Development of Novel Chitosan Microcapsules for Pulmonary Delivery of Dapsone: Characterization,Aerosol Performance,and In Vivo Toxicity Evaluation

Pneumocystis carinii pneumonia (PCP) is a major opportunistic infection that affects patients with human immunodeficiency virus. Although orally administered dapsone leads to high hepatic metabolism, decreasing the therapeutic index and causing severe side effects, this drug is an effective alternative for the treatment of PCP. In this context, microencapsulation for pulmonary administration can offer an alternative to increase the bioavailability of dapsone, reducing its adverse effects. The aim of this work was to develop novel dapsone-loaded chitosan microcapsules intended for deep-lung aerosolized drug delivery. The geometric particle size (D_4,3) was approximately 7 μm, the calculated aerodynamic diameter (d_aero) was approximately 4.5 μm, and the mass median aerodynamic diameter from an Andersen cascade impactor was 4.7 μm. The in vitro dissolution profile showed an efficient dapsone encapsulation, demonstrating the sustained release of the drug. The in vitro deposition (measured by the Andersen cascade impactor) showed an adequate distribution and a high fine particles fraction (FPF = 50%). Scanning electron microscopy of the pulmonary tissues demonstrated an adequate deposition of these particles in the deepest part of the lung. An in vivo toxicity experiment showed the low toxicity of the drug-loaded microcapsules, indicating a protective effect of the microencapsulation process when the particles are microencapsulated. In conclusion, the pulmonary administration of the novel dapsone-loaded microcapsules could be a promising alternative for PCP treatment.KEY WORDS: dapsone, dry powders inhalers, in vivo toxicity, microparticles, pulmonary drug delivery     

Rosin Nanoparticles as a Drug Delivery Carrier for the Controlled Release of Hydrocortisone

Hydrocortisone (HC)-loaded rosin nanoparticles were prepared by a dispersion and dialysis method without addition of surfactant. They were spherical: 167–332 nm diam. The drug was loaded approximately 50% of initial feeding amount in all formulation. Release of hydrocortisone from the nanoparticles in vitro gradually decreased with increasing initial rosin content at pH 7.4. HC was also released very slowly at pH 1.2. Nanoparticles based on rosin thus are potentially useful as a drug delivery system.     

Preparation and Characterization of Microcapsules Based on Biodegradable Polymers: Pectin/Casein Complex for Controlled Drug Release Systems

Controlled release of drugs is an important strategy to diminish the drug dose and adverse side effects. Aqueous mixtures of polysaccharides and proteins are usually unstable above a certain biopolymer concentration and phase separation occurs either because of repulsive (segregative) or attractive (associative) interactions. Herein, pectin/casein microcapsules were prepared by complex coacervation aiming at prolonged drug release. The morphological characteristics, particle size, distribution, and release kinetics of microcapsules were studied using as a model the hydrophilic drug acetaminophen. It was detected that complexation of pectin/casein particles occurs at pH values lower than 6, resulting in the formation of spherical particles after spray drying. Microcapsules had a mean diameter of 3.138 and 4.929 μm without drug, and of 4.680 and 5.182 μm with drug using USP and 8003 pectin, respectively. The in vitro release of acetaminophen from microcapsules was slow and the drug release mechanism was controlled by diffusion following first-order kinetics. There was greater release of acetaminophen in simulated gastric fluid than simulated intestinal fluid conditions. Concluding, the polymeric system present herein seemed to be appropriate for a prolonged release of acetaminophen throughout the gastrointestinal tract. Nevertheless, it is likely that it is a promising pectin/casein complex for lipossoluble drugs, which merits further investigation.KEY WORDS: casein, complex coacervation, microcapsules, pectin, release kinetics     

Targeting the Giant Cell Tumor Stromal Cell: Functional Characterization and a Novel Therapeutic Strategy

Giant cell tumor of bone (GCTB) is a benign, locally destructive neoplasm, with tumors comprised of mesenchymal fibroblast-like stromal cells; monocytic, mononuclear cells of myeloid lineage; and the characteristic osteoclast-like, multinucleated giant cells. Hampering the study of the complex interaction of its constituent cell types is the propensity of longstanding, repeatedly passaged cell cultures to undergo phenotypic alteration and loss of osteoclast-inducing capacities. In this study, we employed a novel, single-step technique to purify freshly harvested stromal cells using a CD14-negative selection column. Using 9 freshly harvested GCTB specimens and the purified stromal cell component, we performed analyses for markers of osteoblast lineage and analyzed the capacity of the stromal cells to undergo osteoblastic differentiation and induce osteoclastogenesis in co-cultures with monocytic cells. Successful purification of the CD14-negative stromal cells was confirmed via flow cytometric analysis and immunocytochemistry. Osteogenic media upregulated the expression of osteocalcin, suggesting an osteoblastic lineage of the GCTB stromal cells. The effects of the Wnt pathway agonist, SB415286, and recombinant human bone morphogenetic protein (BMP)-2 on osteoblastogenesis varied among samples. Notably, osteogenic media and SB415286 reversed the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) expression ratio resulting in diminished osteoclastogenic capacity. Recombinant human BMP2 had the opposite effect, resulting in enhanced and sustained support of osteoclastogenesis. Targeting the giant cell tumor stromal cell may be an effective adjunct to existing anti-resorptive strategies.     

