Leaf Huang

Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes

Incorporation of dioleoyl N‐(monomethoxy polyethyleneglycol succinyl)phosphotidylethanolamine (PEG‐PE) into large unilamellar liposomes composed of egg posphatidylcholine:cholesterol (1:1) does not significantly increase the content leakage when the liposomes are exposed to 90% human serum at 37°C, yet the liposomes show a significant increase in the blood circulation half‐life (t = 5 h) as compared to those without PEG‐PE(t <30 min). The PEG‐PEs activity to prolong the circulation time of liposomes is greater than that of the ganglioside GM1, awell‐described glycolipid with this activity. Another amphipathic PEG derivative, PEG stearate, also prolongs the liposome circulation time, although its activity is less than that ofGM1. Amphipathic PEGs may be useful for the sustained release and the targeted drug delivery by liposomes.

Pharmacokinetics and Biodistribution of Nanoparticles

Nanoparticles show their promise for improving the efficacy of drugs with a narrow therapeutic window or low bioavailability, such as anticancer drugs and nucleic acid-based drugs. The pharmacokinetics (PK) and tissue distribution of the nanoparticles largely define their therapeutic effect and toxicity. Chemical and physical properties of the nanoparticles, including size, surface charge, and surface chemistry, are important factors that determine their PK and biodistribution. The intracellular fate of the nanoparticles after cellular internalization that affects the drug bioavailability is also discussed. Strategies for overcoming barriers for intracellular delivery and drug release are presented. Finally, future directions for improving the PK of nanoparticles and perspectives in the field are discussed.

Gene Therapy Progress and Prospects: Nonviral vectors

The success of gene therapy is largely dependent on the development of the gene delivery vector. Recently, gene transfection into target cells using naked DNA, which is a simple and safe approach, has been improved by combining several physical techniques, for example, electroporation, gene gun, ultrasound and hydrodynamic pressure. Chemical approaches have been utilized to improve the efficiency and cell specificity of gene transfer. Novel gene carrier molecules, which facilitate DNA escape from the endosome into the cytosol, have been developed. Several functional polymers, which enable controlled release of DNA in response to an environmental change, have also been reported. Plasmids with reduced number of CpG motifs, the use of PCR fragments and the sequential injection method have been established for the reduction of immune response triggered by plasmid DNA. Construction of a long-lasting gene expression system is also an important theme for nonviral gene therapy. To date, tissue-specific expression, self-replicating and integrating plasmid systems have been reported. Improvement of delivery methods together with intelligent design of the DNA itself has brought about large degrees of enhancement in the efficiency, specificity and temporal control of nonviral vectors.

Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis

We report the results of a double-blind, placebo-controlled trial in nine cystic fibrosis (CF) subjects receiving cationic liposome complexed with a complementary DNA encoding the CF transmembrane conductance regulator (CFTR), and six CF subjects receiving only liposome to the nasal epithelium. No adverse clinical effects were seen and nasal biopsies showed no histological or immuno-histological changes. A partial restoration of the deficit between CF and non-CF subjects of 20% was seen for the response to low Cl− perfusion following CFTR cDNA administration. This was maximal around day three and had reverted to pretreatment values by day seven. In some cases the response to low Cl− was within the range for non-CF subjects. Plasmid DNA and transgene-derived RNA were detected in the majority of treated subjects. Although these data are encouraging, it is likely that transfection efficiency and the duration of expression will need to be increased for therapeutic benefit.

Nonviral Vectors in the New Millennium: Delivery Barriers in Gene Transfer

Development of an efficient method for introducing a therapeutic gene into target cells in vivo is the key issue in treating genetic and acquired diseases by gene therapy. To this end, various nonviral vectors have been designed and developed, and some of them are in clinical trials. The simplest approach is naked DNA injection into local tissues or systemic circulation. Physical (gene gun, electroporation) and chemical (cationic lipid or polymer) approaches have also been utilized to improve the efficiency and target cell specificity of gene transfer by plasmid DNA. After administration, however, nonviral vectors encounter many hurdles that result in diminished gene transfer in target cells. Cationic vectors sometimes attract serum proteins and blood cells when entering into blood circulation, which results in dynamic changes in their physicochemical properties. To reach target cells, nonviral vectors should pass through the capillaries, avoid recognition by mononuclear phagocytes, emerge from the blood vessels to the interstitium, and bind to the surface of the target cells. They then need to be internalized, escape from endosomes, and then find a way to the nucleus, avoiding cytoplasmic degradation. Successful clinical applications of nonviral vectors will rely on a better understanding of barriers in gene transfer and development of vectors that can overcome these barriers.

DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action

The ability of a polycationic lipid, lipopoly(L-lysine) (LPLL), to mediate efficient DNA transfection depended on scraping of the treated cells (Zhou et al. (1991) Biochim. Biophys. Acta 1065, 8-14). It was found that the mechanical treatment could be avoided by including a helper lipid to the liposome composition. Among the helper lipids tested, a hexagonal phase forming lipid, dioleoylphosphatidylethanolamine (DOPE), gave rise to the highest activity. The transfection efficiency was further optimized by varying the lipophilicity of the LPLL and the ratio of the cationic liposome to DNA. Transfection activity of the optimal DNA-liposome complexes was enhanced by up to 6-fold if cells were pretreated with agents interfering with the process of endocytosis. Meanwhile, pretreatment of cells with a peptide which inhibits membrane fusion decreased the activity by about 60%. These results indicated that DNA-liposome complexes are taken up by an endocytosis mechanism and that cytoplasmic delivery of DNA involves a fusion-related event probably in the endosome compartment. The transfection process was visualized by thin-section electron microscopy. It was found that the complexes entered the cytoplasm mainly by destabilizing endosomes and occasionally by penetrating through the plasma membrane. Therefore, our findings differ from a previous hypothesis which suggests that transfection is mediated by fusion of the liposomes with the plasma membrane of the treated cells.

