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Biomaterials 35 (2014) 9187e9198

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Biomaterials

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Synergistic effect of amino acids modified on dendrimer surface ingene delivery

Fei Wang, Yitong Wang, Hui Wang, Naimin Shao, Yuanyuan Chen, Yiyun Cheng*

Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai 200241, People's Republic of China

a r t i c l e i n f o

Article history:Received 12 July 2014Accepted 19 July 2014Available online 8 August 2014

Keywords:DendrimerAmino acidGene deliverySynergistic effect

* Corresponding author.E-mail address: yycheng@mail.ustc.edu.cn (Y. Che

http://dx.doi.org/10.1016/j.biomaterials.2014.07.0270142-9612/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence ofmultiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the bar-riers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers witharginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acidsplay different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanineimproves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cyto-toxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a syn-ergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimalcytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triple-functionalized dendrimers with comparable transfection efficacy to several commercial transfectionreagents. Such a combination strategy should be applicable to the design of efficient and biocompatiblegene vectors for gene delivery.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Gene therapy remains a promising strategy in the treatmentof hereditary and acquired diseases in recent years. However, thelargest obstacle in gene therapy is to develop high efficient andnontoxic gene carriers [1,2]. An ideal gene carrier should possessmultiple functions to overcome the barriers at different stages ingene transfection process [3]. First, the vector should condensegenes into nanoparticles and the formed nanoparticles should bestable in serum. Then, the nanoparticles can be internalized intocells via specific endocytosis pathways and escaped from acidicvesicles such as endosomes and lysosomes. Finally, the vectorshould release the bound genes in the cytoplasm or nucleus [1].Cationic polymers such as polyethylenimine (PEI) [4], chitosan[5], poly-L-lysine [6], diethylaminoethyl-dextran (DEAE-dextran)[7] and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA)[8] are widely used as nonviral gene carriers due to their ver-satile structures and unique properties, but inherent cytotoxicityand relatively low transfection efficiency are associated withthese polymers, which limit their applications in gene therapy[9].

ng).

Dendrimers are a class of synthetic polymers with uniqueproperties such as well-defined structure, spherical shape, lowpolydispersity, excellent solubility, and large number of surfacefunctionalities and interior cavities [10e12]. Cationic dendrimerscan effectively condense DNA into stable complexes due to themultivalency effect of the positive charges on dendrimer surface[13]. In addition, there is a high density of protonable tertiaryamine groups in dendrimer interior, providing the “protonsponge effect” during endosomal escape process [14]. As a result,dendrimers and their conjugates were widely used as genevectors during the past decade [15]. Dendrimer-based trans-fection reagents such as SuperFect and PolyFect have alreadyentered the market. To further improve their transfection effi-ciency, dendrimers were modified with cyclodextrins [16], lipids,sugars, peptides, fluorous compounds [17,18], amino acids[19e24], mitochondrial targeting ligands [25] and nanoparticles[26]. Among these functionalized dendrimers, amino acid-dendrimer conjugates are of great interest to the researchers[20e24,27]. Amino acids have the same fundamental structure,differing only in their residues. They can be sorted into cationic,anionic and neutral amino acids, or hydrophilic and hydrophobicamino acids. Dendrimers can be modified with amino acids viafacile condensation reactions. Conjugation of cationic aminoacids such as arginine (Arg) and lysine (Lys) to dendrimerdirectly tailors the charge density on dendrimer surface

F. Wang et al. / Biomaterials 35 (2014) 9187e91989188

[19e23,28,29]. Besides, the residues of these amino acids such asguanidinium and imidazole groups play essential roles in thegene delivery processes. The guanidine group has specific affinitywith cell membranes, while the imidazole group provides addi-tional pH-buffering capacity during endosomal escape [24,30].Arginine-rich or histidine (His)-rich peptides can be directly usedas gene vectors [31]. Conjugation of hydrophobic amino acidssuch as phenylalanine (Phe) and leucine to dendrimer surfacetailors the hydrophobicity of dendrimer surface, which isessential in the endocytosis process [32,33]. Conjugation of theseamino acids can increase the transfection efficacy of dendrimersthrough different mechanisms, however, there are multiplebarriers in gene transfection and single-functionalization cannotovercome all the barriers.

