Natural Particulates Inspired Specific-Targeted Codelivery of siRNA

Article pubs.acs.org/molecularpharmaceutics

Natural Particulates Inspired Specific-Targeted Codelivery of siRNA and Paclitaxel for Collaborative Antitumor Therapy Ruoning Wang, Ziqiang Zhao, Yue Han, Shihao Hu, Yaw Opoku-Damoah, Jianping Zhou,* Lifang Yin, and Yang Ding* State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China S Supporting Information *

ABSTRACT: The effective combination of drugs promoting antiangiogenesis and apoptosis effects has proven to be a promising collaborative tumor antidote; and the codelivery of small interfering RNA (siRNA) and chemotherapy agents within one efficient vehicle has gained more attention over single regimen administration. Herein, vascular endothelial growth factor specific siRNA (siVEGF) and paclitaxel (PTX) were introduced as therapeutic companions and coencapsulated into naturally mimic highdensity lipoproteins (rHDL/siVEGF-PTX), so that various mechanisms of treatment can occur simultaneously. The terminal nanoparticles share capacity of specific-targeting to tumor cells overexpressed scavenger receptor class B type I (SR-BI) and deliver siVEGF and PTX into cytoplasm by a nonendocytosis mechanism. By exchanging HDL core lipids with hydrophobic therapeutics, rHDL/siVEGF-PTX possessed particle size of ∼160 nm, surface potential of ∼−20 mV, and desirable long-term storage stability. In vitro results confirmed that the parallel activity of siVEGF and PTX displayed enhanced anticancer efficacy. The half-maximal inhibitory concentration (IC50) of rHDL/ siVEGF-PTX toward human breast cancer MCF-7 cell is 0.26 μg/mL (PTX concentration), which presents a 14.96-fold increase in cytotoxicity by taking Taxol as comparison. Moreover, in vivo results further demonstrated that rHDL/siVEGF-PTX performed enhanced tumor growth inhibition via natural targeting pathway, accompanied by remarkable inhibition of neovascularization in situ caused by siVEGF silencing in down-regulation of VEGF proteins. On the premise of effective drug codelivery, rHDL/siVEGF-PTX demonstrated high tumor targeting for collaborative antitumor efficacy without side effects after systemic administration, and this bioinspired strategy could open an avenue for exploration of combined anticancer therapy. KEYWORDS: reconstituted high density lipoproteins, codelivery of siRNA and paclitaxel, vascular endothelial growth factor, direct cytosolic delivery, collaborative antitumor efficacy

1. INTRODUCTION The development of tumor occurs as the consequence of accumulative acquisition of genetic alterations affecting oncogenes. The arising malignant tumor obtains fatal features such as increased proliferation of tumor cells accompanied by improved intratumoral pressure and impossible tumor penetration of xenobiotics.1 Therefore, a diverse spectrum of mechanisms have been applied in cancer therapy, such as antiangiogenesis,2 nucleic acid synthesis irritation,3 DNA structure destruction,4 RNA transcription inhibition,5 protein function disturbance,6 and immunity stimulation.7 To date, cancer treatment via the single therapeutic strategy stays suboptimal, in most therapies, not all mechanisms of tumor progression are under suppression, and therapeutic companion, therefore, via various principles could collectively kill off tumor cells with synergistic effects.8 Hence, it is imperative to perform dual-mode-therapy in one single carrier, including antiangiogenesis and tumor cell antiproliferation simultaneously, which is much more effective in treating with cancers than two or more drugs administrated separately.9 © 2017 American Chemical Society

RNA interference (RNAi) technology has displayed great potential for anticancer therapy due to its unique sequencespecific gene silencing effect, which is based on small interfering RNA (siRNA).10 Numerous drug targets including multiple oncogenic proteins were identified as RNAi candidates for potential cancer treatment. Vascular endothelial growth factor (VEGF), which is overexpressed in most tumor cells, could stimulate endothelial cells proliferation so as to initiate the angiogenic process for the supply of nutrients as well as oxygen.11 VEGF is identified as the ideal RNAi candidate for breast cancer treatment, which is the angiogenic growth factor and the most potent proangiogenic signal in breast cancer.12 Moreover, the clinical data demonstrated that anti-VEGF therapies could normalize tumor vessels. Vascular normalization restores blood vessels, leading them homogeneous with Received: Revised: Accepted: Published: 2999

March 9, 2017 July 24, 2017 July 28, 2017 July 28, 2017 DOI: 10.1021/acs.molpharmaceut.7b00192 Mol. Pharmaceutics 2017, 14, 2999−3012

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Molecular Pharmaceutics Scheme 1. Construction of Naturally Inspired Nanosystem rHDL/siVEGF-PTXa

After i.v. administration, rHDL/siVEGF-PTX was recognized by SR-BI, followed by membrane reorganization, formation of nonaqueous “channel”, and subsequent cross-membrane delivery of siVEGF and PTX to cells to pursue a strategic combination therapy. a

less leaking. Additionally, interstitial fluid pressure is reduced, which restores a trans-vascular fluid pressure difference, resulting in improved blood flow and anticancer agent penetration in tumors.13 RNAi-mediated silencing of VEGF has demonstrated great capability of VEGF protein expression inhibition;14 nevertheless, the inherent drawbacks of siRNA, such as poor membrane penetration and low targeting efficiency, limit its clinical application. Therefore, it is imperative to develop efficient delivery vehicles in order to overcome the bottlenecks in efficient siRNA delivery to target cells.10,15 Myriad promising approaches based on cationic liposomes,16 polymers,17 or peptides18 have been optimized for delivering siRNA and in vitro applications. However, cationic gene vectors would induce cytotoxicity, accompanied by comparatively poor transfection efficiency and low targetability.19 Therefore, design of biocompatible platforms for siRNA systemic delivery to diseased cells would be much more promising on the way to the clinic.20,21 So far, bioparticulates from mammalian cells to native biomolecules have been exploited for anticancer therapy, and they are designed with specific functions and shared several desirable characters in gene delivery in vivo.22,23 Taking the mimic endogenous structure of native high density lipoprotein (nHDL), its recombinant form (rHDL) possess a hydrophobic core with cholesteryl esters (CE) and triglycerides with a monolayer of phospholipids and apolipoprotein A-I (apoA-I).24 ApoA-I is the most important and abundant apolipoproteins

constituent of HDL, which is responsible for scaffolding the size and shape of HDL species.25 Furthermore, rHDL could provide an effective approach to transfer therapeutic cargos directly into the cytoplasm via a receptor-associated nonendocytotic mechanism after apoA-I bound, thereby bypassing the endosomal compartment.26 As naturally inspired nanoparticles, rHDL remains in extended circulation while eluding the reticuloendothelial system (RES) and targeting their endogenous scavenger receptor BI (SR-BI), which is likely to become an attractive naturally cell-specific strategy for anticancer treatment. In the past decades, rHDL has been optimized for an ideal carrier for multimodal drug-loading patterns, including covalent modification with monolayer, surface intercalation into phospholipid shell, and physical encapsulation into hydrophobic core.27,28 Loading agents at specific location on reconstituted nanoparticles resulted from methodologies of loading as well as features of the molecule. In our previous research, lipophilic siRNAs were synthesized and intercalated into the phospholipid monolayer with cholesterol anchor;29 however, the amphiphilicity of cholesterol-conjugated siRNA allows its hydrophilic face partially to the aqueous surroundings, and the stability of molecule circulation was confined by this loading pattern.30 To ameliorate the situation, materials provided with condensability, e.g., protamine9 and cationic lipids,31 could be designed to package siRNA into the lipid core of rHDL via electrostatic32 and hydrophobic33 interactions, 3000

