Article Cite This: Biomacromolecules XXXX, XXX, XXX−XXX
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Tumor Chemosensitization through Oncogene Knockdown Mediated by Unique α‑Tocopherylated Cationic Geminis Mohini Kamra,† Bappa Maiti,†,‡ Akanksha Dixit,# Anjali A. Karande,# and Santanu Bhattacharya*,†,‡,§ †
Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India School of Applied and Interdisciplinary Sciences, Indian Association for the Cultivation of Science, Kolkata 700 032, India # Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India § Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India Biomacromolecules Downloaded from pubs.acs.org by UNIV OF EDINBURGH on 03/26/19. For personal use only.
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ABSTRACT: Herein, siRNA transfection efficiency of a unique set of α-tocopherylated gemini lipids has been established in vitro and in vivo. High efficacy of oncogene silencing achieved using the biomacromolecular assembly, formed from siRNA complexes of co-liposomes containing an α-tocopherylated gemini lipid, has been utilized for tumor regression via chemosensitization. Delivery studies with the gemini bearing hydroxyethyl headgroup with octamethylene spacer (TH8S) pointed to a higher siRNA transfection efficacy than its analog without hydroxyethyl group (T8S). Owing to p53 upregulation, transfected cells showed enhanced sensitivity to the chemotherapeutic agent, doxorubicin. Studies in murine model revealed significantly low levels of survivin mRNA in xenograft tumors injected with siRNA lipoplexes, leading to effective inhibition of tumor growth and an increase in sensitivity of the tumors toward doxorubicin. These findings enable us to propose the anti-survivin siRNA carrying TH8S co-liposomes as a potent member of cancer management strategies using suicide gene therapy.
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INTRODUCTION Gene therapy has been looked upon as a powerful tool for the development of an efficient anticancer regimen. The ability to maneuver gene expression by means of RNA interference (RNAi) has strengthened the ground for gene therapists worldwide.1,2 The strategy involves the use of small interfering RNA fragments (siRNA) of 21−23 nucleotides that induce the sequence-specific destruction of complementary mRNA.3,4 Although siRNA delivery has achieved therapeutic silencing,5−7 the translation of the RNAi mechanism to successful cancer treatment at the clinical level is impeded by their small size and high negative charge.8 Various efforts were made in order to circumvent these limitations by the development of intracellular delivery systems9 belonging to the league of cationic lipids and polymers.10−15 Although these have proved to be efficient in vitro, the therapeutic importance is limited in vivo by their cellular toxicity, serum intolerance, rapid clearance by renal pathway and instability.16−19 These limitations can be overcome by the use of gemini lipids that are prepared by linking the monomeric lipids using a suitable spacer.20−22 Optimization of the nature and length of spacer have resulted in various gemini lipids showing highly efficacious nucleic acid delivery.23−27 Besides their minimal toxicity, gemini lipids owe their excellent transfection properties to their ability to efficiently complex and release nucleic acid molecules.28−36 Most of these formulations consist of the helper lipid, DOPE, © XXXX American Chemical Society
i.e., 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine. DOPE, a zwitterionic phospholipid, is well explored for its pH dependent structural transition property. Due to its preference for the formation of inverted hexagonal (HII) phase in acidic pH, the fusogenicity of the co-liposomes containing DOPE is enhanced.29 This property enables the internalized nucleic acid complexes of DOPE containing coliposomes to disrupt the endosomal membranes and efficiently release the nucleic acid cargo in the cytoplasm of the cell.37 Since vectors of synthetic origin exert immunostimulatory effects, it was hypothesized that a molecule which is essential for the target tissue/cells and which does not have an intracellular biosynthetic pathway, can serve as a highly effective in vivo gene carrier.38 These criteria are fulfilled by vitamins and vitamin E (α-tocopherol) in particular, being nontoxic even at very high doses. It is a lipophilic vitamin, abundant in the body and has various physiological routes of transportation from serum to the liver.39,40 Tocopherol backbone containing monomeric lipids have been reported to deliver pDNA41−43 but with poor transfection efficiency which was overcome by the introduction of hydroxyethyl functionality at the level of headgroup.44 The hydroxyethyl Received: December 9, 2018 Revised: February 11, 2019
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DOI: 10.1021/acs.biomac.8b01751 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamycin and 5 U/mL nystatin. Cultures were maintained at 37 °C with 5% CO2 and relative humidity >95% while passaging 2−3 times a week using 0.25% Trypsin/EDTA. Preparation of Co-liposomes. Autoclaved Wheaton glass vials were taken in which the cationic lipids were dissolved in chloroform and mixed with the chloroform solution of DOPE at the desired molar ratio. Slow evaporation of chloroform, upon rotation under a stream of nitrogen gas, produced thin films which were dried in vacuum for 6 h to remove any traces of the organic solvent. The dried mixed lipid films were hydrated using autoclaved deionized water (Milli-Q) added in a volume so as to prepare a final lipid concentration of 0.3 mM. After hydration at 4 °C overnight, the samples were subjected to repetitive freeze−thaw cycles involving vortexing, chilling on ice and heating at 60 °C. The samples were finally sonicated at 60 °C to yield colloidal suspensions comprising unilamellar