Synthesis of polyethylenimine grafted with copolymers of polyethylene glycol and polycaprolactone an

Available online at www.sciencedirect.comCHINES EScienceDirectCHEMICALL ETTERSEL SEVIERChinese Chemical Letters 22 (2011) 749-752www.elsevier.com/ocate/ccletSynthesis of polyethylenimine grafted with copolymersof polyethylene glycol and polycaprolactone and its potentialfor siRNA deliveryWei Huang, Ming Lv, Zhong Gao GaoInstitute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Bejing 100050, ChinaReceived 8 November 2010AbstractCopolymers mPEG-PCL were prepared and grafted onto polyethylenimine (PEI) to synthesize copolymers mPEG-PCL-g-PEIwith low cytotoxicity. The mPEG-PCL-g-PEI could condense siRNA to form nanoparticles with positive zeta potential. Thesenanoparticles could delivery siRNA into cells to effectively inhibit the expression of target gene, which suggested that mPEG-PCL-g-PEI could serve as a highly eficient vector for siRNA delivery.◎2011 Zhong Gao Gao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.Keywords: Copolymers; Nanoparticles; Vector; SynthesisPolyethylenimine (PED) is a highly eficient vector for siRNA delivery [1], but the high density of positive charge on thesurfaces of PEI molecules can result in severe cytotoxicity, which has become a limiting factor for PEI application [2]. So,study on various derivatives of PEI became a currently hot topic [3]. The modification of PEI with polyethylene glycol(PEG) not only improved the solubility of PEI/DNA complexes, but also decreased the cytotoxicity of PEI [4,5]. However,PEG-PEI copolymers cannot be biodegraded because the urethane linkages between PEI and PEG cannot be hydrolyzedunder physiological conditions. Moreover, copolymers with too high molecular weight cannot be excreted by kidney,which can lead to organ damage after a long-tem usage [6]. It was demonstrated that the introduction of hydrophobicmoieties could enhance tissue permeability and cellular affinity of gene delivery vectors [7]. In addition, it is well knownthat polycaprolactone (PCL) has good biocompatibility and biodegradability. Therefore, we want to try to introduce PCLinto PEG-PEI to construct termnary copolymers PEG-PCL -PEI with good biodegradability for siRNA delivery. Moreover,PCL can also shield positive charges todecrease PEI cytotoxicity. In this article, mPEG-PCL were synthesized and graftedonto the branched PEI to prepare copolymers mPEG-PCL -g-PEL. The cytotoxicity and siRNA delivery potential ofmPEG-PCL-g-PEI were explored.1. ExperimentalPEI (Mw 10 kDa) was obtained from Polysciences; Monomethoxy polyethylene glycol (mPEG) (Mw 5000 Da)was purchased from Fluka; Human breast cancer cell line MCF-7 was obtained from the Cell Resource Center in中国煤化工* Coresponding author.E-mail address: zqgao@imm.ac cn (Z.G. Gao).YHCNMHG1001-8417/$ - see front matter心2011 Zhong Gao Gao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. AlI rights reserved.doi:10.1016j cclet.2010.12.057750W. Huang et al./Chinese Chemical Ltters 22 (2011) 749 -752IBMS in CAMS/PUMC; GL3_ Luc. _siRNA was synthesized by Dharmacon; Other chemicals were analyticalgrade.Copolymers mPEG-PCL-g-PEI was prepared according to the following steps. Firstly, mPEG-PCL-OH wasprepared through the ring-opening polymerization of ε-caprolactone using mPEG as a macroinitiator [8]. Then,mPEG-PCL COOH was synthesized by hydroxyl esterification using mPEG-PCL-OH and succinic anhydride. The N-hydroxysuccinimide (NHS) was used to react with mPEG-PCI -COOH to prepare mPEG-PCI -NHS. Finally, thebranched PEI was reacted with mPEG-PCL NHS to synthesize mPEG-PCL-g-PEI. The structure of mPEG-PCL-g-PEI was characterized with Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR) andgel permeation chromatography (GPC). GPC analysis was performed using a Waters 515 pump with tetrahydrofuranas an eluent (1 mL /min), and the eluent were analyzed using a Waters 