Hyaluronic Acid-Modified Cationic LipidâPLGA Hybrid Nanoparticles

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Hyaluronic acid-modified cationic lipid-PLGA hybrid nanoparticles as a nanovaccine induce robust humoral and cellular immune responses Lanxia Liu, Fengqiang Cao, Xiaoxuan Liu, Hai Wang, Chao Zhang, Hongfan Sun, Chun Wang, Xigang Leng, Cunxian Song, Deling Kong, and Guilei Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01135 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 23, 2016

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ACS Applied Materials & Interfaces

Hyaluronic acid-modified cationic lipid-PLGA hybrid nanoparticles as a nanovaccine induce robust humoral and cellular immune responses

Lanxia Liu1, Fengqiang Cao1, Xiaoxuan Liu1, Hai Wang1, Chao Zhang1, Hongfan Sun1, Chun Wang1, 2, Xigang Leng1, Cunxian Song1, Deling Kong1, Guilei Ma1*

The Tianjin Key Laboratory of Biomaterials, Institute of Biomedical Engineering, Peking Union Medical College & Chinese Academy of Medical Sciences, Tianjin 300192, China 2

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN,

USA

*

Correspondence to: Guilei Ma (E-mail: [email protected])

ACS Paragon Plus Environment


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ABSTRACT Here, we investigated the use of hyaluronic acid (HA)-decorated cationic lipid-poly(lactide-co-glycolide) acid (PLGA) hybrid nanoparticles (HA-DOTAP-PLGA NPs) as vaccine delivery vehicles, which were originally developed for the cytosolic delivery of genes. Our results demonstrated that after the NPs uptake by dendritic cells (DCs), some of the antigens that were encapsulated in HA-DOTAP-PLGA NPs escaped to the cytosolic compartment, and whereas some of the antigens remained in the endosomal/lysosomal compartment, where both MHC-I and MHC-II antigen presentation occurred. Moreover, HA-DOTAP-PLGA NPs led to the up-regulation of MHC, co-stimulatory molecules and cytokines. In vivo experiments further revealed that more powerful

immune

responses

were

induced

from

mice

immunized

with

HA-DOTAP-PLGA NPs when compared with cationic lipid-PLGA nanoparticles and free ovalbumin (OVA); the responses included antigen-specific CD4+ and CD8+ T-cell responses, the production of antigen-specific IgG antibodies and the generation of memory CD4+ and CD8+ T cells. Overall, these data demonstrate the high potential of HA-DOTAP-PLGA NPs for use as vaccine delivery vehicles to elevate cellular and humoral immune responses.

KEYWORDS: vaccine, adjuvant, immune response, hyaluronic acid, cationic-lipid PLGA hybrid nanoparticles


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1. INTRODUCTION It is thought that many infectious diseases can be prevented by vaccines that induce a strong antigen-specific immune response to provide long-term protection and therapy. Compared with traditional vaccines, which primarily consist of attenuated pathogens and whole inactivated organisms, many vaccines currently in development have well-defined components and are considered to be safer.1-3 However, there is a general need for vaccines delivery vehicles that boost immunogenicity and induce more potent humoral and cell-mediated immunity. It has proven to be especially challenging to stimulate antigen-specific cytotoxic T-lymphocytes (CTL) response, which is essential for the effectiveness of vaccination against HIV, hepatitis C, malaria, and cancers.4, 5 The presentation of exogenous antigens via class I major histocompatibility complex (MHC-I) molecules by dendritic cells (DCs), known as cross-presentation, is essential for the initiation of CD8+ T-cell responses.6-8 However, vaccination with exogenous antigens often fails to generate strong CD8+ T-cell responses due to the insufficient access of exogenous antigens to cytosol and the MHC-I machinery necessary for antigen presentation. Exogenous antigens are endocytosed by antigen-presenting cells and are generally degraded in specialized endo- or lysosomal compartments, resulting in preferential MHC-II presentation. Enhancing the targeted delivery of exogenous antigens to cytosol for processing via the cross-presentation pathway may help to promote MHC-I-restricted antigen presentation for CD8+ T-cell activation.9-11 Nanotechnology has increasingly played a significant role in vaccine development. Several nanoparticle carriers, such as liposomes, polymeric nanoparticles, and inorganic nanoparticles, have been shown to possess significant potential as vaccine delivery systems and immunostimulatory adjuvants.12-14 Among the particulate vaccine delivery systems, cationic lipid-poly (lactide-co-glycolide) acid (PLGA) hybrid microparticles based on a core of FDA-approved biodegradable polymer (PLGA) enveloped by a cationic lipid membrane have exhibited great potential as an antigen delivery system.15,16



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In

particular,

cationic

lipid membranes

composed

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of 1,2-dioleoyl-3-trimethyl

ammonium-propane (DOTAP) have been extensively studied because they can readily form nanocomplexes with anionic peptides, proteins, and plasmid DNA encoding for antigens and can induce immune responses in vivo.17-21 However, when used at high concentrations, their high positive charges result in high cytotoxicity that can negatively affect immune responses, reduce biocompatibility and limit in vivo applications. A good strategy for shielding this positive charge and producing a stable delivery system involves coating these particles with a layer of anionic polymer, such as hyaluronic acid (HA), by electrostatic interaction. HA is a linear anionic polysaccharide, it can interact with cationic materials to form complexes and improve the biocompatibility and stability of the complexes.22-24 Moreover, the specific affinity to CD44 makes HA a potential material for targeted delivery, because CD44, the major cell-surface receptor for HA, is highly expressed on cells such as tumor cells, smooth muscle cells, epithelial cells and DCs.25,

