolysis assay was measured according to the method reported by O’Leary and Guess.27 Red blood cell (RBC) suspension (0.1 mL, 16%) was added into 5 mL of PBS that contained HPG-C18-PLLD with different concentrations. PBS and distilled water were, respectively, used as the negative and positive controls. All samples were incubated for 4 h. Then, the suspensions were centrifuged at 1000 rpm for 5 min, and the absorbance values of the released hemoglobin were tested at 540 nm with a microplate reader. The experiment was performed three times. The hemolysis was calculated as the formula:28

of fresh media was added back. The results of release experiment were analyzed by HPLC. All of the results were studied in triplicate. 2.5. MMP-9 siRNA Binding. For the preparation of HPGC18-PLLD/MMP-9 complexes, HPG-C18-PLLD was first dissolved with required concentrations, and then MMP-9 was added. The mixture was centrifuged for 5 min and stirred for a further 20 min to allow the formation of HPG-C18-PLLD/ MMP-9 complexes. The gel electrophoresis assay was used to evaluate the binding ability of HPG-C18-PLLD to MMP-9. Briefly, HPGC18-PLLD/MMP-9 and free MMP-9 were separated by 1.5% agarose gel electrophoresis containing GoldView II (Sigma) at 120 V for 25 min. After that, a gel imaging analysis system (BioRad, BioDoc-ItTM, USA) was used to capture image. Before experiment, the HPG-C18-PLLD/MMP-9 complexes were mixed at 37 °C for 20 min and then diluted by ultrapure water. A nanoparticle analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., U.K.) was used to measure the particle size and zeta potential. The morphology of the complex was observed by a transmission electron microscope (TEM, JEM2010HR, Japan) after treatment with phosphotungstic acid. 2.6. In Vitro Transfection. Transfection efficiency of combination formulations were determined at different weight ratios in MCF-7 cells using PEI-25k/MMP-9 (w/w = 1.3) as the control. In brief, before transfection, MCF-7 cells were cultured into a 24-well plate in a 5% CO2 atmosphere at 37 °C. Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS) supplemented was used as the medium. After 12 h of incubation, DMEM media was substituted by fresh Opti-MEM containing various HPG-C18-PLLD/MMP-9 complexes (w/w = 40, 60, and 80, respectively, MMP-9:2.0 μg/ well). After another 6 h of incubation, all media were removed and replaced with fresh 10% serum-supplemented DMEM. The experiments were then continued for another 24 h and the then analyzed for green fluorescent protein expression by fluorescence microscope observation (Nikon-2000U, Japan). For transfection efficiency analysis, the media were replaced, and cells were trypsinized and measured by flow cytometry (Beckman Gallios, U.S.A). 2.7. MMP-9 Protein Expression. Briefly, when MCF-7 cells reached 70% confluence in culture plate, various samples including blank HPG-C18-PLLD, PEI-25k/MMP-9 (w/w = 1.3), and HPG-C18-PLLD/MMP-9 (w/w = 80) were added, and the cells were cultured for another 48 h. After that, the lysis buffer with Roche’s protease inhibitor and phenylmethanesulfonyl fluoride was added into the cells, and the collected lysis buffer was put in vortex instrument for 30 s of shaking every 5 min. After centrifugation at 4 °C and 13 000 rpm/min, the supernatant was sucked carefully to obtain the protein. The concentration of protein was tested by BCA protein assay kit (Thermo scientific, U.S.A). Total protein was boiled when loading buffer was added, and it was treated on 10% PAGESDS gels electrophoresis and subsequently transferred with the wet method to polyvinylidene fluoride membranes (Bio-Rad). The membrane that was sealed by 5% skim milk was immersed into the Tris-buffered saline with Tween-20 (TBST) with MMP-9 antibody (1:1000) solution for the incubation at 4 °C overnight. It was then immersed into the TBST with goat antirabbit IgG-HRP antibody (1:2000) at 25 °C for another 1 h. Afterward, the sample was treated with ECL reagent (Bio-Rad) and visualized by the gel imaging system.

hemolysis(%) = (A − C)/(B − C) × 100 C

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ACS Applied Materials & Interfaces Scheme 1. Synthesis Routes to HPG-C18-PLLD

Figure 1. (a) 1H NMR spectrum of HPG-C18-PLLD (D2O, 25 °C). (b) Structure formula of HPG-C18-PLLD. (c) FT-IR spectra of PLLD, HPG− C18-N3, and HPG-C18-PLLD. (d) The GPC chromatogram of HPG-C18-PLLD (using PEG as the standard sample; 0.8 mol/L NaNO3; 35 °C).

