Avidin–Biotin Interaction Mediated Peptide Assemblies as Efficient

Nov 12, 2012 - avidin/biotinylated cell-penetrating peptide (TAC), was designed and ... assembled vector TAC developed is a promising gene vector for ...
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Avidin−Biotin Interaction Mediated Peptide Assemblies as Efficient Gene Delivery Vectors for Cancer Therapy Wei Qu, Wei-Hai Chen, Ying Kuang, Xuan Zeng, Si-Xue Cheng, Xiang Zhou, Ren-Xi Zhuo, and Xian-Zheng Zhang* Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China ABSTRACT: Gene therapy offers a bright future for the treatment of cancers. One of the research highlights focuses on smart gene delivery vectors with good biocompatibility and tumor-targeting ability. Here, a novel gene vector self-assembled through avidin−biotin interaction with optimized targeting functionality, biotinylated tumor-targeting peptide/ avidin/biotinylated cell-penetrating peptide (TAC), was designed and prepared to mediate the in vitro and in vivo delivery of p53 gene. TAC exhibited efficient DNA-binding ability and low cytotoxicity. In in vitro transfection assay, TAC/p53 complexes showed higher transfection efficiency and expression amount of p53 protein in MCF-7 cells as compared with 293T and HeLa cells, primarily due to the specific recognition between tumor-targeting peptides and receptors on MCF-7 cells. Additionally, by in situ administration of TAC/p53 complexes into tumor-bearing mice, the expression of p53 gene was obviously upregulated in tumor cells, and the tumor growth was significantly suppressed. This study provides an alternative and unique strategy to assemble functionalized peptides, and the novel selfassembled vector TAC developed is a promising gene vector for cancer therapy. KEYWORDS: avidin−biotin interaction, peptide assemblies, gene vectors, tumor targeting, cancer therapy

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INTRODUCTION Traditional chemotherapy for cancer treatment suffers from limitations such as serious side effects1−3 and difficulties in curing advanced cancers.4,5 As a promising alternative treatment, gene therapy provides a more efficient way to heal cancers with minimum side effects. In recent years, there have already been progressive clinical applications in gene therapy for ovarian cancer, colorectal cancer, and prostate cancer.6−8 Currently, gene therapy mainly focuses on nonviral vectors for their ease of preparation and relatively low cytotoxicity.9−11 Attributed to the good biocompatibility and inherent biodegradability, peptides fabricated from natural amino acids were considered to be promising candidates for gene delivery among their counterparts, such as liposomes,12 dendrimers,13 and cationic polymers.14 Until now, many peptides have been identified from protein domains with unique biochemical properties. Cell-penetrating peptides, which are able to condense plasmid DNA through charge interaction, are proven carriers for intracellular gene delivery through the cell membrane.15,16 Arginine-rich peptides have been proven to be favorable cell-penetrating peptides, for the hydrogen-bond formation of guanidino moiety in arginine with phosphates, sulfates, and carboxylates on cellular components, which are proposed to be crucial for achieving internalization efficacy.17 The R8 (octaarginine) peptide is utilized as one of the most popular cell-penetrating peptides due to its definite efficient internalization.18,19 And according to the specific recognition between protein ligands and cancer cell receptors, tumortargeting peptides were screened out and applied to enhance the selectivity of drug and gene vehicles.20,21 As an attractive linear tumor-targeting peptide with only 5 amino acids, CREKA © XXXX American Chemical Society

(Cys-Arg-Glu-Lys-Ala) was identified by phage display technique.22 CREKA shows excellent targeting ability to human breast cancer cells in vitro and to tumor stroma in vivo.23,24 Although peptides possess inherent advantages as gene vectors, the synthesis of peptides consisting of complicated sequences is usually relatively tough work with a low yield. The p53 gene is one of the most popular tumor suppressor genes. When deficiency, inactivation, or mutation appears, p53 gene cannot induce cell-cycle arrest and cell apoptosis, which may lead to infinite cell proliferation.25−27 More than 50% of human cancers are caused by mutation and dysfunction of p53 gene.28 Therefore, replacement of aberrant p53 gene is an effective way to recover the normal function of cancer cells. Recently, research has been reported that in vivo cell apoptosis and tumor regression could be accomplished by the introduction of p53 gene, indicating that p53 gene is practical in clinical gene therapy.6,29,30 Avidin is a 66 kDa egg-white glycoprotein, which exhibits strong affinity with a dissociation constant of 10−15 M for biotin. Based on this extraordinarily high affinity, avidin−biotin interaction has been widely utilized for biochemical detection and analysis.31,32 However, the exploration of this unique interaction for biomedical materials has rarely been reported by contrast. Combined with their natural characteristics, avidin and biotin are worthy of further applications in biological medicine. Received: July 18, 2012 Revised: October 3, 2012 Accepted: November 12, 2012

