Polyethylenimine (PEI)-g-comb-poly(ethylene glycol)-transferrin(Ⅰ):Tumor-targeted Vector for Gene De

Chinese Chemical Letters Vol. 17, No.1, pp 113-116, 2006113http://www.imm.ac.cn/joumal/ccl.htmlPolyethylenimine (PEI)- g-comb-poly(ethylene glycol)-transferrin(I):Tumor-targeted Vector for Gene Delivery In-vitroGu Ping TANG12*. Zhi Yu WANG''Collge of Life Science. ZheJiang University, Hangzhou 3100282 Institute of Materials Research & Engineering. Singapore, 3 Research Link 117602The Second Hospital of Zhejiang University, Hangzhou 310006Abstract: The work described thesynthesis and evaluation of PEI -g-comb- PEG-transferrin as a .potential system for gene therapy in vitro. The MW of PEG was 10KDa, and PEI was 2KDa.Its structure was identified by NMR, FT-IR and TGA spectroscopy. MTT assay found that atconcentration up to 4000 n molL of the polymer, cell viability was over 85%. The bio characterof polymer/DNA complex was characterized by agarose gel electrophoresis, ethidium bromideexclusion and zeta- potential assay.The polymer could retardate DNA at N/P ratio 3.0-3.5(mol/mol). The particle size of the polymer/DNA complex was less than 300 nm. Transfectionefficiency of the complex was studied in COS7 and NT2 cell lines.Keywords: PEI-g-Comb PEG, transferrin, gene therapy, transfection efficiency.A new vector, PEG as a core, low Mw PEI was grafted to PEG, and transferrin wasconjugated the co-polymer to form PEIg-PEG-transferrin'2. NMR, FT-IR and TGAspectroscopy confirmed the structures of activated PEG and the final products. TheMW of PEI-g-PEG was 26KDa. DNA condensation with polymer was studied usingagarose gel electrophoresis, ethidium bromide (EtBr) exclusion assay and zeta-potential.The particle size of comb-PEG-PEI/DNA complex was less than 300 nm and it wasstable in 5% glucose solution. The synthetic route of the copolymer is shown inScheme 1.0.2 g Comb-PEG (SHEARWATER USA, Mw= 10KDa, 0.02 mmol) was dissolvedin 3 mL CH2Cl2, 52 mg 1,1-carbonyldiimidazole (CDI, 0.32 mmol) was dissolved in 3mL CH2Cl2, and reacted at RT for 1.5 h, precipitated from ether, dissolved in 5 mLDMSO.64 mg PEI (0.32 mmol ) was dissolved in 5 mL CH2Cl2, and then added toabove reaction solution. The mixture was stirred at RT for 45 min. The reactionproduct was purified by PD- 10 column eluting with PBS solution (PBS containing 5mmol/L EDTA). The ratio of PEG and PEI in the copolymer was determined by 'HNMR spectrum according to the ratio of the integral values for the protons of-CH2CH2O- in PEG and protons of -CH2NH-中国煤化工A cold solution of 26 mg of human hol， 0.32 mmol) in|YHCNMHG'"E-mailtangguping@ yahoo.com.cn114Gu Ping TANG et al..5 mL of 0.1 mol/L PBS (pH 7.6) was mixed with 120 μL of 2-iminothiolane solution (5mg/mL in water). The mixture was reacted at RT for 30 min. The modifiedtransferring was isolated by eluting with PBS buffer solution through a PD- 10 column,filtered and dialyzed for next step reaction. Active PEG-PEI was then slowly added to theabove solution for4h at RT. After reaction, dialysis it overnight.Scheme 1 The synthetic rout of PEI g-comb- PEG transferrinHQOHPEG-01INNHNHLN4:s -NH,HNDNH. ;H0OIOco0CO,oCo, NH2HN、oρ'NH,HNH，PEIOCOOCCHy.N-CNH.82+-(CH);- -CO-N十PEG-NH-CCH-CH2-NH PEI-0-C-0-(CH)5 -NTransferrin +Tfn-C-CH2CH2CH.