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.     

Bifunctional magnetic silica nanoparticles for highly efficient human stem cell labeling

A superparamagnetic iron oxide (SPIO) nanoparticle is emerging as an ideal probe for noninvasive cell tracking. However, its low intracellular labeling efficiency has limited the potential usage and has evoked great interest in developing new labeling strategies. We have developed fluorescein isothiocyanate (FITC)-incorporated silica-coated core-shell SPIO nanoparticles, SPIO@SiO2(FITC), with diameters of 50 nm, as a bifunctionally magnetic vector that can efficiently label human mesenchymal stem cells (hMSCs), via clathrin- and actin-dependent endocytosis with subsequent intracellular localization in late endosomes/lysosomes. The uptake process displays a time- and dose-dependent behavior. In our system, SPIO@SiO2(FITC) nanoparticles induce sufficient cell MRI contrast at an incubation dosage as low as 0.5 microg of iron/mL of culture medium with 1.2x105 hMSCs, and the in vitro detection threshold of cell number is about 1x104 cells. Furthermore, 1.2x105 labeled cells can also be MRI-detected in a subcutaneous model in vivo. Labeled hMSCs are unaffected in their viability, proliferation, and differentiation capacities into adipocytes and osteocytes which can still be readily MRI detected. This is the first report that hMSCs can be efficiently labeled with MRI contrast nanoparticles and can be monitored in vitro and in vivo with a clinical 1.5-T MRI imager under low incubation concentration of iron oxide, short incubation time, and low detection cell numbers at the same time.     

Multiple emulsion microbubbles for ultrasound imaging

In order to improve the sensitivity of ultrasound imaging, the contrast agents, a powerful non-invasive and real-time medical imaging technique, are used. However, air or N₂ or perfluorocarbon only encapsulated microbubbles which are currently used have lower efficiency and short imaging time. So the novel contrast agents with a higher efficiency are required. To achieve this objective, the strategy that we have explored involves the use of superparamagnetic iron oxide (SPIO) Fe₃O₄ nanoparticles multilayer emulsion microbubbles. This multilayer structure consists of three layers. The core is poly-d, l-lactide (PLA) encapsulated N₂ nanobubble with the SPIO nanoparticles forming oil-in-water (W/O) layer. The outermost is water-in-oil-in-water ((W/O)/W) emulsion layer with PVA solution. Herein we describe the synthesis and characterization of ultrasound imaging microstructure with an overall diameter of around 2μm-8μm. On the one hand, the stable gas encapsulated microstructure can provide a high scattering intensity resulting in high echogenicity, On the other hand, SPIO nanoparticles have shown the potential of high-resolution sonography. So the multiple emulsion microbubbles with SPIO can have double action to enhance the ultrasound imaging. Besides, because SPIO can also serve as magnetic resonance imaging (MRI) contrast agents, such microstructure may be useful for multimodality imaging studies in ultrasound imaging and MRI.     

DNA‐Assembled Core‐Satellite Upconverting‐Metal–Organic Framework Nanoparticle Superstructures for Efficient Photodynamic Therapy

DNA‐mediated assembly of core–satellite structures composed of Zr(IV)‐based porphyrinic metal‐organic framework (MOF) and NaYF₄,Yb,Er upconverting nanoparticles (UCNPs) for photodynamic therapy (PDT) is reported. MOF NPs generate singlet oxygen (¹O₂) upon photoirradiation with visible light without the need for additional small molecule, diffusional photosensitizers such as porphyrins. Using DNA as a templating agent, well‐defined MOF–UCNP clusters are produced where UCNPs are spatially organized around a centrally located MOF NP. Under NIR irradiation, visible light emitted from the UCNPs is absorbed by the core MOF NP to produce ¹O₂ at significantly greater amounts than what can be produced from simply mixing UCNPs and MOF NPs. The MOF–UCNP core–satellite superstructures also induce strong cell cytotoxicity against cancer cells, which are further enhanced by attaching epidermal growth factor receptor targeting affibodies to the PDT clusters, highlighting their promise as theranostic photodynamic agents.     

