Tailored Polymers with Complement Activation Ability To Improve

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

pubs.acs.org/molecularpharmaceutics

Tailored Polymers with Complement Activation Ability To Improve Antitumor Immunity Chenxi Li,† Yue Lu,† Qing Chen,† Haiyang Hu,† Xiuli Zhao,† Mingxi Qiao,*,† and Dawei Chen*,†,‡ †

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China School of Pharmacy, Soochow University, Suzhou 215123, China

‡

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S Supporting Information *

ABSTRACT: The complement system plays an important role in host innate immunity, and its activation can be exploited as a potential strategy for vaccine adjuvants. Herein, a pH-responsive micellar vaccine platform (COOH-NPs) was developed using a carboxyl-modified diblock copolymer of poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) (COOH-PEOzPLA). The copolymer self-assembled into micelles with hydroxyl groups shielding on the surface, which activated the complement system for the enhanced immune responses. Compared with the control nanoparticles (OCH3-NPs), COOH-NPs significantly enhanced lymph node-resident dendritic cell maturation, antigen-specific IgG production, antigen-specific CD4+ and CD8+ T-cell activation, and the amount of memory T-cell generation in vivo. Furthermore, immunization with COOH-NPs/OVA in E.G7-OVA tumor-bearing mice not only remarkably inhibited tumor growth but also prolonged the survival of tumor-bearing mice. These results indicated that COOH-NPs with the capability of complement activation efficiently boosted the immune responses for the antitumor effect. The study demonstrated the significance of taking advantage of a complement-activating vaccine platform for cancer immunotherapy. KEYWORDS: nucleophilic polymers, pH-responsive, complement activation, lymph node, immune responses

1. INTRODUCTION Immunotherapy is a treatment that recognizes and kills tumor cells effectively by activating the patient’s own immune system.1 Immunotherapy has been well recognized as a nextgeneration cancer treatment because of its great advantage over chemotherapy, such as avoiding toxic effects, inhibiting tumor metastasis, and preventing tumor recurrence.1,2 Among numerous immunotherapy strategies, dendritic cell (DC)based nanovaccines have gained great attention because DCs are the most powerful antigen-presenting cells in vivo.2,3 The common approach to develop nanovaccines is to codeliver antigens and immunostimulatory molecules to peripheral DCs for the activation of DCs. The activated DCs migrate to lymph nodes (LNs), present antigens to naive T cells, and stimulate the cytotoxic T lymphocyte (CTL) response to eliminate tumor cells.4−6 However, several flaws with this approach severely compromised the efficacy of vaccines and limited their clinical applications.4 First, the preparation of antigen and adjuvant coincorporated vaccines often requires labor-intensive and sophisticated procedures, in which the epitopes of the antigen may be impaired.7 Second, the unexpected leakage of immune adjuvants in vivo poses a risk of triggering strong systemic toxicity.8 Third, few numbers of DCs reside in peripheral sites and even fewer DCs can migrate to LNs, leading to insufficient T-cell responses.4 Furthermore, © XXXX American Chemical Society

premature antigen presentation (i.e., a migrating DC that expresses an antigen on its surface prior to reaching a LN) may lead to immune cell tolerance.9 Because of these limitations, it is highly demanded to explore new strategies to develop effective DC-based vaccines. LNs are the primary lymphoid organs where strong immune responses are stimulated.4,10,11 Direct delivery of antigens to LNs can effectively prevent premature DC activation during migration.9 Moreover, LNs are resident with a higher number of DCs, which make it easier to achieve efficient antigen uptake.8 Previous literature indicated that effective LN targeting was strongly dependent on the particle size and smaller particles tended to target LNs more efficiently than larger ones.10−12 In addition, the negative charge and hydrophilic surface of vaccine vehicles improved the lymphatic drainage of nanoparticles (NPs) to LNs.10,11,13 To initiate effective immune response, nanovaccines must be able to induce DC maturation besides the efficient LN targeting.6,12,13 DCs are often matured by “danger signals” that worked through pathways of innate immunity, such as the Received: February 14, 2019 Revised: April 13, 2019 Accepted: April 22, 2019

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DOI: 10.1021/acs.molpharmaceut.9b00195 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 1. Schematic illustration of antigen-incorporated micelles activating complement to enhance antitumor immune responses. (A) Schematic illustration of self-assembled COOH-PEOz-PLA micelles and the antigen-incorporated vaccine delivery system. (B) Hypothesis of the immune function of COOH-NPs/OVA in vivo. COOH-NPs/OVA is expected to reach DLNs via lymphatic drainage after administration. The surface carboxylic groups of the NPs activate the complement cascade, generating a “danger signal” in situ, which would promote DC maturation, followed by induction of a strong CTL response against cancer.

significantly promoted the antitumor effects in vivo.10 In this study, we developed a micellar platform for vaccine delivery based on the pH-sensitive amphiphilic diblock copolymer of poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) (PEOz-PLA). PLA is an extensively investigated biodegradable polymer for drug delivery because of its biodegradability and biocompatibility in vivo.30 PEOz has been approved by the US Food and Drug Administration (FDA) as a food additive because of its low toxicity and high hydrophilicity.31 The good self-assembly behavior of PEOz-PLA in water ensured a simple and mild process for the preparation of nanovaccines, preventing the antigens from decomposition or denaturation during preparation. In addition, the tertiary amine groups in the PEOz backbone became protonated under acidic conditions, imparting the micelle payloads with endosomal escape via “proton sponge” effect.32−34 This endosomolytic activity of PEOz greatly facilitated the antigen presentation through the MHC I pathway for the CTL-based cellular immune responses. To endow the nanovaccine with intrinsic immunogenic activity, we further chemically modified the ends of PEOz blocks with carboxylic groups. The carboxylic-substituted PEOz would self-assemble into micelles covered with hydroxyl groups, which possessed the ability to attack the thioester group of C3b for the activation of complement to promote immune responses. Utilizing carriers with intrinsic immunogenicity to boost immunity not only avoids the potential systemic toxicity induced by the application of immune adjuvant but also simplifies the procedure for vaccine manufacture. After subcutaneous injection, this nanovaccine platform, denoted COOH-NPs/OVA, is expected to reach draining LNs (DLNs) through lymphatic drainage and to enhance the antigen accumulation in DLNs as well as the subsequent uptake by LN resident-DCs. Meanwhile, the intrinsic adjuvant properties of COOH-NPs are expected to effectively activate DCs and to present antigen peptides to CD8+ T cells through the MHC I pathway for eliciting a robust CTL response against cancer. The schematic of the nanovaccine is shown in Figure 1. The results of this study will elucidate the significance of NP surface characteristics in inducing the immune responses.

