In vivo Gold Nanoparticle Delivery of Peptide Vaccine Induces Anti‐Tumor Immune Response in Prophylactic and Therapeutic Tumor Models

Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, it is hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16‐OVA tumor model. AuNP delivery of OVA (AuNP‐OVA) and of CpG (AuNP‐CpG) enhanced the efficacy of both agents and induced strong antigen‐specific responses. In addition, it is found that AuNP‐OVA delivery alone, without CpG, is sufficient to promote significant antigen‐specific responses, leading to subsequent anti‐tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy is likely due to the adjuvant effect of peptide coated AuNPs, as they induce inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, AuNP‐mediated OVA peptide delivery can produce significant therapeutic benefits without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination.     

Adjuvant properties of mesoporous silica particles tune the development of effector T cells

Alum is the most frequently used adjuvant today, primarily inducing Th2 responses. However, Th1‐type responses are often desirable within immune therapy, and therefore the development of new adjuvants is greatly needed. Mesoporous silica particles with a highly ordered pore structure have properties that make them very interesting for future controlled drug delivery systems, such as controllable particle and pore size; they also have the ability to induce minor immune modulatory effects, as previously demonstrated on human‐monocyte‐derived dendritic cells (MDDCs). In this study, mesoporous silica particles are shown to be efficiently engulfed by MDDCs within 2 h, probably by phagocytic uptake, as seen by confocal microscopy and transmission electron microscopy. A co‐culture protocol is developed to evaluate the capability of MDDCs to stimulate the development of naïve CD4⁺ T cells in different directions. The method, involving ELISpot as a readout system, demonstrates that MDDCs, after exposure to mesoporous silica particles (AMS‐6 and SBA‐15), are capable of tuning autologous naïve T cells into different effector cells. Depending on the size and functionalization of the particles added to the cells, different cytokine patterns are detected. This suggests that mesoporous silica particles can be used as delivery vehicles with tunable adjuvant properties, which may be of importance for several medical applications, such as immune therapy and vaccination.     

TGF‐β1 Conjugated to Gold Nanoparticles Results in Protein Conformational Changes and Attenuates the Biological Function

Gold nanoparticles (AuNPs) are widely used as carriers or therapeutic agents due to their great biocompatibility and unique physical properties. Transforming growth factor‐beta 1 (TGF‐β1), a member of the cysteine‐knot structural superfamily, plays a pivotal role in many diseases and is known as an immunosuppressive agent that attenuates immune response resulting in tumor growth. The results reported herein reflect strong interactions between TGF‐β1 and the surface of AuNPs when incubated with serum‐containing medium, and demonstrate a time‐ and dose‐dependent pattern. Compared with other serum proteins that can also bind to the AuNP surface, AuNP–TGFβ1 conjugate is a thermodynamically favored compound. Epithelial cells undergo epithelial–mesenchymal transition (EMT) upon treatment with TGF‐β1; however, treatment with AuNPs reverses this effect, as detected by cell morphology and expression levels of EMT markers. TGF‐β1 is found to bind to AuNPs through S–Au bonds by X‐ray photoelectron spectroscopy. Fourier transform infrared spectroscopy is employed to analyze the conformational changes of TGF‐β1 on the surface of AuNPs. The results indicate that TGF‐β1 undergoes significant conformational changes at both secondary and tertiary structural levels after conjugation to the AuNP surface, which results in the deactivation of TGF‐β1 protein. An in vivo experiment also shows that addition of AuNPs attenuates the growth of TGF‐β1‐secreting murine bladder tumor 2 cells in syngeneic C3H/HeN mice, but not in immunocompromised NOD‐SCID mice, and this is associated with an increase in the number of tumor‐infiltrating CD4⁺ and CD8⁺ T lymphocytes and a decrease in the number of intrasplenic Foxp3(+) lymphocytes. The findings demonstrate that AuNPs may be a promising agent for modulating tumor immunity through inhibiting immunosuppressive TGF‐β1 signaling.     

