Activatable Protein Nanoparticles for Targeted Delivery of Therapeutic Peptides

Clinical translation of therapeutic peptides, particularly those that require penetration of the cell membrane or are cytolytic, is a major challenge. A novel approach based on a complementary mechanism, which has been widely used for guided synthesis of DNA or RNA nanoparticles, for de novo design of activatable protein nanoparticles (APNPs) for targeted delivery of therapeutic peptides is described. APNPs are formed through self‐assembly of three independent polypeptides based on pairwise coiled‐coil dimerization. They are capable of long circulation in the blood and can be engineered to target diseases. Peptides to be delivered are incorporated into APNPs and released into the disease microenvironment by locally enriched proteases. It is demonstrated that APNPs mediate efficient delivery of NR2B9c, a neuroprotective peptide that functions after cell penetration, and melittin, a cytolytic peptide that perturbs the lipid bilayer, for effective treatment of stroke and cancer, respectively. Due to their robust properties, simple design, and economic costs, APNPs have great potential to serve as a versatile platform for controlled delivery of therapeutic peptides.     

Mixed monolayer-protected gold nanoclusters as selective peptide extraction agents for MALDI-MS analysis

Cationic and anionic nanoparticles selectively target peptides with low and high isoelectric points, respectively. Additionally, their high surface area-to-volume ratios make these nanoparticles (approximately 2-nm core diameter) very efficient extraction and concentration agents. Upon extraction, the peptide-bound nanoparticles can be analyzed by MALDI-MS to provide highly sensitive detection of the targeted peptides. We demonstrate that MALDI-MS can detect peptide concentrations as low as 500 pM from 250-microL solutions using these nanoparticle scaffolds as extraction and concentration agents.     

Bioassisted capture and release of nanoparticles on nanolithographed ZnO films

Using an artificial peptide library, we have identified a peptide that has strict selective affinity for ZnO surfaces. The binding affinity of the peptide on the ZnO surface can be controlled simply through changes in phosphate concentration at constant pH and temperature. In this study, we functionalized inorganic nanoparticles by orderly conjugating ZnO-binding peptides (ZnOBPs) on the surface of cadmium selenide (CdSe) nanoparticles and performed spontaneous and reversible nanopatterning of ZnOBP-displayed nanoparticles on lithographed ZnO films. Conjugation of ZnOBPs on CdSe nanoparticles caused spontaneous adsorption of the nanoparticles on a ZnO film, and fluorescence and cathodoluminescence images clearly showed specific adsorption of nanoparticles on the ZnO films lithographed on nano-?and micrometer scales. The selectively bound nanoparticles on ZnO films were completely released by changing the phosphate concentration in solution; such release did not require heat or mechanical applications. Repeated capture and release of nanoparticles were achieved on the micrometer scale. Our results show the potential of material-binding peptides for nanopatterning and dynamic microarrays.     

Targeted biodegradable nanoparticles for drug delivery to smooth muscle cells

Targeted delivery of therapeutic agents to prevent smooth muscle cell (SMC) proliferation is important in averting restenosis (a narrowing of blood vessels). Since platelet derived growth factor (PDGF) receptors are over-expressed in proliferating SMCs after injury from cardiovascular interventions, such as angioplasty and stent implantation, our hypothesis is that conjugation of PDGF-BB (platelet-derived growth factor BB (homodimer)) peptides to biodegradable poly (D,L-lactic-co-glycolide) (PLGA) nanoparticles (NPs) would exhibit an increased uptake of these NPs by proliferating SMCs. In this study, poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles containing dexamethasone were formulated and conjugated with PDGF-BB peptides. These NPs were stable, biocompatible, and exhibited a sustained drug release over 14 days. Various particle uptake studies using HASMCs (human aortic smooth muscle cells) demonstrated that PDGF-BB peptide-conjugated nanoparticles significantly increased cellular uptake and decreased proliferation of HASMCs compared to control nanoparticles (without conjugation of PDGF-BB peptides). These NPs were internalized primarily by clathrin-mediated endocytosis and macropinocytosis. Our in vitro results suggest that PDGF-BB peptide-conjugated NPs could represent as an effective targeted, sustained therapeutic delivery system to reduce restenosis and neointimal hyperplasia.     