activation of Toll-like receptors (TLRs) and inflammatory cytokine receptors.3,8,14 Codelivery of antigen and adjuvant to one immune cell has been widely applied in the development of various nanovaccines.3,15,16 However, the conjugation or encapsulation of adjuvants into NPs would expose adjuvants to harsh conditions such as acidity, high temperature, water−oil interfaces, and heat, leading to the decomposition of the adjuvants. Moreover, the high cost and the complexity of the manufacturing procedures remained as the limiting issues.8 Some polymers that inherently possess immunostimulatory effects offer an alternative approach to activate DCs.17−19 Complement primarily serves as the first line of the defense system to nonspecifically clear pathogens.8,20,21 It has become clear that complement played an important role in promoting antigen-specific immune responses.22 Synthetic polymers carrying nucleophiles such as −OH or −NH2 are capable of activating complement system and producing a “danger signal” in situ, leading to the DC maturation and induction of enhanced immune responses.15,21,23 Antigen molecules are usually processed through the major histocompatibility complex (MHC) I or II pathways for presentation after internalization.24,25 When the antigens are degraded in the endo-/lysosome compartments, they are processed through the MHC II pathway and are presented to CD4+ T cells, which triggers helper T cells-based humoral immune responses. However, when the antigens are degraded in the cytosol of DCs, they are processed through the MHC I pathway and are presented to CD8+ T cells, which triggers CTL-based cellular immune responses.16,24,26 Because CD8+ CTLs are crucial for eliminating cancer cells, the delivery of antigens to the cytosol for MHC I cross-presentation is preferred for achieving desirable antitumor efficacy.24,27,28 Polymeric micelles self-assembled from amphiphilic polymers have been demonstrated as an effective platform for biomacromolecules’ delivery such as antigens.5,19,29 Zeng et al. prepared a hybrid micelle composed of poly-(ethylene-glycol) phosphorethanolamine/polyethylenimine-stearic acid conjugate for the codelivery of melanoma antigen peptide Trp2 and Toll-like receptor-9 (TLR-9) agonist CpG ODN. The polymeric micelles not only enhanced the encapsulation of the antigen peptide Trp2 and adjuvant CpG ODN but also B


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2. MATERIALS AND METHODS 2.1. Materials. 2-Ethyl-2-oxazoline (EOz), D,L-lactide, stannous octoate, ovalbumin (OVA), OVA 257−264, and OVA323−339 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethyl-3-bromopropionate and KI were obtained from J&K Scientific Ltd. (Beijing, China). Hoechst 33258 and LysoTracker Red were purchased from Invitrogen (Rockford, IL, USA). All fluorescence-conjugated antibodies used in this study (CD3, CD4, CD8, CD44, CD62L, CD11c, CD40, and CD86) and BD Cytofix/Cytoperm Plus (with GolgiPlug) were provided by BD Biosciences (San Jose, CA, USA). All other chemical and buffer solutions were of analytical grade. 2.2. Synthesis and Characterization of COOH-PEOzPLA. The amphiphilic diblock copolymer of COOH-PEOzPLA was synthesized according to a previous publication.35 Briefly, COOH-PEOz-OH was first synthesized by the cationic ring-opening polymerization of EOz using ethyl 3-bromopropionate as the initiator and KI as the catalyst, respectively. The reaction was carried out at 120 °C for 24 h. Then, the mixture was treated with 0.1 N methanolic KOH to introduce hydroxyl groups at the end of the polymer. The product was purified by dialysis with a dialysis membrane (molecular weight cutoff 3500) against Milli-Q water for 2 days and subsequently lyophilized to obtain COOH-PEOz-OH as a white powder. The obtained COOH-PEOz-OH was reacted with D,Llactide in chlorobenzene at 140 °C for 24 h using stannous octoate as the catalyst. The product was purified by precipitation into diethyl ether twice and dried in vacuum overnight. The successful synthesis of COOH-PEOz-OH and COOH-PEOz-PLA was confirmed by 1H NMR. 2.3. Synthesis and Characterization of OVA-ss-PEOzPLA. To enable the covalent conjugation of antigens onto the NPs, a portion of COOH-PEOz-PLA was modified with pyridyl disulfide (PDS) functional groups (PDS-PEOz-PLA), and then the antigen molecules were conjugated onto the surfaces of NPs via a disulfide exchange reaction. The successful conjugation was determined by SDS-PAGE. The loading content was evaluated by the bicinchoninic acid (BCA) protein assay. For the preparation of fluorescein isothiocyanate (FITC)-labeled OVA-ss-PEOz-PLA, OVA was coupled with FITC in NaHCO3 (pH = 9.5) for 48 h prior to conjugation with COOH-PEOz-PLA. 2.4. Preparation and Characterization of COOH-NPs/ OVA. COOH-NPs/OVA was prepared by the thin-film hydration method. COOH-PEOz-PLA and PDS-PEOz-PLA were dissolved in methanol and sonicated for 10 min for complete dissolution. The solvent was evaporated by rotary evaporation at 60 °C to obtain a thin film. Then, the resultant thin film was hydrated with phosphate-buffered saline (PBS) (pH 7.4) for 30 min to obtain a clear solution. The antigen was covalently attached to the surface of the micelle through the disulfide exchange reaction as described above. OCH3-NPs/ OVA was prepared with the same method except COOHPEOz-PLA was replaced by COOCH3-PEOz-PLA. The critical micelle concentration (cmc) value of COOHNPs was determined by fluorescence spectroscopy with pyrene as the fluorescence probe. Briefly, pyrene solution (6.0 × 10−5 M) was dropped into the brown volumetric flask, and the organic solvent was evaporated under nitrogen flow. Copolymer micelle solutions with concentration ranging from 0.5 to 100 μg/mL were added to the flasks. The final concentration of pyrene solution was fixed at 6.0 × 10−7 M. All

