Iron Oxide Nanoparticles-Based Vaccine Delivery for Cancer Treatment

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Iron Oxide Nanoparticles-Based Vaccine Delivery for Cancer Treatment Yi Zhao, Xiaotian Zhao, Yuan Cheng, Xiaoshuang Guo, and Weien Yuan* School of Pharmacy, Shanghai Jiao Tong University, No. 800 Dongchuan Road, Shanghai 200240, China ABSTRACT: Modern therapeutic cancer vaccines need simple and effective formulations to enhance both humoral and cellular immune responses. Nanoparticles have obtained more and more attention in the development of vaccine delivery platforms. Moreover, nanoparticles-based vaccine delivery platform has high potential for improving the immunogenicity of vaccine. The Food and Drug Administration (FDA) has approved many types of iron oxide nanoparticles for clinical use, such as treating iron deficiency, contrast agents for magnetic resonance imaging (MRI) and drug delivery platforms. In this study, we explored a novel combined use of iron oxide nanoparticles (superparamagnetic Fe3O4 nanoparticles) as a vaccine delivery platform and immune potentiator, and investigated how this formulation affected cytokine expression in macrophages and dendritic cells (DCs) in vitro and tumor growth in vivo. Comparing with soluble OVA alone and iron oxide nanoparticles alone, we found significant differences in immune responses and tumor inhibition induced by OVA formulated with iron oxide nanoparticles. Our iron oxide nanoparticles greatly promoted the activation of immune cells and cytokine production, inducing potent humoral and cellular immune responses. These results suggest that this nanoparticle-based delivery system has strong potential to be utilized as a general platform for cancer vaccines. KEYWORDS: iron oxide nanoparticles, adjuvant, vaccine, cancer, vaccine delivery

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mers, (e.g., PGA, PLGA, PLA12−15), inorganic particles,16 immune-stimulating complexes (ISCOMs),17,18 and chitosan.19−21 Among all the nanoparticles used as vaccine delivery platforms, few studies have been performed to exploit solid inorganic nanoparticles, though they have been tested to be safe for drug delivery.16 Some inorganic nanoparticles such as Au, C, Ag, SiO2, and Pt have been studied as adjuvants. Gold nanoparticles (AuGNPs) are of interest for the usage as multivalent antigen carrier scaffoldings22−24 because of their good biocompatibility and lack of immunogenicity.25,26 AuGNPs can be easily fabricated from H[AuCl4] in water with controllable particle sizes (ranging from 1 to 100 nm).27−30 There are studies that utilized AuGNPs for the delivery of viruses antigens for influenza31 and foot-and-mouth disease.32 Moreover, AuGNPs can act as a DNA vaccine adjuvant for human immunodeficiency virus (HIV).33 In addition, some very simple tumor vaccine modalities were invented by exploiting the intrinsic biophysiochemical properties of graphene oxide (GO). The prepared GO vaccines have ultrahigh antigen loading efficiency and can self-produce cytokines and reserve antigen. Moreover, GO can also induce autophagy, leading to more immune cell activation and efficient tumor inhibition.34

INTRODUCTION With the development of the modern vaccine, there is a trend that vaccine contains less and less immunological components, thus leading to the need of highly effective adjuvants and efficient delivery systems.1 Although a handful of vaccine adjuvants have been used in the clinic,2,3 the big gap between the increasing demand for novel adjuvants use in vaccines and the development of effective adjuvants cannot be ignored. It is well-known that adjuvant can influence immune responses,2 and there is no adjuvant formulation fitting for every vaccine. Therefore, exploring novel and alternative strategies can enlarge the set of vaccine adjuvants and vaccine delivery systems. Nowadays, nanotechnology gets more and more attention in the development of vaccines. Nanotechnology offers the opportunity to design different nanoparticles easily, which varying in shape, size, surface properties, and compositions. Nanoparticles, owing to their size similarity to cellular compositions, can enter cells by cellular endocytosis mechanisms, especially pinocytosis,4 so they can be applied to the field of medicine.5,6 Their large surface areas enable multiple antigens displayed on the surface, which lead to high affinity with immune cells through multivalent interactions, therefore enhancing the immune responses.7 Nanoparticles in vaccine formulations cannot only enhance the immunogenicity and stability of antigens, but they can also achieve targeting delivery and delayed release. It has been proved that particles can increase immune responses (e.g., IgE levels) both in animal and human models.8,9 The following types of nanoparticles have been explored in vaccine formulations: virus-like particles (VLPs),10,11 synthetic poly© XXXX American Chemical Society

