Microbial Genetic Composition Tunes Host Longevity.

Homeostasis of the gut microbiota critically influences host health and aging. Developing genetically engineered probiotics holds great promise as a new therapeutic paradigm to promote healthy aging. Here, through screening 3,983 Escherichia coli mutants, we discovered that 29 bacterial genes, when deleted, increase longevity in the host Caenorhabditis elegans. A dozen of these bacterial mutants also protect the host from age-related progression of tumor growth and amyloid-beta accumulation. Mechanistically, we discovered that five bacterial mutants promote longevity through increased secretion of the polysaccharide colanic acid (CA), which regulates mitochondrial dynamics and unfolded protein response (UPRmt) in the host. Purified CA polymers are sufficient to promote longevity via ATFS-1, the host UPRmt-responsive transcription factor. Furthermore, the mitochondrial changes and longevity effects induced by CA are conserved across different species. Together, our results identified molecular targets for developing pro-longevity microbes and a bacterial metabolite acting on host mitochondria to promote longevity.

Preparation and Characterization of Photo-Cross-Linkable Methacrylated Silk Fibroin and Methacrylated Hyaluronic Acid Composite Hydrogels.

Composite biomaterials with excellent biocompatibility and biodegradability are crucial in tissue engineering. In this work, a composite protein and polysaccharide photo-cross-linkable hydrogel was prepared using silk fibroin methacrylate (SFMA) and hyaluronic acid methacrylate (HAMA). SFMA was obtained by the methacrylation of degummed SF with glycidyl methacrylate (GMA), while HA was methacrylated by 2-aminoethyl methacrylate hydrochloride (AEMA). We investigated the effect of the addition of 1 wt % HAMA to 5, 10, and 20 wt % SFMA, which resulted in an increase in both static and cycling mechanical strengths. All composite hydrogels gelled under UV light in <30 s, allowing for rapid stabilization and stiffness increases. The biocompatibility of the hydrogels was confirmed by direct and indirect contact methods and by evaluation against the NIH3T3 and MC3T3 cell lines with a live-dead assay by confocal imaging. The range of obtained mechanical properties from developed composite and UV-cross-linkable hydrogels sets the basis as possible future biomaterials for various biomedical applications.

Nanofluidic microsystem for sustained intraocular delivery of therapeutics.

Globally, 145.2 million people suffer from moderate to severe vision impairment or blindness due to preventable or treatable causes. However, patient adherence to topical or intravitreal treatment is a leading cause of poor outcomes. To address this issue, we designed an intraocularly implantable device called the nanofluidic Vitreal System for Therapeutic Administration (nViSTA) for continuous and controlled drug release based on a nanochannel membrane that obviates the need for pumps or actuation. In vitro release analysis demonstrated that our device achieves sustained release of bimatoprost (BIM) and dexamethasone (DEX) at concentrations within clinically relevant therapeutic window. In this proof of concept study, we constructed an anatomically similar in silico human eye model to simulate DEX release from our implant and gain insight into intraocular pharmacokinetics profile. Overall, our drug-agnostic intraocular implant represents a potentially viable platform for long-term treatment of various chronic ophthalmologic diseases, including diabetic macular edema and uveitis.

The N-Terminal Domain of ALS-Linked TDP-43 Assembles without Misfolding.

Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) misfolding is implicated in several neurodegenerative diseases characterized by aggregated protein inclusions. Misfolding is believed to be mediated by both the N- and C-terminus of TDP-43; however, the mechanistic basis of the contribution of individual domains in the process remained elusive. Here, using single-molecule fluorescence and ensemble biophysical techniques, and a wide range of pH and temperature conditions, we show that TDP-43NTD is thermodynamically stable, well-folded and undergoes reversible oligomerization. We propose that, in full-length TDP-43, association between folded N-terminal domains enhances the propensity of the intrinsically unfolded C-terminal domains to drive pathological aggregation.

Genetically anchored fluorescent probes for subcellular specific imaging of hydrogen sulfide.

Imaging hydrogen sulfide (H2S) at the subcellular resolution will greatly improve the understanding of functions of this signaling molecule. Taking advantage of the protein labeling technologies, we report a general strategy for the development of organelle specific H2S probes, which enables sub-cellular H2S imaging essentially in any organelles of interest.

