(M1530-07-42) Bilayer Buccal Vaccine Film with 3D-Printed Application Control for Ambient-Stable Delivery and Mucosal Immune Activation

Purpose: Needle-free film-based vaccines provide an excellent new platform for improving public health and patient compliance. We have previously shown that adenoviral vectors can be stored, without loss of infectivity, within polymeric films at ambient temperature, eliminating the need for cold chain storage1. It is also known that mucosal vaccines containing viral antigens alone typically do not generate robust, protective immune responses2,3. This limited response can be partly attributed to the variability in placement of the film within the cheek, poor antigen retention due to saliva washout, and lack of sustained adjuvant stimulation4,5. To address these issues, we engineered a bilayer vaccine film system composed of a structurally rigid 3D-printed backing layer (BL) and a sustained-release (SR) layer containing both adjuvant and antigen.



Methods: The BL, which functions as a film casting mold and administration tool, was fabricated by fused deposition modeling (FDM) using biodegradable/biocompatible filaments. The SR layer was cast via semi-solid extrusion. Four model adjuvants were examined: These consisted of two model oil-in-water emulsions (AddaVax, AS03) and two novel small-molecule TRP agonists (capsaicin, cinnamaldehyde). Twelve compositions of the SR-layer each containing an incremental emulsion adjuvant load (0-50% (v/v)), were assessed for mechanical integrity, moisture content, and dissolution behavior. TRP-agonist films were characterized for content and release kinetics by HPLC. Emulsion-loaded films were assessed in terms of physical compatibility and droplet stability using DLS (Z-average, PDI). Fourier Transform Infrared Spectroscopy (FTIR) determined excipient-adjuvant compatibility. Surface morphology of films was analyzed by scanning electron microscopy (SEM). X-ray diffraction analysis (XRD) was performed to assess the crystallinity of adjuvant-containing films. Film structural parameters (weight, thickness) were also recorded. Cytotoxicity of test formulations was assessed 24 hours after treatment of HeLa cells with rehydrated film solutions. The ability of formulations to preserve adenovirus integrity during the film forming process and during storage at ambient temperature was assessed using an infectious titer assay¹.

Results: The 3D-printed BL maintained its structural integrity during hydration and handling, effectively serving as a mold for manufacture of the sustained-release layer and potential administration tool. For the SR layer: All adjuvant-containing films displayed consistent weight and thickness profiles (RSD < 7%), with >90% content uniformity noted across replicates. Release rates for capsaicin and cinnamaldehyde films were 0.0147 ± 0.0005 μg/min and 0.00067 ± 0.000001 μg/min, respectively, with steady-state release maintained over 2 hours. Films containing up to 25% v/v AddaVax or AS03 adjuvant retained structural integrity, with PDI values < 0.23, indicating stable emulsion dispersion in the polymer matrix solution. The previously reported film formulation by Bajrovic¹ was limited to ≤10% emulsion adjuvant loading due to film collapse. Our optimized higher-viscosity formulation allowed a 2.5x; increase in loading capacity without compromising uniformity. FTIR spectra exhibited no significant shifts in peaks corresponding to key functional groups, indicating a lack of molecular incompatibility between adjuvants and components of the film matrix. SEM imaging revealed homogenous amorphous matrices for TRP containing films and well-dispersed microstructures in emulsified films. XRD confirmed an amorphous structure for all adjuvant-containing films, except those loaded with AS03, which exhibited discrete peaks, indicative of partial crystallinity or phase separation within the film matrix. Optimized films for each adjuvant preserved >80% cell viability. All films retained >97% adenovirus infectivity immediately after drying, and over 85% recovery after 28 days at 20°C.

Conclusion: The 3D printed multipurpose backing layer could effectively produce films capable of releasing adjuvants in a tunable manner as well as films that support storage of live recombinant adenovirus at ambient temperature. Incorporation of adjuvants within the same matrix of the virus did not compromise this stability. Studies are currently underway to model the ability of the BL and optimized formulations to accelerate permeability of both antigens and adjuvants through the buccal mucosa. The work summarized here represents progress toward thermostable, needle-free mucosal vaccines that could elicit immune responses similar to injectables with potential for rapid field deployment in low-resource settings.

References: Bajrovic I, Schafer SC, Romanovicz DK, Croyle MA. Novel technology for storage and distribution of live vaccines and other biological medicines at ambient temperature. Sci Adv. 2020;6(10):eaau4819.
Yamamoto T, Tanji M, Mitsunaga F, Nakamura S. SARS-CoV-2 sublingual vaccine with RBD antigen and poly(I:C) adjuvant: Preclinical study in cynomolgus macaques. Biol Methods Protoc. 2023;8(1):bpad017.
Mangla B, Javed S, Sultan MH, Ahsan W, Aggarwal G, Kohli K. Nanocarriers-Assisted Needle-Free Vaccine Delivery Through Oral and Intranasal Transmucosal Routes: A Novel Therapeutic Conduit. Front Pharmacol. 2022;12:757761.
Jin Z, Gao S, Cui X, Sun D, Zhao K. Adjuvants and delivery systems based on polymeric nanoparticles for mucosal vaccines. Int J Pharm. 2019;572:118731.
Coffey JW, Gaiha GD, Traverso G. Oral Biologic Delivery: Advances Toward Oral Subunit, DNA, and mRNA Vaccines and the Potential for Mass Vaccination During Pandemics. Annu Rev Pharmacol Toxicol. 2021;61:517-540. doi:10.1146/annurev-pharmtox-030320-092348

Acknowledgements: This work was supported by an American Association of Pharmacology and Experimental Therapeutics (ASPET) David Lehr Research Award and a Lyda Hill Foundation Impact Prize in Engineering (MAC). Student support was through a Building Our Own Talent (BOOT) Graduate Fellowships from the University of Texas at Austin College of Pharmacy, a Phi Lambda Sigma First Year Graduate Research Fellowship from the American Foundation of Pharmaceutical Education (AFPE) and the Nigerian Association of Pharmacists & Pharmaceutical Scientists in the Americas (NAPPSA) (A.J.E).


Graphical Abstract


Figure 2 - Scanning Electron Microscopy of Adjuvanted Films


Figure 3 - Biocompatibility Test