Application of Deacetylated Poly-N-Acetyl Glucosamine Nanoparticles for the Delivery of miR-126 for the Treatment of Cecal Ligation and Puncture-Induced Sepsis.

Sepsis is an acute inflammatory syndrome in response to infection. In some cases, excessive inflammation from sepsis results in endothelial dysfunction and subsequent increased vascular permeability leading to organ failure. We previously showed that treatment with endothelial progenitor cells, which highly express microRNA-126 (miR-126), improved survival in mice subjected to cecal ligation and puncture (CLP) sepsis. miRNAs are important regulators of gene expression and cell function, play a major role in endothelial homeostasis, and may represent an emerging therapeutic modality. However, delivery of miRNAs to cells in vitro and in vivo is challenging due to rapid degradation by ubiquitous RNases. Herein, we developed a nanoparticle delivery system separately combining deacetylated poly-N-acetyl glucosamine (DEAC-pGlcNAc) polymers with miRNA-126-3p and miRNA-126-5p and testing these combinations in vitro and in vivo. Our results demonstrate that DEAC-pGlcNAc polymers have an appropriate size and zeta potential for cellular uptake and when complexed, DEAC-pGlcNAc protects miRNA from RNase A degradation. Further, DEAC-pGlcNAc efficiently encapsulates miRNAs as evidenced by preventing their migration in an agarose gel. The DEAC-pGlcNAc-miRNA complexes were taken up by multiple cell types and the delivered miRNAs had biological effects on their targets in vitro including pERK and DLK-1. In addition, we found that delivery of DEAC-pGlcNAc alone or DEAC-pGlcNAc:miRNA-126-5p nanoparticles to septic animals significantly improved survival, preserved vascular integrity, and modulated cytokine production. These composite studies support the concept that DEAC-pGlcNAc nanoparticles are an effective platform for delivering miRNAs and that they may provide therapeutic benefit in sepsis.

pGlcNAc Nanofiber Treatment of Cutaneous Wounds Stimulate Increased Tensile Strength and Reduced Scarring via Activation of Akt1.

Treatment of cutaneous wounds with poly-N-acetyl-glucosamine containing nanofibers (pGlcNAc), a novel polysaccharide material derived from a marine diatom, results in increased wound closure, antibacterial activities and innate immune responses. We have shown that Akt1 plays a central role in the regulation of these activities. Here, we show that pGlcNAc treatment of cutaneous wounds results in a smaller scar that has increased tensile strength and elasticity. pGlcNAc treated wounds exhibit decreased collagen content, increased collagen organization and decreased myofibroblast content. A fibrin gel assay was used to assess the regulation of fibroblast alignment in vitro. In this assay, fibrin lattice is formed with two pins that provide focal points upon which the gel can exert force as the cells align from pole to pole. pGlcNAc stimulation of embedded fibroblasts results in cellular alignment as compared to untreated controls, by a process that is Akt1 dependent. We show that Akt1 is required in vivo for the pGlcNAc-induced increased tensile strength and elasticity. Taken together, our findings suggest that pGlcNAc nanofibers stimulate an Akt1 dependent pathway that results in the proper alignment of fibroblasts, decreased scarring, and increased tensile strength during cutaneous wound healing.

Poly-N-acetyl glucosamine nanofibers for negative-pressure wound therapies.

The wound healing promoting effect of negative wound pressure therapies (NPWT) takes place at the wound interface. The use of bioactive substances at this site represents a major research area for the development of future NPWT therapies. To assess wound healing kinetics in pressure ulcers treated by NPWT with or without the use of a thin interface membrane consisting of poly-N-acetyl glucosamine nanofibers (sNAG) a prospective randomized clinical trial was performed. The safety of the combination of NPWT and sNAG was also assessed in patients treated with antiplatelet drugs. In the performed study, the combination of NPWT and sNAG in 10 patients compared to NPWT alone in 10 patients promoted wound healing due to an improved contraction of the wound margins (p = 0.05) without a change in wound epithelization. In 6 patients treated with antiplatelet drugs no increased wound bleeding was observed in patients treated by NPWT and sNAG. In conclusion, the application of thin membranes of sNAG nanofibers at the wound interface using NPWT was safe and augmented the action of NPWT leading to improved wound healing due to a stimulation of wound contraction.

