Introduction
Vaccines are a promising approach to prevent or cure cancer [
1,
2] but generally require a tumor antigen and an immune-stimulatory adjuvant. Breast cancers that express the human epidermal growth factor receptor 2 (HER2) have been treated with some success by immunotherapies that target that antigen [
3]. Vaccines can be potentiated by their method of administration and formulation. For example, nanoparticles (NPs) can protect sensitive/and or unstable antigens such as peptides from degradation and potentially increase the immune response to vaccines. It has been shown that encapsulation of antigen into biodegradable spheres leads to enhanced humoral and cellular immune responses [
4,
5]. Poly(D,L-lactic-co-glycolic) acid nanoparticles (PLGA-NPs) have been used to deliver the cancer-associated antigen MUC1,5,6 as well as tetanus toxoid to enhance immune responses [
6]. PLGA is a biodegradable and biocompatible polymer [
7,
8] with good stability in the gastrointestinal tract [
9] and is used for numerous
in vivo applications [
10,
11]. NPs also have the advantage that, by using different polymer compositions, one can control the release of cargo allowing for antigen depot formation at the injection site. These manipulations might provide enabling technologies to the vaccine as well as drug development field.
Dendritic cells (DCs) are the most potent antigen-presenting cells and are critical for the initiation of adaptive immune responses. Vaccines need to stimulate DCs to induce potent immune responses. DCs must receive a maturation signal to present antigen, upregulate costimulatory and adhesion molecules, and become potent activators of T cells [
12]. The immunostimulatory peptide Hp91, which is derived from the endogenous protein high-mobility group box protein 1 (HMGB1), activates DCs [
13] and primes antigen-specific cytotoxic T lymphocyte (CTL) responses
in vitro [
13] and
in vivo [
14]. Hp91 packaged inside of PLGA-NPs is more potent in activating DCs as compared to free peptide [
15]. In our previous study, the PLGA-NPs were synthesized using an emulsion method yielding non-homogeneous particles. In the current study, we used a precipitation method that yields homogeneous NPs, to package Hp91 inside PLGA-NPs. We evaluated the extent to which Hp91-PLGA-NPs protect against breast cancer using a HER2 breast cancer mouse model [
16]. Our results demonstrate that the delivery of the immunostimulatory peptide Hp91 inside the PLGA-NPs enhances the efficacy of this breast cancer vaccine.
Materials and methods
Peptides
The adjuvant peptide Hp91 (DPNAPKRPPSAFFLFCSE) and MHC class I (H2-Dq)-restricted rat HER-2/neu-derived peptide (PDSLRDLSVF) were both purchased from CPC Scientific (San Jose, CA, USA). The Hp91 peptide was synthesized with an N-terminal biotin and dissolved in RPMI for in vitro studies and phosphate-buffered saline (PBS) for immunizations. The HER2 peptide was dissolved in 3% dimethyl sulfoxide (DMSO)/PBS. Peptides were routinely synthesized with greater than 95% purity.
Animals
FVB.N/neu-tg mice were derived from in-house breeding stocks at the University of California, San Diego (UCSD) Moores Cancer Center animal facility. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of California, San Diego and performed in accordance with the institutional guidelines.
Synthesis of peptide-loaded lipid-polymer hybrid nanoparticles
Ester-terminated poly-lactic-co-glycolic acid, or PLGA (50:50, 0.82 dl/g IV, DURECT Corporation, Cupertino, CA, USA) was dissolved at 1 mg/ml in dimethylformamide (DMF). Hp91 was also dissolved in DMF with the PLGA at concentrations of 1 to 5 mg/ml. Lecithin (molecular weight (MW) 330 Da, Alfa Aesar, Ward Hill, MA, USA) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(carboxy(polyethylene glycol)2000) (ammonium salt) (DSPE-PEG-Carboxy, MW 2,849.54 Da, Avanti Polar Lipids, Alabaster, AL, USA) were dissolved together in 2 ml of 4% ethanol per mg PLGA to be used at a ratio of 9% of total PLGA weight for lecithin and 52% of total PLGA weight for DSPE-PEG-Carboxy. All stock solutions were made using sterile solvents or endotoxin-free water. The aqueous lipid mixture was heated to 68°C while stirring for 3 min. The PLGA-peptide solution was added dropwise to the heated lipid solution while stirring. The solution was then vortexed at 3,000 RPM for three minutes. An additional 1 ml of water per mg of PLGA used was added dropwise to the NP solution while stirring. The NP solution was stirred without cap for 2 h to allow solvent evaporation. The particles were then washed three times using Amicon Ultra centrifugal filter devices by EMD Millipore (Billerica, MA, USA) with 100 Kd cutoff. Particles were suspended in 10% sucrose and flash frozen for later use.
