Self-assembling Protein Nanoparticles Research Articles

Nanoparticle Vaccine for Avian Influenza Virus: A Challenge Study against Highly Pathogenic H5N2 Subtype

We used a platform of self-assembling protein nanoparticles (SAPNs) to develop a self-adjuvanted vaccine for avian influenza virus (AIV). A SAPNs named SA-MC-Penn was designed to display the ectodomain of influenza matrix protein 2 (M2e) as a tetramer and Helix C (Hel C) of hemagglutinin as a trimer. Flagellin domainsexpressed within a peptide chain were co-assembled into the SAPN core serving as a built-in adjuvant. SA-MC-Penn was administered every two weeks for three times via the intramuscular (IM) or eye drop (ED) routes in chickens starting at four-weeks of age. Sera were collected from immunized chickens at twelve-weeks of age prior to challenge with a highly pathogenic (HP) H5N2 strain of AIV. ELISA results demonstrated that high titers of serum antibodies were induced in the IM immunized SAPNs. Upon challenge with HP AIV, 63 % and 30% of chickens the SAPNs IM and SAPN ED groups, respectively, survived. A reduction in challenge virus shedding from oropharyngeal swabbings of the SAPNs IM vaccinates was observed on days two and 14-post challenge. These results show the SAPNs vaccine prototype was partially protective against HPAIV.

Self-assembling protein nanoparticles with built-in flagellin domains increases protective efficacy of a Plasmodium falciparum based vaccine

To eliminate the problems associated with the use of extraneous adjuvants we have designed a Self-Assembling Protein Nanoparticle (SAPN) containing epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) (designated FMP014) and portions of the TLR5 agonist flagellin (designated FMP014D0D1) as an intrinsic adjuvant. By combining different molar ratios of FMP014 to FMP014D0D1 monomers before self-assembly, we generated multiple nanoparticles and investigated their biophysical characteristics, immunogenicity and protective efficacy. Immunization with the construct formulated with the ratio 58:2 of FMP014 to FMP014D0D1 had the highest protective efficacy against a challenge with a transgenic P. berghei sporozoite expressing PfCSP. Increasing the proportion of flagellin per particle resulted in an inverse relationship with levels of both antibody titers and protection. The cytokine profiles of the various immunization groups were evaluated and quantitative amounts of the cytokines IL-2, IFN-γ, IL-12/p70 (Th1); IL4, IL5 (Th2); TNF-α, IL1β, IL-6, KC/GRO (pro-inflammatory), and IL-10 (immunomodulatory) were measured. The relationship of the cytokines to each other revealed a strong immunomodulatory effect depending on the proportion of flagellin in the construct. Our results demonstrate that SAPNs with flagellin may be a promising strategy for the development and delivery of a safe vaccine for infectious diseases.

The use of a P. falciparum specific coiled-coil domain to construct a self-assembling protein nanoparticle vaccine to prevent malaria

BackgroundThe parasitic disease malaria remains a major global public health concern and no truly effective vaccine exists. One approach to the development of a malaria vaccine is to target the asexual blood stage that results in clinical symptoms. Most attempts have failed. New antigens such as P27A and P27 have emerged as potential new vaccine candidates. Multiple studies have demonstrated that antigens are more immunogenic and are better correlated with protection when presented on particulate delivery systems. One such particulate delivery system is the self-assembling protein nanoparticle (SAPN) that relies on coiled-coil domains of proteins to form stable nanoparticles. In the past we have used de novo designed amino acid domains to drive the formation of the coiled-coil scaffolds which present the antigenic epitopes on the particle surface.ResultsHere we use naturally occurring domains found in the tex1 protein to form the coiled-coil scaffolding of the nanoparticle. Thus, by engineering P27A and a new extended form of the coiled-coil domain P27 onto the N and C terminus of the SAPN protein monomer we have developed a particulate delivery system that effectively displays both antigens on a single particle that uses malaria tex1 sequences to form the nanoparticle scaffold. These particles are immunogenic in a murine model and induce immune responses similar to the ones observed in seropositive individuals in malaria endemic regions.ConclusionsWe demonstrate that our P27/P27A-SAPNs induce an immune response akin to the one in seropositive individuals in Burkina Faso. Since P27 is highly conserved among different Plasmodium species, these novel SAPNs may even provide cross-protection between Plasmodium falciparum and Plasmodium vivax the two major human malaria pathogens. As the SAPNs are also easy to manufacture and store they can be delivered to the population in need without complication thus providing a low cost malaria vaccine.

