Siddhesh D. Patil

DNA-based Therapeutics and DNA Delivery Systems: A Comprehensive Review

The past several years have witnessed the evolution of gene medicine from an experimental technology into a viable strategy for developing therapeutics for a wide range of human disorders. Numerous prototype DNA-based biopharmaceuticals can now control disease progression by induction and/or inhibition of genes. These potent therapeutics include plasmids containing transgenes, oligonucleotides, aptamers, ribozymes, DNAzymes, and small interfering RNAs. Although only 2 DNA-based pharmaceuticals (an antisense oligonucleotide formulation, Vitravene, (USA, 1998), and an adenoviral gene therapy treatment, Gendicine (China, 2003), have received approval from regulatory agencies; numerous candidates are in advanced stages of human clinical trials. Selection of drugs on the basis of DNA sequence and structure has a reduced potential for toxicity, should result in fewer side effects, and therefore should eventually yield safer drugs than those currently available. These predictions are based on the high selectivity and specificity of such molecules for recognition of their molecular targets. However, poor cellular uptake and rapid in vivo degradation of DNA-based therapeutics necessitate the use of delivery systems to facilitate cellular internalization and preserve their activity. This review discusses the basis of structural design, mode of action, and applications of DNA-based therapeutics. The mechanisms of cellular uptake and intracellular trafficking of DNA-based therapeutics are examined, and the constraints these transport processes impose on the choice of delivery systems are summarized. Finally, the development of some of the most promising currently available DNA delivery platforms is discussed, and the merits and drawbacks of each approach are evaluated.

Controlled release of dexamethasone from PLGA microspheres embedded within polyacid-containing PVA hydrogels

The development of zero-order release systems capable of delivering drug(s) over extended periods of time is deemed necessary for a variety of biomedical applications. We hereby describe a simple, yet versatile, delivery platform based on physically cross-linked poly(vinyl alcohol) (PVA) microgels (cross-linked via repetitive freeze/thaw cycling) containing entrapped dexamethasone-loaded poly(lacticco-glycolic acid) (PLGA) microspheres for controlled delivery over a 1-month period. The incorporation of polyacids, such as humic acids, Nafion, and poly(acrylic acid), was found to be crucial for attaining approximately zero-order release kinetics, releasing 60% to 75% of dexamethasone within 1 month. Microspheres alone entrapped in the PVA hydrogel resulted in negligible drug release during the 1-month period of investigation. On the basis of a comprehensive evaluation of the structure-property relationships of these hydrogel/microsphere composites, in conjunction with their in vitro release performance, it was concluded that these polyacids segregate on the PLGA microsphere surfaces and thereby result in localized acidity. These surface-associated polyacids appear to cause acid-assisted hydrolysis to occur from the surface inwards. Such systems show potential for a variety of localized controlled drug delivery applications such as coatings for implantable devices.

Anionic liposomal delivery system for DNA transfection.

The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17∶83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofect Amine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.

Recent progress in non-viral nucleic acids delivery.

A major goal of nucleic acid based therapies is to treat inherted and acquired disorders by adding, correcting, suppressing or eplacing genes (Abbas et al., 2008). Advantages of non-viral vectors or delivering nucleic acid based therapies include ease of scale-up, torage stability and improved quality control. The most promisng non-viral vectors have been liposomes and cationic polymers hich complex with nucleic acids such as siRNA and plasmid DNA o form lipoplexes or polyplexes (Wang et al., 2011). Advances in he development of this technology have included pegylation to ncrease circulation times and impart stealth-like properties to the ector and attachment of cell targeting or cell binding ligands to ncrease nucleic acid delivery efficiency. Other improvements have ncluded new formulation strategies to enhance protection of the ucleic acids against enzymatic degradation, to improve stability in he presence of serum and to optimize eventual release capabilities. n additional major goal of researchers has been to design non-viral ectors with desirable cytotoxicity and reduced immunogenicity haracteristics. In this special theme issue, we have assembled manuscripts rom an outstanding group of researchers that are leaders in the ffort to optimize non-viral vectors and overcome several chalenges and barriers to translating use of non-viral vectors into the linic. This theme issue includes comprehensive reviews by Amiji Xu et al., 2011), Torchilin (Wang et al., 2011) and Patil (Kapoor et al., 011) that present recent progress in non-condensing polymeric anoparticles, condensing cationic polymer based nanoplexes and he physico-chemical characterization of lipid based delivery sysems for siRNA. Original research articles in this theme issue focus n the development and testing of nucleic acid delivery systems. or example, Pun and colleagues discuss the development of a educible HPMA-co-oligolysine copolymer for nucleic acid delivery Shi et al., 2011). Mallapragada and colleagues discuss the develpment a temperature-responsive pentablock copolymer that is esigned to deliver DNA and prolong gene expression by forming thermogelling release depot after subcutaneous or intratumoral njection (Zhang et al., 2011). Kwon and colleagues present a iodegradable hybrid recombinant block copolymer for non-viral ene delivery that is capable of appreciable transfections with ow toxicity (Chen et al., 2011). Ghandehari and colleagues carry ut a comparative study between silk-elastin like protein polymer ydrogels and poloxamer gels for matrix-mediated viral gene delivry (Price et al., 2011). Berkland and colleagues demonstrate the tility of calcium condensed cell penetrating peptide (TAT) comlexes for efficient gene silencing with reduced toxicity (Baoum t al., 2011). Salem and colleagues describe the development of a annosylated pegylated polyethylenimine for siRNA delivery and he development of optimized dextran–polyethylenimine conjuates for plasmid DNA delivery (Jiang and Salem, 2011; Kim et al.,