Dilution-Stable PAMAM G1-Grafted Polyrotaxane Supermolecules

Jun 14, 2014 - Numerous preclinical studies have demonstrated that polycation mediated gene delivery systems successfully achieved efficient gene tran...
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Dilution-Stable PAMAM G1-Grafted Polyrotaxane Supermolecules Deliver Gene into Cells through a Caveolae-Dependent Pathway Huan Huang,† Duanwen Cao,‡ Linghao Qin,†,§ Shouqin Tian,† Yang Liang,† Shirong Pan,*,† and Min Feng*,† †

School of Pharmaceutical Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, P. R. China Department of Pharmaceutical Science, Nanfang Hospital, Southern Medical University, Guangzhou 510515, P. R. China § Key Laboratory on Assisted Circulation, Ministry of Health, Guangzhou 510080, P. R. China ‡

ABSTRACT: Numerous preclinical studies have demonstrated that polycation mediated gene delivery systems successfully achieved efficient gene transfer into cells and animal models. However, results of their clinical trials to date have been disappointing. That self-assembled gene and polycation systems should be stable undergoing dilution in the body is one of the prerequisites to ensuring efficiency of gene transfer in clinical trials, but it was neglected in most preclinical studies. In this account, we developed the dilution-stable PAMAM G1grafted polyrotaxane (PPG1) supermolecules in which PAMAM G1-grafted α-cyclodextrins are threaded onto a PEG chain capped with hydrophobic adamantanamine. The PPG1/ pDNA polyplex (approximate 100 nm in diameter) was very stable and kept its initial particle size and a uniform size distribution at ultrahigh dilution, whereas DNA/PEI 25K polyplex was above three times bigger at a 16-fold dilution than the initial size and their particle size distribution indicated multiple peaks mainly due to forming loose and noncompacted aggregates. PPG1 supermolecules showed significantly superior transfection efficiencies compared to either PEI 25K or Lipofectamine 2000 in most cell lines tested including normal cells (HEK293A) and cancer cells (Bel7402, HepG2, and HeLa). Furthermore, we found that the PPG1 supermolecules delivered DNA into HEK293A through a caveolae-dependent pathway but not a clathrindependent pathway as PEI 25K did. These findings raised the intriguing possibility that the caveolae-dependent pathway of PPG1 supermolecule/pDNA polyplex avoiding lysosomal degradation was attributed to their high transfection efficiency. The dilutionstable PPG1 supermolecule polyplex facilitating caveolae-dependent internalization has potential applications to surmount the challenges of high dilutions in the body and lysosomal degradation faced by most gene therapy clinical trials. KEYWORDS: PAMAM dendrimer derivatives, polyrotaxane, supramolecular structures, dilution-stable, caveolae-dependent pathway

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INTRODUCTION

The low dilution stability of DNA/polycation polyplex is not a problem in cell culture in vitro when it is performed in culture vessels containing a small volume of growth medium, but it is a key problem for systemic in vivo delivery. Because DNA/ polycation polyplex is a self-assembled system constructed mainly through electrostatic interactions, loosening and further disassembly of polyplex can be induced by high dilutions. The loose and noncompacted DNA/polycation polyplex forming very large aggregates certainly led to decreasing their cellular uptake, and consequently, their gene transfection was compromised. Therefore, it is thought that stability of the polyplex undergoing dilution in the body is a necessary prerequisite for achieving high delivery efficiency in clinical trials. Recent studies have indicated that hydrophobic modification of polycation, such as hexyl acrylate,8 dodecanoyl

Over the past decade, polycation-mediated gene delivery approaches have been widely tested in preclinical studies and successfully achieved efficient gene transfer into cells and animal models.1,2 However, results of their clinical trials to date have been disappointing.3,4 To explore the gap between preclinical and clinical studies is the critical strategic solution for the development of polycation-mediated gene delivery systems. Most research is focused on synthesizing novel polycations or improving some of the current drawbacks of polycations to overcome various intracellular barriers, such as cellular internalization and endosomal escape based on the clathrin-mediated endocytosis pathway, for gene delivery in high efficiency.3−5 Furthermore, to prevent the serum inhibition of the transfection polyplex is another hot spot investigation.6,7 However, dilution stability of DNA/polycation polyplex in biological environments is one of the most important but often neglected factors to consider when designing a gene delivery carrier. © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2323

December 30, 2013 June 8, 2014 June 14, 2014 June 14, 2014 dx.doi.org/10.1021/mp5002608 | Mol. Pharmaceutics 2014, 11, 2323−2333