Immunomodulatory effects of IL-12 released from poly-N-acetyl glucosamine gel matrix during schistosomiasis infection.

We have reported recently that Interleukin-12 (IL-12) released from poly-N-acetyl glucosamine gel matrix (F2 gel/IL-12) is more effective than free IL-12 to enhance vaccination of mice with Schistosoma soluble worm antigen preparation. The aim of this study is to evaluate the effect of F2 gel/IL-12 on the inflammatory responses in mice undergoing schistosomiasis infection in absence of vaccination. To achieve this, mice undergoing Schistosoma mansoni infection or cured from this infection, after treatment with praziquantil (PZQ), were treated with subcutaneous injection of IL-12 for 3 consecutive days or once with F2 gel loaded with IL-12 (F2 gel/IL-12). The treatment was started on day 35 days after infection. For infection, mice were infected with 100 cercariae of S. mansoni using tail immersion method. We found that treatment with F2 gel/IL-12 induced significant decreases in the egg burden with a moderate reduction in the size of granuloma and decrease in the cellular granulomatous reaction in the lung as compared to infected mice treated with IL-12. These effects of F2 gel/IL-12 were more pronounced in infected mice previously treated with the anti-schistosomal drug PZQ. The total numbers of white blood cells in all treated mice showed similar profile. Treatment with IL-12 or F2 gel/IL-12, however, showed significant reduction in the number of mononuclear cells when compared with non-treated infected mice. In conclusion, this study showed the ability of IL-12 released from F2 gel to lower the inflammatory response to Schistosoma infection even in absence of vaccination.

Evaluation of a novel poly N-acetyl glucosamine (pGlcNAc) hydrogel for treatment of the degenerating intervertebral disc.

AIMS: The early stages of degenerative disc disease (DDD) primarily affect the disc nucleus pulposus (NP). Tissue-engineered strategies may enhance intervertebral disc (IVD) functionality. The aim of this study was to develop and evaluate a novel deacetylated poly-N-acetyl glucosamine (pGlcNAc) hydrogel characterizing its biochemical effect on human IVD cells as well as material biomechanical properties. MAIN METHODS: A novel deacetylated derivative of a marine diatom-derived glycosaminoglycan was developed into a hydrogel formulation as a potential therapy to treat degenerating IVD NP. In vitro biochemical studies were conducted using primary human disc cell cultures to evaluate cell viability, metabolic activity, proteoglycan and extracellular matrix protein expression. The biomechanical hydration kinetics and viscoelastic behavior of the hydrogel were determined and compared with the behavior of human lumbar NP. KEY FINDINGS: Disc cell viability, metabolic activity, and proteoglycan content of the treated cells were observed to be significantly greater in experimental samples when compared to untreated control groups. RT-PCR and immunohistochemical data corroborated the expression of characteristic NP disc markers, aggrecan and type II collagen in cultured cells. Rheological data demonstrated that the elastic component of the hydrogel dominated the viscous component over a frequency range of 0.1 to 15.85rad/s. Of several formulations evaluated, a sulphated, deactylated derivative of the nanofiber derived pGlcNAc hydrogel demonstrated the most robust biologic effects on cell viability, metabolic activity, and proteoglycan expression. SIGNIFICANCE: This in vitro study using human disc cells demonstrates that a sulphated deacetylated glycosaminoglycan derivative hydrogel possesses promising characteristics motivating further evaluation as a potential therapy for NP degeneration.

Integrin-dependent Akt1 activation regulates PGC-1 expression and fatty acid oxidation.