Received: December 8, 2017 Revised: February 10, 2018 Accepted: March 16, 2018

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DOI: 10.1021/acs.molpharmaceut.7b01103 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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from Servicebio (Wuhan, China), were maintained in MEM Alpha Modification (Thermo-Fisher Scientific), supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1× nonessential amino acids, 50 μm 2mercaptoethanol, and 100 U/mL penicillin/streptomycin. The murine RAW264.7 macrophage cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and grown in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 4.5 g/L glucose, 10 mM HEPES, 50 μM 2mercaptoethanol, and 100 U/mL penicillin/streptomycin. BALB/c mice (male, 6-week old, weight 20 ± 2 g) were purchased from the Institute of Zoology, Chinese Academy of Sciences (Beijing, China). All animals were housed under specific pathogen free (SPF) conditions in the laboratory animal facility in the School of Pharmacy, Shanghai Jiao Tong University. The animals were maintained at a relative humidity of 50 ± 20%, noise level under 60 dB, 12-h light/dark cycle, and at the temperature range from 20 to 28 °C. All animal experiments were approved by the Regulations for the Administration of Affair Concerning Laboratory Animals for Shanghai Jiao Tong University, the National Institutes of Health Guide for Care and Use of Laboratory Animals (GB14925-2010), and the Regulations for the Administration of Affairs Concerning Experimental Animals (China, 2014). Preparation of Fe3O4−OVA Nanoparticles. The preparation method was referred to the previously reported method.52 Fe3O4−OVA nanoparticles were prepared in various ratios. Superparamagnetic Fe3O4 nanoparticles with diameters of 28 nm were diluted with deionized water into stock solutions. OVA was diluted into different concentrations. Fe3O4−OVA conjugation was simply obtained by incubating 900 μL of Fe stock solutions with 100 μL of different concentration OVA solutions overnight at 4 °C with mild vortexing. Characterization of Nanoparticles. The particle sizes of different ratios of Fe3O4−OVA nanoparticles were measured by particle-size analyzer (Brookhaven Instruments) at 25 °C.The respective zeta potential of Fe3O4−OVA nanoparticles was determined by dynamic light scattering (DLS) using a Zetasizer Nano range system (Malvern, Worcestershire, U.K.) at the ambient temperature. In Vitro Cytotoxicity. In vitro cytotoxicity of Fe3O4−OVA nanoparticles against CT26 cells was measured by Cell Counting Kit-8 (CCK8) reagent. Briefly, CT26 cells were plated into 96-well plates at 1 × 104 cells per well and incubated for 24 h. Then, cells were treated with PBS, Fe 3 O 4 nanoparticles (70, 140, and 1400 μg/mL), and Fe3O4−OVA nanoparticles (1.5, 5, 15, 25, and 50 μg/mL OVA) for 24 h. At last, 10 μL CCK8 reagent was added into each well and incubated for additional 2 h. After that, to eliminate the background absorbance caused by cellular Fe3O4−OVA nanoparticles uptake, culture supernatant was transferred to a clean 96-well plate. Their absorbance at 450 nm was measured by the multifunctional microplate reader (SpectraMax M3Multi-Mode Microplate Reader) and reference at 630 nm. PBS solution was considered as the control. The blank control group was considered as 100% cell viability. Cell viability was calculated with the formula below:

