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Oct 14, 2016 - Supramolecular Aggregate as a High-Efficiency Gene Carrier. Mediated with Optimized Assembly Structure. Yi Zhang, Junkun Duan, ...
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Supramolecular Aggregate as a High-Efficiency Gene Carrier Mediated with Optimized Assembly Structure Yi Zhang, Junkun Duan, Lingguang Cai, Dong Ma,* and Wei Xue* Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China S Supporting Information *

ABSTRACT: For cancer gene therapy, a safe and highefficient gene carrier is a must. To resolve the contradiction between gene transfection efficiency and cytotoxicity, many polymers with complex topological structures have been synthesized, although their synthesis processes and structure control are difficult as well as the high molecular weight also bring high cytotoxicity. We proposed an alternative strategy that uses supramolecular inclusion to construct the aggregate from the small molecules for gene delivery, and to further explore the relationship between the topological assembly structure and their ability to deliver gene. Herein, PEI-1.8kconjugating β-CD through 6-hydroxyl (PEI-6-CD) and 2hydroxyl (PEI-2-CD) have been synthesized respectively and then assembled with diferrocene (Fc)-ended polyethylene glycol (PEG-Fc). The obtained aggregates were then used to deliver MMP-9 shRNA plasmid for MCF-7 cancer therapy. It was found that the higher gene transfection efficiency can be obtained by selecting PEI-2-CD as the host and tuning the host/guest molar ratios. With the rational modulation of supramolecular architectures, the aggregate played the functions similar to macromolecules which exhibit higher transfection efficiency than PEI-25k, but show much lower cytotoxicity because of the nature of small/low molecules. In vitro and in vivo assays confirmed that the aggregate could deliver MMP-9 shRNA plasmid effectively into MCF-7 cells and then downregulate MMP-9 expression, which induced the significant MCF-7 cell apoptosis, as well inhibit MCF-7 tumor growth with low toxicity. The supramolecular aggregates maybe become a promising carrier for cancer gene therapy and also provided an alternative strategy for designing new gene carriers. KEYWORDS: gene carrier, supramolecular aggregate, cancer therapy, assembly structure optimization, MMP-9 shRNA delivery, host−guest interactions

1. INTRODUCTION Gene therapy has been a promising strategy to cancer treatment for the past decades.1 For this strategy, a safe and high-efficient gene carrier is a must. The viruses were first reported as the carrier for gene therapy in 1990s and showed excellent gene delivery ability.2 However, its safety concern is always the sword of Damocles. Different from viral carriers, nonviral gene carriers can avoid the risks from toxicity and immune responses in clinic.3,4 Then, outstanding efforts have been performed in designing and synthesizing the nonviral carriers with higher gene transfection efficiency. Generation after generation of gene carriers have been developed from cationic liposomes to linear cationic polymers, and now to topological (branched, dendritic and hyperbranched) cationic polymers.5,6 To improve the transfection efficiency of the synthetic gene carriers, many polymers with topological structures, such as branched polyethylenimine (PEI),7 dendritic polyamidoamine (PAMAM),8 or poly(L-lysine) (PLLD)9 have been designed and showed better gene delivery ability compared with the linear polymers.10 Nowadays, the biggest challenge for synthetic gene carriers is how to enhance their © XXXX American Chemical Society

gene delivery ability meanwhile keep their good biocompatibility. Realizing that, most scientists strove to design polymers with some complex structures to increasing the amines number and charge density which are attributed to their gene transfection, although the synthesis processes and structure control are difficult as well as high molecular weight and high charge density also bring with high toxicity.11,12 We proposed an alternative strategy that using supramolecular inclusion interaction to construct the aggregates, rather than polymers, from the molecules with low-molecularweight. These supramolecular aggregates are formed by physical interaction and their property can be modulated by the supramolecular architectures.13,14 The aggregates may form the compacter structure with gene and have the better gene binding ability due to the supramolecular architectures. Meanwhile, the physical interaction makes their structure reversible and adjustable, which may be convenient for Received: September 8, 2016 Accepted: October 14, 2016

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DOI: 10.1021/acsami.6b11390 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Diagram Illustrating the Processes of Preparation and Structure Optimization of PEI-CD/PEG-Fc Assembly and Its Delivery of the Therapeutic Gene to Tumor Cells

intracellular gene release and then enhance the gene delivery. βCyclodextrin (β-CD) is now widely used to build topological polymers with complex structures15−17 and some works have been reported to use β-CD as host molecules to construct cationic supramolecular polymer for gene delivery.18,19 These reported works usually construct the polymers using 6-β-CD conjugation as the host molecules,20,21 in which β-CD was coupled through its 6-hydroxyl. Little works was reported to synthesize the host β-CD-conjugating molecular through 2hydroxyl. In addition to host/guest molecule ratio, whether the aggregate architecture would be altered by coupling β-CD through different positions and then influence the gene transfection efficiency? Moreover, many polymers are constructed to bind gene to form the complexes, less attention is focused on the aggregate, which may become a good candidate of gene carrier. Better form the aggregate directly to deliver gene than construct the complex polymers through a variety of methods?15,22 To explore the relationship between the aggregate structure and the ability to deliver gene, PEI-conjugating β-CD through 6-hydroxyl (PEI-6-CD) and 2-hydroxyl (PEI-2-CD) have been synthesized respectively and then assembled with diferrocene (Fc)-ended polyethylene glycol (PEG-Fc) at various host/guest ratios. The obtained supramolecular aggregates were then used to deliver MMP-9 shRNA plasmid for cancer therapy (Scheme 1). The low molecular weight PEI (1.8 kDa) was used in this work to avoid the significant cytotoxicity of the aggregate, and Fc displayed the invertible inclusion with β-CD was set as the guest molecule which helped us confirm the difference in transfection efficiency before and after the assembly. Our strategy of constructing a delicate supramolecular aggregate for gene delivery not only take advantage facile self-assembly process to fabricate aggregates with small molecule via host− guest interactions, but also further improve the gene transfection efficiency through optimizing assembly structure, which provided a new perspective for the design of a new gene delivery system.

