Therapeutic Platform Research Articles

Bioengineered therapeutic systems for improving antitumor immunity

Abstract Immunotherapy, a monumental advancement in antitumor therapy, still yields limited clinical benefits owing to its unguaranteed efficacy and safety. Therapeutic systems derived from cellular, bacterial, and viral sources possess inherent properties that are conducive to antitumor immunotherapy. However, crude biomimetic systems have restricted functionality and may produce undesired toxicity. With the advances in biotechnology, various toolkits are available to add or subtract certain properties of living organisms to create flexible therapeutic platforms. This review elaborates on the creation of bioengineered systems by gene editing, synthetic biology, and surface engineering to enhance immunotherapy. The modifying strategies on the systems are discussed, including equipment of navigation and recognition systems to improve therapeutic precision, introduction of controllable components to control the duration and intensity of treatment, addition of immunomodulatory components to amplify immune activation, and removal of toxicity factors to ensure biosafety. Finally, we summarize the advantages of bioengineered immunotherapeutic systems and possible directions for their clinical translation.

SURG-27. DIRECTIONALLY NON-ROTATING ELECTRIC FIELD THERAPY (DNEFT) DELIVERED THROUGH IMPLANTED ELECTRODES: A NOVEL SURGICAL PLATFORM FOR GLIOBLASTOMA IMMUNOTHERAPY

Abstract BACKGROUND While directionally rotating Tumor Treating Fields (TTF) therapy has garnered considerable clinical interest in recent years, there has been comparatively less focus on directionally non-rotating electric field therapy (dnEFT). OBJECTIVE Here, we explore dnEFT generated through implanted electrodes as a glioblastoma therapeutic platform, with the goal of facilitating clinical translation. METHODS Using custom-designed electrode arrays to study the effect of dnEFT using in vitro and in vivo glioblastoma models. RESULTS In vitro, dnEFT generated using a clinical grade spinal cord stimulator showed anti-neoplastic activity against independent glioblastoma cell lines. In support of the results obtained using the clinical grade electrode, dnEFT delivered through a customized, two-electrode array induced glioblastoma apoptosis. To characterize this effect in vivo, a custom-designed four-electrode array was fabricated such that tumor cells can be implanted into murine cerebrum through a center channel equidistant from the electrodes. After implantation with this array and luciferase expressing murine GL261 glioblastoma cells, mice were randomized to dnEFT or placebo. Relative to placebo treated mice, dnEFT reduced tumor growth (measured by bioluminescence) and prolonged survival (median survival gain of 6.5 days). Analysis of brain sections following dnEFT showed a notable increase in the accumulation of peri-tumoral macrophage/microglia with increased expression of M1 genes (IFNγ, TNFα, IL-6) and decreased expression of M2 genes (CD206, Arg, IL-10) relative to placebo tumors. CONCLUSIONS Our results suggest therapeutic potential in glioblastoma for dnEFT delivered through implanted electrodes as a novel immunotherapy platform, supporting the concept of a proof-of-principle clinical trial using commercially available deep brain stimulator electrodes.

Ligand-Tethered Extracellular Vesicles Mediated RNA Therapy for Liver Fibrosis.

Liver fibrosis poses a significant global health burden, in which hepatic stellate cells (HSCs) play a crucial role. Targeted nanomedicine delivery systems directed at HSCs have shown immense potential in the treatment of liver fibrosis. Herein, a bioinspired material, engineered therapeutic miR-181a-5p (a miRNA known to inhibit fibrotic signaling pathways) and targeted moiety hyaluronic acid (HA) co-functionalized extracellular vesicles (EVs) are developed. HA is incorporated onto the surface of EVs using DSPE-PEG as a linker, allowing preferential binding to CD44 receptors, which are overexpressed on activated HSCs. Our results confirmed enhanced cellular uptake and improved payload delivery, as evidenced by the increased intracellular abundance of miR-181a-5p in activated HSCs and fibrotic livers. HA-equipped EVs loaded with miR-181a-5p (DPH-EVs@miR) significantly reduce HSC activation and extracellular matrix (ECM) deposition by inhibiting the TGF-β/Smad signaling pathway, thus alleviating the progression of liver fibrosis. Additionally, DPH-EVs@miR improves liver function, ameliorates inflammatory infiltration, and mitigates hepatocyte apoptosis, demonstrating superior hepatic protective effects. Collectively, this study reports a prospective nanovesicle therapeutic platform loaded with therapeutic miRNA and targeting motifs for liver fibrosis. The biomarker-guided EV-engineering technology utilized in this study provides a promising tool for nanomedicine and precision medicine.

