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Development and Characterization of siRNA Lipoplexes: Effect of Different Lipids, In Vitro Evaluation in Cancerous Cell Lines and In Vivo Toxicity Study

  • Research Article
  • Theme: Translational Application of Nano Delivery Systems: Emerging Cancer Therapy
  • Published:
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ABSTRACT

Cationic liposomes have long been used as non-viral vectors for small interfering RNA (siRNA) delivery but are associated with high toxicity, less transfection efficiency, and in vivo instability. In this investigation, we have developed siRNA targeted to RRM1 that is responsible for development of resistance to gemcitabine in cancer cells. Effect of different lipid compositions has been evaluated on formation of stable and less toxic lipoplexes. Optimized cationic lipoplex (D2CH) system was comprised of dioleoyl-trimethylammoniumpropane (DOTAP), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hydrogenated soya phosphocholine (HSPC), cholesterol, and methoxy(polyethyleneglycol)2000–1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG2000–DSPE). D2CH lipoplexes have shown particle size (147.5 ± 2.89 nm) and zeta potential (12.26 ± 0.54 mV) characteristics essential for their in vivo use. In vitro cytotoxicity study has shown low toxicity of developed lipoplexes as compared with lipofectamine-2000 up to N/P ratio as high as 7.5. Cell uptake studies and gene expression studies have confirmed intracellular availability of siRNA. In addition, developed lipoplexes also showed ~3 times less hemolytic potential as compared with DOTAP/DOPE lipoplexes at lipid concentration of 5 mg/mL. Lipoplexes also maintained particle size less than 200 nm on exposure to high electrolyte concentration and showed >70% siRNA retention in presence of serum showing siRNA protection conferred by lipoplexes. Furthermore, in vivo acute toxicity studies in mice showed that formulation was non-toxic up to a dosage of 0.75 mg of siRNA/kg as lipoplexes and 300 mg lipid/kg as blank liposomes indicating tolerability of lipoplexes at a dose much higher than required for therapeutic use. Promising results of this study warrant further investigation of developed siRNA lipoplexes for cancer treatment.

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ACKNOWLEDGMENTS

The authors acknowledge the TIFAC CORE in NDDS, Government of India, New Delhi, for providing the research facilities to the team and Department of Biotechnology (DBT-SBIRI), New Delhi, India for financial assistance. The authors also acknowledge Dr. Vikram Sarabhai Science Block, DBT-ILSPARE, Faculty of Science, M. S. University, Vadodara for providing facility to carry out cell uptake studies.

Conflict of Interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

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Authors and Affiliations

  1. Pharmacy Department, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara, 390001, Gujarat, India

    Nirav Khatri, Dipesh Baradia, Imran Vhora, Mohan Rathi & Ambikanandan Misra

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  1. Nirav Khatri
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  2. Dipesh Baradia
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Correspondence to Ambikanandan Misra.

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Guest editors: Mahavir B. Chougule and Chalet Tan

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Khatri, N., Baradia, D., Vhora, I. et al. Development and Characterization of siRNA Lipoplexes: Effect of Different Lipids, In Vitro Evaluation in Cancerous Cell Lines and In Vivo Toxicity Study. AAPS PharmSciTech 15, 1630–1643 (2014). https://doi.org/10.1208/s12249-014-0193-9

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