Emerging Role of miR-345 and Its Effective Delivery as a Potential Therapeutic Candidate in Pancreatic Cancer and Other Cancers.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with high mortality, poor prognosis, and palliative treatments, due to the rapid upregulation of alternative compensatory pathways and desmoplastic reaction. miRNAs, small non-coding RNAs, have been recently identified as key players regulating cancer pathogenesis. Dysregulated miRNAs are associated with molecular pathways involved in tumor development, metastasis, and chemoresistance in PDAC, as well as other cancers. Targeted treatment strategies that alter miRNA levels in cancers have promising potential as therapeutic interventions. miRNA-345 (miR-345) plays a critical role in tumor suppression and is differentially expressed in various cancers, including pancreatic cancer (PC). The underlying mechanism(s) and delivery strategies of miR-345 have been investigated by us previously. Here, we summarize the potential therapeutic roles of miR-345 in different cancers, with emphasis on PDAC, for miRNA drug discovery, development, status, and implications. Further, we focus on miRNA nanodelivery system(s), based on different materials and nanoformulations, specifically for the delivery of miR-345.

Polymeric Nanoparticle-Based Vaccine Adjuvants and Delivery Vehicles.

As vaccine formulations have progressed from including live or attenuated strains of pathogenic components for enhanced safety, developing new adjuvants to more effectively generate adaptive immune responses has become necessary. In this context, polymeric nanoparticles have emerged as a promising platform with multiple advantages, including the dual capability of adjuvant and delivery vehicle, administration via multiple routes, induction of rapid and long-lived immunity, greater shelf-life at elevated temperatures, and enhanced patient compliance. This comprehensive review describes advances in nanoparticle-based vaccines (i.e., nanovaccines) with a particular focus on polymeric particles as adjuvants and delivery vehicles. Examples of the nanovaccine approach in respiratory infections, biodefense, and cancer are discussed.

Fabrication of Two-Dimensional and Three-Dimensional High-Resolution Binder-Free Graphene Circuits Using a Microfluidic Approach for Sensor Applications.

In this study, a simple microfluidic method, which can be universally applied to different rigid or flexible substrates, was developed to fabricate high-resolution, conductive, two-dimensional and three-dimensional microstructured graphene-based electronic circuits. The method involves controlled and selective filling of microchannels on substrate surfaces with a conductive binder-free graphene nanoplatelet (GNP) solution. The ethanol-thermal reaction of GNP solution at low temperatures (∼75 °C) prior to microchannel filling (preheating) can further reduce the GNP andprovide a homogeneous GNP solution, which in turn enhances conductivity, reduces sheet resistance (∼0.05 kΩ sq-1), enables room-temperature fabrication, and eliminates harsh postprocessing, which makes this fabrication technique compatible with degradable substrates. This method can also be used in combination with 3D printing to fabricate 3D circuits. The feature sizes of the graphene patterns can range from a few micrometers (down to ∼15 µm in width and ∼5 µm in depth) to a few millimeters and use very small amounts of GNP solution (∼2.5 mg of graphene to obtain ∼0.1 kΩ sq-1 of sheet resistance for 1 cm2). This microfluidic approach can also be implemented using other conductive liquids, such as conductive graphene-silver solutions. This technology has the potential to pave the way for low-cost, disposable, and biodegradable circuits for a range of electronic applications including near-field communication antennas and pressure or strain sensors.

Development of a High-Flux Thin-Film Composite Nanofiltration Membrane with Sub-Nanometer Selectivity Using a pH and Temperature-Responsive Pentablock Co-Polymer.

Producing block co-polymer-based nanofiltration (NF) membranes with sharp molecular weight cutoffs via an efficient method exhibiting persistent size-based separation quality is challenging. In this study, this challenge was addressed by reporting a facile approach to fabricate pentablock co-polymer (PBC)-based thin-film composite (TFC) NF membranes. The PBC, consisting of temperature-responsive Pluronic F127 (PEO-b-PPO-b-PEO) middle blocks and pH-responsive poly(N,N-(diethylamino)ethyl methacrylate) end blocks, were synthesized by atom-transfer radical polymerization. This polymer was then attached electrostatically to the surface of polysulfone/sulfonated polyether-sulfone support membranes fabricated using a non-solvent-induced phase separation technique. The conformational changes of the PBC chains in response to pH and temperature determined the pure water flux and neutral solute (PEG 1000) rejection performance of TFC membranes. Permeability of the membranes increased from 13.0 ± 0.63 to 15.9 ± 0.06 L/m2·h·bar and from 6.7 ± 0.00 to 13.9 ± 0.07 L/m2·h·bar by changing the solution pH from 4 to 8.5 and temperature from 4 to 25 °C, respectively. The pH- and temperature-responsive conformational changes did not affect the PEG 1000 rejection and membrane pore radius, which remained constant at ∼89% and ∼0.9 nm, respectively. This important finding was attributed to the high grafting density of co-polymer chains, resulting in spatial limitations among the grafted chains. The pore size of ∼0.9 nm achieved with the proposed membrane design is the smallest size reported so far for membranes fabricated from block co-polymers. TFC membranes demonstrated high stability and maintained their flux and rejection values under both static (storage in an acidic solution for up to 1 month) and dynamic (filtering PEG 1000 solution over 1 week) conditions. Pentablock co-polymers enable a NF membrane with a sharp molecular weight cutoff suitable for size-selective separations. The membrane fabrication technique proposed in this study is a scalable and promising alternative that does not involve complex synthetic routes.