solutions were kept in the dark for 24 h prior to measurement. Then, the fluorescence excitation spectra were determined with a fluorescence spectrophotometer (LS55, PerkinElmer, USA). The peak intensities at 334 and 336 nm from the excitation spectra were monitored with the emission wavelength set at 390 nm. The cmc was determined as the intersection of two straight lines drawn through the points of the flat and drastically increasing region from the concentration−intensity ratio (I336/I334) plots. The size and size distribution of the micelles were determined by dynamic light scattering (DLS), and the zeta potential of the micelles was measured in deionized water. All the measurements were performed using Zetasizer (Nano ZS, Malvern Co. Ltd., UK). The morphology of the micelles was observed using transmission electron microscopy (TEM) (TecnaiG220, FEI, USA). 2.5. Generation of Murine Bone Marrow-Derived DCs. Bone marrow-derived DCs (BMDCs) were extracted from C57BL/6 mice as previously reported.36 In brief, femurs and tibiae were collected from C57BL/6 mice. Both ends of the bones were cut off with scissors, and marrow cells were flushed with 1640 media using a 1 mL syringe. The obtained marrow cells were filtered through a nylon screen (70 μm cell strainer) to prepare a single-cell suspension. Then, the marrow cells were centrifuged at 1400 rpm for 4 min, and the red blood cells were lysed with NH4Cl buffer. After being washed with PBS solution, the remaining marrow cells were seeded in a sixwell plate and incubated with DC complete medium [containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 10 ng/mL GM-CSF]. On day 2, the culture medium was completely removed and replaced with fresh medium. On day 4, half of the medium was replaced with an equal volume of DC complete medium. On day 6, the nonadherent and loosely adherent cells (imDCs) were harvested and subjected to flow cytometry analysis for the expression level of CD11c. The results showed that the purity of the DC population was between 80 and 85%. 2.6. Antigen Uptake and Presentation. BMDCs were incubated with FITC-labeled free OVA or a physical mixture of OVA with COOH-NPs (hereafter denoted “OVA + COOHNPs”), OCH3-NPs/OVA, or COOH-NPs/OVA (different formulations all containing 50 μg/mL OVA) in a six-well plate at 37 °C for 3 h. After being washed three times with PBS, the uptake efficiency was determined by measuring FITC-OVA positive cells using flow cytometry. Antigen presentation assay was conducted using CD8-OVA 1.3 T cells. BMDCs were treated with different formulations at 37 °C for 6 h. Then, the cells were washed three times with PBS. The treated BMDCs were subsequently cocultured with CD8-OVA 1.3 T cells in a 96-well plate. After incubation for 24 h, the cell supernatant was collected to measure the level of IL-2 using an ELISA kit according to the manufacturer’s instructions. The intracellular localization of the antigen in BMDCs was detected by confocal laser scanning microscopy (CLSM). After incubation of BMDCs with different formulations for 3 h, the cells were labeled with LysoTracker Red to identify lysosomes, followed by fixation with 4% paraformaldehyde for 15 min. After being washed with cold PBS for 15 min, the cells were stained with Hoechst 33258 to identify the nucleus. The subcellular localization of the nanovaccines was observed by CLSM. C


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Molecular Pharmaceutics 2.7. In Vivo Delivery of Antigens by Nanovaccines into DLNs. To evaluate the targeting ability of nanovaccines to LNs, two FITC-labeled OVA and fluorescent dye Dil coencapsulated micelles (hereafter denoted “COOH-NPs/ FITC-OVA/Dil” and “OCH3-NPs/FITC-OVA/Dil”) and the physical mixture of OVA with Dil (hereafter denoted “FITCOVA + Dil”) were injected into the tail base of mice, respectively. After 3 h, the mice were sacrificed for isolation of the inguinal LNs. The obtained LNs were digested with 2 mg/ mL collagenase IV and 0.1 mg/mL DNase I at 37 °C for 8 h, followed by filtration with a cell strainer (70 μm). The obtained cells were washed with PBS and incubated with Fcblock (anti-CD16/CD32, BD Bioscience) for 15 min at 4 °C. After incubation, the cells were washed again and stained with anti-mouse APC-conjugated CD11c antibody (eBioscience) for 30 min. After another wash, the cells were fixed with BD Cytofix Fixation Buffer (BD Bioscience) at 4 °C for 30 min. Then, the cells were washed again and resuspended in PBS solution for flow cytometry analysis. Samples were analyzed using FlowJo software (Tree Star, Inc.). CD11c was used as the basis for determination of the percentage of double FITC+/Dil+ cells in DCs. The quadrants were set based on the single positive cells. The in vivo distribution of COOH-NPs/FITC-OVA/Dil was monitored using an optical image system (Carestream Health, Inc., USA) at 6 h, 6 d, and 10 d post-injection. 2.8. In Vivo Adjuvanticity of COOH-NPs/OVA. To determine the intrinsic adjuvant properties of nanovaccines, the mice were injected with different formulations in their tail bases. After 24 h, the cells were isolated from inguinal LNs in the same manner as described above. The obtained cells were placed in tubes and first incubated with Fc-block (anti-CD16/ CD32, BD Bioscience) for 20 min at 4 °C. After being washed three times with PBS, the cells were stained with anti-mouse APC-conjugated CD11c antibody, anti-mouse phosphorethanolamine (PE)-conjugated CD40 antibody, or anti-mouse PEconjugated CD86 antibody for 30 min at 4 °C. After being washed another time, the cells were fixed with BD Cytofix Fixation Buffer (BD Bioscience) for 30 min at 4 °C. Then, the cells were washed and suspended in PBS solution for flow cytometry analysis. Samples were analyzed using FlowJo software (Tree Star, Inc.). The cells were gated based on the CD11c-positive cells, and the percentages of cells that showed positive staining for each surface protein were recorded. The amount of C3a released into the supernatant was measured by C3a sandwich ELISA. Mouse serum was incubated with PBS or various formulations at 37 °C for 1 h. Then, the serum samples were added to the anti-mouse C3a monoclonal antibody-precoated 96-round-bottom well plate. After 3 h incubation, anti-C3a-biotinylated antibodies were added to each well, and the plate was incubated for 50 min. Streptavidin-horseradish peroxidase (HRP) and 3,3′,5,5′tetramethylbenzidine substrate solution were then sequentially added. The absorbance at 450 nm was measured using a microplate reader. 2.9. Immunization of Mice. C57BL/6 mice (20 ± 2 g) were supplied by the Department of Experimental Animal Research, Shenyang Pharmaceutical University (Shenyang, China). All animal procedures were performed in accordance with the guidelines evaluated and approved by the Ethics Committee of Shenyang Pharmaceutical University. C57BL/6 mice aged 6−8 weeks were randomly divided into five groups and immunized with PBS, free OVA, or a physical