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In this study, we present a feasible and flexible strategy for the synthesis of complicated sequence peptides. A novel tumortargeting gene vector, biotinylated tumor-targeting peptide/ avidin/biotinylated cell-penetrating peptide (TAC), developed herein is assembled through avidin−biotin interaction between avidin and biotin-functionalized peptides. The cytotoxicity and tumor-targeting transfection efficiency of TAC were evaluated in different cell lines. In addition, the tumor inhibition effects of TAC/p53 complexes were investigated in tumor-bearing mice preliminarily. We demonstrate that prepared vectors were not only highly efficient in tumor-targeting transfection in vitro but also potently inhibitory against solid tumor proliferation in vivo, suggesting the promising potential of TAC as a gene carrier for cancer therapy.

chloride resin was carried out in a DMF solution with 2 equiv (relative to the substitution degree of resin) of Fmoc-protected amino acid and 4 equiv of DiEA for 2 h at room temperature. After deprotecting of Fmoc groups with 20% piperidine/DMF (v/v), the rest part of peptide residues were coupled in turn by reacting with 2 equiv of Fmoc-protected amino acid, and 4 equiv of HBTU, HOBt, and DiEA, respectively, for 3 h. Biotin was conjugated to the peptide segments as Fmoc-protected amino acids in a mixture of DMF−DMSO. After the biotinylated peptides were synthesized, the resin was washed with DMF and DCM, and dried under vacuum overnight. To deprotect peptide side chains and detach the expected peptides from the solid support, the dried resin was stirred with a mixture containing TFA (83%), phenol (6.3%), thioanisole (4.3%), H2O (4.3%), and EDT (2.1%) for 2 h at room temperature. The supernatant was collected after filtration and further concentrated to a viscous solution by rotary evaporation. The crude product was obtained by precipitating in cold ether. After vacuum-drying overnight, the precipitate was dissolved in distilled water and freeze-dried. The molecular weights of biotinylated peptides were confirmed by electrospray ionization mass spectrometry (ESI-MS). The obtained biotinylated peptides, bio-CREKA and bio-R8, were assembled by avidin in a mild aqueous condition. First, 20-fold weight excess of biotinylated cell-penetrating peptide (bio-R8) was added into the aqueous solution containing appropriate amount of avidin to prepare bio-R8/avidin bioconjugates. The bio-R8/avidin solution was incubated at room temperature for 20 min after vortexing. Then, bioCREKA solution (80 μL, 0.2 mg/mL) was added to the bioR8/avidin solution, and the TAC solution was acquired after reacting at room temperature for another 20 min. Agarose Gel Retardation Assay. The bio-R8/DNA and TAC/DNA complexes were prepared at varying weight ratios ranging from 0 to 30 by adding appropriate volumes of peptide solution to pGL-3 plasmid (0.5 μL, 200 ng/μL in water). The complexes were diluted to a total volume of 8 μL with NaCl solution (150 mM), and then the complexes were incubated at 37 °C for 30 min. After that the complexes were electrophoresed in the 0.7% (w/v) agarose gel containing GelRed and with Tris-acetate (TAE) running buffer at 80 V for 60 min. DNA was visualized with a UV lamp by the Vilber Lourmat imaging system (France). Particle Size and Zeta Potential Measurement. The particle size and zeta potential were measured by Nano-ZS ZEN3600 (MALVERN Instruments) at 25 °C. The TAC/ DNA complexes were prepared at varying weight ratios ranging from 5 to 80 by adding appropriate volumes of peptide solution to pGL-3 plasmid (5 μL, 200 ng/μL in water). After incubation at 37 °C for 30 min, the complexes were diluted to 1 mL with distilled water or NaCl solution (150 mM) for zeta potential and size measurement respectively. In Vitro Cytotoxicity Assay. The cytotoxicity assay of bioR8 and TAC bioconjugates was performed with 293T, HeLa, and MCF-7 cells by MTT assay. 25 kDa PEI was used as the control. Briefly, cells were seeded respectively in 96-well plates at a density of 6000 cells/well and cultured in DMEM (100 μL) containing 10% FBS for 24 h. Then peptide solutions with different concentrations were added to wells. The culture medium was replaced with fresh medium (200 μL) after 48 h, and MTT solution (20 μL, 5 mg/mL) was added to each well and incubated for 4 h. After that, the medium was removed and DMSO (200 μL) was added. The optical density (OD) was

