(M1430-06-34) Development of a Novel Fast Dissolvable Microneedle-Based Delivery System for the Next-Generation Influenza Nanovaccine

Purpose: Influenza is a highly transmissible respiratory virus of global health concern, with frequent mutations and over 8 million cases reported annually. Although a couple of vaccines have been approved, most of these vaccines are administered via the intramuscular route which is painful and has a potential for vaccine hesitancy. Exploring a non-invasive transdermal vaccination approach using fast-dissolving microneedles may enhance influenza vaccine compliance and immune response. While microneedle technology has significantly advanced transdermal drug and protein delivery, its application in controlled antigen release remains largely unexplored for microparticle-based influenza vaccines targeting the H1N1 and H3N2 strains. Thus, we developed a novel fast dissolvable microneedles-based delivery system for the next-generation Influenza Nanovaccine. Our central hypothesis is that antigen-loaded polymeric microparticles with fast-dissolving microneedles will induce a robust immune response. From a manufacturing standpoint, conventional microneedle patch fabrication typically involves solvent casting, where the vaccine antigen is incorporated into a mold and allowed to dry, often assisted by vacuum or heat. Lyophilization, however, is generally challenging as it results in mechanically weak and porous structures due to void formation during sublimation. In this proof-of-concept study, for the first time, we successfully developed lyophilized H1N1 and H3N2-loaded polymeric microparticles and incorporated the payloads into fast-dissolving microneedles that induce a robust and controlled immune response compared to the traditional and more painful intramuscular IM injection.

Methods: Herein, we encapsulated the vaccine antigens, Inactivated Influenza A H1N1 virus (i-Influenza A H1N1) and Inactivated Influenza A H3N2 virus (i-Influenza A H3N2) in a biodegradable Poly (Lactic-co-glycolic acid) (PLGA) polymeric matrix. The adjuvant MF59(AddaVax™) was selected to enhance the immunogenicity of the antigen. The antigen PLGA MPs were prepared using a double emulsion (w/o/w) method, optimized for lyophilization processes, and characterized for their size (nm) and polydispersity index (PDI), and charge (mV) using a dynamic light scattering (DLS) particle size analyzer. The % encapsulation efficiency (%EE) was determined as the amount of antigen successfully encapsulated in the PLGA MPs, which was assessed using a Micro BCA™ (Bicinchoninic acid) Protein Assay. The vaccine MPs were assessed in vitro in dendritic cells (DC 2.4) for immunogenicity and safety using Griess’ nitrite release assay and MTT (3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide) reagent cytotoxicity assay respectively. The dissolving microneedle containing the microparticulate-based vaccine was fabricated by the spin-casting method. Briefly, the microneedle gel base consists of 5% trehalose solution and 10% sodium hyaluronate. The required amount of microparticles equivalent to 20 µg of antigen was weighed and suspended in the trehalose solution and was mixed thoroughly after the addition of sodium hyaluronate. Finally, the pre-weighted PDMS MN mold, 25 mg of gel base, was subjected to centrifugation at 4000 RPM for 15 min at 15 °C and dried in a desiccator overnight. The following morning, a backing layer consisting of 10% sodium hyaluronate solution was added. The dissolving microneedles were characterized before and after administration using the Scanning Electron Microscope (SEM). The adjuvanted vaccine microparticle was administered to mice as one prime (w0) and one boost (w3) via the transdermal route using the dissolving microneedles to test the in vivo vaccine efficacy. The virus-specific serum antibody (IgG, IgG1, IgG2a, IgA) levels were assessed bi-weekly using an enzyme-linked immunosorbent assay (ELISA).

Results: The recovery yield of MPs was greater than 80%w/w. The characterization assessments revealed the following 1.) size: less than 2μm. 2.) PDI range: 0.1 to 0.5. 3.) Charge: -20mV to -30mV 4.) %EE: 80% to 90% (values are expressed within a range to account for the different MP preparations). The in vitro nitric oxide (NO) released by the DCs upon exposure to the vaccine MPs was assessed as a marker for the innate immune response. The in vitro cytotoxicity assessment showed that the MPs were non-cytotoxic to cells. The height of the dissolving microneedles was 486 µm and the vaccine-loaded dissolving microneedles were very sharp, formed pores on the skin, and quickly dissolved completely within 10 mins after administration. More importantly, following in vivo immunization, we found that the Influenza A H1N1 specific serum IgG and IgA levels and Influenza A H3N2 specific serum IgG and IgA increased significantly (p≤0.0001) compared to the No treatment group. The IgG subtype analyses showed both significantly high levels of serum IgG1 (Th-2/antibody mediated response) and IgG2a (Th-1/cytotoxic mediated response) antibodies specific to both strains (p≤0.01). Additionally, we found significant antibody levels (IgG and IgA) in the lungs which show mucosal immunity (p≤0.0001). We also found a significant expression of CD4+ and CD8+ cells in Spleen and Lymph Node (p≤0.01).

Conclusion: This work, for the first time, shows that our delivery system generates robust antigen-specific antibodies in serum and lung samples and cellular responses. This work could not only highlight the role of fast-dissolving microneedles but also provide practical benefits of using dissolving microneedles as an alternative to current injections.

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