Functionalized multifunctional nanovaccine for targeting dendritic cells and modulation of immune response

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Abstract

Multifunctional gelatin nanoparticles modified by NIR-emitting gold/silver alloy nanoclusters and loaded with ovalbumin (OVA) as a model antigen were developed. Two different designs of nanoparticles were introduced; positively charged NPs with OVA displayed over the surface (S-NPs) versus OVA encapsulated in the NPs’ matrix and the surface is functionalized by dextran (Dex-NPs) for dendritic cell targeting. The nanoparticles showed average particle sizes of 210 and 305 nm and zeta potentials of +25.6 and −23.9 mV, for S-NPs and Dex-NPs, respectively. Both types of NPs succeeded to induce maturation of murine bone marrow-derived dendritic cells (BMDCs) as indicated by the upregulated surface expression of MHC-II and co-stimulatory molecules CD86, CD80 and CD40. Dex-NPs induced no cytotoxicity in BMDCs, in contrast to S-NPs. Functionalization of NPs with dextran increased their uptake by BMDCs, enhanced secretion of immune stimulatory chemokines, and boosted their T cell stimulation capacity. Co-culture of NP loaded BMDCs with OVA-specific CD4 or CD8 T cells, induced enhanced T cell proliferation and release of IL-2 from both CD8 and CD4 cells and IFN-γ from CD8 T cells. This highlights the potential of the developed NPs as vaccines for inducing T helper 1 type CD4 as well as CD8 responses, such as vaccines for cancer or viral infections.

Introduction

Vaccines have been an effective tool for protection against diseases by activating and building the memory of immune cells to fight microbes and aberrant cells. However, diseases caused by intracellular pathogens (e.g., viruses) and cancer cells which have evolved to evade host cell immunity require the development of more effective and safe vaccines (Salem, 2015). Antigen-presenting cells (APCs), especially dendritic cells (DCs), can present exogenous and endogenous antigens on major histocompatibility complex (MHC) class II and class I proteins to interact with helper T-cells (TH, CD4) and cytotoxic T-cells (CTL, CD8), respectively. In turn, CD4 cells activate B-cells to produce antibodies against the antigen, while CD8 cells attack and destroy virus-infected or cancer cells (Paulis et al., 2013). Despite being challenging, the development of tumor vaccines using exogenous antigens can be achieved by enhanced uptake of the antigen and/or increased cross-presentation through endolysosomal escape (Embgenbroich and Burgdorf, 2018). As dendritic cells are key players in antigen cross-presentation, targeting dendritic cells has become a core concept in the field of cancer vaccines (Santos and Butterfield, 2018). While early approaches relied on the generation and ex vivo antigen loading of large amounts of dendritic cells (Palucka and Banchereau, 2013, Schreibelt et al., 2015), novel concepts aim at delivering antigen directly to dendritic cells in vivo (Bonifaz et al., 2002, Geijtenbeek and Gringhuis, 2016, Volckmar et al., 2017).

Nowadays, nanoparticles provide an attractive strategy for enhancing antigen delivery. They offer several advantages that overcome the demerits of soluble antigen application (Gregory et al., 2013); 1) antigen encapsulation provides protection of the antigen against premature undesired degradation, 2) displaying repetitive antigen on the nanoparticles’ surface mimics the natural way of antigen presentation by pathogens, 3) controlling and prolonging the release of the antigen enhances its exposure to the immune system, 4) concomitant loading of the antigen together with an adjuvant can enhance the limited immunogenicity of most proteins (Mönkäre et al., 2018, Paulis et al., 2013). Immune response to an antigen can be modulated by modification of nanoparticle size and surface properties. For instance, decoration of particles surface with moieties recognized by APCs may enhance receptor-mediated uptake of the antigen (Paulis et al., 2013). Another example is the surface modification with polyethyleneimine (PEI), which acts as a proton-sponge and can enhance the lysosomal escape of the antigen to the cytosol (Chen et al., 2011). Both strategies can help to induce antigen presentation to MHC class I and thus the activation of CTL.

Different nanocarriers have been introduced for loading and antigen delivery, among which polymeric nanoparticles have gained interest. Different categories of polymers have been tested including poly (esters), poly (α-hydroxy acids) (Mönkäre et al., 2018, Rietscher et al., 2016a, Rietscher et al., 2016b), proteins (da Silva et al., 2018, Lin et al., 2019) and polysaccharides (Gao et al., 2016, Gregory et al., 2013, Zhang et al., 2017). Gelatin is a hydrophilic biodegradable polypeptide polymer which provides a good opportunity for encapsulating high payloads of antigens under non-harsh conditions (Sahoo et al., 2015). This is important to preserve the proteins’ conformation or fold stability which, in turn, influences the intracellular processing of antigens and thus their immunogenic activity (Scheiblhofer et al., 2017). Additionally, gelatin is of low immunogenicity (Tondera et al., 2016) which is an important characteristic for an optimal vaccine nanocarrier. Gelatin nanoparticles (GNPs) have been investigated for the delivery of (model) antigens such as tetanus toxoid (Sudheesh et al., 2011) and ovalbumin (OVA), which were immobilized over the surface of the nanoparticles (Du et al., 2017, Lin et al., 2019). Further, changing the approach of antigen loading and the surface properties of the nanoparticles can manipulate the immune response to the antigen as investigated in our study.

