Single-Particle Spectroscopic Chromatography Reveals Heterogeneous RNA Loading and Size Correlations in Lipid Nanoparticles.

Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and development usually assumed that LNPs are homogeneous in population, loading density, and composition. Such perspectives are difficult to examine due to the lack of suitable tools to characterize these physicochemical properties at the single-nanoparticle level. Here, we report an integrated spectroscopy-chromatography approach as a generalizable strategy to dissect the complexities of multicomponent LNP assembly. Our platform couples cylindrical illumination confocal spectroscopy (CICS) with single-nanoparticle free solution hydrodynamic separation (SN-FSHS) to simultaneously profile population identity, hydrodynamic size, RNA loading levels, and distributions of helper lipid and PEGylated lipid of LNPs at the single-particle level and in a high-throughput manner. Using a benchmark siRNA LNP formulation, we demonstrate the capability of this platform by distinguishing seven distinct LNP populations, quantitatively characterizing size distribution and RNA loading level in wide ranges, and more importantly, resolving composition-size correlations. This SN-FSHS-CICS analysis provides critical insights into a substantial degree of heterogeneity in the packing density of RNA in LNPs and size-dependent loading-size correlations, explained by kinetics-driven assembly mechanisms of RNA LNPs.

Optimization of lipid nanoparticles for gene editing of the liver via intraduodenal delivery.

Lipid nanoparticles (LNPs) have recently emerged as successful gene delivery platforms for a diverse array of disease treatments. Efforts to optimize their design for common administration methods such as intravenous injection, intramuscular injection, or inhalation, revolve primarily around the addition of targeting ligands or the choice of ionizable lipid. Here, we employed a multi-step screening method to optimize the type of helper lipid and component ratios in a plasmid DNA (pDNA) LNP library to efficiently deliver pDNA through intraduodenal delivery as an indicative route for oral administration. By addressing different physiological barriers in a stepwise manner, we down-selected effective LNP candidates from a library of over 1000 formulations. Beyond reporter protein expression, we assessed the efficiency in non-viral gene editing in mouse liver mediated by LNPs to knockdown PCSK9 and ANGPTL3 expression, thereby lowering low-density lipoprotein (LDL) cholesterol levels. Utilizing an all-in-one pDNA construct with Strep. pyogenes Cas9 and gRNAs, our results showcased that intraduodenal administration of selected LNPs facilitated targeted gene knockdown in the liver, resulting in a 27% reduction in the serum LDL cholesterol level. This LNP-based all-in-one pDNA-mediated gene editing strategy highlights its potential as an oral therapeutic approach for hypercholesterolemia, opening up new possibilities for DNA-based gene medicine applications.

In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix.

T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies.

Liter-scale manufacturing of shelf-stable plasmid DNA/PEI transfection particles for viral vector production.

The transfection efficiency and stability of the delivery vehicles of plasmid DNA (pDNA) are critical metrics to ensure high-quality and high-yield production of viral vectors. We previously identified that the optimal size of pDNA/poly(ethylenimine) (PEI) transfection particles is 400-500 nm and developed a bottom-up assembly method to construct stable 400-nm pDNA/PEI particles and benchmarked their transfection efficiency in producing lentiviral vectors (LVVs). Here, we report scale-up production protocols for such transfection particles. Using a two-inlet confined impinging jet (CIJ) mixer with a dual syringe pump set-up, we produced a 1-L batch at a flow rate of 100 mL/min, and further scaled up this process with a larger CIJ mixer and a dual peristaltic pump array, allowing for continuous production at a flow rate of 1 L/min without a lot size limit. We demonstrated the scalability of this process with a 5-L lot and validated the quality of these 400-nm transfection particles against the target product profile, including physical properties, shelf and on-bench stability, transfection efficiency, and LVV production yield in both 15-mL bench culture and 2-L bioreactor runs. These results confirm the potential of this particle assembly process as a scalable manufacturing platform for viral vector production.

Screening for lipid nanoparticles that modulate the immune activity of helper T cells towards enhanced antitumour activity.

