Biomimetic polyelectrolyte coating of stem cells suppresses thrombotic activation and enhances its survival and function.

Mesenchymal stem cells (MSCs) therapy is a promising approach for treating inflammatory diseases due to their immunosuppressive and tissue repair characteristics. However, allogenic transplantation of MSCs induces thrombotic complications in some patients which limits its potential for clinical translation. To address this challenge, we have exploited the bioactivity of heparin, a well-known anticoagulant and immunosuppressive polysaccharide that is widely used in clinics. We have developed a smart layer-by-layer (LbL) coating strategy using gelatin and heparin polymers exploiting their overall positive and negative charges that enabled efficient complexation with the MSCs' glycocalyx. The stable coating of MSCs suppressed complement attack and mitigated thrombotic activation as demonstrated in human whole blood. Gratifyingly, the MSC coating retained its immunosuppressive properties and differentiation potential when exposed to inflammatory conditions and differentiation factors. We believe the simple coating procedure of MSCs will increase allogenic tolerance and circumvent the major challenge of MSCs transplantation.

Hyaluronic acid based next generation bioink for 3D bioprinting of human stem cell derived corneal stromal model with innervation.

Corneal transplantation remains gold standard for the treatment of severe cornea diseases, however, scarcity of donor cornea is a serious bottleneck. 3D bioprinting holds tremendous potential for cornea tissue engineering (TE). One of the key technological challenges is to design bioink compositions with ideal printability and cytocompatibility. Photo-crosslinking and ionic crosslinking are often used for the stabilization of 3D bioprinted structures, which can possess limitations on biological functionality of the printed cells. Here, we developed a hyaluronic acid-based dopamine containing bioink using hydrazone crosslinking chemistry for the 3D bioprinting of corneal equivalents. First, the shear thinning property, viscosity, and mechanical stability of the bioink were optimized before extrusion-based 3D bioprinting for the shape fidelity and self-healing property characterizations. Subsequently, human adipose stem cells (hASCs) and hASC-derived corneal stromal keratocytes were used for bioprinting corneal stroma structures and their cell viability, proliferation, microstructure and expression of key proteins (lumican, vimentin, connexin 43,α-smooth muscle actin) were evaluated. Moreover, 3D bioprinted stromal structures were implanted intoex vivoporcine cornea to explore tissue integration. Finally, human pluripotent stem cell derived neurons (hPSC-neurons), were 3D bioprinted to the periphery of the corneal structures to analyze innervation. The bioink showed excellent shear thinning property, viscosity, printability, shape fidelity and self-healing properties with high cytocompatibility. Cells in the printed structures displayed good tissue formation and 3D bioprinted cornea structures demonstrated excellentex vivointegration to host tissue as well asin vitroinnervation. The developed bioink and the printed cornea stromal equivalents hold great potential for cornea TE applications.

Interpenetrating gallol functionalized tissue adhesive hyaluronic acid hydrogel polarizes macrophages to an immunosuppressive phenotype.

Innovative scaffold designs that modulate the local inflammatory microenvironment through favorable macrophage polarization and suppressing oxidative stress are needed for successful clinical translation of regenerative cell therapies and graft integration. We herein report derivation of a hydrazone-crosslinked gallol functionalized hyaluronic acid (HA-GA)-based hydrogel that displayed outstanding viscoelastic properties and immunomodulatory characteristics. Grafting of 6% gallol (GA) to a HA-backbone formed an interpenetrative network by promoting an additional crosslink between the gallol groups in addition to hydrazone crosslinking. This significantly enhanced the mechanical stability and displayed shear-thinning/self-healing characteristics, facilitated tissue adhesive properties to porcine tissue and also displayed radical scavenging properties, protecting encapsulated fibroblasts from peroxide challenge. The THP-1 human macrophage cell line or primary bone-marrow-derived murine macrophages cultured within HA-GA gels displayed selective polarization to a predominantly anti-inflammatory phenotype by upregulating IL4ra, IL-10, TGF-ß, and TGF-ßR1 expression when compared with HA-HA gels. Conversely, culturing of pro-inflammatory activated primary murine macrophages in HA-GA gels resulted in a significant reduction of pro-inflammatory TNF-α, IL-1ß, SOCS3 and IL-6 marker expression, and upregulated expression of anti-inflammatory cytokines including TGF-ß. Finally, when the gels were implanted subcutaneously into healthy mice, we observed infiltration of pro-inflammatory myeloid cells in HA-HA gels, while immunosuppressive phenotypes were observed within the HA-GA gels. Taken together these data suggest that HA-GA gels are an ideal injectable scaffold for viable immunotherapeutic interventions. STATEMENT OF SIGNIFICANCE: Host immune response against the implanted scaffolds that are designed to deliver stem cells or therapeutic proteins in vivo significantly limits the functional outcome. For this reason, we have designed immunomodulatory injectable scaffolds that can favorably polarize the recruited macrophages and impart antioxidant properties to suppress oxidative stress. Specifically, we have tailored a hyaluronic acid-based extracellular matrix mimetic injectable scaffold that is grafted with immunomodulatory gallol moiety. Gallol functionalization of hydrogel not only enhanced the mechanical properties of the scaffold by forming an interpenetrating network but also induced antioxidant properties, tissue adhesive properties, and polarized primary murine macrophages to immunosuppressive phenotype. We believe such immunoresponsive implants will pave the way for developing the next-generation of biomaterials for regenerative medicine applications.