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MATERIALS AND METHODS Materials. Avidin, biotin, and polyethylenimine (branched PEI, Mw 25 kDa) were purchased from Sigma-Aldrich and used as received. Thioanisole was purchased from Acros and used as supplied. N-Fluorenyl-9-methoxycarbonyl (Fmoc) protected Lamino acids (Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Lys(Boc)-OH), 2-chlorotrityl chloride resin (100−200 mesh, loading: 1.32 mmol/g), o-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU), and N-hydroxybenzotriazole (HOBt) were purchased from GL Biochem Ltd. (Shanghai, China) and used without further purification. N,N′-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), and diisopropylethylamine (DiEA) were obtained from Shanghai Reagent Chemical Co. (China) and distilled prior to use. Phenol, piperidine, trifluoroacetic acid (TFA), and 1,2-ethanedithiol (EDT) were provided by Shanghai Reagent Chemical Co. (China), and used directly. QIAfilter plasmid purification Giga Kit (5) was purchased from Qiagen (Hilden, Germany). GelRed was provided by Biotium (Hayward, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate buffered saline (PBS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT), fetal bovine serum (FBS), penicillin− streptomycin, and trypsin were purchased from Invitrogen Corp. The Micro BCA protein assay kit was purchased from Pierce. All other reagents were of analytical grade and used as received. Cell Culture and Amplification of Plasmid DNA. Human embryonic kidney transformed 293 cells (293T), human cervix carcinoma cells (HeLa), and human breast cancer cells (MCF-7) were incubated in DMEM medium with 10% FBS and 1% antibiotics (penicillin−streptomycin, 10,000 U/ mL) at 37 °C in a humidified atmosphere containing 5% CO2. pGL-3 plasmid used as luciferase reporter gene was transformed in Escherichia coli JM109, and red fluorescent protein-tagged p53 expression plasmid (pDsRed2-N1-p53) was transformed in E. coli DH5α. Both plasmids were amplified in LB medium at 37 °C overnight and purified by an EndoFree QiAfilter Plasmid Giga Kit (5). Then the purified plasmids were dissolved in deionized water and stored at −20 °C. The concentration and purity of plasmids were measured by ultraviolet (UV) absorbance at 280 and 260 nm. Synthesis of Biotinylated Peptides and Preparation of TAC. Both biotinylated peptides, bio-CREKA and bio-R8, were prepared manually by employing solid phase peptide synthesis technique based on classical strategy of Fmoc chemistry.33 Briefly, the loading of the first peptide residue to 2-chlorotrityl B

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caliper every day. Tumor volume (V) was calculated as V = length × width2 × 0.5.34 After 15 days of treatment, the mice of each group were sacrificed to resect tumors. The harvested tumors were weighed and frozen at −70 °C for RT-PCR analysis. Reverse Transcription Polymerase Chain Reaction (RT-PCR). The expression of p53 transcript in tumors was determined by RT-PCR analysis, and β-actin gene was used as the internal control. The harvested tumor tissues were treated with TRIzol (Invitrogen) according to the manufacturer's protocols. The isolated total RNA was used as templates. Total RNA (2 μg) was reverse-transcribed with First Strand cDNA Synthesis Kit (TOYOBO) according to the manufacturer's instructions. The PCR reactions were carried out using THUNDERBIRD SYBR qPCR Mix (TOYOBO), and cDNA was amplified through 95 °C for 1 min followed by 40 cycles. Each cycle consisted of denaturation at 95 °C for 15 s, annealing at 58 °C for 20 s, and extension at 72 °C for 20 s. The PCR products were analyzed by 2% agarose gel electrophoresis (110 V, 30 min) with ethidium bromide staining. Statistical Analysis. The resulting values were expressed as mean ± SD. Statistical analyses were performed with Student’s t test or one-way ANOVA.

