Integrin-targeting block copolymer probes for two-photon fluorescence bioimaging

Targeted molecular imaging with two-photon fluorescence microscopy (2PFM) is a powerful technique for chemical biology and, potentially, for noninvasive diagnosis and treatment of a number of diseases. The synthesis, photophysical studies, and bioimaging are reported for a versatile norbornene-based block copolymer multifunctional scaffold containing biocompatible (PEG), two-photon fluorescent dyes (fluorenyl) and targeting (cyclic-RGD peptide) moieties. The two bioconjugates, containing two different fluorenyl dyes and cRGDfK covalently attached to the polymer probe, formed a spherical micelle and self-assembled structure in water, for which size was analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cell viability and 2PFM imaging of human epithelial U87MG cell lines that overexpress α(v)β(3) integrin was performed via incubation with the new probes, along with negative control studies using MCF-7 breast cancer cells and blocking experiments. 2PFM microscopy confirmed the high selectivity of the biocompatible probe in the integrin-rich area in the U87MF cells while blocking as well as negative control MCF-7 experiments confirmed the integrin-targeting ability of the new probes.     

Dual-mode probe based on mesoporous silica coated gold nanorods for targeting cancer cells

A dual-mode imaging probe for targeting cancer cells has been fabricated based on mesoporous silica coated gold nanorods (MS-GNRs) for the first time. In this probe, fluorescence and surface enhanced Raman scattering (SERS) signals can be generated independently by using different excitation wavelengths. To investigate the targeting performance of the probe, folic acid (FA) is conjugated on the outer surfaces of MS-GNRs as a targeting ligand and HeLa cells were used as model cancer cells because they overexpress folate receptors (FRs). The endocytosis mechanism was verified by competing experiments with free FA through both fluorescence images and SERS mappings. Moreover, the cytotoxicity of the probe was remarkably reduced in comparison with the GNRs without the silica shell as proved by the results of MTT assay. Compared with traditional imaging probes, this new type of nanoprobe has great potential for multiplexed imaging in living cells, which can be easily realized by using fluorescence and SERS signals.     

Artificial cell membrane-covered nanoparticles embedding quantum dots as stable and highly sensitive fluorescence bioimaging probes

To obtain a stable and highly sensitive bioimaging fluorescence probe, polymer nanoparticles with embedded quantum dots were covered with an artificial cell membrane. These nanoparticles were designed by assembling phospholipid polar groups as a platform, and oligopeptide was immobilized as a bioaffinity moiety on the surface of the nanoparticles. The polymer nanoparticles showed resistance to cellular uptake from HeLa cells owing to the nature of the phosphorylcholine groups. When arginine octapeptide was immobilized at the surface of the nanoparticles, they were able to penetrate the membrane of HeLa cells effectively. Cytotoxicity of the nanoparticles was not observed even after immobilization of oligopeptide. Thus, we obtained stable fluorescent polymer nanoparticles covered with an artificial cell membrane, which are useful as an excellent bioimaging probe and as a novel evaluation tool for oligopeptide functions in the target cells.     

Vibrational two-photon microscopy for tissue imaging: Short-wave infrared surface-enhanced resonance hyper-Raman scattering

Multiphoton microscopy using short-wave infrared (SWIR) radiation offers nondestructive and high-resolution imaging through tissue. Two-photon fluorescence (TPF), for example, is commonly employed to increase the penetration depth and spatial resolution of SWIR imaging, but the broad spectral peaks limit its multiplexing capabilities. Hyper-Raman scattering, the vibrational analog of TPF, yields spectral features on the order of 20 cm^?1 and reporter-functionalized noble metal nanoparticles (NPs) provide a platform for both hyper-Raman signal enhancement and selective targeting in biological media. Herein we report the first tissue imaging study employing surface-enhanced resonance hyper-Raman scattering (SERHRS), the two-photon analog of surface-enhanced resonance Raman scattering. Specifically, we employ multicore gold-silica NPs (Au@SiO₂ NPs) functionalized with a near infrared-resonant cyanine dye, 3,3′-diethylthiatricarbocyanine iodide as a SERHRS reporter. SWIR SERHRS spectra are efficiently acquired from mouse spleen tissue. SWIR SERHRS combines two-photon imaging advantages with narrow vibrational peak widths, presenting future applications of multitargeted bioimaging.     

Deep Vascular Imaging in Wounds by Two-Photon Fluorescence Microscopy

Deep imaging within tissue (over 300 μm) at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM). The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels) targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 μm within wound (neodermis)/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D) architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.     

