Immunomodulatory effects of Alhagi honey polysaccharides encapsulated into PLGA nanoparticles

Accepted Manuscript Title: Immunomodulatory effects of Alhagi honey polysaccharides encapsulated into PLGA nanoparticles Authors: Adelijiang Wusiman, Shuwen Xu, Haiyu Ni, Pengfei Gu, Zhenguang Liu, Yue Zhang, Tianxin Qiu, Yuanliang Hu, Jiaguo Liu, Yi Wu, Deyun Wang, Yu Lu PII: DOI: Reference:

S0144-8617(19)30115-8 https://doi.org/10.1016/j.carbpol.2019.01.102 CARP 14560

To appear in: Received date: Revised date: Accepted date:

13 December 2018 21 January 2019 29 January 2019

Please cite this article as: Wusiman A, Xu S, Ni H, Gu P, Liu Z, Zhang Y, Qiu T, Hu Y, Liu J, Wu Y, Wang D, Lu Y, Immunomodulatory effects of Alhagi honey polysaccharides encapsulated into PLGA nanoparticles, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.01.102 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Immunomodulatory effects of Alhagi honey polysaccharides encapsulated into PLGA nanoparticles

Adelijiang Wusimanab, Shuwen Xuab, Haiyu Niab, Pengfei Guab, Zhenguang Liuab, Yue

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Zhangab, Tianxin Qiuab, Yuanliang Huab, Jiaguo Liuab, Yi Wuab, Deyun Wangab*, Yu

a

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Luc*

: Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine,

: MOE Joint International Research Laboratory of Animal Health and Food Safety,

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b

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Nanjing Agricultural University, Nanjing 210095, PR China.

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College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR

c

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China.

: National Research Center of Veterinary Biologicals Engineering and Technology,

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*

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Jiangsu Academy of Agricultural Science, Nanjing 210014,China

Correspondence and reprint requests: Deyun Wang, Ph.D.; Institute of Traditional

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Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P R China; E-mail: [email protected]; Tel:

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0086-25-84395203; Fax: 0086-25-84398669. Yu Lu, Ph.D, National Research Center of Veterinary Biologicals Engineering and Technology, Jiangsu Academy of Agricultural Science, Nanjing 210014,China. E-mail: [email protected]; Tel: 0086-25-84392027; Fax: 0086-25-84392018.

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Highlights 

FT-IR and GPC has been employed to characterize the Alhagi honey polysaccharides (AHP).



AHP and OVA encapsulated in PLGA to form a stable and high encapsulated

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nanoparticles.

AHPP significantly stimulated surface molecule expression in macrophages.



AHPP/OVA significantly increased the level of IgG and Th-associated

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

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cytokines.

honey

polysaccharides

(AHP)

have

been

widely

studied

as

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Alhagi

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Abstract

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immunomodulators. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles have been frequently used to control the release of drugs. In this study, AHP was extracted and

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encapsulated within PLGA (AHPP). Enhancement of immune activity in vitro and the

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adjuvanticity when inoculated with OVA were evaluated. The results demonstrated that the average molecular weight of AHP was 46.8 kDa and possessed typical polysaccharide absorption peaks. The entrapment efficiency for AHP within AHPP

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was 65.76±3.31%. AHPP significantly stimulated phagocytic activity, MHCII and CD86 expression in macrophages. Further investigation showed that AHPP/OVA significantly enhanced lymphocyte proliferation and improved the CD4+/CD8+ T cell ratio. Moreover, AHPP/OVA treatment significantly increased IgG levels and 2

up-regulated Th-associated cytokines with overall Th1 polarization. These studies demonstrated that AHP encapsulated within PLGA as a vaccine delivery system enhanced adaptive immunity.

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Abbreviations: AHP, Alhagi honey polysaccharides; cAHP, crude Alhagi honey polysaccharides; PLGA, Poly(lactic-co-glycolic acid); EE, encapsulation efficiency;

lipopolysaccharide;

PBS,

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TEM, transmission electron; PDI, poly dispersity index; OVA, ovalbumin; LPS, phosphate

buffer

solution;

MTT,

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, dimethyl

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sulfoxide; APC, antigen-presenting cell; OVA-FITC, fluorescein isothiocyanate

CD86,

Anti-Mouse-CD86-PE

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Anti-Mouse-MHCII-APC;

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labeled ovalbumin; ELISA, enzyme-linked immunosorbent assay; MHCII, antibodies;

CD80,

Alhagi

honey

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Keywords:

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Anti-Mouse-CD80-FITC antibodies.

polysaccharides;

Poly(lactic-co-glycolic

acid);

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Macrophages; OVA; Immune response; Adjuvant

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1. Introduction Herbal polysaccharides are natural polymers that exert multiple pharmacological

activities involving immune enhancement (Fang & Huang, 2018), and anti-tumor (Delma, Srinivasan, Aravindan, Natarajan, Herman & Aravindan, 2013; Wang et al., 2016), antioxidant (Wang, Zhao, Sun & Yang, 2014; Wang, Liu & Qin, 2017), and 3

anti-diabetic (Cunha et al., 2016; Wang, Chen, Pan, Gao & Chen, 2017) effects. Polysaccharides have received increased attention in new drug development due to strong immunomodulatory effects combined with a relative lack of toxicity (Bolhassani, Javanzad, Saleh, Hashemi, Aghasadeghi & Sadat, 2014). As such,

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polysaccharides represent a promising therapeutic avenue as vaccine adjuvants. Alhagi honey is a light yellow granulated sugar condensed from secreted fluid of

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the leaves of Alhagi pseudalhagi Desv. Alhagi pseudalhagi Desv., which belongs to the family Fabaceae, has been traditionally used as a medicinal plant in Northwest

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China, Northern India, and other Central Asian countries for centuries (G, Y, J & J,

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2018; Ghosal & Srivastava, 2010; Srivastava, Sharma, Dey, Wanjari & Jadhav, 2014).

