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Polycation liposomes combined with calcium phosphate nanoparticles as a non-viral carrier for siRNA delivery
Journal of Drug Delivery Science and Technology 30 (2015) 1e6
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Research paper
Polycation liposomes combined with calcium phosphate nanoparticles as a non-viral carrier for siRNA delivery Jianjun Zhang a, b, Xiaoyi Sun c, Rong Shao d, Wenquan Liang a, Jianqing Gao a, **, Jinliang Chen d, * a
Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China Department of Pharmaceutics, Zhejiang Hospital of TCM, Hangzhou, Zhejiang, China Department of Pharmacy, Zhejiang University City College, Hangzhou, Zhejiang, China d Center of Clinical Pharmacology, 2nd Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, Zhejiang, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 June 2015 Received in revised form 7 September 2015 Accepted 9 September 2015 Available online 14 September 2015
Low transfection efficiency and instability are the main barriers for siRNA delivery. To overcome these barriers, a core-membrane siRNA delivery system was designed. Calcium phosphate/siRNA nanoparticles were combined with liposomes, and the surface was modified with polyethylenimine-cholesterol (PEIChol). The mean particle size of the polycation liposomes/calcium phosphate/siRNA complexes (PLCP) was approximately 260.4 nm and the zeta potential was 0.3 ± 0.2 mV. The buffer capacity and cellular uptake of PLCP were studied. The results indicated that PEI-Chol and CaP can synergistically improve endosomal escape by swelling and disrupting the endosome. Anti-green fluorescent protein (GFP) siRNA was used to evaluate the gene silencing effect in MCF-7 cells that stably express GFP. PLCP could down regulate approximately 70% of GFP expression in 24 h, which is significantly higher than that of Lipofectamine™ 2000. Additionally, the silencing effect lasted for at least 72 h. This delivery system can improve intracellular uptake and enhance gene silencing, with low cytotoxicity. © 2015 Elsevier B.V. All rights reserved.
Keywords: Calcium phosphate nanoparticles Polycation liposomes siRNA Green fluorescent protein Gene silencing
1. Introduction The development of RNA interference (RNAi) technology has experienced much excitement for more than ten years, providing new methods for potential treatment of genetic related diseases [1e4]. Small interfering RNA (siRNA) incorporates into the RNAiinduced silencing complex (RISC) and allows for the cleavage of its complementary target mRNA in the cytoplasm to reduce related protein expression. siRNA in practical applications, however, have been limited by poor intracellular uptake and instability in the physiological environment [5]. The development of safe and effective delivery systems for siRNA would greatly enhance the gene-silencing ability.
* Corresponding author. Center of Clinical Pharmacology, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Jiefang Road 88, Hangzhou, Zhejiang, 310009, China. ** Corresponding author. Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Yuhangtang Road 388, Hangzhou, Zhejiang, 310058, China. E-mail addresses: [email protected] (J. Gao), [email protected] (J. Chen). http://dx.doi.org/10.1016/j.jddst.2015.09.005 1773-2247/© 2015 Elsevier B.V. All rights reserved.
Calcium phosphate (CaP) has been widely used as a non-viral gene delivery system since the 1970s, because it is considered to be biodegradable and biocompatible [6]. CaP nanoparticles can bind negatively charged nucleic acids by calcium ion chelation to deliver nucleic acids into cells and protect from enzymatic degradation [7]. One of the major limitations for CaP nanoparticles, however, is the uncontrollable rapid growth of CaP crystals after preparation, which can lead to large aggregation and significantly reduce stability and the transfection efficiency [8]. Many approaches have been reported to stabilize CaP precipitates. Some strategies improved the manufacturing process by adding different stabilizers, such as increasing concentrations of magnesium ions to slow down aggregation [9] or adding bisphosphonate to enhance calcium ion chelation [10]. Others have attempted to encapsulate CaP nanoparticles with lipids [11], hyaluronic acid [12] or chitosan [13]. In previous work, we have successfully developed polycation liposomes (PCL) and polycation liposomes/protamine/DNA complexes (PLPD) as gene transfection vectors to overcome intracellular barriers and achieve high transfection efficiency [14e17]. Thus, in this study, we developed a siRNA delivery system using CaP/siRNA
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nanoparticles combined with PCL, which we termed polycation liposomes/calcium phosphate/siRNA complexes (PLCP). Pluronic F68 and sodium citrate were used to stabilize CaP nanoparticles, and PCL combining reduced their aggregation. The particle sizes, zeta potential, cytotoxicity and cellular internalization ability of PLCP were investigated. The gene silencing effect was assessed in MCF-7 cells stably expressing GFP using anti-GFP siRNA mediated by PLCP. 2. Materials and methods 2.1. Materials Pluronic F68, polyethylenimine (PEI, MW of 800 Da and 25 kDa), cholesteryl chloroformate and 1,2-dioleoyl-sn-glycero-3-phosphoe thanolamine (DOPE) were purchased from SigmaeAldrich (St. Louis, MO, USA). Polyethylenimine-cholesterol (PEI 800-Chol) was synthesized as previously reported [14]. Thiazolyl blue tetrazolium bromide (MTT) was purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Dulbecco's modified eagle's medium (DMEM), trypsin, fetal bovine serum (FBS) and Lipofectamine™ 2000 were purchased from Life Technologies (Carlsbad, CA, USA). Anti-green fluorescent protein (GFP) siRNA (sense, 50 -GCCACAACGUCUAUAUCAUGG-30 ; antisense, 50 AUGAUAUAGACGUUGUGGCUG-30 ), random siRNA (sense, 50 UCCUCCGAACGUGUCACGUTT-30 ; antisense, 50 -ACGUGACACGUUC GGAGAATT-30 ) and FAM-labeled random siRNA were synthesized, modified and purified by 100 Biotech Company (Hangzhou, Zhejiang, China). All other chemical reagents were analytical grade from Sinopharm Chemical Reagent Company (Shanghai, China). 2.2. Cell lines Human breast adenocarcinoma cell lines (MCF-7) were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences (Shanghai, China). MCF-7 cells stably expressing GFP were established by 100 Biotech Company (Hangzhou, Zhejiang, China). Cells were cultured as a monolayer in DMEM containing 10% FBS at 37 C in 5% CO2. Cells were regularly passaged and reseeded 24 h before transfection experiments. 2.3. Preparation and characterization of CaP/siRNA nanoparticles, PCL and PLCP CaP/siRNA nanoparticles were prepared by a modified coprecipitation method. Briefly, 30 mL of 75 mM CaCl2, 70 mL of 1% Pluronic F68 and 100 mL of 0.1 mg/mL siRNA solution were mixed and incubated for 10 min at room temperature as calcium moieties. The phosphate moieties were prepared as follows: firstly mixing 120 mL of 6 mM Na2HPO4 and 40 mL of 1% Pluronic F68, and then mixing with 40 mL of 24 mM sodium citrate. CaP/siRNA nanoparticles were prepared by adding the phosphate moieties dropwisely to the calcium moieties, and incubating for another 10 min. PCL were prepared by film dispersion method with a lipid mixture of PEI 800-Chol and DOPE (molar ratio 1:1) as previously descripted [17]. PLCP complexes were prepared by mixing an equal volume of PCL and CaP/siRNA nanoparticles at various N/P ratios (1 mol PEI 800-Chol nitrogen per mol siRNA phosphate), and then incubating for 30 min at room temperature. Particle size and zeta-potential (z) of the vectors were determined by laser diffraction spectrometry (Malvern Zetasizer 3000HS, UK), and the morphologies of the complexes were observed using transmission electron microscopy (TEM, JEM1200EX, Japan).
