Vaccine 33 (2015) 3164–3170

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MPG-based nanoparticle: An efficient delivery system for enhancing the potency of DNA vaccine expressing HPV16E7 Tayebeh Saleh a , Azam Bolhassani b,∗,1 , Seyed Abbas Shojaosadati c,∗,1 , Mohammad Reza Aghasadeghi b a

Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran c Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 7 February 2015 Received in revised form 20 April 2015 Accepted 9 May 2015 Available online 19 May 2015 Keywords: Gene delivery system Cell-penetrating peptide DNA vaccine Human papillomavirus E7

a b s t r a c t DNA vaccines against human papillomavirus (HPV) type 16 have not been successful in clinical trials, due to the lack of an appropriate delivery system. In this study, a peptide-based gene delivery system, MPG, which forms stable non-covalent nanoparticles with nucleic acids, was used for in vitro and in vivo delivery of HPV16 E7 DNA as a model antigen. The results demonstrated that at Nitrogen/Phosphate (N/P) ratio over 10:1, this peptide can effectively condense plasmid DNA into stable nanoparticles with an average size of 180–210 nm and a positive surface charge. The transfection efficiency of MPG-based nanoparticles was shown to be comparable with Polyethyleneimine (PEI). The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPG-based nanoparticles as a potent delivery system in DNA vaccine formulations. Immunization with MPG/E7DNA nanoparticles at an N/P ratio of 10:1 induced a stronger Th1 cellular immune response with a predominant interferon-␥ (IFN-␥) profile than those induced by E7DNA alone in a murine tumor model. These findings suggest that MPG peptide as a novel gene delivery system could have promising applications in improving HPV therapeutic vaccines. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Human papillomavirus (HPV) infection is etiologically related with a subset of cancers. A high proportion of cervical and noncervical cancers, 70–76% and 63–95%, respectively, are caused by HPV types 16 and 18, which emphasizes the potential for prevention or treatment of a majority of HPV-related cancers through HPV vaccination [1]. HPV E7 oncogene, is constantly expressed by the HPV-infected tumor cells and all pre-cancerous lesions. Therefore, it represents an ideal target for tumor-specific immunotherapy [2]. DNA vaccine is an attractive form of immunization with many advantages over conventional therapies. Intracellular production of antigens from DNA can result in coordinated activation of both humoral and cell-mediated responses, hence DNA vaccines

∗ Corresponding authors at: Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran. Tel.: +98 21 82883341; fax: +98 21 82883341 (Seyed Abbas Shojaosadati); Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran. Tel.: +98 21 66953311; fax: +98 21 66465132 (Azam Bolhassani). E-mail addresses: A [email protected] (A. Bolhassani), shoja [email protected] (S.A. Shojaosadati). 1 Equally are corresponding authors. http://dx.doi.org/10.1016/j.vaccine.2015.05.015 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

potentially allow for both prophylactic and therapeutic vaccination strategies [3,4]. Despite their successful applications in some preclinical models, their potency in clinical trials has been insufficient to generate effective immunity. The low immunogenicity may be related to poor delivery of DNA to antigen presenting cells (APCs) and insufficient uptake of DNA plasmids by cells upon injection [4]. Recently, nanoparticulate delivery systems based on cationic polymers and lipids have been developed and promised for gene delivery [4–8]. Among the various nano-carriers, cell-penetrating peptides (CPPs) were found to bypass the problem of poor membrane permeability, protection and nuclear entry of nucleic acids. Typically, these peptides consist of less than 30 amino acids; most possess cationic and hydrophobic residues that facilitate interactions with the cell surface [9–11]. Among them, a short amphipathic peptide, MPG, have been known as an efficient delivery system [12,13]. The main objective of this study is the enhancement of DNA vaccine potency using MPG for the development of future therapeutic HPV vaccines. For this purpose, MPG/E7DNA complexes were prepared and then characterized at different N/P ratios. The plasmid DNA stability during formulation and protection of its structure in serum was evaluated in the presence of DNase and serum. Next, the delivery of plasmid DNA encoding HPV16 E7 gene using MPG-based