DOI: 10.1021/acs.molpharmaceut.7b00192 Mol. Pharmaceutics 2017, 14, 2999−3012

Article

Molecular Pharmaceutics

2.2. Preparation of DOTAP/siRNA Nanocomplexes. siRNA was dissolved in DEPC-treated water (0.5 mg/mL), and DOTAP was dissolved in ethanol (10 mg/mL). These solutions were mixed at different ratios of N/P (0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10) and total volume of 50 μL with gentle vortexing for 2 min. Formation analysis of DOTAP/siRNA nanocomplexes at various N/P ratios was confirmed with agarose gel electrophoresis. The final optimal N/P ratio for DOTAP/ siRNA nanocomplexes were collected and stored. 2.3. Preparation of siRNA and/or PTX Loaded Nanoparticles. PTX and siRNA encapsulated rHDL (rHDL/ siRNA-PTX) were obtained by using the thin film dispersion method as previously described.29 In brief, 1.5 mg of PTX and 10 mg of phospholipids (PC) and cholesterol (Chol) (5:1, w/ w) were dissolved in 200 μL of organic mixture solutions (chloroform/ethanol = 1:1, v/v). Then the thin lipid film was yielded by evaporating under nitrogen, and it was dried under vacuum to remove organic solvent. Subsequently, the film was hydrated with 200 μL of DOTAP/siRNA complexes with the optimal ratio according to section 2.2 and 800 μL of distilled water, then sonicating, which was injected dropwise into 1 mL of apolipoprotein A-I (apoA-I) solution (4 mg/mL). The suspension was sonicated (300 W) at 4 °C with a probe sonicator. Then the resultant product was transferred to a rotary evaporator and filtered via a microporous membrane (0.8 μm) and collected. PTX-loaded rHDL (rHDL/PTX) was obtained in the same manner without DOTAP/siRNA involvement. siRNA and PTX coloaded liposomes (Lipos/ siRNA-PTX) were prepared in the same manner without apoAI incubation. Cy3-siRNA/FAM-siRNA and C6/DiR-loaded nanoparticles were also obtained according to the same procedures for targeting efficacy assay in vitro and in vivo. For control, Taxol was prepared through dissolving 12 mg of PTX in 2.0 mL of ethanol and Cremophor EL (1:1, v/v) and sonicated for 30 min. 2.4. Characterization of Nanoparticles. The diameter, polydispersity index (PDI), and zeta potential (ZP) of rHDL/ PTX, Lipos/siRNA-PTX, and rHDL/siRNA-PTX were detected by dynamic light scattering (DLS, Nano-ZS90, Malvern instruments, UK). The morphology of rHDL/siRNA-PTX was also observed by transmission electron microscopy (TEM). The amount of siRNA in nanoparticles was measured from free siRNA in the supernatant recovered after centrifugation via absorbance measurement by Nano-100 Spectrophotometer instrument (Allsheng, Hangzhou, China). Entrapment efficiency (EE) of siRNA was calculated from the totally added amount of siRNA (T[siRNA]) and that of the supernatant (S[siRNA]) by the following formula: EE% = (T[siRNA] − S[siRNA])/(T[siRNA]) × 100%. The amount of PTX in nanoparticles was detected by highperformance liquid chromatography (HPLC, Shimadzu LC2010 system, Kyoto, Japan) with UV detection at 227 nm. The mobile phase was a 75/25 (v/v) mixture of methanol and water at a flow rate of 1.0 mL/min, and method verification was performed as previously reported.40 EE and loading efficiency (LE) of PTX in different nanoparticles were measured using the following formulas:

wherein cationic lipids including N-[1-(2,3-dioleyloxy)propyl]N,N,N-trimethylammonium chloride (DOTMA),34 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA),35 and (2,3-dioleoyloxy-propyl)-trimethylammonium chloride (DOTAP)36 seem to be great potentials in effective gene silencing therapy of this application. Particularly, a vast amount of data accumulated on DOTAP suggested that this commercial cationic lipid is an ideal candidate because of its facile and economical chemistry in terms of greater efficiency and less toxicity for various in vivo applications, even in recent human clinical trials.37 To this end, the cocktail therapy that combines antiangiogenesis genes with paclitaxel (PTX) molecules within one single vehicle would be adopted to simultaneously block vascularization and kill off cancer cells for collaborative antitumor outcomes.38 With the above-mentioned design inspiration, a new paradigm, rHDL-mediated specific-targeted codelivery of siRNA targeting VEGF (siVEGF) and PTX nanosystem (rHDL/siVEGF-PTX), was formulated to integrate multiple essential attributes in this novel design (Scheme 1). Condensed siRNA nanocomplexes (DOTAP/siRNA) were fabricated through electrostatic interactions between cationic DOTAP and siRNA; PTX and DOTAP/siRNA were synchronously incorporated into the hydrophobic core of rHDL according to theory of similarity and intermiscibility, and apoA-I was finally attached to obtain a stable assembly of this system. The spatial structure of lipid monolayer of rHDL provide enough space for drug loading. After i.v. administration, rHDL/siVEGF-PTX could accumulate in and attach to SR-BI receptor overexpressing malignant tumors, followed by cell membrane reorganization, formation of nonaqueous “channel”, and direct delivery of siVEGF and PTX into cytoplasm by cross-membrane, resulting in simultaneous tumor antiangiogenesis and apoptosis. Finally, to assess the tolerability and toxicity of the biomimetic nanoparticle, blood samples from the nude mice were collected for biochemistry tests, and vital organs were harvested for histological analysis.

2. EXPERIMENTAL SECTION 2.1. Materials. (2,3-Dioleoyloxy-propyl)-trimethylammonium chloride (DOTAP) was obtained from CordenPharma (Liestal, Switzerland). Nontargeted control siRNA (siRNA), Cy3 or FAM labeled siRNA, and siRNA targeting VEGF (siVEGF, 5′-GGA GUA CCC UGA UGA GAU CdTdT-3′) were obtained from Guangzhou RiboBio Co., Ltd. (Shenzhen, China). Apolipoprotein A-I (apoA-I) was purified from the albumin byproduct in our laboratory on the basis of an established protocol with some modifications.39 Paclitaxel (PTX) was obtained from Shanghai Zhongxi Pharmaceutical (Group) Co., Ltd. (Shanghai, China). Soybean phospholipids (PC, purity 90%) were purchased from Shanghai Tywei Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol (Chol) was obtained from Huixing Biochemical Reagent Co., Ltd. (Shanghai, China). High density lipoprotein (HDL), 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) dye, DAPI, and coumarin-6 (C6) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The near-infrared dye DiR was purchased from Beijing Fanbo Science and Technology Co., Ltd. (Beijing, China). IRDye-800-conjugated second antibody was obtained from Rockland Inc. (PA, USA). CD31-antibody was obtained by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals were reagent grade or better.