26

Gene delivery systems consisting of

HA-coated cationic nanoparticles can be therefore used to enhance the gene delivery efficiency of cationic nanoparticles, which is attributed to HA-CD44 receptor-mediated endocytosis and at the same time maintaining the endosomal escape capacity of the cationic nanoparticles through the hyaluronidases-catalyzed degradation of HA in the endosome or lysosome. 27, 28 Taking the advantages of HA-coated cationic nanoparticles with HA-CD44 receptor-mediated endocytosis and endosomal escape capacity, in this study, we utilized ionic

complexation

between

cationic

DOTAP-PLGA

hybrid

nanoparticles

(DOTAP-PLGA NPs) and anionic HA to form HA-DOTAP-PLGA hybrid nanoparticles (HA-DOTAP-PLGA NPs). It is hypothesized that HA-DOTAP-PLGA NPs would facilitate better cell uptake efficiency of DCs than DOTAP-PLGA NPs. Plain cationic DOTAP-PLGA NPs resulted from hyaluronidases-catalyzed degradation of HA in endosome and lysosome could promote the cytosolic release of antigens. Our objectives


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were to investigate whether the use of HA-DOTAP-PLGA NPs facilitates the cytosolic delivery of antigens and hence promotes MHC-I-restricted CD8+ T-cell responses and to evaluate these resultant antigen-specific immune responses.

2. MATERIALS and METHODS 2.1. Materials and reagents PLGA (50/50, molecular weight 7,000-17,000), ovalbumin (OVA), polyvinyl alcohol (PVA) (molecular weight 30,000-70,000), fluorescein isothiocyanate (FITC), OVA257-264 and OVA323-339 were supplied by Sigma-Aldrich (St. Louis, MO, USA). DOTAP was supplied byAvanti Polar Lipids (Alabaster, AL, USA). HA (Mw=46 kDa, pharmaceutical grade) was supplied by Freda Biochem Co., Ltd. (Shangdong, China) and the endotoxin of HA tested by Charles River is less than 0.0025 IU/mg. LysoTracker probes were supplied by Life Technologies (Grand Island, NY, USA). The mouse cytokine ELISA kits, PE-labeled anti-mouse OVA-derived (H2kb, SIINFEKL) specific MHC-I tetramer, eFluor 780, and fluorochrome-labeled MHC-I, MHC-II, CD86, CD40, CD11c, CD44, CD62L, CD4, and CD8 antibodies were obtained from eBioscience (San Diego, CA, USA). GolgiPlug, the anti-CD16/CD32 antibody and PE-labeled anti-mouse intracellular interferon-γ (IFN-γ) antibody were obtained from BD Biosciences (San Jose, CA, USA). Other chemicals used were of reagent grade. 2.2. Preparation of OVA-loaded DOTAP-PLGA NPs with or without HA coatings DOTAP-PLGA NPs encapsulated in OVA were formulated using a double emulsion (W/O/W)/solvent evaporation method. Briefly, the first aqueous solution was mixed with OVA. After the addition of 1 mL of dichloromethane containing 30 mg of PLGA and 6.5 mg of DOTAP, the water-in-oil (W/O) emulsion was sonicated over an ice bath using a microtip probe sonicator (VCX-130-PB, Sonics & Material Inc., Connecticut, USA) at 30 kW of power output for 1 min. The primary emulsion was then further emulsified with a secondary aqueous phase (5 mL of 2% w/v PVA in de-ionized water) for 5 min to form a



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secondary (W/O/W) emulsion. The resultant emulsion was agitated overnight using a magnetic stirrer at room temperature until the dichloromethane completely evaporated. DOTAP-PLGA NPs were washed with distilled water by centrifugation at 21,000 rpm for 30 min. For the preparation of HA-DOTAP-PLGA NPs, DOTAP-PLGA NPs were mixed under gentle stirring with HA solution (0.04% w/v in PBS pH 7.4) for 4 h to achieve effective surface coating. The HA-coated nanoparticles were washed three times with distilled water by centrifugation at 21,000 rpm for 30 min and lyophilized.

2.3. Nanoparticle characterization The average size and zeta potential of the prepared nanoparticles were measured using a NY 90-Plus particle size analyzer (Brookhaven Instruments Corporation, NY, USA) in triplicate. Transmission electron microscopy (TEM) (Tecnai-F20, FEI, the Netherlands) was employed to observe the morphology of the prepared nanoparticle. For quantification of the loaded OVA, the prepared nanoparticle formulations were dissolved in 0.1 M NaOH and 0.1% SDS, incubated overnight at room temperature and measured using a Micro-BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s protocol.29 A standard curve was established by dissolving OVA in 0.1 M NaOH and 0.1% SDS, and blank DOTAP-PLGA NPs and HA-DOTAP-PLGA NPs were used as controls. The in vitro release of OVA from the prepared nanoparticle formulations was measured in PBS (pH 7.4) at 37 °C. Briefly, the prepared nanoparticles in 10 mL of PBS (pH 7.4) were placed into test tubes in a shaking bed at 120 rpm at 37 °C. At predetermined time intervals, the whole medium was withdrawn and 10 mL of fresh medium was added. The amount of OVA released into the supernatant from the prepared nanoparticle formulations was determined using the Micro-BCA protein assay kits described above. Data are expressed as the mean value and standard deviation from three independent experiments.