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nanoparticles was performed in vitro to achieve the best conditions for cell transfection and protein expression. After that, the protective and therapeutic efficacy of this novel nanovaccine was assessed in mice challenged with a TC-1 cancerous cell line. 2. Materials and methods 2.1. Peptide synthesis The MPG peptide was purchased from Biomatik Corporation (Cambridge, Canada). MPG (27 residues: GALFLGFLGAAGSTMGAWSQPKKKRKV) is a primary amphipathic peptide, composed of three domains: an N-terminal hydrophobic domain derived from the fusion sequence of HIV gp41, a hydrophilic lysine-rich domain derived from the nuclear localization sequence (NLS) of SV40 large T-antigen (KKKRKV), and a spacer domain (WSQP) [12,14]. 2.2. Preparation of endotoxin-free plasmid DNA expressing E7 protein Purification of pEGFP-E7 and pcDNA-E7 was performed by ion exchange chromatography with an Endo-free plasmid Giga kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. 2.3. Preparation of peptide/DNA complexes The peptide solution was added dropwise to 1 ␮g of plasmid DNA at different molar ratios of basic amino acid residues in the MPG peptide to DNA phosphates (N/P ratio) in PBS (pH 7.4), and incubated for 60 min at room temperature. The N/P ratio was determined as previously reported [15,16]. The condensation between peptide and DNA was assessed by gel retardation assay. 2.4. Physicochemical characterization The size and zeta-potential of the peptide/DNA complexes at different N/P ratios were measured by a Zetasizer Nano ZS instrument (Malvern Instruments, UK) at 25 ◦ C. The MPG/DNA nanoparticles were prepared at an N/P ratio of 10:1 and the size and morphology of nanoparticles were analyzed with a scanning electron microscope (SEM) (KYKY-EM3200 model, China).

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2.6. Transfection assay The cytotoxicity of both MPG peptide and MPG/plasmid complexes were investigated in COS-7 cell line using the MTT assay [18]. The COS-7 cell line (CRL-1651) was placed at a density of 0.5 × 105 cells/well in 4-well plates (Greiner, Germany) in complete RPMI-1640 supplemented with 5% heat-inactivated FCS. Peptide/pEGFP-E7 nanoparticles at an N/P ratio of 10:1 were prepared and incubated for 1 h at room temperature as a transfection reagent in a total volume of 100 ␮l; MPG/pEGFP-E7 nanoparticles were added to the cells in the presence of 10% serum. The medium was replaced after 8 h incubation at 37 ◦ C with complete RPMI 10% FCS. Polyethyleneimine (LINPEI 25 kDa, Polyscience, N/P: 7) was used as a positive control. The level of GFP expression (transfection efficacy) was monitored by fluorescence microscopy (Envert Fluorescent Ceti, Korea) at 24 and 48 h after transfection and quantified by a FACS Calibur flow cytometer (Partec, Germany) at 48 h post-transfection. Furthermore, E7-GFP expression was detected by Western blot analysis using anti-HPV16 E7 monoclonal. 2.7. In vivo experiments 2.7.1. Protection against tumor growth Inbred C57BL/6 female mice, 5–7 week old, were obtained from the breeding stocks maintained at the Pasteur Institute of Iran. Five groups of 6 female C57BL/6 mice were immunized with 10 ␮g pcDNA-E7 in the right footpad subcutaneously as indicated in Table 1. A booster was performed in all groups with the same dose and formulation as the first immunization after a 3-week interval. Two weeks after the booster vaccination, mice were subcutaneously challenged in the right flank with 1 × 105 TC-1 tumor cells and then monitored for tumor growth and survival rates by palpation twice a week. At each time point, tumor volume was calculated using the formula: V = (a2 b)/2 [19]. 2.7.2. In vivo tumor treatment assay To determine the therapeutic efficacy, 1 × 105 TC-1 tumor cells were inoculated subcutaneously in the right flank of C57BL/6 mice (6 per group). One week after TC-1 inoculation, 5 groups of 5-weekold to 7-week-old C57BL/6 mice were immunized with the same vaccines as described in Section 2.7.1 (Table S1). The booster dose was given to the mice two weeks after the first therapeutic dose. Tumor growth was monitored twice a week by inspection and palpation.

