Gold nanoparticles and vaccine development.

Review

Gold nanoparticles and vaccine development Expert Review of Vaccines Downloaded from informahealthcare.com by Nyu Medical Center on 07/09/15 For personal use only.

Expert Rev. Vaccines Early online, 1–15 (2015)

Jorge Alberto Salazar-Gonza´lez, Omar Gonza´lez-Ortega and Sergio Rosales-Mendoza* Laboratorio de Biofarmaceuticos Recombinantes, Facultad de Ciencias Quı´micas, Universidad Autonoma de San Luis Potosı´, Av. Dr. Manuel Nava 6, SLP, 78210, Mexico *Author for correspondence: Tel.: +444 826 2440 Fax: +444 826 2440 [email protected]

Mucosal vaccines constitute an advantageous immunization approach to achieve broad immunization against widespread diseases; however, improvements in this field are still required to expand their exploitation. As gold nanoparticles are biocompatible and can be easily functionalized with antigens, they have been proposed as carriers for the delivery of vaccines. The study of gold nanoparticles (AuNPs) in vaccinology has been of interest for a number of research groups in recent years and important advances have been made. This review provides a summary of the AuNPs synthesis methodologies and an updated overview of the current AuNPs-based vaccines under development. The implications of these advances for the development of new mucosal vaccines as well as future prospects for the field are discussed. KEYWORDS: antigen uptake . delivery vehicle . functionalization . immunogenicity . intranasal immunization .

mucosal vaccine . nanoparticles

.

oral immunization . toxicity

The current challenges for the vaccinology field comprise the identification of new immunoprotective targets as well as the development of formulations and immunization schemes capable of inducing robust immune responses conferring immunoprotection without undesired serious side effects. These goals are yet to be accomplished for several infectious and non-communicable diseases, and constitute an urgent need due to the sustained epidemiologic impact associated with these diseases [1,2]. Mucosal vaccines constitute a priority as they are of easy, painless, and minimal risk administration in comparison to parenteral vaccines; they can protect mucosal tissues, which are the main port of entry for most pathogens. Although the majority of the licensed vaccines are administered parenterally, even those that protect against pathogens whose transmission occurs at mucosal tissues [3]; they are effective in reducing the impact of infections caused by enteric pathogens, such as rotavirus, V. cholerae, and S. typhi [4]. In particular, oral immunization is a key goal for vaccinology facing a number of challenges. The immune system associated to the gastrointestinal tract is characterized by unresponsiveness and thus, strong adjuvants are typically included in oral formulations to override immune tolerance mechanisms. Unfortunately, only few adjuvants are considered safe and informahealthcare.com

10.1586/14760584.2015.1064772

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vaccine cost

with the potential to be approved for human use [5,6]. Developing effective oral vaccines also requires minimizing antigen degradation by digestive enzymes, enhancing antigen uptake by M cells or dendritic cells, and achieving proper immune response polarization. For example, the induction of cytotoxic lymphocyte responses, which are of key relevance for the defense against highly serious epidemic diseases such as HIV/AIDS, tuberculosis, and cancer, is also a challenging objective [7]. As these mentioned aspects are critical to attain strong immunogenicity, and thus efficacious vaccines, a myriad of efforts have been performed to develop delivery vehicles overriding these obstacles [5,8]. The notion of using new vaccine delivery approaches based on materials that, besides serving as delivery vehicles, could exert adjuvant activities has been explored over the past years. Liposomes, genetically engineered bacteria, plant cells, and algae are some of the alternatives that have been evaluated by several research groups with several candidates under clinical evaluation [9–11]. A number of efforts have been focused on using particles at the micro- and nanoscales to develop convenient vaccine delivery vehicles. Among these PLGA and chitosan particles, liposomes, and ISCOMS have shown good functionality promises and some integrated reviews

2015 Informa UK Ltd

ISSN 1476-0584

1

Review

´ lez, Gonza ´ lez-Ortega & Rosales-Mendoza Salazar-Gonza

on this matter have been previously published [12–16]. Among these vaccine prototypes, gold nanoparticles (AuNPs) constitute a suitable platform for delivering drugs and biopharmaceuticals since these can be easily synthesized, functionalized, and are biocompatible [17]. AuNPs have gained substantial popularity in the biomedical field. Although recent reviews have been published in the topic of gold particles and medical applications [18–20], this review provides a description of synthesis methodologies and an updated overview on the vaccines developed thus far, emphasizing the role of these technologies in the development of mucosal vaccines.

regulator of this phenomenon having the production of antiinflammatory cytokines and a subsequent depression of the DCs activity as the main effector mechanisms [36,37]. Successful immunization schemes intended to induce active immunity should combine proper delivery vehicles, doses, and immunization regime to avoid tolerogenic responses. A general criterion to achieve active responses is avoiding high doses administered at high frequency. In contrast, the immunization schemes aimed at inducing immunological tolerance associated with therapeutic effects are designed in an opposite manner [38].

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Synthesis of AuNPs Mucosal immunity

The efficiency of mucosal vaccines is governed by complex mechanisms of the mucosal associated lymphoid tissues (MALT). The heterogeneity of the mucosal compartments in terms of the immune system components represents a determinant factor for the type and magnitude of the immune responses. However, common mechanisms behind the elicitation of immune responses in mucosal tissues are well known [21]. Antigens at the gastrointestinal tract can be sampled by either M cells at the Peyer’s patches or DCs in a process mediated by dendrites. Once at the subepitelium, the antigen processing phase is critical since it constitutes the interphase between the innate and adaptive immunities [22,23]. In terms of antigen processing, a common processing step involves the action of dendritic cells, which act as antigen presenting cells that subsequently migrate to the draining lymph nodes to mediate the activation of lymphocytes as the key step to initiate the induction of adaptive immune responses [24]. Lymphocyte activation is the subsequent step allowing for the expansion and maturation of B, Th or CTL responses. These lymphocytes can migrate to the systemic circulation, the priming mucosal site, or distant mucosal compartments, which open the possibility for achieving immunoprotection at both the systemic and mucosal levels, even covering different mucosal compartments to those where the antigen was administered [24]. Developing vehicles able to efficiently activate these immune mechanisms to elicit the desired immune response through the mucosa is critical for the progress of mucosal vaccines [25,26]. The parameters that influence the quality of these formulations are antigen stability, antigen bioavailability, antigen uptake rate, and adjuvant activity [27,28]. As particulate antigens are sampled more efficiently by DCs, several types of adjuvants rely on insoluble carriers capable to be internalized with the subsequent activation of inflammatory responses that lead to an efficient antigen presentation and the induction of robust adaptive immune responses [29,30]. In fact, most of the adjuvants enhance T and B cell responses by engaging components of the innate immune system, rather than exerting direct effects on lymphocytes [31–35]. On the other hand, tolerogenic responses consist of the suppression of the immune effector mechanisms mediated by CTL and Th lymphocytes. The regulatory T cells are the main doi: 10.1586/14760584.2015.1064772

