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Biomedical subjects

Leaf Huang

Publications and source records attributed to Leaf Huang.

At least 37 records · Page 2Linked to original sources
PubMed2004

Sustained intravesical drug delivery using thermosensitive hydrogel.

PURPOSE: Direct instillation of drug solutions into the bladder through a urethral catheter (i.e., intravesical therapy) evades systemic adverse effects of drugs used for bladder diseases. However, conventional vehicles for these drugs fail to extend duration of drug exposure in the bladder beyond the first voiding of urine postinstillation. The current study seeks to overcome the aforementioned inherent limitation of intravesical drug administration by using thermosensitive hydrogel as a matrix for sustained intravesical drug delivery. METHODS: Under halothane anesthesia, normal adult female Sprague-Dawley rats were catheterized with PE-50 tubing to instill either 0.02% w/v solution of fluorescein isothiocyanate (FITC) or the same amount of FITC in a 30% w/v dispersion of thermosensitive [Poly(ethylene glycol)-Poly[lactic acid-co-glycolic acid]-Poly(ethylene glycol)) (PEG-PLGA-PEG) polymer in a 0.1 M phosphate buffer. After instillations, rats were kept in metabolic cages for urine collection. Fluorescence emanating from FITC was measured in the urine at various time points up to 24 h after instillation. A rat model of cyclophosphamide-induced cystitis was chosen for the efficacy study using misoprostol as a model drug entrapped in the thermosensitive hydrogel in place of FITC. Efficacy of hydrogel containing misoprostol was compared against rat groups instilled with saline, hydrogel, and misoprostol independently. RESULTS: Prolonged drug exposure to the bladder afforded by hydrogel was evident from the time course of FITC elimination in the urine and by the green fluorescence of FITC seen at the bladder surface when isolated 24 h after instillation. Rats instilled with free FITC voided almost all of the fluorescence in the urine within the first 8 h, whereas rats instilled with hydrogel encapsulated FITC showed sustained release up to 24 h after instillation. Using a cyclophosphamide-induced cystitis model, rats instilled with misoprostol, a synthetic PGE1 analog, showed significantly reduced frequency of urine voiding (p < 0.05) as compared to the rats instilled with saline. Histological examination of the urothelium showed near normal morphology in rats instilled with misoprostol in hydrogel, whereas extensive tissue damage was observed in rats instilled with saline. CONCLUSION: Our study showed that PEG-PLGA-PEG polymer could be used as a viable sustained drug delivery system for intravesical therapy of diseases of the bladder such as cystitis using misoprostol.

Animals↗
PubMed2004

Coating of mannan on LPD particles containing HPV E7 peptide significantly enhances immunity against HPV-positive tumor.

PURPOSE: Previously, our laboratory has reported that liposome-protamine-DNA (LPD) nanoparticle is an effective delivery system for tumor-associated antigens. Mannan, which potentially targets antigen-presenting cells, was coated on LPD to further enhance its antitumor activity. METHODS: Cholesterol-conjugated mannan was coated on LPD. The abilities of mannan-coated LPD to target antigen-presenting cells, to activate dendritic cells, and to induce antitumor immunity were investigated and compared to those of LPD alone. RESULTS: Both in vitro and in vivo uptake of LPD showed that mannan-coated LPD particles were preferably taken up by dendritic cells and macrophages. In addition, the expression of co-stimulatory molecules CD80/CD86 on DC2.4 cells after co-incubation with mannan-coated LPD was significantly higher than that after co-incubation with LPD. A model major histocompatibility complex class I-restricted peptide antigen from HPV 16 E7 protein was incorporated into LPD to immunize mice against the growth of TC-1 tumor cells expressing E7 protein. Coating with mannan significantly enhanced both preventive and therapeutic activities of LPD/E7. Finally, the release of IFN-gamma from isolated splenocytes was significantly enhanced when mice were immunized with mannan-coated LPD/E7 than with LPD/E7 alone. CONCLUSION: Targeting of the LPD/E7 to local draining lymph nodes by mannan is partially responsible for the enhanced anti-tumor activity.

Animals↗
PubMed2004

Urodynamic and immunohistochemical evaluation of intravesical capsaicin delivery using thermosensitive hydrogel and liposomes.

