Biomedical subjects

Leaf Huang

Publications and source records attributed to Leaf Huang.

At least 19 recordsLinked to original sources

PubMed2006

Nanostructured calcium phosphates (NanoCaPs) for non-viral gene delivery: influence of the synthesis parameters on transfection efficiency.

Calcium phosphate (CaP) based approaches remain an attractive option for delivering plasmid DNA (pDNA) into cultured cells. However, despite their appeal, current synthesis methodologies typically yield lower, less consistent transfection efficiencies when compared to viral approaches. Therefore, we report here a novel method to consistently synthesize efficient, nano-sized, mono-dispersed CaP-pDNA particles; accomplished by optimizing both the stoichiometry (Ca/P ratio) of the CaP particles as well as the mode in which the calcium and phosphate precursor solutions are mixed. Our results indicate that calcium and phosphate precursors when mixed in a controlled and regulated manner reproducibly result in nano-sized particles that consistently yield higher transfection efficiencies when compared to particles synthesized via manual mixing (a two-fold increase was observed). Also, maximum transfection efficiencies in both HeLa and MC3T3-E1 cells lines were obtained when a Ca/P ratio between 100 and 300 was used. Particles synthesized within this optimum Ca/P ratio range were between 25 and 50 nm. Our data suggests that these maximized transfection efficiencies were obtained because these particles not only effectively condensed (70% efficient) but also efficiently bound (90% efficient) the pDNA. In addition, X-ray diffraction and Fourier transform infrared spectroscopy analyses confirmed that all of the synthesized CaP structures exhibited the hydroxyapatite phase.

3T3 Cells↗

PubMed2006

Mechanism of in vivo DNA transport into cells by electroporation: electrophoresis across the plasma membrane may not be involved.

BACKGROUND: Recently, in vivo gene transfer with electroporation (electro-gene transfer) has emerged as a leading technology for developing nonviral gene therapies and nucleic acid vaccines. The widely hypothesized mechanism is that electroporation induces structural defects in the membrane and provides an electrophoretic force to facilitate DNA crossing the permeabilized membrane. In this study, we have designed a device and experiments to test the hypothesis. METHODS: In this study, we have designed a device that alternates the polarity of the applied electric field to elucidate the mechanism of in vivo electro-gene transfer. We also designed experiments to challenge the theory that the low-voltage (LV) pulses cannot permeabilize the membrane and are only involved in DNA electrophoresis, and answer the arguments that (1) the reversed polarity pulses can cause opposing sides of the cell membrane to become permeabilized and provide the electrophoresis for DNA entry; or (2) once DNA enters cytoplasmic/endosomal compartments after electroporation, it may bind to cellular entities and might not be reversibly extracted. Thus a gradual buildup of the DNA in the cell still seems quite possible even under the condition of the rapid reversal of polarity. RESULTS: Our results indicate that electrophoresis does not play an important role in in vivo electro-gene transfer. CONCLUSIONS: This study provides new insights into the mechanism of electro-gene transfer, and may allow the definition of newer and more efficient conditions for in vivo electroporation.

Animals↗

PubMed2006

Local drug delivery to bladder using technology innovations.

Local delivery of drugs directly into the bladder by way of a urethral catheter is a clever approach to optimize drug delivery to the disease site while reducing systemic bioavailability. Pharmacotherapy by this route is referred to as intravesical delivery. In recent years, intravesical delivery has been used in combination with and oral regimen of drugs or as second-line treatment for neurogenic bladder and detrusor overactivity. Negligible absorption of instilled drugs into the systemic circulation explains the near-minimal adverse toxicity reported with this form of therapy. The authors discuss shortcomings of the current options available for intravesical delivery and provide a broad overview of the latest advances through technology innovation to overcome these drawbacks.

Adhesiveness↗

PubMed2006

Surface-modified LPD nanoparticles for tumor targeting.

