Core/Shell Gene Carriers with Different Lengths of PLGA Chains to Transfect Endothelial Cells

Abstract

In order to improve the transfection efficiency and reduce the cytotoxicity of gene carriers, many strategies have been used to develop novel gene carriers. In this study, five complex micelles (MSP(2 k), MSP(4 k), MSP(6 k), MSP(8 k), and MSP(10 k)) were prepared from methoxy-poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) (mPEG-b-PLGA) and sorbitol-poly(d,l-lactide-co-glycolide)-graft-PEI (sorbitol-PLGA-g-PEI, where the designed molecular weights of PLGA chains were 2 kDa, 4 kDa, 6 kDa, 8 kDa, and 10 kDa, respectively) copolymers by a self-assembly method, and the mass ratio of mPEG-b-PLGA to sorbitol-PLGA-g-PEI was 1/3. These complex micelles and their gene complexes had appropriate sizes and zeta potentials, and pEGFP-ZNF580 (pDNA) could be efficiently internalized into EA.hy926 cells by their gene complexes (MSP(2 k)/pDNA, MSP(4 k)/pDNA, MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA). The MTT assay results demonstrated that the gene complexes had low cytotoxicity in vitro. When the hydrophobic PLGA chain increased above 6 kDa, the gene complexes showed higher performance than that prepared from short hydrophobic chains. Moreover, the relative ZNF580 protein expression levels in MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA) groups were 79.6%, 71.2%, and 73%, respectively. These gene complexes could promote the transfection of endothelial cells, while providing important information and insight for the design of new and effective gene carriers to promote the proliferation and migration of endothelial cells.

CAGW and TAT‐NLS peptides functionalized multitargeting gene delivery system with high transfection efficiency

“…The representative structures and 1 H NMR spectra of mP, PP, and PC copolymers were shown in Figure S1. The results demonstrated that these amphiphilic polymers were successfully synthesized …”

“…N‐Hydroxy succinimide (NHS), 1‐ethyl‐3‐(3‐(dimethylamino)propyl)‐carbodiimide hydrochloride (EDC), succinic anhydride, diallylcarbamyl chloride, and 2,2‐dimethoxy‐2‐phenylacetophenone (DMPA) were obtained from Tianjin Heowns Biochemical Technology Co., Ltd. CAGW (Cys‐Ala‐Gly‐Trp) and TAT‐NLS (Gly‐Arg‐Lys‐Lys‐Arg‐Arg‐Gln‐Arg‐Arg‐Arg‐Pro‐Lys‐Lys‐Lys‐Arg‐Lys‐Val) peptides were purchased from GL Biochem (Shanghai) Ltd. EA.hy926 cells (human umbilical vein endothelial cells) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and cultured as described before . Other chemicals and biological reagents were supplied by the companies referred in our previous study …”

“…However, the physical and biological functions of gene delivery micelles formed from a single copolymer are difficult to control. Recently, the complex micelles were prepared from the co‐assembly of methoxy‐poly(ethylene glycol)‐b‐poly‐( d , l ‐lactide‐co‐glycolide) (mPEG‐b‐PLGA) and sorbitol‐poly(D,L‐lactide‐co‐glycolide)‐graft‐PEI (sorbitol‐PLGA‐g‐PEI) in our group . mPEG‐b‐PLGA and sorbitol‐PLGA‐g‐PEI had the same hydrophobic segments, but different hydrophilic segments.…”

“…In present study, the multitargeting gene complexes based on mPEG‐b‐PLGA, PLGA‐g‐PEI‐g‐CAGW, TAT‐NLS peptide, and pEGFP‐ZNF580 (pDNA) were developed to improve the transfection of ECs. pEGFP‐ZNF580 (pDNA) was used as target gene as described before . CAGW peptide could endow the multitargeting gene complexes with selective ECs‐targeting ability, and TAT‐NLS peptide enables them with cell‐penetrating ability and nuclear accumulation capacity.…”

“…Recently, the complex micelles were prepared from the co-assembly of methoxy-poly(ethylene glycol)-b-poly-(D,L-lactide-co-glycolide) (mPEG-b-PLGA) and sorbitol-poly(D,L-lactide-co-glycolide)-graft-PEI (sorbitol-PLGA-g-PEI) in our group. 25 mPEG-b-PLGA and sorbitol-PLGA-g-PEI had the same hydrophobic segments, but different hydrophilic segments. The cytotoxicity and physicochemical properties of complexes can be easily controlled by adjusting the copolymer ratios.…”