A Novel Peptide Prevents Enterotoxin- and Inflammation-Induced Intestinal Fluid Secretion by Stimulating Sodium-Hydrogen Exchanger 3 Activity.

BACKGROUND & AIMS: Acute diarrheal diseases are the second most common cause of infant mortality in developing countries. This is contributed to by lack of effective drug therapy that shortens the duration or lessens the volume of diarrhea. The epithelial brush border sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) accounts for a major component of intestinal Na+ absorption and is inhibited in most diarrheas. Because increased intestinal Na+ absorption can rehydrate patients with diarrhea, NHE3 has been suggested as a potential druggable target for drug therapy for diarrhea. METHODS: A peptide (sodium-hydrogen exchanger 3 stimulatory peptide [N3SP]) was synthesized to mimic the part of the NHE3 C-terminus that forms a multiprotein complex that inhibits NHE3 activity. The effect of N3SP on NHE3 activity was evaluated in NHE3-transfected fibroblasts null for other plasma membrane NHEs, a human colon cancer cell line that models intestinal absorptive enterocytes (Caco-2/BBe), human enteroids, and mouse intestine in vitro and in vivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. RESULTS: N3SP uptake stimulated NHE3 activity at nmol/L concentrations under basal conditions and partially reversed the reduced NHE3 activity caused by elevated adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and Ca2+ in cell lines and in in vitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine in vivo and prevented cholera toxin-, Escherichia coli heat-stable enterotoxin-, and cluster of differentiation 3 inflammation-induced fluid secretion in a live mouse intestinal loop model. CONCLUSIONS: These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.

Polymeric nanoparticle gel for intracellular mRNA delivery and immunological reprogramming of tumors.

Immuno-oncology therapies have been of great interest with the goal of inducing sustained tumor regression, but clinical results have demonstrated the need for improved and widely applicable methods. An antigen-free method of cancer immunotherapy can stimulate the immune system to recruit lymphocytes and produce immunostimulatory factors without prior knowledge of neoantigens, while local delivery reduces the risk of systemic toxicity. To improve the interactions between tumor cells and cytotoxic lymphocytes, a gene delivery nanoparticle platform was engineered to reprogram the tumor microenvironment (TME) in situ to be more immunostimulatory by inducing tumor-associated antigen-presenting cells (tAPCs) to activate cytotoxic lymphocytes against the tumor. Biodegradable, lipophilic poly (beta-amino ester) (PBAE) nanoparticles were synthesized and used to co-deliver mRNA constructs encoding a signal 2 co-stimulatory molecule (4-1BBL) and a signal 3 immuno-stimulatory cytokine (IL-12), along with a nucleic acid-based immunomodulatory adjuvant. Nanoparticles are combined with a thermoresponsive block copolymer for gelation at the injection site for local NP retention at the tumor. The reprogramming nanoparticle gel synergizes with immune checkpoint blockade (ICB) to induce tumor regression and clearance in addition to resistance to tumor rechallenge at a distant site. In vitro and in vivo studies reveal increases in immunostimulatory cytokine production and recruitment of immune cells as a result of the nanoparticles. Intratumoral injection of nanoparticles encapsulating mRNA encoding immunostimulatory agents and adjuvants via an injectable thermoresponsive gel has great translational potential as an immuno-oncology therapy that can be accessible to a wide range of patients.

Machine learning guided structure function predictions enable in silico nanoparticle screening for polymeric gene delivery.

Developing highly efficient non-viral gene delivery reagents is still difficult for many hard-to-transfect cell types and, to date, has mostly been conducted via brute force screening routines. High throughput in silico methods of evaluating biomaterials can enable accelerated optimization and development of devices or therapeutics by exploring large chemical design spaces quickly and at low cost. This work reports application of state-of-the-art machine learning algorithms to a dataset of synthetic biodegradable polymers, poly(beta-amino ester)s (PBAEs), which have shown exciting promise for therapeutic gene delivery in vitro and in vivo. The data set includes polymer properties as inputs as well as polymeric nanoparticle transfection performance and nanoparticle toxicity in a range of cells as outputs. This data was used to train and evaluate several state-of-the-art machine learning algorithms for their ability to predict transfection and understand structure-function relationships. By developing an encoding scheme for vectorizing the structure of a PBAE polymer in a machine-readable format, we demonstrate that a random forest model can satisfactorily predict DNA transfection in vitro based on the chemical structure of the constituent PBAE polymer in a cell line dependent manner. Based on the model, we synthesized PBAE polymers and used them to form polymeric gene delivery nanoparticles that were predicted in silico to be successful. We validated the computational predictions in two cell lines in vitro, RAW 264.7 macrophages and Hep3B liver cancer cells, and found that the Spearman's R correlation between predicted and experimental transfection was 0.57 and 0.66 respectively. Thus, a computational approach that encoded chemical descriptors of polymers was able to demonstrate that in silico computational screening of polymeric nanomedicine compositions had utility in predicting de novo biological experiments. STATEMENT OF SIGNIFICANCE: Developing highly efficient non-viral gene delivery reagents is difficult for many hard-to-transfect cell types and, to date, has mostly been explored via brute force screening routines. High throughput in silico methods of evaluating biomaterials can enable accelerated optimization and development for therapeutic or biomanufacturing purposes by exploring large chemical design spaces quickly and at low cost. This work reports application of state-of-the-art machine learning algorithms to a large compiled PBAE DNA gene delivery nanoparticle dataset across many cell types to develop predictive models for transfection and nanoparticle cytotoxicity. We develop a novel computational pipeline to encode PBAE nanoparticles with chemical descriptors and demonstrate utility in a de novo experimental context.

