Spatially Defined Post-manufacturing Loading of Nanoparticles into Hydrogel-Forming Microneedles.

Efficient transdermal delivery of the genetic material remains a key challenge for noninvasive gene therapy due to skin's barrier properties. While lipid nanoparticles (LNPs) effectively encapsulate and protect mRNA, they cannot freely penetrate the skin. Hydrogel-forming microneedle (HFMN) patches, which swell upon skin insertion, offer a promising strategy to overcome this limitation. However, integrating fragile LNPs into HFMNs without compromising the patch integrity or nanoparticle function remains an unresolved issue. Here, we present a method for the spatially controlled, post-manufacturing loading of mRNA-encapsulated LNPs into HFMN patches. Key parameters─including the HFMN patch height (250, 500, and 800 μm), insertion depth, duration (15-60 s), and repetition (up to six times)─were systematically evaluated in an agarose gel containing a dye model to optimize loading while preserving the microneedle and overall patch integrity. Under optimized conditions, 500 μm HFMN patches loaded with either MC3-DOPE-DiI-LNPs or MC3-DOPE-eGFP-LNPs, at 400 μm insertion depth and 15 s hold time, achieved up to 140 μg of payload after six insertions. Ex vivo experiments using fluorescently labeled empty LNPs confirmed the nanoparticle release. Despite modest recovery, functional studies demonstrated the successful delivery and cellular uptake of functional LNPs into human skin, as confirmed by IVIS imaging. This approach offers a robust and minimally invasive method to load and deliver the genetic material through the skin, supporting the advancement of the microneedle-based transdermal gene therapy.