Fluorinated poly(propylenimine) dendrimers as gene vectors

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Biomaterials xxx (2014) 1e7

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Biomaterials

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Fluorinated poly(propylenimine) dendrimers as gene vectors

Hongmei Liu a, Yu Wang b, Mingming Wang a, Jianru Xiao b, Yiyun Cheng a,*

a Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241, PR Chinab The Second Military Medical University, Changzheng Hospital, Department of Orthopedic Oncology, Shanghai PR China

a r t i c l e i n f o

Article history:Received 20 January 2014Accepted 17 March 2014Available online xxx

Keywords:DendrimerPoly(propylenimine)Gene transfectionFluorination

* Corresponding author.E-mail address: [email protected] (Y. Che

http://dx.doi.org/10.1016/j.biomaterials.2014.03.0400142-9612/� 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Liu H, edx.doi.org/10.1016/j.biomaterials.2014.03.04

a b s t r a c t

The use of poly(propylenimine) (PPI) dendrimers as gene delivery vectors is limited by their lowtransfection efficacy and serious cytotoxicity. In this study, we prepare a series of efficient and lowcytotoxic gene vectors based on PPI dendrimers using a facile fluorination strategy. Fluorinated G3, G4,and G5 PPI dendrimers show comparable or superior transfection efficacies to six representativetransfection reagents such as Lipofectamine 2000, jetPEI, branched poly(ethyleneimine), SuperFect,PolyFect, as well as arginine-modified dendrimer on both HEK293 and HeLa cells. These fluorinated PPIdendrimers exhibit low cytotoxicity on the transfected cells because they achieve optimal transfectionefficacy at extremely low nitrogen to phosphorus (N/P) ratios. In addition, the synthesized materialsshow high transfection efficacy on 3D multicellular spheroids. These results demonstrate that fluori-nation is a promising strategy to improve the transfection efficacy of PPI dendrimers while reducing thecytotoxicity of these cationic polymers.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Gene therapy is promising as an approach to treat various dis-eases ranging from viral infections to hereditary disorders andcancers [1]. A key challenge to clinical gene therapy is the need forsafe and effective gene vectors [2e5]. Compared to viral genevectors, non-viral vectors for gene therapy have attracted greatattention in the past decade because of their limited immunoge-nicity, easy-to-manufacture, and ability to deliver large-sized ge-netic materials [6e8]. The major categories of non-viral genevectors include cationic lipids and polymers. Cationic lipids arecytotoxic and often cause acute inflammatory responses in animalsand humans [6], thus polymers are widely used as gene vectorcandidates in recent years. Among the polymers, dendrimers areemerging as a new class of non-viral gene vectors due to theirunique well-defined architecture, high density of surface charges,feasibility to surface modification and multivalent effect confinedwithin a nanostructure [9e20]. Dendrimer-based gene transfectionreagents such as SuperFect� and PolyFect� have already enteredthe market. However, these reagents are criticized by moderatetransfection efficacy and serious cytotoxicity.

ng).

t al., Fluorinated poly(propy0

Poly(propylenimine) (PPI) dendrimer is the first dendrimerfamily that realize industrial kilogram scale synthesis [21]. Thesedendrimers are intensively characterized and widely explored aspotential drug or gene vehicles [22,23]. However, PPI dendrimersare associated with serious cytotoxicity due to their hydrophobicinterior as well as high surface charge density [24]. Transfectionefficacies of PPI dendrimers are not as high as other representativecationic polymers such as linear or branched poly(ethylenimine)(PEI) and poly(amidoamine) (PAMAM) dendrimer. Different stra-tegies were applied to improve the transfection efficacy and toreduce the cytotoxicity of PPI dendrimers. They were functional-ized with gold nanoparticle [25], transferrin [26,27], galactose [28],peptide [29], oligoethylenimine [30,31], amino acids [32,33], qua-ternary ammonium [34], and guanidinium groups [35]. These ap-proaches improve the transfection efficacy of PPI dendrimers orreduce their cytotoxicity to some extent. Most of these gene vectorsform polyplexes with DNA or siRNA by electrostatic interactions,and the resulting polyplexes can be easily destabilized by poly-anionic biomolecules. Although cationic dendrimers with highcharge density and high molecular weight can make the complexesmore stable in physiological conditions, it also leads to increasedcytotoxicity because the excess amount of positive charges on thepolyplex destabilizes cell membrane and causes several undesir-able effects [36]. There is an urgent need to design efficient poly-meric gene vector that has a lower charge density or achievesdesirable transfection efficacy at an extremely low nitrogen tophosphorus (N/P) ratio [6].

