Additive-free synthesis of fused tricyclic cyanoisoxazolidines using in situ formed cyanonitrones.

Herein, we disclose the first report on the generation of cyanonitrone in situ from diazoacetonitrile and nitrosoarene, and its subsequent [3+2] cycloaddition with oxabicyclic alkenes to access fused tricyclic cyanoisoxazolidines. Further, this methodology could be extended to access fused tricyclic trifluoromethylated and phosphonylated isoxazolidines. Surprisingly, the reductive ring-opening of cyanoisoxazolidines was followed by a spontaneous lactonization to produce fused tricyclic amino lactones. Moreover, the N-O bond of the obtained tricyclic trifluoromethylated isoxazolidines could be cleaved to obtain 1,3-amino alcohols.

Curcumin inhibition of bleomycin-induced changes in lung collagen synthesis, deposition and assembly.

BACKGROUND: Idiopathic pulmonary fibrosis is characterized by progressive lung tissue remodeling and disproportionate deposition of collagenous proteins with limited therapeutic interventions. The purpose of this study was to determine whether curcumin inhibits bleomycin (BLM)-induced increases in synthesis, degradation and cross-linking of lung collagen in rats. METHODS AND RESULTS: Following a single intratracheal instillation of BLM to rats (0.75 U/100 g, sacrificed 3, 5, 7, 14 and 28 days post-BLM), lung collagen synthesis (determined by incorporation of 3H-proline) and deposition (determined by lung hydroxyproline content) progressively increased at days 7, 14 and 28 post-BLM injection. Lung lavage fluid hydroxyproline and collagenase levels (a measure of collagen turnover) were increased in BLM rats compared with control groups. In addition, BLM instillation resulted in increased concentrations of collagenase and collagenolytic cathepsin in the lungs. Furthermore, increased cross-linking (as determined by aldehyde content of acid soluble collagen), and decreased susceptibility of fibrotic lung insoluble collagen to denaturing agents occurred in BLM-injured lungs. Significant increases in alveolar macrophage (AM) release of transforming growth factor-ß1 (TGF-ß1) were noted at various time points (days 3, 5, 7, 14 and 28 post-BLM) during the development and progression of lung fibrosis in rats. Curcumin treatment to BLM rats (300 mg/kg 10 days before and daily thereafter throughout the experimental time period) was associated with marked reductions in lung collagen synthesis and deposition, BALF and lung collagenase activity, BALF hydroxyproline content and lung collagenolytic levels. Additionally, reduced levels of collagen cross-linking and enhanced susceptibility of insoluble lung collagen to denaturing agents were observed in curcumin-treated BLM rats. Finally, curcumin inhibited BLM-induced increases in AM production of TGF-ß1. CONCLUSIONS: Our data demonstrate for the first time that curcumin prevents fibrotic deposits by modulating collagen turnover, assembly and deposition in BLM-instilled rat lungs, and that curcumin treatment protects against BLM activation of macrophages by suppressing the release of TGF-ß1.

Protective effects of curcumin on bleomycin-induced changes in lung glycoproteins.

The present study investigated the therapeutic effect of curcumin on bleomycin (BLM)-induced alterations in glycoprotein components in the fibrotic lungs. Analysis of the bronchoalveolar lavage fluid (BALF) demonstrated increased fibronectin content at 3, 5, 7, and 14 days after BLM administration. Similarly, lung tissue fibronectin content revealed a progressive increase at various times (days 3, 5, 7, 14, and 28) during the development of lung fibrosis. In addition, alveolar macrophage release of fibronectin was also elevated in BLM-treated rats. Analysis of carbohydrate moieties of glycoproteins revealed an increase in total hexose, fucose, sialic acid and hexosamine levels at 7, 14, and 28 days after BLM treatment. Furthermore, the activities of lung glycosidases such as N-acetyl-ß-D-glucosaminidase, ß-glucosidase, ß-galactosidase, and ß-fucosidase in the fibrotic rats were elevated. Importantly, curcumin significantly inhibited the BLM-induced increases in BALF and lung fibronectin levels. Treatment of BLM rats with curcumin dramatically suppressed alveolar macrophage release of fibronectin. Curcumin also inhibited the increases in complex carbohydrates and glycosidases in the fibrotic lungs. These findings suggest that BLM-induced lung fibrosis is associated with accumulation of glycoproteins, and curcumin has the ability to suppress the enhanced deposition of glycoproteins in the fibrotic lung.

