Three‐Dimensional Gold Nanoparticle Clusters with Tunable Cores Templated by a Viral Protein Scaffold

Assembling nanoparticles (NPs) into ordered architectures remains a challenge in the field of nanotechnology. Templated strategies have been widely utilized for NP assembly. As typical biological nanostructures, virus‐based NPs (VNPs) have shown great promise in templating NP assembly. Here it is illustrated that the VNP of simian virus 40 (SV40) is a powerful scaffold in directing the assembly of 3D hybrid nanoarchitectures with one NP encapsulated inside as a core and a cluster of gold NPs (AuNPs) on the outer surface of the SV40 VNP as a shell, in which the core NPs can be CdSe/ZnS quantum dots (QDs), Ag₂S QDs, or AuNPs. The assembling of AuNPs onto the SV40 VNP surface is determined by the interactions between the AuNPs and the amine groups on the outer surface of SV40 VNPs. It is expected that the VNP guided 3D hybrid nanoarchitectures provide ideal models for NP interaction studies and open new opportunities for integrating various functionalities in NP assemblies.     

Insights into Stabilization of a Viral Protein Cage in Templating Complex Nanoarchitectures: Roles of Disulfide Bonds

As a typical protein nanostructure, virus‐based nanoparticle (VNP) of simian virus 40 (SV40), which is composed of pentamers of the major capsid protein of SV40 (VP1), has been successfully employed in guiding the assembly of different nanoparticles (NPs) into predesigned nanostructures with considerable stability. However, the stabilization mechanism of SV40 VNP remains unclear. Here, the importance of inter‐pentamer disulfide bonds between cysteines in the stabilization of quantum dot (QD)‐containing VNPs (VNP‐QDs) is comprehensively investigated by constructing a series of VP1 mutants of cysteine to serine. Although the presence of a QD core can greatly enhance the assembly and stability of SV40 VNPs, disulfide bonds are vital to stability of VNP‐QDs. Cysteine at position 9 (C9) and C104 contribute most of the disulfide bonds and play essential roles in determining the stability of SV40 VNPs as templates to guide assembly of complex nanoarchitectures. These results provide insightful clues to understanding the robustness of SV40 VNPs in organizing suprastructures of inorganic NPs. It is expected that these findings will help guide the future design and construction of protein‐based functional nanostructures.     

Imaging Viral Behavior in Mammalian Cells with Self‐Assembled Capsid–Quantum‐Dot Hybrid Particles

Unique spectral properties of quantum dots (QDs) enable ultrasensitive and long‐term biolabeling. Aiming to trace the infection, movement, and localization of viruses in living cells, QD‐containing virus‐like particles (VLPs) of simian virus 40 (SV40), termed SVLP‐QDs, are constructed by in vitro self‐assembly of the major capsid protein of SV40. SVLP‐QDs show homogeneity in size (≈24 nm), similarity in spectral properties to unencapsidated QDs, and considerable stability. When incubated with living cells, SVLP‐QDs are shown to enter the cells by caveolar endocytosis, travel along the microtubules, and accumulate in the endoplasmic reticulum. This process mimics the early infection steps of SV40. This is the first paradigm of imaging viral behaviors with encapsidated QDs in living cells. The method may provide a new alternative for various purposes, such as tracing viruses or viral components, targeted nanoparticle delivery, and probing of drug delivery.     

Fabrication of Nanoarchitectures Templated by Virus‐Based Nanoparticles: Strategies and Applications

Biomolecular nanostructures in nature are drawing increasing interests in the field of materials sciences. As a typical group of them, virus‐based nanoparticles (VNPs), which are nanocages or nanorods assembled from capsid proteins of viruses, have been widely exploited as templates to guide the fabrication of complex nanoarchitectures (NAs), because of their appropriate sizes (ca. 20–200 nm), homogeneity, addressable functionalization, facile modification via chemical and genetic routes, and convenient preparation. Foreign materials can be positioned in the inner cavity or on the outer surface of VNPs, through either direct synthesis or assembling preformed nanomaterials. Simultaneous use of the inner and outer space of VNPs facilitates integration of multiple functionalities in a single NA. This review briefly summarizes the strategies for fabrication of NAs templated by VNPs and wide applications of these NAs in fields of catalysis, energy, biomedicine, and nanophotonics, etc.     