Characterization of lipid-polymer polylactic-co-glycolic acid hybrid nanoparticles (PLGA-NPs)
The NP formation was analyzed for particle size by dynamic light scattering (DLS) using a zetasizer (Zetasizer Nano ZS, Malvern Instruments Ltd, Malvern, UK). To quantify the amount of peptide loaded into the hybrid NPs, the NPs were dissolved in DMF for 30 min under constant shaking at room temperature and peptide content was quantified by high-performance liquid chromatography (HPLC) (column: Waters Delta-Pak C18 5 microns, Waters Corporation, Milford, MA, USA) at 211 nm in comparison to a Hp91 peptide standard curve. To measure the release rate of the peptide from the NPs, 100 μL of Hp91-loaded NP solution was added to microdialysis cassettes with a MW cutoff of 10,000 and dialyzed against 1 L of PBS buffer at pH 7.4 or potassium hydrogen phthalate buffer at pH 5. At each time point, two samples for each buffer condition were recovered from the microdialysis cassettes, and the volumes were brought up to 125 μL to keep all volumes constant. To each sample, 125 μL of DMF was added to dissolve the NPs and release the remaining Hp91 peptide. The samples were shaken for 60 min, and then the total amount of Hp91 in each sample was quantified using HPLC. The amounts were normalized against the starting concentration of peptide before dialysis, which was set at 100% to calculate the percentage released.
Generation of mouse bone marrow-derived DCs
Bone marrow-derived dendritic cells (BM-DCs) were prepared from HER-2/
neu transgenic mice (H-2
q), as described by Inaba
et al. [
17] with minor modifications. Briefly, single bone marrow cell suspensions were obtained from femurs and tibias, depleted of lymphocytes, granulocytes, and Ia + cells using a mixture of monoclonal antibodies (mAbs; anti-CD4, anti-CD8, anti-B220/CD45R, and anti-Ia) for 45 min on ice, followed by incubation with low-toxicity rabbit complement for 30 min at 37°C. Cells were resuspended at a concentration of 10
6 cells/mL in medium supplemented with recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/mL). Fresh medium (5% vol/vol fetal calf serum (FBS)-RPMI) containing GM-CSF was added on day 2 and 4 of culture. On day 6, cells were collected for the experiments.
Antigen presentation assays
Immature BM-DCs (105) were stimulated with media alone, similar amounts of Hp91 free peptide, NP-encapsulated Hp91, or 10 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich, St Louis, MO, USA). Forty-eight hours after activation, the cells were incubated with 100 ng/mL HER2 peptide for 1 h at 37°C. The cells were then washed twice to remove excess peptide and plated with HER2-specific CTL clones (kindly provided to us by Professor E. Jaffee (John Hopkins Medical Institute) at a 103:104 DC to T cell ratio in wells of a nitrocellulose bottom enzyme-linked immunospot (ELISPOT) plate (EMD Millipore) that had been previously coated overnight with 5 μg/mL monoclonal anti-mouse interferon gamma (IFN-γ) antibody (Mabtech, Stockholm, Sweden). After 18 h, the ELISPOT plates were developed using 1 μg/ml biotinylated anti-mouse IFN-γ antibody (Mabtech), Streptavidin-horseradish peroxidase (HRP) (Mabtech), and 3,3',5,5'-Tetramethylbenzidine (TMB) substrate (Mabtech). The plate was scanned and the spots were counted using an automated ELISpot Reader System (CTL ImmunoSpot, Shaker Heights, OH, USA).
Immunizations and spleen cell preparation
The HER-2/neu peptide antigen was co-administered subcutaneously with either PBS, soluble Hp91, or NP-encapsulated Hp91 on the right flank. Spleens were collected 8 days after the final immunization. Single cell suspensions of splenocytes were prepared by mechanical disruption and separation through a 70-μm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Red blood cells were lysed using ammonium chloride buffer (Roche Diagnostics, Indianapolis, IN, USA) and the splenocytes were subsequently resuspended in complete medium (RPMI 1640 with 10% FBS, L-glutamine, penicillin, streptomycin, and HEPES) supplemented with 20 U/mL of recombinant mouse interleukin (IL)-2) (R&D Systems, Minneapolis, MN, USA) and 10 μg/mL of PDSLRDLSVF peptide for expansion. Splenocytes were expanded for 5 days prior to use in ELISPOT experiments.
