Abstract
INTRODUCTION
RALA peptide has significantly improved the potency of a DNA vaccine where the E6/E7 antigens were delivered via polymeric microneedle (MN) patches; successfully inducing potent cytolytic T-cell responses, delaying tumour initiation in a prophylactic model, and slowing tumour growth in a therapeutic model of cervical cancer [1–3]. Messenger RNA (mRNA) vaccines are more potent than DNA, with smaller quantities stimulating greater immune responses [4,5]. In this study, mRNA has been designed to correlate with the stages of CRPC and formulated into nanoparticles (NPs) with RALA, lyophilised and loaded into MN patches for delivery to resident immune cells in the skin.
METHODS
RALA complexed mRNA in NPs, lyophilised and characteristics of NPs were assessed by DLS. In vitro functionality was assessed by transfection in HaCaT keratinocyte, NCTC 929 fibroblast and DC2.4 dendritic cells. Following loading of RALA/mRNA NPs into polyvinyl alcohol (PVA) MN patches, strength and penetration across the stratum corneum were analysed using optical coherence tomography, and NP integrity and functionality assessed. In vivo NP release from MN patches and gene expression were analysed in C57/BL6 mice.
RESULTS AND DISCUSSION
RALA/mRNA formed stable NPs and transfected HaCaT, NCTC 929 and DC2.4 cells successfully, with negligible toxicity. NP loaded MN patches were resistant to compression with no fracturing observed; indicating MN/NP patches retain integrity under application pressure. Lyophilisation of NPs facilitated increased mRNA dose delivery, and MN/NP integrity and functionality confirmed that RALA protects genetic cargo in the MN polymeric matrix. Following in vivo application, NPs were released from the MN patch resulting in localised gene expression, demonstrating the functionality of the MN/RALA/mRNA.
CONCLUSION
This technology consists of i) RALA peptide, to condense mRNA into NPs, protect from degradation, facilitate intracellular delivery of mRNA; and ii) a polymeric MN patch encapsulating NPs, dissolving upon intradermal insertion, releasing mRNA cargo to skin-resident dendritic cells. Future work will utilise the MN/RALA/mRNA platform to deliver clinically relevant tumour-associated antigens (TAAs); stimulating an immune response for both prophylactic and therapeutic vaccination against castrate resistant prostate cancer.
RALA peptide has significantly improved the potency of a DNA vaccine where the E6/E7 antigens were delivered via polymeric microneedle (MN) patches; successfully inducing potent cytolytic T-cell responses, delaying tumour initiation in a prophylactic model, and slowing tumour growth in a therapeutic model of cervical cancer [1–3]. Messenger RNA (mRNA) vaccines are more potent than DNA, with smaller quantities stimulating greater immune responses [4,5]. In this study, mRNA has been designed to correlate with the stages of CRPC and formulated into nanoparticles (NPs) with RALA, lyophilised and loaded into MN patches for delivery to resident immune cells in the skin.
METHODS
RALA complexed mRNA in NPs, lyophilised and characteristics of NPs were assessed by DLS. In vitro functionality was assessed by transfection in HaCaT keratinocyte, NCTC 929 fibroblast and DC2.4 dendritic cells. Following loading of RALA/mRNA NPs into polyvinyl alcohol (PVA) MN patches, strength and penetration across the stratum corneum were analysed using optical coherence tomography, and NP integrity and functionality assessed. In vivo NP release from MN patches and gene expression were analysed in C57/BL6 mice.
RESULTS AND DISCUSSION
RALA/mRNA formed stable NPs and transfected HaCaT, NCTC 929 and DC2.4 cells successfully, with negligible toxicity. NP loaded MN patches were resistant to compression with no fracturing observed; indicating MN/NP patches retain integrity under application pressure. Lyophilisation of NPs facilitated increased mRNA dose delivery, and MN/NP integrity and functionality confirmed that RALA protects genetic cargo in the MN polymeric matrix. Following in vivo application, NPs were released from the MN patch resulting in localised gene expression, demonstrating the functionality of the MN/RALA/mRNA.
CONCLUSION
This technology consists of i) RALA peptide, to condense mRNA into NPs, protect from degradation, facilitate intracellular delivery of mRNA; and ii) a polymeric MN patch encapsulating NPs, dissolving upon intradermal insertion, releasing mRNA cargo to skin-resident dendritic cells. Future work will utilise the MN/RALA/mRNA platform to deliver clinically relevant tumour-associated antigens (TAAs); stimulating an immune response for both prophylactic and therapeutic vaccination against castrate resistant prostate cancer.
| Original language | English |
|---|---|
| Publication status | Published - 27 Oct 2019 |
| Event | International Society for Vaccines Annual Congress 2019 - EU, Ghent, Belgium Duration: 27 Oct 2019 → 29 Oct 2019 https://www.immunology.org/events/international-society-for-vaccines-annual-congress-2019 |
Conference
| Conference | International Society for Vaccines Annual Congress 2019 |
|---|---|
| Country/Territory | Belgium |
| City | Ghent |
| Period | 27/10/2019 → 29/10/2019 |
| Internet address |