Red Blood Cells Aggregation. Morphology of RBCs was studied by incubating 10 μL of different concentrations of

A, B, and C, respectively, represent the absorbance of HPGC18PLLD, the positive control, and the negative control. D

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ACS Applied Materials & Interfaces aqueous HPG-C18-PLLD with 90 μL of RBCs for 10 h at 37 °C. The same volume of RBCs incubated with PBS or 0.01 mg/ mL PEI-25k was used as the control. After that, the RBCs were obtained by centrifuge and fixed by 4% paraformaldehyde overnight at 25 °C and then dehydrated with ethanol. Then the RBCs were observed with the scanning electron microscope (SEM, Philips XL-30). Thromboelastography. Briefly, 360 μL of fresh anticoagulant blood was incubated with 40 μL of aqueous HPG-C18PLLD in a kaolin-containing tube. Then the samples were transferred into the thromboelastography (TEG) cup, and 20 μL of aqueous CaCl2 (0.2 mol/L) was added. PBS was used as the control. The coagulation process was studied at 37 °C using a Thromboelastograph Hemostasis System 5000 (Hemoscope Corporation, U.S.A). 2.11. Statistical Analysis. The data were presented as means ± standard deviation and analyzed by one-way ANOVA with SPSS 16.0. Differences were considered to be significant when P < 0.05.

The molecular weight and its distribution of the HPG-C18PLLD were characterized by GPC analysis. As the Figure 1d shows, the GPC curve showed the Mw of HPG-C18-PLLD was 21194 and the Mn was 15699, then its Mw/Mn was calculated to be 1.35, suggesting a narrow molecular weight distribution of HPG-C18-PLLD. Moreover, combining the result of GPC and elemental analysis, it was calculated that each star-shaped HPGC18-PLLD molecule contained five PLLD arms. 3.2. Docetaxel Loading and MMP-9 Binding. As the drug and gene codelivery carrier, HPG-C18-PLLD should first have the ability to encapsulate drug and gene. Because of its hydrophobic C18 segments and hydrophilic HPG/PLLD segments, the amphiphilic HPG-C18-PLLD could form the micelle in aqueous solution and load the hydrophobic drugs in its hydrophobic core. In this work, DOC was encapsulated in HPG-C18-PLLD micelle to form the HPG-C18-PLLD/DOC complexes using a dialysis method. The loading amount of DOC in the complexes was calculated to be 0.5 mg/g through HPLC analysis. The DOC release profile from the HPG-C18PLLD/DOC complexes was investigated through immersing HPG-C18-PLLD/DOC complexes into PBS, and the result was shown in Figure 2. The release of DOC from complexes was

