Article pubs.acs.org/journal/abseba
Potent Immune Stimulation from Nanoparticle Carriers Relies on the Interplay of Adjuvant Surface Density and Adjuvant Mass Distribution Jeffery Noble, Anthony Zimmerman, and Catherine A. Fromen* Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *
ABSTRACT: Development of novel adjuvant delivery approaches which provide safe and effective immune stimulation are critical for prophylactic and therapeutic advances in a wide range of diseases. Toll-like receptor agonists (TLRas) have been identified as potent stimulators of antigen presenting cells (APCs) and are capable of inducing proinflammatory immune responses desirable for vaccine and immunostimulatory applications. Although TLRas have been successfully incorporated into nanoparticle platforms, minimal work has been done to evaluate the direct role of the adjuvant incorporation in these formulations in directing the immune response. Here, we developed a series of nanoparticle carriers with controlled surface densities of two TLRas, lipopolysaccharide (LPS), corresponding to TLR-4, and CpG oligodeoxynucleotide, corresponding to TLR-9. The proinflammatory cytokine production and expression of costimulatory molecules on APCs were evaluated following a 24 h particle incubation period in vitro using bone marrow derived macrophages and in vivo following particle instillation in the airway of mice. Results demonstrate that proinflammatory cytokine production is predominantly driven by the distribution of the adjuvant dose to a maximal number of cells, whereas the upregulation of costimulatory molecules needed to drive APC maturation and promote adaptive responses indicate the requirement of an optimal density of TLRa on the particle surface. These results indicate that adjuvant surface density is an important parameter for tight control of immune stimulation and provide a foundation for pathogen mimicking particle (PMP) vaccines and immunostimulatory therapeutics. KEYWORDS: adjuvant, toll-like receptor agonist, polyvalency, immunoengineering
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INTRODUCTION Controlled immune stimulation is a key goal for the development of vaccines and immunotherapies focused on the prevention and treatment of cancer, autoimmune disorders, and infectious diseases.1,2 Vaccinations, for example, rely on establishing target antigen recognition while simultaneously engendering a sufficient and directed response, achieved through the delivery of both the antigen for specificity and an adjuvant for enhanced immunogenicity.1−4 This need for adjuvants is especially true in the case for subunit and synthetic antigens5,6 that tend to have low immunogenicity at such an extent that delivery of the antigen without sufficient stimulation can instead result in a tolerance toward the foreign agent.7 Although adjuvants are needed for sufficient immune stimulation, adjuvanticity and toxicity are inseparable, hindering significant advances in adjuvant development.5,8,9 To restrict harmful side effects, nanoparticulate-based methods are being investigated as a way to target the adjuvant dosage while maintaining sufficient and well controlled immune stimulation.3 Recent work in the development of new adjuvants9,10 has identified immunomodulatory agents that stimulate innate © XXXX American Chemical Society
immune cells through evolutionary conserved features called pathogen associated molecular patterns (PAMPs).11 This family of molecules interact with cells’ pathogen-recognition receptors (PRRs), including the membrane-spanning toll-like receptor (TLR) family.9,12,13 To date, ten human and 12 murine TLRs have been identified,14 which initiate the production of TH1-type proinflammatory cytokines and stimulate antigen presenting cell (APC) maturation.12,13 Corresponding PAMPs include lipopolysaccharides (LPS, TLR-4), present on the bacterial wall of Gram-negative bacteria, and unmethylated CpG oligonucleotides (TLR-9), common in bacterial DNA.13 Although effective TLR-agonist (TLRa) delivery has been achieved as isolated soluble molecules,15,16 the delivery of TLRa on a particle surface17,18 or codelivered with an antigen19 results in a stronger, more directed immune response. These surface-dependent responses are hypothesized to be superior over free ligand responses Received: December 6, 2016 Accepted: February 6, 2017 Published: February 6, 2017 A
DOI: 10.1021/acsbiomaterials.6b00756 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering through pathogen mimicry20 by taking advantage of polyvalent stimulation achieved by delivering multiple adjuvant molecules on a single platform.21,22 There have been many investigations tuning the physical properties of particulate carriers for vaccine applications, including manipulations of size,23 shape,24 charge,25 and specifically the antigen type and incorporation.26,27 However, despite considerable achievements in antigen development, including the investigation of new antigens, polyvalent display platforms,28 or stimulus-dependent antigen release,29 surprisingly little work has investigated the adjuvant contribution needed to direct the stimulation level and type of response. In this work, we developed a series of 22 particles with controlled TLRa density on the particle surface attached via biotin-NeutrAvidin interactions. Exploring varied surface densities of both LPS and CpG, we evaluated the resultant in vitro and in vivo immune stimulation following particle administration by analyzing cytokine production and APC costimulatory molecule expression. We also assessed variations in particle surface chemistry and linker orientation. TLRa were conjugated to either carboxlyated or aminated particle surfaces using NeutrAvidin through different residues, further testing the consistency of resultant trends on platforms with varied surface properties. Overall, this work provides a set of design rules that informs particulate vaccine and immunotherapy design to ensure both potency and safety, as well as laying the groundwork for future investigations modulating immune response with pathogen mimicking particles (PMPs).