Engineering Injectable and Highly Interconnected Porous Silk Fibroin Microspheres for Tissue Regeneration.

Injectable porous microspheres represent a promising therapeutic platform for cell delivery, drug delivery, and tissue regeneration. Yet, the engineering of silk fibroin microspheres with a highly interconnected porous structure remains an unsolved challenge. In this study, a simple and efficient method is developed that does not require the use of organic solvents to prepare silk fibroin microspheres with a predictable structure. Through extensive screening, the addition of glucose is found to direct the formation of a highly interconnected porous structure from the interior to the surface of silk fibroin microspheres. Compared to silk fibroin microspheres (SF microspheres) produced through a combination of electro-spray, cryopreservation, and freeze drying, silk fibroin-glucose microspheres (SF-Glu microspheres) demonstrates enhanced capabilities in promoting cell adhesion and proliferation in vitro. Both SF-Glu and SF microspheres exhibit the capacity to maintain the sustained release kinetics of the loaded model drug. Furthermore, SF-Glu microspheres facilitate the recruitment of endogenous cells, capillary migration, and macrophage phenotype switch following subcutaneous injection in the rats. This study opens a new avenue for the construction of porous silk fibroin microspheres, which could lead to a broader range of applications in regenerative medicine.

Triggered Cascade-Activation Nanoplatform to Alleviate Hypoxia for Effective Tumor Immunotherapy Guided by NIR-II Imaging.

Hypoxia is one of the most typical features among various types of solid tumors, which creates an immunosuppressive tumor microenvironment (TME) and limits the efficacy of cancer treatment. Alleviating hypoxia becomes a key strategy to reshape hypoxic TME which improves cancer immunotherapy. However, it remains challenging to perform tumor precision therapy with controllable switches through hypoxia-activated gene editing and prodrugs to alleviate hypoxia. In this study, silica-coated second near-infrared window (NIR-II) emitting silver sulfide quantum dots are used as the carrier to load the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) system to target hypoxia-inducible factor-1 (HIF-1α) and guide tumor-targeted imaging. To reduce the off-target effects in nontumor cells and better control safety risks, a TME-triggered cascade-activation nanodiagnostic and therapeutic platform (AA@Cas-H@HTS) is designed, which achieves the hypoxia activation of prodrug tirapazamine (TPZ) and spatiotemporal release of CRISPR/Cas9 ribonucleoprotein. Tumor hypoxia is greatly alleviated by the synergistic function of HIF-1α depletion by gene editing and TPZ activation. Importantly, targeting HIF-1α disrupts the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) signaling pathway, which effectively reshapes the immune-suppressive TME and activates T cell-mediated antitumor immunity. Taken together, we have provided a TME-triggered cascade-activation nanoplatform to alleviate hypoxia for improved cancer immunotherapy.

Nanomaterial-enabled drug transport systems: a comprehensive exploration of current developments and future avenues in therapeutic delivery.