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groups,9 and C12−16 alkyl groups,10 remarkably increased the colloidal stability of DNA/polycation polyplex. Moreover, increasing hydrophobicity of polycations also tended to improve their biocompatibility, cellular uptake, and transfection efficiency. Cyclodextrin-based (CD-based) polyrotaxanes are a class of necklacelike supramolecular polymers constructed by numbers of CD molecules, a family of cyclic oligosaccharides, threaded onto a linear poly(ethylene glycol) (PEG) chain, which has bulky end groups preventing dissociation of the CD rings.11 CD-based polyrotaxanes are highly polyfunctional molecules with high mobility of CD.12 These unique structural characteristics are very promising for drug and gene delivery.13−15 The first cationic CD-based polyrotaxanes for gene delivery were reported by Li’s and Harashima’s research groups in 2006.16,17 Cationic oligoethylenimine (oligo-PEI) grafting onto CD rings of polyrotaxanes condensed negatively charged DNA or siRNA into nanosized particles and indicated lower cytotoxicity and higher transfection efficiency than PEI 25K.17 Furthermore, cytocleavable polyrotaxanes end-capped with stopper via disulfide linkages utilizing both CD mobility and polyrotaxane dissociation to timely release gene into cells achieve effective gene delivery.16,18 In our early work, we have developed polyamidoamine (PAMAM) dendrimer derivatives as gene delivery carriers. The main advantage of PAMAM dendrimer derivative polyplex showed obviously higher colloidal stability under serumcontaining and phosphate-buffered saline conditions than either PEI 25K or Lipofectamine 2000 gene complexes.19−21 In the present study the dilution-stable PAMAM G1-grafted polyrotaxane (PPG1) supermolecules was designed and explored for gene delivery. PPG1 was constructed by PAMAM G1-modified-α-cyclodextrins threading onto a PEG chain capped with hydrophobic adamantanamine. That the combined use of polyrotaxanes with the high mobility of CD and PAMAM dendrimer derivatives would achieve a dilutionstable and effective PPG1 supermolecule gene delivery system was anticipated. Colloidal stability of DNA/PPG1 polyplex at high dilutions was specifically evaluated in terms of particle size and size distribution undergoing serial dilutions. Transfection efficiencies of PPG1 supermolecules were investigated in four cell lines including normal cells (HEK293A) and cancer cells (Bel7402, HepG2, and HeLa). Furthermore, the endocytic pathway of PPG1 supermolecules delivering gene into cells was explored.

Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone. Penicillin− streptomycin (PS; 10 000 U mL−1) and nonessential amino acids were obtain from Gibco. MTT, amiloride, chlorpromazine, geneistein, chloroquine, and Hoechst 33258 were purchased from Sigma-Aldrich. PEI 25 kDa, Lipofectamine 2000, and LysoTracker Red-DND-99 were purchased from Invitrogen. Synthesis of PAMAM G1-Grafted Polyrotaxane (PPG1) Supermolecules. PPG1 supermolecules were synthesized as previously reported with some modifications.23 First, polyrotaxane with PEG-carboxylic acid (PEG-COOH) was prepared. Briefly, 1.26 g (6.30 × 10−4 mol) of PEG 2000 was oxidized in 100 mL of water by TEMPO (0.21 g, 1.34 × 10−3 mol), NaBr (0.22 g, 2.14 × 10−3 mol), and NaClO (20 mL) at pH 10−11 at room temperature for 15 min.The oxidation was quenched by 10 mL of methanol, followed by acidification with HCl to pH < 2. Then PEG-COOH was extracted by CH2Cl2 and recrystallized with methanol at −20 °C overnight after CH2Cl2 was volatilized by reduced pressure distillation. 0.22 g (1.08 × 10−4 mol) of PEG-COOH was added to 15 mL of αCD saturated aqueous solution (0.145 g/mL). The mixture was ultrasonicated for 15 min and stirred at room temperature overnight to obtain pseudorotaxanes, α-CDs threaded onto a linear PEG-COOH chain. The pseudorotaxanes were freezedried for 24 h. Then pseudorotaxanes were dissolved in 4.5 mL of DMF and mixed with adamantanamine (0.19 g, 1.31 × 10−3 mol), BOP reagent (0.48 g, 1.10 × 10−3 mol), and EDIPA (0.19 g, 1.20 × 10−3 mol). The end-capping reaction with adamantanamine was carried out at 4 °C overnight, and product was purified by washing two times with DMF/ methanol (1:1) and two times with methanol. Adamantanamine end-capped polyrotaxane as a white solid was obtained by freeze-drying for 24 h. PPG1 supermolecules were prepared by cross-linking reaction using 1,1′-carbonyldiimidazole (CDI) to couple primary amines of PAMAM G1 molecules and hydroxyl groups of α-cyclodextrins. Briefly, 0.18 g of adamantanamine endcapped polyrotaxane was dissolved in 30 mL of DMSO under nitrogen. Then the polyrotaxane solution was added dropwise to 2.6 g of CDI in 30 mL of DMSO under nitrogen. The reaction mixture was stirred at room temperature for 24 h and then was poured into 360 mL of tetrahydrofuran/diethyl ether (1:2) mixed solvent to precipitate the product. The resulting solid was dissolved in 20 mL of DMSO, and then added dropwise to PAMAM G1 solution (14.89 g in 20 mL of DMSO) under nitrogen. The reaction mixture was stirred at room temperature for 24 h and then poured into 240 mL of tetrahydrofuran/diethyl ether (2:1) mixed solvent to precipitate PPG1 supermolecules. The aqueous solution of PPG1 supermolecules was dialyzed with dialysis tubing (MWCO 14000) for 3 d and then freeze-dried to obtain the solid PPG1 supermolecule product. Preparation of PPG1 Supermolecule/pDNA Polyplex. The PPG1 supermolecules were self-assembled with pEGFPC1 to form polyelectrolyte complexes (PPG1 supermolecule/ pDNA polyplex). Briefly, pEGFP-C1 (50 μg/mL) was mixed with equal volume of PPG1 supermolecules in deionized water based on various weight ratios of PPG1 to pDNA from 1:1 to 60:1. The resulting mixture was incubated at room temperature for 30 min prior to use. PEI 25K/pDNA polyplex at its optimal N/P ratio of 10 as a control was also prepared.

