The objective of this work was to obtain gene delivery vectors with high efficiency induced by application of local hyperthermia. As a building construct for the polyplex particles, block copolymers ...were used, in which one block represents poly(ethyleneimine) (PEI) and another block a statistical copolymer of poly(N-isopropylacryamide) (PNIPAM) and different hydrophilic monomers (acrylamide or vinylpyrrolidinone). The block copolymers were synthesizized by radical polymerization of the corresponding monomers directly onto PEI. The complexation of DNA with these copolymers led to small, charge neutral particles, which aggregated upon increasing the temperature from 37 °C to 42 °C. This aggregation was found to be responsible for the enhanced transfection efficiency of these formulations under hyperthermic conditions. Gene expression in cells treated by hyperthermia was found to be nearly 2 orders of magnitude higher in comparison to cells transfected at physiological temperature. The mechanism by which hyperthermia influences the gene transfection efficiency is proposed.
Search for neutral MSSM Higgs bosons at LEP Casado, M.P.; Ouyang, Q.; Xu, R. ...
The European physical journal. C, Particles and fields,
09/2006, Letnik:
47, Številka:
3
Journal Article
Recenzirano
Odprti dostop
The four LEP collaborations, ALEPH, DELPHI, L3 and OPAL, have searched for the neutral Higgs bosons which are predicted by the Minimal Supersymmetric standard model (MSSM). The data of the four ...collaborations are statistically combined and examined for their consistency with the background hypothesis and with a possible Higgs boson signal. The combined LEP data show no significant excess of events which would indicate the production of Higgs bosons. The search results are used to set upper bounds on the cross-sections of various Higgs-like event topologies. The results are interpreted within the MSSM in a number of “benchmark” models, including CP-conserving and CP-violating scenarios. These interpretations lead in all cases to large exclusions in the MSSM parameter space. Absolute limits are set on the parameter cosβ and, in some scenarios, on the masses of neutral Higgs bosons.
The dissolution of highly aggregated polyelectrolyte complex particles formed in water after addition of salt was studied. The dissolution of aggregates proceeded to soluble complexes on the ...molecular level of the long-chain component. The driving force of the process is the polyelectrolyte exchange reaction between the aggregates and the free long chains in excess. The kinetics of the process was studied by different light scattering techniques. The rate of dissolution showed a strong dependence on the salt concentration in the solution and on the concentration of the species. The dependence on concentration of the species in solution weakened with increasing salt concentration. Investigations of the structural changes during the dissolution process revealed the presence of only two generations of particles in solution: aggregates and soluble complexes. While the scattering intensity decreased strongly, the dimensions of the aggregates changed only slightly during dissolution, indicating a spontaneous disaggregation of the particles. A mechanism of the dissolution process was proposed, which is in agreement with the experimental findings and previous results in the literature. The process represents a two-step reaction: The first step consists of the release of the short-chain component from the aggregates by an exchange reaction via the free long-chain component in solution (second-order reaction). The second step is the destruction of the aggregates by increasing osmotic pressure in the particle (first-order reaction). The dissolution process may be understood as a model process for the release of DNA from polyelectrolyte complexes in gene therapy.
Locoregional hyperthermia (HT) can be used for site-directed activation of macromolecular drug delivery systems. We have developed a gene delivery system based on thermosensitive block copolymers ...(TSCs) with a phase transition temperature of 42 degrees C Zintchenko, A., Ogris, M., and Wagner, E. (2006). Bioconjug. Chem. 17, 766-772, in which the statistical copolymer of vinylpyrrolidinone and N-isopropylacryamide is grafted on polyethylenimine (PEI). Here we applied polyplexes consisting of plasmid DNA and TSCs systemically in A/J mice bearing a syngeneic Neuro2A neuroblastoma tumor subcutaneously in each hind limb. One limb was selectively treated by HT at 42 degrees C, at the same time that polyplexes were injected via the tail vein. Hyperthermia led to increased accumulation of thermosensitive polymer and aggregation of thermosensitive polyplexes in HT-treated tumors, resulting in up to 10-fold increased DNA deposition compared with non-HT-treated tumor. The level of transgene expression induced by TSC polyplexes in HT-treated tumors was significantly higher and selective for tumor tissue. With nonthermosensitive PEI polyplexes HT did not influence transgene deposition or expression in tumor.
Anionic and cationic block copolymers containing different fractions and chain lengths of poly(ethylene glycol) (PEG) blocks were synthesized and characterized. Polyelectrolyte complex (PEC) ...formation between these copolymers and homopolyelectrolytes and the stability of these complexes in salt solutions were studied by static and dynamic light scattering and laser Doppler electrophoresis. Complex formation led to particle systems with a high level of aggregation. For the anionic copolymers, even a low content of the short PEG block (M = 2 kDa) stabilized the PECs near the 1:1 mixing ratio. Independent of the content of the neutral block, the stability of complexes with short PEG blocks with regard to subsequent addition of salt was very low. When copolymers with long PEG blocks were used in PEC formation, high salt stability was observed.