Efficacy of Recombinant Human Epidermal Growth Factor (Regen-D 150) in Healing Diabetic Foot Ulcers: A Hospital-Based Randomized Controlled Trial.

To validate the efficacy of recombinant human epidermal growth factor (hEGH) in healing diabetic foot ulcers (DFUs) at biochemical and molecular levels. A total of 50 noninfected DFU subjects were recruited for the study and divided into 2 groups based on the treatment application on the subjects. Group 1: DFU subjects treated with hEGH gel-based product called Regen-D 150 (n = 27) and group 2: DFU subjects treated with alternative placebo as the control group (n = 23). Patients were observed for 30 days and punch biopsy was taken at days 0 and 14. Histologic analysis was done to study the matrix alignment, cellular infiltration, and differentiation of epithelial layers. Biochemical analysis was done to quantitatively estimate the amount of collagen and proteoglycans regenerated in the wound area. Complete healing of ulcers was observed in 21 (78%) subjects in group 1, whereas only 12 (52%) subjects among group 2 reported of complete healing of ulcer after completion of the study period of 30 days. Collagen and fibroblasts were significantly developed in group 1 when observed in the follow-up samples. Healing time of the wound among the group 1 subjects was significantly less than the group 2 subjects (45 ± 12 vs 72 ± 18 days, P < .0001) and even showed a better blood glucose level. Early and regular application of the hEGH on DFUs will lead to prevention of leg amputations and would serve to act as a major treatment therapy for healing of chronic wounds.

Efficacy of desulfated heparin mitigating inflammation in rat burn wound model.

During skin repair, leukocyte infiltration is the principal inflammatory response which is instrumental in triggering growth factor and cytokine signals that orchestrate together to recruit cells necessary for healing. In severe wounds like burn, when acute inflammation becomes chronic, intervention is required to control inflammation so as to hasten the process of healing. Heparin, a known anticoagulant also possesses anti-inflammatory activity by its ability to interfere with the adhesion of leukocytes to the endothelium. Desulfated heparins (DSH) have subdued anticoagulant activity while possessing increased anti-inflammatory activity. Among which 2,3 DSH is found to have marked potency as an anti-inflammatory agent and has been utilized for this study. In this investigation, a controlled delivery system was designed by incorporating 2,3 DSH in microspheres and embedding in collagen matrix which could serve as a wound dressing in burns. In vivo evaluation of healing process was ascertained in rat burn wound model by qualitative and quantitative estimation of proinflammatory cytokines in serum and granulation tissue and collagen turnover was also assessed as healing progressed. The results of this study suggests that 2,3 DSH could be delivered in a controlled manner to regulate inflammatory events to hasten healing of burn wounds.

ISSLS prize winner: a study of effects of in vivo mechanical forces on human lumbar discs with scoliotic disc as a biological model: results from serial postcontrast diffusion studies, histopathology and biochemical analysis of twenty-one human lumbar scoliotic discs.

