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Biomaterials 30 (2009) 5793–5803

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Biomaterials

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The gene transfection efficiency of a folate–PEI600–cyclodextrin nanopolymer

Hong Yao a,b, Samuel S. Ng b, Wesley O. Tucker b, Yuk-Kai-Tiu Tsang b, Kwan Man c, Xiao-mei Wang d,Billy K.C. Chow e, Hsiang-Fu Kung a,g, Gu-Ping Tang b,f,**, Marie C. Lin a,b,*

a Biomedical Engineering Research Centre, Kunming Medical University, Kunming, PR Chinab Integrative Chemical Biology Laboratory, Department of Chemistry, The University of Hong Kong, Hong Kongc Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kongd Medical College, Shenzhen University, Shenzhen, PR Chinae School of Biological Sciences, The University of Hong Kong, Hong Kongf Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, PR Chinag Stanley Ho Center for Emerging Infectious Diseases, The State Key Laboratory in Oncology in South China, The Chinese University of Hong Kong, Hong Kong

a r t i c l e i n f o

Article history:Received 30 April 2009Accepted 26 June 2009Available online 16 July 2009

Keywords:Polyethylenimineb-CyclodextrinFolateGene therapyBiodegradationIn vivo test

* Corresponding author. Department of ChemistryBiological Sciences Building, The University of HongKong. Tel.: þ852 2299 0776.** Corresponding author. Institute of Chemical B

Chemistry, Zhejiang University, Hangzhou 310028, PRE-mail addresses: tangguping@yahoo.com.cn

sua.hku.hk (M.C. Lin).

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.06.051

a b s t r a c t

The success of gene therapy relies on a safe and effective gene delivery system. In this communication,we describe the use of folate grafted PEI600–CyD (H1) as an effective polyplex-forming plasmid deliveryagent with low toxicity. The structures of the polymer and polyplex were characterized, and the in vitrotransfection efficiency, cytotoxicity, and in vivo transfection of H1 were examined. We found that folatemolecules were successfully grafted to PEI600–CyD. At N/P ratios between 5 and 30, the resulting H1/DNApolyplexes had diameters less than 120 nm and zeta potentials less than 10 mV. In various tumor celllines examined (U138, U87, B16, and Lovo), the in vitro transfection efficiency of H1 was more than 50%,which could be improved by the presence of fetal bovine serum or albumin. The cytotoxicity of H1 wassignificantly less than high molecular weight PEI-25 kDa. Importantly, in vivo optical imaging showedthat the efficiency of H1-mediated transfection (50 mg luciferase plasmid (pLuc), N/P ratio¼ 20/1) wascomparable to that of adenovirus-mediated luciferase transduction (1� 109 pfu) in melanoma-bearingmice, and it did not induce any toxicity in the tumor tissue. These results clearly show that H1 is a safeand effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA and itsapplication warrants further investigation.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Gene therapy has evolved as a promising therapeutic strategy forcancer and various intractable human diseases. The efficacy andsafety of gene therapy depend not only on gene construct beingdelivered, but more importantly on the delivery vehicle itself.Previously, viral vectors have been a primary focus for cancer genedelivery due to their excellent transfection efficiency in vitro and invivo [1,2]. However, because of their potential safety risks [3,4] andimmunogenicity [5], there is an urgent need to develop alternativenon-viral gene carriers. Hence, cationic liposomal and polymericvectors are common non-viral vectors currently being developed.

, Room 8N11, 8/F KadoorieKong, Pokfulam Road, Hong

iology and PharmaceuticalChina.

(G.-P. Tang), mcllin@hku-

All rights reserved.

Unlike viral vectors, these non-viral alternatives offer severaladvantages over viral vectors. These include increased safety, ease ofdesign and synthesis, low production cost, and flexibility in chem-ical modifications for improved biocompatibility and target-speci-ficity. A number of cationic polymers have been demonstrated todisplay considerable transfection efficiency in vivo and in vitro in thepast decade.

Among those cationic polymers, polyethylenimine (PEI) hasemerged as a promising delivery reagent, primarily owing to itsexcellent transfection efficiency in a wide range of cell types [6]. Byvirtue of its abundant primary amines, PEI readily forms polyplexeswith negatively charged DNA and subsequently buffers the endo-somal environment, facilitating the release of DNA in the cytosol[7,8]. It should be noted that the transfection efficiency and cyto-toxicity of PEI are molecular weight-dependent. In general, PEI withhigher molecular weight has higher transfection efficiency, but alsohigher cytotoxicity. Furthermore, PEI has a propensity to formaggregates in physiological conditions [7,9]. To address this issue,polyethylene glycol (PEG) grafting to PEI has been found useful inreducing cytotoxicity, although PEGylation did not enhance

H. Yao et al. / Biomaterials 30 (2009) 5793–58035794

transfection efficiency [10–12]. Also, the PEGylated PEIs were notefficiently excreted and were found to be non-biodegradable. Giventhese caveats, efforts have been made to further develop biode-gradable copolymers to make it more amenable to safe andeffective gene delivery.

Low molecular weight PEI (LMW PEI) has been utilized for thesynthesis of biodegradable copolymers owing to its high biode-gradability [9,13,14]. Many PEI600-grafted copolymers have beensynthesized, including cross-linking LMW PEI with biodegradablelinkers [15], bifunctional PEG, or degradable oligo(L-lactic acid-cosuccinic acid) [16,17]. Although these polymers showed reducedcytotoxicity, they had lower transfection efficiency than PEI-25 kDa.Forrest et al. synthesized a series of degradable polycations byconjugating PEI 800 Da to small diacrylate cross-linkers. Theyshowed that the polycations could mediate transgene expression2-to 16-fold more efficiently than PEI-25 kDa and was essentiallynontoxic to the mouse myoblast C2C12 cells [18]. However, a biode-gradable polymer with high in vivo transfection efficiency has beenlacking.