lenimine) dendrimers as gene vectors, Biomaterials (2014), http://

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Table 1Characterization of non-modified PPI dendrimers (Gn) and fluorinated PPI den-drimers (Gn-F7x).

Polymer xa x0 Mwb (Da) Charge per dendrimer

G3 PPI 0 0 1687 16G4 PPI 0 0 3514 32G5 PPI 0 0 7168 64G3-F79 9.4224 � 0.0443 9 3451 7G3-F712 11.9798 � 0.0405 12 4039 4G4-F716 15.7488 � 0.2418 16 6650 16G4-F720 20.3318 � 0.3158 20 7434 12G5-F726 25.7254 � 0.1712 26 12264 38G5-F735 34.7321 � 0.5973 35 14028 29

a x is the average number of heptafluorobutyric acid groups modified on each PPIdendrimer determined by the ninhydrin assay (average � S.D.). x0 is the approxi-mate number of x. x0 is used to calculate the molecular weight (Mw) of the fluori-nated dendrimer and the residual primary amine groups on each fluorinated PPIdendrimer.

b Molecular weights of the fluorinated PPI dendrimers were calculated accordingto the number of heptafluorobutyric acid modified on each dendrimer and thetheoretical molecular weight of G3, G4, and G5 PPI dendrimers, respectively.

H. Liu et al. / Biomaterials xxx (2014) 1e72

Recently, we developed a fluorination strategy to improve thetransfection efficacy of PAMAM dendrimers [37]. Fluorinated G5PAMAM dendrimer achieves dramatic transfection efficacy at lowN/P ratios. Fluorination effectively improves the cellular uptake aswell as endosomal escape of G5 PAMAM/DNA polyplexes. In thisstudy, we investigated the effect of fluorination on the transfectionefficacy and cytotoxicity of PPI dendrimers. G3, G4 and G5 PPIdendrimers were modified with heptafluorobutyric acid using afacile route. The transfection efficacies of these materials oncommonly used cell lines as well as 3D multicellular spheroidswere investigated. The major aim of this study is to check iffluorination also works well on PPI dendrimers and to generate alist of high efficient and low cytotoxic PPI dendrimer-based genevectors.

2. Materials and methods

2.1. Materials

G3 (molecular weight: 1687 Da; number of surface amine groups: 16), G4(molecular weight: 3514 Da; number of surface amine group: 32) and G5 (molecularweight: 7618 Da; number of surface amine group: 64) PPI dendrimers were pur-chased from SyMO-Chem (Eindhoven, Netherlands). G5 PAMAM dendrimer (core:ethylenediamine; molecular weight: 28826 Da; number of surface amine groups:128) was obtained from Dendritech (Midland, MI). Heptafluorobutyric anhydrideand branched PEI with a molecular weight of 25000 (bPEI25K) were purchasedfrom SigmaeAldrich (St. Louis, MO). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sangon Biotech Co., Ltd

Fig. 1. Synthesis of fluorinated G3, G4, and G5 PPI dendrimers. Gn-F7x represents the yieldenotes the average number of the heptafluorobutyric acid modified on each PPI dendrime

Please cite this article in press as: Liu H, et al., Fluorinated poly(propydx.doi.org/10.1016/j.biomaterials.2014.03.040

(Shanghai, China). Lipofectamine 2000 (Lipo 2000) was purchased from Life Tech-nologies (Shanghai, China). SuperFect and PolyFect were obtained from Qiagen(Germany). jetPEI was purchased from Polyplus-Transfection (France). All thechemicals were used as received without further purification. Arginine-modified G5PAMAM dendrimer was synthesized according to the method described by Parket al. [38]. An average number of 110 L-arginine was conjugated to each G5 PAMAMdendrimer calculated by 1H NMR (699.804 MHz) and the product was termed G5-Arg110.