Iron nanoparticles are one of the most important classes of inorganic nanoparticles, especially superparamagnetic Fe3O4 nanoparticles. They are highly biocompatible, and the Food and Drug Administration (FDA) has approved ferumoxytol (one of Fe3O4 nanoparticles) as an iron supplement. Other iron nanoparticles can be used as contrast agents for MRI and drug carriers in preclinical and clinical settings.35−39 Owing to these characteristics, iron nanoparticles make in vivo monitoring and tracking much easier, which enables us to study the mechanism of immune activation.40,41 The utilization of nanoparticles in tumor therapy is a powerful technology, because tumorassociated macrophages in tumor microenvironment can internalize systemically or locally administered nanoparticles. Diagnostic nanoparticles can be used to image tumor responses of immune-modulating cancer therapies.35,38,42 Conjugated with drugs, nanoparticles can shuttle therapeutic drugs to tumors.43,44 Recently, Heike’s group45 revealed the intrinsic therapeutic effect of the iron oxide nanoparticles on tumors. This finding showed that tumor cells treated with iron oxide nanoparticles exhibited the much slower growth rate compared with tumor cells without addition of iron oxide nanoparticles. Usually, there are two types of nanoparticle-adjuvant vaccine formulations. The first type is using nanoparticles as a delivery system. Nanoparticles can deliver antigen to immune system, either being congested by the immune cell or releasing antigen at the target location.46 For this type, the association of antigen and nanoparticles is necessary. The other type is using nanoparticles as immune potentiator to enhance antigen processing. In this research, we explored a novel combined use of iron oxide nanoparticles (superparamagnetic Fe3O4 nanoparticles) as vaccine delivery platform and immune potentiator. We hypothesize that using iron oxide nanoparticles as an adjuvant of cancer vaccine will significantly activate the immune system and thus decrease the growth rate of the tumor. On one hand, iron oxide nanoparticles can act as a delivery carrier since the native antigens are naturally displayed on a particle surface. On the other hand, the naked iron oxide nanoparticles can act as immune potentiator as previous studies revealed. There is many researches that showed the enhancement of antigen delivery efficiency to antigen presenting cells by incorporating antigens into nanoparticles, which further induced immune cells maturation and cross-presentation of antigens to activate a strong immune response.47−51 In this research, we will describe the characterization, in vitro cytotoxicity, in vitro cellular uptake, assessment of immune responses, and antitumor efficacy of Fe3O4−OVA nanoparticle vaccines.

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MATERIALS AND METHODS Ovalbumin EndoFit was obtained from InvivoGen (San Diego, CA). Mouse IL-2 ELISA kit, Mouse IL-6 ELISA kit, Mouse TNF-α ELISA kit, and Mouse IFN-γ ELISA kit were purchased from Dakewe Biotech (Shenzhen, China). Superparamagnetic Fe3O4 nanoparticles were purchased from Meilunbio (Dalian, China). Animals and Cells. The mouse colon adenocarcinoma (CT26) cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and grown in RPMI1640 medium (Thermo-Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Gibco, CA), 1 mM sodium pyruvate, 2 mM L-glutamine, 4.5 g/L glucose, 10 mM HEPES, 50 μM 2-mercaptoethanol, and 100 U/mL penicillin/ streptomycin. The DC2.4 murine dendritic cells, purchased B

DOI: 10.1021/acs.molpharmaceut.7b01103 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 1. Physical characterization of Fe3O4−OVA nanoparticles. (A) Particle size and (B) zeta potential of Fe3O4−OVA nanoparticles of different OVA concentrations at 37 °C measured in PBS. Data are shown as mean ± SD (n = 3). (C) TEM images of Fe3O4−OVA nanoparticles with 1.5 μL/ mg OVA. (D) CD spectrometer detection of solutions of OVA (D1) and Fe3O4−OVA nanoparticles (D2).

mice were randomly divided into 4 groups (6 mice per group): saline, Fe3O4 nanoparticles, soluble OVA (10 μg OVA/mouse), Fe3O4−OVA nanoparticles (containing 15 mg OVA/mouse). For all groups, 0.1 mL solutions were injected intratumorally to the mice. In the subcutaneous tumor model, BALB/c mice were immunized with Fe3O4−OVA nanoparticles (containing 15 mg OVA/mouse) three times (every 7 days as a cycle), and saline, soluble OVA (15 mg/mouse), and empty Fe3O4 nanoparticles were utilized as controls. Tumor growth was measured by a vernier caliper every 3 days, and the volume of tumors was calculated using the formula: Volume = 0.5 × length × width2. On the 22nd day, the mice were executed by cervical dislocation and their sera were extracted from the eyes, and their tumors and organs were harvested for weight determination and imaging. Statistical Analysis. All data were representative of at least three independent measurements and presented as mean ± standard deviation (S.D.). The statistical significance of differences was tested by Student’s unpaired t test or one-way ANOVA (Graph Pad Prism 5 software), and a value for *P < 0.05, **P < 0.01, or ***P < 0.001 was considered as statistically significant.