Facile synthesis of anisotropic gold nanoparticles and its synergistic effect on breast cancer cell lines

Gold nanoparticles (AuNPs) possess colourful light‐scattering properties due to different composition, size and shape. Their unique physical, optical and chemical properties coupled with advantages, have increased the scope of anisotropic AuNPs in various fields. This study reports a green methodology developed for the synthesis of anisotropic AuNPs. The aqueous extracts of Alternanthera sessilis (PGK), Portulaca oleracea (PAK) and Sterculia foetida (SF) with gold ions produced violet, purple and pink coloured AuNPs, respectively, under sonication and room temperature methods revealing the formation of different shapes of AuNPs. The results of TEM analysis of AuNPs confirmed the formation of triangular plate AuNPs of the size 35 nm for PAK extract. Spherical‐shaped AuNPs (10–20 nm) were obtained using an extract of PGK. SF extract produced rod, hexagon, pentagon‐shaped AuNPs and nanorice gold particles. The cell viability studies of the PGK, PAK and SF‐mediated AuNPs on MCF‐7 cell lines by MTT assay revealed the cytotoxic activity of AuNPs to depend on the size, shape and the nature of capping agents. The synthesised AuNPs significantly inhibited the growth of cancer cells (MCF‐7) in a concentration‐dependent manner. The size and shape of these anisotropic AuNPs also reveal its potency to be used as sensors, catalysis, photothermal and therapeutic agents.Inspec keywords: toxicology, gold, transmission electron microscopy, catalysis, nanofabrication, biomedical materials, nanomedicine, particle size, cellular biophysics, nanoparticles, cancer, biological organsOther keywords: Au, size 10.0 nm to 20.0 nm, temperature 293.0 K to 298.0 K, size 35.0 nm, TEM analysis, Sterculia foetida, Portulaca oleracea, Alternanthera sessilis, chemical properties, colourful light‐scattering properties, anisotropic AuNP, triangular plate AuNP, spherical‐shaped AuNP, SF‐mediated AuNP, cancer cells, MCF‐7 cell lines, cell viability, nanorice gold particles, gold ions, optical properties, breast cancer cell lines, anisotropic gold nanoparticles     

PEI-conjugated AuNPs as a sensing platform for arsenic (AS-III)

We have developed a simple method for synthesis of spherical gold nanoparticles (AuNPs) with enhanced surface properties. The polyethyleneimine (PEI) has good potential to minimise the size of the precursor. The UV–vis spectra of synthesised AuNPs with reducing agents (PEI) have been characterised with a peak at 530?nm. The size and shape measurement of AuNPs was confirmed by transmission electron microscopy (TEM) which shows that the mean diameter is 3.9?nm. The optimal concentration of reducing agents was found to be 1% for synthesis of AuNPs. PEI-conjugated AuNP shows binding with arsenic III (0.1?ppm) as confirmed by scanning electron microscope (SEM)/energy dispersive X-rays mapping. TEM revealed the particle shape and size. Zeta potential, zeta deviation, effective particle size, Z-average diameter, polydispersity index and electrophoretic mobility have been observed in order to understand the stability of AuNPs. The image of SEM confirmed that As (III) particles were eventually distributed in PEI-conjugated AuNPs matrix. Further, this study demonstrated that PEI-conjugated AuNPs is a sensing platform of As (III).     