PURPOSE: Aqueous insolubility of the vanilloids such as capsaicin is a major disincentive in their intravesical therapy of detrusor hyperreflexia. We sought to overcome the delivery of this hydrophobic neurotoxin by entrapping it in a lipid bilayer of positively charged multilamellar lipid vesicles (liposomes) or in a hydrophobic polymer matrix of thermosensitive hydrogel. MATERIALS AND METHODS: Liposomes, hydrogel and 30% ethanol/normal saline were prepared with or without 1 mM capsaicin (0.5 ml) and administered intravesically for 30 minutes to 7 groups of age matched, normal female adult Sprague-Dawley rats under halothane anesthesia. At 48 hours after intravesical instillation cystometric studies were performed using urethane anesthesia (0.04 ml per minute). The animals were subsequently sacrificed and whole bladders were harvested for histology and immunohistochemistry. RESULTS: In normal urethane anaesthetized rats capsaicin in 30% ethanol and liposomes completely blocked micturition reflexes. Capsaicin in hydrogel did not completely block the micturition reflex but it significantly decreased bladder contraction frequency compared with vehicle controls. The results of cystometry with capsaicin in liposomes and capsaicin in 30% ethanol correlated with a significant decrease in calcitonin gene-related peptide staining of afferent nerves in the bladder wall. Photographs taken after hematoxylin and eosin staining of the bladder treated with liposomes and hydrogel in the absence of capsaicin did not reveal any adverse histological changes. There were significant histological changes in bladders treated with 30% ethanol alone. CONCLUSIONS: In comparison with 30% ethanol liposomes are a superior vehicle for the intravesical administration of capsaicin, producing comparable efficacy with less tissue damage. Hydrogel can also serve as safe alternative option for capsaicin delivery.

Administration, Intravesical↗
PubMed2004

Electroporatic delivery of TGF-beta1 gene works synergistically with electric therapy to enhance diabetic wound healing in db/db mice.

Electrical stimulation (ES) is a therapeutic treatment for wound healing. Electroporation, a type of ES, is a well-established method for gene delivery. We hypothesize that proper conditions can be found with which both electrical and gene therapies can be additively applied to treat diabetic wound healing. For the studies of transforming growth factor-beta1 (TGF-beta1) local expression and therapeutic effects, full thickness excisional wound model of db/db mice was used, we measured TGF-beta1 cytokine level at 24 h postwounding and examined wounds histologically. Furthermore, wound closure was evaluated by wound-area measurements at each day for 14 d. We found that syringe electrodes are more effective than the conventional caliper electrodes. Furthermore, diabetic skin was more sensitive to the electroporative damage than the normal skin. The optimal condition for diabetic skin was six pulses of 100 V per cm for 20 ms. Under such condition, the healing rate of electrically treated wound was significantly accelerated. Furthermore, when TGF-beta1 gene was delivered by electric pulses, the healing rate was further enhanced. Five to seven days postapplication of intradermal injection of plasmid TGF-beta1 followed by electroporation, the wound bed showed an increased reepithelialization rate, collagen synthesis, and angiogenesis. The data indicates that indeed the electric effect and gene effect work synergistic in the genetically diabetic model.

Animals↗
PubMed2003

Lipid-mediated delivery of peptide nucleic acids to pulmonary endothelium.

Peptide nucleic acid (PNA) is a DNA/RNA mimic in which the phosphodiester (PO) linkage is replaced with a peptide bond. It has a number of unique properties compared to currently used oligonucleotides including higher affinity towards RNA or DNA target, resistance to nucleases or proteases, and minimal non-specific interactions with proteins. Clinical applications of PNA, however, are limited by its inefficient intracellular delivery. In this study, we have shown that delivery of PNA to pulmonary endothelium in intact mice can be greatly improved via hybridization with a short PO oligonucleotide that serves as a carrier to form complexes with cationic liposomes. We have also shown for the first time that unlike a CpG DNA oligo that is highly proinflammatory, a CG-containing PNA is inert in triggering TNF-alpha response in cultured macrophages and in mice. Thus delivery of PNA to pulmonary endothelium may prove to be a therapeutically useful for the treatment of pulmonary vascular diseases.