Protein nanovaccine confers robust immunity against Toxoplasma

We designed and produced a self-assembling protein nanoparticle. This self-assembling protein nanoparticle contains five CD8+ HLA-A03-11 supertypes-restricted epitopes from antigens expressed during Toxoplasma gondii’s lifecycle, the universal CD4+ T cell epitope PADRE, and flagellin as a scaffold and TLR5 agonist. These CD8+ T cell epitopes were separated by N/KAAA spacers and optimized for proteasomal cleavage. Self-assembling protein nanoparticle adjuvanted with TLR4 ligand-emulsion GLA-SE were evaluated for their efficacy in inducing IFN-γ responses and protection of HLA-A*1101 transgenic mice against T. gondii. Immunization, using self-assembling protein nanoparticle-GLA-SE, activated CD8+ T cells to produce IFN-γ. Self-assembling protein nanoparticle-GLA-SE also protected HLA-A*1101 transgenic mice against subsequent challenge with Type II parasites. Hence, combining CD8+ T cell-eliciting peptides and PADRE into a multi-epitope protein that forms a nanoparticle, administered with GLA-SE, leads to efficient presentation by major histocompatibility complex Class I and II molecules. Furthermore, these results suggest that activation of TLR4 and TLR5 could be useful for development of vaccines that elicit T cells to prevent toxoplasmosis in humans.

Classification of self-assembling protein nanoparticle architectures for applications in vaccine design.

We introduce here a mathematical procedure for the structural classification of a specific class of self-assembling protein nanoparticles (SAPNs) that are used as a platform for repetitive antigen display systems. These SAPNs have distinctive geometries as a consequence of the fact that their peptide building blocks are formed from two linked coiled coils that are designed to assemble into trimeric and pentameric clusters. This allows a mathematical description of particle architectures in terms of bipartite (3,5)-regular graphs. Exploiting the relation with fullerene graphs, we provide a complete atlas of SAPN morphologies. The classification enables a detailed understanding of the spectrum of possible particle geometries that can arise in the self-assembly process. Moreover, it provides a toolkit for a systematic exploitation of SAPNs in bioengineering in the context of vaccine design, predicting the density of B-cell epitopes on the SAPN surface, which is critical for a strong humoral immune response.

Development of a self-assembling protein nanoparticle vaccine targeting Plasmodium falciparum Circumsporozoite Protein delivered in three Army Liposome Formulation adjuvants.

We have developed FMP014, a vaccine candidate against Plasmodium falciparum malaria, which is comprised of 60 identical monomer protein chains that form an icosahedral shaped self-assembling protein nanoparticle (SAPN). Each monomer contains selected P. falciparum Circumsporozoite Protein (PfCSP) CD4+ and CD8+ epitopes, universal TH epitopes, portions of the α-TSR domain, and 6 repeats of the NANP motifs of the PfCSP. Here we describe the conditions that are required for successful scale-up and cGMP manufacturing of FMP014 with a yield of ≈1.5g of drug substance per 100g of wet bacterial paste. When adjuvanted with an Army Liposomal Formulation (ALF) based adjuvant, the nanoparticle vaccine is highly immunogenic and prevents infection of mice by an otherwise lethal dose of transgenic P. berghei sporozoites expressing the full-length PfCSP.

Vaccination with self-adjuvanted protein nanoparticles provides protection against lethal influenza challenge

Current influenza vaccines should be improved by the addition of universal influenza vaccine antigens in order to protect against multiple virus strains. We used our self-assembling protein nanoparticles (SAPNs) to display the two conserved influenza antigens M2e and Helix C in their native oligomerization states. To further improve the immunogenicity of the SAPNs, we designed and incorporated the TLR5 agonist flagellin into the SAPNs to generate self-adjuvanted SAPNs. We demonstrate that addition of flagellin does not affect the ability of SAPNs to self-assemble and that they are able to stimulate TLR5 in a dose-dependent manner. Chickens vaccinated with the self-adjuvanted SAPNs induce significantly higher levels of antibodies than those with unadjuvanted SAPNs and show higher cross-neutralizing activity compared to a commercial inactivated virus vaccine. Upon immunization with self-adjuvanted SAPNs, mice were completely protected against a lethal challenge. Thus, we have generated a self-adjuvanted SAPN with a great potential as a universal influenza vaccine.

Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli.