Advances in targeted nanotherapeutics: From bioconjugation to biomimicry

Since the emergence of cancer nanomedicine, researchers have had intense interest in developing nanoparticles (NPs) that can specifically target diseased sites while avoiding healthy tissue to mitigate the off-target effects seen with conventional treatments like chemotherapy. Initial endeavors focused on the bioconjugation of targeting agents to NPs, and more recently, researchers have begun to develop biomimetic NP platforms that can avoid immune recognition to maximally accumulate in tumors. In this review, we describe the advantages and limitations of each of these targeting strategies. First, we review developments in bioconjugation strategies, where NPs are coated with biomolecules such as antibodies, aptamers, peptides, and small molecules to enable cell-specific binding. While bioconjugated NPs offer many exciting features and have improved pharmacokinetics and biodistribution relative to unmodified NPs, they are still recognized by the body as “foreign”, resulting in their clearance by the mononuclear phagocytic system (MPS). To overcome this limitation, researchers have recently begun to investigate biomimetic approaches that can hide NPs from immune recognition and reduce clearance by the MPS. These biomimetic NPs fall into two distinct categories: synthetic NPs that present naturally occurring structures, and NPs that are completely disguised by natural structures. Overall, bioconjugated and biomimetic NPs have substantial potential to improve upon conventional treatments by reducing off-target effects through site-specific delivery, and they show great promise for future standards of care. Here, we provide a summary of each strategy, discuss considerations for their design moving forward, and highlight their potential clinical impact on cancer therapy.     

Controlled aggregation of superparamagnetic iron oxide nanoparticles for the development of molecular magnetic resonance imaging probes

A method for synthesizing superparamagnetic iron oxide (SPIO) multi-nanoparticle aggregates as molecular magnetic resonance imaging (MRI) contrast agents is described. The approach utilizes organic acid/base interactions in the colloid to induce highly controllable nanoparticle aggregation. Monodisperse aggregates with diameters as large as 100?nm are synthesized by manipulating the interfacial surface chemistry of the SPIO nanoparticles in tetrahydrofuran solvent. Subsequent phospholipid micelle encapsulation yields micellar multi-SPIO (mmSPIO) aggregates with enhanced T(2) relaxivity (368.0?s(-1)?mmol(-1)?Fe) as compared to micellar single particle SPIO (302.0?s(-1)?mmol(-1)?Fe). mmSPIO conjugated to anti-CA125 monoclonal antibodies were incubated with ovarian carcinoma cell lines to demonstrate targeted in vitro molecular MRI, resulting in a?66% shortening in T(2) time for CA125 positive NIH:OVCAR-3 cells and a less than?3% change in T(2) time for CA125 negative SK-OV-3 cells. The controllable aggregation of mmSPIO shows potential for the development of molecular MRI contrast agents with optimal sizes for specific diagnostic imaging applications.     

Measurement of immunotargeted plasmonic nanoparticles' cellular binding: a key factor in optimizing diagnostic efficacy

In this study, we use polarized light scattering to study immunotargeted plasmonic nanoparticles which bind to live SK-BR-3 human breast carcinoma cells. Gold nanoparticles can be conjugated to various biomolecules in order to target specific molecular signatures of disease. This specific targeting provides enhanced contrast in scattering-based optical imaging techniques. While there are papers which report the number of antibodies that bind per nanoparticle, there are almost no reports of the key factor which influences diagnostic or therapeutic efficacy using nanoparticles: the number of targeted nanoparticles that bind per cell. To achieve this goal, we have developed a 'negative' method of determining the binding concentration of those antibody/nanoparticle bioconjugates which are targeted specifically to breast cancer cells. Unlike previously reported methods, we collected unbound nanoparticle bioconjugates and measured the light scattering from dilute solutions of these particles so that quantitative binding information can be obtained. By following this process, the interaction effects of adjacent bound nanoparticles on the cell membrane can be avoided simply by measuring the light scattering from the unbound nanoparticles. Specifically, using nanoshells of two different sizes, we compared the binding concentrations of anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates targeted to HER2-positive SK-BR-3 breast cancer cells. The results indicate that, for anti-HER2/nanoshell bioconjugates, there are approximately 800-1600 nanoshells bound per cell; for anti-IgG/nanoshell bioconjugates, the binding concentration is significantly lower at nearly 100 nanoshells bound per cell. These results are also supported by dark-field microscopy images of the cells labeled with anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates.     

