Research Paper
Protamine-based nanoparticles as new antigen delivery systems

https://doi.org/10.1016/j.ejpb.2015.09.019Get rights and content

Abstract

The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen.

The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine–polysaccharide nanoparticles as antigen delivery systems.

Introduction

Since their implementation in 1926, aluminum salts have had a crucial role in making vaccination the most important human intervention in the improvement of global health. But despite its great value, alum has important shortcomings, such as (i) insufficient adjuvant effect for subunit vaccines and peptides, (ii) lack of effect when administered via non-parenteral routes, (iii) unwanted side effects, i.e. local reactions or hypersensitization in allergic patients and (iv) limited thermostability [1], [2]. Due to these limitations, significant efforts have been made to develop alternative adjuvants.

In a broad sense, an adjuvant could be defined as a molecule or structure that can increase and/or modulate the immunogenicity of an antigen, allowing it to induce a potent and persistent immune response at low doses [3]. Some authors divided the adjuvants into two groups: immunostimulants (adjuvants that interact with specific receptors of antigen presenting cells) and delivery systems [4]. Particulate delivery systems may act as adjuvants because they can modify the uptake, trafficking and processing of antigens, which results in better and more adequate immune responses [5], [6].

In the design of novel “alum-free” vaccine particulate delivery systems, it is necessary to consider the nature of the antigen and its intrinsic immunogenicity, the administration route and the availability of biomaterials with an adequate safety profile. On the other hand, optimal particle size in antigen delivery still remains a controversial issue [4]. Nanometric systems have raised hope for better adjuvants because of their ability to control the release and increase the trans-epithelial transport of antigens, thus being considered as promising strategies for the development of single dose and needle-free vaccines [7], [8].

Different materials have been studied in order to develop particulate antigen delivery systems, lipids, polymers and polysaccharides among them. In particular, chitosan, polylactic and polylactic-co-glycolic acid have been the polymers most widely applied in the development of vaccine nanocarrier [9], [10]. More recently, polyaminoacids and polypeptides have been considered for their versatility and biocompatibility [11]. In particular, protamine, an arginine-rich peptide with cell-penetrating properties, has shown a synergistic adjuvant effect with other immunomodulatory molecules, i.e. CpG [12]. This effect was also observed when protamine was used in combination with PLGA microparticles. The resulting system was able to stimulate the proliferation of antigen-specific T cells and the secretion of IFN-γ [13]. So far, this polypeptide has been used as biomaterial for antigen delivery and immunomodulation in microparticles [14], DNA-loaded liposomes (LPD) [15] and complexes [16]. In addition to antigen delivery, protamine has also been combined with other biopolymers such as modified polyacrylic acids for mucosal delivery [17], [18].

Other materials, such as hyaluronic acid (HA) and alginate (ALG) have also shown immunoadjuvant activity, as indicated by macrophage and dendritic cell recruitment plus activation and/or induction of cytokine production [19], [20], [21], [22].

Based on this information, the aim of this work was to develop and characterize a new protamine based nanometric antigen delivery system to harness its adjuvant properties in association with a polysaccharide such as alginate or hyaluronic acid. The combination of these biomaterials, in a nanoparticulated form, generates new nanocarriers with safer materials and great adjuvant activity.

The ability of these systems to encapsulate and deliver antigens was assessed using recombinant hepatitis B surface antigen (rHBsAg) as a model antigen. Finally, the in vitro and in vivo performance of this novel delivery approach was assessed in macrophage cell cultures and upon administration to mice either by intramuscular or by nasal administration.