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2.4. Preparation and viability of bone marrow-derived dendritic cells (BMDCs) DCs were generated from bone marrow cells of C57BL/6 mouse femur and tibia as previously described.

23

A CCK-8 assay was used to assess the cell viability in the

presence of HA-DOTAP-PLGA NPs and DOTAP-PLGA NPs. DCs were seeded into 96-well culture plates (1×105 cells/well) in growth medium. Then, the cells were incubated with OVA-loaded nanoparticles formulation with various nanoparticles concentrations for 24 h. Finally, cells were incubated with CCK-8 solution for 4 h and OD450 was measured using a Thermo Varioskan Flash Multifunction Microplate Reader.

2.5. Antigen uptake and intracellular localization in BMDCs For flow cytometry, cells were seeded in six-well culture plates (1×105 cells/well) and cultured for 1 h. The cells were treated with OVA-loaded HA-DOTAP-PLGA NPs and DOTAP-PLGA NP formulations containing FITC-labeled OVA (equivalent OVA concentration: 10 µg/mL) for 16 h. Then, the cells were washed with PBS and harvested for FACS analysis using a BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA). For confocal laser scanning microscopy (CLSM), cells were cultured in a confocal dish

(1×105 cells)

for

1

h.

The

cells

were

incubated

with

OVA-loaded

HA-DOTAP-PLGA NPs and DOTAP-PLGA NP formulations containing FITC-labeled OVA (equivalent OVA concentration: 10 µg/mL) for 16 h. Then, the cells were washed 3 times with PBS. For lysosome labeling, the cells were incubated with LysoTracker Red DND-99 for 0.5 h and rinsed 2 times with PBS. Finally, CLSM (CLSM 410; Zeiss, Jena, Germany) was used to observe the cells using Fluoview FV500 imaging software.

2.6. Activation and maturation of BMDCs OVA-loaded HA-DOTAP-PLGA NPs and DOTAP-PLGA NP formulations or free OVA were incubated with the BMDCs for 24 h. The cytokine concentrations in the culture supernatants of the BMDCs were measured using IL-6, IL-12 (p70) and tumor



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necrosis factor-α (TNF-α) ELISA kits according to the manufacturer's protocol. The BMDCs were harvested and labeled with fluorochrome-labeled antibodies against CD11c, CD40, CD86, MHC-I or MHC-II. The expression of CD40, CD86, MHC-I and MHC-II on CD11c+ DCs was determined by a BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA).

2.7. In vivo immunization studies Six-week-old female C57BL/6 mice (n = 6 mice/group) were intramuscularly injected with a 100 µL suspension of OVA-loaded HA-DOTAP-PLGA hybrid nanoparticles, OVA-loaded DOTAP-PLGA hybrid nanoparticles, or free OVA in saline (each containing 30 µg of OVA). The mice were immunized at weeks 0, 2 and 4. Seven days after the third immunization, the mice were sacrificed, and their spleens were collected for immunological tests.

2.8. ELISA analysis of OVA-specific IgG and isotypes OVA-specific IgG, IgG1 and IgG2a were determined using ELISA. Briefly, a 96-well ELISA plate was coated with 2 µg per well of OVA overnight at 4 °C. After six washes, each well of the plate was blocked with 1% (w/v) BSA-containing PBS and then covered and incubated at 37 °C for 1.5 h. The plates were washed twice. The samples were serially diluted in blocking buffer, added to the plates, and incubated for 1 h at 37 °C. After six washes, the plates were incubated with HRP-conjugated anti-mouse IgG (IgG total, IgG1 or IgG2a) antibodies for 40 min at 37 °C. Finally, the HRP was quantified through the addition of TMB solution. After a 15-min incubation at room temperature, the enzymatic reaction was stopped with 2 M H2SO4, and the OD450 values were measured using a microplate reader. The titers represent the highest dilution of samples with an OD450 value that was two-fold higher than the value of the background controls. Samples were measured in duplicate.


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2.9. Intracellular cytokine staining Splenocytes (2×106 cells) from each experimental group were incubated with OVA257-264 (20 µg/mL) or OVA323-339 (20 µg/mL) at 37 °C. After 2 h, a protein transport inhibitor, GolgiPlug, was added for an additional 8 h. Cells were subsequently washed and stained with antibodies as previously described.10 Cells were analyzed by flow cytometry using a Canto II flow cytometer (BD Biosciences, San Jose, CA) and FlowJo software (FlowJo, Eugene, OR).

2.10. Flow cytometry evaluation of T-cell response Splenocytes (2×106 cells) from each experimental group were restimulated with OVA (50 µg/mL) for 72 h at 37 °C. After washing, cells were stained with fluorescein -labeled anti-mouse antibodies against CD4, CD8, CD44, and CD62L and a PE-labeled anti-mouse OVA-derived (H2kb, SIINFEKL) specific MHC-I tetramer for 30 min at 4 °C. A BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA) was used to measure the percentages of effector memory T cells (CD44Hi CD62LLo), central memory T cells (CD44Hi CD62LHi) and antigen-specific CD8+ T cells (CD8+ SIINFEKL-MHC-I+). Data were analyzed using BD Accuri™ C6 software. 2.11. Statistical Analysis Data were expressed as the mean ± standard deviation (SD). Difference analysis between groups was made by Student's t-test or ANOVA followed by Tukey's multiple comparison. P values of 0.05 or less were considered to be statistically significant.