We have developed a tumor-targeted LPD formulation (liposome-polycation-DNA complex) for siRNA. With surface modification, the targeted, PEGylated LPD increased the delivery efficiency by four-fold and the gene-silencing effect by two- to three-fold. Downregulation of survivin in human lung cancer cells by targeted LPD induced 90% of apoptosis and sensitized the cells to cisplatin by four-fold. PEGylated LPD formulation also significantly improved the tumor localization of siRNA in the NCI-H460 human lung cancer xenograft model. The tumor appeared to be the major uptake organ for siRNA formulated in surface-modified LPD. Our encouraging results indicate that surface-modified LPD may be a potent carrier for RNAi-based tumor therapy.

Animals↗

PubMed2005

Traditional Chinese medicines Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch) activate pregnane X receptor and increase warfarin clearance in rats.

The traditional Chinese medicines (TCMs) are essential components of alternative medicines. Many TCMs are known to alter the expression of hepatic drug-metabolizing enzymes and transporters. The molecular mechanism by which TCMs and/or their constituents regulate enzyme and transporter expression, however, has remained largely unknown. In this report, we show that two TCMs, Wu Wei Zi (Schisandra chinensis Baill) and Gan Cao (Glycyrrhiza uralensis Fisch), and their selected constituents activate the xenobiotic orphan nuclear receptor pregnane X receptor (PXR). Treatment with TCM extracts and the Schisandrol and Schisandrin constituents of Wu Wei Zi induced the expression of drug-metabolizing enzymes and transporters in reporter gene assays and in primary hepatocyte cultures. The affected enzymes and transporters include CYP3A and 2C isozymes and the multidrug resistance-associated protein 2. In transient transfection and reporter gene assays, the Schisandrin constituents of Wu Wei Zi had an estimated EC50 of 2 and 1.25 microM on hPXR and mPXR, respectively. Interestingly, mutations that were intended to alter the pore of the ligand-binding cavity of PXR had species-specific effects on the activities of the individual Schisandrols and Schisandrins. In rats, the administration of Wu Wei Zi and Gan Cao increased the metabolism of the coadministered warfarin, reinforcing concerns involving the safe use of herbal medicines and other nutraceuticals to avoid PXR-mediated drug-drug interactions. Meanwhile, the activation of PXR and induction of detoxifying enzymes provide a molecular mechanism for the hepatoprotective effects of certain TCMs.

Animals↗

PubMed2005

Mechanistic studies of sequential injection of cationic liposome and plasmid DNA.

We previously reported that sequential injection of cationic liposome and plasmid DNA leads to notably reduced inflammatory toxicity and improved transfection in the lung (Y. Tan et al., 2001, Mol. Ther. 3, 673-682). The purpose of the current study was to explore the mechanism involved in sequential injection. It was observed that sequential injection resulted in dramatically lower DNA uptake by the liver and higher DNA levels in the lung than the lipoplex injection. In vitro experiments with macrophage cells further showed that sequential addition of liposomes and DNA could diminish the cellular uptake of DNA by these cells. The contributions of serum to the enhanced bioactivity and decreased toxicity were examined by injecting mice with samples of premixed liposome with serum and then DNA (LSD sample), and the resulting activities were compared to those obtained with injection of lipoplex-serum mixtures (LDS sample). LSD yielded 80% lower TNF-alpha levels and over 10-fold higher transfection than lipoplex, which is consistent with the reported findings with sequential injection. In contrast, LDS resulted in the same TNF-alpha levels and comparable transfection with lipoplex. Thus, the results suggest that the primary interaction of serum with liposome is a critical factor contributing to the superior activity and reduced toxicity of sequential injection. Studies on the interaction between mouse serum, liposomes, and DNA showed that DNA could bind negatively charged liposome-serum complex to form a ternary complex, which has a density similar to that of the ternary complex formed between lipoplex with serum. Further in vitro tests showed that LSD and LDS were similar in particle size and protein content, but different in protein composition as observed by 2-D gel electrophoresis. In addition, DNA in LSD was more readily displaced by dextran sulfate, an anionic polymer, than in LDS. The above findings suggest that the inhibition of opsonin protein binding on the particle surface with the sequential injection may contribute to the reduced macrophage uptake and cytokine induction and that the high ability of DNA release from the particles formed after sequential injection may contribute to the improved lung gene transfection.

Animals↗

PubMed2005

Liposome-polycation-DNA (LPD) particle as a carrier and adjuvant for protein-based vaccines: therapeutic effect against cervical cancer.