Fabrication of High-resolution Graphene-based Flexible Electronics via Polymer Casting.

In this study, a novel method based on the transfer of graphene patterns from a rigid or flexible substrate onto a polymeric film surface via solvent casting was developed. The method involves the creation of predetermined graphene patterns on the substrate, casting a polymer solution, and directly transferring the graphene patterns from the substrate to the surface of the target polymer film via a peeling-off method. The feature sizes of the graphene patterns on the final film can vary from a few micrometers (as low as 5 µm) to few millimeters range. This process, applied at room temperature, eliminates the need for harsh post-processing techniques and enables creation of conductive graphene circuits (sheet resistance: ~0.2 kΩ/sq) with high stability (stable after 100 bending and 24 h washing cycles) on various polymeric flexible substrates. Moreover, this approach allows precise control of the substrate properties such as composition, biodegradability, 3D microstructure, pore size, porosity and mechanical properties using different film formation techniques. This approach can also be used to fabricate flexible biointerfaces to control stem cell behavior, such as differentiation and alignment. Overall, this promising approach provides a facile and low-cost method for the fabrication of flexible and stretchable electronic circuits.

Single dose combination nanovaccine provides protection against influenza A virus in young and aged mice.

Immunosenescence poses a formidable challenge in designing effective influenza vaccines for aging populations. While approved vaccines against influenza viruses exist, their efficacy in older adults is significantly decreased due to the diminished capabilities of innate and adaptive immune responses. In this work, the ability of a combination nanovaccine containing both recombinant hemagglutinin and nucleoprotein to provide protection against seasonal influenza virus infection was examined in young and aged mice. Vaccine formulations combining two nanoadjuvants, polyanhydride nanoparticles and pentablock copolymer micelles, were shown to enhance protection against challenge compared to each adjuvant alone in young mice. Nanoparticles were shown to enhance in vitro activation of dendritic cells isolated from aged mice, while both nanoadjuvants did not induce proinflammatory cytokine secretion which may be detrimental in aged individuals. In addition, the combination nanovaccine platform was shown to induce demonstrable antibody titers in both young and aged mice that correlated with the maintenance of body weight post-challenge. Collectively, these data demonstrate that the combination nanovaccine platform is a promising technology for influenza vaccines for older adults.

Dual delivery nanoscale device for miR-345 and gemcitabine co-delivery to treat pancreatic cancer.

A polymeric dual delivery nanoscale device (DDND) was designed for combined delivery of microRNA (miR-345) and gemcitabine (GEM) to treat pancreatic cancer (PC). This temperature and pH-responsive pentablock copolymer system was able to restore miR-345, making xenograft tumors more susceptible to GEM, the standard therapy for PC. Restoration using DDND treatment results in sonic hedgehog signaling down regulation, which decreases desmoplasia, thereby resulting in improved GEM perfusion to the tumor and better therapeutic outcomes. The release of miR-345 and GEM could be tuned by using the DDND in the form of micelles or in the form of thermoreversible gels, based on polymer concentration. The DDNDs enabled miR-345 stability and sustained co-release of miR-345 and GEM, thereby facilitating dose-sparing use of GEM. Further, enhanced in vitro cellular uptake due to amphiphilic character, and endosomal escape because of the cationic end blocks led to efficient transfection with DDNDs. The combined DDND treatment enabled efficient reduction in cell viability of Capan-1 and CD18/HPAF cells in vitro compared with either GEM or miR-345 treatment alone. Mice carrying xenograft tumors treated with DDNDs carrying both miR-345 and GEM combination therapy displayed reduced tumor growth and less metastasis in distant organs compared to individual drug treatments. Immunohistochemical analysis of the xenograft tissues revealed significant down regulation of desmoplastic reaction, SHH, Gli-1, MUC4, and Ki67 compared to control groups.

Advances in Controlling Differentiation of Adult Stem Cells for Peripheral Nerve Regeneration.