3. RESULTS 3.1. Preparation and characterization of OVA-loaded DOTAP-PLGA NPs with or without HA coatings



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Table I. Characterization of OVA-loaded HA-DOTAP-PLGA NPs and DOTAP-PLGA NPs. Data are presented as the mean ± SD (n = 3). Formulation DOTAP-PLGA NPs HA-DOTAP-PLGA NPs a

Size (nm)

PDIa

203.2±10.5 0.10±0.002 236.4±13.2 0.095±0.008

Zeta potential (mV)

OVA loading

31.5±1.4 -14.7±0.9

24.8±3.2 22.3±1.7

(µ µg/mg)

PDI, polydispersity index fromDLS.

Figure 1. Development of OVA-loaded DOTAP-PLGA NPs with or without HA coatings. (A) Schematic illustration shows the formulation of OVA-loaded HA-DOTAP-PLGA NPs. Typical TEM images and size distributions of (B) DOTAP-PLGA NPs and (C) HA-DOTAP-PLGA NPs. Zeta potential (D) and in vitro OVA release profile (E) of OVA-loaded nanoparticle formulations. Data are presented as the mean ± SD (n = 3)


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We formulated DOTAP-PLGA NPs using a double emulsion (W/O/W)/solvent evaporation method with PLGA and DOTAP co-dissolved in the organic phase. Specifically, the PLGA polymer precipitates to form a hydrophobic core that encapsulates the OVA, and the DOTAP self-assembles around the PLGA core to form a positively charged surface to adsorb the anionic HA (Figure 1A). Representative TEM images of the DOTAP-PLGA NPs and the HA-DOTAP-PLGA NPs are shown in Figure 1B and Figure 1C. The TEM images clearly show the well-defined core-shell structures of the DOTAP-PLGA NPs and the HA-DOTAP-PLGA NPs. The DOTAP-PLGA NPs had a mean diameter of 203.2 ± 10.5 nm (Figure 1B) and a zeta potential of 34.9 ± 1.4 mV (Figure 1D). The mean diameter of the HA-DOTAP-PLGA NPs slightly increased to 236.4 ± 13.2 nm (Figure 1C), and the zeta potential dramatically decreased to -14.7 ± 0.9 mV (Figure 1D), indicating that HA was successfully coated on the surface of the DOTAP-PLGA NPs. The OVA-loaded contents of the DOTAP-PLGA NPs and the HA-DOTAP-PLGA NPs were 24.8 µg/mg and 22.3 µg/mg, respectively (Table I). The in vitro release profile of OVA from the different OVA-loaded hybrid nanoparticle formulations is shown in Figure 1E. As shown in Figure 1E, we observed a high initial burst release of more than 20% during day 1 for OVA-loaded DOTAP-PLGA NPs, followed by a sustained release for 19 days to achieve a cumulative OVA release of almost 80%. Interestingly, OVA-loaded HA-DOTAP-PLGA NPs suppressed the initial burst release by less than 11% and had a slower release than OVA-loaded DOTAP-PLGA NPs during the test period, indicating that HA coatings on the surface of the nanoparticles retarded the release of OVA from the nanoparticles.

3.2. Enhanced biocompatibility of HA-DOTAP-PLGA NPs compared with DOTAP-PLGA NPs



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Figure 2. Cytotoxicity of OVA-loaded DOTAP-PLGA NPs and OVA-loaded HA-DOTAP-PLGA NPs after 24 h of incubation with BMDCs. Data are presented as the mean ± SD (n = 3). Cationic nanoparticles are often associated with significant cytotoxic effects, due to their electrostatic interactions with negatively charged cell membrane.30,

31

The HA

coating will shield the positive charges of DOTAP-PLGA NPs, thus minimizing the cytotoxicity.

To

compare

the

cytotoxicity

of

DOTAP-PLGA

NPs

and

HA-DOTAP-PLGA NPs against cultured BMDCs, the cytotoxic effects of various concentrations of OVA-loaded nanoparticles with or without HA coatings were studied by using the CCK-8 assay method. As shown in Figure 2, DOTAP-PLGA NPs exhibited significant cytotoxicity toward BMDCs, particularly at high nanoparticle concentrations. In contrast, even when the total concentration of HA-DOTAP-PLGA NPs reached 0.5 mg/mL, cells treated with these particles showed 90% viability relative to untreated cells. These results showed that HA coatings could improve the cell biocompatibility of DOTAP-PLGA NPs. Thus, we suspected that the OVA-loaded HA-DOTAP-PLGA NPs potently activated the DCs with significantly reduced cytotoxicity compared with that of the OVA-loaded DOTAP-PLGA NPs. 3.3. Cellular uptake and intracellular localization of OVA-loaded DOTAP-PLGA NPs with or without HA coatings


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Figure 3. (A) Cellular uptake of FITC-labeled OVA-loaded DOTAP-PLGA NPs with or without HA coatings in BMDCs was quantified by a flow cytometer. Results are presented as the mean ± SD (n = 3). (B) CLSM images of BMDCs after a 16-h incubation with FITC-labeled OVA-loaded DOTAP-PLGA NPs with or without HA coatings. OVA were labeled by FITC (green points) and lysosomes were labeled by LysoTracker (red points).

