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Biomedical subjects

K Y Kwok

Publications and source records attributed to K Y Kwok.

12 recordsLinked to original sources
PubMed2001

Evolution of cross-linked non-viral gene delivery systems.

Developing a non-viral gene delivery system that functions in vivo raises the challenge of finding solutions to efficiently deliver DNA to the cell surface that are also compatible with the efficient release of DNA into the cytosol. The stability, particle size and charge of DNA polyplexes and lipoplexes may be optimized to mediate efficient in vitro transfection only to find that different properties are necessary for successful in vivo transfection. Despite their versatility and improved safety, non-viral gene vectors still lack appreciable in vivo transfection efficiency compared to viral vectors. An emerging theme in recent studies is the use of cross-linking to achieve balance between the stability of polyplexes and lipoplexes in the blood and the controlled release of DNA in the cytosol. This review evaluates the evolution of cross-linking strategies aimed at transiently stabilizing non-viral gene delivery systems.

Cross-Linking Reagents↗
PubMed2000

Strategies for maintaining the particle size of peptide DNA condensates following freeze-drying.

The particle size of peptide DNA condensates were studied after freeze-drying and rehydration as a function of sugar excipient, concentration, pH, DNA concentration, and peptide condensing agent. In the absence of an excipient, freeze-dried 50 microg/ml AlkCWK(18) (iodoacetic acid alkylated Cys-Typ-Lys(18)) DNA condensates formed large fibrous flocculates on rehydration. Of the sugars tested as lyoprotectants, sucrose proved most effective at preserving particle size during rehydration. The addition of 5 wt/vol% sucrose preserved a mean particle diameter of less than 50 nm during rehydration of AlkCWK(18) DNA condensates prepared at DNA concentrations up to 200 microg/ml; however, higher DNA concentrations led to the formation of insoluble fibrous flocculates. Substitution of polyethylene glycol (PEG)-CWK(18) as a DNA condensing peptide eliminated the need for sucrose, resulting in peptide DNA condensates that retained particle size when rehydrated in water or normal saline at concentrations up to 5 mg/ml. The results suggest that sucrose functions primarily as a bulking agent during freeze-drying that only preserves the particle size of AlkCWK(18) DNA condensates up to a maximum concentration of 200 microg/ml. Alternatively, the steric layer created on the surface of PEG-CWK(18) DNA condensates provides far more efficient lyoprotection, preserving their particle size at a concentration of 5 mg/ml without a bulking agent.

DNA↗
PubMed2000

A potent new class of reductively activated peptide gene delivery agents.

A new class of peptide gene delivery agents were developed by inserting multiple cysteine residues into short (dp 20) synthetic peptides. Substitution of one to four cysteine residues for lysine residues in Cys-Trp-Lys(18) resulted in low molecular weight DNA condensing peptides that spontaneously oxidize after binding to plasmid DNA to form interpeptide disulfide bonds. The stability of cross-linked peptide DNA condensates increased in proportion to the number of cysteines incorporated into the peptide. Disulfide bond formation led to a decrease in particle size relative to control peptide DNA condensates and prevented dissociation of peptide DNA condensates in concentrated sodium chloride. Cross-linked peptide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS 7 cells relative to uncross-linked peptide DNA condensates. The enhanced gene expression was dependent on the number of cysteine residues incorporated, with a peptide containing two cysteines mediating maximal gene expression. Cross-linking peptides caused elevated gene expression without increasing DNA uptake by cells, suggesting a mechanism involving intracellular release of DNA triggered by disulfide bond reduction. The results establish cross-linking peptides as a novel class of potent gene delivery agents that enhance gene expression through a new mechanism of action.

Amino Acid Sequence↗
PubMed2000

Biodistribution, metabolism, and in vivo gene expression of low molecular weight glycopeptide polyethylene glycol peptide DNA co-condensates.

The biodistribution, metabolism, cellular targeting, and gene expression of a nonviral peptide DNA gene delivery system was examined. (125)I-labeled plasmid DNA was condensed with low molecular weight peptide conjugates and dosed i.v. in mice to determine the influence of peptide DNA formulation parameters on specific gene targeting to hepatocytes. Optimal targeting to hepatocytes required the combined use of a triantennary glycopeptide (Tri-CWK(18)) and a polyethylene glycol-peptide (PEG-CWK(18)) to mediate specific recognition by the asialoglycoprotein receptor and to reduce nonspecific uptake by Kupffer cells. Tri-CWK(18)/PEG-CWK(18) DNA co-condensates were stabilized and protected from metabolism by glutaraldehyde crosslinking. An optimized formulation targeted 60% of the dose to the liver with 80% of the liver targeted DNA localized to hepatocytes. Glutaraldehyde crosslinking of DNA condensates reduced the liver elimination rate from a t((1/2)) of 0.8 to 3.6 h. An optimized gene delivery formulation produced detectable levels of human alpha1-antitrypsin in mouse serum which peaked at day 7 compared to no expression using control formulations. The results demonstrate the application of formulation optimization to improve the targeting selectivity and gene expression of a peptide DNA delivery system.

Animals↗
PubMed1999

Formulation of highly soluble poly(ethylene glycol)-peptide DNA condensates.

