Human single-donor composite skin substitutes based on collagen and polycaprolactone copolymer.

The development and characterization of an enhanced composite skin substitute based on collagen and poly(epsilon-caprolactone) are reported. Considering the features of excellent biocompatibility, easy-manipulated property and exempt from cross-linking related toxicity observed in the 1:20 biocomposites, skin substitutes were developed by seeding human single-donor keratinocytes and fibroblasts alone on both sides of the 1:20 biocomposite to allow for separation of two cell types and preserving cell signals transmission via micro-pores with a porosity of 28.8 +/- 16.1 microm. The bi-layered skin substitute exhibited both differentiated epidermis and fibrous dermis in vitro. Less Keratinocyte Growth Factor production was measured in the co-cultured skin model compared to fibroblast alone condition indicating a favorable microenvironment for epidermal homeostasis. Moreover, fast wound closure, epidermal differentiation, and abundant dermal collagen deposition were observed in composite skin in vivo. In summary, the beneficial characteristics of the new skin substitutes exploited the potential for pharmaceutical screening and clinical application.

Erythropoiesis-stimulating protein delivery in providing erythropoiesis and neuroprotection.

Erythropoietin (EPO), a glycoprotein, plays an important role in erythropoiesis and neuroprotection. EPO therapies for anemia or neurodegenerative diseases require frequent injections or high-dose systemic administration which may cause unwanted side effects. Various strategies for EPO delivery have been investigated for increasing EPO bioavailability and decreasing side effects, including nano/micro particles, PEGylation of EPO and transport-mediated delivery systems. Nano/micro particles provide EPO with long-term effect and protect EPO against proteolytic cleavage. PEGylated EPO prolong circulating time and reduce injection frequency of anemia treatment. A transport-mediated delivery system enables protein to cross biological barriers. Presently, there is no report about an effective delivery system of EPO for neuroprotection. This review focuses on EPO delivery systems for erythropoiesis or neuroprotection with prolonged duration and enhanced bioavailability.

Evaluating the effectiveness of a novel atomized liquid needle-free transdermal delivery system.

Needle-free jet injections constitute a crucial method for drug delivery. A novel liquid drug delivery system has been proposed recently, in which pressure atomizes liquid before delivering that atomized liquid to the patient's body; however, the mechanism and efficiency of the system are unclear. This study explored the shot delivery pressure, penetration depth, and cumulative amount of permeation of this system. This system was used to deliver 0.5% (w/v) methylene blue to agarose phantoms at various shot delivery pressures. Shots of methylene blue were also delivered to porcine skin samples at different shot delivery frequencies for light microscopy evaluation. A commercial microneedle array was used for comparing the effectiveness of the skin penetration depths. The array was gently stamped against porcine skin; methylene blue was subsequently applied to the area for different time points, followed by microscopic observations. In vitro skin penetration was tested using static Franz diffusion cells over 8 h. Finally, the feasibility of the system's clinical application was evaluated by analyzing the local analgesic effect in a heat nociceptive animal model. The penetration depths created using 100 shots at 100 psi were similar to those created using the commercial microneedle array for 2 h. Thermal stimulation responses showed that 15 min after diclofenac sodium was delivered by the system, heat nociception was significantly attenuated for 60 min. Our study presents a novel delivery system that may be useful for future clinical applications.

Novel Anthra[1,2-c][1,2,5]Thiadiazole-6,11-Diones as Promising Anticancer Lead Compounds: Biological Evaluation, Characterization & Molecular Targets Determination.

