journal of materials chemistry b - colcom€¦ · j mater chem b this journal is ' the royal...

This journal is©The Royal Society of Chemistry 2016 J. Mater. Chem. B

Cite this:DOI: 10.1039/c5tb02224j

Harnessing the PEG-cleavable strategy to balancecytotoxicity, intracellular release and thetherapeutic effect of dendrigraft poly-L-lysine forcancer gene therapy

Min Tang,a Haiqing Dong,*b Yongyong Lib and Tianbin Ren*a

Dendrimer catiomers like dendrigraft poly-L-lysine (DGL) have been very popular vectors for gene

delivery recently; however, they generally suffer from serious cytotoxicity for high density of positive

charge. PEGylated DGL engineered using the PEG cleavable mechanism (DGL(R)-SS-mPEG) was first

developed as a non-viral gene vector for cancer intervention. Cleavable PEGylation of the DGL catiomer

in tumor relevant glutathione (GSH) conditions enables us to dramatically decrease the cytotoxicity as

well as to promote the intracellular release and expression of the genetic payload. Like DGL, DGL(R)-SS-

mPEG is capable of efficiently complexing with plasmid DNA (pDNA) to afford homogeneous compact

nano-complexes. Those gene carrying nanostructures could be stably dispersed in the regular serum

medium without GSH, but with fast PEG dis-assembly if subject to 10 mM GSH. Compared with the

non-cleavable counterpart, PEG-cleavable dendrigraft poly-L-lysine exhibited significantly higher

enhanced green fluorescence protein (EGFP) expression against 293T cells. By using small interfering

RNA (siRNA-VEGF) as the therapeutic gene payload, the complex nanoparticles demonstrated the

pronounced inhibition effect on cell growth in vitro and tumor growth in vivo. The promising results

revealed a universal strategy to balance disadvantages and advantages of dendrimer catiomers for future

non-viral gene delivery vector.

Introduction

Gene therapy has been developed over the past two decades forcancer treatment, which is designed to transfer therapeuticgenes to targeted cancer cells using vector systems. Vital torealize gene efficacy, the vector systems remedy the defectsof naked gene which can be degraded in vivo quickly with-out protection. Non-viral vectors, especially cationic polymers,are highly promising vehicles for their weak immunogenicity,controlled synthesis, straightforward production and respect-able gene-loading capacity.1,2 However, excess positive chargeson the surface of cationic polymers may disturb the structure ofcell membranes, leading to unintended toxicity.3 Excess posi-tive charges also lead to limited stability of the gene complexesdue to the reticuloendothelial system (RES).4,5 Therefore, surfacemodification and functionalization is essential to yield improvedgene delivery systems.

PEGylation has become a leading coating strategy to reducethe toxicity of vector systems, enhance biocompatibility, aswell as escape the recognition by RES.6,7 A significant benefitof PEGylation has been the prolonged in vivo circulation timein the cardiovascular system,8 which is crucial for improvingdrug/gene bioavailability. Permanent PEGylation, however,will weaken the interaction between gene vectors and cellmembranes, and pose a significant diffusion barrier to intra-cellular release of genes. So considerable efforts have beendevoted to solve this problem. Ooya and co-operators designeda biocleavable polyrotaxane with a disulfide-introducedpoly(ethylene glycol) (PEG). The cleavage of disulfide linkagesleads to triggering pDNA decondensation (pDNA release).9

Our group has developed cleavable PEGylation on linearpolylysine as a gene vector for gene therapy.10–14 In ourprevious studies, cleavable PEGylation was achieved by incor-poration of the disulfide bond between the PEG segment andpolylysine. During blood circulation, the disulfide bondremains stable. However, the detachment of the PEG layertakes place selectively via disulfide bond cleavage takingadvantage of tumor relevant GSH conditions (four-fold higherthan normal cells and hundred times difference betweenintracellular and extracellular parts).15 The rapid degradation

a School of Material Science and Engineering and Institute for Biomedical

Engineering & Nano Science, Tongji University, Shanghai, P. R. China.

E-mail: [email protected] Shanghai East Hospital, The Institute for Biomedical Engineering & Nano Science

(iNANO), Tongji University School of Medicine, Shanghai 200092, P. R. China.

E-mail: [email protected]

Received 24th October 2015,Accepted 14th January 2016

DOI: 10.1039/c5tb02224j

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occurring in reducing environments could selectively increaseintracellular drug release.16

Dendrimers, with a unique three-dimensional, highly branchedbut monodispersed structure, are being widely investigated forgene delivery.17 High amine density on the surface of dendrimersaffords strong gene binding capacity.18 These reactive groups cannot only interact with nucleic acids, but also be modified orlabelled for specific biomedical applications.19,20 Among them,the polyamidoamine (PAMAM) dendrimer is the first dendrimerused for gene transfection due to their high efficacy,21 but it iscytotoxic and non-biodegradable.22 In contrast, dendrigraft poly-L-lysine (DGL) constituted by naturally derived lysine is relativelybiocompatible and biodegradable. Therefore, DGL has beenrecently explored for gene delivery by different chemical andbiological modifications including PEGylation, bioconjugationwith the doxorubicin (DOX) drug,23 the chlorotoxin (CTX)ligand,24 or the peptide.25 However, as a member of cationicpolymers, inherent cytotoxicity and limited bio-stability of DGLare still the concerns of the application in vivo. In particular,DGL significantly inhibits the cell viability at extremely lowconcentration (10 mg mL�1, data shown below) insufficient forthe regular gene transfection study.