BACKGROUND: Poly-N-acetyl glucosamine nanofibers derived from a marine diatom have been used to increase cutaneous wound healing. These nanofibers exert their activity by specifically activating integrins, which makes them a useful tool for dissecting integrin-mediated pathways. We have shown that short-fiber poly-N-acetyl glucosamine nanofiber (sNAG) treatment of endothelial cells results in increased cell motility and metabolic rate in the absence of increased cell proliferation. RESULTS: Using a Seahorse Bioanalyzer to measure oxygen consumption in real time, we show that sNAG treatment increases oxygen consumption rates, correlated with an integrin-dependent activation of Akt1. Akt1 activation leads to an increase in the expression of the transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). This is not due to increased mitochondrial biogenesis, but is associated with an increase in the expression of pyruvate dehydrogenase kinase 4 (PDK4), suggesting regulation of fatty acid oxidation. Blockade of fatty acid oxidation with etomoxir, an O-carnitine palmitoyltransferase-1 inhibitor, blocks the sNAG-dependent increased oxygen consumption. (3)H-palmitate uptake experiments indicate a PDK4-dependent increase in fatty acid oxidation, which is required for nanofiber-induced cell motility. CONCLUSIONS: Our findings imply a linear pathway whereby an integrin-dependent activation of Akt1 leads to increased PGC-1α and PDK4 expression resulting in increased energy production by fatty acid oxidation.

mRDH bandage for surgery and trauma: data summary and comparative review.

BACKGROUND: Bleeding often poses significant life-threatening situations to surgeons. After trauma, a one-third of civilian casualties and one-half of combat casualties die as a result of exsanguination. Recent advances have provided promising new hemostatic dressings that are applied directly to severely bleeding wounds in the pre-hospital period. METHODS: The modified Rapid Deployment Hemostat (mRDH) trauma/surgery bandage, containing fully acetylated, diatom-derived, poly-N-acetyl-glucosamine fibers, has a unique multifactorial hemostatic action that incorporates vasoconstriction, erythrocyte agglutination, and platelet and RBC activation. RESULTS: Animal studies have shown that the mRDH bandage quickly and completely stops both venous and arterial bleeding, even in the presence of a coagulopathy. A prospective study in humans is in accord with these findings. CONCLUSION: The mRDH trauma/surgery bandage was able to increase survival of patients after high-grade liver trauma with an associated coagulopathy. Additional clinical studies support this result.

Poly-N-acetyl glucosamine fibers activate bone regeneration in a rabbit femur injury model.

BACKGROUND: The purpose of this study was to evaluate the ability of a membrane material, consisting only of short poly-N-acetyl glucosamine (sNAG) nanofibers, to regenerate bone tissue after implantation into circular holes in the rabbit femur. METHODS: Three circular holes were created in the femurs of five male New Zealand white rabbits. The holes were ∼ 2.0 mm in diameter. Three holes in the left femur were implanted with the comparative control substance (Bone Wax; Ethicon, Inc.); three holes in the right femur were implanted with the sNAG membrane test article. Animals were killed 4 weeks after surgery, and macroscopic evaluation of the implant sites was made. Hematoxylin and eosin histology was performed on both control and test sites. RESULTS: All control (bone wax) sites had visible holes (defects) at the 28-day end point of the study and showed no evidence of new bone formation. All the 15 sNAG test sites were found to have new bone tissue present in the bone hole defects. Hematoxylin and eosin histology of the sNAG-treated test sites showed the presence of osteoblasts, osteocytes, and trabecula of new bone formation at the 28-day end point of the study. CONCLUSIONS: The sNAG membrane test material activated the regeneration of new bone tissue in a rabbit femur bone model after 28 days of implantation, whereas the control bone wax material did not.

Anti-bacterial effects of poly-N-acetyl-glucosamine nanofibers in cutaneous wound healing: requirement for Akt1.