The novel compounds NSC745885 and NSC757963 developed at our laboratory were tested against a panel of 60 cancer cell lines at the National Cancer Institute, USA, and a panel of 39 cancer cell lines at the Japanese Foundation of Cancer Research. Both compounds demonstrated selective unique multi-log differential patterns of activity, with GI50 values in the sub-micro molar range against cancer cells rather than normal cardiac cells. NSC757963 showed high selectivity towards the leukemia subpanel. Activities of both compounds strongly correlated to expression of NFKB1 and CSNK2B genes, implying that they may inhibit the NF-κB pathway. Immunocytochemical microscopy of OVCAR-3 cells showed clear cytosolic accumulation of the NF-κB p65 subunit following treatment. Western blotting showed dose dependent inhibition of the nuclear expression of the NF-κB p65 subunit with subsequent accumulation in the cytosol following treatment. Docking experiments showed binding of both compounds to the NF-κB activator IKKß subunit preventing its translocation to the nucleus. Collectively, these results confirm the ability of our compounds to inhibit the constitutively active NF-κB pathway of OVCAR-3 cells. Furthermore, COMPARE analysis indicated that the activity of NSC757963 is similar to the antituberculosis agent rifamycin SV, this was confirmed by testing the antimycobacterial activity of NSC757963 against Mycobacterium tuberculosis, results revealed potent activity suitable for use in clinical practice. Molecular properties and Lipinski's parameters predicted acceptable bioavailability properties with no indication of mutagenicity, tumorigenicity, irritability and reproductive effects. Oral absorption experiments using the human Caco-2 model showed high intestinal absorption of NSC745885 by passive transport mechanism with no intestinal efflux or active transport mechanisms. The unique molecular characterization as well as the illustrated anticancer spectra of activity and bioavailability properties warrant further development of our compounds and present a foundation brick in the pre-clinical investigations to implement such compounds in clinical practice.

Erlotinib-Conjugated Iron Oxide Nanoparticles as a Smart Cancer-Targeted Theranostic Probe for MRI.

We designed and synthesized novel theranostic nanoparticles that showed the considerable potential for clinical use in targeted therapy, and non-invasive real-time monitoring of tumors by MRI. Our nanoparticles were ultra-small with superparamagnetic iron oxide cores, conjugated to erlotinib (FeDC-E NPs). Such smart targeted nanoparticles have the preference to release the drug intracellularly rather than into the bloodstream, and specifically recognize and kill cancer cells that overexpress EGFR while being non-toxic to EGFR-negative cells. MRI, transmission electron microscopy and Prussian blue staining results indicated that cellular uptake and intracellular accumulation of FeDC-E NPs in the EGFR overexpressing cells was significantly higher than those of the non-erlotinib-conjugated nanoparticles. FeDC-E NPs inhibited the EGFR-ERK-NF-κB signaling pathways, and subsequently suppressed the migration and invasion capabilities of the highly invasive and migrative CL1-5-F4 cancer cells. In vivo tumor xenograft experiments using BALB/c nude mice showed that FeDC-E NPs could effectively inhibit the growth of tumors. T2-weighted MRI images of the mice showed significant decrease in the normalized signal within the tumor post-treatment with FeDC-E NPs compared to the non-targeted control iron oxide nanoparticles. This is the first study to use erlotinib as a small-molecule targeting agent for nanoparticles.

Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions.

Constitutive functional HIF-2α was recently identified in cancer and stem cell lines under normoxia. In this study, BEAS-2B, a bronchial epithelial cell line, was shown to constitutively express active HIF-2α under normoxia and exhibit markers of pluripotency including Oct-4, Nanog, and sphere formation. Oct-4 expression was reduced after knockdown of HIF-2α under normoxia. Global enrichment analysis of HIF-2α demonstrated the diverse functions of HIF-2α under normoxia. Bioinformatics analysis of the enriched loci revealed an enhancer role of HIF-2α binding sites, involvement of HIF-2α interacting proteins, and enriched de novo motifs which suggest the diverse role of HIF-2α in pseudohypoxia. The low ratio of the discovered loci overlapping with those revealed in cancer cell lines 786-O (16.1%) and MCF-7 (15.9%) under hypoxia indicated a prevailing non-canonical mechanism. Hypoxia had positive, marginal or adverse effects on the enrichment of the selected loci in ChIP-PCR assays. Deletion of the N-terminal activation domain (N-TAD) of HIF-2α disrupted the reporting activity of two of the loci annotated to ELN and ANKRD31. Hypoxia incurring abundance variation of HIF-2α may misrepresent the N-TAD functions as canonical hypoxia inducible features via C-TAD activation. Elucidation of the pseudohypoxia functions of constitutive HIF-2α is useful for resolving its role in malignancy and pluripotency.

Physicochemical properties and in vivo assessment of timolol-loaded poly(D,L-lactide-co-glycolide) films for long-term intraocular pressure lowering effects.