Herein, we reported and developed a novel DGL vector withcleavable PEGylation for pDNA and siRNA delivery. The primaryamines of DGL were partly guanidinylated to promote cellularuptake.26 Disulfide bonds were introduced between the DGLmatrix and PEG chains by thiol-disulfide exchange reactionto achieve cleavable PEGylation. The chemical modificationsdecreased the cytotoxicity of DGL and improved the bio-stability of genetic payloads. The cleavage of the PEG layer undertumor relevant GSH conditions was investigated in terms ofcellular uptake, intracellular release and gene expression. Theprocess is shown in Fig. 1. The cytotoxicity, redox-sensitivity,transfection efficiency, bio-distribution, and gene therapy effectin vivo were evaluated.

Results and discussionSynthesis and characterization of dendrimers with/withoutcleavable PEGylation

With increase of generation, DGL carries more cationic chargeswhich can deteriorate bio-stability in bloodstream. And it isreported that transfection efficiency will decrease if DGL haslow generation (such as G2).27 So G3 has been chosen as a usualscaffold by some reported studies.23,28,29 Guanidinylation of DGL-G3 was first performed with 1H-pyrazole-1-carboxiamidine hydro-chloride and DIPEA. Previous studies have demonstrated thatguanidinylation is able to promote the cellular uptake of polyplexnanoparticles, leading to an improved transfection efficiency.30–33

For cleavable PEGylation, disulfide bonds were introduced bythe thiol-disulfide exchange reaction between SPDP modifiedDGL and mPEG-SH. The DGL(R) without cleavable PEGylation,DGL(R)-mPEG, was synthesized by conventional condensationreaction between amino groups of DGL(R) and carboxyl groupterminated PEG, which was employed for control experiments.The synthesis route of dendrimers with/without cleavable PEGyla-tion is schematically illustrated in Fig. 2.

1HNMR spectra are shown in Fig. 3. Peaks between 1 and1.8 ppm are assigned to three –CH2– in lysine units. The peak at2.9 ppm represents –CH2–NH2, while that near 4.2 ppm isrepresentative to H on the chiral carbon in the lysine structure.All of the DGL derivatives possess a peak at around 3.05 ppmattributed to the protons next to guanidine groups and a sharppeak near 3.52 ppm owing to the –OCH2– in the repeat mono-mers of mPEG. The integrated areas of 1HNMR signals werecalculated to quantify the quantities of guanidine groups andPEG chains per DGL-G3 assuming 2 protons in –CH2– next toone guanidine group, 44 repeated monomers of PEG, and 123lysine groups per DGL-G3. The resulted molecular ratios of[DGL]: [guanidine groups]: [PEGylation] are shown in Fig. 3.DGL(R)-mPEG without cleavable PEGylation have similar

Fig. 1 Schematic illustrations of disulfide bridged PEG-cleavable DGL(R)-SS-mPEG complexes for the EPR effect, cellular uptake, intracellular releaseand subsequent gene silence (a). The assembled formulation and compositions of DGL(R)-SS-mPEG complexes is also shown in panel (b).

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composition with DGL(R)-SS-mPEG1. DGL(R)-SS-mPEG2 withlower PEGylation was also set to investigate the effect ofPEGylation content. The guanidinylation degree was kept thesame for all of three PEGylated DGL.

Agarose gel electrophoretic analysis

The gene compact capacity of different dendrimers as wellas resistance to DNase I degradation after gene loadingwere evaluated by agarose gel electrophoretic analysis. Asshown in Fig. 4, the pDNA could be both effectively loadedby DGL(R)-SS-mPEG1 and DGL(R)-SS-mPEG2. The mobility ofpDNA in the gel was completely retarded at the weight ratiogreater than two. For siRNA complexation (Fig. 4b and d), a

slightly higher weight ratio is necessary for complete retarda-tion by DGL(R)-SS-mPEG1, due to the higher PEGylation extentcompromising the gene compaction capability.34 This resultalso interpreted the better binding ability of unmodified DGL(Fig. 4e) with the most positive charges. DGL(R)-SS-mPEG2

showed similar capacity for both pDNA and siRNA.

Fig. 2 Synthetic illustration of DGL(R)-mPEG (a) and DGL(R)-SS-mPEG (b). Rm represents the guanidinylated part.

Fig. 3 The 1HNMR spectra of DGL-G3, DGL(R)-mPEG and DGL(R)-SS-mPEGin D2O. The ratios represent the calculated molecular ratio of [DGL]: [guanidinegroups]: [PEGylation]. To distinguish different ratios, DGL(R)-SS-mPEG weremarked with subscripts 1 and 2 after PEG. The integrated area at 3.05 ppm inDGL-G3 was considered as the background peak to be subtracted.

Fig. 4 Agarose gel electrophoretic analysis (a and f) DGL(R)-SS-mPEG1/pDNA; (b) DGL(R)-SS-mPEG1/siRNA; (c and g) DGL(R)-SS-mPEG2/pDNA;(d) DGL(R)-SS-mPEG2/siRNA; (e) DGL/pDNA complexes. The running last40 min for pDNA and 15 min for siRNA at 100 V. The enzyme in (f) and (g)denotes DNase I.

