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21-Arm star polymers with different cationic groups based on cyclodextrin core for DNA delivery

Jianshu Li a ,Zhizhang Guo a ,Jianyu Xin a ,Guanglei Zhao b ,Huining Xiao c,*

College of Polymer Science and Engineering,Sichuan University,Chengdu 610065,China

College of Light Ind.and Food Sci.,South China University of Technology,Guangzhou 510640,China c

Department of Chemical Engineering,University of New Brunswick,P.O.Box 4400,Fredericton,Canada NB E3B 5A3

a r t i c l e i n f o Article history:

Received 1July 2009

Received in revised form 24July 2009Accepted 3August 2009

Available online 9August 2009Keywords:

b -Cyclodextrin Star polymer

Gene delivery system Cell transfection Cytotoxicity

a b s t r a c t

A series of cationic star polymers with 21arms (21ACSPs)were synthesized through atom transfer radical polymerization using a b -cyclodextrin initiator with 21initiation sites.Monomers containing primary,tertiary amino and quaternary ammonium groups were polymerized using 21Br-b -CD,Cu(I)Br and 2,20-dipyridyl as initiator,catalyst and ligand,respectively.It was found that a co-solvent of 1-methyl-2-pyrrolidione and water (1:1)could facilitate the reaction,resulting in a well-controlled living polymer-ization.The conversion was up to 95%compared to the previously reported method (60%).AFM and DLS measurement revealed that 21ACSPs have the ability to condense the plasmid DNA (pCMV-Luc)to 80–180nm.21ACSPs with primary and tertiary amino groups exhibited higher cell transfection ef?ciency to CHSE-214cells than 21ACSPs with quaternary ammonium groups.Meanwhile,all of the reported star polymers have no obvious cytotoxicity.The ?ndings from this work are expected to be helpful for the development of ef?cient DNA delivery system.

1.Introduction

It has been realized that the lack of non-toxic and ef?cacious delivery systems is the greatest hurdle in exploiting the full poten-tial of gene therapy.Over the past decade,nonviral DNA vectors have been demonstrated to be a safety-reliable substitute of viral vector for use in gene therapy (Li &Loh,2008;Ooya et al.,2006;Opanasopit et al.,2009;Pack,Hoffman,Pun,&Stayton,2005;Shen,Li,Tu,&Zhu,2009).In addition to cationic lipids,cationic polymer is one of the typical kinds of nonviral DNA vector due to its capa-bility to condense DNA into small particles termed polyplexes through electrostatic self-assembly (Merdan,Kopecek,&Kissel,2002).The polyplex can bind to cell surfaces and be internalized into the cell through endocytosis.However,both extracellular and intracellular biological barriers can inhibit the approach of the therapeutic gene into the cell nucleus.Currently,the identi?ed biological barriers include the systemic barriers (e.g.,the gene tar-geting (Bellocq,Pun,Jensen,&Davis,2003))and the cellular barri-ers (e.g.,the endosomal escape after endocytosis (Kichler,Leborgne,Coeytaux,&Danos,2001)and the nuclear barrier (Brem-ner,Seymour,Logan,&Read,2004)).

Synthetic cationic polymers can be tailored for various applica-tions by choosing appropriate molecular weight (Yang,Li,Goh,&

Li,2007),suitable charge density (Reineke &Davis,2003),coupling of cell or tissue speci?c targeting moieties,and performing other modi?cation that afford them speci?c physicochemical or physio-logical properties (Merdan et al.,2002).Polyethylenimines (PEI),both branched and linear types,provide ef?cient transfection due to the well known ‘‘proton sponge property”(Boussif,Zanta,&Behr,1996).However,it was found that the cytotoxicity of PEI is rather high,which may limit its further applications (Fischer,Bieber,Li,Elsasser,&Kissel,1999).Poly(L -lysine)-based polymeric vectors are also widely investigated (Wolfert et al.,1999)due to their biodegradable property contributed by the peptide structure.Nevertheless,the DNA transfection ef?ciency of poly(L -lysine)based polymers is poor unless conjugating with speci?c ligands,such as transferrin or folate (Cho,Kim,Jeong,&Park,2005),or with the help of endosomolytic or lysosomotropic agents such as chlo-roquine (Pouton et al.,1998).Dendrimers,which are spherical and hyperbranched,are also the effective cationic polymers for gene delivery.It was believed that the star shape of dendrimers determined their excellent transfection performance:DNA interact with the surface amino groups only,leaving the internal amino groups available for the neutralization of the acid pH within the endosomal or lysosomal compartment (Lee,Wang,&Low,1996).It was also found the fractured dendrimers exhibit much higher levels (>50-fold)of reporter gene expression than that of intact dendrimers due to the better ?exibility to complex DNA (Tang,Redemann,&Szoka,1996).However,from a molecular structural

