Ring-Opening Polymerization for Hyperbranched Polycationic Gene Delivery Vectors with Excellent Serum Tolerance

Abstract

In order to improve the transfection efficiency (TE) and biocompatibility, we synthesized a series of hyperbranched cationic polymers by ring-opening polymerization between diepoxide and several polyamines. These materials can condense plasmid DNA efficiently into nanoparticles that have much lower cytotoxicity than those derived from bPEI. In vitro transfection experiments showed that polymers prepared from branched or cyclic polyamine (P1 and P5) exhibited TE several times higher than 25KDa bPEI. More significantly, serum seemed to have no negative effect on P1-P5 mediated transfection. On the contrary, the TE of P1 improved, even when the serum concentration reached 70%. Several assays demonstrated the excellent serum tolerance of such polycationic vectors: bovine serum albumin (BSA) adsorption assay revealed considerably lower protein adsorption of P1-P5 than PEI; P1 showed better DNA protection ability from degradation by DNase I than PEI; flow cytometry results suggested that any concentration of serum may not decrease the cellular uptake of P1/DNA polyplex; and confocal laser scanning microscopy also found that serum has little effect on the transfection. By using specific cellular uptake inhibitors, we found that the polyplexes enter the cells mainly via caveolae and microtubule-mediated pathways. We believe that this ring-opening polymerization may be an effective synthetic approach toward gene delivery materials with high biological activity.

Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors

“…TEM and/or cryo-TEM studies have reported many micrographs to characterize the size, shape, and morphology of a wide variety of lipoplexes as: (a) pDNA with GCLs [45,54,57,62,64,75,93,115,117,118,120], MVCLs [63], cationic polymers [107,133,[169][170][171] or polycations [40,84,100,136]; and (b) siRNA with cationic polymers [90,104]. Fig.…”

“…(ii) multivalent cationic lipids [67,78,139,178,181], derivated oligoamines [182] and tripeptide multivalent lipids [122]; (iii) cationic polymers derivatives from PEI [124,127,183,184], PAMAM [132,140], PDMAEMA [123,133,185], PAA [103,131,186,187], chitosan [188], polyphosphoramidates [189], polyhydroxyalkanoate [92], polyamines [107], polyaminoacids [127,190] or diblock copolymers [191]; (iv) polycations from calixarene [40,136,165,192] or cyclodextrin [134,135,142,164,193] derivatives; (v) lipopolymers from lysine derivatives [170]; (vi) pendant polymercationic cyclodextrins [129,130,163]; and (vii) cationic polymer-cyclodextrin polyrotaxanes [89]. The picture that results from these transfection studies is that many of the polyplexes show efficacies comparable to that reported by the Lipo2000 positive control and also by most of the commercial or synthezised univalent CLs, but its efficacy as transfecting agents of DNA is still below than that reported by most of t...…”

“…In addition to the previous TE and KE biological studies, microscopic methods have been frequently used to visualize the level of % GFP expression inside the cells after MVCLs containing dendritic headgroups [72,121] or cyclen-based [120] lipids; (c) cationic polymers based on PEI and PAA derivatives [124,183], phospholipid-polymer [203], aminoacid-based [126,127,190], chitosan [204], PDMAEMA [133], dendrimers as PAMAM derivatives [132,201], others cationic polymer-type [107,164] or PEG-PPG-PEG/cyclodextrin polyrotaxanes [89]; and (d) polycations based on calixarenes [166,205] or cyclodextrins [106,136,142]. With respect to gene silencing/knockdown siRNA studies, micrographs and/or images have been reported for systems where the following gene vectors have been used: (a) MVCLs based on dendritic lipids [104]; and (b) cationic polymers based on dendrimers [199], diblock copolymers [105] or POEAA [144], last one in vivo studies.…”

“…The results confirm that all these optimized formulations did not report any toxicity to the cell lines studied, the cell viability after transfection being considerably better than the commercial control Lipo2000. The cell viability/cytotoxicity of several complexes between nucleic acids and a wide variety of recent synthesized MVCVs has been analyzed: (i) GCLs as cholesterol-based with alkyl or oligo-oxyethylene spacers [118,119,121,206,207], aromatic-based with alkyl or oligo-oxyethylene spacers [46], ammonium derivatives [54,71,93] imidazolium derivatives [45,75,99] and pyridinium derivatives [57] with alkyl type spacers, aminoacid-based [64,208], SS14 linkers [66], gemini-like amphiphilic peptides [150] or cyclen-based CLs [120,180]; (ii) several MVCLs [67,72,122,181,182]; (iii) cationic polymer-derivatives from PEI [124,127,183,209], PAMAM [140], PDMAEMA [123,133], chitosan [188], polyphosphoramidates [189], polyhydroxyalkanoates [92], polyamines [107,187] or polyaminoacids [127,190]; (iv)…”

“…In order to understand better each one of these processes and/or to elucidate the determinant step of the biological activity, several biochemical analysis have been recently reported, such as internalization, intracellular uptake and traffic, localization or colocalization, endosomal escape, and so on. The methods used in most of these studies are based in the use of specific fluorescent markers[105][106][107].…”