Poly(β-amino ester)-DNA complexes: Time-resolved fluorescence and cellular transfection studies

A large number of different polymers have been developed and studied for application as DNA carriers for non-viral gene delivery, but the DNA binding properties are not understood. This study describes the efficiency of nanoparticle formation by time-resolved fluorescence measurements for poly(β-amino esters), cationic biodegradable polymers with DNA complexation and transfection capability. From the large library of poly(β-amino esters) ten polymers with different transfection efficacies were chosen for this study. The binding constants for nanoparticle formation were determined and compared to with the same method. Although the DNA binding efficiency of the amine groups are similar for both types of polymers, the overall binding constants are an order of magnitude smaller for poly(β-amino esters) than for 25 kDa polyethylenimines, yet poly(β-amino esters) show comparable DNA transfection efficacy with polyethylenimines. Within this series of polymers the transfection efficacy showed increasing trend in association with relative efficiency of nanoparticle formation.     

Influence of ionic strength on drug adsorption onto and release from a poly(acrylic acid) grafted poly(vinylidene fluoride) membrane

Ion exchange resins have several applications in pharmacy for controlled or sustained release of drugs. In the present study, effects of the ionic strengths of adsorption medium and dissolution medium on drug adsorption onto and release from a acrylic acid grafted poly(vinylidene fluoride) (PAA-PVDF) were studied. Despite their porosity, PAA-PVDF membranes act reasonable well as cation exchange membranes. It was observed, that ionic strength of adsorption medium, degree of grafting and concentration of propranolol-HCl in adsorption medium affect propranolol-HCl adsorption onto the membrane. The fluxes of smaller molecules (MW < 500) across the membrane decreased with ionic strength of buffer solution, whereas the fluxes of the large molecules (FITC-dextran, MW 4400) increased with ionic strength. Release rate of adsorbed propranolol-HCl from the membrane into phosphate buffer was greatly affected by ionic strength of adsorption medium. These results can be explained by a cation exchange process between membrane and cations present in the buffer solution and swelling behavior of the grafted PAA chains.     

Challenges of Using In Vitro Data for Modeling P-Glycoprotein Efflux in the Blood-Brain Barrier

The efficacy of central nervous system (CNS) drugs may be limited by their poor ability to cross the blood-brain barrier (BBB). Transporters, such as p-glycoprotein, may affect the distribution of many drugs into the CNS in conjunction with the restricted paracellular pathway of the BBB. It is therefore important to gain information on unbound drug concentrations in the brain in drug development to ensure sufficient drug exposure from plasma at the target site in the CNS. In vitro methods are routinely used in drug development to study passive permeability and p-glycoprotein efflux of new drugs. This review discusses the challenges in the use of in vitro data as input parameters in physiologically based pharmacokinetic (PBPK) models of CNS drug disposition of p-glycoprotein substrates. Experience with quinidine demonstrates the variability in in vitro parameters of passive permeability and active p-glycoprotein efflux. Further work is needed to generate parameter values that are independent of the model and assay. This is a prerequisite for reliable predictions of drug concentrations in the brain in vivo.     

Design and synthesis of lipid-mimetic cationic iridium complexes and their liposomal formulation for in vitro and in vivo application in luminescent bioimaging

Two iridium [Ir(N^C)₂(N^N)]⁺ complexes with the diimine N^N ligand containing a long polymethylene hydrophobic chain were synthesized and characterized by using NMR and ESI mass-spectrometry: N^N – 2-(1-hexadecyl-1H-imidazol-2-yl)pyridine, N^C – methyl-2-phenylquinoline-4-carboxylate (Ir1) and 2-phenylquinoline-4-carboxylic acid (Ir2). These complexes were used to prepare the luminescent PEGylated DPPC liposomes (DPPC/DSPE-PEG2000/Ir-complex = 95/4.5/1 mol%) using a thin film hydration method. The narrowly dispersed liposomes had diameters of about 110 nm. The photophysics of the complexes and labeled liposomes were carefully studied. Ir1 and Ir2 give red emission (λ_em = 667 and 605 nm) with a lifetime in the microsecond domain and quantum yields of 4.8% and 10.0% in degassed solution. Incorporation of the complexes into the liposome lipid bilayer results in shielding of the emitters from interaction with molecular oxygen and partial suppression of excited state nonradiative relaxation due to the effect of the relatively rigid bilayer matrix. Delivery of labeled liposomes to the cultured ARPE-19 cells demonstrated the usefulness of Ir1 and Ir2 in cellular imaging. Labeled liposomes were then injected intravitreally into rat eyes and imaged successfully with optical coherence tomography and funduscopy. In conclusion, iridium complexes enabled the successful labeling and imaging of liposomes in cells and animals.