Redox responsive Pluronic micelle mediated delivery of functional siRNA: a modular nano-assembly for targeted delivery.

There is an unmet need to develop strategies that allow site-specific delivery of short interfering RNA (siRNA) without any associated toxicity. To address this challenge, we have developed a novel siRNA delivery platform using chemically modified pluronic F108 as an amphiphilic polymer with a releasable bioactive disulfide functionality. The micelles exhibited thermoresponsive properties and showed a hydrodynamic size of ∼291 nm in DLS and ∼200-250 nm in SEM at 37 °C. The grafting of free disulfide pyridyl groups enhanced the transfection efficiency and was successfully demonstrated in human colon carcinoma (HCT116; 88%) and glioma cell lines (U87; 90%), non-cancerous human dermal fibroblast (HDF; 90%) cells as well as in mouse embryonic stem (mES; 54%) cells. To demonstrate the versatility of our modular nanocarrier design, we conjugated the MDGI receptor targeting COOP peptide on the particle surface that allowed the targeted delivery of the cargo molecules to human patent-derived primary BT-13 gliospheres. Transfection experiments with this design resulted in ∼65% silencing of STAT3 mRNA in BT-13 gliospheres, while only ∼20% of gene silencing was observed in the absence of the peptide. We believe that our delivery method solves current problems related to the targeted delivery of RNAi drugs for potential in vivo applications.

Pluronic Micelle-Mediated Tissue Factor Silencing Enhances Hemocompatibility, Stemness, Differentiation Potential, and Paracrine Signaling of Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) evoke great excitement for treating different human diseases due to their ability to home inflamed tissues, suppress inflammation, and promote tissue regeneration. Despite great promises, clinical trial results are disappointing as allotransplantation of MSCs trigger thrombotic activity and are damaged by the complement system, compromising their survival and function. To overcome this, a new strategy is presented by the silencing of tissue factor (TF), a transmembrane protein that mediates procoagulant activity. Novel Pluronic-based micelles are designed with the pendant pyridyl disulfide group, which are used to conjugate TF-targeting siRNA by the thiol-exchange reaction. This nanocarrier design effectively delivered the payload to MSCs resulting in ∼72% TF knockdown (KD) without significant cytotoxicity. Hematological evaluation of MSCs and TF-KD MSCs in an ex vivo human whole blood model revealed a significant reduction in an instant-blood-mediated-inflammatory reaction as evidenced by reduced platelet aggregation (93% of free platelets in the TF-KD group, compared to 22% in untreated bone marrow-derived MSCs) and thrombin-antithrombin complex formation. Effective TF silencing induced higher MSC differentiation in osteogenic and adipogenic media and showed stronger paracrine suppression of proinflammatory cytokines in macrophages and higher stimulation in the presence of endotoxins. Thus, TF silencing can produce functional cells with higher fidelity, efficacy, and functions.

Discrepancies on the Role of Oxygen Gradient and Culture Condition on Mesenchymal Stem Cell Fate.

Over the past few years, mesenchymal stem (or stromal) cells (MSCs) have garnered enormous interest due to their therapeutic value especially for their multilineage differentiation potential leading to regenerative medicine applications. MSCs undergo physiological changes upon in vitro expansion resulting in expression of different receptors, thereby inducing high variabilities in therapeutic efficacy. Therefore, understanding the biochemical cues that influence the native local signals on differentiation or proliferation of these cells is very important. There have been several reports that in vitro culture of MSCs in low oxygen gradient (or hypoxic conditions) upregulates the stemness markers and promotes cell proliferation in an undifferentiated state, as hypoxia mimics the conditions the progenitor cells experience within the tissue. However, different studies report different oxygen gradients and culture conditions causing ambiguity in their interpretation of the results. In this progress report, it is aimed to summarize recent studies in the field with specific focus on conflicting results reported during the application of hypoxic conditions for improving the proliferation or differentiation of MSCs. Further, it is tried to decipher the factors that can affect characteristics of MSC under hypoxia and suggest a few techniques that could be combined with hypoxic cell culture to better recapitulate the MSC tissue niche.

