Enhanced Immune Adjuvant Activity of Aluminum Oxyhydroxide

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Enhanced Immune Adjuvant Activity of Aluminum Oxyhydroxide Nanorods through Cationic Surface Functionalization Bingbing Sun, Zhaoxia Ji, Yu-Pei Liao, Chong Hyun Chang, Xiang Wang, Justine Ku, Changying Xue, Vahid Mirshafiee, and Tian Xia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 8, 2017

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ACS Applied Materials & Interfaces

Enhanced Immune Adjuvant Activity of Aluminum Oxyhydroxide Nanorods through Cationic Surface Functionalization Bingbing Suna,b,*, Zhaoxia Jic, Yu-Pei Liaob, Chong Hyun Changc, Xiang Wangb, Justine Kud, Changying Xuee, Vahid Mirshafieeb, and Tian Xiab* a

State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Dalian University of Technology, 2 Linggong Road, 116024, Dalian, China b Division of NanoMedicine, Department of Medicine, University of California, Los Angeles, CA 90095, United States; c California NanoSystems Institute, University of California, Los Angeles, CA 90095, United States; d Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095, United States; e School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, 116024, Dalian, China; *Address correspondence to [email protected] and [email protected].

1 ACS Paragon Plus Environment


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ABSTRACT Aluminum salt-based vaccine adjuvants are prevailingly used in FDA-approved vaccines for the prevention of infectious diseases for over eight years. Despite of their safe applications, the mechanisms regarding how the material characteristics affect the interactions at nano-bio interface and immunogenicity still remain unclear. Recently, studies have indicated that NLRP3 inflammasome activation plays a critical role to induce adjuvant effects that is controlled by the inherent shape and hydroxyl contents of aluminum oxyhydroxide (AlOOH) nanoparticles, however, the detailed relationship between surface properties and adjuvant effects for these materials remain unknown. Thus, we engineered AlOOH nanorods (ALNRs) with controlled surface functionalization and charge to assess their effects on the activation of NLRP3 inflammasome in vitro and potentiation of immunogenicity in vivo. It is demonstrated that NH2functionalized ALNRs exhibited higher levels of cellular uptake, lysosomal damage, oxidative stress, and NLRP3 inflammasome activation than pristine and SO3H functionalized ALNRs in cells. This structure-activity relationship (SAR) also correlates with material’s adjuvant activity using ovalbumin (OVA) in a mouse vaccination model. This study demonstrates that surface functionalization of ANLRs is critical for rational design of aluminum-based adjuvants to boost antigen-specific immune responses for more effective and long-lasting vaccination.

KEYWORDS: surface functionalization, oxyhydroxide, vaccine adjuvant

IL-1β,

NLRP3


inflammasome,

aluminum

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1. INTRODUCTION Vaccinations stimulate individual’s host immune responses and have remained as an essential way for preventing infectious diseases and infection that could lead to cancers.1 To establish long-lasting immunogenicity against antigens, adjuvants are required in vaccine formulations.2 Thus far, only four adjuvants e.g., aluminum salts, AS03 (squalene-based adjuvant), AS04 (aluminum hydroxide plus monophosphoryl lipid A), and MF59 (squalene-based adjuvant) have been used in the FDA-approved vaccines.3-6 Among these, aluminum salt-based nanoparticles have been acting as an important component in majority of vaccines to induce antigen-specific immune responses, especially the Th2-biased humoral immune responses.2 Thus far, they have been included in inactivated (e.g., hepatitis A), toxoid (e.g., tetanus), and subunit (e.g., pneumococcal) human vaccines.7-8 Though these particles are widely known for their ability to promote the immune responses, which involves various mechanisms including the controlled release of antigens,9-10 local inflammation induction,11 antigen-presenting cell recruitment,12-13 the NLRP3 inflammasome activation,13-14 increased uptake of antigens through phagocytosis,15 and dendritic cell membrane perturbation.16 However, there are still many aspects of their physicochemical properties that determines the immunogenicity are not thoroughly understood.2 Further elucidating the characteristics of aluminum-based adjuvants that determine the immunogenicity will shed light on developing more effective vaccines.8 Characteristics of nanomaterials including size, shape, crystal structure, and surface coatings are known to determine the nano-bio interactions17-18 and induction of immune responses.13, 19-20 A recent study by us has shed light on the structure-activity relationships (SARs), which showed aluminum oxyhydroxide (AlOOH) nanoparticle’s shape and crystallinity affected the level of NLRP3 inflammasome activation in vitro and immune response in vivo,14 demonstrating the potential of AlOOH nanorods with enhanced adjuvancy.14 In addition, size21 and surface phospholipid bilayer coatings22 have already been considered to play an important role in Alumbased immunostimulation due to their effects on antigen uptake. In addition to these properties, 3 ACS Paragon Plus Environment