toluenesulfonyl chloride (PTSC), and all solvents used in this study were purchased from Aladdin Ltd. (Shanghai). Branched polyethylenimine (PEI) with the average molecular weight of 1.8 and 25 kDa were purchased from Alfa Aesar (USA). Bis(3-aminopropyl)terminated poly(ethylene glycol) (PEG-NH2, 1.5 kDa) was purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s Medium (DMEM), Opti-MEM (Reduced Serum Media), fetal bovine serum (FBS), and penicillin−streptomycin were purchased from Gibco Corp. A pcDNA3 plasmid was used for construction of vectors expressing short hairpin RNA (shRNA) for metal matrix proteinase 9 (MMP-9) and green fluorescence protein (GFP) by Invitrogen Corp (Shanghai). A commercial cell counting kit-8 (CCK-8) were purchased from Dojindo Laboratories (Japan) and used as received. The cell line of human breast cancer MCF-7 cells used in this study was supplied by Southern Medical University. 2.2. Preparation of PEI-CD/PEG-Fc Aggregate. 2.2.1. Synthesis of PEI-CD. As shown in Scheme 2a, before conjugating β-CD with branched PEI, β-CDs were monotosylated at the 6-position on their primary faces and at 2-position on their secondary faces respectively to obtain two different molecules, termed as CD-6-OTs and CD-2-OTs. The synthesis of CD-6-OTs23,24 and CD-2-OTs25,26 was according to the previous reports. PEI-CDs (including PEI-6-CD and PEI-2-CD) were synthesized in terms of the same procedure. Briefly, branched PEI (0.36 g) was dissolved in 5 mL of distilled DMSO, and then 5 mL of DMSO containing 0.504 g of CD-6-OTs or 1.2 g of CD-2-OTs was added, respectively. The mixture was stirred for 5 days at 70 °C under N2 atmosphere, and the product was purified by dialysis. 2.2.2. Synthesis of PEG-Fc. For PEG-Fc synthesis (shown in Scheme 2b), ferrocenecarboxylic acid (50.7 mg), PEG-NH2 (150 mg), and HOBt (29.7 mg) were dissolved in DMF, and the mixture was placed in ice-bath. Then TEA (66.8 mg) and HBTU (83.4 mg)/DMF solution were added dropwise to the above mixture in order. After that, the ice bath was removed and the reaction mixture was stirred at room temperature under N2 atmosphere for 2 days. The reaction product was then dialyzed against distilled water [molecular weight cutoff (MWCO) of 1000] for 3 days. Finally, the dialyzate was centrifuged, and the supernatant was freeze-dried to yield dark brown solids. 2.2.3. Construction of Supramolecular PEI-CD/PEG-Fc Aggregate. Solution of PEI-CD in deionized water was prepared with a concentration of 10 mg/mL. According to mole ratio of PEI-CD to PEG-Fc = 2:1, 1:1, 1:2, 1:3, and 1:4, determined amount of PEG-Fc in deionized water (10 mg/mL) was added into PEI-CD solution with moderate stirring. The mixture solutions were stirred at room temperature for 24 h, followed by dialysis against distilled water in a dialysis membrane filter (molecular weight cutoff (MWCO) of 3000)

2. MATERIALS AND METHODS 2.1. Materials. β-Cyclodextrin (β-CD), ferrocenecarboxylic acid (Fc-COOH), poly(ethylene glycol) (PEG, 2 kDa), triethylamine (TEA), 1-hydroxybenzotriazole (HOBt), and O-benzotriazoleN,N,N′,N′-tetramethyluromium hexafluorophosphate (HBTU), pB

DOI: 10.1021/acsami.6b11390 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 2. Synthesis Route to (a) PEI-CDs and (b) PEG-Fc

for 24 h. Finally, the solution was lyophilized to obtain the PEI-CD/ PEG-Fc aggregate. 2.3. Aggregate Characterization. 1H nuclear magnetic resonance (NMR) was recorded on a Bruker Fourier-300 spectrometer operating at 300 MHz in D2O, and tetramethylsilane (TMS) served as the internal standard. To confirm the formation of host−guest assemblies, two-dimensional nuclear overhauser effect (NOE) spectroscopy nuclear magnetic resonance (2D-NOESY NMR) was performed at 400 MHz in D2O on a Bruker Avance NanoBay 400 NMR spectrometer. The molecular weight of PEI-CD/PEG-Fc assembled at 1:1 molar ratio, PEI-25k and PEI-1.8k were measured by a gel permeation chromatography (GPC) on a Malvern Viscotek270 system equipped with a dual-detector system consisting of a viscometer detector and a modular differential refractive index detector. A 0.8 mol/L NaNO3 solution was used as the eluent at a flow rate of 1 mL/min. The hydrodynamic diameter of the PEI-CD/ PEG-Fc aggregates in aqueous solution were performed with a Malvern Zetasizer Nano ZS instrument equipped with a 4.0 mW laser operating at λ = 633 nm at 25 °C. Specifically, particle sizes of PEICD/PEG-Fc aggregates at 2:1, 1:1, 1:2, 1:3, and 1:4 molar ratio were

all determined by Dynamic Light Scattering at 173° scattering with the concentration at 1 mg/mL. 2.4. Cytotoxicity Assay in Vitro. Human breast adenocarcinoma (MCF-7) cells were cultured at 37 °C in DMEM containing 10% FBS and 1% antibiotics (penicillin−streptomycin, 10000 units/mL) in a humidified atmosphere containing 5% CO2. The in vitro relative cytotoxicity of PEI-CD/PEG-Fc aggregates at 2:1, 1:1, 1:2, 1:3 and 1:4 molar ratio was measured with MCF-7 cells by the Cell Counting Kit8 (CCK-8) method. Briefly, 100 μL of the cell suspension with a density of 10000 cells per well was plated into a 96-well plate and subjected to a 24 h incubation in 100 μL of DMEM containing 10% FBS. The PEI-6-CD/PEG-Fc and PEI-2-CD/PEG-Fc aggregates dissolved in serum-free Opti-MEM respectively with concentrations of 1, 5, 20, 50, 100, 200, and 500 μg/mL were then added to each well. After 4 h incubation, the medium was removed and the microplates were washed with PBS twice, then replaced with 100 μL of fresh medium and continually cultured for another 20 h. The assay solution was prepared by mixing CCK-8 solution and culture medium containing FBS at volume ratio 1:9. After the microplates were washed with PBS twice, 100 μL of assay solution was added to each well for further 2 h incubation. The optical density (OD) of the wells C