The synthesis of gold nanoparticles is a redox reaction in which Au(III) ions are reduced to metallic gold. The most common oxidized gold compound is HAuCl4, whereas the most common reducing agents are sodium citrate and NaBH4, although several others had been used, such as ascorbic [39], formaldehyde [40], hydrazine [41], and borohydride [42]. Thus, many protocols are available to produce gold nanoparticles with varying size, shape and surface ligand composition. The molecules present at the gold nanoparticle surface are important as they allow for nanoparticle stabilization and are the basis for nanoparticle bioconjugation. In general, the protocols produce either gold nanoparticles dispersed in aqueous or in organic solvents. As biomedical applications use gold nanoparticles soluble in aqueous solvents, this review will focus in their synthesis. Synthesis using citrate

Synthesis of gold nanoparticles using sodium citrate as reducing agent is by far the most employed procedure to generate aqueous soluble nanoparticles due to its simplicity. The protocol was first described by Turkevich et al. [43] and produces gold nanoparticles with a size of 15 nm. Citrate not only reduces the gold ions but also adsorbs to the surface of the gold nanoparticles imparting a negative charge making them stable. The general protocol is as follows: Tetrachloroauric acid is dissolved in water and the solution is heated until boiling, sodium citrate is the added under vigorous stirring and the mixture is refluxed. The gold nanoparticles are synthesized within minutes, the aqueous solution changes coloration following the sequence: yellow, colorless, purple and red. Several modifications of this protocol had been followed to generate gold nanoparticles of different size. The redox reaction for the synthesis of gold nanoparticles using citrate is depicted in FIGURE 1 where acetone dicarboxylate and carbon dioxide are formed. The citrate ions are added in excess to speed up the reaction and become adsorbed on the gold nanoparticle surface for stabilization purposes. If positively charged gold nanoparticles are desirable, three approaches can be followed. The first approach consists of synthesizing negatively charged gold nanoparticles, using citrate as described, and performing a ligand exchange reaction to replace the citrate ions with molecules bearing positive charges. For this, it is a common practice to use, as replacing ligand, a molecule bearing a positive charge with a thiol moiety, such as Expert Rev. Vaccines

Gold nanoparticles and vaccine development

O– 3

O–

OH

O

O

O –

+

2AuCl4–

O

O

Citrate

Review

O–

3

O

+ 3CO2 + 3H+ + 2Au0 + 8Cl–

-

O O Acetone dicarboxylate

Growth, stabilizer


Figure 1. The redox reaction for the synthesis of gold nanoparticles using citrate. Acetone dicarboxylate and carbon dioxide are formed. The citrate ions are added in excess to speed up the reaction and to become adsorbed on the gold nanoparticle surface for stabilization purposes.

cysteamine [44]. The second approach, with the same result as the first one, consists of synthesizing the gold nanoparticles using a stronger reducing agent, such as borohydride, in the presence of the molecule bearing a positive charge with a thiol moiety; thus obtaining gold nanoparticles capped with groups having positive charges [45]. The third approach consists of synthesizing gold nanoparticles using a molecule for reduction and stabilization purposes, such as polyethyleneimine (PEI). Some of the PEI molecules are used for reduction of the gold ions, whereas other PEI molecules become adsorbed on the surface of the gold nanoparticle thus imparting a positive charge [46]. Protein functionalization (bioconjugation) of AuNPs

The placement of a protein molecule over a gold nanoparticle surface can be achieved following direct or indirect methodologies. In the direct methodology, when protein molecules are added to a gold nanoparticle suspension, these can be adsorbed on the surface of the particles by electrostatic, hydrophobic, Van der Waals, and coordination interactions (FIGURE 2). In the electrostatic interaction, a positively charged protein molecule is attached by the negatively charged surface of a gold nanoparticle. Hydrophobic interaction comprises the adsorption of protein on the surface of the gold nanoparticle involving the hydrophobic patches on the surface of the protein. The adsorption of protein molecules to the surface of a gold nanoparticle involving dipoles are contained in the Van der Waals interactions. Coordination interactions consider the establishment of a coordination bond between the protein molecule and the surface of the gold nanoparticle. In the indirect methodology, also shown in FIGURE 2, a linker is used between the gold nanoparticle surface and the protein molecule. The linker and the protein molecule are covalently joined. A molecule containing polyethylene glycol (PEG) can be used as the linker to impart stability to the gold nanoparticle as later described. To functionalize a gold nanoparticle with a protein molecule through electrostatic interactions, two aspects are vital. The surface of the gold nanoparticles is negatively charged due to the presence of adsorbed AuCl4 ions or some other negative ions. To interact with these negative ions, the protein molecules need to be positively charged. To ensure that most of the protein molecules are positively charged, the pH of the solution has to be at least one unit below the isoelectric point of the informahealthcare.com

protein. The positive charge in a protein molecule is due to the presence of basic residues, such as lysine. Thus, a higher amount of lysine residues favors the possibility of electrostatic interaction between the protein molecule and the gold nanoparticle surface. The second aspect deals with the stability of the gold nanoparticles. Depending on the synthesis methodology of the gold nanoparticles, these are generally stable (from aggregation) due to the presence of AuCl4 ions or other molecules that contribute toward the negative charge of the gold nanoparticle. These negative charges on the surface of the gold nanoparticles produce a particle–particle repulsion thus stabilizing the suspension. Nonetheless, if this particle–particle repulsion is somehow reduced, gold nanoparticles will enter the region of the Van der Waals attraction and start to agglomerate. When protein molecules are then added to a gold nanoparticle suspension, they can bind electrostatically to the particles creating a new entity that offers new regions for Van der Waals attractions that can promote the aggregation. To prevent this, the number of protein molecules on the surface of the gold nanoparticles must continue to provide proper particle–particle repulsion. To ensure that this situation prevails, it is common Van der Waals interaction

Electrostatic interaction

Gold nanoparticle H S

Coordination interaction

Hydrophobic interaction

Figure 2. Schematic representation of protein placement over a gold nanoparticle surface through direct or indirect methodologies.

doi: 10.1586/14760584.2015.1064772

Review


A.