*Corresponding author.Tel.:+15064533532;fax:+15064533591.E-mail address:hxiao@unb.ca (H.Xiao).Carbohydrate Polymers 79(2010)

277–283

Contents lists available at ScienceDirect

Carbohydrate Polymers

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p o

design point of view,the structure of dendrimers is rather con-gested;and the generation of the dendrimers is thus limited to form a star with more cationic groups.Also,the?exibility of den-drimers will decrease with the increase of their generations, whereas the star polymer have better?exibility due to the lack of the connection between each generation as that of dendrimers. This literally raises the demand on the synthesis of star polymers with a star shape similar to dendrimers but does not have a limita-tion of the length of its branches.Georgiou et al.(Georgiou,Phylac-tou,&Patrickios,2006;Georgiou,Vamvakaki,Patrickios,Yamasaki, &Phylactou,2004)did the?rst study about star polymers based on 2-(dimethylamino)ethyl methacrylate(DMAEMA)as gene vectors and compared them to a commercially available dendrimer(Super-Fect).The overall transfection ef?ciency of the optimum star poly-mer to human cervical HeLa cancer cells was comparable to the SuperFect.A recent work reported by Yang et al.,have revealed that a kind of cationic star polymer consisting of a-cyclodextrin core and oligoethylenimine arms could reduce the cytotoxicity but retain or even enhance the gene transfection ef?ciency of PEI(25K)(Yang et al.,2007).Xu et al.(2009)also prepared star-shaped copolymer consisting of b-CD core and DMAEMA arms, with poly(poly(ethylene glycol)ethyl ether methacrylate))P(PEGE-EMA)as end blocks.The unique star-shaped architecture can re-duce cytotoxicity and enhance the gene transfection ef?ciently compared with that of PEI(25K).Thus further research on star polymer is expected to be promising as an effective DNA vector due to the various reasons addressed above.

Living polymerization,including atom transfer radical polymer-ization(ATRP),has been often used for synthesizing star polymers with well-de?ned structure,arm chain length in particular(Simms &Spinelli,1997).In our previous study,a special ATRP initiator with 21initiation sites(21Br-b-CD)was synthesized(Li&Xiao,2005), and followed by the success in synthesizing a range of21-arm cat-ionic star polymer(Star-p(MeDMA))with well-de?ned structures using the initiator(Li,Xiao,Kim,&Lowe,2005).The polymerization was carried out in aqueous solution and the resulting star polymers are suitable for potential biomedical applications.

The Star-p(MeDMA)was synthesized from monomer with qua-ternary ammonium groups,which will not have the‘‘proton sponge property”(Boussif et al.,1995)at physiological pH.Also, the moderate water solubility of the21Br-b-CD was proved to be a limitation to obtain21arms star polymers(Li et al.,2005).In this work,we utilized a mixed solvent of1-methyl-2-pyrrolidione and water to facilitate the living polymerization of a series of novel cat-ionic star polymers with21arms(21ACSPs)through ATRP using 21Br-b-CD initiator.In addition,monomers bearing primary,ter-tiary amino and quaternary ammonium groups,which have been polymerized into linear polymers as DNA vectors and with suitable p K a values(Rémy-Kristensen,Clamme,Vuilleumier,Kuhry,&Mély, 2001),were all used in an attempt to identify the polymer struc-ture–bioactivity relation in terms of physicochemical properties, cell transfection ef?ciency and cytotoxicity.

2.Experimental

2.1.Materials

Heptakis[2,3,6-tri-O-(2-bromo-2-methylpropionyl)]-b-cyclodex-trin(21Br-b-CD)was synthesized using an improved synthetic-route developed in our previous work.Ethylenediamine,glycidyl methacrylate,2-(Dimethylamino)ethyl methacrylate(DMAEMA), N-[3-(Dimethylamino)propyl]methacrylamide(DMAPMAm)and [2-(methacryloyloxy)ethyl]trimethyl ammonium chloride(MeD-MA)were obtained from Aldrich.Cu(I)Br,2,20-bipyridine(bpy), 1-methyl-2-pyrrolidione(NMP),branched polyethylenimine(PEI,25KDa),chloroquine and other regents were purchased from Al-drich and used without further puri?cation.GPC standard samples, Pullulan(P-82,a linear macromolecular polysaccharide that con-sists of links of maltotriose)from P-5to P-800,were obtained from Shodex.Spectra/Por dialysis membrane(MWCO1000)was pur-chased from Spectrum Laboratories Inc.Distilled–deionized water was used in all the experiments.