Two-photon fluorescence vascular bioimaging with new bioconjugate probes selective toward the vascular endothelial growth factor receptor 2

We report the synthesis and characterization of two amine reactive fluorescent dyes with efficient two-photon absorption (2PA) properties and high fluorescence quantum yields. Bioconjugation of these dyes with the DC-101 antibody proved to be useful for selectively imaging the vascular endothelial growth factor receptor 2 (VEGFR-2) in cells expressing this receptor in vitro and in "whole" mounted excised tumors (ex vivo) by two-photon fluorescence microscopy (2PFM). The penetration depths reached within the tumors by 2PFM was over 800 μm. In addition, the concentration of dye required for incubation of these bioconjugates was in the picomolar domain, the probes possessed very good photostability, and the 2PFM setup did not require any additional means of increasing the collection efficiencies of fluorescent photons to achieve the relatively deep tissue imaging that was realized, due, in large part, to the favorable photophysical properties of the new probes.     

Quantum dots bearing lectin-functionalized nanoparticles as a platform for in vivo brain imaging

Delivery of imaging agents to the brain is highly important for the diagnosis and treatment of central nervous system (CNS) diseases, as well as the elucidation of their pathophysiology. Quantum dots (QDs) provide a novel probe with unique physical, chemical, and optical properties, and become a promising tool for in vivo molecular and cellular imaging. However, their poor stability and low blood-brain barrier permeability severely limit their ability to enter into and act on their target sites in the CNS following parenteral administration. Here, we developed a QDs-based imaging platform for brain imaging by incorporating QDs into the core of poly(ethylene glycol)-poly(lactic acid) nanoparticles, which was then functionalized with wheat germ agglutinin and delivered into the brain via nasal application. The resulting nanoparticles, with high payload capacity, are water-soluble, stable, and showed excellent and safe brain targeting and imaging properties. With PEG functional terminal groups available on the nanoparticles surface, this nanoprobe allows for conjugation of various biological ligands, holding considerable potential for the development of specific imaging agents for various CNS diseases.     

High-speed, low-photodamage nonlinear imaging using passive pulse splitters

Pulsed lasers are key elements in nonlinear bioimaging techniques such as two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a percent or less of the laser power available can be delivered to the sample before photoinduced damage becomes excessive. Here we describe a passive pulse splitter that converts each laser pulse into a fixed number of sub-pulses of equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices labeled with GFP and show that, in different power regimes, the splitter can be used either to increase the signal rate more than 100-fold or to reduce the rate of photobleaching by over fourfold. In living specimens, the gains were even greater: a ninefold reduction in photobleaching during in vivo imaging of Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of photodamage during calcium imaging of rat hippocampal brain slices.     

Gold nanoparticles conjugated to [Tyr3]octreotide peptide

A multifunctional system of gold nanoparticles (AuNP) capped by the [Tyr(3)]Octreotide (TOC) peptide was prepared and characterized by transmission electron microscopy (TEM) and UV-Vis, infrared and fluorescence spectroscopy. AuNP and AuNP-TOC fluorescence emission spectra were obtained both in solution and in murine AR42J-tumor tissues. Results suggest that AuNP were functionalized with TOC through interactions with the N-terminal amine of the phenylalanine, the amide groups and possibly with the indole group of the tryptophan residue. The fluorescence analyses in tissue revealed a recognition of the AuNP-TOC conjugate for the neuroendocrine tumor because of the lower energy position of the fluorescence resonance (692 nm) with respect to that of the AuNP in the same tumoral tissue (684 nm). The emission band observed in the near-infrared region (692 nm) opens the possibility for AuNP-TOC use in bioimaging.     

Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.     

Oleyl-chitosan nanoparticles based on a dual probe for optical/MR imaging in vivo

Oleic acid-conjugated chitosan (oleyl-chitosan) is a powerful platform for encapsulating oleic acid-decorated iron oxide nanoparticles (ION), resulting in a good magnetic resonance imaging (MRI) probe. Oleyl-chitosan could self-assemble into core-shell structures in aqueous solution and provide the effective core compartment for loading ION. ION-loaded oleyl-chitosan nanoparticles showed good enhanced MRI sensitivity in a MR scanner. Cy5.5 dye was accessed to the oleyl-chitosan conjugate for near-infrared (NIR) in vivo optical imaging. After intravenous injection of ION-loaded Cy5.5-conjugated oleyl-chitosan (ION-Cy5.5-oleyl-chitosan) nanoparticles in tumor-bearing mice, both NIRF and MR imaging showed the detectable signal intensity and enhancement in tumor tissues via enhanced permeability and retention (EPR) effect. Tumor accumulation of the nanoparticles was confirmed through ex vivo fluorescence images and Prussian blue staining images in tumor tissues. It is concluded that ION-Cy5.5-oleyl-chitosan nanoparticle is highly an effective imaging probe for detecting tumor in vivo.     