Animals↗
PubMed2003

Gene transfer to subdermal tissues via a new gene gun design.

Although particle-mediated gene transfer technology (gene gun) has been applied for gene transfer to external tissues, the application of this technology to other tissues has met with limited success. Here we report the development of a new design of a gene gun that uses helium discharge to propel DNA-coated gold beads that are suspended in liquid. Higher discharge pressures allow for the delivery of DNA to deeper tissues. Using the new gene gun to deliver a luciferase expression plasmid resulted in higher levels of gene expression in the skin than observed with conventional guns, as well as in subdermal tissues, including subcutaneous tumors. Even when using as little as 125 ng of DNA, gene expression in skin and muscle reached its peak level at 24 hr postbombardment and remained for at least 1 week. The use of a LacZ expression plasmid showed that gene expression was distributed throughout the skin with no observable pathology. The new gene gun was used to deliver a model tumor rejection antigen (a modified human papilloma virus [HPV] E7 gene) to mice. All of the treated animals developed protective immunity against HPV-positive tumors. These results demonstrate that our new design can be used in standard gene gun applications and extends the reach of gene gun technology to tissues that were previously unavailable.

Animals↗
PubMed2003

Intravesical protamine sulfate and potassium chloride as a model for bladder hyperactivity.

OBJECTIVES: An acute animal model for hyperactive bladder in rats was developed using intravesical infusion of protamine sulfate (PS), an agent thought to break down urothelial barrier function, and physiologic concentrations of potassium chloride (KCl). METHODS: Continuous cystometrograms (CMGs) were performed in urethane-anesthetized female rats by filling the bladder (0.04 mL/min) with normal saline followed by intravesical infusion of a test solution consisting of either KCl (100 or 500 mM) or PS (10 or 30 mg/mL) for 60 minutes. Subsequently, the 10 mg/mL PS-treated animals were infused intravesically with 100, 300, or 500 mM KCl. Some animals were pretreated with capsaicin (125 mg/kg subcutaneously) 4 days before the experiments. RESULTS: Unlike KCl (100 or 500 mM) or a low concentration of PS (10 mg/mL) alone, the intravesical administration of a high concentration of PS (30 mg/mL) produced irritative effects with a decreased intercontraction interval (by 80.6%). After infusion of a low concentration of PS, infusion of 300 or 500 mM KCl produced irritative effects (intercontraction interval decreased by 76.9% or 82.9%, respectively). The onset of irritation occurred more rapidly after 500 mM KCl (10 to 15 minutes) than after 300 mM KCl (20 to 30 minutes). Capsaicin pretreatment delayed the onset (approximately 60 minutes) and reduced the magnitude (intercontraction interval decreased by 35.5%) of irritative effects. CONCLUSIONS: Intravesical administration of KCl after PS treatment activates capsaicin-sensitive afferents and detrusor muscle and presumably capsaicin-resistant afferents. Modest, noncytotoxic affronts to urothelial barrier function can result in dramatic irritative responses. This model may be useful in the study of bladder irritation and hyperactivity.

Administration, Intravesical↗
PubMed2003

Intravesical liposome administration--a novel treatment for hyperactive bladder in the rat.