3. RESULTS AND DISCUSSION 3.1. Synthesis of HPG-C18-PLLD. For the codelivery of DOC and MMP-9 efficiently in MCF-7 tumor treatment, an amphiphilic star-shaped copolymer with high gene transfection efficiency and good blood compatibility was synthesized according to the synthesis routes shown in Scheme 1. The amphiphilic HPG-C18 was first synthesized through the onepot reaction and then modified by azido ending. Meanwhile, the propargyl focal point PLLD-G4 was synthesized through the divergent and convergent approaches of lysine (Lys). Then, the azido-ended HPG-C18 (HPG−C18-N3) was conjugated with the propargyl focal point PLLD through a click reaction to form an amphiphilic star-shaped HPG-C18-PLLD. To confirm its chemical structure, HPG-C18-PLLD was characterized by 1H NMR and FT-IR analysis. From Figure 1a, it was clearly seen that the peak at 3.6 ppm belonged to the protons from the methylene and methenyl of HPG segments, and C18 segments showed their characterized signals at 1.2 ppm. For PLLD segments, its characteristic peaks of methylene appeared at 1.5−1.9 and 3.0 ppm, and that of methenyl appeared at 4.1 ppm. All signals were marked according to the chemical structure shown in Figure 1b. More important, the signal at 8.05 ppm means the presence of the triazole group, which was ascribed to the click reaction by HPG−C18-N3 with PLLD.29 This result indicated that HPG-C18 was conjugated with PLLD successfully. Moreover, the PLLD content in HPGC18-PLLD was calculated as 46.9% by elemental analysis. Figure 1c gave the FT-IR spectra of PLLD, HPG−C18-N3, and HPG-C18-PLLD, respectively. It was found that PLLD and HPG−C18-N3 displayed their characteristic absorption bands before the click conjugation. In particular, the HPG−C18-N3 displayed the distinct band of azido group at 2100 cm−1.30 However, the result from HPG-C18-PLLD exhibited the main characteristic absorption bands of both PLLD and HPG−C18N3 and also showed that the band of azido group decreased obviously. This result indicated that partial azido group of HPG−C18-N3 was reacted with propargyl focal point PLLD, and the conjugation was obtained successfully. However, compared with our previous click reaction,25 not all azido groups were reacted with PLLD in this work, and the reaction efficiency was lower. This result may be ascribed to the larger steric hindrance of PLLD-G4, which hindered the click reaction.31

Figure 2. Cumulative DOC release profile from the HPG-C18-PLLD/ DOC complex (37 °C, PBS, pH = 7.4, n = 3).

fast in the beginning of 8 h, but it was slower later. After 48 h of in vitro assay, ∼70% DOC was released from the HPG-C18PLLD/DOC complexes. For MMP-9 siRNA plasmid binding, the outer cationic PLLD could bind with MMP-9 in aqueous solution to form the HPG-C18-PLLD/MMP-9 complexes by electrostatic interactions. The gel electrophoresis assay was used to assess the binding ability of HPG-C18-PLLD to MMP-9. As shown in Figure 3a, at the low weight ratios (lower than 0.2), HPG-C18PLLD could not bind MMP-9 siRNA plasmid entirely, and the MMP-9 migrated out under the electric field. When the w/w ratio was equal to or above 0.5, HPG-C18-PLLD could dispute entirely the mobility of MMP-9, demonstrating a great binding ability to MMP-9. These results showed HPG-C18-PLLD could load both hydrophobic drugs and genes efficiently, indicating the potential value in drug/gene codelivery. Surface charge and particle size are crucial importance for the complexes gene transfection. It is also reported that most cells uptake nanoparticles from dozens of nanometers to several hundred nanometers.32 So, the complex HPG-C18-PLLD/ MMP-9 was expected to form the compact nanoparticle with proper particle size, which was favorable for endocytosis. In Figure 3b, the complexes showed the big particle size of ∼175 nm and negative zeta potential of ca. −26 mV at the low weight ratio of 0.2. Beyond the weight ratio of 1.0, the complexes E