cell viability =

sample group(OD450) − sample group(OD630) blank group(OD450) − blank group(OD630) × 100%

In Vitro Cellular Uptake Studies. To further assess the cellular uptake of Fe3O4−OVA particles, CT26 cells were first seeded into a 6-well plate (1 × 106 cells/well) and cultured for 24 h. Cells were then treated with Fe3O4−OVA nanoparticles (1.5, 5, 15 μg/mL OVA) for 24 h. Before cells were harvested, using PBS washed for three times to clean the residue of media. Using cell scraper to remove the cells, and the amount of Fe3O4 within cells was measured by ICP after acid digestion using boiling HNO3 and aqua regia. In Vitro Effects on DC2.4 Cells and Macrophages Following Fe3O4−OVA Stimulation. To determine if Fe3O4−OVA nanoparticles induced immune response, we chose DC2.4 cells and murine RAW264.7 macrophage cells as in vitro cell models. DC2.4 cells and RAW264.7 macrophage cells were first seeded into 12-well plates and cultured for 24 h. Cells were then treated with PBS, Fe3O4 nanoparticles, soluble OVA (1.5 and 15 μg/mL), Fe3O4−OVA nanoparticles (1.5, 5, and 15 μg/mL OVA), and LPS (10 μg/mL) for 24 h. The cell supernatant was collected for IFN-γ, TNF-α, and IL-6 measurements with cytokine-specific ELISA (enzyme-linked immunosorbent assay) kits according to the manufacturer’s instructions. In Vivo Antitumor Treatment. To test the efficacy of Fe3O4-OVA nanoparticles in tumor treatment, BALB/c mice were first injected with 0.1 mL of CT26 cell suspension (5 × 106/mL) into the right flank region. Then the mice were raised in SPF environment waiting for the volume of tumor to reach 150 mm3 to 200 mm3. When enough mice met the criterion,

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RESULTS Preparation and Characterization of Fe3O4−OVA nanoparticles vaccines. According to the method described before, Fe3O4−OVA nanoparticle vaccines with different OVA concentrations were made. Adsorption of OVA antigen onto a Fe3O4 nanoparticle is generally based on hydrophobic interaction or charge. Incorporation of different concentrations of OVA onto the Fe3O4−OVA nanoparticles had effects on nanoparticle sizes. As shown in Figure 1A, the average particle size of Fe3O4−OVA nanoparticles significantly decreased compared with Fe3O4 nanoparticles, suggesting OVA had C

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Molecular Pharmaceutics conjunct onto Fe3O4 nanoparticles and then broke the magnetic adsorption of Fe3O4, and consequently the size decreased. Among three concentrations, average sizes of Fe3O4−OVA nanoparticles with 1.5 μg/mL were the smallest, suggesting there were more antigen attached. The measured nanoparticle sizes were much larger than expected (single Fe3O4 nanoparticle size is 28 nm), and it was caused by magnetic adsorption of Fe3O4. Zeta potential was unaffected by concentrations of OVA (Figure 1B), and the average zeta potential was −22 mV. Transmission electron micrographs (TEM) of dried Fe3O4−OVA nanoparticles demonstrated a roughly graininess morphology and appeared to have size distributions (200−250 nm) much smaller than those obtained by DLS in the hydrated state (Figure 1C). Circular dichroism (CD) spectrometry detection revealed that conjugation to Fe3O4 nanoparticles did not change the property of OVA (Figure 1D). Since the U.S. FDA has approved iron oxide nanoparticles as iron supplements, the iron oxide nanoparticles vaccine presented a high possibility for safe human use in the future. In Vitro Cytotoxicity and in Vitro Cellular Uptake Studies. Before examining in vitro cellular uptake and maturation, the Cell Counting Kit 8 (CCK-8) assay was used to measure the cytotoxicity of Fe3O4−OVA nanoparticles in the CT26 cell line. The assay showed no signs of toxicity, even at the high concentration of nanoparticles (Figure 2). An ICP

Figure 3. Uptake of Fe3O4−OVA in DCs was measured by ICP (n = 3). Data are shown as mean ± SD (n = 6).

