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Background: Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. It affects an estimated five million Americans. There is no cure for this disease. Assessing lymphatic growth is essential in developing novel therapeutics. Intravital microscopy (IVM) is a powerful imaging tool for investigating various biological processes in live animals. Tissue nanotransfection technology (TNT) facilitates a direct, transcutaneous nonviral vector gene delivery using a chip with nanochannel poration in a rapid (<100 ms) focused electric field. TNT was used in this study to deliver the genetic cargo in the murine tail lymphedema to assess the lymphangiogenesis. The purpose of this study is to experimentally evaluate the applicability of IVM to visualize and quantify lymphatics in the live mice model. Methods and Results: The murine tail model of lymphedema was utilized. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: TNTpCMV6 group receives pCMV6 (expression vector backbone alone) (n = 6); TNTProx1 group receives pCMV6-Prox1 (n = 6). Lymphatic vessels (fluorescein isothiocyanate [FITC]-dextran stained) and lymphatic branch points (indicating lymphangiogenesis) were analyzed with the confocal/multiphoton microscope. The experimental group TNTProx1 exhibited reduced postsurgical tail lymphedema and increased lymphatic distribution compared to TNTpCMV6 group. More lymphatic branching points (>3-fold) were observed at the TNT site in TNTProx1 group. Conclusions: This study demonstrates a novel, powerful imaging tool for investigating lymphatic vessels in live murine tail model of lymphedema. IVM can be utilized for functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy.

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Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. There is no cure for the disease. Clinically, a preventive surgical approach called immediate lymphatic reconstruction (ILR) has gained traction. Experimental gene-based therapeutic approaches (e.g., using viral vectors) have had limited translational applicability. Tissue nanotransfection (TNT) technology uses a direct, transcutaneous nonviral vector, gene delivery using a chip with nanochannel poration in response to a rapid (<100 ms) focused electric field. The purpose of this study was to experimentally prevent lymphedema using focal delivery of a specific gene Prox1 (a master regulator of lymphangiogenesis). TNT was applied to the previously optimized lymphedematous mice tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: group I (sham) was given pCMV6 (expression vector backbone alone) and group II was treated with pCMV6-Prox1. Group II mice had decreased tail volume (47.8%) compared to sham and greater lymphatic clearance on lymphangiography. Immunohistochemistry showed greater lymphatic vessel density and RNA sequencing exhibited reduced inflammatory markers in group II compared to group I. Prox1 prophylactically delivered using TNT to the surgical site on the day of injury decreased the manifestations of lymphedema in the murine tail model compared to control.

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Tissue nanotransfection (TNT), a cutting-edge technique of in vivo gene therapy, has gained substantial attention in various applications ranging from in vivo tissue reprogramming in regenerative medicine, and wound healing to cancer treatment. This technique harnesses the advancements in the semiconductor processes, facilitating the integration of conventional transdermal gene delivery methods-nanoelectroporation and microneedle technologies. TNT silicon chips have demonstrated considerable promise in reprogramming fibroblast cells of skin in vivo into vascular or neural cells in preclinical studies to assist in the recovery of injured limbs and damaged brain tissue. More recently, the application of TNT chips has been extended to the area of exosomes, which are vital for intracellular communication to track their functionality during the wound healing process. In this review, we provide an in-depth examination of the design, fabrication, and applications of TNT silicon chips, alongside a critical analysis of the electroporation-based gene transfer mechanisms. Additionally, the review discussed the existing limitations and challenges in the current technique, which may project future trajectories in the landscape of gene therapy. Through this exploration, the review aims to shed light on the prospects of TNT in the broader context of gene therapy and tissue regeneration.

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Fetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition in vitro and in vivo. Increased NPGPx expression induced increased SOX2 expression and ß-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.

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Hollow needle array-based tissue nanotransfection (TNT) presents an in vivo transfection approach that directly translocate exogeneous genes to target tissues by using electric pulses. In this work, the gene delivery process of TNT was simulated and experimentally validated. We adopted the asymptotic method and cell-array-based model to investigate the electroporation behaviors of cells within the skin structure. The distribution of nonuniform electric field across the skin results in various electroporation behavior for each cell. Cells underneath the hollow microchannels of the needle exhibited the highest total pore numbers compared to others due to the stronger localized electric field. The percentage of electroporated cells within the skin structure, with pore radius over 10 nm, increases from 25% to 82% as the applied voltage increases from 100 to 150 V/mm. Furthermore, the gene delivery behavior across the skin tissue was investigated through the multilayer-stack-based model. The delivery distance increased nonlinearly as the applied voltage and pulse number increased, which mainly depends on the diffusion characteristics and electric conductivity of each layer. It was also found that the skin is required to be exfoliated prior to the TNT procedure to enhance the delivery depth. This work provides the foundation for transition from the study of murine skin to translation use in large animals and human settings.