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glycine (7.5 mg/mL) was added for 15 min to quench the reaction. NeutrAvidin conjugated particles were then washed with a PBS buffer (50 mM) and stored at 4 °C until adjuvant conjugation. Amine-PS particles will react via carboxylate residues primarily located on the distal ends of the Neutravidin protein structure,31 whereas carboxylatePS carriers have the availability for lysine residue conjugation within the core of protein, yielding the immobilization of avidin almost perpendicularly to the surface.32 NeutrAvidin conjugated particles were washed at 2 × 107 particles/ aliquot and resuspended in 200 μL reaction volumes containing varying concentrations of the biotinylated adjuvant. For LPS-modified particles, the soluble LPS-biotin concentrations ranged from 5−0.01 μg/mL for particle types B−F, respectively. In a similar manner, the soluble CpG reaction volume concentrations ranged from 1−0.01 μg/ mL for particle types G - K, respectively. Particle type A was resuspended in 200 μL reaction volumes of adjuvant-free buffer for use as the control particle. Optimized reaction conditions were scaled to larger volumes for in vivo studies. The zeta potentials of the particles at 1 × 108/mL in 0.2 μm filtered distilled water were measured by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, Ltd.). Quantification of adjuvant ligand density was accomplished indirectly by use of a flow cytometry particle backfill method using biotin-PE (see Figure S1). A higher density of adjuvants on the particle surface can be correlated to lower amount of biotin-PE backfill. Thus, assuming similar possible binding sites for both adjuvant and biotinPE, the number of adjuvant molecules can be quantified by comparing the fluorescent intensity of biotin-PE with the maximum amount of fluorescence using a particle with solely biotin-PE. The backfill method was tested on an equivalent sialyl Lewis A system and confirmed with a direct fluorescent antibody method as described previously.30 Adjuvant density was confirmed for each individual batch of particles, which was used to determine total adjuvant dosed. In Vitro Bone-Marrow-Derived Macrophage (BMM) Studies. Murine BMMs were cultured from bone marrow progenitor cells of untreated C57BL/6 mice as described previously.33 Bone marrow cells were collected by flushing the marrow cavity of the femurs and tibias with PBS + 2% FBS. Cells were then filtered and seeded with L929conditioned medium (containing GM-CSF). Additional media was added on day 3. Cells were harvested on day 6 or 7 by cell scraping and seeded in 96-well plates at 1−2 × 105 cells/well in supplemented DMEM media for in vitro studies. Respective particles dosages were resuspended in supplemented DMEM media and added to the 96-well plate. To evaluate the role of adjuvant distribution and density-dependent immune stimulation, we dosed cells based on an equivalent adjuvant dosage or an equivalent particle dosage. For the constant adjuvant dosing of BMMs, LPS was fixed at 25 ng/mL, whereas CpG was fixed at 10 ng/mL, which resulted in varied particles concentrations per dose. On a similar note, the constant particle study used a concentration of 1 × 107 particles/mL, resulting in varied adjuvant amounts per dose. Incubation conditions were specified at 200 μL volumes per well with a constant cellular concentration of 1−2 × 105 cells/well. Following the 24 h incubation, supernatants were removed from each well and assayed for cytokine production. BMMs were detached following incubation with trypsin at 37 °C for 5−10 min. Cells were then stained for costimulatory expression of CD40, CD80, and CD86, and fixed with 4% paraformaldehyde (PFA). In Vivo Lung Instillation of Adjuvants. Following anesthesia using isoflurane, soluble and nanoparticle-attached adjuvants were delivered to the lungs of male C57BL/6 mice through an orotracheal instillation in a 50 μL volume in PBS, as previously described in detail elsewhere.34 We instilled both the aminated and carboxylated forms of particle types B and F to represent high and low density versions of LPS-modified particles. Accounting for an equivalent LPS dosage of 10 μg/mouse, the scaled-up carboxylated particle B was dosed at 3.9 × 109/mouse and particles F and A were dosed at 2.0 x1010/mouse; aminated particle B was dosed at 6.7 × 109/mouse and particles F and A dosed at 2.0 x1010/mouse. Similarly, both the aminated and carboxylated forms of CpG-modified particles were dosed at 2.5 μg/