2.4. Determination of buffer capacity The buffer capacity of vectors was determined by acidebase titration. Thirty milliliters of PEI 25 K solution, PCL and PLCP were prepared. The concentrations of PEI 25 or PEI-Chol in PCL and PLCP were 0.3 mg/mL. The pH value of every group was raised to 10 using 1 M NaOH, and then titrated with 0.1 M HCl. The volumes of HCl consumed and pH values were recorded. The 0.1 M NaCl solution was used as a negative control. 2.5. Cytotoxicity assay Cellular viability was determined using MTT assay. MCF-7 cells were plated in 96-well plates at a density of 1 104 cells/well and incubated for 24 h at 37 C in CO2 incubator. The cells were washed with PBS and exposed to 20 mL PLCP or PEI 25 K/siRNA (0.2 mg siRNA/well) with various N/P ratios (N/P ¼ 1, 2.5, 5, 10, 20, 30, 40 and 50) in serum-free medium. Serum-free medium was added at an equal volume as a control group. After transfection for another 24 h, the culture medium was replaced with the fresh DMEM medium, and then 20 mL of MTT (5 mg/mL) was added to each well and incubated for 4 h at 37 C. The reaction was stopped by lysing the cells with 150 mL of DMSO for 10 min. The absorbance was measured at 570 nm in microplate reader (Bio-Tek, ELx800, USA). 2.6. Cellular uptake assay MCF-7 cells were seeded at 1 105 cells/well in a 24-well plate and incubated overnight at 37 C with 5% CO2. Lipofectamine™ 2000/siRNA complexes, CaP/siRNA nanoparticles and PLCP/siRNA complexes were added after changing cells to serum-free DMEM (1 mg siRNA/well). After incubation for another 4 h, cells were washed with PBS and the medium was changed to DMEM containing 10% FBS. Green fluorescence was observed using an inverted fluorescent microscope (HBO 100, Zeiss, Germany). 2.7. In vitro gene silencing activity To evaluate the gene silencing efficiency of different carriers, MCF-7 cells with stable GFP expression were used. These cells were seeded with 2 mL of DMEM containing 10% FBS on 12-well plates at 1.5 104 cells/well, overnight before transfection. Upon reaching 60% confluency, anti-GFP siRNA was transfected using Lipofectamine™ 2000, CaP and PLCP into cells for 5 h, following which the medium was replaced with fresh medium. The cells were then cultured for another 24, 48 or 72 h at 37 C. The dosage of siRNA in each well was 1 mg. The fluorescence of GFP expression in MCF-7 cells was observed by inverted fluorescent microscopy, and related GFP mRNA expression was determined by RT-qPCR, using hGAPDH as an internal standard. The 2DDCT method was used and CT value was defined as the cycle number when the fluorescent signal of each reaction tube reached its set threshold [18]. 2.8. Statistical analysis Statistical analysis was performed using Student's t-test. 3. Results 3.1. Characterization of PCL, CaP/siRNA nanoparticles and PLCP The mean particle sizes of PCL and CaP/siRNA nanoparticles were approximately 129.6 nm and 148.9 nm, respectively (Table 1). The PCL composed of PEI 800-Chol and DOPE had a positive zeta potential about 39.3 mV, while the CaP/siRNA nanoparticles had
J. Zhang et al. / Journal of Drug Delivery Science and Technology 30 (2015) 1e6
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Table 1 Particle sizes and zeta potential (z) of PCL/siRNA, CaP nanoparticles/siRNA and PLCP.
PCL/siRNAa CaP nanoparticles/siRNA PLCPa a
Particle sizes (nm)
PDI
Zeta potential (z, mV)
129.6 ± 14.0 148.9 ± 21.8 260.4 ± 23.4
0.200 0.248 0.188
39.3 ± 6.2 13.6 ± 1.0 0.3 ± 0.2
The N/P ratio of the complexes was 20.
negative zeta potentials about 13.6 mV. After encapsulating CaP/ siRNA nanoparticles with PCL, the sizes of PLCP increased to approximately 260.4 nm and zeta potential reached 0.3 mV, which was nearly neutral. We also investigated the morphological characteristics of the complexes using TEM. For PCL, spherical particles with an inner phase structure were observed (Fig. 1A). CaP/siRNA nanoparticles showed a spherical solid structure, with a smooth surface (Fig. 1B). The PLCP complexes appeared to have an irregular form (Fig. 1C). CaP and PCLs both have high nucleic acids binding ability [6,14]. A gel retardation assay was used to semi-quantify the siRNA binding rate of PLCP. Those results indicated that the siRNA binding rate exceed 90% when the N/P rate is larger than 2 (data not shown).
Fig. 2. Determination of the buffer capacity of NaCl, PEI 25 K, PCL and PLCP by acidebase titration.