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Anti-DNase I ability of DGL(R)-SS-mPEG1 and DGL(R)-SS-mPEG2

(Fig. 4f and g) was evaluated to investigate the stability of thecomplexes against endonuclease degradation, which was crucial forintravenous administration.35 As shown in the figure, there was noresidue of naked DNA observed when exposed to endonucleases,which indicated the poor stability of naked DNA. In contrast, theDNA condensed by DGL(R)-SS-mPEG (1 and 2) at a weight ratio of 4showed almost the same migration of DNA bond as control. Thefindings indicated the efficiency of DGL(R)-SS-mPEG in protectingthe DNA from enzymatic degradation.

Size and f-potential of DGL(R)-SS-mPEG/pDNA complexes

The particle size distribution and zeta potential of DGL(R)-SS-mPEG/pDNA complexes were measured by dynamic lightscattering (DLS). In a typical weight ratio of 4, as shown inFig. 5(c), the mean particle size of the DGL(R)-SS-mPEG1/pDNAcomplex was around 210 nm with a gaussian distribution. Butwith the increase of the weight ratio over 4, the mean particlesize decreased and is kept below 200 nm (Fig. 5a), which wasconsidered favorable for endocytic intracellular uptake.36 Thetypical image of TEM (Fig. 5d) showed the complex possessinghomogeneous spherical shape with approximate 50 nm indiameter. This discrepancy of the results is presumably attribut-able to the shrinkage effect of samples upon drying, owing to thehighly hydrophilic structure of DGL-PEG. For the TEM observa-tion, the samples on the copper mesh were subjected to vacuumdried before detection, whereas DLS measured the size in aqueoussolution. Therefore the nanoparticles showed a larger hydro-dynamic size due to the solvent effect in the hydrated state.37

The z-potential values of two DGL(R)-SS-mPEG/pDNA com-plexes (Fig. 5b) ascended with the elevated weight ratios. At theweight ratio lower than 2, the z-potentials were negative, indicat-ing that pDNA was condensed by dendrimers incompletely.At the weight ratio higher than 4, the z-potentials were around

30 mV. The available z-potentials could allow the complexes tobe uptaken easily by cells, for the effective electrostatic inter-action of negatively charged membranes and positively chargedcomplexes.38 It should be mentioned that, although theDGL(R)-SS-mPEG1/pDNA complexes possessed positive chargeat a weight ratio of 2, the DNA band was still migrated to thecathode (Fig. 4a and c). This can be explicated with the loose,instable structure of the complex at a low weight ratio. Further-more, the z-potential values of DGL(R)-SS-mPEG1/pDNA complexeswere slightly lower than DGL(R)-SS-mPEG2/pDNA complexes atthe same weight ratio. This result may be interpreted by theprevious report that increasing the PEGylation extent ofdendrimers decreased zeta potentials.34

Bio-stability and redox-sensitivity

For structural stability during circulation, PEG-shieldinghas become a leading solution which can prolong the in vivocirculation capability.8,39,40 As shown in Fig. 6, the size changeof DGL(R)-SS-mPEG/pDNA complexes in PBS (a) or added 10%FBS (b) was negligible, due to the protection capability of thePEG layer. A new peak appeared in Fig. 6(b) at around 20 nmascribing to FBS.

For the disassembling of complexes, redox-sensitive disulfidebonds were introduced into the dendrimer. To investigatestructural alterations triggered by GSH, the size change ofDGL(R)-SS-mPEG/pDNA complexes exposed to 10 mM GSH(simulating the GSH concentration in cancer cells) in PBS wasmeasured. As shown in Fig. 6(c), there was no obvious sizealteration in 0.25 h. But as time extended, the shedding of thePEG layer occurred and the size lower than 40 nm was detectedafter 6 h. This change of particle sizes was likely due to therearrangement of the structure of polymer/pDNA complexes accom-panying the release of pDNA during the detachment of PEG. Ourprevious work containing a similar PEG detachable mechanism onpolylysine also revealed that the detachment of the PEG layer wouldaffect the structure of gene complexes and promote the generelease.13 From this point of view, the disulfide-bridged cleavageof PEGylation would result in significant structural dis-assembly ofthe complexes favorable for selective gene release.

Cytotoxicity assay

The toxic effect of dendrimer gene delivery vectors is mainlydetermined by their cationic nature for a cationic gene vector.41

The excessive cationic charges of catiomers can affect theintegrity of outer and inner cell membranes.42 So MTTassay was conducted in order to examine the cytotoxicity of

Fig. 5 Mean particle size (a) and z-potential (b) of the DGL(R)-SS-mPEG/pDNA complexes at various weight ratios. The particle size distribution byintensity of DGL(R)-SS-mPEG1/pDNA at a weight ratio of four is shown inpanel (c) and the TEM image (bar: 50 nm) in panel (d).

Fig. 6 DLS measurement for the particle size change of the DGL(R)-SS-mPEG1/pDNA complexes during 24 h storage (a) in PBS (pH 7.4), (b) inadded 10% FBS and (c) PBS with 10 mM GSH for 6 h.