BackgroundProduction of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free proteins, no data has been so far recorded regarding how the system performs in the fabrication of smart nanostructured materials.ResultsWe have here explored the nanoarchitecture and in vitro and in vivo functionalities of CXCR4-targeted, self-assembling protein nanoparticles intended for intracellular delivery of drugs and imaging agents in colorectal cancer. Interestingly, endotoxin-free materials exhibit a distinguishable architecture and altered size and target cell penetrability than counterparts produced in conventional E. coli strains. These variant nanoparticles show an eventual proper biodistribution and highly specific and exclusive accumulation in tumor upon administration in colorectal cancer mice models, indicating a convenient display and function of the tumor homing peptides and high particle stability under physiological conditions.DiscussionThe observations made here support the emerging endotoxin-free E. coli system as a robust protein material producer but are also indicative of a particular conformational status and organization of either building blocks or oligomers. This appears to be promoted by multifactorial stress-inducing conditions upon engineering of the E. coli cell envelope, which impacts on the protein quality control of the cell factory.

Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles

Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications.

Metallochelate Coupling of Phosphorescent Pt-Porphyrins to Peptides, Proteins, and Self-Assembling Protein Nanoparticles.

Specific and reversible metallochelate coupling via nitrilotriacetate (NTA) moiety is widely used for immobilization, purification, and labeling of oligo(histidine)-tagged proteins. Here, we evaluated this strategy to label various peptides and proteins with phosphorescent Pt-porphyrin derivatives bearing NTA group(s). Zn(2+) complexes were shown to have minimal effect on the photophysics of the porphyrin moiety, allowing quenched-phosphorescence sensing of O2. We complexed the PtTFPP-NTA conjugate with His-containing peptide that can facilitate intracellular loading, and observed efficient accumulation and phosphorescent staining of MEF cells. The more hydrophilic PtCP-NTA conjugate was also seen to form stable complexes with larger polypeptide constructs based on fluorescent proteins, and with subunits of protein nanoparticles, which retained their ability to self-assemble. Testing in phosphorescence lifetime based O2 sensing assays on a fluorescence reader and PLIM microscope revealed that phosphorescent metallochelate complexes perform similarly to the existing O2 probes. Thus, metallochelate coupling allows simple preparation of different types of biomaterials labeled with phosphorescent Pt-porphyrins.

Self-assembling protein nanoparticles in the design of vaccines

For over 100years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estimated to save millions of lives globally each year. Nevertheless, many diseases are not yet preventable by vaccination. This large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety. Compared to the 19th and early 20th century vaccines that were made of killed, inactivated, or live-attenuated pathogens, modern vaccines containing isolated, highly purified antigenic protein subunits are safer but tend to induce lower levels of protective immunity. One strategy to overcome the latter is to design antigen nanoparticles: assemblies of polypeptides that present multiple copies of subunit antigens in well-ordered arrays with defined orientations that can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions. Such nanoparticles offer a collective strength of multiple binding sites (avidity) and can provide improved antigen stability and immunogenicity. Several exciting advances have emerged lately, including preclinical evidence that this strategy may be applicable for the development of innovative new vaccines, for example, protecting against influenza, human immunodeficiency virus, and respiratory syncytial virus. Here, we provide a concise review of a critical selection of data that demonstrate the potential of this field. In addition, we highlight how the use of self-assembling protein nanoparticles can be effectively combined with the emerging discipline of structural vaccinology for maximum impact in the rational design of vaccine antigens.

Optimizing the design of protein nanoparticles as carriers for vaccine applications

Successful vaccine development remains a huge challenge for infectious diseases such as malaria, HIV and influenza. As a novel way to present antigenic epitopes to the immune system, we have developed icosahedral self-assembling protein nanoparticles (SAPNs) to serve as a prototypical vaccine platform for infectious diseases. Here we examine some biophysical factors that affect the self-assembly of these nanoparticles, which have as basic building blocks coiled-coil oligomerization domains joined by a short linker region. Relying on in silico computer modeling predictions, we selected five different linker regions from the RCSB protein database that connect oligomerization domains, and then further studied the self-assembly and stability of in vitro produced nanoparticles through biophysical characterization of formed particles. One design in particular, T2i88, revealed excellent self-assembly and homogeneity thus paving the way toward a more optimized nanoparticle for vaccine applications. From the Clinical EditorDespite the widespread use of vaccines worldwide, successful development of vaccines against some diseases remains a challenge still. In this article, the authors investigated the physic-chemical and biological properties of icosahedral self-assembling protein nanoparticles (SAPNs), which mimic viral particles, in order to utilize this technology as potential platform for future design of vaccines.

