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Silica Nanoparticle as a Lymph Node Targeting Platform for Vaccine Delivery Myunggi An,† Meng Li,† Jingchao Xi,† and Haipeng Liu*,†,‡,§ †
Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, Michigan 48202, United States Department of Oncology, Wayne State University, Detroit, Michigan 48201, United States § Tumor Biology and Microenvironment Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201, United States ‡
S Supporting Information *
ABSTRACT: Nanoparticles have emerged as the platform of choice to improve the efficacy and safety of subunit vaccines. A major challenge underlying the use of nanomaterials in vaccines lies in the particle designs that can efficiently target and activate the antigen-presenting cells, especially dendritic cells. Here we show a toll-like receptor 9 (TLR-9) agonist and antigen coloaded, silica nanoparticles (SiNPs) are able to accumulate in antigen presenting cells in the draining lymph nodes after injection. Vaccine loaded SiNPs led to dramatically enhanced induction of antigen-specific B and T cell responses as compared to soluble vaccines, which in turn drove a protective antitumoral immunity in a murine tumor model. Additionally, SiNP vaccines greatly reduced the production of systemic proinflammatory cytokines and completely abrogated splenomegaly, key systemic toxicities of TLR-9 agonists that limit their advances in clinical applications. Our results demonstrate that structureoptimized silica nanocarriers can be used as an effective and safe platform for targeted delivery of subunit vaccines. KEYWORDS: silica nanoparticle, electrostatic binding, vaccine delivery, lymph node targeting, cancer
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INTRODUCTION Vaccination aims to elicit the host immune responses by administration of antigenic materials. It represents the single most effective medical intervention in modern medicine.1−3 Traditionally, attenuated live or killed whole pathogens are administrated into the human body to trigger protective immune responses without causing illness. Recent advances in vaccinology have led to the development of more defined synthetic subunit antigens (proteins, peptides, lipids, or nucleic acids) to improve vaccine stability, safety, tolerability, and reduce cost.4 More importantly, subunit vaccines are able to elicit selective immunity to particular antigens, avoiding activation of unrelated immunity.5 Unfortunately, subunit vaccines are usually poorly immunogenic and require coadministration of adjuvants and/or delivery carrier to generate an effective immune response. Another challenge in subunit vaccine is that, while induction of IgG response has been mostly successful for prophylactic vaccines, fewer studies have demonstrated the induction of antigen-specific CD4+ and CD8+ T cell responses, which are particularly important for therapeutic settings such as chronic infectious diseases and cancer.6,7 The induction of antigen-specific B cell and T cell immunity occurs exclusively in secondary lymphoid organs, especially in the lymph nodes (LNs).7,8 Vaccines fail to reach the LNs are largely ignored by the immune system, leading to unresponsiveness. However, failure to confine vaccines to the LNs often © XXXX American Chemical Society
leads to unacceptable systemic side-effects. Thus, strategies that target vaccines to the LNs are critical for improving both the efficacy and safety of vaccines. Parentally injected vaccines are transported to LNs by two distinct pathways: vaccines can drain to LNs along with interstitial flow (passive targeting); alternatively, vaccines can be taken up and carried to the LNs by migratory dendritic cells (active targeting).8 The latter pathway is believed to be less efficient as only a small fraction of the antigens is taken up by DC.9 Synthetic particulate delivery systems combined with immunological cues that mimic various properties of natural pathogens are of particular interest in subunit vaccine applications.10,11 In the past three decades numerous delivery systems based on nano- or microsized particles have been investigated preclinically and technologies have already been introduced to the clinic.12 Particle-based vaccines have significant advantages over their nonformulated counterparts. For example, nanoparticles can protect antigen from degradation, codeliver antigen and costimulatory context signals, provide multivalent stimulation and enhance antigen uptake by targeting the lymphoid organs.10,13−17 Targeting draining LNs by nanoparticles has been shown to produce a significantly improved humoral immune response.18 Further, targeting vaccines to lymphoid tissue residing APCs is also Received: April 29, 2017 Accepted: June 22, 2017 Published: June 22, 2017 A