Loading and Release Mechanism of Red Clover Necrotic Mosaic Virus Derived Plant Viral Nanoparticles for Drug Delivery of Doxorubicin

Loading and release mechanisms of Red clover necrotic mosaicvirus (RCNMV) derived plant viral nanoparticle (PVN) are shown for controlled delivery of the anticancer drug, doxorubicin (Dox). Previous studies demonstrate that RCNMV's structure and unique response to divalent cation depletion and re‐addition enables Dox infusion to the viral capsid through a pore formation mechanism. However, by controlling the net charge of RCNMV outer surface and accessibility of RCNMV interior cavity, tunable release of PVN is possible via manipulation of the Dox loading capacity and binding locations (external surface‐binding or internal capsid‐encapsulation) with the RCNMV capsid. Bimodal release kinetics is achieved via a rapid release of surface‐Dox followed by a slow release of encapsulated Dox. Moreover, the rate of Dox release and the amount of released Dox increases with an increase in environmental pH or a decrease in concentration of divalent cations. This pH‐responsive Dox release from PVN is controlled by Fickian diffusion kinetics where the release rate is dependent on the location of the bound or loaded active molecule. In summary, controllable release of Dox‐loaded PVNs is imparted by 1) formulation conditions and 2) driven by the capsid's pH‐ and ion‐ responsive functions in a given environment.     

M13 Virus‐Based Framework for High Fluorescence Enhancement

Fluorescence imaging is a powerful tool for studying biologically relevant macromolecules, but its applicability is often limited by the fluorescent probe, which must demonstrate both high site‐specificity and emission efficiency. In this regard, M13 virus, a versatile biological scaffold, has previously been used to both assemble fluorophores on its viral capsid with molecular precision and to also target a variety of cells. Although M13‐fluorophore systems are highly selective, these complexes typically suffer from poor molecular detection limits due to low absorption cross‐sections and moderate quantum yields. To overcome these challenges, a coassembly of the M13 virus, cyanine 3 dye, and silver nanoparticles is developed to create a fluorescent tag capable of binding with molecular precision with high emissivity. Enhanced emission of cyanine 3 of up to 24‐fold is achieved by varying nanoparticle size and particle‐fluorophore separation. In addition, it is found that the fluorescence enhancement increases with increasing dye surface density on the viral capsid. Finally, this highly fluorescent probe is applied for in vitro staining of E. coli. These results demonstrate an inexpensive framework for achieving tuned fluorescence enhancements. The methodology developed in this work is potentially amendable to fluorescent detection of a wide range of M13/cell combinations.     

Site‐Specific Modification of Adeno‐Associated Viruses via a Genetically Engineered Aldehyde Tag

As a consequence of their well‐defined nanostructure and intrinsic bioactive functionality, virus‐based nanoparticles have shown promise for mediating gene delivery. Adeno‐associated virus (AAV) nanoparticles, which possess an excellent safety profile and therapeutic potential, hold potential for use in human gene therapy. However, because of their native tropisms, the applicability of AAV nanoparticles is often limited to restricted ranges of cells or tissues. Thus, retargeting AAV particles to the desired cell populations has continued to be a major research focus in many gene therapy applications. In this study, a general strategy is reported for nanoparticle targeting. This involves the site‐specific modification of AAV type 2 (AAV2) by genetically incorporating a short peptide, in this case an aldehyde tag, in the viral capsid. Such a tag can be exploited for site‐specific attachment of targeting molecules and allows for further introduction of targeting antibodies or ligands. It is shown that this modification neither affects the level of infectious viral titer nor intracellular trafficking properties. Furthermore, the site‐specific conjugation of targeting antibodies could significantly enhance viral transduction to those target cells that have otherwise exhibited very low permissiveness to AAV2 infection. This method also allows the functional incorporation of RGD peptides onto AAV2 for enhanced delivery with implications for cancer gene therapy.     