2.5. Stability and protection assay of nanoparticles To assess the stability of MPG/DNA complexes against DNA nucleases, DNase I was added to the complexes (at different N/P ratios of 2:1 to 25:1) with a final concentration of 1.37 U enzyme per 1 ␮g DNA and the mixtures were incubated at 37 ◦ C for 1 h followed by the addition of stop solution (200 mM sodium chloride, 20 mM EDTA and 1% SDS) [17]. To evaluate the serum stability, the nanoparticles at an N/P ratio of 10:1 were exposed to 10% serum and incubated for 5 h at 37 ◦ C. Then, DNA plasmids were released from peptide by adding 10% SDS solution for 2 h and analyzed with electrophoresis on agarose gel 1% [18].

2.7.3. Monitoring humoral immune response by ELISA The mice in preventive groups were bled from retro-orbital at two and six weeks after the booster injection. Then, the sera were pooled for each group. The production of IgG1 and IgG2a antibodies was assayed by ELISA as previously described [20]. The coated antigens were the synthetic MPG peptide (10 ␮g/ml) and the recombinant E7 (rE7, 5 ␮g/ml) purified as previous protocol [20]. 2.7.4. Cytokine assay Two mice from each preventive group and also therapeutic group were sacrificed randomly before challenge and 3 weeks after

Table 1 Immunization schedule in preventive study. Groups

Vaccine modality

Priming

Booster (three weeks after priming)

Challenge with TC-1 (2 weeks after booster)

1 2 3 4 5

Control (–) DNA/DNA Peptide/peptide (control) Peptide-DNA/peptide-DNA Peptide-DNA/peptide-DNA

PBS pcDNA-E7 MPG MPG/DNA (N/P: 10:1) MPG/DNA (N/P: 5:1)

PBS pcDNA-E7 MPG MPG/DNA (N/P: 10:1) MPG/DNA (N/P: 5:1)

TC-1 TC-1 TC-1 TC-1 TC-1

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Fig. 1. Physiochemical characterization of the MPG/DNA nanoparticles. (A) Representative gel retardation assay of MPG peptide complexed with pcDNA-E7 at different N/P ratios; Lane 1: naked plasmid DNA as a control (pcDNA-E7), Lane 2: N/P = 1:1, Lane 3: N/P = 2:1, Lane 4: N/P = 5:1, Lane 5: N/P = 10:1, Lane 6: N/P = 15:1, Lane 7: N/P = 20:1, lane 8: N/P = 25:1, and lane 9: N/P = 30:1. The DNA complexed with MPG that was not able to migrate into the gels was observed at an N/P ratio of 10:1. (B) The particle hydrodynamic diameter and zeta potential analysis (mean ± SD) of MPG/pcDNA-E7 complexes over a range of N/P ratios with dynamic light scattering. (C) Stability analysis of MPG-based nanoparticles against DNase I. Lane 1: naked plasmid DNA without DNase, Lane 2: naked plasmid DNA with DNase, Lane 3: N/P = 2:1, Lane 4: N/P = 5:1, Lane 5: N/P = 10:1, Lane 6: N/P = 15:1, Lane 7: N/P = 20:1, Lane 8: N/P = 25:1. (D) The SEM micrograph of the spherical nanoparticles formed at N/P = 10:1 at 40,000× magnification.

the booster injection, respectively. The spleens were removed and the red blood cell-depleted pooled splenocytes (2 × 106 cells/ml) were cultured in U-bottomed, 96-well plates (Costar, Cambridge, MA) for 72 h in the presence of 10 ␮g/ml of rE7 protein, RPMI 5% as negative control and 5 ␮g/ml of concanavalin A (ConA) as positive control in complete culture medium. The supernatants were harvested and frozen at −70 ◦ C, until the samples were analyzed. Production of IFN-␥ and IL-4 were measured with the sandwichbased ELISA method using a DuoSet ELISA system (R&D Systems) according to the manufacturer’s instructions. All data are represented as mean ± SD for each set of samples. The lower detection limit was 2 pg/ml and 7 pg/ml for IFN-␥ and IL-4, respectively. 2.7.5. Statistical analysis The differences between the control and experimental groups were assessed using Student’s t-test or one-way ANONA (Graphpad Prism, GraphPad Software, La Jolla, CA). Survival was evaluated using Graph Prism Pad version 5 soft-ware and statistical significance calculated using the log-rank (Mantel–Cox) test. 3. Results 3.1. Formation of MPG/DNA complexes The negatively charged plasmid interacted with the cationic peptide for generation of nanoparticles. As shown in Fig. 1A, the polynucleotide molecule did not migrate into the agarose gel at N/P ratio of 10:1, indicating the formation of the MPG/DNA complex. 3.2. Physicochemical characterization of nanoparticles Our results showed that the size of MPG/DNA complexes was reduced as the N/P ratios increased from 2:1 to 30:1 up to ∼150 nm (Fig. 1B). These data are in agreement with the gel retardation assays indicating intense interaction of MPG with DNA in high

