Article pubs.acs.org/journal/abseba
Polyanhydride Nanoparticle Interactions with Host Serum Proteins and Their Effects on Bone Marrow Derived Macrophage Activation Julia E. Vela Ramirez,† Paola M. Boggiatto,‡ Michael J. Wannemuehler,‡ and Balaji Narasimhan*,† †
Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, United States Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa 50011, United States
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ABSTRACT: An in-depth understanding of the interactions of vaccine delivery vehicles with antigen presenting cells is important for tailoring optimal adjuvant properties. Polymeric nanoparticles have been widely studied as adjuvants and delivery vehicles; however, there is little information regarding the effect of serum protein adsorption onto biomaterials and the effect of this adsorption upon interactions with antigen presenting cells. The current studies analyzed effects of polyanhydride chemistry on serum adsorption to nanoparticles with respect to their uptake by and activation of bone marrow-derived macrophages. Differential effects of serum adsorption based on nanoparticle chemistry were shown to enhance (for 1,6-bis(pcarboxyphenoxy)hexane and sebacic anhydride-based) or reduce (for 1,6-bis(p-carboxyphenoxy)hexane and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane-based) nanoparticle uptake. The observed complex interdependence between nanoparticle chemistry and serum protein adsorption on macrophage activation provided insights that will facilitate the rational design of single-dose nanovaccines developed to induce robust immune responses. KEYWORDS: vaccine delivery vehicles, serum, nanoparticles, macrophage activation
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INTRODUCTION
Among the key components of the innate immune system are surface receptors that facilitate the interactions between the cell and antigen−antibody complexes, aggregated immunoglobulins, and opsonized particles and result in engulfment of the opsonized particles into a phagosome.9,10 To facilitate the rational design of pathogen mimicking vaccine delivery systems, a systematic examination of whether nanoparticle formulations interact differently with serum proteins and the subsequent change in their ability activating APCs is necessary. Polymeric nanoparticle-based adjuvants/delivery systems have been studied for the past several decades for the development of efficacious vaccine formulations.11,12 In particular, biodegradable polyanhydride particles based on 1,6-bis(p-carboxyphenoxy)hexane (CPH), 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG), and sebacic anhydride (SA) have demonstrated excellent capabilities with respect to antigen stabilization, sustained antigen release, and immune stimulation that make them promising candidates for vaccine adjuvants and/or delivery vehicles.13−15 In addition, polyanhydride particles have the following attributes: (i) uptake by and activation of APCs;16,17 and (ii) surface chemistry and polymer
In order to elicit robust immune responses and confer protective immunity, activation of both the innate and adaptive immune systems is critical.1,2 A key component of innate immunity is the detection and recognition of foreign materials by antigen presenting cells (APCs) via multiple signaling receptors on or within APCs.3 The interactions between APCs and antigen delivery systems, which can target specific receptors on APCs, can be tailored to enhance antigen presentation and processing, thus affecting the downstream immune response.4,5 Therefore, in order to rationally design vaccine delivery vehicles that possess adjuvant activity, an indepth understanding of how delivery vehicles interact with APCs is necessary.4,6 When pathogens enter the body, plasma proteins adsorb to their surface.7 Binding of serum proteins can result in increased immune system recognition (i.e., opsonization) via binding or adsorption of complement proteins or immunoglobulins. However, pathogens have also developed ways to utilize proteins to hide from the immune system.8 Similarly, nanoparticle interactions with macrophages and dendritic cells are also subject to modifications by host protein adsorption, and given that the nanoparticle chemistry formulations can be modulated to affect their hydrophobicity and/or charge, adsorption of different groups of host proteins may be expected. © XXXX American Chemical Society
Special Issue: Biomaterials for Immunoengineering Received: July 13, 2016 Accepted: September 23, 2016
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DOI: 10.1021/acsbiomaterials.6b00394 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering chemistry-dependent serum protein adsorption patterns.4,18 These attributes have important implications for adjuvant design and for the induction of efficacious immune responses.5 In this study, serum adsorption patterns on two formulations of polyanhydride nanoparticles and their effects on macrophage uptake and activation were investigated. These two formulations, specifically 20:80 CPTEG:CPH and 20:80 CPH:SA, were chosen because previous studies have shown them to be potent adjuvants, based on their ability to enhance both humoral and cell-mediated immunity.13,19−21 Since both formulations are hydrophobic, with 20:80 CPTEG:CPH being more so,22 the insights gained from these studies can help unravel the role of particle/protein and particle/cell interactions on the activation of macrophages and aid in the rational design of efficacious vaccine adjuvants/delivery systems.