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It is normally used as an immunomodulator to enhance immunity, and as a remedy for

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cough and diarrhea in China and other Central Asian countries (Marashdah &

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Al-Hazimi, 2010; Marashdah & Farraj, 2010; Muhammad, Hussain, Anwar, Ashraf & Gilani, 2015). Modern studies revealed that the major constituent of Alhagi honey is

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polysaccharides, which exert various pharmacological effects such as enhanced

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immunity, and anti-tumor, anti-bacterial, and hepatoprotective effects (Ghosal & Srivastava, 2010; Mamatkulova, Nishanbaev, Mukarramov & Khidyrova, 2012; Atta & Abo, 2004). AHP is composed of rhamnose, mannose, glucose, and galactose, with

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an MW of 47479 Da and a molar monosaccharide ratio of 2.11: 3.80: 4.19: 7.43. The main chain of AHP is primarily composed of -2,3)‒α‒L-Rhap‒(1,2,6)α‒D‒Manp‒ (1,3)‒β‒D‒Galp-(1,3,6) α‒D-Galp‒ (1-, while the side chain is composed of α-D‒ Glcp‒(1‒ (Jian, Chang, Ablise, Li & He, 2014). However, AHP also have some 4

disadvantageous characteristics, such as brief biological half-life, low bioavailability and non-focused action scope, which limit its long-lasting immune enhancement in the body. To address these limitations, further research should be conducted here to improve its bioavailability.

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Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable polymer that has been widely used an adjuvant to carry antigens and drugs (Shen, Lee, Choi, Wen, Wang &

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Burgess, 2016; Singh, Alkie, Nagy, Kulkarni, Hodgins & Sharif, 2016). Favorable

characteristics of PLGA include safety, biodegradability, and stability (Arias et al.,

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2015; Peters et al., 2017). A number of studies have demonstrated that encapsulation

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of antigens or polysaccharides into PLGA nanoparticles increased antigen-presenting

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cell (APC) antigen uptake, controlled drug release, and also enhanced cellular and

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humoral immune responses (Luo et al., 2017; Xia et al., 2018). In order to improve

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the time and intensity of the immunomodulatory effects of AHP in the body, this study encapsulated the AHP in PLGA nanoparticles to improve its adjuvant activity.

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This study investigated whether encapsulation of AHP in PLGA could improve

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immunoenhancement effects of AHP. In this study, AHP was extracted and purified, and a series of verifications were performed to confirm polysaccharide homogeneity and structural conformation. AHP was encapsulated within PLGA nanoparticles and

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activity of AHP was tested in vitro. Enhancement of antigen-specific immune responses by AHPP/OVA was assessed in vivo.

2. Materials and methods 2.1. Materials 5

Alhagi honey was purchased from the hospital of Xinjiang traditional Uyghur medicine in Urumqi, China. PLGA (75:25, MW 18KDa) was purchased from Jinan Daigang Biomaterial (Shandong, China). Pluronic F68 (F68) was purchased from Shanghai Yuanye Biotechnology (Shanghai, China). AB-8 Macroporous Adsorption

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Resin, DEAE-52, Sephadex G-100, Micro-BCA Protein Assay Kit, and OVA-FITC were purchased from Solarbio Science & Tecnology Co. (Beijing, China).

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Phytohaemagglutinin (PHA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Lipopolysaccharide (LPS), Freund’s complete adjuvant (FCA), and

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albumin from chicken egg white (OVA) were purchased from Sigma-Aldrich Co.

Engineering (Suzhou,

Mouse-CD86-PE,

China).

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Chemical

Anti-Mouse-CD3e-FITC,

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of

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(MO, USA). Dimethyl sulfoxide (DMSO) were purchased from Zhengxing Institute Anti-Mouse-MHCII-APC,

anti-

Anti-Mouse-CD4-APC

and

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Anti-Mouse-CD8a-FITC antibodies were all purchased from eBioscience Inc. (San Diego, CA, USA). Assay kits for OVA-specific IgG, IgG1, IgG2a, IL-4, IL-6, IFN-γ,

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and TNF-α were purchased from Wuhan Boster Biological Technology (Wuhan,

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China). All other chemicals used were of analytical grade. 2.2. Preparation and identification of AHP AHP was extracted using a column chromatography method as previously