Inhibition of HSV‐1 Attachment,Entry, and Cell‐to‐Cell Spread by Functionalized Multivalent Gold Nanoparticles

The use of modified nanoparticles in interactions with biological targets is attracting rapidly increasing attention. In this Full Paper, the application of gold nanoparticles capped with mercaptoethanesulfonate (Au‐MES NPs) as effective inhibitors of Herpes simplex virus type 1 infection based on their ability to mimic cell‐surface‐receptor heparan sulfate is described. Mechanistic studies reveal that Au‐MES NPs interfere with viral attachment, entry, and cell‐to‐cell spread, thereby preventing subsequent viral infection in a multimodal manner. The ligand multiplicity achieved with carrier nanoparticles is crucial in generating polyvalent interactions with the virus at high specificity, strength, and efficiency. Such multivalent‐nanoparticle‐mediated inhibition is a promising approach for alternative antiviral therapy.     

Probing Cell‐Type‐Specific Intracellular Nanoscale Barriers Using Size‐Tuned Quantum Dots

The compartmentalization of size‐tuned luminescent semiconductor nanocrystal quantum dots (QDs) in four distinctive cell lines, which would be representative of the most likely environmental exposure routes to nanoparticles in humans, is studied. The cells are fixed and permeabilized prior to the addition of the QDs, thus eliminating any cell‐membrane‐associated effects due to active QD uptake mechanisms or to specificity of signaling routes in different cell types, but leaving intact the putative physical subcellular barriers. All quantitative assays are performed using a high content analysis (HCA) platform, thereby obtaining robust data on large cell populations. While smaller QDs 2.1 nm in diameter enter the nuclei and localize to the nucleoli in all cell types, the rate and dynamics of their passage vary depending on the cell origin. As the QD size is increased to 4.4 nm, penetration into the cell is reduced but each cell line displays its own cutoff size thresholds reflecting cell‐type‐determined cytoplasmic and nuclear pore penetration specificity. These results give rise to important considerations regarding the differential compartmentalization and susceptibility of organs, tissues, and cells to nanoparticles, and may be of prime importance for biomedical imaging and drug‐delivery research employing nanoparticle‐based probes and systems.     

Programmed Nanoparticle‐Loaded Nanoparticles for Deep‐Penetrating 3D Cancer Therapy

Tumors are 3D, composed of cellular agglomerations and blood vessels. Therapies involving nanoparticles utilize specific accumulations due to the leaky vascular structures. However, systemically injected nanoparticles are mostly uptaken by cells located on the surfaces of cancer tissues, lacking deep penetration into the core cancer regions. Herein, an unprecedented strategy, described as injecting “nanoparticle‐loaded nanoparticles” to address the long‐lasting problem is reported for effective surface‐to‐core drug delivery in entire 3D tumors. The “nanoparticle‐loaded nanoparticle” is a silica nanoparticle (≈150 nm) with well‐developed, interconnected channels (diameter of ≈30 nm), in which small gold nanoparticles (AuNPs) (≈15 nm) with programmable DNA are located. The nanoparticle (AuNPs)‐loaded nanoparticles (silica): (1) can accumulate in tumors through leaky vascular structures by protecting the inner therapeutic AuNPs during blood circulation, and then (2) allow diffusion of the AuNPs for penetration into the entire surface‐to‐core tumor tissues, and finally (3) release a drug triggered by cancer‐characteristic pH gradients. The hierarchical “nanoparticle‐loaded nanoparticle” can be a rational design for cancer therapies because the outer large nanoparticles are effective in blood circulation and in protection of the therapeutic nanoparticles inside, allowing the loaded small nanoparticles to penetrate deeply into 3D tumors with anticancer drugs.     