Microbial Biofabrication for Nanomedicine: Biomaterials, Nanoparticles and Beyond

Author for correspondence: Institut de Biotecnologia i de Biomedicina, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain and Department de Genetica i de Microbiologia, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona, Spain and CIBER en Bioingenieria, Biomateriales y Nanomedicina, Bellaterra, 08193 Barcelona, Spain antoni.villaverde@uab.cat Microbial biofabrication for nanomedicine: biomaterials, nanoparticles and beyond

The biodistribution of self-assembling protein nanoparticles shows they are promising vaccine platforms

BackgroundBecause of the need to limit side-effects, nanoparticles are increasingly being studied as drug-carrying and targeting tools. We have previously reported on a scheme to produce protein-based self-assembling nanoparticles that can act as antigen display platforms. Here we attempted to use the same system for cancer-targeting, making use of a C-terminal bombesin peptide that has high affinity for a receptor known to be overexpressed in certain tumors, as well as an N-terminal polyhistidine tag that can be used for radiolabeling with technetium tricarbonyl.ResultsIn order to increase circulation time, we experimented with PEGylated and unPEGylated varities typo particle. We also tested the effect of incorporating different numbers of bombesins per nanoparticle. Biophysical characterization determined that all configurations assemble into regular particles with relatively monodisperse size distributions, having peaks of about 33 – 36 nm. The carbonyl method used for labeling produced approximately 80% labeled nanoparticles. In vitro, the nanoparticles showed high binding, both specific and non-specific, to PC-3 prostate cancer cells. In vivo, high uptake was observed for all nanoparticle types in the spleens of CD-1 nu/nu mice, decreasing significantly over the course of 24 hours. High uptake was also observed in the liver, while only low uptake was seen in both the pancreas and a tumor xenograft.ConclusionsThe data suggest that the nanoparticles are non-specifically taken up by the reticuloendothelial system. Low uptake in the pancreas and tumor indicate that there is little or no specific targeting. PEGylation or increasing the amount of bombesins per nanoparticle did not significantly improve targeting. In particular, the uptake in the spleen, which is a primary organ of the immune system, highlights the potential of the nanoparticles as vaccine carriers. Also, the decrease in liver and spleen radioactivity with time implies that the nanoparticles are broken down and cleared. This is an important finding, as it shows that the nanoparticles can be safely used as a vaccine platform without the risk of prolonged side effects. Furthermore, it demonstrates that technetium carbonyl radiolabeling of our protein-based nanoparticles can be used to evaluate their pharmacokinetic properties in vivo.

Erratum to: Mechanisms of protective immune responses induced by the Plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine

Erratum to: Mechanisms of protective immune responses induced by the Plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine

Mechanisms of protective immune responses induced by the Plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine

BackgroundA lack of defined correlates of immunity for malaria, combined with the inability to induce long-lived sterile immune responses in a human host, demonstrate a need for improved understanding of potentially protective immune mechanisms for enhanced vaccine efficacy. Protective sterile immunity (>90%) against the Plasmodium falciparum circumsporozoite protein (CSP) has been achieved using a transgenically modified Plasmodium berghei sporozoite (Tg-Pb/PfCSP) and a self-assembling protein nanoparticle (SAPN) vaccine presenting CSP epitopes (PfCSP-SAPN). Here, several possible mechanisms involved in the independently protective humoral and cellular responses induced following SAPN immunization are described.MethodsInbred mice were vaccinated with PfCSP-SAPN in PBS. Serum antibodies were harvested and effects on P. falciparum sporozoites mobility and integrity were examined using phase contrast microscopy. The functionality of SAPN-induced antibodies on inhibition of sporozoite invasion and growth within primary human hepatocytes was also examined. The internal processing of SAPN by bone marrow-derived dendritic cells (BMDDC), using organelle-specific, fluorescent-tagged antibody or gold-encapsulated SAPN, was observed using confocal or electron microscopy, respectively.ResultsThe results of this work demonstrate that PfCSP-SAPN induces epitope-specific antibody titers, predominantly of the Th2 isotype IgG1, and that serum antibodies from PfCSP-SAPN-immunized mice appear to target P. falciparum sporozoites via the classical pathway of complement. This results in sporozoite death as indicated by cessation of motility and the circumsporozoite precipitation reaction. Moreover, PfCSP-SAPN-induced antibodies are able to inhibit wild-type P. falciparum sporozoite invasion and growth within cultured primary human hepatocytes. In addition, the observation that PfCSP-SAPN are processed (and presented) to the immune system by dendritic cells in a slow and continuous fashion via transporter associated with antigen processing (TAP) recruitment to the early endosome (EE), and have partially delayed processing through the endoplasmic reticulum, has the potential to induce the long-lived, effector memory CD8+ T-cells as described previously.ConclusionThis paper describes the examination of humoral and cellular immune mechanisms induced by PfCSP-SAPN vaccination which result in sterile host protection against a transgenic P. berghei malaria sporozoite expressing the P. falciparum CSP, and which significantly inhibits native P. falciparum sporozoites from invading and developing within cultured human hepatocytes. These results may indicate the type and mode of action of protective antibodies needed to control P. falciparum sporozoites from infecting humans as well as a potential mechanism of induction of protective long-lived effector memory CD8+ T-cells.