3.2. Buffer capacity of PLCP To evaluate the buffer capacity of PLCP under acidic conditions, the environment of endosomes was simulated. The acidebase titration profiles obtained for NaCl, PEI 25 K, PCL and PLCP are shown in Fig. 2. The pH changed from neutral to ~5.0, like in endosomes/lysosomes after cellular internalization. Compared with PCL and negative control, the pH value of PLCP showed a slower descent. The buffer capacity of PLCP fell between that of PCL and PEI 25 K, which suggested that PLCP could also swell and disrupt the endosome through the proton-sponge mechanism. 3.3. Cellular toxicity and internalization ability of PLCP The cytotoxicity of PLCP against MCF-7 cells was determined by MTT assay using PEI 25 K as the control. As shown in Fig. 3, PLCP exhibited no cytotoxicity over the whole N/P ratio range; however, cell viability dramatically decreased to approximately 40% when treated with PEI 25 K at similar N/P ratio. FAM-labeled siRNA was used to evaluate the internalization ability of CaP nanoparticles, PCL and PLCP in MCF-7 cells. Lipofectamine™ 2000/siRNA complexes were used as positive control. The strongest fluorescence intensity was observed when FAM-siRNA was transfected using PLCP
Fig. 3. Cytotoxicity assay of PLCP and PEI 25 K with siRNA (0.2 mg/well siRNA) at various N/P ratios (N/P ¼ 1, 2.5, 5, 10, 20, 30, 40 and 50).
(Fig. 4D). From the results, we also found that CaP nanoparticles were capable of facilitating higher delivery of siRNA into cells than PCL (Fig. 4B and C).
Fig. 1. Transmission electron micrograph of (A) PCL, (B) CaP nanoparticles and (C) PLCP. Scale bar, 0.5 mm.
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Fig. 4. Fluorescence image observations of cellular uptake in MCF-7 cells, mediated by Lipofectamine™ 2000 (A), CaP nanoparticles (B), PCL (C) and PLCP (D). 1 mg siRNA/well. Magnification ¼ 200. Dimensions of each picture is 500 400 pixels (length width).
3.4. In vitro gene silencing activity Anti-GFP siRNA was constructed to cleave GFP mRNA for reduced protein production. Thus, the fluorescence intensity represented the final silencing efficiency of the siRNA delivery system. Fig. 5 shows the green fluorescence intensity of MCF-7 cells stably expressing GFP following transfection with anti-GFP siRNA using four different vectors for different transfection times. Negligible silence effect was found in the CaP group (Fig. 5B), and the fluorescence intensity of cells increased with longer transfection times compared with the negative control. All other vectors had sufficient silencing effect to inhibit GFP expression after 24 h transfection. Anti-GFP siRNA mediated by PLCP (Fig. 5D) and Lipofectamine™ 2000 (Fig. 5A) could significantly inhibit GFP expression over the whole investigation time (0e72 h). GFP expression in MCF-7 cells could be efficiently inhibited by PCL, however, the inhibition efficiency decreased as the culture time increased, especially after 72 h transfection (Fig. 5C). The RT-qPCR assay was used to quantitate GFP mRNA in MCF-7 cells, and the results are presented in Fig. 6. In the first 24-h transfection, approximately 70%, 50% and 25% GFP mRNA were down regulated by PLCP, PCL and CaP, respectively. The silencing efficiency of PLCP was improved to 80% in the following 48-h transfection, which was obviously higher than that of Lipofectamine™ 2000 (~60%), however, the silencing abilities of PCL and CaP were reduced. 4. Discussion Unlike DNA which requires nuclear entry for transfection, siRNA can specifically silence its target mRNA when it is successfully delivered into the cytoplasm [19]. Thus, enhancing the localization of siRNA into the cytoplasm is an important step to promote gene silencing efficiency. In this study, we designed a siRNA delivery system called PLCP, which was composed of CaP nanoparticles and PCL.
CaP nanoparticles have been identified as potentially useful biomaterials for many biomedical applications, particularly in gene delivery applications. They can dissolve in acidic environments and release nucleic acids in cytoplasm [20]. However, the drawback of precipitated CaP is the low transfection efficiency compared with other vectors. Many studies reported that large particle size of CaP precipitate (usually larger than 300 nm) would be one of the major impediments. Researchers have developed various approaches to stabilize CaP precipitates [9,10]. In this study, siRNA is adsorbed to CaP by calcium ion chelation. Pluronic F68, a nontoxic surfactant, was used as a stabilizer to slow down the CaP aggregation process. The optimal CaP/siRNA nanoparticles were fairly homogenous and spherically shaped, with a diameter of approximately 149 nm (Table 1 and Fig. 1B). Strong fluorescence intensity was observed in cells after internalizing negative CaP/siRNA nanoparticles (Fig. 4B). We believe that Pluronic F68 increased cell viability and enabled highly efficient intracellular delivery, as previously reported by Sengupta et al. [21]. On the other hand, encapsulating CaP nanoparticles with functional materials was an alternate method [11e13]. Though cellular internalization ability seemed weaker than other vectors (Fig. 4C), PCL was developed by us to improve the ability of endosomal escape by combining the protonation of PEI and the membrane destabilization of DOPE. These materials could lead to high transfection efficiency [14]. Combined with the results of gene silencing study, endosomal escape ability of PCL should play an important role in mRNA down regulation. Since the nanoparticles were encapsulated with a positively charged liposome, the inner CaP/ siRNA nanoparticles needed to contain a certain negative charge. Sodium citrate addition could not only increase the stability of CaP nanoparticles, but also change the surface charge of the CaP nanoparticles to negative (~13.6 mV). Thus, PCL could effectively encapsulated CaP/siRNA nanoparticles to form PLCP, with a neutral potential (0.3 mV) and mean particle sizes of approximately 260 nm (Table 1 and Fig. 1C), which is similar to our previous report on PLPD containing protamine/DNA nanoparticles as inner cores
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Fig. 5. GFP expression inhibition in MCF-7 cells stably expressing GFP, following treatment with anti-GFP siRNA mediated by Lipofectamine™ 2000 (A), CaP nanoparticles (B), PCL (C), PLCP (D) and free anti-GFP siRNA (E), for various times. 1 mg siRNA/well. Magnification ¼ 200. Dimensions of each picture is 500 400 pixels (length width).
Fig. 6. GFP mRNA expression relative to human GAPDH mRNA in MCF-7 cells for 24 h, 48 h and 72 h Lipofectamine™ 2000 was used as a positive control (1 mg siRNA/well). Values represent the mean ± SD (n ¼ 3). PCLP group compared with PCL and CaP groups: *P < 0.05, **P < 0.01. Lipofectamine™ 2000 group compared with the PCLP group: #P < 0.05.