DOI: 10.1021/acsami.7b06024 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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was determined by 1% agarose gel electrophoresis at 75 V for 30 min in 1× TBE buffer. All the materials showed undetectable endotoxin contamination as measured by a ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript). Size and Zeta Potential Measurements. To measure the size and zeta potential of nanoparticles, dynamic light scattering (DLS, Zetasizer, Malvern) was used with an He−Ne laser (633 nm) at 90° collecting optics at 25 °C. The surface morphology and microstructures were analyzed using a high-resolution transmission electron microscopy (HR-TEM, JEOL 2010). For TEM analysis, microfilms were made by placing a drop of the respective nanoparticle suspensions onto a 200 mesh copper TEM grid (Ted Pella, CA) and then dried at room temperature for 3 h. A minimum of four images for each sample were captured, and representative images were included. In Vitro TLR Activation. HEK-Blue-mTLR9 and RAW-Blue cells were purchased from InvivoGen and were used to evaluate adjuvant activity in vitro. The cells were cultured in RPMI (Thermo Fisher), containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. Inducible SEAP (Secreted Embryonic Alkaline Phosphatase) levels were detected by HEK-Blue detection kit from InvivoGen. Cells were cultured in an incubator at 37 °C and then were transferred into a 96-well plate. Nanoparticles or soluble CpG were added to 2 × 105 cells per well of the 96-well plate and after 24 h incubation, the stimulation of the cells was assessed by measuring the levels of SEAP secretion using QUANTI-Blue quantitatively measured by a spectrophotometer at 650 nm. In Vitro Cellular Uptake. DC 2.4 cells were kindly provided by Dr. Wei (Barbara Ann Karmanos Cancer Institute, Detroit, MI). To determine the intracellular delivery capacity of nanoparticles, the DC2.4 cells were seeded on glass coverslips in 6-well microscopy chamber at a density of 2 × 104 cells per well for 24 h at 37 °C. After 24 h, cells were treated with 10 μg/mL of fluorescence labeled CpG in soluble or nanoparticle form for 2 h, and then washed with saline. For fixation, the glass that cells adhered on was immersed in 4% paraformaldehyde in saline for 10 min at room temperature. Following fixation, the glass was washed with saline and mounted on a slide with nuclei staining by DAPI. Fluorescence images were obtained using a confocal microscope (Zeiss LSM-510) with a filter set of DAPI, FITC, and Alexa Fluor 647 excitation/emission. For the FACS analysis, DC2.4 cells were seeded in 6-well plates at a density of 4.5 × 105 cells/ well in the culture medium. Fluorescently labeled CpG in soluble or nanoparticle form were added to each well and incubated for 0.5 h, 1h, and 2 h, respectively. After being washed with saline, the cells were analyzed using a flow cytometer (Attune Focus). A minimum of 1000 events were collected. Lymph Node Imaging and Cellular Uptake. Groups of C57BL/6 (n = 4 LNs per group) were injected subcutaneously at the tail base with 1.24 nmol of fluorescein-labeled CpG in soluble or in nanoparticle formulations. After 24 h, animals were sacrificed and inguinal and axillary LNs were excised and imaged using In-Vivo Xtreme (Bruker) imaging system. LNs were then digested with 1.5 mL of freshly prepared enzyme mix composed of RPMI-1640 containing 0.8 mg/mL Collagenase/Dispase (Roche Diagnostics) and 0.1 mg/mL DNase (Roche Diagnostics) and LN cells were stained with antibodies against F4/80 and CD11c versus CpG fluorescence in viable cells. Percentages of CpG+ cells in the LNs were determined by flow cytometry. Immunizations. Groups of C57BL/6 mice (n = 3 per group) were immunized by subcutaneous injection on day 0 and day 14 with 5 μg ovalbumin (OVA) plus 0.62 nmol CpG in soluble or in nanoparticlebased adjuvants. Seven days after the final immunization (day 21), mice were bled, and peripheral blood mononuclear cells were evaluated by SIINFEKL/H-2Kb peptide-MHC tetramer staining and intracellular cytokines (IFN-γ and TNF-α) staining. To assess the functionality of primed CD8+ T cells, peripheral blood mononuclear cells were stimulated ex vivo with 10 μg/mL OVA peptide SIINFEKL for 6 h with Brefeldin-A, fixed, permeabilized, stained with anti-IFN-γ, anti-TNF-α, and anti-CD8α, and analyzed by flow cytometry. AntiOVA IgG titers, defined as the dilution of serum at which 450 nm OD
crucial for inducing cytotoxic T lymphocytes (CTLs) response19 as LN CD8+ DCs are the major DCs capable of cross-presentation, a process required for presenting extracellular antigens within MHC class I molecules to CD8+ T cells.20−22 To date, there are considerable efforts in the design of nanoparticles to improve LN accumulation for vaccines. For example, Ilyinskii et al.23 developed a synthetic polymer nanoparticles that enabled antigen and adjuvants (TLR7/8 or TLR9 agonist) codelivery into the draining LNs. This approach resulted in strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. Nanoparticle characteristics such as size, shape, and surface properties can significantly influence their LN drainage in vivo.4,5 Despite intensive research, only a few of the particulate system reach clinical trials and the results are rather disappointing, especially for cancer vaccines.24−26 This highlights the needs for a new system which can efficiently target vaccine components to antigen presenting cells (APCs) and elicit the desired immune responses and minimize side effects. Here we describe a silica nanoparticle (SiNP) delivery system which combines the loading advantages of solid particles and biomimetic structure advantages. We show that cationic silica nanoparticles can efficiently coload negatively charged oligonucleotide adjuvant and Ovalbumin (OVA) antigen through electrostatic interactions, which provide a multivalent presentation that can more strongly engage and enhance immunogenic responses. Immunization with SiNPs potentiates the in vivo generation of antigen-specific cytotoxic T cells and humoral response, which in turn, lead to enhanced antitumor efficacy. LN targeting SiNPs approach also greatly reduces the vaccine induced toxicity by minimizing systemic exposure. Our findings provide a simple, efficient, and safe method to target molecular therapeutics to LN to locally modulate the immune system.