2.2. Synthesis and characterization of the fluorinated PPI dendrimers

Fluorinated PPI dendrimers were synthesized according to reference 37 and thedetailed method is described in Appendix. Supplementary data. All the fluorinateddendrimers show excellent aqueous solubility. The number of heptafluorobutyricacid modified on PPI dendrimer was determined using a well-established ninhydrinassay to measure the residual primary amine groups on each fluorinated dendrimer.Generally, 85 mg ninhydrin and 15 mg hydrindantin were dissolved in 10 mLethylene glycol-monomethyl ether. 100 mL of the solution was mixed with 100 mLsodium acetate buffer (0.2 M, pH ¼ 5.4). The mixtures were added with fluorinatedG3, G4 and G5 PPI dendrimers at proper concentrations and the final volume of thesolutions was fixed to 300 mL using distilled water. The solutions were then incu-bated in boiling water for 10 min. After cooling to room temperature, 300 mLethanol/water (v/v ¼ 60/40) mixture was added to each sample. Absorbance of thesamples was measured at 570 nm using a UV-Vis spectrophotometer. G3, G4 and G5PPI dendrimers at different concentrations were tested according to the same pro-tocol and linear relationships between amine concentration and absorbance wereused as standard curves for the fluorinated dendrimers (Abs ¼ 22.061Ce0.0112,R2 ¼ 0.9997 for G3 PPI dendrimer; Abs ¼ 18.21Ce0.0253, R2 ¼ 0.9952 for G4 PPIdendrimer; Abs ¼ 16.43Ce0.0423, R2 ¼ 0.9983 for G5 PPI dendrimer. Abs is theabsorbance of the sample at 570 nm, C is the concentration of primary amine groups,mM). The numbers of fluorinated groups modified on each PPI dendrimer werecalculated and listed in Table 1. The synthesized fluorinated PPI dendrimers werealso characterized by 19F NMR (500.132 MHz).

2.3. Preparation and characterization of dendrimer/DNA polyplexes

Polyplexes consisted of fluorinated PPI dendrimers and EGFP plasmid wereprepared at N/P ratios of 0.5:1, 1:1, 2:1, 4:1 and 8:1, respectively. N here representsthe number of residual primary amine groups on the surface of fluorinated PPIdendrimers; P here represents the number of phosphate anions in the plasmidbackbone. The polyplexes were incubated for 30 min before characterization. DNAbinding and condensation capability of the fluorinated PPI dendrimers were firstlyevaluated by an agarose gel retardation assay. Generally, the polyplexes werediluted with DNA loading buffer and the samples were run on a 1% (w/v) agarosegel at 100 V and stained by ethidium bromide for 40 min. The DNA bands werevisualized by a UV illuminator and photographed using an UVIpro Gel documen-tation system.

The size and zeta-potential of the prepared polyplexes were further measuredby dynamic light scattering at 25 �C (Zetasizer Nano ZS90, Malvern).

2.4. Cell culture and gene transfection experiments

HEK293 (a human embryonic kidney cell line, ATCC) and HeLa (a human cervicalcarcinoma cell line, ATCC) cells were used to evaluate the transfection efficacy andcytotoxicity of the synthesized fluorinated PPI dendrimers. The cells were

ding fluorinated dendrimers. n denotes the dendrimer generation (n ¼ 3, 4, 5) and xr.


Fig. 2. Gel retardation assay used to determine the DNA binding capacity of G3, G4,and G5 PPI dendrimers and their fluorinated products (G3-F79, G4-F716 and G5-F726).The amount of DNA in each lane was 0.8 mg. The polyplexes were prepared at N/P ratiosof 0.5:1, 1:1, 2:1, 4:1 and 8:1, respectively.

H. Liu et al. / Biomaterials xxx (2014) 1e7 3

maintained in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO Inc.) containingpenicillin sulfate (100 units/mL), streptomycin (100 mg/mL) and 10% heat-inactivedfetal bovine serum (FBS, GIBCO Inc.) at 37 �C and 5% CO2.

