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.

Microwave photonic I/Q mixer for wideband frequency downconversion with serial electro-optical modulations.

In this paper, we propose a serial electro-optical (EO)-modulation-based microwave photonic in-phase and quadrature (I/Q) mixer and investigate its performance for wideband frequency downconversion. The proposed I/Q mixer uses two EO modulators and a programmable optical processor in a serially cascaded structure, which ensures good phase stability and flexibility to achieve high-performance broadband frequency downconversion. A proof-of-concept experiment is carried out in which the frequency downconversion of the RF signals in the range from 10 to 40 GHz is demonstrated with an average image rejection ratio of 38.66 dB. The spurious-free dynamic range (SFDR) measured at around 15 GHz is 86 dBc·Hz2/3. Based on this microwave photonic I/Q mixer, a broadband radar receiver is established and de-chirping of linearly frequency modulated (LFM) radar echoes with an instantaneous bandwidth of 8 GHz (10-18 GHz) is achieved. The results verify its feasibility for high-performance I/Q mixing in practical applications.

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.

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.

Influence of Underground Mining Direction Based on Particle Flow on Deformation and Failure of Loess Gully Area.

Due to unique landform and lithological features of the loess gully area, the geological disasters caused by coal mining have become more complex. Moreover, the advancing direction of the working face has an important influence on the deformation of the slope on both sides of the gully. In this paper, combined with the specific conditions of a coal mine working face in western China, we use the method of particle flow numerical simulation and theoretical analysis to examine and observe the deformation, as well as characteristics of failures in the loess gully area under different mining directions of the working face. The deformation process of the gully area can be obtained by combining the numerical simulation results. According to different mining directions of the working face, the failure mode of mining in the loess gully area was divided into the back slope and along slope advancing failure modes. Through empirical evaluation, our investigation demonstrates that the bottom of the gully was damaged seriously by both along slope mining and back slope mining. Albeit the influence of coal seam excavation on the slope surface was relatively small; however, it is greater on the flat ground of the upper slope. Under the same mining conditions, the advancing direction of the working face affected the horizontal movement of the loess gully area but had less effect on the subsidence. Furthermore, we observed that the failure mode of the mining slope is highly correlated with the mining direction and relative position of the working face. The results obtained from this research can provide useful information for deformation and failure prediction, working face mining method, and geological disaster assessment in the loess gully area.

Biomaterials strategies to balance inflammation and tenogenesis for tendon repair.

Adult tendon tissue demonstrates a limited regenerative capacity, and the natural repair process leaves fibrotic scar tissue with inferior mechanical properties. Surgical treatment is insufficient to provide the mechanical, structural, and biochemical environment necessary to restore functional tissue. While numerous strategies including biodegradable scaffolds, bioactive factor delivery, and cell-based therapies have been investigated, most studies have focused exclusively on either suppressing inflammation or promoting tenogenesis, which includes tenocyte proliferation, ECM production, and tissue formation. New biomaterials-based approaches represent an opportunity to more effectively balance the two processes and improve regenerative outcomes from tendon injuries. Biomaterials applications that have been explored for tendon regeneration include formation of biodegradable scaffolds presenting topographical, mechanical, and/or immunomodulatory cues conducive to tendon repair; delivery of immunomodulatory or tenogenic biomolecules; and delivery of therapeutic cells such as tenocytes and stem cells. In this review, we provide the biological context for the challenges in tendon repair, discuss biomaterials approaches to modulate the immune and regenerative environment during the healing process, and consider the future development of comprehensive biomaterials-based strategies that can better restore the function of injured tendon. STATEMENT OF SIGNIFICANCE: Current strategies for tendon repair focus on suppressing inflammation or enhancing tenogenesis. Evidence indicates that regulated inflammation is beneficial to tendon healing and that excessive tissue remodeling can cause fibrosis. Thus, it is necessary to adopt an approach that balances the benefits of regulated inflammation and tenogenesis. By reviewing potential treatments involving biodegradable scaffolds, biological cues, and therapeutic cells, we contrast how each strategy promotes or suppresses specific repair steps to improve the healing outcome, and highlight the advantages of a comprehensive approach that facilitates the clearance of necrotic tissue and recruitment of cells during the inflammatory stage, followed by ECM synthesis and organization in the proliferative and remodeling stages with the goal of restoring function to the tendon.