EE% = (weight of PTX in nanoparticles) /(weight of PTX fed initially) × 100 3001


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final concentration of Cy3-siRNA and C6 was 100 nM and 50 ng/mL, respectively. Flow cytometric analyses was performed to confirm SR-BI receptor-specific targeted uptake of fluorescence probe. MCF-7 cells (SR-BI+) and HT1080 cells (SR-BI−) were seeded into 6-well plates (1 × 105 cells/well) and proliferated for 24 h. Then the medium with Lipos/Cy3siRNA-C6 or rHDL/Cy3-siRNA-C6 was substituted and incubated for 4 h. The fluorescence intensity of C6 and Cy3siRNA positive cells percentage were detected by the flow cytometer with exciting wavelength (Ex = 488 nm) for C6 and (Ex = 532 nm) for Cy3. MCF-7 cells were cultured for 24 h in glass bottom culture dishes (1 × 104 cells/well). After incubation for 4 h with Lipos/Cy3-siRNA-C6 or rHDL/Cy3siRNA-C6, cells were washed thrice and fixed, and then were observed with CLSM at 532 nm (exciting Cy3) and 488 nm (exciting C6). For the competition assay, cells were pretreated with free native HDL (nHDL, 5 mg/mL) for 2 h before substituting for corresponding payloads of rHDL/Cy3-siRNAC6. The cells were treated for 4 h, and fluorescence intensity was detected as described above. 2.9. Cytotoxicity Assay. MTT assay was carried out for detecting the induced cytotoxicity by different formulations. MCF-7 cells were seeded in 96-well plates (5 × 103 cells/well). On achieving 70−80% confluence, cells were treated with blank nanoparticles (Lipos and rHDL) at a concentration of 5−500 μg/mL, siRNA-loaded formulations (free siRNA, rHDL/ siRNA, rHDL/siVEGF) with 100 or 200 nM siRNA concentration, and PTX-loaded formulations (Taxol, Lipos/ siVEGF-PTX, rHDL/PTX and rHDL/siVEGF-PTX) with PTX concentration of 0.1−100 μg/mL at 100 μL/well. After incubation for 48 h, MTT was added for culturing another 4 h. The supernatant was discarded with the addition of DMSO. Then the absorbance was detected by a microplate reader at 570 nm (Multiskan Mk3, Thermo Labsystems, Beverly, MA, USA). Untreated cells were used as 100% viability control and cells without MTT took as control to calibrate spectrophotometer to zero absorbance. 2.10. In Vivo Antitumor Efficacy. BALB/c nude mice (female, 6−7 weeks of ages, 18−23 g) were selected for in vivo antitumor research. The animal use protocol was approved by the China Pharmaceutical University Ethics Committee and was operated in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. MCF-7 cells (2.0 × 107 cells in 50 μL of PBS) mixed with 50 μL of matrigel were transplanted into the armpit region of mice. The tumor size was measured with a vernier caliper, and the tumor volume was calculated as V = A × B2/2, where A is the largest diameter, B is the perpendicular diameter, and V is given in mm3. For in vivo distribution, when the tumor size of BALB/c nude mice reached around 500 mm3, the mice were administrated intravenously with FAM-labeled siRNA and DiR coloadedLipos (Lipos/FAM-siRNA-DiR) and rHDL (rHDL/FAMsiRNA-DiR) at the dose of 2 mg/kg FAM-siRNA and 50 μg/ kg DiR. The nude mice were anesthetized, and images were captured at various time intervals (6 h, 24 h) by in vivo Maestro EX fluorescence imaging system (Cambridge Research and Instrumentation, USA). To evaluate the biodistribution of FAM-siRNA fluorescence, the bandpass filter was fixed at Ex = 470 nm and Em = 530 nm. Additionally, the filter also was equipped at Ex = 720 nm and Em = 790 nm for DiR detection. The mice were sacrificed, and vital organs of the rHDL group were excised to compare the relative accumulation for both

LE% = (weight of PTX in nanoparticles) /(weight of PTX − loaded nanoparticles) × 100

2.5. Stability Assay. To evaluate storage stability of rHDL/ siRNA-PTX in PBS at 4 °C for 20 days, diameter, ZP, and leakage percentage of nanoparticles were investigated at predetermined intervals, respectively. Diameter and ZP were measured using DLS; and the leakage percentage was calculated using the following equation by HPLC and spectrometer: leakage ratio (%) = [EE(0) − EE(t )]/EE(0) × 100

where EE(0) and EE(t) represent the EE of PTX or siRNA in rHDL/siRNA-PTX at 0 h or prearranged time (t), respectively. Serum stability assay was operated to detect the rHDL on protecting siRNA and PTX against serum degradation. The free siRNA and rHDL/siRNA-PTX were incubated with FBS (1:1, v/v) at 37 °C. To release siRNA from rHDL nanoparticles, 20% Triton X-100 (v/v) and 20% heparin (w/v) were added to rHDL for 1 h incubation.41 Then samples were evaluated by agarose gel electrophoresis. Furthermore, diameter, ZP, and EE(PTX) of rHDL/siRNA-PTX were investigated at predetermined intervals, respectively. 2.6. SiRNA and PTX Release from Nanoparticles. 2.6.1. In Vitro Release of siRNA. rHDL/siRNA-PTX (1.0 mL) was prepared at the optimal ratio of N/P in PBS buffer (pH 7.4) and gently shook with the speed of 100 rpm/min to evaluate the release pattern of siRNA in the water bath. Lipos/ siRNA-PTX with the equivalent siRNA was prepared as control. The supernatant was periodically (0, 2, 4, 8, 12, 24 h) collected by centrifugation. The amount of siRNA in supernatant release from nanoparticles was determined by Nano-100 Spectrophotometer instrument, and release percentage of siRNA (%) in nanoparticles was determined as a ratio (%) to the total amount of siRNA. 2.6.2. In Vitro Release of PTX. The PTX release from rHDL/siRNA-PTX and Lipos/siRNA-PTX was detected by the dialysis method.42 Briefly, 1 mL of rHDL/siRNA-PTX or Lipos/siRNA-PTX containing 0.5 mg of PTX was placed in a clamped dialysis bag (MWCO 3500 Da) and immersed in 150 mL of PBS buffer (0.1 M, pH 7.4) containing 0.1% (w/v) Tween 80 in an incubation shaker at 100 rpm. At selected time intervals (0, 2, 4, 8, 12, 24 h), 1 mL of medium was taken for HPLC analysis as discussed in section 2.4, and the whole media were refreshed. 2.7. Cell Culture. Human breast cancer cell line MCF-7 and fibrosarcoma cell line HT1080 were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cell line MCF-7 with SR-BI overexpression (SR-BI+) and HT1080 cells without SR-BI (SRBI−) were used for cellular uptake comparison.43 The DMEM medium with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin was utilized as cell culture medium. Cells were cultivated at 37 °C with 5% CO2. 2.8. Cell Uptake and Intracellular Trafficking of Nanoparticles. The cellular uptake and intracellular trafficking of nanoparticles were investigated by flow cytometry (BD FACS Calibur, Franklin, USA) and confocal laser scanning microscopy (CLSM, Leica TCS SP5, Heidelberg, Germany), respectively. Cy3 labeled siRNA (Cy3-siRNA) and coumarin 6 (C6) loaded-Lipos or rHDL abbreviated as Lipos/Cy3-siRNAC6 and rHDL/Cy3-siRNA-C6 were prepared for cytosolic localization and targeting efficacy assay in vitro, of which the 3002