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DGL(R)-SS-mPEG. Unmodified DGL and bPEI (25k) were usedfor control, with HeLa cells (Fig. 7a) and 293T cells (Fig. 7b)as the models. Compared with bPEI (25k) and primitive DGL,the cytotoxicity of DGL(R)-SS-mPEG1 and DGL(R)-SS-mPEG2

exhibits remarkable improvement. The DGL(R)-SS-mPEG1 evenexhibited high relative cell viability (over 80%) at 200 mg mL�1

concentration. Less than 10 percentage of cells remained at thisconcentration for bPEI, while more than 70 percentage underwentapoptosis for primitive DGL. Compared with DGL(R)-SS-mPEG2,one can further deduce higher PEGylation extent is favorable forcell viability, for that low PEGylation extent is not competent toshield detrimental cationic charges. Both unmodified DGL andbPEI significantly inhibited cell viability at the concentration ofor even lower than 10 mg mL�1. These findings imply that thePEG-shielding effect significantly decreased cytotoxicity.

In vitro transfection

For gene delivery vectors, transfection needs to be performedto evaluate gene expression efficiency in host cells. The trans-fection efficiency of DGL(R)-SS-mPEG1, DGL(R)-SS-mPEG2 andDGL(R)-mPEG was investigated by the BCA method. bPEI(25k)/pEGFP complexes at the optimal weight ratio (1.3 : 1) as positivecontrol. PEI (especially with Mw of 25 000) has been known as agold standard of gene transfection due to its high transfectionefficiency,43 and many research studies choose bPEI (25k) aspositive control.44,45 In 293T cells, all the experimental groupswere detected for GFP expression, though the intensities of theDGL(R)-SS-mPEG group were somewhat weaker than the posi-tive control (Fig. 8). An important finding is DGL with cleavablePEGylation exhibited obvious higher expression compared withnon-cleavable DGL(R)-mPEG for all of the investigated weightratios. This result dictates the effect of intracellular cleavageof PEGylation promoting the gene expression, in line withprevious studies.11,12,14 Moreover, DGL(R)-SS-mPEG1 showedthe higher efficiency than that of DGL(R)-SS-mPEG2, whichindicates PEGylation may improve transfection activity. Thisresult is consistent with the finding that L-arginine basedpoly(ether ester amide)s having more flexible chains showedbetter transfection efficiency than that having more rigidchains, in which PEG was introduced to develop the flexibilityof polymers.46

To visualize the cells with transgene expression, the typicalfluorescence images of post-transfected 293T cells were captured.As shown in Fig. 9, GFP was successfully expressed. In line withabove quantitative data, DGL(R)-SS-mPEG groups show obvioushigher fluorescence than the DGL(R)-mPEG group. GFP fluores-cence by DGL(R)-SS-mPEG expressed even was a comparableamount of GFP in contrast with bPEI.

Intracellular distribution

The cellular entry is a prerequisite for gene expression. Intra-cellular trafficking of the complexes containing FAM-labeledsiRNA was examined by means of confocal microscopy in

Fig. 7 Percentage viability of HeLa cells (a) and 293T cells (b) treated withDGL(R)-SS-mPEG at various concentrations using the MTT method afterincubation for 4 h at 37 1C. bPEI (25k) and DGL were used as control. Datawere shown as mean � SD (n = 5).

Fig. 8 Quantity for GFP expression of dendrimer/pEGFP complexes in293T cells by the BCA assay. *p o 0.05, **p o 0.01, ***p o 0.001. Thedata in the groups of 4 and 10 without labelling denote p Z 0.05.

Fig. 9 Fluorescence images of GFP expression in 293T cells. The w/wratios of DGL(R)-SS-mPEG1/pDNA, DGL(R)-SS-mPEG2/pDNA, DGL(R)-mPEG/pDNA and bPEI/pDNA were 20 : 1, 20 : 1, 20 : 1 and 1.3 : 1. The cellswere incubated with different complexes for 4 h and the images weretaken 24 h post-transfection at a magnification of 20.


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293T cells. The images of DGL(R)-SS-mPEG1/siRNA (a),DGL(R)-SS-mPEG2/siRNA (b) and DGL(R)-mPEG/siRNA (c) areshown in Fig. 10. For the three complexes, siRNA fluorescencewas observed in the cytoplasm and predominantly localized inthe perinuclear area, which confirmed the successful delivery ofsiRNA by the complexes. Importantly, siRNA fluorescence forgroups of DGL(R)-SS-mPEG1/siRNA and DGL(R)-SS-mPEG2/siRNA tended to widely distribute around the nucleus inan irregular strip-like shape but siRNA fluorescence in theDGL(R)-mPEG group held particle-like distribution, indicatinglower intracellular release of siRNA for the non-cleavable group.Furthermore, the former two which have cleavable PEGlyationshowed more fluorescence than the last one.

Cell growth inhibition effect

Using siRNA-VEGF as the therapeutic gene, the cell growthinhibition effect on 293T cells by DGL(R)-SS-mPEG was evalu-ated by the MTT study. As shown in Fig. 11, both two complexesshowed obvious dose-dependent cell growth inhibition com-pared to negative control. Both two DGL(R)-SS-mPEG complexeswith different PEGylation contents showed similar tendencyon cell inhibition efficacy. At siRNA-VEGF of 8 mg mL�1, cellviabilities of both DGL(R)-SS-mPEG complexes can be as low as20%. In view of the low cytotoxicity of DGL(R)-SS-mPEG themselvesat this vector concentration, the above results clearly demonstratesthe major role of successful delivery in siRNA-VEGF into cytoplasmresulting in the effective cell inhibition.