B.

Linker

Linker Protein

Gold nanoparticle


Protein

Gold nanoparticle conjugate

Gold nanoparticle

Linker-Protein

Gold nanoparticle conjugate

Figure 3. Functionalization of the surface of gold nanoparticle through coordination bonds. (A) Direct methodology. The linker is attached to the surface of the gold nanoparticle through coordination bonds. (B) Indirect methodology. The second approach first attaches the protein molecule to the linker and then the complex linker protein is attached to the surface of the gold nanoparticle using again coordination bonds.

to add NaCl to the functionalized gold nanoparticles to verify their stability as this addition will promote the Van der Waals attraction. A situation that also needs to be prevented is the possibility that a single protein molecule attaches to several gold nanoparticles promoting particle aggregation. To avoid this, the amount of protein molecules has to be large enough to guarantee that protein molecules only attach to one gold nanoparticle. Brewer et al. [47] evaluated the interaction between citrate-coated gold nanoparticles (negatively charged) with BSA molecules and determine the interaction as being electrostatic even when working at the isoelectric point due the presence of protonated lysine residues. If we are interested in functionalizing gold nanoparticles regardless of the adsorption mechanism, the gold nanoparticle suspension can be contacted with the protein molecules at a pH close to their isoelectric point. This situation will favor the hydrophobic and Van der Waals attractions between the protein molecule and the surface of the gold nanoparticle. The other two types of interactions could also occur as part of the final adsorption process. An important aspect is to ensure that the gold nanoparticles are complete covered by protein molecules such that particle–particle interactions are reduced. To ensure this, it is a common practice to contact the gold nanoparticle suspensions with different amounts of protein and add NaCl. Those batches were particle agglomeration occurs are then discarded. The agglomeration process can be followed visually, if a blue coloration appears agglomeration exists. This process was used by Paciotti et al. [48] to conjugate colloidal gold (33 nm in diameter) with the TNF where the pH for maximum protein adsorption was determined. Similarly, Kamnev et al. [49] prepared gold bioconjugates using staphylococcal protein A by simply mixing the colloidal gold (30 nm) suspension with the protein solution. El-Sayed et al. [50] adsorbed anti-EGFR on the surface of gold nanoparticles under the presence of polyethylene glycol to prevent particle aggregation.

doi: 10.1586/14760584.2015.1064772

When using electrostatic interactions to functionalize gold nanoparticles, negatively charged particles can be modified with positively charged biomolecules, creating a positively charged entity which, in theory, can interact with the negatively charged cell surface. If positively charged gold nanoparticles are modified with negatively charged biomolecules, a negatively charged functionalized gold nanoparticle is created and when in contact with negatively charged cells a poor interaction takes place impairing the entrance of the particles [51]. It is important to mention that when a protein molecule interacts hydrophobically with the gold nanoparticle, the protein could undergo conformational changes that could affect their activity (if any). For instance, Shang et al. [52] determine that the bioconjugation of BSA on the surface of gold nanoparticles resulted in conformational changes in the protein molecule at secondary and tertiary structure levels. 25-nm gold nanoparticles conjugated to monoclonal antibodies specific to HER2 overexpressing SKBR3 breast carcinoma cells were prepared by Rayavarapu et al. [53] using a combination of electrostatic and hydrophobic interactions. The coordination process between a protein molecule and a gold nanoparticle surface is considered to be mediated by free thiol groups on the surface of the protein molecule. The sulfur atom can fill a free orbital from gold atoms on the surface of the gold nanoparticle, establishing a coordination bond sometimes called a coordinate covalent bond. Other groups from the protein molecule can coordinate with the gold surface, such as amino groups and carboxylic groups; nonetheless thiolcontaining molecules are used routinely to coat the surface of metallic particles. For example, Tsai et al. [54] attached BSA to gold nanoparticles (10, 30, and 60 nm in diameter) and concluded that the thiol group from a cysteine residue was responsible for the adsorption of protein on the surface of the gold nanoparticle. As the coordination bond is stronger than, under proper circumstances, electrostatic interactions, hydrophobic interactions,

Expert Rev. Vaccines



or Van der Waals interactions, it is exploited to functionalize the surface of gold nanoparticle using an indirect methodology. For this, two approaches can be followed. The first approach consists in attaching a molecule (linker) to the surface of the gold nanoparticle through coordination bonds (FIGURE 3A). The molecule presents at one end a thiol group and at the other end a reactive group. Once the molecule is attached to the gold nanoparticle, the reactive group is used to covalently bind a protein molecule. If the reactive group is a carboxylic moiety, free amino groups from the protein molecule can be reacted with the carboxylic group to form a covalent bond using carbodiimide chemistry. If the reactive group is an aldehyde group, free amino groups from the protein molecule can be reacted with the aldehyde group to form a covalent bond using reductive amination chemistry. It is also possible to modify the surface of the gold nanoparticle with charged molecules that act as adsorption sites for the electrostatic interaction with protein molecules. In this regard, AubinTam and Hamad-Schifferli [55] modified the surface of gold nanoparticles (1.5 nm in diameter) with aminoethanethiol. The thiol group interacted with the surface of the gold nanoparticle while exposing a positive charge (from the amino group). Then the nanoparticles were exposed to cytochrome c for conjugation through electrostatic interactions with negatively charged regions on the surface of the protein. An alternative methodology using the affinity of thiol groups to the gold nanoparticle surface and the affinity of proteins toward immobilized metal ions was reported by Abad et al. [56]. These researchers first attached lipoic acid to the surface of gold nanoparticles, afterwards a chelating agent (amino-nitrile-triacetic acid) charged with Co(II) ions was reacted with the carboxylic groups from the lipoid acid using carbodiimide chemistry. Finally, two recombinant proteins (horseradish peroxidase and ferredoxinNADP+ reductase) were then attached to the gold nanoparticle by interaction of their His-tag moieties with the immobilized metal ions by means of coordination bonds. The same approach, but using Ni(II) ions, was followed by Hu et al. [57] to assemble histidine-tagged proteins: adenovirus serotype 12 knob and mycobacterium tuberculosis 20S proteasome. Pandey et al. [58] also bioconjugate an enzyme on the surface of gold nanoparticles. First, the gold nanoparticles were modified with mercaptoundecanoic acid (as linker) and then the enzyme was covalently attached using carbodiimide chemistry mediated by N-hydroxysuccinimide. The same last procedure was employed by Li et al. [59] to immobilize glucose oxidase on the surface of gold nanoparticles. An alternative procedure for bioconjugation was reported by Brennan et al. [60]. In this approach, a gold nanoparticle is modified with a linker bearing azide groups, an acetylene-functionalized lipase molecules was then reacted with the azide groups using Cu(I) as a catalyst following a click chemistry. For this process, it is necessary to previously modify the desired protein to bear an acetylene moiety. This prior modification can be accomplished through carbodiimide chemistry. Cao and Sim [61] used a bifunctional oligoethylene glycol thiol as linker to modify, and informahealthcare.com

