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Drug Delivery

Drug Delivery - Poster Session D

Poster J5 - PolyCatechin Nanoparticle Drug Delivery Platform

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Given the ability of polymeric nanoparticles (NPs) to encapsulate multiple immunity activating molecules, NPs are attractive platforms for improving the immunogenicity of subunit vaccines, which are composed of protein antigens and adjuvants. Here, we introduce a nanoparticle (NP) subunit vaccine platform comprised of PolyCatechin (pCAT) nanoparticles (CNP) decorated with the model antigen ovalbumin (OVA), a toll-like receptor 7 agonist, and loaded with Resiquimod (R848). Mice vaccinated with R848-loaded OVA-decorated CNPs (O-RCNPs) on days 0 and 21 experienced a more robust humoral and cellular immune response (i.e., OVA-specific IgG concentration, CD4 and CD8 T cell response) than mice vaccinated with OVA admixed with R848-loaded bovine serum albumin (BSA)-decorated particles (B-RCNPs) or OVA admixed with free R848. Given their ease of formulation and ability to induce both humoral and cellular immune responses and ability to be decorated with various protein antigens, we anticipate that antigen-decorated RCNPs have the potential to generate immunity against numerous infectious diseases and malignancies.

Materials and

Methods:  Synthesis of nanoparticles:

The synthesis of pCAT was catalyzed by acetaldehyde after dissolving (+)-catechin hydrate in acetic acid and ethanol solution (Fig. 1) and then precipitated via centrifuge, wash, and dried in vacuum. H1NMR spectroscopy and gel permeation chromatography confirmed catechin polymerization.

CNPs were formed via solvent displacement method by adding a methanol/R848/pCAT solution dropwise into rapidly stirred PBS. CNPs were then purified through centrifugation, wash, and lyophilized. Nanoparticle size was measured using dynamic light scattering and R848 loading per particle was determined by HPLC.

As described in a previous report (Zhou D. et al., ACS Appl Mater Interfaces, 2020), to covalently attach proteins to CNP surfaces, NaIO4 was used to oxidize their surfaces to bind OVA or BSA in pH 8.5 buffer (Fig. 2). Protein binding was characterized by gel electrophoresis and NanoOrange® protein quantification assay.

In vitro and in vivo trials:

Bone marrow-derived dendritic cells (BMDCs) from C56BL/6 mice were treated for 8 hours with various concentrations of O-RCNPs or R848. Expression of CD80 and CD40 was determined via flow cytometry.

To demonstrate efficacy in vivo, five groups of mice (n=5 each) were injected subcutaneously with different formulations on day 0 and 21: saline, O-RCNPs, free R848 and free OVA, B-RCNPs and free OVA, or OVA admixed with alum. Mice were bled on days 14 and 28, and OVA-specific IgG titer was quantified by ELISA assay. Mice were sacrificed on day 29 to assess OVA-specific T cell stimulation and CD4 and CD8 T cell activation.

Results, Conclusions, and Discussions: RCNPs have a diameter of 250 ± 20nm. The O-RCNPs had an R848 encapsulation efficiency of 5.1 ± 0.4% and were decorated with 42.8 µg of protein per mg of RCNP. Our in vitro activation assay performed with BMDCs showed that RCNPs elicited increased expression of CD40 and CD80 in a dosage-dependent manner with minimal cytotoxicity (data not shown). Moreover, the mice treated with O-RCNPs induced a comparable OVA-specific IgG response compared to B-RCNPs and free OVA and OVA admixed with alum— an FDA-approved adjuvant that is used in various vaccine formulations (Fig. 3). Analysis of the T cell response in the spleen showed that O-RCNPs induced a more robust CD8 and CD4 T cell response than the other formulations, as evidenced by cytokine production by CD8 and CD4 T cells in ex vivo antigen-restimulated splenocytes (Fig. 4). The percentage of IFN-γ+ and TNF-α+ CD4 T cells treated with O-RCNPs was 18.7-fold higher than that of OVA admixed with alum and 10.6-fold higher than B-RCNPs and free OVA. Additionally, the percentage of IFN-γ+ CD8 T cells from mice treated with O-RCNPs was 5.5-fold higher than that of mice treated with OVA admixed with alum and 4-fold higher than mice treated with B-RCNPs and free OVA.

In conclusion, CNPs provide an effective platform for subunit vaccine delivery. They elicit robust humoral and cellular immune responses, which are both required for protection from numerous infectious diseases, including HIV, malaria, and herpes. Given that protein binding to the surface is mediated by protein surface amines, we anticipate that RCNPs can be decorated with amine-bearing antigens from other infectious diseases. Further investigation is undergoing to optimize the formulation and study the biodistribution and cellular uptake of this platform.

Acknowledgements (Optional):