Peptide-conjugated Gold Nanoparticles Research Articles

Detection of Femtomolar Amyloid-β Peptides for Early-Stage Identification of Alzheimer's Amyloid-β Aggregation with Functionalized Gold Nanoparticles.

Progressive amyloid-β (Aβ) fibrillar aggregates have long been considered as the pathogenesis of Alzheimer's disease (AD). Biocompatible and stable cysteine-Aβ peptide-conjugated gold nanoparticles (Cys-Aβ@AuNP) are demonstrated as suitable materials for detecting subfemtomolar Aβ peptides in human plasma. Incubation with Aβ peptides causes the Cys-Aβ@AuNP to aggregate and changes its absorption spectra. The spectral change is especially apparent and noticeable when detecting subfemtomolar Aβ peptides, and the aggregates contain only two or three AuNPs. Cys-Aβ@AuNP can also be used to identify early-stage Aβ oligomerization, which is not possible using the conventional method, in which the fluorescence of thioflavin-T is measured. The ability to detect Aβ oligomerization can facilitate therapeutics for AD. In addition, the binding of Aβ peptides by Cys-Aβ@AuNP in combination with centrifugation redirects the conventional Aβ aggregation pathway and can effectively inhibit the formation of toxic Aβ oligomers or fibrils. Therefore, the proposed Cys-Aβ@AuNP can also be used to develop effective therapeutic agents to inhibit Aβ aggregation. The results obtained in this study are expected to open revolutionary ways to both detect and inhibit Aβ aggregation at an early stage.

Development of a rapid dot-blot assay for ochratoxin A (OTA) detection using peptide conjugated gold nanoparticles for bio-recognition and detection

Ochratoxin A is a secondary metabolite produced by fungi and a major mycotoxin contaminating cereal grains and cereal-based products. Although, ochratoxin A is hepatotoxic, nephrotoxic and carcinogenic, its detection is limited due to low awareness, time-consuming conventional methods and false positive results. In this study, a rapid, user-friendly dot-blot assay was developed using peptide conjugated gold nanoparticles (AuNPs) as a detection agent. The peptide binding to ochratoxin A was evaluated by the apparent dissociation constant ( K d app ) measurement using indirect enzyme linked immunoabsorbent assay. A low K d value (1.046 μM) suggested high binding of peptide with ochratoxin A. The potential of peptide-based ochratoxin A recognition was translated to a portable platform using streptavidin functionalized AuNPs conjugated to a biotinylated 11-mer peptide. Dot-blot assay was developed for detection of ochratoxin A with a limit of detection of 0.49 μg/kg. Negligible cross-reactivity with low spot intensities were observed for high concentrations of other mycotoxins like aflatoxin B1 and citrinin, in comparison to control. Further, percent recovery of ochratoxin A from spiked wheat samples by dot-blot was comparable to the conventional high performance liquid chromatography (HPLC). Moreover, detection of ochratoxin A by dot-blot was validated by HPLC in 65 whole wheat, crushed wheat coarse, crushed wheat fine and wheat flour samples with a high correlation with R 2 = 0.93. HPLC and dot-blot confirmed the presence of ochratoxin A ˂ 5 μg/kg in the samples. Therefore, a portable, easy-to-use dot-blot assay for ochratoxin A detection with potential of application at each stage of harvesting, storage, production and distribution can contribute to safe and healthy food and feed. • A rapid, on-site, sensitive visual Dot-blot assay was developed for ochratoxin detection. • Peptide conjugated gold nanoparticles were employed as a recognition label to replace expensive antibodies. • The developed Dot-blot assay had a limit of detection of 0.49 μg/kg for ochratoxin. • High sensitivity and specificity for ochratoxin detection.

Novel Approaches of Dysregulating Lysosome Functions in Cancer Cells by Specific Drugs and Its Nanoformulations: A Smart Approach of Modern Therapeutics.