Biodistribution of DGL(R)-SS-mPEG in vivo

Biodistribution of DGL(R)-SS-mPEG in vivo was investigated bythe real-time fluorescence imaging technique. The biodistribu-tion profile will provide important insights into how much thevector accumulates in tumors and various organs at differenttime scales. For this purpose, DGL(R)-SS-mPEG1 which haslower cytotoxicity, greater transfection efficiency and betterintracellular release was selected to be labelled by BODIPYand intravenously injected into mice bearing HeLa tumor. InFig. 12(b–d), compared with control (treated with pure PBS), themice displayed significant fluorescence which mostly concen-trated on liver. As time goes on, DGL(R)-SS-mPEG1 graduallyaccumulated in the tumor site, especially at 24 h after injection.This result can be interpreted by the enhanced permeability

and retention (EPR) effect, which strengthens passive target-ing47 of DGL(R)-SS-mPEG1 nanoparticles towards tumor tissue.Furthermore, modification by PEG may extend the circulationtime and reduce phagocytosis of the reticuloendothelialsystem, making stealth property of the complexes.48,49 As aresult, DGL(R)-SS-mPEG1 gradually accumulated in the tumorsite and lasted for a long considerable time magnitude, whichprovides a robust foundation to deliver gene towards tumor sitein vivo.

Fig. 10 Confocal microscopy images of cells after treatment with FAM-siRNA formulated in DGL(R)-SS-mPEG1 (a), DGL(R)-SS-mPEG2 (b) andDGL(R)-mPEG (c) at the weight ratio of 20 for 4 h. The nucleus wasstained blue by DAPI and the siRNA appeared as green labelled by FAM.Bar: 20 mm.

Fig. 11 The cell growth inhibition effect is shown as the cell viabilities in293T cells which were transfected with DGL(R)-SS-mPEG1/siRNA-VEGF orDGL(R)-SS-mPEG2/siRNA-VEGF complexes at a weight ratio of 16 for 4 hat different amounts of siRNA-VEGF. bPEI/NC-siRNA (w : w = 1.3 : 1) wasused for negative control. ***p o 0.001 as compared with respectivecontrol.

Fig. 12 In vivo imaging of tumor-bearing nude mice at 4 h (b), 8 h (c), and24 h (d) after administration with BODIPY-labelled DGL(R)-SS-mPEG1

(right in every panel). The left mouse in every panel was treated with PBSas control and the image deducted fluorescence is shown in panel (a) ascontrast. The tumor site was marked by the red circle in every panel.


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In vivo anti-tumor effect

VEGF is a critical regulator for angiogenesis, necessary forthe proliferation of cancer cells.50 Anti-angiogenesis to inhibitVEGF expression has been developed as an intriguing strategyfor anti-cancer therapy. It has been reported using siRNArealized inhibition of VEGF expression.11,51 To further explorethe potential in cancer therapy, a therapeutic attempt ofDGL(R)-SS-mPEG1/siRNA-VEGF complexes for HeLa tumor-bearing mice was conducted. In the experiment, three groupswere set for evaluation, which were treated respectively by PBS(control group), DGL(R)-mPEG/siRNA-VEGF complexes, andDGL(R)-SS-mPEG1/siRNA-VEGF complexes. The results aresummarized in Fig. 13(a), where significant inhibition of tumorgrowth was observed in groups of the complexes with or with-out cleavable PEGylation, in comparison with the controlgroup. The DGL(R)-SS-mPEG1 group exhibited maximal sup-pression of tumor, with the relative tumor volume nearly 4-foldsmaller than the control group at the 16th day. Comparedwith the DGL(R)-PEG group without cleavable PEGylation, theinhibition effect of the DGL(R)-SS-mPEG1 group was also morepronounced (40% smaller than that without cleavable PEGyla-tion). Similar differences were found in tumor weight (Fig. 13b).Mice weight showed no obvious alteration (Fig. 13c) aftertreatment of both complexes, indicating the preliminary bio-compatibility of the developed new gene vector.

After the mice being sacrificed, HeLa tumors were photo-graphed and representative tumors of each group are shown inFig. 13(d). It is noteworthy that the surface morphology of thetumors of the DGL(R)-SS-mPEG1 group clearly distinguishedfrom the control group and the DGL(R)-mPEG group. From theimages, the control group and the DGL(R)-mPEG group werefound to be covered with obvious red blood vessels on thesurface, though fewer is found in the latter group. In a sharp

difference, the DGL(R)-SS-mPEG1 group exhibited no visible redblood vessel by naked eye, which indicated the effective VEGFinhibition effect. Cleavable PEGylation under tumor relevant con-dition is possibly to facilitate the accumulation of the complexeswithin tumor, intracellular uptake and release of siRNA-VEGF. Thereleased siRNA then exerts effective silencing of VEGF necessaryfor blood vessel growth and inhibits the tumor growth.51