Active Targeting Using HER‐2‐Affibody‐Conjugated Nanoparticles Enabled Sensitive and Specific Imaging of Orthotopic HER‐2 Positive Ovarian Tumors

Despite advances in cancer diagnosis and treatment, ovarian cancer remains one of the most fatal cancer types. The development of targeted nanoparticle imaging probes and therapeutics offers promising approaches for early detection and effective treatment of ovarian cancer. In this study, HER‐2 targeted magnetic iron oxide nanoparticles (IONPs) are developed by conjugating a high affinity and small size HER‐2 affibody that is labeled with a unique near infrared dye (NIR‐830) to the nanoparticles. Using a clinically relevant orthotopic human ovarian tumor xenograft model, it is shown that HER‐2 targeted IONPs are selectively delivered into both primary and disseminated ovarian tumors, enabling non‐invasive optical and MR imaging of the tumors as small as 1 mm in the peritoneal cavity. It is determined that HER‐2 targeted delivery of the IONPs is essential for specific and sensitive imaging of the HER‐2 positive tumor since we are unable to detect the imaging signal in the tumors following systemic delivery of non‐targeted IONPs into the mice bearing HER‐2 positive SKOV3 tumors. Furthermore, imaging signals and the IONPs are not detected in HER‐2 low expressing OVCAR3 tumors after systemic delivery of HER‐2 targeted‐IONPs. Since HER‐2 is expressed in a high percentage of ovarian cancers, the HER‐2 targeted dual imaging modality IONPs have potential for the development of novel targeted imaging and therapeutic nanoparticles for ovarian cancer detection, targeted drug delivery, and image‐guided therapy and surgery.     

Mesoporous silica nanoparticles as a delivery system of gadolinium for effective human stem cell tracking

The progress of using gadolinium (Gd)-based nanoparticles in cellular tracking lags behind that of superparamagnetic iron oxide (SPIO) nanoparticles in magnetic resonance imaging (MRI). Here, dual functional Gd-fluorescein isothiocyanate mesoporous silica nanoparticles (Gd-Dye@MSN) that possess green fluorescence and paramagnetism are developed in order to evaluate their potential as effective T1-enhancing trackers for human mesenchymal stem cells (hMSCs). hMSCs are labeled efficiently with Gd-Dye@MSN via endocytosis. Labeled hMSCs are unaffected in their viability, proliferation, and differentiation capacities into adipocytes, osteocytes, and chondrocytes, which can still be readily MRI detected. Imaging, with a clinical 1.5-T MRI system and a low incubation dosage of Gd, low detection cell numbers, and short incubation times is demonstrated on both loaded cells and hMSC-injected mouse brains. This study shows that the advantages of biocompatibility, durability, high internalizing efficiency, and pore architecture make MSNs an ideal vector of T1-agent for stem-cell tracking with MRI.     

Multifunctional nanoparticles, nanocages and degradable polymers as a potential novel generation of non-invasive molecular and cellular imaging systems

In recent years, polymeric scaffolds have been used in several biomedical applications for delivery of drugs or other biologically relevant molecules. Polymeric nanostructures display different (and in some cases more powerful) properties respect to bulk materials. This, lead academic researchers and industry to cooperate in developing pioneering nanostructured materials for industrial and biomedical applications. Moreover, the preparation and use of systems with multiple (multifunctional) properties (i.e., bioconjugation with superparamagnetic, fluorescent or targeting molecules) is positioned to become a viable and innovative tool for application in several clinical fields. Other nanostructured systems like nanocages and degradable nanoparticles, are emerging as potential innovative systems that could be exploited as multifunctional delivery vectors. This brief critical review is aimed at collecting and discussing some recent patents dealing with the preparation and use of multifunctional nanoparticles, nanocages and degradable nanoparticles in biomedicine and non-invasive bioimaging applications. Perspectives for a potential use of these multifunctional nanosystems in pediatries have been also discussed.     