Plasmonic circular dichroism of Peptide-functionalized gold nanoparticles

Nature is remarkable at tailoring the chirality of different biomolecules to suit specific functions. Chiral molecules can impart optical activity to achiral materials in the form of the particle's electronic transition frequency. Herein, we used peptides of differing secondary structures (random coil and α-helix) to artificially create optically active chiral gold nanoparticles through peptide-nanoparticle interactions as observed by circular dichroism (CD) spectroscopy. This interaction produces a CD signal at the plasmon resonance frequency (～520 nm) of the chiral peptide-nanoparticle complex. Aggregation of the peptide-coated nanoparticles using metal ions results in a red-shifted plasmonic CD response. Our results suggest that chiroptical properties of nanomaterials can be engineered using peptides.     

Protein oriented ligation on nanoparticles exploiting O6-alkylguanine-DNA transferase (SNAP) genetically encoded fusion

A bimodular genetic fusion comprising a delivery module (scFv) and a capture module (SNAP) is proposed as a novel strategy for the site-specific covalent conjugation of targeting peptides to nanoparticles. An scFv mutant selective for HER2 tumor antigen is chosen as the targeting ligand. SNAP-scFv is immobilized on magnetofluorescent nanoparticles and its targeting efficiency against HER2-positive cells is assessed by flow cytometry and immunofluorescence.     

Exploration of possible binding sites of nanoparticles on protein by cross-linking chemistry coupled with mass spectrometry

For the first time, the possible binding site of nanoparticles on protein was revealed by cross-linking chemistry coupled with mass spectrometry. The peptides located very close to the poly(acrylic acid) (PAA)-coated Fe(3)O(4) nanoparticles (NPs) during interaction with human serum albumin (HSA) were cross-linked to the surface of NPs. Following protease digestion, the attached peptides were cleaved off the particle surface and identified by matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). The peptides were found to be part of the so-called drug binding site 2 of HSA; and the competitive binding to HSA between the corresponding drug, ibuprofen, and the NPs was observed. Our results demonstrated that cross-linking chemistry coupled with MS was a quick and simple method for locating the possible binding sites of NPs on protein. Information on NP-protein binding interface will benefit the study of how the interactions are governed by the physicochemical properties of NPs, for guiding the design of functional bionano constructs. It can also help to predict the biological consequence of protein adsorption on NPs, for obtaining more knowledge on nanotoxicity.     

Advances in Peptide Functionalization on Mesoporous Silica Nanoparticles for Controlled Drug Release

During the last decade, using versatile, promising, and fascinating mesoporous silica nanoparticles (MSNs) as site‐specific and stimuli‐responsive drug delivery systems (DDSs) has received concentrated research interest. As one of the most attractive surface modification units, peptides have inherent bioactivity, biodegradability and biocompatibility. Recent progresses in the utilization of versatile peptides for surface functionalization of MSNs to achieve cell‐specific targeting, fluorescence imaging, and intracellular diagnosis and treatment of tumors are summarized in this review. The various functional peptides decorated on the MSNs are introduced and classified into three types, including targeting peptides, stimuli‐responsive peptides and multifunctional chimeric peptides. The limitations and challenges of peptide modified MSNs and their potential applications are further discussed.     

肿瘤靶向纳米四氧化三铁造影剂的制备及其磁共振成像应用

环氧氯丙烷交联法制备交联葡聚糖与多肽偶联的肿瘤新生血管靶向的纳米Fe3O4造影剂,考察其体内肿瘤靶向性并进行磁共振成像试验。以共沉淀法制备6～8nm的Fe3O4粒子,采用油酸钠和葡聚糖二次包覆,用环氧氯丙烷使葡聚糖交联并与靶向多肽偶联,进行了肿瘤细胞结合实验和荷瘤动物磁共振成像实验。结果表明,包覆后纳米Fe3O4复合粒子为20～30nm,水动力学粒径小于80nm,仍表现为超顺磁性;葡聚糖交联的时间4～8h,造影剂在体内血浆半衰期从2.8h延长到6.2h;主要通过肝脏和肾脏代谢。与无靶的比较,靶向多肽偶联后与肿瘤细胞特异性结合能力提高了10～30倍,MR成像信号密度是无靶的3.68倍。     