Plasmid pCMV-Luc containing the luciferase as a reporter gene was obtained from Plasmid Factory,Bielefeld,Germany.CHSE-214 (Chinook salmon embryo)cell line was purchased from American Type Culture Collection(ATCC).Both luciferase assay kit and pro-tein BCA assay kit were purchased from Sigma.

2.2.Synthesis and characterization of21arms cationic star polymers (21ACSPs)

First,the cationic monomer with primary amino group,i.e.,[2-hydroxy-3-(2-aminoethyl)amino]propyl methacrylate(HAE-APMA),was synthesized as follows:Dropwise add14.66g glycidyl methacrylate into the mixture of6.02g ethylenediamine in30ml H2O.The reaction was then carried out at room temperature for additional45min.The obtained HAEAPMA was precipitated and washed by cold diethyl ether(3?50ml)(Anal.C9H18N2O3,C,H,N; FTIR(zinc selenide):1717cmà1(m C=O),1042cmà1(m C–N);1H NMR (D2O,300MHz):d2.72(N–H),d1.3(C–H).).The polymerizations of HAEAPMA as well as other cationic monomers with tertiary amino or quaternary ammonium groups,i.e.,2-(Dimethylamino)ethyl methacrylate(DMAEMA),N-[3-(Dimethylamino)propyl]meth-acrylamide(DMAPMAm)and[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride(MeDMA),were conducted under the same conditions as those addressed in our previous paper(Li et al., 2005).The only difference was that the polymerization proceeded in a co-solvent of1-methyl-2-pyrrolidione(NMP)and water at a ratio of1:1instead of water alone.The resulting21ACSPs were puri-?ed with dialysis membrane(MWCO1000)against water for24h prior to use.

1H NMR spectra of the samples in D

O were acquired using a Var-ian Unity400spectrometer operated at300MHz in order to quantify the monomer conversion in ATRP.The conversion of monomer was determined by comparing the peak integrals of the monomer vinyl signals at d5.5and d6.0to those of the methacrylate backbone at d0.5–1.1and d1.5–2.0(Li,Armes,Jin,&Zhu,2003).Apparent charge density of the21ACSPs was determined via colloidal titration using a Particle Charge Detector Mütek PCD03(Herrsching,Germany).

0.2ml of0.1%polymer solution was added into the sample cell containing9.8ml water.The solution was titrated with a standard anionic polyelectrolyte(potassium polyvinyl sulphate(PVSK)solu-tion)(concentration=1mM).The charge densities of the star poly-mers were estimated from the volume of the anionic polymer solution required to reach the end point during titration.Three re-peats were conducted to acquire an average value for each sample. Gel permeation chromatography(GPC)(Pump:Waters600E System Controller;Detector:Waters410Differential Refractometer)was carried out to determine their molecular weights with Ultrahydrogel 250and Ultrahydrogel500columns at40°C and the?ow rate at 0.7ml/min.Water was used as an eluent.Calibration was made using standard Pullulan samples from P-5to P-800.The standard sample aqueous solutions(0.2%w/v)were?ltered with a0.45l m Nylon Cameo?lter-syringe before the test.

2.3.Preparation of21ACSPs/DNA polyplexes

Stock21ACSPs and luciferase plasmid(pCMV-Luc)solutions were prepared in PBS buffer at pH=7.4at various molar ratios of N/P up to10prior to each experiment.Polyplex formation always utilized the solutions of equal volumes with the polymer solution

278J.Li et al./Carbohydrate Polymers79(2010)277–283

being added to the DNA solution,not the opposite sequence(Bous-sif et al.,1996).The mixed solution was vortexed and incubated at room temperature for30min before use.Polyplexes were freshly prepared before each individual measurement.

2.4.Zeta-potential measurements of21ACSPs/DNA polyplexes

The DNA concentration was held constant at20l g/ml,whereas the N/P ratios of the21ACSPs/pCMV-Luc polyplexes were varied. An electrophoresis meter DELSA440SX(Coulter,USA)was used to determine the zeta-potential of the polyplexes in the presence of0.1mM NaCl in aqueous solution at25°C.The system was rou-tinely calibrated using aà55mV standard.Three repeats were conducted to acquire an average value for each test.

2.5.Particle characterization by atomic force microscopy(AFM)and dynamic light scattering(DLS)

Imaging samples of21ACSP alone were prepared at a concentra-tion of100l g/ml.Samples containing polyplexes in water were ?xed at a?nal DNA concentration of10l g/ml.Samples were deposited onto the center of a freshly split untreated mica disk (Agar Scienti?c Inc.).Following the adsorption for1–2min at room temperature,excess?uid was taken off by absorption onto?lter paper.The mica surface was dried at room temperature before imaging.A Nanoscope IIIa controller with a Multimode Pico-Force AFM(Veeco Instruments Inc.,Santa Barbara,CA)was used.AFM imaging was performed in air in Multimode using silicon tapping tips(NP-S20,Veeco Instruments).