Antigens uptake efficiency and intracellular localization in DCs dramatically influence DC activation and antigen processing and presentation.32 Generally, positively charged nanoparticles can efficiently attatch to the cells surface by electrostatic interactions with negatively charged cell membranes. Here, we evaluated the ability of DOTAP-PLGA NPs and HA-DOTAP-PLGA NPs to deliver OVA into BMDCs. As shown in Figure 3A, the flow cytometry data illustrated that despite the zeta potential of the HA-DOTAP-PLGA NPs corresponding to negatively charged NPs, the relative fluorescence intensity was much higher in cells treated with HA-DOTAP-PLGA NPs than in cells treated with DOTAP-PLGA NPs. This results indicated that the HA coatings improved the cellular uptake of DOTAP-PLGA NPs, which is attributed to HA-CD44 receptor-mediated endocytosis.24 The final intracellular location of antigens after uptake by DCs is critical for subsequent antigen processing and presentation. Efficient exogenous antigen endosomal escape is essential for immune responses because it can facilitate cross-presentation in



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immune responses and further assist antigens to simultaneously activate CD4+ and CD8+ T cells.9,11 We next investigated the location of different FITC-OVA-loaded hybrid nanoparticle formulations in cells by staining the acidic organelles (including endosomes and early lysosomes). Confocal images revealed that the majority of the FITC-OVA (green) was co-localized with the LysoTracker Red-stained organelles in the FITC-OVA-loaded DOTAP-PLGA NPs-treated cells, indicating that these hybrid nanoparticles mainly resided in endosomes or early lysosomes (Figure 3B). In the FITC-OVA-loaded HA-DOTAP-PLGA NPs-treated cells, many FITC-OVA molecules did not co-localize with endosomal vesicles, and the separation of the green and red fluorescence was more significant, indicating that FITC-OVA encapsulated in HA-DOTAP-PLGA NPs more efficiently escaped from endosomes. Interestingly, in addition to appearing in large numbers in a location separate from the endosomal vesicles, some of the FITC-OVA molecules encapsulated in HA-DOTAP-PLGA NPs also co-localized with endosomes. The diverse trafficking fates of the FITC-OVA encapsulated in HA-DOTAP-PLGA NPs indicated that HA-DOTAP-PLGA NPs are able to process exogenous antigens via both the MHC-I and MHC-II antigen presentation pathways.

3.4. Efficient activation of BMDCs with OVA-loaded HA-DOTAP-PLGA NPs


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Figure 4. The effect of OVA-loaded DOTAP-PLGA NPs with or without HA coatings on BMDC activation. Expression of the co-stimulatory molecules CD40 (A) and CD86 (B) and of MHC-I (C) and MHC-II (D) on CD11c+ BMDCs. The percentages of CD40, CD86, MHC-I and MHC-II expression on CD11c+ BMDCs were determined by flow cytometry. (E-G) Cytokine secretion by BMDCs. Results are expressed as the mean ± SD (n = 6) (*p < 0.05; **p < 0.01). To initiate immune responses, DCs must ﬁrst be activated into maturation, a process that has been widely studied.33 Fully mature DCs exhibit an increase in the surface expression of MHC-I and MHC-II and co-stimulatory molecules, such as CD40 and



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CD86, together with cytokine production. Therefore, we investigated BMDC activation by incubating BMDCs with OVA-loaded hybrid nanoparticles with or without HA coatings. The levels of the cell surface expression of MHC-I and MHC-II and the co-stimulatory molecules CD40 and CD86 were measured by flow cytometry. Moreover, the release of the Th1-polarizing cytokines TNF-α and IL-12 and the Th2-polarizing cytokine IL-6 from BMDCs was also determined. DCs expressed significantly higher levels of the co-stimulatory molecules CD40 and CD86 on their surfaces after incubation with OVA-loaded DOTAP-PLGA NPs and OVA-loaded HA-DOTAP-PLGA NPs than after incubation with free OVA (p < 0.05) (Figure 4A-4B). Compared with the DOTAP-PLGA NPs and free OVA, the HA-DOTAP-PLGA NPs dramatically increased the expression of MHC-I (p < 0.01) (Figure 4C). Additionally, there was no significant difference in MHC-II expression between the OVA-loaded HA-DOTAP-PLGA NPs and the DOTAP-PLGA NPs, but both induced significantly higher expression than free OVA alone (p < 0.05) (Figure 4D). IL-12 is a T-cell growth and stimulating factor and is known to be a strong inducer of INF-γ production. In addition, TNF-α, similar to INF-γ, is important for eliciting protective cellular immune responses.34, 35 As shown in Figure 4E and 4F, the levels of both TNF-α and IL-12 induced by HA-DOTAP-PLGA NPs showed significantly higher than DOTAP-PLGA NPs and free OVA (p < 0.01). The production of IL-6 was also significantly elevated in DCs treated with HA-DOTAP-PLGA NPs (HA-DOTAP-PLGA NPs vs. free OVA: p < 0.01; HA-DOTAP-PLGA NPs vs. DOTAP-PLGA NPs: p < 0.05). Together, these results indicate that the HA-DOTAP-PLGA NPs had stronger efficacy in DC activation than did DOTAP-PLGA NPs and free OVA. 3.5. HA coatings enhance antigen-specific CD8+ and CD4+ T-cell responses in vivo