Timolol-loaded poly(D,L-lactide-co-glycolide; PLGA) films were prepared for achieving the long-term intraocular pressure (IOP) lowering effect on glaucoma treatment. The physicochemical properties and in vivo effects of films were determined and characterize the delivery system. PLGA, span 20, propylene glycol (PG), and timolol base were dissolved in dichloromethane (DCM) and kept in a stainless mold; timolol films were prepared after the evaporation of DCM. Timolol disc-shape film preparation (TDF), containing 1 mg of timolol in 0.37 cm(2) area, was fabricated by using a trephine and placed onto the cul de sac of alpha-chymotrypsin-induced ocular hypertension rabbits for assessing the IOP lowering effect. The prepared films characterized a Young's modulus ranged from 1.13 to approximately 2.49 MPa, and related to the content percentages of PLGA, PG, and the residual DCM. The timolol film could maintain drug release for 1 week. Following a single-dose application in ocular hypertension rabbits, the prepared TDF could achieve a long-term IOP lowering effect and maintain the IOP change (in comparison with baseline) of approximately 7 mmHg within 1 week. The aqueous humor levels of timolol were low within a range of 0.8 to approximately 0.24 microg/mL for the initial 24 h and less than 0.15 microg/mL for 4-7 days. The investigated film formulation might be potentially developed for the application of long-term ocular delivery systems.

Contribution of poly(amino acids) to advances in pharmaceutical biotechnology.

Recently, protein biotechnology generates tremendous impacts in therapeutic products. These products include enzymes, antibodies, hormones, blood factors, growth factors and regulatory factors. Protein, vaccine and gene therapy drugs could be formulated with suitable biomaterials to deliver active agents to their target sites at the right time and maintain therapeutic effects for proper durations. In this review article, we focus on poly(amino acids) or polymerized amino acids for their applications in drug delivery systems, vaccines, and gene therapy. The nomenclatures of poly(amino acids) are briefly introduced to systematically express synthetic polypeptides. In drug delivery systems, we introduce two applications of poly(amino acids) in pharmaceutical biotechnology, either as carriers to facilitate drug delivery, or as biomaterials to be formulated as suitable delivery systems for application in tissue engineering. Many short polypeptides are mapped from antigen motifs and used for vaccination. These poly(amino acids) provide protective effects in animal challenge tests and potential application in vaccine development to be briefly introduced. Finally, some reports related to new developed poly(amino acids) as DNA carriers for achieving gene delivery are also described in the text.

Oral immunogenicity of the inactivated Vibrio cholerae whole-cell vaccine encapsulated in biodegradable microparticles.

Vibrio cholerae (VC)-loaded microparticles as an oral vaccine delivery system were prepared with 6% w/v poly(DL-lactide-co-glycolide)(PLG) in the oil phase as well as 10% w/v PVP and 5% w/v NaCl in the aqueous phase, by an water-in-oil-in-water emulsion/solvent extraction technique. VC was successfully entrapped in the microparticles with trapping efficiencies up to 97.8% and a loading level of 55.4+/-6.9 microg/mg. The microparticle delivery system with a particle size of 3.8 microm had different distribution of VC content in the core region (25.7+/-1.9 microg/mg) and surface (6.2+/-0.9 microg/mg). The immunogenic potential of VC-loaded microparticles in comparison with PLG microparticles or VC solution was evaluated in adult mice by oral immunization, in which mice received one dose of 20 mg VC-loaded microparticles or 20 mg VC-loaded microparticles physical mixed with amphotericin B. The control group received 20 mg PLG microparticle or VC solution. Serum samples were collected from all tested mice on the day of immunization and at 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 weeks postimmunization. Sera were examined for vibriocidal antibodies by microtitration and Vibrio-specific serum IgG and IgM antibodies were assessed by the ELISA method. IgG and IgM antibodies to intact VC were detected in sera from all animals immunized with VC. The response was specific and of high magnitude. Significantly higher antibody responses were obtained when sera from both VC-loaded microparticles and VC-loaded microparticles physical mixed with amphotericin B immunized mice were titrated against VC. The immunogenicity of VC-loaded microparticles mixed with amphotericin B in evoking serum IgG and IgM responses was higher than that of VC-loaded microparticles only. These results demonstrate that VC-loaded microparticles physical mixed with amphotericin B and VC-loaded microparticles orally administered evoke Vibrio-specific serum IgG and IgM responses as well as vibriocidal antibody activity in mice. The VC incorporation, physicochemical characterization data, and the animal results obtained in this study may be relevant in optimizing the vaccine incorporation and delivery properties of these potential vaccine targeting carriers.

Corneal and scleral permeability of quinolones--a pharmacokinetics study.