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EXPERIMENTAL SECTION
Materials and Methods. All chemicals were purchased from Sigma-Aldrich unless noted otherwise. CpG oligonucleotides were synthesized using an ABI 394 synthesizer (Applied Biosystems, Inc.) on 1.0 μmol scale. Murine MHC class I tetramers were obtained from MBL international Corporation (Woburn, MA). Antibodies were purchased from eBioscience (San Diego, CA) or BD Bioscience (San Jose, CA). Animals and Cells. Animals were housed in the USDA-inspected WSU Animal Facility under federal, state, local, and NIH guidelines for animal care. Female C57BL/6 mice (6−8 weeks) were obtained from the Jackson Laboratory. EG.7-OVA cells were obtained from American Type Culture Collection (ATCC), RAW-blue and HEK-Blue-mTLR9 reporter cell lines were purchased from invivogen (San Diego, California). Cells were cultured in complete medium (MEM, 10% fetal bovine serum (Greiner Bio-one), 100 U/mL penicillin G sodium and 100 μg/mL streptomycin (Pen/Strep), MEM sodium pyruvate (1 mM), NaH2CO3, MEM vitamins, MEM nonessential amino acids (all from Invitrogen), and 20 μM β-mercaptoethanol (β-ME)). Preparation of Vaccine Loaded SiNPs. Positively charged SiNPs were purchased from Sigma-Aldrich that were initially functionalized by triethoxypropylaminosilane. The size of the purchased SiNPs was about 30 nm and was confirmed by dynamic light scattering (DLS) measurements (Malvern Zetasizer). The SiNP solution was diluted 10 times using saline to decrease the nanoparticle density (0.116 g/mL) and mixed with the desired amount of CpG DNA or CpG DNA along with OVA to prepare CpG-loaded SiNPs or CpG-OVA coloaded SiNPs with different mass ratios (SiNPs/CpG or SiNPs/CpG plus OVA) such as 10, 30, 60, 90, and 120. To facilitate the complexation process, probe-sonication was performed for 1 min with 2/2s on/off working cycle at a power output of 4 J. The CpG DNA condensation B
DOI: 10.1021/acsami.7b06024 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces were determined by an enzyme-linked immunosorbent assay (ELISA) analysis. To determine antibody titers, ELISA plates (eBioscience) were coated with 10 μg/mL of OVA in saline overnight at room temperature. The plates were blocked with 200 μL of 1% bovine serum albumin (BSA) in saline for 1 h. Serum was serially diluted in saline between 1:102 and 1:107, and applied to the plate and incubated for 1 h at room temperature. Peroxidase-conjugated goat antimouse IgG (H+L) (1:5000 in 1% BSA-PBST, 100 μL/well) was then applied for 1 h, and the plates were developed using TMB substrate (100 μL/ well, eBioscience). The reaction was stopped using 50 μL of 1 M sulfuric acid and absorbance values were measured at 450 nm. Interleukin (IL)-6 and IL12 were also analyzed using cytokine-specific ELISA (BD Biosciences) according to the manufacturer’s instructions. Splenomegaly Measurements. Groups of C57BL mice (n = 3 per group) were injected with 1.24 nmol, 2.48 nmol, and 2.48 nmol CpGs in soluble or nanoparticle form subcutaneously at the tail base at day 0, day 2, and day 4, respectively; on day 6, animals were sacrificed and spleens were collected, weighed, and photographed. Splenomegaly was normalized to individual animal’s body weight. Proinflammatory Cytokines Assays. Groups of C57BL mice (n = 3 per group) were administered a single dose (6.2 nmol) of CpGs in soluble or nanoparticle form subcutaneously at the tail base. Serum samples were collected at 2 and 24 h post injection, and cytokines present were quantified using cytokine-specific ELISA (BD Biosciences) according to the manufacturer’s instructions. Tumor Model. EG.7-OVA cells (Mouse thymoma EL4 cells) were purchased from ATCC and 2 × 106 cells were subcutaneously inoculated into the right flank of 5−6-week-old C57BL/6 mice. When the tumor mass became palpable (7−8 mm, typically 5 days later), mice were divided into six treatment groups (n = 6), and the tumorbearing mice were subcutaneously injected with 20 μg OVA plus 1.24 nmol CpG (4 μg) in nanoparticle form. Survival and tumor size were measured everyday using a sliding caliper. Statistical Analysis. Comparisons of mean values of two groups were performed using unpaired Student’s t tests. To analyze the statistical difference between groups, a one-way analysis of variance (ANOVA) with Bonferroni post-test was used. All of the values were expressed as means ± standard deviations. GraphPad Prism software was used for all the statistical analyses. ***P < 0.001, **P < 0.01, *P < 0.05. NS, not significant.