A solution to this problem is multiple-functionalization ofdendrimers with different amino acids such as Arg, His and Phe.Combination of these amino acids on dendrimer may simulta-neously improve the membrane affinity, endocytosis and endo-somal escape of the complexes. A recent study found that acombination of His and hydrophobic amino acids such as Phe andtyrosine can significantly improve the siRNA interference efficacy ofa reduction-sensitive polymer [34]. Also, dual-functionalization ofArg and His on dendrimer surface allows high transfection efficacy[35]. However, these dual-functionalized polymers were synthe-sized in multi-steps and the synergistic effect of amino acids onthese polymers still needs in-depth investigations.

Here, we systematically investigate the synergistic effect ofArg, Phe and His with distinct functions on dendrimer surface ingene delivery. A one-pot approach was adopted to constructsingle-, dual- and triple-functionalized dendrimers (Fig. 1).Generation 5 (G5) polyamidoamine (PAMAM) dendrimer with amolecular weight of 28826 Da was used as the scaffold material.A total number of 15 dendrimer-amino acid conjugates including5 single-, 5 dual- and 5 triple-functionalized dendrimers,respectively were synthesized. The physicochemical properties,complex formation, transfection efficacy, transfection mecha-nisms and cytotoxicity of these amino acid-modified dendrimerswere investigated. The aims of this study are to reveal the syn-ergistic effect of amino acids on the transfection efficacy ofamino acid-modified dendrimers and to prepare multiple-functionalized dendrimers as efficient gene vectors using afacile one-pot strategy.

Fig. 1. Synthesis of multi-functionalized d

2. Materials and methods

2.1. Materials

G5 PAMAM dendrimer with an ethylenediamine core and surface primaryamine groups was purchased fromDendritech (Midland, MI). Boc-Arg(Pbf)-OH, Boc-His(Trt)-OH and Boc-Phe-OH were purchased from GL Biochem (Shanghai, China)Ltd. YOYO-1 and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad,California). PolyFect was purchased from Qiagen (German). JetPEI was purchasedfrom Polyplus-Transfection (France). Fetal bovine serum (FBS) and Dulbecco'smodified Eagle's medium (DMEM) were purchased from GIBCO (Gaithersburg, MD).G5 dendrimer was received in methanol solution and the solvent was distilledbefore use. The dendrimer was characterized by 13C NMR and polyacrylamide gelelectrophoresis. All the other chemicals were used as received without furtherpurification.

2.2. Synthesis and characterization of the single-, dual- and triple-functionalizeddendrimers

Amino acid-modified G5 PAMAM dendrimers were synthesized by a facilecondensation reaction as described elsewhere [28]. Briefly, different amounts ofBoc-Arg(pbf)-OH, Boc-Phe-OH and Boc-His(Trt)-OH were dissolved in 1.5 mLdehydrated N,N-dimethyl formamide (DMF), followed by addition of dicyclohex-ylcarbodiimide (DCC,1.3 molar equivalents of carboxyl group in the protected aminoacids) and N-hydroxysuccinimide (NHS, 1.2 molar equivalents of carboxyl group inthe protected amino acids) to activate the carboxyl groups of amino acids for 6 h.50 mg G5 PAMAM dendrimer was dissolved in 2 mL anhydrous dimethyl sulfoxide(DMSO) and added dropwise into the activated amino acid solution. After that, thereaction mixture was stirred at room temperature for 7 d. The molar ratios of fedamino acids to each G5 dendrimer are listed in Table 1. The reaction mixture wasdialyzed against DMSO (500 mL) for two times and freeze-dried, the obtained solidwas dissolved in 2 mL trifluoroacetic acid (TFA) and stirred at room temperature for6 h to de-protect the protected groups such as Boc, Pbf and Trt. Then, TFA wasremoved by rotary evaporation and the crudematerials were dialyzed against DMSO(500 mL, three times), PBS buffer (500 mL, three times) and distilled water (500 mL,ten times). The purified product was freeze-dried as white powders. The yieldingproducts were characterized by 1H NMR in D2O (Varian 699.804 MHz).