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Figure 5. HA coatings enhanced antigen-specific T-cell responses. (A) Immunization schedule; mice (n = 6) were immunized at weeks 0, 2 and 4. (B) The proportion of OVA-specific CD8+ T cells in the spleen was determined by SIINFEKL-MHC-I tetramer staining and flow cytometry. (C) and (D) Splenocytes were collected and restimulated ex vivo with OVA257-264 (CD8+ response) or OVA323-339 (CD4+ response). The proportion of IFN-γ-producing CD8+ T cells and CD4+ T cells was determined after 4 h of restimulation by intracellular staining and flow cytometry. Representative FACS plots are shown. Data for the percentages of IFN-γ+ CD8+ T cells and IFN-γ+ CD4+ T cells are expressed as the mean ± SD (n = 6) (*p < 0.05, **p < 0.01). We first evaluated the induction of OVA-specific CD8+ T-cell responses in immunized mouse splenocytes 7 days after the third immunization; this induction is a pre-requisite for cellular immunity. The OVA specificity of CD8+ T cells was determined by staining with a fluorescently tagged tetramer capable of binding to CD8+ T-cell



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receptors

that

recognize

MHC-I-restricted

antigens

in

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association

with

an

immunodominant epitope of OVA (OVA257-264, SIINFEKL). The proportion of OVA-specific CD8+ T cells was very significantly enhanced in mice immunized with HA-DOTAP-PLGA NPs compared with DOTAP-PLGA NPs and free OVA, suggesting the high potential of HA-DOTAP-PLGA NPs in effector CD8+ T-cell proliferation (p < 0.001) (Figure 5B). To evaluate the activation of OVA-specific CD8+ T cells, we restimulated splenocytes ex vivo for 2 h with SIINFEKL and analyzed the activation of CD8+ T cells by measuring the production of IFN-γ. Intracellular cytokine staining showed that immunization with HA-DOTAP-PLGA NPs resulted in high levels of IFN-γ+ CD8+ T cells (6.01 ± 0.7%), a significantly stronger response than that elicited by free OVA (2.6 ± 0.8%) or DOTAP-PLGA NPs (3.54 ± 0.9%) (p < 0.01) (Figure 5C). Thus, immunization with HA-DOTAP-PLGA NPs was able to generate robust CD8+ T-cell responses, which is likely attributed to the advantages of HA-DOTAP-PLGA NPs with high cellular uptake efficiency and cytoplasmic delivery of antigens in BMDCs. Effector CD4+ T-cell responses are also important in the context of vaccination and the induction of cellular immunity because they can modulate both cellular and humoral immunity.36, 37 We thus restimulated splenocytes ex vivo for 2 h with the class II OVA peptide (OVA323-339) and analyzed the activation of CD4+ T cells by measuring the production of IFN-γ. Intracellular cytokine staining showed that the levels of IFN-γ+ CD4+

T

cells

were

significantly

enhanced

in

the

mice

immunized

with

HA-DOTAP-PLGA NPs (5.13 ± 0.7%) compared with the other groups (vs. DOTAP-PLGA-NPs (3.85 ± 0.6%): p < 0.05; vs. free OVA (2.13 ± 0.6%): p < 0.001) (Figure 5D). In summary, HA-DOTAP-PLGA NPs enhanced both OVA-specific CD8+ T and CD4+ T-cell responses, which may induce robust cellular and humoral immune responses. 3.6. HA coatings enhance antibody responses in vivo


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Figure 6. Antigen-specific antibody production in vivo. (A) Antigen-specific total IgG and (B) the IgG2a/IgG1 ratio was measured on day 35 in sera from immunized mice, and data are expressed as the mean ± SD (n = 6) (*p < 0.05, **p < 0.01).

To investigate the effect of DOTAP-PLGA NPs with or without HA coatings on antibody responses, we performed ELISA 7 days after the third immunization to determine anti-OVA IgG levels (Figure 6A). As shown in Figure 6A, the OVA-loaded HA-DOTAP-PLGA NPs induced significantly higher antigen-specific IgG titers than did the other two groups (p < 0.05). We also measured the IgG2a and IgG1 levels of each group to determine the types of T helper (Th) cell immune responses. In general, Th1 cells direct IgG2a antibodies and Th2 cells direct IgG1 antibodies, and a high IgG2a/IgG1 ratio is indicative of a Th1-biased immune response.38, 39 Thus, we analyzed the IgG2a/IgG1 ratios in sera from mice sacrificed 7 days after the third immunization. As shown in Figure 6B, the OVA-loaded HA-DOTAP-PLGA NPs induced significantly higher IgG2a/IgG1 ratios than did the free OVA and OVA-loaded DOTAP-PLGA NPs, indicating a more balanced Th1/Th2 response that still favored a Th1 response (p < 0.001), which was consistent with stronger IFN-γ production induced by OVA-loaded HA-DOTAP-PLGA NPs.



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3.7. HA coatings enhance memory T-cell responses in vivo We evaluated development of memory T-cell responses following immunization with DOTAP-PLGA NPs with or without HA coatings; these responses are essential for long-term vaccine efficacy. Memory T cells can segregate into effector and central memory subsets based on their expression of lymph node homing receptors; the former (TEM, CD44Hi CD62LLo) exhibit rapid effector function, and the latter (TCM, CD44Hi CD62LHi) demonstrate potent proliferative and lymph node homing properties.40 As shown in Figure 7B and 7D, the TCM proportions of CD4+ and CD8+ T cells were both significantly higher for the mice immunized with HA-DOTAP-PLGA NPs than for those incubated with free OVA (p < 0.01) and DOTAP-PLGA hybrid NPs (p < 0.05). The TEM production was similar to the TCM production, and the HA coating offered advantages over free OVA and DOTAP-PLGA hybrid NPs (Figure 7C and 7E). In summary, these data highlight the remarkable memory T-cell responses elicited by HA-DOTAP-PLGA NPs, which may be key to protect against reinfection.