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ACS Applied Materials & Interfaces was measured at 450 nm by a microplate reader (Multiskan MK3, Thermo Scientific). The relative cell viability (RCV) was defined as RCV (%) = (ODsamples − ODbackground)/(ODcontrol − ODbackground) × 100, where ODsample and ODcontrol were measured in the presence and absence of PEI-CD/PEG-Fc. Data are shown as means ± the standard deviation (SD) on the basis of independent experiments. 2.5. In Vitro Transfection. The in vitro transfection mediated by the EGFP-N1 plasmid-containing complexes were conducted in MCF7 cells at 37 °C, using PEI-25k complexes as the positive control prepared at the optimal mass ratio. To select a better supramolecular assembly structure and determine the optimal host/guest molar ratio for gene delivery, both PEI-6-CD/PEG-Fc and PEI-2-CD/PEG-Fc aggregates with host/guest molar ratio of 2:1, 1:1, 1:2, 1:3, and 1:4 at vector/EGFP mass ratio of 10, 20, 40, 80, and 120 were all employed to measure the transfection efficiency. Moreover, the maximum vector/pDNA mass ratio of each host/guest molar ratio as depicted in Figures S3 and S4 was determined according to the cytotoxicity results in Figure 5 to ensure the aggregates were noncytotoxicity.27 Transfection procedures were described as follow: cells (5 × 104 cells/well) were seeded in 24-well plates and cultured with 0.5 mL of DMEM containing 10% FBS for 24 h. Then the culture media were replaced with the complex solution (0.5 mL) in serum-free OptiMEM. After a 4 h coincubation, the medium containing sample was removed and the microplate were washed with PBS twice, then replaced with 0.5 mL of fresh 10% FBS containing DMEM and continually cultured for further 20 h. The levels of GFP expression were observed using fluorescence microscopy (Zeiss, German). After that, the cells were washed with PBS and digested with pancreatin for further flow cytometry test, and the transfection efficiency was quantified by the percentage of cells expressing GFP, analyzed by flow cytometry using a Gallios system (Beckman, USA). 2.6. Disassembly of PEI-CD/PEG-Fc Aggregates. Normally, uncharged Fc species or its derivatives are strongly bound in the cavity of β-CD, whereas the charged species (Fc+) dissociate rapidly from the cavity, and this process can be reversibly switched under redox agents28,29 or external voltage.30 In this regard, supramolecular PEICD/PEG-Fc aggregates connected through β-CD/Fc inclusion would undergo a dissociation effect, inducing a desirable disassembly of vector upon oxidation. Therefore, hydrogen peroxide (H2O2) was selected as the model oxidant to examine the disassembly property of PEI-CD/PEG-Fc aggregates. The PEI-2-CD/PEG-Fc aggregate solution (10 mM) with the addition of H2O2 was placed in the incubator shaker at 37 °C for 30 min, and then the mixture was dialyzed against distilled water for 24 h to remove excessive H2O2. After that, the disassembly of this aggregate at host/guest ratio of 1:3 was evaluated by DLS, TEM and GPC measurements. To further clarify the effect of host−guest assembly on gene transfection efficiency, the disassembled PEI-2-CD/PEG-Fc aggregates at various host/guest ratios were employed to measure the transfection efficiency according to above procedures. 2.7. Characterization of PEI-2-CD/PEG-Fc Aggregates at Various Host/Guest Ratios. 2.7.1. Aggregate Sizes and Zeta Potentials. Using a method similar to that described above, particle sizes of PEI-2-CD and PEI-2-CD/PEG-Fc aggregates at 2:1, 1:1, 1:2, 1:3, and 1:4 host/guest ratio were all determined by Dynamic Light Scattering at 173° scattering with the concentrations at 1 mg/mL. For zeta potential measurement, ultrapure water was used as the solvent and the measurements were performed in the zeta-model for a minimum of 10 cycles and a maximum of 100 cycles. 2.7.2. Transmission Electron Microscopy (TEM). TEM observation was carried out on a JEOL-3011 high-resolution electron microscope operating at an acceleration voltage of 300 kV. Samples of 1 mg/mL were prepared at by dipping the grid into the aqueous solution of PEI2-CD and PEI-2-CD/PEG-Fc aggregates at host/guest molar ratio of 2:1, 1:1, 1:2, 1:3, and 1:4, and extra solution was blotted with filter paper. After the water was evaporated at room temperature for several days, samples were observed directly without any staining. 2.7.3. UV−vis of the Aggregates. The UV−vis absorption of the PEI-2-CD/PEG-Fc aggregates at host/guest molar ratio of 2:1, 1:1, 1:2, 1:3, and 1:4 were measured at room temperature by using a