Nanoparticle‐Induced Folding and Fibril Formation of Coiled‐Coil‐Based Model Peptides

Nanomedicine is a rapidly growing field that has the potential to deliver treatments for many illnesses. However, relatively little is known about the biological risks of nanoparticles. Some studies have shown that nanoparticles can have an impact on the aggregation properties of proteins, including fibril formation. Moreover, these studies also show that the capacity of nanoscale objects to induce or prevent misfolding of the proteins strongly depends on the primary structure of the protein. Herein, light is shed on the role of the peptide primary structure in directing nanoparticle‐induced misfolding by means of two model peptides. The design of these peptides is based on the α‐helical coiled‐coil folding motif, but also includes features that enable them to respond to pH changes, thus allowing pH‐dependent β‐sheet formation. Previous studies showed that the two peptides differ in the pH range required for β‐sheet folding. Time‐dependent circular dichroism spectroscopy and transmission electron microscopy are used to characterize peptide folding and aggregate morphology in the presence of negatively charged gold nanoparticles (AuNPs). Both peptides are found to undergo nanoparticle‐induced fibril formation. The determination of binding parameters by isothermal titration calorimetry further reveals that the different propensities of both peptides to form amyloid‐like structures in the presence of AuNPs is primarily due to the binding stoichiometry to the AuNPs. Modification of one of the peptide sequences shows that AuNP‐induced β‐sheet formation is related to the structural propensity of the primary structure and is not a generic feature of peptide sequences with a sufficiently high binding stoichiometry to the nanoparticles.     

Synthesis, stability, and cellular internalization of gold nanoparticles containing mixed peptide-poly(ethylene glycol) monolayers

Gold nanoparticles have shown great promise as therapeutics, therapeutic delivery vectors, and intracellular imaging agents. For many biomedical applications, selective cell and nuclear targeting are desirable, and these remain a significant practical challenge in the use of nanoparticles in vivo. This challenge is being addressed by the incorporation of cell-targeting peptides or antibodies onto the nanoparticle surface, modifications that frequently compromise nanoparticle stability in high ionic strength biological media. We describe herein the assembly of poly(ethylene glycol) (PEG) and mixed peptide/PEG monolayers on gold nanoparticle surfaces. The stability of the resulting bioconjugates in high ionic strength media was characterized as a function of nanoparticle size, PEG length, and monolayer composition. In total, three different thiol-modified PEGs (average molecular weight (MW), 900, 1500, and 5000 g mol-1), four particle diameters (10, 20, 30, and 60 nm), and two cell-targeting peptides were explored. We found that nanoparticle stability increased with increasing PEG length, decreasing nanoparticle diameter, and increasing PEG mole fraction. The order of assembly also played a role in nanoparticle stability. Mixed monolayers prepared via the sequential addition of PEG followed by peptide were more stable than particles prepared via simultaneous co-adsorption. Finally, the ability of nanoparticles modified with mixed PEG/RME (RME = receptor-mediated endocytosis) peptide monolayers to target the cytoplasm of HeLa cells was quantified using inductively coupled plasma optical emission spectrometry (ICP-OES). Although it was anticipated that the MW 5000 g mol-1 PEG would sterically block peptides from access to the cell membrane compared to the MW 900 PEG, nanoparticles modified with mixed peptide/PEG 5000 monolayers were internalized as efficiently as nanoparticles containing mixed peptide/PEG 900 monolayers. These studies can provide useful cues in the assembly of stable peptide/gold nanoparticle bioconjugates capable of being internalized into cells.     

