Peptide Functionalized Gold Nanoparticles Research Articles

A sandwich FRET biosensor for lysozyme detection based on peptide-functionalized gold nanoparticles and FAM-labeled aptamer

Lysozyme (LYZ) plays a crucial role in the body's immune defense system. Monitoring LYZ levels can provide valuable insights into the diagnosis and severity assessment of various diseases. Traditionally, antibody-based sandwich assays are employed for LYZ detection, but they are often time-consuming and operationally complicated. In this research, a novel sandwich FRET biosensor was developed, which enables rapid detection of LYZ based on peptide-functionalized gold nanoparticles (pAuNPs) and FAM-labeled aptamer (Apt-FAM). Initially, a mixture of Apt-FAM and pAuNPs resulted in partial quenching of the Apt-FAM fluorescence emission through an inner filter effect (IFE), with negligible energy transfer because of the electrostatic repulsion between the negatively charged pAuNPs and Apt-FAM. The introduction of LYZ into the mixture drove the specific binding of Apt-FAM and pAuNPs to LYZ, facilitating the formation of a pAuNPs-LYZ-aptamer sandwich structure. The formation of this complex drew the pAuNPs and Apt-FAM into close enough proximity to enable FRET to occur, which in turn effectively quenched the fluorescence emission of FAM. The decrease in FAM fluorescence intensity was correlated with the increasing concentration of LYZ. Thus, a sandwich FRET biosensor was successfully developed for LYZ detection with a linear detection range of 0–1.75 μM and a detection limit of 85 nM. Additionally, the biosensor allowed visual detection of LYZ in a 96-well microplate, with a rapid response time of just 15 s. This study introduces a innovative sandwich FRET biosensor that combines aptamer and peptide recognition elements, offering a fast and antibody-free method for protein detection.

Harnessing Peptide-Functionalized Multivalent Gold Nanorods for Promoting Enhanced Gene Silencing and Managing Breast Cancer Metastasis.

Small interfering RNA (siRNA) has become the cornerstone against undruggable targets and for managing metastatic breast cancer. However, an effective gene silencing approach is faced with a major challenge due to the delivery problem. In our present study, we have demonstrated efficient siRNA delivery, superior gene silencing, and inhibition of metastasis in triple-negative breast cancer cells (MDA-MB-231) using rod-shaped (aspect ratio: 4) multivalent peptide-functionalized gold nanoparticles and compared them to monovalent free peptide doses. Multivalency is a new concept in biology, and tuning the physical parameters of multivalent nanoparticles can enhance gene silencing and antitumor efficacy. We explored the effect of the multivalency of shape- and size-dependent peptide-functionalized gold nanoparticles in siRNA delivery. Our study demonstrates that peptide functionalization leads to reduced toxicity of the nanoparticles. Such designed peptide-functionalized nanorods also demonstrate antimetastatic efficacy in Notch1-silenced cells by preventing EMT progression in vitro. We have shown siRNA delivery in the hard-to-transfect primary cell line HUVEC and also demonstrated that the Notch1-silenced MDA-MB-231 cell line has failed to form nanobridge-mediated foci with the HUVEC in the co-culture of HUVEC and MDA-MB-231, which promote metastasis. This antimetastatic effect is further checked in a xenotransplant in vivo zebrafish model. In vivo studies also suggest that our designed nanoparticles mediated inhibition of micrometastasis due to silencing of the Notch1 gene. The outcome of our study highlights that the structure-activity relationship of multifunctional nanoparticles can be harnessed to modulate their biological activity.

Peptide mediated targeted delivery of gold nanoparticles into the demyelination site ameliorates myelin impairment and gliosis

Drug development for multiple sclerosis (MS) clinical management focuses on both neuroprotection and repair strategies, and is challenging due to low permeability of the blood-brain barrier, off-target distribution, and the need for high doses of drugs. The changes in the extracellular matrix have been documented in MS patients. It has been shown that the expression of nidogen-1 increases in MS lesions. Laminin forms a stable complex with nidogen-1 through a heptapeptide which was selected to target the lesion area in this study. Here we showed that the peptide binding was specific to the injured area following lysophosphatidylcholine (LPC) induced demyelination. In vivo data showed enhanced delivery of the peptide-functionalized gold nanoparticles (Pep-GNPs) to the lesion area. In addition, Pep-GNPs administration significantly enhanced myelin content and reduced astrocyte/microglia activation. Results demonstrated the possibility of using this peptide to target and treat lesions in patients suffering from MS.