mixture of OVA + COOH-NPs, OCH3-NPs/OVA, or COOHNPs/OVA (all the formulations contained 50 μg/mL OVA) on days 0, 7, and 14. Subcutaneous immunization was delivered in 200 μL every time. 2.10. Assessment of T-Cell Immunity. To measure the number of IFN-γ+ CD8+ T cells, spleens were excised from the above-mentioned immunized mice 7 days after the last immunization. The organs were mechanically homogenized with the end of sterile syringe and then filtered with a cell strainer (70 μm) to prepare single-cell suspensions. The obtained splenocytes were subsequently incubated with lysis buffer at room temperature for 2 min to lyse the red blood cells. The reaction was quenched with PBS, and then the cells were washed, seeded in 24-well plates, and restimulated with 100 μg/mL OVA257−264 for 72 h. A protein transport inhibitor (GolgiPlug; BD Bioscience) was added to each well for an additional 6 h incubation. The cells were washed with PBS and incubated with Fc-block (anti-CD16/CD32; BD Bioscience) for 15 min at 4 °C. After incubation, the cells were washed and stained with anti-mouse APC-conjugated CD3 and anti-mouse FITC-conjugated CD8 antibody at 4 °C for 30 min. Then, the cells were washed and fixed, followed by permeabilization with BD Cytofix/Cytoperm (BD Bioscience) for 20 min. After being washed with BD Perm/Wash buffer, the cells were incubated with anti-mouse PE-conjugated IFN-γ antibody (BD Bioscience) in permeabilization buffer for 60 min. The cells were washed three times with Perm/Wash buffer and suspended in stain buffer for analysis with flow cytometry. Samples were analyzed using FlowJo software (Tree Star, Inc.). CD3 was used as the basis for determining the percentages of double-positive cells (CD8+ IFN-γ+). To measure the number of IFN-γ+ CD4+ T cells, splenocytes were collected as mentioned above and seeded in a 96-well plate precoated with anti-mouse IFN-γ monoclonal antibody (Dakewe Biotech). After incubation with 100 μg/mL OVA323−339 for 72 h, the culture medium was removed. The cold deionized water was added to each well to lyse the cells. The plates were washed and incubated with biotin-conjugated anti-mouse IFN-γ detection antibody at 37 °C for 1 h. After incubation, the plates were washed again and subsequently incubated with avidin-HRP at 37 °C for 1 h. Then, the AEC peroxidase substrate was added to the plates for 1 h incubation, followed by addition of deionized water to quench the reaction. The plates were dried overnight, and the number of spots was counted by Biosys Bioreader. For quantification of memory T-cell phenotypes, splenocytes were harvested and restimulated with an antigen. The Fc surface receptors were blocked as described above. Then, the cells were stained with anti-mouse PE-Cy7-conjugated CD3, anti-mouse APC-conjugated CD8, anti-mouse APC-conjugated CD4, anti-mouse PE-conjugated CD62L, or antimouse FITC-conjugated CD44 at 4 °C for 30 min. The cells were washed with PBS and subsequently suspended in BD Cytofix Fixation Buffer (BD Bioscience) at 4 °C for 30 min. The cells were washed three times and resuspended in stain buffer for analysis with flow cytometry. Samples were analyzed using FlowJo software (Tree Star, Inc.). The dot plots were gated on T-cell events, and the quadrants were set based on the single positive cells. 2.11. Effectiveness of COOH-NPs/OVA To Induce Humoral Immune Response. Sera from immunized mice were collected 7 days after the last immunization. The level of D


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Figure 2. (A) Particle size distribution and TEM image of COOH-NPs/OVA. (B,C) Fluorescence intensity ratio of the I336/I334 ratio from emission spectra and the log concentration of COOH-NPs/OVA at pH 7.4.

Table 1. Characterization of Antigen-Incorporated Micelles formulation

size (nm)a

PDI

Z-potential (mV)

OVA content (μg/mg)b

cmc (μg/mg)

COOH-NPs/OVA OCH3-NPs/OVA

58.4 ± 1.8 54.5 ± 2.3

0.11 ± 0.02 0.13 ± 0.03

−22.3 ± 2.4 −7.5 ± 1.9

62.7 ± 9.2 64.5 ± 8.9

1.15 2.13

Data are presented as the mean diameter ± SD, n = 3. bIncorporation amount per 1 mg of micelles.

a

by SDS-PAGE. As shown in Figure S3, the antigen remained conjugated on the surface of COOH-NPs after incubation with complete medium containing 10% FBS for 72 h, suggesting the good antigen retaining performance of COOH-NPs. It could be assumed that COOH-NPs were capable of carrying the antigen molecules to the targeting cells without premature release. However, incubation of COOH-NPs/OVA with cytosolic levels of glutathione triggered the burst release of antigen molecules, indicating the redox-responsive properties of COOH-NPs. This tunable release of antigens in response of the redox cytosol was crucial for triggering effective antigen cross-presentation. According to the literature, antigen conjugation to the NPs with a reducible bond (disulfide) showed much more cross-presentation than those conjugated with nonreducible bonds.37 The antigen incorporation content was determined by the BCA protein assay, giving 0.0627 mg of conjugated OVA per milligram of micelles. The particle sizes of COOH-NPs and COOH-NPs/OVA measured by DLS were approximately 55 and 60 nm, respectively (Figures S5 and 2A). These results were consistent with the coating of the micellar surface with OVA because each protein had a diameter of approximately 5 nm.38 The zeta potential of the nanovaccines was −22.3 mV, which could be ascribed to the ionization of the carboxylic group in the micellar shell. The cmc value of the copolymer was measured by fluorescence spectroscopy using pyrene as a hydrophobic fluorescence probe. As shown in Figure 2B, as the copolymer concentration increased, the fluorescence intensity increased. The maximum excitation wavelength of pyrene shifted from 334 to 336 nm. The intensity ratio (I336/I334) of pyrene as a function of the copolymer concentration is shown in Figure 2C. When the copolymer self-assembled into micelles, a remarkable increase in the intensity ratio could be found. The cmc value of

anti-OVA antibody was determined by ELISA kit as previously reported.32 2.12. In Vivo Tumor Challenge. To examine the preventive effect, the C57BL/6 mice were immunized three times with PBS and different formulations at the tail bases. After 7 days post-immunization, 2 × 105 E.G7-OVA cells were injected into the right flanks of the C57BL/6 mice. To assess the therapeutic effect, the mice were inoculated of 2 × 105 E.G7-OVA cells in the right flanks to establish tumor models on day 0. After 5 days, the mice were immunized with PBS and different formulations on days 5, 11, and 17. Tumor volume and body weight were monitored every 3 days. The tumor volume was calculated using the following equation Tumor volume (mm 3) = length × (width2) × 0.5

2.13. Statistical Analysis. All data in this paper are shown as the mean ± SD (n = 3−5). Comparisons of mean values between treatment groups were assessed using one-way ANOVA, followed by Tukey’s post-test, *, p < 0.05; **, p < 0.01; ***, p < 0.001. Statistical analysis was performed using GraphPad Prism software.