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surface functionalization is considered as a crucial feature in nanoparticle designs that may affect their efficiency in vaccine delivery and promotion of an immune response,23-25 owing to their ability to promote antigen uptake, dendritic cell maturation and ROS generation. Thus, it is critical to elucidate how differently functionalized aluminum nanoparticles will affect the efficiency of immunostimulation and to establish SARs for the design of optimal Alum-based adjuvants for vaccination. In our recent study, aluminum oxyhydroxide (AlOOH) nanorods (ALNRs) with different adjuvant activities were prepared through precise tuning of crystal structure and shape and only nanorods with crystallinity (5-21%) showed the highest adjuvant activity. The small operation window for the synthesis (2-3 hrs at 200 °C by hydrothermal method) makes the potential scaleup of this adjuvant challenging. As an alternative, in this study we performed a simple surface functionalization on fully crystallized ALNRs using either -NH2 or -SO3H groups. These highly crystallized ALNRs exhibited low hydroxyl content and low immunostimulatory effects both in vitro and in animal models based on our previous report.14 For comparison purposes, the low crystallinity ALNR-C (21%) that showed high adjuvant activities in vivo was included as a positive control.14 We determined whether surface functionalization could impact the NLRP3 inflammasome activation in cells and the potentiation of humoral immune responses including OVA-specific antibody productions in mice. We demonstrated that the NH2-functionalized ALNRs (ALNR-NH2) exhibited higher level of cellular uptake, lysosomal damage and oxidative stress that determined the level of NLRP3 inflammasome activation and IL-1β section. As a result, ALNR-NH2 showed higher levels of OVA-specific antibody titers compared to pristine and SO3H-functionalized particles. This study provides rational material design strategies for aluminum-based adjuvants that may further increase the effectiveness of human vaccination.


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2. EXPERIMENTAL SECTION 2.1. Synthesis and Surface Functionalization of Aluminum Oxyhydroxide Nanorods. The synthesis of aluminum oxyhydroxide nanorods (ALNRs) was described previously, and the pristine crystalline ALNRs were synthesized at 200 °C for 16 h.14 Pristine ALNRs were washed and dried before use. Pristine ALNRs were further functionalized with APTES (Sigma) or HSPSA (Sigma).26 Briefly, pristine particles were mixed with 0.1 M of APTES or HSPSA aqueous solution. The mixtures were incubated for 60 min @ 25 °C with constant stirring, followed by an additional 90 min @ 115 °C with constant stirring. After reaction, functionalized ALNRs were washed three times and kept in deionized water before use. 2.2. Characterization of Aluminum Oxyhydroxide Nanorods. The particles’ primary size and morphology were determined using transmission electron microscopy (JEOL 1200EX). The hydrodynamic sizes and zeta potentials in exposure media were measured with high-throughput dynamic light scattering (DLS) instrument (Wyatt Technology) and ZetaPALS (Brookhaven), respectively. The surface functional groups were assessed using Fourier transform infrared spectroscopy (FTIR) (Bruker). The absorbance, fluorescence and luminescence in the following experimental procedures were measured using a SpectraMax M5 microplate reader. 2.3. Abiotic ROS Generation Quantification. Abiotic ROS generation by the nanorods was assessed by DCF assay. The preparation of H2DCF reagent (Thermo Scientific) was described by us as reported previously.14 To determine the total abiotic ROS generation, ALNRs (25 µg/mL) were incubated with H2DCF reagent in 100 µL with constant shaking for 3h at room temperature (RT). The emission of fluorescence at 527 nm was determined with an excitation wavelength of 492 nm. 2.4. Determination of the Endotoxin Levels. The endotoxin levels in ALNR particles were assessed by LAL assay (Lonza). ALNR particles (500 µg/mL) were mixed with LAL reagent in 50 µL for 10 min at 37 °C. Next, 100 µL of chromogenic substrate solution was introduced to



