Natural Particulates Inspired Specific-Targeted ... - ACS Publications

Subscriber access provided by UNIVERSITY OF THE SUNSHINE COAST

Article

Natural particulates inspired specific-targeted co-delivery 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 Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00192 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on July 29, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Natural particulates inspired specific-targeted co-delivery of siRNA and paclitaxel for collaborative antitumor therapy Ruoning Wang, Ziqiang Zhao, Yue Han, Shihao Hu, Yaw Opoku-Damoah, Jianping Zhou,* Lifang Yin, Yang Ding* State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 53

ABSTRACT: The effective combination of drugs promoting anti-angiogenesis and apoptosis effects has proven to be a promising collaborative tumor antidote; and the co-delivery of small interfering RNA (siRNA) and chemotherapy agents within one efficient vehicle has gained more attentions over single regimen administration. Herein, vascular endothelial growth factor specific siRNA (siVEGF) and paclitaxel (PTX) were introduced as therapeutic companions and co-encapsulated into naturally mimic high-density 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 non-endocytosis 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 towards 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 comparision. Moreover, in vivo results further demonstrated that rHDL/siVEGF-PTX performed enhanced tumor growth inhibition via natural targeting pathway, accompanied with 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; Co-delivery of siRNA and paclitaxel;

Vascular

endothelial

growth

factor;

Direct

Collaborative antitumor efficacy


cytosolic

delivery;

Page 3 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 companied 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 sub-optimal, 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 RNA interference (RNAi) technology has displayed great potential for anticancer therapy due to its unique sequence-specific 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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 53

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 homogenous with less leaky. Additionally, interstitial fluid pressure is reduced which restores a trans-vascular fluid pressure difference, resulting in improved blood flow and anti-cancer agents 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 mimick endogenous structure of native high density lipoprotein (nHDL), its recombinant form (rHDL) possess a hydrophobic core


Page 5 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 non-endocytotic mechanism after apoA-I bound, thereby bypassing 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.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Wherein,

cationic

lipids

N-trimethylammonium

including chloride

N-[1-(2,

Page 6 of 53

3-dioleyloxy)

(DOTMA),34

2,

propyl

]-N,

N,

3-dioleyloxy-N-[2

(sperminecarboxamido) ethyl]-N, N-dimethyl-1-propanammonium trifl uoroacetate (DOSPA),35 and (2, 3-Dioleoyloxy-propyl)- trimethylammoniumchloride (DOTAP),36 seem to be great potentials in effective gene silencing therapy of this application. Particularly, 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 abovementioned design inspiration, a new paradigm, rHDL-mediated specific-targeted co-delivery 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


Page 7 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


attach to SR-BI receptor over-expressing malignant tumors, followed by cell membrane re-organization, formation of non-aqueous “channel” and directly deliver 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.

Scheme 1. Construction of naturally inspired nanosystem rHDL/siVEGF-PTX. After i.v. administration, rHDL/siVEGF-PTX was recognized by SR-BI, followed by membrane re-organization, formation of non-aqueous “channel” and subsequent cross-membrane deliver siVEGF and PTX to cells to pursue a strategic combination therapy.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2. EXPERIMENTAL SECTION 2.1. Materials. (2, 3-Dioleoyloxy-propyl)-trimethylammoniumchloride (DOTAP) was obtained from CordenPharma (Liestal, Switzerland). Non-targeted 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 by-product 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.

2.2. Preparation of DOTAP/siRNA nanocomplexes. siRNA dissolved in DEPC-treated water (0.5 mg/mL), and DOTAP dissolved in ethanol (10 mg/mL).