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BACKGROUND: Tissue ischemia contributes to necrosis and infection. While angiogenic cell therapies have emerged as a promising strategy against ischemia, current approaches to cell therapies face multiple hurdles. Recent advances in nuclear reprogramming could potentially overcome some of these limitations. However, under severely ischemic conditions necrosis could outpace reprogramming-based repair. As such, adjunctive measures are required to maintain a minimum level of tissue viability/activity for optimal response to restorative interventions. METHODS: Here we explored the combined use of polymerized hemoglobin (PolyHb)-based oxygen nanocarriers with Tissue Nano-Transfection (TNT)-driven restoration to develop tissue preservation/repair strategies that could potentially be used as a first line of care. Random-pattern cutaneous flaps were created in a mouse model of ischemic injury. PolyHbs with high and low oxygen affinity were synthesized and injected into the tissue flap at various timepoints of ischemic injury. The degree of tissue preservation was evaluated in terms of perfusion, oxygenation, and resulting necrosis. TNT was then used to deploy reprogramming-based vasculogenic cell therapies to the flaps via nanochannels. Reprogramming/repair outcomes were evaluated in terms of vascularity and necrosis. RESULTS: Flaps treated with PolyHbs exhibited a gradual decrease in necrosis as a function of time-to-intervention, with low oxygen affinity PolyHb showing the best outcomes. TNT-based intervention of the flap in combination with PolyHb successfully curtailed advanced necrosis compared to flaps treated with only PolyHb or TNT alone. CONCLUSIONS: These results indicate that PolyHb and TNT technologies could potentially be synergistically deployed and used as early intervention measures to combat severe tissue ischemia.

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While gene and cell therapies have emerged as promising treatment strategies for various neurological conditions, heavy reliance on viral vectors can hamper widespread clinical implementation. Here, the use of tissue nanotransfection as a platform nanotechnology to drive nonviral gene delivery to nerve tissue via nanochannels, in an effective, controlled, and benign manner is explored. TNT facilitates plasmid DNA delivery to the sciatic nerve of mice in a voltage-dependent manner. Compared to standard bulk electroporation (BEP), impairment in toe-spread and pinprick response is not caused by TNT, and has limited to no impact on electrophysiological parameters. BEP, however, induces significant nerve damage and increases macrophage immunoreactivity. TNT is subsequently used to deliver vasculogenic cell therapies to crushed nerves via delivery of reprogramming factor genes Etv2, Foxc2, and Fli1 (EFF). The results indicate the TNT-based delivery of EFF in a sciatic nerve crush model leads to increased vascularity, reduced macrophage infiltration, and improved recovery in electrophysiological parameters compared to crushed nerves that are TNT-treated with sham/empty plasmids. Altogether, the results indicate that TNT can be a powerful platform nanotechnology for localized nonviral gene delivery to nerve tissue, in vivo, and the deployment of reprogramming-based cell therapies for nerve repair/regeneration.

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Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT), and they were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low-base-pair RNA and was selectively engulfed by wound macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFPin vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to the murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to the TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, the TLNPκ/si-hnRNPA2B1 treated group showed a significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to the TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP is recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties postinjury.

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This work rests on our recent report on the successful use of tissue nanotransfection (TNT) delivery of Ascl1, Brn2, and Myt1l (TNTABM) to directly convert skin fibroblasts into electrophysiologically active induced neuronal cells (iN) in vivo. Here we report that in addition to successful neurogenic conversion of cells, TNTABM caused neurotrophic enrichment of the skin stroma. Thus, we asked whether such neurotrophic milieu of the skin can be leveraged to rescue pre-existing nerve fibers under chronic diabetic conditions. Topical cutaneous TNTABM caused elevation of endogenous NGF and other co-regulated neurotrophic factors such as Nt3. TNTABM spared loss of cutaneous PGP9.5+ mature nerve fibers in db/db diabetic mice. This is the first study demonstrating that under conditions of in vivo reprogramming, changes in the tissue microenvironment can be leveraged for therapeutic purposes such as the rescue of pre-existing nerve fibers from its predictable path of loss under conditions of diabetes.

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