[16]. From the results of TEM, PLCP complexes appeared to be irregular shape. We presumed that CaP of smaller particle size might locate on the surface of the PLCP which cause the zeta potential reduction as well [22]. Combined the advantage of CaP and PCL, high intracellular activity of PLCP was observed (Fig. 4D) and
the surface CaP was suggested to be important during the intracellular process due to fusogenic and membranolytic activity of calcium [23]. The cytotoxicity of PLCP was determined by MTT assay. The results indicated that PLCP had significantly lower cytotoxicity compared with PEI 25 K (Fig. 3), a common commercial transfection agent, at the same N/P ratio level. There were two possible reasons for low cytotoxicity of PLCP, the neutral surface charge and biocompatibility of component. Generally, the cytotoxicity of the polycations was caused by the increased molecular weight and the positive charge density [24]. PLCP showed slightly positive zeta potential compared with high positive charge PEI 25 K, which might reduce the cytotoxicity of the complexes. In addition, CaP has been generally regarded as safety biomaterials by the FDA and showed no significant toxicity in most cell types [6]. In the gene silencing study, anti-GFP siRNA mediated by PLCP could down regulate mRNA in cytoplasm effectively and quickly (Fig. 6), and resulted in obvious GFP expression reductions (~70% inhibition, Fig. 5D). The comparison of PLCP with PCL and CaP nanoparticles strongly suggested that the core-membrane structure of PLCP contributed to the significant gene silencing. Because most non-viral vectors are internalized by endocytosis, the endosomal escape capability and siRNA release from vectors are critical for effective gene silencing activity. We have found that PCL have endosomal escape capability in different cell lines [14,17]. The buffer capacity test results in this study also showed that the PLCP could accept more protons than PCL, when the pH value decreased from neutral to 5.0 (Fig. 2). We presumed that the partial CaP located on the surface of PLCP dissolve in the acidic medium of the endosome would lead to enhance destabilization of the endosomal membrane by osmotic imbalance [20]. Furthermore, residual CaP
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nanoparticles retained the silencing activity for long time periods (72 h). The results of this study indicated that PLCP can deliver the therapeutic siRNA into the targeted tumors by intratumoral injection. 5. Conclusion In this study, we demonstrated a new approach for RNAi gene silencing using a PLCP delivery system. The CaP nanoparticle core were prepared by a co-precipitation method and further combined with PCL to form PLCP. The vector has a slightly positive zeta potential, with a size of about 260.4 nm. Because they exhibited considerable endosomal escape capability, low cytotoxicity and high efficiency in target gene silencing, PLCP nanoparticles have good potential for applications delivering therapeutic siRNA. Acknowledgments This study was supported by the National Natural Science Foundation of China (NSFC No. 81001409 and 8140287) and Zhejiang Provincial Natural Science Foundation of China (No. LQ12H30004). References [1] S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature 411 (6836) (2001) 494e498. [2] Y. Dorsett, T. Tuschl, siRNAs: applications in functional genomics and potential as therapeutics, Nat. Rev. Drug Discov. 3 (4) (2004) 318e329. [3] N.S. Gandhi, R.K. Tekade, M.B. Chougule, Nanocarrier mediated delivery of siRNA/miRNA in combination with chemotherapeutic agents for cancer therapy: current progress and advances, J. Control. Release 194C (2014) 238e256. [4] T.E. Park, B. Singh, H. Li, J.Y. Lee, S.K. Kang, Y.J. Choi, C.S. Cho, Enhanced BBB permeability of osmotically active poly(mannitol-co-PEI) modified with rabies virus glycoprotein via selective stimulation of caveolar endocytosis for RNAi therapeutics in Alzheimer's disease, Biomaterials 38 (2015) 61e71. [5] A. Sato, M. Takagi, A. Shimamoto, S. Kawakami, M. Hashida, Small interfering RNA delivery to the liver by intravenous administration of galactosylated cationic liposomes in mice, Biomaterials 28 (7) (2007) 1434e1442. [6] A. Maitra, Calcium phosphate nanoparticles: second-generation nonviral vectors in gene therapy, Expert Rev. Mol. Diagn. 5 (6) (2005) 893e905. [7] M. Okazaki, Y. Yoshida, S. Yamaguchi, M. Kaneno, J.C. Elliott, Affinity binding phenomena of DNA onto apatite crystals, Biomaterials 22 (18) (2001) 2459e2464.
[8] M. Jordan, A. Schallhorn, F.M. Wurm, Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation, Nucleic Acids Res. 24 (4) (1996) 596e601. [9] E.H. Chowdhury, M. Kunou, M. Nagaoka, A.K. Kundu, T. Hoshiba, T. Akaike, High-efficiency gene delivery for expression in mammalian cells by nanoprecipitates of CaeMg phosphate, Gene 341 (2004) 77e82. [10] E.C. Giger, J. Puigmarti-Luis, R. Schlatter, B. Castagner, P.S. Dittrich, J.C. Leroux, Gene delivery with bisphosphonate-stabilized calcium phosphate nanoparticles, J. Control. Release 150 (1) (2011) 87e93. [11] J. Li, Y. Yang, L. Huang, Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor, J. Control. Release 158 (1) (2012) 108e114. [12] M.S. Lee, J.E. Lee, E. Byun, N.W. Kim, K. Lee, H. Lee, S.J. Sim, D.S. Lee, J.H. Jeong, Target-specific delivery of siRNA by stabilized calcium phosphate nanoparticles using dopa-hyaluronic acid conjugate, J. Control. Release 192 (2014) 122e130. [13] J. Wang, B. Chen, D. Zhao, Y. Peng, R.X. Zhuo, S.X. Cheng, Peptide decorated calcium phosphate/carboxymethyl chitosan hybrid nanoparticles with improved drug delivery efficiency, Int. J. Pharm. 