measured at 570 nm using a microplate reader (Bio-Rad, model 550, USA). The relative cell viability was calculated as follows: cell viability (%) = (OD570sample/OD570control) × 100, where OD570control was obtained in the absence of peptides and OD570sample was obtained in the presence of peptides. In Vitro Transfection. The transfection efficiency of bio-R8 and TAC bioconjugates was evaluated with pGL-3 plasmid. 25 kDa PEI was used as the control. 293T, HeLa, and MCF-7 cells were seeded in 24-well plates at a density of 6 × 104 cells/well and cultured with 1 mL of DMEM containing 10% FBS for 24 h respectively. The complexes of different weight ratios were prepared by mixing 1 μg of plasmid DNA and corresponding volumes of peptide solution at 37 °C for 30 min. Then the complexes were added into plates with serum-free DMEM and incubated for 4 h at 37 °C. Thereafter, the medium was replaced with fresh DMEM containing 10% FBS and the cells were cultured for another 48 h. For luciferase assay, cells were washed by PBS after removing the medium. Then the cells were lysed using 200 μL of reporter lysis buffer (Pierce). The relative light units (RLUs) were measured with a chemiluminometer (Lumat LB9507, EG&G Berthold, Germany). The total protein was measured according to a BCA protein assay kit (Pierce), and luciferase activity was expressed as RLU/mg protein. For detecting pDsRed2-N1-p53 plasmid expression, transfected cells with red fluorescent proteins were observed under confocal laser scanning microscopy (Nikon C1-si TE2000, Japan) directly at the excitation length of 543 nm. The cells were viewed at the magnification of 100× to obtain and record micrographs using EZ-C1. Western Blotting Analysis. After transfection with pDsRed2-N1-p53 plasmid loaded complexes, cells were harvested and lysed in 50 μL of RIPA buffer (1× PBS, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 10 μg/mL PMSF, 2 μg/mL aprotinin, 100 mM Na-orthovanadate) and resuspended in 50 μL of 2× SDS sample buffer containing 1% βmercaptoethanol. Samples were boiled for 5 min and loaded on 10% SDS−PAGE (10 μL per lane). After separation by electrophoresis, the proteins were transferred onto a PVDF membrane (Millipore). The membranes were blocked in 5% skim milk for 1 h. Proteins were detected using enhanced chemiluminescence kit (ECL; Pierce) after incubation with the primary antibody rabbit anti human p53 (1:2000 dilution, Santa Cruz Biotechnology) overnight at 4 °C and then with the secondary antibody HRP-labeled goat anti rabbit IgG (1:500 dilution, Santa Cruz Biotechnology) at room temperature for 1 h. Mouse monoclonal anti-β-actin antibody (Santa Cruz Biotechnology) was used as protein loading control. Inhibition of Tumor Growth. Normal male Chinese Kun Ming (KM) mice, 4−6 weeks old and weighing 18−22 g, were supplied by Wuhan University Center for Animal Experiment (Wuhan, China). All animal studies involving their handling and care were performed in compliance with the National Guideline on the Care and Use of Laboratory Animals. Mice were subcutaneously inoculated with H22 cells (5 × 106 cells/mouse) at the axillary region to establish a solid tumor model. When tumor volume reached about 100 mm3, the mice were randomly divided into three groups of ten mice each. Two groups of tumor-bearing mice were treated every day with p53 loaded complexes or p53 solution (3 μg of p53 plasmid/ mouse) by injection subcutaneously adjacent to tumors, respectively. The third group received sterile PBS injection as the negative control. Tumor size was monitored with a vernier

