Injectable Biodegradable Chitosan-Alginate 3D Porous Gel Scaffold for mRNA Vaccine Delivery

Abstract

mRNA vaccines have proven to be more stable, effective, and specific than protein/peptide-based vaccines in stimulating both humoral and cellular immune response. However, mRNA's fast degradation rate and low-transfection efficiency in vivo impede its potential in vaccination. Recent research in gene delivery has focused on nonviral vaccine carriers and either implantable or injectable delivery systems to improve transgene expression in vivo. Here, an injectable chitosan-alginate gel scaffold for the local delivery of mRNA vaccines is reported. Gel scaffold biodegradation rates and biocompatibility are quantified. Scaffold-mediated mRNA in vivo transgene expression as well as ovalbumin antigen specific cellular and humoral immune responses are evaluated in vivo. Luciferase reporter protein expression resulting from mRNA lipoplex-loaded gel scaffolds is five times higher than systemic injection. Compared to systemic injections of naked mRNA or mRNA:lipoplexes, elevated levels of T cell proliferation and IFN-γ secretion are seen with in vivo scaffold-mediated mRNA lipoplex delivery. Furthermore, a humoral response (ovalbumin antigen specific IgG levels) is observed as early as week 1 for scaffold-mediated mRNA lipoplex delivery, while protein-based immunization did not elicit IgG production until 2 weeks post-injection. Results suggest that injectable scaffold mRNA vaccine delivery maybe a viable alternative to traditional nucleic acid immunization methods.

Keywords: biodegradable; injectable immunizing scaffold; mRNA vaccine delivery.

Figure 1

Schematic illustration of chitosan-alginate 3D gel scaffold formation. Hydrogel (10CS/50Alg) was first prepared via Schiff-base reaction in an injector by mixing N-succinyl chitosan (S-CS) and oxidized alginate (O-Alg) solutions A). Scaffold was formed by lyophilization of hydrogel overnight B). mRNA lipoplexes were loaded by rehydration of scaffold, which transferred to gel state and was able to be injected through needle (25G × 5/8″) at room temperature C). By either diffusion or degradation of scaffold, mRNA lipoplexes released from gel and were taken by cells in surrounding environment D).

Figure 2

A) Swelling ratio of a chitosan:alginate gel scaffold (10CS/50Alg) over 2 h in PBS at 37 °C, B) in vitro degradation of gel scaffold (with or without mRNA), in PBS-BSA (20% w/v) at 37 °C. C) Concomitant mRNA released (either naked mRNA or Stemfect:mRNA) during gel degradation in PBS-BSA (20% w/v) at 37 °C. Values reported are an average n = 3, ± standard deviation.

Figure 3

A) BHK, B) DC 2.4, and C) JAWsII cell viability over time as indicated by Live/Dead assay. Cells were seeded at 150 000 cells well⁻¹ and 50 μL of 10CS/50Alg hydrogel was injected. Pictures of cells only (left) and cells with gel (right) were taken by inverted microscope (20×) at 1 day, 3 days, and 5 days. The number of live and dead cells were counted by ImageJ software; and cell viability were quantified by calculating the percentage of live cells. pH value of BHK D), DC 2.4 E) and JAWsII F) cell culture medium was also monitored at each time point. Scale bar is 50 μm. Results were averages of three independent experiments carried out in triplicate.

Figure 4

In vivo mRNA protein expression. Individual mice were injected subcutaneously in the back with either 200 μL PBS, naked mRNA (mRNA s.c.), Stemfect:mRNA lipoplexes (LP s.c.), naked mRNA loaded gel (mRNA gel) or Stemfect:mRNA lipoplex-loaded gel (LPgel). A) The mRNA-Luc reporter expression was imaged over 2 days by IVIS imaging. B) The luminescence for LP gel injections were quantified at each time point by Xenogen IVIS Living Image software.

Figure 5

Ovalbumin-specific humoral IgG responses. Individual mice were injected once subcutaneously in the dorsal right flank with either 200 μL PBS, ovalbumin protein solution 100 μg mL⁻¹(Protein), naked mRNA (naked mRNA), mRNA/Stemfect lipoplexes (LP), naked mRNA loaded gel scaffold (mRNA gel) or mRNA:lipoplex-loaded gel scaffold (LPgel). The amount of mRNA applied per mouse 4 μg. Serum samples were collected weekly for a month and analyzed for anti-ovalbumin IgG by ELISA.

Figure 6

Ovalbumin immunization T cell response. Individual mice were injected once subcutaneously in the dorsal right flank with either 200 μL PBS, ovalbumin protein solution (Protein), 4 μg naked mRNA (naked mRNA), mRNA/Stemfect lipoplexes (LP), mRNA loaded gel scaffold (mRNA gel) or mRNA lipoplex-loaded gel scaffold (LP gel). Cells were isolated from recovered lymph nodes 5 days post immunization and analyzed for: T lymphocyte proliferation based on CFSE stain dilution A–C) and quantification of T cell IFN-γ secretion D) upon in vitro stimulation with OVA protein. (n.s., nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).