Enriched Au nanoclusters with mesoporous silica nanoparticles for improved fluorescence/computed tomography dual‐modal imaging

ObjectivesAu nanoclusters (AuNCs) have been used widely in fluorescence bio‐imaging because of their good fluorescence, small particle size and non‐cytotoxicity. AuNCs are also efficient in computed tomography (CT) imaging. Hence, a dual‐modal imaging probe can be constructed without any complicated modification processes by exploiting the excellent performance of AuNCs. In the present study, AuNCs were enriched with mesoporous silica nanoparticles (MSNs) to obtain enhanced fluorescence/CT dual‐modal imaging, which was capable of acquiring more imaging information for diseases compared with single‐mode imaging.Materials and methodsBiocompatible bovine serum albumin (BSA)‐capped AuNCs were prepared and loaded into amine‐functionalized MSNs to form MSN@AuNCs. BSA‐AuNCs, MSNs, and MSN@AuNCs were characterized by ultraviolet‐visible (UV‐vis) spectra, transmission electron microscopy (TEM), fluorescence spectra, and zeta potential. CT imaging was recorded using micro‐CT scanning. Fluorescence imaging was measured using confocal laser scanning microscopy and flow cytometry.ResultsThe prepared AuNCs and MSNs possessed good properties as previously reported. The fluorescence intensity and CT value of the AuNCs were enhanced after being enriched with MSNs. The nanoparticles were both non‐cytotoxic. Confocal laser scanning microscopy and flow cytometry indicated that MSN@AuNCs in CAL‐27 cells showed improved fluorescence imaging compared with simple AuNCs at the same concentration.ConclusionsThe results revealed that the strategy of enriching AuNCs with MSNs can obtain highly sensitive fluorescence/CT dual‐modal imaging, which indicated the potential of this nanoparticle in the diagnosis and treatment of disease.     

In vivo two-photon laser-scanning microscopy of Ca2+ dynamics in visual motion-sensitive neurons

We applied two-photon laser-scanning microscopy (TPLSM) to motion-sensitive visual interneurons of the fly to study Ca(2+) dynamics in vivo at a higher spatial and temporal resolution than possible with conventional fluorescence microscopy. Based on a custom-built two-photon microscope, we performed line scans to measure changes in presynaptic Ca(2+) concentrations elicited by visual stimulation. We used a fast avalanche photodiode (APD) with a high quantum efficiency to detect even low levels of emitted fluorescence. Our experiments show that our in vivo preparation is amenable to TPLSM: with excitation intensities low enough not to cause photodamage, activity-dependent fluorescence changes of Ca(2+)-sensitive dyes can be detected in small neuronal branches. The performance of two-photon and conventional Ca(2+) imaging carried out consecutively at the same neuron is compared and it is demonstrated that two-photon imaging allows us to detect differences in Ca(2+) dynamics between individual neurites.     

Preparation and fluorescence properties of fluorophore-labeled avidin–biotin system immobilized on Fe3O4 nanoparticles through functional indolequinone linker

We prepared and characterized a new class of fluorophore-labeled magnetic nanoparticles (MNPs) possessing a hypoxia-responsive unit to construct a hypoxia-selective emission system. The indolequinone derivative as a hypoxia-response unit bearing biotin was synthesized and immobilized on Fe₃O₄ MNP. Subsequent complexation of this functionalized MNP with fluorescein-labeled avidin formed fluorophore-labeled nanoparticles (AF-QB@MNP). The fluorescence intensity of AF-QB@MNP was suppressed because of the adjacent quenching function of the indolequinone moiety and MNP. Upon hypoxic treatment by NADPH:cytochrome P450 reductase, AF-QB@MNP was activated to liberate a fluorescence unit, leading to the significant enhancement of fluorescence emission, while a smaller enhancement in fluorescence emission occurred upon aerobic treatment. The AF-QB@MNP has a indispensable properties as a fluorescent probe for imaging of disease relevant hypoxic microenvironments.     

In vivo detection of phospholipase C by enzyme-activated near-infrared probes

In this article, the characterization of the first near-infrared (NIR) phospholipase-activated molecular beacon is reported, and its utility for in vivo cancer imaging is demonstrated. The probe consists of three elements: a phospholipid (PL) backbone to which the NIR fluorophore, pyropheophorbide a (Pyro), and the NIR Black Hole Quencher 3 (BHQ) were conjugated. Because of the close proximity of BHQ to Pyro, the Pyro-PtdEtn-BHQ probe is self-quenched until enzyme hydrolysis releases the fluorophore. The Pyro-PtdEtn-BHQ probe is highly specific to one isoform of phospholipase C, phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for catabolizing phosphatidylcholine directly to phosphocholine. Incubation of Pyro-PtdEtn-BHQ in vitro with PC-PLC demonstrated a 150-fold increase in fluorescence that could be inhibited by the specific PC-PLC inhibitor tricyclodecan-9-yl xanthogenate (D609) with an IC(50) of 34 ± 8 μM. Since elevations in phosphocholine have been consistently observed by magnetic resonance spectroscopy in a wide array of cancer cells and solid tumors, we assessed the utility of Pyro-PtdEtn-BHQ as a probe for targeted tumor imaging. Injection of Pyro-PtdEtn-BHQ into mice bearing DU145 human prostate tumor xenografts followed by in vivo NIR imaging resulted in a 4-fold increase in tumor radiance over background and a 2 fold increase in the tumor/muscle ratio. Tumor fluorescence enhancement was inhibited with the administration of D609. The ability to image PC-PLC activity in vivo provides a unique and sensitive method of monitoring one of the critical phospholipase signaling pathways activated in cancer, as well as the phospholipase activities that are altered in response to cancer treatment.     