N/P ratios with complete DNA condensation (Fig. 1A). Indeed, the nanoparticle size and positive charge were obtained at an N/P ratio of 10:1 and could be selected for in vitro and in vivo delivery of DNA plasmids. SEM analysis of nanoparticles at an N/P ratio of 10:1 showed a spherical and regular shape with a narrow size distribution (Fig. 1D). 3.3. Stability of MPG-based nanoparticles After DNase I treatment, the naked DNA was quickly degraded (Fig. 1C, lane2), while the DNA-peptide complex protected the DNA from DNase I degradation at the N/P ratios more than 10:1 (Fig. 1C). For serum protection assay, the N/P ratio of 10:1 was selected based on the analysis of DNase I stability. Agarose gel electrophoresis showed that unprotected plasmid DNA was degraded in the presence of serum after 5 h incubation with FCS (data not shown). In contrast, recovered DNA from nanoparticles remained intact. 3.4. In vitro cytotoxicity and cell transfection assay According to MTT results, MPG did not induce any cytotoxic effects up to a concentration of 30 ␮M (42 ␮g) over a period of 48 h. Our results showed that MPG is not cytotoxic at the concentrations used for gene delivery, but actually the combination of MPG with DNA has been shown to reduce cytotoxicity of MPG at higher concentrations (Fig. S1). The GFP expression efficiency induced by MPG/DNA nanoparticle was investigated at an N/P ratio of 10:1 against the COS-7 cell line for 8 h with plasmid DNA encoding E7-GFP. The results were compared with expression efficiency induced by naked plasmid DNA and PEI/DNA at an N/P ratio of 7. Incubation with naked plasmid DNA (control) showed no fluorescence at 24 and 48 h after transfecting cells. Our results indicated that MPG/DNA nanoparticles facilitate uptake of DNA by cells, which leads to the protein expression. The transfection efficiency of the E7-GFP gene using

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Fig. 2. Transfection efficiency of pEGFP-E7 using MPG-based nanoparticles at an N/P ratio of 10:1 in the COS-7 cells after 48 h: The expression of E7-GFP using GFP reporter was monitored with Epi-fluorescent microscopy (A) and flow cytometry (B). E7-GFP expression in the COS-7 cells was 66% at an N/P ratio of 10:1 compared with the negative control. Western blot analysis (C) using an anti-E7 monoclonal antibody showed expression of the full-length E7-GFP protein (50 kDa) in transfected cells by PEI (N/P = 7) and MPG-based nanoparticles (Lane 2 and 3), respectively, as compared to the untransfected cells (Lane 1). MW is the molecular weight marker (14.4–97.4 kDa, Fermentase).