Bright Single‐Chain Conjugated Polymer Dots Embedded Nanoparticles for Long‐Term Cell Tracing and Imaging

Single‐chain conjugated polymer (CP) dots embedded nanoparticles (NPs) bearing cell penetration peptide (TAT) as surface ligands are synthesized for long term cancer cell tracing applications. The CPNPs are fabricated by matrix‐encapsulation method and the embedded CPs can be modulated into spherical dots with different size upon alteration of feed concentrations. Single‐chain CP dots are formed upon decreasing feed concentration to 0.2 mg/mL, where CPNPs exhibit highest fluorescence quantum yield of 32%. Maleimide is introduced as the new NP surface functional group, which favors easy conjugation with cell penetration peptide via click chemistry to preserve its biofunctions. The obtained CPNPs show high brightness and good biocompatibility, which allow cell tracing for over 9 generations, superior to commercial cell tracker Qtracker 585.     

Exploring Protein–Nanoparticle Interactions with Coarse‐Grained Protein Folding Models

Understanding the fundamental biophysics behind protein–nanoparticle (NP) interactions is essential for the design and engineering bio‐NP systems. The authors describe the development of a coarse‐grained protein–NP model that utilizes a structure centric protein model. A key feature of the protein–NP model is the quantitative inclusion of the hydrophobic character of residues in the protein and their interactions with the NP surface. In addition, the curvature of the NP is taken into account, capturing the protein behavior on NPs of different size. The authors evaluate this model by comparison with experimental results for structure and adsorption of a model protein interacting with an NP. It is demonstrated that the simulation results recapitulate the structure of the small α/β protein GB1 on the NP for data from circular dichroism and fluorescence spectroscopy. In addition, the calculated protein adsorption free energy agrees well with the experimental value. The authors predict the dependence of protein folding on the NP size, surface chemistry, and temperature. The model has the potential to guide NP design efforts by predicting protein behavior on NP surfaces with various chemical properties and curvatures.     

Virus‐Templated Plasmonic Nanoclusters with Icosahedral Symmetry via Directed Self‐Assembly

The assembly of plasmonic nanoparticles with precise spatial and orientational order may lead to structures with new electromagnetic properties at optical frequencies. The directed self‐assembly method presented controls the interparticle‐spacing and symmetry of the resulting nanometer‐sized elements in solution. The self‐assembly of three‐dimensional (3D), icosahedral plasmonic nanosclusters (NCs) with resonances at visible wavelengths is demonstrated experimentally. The ideal NCs consist of twelve gold (Au) nanospheres (NSs) attached to thiol groups at predefined locations on the surface of a genetically engineered cowpea mosaic virus with icosahedral symmetry. In situ dynamic light scattering (DLS) measurements confirm the NSs assembly on the virus. Transmission electron micrographs (TEM) demonstrate the ability of the self‐assembly method to control the nanoscopic symmetry of the bound NSs, which reflects the icosahedral symmetry of the virus. Both, TEM and DLS show that the NCs comprise of a distribution of capsids mostly covered (i.e., 6–12 NS/capsid) with NSs. 3D finite‐element simulations of aqueous suspensions of NCs reproduce the experimental bulk absorbance measurements and major features of the spectra. Simulations results show that the fully assembled NCs give rise to a 10‐fold surface‐averaged enhancement of the local electromagnetic field.     