The smart strategy of cancer cells to bypass the caspase-dependent apoptotic pathway has led to the discovery of novel anti-cancer approaches including the targeting of lysosomes. Recent discoveries observed that lysosomes perform far beyond just recycling of cellular waste, as these organelles are metabolically very active and mediate several signalling pathways to sense the cellular metabolic status. These organelles also play a significant role in mediating the immune system functions. Thus, direct or indirect lysosome-targeting with different drugs can be considered a novel therapeutic approach in different disease including cancer. Recently, some anticancer lysosomotropic drugs (eg, nortriptyline, siramesine, desipramine) and their nanoformulations have been engineered to specifically accumulate within these organelles. These drugs can enhance lysosome membrane permeabilization (LMP) or disrupt the activity of resident enzymes and protein complexes, like v-ATPase and mTORC1. Other anticancer drugs like doxorubicin, quinacrine, chloroquine and DQ661 have also been used which act through multi-target points. In addition, autophagy inhibitors, ferroptosis inducers and fluorescent probes have also been used as novel theranostic agents. Several lysosome-specific drug nanoformulations like mixed charge and peptide conjugated gold nanoparticles (AuNPs), Au-ZnO hybrid NPs, TPP-PEG-biotin NPs, octadecyl-rhodamine-B and cationic liposomes, etc. have been synthesized by diverse methods. These nanoformulations can target cathepsins, glucose-regulated protein 78, or other lysosome specific proteins in different cancers. The specific targeting of cancer cell lysosomes with drug nanoformulations is quite recent and faces tremendous challenges like toxicity concerns to normal tissues, which may be resolved in future research. The anticancer applications of these nanoformulations have led them up to various stages of clinical trials. Here in this review article, we present the recent updates about the lysosome ultrastructure, its cross-talk with other organelles, and the novel strategies of targeting this organelle in tumor cells as a recent innovative approach of cancer management.

Effective ACE2 peptide-nanoparticle conjugation and its binding with the SARS-Cov-2 RBD quantified by dynamic light scattering.

The infection of coronavirus initiates with the binding between its spike protein receptor binding domain (RBD) and a human cellular receptor called angiotensin-converting enzyme 2 (ACE2). Here, we construct truncated ACE2 peptide-conjugated gold nanoparticles as antiviral scaffolds and study their binding with the SARS-CoV-2 RBD using dynamic light scattering (DLS). Systematic DLS analysis identifies the effective peptide-nanoparticle conjugation and its efficient, specific, and long-lasting multivalent binding towards the RBD with a binding affinity of 41 nM, indicating the potential of this antiviral platform to compete with natural ACE2-RBD interactions for viral blocking and showcasing an accessible approach to measure the binding constants and kinetics.

Bifunctional Peptide-Conjugated Gold Nanoparticles for Precise and Efficient Nucleus-Targeting Bioimaging in Live Cells.

Real-time in situ imaging of organelles is increasingly important in modern biomedical analysis and diseases diagnosis. To realize this goal, organelle-targeting nanoparticles as one of the most commonly used technologies in subcellular sensing and imaging has attracted a lot of interest. The biocompatibility, specificity, and binding efficiency are especially critical for efficient organelle-targeting bioimaging. Gold nanoparticles (AuNPs) fabricated with bifunctional peptides constructed with both Au-binding affinity and nucleus-targeting ability were designed and examined for efficient nucleus-targeting bioimaging. Such a design is expected to achieve an oriented assembling of peptides by the medium of the Au-binding peptides specifically assembled on the surface of AuNPs, with the nucleus-targeting end open for accessibility. The bifunctional peptides showed strong binding affinity toward AuNPs and led to a binding capability ∼1.5 times higher than that of the bare nucleus-targeting peptides, ensuring good surface coverage of the nanoparticles for enhanced nucleus-targeting ability. Such fabricated AuNPs demonstrated over 90% cell viability after incubation for 24 h with HepG2 cells, which were highly biocompatible. Precise and efficient bioimaging of the nucleus was achieved for HepG2 cells by using the fabricated AuNPs as observed with a confocal laser scanning microscope, a dark-field/fluorescence microscope, and a transmission electron microscope. The high surface coverage and oriented binding pattern appeared to be a promising strategy for construction of organelle-targeting agencies.

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles.

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.

