Abstract
INTRODUCTION
Nucleic acid vaccination holds tremendous potential, possessing both prophylactic and therapeutic application. The 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]. Messenger RNA (mRNA) vaccines are much more potent than DNA, with smaller quantities stimulating greater immune responses [2]. In this study, mRNA has been designed to correlate with the stages of CRPC, as evidenced by clinical biopsies and formulated into nanoparticles (NPs) with RALA, lyophilised and loaded into MN patches for delivery to the abundant resident immune cells in the skin.
METHODS
RALA complexed mRNA at a range of N:P ratios and characteristics of NPs were assessed by dynamic light scattering. In vitro functionality was assessed by transfection studies in HaCaT human keratinocyte cells and DC2.4 murine dendritic cells. Following loading of RALA/mRNA NPs into polyvinyl alcohol (PVA) MN patches, MN strength and penetration across the stratum corneum, and NP integrity and functionality were assessed. In vivo NP release from MN patches and gene expression were then analysed in C57/BL6 mice.
RESULTS AND DISCUSSION
RALA formed stable nanoparticles with mRNA (~100 nm diameter and ~20 mV zeta potential) and transfected HaCaT and DC2.4 cells successfully, with negligible toxicity. MN patches loaded with NPs were resistant to compression with no fracturing observed; indicating NP loaded MN patches retain integrity under the pressure of application. NP integrity and functionality upon loading into MN patches confirmed that RALA protects the genetic cargo in the MN polymeric matrix. Following application to mouse ears, NPs were released from the MN patch resulting in localised gene expression, demonstrating the functionality of the MN/RALA/mRNA in vivo.
CONCLUSION
This technology consists of i) RALA peptide delivery system, to condense mRNA into NPs, protect from degradation, facilitate cellular entry and deliver mRNA into the cytoplasm; and ii) a MN patch that encapsulates the NPs within the polymeric matrix, dissolves upon intradermal insertion, releasing the cargo to the skin-resident dendritic cells. Future work will utilise this MN/RALA/mRNA platform to deliver clinically relevant tumour-associated antigens (TAAs); stimulating an immune response against cancer cells expressing the TAAs for both prophylactic and therapeutic vaccination against castrate resistant prostate cancer.
Nucleic acid vaccination holds tremendous potential, possessing both prophylactic and therapeutic application. The 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]. Messenger RNA (mRNA) vaccines are much more potent than DNA, with smaller quantities stimulating greater immune responses [2]. In this study, mRNA has been designed to correlate with the stages of CRPC, as evidenced by clinical biopsies and formulated into nanoparticles (NPs) with RALA, lyophilised and loaded into MN patches for delivery to the abundant resident immune cells in the skin.
METHODS
RALA complexed mRNA at a range of N:P ratios and characteristics of NPs were assessed by dynamic light scattering. In vitro functionality was assessed by transfection studies in HaCaT human keratinocyte cells and DC2.4 murine dendritic cells. Following loading of RALA/mRNA NPs into polyvinyl alcohol (PVA) MN patches, MN strength and penetration across the stratum corneum, and NP integrity and functionality were assessed. In vivo NP release from MN patches and gene expression were then analysed in C57/BL6 mice.
RESULTS AND DISCUSSION
RALA formed stable nanoparticles with mRNA (~100 nm diameter and ~20 mV zeta potential) and transfected HaCaT and DC2.4 cells successfully, with negligible toxicity. MN patches loaded with NPs were resistant to compression with no fracturing observed; indicating NP loaded MN patches retain integrity under the pressure of application. NP integrity and functionality upon loading into MN patches confirmed that RALA protects the genetic cargo in the MN polymeric matrix. Following application to mouse ears, NPs were released from the MN patch resulting in localised gene expression, demonstrating the functionality of the MN/RALA/mRNA in vivo.
CONCLUSION
This technology consists of i) RALA peptide delivery system, to condense mRNA into NPs, protect from degradation, facilitate cellular entry and deliver mRNA into the cytoplasm; and ii) a MN patch that encapsulates the NPs within the polymeric matrix, dissolves upon intradermal insertion, releasing the cargo to the skin-resident dendritic cells. Future work will utilise this MN/RALA/mRNA platform to deliver clinically relevant tumour-associated antigens (TAAs); stimulating an immune response against cancer cells expressing the TAAs for both prophylactic and therapeutic vaccination against castrate resistant prostate cancer.
| Original language | English |
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| Publication status | Published - 09 Sept 2018 |
| Event | 29th Annual meeting of the European Society for Biomaterials (ESB) - Europe, Maastricht , Netherlands Duration: 09 Sept 2018 → 13 Sept 2018 http://www.esb2018maastricht.org |
Conference
| Conference | 29th Annual meeting of the European Society for Biomaterials (ESB) |
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| Country/Territory | Netherlands |
| City | Maastricht |
| Period | 09/09/2018 → 13/09/2018 |
| Internet address |