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stabilize at the same time, gold nanoparticles. The carboxylic group from the linker was then used to couple an anti IgG–HRP complex. The second approach first attaches the protein molecule with the linker and then the complex linker–protein is attached to the surface of the gold nanoparticle using again coordination bonds (FIGURE 3B). This approach was used by Loo et al. [62] to bioconjugate gold nanoparticles (gold nanoshells) to attach either anti-IgG or anti-HER2. For this, the antibody was first covalently attached to the linker (orthopyridyldisulfide-polyethylene glycol-N-hydroxysuccinimide) using the hydroxysuccinimide group and free amino groups from the antibody. Afterwards the linker–protein complex was attached to the gold surface using the disulfide group. Hirsh et al. [63] applied this approach to attach anti-rabbit IgG on a gold surface. Kumar et al. [64] used as linker a dithioalkanearomatic polyethylene glycol derivative having a hydrazide moiety. This molecule was attached to a glycosylated antibody (previously oxidized to create an aldehyde reactive group) forming a hydrazine bond. The thiol groups from the linker were then used to attach the linker–protein complex to 20 nm gold nanoparticles. The last approach was also used by Mallidi et al. [65] to bioconjugate anti-EGFR monoclonal antibodies to 50 nm gold nanoparticles. Following a similar approach, Chen et al. [66] first modified antibodies with succinimidyl propionyl polyethylene glycol disulfide (linker). This bifunctional linker was able to incorporate two antibody molecules to its ends. Then the disulfide moiety was used to interact and modify hollow gold nanoparticles. The following section describes the current knowledge on the properties of gold nanoparticles in terms of uptake by mammalian cells and triggering inflammatory responses, factors that are directly related to immunogenicity and adjuvanticity. AuNPs uptake & immune function

AuNPs have been researched for biomedical applications due to their biocompatibility and singular physicochemical properties, such as size and surface area, making site-specific delivery and size-specific deposition possible while increasing uptake efficiency. These attributes have implications on the design of therapies based on sensing, photothermal processes, tracking, and drug delivery [67,68]. The interactions of AuNPs with the cell have been studied in several cell types including liver cells [69], human endothelial, and epithelial cells [70], as well as in cell lines comprising bronchial epithelial cell line BEAS-2B, the Chinese hamster ovary cell line CHO, and the human embryonic kidney cell line HEK 293 [67]. It has been found that particle size, functionalization, cell type, AuNPs shape, orientation of the process (with/against the gravity), and concentration are factors that influence AuNPs uptake. Vetten et al. [71] observed an efficient uptake of 14 and 20 nm AuNPs by BEAS-2B cells in a short period of time (1 h) with minimal toxicity. Interestingly, these particles aggregated once internalized by these cells. Similar findings were found for HEK 293 cells. In contrast, CHO cells doi: 10.1586/14760584.2015.1064772


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showed a size-dependent uptake where the 14 nm and the 20 nm AuNPs could enter cells in a time-dependent manner with higher toxicity found for the 20 nm AuNPs. Differences in uptake by HeLa, A549, and 1321N1 cell lines have also been shown for 40 and 200 nm nanoparticles synthesized with other nanomaterials (e.g., polystyrene) [72]. Regarding uptake mechanisms, it has been hypothesized that serum proteins are absorbed onto negatively charged AuNPs, an event that allows for nanoparticle entry via receptor-mediated endocytosis [73]. In the case of immunotherapies, studies on the interactions and effects of AuNPs on DCs is critical due to their role on the activation of the adaptive immune system function; thus targeting DCs through specific surface properties could make the effective induction of proper immune responses in terms of potency and polarization possible [74]. Several reports on the safety of AuNPs for DCs in terms of cytotoxicity, cytokine production, activation, and phenotype changes in vitro have been published [75,76]. However, the detailed knowledge of AuNPs and immune cell interaction and their potential immunostimulation or immunosuppression effects remains to be explored. It has been reported that coating AuNPs with PEG, polyvinyl alcohol (PVA), or a mixture of both influence the interaction with monocyte-derived dendritic cells. PEG-COOH AuNPs showed limited uptake but induced significant TNF-a production, whereas (PEG+PVA)-NH2 and PVA-NH2 AuNPs were introduced at higher rates inducing IL-1b secretion. Surprisingly, none of the AuNPs caused changes in phenotype, activation, or immunological properties of MDDC [77]. The lack of correlation between uptake magnitude and cytokine secretion, in this particular study, raises new questions on the mechanisms involved in the differential behavior of these functionalized AuNPs and how these nanomaterials may induce adaptive immune responses in a DC-independent manner, which indeed will be relevant aspects for the design of AuNPsbased vaccines. Studies with AuNPs (2.4 to 89 nm in diameter), functionalized with cell penetrating peptides and poly(vinyl alcohol) and poly(ethylene glycol)-based polymers, evaluated the uptake in human monocyte derived dendritic cells. A differential functionalization was also associated with changes on cellular uptake [78]. Intracellular localization was correlated with the AuNPs diameter: the smallest AuNPs (2.4 nm) localized in the nucleus, whereas intermediate size particles (5.5 and 8.2 nm) were partially found in the cytoplasm, and large nanoparticles (>16 nm) did not enter the cells [79]. Interestingly, the behavior of AuNPs in mucosal tissues has also been studied. Developing oral vaccines demands the design of particulate delivery vehicles capable of resisting an aggressive environment at the gastrointestinal tract and being efficiently uptaken by DCs located at the gut-associated lymphoid tissues. Adjuvanticity of particulate antigens is well described and is mainly due to their higher uptake rate by DCs. Remarkably, the gastrointestinal uptake and subsequent distribution of metallic colloidal gold particles of distinct sizes (ranging from 4 to 58 nm) have been studied in mice. Uptake is dependent doi: 10.1586/14760584.2015.1064772