Lipid nanoparticles (LNPs) can be designed to potentiate cancer immunotherapy by promoting their uptake by antigen-presenting cells, stimulating the maturation of these cells and modulating the activity of adjuvants. Here we report an LNP-screening method for the optimization of the type of helper lipid and of lipid-component ratios to enhance the delivery of tumour-antigen-encoding mRNA to dendritic cells and their immune-activation profile towards enhanced antitumour activity. The method involves screening for LNPs that enhance the maturation of bone-marrow-derived dendritic cells and antigen presentation in vitro, followed by assessing immune activation and tumour-growth suppression in a mouse model of melanoma after subcutaneous or intramuscular delivery of the LNPs. We found that the most potent antitumour activity, especially when combined with immune checkpoint inhibitors, resulted from a coordinated attack by T cells and NK cells, triggered by LNPs that elicited strong immune activity in both type-1 and type-2 T helper cells. Our findings highlight the importance of optimizing the LNP composition of mRNA-based cancer vaccines to tailor antigen-specific immune-activation profiles.

Machine Learning Elucidates Design Features of Plasmid DNA Lipid Nanoparticles for Cell Type-Preferential Transfection.

For cell and gene therapies to become more broadly accessible, it is critical to develop and optimize non-viral cell type-preferential gene carriers such as lipid nanoparticles (LNPs). Despite the effectiveness of high throughput screening (HTS) approaches in expediting LNP discovery, they are often costly, labor-intensive, and often do not provide actionable LNP design rules that focus screening efforts on the most relevant chemical and formulation parameters. Here we employed a machine learning (ML) workflow using well-curated plasmid DNA LNP transfection datasets across six cell types to maximize chemical insights from HTS studies and has achieved predictions with 5-9% error on average depending on cell type. By applying Shapley additive explanations to our ML models, we unveiled composition-function relationships dictating cell type-preferential LNP transfection efficiency. Notably, we identified consistent LNP composition parameters that enhance in vitro transfection efficiency across diverse cell types, such as ionizable to helper lipid ratios near 1:1 or 10:1 and the incorporation of cationic/zwitterionic helper lipids. In addition, several parameters were found to modulate cell type-preferentiality, including the ionizable and helper lipid total molar percentage, N/P ratio, cholesterol to PEGylated lipid ratio, and the chemical identity of the helper lipid. This study leverages HTS of compositionally diverse LNP libraries and ML analysis to understand the interactions between lipid components in LNP formulations; and offers fundamental insights that contribute to the establishment of unique sets of LNP compositions tailored for cell type-preferential transfection.

Gelatin nanofiber-reinforced decellularized amniotic membrane promotes axon regeneration and functional recovery in the surgical treatment of peripheral nerve injury.

Effective recovery of peripheral nerve injury (PNI) after surgical treatment relies on promoting axon regeneration and minimizing the fibrotic response. Decellularized amniotic membrane (dAM) has unique features as a natural matrix for promoting PNI repair due to its pro-regenerative extracellular matrix (ECM) structure and anti-inflammatory properties. However, the fragile nature and rapid degradation rate of dAM limit its widespread use in PNI surgery. Here we report an engineered composite membrane for PNI repair by combining dAM with gelatin (Gel) nanofiber membrane to construct a Gel nanofiber-dAM composite membrane (Gel-dAM) through interfacial bonding. The Gel-dAM showed enhanced mechanical properties and reduced degradation rate, while retaining maximal bioactivity and biocompatibility of dAM. These factors led to improved axon regeneration, reduced fibrotic response, and better functional recovery in PNI repair. As a fully natural materials-derived off-the-shelf matrix, Gel-dAM exhibits superior clinical translational potential for the surgical treatment of PNI.

A combination therapy of bortezomib, CXCR4 inhibitor, and checkpoint inhibitor is effective in cholangiocarcinoma in vivo.

Cholangiocarcinoma (CCA) is a biliary tree malignancy with a dismal prognosis. Tumor microenvironment (TME), including cancer-associated fibroblasts (CAFs) has been shown to be involved in drug resistance. To model the interactions between cancer cells and the TME, we established CCA complex patient-derived organoids (cPDOs) to include epithelial PDO (ePDOs) and matched CAFs. While ePDOs were sensitive to bortezomib, we found the matched cPDOs were relatively resistant. Mechanistically, this resistance was correlated with over-expression of CXCR4 in the CAF component of cPDOs. In accord with the role of CXCR4 in the resistance to bortezomib, we found that a CXCR4 inhibitor can reverse the resistance to bortezomib in vivo. Furthermore, we found that the inhibition of CXCR4 allowed bortezomib to sensitize CCA to anti-PD1 treatment, with a significant reduction of tumor burden and long-term overall survival. This novel cancer/stroma/immune triple treatment holds great promise for the treatment of CCA.

