As the designs of polymer systems used to deliver nucleic acids continue to evolve, it is becoming increasingly apparent that the basic bulk manufacturing techniques of the past will be insufficient to produce polymer-nucleic acid nanocomplexes that possess the uniformity, stability, and potency required for their successful clinical translation and widespread commercialization. Traditional bulk-prepared products are often physicochemically heterogeneous and may vary significantly from one batch to the next. Here we show that preparation of bioreducible nanocomplexes with an emulsion-based droplet microfluidic system produces significantly improved nanoparticles that are up to fifty percent smaller, more uniform, and are less prone to aggregation. The intracellular integrity of nanocomplexes prepared with this microfluidic method is significantly prolonged, as detected using a high-throughput flow cytometric quantum dot Förster resonance energy transfer nanosensor system. These physical attributes conspire to consistently enhance the delivery of both plasmid DNA and messenger RNA payloads in stem cells, primary cells, and human cell lines. Innovation in processing is necessary to move the field toward the broader clinical implementation of safe and effective nonviral nucleic acid therapeutics, and preparation with droplet microfluidics represents a step forward in addressing the critical barrier of robust and reproducible nanocomplex production.

Microemulsion represents an attractive platform for fundamental and applied biomedical research because the emulsified droplets can serve as millions of compartmentalized micrometer-sized reactors amenable to high-throughput screening or online monitoring. However, establishing stable emulsions with surfactants that are compatible with biological applications remains a significant challenge. Motivated by the lack of commercially available surfactants suitable for microemulsion-based biological assays, this study describes the facile synthesis of a biocompatible fluorosurfactant with nonionic tris(hydroxymethyl)methyl (Tris) polar head groups. We have further demonstrated compatibility of the developed surfactant with diverse emulsion-based applications, including DNA polymeric nanoparticle synthesis, enzymatic activity assay, and bacterial or mammalian cell culture, in the setup of both double- and multiphases of emulsions.

Cellular reprogramming holds tremendous potential for cell therapy and regenerative medicine. Recently, fibroblasts have been directly converted into induced neurons (iNs) by overexpression of the neuronal transcription factors Ascl1, Brn2 and Myt1L. Hypothesizing that cell-topography interactions could influence the fibroblast-to-neuron reprogramming process, we investigated the effects of various topographies on iNs produced by direct reprogramming. Final iN purity and conversion efficiency were increased on micrograting substrates. Neurite branching was increased on microposts and decreased on microgratings, with a simplified dendritic arbor characterized by the reduction of MAP2(+) neurites. Neurite outgrowth increased significantly on various topographies. DNA microarray analysis detected 20 differentially expressed genes in iNs reprogrammed on smooth versus microgratings, and quantitative PCR (qPCR) confirmed the upregulation of Vip and downregulation of Thy1 and Bmp5 on microgratings. Electrophysiology and calcium imaging verified the functionality of these iNs. This study demonstrates the potential of applying topographical cues to optimize cellular reprogramming.

Oral nonviral gene delivery is the most attractive and arguably the most challenging route of administration. To identify a suitable carrier, we studied the transport of different classes (natural polymer, synthetic polymer and synthetic lipid-polymer) of DNA nanoparticles through three well-characterized cellular models of intestinal epithelium (Caco2, Caco2-HT29MTX and Caco2-Raji). Poly(phosphoramidate-dipropylamine) (PPA) and Lipid-Protamine-DNA (LPD) nanoparticles consistently showed the highest level of human insulin mRNA expression and luciferase protein expression in these models, typically at least three orders of magnitude above background. All of the nanoparticles increased tight junction permeability, with PPA and PEI having the most dramatic transepithelial electrical resistance (TEER) decreases of (35.3±8.5%) and (37.5±1.5%) respectively in the first hour. The magnitude of TEER decrease correlated with nanoparticle surface charge, implicating electrostatic interactions with the tight junction proteins. However, confocal microscopy revealed that the nanoparticles were mostly uptaken by the enterocytes. Quantitative uptake and transport experiments showed that the endocytosed, quantum dot (QD)-labeled PPA-DNA nanoparticles remained in the intestinal cells even after 24h. Negligible amount of quantum dot labeled DNA was detected in the basolateral chamber, with the exception of the Caco2-Raji co-cultures, which internalized nanoparticles 2 to 3 times more readily compared to Caco2 and Caco2-HT29MTX cultures. PEGylation decreased the transfection efficacy by at least an order of magnitude, lowered the magnitude of TEER decrease and halved the uptake of PPA-DNA nanoparticles. A key finding was insulin mRNA being detected in the underlying HepG2 cells, signifying that some of the plasmid was transported across the intestinal epithelial layer while retaining at least partial bioactivity. However, the inefficient transport suggests that transcytosis alone would not engender a significant therapeutic effect, and this transport modality must be augmented by other means in vivo to render nonviral oral gene delivery practical.