Inorganic Nanoparticles for MRI Contrast Agents

Various inorganic nanoparticles have been used as magnetic resonance imaging (MRI) contrast agents due to their unique properties, such as large surface area and efficient contrasting effect. Since the first use of superparamagnetic iron oxide (SPIO) as a liver contrast agent, nanoparticulate MRI contrast agents have attracted a lot of attention. Magnetic iron oxide nanoparticles have been extensively used as MRI contrast agents due to their ability to shorten T2* relaxation times in the liver, spleen, and bone marrow. More recently, uniform ferrite nanoparticles with high crystallinity have been successfully employed as new T2 MRI contrast agents with improved relaxation properties. Iron oxide nanoparticles functionalized with targeting agents have been used for targeted imaging via the site‐specific accumulation of nanoparticles at the targets of interest. Recently, extensive research has been conducted to develop nanoparticle‐based T1 contrast agents to overcome the drawbacks of iron oxide nanoparticle‐based negative T2 contrast agents. In this report, we summarize the recent progress in inorganic nanoparticle‐based MRI contrast agents.     

Magnetic resonance imaging probes for labeling of chondrocyte cells

Recent progress in cell therapy research has raised the need for non-invasive monitoring of transplanted cells. Magnetic resonance imaging (MRI) of superparamagnetic iron oxide (SPIO) labeled cells have been widely used for high resolution monitoring of the biodistribution of cells after transplantation. Here we report that self-assembly of amphiphilic polyethylenimine (PEI)/SPIO nanocomposites can lead to the formation of ultrasensitive MRI probes, which can be used to label chondrocyte cells with good biocompatibility. The labeled cells display strong signal contrast compared to unlabeled ones in a clinical MRI scanner. This probe may be useful for noninvasive MR tracking of implanted cells for tissue regeneration.     

Molecular Imaging Using Nanoparticle Quenchers of Cerenkov Luminescence

Cerenkov luminescence (CL) imaging is an emerging technique that collects the visible photons produced by radioisotopes. Here, molecular imaging strategies are investigated that switch the CL signal off. The noninvasive molecularly specific detection of cancer is demonstrated utilizing a combination of clinically approved agents, and their analogues. CL is modulated in vitro in a dose dependent manner using approved small molecules (Lymphazurin), as well as the clinically approved Feraheme and other preclinical superparamagnetic iron oxide nanoparticles (SPIO). To evaluate the quenching of CL in vivo, two strategies are pursued. [¹⁸F]‐FDG is imaged by PET and CL in tumors prior to and following accumulation of nanoparticles. Initially, non‐targeted particles are administered to mice bearing tumors in order to attenuate CL. For targeted imaging, a dual tumor model (expressing the human somatostatin receptor subtype‐2 (hSSTr2) and a control negative cell line) is used. Targeting hSSTr2 with octreotate‐conjugated SPIO, quenched CL enabling non‐invasive distinction between tumors' molecular expression profiles is demonstrated. In this work, the quenching of Cerenkov emissions is demonstrated in several proof of principle models using a combination of approved agents and nanoparticle platforms to provide disease relevant information including tumor vascularity and specific antigen expression.     