OBJECTIVES: To examine the effect of intravesical administration of liposomes (LPs) on chemically induced bladder hyperactivity in the rat. It has been suggested that interstitial cystitis (IC) is associated with a dysfunctional or leaky epithelium. Thus, enhancement of epithelial barrier function might be useful in the treatment of IC. LPs are vesicles that are concentric phospholipid bilayers separated by an aqueous compartment and can fuse with cells to provide a molecular film that can promote wound healing. METHODS: The intravesical pressure was recorded using a transurethral catheter in adult female Sprague-Dawley rats anesthetized with urethane (1.2 g/kg subcutaneously). Some animals were pretreated with capsaicin (125 mg/kg subcutaneously) 4 days before the experiments. Continuous cystometrograms were performed by slowly filling the bladder (0.04 mL/min) with solutions of varying compositions, including saline, acetic acid (AA, 0.1%), potassium chloride (KCl, 500 mM), protamine sulfate (PS, 10 mg/mL), LPs, PS/KCl, or LPs/KCl. The parameters measured included the intercontraction interval (ICI), amplitude of bladder contractions, compliance, and micturition pressure threshold. RESULTS: The ICI was decreased after exposure to AA (79.8% decrease) or PS/KCl (81% decrease); however, the ICI was not changed after LPs, PS, or KCl alone. The decreased ICI was partially reversed after infusion of LPs (172.8% increase) or LPs/KCl (63% increase), but was not significantly changed after switching to saline or KCl administration. Pretreatment with capsaicin delayed the onset of the irritative effects of AA by approximately 30 to 60 minutes, but had not changed the magnitude after 2 hours of infusion. CONCLUSIONS: Intravesical administration of PS/KCl or AA activates capsaicin-sensitive and capsaicin-resistant afferents in a time-dependent sequence that is partially reversed by LP infusion. We hypothesize that LPs might enhance the barrier properties of a dysfunctional uroepithelium and increase resistance to irritant penetration. Thus, intravesical LP administration could be a novel treatment of patients with IC.

Administration, Intravesical↗
PubMed2003

Lipid-protamine-DNA-mediated antigen delivery to antigen-presenting cells results in enhanced anti-tumor immune responses.

Vaccination with antigenic peptides encoding tumor antigens has the potential to be an effective treatment for cancer. To induce tumor-specific cellular immune responses, a peptide antigen must be presented by antigen-presenting cells (APCs) to T-cells in the lymphatic tissues. Effective in vivo delivery of peptide antigens to APCs has been problematic. Here we use a model antigen from the HPV16 E7 protein to formulate LPD/E7 particles that upon iv administration are internalized by CD11c(+) and CD11b(+) cells in the marginal zone of the spleen. Either iv or sc vaccination with LPD/E7 particles induces E7-specific CTL responses stronger than those obtained using previously described liposome/peptide strategies and prevents the establishment of E7-expressing tumors. Furthermore, the administration of LPD/E7 particles to tumor-bearing mice caused complete tumor regression in 100% of the treated animals. Based on these studies, the entrapment of peptide antigens inside LPD particles may be an effective and generally applicable strategy for the enhancement of peptide vaccine potency.

Animals↗
PubMed2003

Intradermal injection of transforming growth factor-beta1 gene enhances wound healing in genetically diabetic mice.

PURPOSE: To evaluate the biologic effect of direct cutaneous TGF-beta1 gene delivery on impaired wound healing models using genetically diabetic mice. METHODS: Diabetic mice (C57BKS.Cg-m +/+ Leprdb female mice) with 1 cm x 1 cm excisional wounds were intradermally injected with 60 microg of plasmid DNA encoding TGF-beta1 gene. The wound closure was measured up to 14 days postwounding. At days 7 and 14 postwounding, sections of skin were taken for hematoxylin and eosin and Masson's trichome staining to examine the morphology and collagen deposition. The cell proliferation and TGF-beta1 gene expression were studied using immunohistochemical stainings for 5-bromo-2-deoxyuridine and for TGF-beta1. RESULTS: A higher cell proliferation rate and a denser and more organized new extracellular matrix were observed in the treated wound site. Complete wound closure was detected as early as 7 days for TGF-beta1-treated group in comparison with 11-14 days for the untreated, control plasmid DNA- and PBS-treated groups. CONCLUSION: A single intradermal injection of TGF-beta1 plasmid DNA was sufficient to enhance wound healing. This approach represents a new strategy that may be applied to the treatment of excisional wounds in human diabetic patients.

Animals↗
PubMed2003

Controlled gene delivery system based on thermosensitive biodegradable hydrogel.