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increased with the increase of weight ratio and then remained a constant (+19 mV) when the weight ratio was higher than 10. The results showed that HPG-C18-PLLD could form stable and compact complexes with MMP-9 when the weight ratio was higher than 10, so the following transfection assay was performed at the weight ratio higher than 10. Moreover, the typical topology morphology of the complexes was observed by TEM using the HPG-C18-PLLD/MMP-9 complexes (w/w = 20). As shown in Figure 3c, the complex displayed a compact spherical shape ∼100 nm. 3.3. In Vitro Transfection. To confirm the best conditions for MMP-9 siRNA plasmid delivery, the gene transfection efficiency of HPG-C18-PLLD/MMP-9 complexes at different weight ratios was evaluated in vitro. PEI-25k was used as the standard sample for gene delivery. It is reported that the PEI25k/MMP-9 displays its best transfection ability with an N/P ratio of 10.33 As shown in Figure 4, for PEI-25k/MMP-9, the efficiency of gene transfection was ∼48% in this work. Although HPG-C18-PLLD/MMP-9 complexes showed lower transfection efficiency compared with PEI-25k under this assay condition, it also displayed the receivable transfection efficiency at the weight ratio of 80 with the efficiency of more than 32%. This result was much better than our previous work, in which the star-shaped porphyrin−PLLD only displayed 23% of transfection efficiency when delivering MMP-9 to MCF-7 cells.18 The reasons for higher transfection efficiency may be resulted from the higher generation of PLLD as well as the hyperbranched structure of HPG-C18, which provided many more conjugation points and then improved the local cationic charge density. Moreover, although it was found that the transfection efficiency increased with the increase of weight ratios, HPG-C18-PLLD/MMP-9 complexes at the weight ratio of 100 or higher were not recommended in this work because of their significant cytotoxicity. Therefore, the weight ratio of 80 for HPG-C18-PLLD/MMP-9 was performed for the following assays. To verify further the transfection ability of HPG-C18-PLLD to MMP-9 into MCF-7 cells, the expression quantity of MMP-9 protein under the weight ratio of 80 was analyzed by western blot assay. Figure 5a showed the image of western blot result, and its relatively quantitative expression of MMP-9 protein was shown in Figure 5b. It was found that the blank HPG-C18PLLD could not reduce MMP-9 expression, and there was no significant difference with PBS control. While after transfected with MMP-9 siRNA plasmid, the expression quantity of MMP9 reduced obviously. PEI-25k/MMP-9 mediated more than

Figure 3. (a) Agarose gel electrophoresis retardation assay of HPGC18-PLLD/MMP-9 complexes at various weight ratios. (b) The particle sizes and zeta potentials of HPG-C18-PLLD/MMP-9 complexes formed at various weight ratios. (c) Typical TEM image of the HPG-C18-PLLD/MMP-9 (w/w = 20:1) complex.

showed the positive zeta potentials. The particle size of the complexes slightly change; it remained 110−130 nm. But the zeta potential of the complexes has significant variation, it

Figure 4. (a) Result histogram of transfected MCF-7 cells by HPG-C18-PLLD/MMP-9 complexes. (b) The representative photographs (×10) of transfected MCF-7 cells by fluorescence microscope. 1: PEI-25k/MMP-9 (w/w = 1.3), 2−4: HPG-C18-PLLD/MMP-9 (w/w = 40, 60, and 80, respectively). F

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Figure 5. (a) Representative MMP-9 protein expression determined by western blot analysis; (b) Analysis of light intensities of MMP-9 protein expression as the ratio of MMP-9 to β-actin from western blot results. 1: HPG-C18-PLLD; 2: PEI-25k/MMP-9 (w/w = 1.3); 3: HPG-C18-PLLD/MMP-9 (w/w = 80). PBS was set as the control.