Precisely tunable engineering of sub-30 nm monodisperse oligonucleotide nanoparticles.

Advancement of RNAi therapies is mainly hindered by the development of efficient delivery vehicles. The ability to create small size (<30 nm) oligonucleotide nanoparticles is essential for many aspects of the delivery process but is often overlooked. In this report, we describe diblock star polymers that can reproducibly complex double-stranded oligonucleotides into monodisperse nanoparticles with 15, 23, or 30 nm in diameter. The polymer-nucleic acid nanoparticles have a core-shell architecture with dense PEG brush coating. We characterized these nanoparticles using ITC, DLS, FRET, FCS, TIRF, and TEM. In addition to small size, these nanoparticles have neutral zeta-potentials, making the presented polymer architecture a very attractive platform for investigation of yet poorly studied polyplex size range for siRNA and antisense oligonucleotide delivery applications.

Tunable thioesters as "reduction" responsive functionality for traceless reversible protein PEGylation.

Disulfide has been the only widely used functionality to serve as a reduction responsive trigger in drug delivery. We introduce thioester as a novel thiol responsive chemistry for drug delivery, whose reactivity can be conveniently modulated by choosing the appropriate steric environment around the thioester. Compared with disulfides, thioesters are facile to synthesize and have an order of magnitude broader kinetic tunability. A novel traceless reversible protein PEGylation reagent is developed based on thioester chemistry.

Poly(trehalose): sugar-coated nanocomplexes promote stabilization and effective polyplex-mediated siRNA delivery.

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly(methacrylamidotrehalose) or "poly(trehalose)" and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (n = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P(AEMA) and P(AEMA)]. First, we demonstrate that the poly(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC50 of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications.

Effects of trehalose polycation end-group functionalization on plasmid DNA uptake and transfection.

In this study, we have synthesized six analogs of a trehalose-pentaethylenehexamine glycopolymer (Tr4) that contain (1A) adamantane, (1B) carboxy, (1C) alkynyl-oligoethyleneamine, (1D) azido trehalose, (1E) octyl, or (1F) oligoethyleneamine end groups and evaluated the effects of polymer end group chemistry on the ability of these systems to bind, compact, and deliver pDNA to cultured HeLa cells. The polymers were synthesized in one-pot azide-alkyne cycloaddition reactions with an adaptation of the Carothers equation for step-growth polymerization to produce a series of polymers with similar degrees of polymerization. An excess of end-capping monomer was added at the end of the polymerizations to maximize functionalization efficiency, which was evaluated with GPC, NMR, and MALDI-TOF. The polymers were all found to bind and compact pDNA at similarly low N/P ratios and form polyplexes with plasmid DNA. The effects of the different end group structures were most evident in the polyplex internalization and transfection assays in the presence of serum as determined by flow cytometry and luciferase gene expression, respectively. The Tr4 polymers end-capped with carboxyl groups (1B) (N/P = 7), octyne (1E) (N/P = 7), and oligoethyleneamine (1F) (N/P = 7), were taken into cells as polyplex and exhibited the highest levels of fluorescence, resulting from labeled plasmid. Similarly, the polymers end-functionalized with carboxyl groups (1E at N/P = 7), octyl groups (1E at N/P = 15), and in particular oligoethyleneamine groups (1F at N/P = 15) yielded dramatically higher reporter gene expression in the presence of serum. This study yields insight into how very subtle structural changes in polymer chemistry, such as end groups can yield very significant differences in the biological delivery efficiency and transgene expression of polymers used for pDNA delivery.

Extending drug release from implants via transcutaneous refilling with solid therapeutics.

