Delivery of Plasmid DNA into Tumors by Intravenous Injection of PEGylated Cationic Lipoplexes into Tumor-Bearing Mice

For systemic injection of cationic liposome/plasmid DNA (pDNA) complexes (cationic lipoplexes), polyethylene glycol (PEG)-modification (PEGylation) of lipoplexes can enhance their systemic stability. In this study, we examined whether intravenous injection of PEGylated cationic lipoplexes into tumor-bearing mice could deliver pDNA into tumor tissues and induce transgene expression. PEGylation of cationic liposomes could prevent their agglutination with erythrocytes. However, when PEGylated cationic lipoplexes were injected intravenously into tumor-bearing mice, they accumulated in tumor vascular vessels and did not exhibit transgene expression in tumors with both poor and well-developed vascularization. Furthermore, PEGylated cationic lipoplexes of CpG- free pDNA could not increase transgene expression in tumors after intravenous injection. These results suggested that PEGylation could not extravasate cationic lipoplexes from vascular vessels in tumors and abolished transgene expression although it enhanced the systemic stability of cationic lipoplexes by avoiding interactions with blood components such as erythrocytes. Successful delivery of pDNA to tumors by PEGylated cationic liposomes will require a rational strategy and the design of liposomal delivery systems to overcome the issue associated with the use of PEG.

Synthesis, Biophysical Characterization and <i>in Vitro</i>Transfection Activity of Novel Bivalent Amine Cationic Lipids in the Absence of Dioleoylphosphatidylethanolamine (DOPE)

In this paper, a novel series of bis [(aminoethyl)]-amine cationic lipid derivatives have been synthesized and identified to purity by NMR and Elemental analysis. B16-F0 cells were transfected with cationic lipid/pEGFP-N1 and cationic lipid/&#223-gal lipoplexes complexed at +/&#45 charge ratios of 1:1, 2:1, and 4:1. Dimyristoyl derivative showed highest activity at charge ratio 2:1 and both dimyristoyl and dioleoyl derivatives showed similar &#223-gal activity at charge ratios 4:1. In 40 mM tris buffer pH 7.2 the dioleoyl derivative was able to fully complex with and retard pDNA at charge ratios above 2:1. None of the other lipid derivatives, dilauroyl, dimyristoyl, dipalmitoyl and distearoyl were able to fully neutralize the plasmid DNA at charge ratios similar to those used in the transfection experiment. The gel-to-liquid phase transition temperatures for dimyristoyl, dipalmitoyl and distearoyl were determined by a fluorescence anisotropy method to be 27.5&degC, 32.5&degC and 39&degC, respectively. A gel-to-liquid crystalline phase transition temperature below 37&degC, appears to be the crucial property that cationic lipids have to possess in order to mediate high levels of in vitro transfection activity in the absence of other helper lipids.

Bone-targeted lipoplex-loaded three-dimensional bioprinting bilayer scaffold enhanced bone regeneration

Clinical bone-morphogenetic protein 2(BMP2)treatment for bone regeneration,often resulting in complications like soft tissue inflammation and ectopic ossification due to high dosages and non-specific delivery systems,necessitates research into improved biomaterials for better BMP2 stability and retention.To tackle this challenge,we introduced a groundbreaking bone-targeted,lipoplex-loaded,three-dimensional bioprinted bilayer scaffold,termed the polycaprolactone-bioink-nanoparticle(PBN)scaffold,aimed at boosting bone regeneration.We encapsulated BMP2 within the fibroin nanoparticle based lipoplex(Fibroplex)and functionalized it with DSS6 for bone tissue-specific targeting.3D printing technology enables customized,porous PCL scaffolds for bone healing and soft tissue growth,with a two-step bioprinting process creating a cellular lattice structure and a bioink grid using gelatin-alginate hydrogel and DSS6-Fibroplex,shown to support effective nutrient exchange and cell growth at specific pore sizes.The PBN scaffold is predicted through in silico analysis to exhibit biased BMP2 release between bone and soft tissue,a finding validated by in vitro osteogenic differentiation assays.The PBN scaffold was evaluated for critical calvarial defects,focusing on sustained BMP2 delivery,prevention of soft tissue cell infiltration and controlled fiber membrane pore size in vivo.The PBN scaffold demonstrated a more than eight times longer BMP2 release time than that of the collagen sponge,promoting osteogenic differentiation and bone regeneration in a calvarial defect animal.Our findings suggest that the PBN scaffold enhanced the local concentration of BMP2 in bone defects through sustained release and improved the spatial arrangement of bone formation,thereby reducing the risk of heterotopic ossification.

Intranasal application of Epstein-Barr virus/lipoplex to abrogate eosinophillia in murine model of allergic rhinitis

Background Currently anti-inflammatory therapy with steroids for allergic rhinitis need long-term repeated administration, although it is effective. Gene therapy is being suggested to substitute it. The aim of this study was to investigate nonviral vector mediated exogenous gene expression in COS-7 cells in vitro and the effect of intranasal mouse interleukin （mIL）-12 transgene expression on allergen induced eosinophil infiltration of nasal mucosa in a murine model of allergic rhinitis. Methods In vitro COS-7 cells were infected with Epstein-Barr virus （EBV）/lipoplex. The expression of IL-12 p70 in cell culture supernatant was examined by enzyme-linked immunosorbent assay （ELISA）. In mice with ovalbumin （OVA） induced allergic rhinitis, EBV/lipoplex was administered by nasal drops before OVA challenge once a day from day 1 to day 10. The expression of IL-12 mRNA and protein, the change of eosinophil count in nasal mucosa and serum total IgE were measured 24 hours after the last challenge. Results EBV/lipoplex could effectively transfect COS-7 cells. The expression of IL-12 p70 in cell culture supernatant was significantly more than in blank control. IL-12 via EBV plasmid vector transduction could be overexpressed in vivo. In pGEGmIL-12 treated models, the nasal mucosa revealed a high level of widespread raiL-12 transduction by immunohistochemistry and in situ hybridization. Histological evaluation revealed marked suppression of eosinophil infiltration in nasal mucosa. The eosinophil count in allergic rhinitis group [（26.5±9.8）/high-power field （HPF）] was significantly increased over control group [（0.40±0.52）/HPF] （F=56.94, P〈0.01）, while the count in IL-12 gene therapy group [（4.60±2.63）/HPF] was significantly less than that of allergic group （F=56.9, P〈0.01）. Serum total IgE between in gene therapy mice [（88.83±6.71） ng/ml] and allergic rhinitis mice [（103.1±5.7） ng/ml] showed a significant difference （F= 1216, P〈0.05）. Conclusions Nonviral EBV plasmid vector, pGEGmIL-12 was able to overexpress exogenous gene both in vitro and in murine nasal mucosa in vivo. IL-12 overexpression via EBV/lipoplex could stem allergen induced eosinophil infiltration in nasal mucosa in murine models of allergic rhinitis, which may suggest a new cytokine immunogenetic therapy for allergic rhinitis.