Adult stems cells, possessing the ability to grow, migrate, proliferate, and transdifferentiate into various specific phenotypes, constitute a great asset for peripheral nerve regeneration. Adult stem cells' ability to undergo transdifferentiation is sensitive to various cell-to-cell interactions and external stimuli involving interactions with physical, mechanical, and chemical cues within their microenvironment. Various studies have employed different techniques for transdifferentiating adult stem cells from distinct sources into specific lineages (e.g., glial cells and neurons). These techniques include chemical and/or electrical induction as well as cell-to-cell interactions via co-culture along with the use of various 3D conduit/scaffold designs. Such scaffolds consist of unique materials that possess controllable physical/mechanical properties mimicking cells' natural extracellular matrix. However, current limitations regarding non-scalable transdifferentiation protocols, fate commitment of transdifferentiated stem cells, and conduit/scaffold design have required new strategies for effective stem cells transdifferentiation and implantation. In this progress report, a comprehensive review of recent advances in the transdifferentiation of adult stem cells via different approaches along with multifunctional conduit/scaffolds designs is presented for peripheral nerve regeneration. Potential cellular mechanisms and signaling pathways associated with differentiation are also included. The discussion with current challenges in the field and an outlook toward future research directions is concluded.

Proteomic analysis of mesenchymal to Schwann cell transdifferentiation.

While transplantation of Schwann cells facilitates axon regeneration, remyelination and repair after peripheral nerve injury clinical use is limited by cell bioavailability. We posit that such limitation in cell access can be overcome by the use of autologous bone-marrow derived mesenchymal stem cells (MSCs). As MSCs can transdifferentiate to Schwann cell-phenotypes and accelerate nerve regeneration we undertook proteomic evaluation of the cells to uncover the protein contents that affects Schwann cell formulation. Transdifferentiated MSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in cell-conditioned media that facilitated neurite outgrowth. MSC proteins significantly regulated during Schwann cell transdifferentiation included, but were not limited to, GNAI2, MYL9, ACTN4, ACTN1, ACTB, CAV-1, HSPB1, PHB2, TBB4B, CTGF, TGFI1, ARF6, EZR, GELS, VIM, WNT5A, RTN4, EFNB1. These support axonal guidance, myelination, neural development and neural growth and differentiation. The results unravel the molecular events that underlie cell transdifferentiation that ultimately serve to facilitate nerve regeneration and repair in support of cell transplantation. SIGNIFICANCE STATEMENT: While Schwann cells facilitate axon regeneration, remyelination and repair after peripheral nerve injury clinical use is limited by cell bioavailability. We posit that such limitation in cell access can be overcome by the use of bone-marrow derived mesenchymal stem cells (MSCs) transdifferentiated to Schwann cell-phenotypes. In the present study, we undertook the first proteomic evaluation of these transdifferentiated cells to uncover the protein contents that affects Schwann cell formulation. Furthermore, these transdifferentiated MSCs secrete significant amounts of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in cell-conditioned media that facilitated neurite outgrowth. Our results demonstrate that a number of MSC proteins were significantly regulated following transdifferentiation of the MSCs supporting roles in axonal guidance, myelination, neural development and differentiation. The conclusions of the present work unravel the molecular events that underlie cell transdifferentiation that ultimately serve to facilitate nerve regeneration and repair in support of cell transplantation. Our study was the first proteomic comparison demonstrating the transdifferentiation of MSCs and these reported results can affect a wide field of stem cell biology, tissue engineering, and proteomics.

Electrical Differentiation of Mesenchymal Stem Cells into Schwann-Cell-Like Phenotypes Using Inkjet-Printed Graphene Circuits.

Graphene-based materials (GBMs) have displayed tremendous promise for use as neurointerfacial substrates as they enable favorable adhesion, growth, proliferation, spreading, and migration of immobilized cells. This study reports the first case of the differentiation of mesenchymal stem cells (MSCs) into Schwann cell (SC)-like phenotypes through the application of electrical stimuli from a graphene-based electrode. Electrical differentiation of MSCs into SC-like phenotypes is carried out on a flexible, inkjet-printed graphene interdigitated electrode (IDE) circuit that is made highly conductive (sheet resistance < 1 kΩ/sq) via a postprint pulse-laser annealing process. MSCs immobilized on the graphene printed IDEs and electrically stimulated/treated (etMSCs) display significant enhanced cellular differentiation and paracrine activity above conventional chemical treatment strategies [≈85% of the etMSCs differentiated into SC-like phenotypes with ≈80 ng mL-1 of nerve growth factor (NGF) secretion vs. 75% and ≈55 ng mL-1 for chemically treated MSCs (ctMSCs)]. These results help pave the way for in vivo peripheral nerve regeneration where the flexible graphene electrodes could conform to the injury site and provide intimate electrical simulation for nerve cell regrowth.