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EXPERIMENTAL SECTION Materials. Poly(ethylene glycol) 2000 (PEG 2000) was purchased from Tianjin Damao Chemical reagent Co. Ltd. (Tianjin, China). α-Cyclodextrin (α-CD) was purchased from Shandong Binzhou Zhiyuan Bio-Technology Co. Ltd. (Shandong, China). 1,1′-Carbonyldiimidazole (CDI), 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO), ethyldiisopropylamine (EDIPA), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), and pyrene (a purity of 99%) were purchased from Aladdin Inc. (Shanghai, China). Adamantanamine hydrochloride was purchased from Chengdu Xiya Reagent, Inc. (Chengdu, China). NaBr, NaClO, DMSO, DMF, methanol, tetrahydrofuran, and diethyl ether were purchased from Guangzhou Reagent Inc. (Guangzhou, China). PAMAM G1 was synthesized in our laboratories according to the procedure detailed in ref 22. All solvents were distilled to remove any traces of water before use. 2324

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Characterization of PPG1 Supermoleculs and PPG1 Supermolecule/pDNA Polyplex. Particle size and zeta potential of PPG1 supermolecules at various concentrations and PPG1 supermolecule/pDNA polyplex at various weight ratios were measured by Malvern Zetasizer Nano ZS90. The morphologies of PPG1 supermolecules and PPG1 supermolecule/pDNA polyplex at the weight ratio of 5 were visualized by SPM-300HV atomic force microscope (Seiko Instrument, Inc., Japan). A drop of freshly prepared sample solution was immediately dropped onto the surface of freshly prepared mica. The sample was allowed to dry at room temperature for 24 h, and the images were scanned in the tapping mode at a scan speed of approximately 3 Hz with 512 pixel ×512 pixel resolution. The self-assembly of PPG1 nanoparticles was examined by fluorescence experiments with pyrene as a probe. Fluorescence spectra were recorded on a SAFAS Spectrofluorometer flx spectrometer. A serial dilution sample of PPG1 dispersion was prepared with a pyrene concentration of 6 × 10−7 M. Both the excitation and emission spectra were measured. After repeated testing and debugging, the optimal conditions for determining pyrene fluorescence were established as follows: excitation wavelength, 333 nm; emission wavelength were set at 373 and 384 nm; excitation slit width, 2 nm; emission slit width, 2 nm; and high scanning speed of 250 nm/min. The ratio of intensity at 373 and 384 nm was recorded. Dilution Stability Evaluation. Particle size and size distribution measurements of diluted DNA polyplex samples were performed to evaluate their dilution stability. Serial dilutions (up to 256-fold dilution) of PPG1 supermolecule/ pDNA polyplex at the weight ratio of 5 were carried out. Then their particle size and size distribution were measured by using Malvern Zetasizer Nano ZS90. PEI 25K/pDNA polyplex was used as a control. Dilution-induced DNA dissociation from polyplex was assayed by agarose gel electrophoresis. Serial dilution samples of PPG1 supermolecule/pDNA polyplex at the weight ratio of 5 were prepared. Samples of 10 μL each were loaded into wells of a 1.0% agarose gel prepared in TAE buffer containing 0.5 μg/mL ethidium bromide. The samples were subsequently electrophoresed in an electric field of 110 V for 50 min, and bands corresponding to pDNA were visualized on a UV illuminator and photographed by a UV Ipro gel image system (UV Itec). Naked pDNA and PEI 25K polyplex of the same dilution were used as controls. Evaluation of Transfection Efficiency. HEK293A cells (1.0 × 104 cells/well) were seeded in 24-well plates with 500 μL of DMEM complete medium with 10% FBS. After the confluence of the cells reached 70−85%, the culture medium was placed with 600 μL of serum-free medium, and PPG1 supermolecule/pDNA polyplex at various weight ratios from 2 to 30. The final pDNA (pEGFP-C1) concentration was 1.0 μg/ well. After transfection for 4 h, the culture media were changed back to complete medium containing 10% serum. After 48 h incubation to enhance green fluorescent protein expression, the cells were rinsed and the green fluorescence was observed by fluorescence spectroscopy (Olympus BX51 fluorescence spectroscope, Japan). Subsequently the cells were treated with 0.25% trypsin/EDTA for 1 min, collected by centrifugation, and suspended in 0.3 mL of PBS. The harvested cells were analyzed by flow cytometry (Becton-Dickinson) to determine the percentage of fluorescently labeled HEK293A cells for each treatment. The transfection efficiency of PPG1 supermolecule/