80% reduction in protein expression due to its excellent gene delivery ability. HPG-C18-PLLD/MMP-9 complex at the weight ratio of 80 also makes MMP-9 protein expression reduce by 75%. These results suggested that HPG-C18-PLLD could deliver MMP-9 siRNA effectively into MCF-7 cells and reduced MMP-9 protein expression. Although showing a slightly poorer ability to deliver MMP-9 siRNA plasmid compared with PEI-25k, HPG-C18-PLLD was still considered as a promising gene carrier due to its following advantages in biocompatibility. 3.4. In Vitro and in Vivo Therapy. The effect of the combined therapy was studied by apoptosis assay, and the percentage of MCF-7 cells apoptosis was measured by flow cytometer. Annexin V-PE and 7-AAD can differentiate the early or late apoptosis and living cells or necrotic cells.34 As shown in Figure 6a,b, after incubation with blank HPG-C18-PLLD for 48 h, the cells showed negligible apoptosis compared to PBS control, suggesting nontoxicity to MCF-7 cells at the assay concentration. Cells treated with DOC or MMP-9 showed the distinct apoptosis regardless of early or late cell apoptosis. The percentage of early and late apoptosis for HPG-C18-PLLD/ DOC treated cells was calculated as 23.8%. Similarly, HPGC18-PLLD/MMP-9 induced 22.8% of cell apoptosis. For the coloaded HPG-C18-PLLD/DOC/MMP-9 complexes, the apoptosis percentage of MCF-7 cells reached 47.2%. This result indicated that HPG-C18-PLLD/DOC/MMP-9 could induce significant higher apoptosis percentage to MCF-7 cells than that of only DOC or MMP-9 used alone; in other words, MMP-9 combined with DOC could enhance the MCF-7 cell apoptosis. For the codelivery strategy, the better inhibition effect to MCF-7 cells may be resulted from that the released DOC could damage the mitosis and proliferation of MCF-7 cells, and meanwhile MMP-9 siRNA could instigate mRNA to down-regulate protein expression.35 The in vivo combined therapy to MCF-7 tumor was also explored, and PBS and blank HPG-C18-PLLD were set as the control. The tumor growth profiles and representative tumor image treated with various formulations were given as Figure 7. Seeing from Figure 7a, for the tumor treated with the blank HPG-C18-PLLD, its volume increased rapidly after 21 d of treatment. There was no obvious difference in volume compared to PBS control, which suggested that the blank HPG-C18-PLLD could not inhibit MCF-7 tumor. Other

Figure 6. Apoptosis analysis (a, b) by flow cytometry after MCF-7 cells incubated with various samples. 1: HPG-C18-PLLD; 2: HPGC18-PLLD/DOC (DOC: 0.08 μg/well); 3: HPG-C18-PLLD/MMP-9 (w/w = 80:1; MMP-9:2 μg/well); 4: HPG-C18-PLLD/DOC/MMP-9 (w/w = 80:1; DOC: 0.08 μg/well; MMP-9:2 μg/well).

Figure 7. Representative image of MCF-7 tumors at the 21st day (a) and tumor growth profiles (b) after treatment with various formulations (P < 0.01). 1: PBS control; 2: Blank HPG-C18-PLLD; 3: PEI-25k/MMP-9; 4: HPG-C18-PLLD/MMP-9; 5: HPG-C18PLLD/DOC; 6: HPG-C18-PLLD/MMP-9/DOC.

G

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mL, the blank HPG-C18-PLLD showed negligible cytotoxicity compared to PBS control. Contrarily, PEI-25k showed the significant cytotoxicity, and no more than 20% MCF-7 cells were available after treatment with 50 μg/mL PEI-25k. The huge difference in cytotoxicity between HPG-C18-PLLD and PEI-25k suggested that HPG-C18-PLLD has greater advantage in gene delivery in clinical. Furthermore, in vivo toxicity was also studied to prove further the good biocompatibility of HPG-C18-PLLD by the histological analysis. As observed from Figure 9, there was no significant histological difference between HPG-C18-PLLD group and PBS control group. It has been reported that the toxicity of biomaterials is connected with many characterizations including chemical structures, size, nature of the surface, terminal groups, biodistribution, metabolism, and so on.36 The nontoxicity observed in this work of star-shaped HPG-C18-PLLD may be ascribed to its chemical structure as well as its nature biocompatibility of HPG, C18, and PLLD segments. Its characteristic star-shaped and dendritic structures could reduce its toxicity compared with the linear cationic polymers having the similar molecular weights.37 Its biodegradability and nature biocompatibility of HPG, C18, and PLLD segments maybe promote the elimination effect from cells or organisms and then resulted in the improvement of biocompatibility. 3.5.2. Blood Compatibility. As the nanocomplexes, HPGC18-PLLD was usually expected to codeliver DOC/MMP-9 through intravenous injection in clinical. Then, the blood compatibility of HPG-C18-PLLD was assessed by hemolysis analysis, RBCs observation, and TEG assay. The percentage of hemolysis is the most commonly used evaluation for the blood compatibility of biomaterials. Most biomaterials interact with RBCs in the blood and then destroy the integrity of RBCs membrane, which results in the hemoglobin release from RBCs and erythrocyte death. Therefore, hemolysis was used in the biosafety evaluations of HPG-C18-PLLD with different concentrations. As shown in Figure 10, 1 mg/mL of HPG-C18-PLLD showed nonhemolytic; the percent of hemolysis was lower than 5%.28 In