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reported (Jian, Chang, Ablise, Li & He, 2014). Briefly, Alhagi honey (100 g) was refluxed several times using petroleum ether and ethanol to remove lipids and impurities. The processed sample was submitted to aqueous extraction, with 1000 mL of hot water at 70 °C (1.5 hours). The remaining aqueous extract was filtrated and 6

concentrated to 1/4 of the original volume by rotary vacuum evaporator. The filtrate was then precipitated with anhydrous ethanol to 50% at 4 °C for overnight. After extraction of polysaccharides, the precipitate was dissolved in water (200 mg/mL) and deproteinated by the Sevag method (1-butanol/chloroform, V/V 1:4) then further

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processed using a macroporous resin AB-8 column (3×30 cm, flow rate of 0.5 mL/min) for decolorization. Finally the supernatant was lyophilized to give crude

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Alhagi honey polysaccharides (cAHP). cAHP was further separated in a fiber gel

DEAE-52 column (2.6×50 cm, flow rate of 1 mL/min ) with gradient elutions

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(deionized water, 0.1, 0.2, 0.5 M NaCl solutions). Water elution part was collected

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and further purified with a Sephadex G-100 column (2.6×70 cm, flow rate of 0.25

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mL/min) to obtain pure and homogeneous AHP.

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The elution curve of polysaccharide from the Sephadex G-100 column and the

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carbohydrate content of purified AHP was determined using the phenol sulfuric acid method (Tatsuya et al., 2016). The protein and impurity content of the polysaccharide

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was determined by UV absorption. The homogeneity and molecular weight was

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determined by gel permeation chromatography (GPC). FT-IR spectra of AHP (2 mg of AHP per 200 mg of KBr powder) was investigated with the KBr pellets method (Shi et al., 2007).

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2.3. Preparation of AHPP and AHPP/OVA nanoparticles AHPP nanoparticles were prepared according to a previous study (Gu et al., 2017). Briefly, 10 mg of AHP was dissolved in 125 μL of ultrapure water (internal water phase W1) and added to 1.25 mL of dichloromethane containing 100 mg of 7

PLGA (organic phase, O). The mixture was probed sonicated (5%, amplitude) for 90 seconds in an ice bath to obtain the primary emulsion (W1/O). The primary emulsion was then mixed with 10 mL of ultrapure water containing 88 mg of Pluronic F68 (F68) (W2) for 2 min (10%, amplitude) and stirred to evaporate the organic solvent and form

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a stable W1/O/W2 emulsion (PH=7.0). AHPP/OVA (PH=7.0) was produced using the same method except that W1

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contained both OVA and AHPP in ultrapure water. Blank/OVA PLGA nanoparticles (BP/OVA, PH=7.0) were prepared in the same way but without AHP.

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2.4 Entrapment efficiency and morphological characterization

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AHP and OVA-entrapment efficiency in the AHPP and AHPP/OVA preparations

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were measured using the phenol sulfuric acid method and Micro-BCA Protein Assay

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Kit (Solarbio Science & Tecnology Co, Beijing, China). The nanoparticles were

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collected by centrifugation at 12,000 rpm for 30 min at 4˚C, un-encapsulated AHP and OVA were measured in the supernatant (Arasoglu et al., 2016). Each batch was

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analyzed in triplicate. Morphological examination of the resultant nanoparticles was

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performed using transmission electron microscopy (TEM, Tecnai 12, Philips, Holland).

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2.5. Stability and in vitro release of nanoparticles Physical stability of nanoparticles was assessed by monitoring changes in

particle size, PDI, and zeta potential using a laser particle size analyzer (Hydro2000Mu,

MAL1009117,

Malvern

measurement was performed in triplicate. 8

Instruments,

UK).

Each

sample

Release of AHP and OVA from AHPP/OVA in vitro was performed by monitoring changes of the free AHP and OVA in solution. Three milligram of AHPP/OVA were dispersed in 30 mL of deionized water (PH=7.0) and placed in a shaker bath (37 ℃, 80 rpm). At predetermined time intervals (2, 4, 6, 8, 12 hours and

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1, 2, 4, 8, 12, 16, 20, 24, 30 days), the suspension was centrifuged at 12,000 rpm for 30 min. The contents of free AHP and OVA in supernatant were determined by

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phenol sulfuric acid method and Micro-BCA Protein Assay Kit, and each batch was analyzed in triplicate.

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2.6. Cell viability

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Peritoneal macrophages were isolated and purified from ICR mice according to

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previous reports (Zhang, Ma, Zhang, Song, Sun & Fan, 2017). Macrophages were

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plated at 5×105 cells/mL in a 96-well plate and cultured for 4 hours in a humid

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atmosphere with 5% CO2 at 37 ◦C (Thermo, Waltham, USA). Non-adherent cells were removed by washing with PBS, and adherent cells were pretreated with AHP and

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AHPP at various concentrations. Cells cultured in DMEM were used as controls.