Transfection of DNA vaccines with chemokines may recruit dendritic cells (DCs) locally to capture the antigenic genes and their gene products to generate enhanced CD8(+) cytotoxic T lymphocytes (CTLs). In this study, we investigated the effects of macrophage inflammatory protein (MIP)-1 alpha, MIP-3 alpha, and MIP-3beta on human immunodeficiency virus (HIV) Gag DNA vaccination. The chemokine plasmids markedly enhanced the local infiltration of inflammatory cells and increased the presence of CD11c(+) B7.2(+)-activated DCs. MIP-1 alpha and MIP-3 alpha were potent adjuvants in augmenting CTLs and afforded strong protection to immunized animals against challenge with vaccinia virus expressing Gag (vv-Gag). However, decreased humoral response was observed. MIP-3beta plasmid did not dramatically alter immunity. The chemokine inoculation time with respect to DNA vaccine priming was also investigated. The injection of pMIP-3 alpha three days before Gag plasmid (pGag) vaccination markedly increased specific CTLs compared with simultaneous injection and led to higher protection against vv-Gag. Immunity was also shifted toward a T-helper type-1 (Th1) response. In contrast, inoculation with pMIP-3 alpha three days after pGag vaccination shifted immunity toward a Th2 response. Our data suggest that administration of a chemokine with DNA vaccines offers a valuable strategy to modulate the efficacy and polarization of specific immunity and that chemokine-antigen timing is critical in determining overall biological effects.

Cartilage defect is difficult to heal due to its avascular properties. Implantation of mesenchymal stem cell is one of the most promising approach for regenerating cartilage defects. Here we prepared polymeric nanofibrils decorated with cartilage-derived decellularized extracellular matrix (dECM) as a chondroinductive scaffold material for cartilage repair. To fabricate nanofibrils, eletrospun PCL nanofibers were fragmented by subsequent mechanical and chemical process. The nanofibrils were surface-modified with poly(glycidyl methacrylate) (PGMA@NF) via surface-initiated atom transfer radical polymerization (SI-ATRP). The epoxy groups of PGMA@NF were subsequently reacted with dECM prepared from bovine articular cartilage. Therefore, the cartilage-dECM-decorated nanofibrils structurally and biochemically mimic cartilage-specific microenvironment. Once adipose-derived stem cells (ADSCs) were self-assembled with the cartilage-dECM-decorated nanofibrils by cell-directed association, they exhibited differentiation hallmarks of chondrogenesis without additional biologic additives. ADSCs in the nanofibril composites significantly increased expression of chondrogenic gene markers in comparison to those in pellet culture. Furthermore, ADSC-laden nanofibril composites filled the osteochondral defects compactly due to their clay-like texture. Thus, the ADSC-laden nanofibril composites supported the long-term regeneration of 12 weeks without matrix loss during joint movement. The defects treated with the ADSC-laden PGMA@NF significantly facilitated reconstruction of their cartilage and subchondral bone ECM matrices compared to those with ADSC-laden nanofibrils, non-specifically adsorbing cartilage-dECM without surface decoration of PGMA.