Expression, purification and refolding of a self-assembling protein nanoparticle (SAPN) malaria vaccine

There are many ways to present antigens to the immune system. We have used a repetitive antigen display technology that relies on the self-assembly of 60 protein chains into a spherical self-assembling protein nanoparticle (SAPN) to develop a vaccine against Plasmodium falciparum malaria. The protein sequence contains selected B- and T-cell epitopes of the circumsporozoite protein of P. falciparum (PfCSP) and, when assembled into a nanoparticle induces strong, long-lived and protective immune responses against the PfCSP. Here we describe the conditions needed for promoting self-assembly of a P. falciparum vaccine nanoparticle, PfCSP-KMY-SAPN, and note pitfalls that may occur when determining conditions for other SAPN vaccines. Attention was paid to selecting processes that were amenable to scale up and cGMP manufacturing.

Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

BackgroundThe worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8+ and CD4+ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects.Methodology/Principal FindingsTo establish the basis for a SAPN-based vaccine, B- and CD8+ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ+, IL-2+) long-lived central memory CD8+ T-cells. Furthermore, we demonstrated that these Ab or CD8+ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites.ConclusionThe SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP.

Conformation‐specific Display of 4E10 and 2F5 Epitopes on Self‐assembling Protein Nanoparticles as a Potential HIV Vaccine

The self-assembling protein nanoparticle (SAPN) is an antigen-presenting system that has been shown to be suitable for use as a vaccine platform. The SAPN scaffold is based on the principles of icosahedral symmetry, beginning from a monomeric chain that self-assembles into an ordered oligomeric state. The monomeric chain contains two covalently linked α-helical coiled-coil domains, an N-terminal de novo-designed pentameric tryptophan zipper and a C-terminal de novo-designed trimeric leucine zipper, which assemble along the internal symmetry axes of an icosahedron. In this study, we incorporated the membrane proximal external region (MPER) of HIV-1 gp41 from HXB2 into the N-terminal pentamer, referred to as MPER-SAPN, attempting to reproduce the α-helical state of the 4E10 epitope while maintaining a structurally less-constrained 2F5 epitope. Sprague-Dawley rats were immunized with MPER-SAPNs, and their sera were analyzed for induced humoral anti-HIV-1 responses. We show that high membrane proximal external region-specific titers can be raised via the repetitive antigen display of MPER on the SAPN without the need for adjuvant. However, none of the sera displayed a detectable neutralizing activity against HIV-1. Thus, 4E10- and 2F5-like neutralizing antibodies could not be elicited by MPER conformationally restrained in the SAPN context.

Encapsulation of gold nanoparticles into self-assembling protein nanoparticles

BackgroundGold nanoparticles are useful tools for biological applications due to their attractive physical and chemical properties. Their applications can be further expanded when they are functionalized with biological molecules. The biological molecules not only provide the interfaces for interactions between nanoparticles and biological environment, but also contribute their biological functions to the nanoparticles. Therefore, we used self-assembling protein nanoparticles (SAPNs) to encapsulate gold nanoparticles. The protein nanoparticles are formed upon self-assembly of a protein chain that is composed of a pentameric coiled-coil domain at the N-terminus and trimeric coiled-coil domain at the C-terminus. The self-assembling protein nanoparticles form a central cavity of about 10 nm in size, which is ideal for the encapsulation of gold nanoparticles with similar sizes.ResultsWe have used SAPNs to encapsulate several commercially available gold nanoparticles. The hydrodynamic size and the surface coating of gold nanoparticles are two important factors influencing successful encapsulation by the SAPNs. Gold nanoparticles with a hydrodynamic size of less than 15 nm can successfully be encapsulated. Gold nanoparticles with citrate coating appear to have stronger interactions with the proteins, which can interfere with the formation of regular protein nanoparticles. Upon encapsulation gold nanoparticles with polymer coating interfere less strongly with the ability of the SAPNs to assemble into nanoparticles. Although the central cavity of the SAPNs carries an overall charge, the electrostatic interaction appears to be less critical for the efficient encapsulation of gold nanoparticles into the protein nanoparticles.ConclusionsThe SAPNs can be used to encapsulate gold nanoparticles. The SAPNs can be further functionalized by engineering functional peptides or proteins to either their N- or C-termini. Therefore encapsulation of gold nanoparticles into SAPNs can provide a useful platform to generate a multifunctional biodevices.