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the mixture, which was further incubated for 6 min at 37 °C. Finally, 100 µL of acetic acid (25%) was applied to the mixture and the absorbance was measured at 405 nm. 2.5. Cell Culture and Particle Treatment. THP-1 cells from ATCC were maintained in medium composed of RPMI 1640 (Corning), fetal bovine serum (10%, Corning), penicillinstreptomycin (100 U/mL-100 µg/mL, Thermo Scientific) and beta-mercaptoethanol (50 µM, Sigma). NLRP3 and ASC knockdown THP-1 cells were purchased from InvivoGen, and were cultured in medium composed of RPMI 1640, fetal bovine serum (10%), Normocin (100 µg/mL, InvivoGen), and HygroGold (200 µg/mL, InvivoGen). Cells were seeded in a well of 96-well plate at 3×104 cells/100 µl of medium that was supplemented with phorbol, 12-myristate, 13acetate (1 µg/mL, PMA, Sigma) to initiate their differentiation (dTHP-1). The medium was replaced after 16 h, and the differentiated cells were exposed to 500 µg/mL of ALNR particles with LPS (10 ng/mL, Sigma) for 6 h with 5% CO2 at 37 °C. Next, cell supernatant was collected to determine IL-1β production using human IL-1β ELISA kit (R&D Systems).27 For the inhibitor studies, dTHP-1 cells were treated with particles with or without N-acetylecystine (NAC) at 25 mM or cathepsin B inhibitor CA-074-Me (20 µM), followed by IL-1β quantification using ELISA. CellTiter 96 Aqueous One Solution Cell Proliferation assay kit (MTS, Promega) was used to determine the cytotoxicity of ALNRs. Briefly, after exposure to ALNR particles, the cell culture media was replaced with fresh media containing 16.7% of MTS stock solution, and cells were maintained at 37 °C with 5% CO2. Following 1h incubation, the absorbance was measured at 490 nm. 2.6. Determination of Cellular GSH Level. Cellular GSH was determined by GSH-Glo Glutathione assay (Promega). First, dTHP-1 cells were treated with 250 µg/mL of ALNR particles for 24 h with the addition of LPS (10 ng/mL). After exposure, the media was replaced with 100 µL of reaction mixture including Luciferin-NT (100X dilution) and glutathione Stransferase (100X dilution), and incubated at RT. After 30 min, Luciferin D detection reagent


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(100 µL) was added, and the cells were maintained at RT for another 15 min. Finally, the luminescence was determined. 2.7. Determination of Lysosomal Damage. dTHP-1 cells were treated with 500 µg/mL of ALNR particles in a Nunc Lab-Tek chamber slide (8-well) for 5 h with the addition of LPS (10 ng/mL). Next, the cells were stained with Magic Red (ImmunoChemistry Technologies) solution (420 µL) for 1 h with 5% CO2 at 37 °C. The cells were treated with PFA (4%) for 15 min at RT, followed by cell membrane and nuclei staining with WGA-Oregon Green® 488 (1 µg/mL, Thermo Scientific) and Hoechst 33342 (10 µM, Thermo Scientific) at RT. After 20 min incubation, cells were examined using confocal microscope (Leica Confocal SP2 1P-FCS). 2.8. Animal Vaccination. C57BL/6 mice (female, eight week old) were obtained from Charles River Laboratories. Mice were maintained following UCLA and NIH guidelines. On day 0, mice were vaccinated with 400 µg of ovalbumin (OVA, BioVendor R&D) or 400 µg/2mg of OVA/ALNRs through intraperitoneal administration. On day 7, mice were boosted with 200 µg of OVA through intraperitoneal administration. On day 14, animals were anesthetized, and the whole blood was collected. The serum was collected in a CAPIJECT blood collection tube by centrifugation at 1500 rpm for 5 min. The OVA-specific IgG1 and IgE were measured by ELISA following the procedures in our previous study.28 2.9. Statistical Analysis. In figures and tables, the values are represented as mean ± standard deviation (SD). For size measurement, zeta potential measurement, and all in vitro experiments, three replicate samples were included. For in vivo experiments, 6 animals were included. Statistical analysis was performed using two-tailed Student's t-test (two-group comparison) or Tukey’s test (multiple group comparison).



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3. RESULTS 3.1. Synthesis, Surface Functionalization and Characterization of Aluminum Oxyhydroxide Nanorods. Highly crystallized aluminum oxyhydroxide (γ-AlOOH, boehmite) nanorods (ALNRs) were synthesized using a hydrothermal method,14 and they exhibited low hydroxyl content and low adjuvant effect as described previously.14 Functionalization of ALNRs with either (3-aminopropyl) triethoxysilane (APTES) or 3-(Trihydroxysilyl)-1-propanesulfonic acid (HSPSA) was performed using a wet chemical process (Figure 1A).26 We included ALNRC, a positive control with higher hydroxyl content and high adjuvant effect,14 to study the effectiveness of surface functionalization. The pristine long aspect ratio (LAR) ALNRs exhibited 20 nm in diameter and 150-200 nm in length, and the functionalization of the ALNRs with APTES or HSPSA did not change their primary size or morphology (Figure 1B). The hydrodynamic size measurement showed that the sizes of ALNRs ranged from 203 to 618 nm in water and protein-containing cell culture medium or PBS (Table 1). Although hydrodynamic size does not represent the actual size of high aspect ratio material in solutions, studies have demonstrated that they could be used for size comparison purposes. Surface charge measurement showed that ALNRs exhibited zeta potential of 40±2 mV in water. In contrast, ALNR-NH2 was more positive at 61±7 mV, and ALNR-SO3H was negative at -24±3 mV, suggesting the presence of NH2 and SO3H functional groups, respectively. In both RPMI and PBS, their zeta potentials all became negative and in the range of -28 to -7 mV, due to the presence of high salt concentration and OVA (Table 1).


