Sterically Polymer-Based Liposomal Complexes ... - ACS Publications

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Sterically Polymer-Based Liposomal Complexes with Dual-Shell Structure for Enhancing the siRNA Delivery Shuian-Yin Lin,† Wei-Yu Zhao,‡ Hsieh-Chih Tsai,§ Wei-Hsin Hsu,∥ Chun-Liang Lo,*,⊥ and Ging-Ho Hsiue*,†,# †

Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, Republic of China Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan, Republic of China § Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, 106 Taipei, Taiwan, Republic of China ∥ Department of Biomedical Engineering and Environmental Science, National Tsing Hua University, Hsinchu 300, Taiwan, Republic of China ⊥ Department of Biomedical Engineering, National Yang Ming University, Taipei, Taiwan 112, Republic of China # Department of Chemical Engineering/R&D Center for Membrane Technology, Chung Yuan University, Chung Li, 320 Taiwan, Republic of China ‡

S Supporting Information *

ABSTRACT: The sterically polymer-based liposomal complexes (SPLexes) were formed by cationic polymeric liposomes and pHsensitive diblock copolymer were studied for their capabilities in improving the stability with high efficiency of siRNA delivery. The SPLexes were formed a dual-shelled structure and uniform size distribution. The PEGylated outer shell could mitigate the phagocytosis and reduce the cytotoxicity. Moreover, the folated SPLexes improved 42.9× accumulation in vitro and 1.7× tumor uptake in vivo in contrast with nonfolated SPLexes. The protonated copolymer at low pH would improve the siRNA released into cytoplasm following SPLexes fusion with the endo/lysosome membrane and inhibited the protein expression to 75.6 ± 4.5% efficiently. Results of this study significantly contribute to efforts to develop lipoplexes based siRNA delivery systems.

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INTRODUCTION As a leading health concern globally, cancer originates from genetic abnormalities that typically cause the affected cells to behave differently at the molecular level. Some gene expressions involving control of cellular differentiation, proliferation, and apoptosis are overactivation in tumor. Capable of regulating and modulating gene expression, small interfering RNA (siRNA) gene therapy has received considerable attention as a novel therapeutic modality. Besides controlling expression of the RNA of specific genes, siRNA is a highly promising therapeutic strategy for treating infections, cancer, neurodegenerative diseases, and other illnesses by reducing the expression of problem genes.1−3 Among the subsequent effects of the releasing siRNA interference include posttranscriptional gene silencing (PTGS), apoptosis and proliferation inhibition, thus, achieving successful cancer therapy after endogenous production or artificial introduction into a cell.4,5 Despite some successful clinical case studies, clinical trials with siRNA have failed because of its inherent instability in biological fluids and inability to delivery therapeutic genes in vivo efficiently and specifically.6 Various strategies of siRNA delivery have been investigated, ranging © 2012 American Chemical Society

from injection of the naked siRNA into the target organ to systemic delivery in a nanocarrier.7 Cholesterol groups and aptamer can be linked to modified siRNAs to improve their stability and the target capability.8,9 The cationic and polymerconjugating liposomes show a high transfection, which can be attributed partially to interaction with negative charge of cell membranes and improvement in stability.10−12 Additionally, the cationic nanopartiles encapsulated with siRNA have also been examined to deliver specific cells by using surface ligands that bind receptors on target cells.13,14 Within gene delivery nanocarriers, the therapeutic gene is taken into the cell by endocytosis. For many of these systems, especially non-virusbased ones, the lack of efficient mechanisms for translocating the gene out of the endocytic vesicles represents a major obstacle to achieving the therapeutic potential of the therapeutic gene.15 Importantly, specificity of a treatment must be improved in cancer therapy and the treatment of many other diseases. Received: October 25, 2011 Revised: January 24, 2012 Published: January 30, 2012 664

dx.doi.org/10.1021/bm201746t | Biomacromolecules 2012, 13, 664−675

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Scheme 1. Procedure Involved in Preparing SPLexes Entrapping siRNAa

a The cationic lipid film composed of DOPE and PI was prepared and rehydrated by siRNA solution, generating cationic SPLexes entrapping siRNA. After extruding to form unilamellar nanoscale cationic SPLexes, the PII (PII, folate-PII, 99mTc, or Cy 7-PII) cross-linked to the surface of SPLexes for targeting and image function.