Experimental sectionMaterials

Dendrigraft poly-L-lysine (DGL-G3 or DGL, generation 3, Mw =22 000, 123 of lysine groups) was purchased from Colcom(France). Sulfhydryl terminated methoxy poly(ethylene glycol)(mPEG-SH, MW 2000), carboxyl terminated methoxy poly-(ethylene glycol) (mPEG-COOH, MW 2000) were purchasedfrom Yare Biotech Inc (China). 3-(2-Pyridyldithio) propionicacid N-hydroxysuccinimide ester (SPDP) was purchased fromHeowns (China). 1H-Pyrazole-1-carboxamidine (99%), N, N-diiso-propylethylamine (DIPEA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCHCl), N-hydroxy succinimide(NHS) and glutathione (GSH) were purchased from Aladdin(China). Dialysis bags (regenerated cellulose, MWCO 3500 Daand 8–14k Da) were purchased from Genestar Biotech (China).

Dulbecco’s modified Eagle’s medium (DMEM) and Dulbecco’sphosphate buffered saline (PBS) were supplied from Hyclone(Thermo, USA). Fetal bovine serum (FBS), penicillin–streptomycin(PS), trypsin and BODIPY 650/665-X succinimidyl ester wereobtained from Gibco (Invitrogen, USA). Deoxyribonuclease I(DNase I), diethyl pyrocarbonate water (DEPC H2O), 3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyltetrazoium bromide (MTT), celllysis buffer and 40,6-diamidino-2-phenylindole (DAPI) were pur-chased from Beyotime Institute of Biotechnology (China). Thebicinchoninic acid (BCA) protein assay kit was purchased fromPierce (Thermo, USA). pEGFP was obtained from Invitrogen, andsiRNA-VEGF and FAM-labelled siRNA-VEGF were synthesized byGenePharma (China). Branched poly(ethylenimine) (bPEI-25k,Mw = 25 000 g mol�1) was obtained from Sigma-Aldrich (China).

Synthesis of DGL(R)

Guanidinylation of DGL (0.005 mmol) was performed inN, N-dimethyl formamide (DMF) with drop addition of DMFsolution containing equivalent moles (0.2 mmol) of 1H-pyrazole-1-carboxamidine and DIPEA. The reaction was kept for 24 hunder a nitrogen atmosphere, followed by precipitation usingcold diethyl ether at 4 1C overnight. After removal of diethylether, the residual was subject to dialysis to remove any impu-rities (MWCO 3500 Da), before lyophilization. The degree ofguanidinylation was determined by 1H NMR (500 MHz, D2O).

Synthesis of DGL(R)-mPEG

To activate carboxyl groups, EDCHCl (0.03 mmol) and NHS(0.03 mmol) were reacted with mPEG-COOH (0.027 mmol) inpure water at 0 1C for 4 h. Then the mixture was dropped intosolution of DGL(R) (27 mg, 0.0012 mmol), and the reaction was

Fig. 13 Antitumor application of vector/siRNA-VEGF complexes in vivo.PBS was used for control. (a) Relative tumor volume (V/V0, V0 means theinitial volume and V means the volume measured on specific day) and (b)tumor weight in nude mice bearing HeLa tumor after treatment withdifferent complexes; (c) mice weight and (d) representative photographof tumors. **p o 0.01, ***p o 0.001.


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kept for 24 h. Dialysis (MWCO 8–14k Da) was employed toremove the impurities, and DGL(R)-mPEG was obtained byfreeze-drying. The substitution degree of PEG was characterizedby 1H NMR (500 MHz, D2O).

Synthesis of DGL(R)-SS-mPEG

SPDP (0.027, 0.015 mmol) dissolved in DMF was addedto DGL(R) (27 mg, 0.0012 mmol) dissolved in H2O : DMF(v : v = 1 : 1) to modify the part of amino groups of the dendrigraftpolymer with pyridyl disulfide groups (DGL(R)-PDP). Thereaction was allowed to react for 24 h, followed by dialysis(MWCO 3500 Da). For the disulfide bridged cleavable PEGyla-tion, mEPG-SH (0.027, 0.015 mmol) dissolved in water wasadded into the DGL(R)-PDP solution, and the thiol-disulfideexchange reaction was carried out by adding a catalytic amountof acetic acid under a N2 atmosphere for 24 h. Subsequently, themixture was dialyzed (MWCO 8–14k Da) and DGL(R)-SS-mPEGwas obtained by freeze-drying. The substitution of disulfidebridged PEG was characterized by 1H NMR (500 MHz, D2O).

Characterization1H NMR spectra of different dendrimers were recorded. Sam-ples were dissolved in D2O and performed by a 500 MHzspectrometer (Avance, Switzerland) using TMS as the standard.Copper meshes were infiltrated in sample solution and dried bynatural ventilation, and then stained negatively by phospho-tungstic acid (1 w/v%). The morphology of dendrimer/pDNAcomplexes was characterized by H7100 transmission electronmicroscopy (TEM) with an acceleration voltage of 100 kV.The representative TEM micrograph of DGL(R)-SS-mPEG/pDNAcomplexes was shown.