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RESULTS AND DISCUSSION Synthesis and Characterization of Biotinylated Peptides. Based on the high affinity of avidin−biotin interaction, TAC bioconjugates were designed by the connection of biotinfunctionalized peptides bio-CREKA and bio-R8 mediated by avidin, as illustrated in Figure 1. Biotinylated peptides were

Figure 1. Design of peptide-based TAC bioconjugates. Schematic of TAC with (a) CREKA, for specific interaction with receptors on tumor cells, (b) biotin, for avidin bioconjugation, and (c) R8, for DNA binding and membrane penetration. Atoms of biotinylated peptides are represented by different colors: white, H; gray, C; blue, N; red, O; and yellow, S.

synthesized manually employing a classical strategy of Fmoc chemistry.33 The molecular weight of bio-CREKA and bio-R8 found in ESI-MS (LCQ Advantage, Finigan, USA) was 833.5 [M+ + H] and 785.5 [2M+ + K], respectively. Vector−DNA Complexation and Characterization. TAC bioconjugates were prepared by adding appropriate amounts of avidin and bio-CREKA solution into bio-R8 solution subsequently. After incubation, TAC bioconjugates were formed due to the avidin−biotin interaction. The whole procedure was performed at room temperature with mild conditions owing to the particular affinity between avidin and biotin. For the comparison, the property of bio-R8 in mediating gene transfection was also studied. For gene delivery, the binding capability to condense DNA is a prerequisite.28 Agarose gel electrophoresis was carried out to estimate the association between vectors and DNA with increasing vector/DNA ratios. DNA was condensed compactly at w/w ratios of 15 and 20, for bio-R8/DNA and TAC/DNA complexes, respectively (Figure C

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the high molecular weight of avidin. The zeta potential of both complexes increased slightly with the growth of vector/DNA ratios (Figure 3B). At each particular ratio, the TAC/DNA complexes possessed a much higher zeta potential compared with bio-R8/DNA complexes, indicating the effect of avidin with positive charges (at pH = 7.4). In the process of gene delivery, positively charged complexes are preferred by interaction with the negatively charged cell membrane, which is crucial for cellular uptake and enhancing endocytosis.10 In addition, vector/DNA complexes with unsuitable particle size and zeta potential will be cleared by the reticuloendothelial system (RES), which will hinder the effective gene delivery. Therefore, in order to ensure efficient gene delivery, weight ratios of TAC/DNA complexes should be controlled to obtain particles with appropriate size and zeta potential. Cytotoxicity. The cytotoxicity of bio-R8 and TAC was evaluated with 293T, HeLa, and MCF-7 cells by MTT assay, using 25 kDa PEI as the control. Both peptide-based vectors exhibited significantly higher cell viabilities in the above cell lines as compared with PEI (P < 0.05); and bio-R8 and TAC did not show obvious differences in cytotoxicity for different cell lines (Figure 4), indicating the good biocompatibility of both peptide based vectors. In addition, the relative cell viabilities after the treatment of TAC were slightly higher than those of bio-R8, which might be caused by the conjugation of natural avidin molecules. The relatively high cell viability showed that TAC would be safe for advanced research of gene therapy. In Vitro Transfection. The transfection efficiency of luciferase mediated by both peptide-based vectors was assessed in 293T, HeLa, and MCF-7 cells, and 25 kDa PEI/DNA complexes with the optimal weight ratio of 1.33 were used as the control. As illustrated in Figure 5, the luciferase expression level was influenced by the vector/DNA weight ratio and the cell type. In 293T and HeLa cells, the transfection efficiency of bio-R8/DNA and TAC/DNA complexes showed a similar trend with peak values at the ratio of 40 within w/w ratios ranging from 5 to 80. However, those peak values were lower than the transfection efficiency of 25 kDa PEI/DNA complexes (1.25 × 108 RLU/mg protein in 293T cells and 1.30 × 107 RLU/mg protein in HeLa cells). With respect to MCF-7 cells, the transfection efficiency of bio-R8/DNA complexes was similar to that in HeLa cells, while TAC/DNA complexes showed a significant enhancement of transfection efficiency (P < 0.1). At w/w ratio higher than 20, the transfection efficiency of TAC/DNA complexes was comparable to that of 25 kDa PEI/DNA complexes (2.23 × 107 RLU/mg protein). More-

2). It was inferred that the bioconjugation of bio-CREKA mediated by avidin weakens the DNA binding capability of

Figure 2. Agarose gel electrophoresis retardation assay of (A) bio-R8/ DNA complexes and (B) TAC/DNA complexes at w/w ratios ranging from 0 to 30.