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Figure 1. (A) Schematic diagram of the surface functionalization of AlOOH with APTES and HSPSA. (B) Representative TEM images of ALNR, ALNR-NH2, and ALNR-SO3H nanorods. ALNR-C was used as control. The scale bar is 200 nm. Table 1. Characterization of aluminum oxyhydroxide nanorods in exposure media. Sample ALNR ALNR-NH2 ALNR-SO3H ALNR-C Imject Alum (Alum)

Hydrodynamic size (nm) RPMI plus 10% FBS 202.77 ± 7.76 311.30 ± 9.35 618.23 ± 10.72 414.97 ± 10.70 202.90 ± 1.10 338.73 ± 6.38 367.57 ± 8.83 512.30 ± 9.59 1144.80 ± 38.56 607.23 ± 51.87 Water

PBS plus 0.2% OVA 422.90 ± 15.48 389.20 ± 43.32 346.20 ± 5.01 475.60 ± 33.36 379.67 ± 31.84

Water 40.32 ± 1.79 61.44 ± 7.22 -23.80 ± 2.73 39.34 ± 1.83 -4.05 ± 1.43

Zeta potential (mV) RPMI plus PBS plus 10% FBS 0.2% OVA -9.64 ± 2.17 -20.62 ± 4.78 5.51 ± 17.79 -21.54 ± 2.35 -2.87 ± 13.23 -28.36 ± 11.38 -7.43 ± 2.95 -9.25 ± 2.63 -8.78 ± 13.02 -21.47 ± 8.01

FTIR was used to further analyze the functionalization of particles. Characteristic bands confirming the presence of OH groups on ALNRs were identified, including symmetric OH deformation (1067 cm-1), asymmetric OH deformation (1156 cm-1), symmetric stretching vibration (3095 cm-1), and asymmetric stretching vibration (3300 cm-1) of OH groups (Figure 2 and Table S1).29 By modifying the particle surfaces with either APTES or HSPSA, the intensities of these four bands decreased (Figure 2 and Table S1). For APTES-modified nanorods, bending vibration of N-H and deformation vibration of N-H bond were observed at 1539 and 1558 cm-1, respectively (Figure 2A). While for HSPSA-modified nanorods, asymmetric stretching vibration of SO2 was found at 1335 and 1413 cm-1 (Figure 2C). Together



with zeta potential measurements, the FTIR analysis suggests successful grafting of NH2- or SO3H- groups on pristine ALNR surfaces.

3500

Presence of SO2

1335

1500140013001200

ALNR

ALNR-SO3H

3500

Wavenumber (cm ) -1

1200

1067 1000

Wavenumber (cm-1)

Decrease of OH

D

ALNR-SO3H

1156

3095

2500

Wavenumber (cm-1)

Absorbance (a.u.)

ALNR

1413

Absorbance (a.u.)

C

3000

ALNR-NH2

3000

Wavenumber (cm )

ALNR-SO3H 1200

2500 -1

ALNR

1067

1300

Wavenumber (cm-1)

ALNR

1156

1400

3095

1500

ALNR-NH2

3300

1600

Absorbance (a.u.)

ALNR-NH2

ALNR

3300

Absorbance (a.u.)

1558 1539

ALNR

1700

Decrease of OH

B

Absorbance (a.u.)

Presence of NH 2

A Absorbance (a.u.)

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1000

Wavenumber (cm-1)

Figure 2. FTIR characterization of ALNRs. FTIR analysis of (A, B) ALNR-NH2 and (C, D) ALNR-SO3H showing the functionalization of ALNRs with either -NH2 or -SO3H group. Functional groups on synthesized ALNR have been shown to be capable of producing reactive oxygen species (ROS) and biological activities.14, 30-31 Considering the possibility of different species of ROS, we used DCF assay to determine the total abiotic ROS generation. It is demonstrated that APTES functionalization greatly enhanced the ROS generation potential of ALNRs, even higher than that of the positive control, ALNR-C;14 while functionalization with HSPSA did not significantly change the oxidation potential (Figure 3).


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Abiotic ROS generation potential 3000

DCF Fluorescence Intensity

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c b

2000 d 1000

a

a

0

Figure 3. Abiotic analysis of total ROS production induced by ALNRs. ALNRs (25 µg/mL) were incubated with H2DCF reagent in a volume of 100 µL for 3h at RT. The fluorescence emission at 527 nm was determined with an excitation of 492 nm. Statistical analysis was performed using Tukey’s test. Values that do not share the same letter indicate statistical differences at p

Enhanced Immune Adjuvant Activity of Aluminum Oxyhydroxide

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