Transdermal delivery of PeptiCRAd cancer vaccine using microneedle patches

Abstract

Microneedles (MNs) are a prospective system in cancer immunotherapy to overcome barriers regarding proper antigen delivery and presentation. This study aims at identifying the potential of MNs for the delivery of Peptide-coated Conditionally Replicating Adenoviruses (PeptiCRAd), whereby peptides enhance the immunogenic properties of adenoviruses presenting tumor associated antigens. The combination of PeptiCRAd with MNs containing polyvinylpyrrolidone and sucrose was tested for the preservation of structure, induction of immune response, and tumor eradication. The findings indicated that MN-delivered PeptiCRAd was effective in peptide presentation in vivo, leading to complete tumor rejection when mice were pre-vaccinated. A rise in the cDC1 population in the lymph nodes of the MN treated mice led to an increase in the effector memory T cells in the body. Thus, the results of this study demonstrate that the combination of MN technology with PeptiCRAd may provide a safer, more tolerable, and efficient approach to cancer immunotherapy, potentially translatable to other therapeutic applications.

Keywords: Adenoviral vector; Cancer therapy; Microneedles; Vaccine.

Graphical abstract

Fig. 1

Characterization and Mechanical Assessment of PVP-Sucrose MNs. (A) Assessment of MN detachment from the base after 60-s insertion into ex vivo porcine skin, demonstrating the dissolution of the MN tips by SEM. (B) Comparison of piercing abilities between PVP and PVP-Sucrose MNs through multiple layers of Parafilm (approximately 100 μm thick each) to ensure that sucrose addition does not compromise mechanical properties or penetration capability. (C) Force-deformation curves for both PVP and PVP-Sucrose MNs, indicating similar mechanical integrity and elasticity, confirming that sucrose addition does not affect the MNs' mechanical suitability for transdermal applications.

Fig. 2

Assessment of Immune Activation by PVP-Sucrose MNs. (A) Schematic representation of the adenovirus infection leading to RFP expression. RFP expression in A549 cells treated with solutions from PVP MNs, PVP-Sucrose MNs, and direct virus application. (B) Cell viability assay demonstrating the effects of viral particle concentration on cell viability. (C) Flow cytometry analysis of CD86 expression in JAWS II dendritic cells, showing significantly higher levels in cells treated with Virus MNs and PeptiCRAd MNs. (D) Presentation of SIINFEKL peptide on MHC class I molecules, observed in Peptide MNs and PeptiCRAd MNs but absent in Baseline and Virus MN groups. ANOVA was used for statistical analysis, and p-values were set at probabilities ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Fig. 3

Immunogenicity of PeptiCRAd Delivered via MNs and Subcutaneous Injection. (A) Schematic representation of the in vivo experiment. (B) ELISA results measuring Ig specific for the adenovirus component of PeptiCRAd. (C) ELISpot assay results focusing on the T-cell response. (D) Visualization of the ELISpot assay showing the frequency of T-cells activated in response to the SIINFEKL peptide. The MN group displayed a higher density of spot-forming cells, indicative of robust T-cell activation. Statistical analysis was conducted using ANOVA, with p-values set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Fig. 4

Evaluation of PeptiCRAd Delivered via MNs in Murine Melanoma Model. (A) Schematic representation of the in vivo experiment setup, including vaccine preparation, dosing schedule, tumor cell injection, tumor growth monitoring, boosting dose administration, and analysis. (B) Tumor rejection data showing complete tumor rejection in the PeptiCRAd group. (C) Average tumor growth curves for each treatment group. (D) ELISpot assay results measuring T-cell responses against the AD5/3d24 - OX40-CD40L antigen. (E) ELISpot assay results measuring IFN-g as T-cell responses against the SIINFEKL peptide. Statistical analysis was conducted using ANOVA, with p-values set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Fig. 5

Assessment of dendritic cell subsets post PeptiCRAd MN vaccination. (A) Quantification of dendritic cell subsets, showing an increased percentage of cDC1 cells and a decrease in cDC2 cells in the MN containing PeptiCRAd group compared to other groups. (B) Contour plots from flow cytometry analysis depicting the distribution of cDC1 and cDC2 cells across different treatment groups. (C) t-SNE maps illustrating the higher number of cDC1 populations in the MN containing PeptiCRAd group. Statistical analysis was conducted using ANOVA, with p-values set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Fig. 6

Analysis of T Cell Responses in PeptiCRAd MN Vaccinated Mice. (A) Quantification of IFN-g release by CD8⁺ T cells after stimulation with media or SIINFEKL. (B) Quantification of TNF-a release by CD8⁺ T cells. (C) Contour plots from flow cytometry analysis depicting IFN-g producing CD8⁺ T cells. (D) t-SNE plots illustrating the distribution of CD8⁺ T cell populations in different treatment groups. (E) Quantification of memory T cell phenotypes, showing an increase in effector memory T cells and a decrease in naïve memory T cells in the MN containing PeptiCRAd group. (F) Heat map representing the memory T cell phenotypes in different treatment groups. Statistical analysis was conducted using ANOVA, with p-values set at ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.