STUDY DESIGN: A comprehensive study of 21 lumbar scoliotic discs by in vivo serial post contrast diffusion magnetic resonance imaging (MRI), histopathological, and biochemical analysis. OBJECTIVE: To investigate the in vivo effects of compressive and tensile mechanical stress on the lumbar discs with scoliotic disc as the biologic model. SUMMARY OF BACKGROUND DATA: Most studies implicating mechanical stress in degenerative disc disease (DDD) are on animals, in vitro conditions and cadavers. They are also restricted to histopathological or biochemical evaluation without analyzing the endplate (EP) and nucleus pulposus (NP) separately. The few human studies have not analyzed diffusion changes which is the final pathway for DDD. Adolescent scoliotic disc offer a perfect model to study the effects of mechanical stress. METHODS: Twenty-one discs from 6 patients with adolescent idiopathic scoliosis undergoing anterior corrective surgery were assessed before surgery by postcontrast MRI to document the EP diffusion patterns. The same discs harvested during surgery were analyzed histologically and biochemically. The results were correlated to clinical and radiologic parameters. RESULTS: Altered diffusion patterns was seen in all discs with site specific breaks in 2, double peak pattern in 3, high intensity pattern in 14, and frank contrast leak in 2. There was marked decrease in cell density and viability in all discs on both convex and concave sides compared to the control disc (P = 0.001). Neovascularization, calcification, and matrix degeneration were observed to varying extent in different regions of NP and EP. There was a decrease in water content with increasing severity of curves with significant difference between mild and severe curves (NP: P = 0.000, EP: P = 0.002). Lactate was significantly higher in caudal EP (P = 0.035) and discs with coronal migration of more than 15 mm (P = 0.007). Regression analysis showed that truncal decompensation was a main factor for decrease in cell density, matrix degeneration, calcification, and water content. CONCLUSION: The study documents widespread changes in the EP and NP even in discs with minimal wedging. EP damage and alterations in diffusion were observed earlier than MRI changes and could indicate nutritional factors as the primary mechanism of degeneration induced by mechanical stress. Degeneration was more severe in caudal discs and those with truncal decompensation. Its implications on the timing and choice of surgery in scoliosis are discussed.

Efficacy of frog skin lipids in wound healing.

BACKGROUND: Frog skin has been sequentially and scientifically evaluated by our group for its wound healing efficiency. Owing to the complex structure of skin, attempts were being made to analyse the role of individual constituents in different phases of healing. Our earlier papers have shown the significance of frog skin not only in wound healing but also enhancing the proliferating activity of the epidermal and dermal cells which are instrumental for normal healing process. We also have identified for the first time novel antimicrobial peptides from the skin of Rana tigerina and thereby reduce the complications involved in the sepsis. PURPOSE OF THE STUDY AND RESULTS: The current study envisages the role of frog skin lipids in the inflammatory phase of wound healing. The lipid moiety of the frog skin dominated by phospholipids exhibited a dose dependent acceleration of healing irrespective of the mode of application. The efficiency of the extract is attributed partially to the anti-inflammatory activity as observed by the histochemical and immunostimulatory together with plethysmographic studies. CONCLUSIONS: Thus, frog skin for the first time has been demonstrated to possess lipid components with pharmaceutical and therapeutic potential. The identification and characterization of such natural healing molecules and evaluating their mechanism of action would therefore provide basis for understanding the cues of Nature and hence can be used for application in medicine.

Controlled release of 2, 3 desulfated heparin exerts its anti-inflammatory activity by effectively inhibiting E-selectin.

Control of inflammation using appropriate anti-inflammatory agent prevents wound from becoming chronic. Heparin is a heterogeneous mixture of polysaccharide molecules with a mean molecular weight between 12-30 kDa containing 200-300 disaccharide units of glycosaminoglycan chains. Chemical modifications leading to generation of a unique pentasaccharide sequence effectively reduces its anticoagualant activity, while retaining its anti-inflammmatory property. In this study, Standard heparin was partially desulfated to 2, 3 desulfated heparin (2, 3 DSH) and its anti-inflammatory property was determined by assessing its ability to prevent static adhesion of leukocytes to endothelium and clotting assay. The effectiveness of 2, 3 DSH to down regulate E-selectin and key proinflammatory cytokines (IL-1beta and IL-6) was assessed by western blot and immunocytochemistry. These results were compared with commercially available 2-Desulfated Heparin (2DSH) and standard heparin (SH). Finally, a controlled delivery system of 2, 3 DSH was designed using chitosan microspheres and collagen as scaffold. Optimal loading of 2, 3 DSH was achieved and the release kinetics were tuned to follow a controlled release pattern. The steady state concentration of 2, 3 DSH was found to be optimal to elicit anti-inflammatory property and could achieve inhibition of E-selectin expression while unaffecting the normal clotting cascade.