Cyclodextrins (CyD) are cyclic molecules, comprising 6, 7 or 8glucose units (called a-, b- and g-CyD, respectively). As a biocom-patible material, CyD do not elicit immune responses and have lowtoxicities in animals and humans [6,19]. Gonzalez et al. firstsynthesized b-CyD-containing polymers for gene delivery in 1999[20]. Since then, numerous CyD-containing polymers have beenused as delivery vehicles for nucleic acids. One attractive feature ofCyD-containing polycations is their low in vivo toxicities comparedwith other non-CyD-containing polycations [21]. Therefore, CyDare suitable to be used as biocompatible and solubility-enhancingmoieties for the synthesis of biodegradable polymers.

In addition to modifying and optimizing the polymeric genecarrier backbones, different target-specific ligands, includingvarious signal transduction proteins, transfer proteins [22,23], andsmall molecules like folic acid [24,25] have also been explored topromote target-specific gene delivery in vitro and in vivo. Folate, inparticular, has been found to be an optimal ligand for targetingtumor cells due to its low immunogenicity, low toxicity, and highaffinity to the folate receptor, which is over-expressed in certaincancer cells [26].

Previously, we reported a PEI-based polymer which is composedof low molecular weight PEI (600 Da) crosslinked by CyD molecules(PEI600–CyD) [27]. PEI600–CyD is biodegradable and it displays hightransfection efficiency in cultured neurons. In this study, wesynthesized and characterized a new nanopolymer, folic acid-grafted PEI600–CyD (named H1), which exhibits higher transfectionefficiency and lower cytotoxicity in various tumor cell lines exam-ined and in a mouse model of melanoma.

2. Materials and methods

2.1. Materials

2.1.1. InstrumentsThe structure of H1 was characterized by 1H NMR (400 MHz; Bruker Corporation,

Germany), UV–visible spectrometer (Perkin Elmer Lambda Bio 40, USA) and X-raydiffractometer (Siemens D5005, Karlsruhe, Germany). The morphology of polymer/DNA polyplexes was observed by transmission electron microscope (Philips 201, USA).Both the particle size and zeta potential of H1/DNA polyplexes were measured bylaser-light scattering (Zetasizer 3000, Malvern Instruments, UK). Green fluorescenceprotein expression was monitored by a fluorescence microscope (Olympus, IX71,Germany). Luciferase assay was performed by a microplate luminometer (LB 96V,EG&G, Berthold). In vivo optical imaging was performed with an IVIS 100 seriesimaging system (Caliper Life Sciences, USA).

2.1.2. ChemicalsPolyethylenimine (PEI, MW 600 or 25 000 Da), b-cyclodextrin (MW 1135), folic acid

(MW¼ 441.4), N,N-dimethylformanide (DMF), triethylamine (Et3N), dimethyl sulf-oxide (DMSO), and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]

(MTT), were obtained from Sigma–Aldrich (St. Louis, MO, USA). 1,10-Carbonyl-diimidazole (CDI) was obtained from Pierce (Rockford, IL, USA). PEI-25 kDa was purifiedby dialysis in three changes of a 100-fold volume excess of water, lyophilized, and re-hydrated before use. One gram of D-luciferin potassium salt (syn) (Caliper Life Sciences,USA) was dissolved in 35 ml Dulbecco’s PBS and was then filter sterilized througha 0.45-mm sterile filter. Reconstituted substrate was stored in 1 ml aliquots at�80 �C.

2.1.3. PlasmidscDNAs encoding the enhanced green fluorescent protein (EGFP) and firefly

luciferase were obtained by polymerase chain reaction (PCR) using the pEGFP-N1(BD Biosciences, San Jose, CA) and pGL3 Luciferase reporter vector (PromegaCorporation, USA) as templates, respectively. The sequences of the PCR productswere confirmed by DNA sequencing. The EGFP and luciferase cDNAs were subclonedinto the EcoRI and XhoI sites of the pAM/CAG-WPRE-BGHpolyA plasmid to generatepAM/CAG-EGFP (pEGFP) and pAM/CAG-Luc (pLuc), respectively. The pEGFP or pLucplasmid was transformed into competent DH5a cells, propagated in LB brothsupplemented with 100 mg/mL ampicillin, and purified with PureLink� HipurePlasmid Maxiprep kit (Invitrogen). The quantity and quality of the purified plasmidDNAs were assessed by measuring its optical density at 260 and 280 nm.

2.1.4. Cell cultureMinimum Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium

(DMEM), RPMI 1640 medium, DMEM/F12 medium, fetal bovine serum (FBS),trypsin–EDTA (0.25% trypsin–EDTA), Opti-MEM� I Reduced-Serum Medium, andpenicillin–streptomycin were purchased from Invitrogen. Albumin solution frombovine serum (5%) was purchased from Sigma (A4628-100 ml). Human glioblastomaU87 and U138 cells, mouse melanoma B16 cells, human colorectal cancer Lovo cellsand human lung carcinoma A549 cell lines were obtained from ATCC (AmericanType Culture Collection).

2.1.5. AnimalsAll aspects of the animal studies were performed in accordance with the protocol

approved by Committee on the Use of Live Animals in Teaching and Research of theUniversity of Hong Kong. Normal female Balb/c and C57/BL6 mice (6–8 weeks) wereobtained from Laboratory Animal Unit of the University of Hong Kong.