groups containing DOC or MMP-9 exhibited the obvious inhibition effect, and group combining DOC and MMP-9 displayed the best inhibition effect with the smallest tumor volume shown. Moreover, it is noteworthy that although HPGC18-PLLD showed the lower transfection efficiency than PEI25k in vitro, it showed the comparative MMP-9 delivery ability in vivo, and there was no obvious difference in volume between HPG-C18-PLLD/MMP-9 group and PEI-25k/MMP-9 group. This may be attributed to the better blood compatibility of HPG-C18-PLLD compared with that of PEI-25k, which makes the complexes more stable in the blood and avoided the gene leakage and inactivation caused by the interactions between the complexes and blood proteins. Figure 7b gave the MCF-7 tumor growth profiles after treated with various groups, and the same results as Figure 7a were obtained. The tumor treated with HPG-C18-PLLD/DOC/MMP-9 showed the smallest tumor volume and slowest tumor growth rate, indicating the codelivering strategy is a promising approach for tumor therapy. 3.5. Biocompatibility. 3.5.1. Toxicity. The cytotoxicity of the blank HPG-C18-PLLD was assessed by CCK-8 assay, and the PEI-25k and PBS were used as control. The results were shown in Figure 8. As seen, below the concentration of 200 μg/

Figure 8. CCK-8 results of HPG-C18-PLLD and PEI-25k at different concentrations on MCF-7 cells.

Figure 9. Representative organ histology for control (top) and HPG-C18-PLLD (bottom) injected mice. H

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According to report, the interactions of polymeric biomaterials and RBCs are mediated by the electrostatic and the hydrophobic interactions.38 The introduction of HPG segments improved the blood compatibility of HPG-C18-PLLD and made the resultant codelivery carrier safer to the blood. Besides the RBCs, to study further the effect of HPG-C18PLLD on the whole blood clotting process, a TEG assay was performed to provide the information about the activity of the clotting system through recording the process of the blood clot. Four key parameters in the TEG assay were as follows: time R, time K, α angle, and maximum amplitude (MA); these parameters represent the time from the test to the formation of the fibrin, the dynamics of clot formation, the rate of clot aggregating, and the maximum clot strength, respectively.39 Figure 12 gave the TEG traces of the samples of the whole blood mixed with HPG-C18-PLLD. It was found that the TEG traces of formulations containing 0.01 and 0.1 mg/mL HPGC18-PLLD were similar to PBS control, suggesting the safety to the whole blood. However, the TEG trace displayed some difference compared to PBS control when the concentration of HPG-C18-PLLD was 1 mg/mL, in which the parameters of R and MA were observed to be abnormal. The corresponding data of these TEG trances were listed in Table 1. It was found that all TEG parameters for 0.01 and 0.1 mg/mL HPG-C18PLLD were in the normal range, which indicated that 0.01 and 0.1 mg/mL HPG-C18-PLLD were safe to the whole blood. On the contrary, 1 mg/mL HPG-C18-PLLD may be not safe enough because all of four parameters were out of the normal range, suggesting that this concentration may impair the activity of the clotting system. All in all, HPG-C18-PLLD displayed much better blood compatibility compared with PEI-25k. Furthermore, the used concentration of HPG-C18-PLLD in this work was lower than 1 mg/mL, suggesting more suitable for drug/gene delivery through intravenous injection. A further stability of nanocomplex in the blood was also studied by recoding its particle size and zeta potential within the experimental 48 h. The result displayed that nanocomplex showed good stability in the blood, and its particle size changed little within 48 h. However, because of its positive surface, nanocomplex inevitably interacted with the proteins in the blood. After mixed with the blood, nanocomplex gave the increase in particle size and decline in zeta potential. This interaction was also reflected in above TEG analysis and is a challenge in our following works.