TAC at identical weight ratios compared with bio-R8. This could be ascribed to the introduction of a targeting segment without obvious DNA binding ability to the biotinylated cellpenetrating peptide/DNA complexes. To investigate the efficiency of cellular uptake for bio-R8/ DNA and TAC/DNA complexes at different weight ratios, the particle size and zeta potential of the complexes are important parameters. With increasing weight ratios, the particle size of bio-R8/DNA complexes decreased sharply before the bio-R8/ DNA ratio reached 40. For higher w/w ratios, the particle size increased slightly, which might imply the appearance of aggregation between particles. For TAC/DNA complexes, the particle size exhibited a similar trend to bio-R8/DNA complexes with increasing w/w ratios, whereas the turning point was at 20 (Figiure 3A). However, the size of TAC/DNA complexes was much larger than that of bio-R8/DNA complexes at each w/w ratio, which could be attributed to

Figure 3. (A) Particle size and (B) zeta potential of bio-R8/DNA and TAC/DNA complexes at w/w ratios ranging from 5 to 80. Data are shown as mean ± SD (n = 3). (*P < 0.05 as compared with complexes at the same w/w ratio.) D

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Figure 5. In vitro transfection efficiency of bio-R8/DNA and TAC/ DNA complexes at w/w ratios ranging from 5 to 80 in (A) 293T, (B) HeLa, and (C) MCF-7 cells. 25 kDa PEI/DNA complex at w/w = 1.33 was used as the control. Data are shown as mean ± SD (n = 3). (*P < 0.1 as compared with complexes at the same w/w ratio.)

Figure 4. Cytotoxicity of bio-R8 and TAC in (A) 293T, (B) HeLa, and (C) MCF-7 cells. 25 kDa PEI was used as the control. Data are shown as mean ± SD (n = 3). (*P < 0.05 as compared with solutions of the same concentration.)

over, TAC/DNA complexes with w/w ratio of 30, 40, and 50 were superior to 25 kDa PEI/DNA complexes in transfection ability. Consequently, compared with bio-R8/DNA and 25 kDa PEI/DNA complexes, TAC/DNA complexes exhibited a particular transfection capability in MCF-7 cells. This demonstrated the targeting capability of bio-CREKA for human breast cancer cells in vitro, which was in agreement with the results of early reports.23 The possible reasons for the low gene expression levels for TAC/DNA complexes in 293T and HeLa cells might be lack of specific receptors on the cell surfaces; and this ligand−receptor recognition was usually considered to be dominant in cellular uptake and gene expression process, which could efficiently facilitate targeted delivery of complex DNA.28 Among the studied cell lines, bio-R8/DNA complexes performed with relatively low transfection efficiencies, which were consistent with our previous conclusion that the transfection effect of R8 alone was limited as a cell-penetrating peptide.35 To improve tumor-targeting transfection efficiency, avidin was brought to bridge bio-CREKA and bio-R8 to construct TAC assemblies. As a bioactive moiety, bio-CREKA could specifically interact with the receptors on MCF-7 cells and induce further cellular uptake. Combined with inherent internalization capability of bio-R8, TAC/p53 complexes were

prepared as an efficient delivery system for tumor-targeting gene therapy. A schematic illustration of TAC as a tumortargeting gene vector for expression of p53 protein mediated by specific receptor recognition in cancer cells is presented in Figure 6.