Dynamic light scattering(DLS)size determination:21ACSP/ DNA polyplexes were prepared as previous procedure and then di-luted with PBS to a?nal concentration of1l g DNA/ml.Polyplex size determination was performed on a DynaPro-MS800(Protein Solutions,Lakewood,NJ)with Dynamics v6.3software.Samples were run in triplicate.

2.6.Transfection in vitro and cell viability

CHSE-214cells were seeded in12well plates at a density of 50,000cells per well one day before transfection.At the day of transfection,the cells have grown to approximately60–80%con-?uence.Transfection experiments were performed with4l g of pCMV-Luc per well as follows:prepare polyplexes with adding appropriate amount of21ACSP in300l l PBS buffer to4l g plasmid in300l l PBS buffer(pH=7.4).Remove the medium and rinse the cells with4°C PBS(3?5min?2ml).Add the polyplexes in1ml of fresh medium without FBS.Then the cells were incubated at room temperature for4h.After that,the medium was exchanged and the cells were incubated for additional40h.Luciferase gene expression was quanti?ed using a Luciferase Assay Kit(Sigma) with a POLARstar OPTIMA luminometer(BMG LABTECH,USA).Re-sults in relative light units per second(RLU/s)were converted into nanograms of luciferase by creating a calibration curve with lucif-erase standard(Sigma).Protein concentration in each sample indi-cating cell viability was determined using BCA Protein Assay Kit. The?nal transfection results were expressed in nanograms of lucif-erase per milligram of protein.pCMV-Luc alone was used as a con-trol and data is reported from six separate experiments.

3.Results and discussion

3.1.Synthesis of21ACSPs by ATRP in co-solvent

Living polymerization,including atom transfer radical polymer-ization(ATRP),has often been used for synthesizing star polymers with well-de?ned structure by both core-?rst and arm-?rst meth-ods(Simms&Spinelli,1997).In our previous work(Li&Xiao, 2005),we developed a novel ATRP initiator(21Br-b-CD)with21 initiation sites based on b-cyclodextrin(b-CD).The21sites in b-CD are due to its cyclic oligosaccharide consisting of seven glu-cose units linked by a-1,4-glucosidic bonds and each unit having three substitutable hydroxyl groups.After preparing the initiator, we synthesized a cationic star polymer(Star-p(MeDMA))in aque-ous medium and obtained some positive results(Li et al.,2005). However,we found that the limited water solubility of21Br-b-CD restricted its induction of ATPR in preparing the star poly-mers with a wide range of molecular weights.For example,at a higher initiator concentration of100/1/2/5([M]0/[I]0/[Cu(I)0/ [L]0])at40or60°C which was aimed at acquiring a star polymer with low molecular weight,the ATRP process,however,lost con-trol and resulted in the polymer with very high molecular weight. The reason is simple:the21Br-b-CD initiator could not be com-pletely dissolved at this condition and the low initiator ef?ciency would generate much higher molecular weight with broad polydis-persity.A lower initiator concentration at500/1/2/5([M]0/[I]0/ [Cu(I)0/[L]0])and a higher reaction temperature at90°C have been proved feasible to synthesize a star polymer with high molecular weight,e.g.,79,400,in our previous work.

However,star polymers with lower molecular weight(thus smaller star size)could facilitate the formation of DNA/polymer polyplexes with smaller size,which has been believed to be more desirable to achieve a high transfection ef?ciency(Kunath,von Harpe,Fischer,&Kissel,2003);.Thus,we proposed to optimize the polymerization conditions based on21Br-b-CD ATRP initiator by utilizing1-methyl-2-pyrrolidione(NMP)as solvent.Preliminary experiment was performed in NMP alone,which led to a successful polymerization in homogeneous system and resulted in a star polymer with well-controlled structure.Having considered the sol-ubility of hydrophilic cationic monomers,we tried NMP/water co-solvent and gradually reduced the NMP content in the entire sys-tem until the lowest ratio of NMP to water(1:1)was reached. Fig.1shows the chemical structures of these21arms cationic star polymers(21ACSPs)synthesized in the mixed solvent using vari-ous cationic monomers.As can be seen,all the21ACSPs radiate from a common b-cyclodextrin core but have different types of amino functional groups,i.e.,primary amino,tertiary amino and quaternary ammonium groups.It is also worth noting that the per-formance of Star-p(HAEAPMA)might be contributed from the sec-ondary amino groups although they may not be as important as the primary amino ones due to the steric hindrance.