With the successful identification of many tumor-specific antigens, tumor-associated antigens, and the potential of using unfractioned tumor cell derivatives as tumor antigens, a system and/or adjuvant that can deliver these antigens and help them to induce strong and effective anti-tumor immune responses is greatly needed. Previously, we reported that a MHC class I-restricted peptide epitope derived from human papillomavirus (HPV) 16 E7 protein, when incorporated into a clinically proven safe LPD (liposome-polycation-DNA) particle, was able to effectively eradicate tumors established in mice. Cervical cancer is the second most common cancer among women worldwide. HPV infection is clearly linked to this cancer. Vaccines based on the early (E) gene products of HPV could be effective in controlling it. However, besides the fact that epitope vaccines have many limitations particularly, concerning the diverse HLAs in humans, the use of the epitope as an antigen prevented us from fully characterizing the immune responses induced by the LPD as a vaccine carrier and/or adjuvant in previous studies. In the present study, by using the HPV 16 E7 protein as an antigen, we first showed that LPD, as a vaccine carrier and adjuvant induced strong and robust immune responses, both cellular and antibody. We then showed that immunization with LPD particles incorporated with either the wild type HPV 16 E7 protein or a potentially safer mutant induced strong immune responses that caused complete regressions of a model cervical cancer tumor established in murines. LPD could be a potent vaccine carrier and/or adjuvant for many antigens.

Adjuvants, Immunologic↗

PubMed2005

Implications of pharmacokinetic behavior of lipoplex for its inflammatory toxicity.

Inflammatory toxicity represents a typical toxicity associated with systemic administration of cationic liposome/DNA complex (lipoplex). Collected information indicates that the lipoplex gene delivery system mediates an uptake of plasmid DNA by the liver, mainly by Kupffer cells, in which a large amount of cytokine is produced. Therefore, many efforts have been made to overcome this problem. Previous reports by our laboratory demonstrated that sequential injection of cationic liposome and DNA could dramatically decrease the toxicity. In comparison with lipoplex injection, this method significantly suppresses the uptake of DNA by the liver. Opsonization effect in the stimulation of Kupffer cell uptake is proposed as an explanation for the differences in the pharmacokinetic properties of plasmid DNA after lipoplex injection and sequential injection. In this review, we cover the current understanding of the mechanisms underlying inflammatory toxicity and the several attempts to overcome this toxicity. The mechanism related to the pharmacokinetic properties of the lipoplex is focused on here for discussion.

Animals↗

PubMed2005

Recent advances in non-viral gene delivery.

Gene therapy has been deemed the medicine of the future due to its potential to treat many types of diseases. However, many obstacles remain before gene delivery is optimized to specific target cells. Over the last several decades, many approaches to gene delivery have been closely examined. By understanding the factors that determine the efficiency of gene uptake and expression as well as those that influence the toxicity of the vector, we are better able to develop new vector systems. This chapter will provide a brief overview of recent advances in gene delivery, specifically on the development of novel non-viral vectors. The following chapters will provide additional details regarding the evolution of non-viral gene delivery systems.

Cations↗

PubMed2005

Targeted delivery of oligodeoxynucleotides to mouse lung endothelial cells in vitro and in vivo.

Pulmonary endothelium plays an important role in the maintenance of normal pulmonary physiology and its dysfunction is involved in a number of pulmonary diseases. Correction of endothelial dysfunction via antisense oligodeoxynucleotides (ODN) is dependent on the development of a delivery vehicle that can efficiently deliver the ODN to pulmonary endothelium with minimal toxicity. To this end, we have developed a novel lipidic vector that is highly efficient in targeted delivery of ODN to pulmonary endothelium. This is based on a method that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane) and an ethanol-containing buffer system for encapsulating large quantities of polyanionic ODN in lipid vesicles. An endothelium-specific antibody (273-34A) is incorporated into the lipid vesicles via a distearoylphosphatidylethanolamine-poly(ethylene glycol) spacer. The 273-34A antibody efficiently mediated delivery of ODN to mouse lung endothelial cells in vitro and in vivo. Furthermore, systemic administration of this formulation is associated with minimal hematological toxicities and induces little acute change in systemic and pulmonary hemodynamics. These results provide a basis for lipid-mediated delivery of ODN for the treatment of pulmonary diseases. They also suggest the utility of this approach as a research tool to characterize the function of genes in the pulmonary endothelium.