1,4-dioxane and added to a two-necked round-bottle flask with magnetic stirring. Succinic acid (SA, 1.3 mmol) was dissolved in 1,4dioxane and poured into an addition funnel. The reaction was then performed in a 0 °C ice bath with slow dripping of SA solution. The reaction lasted 24 h. The crude product was vacuum-dried and resolved in DCM. Next, carboxyl-cholesterol was washed with ROwater to remove the unreactive SA, catalyst (DPTS), and TEA, followed by vacuum-drying. Additionally, the product was characterized by 1H NMR (chloroform-d): δ 2.59−2.65 (m, CCH2CH2C from succinic acid), 4.58−4.64 (m, CH from cholesterol), 5.58−5.64 (m, CH2O from cholesterol). To prepare cholesterol-NHS ester, carboxyl-cholesterol (1 mmol), N-hydroxylsuccinimide (NHS, 1 mmol), and DPTS (0.1 mmol) were dissolved in dry dichloromethane (DCM) and added to a two-necked round-bottle flask with magnetic stirring. N,N′-Dicyclohexyl carbodiimide (DCC, 3 mmol) was then dissolved in dry DCM and poured into an addition funnel. Next, the reaction was undertaken in a 0 °C ice bath with a slow dripping DCC solution. Following a reaction of 24 h, the product was filtered to remove the byproduct DUU and then freeze-dried. Finally, the product was characterized by 1H NMR (dchloroform): δ 2.59−2.65 (m, CCH2CH2C from succinic acid); δ 2.843 (o-H from NHS); δ 4.58−4.64 (m, CH from cholesterol); and δ 5.58−5.64 (m, CH2O from cholesterol). Synthesis of Fmoc-Lysine (Fmoc)-EMA. EDC (3.0 mmol), HEMA (1 mmol), and DMAP (0.1 mmol) were dissolved in 30 mL of DCM (the mixture cannot dissolve completely and a whitish color also appears) and added to a two-necked round-bottle flask with a magnetic stirrer and an addition funnel. Fmoc-Lys(Fmoc) (1 mmol) was then dissolved in THF and added to the addition funnel. The reaction was then conducted for 18 h in a 0 °C ice bath with a slow

Therefore, this study establishes the carriers for VEGFsiRNA delivery, with the sterically polymer-based liposomal complexes (SPLexes) system consisting of 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) and cholesterol-P (HEMA-lysine; abbreviated as PI). Capable of stabilizing liposome mechanical strength, cationic polymers have the potential to encapsulate with VEGF-siRNA by the electronic interaction between lysine and VEGF-siRNA and adsorbed on the external and inside surface of SPLexes liposome-like structure. Moreover, the versatility of polymer, folate-PEGP(HEMA-His-co-MAAc) (abbreviated as PII), cross-links to the surface of cationic SPLexes (Scheme 1) and the higher negative charge competes with siRNA instead of the ones on the surface of cationic SPLexes. Capable of extending the half-life of the SPLexes in circulation, PII can target the cancer cells by EPR effect. Furthermore, the folate molecules can also actively target the cancer cells whose folate receptors are overexpressed. Notably, PII from the SPLexes surface is disassociated by intracellular pH changes after cancer cell uptakes. The cationic SPLexes can then directly fuse endo/lysosomes into the cytoplasm. Early endosome escape of lipoplexes likely occurs mainly through local destabilization of the cationic liposomes.16

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EXPERIMENTAL SECTION

Synthesis of Cholesterol-NHS Ester. Cholesterol (1 mmol), tetraethylammonium (TEA, 1 mmol), and (dimethylamino)pyridinium-4-toluenesulfonate (DPTS, 0.1 mmol) were dissolved in 665


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Preparation and Optimization of SPLexes. DOPE and PI in an adequately designed molar ratio were dissolved in 3 mL of chloroform/methanol (volume ratio: 1:1) and mixed in a round bottomed flask. A lipid film was formed by rotary evaporation at room temperature. The lipid film conjugated siRNA was rehydrated directly with 2.5 nmol of siRNA in PBS solution (5 mL, pH value 7.4, temperature 65 °C); the solution was treated in sonication for 6 min. Next, the cationic SPLexes were extruded three times (at room temperature) through a polycarbonate membrane filter with 100 nm pores. The PII (dissolved in pH 7.4 PBS) in a well-designed molar ratio was added to the cationic SPLexes and mixed thoroughly for 5 min. The size of PEGylated SPLexes distribution was determined by dynamic light scattering (DLS, Zetasizer 3000HS, Malvern), and the structure was identified by transmission electron microscopy (TEM, Hitachi, H-7500) with 2 wt % uranyl acetate staining. An attempt was made to create the optimal process for a smaller sized SPLex by using STATISTICA software and experimental design as “mixture designs triangular surfaces” and identifying the optimization. Before the optimal experiment, all of the above components are of minimum and effective concentration has to be ranged and amounted. Cross-test design is a relatively simple means of ensuring an effective concentration range of components. The Supporting Information describes in detail the design. According to cross-testing results, in the experimental design of this study, the proportions of the original components ranged from 4 to 7 mg, 2 to 5 mg, and 2 to 4 mg for DOPE, PI, and PII, respectively. Mixture design for the optimal process suggested six experimental points, each one corresponding to a mixture composition. Stability and pH Stimuli of SPLexes. The stabilities of SPLexes were determined by DLS. The SPLexes in PBS were mixed with an equal volume of 10 wt % BSA in PBS solution. The mixture was incubated at 37 °C and determined at time interval ti. The average diameter of SPLexes in PBS before adding BSA was evaluated at t0. The relative size was calculated by the ratio (ti/t0) of particles size. To observe pH stimuli, the pH 7.4 of SPLexes were prepared, adjusted to pH5.0, and analyzed by Gel Chromatography with Sephadex G-50. The eluate solution was determined the zeta potential (Zetasizer 3000HS, Malvern). In addition, gel electrophoresis analysis was also used to identify the pH stimuli of SPLexes. SPLexes encapsulated with ODNs (N/P = 1.4) in various conditions (pH 7.4, 5.0, 4.5, 4.0) were transferred to a 15% tris-borate urea (7 M) denaturing polyacrylamide gel, stained with EtBr, and observed under a UV transilluminator attached with a gel documentation system. Capacity of Encapsulation with siRNA. The optimal N/P ratio (amines (N) of polycation (PI) per phosphate groups (P) of oligodeoxynucleotides (ODNs), in which the sequence is the same with VEGF-siRNA) was varied for encapsulating completely by gel electrophoresis analysis.21,22 SPLexes/ODNs complexes with varying complexation ratios were loaded into the wells of a 15% tris-borate urea (7 M) denaturing polyacrylamide gel. Next, a constant voltage (100 V) was applied to the SPLex-loaded gel in TBE buffer for 60 min. Finally, unannealed ODNs or exposed ODNs in the SPLexes was detected with a UV illuminator after the gel was supplemented with EtBr and calculated using the public domain NIH ImageJ software. Phagocytosis and Cytotoxic. MHS, HeLa, and HCT-116 cells (from FIRDI, Taiwan) were culture in RPMI 116, DMEM, and McCoy’s 5A medium, respectively. All mediums were supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a 5% CO2 atmosphere. The scope of the initial phagocytosis was carried out using a Carl Zeiss LSM 5 PASCAL confocal laser scanning microscope (CLSM). The MHS cells were seeded on coverslides for 24 h and PII, (PII−)-SPLexes, and (PII+)SPLexes were coincubated with 0.1 mg/mL of drug content at 37 °C for 0.5 or 6 h. After mounting the cells with 4 wt % paraformaldehyde, the cells were washed with PBS and permeabilized with 0.5% Triton X100 for 3 min. The cells were washed again with PBS, and 2.5 U/mL rhodamine phalloidin (Molecular Probes) and 2 μg/mL DAPI were added to each dish for 15 min to stain polymerized actin filaments and locate the nucleus. Fluorescence was observed at 532 nm for excitation and an LP filter of 590 nm for actin polymerization detection;