pDNA polyplex at its optimal weight ratio was also tested in Bel-7402, HepG-2, and HeLa cell lines. PEI 25K and Lipofectamine 2000 were used as controls. Endocytosis Assays. Endocytic inhibitor was used to identify the possible endocytotic pathways. According to previous studies, amiloride, chlorpromazine, and geneistein can inhibit macropinocytosis and clathrin-mediated and caveolae-mediated endocytosis, respectively.24 To compare the endocytotic pathway of PPG1 supermolecule/pDNA polyplex and PEI 25K/pDNA polyplex in HEK293A cells, the cells were pretreated with 1.0 mM amiloride, 0.014 mM chlorpromazine, 0.2 mM genistein, or coinhibitors (0.014 mM chlorpromazine and 0.2 mM genistein) for 1 h before transfection. The PPG1 supermolecule/pDNA polyplex at the weight ratio of 5 and PEI 25K/pDNA polyplex at the N/P ratio of 10 containing 1.0 μg of pDNA were added to cells in the presence of the inhibitors according to the transfection experiment mentioned above. Cytotoxicity of PPG1 Supermolecules and Endocytotic Inhibitors. Cytotoxicity of PPG1 supermolecules and endocytotic inhibitors was tested by MTT assay. HEK293A cells were seeded at 2 × 104 cells/well in a 96-well plate in 150 μL of DMEM medium containing 10% FBS for 24 h. The culture media were replaced by fresh medium containing various amounts of PPG1 supermolecules, PAMAM G1, PEI 25K, or a variety of endocytotic inhibitors (1.0 mM amiloride, 0.014 mM chlorpromazine, 0.2 mM genistein, or coinhibitors (0.014 mM chlorpromazine and 0.2 mM genistein)), respectively. After 4 h incubation, the medium was replaced with 200 μL of complete medium and cells were incubated for another 48 h. Then 20 μL of stock solution of MTT (5 mg/mL in PBS) was added to each well and incubated for 4 h. After that, medium was changed with 150 μL/well DMSO to dissolve the formazan crystal produced by live cells. The plate absorbance was read at 570 nm by enzyme-linked immunosorbent assay (ELISA) microplate reader (Bio-Rad). Cell viability (%) was calculated according to the following equation: A(sample)/A(control) × 100%. Inspection of Intracellular Trafficking by Confocal Laser Scanning Microscopy (CLSM). HEK293A cells were used as the cell model to monitor the intracellular trafficking of PPG1 supermolecule/pDNA polyplex by using CLSM. HEK293A cells (2 × 104 cells/well) were seeded in a LabTek chambered coverglass system (8 wells/chamber) with 300 μL of DMEM complete medium with 10% FBS for 24 h. Fluorescein-labeled plasmid DNA was prepared according to the manufacturer’s protocol (Mirus, Madison, WI). Polycation/ flourescein-labeled pDNA polyplex containing 0.5 μg of flourescein-labeled pDNA was transfected to cells according to the procedure mentioned above. The lysosomes were stained by Lyso-Tracker Red DND-99 (150 nM) at specific time points during transfection or post-transfection for 45 min. After 0.5 h or 4 h incubation, the cells were rinsed three times with PBS and fixed by 4% paraformaldehyde. The nucleus was stained using 10 μg/mL of Hoechst 33258 for 15 min. The intracellular distribution of flourescein-labeled pDNA was observed and photographed by a Zeiss LSM-510 confocal laser scanning microscope. The excitation wavelength of 365 nm was for Hoechst 33258, 470 nm for flourescein-labeled pDNA, and 546 nm for late endosome/lysosome. Effects of Chloroquine on Transfection. Chloroquine, a lysosomotropic agent, was used to study the difference in intracellular trafficking of PPG1 supermolecules and PEI 25K. 2325

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proton signals of α-CD were also present in the spectrum of pseudorotaxane, α-CDs threaded onto a linear PEG-COOH chain (Figure 1D), whereas these proton signals became almost negligible in PPG1 because of the restriction of the molecular motion by grafting PAMAM G1 to α-CD core and only proton signals of PAMAM G1 were observed (3.48−3.20 ppm (bm, C(f)H), 2.94−2.64 (m, C(e)H and C(g)H), 2.61−2.49 ppm (m, C(d)H)) (Figure 1F). The 1H NMR spectra analysis indicated that the PPG1 supermolecules were synthesized successfully. The average number of α-CDs threaded onto the PEG chain was 15.16, which was calculated by the integral intensities of the α-CD C1−H (5.12 ppm) and −CH2CH2O− repeat unit of PEG (3.77 ppm) signals in 1H NMR spectrum of pesudorotaxane (Figure 1D). Polycations as gene carriers were usually found to have their cytotoxicity increased with an increase in molecular weight, although their transfection efficiencies appeared to increase with increasing polymer size. The molecular weight of a PPG1 supermolecule depends mostly on the molecular weight of linear PEG. The higher molecular weight of PEG could thread more α-CD rings as a result of the bigger molecule of PPG1. Here, PEG with average molecular weight of 2000 was used as the linear molecule for preparation of PPG1 supermolecules. The cytotoxicity assay of PPG1 was carried out in HEK293A cells. A commercial PEI 25K was used as a positive control to compare the relative toxicity of these PPG1 supermolecules. The viability of HEK293A cells incubated with PPG1 supermolecules at a concentration of 80 μg/mL was more than 80%, as shown in Figure 2. In contrast, less than 30% of cells incubated with PEI 25K were viable. The IC50 value of PPG1 calculated based on MTT assay was 258.98 μg/mL, which was about 5 times higher than that of PEI 25K. The results demonstrated that PPG1 supermolecules were considerably less toxic than PEI 25K. The relatively low cytotoxic PPG1 supermolecules can be beneficial in further biomedical applications. The Nanostructure of PPG1 Supermolecules and PPG1/DNA Complexes. Particle size and surface charge are important physicochemical properties of nonviral gene delivery system that could affect biological performance, including cellular uptake and transfection efficiency. Figure 3A indicates that the particle size and zeta potential of PPG1 supermolecules were in the range of 200 to 250 nm and 18 to 28 mV respectively, with varying PPG1 concentrations (100−2000 μg/ mL). Increases in PPG1 concentration led to a decrease in particle size and reached a plateau corresponding to about 200 nm in diameter when the PPG1 concentration was raised beyond 200 μg/mL. The self-assembly of PPG1 supermolecules into nanoparticles was monitored by fluorescence experiments with pyrene as a probe, as shown in Figure 3C. The ratio of vibronic bands in the pyrene fluorescence spectra (I373/I384) calculated from the intensities at 373 and 384 nm was plotted at serially diluted PPG1 supermolecule concentrations. The I373/ I384 value was 1.05 when the concentration of PPG1 supermolecules was 0.1 mg/mL, which confirmed the formation of supermolecular objects with a hydrophobic environment. With further dilution of the PPG1 dispersion (below 0.05 mg/mL), the I373/I384 value was gradually increased to 1.45, which was almost the same as the value of pyrene dissolved in water. The shift of I373/I384 indicated the slow opening of the self-assembled supermolecular objects into free polymer chains and reduction in hydrophobic environment within the PPG1 nanoparticles. The formation of PPG1