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Cells were incubated for 48 hours and then the cell viability was assayed by the MTT method. Briefly, 30 μL of MTT solution (5 mg/mL) was added to each well. After incubation at 37 °C for 4 h, the plates was centrifuged (4000 rpm, 5 min) and the

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untransformed MTT in the supernatant was removed and then 100 μL of DMSO was added. The solution was shaken with a micro-oscillator (Shanghai Yairong Biochemical Instrument Factory, Shanghai, China) for 10 min to completely dissolve the crystals, and the absorbance was measured using a microplate reader (Thermo, 9

Waltham, USA) at 570 nm (Liu et al., 2015). 2.7. Phagocytic assay Macrophages were plated on 24-well plates (5×105 cells per well) as described above. OVA-FITC, OVA-FITC-loaded BP (BP/OVA-FITC), and OVA-FITC-loaded

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AHPP (AHPP/OVA-FITC) were subsequently added to maintain the final OVA-FITC concentration at 20 μg/mL. Cells were incubated for 12 hours in a CO2 incubator.

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Cells were washed twice with PBS, then fixed with 4% paraformaldehyde prior to evaluation of phagocytic activity using flow cytometry (FACSCalibur, BD

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Biosciences, San Jose, CA, USA). Experiments were conducted in quadruplicate.

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2.8. Immunophenotypic analysis

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Peritoneal macrophages were plated onto a 24-well plate at a concentration of

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5×105 cells per well and purified as described above. After treatment with AHP, AHPP,

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or BP for 24 hours, the cells were collected and washed twice with PBS, then stained using Anti-Mouse-MHCII-APC and Anti-Mouse-CD86-PE antibodies (eBioscience,

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San Diego, CA, USA) for 30 min at 4 °C without light. The cells were washed twice

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with PBS, then fixed with 4% paraformaldehyde prior (Liu et al., 2015). Expression of CD86 and MHCII on the surface of macrophages was measured by fluorescence-activated cell sorting. (FACSCalibur, BD Biosciences, San Jose, CA,

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USA). Experiments were conducted in quadruplicate. 2.9. Animal immunization ICR mice were obtained from Comparative Medicine Centre of Yangzhou University. All animal experiments were treated in accordance with the guideline of 10

Institutional Animal Care and Use Committee (IACUC), Nanjing Agricultural University IACUC, and the protocol was approved by the IACUC (No.: 2011BAD34B02). ICR mice (8 weeks old) were randomly separated into six groups and vaccinated

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subcutaneously with AHP/OVA (50 μg OVA+50 μg AHP in 0.2 mL), AHPP/OVA (50 μg OVA+50 μg AHPP in 0.2 mL), or BP/OVA (50 μg OVA+50 μg BP in 0.2 mL).

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Mice vaccinated subcutaneously with PBS, OVA, or FCA/OVA were used as controls. Following initial immunization, animals received equivalent booster doses at day 14.

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Mice were sacrificed at days 7, 21, and 35 after the second immunization, and blood

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2.10. Splenocytes proliferation assay

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and spleens were collected. Serum samples were separated and stored at -80˚C.

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Splenic lymphocytes were isolated from the immunized mice on day 21 after the

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second immunization. Splenocytes (2.5×105 cells/mL) were re-suspended in complete medium and re-stimulated with OVA (50 μg/mL), then incubated for 48 hours. Cells

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in the blank cells group were used as controls. MTT assay was used to assess cell 570

(control group).

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proliferation. Proliferation index = (A570 (experimental group) /A

Each test was analyzed using four repeated measurements (Bo et al., 2017).

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2.11. Lymphocyte immunophenotype analysis To investigate the T lymphocyte subpopulation, splenic lymphocytes were

harvested from the immunized mice on day 21 after the second immunization. Lymphocytes were incubated in 24-well plates (1×106 cells per well), re-stimulated with OVA (50 μg/mL), and incubated for 48 hours. Cells were collected and stained 11

with anti-CD3e-FITC, anti-CD4-APC, and anti-CD8a-PE antibodies (eBioscience, San Diego, CA, USA) for 30 min at 4 °C without light. The cells were washed twice with PBS, then fixed with 4% paraformaldehyde prior (Bo et al., 2017). Lymphocyte subpopulations were analyzed by flow cytometry (FACSCalibur, BD Biosciences, San

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Jose, CA, USA). Experiments were conducted in quadruplicate. 2.12. Determination of antibodies and cytokine levels

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OVA-specific IgG antibody, IgG1 and IgG2a isotypes in serum were measured

by enzyme-linked immunosorbent assay (ELISA) on days 7, 21, and 35 after the

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second immunization, while IL-4, IL-6, TNF-α, and IFN-γ levels were analyzed on

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day 35 by ELISA.

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2.13. Statistical analysis

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The quantitative data collected were expressed as means ± SEM Statistical

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significance was analyzed using Duncan’s multiple range test. A probability value (P)

3. Results

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less than 0.05 was considered to identify statistically significant.

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3.1. Extraction, isolation and purification of AHP The crude polysaccharides cAHP was isolated from Alhagi honey by hot-water

extraction, ethanol precipitation, deproteinization, and decolorization. The yield of

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cAHP from Alhagi honey was about 10.5% of the dried material. The crude polysaccharide was separated by the fiber gel DEAE-52 column and purified by Sephadex G-100 column. The main fraction was collected by DEAE-52 column accounted for 51.2% of the total cAHP content (Fig. 1A), indicating that this is the 12

main part of the cAHP. The main part of the cAHP was further purified through Sephadex G-100 column and detected by UV spectrometric analysis. The carbohydrate contents of AHP was 99.2% by the phenol-sulfuric acid method. As shown in Fig. 1B, the purified polysaccharides appear to be a single, symmetrical

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sharp peak, suggesting that AHP was homogeneous samples. There is no obvious absorption peak at 260 nm and 280 nm, so it can be judged that the AHP was free

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from nucleic acid and protein.