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.     

Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy

Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack.     

Synthesis of tunable and multifunctional Ni-doped near-infrared QDs for cancer cell targeting and cellular sorting

Here, we report the facile preparation of tunable magnetic Ni-doped near-infrared (NIR) quantum dots (MNIR-QDs) as an efficient probe for targeting, imaging, and cellular sorting applications. We synthesized the MNIR-QDs via a hot colloidal synthesis approach to yield monodisperse and tunable QDs. These hydrophobic QDs were structurally and compositionally characterized and further functionalized with amino-PEG and carboxyl-PEG to improve their biocompatibility. Since QDs are known to be toxic due to the presence of cadmium, we have evaluated the in vitro and in vivo toxicity of our surface-functionalized MNIR-QDs. Our results revealed that surface-functionalized MNIR-QDs did not exhibit significant toxicity at the concentrations used in the experiments and are therefore suitable for biological applications. For further in vitro applications, we covalently linked folic acid to the surface of amino-PEG-coated MNIR-QDs through NHS chemistry to target the folate receptors largely present in the HeLa cells to demonstrate the specific targeting and magnetic behavior of these MNIR-QDs. Improved specificity has been observed with treatment of HeLa cells with the folic acid-linked amino PEG-coated MNIR QDs (FA-PEG-MNIR-QDs) compared to the one without folic acid. Since the synthesized probe has magnetic property, we have also successfully demonstrated sorting between the cells which have taken up the probe with the use of a magnet. Our findings strongly suggest that these functionalized MNIR-QDs can be a potential probe for targeting, cellular sorting, and bioimaging applications.     

Structural and optical properties of dysprosium-doped hydroxyapatite nanoparticles and the use as a bioimaging probe in human cells

In this work, the hydroxyapatite nanoparticles doped with trivalent dysprosium ions were synthesized by a co-precipitation method. The characterization techniques such as X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX) were carried out to determine the crystalline and structural properties. The Rietveld structural refinement of the XRD patterns confirmed the purity of the phase formation of the synthesized nanoparticles. The photoluminescence emission spectra exhibited intense emissions in the blue region at 450 nm and 476 nm along with less intense yellow emission at 573 nm which can be attributed to the magnetic dipole and electric dipole transitions of dysprosium respectively. In order to evaluate the colour tunability of the emitted light CIE chromaticity coordinate values were calculated. The intense blue emissions from the synthesized sample were found to be favourable for bioimaging. The images obtained from the fluorescence microscopy revealed that the dysprosium-doped hydroxyapatite nanoparticles are potential bioimaging probes in human cells.     

Biocompatibility of amorphous silica nanoparticles: Size and charge effect on vascular function, in vitro

Synthetic amorphous silica is gaining popularity as the material of choice in the fabrication of nanoparticles for use in imaging diagnostics, medical therapeutics, and tissue engineering because of its biocompatible nature. However, recent evidence suggests that silica nanoparticles (SiNPs) show a concentration- and size-dependent toxic effect that is cell specific. We investigated the direct influence of SiNP uptake on the vasodilator responses of rat aortic vessels, in vitro, using fabricated SiNPs of defined size (97 ± 7.60 and 197 ± 7.50 nm) and charge (positive and nonmodified). Dilator responses to cumulative doses of endothelial-dependent [acetylcholine (Ach); 0.01 μM-1.0 mM] and endothelial-independent (sodium nitroprusside; 0.01-10 μM) agonists were determined before and 30 Min after incubation in SiNPs (at 1.1 × 10(11) nanoparticles/mL). Acute exposure to SiNPs led to their rapid uptake by the lining endothelial cells (as verified by transmission electron microscopy). SiNP uptake had no significant influence on dilator responses, although a greater degree of attenuation was evident after uptake of the 100 nm and positively charged SiNPs (significant at the highest 1.0 mM Ach concentration between positive and nonmodified 200 nm SiNPs; P < 0.05). In summary, our findings suggest that SiNP surface interactions, rather than mass, affect vasodilator function of aortic vessels.