3. RESULTS 3.1. Preparation and Characterization of COOH-NPs/ OVA. The carboxylic-decorated amphiphilic pH-sensitive copolymer COOH-PEOz-PLA was synthesized as previously reported in the literature.35 The details of the synthesis and characterization of COOH-PEOz-PLA are shown in the Supporting Information (Figures S1 and S2). A part of COOH-PEOz-PLA was modified with PDS functional groups in order to conjugate the antigens onto the surface of NPs. The stability of the antigen-conjugated COOH-NPs was evaluated E


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Figure 3. COOH-NPs/OVA-enhanced antigen uptake and cross-presentation via the MHC I pathway. (A) BMDCs were incubated with FITClabeled free OVA or a physical mixture of OVA + COOH-NPs, OCH3-NPs/OVA, or COOH-NPs/OVA (all cases containing 50 μg/mL OVA) for 3 h. Antigen uptake was measured by quantifying FITC-OVA positive cells using flow cytometry (MFI = mean fluorescence intensity). (B) BMDCs were incubated with the nanovaccines for 6 h and subsequently cocultured with CD8-OVA1.3 cells for 24 h. The amount of IL-2 released by the CD8-OVA1.3 cells was measured using ELISA kits (data are presented as the mean ± SD, n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001). (C) CLSM images of BMDCs incubated with FITC-labeled nanovaccines for 3 h. Hoechst 33258 was used to stain nuclei, and LysoTracker Red was used to identify the endo-/lysosomes.

NPs by BMDCs was first measured by flow cytometry. As shown in Figure 3A, both free OVA and the physical mixture of OVA + COOH-NPs showed modest internalization in BMDCs after 3 h incubation, suggesting that free antigens were difficult to translocate across the cell plasma membrane efficiently. As expected, COOH-NPs/OVA and OCH3-NPs/ OVA significantly increased the uptake of antigens by BMDCs, indicating that the NPs served as carriers for enhanced internalization of the antigen. A coculture assay was performed to assess the ability of COOH-NPs to activate CD8+ T cells through the MHC I pathway as previously reported.32 As compared to the reference groups (free OVA and OVA + COOH-NPs), COOH-NPs/OVA and OCH3-NPs/OVA dramatically increased the production of IL-2 from CD8-OVA1.3 cells (Figure 3B), suggesting the positive role of NPs in enhancing the antigen uptake and antigen cross-presentation. The subcellular distribution of the different formulations was

COOH-NPs estimated from the plots was 1.15 μg/mL. The relatively low cmc value indicated the good self-assembly of the copolymer and the good stability of the micelles under dilution.32 The particle size variation of COOH-NPs/OVA in 1640 medium containing 10% FBS was further investigated to evaluate the stability of COOH-NPs/OVA in vivo. As shown in Figure S4, the particle size of COOH-NPs/OVA remained almost unchanged for 72 h. The cmc and stability results indicated the excellent stability of COOH-NPs/OVA in the extracellular matrix after subcutaneous administration. The physical characterization results of COOH-NPs/OVA are summarized in Table 1. 3.2. COOH-NPs/OVA Enhances Antigen Uptake and Cross-Presentation via the MHC I Pathway. To elicit potent antitumor immunity, antigen molecules were expected to be efficiently internalized and transported into the cytosol for MHC class I presentation, resulting in the activation of CD8+ T cells.2 The cellular uptake of antigen-incorporated F


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CD11c+ cells confirmed the stability of the micelles in vivo because free OVA and Dil hardly migrated into the LNs. The retention of COOH-NPs/FITC-OVA/Dil in LNs was monitored using an optical imaging system. The fluorescence of Dil could be detected in the inguinal LNs of the mice treated with COOH-NPs/FITC-OVA/Dil until 10 days postinjection, suggesting the long-term retention of nanovaccines in LNs. The long residence of nanovaccines in LNs helped to increase the interaction between antigens and DCs, which was important for eliciting an antigen-specific immune response.40 Additionally, COOH-NPs/FITC-OVA/Dil at the injection site gradually decayed as time elapsed, eliminating the possibility of inducing autoimmune diseases, which was commonly associated with Alum adjuvant.7 3.4. In Vivo Adjuvanticity of COOH-NPs. To evaluate the ability of COOH-NPs/OVA in promotion of DC maturation, free OVA or a physical mixture of OVA + COOH-NPs, OCH3-NPs/OVA, or COOH-NPs/OVA was administered into the mice. The expression of the costimulatory molecules CD40 and CD86 in CD11c+ cells was analyzed by flow cytometry. As shown in Figure 5A−D, COOH-NPs/OVA-treated mice exhibited significantly higher levels of CD86 and CD40 expression than free OVA and the physical mixture of OVA + COOH-NPs-treated mice, demonstrating the more efficient antigen delivery for DC maturation. Compared to free OVA, the physical mixture of OVA + COOH-NPs moderately enhanced the CD40 and CD86 expression, suggesting that the blank COOH-NPs might possess the intrinsic adjuvant properties. The adjuvant effect of COOH-NPs could be further confirmed by comparison of OCH3-NPs/OVA and COOH-NPs/OVA. Although both nanovaccines showed the enhanced antigen accumulation in DLNs, OCH3-NPs/OVA demonstrated much less performance than COOH-NPs/OVA. The complement system consists of over 30 soluble and cellsurface proteins that are present in animal body fluids as well as on cell surfaces. The system can be activated via classical, lectin, and alternative pathways.22 Biomaterials carrying nucleophilic groups, such as hydroxyl (OH) and amino (NH2) groups, are capable of activating the complement system via the alternative pathway.21−23 When the complement system is activated, complement protein C3 is cleaved into C3a and C3b fragments. C3b exposes a highly reactive thioester group because of the conformation change, leading to the formation of a stable C3b−surface complex. Because C3a release is an indicator of the generation of the C3b−surface complex, C3a secretion can be used as an indicator of the complement activation.41 Hence, we incubated mice serum with the nanovaccines to examine whether COOH-NPs could activate the complement system. As compared to the free OVA and OCH3-NPs/OVA, the physical mixture of OVA + COOHNPs and COOH-NPs/OVA showed remarkable production of C3a (Figure 5E), suggesting that COOH-NPs were involved in the activation of the complement system because of the hydroxyl groups on the micelle surface. 3.5. COOH-NPs/OVA Promotes Cellular and Humoral Immunity in Vivo. To investigate whether the activated complement system could improve the T-cell immunity, the elicitation of cytotoxic CD8+ T-cell immune response was assessed. As shown in Figure 6A, COOH-NPs/OVA-treated mice produced higher levels of IFN-γ+ CD8+ T cells than free OVA and the physical mixture of OVA + COOH-NP-treated mice. Furthermore, COOH-NPs/OVA was superior to OCH3-