Figure 1. Silica nanoparticles efficiently accumulate in the draining lymph nodes and promote antigen presenting cell uptake after subcutaneous injection. The efficacy of nanoparticles in immunization strongly lies in the ability to traffic into draining LNs and activate the APCs. The antigen and stimulatory molecules coloaded silica nanoparticles can mimic the viral particles and accumulate in the antigen presenting cells in the LNs and can boost the antigen-induced immune responses.
The pathogen mimicking nanoparticles were prepared from cationic silica nanoparticles (SiNPs) (Figure 2a). Cationic SiNPs were selected primarily due to their ability to condense nucleic acids27−31 and more importantly, due to their intrinsic adjuvant activities.32−34 Briefly, amine modified SiNPs (30 nm) were complexed with CpG DNA, a single-stranded synthetic oligodeoxynucleotide with cytosine-phosphate-guanine (CpG) motifs that activate the APCs expressing pathogen recognition receptor Toll-like receptor 9 (TLR-9). We first prepared fluorescein amidite (FAM)-labeled CpG-loaded SiNPs (CS) with mass ratios (SiNPs/CpG) of 10, 30, 60, 90, and 120 to find the optimum CpG DNA loading on SiNPs, focusing on the nanoparticle’s stability. The desired amount of SiNPs were simply added to fluorescence labeled CpG solution and briefly probe-sonicated, following 1 h incubation at room temperature. The 10, 30, 60, and 90 ratio CS showed a precipitate layer on the bottom of 1.5 mL microcentrifuge tubes, emitting green fluorescence under the UV lamp, implying that the interaction between SiNPs and CpG DNA was sufficient to promote significant aggregation of nanoparticles (Figure S1a of the Supporting Information, SI). In contrast, soluble 3′-FAM CpG DNA and 120 ratios CS showed no precipitation and uniform green fluorescence was observed in the supernatant, indicating formation of stabilized NPs. To further confirm the stability of SiNP and CpG complexes, brief centrifugation (5000 rpm for 10 s) was performed for all samples, and 120 ratio CS showed excellent stability (no precipitate layer) that was similar to soluble 3′-FAM CpG DNA (Figure 2b). Measuring the CpG in the supernatant revealed a saturation point at 60 ratios CS (Figures S1b and 2c). At this ratio, SiNPs appeared to be fully covered by CpG DNA, enabling high loading of CpG DNA per nanoparticle. The 120 ratio CS was, however, monodisperse in size (47 nm, Figures S1c) and exhibited excellent colloidal stability, two vital features required for vaccine application. Zeta-potential measurements of these particles showed a
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RESULTS AND DISCUSSION Design of LN-Targeting CpG Loaded SiNPs (CS). Vaccine delivery by nanocarriers could make a big impact on the treatment of infectious diseases as well as on cancer immunotherapy. Properties of nanosized vaccine carrier are highly tunable. For example, the surface of nanocarriers can be easily modified to expose either antigen or adjuvant (or both), allowing codelivery of antigen and immune signal to the same antigen presenting cells. Although vaccine delivery systems via nanoparticles have been gaining momentum in the past decade,9−11 the development of simple and efficient delivery vehicles for in vivo applications, especially for delivery to LNs, has remained a major challenge. The efficacy of nanoparticles in immunization strongly lies in their ability to traffic into draining LNs and activate the APCs. Naturally occurring pathogens, especially virus can induce robust T-helper and CTL immune responses. This is in part because the size and surface features of viral particles allow efficient in vivo LN drainage. We hypothesize that antigen and stimulatory molecules loaded SiNPs can mimic the viral particles and accumulate in the APCs in the LNs (Figure 1). These LN-targeting nanoparticles can subsequently boost the antigen-induced immune responses. Furthermore, we hypothesize that LN-targeting would also confine the vaccine components within the lymphoid organs, minimizing the systemic dissemination and reducing vaccineinduced toxicity. C
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Figure 2. Synthesis and characterization of CpG loaded silica nanoparticles. (a) Illustration of CpG-loaded SiNPs (CS) preparation via electrostatic charge interactions between SiNPs and CpG DNA. (b) Fluorescent image of CS with different mass ratios after brief centrifugation (5000 rpm for 10 s), illuminated with a UV lamp. (c) CpG concentration measured from the supernatant of soluble CpG or CS samples. (d) Zeta potential of CpG and CS with different SiNPs/CpG mass ratios. (e) Expected distance between two CS nanoparticles with low and high mass ratio, and its Debye screening length. (f) Transmission electron microscopy (TEM) images of SiNPs and CS. (g) Mean percentages of CpG+ APCs in inguinal and axillary LNs determined by flow cytometry at 24 h after injection. (h) Fluorescent images and (i) quantifications of lymph node fluorescence in inguinal and axillary nodes at 24 h. ***P < 0.001, **P < 0.01, *P < 0.05. NS, not significant by Students t test (g) or one-way analysis of variance (ANOVA) with Bonferroni post-test (i). Data represent mean ± standard error of the mean (s.e.m.) of 2−3 independent experiments.