EXPERIMENTAL SECTION
Materials and Methods. Animal Study Approvals. All animal studies were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and approved by the Institutional Committee on Use and Care of Animals (ICUCA) at the University of Michigan. Male and female C57BL/6 mice 4−5 weeks in age were purchased from Jackson Laboratory and were maintained in pathogen-free facilities at the University of Michigan until use at 6−10 weeks in age. Materials. Unless noted, all chemicals were obtained from SigmaAldrich and buffers were obtained from Fisher. 500 nm Fluoresbrite YG Carboxylate Polystyrene Microspheres and 500 nm Polybead Amino Microspheres were purchased from Polysciences, Inc. Neutravidin Biotin-Binding Protein was purchased from Thermo Scientific, Biotin-Phycoerythrin (PE) were purchased from Fisher. Murine TLR 9 agonist, ODN 1826 Biotin labeled CpG 20-mer oligodeoxynucleotide (5′-TCCATGACGTTCCTGACGTT-3′−20 mer) (MW 6383 g/mol), and TLR 4 agonist, biotinylated lipopolysaccharide (LPS-EB, MW ≈ 15 000 g/mol) from E. coli 0111:B4, were both obtained from Invivogen. Antibodies for CD40PE/Cy7, CD45-APC/Cy7, CD80-APC, CD86-PE/Cy7, Ly6G-APC, CD11c-PE/Cy7, MHC II (Ia/Ie)-PE were purchased from Biolegend. DMEM media (Gibco) was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Collagenase was obtained from Worthington. Lymphoprep was obtained from Stemcell Technologies. Enzyme-linked immunosorbent assay (ELISA) kits were purchased from BD Biosciences (IL-6) and R&D Systems (TNF-α, MIP-2, and KC). Particle Functionalization and Characterization. Five-hundred nanometer carboxylated and aminated polystyrene (PS) particles were covalently modified with NeutrAvidin Biotin-Binding Protein (Thermo Scientific) via carbodiimide chemistry.30 Particles were washed with 50 mM MES buffer and incubated with a NeutrAvidin solution (5 mg/mL) for 15 min at room temperature. Then an equal volume of N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, 75 mg/mL) was added and pH was adjusted to 9.0 using 1 M NaOH. After incubating for 24 h at room temperature, B
DOI: 10.1021/acsbiomaterials.6b00756 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 1. Particle characterization of carboxyl-PS and amine-PS particles with varied LPS or CpG densities. (A) Quantified LPS surface density for particles B−F following varied biotinylated-LPS reaction conditions. (B) Quantified CpG surface density for particles G-K following varied biotinylated-CpG reaction conditions. Zeta potentials for (C) LPS-modified and (D) CpG-modified particles. N = 5 independent particle batches, error bars represent standard error. mouse. Carboxylated particle G was dosed at 8.2 × 108 /mouse and particle K was dosed at 2.5 × 109 /mouse; aminated particle G was dosed at 7.8 x108 /mouse and particle K dosed at 1.7 × 109 /mouse. Twenty-four hours after instillation, mice were euthanized and blood was collected via cardiac puncture. Bronchoalveolar lavage fluid (BALF) was collected by inserting a cannula in an incision in the trachea. The lungs were flushed with three sequential 1 mL washes of PBS and the BALF cells were obtained by centrifugation. Furthermore, the lung was harvested and homogenized into single cell suspensions using 5 mg/mL collagenase, as described previously.34 Cell and Tissue Analysis. BALF and BMM supernatants were evaluated via ELISA following manufacturer’s instructions. Cell lysates from in vitro BMM incubation studies were evaluated via CellTiterGlo Luminescent Cell Viability Assay (Promega) following manufacturer’s instructions. Fixed BALF and lung cells from in vivo studies and in vitro BMMs were evaluated via flow cytometry. BALF cells were stained with Ly6G and CD11c to identify neutrophils and macrophages in the BALF. Single-cell lung suspensions were stained with CD45, CD11c, and MHC II (Ia/Ie) to identify dendritic cells. Cells were then fixed in 4% PFA until flow cytometry. Flow cytometry data was collected on an Attune NxT Focusing flow cytometer (Life Technologies) and analyzed using FlowJo software (Tree Star). Statistics. For all studies, the entire data set was included in the analyses and no outliers were excluded in the calculations of means or statistical significance. Data are plotted with standard error bars and analyzed as indicated in figure legends. Asterisks indicate p values of *