on particle size with the smaller the better. Particle uptake occurred in the small intestine by persorption through holes created by extruding enterocytes [80]. Kingston et al. [81] have generated evidence on the lack of toxicity of 50 nm AuNPs coated with poly-N-vinylpyrrolidone and their effect on cellular responses to infection or inflammation by altering the balance of cytokines. In mouse macrophage, upon LPS treatment, increasing AuNPs concentrations led to a decrease in the release of TNF-a. The reactive oxygen species (ROS) were reduced upon AuNPs presence, suggesting oxygen radical scavenging. In splenocyte cultures, the production of IL-17 and TNF-a upon LPS treatment was reduced. These studies will be useful to achieve the design of new AuNPs that are successfully delivered at the GALT for the formulation of efficient oral vaccines. AuNPs functionalized with chitosan have been explored as delivery vehicle for insulin through the intranasal and oral routes with promising results [82]. Brandenberger et al. [83] used 15 nm AuNPs for evaluating their safety when administered through the airways. The experimental model consisted of a triple cell co-culture aimed at stimulating the alveolar lung epithelium. Measurements of pro-inflammatory (TNF-a, IL-8, iNOS) and oxidative stress markers (HO-1, SOD2) revealed that no adverse effects were induced by AuNPs, supporting the use of AuNPs as safe vehicles for the delivery of antigens through the airways. Other reports on the literature indicate the effect of AuNPs on the immune system function. It deserves our attention that AuNPs have shown effects on the inflammatory process associated to obesity. The treatment of mice with spherical AuNPs (21 nm in diameter) correlated with a significant fat loss and inhibition of inflammatory effects. This report pointed out the possible use of AuNPs in the fight against chronic diseases where inflammatory processes play a key role [84]. Parenteral vaccine prototypes Targeting infectious diseases

Niikura et al. [85] demonstrated that the shape and size of AuNPs affect the immune response polarization in an immunization model against the West Nile virus. AuNPs with three different shapes (spherical, rod-like, and cubical) and different sizes (20 and 40 nm spheres, 40 nm long 10 nm wide for rods, and 40 nm in edges cubes) were conjugated with the West Nile virus envelope (WNVe) protein. Mice immunized with these formulations developed humoral responses when intraperitoneally administered in the absence of adjuvants. The 40 nm spherical AuNPs induced the highest level of specific West Nile virus envelope (WNVe)-IgGs (1200 titters), whereas rod AuNPs induced a half antibody titter. In vitro assays to estimate AuNPs uptake and interleukins production in RAW264.7 cells, which are used as a model for primary macrophages and bone marrow-derived dendritic cells (BMDCs), respectively, revealed that particle uptake is more efficient for the rod-AuNPs suggesting that the antibody production is not dependent on the uptake efficiency. As for the cytokine production from bone marrow-derived dendritic cells (BMDCs) Expert Rev. Vaccines



treated with the AuNPs, only rod-AuNPs-treated cells produced significant levels of IL-1b and IL-18; meanwhile 40 nm spherical-AuNPs and cube-AuNPs significantly induced inflammatory cytokine production including TNF-a, IL-6, IL-12, and granulocyte macrophage colony-stimulating factor (GM-CSF). Therefore, these results provide evidence on the adjuvant activity of AuNPs and a relevant size/shape-dependent response which can be applied to polarize the immune response in specific applications. Similar findings have been reported by Chen et al. [86], where spherical AuNPs (ranging from 8 to 17 nm in diameter) were conjugated to an epitope of the Foot and Mouth disease virus viral protein 1. Specific IgGs were induced after intraperitoneal or subcutaneous immunization of mice. Besides peptide-conjugated AuNPs vaccines, carbohydrateconjugated AuNPs have also been developed showing similar immunogenicity. Safari et al. [87] fused the Streptococcus pneumoniae type 14 capsular polysaccharide (Pn14PS) to 2 nmspherical AuNPs and administered this candidate vaccine to mice by the subcutaneous route. Specific anti-Pn14PS IgG antibodies (titers of 1000) were successfully induced. TNF-a, IL-2, and IL-5 production by spleen cells of the vaccine-treated mice group was also observed, which is a characteristic profile of memory T-cells activation. A novel approach to formulate vaccines using AuNPs consists on coating them with native bacterial membranes collected from bacterium-secreted outer membrane vesicles, which was called BM-AuNPs. This formulation was shown to be highly stable, and in mice subcutaneously immunized with BMAuNPs, a rapid activation and maturation of DCs in lymph nodes was induced. Strong and durable antibody responses were also induced in a superior magnitude than those attained with OMVs alone. The same was observed for the production of IFN-g and IL-17 but not IL-4, indicating their polarization to Th1 and Th17 biased cell responses [88]. Burkholderia mallei, which is the causative bacterial agent of glanders disease and a potential bioweapon, has also been targeted through a AuNPs-based vaccine approach. AuNPs were functionalized with LPS from B. thailandensis and their immunoprotective potential was evaluated initially in mice using AuNPs covalently coupled to protein carriers (TetHc, Hcp1, or FliC). The latter were subsequently conjugated to LPS from B. thailandensis, which is a non-virulent clonal relative. This vaccine was administered to mice by the intranasal route threetimes at 2-week intervals (LPS dose of 0.93 mg plus alhydrogel), generating significantly higher antibody titers than those induced by LPS alone. This immune response proved to be immunoprotective as immunized animals showed improved protection against a lethal inhalation challenge of B. mallei [89]. A further investigation provided evidence on the immunoprotective potential of this vaccine in rhesus macaque as an advanced biomodel. Although not full protection was achieved in animals immunized with the AuNPs-based vaccine, half of the immunized animals produced IgG responses against LPS and survived to a challenge with an aerosol B. mallei. One informahealthcare.com

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month after challenge, the surviving animals did not present bacterial load in multiple organs in comparison to nonvaccinated animals [90]. Targeting non-infectious diseases