2.3. Preparation of polymer/DNA complexes

All the polymer/DNA complexes were freshly prepared before characterizationor gene transfection experiments. Generally, amino acid-dendrimer conjugateswereadded into 0.8 mg plasmid DNA (EGFP or luciferase plasmid) at different N/P ratiosand the sample was incubated for 30 min at room temperature. The N/P ratio wascalculated according to the cationic groups (N number) on the dendrimer surface toanionic phosphate groups (P number) in plasmid DNA. The corresponding polymer/DNA weight ratios for the complexes at different N/P ratios are also shown inTable S1. For His- and Phe-functionalized dendrimers, the N number for each con-jugate was a constant of 128. For Arg-functionalized dendrimers, the N number wasa sum of 128 and the number of conjugated Arg moieties since Arg has two cationicgroups. Since the imidazole group in His and the tertiary amine group in dendrimerare not protonated at pH 7.4, these groups were not considered when calculating theN numbers. For the commercial transfection reagents such as Lipofectamine 2000,

endrimers using a one-pot approach.

Table 1Characterizations of the single-, dual- and triple-functionalized dendrimers.

Functionalized dendrimers Dendrimer conjugates Number of amino acid per G5 dendrimer Molecular weight (Da)

Fed Conjugated

Arg Phe His Arg Phe His

Single-functionalized dendrimers G5-Arg110 166 0 0 110 0 0 46,008G5-Arg33 0 45 0 33 0 0 33,981G5-Phe99 0 166 0 0 99 0 43,398G5-Phe71 0 83 0 0 71 0 39,276G5-His113 0 0 166 0 0 113 44325

Dual-functionalized dendrimers G5-Arg34Phe71 83 83 0 34 71 0 44,587G5-Arg51Phe53 111 55 0 51 53 0 44,593G5-Arg82Phe34 124 42 0 82 34 0 46,639G5-Arg44His53 83 0 83 44 0 53 42968G5-Phe64His40 0 83 83 0 64 40 43733

Triple-functionalized dendrimers G5-Arg24Phe20His40 48 24 95 24 20 40 41,005G5-Arg33Phe49His14 85 64 43 33 49 14 43113G5-Arg35Phe29His28 67 33 67 35 29 28 42,402G5-Arg47Phe24His25 83 28 55 47 24 25 43,129G5-Arg56Phe22His22 95 24 48 56 22 22 43,829

F. Wang et al. / Biomaterials 35 (2014) 9187e9198 9189

PolyFect and JetPEI, the complexes were prepared according to the protocols at theiroptimal conditions.

2.4. Characterization of the polymer/DNA complexes

The DNA binding capacity of amino acid-dendrimer conjugates was investigatedby agarose gel (Biowest, Spain) electrophoresis. Generally, the polymer/DNA com-plexes at N/P ratios of 0.5:1, 1:1, 2:1 and 4:1 were prepared in deionized water anddiluted with DNA loading buffer. The samples were electrophoresed on a 1% (w/v)agarose gel at 100 V for 1 h. The DNA in the gels were stained by ethidium bromideand visualized under UV illumination (Tanon-2500, China). For the DNA releaseassay, polymer/DNA complexes were prepared a specific molar concentration anddifferent concentrations of heparin were added to release the bound DNA in thecomplexes. The resulting solutions were further analyzed by agarose gel electro-phoresis as described above.

Fig. 2. 1H NMR spectra of single-, dual- and t

The size and zeta potential analysis of polymer/DNA complexes prepared indeionized water at N/P ratios of 0.5:1, 5:1, 10:1, 20:1 and 40:1 were carried out bydynamic light scattering (DLS) using Malvern Zetasizer Nano ZS90 (Malvern, UK) at25 �C.

2.5. Cell culture and in vitro gene transfection experiments

HeLa (ATCC, a human cervical carcinoma cell line), HEK293 (a human embryonickidney cell line, ATCC) and NIH 3T3 (ATCC, a mouse embryonic fibroblast cell line)cells were cultured with DMEM containing 100 units/mL penicillin sulfate andstreptomycin and 10% (v/v) heat-inactivated FBS at 37 �C under a humidified at-mosphere containing 5% CO2. Before gene transfection experiments, HeLa and NIH3T3 cells were seeded in 24-well plates and cultured overnight to reach an appro-priate cell density. The polymer/DNA complexes containing 0.8 mg plasmid DNAwere diluted with 100 mL DMEM containing 10% FBS and incubated 30 min at room

riple-functionalized dendrimers in D2O.