Site-directed dual bioprobes inducing single-step nano-sandwich assay for the detection of cardiac troponin I.

Bioreceptor functionalized metallic nano-colloids have been identified as effective nanobioprobes to realize the detection of an analyte based on a common phenomenon of salt-induced aggregation. In marked contrast to this, we describe a nano-sandwich assay integrating the novel match-pair of aptamer and peptide functionalized gold nanoparticles. The site-directed biomolecular interaction of high affinity aptamer and peptide bioreceptors directed towards distinct sites of cardiac biomarker troponin I; this was found to form a nano-sandwich assay in a peculiar manner. The gold nanoconjugates interact with specific and distant regions of troponin I to result in collision of probes upon target identification. In the presence of TnI, both nanobioprobes bind at their respective sites forming a nano-sandwich pair providing a visual color change from red to blue. Thus, the presence of target TnI itself causes instant agglomeration in just a single-step without addition of any external aggregator. The assay imparts 100% specificity and 90% sensitivity in a dynamic concentration range of 0.1-500ng/mL troponin I with detection limit as low as 0.084ng/mL. The applicability of the assay has been validated in clinical samples of acute myocardial infarction patients thus establishing a promising point-of-care detection of TnI.

Active targeting of CD4+ T lymphocytes by PEI-capped, peptide-functionalized gold nanoparticles

Active targeting is a promising approach for the treatment of viral infections. In particular, site-specific formulations for the treatment of HIV infection may overcome challenges associated with current ARV regimens. In this study we explored active targeting by synthesizing a gold nanoparticle construct decorated with an anti-CD4 cyclic peptide. The aim was to demonstrate selectivity of the system for the CD4 receptor and to deliver the RNA payload into T-lymphocytes. Colloidal gold nanoparticles functionalized with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) were formed by a one-pot synthesis method where thiol modified polyethyleneimine (PEI) was mixed with chloroauric acid. PEI-SPDP AuNPs (gold nanoparticles) were conjugated to an anti-CD4 peptide and loaded with RNA. We measured toxicity and uptake using TZM-bl and HeLa cells. Our findings show that the nanoparticles bind selectively to CD4 + cells. UV–vis characterisation of the nanoparticles revealed a surface plasmon resonance (SPR) peak at 527 nm, corresponding to a 6 nm diameter. HRTEM of the complete nanoparticles visualised circular shaped particles with average diameter of ∼7 nm. The polydispersity index was calculated to be 0.08, indicating monodispersity of complete NPS in solution. Through the pyridine-2-thione assay each nanoparticle was calculated to carry 1.37 × 105 SPDP molecules available for peptide binding. Flow cytometry showed that 13.6% of TZM-bl cells, and 0.14% of HeLa cells retained fluorescence after an overnight incubation, an indication of system binding. No internal RNA delivery was demonstrated. Further work is required to improve internalization.

Melatonin Loaded Cardiac Homing Peptide-Functionalized Gold Nanoparticles for the Care of Anti-Cardiac Hypertrophy

Melatonin Loaded Cardiac Homing Peptide-Functionalized Gold Nanoparticles for the Care of Anti-Cardiac Hypertrophy

Lateral flow immunoassay with peptide-functionalized gold nanoparticles for rapid detection of protein tyrosine phosphatase 1B