Nanoparticles for generating antigen-specific T cells for immunotherapy.

T cell therapy shows promise as an immunotherapy in both immunostimulatory and immunosuppressive applications. However, the forms of T cell-based therapy that are currently in the clinic, such as adoptive cell transfer and vaccines, are limited by cost, time-to-treatment, and patient variability. Nanoparticles offer a modular, universal platform to improve the efficacy of various T cell therapies as nanoparticle properties can be easily modified for enhanced cell targeting, organ targeting, and cell internalization. Nanoparticles can enhance or even replace endogenous cells during each step of generating an antigen-specific T cell response - from antigen presentation and T cell activation to T cell maintenance. In this review, we discuss the unique applications of nanoparticles for antigen-specific T cell therapy, focusing on nanoparticles as vaccines (to activate endogenous antigen presenting cells (APCs)), as artificial Antigen Presenting Cells (aAPCs, to directly activate T cells), and as drug delivery vehicles (to support activated T cells).

Non-viral gene delivery of HIF-1α promotes angiogenesis in human adipose-derived stem cells.

Stable and mature vascular formation is a current challenge in engineering functional tissues. Transient, non-viral gene delivery presents a unique platform for delivering genetic information to cells for tissue engineering purposes and to restore blood flow to ischemic tissue. The formation of new blood vessels can be induced by upregulation of hypoxia-inducible factor-1α (HIF-1), among other factors. We hypothesized that biodegradable polymers could be used to efficiently deliver the HIF-1α gene to human adipose-derived stromal/stem cells (hASCs) and that this treatment could recruit an existing endogenous endothelial cell population to induce angiogenesis in a 3D cell construct in vitro. In this study, end-modified poly(ß-amino ester) (PBAE) nanocomplexes were first optimized for transfection of hASCs and a new biodegradable polymer with increased hydrophobicity and secondary amine structures, N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine end-modified poly(1,4-butanediol diacrylate-co-4-amino-1-butanol), was found to be most effective. Optimal PBAE nanocomplexes had a hydrodynamic diameter of approximately 140 nm and had a zeta potential of 30 mV. The PBAE polymer self-assembled with HIF-1α plasmid DNA and treatment of hASCs with these nanocomplexes induced 3D vascularization. Cells transfected with this polymer-DNA complex were found to have 106-fold upregulation HIF-1α expression, an approximately 2-fold increase in secreted VEGF, and caused the formation of vessel tubules compared to an untransfected control. These gene therapy biomaterials may be useful for regenerative medicine. STATEMENT OF SIGNIFICANCE: Not only is the formation of stable vasculature a challenge for engineering human tissues in vitro, but it is also of valuable interest to clinical applications such as peripheral artery disease. Previous studies using HIF-1α to induce vascular formation have been limited by the necessity of hypoxic chambers. It would be advantageous to simulate endogenous responses to hypoxia without the need for physical hypoxia. In this study, 3D vascular formation was shown to be inducible through non-viral gene delivery of HIF-1α with new polymeric nanocomplexes. A biodegradable polymer N'-(3-aminopropyl)-N,N-dimethylpropane-1,3-diamine end-modified poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) demonstrates improved transfection of human adipose-derived stem cells. This nanobiotechnology could be a promising strategy for the creation of vasculature for tissue engineering and clinical applications.

Bioprinted trachea constructs with patient-matched design, mechanical and biological properties.

Tracheal stenosis is a rare but life-threatening disease. Primary clinical procedures for treating this disease are limited if the region requiring repair is long or complex. This study is the first of its kind to fabricate bioprinted tracheal constructs with separate cartilage and smooth muscle regions using polycaprolactone (PCL) and human mesenchymal stem cell (hMSC)-laden hydrogels. Our final bioprinted trachea showed comparable elastic modulus and yield stress compared to native tracheal tissue. In addition, both cartilage and smooth muscle formation were observed in the desired regions of our bioprinted trachea through immunohistochemistry and western blot after two weeks of in vitro culture. This study demonstrates a novel approach to manufacture tissue engineered trachea with mechanical and biological properties similar to native trachea, which represents a step closer to overcoming the clinical challenges of treating tracheal stenosis.

Polymeric micro- and nanoparticles for immune modulation.

New advances in biomaterial-based approaches to modulate the immune system are being applied to treat cancer, infectious diseases, and autoimmunity. Particulate systems are especially well-suited to deliver immunomodulatory factors to immune cells since their small size allows them to engage cell surface receptors or deliver cargo intracellularly after internalization. Biodegradable polymeric particles are a particularly versatile platform for the delivery of signals to the immune system because they can be easily surface-modified to target specific receptors and engineered to release encapsulated cargo in a precise, sustained manner. Micro- and nanoscale systems have been used to deliver a variety of therapeutic agents including monoclonal antibodies, peptides, and small molecule drugs that function to activate the immune system against cancer or infectious disease, or suppress the immune system to combat autoimmune diseases and transplant rejection. This review provides an overview of recent advances in the development of polymeric micro- and nanoparticulate systems for the presentation and delivery of immunomodulatory agents targeted to a variety of immune cell types including APCs, T cells, B cells, and NK cells.