Chloroquine (75 μM) with DMEM was pretreated to cells for 1 h before the addition of DNA polyplex to make sure that endosome membrane could disrupt and facilitate pDNA escape into cytosome.20 Then PPG1 supermolecule/pDNA polyplex and PEI 25K/pDNA polyplex were added to cells in the presence of the chloroquine as in the transfection experiment mentioned above. Statistical Analysis. Data were expressed as the mean ± the standard error of the mean. Statistical comparisons were performed using one-way analysis of variance (ANOVA). P values smaller than 0.05 and 0.01 were statistically significant and highly statistically significant, respectively.

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RESULTS Design and Synthesis of PAMAM G1-Grafted Polyrotaxane (PPG1) Supermolecules. As described in the Introduction, we aimed to synthesize a PAMAM G1-grafted polyrotaxane (PPG1) supermolecule, composed of a polyfunctional polyrotaxane with high mobility of CD and PAMAM dendrimers. The PPG1 supermolecules were synthesized in two steps as shown in Scheme 1: (1) The CD-based polyrotaxanes Scheme 1. Schematic Diagram of PAMAM G1-Grafted Polyrotaxane (PPG1) Supermolecules

were prepared by threading many α-cyclodextrins onto a PEG chain end-capped with bulky 1-adamantanamine. (2) PAMAM G1-grafted CD-based polyrotaxane was synthesized by crosslinking reaction using CDI to couple primary amines of PAMAM G1 molecules and hydroxyl groups of α-cyclodextrins. Figure 1 shows the 1H NMR spectra of PEG, PEG-COOH, αCD, pesudorotaxane, polyrotaxane, and PPG1. Figure 1C shows the proton signals of α-CD (5.14 ppm (s, C(1)H of αCD), 4.09−3.87 ppm (m, C(3)H, C(6)H, and C(5) of α-CD), 3.73−3.65 ppm (m, C(2), and C(4)H of α-CD)). These 2326

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Figure 1. 1H NMR spectra (400 MHz) and their assignments of (A) PEG2000 (D2O), (B) PEG-COOH (D2O), (C) α-CD (D2O), (D) pseudorotaxanes (D2O), (E) polyrotaxane ((CD3)2SO), and (F) PPG1 (D2O).

supermolecule nanoparticles showed the concentration-dependent self-assembly behavior. It was interesting to note that the PPG1 supermolecules complexed with pDNA (pEGFP-C1, 4.8 kbp) at varying weight ratios showed condensed particles with smaller particle size of approximate 100 nm diameter than PPG1 supermolecule particles, whereas zeta potential of PPG1 supermolecule/pDNA complexes increased to 31−34 mV, as shown in Figure 3B. Particle size observed by AFM confirmed the results of PCS determination (Figure 3A,B). Dilution Stability of PPG1 Supermolecule/DNA Polyplex. Dilution stability of polycation/DNA polyplex for systemic in vivo delivery is one of the most important but often neglected factors to consider when designing a gene carrier. Figures 4A and 4C showed that both the PPG1 supermolecules (approximately 200 nm in diameter) and PPG1

Figure 2. Cell viabilities of PPG1 supermolecules at various concentrations.

