ABSTRACT
The pig was introduced more than 20 years ago in drug development following attempts of finding a species that shares better homology with human than the dog, based on biophysiological parameters. However, miniaturization, standardized breeding, and health status control were required before the pig could find a broader than niche application in pharmaceutical industry. During the years of experience with minipigs in pharmaceutical research and the science evolving rapidly, the selection of a nonrodent animal species for preclinical safety testing became primarily driven by pharmacological (target expression homologous function), pharmacokinetic, and biophysiological considerations. This offered a broad field of application for the minipig, besides the well-established use in dermal projects in all areas of drug development but also in novel approaches including genetically modified animals. In this article, we look at recent approaches and requirements in the optimal selection of a nonrodent model in pharmaceutical development and critically ask how good a choice the minipig offers for the scientist, how did the testing environment evolve, and what are the key requirements for a broader use of the minipig compared to the other well-established nonrodent species like dog or monkey.
Subject(s)
Biomedical Research/methods , Drug Discovery , Drug Evaluation, Preclinical , Swine, Miniature , Animals , Drug Discovery/methods , Drug Discovery/standards , Drug Evaluation, Preclinical/methods , Drug Evaluation, Preclinical/standards , Models, Animal , SwineABSTRACT
The synthesis and biophysical characterization of four prodrug ether phospholipid conjugates are described. The lipids are prepared from the anticancer drug chlorambucil and have C16 and C18 ether chains with phosphatidylcholine or phosphatidylglycerol headgroups. All four prodrugs have the ability to form unilamellar liposomes (86-125 nm) and are hydrolyzed by phospholipase A(2), resulting in chlorambucil release. Liposomal formulations of prodrug lipids displayed cytotoxicity toward HT-29, MT-3, and ES-2 cancer cell lines in the presence of phospholipase A(2), with IC(50) values in the 8-36 microM range.
Subject(s)
Antineoplastic Agents, Alkylating/chemical synthesis , Chlorambucil/analogs & derivatives , Prodrugs/chemical synthesis , Antineoplastic Agents, Alkylating/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Chlorambucil/chemical synthesis , Chlorambucil/pharmacology , Drug Screening Assays, Antitumor , Humans , Hydrolysis , Inhibitory Concentration 50 , Liposomes , Phospholipases A2/metabolism , Phospholipid Ethers/chemical synthesis , Prodrugs/metabolismABSTRACT
Combining supramolecular self-assembly of lipids with enzymatic triggered DNA interfacial polymerization allows construction of composite nanocapsules. Covalent grafting of oligonucleotides functionalizes the surface of liposomes. Subsequent addition of an enzyme called terminal deoxynucleotidyl transferase elongates the single-stranded DNA. The elongated DNA hybridizes, creating a random network. The short segments of double-stranded DNA provides a substrate for the Klenow fragment of E. coli DNA polymerase, which synthesizes a double-strand DNA, reinforcing the network. Alternate action of both enzymes leads to a three-dimensional network anchored on the liposome surface.
Subject(s)
Cross-Linking Reagents/chemistry , DNA Nucleotidylexotransferase/chemistry , DNA Polymerase I/chemistry , DNA/chemistry , Liposomes/chemistry , Amides/chemistry , Diglycerides/chemistry , Fluorescent Dyes , Maleimides/chemistry , Microscopy, Confocal , Oligonucleotides/chemistry , Phosphatidylcholines/chemistryABSTRACT
Conversion of a DNA chip to a nanocapsule array was performed by grafting on a liposome an oligonucleotide complementary to an oligonucleotide bound to the array. Each liposome may be loaded by a soluble molecule or may present a hydrophobic or amphiphilic molecule inserted in its wall. To detect liposomes on the chip, we used fluorescent dyes encapsulated in the liposome internal volume or fluorescent lipids. We observed that an oligonucleotide-grafted liposome containing a defined dye specifically accumulated on the area where its complementary oligonucleotide had been spotted on the array. The virtually unlimited amount of addresses allows the specific binding of large amounts of liposomes in one single batch.
Subject(s)
Liposomes/chemistry , Oligonucleotides/chemistry , Nanotechnology , Oligonucleotide Array Sequence AnalysisABSTRACT
BACKGROUND: Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void. RESULTS: Here we show that intact liposomes and their contents are retained in the exclusion gel. Retention depends on the pore size, the smaller the pores, the higher the retention. Retained liposomes are not tightly fixed to the beads and are slowly released from the gels upon direct or inverted eluent flow, long washing steps or column repacking. Further addition of free liposomes leads to the elution of part of the gel-trapped liposomes, showing that the retention is transitory. Trapping reversibility should be related to a mechanism of partitioning of the liposomes between the stationary phase, water-swelled polymeric gel, and the mobile aqueous phase. CONCLUSION: Retention of liposomes by size exclusion gels is a dynamic and reversible process, which should be accounted for to control lipid loss and sample contamination during chromatography.
Subject(s)
Biotechnology/methods , Chromatography/methods , Liposomes/chemistry , Acetylcholinesterase/chemistry , Animals , Chemistry, Physical/methods , Chromatography, Gel/methods , Drosophila melanogaster/enzymology , Drug Carriers/chemistry , Fluoresceins/chemistry , Gels/chemistry , Kinetics , Lipids/chemistry , Membranes, Artificial , Microscopy, Electron, Scanning , Microscopy, Fluorescence , PermeabilityABSTRACT
Amphiphilic ABA triblock copolymers, such as poly(2-methyloxazoline)-block-poly(dimethylsiloxan)-block-poly(2-methyloxazoline) (PMOXA-PDMS-PMOXA), form vesicular structures. Here, the interaction of these ABA molecules with lipids is investigated by electron microscopy, fluorescence spectroscopy, light scattering, and differential scanning calorimetry. Our observations suggest the formation of homogeneous mixed polymer-lipid composites, independent of preparation method, i.e. film hydration, dispersion, or detergent removal. When ABA polymersomes and liposomes are mixed, we observed monomer exchanges on a time scale of minutes. The possibility of forming mixed structures and the exchanges between preformed structures allow the combination of the properties of lipids and polymers such as stability and loading encapsulation capacity.
Subject(s)
Dimethylpolysiloxanes/chemistry , Liposomes/chemistry , Nanostructures/chemistry , Oxazoles/chemistry , Polymers/chemistry , Calorimetry, Differential Scanning , Fluorescence Resonance Energy Transfer , Light , Lipids/chemistry , Microscopy, Electron, Transmission , Scattering, RadiationABSTRACT
Here we present three different types of mechanically stable nanometer-sized hollow capsules. The common point of the currently developed systems in our laboratory is that they are liposome based. Biomolecules can be used to functionalize lipid vesicles to create a new type of intelligent material. For example, insertion of membrane channels into the capsule wall can modify the permeability. Covalent binding of antibodies allows targeting of the capsule to specific sites. Liposomes loaded with enzymes may provide an optimal environment for them with respect to the maximal turnover and may stabilize the enzyme. However, the main drawback of liposomes is their instability in biological media as well as their sensitivity to many external parameters such as temperature or osmotic pressure. To increase their stability we follow different strategies: 1) polymerize a two-dimensional network in the hydrophobic core of the membrane; 2) coat the liposome with a polyelectrolyte shell; or 3) add surface active polymers to form mixed vesicular structures.