vesicles. These were used under sterile conditions and stored at 4 °C when not in use. GFP Knockdown Assay. Flow cytometry was used for assessing the efficiency of siRNA transfection, the GFP silencing capability of different lipoplexes. HEK 293T cells were counted and seeded in a 24well plate at a density of 5 × 104 cells/300 μL/well. Leaving a set of wells as an untreated control, the other wells were treated with Lipofectamine 2000/pEGFP complexes according to the manufacturer’s protocol. After 24 h, a set of wells to be kept as a positive control was supplied with fresh culture medium while the others were treated with anti-GFP siRNA containing lipoplexes at various molar ratios. After another 24 h, cells were harvested by trypsinization, suspended in PBS and their fluorescence emission was measured using the cytometer, BD FACSCanto II. The percentage of cell population exhibiting green fluorescence due to GFP expression when plotted was found to decrease significantly for the gemini coliposomal formulations. Characterization of Lipid/siRNA Complexes. Agarose Gel Electrophoresis (AGE) was used for the assessment of siRNA binding ability of DOPE: TH8S (2:1) liposomes. Aliquots of 300 ng of antiGFP siRNA in 10 μL of RNase free water were mixed with the coliposomal formulations (10 μL) at different molar ratios and incubated at room temperature for about 30 min. The samples were then mixed with loading buffer, loaded onto a 2% agarose gel prepared in 1× TBE buffer and electrophoresed at 60 V for about an hour. The electrophoretic mobility of free siRNA is impeded upon binding with the cationic lipid vesicles and this was visualized by transillumination of the gel using EtBr staining in ImageQuant 4000 (GE) in the fluorescence mode. Similarly, lipid/siRNA complexes at various molar ratios were prepared and analyzed for their size and ζpotential using Malvern Zetasizer NanoZS at 173° backscatter. Since complete siRNA binding at a molar ratio of 40 was observed, the lipoplexes at this molar ratio along with the uncomplexed liposomes were coated on a cleaved mica sheet in a dust free environment. The samples were then air-dried and examined under the Atomic force microscope (JPK instruments). All AFM, DLS and ζ-potential measurements were performed at an siRNA concentration of 50 μg/mL. RT-PCR for Post-Transcriptional Gene Silencing of GFP. Expression of GFP was traced using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). For this, primers were first designed from the known sequence of the GFP gene using NCBI primer blast database, allowing an amplicon size of ∼150 bp. The chosen set of primers were screened for their 3′ self-complementarity, melting temperature etc. after which the final set of primers were selected as GFP FP: 5′-TTCCGCGCGGGTTTTATAGT-3′; GFP RP: 5′CAGTGCTTTAGCCGCTATCC-3′ and were procured after synthesis by Sigma Genosys. HEK 293T cells, grown in 60 mm dishes were treated with siRNA/co-liposome complexes as described earlier, harvested in TRI Reagent 24 h post transfection and total cellular RNA was isolated. 5 μg of RNA was then taken for the preparation of cDNA by reverse transcription reaction using cDNA synthesis kit (Applied Biosystems). Using these cDNA as templates, PCR was carried out using Taq DNA polymerase at an annealing temperature
functionalities are known to improve transfection ability by two plausible mechanisms. One, via hydrogen bonding interactions with the nucleic acids and second, by modifying the extent of hydration of the DOPE lipids in co-liposomal membranes.45 The gemini lipid containing tocopherol backbone was designed and synthesized while achieving the aim of significantly high transfection efficiencies for both pDNA and siRNA.46,47 Tocopherol gemini lipids carrying hydroxyethyl functionality were found to exhibit significantly high pDNA transfection when used as DOPE co-liposomes.48 The present study utilizes the gemini tocopherol lipids with hydroxyethyl headgroup for delivery of an siRNA of therapeutic importance. The survivin gene, belonging to the inhibitor of apoptosis protein (IAP) family,49 plays an essential role in maintaining the viability of cancer cells and has been used as a target for their suicide.50−56 RNAi based anti-survivin approaches have been utilized for not just promoting programmed cell death in cancer cells57−63 but also for enhancing their chemosensitivity.64−68 Chemosensitization refers to the use of a substance that increases the sensitivity of cancer cells toward a particular chemotherapeautic agent.69 It is a known strategy to overcome drug resistance via lowering of the required doses of an anticancer drug, thereby avoiding nontarget cytotoxicity and reducing the negative side-effects. The downregulation of IAP family of proteins for activation of apoptotic pathways is a suitable approach to elicit chemosensitization.70 Having a wellcharacterized target gene at hand, we proceeded with the aim of achieving a strong interference response in cell culture and in the nude mouse model using a stable, effective, and biocompatible vector for anti-survivin siRNA. In this study, siRNA binding and transfection properties of DOPE co-liposomes comprising of the TH8S gemini having octamethylene [−(CH2)8−] spacer, have been explored by means of GFP knockdown. After biophysical characterizations, siRNA lipoplexes at the optimum molar ratio were used for survivin knockdown resulting in the induction of apoptosis in cancer cells. In vivo delivery of anti-survivin siRNA was assessed by measuring the size and weight of xenograft tumors. Successful downregulation of the anti-apoptotic gene in the tumor was then coupled with doxorubicin treatment in order to envisage the chemosensitization effect. A detailed study of this therapeutic siRNA delivery system, its in vitro and in vivo efficacy along with its ability to chemosensitize tumor cells in a nude mouse model is described herein.