dripping Fmoc-Lys(Fmoc) solution. Following the coupling reaction for 2 h, the solution color became transparent. Finally, the product was washed with saturated NaCl solution three times and characterized by 1 H NMR (DMSO-d6): δ 1.22−1.39 (m, CH2 form lysine (Fmoc)), 1.86 (s, CH3 from HEMA), 2.07−2.09 (m, CH2 from lysine (Fmoc)), 2.86 (t, CH2 from lysine (Fmoc)), 4.08−4.10 (t, CH from lysine (Fmoc)), 4.25−4.30 (m, OCH2CH2O from HEMA), 5.69−5.69, 6.02−6.05 (m, CH2C from HEMA), 7.32−7.35, 7.39−7.42, 7.83− 7.84, 7.87−7.88 (m, from the Fmoc group). Synthesis of Chol-P(HEMA-Lys) (abbreviated as PI). Aminoterminated P(HEMA-Lys(Fmoc)) was prepared by radical polymerization using 2,2′-azo-bis-isobutyrylnitrile (AIBN) and AET-HCl as a initiator and chain transfer agent, respectively. HEMA, Lys (Fmoc)EMA, AET-HCl, and AIBN in methanol/THF (volume ratio was 1:1) were placed in a two-necked, round-bottle flask with a magnetic stirrer. The reaction was conducted at 70 °C for 24 h under nitrogen and then purified by dialysis (Millipore MWCO 1 K). Cholesterol-NHS ester (1 mmol) and NH2-P(HEMA-Lys(Fmoc)) were then dissolved in dry DCM and reacted for 2 h at room temperature. Next, the product was added piperidine until the final conc. 20−25% to remove the Fmoc group. Following the de-Fmoc reaction, the structure of cholP(HEMA-lys) copolymer was determined by 1H NMR (CDCl3 and DMSO-d6, ppm): δ 2.88−2.90 (m, CCH2CH2C from succinic acid); δ 3.00−311 (t, CH2 from lysine); δ 4.86−4.91 (t, CH2 form lysine); δ 4.15−4.19 (t, CH from lysine); δ 4.21−4.23 (m, OCH2CH2O from HEMA); δ 4.21−4.32 (m, CHO from cholesterol); and δ 5.21−5.23(t, CHC from cholestrol). PDI of the copolymer was 1.2 by GPC measurement. Next, the products were determined by attenuated total reflection-Fourier transform infrared (ATR-FTIR). Cholesterol showed revealed the anticipated transmission at 1657 for endodouble bond. Finally, transmission bond of chol-P(HEMA-lys) was obtained at 755 cm −1 , corresponding to C−S−C, and 2361 cm −1 , corresponding to S−H. Synthesis of PEG-P(HEMA-His-co-MAAc), abbreviated as PII. These macroinitiators, ABCPA-Boc-PEG2 and ABCPA-mPEG2 were synthesized by esterification reaction and collected by precipitation twice from diethyl ether.17 The monomer, Boc-histidine-EMA (BocHis-EMA), was esterified by EDC activation.18 To prepare PEGP(HEMA-His-co-MAAc), ABCPA-PEG2, HEMA, histindine-EMA, and MAAc were placed in a two-necked, round-bottom flask with a magnetic stirrer; the mixture was then dissolved in methanol. Next, the reaction was conducted at 70 °C for a 24 h reaction under nitrogen. Following free radical polymerization, the product was purified by precipitation from diethyl ether twice to obtain polymers. Synthesis of Cy7-PII and Folate-PII. The Boc-PEG-P(HEMAHis(Boc)-co-MAAc) block copolymer was dissolved in ethanol/DMF (volume ratio: 1:1) in the presence of Pd/C catalyst and reacted with hydrogen at room temperature for 24 h. The final product, H2N-PEGP(HEMA-His-co-MAAc) was characterized by 1H NMR (DMSO-d6): δ 0.94−0.97 (d, CH3 from HEMA), 1.34 (s, (CH3)C from Boc), 2.07 (s, CH from HEMA), 2.81−2.93 (d, CH2 from his), 3.49 (m, OCH2CH2O from PEG), 3.57 (m, OCH2CH2O from HEMA), 3.87 (t, CH2 from HEMA), 4.19−4.21 (m, NH from his), 7.16 (s, m-H of imidazole), 7.53 (s, o-H of imidazole). The PDI of the copolymer was 1.1 by GPC. Additionally, the products were determined by ATRFTIR: three transmission bonds were obtained at 2880, 2566, and 1715 cm−1, corresponding to CCO, COOH, and COOH. Subsequently, the modified reactions with folate and Cy 7 were carried out by adding folate-NHS and Cy 7-NHS into a pH 7.4 NH2-PII solution, respectively. The reaction was performed at room temperature for 16 h under nitrogen. The final product was purified by dialysis against deionized water for 24 h (molecular weight cutoff 2000 Da), freezedried, and characterized by using a UV−U-3300 spectrophotometer. Synthesis of 99mTc-PII. [99mTc(OH2)3(CO)3]+ was synthesized by direct reduction.19 In total, 2 mg PII was added to 0.5 mL of (30 mCi) [99mTc(OH2)3(CO)3]+ solution and heated at 60 °C for 30 min.20 The purity of product, PEG-P(HEMA-99mTc(CO)3-his-co-MAAc), was identified by thin-layer chromatography (TLC) and GPC (PD-10 columns) with mobile phase saline. An auto γ-counter was used to determine eluate radioactivities. 666