2.2. Methods

2.2.1. Synthesis and characterization of H1

The PEI600–CyD backbone was synthesized by a method reported previously byTang et al. [27]. Briefly, b-Cyclodextrin (0.42 g, 0.37 mmol) and 1,10-carbon-yldiimidazole (CDI, 0.5 g, 3 mmol) were dissolved in N,N-dimethylformamide(DMF). The mixture was stirred at room temperature for 1 h and then precipitated incold diethylether. The resulting CyD–CDI was filtered, dissolved in dimethylsulfoxide (DMSO), and stored at 4 �C. PEI600 (1.80 g, 3 mmol) was dissolved in DMSO.The described CyD–CDI in DMSO and triethylamine (Et3N) was added dropwise tothe PEI solution over 1.5 h with stirring, followed by reaction for 5 h. After thereaction, the mixture was dialyzed with dialysis tubing (MWCO, 12 kDa) in water for2 days and freeze-dried for another 2 days.

Similarly, folic acid (0.053 g, 0.12 mmol) in DMSO was activated by CDI (0.02 g,0.12 mmol) using Et3N as the catalyst under nitrogen atmosphere. The reactionmixture was added to the DMSO solution of PEI600–CyD (0.12 g), and stirred for 24 hto obtain the crude product of H1, which was purified by dialysis in water for 3 dayswith dialysis tubing (MWCO, 12 kDa). Pale yellow powder of H1 was obtained afterlyophilization for 2 days.

For NMR analysis, samples of H1 (1 mg) were dissolved in 0.5 ml of D2O. The 1Hspectrum of each sample was recorded with a NMR spectrometer (400 MHz; BrukerCorporation). For spectrometric analysis, all samples were dissolved in 1 ml of waterand scanned by UV–visible spectrometer from 200 nm to 400 nm. Cary 300 software(Varian, Palo Alto) was used to analyze the signals. The diffraction pattern was takenon a Siemens D5005 X-ray diffractometer with a scanning speed at 0.6� per minunder nickel-filtered Cu-Ka radiation (l¼ 1.542 Å) with voltage at 40 kV and currentat 40 mA.

2.2.2. Characterization of H1/pDNA polyplexesAccording to the N/P ratio (where ‘‘N’’ is the amount of nitrogen in PEI and ‘‘P’’ is

the amount of phosphate in 1 mg DNA), varying concentration of polymer solutionwas added to pDNA solution in equivalent volume to form the polyplexes. Polyplexeswere allowed to incubate for 20 min at room temperature and were filtered with0.45 mm syringe filters prior to measurement. The hydrodynamic particle size andzeta potential of the polyplexes were measured at 25 �C by dynamic light scattering(DLS) using Zetasizer 3000 (Malvern Instruments, Worcestershire, UK). Polyplexesof different N/P ratios had a final pDNA concentration of 10 mg/ml. Z Average Mean(nm) and Zeta Potential (mV) were used for data analysis. The presented data aremeans of three independent measurements.

The H1/pDNA polyplexes were characterized by transmission electronmicroscopy (TEM). Briefly, 50 ml of H1/pDNA polyplexes solution was dropped toglow-discharged, 400-mesh carbon-coated copper grids for 45 s, after which excess

H. Yao et al. / Biomaterials 30 (2009) 5793–5803 5795

liquid was removed by blotting. After drying at room temperature for 5 min, imageswere recorded by a Philips 201 electron microscope operated at 80 kV.

2.2.3. Production of recombinant adenovirus harboring luciferaseA full-length cDNA encoding luciferase was amplified from pGL3 Luciferase

reporter vector (Promega Corporation, USA). The cDNA was subcloned into the pAd/CMV/DEST� plasmid (Invitrogen). The recombinant plasmid was then transfectedinto 293 Å cells using Lipofectamine� 2000 Reagent (Invitrogen) to producerAdv-Luc. The rAdv-Luc viruses were purified by cesium chloride gradient centri-fugation and their titers were determined by the tissue culture infectious dose 50(TCID50) method (AdEasy� Vector System Application manual, Qbiogene). Theviruses were stored at �80 �C prior to use.

2.2.4. In vitro transfection efficiency assay2.2.4.1. Procedure for in vitro cell transfection. Various cancer cell lines (U138, U87,B16, Lovo, and A549) were separately seeded at a density of 1�105 cells/well ina 24-well plate 24 h before transfection. The polyplexes of H1/pEGFP or H1/pLuc,which had pDNA concentration of 1 mg/well, were prepared at varying N/P ratios andincubated for 15 min at room temperature. The original cell culture media wasreplaced with the complex solution containing the polyplexes and an additional400 ml of Opti-MEM in each well. After incubation for 4 h at 37 �C, the transfectionmedium was replaced with fresh growth medium and then the cells were incubatedfor 24 h or 48 h. To determine the effect of FBS or albumin on transfection efficiency,Opti-MEM containing various concentrations of FBS or albumin was also used as thetransfection media. To visualize the expression of EGFP in the cancer cells transfectedwith H1/pEGFP, a microscopic evaluation of the EGFP expression was performed at48 h post transfection. To detect the expression of luciferase in the cancer cellstransfected by H1/pLuc, luciferase assay was conducted at 48 h post transfection.

2.2.4.2. Microscopic evaluation of the EGFP expression. Microscopic evaluation of theEGFP expression in different cancer cell lines was performed as described previously[28]. Transfection of U138, U87, B16, Lovo, and A549 cells with H1/pEGFP polyplexeswere observed at 48 h post transfection under a phase contrast microscope anda fluorescent microscope. The percentage of transfected cells was determined bycounting the number of green fluorescent cells in 10 randomly selected fields at200�magnifications and dividing by the total number of cells in the same 10 fields.