In this work,we?xed the ratio of monomer:initiator:catalyst: ligand at100/1/2/5for all the21ACSPs except for Star-p(MeDMA) which was still synthesized at various ratios.Polymerization tem-perature was kept at60°C for all.An obvious change observed

was

Fig.1.Chemical structures of21ACSPs.(a)Star-p(HAEAPMA);(b)Star-p(DMA-EMA);(c)Star-p(DMAPMAm);(d)Star-p(MeDMA).

J.Li et al./Carbohydrate Polymers79(2010)277–283279

that the reaction system became homogenous rather than hetero-geneous as previous systems due to the incomplete dissolvability of the initiator in water at 60°C.

Table 1lists the results of the polymerizations.As it can be seen,the homogenous polymerization system brought by NMP/water co-solvent resulted in two signi?cant improvements compared with our previous report (Li et al.,2005):First,the molecular weights of the polymers with well-de?ned structure are close to the designed values at wide temperature and monomer concentra-tion ranges.The previous reaction system in water can only obtain the well-de?ned star polymer at a high monomer concentration and a high temperature,i.e.,500/1/2/5([M]0/[I]0/[Cu(I)0/[L]0])at 90°C,in which all the initiator can be totally dissolved at the beginning of the polymerization.Otherwise at low monomer con-centration (thus high initiator concentration)and low tempera-ture,the incomplete dissolved initiator would cause low initiation ef?ciency and result in unexpected high molecular weight (e.g.,polymerization of MeDMA at 100/1/2/5and 60°C lead to a star polymer with molecular weight as high as 500,500instead of the theoretical value of 15,600)(Li et al.,2005).As shown in Ta-ble 1,we synthesized MeDMA in the 1:1NMP/water co-solvent at different monomer:initiator ratios,ranging from 20/1to 250/1;and produced all Star-p(MeDMA)s with the molecular weights close to the designed values.Secondly,the monomer conversion is improved remarkably.The conversion of MeDMA polymeriza-tion in water alone varied from 39%to 70%under different condi-tions in our previous study (Li et al.,2005).The current co-solvent polymerization system enabled us to obtain star polymers with high monomer conversion up to 95%,which is comparable to the high conversion reported for MeDMA linear polymer (Li et al.,2003).The monomer conversion in the synthesis of star polymers is strongly dependent on the steric hindrance.Low initiation ef?-ciency could lead to undesirable high molecular weight and con-gest star branches,thus preventing the monomers from approaching to initiation site and reducing the conversion.The polymerization in co-solvent eliminated the uncontrolled growth of the star branches,and thus increasing the accessibility of mono-mers toward initiation sites.It should also be noted that an appro-priate polymerization time (e.g.,6h)could decrease the coupling termination between two or more star polymers,which may gen-erate a multiple-core star polymer as addressed elsewhere (Li et al.,2003;Ohno,Wong,&Haddleton,2001).

Meanwhile,we found the charge densities of the 21ACSP were in?uenced signi?cantly by their functional groups as shown in Ta-ble 1.The star polymers with primary amino groups and tertiary amino groups,i.e.,Star-p(HAEAPMA),Star-p(DMAEMA)and Star-p(DMAPMAm),have much lower charge density than that of Star-p(MeDMA)with quaternary ammonium groups.The differ-ence originates from their different principles in rendering the polymers cationic.The quaternary ammonium group carries a per-manent cationic –N +(CH 3)3Cl àon its structure,whereas only part

of the primary and tertiary amino groups in the star polymer could be positively charged by protonation process.It is to be noted here that direct titration method was used instead of reverse titration method to measure the surface charges of star polymers since the information is more important to evaluate their abilities to condense DNA.

Figs.2and 3present the kinetic data of Star-p(MeDMA)3at an initial ratio of 250/1/2/5([M]0/[I]0/[Cu(I)0/[L]0])synthesized at 60°C.A good linear relationship of the semilogarithmic plot of monomer concentration vs.time was observed,which implies that the radical concentration was constant throughout the polymeri-

Table 1

Conditions and results of ATRP for 21ACSPs.Samples

[M]0/[I]0/[Cu]0/[L]0a Temp.(°C)Time (h)Conv.(%)b M n ,th c M n ,exp M W /M n Charge density (?10à3eq/g)d Star-p(MeDMA)120/1/2/560695.780606880 1.15 5.10Star-p(MeDMA)2100/1/2/560692.82322021450 1.26 5.98Star-p(MeDMA)3250/1/2/560695.55365049600 1.30 6.12Star-p(HAEAPMA)100/1/2/560690.62220020800 1.180.262Star-p(DMAEMA)100/1/2/560691.21822017400 1.190.305Star-p(DMAPMAm)

100/1/2/5

93.0

19800

18700

1.22

0.440

a [M]0,[I]0,[Cu]0and [L]0represent initial monomer concentration,21Br-

b -CD,Cu(I)Br and ligand (2,20-bipyridine),respectively.b Determined by 1H NMR.

c Calculate

d from th

e [I]0and monomer conversion.d

Tested without pH

adjustment.