Self‐Assembly of Poly‐Adenine‐Tailed CpG Oligonucleotide‐Gold Nanoparticle Nanoconjugates with Immunostimulatory Activity

Synthetic unmethylated cytosine–guanine (CpG) oligodeoxynucleotides (CpG ODNs) possess high immunostimulatory activity and have been widely used as a therapeutic tool for various diseases including infection, allergies, and cancer. A variety of nanocarriers have been developed for intracellular delivery of CpG ODNs that are otherwise nonpermeable through the cellular membrane. For example, previous studies showed that gold nanoparticles (AuNPs) could efficiently deliver synthetic thiolated CpG ODNs into cultured cells and induce expression of proinflammatory cytokines. Nevertheless, the necessity of using thiolated CpG ODNs for the modification of AuNPs inevitably complicates the synthesis of the nanoconjugates and increases the cost. A new approach is demonstrated for facile assembly of AuNP‐CpG nanoconjugates for cost‐effective drug delivery. It is found that non‐thiolated, diblock ODNs containing a CpG motif and a poly‐adenine (polyA) tail can readily self‐assemble on the surface of AuNPs with controllable and tunable density. Such nanoconjugates are efficiently delivered into RAW264.7 cells and induce immune response in a Toll‐like receptor 9 (TLR9)‐dependent manner. Under optimal conditions, polyA‐CpG‐AuNPs show significantly higher immunostimulatory activity than their thiolated counterpart. In addition, the immunostimulatory activity of CpG‐AuNPs can be modulated by varying the length of the polyA tail. In vivo induction of immune responses in mice is demonstrated by using polyA‐tailed CpG‐AuNP nanoconjugates.     

Combined Positron Emission Tomography and Cerenkov Luminescence Imaging of Sentinel Lymph Nodes Using PEGylated Radionuclide‐Embedded Gold Nanoparticles

New imaging probes with high sensitivity and stability are urgently needed to accurately detect sentinel lymph nodes (SLNs) for successful cancer diagnosis. Herein, the use of highly sensitive and stable PEGylated radionuclide‐embedded gold nanoparticles (PEG‐RIe‐AuNPs) is reported for the detection of SLNs by combined positron emission tomography and Cerenkov luminescence imaging (PET/CLI). PEG‐RIe‐AuNPs show high sensitivity and stability both in vitro and in vivo, and are not toxic to normal ovarian and immune cells. In vivo PET/CLI imaging clearly reveals SLNs as early as 1 h post PEG‐RIe‐AuNP‐injection, with peak signals achieved at 6 h postinjection, which is consistent with the biodistribution results. Taken together, the data provide strong evidence that PEG‐RIe‐AuNPs are promising as potential lymphatic tracers in biomedical imaging for pre and intraoperative surgical guidance.     

Mass Cytometry and Single‐Cell RNA‐seq Profiling of the Heterogeneity in Human Peripheral Blood Mononuclear Cells Interacting with Silver Nanoparticles

Understanding the interactions between nanoparticles (NPs) and human immune cells is necessary for justifying their utilization in consumer products and biomedical applications. However, conventional assays may be insufficient in describing the complexity and heterogeneity of cell–NP interactions. Herein, mass cytometry and single‐cell RNA‐sequencing (scRNA‐seq) are complementarily used to investigate the heterogeneous interactions between silver nanoparticles (AgNPs) and primary immune cells. Mass cytometry reveals the heterogeneous biodistribution of the positively charged polyethylenimine‐coated AgNPs in various cell types and finds that monocytes and B cells have higher association with the AgNPs than other populations. scRNA‐seq data of these two cell types demonstrate that each type has distinct responses to AgNP treatment: NRF2‐mediated oxidative stress is confined to B cells, whereas monocytes show Fcγ‐mediated phagocytosis. Besides the between‐population heterogeneity, analysis of single‐cell dose–response relationships further reveals within‐population diversity for the B cells and naïve CD4⁺ T cells. Distinct subsets having different levels of cellular responses with respect to their cellular AgNP doses are found. This study demonstrates that the complementary use of mass cytometry and scRNA‐seq is helpful for gaining in‐depth knowledge on the heterogeneous interactions between immune cells and NPs and can be incorporated into future toxicity assessments of nanomaterials.     