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Figure 1. (A) Analysis of DOTAP/siRNA at different ratios of N/P. Lane 1, naked siRNA; Lane 2−10, siRNA with increasing amount of DOTAP. (B) Diameter and ZP of DOTAP/siRNA in various ratios of N/P. (C) TEM image and (D) diameter graph of rHDL/siRNA-PTX. Data are presented as mean ± SD (n = 3).

of tumor tissues were isolated and quantified by the BCA protein assay. The samples were separated with SDSpolyacrylamide gel and transferred onto the PVDF membranes.44 The formation of immune complexes is incubated by proteins with primary antibodies (rabbit anti-VEGF) overnight. Blots were washed and treated with IRDyeTM-800-conjugated antirabbit second antibody. Immunoreactive protein bands were analyzed with an odyssey scanning system (LI-COR Inc., USA). 2.12. Safety Profiles. BALB/c nude mice (female, 6−7 weeks) were used to evaluate in vivo adverse effect upon i.v. administration of rHDL/siVEGF-PTX with doses of 7 mg/kg PTX and 1 mg/kg siRNA every 2 days for 2 weeks. Taxol and saline were used as controls. Animal behaviors were recorded during the entire procedure. The animals were sacrificed 24 h post-last-dosing, and blood samples were subjected to hematological and biochemistry analyses; main organs were excised for H&E analysis. 2.13. Statistical Analysis. All data were expressed as mean ± SD, and statistical significance was tested using one-way ANOVA. All comparisons were made relative to corresponding controls, and significance of difference was indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

FAM-siRNA and DiR 24 h after injection. All the images were analyzed using Living Image Software. BALB/c nude mice bearing MCF-7 tumors were established as described above, and the antitumor efficacy was assessed when the size of tumor reached approximately 200 mm3. The nude mice were divided into five groups (n = 12), then administrated with saline, Taxol, rHDL/PTX, Lipos/siVEGFPTX, and rHDL/siVEGF-PTX with doses of 7 mg/kg PTX and 1 mg/kg siRNA via tail vein on day 0, 3, 6, 9, 13 and 15, respectively. The therapeutic efficacy on these nude mice was evaluated by measuring tumor volume, body weight, and tumor weight in every group. Six nude mice in each group were sacrificed 3 days post-final-injection, and tumor tissues were harvested for hematoxylin and eosin (H&E) staining to assess the histological features. The tumor weight inhibition (% TWI) was calculated according to the equation: TWI (%) = (WC − WD)/WC × 100, where WC and WD represent the control group and treated groups of mean tumor weight. The survival rates were monitored throughout the study after the final injection. 2.11. In Vivo Tumor Angiogenesis Suppression. Immunohistochemical staining for CD31 were carried out to evaluate the mechanism of tumor angiogenesis suppression. In brief, at day 18, three mice in each group were sacrificed to obtain tumors, fixed in 10% formaldehyde, and embedded. Histological sections were obtained by Freezing Microtome. After blocking with goat serum, sections were incubated with CD31-antibody, which was followed by secondary antibody. Then tissue images were captured at 200× magnification. Microvessel density represented the average quantity of microvessels (MVD, the number of CD31-positive vessel/ mm2) in each field measured as the number of CD31-positive objects identified, and four fields per section were calculated. To determine VEGF amount in the solid tumor region of each group, tumors were excised after final treatment. Proteins

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterizations of DOTAP/ siRNA Complexes. siRNA was condensed with cationic lipid DOTAP by electrostatic interactions at different N/P (quaternary nitrogen of DOTAP/phosphate of siRNA) ratios ranging from 0.5 to 10.0 to obtain DOTAP/siRNA complexes. As documented in the results obtained from Figure 1A,B, N/P ratio at 4 was chosen to achieve complete protection of siRNA via the proper amount of DOTAP without any free siRNA bands in agarose gel electrophoresis assays, and the compact complexes exhibited the highly positive charge of +27.83 mV 3003


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Molecular Pharmaceutics Table 1. Formulation Characteristics of rHDL/PTX, Lipos/siRNA-PTX, and rHDL/siRNA-PTXa

a

samples

size (nm)

PDI

ZP (mV)

EE of PTX (%)

EE of siRNA (%)

LE of PTX (%)

rHDL/PTX Lipos/siRNA-PTX rHDL/siRNA-PTX

126.22 ± 21.21 137.83 ± 18.93 161.21 ± 15.21

0.16 ± 0.08 0.22 ± 0.05 0.12 ± 0.06

−24.73 ± 11.07 −27.19 ± 10.31 −23.21 ± 8.43

83.12 ± 3.96** 96.13 ± 3.61 95.81 ± 2.34

98.52 ± 6.78 93.45 ± 1.23

9.25 ± 2.09 9.41 ± 1.83 9.47 ± 1.12

Data are presented as mean ± SD. **p < 0.01 vs rHDL/siRNA-PTX.