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Scheme 2. Synthesis of Cholesterol-P(HEMA-Lysine)

Apoptosis Analysis. Cells treated with SPLexes encapsulated with VEGF-siRNA were harvested at a density of 1 × 106/mL and then washed with phosphate buffer saline (PBS). After cells were stained simultaneously with FITC-Annexin V (green fluorescence) and the nonvital dye propidium iodide (red fluorescence), they were analyzed by flow cytometry. Biodistribution. The HeLa tumor cell xenograft-bearing BALB/c nude mice (tumor grew to reach 300 mm3)23 were injected intravenously with of 99mTc-Labled (FA±)-SPLexes (∼1 mg/mL) in 100 μL via the tail vein and a trace of 99mTc(CO)3 (1 mCi). The mice were sacrificed at 48 h. Organs of interest were removed and weighed, and sample radioactivity was measured. Standards were prepared and measured along with the sample to calculate the percentage injected dose per gram of tissue (%ID/g).

fluorescence observation was carried out was observed at 405 nm for excitation and LP filter of 461 for detecting the nucleus. MHS, HeLa, and HCT-116 cells were seeded and incubated in 96 well assay plate for 24 h before treating with PII, (PII−)-SPLexes, and (PII+)-SPLexes from 0 to 2 mg/mL for 24 h at 37 °C. Next, the cells were washed and incubated for 24 h. Pipet 20 μL of the combined MTS/PMS solution into each well and incubate the plate for 1 h at 37 °C. Then, the 96-well plate was recorded with the absorbance at 490 nm using an ELISA plate reader. The cells without test were used as negative control test and the method for calculating the cell viability was as below: %cell viability = (Abssample − Abscell free sample)/Abscontrol test

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RESULTS AND DISCUSSION Characteristics of Polymer and SPLexes. Besides safety issues, various biological barriers to efficient gene transfer include biocompatibility, escape of endolysosomal pathway, and role of gene/vector release.24 In this study, the design of SPLexes integrated with a polymeric and liposomal system are expected to be a novel candidate of nanocarrier for gene delivery agents in cancer treatment. Following the preparation, the cholesteric PI can stabilize the structure of SPLexes and showed the cationic charge. The cationic (PII−)-SPLexes that cause an extremely high pKa of ε- and α-amino groups (values are 10.5 and 9.0) from the lysine are potential candidates related to the encapsulation with a negatively charged gene.6 Additionally, cross-linking PII on the surface of SPLexes was designed for prolonging the blood residence time owing to its uncharged, hydrophilic and nonimmunogenic of PEG segment.25,26 Notably, PII is dissociated from the SPLex surface by