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP⁺ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP⁺ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H₂O₂ exposure, implying that Yhm2p has an antioxidant function.     

Antimicrobial Peptides,a Pool for Novel Cell Penetrating Peptides Development and Vice Versa

International Journal of Peptide Research and Therapeutics - Membrane active peptides are a family of peptides with ability to interact with plasma membrane. Cell penetrating peptides (CPPs)...     

七甲川花菁染料作为肿瘤靶向和光动力治疗载体的研究进展

七甲川花菁近红外荧光染料(NIRF)可直接被肿瘤细胞特异性吸收,具有肿瘤靶向性。与化疗药物偶联后,该类染料可通过血脑屏障将药物转运至肿瘤部位,不仅可以减少化疗药物使用剂量,降低药物的毒副作用,也可通过近红外荧光成像实现对肿瘤治疗的实时监控。七甲川花菁染料所展示的线粒体毒性和光敏特性,可直接杀死肿瘤细胞,抑制肿瘤新生血管的形成。通过纳米包裹,能够显著增强该类染料的肿瘤靶向能力,实现实时跟踪药物释放情况。七甲川花菁染料特异性识别肿瘤细胞的能力与有机阴离子转运肽的作用密切相关,缺氧和线粒体膜电位也参与了染料吸收的调控。这些发现有利于将近红外荧光染料应用于肿瘤的靶向治疗。     

Porous Starch: a Novel Carrier for Solubility Enhancement of Carbamazepine

To circumvent the solubility-related issues associated with Biopharmaceutics Classification System class II drugs, a novel porous carrier has been developed. In the present study, a process for preparation of porous starch (PS) is demonstrated. The process briefly comprises of translucent gel preparation followed by solvent replacement, drying, and sizing. Carbamazepine (CBZ) was used as a drug candidate to exhibit solubility enhancement potential of PS. PS and CBZ-loaded PS (CBZ-PS) systems were characterized with respect to IR, DSC, XRD, SEM, and dissolution kinetic studies. PS-CBZ was found to follow a Fickian behavior during dissolution. In vivo studies conducted in mice displayed a superior performance of CBZ-PS as compared to neat CBZ.     

Plastic Compressed Collagen as a Novel Carrier for Expanded Human Corneal Endothelial Cells for Transplantation

Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.     

Fibrous Hydrogels for Cell Encapsulation: A Modular and Supramolecular Approach

Artificial 3-dimensional (3D) cell culture systems, which mimic the extracellular matrix (ECM), hold great potential as models to study cellular processes under controlled conditions. The natural ECM is a 3D structure composed of a fibrous hydrogel that provides both mechanical and biochemical cues to instruct cell behavior. Here we present an ECM-mimicking genetically engineered protein-based hydrogel as a 3D cell culture system that combines several key features: (1) Mild and straightforward encapsulation meters (1) ease of ut I am not so sure.encapsulation of the cells, without the need of an external crosslinker. (2) Supramolecular assembly resulting in a fibrous architecture that recapitulates some of the unique mechanical characteristics of the ECM, i.e. strain-stiffening and self-healing behavior. (3) A modular approach allowing controlled incorporation of the biochemical cue density (integrin binding RGD domains). We tested the gels by encapsulating MG-63 osteoblastic cells and found that encapsulated cells not only respond to higher RGD density, but also to overall gel concentration. Cells in 1% and 2% (weight fraction) protein gels showed spreading and proliferation, provided a relative RGD density of at least 50%. In contrast, in 4% gels very little spreading and proliferation occurred, even for a relative RGD density of 100%. The independent control over both mechanical and biochemical cues obtained in this modular approach renders our hydrogels suitable to study cellular responses under highly defined conditions.     

Liz1p, a Novel Fission Yeast Membrane Protein, Is Required for Normal Cell Division When Ribonucleotide Reductase Is Inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1⁺, which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1⁻ cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1⁺ encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.     