Fig. 1. (A) Elution curve of cAHP on a DEAE-52 column. (B) Elution curve of AHP 13

on a Spendex-100 column and UV spectrum. 3.2. Molecular weight and FT-IR spectra of AHP As shown in Fig. 2A, a single, symmetrical peak was also detected by GPC analysis, indicating that AHP was a homogeneous polysaccharide. According to GPC

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analysis, the estimated average molecular weight of AHP was 46.8 kDa, which was close to the reported molecular weight for Alhagi honey polysaccharides (47,479 Da)

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(Jian, Chang, Ablise, Li & He, 2014).

FT-IR spectra of AHP in the ranges of 4000-400 cm-1 are illustrated in Fig. 2B.

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FT-IR spectra of AHP displayed a strong absorption peak at 3431.31 cm-1 and a small

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band at 2936.76 cm-1, consistent with the presence of hydroxyl groups (-OH) and C-H

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stretching, respectively. Absorption peaks observed at approximately 1419.91 cm-1

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was consistent with the presence of carbonyl groups. The broad band at 1617.5 cm-1

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was assigned to the bending vibration of the water. The spectral characteristics in the region ranging from 1300-1000 cm-1 are consistent with C-O and C-C stretching

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vibrations in polysaccharides (Cai, Zou, Liang & Luan, 2018). These results indicated

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that AHP possessed typical absorption peaks of polysaccharides.

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Fig. 2. (A) Molecular weight of AHP was determined using GPC. (B) Infrared spectrum of AHP. 3.3. Characterization and stability of nanoparticles

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AHPP was prepared using the double emulsion technique. EE of AHP and

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AHPP/OVA were measured, as shown in Fig. 3A. The EE for AHP within AHPP was

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65.76±3.31%, while the EE for AHP and OVA within AHPP/OVA were 60.37±1.79% and 59.87±2.03%, respectively. The lower value for AHP within AHPP/OVA may be

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associated with the distribution of polysaccharides and antigens in PLGA

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nanoparticles. Particle size, zeta potential, and PDI of nanoparticles are important measurements that determine physiological distribution and stability (Xia et al., 2018).

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As shown in Fig. 3B, changes in PDI and zeta potential were negligible for 14 days, but were increased at days 21 and 28. AHPP particle size changed by day 14 and more

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extensive aggregation occurred by days 21 and 28 (Fig. 3C). The above results indicate AHPP/OVA activity maintained intact after storage at 37°C for 21 days. As shown in Fig. 3D, the cumulative releases of AHP and OVA in AHPP/OVA showed the same trend. During the first 6 hours, AHP and OVA were rapidly released from AHPP/OVA, and the cumulative releases of AHP and OVA were 24% and 17%, 15

respectively. However, from 8 hours to 12 days, the cumulative releases of AHP and OVA became slowly and the cumulative releases of AHP and OVA were less than 15%. From 12 days to 16 days, AHP and OVA boosted released again, and the cumulative releases of AHP and OVA were 33% and 31%, respectively. After 17 days,

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the cumulative releases of AHP and OVA became slowly again. The results of cumulative release demonstrated that AHPP had the effect of slow release.

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Morphologies of AHPP/OVA and BP/OVA were evaluated using TEM. As shown in Fig. 3E, AHPP/OVA was approximately spherical in shape and was composed of

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monodispersed particles with uniform size. The average particle sizes of AHPP/OVA

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and BP/OVA were approximately 220 nm and 200 nm, respectively. The particle size

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of OVA in the AHPP/OVA (Fig. 3F).

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of AHPP/OVA was slightly larger than BP/OVA, which may be caused by the amount

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Fig. 3. Characterization of AHPP and AHPP/OVA. (A) Encapsulation efficiency (EE) of AHPP and AHPP/OVA dispersions stored at 37˚C. (B and C) Zeta potential,

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PDI, and particle size of AHPP/OVA dispersions stored at 37˚C. (D) AHP and OVA

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release from the AHPP/OVA were incubated in deionized water (PH=7.0) for 35 days.

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(E and F) TEM micrographs of AHPP/OVA and BP/OVA. Results were expressed as

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means ± SEM (n=3).

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3.4. Cytotoxicity and in vitro uptake study

To test potential toxicity of AHP and AHPP nanoparticles, macrophages were

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incubated with different concentrations of AHP and AHPP. Cell viability was assessed

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by MTT assay. As shown in Fig. 4A, AHP displayed little toxicity against macrophages at 250 µg/mL, while AHPP was not toxic at 125 µg/mL. Although PLGA has the properties of nontoxicity and good biocompatibility, a certain amount

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of F68 was required as the surfactant in the preparation of AHPP nanoparticles, and the toxicity of AHPP probably resulted from the low toxicity of F68. To allow for direct comparison 125 µg/mL was chosen for both nanoparticles as the non-toxic dosage. 17

Cellular uptake of free OVA-FITC, BP/OVA-FITC, and AHPP/OVA-FITC nanoparticles by macrophages was investigated by pre-labeling OVA with FITC and performing flow cytometry analysis. As shown in Fig. 4B and C, the efficiency ratio of positive fluorescent cells for two AHPP/OVA-FITC groups (62.5 μg/mL and 31.25 was greater than for BP/OVA-FITC and OVA-FITC groups (P<0.05). These

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results suggested that AHPP facilitated antigen uptake in macrophages.