NIR‐Emitting Quantum Dot‐Encoded Microbeads through Membrane Emulsification for Multiplexed Immunoassays

NIR‐emitting CdSeTe/CdS/ZnS core/shell/shell QD‐encoded microbeads are combined with common flow cytometry with one laser for multiplexed detection of hepatitis B virus (HBV). A facile one‐pot synthetic route is developed to prepare CdSeTe/CdS/ZnS core/shell/shell QDs with high photoluminescence quantum yield and excellent stability in liquid paraffin, and a Shirasu porous glass (SPG) membrane emulsification technique is applied to incorporate the QDs into polystyrene–maleic anhydride (PSMA) microbeads to obtain highly fluorescent QD‐encoded microbeads. The relatively wide NIR photoluminescence full width half maximum of the CdSeTe/CdS/ZnS QDs is used to develop a ‘single wavelength’ encoding method to obtain different optical codes by changing the wavelengh and emission intensity of the QDs incorporated into the microbeads. Moreover, a detection platform combining NIR‐emitting CdSeTe/CdS/ZnS QD‐encoded microbeads and Beckman Coulter FC 500 flow cytometry with one laser of 488 nm is successfully used to conduct a 2‐plex hybridization assay for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and a 3‐plex hybridization assay for hepatitis B surface antibody (HBsAb), hepatitis B e antibody (HBeAb), and hepatitis B core antibody (HBcAb), which suggests the promising application of NIR QD‐encoded microbeads for multiplex immunoassays.     

Shuttle‐Mediated Nanoparticle Delivery to the Blood–Brain Barrier

Many therapeutic drugs are excluded from entering the brain due to their lack of transport through the blood–brain barrier (BBB). The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous diseases. To overcome this problem, a viral fusion peptide (gH625) derived from the glycoprotein gH of Herpes simplex virus type 1 is developed, which possesses several advantages including high cell translocation potency, absence of toxicity of the peptide itself, and the feasibility as an efficient carrier for delivering therapeutics. Therefore, it is hypothesized that brain delivery of nanoparticles conjugated with gH625 should be efficiently enhanced. The surface of fluorescent aminated polystyrene nanoparticles (NPs) is functionalized with gH625 via a covalent binding procedure, and the NP uptake mechanism and permeation across in vitro BBB models are studied. At early incubation times, the uptake of NPs with gH625 by brain endothelial cells is greater than that of the NPs without the peptide, and their intracellular motion is mainly characterized by a random walk behavior. Most importantly, gH625 peptide decreases NP intracellular accumulation as large aggregates and enhances the NP BBB crossing. In summary, these results establish that surface functionalization with gH625 may change NP fate by providing a good strategy for the design of promising carriers to deliver drugs across the BBB for the treatment of brain diseases.     

Mechanotargeting: Mechanics‐Dependent Cellular Uptake of Nanoparticles

Targeted delivery of nanoparticle (NP)‐based diagnostic and therapeutic agents to malignant cells and tissues has exclusively relied on chemotargeting, wherein NPs are surface‐coated with ligands that specifically bind to overexpressed receptors on malignant cells. Here, it is demonstrated that cellular uptake of NPs can also be biased to malignant cells based on the differential mechanical states of cells, enabling mechanotargeting. Owing to mechanotransduction, cell lines (HeLa and HCT‐8) cultured on hydrogels of various stiffness are directed into different stress states, measured by cellular force microscopies. In vitro NP delivery reveals that increases in cell stress suppress cellular uptake, counteracting the enhanced uptake that occurs with increases in exposed surface area of spread cells. Upon prolonged culture on stiff hydrogels, cohesive HCT‐8 cell colonies undergo metastatic phenotypic change and disperse into individual malignant cells. The metastatic cells are of extremely low stress state and adopt an unspread, 3D morphology, resulting in several‐fold higher uptake than the nonmetastatic counterparts. This study opens a new paradigm of harnessing mechanics for the design of future strategies in nanomedicine.     