Novel lipoidal Ir(iii) phosphorescent labels were embedded into liposomes and used for imaging in cells and animals.     

Extracellular and intracellular factors influencing gene transfection mediated by 1,4-dihydropyridine amphiphiles

Double-charged 1,4-dihydropyridine (1,4-DHP) amphiphiles have been shown to condense DNA and efficiently transfect it into cells in vitro [Hyvönen et al., Biochim. Biophys. Acta 1509 (2000) 451]. Alkyl chain length and buffering capacity at endosomal pH range (5.0–7.4) affected complexation and transfection activity. In this study we examined how those chemical modifications of amphiphile–DNA complexes (amphiplexes) affect their interactions with extracellular polyanions (glycosaminoglycans, albumin) and lipid bilayers, their cellular uptake and intracellular distribution. To evaluate cellular uptake, CV1-P cells were incubated with labeled DNA–amphiphile complexes and analyzed by flow cytometry. Confocal laser fluorescence microscopy was used to investigate the intracellular distribution of amphiplexes. The results showed that biophysical properties of compounds can be changed by slight structural modifications. These factors determine the intracellular kinetics and transfection efficacy of the compounds. Some extracellular glycosaminoglycans and serum interfere with 1,4-DHP-amphiphile-mediated transfection by destabilizing the amphiplexes. Neither high cellular uptake, membrane destabilizing activity nor buffering capacity alone is adequate for high transfection efficacy. The activity results from complex interplay of various factors that determine intracellular kinetics and, consequently, transfection.     

Different synergistic roles of small polyethylenimine and Dosper in gene delivery.

Low-molecular-weight PEIs and cationic liposomes can be combined resulting in a synergistic increase in transfection efficiency as we have reported earlier. Here, we have further investigated the potential mechanisms of this synergy. Complex morphology, complex sizes and DNA condensation were studied using transmission electron microscopy, light scattering methods and ethidium bromide exclusion, respectively. Cellular uptake, transfection efficiency, and effect of proton pump inhibitor bafilomycin A1 were examined in cell cultures. The cellular uptake of DNA was negligible with PEI2K-DNA complexes, whereas the uptake of the PEI2K-DNA-Dosper or the Dosper-DNA complexes was maximally about 40%. The number of transfected cells was two times higher with PEI2K-DNA-Dosper complexes than with Dosper-DNA complexes. The PEI2K-DNA-Dosper combination was slightly less sensitive to bafilomycin A1 than the PEI25K-DNA or Dosper-DNA complexes. There were no differences between PEI2K and PEI25K in DNA condensation. Dosper condensed DNA slightly more in PEI2K complexes. The PEI25K-DNA complexes were much smaller (<250 nm) than the PEI2K-DNA complexes (0.5-12 micro m) which were also rather polydisperse. It is suggested that two independent mechanisms would lead to synergistic transfection efficiency: (1) Dosper improves the cellular uptake of PEI2K-DNA complexes, and (2) PEI2K improves a transfer of the complexes from lysosomes to nucleus.     

Pharmacokinetics and safety of deramciclane during multiple oral dosing

Deramciclane is a new putative non-benzodiazepine-type anxiolytic compound. It is a selective serotonin 5-HT(2A) and 5-HT(2C) receptor antagonist and has also inverse agonist properties. The aim of this study was to reveal the pharmacokinetics and tolerability of deramciclane during repeated oral dosing in healthy male volunteers. SUBJECTS, MATERIAL AND METHODS: A randomized double-blind, placebo-controlled design was used. The study had three consecutive groups that received first a single oral dose of 10, 30 and 60 mg of deramciclane followed by twice a day administration for seven days. The total number of subjects was 28. The pharmacokinetic parameters were calculated for a single dose and after repeated administration. Tolerability was assessed by monitoring safety laboratory variables, electrocardiogram, heart rate, blood pressure and adverse events. RESULTS: The steady-state was reached during the seven-day administration. The pharmacokinetics of deramciclane was dose-proportional at steady-state at each dose level. Deramciclane accumulated about three-fold during repeated administration. The relative bioavailability of deramciclane increased about 1.4-fold compared to that of a single dose at each dose level. The mean elimination half-life of deramciclane for 10, 30 and 60 mg doses prolonged from 24.3, 20.9 and 22.9 h after a single dose to 30.5, 25.6 and 28.7 h at steady-state, respectively. Only few adverse events were reported, all mild and transient in nature. The most frequently reported adverse drug reactions were tiredness and headache. There were no deramciclane-induced changes in the clinical chemistry or hematology variables, blood pressure, heart rate or in electrocardiogram. CONCLUSIONS: In conclusion, the pharmacokinetics of deramciclane is linear over the dose range of 10 - 60 mg at steady-state. The slight non-linearity within the dose levels during repeated administration of seven days was regarded as clinically irrelevant. Deramciclane was safe and well tolerated up to doses of 60 mg b.i.d. for seven days.     

