Comblike Polyethylenimine-Based Polyplexes: Balancing Toxicity, Cell Internalization, and Transfection Efficiency via Polymer Chain Topology.

Comblike polyethylenimines with varying degrees of polymerization of both the main and side chains as well as different grafting densities were evaluated as gene delivery vectors. They were able to condense linear and plasmid DNA into nanosized polyplex particles with dimensions and surface potentials in the 130-330 nm and -30 to +15 mV ranges, respectively, depending on the amine/phosphate (N/P) ratio. The polyplexes remained stable in aqueous and buffer solutions from several hours up to several days. The moderate colloidal stability was also manifested in a relatively broad size distribution (PDI typically above 0.2) and structural polymorphism observed by transmission electron microscopy. Both the neat polymers and polyplexes displayed low cytotoxicity in WISH cells as the relative cell viability was more than 60%. Experiments with lysosomal fluorescence staining revealed that the internalization pathways and, in turn, transfection efficiency of the polyplex nanoparticles depended on the polymer chain topology. The vector systems based on the polymers of denser structure can be considered to be promising systems for gene transfection in eukaryotic cells.

Enhanced gene expression promoted by hybrid magnetic/cationic block copolymer micelles.

We report on novel gene delivery vector systems based on hybrid polymer-magnetic micelles. The hybrid micelles were prepared by codissolution of hydrophobically surface modified iron oxide and amphiphilic polystyrene-b-poly(quaternized 2-vinylpyridine) block copolymer (PS-b-P2QVP) in organic solvent. After extensive dialysis against water, micelles with positively charged hydrophilic corona of PQVP and hydrophobic PS core were prepared, in which magnetic nanoparticles were randomly distributed. The hybrid micelles were used to form complexes with linear (salmon sperm, 2000 bp, corresponding to M(w) of 1.32 × 10(6) Da) and plasmid (pEGFP-N1, 4730 bp, corresponding to M(w) of 3.12 × 10(6) Da) DNA. The resulting magnetopolyplexes of phosphate:amine (P/N) ratios in the 0.05-20 range were characterized by light scattering, ζ-potential measurements, and transmission electron microscopy as well as cytotoxicity and gel retardation assays. The investigated systems displayed a narrow size distribution, particle dimensions below 360 nm, whereas their ζ-potential values varied from positive to negative depending of the P/N ratio. The resulting vector nanosystems exhibited low toxicity. They were able to introduce pEGFP-N1 molecules into the cells. The application of a magnetic field markedly boosted the transgene expression efficiency of the magnetopolyplexes, which was even superior to those of commercial transfectants such as Lipofectamine and dendritic polyethylenimine.

Electrochemotherapy of Mycosis fungoides by interferon-alpha.

Eight patients with 29 lesions of histologically verified 1st stage of Mycosis fungoides were successfully treated by electrochemotherapy with interferon-alpha. For this purpose 8 biphasic pulses were used, each of 50+50 micros duration with 900 micros interpulse intervals, resulting in a burst of 7.1 ms total duration. Compared to the traditional monoimmunotherapy with interferon-alpha applied three times weekly for a total of 4 weeks, the electrochemotherapy was very efficient. Complete response (CR) was observed in 25 (86%) of the 29 treated lesions by single-act electrochemotherapy with interferon-alpha. At the end of the 12-month period, all 29 lesions showed 100% complete response (CR). New lesions for a period of 12 months were not observed. The expected mechanism involved in multiple cytotoxic action of interferon-alpha could be the local increased concentration in the tumour and prolongation of the time of its action after the application of pulses.

Electrochemotherapy of skin tumours: comparison of two electroporation protocols.

PURPOSE: To study the effect of electric pulses duration and number in a sequence for electrochemotherapy of primary non-melanoma skin tumours. PATIENTS AND METHODS: Two electroporation sequences were applied: sequence A, consisting of 16 biphasic pulses of 25+25 mus duration, spaced at 0.6 ms, with a duration-number product of 0.8 ms; and sequence B, consisting of 8 50+50 mus biphasic pulses, spaced at 1.0 ms, with the same duration-number product of 0.8 ms. The two sequences were used for electrochemotherapy of clinical stage I (T1N0M0) basal cell and spindle-squamous cell carcinoma lesions in two groups of patients. Sequence A was applied to 59 patients (group A; subgroup A1 with 45 patients with basal cell carcinoma, and subgroup A2 with 14 patients with spindle-squamous cell carcinoma) and sequence B to 54 patients (group B; subgroup B1 with 40 patients with basal cell carcinoma, and subgroup B2 with 14 patients with spindle-squamous cell carcinoma). Intratumoral bleomycin was administered to all patients. RESULTS: All group A and B patients achieved initially complete response (CR). The sustained CR rate in subgroup A1 patients was 80%, due to 9 recurrences and for subgroup A2 patients it was 71.4% because of 4 recurrences. No subgroup B1 patient developed recurrence. Three subgroup B2 patients showed disease recurrence, for a sustained CR rate of 78.6%. CONCLUSION: The efficiency of electrochemotherapy in cutaneous non-melanoma tumours depends on the duration of electric pulses. For biphasic pulses, duration below 50 mus per phase (or 50+50 mus for the total pulse) is not recommended, in spite of a markedly reduced pain sensation. The rule about equal efficiency of larger number of shorter pulses and longer pulses of lesser number has its limits for external application, especially through the skin.