Over the years, nanotechnology has gained popularity as a viable solution to address gene and drug delivery challenges over conventional methods. Extensive research has been conducted on nanosystems that consist of organic/inorganic materials, drugs, and its biocompatibility become the primary goal of improving drug delivery. Various surface modification methods help focus targeted and controlled drug release, further enabling multidrug delivery also. This newer technology ensures the stability of drugs that can unravel the mechanisms involved in cellular processes of disease development and its management. Tailored medication delivery provides benefits such as therapy, controlled release, and reduced adverse effects, which are especially important for controlling illnesses like cancer. However, multifunctional nanocarriers that possess high viscoelasticity, extended circulation half-life, biocompatibility, and biodegradability face some challenges and limitations too in human bodies. To produce a consistent therapeutic platform based on complex three-dimensional nanoparticles, careful design and engineering, thorough orthogonal analysis methods, and reproducible scale-up and manufacturing processes will be required in the future. Safety and effectiveness of nano-based drug delivery should be thoroughly investigated in preclinical and clinical trials, especially when considering biodistribution, targeting specific areas, and potential immunological toxicities. Overall, the current review article explores the advancements in nanotechnology, specific to nanomaterial-enabled drug delivery systems, carrier fabrication techniques and modifications, disease management, clinical research, applications, limitations, and future challenges. The work portrays how nanomedicine distribution affects healthcare with an emphasis on the developments in drug delivery techniques.

Virulence‐Free Reconstituted Synthetic Nanopathogen Empowered by Timely‐Activating TLR Agonist Promotes Heterologous Cancer Immunotherapy with Depletion of Tumor‐Specific Treg Cells

AbstractThe heterologous immunity of live pathogens leads to the emergence of this approach as a pivotal component in cancer immunotherapy. However, virulence, inflammatory‐related toxicity, and the induction of Treg cells after treatment hinder their clinical translation. Here, to exploit the heterologous immunity of live pathogens without risks, a virulence‐free reconstituted synthetic nanopathogen (RSnP) is developed by the cell wall skeleton of disrupted Mycobacterium bovis and the incorporation of timely‐activating Toll‐like receptor 7/8 agonists of which multifaceted activities can be promoted by endolysosomal enzymes. Immunization with RSnP, even without tumor‐specific antigens, exhibits potent antitumor efficacy against melanoma, breast cancer, and bladder cancer by promoting antitumor effector cells (CD8+ T cells, NK cells, M1 macrophages, and Th17 cells) and proinflammatory cytokines (IL‐12p70, TNF‐α, and IL‐6) while simultaneously mitigating immunosuppressive myeloid cells (MDSCs and M2 macrophages) in the tumor microenvironment, surpassing the therapeutic efficacies of approved live‐BCG drug and mRNA vaccine. The increase in CCR8+Foxp3+ Treg cells induced to counteract RSnP treatment can be attenuated by anti‐CCR8 antibody, a depletion antibody for tumor‐specific Treg cells, to synergize therapeutic efficacy with relieved autoimmunity. RSnP can be a therapeutic nanomedicine platform across heterologous cancers and emerging infectious virus variants with minimized toxicity.

Advancements in Engineering Tetrahedral Framework Nucleic Acids for Biomedical Innovations.

Tetrahedral framework nucleic acids (tFNAs) are renowned for their controllable self-assembly, exceptional programmability, and excellent biocompatibility, which have led to their widespread application in the biomedical field. Beyond these features, tFNAs demonstrate unique chemical and biological properties including high cellular uptake efficiency, structural bio-stability, and tissue permeability, which are derived from their distinctive 3D structure. To date, an extensive range of tFNA-based nanostructures are intelligently designed and developed for various biomedical applications such as drug delivery, gene therapy, biosensing, and tissue engineering, among other emerging fields. In addition to their role in drug delivery systems, tFNAs also possess intrinsic properties that render them highly effective as therapeutic agents in the treatment of complex diseases, including arthritis, neurodegenerative disorders, and cardiovascular diseases. This dual functionality significantly enhances the utility of tFNAs in biomedical research, presenting valuable opportunities for the development of next-generation medical technologies across diverse therapeutic and diagnostic platforms. Consequently, this review comprehensively introduces the latest advancements of tFNAs in the biomedical field, with a focus on their benefits and applications as drug delivery nanoplatforms, and their inherent capabilities as therapeutic agents. Furthermore, the current limitations, challenges, and future perspectives of tFNAs are explored.