Transfection. HeLa cells were seeded at a density of 1 × 105 per well into 6-well plates and incubated 1 day before transfection. The cells were transfected with SPLexes encapsulating VEGF-siRNA against 1 nmol siRNA. Following approximately 24 h of posttransfection, the supernatant was removed and cell pellets were collected for Western blotting and flow cytometer analyses. Western Blotting. The protein level was determined by extracting cellular proteins through 1 mL of lysis buffer, followed by incubation on ice for 30 min. After centrifugation, the supernatant was collected and separated by SDS-polyacrylamide gel electrophoresis with 30 μg of total proteins following transferal to PVDF membrane. The membranes were hybridized with anti-VEGF antibody (purchased from Abcam), and then incubated within secondary antibodies conjugated with horseradish peroxides (purchased from Abcam). The western lighting was developed by using chemiluminescence reagents (Blossom Biotechnologies Inc.). Expose blot with film. Finally, the relative expression of proteins was defined by comparing the band density of each sample with one another after normalization with an internal control (GAPDH protein expression). 667


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Scheme 3. Synthesis of mPEG-P(HEMA-histidine-co-MAAc), mPEG-P(HEMA-99mTc(CO)3-His-co-MAAc), Folate-PEGP(HEMA-histidine-co-MAAc), and Cy7-PEG-P(HEMA-histidine-co-MAAc)

around 1.5, 2.0, and 1.5 mg/mL against Hela and MH-S, as well as HCT116 cells after coincubating 72 h) provided the potential for gene delivery. At a sufficient nitrogen to phosphate (N/P) ratio, the level of copolymers (capable of condensing siRNA to gene interference compatibility) was significantly lower than that of IC50. Experimental Design To Optimize the Size of SPLexes. Many studies have adopted the design of experiments with mixtures and the applied response surface analysis to obtain product compositions or formulations with optimized properties. The mixture design and response surface methodology (RSM) can reduce the number of experimental trials and respond to the effect-related interaction between factors. Size of the optimal particles was incorporated in the preparation of SPLexes by using triangular contour diagrams and triangular three-dimensional surfaces of particles size. Figure 1 shows the experimental design and results, along with calculations from regression analysis made based on the program of “STATISTA”. The numerical results in Figure 1 are represented in a triangular graph, indicating the level curves of modulus of

intracellular pH changes owing to the pKa of MAAc (5.35) and imidazole (6.0) in the histidine after cancer cell uptakes (Scheme 1).27 Amino-terminated P(HEMA-Lys(Fmoc)) was prepared by radical polymerization through use of AET-HCl as a chain transfer agent and coupled with cholesterol-NHS ester to chol-P(HEMA-Lys) (Scheme 2). Next, PEG-P(HEMA-Hisco-MAAc) was synthesized by free radical polymerization; the 99m Tc radiolabeling and Cy7/folate moiety coupling were prepared and used for targeting and imaging (Scheme 3). Two copolymers, (chol-P(HEMA15-Lys10)) and PEG1-P (HEMA27his15-co-MMAc15), were identified by 1H NMR, IR, and GPC (Supporting Information); in addition, their molecular weights were 8410 and 13274, respectively. Positive-charge for the strategy of siRNA packaging was identified and showed the zeta potential was 28.9 ± 2.9 mV. Notably, PI provides the amino group of lysine with the ability to drive the electrostatic self-assembly with siRNA. Additionally, PII with −13.8 ± 1.5 mV zeta potential was associated the surface of cationic SPLexes; in addition, a neutral charge occurred around −2.4 ± 0.2 mV. According to the cell viability assay, PEGylated SPLexes prepared from PI and PII (IC50 were 668


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coefficients, thus, corresponding to the interactions between XY, XZ, and YZ while increasing two or more composition concentrations. The values, that is, 4.8 mg of DOPE, 3.0 mg of PI, and 2 mg of PII, were approximately the optimal conditions to minimize the size of SPLexes. In this case, the minimum value modulus of particle should be expected to be around 71.8 nm; in this experiment, the actual value was 73.5 nm. Image from Transmission Electron Microscopy. The most direct evidence of SPLexes structure comes from TEM, as shown in Figure 2, which is often used to differentiate the core−shell structure of SPLexes. TEM image of the cationic (PII−)-SPLexes suggests that the dark region was inner core of stain agent diffusion (Figure 2a). The lipid bilayer of the SPLexes emerged from the light region. After associating with PII, methacrylic acid groups (from MAAc of PII) and ester bond (from lysine-HEMA of PI and histidine-HEMA of PII) were stained with uranyl acetate; in addition, a thin and dark line were generated outside of lipid bilayer (Figure 2b). The segment of mPEG extended out-shell of SPLexes contained a light region, demonstrating that the anionic PII covered the surface of SPLexes completely. According to the TEM image, SPLexes are dual-shell spherical with a uniform size and the size of the particles resembles that determined by DLS. Stability and pH Stimuli of SPLexes. Liposomes without polymers stabilize in liquid formulation are not stored for extended periods without aggregation and loss of transfection efficiency.30 Stable liposomes with PEG-ligands coupled,31 however, appears to diminish with interaction with the cells and subsequent transfection efficiency. The stable, efficacious, and safe issues are most concerned in gene delivery. For gene packaging and inducement of transfection efficiency, the positive surface charge of SPLexes w/o PII was carried out along with cationic SPLexes in the stable state; meanwhile, the diameter was around 59 ± 0.32 nm in pH 7.4 PBS. After immersing in 5% BSA at 37 °C and pH 7.4, the aggregation was showed while the cationic SPLexes interacted with BSA (Figure 3). As for electronic interaction with the protein, the structure of cationic SPLexes shears to fracture and follows the high degree of cytotoxicity typically associated with cationic charge. The SPLexes associated with PII can be PEG-coated to prevent inactivation with proteins; with our results indicating that size/ PDI of SPLexes preserved the stability over 48 h. Capable of avoiding protein aggregation due to the steric repulsion, the