progressive decrease in surface potential from 31 mV for bare SiNPs to 4 mV for the 60 ratios CS (Figure 2d). The negatively charged CpG DNA induced the formation of big size CS, making their surface charge negative or near zero. The 120 ratio CS covered partially by CpG DNA has the positively charged surface (15 mV) and creates an electrostatic field that affects the ions in the bulk of the liquid, creating an electric double layer. Thus, particles tend to segregate into a layer adjacent to the layer of surface charges in SiNPs, showing better colloidal stability (Figure 2e). Since the 10, 30, 60, and 90 ratios CS gave large aggregates, they are not suitable for vaccine delivery, the 120 ratios CS was selected as a nanoparticle delivery platform of CpG DNA for further vaccine studies. The surface morphology and microstructures of nanoparticles were analyzed using HR-TEM (Figure 2f). The CS showed no difference in morphology compared to bare SiNPs. The formation of CpG-SiNPs complex was confirmed by inhibition of CpG migration in gel electrophoresis (Figures S1d). Despite a positive charged surface, CS are also relatively stable in the presence of serum protein, showing no sign of aggregation and releasing only 18.7% of the encapsulated CpG DNA when incubated in the presence of 10% serum at 37 °C for 3 days (Figures S1e). This result implies that nanoparticles play roles in not only retention of CpG DNA via electrostatic binding but also sustainable release of CpG DNA over time. SiNPs Enhances Cellular Uptake and Immune Stimulation of CpG in Vitro. The uptake of vaccine components
by immune cells is crucial in antigen-processing and immune activation. Nanomaterials are known to promote the uptake by a wide variety of immune cells, including macrophage and DCs. To determine the efficiency of cellular uptake of SiNPs, we incubated DC2.4 cells with CS, using unformulated CpG DNA as control. Confocal microscopic and flow cytometry analyses showed that the uptake of free CpG DNA in DC2.4 cells was detectable but low (Figure S2a,b), suggesting the unformulated CpG was unable to translocate across the cell plasma membrane efficiently. By contrast, CS greatly enhance the cellular uptake of CpG as demonstrated by the significantly stronger fluorescent intensity in the cells treated with CS. Two hours after incubation, CpG DNA was observed in the confined intracellular area, most likely in the endosomes, where TLR-9 is located (Figure S2a). These results suggested that CpG DNA in the nanoparticle form allowed more efficient uptake with minimal leakage into the culture medium. Despite their differences in surface charge and molecular pattern, CS showed extensive intracellular uptake by DC2.4 cells. Although distinct mechanisms might be involved,35−38 the particulate nature of these CpG containing SiNPs may facilitate their uptake by dendritic cells.11 SiNPs have been extensively used in the biomedical field over the past two decades due to their biocompatibility, and it is expected that SiNPs can act as an antigen and adjuvant delivery vehicle and an immune potentiator.39−41 To evaluate the adjuvant activity of the CpG/SiNP complex, mouse TLR9 D
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Figure 3. Co-delivery of CpG DNA and OVA antigen with SiNPs. (a) Illustration of CpG and OVA coloaded SiNP (COS) preparation. (b) Image showing COS with increasing mass ratios of SiNPs/OVA plus CpG after brief centrifugation. (c) Percentage of loading of OVA and CpG with increasing mass ratios. (d) The size and zeta potential of CS and COS prepared by 120 mass ratios. Groups of C57BL/6 mice were injected with different formulations of fluorescent-labeled CpG plus OVA and draining lymph nodes (LNs) were analyzed, showing (e) mean percentages and (f) representative flow cytometry plots of CpG+ or OVA+ among CD11c+ DCs or F4/80+ macrophages. (g) Immunohistochemistry of inguinal LNs at 24 h (CpG, green; OVA, red) after injection of CpG and OVA in soluble or nanoparticle forms. (h) Representative confocal microscopy images of DC2.4 cells incubated with CpG and OVA in soluble or nanoparticle formulations. Cell nuclei were stained with DAPI (blue) and fluorescentlabeled CpG (green) and OVA (red) were used to detect CpG and OVA uptake. ***P < 0.001, **P < 0.01, *P < 0.05. NS, not significant by Students t test.