Shimadzu UV-2550 UV−vis spectrophotometer, in which PEG-Fc concentration was kept constant at 0.01 mg/mL and the PEI-2-CD concentration was varied with molar ratio. The slit-width was set as 2 nm, and scan speed was set as 480 nm/min. The aqueous solution of PEG-Fc displays a diagnostic absorption of ferrocenyl group at 268 nm. 2.8. In Vitro MMP-9 Delivery by Optimized Aggregate. 2.8.1. MMP-9 pDNA Condensation Ability. To evaluate MMP-9 pDNA condensation ability of PEI-2-CD/PEG-Fc aggregates, the agarose gel electrophoresis was used. The aggregate/pDNA complexes were prepared at optimal ratio (host/guest molar ratio at 1:3), using 1.5 μL of MMP-9 pDNA (330 ng/μL). All the complex solutions were added with a 2 μL of loading buffer (6×) after incubation at 37 °C for 30 min. Then, the complexes were electrophoresed on the 1% (w/v) agarose gel containing SYBR Safe DNA Gel Stain (Invitrogen) using Tris-acetate (TAE) running buffer at 150 V for 30 min. The migration of DNA was visualized and photographed on a UV lamp using a Biorad Gel Doc XR+ System. Besides aggregate/pDNA complex at optimal ratio was also observed by high-resolution TEM without any staining. Further, particle sizes and zeta potentials of MMP-9 pDNA in the absence and presence of PEI-CD/PEG-Fc were measured with a Malvern Zetasizer Nano ZS instrument and the aggregate/pDNA complex at optimal ratio (host/guest molar ratio at 1:3 and aggregate/ pDNA mass ratio at 120) was selected to assess DNA condensation capacity. 2.8.2. Western Blot Assay. To measure the MMP-9 protein expression level in MCF-7 cell after transfection, Western blotting was performed. The procedures were described as follow: MCF-7 cells (1 × 105) were seeded in 6-well plates and cultured in complete medium for 24 h. Various formulations (PBS, PEI-25k/MMP-9, PEI-2-CD/ MMP-9, and PEI-2-CD/PEG-Fc (1/3) aggregate/MMP-9 at different host/guest ratios) were added and incubated with the cells for 48 h (for protein extraction). After that, transfected cells were washed twice with cold PBS and added SDS lysis buffer freshly supplemented with Roche’s Protease Inhibitor PMSF. The lysates were then clarified by centrifugation for 30 min at 12000 rpm. The concentration of the lysate supernatant was determined by a BCA protein assay kit (Thermo Scientific). Target protein concentrations were determined by Western blot analysis. First, proteins (50 μg per lane) were separated using 10% sodium dodecyl sulfate polyacrylamide gel (at 100 V for 2 h). Second, the proteins were transferred (at 100 V for 90 min) onto a PVDF membranes (Bio-Rad). Third, after incubation in 5% skim milk powder in TBST (Tris-buffered saline, 0.1% Tween-20) for 1 h, the membranes were hybridized with rabbit antihuman MMP9 antibody (1:1000) overnight. Fourth, goat antirabbit HRPconjugated secondary antibody (1:2000) was added for an hour at room temperature followed by washes with TBST. Finally, bands were visualized using the enhanced chemiluminescence (ECL) system. The β-actin antibody was used as a control. All bands were analyzed by “‘ImageJ’” software for their areas and mean gray values. 2.8.3. Cell Apoptosis Assay. To investigate whether aggregate/ MMP-9 complexes transfection induced MCF-7 cell apoptosis, the status of MMP-9 transfected cells were analyzed 48 h post transfection using CCK-8 cytotoxicity assay and PE-Annexin V/7-AAD Dead Cell Apoptosis Kit (BD) by flow cytometry, respectively. The CCK-8 assay was conducted according to above procedures. The transfection process of Annexin V-PE/7-AAD double-staining cell apoptosis assay was similar to in vitro transfection measurement. After transfection, the cells were harvested and washed three times with cold PBS and resuspended in 500 μL of binding buffer. Then, 5 μL of Annexin V-PE and 5 μL of 7-AAD were added and incubated with the cells for 15 min in the dark. Finally, the stained cells were analyzed by a flow cytometer (BD FACS Calibur). 2.9. In Vivo MMP-9 Delivery by Optimized Aggregate. Female BALB/c mice (initially weighing 18−20 g) were used to establish the subcutaneous breast tumor model and the experiment of animals was conducted according to the guideline established by Animal Care and Use Committee of Jinan University. To investigate the gene therapy effect using the aggregate as the carrier in vivo, the mice were inoculated with 1 × 107 MCF-7 cells. After 10 days, mice D

DOI: 10.1021/acsami.6b11390 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. 1H NMR spectrum of PEI-1800 (a), PEI-6-CD (b), PEI-2-CD (c), and PEG-Fc (d) in D2O. were randomly assigned to four groups (5 mice in each group), and were administrated with saline control, blank PEI-2-CD/PEG-Fc(1/3) aggregate (600 mg/kg aggregate), PEI-25k/MMP-9 (5 mg/kg MMP9), and PEI-2-CD/PEG-Fc(1/3) aggregate/MMP-9 (5 mg/kg MMP9) respectively via tail vein injection. All groups were injected everyday. After 21 days, the mice were sacrificed by cervical dislocation, and the major tissues including heart, liver, spleen, lung and kidney as well as tumor were removed. The tumor sizes from each group were imaged by a camera and the separated major tissues were fixed in 10% (v/v) formalin saline for routine histopathological procedures. Paraffin-embedded specimens were cut into 5 μm sections and stained with hematoxylin and eosin (H&E) for histopathological evaluations. To further quantify the tumor inhibition in vivo, BALB/c mice were injected subcutaneously with MCF-7 cell suspension containing 1 × 107 cells in the right flank and the mice were randomized into four treatment groups as above. The tumor sizes at days of 3, 6, 9, 12, 15, 18, and 21 were measured using vernier calipers, and the tumor volume (mm3) was calculated using V = L × W2/2, where L and W (mm) are the longest and shortest diameters of the tumors, respectively. 2.10. Statistical Analysis. All the quantitative experiments were carried out in replicates, mean and standard deviation were calculated and the results are expressed as mean ± SD. For comparison of quantitative results, significance of difference between two groups was detected using analysis of variance (ANOVA single factor); p < 0.05 was considered as statistically significant.