Figure 1. STATISTICA software and experiment design as “mixture designs and triangular surfaces” were used to identify the optimal SPLexes size.

particle size as a function of the composition, obtained from a quadratic regression. The graph in this figure also represents the experimental points. Notably, an optimal size can be observed near the PII axis and central area of this graph. The smallest value of size of modulus, above 71.8 nm, is around point 6. A second-degree polynomial model to correlate with the above experimental data can be expressed as follows according to the original components:28,29 MR = 133X + 84.7Y + 80.7Z − 61.4XY − 59XZ − 35.2YZ

where MR denotes the expected modulus of particle size of PEGlyated SPLexes under simple compression; X represents the amount of DOPE in weight (mg); Y refers to the amount of polymer PI in weight (mg); and Z is the amount of polymer PII in weight (mg). Based on this quadratic model, the response surface can be obtained for analysis. In this study, the size of SPLexes increased following the presentation of negative

Figure 2. TEM image: (a) (PII−)-SPLexes; (b) (PII+)-SPLexes. 669


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Figure 3. (a) Relative particle size and (b) distribution of (PII−)-SPLexes, (PII+)-SPLexes, and liposome in 5%, 37 °C and pH 7.4 BSA (the initial particles size was 73.5 ± 0.6, 59.6 ± 0.32, and 82.6 ± 7.2 nm for (PII−)-SPLexes, (PII+)-SPLexes, and liposome, respectively, before adding BSA).

the well again. Moreover, the MAAc molecules in PII were protonated to become the uncharged form and detach themselves from the surface of SPLexes in an acidified environment. Simulating the role for leading the SPLexes into the cell via the endocytosis results in PII detaching from the SPLexes; in addition, cationic SPLexes interact with the positive membrane of endo/lysosome. After fusion, leaking siRNA would release into the cytoplasm and trigger gene silencing for treating diseases. Capacity of Encapsulation with siRNA. SPLexes were prepared by identifying the variation of entrapment efficiency on the ODN-to-PI ratio (N/P value) through means of the polyacrylamide gel electrophoresis and using the public domain NIH ImageJ software. Experimental results indicated that both the size of the SPLexes entrapping ODNs (ODNs exist with a similar amount of charge comparison of the same base sequence of VEGF-siRNA) and entrapment efficiency depending on the initial ODN-to-PI ratio (P/N value); in addition, a higher ODN-to-PI ratio increased entrapment efficiency (Figure 5). The binding ratio at maximum entrapment is 0.059 mg ODNs per mg of PI (0.075 mol/mol, negative-topositive charge ratio, i.e., P/N = 1.4). Despite theoretical charge neutralization against P/N 1, the excessive ODNs around 4.2% were not encapsulated with SPLexes. A phenomenon may subsequently occur in which poly-HEMA incorporated with an amino group (lysine) contains the random coil and provides steric hindrance to interact with negatively charged ODNs. Once the ODNs-to-PI ratio was increased, the size of (PII+)SPLexes increased slightly from 73.5 nm for SPLexes along 93.0 nm for a P/N ratio of 1.4. In comparison, with respect to the zeta potential of PEG, the SPLexes with PII maintained an identical value (2−3 mv) while incorporated with siRNA, which is protected with this delivery system. Phagocytosis and Cytotoxicity. Successfully delivering particulate drug formulations to the target sites heavily depends on the ability to prolong the blood circulation by escaping phagocytosis through means of macrophages of the reticuloendothelial system (RES). The principal mechanism is actin polymerization, in which macrophages push the leading membrane edge and engulf particles.34 The actin is transformed into a ring type around the particle and drives the membrane along the particle until it is internalized. Actin polymerization

PEG segment of PII can evade immunity to enhance the carrier accumulation in the target tumor located outside the mononuclear phagocyte system (MPS) regions.32 Additionally, this segment can also provide steric protection from the nuclease degradation. After separation using size-exclusion chromatography, PD-10 column (GE Healthcare), the elute solutions were analyzed by DLS, as shown in Figure 4a. The plots were used to model the signal profile as a function of kilo counts per second (KCPs; i.e., as obtained from the number of particles delivered to the detection unit). An increasing number of KCPs were obtained among the fractions between 7.5 to 12 mL of elute, followed by the zeta potential rising to 8 ± 1.3 mV. The fractions, a relatively low zeta between 16.5 and 18 mL of elute, were collected and purified by centrifugation and a similar molecular weight against PII was identified by GPC. The zeta potential from the PII elute has the same intensity as the zeta potential of (PII+)-SPLexes. This intensity could be created by a small amount of negatively charged PEG.14,33 The particle size decreased from 75.3 ± 1.8 to 61.9 ± 0.4 nm while acidifying for 12 h. The change of size was caused by the protonated and detached PII. These results also related the diameter from the (PII±)-SPLexes (59.6 ± 0.32 and 73.5 ± 0.6 for (PII−)- and (PII+)-SPLexes, respectively). The PII came off from the cationic SPLexes at low pH and the system remained stable because the charges repelled each other and showed the dispersion. According to Figure 4c, the SPLexes that encapsulated the oligodeoxynucleotides (ODNs) and had a P/N (ODN-to-PI) value of 1.5, where the entrapment efficiency is illustrated in section 3.5, were demonstrated to respond to pH stimuli after immersing at pH 7.4, 5.0, 4.5, and 4.0 of PBS. Additionally, the (PII−)-SPLexes were retained in wells without the migration, indicating complete complexation of ODNs. After associating with PII, the SPLexes were not observed in the well, indicating that the PEG of PII provides the steric protection and shield outside of ODNs. Otherwise, the higher negative charge of the P(HEMA-His-co-MAAc) of PII competes with the external surface of ODNs instead of the ones on the surface of cationic SPLexes. The dissociated ODNs migrated to the site of 19 bp against the DNA marker. Following acidification, the PII dissociated from the surface of SPLex and was observed in 670