Novel route for rapid biosynthesis of lead nanoparticles using aqueous extract of Jatropha curcas L. latex

We are reporting a novel, low-cost and eco-friendly route for rapid synthesis of lead nanoparticles by using 0.5% aqueous extract of Jatropha curcas L. latex. Lead nanoparticles were characterized initially by UV–vis spectroscopy and shown distinct peak at 218 nm. This peak was highly specific for lead nanoparticles. Formation of Pb (0) was confirmed by X-ray diffraction technique (XRD).Transmission electron microscopy (TEM) was performed for estimating the size and shape of nanoparticles. The average size of lead nanoparticles was found to be in the range of 10 to 12.5 nm. Energy dispersive analysis of X-rays (EDAX) showed distinct peaks of lead. Fourier Transform Infrared Spectroscopy (FTIR) were performed to find the role of cyclic peptides namely curcacycline A (an octapeptide) and curcacycline B (a nonapeptide) as a possible reducing and capping agents present in the latex of Jatropha curcas L. Lead nanoparticles formed by the above method were monodisperse.     

Peptide synthesis of gold nanoparticles: the early steps of gold reduction investigated by density functional theory

Gold nanoparticles can be synthesized by reducing chloroaurate(III) ions in the presence of peptides. Here, such reduction for serine and tyrosine is studied by density functional theory including solvent effects. We find that the formation of chloroaurate complexes of these amino acids is thermodynamically viable and facilitates the reduction of Au(III), to a greater degree for tyrosine as found in experiments. Our results also suggest a rationale for the behavior of tyrosine-intercalated peptides.     

Magnetite/Ceria Nanoparticle Assemblies for Extracorporeal Cleansing of Amyloid‐β in Alzheimer's Disease

Accumulation of amyloid‐β (Aβ) peptides in the brain is regarded as a major contributor to the pathogenesis and progression of Alzheimer's disease (AD). However, development of clinically relevant techniques to reduce Aβ levels in AD patients is hindered by low efficiency and/or side effects. Here, an extracorporeal Aβ cleansing system, where multifunctional magnetite/ceria nanoparticle assemblies are used to remove Aβ peptides from flowing blood by specific capture and magnetic separation, is reported. The magnetite nanoparticles in the nanoassembly core enable the magnetic isolation of the captured Aβ peptides by generating a large attraction force under an external magnetic field. The ceria nanoparticles in the nanoassembly shell relieve oxidative stress by scavenging reactive oxygen species that are produced by immune response during the process. Blood Aβ cleansing treatment of 5XFAD transgenic mice not only demonstrates the decreased Aβ levels both in the blood and in the brain but also prevents the spatial working memory deficits, suggesting the potential of the method for AD prevention and therapy.     

Gold nanoparticles capped by peptides

Two dipeptides (GK and GC) and two 15-aminoacid peptides (GK15 and GC15) were used as capping agents in the preparation of monolayer-protected gold nanoparticles (MPCs). They were characterized by TEM microscopy, UV–vis, NMR and IR spectroscopy.     

Identification of peptides using gold nanoparticle-assisted single-drop microextraction coupled with AP-MALDI mass spectrometry

A novel technique, gold nanoparticle-assisted single-drop microextraction (SDME) combined with atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) for the identification of peptides has been described. The SDME of peptides from aqueous solution was achieved using gold nanoparticles prepared in toluene as the acceptor phase. A simple phenomenon of isoelectric point (pI) of the peptides has been utilized successfully to extract the peptides into a single drop of nanogold in toluene. After extraction, a single-drop nano gold solution was directly spotted onto the target plate with an equal volume of matrix, proportional, variant-cyanohydroxy cinnamic acid ( proportional, variant-CHCA) and analyzed in AP-MALDI-MS. The parameters, such as solvent selection, extraction time, agitation rate, and pH effect, were optimized for the SDME technique. Using this technique, in aqueous solution, the lowest concentration detected for Met- and Leu-enkephalin peptides was 0.2 and 0.17 microM, respectively. In addition, the application of this technique to obtain the signal for the selected peptides in a mass spectrum in the presence of matrix interferences such as 1% Triton X-100 and 6.5 M urea has been showed. The application was extended to identify the peptides spiked into urine.     