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Nanoparticle Synthesis. Polyanhydride nanoparticles were synthesized using an antisolvent nanoencapsulation method as described previously.28 Briefly, polymer (20 mg/mL) was dissolved in methylene chloride (at 4 °C for 20:80 CPTEG:CPH and at room temperature (RT) for 20:80 CPH:SA). The polymer solution was sonicated at 40 Hz for 30 s using a probe sonicator (Ultra Sonic Processor VC 130PB, Sonics Vibra Cell, Newtown, CT) and rapidly poured into a pentane bath (at −40 °C for CPTEG:CPH and at RT for CPH:SA) at a solvent to nonsolvent ratio of 1:250. Particles were collected by filtration and dried under vacuum for 1 h. Nanoparticles were characterized using scanning electron microscopy (SEM, FEI Quanta 250, Kyoto, Japan). Mice. C57BL/6 mice were purchased from Harlan Laboratories (Indianapolis, IN). Mice were housed in specific pathogen-free conditions where all bedding, caging, and feed were sterilized prior to use. All animal procedures were conducted with the approval of the Iowa State University Institutional Animal Care and Use Committee. Murine Serum Protein Adsorption onto Polyanhydride Nanoparticles. Blood was obtained via cardiac puncture after euthanasia of the mice, samples were held at room temperature for less than 30 min prior to centrifugation (10,000 rcf for 10 min), and serum was removed and stored at −20 °C. For the adsorption studies, 13.3% w/v suspensions of polyanhydride nanoparticles were prepared in phosphate buffer saline (PBS, pH 7.4). Nanoparticle suspensions were incubated with serum in a 1:4 volume ratio. The nanoparticle/ serum suspension was sonicated for 30 s at 40 Hz and incubated for 30 min at 37 °C under constant agitation. Next, the suspension was centrifuged at 10,000 rcf for 10 min to pellet the particles. Supernatants were removed, nanoparticles were resuspended in PBS, and the centrifugation step was repeated. This washing process was repeated three times, and the nanoparticles were dried under vacuum for 1 h and stored for characterization and further use. Nanoparticle Surface Analysis. To assay for the presence of serum protein on the nanoparticles, energy-dispersive X-ray spectroscopy (EDS) analysis of elemental composition was performed. Samples of serum-coated nanoparticles were prepared in carbon stubs and analyzed using an AZtec EDS system incorporated in a scanning electron microscope (Oxford Instruments, Concord, MA). The quantified elements are presented as percent of total content, and the experiments were performed in triplicate. Macrophage Culture and Stimulation. Bone marrow-derived macrophages (BMDM) were derived from bone marrow cells of C57BL/6 wild-type mice as described previously in published protocols.16,29,30 Briefly, mice were euthanized using carbon dioxide inhalation, and cardiac puncture was performed. Bone marrow cells were harvested from tibias and femurs and cultured in 25 mL of media containing Dulbecco’s modified Eagle’s medium (high glucose), with 20% fetal bovine serum, 30% L-cell conditioning supernatant, 1% penicillin/streptomycin, 1% 2 mM L-glutamine, and 1% sodium pyruvate in 150 × 15 mm cell culture grade Petri dishes at a cell density of 20 × 106 cells/25 mL of media in an incubator at 37 °C with 5% CO2 for 6 days. At day 2, 20 mL of fresh bone marrow macrophage media were added. On day 6, adherent cells were harvested by first discarding the cell media and placing dishes on ice for 30 min, 20 mL of sterile phosphate buffer saline were added, and cell scrapers were used to remove all adherent cells. Cells were then centrifuged at 250 rcf for 10 min, resuspended in fresh complete tissue culture media (CTCM) containing Dulbecco’s modified Eagle’s medium (high glucose), with 10% of fetal bovine serum, 1% penicillin/streptomycin, 1% 2 mM L-glutamine, 0.1% 50 mM β-mercaptoethanol, 2.5% HEPES buffer, and counted for plating. Cells were plated in 24 well plates using a concentration of 5 × 105 cells per well in CTCM. Macrophages were stimulated with 0.1 mg/mL per well of polyanhydride nanoparticles, based on previous studies.17,18 Nonstimulated (NS) cells were used as a negative control, and 200 ng/mL of Escherichia coli LPS was used as a positive control. Internalization of Serum-Coated Nanoparticles. Cadmium selenide quantum dot (QD)-loaded polyanhydride nanoparticles were synthesized using antisolvent nanoencapsulation.28 Briefly, 1% w/w QDs (630 nm emission) were suspended with the respective polymer