Mechanobiological Mimicry of Helper T Lymphocytes to Evaluate Cell–Biomaterials Crosstalk

The unique properties of immune cells have inspired many efforts in engineering advanced biomaterials capable of mimicking their behaviors. However, an inclusive model capable of mimicking immune cells in different situations remains lacking. Such models can provide invaluable data for understanding immune–biomaterial crosstalk. Inspired by CD4+ T cells, polymeric microparticles with physicochemical properties similar to naïve and active T cells are engineered. A lipid coating is applied to enhance their resemblance and provide a platform for conjugation of desired antibodies. A novel dual gelation approach is used to tune the elastic modulus and flexibility of particles, which also leads to elongated circulation times. Furthermore, the model is enriched with magnetic particles so that magnetotaxis resembles the chemotaxis of cells. Also, interleukin‐2, a proliferation booster, and interferon‐γ cytokines are loaded into the particles to manipulate the fates of killer T cells and mesenchymal stem cells, respectively. The penetration of these particles into 3D environments is studied to provide in vitro models of immune‐biomaterials crosstalk. This biomimicry model enables optimization of design parameters required for engineering more efficient drug carriers and serves as a potent replica for understanding the mechanical behavior of immune cells.     

The Role of Temperature and Lipid Charge on Intake/Uptake of Cationic Gold Nanoparticles into Lipid Bilayers

Understanding the molecular mechanisms governing nanoparticle–membrane interactions is of prime importance for drug delivery and biomedical applications. Neutron reflectometry (NR) experiments are combined with atomistic and coarse‐grained molecular dynamics (MD) simulations to study the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes composed of a mixture of zwitterionic di‐stearoyl‐phosphatidylcholine (DSPC) and anionic di‐stearoyl‐phosphatidylglycerol (DSPG). MD simulations show that the interaction between AuNPs and a pure DSPC lipid bilayer is modulated by a free energy barrier. This can be overcome by increasing temperature, which promotes an irreversible AuNP incorporation into the lipid bilayer. NR experiments confirm the encapsulation of the AuNPs within the lipid bilayer at temperatures around 55 °C. In contrast, the AuNP adsorption is weak and impaired by heating for a DSPC–DSPG (3:1) lipid bilayer. These results demonstrate that both the lipid charge and the temperature play pivotal roles in AuNP–membrane interactions. Furthermore, NR experiments indicate that the (negative) DSPG lipids are associated with lipid extraction upon AuNP adsorption, which is confirmed by coarse‐grained MD simulations as a lipid‐crawling effect driving further AuNP aggregation. Overall, the obtained detailed molecular view of the interaction mechanisms sheds light on AuNP incorporation and membrane destabilization.     

Chemical alignment of DNA origami to block copolymer patterned arrays of 5 nm gold nanoparticles

We have used block copolymer patterned arrays of 5 nm gold nanoparticles (AuNPs) for chemically aligned surface attachment of DNA origami. Addition of single-stranded DNA-thiol to AuNPs allowed a base paired attachment of sticky end modified DNA origami. Results indicate a stable, selective attachment between the DNA origami and ssDNA modified AuNPs. Yield data showed 74% of AuNP binding sites forming an attachment with a DNA origami rectangle, and control surfaces showed less than 0.5% nonspecific adsorption.     

Asymmetrically functionalized gold nanoparticles organized in one-dimensional chains

Organized assembly remains a major challenge for optimizing and extending the application of nanoparticles. Here we report a simple method to assemble spherical gold nanoparticles (AuNPs) in one-dimensional (1D) chains. The chain-forming process takes advantage of asymmetrically functionalized AuNPs that serve as building blocks. The 1D AuNP chains were prepared by covalent attachment of spatially localized functional groups on the AuNPs to polymer backbone pendent groups. We demonstrate control of interparticle spacing and the preparation of 1D chains containing AuNPs of different sizes.     

3D nanometer images of biological fibers by directed motion of gold nanoparticles

Using near-infrared femtosecond pulses, we move single gold nanoparticles (AuNPs) along biological fibers, such as collagen and actin filaments. While the AuNP is sliding on the fiber, its trajectory is measured in three dimensions (3D) with nanometer resolution providing a high-resolution image of the fiber. Here, we systematically moved a single AuNP along nanometer-size collagen fibers and actin filament inside chinese hamster ovary K1 living cells, mapping their 3D topography with high fidelity.     