These molecules play important roles in immune systems.53−56 As shown in Figure 4, after incubation with nanoparticles for 24 h, the Fe3O4−OVA nanoparticles greatly enhanced the concentration of secreted TNF-α, IL-6, and IFN-γ in the cell supernatant, and the secretion level was comparative to LPS treatment group (as a positive control). In experiment groups, IFN-γ secretion of the Fe3O4−OVA nanoparticles group was even higher than the LPS group (Figure. 4C). As shown in Figure 4 and 5, the Fe3O4−OVA nanoparticles had been proved and can significantly activate immune response compared with free OVA and Fe3O4 nanoparticles (P < 0.05). These cytokines are recognized as Th-1 bias immune cytokines and can help induce Th-1 bias polarization of macrophages and naive T cells. Therefore, Fe3O4−OVA nanoparticles had the ability to induce Th-1 biased cytokine secretion from macrophages and DC2.4 cells, but free Fe3O4 nanoparticles and soluble OVA could not (P < 0.05). In Vivo Antitumor Studies. To assess the therapeutic capability of Fe3O4−OVA nanoparticles against tumors in tumor-bearing mice, we investigated their antitumor efficacy in subcutaneous tumor models with intratumoral injection. For tumor implantation, 0.1 mL of CT26 cell suspension (5 × 106/ mL) was injected into the right flank region of BALB/c mice. When the volume of tumor reached 150 mm3 to 200 mm3, BALB/c mice were immunized with different formulations, including PBS, soluble OVA, Fe3O4 nanoparticles, Fe3O4−OVA nanoparticles. The whole immune therapy was administrated every 7 days for three times (Figure 6A). Saline, soluble OVA (15 mg/mouse), empty Fe3O4−OVA nanoparticles were utilized as controls. As shown in the tendency chart (Figure 6D), tumor growth was remarkably inhibited in mice immunized with Fe3O4−OVA nanoparticles, and single Fe3O4 nanoparticle group slightly retarded tumor growth, whereas soluble OVA did not inhibit the growth of tumors, which had a size similar to that in the PBS control group. On the 22nd day, subcutaneous tumors were peeled off and the photo demonstrated that mice immunized with Fe3O4−OVA nanoparticles had much smaller sizes (P < 0.05) of the tumor masses compared with mice of the other groups (Figure 6B). The subcutaneous tumors were weighed (Figure 6C), the Fe3O4− OVA nanoparticle treatment group had much lighter weight compared with mice without treatment (P < 0.01). Throughout the animal study, no visible inflammatory responses had been seen at the injection sites. To further study the potential immune activation nature of the Fe3O4−

Figure 2. Cell viability (%) of CT26 cells treated by Fe3O4−OVA nanoparticles and single Fe3O4 nanoparticles at different concentrations. Data are shown as mean ± SD (n = 6).

mass analysis for Fe in cell digestion solutions revealed that DC2.4 cell line had high efficiency of uptake of Fe3O4−OVA nanoparticles (Figure 3). Fe3O4−OVA nanoparticles with 1.5 μg/mL OVA had the maximum efficiency of uptake by DCs, and it has a statistical significance of differences with the other two (P < 0.01) and may be caused by its lowest size, revealing DCs uptake bias. A similar trend of size-dependent uptake was also reported for murine macrophage-like cells with GNPs.52 In Vitro Effects on Macrophages and DC2.4 Cells Following Fe3O4−OVA Nanoparticles Stimulation. The immunostimulatory effect of Fe3O4−OVA nanoparticles on immune cells was investigated after treating murine RAW264.7 macrophages and DC2.4 cells with Fe3O4−OVA nanoparticles and three control treatments. After 24 h of incubation with Fe3O4−OVA nanoparticles or empty NPs, free OVA, or saline only as controls, cell culture supernatants were collected. Then, enzyme linked immunosorbent assays were used to evaluate the expression levels of pro-inflammatory cytokines, such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). D

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Figure 4. Effects on macrophages following Fe3O4−OVA nanoparticle stimulation: TNF-α (A), IL-6 (B), and IFN-γ (C) production in response to Fe3O4−OVA nanoparticles were assessed by an ELISA kit. Saline = negative control, LPS = positive control.

Figure 5. Effects on DC2.4 cells following Fe3O4−OVA nanoparticle stimulation. IL-6 (A), TNF-α (B), and IFN-γ (C) production in response to Fe3O4−OVA nanoparticles were assessed by an ELISA kit. Saline = negative control, LPS = positive control.