Animals↗

PubMed2005

Recent Advances in Non-viral Gene Delivery.

Journal Article↗

PubMed2005

Non-viral vector as vaccine carrier.

Over the last several years, advances in gene-based delivery technology arising from the field of gene therapy have helped revitalize the field of vaccine development. Genetic vaccination encoding antigen from bacteria, virus, and cancer has shown promise in protective humoral and cellular immunity; however, the potential disadvantages of naked DNA vaccine have reduced the value of the approach. To optimize antigen delivery efficiency as well as vaccine efficacy, the non-viral vector as vaccine carrier, for example, the cationic liposome, has shown particular benefits to circumvent the obstacles that both peptide/protein- and gene-based vaccines have encountered. Liposome-mediated vaccine delivery provides greater efficacy and safer vaccine formulation for the development of vaccine for human use. The success of the liposome-based vaccine has been demonstrated in clinical trials and further human trials are also in progress.

Animals↗

PubMed2005

Lipid-protamine-DNA-mediated antigen delivery.

The development of novel 'new generation' vaccine systems that is based on proteins, peptides or DNA is of great current interest. However, due to the lower efficiencies of these new generation vaccines, they are seldomly used alone. Rather, their formulations often contain adjuvants, either to enhance the immune responses or to reduce dosing. The present chapter will provide a brief overview of the recent advances in peptide-based cancer vaccine adjuvants, focusing mainly on Liposome-Protamine-DNA (LPD) nanoparticle-mediated antigen delivery.

Adjuvants, Immunologic↗

PubMed2004

Anisamide-targeted stealth liposomes: a potent carrier for targeting doxorubicin to human prostate cancer cells.

Certain human malignancies including prostate cancer overexpress sigma receptor, a membrane bound protein that binds haloperidol and various other neuroleptics with high affinity. An anisamide derivatized ligand possesses high affinity for sigma receptors and we hypothesized that its incorporation into the liposomes encapsulating doxorubicin (DOX) can specifically target and deliver the drug to prostate cancer cells that overexpress sigma receptors. A polyethylene glycol phospholipid was derivatized with an anisamide ligand, which was then incorporated into the DOX-loaded liposome. The resulting anisamide-conjugated liposomal DOX showed significantly higher toxicity to DU-145 cells than non-targeted liposomal DOX, the IC50 being 1.8 microM and 14 microM respectively. The cytotoxicity of the targeted liposomal DOX, however, was significantly blocked by haloperidol, suggesting that the enhanced cytotoxicity was specifically mediated by the sigma receptors. Fluorescence imaging studies after intravenous (i.v.) administration showed that incorporation of anisamide into liposomes significantly improved their accumulation into the tumor. A weekly injection of the targeted liposomal DOX for 4 weeks at a dose of 7.5 mg/kg led to a significant growth inhibition of established DU-145 tumor in nude mice with minimal toxicity. Free DOX was effective, but associated with significant toxicities. The present study is the first to demonstrate the use of small molecular weight ligand for mediating efficient targeting of liposomal drugs to sigma receptor expressing prostate cancer cells both in vitro and in vivo.

Adenocarcinoma↗

PubMed2004

HIV-1 Tat protein transduction domain peptide facilitates gene transfer in combination with cationic liposomes.

The protein transduction domain (PTD) of the HIV-1 Tat protein can facilitate the cellular and nuclear uptake of macromolecular particles. Here, we demonstrate that incorporation without covalent linkage of a 17-amino acid PTD peptide into gene delivery lipoplexes improves gene transfer. Tat/Liposome/DNA (TLD) transfection, as evaluated by Fluorescence Activated Cell Scan analysis of a Green Fluorescence Protein expression plasmid, enabled transfection of highly recalcitrant primary cells in the form of air/liquid interface cultures of sheep tracheal epithelium. Treatment with chloroquine increased, and incubation at low temperature decreased, TLD transfection, suggesting that the endocytosis uptake pathway is involved.