BACKGROUND: Treatment of cutaneous wounds with poly-N-acetyl-glucosamine nanofibers (sNAG) results in increased kinetics of wound closure in diabetic animal models, which is due in part to increased expression of several cytokines, growth factors, and innate immune activation. Defensins are also important for wound healing and anti-microbial activities. Therefore, we tested whether sNAG nanofibers induce defensin expression resulting in bacterial clearance. METHODOLOGY: The role of sNAG in defensin expression was examined using immunofluoresence microscopy, pharmacological inhibition, and shRNA knockdown in vitro. The ability of sNAG treatment to induce defensin expression and bacterial clearance in WT and AKT1-/- mice was carried out using immunofluoresent microscopy and tissue gram staining. Neutralization, using an antibody directed against ß-defensin 3, was utilized to determine if the antimicrobial properties of sNAG are dependent on the induction of defensin expression. CONCLUSIONS/FINDINGS: sNAG treatment causes increased expression of both α- and ß-type defensins in endothelial cells and ß-type defensins in keratinocytes. Pharmacological inhibition and shRNA knockdown implicates Akt1 in sNAG-dependent defensin expression in vitro, an activity also shown in an in vivo wound healing model. Importantly, sNAG treatment results in increased kinetics of wound closure in wild type animals. sNAG treatment decreases bacterial infection of cutaneous wounds infected with Staphylococcus aureus in wild type control animals but not in similarly treated Akt1 null animals. Furthermore, sNAG treatment of S. aureus infected wounds show an increased expression of ß-defensin 3 which is required for sNAG-dependent bacterial clearance. Our findings suggest that Akt1 is involved in the regulation of defensin expression and the innate immune response important for bacterial clearance. Moreover, these findings support the use of sNAG nanofibers as a novel method for enhancing wound closure while simultaneously decreasing wound infection.

Poly-N-acetyl glucosamine gel matrix as a non-viral delivery vector for DNA-based vaccination.

Intramuscular administration of plasmid DNA vaccines is one of the main delivery approaches that can generate antigen specific T cell responses. However, major limitations of the intramuscular delivery strategy are the low level of myocyte transfection, resulting in a minimal level of protein expression; the inability to directly target antigen presenting cells, in particular dendritic cells, which are critical for establishment of efficacious antigen-specific immune responses. Although several viral vectors have been designed to improve plasmid DNA delivery, they have limitations, including the generation of neutralizing antibodies in addition to lacking the simplicity and versatility required for universal clinical application. We have developed an inexpensive non-viral delivery vector based on the polysaccharide polymer poly-N-acetyl glucosamine with the capability to target dendritic cells. This vector is fully biocompatible, biodegradable, and nontoxic. The advantage of the application of this delivery system relative to other approaches is discussed.

Using poly-N-acetyl glucosamine gel matrix to deliver IL-12 with anti-schistosomasis vaccination.

BACKGROUND: Interleukin (IL)-12 is a potential adjuvant in a variety of diseases including schistosomiasis. The clinical use of IL-12, however, is limited by the toxicity associated with its systemic administration. We have developed a novel delivery system (designated F2 gel matrix) composed of poly-N-acetyl glucosamine that has the dual properties of sustaining the release of proteins (e.g. interleukins) and adjuvant effects. The main aim of this study was to use a mouse model to test whether IL-12 released from F2 gel can induce adjuvant effects in the schistosomiasis setting as compared to those obtained after systemic delivery of IL-12. METHODOLOGY: First, we compared the toxicity induced by paracrine (delivered by F2 gel) and systemic IL-12. Second, we compared the induction of cytokines induced by paracrine and systemic IL-12. Third, we compared the adjuvant effects of paracrine and systemic IL-12-based prophylactic vaccination against schistosomiasis using soluble worm antigen preparation (SWAP). RESULTS: IL-12 released from F2 gel did not induce significant toxicity measured by alanine aminotransferase (ALT). We found similar serum levels of IFN-gamma, TNF-alpha and IL-2 after paracrine and systemic IL-12 treatments. We also found that vaccination with F2 gel/SWAP/IL-12 induced higher anti-schistosomal effects than IL-12/SWAP as evidenced by 1) the decrease in the total liver egg counts; 2) the reduction in the granuloma size and fibrotic reaction in the liver; and 3) the amelioration of the liver functions. CONCLUSION: Collectively, these results indicate that IL-12-F2 gel delivery approach could be considered as a potential strategy for the treatment of schistosomiasis.