Gum arabic as a phytochemical construct for the stabilization of gold nanoparticles: in vivo pharmacokinetics and X-ray-contrast-imaging studies

Gold nanoparticles (AuNPs) have exceptional stability against oxidation and therefore will play a significant role in the advancement of clinically useful diagnostic and therapeutic nanomedicines. Despite the huge potential for a new generation of AuNP-based nanomedicinal products, nontoxic AuNP constructs and formulations that can be readily administered site-specifically through the intravenous mode, for diagnostic imaging by computed tomography (CT) or for therapy via various modalities, are still rare. Herein, we report results encompassing: 1) the synthesis and stabilization of AuNPs within the nontoxic phytochemical gum-arabic matrix (GA-AuNPs); 2) detailed in vitro analysis and in vivo pharmacokinetics studies of GA-AuNPs in pigs to gain insight into the organ-specific localization of this new generation of AuNP vector, and 3) X-ray CT contrast measurements of GA-AuNP vectors for potential utility in molecular imaging. Our results demonstrate that naturally occurring GA can be used as a nontoxic phytochemical construct in the production of readily administrable biocompatible AuNPs for diagnostic and therapeutic applications in nanomedicine.     

A multifunctional core-shell nanoparticle for dendritic cell-based cancer immunotherapy

Dendritic cell-based cancer immunotherapy requires tumour antigens to be delivered efficiently into dendritic cells and their migration to be monitored in vivo. Nanoparticles have been explored as carriers for antigen delivery, but applications have been limited by the toxicity of the solvents used to make nanoparticles, and by the need to use transfection agents to deliver nanoparticles into cells. Here we show that an iron oxide-zinc oxide core-shell nanoparticle can deliver carcinoembryonic antigen into dendritic cells while simultaneously acting as an imaging agent. The nanoparticle-antigen complex is efficiently taken up by dendritic cells within one hour and can be detected in vitro by confocal microscopy and in vivo by magnetic resonance imaging. Mice immunized with dendritic cells containing the nanoparticle-antigen complex showed enhanced tumour antigen specific T-cell responses, delayed tumour growth and better survival than controls.     

Biomimetic high density lipoprotein nanoparticles for nucleic acid delivery

We report a gold nanoparticle-templated high density lipoprotein (HDL AuNP) platform for gene therapy that combines lipid-based nucleic acid transfection strategies with HDL biomimicry. For proof-of-concept, HDL AuNPs are shown to adsorb antisense cholesterylated DNA. The conjugates are internalized by human cells, can be tracked within cells using transmission electron microscopy, and regulate target gene expression. Overall, the ability to directly image the AuNP core within cells, the chemical tailorability of the HDL AuNP platform, and the potential for cell-specific targeting afforded by HDL biomimicry make this platform appealing for nucleic acid delivery.     

Design of multicomponent microgels by selective deposition of nanomaterials

In the present paper a method for the targeted deposition of different nanomaterials on aqueous microgels is described. In the first stage poly(3,4-ethylenedioxythiophene) (PEDOT) nanorods are introduced into the microgel structure by in situ oxidative polymerization. In the second stage hydrogen tetrachloroaurate is used to transform PEDOT chains to an oxidized state in the microgel structure, leading to the fixation of chloroaurate anions on the surface of the PEDOT nanorods. The reduction of chloroaurate ions induces the formation of gold nanoparticles (AuNPs) predominantly located on the PEDOT surface. Obtained microgel/PEDOT/AuNP hybrid particles with different nanoparticle loadings exhibit superior colloidal stability and temperature sensitivity. The microgel/PEDOT/AuNP hybrid microgels exhibit extraordinary catalytic activity in aqueous media.     