F. Wang et al. / Biomaterials 35 (2014) 9187e91989190

temperature. Then, the complexes were further diluted with 150 mL medium andincubated with the cells for 6 h. After that, 500 mL fresh mediumwas added to eachwell and the cells were further cultured for 42 h. EGFP expressions in the cells wereobserved by a fluorescence microscopy (Olympus, Japan) and quantitativelyanalyzed by flow cytometry (BD FACSCalibur, San Jose). Luciferase expressions in thecells were analyzed according to the manufacturer's protocols (Promega). Thecommercial gene transfection reagents including JetPEI, PolyFect and Lipofectamine2000 were used as positive controls.

2.6. Cytotoxicity of the amino acid-dendrimer conjugates and their DNA complexes

The cytotoxicity of amino acid-dendrimer conjugates and their DNA complexeswere examined on NIH 3T3 cells using a well-established MTT assay. The cells wereseeded in 96-well plates at a density around 104 cells per well and cultured for 12 hin 100 mL DMEM containing 10% FBS. The cells were treated with polymers orpolymer/DNA complexes at different concentrations for 48 h. The chosen polymerconcentrations (0.55 mM) and DNA concentration (3.2 mM) equal to those in the genetransfection experiments.

2.7. Cellular uptake of polymer/DNA complexes

To analyze the cellular uptake of polymer/DNA complexes, the plasmid DNAwaslabeled with a green fluorescent dye YOYO-1 for 10 min according to the manu-facturer's protocol (Invitrogen). The polymer/DNA complexes were then prepared asdescribed above (the dendrimer concentration is fixed for different amino acid-dendrimer conjugates in the complexes). Generally, NIH 3T3 cells was seeded in

Fig. 3. DNA retardation assay of amino acid-dendrimer conjugates. The polymer/DNA comratios of 0.5:1, 1:1, 2:1 and 4:1, respectively. (a) single-functionalized dendrimers, (b) dual-

24-well plates and cultured for 24 h to reach an appropriate cell density, and YOYO-1(excitation at 491 nm and emission at 509 nm) labeled complexes were incubatedwith the cells for 1 and 2 h. After that, the cells werewashedwith 500 mL cold PBS forthree times. Cellular uptake of the complexes was analyzed by flow cytometry.

2.8. pH-buffering capacity assay

The pH buffering capacity of amino acid-dendrimer conjugates was determinedas described below. Briefly, the concentration of different conjugates were fixed at aconstant dendrimer concentration (22.6 nM), and pH value of the conjugate solu-tionswas adjusted to 7.4. Then the samples were titratedwith 0.12 M HCl. pH value ofthe conjugate solution after each titration (1 mL) was measured using a pH meter(Mettler-Toledo). The titration experiment was continued until the pH value of thesolution reaches 5.0 (pH range of 7.4e5.0 mimics the endosome acidification).

2.9. Confocal microscopy

NIH 3T3 cells were seeded on glass slides in 24-well plates and cultured for 24 hat 37 �C. The cells were incubated with YOYO-1-labeled polymer/DNA complexes for2 and 4 h. Themediumwere removed and the cells werewashed with 500 mL PBS forthree times. The cells were further incubated with PBST (PBS containing 0.1% Tween20) for 5 min, and then with 0.2% BSA at room temperature for 30 min. The acidicvesicles of NIH 3T3 cells was stained with LAMP-2 (2 mg/mL) antibody conjugatedwith Alexa Fluor 647 (excitation at 650 nm and emission at 665 nm) for 1 h at 37 �C.The cells were washed with PBS for three times and nuclei of the cells were stainedby Hoechst 33342 (excitation at 346 nm and emission at 460 nm, 5 mg/mL) for

plexes were prepared by mixing the synthesized polymers with EGFP plasmid at N/Pfunctionalized dendrimers and (c) triple-functionalized dendrimers.

F. Wang et al. / Biomaterials 35 (2014) 9187e9198 9191

10 min at room temperature. After the cells were washed with PBS for three times,co-localization of the complexes with acidic vesicles was observed by confocal mi-croscopy (Leica SP5, Germany).