PURPOSE: To investigate the corneal and scleral permeability of nalidixic acid and synthesized fluoroquinolones and their in vivo pharmacokinetics in rabbits. METHODS: The corneal and scleral permeability coefficients of ciprofloxacin, norfloxacin, cinoxacin, enoxacin, and ofloxacin were determined in rabbits using high performance liquid chromatography (HPLC). The aqueous humor levels of norfloxacin and ciprofloxacin were measured separately by topical instillation of 0.3% solutions of the two drugs onto rabbit eyes. RESULTS: Nalidixic acid had a higher corneal permeability coefficient (17.3 +/- 3.56 x 10(-6) cm/second) than all other drugs tested (p < 0.01). Corneal permeability coefficients in rabbits among ciprofloxacin, norfloxacin, cinoxacin, enoxacin, and ofloxacin were not significantly different (p > 0.1). Comparing the corneal and scleral permeability coefficients, only values for nalidixic acid were not significantly different (17.35 +/- 3.56 x l0(-6) cm/second versus 22.69 +/- 5.19 x 10(-6) cm/second, p > 0.05), while all other drugs had scleral permeability coefficients 8 to 10 times greater than corneal permeability coefficients. The mean aqueous humor concentration of norfloxacin and ciprofloxacin at 60 minutes to 180 minutes after instillation was around 0.3 microg/mL, a value higher than MIC90 of most bacteria.

Nitric oxide levels in the aqueous humor vary in different ocular hypertension experimental models.

This study investigated the relationships among intraocular pressure (IOP), nitric oxide (NO) levels, and aqueous flow rates in experimental ocular hypertension models. A total of 75 rabbits were used. One of four different materials [i.e., α-chymotrypsin, latex microspheres (Polybead), red blood cell ghosts, or sodium hyaluronate (Healon GV)] was injected into the eyes of the 15 animals in each experimental group; the remaining 15 rabbits were reserved for a control group. The IOP changes in the five groups were recorded on postinduction Days 1-3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120. On postinduction Day 7, the dynamics and NO levels in the aqueous humor were recorded. Significant IOP elevations were induced by α-chymotrypsin (p < 0.01) and Polybead (p < 0.01) on each postinduction day. In the red blood cell ghosts model, significant elevations (p < 0.01) were found on postinduction Days 1-3; Healon GV significantly elevated IOP (p < 0.01) on postinduction Day 1 and Day 2. On postinduction Day 7, the aqueous humor NO levels increased significantly in the models of α-chymotrypsin, Polybead, and red blood cell ghosts (all p < 0.01), while the aqueous flow rates were significantly reduced in the models of α-chymotrypsin and Polybead (p < 0.005). Persistent ocular hypertension models were induced with α-chymotrypsin and Polybead in the rabbits. The Polybead model exhibited the characteristic of an increased aqueous humor NO level, similar to human eyes with acute angle-closure glaucoma and neovascular glaucoma.

Efficient downregulation of VEGF in retinal pigment epithelial cells by integrin ligand-labeled liposome-mediated siRNA delivery.

BACKGROUND: The purpose of this study was to demonstrate the effectiveness of an integrin peptide ligand-labeled liposomal delivery system loaded with vascular endothelial growth factor (VEGF)-siRNA in a model study of gene therapy for retinopathy using human retinal pigment epithelial cells. METHODS: Arg(R)-Gly(G)-Asp(D) motif peptide conjugating polyethylene glycol modified (RGD-PEGylated) liposomes were prepared using a thin-film hydration method and optimized for surface charge, particle size, small interfering RNA (siRNA) load, and entrapment efficiency. Reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine VEGF levels in retinal pigment epithelial cells. Cytotoxicity was determined using the 3-[4, 5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and flow cytometry. RESULTS: Physicochemical properties, including particle size, zeta potential, and siRNA load, of the prepared RGD-PEGylated liposomes and their entrapment efficiency were determined to be within the following ranges: 123.8-234.1 nm, 17.31-40.09 m V, 5.27%-6.33%, and >97%, respectively. RGD-PEGylated liposome-mediated fluorescent-labeled siRNA delivery demonstrated significantly enhanced cellular uptake, and 3 mol% RGD-PEGylated liposomes (having 3ß-[N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol) DSPE and DSPE-PEG(2000)-RGD with molar ratio of 50/47/3) were shown to have better efficacy with regard to specificity for retinal pigment epithelial cells, reduced cytotoxicity, and knockdown of the target molecule. CONCLUSION: By integrin receptor-mediated endocytosis, 3 mol% RGD-PEGylated liposomes were shown to be a suitable vector when loaded with VEGF-siRNA for efficient downregulation of VEGF in retinal pigment epithelial cells at both the protein and gene levels. This integrin ligand-modified liposomal delivery system has therapeutic potential for ocular gene therapy.