(mTLR9) reporter cells that express TLR9 and a NF-κBinducible SEAP reporter gene were incubated with CpG and CS, and the levels of NF-κB-induced SEAP were determined in 24 h. CS stimulated higher levels of SEAP than soluble CpG DNA, suggesting that the complexation between CpG and SiNPs does not compromise the immunostimulatory activities of CpG DNA, but instead enhances the level of stimulation (Figure S2c). This observation is consistent with previous findings, where CpG oligonucleotide−gold nanoparticles shown enhanced activities in vitro.41 To test whether SiNPs possess intrinsic adjuvant property, SiNPs were incubated with RAW-Blue mouse macrophage reporter cells, which secrete SEAP upon TLR, NOD, or Dectin-1 stimulation. Bare SiNPs induced a dose-dependent SEAP secretion (Figure S2d), suggesting that SiNPs possess intrinsic adjuvant properties. Thus, in vitro, SiNP formulated CpG augment the immune cell activation by a combination of enhancement in cellular uptake and intrinsic adjuvant activities exerted by SiNPs. Nanoparticle Formulation Targets CpG to the Draining Lymph Nodes. To test whether nanoparticles lead to
efficient transport of CpG DNA to draining LNs, we assessed LN accumulation of CpG DNA following subcutaneous injection. CpG and CS were injected subcutaneously into C57BL/6 mice (n = 4 LNs per group), and 24 h later, inguinal and axillary LNs were excised and analyzed by flow cytometry. Draining LNs increased in size in mice following injection with CpG loaded nanoparticles compared to those in mice injected with soluble CpG DNA or saline (Figure S3a,b). By flow cytometry, uptake of CS in the draining LNs was the highest in APCs, with the majority of CpG accumulated in CD11c+ DCs and F4/80+ macrophages (Figures 2g and S3c). Surprisingly, despite the positive surface charge, 120 ratio CS also exhibited high LN accumulation after injection. We suspected the in situ complexation between CS and albumin protein from interstitial fluids alleviates the nonspecific interaction with local tissue and thus, facilitates the LN drainage. To test this hypothesis, CS was incubated with albumin protein or serum. CS showed an increased size in the presence of albumin but were otherwise stable without forming aggregation (Figure S4). Nevertheless, CpG delivered via SiNPs promotes LN accumulation, as E
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Figure 4. SiNP formulations trigger potent antigen-specific immune responses. Groups of C57BL/6 mice (n = 3 per group) were immunized subcutaneously on day 0 and day 14 with 5 μg OVA and 0.62 nmol CpG (4 μg), in different formulations. (a) Mean percentages of OVA-specific CD8+ T cell. (b) Representative flow cytometry plots of OVA-specific CD8+ T cells. Peripheral blood mononuclear cells were restimulated ex vivo with CD8+ or CD4+ OVA epitopes and analyzed by flow cytometry for intracellular cytokine staining, showing (c) mean percentages of TNF-α+ or IFN-γ+ CD8+ T cell frequencies. (d) Mean percentages of IFN-γ+ CD4+ T cell frequencies. (e) Levels of anti-OVA IgG in mice sera. C57BL/6 mice (n = 6 per group) were inoculated with 2 × 106 EG.7-OVA cells subcutaneously on day 0 and then vaccinated with 5 μg OVA and 0.62 nmol nanoparticle-based CpG formulation on days 5, 11, and 18. (f) Tumor sizes over time. (g) Kaplan−Meier survival curves. (h) Image represent the progression in tumor volume on day 15. (i) Individual tumor growth curves. ***P < 0.001, **P < 0.01, *P < 0.05. NS, not significant by one-way analysis of variance (ANOVA) with Bonferroni post-test. Data represent mean ± standard error of the mean (s.e.m.) of 2−3 independent experiments.
supported by fluorescent images of intact inguinal and axillary nodes (Figure 2h) and LN fluorescence of inguinal and axillary nodes at 24 h post injection (Figure 2i). SiNP Formulation Efficiently Targets Adjuvant and Antigen to the Draining Lymph Nodes. Co-delivery of antigens with appropriate adjuvants is often required to amplify the antigen-specific responses and alleviate side effects.42−44 To prepare the antigen and CpG adjuvant coloaded SiNPs, ovalbumin (OVA), an antigen which is widely used for the characterization of immune responses was chosen as a model antigen (Figure 3a). The desired amount of SiNPs was added to OVA and CpG mixed phosphate-buffered saline (PBS) solution, prepared at a pH 7 where CpG and OVA net charges were negative (−12.8 mV and −17.4 mV, Figure S5a). With increasing mass ratio of SiNPs/OVA plus CpG (Figure 3b), the percentages of both OVA and CpG loading ratio was increased and reached up to 99% at the 120 mass ratios (Figures S5b and 3c). However, vaccine loaded SiNPs with lower mass ratios exhibited poor colloid stability (Figure 3b), as these nanoparticles quickly precipitated after brief centrifugation. In
contrast, nanoparticles with 120 mass ratios showed no signs of precipitation after long time storage at 4 °C. We therefore choose 120 mass ratios to design efficient LNs targeting nanoparticles. The CpG and OVA coloaded SiNPs (COS) showed a mean 77 nm diameters, which are slightly bigger than CS (Figure 3d). The COS showed similar stability with a little bit larger size against the CS when they were incubated with albumin protein or serum (Figure S4). The COS were also relatively stable in the presence of serum protein, releasing only 8.3% of loaded OVA from the surface of NPs when incubated in the presence of 10% serum at 37 °C for 3 days (Figure S1e). This OVA kinetic profile was relatively slower than that of CpG probably due to stronger charge interactions between OVA and SiNPs than those between CpG and SiNPs (Figure S5a). The lymph node draining after s.c. injection of the above COS was determined by flow cytometry (Figure 3e,f), the soluble CpG and OVA group exhibited less accumulation of CpG and OVA in CD11c+ DCs than that of COS, reflecting the enhanced draining ability of encapsulated CpG and OVA via the form of nanoparticles into draining LNs. Histological sections of F
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Figure 5. LN targeting reduces systemic toxicity of CpG. C57BL/6 mice (n = 3 per group) were injected with 1.24 nmol CpGs subcutaneously on day 0 and 2.48 nmol CpG on days 2 and 4. On day 6 mice were euthanized, and splenomegaly and LNs were isolated and photographed with a digital camera. The (a) image and (b) size of excised splenomegaly. BW, body weight. The (c) image and (d) size of excised inguinal and axillary LNs. The size of splenomegaly and LNs were determined by image J. (e) Serum cytokines after a single injection (n = 3 per group) of 6.2 nmol CpG. ***P < 0.001, **P < 0.01, *P < 0.05. NS, not significant by one-way analysis of variance (ANOVA) with Bonferroni post-test. Data represent mean ± standard error of the mean (s.e.m.) of 2−3 independent experiments.