2.2.4.3. Luciferase assay. Luciferase assay was performed according to the recom-mended protocol from the manufacturer (technical bulletin No. 281, Promega).Briefly, cells were washed with PBS and lysed by the addition of 100 ml of cell cultureLysis Buffer (Promega, Madison, WI). The cell lysates from each well (20 ml) wereused for luciferase activity assay with the Promega’s luciferase reagent. Light unitswere measured with a Microplate luminometer LB 96V (EG&G, Berthold). Proteinassay kit (Bio-Rad, 500-0122) was used to determine the protein concentrations ofcell lysates [luciferase activity (RLU/mg)¼ value of relative light unit (RLU)/20 mltotal protein quantity (mg)].

2.2.5. MTT assayThe method of MTT assay was the same as the one described previously [27].

Briefly, U87 cells or B16 cells were seeded at 1�104 cells/well in 96-well plates andincubated for 24 h. The culture medium was replaced with 200 ml serial dilutions ofpolymer solutions (the concentrations of nitrogen in polymers from 0.05 to 1 mM)prepared in complete culture media, and then the cells were incubated for 8 h. Thepolymer solutions were replaced with 200 ml polymer-free complete culture mediaand the cells were incubated for another 16 h. Each concentration was replicated in 5wells. Filtered MTT reagent (20 ml) at a concentration of 0.5 mg/ml was added toeach well and the cells were incubated for 4 h. The un-reacted dyes were removedby aspiration. The violet crystals in each well were dissolved in 100 ml DMSO and theoptical density (OD) was measured by an ELISA reader at a wavelength of 595 nm.Cell viability (%) in each well was calculated by OD595 test/OD595 control� 100.

2.2.6. The transfection efficiency of H1 in xenografted C57/BL6 mice2.2.6.1. Establishment of the B16 melanoma-bearing C57/BL6 mouse model. Mousemelanoma B16 cells in exponential growth phase were harvested and washed twicein PBS, resuspended in PBS at a density of 1�107 cells/ml, and injected subcuta-neously (0.1 ml injection volume; 1�106 cells) at the right dorsal flank of C57/BL6mice, which were previously shaved to clear the local fur at the injection site. Whenthe tumor size reached 0.3 cm in diameter, mice were injected with H1/pLucpolyplexes (50 mg plasmid DNA, N/P ratio¼ 20/1), pLuc plasmid alone (50 mg) orrAdv-Luc (1�109 pfu) by intratumoral injection, respectively. Each treatment groupwas composed of three mice (n¼ 3). The luciferase gene expression levels in thetumor tissues were determined by in vivo optical imaging system (IVIS ImagingSystem 100 Series, Xenogen, MA, USA) and a luciferase assay of tumor tissuehomogenates.

2.2.6.2. In vivo optical imaging of living C57/BL6/mice bearing B16 cells tumor. Micewere anesthetized, and D-luciferin (Xenogen) at a dose of 150 mg per gram mousebody weight was injected intraperitoneally 5 min before the images were taken.Images were obtained at exposure time from 1 min to 10 min depending on the

intensity of the emitted protons. The total photon flux (photons/second) in a regionof interest (ROI) was quantitated by the image software (Living Image� Software,Xenogen MA, USA).

2.2.6.3. Luciferase assay of tumor tissue homogenates. Mice were scarified by CO2

inhalation and perfused by ice-cold PBS via cardiac puncture. Tumor tissues werethen extracted and homogenized in Lysis Buffer (Promega, Madison, WI) on ice. Thehomogenates were centrifuged and the luciferase activities in the supernatants wereassayed by a luminometer. The following processes were the same as the luciferaseassay as described in Section 2.2.4.3.

2.2.6.4. Hematoxylin and eosin (HE) staining. The tumor-bearing mice were injectedintratumorally with either 5% glucose solution containing the H1/pLuc polyplexes(50 mg of DNA, N/P ratio 20/1) or an equal volume of polymer-free 5% glucosesolution (as a control). The tumors were extracted from the mice sacrificed at 48 hpost-injection. The tumors were fixed with 4% formaldehyde and embedded inparaffin. Five-micron thick sections were cut, deparaffinized, rehydrated, and thenstained with hematoxylin and eosin (HE) prior to further histological analysis undera light microscope.

3. Results

3.1. Synthesis and characterization of H1

3.1.1. Conjugation of folate to PEI600–CyD backbone and synthesisof H1

Based on PEI600–CyD polymeric backbone, we succeeded inconjugating the targeting folate molecule via its terminal carboxy-late group onto the primary amine groups using a 1,10-carbonyl-diimidazole (CDI) coupling reaction to afford PEI600–CyD–folate(Fig. 1). It should be noted that the structure of H1 shown in Fig. 1 isputative. Nevertheless, the successful conjugation of folate toPEI–CyD was duly confirmed by NMR, UV–visible spectral analysis,and X-ray diffraction studies.

3.1.2. NMR studiesThe successful conjugation of folate onto PEI600–CyD backbone

was confirmed using 1H nuclear magnetic resonance (1H NMR)(Fig. 2A). For both PEI600–CyD and H1, the signals from PEI ethyleneprotons (–CH2CH2NH–) appeared at 2.4–3.0 ppm. Also, the C1

proton and C2–C6 protons of b-CyD appeared at 4.9 ppm and 3.0–4.0 ppm, respectively. Three small peaks corresponding to thearomatic protons (ranged from 5.5 to 8.5 ppm) were only observedin the NMR spectrum of H1, indicating the successful conjugation ofthe folate moiety to PEI600–CyD. Furthermore, quantitative analysisof peak integral values showed that around 5% of the total primaryamine groups on PEI600–CyD were conjugated (data not shown).

3.1.3. UV–visible spectral analysisThe conjugation of folate onto PEI600–CyD was also supported

by UV–visible spectral analysis, which could display differences inthe UV absorption patterns between the aqueous solutions ofPEI600–CyD, folic acid, and H1 (Fig. 2B). Folic acid had absorptionpeaks at both 280 and 350 nm. The UV spectrum of H1 was similarto that of folic acid but displayed a bathochromic shift where theabsorption peaks at 280 and 350 nm were shifted to 288 and390 nm, respectively. This could be explained by the modificationof folic acid between the free and conjugated states. On the otherhand, the PEI600–CyD backbone did not show any distinct absorp-tion peak from 200 to 400 nm. These results suggested that thefolate moiety was responsible for the differences between the UVspectrum of PEI600–CyD and that of H1.

3.1.4. X-ray diffractogram studiesAs shown in the X-ray diffractogram (Fig. 2C), folic acid was

crystalline because a number of sharp reflection peaks at various2-theta (2q) angles (5.4�, 10.9�, 11.6�, 13.1�, 16.4�, 17.1�, 19.3�, and soon) were observed. However, the reflection peaks of PEI600–CyD at

Fig. 1. Synthesis and putative structure of H1. CDI-coupled b-cyclodextrin (step 1) was reacted with branched PEI (step 2) to form PEI–CyD. Similarly, CDI-coupled folic acid (notshown) was reacted with the established PEI–CyD to generate H1 (Step 3).

H. Yao et al. / Biomaterials 30 (2009) 5793–58035796

2q¼ 12.4� and 18.9� were broad, suggesting that PEI600–CyD wasamorphous. H1 showed a range of reflection peaks at 2q from 19.4�

to 27.0�, but no sharp reflection peak was detected. These findingssuggested that folic acid was successfully conjugated to thePEI600–CyD backbone because the crystalline form of folic acid hadbeen transformed to an amorphous state in H1.

3.2. Characterization of H1/DNA polyplexes

3.2.1. Particle sizeParticle size is a crucial factor for determining the rate of

cellular uptake of DNA polyplexes [22]. The transmission electronmicroscopy (TEM) image of H1/DNA polyplex at an N/P ratio of 20is shown in Fig. 3A. According to the TEM image, H1/DNA poly-plexes had a compact structure and displayed a characteristicglobular morphology. Dynamic light scattering showed that theparticle sizes were less than 120 nm when N/P ratios rangedbetween 10 and 40 (Fig. 3B). At N/P ratios between 1 and 20, theparticle size of H1/DNA polyplexes decreased significantly. Thiswas likely due to the condensation of the plasmid DNA by anincreasing amount of H1. At N/P ratio of 20, the particle size wasminimal because the positive charge on H1 might be balanced bythe negative charge of the plasmid DNA and hence polyplexeswith the most condensed structures were formed. At N/P ratiosbetween 20 and 40, the particle size increased slightly becausethe excess positive charge from H1 could lead to the formationloosely packed polyplexes.

3.2.2. Zeta potentialZeta potential is another important factor affecting the cellular

uptake of polyplexes [29]. As shown in Fig. 3C, the zeta potential

of plasmid DNA alone was �5 mV, which could be attributed tothe negatively charged phosphate groups in the plasmid DNA.When H1 was mixed with the plasmid DNA at N/P ratio of 5, thezeta potential was changed from negative to positive, implyingthat the positive charge contributed by the amine groups of H1

exceeded the negative charge of the plasmid DNA. At N/P ratiosbetween 5 and 30, the zeta potential increased slightly becausethe positive charge of H1 could be balanced by the negativecharge of the plasmid DNA. At N/P ratios between 30 and 40, thezeta potential increased significantly, suggesting that all theplasmid DNA was bound by H1 and an excessive amount of H1 waspresent in the free form.

3.3. In vitro transfection

3.3.1. pLuciferace expression assaysFirst we investigated the effect of N/P ratio on the transfection

efficiency of H1 and assessed the difference in luciferase activitiesbetween H1/pLuc- and PEI600–CyD/pLuc-transfected B16 cells(Fig. 4A). We found that at N/P ratios of 10, 20, and 30, the luciferaseactivity in H1/pLuc- transfected cells was approximately 5-fold,17-fold, and 33-fold of that in PEI600–CyD/pLuc-transfected cells,respectively (**p< 0.001, n¼ 4, Student’s t test). Next, we tested theeffect of FBS or albumin (ALB) on the transfection efficiency of H1

and PEI-25 kDa (Fig. 4B). Our results showed that at a serum-freecondition, B16 cells transfected with PEI-25 kDa/pLuc showeda 5-fold higher luciferase activity than those transfected with H1/pLuc. The luciferase activity in H1/pLuc-transfected cells in thepresence of 10% FBS and 0.1% ALB was respectively 7-fold and 9-foldof that in the absence of FBS (**p< 0.001, n¼ 4, Student’s t test).On the contrary, the luciferase activity in PEI-25 kDa/pLuc

Fig. 2. Characterization of H1 polymer. (A) NMR analysis revealed the aromatic protons in folic acid. (B) The UV spectra of folic acid (FA), PEI600–CyD (PEI–CyD), and H1 (PEI600–CyD–FA). (C) An X-ray diffractogram showing the reflection peaks of FA, PEI–CyD, and H1.

H. Yao et al. / Biomaterials 30 (2009) 5793–5803 5797

transfected-cells in the presence of 10% and 0.1% ALB was almost 5-fold of that in the serum-free condition.

3.3.2. Microscopic evaluation of EGFP expressionEGFP expression analysis is the most convenient method to

evaluate the transfection efficiency in vitro. Mouse melanoma B16 orhuman lung carcinoma A549 cells were transfected with H1/pEGFPpolyplexes in vitro at an N/P ratio of 20. Fig. 4C summarizes theexpression levels of EGFP in the two cell lines as normalized to cellcount (% EGFP positive). We found that H1 had superior transfectionability compared to PEI600–CyD and PEI-25 kDa in B16 cells, but notA549 cells. For B16 cells, various transfection media with either FBSor different concentrations of albumin were also tested (Fig. 4D).Cell counts were taken and percent EGFP positive cells were used toassess the effect of transfection media. Our results showed that thepresence of 10% FBS and albumin (0.05–0.2%) significantly enhancedthe transfection efficiency by 2–3-fold compared to Opti-MEMalone (blank) (**p< 0.001, n¼ 3, Student’s t test).

Using the optimal transfection conditions (N/P ratio¼ 20,10% FBSor 0.1% ALB) as determined by the above experiments, we examinedthe transfection efficiency of H1 in various tumor cell lines (U138,U87, B16, Lovo, and A549). The percentage of EGFP positive cells ofthese tumor cell lines was counted and is shown in Fig. 4E. Our datashowed that the transfection efficiency of H1/pEGFP polyplexes, asreflected by the percentage of EGFP positive cells, was around 50–

70% in U138, U87, B16, and Lovo cells, but was merely 16% in A549cells. Fig. 4F shows the representative photos of EGFP expression,which were taken at 48 h after transfection with H1/pEGFP.

3.4. Cytotoxicity of H1

We next used MTT assay to evaluate the cytotoxicity of H1 indifferent tumor cell lines. Fig. 5A and B shows the cell viability ofU87 and B16 cells incubated with various concentrations ofPEI-25 kDa, PEI600–CyD, and H1, respectively. PEI-25 kDa wasconsiderably more toxic than PEI600–CyD and H1. At all theconcentrations tested, the viability of cells incubated with H1 wassimilar to those incubated with PEI600–CyD backbone. The medianlethal dose (LD50) of PEI-25 kDa in U87 and B16 cells were found tobe 0.2 mM and 0.3 mM, respectively. In contrast, in both cell lines,the LD50 values of H1 were estimated to be about 1.0 mM.

3.5. The transfection efficiency and toxicity of H1 in vivo

To evaluate the dynamic change of gene expression over time intransfected tumor cells in vivo, C57/BL6 mice bearing B16 tumorswere injected intratumorally with pLuc (50 mg), H1/pLuc (DNA50 mg, N/P ratio 20:1) or rAdv-Luc (1�109 pfu). The expression ofluciferase was then monitored by a cooled CCD camera at 24 h, 48 hand 72 h post-injection. The representative photos of the mice are

Fig. 3. Characterization of H1/DNA polyplexes. (A) A representative transmission electron microscope (TEM) image of the polyplex at N/P ratio of 20:1. (B) Polyplex diametersmeasured by dynamic light scattering showed that particles sizes were around 100 nm at N/P ratios ranging from 10:1 to 40:1. (C) Zeta potential was found to be 0–10 mV at N/Pratios ranging from 5:1 to 30:1.

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shown in Fig. 6A. At 1 min exposure time, the luminescence signalsinduced by H1/pLuc and rAdv-Luc could be easily captured by a CCDcamera. However, the luminescence signals of pLuc could bedetected only when the exposure time was extended to 10 min.From 24 h to 48 h post-injection, total photon flux (TPF) inpLuc-injected mice was reduced by half and was not detected at72 h. However, the TPF in mice injected with H1/pLuc was morerobust and almost equivalent to those elicited by rAdv-Luc trans-duction at 24 h post injection. At 48 h post-injection, The TPF in H1/pLuc-injected mice was reduced by 20–30% and 50% at 48 h and72 h post-injection, respectively (Fig. 6B). In these mice, the lumi-nescence signals could still be detected for 4–6 days, when theexposure time was extended to 5 min (Fig. 6A). On the other hand,the TPF in rAdv-Luc-injected mice was maximal at 48 h (Fig. 6A andB). After that, the signals reduced substantially from day 7 to day 10post-injection (Fig. 6A).

To further examine the levels of luciferase expression in thetumor-bearing mice, tumor tissue homogenates were preparedfrom pLuc-, H1/pLuc, and rAdv-Luc-injected tumor masses, respec-tively, at 24 h and 48 h post injection. At 24 h, the luciferase activityin H1/pLuc-injected tumor was almost 100-fold higher than that inpLuc-injected tumor, and it was of the same order of magnitudecompared with that in rAdv-luc-injected tumor (Fig. 6C). At 48 hpost-injection, the luciferase activity in H1/pLuc-injected tumor wasreduced 20-fold and that in rAdv-Luc-injected tumor was furtherincreased by more than 10-fold.

To visualize the histology of the tumor tissues after injectionwith H1/pLuc, we performed hematoxylin & eosin (HE) staining ofB16 tumor tissues at 48 h after H1/pLuc (50 mg DNA, N/P 20/1, 5%glucose) injection (Fig. 6D). Tissues injected with H1/pLuc displayeda similar histology to those injected with 5% glucose. This obser-vation is in agreement with the in vitro cytotoxicity data wherebyH1 did not exhibit substantial toxicity at the administered dose(Fig. 5).

4. Discussion

The toxicity of a gene delivery vector is the primary concern forits application in vivo. Although high molecular weight PEI(PEI-25 kDa) has demonstrated high efficacy for gene delivery indifferent administration routes in vivo [30–32], the high mortalityand serious tissue damage associated with PEI-25 kDa indicatedthat it is not suitable for in vivo gene transfer [33,34]. It has beendocumented that the toxicity of PEI can be reduced by the graftingof b-cyclodextrin [35–37]. To further improve the efficiency ofPEI600–CyD, we conjugated folic acid to the PEI600–CyD backbonein the synthesis of H1. The low zeta potential of H1/DNA poly-plexes may contribute to the low toxicity of H1 in vivo and in vitro.The results of our cell viability assays showed that the apparentIC50 of H1 was more than 5-fold and 3.3-fold that of PEI-25 kDa inU87 cells and B16 cells, respectively (Fig. 5A and B). The cyto-toxicity of H1 was similar to the PEI600–CyD backbone in both cell

Fig. 4. Luciferase expression and H1-mediated EGFP in vitro. (A) The optimal N/P ratios were assessed by pLuciferase assay for PEI–CyD and H1 (10% FBS). A significant difference wasobserved between H1 and its precursor, PEI600–CyD, at N/P ratios of 20:1 and 30:1 (**p< 0.001, n¼ 4, Student’s t-test). (B) The effects of fetal bovine serum (FBS) or albumin (ALB) inthe formulation were further shown by luciferase expression assays for H1 (DNA 1 mg, N/P ratio: 20/1) and PEI-25 kDa (DNA 1 mg, N/P ratio 10/1). Significant differences in luciferaseexpression among serum-free, FBS and ALB preparations were detected for both polymer types (**p< 0.001, n¼ 4, Student’s t-test). (C) A summary of percent EGFP expression inB16 melanoma and A549 alveolar basal epithelial cells. (D) The advantage of FBS (10%) and various ALB concentrations in the formulation is shown as EGFP expression issignificantly higher than that FBS-free formulation (**p< 0.001, n¼ 3, Student’s t-test). (E) A panel of five different cell types was also evaluated for EGFP expression. More than 50%of U138 and U87 glioblastoma cells and B16 melanoma cells were EGFP positive 48 h after transfection by H1/pEGFP polyplexes. (F) Representative photos of EGFP expression in (a)U138, (b) U87, (c) B16, and (d) A549 cells.

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lines. Importantly, we did not detect the toxicity or tissue damagein HE-stained tumor tissues at 48 h after intratumoral injection ofH1/pLuc (50 mg, N/P¼ 20/1). The mechanism of PEI-25 kDatoxicity was referred to the membrane disruptive behavior ofdense cationic polymers [38]. The zeta potential of the PEI-

25 kDa/DNA polyplexes was usually more than þ30 mV at an N/Pratio more than 10/1. However, the zeta potential of H1/DNApolyplexes was less than þ10 mV for N/P ratios smaller than 40/1.This finding suggests that the lower cytotoxicity of H1 may beattributed to the decreased charge density of H1/DNA polyplexes.

Fig. 5. Cytotoxicity of H1 was determined by MTT assays. The percent cell viability of PEI600–CyD-, PEI-25 kDa-, or H1-transfected (A) U87 cells and (B) B16 cells was plotted as theconcentrations of nitrogen in polymers (mM).

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The transfection efficiency and transgene expression level arethe most important features of copolymers. In vitro, we found thatthe transfection efficiency of H1 in B16 cells, where the folic receptoris over-expressed, was significantly enhanced compared with thePEI600–CyD backbone (Fig. 4A and E) [39]. Consistently, the trans-fection efficiency of H1 was markedly attenuated in A549 cellswhere the folic receptor expression is low [40]. These results indi-cate that folic receptor-mediated endocytosis may contribute to thehigh transfection efficiency mediated by H1. To date, three isoformsof folate receptors (FR) have been identified, namely FRa, FRb andFRg [41]. Among them, FRa has gained the most attention because itis primarily expressed in cancerous cells such as malignant naso-pharyngeal, colon, ovarian, breast, renal, and testicular carcinomas[26,42–44]. The mechanism of folate-mediated targeting of nano-particles to FRs and the subsequent cellular uptake is perceived to bethe same as that for the free folate, which is internalized into thecells via receptor-mediated endocytosis [45]. The same reason canalso be given to explain the discrepancy of the transfection effi-ciency of H1 in various tumor cell lines. U138 and U87 cells, whichshowed the highest transfection efficiency of round 60–70%, werederived from malignant gliomas with higher folate receptorexpression [38,46].

Serum usually has negative impact on the transfection efficiencyof cationic liposomes and the interactions of cationic polymers withserum may serve as a predictive model for the in vivo efficiency ofa cationic polymer [47]. Contrarily, our in vitro data show that thetransfection efficiency of H1 can be significantly increased whenadministered in the presence of 5% FBS and 10% FBS. This obser-vation urged us to further investigate why serum could increase thetransfection efficiency of H1. Subsequently, we obtained an inter-esting result, in which the transfection efficiency of H1 could also beenhanced by albumin, and the enhancement effect of 0.1% or 0.2%albumin was even higher than that of 10% FBS. As the most abun-dant protein in serum, albumin serves as a transporter of vitamins,ions and drugs in the blood and acts as a major pH buffering protein[48]. Based on this biological function, a rational explanation wouldbe that the cationic H1/DNA polyplexes can bind to the anionicalbumin by ionic interaction. This ionic interaction may thenneutralize the potential of polyplexes, speed up the process ofendocytosis, and reduce the cytotoxicity induced by the cationicpolyplexes in vitro. The improvement of H1-mediated transfectionby albumin also implies a compatibility with intravenous injectionand localization tissue injection of H1/DNA polyplexes in vivo.

Creating synthetic gene delivery vectors with viral-like trans-fection efficiency has been an important goal in gene therapyresearch. Among various viral vectors, adenovirus has been one ofthe most commonly used vectors in gene therapy. It is a double-stranded DNA virus that can transfer exogenous gene to bothdividing and non-dividing cells and its genome will not integrateinto the host cell genetic material. Thus, adenovirus can be used asa standard for comparing the efficiency of non-viral synthetic genedelivery vectors. In this study, we found that the luciferase activityof H1/pLuc-mediated transfection was 13 times higher than that ofpLuc alone, and was almost comparable to that of rAdv-Luc(1�109 pfu) in B16 melanoma-bearing C57/BL6 mice at 24 h post-intratumoral injection. Similar results were observed in assayingthe luciferase activity in the tissue homogenates prepared from theinjected melanoma xenografts. Based on our knowledge, this is thefirst report that the transfection efficiency mediated by a non-viralgene delivery vector can achieve a transgene expression levelsimilar to that mediated by a recombinant adenoviral vector.

In addition to the advantageous effects contributed by graftingfolic acid and the enhancement effects from FBS or albumin, thephysical properties of H1/pDNA polyplexes also determine its effi-cacy in vivo. The particle size of pDNA is generally more than700 nm, which is easy to be cleared by macrophages located withinthe tumor and decomposed by cellular DNase. These disadvantageslead to the low transfection efficiency of naked pDNA in vivo. In ourstudy, we found that H1 was able to condense DNA into a globularpolyplex with a particle size of about 92 nm, which is similar to thesize of rAdv. In vivo, the small particle size of H1 can prevent DNAfrom being phagocytosed by macrophages and accelerate thepenetration from intercellular space. Furthermore, the positive zetapotential of H1/DNA polyplexes is likely to have an affinity for thenegatively charged cell surface. Thus, the small size and the positivecharge of H1/DNA polyplexes help H1 achieve a high level of trans-fection efficiency in various cell lines. With reference to the particlesize data (Fig. 3B) and luciferase expression data (Fig. 5A), theoptimal N/P ratio of H1 for in vitro and in vivo delivery should be 20/1.

It should be pointed out that peak value and duration oftransgene expression induced by recombinant adenovirus are stillbetter than that of H1. A possible mechanistic explanation is that itcould be due to methylation of episomal plasmid DNA, which mayturn off transgene expression [49,50]. On the other hand, previousstudies have shown that neither free intracellular adenovirus DNAnor its integrated form is methylated in productively infected cells

Fig. 6. The transfection efficiency and toxicity of H1 in vivo. (A) Representative images of C57BL/6 mice with B16 xenografts exhibiting in vivo bioluminescence on days (D) 1, 2, 3, 6,and 10 following intratumoral injection with plasmid DNA expressing luciferase (pLuc; 50 mg), H1/pLuc (50 mg pLuc, N/P ratio 20:1), and adenovirus-Luc (rAdv-Luc; 1�109 pLuc),respectively. The total photon flux (photons/second) in a region of interest (ROI) was shown in the red boxes. (B) The total flux of ROI over time (photons/minute) was used asa parameter to compare the transfection efficiency of pLuc injection, H1-mediated pLuc transfection, and rAdv-Luc transduction on various time points. (C) Determination of relativeluciferase unit (RLU) of tumor tissue homogenates at 24 h after transfection. The level of luciferase expression induced by H1/pLuc transfection was similar to that mediated by rAdv-Luc transduction. (D) HE staining did not detect any toxic response in tumor tissue injected with (a) H1/pLuc (50 mg; N/P ratio 20:1) and (b) polymer-free 5% glucose solution.

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[51,52]. Another explanation is that adenoviral viral proteins mayfacilitate the recruitment of transcription factors, nuclear importprotein binding terminal protein, and various proteins derived fromE4 region, which may enhance transcription and nuclear stability ofadenovirus [53–55]. Thus, structural modifications aiming atimproving the transcription and nuclear stability of the H1-medi-ated gene delivery system should be pursued in the future.

5. Conclusions

H1, a folate–PEI–CyD polymer, was synthesized for gene deliveryand it allows efficient transfection of various cancer cell types withlow cytotoxicity. The transfection efficiency of H1 is superior to thatof PEI600–CyD as previously described [13]. Importantly, with itshigh in vivo transfection efficiency and capacity to target tumorcells, it is envisaged that H1 is worthy of further development as anin vitro and in vivo gene delivery agent for cancer gene therapy.

Acknowledgments

We would like to thank Wing-Fu Lai and Ka-Chun WilliamCheung for critical reading of the manuscript, King-Sun Siu forsupport in chemical synthesis, Fang Yu and Gao Yi for support invitro experiments, Zan Shen and Long-Fei Huo for support in animalexperiments. This work was supported by the Focus Budget Schemeproject at the Chinese University of Hong Kong (Project PI: Hsiang-fu Kung) and the National Nature Science Foundation of China(30571068).

Appendix

Figures with essential colour discrimination. Certain figures inthis article, in particular Figs. 4 and 6, are difficult to interpret inblack and white. The full colour images can be found in the on-lineversion, at doi:10.1016/j.biomaterials.2009.06.051.

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