Aniruddha Ray

Targeted blue nanoparticles as photoacoustic contrast agent for brain tumor delineation

AbstractDistinguishing a tumor from non-neoplastic tissue is a challenging task during cancer surgery. Several attempts have been made to use visible or fluorescent agents to aid in the visualization of a tumor during surgery. We describe a novel method to delineate brain tumors, using a highly sensitive photoacoustic imaging technique that is enhanced by tumor-targeting blue nanoparticles serving as a contrast agent. Experiments on phantoms and on rat brains, ex vivo, demonstrate the high sensitivity of photoacoustic imaging in delineating tumors containing contrast agent at a concentration much lower than needed for visualization by the naked eye. The limit of detection of the system for the nanoparticles is about 0.77 μg/mL in water (equivalent to 0.84 μmol/L Coomassie Blue dye). The present exploratory study suggests that photoacoustic imaging, when used with strongly optical absorbing contrast agents, could facilitate cancer surgery intraoperatively by revealing the distribution and extent of the tumor.

Two-photon nano-PEBBLE sensors: subcellular pH measurements

Intracellular pH mapping is of great importance as it plays a critical role in many cellular events. Also, in tissue, pH mapping can be an indicator for the onset of cancer. Here we describe a biocompatible, targeted, ratiometric, fluorescent, pH sensing nano-PEBBLE (Photonic Explorer for Biomedical use with Biologically Localized Embedding) that is based on two-photon excitation. Two-photon excitation minimizes the photobleaching and cell autofluorescence drastically, leading to an increase in the signal-to-noise ratio. PEBBLE nanosensors provide a novel approach for introducing membrane impermeant dyes, like HPTS, into cells. We use both non-targeted and F3 peptide targeted PEBBLE nanosensors for intracellular pH measurement of 9L cells. The intracellular measurements suggest that the non-targeted nanosensors are mostly trapped in endosomes, whereas the F3 peptide targeting enables them to escape/avoid these acidic compartments. Combining the advantages of pH sensitive PEBBLE nanoparticles, including their specific targeting, with the advantages of two-photon microscopy provides an attractive and promising prospect for non-invasive real-time monitoring of pH inside cancer cells and tissues.

Hydrogel Nanoparticles with Thermally Controlled Drug Release

Improving the therapeutic efficacy and reducing systemic side effects of drugs is an important aspect in chemotherapy. The strategy presented here is the use of cisplatin loaded, temperature-sensitive, hydrogel nanoparticles (CisPt-NPs) and their ability to deliver and release chemodrugs selectively, based on thermal stimuli. The specially synthesized CisPt-NPs show a temperature-dependent increase of cisplatin release, at neutral pH (as in blood and normal tissue), in both the presence and absence of common metallic ions, as well as at the low pH found in lysosomes, where endocytosed NPs often localize. These CisPt-NPs were uptaken by breast cancer MDA-MB-435 cells, via endocytosis, and then mostly localized in the lysosomes. The in vitro cytotoxicity tests show that these CisPt-NPs have a significantly better efficacy at the slightly elevated temperatures. Potential applications are discussed.

Lifetime-based photoacoustic oxygen sensing in vivo

The determination of oxygen levels in blood and other tissues in vivo is critical for ensuring proper body functioning, for monitoring the status of many diseases, such as cancer, and for predicting the efficacy of therapy. Here we demonstrate, for the first time, a lifetime-based photoacoustic technique for the measurement of oxygen in vivo, using an oxygen sensitive dye, enabling real time quantification of blood oxygenation. The results from the main artery in the rat tail indicated that the lifetime of the dye, quantified by the photoacoustic technique, showed a linear relationship with the blood oxygenation levels in the targeted artery.