Adipose-derived stem cell delivery into collagen gels using chitosan microspheres.

Integration of stem cells to injured tissues requires an appropriate delivery device and scaffolding system. In the present study we have developed an in vitro strategy to load and release adipose-derived mesenchymal stem cells (ASC) from chitosan microspheres (CSM) into a collagen gel scaffold. Porous CSM of uniform size and composition were prepared and used as a stem cell carrier. ASC were allowed to attach to the microspheres and infiltrate through the microsphere pores. The number of viable cells was counted in vitro, using MTT and Calcein acetoxymethyl ester (AM) assays, and it showed a proportional increase with seeding density and reached a maximum cell number by 24 h. The cells inside the microspheres remained metabolically active and viable, could be retrieved from the spheres, and maintained expression of stem-cell-specific markers. Electron microscopic evaluation of the cell-microsphere complex showed that the CSM were able to support cell attachment and that the cells had infiltrated into the pores of the microspheres. The ability of the cells to proliferate and differentiate into adipogenic- and osteogenic-like precursors indicates that the cells have maintained their multipotency after migration out of the microspheres. To mimic cell delivery into a tissue, ASC-loaded CSM were embedded in type-1 collagen scaffold by mixing them with type-1 collagen solution while inducing gelation. By 14 days the cells released into the collagen gel and were able to populate the entire scaffold. When observed through transmission electron microscopy, the cells align along the collagen fibrils with a characteristic fibroblast-like morphology. This study provides a model to capture pluripotent stem cells, expand their cell number within a biomaterial scaffold in vitro, and deliver within an appropriate matrix to repair damaged tissue.

Collagen-based wound dressing for doxycycline delivery: in-vivo evaluation in an infected excisional wound model in rats.

OBJECTIVES: A novel collagen-based dressing consisting of 2,3-dihydroxybenzoic-acid-modified gelatin microspheres loaded with doxycycline has previously been reported to address both infection and matrix degradation. In the present study the potential benefits of the dressing were investigated in an excisional wound model in rats challenged with Pseudomonas aeruginosa. METHODS: A full-thick excisional wound (1.5 x 1.5 cm) was created on the dorsum of the rats and infection induced by injecting 10(5) colony-forming units (CFU) of P. aeruginosa. The healing pattern was assessed from wound reduction, matrix metalloprotease (MMP) levels, CFU reduction and histological and biochemical analysis. KEY FINDINGS: The treated group exhibited complete healing by day 15, compared with day 24 in the control group. Early subsidence of infection (99.9% by day 9) resulted in faster epidermal resurfacing and fibroplasias, whereas the microbial load exceeded 10(3) CFU even on day 15 in the control group and caused severe inflammation. Biochemical analysis showed that the expression of both collagen and hexosamine was significantly increased in the treated group. Gelatin zymography revealed prolonged expression of MMPs 2, 8 and 9 in the control group compared with the treated group. CONCLUSIONS: The study indicates that the developed dressing attenuated both infection and metalloprotease levels, and may therefore have potential application in wound healing.

Low decorin expression along with inherent activation of ERK1,2 in ear lobe keloids.