2.10. In vivo transfection

HeLa tumor xenograft model was established as described below. 6-week-oldfemale BALB/c nude mice at an average weight of 22.0 g were obtained from SLACLaboratory Animal Co. Ltd. (Shanghai, China). The animals were housed underspecific-pathogen-free conditions within the animal care facility at East ChinaNormal University. The animal experiments were carried out according to the Na-tional Institutes of Health guidelines for care and use of laboratory animals andapproved by the ethics committee of East China Normal University. HeLa cells werecultured in DMEM containing 10% FBS. 100 mL cell suspensions in PBS (containing107 cells) were subcutaneously injected into the BALB/c nudemice.When tumor sizereaches 50 mm3, the mice were administrated with 25 mL G5-Arg47Phe24His25/DNAor G5-Arg110/DNA complexes in 5% glucose solution (containing 3.3 mmol polymerand 10 ug luciferase plasmid) by injection into the tumor. The animal treatedwith 5%glucose solution was used as a control. The treatments were repeated every day anda total number of three injections were administrated for each animal. The in vivotransfection experiments were continued for another two days. After that, 200 mL D-luciferin potassium salt (15 mg/mL) was intraperitoneally injected into the anes-thetized mice. 5 min later, the mice were placed into an in vivo imaging system(Xenogen IVIS-200, Caliper Life Sciences, Hopkinton), and the luminescence at tu-mor site was recorded with an exposure time of 5 min.

Fig. 4. Size (a) and zeta potential (b) analysis of polymer/DNA complexes

3. Results and discussion

3.1. Synthesis and characterization of single-, dual- and triple-functionalized dendrimers and their complexes with DNA

To investigate the synergistic effect of amino acids in gene de-livery, G5 PAMAM dendrimer was functionalized with Arg, Phe andHis using a facile condensation reaction. For dual- and triple-functionalized dendrimers, mixtures of specific amino acids atdifferent molar ratios were added to the dendrimer solution andthe functionalized dendrimers were obtained by a one-pot strategy.The number of each amino acid conjugated to dendrimer wascharacterized by 1H NMR. As shown in Fig. 2 and Fig. S1, the fourbroad peaks (Ha, Hb, Hc,b0 and Hd,d0) in the chemical shift range of2.0e3.5 ppm correspond to protons on dendrimer scaffold andother peaks in the spectra are assigned to protons of Arg, Phe andHis, respectively [36]. The average number of amino acid conju-gated to dendrimer was calculated according to the peaks areas ofdendrimer protons and amino acid protons, respectively. Thecomponents of the 15 amino acid-dendrimer conjugates are listedin Table 1. For single-functionalized dendrimer, the conjugate G5-

prepared at N/P ratios of 0.5:1, 5:1, 10:1, 20:1 and 40:1, respectively.

F. Wang et al. / Biomaterials 35 (2014) 9187e91989192

Arg110 represents a G5 PAMAM dendrimer conjugated with anaverage number of 110 Arg molecules on its surface. For dual-functionalized dendrimer, the conjugate G5-Arg34Phe71 repre-sents a G5 dendrimer modified with 34 Arg and 71 Phe moieties.For triple-functionalized dendrimer, the conjugate G5-Arg35Phe29His28 means a conjugate with 35 Arg, 29 Phe and 28His moieties.

Gel retardation experiments were used to evaluate the DNAbinding ability of the synthesized conjugates. As shown in Fig. 3, allthe conjugates except G5-Phe99 successfully reduce DNA mobilityabove an N/P ratio of 1:1. G5-Phe99 even fails to condense DNA at anN/P ratio of 4:1. The lower DNA binding capacity of G5-Phe99 isprobably due to the charge shielding effect of aromatic rings pre-sent on dendrimer surface [33]. For gene delivery, the size ofpolymer/DNA polyplexes is essential for efficient gene transfection.As shown in Fig. 4 and Fig. S2, the conjugates containing Argmoieties can condense plasmid DNA into nanoparticles around200 nm (Fig. 4 and Fig. S2), while those without Arg moieties suchas G5-Phe99, G5-Phe71, G5-His113 and G5-Phe64His40 fail to formsmall nanoparticles below 400 nm with DNA at N/P ratios of 5:1,

Fig. 5. Synergistic effect of Arg and Phe on G5-Arg34Phe71. The transfection efficacy of G5-AG5-Phe99, respectively. The dendrimer concentration is fixed at 0.36 mM for all the conjugatein HeLa cells measured by flow cytometry (n ¼ 3). Statistically significant differences are d