High-throughput screening for myelination promoting compounds using human stem cell-derived oligodendrocyte progenitor cells.

Promoting myelination capacity of endogenous oligodendrocyte precursor cells (OPCs) is a promising therapeutic approach for CNS demyelinating disorders such as Multiple Sclerosis (MS). To aid in the discovery of myelination-promoting compounds, we generated a genome-engineered human pluripotent stem cell (hPSC) line that consists of three reporters: identification-and-purification tag, GFP, and secreted-NanoLuc, driven by the endogenous PDGFRA, PLP1, and MBP genes, respectively. Using this cell line, we established a high-throughput drug screening platform and performed a small-molecule screen, which identified at least two myelination-promoting small-molecule (Ro1138452 and SR2211) that target prostacyclin (IP) receptor and retinoic acid receptor-related orphan receptor γ (RORγ), respectively. Single-cell-transcriptomic analysis of differentiating OPCs treated with these molecules further confirmed that they promote oligodendrocyte differentiation and revealed several pathways that are potentially modulated by them. The molecules and their target pathways provide promising targets for the possible development of remyelination-based therapy for MS and other demyelinating disorders.

A nanofiber-hydrogel composite improves tissue repair in a rat model of Crohn's disease perianal fistulas.

Perianal fistulas (PAFs) represent a severe complication of Crohn's disease (CD). Despite the advent of biologic and small-molecule therapeutics for luminal disease, PAFs in CD (CD-PAF) are relatively resistant to treatment, with less than 50% responding to any therapy. We report an injectable, biodegradable, mechanically fragmented nanofiber-hydrogel composite (mfNHC) loaded with adipose-derived stem cells (ADSCs) for the treatment of fistulas in a rat model of CD-PAF. The ADSC-loaded mfNHC results in a higher degree of healing when compared to surgical treatment of fistulas, which is a standard treatment. The volume of fistulas treated with mfNHC is decreased sixfold compared to the surgical treatment control. Molecular studies reveal that utilization of mfNHC reduced local inflammation and improved tissue regeneration. This study demonstrates that ADSC-loaded mfNHC is a promising therapy for CD-PAF, and warrants further studies to advance mfNHC toward clinical translation.

A Novel Patient-like Swine Model of Perianal Crohn's Disease.