Fig.2.Conversion vs.time data and semilogarithmic plot of monomer concentra-tion vs.time for Star-p(MeDMA)3synthesized at 250/1/2/5and 60°

Fig.3.Evolution of M n and M W /M n as a function of monomer conversion for the polymerization of Star-p(MeDMA)3at 250/1/2/5and 60°C.

280J.Li et al./Carbohydrate Polymers 79(2010)277–283

zation process (see Fig.2).The maintaining of free radical concen-tration constant was affected by three aspects:(1)the well-de?ned and extended con?guration of star chains.In the process of ATRP,the initiation sites,i.e.,–(CH 3)2Br,are supposed to locate on the outer surface of the initiator core.However,the initiation site could be entangled into the internal of the star due to the substantial increasing of the star branch chain length if the reaction is out of control.As a result,the initiation of further polymerization be-comes impossible (Li et al.,2005).The unexpected increasing of star branches can be avoided by the using of NMP/water co-solvent as addressed above,and thus helping to keep the radical concen-tration constant.(2)The side reactions due to the polar polymeri-zation medium (water).It has been found that the poor control of the aqueous ATRP of hydrophilic methacrylates could be brought by the side reactions in the water because water can displace the halide ligand from the copper(I)-mediated ATRP catalyst and dete-riorate the stability of the living radicals (Tsarevsky,Pintauer,&Matyjaszewski,2004).The decreasing amount of water medium in this system is bene?cial to this aspect.(3)Coupling termination between star polymers.In our previous study using gel permeation chromatography (GPC),we found that prolonged polymerization at a high temperature (13.5h at 90°C)resulted in a M W doubling the designed value.We also found some splits or small peaks corre-sponding to high M W in the trace chromatography of GPC in some cases.

The phenomena above indicated the importance of controlling the coupling termination in synthesizing well-de?ned star poly-mers.The homogeneous system provided by NMP/water solvent made it possible to synthesize the 21ACSPs at a lower temperature and potentially eliminated the combination termination of free radicals among star polymer chains.

As can be seen in Fig.3,in the range up to 95%conversion,the M n values increased almost linearly with increasing conversion and the polydispersity remained relatively constant.Otherwise,there could be a sharp increasing point of the M n as well as for the polydispersity if a large amount of coupling termination hap-pened.The result indicated that the polymerization proceeded in a good living protocol and the side reactions such as termination and transfer reactions were thus negligible.

3.2.21ACSPs/DNA polyplexes

The zeta-potential of the polyplexes was determined as a func-tion of N/P ratio and the results are presented in Fig.4.It is well known that the surface charge of the polymer/DNA polyplex has great impact on its biological functions:usually a high surface zeta-potential makes it easier for the polyplexes to be taken into

the cells by endocytosis (Rémy-Kristensen et al.,2001),resulting in high transfection ef?ciency,especially in vitro (Takeuchi et al.,1996).However,it may also bring some undesired effects,e.g.,non-selectivity to any cells when applied in vivo (Ooya et al.,2006)and higher cytotoxicity (Ogris,Brunner,Schüller,Kircheis,&Wagner,1999).Thus it would be desirable if we can control the zeta-potential of the DNA polyplexes in a wide range.Star-p(MeDMA)2was chosen to compare with other 21ACSPs in order to keep the same monomer/initiator ratio and similar molecular weight.As can be seen in Fig.4,Star-p(MeDMA)2/DNA complex has the highest zeta-potential value at each N/P ratio.This should be due to its high charge density contributed from quaternary ammonium groups as we presented in Table 1.All the 21ACSPs are capable of forming polyplexes with DNA and the zeta-poten-tials of these polyplexes rang from negative,slightly positive and relatively strong positive,depending on different N/P ratios.

The results above provide useful information for further various transfection applications in vitro or in vivo.Star-p(DMAEMA),Star-(DMAPMAm)and Star-p(MeDMA)2all can complex DNA and alter the negative zeta-potential surface (about à40mV)of DNA to po-sitive at a N/P ratio around 3.Star-p(HAEAPMA)/DNA exhibited the weakest capability for this purpose and it needs a N/P ratio of 6to reverse the surface charge.It seems that 21ACSP converts the sur-face charge at a higher N/P ratio compared with linear polymers synthesized from a same monomer.For instance,the charge rever-sal for linear p-(DMAEMA)/DNA and p(DMAPMAm)/DNA occurred at N/P ratio around 2:1(van de Wetering,Moret,Schuurmans-Nieuwenbroek,van Steenbergen,&Hennink,1999).As for the star shape,some functional groups,i.e.,cationic amino ones,locate in the internal of the star and cannot be utilized to bind with DNA.Only the cationic groups on the peripheral surface can conjugate with DNA;thus it is reasonable that more star polymers are re-quired to convert the surface charge of DNA.However,the excess amino groups in the internal of the star structure might be useful to act as the ‘‘proton sponge”for pH neutralization as those in den-drimers (Lee et al.,1996).