MPG is shown in Fig. 2A. In addition, we examined the efficiency of gene transfer mediated by MPG-based nanoparticles using flow cytometry and western blot analysis. As shown in Fig. 2B, strong E7GFP expression was detected in approximately 66% of COS-7 cells treated with an N/P ratio of 10:1. The transfection efficiency of E7 gene using PEI 25 kDa as a positive control was 71.5% for COS-7 cells comparable with MPG-based nanoparticles (data not shown). E7 expression was detectable in COS-7 cells transfected with either PEI/DNA or MPG/DNA nanoparticles at an N/P ratio of 10:1 with western blotting at 48 h after transfection. The dominant bands of 50 kDa (i.e., 23 kDa E7 + 27 kDa GFP) were detected in transfected cells expressing E7-GFP using the anti-HPV16 E7 monoclonal antibody (Fig. 2C). The corresponding bands were not detected in the untransfected cells, indicating that expression of the E7 protein was specifically induced in the transfected cells. 3.5. Antibody responses As shown in Fig. 3A, the anti-E7 IgG1 level in mice that received the nanoparticle (GP4 and GP5) was significantly higher than those in group GP2 immunized with pcDNA-E7 (p < 0.01), before challenge. The evaluation of IgG2a indicated that there was no significant response against E7-antigen and MPG peptide, before and after TC-1 challenge. 3.6. Cytokine profile In the preventive study, immunization with MPG/DNA nanoparticles effectively enhanced the IFN-␥ level compared to the GP2 group immunized with pcDNA-E7 alone (GP4 vs. GP2: p < 0.001, GP5 vs. GP2: p < 0.05, Fig. 3B). The groups vaccinated with MPG/DNA nanoparticles showed higher IL-4 responses than control groups (p < 0.05) (Data not shown). In the therapeutic study, mice vaccination with the MPG/DNA nanoparticles after the TC-1 challenge resulted in a strong IFN-␥ response, similar to that observed in the preventive study (GT5 vs. GT2: p < 0.001 and GT4 vs. GT2: p < 0.01). Notably, the MPG/DNA nanoparticles at an N/P ratio of 10:1 (G4) induced IFN-␥ production more potently than an N/P ratio of 5:1 (G5) in both the preventive and therapeutic study (GP4 vs. GP5: p < 0.01 and GT4 vs. GT5:

p < 0.001). In the therapeutic study, there was no significant difference in IL-4 level between different groups (data not shown). The level of IFN-␥ and IL-4 with respect to the MPG peptide was not detectable in all the groups (data not shown). Interestingly, the IFN-␥/IL-4 ratio in GP4 (mice immunized with MPG/E7DNA nanoparticles at an N/P ratio of 10:1) was about 2-fold more than that in GP2 receiving pcDNA-E7 in the preventive study (Table 2). This ratio was much higher (>5-fold) in GT4 compared to GT2 in the therapeutic test. 3.7. MPG-based nanovaccine enhances mice protection against E7-expressing tumor cell In order to study the potency of the nanovaccine, tumor growth was measured in different groups. As shown in Fig. 4A, there was a significant decrease in tumor growth induced by DNA immunization with MPG/DNA nanoparticles in comparison with E7 DNA immunization (GP4 vs. GP2: p < 0.01 and GP5 vs. GP2: p < 0.05 on day 55 post-challenge). Immunization with MPG/DNA nanoparticles at an N/P ratio of 10:1 (GP4) protected all mice from tumor growth. In contrast, all mice in GP1, GP2, and GP3 developed tumor growth on approximately days 21, 35, and 25, respectively. As indicated in Fig. 4B, mice immunized with MPG/E7 DNA nanoparticles demonstrated a significant difference in tumor-free percentage compared to E7 DNA alone (100% and 80% survival in GP4 and GP5, respectively). 3.8. Treatment with MPG-based nanoparticles effectively inhibited the tumor growth in mice Mice with pre-established E7-expressing tumors were treated with MPG/DNA-nanoparticles or naked pcDNA-E7 one week after tumor cell injection. Tumor size and animal survival were Table 2 The ratio of IFN-␥/IL-4 in the preventive and therapeutic studies. Groups

G1

G2

G3

G4

G5

Preventive study Therapeutic study

0.06 0.3

3.8 4.84

1.35 0.8

8 27

4.5 21.7

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Fig. 3. (A) IgG1 antibody isotype induced by immunization with pcDNA-E7 with or without MPG peptide in the preventive experiment. The results from the 1:50 dilution are shown as mean absorbance at 492 nm ± SD. (B) IFN-␥ levels in preventive and therapeutic studies. Different groups were immunized with various formulations. Pooled splenocyte cultures were prepared and restimulated with rE7 in vitro. The levels of IFN-␥ were determined in supernatant with ELISA. The graph represents the levels of cytokine in E7-stimulated splenocytes. All analyses were performed in duplicate for each sample. Significant difference is indicated by * p < 0.05, ** p < 0.01 and *** p < 0.001.