Figure 6. Schematic illustration of TAC as a tumor-targeting gene vector for expression of p53 protein. E

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Figure 7. Confocal microscopy images of 293T, HeLa, and MCF-7 cells transfected by bio-R8/p53 and TAC/p53 complexes at w/w = 40. The red fluorescence images of bio-R8 and TAC are (a, c, e) and (b, d, f), respectively, and the overlapped images of bio-R8 and TAC are (a′, c′, e′) and (b′, d′, f′), respectively (scale bar = 200 μm).

Figure 8. Western blotting analysis. A: Expression of p53 protein in MCF-7 cells transfected by complexes of TAC/p53 (w/w = 40) (a), 25 kDa PEI/p53 (w/w = 1.33) (b), and bio-R8/p53 (w/w = 40) (c). B: Expression of p53 protein in HeLa (a), MCF-7 (b), and 293T cells (c) transfected by TAC/p53 complexes (w/w = 40).

induced an obvious expression amount of p53 protein superior to that of 25 kDa PEI. The results were in accordance with the above transfection assay outcome and further suggested that the mammary tumor-targeting ability was imparted to TAC by the introduction of bio-CREKA, which had been proven by previous studies.23 In addition, the expression amounts of p53 protein transfected by TAC/p53 complexes in 293T, HeLa, and MCF-7 cells were determined by Western blotting assay as well. As shown in Figure 8B, the expression levels of p53 protein in 293T and HeLa cells were similar, which were obviously lower than that in MCF-7 cells. The increased expression of p53 protein in MCF-7 cells illustrated that the cell-type specificity of bio-R8 was enhanced by the tumor-targeting moiety. The efficient expression of p53 gene in MCF-7 cells mediated by TAC in vitro suggested that TAC would be a promising candidate for cancer therapy application. Inhibition of Tumor Growth. To further study the transfection efficiency of TAC/p53 complexes in a natural physiological condition, inhibition effects of tumor growth were

Based on the above transfection results of vector/pGL-3 complexes, the optimal weight ratio of 40 was adopted to study the expression level of p53 protein mediated by bio-R8/p53 and TAC/p53 complexes. The red fluorescent protein expressions of pDsRed2-N1-p53 plasmid in 293T, HeLa, and MCF-7 cells are shown in Figure 7. For 293T and HeLa cells, the images of TAC/p53 complexes exhibited similar density and luminance of red fluorescence to those of bio-R8/p53 complexes, respectively. On the contrary, MCF-7 cells transfected by TAC/p53 complexes presented a higher density of red fluorescence compared with that of bio-R8/p53 complexes. These results were consistent with the above transfection assay of luciferase and further confirmed the specific tumor-targeting ability of TAC improved by the bioCREKA moiety. Furthermore, Western blotting assay was applied to evaluate the transfection abilities of bio-R8, TAC, and 25 kDa PEI for pDsRed2-N1-p53 plasmid in MCF-7 cells, and β-actin was used as the internal control. According to Figure 8A, the expression of p53 protein mediated by bio-R8 was lowest, while TAC F

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Figure 9. Inhibition effects of tumor growth by injection of TAC/p53 complexes (w/w = 40) and p53 solution in tumor-bearing mice. (A) The development of tumor volumes during the treatment period. (B) The tumor weights of sacrificed mice in each treatment group. Data are shown as mean ± SD (n = 10). (*P < 0.05 as compared with weights of other groups.) (C) Images of tumors harvested from each group at the end time point of the treatment. PBS was used as the control.

estimated in vivo. Tumor-bearing mice were used by inoculation with H22 cells. As one of the most common cells for building model of tumor-bearing mice, H22 cells can form stable tumors efficiently, which is important for long-term investigation plans with minimal interference brought by model animals.36,37 In this work, high success rate of modeling cancer in mice (over 90%) contributed a convincing outcome of transfection efficiency evaluation for TAC/p53 complexes. In this study, p53 solution was selected for comparison with TAC/p53 complexes, and sterile PBS was used as the negative control. The results showed that TAC/p53 complexes (w/w = 40) and p53 solution had an efficient antitumor activity compared with PBS group through the monitoring period (Figure 9A). After the initial several days, tumor volumes of PBS group increased dramatically, whereas the curve of TAC/ p53 complex injection group ascended gently through the treatment period. Between groups administered TAC/p53 complexes and p53 solution, the former exhibited a superior inhibition effect against tumor growth. At the final day of the treatment period, tumors were harvested from sacrificed mice and weighed. The average weights of three groups are shown in Figure 9B. The average weight value of the group treated by TAC/p53 complexes was the lowest (P < 0.05 as compared with p53 and PBS groups at the same time), which was consistent with the observation of tumor volumes. During the treatment period, ulceration appeared accompanying the growth of some tumors within the PBS group since PBS did not have an inhibition effect. With further observation of harvested tumors (Figure 9C), the tumors of the PBS group were compact and rigid in texture, and with abundant distribution of microvessels. In contrast, fewer or no microvessels were observed in tumors of the TAC/p53 complexes or p53 solution injection groups, and the texture was loose. Moreover, as affected by increasing growth of adjacent tumors, necrosis of limb tissues appeared at the late period of treatment