Figure 10. Effect of HPG-C18-PLLD and PEI-25k with different concentrations on the hemolysis.

other words, 1 mg/mL of HPG-C18-PLLD was safe to RBCs. However, the obvious hemolysis was observed when PEI-25k was used, even if its concentration was only 0.1 mg/mL. This result indicated that HPG-C18-PLLD was more suitable for drug/gene delivery through intravenous injection. Moreover, the morphological changes of RBCs were observed by SEM to explore the effect of HPG-C18-PLLD on RBCs more intuitively. Seeing from Figure 11, it was found

4. CONCLUSION In summary, the star-shaped copolymer of dendritic poly(Llysine)-modified amphiphilic hyperbranched polyglycerol derivative was synthesized and then used to codeliver DOC/ MMP-9 for breast cancer treatment. The obtained complexes displayed good drug and gene delivery ability, and also could obviously decrease the expression of MMP-9 protein in MCF-7 cells. The coloaded complexes could induce a more significant apoptosis to MCF-7 cells than that of only DOC or MMP-9 used alone. In vivo assay indicated that the combination strategy displayed a better effect on tumor inhibition. Moreover, HPG-C18-PLLD showed lower toxicity and better blood compatibility compared to PEI-25k, which may be the result of that HPG-C18-PLLD showed the comparative MMP9 delivery ability in vivo compared with PEI-25k even if it showed the slightly lower transfection efficiency in vitro. Therefore, HPG-C18-PLLD is a promising safe and effective

Figure 11. Effect of HPG-C18-PLLD with different concentrations on the aggregation and morphology of RBCs. (a: PBS control; b: 0.01 mg/mL PEI-25k; c−f: HPG-C18-PLLD with the concentrations of 0.01, 0.1, 1, and 10 mg/mL, respectively).

that the morphology of the RBCs did not change within the range from 0.01 to 0.1 mg/mL of the HPG-C18-PLLD compared to PBS control. However, the RBCs had slight change in shape in 1 mg/mL of the aqueous HPG-C18-PLLD, which indicated that HPG-C18-PLLD affected a little to RBCs morphology. The hedgehog-like morphology of RBCs appeared when RBCs mixed with 10 mg/mL HPG-C18-PLLD or 0.01 mg/mL PEI-25k, suggesting the obvious effect to RBCs. This result was in accordance with that of hemolysis analysis. I