446 (1e2) (2013) 205e210. [14] J.L. Chen, H. Wang, J.Q. Gao, H.L. Chen, W.Q. Liang, Liposomes modified with polycation used for gene delivery: preparation, characterization and transfection in vitro, Int. J. Pharm. 343 (1e2) (2007) 255e261. [15] J.L. Chen, Y. Hu, W.P. Shuai, H.L. Chen, W.Q. Liang, J.Q. Gao, Telomerase-targeting antisense oligonucleotides carried by polycation liposomes enhance the growth inhibition effect on tumor cells, J. Biomed. Mater. Res. Part B, Appl. Biomater. 89 (2) (2009) 362e368. [16] J. Chen, Z. Yu, H. Chen, J. Gao, W. Liang, Transfection efficiency and intracellular fate of polycation liposomes combined with protamine, Biomaterials 32 (5) (2011) 1412e1418. [17] J. Chen, X. Sun, Z. Yu, J. Gao, W. Liang, Influence of lipid components on gene delivery by polycation liposomes: transfection efficiency, intracellular kinetics and in vivo tumor inhibition, Int. J. Pharm. 422 (1e2) (2012) 510e515. [18] Q.Q. Li, J. Skinner, J.E. Bennett, Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment, BMC Mol. Biol. 13 (22) (2012). [19] H.R. Kim, I.K. Kim, K.H. Bae, S.H. Lee, Y. Lee, T.G. Park, Cationic solid lipid nanoparticles reconstituted from low density lipoprotein components for delivery of siRNA, Mol. Pharm. 5 (4) (2008) 622e631. [20] J. Li, Y.C. Chen, Y.C. Tseng, S. Mozumdar, L. Huang, Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery, J. Control. Release 142 (3) (2010) 416e421. [21] A. Sengupta, N. Dwivedi, S.C. Kelly, L. Tucci, N.N. Thadhani, M.R. Prausnitz, Poloxamer surfactant preserves cell viability during photoacoustic delivery of molecules into cells, Biotechnol. Bioeng. 112 (2) (2015) 405e415. [22] M. Junghans, S.M. Loitsch, S.C.J. Steiniger, J. Kreuter, A. Zimmer, Cationic lipidprotamine-DNA (LPD) complexes for delivery of antisense c-myc oligonucleotides, Eur. J. Pharm. Biopharm. 60 (2005) 287e294. [23] A. Kovtun, R. Heumann, M. Epple, Calcium phosphate nanoparticles for the transfection of cells, Biomed. Mater. Eng. 19 (2e3) (2009) 241e247. [24] D. Fischer, Y. Li, B. Ahlemeyer, J. Krieglstein, T. Kissel, In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis, Biomaterials 24 (7) (2003) 1121e1131.
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Research paper
Polycation liposomes combined with calcium phosphate nanoparticles as a non-viral carrier for siRNA delivery Jianjun Zhang a, b, Xiaoyi Sun c, Rong Shao d, Wenquan Liang a, Jianqing Gao a, **, Jinliang Chen d, * a
Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China Department of Pharmaceutics, Zhejiang Hospital of TCM, Hangzhou, Zhejiang, China Department of Pharmacy, Zhejiang University City College, Hangzhou, Zhejiang, China d Center of Clinical Pharmacology, 2nd Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, Zhejiang, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 June 2015 Received in revised form 7 September 2015 Accepted 9 September 2015 Available online 14 September 2015
Low transfection efficiency and instability are the main barriers for siRNA delivery. To overcome these barriers, a core-membrane siRNA delivery system was designed. Calcium phosphate/siRNA nanoparticles were combined with liposomes, and the surface was modified with polyethylenimine-cholesterol (PEIChol). The mean particle size of the polycation liposomes/calcium phosphate/siRNA complexes (PLCP) was approximately 260.4 nm and the zeta potential was 0.3 ± 0.2 mV. The buffer capacity and cellular uptake of PLCP were studied. The results indicated that PEI-Chol and CaP can synergistically improve endosomal escape by swelling and disrupting the endosome. Anti-green fluorescent protein (GFP) siRNA was used to evaluate the gene silencing effect in MCF-7 cells that stably express GFP. PLCP could down regulate approximately 70% of GFP expression in 24 h, which is significantly higher than that of Lipofectamine™ 2000. Additionally, the silencing effect lasted for at least 72 h. This delivery system can improve intracellular uptake and enhance gene silencing, with low cytotoxicity. © 2015 Elsevier B.V. All rights reserved.
Keywords: Calcium phosphate nanoparticles Polycation liposomes siRNA Green fluorescent protein Gene silencing
1. Introduction The development of RNA interference (RNAi) technology has experienced much excitement for more than ten years, providing new methods for potential treatment of genetic related diseases [1e4]. Small interfering RNA (siRNA) incorporates into the RNAiinduced silencing complex (RISC) and allows for the cleavage of its complementary target mRNA in the cytoplasm to reduce related protein expression. siRNA in practical applications, however, have been limited by poor intracellular uptake and instability in the physiological environment [5]. The development of safe and effective delivery systems for siRNA would greatly enhance the gene-silencing ability.
* Corresponding author. Center of Clinical Pharmacology, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Jiefang Road 88, Hangzhou, Zhejiang, 310009, China. ** Corresponding author. Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Yuhangtang Road 388, Hangzhou, Zhejiang, 310058, China. E-mail addresses: [email protected] (J. Gao), [email protected] (J. Chen). http://dx.doi.org/10.1016/j.jddst.2015.09.005 1773-2247/© 2015 Elsevier B.V. All rights reserved.