Over production of collagen and its abnormal assembly is the hallmark of abnormal scars such as keloids, a condition which reflects fibrosis of the skin. Fibrotic conditions of organs such as liver, lungs and eyes are characterized by low levels of decorin, a member of the small leucine proteoglycan family, which is an indispensable modulator of collagen assembly. Involvement of decorin in keloids is not well described, therefore, its expression in keloids was studied here. We showed low decorin levels in the total tissue extract of keloids as compared to normal skin. Further, we showed that this low level is due to reduced transcription of decorin by keloid fibroblasts. As decorin can also act as an intermediate signaling molecule involved in the ERK1,2 pathway, levels of both ERK1,2 and its activated counterpart were studied and found to be up-regulated in keloids. Through this study a novel attempt has been made to understand the implications of decorin expression and a possible cause of over production of collagen and its aberrant assembly in keloids is suggested.

Functionally modified gelatin microspheres impregnated collagen scaffold as novel wound dressing to attenuate the proteases and bacterial growth.

An attempt was made to develop a new therapeutic delivery system which would play a dual role of attenuating MMP activity in the wounds and also prevent infection by controlled delivery of antimicrobials. A catechol type MMP inhibitor 2,3-dihydroxybenzoic acid (DHBA) was conjugated to gelatin microspheres using EDC/NHS as coupling agents. The potential of the modified gelatin microspheres (DHB-MS) to attenuate the proteases such as MMP 2 and MMP 9 in the diabetic wound tissues was investigated by gelatin zymography. Further the modified microspheres were loaded with doxycycline and impregnated in a reconstituted collagen scaffold as novel wound dressing. The in vitro release behavior of doxycycline from both DHB-MS and DHB-MS impregnated collagen scaffold was investigated. DHB-MS when incubated with the tissue lysate for 6h displayed the complete inhibition of the MMPs in the tissue lysate. The in vitro drug release studies from the collagen scaffold exhibited the burst release of 44%, exerted immediate chemo prophylaxis and sustained delivery for 72 h. The MTT assay and fluorescent labeling with calcein AM indicated that the DHB-MS is biocompatible to human foreskin fibroblasts. Thus the system developed provides wider scope to control the pathogens involved in infection and also the excess matrix degradation.

Efficiency of controlled topical delivery of silver sulfadiazine in infected burn wounds.

The present study is designed to assess the potential benefits of controlled delivery of silver sulfadiazine from collagen scaffold (SSDM-CS) in infected deep partial thickness burn wounds in which epidermis is lost completely and the entire papillary dermis and most of the recticular layer of the dermis is lost. Infection induced by inoculating 10(7) colony forming units (cfu) of Pseudomonas aeruginosa caused significant increase in wound size (20%) till day 15, which decreased significantly from day 9 by SSDM-CS treatment, showing complete healing by day 27 (control > or = 37 days). Early subsidence of infection (<10(2) cfu, day 9) by SSDM-CS resulted in faster epidermal resurfacing and fibroplasia, whereas heavy microbial load (>10(7) cfu, day 9) in controls caused severe inflammatory cellular infiltration. Persistent infection triggered early expression of proinflammatory cytokines intereukin-6, intereukin 1-beta, and tumor necrosis factor-alpha, lasting until day 9, whereas cytokine level decreased in SSDM-CS-treated group by day 6. Infection exacerbated expression of active matrix metalloproteinases (MMPs)-2 and -9 in controls (day 15), while SSDM-CS positively modulated MMP-2 and -9 with faster decline in their levels (day 12). Inherent nature of the dressing to maintain drug level at equilibrium therapeutic concentration (51.2 microg/mL) for prolonged time (72 h), below systemic toxic limits (20 microg/dL, serum level), accelerated the magnitude and sequence of reparative events.

Oxidative stress markers regulating the healing of foot ulcers in patients with type 2 diabetes.