Page 8 of 53

Page 9 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 mins. 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 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) dissolved in 200 µL 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 drop-wise 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), collected. PTX-loaded rHDL (rHDL/PTX) was obtained in the same manner without DOTAP/siRNA involvement. siRNA and PTX co-loaded liposomes (Lipos/siRNA-PTX) were prepared in the same manner without apoA-I incubation. Cy3-siRNA/FAM-siRNA and C6/DiR-loaded nanoparticles were also obtained according to the same procedures for targeting



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

efficacy assay in vitro and in vivo. For control, Taxol was prepared through dissolving 12 mg PTX in 2.0 mL 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. 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 High Performance Liquid Chromatography (HPLC, Shimadzu LC-2010 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 formula: EE% = (weight of PTX in nanoparticles)/(weight of PTX fed initially) ×100%; LE% = (weight of PTX in nanoparticles)/(weight of PTX-Loaded nanoparticles)


Page 10 of 53

Page 11 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


×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. 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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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. 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 PTX were placed in a clamped dialysis bag (MWCO 3,500 Da), and immersed in 150 mL 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 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 (SR-BI-) were used for cellular uptake comparision.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


Page 12 of 53

Page 13 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-siRNA-C6 and rHDL/Cy3-siRNA-C6, were prepared for cytosolic localization and targeting efficacy assay in vitro, of which the 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/Cy3-siRNA-C6 or rHDL/Cy3-siRNA-C6 was substituted and incubated for 4 h. The fluorescence intensity of C6 and Cy3-siRNA 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/Cy3-siRNA-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 pre-treated with free native HDL (nHDL, 5 mg/mL) for 2 h before substituting for corresponding payloads of rHDL/Cy3-siRNA-C6. 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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(5×103 cells/well). On achieving 70-80% confluence, cells were treated with blank nanoparticles (Lipos and rHDL) at concentration of 5-500 µg/mL, siRNA-loaded formulations (free siRNA, rHDL/siRNA, rHDL/siVEGF) with 100 nM 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 adding 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 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


Page 14 of 53

Page 15 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


siRNA

and

DiR

co-loaded-Lipos

(Lipos/FAM-siRNA-DiR)

and

rHDL

(rHDL/FAM-siRNA-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 MaestroTM EX fluorescence imaging system (Cambridge Research & 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 equipped at Ex=720 nm and Em=790 nm for DiR detection. The mice were sacrificed and vital organs of rHDL group were excised to compare the relative accumulation for both FAM-siRNA and DiR after 24 h injection. All the images were analyzed using Living Image Software. BALB/c nude mice bearing MCF-7 tumors were established as described above, and assessed the antitumor efficacy 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/siVEGF-PTX 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 of nude mice in each group were sacrificed 3 days post final injection, and tumor tissues were harvested for haematoxylin 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 represents the control group and treated groups of mean tumor weight. The survival rates were



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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, 3 mice in each group were sacrificed to obtain tumors, fixed in a 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 average quantity of microvessels (MVD, the number of CD31-positive vessel/mm2) in each field was measured as number of CD31-positive objects identified, and four fields per section were calculated. To determine VEGF amount in solid tumor region of each group, tumors were excised after final treatment. Proteins of tumor tissues were isolated and quantified by the BCA protein assay. The samples were separated with SDS-polyacrylamide 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 anti-rabbit 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 carried out to


Page 16 of 53

Page 17 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 whole procedures. The animals were sacrificed 24 h post last dosing, and blood samples were subjected to hematological and biochemistry analysis; main organs were excised for H&E analysis.

2.13. Statistical analysis. All data were expressed as mean ± S.D., 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.

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 and 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 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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

demonstrated that the cationic lipid has completely entangled with negative siRNA, thereby forming the positive-charged complexes; moreover, gradual increase 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”.

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 ± S.D. (n=3).

3.2. Preparation and characterizations of nanoparticles. As illustrated in TEM


Page 18 of 53

Page 19 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 conspicuously 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, apoA-I was inserted to form rHDL/siRNA-PTX by its amphipathic α-helix 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 cellular uptake and therapeutic effect.45 Furthermore, rHDL exhibited expected negative surface charge, and negative ZP guaranteed its stability in plasm, avoiding to interact with negatively charged proteins in the extracellular matrix and blood.46 Thereafter, Table 1 summarizes the characteristics of rHDL/PTX, Lipos/siRNA-PTX, 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/DiR-loaded nanoparticles were



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 53

shown in Table S1 to confirm that the labeling wouldn’t affect the physicochemical property of the nanoparticles.

Table

1.

Formulation

characteristics

of

rHDL/PTX,

Lipos/siRNA-PTX

and

rHDL/siRNA-PTX (Data are presented as mean ± S.D., **p

Natural Particulates Inspired Specific-Targeted ... - ACS Publications

Recommend Documents