The exchange of sodium polymethacrylate (Na‐PMA) for anionic surfactant sodium dodecylsulfate (SDS) in a polyelectrolyte complex of Na‐PMA and poly(L‐lysine) (PLL) was studied by light scattering ...techniques. The exchange reaction proceeds inside the complex particles without any aggregation. It causes swelling of the particles and the formation of thick polymer shell around them. In order to prove the formation of the shell, the colloidal stability of the particles in salt solutions was studied. The addition of SDS to a complex solution dramatically increases colloid stability. The stability was found to be independent of the mixing ratio of the initial complex, depending only on the molecular weight of Na‐PMA and slowly decreasing with time. The mechanism of the exchange reaction was proposed. The addition of SDS causes fast substitution of charged units of Na‐PMA for surfactant molecules in the complex and formation of the PLL/SDS complex stabilized by hydrophobic interactions between aliphatic chains of SDS. The Na‐PMA chains cannot be released sufficiently fast from the particles due to entanglements of the chains inside the particles. They build a thick stabilizing shell around the particles and lead to their swelling. This shell protects the particles against coagulation at relatively high salt concentrations. However, such state of the particles does not correspond to thermodynamic equilibrium. Na‐PMA is very slowly released from the particles, decreasing the density of stabilizing shell and stability of the particles in salt solutions. The possible application of ternary conjugates in drug delivery is discussed.
Schematic representation of changes in structure and stability of Na‐PMA/PLL complex particles upon the exchange of polyanion Na‐PMA for negatively charged surfactant SDS.
Self-assembling systems based on ionic complexes of oligonucleotides (36 base pairs) and model oligophosphates (polymerization degree of 35) with high molecular poly(l-lysine) molecules (M w = 134 ...000) grafted with short polyN-(2-hydroxypropyl)methacrylamide chains (M w = 7000) were studied as systems suitable for gene therapy applications. Poly(l-lysine) and poly(trimethylammonioethyl methacrylate chloride) homopolycations were used for comparison. The physicochemical properties of polyelectrolyte complexes (PEC) were examined by static and dynamic light scattering methods. While PECs prepared with homopolycations tend to aggregate, particularly at high degrees of charge conversions, the complexes prepared with graft copolymers are soluble at any charge conversions in aqueous solutions. The complexes prepared with an excess of oligophosphates were found to be stable in physiological salt conditions and in the bovine serum albumin solutions (1 mg/mL). A formation of PEC/albumin complexes and large aggregates was observed for uncompensated PECs with cationic excess.
The interaction between negatively charged lipid vesicles and positively charged DNA/polylysine complexes was studied. The interaction does not lead to release of DNA from the initial complexes. The ...particles formed are easy to prepare, they have slight negative charge, small dimensions and show good stability in physiological NaCl solution. Such properties might indicate that stabilization of the particles by lipid coating might be a potent strategy, alternative to PEGylation of DNA/polycation complexes.
Interaction of DNA/polycation complex particles with lipid vesicles.
Self‐assembling systems based on ionic complexes of DNA fragments (36 base pairs), bcl‐2 antisense oligonucleotides (octadecamer), oligophosphates (25 phosphate groups) or acrylic oligomers (18 ...groups of phosphonic acid) with poly(L‐lysine) (PLL) ($\overline M _{\rm w}$ = 130 000 and 88 000) grafted with short polyN‐(2‐hydroxypropyl)methacrylamide (PHPMA) chains ($\overline M _{\rm n}$ = 4 300 or 8 600) were studied by static and dynamic light scattering methods as systems suitable for gene therapy applications. The graft copolymers (GPLLs) with shorter PHPMA grafts ($\overline M _{\rm n}$ = 4 300) provide polyelectrolyte complexes (PECs) with smaller $\overline M _{\rm w}$ and RH than the corresponding GPLLs with longer grafts ($\overline M _{\rm n}$ = 8 600) and the same content of PLL. The lowest aggregation number of 2 was observed for PECs prepared from the GPLL with short grafts and 40 wt.‐% of PLL. The complexes of oligonucleotides and DNA fragments with GPLLs showed quite similar behavior to that with oligophosphates and acrylic oligomer. The complexes prepared from GPLLs containing 40 wt.‐% of PLL and at excess of oligophosphate were stable for at least 48 h under physiological conditions (0.15 M NaCl) and in bovine serum albumin solutions (1 mg · mL−1). Additionally, polyanion exchange reactions of the PECs in contact with poly(styrenesulfonate) and DNA were studied in 0.15 M NaCl solutions. The oligophosphates in complexes were at least partially substituted with high‐molecular‐weight polyanions. The structure of the initial PECs dominated the PEC structure after the exchange reaction.
The dependence of the molecular weight $\overline M _{\rm w}$ (a) and the hydrodynamic radius RH (b) of complexes of the oligophosphate (OPP) and four graft copolymers (GPLLi, i = 0–3) on the mixing ratio X.
Different lipase preparations were immobilized at various conditions in polyelectrolyte complexes (PEC) of Na-poly(styrene sulfonates) (as polyanions) and poly(diallyldimethylammonium chloride) (as ...polycation) with high retention of the enzyme activity. The relative activity of the immobilized enzymes strongly depends on the pH value during complex formation, the use of a buffer for the enzyme and polyelectrolyte solutions and the relative concentrations of the polyelectrolytes to lipase. The highest effective enzyme activity of about 62–68% in PEC (as difference between activity in PEC solution and in supernatant) was found for pH 9.0 and a high excess (100-fold) of the polyelectrolyte to lipase during complex formation. The use of a phosphate buffer as solvent for lipase and the polyelectrolytes resulted in activities much higher than obtained for the free lipases. Most likely, these effects are due to both (hydrophobic and electrostatic) interactions between the lipases and the polyelectrolytes.
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