Biomimetic synthesis and patterning of silver nanoparticles

The creation of nanoscale materials for advanced structures has led to a growing interest in the area of biomineralization. Numerous microorganisms are capable of synthesizing inorganic-based structures. For example, diatoms use amorphous silica as a structural material, bacteria synthesize magnetite (Fe3O4) particles and form silver nanoparticles, and yeast cells synthesize cadmium sulphide nanoparticles. The process of biomineralization and assembly of nanostructured inorganic components into hierarchical structures has led to the development of a variety of approaches that mimic the recognition and nucleation capabilities found in biomolecules for inorganic material synthesis. In this report, we describe the in vitro biosynthesis of silver nanoparticles using silver-binding peptides identified from a combinatorial phage display peptide library.     

Magnetoferritin nanoparticles for targeting and visualizing tumour tissues

Engineered nanoparticles have been used to provide diagnostic, therapeutic and prognostic information about the status of disease. Nanoparticles developed for these purposes are typically modified with targeting ligands (such as antibodies, peptides or small molecules) or contrast agents using complicated processes and expensive reagents. Moreover, this approach can lead to an excess of ligands on the nanoparticle surface, and this causes non-specific binding and aggregation of nanoparticles, which decreases detection sensitivity. Here, we show that magnetoferritin nanoparticles (M-HFn) can be used to target and visualize tumour tissues without the use of any targeting ligands or contrast agents. Iron oxide nanoparticles are encapsulated inside a recombinant human heavy-chain ferritin (HFn) protein shell, which binds to tumour cells that overexpress transferrin receptor 1 (TfR1). The iron oxide core catalyses the oxidation of peroxidase substrates in the presence of hydrogen peroxide to produce a colour reaction that is used to visualize tumour tissues. We examined 474 clinical specimens from patients with nine types of cancer and verified that these nanoparticles can distinguish cancerous cells from normal cells with a sensitivity of 98% and specificity of 95%.     

Interactions of Skin with Gold Nanoparticles of Different Surface Charge,Shape, and Functionality

The interactions between skin and colloidal gold nanoparticles of different physicochemical characteristics are investigated. By systematically varying the charge, shape, and functionality of gold nanoparticles, the nanoparticle penetration through the different skin layers is assessed. The penetration is evaluated both qualitatively and quantitatively using a variety of complementary techniques. Inductively coupled plasma optical emission spectrometry (ICP‐OES) is used to quantify the total number of particles which penetrate the skin structure. Transmission electron microscopy (TEM) and two photon photoluminescence microscopy (TPPL) on skin cross sections provide a direct visualization of nanoparticle migration within the different skin substructures. These studies reveal that gold nanoparticles functionalized with cell penetrating peptides (CPPs) TAT and R₇ are found in the skin in larger quantities than polyethylene glycol‐functionalized nanoparticles, and are able to enter deep into the skin structure. The systematic studies presented in this work may be of strong interest for developments in transdermal administration of drugs and therapy.     

Gold nanoparticle-enhanced secondary ion mass spectrometry imaging of peptides on self-assembled monolayers

We demonstrate the use of gold nanoparticles (AuNPs) to enhance the secondary ion emission of peptides in time-of-flight secondary ion mass spectrometry (TOF-SIMS). The signal intensity of peptides adsorbed onto AuNPs was significantly increased when compared to that of self-assembled monolayers (SAMs). This gold nanoparticle-enhanced SIMS, termed NE-SIMS, enabled the sensitive detection of subtle modifications of peptides, such as phosphorylation. From a quantitative analysis of the amounts of adsorbed peptides and AuNPs on SAMs using quartz crystal microbalance and surface plasmon resonance spectroscopy, the ratio of peptide molecule to AuNP on amine-SAMs was revealed to be 18-19:1. When considering the ratio of peptide to matrix (1:10(3)-10(6)) employed in a matrix-enhanced SIMS, the use of AuNPs gave rise to a significantly increased secondary ion emission of peptides. Peptides were adsorbed onto patterned AuNPs on SAMs using a microfluidic system, and well-contrasted molecular ion images were obtained. NE-SIMS is expected to be applied to a chip-based analysis of modification of biomolecules in a label-free manner.