Figure 2. Changes in diameter and ZP (A) and drug leakage ratio (B) of rHDL/siRNA-PTX during 19-day storage at 4 °C. (C) Changes in diameter and ZP of rHDL/siRNA-PTX during serum stability assay. (D) Serum stability assay of rHDL/siRNA-PTX. (E) Changes in EE of PTX from rHDL/ siRNA-PTX in vitro. Data are presented as mean ± SD (n = 3).

cellular uptake and therapeutic effect.45 Furthermore, rHDL exhibited expected negative surface charge, and negative ZP guaranteed its stability in plasm, avoiding interaction with negatively charged proteins in the extracellular matrix and blood.46 Thereafter, Table 1 summarizes the characteristics of rHDL/PTX, Lipos/siRNA-PTX, and rHDL/siRNA-PTX. The high loading capacity of rHDL was demonstrated as desirable EE of siRNA (93.45%) and PTX (95.81%) and LE of PTX (9.47%). We speculate that dual drug-loaded rHDL nanoparticles possessing a promising structural control over nanoparticle size, monodispersity, and high drug loading might result from interactions between self-assembled apoA-I and lipid monolayer, subsequently leading to improving biostability and prolonging blood circulation time. The characterizations of Cy3-siRNA/FAM-siRNA and C6/DiRloaded nanoparticles were shown in Table S1 to confirm that the labeling would not affect the physicochemical property of the nanoparticles. 3.3. Stability of Nanoparticles. To verify physical stability of nanoparticles, the variations in the diameter, ZP, and drug leakage ratio of rHDL/siRNA-PTX were monitored during 19day storage at 4 °C and serum treatment for 24 h. As shown in Figure 2, diameter and ZP of rHDL/siRNA-PTX were almost stable from the beginning to the 19th day, and the size slightly decreased 4.60% compared to the original. In addition, the obtained leakage ratios of both siRNA and PTX were within 6%. The data confirmed that the structure of rHDL/siRNAPTX, where a core was composed of DOTAP/siRNA and PTX covered by a layer of phospholipids, into which amphipathic

and the desirable diameter of 92.50 nm. The surface charge of DOTAP/siRNA complexes inverted from negative to positive beyond N/P = 1.5 demonstrated that the cationic lipid has completely entangled with negative siRNA, thereby forming the positive-charged complexes; moreover, a gradual increase in the amount of DOTAP decreased particle size due to its condensability. Subsequently, N/P ratio was set at 4 for siRNA formulated nanoparticles. These results provided decisive evidence that DOTAP/siRNA complexes were successfully constructed, while the hydrophobic DOTAP domains give a favor for hydrophilic siRNA and hydrophobic PTX packaging into the lipid core according to the theory of “similarity and intermiscibility”. 3.2. Preparation and Characterizations of Nanoparticles. As illustrated in TEM image, rHDL/siRNA-PTX shared a spherical shape and compact structure, which was further confirmed as well-formed intact nanoparticles (Figure 1C). The mean diameter of rHDL/siRNA-PTX determined by DLS analyzer showed diameter around 160 nm with a desirable PDI of 0.119 (Figure 1D). Compared with Lipos/siRNA-PTX, a conspicuous increase in rHDL/siRNA-PTX diameter was contributed to the coronal anchor of apoA-I. Based on the phospholipid monolayer structure of Lipos/siRNA-PTX, apoAI was inserted to form rHDL/siRNA-PTX by its amphipathic αhelix structure embedded between phospholipid molecules with their hydrophobic faces and phospholipid acyl chains. Furthermore, nanoparticles with the size of approximately 100−170 nm were preferred to leading long blood circulation time due to avoidance of the recognition of RES for superior 3004


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Figure 3. (A) PTX cumulative release (%) of rHDL/siRNA-PTX and Lipos/siRNA-PTX in PBS of pH 7.4. (B) In vitro siRNA cumulative release (%) of rHDL/siRNA-PTX and Lipos/siRNA-PTX in PBS of pH 7.4. Data are presented as mean ± SD (n = 3).

Figure 4. (A) Quantitative analysis (fluorescence intensity) of the C6 cellular uptake was performed using flow cytometry. (B) Positive cell percentage of Cy3-siRNA was measured by flow cytometry. (C) Confocal microscopy images of intracellular uptake of rHDL/Cy3-siRNA-C6, Lipos/Cy3-siRNA-C6, and nHDL-pretreated rHDL/Cy3-siRNA-C6 (nHDL + rHDL/Cy3-siRNA-C6) at 4 h in MCF-7 cells. Images from left to right show C6 (green fluorescence), Cy3-siRNA (red fluorescence), bright images, and the merged images (C6, Cy3-siRNA, and the bright). Scale bars are 10 μm. Data are presented as mean ± SD. **p < 0.01, ***p < 0.001.

assay. It was worth noting that the stability of rHDL/siRNAPTX against serum degradation was necessary for an effective in vivo codelivery of siRNA and PTX. Results from stability profiles indicated that natural particulates inspired rHDLs that would be stable during blood circulation for further use within approximately 3 weeks. 3.4. Drug Release of Nanoparticles. To further evaluate the PTX and siRNA release from rHDL/siRNA-PTX upon the

apolipoproteins (apoA-I) are embedded, provided steric stabilization to the vesicles. During serum stability assays, there were few changes in diameter and ZP of rHDL/siRNAPTX. As illustrated in Figure 2D, naked siRNA was degraded completely after 1 h of observation, whereas siRNA packaged in rHDLs stayed intact even when they were treated with high concentration of FBS for 24 h. The changes for EE of PTX from rHDL/siRNA-PTX were negligible during serum stability 3005


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Figure 5. In vitro cytotoxicity treated with various formulations against MCF-7 cells. (A) Cytotoxicity of Lipos and rHDL at tested concentrations (5−500 μg/mL) toward MCF-7 cells after 48 h incubation. (B) Cytotoxicity of free siRNA, rHDL/siRNA, and rHDL/siVEGF with siRNA concentration of 100 or 200 nM toward MCF-7 cells after 48 h. (C) Cytotoxicity of taxol, Lipos/siVEGF-PTX, and rHDL/siVEGF-PTX with various PTX concentration of 0.1−100 μg/mL against cells after 48 h. The data are presented as the means ± SD (n = 6).

efficiency of apoptosis and gene silencing in vivo, 24 h of siRNA or PTX release study was conducted in PBS buffer to simulate in vivo biological environment. As shown in Figure 3A, sustained release of PTX from rHDL was detected rather than Lipos. Approximately 45% PTX was released from Lipos in 24 h, while a sustained release pattern of PTX (∼20%) was displayed in the group of rHDL/siRNA-PTX. It was attributed to the intact structure of rHDL, into which PTX was incorporated, and the close interactions between hydrophobic PTX and the lipid DOTAP, which decelerated PTX release remarkably. As indicated in Figure 3B, no burst release of siRNA from each group was recorded in simulated physiological circumstance, nearly 13% of siRNA was released from the Lipos group within 24 h; meanwhile, the resemblant release tendency of siRNA (∼9%) was observed from the rHDL/ siRNA-PTX. It likely accounted for the relatively high cationic charge density of DOTAP, resulting in tightly electrostatic association with negative siRNA and hindering disassociation of it from nanoparticles. Overall, the sustained-release pattern of PTX and siRNA from rHDLs when circulated in bloodstream would bring more PTX and siRNA accumulation in tumors for efficient combination antitumor therapy. 3.5. Cell Uptake and Intracellular Trafficking. The employment of Cy3 labeled siRNA (Cy3-siRNA) and hydrophobic fluorescence probe (C6) offers an effective methodology to illuminate the intracellular codelivery and localization of siRNA and PTX mediated via rHDL-based nanosystems. To examine whether siRNA and PTX transport by rHDL was indeed SR-BI-specific, flow cytometer was applied to assessing the percentage of fluorescence intensity of C6 or Cy3-siRNA positive cells in SR-BI high-expressing MCF-7 cells (SR-BI+) and low-expressing HT1080 cells (SR-BI−). MCF-7 cells that were incubated by rHDL/Cy3-siRNA-C6 exhibited the strongest intracellular fluorescence intensity, supported by the mean fluorescence intensity of C6 at 900 from flow cytometry (Figure 4A), and took up nearly three times more than that in HT1080 cells. Moreover, rHDL gave almost two times more fluorescence intensity of C6 than Lipos in MCF-7 cells. The results confirmed that the uptake of C6 was favored by SR-BI overexpression. Moreover, positive cell percentages of Cy3siRNA by MCF-7 and HT1080 cells were consistent with the intracellular uptake of C6 in each group through FACS measurements (Figure 4B). rHDL facilitated more than 60% of MCF-7 cells in cellular uptake of Cy3-siRNA, which was superior to that of 3% observed in HT1080 cells under the same condition of incubation. To verify the targetability of apoA-I in facilitating cellular internalization of drugs via the natural routing of HDL, cells were pretreated with excessive