Optical Detection of Human Papillomavirus Type 16 and Type 18 by Sequence Sandwich Hybridization With Oligonucleotide-Functionalized Au Nanoparticles

The importance of detecting and subtyping human papillomaviruses (HPVs) in clinical and epidemiological studies has been well addressed. In detecting the most common types of HPV, type 16 (HPV-16) and type 18 (HPV-18), in the cervical mucous of patients in a simple and rapid manner, the assay of a label-free colorimetric DNA sensing method based on sequence sandwich hybridization with oligonucleotide-functionalized Au nanoparticles (AuNPs) was fabricated in this study. Specific oligonucleotide probes were designed for the sequence detection within the L1 gene of HPV-16 and HPV-18, and the probes were capped onto AuNPs, as AuNP probes. The target HPV sequences in clinical specimens were obtained by an asymmetric polymerase chain reaction (PCR) with universal primers, which can amplify the target sequences from several HPV serotypes, including HPV-16 and HPV-18. The DNA sandwich hybridization between the target sequences and the specific AuNP probes was performed at a temperature closer to the theoretical melting temperature of the DNA hybridization. Next, the procedure of increasing salt concentration and cooling the hybridizing solution was immediately utilized to discriminate the target sequences of HPV-16 or HPV-18. If the target sequences were not complementary to sequences of AuNP probes, the AuNPs would aggregate because no duplex DNA formation occurred such that the color of the reaction solution changed from red to purple. If the AuNP probes were a perfect match to the target sequences and a full DNA sandwich hybridization occurred, the reaction solution maintained its red color. A total of 70 mucous specimens from patients with cervical intraepithelial neoplasia were tested by the AuNP probes sandwich hybridization.      

Fabrication of Flexible and Transparent Conductive Nanosheets by the UV‐Irradiation of Gold Nanoparticle Monolayers

Conductive films that are highly transparent and flexible are extremely attractive for emerging optoelectronic applications. Currently, indium‐doped tin oxide films are the most widely used transparent conductive films and much research effort is devoted to developing alternative transparent conductive materials to overcome their drawbacks. In this work, a novel and facile approach for fabricating transparent conductive Au nanosheets from Au nanoparticles (AuNPs) is proposed. Irradiating an AuNP monolayer at the air–water interface with UV light results in a nanosheet with ≈3.5 nm thickness and ≈80% transparency in the UV–visible region. Further, the so‐fabricated nanosheets are highly flexible and can maintain their electrical conductivity even when they are bent to a radius of curvature of 0.6 mm. Fourier‐transform infrared and X‐ray photoelectron spectroscopy characterizations reveal that the transformation of the monolayer of AuNPs into the nanosheet is induced by the photodecomposition and/or photodetachment of the dodecanethiol ligands capping the AuNPs. Further, the UV‐irradiation of a hybrid monolayer consisting of AuNPs and silica particles affords the patterning of Au nanosheets with periodic hole arrays.     

Adjuvant‐Loaded Subcellular Vesicles Derived From Disrupted Cancer Cells for Cancer Vaccination

Targeted subunit vaccines for cancer immunotherapy do not capture tumor antigenic complexity, and approaches employing tumor lysate are often limited by inefficient antigen uptake and presentation, and low immunogenicity. Here, whole cancer cells are processed to generate antigen‐rich, membrane‐enclosed subcellular particles, termed “reduced cancer cells”, that reflect the diversity and breadth of the parent cancer cell antigen repertoire, and can be loaded with disparate adjuvant payloads. These vesicular particles enhance the uptake of the adjuvant payload, and potentiate the activation of primary dendritic cells in vitro. Similarly, reduced cancer cell‐associated antigens are more efficiently presented by primary dendritic cells in vitro than their soluble counterparts or lysate control. In mice, vaccination using adjuvant‐loaded reduced cancer cells facilitates the induction of antigen‐specific cellular and humoral immune responses. Taken together, these observations demonstrate that adjuvant‐loaded reduced cancer cells could be utilized in cancer vaccines as an alternative to lysate.