in the PBS group, which seriously influenced the ordinary physiological activities of mice. These results indicated that TAC/p53 complexes could suppress the in vivo tumor development efficiently through the whole treatment period. Although p53 solution also exhibited an inhibition capability of tumors in vivo, after complexing with TAC, p53 gene was transfected into solid tumors more efficiently, which could be attributed to the enhancement of internalization efficacy conferred by TAC; and the in vivo results were in accordance with previous reports.22,24 Overall, it was inferred that TAC could be considered as a potential vector for delivering p53 gene in future clinical research. p53 Gene Expression in Tumors. After injection of TAC/ p53 complexes or p53 solution into tumor-bearing mice, the expression of p53 transcript in tumors was detected by RTPCR, and β-actin gene was used as the internal control. Among investigated groups, TAC/p53 complex injection group showed a higher expression level of p53 transcript compared with that of p53 solution injection group, while PBS group exhibited the lowest expression amount from the observation of RT-PCR analysis (Figure 10). As the reverse transcription outcome, the detected amount of p53 transcript was consistent with the expression amount of p53 gene. Hence, the results of RT-PCR analysis implied that TAC could obviously enhance the delivery and transfection of p53 gene in vivo, which subsequently activated the expression of p53 protein to induce tumor cell apoptosis. Furthermore, considering combination with the inhibitory effects of tumor growth in tumor-bearing mice, TAC/p53 complexes were proven to be a promising gene delivery system for clinical application of tumor therapy.

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CONCLUSIONS This study developed an alternative approach to form peptide based tumor-targeting gene vectors through avidin−biotin G

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Figure 10. RT-PCR analysis of PBS group (a), p53 solution group (b), and TAC/p53 complex group (c) for expression of p53 transcript in vivo.

interaction. To the best of our knowledge, this is the first study of fabricating peptide assemblies for tumor-targeting gene delivery by employing avidin−biotin interaction. According to this principle, a novel gene vector, TAC, was prepared by the bioconjugation between avidin and two biotinylated peptides with different bioactivities. The arginine-rich segment, bio-R8, condensed DNA effectively and prompted the internalization efficacy; and the tumor-targeting segment, bio-CREKA, was in charge of specific recognition and binding with receptors on MCF-7 cells in vitro. Combined with p53 gene, TAC presented remarkable suppression effects on the growth of solid tumors in tumor-bearing mice. Further in vivo tumor-targeting evaluation of TAC with MCF-7 bearing nude mice was still under study. In summary, different peptides could be easily self-assembled through the avidin−biotin interaction to form novel gene vector with combined functions. Moreover, by introduction of biotinylated ligands with versatile functionalities, the vectors can be endowed with other characteristics such as nuclear localization and multitarget capability, which facilitate their applications as potential gene vectors for cancer therapy.

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AUTHOR INFORMATION

Corresponding Author

*Tel and fax: + 86 27 6875 4059. E-mail: [email protected]. cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology of China (2011CB606202), National Natural Science Funds for Distinguished Young scholar (51125014), Program for Changjiang Scholars and Innovative Research Team in University (IRT1030) and the Fundamental Research Funds for the Central Universities.

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Molecular Pharmaceutics

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dx.doi.org/10.1021/mp300392z | Mol. Pharmaceutics XXXX, XXX, XXX−XXX