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.

Gelatin-based 3D conduits for transdifferentiation of mesenchymal stem cells into Schwann cell-like phenotypes.

In this study, gelatin-based 3D conduits with three different microstructures (nanofibrous, macroporous and ladder-like) were fabricated for the first time via combined molding and thermally induced phase separation (TIPS) technique for peripheral nerve regeneration. The effects of conduit microstructure and mechanical properties on the transdifferentiation of bone marrow-derived mesenchymal stem cells (MSCs) into Schwann cell (SC) like phenotypes were examined to help facilitate neuroregeneration and understand material-cell interfaces. Results indicated that 3D macroporous and ladder-like structures enhanced MSC attachment, proliferation and spreading, creating interconnected cellular networks with large numbers of viable cells compared to nanofibrous and 2D-tissue culture plate counterparts. 3D-ladder-like conduit structure with complex modulus of ∼0.4×106Pa and pore size of ∼150µm provided the most favorable microenvironment for MSC transdifferentiation leading to ∼85% immunolabeling of all SC markers. On the other hand, the macroporous conduits with complex modulus of ∼4×106Pa and pore size of ∼100µm showed slightly lower (∼65% for p75, ∼75% for S100 and ∼85% for S100ß markers) immunolabeling. Transdifferentiated MSCs within 3D-ladder-like conduits secreted significant amounts (∼2.5pg/mL NGF and ∼0.7pg/mL GDNF per cell) of neurotrophic factors, while MSCs in macroporous conduits released slightly lower (∼1.5pg/mL NGF and 0.7pg/mL GDNF per cell) levels. PC12 cells displayed enhanced neurite outgrowth in media conditioned by conduits with transdifferentiated MSCs. Overall, conduits with macroporous and ladder-like 3D structures are promising platforms in transdifferentiation of MSCs for neuroregeneration and should be further tested in vivo. STATEMENT OF SIGNIFICANCE: This manuscript focuses on the effect of microstructure and mechanical properties of gelatin-based 3D conduits on the transdifferentiation of mesenchymal stem cells to Schwann cell-like phenotypes. This work builds on our recently accepted manuscript in Acta Biomaterialia focused on multifunctional 2D films, and focuses on 3D microstructured conduits designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. The comparison between conduits fabricated with nanofibrous, macroporous and ladder-like microstructures showed that the ladder-like conduits showed the most favorable environment for MSC transdifferentiation to Schwann-cell like phenotypes, as seen by both immunolabeling as well as secretion of neurotrophic factors. This work demonstrates the importance of controlling the 3D microstructure to facilitate tissue engineering strategies involving stem cells that can serve as promising approaches for peripheral nerve regeneration.

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.

Development of multifunctional films for peripheral nerve regeneration.

In this study, a poly(lactic acid) (PLLA) porous film with longitudinal surface micropatterns was fabricated by a dry phase inversion technique to be used as potential conduit material for peripheral nerve regeneration applications. The presence of a nerve growth factor (NGF) gradient on the patterned film surface and protein loaded, surface-eroding, biodegradable, and amphiphilic polyanhydride (PA) microparticles within the film matrix, enabled co-delivery of neurotrophic factors with controlled release properties and enhanced neurite outgrowth from PC12 cells. The protein loading capacity of PA particles was increased up to 80% using the spray drying technique, while the surface loading of NGF reached 300ng/cm2 through ester-amine interactions. The NGF surface gradient provided initial fast release from the film surface and facilitated directional neurite outgrowth along with the longitudinal micropatterns. Furthermore, the variable backbone chemistry and surface eroding nature of protein-loaded PA microparticles within the film matrix ensured protein stability and enabled controlled protein release. This novel co-delivery strategy yielded tunable diffusion coefficients varying between 6×10-14 and 1.67×10-10cm2/min and dissolution constants ranging from 1×10-4 to 1×10-3min-1 with released amounts of ∼100-300ng/mL. This strategy promoted guided neurite extension from PC12 cells of up to 10µm total neurite length per cell in 2days. Overall, this unique strategy can potentially be extended for individually programmed delivery of multiple growth factors through the use of PA microparticle cocktails and can further be investigated for in vivo performance as potential conduit material for peripheral nerve regeneration applications. STATEMENT OF SIGNIFICANCE: This manuscript focuses on the development of multifunctional degradable polymer films that provide topographic cues for guided growth, surface gradients of growth factors as well as nanoparticles in the films for tunable release of growth factors to enable peripheral nerve regeneration. The combination of cues was designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. These multifunctional films successfully provided high protein loading capacities while persevering activity, protein gradients on the surface, and tunable release of bioactive nerve growth factor that promoted directional and guided neurite extension of PC12 cells of up to 10µm in 2days. These multifunctional films can be made into conduits for peripheral nerve regeneration.

Effect of PEG Grafting Density and Hydrodynamic Volume on Gold Nanoparticle-Cell Interactions: An Investigation on Cell Cycle, Apoptosis, and DNA Damage.

In this study, interactions of polyethylene glycol (PEG)-coated gold nanoparticles (AuNPs) with cells were investigated with particular focus on the relationship between the PEG layer properties (conformation, grafting density, and hydrodynamic volume) and cell cycle arrest, apoptosis, and DNA damage. Steric hindrance and PEG hydrodynamic volume controlled the protein adsorption, whereas the AuNP core size and PEG hydrodynamic volume were primary factors for cell uptake and viability. At all PEG grafting densities, the particles caused significant cell cycle arrest and DNA damage against CaCo2 and PC3 cells without apoptosis. However, at a particular PEG grafting density (∼0.65 chains/nm(2)), none of these severe damages were observed on 3T3 cells indicating discriminating behavior of the healthy (3T3) and cancer (PC3 and CaCo2) cells. It was concluded that the PEG grafting density and hydrodynamic volume, tuned with the PEG concentration and AuNP size, played an important role in particle-cell interactions.