24 10 differential refractive index detector.The (mPEGsk-PCL1.2k).4-g-PEIo/GL3. Luc_ siRNA nanoparticles were prepared by complex coacervation. The(mPEGsk-PCL1.zk)1.4-g-PEI1ok was dissolved in double distilled water at various concentrations, and then filtered forsterilization using 0.22 μm membranes. The GL3_ Luc_ siRNA was diluted with Rnase-free water at a specificconcentration. Finally, the equal volume of copolymers and GL3_ Luc_ siRNA solutions were mixed and vortexed for0.5 min to generate nanoparticles suspensions. In addition, the prepared nanoparticles suspensions were diluted withdouble distilled water to measure particle size and zeta potential using a Nicomp380/ZLS analyzer.In MTT assay, MCF-7 cells were seeded into a 96-well plate at a density of 5000 cells/well. After 24-h cell culture,(mPEGsk-PCL.2x)1.4-g-PEIo/GL3_ Luc_ siRNA and PEIow/GL3_ Luc_ siRNA nanoparticles at various N/P ratioswere respectively added in cell wells. After another 24-h cell culture, MTT assay was performed to determine cellviability to evaluate the cytotoxicity of (mPEGsk-PCL1.2k)1.4-g-PEIok and PEI1ok. .For siRNA delivery in vitro, MCF-7 cells were seeded into a 24 well plate at a density of 6 x 104 cells/well. After24-h cell culture, cells were co-transfected with 400 ng pGL3-Control reporter plasmid and 1 ng pGL4.75 referenceplasmid using Lipofectamine 2000 reagent. After 4-h transfection, (mPEGsk-PCL1.2x)1.4-g-PE1o/GL3_ Luc_ siRNAnanoparticles were added into wells in test groups. Lipofectamine 2000, naked GL3_ Luc. siRNA and Rnase-free waterwere respectively used as positive, negative and blank controls. The amount of GL3 Luc. _siRNA added into each wellwas 4 pmol. After another 36-h cell culture, dual-luciferase reporter gene assay was performed to measure theactivities of frefly and Renilla luciferases in each well.2. Results and discussionPEI molecules have high density of positive charge due to protonation of its primary amine groups. By electrostaticinteraction, PEI can condense the negatively charged nucleic acid molecules to form complexes for cell transfection.However, the high density of positive charge exerts great toxic effect on cells. Although the modification of PEI withPEG can decrease PEI cytotoxicity, PEG-PEI copolymers cannot be biodegraded in vivo because the urethane linkagescannot be hydrolyzed. To enhance the biodegradability of PEG-PEI, the biodegradable PCL was introduced into PEG-PEI. Consequently, diblock copolymers mPEG-PCL were firstly synthesized and then used to replace the primaryamine groups on PEI molecules to prepare temary copolymers mPEG-PCL-g-PEI, which can also reduce the highdensity of positive charge of PEI at the same time.PEI with molecular weight ranging from 1 to 25 kDa is widely used as gene delivery vector. The transfectionefficiency of PEI enhances with molecular weight, but the cytotoxicity also increases [9]. Although PEI with lowmolecular weight shows lower cytotoxicity, its transfection efficiency also decreases. So, we selected the PEI with amoderate Mw of 10 kDa (PEIo) to synthesize mPEG-PCL-g-PEI. FTIR, NMR and GPC results showed that mPEG-PCL-g-PEI had controllable composition and molecular weight. According to the results of 'H NMR, the structuralcomposition of synthesized copolymers was identified as (mPEGsk-PCL1.zk)1.4-g-PEI1ok. GPC analysis showed that(mPEGsx-PCL1.zx).4-g-PEIok have a Mw of 19.5 kDa with a polydispersity index of 1. 