BACKGROUND: Perianal Crohn's disease is associated with poor outcomes and high medical costs. It is notoriously difficult to treat despite therapeutic advancements for luminal disease. A large animal model that mimics human perianal disease is needed to test innovative therapies. OBJECTIVE: This study aimed to create a swine model that replicates the inflammatory component and therapeutic challenges found in patients with perianal Crohn's disease. DESIGN: This was an animal preclinical study. SETTINGS: The experiments were performed at the animal laboratory at the Johns Hopkins University. PATIENTS: Four sus scrufus female pigs were included in the study. INTERVENTIONS: Four female pigs underwent creation of 3 surgical perianal fistulas each, 1 rectovaginal and 2 perianal. Size 24 French setons were placed to maintain patency of the fistula tracts for 4 weeks. After removal of the setons, trinitrobenzene sulfonic acid was administered into the fistula tract to create and maintain local inflammation mimicking perianal Crohn's disease. MAIN OUTCOMES MEASURES: An MRI was obtained to assess the fistulas and the pigs were euthanized to review histopathology. RESULTS: Three inflammatory chronic fistula tracts were successfully created in each pig as confirmed by MRI and examination under anesthesia. This is the first report of maintaining patent fistulas in swine 2 weeks after removal of setons. For the first time, we reported that 2 pigs developed branching fistulas and small abscesses reminiscent of human perianal Crohn's disease. The corresponding histopathologic examination found significant chronic active inflammation on standard hematoxylin and eosin staining. LIMITATIONS: The fistulas were surgically induced and did not occur naturally. CONCLUSIONS: A chronic perianal fistula model in pigs that strongly resembles human perianal Crohn's disease was successfully created. This model can be used to test novel therapeutics and techniques to pave the path for human trials. See Video Abstract at http://links.lww.com/DCR/B969 . UN NUEVO MODELO PORCINO SIMILAR A UN PACIENTE DE LA ENFERMEDAD DE CROHN PERIANAL ANTECEDENTES: La enfermedad de Crohn perianal se asocia con malos resultados y altos costos médicos. Es notoriamente difícil de tratar a pesar de los avances terapéuticos para la enfermedad luminal. Se precisa de un modelo animal grande que imite la enfermedad perianal humana para probar terapias innovadoras.OBJETIVO:Nuestro objetivo de este estudio fue crear un modelo porcino que replique el componente inflamatorio y los desafíos terapéuticos que se encuentran en los pacientes con enfermedad de Crohn perianal.DISEÑO:Este fue un estudio preclínico en animales.AJUSTES:Los experimentos se realizaron en el laboratorio de animales de la Universidad Johns Hopkins.PACIENTES:Se incluyeron en el estudio cuatro cerdas sus scrofa.INTERVENCIONES:Cuatro cerdas fueron sometidas a la creación de 3 fístulas perianales quirúrgicas cada una: 1 recto vaginal y 2 perianales. Se colocaron sedales de 24 French para mantener la permeabilidad de los trayectos fistulosos durante 4 semanas. Tras el retiro de los sedales, se administró ácido trinitrobenceno sulfónico en el trayecto de la fístula para crear y mantener la inflamación local simulando la enfermedad de Crohn perianal.PRINCIPALES MEDIDAS DE RESULTADOS:Se obtuvo una resonancia magnética para evaluar las fístulas y los cerdos fueron sacrificados para revisar la histopatología.RESULTADOS:Se crearon de manera exitosa tres trayectos fistulosos inflamatorios crónicos en cada cerdo, confirmados por imágenes de resonancia magnética y examen bajo anestesia. Este es el primer informe de preservación de fístulas permeables en cerdos 2 semanas tras el retiro de los setones. Por primera vez, informamos que dos cerdos desarrollaron fístulas ramificadas y pequeños abscesos que recuerdan a la enfermedad de Crohn perianal humana. El examen histopatológico correspondiente encontró una significativa inflamación crónica activa en la tinción estándar de hematoxilina y eosina.LIMITACIONES:Las fístulas se indujeron quirúrgicamente y no se produjeron de forma natural.CONCLUSIONES:Se logro recrear con éxito un modelo de fístula perianal crónica en cerdos que se asemeja mucho a la enfermedad de Crohn perianal humana. Este modelo se puede utilizar para probar nuevas terapias y técnicas para allanar el camino para los ensayos en humanos. Consulte Video Resumen en http://links.lww.com/DCR/B969 . (Traducción-Dr Osvaldo Gauto).

Translational bioengineering strategies for peripheral nerve regeneration: opportunities, challenges, and novel concepts.

Peripheral nerve injuries remain a challenging problem in need of better treatment strategies. Despite best efforts at surgical reconstruction and postoperative rehabilitation, patients are often left with persistent, debilitating motor and sensory deficits. There are currently no therapeutic strategies proven to enhance the regenerative process in humans. A clinical need exists for the development of technologies to promote nerve regeneration and improve functional outcomes. Recent advances in the fields of tissue engineering and nanotechnology have enabled biomaterial scaffolds to modulate the host response to tissue repair through tailored mechanical, chemical, and conductive cues. New bioengineered approaches have enabled targeted, sustained delivery of protein therapeutics with the capacity to unlock the clinical potential of a myriad of neurotrophic growth factors that have demonstrated promise in enhancing regenerative outcomes. As such, further exploration of combinatory strategies leveraging these technological advances may offer a pathway towards clinically translatable solutions to advance the care of patients with peripheral nerve injuries. This review first presents the various emerging bioengineering strategies that can be applied for the management of nerve gap injuries. We cover the rationale and limitations for their use as an alternative to autografts, focusing on the approaches to increase the number of regenerating axons crossing the repair site, and facilitating their growth towards the distal stump. We also discuss the emerging growth factor-based therapeutic strategies designed to improve functional outcomes in a multimodal fashion, by accelerating axonal growth, improving the distal regenerative environment, and preventing end-organs atrophy.