UNLABELLED:  Objective. This study was aimed at identifying factors that affect the healing of foot ulcers among patients with type 2 diabetes, focusing on the evaluation of oxidative stress-one marker of the inflammatory response. METHODS: A cross sectional study comprised of 96 subjects who were divided into 6 groups (16 subjects in each group). The groups were classified as non-diabetic control (group I), diabetic subjects without foot ulcer (group II), diabetic subjects with foot ulcers were sub-divided as neuropathic ulcer-noninfected (group III), neuropathic ulcer-infected (group IV), neuroischemic ulcer-noninfected (group V), and neuroischemic ulcer-infected (group VI). Oxidative stress markers such as lipid peroxidation, thiobarbituric acid reactive substance (TBARS), superoxide dismutase (SOD), catalase, G-peroxidase, GS-peroxidase, and plasma total antioxidant status were assayed in the blood samples. RESULTS: Lipid peroxidation increased progressively from group I to group VI subjects (P < 0.001). The TBARS in erythrocyte membrane was higher than in plasma. A progressive decrease of the total antioxidant status in plasma from group III to group VI (P < 0.01) was noted. There was a triggering increase in the antioxidative enzymes SOD and catalase in group V and group VI. CONCLUSION: There is a high level of lipid peroxidation with insufficient antioxidant enzymes and decreased total antioxidant status in plasma that leads to chronic ulceration and an extended inflammatory reaction. Thus, oxidative stress may be regarded as an important factor in nonhealing diabetic foot ulcers among patients with type 2 diabetes. .

Cytotoxic effect of achatinin(H) (lectin) from Achatina fulica against a human mammary carcinoma cell line (MCF7).

The hemolymph-derived achatinin(H) (lectin) from Achatina fulica showed a marked cytotoxic effect on MCF7, a human mammary carcinoma cell line. IC(50) values as measured by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay for achatinin(H) ranged from 6 to 10 microg/ml in the MCF7 cells. MCF7 cells showed significant morphological changes leading to cell death. The above cell death was observed after 48 h of treatment with 8 microg/ml when compared to untreated cells. Alterations in the tumor marker enzymes, as well as in antioxidant enzymes, were observed after achatinin(H) treatment. The specificity and purity of the achatinin(H) was confirmed by the Western blot assay. Achatinin(H) binding to MCF7 cells was detected by anti-achatinin(H), and visualization of the achatinin(H) binding sites on confluent MCF7 cells was confirmed by flourescein isothiocyanate conjugated secondary antibody. MCF7-treated cells fluoresced, indicating the presence of achatinin(H) binding sites. Fluorescence-activated cell sorting analysis of the cell cycle showed a significant increase in S-phase in MCF7 cells after 48 h of achatinin(H) treatment. The cells were arrested in G(2)/M phase of the cell cycle after 48 h with significant changes in cell viability. Cellular damage was confirmed by agarose gel electrophoresis with the characteristic appearance of a DNA streak in treated MCF7 cells indicating the ongoing apoptosis.

Gelatin microspheres cross-linked with EDC as a drug delivery system for doxycyline: development and characterization.

Chronic wounds express elevated levels of proteases, in particular matrix metalloproteinases (MMPs), which degrades de novo granulation tissue and endogenous biologically active proteins. An effective therapeutic approach for chronic wounds would be to modify this hostile environment and reduce the proteolytic imbalance. Doxycycline has been proved recently to inhibit MMPs and used topically for chronic wound ulcers, beyond their antimicrobial profile. To this end, a carrier system for controlled release of doxycycline, suitable for incorporation into various wound dressings like membranes and sponges was developed. In the present study gelatin microspheres, cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was proposed. The cross-linking was carried out with different concentrations of EDC (10 mM, 50 mM and 100 mM) and for different time periods (3-24 h). The cross-linked microspheres were characterized by evaluating the extent of cross-linking, the morphology, swelling behaviour and drug loading and in vitro studies of drug release, enzymatic degradation and biocompatibility. The extent of cross-linking increased as a function of both EDC concentration and the cross-linking time periods. It is found that the extent of cross-linking greatly influences the swelling and drug release behaviour of the microspheres. The cross-linked microspheres were found to be biocompatible to NIH 3T3 mouse embryonic fibroblast. The overall study indicates that the zero length cross-linker EDC can be considered as a potential alternative for cross-linking the gelatin microspheres.