investigated with CLSM (Figure 3C). The free OVA and the physical mixture of OVA + COOH-NPs-incubated BMDCs revealed weak FITC-OVA fluorescence and the fluorescence was predominantly colocalized with lysosomes, as indicated by the yellow fluorescence in the merged images. This indicated that the OVA was trapped inside lysosomes after cellular uptake. However, the nanovaccine-incubated BMDCs showed clear fluorescent signal of FITC-OVA in the cytosol, indicating the endo-/lysosomal escape of antigens. This could be attributed to the “proton sponge” effect arising from the pHresponsive property of PEOz-PLA.33,34 The amide groups in PEOz become protonated in the acidic endo-/lysosome environment, keep the VATPase (proton pump) functioning, and lead to the influx of one Cl− ion and one water molecule per proton. The influx of Cl− ions and water molecules into the endo-/lysosome causes an increase in osmotic pressure and a tension on the endo-/lysosomal membrane, facilitating the endo-/lysosomal escape of the antigen.39 Therefore, the endosomolytic activity of PEOz played a key role in facilitating antigen presentation by the MHC I pathway.32 3.3. Ability of COOH-NPs/OVA Targeting DLNs. To investigate the in vivo delivery to DLNs, FITC-labeled OVA and fluorescent dye Dil-coincorporated micelles were prepared (COOH-NPs/FITC-OVA/Dil and OCH3-NPs/FITC-OVA/ Dil, Dil was encapsulated into the micelles) and administered by subcutaneous injection through the tail bases of C57BL/6 mice. The physical mixture of free FITC-OVA with Dil (FITCOVA + Dil) was used as control. The uptake efficiency of the nanovaccines by LN-resident DCs was determined by flow cytometry. As shown in Figure 4, COOH-NPs/FITC-OVA/ Dil and OCH3-NPs/FITC-OVA/Dil-treated mice exhibited higher FITC+/Dil+ double signals in CD11c+ cells than the physical mixture of FITC-OVA + Dil, indicating the enhanced draining ability of the nanovaccines. Furthermore, the colocalization of FITC-OVA and fluorescent dye Dil in

Figure 4. COOH-NPs enhance their cargos (FITC-OVA and Dil) into DLNs after subcutaneous injection. Histogram of the % FITC+/ Dil+ in CD11c+ cells. FITC-OVA and Dil-coloaded COOH-NPs, OCH3-NPs, and the physical mixture of FITC-OVA and Dil (FITCOVA + Dil) were administrated into the mice via subcutaneous injection at the tail base site. Inguinal LNs were isolated 3 h postinjection, and the cells positive for FITC+/Dil+ were analyzed using flow cytometry. Cells were first gated on CD11c positive cells, and then the percentages of FITC+/Dil+ double-positive cells were recorded (data are presented as mean ± SD n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001). G


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Figure 5. COOH-NPs/OVA-enhanced DC maturation in vivo. Free OVA or a physical mixture of OVA + COOH-NPs, OCH3-NPs/OVA, or COOH-NPs/OVA was administered into the mice via subcutaneous injection at the tail base site (50 μg/mL OVA in all cases). After 24 injection, inguinal LNs were separated, and the obtained cells were first gated on positive CD11c cells. Then, the expression of positive CD40 or positive CD86 cells was analyzed based on the positive CD11c cells. (A,B) Representative flow cytometry plots of the percentages of CD40+/CD11c+ or CD86+/CD11c+ double-positive cells. (C,D) Histogram of the % CD40+/CD11c+ or CD86+/CD11c+ double-positive cells (data are presented as the mean ± SD n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001). (E) COOH-NPs activated the complement system. Serum from the C57BL/6 mice was incubated with the nanovaccines at 37 °C for 1 h, and the amount of C3a released into the serum was measured by ELISA (data are presented as the mean ± SD n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

NPs/OVA in terms of the IFN-γ+ CD8+ T-cell production, indicating that the intrinsic adjuvant property of COOH-NPs was more effective for induction of immune responses in vivo. Helper CD4+ T-cell response induced by COOH-NPs was also determined because they could augment the activation of cytotoxic CD8+ T cells or B cells by secreting distinct patterns of cytokine.42 Because CD4+ T cells recognized the dominant MHC II-restricted peptide of OVA, the OVA-specific CD4+ Tcell response was determined by restimulation of splenocytes with MHC II-restricted peptide of OVA (OVA323−339).43,44 The IFN-γ production in splenocytes could be attributed to the production by CD4+ T cells.43,44 As shown in Figure 6B,

COOH-NPs/OVA-treated mice produced the highest IFN-γ, suggesting that the LN-targeting, complement-activating nanovaccine COOH-NPs/OVA was able to elicit potent immune responses from both CD4+ and CD8+ T cells in vivo. An ideal vaccine should not only induce cellular immunity to eliminate cancer cells effectively but also generate memory immunity to protect hosts from the reinfection.43,45,46 Effector memory (TEM, CD44hi CD62Llow) and central memory (TCM, CD44hi CD62Lhi) cells are the two main subsets of memory T cells. The former ones are responsible for eliciting immediate effector functions when re-exposed to an antigen, and the latter ones are responsible for the generation of effector cells to H


