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Nano and Micro Technologies

Nano and Micro Technologies - Poster Session E

Poster Y11 - Innovative mRNA Vaccine Delivery through Dissolvable Microneedles Incorporating Micro-Nano Particles

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  mRNA delivery represents a highly promising area of research due to its key role in the context of vaccine development and gene therapy. However, the inherent instability of mRNA and the challenges associated with successful transfection and achieving the required dosage present significant hurdles in this field.
This study explores dissolvable microneedle patches (DMNPs) for minimally invasive transdermal delivery of mRNA nanocomplex encapsulated in poly(lactic-co-glycolic) acid PLGA microparticles. Currently, lipid nanoparticles LNPs have emerged as successful carriers for mRNA, notably utilized by Moderna and Pfizer during the COVID-19 pandemic. However, LNPs are thermosensitive, limiting further manipulation to achieve a sustained release profile and having poor endosomal escape capability. This study utilizes cell-penetrating peptides CPPs as an alternative mRNA carrier to address these challenges and provide better cell transfection and transcription. Additionally, CPPs/mRNA nanocomplexes (NCs) are encapsulated within poly(lactic-co-glycolic) acid PLGA microparticles (MPs) to develop a sustained release system for mRNA delivery. The multicompartment-designed microneedles lead to initial mRNA release from the DMNP tips and shells containing mRNA NCs, followed by sustained release facilitated by mRNA MPs encapsulated in the microneedle bodies. Furthermore, a microfluidics device is designed to study real-time mRNA cell transfection, serving as a skin-on-a-chip model.

Materials and

Methods:  The microneedle master mold was fabricated using 3D printing laser lithography (Nanoscribe GmbH), featuring an array of 16 by 16 cone-shaped needles with a height of 600 µm. Subsequently, the PDMS mold was prepared using the standard molding method. The mRNA NCs were prepared using a microfluidic device-based micromixer (Dolomite) and characterized by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (cryo-TEM). These NCs were encapsulated in PLGA (38–54 kDa, lactide: glycolide = 50:50, Boehringer Ingelheim) MPs, prepared using the W/O/W double emulsion solvent evaporation technique and characterized using static light scattering (Mastersizer-2000, Malvern, UK).
For microneedle preparation, a combination of polyvinylpyrrolidpillone (PVP, Mw 55 kDa) and hyaluronic acid (HA, Mw 200 kDa) was used along with the mRNA NCs to prepare the microneedle tips, providing a burst release profile. The remaining microneedle cavities were filled with MPs containing mRNA NCs to achieve a sustained release profile. Capillary gel electrophoresis and the Quant-iT RiboGreen assay (Thermo Fisher Scientific) were used to evaluate mRNA concentration and encapsulation efficiency.
A microfluidic device was also designed and fabricated using a micro-milling machine to assay microneedle penetration, mRNA delivery, transfection, and transcription.

Results, Conclusions, and Discussions: mRNA NCs of around 100 nm were prepared with a CPP carrier, the p5RHH peptide sequence, using a microfluidic device by optimizing the flow rate and concentration ratio of CPP and mRNA (Fig. 1a). Characterized by DLS, these NCs showed improved uniformity, compactness, and reproducibility over traditional methods. mRNA stability and encapsulation efficiency were analyzed with electrophoresis Fig. 1b, RiboGreen assay, and fluorimeter spectroscopy, and their fibroblast cell transfection capability in 2D culture was confirmed. Subsequently, these NCs were encapsulated in PLGA MPs to provide a sustained mRNA release profile, leading to prolonged antigen presentation. The MN preparation process as mentioned previously is shown in Fig. 1c. The MPs were also characterized for size and structural criteria (Fig. 1c), and their mRNA encapsulation and cell transfection capabilities were confirmed as in the previous step. In fig.1d the colocalization of Cy5 mRNA and CPP nanocomplexes is depicted.
On the other hand, a multilayer microfluidic platform was designed as a skin-on-a-chip model using human dermis equivalent to study immune cell recruitment and simulate a 3D cell culture environment for fibroblast mRNA transfection. This design features a central hollow part for real-time optical monitoring and a central crown to keep the skin model fixed (Fig. 2a). Immune cell recruitment and differentiation in macrophages as well as the presence of intact cells are shown in Fig. 2b and 2c. This setup provided valuable insights into cellular responses and transfection efficiency, paving the way for advanced studies in potential therapeutic applications and transdermal drug monitoring.

Acknowledgements (Optional):  We acknowledge the grant CN00000041 “National Center for Gene Therapy and Drugs based on RNA Technology” (concession number 1035 of 17 June 2022-PNRR MUR - M4C2 - Investment 1.4 Call "National Centers”, financed by EU- NextGenerationEU), project code (CUP J33C22001130001).