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Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes
Published on: July 23, 2016
PEGylation of lipoplexes: The right balance between cytotoxicity and siRNA effectiveness
Anna Lechanteur1, Tania Furst2, Brigitte Evrard2
1Laboratory of Pharmaceutical Technology and Biopharmacy, CIRM, University of Liège, Liège 4000, Belgium; Laboratory of Experimental Pathology, GIGA-Cancer, University of Liège, Liège 4000, Belgium.
Abstract:
The delivery of small interfering RNA (siRNA) is an attractive therapeutic approach to treat several pathologies, such as viral infections or cancers. However, the stability and the efficacy of these biotherapies are still a major obstacle to their use. Cationic liposomes (DOTAP/Chol/DOPE 1/0.75/0.5M ratio) have been complexed to siRNA (lipoplexes) in order to be administrated by the vaginal route, in the context of HPV16 induced cervical preneoplastic lesions. To overcome the constraint of the cervico-vaginal mucus, PEGylation is required to allow the diffusion of lipoplexes through it. Thereby, PEGylated lipoplexes coated with three types of polyethylene glycol (PEG) as DSPE-PEG2000, DSPE-PEG750 or C8-PEG2000-Ceramide (Ceramide-PEG2000) at different densities have been developed and characterized. PEGylated lipoplexes were successfully prepared and showed a hydrodynamic diameter around 200nm, appropriate for vaginal application. In vitro assays on HPV16 positive cell lines revealed that a positive charge of PEGylated lipoplexes allows a higher mRNA knockdown by siRNA. However, the cationic property is also associated to cytotoxicity. The addition of a high percentage of PEG prevented this toxicity but seemed also to reduce siRNA endosomal escape, probably by steric hindrance. The decreasing of PEG density of Ceramide-PEG2000 to 20% allows the release of siRNA and in consequence, biological activities, contrarily to DSPE-PEG. These results suggest that Ceramide-PEG is more appropriate for siRNA delivery compared to DSPE-PEG. In conclusion, the right balance between cytotoxicity and siRNA effectiveness has been found with the transfection of lipoplexes coated with 20% of Ceramide-PEG2000. This new nanovector could have a high potential against multiple mucosal diseases, such as human papillomavirus-induced genital lesions.
Insights
Developing novel PEGylated lipoplexes for vaginal siRNA delivery improved efficacy against HPV16-induced lesions. Ceramide-PEG demonstrated better siRNA release and therapeutic potential compared to DSPE-PEG.
Area of Science:
- Biotechnology and Nanomedicine
- RNA Therapeutics
- Gynecologic Oncology
Background:
- Small interfering RNA (siRNA) offers therapeutic potential for viral infections and cancers but faces challenges in stability and efficacy.
- Cervico-vaginal delivery of siRNA faces obstacles due to mucus barriers, necessitating advanced delivery systems.
- Human papillomavirus (HPV) type 16 is a primary cause of cervical preneoplastic lesions, highlighting the need for effective local therapies.
Purpose of the Study:
- To develop and characterize PEGylated lipoplexes for vaginal delivery of siRNA targeting HPV16-induced cervical lesions.
- To evaluate the impact of different polyethylene glycol (PEG) coatings and densities on lipoplex properties, including diffusion, toxicity, and siRNA delivery efficacy.
- To identify an optimal PEGylated lipoplex formulation balancing cytotoxicity and therapeutic effectiveness for mucosal diseases.
Main Methods:
- Cationic liposomes (DOTAP/Chol/DOPE) were complexed with siRNA to form lipoplexes.
- Lipoplexes were PEGylated using DSPE-PEG2000, DSPE-PEG750, or C8-PEG2000-Ceramide at varying densities.
- Characterization included hydrodynamic diameter measurement and in vitro assays on HPV16-positive cell lines to assess mRNA knockdown, cytotoxicity, and siRNA release.
Main Results:
- PEGylated lipoplexes exhibited a suitable hydrodynamic diameter (~200nm) for vaginal application.
- Positive charge enhanced siRNA efficacy but increased cytotoxicity; high PEG density reduced both.
- Ceramide-PEG2000 at 20% density facilitated siRNA release and biological activity, outperforming DSPE-PEG formulations.
- A formulation with 20% Ceramide-PEG2000 achieved a balance between reduced cytotoxicity and effective siRNA delivery.
Conclusions:
- PEGylated lipoplexes, particularly those coated with 20% Ceramide-PEG2000, represent a promising nanovector for vaginal siRNA delivery.
- This optimized formulation overcomes mucus barriers and demonstrates potential for treating HPV-induced genital lesions and other mucosal diseases.
- The study successfully balanced siRNA effectiveness and safety, paving the way for advanced mucosal therapies.
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