Preparation of dendrimer/gene complexes

Specifically, the different dendrimers were dissolved in PBS(pH 7.4) to yield the concentration of 0.5 mg mL�1, and thesolutions were filtered using a 0.45 mm microfiltrationmembrane. Respectively, the dendrimer solutions were addedinto pDNA or siRNA (dispersed in DEPC H2O) at various weightratios. After vortexing for seconds, the mixtures were incubatedat 37 1C for 30 min for further experiments.

Particle size and f-potential measurement

The particle size distribution and zeta potential of DGL(R)-SS-mPEG/pDNA complexes were determined by dynamic lightscattering (DLS) using a Nano-ZS 90 Nanosizer (Malvern,U.K.). The complexes (containing 1 mg pDNA per sample) wereprepared as described above and diluted to 1 mL with PBS(pH 7.4) before measurement.

Agarose gel electrophoretic analysis and anti-DNase Ievaluation

Agarose gel electrophoretic analysis was performed to assessthe ability of different dendrimers to load nucleic acids.According to the aforementioned method, the complexes wereprepared at weight ratios of 0.25 : 1, 0.5 : 1, 1 : 1, 2 : 1, 4 : 1, 6 : 1and 8 : 1 (polymer to nucleic acids). The gels were made of

1% (w/v) agarose in tris-acetate (TAE) containing ethidiumbromide (EB). The complexes were loaded with 6� DNA loadingbuffer on the gels and the electrophoresis last 40 min for pDNAand 15 min for siRNA at 100 V in TAE. Then the location ofpDNA or siRNA was visualized under UV excitation.

The stability of the complexes against endonuclease degrada-tion was evaluated by incubating the complexes with DNase I,according to reported procedures with slight modification.52,53

The complexes were treated with DNase I at 37 1C for 10 minbefore termination by 25 mM EDTA at 65 1C for 10 min.

Bio-stability and redox-sensitivity

Firstly, the stability of DGL(R)-SS-mPEG/pDNA complexes in PBSand serum (10% FBS in PBS) was evaluated by measuring theparticle size change. Then the redox-sensitivity of DGL(R)-SS-mPEG/pDNA complexes exposure to GSH (10 mM) was assessed.The size change was monitored at predetermined time points.

Cell culture

Human embryonic kidney transformed 293 (293T) cells andhuman cervical carcinoma (HeLa) cells were gifted from theMedicine School of Tongji University. The cells were main-tained in complete medium (high-glucose DMEM containingwith 10% FBS and 1% penicillin–streptomycin) in a humidified5% CO2 air incubator at 37 1C. The cells were passagedfrequently and at regular intervals to maintain mid-log growth.

Cell viability assay

The cytotoxicity of dendrimers was evaluated by the MTTmethod54 against HeLa and 293T cells. Briefly, the cells wereseeded at a density of 8 � 103 cells per well and cultivated untilreaching 60–70% confluence. Then the medium was replacedby the dendrimers dissolved in serum-free DMEM at variousconcentrations of 3.125, 6.25, 12.5, 25, 50, 100 and 200 mg mL�1.After 4 h incubation, the dendrimers were removed and furtherincubated in fresh serum-containing DMEM for 24 h. Then thecells were exposed to MTT for another 4 h. Then using DMSOto dissolve the formazan produced, the plate was placed on ashaker with gentle shaking. The absorbance measurement wasdetected by a microplate reader (Multiscan MK3, Thermo, USA)at an excitation wavelength of 570 nm and 630 nm as thebackground. The branched polyetherimide (bPEI, 25k) was usedas positive control. Blank values without cells were subtracted.

In vitro transfection

The transfection activity against 293T cells in vitro was evaluatedusing pEGFP as the reporter gene. Before measurement, the cellswere seeded in 24-well plates at a density of 8 � 104 cells per welland cultivated until reaching 60–70% confluence. The dendri-mers/pEGFP complexes (1 mg pEGFP per well) at various weightratios of 4 : 1, 8 : 1, 10 : 1, 16 : 1, 20 : 1 were added to each well inFBS-free medium, and the cells were incubated with thesecomplexes for 4 h. Then the medium was replaced with com-pleted medium and the cells were incubated for further 24 h.

To visualize the GFP expressed, the fluorescent imageswere imaged using the inverted fluorescence microscope


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(Nikon TI-S-EJOY). To quantify the GFP expression, fluores-cence intensity was measured by BCA protein assay. In detail,the cells were washed by PBS, followed by incubation with200 mL of cell lysis buffer at 4 1C for 30 min. The cell lysateswere centrifuged to remove large cellular debris and 100 mL wastransferred to the black 96-well plate to measure the fluores-cence intensity of GFP by fluorospectrophotometer (FluoroskanAscent FL, Thermo) at an excitation wavelength of 488 nm andemission at 509 nm. The total protein was analyzed using a BCAprotein assay kit, taking BSA as the standard. The transfectionefficiency was described as GFP fluorescence intensity (A.U.)per mg of total protein. bPEI (25k)/pEGFP (w : w = 1.3 : 1) wasused for positive control.

Intracellular distribution studies

Confocal laser scanning microscopy (CLSM) assay was carriedout to examine the intracellular distribution of the dendrimers/siRNA complexes. 293T cells were cultivated on glass coverslipsin the 6-well plate until reaching 60–70% confluence. The cellswere incubated with dendrimers complexing FAM-labelledsiRNA at 37 1C for 4 h in FBS-free medium. Then the plateswere washed three times with PBS, and the cells were fixed with4% (v/v) paraformaldehyde for 10 min. After washing threetimes by PBS again, the cells were dyed by DAPI for 10 min andsealed on a glass slide by glycerin. Then the cells were observedby CLSM. FAM-siRNA was imaged at excitation of 488 nm andDAPI of 340 nm.