3. RESULTS AND DISCUSSION 3.1. Synthesis of PEI-CDs and PEG-Fc. For the host− guest inclusion, PEI-2-CD and PEI-6-CD as the host molecules were synthesized respectively through the conjugation of PEI1.8k with 6-monotosyl and 2-monotosyl β-CDs. Their chemical structures were confirmed by 1H NMR analysis shown in Figure 1. The signals at 2.4−3.0 ppm belong to the protons from PEI shown in Figure 1b and 1c, and the signals at 3.2−4.0 and 5.0 ppm are the characteristic peaks from β-CD. These results suggested that PEI-1.8k has been conjugated with 6monotosyl and 2-monotosyl β-CDs to form PEI-2-CD and PEI-6-CD. Moreover, by calculating the integral area ratio between PEI and β-CD, both PEI-6-CD and PEI-2-CD were confirmed that each PEI chain was conjugated with two β-CD units in average. To further confirm that β-CD was conjugated with PEI by the covalent bond, GPC traces of PEI-1.8k and PEI-CDs were recorded and presented in Figure S1. It was found that the molecular weights of both PEI-6-CD and PEI-2CD increased after conjugated with CD, indicating CD was connected with PEI by the covalent bond. Moreover, the two curves of PEI-6-CD and PEI-2-CD overlapped in GPC traces, suggesting the same molecular weight for them. This result also further confirmed that for both PEI-6-CD and PEI-2-CD, each PEI chain was conjugated with two β-CD units in average. E

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Figure 2. (a) 2D-NOESY NMR spectra of the PEI-2-CD/PEG-Fc inclusion aggregate (molar ratio of 1:1) in D2O (400 Hz). (b) Representative of GPC traces of PEI-2-CD/PEG-Fc, PEI-6-CD/PEG-Fc, PEI-25k, and PEI-2-CD. (c) Particle sizes of PEI-6-CD/PEG-Fc and PEI-2-CD/PEG-Fc aggregates at different PEI-CD/PEG-Fc molar ratios (n = 3).

that both PEI-2-CD and PEI-6-CD could assemble with PEGFc and the molecular weights of the resultant aggregates were much higher than PEI-2-CD but lower than PEI-25k. The results from Figure 2c indicated that PEI-CDs could form the nanoaggregate with PEG-Fc and their sizes increased with the decrease of PEI-CD/PEG-Fc molar ratios. Therefore, the supramolecular aggregates were constructed from small molecules by the host−guest interaction. Moreover, PEI-2-CD/PEG-Fc aggregate displayed the higher molecular weight than PEI-6-CD/PEG-Fc aggregate, suggesting that there was some difference in the inclusion interactions with PEG-Fc between PEI-2-CD and PEI-6-CD. Similar result was also found through their particle sizes, and PEI-2-CD/PEG-Fc aggregate always showed much smaller particle sizes than PEI6-CD/PEG-Fc aggregate at the same molar ratio. These results implied that PEI-2-CD may possess the better assemble ability with PEG-Fc than PEI-6-CD. To confirm this, the association constant for PEI-CDs with PEG-Fc was determined by UV−vis spectroscopy analysis, and the results shown in Figure S2 were calculated to be 1.93 × 104 M−1 for PEI-2-CD/PEG-Fc and 1.23 × 104 M−1 for PEI-6-CD/PEG-Fc according to the modified Hildebrand−Benesi equation.32 This result indicated that PEI-2-CD could assemble with PEG-Fc more efficiently to form the more compacter aggregate. 3.3. Cytotoxicity and Transfection. The cytotoxicity of gene carriers has great influence on gene delivery. It is reported

PEG-Fc was synthesized as the guest molecule by amidation reaction between NH2−PEG−NH2 and ferrocenecarboxylic acid, and its 1H NMR spectrum was shown in Figure 1d. The protons of Fc moiety displayed the signals at 4.46 and 4.21 ppm, distinct from those of PEG at 3.62 ppm. By calculating the integral area ratio between PEG and Fc, it was found that more than 90% hydroxyl groups of PEG were converted to Fcended groups. 3.2. Supramolecular PEI-CD/PEG-Fc Aggregates. After mixing PEI-CDs with PEG-Fc, the assembled aggregates were prepared by the supramolecular inclusion between β-CD moiety from PEI-CD and Fc moiety from PEG-Fc. To confirm this, 2D-NOESY NMR analysis of PEI-2-CD/PEG-Fc aggregate was carried out and the result was shown in Figure 2a. The correlative signals in the 2D-NOESY spectrum are usually used to measure the intermolecular correlations between internal C(3)H and C(5)H protons of β-CD moiety and the protons of guest molecules.28,31 It was found that the signals from H protons of Fc were obviously correlated with the signals from inner protons (H-3 and H-5) of β-CD, indicating the Fc moiety has entered into the hydrophobic cavity of β-CD through the supramolecular inclusion. GPC and particle size results also confirmed that PEI-CDs could assemble with PEG-Fc to form the aggregates. As shown in Figure 2b, PEI-CD/PEG-Fc aggregates showed their retention volumes between PEI-2-CD and PEI-25k, suggesting F

DOI: 10.1021/acsami.6b11390 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 3. (a) MCF-7 cell viabilities of PEI-6-CD/PEG-Fc and PEI-2-CD/PEG-Fc aggregates at different PEI-CD/PEG-Fc ratios (n = 5). Results of GFP expression (b) and gene transfection efficiencies (c) mediated by PEI-2-CD/PEG-Fc and PEI-6-CD/PEG-Fc aggregates at their optimal ratios in MCF-7 cell (n = 3).