10:1 and 20:1. The polyplexes with sizes around 200 nm areappropriate for cellular uptake and gene delivery. This result sug-gests that Arg is essential in the formation of stable complexes forgene transfection [37e39]. Plasmid DNA is negatively charged atneutral conditions, and the addition of all the conjugates can turnDNA charge state from negative to positive at an N/P ratio of5:1. The biophysical properties of the polymer/DNA complexes willbe further discussed to analyze the transfection mechanisms ofsingle-, dual- and triple-functionalized dendrimers in Section 3.3.

3.2. Synergistic effect of amino acids modified on dendrimer surfacein gene delivery

The transfection efficacy of single-, double- and triple-functionalized dendrimers were tested on HeLa, HEK293 or NIH3T3 cells using EGFP and luciferase plasmid as reporter genes. Asshown in Fig. 5, a dual-functionalized dendrimer G5-Arg34Phe71successfully transfected 75.2% HeLa cells at its optimal N/P ratio,while the single-functionalized conjugates G5-Arg33 and G5-Phe71only transfected 21.8% and 12.1% HeLa cells at equivalent dendrimer

rg34Phe71 on HeLa cells was compared with those of G5-Arg33, G5-Phe71, G5-Arg110 ands. (a) Fluorescence microscopy images of the transfected cells and (b) EGFP expressionsenoted by ***p < 0.001 using student's t-test.

Fig. 6. Transfection efficacies of single-, dual- and triple-functionalized dendrimers on NIH 3T3 cells for 48 h. The dendrimer concentration is fixed at 0.55 mM for all the conjugates.(a) Fluorescence microscopy images of the transfected cells and (b) EGFP expressions in NIH 3T3 cells measured by flow cytometry (n ¼ 3).

Fig. 7. Luciferase activities of NIH 3T3 cells transfected by single-, dual- and triple-functionalized dendrimers for 48 h. The dendrimer concentration is fixed at 0.55 mM for all theconjugates. The luciferase activity is expressed as relative luciferase light units per mg protein (RLU/mg protein, n ¼ 3).

F. Wang et al. / Biomaterials 35 (2014) 9187e91989194

and EGFP plasmid molar concentrations. Also, G5-Arg34Phe71 ismuch more efficient (both EGFP positive cells and mean fluores-cence intensity) than G5-Arg110 or G5-Phe99. Not limited to theoptimal N/P ratio, G5-Arg34Phe71 shows much higher efficacy thanG5-Arg110 on HeLa cells at all the N/P ratios (Fig. S3). These resultssuggest synergistic effect of Arg and Phe on the dual-functionalizeddendrimer in gene delivery. Similar results were obtained onHEK293 cells (Fig. S4).

The synergistic effect amino acids in gene delivery were furtherconfirmed on NIH 3T3 cells. As shown in Fig. 6 and Fig. S5, all thedual- and triple-functionalized dendrimers except G5-Phe64His40exhibit higher transfection efficacy than single-functionalizeddendrimers. The triple-functionalized dendrimers G5-Arg47Phe24His25 and G5-Arg56Phe22His22 are more efficient ondelivering EGFP plasmid than the dual-functionalized ones. G5-Phe64His40 shows poor transfection efficacy due to its weak DNAbinding capacity as revealed in Fig. 4. Similar results were obtainedon NIH 3T3 cells using a luciferase reporter gene (Fig. 7). The dual-and triple-functionalized dendrimers show higher luciferase

Fig. 8. Cytotoxicities of single-, dual- and triple-functionalized dendrimers (a) and their DNAequal to those in gene transfection experiments.

activity than single-functionalized ones. The synthesized conju-gates except G5-Phe99 show minimal cytotoxicity on NIH 3T3 cellsat transfection concentrations (Fig. 8a). The relatively high toxicityof G5-Phe99 is due to the hydrophobic character of dendrimersurface after Phe modification. This phenomenon is also observedin a previous result [32]. Though it seems to introduce cytotoxicityafter Phe modification, the dual- and triple-functionalized den-drimers containing Phe moieties showminimal cytotoxicity on NIH3T3 cells. Even in the presence of plasmid DNA, these materialsmaintain high cell viability above 90% (Fig. 8b), suggesting that thedual- and triple-functionalized dendrimers can achieve hightransfection efficacy with low cytotoxicity on the transfected cells.