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MATERIALS AND METHODS
Materials. The L-α-1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) for the preparation of co-liposomes was purchased from Avanti Polar Lipids. For the culturing of cells, Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Sigma and nystatin was bought from Calbiochem and Gentamycin from Abott. Fetal bovine serum (FBS, Gibco), Trypsin-EDTA (0.25%, Gibco) and 100× Penstrep were all obtained from Invitrogen. Lipofectamine 2000, anti-survivin siRNA for transfection and Prolong Gold Antifade reagent for microscopy were also purchased from Invitrogen. AntiGFP siRNA (GFP duplex) from Dharmacon and Apoptosis detection kit (annexin V-FITC and PI) from BD were used. MTT, TRI reagent, along with primers for GFP, survivin, and p53 were obtained from Sigma. For probing the Western blots, monoclonal antibodies were procured from the respective sources and Chemiluminescence HRP substrate was purchased from Millipore. Cell Culture. HEK 293T (transformed human embryonic kidney cells), HepG2 (human hepatocellular carcinoma) and HepG2e (human hepatocellular carcinoma explant) cells71 were cultured in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with B
DOI: 10.1021/acs.biomac.8b01751 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules of 48.5 °C (optimized based on an initial gradient PCR). In parallel, PCR of the cDNA templates using primers for 18S rRNA (annealing temperature 59 °C) served as an internal control. Amount of PCR product observed was directly correlated with the amount of GFP mRNA present in the cells, which was in turn indicative of the degree of GFP silencing achieved. Analysis of Reduction of Cellular GFP Using Western Blot. Equal numbers of HEK 293T cells were grown in separate 100 mm dishes and the respective pEGFP and anti-GFP siRNA transfections were performed as required. After 48 h of transfection, cells were lysed using NP40 lysis buffer containing Protease inhibitor cocktail. After protein estimation by Bradford Assay, these cell lysates (containing 100 μg of total protein) were electrophoresed in a 12.5% SDS-PAGE gel under reducing conditions along with a prestained protein marker. This was then transferred onto an activated nitrocellulose membrane and blocked for 3 h at room temperature using PBS containing 3% BSA. Primary antibody for GFP, obtained from the culture supernatant of the specific hybridoma clone (1B3A8), was added to the blocking buffer at a dilution of 1:1000 and incubated with the required portion of the blot at 4 °C overnight on a slow rocker. After washing the blot using PBST followed by PBS, HRP conjugated antimouse secondary antibody diluted, 1:3000 in RIA buffer was incubated with the blot for 50 min at room temperature. The blot was again washed with PBST followed by PBS and then probed using Immobilon Enhanced Chemiluminescence (ECL) substrate and imaged using ImageQuant 4000 (GE Healthcare Life Sciences) gel documentation machine. For establishing a loading control, the blot was incubated with HRP conjugated anti-β-actin antibody (Sigma) for 1 h at room temperature before being imaged using ECL reagents. Visualization of Loss in GFP Expression Using Confocal Microscopy. HEK 293T cells were seeded on 18 mm glass coverslips at a density of 2 × 104 cells/well a day prior to treatment. In a protocol similar to the one described above, cells were transfected with pEGFP were treated with TH8S and T8S lipoplexes bearing the siRNA for GFP knockdown. After the treatment was complete, the cells were washed with PBS and mounted onto glass slides using Prolong Gold Antifade (Invitrogen). The slides were then imaged in Leica TCS SP5 inverted microscope under 63× oil immersion objective. Evaluation of Anti-Survivin siRNA Delivery by Cell Viability Assay. Hep G2 cells were counted and seeded in a 96-well plate (1 × 4 10 cells/well) 24 h prior to the time of treatment. These were then incubated with anti-survivin siRNA (sense sequence: 5′-GGACCACCGCAUCUCUAC-A55-3′) at varying concentrations after complexation