Morphological observations on 21ACSP and 21ACSP/DNA poly-plexes using AFM are presented in Fig.5.AFM scanning in a tap-ping mode was performed for all the 21ACSPs synthesized in this work.A typical AFM image is shown in Fig.5(a)(Star-p(MeDMA)2),in which the star shape of the polymers is visible but the exact 21arms of the polymer cannot be identi?ed.This result seems to be con?icted with the design of the 21star branches.Following the previous method (Li et al.,2005),we hydrolyzed the star polymers under 1%potassium hydroxide for 60min to cleave the ester link-age between the b -CD core and arms;and then measured the molecular weight and M W /M n of the star arms to determined the number of arms.It was proved that the 21ACSPs indeed have 21arms.Therefor,the failure in distinguishing each arm might be associated with the sample preparation process for AFM.The star polymers based on b -cyclodextrin core should have a three-dimen-sional structure,attributed to the three-dimensional feature of cyclodextrin.After 21ACSPs were dried on the surface of the mica disk,the 21arms cannot maintain well extended in three dimen-sions as in medium.Most of the arms aggregate together thus we can only view a star shape with several arms.

The size of the Star-p(MeDMA)2is around 1l m in diameter as shown in Fig.5(a).However,since the molecular weight of Star-p(MeDMA)2is only 21,450,this AFM image could result from the aggregates of several star polymers.The aggregation likely oc-curred when the star polymer was dried on the mica disk prior to being observed under AFM.Apparently,the sample preparation is crucial for acquiring AFM image with high resolution.The origi-nal size of plasmid DNA is around 600nm.After having conjugated with DNA at a ratio of N/P =6,Star-p(MeDMA)2condense the DNA to a size of 80–180nm as shown in the phase image of

AFM

Fig.4.Zeta-potential of 21ACSPs/DNA polyplexes at different N/P ratios (j :Star-p(HAEAPMA)/DNA;h :Star-p(DMAEMA)/DNA;N :Star-p(DMAPMAm)/DNA;4:Star-p(MeDMA)2/DNA).

J.Li et al./Carbohydrate Polymers 79(2010)277–283281

(Fig.5b).Dynamic light scattering measurement also provide addi-tional information as the hydrodynamic radii of Star-(MeDMA)2/DNA polyplex at N/P =6in PBS (pH 7.4)was 71.0±4.5nm.The small size of the polyplexes is essential for transferring plasmid DNA into cell membranes and nucleoli.

3.3.In vitro transfection and cytotoxicity

The polyplexes used for cell transfection experiment were formed at N/P =3,6,10based on the results of zeta-potential mea-surements.Also,Star-p(MeDMA)2was selected to maintain the same monomer/initiator ratio and the molecular weight similar to other 21ACSPs.All the polyplexes are either slightly or strongly positively-charged,except for Star-p(HAEAPMA)/DNA at N/P =3which is slightly negative.The CHSE-214cell transfection ef?cien-cies reached by the polyplexes above are presented in Fig.6in terms of ng luciferase per mg https://www.wendangku.net/doc/bf1973469.html,bining the results in Table 2and Fig.6,it can be found that N/P ratio has profound impact on the cytotoxicity and cell transfection ef?ciency for each 21ACSP/DNA polyplex.The similar trend,i.e.,the transfection

ef?ciency increased but the cell viability decreased with the in-crease of the N/P ratio,was observed for all polyplexes.This phe-nomenon is well known,and has been attributed to the increasing amount of free polycation of the polyplexes,as well as to an increase of polyplex uptake (Shuai,Merdan,Unger,Wittmar,&Kissel,2003;Takeuchi et al.,1996).Meanwhile,the cytotoxicity of all the polyplexes to CHSE-214cell are lower than that of PEI as shown in Table 2,demonstrating their excellent cell biocompatibil-ity and great potential as DNA vectors.However,additional in vivo toxicity measurements are still necessary in further work for its application in the body.