Calcium phosphate (CaP) has been widely used as a non-viral gene delivery system since the 1970s, because it is considered to be biodegradable and biocompatible [6]. CaP nanoparticles can bind negatively charged nucleic acids by calcium ion chelation to deliver nucleic acids into cells and protect from enzymatic degradation [7]. One of the major limitations for CaP nanoparticles, however, is the uncontrollable rapid growth of CaP crystals after preparation, which can lead to large aggregation and significantly reduce stability and the transfection efficiency [8]. Many approaches have been reported to stabilize CaP precipitates. Some strategies improved the manufacturing process by adding different stabilizers, such as increasing concentrations of magnesium ions to slow down aggregation [9] or adding bisphosphonate to enhance calcium ion chelation [10]. Others have attempted to encapsulate CaP nanoparticles with lipids [11], hyaluronic acid [12] or chitosan [13]. In previous work, we have successfully developed polycation liposomes (PCL) and polycation liposomes/protamine/DNA complexes (PLPD) as gene transfection vectors to overcome intracellular barriers and achieve high transfection efficiency [14e17]. Thus, in this study, we developed a siRNA delivery system using CaP/siRNA
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nanoparticles combined with PCL, which we termed polycation liposomes/calcium phosphate/siRNA complexes (PLCP). Pluronic F68 and sodium citrate were used to stabilize CaP nanoparticles, and PCL combining reduced their aggregation. The particle sizes, zeta potential, cytotoxicity and cellular internalization ability of PLCP were investigated. The gene silencing effect was assessed in MCF-7 cells stably expressing GFP using anti-GFP siRNA mediated by PLCP. 2. Materials and methods 2.1. Materials Pluronic F68, polyethylenimine (PEI, MW of 800 Da and 25 kDa), cholesteryl chloroformate and 1,2-dioleoyl-sn-glycero-3-phosphoe thanolamine (DOPE) were purchased from SigmaeAldrich (St. Louis, MO, USA). Polyethylenimine-cholesterol (PEI 800-Chol) was synthesized as previously reported [14]. Thiazolyl blue tetrazolium bromide (MTT) was purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Dulbecco's modified eagle's medium (DMEM), trypsin, fetal bovine serum (FBS) and Lipofectamine™ 2000 were purchased from Life Technologies (Carlsbad, CA, USA). Anti-green fluorescent protein (GFP) siRNA (sense, 50 -GCCACAACGUCUAUAUCAUGG-30 ; antisense, 50 AUGAUAUAGACGUUGUGGCUG-30 ), random siRNA (sense, 50 UCCUCCGAACGUGUCACGUTT-30 ; antisense, 50 -ACGUGACACGUUC GGAGAATT-30 ) and FAM-labeled random siRNA were synthesized, modified and purified by 100 Biotech Company (Hangzhou, Zhejiang, China). All other chemical reagents were analytical grade from Sinopharm Chemical Reagent Company (Shanghai, China). 2.2. Cell lines Human breast adenocarcinoma cell lines (MCF-7) were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences (Shanghai, China). MCF-7 cells stably expressing GFP were established by 100 Biotech Company (Hangzhou, Zhejiang, China). Cells were cultured as a monolayer in DMEM containing 10% FBS at 37 C in 5% CO2. Cells were regularly passaged and reseeded 24 h before transfection experiments. 2.3. Preparation and characterization of CaP/siRNA nanoparticles, PCL and PLCP CaP/siRNA nanoparticles were prepared by a modified coprecipitation method. Briefly, 30 mL of 75 mM CaCl2, 70 mL of 1% Pluronic F68 and 100 mL of 0.1 mg/mL siRNA solution were mixed and incubated for 10 min at room temperature as calcium moieties. The phosphate moieties were prepared as follows: firstly mixing 120 mL of 6 mM Na2HPO4 and 40 mL of 1% Pluronic F68, and then mixing with 40 mL of 24 mM sodium citrate. CaP/siRNA nanoparticles were prepared by adding the phosphate moieties dropwisely to the calcium moieties, and incubating for another 10 min. PCL were prepared by film dispersion method with a lipid mixture of PEI 800-Chol and DOPE (molar ratio 1:1) as previously descripted [17]. PLCP complexes were prepared by mixing an equal volume of PCL and CaP/siRNA nanoparticles at various N/P ratios (1 mol PEI 800-Chol nitrogen per mol siRNA phosphate), and then incubating for 30 min at room temperature. Particle size and zeta-potential (z) of the vectors were determined by laser diffraction spectrometry (Malvern Zetasizer 3000HS, UK), and the morphologies of the complexes were observed using transmission electron microscopy (TEM, JEM1200EX, Japan).
2.4. Determination of buffer capacity The buffer capacity of vectors was determined by acidebase titration. Thirty milliliters of PEI 25 K solution, PCL and PLCP were prepared. The concentrations of PEI 25 or PEI-Chol in PCL and PLCP were 0.3 mg/mL. The pH value of every group was raised to 10 using 1 M NaOH, and then titrated with 0.1 M HCl. The volumes of HCl consumed and pH values were recorded. The 0.1 M NaCl solution was used as a negative control. 2.5. Cytotoxicity assay Cellular viability was determined using MTT assay. MCF-7 cells were plated in 96-well plates at a density of 1 104 cells/well and incubated for 24 h at 37 C in CO2 incubator. The cells were washed with PBS and exposed to 20 mL PLCP or PEI 25 K/siRNA (0.2 mg siRNA/well) with various N/P ratios (N/P ¼ 1, 2.5, 5, 10, 20, 30, 40 and 50) in serum-free medium. Serum-free medium was added at an equal volume as a control group. After transfection for another 24 h, the culture medium was replaced with the fresh DMEM medium, and then 20 mL of MTT (5 mg/mL) was added to each well and incubated for 4 h at 37 C. The reaction was stopped by lysing the cells with 150 mL of DMSO for 10 min. The absorbance was measured at 570 nm in microplate reader (Bio-Tek, ELx800, USA). 2.6. Cellular uptake assay MCF-7 cells were seeded at 1 105 cells/well in a 24-well plate and incubated overnight at 37 C with 5% CO2. Lipofectamine™ 2000/siRNA complexes, CaP/siRNA nanoparticles and PLCP/siRNA complexes were added after changing cells to serum-free DMEM (1 mg siRNA/well). After incubation for another 4 h, cells were washed with PBS and the medium was changed to DMEM containing 10% FBS. Green fluorescence was observed using an inverted fluorescent microscope (HBO 100, Zeiss, Germany). 2.7. In vitro gene silencing activity To evaluate the gene silencing efficiency of different carriers, MCF-7 cells with stable GFP expression were used. These cells were seeded with 2 mL of DMEM containing 10% FBS on 12-well plates at 1.5 104 cells/well, overnight before transfection. Upon reaching 60% confluency, anti-GFP siRNA was transfected using Lipofectamine™ 2000, CaP and PLCP into cells for 5 h, following which the medium was replaced with fresh medium. The cells were then cultured for another 24, 48 or 72 h at 37 C. The dosage of siRNA in each well was 1 mg. The fluorescence of GFP expression in MCF-7 cells was observed by inverted fluorescent microscopy, and related GFP mRNA expression was determined by RT-qPCR, using hGAPDH as an internal standard. The 2DDCT method was used and CT value was defined as the cycle number when the fluorescent signal of each reaction tube reached its set threshold [18]. 2.8. Statistical analysis Statistical analysis was performed using Student's t-test. 3. Results 3.1. Characterization of PCL, CaP/siRNA nanoparticles and PLCP The mean particle sizes of PCL and CaP/siRNA nanoparticles were approximately 129.6 nm and 148.9 nm, respectively (Table 1). The PCL composed of PEI 800-Chol and DOPE had a positive zeta potential about 39.3 mV, while the CaP/siRNA nanoparticles had
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Table 1 Particle sizes and zeta potential (z) of PCL/siRNA, CaP nanoparticles/siRNA and PLCP.