Uveal melanoma (UM) is the most common intraocular malignant tumor in adults and has a low survival rate following metastasis; it is derived from melanocytes susceptible to reactive oxygen species (ROS). Carbon dot (Cdot) nanoparticles are a promising tool in cancer detection and therapy due to their unique photophysical properties, low cytotoxicity, and efficient ROS productivity. However, the effects of Cdots on tumor metabolism and growth are not well characterized. Here, the effects of Cdots on UM cell metabolomics, growth, invasiveness, and tumorigenicity are investigated in vitro and in vivo zebrafish and nude mouse xenograft model. Cdots dose-dependently increase ROS levels in UM cells. At Cdots concentrations below 100 µg mL-1, Cdot-induced ROS promote UM cell growth, invasiveness, and tumorigenicity; at 200 µg mL-1, UM cells undergo apoptosis. The addition of antioxidants reverses the protumorigenic effects of Cdots. Cdots at 25-100 µg mL-1 activate Akt/mammalian target of rapamycin (mTOR) signaling and enhance glutamine metabolism, generating a cascade that promotes UM cell growth. These results demonstrate that moderate, subapoptotic doses of Cdots can promote UM cell tumorigenicity. This study lays the foundation for the rational application of ROS-producing nanoparticles in tumor imaging and therapy.

Atherosclerosis is a primary precursor of cardiovascular disease (CVD), the leading cause of death worldwide. While proprotein convertase subtilisin/kexin 9 (PCSK9) contributes to CVD by degrading low-density lipoprotein receptors (LDLR) and altering lipid metabolism, PCSK9 also influences vascular inflammation, further promoting atherosclerosis. Here, we utilized a vascular microphysiological system to test the effect of PCSK9 activation or repression on the initiation of atherosclerosis and to screen the efficacy of a small molecule PCSK9 inhibitor. We have generated PCSK9 over-expressed (P+) or repressed (P-) human induced pluripotent stem cells (iPSCs) and further differentiated them to smooth muscle cells (viSMCs) or endothelial cells (viECs). Tissue-engineered blood vessels (TEBVs) made from P+ viSMCs and viECs resulted in increased monocyte adhesion compared to the wild type (WT) or P- equivalents when treated with enzyme-modified LDL (eLDL) and TNF-α. We also found significant viEC dysfunction, such as increased secretion of VCAM-1, TNF-α, and IL-6, in P+ viECs treated with eLDL and TNF-α. A small molecule compound, NYX-1492, that was originally designed to block PCSK9 binding with the LDLR was tested in TEBVs to determine its effect on lowering PCSK9-induced inflammation. The compound reduced monocyte adhesion in P+ TEBVs with evidence of lowering secretion of VCAM-1 and TNF-α. These results suggest that PCSK9 inhibition may decrease vascular inflammation in addition to lowering plasma LDL levels, enhancing its anti-atherosclerotic effects, particularly in patients with elevated chronic inflammation.

Light sheet fluorescence microscopy (LSFM) is a widely used imaging technique for living and large cleared samples. However, high-performance LSFM systems are often prohibitively expensive and not easily scalable for high-throughput applications. Here, we introduce a cost-effective, scalable, and versatile high-resolution imaging framework, called projected Light Sheet Microscopy (pLSM), which repurposes readily available off-the-shelf consumer-grade components and an over-the-network control architecture to achieve high-resolution imaging of living and cleared samples. We extensively characterize the pLSM framework and showcase its capabilities through high-resolution, multi-color imaging and quantitative analysis of mouse and post-mortem human brain samples cleared using various techniques. Moreover, we show the applicability of pLSM for high-throughput molecular phenotyping of human induced pluripotent cells (iPSC)-derived brain and vessel organoids. Additionally, we utilized pLSM for comprehensive live imaging of bacterial pellicle biofilms at the air-liquid interface, uncovering their intricate layered architecture and diverse cellular dynamics across different depths. Overall, the pLSM framework has the potential to further democratize LSFM by making high-resolution light sheet microscopy more accessible and scalable.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain.

Mesenchymal stem/stromal cells respond to physical cues present in their microenvironment such as substrate elasticity, geometry, or topography with respect to morphology, proliferation, and differentiation. Although studies have demonstrated the role of focal adhesions in topography-mediated changes of gene expression, information linking substrate topography to the nucleus remains scarce. Here we show by two-dimensional gel electrophoresis and western blotting that A-type lamins and retinoblastoma protein are downregulated in mesenchymal stem/stromal cells cultured on 350 nm gratings compared to planar substrates; these changes lead to a decrease in proliferation and changes in differentiation potential.