aggregates, due to the decrease of actual PEI contents in the aggregates, both PEI-2-CD/PEG-Fc and PEI-6-CD/PEG-Fc aggregates showed much lower cytotoxicity. They displayed noncytotoxicity at the molar ratios of 1:3 and 1:4, even their concentrations reached up to 500 μg/mL. At other ratios from 2:1 to 1:2, the aggregates showed the obvious cytotoxicity at high concentrations. The following gene transfection assays were then performed under their maximum safe concentration,

that the transfection efficiency of nonviral carriers increases with the increased mass ratio of carriers to gene, until the carriers show significant cytotoxicity at high concentrations.33 Then, the cytotoxicity of both PEI-2-CD/PEG-Fc and PEI-6CD/PEG-Fc aggregates with different PEI-CD/PEG-Fc molar ratios has been first analyzed using MCF-7 cells and the results were shown as Figure 3a. It was found that both PEI-25k and PEI-CDs showed the obviously high and concentrationdependent cytotoxicity to MCF-7 cells. For the assembly G

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ACS Applied Materials & Interfaces under which the aggregates displayed no significant cytotoxicity to MCF-7 cells in this work. To explore further the relationship between the compact topological structure of the assembly aggregates and their ability to deliver gene, in vitro gene transfection assay was carried out. The gene transfection results of the aggregates with different PEI-CD/PEG-Fc molar ratios, as well as aggregate/ gene weight ratios, were given as Figures S3 and S4. It was found that nearly for all formulations, PEI-2-CD/PEG-Fc aggregates always showed the better gene delivery result than PEI-6-CD/PEG-Fc aggregates. Figure 3b and c displayed the top transfection results of both PEI-2-CD/PEG-Fc/EGFP and PEI-6-CD/PEG-Fc/EGFP complexes under their respective optimized conditions. PEI-CDs alone showed the poor transfection efficiency because of their low molecular weights and low charge density. After assembled with PEG-Fc, their aggregates showed the significant enhancement in gene transfection efficiency. Particularly, PEI-2-CD/PEG-Fc aggregate showed much better gene transfection ability than PEI-6CD/PEG-Fc aggregate, even better than PEI-25k at the PEICD/PEG-Fc molar ratio of 1:3. This can be confirmed that the aggregate formed by PEI-2-CD showed better gene delivery ability compared with PEI-6-CD due to the better and compacter assembly interactions between PEI-2-CD and PEG-Fc. It has been reported that the methods of incorporating cell-penetrating peptides,34 targeting moieties,19 hydrophobic moieties,35 and disulfide linkages33 can mediate efficient gene transfection, whereas our work proposed a new strategy to achieve high gene transfection through screening optimized supramolecular assembly structure. 3.4. Disassembly of PEI-2-CD/PEG-Fc Aggregate. Furthermore, to confirm that the high transfection efficiency of PEI-2-CD/PEG-Fc aggregate derived from its supramolecular inclusion structure, hydrogen peroxide was added to disassemble PEI-2-CD/PEG-Fc (1/3, molar ratio) aggregate. GPC result from Figure 4a displayed that PEI-2-CD/PEG-Fc (1/3) aggregate disassembled and its molecular weight decreased obviously after hydrogen peroxide was added. TEM observation shown as Figure 4b also reflected that the aggregate disassembled with the smaller size and irregular topography. Under this disassembly condition, its gene transfection efficiency decreased obviously and the result was shown as Figure 4c. It was found that the disassembled aggregate showed much lower transfection efficiency compared with the former aggregate, which decreased nearly to that of free PEI-2-CD polymer. This result confirmed that the high gene transfection efficiency of PEI-2-CD/PEG-Fc (1/3) aggregate came from the supramolecular topography formed by the host−guest inclusion interaction. It is worth noting that tumor cells exhibit an elevated level of H2O2 up to 0.5 nmol (104 cells)−1 h−1 compared with normal cells.36 Thus, the redox-responsive aggregates can be used as smart carriers for controllable gene delivery in tumor cells. Generally, low molecular weight cationic polymers (such as PEI-1.8k used in this work) usually showed the low gene transfection efficiency with low cytotoxicity, while high molecular weight cationic polymers (such as PEI-25k) showed the high gene transfection efficiency but high cytotoxicity.37,38 The way out of this dilemma to obtain a high-efficient gene carrier with low cytotoxicity is the focus for all researchers about gene delivery.39,40 The host/guest interactions formed by CDs and guest molecules provide a new strategy to construct

Figure 4. GPC (a) and TEM (b) results for PEI-2-CD/PEG-Fc (10 mM) (1/3) aggregate before and after addition of hydrogen peroxide (40 mM). (c) In vitro gene transfection efficiencies mediated by disassembled PEI-2-CD/PEG-Fc at different molar ratios (n = 3).

topological polymers from small/low molecules, and the resultant polymers are formed by physical interactions with reversible structures. The resultant polymers play the functions similar to macromolecules, but show much lower cytotoxicity than them due to the nature of small/low molecules. Some works have been reported on the construction of topological polymers for drug or gene delivery using CDs as the host molecules. However, most of them need to improve their delivery efficiency.32 Two hypotheses are given in this work for the high gene delivery efficiency of PEI-2-CD/PEG-Fc aggregate. One is that most works synthesize the host molecules by conjugating 6-hydroxyl of CD rather than 2hydroxyl. It has been verified that 6-CD conjugation displayed the lower inclusion efficiency and then resulted into the low gene transfection efficiency in this work. The other is that most works usually construct various topological polymers rather H

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Figure 5. (a) Particle sizes and zeta potentials of PEI-2-CD/PEG-Fc aggregates at different host/guest ratios (n = 3). (b) TEM images of PEI-2-CD (1 mg/mL) and PEI-2-CD/PEG-Fc (1 mg/mL) at different host/guest molar ratios. (c) UV−vis spectra of PEI-2-CD/PEG-Fc aggregates at different host/guest ratios.