-SHNH,CI9PEG-NHC-CH,CH,-NH-PEI-OC-O-(CH)- N( ] + Ti.CH2CH-CH-SH，PEG-PEI-Tfnoo'Result and Discussion中国煤化工'H NMR spectroscopic data were carried3.6 ppm wereassigned to protons of PEG and the peaks at.MHC N M H GPEI (Figure 1).According to the NMR data the molecular ratio of PEG and PEI in the polymeris 1 to 8.It means each finger of comb in PEG bore one PEI.Polyethylenimine (PEID)- g-comb-poly(ethylene glycol)-transferrin(I)115Figure 1 'H NMR spectrum of PEG-g-PEIFigure 2 Agarose gel electrophoresis andethidium bromide displacement assayfor PEG-g-PEI/DNA complexesPraio DNA.62 2.!53.04.2521.2- -PEGPEI0.7.06.0504.03.0201.0 0.00.41 1.67 2.5 3.5 5 7 12 17.5 NTP RatioAfter coupling with PEI, a stretching absorption at 1624 cm:' was appeared in FT-IRspectrum.The TGA was carried out for the polymer.At temperature higher than 200 C,PEG-PEI began to degrade and coursed the first degradation step. A strong thermaldegradation was monitored above 300° C when the PEI began to degrade. Theformation of PEI-PEG/DNA complexes was examined by gel retardation assay andethidium bromide (EtBr) exclusion assay (Figure 2). For PEG-PEI polymer, DNA wasretarded at N/P ratio of 3-3.5. The assay established the complexes ratio required forcomplete condensation of DNA.MTT assay in COS7 cell lines was used for evaluating the cytotoxicty ofPEI-g-PEG. Considering the dependence of cytotoxicy on the molecular weight of PEI,low cytotoxicity was expected from the degradation of PEI-g-PEI polymer into less toxiclow MW PEI. In addition, the introduction of PEG contributed to reduce thecytotoxicity. Comparing with high MW PEI (25KDa) and low MW PEI (2KDa), theaverage cell viability was obtained over 85% for PEG-g-PEI in concentration of 120 to4000 n mol/L. The level was the same as the low MW PEI (2KDa).Size of PEG-PEI/DNA complex was measured with N4 Plus Submicron ParticleSizer (COULTER, USA) at RT. The PEG-g PEI/DNA complexes were preparedaccording to that plasmid DNA was diluted in 5% glucose to the chosen concentration(usually 0.5-2 μg/uL). After vortexing, the appropriate amount of 0.1 mol/L PEG-g-PEI was added dropwise into DNA solutimg 2nd tha plutinn re :vortexed. Thecomplexes size remained small and stable les中国煤化工at NIP ratio 30/1.No aggregation was occurred in the solution.YHC N M H Garticle size wasnearly 500 nm.116Gu Ping TANG et al.Figure 3 Transfection efficiency of PEI-g-PEG/DNA, COS7(A), NT2 (B) cell lines1.40E+108.00E+071.20E+ 100 PEI25KDaI PEG-g-PEI哥6.00E+07 t | 0 PEG-g-PEI信100E+10 .包8.00E+094.00E+0726.00E+094.00E+092.00E+072.00E+090.00E+001:03(120N/PNPTo estimate the surface charge of the PEI-g-PEG/DNA complexes, its zeta potentialwas tested. High zeta potentials of more than +25 mV were measured for PEI-g-PEI atN/P ratio over 20/1 at 0.1 mmol/L KCl. It explored that after grafting PEI to comb PEG,the MW was increased and the density of amine was also increased, so the surface chargeof the PEI-g-PEG/DNA was increased.In our study, the transfection activity of the complex was tested using COS7 andNT2 cell lines.The transfection efficiency was expressed by amount of luciferase atN/P ratios of 15/1, 20/1 and 30/1, respectively (Figure 3). The cytotoxicity for highMW PEI (25KDa) was high, the transfection efficiency showed reduced, when N/P ratioincreased, no matter in COS7 or NT2 cell lines. In contract, for PEI-g- PEG polymer,the transfection activity was higher than PEI (25KDa) at high N/P ratio. As expectedfrom an increased MW of the copolymer， the toxicity as the same as PEI2KDa,transfection efficiency increased 1000 times than that of PEI with the initial MW (data .not shown). The result of our experiment showed that the transection efficiency withgrafting low MW PEI to comb PEG improved, and the toxicity of PEI reduced. Furthermore it gave sites to conjugated target groups to matrix. The detail investigation ofPEI-g-PEI-Tfn in vitro is in progressing.AcknowledgmentThis work was supported by the grants from the National High Biotechnology DevelopmentProgram of China (2003AA216041), and the National Key Basic Research Program of China(2004CB518802).References1. C. L Gebhart, A V. Kabanov, J of Controlled Release, 2001, 73, 401.2. T Nidome, L Huang, Gene Therapy, 2002, 9, 1647.3.G.P.Tang,J.M.Zeng,S.J.Gao,YX.Ma,et中国煤化工51.Received 24 April, 2005TYHCNM HG