observation is consistent with our in vitro adjuvant activity study, where bare SiNPs exhibit a dose-dependent activation of RAW-Blue cells. The administered OS was prepared at pH 7 with 99% of OVA encapsulation efficiency (Figure S6). Mice immunized with COS particles generated 8-fold higher OVAspecific CD8+ T cell proliferation than free CpG plus OVA (Figure 4a,b). Intracellular cytokine staining was also carried out at 7 days after boost on peripheral blood mononuclear cells restimulated with SIINFEKL peptide or OVA protein to identify cytokine-producing antigen-specific T cells. COS vaccines induced 3-fold and 3.5-fold greater total cytokine+ CD8+ and CD4+ T cell frequencies, respectively, compared with vaccination with soluble CpG DNA adjuvant (Figure 4c,d). These results imply that COS generate a higher frequency of CD8+ T cells producing both IFN-γ and TNFα, which are critical for innate and adaptive immunity against viral infections.47 Additionally, we measured the level of OVAspecific IgG in the sera of immunized mice to assess the formulation’s capacity for generating humoral immunity. Measurement of the IgG level gives insight into the types of T helper cell immune responses. Sera from immunized mice were collected on day 21 following a prime on day 0 and boost on day 14. ELISA measurements of serum titers of OVAspecific IgG showed the higher levels of anti-OVA IgG in mice immunized by nanoparticle delivery (Figure 4e). Together, these data demonstrated that SiNPs greatly enhance antigeninduced humoral and cellular immunities in mice.
draining LN from mice injected with soluble CpG DNA and OVA showed undetectable CpG and OVA uptakes, whereas LN from mice injected with nanoparticles represented both accumulated CpG and OVA in the subcapsular sinus and interfollicular areas (Figure 3g). Confocal microscopic analyses exhibited that the uptake of soluble CpG DNA and OVA in DC2.4 cells was low (Figure 3h). By contrast, cells treated with COS showed overlapped green and red fluorescence, suggesting that CpG and OVA were incorporated concurrently as expected endocytosis in the DCs. SiNPs Formulations Trigger Potent Antigen-Specific Immune Responses. CpG DNA has been reported to activate cells expressing TLR9 receptor and to promote expression of costimulatory molecules from APCs that can activate T-cell responses.45,46 To first measure the impact of nanoparticle delivery on T cell priming, C57BL/6 mice were immunized subcutaneously on day 0 and day 14 with 5 μg OVA antigen alone or adjuvanted by SiNPs, 0.62 nmol CpG DNA (4 μg) in soluble or in nanoparticle form. The cellular immunity was monitored by measuring OVA-specific CD8+ T-cell proliferation in peripheral blood using SIINFEKL/H-2Kb peptide-MHC tetramers. As expected, soluble CpG DNA induced weak antigen-specific CD8+ T cell responses at a level that was at or barely above background. OVA-loaded SiNPs (OS) immunized mice showed slightly increased frequency of OVA-specific CTLs without CpG (Figure 4a,b), suggesting SiNPs themselves can act as immune-potentiators. This G
DOI: 10.1021/acsami.7b06024 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces SiNPs Vaccination Elicit a Protective Antitumor Immune Response. Several studies have previously shown that in the EG.7-OVA murine lymphoma model, CpG DNA vaccination resulted in significant regression of tumor growth.48−54 Therefore, we next evaluated the antitumor activities of CpG DNA in nanoparticle forms by therapeutically vaccinating C57BL/6 mice bearing OVA-expressing EG.7 thymoma tumors. A total of 2 × 106 EG.7-OVA cells were subcutaneously inoculated into mice. When the tumor mass became palpable (7−8 mm), mice were vaccinated on day 5, and 11 with 20 μg OVA plus 1.24 nmol CpG DNA in nanoparticle forms or controls. The administration of COS resulted in similar tumor growth initially as no treatment group but triggered sustained regression over time (Figure 4f−i). The corresponding survival rate also supports the potential utility of our nanoparticle strategy for therapeutic cancer vaccines (Figure 4g). These data demonstrated that the nanoparticle formulation was more effective than soluble vaccine in stimulating antigen-specific cytotoxic T cells that can lead to tumor regression. SiNPs Formulations Dramatically Reduce CpG-Induced Toxicity. Subcutaneous injection of unformulated CpG DNA diffuses into blood circulation.19,55,56 Systemic exposure of CpG DNA is known to generate nonspecific immune activation, characterized by lymphocyte proliferation in the spleen and release of proinflammatory cytokines.19,55,56 Our previous data demonstrated CpG formulated with SiNPs enhanced lymph node accumulation. Target CpG into lymph