Abstract LB-191: Photoacoustic detection and imaging of single cancer cells with peptide-conjugated gold nanoparticles

Abstract Photoacoustic imaging (PAI), which is based on the conversion of light into sound, is an emerging, hybrid and noninvasive imaging modality with highly scalable resolution. PAI has shown tremendous potential in early tumor detection and imaging by providing structural, functional and metabolic information. But to date, most work has been focused on tumor detection at the macroscopic level (i.e. tissue, organ). For early tumor diagnosis, microscopic detection of individual cancer cells is highly desirable and of special significance. However, detection and imaging of single cancer cells using PAI is very challenging because most cancer cells are colorless, thus exhibiting little endogenous contrast. Gold nanoparticles (AuNPs), which have good biocompatibility, low toxicity and high optical absorption, are an exogenous contrast agent that can facilitate PAI of single cancer cells. In this study, we synthesized peptide-conjugated AuNPs to selectively target DU145 human prostate cancer cells, and employed a laser-scanning photoacoustic microscopy (PAM) to detect and image individual cells. The used AuNPs had an average diameter of 30 nm and were physically fabricated by ultrafast pulsed laser ablation of gold target in ultrapure water. Their surface chemistry was modified through a sequential conjugation process, in which a predetermined amount of thiolated PEG 5000 molecules and RGD peptides were successively bound to the AuNPs with quantitative control and high efficiency. After incubation with DU145 cells, the functionalized PEG-RGD-AuNPs were endocytosed with the help of membrane receptors and produced sharp absorption contrast. The internalized AuNPs generated strong photoacoustic signals after absorbing excitation laser and the signals were captured by a highly sensitive hydrophone to form cell images. Combing functional AuNPs and high-resolution PAM, we successfully detected and imaged single, live DU145 cancer cells. Though transparent, the cancer cells were visibly delineated by intracellular AuNPs and were clearly distinguishable. Moreover, to achieve optimum detection efficiency, we also studied the relationship between the density of RGD peptide and the amplitude of photoacoustic signal. Experimental results reveal that, although the amount of cellular uptake of AuNPs was proportional to RGD density (from radioactivity analysis using a Gamma counter), the amplitude of photoacoustic signal did not. Photoacoustic detection efficiency reached a maximum when the molar ratio of RGD peptides to AuNPs was approximately 1000. To our best knowledge, this is the first report on photoacoustic detection and imaging of specific cancer cells using peptide-conjugated AuNPs at single-cell resolution. By translating PAI from macro to micro, the work is expected to open a new avenue for early cancer detection, imaging, diagnosis and even treatment. Citation Format: Chao Tian, Wei Qian, Xia Shao, Zhixing Xie, Xu Cheng, Shengchun Liu, Qian Cheng, Bing Liu, Xueding Wang. Photoacoustic detection and imaging of single cancer cells with peptide-conjugated gold nanoparticles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-191.

One-step synthesis of high-density peptide-conjugated gold nanoparticles with antimicrobial efficacy in a systemic infection model

The increase in antibiotic drug resistance and the low number of new antibacterial drugs approved in the last few decades requires the development of new antimicrobial strategies. Antimicrobial peptides (AMPs) are very promising molecules to fight microbial infection since they kill quickly bacteria and, in some cases, target bacterial membrane. Although some AMPs may be stable against proteolytic degradation by chemical modification, in general, low AMP activity and stability in the presence of serum and proteolytic enzymes as well as their cytotoxicity have impaired their clinical translation. Here, we describe a one-step methodology to generate AMP-conjugated gold nanoparticles (Au NPs), with a high concentration of AMPs (CM-SH) (≈240 AMPs per NP), controlled size (14 nm) and low polydispersity. AMP-conjugated Au NPs demonstrated higher antimicrobial activity and stability in serum and in the presence of non-physiological concentrations of proteolytic enzymes than soluble AMP, as well as low cytotoxicity against human cells. Moreover, the NPs demonstrated high antimicrobial activity after in vivo administration in a chronic wound and in an animal model of systemic infection.

Targeting B16 tumors in vivo with peptide-conjugated gold nanoparticles

This study examines the effects of polyethylene glycol (PEG) and peptide conjugation on the biodistribution of ultrasmall (2.7 nm) gold nanoparticles in mice bearing B16 melanoma allografts. Nanoparticles were delivered intravenously, and biodistribution was measured at specific timepoints by organ digestion and inductively coupled plasma mass spectrometry. All major organs were examined. Two peptides were tested: the cyclic RGD peptide (cRGD, which targets integrins); and a recently described peptide derived from the myxoma virus. We found the greatest specific tumor delivery using the myxoma peptide, with or without PEGylation. Un-PEGylated cRGD performed poorly, but PEGylated RGD showed a significant transient collection in the tumor. Liver and kidney were the primary targets of all constructs. None of the particles were able to cross the blood–brain barrier. Although it was able to deliver Au to B16 cells, the myxoma peptide did not show any cytotoxic activity against these cells, in contrast to previous reports. These results indicate that the effect of passive targeting by PEGylation and active targeting by peptides can be independent or combined, and that they should be evaluated on a case-by-case basis when designing new nanosystems for targeted therapies. Both myxoma peptide and cRGD should be considered for specific targeting to melanoma, but a thorough investigation of the cytotoxicity of the myxoma peptide to different cell lines remains to be performed.