Most cancer and cancer-associated antigens are self-antigens, to which cytotoxic T-lymphocyte (CTL) responses are difficult to induce. Thus, the effectiveness of vaccines against such cancer antigens is limited by the fact that tumor cells easily evade the immune system effector mechanisms, a process named immune surveillance. Therefore, other biomedical application based on AuNPs are anticancer immunotherapies that rely on the use of peptides, DNA, or carbohydrates conjugated to NPs. This aim is of particular relevance as current therapies are limited on safety and efficacy due to a poor antigen delivery to dendritic cells (DCs) or lymph nodes (LN) and a low efficiency in eliciting antigen-specific CTL responses. Tumor-associated carbohydrate antigens (TACA’s) are glycan chains specifically and aberrantly expressed on the surface of malignant cells [91]. Therefore, antibody production mediated by glycopeptide chains coupled to AuNPs has been explored as a strategy to eliminate tumor cells by the action of antibody mediated cytotoxicity. Brina˜s et al. [92] conjugated tumor associated glycopeptide antigens to 3–5 nm spherical AuNPs. The subcutaneous immunization with 50 mM particle concentration with boosts every 2 weeks for 12 weeks in mice produced both IgM and IgG responses against the target glycopeptides. The therapeutic potential of this approach is yet to be fully determined. The intramuscular administration of AuNPs directly to local LN in mice, which contains a high population of APCs, has proven to be an effective approach to induce humoral and CTL responses [93]. Intratumor administration of AuNPsderived vaccines has also been explored as this administration mode is more efficient than intravenous or subcutaneous injections [94,95]. The administration of 1013 spherical AuNPs of 15 nm, induced TNF-a, IL-6, and G-CSF secretion and induced CD8 (cytotoxic T cells), CD4 (helper T cells), CD11c (DCs), and CD11b (macrophage) proliferation. Also tumor infiltration significantly inhibited tumor growth by 70% and promoted survival in immunized mice implanted with tumor cells [96]. AuNPs were recently assessed for the delivery of an ovalbumin (OVA) peptide using the CpG adjuvant to evaluate the therapeutic effect in a B16-OVA tumor model. The subcutaneous administration of 2 1011 AuNP-OVA and 1012 AuNPCpG induced strong antigen-specific responses in mice and IL-6 release in bone marrow-derived dendritic cells cultures. AuNP-OVA without adjuvant was sufficient to promote significant antigen-specific responses, leading to a subsequent antitumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This findings support the use of AuNPs as effective peptide vaccine carriers with the potential of using lower and safer adjuvant doses during vaccination. These administration approaches will positively impact doi: 10.1586/14760584.2015.1064772

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Table 1. Overview of current research on AuNPs vaccine-approaches. Molecule coupled to the AuNPs

Size/ shape

Adjuvant

Dose

Antigen/ disease

Test animal/ administration route

Observations

Protein

30 nm/ Spherical

None

6–24 mg

TNF/Cancer

Mice Intravenous

The dose of 15 mg of Au-TNF causes tumor regression by 79% with 100% of mice survival

[48]

DNA

1–9 nm/ Spherical

Chitosan

1–10 mg

HBsAg/HBV

Mice Intramuscular

Induced potent cytotoxic T lymphocyte responses in mice and specific IgG serum antibodies with a 10-fold potency when compared with the naked DNA vaccine

[118]

Peptide

2 to 50 nm/ Spherical

Freund’s adjuvant

10 ng/mL

VP1e/FMDV

Mice Subcutaneous and Intraperitoneal

Maximal antibody binding was noted for AuNPs of diameter 8–17 nm and serum specific IgG in mice was induced

[86]

Protein

5 nm/ Spherical

None

200 mM

OVA

Mice Percutaneous

Specific IgG serum antibodies in mice were induced

[102]

Carbohydrate

2 nm/ Spherical

Quil-A

3 mg

Pn14PS/S. pneumoniae

Mice Subcutaneous

Induced titers of up to 1000 of specific IgG in mice and the production of TNF-a, IL-2, IL-5

[87]

Peptide

25 nm/ Spherical

None

40 Lf

TT/C. tetani

Mice Oral

Induced specific IgA titers in intestinal mucosa of mice

Carbohydrate

3 to 5 nm/ Spherical

C3d

50 mM

Tumorassociated glycopeptides antigens/Cancer

Mice Subcutaneous

Mice immunized with these particles produced both IgM and IgG isotypes against each glycopeptide antigen

[92]

DNA

15, 30, and 80 nm/ Spherical

CpG

6.4 mg

Cancer

Mice Intratumor injection

15 nm AuNPs induced macrophage and dendritic cell tumor infiltration, significantly inhibited tumor growth, and promoted survival in mice

[96]

Peptide

40 nm/ Spherical

None

100 ng

Envelope protein/WNV

Mice Intraperitoneal

Induced titers of up to 1200 of specific IgG in mice and production of TNF-a, IL-6, IL-12, and GM-CSF in DCs

[85]

42 nm

[103]

Rod-NPs can deliver WNV antigens more efficiently into macrophages and induce IL-1b and IL-18 secretion in DCs

36 nm long, 10 nm wide/Rod Bacterial Outer membrane vesicles

Ref.

None

LPS

0.2 or 0.02 mg

0.93 g

Escherichia coli

Mice Subcutaneous

Rapidly activated dendritic cells residing in the lymph nodes. Strong and durable antibody responses and production of IFN-g and IL-17 but not IL-4 (Th1 and Th17 responses)

[88]

[89]

Abbreviations: AuNPs: Gold nanoparticles; DCs: Dendritic cells; FliC: Flagellin; FMDV: Foot and mouth disease virus; GM-CSF: Granulocyte macrophage colony-stimulating factor; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus; Hcp1: Haemolysin co-regulated protein; IgG: Immunoglobulin G; IgM: immunoglobulin M; IgA: immunoglobulin A; IL-6: Interleukin-6; IL-12: Interleukin-12; IL-1 b: Interleukin-1 b; M2e: Influenza matrix protein 2; OVA: ovalbumin; Pn14PS: Type 14 capsular polysaccharide; TetHc: Hc fragment of tetanus toxin; TNF: Tumor necrosis factor; TT: Tetanus toxoid; VP1e: Viral protein epitope 1; WNV: West Nile virus.

doi: 10.1586/14760584.2015.1064772



Review

Table 1. Overview of current research on AuNPs vaccine-approaches (cont.). Antigen/ disease

Test animal/ administration route

Observations

TetHc, Hcp1 and FliC/ Burkholderia mallei

Mice Intranasal

Induced higher antibody titers when compared to LPS alone and improved protection against a lethal inhalation challenge

20 mg

FliC/Burkholderia malle

Rhesus macaque Subcutaneous

Aerosol challenge produced mortality in 50% of the animals. serum LPS-specific IgG titers were significantly higher in 3 vaccinated animals who survived compared with 3 vaccinated animals who died.