Protection from acute and chronic lung diseases by curcumin.

The aim of this review has been to describe the current state of the therapeutic potential of curcumin in acute and chronic lung injuries. Occupational and environmental exposures to mineral dusts, airborne pollutants, cigarette smoke, chemotherapy, and radiotherapy injure the lungs, resulting in acute and chronic inflammatory lung diseases. Despite major advances in treating lung diseases, until now disease-modifying efficacy has not been demonstrated for any of the existing drugs. Current medical therapy offers only marginal benefit; therefore, there is an essential need to develop new drugs that might be of effective benefit in clinical settings. Over the years, there has been increasing evidence that curcumin, a phytochemical present in turmeric (Curcuma longa), has a wide spectrum of therapeutic properties and a remarkable range of protective effects in various diseases. Several experimental animal models have tested curcumin on lung fibrosis and these studies demonstrate that curcumin attenuates lung injury and fibrosis caused by radiation, chemotherapeutic drugs, and toxicants. The growing amount of data from pharmacological and animal studies also supports the notion that curcumin plays a protective role in chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, and allergic asthma, its therapeutic action being on the prevention or modulation of inflammation and oxidative stress. These findings give substance to the possibility of testing curcumin in patients with lung diseases.

Design and delivery of silver sulfadiazine from alginate microspheres-impregnated collagen scaffold.

A reconstituted collagen scaffold impregnated with silver sulfadiazine (SSD) loaded alginate microspheres, capable of delivering the drug in a controlled manner has been developed. SSD-loaded alginate microspheres were prepared by modified water-in-oil emulsion technique through interfacial ionic gelation of alginate using CaCl2. The SSD-loaded microspheres were impregnated in pepsin-solubilized collagen, in situ, while inducing fibrillation and cast as thin scaffold. Morphological features of microspheres and microsphere-impregnated collagen were analyzed through SEM. Distribution homogeneity of impregnated microspheres, their in vitro behavior in (Dulbecco's modified minimal essential media) DMEM, and antibacterial efficiency against ATCC pathogens were determined. Initial drug load of 20% (w/w) with respect to alginate and 40% (v/v) of 2% alginate with respect to oil phase were found to produce microspheres of optimum drug entrapment (3%) and required size range (300-370 microm). In vitro drug release studies from the scaffold showed an initial burst release of 47.5% and a controlled release for 72 h with equilibrium concentration of 68.8%. SSD-loaded microspheres exhibited minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) levels of 32 and 40.2 microg/mL to both K. pneumoniae and E. coli respectively. P. aeruginosa showed MIC and MBC levels of 44.8 and 51.2 microg/mL respectively, while Staphylococcus aureus exhibited MIC and MBC at the same concentration range (57.6 microg/mL). The collagen-based scaffold impregnated with SSD-loaded alginate microspheres can deliver SSD in a controlled fashion, can control infection for extended time period with lesser dressing frequencies, and will enable easier assessment of wound.

Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica.

Collagenase (matrix metalloproteinase-1, EC:3.4.24.7) was isolated from the hepatopancreas of Achatina fulica and characterized for its enzymatic activity and immunological properties. Procollagenase was isolated using ammonium sulphate precipitation and gel filtration, followed by purification by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid and by dialysis in neutral buffer. In the presence of SDS and beta-mercaptoethanol, the procollagenase resolved into two subunits with molecular masses of 63 and 28 kDa, respectively. The 63 kDa fragment retained its ability to bind and degrade gelatin, but the 28 kDa was inactive. Analysis by 2D gel electrophoresis revealed that the 63 kDa fragment was basic (pIs 7.6, 7.8 and 8.15), while the 28 kDa fragment was acidic (pI 4.7 and 5.1). Western blot analysis confirmed the identity of collagenase, as only matrix metalloproteinase-1 rabbit antibodies against human matrix metalloproteinase-1 (N-terminal region) recognized both the isolated procollagenase and the 63 kDa fragment.