Targeted and Efficient Delivery of siRNA Using Tunable Polymeric Hybrid Micelles for Tumor Therapy

Materials and Methods

Materials. Branched polyethylenimine (PEI, 25 kDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-DEimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Shanghai Yuanye Biological Technology (Shanghai, China). mPEG-NH₂ (2,000Da) was purchased from Yarebio (Shanghai, China). Linolyl chloride (LC) was obtained from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). Survivin siRNA: Sense (5’-3’): ^mGCAGGUUCCU^mUAUCUGUCAdTdT; Antisense (5’-3’): UGA^mCAGA^mUAAGGAACCUG^mC dTdT and fluorescence-labeled survivin siRNA that had Cy3 or Cy5 conjugated to the 5’ end for cellular uptake and bio-distribution studies were synthesized by Ribo Biochemistry (Guangzhou, China). A549 cells were purchased from ATCC (Rockefeller, MD, USA). All chemical reagents used were of analytical grade.

Synthesis and characterization of the synthesized amphiphilic polymers. LA was separately conjugated to the mPEG and PEI using a previously published method (20-22). Briefly, LC dissolved in anhydrous dichloromethane (DCM) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, PR China) was added dropwise to the mPEG₂₀₀₀-NH₂ and PEI_25kDa anhydrous DCM solution, respectively. After 12 h, the reaction mixture was precipitated and washed three times by diethyl ether. The products mPEG-LA and bPEI-LA were obtained by removing diethyl ether by a rotary evaporator (Shanghai Yukang Scientific Instrument Co., Ltd., Shanghai, PR China) and vacuum drying.

Preparation of polymeric hybrid micelle (PHM). PHM was prepared by self-assembly of PEI-LA and mPEG-LA. Different weight ratios PEI-LA and mPEG-LA were mixed together in DCM and sonicated. Then, the mixture solution was evaporated by a rotary evaporator to remove the organic solvent and further vacuumed for 2 h to remove residual organic solvents. To prepare PHM, diethyl pyrocarbonate (DEPC)-treated water (Coolaber, Beijing, PR China) was added to the flasks and sonicated for 2 min. Particle size, zeta potential, and polydispersity index (PDI) of PHM were measured on a Zeta-sizer Nano ZS90 from Malvern Instruments (Malvern, UK) at 25°C.

Hemolytic analysis of PHM. Fresh blood samples from healthy mice were collected from the orbital sinus in heparin-coated tubes. Red blood cells (RBCs) were collected by centrifuging at 3000 rpm for 5 min and washed three times with physiological saline solution. Then, the RBCs were dispersed in the physiological saline solution to obtain a 2% RBCs standard dispersion (v/v). PHM (100 μl, 5 mg/ml) was incubated with 0.5 ml RBCs standard dispersion for 3 h. The suspensions were centrifuged, and the absorbance of supernatant was measured at 450 nm.

Gel retardation assay. Gel retardation assay was conducted to study the ability of PHM to complex siRNA using agarose gel electrophoresis. PHM was first incubated with survivin siRNA for 10 min at different PEI nitrogen-to-RNA phosphate (N/P) ratios. Then, 10 μl of the PHM/siRNA complexes with different N/P ratios were loaded onto an agarose gel (2%, w/v). The electrophoresis was set up at 100 V and run for 10 min in a Tris-acetate-EDTA (TAE) buffer (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, PR China). The gel was stained with ethidium bromide (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, PR China) and imaged by a UV transilluminator (Analytik Jena US LLC., Upland, CA, USA).

Cellular uptake of the PHM/Cy3-siRNA. A549 cells were cultured in Dulbecco's modified eagle medium (DMEM) (Invitrogen Co., Carlsbad, CA, USA) which contained 10% fetal bovine serum (FBS) (Gemini, Woodland, CA, USA) and 1% penicillin-streptomycin (Carlsbad, CA, USA) at 37°C in a humidified atmosphere of 5% CO₂. A549 cells were seeded in 12-well cell culture plates with 1×10⁵ cells/well and cultured for 24 h. Naked Cy3-siRNA and PHM/Cy3-siRNA with an equivalent siRNA concentration of 100 nM were then added to the wells and incubated with the cells for 4 h. A549 cells were then trypsinized, harvested and fixed with 4% (w/v) cold formaldehyde solution (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, PR China). The fluorescence intensity of the cells was measured on a Beckman Coulter EPICS XL flow cytometer (Brea, CA, USA).