Engineering Skeletal Muscle Grafts with PAX7::GFP-Sorted Human Pluripotent Stem Cell-Derived Myogenic Progenitors on Fibrin Microfiber Bundles for Tissue Regeneration.

Tissue engineering strategies that combine human pluripotent stem cell-derived myogenic progenitors (hPDMs) with advanced biomaterials provide promising tools for engineering 3D skeletal muscle grafts to model tissue development in vitro and promote muscle regeneration in vivo. We recently demonstrated (i) the potential for obtaining large numbers of hPDMs using a combination of two small molecules without the overexpression of transgenes and (ii) the application of electrospun fibrin microfiber bundles for functional skeletal muscle restoration following volumetric muscle loss. In this study, we aimed to demonstrate that the biophysical cues provided by the fibrin microfiber bundles induce hPDMs to form engineered human skeletal muscle grafts containing multinucleated myotubes that express desmin and myosin heavy chains and that these grafts could promote regeneration following skeletal muscle injuries. We tested a genetic PAX7 reporter line (PAX7::GFP) to sort for more homogenous populations of hPDMs. RNA sequencing and gene set enrichment analyses confirmed that PAX7::GFP-sorted hPDMs exhibited high expression of myogenic genes. We tested engineered human skeletal muscle grafts derived from PAX7::GFP-sorted hPDMs within in vivo skeletal muscle defects by assessing myogenesis, engraftment and immunogenicity using immunohistochemical staining. The PAX7::GFP-sorted groups had moderately high vascular infiltration and more implanted cell association with embryonic myosin heavy chain (eMHC) regions, suggesting they induced pro-regenerative microenvironments. These findings demonstrated the promise for the use of PAX7::GFP-sorted hPDMs on fibrin microfiber bundles and provided some insights for improving the cell-biomaterial system to stimulate more robust in vivo skeletal muscle regeneration.

Payload distribution and capacity of mRNA lipid nanoparticles.

Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are critical to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence analysis of fluorescent tags on different LNP components, and quantitatively resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%-80% empty LNPs depending on the assembly conditions. Systematic analysis of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the molecular assembly of mRNA LNPs.

Off-the-shelf, heparinized small diameter vascular graft limits acute thrombogenicity in a porcine model.

Thrombogenicity poses a challenge to the clinical translation of engineered grafts. Previously, small-diameter vascular grafts (sdVG) composed of fibrin hydrogel microfiber tubes (FMT) with an external poly(ε-caprolactone) (PCL) sheath supported long-term patency in mice. Towards the development of an sdVG with off-the-shelf availability, the FMT's shelf stability, scale-up, and successful conjugation of an antithrombotic drug to the fibrin scaffold are reported here. FMTs maintain mechanical stability and high-water retention after storage for one year in a freezer, in a refrigerator, or at room temperature. Low molecular weight heparin-conjugated fibrin scaffolds enabled local and sustained delivery during two weeks of enzymatic degradation. Upscaled fabrication of sdVGs provides natural biodegradable grafts with size and mechanics suitable for human application. Implantation in a carotid artery interposition porcine model exhibited no rupture with thrombi prevented in all heparinized sdVGs (n = 4) over 4-5 weeks. Remodeling of the sdVGs is demonstrated with endothelial cells on the luminal surface and initial formation of the medial layer by 4-5 weeks. However, neointimal hyperplasia at 4-5 weeks led to the stenosis and occlusion of most of the sdVGs, which must be resolved for future long-term in vivo assessments. The off-the-shelf, biodegradable heparinized fibrin sdVG layer limits acute thrombogenicity while mediating extensive neotissue formation as the PCL sheath maintains structural integrity. STATEMENT OF SIGNIFICANCE: To achieve clinical and commercial utility of small-diameter vascular grafts as arterial conduits, these devices must have off-the-shelf availability for emergency arterial bypass applications and be scaled to a size suitable for human applications. A serious impediment to clinical translation is thrombogenicity. Treatments have focused on long-term systemic drug therapy, which increases the patient's risk of bleeding complications, or coating grafts and stents with anti-coagulants, which minimally improves patient outcomes even when combined with dual anti-platelet therapy. We systematically modified the biomaterial properties to develop anticoagulant embedded, biodegradable grafts that maintain off-the-shelf availability, provide mechanical stability, and prevent clot formation through local drug delivery.