Long-acting (LA) implantable drug delivery systems (IDDS) offer an effective approach for the management or prevention of chronic conditions by sustained parenteral therapeutic administration. LA IDDS can and improve adherence to treatment regimens by minimizing dosing frequency. However, their clinical deployment is challenged by factors such as poor drug loading capacity, which limit their lifespan and require repeated surgical replacement for continued therapy. To address these challenges, and by leveraging previous work on nanofluidic systems, a reservoir-based IDDS that enables transcutaneous refilling of solid drug formulations through minimally invasive needle injection is presented. With thousand-fold higher drug loading efficiency, the implant affords minimal volume and aspect ratio suitable for discrete subcutaneous deployment. Key parameters for clinical acceptability, namely implant safety, access port robustness, and refilling method were systematically evaluated. The implant and refilling procedure are studied in rats and nonhuman primates with therapeutics used clinically for type 2 diabetes and human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP). The ability to extend drug release and maintain equivalent pharmacokinetics (PK) profiles pre- and post-drug refilling is demonstrated. This technology presents a clinically viable LA approach to prolong drug release for lifelong prevention or management of chronic conditions.

Diblock glycopolymers promote colloidal stability of polyplexes and effective pDNA and siRNA delivery under physiological salt and serum conditions.

A series of glycopolymers composed of 2-deoxy-2-methacrylamido glucopyranose (MAG) and the primary amine-containing N-(2-aminoethyl) methacrylamide (AEMA) were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The colloidal stability of the polyplexes formed with three diblock glycopolymers and pDNA was assessed using dynamic light scattering, and the polyplexes were found to be stable against aggregation in the presence of salt and serum over the 4 h time period studied. Delivery experiments were performed in vitro to examine the cellular uptake, transfection efficiency, and cytotoxicity of the glycopolymer/pDNA polyplexes in cultured HeLa cells and the diblock copolymer with the shortest AEMA block was found to be the most effective. Additionally, the ability of the diblock glycopolymers to deliver siRNA to U-87 (glioblastoma) cells was screened, and the diblock copolymer with the longest AEMA block was found to have gene knockdown efficacy similar to Lipofectamine 2000.

Carbon fiber reinforced polymers for implantable medical devices.

Carbon fibers reinforced polymers (CFRPs) are prolifically finding applications in the medical field, moving beyond the aerospace and automotive industries. Owing to its high strength-to-weight ratio, lightness and radiolucency, CFRP-based materials are emerging to replace traditional metal-based medical implants. Numerous types of polymers matrices can be incorporated with carbon fiber using various manufacturing methods, creating composites with distinct properties. Thus, prior to biomedical application, comprehensive evaluation of material properties, biocompatibility and safety are of paramount importance. In this study, we systematically evaluated a series of novel CFRPs, aiming at analyzing biocompatibility for future development into medical implants or implantable drug delivery systems. These CFRPs were produced either via Carbon Fiber-Sheet Molding Compound or Fused Deposition Modelling-based additive manufacturing. Unlike conventional methods, both fabrication processes afford high production rates in a time-and cost-effective manner. Importantly, they offer rapid prototyping and customization in view of personalized medical devices. Here, we investigate the physicochemical and surface properties, material mutagenicity or cytotoxicity of 20 CFRPs, inclusive of 2 surface finishes, as well as acute and sub-chronic toxicity in mice and rabbits, respectively. We demonstrate that despite moderate in vitro physicochemical and surface changes over time, most of the CFRPs were non-mutagenic and non-cytotoxic, as well as biocompatible in small animal models. Future work will entail extensive material assessment in the context of orthopedic applications such as evaluating potential for osseointegration, and a chronic toxicity study in a larger animal model, pigs.

Preventive efficacy of a tenofovir alafenamide fumarate nanofluidic implant in SHIV-challenged nonhuman primates.

Pre-exposure prophylaxis (PrEP) using antiretroviral oral drugs is effective at preventing HIV transmission when individuals adhere to the dosing regimen. Tenofovir alafenamide (TAF) is a potent antiretroviral drug, with numerous long-acting (LA) delivery systems under development to improve PrEP adherence. However, none has undergone preventive efficacy assessment. Here we show that LA TAF using a novel subcutaneous nanofluidic implant (nTAF) confers partial protection from HIV transmission. We demonstrate that sustained subcutaneous delivery through nTAF in rhesus macaques maintained tenofovir diphosphate concentration at a median of 390.00 fmol/106 peripheral blood mononuclear cells, 9 times above clinically protective levels. In a non-blinded, placebo-controlled rhesus macaque study with repeated low-dose rectal SHIVSF162P3 challenge, the nTAF cohort had a 62.50% reduction (95% CI: 1.72% to 85.69%; p=0.068) in risk of infection per exposure compared to the control. Our finding mirrors that of tenofovir disoproxil fumarate (TDF) monotherapy, where 60.00% protective efficacy was observed in macaques, and clinically, 67.00% reduction in risk with 86.00% preventive efficacy in individuals with detectable drug in the plasma. Overall, our nanofluidic technology shows potential as a subcutaneous delivery platform for long-term PrEP and provides insights for clinical implementation of LA TAF for HIV prevention.