native HDL (nHDL) prior to the incubation of rHDL/Cy3siRNA-C6. As a competition-blocking study, we learned from FACS analysis that the percentages of fluorescence intensity of C6 and Cy3-siRNA positive cell were significantly reduced, while SR-BI receptors were competitively enclosed by nHDL. Therefore, rHDL inspired tumor specific targeting appears to occupy a leading position in the cellular internalization enhancement. To validate the cytosolic-specific delivery of Cy3-siRNA and C6 from rHDL into SR-BI positive cells, MCF-7 cells were separately incubated with formulations of Lipos/Cy3-siRNAC6, rHDL/Cy3-siRNA-C6, and rHDL/Cy3-siRNA-C6 (pretreatment with nHDL), and the subcellular distribution of fluorescent probes was detected by using confocal laser scanning microscope (CLSM). Compared to Lipos/Cy3siRNA-C6 group, an obvious intracellular fluorescence intensity of Cy3-siRNA and C6 were visibly stronger via incubation of rHDL/Cy3-siRNA-C6 in MCF-7 cells (Figure S1), further confirming rHDL-mediated SR-BI affinity for drug cellular internalization. Taking into account the broad cytosolic distribution of Cy3-siRNA and C6, we hypothesized that the HDL-CE-like transport process, i.e., nonendocytosis cellular internalization mechanism, accounted for the direct cytosolic delivery of cargos via rHDL into SR-BI high-expressing cancer cells.23 As illustrated in Figure 4C, rHDL group mediated a considerable cytoplasmic distribution of Cy3-siRNA and C6, judging from separate location of red and green fluorescence. In sharp contrast, Lipos/Cy3-siRNA-C6 was primarily accumulated within endo/lysosomes according to fluorescence colocalization. Hence, the data supported the hypothesis that cargoes in rHDLs transported into cytoplasm were possibly caused by SR-BI-mediated direct transmembrane delivery. Interestingly, when the excessive nHDL was preincubated, the intracellular distribution of fluorescence was similar to that of the Lipos/Cy3-siRNA-C6 group. The results indicated that SR-BI receptors on the cell surface were blocked, which probably inhibited the endocytosis-independent process for cellular uptake of Cy3-siRNA and C6 due to the affinity of excessive nHDL with SR-BI receptors. According to the results above, it suggested that rHDL facilitated efficient cellular uptake of hydrophobic contents, which is due to the interactions between apoA-I anchor and the receptor SR-BI on MCF-7 cells. 3.6. Cytotoxicity Assay. The cytotoxicity of Lipos and rHDL toward MCF-7 cells was evaluated by MTT assay. Cells incubated with Lipos and rHDL at various concentrations ranging from 5 to 500 μg/mL yielded negligible toxicities toward cells within tested concentrations for 48 h, demonstrat3006


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Molecular Pharmaceutics Table 2. Inhibitory Concentration (IC50, 48 h) in MCF-7 Cellsa

a

samples

taxol

rHDL/PTX

Lipos/siVEGF-PTX

rHDL/siVEGF-PTX

IC50 (μg/mL)

4.15 ± 1.12

0.31 ± 1.39

6.69 ± 1.73

0.26 ± 0.11***

Data are represented as mean ± SD. ***p < 0.001 compared to other groups.

Figure 6. (A) In vivo imaging of tumor-bearing mice after administration of Lipos/FAM-siRNA-DiR and rHDL/FAM-siRNA-DiR at 6 and 24 h under DiR channel (Ex = 720 nm, Em = 790 nm). The tumor site is marked with a red circle. (B) Ex vivo fluorescence images of tissues and tumors collected at 24 h post-injection of rHDL/FAM-siRNA-DiR under FAM channel (Ex = 470 nm, Em = 530 nm) and DiR channel mentioned above, respectively. (C) Quantification of ex vivo tumors and other main organs in mean fluorescence intensity 24 h post-injection (n = 3), respectively.

demonstrated in cells incubated with rHDL/siVEGF, compared to those of Lipos/siVEGF, confirming that apoA-I incubation could greatly enhance the gene silencing effect on rHDL. From the above results, it was suggested that a SR-BI receptormediated targeting delivery system would assist in enhancing intracellular uptake of siRNA, thus promoting its gene silencing efficacy. Consequently, rHDL could make an advantage of direct transmembrane pathway for accumulation of siRNA and PTX in cytoplasm, where the targets (e.g., siRNA for mRNAs and PTX for microtubules) are located for taking effect. 3.7. In Vivo Distribution Analysis. To estimate the targetability of rHDL in vivo and effective drug codelivery to tumors, the biodistributions of FAM-labeled siRNA (FAMsiRNA) and DiR coloaded rHDL and Lipos (rHDL/FAMsiRNA-DiR and Lipos/FAM-siRNA-DiR) were injected into the MCF-7 tumor-implanted nude mice via tail vein and then monitored by the near-infrared optical imagining technique. Real-time images of nanoparticles in the living tumor-bearing mice were only conducted at 6 and 24 h against DiR fluorescence intensity (Figure 6A) due to the massive background fluorescence (Ex = 470 nm for fluorescein FAM). Six hours post-administration, saturated levels of fluorescence displayed in the tumor, liver, and kidney of the rHDL group, indicating that accumulation of rHDL particulates was entirely consistent with the distribution and high