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Figure 6. COOH-NPs/OVA-enhanced T cell immunity in vivo. Splenocytes were collected on day 21 from the mice immunized with free OVA or a physical mixture of OVA + COOH-NPs, OCH3-NPs/OVA, or COOH-NPs/OVA. (A) Cells were restimulated with OVA257−264 for 3 d, and the percentage of IFN-γ+ CD8+ T cells was measured by flow cytometry. (B) Collected splenocytes were restimulated with OVA323−339 for 3 d because CD4+ T cells recognized MHC II-restricted peptide of OVA (OVA323−339), the IFN-γ production in splenocytes could be attributed to the production by CD4+ T cells. The number of IFN-γ+ CD4+ T cells was determined by the ELISPOT assay. (C,D) Percentages of CD44hi CD62Lhi/ CD4+ and CD44hi CD62Llow/CD4+ cells were measured by flow cytometry. The cells were first gated on CD3 and CD4 positive cells, and then, the percentages of double-positive cells (CD44+ CD62L+) were recorded based on the gated CD3+ CD4+ cells. (E,F) Percentages of CD44hi CD62Lhi/ CD8+ and CD44hi CD62Llow/CD8+ were measured by flow cytometry. The cells were first gated on CD3 and CD8 positive cells, and then, the percentages of double-positive cells (CD44+ CD62L+) were recorded based on the gated CD3+ CD8+ cells (data are presented as the mean ± SD n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

guarantee long-lasting cellular memory.43,45,46 As shown in Figure 6C−F, the mice treated with two nanovaccines (COOH-NPs/OVA and OCH3-NPs/OVA) induced higher levels of the TEM and TCM of both CD4+ and CD8+ T cells than those treated with the physical mixture of OVA + COOH-NPs and free OVA, indicating that the high antigen delivery efficiency of the nanovaccines was necessary for enhancing immunity. Furthermore, COOH-NPs/OVA produced higher levels of CD4+ and CD8+ TEM and TCM cells, implying the positive role of intrinsic adjuvant effect of COOH-NPs in vivo. To investigate the humoral immunity of COOH-NPs/OVA in vivo, the IgG level was measured after the C57BL/6 mice were immunized three times. As expected, the mice vaccinated with COOH-NPs/OVA significantly induced higher IgG production than the other experimental groups (Figure 7). Previous studies have demonstrated that the complement system was the “instructor” of the humoral immune response; it could lower the threshold for B-cell activation and facilitate the localization of antigen to follicular DCs (FDCs) in lymphoid follicles as well as promote the development of optimal B-cell memory.17,47

Figure 7. COOH-NPs/OVA-enhanced humoral immune response in vivo. The amount of antigen-specific IgG production was measured on day 21 from the mice immunized with different formulations (data are presented as the mean ± SD n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

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Figure 8. COOH-NPs/OVA-induced potent antitumor immunity in vivo. C57BL/6 mice were subcutaneously injected with free OVA or a physical mixture of OVA + COOH-NPs, OCH3-NPs/OVA, or COOH-NPs/OVA three times at the tail base. One week after the last immunization, E.G7-OVA tumor cells were inoculated for the tumor challenge. The tumor volumes over time are shown in (A). To assess the therapeutic effect of COOH-NPs, the mice were inoculated with 2 × 105 E.G7-OVA cells in the right flanks before immunization to establish tumor models on day 0 and then treated with the nanovaccines on days 5, 11, and 17. The tumor sizes during immunization are shown in (B), and the tumor weights after 27 d of inoculation are shown in (C). Survival data in tumor-bearing mice are shown in (D) and the mice body weight during vaccination are shown in (E) (data are presented as the mean ± SD n = 5; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

3.6. Antitumor Immunity of COOH-NPs/OVA in Vivo. The antitumor immunity of COOH-NPs/OVA was evaluated with tumor challenge experiments on both preventive model and therapeutic model. On the preventive model, the C57BL/ 6 mice were immunized with different vaccine formulations and the tumor growth was evaluated. As shown in Figure 8A, the mice immunized with COOH-NPs/OVA showed potent inhibition effect on tumor growth compared with the other formulations, indicating a better protective immunity to the mice. On the therapeutic model, the C57BL/6 mice were vaccinated with the different vaccine formulations on days 5, 11, and 17 after E.G7-OVA tumor inoculation. As shown in Figure 8B, COOH-NPs/OVA significantly inhibited the tumor growth compared to the other vaccine formulations. COOHNPs/OVA showed more effective antitumor immune responses than OCH3-NPs/OVA, although OCH3-NPs/ OVA was also able to effectively promote MHC I antigen presentation. This comparison demonstrated that the surface characteristics of the nanovaccines played an important role in modulating the immune responses. Moreover, the mice immunized with COOH-NPs/OVA markedly prolonged the

50% survival of mice to day 40, which was significantly longer than the other vaccine formulations (Figure 8D). In addition, the mice treated with COOH-NPs/OVA showed no significant body weight loss compared with the PBS-treated mice (Figure 8E), indicating the minimal adverse effect induced by COOHNPs/OVA. It was reported that the complement activation was a double-edged sword that not only helped the host to protect against invaders but also had the potential to induce damage to self-tissues.17 Hematoxylin and eosin (H&E) staining (Figure S6) showed little damage of COOH-NPs/OVA vaccination, indicating the safety of the nanovaccine in vivo.

4. DISCUSSION DCs are the most powerful antigen-presenting cells for the induction of antigen-specific T-cell response; therefore, DCbased vaccine is very promising in the tumor immunotherapy. The strategy for ex vivo matured DCs pulsed with tumor lysates or specific antigens failed to achieve desirable clinical results because more than 90% of the transplanted DCs died or failed to reach lymphoid organs after administration.4,48 Thus, J