Two poly(ethylene glycol) (PEG)-peptides were synthesized and tested for their ability to bind to plasmid DNA and form soluble DNA condensates with reduced spontaneous gene expression. PEG-vinyl sulfone or PEG-orthopyridyl disulfide were reacted with the sulfhydryl of Cys-Trp-Lys(18) (CWK(18)) resulting in the formation of nonreducible (PEG-VS-CWK(18)) and reducible (PEG-SS-CWK(18)) PEG-peptides. Both PEG-peptides were prepared on a micromole scale, purified by RP-HPLC in >80% yield, and characterized by (1)H NMR and MALDI-TOF. PEG-peptides bound to plasmid DNA with an apparent affinity that was equivalent to alkylated (Alk)CWK(18), resulting in DNA condensates with a mean diameter of 80-90 nm and zeta (zeta) potential of +10 mV. The particle size of PEG-peptide DNA condensates was constant throughout the DNA concentration range of 0. 05-2 mg/mL, indicating these to be approximately 20-fold more soluble than AlkCWK(18) DNA condensates. The spontaneous gene transfer to HepG2 cells mediated by PEG-VS-CWK(18) DNA condensates was over two orders of magnitude lower than PEG-SS-CWK(18) DNA condensates and three orders of magnitude lower than AlkCWK(18) DNA condensates. PEG-VS-CWK(18) efficiently blocked in vitro gene transfer by reducing cell uptake. The results indicate that a high loading density of PEG on DNA is necessary to achieve highly soluble DNA condensates that reduce spontaneous in vitro gene transfer by blocking nonspecific uptake by HepG2 cells. These two properties are important for developing targeted gene delivery systems to be used in vivo.

Cell Line↗
PubMed1998

Stability of peptide-condensed plasmid DNA formulations.

Low molecular weight homogeneous peptides were used to form peptide/DNA condensates. A peptide possessing 18 lysines was found to protect plasmid DNA from serum endonuclease and sonicative-induced degradation whereas a shorter peptide possessing 8 lysines dissociated in 0.1 M sodium chloride and failed to protect DNA from enzymatic degradation. Peptide-condensed DNA showed no change in the ratio of supercoiled to circular DNA following 100 W sonication for up to 60 s and was able to transfect HepG2 cells with equivalent efficiency as untreated condensed plasmid DNA. Alternatively, uncondensed plasmid DNA was rapidly fragmented by sonication and serum endonucleases and resulted in negligible gene expression following condensation with peptide. Cationic lipid/DNA complexes were only partially effective at stabilizing DNA in serum compared to the complete stabilization afforded by peptide/DNA condensation. These results indicate that the stabilization afforded by condensation with a peptide protects DNA during formulation and preserves its structure in serum. These functions are important to achieve optimal gene expression from a nonviral gene delivery system.

Animals↗
PubMed1986

A prospective study of bladder function following aortic surgery.

Twenty-four patients undergoing aortic surgery for aneurysmal or occlusive aortic disease were studied prospectively to assess the effect on bladder function. Thirteen patients completed the study with post-operative follow-up at 3 months. Aortic surgery was found to be unlikely to cause damage to the parasympathetic nerves. There was no change in the detrusor stability/instability status in five of seven patients undergoing surgery for occlusive disease. Fifty-seven per cent of patients undergoing surgery for aneurysmal disease and 83% of those undergoing surgery for occlusive disease noted improved urinary flow with decreased maximal urethral pressure following surgery, suggesting damage to the sympathetic nerve supply to the lower urinary tract. The improvement in urinary flow did not relate either to the assessed extent of damage to the sympathetic nerves at operation or to the type of operation or vascular anastomosis. It is concluded that aortic surgery damages the sympathetic rather than the parasympathetic nerves and that any effect on bladder function is of no serious significance.

Aged↗
PubMed1981

Group-specific component (Gc) subtypes in the Chinese population of Hong Kong.

The distribution of Gc subtypes in a sample of the Chinese population of Hong Kong was studied using isoelectric focusing followed by immunofixation. A sensitive modification of this technique is described. Nine distinct phenotypes were observed which appear to result from the three common alleles Gc1F, Gc1S, and Gc2, which are found in most populations. The respective gene frequencies were 0.494, 0.258, and 0.247. In addition, two rare phenotypes were observed which appear to be due to a rare allele tentatively identified as Gc2C2.

Adult↗
PubMedDate not supplied

Low molecular weight disulfide cross-linking peptides as nonviral gene delivery carriers.

Cross-linking peptides have been developed by inserting multiple Cys residues into a 20 amino acid condensing peptide that polymerizes through disulfide bond formation when bound to DNA resulting in small, highly stable DNA condensates that mediate efficient in vitro gene transfer [McKenzie et al. (2000) J. Biol. Chem. 275, 9970-9977]. In the present study, a minimal peptide of four Lys and two terminal Cys residues was found to substitute for Cys-Trp-(Lys)(17)-Cys, resulting in DNA condensates with similar particle size and gene expression in HepG2 cells. Substitution of His for Lys residues resulted in an optimal peptide of Cys-His-(Lys)(6)-His-Cys that, in addition to the attributes described above, also provided buffering capacity to enhance in vitro gene expression in the absence of chloroquine. The reported structure-activity relationships systematically explore peptides with combinations of Lys, Cys, and His residues resulting in low molecular weight peptides with improved gene transfer properties.

Amino Acid Sequence↗