monitored for 70 days following the challenge. As shown in Fig. 4C, mice in GT4 displayed complete regression and remained tumorfree >70 days following treatment. Mice in GT5 demonstrated significant tumor reduction following TC-1 challenge in comparison with mice in GT2 which received E7DNA alone [GT5 vs. GT2: p < 0.01]. As demonstrated in Fig. 4D, tumor-free curves displayed that at 70 days post-challenge, all mice in GT4 remained free of tumors (100% survival). The tumor growth of mice in GT5 delayed until on day 56, and the tumor-free percentage of mice in GT5 was significantly higher than the other groups (75% survival). All animals in other groups (GT1, GT2, and GT3) developed tumors by 45 days after injection. Mice were euthanized when tumor diameter exceeded >5% of body weight. 4. Discussion The main criteria for an effective DNA vaccine is the delivery of genetic materials into target cells through the plasma and nuclear membranes in order to reach the transcription machinery of antigen-presenting cells, followed by processing, presentation and induction of an immune response [4]. CPPs represent promising non-viral delivery vectors in preference to the polymer and lipidbased DNA delivery systems, because they are relatively stable, easy to synthesize and functionalize, and less toxic or immunogenic than other vectors [9]. Amongst them, there are several properties of MPG that made it ideal for use in gene delivery. One of the foremost requirements for a non-viral gene delivery vector is the ability to interact and form compacted small size particles with DNA. This property was demonstrated in MPG when it was used to complex with pcDNA-E7 and pEGFP-E7 to form particles of a spherical nature with a size range of 150 nm to 1 ␮m in diameter at different

N/P ratios. Furthermore, the condensation of DNA with MPG peptide protects DNA during formulation and preserves its structure in serum. This study indicates that the MPG/DNA nanoparticles were stable in transfection media containing serum and overcame the intracellular barriers, which led to significant E7 expression in transfected cells. One of the unique and essential features of MPG in comparison with other non-viral delivery systems is the presence of a nuclear localization signal (NLS) which plays a crucial role in both electrostatic interactions with DNA and nuclear uptake [21]. Western blot and flow cytometry analysis revealed that the E7 protein expression was detected following MPG-mediated transfection. It was reported that the cellular uptake, antigen processing and the presentation by antigen-presenting cells depend on particle characteristics, such as size and surface charge [22,23]. Cationic particles are particularly effective for uptake by DCs and macrophages. As observed in this study, MPG/E7DNA nanoparticles with a positive surface charge enhanced the antigen expression, resulting in the strong immune effects. The results indicated that there is strong IgG1 response against rE7 in groups 4 and 5 in comparison with the control groups. This result confirmed that MPG/E7DNA could enhance humoral immune responses in vivo. A similar result was obtained in our previous studies. It was indicated that co-administration of naked E7DNA with heat shock protein (Gp96) or cationic polymer–peptide (PEI600-Tat) as a DNA immunization generates IgG1 as the predominant isotype of antibody [20,24]. Interestingly, in this study, it also becomes evident that the size of the particles influences the immune responses induced as well. It has been reported that microparticles promote humoral immune responses, whereas nanoparticles may favor the induction of

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Fig. 4. Preventive study against TC-1 tumor cells in mice immunized with different DNA vaccine formulations: Five groups of female C57BL/6 mice (6 mice/group) were vaccinated two times with a three-week interval. The mice were challenged with 1 × 105 TC-1 in the right flank three weeks later. (A) Tumor volumes were measured twice a week. (B) The percentage of tumor-free mice over time in various groups. In vivo tumor treatment experiment: C57BL/6 mice (6 mice/group) were subcutaneously challenged with 1 × 105 TC-1 cells/mouse. One and two weeks later, the mice were treated with the various regimens of DNA vaccines. (C) Tumor volume was measured twice a week using a caliper. (D) Tumor-free percentage mice over time in various groups.