ing that rHDL was a nontoxic and biocompatible vehicle for clinical use of drug delivery (Figure 5A). Cells treated with various siRNA formulations demonstrated that neither nontargeted control siRNA involved samples nor rHDL/siVEGF showed any cytotoxicity at siRNA concentration of 100 or 200 nM (Figure 5B). The cytotoxicity of rHDL/PTX, Lipos/ siVEGF-PTX, and rHDL/siVEGF-PTX at different PTX concentrations was also conducted after cell incubation for 48 h, while Taxol was taken as control. As shown in Figure 5C, compared to Lipos/siVEGF-PTX and Taxol groups, an obvious inhibitory effect on rHDL/siVEGF-PTX treated cells were observed. Specifically, rHDL/siVEGF-PTX applied group demonstrated significantly lower half maximal inhibitory concentration (IC50) of 0.26 μg/mL (PTX concentration), which presents a 14.96-fold increase in cytotoxicity when taking Taxol as comparison (Table 2). More importantly, IC50 of rHDL/siVEGF-PTX showed 24.73-fold increase compared to 6.69 μg/mL of Lipos/siVEGF-PTX without apoA-I incubation, indicating that rHDLs promote the efficient intracellular delivery for enhancing cell antiproliferation. Therefore, the antiproliferation effect was mainly caused by PTX rather than siVEGF. Such targeted and bioinspired particulates inevitably possessed the capacity management of efficient antitumor efficacy through the receptor-mediated pathway. Moreover, as shown in Figure S2, a higher VEGF downregulation effect was 3007


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Figure 7. In vivo antitumor efficacy and pathological data in nude mice bearing MCF-7 tumors. (A) Xenograft MCF-7 tumor growth curves (n = 12). (B) Changes of tumor weight (n = 6). (C) Survival rates (n = 6). (D) Body weight variation curves (n = 12). (E) Histological images of tumor tissues. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

3.8. In Vivo Antitumor Efficacy. To verify the feasible application of rHDL/siVEGF-PTX on the inhibition of tumor growth and neovasculature in vivo, antitumor activity was also evaluated among groups of saline, taxol, rHDL/PTX, Lipos/ siVEGF-PTX, and rHDL/siVEGF-PTX using MCF-7 tumorbearing xenograft models. The successive intravenous injection of rHDL/siVEGF-PTX was, therefore, shown to work efficiently, resulting from the combination of tumor specific targetability, gene silencing, and cell apoptosis (Figure 7A). TWI was calculated based on the weight of excised tumors (Figure 7B). rHDL/siVEGF-PTX generated the noticeable antitumor efficacy (TWI of 76.74%), which was 1.53-, 1.38-, and 1.10-fold higher than that of taxol (30.23%), rHDL/PTX (32.21%), and Lipos/siVEGF-PTX (36.51%), further confirming the synergistic effect on active-targeting with collaborative antitumor outcomes of siVEGF and PTX. To further estimate the antitumor efficacy, the overall survival period and the body weight of the tumor-bearing nude mice were evaluated. As presented in Figure 7C, rHDL/siVEGF-PTX displayed a dominant effect on extending the survival period of tumorbearing nude mice. Moreover, there was no significant change of body weight in the mice except the saline group, and slight weight gain in some groups may result from the tumor growth (Figure 7D), whereas mice receiving taxol showed remarkable body weight loss, of which Cremophor EL and ethanol utilized would induce serious and dose-dependent toxicity. The results substantiated that rHDL/siVEGF-PTX produced the prominent capability of suppressing the tumor growth, owing to the

expression level of SR-BI receptors, in agreement with what had been observed in liposomes and nHDLs.28,29 This biodistribution pattern was responsible for the receptor-mediated targeting of siVEGF and PTX directed by rHDLs. However, when time passed by, the accumulation of fluorescence in the rHDL group was realized in tumor rather than liver or other vital tissues 24 h post-injection. On the contrary, images of Lipos exhibited a major fluorescence distribution in liver rather than tumor tissues 24 h post-injection, demonstrating a high hepatic uptake and a rapid clearance of Lipos. These results provide decisive evidence that rHDL is available for the receptor-mediated targeting of drugs.29,30 Moreover, the animals were sacrificed at 24 h, and main organs of mice treated by rHDL/FAM-siRNA-DiR were collected for ex vivo imaging to further verify in vivo biodistribution. The fluorescence intensity of FAM and DiR was detectable through their individual channels. In the quantitative analyses, an effective fluorescence accumulation of FAM (∼1500/mm2) and DiR (∼4150/mm2) in the tumors was observed and is shown in Figure 6B,C, which also demonstrates that rHDL could be a highly efficient siRNA and small-molecules codelivery vehicle, and rHDL mediated dual-drug nanosystem remained intact during blood circulation for collaborative tumor-targeted therapy. Considering these results above, we could speculate that the tumor-targeted accumulation of rHDL mainly ascribed to its improved biostability, prolonged circulation, and natural affinity with SR-BI receptors overexpressed on the tumor surface for further antitumor efficacy. 3008


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Figure 8. (A) Representative immunohistochemical staining of MCF-7 tumor bearing nude mice. Blood vessels in microphotographs are marked by a red-brownish color (CD31-antibody). (B) Tumor was excised, sectioned, and stained with CD31-antibody. Intratumoral microvessel density in the tumor tissue was quantified as the number of microvessels/mm2. Results were expressed as mean ± SD. **p < 0.01, ***p < 0.001, vs. rHDL/ siVEGF-PTX. (C) Representative immunoblots of VEGF protein expression in MCF-7 cells in vivo after intravenous administration assessed by Western blot. Lane 1, control; Lane 2, Lipos/siVEGF-PTX; Lane 3, rHDL/PTX; Lane 4, rHDL/siVEGF-PTX (n = 3).

ideal site-specific tumor-targeting biofunction and effective cellular internalization within tumors. H&E staining was further used to analyze the histological tumors feature caused by various nanoparticles. As illustrated in Figure 7E, the pattern of cell apoptosis in tumor tissues showed a considerable evidence of in vivo antitumor activity of rHDL/siVEGF-PTX, which confirmed the results in the study of tumor growth inhibition. All therapeutic groups exhibited different signs of tumor growth inhibition, while the rHDL/siVEGF-PTX treatment obtained the highest level of cell toxicity and the maximum tumor necrosis. Inspiringly, the rHDL/PTX group achieved less tumor growth inhibition efficacy than that of rHDL/siVEGF-PTX, which resulted from a synergetic effect by codelivery of siVEGF and PTX. The internalized siVEGF could selectively and successfully down-regulate the expression of VEGF protein via RNAi and thereby induce the inhibition of neovasculature. The antiangiogenic therapy via RNAi could normalize abnormal tumors blood vessels by making them less leaky, leading to spatially and temporally increased blood perfusion in tumors with improving chemotherapy agents delivery efficiency.47,48 Overall, rHDL/siVEGF-PTX exhibited the maximum inhibition on tumor growth and prolonged survival period, owing to the specific-targeted natural rHDL particulates, drug accumulation in tumor tissues, and the collaborative antitumor efficacy of antiangiogenesis and cell antiproliferation in vivo. 3.9. In Vivo Tumor Angiogenesis Suppression. Angiogenesis is the process by which new vessels are formed within the region of growing tumors so as to improve the supply of nutrients for cells. In order to disrupt the angiogenesis signal network, we chose siRNA for sequence-specific silencing of VEGF, the important angiogenic factor associated with tumor growth of solid tumors, especially in the early stage of breast cancer. Since the intratumoral VEGF content was relevant to neovascularization, inhibition of VEGF expression would induce tumor antiangiogenesis, which resulted in the reduced number of intratumoral microvessels. Taking these into consideration, the efficiency of VEGF gene silencing, including Lipos/siVEGF-PTX, rHDL/PTX, and rHDL/siVEGF-PTX, was analyzed by intratumoral microvessels and Western blot analysis. Correlating to the antitumor effects of treated groups, the formation of microvessels was inhibited by intravenous administration of rHDL/siVEGF-PTX remarkably. For immu-