In this work, a lateral flow immunoassay (LFIA) with peptide functionalized gold nanoparticles (termed as biotin-ppeptide-AuNPs) has been developed for rapid, semi-quantitative detection of PTP1B activity without using any sophisticated equipment. In this method, the anti-phosphotyrosine (anti-pY) monoclonal antibody and streptavidin were used as test line and control line, respectively. The biotin-ppeptide-AuNPs contain 10% biotinylated peptide ligand carry a motif SDGHEpYIYVDP with pY (phosphotyrosine) and 90% pentapeptide (CALNN) ligand, which are used as PTP1B substrates and LFIA labelling probes. The experimental results demonstrate that the as-proposed LFIA with biotin-ppeptide-AuNPs exhibits a wide linear range (from 50 ng/mL to 10 μg/mL), a relatively low limit of detection (LOD, 44 ng/mL), and good specificity. In addition, the LFIA with biotin-ppeptide-AuNPs has been successfully used to evaluate activity levels of PTP1B in four cell lysates and the detection results exhibit a consistent trend with that of commercial kit.

Controlled 3D assembly and stimuli responsive behavior of DNA and peptide functionalized gold nanoparticles in solutions

Self assembly of DNA and peptide functionalized gold nanoparticles.

Self-assembled chitosan polymer intercalating peptide functionalized gold nanoparticles as nanoprobe for efficient imaging of urokinase plasminogen activator receptor in cancer diagnostics

Targeting cell surface receptors for specific drug delivery in cancer has garnered lot of attention. Urokinase plasminogen activator receptor (uPAR), a surface biomarker, is overexpressed on many tumours including breast, colorectal, prostate, and ovarian cancers. Binding of growth factor domain (GFD) of urokinase plasminogen activator (uPA) with uPAR lead to its close conformation, and allow somatomedin B domain (SMB) of vitronectin binding by allosteric modulation. In-silico docking of uPAR with GFD and SMB peptides was performed to identify potential binding affinity. Herein, we report fluorescently labeled peptide functionalized AuNPs with a mixed self-assembled monolayer of intercalating chitosan polymer for efficient targeting and imaging of uPAR-positive cells. The biophysical characterization of nanoconjugates and uPAR-specific targeting was assessed by FACS, cell adhesion, and fluorescence imaging. AuNPs/chitosan/GFD+SMB peptides showed higher uptake as compared to AuNPs/chitosan/GFD, and AuNPs/chitosan/SMB that can be utilized as a tool for molecular targeting and imaging in metastasis.

In Situ, Noncovalent Labeling and Stimulated Emission Depletion-Based Super-Resolution Imaging of Supramolecular Peptide Nanostructures.

Supramolecular materials have gained substantial interest for a number biological and nonbiological applications. However, for optimum utilization of these dynamic self-assembled materials, it is important to visualize and understand their structures at the nanoscale, in solution and in real time. Previous approaches for imaging these structures have utilized super-resolution optical imaging methods such as STORM, which has provided important insights, but suffers from drawbacks of complex sample preparation and slow acquisition times, thus limiting real-time in situ imaging of dynamic processes. We demonstrate a noncovalent fluorescent labeling design for STED-based super-resolution imaging of self-assembling peptides. This is achieved by in situ, electrostatic binding of anionic sulfonates of Alexa-488 dye to the cationic sites of lysine (or arginine) residues exposed on the peptide nanostructure surface. A direct, multiscale visualization of static structures reveals hierarchical organization of supramolecular fibers with sub-60 nm resolution. In addition, the degradation of nanofibers upon enzymatic hydrolysis of peptide could be directly imaged in real time, and although resolution was compromised in this dynamic process, it provided mechanistic insights into the enzymatic degradation process. Noncovalent Alexa-488 labeling and subsequent imaging of a range of cationic self-assembling peptides and peptide-functionalized gold nanoparticles demonstrated the versatility of the methodology for the imaging of cationic supramolecular structures. Overall, our approach presents a general and simple method for the electrostatic fluorescent labeling of cationic peptide nanostructures for nanoscale imaging under physiological conditions and probe dynamic processes in real time and in situ.