Carbohydrate polymers for nonviral nucleic acid delivery.

Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed significantly to progress in the field of non-viral DNA delivery, and these new discoveries are impactful for developing new vehicles and materials for treatment of human disease.

Electrostatically gated nanofluidic membrane for ultra-low power controlled drug delivery.

Patient-centered therapeutic management for chronic medical conditions is a desired but unmet need, largely attributable to the lack of adequate technologies for tailored drug administration. While triggered devices that control the delivery of therapeutics exist, they often rely on impractical continuous external activation. As such, next generation continuously tunable drug delivery systems independent of sustained external activation remain an elusive goal. Here we present the development and demonstration of a silicon carbide (SiC)-coated nanofluidic membrane that achieves reproducible and tunable control of drug release via electrostatic gating. By applying a low-intensity voltage to a buried electrode, we showed repeatable and reproducible in vitro release modulation of three model analytes. A small fluorophore (Alexa Fluor 647), a large polymer poly(sodium 4-styrenesulfonate) and a medically relevant agent (DNA), were selected as representatives of small molecule therapeutics, polymeric drug carriers, and biological therapeutics, respectively. Unlike other drug delivery systems, our technology performed consistently over numerous cycles of voltage modulation, for over 11 days. Importantly, low power consumption and minimal leakage currents were achieved during the study. Further, the SiC coating maintained integrity and chemical inertness, shielding the membrane from degradation under simulated physiological and accelerated conditions for over 4 months. Through leveraging the flexibility offered by electrostatic gating control, our technology provides a valuable strategy for tunable delivery, setting the foundation for the next generation of drug delivery systems.

Trans-urocanic acid enhances tenofovir alafenamide stability for long-acting HIV applications.

Long-acting (LA) pre-exposure prophylaxis (PrEP) for HIV prevention is poised to address non-adherence and implementation challenges by alleviating the burden of user-dependent dosing. Due to its potency, tenofovir alafenamide (TAF) is a viable candidate for LA PrEP. However, the inherent hydrolytic instability of TAF presents a challenge for application in LA systems. In this work, we examined the mechanism of TAF hydrolysis in a reservoir-based implant system and characterized TAF degradation kinetics as a function of the solution pH. We determined a pH "stability window" between pH 4.8 - 5.8 in which TAF degradation is substantially mitigated, with minimal degradation at pH 5.3. In a pursuit of a TAF formulation suitable for LA PrEP, we studied trans-urocanic acid (UA) as a buffer excipient. Here we show that UA can maintain the pH of TAF free base (TAFfb) solution inside a surrogate implant model at approximately pH 5.4. Through in vitro analysis, we demonstrated preservation of released TAF purity above 90% for over 9 months. Further, we performed an in vivo assessment of TAFfb-UA formulation in a reservoir-based nanofluidic implant inserted subcutaneously in non-human primates. Preventive levels of tenofovir diphosphate above 100 fmol/106 peripheral blood mononuclear cells were achieved in 2 days and sustained over 35 days. Fluid retrieved from implants after 60 days of implantation showed that UA preserved the aqueous phase in the implant at ~ pH 5.5, effectively counteracting the neutralizing action of interstitial fluids. Moreover, residual TAF in the implants maintained > 98% purity. Overall, TAF-UA represents a viable formulation applicable for LA HIV PrEP.

Neovascularized implantable cell homing encapsulation platform with tunable local immunosuppressant delivery for allogeneic cell transplantation.