Enzyme-linked immunospot assay
The expanded splenocytes were collected and washed twice before being plated in duplicate 106 cells to wells of an ELISPOT plate that had been previously coated overnight with 5 μg/mL monoclonal anti-mouse IFN-γ antibody. Splenocytes were cultured overnight at 37°C with 2.5 μg/mL HER2 peptide, 5 μg/ml concavalin A (Sigma-Aldrich) as positive control or left unstimulated (medium only). After 18 h, ELISPOT plates were developed using 1 μg/ml biotinylated anti-mouse IFN-γ antibody, streptavidin-HRP, and TMB substrate. The plate was scanned and the spots were counted using an automated ELISpot Reader System.
Tumor prevention experiments
Female HER-2/neu mice, 8 weeks of age, were immunized with 5 μg of HER2 antigen mixed with either PBS only, 25 μg of Hp91 free peptide, or 25 μg of Hp91 delivered in PLGA-NPs. Mice received their first boost 2 weeks post-prime, and a second boost 1 month thereafter. All injections were performed subcutaneously on the right flank of the mice. The incidence and growth of tumors were evaluated twice a week by measuring palpable tumors, defined as tumors with diameters that exceed 3 mm, with calipers in two perpendicular diameters. Calipers were used to measure tumor length and width and the volume was calculated as volume (mm3) = (width)2 × length/2. All mice bearing tumor masses exceeding 1.5 cm mean diameter were sacrificed.
Statistical analysis
Data represented are mean ± standard error of the mean (SEM). Data were analyzed for statistical significance using unpaired Student’s t test. Statistical analyses were done using GraphPad software version 5.01 for Windows (GraphPad Software, San Diego, CA, USA). A P value <0.05 was considered statistically significant for these analyses.
Discussion
The activation of potent immune responses by vaccines or therapies is essential and not always achieved by traditional vaccines or therapies. The use of NPs to deliver adjuvant and antigen is now being thoroughly investigated. Tumor-associated antigens have been identified for a variety of cancers but so far very few clinical trials have shown great efficacy. This could have many reasons including immune suppression by the tumor microenvironment, poorly immunogenic antigens, limited T cell repertoire and so on. Another possibility could be deficiency in the vaccine delivery systems. The immune system generally responds to viruses and bacteria, which are particles and not isolated molecules and they contain the antigen in close proximity to the adjuvant. Various NPs are being explored as delivery tools for drugs, vaccine antigens and adjuvants. NPs are a delivery platform that can be tuned for different needs. The material used to make NPs can in itself be immunogenic, for instance poly(γ-glutamic acid) activates the immune system via TLR4/MyD88 NP [
19]. Our goal was to use inert materials like PLGA-NPs as a neutral carrier, which can be loaded with immunogenic moieties, in this case the Hp91 peptide, to trigger desired immune responses. One reason for using non-stimulatory materials and loading them with adjuvant is that we believe understanding the mechanism behind material-induced immune responses is difficult, and using characterized adjuvants should be of advantage. The PLGA-NPs used in this study are biodegradable and biocompatible polymer10-13 that has been employed for numerous
in vivo applications. We have not observed immune activation with the PLGA-NPs alone [
16]. The PLGA-NPs used were 100 nm (range 30 to 200 nm), a size which has been shown to be preferentially taken up by professional antigen-presenting cells (APCs) and are expected to enter DCs or Langerhans cells (LCs) when given transcutaneous or s.c..
We found that Hp91, when packaged inside of PLGA-NPs, activated both mouse and human DCs in vitro and induced increased CD8+ T cell responses as compared to free peptide. One possible explanation is that the delivery is more efficient, because the NPs are readily taken up by DCs and each NP will deliver many peptides, whereas free peptide will diffuse around the cells and the uptake is much less effective. Interestingly, the intensity of CTL responses seen in the immunized mice mirrored protection from tumor development.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DFC isolated DC and performed immune assays in vitro, synthesized nanoparticles, immunized mice and performed immune readouts after vaccination, plotted data and drafted the manuscript. RS helped with immunization, isolation of immune cells, and critical revision of the manuscript. ISB bred the mouse colony, helped with immunizations and was involved in drafting the manuscript. DS synthesized nanoparticles and was involved in drafting the manuscript. LZ designed nanoparticles and was involved in drafting the manuscript. SE helped with nanoparticle analysis and characterization and was involved in drafting the manuscript. BM helped with experimental design, wrote parts of the manuscript and was involved in critical revision of the manuscript. ML helped with experimental design and analysis, wrote parts of the manuscript and was involved in critical revision of the manuscript. DM conceived of the study, guided the project, designed experiments, analyzed data, supervised the team, drafted, and wrote the manuscript. All authors have given final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
D.F. Campbell was formerly Futalan.