PURPOSE: Currently, most pDNA delivery systems based on synthetic polymers are either nonbiodegradable or not sensitive to the release environment. The primary objective of this study was to develop and evaluate an aqueous-based, thermosensitive, biodegradable and biocompatible triblock copolymer to control pDNA delivery in vitro and in vivo. METHODS: The triblock copolymers, poly[ethylene glycol-b-(D, L-lactic acid-co-glycol acid)-b-ethylene glycol] (PEG-PLGA-PEG), were synthesized as previously described. The molecular weight and polydispersity of PEG-PLGA-PEG were monitored by gel permeation chromatography (GPC). The cytotoxicity of PEG-PLGA-PEG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The release of 32P-labeled pDNA entrapped in aqueous dispersion of PEG-PLGA-PEG in 0.1 mol/L sodium phosphate buffer solution (pH 7.4) was studied at 37 degrees C under agitation. Gene transfection efficiency was evaluated in a skin wound model in CD-1 mice. RESULTS: The aqueous dispersion of PEG-PLGA-PEG flows freely at room temperature but form a gel at 37 degrees C body temperature. The in vitro degradation of PEG-PLGA-PEG lasted for more than 30 days. The cytotoxicity of PEG-PLGA-PEG evaluated in HEK 293 cells was significantly lower than that of poly-L-lysine hydrochloride. The release profile of supercoiled pDNA from the polymer followed the zero-order kinetics up to 12 days. Maximal gene expression of luciferase was at 24 h in the skin wound of CD-1 mice and by 72 h, the expression dropped by nearly 94%. CONCLUSION: These results suggest hydrogel formed by PEG-PLGA-PEG could be a promising platform for delivery of pDNA, which represents a novel strategy that may serve as a non-viral vector for gene therapy in wound healing.

Animals↗
PubMed2003

Thermosensitive hydrogel as a Tgf-beta1 gene delivery vehicle enhances diabetic wound healing.

PURPOSE: To accelerate diabetic wound healing with TGF-beta1 gene delivery system using a thermosensitive hydrogel made of a triblock copolymer, PEG-PLGA-PEG. METHODS: Two 7 x 7 mm full thickness excisional wounds were created in parallel at the back of each genetically diabetic mouse. The hydrogel containing plasmid TGF-beta1 was administered to the wound and formed an adhesive film in situ. Controls were either untreated or treated with the hydrogel without DNA. We used a commercial wound dressing, Humatrix, either with or without DNA, to compare the therapeutic effect with the thermosensitive hydrogel. RESULTS: We found that thermosensitive hydrogel alone is slightly beneficial for reepithealization at early stage of healing (day 1-5), but significantly accelerated repithelializaion, increased cell proliferation, and organized collagen were observed in the wound bed treated with thermosensitive hydrogel containing plasmid TGF-beta1. The accelerated reepithelialization was accompanied with enhanced collagen synthesis and more organized extracellular matrix deposition. Humatrix alone or with plasmid TGF-beta1, had little effect. CONCLUSIONS: Thermosensitive hydrogel made of PEG-PLGA-PEG triblock copolymer provides excellent wound dressing activity and delivers plasmid TGF-beta1 to promote wound healing in a diabetic mouse model.

Animals↗
PubMed2002

Systemic gene therapy in human xenograft tumor models by liposomal delivery of the E1A gene.

The adenovirus type 5 E1A protein has been demonstrated to elicit antitumor effects through the induction of apoptosis, inhibition of cell cycle progression, induction of differentiated epithelial phenotypes, repression of oncogene expression and function, and sensitization to chemotherapeutic agents and radiation. These unique properties have led to use of the E1A gene in adenoviral and lipid-based gene therapy systems, and it has demonstrated antitumor effects in tumor xenograft model systems. However, the delivery systems used in those studies are best suited for local or intratumoral delivery rather than systemic delivery. Because the effective treatment of many primary tumors as well as metastatic disease requires systemic delivery systems, a novel gene delivery system composed of liposome/protamine/DNA (LPD) was investigated for systemic delivery of the E1A gene. Athymic nude mice bearing human breast (MDA-MB-361) or head and neck (WSUHN-31) tumor xenografts were treated i.v. with LPD-E1A, and the expression of E1A protein and effects on tumor growth were assessed. In the MDA-MB-361 breast model, expression of E1A protein was detected in the tumors after LPD-E1A treatment, which was associated with down-regulation of HER-2/neu protein expression and the presence of apoptotic cells. Tumor volume was also smaller in mice treated with LPD-E1A than in controls in both of the xenograft models. Lastly, LPD-E1A in combination with paclitaxel was more effective than LPD-E1A or paclitaxel alone in the MDA-MB-361 model. Additional preclinical and clinical development of LPD-E1A is warranted for the treatment of advanced or metastatic cancer.