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Figure 12. TEG traces of the whole blood in the presence of different concentrations of HPG-C18-PLLD. PBS was set as the control. (a: PBS control; b: 0.01 mg/mL HPG-C18-PLLD; c: 0.1 mg/mL HPG-C18-PLLD; d: 1 mg/mL HPG-C18-PLLD.). (6) Koirala, A.; Conley, S. M.; Naash, M. I. A Review of Therapeutic Prospects of Non-Viral Gene Therapy in the Retinal Pigment Epithelium. Biomaterials 2013, 34, 7158−7167. (7) Creixell, M.; Peppas, N. A. Co-Delivery of siRNA and Therapeutic Agents Using Nanocarriers to Overcome Cancer Resistance. Nano Today 2012, 7, 367−379. (8) Yin, T. J.; Wang, L.; Yin, L. F.; Zhou, J. P.; Huo, M. R. CoDelivery of Hydrophobic Paclitaxel and Hydrophilic AURKA Specific siRNA by Redox-Sensitive Micelles for Effective Treatment of Breast Cancer. Biomaterials 2015, 61, 10−25. (9) Gaspar, V. M.; Baril, P.; Costa, E. C.; de Melo-Diogo, D.; Foucher, F.; Queiroz, J. A.; Sousa, F.; Pichon, C.; Correia, I. J. Bioreducible Poly(2-ethyl-2-oxazoline)-PLA-PEI-SS Triblock Copolymer Micelles for Co-Delivery of DNA Minicircles and Doxorubicin. J. Controlled Release 2015, 213, 175−191. (10) Xu, C. N.; Wang, P.; Zhang, J. P.; Tian, H. Y.; Park, K.; Chen, X. S. Pulmonary Codelivery of Doxorubicin and siRNA by pH-Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer. Small 2015, 11, 4321−4333. (11) Kaneshiro, T. L.; Lu, Z. R. Targeted Intracellular Codelivery of Chemotherapeutics and Nucleic Acid with a Well-Defined DendrimerBased Nanoglobular Carrier. Biomaterials 2009, 30, 5660−5666. (12) Sousa-Herves, A.; Riguera, R.; Fernandez-Megia, E. PEGDendritic Block Copolymers for Biomedical Applications. New J. Chem. 2012, 36, 205−210. (13) Al-Jamal, K. T.; Al-Jamal, W. T.; Akerman, S.; Podesta, J. E.; Yilmazer, A.; Turton, J. A.; Bianco, A.; Vargesson, N.; Kanthou, C.; Florence, A. T.; Tozer, G. M.; Kostarelos, K. Systemic Antiangiogenic Activity of Cationic Poly-L-lysine Dendrimer Delays Tumor Growth. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 3966−3971. (14) Samal, S. K.; Dash, M.; Van Vlierberghe, S.; Kaplan, D. L.; Chiellini, E.; van Blitterswijk, C.; Moroni, L.; Dubruel, P. Cationic Polymers and Their Therapeutic Potential. Chem. Soc. Rev. 2012, 41, 7147−7194. (15) Gao, Y.; Gao, G.; He, Y.; Liu, T. L.; Qi, R. Recent Advances of Dendrimers in Delivery of Genes and Drugs. Mini-Rev. Med. Chem. 2008, 8, 889−900. (16) Inoue, Y.; Kurihara, R.; Tsuchida, A.; Hasegawa, M.; Nagashima, T.; Mori, T.; Niidome, T.; Katayama, Y.; Okitsu, O. Efficient Delivery of siRNA Using Dendritic Poly(L-lysine) for Loss-of-Function Analysis. J. Controlled Release 2008, 126, 59−66. (17) Luo, K.; Li, C. X.; Li, L.; She, W. C.; Wang, G.; Gu, Z. W. Arginine Functionalized Peptide Dendrimers as Potential Gene Delivery Vehicles. Biomaterials 2012, 33, 4917−4927. (18) Ma, D.; Lin, Q. M.; Zhang, L. M.; Liang, Y. Y.; Xue, W. A StarShaped Porphyrin-Arginine Functionalized Poly(L-lysine) Copolymer for Photo-Enhanced Drug and Gene Co-Delivery. Biomaterials 2014, 35, 4357−4367. (19) Liu, T.; Xue, W.; Ke, B.; Xie, M. Q.; Ma, D. Star-Shaped Cyclodextrin-Poly(l-lysine) Derivative Co-Delivering Docetaxel and MMP-9 siRNA Plasmid in Cancer Therapy. Biomaterials 2014, 35, 3865−3872. (20) Stiriba, S. E.; Frey, H.; Haag, R. Dendritic Polymers in Biomedical Applications: From Potential to Clinical Use in Diagnostics and Therapy. Angew. Chem., Int. Ed. 2002, 41, 1329−1334.

Table 1. Clotting Kinetics Parameters of Human Whole Blood Mixed with Aqueous HPG-C18-PLLD Solutions at Different Concentrations samplesa

R (min)

K (min)

α (deg)

MA (mm)

normal range PBS control 1.0 mg/mL HPG-C18-PLLD 0.1 mg/mL HPG-C18-PLLD 0.01 mg/mL HPG-C18PLLD

5−10 5.6 15.3↑ 9.8 6.8

1−3 2.0 4.6↑ 2.3 1.8

53−72 63.2 39.1↓ 58.3 64.0

50−70 61.2 47.8↓ 59.2 55.4

The sign ↓ indicates a low value, and ↑ indicates a high value compared with the normal range provided by the TEG analyzer.

a

carrier for drug/gene codelivery, which has a potential application in tumor treatment.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (D.M.) *E-mail: [email protected]. (W.X.) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by the National Natural Science Foundation of China (31271019 and 51573071), Natural Science Foundation of Guangdong Province (S2013030013315 and 2014A030313361) as well as the fund from Pearl River S&T Nova Program of Guangzhou (201506010069).

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