Detection of magnetic nanoparticles in tissue using magneto-motive ultrasound

The purpose of this study was to demonstrate the magneto-motive ultrasonic detection of superparamagnetic iron oxide (SPIO) nanoparticles as a marker of macrophage recruitment in tissue. The capability of ultrasound to detect SPIO nanoparticles (core diameter ～20?nm) taken up by murine liver macrophages was investigated. Eight mice were sacrificed two days after the intravenous administration of four SPIO doses (1.5, 1.0, 0.5, and 0.1?mmol Fe/kg body weight). In the iron-laden livers, ultrasound Doppler measurements showed a frequency shift in response to an applied time-varying magnetic field. M-mode scan and colour power Doppler images of the iron-laden livers also demonstrated nanoparticle movement under focused magnetic field excitation. In the livers of two saline injected control mice, no movement was observed using any ultrasound imaging modes. The results of our experiments indicate that ultrasound imaging of magneto-motive excitation is a candidate imaging modality to identify tissue-based macrophages containing SPIO nanoparticles.     

M2 macrophage-targeted iron oxide nanoparticles for magnetic resonance image-guided magnetic hyperthermia therapy

Tumor-associated macrophages (TAMs) play an important role in tumor development and progression.In particular,M2 TAMs can promote tumor growth by facilitating tumor progression and malignant behav-iors.Selectively targeted elimination of M2 TAMs to inhibit tumor progression is of great significance for cancer treatment.Iron oxide nanoparticles based magnetic hyperthermia therapy (MHT) is a classical approach to destroy tumor tissue with deep penetration depth.In this study,we developed a typical M2 macrophage-targeted peptide (M2pep) functionalized superparamagnetic iron oxide nanoparticle(SPIO) for magnetic resonance imaging (MRI)-guided MHT in an orthotopic breast cancer mouse model,The obtained multifunctional SPIO-M2pep with a hydrodynamic diameter of 20 nm showed efficient targeting capability,high transverse relaxivity (149 mM-1 s-1) and satisfactory magnetic hyperthermia performance in vitro.In vivo studies demonstrated that the SPIO-M2pep based MRI can monitor the distri-bution of nanoparticles in tumor and indicate the suitable timing for MHT.The M2 macrophage-targeted MHT significantly reduced the tumor volume and the population of pro-tumoral M2 TAMs in tumor.In addition,the SPIO-M2pep based MHT can remodel the tumor immune microenvironment (TIME).The multifunctional SPIO-M2pep with M2 macrophage-targeting ability,high magnetic hyperthermia effi-ciency,MR imaging capability and effective role in remodeling the TIME hold great potential to improve clinical cancer therapy outcomes.     

In Situ Coupling of CoP Polyhedrons and Carbon Nanotubes as Highly Efficient Hydrogen Evolution Reaction Electrocatalyst

Hydrogen evolution reaction (HER) from water electrolysis is an attractive technique developed in recent years for cost‐effective clean energy. Although considerable efforts have been paid to create efficient catalysts for HER, the development of an affordable HER catalyst with superior performance under mild conditions is still highly desired. In this work, metal–organic frameworks (MOFs)‐templated strategy is proposed for in situ coupling of cobalt phosphide (CoP) polyhedrons nanoparticles and carbon nanotubes (CNTs). Due to the synergistic catalytic effect between CoP polyhedrons and CNTs, the as‐prepared CoP–CNTs hybrids show excellent HER performance. The resultant CoP–CNTs demonstrate excellent HER activity in 0.5 m H₂SO₄ with Tafel slope of 52 mV dec^?1, small onset overpotential of ≈64 mV, and a low overpotential of ≈139 mV at 10 mA cm^?2. Additionally, the catalyst also manifests superior durability in acid media. Considering the structure diversity of MOFs, the strategy presented here can be extended for synthesizing other well‐defined metal phosphides–CNTs hybrids, which may be used in the fields of catalysis, energy conversion and storage.     