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Figure 3. Nanostructure of PPG1 supermolecules and PPG1/DNA polyplex. (A) Particle size, zeta potential, and AFM images (scan range 10 μm × 10 μm) of PPG1 supermolecules in deionized water. (B) Particle size, zeta potential, and AFM images (scan range 2 μm × 2 μm) of PPG1 supermolecule/DNA polyplex at various weight ratios in deionized water. Data represent the mean ± SD, n = 3. (C) Normalized pyrene fluorescence intensities (I373/I384) for PPG1 supermolecule nanoparticles with serially diluted concentrations.

fection reagents such as PEI 25K and Lipofectamine 2000, the PPG1 supermolecule/pDNA polyplex was assessed for in vitro transfection efficiency in a variety of cell lines including one normal cell line (HEK293A) and three cancer cell lines (Bel7402, HepG2, and HeLa). Figure 5A shows that PPG1 supermolecule/pDNA polyplex at the weight ratio of 5:1 had the best performance in transfection efficiency in serum-free medium. Furthermore, transfection efficiencies of PPG1 supermolecule/pDNA polyplex at its optimal weight ratio were significantly superior to those of either PEI 25K in HEK293A, HepG2, and HeLa cells or Lipofectamine 2000 in Bel7402 and HeLa cells under their respective optimal conditions, as shown in Figure 5B. Endocytic Pathway of PPG1 Supermolecule/pDNA Polyplex. For efficient optimization of the gene delivery carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency.25 Here the endocytic pathway of PPG1 supermolecules was assayed to address their relatively high efficiency of gene transfer based on data following the use of

supermolecule/pDNA polyplex at the weight ratio of PPG1 to pDNA of 5:1 (approximately 100 nm in diameter) were very stable and kept their initial size and size distribution undergoing up to 256-fold dilution. However, PEI 25K/pDNA polyplex at 16-fold dilution was above three times bigger than its initial size, and their size distribution indicated multiple peaks corresponding to their polydispersion index (PDI) rising to 0.5 as shown in Figure 4B. Figure 4E indicates the retardation assay images of polycations/pDNA polyplex with a serial dilution. Compared with naked pDNA, no free pDNA bands were detected in either PPG1/pDNA polyplex or PEI 25K/ pDNA polyplex, demonstrating that the dilution process did not cause any decomposition of tested polycations/pDNA polyplex. These results suggested that the condensation of pDNA and well-defined nanoparticles of PPG1/pDNA polyplex are maintained during a high-dilution process, whereas PEI 25K/pDNA polyplex is likely to form loose and noncompacted aggregates at a 16-fold dilution. In Vitro Transfection Studies. To investigate whether the PPG1 supermolecule is superior to commercial gene trans2328

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Figure 4. Dilution stability of PPG1 supermolecule/DNA polyplex. (A) Particle size and (B) the polydispersity index (PDI) of polycation/DNA polyplex at a serial dilution. Data represent the mean ± SD, n = 3. Size distribution of (C) PPG1 supermolecule/DNA polyplex and (D) PEI25K/ DNA polyplex before and after undergoing 16-fold dilution. (E) Agarose gel electrophoresis retardation assay for detection of loosening or disassembly of DNA polyplex in a serial dilution process.

was not via macropinocytosis. After treatment of the HEK293A cells with an inhibitor of chlorpromazine, transfection efficiency and green-fluorescent protein (GFP) expression of PPG1 supermolecule/pDNA polyplex significantly increased, whereas treatment of the cells with chlorpromazine strongly reduced the transfection efficiency and GFP expression of PEI 25K/pDNA polyplex (Figure 6C). The results suggested that the endocytic pathway of PPG1 supermolecules delivering gene was different from that of PEI 25K. Chlorpromazine is believed to inhibit clathrin-coated pit formation by a reversible translocation of clathrin and its adapter proteins from the plasma membrane to intracellular vesicles.27 Our results revealed that the caveolindependent pathway is not essential for the cellular uptake of PPG1 supermolecule/pDNA polyplex, in contrast to PEI 25K/ pDNA polyplex transfer which is associated with clathrin dependent endocytosis. Interestingly, with treatment of the cells with genistein, a tyrosine kinase inhibitor known to disrupt caveolae-dependent endocytosis,28 the PPG1 supermoleculemediated gene transfection efficiency represented a reduction

three chemical inhibitors of endocytosis including amiloride, chlorpromazine, and genistein. We initially investigated the cytotoxicity of three inhibitors in HEK293A cells, as shown in Figure 6B. After exposure to these inhibitors at their working concentrations, all of the HEK293A cells retained >85% viability, demonstrating that these inhibitors at the concentrations used for endocytosis studies did not have a noticeable effect on the viability of the tested cell line. Subsequently, the endocytic pathway of PPG1 supermolecule/pDNA polyplex was investigated by using a combination of flow cytometry, fluorescence microscopy, and confocal laser scanning microscopy. Figure 6A shows relative transfection efficiencies of PPG1 supermolecule/pDNA polyplex and PEI 25K/pDNA polyplex in HEK293A cells after treatment with these inhibitors of endocytosis. Amiloride, a specific inhibitor of macropinocytosis,26 did not cause any obvious change in transfection efficiencies of either PPG1 supermolecules or PEI 25K. It indicates that the cellular internalization pathway into HEK293A cells of either PPG1 supermolecules or PEI 25K 2329

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Clathrin-mediated endocytosis of gene delivery systems involves endosomal escape, which is well-known to be crucial to efficient gene delivery.20 Chloroquine, a lysosomotropic agent, can promote endosomal escape resulting in improvement of transfection efficiencies.30 As evidenced in Figure 7B and C, after treatment with chloroquine, PPG1 supermolecule polyplex did not show any significant change in transfection efficiency, whereas a distinct increase transfected by PEI 25K polyplex was observed. Enhancing endosomal escape having no effects on transfection efficiency of PPG1 supermolecule polyplex provided further evidence that the endocytic pathway of PPG1 supermolecules was not clathrin-dependent endocytosis as was that of PEI 25K. The results of transfection together with the endocytic pathway of PPG1 supermolecule/pDNA polyplex substantiated our hypothesis that PPG1 supermolecules transfer gene into HEK293A cells mainly via caveolae-dependent endocytosis and bypassing lysosomes, the major biodegradative compartments within cells, resulting in higher transfection efficiency compared with PEI 25K that mediated lysosomal delivery via clathrindependent endocytosis.