Protease functional assay on membrane

Targeting enzymes, proteases in particular, using various assay formats has attracted considerable interest for early disease diagnosis. While affinity-based assays that report the presence of proteases in samples have been widely explored, it is of utmost importance to develop functional assays that provide information on the activity of proteases for a more precise indication of the disease state and progression. Herein, we report a paper-based functional assay that yields naked eye observable and concentration dependent responses for the active form of proteases. The paper-based assay involves a facile single step fabrication process of depositing protease specific peptide functionalized gold nanoparticles on paper membranes. Matrilysin is used as a model protease to validate the proposed methodology. The proteolysis-driven aggregation of nanoparticles on the membrane yields a colorimetric response that has limit of detection that is ∼4 times lower (3.1 μg/mL) than for the same assay performed in homogeneous solution (12.5 μg/mL). To the best of our knowledge, this is the first report on heterogeneous protease assay on paper relying on aggregation of peptide-functionalized nanoparticles. The approach easily can be extended to assay other enzymes by functionalizing the gold nanoparticles using specific peptides.

Rapid Preparation of Au–Se–Peptide Nanoprobe Based on a Freezing Method for Bioimaging

Fluorescent nanoprobes based on peptide-functionalized gold nanoparticles (AuNPs) have been widely used in bioassays. The Au-Se bond is considered as a better candidate than the Au-S bond to link the peptides and AuNPs due to the stronger ability against interference of intracellular thiol. However, the current synthetic methods for preparing peptide/AuNPs nanoprobes are always complex and time-consuming. Developing a convenient and rapid method to synthesize the Au-Se bond based nanoprobes is expected to further facilitate their application in fundamental research. Herein, we present a facile and rapid approach to prepare the Au-Se-peptide nanoprobes through a direct freezing process, which is easy-to-operate, time-saving, and surfactant-free. Compared with the traditional method, the amount of peptide loaded on AuNPs by freezing method is also promoted with 20-30%. Furthermore, the obtained nanoprobe was successfully applied to identify autophagy and apoptosis in chemotherapeutic drug treated cancer cells.

Highly sensitive surface-enhanced Raman spectroscopy (SERS) platform using core/double shell (Ag/polymer/Ag) nanohorn for proteolytic biosensor

Sensitive detection platforms have recently gathered more interest due to its ability to make an early diagnosis of several diseases, which increases the chances of successful treatment. Although several highly sensitive platforms have been developed, improvements are needed to reduce their reaction time and cost. In this study, we established a surface-enhanced Raman spectroscopy (SERS)-based ultrasensitive platform by using SERS-active hollow polypyrrole (hPPy) nanohorn and peptide-functionalized gold nanoparticles (AuNPs). To create a SERS-active platform, we deposited silver on the inside and outside of the hPPy nanohorn surface; this resulted in the formation of core/double-shells Ag/polymer/Ag nanostructures. In addition, peptide-functionalized AuNPs functionalized with Raman dye was pre-immobilized on the Ag/hPPy/Ag nanohorn to enhance the signals and skip additional steps for the biomolecular detection. Using this SERS-sensitive novel core/double-shell nanohorn platform, we enhanced the Raman signal and improved the sensitivity of caspase-3, which is associated with several diseases, and achieved a broad detection range from 10 pg/ml to 10 µg/ml. We suggest that the highly sensitive and simple detection of this nanohorn platform can be widely applied for sensitive detection of proteolytic biomarkers and early diagnosis of several diseases.

Self-Assembly of a Structurally Defined Chiro-Optical Peptide-Oligothiophene Hybrid Material.

Conducting polymers are routinely used in optoelectronic biomaterials, but large polymer polydispersity and poor aqueous compatibility complicate integration with biomolecular templates and development of discrete and defined supramolecular complexes. Herein, we report on a chiro-optical hybrid material generated by the self-assembly of an anionic peptide and a chemically defined cationic pentameric thiophene in aqueous environment. The peptide acts as a stereochemical template for the thiophene and adopts an α-helical conformation upon association, inducing optical activity in the thiophene π–π* transition region. Theoretical calculations confirm the experimentally observed induced structural changes and indicate the importance of electrostatic interactions in the complex. The association process is also probed at the substrate–solvent interface using peptide-functionalized gold nanoparticles, indicating that the peptide can also act as a scaffold when immobilized, resulting in structurally well-defined supramolecular complexes. The hybrid complex could rapidly be assembled, and the kinetics of the formation could be monitored by utilizing the local surface plasmon resonance originating from the gold nanoparticles. We foresee that these findings will aid in designing novel hybrid materials and provide a possible route for the development of functional optoelectronic interfaces for both biomaterials and energy harvesting applications.

