Spinal cord injury commonly leads to permanent motor and sensory deficits due to the limited regenerative capacity of the adult central nervous system (CNS). Nucleic acid-based therapy is a promising strategy to deliver bioactive molecules capable of promoting axonal regeneration. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to its cytotoxicity and low transfection efficiency in the presence of serum proteins. In this study, we synthesized cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP), by grafting low molecular weight PLGA (4kDa) to bPEI (25kDa) at approximately a 3:1 ratio as an efficient nonviral vector. We show that PgP micelle is capable of efficiently transfecting plasmid DNA (pDNA) and siRNA in the presence of 10% serum in neuroglioma (C6) cells, neuroblastoma (B35) cells, and primary E8 chick forebrain neurons (CFN) with pDNA transfection efficiencies of 58.8%, 75.1 %, and 8.1 %, respectively. We also show that PgP provides high-level transgene expression in the rat spinal cord in vivo that is substantially greater than that attained with bPEI. The combination of improved transfection and reduced cytotoxicity in vitro in the presence of serum and in vivo transfection of neural cells relative to conventional bPEI suggests that PgP may be a promising nonviral vector for therapeutic nucleic acid delivery for neural regeneration.

To serve as artificial matrices for therapeutic cell transplantation, synthetic hydrogels must incorporate mechanisms enabling localized, cell-mediated degradation that allows cell spreading and migration. Previously, we have shown that hybrid semi-interpenetrating polymer networks (semi-IPNs) composed of hydrolytically degradable PEG-diacrylates (PEGdA), acrylate-PEG-GRGDS, and native hyaluronic acid (HA) support increased cell spreading relative to fully synthetic networks that is dependent on cellular hyaluronidase activity. This study systematically investigated the effects of PEGdA/HA semi-IPN network composition on 3D spreading of encapsulated fibroblasts, the underlying changes in gel structure responsible for this activity, and the ability of optimized gel formulations to support long-term cell survival and migration. Fibroblast spreading exhibited a biphasic response to HA concentration, required a minimum HA molecular weight, decreased with increasing PEGdA concentration, and was independent of hydrolytic degradation at early time points. Increased gel turbidity was observed in semi-IPNs, but not in copolymerized hydrogels containing methacrylated HA that did not support cell spreading; suggesting an underlying mechanism of polymerization-induced phase separation resulting in HA-enriched defects within the network structure. PEGdA/HA semi-IPNs were also able to support cell spreading at relatively high levels of mechanical properties (~10 kPa elastic modulus) compared to alternative hybrid hydrogels. In order to support long-term cellular remodeling, the degradation rate of the PEGdA component was optimized by preparing blends of three different PEGdA macromers with varying susceptibility to hydrolytic degradation. Optimized semi-IPN formulations supported long-term survival of encapsulated fibroblasts and sustained migration in a gel-within-gel encapsulation model. These results demonstrate that PEGdA/HA semi-IPNs provide dynamic microenvironments that can support 3D cell survival, spreading, and migration for a variety of cell therapy applications.

The surgical connection of blood vessels, anastomosis, is a critical procedure in many reparative, transplantation, and reconstructive surgical procedures. However, effective restoration of circulation is complicated by pathological clotting (thrombosis) or progressive occlusion due to excess cell proliferation that often leads to additional surgeries and increases morbidity and mortality risk for patients. Pharmaceutical agents have been tested to prevent these complications, but many have unacceptable systemic side effects. Therefore, an alternative approach to deliver these drugs at the site of injury in a controlled manner is necessary. The objective of this study was to develop electrospun nanofibers composed of polyester poly(lactide- co-glycolide) (PLGA), poly(ethylene oxide) (PEO), and positively charged copolymer, poly(lactide- co-glycolide)- graft-polyethylenimine (PgP) for electrostatic binding and release of heparin for application as an antithrombotic microvascular suture. PgP was synthesized with different coupling ratios between PLGA and branched polyethylenimine (bPEI) to obtain PgP (∼1 PLGA grafted to 1 bPEI) and PgP (∼3.7 PLGA grafted to 1 bPEI). Nanofiber yarns (PLGA/PEO/PgP and PLGA/PEO/PgP) were fabricated by electrospinning. Heparin immobilization on the positively charged nanofiber yarns was visualized using fluorescein-conjugated heparin (F-Hep), and the amount of immobilized F-Hep was higher on both PLGA/PEO/PgP and PLGA/PEO/PgP than yarns without PgP (PLGA/PEO). We also found that F-Hep was released from both PgP-containing yarns in a sustained manner over 20 days, while over 60% of F-Hep was released within 4 h from PLGA/PEO. Finally, we observed that heparin-eluting nanofiber yarns with both PgP and PgP showed significantly longer clotting times than nanofiber yarns without PgP. The clotting time of PLGA/PEO/PgP was not significantly different than that of free heparin (0.5 μg/mL). These results show that heparin-eluting electrospun nanofiber yarns may offer a basis for the development of microvascular sutures with anticoagulant activity.