Adenovirus E1A Proteins↗
PubMed2002

Systemic administration of naked DNA encoding interleukin 12 for the treatment of human papillomavirus DNA-positive tumor.

Interleukin 12 (IL-12) is one of the most effective and promising cytokines for cancer therapy. Its therapeutic effects have been demonstrated in a variety of tumors in animal models when it is administrated locally or systemically. We describe here a systemic delivery of naked murine IL-12 (mIL-12) gene in vivo. Dose-dependent systemic production of mIL-12, with a serum level up to approximately 20 microg/ml, was observed 24 hr after systemic gene delivery. The apparent half-life in the circulation was about 5 hr. The result of a bioactivity assay (in vitro interferon gamma [IFN-gamma] release) indicated that the gene product in mice was as active as the purified recombinant murine IL-12 protein (rmIL-12). The circulating mIL-12 activated natural killer cells and stimulated IFN-gamma production in vivo. A single administration of mIL-12 gene resulted in prominent regression of established subcutaneous tumor in a human papillomavirus (HPV) DNA-positive tumor model (TC-1) in C57BL/6J mice. The antitumor effect of the single gene dose was comparable to repeated intraperitoneal administration of rmIL-12 (0.5 microg/day for consecutive 5 days). This systemic gene delivery is simple, economical, and highly efficient for the production of large amounts of cytokine in vivo. With this gene delivery method, we have demonstrated the therapeutic potential of IL-12 for the treatment of HPV DNA-positive tumor and the usefulness of the systemic gene delivery for assessing the therapeutic effect of a candidate gene.

Animals↗
PubMed2002

Development of non-viral vectors for systemic gene delivery.

One of the major challenges for gene therapy is systemic delivery of a nucleic acid directly into an affected tissue. This requires developing a vehicle which is able to protect the nucleic acid from degradation, while delivering the gene of interest to the specific tissue and specific subcellular compartment. In this review, we summarize some of the recent advances in new non-viral delivery systems for systemic administration. Two types of gene delivery systems are described: (i) LPD1 (cationic liposome-entrapped, polycation-condensed DNA, type 1), and (ii) retention-time mediated naked DNA delivery. Hypothesized mechanisms for these systemic gene transfers are also discussed.

Animals↗
PubMed2002

Enhanced cutaneous gene delivery following intradermal injection of naked DNA in a high ionic strength solution.

Intradermal injection of naked DNA results in gene transfer to skin cells, but the efficiency of this gene transfer method is relatively low and variable. We have systematically optimized several parameters to obtain reproducible, high-level gene transfer to the mouse skin. Older mice (approximately 7 weeks) showed a significant decrease in gene expression compared with younger mice (4-5 weeks old). The composition of the solvent vehicle (electrolyte versus nonelectrolyte) strongly affected gene expression in the skin. A higher level of gene expression was achieved when naked DNA was dissolved in isotonic phosphate buffered saline solution compared with isotonic dextrose solution. Finally, transfection efficiency in older mice was greatly improved by increasing the ionic strength of the solvent vehicle. The improved transfection efficiency was due to an enhanced DNA uptake by the skin cells. Gene transfer was most evident in the subdermal smooth muscle cells and epidermal cells. With the optimized conditions, gene transfer mediated by intradermal injection of naked DNA was comparable in efficiency to electroporation. However, cellular distributions of the gene transfer of the two methods were different.

Animals↗
PubMed2002

A syringe electrode device for simultaneous injection of DNA and electrotransfer.

Electroporation for gene delivery has attracted considerable attention recently, because of both the site-specific nature of the delivery and the high efficiency of the method. Electrotransfer of genes involves the application of an electric field to cells to enhance their permeability, facilitating exogenous polynucleotide transit across the cytoplasmic membrane. However, the relatively high electric field strength required for electroporation induces tissue damage, thus limiting its widespread application. We describe here a syringe electrode of our design, with which the same transfection efficiency can be achieved by using much lower electric field strength than that of conventional electrodes, such that the tissue damage is minimized.

Animals↗