Optimization of immunolabeled plasmonic nanoparticles for cell surface receptor analysis

Noble metal nanoparticles hold great potential as optical contrast agents due to a unique feature, known as the plasmon resonance, which produces enhanced scattering and absorption at specific frequencies. The plasmon resonance also provides a spectral tunability that is not often found in organic fluorophores or other labeling methods. The ability to functionalize these nanoparticles with antibodies has led to their development as contrast agents for molecular optical imaging. In this review article, we present methods for optimizing the spectral agility of these labels. We discuss synthesis of gold nanorods, a plasmonic nanoparticle in which the plasmonic resonance can be tuned during synthesis to provide imaging within the spectral window commonly utilized in biomedical applications. We describe recent advances in our group to functionalize gold and silver nanoparticles using distinct antibodies, including EGFR, HER-2 and IGF-1, selected for their relevance to tumor imaging. Finally, we present characterization of these nanoparticle labels to verify their spectral properties and molecular specificity.     

Biomedical applications of multifunctional gold-based nanocomposites

Active application of gold nanoparticles for various diagnostic and therapeutic purposes started in recent decades due to the emergence of new data on their unique optical and physicochemical properties. In addition to colloidal gold conjugates, growth in the number of publications devoted to the synthesis and application of multifunctional nanocomposites has occurred in recent years. This review considers the application in biomedicine of multifunctional nanoparticles that can be produced in three different ways. The first method involves design of composite nanostructures with various components intended for either diagnostic or therapeutic functions. The second approach uses new bioconjugation techniques that allow functionalization of gold nanoparticles with various molecules, thus combining diagnostic and therapeutic functions in one medical procedure. Finally, the third method for production of multifunctional nanoparticles combines the first two approaches, in which a composite nanoparticle is additionally functionalized by molecules having different properties.     

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.     

Affibody-attached hyperbranched conjugated polyelectrolyte for targeted fluorescence imaging of HER2-positive cancer cell

A hyperbranched conjugated polyelectrolyte (HCPE) with a core-shell structure is designed and synthesized via alkyne polycyclotrimerization and click chemistry. The HCPE has an emission maximum at 565 nm with a quantum yield of 12% and a large Stokes shift of 143 nm in water. By virtue of its poly(ethylene glycol) shell, this polymer naturally forms spherical nanoparticles that minimize nonspecific interaction with biomolecules in aqueous solution, consequently allowing for efficient bioconjugation with anti-HER2 affibody via carbodiimide-activated coupling reaction. The resulting affibody-attached HCPE can be utilized as a reliable fluorescent probe for targeted cellular imaging of HER2-overexpressed cancer cells such as SKBR-3. Considering its low cytotoxicity and good photostability, the HCPE nanoprobe holds great promise in practical imaging tasks. This study also provides a molecular engineering strategy to overcome the intrinsic limitations of traditional fluorescent polymers (e.g., chromophore-tethered polymers and linear conjugated polyelectrolytes) for bioconjugation and applications.     

Enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides

Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide–nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide–nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3′-thioctic acid modified oligonucleotides by treatment with an N-hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide–nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold.     

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture and detection of circulating tumor cells (CTCs), and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.     

Aptamer-targeted gold nanoparticles as molecular-specific contrast agents for reflectance imaging

Targeted metallic nanoparticles have shown potential as a platform for development of molecular-specific contrast agents. Aptamers have recently been demonstrated as ideal candidates for molecular targeting applications. In this study, we investigated the development of aptamer-based gold nanoparticles as contrast agents, using aptamers as targeting agents and gold nanoparticles as imaging agents. We devised a novel conjugation approach using an extended aptamer design where the extension is complementary to an oligonucleotide sequence attached to the surface of the gold nanoparticles. The chemical and optical properties of the aptamer-gold conjugates were characterized using size measurements and oligonucleotide quantitation assays. We demonstrate this conjugation approach to create a contrast agent designed for detection of prostate-specific membrane antigen (PSMA), obtaining reflectance images of PSMA(+) and PSMA(-) cell lines treated with the anti-PSMA aptamer-gold conjugates. This design strategy can easily be modified to incorporate multifunctional agents as part of a multimodal platform for reflectance imaging applications.     

