Polymer-colloidal systems as MRI-detectable nanocarriers for peptide vaccine delivery
Graphical abstract
Introduction
Vaccine-induced active immunisation is now an indispensable aspect of modern human medicine. Vaccines play a key role, chiefly in the prevention of many types of serious infectious diseases, but recent efforts have extended their use to cancer immunotherapy [1], [2]. Although traditional vaccines based on live attenuated or killed microorganisms have significantly suppressed or even eradicated the spread of many types of serious infectious diseases in the human population, there is still a growing public demand for safer and longer-acting vaccines.
Peptide immunogens derived from the antigenic parts of microbes are highly promising representatives of safe and well-defined vaccines. Nonetheless, they are often characterised by weak immunogenicity [3], [4], [5]. This is mainly due to their small size and lower stability in the blood, which adversely affects their pharmacokinetic properties and ability to effectively crosslink immune cell receptors. Moreover, a significant proportion of peptide immunogens are poorly soluble in aqueous solutions, limiting their therapeutic use [4]. To overcome these obstacles, specific chemical modifications of peptide immunogens [6] or various particulate delivery systems [7] have been proposed to improve their penetration through cell membranes, modulate their persistence in the body or increase the multiplicity of immunogenic elements on their surfaces. The most frequently used delivery systems include supramolecular self-assemblies such as liposomes [8], polymer micelles [9], polymerosomes [10]; water-soluble high-molecular-weight macromolecules [11]; and inorganic and polymer-based colloids [12]. Such systems may also help to improve the solubility of immunogens, protect immunogens from premature degradation, and provide site-specific effects.
In order to maximise the delivery of immunogens and tune their spatial arrangement, the size, morphology and surface chemistry of these particulate carriers must be adapted. Although the optimal vaccine size varies depending on the desired immune response (CD8 or CD4 T-cell response) and the route of administration (intramuscular, intravenous or mucosal), particles in the range of 45–200 nm generally appear to be more advantageous for most types of parenterally administered vaccines [13], [14]. One reason is that larger (>1 μm) particles are trapped in the tissue at the injection site where they degrade before reaching the lymph node, while nanoparticles (<45 nm) have been shown to drain directly into the lymph nodes [15]. Another explanation is the preferential uptake of nanoparticles by various antigen-presenting cells compared to microparticles. In particular, dendritic cells favour taking up particles by macropinocytosis, which, for larger microparticles, is hard to achieve [16], [17].
In addition to size, the immunogenicity of a nanoparticle vaccine is also affected by the number and spatial arrangement of peptide immunogens incorporated into the carrier structure. Of the water-soluble biocompatible polymers, materials with multiple binding sites (e.g. copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA), 2-methacryloyloxyethylphosphorylcholine, 2-ethyl-2-oxazoline, etc.) are the preferred choice because they facilitate the covalent attachment of several immunogen copies attached via a stable or stimuli-responsive spacer [11], [18], [19], [20]. Although these immunogens are readily available, it should be noted that the total number of units attached is limited for steric reasons.
In contrast to hydrophilic polymer carriers, liposomes, polymer micelles and polymer particles are able to incorporate relatively high immunogen payloads, despite usually being encapsulated inside the particles, making them less accessible to antigen-presenting cell (APC) receptors. Therefore, it is necessary to ensure the release of immunogens from the cavity of the delivery system inside the APCs to promote their efficient presentation to CD8 + and CD4 + T cells. As for organic polymer particles, biodegradable aliphatic polyesters such as poly(lactic acid), poly(glycolic acid), poly(ε-caprolactone), poly(hydroxybutyrate) and their copolymers are a popular choice, because they ensure sustained release of the encapsulated immunogen, which is controlled by diffusion through a hydrolytically disrupted polymer matrix [21], [22]. However, the drawbacks are their larger (rather submicron) particle size, lower stability, and difficult surface functionalisation. Regarding liposomes and polymerosomes, pH-sensitive ionic lipids or polymers – prone to destabilisation or conformational changes in the acidic environment of the endocytic pathway – are usually incorporated into their structures to facilitate the release of peptide immunogens from cavities [23], [24], [25]. Although this approach is elegant, the materials used are more difficult to prepare and unsuited to the delivery of hydrophilic immunogens, as their encapsulation efficiency is usually very low [26]. Alternatively, peptide immunogens can be conjugated to the particle surface. In addition to lipid-based vesicles, inorganic micro- and nanoparticles can be used to deliver peptide immunogens that are chemically bound or physically adsorbed on their surface. The most frequently used inorganic delivery systems are nanoparticles based on gold, iron oxide or mesoporous silica [27], [28], [29]. They have the advantage of being reproducible, relatively easy to prepare, and adjustable in size and shape while also having a high immunogen-loading capacity. Unfortunately, they often struggle with increases in cellular toxicity and have poor biodegradability. Moreover, the immunogens that adhere tightly to their surfaces are not readily available to cellular receptors. A promising solution is to modify the surface of the inorganic core with the outer polymeric shell to create a hybrid polymer-inorganic nanomaterial. This strategy combines the benefits of both types of delivery systems and allows the preparation of stable non-toxic nanoparticles with a high content of readily accessible peptide immunogens.