Dual-luminophore-doped silica nanoparticles for multiplexed signaling

We have synthesized dual-luminophore-doped silica nanoparticles for multiplexed signaling in bioanalysis. Two luminophores, Tris(2,2'-bipyridyl)osmium(II)bis(hexafluorophosphate) (OsBpy) and Tris(2,2'-bipyridyl)dichlororuthenium(II)hexahydrate (RuBpy), were simultaneously entrapped inside silica nanoparticles at precisely controlled ratios, with desirable sizes and required surface functionality. Single-wavelength excitation with dual emission endows the nanoparticles with optical encoding capability for rapid and high-throughput multiplexed detection. The nanoparticles can be prepared with sizes ranging from a few nanometers to a few hundred nanometers, with specific ratios of luminescence intensities at two well-resolved wavelengths and with excellent reproducibility. These nanoparticles also possess unique properties of high signal amplification, excellent photostability, and easy surface bioconjugation for highly sensitive measurements when used as signaling markers. A simplified ligand binding system using avidin-biotin and an application extension to immunoassays have been explored, demonstrating the potential use of these easily obtainable bioconjugated nanoparticles for multiplexed signaling and bioassays.     

Detection of bioconjugated quantum dots passivated with different ligands for bio-applications

Bioconjugation of quantum dots has resulted in a significant increase in resolution of biological fluorescent labeling. This intrinsic property of quantum dots can be utilized for sensitive detection of target analytes with high sensitivity; including pathogenic bacteria and cancer monitoring. The quantum dots and quantum dot doped silica nanoparticles exhibit prominent emission peaks when excited at 400 nm but on conjugation to model rabbit antigoat antibodies exhibit diminished intensity of emission peak at 600 nm. It shows that photoluminescence intensity of conjugated quantum dots and quantum dot doped silica nanoparticles could permit the detection of bioconjugation. Samples of conjugated and unconjugated quantum dots and quantum dot doped silica nanoparticles were subjected to enzyme linked immunosorbent assay for further confirmation of bioconjugation. In the present study ligand exchange, bioconjugation, fluorescence detection of bioconjugated quantum dots and quantum dot doped silica nanoparticles and further confirmation of bioconjugation by enzyme linked immunosorbent assay has been described.     

Enhanced electrocatalytic activity of Co@N-doped carbon nanotubes by ultrasmall defect-rich TiO<Subscript>2</Subscript> nanoparticles for hydrogen evolution reaction

Despite being technically possible,splitting water to generate hydrogen is practically unfeasible,mainly because of the lack of sustainable and efficient earth-abundant catalysts for the hydrogen-evolution reaction (HER).Herein,we report a durable and highly active electrochemical HER catalyst based on defect-rich TiO2 nanoparticles loaded on Co nanoparticles@N-doped carbon nanotubes (D-TiO2/Co@NCT) synthesized by electrostatic spinning and a subsequent calcining process.The ultrasmall TiO2 nanoparticles are 1.5-2 nm in size and have a defect-rich structure of oxygen vacancies.D-TiO2/Co@NCT exhibits excellent HER catalytic activity in an acidic electrolyte (0.5 M H2SO4),with a low onset potential of-57.5 mV (1 mA·cm-2),a small Tafel slope of 73.5 mV-dec-1,and extraordinary long-term durability.X-ray photoelectron spectroscopy,electron paramagnetic resonance spectroscopy,and theoretical calculations confirm that the Ti3+ defect-rich structure can effectively regulate the catalytic activity for electrochemical water splitting.     

Gram-scale synthesis and biofunctionalization of silica-coated silver nanoparticles for fast colorimetric DNA detection

A direct silica-coating method has been developed for the gram-scale synthesis of well-dispersed Ag@SiO(2) nanoparticles. Subsequent surface functionalization via the well-established silica surface chemistry provided arching points for straightforward bioconjugation with amino-terminated oligonucleotides. Fast hybridization kinetics of the resulting robust oligo-modified Ag@SiO(2) nanoprobes with complementary target oligonucleotides render themselves very useful for the fast colorimetric DNA detection based on the sequence-specific hybridization properties of DNA. Additionally, the reliable protocols developed in this study for preparing and functionalizing Ag@SiO(2) nanoparticles can be readily extended to other silica-coated nanoparticles, which can also provide a specific platform for the covalent attachment of biomolecules such as amino-rich proteins, enzymes, or amino-terminated oligonucleotides for diverse bioapplications.