Before gene transfection experiments, HEK293 and HeLa cells were cultured in24-well plates overnight. 0.8 mg EGFP or luciferase plasmids were mixed withfluorinated PPI dendrimers at different N/P ratios and the polyplexes were incu-bated for 30 min and diluted with DMEM containing 10% FBS before addition into

Fig. 3. Hydrodynamic size and zeta potential of the polyplexes consisted of fluorinated PPIand 8:1, respectively. DNA in the absence of fluorinated PPI dendrimers were used as a con


the wells (total volume: 250 mL). After incubation for 6 h, 500 mL fresh DMEMmedium containing 10% FBS was added to each well. The gene transfection ex-periments were conducted for 48 h. Expressions of the EGFP plasmid in thetransfected cells were observed by a fluorescent microscopy (Olympus, Japan) andquantitatively analyzed using flow cytometry. The luciferase assay was performedaccording to the manufacturer’s protocols (Promega). Protein concentration in eachwell was determined using a BCA protein assay kit (Beyotime). The luciferase ac-tivities were normalized with respect to the relative luciferase light units permilligram of protein (RLU/mg protein). Unmodified G3, G4 and G5 PPI dendrimers,G5-Arg110, bPEI25K, and commercial representative transfection reagents includingLipo 2000, SuperFect, PolyFect and jetPEI were used as controls. Optimal trans-fection conditions for these materials were screened before use. Three repeatswere conducted for each material.

2.5. Cytotoxicity of the fluorinated PPI dendrimers and their polyplexes

Cytotoxicity of the fluorinated dendrimers and their complexes with DNA wasevaluated by a well-established MTT assay as described elsewhere. Generally,HEK293 and HeLa cells were cultured in 96-well plates at a density around1 � 104 cells per well overnight. The cells were incubated with fluorinated PPIdendrimers or dendrimer/DNA polyplexes at different concentrations (concentra-tions used in the gene transfection experiments) for 48 h. Themediumwas removedand the cells were added with 100 mL MTT solution. The yielding formazan crystalswere dissolved by 150 mL DMSO. Absorbance of the solution in each well wasdetected at 490 nm by a microplate reader. The data were given as means � S.E.Mand analyzed by students’t-test.

2.6. Gene transfection experiments on multicellular spheroids

The multicellular spheroids of HEK293 cells were prepared by a hanging droptechnique. Generally, cell suspensions were diluted in medium containing 0.24% (w/v) methylcellulose at a density around 105 cells per milliliter. Then, 25 mL dilutedcells were dropped on the lids of 10 cm cell culture plates. After 24 h, the spheroidswere formed and transferred to agarose coated 96-well plates with one spheroid ineach well. The spheroids were incubated for another 72 h and harvested for genetransfection experiments using an EGFP reporter gene.

Polyplexes consisted of fluorinated PPI dendrimers and EGFP plasmid wereprepared at their optimal N/P ratios on HEK293 cells. Lipo 2000/EGFP plasmid lip-oplex was prepared according to standard protocol of Lipofectamine 2000 (2.5 mLLipo 2000 complexed with 1 mg DNA). The HEK293 spheroids in agarose coated 96-well plates were treated with the prepared complexes (1 mg DNA in each complex).After incubation for 72 h, the spheroids werewashedwith PBS and EGFP expressionsin the spheroids were observed by confocal microscopy (Leica) using an argon laser(excitation wavelength: 488 nm).

3. Results and discussion

3.1. Characterizations of fluorinated PPI dendrimers and dendrimer/DNA polyplexes

Fluorinated G3, G4 and G5 PPI dendrimers were synthesizedusing a facile strategy as shown in Fig. 1. The average number ofheptafluorobutyric acids (F7) modified on the PPI dendrimers was

dendrimers and DNA. The polyplexes were prepared at N/P ratios of 0.5:1, 1:1, 2:1, 4:1trol. Three repeats were conducted for each sample.