The cellular uptake of PHM/Cy3-siRNA was further imaged by confocal laser scanning microscopy (CLSM). A549 cells were seeded at the bottom of a glass flask and cultured for 12 h. The cells were then treated with naked siRNA or PHM/Cy3-siRNA (100 nM) at 37°C. After 4 h of incubation, the medium was removed and the cells were washed with phosphate buffer solution (PBS, 0.01 M, pH 7.4). Then, the cells were fixed with 4% (w/v) formaldehyde for 15 min at room temperature and stained with 4’,6-Diamidino-2-phenylindole (DAPI) (Invitrogen Co., Carlsbad, CA, USA) for 15 min. Uptake of the PHM/Cy3-siRNA complexes from A549 cells was studied using an LSM710 microscope from Carl Zeiss (Oberkochen, Germany).

Cytotoxicity of the PHM/survivin-siRNA. A549 cells were plated in 96-well microtiter plates (8000 cells per well) and cultured overnight. Naked survivin-siRNA, PHM and PHM/survivin-siRNA were added to the wells (survivin siRNA 40 nM, 80 nM, 100 nM). After cultured for 48 h, 20 μl of MTT solution (5 mg/ml) were added to the well and incubated with the cells for 4 h. The medium was removed and 150 μl DMSO was added. Absorbance values at 490 nm were measured on Bio Tek SYNERGY4 (Winooski, VT, USA). Relative cell viability was presented as a percentage of viability treated with different formulations compared to untreated cell samples.

Western blot assay. A549 cells were seeded in 6-well cell culture plates (1×10⁵ cells /per well) for 24 h at 37°C in 5% CO₂ humidified atmosphere. Naked survivin-siRNA and PHM/survivin-siRNA were added to the wells (survivin siRNA 80 nM). At 48 h later, cells were collected and lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Protein fractions were collected, quantified, subjected to polyacrylamide gel electrophoresis, and finally transferred to polyvinylidene fluoride (PVDF) membrane (0.45 um, Merck Millipore, Billerica, MA, USA). The membrane was blocked with 5% BSA for 4 h at room temperature and incubated with survivin Rabbit mAb (71G4B7E, Cell Signaling Technology Inc, Danvers, MA, USA) and GAPDH Rabbit antibodies (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, PR China) at 4°C overnight. Horseradish peroxidase (HRP)-conjugated secondary antibody (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, PR China) was then added and incubated at 4°C for 4 h. The corresponding protein expression was visualized by an imaging system (BioSpectrum600, Analytik Jena US LLC., Upland, CA, USA).

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Figure 1.

The ¹H NMR of PEI-LA and mPEG-LA. PEI-LA and mPEG-LA were dissolved in CDCl₃ and measured in a nuclear magnetic resonance instrument (400 MHZ). The characteristic peaks of PEI-LA and mPEG-LA were marked with the characters (a, b, c, d).

Establishment of a tumor model. The animal experimental protocol was complied with the institutional guidelines and was approved by the Experimental Animal Ethics Committee of the School of Life Sciences, Jilin University (approval number 201805003). BALB/c nude mice (female, 6-8 weeks) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, PR China). Tumor-bearing mice were established through subcutaneous injection of 5×10⁶ A549 cells into the right rear leg of nude mice after the mice were adapted to the new environment for a week.

Biodistribution of PHM/Cy5-siRNA complexes in tumor-bearing mice. Tumor-bearing mice were injected with Cy5-siRNA and PHM/Cy5-siRNA via the tail vein with an equivalent amount of Cy5-siRNA (0.8 nmol). Mice were anesthetized and the biodistribution of the complexes was imaged on an IVIS® spectrum system from Caliper Life Sciences (Hopkinton, MA, USA) at the first, second, and fourth h after administration with an optimized parameter (Excitation at 640 nm and Emission at 680 nm). After 6 h, internal organs (Heart, Liver, Spleen, Lung, and Kidney) and tumors were dissected and then imaged.

Therapeutic efficacy of PHM/siRNA in vivo. When the average tumor volume of nude mice was grown to approximately 100-150 mm³ (Day 0), animals were randomized into three groups, each containing 5 nude mice. Tumor-bearing mice were then injected with saline, free siRNA and PHM/siRNA via intravenous injection every 3 days. The dose of naked siRNA and PHM/siRNA was 4 nmol per mice. The volume of the tumor and body weight were measured every 3 days using a vernier caliper and scale. On day 20, mice were anesthetized with sodium pentobarbital and sacrificed. Vital organs (Heart, Liver, Spleen, Lung, and Kidney) and tumors were dissected in each group and fixed with 4% paraformaldehyde. Tissue sections were cut into 5 microns thick and stained with hematoxylin and eosin (H&E). The expression of survivin in tumor tissue was analyzed by western blot assay.

Statistical analysis. Data were presented as mean±SEM and graphed by Origin 8.0 (OriginLab Corp., Northampton, MA, USA). Statistical analysis of group differences and correlations was determined using Student's t-test. *p<0.05 was considered statistically significant.