cellular immune responses [4,25,26]. Our results in agreement with what reported before showed that microparticles at N/P ratio of 5:1 (1 ␮m) were elicited higher antibody titers in comparison to nanoparticles at an N/P ratio of 10:1 (200 nm). The anti-tumor activity induced by the microparticles at an N/P ratio of 5:1 was weaker than that induced by the nanoparticles at an N/P ratio of 10:1, but much stronger than that induced by the naked DNA vaccine. Analysis of E7-specific, cell-mediated immune responses revealed that the IFN-␥ level in the supernatant of splenocytes from mice injected with MPG/E7DNA nanoparticles at an N/P ratio of 10:1 (group 4) was significantly higher than the other groups (p < 0.05). The level of IFN-␥/IL-4 showed that MPG-based nanovaccine was 4-fold to 5-fold more efficient than E7DNA vaccine in the therapeutic study. Our results demonstrated that the immune response elicited by MPG/DNA nanoparticles was a dominant Th1 response denoted by the production of IFN-␥. In our previously published data for improving DNA vaccine potency, it was indicated that codelivery of naked E7DNA with heat shock protein (Gp96) or cationic polymer–peptide (PEI600-Tat) induced Th1 response [20,24]. In this study, a similar result was obtained using MPG peptide as a novel delivery system. Importantly, it should be noted that in this approach mice were treated subcutaneously with 10 ␮g of E7DNA in a nanovaccine formulation, achieving significant production of IFN-␥ and complete inhibition of tumor growth. In contrast, in our previous studies similar levels of IFN-␥ were obtained for 50 and 100 ␮g of E7DNA plasmid complexed with PEI600-Tat and Gp96, respectively [20,24].

Subcutaneous injection of MPG/E7DNA nanoparticles was shown to potently inhibit the TC-1 tumor growth during the 70day period, but tumor volume increased in mice immunized with E7DNA. These results showed the potency of CPPs in enhancing DNA delivery, processing and efficacy in both prophylactic and therapeutic preclinical tumor models. Other studies showed that vaccination with HSV1VP22 linked to HPV 16 E7 (VP22/E7) induced Th1 response and stimulated antitumor immunity against the E7expressing tumor model [27]. The advantage of our study is that we have used a non-covalent approach through electrostatic interaction between a cationic peptide and E7DNA to enhance the potency of a DNA vaccine. Altogether, our data showed that immunization of mice with MPG/E7DNA nanoparticles obviously alters the E7 recall responses from IL-4 to IFN-␥ production, suggesting a shift toward Th1 responses and an increase in the protective potency of the nanovaccine in comparison with E7DNA alone. 5. Conclusion Internalization of DNA into live cells using CPPs is a practical and efficient approach for gene therapy through the formation of noncovalent complexes. In the present study, we have implemented MPG for the delivery of HPV16E7 as a tumor antigen in vitro and in vivo. The efficiency of transfection mediated by MPG was comparable with PEI 25 kDa, with some preferences for MPG-based nanoparticles including no cytotoxicity to mammalian cells and

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no sensitivity to serum. These nanoparticles with a lower dose of DNA plasmid enhanced the uptake of the E7DNA and consequently induced both humoral and cellular immune responses in vaccinated mice. Our data illustrates that E7 in nanoparticle formulation drives T cell responses towards a Th1-type and could completely confer protection against tumor-challenged mice (100% tumor free mice) in a certain ratio. However, further studies are required to determine its mechanism in vitro and in vivo. Acknowledgements Financial support of this work was provided by Research Council of Tarbiat Modares University and Pasteur Institute of Iran. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2015.05. 015 References [1] Chaturvedi AK. Beyond cervical cancer: burden of other HPV-related cancers among men and women. J Adolesc Health 2010;46(4):S20–6. [2] Zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002;2(5):342–50. [3] Rice J, Ottensmeier CH, Stevenson FK. DNA vaccines: precision tools for activating effective immunity against cancer. Nat Rev Cancer 2008;8(2):108–20. [4] Nguyen DN, Green JJ, Chan JM, Langer R, Anderson DG. Polymeric materials for gene delivery and DNA vaccination. Adv Mater 2009;21(8):847–67. [5] Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Ther 2000;7(1):31–4. [6] Brown MD, Schätzlein AG, Uchegbu IF. Gene delivery with synthetic (non viral) carriers. Int J Pharm 2001;229(1):1–21. [7] Hartono SB, Gu W, Kleitz F, Liu J, He L, Middelberg AP, et al. Poly-l-lysine functionalized large pore cubic mesostructured silica nanoparticles as biocompatible carriers for gene delivery. ACS Nano 2012;6(3):2104–17. [8] Mao H-Q, Roy K, Troung-Le VL, Janes KA, Lin KY, Wang Y, et al. Chitosan–DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J Control Release 2001;70(3):399–421. [9] Bolhassani A. Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer. Biochim Biophys Acta Rev Cancer 2011;1816:232–46. [10] Morris MC, Deshayes S, Heitz F, Divita G. Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol Cell 2008;100(4):201–17.