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Figure 7. Proportion of central memory (TCM, CD44Hi CD62LHi) and effector memory (TEM, CD44Hi CD62LLo) in CD4+ T cells and CD8+ T cells. Mice (n = 6) were immunized at weeks 0, 2 and 4. Splenocytes were collected and restimulated ex vivo with OVA (50 µg/mL) 7 days after the third boosting. The proportions of the CD44Hi CD62LHi/CD4+ T cells, CD44Hi CD62LLo/CD4+ T cells, CD44Hi CD62LHi/CD8+ T cells and CD44Hi



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CD62LLo/CD8+ T cells were determined by flow cytometry. Representative FACS plots are shown (A). Data for (B) the CD44Hi CD62LHi/CD4+ T cells, (C) CD44Hi CD62LLo/CD4+ T cells, (D) CD44Hi CD62LHi/CD8+ T cells and (E) CD44Hi CD62LLo/CD8+ T cells are expressed as the mean ± SD (n = 6) (*p < 0.05, **p < 0.01).

4. DISCUSSION Strategies to enhance the efficacy of polymeric NP-based adjuvants for boosting immunogenicity, particularly inducing cell-mediated immune responses to cytotoxic damage, are of great interest. In this work, we developed an antigen delivery platform using the ionic interaction between cationic DOTAP-PLGA hybrid NPs and anionic HA to form HA-decorated DOTAP-PLGA hybrid NPs. Previous studies demonstrated that HA-coated cationic NPs could mediate the cytosolic delivery of DNA and siRNA.24, 25 Our work is the first to demonstrate that vaccines based on HA-modified cationic NPs could also enhance cellular uptake efficiency and deliver antigens to the cytoplasm (Figure 3). Our results indicated that HA-DOTAP-PLGA NPs promoted DC activation and maturation (Figure 4), enhanced antigen-specific CD4+ and CD8+ T-cell responses (Figure 5), facilitated robust antigen-specific IgG antibody production (Figure 6), and induced memory T-cell generation (Figure 7). Antigens can be incorporated to nanoparticles through complex processes, such as encapsulation and chemical conjugation, or simple physical adsorption based on charge or hydrophobic interactions.41, 42 Antigen encapsulation into nanoparticles allows for a stronger interaction between nanoparticles and the antigen, which may lead to slow antigen release from the nanoparticles. For example, Zhang et al. showed that antigen-encapsulated nanoparticles create an antigen depot at the injection site, providing a persistent supply of antigen for jmmune activation.43 Kanchan et al. reported that memory antibody response was generated from immunization with single dose of polylactide (PLA) microparticles entrapped antigens.44 In this study, HA-DOTAP-PLGA NPs showed the initial burst release of less than 11% and had a slower release during 19


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days. Thus, we expected that HA-DOTAP-PLGA NPs would provide long-term antigen persistence at the injection site after vaccination. It

was

reported

that

HA coated

nanoparticles

could

share

HA-CD44

receptor-mediated endocytosis in DCs.24 In this study, we further proved for the first time that HA-DOTAP-PLGA hybrid NPs could enhance cellular uptake efficiency as Figure 3A displayed, which might also be attributed to HA receptor-mediated endocytosis. Exogenous antigens are endocytosed by DCs and further transported to the cell surface for MHC-I and MHC-II presentation pathway, which resulted in CD8+ and CD4+T-cell activation, respectively. Previous studies showed that pH-responsive polymer NPs could deliver antigens to the cell cytosol, promote MHC-I-restricted antigen presentation for CD8+ T-cell activation.

10,11,45

However, recent research by Tran et al. suggested that

pH-responsive polymer NPs result in less MHC-II antigen presentation and decrease CD4+ T-cell stimulation.46 Defense against many persistent diseases requires a combination of humoral and cellular responses, which is necessary to target antigens to both the MHC-I and MHC-II antigen presentation pathways. Regarding the intracellular fates of vaccines based on HA-DOTAP-PLGA NPs after DCs uptake, our results showed that some of the antigens were able to escape into the cytosolic compartment, whereas some of the antigens remained in the endosomal/lysosomal compartment (Figure 3B). The diverse trafficking fate of OVA encapsulated in HA-DOTAP-PLGA NPs suggested that HA coatings of DOTAP-PLGA NPs could increase the degree of MHC-I antigen presentation while maintaining MHC-II antigen presentation. Our studies showed that compared with DOTAP-PLGA NPs and free OVA, HA-DOTAP-PLGA NPs were better able to maintain both CD8+ and CD4+ T-cell responses (Figure 5). Thus, in this study, we developed vaccines delivered by HA-decorated DOTAP-PLGA NPs with strong endosomolytic activity; the features to modulate both CD8+ and CD4+ T-cell responses provide new insight for the future design of vaccine.