decreased continuously. It was found that their sizes and zeta potentials changed continuously with the molar ratios from 2:1 to 1:4 (Figure 5a), and the aggregate at 1:3 did not showed the irregular results. TEM observation shown as Figure 5b also displayed no significant difference between the aggregate formed at 1:3 and that at other ratios except of their sizes. Then, above results did not illustrate the difference of aggregates between at 1:3 ratio and others. In other words, PEI-2-CD/PEG-Fc aggregate at 1:3 ratio did not showed any irregular apparent appearance with the continuous molar changes from 2:1 to 1:4. Then, why the aggregate showed its highest gene transfection efficiency at the PEI-2-CD/PEG-Fc

than aggregates. The assembly aggregates with compacter structure form the compacter complexes with gene and showed the better gene binding ability. Meanwhile, the host−guest interaction makes them easily reversible in structure, which may be convenient for cellular gene release and carrier metabolism. 3.5. Effect of Host/Guest Ratio on Gene Delivery. In particular, the PEI-2-CD/PEG-Fc aggregate was found to show its highest gene transfection efficiency at a PEI-2-CD/PEG-Fc molar ratio of 1:3, rather than theoretical 1:1 or others. The reason for this was explored herein. First, the aggregate sizes and zeta potentials were studied with the PEI-2-CD/PEG-Fc molar ratio from 2:1 to 1:4, in which PEI content in aggregate I

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ACS Applied Materials & Interfaces molar ratio of 1:3? One reasonable explanation was that high PEI content (PEI-2-CD/PEG-Fc molar ratio of 2:1) of the aggregate resulted in the high transfection efficiency but high cytotoxicity, and low PEI content (1:4) resulted in the low transfection efficiency but low cytotoxicity. Considering the balance relationship between transfection efficiency and cytotoxicity, PEI-2-CD/PEG-Fc aggregate showed the best result at the ratio of 1:3. Further studies then must be carried out to explore the difference of aggregates at the PEI-2-CD/PEG-Fc molar ratio of between 1:3 and others. It is reported that the inclusion of Fc with CD could enhance the absorbance of Fc itself in UV−vis spectrum, because the inclusion could improve the solubility of Fc in aqueous solution and then shows its spectral characteristics more adequately.41 UV−vis spectroscopy analysis for PEI2-CD/PEG-Fc aggregates with different molar ratios was performed and the result was shown in Figure 5c. As shown, PEG-Fc showed its characteristic maximum absorption peak at 268 nm. After PEI-2-CD was added, the mixture showed an obvious increase of absorbance at 268 nm, indicating the inclusion interaction between PEI-2-CD and PEG-Fc. By dividing the absorbance increment using the PEI-2-CD concentration (ΔA/[PEI-2-CD], in which ΔA denotes the absorbance increment after PEI-2-CD is added and [PEI-2-CD] denotes its concentration), the contribution of per unit PEI-2CD on the increment of the Fc absorbance was defined. The maximum ΔA/[PEI-2-CD] was found at the PEI-2-CD/PEGFc molar ratio of 1:3, suggesting that at this ratio per unit PEI2-CD interacted with PEG-Fc the most effectively. In other words, the most efficient inclusion occurred when the molar ratio of PEI-2-CD/PEG-Fc was 1:3, at which the aggregate realized the dominant structure for the high-efficient gene delivery and low cytotoxicity. The 1H NMR results shown as Figure S5 (Supporting Information) also displayed that the aggregate with the best inclusion effect was found at 1:3 ratio. 3.6. MMP-9 pDNA Condensation. The ability of cationic polymers to condense DNA into stable polyplexes is a prerequisite for applied as the gene carriers.42 Thus, the gene binding ability which can protect the DNA from enzymatic degradation and facilitate cellular internalization was evaluated by agarose gel electrophoresis. Figure 6a gave the gel electrophoresis result of PEI-2-CD/PEG-Fc aggregates. For PEI-2-CD/PEG-Fc (1/3) aggregates, DNA migration was observed at the aggregate/DNA ratio of 2:1. However, no fluorescence bands of free DNA were observed for both PEI-2CD and PEI-2-CD/PEG-Fc above the mass ratio of 5:1 which was lower than actual use, suggesting that the migration of DNA in agarose gel was completely retarded. Other information about the DNA condensation by PEI-2CD/PEG-Fc (1/3) aggregates comes from DLS, zeta potential and HR-TEM. It was found that the aggregates showed the mesoporous structure in TEM image which may be easy to encapsulate gene (Figure 6b). Figure 6b showed that the aggregate efficiently compact DNA into small nanoparticles. Table 1 displayed that the sizes of the DNA nanoparticles decreased from 400 to 250 nm and the zeta potential of DNA nanoparticles increased from −16 to 33 mV. The above results verified the strong DNA condensation ability of PEI-2-CD/ PEG-Fc aggregate. 3.7. MCF-7 Cell Inhibition. The significantly enhanced gene delivery ability of the PEI-2-CD/PEG-Fc (1/3) aggregate was then used to deliver MMP-9 shRNA plasmid for MCF-7 cell inhibition. MMP-9 protein plays an important role in

Figure 6. (a) DNA binding ability of PEI-2-CD/PEG-Fc (1/3) evaluated by gel retardation assay. (b) TEM image of aggregate/MMP9 complexes at molar ratio of 1:3.

Table 1. Particle Sizes and Zeta Potentials of MMP-9 pDNA in the Absence and Presence of PEI-2-CD/PEG-Fc at a Molar Ratio of 1:3 (n = 3) sample

particle sizes (nm)

zeta potentials (mV)

MMP-9 MMP-9/PEI-2-CD/PEG-Fc

387.20 ± 86.32 247.13 ± 2.51

−15.93 ± 3.38 32.93 ± 3.93

promoting angiogenesis and tumor metastasis, and the suppression of MMP-9 protein expression contributes to the tumor inhibition.43,44 Western-blot analysis was carried out to study the therapeutic potential of MMP-9 targeting siRNA plasmid transported by PEI-2-CD/PEG-Fc (1/3) aggregate. As shown in Figure 7a, similar to the gene transfection efficiency result, PEI-2-CD/PEG-Fc (1/3) aggregate/MMP-9 complex showed good effect by inducing the most decrease in MMP-9 protein expression than other groups. PEI-2-CD/MMP-9 showed limited affection on MMP-9 protein expression because of its low gene transfection efficiency. Although PEI-25k/ MMP-9 mediated about 80% reduction of MMP-9 protein expression, still displayed the weaker influence than PEI-2-CD/ PEG-Fc (1/3) aggregate/MMP-9. The best effect of PEI-2CD/PEG-Fc (1/3) aggregate/MMP-9 on reducing MMP-9 protein also resulted in the best MCF-7 cell inhibition. Figure 7b and 7c gave the CCK-8 and apoptosis results which confirmed that PEI-2-CD/PEG-Fc (1/3) aggregate/MMP-9 showed the best inhibition effect to MCF-7 cells (more than 50% cells viability were inhibited) as well as the most apoptosis percent of MCF-7 cells (more than 50% cells apoptosis), which J