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DISCUSSION To date the outcomes of gene therapy clinical trials have been relatively disappointing mainly because of a lack of safe and efficient gene delivery vectors.3,4 Polycations are the nonviral vectors most commonly studied for gene delivery due to their characteristics associated with ease of use, low immune response, and unrestricted gene size.31,32 However, several fundamental problems with polycations for gene delivery have not yet been worked out sufficiently to make the technology therapeutically viable.33 Dilution stability of polycation/DNA polyplex is one of the most important but often neglected factors to consider when designing gene delivery carriers. In the present study the designed polyrotaxane-grafted-PAMAM G1 (PPG1) supermolecules in which PAMAM G1-modified-αcyclodextrins are threaded onto a PEG chain end-capped with adamantanamine were explored for gene delivery. We anticipated that PPG1 supermolecules combining the efficient gene delivery of cationic CD-based polyrotaxane13 and good colloidal stability of PAMAM dendrimer derivatives20 would achieve a dilution-stable and effective gene delivery system. It was found that the nanostructure of PPG1 supermolecules was very different from the most widely studied polycations such as branched PEI, linear polylysine, and chitosan. The results of particle size, AFM observations, and pyrene monitoring indicated that PPG1 supermolecules could spontaneously organize molecular units into nanoparticles by noncovalent interactions with an average size of approximately 200 nm (Figure 3A,C). PPG1 was amphipathic molecules mostly due to their composition of highly hydrophobic end-capping adamantanamine and hydrophilic PAMAM G1 and PEG. Furthermore, the self-assembled PPG1 supermolecules complexed with pDNA into polyplex showed highly colloidal stability (Figure 4). In contrast, PEI 25K/pDNA polyplex was above three times bigger than its initial size and indicated multiple peaks in their size distribution at 16-fold dilution, although PEI 25K polyplex did not show any DNA dissociation from polyplex during dilution (Figure 4E), mainly due to forming loose and noncompacted aggregates. The dilution stability of DNA polyplex is not a problem associated with in vitro cell cultures using a small volume of growth medium, but

Figure 5. (A) Transfection efficiency of PPG1 supermolecule/DNA polyplex at various weight ratios. (B) Transfection efficiency of PPG1 supermolecule/DNA polyplex at its optimal weight ratios in a variety of cell lines, compared with PEI 25K and Lipofectamine 2000. Data represent the mean ± SD, n = 3, *P < 0.05 and **P < 0.01.

of more than half (46.94%) corresponding to remarkably decreased expression levels of GFP. Furthermore, with coblocking with chlorpromazine and genistein, transfection efficiencies of PPG1 indicated a highly statistically significant decrease. The results demonstrated that the cellular internalization of PPG1 supermolecule/pDNA polyplex occurred mainly through caveolae-dependent endocytosis. On the other hand, genistein did not inhibit either transfection efficiency or GFP expression of PEI 25K, indicating that PEI 25K-mediated endocytosis of pDNA polyplex in HEK293A cells was not a caveolae-dependent pathway. A caveola-dependent endocytic entry pathway is involved in early endosome or caveosome which is characterized by neutral pH and unable to accumulate a lysosomal dye (LysoTracker).29 PPG1 supermolecule-mediated gene transfer mainly through caveolae-dependent endocytosis based on the flow cytometry data was visually confirmed by confocal laser scanning microscopy. As shown in Figure 7, PPG1 supermolecule/ pDNA polyplex (green fluorescence due to the fluoresceinlabeled pDNA) were not colocalized with LysoTracker-red stained lysosomes (red fluorescence) at either an early stage (for 0.5 h) or a later stage (for 4 h) in the transfection process, implying that the pDNA polyplex transferred by PPG1 supermolecule was not via the later endosomes/lysosomes into the cytosol. On the contrary, colocalization of PEI 25K/ pDNA polyplex (green fluorescence) and red fluorescent lysosomes during both the 0.5 and 4 h incubation periods were found. The observation by CLSM confirmed that the endocytic pathways of PPG1 supermolecule polyplex and PEI 25K polyplex are different. 2330

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Figure 6. Endocytic pathway of PPG1 supermolecule/DNA polyplex. (A) Relative transfection efficiency of DNA polyplex in Hek293A cells after pretreatment with the endocytic inhibitors. Data represent the mean ± SD, n = 3, **P < 0.01. (B) Cytotoxicity assay of endocytotic inhibitors. (C) Fluorescence images of GFP expression levels in Hek293A cells transfected with DNA polyplex after pretreatment with the endocytic inhibitors.