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Figure 5. Capacity of encapsulation with siRNA.

Figure 6. Confocal images of the phagocytosis from PII, SPLexes w/o PII, and SPLexes with PII. Red fluorescence indicates the location of the actin polymerization of macrophage, and blue fluorescence was localized in the nucleus.

SPLexes. Notably, the critical parameters for mitigating phagocytosis are limited in size, hydrophobicity, electrical charge, and protein adsorption mediated recognition based on RES.35 The MAAc molecules in PII (apparently contained a negative charge in pH 7.4 (−13.8 ± 1.5 mv of zeta potential). The positive charge of (PII−)-SPLexes showed more cytotoxicity, subsequently causing the interaction with a negatively charged cell membrane. Immunological characterizations of nanoparticles (NPs) with varied surface groups influence the levels of complement activation. The complement system would be activated by positively as well as negatively charged nanoparticles (e.g., nanoparticles surface with amine and carboxyl groups) primarily via the alternative pathway, and methoxyl surface groups induced the lowest complement activation.36 The activated complement system would lead to phagocytosis. Despite the cytotoxicity from the charge, associated and neutralized SPLexes were stable and mitigated the phagocytosis, ultimately reducing cytotoxicity. Figure 7 presents the results of MTS assays comparing the cellular toxicity of PII, (PII−)-SPLexes, and (PII+)-SPLexes, with all carrying inactive siRNA. The responses of the cancerous HeLa, HCT116, and noncancerous MHS cells are reported. The (PII+)-SPLexes are substantially less toxic than cationic or anionic materials. In spite of high transfection efficiency by using a cationic vector (e.g., Lipofectamine, DOTAP liposomes), they are known to have marked cytotoxic activity.37−39 When the complement system is stimulated by the charged nanoparticles, the cellkilling membrane attack complex would result and respond to the attacked cell to induce cytotoxicity.40,41

Figure 4. pH stimuli of SPLexes. (a) Gel filtration chromatography for (PII+)-SPLexes solution at pH 5.0. (b) The particles size and distribution of SPLexes in pH 5. (c) SPLexes encapsulated the ODNs were responded after immersing at pH 7.4, 5.0, 4.5, and 4.0 of PBS.

was performed based on fluorescent staining, as shown in Figure 6. Red fluorescence denotes the location of the actin polymerization of macrophage; in addition, blue fluorescence was localized in the nucleus. The cells existed a high intensity of red fluorescence against the coincubation with PII or (PII−)SPLexes and rare intensity in cells coincubated with (PII+)671


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Figure 7. Cell viability by MTS assay. The varieties concentration of PII, (PII−)-SPLexes, and (PII+)-SPLexes cocultured with (a) HeLa, (b) HCT116, and (c) MH-S cells for 72 h.

Figure 8. Inhibition of gene target expression and induction of apoptosis using siSPLexes. (a) Evaluation of silence efficiency by Western blot analysis; apoptosis detection from transfection of (b) nontreatment, (c) free siRNA, (d) (PII−)-siSPLexes, (e) siLipofectamine, (f) (FA−)-siSPLexes, and (g) (FA+)-siSPLexes.

reduces VEGF at the message level.42 Western blot results were quantified using scanning and ImageJ software. Results are expressed as integrated optical density. Each sample was

Inhibition of Gene Target Expression and Induction of Apoptosis Using SPLexes-siRNAVEGF. Previous studies have demonstrated that siRNAVEGF treatment in HeLa cells 672


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Figure 9. Efficiency of accumulation in HeLa cells.