Biostimulatory Micro-Fragmented Nanofiber-Hydrogel Composite Improves Mesenchymal Stem Cell Delivery and Soft Tissue Remodeling.

Functional microgels are preferred stem cell carriers due to the ease of delivery through minimally invasive injection and seamless integration with the surrounding host tissue. A biostimulatory nanofiber-hydrogel composite (NHC) has been previously developed through covalently crosslinking a hyaluronic acid hydrogel network with surface-functionalized poly (ε-caprolactone) nanofiber fragments. The NHC mimics the microarchitecture of native soft tissue matrix, showing enhanced cell infiltration, immunomodulation, and proangiogenic properties. Here, injectability of the pre-formed NHC is improved by mechanical fragmentation, making it into micro-fragmented NHC (mfNHC) in a granular gel form as a stem cell carrier to deliver mesenchymal stem cells (MSCs) for soft tissue remodeling. The mfNHC shows a similar storage modulus but a significantly reduced injection force, as compared with the corresponding bulk NHC. When injected subcutaneously in a rat model, mfNHC-MSC constructs initiate an elevated level of host macrophage infiltration, more pro-regenerative polarization, and subsequently, improved angiogenesis and adipogenesis response when compared to mfNHC alone. A similar trend of host cell infiltration and pro-angiogenic response is detected in a swine model with a larger volume injection. These results suggest a strong potential for use of the mfNHC as an injectable carrier for cell delivery and soft tissue remodeling.

Multi-step screening of DNA/lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression.

Lipid nanoparticles hold great potential as an effective non-viral vector for nucleic acid-based gene therapy. Plasmid DNA delivery can result in extended transgene expression compared to mRNA-based technologies, yet there is a lack of systematic investigation into lipid nanoparticle compositions for plasmid DNA delivery. Here, we report a multi-step screening platform to identify optimized plasmid DNA lipid nanoparticles for liver-targeted transgene expression. To achieve this, we analyze the role of different helper lipids and component ratios in plasmid DNA lipid nanoparticle-mediated gene delivery in vitro and in vivo. Compared to mRNA LNPs and in vivo-jetPEI/DNA nanoparticles, the identified plasmid DNA lipid nanoparticles successfully deliver transgenes and mediate prolonged expression in the liver following intravenous administration in mice. By addressing different physiological barriers in a stepwise manner, this screening platform can efficiently down select effective lipid nanoparticle candidates from a lipid nanoparticle library of over 1000 formulations. In addition, we substantially extend the duration of plasmid DNA nanoparticle-mediated transgene expression using a DNA/siRNA co-delivery approach that targets transcription factors regulating inflammatory response pathways. This lipid nanoparticle-based co-delivery strategy further highlights the unique advantages of an extended transgene expression profile using plasmid DNA delivery and offers new opportunities for DNA-based gene medicine applications.

Quaternary nanoparticles enable sustained release of bortezomib for hepatocellular carcinoma.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related mortality in the world. Over the past two decades, there has been minimal improvement in therapies as well as clinical outcomes for patients with Barcelona Clinic Liver Cancer (BCLC)-B. These patients are treated with local interventions, including transarterial chemoembolization. Current methodologies only allow sustained intratumoral release measured in hours. Methodologies to allow sustained local release of the drug cargo over days to weeks are acutely needed. We hypothesize that tumor response as well as outcomes of patients with BCLC-B can be improved through utilization of a highly cytotoxic agent delivered with a sustained release platform. APPROACH AND RESULTS: High-throughput drug screening across 40 HCC patient-derived organoids identified bortezomib (BTZ) as a highly cytotoxic small molecule for HCC. We designed and manufactured sustained release BTZ nanoparticles (BTZ-NP) using a flash nanocomplexation/nanoprecipitation process. We quantified the release profile and tested the anti-tumoral effects in vivo. The BTZ-NP formulation demonstrated a sustained release of BTZ of 30 days. This BTZ-NP formulation was highly effective in controlling tumor size and improved survival in vivo in three animal models of HCC, including when delivered via the hepatic artery, as we envision its delivery in patients. In addition, the BTZ-NP formulation was superior to treatment with doxorubicin-drug eluting beads. CONCLUSIONS: The BTZ-NP formulation provides a potent and safe treatment of HCC via a localized delivery approach. These results warrant additional preclinical studies to advance this technology to human clinical trials.