nostaining of tumor vasculature distribution in tumor sections, CD31-positive tumor vessels were abundant inside the tumor in the control group, while those of the rHDL/siVEGF-PTX group were significantly reduced, shown in Figure 8A. According to quantitative analysis on microvessel density (MVD), the rHDL/siVEGF-PTX group exhibited the highest inhibition of microvessel formation of 79.31%. Inasmuch, the MVD of tumors reduced to ∼36/mm2 in rHDL/siVEGF-PTX treated mice, which was much lower than that of other groups (rHDL/PTX of ∼110/mm2; Lipos/siVEGF-PTX of ∼100/ mm2) (Figure 8B). This result was consistent with in vivo collaborative antitumor efficacy, persuasively implying that tumor angiogenesis suppression was one of the reasons for tumor growth inhibition in MCF-7 xenograft models treated with rHDL/siVEGF-PTX. The inhibition of intratumoral neovascularization was particularly due to the improved biostability, efficient cellular uptake, and direct cytosolic delivery of siVEGF mediated by rHDL, indicating siVEGF could play a role as an important RNAi strategy for tumor antiangiogenesis. As shown in the data obtained from Figure 8C, the relative expression level of VEGF protein in rHDL/ siVEGF-PTX treated tumors was markedly decreased, whereas that of the Lipos/siVEGF-PTX and rHDL/PTX groups had no significant change in comparison with the control group, confirming that cellular internalized siVEGF via rHDL could down-regulate VEGF protein expression, accounting for RNAimediated degradation of VEGF mRNA. Superior tumor targeting ability of rHDL/siVEGF-PTX in MCF-7 tumor tissues could greatly enhance the gene silencing effect. Meanwhile, we noticed that Lipos/siVEGF-PTX had a relatively lower gene silencing efficiency, similar to the cellular uptake and in vivo distribution assay. From the results above, it was concluded that the bioinspired nanoparticulate would assist in improving cellular uptake of siRNA and chemotherapy agents (PTX), which might promote gene silencing and cell apoptosis activities, thereby ultimately restraining tumorassociated angiogenesis proliferation. 3.10. In Vivo Safety Evaluation. In clinical cancer therapy, chemotherapy would cause severe side effects, thus compromising the therapeutic efficacy. The purpose of combined therapy of siRNA and PTX is to reduce the side effects.49 To assess whether rHDL would cause any adverse effect during 3009


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Figure 9. (A) Hematology data of healthy BALB/c nude mice (RBC, WBC, and PLT) treated with different formulations, and evaluation of liver functions biomarkers (ALT, ALP) and renal functions biomarkers (BUN, CRE). (B) H&E histopathological sections of vital tissues. Data are presented as mean ± SD.

weight loss, as the result of inherent toxicity of Cremophor EL in formulations and the unselective distribution to normal tissues. At the test dose for in vivo administration, rHDL/ siVEGF-PTX was relatively safe based on the results of hematologic indicators and histopathological evaluation. Finally, we have rationally developed biomimetic rHDLs with the facile cationic−lipid condense strategy to inspire efficient codelivery of therapeutic companies (siVEGF and PTX), thus making it a useful tool for collaborative antitumor therapy without potential adverse effects.

treatment, safety assessment including biochemical effects and histochemistry was performed, while saline and taxol were served as control groups, respectively. When compared with the saline group, the rHDL/siVEGF-PTX group showed no measurable adverse effect on blood cells as well as liver and renal functions throughout the profiles (Figure 9A). The peripheral blood cells including red blood cells (RBC), white blood cells (WBC), and platelets (PLT) were all within the normal range, suggesting no syndrome including hemolytic anemia or acute infection after rHDL/siVEGF-PTX application. In blood chemistry analysis, the liver function biomarkers, e.g., alanine aminotransferase (ALT), alkaline phosphatase (ALP), and renal function biomarkers, e.g., urea nitrogen (BUN) as well as creatinine (CRE), were all measured to be normal, indicating that negligible hepatotoxicity and nephrotoxicity of mice were observed after rHDL/siVEGF-PTX treatment. In comparison, taxol-induced acute inflammation in liver was characterized by an ALT increase. As shown in Figure 9B, normal tissues were also harvested for histopathological assessment. No pathological abnormalities were observed in mice treated with rHDL/siVEGF-PTX and saline. Likewise, no apparent signs of dehydration, anorexia, and other symptoms were recorded during the treatment period after rHDL/ siVEGF-PTX injection; and meanwhile, no behavioral abnormalities were observed when compared with saline group. However, the safety evaluation of Taxol demonstrated

4. CONCLUSION Our research provides new insights into an SR-BI targeting biomimic rHDL/siVEGF-PTX system construction for collaborative antitumor efficacy. Coencapsulation of hydrophilic biomolecule (siVEGF) and small hydrophobic molecule (PTX) was simultaneously cellular internalized via nonendocytosic pathways based on rHDL nanoplatform, of which apoA-I was served as the targeting ligand for tumorhoming and penetration. On the premise of spatiotemporal codelivery fashion, such combined therapeutic pattern with antiangiogenesis and apoptosis achieved promising antitumor efficacy in the MCF-7 tumor-bearing nude mice model, including significant tumor growth inhibition, extended survival rates, and decreased MVD and VEGF protein expression level. Collectively, this bioinspired nanosystem holds great promise 3010


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for siRNA and PTX codelivery and collaborative antitumor therapy, at a safe level.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00192. Characteristics of labeled nanoparticles, cell uptake of nanoparticles, and in vitro VEGF gene silencing (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel: +86 25 83271102. Fax: +86 25 83271102. ORCID

Ruoning Wang: 0000-0001-7466-9602 Yang Ding: 0000-0003-2894-7303 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the National Science and Technology Major Project (2014ZX09507001005), National Key Research and Development Program (2017YFD0501403), the National Natural Science Foundation of China (No. 81273469, 81501582, and 81573379) and the Natural Science Foundation of Jiangsu Province (No. BK20171390). The research was also supported by Graduate Cultivation Innovative Project of Jiangsu Province (No. KYLX16_1179), Development Funds for Priority Academic Programs in Jiangsu Higher Education Institutions, and Fostering Plan of University Scientific and Technology Innovation Team of Jiangsu Qing Lan Project (2014).

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REFERENCES

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