MATERIALS AND METHODS
Materials. Chemicals needed for monomer synthesis, polymerization, and nanoparticle synthesis include 1-methyl-2-pyrrolidinone, anhydrous (99+%) p-carboxy benzoic acid (99+%), and sebacic acid (99%), purchased from Aldrich (Milwaukee, WI); 1,6-dibromohexane, 4-p-hydroxybenzoic acid, and triethylene glycol, purchased from Sigma-Aldrich (St. Louis, MO); 4-p-fluorobenzonitrile, obtained from Apollo Scientific (Cheshire, UK); and acetic acid, acetic anhydride, acetonitrile, dimethylformamide (DMF), ethyl ether, hexane, methylene chloride, pentane, petroleum ether, potassium carbonate, sulfuric acid, and toluene, purchased from Fisher Scientific (Fairlawn, NJ). For 1H NMR characterization, deuterated chemicals, including chloroform and dimethyl sulfoxide (DMSO), were purchased from Cambridge Isotope Laboratories (Andover, MA). βMercaptoethanol, E. coli LPS O26:B6, and rat immunoglobulin (IgG) were purchased from Sigma-Aldrich. Materials required for harvest and culture media for macrophages included Dulbecco’s modified Eagle’s medium with high glucose, sodium pyruvate, 1 M HEPES buffer, penicillin−streptomycin, and L-glutamine, purchased from Mediatech (Herndon, VA) and heat inactivated fetal bovine serum, purchased from Atlanta Biologicals (Atlanta, GA). Materials used for flow cytometry included BD stabilizing fixative solution purchased from BD Biosciences (San Jose, CA); unlabeled anti-CD16/32 FcγR, purchased from Southern Biotech (Birmingham, AL); allophycocyanin (APC) antimouse CD40 (clone 1C10), FITC conjugated antimouse MHC Class II (I-A/I-E) (clone M5/114.15.2), and their corresponding isotypes APC-conjugated rat IgG2aκ (clone eBR2a) and FITCconjugated rat IgG2bκ (clone eB149/10H5) were purchased from eBiosciences (San Diego, CA). APC/Cy7 conjugated antimouse F4/ 80 (clone BM8), PE/Cy7 conjugated antimouse CD86 (clone GL-1), and their corresponding isotypes APC/Cy7 conjugated rat IgG2aκ (clone RTK2758) and PE/Cy7 conjugated rat IgG2aκ (clone RTK2758) were purchased from BioLegend (San Diego, CA). Cadmium selenide/zinc sulfide quantum dots (QDs) (emission at 630 nm) were purchased from Sigma-Aldrich. Monomer and Polymer Synthesis. Diacid monomers based on 1,6-bis(p-carboxyphenoxy) hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) were synthesized as described previously.23,24 Sebacic anhydride (SA) and CPH prepolymers were synthesized by the methods described by Shen et al.25 and Conix et al., 23 respectively. Subsequently, 20:80 CPH:SA and 20:80 CPTEG:CPH copolymers were synthesized by melt polycondensation as described by Kipper et al.26 and Torres et al.,17 respectively. The chemical structure of the copolymers was characterized with 1H NMR using a Varian VXR 300 MHz spectrometer (Varian Inc., Palo Alto, CA), and the molecular mass was determined using gel permeation chromatography (GPC) on a Waters GPC chromatograph (Milford, MA) containing PL gel columns (Polymer Laboratories, Amherst, MA). The 20:80 CPH:SA copolymer had a Mw of 14 000 Da and a polydispersity index (PDI) of 1.4, and the 20:80 CPTEG:CPH copolymer had a Mw of 7,800 and a PDI of 1.3. These values are consistent with previous work.4,19,27 B