Cell encapsulation is an attractive transplantation strategy to treat endocrine disorders. Transplanted cells offer a dynamic and stimulus-responsive system that secretes therapeutics based on patient need. Despite significant advancements, a challenge in allogeneic cell encapsulation is maintaining sufficient oxygen and nutrient exchange, while providing protection from the host immune system. To this end, we developed a subcutaneously implantable dual-reservoir encapsulation system integrating in situ prevascularization and local immunosuppressant delivery, termed NICHE. NICHE structure is 3D-printed in biocompatible polyamide 2200 and comprises of independent cell and drug reservoirs separated by a nanoporous membrane for sustained local release of immunosuppressant. Here we present the development and characterization of NICHE, as well as efficacy validation for allogeneic cell transplantation in an immunocompetent rat model. We established biocompatibility and mechanical stability of NICHE. Further, NICHE vascularization was achieved with the aid of mesenchymal stem cells. Our study demonstrated sustained local elution of immunosuppressant (CTLA4Ig) into the cell reservoir protected transcutaneously-transplanted allogeneic Leydig cells from host immune destruction during a 31-day study, and reduced systemic drug exposure by 12-fold. In summary, NICHE is the first encapsulation platform achieving both in situ vascularization and immunosuppressant delivery, presenting a viable strategy for allogeneic cell transplantation.

Viral load Reduction in SHIV-Positive Nonhuman Primates via Long-Acting Subcutaneous Tenofovir Alafenamide Fumarate Release from a Nanofluidic Implant.

HIV-1 is a chronic disease managed by strictly adhering to daily antiretroviral therapy (ART). However, not all people living with HIV-1 have access to ART, and those with access may not adhere to treatment regimens increasing viral load and disease progression. Here, a subcutaneous nanofluidic implant was used as a long-acting (LA) drug delivery platform to address these issues. The device was loaded with tenofovir alafenamide (TAF) and implanted in treatment-naïve simian HIV (SHIV)-positive nonhuman primates (NHP) for a month. We monitored intracellular tenofovir-diphosphate (TFV-DP) concentration in the target cells, peripheral blood mononuclear cells (PBMC). The concentrations of TFV-DP were maintained at a median of 391.0 fmol/106 cells (IQR, 243.0 to 509.0 fmol/106 cells) for the duration of the study. Further, we achieved drug penetration into lymphatic tissues, known for persistent HIV-1 replication. Moreover, we observed a first-phase viral load decay of -1.14 ± 0.81 log10 copies/mL (95% CI, -0.30 to -2.23 log10 copies/mL), similar to -1.08 log10 copies/mL decay observed in humans. Thus, LA TAF delivered from our nanofluidic implant had similar effects as oral TAF dosing with a lower dose, with potential as a platform for LA ART.

2-Hydroxypropyl-ß-cyclodextrin-enhanced pharmacokinetics of cabotegravir from a nanofluidic implant for HIV pre-exposure prophylaxis.

Preexposure prophylaxis (PrEP) with antiretrovirals (ARV) can prevent human immunodeficiency virus (HIV) transmission, but its efficacy is highly dependent on strict patient adherence to daily dosing regimen. Long-acting (LA) ARV formulations or delivery systems that reduce dosing frequency may increase adherence and thus PrEP efficacy. While cabotegravir (CAB) long-acting injectable (CAB LA), an integrase strand transfer inhibitor (INSTI), reduces dosing frequency to bimonthly injections, variable pharmacokinetics (PK) between patients and various adverse reactions necessitate improvement in delivery methods. Here we developed a subcutaneously implantable nanofluidic device for the sustained delivery of CAB formulated with 2-hydroxypropyl-ß-cyclodextrin (ßCAB) and examined the pharmacokinetics (PK) in Sprague-Dawley rats for 3 months in comparison to CAB. Our study demonstrated ßCAB treatment group maintained clinically-relevant plasma CAB concentrations 2 times above the protein-adjusted concentration that inhibits viral replication by 90% (2 × PA-IC90) and drug penetration into tissues relevant to HIV-1 transmission. Further, we successfully fitted plasma CAB concentrations into a PK model (R2 = 0.9999) and determined CAB apparent elimination half-life of 47 days. Overall, our data shows the potential of sustained release of ßCAB via a nanofluidic implant for long-term PrEP delivery, warranting further investigation for efficacy against HIV infections.