Biomaterial-Mediated Metabolic Regulation of Ferroptosis for Cancer Immunotherapy.

Ferroptosis is a lipid peroxidation-driven cell death route and has attracted enormous interest for cancer therapy. Distinct from other forms of regulated cell death, its process is involved with multiple metabolic pathways including lipids, bioenergetics, iron, and so on, which influence cancer cell ferroptosis sensitivity and communication with the immune cells in the tumor microenvironment. Development of novel technologies for harnessing the ferroptosis-associated metabolic regulatory network would profoundly improve our understanding of the immune responses and enhance the efficacy of ferroptosis-dependent immunotherapy. Interestingly, the recent advances in bio-derived material-based therapeutic platforms offer novel opportunities to therapeutically modulate tumor metabolism through the insitu delivery of molecular or material cues, which not only allows the tumor-specific elicitation of ferroptosis but also holds promise to maximize their immunostimulatory impact. In this review, we will first dissect the crosstalk between tumor metabolism and ferroptosis and its impact on the immune regulation in the tumor microenvironment, followed by the comprehensive analysis on the recent progress in biomaterial-based metabolic regulatory strategies for evoking ferroptosis-mediated antitumor immunity. A perspective section is also provided to discuss the challenges in metabolism-regulating biomaterials for ferroptosis-immunotherapy. We envision that this review may provide new insights for improving tumor immunotherapeutic efficacy in the clinic.

Review of Microneedle Technology for Targeted Therapeutics in Vitiligo: Design Principles, Application Prospects.

Vitiligo is a chronic autoimmune disorder characterized by depigmented patches of the skin. The treatment of vitiligo remains challenging, partly owing to the lack of efficient drug delivery system. Microneedles (MNs), an ideal transdermal drug delivery system, have emerged as promising drug delivery platform for vitiligo. Recently, the emergence of novel MNs with increased biocompatibility, including hydrogel and hollow MNs, further enhance the translational value of MNs in the treatment of vitiligo. However, up-to-date review of these advancements remains lacking. This review aims to summarize the most recent studies of MN-based drug delivery systems for vitiligo, highlighting the translational potential of MNs as a therapeutic platform for the treatment of vitiligo in the near future.

Self-adaptive carbon nanozyme regulation of ROS balance for bacteria-infected wound therapy

Nanozymes sterilizing drug-resistant (DR) bacteria by generating reactive oxygen species (ROS) have emerged as a promising approach for treating infected wounds. However, designing biocompatible adaptive ROS balance therapeutic platform for DR bacteria-infected wounds remains a great challenge. Herein, biocompatible lignin-based carbon dot nanomaterials (LCDs-OA) with more CO and COOH functional groups on the surface were prepared by hydrothermal organic acid co-processing and used as a dual enzyme-like active nanozyme for the therapy of wounds infected by DR bacteria. Experimental and theoretical calculations demonstrated that the CO and COOH functional groups in LCDs-OA have site-switching roles in the dual enzyme-like activity, which can balance reactive oxygen species (ROS) according to the changes in microenvironmental pH at the wound site, and realize the adaptive regulation of ROS generation for bactericidal and ROS scavenging for healing. Furthermore, the performance differences between different organic acid treatments were investigated and an adaptive ROS balance therapeutic platform (LCDs-OA@CaO2) was constructed to overcome the major limitation in treating DR bacterial infected wounds. The system achieved >99.6 % in vitro antimicrobial efficiency against DR bacteria and was highly biocompatible while effectively reducing oxidative damage to normal tissues, resulting in satisfactory therapeutic results in repairing wounds infected with DR bacteria.

Development of a Limosilactobacillus reuteri therapeutic delivery platform with reduced colonization potential.