Gold nanoparticles regulate the blimp1/pax5 pathway and enhance antibody secretion in B-cells

Nanoparticles are potential threats to human health and the environment; however, their medical applications as drug carriers targeting cancer cells bring hope to contemporary cancer therapy. As a model drug carrier, gold nanoparticles (GNPs) have been investigated extensively for in vivo toxicity. The effect of GNPs on the immune system, however, has rarely been examined. Antibody-secreting cells were treated with GNPs with diameters ranging from 2 to 50 nm. The GNPs enhanced IgG secretion in a size-dependent manner, with a peak of efficacy at 10 nm. The immune-stimulatory effect reached a maximum at 12 h after treatment but returned to control levels 24 h after treatment. This enhancing effect was validated ex vivo using B-cells isolated from mouse spleen. Evidence from RT-PCR and western blot experiments indicates that GNP-treatment upregulated B-lymphocyte-induced maturation protein 1 (blimp1) and downregulated paired box 5 (pax5). Immunostaining for blimp1 and pax5 in B-cells confirmed that the GNPs stimulated IgG secretion through the blimp1/pax5 pathway. The immunization of mice using peptide-conjugated GNPs indicated that the GNPs were capable of enhancing humoral immunity in a size-dependent manner. This effect was consistent with the bio-distribution of the GNPs in mouse spleen. In conclusion, in vitro, ex vivo, and in vivo evidence supports our hypothesis that GNPs enhance humoral immunity in mouse. The effect on the immune system should be taken into account if nanoparticles are used as carriers for drug delivery. In addition to their toxicity, the immune-stimulatory activity of nanoparticles could play an important role in human health and could have an environmental impact.

Nuclear Targeted Silver Nanospheres Perturb the Cancer Cell Cycle Differently than Those of Nanogold

Plasmonic nanoparticle research has become increasingly active due to potential uses in biomedical applications. However, little is known about the intracellular effects these nanoparticles have on mammalian cells. The aim of this work is to investigate whether silver nanoparticles (AgNPs) conjugated with nuclear and cytoplasmic targeting peptides exhibit the same intracellular effects on cancer cells as peptide-conjugated gold nanoparticles (AuNPs). Nuclear and cytoplasmic targeting spherical AgNPs with a diameter of 35 nm were incubated in a cancer (HSC-3) and healthy (HaCat) cell line. By utilizing flow cytometry, confocal microscopy, and real-time dark field imaging, we were able to analyze how targeting AgNPs affect the cell cycle and cell division. These experiments demonstrated that nuclear-targeting AgNPs cause DNA double-strand breaks and a subsequent increase in the sub G1 (apoptotic) population in our cancer cell model at much lower concentrations than previously reported for nuclear targeting AuNPs. Unlike the M phase accumulation seen in cancer cells treated with AuNPs, an accumulation in the G2 phase of the cell cycle was observed in both cell models when treated with AgNPs. Additionally, real-time dark field imaging showed that cancer cells treated with nuclear targeting AgNPs did not undergo cell division and ultimately underwent programmed cell death. A possible explanation of the observed results is discussed in terms of the chemical properties of the nanoparticles.

Direct Synthesis of Rev Peptide-Conjugated Gold Nanoparticles and Their Application in Cancer Therapeutics

We have developed a simple approach for generating peptide-conjugated gold nanoparticles (AuNPs) from the Rev peptide and gold aqueous solution. The peptide functions as both a reducing agent and a capping molecule. AuNPs of various sizes (20-300 nm) and shapes (spheres, triangular plates, and polygons) can be obtained upon modulating the ratio of gold ions to the Rev peptide. Transmission electron microscopy, X-ray diffraction, and UV-vis spectroscopy were utilized to characterize these nanoparticles. Fourier-transform infrared and X-ray photoelectron spectroscopy measurements were performed to investigate chemical interactions between the Rev peptide and AuNPs. Lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed that the Rev peptide-AuNP nanocomposites exhibited exceptionally high cytotoxic effects toward mouse ovarian surface epithelial cell lines, relative to the effects of equal doses of the free Rev peptide. Our study suggests a new way of utilizing biomolecule-conjugated AuNPs as potentially effective anticancer drugs.