Alginate-based microencapsulation of retinal pigment epithelial cell line for cell therapy

The goals of this study were to evaluate human retinal pigment epithelial cell line (ARPE-19) for cell encapsulation and to optimize the alginate-based microencapsulation. We used immortalized ARPE-19 cells and the transfected sub-line that expresses secreted alkaline phosphatase (SEAP) reporter enzyme. Alginate was cross-linked with different divalent cations (Ca(2+), Ba(2+), Sr(2+) and combination of Ca(2+) and Ba(2+)), coated first with poly-l-lysine (PLL), and then with alginate. Microcapsules with different pore sizes and stability were generated. The pore size of the microcapsules was assessed by the release of encapsulated fluorescein isothiocyanate (FITC)-dextrans. The viability of the cells in the microcapsules was studied in vitro by assessing the secretion rates of SEAP and oxygen consumption by the cells. The best microcapsule morphology, durability and cellular viability were obtained with alginate microcapsules that were cross-linked with Ca(2+) and Ba(2+) ions and then coated with PLL and alginate. Based on FITC-dextran release these microcapsules have porous wall that enables the rapid contents release. The ARPE-19 cells maintained viability in the Ca(2+) and Ba(2+) cross-linked microcapsules for at least 110 days. The alginate microcapsules cross-linked with Ca(2+) and Ba(2+) have sufficiently large pore size for prolonged cell viability and for the release of secreted SEAP model protein (Mw 50 kDa; radius of gyration of 3 nm). ARPE-19 cells show long-term viability and protein secretion within alginate microcapsules cross-linked with Ca(2+) and Ba(2+). This combination may be useful in cell therapy.     

Methylprednisolone esters of hyaluronic acid in ophthalmic drug delivery: in vitro and in vivo release studies

Films and microspheres were prepared from various esters of hyaluronic acid. A model drug, methylprednisolone, was either physically incorporated into the polymer matrix or chemically bound to the polymer backbone through an ester linkage. In vitro release from films with covalently bound drug was much slower (t_50% = 71 h) than that for physically dispersed drug (t_50% = 2.5−17 h). Methylprednisolone concentrations in the tear fluid of New Zealand rabbits were measured after ocular application of drug (approx. 420 μg) in different dosage forms. When methylprenisolone was physically dispersed in the polymer matrix, in vivo drug release from matrices was slower than that observed in vitro. Compared with a suspension control, peak methylprednisolone concentrations in tear fluid were 9–14 times lower after administration of drug in polymer films and AUC_{0–8 h} values were 4–7 times higher. These results imply that hyaluronic acid ester preparations can increase the residence time of methylprednisolone in the tear fluid of rabbits.     

Pilocarpine release from hydroxypropyl-cellulose-polyvinylpyrrolidone matrices

Characteristics of pilocarpine release from cast plasticized hydroxypropylcellulose (HPC) and HPC-polyvinylpyrrolidone (PVP) matrices were studied using tritiated pilocarpine. Increased concentration of PVP and decreased molecular weight of HPC accelerated release of pilocarpine from the matrices. The aqueous solution penetrated rapidly into the matrices, which swelled rapidly to their equilibrium volumes. With increased molecular weight and concentration of HPC in the matrices, the rate of solvent penetration decreased and swollen volume of the matrix increased. Pilocarpine concentration also decreased in the ungelled cores of the matrices, indicating that the solvent had penetrated these cores. Solvent penetration alone did not control the rate of drug release, because penetration was at least twice as rapid as pilocarpine release. In the matrices without polymer dissolution, the best fits of the release data were obtained with diffusional square-root of time dependence, although relaxation of the polymers caused slight deviations from the Fickian diffusion. Thus the rate-limiting step of pilocarpine release was the diffusion of the drug from the matrix. The decreased rate of pilocarpine release with increased molecular weight and concentration of HPC was due to the decreased rate of drug diffusion from the matrix. Retardation of this diffusion was caused by the increased swelling of the matrix and decreased diffusivity of the drug. High initial concentration of PVP resulted in substantial deformation and attrition of the matrices.