In this work, we focused on the development of a novel hybrid delivery system based on maghemite (γ-Fe2O3) nanoparticles covered with a hydrophilic shell of poly[N-(2-hydroxypropyl)methacrylamide] (p(HPMA)) polymers. Various types of p(HPMA) polymers (telechelic polymers, statistical and di-block copolymers) were attached to the surfaces of γ-Fe2O3 particles via iron-chelating deferoxamine (DFA) groups linked either to the ends or along their chains through stable (amide) or bio-responsive (disulphide) bonds; the other sides of the polymer chains were terminated with a reactive group to ensure covalent attachment of the peptide immunogen. The resulting delivery systems formed precisely defined particles distinguished by high colloidal stability, low toxicity and hydrodynamic sizes similar to viruses (∼90–100 nm). The thickness of the polymer shell surrounding the particle and the number of immunogen-binding sites can be controlled by both the polymer selected and the weight ratio of particles to the polymer. While γ-Fe2O3 particles with the polymer chains attached to their surface through amide bonds were stable under different physiological conditions, those with polymers linked via disulphide bonds exhibited rapid removal of the polymer shell upon incubation in solutions mimicking reductive environments inside cells. In addition, a minimal peptide immunogen (V3) derived from the HIV-1 binding site was successfully conjugated to representative polymer-colloidal systems, demonstrating their suitability for vaccine delivery. Finally, the superparamagnetic properties of the nanoparticle vaccine enabled its detection by magnetic resonance imaging (MRI), as documented in both phantoms and mice.
Section snippets
Chemicals and cell-culture materials
(RS)-1-Aminopropan-2-ol, 3-aminopropanoic acid, 4-[(benzenecarbothioyl)sulphanyl]-4-cyanopentanoic acid, N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 2,2′-dipyridyldisulphide, 3-mercaptopropionic acid, methacryloyl chloride, propargylamine, triethylamine and 2-thiazoline-2-thiol (TT) were purchased from TCI Europe, Belgium. 4,4′-Azobis(4-cyanopentanoic acid) (ACVA),
Inorganic core synthesis and characterisation
Iron oxide-based particles have been widely used for numerous in vitro and in vivo biomedical applications [37]. This is principally due to their adjustable size and morphology, controlled surface functionalisation and relatively low toxicity. In addition, unlike commonly used inorganic particles based on gold, silver, silica or carbon, iron oxide particles can also offer superior magnetic properties, which facilitate site-specific targeting by external magnetic fields and sensitive MRI
Conclusions
This study focuses on the design, synthesis and characterisation of novel polymer-colloidal nanocarriers for the delivery of peptide vaccines. The nanocarriers were produced by attaching HPMA-based polymer arms of various types to the surface of an inorganic γ-Fe2O3 core via iron-chelating deferoxamine groups linked either to the ends or along their chains. In all cases, the modification of the γ-Fe2O3 surface was rapid, leading to the formation of well-defined nanoparticles of virus-like size
CRediT authorship contribution statement
Lucie Kracíková: Investigation, Formal analysis, Writing – original draft. Ladislav Androvič: Investigation, Formal analysis, Writing – review & editing. Lucie Schindler: Investigation, Formal analysis. Gabriela Mixová: Investigation, Formal analysis. Michal Babič: Conceptualization, Investigation, Formal analysis, Writing – review & editing. Monika Paúrová: Investigation, Formal analysis. Marcela Filipová: Methodology, Investigation, Formal analysis, Writing – review & editing. Jiřina
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
This work was supported by the Czech Science Foundation [project no. 19-08176S]; the Ministry of Education, Youth and Sport of the Czech Republic [project no. LTAUSA18173]; and the Ministry of Health of the Czech Republic [project no. NU20-08-00095].
References (67)
- et al.
Adjuvants for cancer vaccines
Semin. Immunol.
(2010) - et al.
Composition design and medical application of liposomes
Eur J Med Chem
(2019) - et al.
Liposomes to target the lymphatics by subcutaneous administration
Adv Drug Deliv Rev
(2001) - et al.
Poly-(lactic-co-glycolic-acid)-based particulate vaccines: Particle uptake by dendritic cells is a key parameter for immune activation
Vaccine
(2015) - et al.
Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery
Biomaterials
(2011) - et al.
pH-Responsive Polymersome Microparticles as Smart Cyclodextrin-Releasing Agents
Biomacromolecules
(2019) - et al.
Polymeric drugs based on conjugates of synthetic and natural macromolecules. I. Synthesis and physico-chemical characterisation
J Control Release
(2000) - et al.
Synthesis and properties of new N-(2-hydroxypropyl)methacrylamide copolymers containing thiazolidine-2-thione reactive groups
React. Funct. Polym.
(2006) - et al.
Surface modification of monodisperse magnetite nanoparticles for improved intracellular uptake to breast cancer cells
J Colloid Interface Sci
(2005) - et al.
Biosynthesis of desferrioxamine B in Streptomyces pilosus: Evidence for the involvement of lysine decarboxylase
FEMS Microbiol. Lett.
(1987)