Poly-N-acetyl glucosamine nanofibers: a new bioactive material to enhance diabetic wound healing by cell migration and angiogenesis.

INTRODUCTION: In several fields of surgery, the treatment of complicated tissue defects is an unsolved clinical problem. In particular, the use of tissue scaffolds has been limited by poor revascularization and integration. In this study, we developed a polymer, poly-N-acetyl-glucosamine (sNAG), with bioactive properties that may be useful to overcome these limitations. OBJECTIVE: To develop a scaffold-like membrane with bioactive properties and test the biologic effects in vitro and in vivo in diabetic wound healing. METHODS: In vitro, cells-nanofibers interactions were tested by cell metabolism and migration assays. In vivo, full thickness wounds in diabetic mice (n = 15 per group) were treated either with sNAG scaffolds, with a cellulosic control material, or were left untreated. Wound healing kinetics, including wound reepithelialization and wound contraction as well as microscopic metrics such as tissue growth, cell proliferation (Ki67), angiogenesis (PECAM-1), cell migration (MAP-Kinase), and keratinocyte migration (p 63) were monitored over a period of 28 days. Messenger RNA levels related to migration (uPAR), angiogenesis (VEGF), inflammatory response (IL-1beta), and extracellular matrix remodeling (MMP3 and 9) were measured in wound tissues. RESULTS: sNAG fibers stimulated cell metabolism and the in vitro migratory activity of endothelial cells and fibroblasts. sNAG membranes profoundly accelerated wound closure mainly by reepithelialization and increased keratinocyte migration (7.5-fold), granulation tissue formation (2.8-fold), cell proliferation (4-fold), and vascularization (2.7-fold) compared with control wounds. Expression of markers of angiogenesis (VEGF), cell migration (uPAR) and ECM remodeling (MMP3, MMP9) were up-regulated in sNAG treated wounds compared with controls. CONCLUSIONS: The key mechanism of the bioactive membranes is the cell-nanofiber stimulatory interaction. Engineering of bioactive materials may represent the clinical solution for a number of complex tissue defects.

The design and testing of a dual fiber textile matrix for accelerating surface hemostasis.

The standard treatment for severe traumatic injury is frequently compression and application of gauze dressing to the site of hemorrhage. However, while able to rapidly absorb pools of shed blood, gauze fails to provide strong surface (topical) hemostasis. The result can be excess hemorrhage-related morbidity and mortality. We hypothesized that cost-effective materials (based on widespread availability of bulk fibers for other commercial uses) could be designed based on fundamental hemostatic principles to partially emulate the wicking properties of gauze while concurrently stimulating superior hemostasis. A panel of readily available textile fibers was screened for the ability to activate platelets and the intrinsic coagulation cascade in vitro. Type E continuous filament glass and a specialty rayon fiber were identified from the material panel as accelerators of hemostatic reactions and were custom woven to produce a dual fiber textile bandage. The glass component strongly activated platelets while the specialty rayon agglutinated red blood cells. In comparison with gauze in vitro, the dual fiber textile significantly enhanced the rate of thrombin generation, clot generation as measured by thromboelastography, adhesive protein adsorption and cellular attachment and activation. These results indicate that hemostatic textiles can be designed that mimic gauze in form but surpass gauze in ability to accelerate hemostatic reactions.

Differential effect of materials for surface hemostasis on red blood cell morphology.

The design of devices for surface (topical) hemostasis has been based on maximizing activation of platelets and accelerating coagulation pathways. The studies reported herein examine another aspect of blood contact with topical hemostasis materials, i.e., surface binding of red blood cells (RBCs) and related alterations in RBC morphology. Whole blood was allowed to contact poly-N-acetyl glucosamine (pGlcNAc) containing materials: pGlcNAc nanofibers with parallel polymer alignment (beta-pGlcNAc), chitin, and chitosan. The effect on RBC morphology and function via contact with the artificial surfaces on the cell's morphology was examined with scanning and transmission electron microscopy (TEM). beta-pGlcNAc was found to densely bind RBCs and induce a stomatocytic-like morphology. Chitin and chitosan also bound RBCs, but with approximately 10-fold lower levels and with less distinct general morphologies. beta-pGlcNAc is thus unique in the nature of its interaction with RBCs. These studies indicate that the differential ability of various materials to bind and alter the morphology of RBCs at the artificial surface interface with blood is an important consideration in the design of devices for surface hemostasis.