Figure 6. (A) Schematic illustration of the experimental protocol and tumor challenge. (B) Photograph of tumors in CT26 subcutaneous tumor mice after in vivo immunization. (C) End point tumors were weighted. (D)Tumor volume change in CT26 subcutaneous tumor mice were monitored and analyzed using one-way ANOVA. Results were presented as mean ± SD (n = 6), P < 0.05.

significantly increased (P < 0.01). Other pro-inflammatory mediators, such as IFN-γ detected from Fe3O4−OVA injected mice or control mice did not have much difference (Figure 7). The in vivo cytotoxicity of the Fe3O4−OVA nanovaccine was shown in Figure 8. Compared with the control group, these results suggested our vaccine had relatively good tissue compatibility against main organs after intratumoral injection.

OVA vaccine formulations, a panel of pro-inflammatory cytokines and mediators were screened by an ELISA assay on serum. As shown in Figure 7, in sera from mice injected with Fe3O4−OVA nanovaccine, comparable high levels of IL-6 and TNF-α were detected, which were the major pro-inflammatory cytokines responsible for early immune responses. Compared with sera from control group mice, the concentration level E

DOI: 10.1021/acs.molpharmaceut.7b01103 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 7. Secretion levels of TNF-α (A), IFN-γ (B), and IL-6 (C) treated with saline, single Fe3O4 nanoparticles, soluble OVA, and Fe3O4−OVA nanoparticles from immunized BALB/c mice.

ible materials, especially superparamagnetic Fe3O4 nanoparticles, which have been approved by the FDA for internal use in human as iron supplements. Therefore, like other nanoparticles, iron nanoparticles are potentially acceptable as vaccine components for treating cancer. Previous studies revealed that systemically administered iron nanoparticles were assimilated by the reticuloendothelial system, thus leading to the exposure of immune cells to iron nanoparticles. Although several studies have demonstrated that iron oxide nanoparticles influenced the functionality of macrophages and antigen presenting cells,57−60 evidence pertaining to the effects of iron oxide nanoparticles on tumor inhibition is scarce. In this study, we investigated the in vitro and in vivo effects of iron oxide nanoparticles as the adjuvant of vaccines on macrophages, DC 2.4 cells and the tumor inhibition effect in tumor-bearing mice. The average particle size of Fe3O4−OVA nanoparticles significantly decreased compared with Fe3O4 nanoparticles. It was because OVA had conjunct onto Fe3O4 nanoparticles and then broke the magnetic adsorption of Fe3O4 nanoparticles, and consequently the size decreased. Since the antigen carried the opposite charge to iron oxide nanoparticles, iron oxide nanoparticles would tighten closer by ferromagnetism if there was less antigen attached. This reason also explained why the size measured by DLS was much larger than the TEM showed. Among three concentrations, average sizes of Fe3O4−OVA nanoparticles with 1.5 μg/mL were the smallest, suggesting there were more antigen attached. As is known to all, premature T cells usually need the stimulation of specialized antigen presenting cells (APCs) to turn to matured status. Antigen presenting cells, such as macrophages and dendritic cells, have the ability to activate and maintain both humoral and cellular immune responses; therefore, they are the major targets of iron oxide nanoparticle vaccines. The Fe3O4−OVA nanovaccine had presented a super ability of inducing immune response, for example, Th1 cell responses, Th2 cell responses, and antigen specific CTL responses. There is evidence showing that iron oxide nanoparticles affected the function and apoptosis of macrophages.61−65 Heike’s group45 had demonstrated that iron oxide nanoparticles could induce pro-inflammatory macrophage polarization in tumor sites and consequently inhibit tumor growth. Our data showed that macrophages in vitro induced by Fe3O4−OVA nanoparticles could produce diverse pro-inflammatory cytokines, including TNF-α, IFN-γ, and IL-6. DCs, known as the strongest professional antigen presenting cells, could efficiently ingest, process, and deliver antigen. Immature

Figure 8. Histological sections of organs obtained from saline, single Fe3O4 nanoparticles, soluble OVA, and Fe3O4−OVA nanoparticles treated mice after 23 days. Data are representative of 4 mice.