Section snippets

Chemicals

Protamine sulfate was purchased from Yuki Gosei Kogyo, Ltd. (Japan) and Hyaluronic acid (HA) of 162 kDa and 29 kDa was provided by Bioiberica (Spain) and by Soliance (France), respectively. Sodium alginate (ALG) (PRONOVA UP VLVG) of <75 kDa was supplied by Novamatrix (Norway). Recombinant hepatitis B surface antigen (rHBsAg) was kindly donated by Shantha Biotechnics Limited (Hyderabad, India). Enzyme linked immunosorbent assay (ELISA) kit (Murex rHBsAg Version 3) was obtained from Diasorin (United

Results and discussion

This paper describes the development of novel nanoparticulated antigen delivery systems consisting of protamine and polysaccharides, such as hyaluronic acid (HA) and alginate (ALG). The use of such biocompatible protamine-based carriers is an attractive approach for the development of vaccine nanocarriers. The properties of the protamine are expected to offer the nanoparticles advantageous characteristics over other nanocompositions in terms of enhanced antigen delivery and recognition by

Conclusion

Protamine:polysaccharide nanoparticles are introduced here as novel antigen delivery vehicles exhibiting a number of attractive features, namely: (i) highly versatile physicochemical properties; (ii) easy and mild preparation technique; (iii) ability to associate significant amounts of rHBsAg while keeping their integrity; and (iv) interaction with macrophages and immunostimulating properties both in vitro and in vivo. These results highlight the potential interest that the further development

Acknowledgments

This work was supported by the Xunta de Galicia (Competitive Reference Groups), the Spanish Ministry of Economy and Competitiveness (SAF2011-30337-553 C02-02) and by FP7/REGPOT-2012-2013.1-BIOCAPS-316265. Technical assistance of Jesús Méndez and Rafael Romero is highly appreciated. M. Peleteiro and J.V. Gonzalez-Aramundiz acknowledge their fellowships from the Spanish Ministry of Education (FPU predoctoral grants program) and from MAEC AECID.

References (48)

  • K.Y. Choi et al.

    Hyaluronic acid-based nanocarriers for intracellular targeting: Interfacial interactions with proteins in cancer

    Colloids Surf. B: Biointerf.

    (2012)
  • D. Lochmann et al.

    Albumin–protamine–oligonucleotide nanoparticles as a new antisense delivery system. Part 1: Physicochemical characterization

    Eur. J. Pharm. Biopharm.

    (2005)
  • C. He et al.

    Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles

    Biomaterials

    (2010)
  • S. Vicente et al.

    Co-delivery of viral proteins and a TLR7 agonist from polysaccharide nanocapsules: a needle-free vaccination strategy

    J. Control. Release

    (2013)
  • R. Simón-Vázquez et al.

    Chapter 15 – Nanotoxicology

  • E. Bergfors et al.

    How common are long-lasting, intensely itching vaccination granulomas and contact allergy to aluminium induced by currently used pediatric vaccines? A prospective cohort study

    Eur. J. Pediatr.

    (2014)
  • T.Y.H. Wu et al.

    Rational design of small molecules as vaccine adjuvants

    Sci. Transl. Med.

    (2014)
  • B. Guy

    The perfect mix: recent progress in adjuvant research

    Nat. Rev. Microbiol.

    (2007)
  • J.V. González-Aramundiz et al.

    Nanovaccine: nanocarriers for antigen delivery

    Biologie Aujourd’hui

    (2012)
  • Y.M. Vasiliev

    Chitosan-based vaccine adjuvants: incomplete characterization complicates preclinical and clinical evaluation

    Expert Rev. Vaccines

    (2015)
  • J.V. González-Aramundiz et al.

    Polypeptides and polyaminoacids in drug delivery

    Expert Opin. Drug Deliv.

    (2012)
  • J.M. Gómez et al.

    A protective allergy vaccine based on CpG- and protamine-containing PLGA microparticles

    Pharm. Res.

    (2007)
  • P.Y.J. Hsu et al.

    Encapsulation of poly(d,l-lactide) microparticles with polyelectrolyte multilayers for antigen delivery

    J. Microencapsul.

    (2014)
  • P. Li et al.

    Dendritic cell targeted liposomes-protamine-DNA complexes mediated by synthetic mannosylated cholestrol as a potential carrier for DNA vaccine

    Nanotechnology

    (2013)
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