Activity of amphipathic poly(ethylene glycol) 5000 to prolong the circulation time of liposomes depends on the liposome size and is unfavorable for immunoliposome binding to target

Dioleoyl-N-(monomethoxy polyethyleneglycol succinyl)-phosphatidylethanolamine (PEG-PE) (mol. wt. of PEG = 5000), an amphipathic polymer, can be incorporated into the liposome membrane and significantly prolong the blood circulation time of the liposome. As little as 3.7 mol% of PEG-PE in liposome resulted in maximal enhancement of liposome circulation time. However, this activity of PEG-PE was only seen with relatively small liposomes (d less than or equal to 200 nm); larger liposomes containing PEG-PE showed an unusually high level (approx. 35% injected dose) of accumulation in the spleen. We have tested whether the small, PEG-PE containing liposomes are suitable for immuno targeting by incorporating a lung-specific monoclonal antibody on the liposome surface. While another amphiphile, ganglioside GM1, which is well known for its activity to prolong the liposome circulation time, significantly enhanced the lung binding of the immunoliposomes, PEG-PE incorporation of immunoliposomes resulted in a low level of target binding. To test if the reduced target binding is due to a steric barrier effect of the surface PEG polymer, we have incorporated a small amount of N-biotinaminocaproylphosphatidylethanolamine into the PEG-PE containing liposomes and examined the liposome agglutination induced by the addition of streptavidin. As little as 0.72 mol% PEG-PE in these liposomes completely abolished agglutination. In contrast, incorporation of GM1 in liposomes only reduced the rate, but not the extent, of liposome agglutination. These results strongly support the hypothesis that PEG-PE prolongs liposome circulation time by providing a strong steric barrier which prevents close contact with another liposome or cell. Since GM1 provides only a weak steric barrier effect, its activity to prolong the liposome circulation time must involve another yet unknown mechanism.

In vivo gene transfer via intravenous administration of cationic lipid–protamine–DNA (LPD) complexes

A novel LPD formulation has been developed for in vivo gene transfer. It involves the interaction of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complexes, LPD offers better protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. When a luciferase reporter gene was employed, gene expression was found in all tissues examined including lung, heart, spleen, liver and kidney with the highest expression in the lung. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 μg per mouse at which concentration approximately 20 ng luciferase protein per milligram extracted tissue protein could be detected in the lung. Increasing the DNA to 100 μg per mouse resulted in toxicity and death of the animal. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h and declined thereafter. High expression was also found in the spleen 6 h after injection but dropped very rapidly thereafter. The in vivo gene expression by LPD was dependent upon the route of administration since intraportal injection of LPD led to about a 100-fold decrease in gene expression in the lung as compared with i.v. injection. Using lacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both the lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be nontoxic and only weakly immunogenic in humans, this novel vector may be useful for clinical gene therapy.

New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy

Structures formed during interaction of cationic liposomes and plasmid DNA were studied by freeze—fracture electron microscopy and their morphology was found to be dependent on incubation time and DNA concentration. These structures were formed with liposomes composed of DC‐Chol and DOPE after 30 min incubation at DNA: lipid concentrations encompassing maximal transfection activity. They resembled liposome complexes (meatballs) and additionally bilayer‐covered DNA tubules (spaghetti), whereby the DNA‐tubules were found to be connected to the liposome complexes as well as occurring free in the suspension. At later times and higher DNA‐to‐liposome ratios the complexes grow larger while their membranes become discontinuous, allowing the self‐encapsulation of the DNA. The relative transfection potency of the various morphologically distinct structures is discussed.

Protamine sulfate enhances lipid-mediated gene transfer

A polycationic peptide, protamine sulfate, USP, has been shown to be able to condense plasmid DNA efficiently for delivery into several different types of cells in vitro by several different types of cationic liposomes. The monovalent cationic liposomal formulations (DC-Chol and lipofectin) exhibited increased transfection activities comparable to that seen with the multivalent cationic liposome formulation, lipofectamine. This suggests that lipofectamine’s superior in vitro activity arises from its ability to condense DNA efficiently and that protamine’s primary role is that of a condensation agent, although it also possesses several amino acid sequences resembling that of a nuclear localization signal. While the use of polycations to condense DNA has been previously reported, the use of protamine sulfate, USP as a condensation agent was found to be superior to poly- L-lysine as well as to various other types of protamine. These differences among various salt forms of protamine appear to be attributable to structural differences between the protamines and not due to differences in the net charge of the molecule. The appearance of lysine residues within the protamine molecule correlate with a reduction in binding affinity to plasmid DNA, as well as an observed loss in transfection-enhancing activity. This finding sheds light on the structural requirements of condensation agents for use in gene transfer protocols. Furthermore, protamine sulfate, USP is an FDA-approved compound with a documented safety profile and could be readily used as an adjuvant to a human gene therapy protocol.