Novel chitosan membranes as support for lipases immobilization: Characterization aspects

Membranes of chitosan (QS), chitosan treated with glutaraldehyde (QGA) and chitosan crown ether (QCE) were utilized as carriers for immobilization of Candida antarctica and Candida rugosa lipases. Membrane supports were characterized by several techniques (Raman spectroscopy, elemental analysis by CHN determination and Energy Dispersive X-ray (EDX), water sorption isotherms, and surface area from nitrogen sorption data). To verify the presence of enzymes, some of these techniques were also used for lipase on chitosan biocatalytic systems. Measurements of protein load from Biuret assays and catalytic activity in esterification in nonaqueous media were also made for the immobilized enzymes. Sorption isotherms at 20, 30, 40 and 50 °C for QS, QGA and QCE supports were fitted to the Guggenheim, Anderson and Böer model. GAB monolayer moisture parameter, Xm, varied between 0.029 and 0.051 for QS, 0.039 and 0.058 for QGA and 0.039–0.075 g of water g⁻¹ s.s. for QCE membranes. Elemental analysis and Raman spectra measurements of the lipase, supports and immobilized lipase systems gave evidence of the presence of enzymes on supports. Chitosan supports with internal surface area (m2 g⁻¹) among 3.31 and 1.26 were obtained. Regardless of these low values, acceptable protein load (0.61 to 3.21%) and esterification initial rates were achieved (0.88–2.75 mmol min⁻¹ g of protein⁻¹).     

Liposomal Delivery Systems for Encapsulation of Ferrous Sulfate: Preparation and Characterization

Liposomal delivery systems for water-soluble bioactives were prepared using the pro-liposome and the microfluidization technologies. Iron, an essential micronutrient as ferrous sulfate and ascorbic acid, as an antioxidant for iron were encapsulated in the liposomes. Liposomes prepared by the microfluidization technology using 6% (w/w) concentration of the lipid encapsulated with ferrous sulfate and ascorbic acid had particle size distributions around 150 to 200 nm, whereas liposomes from the pro-liposome technology resulted in particle sizes of about 5 μm. The encapsulation efficiency of ferrous sulfate was 58% for the liposomes prepared by the microfluidization using 6% (w/w) lipid and 7.5% of ferrous sulfate concentrations, and it was 11% for the liposomes from pro-liposome technology using 1.5% (w/v) lipid and 15% of ferrous-sulfate concentration. Both the liposomes exhibited similar levels of oxidative stability, demonstrating the feasibility of microfluidization-based liposomal delivery systems for large-scale food/nutraceutical applications.     

Temporal and Pharmacological Characterization of Angiostatin Release and Generation by Human Platelets: Implications for Endothelial Cell Migration

Platelets play an important role in thrombosis and in neo-vascularisation as they release and produce factors that both promote and suppress angiogenesis. Amongst these factors is the angiogenesis inhibitor angiostatin, which is released during thrombus formation. The impact of anti-thrombotic agents and the kinetics of platelet angiostatin release are unknown. Hence, our objectives were to characterize platelet angiostatin release temporally and pharmacologically and to determine how angiostatin release influences endothelial cell migration, an early stage of angiogenesis. We hypothesized anti-platelet agents would suppress angiostatin release but not generation by platelets. Human platelets were aggregated and temporal angiostatin release was compared to vascular endothelial growth factor (VEGF). Immuno-gold electron microscopy and immunofluorescence microscopy identified α-granules as storage organelles of platelet angiostatin. Acetylsalicylic acid, MRS2395, GPIIb/IIIa blocking peptide, and aprotinin were used to characterize platelet angiostatin release and generation. An endothelial cell migration assay was performed under hypoxic conditions to determine the effects of pharmacological platelet and angiostatin inhibition. Compared to VEGF, angiostatin generation and release from α-granules occurred later temporally during platelet aggregation. Consequently, collagen-activated platelet releasates stimulated endothelial cell migration more potently than maximally-aggregated platelets. Platelet inhibitors prostacyclin, S-nitroso-glutathione, acetylsalicylic acid, and GPIIb/IIIa blocking peptide, but not a P2Y12 inhibitor, suppressed angiostatin release but not generation. Suppression of angiostatin generation in the presence of acetylsalicylic acid enhanced platelet-stimulated endothelial migration. Hence, the temporal and pharmacological modulation of platelet angiostatin release may have significant consequences for neo-vascularization following thrombus formation.     