195, which showed that the Mwdistributed in a narrow range (Fig. 1). Moreover, we found that the cytotoxicity of (mPEGsk-PCL1.2u).4-g-PEI1ok waslower than mPEGsk-PEIjok, which suggested that PCL introducticnihility of PEG-PELCopolymers (mPEGsk-PCL1.2z)1.4-g-PEI1ok could condense GL中国煤化工Eles (<200 nm) wthpositive zeta potential (<20 mV). With the increase of N/P ratio,C N M H Greased, and the zetapotential gradually increased (Fig. 2). In addition, the cytotoxicity ot (mPEUisk-FL1.2x)1.4-g-PEIok/GL3. Luc _siRNA nanoparticles (denoted as test) was evaluated by MTT assay. The results revealed that (mPEGsk-PCL1.2x)1.4g-PEI10:/GL3_ Luc_ siRNA nanoparticles had significantly lower cytotoxicity compared to PEIo:/GL3_ Luc _siRNAW. Huang et al./Chinese Chemical Letters 22 (2011) 749 -752751AB8+64-2十1202530Elution time (min)Fig. 1. GPC cluograms of syubesized copolymers (A) Final product (mPEGs-PCL.x)urgPELxo; (B) mediate product mPEGsrPCL1x-OH.60140 I20 t1500 t5010 t+Pricle 12一+ Zeta potcotal7:x0125NP ratioFig. 2. Particle sizce and zeta potential of (mPEGxrPCL x)4-gPElLox/CL3. Luc sRNA nanoparticles at various NP ratios.nanoparticles (denoted as control) (P< 0.05) (Fig. 3), which demonstrated that the cytotoxicity of (mPEGskPCL1.2x).4-g-PEI1ok was significantly lower than PEI1ok.The siRNA delivery potential of (mPEGsk-PCL1.2x)1 .4-g-PEI1ok was investigated using GL3_ Luc_ siRNAinterfering with the expression of frefly luciferase gene. (mPEGsk-PCL| 2x).4-g-PEI1ok condensed GL3. _Luc_ siRNAinto nanoparticles to transfect MCF-7 cells which were pre-transfected with the plasmids of pGL3-Control (target100丢80王工8 40-◆-Test-★Control中国煤化工75N/P ratio:YHCNMHGFig. 3. Infuence of (mPEGst-PCL| x)4g PEIo/GL3. Luc_ siRNA nanoparticles at various N/P ratios on MCF-7 cell viability. Test: (mPEGgrPCL1 zx).4-g-PEIqo/GL3_ Luc. siRNA nanoparticles; Control: PEIoN/GL3. Luc. siRNA nanoparticles.752W. Huang et al./Chinese Chemical Ltters 22 (20111 749-752! 1.lull100 125 150 P(NC BGroupsFig. 4. Inhibition of frefly luciferse reporter gene expression by GL3. Luc_ siIRNA delivered into MCF-7 cells using (mPEGskPCL.2x)1.4g.PEIo/GL3_ Luc siRNA nanoparticles at dfferent NP ratios (n= 3). PC: positive control; NC: negative control; BC: blank control.gene) expressing firefly luciferase and pGL4.75 (intemal control) expressing Rerilla luciferase. Compared to negativeand blank controls, the expression of frefly luciferase gene was significantly inhibited in positive control group(4p < 0.01), and could be significantly down-regulated at a range of N/P ratio from 50 to 150 in nanoparticles groups(#P < 0.05) (Fig.4). The frefly luciferase gene expression was maximally inhibited at the N/P ratio of 125, which wassimilar to the inhibition efficiency in positive control group ( P > 0.05) (Fig. 4). Therefore, it could be concluded that(mPEGsk-PCL1. 2)1.4-g-PEI1ok could delivery siRNA into cells to inhibit the expression of target gene.In conclusion, we have demonstrated that the synthesized copolymers mPEG-PCL-g-PEI were able to condensesiRNA into nanoparticles, and deliver siRNA into cells to effectively down-regulate the expression of target gene.Moreover, the cytotoxicity of mPEG-PCL-g-PEI was significantly lower than that of PEI, which suggested thatmPEG-PCL-g-PEI as siRNA delivery vector had more advantages. In the future study, the structural composition ofmPEG-PCL-g-PEI will be optimized to improve siRNA delivery efficiency so as to completely inhibit the expressionof target gene, which eventually leads to a kind of siRNA delivery vectors for clinical application.AcknowledgmentThis work was financially supported by the National Natural Science Foundation of China (No. 30873168).References[1] A.C. Grayson, A.M. Doody, D. Putnam, Pharm. Res. 23 (2006) 1868.[2] P. Chollel, M.C. Favrol, A. Hubin, et al. J. Gene Med.4 (2002) 84.[3] WJ. Kim, C.W. Chang, M. Lee, et al. J. Control. Release 118 (2007) 357.[4] A. Malek, F Czubayko, A Aigner, J. Drug Target. 16 (2008) 124.[5] 0M. Merkel, A. Beyerle, D. Librizzi, et al. Mol. Pharm. 6 (2009) 1246.[6] H. Petersen, T. Merdan, K. Kunath, et al. Bioconjug. Chem.13 (2002) 812.[7] C.L. Gebhart, S. Sriadibhatla, S. Vinogradov, et al. Bioconjug. Chem.13 (2002) 937.[8] S.B. Zhou, X.M. Deng, H. Yang, Biomaterials 24 (2003) 3563.[9] S. Hobel, R. Prinz, A. Malek, et al. Eur. J. Pharm. Biopharm. 70 (2008) 29.中国煤化工MYHCNMHG