DOI: 10.1021/acsbiomaterials.6b00394 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 1. Characterization of polyanhydride nanoparticles. Morphology of polyanhydride nanoparticles was characterized using scanning electron microscopy. Energy-dispersive X-ray spectroscopy (EDS) analysis of elemental composition of noncoated and serum-coated polyanhydride nanoparticles was used to confirm serum protein adsorption. Presence or increase in the nitrogen content of polyanhydride nanoparticles represents the serum protein coating effect. Data are reported as the mean ± standard error of the mean (SEM) of three independent experiments performed in triplicate. Noncoated nanoparticles were used as controls to assess the absence of nitrogen. solution, and nanoparticles were synthesized as described previously. Nanoparticles were suspended in PBS (pH 7.4, 37 °C) for 2 h, and supernatants were collected and used as controls to account for released QDs.31 Serum adsorption onto these nanoparticles was performed as described previously and used for macrophage internalization experiments. BMDMs were stimulated with nanoparticle formulations for 48 h and particle-positive cells were quantified using flow cytometry.18 Nonstimulated cells were used as control. Activation of BMDM by Serum-Coated Nanoparticles. BMDMs from wild type (WT) C57BL/6 mice were analyzed using flow cytometry for surface expression of costimulatory molecule expression (CD40, CD86) and major histocompatibility complex (MHC) II as described previously.17 Serum adsorption onto nanoparticles was performed as described previously and used for macrophage activation studies. Samples were analyzed using a BectonDickinson FACSCanto flow cytometer (San Jose, CA), and the data were processed using the FlowJo vX software (TreeStar Inc., Ashland, OR). Cytokine Secretion by BMDM. Supernatants of cultured cells 48 h poststimulation were analyzed for secretion of IL-6, IL-1β, TNF-α, IL-12p40, IFN-γ, and IL-10 using a multiplex cytokine assay with a Bio-Plex 200 system (Bio-Rad, Hercules, CA) as described in previous protocols.16,32 Analysis of Adsorbed Serum Proteins. Analysis of serum proteins adsorbed onto polyanhydride nanoparticles was performed using 2D gel electrophoresis.4,18,33 After adsorption, nanoparticles were dried and incubated with 250 μL of elution buffer with 5% (w/v) sodium dodecyl sulfate (SDS) and 2.3% (w/v) dithioerythritol to elute protein. Nanoparticles were heated at 95 °C for 10 min and centrifuged for 10,000 rcf for 10 min, and the supernatants were removed from the particle pellet for analysis. To quantify and analyze serum proteins, supernatants were passed through SDS removal columns (Pierce, Cat # 87777) to reduce the amount of SDS because of its interference with microBCA and gel electrophoresis. To analyze the eluted proteins, equal amounts of protein in elution buffers were processed for the first dimensional separation. For the first dimension, IPGPhor systems (GE Healthcare, Piscataway, NJ) were used with 7 cm IPG strips (pH 3−10) and a slow voltage ramping protocol: 50 V for 12 h, 500 V for 1 h, 1000 V for 1 h, and 8000 V for 6 h. For the second dimension of the separation analysis the IPG strips were loaded in 4−20% polyacrylamide gels and run for 4 h at 90 V. The gels were incubated in fixative solution (40% methanol, 10% acetic acid) for 3 h at 4 °C. Staining with Flamingo fluorescent gel stain (BioRad Laboratories, Richmond, CA) was performed at 4 °C overnight, following which a destaining process with a 0.1% Tween 20 solution
was carried out for 30 min to reduce background. The gels were scanned using a Typhoon 9410 Variable Mode Imager (GE Healthcare). Images were collected using ImageQuant TL and qualitative analyses were performed using ImageJ (version 1.47, NIH, Bethesda, MD) and Progenesis SameSpots (Nonlinear Dynamics, Durham, NC) to determine the location and intensity of the spots on the gels. The intensity of each spot was normalized with the total fluorescence, and the values are presented as percentages of the total fluorescence of the gels. Identification of each spot was performed by comparison of experimental gels with murine serum protein profiles found in the literature and in protein databases.33−40 Statistical Analysis. Statistical analysis was used to analyze the cell surface marker expression and cytokine secretion data. One-way ANOVA and Tukey’s HSD were used to determine statistical significance among the various treatment groups, and p-values