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groups, which possessed the ability to attack the thioester group of C3b for activation complement to promote immune responses. COOH-NPs/OVA was found to significantly upregulate the expression of maturation markers on LNresident DCs, which was ascribed to the complement activation via the determination of C3a release. Compared to the traditional application of immune adjuvant, utilizing vaccine carrier with intrinsic adjuvant property not only simplified the procedure of vaccine manufacture but also spared the potential systemic toxicity induced by immune adjuvant. Besides the surface chemistry of NPs, which has been investigated in this study, the polymer type, the conformation of NP, the surface density of functional groups, and the interactive forces between polymers such as van der Waals force of attraction may all contribute to the activation of complement. Owing to the limitation of this study, these factors have not been discussed thoroughly and need to be investigated in the future research studies. In addition, it has been demonstrated that the physiochemical properties of NPs are also related to the immunogenicity of NPs.51 The hydrophobicity and the surface charge of nanovaccine may contribute to the enhanced immunity of COOH-NPs. Some previous studies suggested that the less hydrophobic particles could induce higher levels of cytokines than the more hydrophobic particles.52 The complement protein absorbed preferentially onto the negatively charged NPs, leading to the enhanced complement activation.53 In vivo studies showed that the multifunctional nanovaccine of COOH-NPs/OVA could regulate the function of both T cells and B cells. When splenocytes were restimulated with OVA, the mice immunized with COOH-NPs/OVA induced the high level of IFN-γ+ CD8+ T and IFN-γ+ CD4+ T-cell production, indicating the robust cellular immunity induced by the nanovaccine. Furthermore, COOH-NPs/OVA enhanced the formation of the memory T cells, as indicated by the high levels of CD8+ TEM, CD4+ TEM and the CD8+ TCM, CD4+ TCM, which would effectively protect the host from reinfection of specific pathogens for an extended period. Additionally, the level of lgG production in serum demonstrated the effect of COOH-NPs on the humoral immunity. Because of the superior activity of COOH-NPs/OVA in cellular and humoral immunity, the mice treated with COOH-NPs/OVA showed the potent inhibition on tumor growth and remarkably prolonged the survival time of tumor-bearing mice. However, the inhibition of COOH-NPs on tumor growth as the tumor progressed over 30 days has not been investigated in this paper, which required more work in the future. The potent antitumor immunity could be mainly attributed to the excellent LN trafficking, cytoplasmic presentation pathway, as well as the complement activation of COOH-NPs. These findings highlighted the significance of rational design of vaccine platforms for achieving effective anticancer immunity.

more and more researchers tried to develop new strategies able to directly manipulate or recruit DCs in the body.4 Nowadays, most of DC-targeting vaccines have been focused on delivery vaccines to the peripheral DCs, which was impeded by the relatively low numbers of DCs.38 LNs are the organs of the lymphatic system that initiate and regulate the adaptive immune response, which contain a large number of phagocytically active DCs.9 Therefore, delivery of nanovaccines to LN is an alternative approach to enhance immune responses. In this study, we developed a nanovaccine delivery system based on the biodegradable and low toxic amphiphilic diblock copolymer of COOH-PEOz-PLA, which could self-assemble into micelles for incorporation of the vaccine to target LNs. The particle size of vaccine carriers has great effect on the LNtargeting efficiency. It was reported that the particle size in the range of 10−70 nm was preferred for LN targeting.11 The amphiphilic copolymer of COOH-PEOz-PLA self-assembled into micelles with a particle size of around 60 nm, ensuring the targeting efficiency. COOH-NPs also showed long-term retention in LNs, which contributed to increase the interactions between antigen and LN-resident DCs for elicitation of effective antigen-specific immune responses.40 The antigen OVA was covalently conjugated onto the surface of micelles through reducible disulfide bond because the surface shielding of antigens greatly increased the chances of direct antigen cross-presentation to DCs.37,40 The disulfide bond was used to link the antigen OVA with the micelles in order to achieve triggered release of the antigen in response to the cell cytosol environment. The pH-responsive property of the PEOz backbone in the acidic conditions facilitated the endosomal escape of the antigen via “proton sponge” effect, ensuring the antigen presentation through the MHC-I pathway for CTL-based cellular immune responses. Codelivery of antigen and immune adjuvant with NPs was the common approach to promote DC maturation. However, the acidity, heat, and high temperature occurred during the incorporation of vaccine into NPs usually caused the decomposition of the adjuvants. Moreover, this approach was prone to induce unpredicted toxicity after administration. Therefore, new strategies to facilitate immune responses are highly demanded to overcome these limitations. The complement system is the part of innate immunity and serves as the first line to invade pathogens and other foreign substances to enter the body. Recently, studies revealed that it also played an important role in adaptive immunity.49 The complement could be activated by the biomaterials with nucleophiles via the alternative pathway, generating the “danger signal” in situ which greatly facilitated the DC maturation.8 Activation of the complement could further release a variety of biologically active complement peptides, such as C4a, C3a, and C5a.22,50 C4a, C3a, and C5a are known as the anaphylatoxin, and they could enhance the phagocytosis and cytotoxicity of immune cells by initiating potent inflammatory reactions.22 C3a and C5a can also participate in almost all phases of T-cell responses. They could enhance T-cell-mediated immune responses by promoting the cytokine production.22 In addition, C3a can also inhibit the apoptosis and promote the proliferation of immune cells by upregulating the antiapoptotic genes such as Bcl-2.22 Because of the biological activity of complement activation, we chemically modified the end of PEOz blocks with carboxylic groups to endow the nanovaccine with intrinsic immunogenic activity. The carboxylic-substituted PEOz would self-assemble into micelles covered with hydroxyl

5. CONCLUSIONS In this study, we developed a multifunctional COOH-NP nanovaccine based on the amphiphilic diblock copolymer of COOH-PEOz-PLA, which offered a good LN targeting, antigen presentation for CTL-based cellular immune responses, and complement activation for promotion of DCs maturation. COOH-NPs/OVA showed efficient targeting to LNs after subcutaneous administration. The pH-responsive properties of COOH-NPs ensured the endosomal escape of the antigen, facilitating the antigen presentation through the K


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MHC I pathway. Furthermore, COOH-NPs/OVA demonstrated the robust ability to trigger the complement activation, leading to the DC maturation, enhanced the T-cell immunity, antigen-specific antibodies’ production, and the antitumor effect in vivo. COOH-NPs have been demonstrated to be an effective and a promising approach to deliver the antigens for cancer immunotherapy. This study also demonstrated the potential of using complement-activating NPs as the vaccine platforms for future studies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.9b00195. Characterization of COOH-PEOz-OH and COOHPEOz-PLA, SDS-PAGE of different formulations of OVA, stability of antigen-incorporated micelles, particle size distribution and the TEM image of COOH-NPs, and H&E staining of the main organs from COOHNPs/OVA-immunized mice (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-24-23986308. Fax: +86-24-23986306 (M.Q.). *E-mail: [email protected]. Phone: +86-24-23986982. Fax: +86-24-23986980 (D.C.). ORCID

Dawei Chen: 0000-0002-1079-775X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81573370). REFERENCES

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Tailored Polymers with Complement Activation Ability To Improve

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