Liposomes and Other Lipid-based Nanoparticles

Liposomal Gene Delivery

Negatively charged, or classical, liposomes have been used to deliver encapsulated drugs for some time and have also been used as vehicles for gene transfer into cells in culture. Problems with the efficiency of nucleic acid encapsulation, coupled with a requirement to separate the DNA-liposome complexes from "ghost" vesi-cles has lead to the development of positively charged liposomes. Cationic lipids are able to interact sponta-neously with negatively charged DNA to form clusters of aggregated vesicles along the nucleic acid. At a critical liposome density the DNA is condensed and becomes encapsulated within a lipid bilayer, although there is also some evidence that cationic liposomes do not actually encapsulate the DNA, but instead bind along the surface of the DNA, maintaining its original size and shape.
Cationic liposomes are also able to interact with negatively charged cell membranes more readily than classi-cal liposomes. Fusion between cationic vesicles and cell surfaces might result in delivery of the DNA directly across the plasma membrane. This process bypasses the endosomal-lysosomal route which leads to degra-dation of anionic liposome formulations. Cationic liposomes can be formed from a variety of cationic lipids, and they usually incorporate a neutral lipid such as DOPE (dioleoylphosphatidyl-ethanolamine) into the for-mulation in order to facilitate membrane fusion. A variety of cationic lipids have been developed to interact with DNA, but perhaps the best known are DOTAP (N-1(-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniumethyl sulphate) and DOTMA (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride). These are commercially available lipids that are sold as in vitro transfecting agents, with the latter sold as Lipofectin.
There have been several studies on the in vivo, systemic use of liposome/DNA complexes. The factors con-trolling the transfection efficiency of liposome/DNA complexes following intravenous administration are still poorly understood. Complexes formed between the cationic lipid DOTMA and DNA are rapidly cleared from the bloodstream and were found to be widely distributed in the body and expression was detected mainly in the lungs but also in the liver, spleen, heart and kidneys. Similar results were found when DOTAP-based liposomes were used and it was found that the main factors controlling transfection efficiency were the struc-ture of the cationic lipid and the ratio of the cationic lipid to DNA. The type of helper lipid used was also im-portant as the addition of DOPE was found to reduce the in vivo transfection efficiency of DOTAP/DNA com-plexes.

The transfection efficiency of liposome/DNA complexes in vivo has been shown to be relatively low, especially when compared to viral vectors. One study has suggested that the in vivo transfection efficiency of adenovi-ruses is around 200 times greater than that observed with liposomes. One explanation for the relatively poor transfection efficiency of liposome/DNA complexes is that they are susceptible to disruption by serum pro-teins. A variety of proteins are known to bind to liposomes in vitro and in vivo and may membrane destabili-sation. There are now serious efforts being made to develop liposomal vectors that are resistant to serum disruption. Novel cationic lipids are also being developed to try to improve the transfection efficiency of lipo-some/DNA complexes. Targeting of the liposomes to specific cell types has also been investigated as a means of improving the transfection efficiency.

There have been several clinical trials of liposome/DNA complexes, although almost all of these have been involved in the treatment of cystic fibrosis. Most protocols involve the use of DC-chol/DOPE liposomes di-rectly instilled onto the nasal epithelium of CF patients. The effect of gene expression on CFTR function was determined and the presence of the gene in the target cells was determined by PCR (polymerase chain reac-tion) and Southern blot analysis.
Initial clinical studies found no evidence of any safety problems with the use of liposome/DNA complexes. This is surprising as it is well documented that the liposome/DNA complexes used in clinical trials are directly cytotoxic in vitro. Furthermore, studies in mice and macaques have demonstrated that exposure to high doses or to repeat doses of liposome/DNA complexes results in histopathology and gross lung pathology, suggesting that these vectors may not be as clinically safe as previously thought.

http://www.gene-delivery.ox.ac.uk/Gene%20Therapy/Vectors/Synthetic%20Vectors/Liposomes/Liposomal%20Gene%20Delivery.htm