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μg/mL)

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Fig. 4. Cytotoxicity and cellular uptake in macrophages. (A) Viability of murine peritoneal macrophages after 48 hours exposure to AHP and AHPP. (B) Dot plot analyses of mouse peritoneal macrophages incubated with OVA-FITC coated AHPP. (C) Efficiency ratio of the phagocytosis response in each group. Results were

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expressed as means ± SEM (n=3). a–d Bars in the figure without the same superscripts differ significantly (P<0.05).

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3.5. Macrophage surface molecule expression

Up-regulation of MHCII and CD86 is important for activation of macrophages

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and further affects T-cell proliferation and subsequent immune responses. To

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investigate the effects of AHP and AHPP on macrophage expression of MHCII and

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CD86, purified macrophages were cultured in vitro and analyzed by flow cytometry.

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The macrophages were encircled then MHCII and CD86 positive cells treated by

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different formulations were displayed (Fig. 5A). As shown in Fig. 5B and C, compared to AHP and BP, AHPP at 62.5 µg/mL and 31.25 µg/mL induced greater

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MHCII and CD86 expression in each corresponding group (P<0.05).

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Fig. 5. (A) Dot plot analyses of MHCII and CD86 on peritoneal macrophages was

measured by flow cytometry. (B and C) Expression of MHCII and CD86 on a–e

Bars in

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peritoneal macrophages. Results were expressed as means ± SEM (n=3).

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the figures without the same superscripts differ significantly (P<0.05).

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3.6. Splenocyte proliferation and OVA-specific T-cell activation

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Spleen lymphocyte proliferation in response to AHPP/OVA and AHP/OVA-mice

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at 21 days after the second vaccination was measured. As shown in Fig. 6A, following stimulation by OVA, the proliferation index value of the AHPP/OVA group was

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significantly higher than that of the BP/OVA and AHPP/OVA groups (P<0.05), while

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the FCA-OVA group was not significantly different from the AHPP/OVA group (P>0.05). Therefore, the AHPP/OVA vaccine formulation led to more potent

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antigen-specific immune responses than the other formulations. Based on spleen lymphocyte proliferation results, AHPP/OVA-induced activation

of antigen-specific CD4+/CD8+ T cells at 21 days following the final immunization was further tested by flow cytometry analysis. A significantly greater percentage of CD4+/CD8+ T cells was observed in spleens (Fig. 6B and C) of mice vaccinated with 20

AHPP/OVA compared to APP/OVA, BP/OVA, or OVA alone, (P<0.05), while no difference from the FCA-OVA group was observed (P>0.05). T cell activation was elicited by AHPP/OVA, suggesting that the AHPP/OVA formulation had the potential to induce a more effective immune response than AHP/OVA and BP/OVA

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formulations, and CD4+ T cells may play an indispensable role in adjuvant activity.

Fig. 6. (A) Effects of drugs on splenic lymphocyte proliferation. (B) Dot plot analyses of subpopulations of CD4+ and CD8+ T cells. (C) Ratio of CD3+CD4+ to CD3+CD8+ 21

splenocytes harvested from vaccinated mice re-stimulated using OVA. Mice (n=4) were immunized with different vaccine formulations. a–d Bars in the figure without the same superscripts differed significantly (P<0.05). 3.7. Cytokine levels in serum

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Th1 cytokines (TNF-α and IFN-γ) and Th2 cytokines (IL-4 and IL-6) in serum were measured 35 days after the final vaccination. As shown in Fig. 7A and B, the

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AHPP/OVA formulation significantly increased expression of IL-4, IL-6, TNF-α, and IFN-γ compared to the AHP/OVA, BP/OVA, and OVA groups (P<0.05), which

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indicated that AHPP/OVA could enhance both Th1-type and Th2-type immune

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responses. In addition, there was no significant difference in TNF-α values between

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the AHPP/OVA group and the FCA/OVA group.

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Fig. 7. Cytokine secretion. (A) L-4, IL-6, (B) IFN-γ, and TNF-α levels in serum 35 days after the last immunization were measured by ELISA. Mice (n=4) were

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immunized with different vaccine formulations. a–e Bars in the figure without the same

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3.8. Serum antibody in vaccinated mice

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superscripts differ significantly (P<0.05).

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To better understand the effects of various OVA formulations on antibody

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response, levels of OVA-specific serum IgG were determined using indirect ELISA analysis at days 7, 21, and 35 after the last immunization. As shown in Fig. 8A,

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AHP/OVA-induced significantly higher antigen-specific IgG levels than the BP/OVA

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and free OVA groups on day 7 (P<0.05), but the antibody levels on days 21 and 35 decreased

significantly.