Fig. 4. EGFP expressions in HEK293 cells transfected by G3-F79, G4-F716, and G5-F726. Unmodified G3, G4, and G5 PPI dendrimers, G5-Arg110, bPEI25K and four commercialtransfection reagents were used as controls. The transfection experiments were conducted for 48 h. The optimal N/P ratios of G3-F79, G4-F716 and G5-F726 were 1.6:1, 2:1 and 1.8:1,respectively, while the optimal N/P ratios of unmodified G3, G4 and G5 PPI dendrimers, G5-Arg110, and bPEI25K are 10:1, 10:1, 8:1, 10:1, and 8:1, respectively. Optimal transfectionconditions for the four commercial reagents were optimized according to the products’ protocols.


determined by a well-established ninhydrin assay and the resultswere shown in Table 1. According to the numbers of F7 groups andthe dendrimer generation, the yielding fluorinated PPI dendrimersare termed G3-F79, G3-F712, G4-F716, G4-F720, G5-F726 and G5-F735,respectively. The 19F NMR spectra of the products in Fig. S1 furtherconfirmed the successful conjugation of F7 groups on the surface ofPPI dendrimers.

The G3-F79/DNA, G4-F716/DNA and G5-F726/DNA polyplexeswere further characterized by agarose gel electrophoresis. Asshown in Fig. 2, all the fluorinated PPI dendrimers showed similarDNA condensation behaviors to unmodified PPI dendrimers. Thedendrimers are able to effectively retard the electrophoreticmobility of plasmid DNA in gels above an N/P ratio of 1:1. Thefluorinated dendrimers can condense the DNA into polyplexesbelow 300 nm at N/P ratios above 1:1 (Fig. 3a). Above an N/P ratio of2:1, the formed nanoparticles are positively charged which favors

Fig. 5. Quantitatively analysis of EGFP transfection efficacy of the materials onHEK293 cells by flow cytometry. The optimal N/P ratios of the materials were equal tothose listed in the caption of Fig. 4. Optimal N/P ratios of G4-F720 and G5-F735 were 1:1and 1.7:1, respectively. Three repeats were conducted for each transfection. **p < 0.01according to positive EGFP cells (%) analyzed by students’t-test.


efficient cellular uptake of dendrimer/DNA polyplexes (Fig. 3b).These results together confirmed that fluorinated PPI dendrimersmaintained the DNA packing abilities of unmodified PPIdendrimers.

3.2. Transfection efficacies of fluorinated PPI dendrimers ondifferent cell lines

In vitro transfection efficacies of the synthesized fluorinated PPIdendrimers on HEK293 and HeLa cells were conducted using bothEGFP and luciferase reporter genes. As G3-F712 shows extremelyweak transfection efficacy on both cell lines (Fig. S2), we investigatethe other five fluorinated PPI dendrimers in detail in further genetransfection studies. The optimal N/P ratio for each material wasscreened before the transfection experiments (Fig. S3eFig. S5).The fluorinated dendrimers including G3-F79, G4-F716, G4-F720,

Fig. 6. Luciferase activities of the HEK293 cells transfected by fluorinated PPI den-drimers. Unmodified G3, G4, and G5 PPI dendrimers, G5-Arg110, bPEI25K and fourcommercial transfection reagents were used as controls. N/P ratios of the materialsequal to those used in the EGFP transfection experiments on HEK293 cells. Three re-peats were conducted for each transfection. **p < 0.01 analyzed by students’t-test.


Fig. 7. Quantitatively analysis of EGFP transfection efficacies of fluorinated PPI den-drimers on HeLa cells by flow cytometry. Unmodified G3, G4, and G5 PPI dendrimers,G5-Arg110, bPEI25K and four commercial transfection reagents were used as controls.The transfection experiments were conducted for 48 h. The optimal N/P or mass ratiosof materials equal to those in the EGFP transfections on HEK293 cells except those ofG4-F720, G5-Arg110, and bPEI25K (optimal N/P ratios of 1.5:1, 14:1 and 12:1, respec-tively). Three repeats were conducted for each transfection. **p < 0.01 according to thepositive EGFP cells (%) analyzed by students’t-test.


