Cellular uptake and intracellular pathways of PLL-g-PEG-DNA nanoparticles.

Polycationic molecules form condensates with DNA and are used for gene therapy as an alternative to viral vectors. As clinical efficacy corresponds to cellular uptake, intracellular stability of the condensates, and bioavailability of the DNA, it is crucial to analyze uptake mechanisms and trafficking pathways. Here, a detailed study of uptake, stability, and localization of PLL-g-PEG-DNA nanoparticles within COS-7 cells is presented, using FACS analysis to assess the involvement of different uptake mechanisms, colocalization studies with markers indicative for different endocytotic pathways, and immunofluorescence staining to analyze colocalization with intracellular compartments. PLL-g-PEG-DNA nanoparticles were internalized in an energy-dependent manner after 2 h and accumulated in the perinuclear region after >6 h. The nanoparticles were found to be stable within the cytoplasm for at least 24 h and did not colocalize with the endosomal pathway. Nanoparticle uptake was approximately 50% inhibited by genistein, an inhibitor of the caveolae-mediated pathway. However, genistein did not inhibit gene expression, and PLL-g-PEG-DNA nanoparticles were not colocalized with caveolin-1 indicating that caveolae-mediated endocytosis is not decisive for DNA delivery. Clathrin-mediated endocytosis and macropinocytosis pathways were reduced by 17 and 24%, respectively, in the presence of the respective inhibitors. When cells were transfected in the presence of double and triple inhibitors, transfection efficiencies were increasingly reduced by 40 and 70%, respectively; however, no differences were found between the different uptake mechanisms. These findings suggest that PLL-g-PEG-DNA nanoparticles enter by several pathways and might therefore be an efficient and versatile tool to deliver therapeutic DNA.

Design of high-throughput-compatible protocols for microencapsulation, cryopreservation and release of bovine spermatozoa.

With a rate exceeding 90% in cattle, artificial insemination (AI) is the prime reproduction technology in stock farming. AI success is expected to increase with extended persistence of sperms in utero. In order to enable controlled sperm release during artificial insemination we have designed two strategies for the automated microencapsulation of bovine spermatozoa in either alginate-Ca2+ or cellulose sulfate (CS)-poly-diallyldimethyl ammonium chloride (pDADMAC) capsules using standard encapsulation hardware. Animal protein- and citric acid-free sperm extenders and encapsulation protocols have been developed to ensure encapsulation compatible with sperm physiology. Bovine spermatozoa have showed high motility rates inside CS-pDADMAC-based capsules, were preserved by standard cryoconservation and rescued with high viability/motility following disintegration of the thawed capsules. CS-pDADMAC-based capsules break up within 72 h after addition of either purified cellulase or cellulase-filled alignate-Ca2+ capsules. The controlled release, associated with the microencapsulation of bovine spermatozoa, may be a promising approach to increase the success rate of artificial insemination.

The angiogenic response to PLL-g-PEG-mediated HIF-1α plasmid DNA delivery in healthy and diabetic rats.

Impaired angiogenesis is a major clinical problem and affects wound healing especially in diabetic patients. Improving angiogenesis is a reasonable strategy to increase diabetes-impaired wound healing. Recently, our lab described a system of transient gene expression due to pegylated poly-l-lysine (PLL-g-PEG) polymer-mediated plasmid DNA delivery in vitro. Here we synthesized peptide-modified PLL-g-PEG polymers with two functionalities, characterized them in vitro and utilized them in vivo via a fibrin-based delivery matrix to induce dermal wound angiogenesis in diabetic rats. The two peptides were 1) a TG-peptide to covalently bind these nanocondensates to the fibrin matrix (TG-peptide) for a sustained release and 2) a polyR peptide to improve cellular uptake of these nanocondensates. In order to induce angiogenesis in vivo we condensed modified and non-modified polymers with plasmid DNA encoding a truncated form of the therapeutic candidate gene hypoxia-inducible transcription factor 1α (HIF-1α). HIF-1α is the primarily oxygen-dependent regulated subunit of the heterodimeric transcription factor HIF-1, which controls angiogenesis among other physiological pathways. The truncated form of HIF-1α lacks the oxygen-dependent degradation domain (ODD) and therefore escapes degradation under normoxic conditions. PLL-g-PEG polymer-mediated HIF-1α-ΔODD plasmid DNA delivery was found to lead to a transiently induced gene expression of angiogenesis-related genes Acta2 and Pecam1 as well as the HIF-1α target gene Vegf in vivo. Furthermore, HIF-1α gene delivery was shown to enhance the number endothelial cells and smooth muscle cells - precursors for mature blood vessels - during wound healing. We show that - depending on the selection of the therapeutic target gene - PLL-g-PEG nanocondensates are a promising alternative to viral DNA delivery approaches, which might pose a risk to health.

Characterization of PLL-g-PEG-DNA nanoparticles for the delivery of therapeutic DNA.

Local and controlled DNA release is a critical issue in current gene therapy. As viral gene delivery systems are associated with severe security problems, nonviral gene delivery vehicles were developed. Here, DNA-nanoparticles using grafted copolymers of PLL and PEG to increase their biocompatibility and stealth properties were systematically studied. Ten different PLL-based polymers with no, low, and high PEG grafting and PEG molecular weights as well as different PLL backbone lengths were complexed with plasmids containing 3200 to 10,100 base pairs. Stable complexes were formed and selected for cytotoxicity and transfection efficiency. Predominantly, PLL-g-PEG-DNA nanoparticles grafted with 4 or 5% PEG moieties of 5 kDa transfected 40% COS-7 cells without reduction of cell viability when formed at N/P ratios between 0.1 and 12.5. The molecular weight of PLL did not significantly affect transfection efficiency or cytotoxicity indicating that a specific cationic charge-density-to-PEG-ratio is important for efficient transfection and low cytotoxicity. The PLL-g-PEG-DNA nanoparticles were spherical with a diameter of approximately 100 nm and did not aggregate over 2 weeks. Moreover, they protected included plasmid DNA against serum components and DNase I digestion. Therefore, such storage stable and versatile PLL-g-PEG-DNA nanoparticles might be useful to deliver differently sized therapeutic DNA for in vivo applications.