Potential of nonoral α-lipoic acid aqueous formulations to reduce ocular microvascular complications in a streptozotocin-induced diabetic rat model.

PURPOSE: α-Lipoic acid (LA) aqueous formulations were studied for nonoral administration, including intravitreal and intraperitoneal preparations and topical eyedrops. The potential retinoprotective effects of these LA preparations were also evaluated in streptozotocin (STZ)-induced diabetic rats for screening better delivery systems of LA. METHODS: Four LA liquid preparations were prepared and investigated. The short-term accelerated stabilities of LA preparations were investigated at 3 temperatures: 50°C, 70°C, and 90°C. The time courses of LA degradation in the preparations were determined by high-performance liquid chromatography. Furthermore, the potential therapeutic effects of LA preparations in a STZ-induced diabetic rat model were assessed by vitreous fluorophotometry to evaluate the fluorescein leakage from ocular vascular vessels into the vitreous. Capillary lesion in the retina was also examined using hematoxylin-eosin-stained microsections. RESULTS: LA in an aqueous solution was rapidly degraded with the activation energy of 10.4 kcal/mol. The 3 LA preparations had shelf lives of ∼1 month at 25°C. These formulations significantly reduced the vitreous fluorescein level in STZ-induced diabetic rats as evaluated by the fluorescein leakage after tail vein injection. Capillary lesions in the retina of the diabetic rats were remarkably reduced by nonoral administration, particularly the intraperitoneal injection (30 mg/kg/day). CONCLUSIONS: LA could be developed as aqueous preparations with suitable stability for short-term use in nonoral administration. LA preparations could be administered intravitreally or intraperitoneally to reduce ocular microvascular complications, such as retinopathy, in diabetic patients.

Erythropoietin protects adult retinal ganglion cells against NMDA-, trophic factor withdrawal-, and TNF-α-induced damage.

PURPOSE: This study aimed to evaluate the neuroprotective effect of EPO in the presence of N-methyl-d-aspartate (NMDA)-, trophic factor withdrawal (TFW)-, and tumor necrosis factor-alpha (TNF-α)-induced toxicity on total, small, and large retinal ganglion cells (RGCs). METHODS: Retinal cells from adult rats were cultured in a medium containing brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), and forskolin. Expression of RGC markers and EPOR was examined using immunocytochemistry. RGCs were classified according to their morphological properties. Cytotoxicity was induced by NMDA, TFW, or TNF-α. RGC survival was assessed by counting thy-1 and neurofilament-l double-positive cells. RESULTS: EPO offered dose-dependent (EC50 = 5.7 ng/mL) protection against NMDA toxicity for small RGCs; protection was not significant for large RGCs. Time-course analysis showed that the presence of EPO either before or after NMDA exposure gave effective protection. For both small and large RGCs undergoing trophic factor withdrawal, EPO at concentrations of 1, 10, or 100 ng/mL improved survival. However, EPO had to be administered soon after the onset of injury to provide effective protection. For TNF-α-induced toxicity, survival of small RGCs was seen only for the highest examined concentration (100 ng/mL) of EPO, whereas large RGCs were protected at concentrations of 1, 10, or 100 ng/mL of EPO. Time-course analysis showed that pretreatment with EPO provided protection only for large RGCs; early post-treatment with EPO protected both small and large RGCs. Inhibitors of signal transduction and activators of transcription such as (STAT)-5, mitogen-activated protein kinases (MAPK)/extracellular-regulated kinase (ERK), and phosphatidyl inositol-3 kinase (PI3K)/Akt impaired the protective effect of EPO on RGCs exposed to different insults. CONCLUSION: EPO provided neuroprotection to cultured adult rat RGCs; however, the degree of protection varied with the type of toxic insult, RGC subtype, and timing of EPO treatment.

A novel high-content flow cytometric method for assessing the viability and damage of rat retinal ganglion cells.