[11] Laufer S, Restle T. Peptide-mediated cellular delivery of oligonucleotide-based therapeutics in vitro: quantitative evaluation of overall efficacy employing easy to handle reporter systems. Curr Pharm Des 2008;14(34):3637. [12] Morris M, Vidal P, Chaloin L, Heitz F, Divita G. A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res 1997;25(14):2730–6. [13] Morris MC, Chaloin L, Méry J, Heitz F, Divita G. A novel potent strategy for gene delivery using a single peptide vector as a carrier. Nucleic Acids Res 1999;27(17):3510–7. [14] Deshayes S, Morris M, Heitz F, Divita G. Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy. Adv Drug Delivery Rev 2008;60(4–5):537–47. [15] Lee SH, Kim SH, Park TG. Intracellular siRNA delivery system using polyelectrolyte complex micelles prepared from VEGF siRNA-PEG conjugate and cationic fusogenic peptide. Biochem Biophys Res Commun 2007;357(2):511–6. [16] Tang Q, Cao B, Wu H, Cheng G. Cholesterol-peptide hybrids to form liposomelike vesicles for gene delivery. PLoS ONE 2013;8(1):e54460. ´ et al. Stability [17] Moret I, Esteban Peris J, Guillem VM, Benet M, Revert F, DasıF, of PEI–DNA and DOTAP–DNA complexes: effect of alkaline pH, heparin and serum. J Control Release 2001;76(1):169–81. [18] Sadeghian F, Hosseinkhani S, Alizadeh A, Hatefi A. Design, engineering and preparation of a multi-domain fusion vector for gene delivery. Int J Pharm 2012;427(2):393–9. [19] Li Y, Subjeck J, Yang G, Repasky E, Wang X-Y. Generation of anti-tumor immunity using mammalian heat shock protein 70 DNA vaccines for cancer immunotherapy. Vaccine 2006;24(25):5360–70. [20] Bolhassani A, Zahedifard F, Taghikhani M, Rafati S. Enhanced immunogenicity of HPV16E7 accompanied by Gp96 as an adjuvant in two vaccination strategies. Vaccine 2008;26(26):3362–70. [21] Simeoni F, Morris MC, Heitz F, Divita G. Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Res 2003;31(11):2717–24. [22] Hamdy S, Haddadi A, Hung RW, Lavasanifar A. Targeting dendritic cells with nano-particulate PLGA cancer vaccine formulations. Adv Drug Deliv Rev 2011;63:943–55. [23] Silva JM, Videira M, Gaspar R, Préat V, Florindo HF. Immune system targeting by biodegradable nanoparticles for cancer vaccines. J Controlled Release 2013;168(2):179–99. [24] Bolhassani A, Ghasemi N, Servis C, Taghikhani M, Rafati S. The efficiency of a novel delivery system (PEI600-Tat) in development of potent DNA vaccine using HPV16 E7 as a model antigen. Drug Delivery 2009;16(4): 196–204. [25] Oyewumi MO, Kumar A, Cui Z. Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses. Expert Rev Vaccines 2010;9(9):1095–107. [26] Kanchan V, Panda AK. Interactions of antigen-loaded polylactide particles with macrophages and their correlation with the immune response. Biomaterials 2007;28(35):5344–57. [27] Hung C-F, Cheng W-F, Chai C-Y, Hsu K-F, He L, Ling M, et al. Improving vaccine potency through intercellular spreading and enhanced MHC class I presentation of antigen. J Immunol 2001;166(9):5733–40.