Targeting of ovarian cancer cell through functionalized gold nanoparticles by novel glypican-3- binding peptide as a ultrasound contrast agents

Gold nanoparticles (AuNPs) are widely studied nanomaterials for their potential employment in advanced biomedical applications, such as selective molecular imaging and targeted drug delivery. AuNPs are generally low cost and highly biocompatible, can be easily functionalized with a wide variety of functional ligands, and have been demonstrated to be effective in enhancing ultrasound contrast at clinical diagnostic frequencies. Therefore, AuNPs might be used as contrast agents in echographic imaging. In this work, we have developed a AuNPs -based system for the in vitro molecular imaging of ovarian carcinoma cells that express high levels of glypican-3 protein (GPC-3) on their surface. In this regard, a novel GPC-3 targeting peptide was designed and conjugated to fluorescent AuNPs nanoparticles. The physicochemical properties, acoustic behavior, and biocompatibility profile of the functionalized AuNPs were characterized. Then, the binding and uptake of both naked and functionalized AuNPs were analyzed by laser scanning confocal microscopy in human HeLa cells (ovarian carcinoma) cell line. The results obtained showed that GPC-3-functionalized fluorescent AuNPs significantly enhanced the ultrasound contrast and were effectively bound and taken up by HeLa cells without affecting their viability.     

Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber

Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-μg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ～100 pg in a sample volume of ～10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases.     

Surface functionalization of gold nanoparticles using hetero-bifunctional poly(ethylene glycol) spacer for intracellular tracking and delivery

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site.     

Bioconjugated nanoparticle detection of respiratory syncytial virus infection

The integration of nanotechnology with biology has produced major advances in molecular diagnostics, therapeutics, and bioengineering. Recent advances have led to the development of functionalized nanoparticles (NPs) that are covalently linked to biological molecules such as antibodies, peptides, proteins, and nucleic acids. These functionalized NPs allow for development of novel diagnostic tools and methods, particularly for pathogens, as rapid and sensitive diagnostics are essential for defining the emergence of infection, determining the period that preventive measures should be applied, for evaluating drug and vaccine efficacy, and for controlling epidemics. In this study, we show that functionalized NPs conjugated to monoclonal antibodies can be used to rapidly and specifically detect respiratory syncytial virus in vitro and in vivo. These results suggest that functionalized NPs can provide direct, rapid, and sensitive detection of viruses and thereby bridge the gap between current cumbersome virus detection assays and the burgeoning need for more rapid and sensitive detection of viral agents.     

Peptide‐mediated cell penetration and targeted delivery of gold nanoparticles into lysosomes

There is considerable interest in the sub‐cellular targeting and delivery of biomolecules, therapeutic and imaging agents, and nanoparticles and nanoparticle conjugates into organelles for therapeutic and imaging purposes. To date, a number of studies have used sorting peptides for targeted delivery of cargo into different cell organelles but not into lysosomes. In this study, the delivery of 13‐nm gold nanoparticles across the cell membrane followed by targeted localisation into the lysosomes of a mammalian cell line was examined using novel combinations of cell‐penetrating peptides and lysosomal sorting peptides conjugated to the nanoparticles. Using a combination of fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy techniques, we show that these nanoconjugates were efficiently and selectively delivered into the lysosomes with minimal cytotoxic effects. This novel targeted delivery system may underpin the development of a new strategy for the treatment of lysosomal storage diseases by exploiting the large surface area of nanoparticles to deliver drugs or replacement enzymes directly to the lysosomes. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Simultaneous detection of multiple targets for ultrastructural immunocytochemistry

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.     