Liposome


Recent developments in nanotechnology have provided researchers with new tools for cancer imaging and treatment. This technology has enabled the development of nanoscale devices that can be conjugated with several functional molecules simultaneously, including tumor-specific ligands, antibodies, anticancer drugs, and imaging probes. Since these nanodevices are 100 to 1,000-fold smaller than cancer cells, they can be easily transferred through leaky blood vessels and interact with targeted tumor-specific proteins both on the surface of and inside cancer cells. Therefore, their application as cancer cell-specific delivery vehicles will be a significant addition to the currently available armory for cancer therapeutics and
imaging. Nanoparticles can consist of a number of materials, including polymers, metals, and ceramics. Based on their manufacturing methods and materials used, these particles can adopt diverse shapes and sizes with distinct properties. Many types of nanoparticles are under various stages of development as drug delivery systems, including liposomes and other lipid-based carriers (such as lipid emulsions and lipid-drug complexes), polymer-drug conjugates, polymer microspheres, micelles, and various ligand-targeted products (such as immunoconjugates).

Liposomes are self-assembling, spherical, closed colloidal structures composed of lipid bilayers that surround a central aqueous space. Liposomes are the most studied formulation of nanoparticle for drug delivery (Table 1). Several types of anticancer
drugs have been developed as lipid-based systems by using a variety of preparation methods. Liposomal formulations have shown an ability to improve the pharmacokinetics and pharmacodynamics of associated drugs.

Second Generation Liposomal Drugs....Surface-modified liposomes (Stealth) have hydrophilic carbohydrates or polymers, which usually are lipid derivatives of polyethylene glycol (PEG) grafted to the liposome surface. While this surface modification has solved the problem of fast clearance from the circulation, yielding liposomes with a significantly increased half-life in the blood, the challenge remains to attain preferential accumulation of liposomes in tumor tissues. One strategy to achieve tumor-specific targeting is to conjugate a targeting moiety on the outer surface of the lipid bilayer of the liposome that selectively delivers drug to the desired site of action............

2008;58:97–110; Application of Nanotechnology in Cancer Therapy and Imaging; American Cancer Society, Inc., 2008

Superior physiological performance through mild ketosis

During periods of stress, elevated catecholamines, steroids and cytokines increase the metabolism of stored fat in the body. The increase in circulating free fatty acids causes insulin resistance, decreases skeletal and cardiac muscular efficiency and may decrease metabolic fuel for the brain, which cannot metabolize fat, but can metabolize ketones. Ketone bodies contain more recoverable metabolic energy than fatty acids and yield 28% more energy on combustion than glucose. We are testing whether the negative effects of elevated free fatty acids can be overcome by mild ketosis. In collaboration with the National Institutes of Health in the US, we created a diet containing ketone bodies, which caused mild ketosis. We tested the metabolic mechanism underlying the effects of the ketone body diet during extreme exercise, with and without ketosis. Endurance and cognitive function, tested using treadmill exercise and a maze test, respectively, were found to be increased by the ketosis. We propose to further test the ketone diet before during and after 5 days of intense training, in a double-blind placebo-controlled cross-over trial. Exercise testing, cognitive function and skeletal and cardiac muscle energetics will be followed using psychological testing and non-invasive MRI of brain and muscle during exercise. Should subjects on the ketone body diet have greater metabolic efficiency, and therefore greater endurance and cognitive function, during extreme exercise and psychological stress than those on a normal diet, the diet could be used by athletes and to treat metabolic diseases, such as obesity, Alzheimer’s and Parkinson’s diseases.

Kieran Clarke: www.physiol.ox.ac.uk/Research_Groups/Cardiac_Metabolism/




Oxford BioSignals' are designed to automate the monitoring process for complex multiple signal systems. Visensia, a diagnosis technology, provides life-saving medical insight by analyzing five patient vital signs – heart rate, respiration rate, body temperature, oxygen saturation and blood pressure - and fuses this data into a measurable index. The first early warning system for patients created, which is used in Radcliff Hospital at Oxford, takes the vital sign information that is currently monitored such as heart rate, respiration rate and oxygen levels and defines them in a special way. It uses advance special software to look for small changes in several vital signs rather than a big change in just one vital sign. This can give doctors and nurses extra time to respond to an unwell patient. …….The technology has just been introduced on the A380, that’s the Airbus super jumbo, and that will be the first commercial airliner that will use the technology. Rolls Royce had first used the technology but this will be the first time this new generation of monitoring technology has been used on a commercial basis. http://www.oxford-biosignals.com/