with DOPE-TH8S lipoplexes at a molar ratio of 40. In parallel, equivalent amounts of co-liposomal formulations were added to another set of wells to count out any cytotoxicity caused by the lipid vector itself. After 48 h of incubation with the cells, 25 μL of a 4 mg/mL stock solution of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide] reagent was added to each of the wells and incubated at 37 °C for another 4 h. Supernatant medium was then removed from all the wells, 200 μL of DMSO was added to each well and the plate was shaken mildly to dissolve the formazan crystals. Absorbance at 570 nm was then read off using VERSAMAX Microplate reader (Molecular Devices) and percentage of viable cells was computed from the same. Based on each of the triplicate treatments from three separate experiments, a plot of %cell viability vs [siRNA]/nM represented the cytotoxicity of the anti-survivin siRNA containing lipoplexes. Apoptosis Assay. Flow cytometry-based apoptosis detection assay was to examine whether this mechanism is operating in the cells transfected with anti-survivin siRNA using the co-liposomal formulations. HepG2 cells were inoculated in a 24-well plate (1 × 105 cells/well) 1 day before the treatment. After the cells had adhered, lipoplexes prepared using 50 nM anti-survivin siRNA and cationic lipids (T8S and TH8S) at a molar ratio of 40 were added to the cells. After 24 h, cells were harvested by trypsinization, washed with PBS and stained with Annexin V-FITC and PI (BD Apoptosis detection kit). These samples were then recorded in the fluorescent channels for
FITC and PI in the cytometer BD FACS Canto II and analyzed in the FACS Diva software. Assessment of Downregulation of Survivin at mRNA and Protein Level. For comparison of the amount of survivin mRNA and p53 mRNA present in the cells, RT-PCR was carried out. The following primers were used along with the annealing temperatures mentioned in parentheses. Survivin gene, FP: 5′-CGACGTTGCCCCCTGCCTG-3′, RP: 5′-AAGGAAAGCGCAACCGGACGA3′(58 °C); p53 gene: FP, 5′-AGCGATGGTCTGGCCCCTCC-3′, RP-5′-GCGCCGGTCTCTCCAGGA-3′(58 °C); GAPDH, FP: 5′TCACCATCTTCCAGGAGCG-3′, RP: 5′-CTGCTTCACCACCTTCTTGA-3′ (58 °C). PCR product was analyzed on a 1% agarose gel containing ethidium bromide while using PCR of the GAPDH gene as an internal control for normalization. For understanding the loss of gene expression, lysates of HepG2 cells both control and transfected, containing 100 μg of total protein, were electrophoresed on a 12.5% SDS-PAGE gel under reducing conditions along with prestained protein ladder. Western transfer on to a nitrocellulose membrane was followed by blocking using 3% BSA in PBS for 2 h at room temperature. Anti-survivin antibody (3F5H5, Novex, Invitrogen) and anti-p53 antibody (DO1, Calbiochem Inc.) were incubated with the respective portions of the blots overnight at 4 °C with gentle rocking. Washing the blot was followed by incubating it with HRP conjugated anti-mouse secondary antibody (P260) for 50 min at room temperature along with the simultaneous incubation of the required portion of the blot with HRP conjugated anti-β-actin antibody (Sigma). All the blots were washed and probed with ECL reagents just before being imaged in the chemiluminescence mode of ImageQuant4000. Endocytosis Analysis. For the analysis of endocytosis mechanism, flow cytometry was performed using Cy5 labeled anti-survivin siRNA. 24 h prior to treatment, 1 × 105 million cells/well were seeded in a 24-well plate. After the cells adhered, the culture medium was replenished with that containing the respective endocytosis inhibitors, i.e., chlorpromazine (CPZ, 10 μg/mL), genistein (GNT, 400 μM), methyl β-cyclodextrin (m-β-CD, 5 mM) and amiloride (200 μM). The cells were then maintained at 37 °C for 2 h, after which the Cy5siRNA carrying lipoplexes were added. The cells were harvested for FACS measurement after 4 h of incubation at 37 °C. Chemosensitization Assay. For the MTT assay, 2 × 104 HepG2 cells/well were seeded in a 96-well plate. After 24 h, cells were coincubated with 20 μM (