Non-classical processes in surface hemostasis: mechanisms for the poly-N-acetyl glucosamine-induced alteration of red blood cell morphology and surface prothrombogenicity.

It is well established that platelets and the intrinsic plasma coagulation pathway can be activated when blood contacts artificial surfaces. Experiments were performed to assess the effect of hemostatic poly-N-acetyl glucosamine (pGlcNAc) nanofibers on red blood cells. The pGlcNAc nanofibers, isolated from a marine diatom, interact with red blood cells (RBCs) to produce stomatocytes. The stomatocytes could be converted to echinocytes by treatment with echinocytic reagents, as measured by electron microscopy. Electrophoretic and Western blot analysis of RBC surface proteins demonstrated that pGlcNAc fibers were bound to band 3 of the RBC. An important and unique result of the interaction of RBCs with pGlcNAc fibers was the activation of the intrinsic coagulation cascade. This prothrombotic effect was associated with the presentation of phosphatidylserine on the outer layer of the surface membrane of nanofiber bound RBCs. The results demonstrate that RBCs can play a direct and important role in achieving surface hemostasis by accelerating the generation of thrombin, and add to the growing body of evidence that RBCs can strongly interact with hemostatic systems.

Effects of poly-N-acetyl glucosamine (pGlcNAc) patch on wound healing in db/db mouse.

BACKGROUND: Poly-N-acetyl glucosamine (pGlcNAc) nanofiber-based materials, produced by a marine microalga, have been characterized as effective hemostatic agents. In this study, we hypothesized that a pGlcNAc fiber patch may enhance wound healing in the db/db mouse. METHODS: pGlcNAc patches were applied on 1-cm, full-thickness, skin wounds in the db/db mouse model. Wounds (n = 15 per group) were dressed with a pGlcNAc nanofiber patch for 1 hour, 24 hours, or left untreated. After the application time, patches were removed and wounds were allowed to heal spontaneously. The rate of wound closure was evaluated by digital analysis of unclosed wound area as a function of time. At day 10, wounds (n = 7 per group) were harvested and quantified with immunohistochemical markers of proliferation (Ki-67) and vascularization (platelet endothelial cell adhesion molecule). RESULTS: Wounds dressed with pGlcNAc patches for 1 hour closed faster than control wounds, reaching 90% closure in 16.6 days, 9 days faster than untreated wounds. Granulation tissue showed higher levels of proliferation and vascularization after 1-hour treatment than the 24-hour and left-untreated groups. Foreign body reaction to the material was not noted in applications up to 24 hours. DISCUSSION: In addition to its hemostatic properties, the pGlcNAc material also appears to accelerate wound closure in healing-impaired genetically diabetic mice. This material, with its combination of hemostatic and wound healing properties, has the potential to be effective agent for the treatment of complicated wounds.

Poly-N-acetyl glucosamine nanofibers regulate endothelial cell movement and angiogenesis: dependency on integrin activation of Ets1.