While

AHPP/OVA-induced

significantly

higher

antigen-specific IgG levels than AHP/OVA, BP/OVA, and free OVA on days 21 and

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35 after the last immunization (P<0.05), the FCA/OVA group was not significantly different from the AHPP/OVA group. These results indicated that AHPP formulations could induce a large and sustained humoral antibody response. As shown in Fig. 8B, C and D immunization with AHPP/OVA constructs was 23

nearly equal in response to the group injected with FCA on days 7, 21 and 35 after the last immunization. The expression of IgG1 and IgG2a in response to AHPP/OVA was greater compared to the controls at days 7, 21 and 35 after the final vaccination. In addition, AHPP/OVA-induced higher ratios of IgG2a/IgG1 than those induced by

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OVA alone, and equal to that of the FCA group.

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Fig. 8. (A) OVA-specific IgG levels at the indicated time points (B, C and D)

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Th2-associated isotype IgG1 levels, Th1-associated isotype IgG2a levels, and the rate of IgG2a/IgG1 at day 7, 21 and 35 after the final vaccination. Mice (n=4) were immunized with different vaccine formulations.

a–d

Bars in the figure without the

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same superscripts differed significantly (P<0.05).

4. Discussion Polysaccharides exert multiple pharmacological activities and are widely used as immunopotentiators. As such, polysaccharides have recently received a great deal of 25

attention as vaccine adjuvants. However, a major challenge in the use of polysaccharides as vaccine adjuvants is brief biological half-life and non-focused action scope. In recent years, polymer nanoparticles have been extensively investigated as drug delivery systems due to their targeting ability and controlled the

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release properties (Kamaly, Yameen, Wu & Farokhzad, 2016; Saad & Prud’Homme, 2016; Soppimath, Aminabhavi, Kulkarni & Rudzinski, 2001). PLGA is one of the

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most highly synthesized biodegradable polymers, commonly used for drug delivery

systems due to safety, biodegradability, stability in blood, non-inflammatory

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properties, and control the release of encapsulated drugs (Anwer, Almansoor, Jamil,

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Alshdefat, Ansari & Shakeel, 2016; Chen et al., 2014; Heiny & Shastri, 2015). Herein,

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we hypothesized that encapsulation of AHP into PLGA could control the release of

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AHP and slow metabolic rate, thereby enhancing immunomodulatory capacity and

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time of action of AHP. We further hypothesized that AHPP/OVA formulations could be effective adjuvants to induce a strong and long-lasting adaptive immune response.

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In this study, a delivery system with AHP and model antigen OVA encapsulated

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into PLGA nanoparticles was developed. The nanoparticles were stable, approximately spherical in shape, uniform in size and distribution, and showed a high encapsulation rate. Particle size, PDI, and zeta potential of AHPP/OVA did not change

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within 21 days at 37°C (Fig. 3A-C), indicating that AHPP/OVA had good stability for 21 days at 37°C as a vaccine delivery system. The release of AHPP/OVA also showed the same trend. Therefore, the AHPP/OVA formulations could provide long-lasting antigen persistence at the injection site after immunization. 26

Macrophages are the first line of defense against invading pathogens and also are an important bridge between innate and adaptive immunity (Biswas & Mantovani, 2010; Zhang, Ma, Zhang, Song, Sun & Fan, 2017). Macrophages engulf microbes and can present antigens to T-cells to promote adaptive immunity (Gordon & Martinez,

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2010; Murray & Wynn, 2011). Activated macrophages maintain homeostasis (Hume, 2006), and function as professional APC and as effector cells in humoral and cellular

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immunity (Wijburg, Vadolas, Sanders, Strugnell & van Rooijen, 2015). In this study,

phagocytosis by macrophages was detected, which is an important step in the uptake

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of antigens. As shown in Fig. 4B and C compared with free OVA-FITC, significantly

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elevated OVA uptake by macrophages was found in the AHPP/OVA-FITC group.

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These results suggested that AHPP facilitated antigen transport and uptake in

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macrophages. After phagocytic uptake, macrophages act as antigen-presenting cells

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with an expression of higher levels of co-stimulatory molecules. As shown in Fig. 5A, B, and C, AHPP increased MHCII and CD86 expression to levels significantly higher

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than in the AHP and BP groups (P<0.05). Activated macrophages that express

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up-regulated levels of MHCII and CD86 have been shown to be essential during induction of T-cell-mediated immune responses (Lv et al., 2016). Therefore, up-regulation of MHCII and CD86 molecules is an important mechanism underlying

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the adjuvant effect of AHPP. The spleen is an important immune organ. Lymphocyte proliferation is an indicator of immune-stimulation, reflecting the level of cellular immune response (Kuang et al., 2016). Our results demonstrated that the AHPP/OVA formulation 27

significantly increased proliferation of splenic lymphocytes compared with the free OVA, AHP/OVA, and BP/OVA groups (Fig. 6A), indicating that the AHPP/OVA formulation could induce strong cellular immune responses. In the present study, the ratio of CD4+/CD8+ T cells was considered a key immunological event following

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immune activation, and high CD4+/CD8+ lymphocyte ratios are typically observed in individuals with increased immune capacity (Lee et al., 2014; Xu, Zhao, Yang, Zhang,

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Li & Wang, 2013). Splenic lymphocytes were harvested from immunized mice 21

days after the second immunization, and the ratio of CD4+/CD8+ T cells was

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measured using flow cytometry. As shown in Fig. 6B and C, high CD4+/CD8+

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lymphocyte ratios were observed in spleens in the AHPP/OVA group (3.94),

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significantly higher than the free OVA (2.97) and the PBS control groups (2.66),

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which indicated that AHPP/OVA could enhance T cell immune effects.