was induced in HeLa cells after transfection of siRNAVEGF, as shown in Figure 9. The early and undergoing apoptosis assays revealed that (FA+)-SPLexes induced 49.65 and 14.11% of HeLa cells to early and late of apoptosis upon culturing for 24 h. Despite the similar efficiency of gene silence between (FA+)SPLexes and Lipofectamine, the ratios from cells in the stage of early necrosis were 29.95% related to Lipofectamine treatment against the (FA+)-SPLexes (1.2%). In sum, the (FA+)-SPLexes are stable, efficient, and safe carriers for siRNA delivery. Biodistribution. Figure 10 presents a summary of the biodistribution data from the 99mTc-(FA+)-SPLexes and 99mTc(FA−)-SPLexes conjugated in athymic nu/nu mice bearing small HeLa tumor cell xenografts. After intravenous injection, the highest %ID went to organs of RES, after 48 h, and both SPLexes were taken up obviously by the liver (15.3 ± 2.2%ID/ g and 17.1 ± 2.4% ID/g for 99mTc-(FA−)-SPLexes and 99mTc(FA+)-SPLexes) and spleen (9.2 ± 1.0%ID/g and 11.4 ± 3.6% ID/g for 99mTc-(FA−)-SPLexes and 99mTc-(FA+)-SPLexes). The liver expresses high levels of folate receptor,43,44 and higher opsonization and macrophage recognition would activate after folate conjugation to direct recognition by the liver FR (Kuppfer cell receptor−mediated endocytosis). Reasonably, folate modification nanoparticles would be found after rapid uptake in liver immediately. After elimination and clearing for 48 h, the folate SPLexes showed lower accumulation in liver compared with nonfolated SPLexes. Among the non-RES organ (heart, lung, muscle, bladder, and bone), the accumulation showed a similar level of (FA+)- and (FA−)-SPLexes uptake. Moreover, both folated and nonfolated SPLexes were present in the blood at 48 h after injection (2.4 ± 0.4%ID/g and 3.2 ±

normalized to GADPH content. The efficiencies of gene silence were 75.6 ± 3.1, 54.1 ± 4.2, 32.3 ± 5.3, 68.6 ± 6.2, 13.6 ± 2.2, and 0.87 ± 0.0% for (FA+)-, (FA−)-, (PII−)-siSPLexes, lipofectamine 2000, naked siRNA, and (PII+)-SPLexes w/o siRNA, respectively, against the control test (n = 3, mean ± SD; Figure 8a). After entering the endocytic compartment, although the neutralized surface of (PII−)-SPLexes with the siRNA could not easily fuse with lysosome, more gene silence occurred than the free siRNA did. Our results further demonstrated that the internalization of (PII−)-SPLexes was 6.8 ± 1.4, which had a higher efficiency than 0.5 ± 0 of siRNA (the fluorescence formed TAMRA-siRNA; Figure 9a). Despite the stability causing the association of the polyion polymers in serum, the (FA−)-SPLexes showed the lower inhibition of VEGF expression than lipofectamin did. This finding may be owing to the fact that the weak negative charge of PEG on the outer shell of SPLexes has a lower affinity to the cell membrane and lower internalization efficiency (8.4 ± 3.2) than that with the positive charge lipofectamine. The intensity of florescence from TAMRA-siRNA of (FA+)-SPLexes was 35.0 ± 3.3. Capable of targeting tumor cells with an amplified folate receptor, the folate moiety provides the efficiency via the receptor-mediated endocytosis. Those results were also verified from the 30.0 ± 2.8 of Cy7 florescence in (FA+)-SPLexes in contrast with 0.7 ± 0.1 of Cy7 florescence in (FA−)-SPLexes. Moreover, the liopoplex particles can escape the early endosomes and be released into cytoplasm when PII protonated and detached itself in endo/lysosomal vesicles. Additionally, VEGF decreases the gene expression of gene products involved in antiapoptosis and proliferation. Apoptosis 673


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ACKNOWLEDGMENTS

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REFERENCES

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The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financial support (982320-B-033-002-MY2 and 99-2120-M-033-001). National Synchrotron Radiation Research Center is appreciated for kindly providing the CLSM observation.

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Figure 10. Biodistribution data of Tc99m-(FA+)-SPLexes and Tc99m(FA−)-SPLexes in HeLa tumor cell xenograft-bearing athymic nude female mice (n = 4). Data are presented as percentage injected dose per gram (%ID/g).

1.3%ID/g, respectively), with a slightly lower blood level of 99m Tc-(FA+)-SPLexes compared with the nonfolated conjugate due to higher liver uptake. Furthermore, by comparing these results with tumor uptake in mice, the higher level of folate SPLexes (8.9 ± 2.0%ID/g) tumor uptake in FR-overexpressing tumors was observed compared with nonfolate SPLexes (5.3 ± 1.1%ID/g).

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CONCLUSIONS Due to free therapeutic oligonuclestides, that is, siRNA, necessary for treatment and their degradation and rapid clearance from the circulation, their potential nonspecific effects are limited to their ability to investigate certain target tissues. Our findings suggest that down-regulation of VEGF expression encapsulates siRNA efficiently and stably via the dual-shelled structure of SPLexes leads to escape of endo/ lisosome from their markedly pH sensitivity and accumulates in HeLa cells to shutdown gene expression and induce apoptosis. The versatility of this carrier system and their “programmable” feature likely enhance the therapeutic value of antisense oligonuclestides, ribozymes, siRNA, and triple-helix forming oligonuclestides.

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ASSOCIATED CONTENT

* Supporting Information S

Description of the spectrum analysis of copolymers by 1H NMR, ATR-FTIR, UV−visible, and radiochemical purity, the conditions for experiment design to optimize the size of SPLexes, and zeta potential for PII, (PII−)-SPLexes, and (PII+)SPLexes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.-H.H.); [email protected] (C.-L.L.). Notes

The authors declare no competing financial interest. 674


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