Poly-N-acetyl glucosamine (pGlcNAc) nanofiber-derived materials effectively achieve hemostasis during surgical procedures. Treatment of cutaneous wounds with pGlcNAc in a diabetic mouse animal model causes marked increases in cell proliferation and angiogenesis. We sought to understand the effect of the pGlcNAc fibers on primary endothelial cells (EC) in culture and found that pGlcNAc induces EC motility. Cell motility induced by pGlcNAc fibers is blocked by antibodies directed against alphaVbeta3 and alpha5beta1 integrins, both known to play important roles in the regulation of EC motility, in vitroand in vivo. pGlcNAc treatment activates mitogen-activated protein kinase and increases Ets1, vascular endothelial growth factor (VEGF) and interleukin 1 (IL-1) expression. pGlcNAc activity is not secondary to its induction of VEGF; inhibition of the VEGF receptor does not inhibit the pGlcNAc-induced expression of Ets1 nor does pGlcNAc cause the activation of VEGF receptor. Both dominant negative and RNA interference inhibition of Ets1 blocks pGlcNAc-induced EC motility. Antibody blockade of integrin results in the inhibition of pGlcNAc-induced Ets1 expression. These findings support the hypothesis that pGlcNAc fibers induce integrin activation which results in the regulation of EC motility and thus in angiogenesis via a pathway dependent on the Ets1 transcription factor and demonstrate that Ets1 is a downstream mediator of integrin activation.

Hemostatic properties of glucosamine-based materials.

Glucosamine- and N-acetyl glucosamine-containing polymers are being used in an increasing number of biomedical applications, including in products for surface (topical) hemostasis. The studies presented here investigate the relationship between the structure (conformation) and function (activation of hemostasis) of glucosamine-based materials. Several polymer systems were studied, including fibers isolated from a microalgal source containing poly-N-acetyl glucosamine polymers that are organized in a parallel, hydrogen-bonded tertiary structure and can be chemically modified to an antiparallel orientation; and gel formulation derivatives of the microalgal fibers consisting of partially deacetylated (F2 gel) and fully deacetylated (F3 gel) polymers. Comparison of the properties of the poly-N-acetyl glucosamine fiber-derived materials with chitin, chitosan, and commercial chitosan-based products are presented. Several studies were performed with the glucosamine-based materials, including (1) an analysis of the ability of materials to activate platelets and turnover of the intrinsic coagulation cascade, (2) an examination of the viscoelastic properties of mixtures of platelet-rich plasma and the glucosamine-based materials via thromboelastography, and (3) scanning electron microscopic studies to examine the morphology of the glucosamine-based materials. The results presented demonstrate that hemostatic responses to the glucosamine-based materials studied are highly dependent on their chemical nature and tertiary/quaternary structure. The unique natural microalgal fibers were found to have strongly prohemostatic activity compared to the other materials studied.

Review: novel nonviral delivery approaches for interleukin-12 protein and gene systems: curbing toxicity and enhancing adjuvant activity.

It has become increasingly apparent that the ability to generate an optimal host immune response requires effective cross talk between the innate and adaptive components of the immune system. Pro-inflammatory cytokines, in particular those that can induce a danger signal, often called signal 3, are crucial in this role of initiating and augmenting the presentation of exogenous antigen to T cells by dendritic cells. Interleukin-12 (IL-12) in particular has been defined as a "signal 3" cytokine required for the antigen cross priming. Given this unique interactive function, a significant amount of work has been performed to define possible therapeutic applications for IL-12. Systemic IL-12 administration can clearly act as a potent adjuvant for postvaccination T cell responses in a variety of diseases. As an example, in the cancer setting, systemic IL-12 is capable of suppressing tumor growth, metastasis, and angiogenesis in vivo. IL-12, however, has been associated with significant dose- and schedule-dependent toxicity in early clinical trials, results that have proven to be a major obstacle to its clinical application. Recent research has focused on decreasing the toxicity of IL-12 using different delivery approaches, including virus-based and gene-modified cell-based delivery. Although effective, these approaches also have limitations, including the generation of neutralizing antibodies, in addition to lacking the simplicity and versatility required for universal clinical application. Thus, there is a significant interest in the development of alternative delivery approaches for IL-12 administration that can overcome these issues. Several nonviral delivery approaches for IL-12 protein or gene expression vectors are being defined, including alum, liposomes, and polymer-based delivery. These developing approaches have shown promising adjuvant effects with significantly lessened systemic toxicity. This article discusses the potential capabilities of these nonvirus-based IL-12 delivery systems in different disease settings, including allergy, infection, and cancer.