Encapsulation and release of a bacterial carotenoid from hydrogel matrix: Characterization,kinetics and antioxidant study

Various natural polymers with hydrophilic properties have been used to form hydrogels for the encapsulation and delivery of nutrients and drugs in food and pharmaceutical industries. Among them, chitosan (ChiHG)‐ and alginate (AlgHG)‐ based hydrogels have been extensively explored for delivery of several nutraceuticals in recent years. Release of natural canthaxanthin (CX) obtained from Dietzia maris NITD (accession number: HM151403) has been investigated with emphasis on biomedical applications. Significant changes (P < 0.05) in degree of swelling and moisture content (% dry basis) were found after encapsulation of bacterial canthaxanthin (BCX), but the gel content remained unchanged. BCX encapsulation efficiency was calculated to be 55.92% and 60.45% in ChiHG and AlgHG, respectively. A noticeable change in heat of fusion d melting point was recorded in ChiHG and AlgHG after BCX encapsulation. Swelling and BCX release from gel matrix was performed under two different pH (1.2 and 7.4). The results showed that swelling of hydrogel and BCX release was facilitated at higher pH (7.4) than acidic pH (1.2). With regard to the release kinetics data, it was found that BCX is released from bothand AlgHG in a diffusion transport method. In addition, antioxidant activity of BCX encapsulated hydrogels was found significantly higher (P < 0.001) in terms of DPPH, ABTS, nitrite, hydroxyl radical scavenging and reducing power assay. These results indicated that BCX can be successfully encapsulated into a polymeric hydrogel to obtain a dynamic biomaterial that may be used in drug delivery applications in future.     

Development of Hydrogels for Entrapment of Vitamin D3: Physicochemical Characterization and Release Study

In this study two carbohydrate biopolymers were used to entrap vitamin D3. In order to optimize the microencapsulation parameters, response surface methodology was applied to evaluate the effects of three independent variables (alginate percentage, vitamin: alginate weight ratio, and ultrasound time) on the efficiency of microencapsulation and loading capacity. According to the results, 0.23% alginate (W/V), 1: 5 weight ratio of vitamin D3: alginate, and 13.7 min ultrasound time were determined as the optimal conditions for obtaining maximum microencapsulation efficiency (92.86%) and loading capacity (30.1%). Then, the optimized carrier was coated by chitosan followed by the examinations of morphological characteristics, mean particle size, Fourier transform infrared (FTIR) spectrometry, in vitro release characteristics, and release modeling. Scanning electron microscopy examinations showed that the alginate and alginate-chitosan microcapsules had irregular and interlacing forms. The average particle sizes of alginate and alginate-chitosan were 11.3 and 23.3, respectively, which decreased to 9.8 and 14.0 μm after drying. Results of FTIR indicated a physical interaction between alginate and vitamin D3. The Weibull II model was found to be the best one to predict vitamin release behavior. The results of this study showed the potential application of developed carriers to encapsulate hydrophobic compounds.     

Polymeric Nanoparticles as Oral Delivery Systems for Encapsulation and Release of Polyphenolic Compounds: Impact on Quercetin Antioxidant Activity & Bioaccessibility

A delivery system containing polymeric (Eudragit) nanoparticles has been developed for encapsulation and controlled release of bioactive flavonoids (quercetin). Nanoparticles were fabricated using a solvent displacement method. Particle size, morphology, and charge were measured by light scattering, electron microscopy and ??-potential. Encapsulation efficiency (EE) and release profiles were determined using electrochemical methods. Molecular interactions within the particle matrix were characterized by X-ray diffraction, differential scanning calorimetry, and infrared spectroscopy. Antioxidant properties of free and encapsulated quercetin were analyzed by TBARS and fluorescence spectroscopy. Bioaccessibility of quercetin was evaluated using an in vitro digestion model. Relatively small (d????370?nm) anionic polymeric nanoparticles were formed containing quercetin in a non-crystalline form (EE????67?%). The main interaction between quercetin and Eudragit was hydrogen bonding. Encapsulated quercetin remained stable during 6?months storage and maintained its antioxidant activity. Quercetin bioaccessibility within simulated small intestinal conditions was improved by encapsulation. The knowledge obtained from this study will facilitate the rational design and fabrication of polymeric nanoparticles as oral delivery systems for encapsulation, protection, and release of bioactive compounds.     

A Thermoacidophile-Specific Protein Family,DUF3211, Functions as a Fatty Acid Carrier with Novel Binding Mode

STK_08120 is a member of the thermoacidophile-specific DUF3211 protein family from Sulfolobus tokodaii strain 7. Its molecular function remains obscure, and sequence similarities for obtaining functional remarks are not available. In this study, the crystal structure of STK_08120 was determined at 1.79-Å resolution to predict its probable function using structure similarity searches. The structure adopts an α/β structure of a helix-grip fold, which is found in the START domain proteins with cavities for hydrophobic substrates or ligands. The detailed structural features implied that fatty acids are the primary ligand candidates for STK_08120, and binding assays revealed that the protein bound long-chain saturated fatty acids (>C₁₄) and their trans-unsaturated types with an affinity equal to that for major fatty acid binding proteins in mammals and plants. Moreover, the structure of an STK_08120-myristic acid complex revealed a unique binding mode among fatty acid binding proteins. These results suggest that the thermoacidophile-specific protein family DUF3211 functions as a fatty acid carrier with a novel binding mode.