(T1030-06-39) Next-Generation Gene Delivery: Integrating Lipoplex Technology with Trehalose-Stabilized Dissolving Microneedle Arrays

Purpose: Gene delivery via conventional routes faces significant challenges due to physiological, immunological, and technical barriers. Conventional methods often struggle with inefficient transfection, as nucleic acids face rapid degradation by serum nucleases, renal clearance, immunogenicity and reticuloendothelial system recognition before reaching target cells, diminishing therapeutic efficacy. These challenges necessitate innovations like microneedle-based systems to enhance targeting, stability, and patient compliance while mitigating immune risks. Lipoplexes are lipid-based nanoparticles known for enhancing cellular uptake and protecting nucleic acids-the system aims to improve transfection efficiency while minimizing systemic toxicity. While dissolving microneedles is ought to be promising technology for transdermal gene delivery, current literature remains limited in optimizing combinatorial strategies-such as integrating lipid-based carriers (e.g., lipoplexes) into the dissolving microneedle. Current research aims to develop a pain-free gene delivery platform by integrating lipoplex technology with trehalose-stabilized dissolving microneedle arrays, addressing critical challenges via the traditional route. Dissolving microneedles (DMN) strategy enables pain-free, self-administration along with dose-sparing efficiency, leveraging rapid dissolution of polymers ( < 5 minutes) to release lipoplexes containing genetic materials (e.g., siRNA, p-DNA) directly into the dermis of the skin. This enables broad-spectrum utilization of next-generation gene delivery, which is particularly advantageous for immunization and treating localized diseases like psoriasis, alopecia, rheumatoid arthritis, and so on. There are several reports of possible thermal instability of gene delivery via DMN; hence, various sugars were explored to stabilize the lipoplex, preserving genetic cargo during storage and microneedle fabrication, a critical factor for thermal stability in resource-limited settings. This project bridges gap by incorporation of trehalose as a thermal stabilizer alongside lipoplex-microneedle synergy, offering a scalable, painless alternative to traditional injections with its diverse applications in vaccines, autoimmune disorders, and regenerative therapies.

Methods: Liposomes were prepared via ethanol injection method; ethanol-dissolved lipids were injected into a preheated aqueous phase, triggering lipid precipitation and liposome formation. Particle size, polydispersity index(PDI), and zeta potential were assessed using Dynamic Light Scattering(DLS), while morphology was analyzed via cryo-TEM. Lipoplexes were formed through electrostatic complexation between cationic liposomes and the negative charge of the nucleic acid of plasmid-encoded Enhanced Green Fluorescence Protein (p-EGFP) as the model cargo. The ratios of p-EGFP and liposomes were optimized for achieving maximum transfection efficiency in the human colorectal carcinoma(HCT-116) cell line. In-vitro toxicity of polymers used for fabrication of DMN was evaluated via crystal violet staining. DMNs were solvent-casted using polydimethylsiloxane (PDMS) molds (hheight=1000µm), with polymer ratios optimized for fabrication. Lipoplex loading into DMNs was fine-tuned. Mechanical properties(parafilm insertion, fracture force) were tested via texture analyzer, and morphology was assessed via Scanning Electron Microscopy(SEM). Successful insertion of DMNs into rat/porcine skin was confirmed. In-vitro p-EGFP release from trehalose-stabilized DMNs was measured by determining transfection efficiency in HCT116. Ongoing studies include thermal stability enhancement with adjuvants, 2D gene transfection in cell culture inserts, and in-vivo comparisons against conventional route.

Results: Liposomes were characterized by DLS analysis; Fig. 2A revealed liposome hydrodynamic diameter was 88.20 nm, with PDI value of 0.363, indicating monodisperse to moderately polydisperse populations. Zeta potential measurements showed surface charge of +48.8 mV demonstrating colloidal stability; enhanced positive charge could be due to presence of cationic lipids in the liposome. Morphology of the complex liposomal structure was studied by Cryo-TEM imaging (Fig. 2B) confirming spherical morphology, uniformity and unilamellar structure. Pullulan and Polyvinylpyrrolidone K-90 were found to be safe upon treatment with highest concentration of 5%w/v (Fig. 2B) at 48h in HCT116 enabling their selection for fabrication of DMN. DMN morphology was observed using SEM (Fig. 3E) revealing complete MN array formation with sharp tips. Trehalose was added as thermal stabilizer to prevent p-EGFP degradation at room temperature. The mechanical properties of DMNs were characterized; Fig. 3A) illustrates remarkable insertion of trehalose-stabilized lipoplex loaded DMNs with 100% insertions until 600 μm, and >45% until 750 μm depth respectively. Dissolution of polymers (Fig. 3B) within < 5 minutes of lipoplex loaded-DMN upon manual insertion into porcine skin was observed. The optimized trehalose-stabilized lipoplex DMN exhibited the highest fracture force at approximately 44 N indicating sufficient mechanical strength of needle to resist breakage upon manual application. Hematoxylin and eosin imaging of microneedle insertion into excised rat skin as depicted in Fig. 3D a) normal rat skin with intact keratin layer, while b) lipoplex-loaded DMN pierces keratin layer to achieve depth penetration of 596.8µm reaching dermis of the skin. Transfection experiments (Fig. 2C) showed the highest transfection efficiency with lipoplex delivery via trehalose-stabilized DMNs which was comparable to positive control (p-EGFP+ Fugene) at 24h.

Conclusion: Liposomes complexed with p-EGFP formed stable lipoplexes, optimized with trehalose-stabilized DMNs, and confirmed transfection in HCT-116 cell line at 24h. DMNs fabricated via solvent casting demonstrated robust mechanical properties and successful skin insertion. Trehalose-stabilized DMNs enabled efficient in-vitro model cargo release and thermostable patch production, removing reliance on cold-chain storage.


Figure 1. Graphical illustration A) Methodology of liposome preparation via the ethanol injection method and B) Fabrication of a trehalose-stabilized lipoplex-loaded dissolving microneedle.


Figure 2 A) In vitro characterization of liposome. a) particle size and b) zeta potential, B) cryo-TEM imaging, C) in-vitro toxicity of polymers using crystal violet staining in HCT 116 at 48h showing safety at maximum polymer concentration (4x magnification), and D) in-vitro transfection efficiency of Lipoplex-loaded DMNs in the HCT116 cell line at 24h (10x magnification).


Figure 3. In-vitro characterization of Lipoplex loaded DMN A) Parafilm insertion study, B) Dissolution of lipoplex-loaded DMN post-insertion into porcine skin. C) In vitro fracture force analysis of DMN with the highest fracture force of 44 N for trehalose-stabilized DMN and D) hematoxylin and eosin staining and imagining (5x Magnification) of microneedle insertion into excised rat skin showing a) Normal rat skin with intact keratin layer, and b) depth (596.8µm) penetration of Lipoplex-loaded DMN with a pierced keratin layer, and E) scanning electron microscope imaging (40x Magnification) of Trehalose stabilized Lipoplex-loaded DMN.