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In vivo studies based on the outstanding properties mentioned above further revealed that HA-DOTAP-PLGA NPs induced significantly higher antigen-specific IgG titers than did the other two groups (Figure 6A), which may be attributed to their immunostimulatory effects on antigen-specific CD4+ T-cell activation and maturation. Antigen-specific CD4+ T cells can activate B cells for antigen-specific antibody production.47 The CD4+ T-cell activation improved with the HA-DOTAP-PLGA NPs maybe facilitate the generation of functional B cell to produce antibodies. Previous reports have also demonstrated the effect of HA on antigen uptake, T-cell functions and responses, likely mediated via the CD44 receptors, which are consistent with our study.48 The ability to induce long-term immunological memory is an important aspect of effective vaccines, which can completely prevent reinfection or greatly reduce the severity of disease, and memory T cells are the basis of immunological memory. Memory T cells can segregate into TEM cells and TCM cells; the former (TEM, CD44Hi CD62LLo) display rapid effector function, and the latter (TCM, CD44Hi CD62LHi) exhibit potent proliferative and lymph node homing properties.40 However, it is still unclear which subset is most beneficial for conferring long-term protective memory. Here, we found that HA-DOTAP-PLGA NPs are superior to DOTAP-PLGA NPs and free OVA in both memory TEM and TCM cell generation (Figure 7). Our HA-DOTAP-PLGA favored greater memory CD8+ T-cell formation, which may be attributed to the high levels of antigen-specific CD4+ T-cell generation mediated by HA-DOTAP-PLGA NPs. It was previously reported that CD4+ T cells help is required in generating functional CD8+ T cell memory.37 Of the tested formulations, HA-DOTAP-PLGA NPs better favored increased memory T-cell formation, suggesting that HA-DOTAP-PLGA NPs provide better protection against reinfection. Here, we demonstrated a potential effect of HA-DOTAP-PLGA NPs on memory T cells. However, little is known regarding the exact mechanisms of HA-CD44 receptor and T cells, extensive investigation is still required. 48 Based on the above results, Figure 8 exhibits the schematic illustration for the


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induction of the immune response following vaccination by HA-DOTAP-PLGA NPs (Figure 8). Initially, after HA-CD44 receptor-mediated uptake by DCs, some of the antigens encapsulated in the HA-DOTAP-PLGA NPs escaped from the lysosomes into the cytosolic space, whereas some of the antigens remained in the endosomal/lysosomal compartment, where both MHC-I and MHC-II antigen presentation occurred (Figure 3). At the same time, DCs were activated and matured to prime T cells through the up-regulation of MHC-I, MHC-II, the co-stimulatory molecules CD40 and CD86 and secreting cytokines (Figure 4). Subsequently, robust CD4+ and CD8+ T-cell responses were induced (Figure 5), strong antigen-specific IgG antibody titers were elicited (Figure 6), and potent memory T-cell responses were maintained (Figure 7). Therefore, HA-DOTAP-PLGA NPs offer a potential vaccine delivery platform for stimulating a combination of humoral and cell-mediated immune responses.

Figure 8. Schematic representation of the induction of the immune response following vaccination with the OVA-loaded HA-DOTAP-PLGA NPs.

5. CONCLUSIONS In this work, we developed a novel antigen delivery platform using the ionic



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interaction between cationic DOTAP-PLGA hybrid NPs and anionic HA to form HA-decorated DOTAP-PLGA hybrid NPs. Here, we demonstrated that vaccines based on HA-modified DOTAP-PLGA NPs were able to process exogenous antigens via both the MHC-I and MHC-II antigen presentation pathways, promote DC activation and maturation, induce antigen-specific CD4+ and CD8+ T-cell responses, facilitate robust antigen-specific IgG antibody production, and enhance memory T-cell generation, highlighting the promise of HA-decorated DOTAP-PLGA hybrid NPs as a vaccine delivery platform for potent immunity induction. Acknowledgements We acknowledge financial support from the Natural Science Foundation of China (No. 50903093, 31200674 and 51373199), the Tianjin Natural Science Foundation project (No.15JCYBJC18400 and 15JCYBJC47600). Reference [1] Delany, I.; Rappuoli, R.; De Gregorio, E. Vaccines for the 21st Century. EMBO Mol. Med. 2014, 6, 708-720. [2] De Gregorio, E.; Rappuoli, R. Vaccines for the Future: Learning from Human Immunology. Microb. Biotechnol. 2012, 5, 149-155. [3] Ulmer, J. B.; Valley, U.; Rappuoli, R. Vaccine Manufacturing: Challenges and Solutions. Nat. Biotechnol. 2006, 24, 1377-1383. [4] Melief, C. J.; van Hall, T.; Arens, R.; Ossendorp, F.; van der Burg, S. H. Therapeutic Cancer Vaccines. J. Clin. Invest. 2015, 125, 3401-3412. [5] Foged, C.; Hansen, J.; Agger, E. M. License to Kill: Formulation Requirements for Optimal Priming of CD8+ CTL Responses with Particulate Vaccine Delivery Systems. Eur. J. Pharm. Sci. 2012, 45, 482-491. [6] Joffre, O. P.; Segura, E.; Savina, A.; Amigorena, S. Cross-Presentation by Dendritic Cells. Nat. Rev. Immunol. 2012, 12, 557-569.


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for Targeting Antigen to Both Class I and II Antigen Presentation Pathways. Adv. Healthc. Mater. 2014, 3, 690-702. [47] Bishop, G. A.; Haxhinasto, S. A.; Stunz, L. L.; Hostager, B. S. Antigen-Specific B-Lymphocyte Activation. Crit. Rev. Immunol. 2003, 23, 149-197. [48] Baaten,

B.J.;

Tinoco,

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Antigen-Experienced T Cells: Lessons from the Quintessential Memory Marker CD44. Front Immunol. 2012, 3, 23.



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Abstract Graphic


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Hyaluronic Acid-Modified Cationic LipidâPLGA Hybrid Nanoparticles

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