Bacterial biotherapeutic delivery vehicles have the potential to treat a variety of diseases. This approach obviates the need to purify the recombinant effector molecule, allows delivery of therapeutics in situ via oral or intranasal administration, and protects the effector molecule during gastrointestinal transit. Lactic acid bacteria have been broadly developed as therapeutic delivery vehicles though risks associated with the colonization of a genetically modified microorganism have so-far not been addressed. Here, we present an engineered Limosilactobacillus reuteri strain with reduced colonization potential. We applied a dual-recombineering scheme for efficient barcoding and generated mutants in genes encoding five previously characterized and four uncharacterized putative adhesins. Compared with the wild type, none of the mutants were reduced in their ability to survive gastrointestinal transit in mice. CmbA was identified as a key protein in L. reuteri adhesion to HT-29 and enteroid cells. The nonuple mutant, a single strain with all nine genes encoding adhesins inactivated, had reduced capacity to adhere to enteroid monolayers. The nonuple mutant producing murine IFN-β was equally effective as its wild-type counterpart in mitigating radiation toxicity in mice. Thus, this work established a novel therapeutic delivery platform that lays a foundation for its application in other microbial therapeutic delivery candidates and furthers the progress of the L. reuteri delivery system towards human use.IMPORTANCEOne major advantage to leverage gut microbes that have co-evolved with the vertebrate host is that evolution already has taken care of the difficult task to optimize survival within a complex ecosystem. The availability of the ecological niche will support colonization. However, long-term colonization of a recombinant microbe may not be desirable. Therefore, strategies need to be developed to overcome this potential safety concern. In this work, we developed a single strain in which we inactivated the encoding sortase, and eight genes encoding characterized/putative adhesins. Each individual mutant was characterized for growth and adhesion to epithelial cells. On enteroid cells, the nonuple mutant has a reduced adhesion potential compared with the wild-type strain. In a model of total-body irradiation, the nonuple strain engineered to release murine interferon-β performed comparable to a derivative of the wild-type strain that releases interferon-β. This work is an important step toward the application of recombinant L. reuteri in humans.

Cetuximab scFv-Modified 5-FU Loaded Chitosan Nanoparticles: "A Novel Therapeutic Platform."

Colorectal cancer [CRC] is among the most fatal types of cancer. An active targeting delivery system that specifically interacts with CRC cells could improve the therapy's outcomes. Herein, Cetuximab single-chain fragment variable antibody [scFv] fragments were conjugated to the surface of 5-FU encapsulated chitosan nanoparticles [CS NPs] to develop an effective therapeutic platform [scFv-CS/5-FU NPs]. CS/5-FU NPs were synthesized using a special fluidic system. Encapsulation efficiency [EE], loading capacity [LC], and the drug release profile of the particles were determined. scFv fragments were produced recombinantly and tailored on the surface of CS/5-FU NPs. The physicochemical features of scFv-CS/5-FU NPs were also characterized. MTT and flow cytometry assay investigated the toxicity effect of scFv-CS/5-FU NPs on the HCT116 cell line. CS/5-FU NPs had a homogenous spherical shape. They possessed sustainable drugrelease behavior. The produced scFv-CS/5-FU NPs were also spherical. scFv-CS/5-FU NPs significantly decreased the viability of cancerous cells in a dose-dependent manner and induced apoptosis in 97.97% of targeted cells. scFv-CS/5-FU NPs showed remarkable anti-CRC activity. This novel targeting delivery system reduced the effective dose of 5-FU which is of vital importance to decrease the devastating side effects of chemotherapy.

Modification of MWCNTs with Bi2WO6 nanoparticles targeting IL-1β and NLRP3 inflammasome via augmented autophagy.