Excessive and prolonged neuroinflammation leads to neuronal cell death and limits functional recovery after traumatic brain injury (TBI). Dexamethasone (DX) is a steroidal anti-inflammatory agent that is known to attenuate early expression of pro-inflammatory cytokines associated with activated microglia/macrophages. In this study, we investigated the effect of dexamethasone-conjugated hyaluronic acid (HA-DXM) incorporated in a hydrolytically degradable, photo-cross-linkable poly (ethylene) glycol-bis-(acryloyloxy acetate) (PEG-bis-AA) hydrogel on the inflammatory response, apoptosis, and functional recovery in a controlled cortical impact (CCI) rat TBI model. In vitro, DX release from PEG-bis-AA/HA-DXM hydrogel was slow in phosphate-buffered saline without enzymes, but significantly increased in the presence of hyauronidase/esterase enzymes. TBI was generated by a CCI device armed with a 3 mm tip (3.5 m s−1, depth: 2 mm) and treated immediately with PEG-bis-AA/HA-DXM hydrogel. PEG-bis-AA/HA hydrogel without DX was used for comparison and untreated TBI group was used as a control. Significant reductions in cavity size, inflammatory response, and apoptosis were observed in animals treated with PEG-bis-AA/HA-DXM compared to those receiving PEG-bis-AA/HA and untreated. Animals receiving the PEG-bis-AA/HA-DXM hydrogel also exhibited higher neuronal cell survival and improved motor functional recovery compared to the other two groups.

Multiple age-related and injury-induced characteristics of the adult central nervous system (CNS) pose barriers to axonal regeneration and functional recovery following injury. In situ gene therapy is a promising approach to address the limited availability of growth-promoting biomolecules at CNS injury sites. The ultimate goal of our work is to develop, a cationic amphiphilic copolymer for simultaneous delivery of drug and therapeutic nucleic acids to promote axonal regeneration and plasticity after spinal cord injury. Previously, we reported the synthesis and characterization of a cationic amphiphilic copolymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) and its ability to efficiently transfect cells with pDNA in the presence of serum. We also demonstrated the efficacy of PgP as a therapeutic siRhoA carrier in a rat compression spinal cord injury model. In this work, we show that PgP/pDNA polyplexes provide improved stability in the presence of competing polyanions and nuclease protection in serum relative to conventional branched polyethylenimine control. PgP/pDNA polyplexes maintain bioactivity for transfection after lyophilization/reconstitution and during storage at 4 °C for up to 5 months, important features for commercial and clinical application. We also demonstrate that PgP/pDNA polyplexes loaded with a hydrophobic fluorescent dye are retained in local neural tissue for up to 5 days and that PgP can efficiently deliver pβ-Gal in a rat compression SCI model.

Scaffold-based delivery of bioactive molecules capable of directing stem cell differentiation is critical to the development of point-of-care cell therapy for orthopaedic repair. Dexamethasone-conjugated hyaluronic acid (HA-DXM) was synthesized and combined with hydrolytically degradable, photocrosslinkable PEG-bis-(2-acryloyloxy propanoate) (PEG-bis-AP) to form semi-IPNs. Dexamethasone (DX) release was limited in physiological buffer and substantially increased in the presence of encapsulated human mesenchymal stem cells (hMSCs) or exogenous hyaluronidase, confirming that release occurred primarily by a cell-mediated enzymatic mechanism. hMSCs encapsulated in PEG-bis-AP/HA-DXM semi-IPNs increased osteoblast-specific gene expression, alkaline phosphatase activity, and matrix mineralization, attaining levels that were not significantly different from positive controls consisting of hMSCs in PEG-bis-AP/native HA cultured with DX supplementation in the culture medium. These studies demonstrate that PEG-bis-AP/HA-DXM semi-IPNs can provide cell-mediated release of bioactive free DX that induces hMSC osteogenic differentiation. This approach offers an efficient system for local delivery of osteogenic molecules empowering point of care applications.

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium.

Functional recovery following traumatic brain injury (TBI) is limited due to progressive neuronal damage resulting from secondary injury-associated neuroinflammation. Steroidal anti-inflammatory drugs, such as dexamethasone (DX), can reduce neuroinflammation by activated microglia and infiltrated macrophages. In our previous work, we developed hydrolytically degradable poly(ethylene) glycol-bis-(acryloyloxy acetate) (PEG-bis-AA) hydrogels with dexamethasone (DX)-conjugated hyaluronic acid (HA-DXM) and demonstrated that dexamethasone-loaded hydrogels (PEG-bis-AA/HA-DXM) can reduce neuroinflammation, apoptosis, and lesion volume and improve neuronal cell survival and motor function recovery at seven days post-injury (DPI) in a rat mild-TBI model. In this study, we investigate the effects of the local application of PEG-bis-AA/HA-DXM hydrogels on motor function recovery at 7 DPI and cognitive functional recovery as well as secondary injury at 14 DPI in a rat mild-CCI TBI model. We observed that PEG-bis-AA/HA-DXM-treated animals exhibit significantly improved motor functions by the rotarod test and cognitive functions by the Morris water maze test compared to untreated TBI animals. We also observed that PEG-bis-AA/HA-DXM hydrogels reduce the inflammatory response, apoptosis, and lesion volume compared to untreated animals at 14 DPI. Therefore, PEG-bis-AA/HA-DXM hydrogels can be promising a therapeutic intervention for TBI treatment.