Directional conjugation of antibodies to nanoparticles for synthesis of multiplexed optical contrast agents with both delivery and targeting moieties

Molecular optical imaging has shown promise in visualizing molecular biomarkers with subcellular resolution both noninvasively and in real-time. Here, we use gold nanoparticles as optical probes to provide meaningful signal in the presence of targeted biomarkers. We present a novel conjugation technique to control the binding orientation of antibodies on the surface of gold nanoparticles to maximize antibody functionality. Briefly, a heterobifunctional linker, hydrazide-polyethylene glycol-dithiol, is used to directionally attach the Fc, or nonbinding region of the antibody, to the gold nanoparticle surface. The conjugation strategy allows for multiplexing various glycosylated antibodies on a single nanoparticle. We present a method to prepare multifunctional nanoparticles by incorporating targeting and delivery moieties on the same nanoparticle that addresses the challenge of imaging intracellular biomarkers. The time estimate for the entire protocol is approximately 6 h.     

金纳米微粒辅助细胞激光热作用疗法研究

金纳米微粒对可见光的强吸收特性使得光能可以高效地转换为热能.由于金纳米微粒的尺度在几十纳米范围,并且很容易与其他生物体结合,因此可以在局部范围进行激光选择性加热,这非常适合作为分子或细胞的靶向.采用这种金纳米微粒辅助激光热作用方法,对牛肠碱性磷酸酯酶(alkaline phosphatase aP)的选择性破坏,细胞膜的通透性提高以及对细胞的选择性灭活进行了试验并得到了很好的结果.此外,还讨论了用这种方法进行基因转染以及选择性光热治疗一些疾病的可能性.     

Label-free electrochemical immunosensor for the determination of fetoprotein based on core-shell-shell nanocomposite particles

A new approach toward the development of advanced immunosensors based on chemically functionalized core-shell-shell magnetic nanocomposite particles, and the preparation, characteristics, and measurement of relevant properties of the immunosensor useful for the detection of alpha-1-fetoprotein (AFP) in clinical immunoassays. The core-shell NiFe2O4/3-aminopropyltriethoxysilance (APTES) (NiFe2O4@APTES) was initially prepared by covalent conjugation, then gold nanoparticles were adsorbed onto the surface of NiFe2O4@APTES, and then anti-AFP molecules were conjugated on the gold nanoparticles. The core-shell-shell nanocomposite particles not only had the properties of magnetic nanoparticles, but also provided a good biocompatibility for the immobilization of biomolecules. The core-shell-shell nanostructure present good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. The analytical performance of the immunosensor was investigated by using an electrochemical method. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of AFP, and exhibits wide linear range from 0.9 to 110 ng/mL AFP with a detection limit of 0.5 ng/mL. Moreover, the proposed immunosensors were used to analyze AFP in human serum specimens. Analytical results, obtained for the clinical serum specimen by the developed immunosensor, were in accordance with those assayed by the standard ELISA. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

抗人精子蛋白17磁性纳米探针靶向的卵巢癌体内外磁共振成像

用MRI(magnetic resonance imaging)技术探索连接抗人精子蛋白17单克隆抗体(anti-Sp17 mAb)的磁性纳米探针对体外培养及动物体内Sp17+卵巢癌的靶向性。将anti-Sp17mAb连接到表面包覆壳聚糖的超顺磁性氧化铁纳米颗粒上,制成磁性纳米探针anti-Sp17-MNP,用作MRI阴性对比剂。将磁性纳米探针与Sp17+和Sp17-培养的肿瘤细胞共育,进行一系列体外磁共振成像实验。荷瘤小鼠尾静脉注射磁性纳米颗粒,用7T磁共振仪在体成像,观察肿瘤部位的信号变化,并用普鲁士蓝染色肿瘤组织切片,观察有无铁粒子聚集。体外MRI数据显示,anti-Sp17-MNP与细胞靶向结合,并与细胞共育2 h后,Sp17+HO-8910的T2*信号强度比Sp17-HepG2低2倍;anti-Sp17-MNP对肿瘤细胞的靶向作用可被重组人Sp17阻断。7T磁共振仪对动物在体肿瘤成像结果显示,感兴趣区因磁性纳米探针靶向聚集而导致信号降低,并经组织切片普鲁士蓝染色证实。本研究结果表明,用anti-Sp17抗体和新的合成路线制备的纳米探针具有用作MR对比剂进行分子成像的潜能。     

Immobilization of Candida bombicola cells on free-standing organic-gold nanoparticle membranes and their use as enzyme sources in biotransformations

Preparation of chemically functionalized biocompatible surfaces is of current interest, with application in the immobilization of various bioactive species such as DNA, enzymes, whole cells, etc. We report herein the one-step synthesis of a self-supporting gold nanoparticle membrane, its surface modification, and application in the immobilization of Candida bombicola (yeast) cells. The gold nanoparticle membrane is prepared by the spontaneous reduction of aqueous chloroaurate ions by a diamine at a liquid-liquid interface. The gold nanoparticles in the polymeric membrane may be capped with octadecylamine (ODA) molecules, thereby rendering the nanoparticle membrane hydrophobic. Exposure of the hydrophobized organic-gold nanoparticle membrane to C. bombicola yeast cells results in their binding to the membrane, possibly through nonspecific interactions such as hydrophobic interactions between the yeast cell walls and the ODA molecules. The enzyme cytochrome P450 present in the yeast cells immobilized on the organic-gold nanoparticle membrane was then used in the transformation of the arachidonic acid (AA) to sophorolipids followed by acid hydrolysis to form 20-hydroxyeicosatetraneoic acid (20-HETE). The organic-gold nanoparticle membrane-C. bombicola bioconjugate could be easily separated from the reaction medium and reused a number of times.     

Investigation of Gold and Silver Nanoparticles on Absorption Heating and Scattering Imaging

Efficient conversion of absorbed light to heat energy and strong scattering by gold and silver nanoparticles suggest these nanoparticles as the agents of heating and imaging. Absorption efficiency and scattering efficiency of gold and silver nanoparticles were studied through numerical simulation using the discrete dipole approximation method. This study shows that the size of gold and silver nanoparticles can effect gold and silver nanoparticles’ absorption efficiency and scattering efficiency. The gold nanoparticle is found to possess the maximum absorption efficiency when the size of gold nanoparticle is 50 nm and the incident wavelength is 540 nm, and the increasing scattering efficiency with the increasing size of gold nanoparticle in the medium, and refractive index of the medium is around 1.33. However, the silver nanoparticle owns the maximum absorption efficiency when the size of silver nanoparticle is 20 nm and the incident wavelength is 396 nm, and the maximum scattering efficiency when the size of silver nanoparticle is 30 nm and the incident wavelength is 410 nm in the same medium. The conditions for achieving the maximum adsorption efficiency and scattering efficiency of gold and silver nanoparticle can be used for heating and imaging using visible and near-infrared light.     

Dendrimer-based macromolecular MRI contrast agents: characteristics and application

Numerous macromolecular MRI contrast agents prepared employing relatively simple chemistry may be readily available that can provide sufficient enhancement for multiple applications. These agents operate using a approximately 100-fold lower concentration of gadolinium ions in comparison to the necessary concentration of iodine employed in CT imaging. Herein, we describe some of the general potential directions of macromolecular MRI contrast agents using our recently reported families of dendrimer-based agents as examples. Changes in molecular size altered the route of excretion. Smaller-sized contrast agents less than 60 kDa molecular weight were excreted through the kidney resulting in these agents being potentially suitable as functional renal contrast agents. Hydrophilic and larger-sized contrast agents were found better suited for use as blood pool contrast agents. Hydrophobic variants formed with polypropylenimine diaminobutane dendrimer cores created liver contrast agents. Larger hydrophilic agents are useful for lymphatic imaging. Finally, contrast agents conjugated with either monoclonal antibodies or with avidin are able to function as tumor-specific contrast agents, which also might be employed as therapeutic drugs for either gadolinium neutron capture therapy or in conjunction with radioimmunotherapy.