3.3. Synergistic mechanism of amino acids modified on dendrimersurface in gene delivery

The transfection efficacy of a gene material depends on severalparameters such as complex formation and stability, cellular up-take, endosomal escape and intracellular DNA release [1]. As shown

complexes (b) on NIH 3T3 cells for 48 h (n ¼ 5). The polymer concentrations (0.55 mM)

F. Wang et al. / Biomaterials 35 (2014) 9187e9198 9195

in Figs. 5 and 6, Arg plays an essential role in gene transfection. Thetransfection efficacy of dual- and triple-functionalized dendrimersincreases in proportion with the number of conjugated Arg moi-eties. Those conjugates without Arg moieties such as G5-Phe99, G5-His113 and G5-Phe64His40 show extremely low transfection efficacy.First, the conjugated Arg moiety has two positive charged groups,which facilitates DNA condensation. This is evidenced by DNAretardant and DLS results in Figs. 3 and 4. Arg-containing conju-gates show strong DNA binding capacity, while those without Argmoieties fail to condense plasmid DNA into nanoparticles around200 nm at relative lowN/P ratios. Second, the positive charge of theguanidinium group in Arg is delocalized on three nitrogen atoms,thus guanidinium shows better interactions with anions such asphosphates than localized cations such as ammonium [40]. Third,the guanidinium group has strong affinity to cell membranesthrough ionic pairing and hydrogen bonding [41]. Due to thesereasons, Arg-dendrimer conjugates were widely investigated asefficient gene vectors during the past decade [19e23]. However, thedendrimer conjugated with 110 Arg moieties (G5-Arg110) showsmuch lower efficacy compared to the dual- and triple-functionalized dendrimers, suggesting that His and Phe moietiesalso play important roles in gene delivery. This is attributed to thehigh charge density on G5-Arg110, which decreases the penetrationof polymer/DNA complex through cell membrane via endocytosis.The high charge density on polymers may also trigger

Fig. 9. (a) Cellar uptake of polymer/DNA complexes for 1 and 2 h by NIH 3T3 cells (n ¼ 3student's t-test. The polymer concentrations equal to those in gene transfection experimenconcentrations (n ¼ 4). (c) pH-buffering capacities of G5-His113 and G5-Arg110 (n ¼ 3). (d) Concomplex (green) for 2 and 4 h. The acidic vesicles were stained with LAMP-2 conjugated winterpretation of the references to color in this figure legend, the reader is referred to the

destabilization of self-assembled phospholipids in the cell mem-brane, resulting in serious cytotoxicity [9]. Increased cytotoxicity isalso a limiting factor for high transfection efficacy.

One solution to this problem is tailoring the balance of chargeand hydrophobic contents on dendrimer surface [42]. Replacementof partial Arg moieties in G5-Arg110 with hydrophobic amino acidsuch as Phe can improve the complex cellular uptake. As shown inFig. 9a, complexes of G5-Arg51Phe53, G5-Arg47Phe24His25 and G5-Arg56Phe22His22 with DNA show higher cellular uptake than thatof G5-Arg110. As a result, dual-functionalized dendrimer such as G5-Arg51Phe53 and triple-functionalized dendrimer such as G5-Arg47Phe24His25 and G5-Arg56Phe22His22 are more efficient ingene transfection than G5-Arg110. In addition, G5-Arg51Phe53/DNAcomplex is easier to release their bound DNA in the presenceheparin than G5-Arg110/DNA complex (Fig. S6). Such a hydrophobiceffect on improving gene transfection efficacy is also reported inseveral polymeric gene delivery systems [42e45]. Though Pheeffectively tailors the hydrophobic/hydrophilic balance on den-drimer surface, the conjugation of excess Phe moieties to a G5dendrimer also leads to increased cytotoxicity. For example, G5-Phe99 is even more cytotoxic than unmodified G5 PAMAM den-drimer and it kills most of the cells at a concentration of 50 mg/mL(Fig. 9b).