G5-F726 and G5-F735 show much improved transfection efficaciescompared to the unmodified PPI dendrimers on HEK293 cells(Figs. 4 and 5). EGFP transfection efficacies of all the five fluorinatedPPI dendrimers are higher than 80%, while the efficacies of G3, G4and G5 PPI dendrimers are less than 5% on the cells (Fig. 5). Suchefficacies (both percent of EGFP positive cells and mean fluores-cence intensity) are superior to those of two representative trans-fection reagents G5-Arg110 and bPEI25K, as well as four commercialtransfection reagents such as Lipo 2000 (the highest one, 71.1%),SuperFect, PolyFect and jetPEI (both lipid- and polymer-basedtransfection reagents, Lipo 2000 belongs to lipid-based gene vec-tor, while SuperFect, PolyFect and jetPEI belong to polymer-basedones). In addition, the optimal N/P ratios of the fluorinated PPIdendrimers are around 2:1, which is much lower than those of

Fig. 8. Luciferase activities of the HeLa cells transfected by fluorinated PPI dendrimers.Unmodified G3, G4, and G5 PPI dendrimers, G5-Arg110, bPEI25K and four commercialtransfection reagents were used as controls. The optimal N/P ratios of G3-F79, G4-F716,G4-F720, G5-F726 and G5-F735 are 1.2:1, 2:1, 1.5:1, 1.5:1 and 1.7:1, respectively. Threerepeats were conducted for each transfection. **p < 0.01 analyzed by students’t-test.


unmodified PPI dendrimers (10:1, 10:1 and 8:1 for G3, G4 and G5PPI dendrimers, respectively), G5-Arg110 (10:1) and bPEI25K (8:1).This result is in accordance with our recent findings on fluorinatedPAMAM dendrimers [37]. Similarly, fluorination on PPI dendrimersimproves the luciferase transfection efficacy by more than twoorders of magnitude compared to unmodified PPI dendrimers(Fig. 6). These materials are more efficient than G5-Arg110, bPEI25K,SuperFect and PolyFect, and only slightly lower than Lipo 2000 andjetPEI.

The transfection efficacies of fluorinated PPI dendrimers (bothEGFP and luciferase plasmids) were also tested on HeLa cells. Again,fluorination significantly improves the transfection efficacies of PPIdendrimers on the cells (Fig. 7 and Fig. 8). The most efficientfluorinated PPI dendrimer G4-F720 achieves more efficient EGFPtransfection (78.5%) than all the control materials (Fig. 7 andFig. S6). G3-F79 and G5-F726 also show comparable transfection

Fig. 9. Cytotoxicities of fluorinated PPI dendrimers and their complexes with DNA onHeLa (a) or HEK293 (b) cells for 48 h. The concentrations of the fluorinated dendrimerswith or without DNA equal to those in the EGFP gene transfection experiments. Un-modified G3, G4, and G5 PPI dendrimers, G5-Arg110, bPEI25K and four commercialtransfection reagents were used as controls. Four repeats were conducted for eachsample.


Fig. 10. EGFP transfection efficacies of G3-F79, G4-F720 and G5-F726 (optimal N/P ratio of 1.6:1, 1:1 and 1.8:1, respectively) on 3D multicellular spheroids consisted of HEK293 cells.Lipo 2000 was used as control. The transfections were conducted for 72 h. The EGFP expressions in the spheroids were observed by confocal microscopy.


efficacy (similar percent of EGFP positive cells but higher meanfluorescence intensity) to the most efficient commercial reagentLipo 2000. The fluorinated PPI dendrimers also showmore efficientluciferase gene transfection efficacies than G5-Arg110, bPEI25K, Lipo2000 and jetPEI, and slightly lower efficacies compared to Super-Fect and PolyFect. As can be seen, transfection efficacies of thecommercial transfection reagents depend much on the cell linesand the reporter gene. Our fluorinated dendrimers are greatly su-perior to these lipid- or polymer-based transfection reagents as weachieve efficient and reproducible transfections in all cases.

The transfection efficacy of a gene vector depends on multiplekey steps during gene transfection such as DNA packing, serumstability, cellular uptake, endosomal escape, DNA unpacking and soon. As discussed in our previous result, fluorination significantlyimproves the cellular uptake of dendrimer/DNA polyplexes [37].Also, the fluorinated dendrimer/DNA polyplexes show more effi-cient endosomal escape compared to non-modified dendrimer/DNA polyplexes. In addition, fluorination increases the serum sta-bility of dendrimer/DNA polyplexes due the inert properties of themodified fluorinated groups and potentially tailors the intracellularDNA release after the polyplexes reach the cytoplasm or nuclei [37].These findings well explainwhy fluorination dramatically improvesthe transfection efficacies of PPI dendrimers.