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To better understand the effects of the AHPP/OVA formulation on T cells, blood serum was collected from the immunized mice 35 days after the final vaccination, and

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Th1 cytokines (TNF-α, IFN-γ) and Th2 cytokines (IL-4 and IL-6) were measured

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using ELISA kits. Mice immunized with the AHPP/OVA formulation showed higher levels of TNF-α, IFN-γ, IL-4, and IL-6 compared with the free OVA, AHP/OVA, and BP/OVA groups (Fig. 7A and B), which indicated that AHPP/OVA could enhance

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both Th1-type and Th2-type immune responses. In addition, there was no significant difference in IFN-γ values between the AHPP/OVA group and the FCA/OVA group (Fig. 7B). FCA is known to promote a robust antibody and Th1-type response (Luo et al., 2017; Xing et al., 2016). As such, these data suggested that AHPP/OVA 28

formulations mainly induced a stronger Th1 immune response. As shown in Fig. 8A and B, at day 7 expression of OVA-specific IgG, IgG1 and IgG2a in response to AHP/OVA was not significantly different compared with the AHPP/OVA and FCA/OVA groups (P>0.05), but antibody and isotypes levels

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decreased significantly at days 21 and 35. The PLGA/OVA formulation significantly enhanced production of IgG, IgG1 and IgG2a compared with free OVA (P<0.05), but

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produced a similar response as the FCA/OVA formulation at days 7, 21, and 35 after the last immunization. Polysaccharides can stimulate lymphocyte proliferation

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activity and enhance antibody titers (Zhang, Yin & Wei, 2010). However,

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polysaccharides metabolize faster in the body. It is possible that the short biological

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half-life of polysaccharides in the body was responsible for greater expression of

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OVA-specific IgG, IgG1 and IgG2a resulting from the AHP/OVA formulation at day 7.

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IgG1 antibody production is characteristic of a Th2-polarized immune response, while IgG2a antibody production is characteristic of a Th1-polarized immune response. The

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ratio of IgG2a/IgG1 is indicative of a Th1-biased immune response (Wang, Tan,

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Keegan, Barry & Heffernan, 2014). AHPP/OVA formulations could induce a large Th1-associated isotype IgG2a response and Th2-associated isotype IgG1 response. FCA is known to induce strong Th-1 polarized humoral immune responses. The

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IgG2a/IgG1 ratios resulting from AHPP/OVA immunization was nearly equal to that of the FCA/OVA group (Fig. 8B, C and D), further demonstrating that AHPP/OVA generated a stronger Th1-biased immune response compared to a Th2-biased immune response. 29

Safety, stability and the ability to induce immune response are the important factors in the successful preparation of vaccine adjuvants. Some diseases, protection is provided by an adequate antibody response, while other diseases require cellular immunity for protection. Aluminum adjuvant as an effective clinical adjuvant are

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efficiently generating a lasting antibody response, biased towards the Th2-type immune response, but it cannot induce an effective Th1-type immune response. Our

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results of the researches showed AHPP/OVA formulation could induced a mixed Th1/Th2-type immune responses, and biased towards the Th1-type immune response,.

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The long-term immune responses and strong Th1-biased immunity responses of

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AHPP/OVA formulation may provide an effective, safe and broadly applicable

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strategy to enhance cellular immunity against infections and diseases.

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5. Conclusions

In conclusion, AHP and PLGA delivery vehicles are both essential components

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of the adjuvant system, as the humoral and cellular immune responses of the BP/OVA

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and AHP/OVA groups were much lower than those of the AHPP/OVA group. The AHPP/OVA formulation had good colloidal stability and allowed for the sustained release of OVA. Mice immunized with the AHPP/OVA formulation showed greater

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lymphocyte proliferation and an increased ratio of CD4+ to CD8+ T cells. The AHP/OVA formulation only stimulated a short time OVA-specific IgG, IgG1 and IgG2a response. However, AHPP/OVA formulation stimulated a strong and continuous OVA-specific IgG, IgG1 and IgG2a response and cellular immune 30

responses, along with a strong Th1-associated immune response. Thus, encapsulation of AHP in PLGA could improve the immunoenhancement effects of AHP, and the AHPP/OVA formulation could potentially serve as a novel and effective vaccine

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adjuvant to induce strong and long-term immune responses.

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Acknowledgments

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This project was supported by the National Natural Science Foundation of China

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(Grant nos. 31872509, 31672596), the Special Fund for Agro-scientific Research in

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the Public Interest (Grant nos. 201403051), and the Priority Academic Program

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Development of Jiangsu Higher Education Institutions (PAPD). We are grateful to all

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of the other staff members at the Institute of Traditional Chinese Veterinary Medicine

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of Nanjing Agricultural University for their assistance in this study.

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