PURPOSE: The aim of the study was to develop a high-content flow cytometric method for assessing the viability and damage of small, medium, and large retinal ganglion cells (RGCs) in N-methyl-D-aspartic acid (NMDA)-injury model. METHODS/RESULTS: Retinal toxicity was induced in rats by intravitreal injection of NMDA and RGCs were retrogradely labeled with Fluoro-Gold (FG). Seven days post-NMDA injection, flatmount and flow cytometric methods were used to evaluate RGCs. In addition, the RGC area diameter (D((a))) obtained from retinal flatmount imaging were plotted versus apparent volume diameter (D((v))) obtained from flow cytometry for the same cumulative cell number (sequentially from small to large RGCs) percentile (Q) to establish their relationship for accurately determining RGC sizes. Good correlation (r = 0.9718) was found between D((a)) and apparent D((v)). Both flatmount and flow cytometric analyses of RGCs showed that 40 mM NMDA significantly reduced the numbers of small and medium RGCs but not large RGCs. Additionally, flow cytometry showed that the geometric means of FG and thy-1 intensities in three types of RGCs decreased to 90.96±2.24% (P<0.05) and 91.78±1.89% (P>0.05) for small, 69.62±2.11% (P<0.01) and 69.07±2.98% (P<0.01) for medium, and 69.68±6.48% (P<0.05) and 69.91±6.23% (P<0.05) for large as compared with the normal RGCs. CONCLUSION: The established flow cytometric method provides high-content analysis for differential evaluation of RGC number and status and should be useful for the evaluation of various models of optic nerve injury and the effects of potential neuroprotective agents.

Influence of charge on FITC-BSA-loaded chondroitin sulfate-chitosan nanoparticles upon cell uptake in human Caco-2 cell monolayers.

BACKGROUND AND METHODS: Chondroitin sulfate-chitosan (ChS-CS) nanoparticles and positively and negatively charged fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA)-loaded ChS-CS nanoparticles were prepared and characterized. The properties of ChS-CS nanoparticles, including cellular uptake, cytotoxicity, and transepithelial transport, as well as findings on field emission-scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy were evaluated in human epithelial colorectal adenocarcinoma (Caco-2) fibroblasts. ChS-CS nanoparticles with a mean particle size of 250 nm and zeta potentials ranging from -30 to +18 mV were prepared using an ionic gelation method. RESULTS: Standard cell viability assays demonstrated that cells incubated with ChS-CS and FITC-BSA-loaded ChS-CS nanoparticles remained more than 95% viable at particle concentrations up to 0.1 mg/mL. Endocytosis of nanoparticles was confirmed by confocal laser scanning microscopy and measured by flow cytometry. Ex vivo transepithelial transport studies using Caco-2 cells indicated that the nanoparticles were effectively transported into Caco-2 cells via endocytosis. The uptake of positively charged FITC-BSA-loaded ChS-CS nanoparticles across the epithelial membrane was more efficient than that of the negatively charged nanoparticles. CONCLUSION: The ChS-CS nanoparticles fabricated in this study were effectively endocytosed by Caco-2 fibroblasts without significant cytotoxicity at high nanoparticle concentrations. ChS-CS nanoparticles represent a potential novel delivery system for the transport of hydrophilic macromolecules.

The comparison of protein-entrapped liposomes and lipoparticles: preparation, characterization, and efficacy of cellular uptake.

Fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA)-loaded polyethylene glycol (PEG)-modified liposomes and lipoparticles with high protein entrapment were developed. The lipid formula of the liposomes contained PEGylated lipids and unsaturated fatty acids for enhancing membrane fluidity and effective delivery into cells. The preparation techniques, lipid content, and PEG-modified lipoparticle ratios were evaluated. The PEG-modified lipoparticles prepared by ethanol injection extrusion (100 nm pore size) achieve a population of blank liposomes with a mean size of 125 ± 2.3 nm and a zeta potential of -12.4 ± 1.5 mV. The average particle size of the PEG-modified lipoparticles was 133.7 ± 8.6 nm with a zeta potential of +13.3 mV. Lipoparticle conformation was determined using transmission electron microscopy and field-emission scanning electron microscopy. The FITC-BSA encapsulation efficiency was dramatically increased from 19.0% for liposomes to 59.7% for lipoparticles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results confirmed the preparation process, and an 8-hour leaching test did not harm the protein structure. Once prepared, the physical and chemical stability of the PEG-modified lipoparticle formulations was satisfactory over 90 days. In vitro retention tests indicated that the 50% retention time for the protein-containing lipoparticles was 7.9 hours, substantially longer than the liposomes at 3.3 hours. A Caco-2 cell model was used for evaluating the cytotoxicity and cell uptake efficiency of the PEG-modified lipoparticles. At a lipid content below 0.25 mM, neither the liposomes nor the lipoparticles caused significant cellular cytotoxicity (P < 0.01) and FITC-BSA was significantly taken up into cells within 60 minutes (P < 0.01).