3.3. Cytotoxicities of fluorinated PPI dendrimers and theirpolyplexes with DNA

Successful non-viral gene transfection requires efficient trans-fection while minimizing the toxicity [39]. However, most of thepolymeric gene vectors are associated with the “malignant” cor-relation between transfection efficacy and cytotoxicity, whichseems like choosing between Scylla and Charybdis: either a hightransfection efficacy associated with serious cytotoxicity, or a lowtransfection efficacy with better compatibility [40]. The fluorina-tion strategy can efficiently break up such a correlation betweenefficacy and toxicity as the fluorinated dendrimers achieve efficienttransfection efficacy at extremely lowN/P ratios. As shown in Fig. 9,all the fluorinated PPI dendrimers show low cytotoxicity on


HEK293 and HeLa cells at their transfection concentrations. Thepresence of DNA does not affect the cytotoxicity of fluorinated PPIdendrimers on HeLa cells. About 90% of cells survived after genetransfection. The fluorinated PPI dendrimers ensure both efficientgene transfection and 90% cell viability after transfection, which aresuperior to the six representative transfection reagents.

3.4. Transfection efficacies of fluorinated PPI dendrimers onmulticellular spheroids

All the above gene transfection experiments were conducted inthe presence of 10% fetal bovine serum (FBS), suggesting that thefluorinated PPI dendrimers have excellent serum-resistance. Thehigh transfection efficacy, low cytotoxicity and good serum-resistance of fluorinated PPI dendrimers promote us to test thepotential applications of these materials for cancer gene therapy.We further used a 3D multicellular spheroids consisted ofHEK293 cells to test the efficacies of the fluorinated PPI dendrimers.As shown in Fig. 10, G4-F720 and G5-F726 show comparable trans-fection efficacy (EGFP fluorescence intensity) on the multicellularspheroid surface. However, stronger EGFP fluorescence in theinterior of the spheroid transfected with G4-F720/EGFP and G5-F726/EGFP polyplexes compared to Lipo 2000/EGFP lipoplex isobserved. Therefore, the fluorinated dendrimers have higher abilityto deliver plasmid DNA into the interior of 3D spheroids than Lipo2000. Only G3-F79 shows weaker ability to transfect theHEK293 cells in the multicellular spheroids than Lipo 2000. Thisdendrimer generation effect is probably due to the lower chargedensity on the G3-F79 than that of higher generation ones (G4-F720and G5-F726).

4. Conclusions

High transfection efficacy and low cytotoxicity during trans-fection are the two main requirements in the design of polymericgene vectors [41,42]. In this study, we successfully synthesizedseveral fluorinated PPI dendrimers using a facile reaction. Thefluorinated PPI dendrimers show efficient DNA condensation



ability and high transfection efficacy on both HEK293 and HeLacells. Fluorination significantly improves the transfection efficacy ofcationic PPI dendrimers and down-regulates their optimal N/P ra-tios from 8:1 or 10:1 to 2:1. Transfection efficacies of these mate-rials are comparable or superior to four commercial transfectionreagents such as Lipofectamine 2000, jetPEI, SuperFect and Poly-Fect when delivering EGFP or luciferase reporter gene. Since thetransfection experiments for fluorinated PPI dendrimers wereconducted at extremely low N/P ratios, the transfected cellsmaintained high cell viability around 90%. In addition, these fluo-rinated PPI dendrimers show high transfection efficacy on 3Dmulticellular spheroids. Such a fluorination strategy allows us todesign efficient and low cytotoxic gene materials based on cationicPPI dendrimers. Further work will be focused on exploring thein vivo efficacies of these fluorinated PPI dendrimers especially oncancer gene therapy.

Acknowledgment

The authors thank the grants including National Natural ScienceFoundation of China, China (No. 21322405), the Shanghai Rising-star Program (13QA1401500) and the Science and Technology ofShanghai Municipality (11DZ2260300) for financial support.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2014.03.040.

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Fluorinated poly(propylenimine) dendrimers as gene vectors