A Facile Approach for Synthesis and Intracellular Delivery of Size Tunable Cationic Peptide Functionalized Gold Nanohybrids in Cancer Cells.

Peptide-based drug delivery systems have become a mainstay in the contemporary medicinal field, resulting in the design and development of better pharmaceutical formulations. However, most of the available reports employ tedious multiple reaction steps for the conjugation of bioactive cationic peptides with drug delivery vehicles. To overcome these limitations, the present work describes a one-step approach for facile and time efficient synthesis of highly cationic cell penetrating peptide functionalized gold nanoparticles and their intracellular delivery. The nanoconstruct was synthesized by the reduction of gold metal ions utilizing cell penetrating peptide (CPP), which facilitated the simultaneous synthesis of metal nanoparticles and the capping of the peptide over the nanoparticle surface. The developed nanoconstruct was thoroughly characterized and tested for intracellular delivery into HeLa cells. Intriguingly, a high payload of cationic peptide over gold particles was achieved, in comparison to conventional conjugation methods. Moreover, this method also provides the ability to control the size and peptide payload of nanoparticles. The nanoconstructs produced showed enhanced cancer cell penetration (μM) and significant cytotoxic effect compared to unlabeled gold nanoparticles. Therefore, this novel approach may also have significant future potential to kill intracellular hidden dreaded pathogens like the human immunodeficiency virus, Mycobacterium tuberculosis, and so forth.

Development and Evaluation of Peptide-Functionalized Gold Nanoparticles for HIV Integrase Inhibition

The HIV integrase enzyme represents an important target for HIV inhibition because it is essential for viral replication and there is no cellular counterpart. Blocking HIV replication at this early stage of the viral life cycle can also prevent the establishment of viral reservoirs in the form of latently infected cells. Complications such as drug resistance and other adverse side effects associated with HIV treatment necessitate novel approaches for HIV inhibition. Nanoparticle-based systems are fast revolutionizing the biomedical field with applications in infectious disease diagnosis and treatment, and are important tools to investigate for HIV targeting. This study explores the development of gold nanoparticles functionalized with a hexapeptide, previously demonstrated to inhibit integrase at low micromolar concentrations. We also sought to investigate the effect of Tat peptide incorporation to facilitate both cellular and nuclear entry, so that both cytoplasmic and nuclear events mediated by integrase, could be targeted. Therefore, the aim of this study is to synthesize, characterize and evaluate the antiviral activity of nanoparticle-peptide complexes. Our results showed no obvious HIV inhibitory activity, but did provide original and fundamental in vitro data for this potent hexapeptide when challenged with HIV infection. Further insight into the optimization of gold nanoparticle functionalization with peptides is also provided.

Short elastin-like peptide-functionalized gold nanoparticles that are temperature responsive under near-physiological conditions.

Thermally-responsive, short elastin-like peptides (ELPs) of sequence VPGVG (V, P and G represent valine, proline and glycine respectively), bearing different N-terminal functional groups (amino-, N-acetyl and thiol) and a non-ionisable C-terminal group, were prepared by solid phase synthesis. The conformation and aggregation properties of the ELPs were studied in different pH aqueous buffer solutions using UV-vis spectroscopy and circular dichroism (CD). The thiol-capped ELPs were used to prepare functionalized gold nanoparticles (GNPs), which were found to undergo thermally-triggered reversible aggregation at 40 °C. The peptide conformation and nanoparticle aggregation behaviour of the ELP-GNPs in aqueous solution were investigated by transmission electron microscopy (TEM), circular dichroism (CD) and UV-vis spectroscopy. It was found that the ELP-GNP conjugates were capable of reversible, thermally triggered aggregation at near-physiological temperatures (transition temperature of 40 °C at pH = 7.4), opening up applications in photothermal cancer therapy and diagnosis.