In the current work, we present multifunctional functionalized gelatin nanoparticle for simultaneous imaging and modulating the immune response against the model antigen OVA. The NIR-emitting gold/silver nanocluster-labelled gelatin nanoparticles were designed as a novel imaging probe. Two designs for OVA loading have been adapted, direct loading and crosslinking versus surface loading by electrostatic interaction to poly-L-lysine-coated GNPs. OVA-encapsulated nanoparticles were further decorated with dextran, which is known for its high affinity to a group of receptors on DCs; the DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) family receptors: DC-SIGN (CD209) and L-SIGN (the liver and lymphatic endothelium homologue of DC-SIGN), the mannose receptor (CD206), and langerin (CD207) (Pustylnikov et al., 2014). The functionalization of the nanoparticles by dextran was tested for its capability to enhance the NP uptake by bone marrow-derived dendritic cells (BMDCs) and induce activation of these cells. We highlight the effect of the nanoparticle design on the response of the antigen presenting cells and how this affects the further cascade of the immune network (release of chemokines, proliferation and stimulation of T cells and cytokine release). Such in vitro cell models are easily accessible to many researchers and are indispensable tools for the screening of different nanoparticle designs. In compliance with the 3R principles, in vitro correlates for vaccine potency are intensely studied (Azizi, 2018).

Section snippets

Materials

Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O), gelatin type B (from bovine skin, gel strength ~75 Bloom), silver nitrate, albumin from chicken egg white, poly-L-lysine hydrobromide (Mw 30,000–70,000) (PLL), N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), dextran sulphate sodium salt (from Leuconostoc species, Mw 100,000), D-(+)- trehalose dihydrate, methanol were all purchased from Sigma Aldrich, Steinheim, Germany. Endotoxin-free

Design, preparation, and characterization of ovalbumin-loaded nanoparticles

Gelatin has been modified to be fluorescent by using it as a reducing and stabilizing agent for the synthesis of gold/silver alloy nanoclusters (NCs). The formed nanoclusters are ultra-small structures of size less than 1 nm and emit light in the near infra-red region (λem = 700 nm) (El-Sayed et al., 2019). The nanocluster-modified gelatin was further utilized for nanoparticle preparation by two-step desolvation method. The first desolvation step was to precipitate the high molecular weight

Discussion

In the current study we have designed nanoparticulate systems with different properties for the delivery of OVA as a model antigen. We have selected gelatin as biopolymer for the NP preparation. Gelatin is the product of collagen hydrolysis and is generally regarded as safe (GRAS) by the FDA. It is biodegradable and biocompatible with low or negligible antigenicity (Smith et al., 2016). It offers high capacity for delivery of biomacromolecules such as antigens (Sahoo et al., 2015). Due to its

Conclusions

Multifunctional gelatin nanoparticles for delivery of OVA as model antigen were developed. The imaging agent was a novel NIR-emitting gold/silver alloy nanocluster that was synthesized using gelatin as a templating agent and thus attached to the gelatin polymer backbone. Different designs of the nanoparticles were introduced to serve different functions and their effect on the antigen processing pathway by dendritic cells has been investigated. Positively charged nanoparticles displaying the

Disclosure

Graphical abstract was created using BioRender.com.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors acknowledge the Egyptian Ministry of Higher Education, Culture Affairs and Missions Sector (MOHE-CASM) for co-funding as well as the Austrian Science Fund (FWF), project W1213.

References (53)

  • L.E. Paulis et al.

    Dendritic cell-based nanovaccines for cancer immunotherapy

    Curr. Opin. Immunol.

    (2013)
  • R. Rietscher et al.

    Impact of PEG and PEG-b-PAGE modified PLGA on nanoparticle formation, protein loading and release

    Int. J. Pharm.

    (2016)
  • R. Rietscher et al.

    Antigen delivery via hydrophilic PEG-b-PAGE-b-PLGA nanoparticles boosts vaccination induced T cell immunity

    Eur. J. Pharm. Biopharm.

    (2016)
  • N. Sahoo et al.

    Recent advancement of gelatin nanoparticles in drug and vaccine delivery

    Int. J. Biol. Macromol.

    (2015)
  • J.R. Schoenborn et al.

    Regulation of interferon-γ during innate and adaptive immune responses

    Adv. Immunol.

    (2007)
  • A.M. Smith et al.

    Biopolymers as wound healing materials

  • M.D. Turner et al.

    Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease

    BBA

    (2014)
  • M. Yong et al.

    Expression of maturation markers on murine dendritic cells in response to group A streptococcal lipopeptide vaccines

    Vaccine

    (2009)
  • C. Zhang et al.

    Targeted antigen delivery to dendritic cell via functionalized alginate nanoparticles for cancer immunotherapy

    J. Control. Release

    (2017)
  • A. Baseer et al.

    Design and characterization of surface-crosslinked gelatin nanoparticles for the delivery of hydrophilic macromolecular drugs

    Macromol. Chem. Phys.

    (2019)
  • L. Bonifaz et al.

    Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance

    J. Exp. Med.

    (2002)
  • T.Z. Chang et al.

    Effects of ovalbumin protein nanoparticle vaccine size and coating on dendritic cell processing

    Biomater. Sci.

    (2017)
  • J. Chen et al.

    Improved antigen cross-presentation by polyethyleneimine-based nanoparticles

    Int. J. Nanomed.

    (2011)
  • J.H. Choi et al.

    Influence of pH and surface chemistry on Poly(L-lysine) adsorption onto solid supports investigated by quartz crystal microbalance with dissipation monitoring

    J. Phys. Chem. B

    (2015)
  • C.J. Coester et al.

    Gelatin nanoparticles by two step desolvation a new preparation method, surface modifications and cell uptake

    J. Microencapsul.

    (2000)
  • J. Conde et al.

    Revisiting 30 years of biofunctionalization and surface chemistry of inorganic nanoparticles for nanomedicine

    Front. Chem.

    (2014)
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