Harnessing hyaluronic acid-based nanoparticles for combination therapy: A novel approach for suppressing systemic inflammation and to promote antitumor macrophage polarization.

Anti-inflammatory drugs such as dexamethasone (DEX) are commonly administered to cancer patients along with anticancer drugs, however, the effect of DEX on human cancers is poorly understood. In this article, we have tailored self-assembled nanoparticles derived from hyaluronic acid (HA) wherein, anti-inflammatory DEX was used as a hydrophobic moiety for inducing amphiphilicity. The HA-DEX micelles were subsequently loaded with chemotherapeutic agent, doxorubicin (DOX) (HA-DEX-DOX) and was utilized to deliver drug cargo to human cancer cells expressing different levels of CD44 receptors. We found that DEX suppressed the cytotoxicity of DOX in HCT116, while it synergistically enhanced cytotoxicity in MCF-7 cells. When we tested DOX and HA-DEX-DOX in an ex-vivo human whole blood, we found activation of complement and the coagulation cascade in one group of donors. Encapsulation of DOX within the nanoparticle core eliminated such deleterious side-effects. The HA-DEX-DOX also polarized bone-marrow-derived anti-inflammatory M2 macrophages, to pro-inflammatory M1 phenotype with the upregulation of the cytokines TNF-α, iNOS and IL-1ß.

Hyaluronan derived nanoparticle for simvastatin delivery: evaluation of simvastatin induced myotoxicity in tissue engineered skeletal muscle.

Statins are currently the most prescribed hypercholesterolemia-lowering drugs worldwide, with estimated usage approaching one-sixth of the population. However, statins are known to cause pleiotropic skeletal myopathies in 1.5% to 10% of patients and the mechanisms by which statins induce this response, are not fully understood. In this study, a 3D collagen-based tissue-engineered skeletal muscle construct is utilised as a screening platform to test the efficacy and toxicity of a new delivery system. A hyaluronic acid derived nanoparticle loaded with simvastatin (HA-SIM-NPs) is designed and the effect of free simvastatin and HA-SIM-NPs on cellular, molecular and tissue response is investigated. Morphological ablation of myotubes and lack of de novo myotube formation (regeneration) was evident at the highest concentrations (333.33 µM), independent of delivery vehicle (SIM or HA-SIM-NP). A dose-dependent disruption of the cytoskeleton, reductions in metabolic activity and tissue engineered (TE) construct tissue relaxation was evident in the free drug condition (SIM, 3.33 µM and 33.33 nM). However, most of these changes were ameliorated when SIM was delivered via HA-SIM-NPs. Significantly, homogeneous expressions of MMP2, MMP9, and myogenin in HA-SIM-NPs outlined enhanced regenerative responses compared to SIM. Together, these results outline statin delivery via HA-SIM-NP as an effective delivery mechanism to inhibit deleterious myotoxic side-effects.

Exploring membrane permeability of Tomatidine to enhance lipid mediated nucleic acid transfections.

Intracellular delivery of nucleic acids is one of the critical steps in the transfections. Prior findings demonstrated various strategies including membrane fusion, endosomal escape for the efficient cytoplasmic delivery. In our continuing efforts to improve the nucleic acids transfections, we harnessed cell permeable properties of Tomatidine (T), a steroidal alkaloid abundantly found in green tomatoes for maximizing intracellular delivery of lipoplexes. We doped Tomatidine into liposomes of cationic lipid with amide linker (A) from our lipid library. Six liposomal formulations (AT) of Lipid A (1 mM) with varying concentrations of Tomatidine (0-1 mM) were prepared and evaluated for their transfection efficacies. Owing to its signature characteristic of cell membrane permeability, Tomatidine modulated endocytosis process, enhanced the intracellular delivery of the lipoplexes, and in turn increased the transfection efficacy of cationic liposomes. Our findings provide 'proof of concept' for enhancing transfections in gene delivery applications with Tomatidine in cationic liposomal formulations. These findings can be further applied in lipid mediated gene therapy and drug delivery applications.