Together, the data suggested that Fe3O4−OVA nanovaccine formulations induced potent immune responses in vivo and were not harmful to mice.

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DISCUSSION Owing to their special properties, the usage of nanoparticles in vaccine products has been enlarged rapidly in recent years. It is well-known that nanoparticles have similar size to cellular compositions, so nanoparticles can enter cells by the cellular endocytosis mechanism, especially pinocytosis. Moreover, nanoparticles can induce strong allergic responses as vaccine adjuvants and further provide a better method for simplify nanovaccine formulations. In this paper, we developed the method for iron oxide nanoparticles-based vaccine formulations that could inhibit tumor growth. Iron oxide nanoparticles are highly biocompatF

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DCs have potent migration ability, and matured DCs can effectively activate the initial T cells; therefore, they are at the center of initiation, regulation, and maintenance of immune responses. Moreover, they play a vital role in inducing CTL response through cross-presentation. The data showed that DC 2.4 cells activated in vitro by Fe3O4−OVA nanoparticles could secrete multiple pro-inflammatory cytokines, including TNF-α, IFN-γ, and IL-6. As shown in Figure 5, DCs treated with Fe3O4−OVA nanoparticles were able to induce more potent immune responses compared with those treated with soluble OVA antigen and single iron oxide nanoparticles (P < 0.05). Previous studies have established that these cytokines (IFNγ, TNF-α) were significant in the induction of naive T cell differentiation into Th1 cells, and IL-6 is induced by Th2 cells. According to the concentration of these cytokines, our present studies indicated an unequaled sensitivity between Th1 and Th2 cells. With greater production of cytokines, we assumed that Th1 cells were potentially more sensitive to Fe3O4−OVA nanoparticles. Our findings were consistent with a previous research showing that when exposure to iron oxide nanoparticles, antigen-specific IFN-γ and IgG 2a were slightly more sensitive than IgG 1 and IL-4.66 According to this evidence, there was a high possibility that when treated with Fe3O4−OVA nanoparticles, the Th1/Th2 immunobalance may switch toward Th1-dominant immunity; however, this conclusion still needs further investigation. Jin’s group has found that Th1 responses activated cellmediated immunity and primed CTL responses.67 CTLs played a vital role in antitumor immunity because they can specifically target antigen epitopes on the surface of tumor cells and kill them. Hence, the effect of Fe3O4−OVA nanoparticles had profound base. In animal experiments, the data suggested that the prepared Fe3O4−OVA nanoparticles significantly delayed the growth of tumors in mice, which further convinced that our nanoparticle vaccine triggered potent immune responses toward tumor cells.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The study was supported by the Projects of National Science Foundation of China (Grant No. 81570992), SUMHS seed foundation project (Grant No. HMSF-16-21-010), and Science and Technology Development Foundation of Pudong New District, Shanghai, China (Grant Nos. PKJ2016-Y55 and PWZxq2017−03). The study was also partly sponsored by the Interdisciplinary Program of Shanghai Jiao Tong University (Grant Nos. YG2017MS22, YG2015MS06, YG2015QN12, YG2017QN56, and YG2016QN22). We appreciate the help from the faculty of the Instrumental Analysis Center (IAC) of Shanghai Jiao Tong University.

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CONCLUSION In summary, we examined the effect of tumor prevention of iron oxide nanoparticles as vaccine adjuvant. Comparing with soluble OVA and single iron oxide nanoparticles, the results showed that Fe3O4−OVA nanoparticles had significant influence in immunity responses and tumor inhibition. These results indicate that our strategy has strong potential to be utilized as a general platform for cancer vaccines and may also be applicable to other types of vaccine in which strong humoral or cellular immune responses are required or for fast antibody elicitation.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Weien Yuan: 0000-0003-4177-7812 Author Contributions

W. Yuan conceived the initial idea, the conceptualization and the study design, participated in the data extraction and analysis, and revised the manuscript. Y. Zhao, X. Zhao, Y. Cheng, and X. Guo conceived and participated in the study design, searched databases, extracted and assessed studies, and helped draft the manuscript. Y. Zhao wrote the manuscript. All authors have read and approved the final manuscript. G

DOI: 10.1021/acs.molpharmaceut.7b01103 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.molpharmaceut.7b01103 Mol. Pharmaceutics XXXX, XXX, XXX−XXX