A fluorescence sensor for protein kinase activity detection based on gold nanoparticles/copper nanoclusters system

In this work, we develop a novel biosensor for the detection of protein kinase A (PKA) based on the fluorescence inner-filter effect (IFE) between peptide functionalized gold nanoparticles (p-AuNPs) and polyethyleneimine protected copper nanoclusters (PEI-CuNCs). The fluorescence of PEI-CuNCs could be quenched by p-AuNPs through IFE. In the presence of adenosine 5′-triphosphate (ATP) and PKA, the substrate peptide of p-AuNPs was phosphorylated and then selective aggregation of phosphorylated p-AuNPs was triggered by Zr4+ ion coordination. Compared with the monodispersed p-AuNPs, the aggregated p-AuNPs have less fluorescence quenching effect on PEI-CuNCs. So the activity of PKA could be effectively monitored according to the change of fluorescence signal in the range of 0.1–6.0U/mL, and the detection limit (LOD) is 0.038U/mL. In addition, we explored the inhibitory effect of H-89 on protein kinase activity. The developed method was also applied to the investigation of drug-induced PKA activation in HepG-2 cell, which provides one promising tool for kinase-related biochemical fundamental research and inhibitor screening with excellent performance.

A Spectral Probe for Detection of Aluminum (III) Ions Using Surface Functionalized Gold Nanoparticles.

A simple green route has been developed for the synthesis of casein peptide functionalized gold nanoparticles (AuNPs), in which casein peptide acts as a reducing as well as the stabilizing agent. In this report, AuNPs have been characterized on the basis of spectroscopic and microscopic results; which showed selective and sensitive response toward Al3+ in aqueous media, and Al3+ induces aggregation of AuNPs. The sensing study performed for Al3+ revealed that the color change from red to blue was due to a red-shift in the surface plasmon resonance (SPR) band and the formation of aggregated species of AuNPs. The calibration curve determines the detection limit (LOD) for Al3+ about 20 ppb (0.067 μM) is presented using both decrease and increase in absorbance at 530 and 700 nm, respectively. This value is considerably lower than the higher limit allowed for Al3+ in drinking water by the world health organization (WHO) (7.41 μM), representing enough sensitivity to protect water quality. The intensity of the red-shifted band increases with linear pattern upon the interaction with different concentrations of Al3+, thus the possibility of producing unstable AuNPs aggregates. The method is successfully used for the detection of Al3+ in water samples collected from various sources, human urine and ionic drink. The actual response time required for AuNPs is about 1 min, this probe also have several advantages, such as ease of synthesis, functionalization and its use, high sensitivity, and enabling on-site monitoring.

Peptide functionalized gold nanoparticles: the influence of pH on binding efficiency

We report herein on the synthesis of mixed monolayer gold nanoparticles (AuNPs) capped with both polyethylene glycol (PEG) and one of three peptides. Either a receptor-mediated endocytosis peptide, an endosomal escape pathway (H5WYG) peptide or the Nrp-1 targeting RGD peptide (CRGDK) labeled with FITC. All three peptides have a thiol containing cysteine residue which can be used to bind the peptides to the AuNPs. In order to investigate the influence of pH on peptide attachment, PEGylated AuNPs were centrifuged, the supernatant removed, and the nanoparticles were then re-suspended in a range of pH buffer solutions above, below and at the respective isoelectric points of the peptides before co-functionalization. Peptide attachment was investigated using dynamic light scattering, Ultra-violet visible spectroscopy (UV/Vis), FTIR and photo luminescence spectroscopy. UV/Vis analysis coupled with protein assay results and photoluminescence of the FITC tagged RGD peptide concluded that a pH of ∼8 optimized the cysteine binding and stability, irrespective of the peptide used.