This study reports the facile hydrothermal synthesis of pure Bi2WO6 and Bi2WO6\MWCNTs nanocomposite at specific molar ratio 1:2.5 of Bi2WO6:MWCNTs and elucidates their role in modulating the NLRP3 inflammasome pathway via autophagy induction. Comprehensive characterization techniques, including XRD, Raman, UV.Vis PL,FESEM,EDS and TEM, revealed the successful incorporation of MWCNTs into the Bi2WO6 structures, leading to enhanced crystattlinity, reduced band gap energy (2.4 eV) suppressed charge carrier recombination and mitigated nanoparticles aggregation. Notably, the reduced band gap facikitaed improved visible light harvesting, a crucial attribute for photocatalytic applications. Significantly, the nanocompsoite exhibited a remarkable capacity to augment autophagy in bone marrow-derived macrophages (BMDMs), consequently down-regulating the NLRP3 inflammasom activation and IL-1β secretion upon LPS and ATP stimulation. Immunofluorescence assays unveiled increased co-localization of LC3 and NLRP3, suggestion enhanced targeting of NLRP3 by autophagy. Inhibition of autophagy by 3-MA reversed these effects, confirming the pivotal role of autophagy induction. Furthermore, the nanocomposite attenuated caspase-1 activation and ASC oligomerzation, thereby impeding inflammasome assembly. Collectively, these findings underscore the potential of Bi2WO6\MWCNTs nanocompsite as a multifaceted therapeutic platform, levering its tailored optoelectronic properties and sbility to modulate the NLRP3 infalmmasome via autophagy augmentation. This work covers the way for the development of advanced nanomaterials with tunable functionalities for combating inflammatory disorders and antimicrobial applications.

Pluronic F127/lecithin PLGA nanoparticles as carriers of monocyte-targeted jakinibs: a potential therapeutic platform.

Aim: In this study, PLGA nanoparticles (PNPs) emulsified in Pluronic F127 (F127)/Lecithin (LEC) were designed to load Itacitinib (ITA), a selective JAK1 inhibitor, for targeting human monocytes.Materials & methods: The physicochemical characteristics of empty and ITA-loaded F127/LEC PNPs were analyzed. The binding and internalization of NPs in leukocytes were evaluated. The effect of NPs on monocyte activation and JAK1 inhibition was assessed.Results: F127/LEC PNPs were selectively bound and internalized by monocytes, sparing other leukocytes. ITA-F127/LEC PNPs significantly dampened monocyte activation. They also inhibited the monocyte's ability to promote T-cell proliferation and inhibited proinflammatory cytokine production.Conclusion: ITA-loaded F127/LEC PNPs showed potential for monocyte-targeted therapy, offering new avenues for disease treatment.

Injectable Autocatalytic Hydrogel Triggers Pyroptosis to Stimulate Anticancer Immune Response for Preventing Postoperative Tumor Recurrence.

Modulating immunosuppression while eliminating residual microscopic tumors is critical for inhibiting the postoperative recurrence of triple-negative breast cancer (TNBC). Although immunotherapy has shown potential in achieving this goal, due to multiple immunosuppression and poor immunogenicity of apoptosis, a satisfactory anti-recurrence effect still faces the challenge. Herein, an injectable hydrogel-encapsulated autocatalytic copper peroxide (CP@Gel) therapeutic platform is designed and combine it with the clinical-grade DNA methyltransferase inhibitor decitabine (DAC) to effectively inhibit TNBC growth and postoperative recurrence via pyroptosis, killing residual cancer cells that bypass apoptosis resistance while also improving immunogenicity and modulating immunosuppression to achieve an intense anti-tumor immune response. Following injection of the CP@Gel, the sustained release of CP leads to the autocatalytic generation of reactive oxygen species, resulting in caspase-3 activation, and the pre-administered DAC inhibits the methylation of Gsdme to elevate the GSDME protein levels, leading to intense pyroptosis and anti-tumor immune responses. The in vivo results show a 67% elimination of local tumor recurrence via treatment with DAC+CP@Gel, suggesting the successful integration of sustained drug release with autocatalysis and epigenetic modification. The results thus suggest great potential for pyroptosis-based and injectable hydrogel-aided strategies for preventing the postoperative recurrence of TNBC.

CACNA1A haploinsufficiency leads to reduced synaptic function and increased intrinsic excitability.