The different functional groups on the star polymers also play a vital role in their biological performance.The Star-p(MeDMA)2alone exhibited almost no transfection.This result is not surprising because Star-p(MeDMA)2was polymerized from a quaternary ammonium monomer,which cannot be further protonated in the acid environment of the endosome.Thus,Star-p(MeDMA)2/DNA polyplexes might be trapped in endosome and cannot be released or destroyed within this organelle prior to releasing.This problem can be somehow addressed by adding an endosomolytic agent,i.e.,chloroquine,as shown in the Fig.6.Clearly,Star-p(MeDMA)2/DNA at N/P =10achieved a transfection ef?ciency about 0.38ng lucifer-ase/mg protein with the help of chloroquine.The performance of chloroquine in promoting the endosomal escape is consistent with those reported elsewhere (Pouton et al.,1998).However,the ef?-ciency is still signi?cantly lower than that of other 21ACSPs (see Fig.6).Since chloroquine helped the endosomal escape in this case,its low transfection ef?ciency was due to the lower af?nity be-tween quaternary ammonium groups and DNA under physiological conditions as reported by Reschel,Konak,Oupick,Seymour,

and

Fig.5.Atomic force microscopy (AFM)study of 21ACSP and 21ACSP/DNA polyplexes.(a)Star-(MeDMA)2;(b)phase image of Star-(MeDMA)2/DNA polyplexes at N/P =

Fig. 6.CHSE-214cell transfection ef?ciency by 21ACSP/DNA polyplexes (the negative control sample,i.e.,the pCMV-Luc alone,exhibited almost no transfection ef?ciency).

Table 2

CHSE-214cell viability in the transfection with 21ACSP/DNA polyplexes.a

Polyplexes

Cell viability b %N/P =3

N/P =6N/P =10Star-p(MeDMA)2/DNA

100.0±2.594.0±3.384.8±3.0Star-p(MeDMA)2/DNA +Chloroquine c 93.7±3.184.2±2.377.4±2.5Star-p(HAEAPMA)/DNA 79.6±3.579.5±3.077.2±2.0Star-p(DMAEMA)/DNA 100.0±2.892.2±2.588.6±2.2Star-p(DMAPMAm)/DNA 88.1±3.278.2±2.677.8±2.3PEI/DNA

75.9±3.0

52.4±2.2

10.7±2.7

Plasmid DNA without star polymers has a cell viability of 90.5±3.5%in the parallel tests.b

Calculated based on the protein concentration tested by BCA assay.c

Chloroquine was added with the polyplexes.

282J.Li et al./Carbohydrate Polymers 79(2010)277–283

Ulbrich(2002).In addition,the use of chloroquine decreased the biocompatibility of the polyplexes.As shown in Table2,Star-p(MeDMA)2/DNA polyplexes have100%,94%and84.8%cell viabil-ity at N/P=3,6and10,respectively.After conjunction with chloro-quine the corresponding viabilities were reduced to93.7%,84.2% and77.4%,respectively.On the other hand,Star-p(HAEAPMA)/ DNA and Star-p(DMAPMAm)/DNA polyplexes have comparable transfection ef?ciency to that mediated by PEI,but higher than that of Star-p(DMAEMA)/DNA.The difference should be caused by dif-ferent p K a values in physiological pH,which are associated with their structure and functional groups as reported by others(Shuai et al.,2003).It is also of interest that Star-p(HAEAPMA)/DNA at N/ P=3still exhibited the transfection ability to a certain extent although the polymer has a slightly negative zeta-potential.It is likely attributed to the cationic amino groups in the internal sec-tion of the star polymer.Those groups are not bound with DNA while forming the polyplexes but could help the endocytosis pro-cess owing to its ability to neutralize the protons under physiolog-ical conditions,thus leading to the endosomal escape as dendrimers do(Lee et al.,1996;Tang et al.,1996).

4.Conclusions

In this study,a series of21arms cationic star polymers were successfully synthesized by an improved atom transfer radical polymerization performed in the co-solvent of NMP and water at ratio1:1.The living polymerization proceeded in a well-controlled manner,thus allowing all the21ACSPs to possess the well-de?ned structure and the molecular weights close to theoretical ones.The polyplexes formed by21ACSPs and plasmid DNA were character-ized by zeta-potential measurement as well as AFM image.The peripheral surface charges of polyplexes range from negative to positive,depending on different polymer/DNA ratios;and the typ-ical size of polyplexes is within80–180nm.21ACSPs exhibited sat-isfactory transfection ef?ciency of plasmid pCMV-Luc to CHSE-214 cells.In addition,the cytotoxicity of the polyplexes is lower than that of PEI during the transfection.

Acknowledgements

Financial supports for this work from the NSERC Canada and the National Natural Science Foundation of China(20704026)are gratefully acknowledged.

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宽带卫星通信技术的现状与发展 -标准化文件发布号：（9456-EUATWK-MWUB-WUNN-INNUL-DDQTY-KII

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ArcGIS格式的转换方法

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