[90]

CpG

60 mg

M2e antigen/ Influenza virus

Mice Intranasal

Induced specific serum IgG and protection against a lethal challenge in mice Presence of free M2e in the formulation of functionalized particles enhanced immunogenicity and induced long lasting immunoprotection

[105,106]

30 nm/ Spherical

None

2 1011

cancer

Mice subcutaneous

AuNP-OVA elicits significant antigen-specific responses.

Peptide

40 nm/ Spherical

TT

20 Lf/mL

TT/C. tetani

Mice oral

AuNPs elicited both systemical specific IgG and IgA and local IgA was found in intestinal washes and feces

[104]

Peptide

60 nm/ Spherical

None

6 mg

myelin oligodendrocyte glycoprotein/ Multiple sclerosis

Mice intraperitoneal

AuNPs expanded the FoxP3+ Tregs and suppressed the development of experimental autoimmune encephalomyelitis

[101]

Size/ shape

Adjuvant

15 nm Spherical

0.26% (w/v) alhydrogel

LPS

15 nm Spherical

0.26% (w/v) alhydrogel

Peptide

12 nm/ Spherical

Peptide


Molecule coupled to the AuNPs

Dose

Ref.

[97]

Abbreviations: AuNPs: Gold nanoparticles; DCs: Dendritic cells; FliC: Flagellin; FMDV: Foot and mouth disease virus; GM-CSF: Granulocyte macrophage colony-stimulating factor; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus; Hcp1: Haemolysin co-regulated protein; IgG: Immunoglobulin G; IgM: immunoglobulin M; IgA: immunoglobulin A; IL-6: Interleukin-6; IL-12: Interleukin-12; IL-1 b: Interleukin-1 b; M2e: Influenza matrix protein 2; OVA: ovalbumin; Pn14PS: Type 14 capsular polysaccharide; TetHc: Hc fragment of tetanus toxin; TNF: Tumor necrosis factor; TT: Tetanus toxoid; VP1e: Viral protein epitope 1; WNV: West Nile virus.

the implementation of new immunization schemes against cancer [97]. Radiofrequency ablation, cryoablation, photodynamic therapy, and magnetic nanoparticle hyperthermia can induce tumor death following different pathways. In general, heat treatments between 41 and 46 C induce apoptosis in cancer cells, whereas treatments above this range (up to 70 C) induce necrosis [98]. Although, in general, heat treatments have been focused on local tumors relying on physical principles as the main mechanism, it has been shown that these treatments can induce a tumor-specific immune response [99,100]. This response is mediated by the release of antigens and heat shock proteins from dying tumor cells, which are then captured by DCs and other APCs. The immune system can subsequently mount a response against cancer cells in distal, untreated sites. informahealthcare.com

Other non-infectious diseases, such as multiple sclerosis and autoimmune encephalomyelitis have also been targeted using AuNPs-based immunotherapies. Yeste et al. [101] reported the use of AuNPs to generate a tolerogenic vaccine candidate for multiple sclerosis using a T-cell epitope from the myelin oligodendrocyte glycoprotein 35–55 to promote the expansion of Tregs by DCs. DCs treated with AuNPs displayed a tolerogenic phenotype and promoted the differentiation of Tregs using in vitro assays. Moreover, in a murine model of autoimmune encephalomyelitis weekly intraperitoneal immunization with 6 mg of AuNPs led to the expansion of the FoxP3+ Treg cells and suppressed the development of experimental autoimmune encephalomyelitis, an experimental model of multiple sclerosis. doi: 10.1586/14760584.2015.1064772

Review



Mice

Mucosal tissues

Cell lines

Systemic compartment

– Show size and shape-dependent uptake and cytokine secretion – Show acceptable safety – Differential functionalization influences cellular uptake

– Show acceptable safety – Induce local humoral responses (IgA) – Favor antigen stability and uptake – Exert adjuvant activity

– Show acceptable safety – Induce systemic humoral and cellular responses (IgG) – Favor antigen stability and uptake – Exert adjuvant activity – Efficient for cancer vaccines – Activate cytokine secretion – Effectively target DCs located in lymph nodes

Figure 4. Main findings with implications on the use of AuNPs in vaccination.

Needle-free vaccine prototypes

Due to the advantages of vaccine formulations delivered by non-invasive routes, Huang et al. [102] developed a protocol based in percutaneous administration of 5 nm AuNPs in mice. The administration of 50 mM AuNPs elicited specific IgGs and proved the lack of cytotoxicity on keratinocytes at concentrations of up to 200 mM. Following efforts on mucosal vaccines development, Pokharkar et al. [103] have explored an oral immunization scheme against Clostridium tetani. Tetanus toxoid was conjugated to 25 nm spherical AuNPs and administered to test mice by the oral route. IgAs responses were successfully induced at the intestinal mucosa. Recently, the same group developed a novel oral delivery system for tetanus toxoid comprised of chitosan-functionalized gold nanoparticles (CsAuNPs) and a plant extract derived from Asparagus racemosus. The oral administration in mice at days 0, 14, and 28 with 20 Lf/mL elicited a significant increase in TT-specific systemic IgG (34.53-fold) and IgA (43.75-fold); local IgA immune responses for TT also showed a significant increase (106.5-fold) in intestine washes and in feces (99.74-fold) [104]. AuNPs have also been explored for intranasal vaccine approaches. Tao et al. [105] immunized mice intranasally with 12 nm spherical influenza matrix protein 2-conjugated AuNPs, achieving the induction of specific IgGs against the doi: 10.1586/14760584.2015.1064772

influenza peptide. Moreover, protection against a lethal challenge of a PR8-H1N1 influenza virus strain was observed. A further study revealed that free M2e antigen along with M2e immobilized on AuNPs increased the immunogenicity of the vaccine leading to high antibody levels with the subsequent protection against a lethal challenge with influenza virus. This immunization approach also achieved long-lasting immunoprotection [106]. Expert commentary