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Figure 7. In vitro of MMP-9 shRNA plasmid inhibition to MCF-7 cells (1: PBS control, 2: PEI-25k/MMP-9, 3: PEI-2-CD/MMP-9, 4: PEI-2-CD/ PEG-Fc (1/3) aggregate/MMP-9). (a) Western-blot analysis for MCF-7 cells after transfection mediated by various formulations. (b) Cytotoxicity induced by various formulations determined by CCK-8 assay (n = 5). (c) Result of apoptotic cells induced by various formulations (n = 3).

was significantly higher than other groups. These results indicated that PEI-2-CD/PEG-Fc (1/3) aggregate could deliver MMP-9 shRNA effectively and perform effective therapeutic effect. To further confirm the significantly enhanced gene delivery ability of PEI-2-CD/PEG-Fc aggregates, the results of PEI-6-CD/PEG-Fc aggregates delivering MMP-9 shRNA plasmid for MCF-7 cell inhibition have also been given in Supporting Information (Figure S6). The results showed that PEI-2-CD/PEG-Fc/MMP-9 complex showed more remarkable MCF-7 cell inhibition and MMP-9 induced apoptosis than PEI6-CD/PEG-Fc/MMP-9, indicating that PEI-2-CD/PEG-Fc aggregates displayed better gene delivery ability compared with PEI-6-CD/PEG-Fc. 3.8. In Vivo Assays. Besides in vitro assays, tumor inhibition assay was also carried out through injecting complexes into nude mice bearing MCF-7 tumors. From Figure 8a, it was found that both PBS control and blank PEI-2CD/PEG-Fc (1/3) aggregate showed no effect on MCF-7 tumors inhibition, and the tumors grew rapidly with time and more than 40 times increased in volume after 21 days cultivation. PEI-25k displayed good MMP-9 delivery ability in vivo and the 35% decrease in volume of tumor was found compared with PBS control after 21 days. For PEI-2-CD/PEG-

Fc (1/3) aggregate, it showed the best MMP-9 delivery ability and the tumor volume reduced more than 50% compared with PBS control after 21 days, indicating the high efficiency in tumor gene therapy. Moreover, hematoxylin-eosin staining analysis was performed to evaluate the in vivo toxicity of blank PEI-2-CD/PEG-Fc (1/ 3) aggregate. As shown in Figure 8b, histologically, no visible difference was found between PEI-2-CD/PEG-Fc (1/3) aggregate (bottom row) and the PBS control (top row), suggesting the nontoxicity of the aggregate. The toxicity of biomaterials is usually influenced by their chemical structures, size, exposure duration, biodistribution, location, metabolism as well as the nature of the surface and terminal groups.45 PEI-2CD/PEG-Fc aggregate was constructed from low molecules, which remained the low toxicity and easy elimination from organism due to the nature of low molecules.

4. CONCLUSION In conclusion, aggregate, the new promising gene carrier, was constructed from the host−guest interaction between low molecular PEI-conjugating 2-β-CD and diferrocene-ended polyethylene glycol. Significantly, we gained more insight into the relationship between the gene delivery ability and K

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deliver MMP-9 shRNA plasmid for MCF-7 cell inhibition (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Tel./Fax: 86-20-85224338. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (31271019 and 51573071), Natural Science Foundation of Guangdong Province (2014A030313361), the fund from Pearl River S&T Nova Program of Guangzhou (201506010069) and Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering. We thanked Dr. Qiao Tang in our group for the contribution to figures and supplementary data revision, and also thanked Prof. Jiake Xu (University of Western Australia) and Prof. Thomas Brett Kirk (Curtin University) for the contribution to the grammar revision of manuscript.

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Figure 8. In vivo tumor inhibition result (1, PBS; 2, blank aggregate; 3, PEI-25k/MMP-9; 4, PEI-2-CD/PEG-Fc (1/3) aggregate/MMP-9). (a) Representative images and tumor volume profiles of MCF-7 tumor treated with various formulations (n = 5). (b) Representative HE staining of organ histology by PBS control (top row) and PEI-2-CD/ PEG-Fc (1/3) aggregate (bottom row).

topological assembly structure which can be tuned by altering conjugating position on the β-CD and the host/guest ratio. The aggregate played the functions similar to macromolecule which displayed excellent gene delivery ability under optimized experimental conditions, even better than PEI-25k. In vitro and in vivo assays confirmed that the aggregate could deliver MMP-9 shRNA plasmid effectively into MCF-7 cells and then induce the significant MCF-7 cell apoptosis compared with PEI-25k, as well inhibit MCF-7 tumor growth with low toxicity. Considering the difficult in synthesizing the topological polymers with complex structures, the aggregates formed from supramolecular interactions maybe become a promising gene carrier.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b11390. GPC traces of PEI-CDs and PEI-1.8k, association constants of PEI-CDs/PEG-Fc, flow cytometry analysis of in vitro transfection of PEI-2-CD/PEG-Fc and PEI-6CD/PEG-Fc, 1H NMR spectra of PEI-2-CD/PEG-Fc aggregates at different molar ratios, CCK-8 and apoptosis results of PEI-2-CD/PEG-Fc and PEI-6-CD/PEG-Fc to L

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M

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