Dilution-Stable PAMAM G1-Grafted Polyrotaxane Supermolecules Deliver Gene into Cells through a Caveolae-Dependent Pathway

Abstract

Numerous preclinical studies have demonstrated that polycation mediated gene delivery systems successfully achieved efficient gene transfer into cells and animal models. However, results of their clinical trials to date have been disappointing. That self-assembled gene and polycation systems should be stable undergoing dilution in the body is one of the prerequisites to ensuring efficiency of gene transfer in clinical trials, but it was neglected in most preclinical studies. In this account, we developed the dilution-stable PAMAM G1-grafted polyrotaxane (PPG1) supermolecules in which PAMAM G1-grafted α-cyclodextrins are threaded onto a PEG chain capped with hydrophobic adamantanamine. The PPG1/pDNA polyplex (approximate 100 nm in diameter) was very stable and kept its initial particle size and a uniform size distribution at ultrahigh dilution, whereas DNA/PEI 25K polyplex was above three times bigger at a 16-fold dilution than the initial size and their particle size distribution indicated multiple peaks mainly due to forming loose and noncompacted aggregates. PPG1 supermolecules showed significantly superior transfection efficiencies compared to either PEI 25K or Lipofectamine 2000 in most cell lines tested including normal cells (HEK293A) and cancer cells (Bel7402, HepG2, and HeLa). Furthermore, we found that the PPG1 supermolecules delivered DNA into HEK293A through a caveolae-dependent pathway but not a clathrin-dependent pathway as PEI 25K did. These findings raised the intriguing possibility that the caveolae-dependent pathway of PPG1 supermolecule/pDNA polyplex avoiding lysosomal degradation was attributed to their high transfection efficiency. The dilution-stable PPG1 supermolecule polyplex facilitating caveolae-dependent internalization has potential applications to surmount the challenges of high dilutions in the body and lysosomal degradation faced by most gene therapy clinical trials.

Clustering Small Dendrimers into Nanoaggregates for Efficient DNA and siRNA Delivery with Minimal Toxicity

“…Second, low molecular weight dendrimers were grafted on biocompatible polymers or nanoparticles via stimuli-responsive linkages to "temporarily" generate materials with high charge density for effi cient and safe gene delivery. [17][18][19] Third, low molecular weight dendrimers were conjugated with lipids or fl uorous chains to generate amphiphilic materials, and these materials can self-assemble into cationic micelles via hydrophobic interactions or fl uorinefl uorine interactions. [20][21][22][23][24] After gene delivery, the micelles may disassemble into amphiphilic monomers with reduced cytotoxicity.…”

Synthesis of taurine–fluorescein conjugate and evaluation of its retina-targeted efficiency in vitro

“…3 and Table 2 , the transport of F–Tau in cells previously incubated with free taurine did not show the inhibition, but rather an increase in the pentration of ARPE-19 and hRMECs monolayer cells; the resulting P app, F–Tau+Tau to F–Tau values ranged from 7.79 to 7.72 on ARPE-19 and 13.30 to 10.67 on hRMECs. One possible answer for these unexpected results was that free taurine, used as the competitive inhibitor, may have activated other transport pathways to improve the penetrability of F–Tau as reported in other studies 27 , 28 . Identification of such transporters requires further studies 29 .…”

Endothelial cell-targeted pVEGF165 polyplex plays a pivotal role in inhibiting intimal thickening after vascular injury

“…32 CDG2 was prepared by a cross-linking reaction using CDI to couple the primary amines of PAMAM G2 and the hydroxyl groups of α-CDs. Briefly, α-CD (0.5 g, 0.483 mmol) in 25 mL DMSO was added dropwise to CDI (6.5 g, 0.040 mol) in 25 mL DMSO under nitrogen.…”