PCL/siRNAa CaP nanoparticles/siRNA PLCPa a
Particle sizes (nm)
PDI
Zeta potential (z, mV)
129.6 ± 14.0 148.9 ± 21.8 260.4 ± 23.4
0.200 0.248 0.188
39.3 ± 6.2 13.6 ± 1.0 0.3 ± 0.2
The N/P ratio of the complexes was 20.
negative zeta potentials about 13.6 mV. After encapsulating CaP/ siRNA nanoparticles with PCL, the sizes of PLCP increased to approximately 260.4 nm and zeta potential reached 0.3 mV, which was nearly neutral. We also investigated the morphological characteristics of the complexes using TEM. For PCL, spherical particles with an inner phase structure were observed (Fig. 1A). CaP/siRNA nanoparticles showed a spherical solid structure, with a smooth surface (Fig. 1B). The PLCP complexes appeared to have an irregular form (Fig. 1C). CaP and PCLs both have high nucleic acids binding ability [6,14]. A gel retardation assay was used to semi-quantify the siRNA binding rate of PLCP. Those results indicated that the siRNA binding rate exceed 90% when the N/P rate is larger than 2 (data not shown).
Fig. 2. Determination of the buffer capacity of NaCl, PEI 25 K, PCL and PLCP by acidebase titration.
3.2. Buffer capacity of PLCP To evaluate the buffer capacity of PLCP under acidic conditions, the environment of endosomes was simulated. The acidebase titration profiles obtained for NaCl, PEI 25 K, PCL and PLCP are shown in Fig. 2. The pH changed from neutral to ~5.0, like in endosomes/lysosomes after cellular internalization. Compared with PCL and negative control, the pH value of PLCP showed a slower descent. The buffer capacity of PLCP fell between that of PCL and PEI 25 K, which suggested that PLCP could also swell and disrupt the endosome through the proton-sponge mechanism. 3.3. Cellular toxicity and internalization ability of PLCP The cytotoxicity of PLCP against MCF-7 cells was determined by MTT assay using PEI 25 K as the control. As shown in Fig. 3, PLCP exhibited no cytotoxicity over the whole N/P ratio range; however, cell viability dramatically decreased to approximately 40% when treated with PEI 25 K at similar N/P ratio. FAM-labeled siRNA was used to evaluate the internalization ability of CaP nanoparticles, PCL and PLCP in MCF-7 cells. Lipofectamine™ 2000/siRNA complexes were used as positive control. The strongest fluorescence intensity was observed when FAM-siRNA was transfected using PLCP
Fig. 3. Cytotoxicity assay of PLCP and PEI 25 K with siRNA (0.2 mg/well siRNA) at various N/P ratios (N/P ¼ 1, 2.5, 5, 10, 20, 30, 40 and 50).
(Fig. 4D). From the results, we also found that CaP nanoparticles were capable of facilitating higher delivery of siRNA into cells than PCL (Fig. 4B and C).
Fig. 1. Transmission electron micrograph of (A) PCL, (B) CaP nanoparticles and (C) PLCP. Scale bar, 0.5 mm.
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Fig. 4. Fluorescence image observations of cellular uptake in MCF-7 cells, mediated by Lipofectamine™ 2000 (A), CaP nanoparticles (B), PCL (C) and PLCP (D). 1 mg siRNA/well. Magnification ¼ 200. Dimensions of each picture is 500 400 pixels (length width).
3.4. In vitro gene silencing activity Anti-GFP siRNA was constructed to cleave GFP mRNA for reduced protein production. Thus, the fluorescence intensity represented the final silencing efficiency of the siRNA delivery system. Fig. 5 shows the green fluorescence intensity of MCF-7 cells stably expressing GFP following transfection with anti-GFP siRNA using four different vectors for different transfection times. Negligible silence effect was found in the CaP group (Fig. 5B), and the fluorescence intensity of cells increased with longer transfection times compared with the negative control. All other vectors had sufficient silencing effect to inhibit GFP expression after 24 h transfection. Anti-GFP siRNA mediated by PLCP (Fig. 5D) and Lipofectamine™ 2000 (Fig. 5A) could significantly inhibit GFP expression over the whole investigation time (0e72 h). GFP expression in MCF-7 cells could be efficiently inhibited by PCL, however, the inhibition efficiency decreased as the culture time increased, especially after 72 h transfection (Fig. 5C). The RT-qPCR assay was used to quantitate GFP mRNA in MCF-7 cells, and the results are presented in Fig. 6. In the first 24-h transfection, approximately 70%, 50% and 25% GFP mRNA were down regulated by PLCP, PCL and CaP, respectively. The silencing efficiency of PLCP was improved to 80% in the following 48-h transfection, which was obviously higher than that of Lipofectamine™ 2000 (~60%), however, the silencing abilities of PCL and CaP were reduced. 4. Discussion Unlike DNA which requires nuclear entry for transfection, siRNA can specifically silence its target mRNA when it is successfully delivered into the cytoplasm [19]. Thus, enhancing the localization of siRNA into the cytoplasm is an important step to promote gene silencing efficiency. In this study, we designed a siRNA delivery system called PLCP, which was composed of CaP nanoparticles and PCL.