Amino Acid Sequence↗

PubMed2004

Non-immunostimulatory nonviral vectors.

The vectors for gene delivery are usually classified as viral and nonviral vectors. While the viral vectors are very efficient in transducing cells, safety concerns regarding their use in humans make nonviral vectors an attractive alternative. Among the nonviral vectors, the lipoplexes (complexes of cationic liposome/pDNA) are the most studied and represent the most promising approaches for human clinical trials. However, an inflammatory response is invariably associated with administration of the lipoplexes, which must be avoided in the clinical application. Here, we have successfully developed a nonimmunostimulatory vector for gene therapy. The vector possesses dual functions of: 1) efficiently delivering a gene to target cells and 2) codelivering DNA and inflammatory suppressors into the immune cells where the released suppressor can inhibit cytokine production. The inflammatory suppressors successfully delivered by the vector included glucocorticoids, a nonsteroidal anti-inflammatory drug (NSAID), an NF-kappaB inhibitor, and a natural compound from an herbal medicine. Intravenous injection of the vector dramatically suppressed the cytokine production induced by CpG motif pDNA, including TNF-alpha, IL-12 and IFN-gamma. This new gene vector has a great potential in clinical gene therapy. Another potential use of the vector is codelivery of an enhancer candidate, acting at the transcriptional and translational levels to improve the efficiency of gene transfer by the nonviral vector. Moreover, the unique feature of this vector is that it can be used as an easy and powerful tool for in vivo screening of anti-inflammatory drugs.

Animals↗

PubMed2004

Sustained delivery and expression of plasmid DNA based on biodegradable polyester, poly(D,L-lactide-co-4-hydroxy-L-proline).

Gene expression mediated by a non-viral vector usually lasts only a few days. The objective of this study was to synthesize and characterize a non-toxic, polymeric gene carrier, poly(D,L-lactide-co-4-hydroxy-L-proline) (PLHP) for sustained gene delivery. The copolymer was synthesized by ring-opening polymerization of D,L-lactide (DLLA) with N-cbz-4-hydroxy-L-proline (HP) in the presence of stannous octoate (Sn(Oct)(2)). The resulting copolymer was characterized by (1)H nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). Degradation of PLHP was examined by monitoring the medium pH change and molecular weight (MW) of the remaining polymer. It showed a rapid initial degradation and followed by a slower degradation for about 30 days at 37 degrees C. The cytotoxicity of copolymer was significantly lower than polyethylenimine (PEI) and poly-L-lysine hydrochloride (PLL) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The plasmid DNA (pDNA)-loaded microspheres based on the copolymer were prepared by a water-oil-water (w/o/w) solvent evaporation emulsion method. The release profile of pDNA from PLHP microspheres showed an initial burst release, and then a slower and continuous release for about 18 days at 37 degrees C. Gene transfer efficiency of PLHP/pDNA delivery system showed a sustained activity (over a week) when compared with PEI and PLL, and can be further improved by the addition of cationic liposomes. The results suggest that PLHP is a promising candidate for long-term gene delivery with good biocompatibility and biodegradability.

Cell Survival↗

PubMed2004

Mechanism of liver gene transfer by mechanical massage.

Many metabolic diseases are caused by defects in the metabolic pathways in the liver. Others result from the absence of specific proteins normally produced and secreted by the liver. Because these metabolic disorders are usually caused by single gene defect, they are ideal candidates for gene therapy. We have previously shown that mouse liver can be transfected by mechanically massaging the liver (MML) after intravenous injection of naked plasmid DNA. We now show a significant linear relationship between the level of liver gene expression and the venous blood pressure, supporting the idea that gene transfer by MML is due, at least in part, to pressure-mediated effect. Liver transfection could not be blocked by co-injection of excess irrelevant DNA or poly I, suggesting that there is no involvement of receptors, including the scavenger receptor, in MML. Moreover, the level of gene expression could be further enhanced by a combination of MML and an increase in DNA retention-time in the liver. Persistence of gene expression could be significantly improved using an EBV-based plasmid vector. Our data suggest the mechanical massage produces transient membrane defects through which naked DNA can enter into the liver cells by simple diffusion.

Animals↗