nodes might also reduce its systemic exposure. To exam whether nanoparticle formulation could reduce systemic toxicity, mice were repetitively injected with CpG, or nanoparticle CpG, and systemic immune activation was assessed. Remarkably, although mice injected with free CpG induced severe splenomegaly, in SiNP formulated CpG injected animals, spleens were essentially the same as PBS-injected animals (Figure 5a,b). In addition, the size of draining LNs implicated that the local activity of inguinal LNs was markedly increased by nanoparticle formulation as proven by their size increase (Figure 5c,d). However, it was found that the size difference in axillary nodes between PBS and NPs groups was not significant. We suspect part of the reason that NPs following subcutaneous injections moved from the injection site through the inguinal nodes first which was closely located in the injection site and finally into the axillary lymph nodes. Additionally, free CpG, but not SiNP formulated CpG strongly increased serum levels of proinflammatory cytokines IL6, IL12, and TNF-α (Figure 5e). These data suggest SiNPs formulation protects mice from generalized and unspecific immune activation, thus lowers the systemic toxicity of CpG adjuvant. Altogether, the results presented here demonstrated the use of silica nanoparticles as a general strategy to target LN and to enhance the potency and safety of vaccines, an approach that may also be applicable to other immunomodulatory therapeutics.
approach provides rapid and simple guidelines to fabricate effective vaccine carriers and an understanding of how the electrostatic charge interaction affects their vaccine loading, size, and surface charge. The optimum mass ratio between SiNPs and CpG DNA or CpG DNA and OVA was explored, showing their high colloidal stability and efficient CpG DNA and OVA loading with minimal leakage into the medium. Immunization by nanoparticle delivery generated potent cellular and humoral immunity superior to vaccination by soluble CpG DNA and OVA. Nanoparticle delivery acts synergistically in suppressing tumor growth, outperforming soluble vaccine in an animal tumor model. Silica nanoparticles especially mesoporous silica have been extensively studied for vaccine delivery.58−60 These nanoparticles are self-adjuvanting and elicit antigen-specific cellular and humoral immune responses following subcutaneous injections.58 However, whether these SiNPs can target LN resident antigen presenting cells have not been established. The current study uses a surface-loaded amorphous silica NPs for lymph node targeting. Silica nanoparticles revealed in this study can be used as efficient carriers to target CpG DNA adjuvants and OVA antigen to draining LNs, thereby modulating the immune system in safer and effective ways. We expect that the results of our work will contribute to the advancement of vaccine loading via electrostatic charge interactions and will help to develop more efficient therapeutics for treating cancer.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b06024. Figure S1. Preparation and characterization of CpG adjuvant loaded SiNPs (CS); Figure S2. In vitro cellular uptake and immunostimulatory function of SiNPs.; Figure S3. SiNPs enhances CpG uptake in the draining lymph nodes; Figure S4. Nanoparticle size temporal stability in vitro; Figure S5. SiNPs efficiently condense both CpG and OVA; and Figure S6. Preparation of OVA-loaded SiNPs (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (H.L.). ORCID
Haipeng Liu: 0000-0002-4267-237X Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported in part by American Cancer Society (11-053-01-IRG).
CONCLUSIONS We have designed and characterized a system for vaccine delivery that utilizes several advantages of nanoparticles: antigen and adjuvant coloading, efficient lymphatic drainage, and versatile packaging of immunostimulatory adjuvants via strong electrostatic surface charge interactions. We used SiNPs as a platform for the efficient delivery of CpG DNA and OVA into dLNs for ensuring their immunostimulatory activity. This H
ABBREVIATIONS USED SiNPs, silica nanoparticles CS, CpG-loaded silica nanoparticles COS, CpG and OVA coloaded silica nanoparticles OVA, ovalbumin DOI: 10.1021/acsami.7b06024 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
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DC, dendritic cell TLR, toll-like receptor LN, lymph node CTL, cytotoxic T lymphocyte APC, antigen presenting cell IL6, interleukin 6 IL12, interleukin 12 IFN-γ, interferon γ TNF-α, tumor necrosis factor α SEAP, secreted embryonic alkaline phosphatase DLS, dynamic light scattering
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