Novel RGD-lipid conjugate-modified liposomes for enhancing siRNA delivery in human retinal pigment epithelial cells.

BACKGROUND: Human retinal pigment epithelial cells are promising target sites for small interfering RNA (siRNA) that might be used for the prevention and/or treatment of choroidal neovascularization by inhibiting the expression of angiogenic factor; for example, by downregulating expression of the vascular endothelial growth factor gene. METHODS: A novel functional lipid, DSPE-PEG-RGD, a Arg(R)-Gly(G)-Asp(D) motif peptide conjugated to 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[maleimide (polyethylene glycol)-2000], was synthesized for the preparation of siRNA-loaded RGD-PEGylated liposomes to enhance uptake of encapsulated siRNA in retinal pigment epithelial cells. Various liposomes, with 1 mol% and 5 mol% PEGylated lipid or 1 mol% and 5 mol% RGD-PEGylated lipid, were fabricated. RESULTS: Characterization of the liposomes, including siRNA entrapment efficiency, average particle size and ζ-potential, were determined to be as follows: >96%, 129.7 ± 51 to 230.7 ± 60.7 nm, and 17.3 ± 0.6 to 32 ± 1.3 mV, respectively. For the in vitro retinal pigment epithelial cell studies, the RGD-PEGylated liposomes had high delivery efficiency with siRNA delivery, about a four-fold increase compared with the PEGylated liposomes. Comparison of the various liposomes showed that the 1 mol% RGD-modified liposome had less cytotoxicity and higher siRNA delivery efficiency than the other liposomes. The antibody blocking assay confirmed that uptake of the 1 mol% RGD-PEGylated liposome was via integrin receptor- mediated endocytosis in retinal pigment epithelial cells. CONCLUSION: The results of this study suggest that RGD-PEGylated liposomes might be useful for siRNA delivery into retinal pigment epithelial cells by integrin receptor-medicated endocytosis.

The preparation of sustained release erythropoietin microparticle.

PURPOSE: Protein microencapsulation in biodegradable polymers is a promising route to provide for sustained release. The erythropoietin (EPO) microparticles are using human serum albumin (HSA) and poly-L-lysine (PK) as the protection complex to increased EPO integrity, entrapped efficiency and active EPO release by w/o/w solvent evaporation techniques. The optimum formulation development process was also reported by using FITC-OVA as a model protein. METHODS: The model protein FITC-ovalbumin and EPO are protected by human serum albumin and poly-L-lysine complex and encapsulated in 50:50 poly(DL-lactide-co-glycolide) by a w/o/w solvent evaporation method. Protein active integrity and degradation compound is measured by size-exclusion chromatography. Protein-loaded microparticle physical properties and in vitro active and degradation compounds release profile are characterized. RESULTS: High active integrity protein loading efficiency and particle yield of EPO or OVA-HSA/PK-loaded PLG microparticles are successfully produced by a w/o/w solvent evaporation method. Varied protection protein complex formulations and encapsulation processes are investigated. The high OVA model protein loading efficiency (80.2%), FITC-OVA content (0.24 microg mg(-1)) and yield (72.4%) are obtained by adding 100 microg mL(-1) FITC-OVA complex with 10% HSA/0.05% PK (Mw 1.5-3 kD) in the initial solution to protect the model protein. In vitro release profiles show more active OVA release from HSA/PK OVA-loaded than OVA-loaded only microparticles and also the amount of degraded protein that comes out after 3 weeks incubated in the PBS medium for OVA-loaded only microparticles is observed. The same formulation and preparation process resulted in EPO loading efficiency (68.4%), EPO content (0.23 microg mg(-1)) and yield (76.1%) for HSA/PK EPO-loaded microparticles. In vitro release profiles show active EPO sustained release over 7 days. Using HSA/PK as carried in the primary emulsion of EPO-loaded microparticles resulted in less burst release% than EPO-loaded only microparticles.

A co-cultured skin model based on cell support membranes.

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals.