Evaluation of Rod-Shaped Nanoparticles as Carriers for Gene Delivery

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Fabrication of nonspherical particles for gene delivery remains a major chal-lenge. In this study, novel rod-like nanoparticles are prepared for effi cient gene delivery by self-assembly of α-cyclodextrin (α-CD) and polyethylenimine-methoxy poly(ethylene glycol) (PEI-mPEG). The study reveals that the rod-like PEI-mPEG/α-CD particles can bind DNA effectively and the resulting PEI-mPEG/α-CD/DNA complexes show over four times higher gene delivery capability than their spherical counterparts and PEI(25K) due to more effi cient cellular uptake. Furthermore, the cytotoxicity of rod-like PEI-mPEG/α-CD is about fi ve times lower than that of the nanospheres, and 50 times lower than that of DNA/PEI(25K). These results indicate that shape is an important parameter for the design of gene delivery vectors.

different design parameters of nonviral particles should be considered and opti-mized. There exists ample evidence that particle size [ 3 ] and surface chemistry [ 4 ] can dramatically infl uence gene transfection effi ciency. However, there is a striking lack of studies on the effect of particle shape on the gene delivery behavior.

Recently, the importance of the shape of carriers in various biological processes has been realized. Emerging studies show that important biological processes such as biodistribution, cellular internalization, and toxicity exhibit strong dependence on shape. [ 5 ] The majority of the evidence available suggests that rod-like nanopar-

ticles are more favorable for drug delivery applications. [ 6 ] But up to now, most nonviral gene vectors are spherical particles. There are only few reports on the gene delivery behavior of nonspherical nanoparticles, except needle-like histidine-lysine polymers, [ 7 ] inorganic nanotubes, [ 8 ] and some star-shaped poly-meric vectors, [ 9 ] although actually a number of viruses are non-spherical in nature.

The most prominent approach for fabricating rod-like parti-cles has been self-assembly of amphiphilic copolymers. [ 10 ] How-ever, preparing biocompatible rod-like particles with good DNA-binding capability via self-assembly and retention of shape in physiological conditions still remains a challenge. Most rod-like polymeric particles lack the desirable degradable components and only serve as a model system. To date, rare rod-like assem-blies show potential for DNA binding, while good DNA-binding ability is necessary for a good gene delivery vector. [ 11 ]

Our recent research has demonstrated that rod-coil com-plexes formed by CD and polymer chains could self-assemble into hollow spheres or rod-like tubes in aqueous solution by varying the rigid block length fraction. [ 12 ] Earlier research focused on the methodology and control of morphology. In this study, the work is extended to binding DNA using the self-assembly aggregates with different shapes and comparing their gene delivery capability. It was found that the particles prepared by α-cyclodextrin (α-CD) and polyethylenimine-methoxy poly(ethylene glycol) (PEI-mPEG) showed good DNA-binding ability, low cytotoxicity, and high gene transfec-tion effi ciency. The key fi nding was that rod-like vectors show higher gene delivery capability than their spherical counter-parts due to the more effi cient cellular uptake, indicating that shape effect plays an important role for the enhancement of gene transfection.

Dr. M.-M. Fan, [+] W.-Z. Zhang, [+] C. Cheng, Dr. Y. Liu, Prof. S. Zhang State Key Laboratory of Polymer Materials Engineering Polymer Research InstituteSichuan University Chengdu 610065 , PR China E-mail: [email protected] Dr. B.-J. Li Chengdu Institute of Biology Chinese Academy of Sciences Chengdu 610041 , PR China E-mail: [email protected] Prof. X. Sun West China School of PharmacySichuan University Chengdu 610041 , PR China E-mail: [email protected]

DOI: 10.1002/ppsc.201300383

Evaluation of Rod-Shaped Nanoparticles as Carriers for Gene Delivery

Min-Min Fan , Wan-Zhen Zhang , Cong Cheng , Yan Liu , Bang-Jing Li , * Xun Sun , * and Sheng Zhang *

1. Introduction

Gene therapy has been proposed as a promising treatment for many acquired and inherited diseases. Although viral vectors exhibit high transfection effi ciencies, fundamental problems associated with them, including toxicity, immunogenicity, and limitations with respect to scale-up procedures, have encour-aged the investigation of nonviral vectors, which are safer and possess greater potential for large-scale production. [ 1 ] As a result, various nonviral delivery systems, such as cationic lipids, polymers, dendrimers, peptides, and inorganic nanoparticles, have been developed as carriers to deliver DNA systemically. [ 2 ] A great challenge of nonviral delivery vectors is their lower potency than viral vectors. To improve transfection effi ciency,

[+]M.-M. Fan and W.-Z. Zhang contributed equally to this work.

Part. Part. Syst. Charact. 2014, 31, 994–1000

http://doi.wiley.com/10.1002/ppsc.201300383

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2. Results and Discussion

2.1. Preparation and Characterization of PEI-mPEG/α-CD Assemblies

PEI-mPEG was selected as building blocks for the preparation of self-assembled particles since polyethylenimine (PEI) is one of the most prominent examples of cationic polymers capable of gene transfection. [ 2b ] As shown in Scheme S1 (Supporting Information), two PEI-PEG copolymer samples with different PEI-PEG ratio are prepared by bonding PEG oligomers ( M n = 2000) on the chains of branched PEI 10 kDa (PEI(10K)) and linear PEI 2.5 kDa (PEI(2.5K)). [ 13 ] The notations of samples were PEI(10K)-mPEG and PEI(2.5K)-mPEG, respectively. Char-acterization by 1 H NMR is provided in Figures S1–S3 (Sup-porting Information).

PEI-mPEG was water-soluble and formed a transparent solu-tion in water. When an aqueous solution of PEI-mPEG was added to α-CD aqueous solution at room temperature, the solu-tion gradually became turbid, indicating the formation of aggre-gates. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) observations showed that PEI(10K)-mPEG/α-CD aggregates were nanospheres around 200–300 nm in diameter ( Figure 1 A and a) and PEI(2.5K)-mPEG/α-CD aggregates were rod-like particles around 700–1000 nm in length and 100 nm in diameter (Figure 1 C and c).

Our previous paper demonstrated that α-CD should thread on the PEG blocks selectively in the PEI-mPEG copolymer below pH 8.0 due to the energetically unfavorable interaction between protonated PEI and the cavities of α-CD. The CDs in PEG chains were densely stacked forming strong hydrogen bonds with each other and the resulting PEG-α-CD showed poor solubility in water. Consequently, the PEI-PEG-α-CD complexes had both a rigid block (PEG-α-CD) and a coil block (protonated PEI). Due to the propensity of the rigid blocks to pack in parallel, the rod-coil complexes formed hollow parti-cles. Varying the rigid block fraction was an effective way to control the morphology of the aggregates. [ 12 ] In this study, PEI(10K)-mPEG/α-CD had a low rigid block fraction (rod:coil = 1:5) and self-assembled to spheres, while PEI(2.5K)-mPEG/α-CD had a high rigid block fraction (rod:coil = 1:1) and self-assembled to rod-like particles. The expected structure

of these aggregates was an inner PEG-α-CD inclusion block surrounded by the protonated coil-like PEI shell. As shown in Table 1 , the zeta potential of PEI(10K)-mPEG/α-CD and PEI(2.5K)-mPEG/α-CD was 41.9 and 35.0 mV, respectively, indicating that the surface of the particles was covered by pro-tonated PEI blocks.

2.2. DNA Binding of PEI-mPEG/α-CD Assemblies

The ability of a gene delivery vector to form a stable elec-trostatic complex with negatively charged DNA is the basic criterion for effi cient gene transfection. We studied the DNA-binding capabilities of the PEI-mPEG/α-CD assemblies using agarose gel electrophoresis. As shown in Figure 2 , when the molar ratio of PEI nitrogen atoms to DNA phosphate (N/P ratio) was above 15, both rod-like and spherical PEI-mPEG/α-CD assemblies with DNA did not migrate into the gel, which indicated that PEI-mPEG/α-CD could inhibit the migration of pDNA effectively and implied complete complex formation between DNA and PEI-mPEG/α-CD aggregates. According to the literature, [ 14 ] the DNA condensation capacity of PEI corre-lates strongly with its molecular mass. For example, PEI(2.5K) is unable to condense DNA effectively and shows very low transfection effi ciency. Here, the PEI(2.5K)-mPEG/α-CD rods with linear PEI(2.5K) showed excellent DNA condensation ability compared with PEI(2.5K), which could be attributed


Figure 1. A–D) SEM and a–d) TEM images of A,a) PEI(10K)-mPEG/α-CD aggregates, B,b) DNA/PEI(10K)-mPEG/α-CD aggregates, C,c) PEI(2.5K)-mPEG/α-CD aggregates, and D,d) DNA/PEI(2.5K)-mPEG/α-CD aggregates. Samples (a), (b), (c) and (d) were negatively stained with phosphotungstic acid.

Table 1. Size and zeta potential of the PEI(10K)-PEG/α-CD nano-spheres, PEI(2.5K)-PEG/α-CD nanorods and DNA/PEI(25K), DNA/PEI(10K)-PEG/α-CD nanospheres, DNA/PEI(2.5K)-PEG/α-CD nanorods at the N/P ratio of 15 ( n = 3).

Sample Zeta potential [mV] a)

DNA/PEI(25K) 53.1 ± 7.3

PEI(10K)-PEG/α-CD nanospheres 41.9 ± 5.4

DNA/PEI(10K)-PEG/α-CD nanospheres 36.5 ± 3.7

PEI(2.5K)-PEG/α-CD nanorods 35.0 ± 6.1

DNA/PEI(2.5K)-PEG/α-CD nanorods 19.4 ± 5.2

a) Data represented as mean ± SD.

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to the fact that the PEI(2.5K)-mPEG/α-CD rods were aggre-gates rather than individual molecules, which then led to a greater effective molecular weight and higher charge intensity. An N/P ratio of 15, where all DNA were bound to the PEI-mPEG/α-CD assemblies, was chosen and used for subsequent experiments.

Morphology studies showed that the presence of DNA mol-ecules had no effect on the particle shapes. Only the particles size increased a little because of the introduction of DNA (Figure 1 ). The zeta potentials of DNA/PEI-mPEG/α-CD com-plexes were much lower than those of PEI-mPEG/α-CD parti-cles, which further confi rmed that the DNA molecules bonded on the surface of the assemblies (Table 1 ).

2.3. Cell Transfection of DNA/PEI-mPEG/α-CD

An X-gal staining assay was conducted to qualitatively assess the DNA/PEI-mPEG/α-CD-mediated transfection of HepG2 cells. As shown in Figure 3 , effective transfection was not observed for naked DNA and DNA/PEI(2.5K). DNA/PEI(10K) showed a little transfection, which was better than DNA/PEI(2.5K) due to the relatively higher molecular weight of PEI. These results were quite consistent with the other reports. [ 14 ]

It is interesting that both DNA/PEI-mPEG/α-CD complexes showed much high transfection effi ciency, which may be due to the membrane disruptive ability of CD molecules. [ 15 ] It can be seen that many blue spots were detected under an optical microscope from cells transfected with DNA/PEI-mPEG/α-CD complexes, especially for rod-like complexes. We selected DNA/PEI(25K) at the optimal N/P ratio (N/P = 15), the “gold standard” for nonviral gene carriers, [ 16 ] as a blank comparison. It seems that the transfection effi ciency of nanospheres was similar to that of DNA/PEI(25K). The transfection effi ciency of nanorods was much higher than nanospheres and DNA/PEI(25K).

An assay of β-galactosidase was conducted to quantify the effi ciency of DNA delivery of spherical and rod-like DNA/PEI-mPEG/α-CD in the presence of serum, compared with those of DNA/PEI(2.5K), DNA/PEI(10K), DNA/PEI(25K), lipofectamine 2000, and naked DNA ( Figure 4 ). Consistent with the qualiti-tative results, it can be seen that DNA/PEI(2.5K)-mPEG/α-CD nanorods exhibited much better transfection effi ciency (about four times) than DNA/PEI(10K)-mPEG/α-CD nanospheres and DNA/PEI(2.5K). Compared with leading transfection reagent lipofectamine 2000, DNA/PEI(2.5K)-mPEG/α-CD nanorods showed slightly lower transfection effi ciency, which may be attributed to the superior performance of the surface structure or the smaller size of the DNA/lipofectamine 2000 rather than the shape of the vectors.


Figure 2. Binding ability of a) naked DNA and PEI(10K)-mPEG/α-CD nanospheres [b) N/P = 5, c) N/P = 15, d) N/P = 47, e) N/P = 70] at various N/P ratio in comparison with PEI(2.5K)-mPEG/α-CD nanorods [f) N/P = 5, g) N/P = 15, h) N/P = 47, i) N/P = 70] to pDNA.

Figure 3. X-gal staining assay of a) naked DNA, b) PEI(10K)/DNA (N/P = 15), c) PEI(25K)/DNA (N/P = 15), d) PEI(2.5K)/DNA (N/P = 15), e) DNA/PEI(10K)-mPEG/α-CD nanospheres (N/P = 15), and f) DNA/PEI(2.5K)-mPEG/α-CD nanorods (N/P = 15) in the complete medium.

Figure 4. In vitro gene transfection effi ciency of DNA/PEI(2.5K)-mPEG/α-CD nanorods (N/P = 15) and DNA/PEI(10K)-mPEG/α-CD nanospheres (N/P = 15) in comparison with that of PEI(2.5K)/DNA (N/P = 15), PEI(10K)/DNA (N/P = 15), PEI(25K)/DNA (N/P = 15), Lipofectamine/DNA, and naked DNA (ND) in HepG2 cells in the presence of serum. Data represent mean ± standard deviation ( n = 3).

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Considering the similar chemical composition of the nano-spheres and rods, the shape effect was proposed to play a key role for the enhancement of gene transfection.

There are mainly two processes involved in gene transfection medicated by nonviral gene delivery vectors. One is endocytosis of the vector and another is the transfer of DNA from endo-cytotic particles to nucleus. It has been reported that particle shape plays an important role in the cellular internalization of particles via endocytosis. [ 5a – 5e ] For example, Mixson and cow-orkers [ 7 ] reported that needle-like histidine-lysine polyplexes entered the cell through clathrin-mediated endocytosis, in con-trast to spherical ones, which entered primarily through non-clathrin-mediated endocytosis. In this study, cellular uptake of spherical and rod-like DNA/PEI-mPEG/α-CD complexes by HepG2 cells was investigated using fl uorescent labeling and confocal microscopy. As shown in Figure 5 , rod-like complexes exhibited greater fl uorescence intensity of intracellular-labeled plasmid DNA than sphere-like complexes, indicating that the rod-like complexes could be internalized into cells more effi -ciently. The better cellular internalization property of rod-like DNA/PEI-mPEG/α-CD is a possible explanation for their high gene transfection effi ciency.

There are two contradictory evidences for the effect of particle shape on the cellular internalization. Some reports showed that elongated particles enhanced the propensity to be internalized over their spherical coun-terparts. [ 17 ] However, several other studies found that spherical particles were internal-ized to a greater extent than corresponding rod-shaped or cylindrical particles. [ 18 ] There-fore, the contribution and mechanism of particle shapes toward cellular internaliza-tion need to be evaluated carefully. Champion and Mitragotri [ 5a ] pointed out that the point

of nonspherical particles attachment to the macrophage played a key role on the phagocytosis. For instance, a macrophage attached to an ellipse at the pointed end internalized it in a few minutes, while a macrophage attached to a fl at region of the same ellipse did not internalize the particle for over 12 h. In the case of rod-like DNA/PEI(2.5K)-mPEG/α-CD, more positive charges should accumulate at both ends of rod-particles since charge always tends to distribute at the high curvature sur-face, [ 19 ] as such the rod-like DNA/PEI(2.5K)-mPEG/α-CD com-plexes should favor contact with the negative cell membrane at the tips of the rods. In order to confi rm the nonuniform sur-face charge distribution of rod-like complexes, we deposited the rod-like PEI(2.5K)-mPEG/α-CD and spherical PEI(10K)-mPEG/α-CD on a negative plate separately in an electric fi eld. As shown in Figure 6 A, PEI(10K)-mPEG/α-CD complexes were round particles around 200 nm, which were quite similar to the results without electric fi eld, suggesting that the electric fi eld did not infl uence the structure of assemblies. However, the observa-tion of PEI(2.5K)-mPEG/α-CD complexes deposited on the neg-ative plate was very different to that observed without an electric fi eld (Figure 6 B). Although there are a few rod-like particles, a


Figure 5. Confocal microscopy images of HepG2 cells after 2 h incubation at 37 °C with the DNA/PEI(10K)-mPEG/α-CD nanospheres (N/P = 15) and DNA/PEI(2.5K)-mPEG/α-CD nanorods (N/P = 15) in the complete medium. A) Staining with nuclear stain, 4′,6-diamidino-2-phenylindole (DAPI). B) Fluorescence images. C) Bright-fi eld images. D) Merged images. Scale bar: 25 µm.

Figure 6. SEM images of A) PEI(10K)-mPEG/α-CD complexes, B) PEI(2.5K)-mPEG/α-CD com-plexes deposited on negative plate in an electric fi eld.

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rounded shape around 100 nm in diameter was dominant for the PEI(2.5K)-mPEG/α-CD samples deposited on the negative plate, indicating that PEI(2.5K)-mPEG/α-CD oriented vertically in the electric fi eld. This result implied that PEI(2.5K)-mPEG/α-CD complexes showed greater surface charge in the region of the pointed end than in the rod-like fl at region, which in accord-ance with the theory that charge always tends to distribute at the high curvature surface. Therefore, DNA/PEI(2.5K)-mPEG/α-CD complexes should favor to contact with negative cell membrane at the tips, which was benefi cial for the cellular uptake.

The self-assembly procedures of DNA/PEI-PEG/α-CD aggre-gations, and their gene delivery behavior are summarized graphically in Figure 7 . The rod-like shape and surface prop-erty resulted in that the DNA/PEI(2.5K)-mPEG/α-CD complex attached the cell like a needle, which was advantageous to the cellular internalization. Due to the effi cient cellular uptake, the rod-like DNA/PEI(2.5K)-mPEG/α-CD aggregates exhibited high transfection effi ciency.

2.4. Cytotoxicity of the DNA/PEI-mPEG/α-CD Complexes

We further compared the cytotoxicity of the DNA/PEI-mPEG/α-CD complexes with DNA/PEI(2.5K), DNA/PEI(10K), and DNA/PEI(25K) by MTT method in HepG2 cells. As shown in Figure 8 , the cytotoxicity of the nanorods (DNA/PEI(2.5K)-mPEG/α-CD) was about fi ve times lower than that of the nano-spheres (DNA/PEI(10K)-mPEG/α-CD), and 50 times lower than that of DNA/PEI(25K). It is known that the cytotoxicity of PEI is dependent upon their chain length. In most cases, high-molecular-weight PEI caused high cytotoxicity. In rod-like DNA/PEI(2.5K)-mPEG/α-CD complexes, the molecular weight of PEI blocks was only 2500, which may result in their low cytotoxicity. It should be noticed that the cytotoxicity of the nanorods (DNA/PEI(2.5K)-mPEG/α-CD) was also lower than that of DNA/PEI(2.5K), although the molecular weight of PEI was same in both complexes. This result may be attributed to the introduction of PEG and α-CD.

3. Conclusion

A rod-like nanometer-sized carrier, PEI(2.5K)-mPEG/α-CD, was prepared for highly effi cient gene delivery by using simple self-assembly method. The rod-like PEI(2.5K)-mPEG/α-CD can bind DNA effectively and the resulting complex showed over four times higher gene delivery capability than its spherical counterpart and PEI(25K), which was attributed to more effi -cient cellular uptake. Furthermore, the cytotoxicity of rod-like PEI-mPEG/α-CD was found to be about fi ve times lower than that of the nanospheres, and 50 times lower than that of DNA/PEI(25K). These results indicate that shape is an impor-tant parameter for the design of gene delivery vectors, the exact mechanisms of which remains an open question. The


Figure 7. Schematic illustration of the formation of DNA/PEI(10K)-PEG/α-CD nanospheres and DNA/PEI(2.5K)-PEG/α-CD nanorods.

Figure 8. Cell viability assay in HepG2 cells. The cells were treated with various concentrations of PEI(2.5K)/DNA (N/P = 15), PEI(10K)/DNA (N/P = 15), PEI(25K)/DNA (N/P = 15), DNA/PEI(10K)-mPEG/α-CD (N/P = 15), and DNA/PEI(2.5K)-mPEG/α-CD (N/P = 15) for 4 h in complete medium. Cell viability was determined by the MTT assay and expressed as a percentage of control, that is, the untreated cell cultures.

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successful development of nonspherical and biocompatible gene delivery carriers opens up a new avenue to improve the transfection effi ciency for gene therapy.

4. Experimental Section Chemicals : Polyethylenimine (linear M w = 2500), polyethylenimine

(branched M w = 10 000), and polyethylenimine (branched M w = 25 000) were supplied by Polysciences, Inc. (Warrington, PA, USA). Methoxy poly(ethylene glycol) (CH 3 OPEG) ( M n = 2000) was purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Plasmid pORF lacZ (3.54 kb) was purchased from Invivogen (San Diego, CA, USA). Qiagen Giga Endo-free plasmid purifi cation kit was purchased from Qiagen (Valencia, CA, USA). Cell culture media RPMI 1640 was obtained from Gibco Co. (Grand Island, NY, USA). 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), α-cyclodextrin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazodium bromide (MTT), penicillin, streptomycin, 4′,6-diamidino-2-phenylindole (DAPI), lipofectamine 2000, and β-galactosidase reporter gene-staining kit were obtained from Sigma (St Louis, MO, USA). Trypsin was purchased from Bio Basic Inc. (Markham Ontario, Canada). Label IT Tracker nucleic acid-labeling kit was purchased from Mirus (Madison, WI, USA). All the other chemicals and reagents used were of the analytical grade obtained commercially.

Measurements : The structure of CH 3 OPEG-COOH, PEI-mPEG, and PEI-mPEG/α-CD were characterized by 1 H NMR (600 MHz; Bruker Corporation, Germany). Transmission electron microscopy (TEM) observations were performed on a Jeol JEM-100CX electron microscope at an accelerating voltage of 80 kV. Scanning electron microscopy (SEM) observations were performed on a Jeol JSM-5900LV electron microscope at an accelerating voltage of 20 kV. Zeta potential of PEI-mPEG/α-CD and DNA/PEI-mPEG/α-CD complexes were measured by laser-light scattering (Zetasizer 3000; Malvern Instruments, UK). The transfected cells after X-gal staining was monitored by a fl uorescence microscope (Axiovert 40 CFL; Carl Zeiss, Germany). Intracellular localization and expression of labeled plasmid DNA were monitored by confocal laser scanning microscope (510 META; Carl Zeiss, Germany).

Plasmids : Plasmid pORF lacZ (3.54kb) is a eukaryotic expression vector containing the EF-1α/HTLV hybrid promoter within an intron. The lacZ gene codes for the enzyme β-galactosidase, whose activity allows for quick determination of cells expressing the lacZ gene. pORF-lacZ plasmid DNA was isolated and purifi ed from DH5-αE coli using the Qiagen Giga Endo-free plasmid purifi cation kit. DNA concentration and purity were quantifi ed by UV absorbance at 260 and 280 nm on a GBC UV cintra 10e spectrophotometer. The structural integrity and topology of purifi ed DNA were analyzed by agarose gel electrophoresis.

Cell Culture : Human HepG2 (hepatocellular carcinoma) cell line was obtained from the Shanghai Cell Institute, Chinese Academy of Sciences. The cells were maintained in RPMI1640 medium with 10% FBS (fetal bovine serum). In each case, 100 U mL −1 of penicillin and 100 U mL −1 of streptomycin were added.

Synthesis and Characterization of PEI-mPEG : PEI-mPEG was synthesized in a two-step reaction as outlined in Scheme S1 (Supporting Information). CH 3 OPEG (20 mmol primary alcohol), TEMPO (0.13 mmol, 0.02 g), and NaBr (7.8 mmol, 0.8 g) were dissolved in water (100 mL). A sodium hypochlorite solution (15%, 2.2 mmol sodium hypochlorite/mmol primary alcohol, 20% excess) was adjusted to the desired pH by adding aq HCl (4 M ). Both solutions were brought to the desired temperature; the reaction was conducted at 5 ± 1 °C, and added at once to each other. The reaction mixture was stirred for 3 h and then extracted three times with chloroform; the organic phases were combined and dried over Na 2 SO 4 . Filtered the Na 2 SO 4 and evaporated the solvent, the oil residue was dried under vacuum over P 2 O 5 at room temperature for 48 h and then verifi ed by 1 H NMR (Figure S1, Supporting Information).

PEI-mPEG was prepared according to the procedure of Eiselt et al. [ 20 ] A CH 3 OPEG-COOH solution (1% (w/v)) was prepared in a buffer

solution of MES (0.1 M ) and NaCl (0.5 M ), and the pH was adjusted to 6. A sample of NHS (292.2 mg) and of EDC (98.8 mg, molar ration of EDC:NHS:COO − = 1:0.5:1) were added to the solution of CH 3 OPEG-COOH (100 mL) to activate the carboxylic acid groups on the polymer backbone. The solution was agitated to obtain a homogeneous solution followed by the addition of PEI solution (2% (w/v)). The reaction was carried out at room temperature for 24 h. The resulting mixture was dialyzed against pure water for 3 d. The structure of resultant was verifi ed by 1 H NMR (Figures S2 and S3, Supporting Information).

Preparation of Nanoparticles : Filtered PEI-mPEG aqueous solution (1% (w/v), 2.5 mL) was added dropwise to α-CD aqueous solution (10% (w/v), 3.5 mL) under stirring. With the addition of PEI-mPEG, the solution turned to turbid, which indicated the formation of nanoparticles. The assemblies were collected by centrifugation (14 000 rpm, 20 min) and lyophilized for the further utilization. The actual ratio of PEI, mPEG, and α-CD was verifi ed by 1 H NMR (Figures S4 and S5, Supporting Information).

Electrophoretic Deposition of Nanoparticles : The electrophoretic deposition of rod-like PEI(2.5K)-mPEG/α-CD and spherical PEI(10K)-mPEG/α-CD were carried out according to the literature. [ 21 ] The schematic drawing of experimental setup of the electrophoretic cell was shown in Figure S6 (Supporting Information). The aqueous solution of PEI-mPEG/α-CD particles was prepared in a suitable concentration (1‰, w/v). The electrophoretic deposition was performed in direct current electric fi eld of 28 V for 30 min.

Preparation of DNA/PEI-mPEG/α-CD : DNA/PEI-mPEG/α-CD was formed by mixing equal volumes of DNA and PEI-mPEG/α-CD. DNA and PEI-mPEG/α-CD were both diluted from the stock with glucose solution (5%, w/v). After added PEI-mPEG/α-CD dropwise to DNA, the solution was briefl y vortexed, and the resulting polyplexes were incubated for 30 min at room temperature.

In Vitro Transfection Studies : DNA/PEI-mPEG/α-CD complexes, DNA/PEI complexes, DNA/lipofectamine 2000 lipoplex and naked DNA were added to HepG2 cells, an epithelial cell line grown at 37 °C with 5% CO 2 in complete growth media supplemented 10% FBS. Cells were seeded 24 h prior to transfection in a 24-well tissue culture plate at 1.0 × 10 5 cells per well. DNA/PEI-mPEG/α-CD complexes (0.15%, w/v), DNA/PEI complexes, and naked DNA (2 µg of DNA per well) were added to each well and incubated for 4 h at 37 °C in complete RPMI 1640 media, unless otherwise noted. For transfection with lipofectamine 2000, lipofectamine 2000 lipoplex was prepared according to the manufacturer’s protocol. Media were then replaced with fresh complete growth media after 4 h. After 48 h, cells were assayed for gene expression by microscopic lacZ histochemistry or by quantitative detection of β-galactosidase production.

Gel Retardation Experiments : PEI-mPEG/α-CD nanoparticles were examined for its ability to bind pORF lacZ through gel electrophoresis experiments. All the PEI-mPEG/α-CD stock solutions were prepared at a certain nitrogen concentration (1 × 10 −3 M ) in distilled water and the pH was adjusted to 7.4. Solutions were sterile fi ltered (0.2 µm) and stored at 4 °C. pORF lacZ (0.2 g in 2 µL of TE buffer) was mixed with an equal volume of PEI-mPEG/α-CD solution at different nitrogen/phosphate (N/P) ratios. The mixture was vortexed and incubated for 30 min at room temperature. Then, the mixture was analyzed on 1% agarose gel containing goll-views (0.5 µg mL −1 ). Gel electrophoresis was carried out in TAE running buffer (40 × 10 −3 M Tris-acetate, 1 × 10 −3 M EDTA) at 80 V for 40 min in a Sub-Cell system (Bio-Rad Laboratories, CA). DNA bands were visualized and photographed by a UV transilluminator and BioDoc-It imaging system. Both of them were purchased from UVP Inc, USA.

X-Gal Staining : Cells were fi xed with 1% formaldehyde and 0.2% glutaraldehyde in PBS (100 µL) for 10 min at room temperature. After the cells were washed twice with PBS (200 µL), they were incubated at 37 °C in X-Gal (50 µL, 2.5 × 10 −3 M ) (5-bromo-4-chloro-3-indolyl-β-galactosidase, Invitrogen, Grand Island, NY) reaction mixture containing K 4 Fe(CN) 6 (5 × 10 −3 M ), K 3 Fe(CN) 6 (5 × 10 −3 M ), and magnesium chloride (2 × 10 −3 M ) in PBS. After 4 h of incubation, the result was observed under the inversion microscope.


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Cell Viability Assay (MTT assay) : HepG2 cells were cultured in RPMI 1640 medium supplemented with 10% FBS at 37 °C, 5% CO 2 , and 95% relative humidity. For cell viability assay, the cells were seeded at 1 × 10 4 cells per well in 96-well microtiter plates. After 24 h, the culture medium was replaced with serial dilutions of polymer solutions (200 µL) prepared in complete culture media, and then the cells were incubated for 4 h. The polymer solutions were replaced with polymer-free complete culture media (200 µL) and the cells were incubated for another 48 h. Each concentration was replicated in fi ve wells. Filtered MTT reagent (0.5 mg mL −1 , 20 mL) was added to each well and the cells were incubated for 4 h. The unreacted dye was removed by aspiration. The formazan crystals were dissolved in DMSO (200 µL per well) and measured spectrophotometrically in a microplate reader (Model-550; Biorad Co., USA) at a wavelength of 570 nm. The relative cell growth (%) related to control cells cultured in media without polymer was calculated by [A]test/[A]control × 100%.

Assay for β-Galactosidase Enzymatic Activity : At 48 h incubation after transfection, the cells were washed with PBS, lysed with lysis reagent (100 µL), and centrifuged at 10 000 rpm for 10 min. The β-galactosidase activity was measured with an ONPG assay from the supernatant (50 µL, up to 100 µL of H 2 O and 100 µL of ONPG solution were pipetted in a 96-well plate) and incubated up to 30 min at 37 °C. After this, the reaction was stopped with Na 2 CO 3 (1 M ). During the incubation, the β-galactosidase enzyme cleaves the β-bond from ONPG resulting in the formation of the yellow o -nitrophenol molecule. Samples were analyzed by measuring absorbance at 415 nm with the microplate reader (Model-550; Biorad Co., USA). The total protein content of the lysates was measured by a BCA assay (Pierce, USA) using a standard of bovine serum albumin. Transfection effi ciency was expressed as a ratio of β-galactosidase activity to total cellular protein (mU/mg protein). The data represented the mean SD of three wells and were representative of three independent experiments.

Intracellular Localization and Uptake of Labeled Plasmid DNA: Plasmid DNA was labeled with the indicated Label IT Tracker Reagent (at a ratio of 1 µL reagent per 1 µg of DNA) and incubated for 1 h at 37 °C, as described in the Label IT Tracker protocol. The sample was purifi ed by ethanol precipitation, and the labeled DNA pellet was resuspended in sterile, molecular-biology grade water and quantifi ed. Labeled DNA was stored, protected from light, at −20 °C.

The indicated cell line was plated on poly- D -lysine-treated glass coverslips, in 6-well plates at 4.0 × 10 5 cells per well, such that their confl uency would be ≈60% the following day. At this time, rod-like DNA/PEI(2.5K)-mPEG/α-CD complexes (0.15%, w/v) and sphere-like DNA/PEI(10K)-mPEG/α-CD (0.15%, w/v) complexes with DNA (4 µg) per well were added to each well and incubated for 2 h at 37 °C in complete RPMI 1640 media. After 2 h, the cells were fi xed on the coverslips with 4% formaldehyde in PBS for 20 min at room temperature. After fi xing, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, 100 ng mL −1 ), rinsed three times with PBS and mounted on the ends of two partial glass slides, and one drop of PBS was placed on top to keep the cells from drying out. Confocal analysis was performed on each sample of cells for no more than 30 min.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This work was funded by the National Natural Science Foundation of China (grant no. 51373174, 21074138, and 51073107), CAS Knowledge Innovation Program (grant no. KSCX2-EW-J-22), West Light Foundation

of CAS, Program for Changjiang Scholars, Innovative Research Team in University of Ministry of Education of China (IRT1026).

Received: December 27, 2013 Revised: February 24, 2014

Published online: April 9, 2014

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Evaluation of Rod-Shaped Nanoparticles as Carriers for Gene Delivery

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