is a key problem for in vivo systemic delivery in a large volume of biological fluids. Endosomal escape is thought to be a significant intracellular barrier for gene delivery.34 It has been proposed based on many polycation/DNA polyplexes being internalized into cells mainly through a well-recognized clathrin-mediated endocytosis pathway. Gene delivery via clathrin-mediated endocytosis is characterized by the clathrin coated pit internalization and subsequent endolysosomal trafficking of nucleic acid molecules. Lysosomes at the end of clathrin-mediated endocytosis process are acidic membrane-enclosed compartments filled with various nucleases that promote the degradation of the nucleic acid molecules.35 To decrease lysosomal degradation, many strategies that have been developed to enhance endosomal escape, including the use of fusogenic lipids,36 polymers with high buffering capacity,37 and membrane-interacting peptides.38 In the present study, PPG1 supermolecules exhibited significantly superior transfection efficiencies compared to either PEI 25K or Lipofectamine 2000 in most cell lines tested, including normal cells (HEK293A) and cancer cells (Bel7402, HepG2, and HeLa) (Figure 5). The endocytic pathways investigated by using a variety of chemical inhibitors of endocytosis revealed that the PPG1 supermolecules delivered pDNA into HEK293A cells mainly through a caveolae-dependent pathway as shown in Figure 6, but not a clathrin-dependent pathway as PEI 25K did. Furthermore, the

intracellular localizations of PPG1 supermolecule polyplex in the early-stage or later-stage transfection process were observed as separated from acidic later endosomes/lysosomes because of caveolae-dependent endocytosis (Figure 7). On the contrary, PEI 25K polyplex colocalized with the acidic later endosomes/ lysosomes, confirming that the PEI 25K polyplex entered into the cells mainly through a clathrin-mediated endocytosis. Caveolae-mediated endocytosis is thought of as a cellular uptake mechanism parallel to the clathrin-mediated endocytosis. The caveosome is an endosomal compartment with neutral pH, which is in contrast to late endosomes and lysosomes, which have an acidic pH inducing lysosomal enzymes to work most efficiently.39 Some viruses are observed to infect cells by caveolae-mediated endocytosis, resulting in avoiding lysosomal degradation.40 Therefore, the completely different cellular uptake pathway of PPG1 supermolecule polyplex bypassing the clathrin-dependent pathway which is likely to be destined for lysosomal degradation was presumably attributable to their relatively high transfection efficiency, as shown in Scheme 2. The two interesting findings of this study were that PPG1 supermolecule polyplex took advantage of dilution stability and achieved high gene transfection efficiency likely due to caveolae-mediated endocytosis. It is possible for the dilutionstable property of PPG1supermolecule polyplex to overcome the fundamental obstacle to systemic application. Thus, there is 2331

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Scheme 2. Caveolae-Dependent Pathway of PPG1 Supermolecule/DNA Polyplex Avoiding Lysosomal Degradation Was Presumably Attributable to Their Relatively High Transfection Efficiency

mainly through a caveolae-dependent pathway but not a clathrin-dependent pathway as PEI 25K did. These findings raised the intriguing possibility that the caveolae-dependent endocytosis of PPG1/pDNA polyplex avoiding lysosomal degradation was attributable to its high transfection efficiency. The dilution-stable PPG1 supermolecule polyplex facilitating caveolae-dependent internalization has potential applications to surmount obstacles of ultrahigh dilutions in the body and lysosomal degradation faced by most gene therapy clinical trials.

Figure 7. (A) The separation of pDNA from lysosome compartments in Hek293A cells transfected by PPG1 supermolecule/DNA polyplex at an early stage (for 0.5 h) or a later stage (for 4 h) in the transfection process was observed by CLSM. pDNA was labeled by fluorescein (green). Lysosome compartments were stained with LysoTracker Red DND-99 (red). The cell nuclei were stained with Hoechst 33258 (blue). Colocalization of pDNA with lysosome appeared yellow in merged images. (B) Fluorescence images of GFP expression levels and (C) transfection efficiency of DNA polyplex in Hek293A cells after pretreatment with the lysosomotropic agent chloroquine. Data represent the mean ± SD, n = 3, **P < 0.01.

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AUTHOR INFORMATION

Corresponding Authors

concern about their stability to dilution under “working” conditions. The PPG1 supermolecule system will need to be further evaluated in vivo in terms of stability and transfection assay.

*Tel: +8620 39943119. Fax: +8620 87330396. E-mail: gzpshr@ 163.com. *Tel: +8620 39943119. Fax: +8620 87330396. E-mail: [email protected].

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Notes

CONCLUSION In this study, we developed the amphipathic PPG1 supermolecules complexed with pDNA to explore their colloidal stability at a high dilution and the endocytic pathway of PPG1 supermolecules delivering gene into cells. The PPG1supermolecule/pDNA polyplex (approximate 100 nm in diameter) was very stable and kept their initial size and size distribution undergoing up to 256-fold dilution, whereas PEI 25K/pDNA polyplex was above three times bigger than its initial size and indicated multiple peaks in their size distribution at 16-fold dilution mainly due to forming loose and noncompacted aggregates. PPG1 supermolecules showed significantly superior transfection efficiencies in several cell lines compared to either PEI 25K or Lipofectamine 2000. Furthermore, we found that the PPG1 supermolecules delivered pDNA into HEK293A cells

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the National Natural Science Foundation of China (Project No. 31170918) for their financial support of this research.

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REFERENCES

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