The Effects of the Combination of Mesenchymal Stromal Cells and Nanofiber-Hydrogel Composite on Repair of the Contused Spinal Cord.

A bone marrow-derived mesenchymal stromal cell (MSC) transplant and a bioengineered nanofiber-hydrogel composite (NHC) have been shown to stimulate nervous tissue repair in the contused spinal cord in rodent models. Here, these two modalities were combined to assess their repair effects in the contused spinal cord in adult rats. Cohorts of contused rats were treated with MSC in NHC (MSC-NHC), MSC in phosphate-buffered saline (MSC-PBS), NHC, or PBS injected into the contusion site at 3 days post-injury. One week after injury, there were significantly fewer CD68+ cells in the contusion with MSC-NHC and NHC, but not MSC-PBS. The reduction in CD86+ cells in the injury site with MSC-NHC was mainly attributed to NHC. One and eight weeks after injury, we found a greater CD206+/CD86+ cell ratio with MSC-NHC or NHC, but not MSC-PBS, indicating a shift from a pro-inflammatory towards an anti-inflammatory milieu in the injury site. Eight weeks after injury, the injury size was significantly reduced with MSC-NHC, NHC, and MSC-PBS. At this time, astrocyte, and axon presence in the injury site was greater with MSC-NHC compared with MSC-PBS. We did not find a significant effect of NHC on MSC transplant survival, and hind limb function was similar across all groups. However, we did find fewer macrophages at 1 week post-injury, more macrophages polarized towards a pro-regenerative phenotype at 1 and 8 weeks after injury, and reduced injury volume, more astrocytes, and more axons at 8 weeks after injury in rats with MSC-NHC and NHC alone compared with MSC-PBS; these findings were especially significant between rats with MSC-NHC and MSC-PBS. The data support further study in the use of an NHC-MSC combination transplant in the contused spinal cord.

Enrichment of Nanofiber Hydrogel Composite with Fractionated Fat Promotes Regenerative Macrophage Polarization and Vascularization for Soft-Tissue Engineering.

BACKGROUND: Fractionated fat has been shown to promote dermal regeneration; however, the use of fat grafting for reconstruction of soft-tissue defects is limited because of volume loss over time. The authors have developed a novel approach for engineering of vascularized soft tissue using an injectable nanofiber hydrogel composite enriched with fractionated fat. METHODS: Fractionated fat was generated by emulsification of groin fat pads from rats and mixed in a 3:1 ratio with nanofiber hydrogel composite (nanofiber hydrogel composite with fractionated fat). Nanofiber hydrogel composite with fractionated fat or nanofiber hydrogel composite alone was placed into isolation chambers together with arteriovenous loops, which were subcutaneously implanted into the groin of rats (n = 8 per group). After 21 days, animals were euthanized and systemically perfused with ink, and tissue was explanted for histologic analysis. Immunofluorescent staining and confocal laser scanning microscopy were used to quantify CD34+ progenitor cell and macrophage subpopulations. RESULTS: Nanofiber hydrogel composite with fractionated fat tissue maintained its shape without shrinking and showed a significantly stronger functional vascularization compared to composite alone after 21 days of implantation (mean vessel count, 833.5 ± 206.1 versus 296.5 ± 114.1; p = 0.04). Tissue heterogeneity and cell count were greater in composite with fractionated fat (mean cell count, 49,707 ± 18,491 versus 9263 ± 3790; p = 0.005), with a significantly higher number of progenitor cells and regenerative CD163+ macrophages compared to composite alone. CONCLUSIONS: Fractionated fat-enriched nanofiber hydrogel composite transforms into highly vascularized soft tissue over 21 days without signs of shrinking and promotes macrophage polarization toward regenerative phenotypes. Enrichment of injectable nanofiber hydrogel composite with fractionated fat represents a promising approach for durable reconstruction of soft-tissue defects. CLINICAL RELEVANCE STATEMENT: The authors' approach for tissue engineering may ultimately lay the groundwork for clinically relevant applications with the goal of generating large volumes of vascularized soft tissue for defect reconstruction without donor site morbidity.