The study of gold nanoparticles in biomedical applications has been of interest for a number of research groups during the last years, with recent advances in their use as vaccination prototypes (TABLE 1). Considering that AuNPs can be internalized by mammalian cells, depending on the particle size, and have low toxicity, these have been studied as vaccine delivery vehicles. The conjugation of immunogens, such as peptides, polysaccharides or naked DNA encoding a protective antigen, onto AuNPs allows for the presentation of antigens to the immune system resembling the pathogen itself, thereby leading to a high immunogenic activity. Typically, the need for triggering robust immunoprotective responses against the target molecule using a subunit vaccine requires the use of strong adjuvants, where the most commonly used include lipopolysaccharides, toxins, oil in water Expert Rev. Vaccines



emulsions with or without the addition of mycobacteria (Freund’s complete and incomplete adjuvant, respectively), unmethylated cytosine-guanine dinucleotides (CpG), aluminum-based compounds, and immune stimulating complexes (ISCOMs). Interestingly, several studies have revealed the adjuvant properties of AuNPs in vaccines targeting several pathogens thus far. (FIGURE 4) summarizes the main findings with implications on the use of AuNPs in vaccination. These promising strategies have opened a path to explore the use of AuNPs as mucosal vaccine delivery vehicles to favor the induction of local specific immune responses through the mucosal associated lymphoid tissues (MALT). As the current outlook reflects that most of the AuNPs-based vaccines had been evaluated in parenteral immunization schemes, an important need for this field consists on exploring mucosal immunization approaches. Although the interactions between AuNPs and immune system cells have been described and some evidence indicate that these AuNPs are introduced by DCs, important knowledge gaps remain to be addressed in regard to AuNPs as mucosal vaccine delivery vehicle including: the characterization of mucoadhesive properties and the uptake by M cells, the stability of the AuNPs under the digestive process, and the implementation of functionalization strategies to target specific cells at the mucosal epithelium. It is considered that the current knowledge on using other nanomaterials as delivery vehicles for mucosal vaccine formulations will support the accomplishment of this goal by adopting already known criterions and strategies as initial scenario [107,108]. The cost of vaccination is a critical aspect to determine the potential of addressing massive immunization interventions. Perhaps the main drawbacks on the use of AuNPs are their cost and potential toxicity. Although low toxicity is observed for most of the vaccine prototypes, this topic requires further validation, considering that other particulate antigens are non-toxic and biodegradable, such as silicon, chitosan and PLGA [109]. A key perspective is derived from the fact that no comparative studies on the immunogenic or adjuvant potency exist in the literature. Performing this kind of comparative evaluations will provide elements to contrast, in an integrative manner, the distinct delivery systems. Five-year view

Given the promising evidence generated by the preclinical evaluation of several AuNPs-based vaccine candidates, several prospects arise. Although attractive immunogenic properties

informahealthcare.com

Review

have been observed for most of the candidates, the rationale design of AuNPs-based vaccines is still in its infancy. Among the approaches that will contribute to expand the knowledge and technologies in this field, we can list the following: carry out studies to compare distinct nanomaterials looking to provide elements to choose the most advantageous system in terms of cost, efficacy, and safety; the formulation of multi-epitope formulations, which will open the possibility to generate multi-valent or multi-target vaccine prototypes of key importance in the fight against complex or hypervariable pathogens such as the HIV and Influenza virus to which effective vaccines have not been achieved thus far [110]. Cancer is also a pathology in which targeting several antigens is highly desirable [111,112]; the functionalization of AuNPs with ligands targeting specific receptors of DCs, such as CD32, CD40, and CD205, also constitutes a potential approach to obtain highly efficient vaccines [113]. This approach in fact has been applied for other NPs [114]; the use of cytokines in the formulations is also a prospect with relevant implications as specific immune polarization might be achieved for specific diseases [115]; to systematically study the immunization using the distinct mucosal administration routes with AuNPs varying in shape and size to better understand the type and magnitude of the immune responses induced and their safety in terms of the localization and effects of the nanoparticles in the involved tissues. This latter aspect is of especial relevance for the intranasal route since antigen transport to the central nervous system might cause adverse effects [116]. As the current knowledge of these aspects is limited, further research on the behavior of AuNPs administered through mucosal tissues will open new and relevant paths for AuNPs mucosal vaccine development. The development of tolerogenic AuNPs vaccines is also considered an area of opportunity. Regulatory T-cells play a central role in maintaining immune homeostasis, and autoimmune inflammation is frequently associated with a decrease on the number and/or the function of Treg. Nanoparticles-based therapies promoting Tregs expansion to treat autoimmune inflammation has been barely explored [117]. Financial & competing interests disclosure

S Rosales-Mendoza and O Gonza´lez-Ortega were supported by CONACYT/Mexico CONACYT (grant INFR-2014-01- 225843) and PROFOCIES 2014. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

doi: 10.1586/14760584.2015.1064772

Review


Key issues .

The development of new efficacious and affordable vaccines is a priority in vaccinology, a goal that requires efficient delivery vehicles to ensure not only antigen delivery but protection from degradation.

.

Gold nanoparticles (AuNPs) can be used as vaccines delivery vehicles as they are biocompatible and can be easily functionalized with antigens.

.

AuNPs show low toxicity in mammals and after dendritic cells uptake a cytokine production can be induced which is dependent on the


shape and size of the AuNPs. .

A summary of the most common synthesis methodology for AuNPs production is provided.

.

The state of the art on developing AuNPs-based vaccines is presented.

.

Future prospects in the development and evaluation of AuNPs-based candidate vaccines are identified.

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Influence of gold, silver and gold-silver alloy nanoparticles on germ cell function and embryo development.

Nanoparticles in vaccine delivery.

Engineered Gold Nanoparticles and Plant Adaptation Potential.

Coalescence and Collisions of Gold Nanoparticles.

Functionalized Gold Nanoparticles and Their Biomedical Applications.

Surface Interactions between Gold Nanoparticles and Biochar.

Gold nanoparticles for photoacoustic imaging.

Chemistry for oncotheranostic gold nanoparticles.

Toxicity and Biokinetics of Colloidal Gold Nanoparticles.

Therapeutic gold, silver, and platinum nanoparticles.

Glyco-gold nanoparticles: synthesis and applications.

Structural development of gold and silver nanoparticles within hexagonally ordered spherical micellar diblock copolymer thin films.

Development of uniform density control with self-assembled colloidal gold nanoparticles on a modified silicon substrate.

Development of a mercury detection kit based on melamine-functionalized gold nanoparticles.

Gold nanoparticles for in vivo cell tracking.

Gold nanoparticles for photothermally controlled drug release.

Trapping Iron Oxide into Hollow Gold Nanoparticles.

Cardioprotective potential of biobased gold nanoparticles.

Gold nanoparticles to improve HIV drug delivery.

Gold nanoparticles for nucleic acid delivery.

Antibacterial Activity and Cytotoxicity of Gold (I) and (III) Ions and Gold Nanoparticles.

Colorimetric biosensing of pathogens using gold nanoparticles.

Luminescent Gold Nanoparticles with Size-Independent Emission.

Applications of gold nanoparticles in cancer nanotechnology.