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

Direct in vivo administration of messenger RNA (mRNA) delivered in both naked and nanoparticle formats are actively investigated because the use of dendritic cells transfected ex vivo with mRNA for cancer therapy is expensive and needs significant infrastructure. Notably, intravenous and subcutaneous injections are the only routes of administration tested for mRNA nanoparticle tumor vaccination. In this report, we demonstrate that tumor immunity can be achieved via nasal administration of mRNA. Mice nasally immunized with mRNA delivered in nanoparticle format demonstrate delayed tumor progression in both prophylactic and therapeutic immunization models. The observed tumor immunity correlates with splenic antigen-specific CD8+ T cells and is achieved only when mRNA is delivered in nanoparticle but not in naked format. In conclusion, we demonstrate, as a proof-of-concept, a non-invasive approach to mRNA tumor vaccination, increasing its potential as a broadly applicable and off-the-shelf therapy for cancer treatment.

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs) on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3), hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

In critical limb ischemia (CLI), overproduction of reactive oxygen species (ROS) and impairment of neovascularization contribute to muscle damage and limb loss. Cerium oxide nanoparticles (CNP, or 'nanoceria') possess oxygen-modulating properties which have shown therapeutic utility in various disease models. Here we show that CNP exhibit pro-angiogenic activity in a mouse hindlimb ischemia model, and investigate the molecular mechanism underlying the pro-angiogenic effect. CNP were injected into a ligated region of a femoral artery, and tissue reperfusion and hindlimb salvage were monitored for 3 weeks. Tissue analysis revealed stimulation of pro-angiogenic markers, maturation of blood vessels, and remodeling of muscle tissue following CNP administration. At a dose of 0.6 mg CNP, mice showed reperfusion of blood vessels in the hindlimb and a high rate of limb salvage (71%, n = 7), while all untreated mice (n = 7) suffered foot necrosis or limb loss. In vitro, CNP promoted endothelial cell tubule formation via the Ref-1/APE1 signaling pathway, and the involvement of this pathway in the CNP response was confirmed in vivo using immunocompetent and immunodeficient mice and by siRNA knockdown of APE1. These results demonstrate that CNP provide an effective treatment of CLI with excessive ROS by scavenging ROS to improve endothelial survival and by inducing Ref-1/APE1-dependent angiogenesis to revascularize an ischemic limb.

Controlled release of CRISPR-Cas9 ribonucleoprotein (RNP) and codelivery with other drugs remain a challenge. We demonstrate controlled release of CRISPR-Cas9 RNP and codelivery with antitumor photosensitizer chlorin e6 (Ce6) using near-infrared (NIR)- and reducing agent-responsive nanoparticles in a mouse tumor model. Nitrilotriacetic acid-decorated micelles can bind His-tagged Cas9 RNP. Lysosomal escape of nanoparticles was triggered by NIR-induced reactive oxygen species (ROS) generation by Ce6 in tumor cells. Cytoplasmic release of Cas9/single-guide RNA (sgRNA) was achieved by reduction of disulfide bond. Cas9/sgRNA targeted the antioxidant regulator Nrf2, enhancing tumor cell sensitivity to ROS. Without NIR irradiation, Cas9 was degraded in lysosomes and gene editing failed in normal tissues. The synergistic effects of Ce6 photodynamic therapy and Nrf2 gene editing were confirmed in vivo. Controlled release of CRISPR-Cas9 RNP and codelivery with Ce6 using stimuli-responsive nanoparticles represent a versatile strategy for gene editing with potentially synergistic drug effects.

Cancer immunotherapy using cancer vaccines has shown great potential in the prevention and treatment of cancer. Here, we report an implantable autologous blood clot scaffold for enhanced cancer vaccination. It comprises a gel-like fibrin network formed by coagulation of blood to trap a large number of red blood cells. Upon implantation, the cross-linked RBCs in the blood clot can attract and recruit a great number of immune cells, leading to the formation of an "immune niche." Encapsulated with tumor-associated antigen and adjuvant, the blood clot vaccine (BCV) can induce a robust anticancer immune response. The BCV combined with immune checkpoint blockade effectively inhibits tumor growth in B16F10 and 4T1 tumor models. The proposed implantable blood clot cancer vaccine can be readily made by mixing the blood from patients with cancer with immunomodulating agents ex vivo, followed by reimplantation into the same patient for personalized cancer immunotherapy in future clinical translation.