CaP nanoparticles have been identified as potentially useful biomaterials for many biomedical applications, particularly in gene delivery applications. They can dissolve in acidic environments and release nucleic acids in cytoplasm [20]. However, the drawback of precipitated CaP is the low transfection efficiency compared with other vectors. Many studies reported that large particle size of CaP precipitate (usually larger than 300 nm) would be one of the major impediments. Researchers have developed various approaches to stabilize CaP precipitates [9,10]. In this study, siRNA is adsorbed to CaP by calcium ion chelation. Pluronic F68, a nontoxic surfactant, was used as a stabilizer to slow down the CaP aggregation process. The optimal CaP/siRNA nanoparticles were fairly homogenous and spherically shaped, with a diameter of approximately 149 nm (Table 1 and Fig. 1B). Strong fluorescence intensity was observed in cells after internalizing negative CaP/siRNA nanoparticles (Fig. 4B). We believe that Pluronic F68 increased cell viability and enabled highly efficient intracellular delivery, as previously reported by Sengupta et al. [21]. On the other hand, encapsulating CaP nanoparticles with functional materials was an alternate method [11e13]. Though cellular internalization ability seemed weaker than other vectors (Fig. 4C), PCL was developed by us to improve the ability of endosomal escape by combining the protonation of PEI and the membrane destabilization of DOPE. These materials could lead to high transfection efficiency [14]. Combined with the results of gene silencing study, endosomal escape ability of PCL should play an important role in mRNA down regulation. Since the nanoparticles were encapsulated with a positively charged liposome, the inner CaP/ siRNA nanoparticles needed to contain a certain negative charge. Sodium citrate addition could not only increase the stability of CaP nanoparticles, but also change the surface charge of the CaP nanoparticles to negative (~13.6 mV). Thus, PCL could effectively encapsulated CaP/siRNA nanoparticles to form PLCP, with a neutral potential (0.3 mV) and mean particle sizes of approximately 260 nm (Table 1 and Fig. 1C), which is similar to our previous report on PLPD containing protamine/DNA nanoparticles as inner cores
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Fig. 5. GFP expression inhibition in MCF-7 cells stably expressing GFP, following treatment with anti-GFP siRNA mediated by Lipofectamine™ 2000 (A), CaP nanoparticles (B), PCL (C), PLCP (D) and free anti-GFP siRNA (E), for various times. 1 mg siRNA/well. Magnification ¼ 200. Dimensions of each picture is 500 400 pixels (length width).
Fig. 6. GFP mRNA expression relative to human GAPDH mRNA in MCF-7 cells for 24 h, 48 h and 72 h Lipofectamine™ 2000 was used as a positive control (1 mg siRNA/well). Values represent the mean ± SD (n ¼ 3). PCLP group compared with PCL and CaP groups: *P < 0.05, **P < 0.01. Lipofectamine™ 2000 group compared with the PCLP group: #P < 0.05.
[16]. From the results of TEM, PLCP complexes appeared to be irregular shape. We presumed that CaP of smaller particle size might locate on the surface of the PLCP which cause the zeta potential reduction as well [22]. Combined the advantage of CaP and PCL, high intracellular activity of PLCP was observed (Fig. 4D) and
the surface CaP was suggested to be important during the intracellular process due to fusogenic and membranolytic activity of calcium [23]. The cytotoxicity of PLCP was determined by MTT assay. The results indicated that PLCP had significantly lower cytotoxicity compared with PEI 25 K (Fig. 3), a common commercial transfection agent, at the same N/P ratio level. There were two possible reasons for low cytotoxicity of PLCP, the neutral surface charge and biocompatibility of component. Generally, the cytotoxicity of the polycations was caused by the increased molecular weight and the positive charge density [24]. PLCP showed slightly positive zeta potential compared with high positive charge PEI 25 K, which might reduce the cytotoxicity of the complexes. In addition, CaP has been generally regarded as safety biomaterials by the FDA and showed no significant toxicity in most cell types [6]. In the gene silencing study, anti-GFP siRNA mediated by PLCP could down regulate mRNA in cytoplasm effectively and quickly (Fig. 6), and resulted in obvious GFP expression reductions (~70% inhibition, Fig. 5D). The comparison of PLCP with PCL and CaP nanoparticles strongly suggested that the core-membrane structure of PLCP contributed to the significant gene silencing. Because most non-viral vectors are internalized by endocytosis, the endosomal escape capability and siRNA release from vectors are critical for effective gene silencing activity. We have found that PCL have endosomal escape capability in different cell lines [14,17]. The buffer capacity test results in this study also showed that the PLCP could accept more protons than PCL, when the pH value decreased from neutral to 5.0 (Fig. 2). We presumed that the partial CaP located on the surface of PLCP dissolve in the acidic medium of the endosome would lead to enhance destabilization of the endosomal membrane by osmotic imbalance [20]. Furthermore, residual CaP
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nanoparticles retained the silencing activity for long time periods (72 h). The results of this study indicated that PLCP can deliver the therapeutic siRNA into the targeted tumors by intratumoral injection. 5. Conclusion In this study, we demonstrated a new approach for RNAi gene silencing using a PLCP delivery system. The CaP nanoparticle core were prepared by a co-precipitation method and further combined with PCL to form PLCP. The vector has a slightly positive zeta potential, with a size of about 260.4 nm. Because they exhibited considerable endosomal escape capability, low cytotoxicity and high efficiency in target gene silencing, PLCP nanoparticles have good potential for applications delivering therapeutic siRNA. Acknowledgments This study was supported by the National Natural Science Foundation of China (NSFC No. 81001409 and 8140287) and Zhejiang Provincial Natural Science Foundation of China (No. LQ12H30004). References [1] S.M. Elbashir, J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl, Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature 411 (6836) (2001) 494e498. [2] Y. Dorsett, T. Tuschl, siRNAs: applications in functional genomics and potential as therapeutics, Nat. Rev. Drug Discov. 3 (4) (2004) 318e329. [3] N.S. Gandhi, R.K. Tekade, M.B. Chougule, Nanocarrier mediated delivery of siRNA/miRNA in combination with chemotherapeutic agents for cancer therapy: current progress and advances, J. Control. Release 194C (2014) 238e256. [4] T.E. Park, B. Singh, H. Li, J.Y. Lee, S.K. Kang, Y.J. Choi, C.S. Cho, Enhanced BBB permeability of osmotically active poly(mannitol-co-PEI) modified with rabies virus glycoprotein via selective stimulation of caveolar endocytosis for RNAi therapeutics in Alzheimer's disease, Biomaterials 38 (2015) 61e71. [5] A. Sato, M. Takagi, A. Shimamoto, S. Kawakami, M. Hashida, Small interfering RNA delivery to the liver by intravenous administration of galactosylated cationic liposomes in mice, Biomaterials 28 (7) (2007) 1434e1442. [6] A. Maitra, Calcium phosphate nanoparticles: second-generation nonviral vectors in gene therapy, Expert Rev. Mol. Diagn. 5 (6) (2005) 893e905. [7] M. Okazaki, Y. Yoshida, S. Yamaguchi, M. Kaneno, J.C. Elliott, Affinity binding phenomena of DNA onto apatite crystals, Biomaterials 22 (18) (2001) 2459e2464.
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