The incorporation of His into the conjugates can alleviate thetoxicity issues of Arg and Phe. As shown in Fig. 9b, G5-Arg44His53

), statistically significant differences are denoted by *p < 0.05 and ***p < 0.001 usingts. (b) Cytotoxicities of amino acid-dendrimer conjugates on NIH 3T3 cells at differentfocal images of NIH 3T3 cells incubated with YOYO-1-labeled G5-Arg47Phe24His25/DNAith Alexa Fluor 647 (red) and the nuclei were stained with Hoechst 33342 (blue). (Forweb version of this article.)

F. Wang et al. / Biomaterials 35 (2014) 9187e91989196

and G5-Arg47Phe24His25 are less cytotoxic than G5-Arg110 on NIH3T3 cells. G5-His113 is non-toxic at concentrations up to 600 mg/mL.Besides the role of reducing cytotoxicity, the imidazole group(pKa ~ 6.04) in His is protonable at mild acidic conditions. As shownin Fig. 9c, G5-His113 shows better pH-buffering capacity than G5-Arg110 within the pH range of 7.4 to 5.0 (endosome acidification),suggesting that the incorporation of His moieties to the conjugatescan facilitate endosomal escape [24]. The triple-functionalizedconjugate G5-Arg47Phe24His25 shows excellent endosomal escapeability and the complex is not co-localized with acidic vesicleswithin 2 h (Fig. 9d).

It is worth noting that the molar ratios of Arg, Phe and Hisshould be carefully chosen to obtain an efficient triple-functionalized dendrimer. For example, G5-Arg47Phe24His25 andG5-Arg56Phe22His22 are much more efficient in delivering EGFPplasmid on NIH 3T3 cells than the other three triple-functionalizeddendrimers (Fig. 6). G5-Arg47Phe24His25 and G5-Arg56Phe22His22show comparable efficacy with several commercial transfection

Fig. 10. Comparisons of triple-functionalized dendrimers with three commercial transfectiofor G5-Arg47Phe24His25 and G5-Arg56Phe22His22 are 30:1 and 32:1, respectively. Gene tramanufacturer's protocols. (a) Fluorescence microscopy images of the transfected cells and (significant differences are denoted by *p < 0.05 and **p < 0.01 using student's t-test.

reagents such as JetPEI, PolyFect and Lipofectamine 2000 at theiroptimal conditions (Fig. 10). The triple-functionalized dendrimerG5-Arg47Phe24His25 also shows higher in vivo transfection efficacythan the single functionalized dendrimer G5-Arg110 when deliv-ering a luciferase plasmid (Fig. 11). These results suggest that thetriple-functionalized dendrimers can be developed for commercialpurpose in the future.

4. Conclusions

In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with Arg, Phe and His for gene de-livery. The amino acids show synergistic effects on dual- and triple-functionalized dendrimers. Arg in the conjugates is essential forcomplex formation. Phe modulates the balance of hydrophobic andhydrophilic contents on dendrimer surface, thereby facilitates thecellular uptake process. His improves pH-buffering capacity andreduces cytotoxicity of the cationic dendrimers. These amino acids

n reagents (JetPEI, PolyFect and Lipofectamine 2000) on transfection efficacy. N/P ratiosnsfection experiments for the commercial reagents were optimized according to theb) EGFP expressions in NIH 3T3 cells measured by flow cytometry (n ¼ 3). Statistically

Fig. 11. In vivo gene transfection on a HeLa tumor xenograft model. The animals were treated with 5% glucose solution, G5-Arg47Phe24His25/DNA and G5-Arg110/DNA complexes,respectively. Luciferase plasmid was used as the reporter gene. The polyplexes were prepared in 5% glucose solution.

Fig. 12. Transfection mechanisms of single-, dual- and triple-functionalized dendrimers.

F. Wang et al. / Biomaterials 35 (2014) 9187e9198 9197

F. Wang et al. / Biomaterials 35 (2014) 9187e91989198

have different functions in the gene delivery process and hence acombination of them generates synergistic effects in gene delivery(Fig. 12). The efficient triple-functionalized dendrimers achievecomparable transfection efficacy to several commercial trans-fection reagents on NIH 3T3 cells. In addition, the dual- and triple-functionalized dendrimers show low cytotoxicity on the trans-fected cells. This study provides a new insight into the design ofefficient and low cytotoxic gene vectors using a facile strategy.

Acknowledgment

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

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2014.07.027.

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