Metal Ion‐Induced Self‐Assembly of a Multi‐Responsive Block Copolypeptide into Well‐Defined Nanocapsules

Protein cages are an interesting class of biomaterials with potential applications in bionanotechnology. Therefore, substantial effort is spent on the development of capsule‐forming designer polypeptides with a tailor‐made assembly profile. The expanded assembly profile of a triblock copolypeptide consisting of a metal ion chelating hexahistidine‐tag, a stimulus‐responsive elastin‐like polypeptide block, and a pH‐responsive morphology‐controlling viral capsid protein is presented. The self‐assembly of this multi‐responsive protein‐based block copolymer is triggered by the addition of divalent metal ions. This assembly process yields monodisperse nanocapsules with a 20 nm diameter composed of 60 polypeptides. The well‐defined nanoparticles are the result of the emergent properties of all the blocks of the polypeptide. These results demonstrate the feasibility of hexahistidine‐tags to function as supramolecular cross‐linkers. Furthermore, their potential for the metal ion‐mediated encapsulation of hexahistidine‐tagged proteins is shown.     

Cancer‐Cell‐Phenotype‐Dependent Differential Intracellular Trafficking of Unconjugated Quantum Dots

A diverse array of nanoparticles, including quantum dots (QDs), metals, polymers, liposomes, and dendrimers, are being investigated as therapeutics and imaging agents in cancer diseases. However, the role of the cancer‐cell phenotype on the uptake and intracellular fate of nanoparticles in cancer cells remains poorly understood. Reported here is that differences in cancer‐cell phenotypes can lead to significant differences in intracellular sorting, trafficking, and localization of nanoparticles. Unconjugated anionic QDs demonstrate dramatically different intracellular profiles in three closely related human‐prostate‐cancer cells used in the investigation: PC3, PC3‐flu, and PC3‐PSMA. QDs demonstrate punctated intracellular localization throughout the cytoplasm in PC3 cells. In contrast, the nanoparticles localize mainly at a single juxtanuclear location (“dot‐of‐dots”) inside the perinuclear recycling compartment in PC3‐PSMA cells, where they co‐localize with transferrin and the prostate‐specific membrane antigen. The results indicate that nanoparticle sorting and transport is influenced by changes in cancer‐cell phenotype and can have significant implications in the design and engineering of nanoscale drug delivery and imaging systems for advanced tumors.     

Translocation Pathway‐Dependent Assembly of Streptavidin‐ and Antibody‐Binding Filamentous Virus‐Like Particles

Compared to well‐tolerated p3 fusion, the display of fast‐folding proteins fused to the minor capsid p7 and the major capsid p8, as well as in vivo biotinylation of biotin acceptor peptide (AP) fused to p7, are found to be markedly inefficient using the filamentous phage. Here, to overcome such limitations, the effect of translocation pathways, amber mutation, and phage and phagemid display systems on p7 and p8 display of antibody‐binding domains are examined, while comparing the level of in vivo biotinylation of AP fused to p7 or p3. Interestingly, the in vivo biotinylation of AP occurs only in p3 fusion and the fast‐folding antibody‐binding scaffolds fused to p7 and p8 are best displayed via a twin‐arginine translocation pathway in TG1 cells. The lower the expression level of the wild‐type p8 and the smaller the size of the guest protein, the better the display of Z‐domain fused to the recombinant p8. The in vivo biotinylated multifunctional filamentous virus‐like particles can be vertically immobilized on streptavidin (SAV)‐coated microspheres to resemble cellular microvilli‐like structures, which reportedly enhance protein–protein interactions due to dramatically expanded flexible surface area.     

New Strategies for the Preparation of Sinter‐Resistant Metal‐Nanoparticle‐Based Catalysts

Supported metal nanoparticles are widely used as catalysts in the industrial production of chemicals, but still suffer from deactivation because of metal leaching and sintering at high temperature. In recent years, serious efforts have been devoted to developing new strategies for stabilizing metal nanoparticles. Recent developments for preparing sinter‐resistant metal‐nanoparticle catalysts via strong metal–support interactions, encapsulation with oxide or carbon layers and within mesoporous materials, and fixation in zeolite crystals, are briefly summarized. Furthermore, the current challenges and future perspectives for the preparation of highly efficient and extraordinarily stable metal‐nanoparticle‐based catalysts, and suggestions regarding the mechanisms involved in sinter resistance, are proposed.