Periodontitis is a common type of inflammatory bone loss and a risk factor for systemic diseases. The pathogenesis of periodontitis involves inflammatory dysregulation, which represents a target for new therapeutic strategies to treat periodontitis. After establishing the correlation of cell-free DNA (cfDNA) level with periodontitis in patient samples, we test the hypothesis that the cfDNA-scavenging approach will benefit periodontitis treatment. We create a nanoparticulate cfDNA scavenger specific for periodontitis by coating selenium-doped hydroxyapatite nanoparticles (SeHANs) with cationic polyamidoamine dendrimers (PAMAM-G3), namely G3@SeHANs, and compare the activities of G3@SeHANs with those of soluble PAMAM-G3 polymer. Both G3@SeHANs and PAMAM-G3 inhibit periodontitis-related proinflammation in vitro by scavenging cfDNA and alleviate inflammatory bone loss in a mouse model of ligature-induced periodontitis. G3@SeHANs also regulate the mononuclear phagocyte system in a periodontitis environment, promoting the M2 over the M1 macrophage phenotype. G3@SeHANs show greater therapeutic effects than PAMAM-G3 in reducing proinflammation and alveolar bone loss in vivo. Our findings demonstrate the importance of cfDNA in periodontitis and the potential for using hydroxyapatite-based nanoparticulate cfDNA scavengers to ameliorate periodontitis.

Silver nanoclusters (AgNCs) have outstanding physicochemical characteristics, including the ability to interact with proteins and DNA. Given the growing number of diagnostic and therapeutic applications of AgNCs, we evaluated the impact of AgNCs on DNA replication and DNA damage response in cell-free extracts prepared from unfertilized Xenopus laevis eggs. We find that, among a number of silver nanomaterials, AgNCs uniquely inhibited genomic DNA replication and abrogated the DNA replication checkpoint in cell-free extracts. AgNCs did not affect nuclear membrane or nucleosome assembly. AgNCs-supplemented extracts showed a strong defect in the loading of the mini chromosome maintenance (MCM) protein complex, the helicase that unwinds DNA ahead of replication forks. FLAG-AgNCs immunoprecipitation and mass spectrometry analysis of AgNCs associated proteins demonstrated direct interaction between MCM and AgNCs. Our studies indicate that AgNCs directly prevent the loading of MCM, blocking pre-replication complex (pre-RC) assembly and subsequent DNA replication initiation. Collectively, our findings broaden the scope of silver nanomaterials experimental applications, establishing AgNCs as a novel tool to study chromosomal DNA replication.

Burn, trauma, and various medical conditions including bacterial infection, diabetes complication, and surgery could lead to an acute cutaneous wound and scar formation. Application of tissue glues instead of sutures could minimize the additional trauma and scar formation. Despite the countless efforts devoted to the development of high-strength tissue glues, little attention has been paid to their influence on the scar formation. Here, we report the development of a new tissue glue with excellent biocompatibility and tunable degradability for scarless wound closure. A series of catechol-containing poly(amidoamine) (CPAA) polymers were synthesized via the one-step Michael addition of dopamine and bisacrylamide. The tertiary amino group in the polymer backbone was used to introduce a zwitterionic sulfobetaine group by one-step ring-opening polymerization. The introduction of the zwitterionic sulfobetaine group could easily tune the hydrophilicity and the degradability of CPAA without influencing the density of the catechol group in the polymer. Lap-shear tests on the porcine skin demonstrated a high adhesion strength of 7 kPa at 1 h, rising to 24 kPa by 12 h. Addition of silica nanoparticles could further enhance the adhesion strength by 50%. In vivo studies further confirmed that the CPAA tissue glue could effectively accelerate the healing process of incisional wounds on the back of Sprague Dawley rats compared with suture and reduce the scar formation.