Dark-field light scattering imaging of living cancer cell component from birth through division using bioconjugated gold nanoprobes

Novel methods and technologies that could extend and complement the capabilities of the prevailing fluorescence microscope in following the cell cycle under different perturbations are highly desirable in the area of biological and biomedical imaging. We report a newly designed instrument for long-term light scattering live cell imaging based on integrating a homebuilt environmental cell incubation minichamber and an angled dark-field illumination system into a conventional inverted light microscope. Peptide-conjugated gold nanoparticles that are selectively delivered to either the cytoplasmic or nuclear region of the cell are used as light scattering contrast agents. The new system enables us to carry out continuous and intermittence-free dark-field live cell imaging over several tens of hours. A variety of applications of this imaging system are demonstrated, such as monitoring the nuclear uptake of peptide-conjugated gold nanoparticles, tracking the full cycle of cancer cells from birth to division, following the chromosome dynamics during cell mitosis, and observing the intracellular distribution of gold nanoparticles after cell division. We also discuss the overall effect of nuclear targeting gold nanoparticles on the cell viability of parent and daughter cells.

Activity-Based Assay of Matrix Metalloproteinase on Nonbiofouling Surfaces Using Time-of-Flight Secondary Ion Mass Spectrometry

A label-free, activity-based assay of matrix metalloproteinase (MMP) and its inhibition was demonstrated on peptide-conjugated gold nanoparticles (AuNPs) with nonbiofouling poly(oligo(ethylene glycol) methacrylate) (pOEGMA) films using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Following surface-initiated atom-transfer radical polymerization of OEGMA on a Si/SiO2 substrate, the MMP activity was determined by analyzing the cleaved peptide fragments using TOF-SIMS on the peptide-conjugated AuNPs. The use of nonbiofouling pOEGMA films in conjunction with AuNPs synergistically enhanced the sensitivity of assays for MMP activity and its inhibition in human serum. The detection sensitivity of MMP-7 in serum was as low as 20 ng mL(-1) (1 pmol mL(-1)), and the half-maximal inhibitory concentration (IC50) of minocycline, which is a MMP-7 inhibitor, was estimated to be 450 nM. It is anticipated that the developed system will be broadly useful for conducting activity-based assays of serum proteases, as well as for screening of their inhibitors, with high sensitivity in a high-throughput manner.

Protein kinase assay on peptide-conjugated gold nanoparticles

We demonstrate that protein kinase can be assayed with high sensitivity on peptide-conjugated gold nanoparticles (AuNPs). Phosphorylation of peptides on the AuNP-monolayers was detected by using an anti-phosphotyrosine antibody (α-pY) and Cy3-labeled secondary antibody (Cy3-α-mIgG) as a probing molecule. When compared to conventional self-assembled monolayers (SAMs), spherical and three-dimensional geometry of AuNPs led to high surface density of peptide substrate and easy accessibility to enzyme, and consequently the resulting AuNP monolayers gave rise to improved detection sensitivity. Blocking of peptide-conjugated AuNPs with a poly(ethylene glycol) (PEG) also contributed to a higher signal-to-background ratio in kinase and its inhibition assays. The use of AuNPs as the platform surface will enable highly sensitive detection of protein kinases in a high-throughput manner.

Protein Kinase Assay on Peptide‐Conjugated Gold Nanoparticles by Using Secondary‐Ion Mass Spectrometric Imaging

Spot the mass of peptides: A label-free protein kinase assay that uses secondary-ion mass spectrometric imaging has been demonstrated with peptide-conjugated gold nanoparticles (AuNPs). With detection of the mass change of peptide substrates in a kinase reaction, AuNP-enhanced peptide signals enabled the assaying of both the protein kinase and its inhibition by chemical imaging of peptide substrates on a surface (see picture).