Cell growth inhibition effect

A siRNA sequence (sense, 50-GGAGUACCCUGAUGAGAUCdTdT-30; antisense, 50-GAUCUCAUCAGGGUACUCCdTdT-3 0) that cansilence VEGF was chosen to evaluate the role of DGL(R)-SS-mPEG to deliver RNA. The negative control (NC) sequence is50-UUCUCCGAACGUGUCACGUdTdT-3 0 and 50-ACGUGACACGUUCGGAGAAdTdT-3 0. 293T cells were exposed to differentconcentrations of DGL(R)-SS-mPEG/siRNA-VEGF complexesand the assay was carried out according to in vitro transfection.The complexes were prepared at the same weight ratio of 16 : 1with 0.5, 1, 2, 4, 8 mg siRNA per sample and the concentrationsof DGL(R)-SS-mPEG used were 8, 16, 32, 64, 128 mg mL�1

respectively. The cell growth inhibition effect was evaluatedby the MTT method according to the aforementioned assay.bPEI/NC-siRNA was used for negative control.

Animal models

All animal experiments were carried out in accordance with theAnimal Care and Use guidelines approved by the Committeeof Tongji University. Healthy female nude mice (4–5 weeks,Slaccas, China) were housed at specific pathogen free (SPF) lab.The mice were received subcutaneous inoculation of 7 � 106

HeLa cells dispersed in 200 mL of PBS and used for the in vivotest after tumor volume reaching 50 mm3.

Biodistribution of DGL(R)-SS-mPEG in vivo

To synthesize the BODIPY-labelled material,55 DGL(R)-SS-mPEG1

was dissolved in 100 mM NaHCO3 solution and added with

BODIPY (10 equiv.) in DMSO. The reaction was allowed for 1 h atthe room temperature and the product was purified via ultra-filtration using a membrane (molecular weight cutoff = 3000).The linkage can be implemented by the reaction betweensuccinimidyl ester (NHS ester) of BODIPY and amino groupsof DGL.

For the experiment of biodistribution in vivo, the tumor-bearing mice were injected intravenously via the tail vein with200 mg BODIPY-labelled DGL(R)-SS-mPEG1 (in 150 mL of PBS)per mouse respectively. At 4 h, 8 h, 24 h after administration,the mice were anesthetized and imaged with 589 nm as theexcitation wavelength (Maestro IN-VIVO imaging system, CRI,MA). The mice injected with PBS were used as control.

Antitumor evaluation of dendrimer/siRNA-VEGF complexes

To further study the antitumor effect of siRNA-VEGF delivered bymodified DGL in vivo, the tumor-bearing mice were treated andtumor growth was observed for 16 days. The mice were randomlyassigned to three groups and marked. The first group was injectedintravenously with 200 mL of DGL(R)-SS-mPEG1/siRNA-VEGF(w/w = 10, 20 mg siRNA), the second group was injected with200 mL of DGL(R)-mPEG/siRNA-VEGF (w/w = 10, 20 mg siRNA), andthe last was injected with 200 mL of PBS as control. The micewere treated every other day, and, before treatment, the weightof mice and the size of tumor were recorded. Then tumor sizeswere calculated using the following formula: V(mm3) = (lengthof tumor) � (width of tumor)2/2. After 16 days, mice were put todeath by cervical vertebra dislocation, and their tumors wereimmediately harvested, weighed, and photographed.

Statistical analysis

Data were analyzed using one-factor analysis of variance. Theresults were expressed as mean � standard deviation (SD). Thestatistical significance of the results was judged at *p o 0.05,**p o 0.01, ***p o 0.001.

Conclusions

In this study, cleavable PEGylation was incorporated ontodendrigraft poly-L-lysine via thiol-disulfide exchange reaction.These dendrigraft poly-L-lysines with cleavable PEGylationcould efficiently compact both pDNA and siRNA, and protectthem from DNase I degradation. Behavior of PEGylation wasinvestigated by monitoring size alterations by gluthationeexposure, in which size underwent significant reduction dueto PEG layer detachment. PEGylation incorporation signifi-cantly decreased cytotoxicity as well as promoted the inter-cellular release and expression of the genetic payload. The genetransfection study found the PEG-cleavable catiomer resultedin significantly higher GFP expression against 293T cells. Afterloading small interfering RNA (siRNA-VEGF), the complexnanoparticles demonstrated the pronounced inhibition effecton cell growth in vitro and tumor growth in vivo. The aboveresults revealed a universal strategy to balance disadvantages


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and advantages of dendrimer catiomers for the future non-viralgene delivery vector.

Acknowledgements

This work was financially supported by the 973 program(2013CB967500), research grants from the National Natural ScienceFoundation of China (NSFC51473124, 81470390, 81571801 and51173136), the Fundamental Research Funds for the Central Uni-versities (2013KJ038), and ‘‘Chen Guang’’ project (12CG17) foundedby Shanghai Municipal Education Commission and ShanghaiEducation Development Foundation.

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