Haploinsufficiency of the CACNA1A gene, encoding the pore-forming α1 subunit of P/Q-type voltage-gated calcium channels, is associated with a clinically variable phenotype ranging from cerebellar ataxia, to neurodevelopmental syndromes with epilepsy and intellectual disability. To understand the pathological mechanisms of CACNA1A loss-of-function variants, we characterized a human neuronal model for CACNA1A haploinsufficiency, by differentiating isogenic induced pluripotent stem cell lines into glutamatergic neurons, and investigated the effect of CACNA1A haploinsufficiency on mature neuronal networks through a combination of electrophysiology, gene expression analysis, and in silico modeling. We observed an altered network synchronization in CACNA1A+/- networks alongside synaptic deficits, notably marked by an augmented contribution of GluA2-lacking AMPA receptors. Intriguingly, these synaptic perturbations coexisted with increased non-synaptically driven activity, as characterized by inhibition of NMDA and AMPA receptors on micro-electrode arrays. Single-cell electrophysiology and gene expression analysis corroborated this increased intrinsic excitability through reduced potassium channel function and expression. Moreover, we observed partial mitigation of the CACNA1A+/- network phenotype by 4-aminopyridine, a therapeutic intervention for episodic ataxia type 2. Positive modulation of KCa2 channels could reverse the CACNA1A+/- network electrophysiological phenotype. In summary, our study pioneers the characterization of a human induced pluripotent stem cell-derived neuronal model for CACNA1A haploinsufficiency, and has unveiled novel mechanistic insights. Beyond showcasing synaptic deficits, this neuronal model exhibited increased intrinsic excitability mediated by diminished potassium channel function, underscoring its potential as a therapeutic discovery platform with predictive validity.

Efficient electroporation in primary cells with PEDOT:PSS electrodes.

Precise and efficient delivery of macromolecules into cells enhances basic biology research and therapeutic applications in cell therapies, drug delivery, and personalized medicine. While pulsed electric field electroporation effectively permeabilizes cell membranes to deliver payloads without the need for toxic chemical or viral transduction agents, conventional bulk electroporation devices face major challenges with cell viability and heterogeneity due to variations in fields generated across cells and electrochemistry at the electrode-electrolyte interface. Here, we introduce the use of microfabricated electrodes based on the conducting polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS), which substantially increases cell viability and transfection efficiency. As a proof of concept, we demonstrate the enhanced delivery of Cas9 protein, guide RNA, and plasmid DNA into cell lines and primary cells. This use of PEDOT:PSS enables rapid modification of difficult-to-transfect cell types to accelerate their study and use as therapeutic platforms.

Mesenchymal Stem Cells Engineered by Multicomponent Coassembled DNA Nanofibers for Enhanced Wound Healing.

A major challenge for stem cell therapies, such as using mesenchymal stem cells to treat skin injuries, is the stable engraftment of exogenous cells and the maintenance of their regenerative capacities in the wound areas. DNA-based self-assembly strategies can be used for artificial and multifunctional cell surface engineering to stabilize and enhance their functions for therapeutic applications. Here, we developed DNA nanofiber-decorated stem cells, in which DNA-based, multivalent fiber-like structures were self-assembled in situ on the cell surfaces. These engineered stem cells have demonstrated robust reactive oxygen species (ROS) scavenging effects, specific adhesion to damaged vascular endothelial cells, and the ability to enhance angiogenesis, which were effective and safe for acute or chronic wound healing in a mouse model with excisional skin injury. This DNA nanostructure-engineered stem cell provides a novel therapeutic platform for the treatment of tissue damage.

An extracellular vesicle delivery platform based on the PTTG1IP protein

Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues. Chimeric proteins consisting of PTTG1IP fused with various cargo proteins, and separated by self-cleaving sequences (to promote cargo release), were shown to enable highly efficient functional delivery of Cre protein to recipient cell cultures and mouse xenograft tumors, and delivery of Cas9-sgRNA complexes to recipient reporter cells. The favorable membrane topology of PTTG1IP enabled facile engineering of further variants with improved properties, highlighting its versatility and potential as a platform for EV-based therapeutics.