Polymer–protein hydrogel nanomatrix for stabilization of indocyanine green towards targeted fluorescence and photoacoustic bio-imaging

Indocyanine green (ICG) is an optical contrast agent commonly used for a variety of imaging applications. However, certain limitations of the free dye molecule, concerning its low stability, uncontrolled aggregation and lack of targeting ability, have limited its use. Presented here is a method of embedding ICG in a novel polymer/protein hybrid nanocarrier so as to overcome the above inherent drawbacks of the free molecule. The hybrid nanocarrier consists of a non-toxic and biocompatible polyacrylamide nanoparticle (PAA NP) matrix that incorporates human serum albumin (HSA). This nanocarrier was synthesized through pre-conjugation with HSA and amine functionalized monomer, followed by polymerization using biodegradable cross-linkers, in a water-in-oil emulsion. The ICG dye is loaded into the HSA conjugated PAA nanoparticles (HSA-PAA NPs) through post-loading. Compared to the PAA polymer matrix, the presence of hydrophobic pockets in the HSA-PAA NPs further increases the chemical and physical stability of ICG. This is manifested by lowering the chemical degradation rates under physiological conditions, as well as by improving the thermal- and photo-stability of the dye. A targeting moiety, F3-Cys peptide, was attached to the surface of the NPs, for selective delivery to specific cancer cell lines. The suitability of these NPs for optical imaging applications was demonstrated by performing fluorescence imaging on a rat gliosarcoma cell line (9L). We also present the photoacoustic response of the HSA-PAA NPs, used as imaging contrast agents, in the spectral window of 700 nm to 800 nm.

Two‐Photon Fluorescence Imaging Super‐Enhanced by Multishell Nanophotonic Particles, with Application to Subcellular pH

A novel nanophotonic method for enhancing the two-photon fluorescence signal of a fluorophore is presented. It utilizes the second harmonic (SH) of the exciting light generated by noble metal nanospheres in whose near-field the dye molecules are placed, to further enhance the dyes fluorescence signal in addition to the usual metal-enhanced fluorescence phenomenon. This method enables demonstration, for the first time, of two-photon fluorescence enhancement inside a biological system, namely live cells. A multishell hydrogel nanoparticle containing a silver core, a protective citrate capping, which serves also as an excitation quenching inhibitor spacer, a pH indicator dye shell, and a polyacrylamide cladding are employed. Utilizing this technique, an enhancement of up to 20 times in the two-photon fluorescence of the indicator dye is observed. Although a significant portion of the enhanced fluorescence signal is due to one-photon processes accompanying the SH generation of the exciting light, this method preserves all the advantages of infrared-excited, two-photon microscopy: enhanced penetration depth, localized excitation, low photobleaching, low autofluorescence, and low cellular damage.

Overcoming cancer multidrug resistance by codelivery of doxorubicin and verapamil with hydrogel nanoparticles.

The efficacy of chemotherapy is often inhibited by multidrug resistance (MDR). A highly engineerable hydrogel nanoparticle (NP) serves as a carrier for the optimal codelivery to tumor cells of the chemodrug, doxorubicin (Dox) and the chemosensitizer, verapamil (Vera), aiming at alleviating tumor MDR. The hydrogel NPs are prepared via the copolymerization of acrylamide and 2-carboxyethyl acrylate. Dox and Vera are post-loaded into the respective NPs, with drug loading around 7.7 wt% and 8.0 wt%, respectively. The codelivery of Dox-NPs and Vera-NPs increases the intracellular accumulation of Dox, and significantly enhances the cell killing ability of Dox with respect to NCI/ADR-RES cells in vitro. These findings suggest that such codelivery nanoplatforms provide a promising route for overcoming tumor MDR.

Sonophoric nanoprobe aided pH measurement in vivo using photoacoustic spectroscopy

Presented here is a novel method of in vivo pH sensing utilizing a hybrid optical imaging technique, photoacoustic imaging (PAI), and pH sensitive polymeric nanoprobes. Nanoprobes with hydrophobic core containing a pH sensitive dye were synthesized and used to measure the pH level ex vivo first and then in vivo by performing experiments on a rat joint model, with an achieved precision of less than 0.1 pH units. The ability of the hydrophobic functional groups in the polyacrylamide matrix to shield the molecular dye from being affected by the proteins in the plasma, and prevent the dye from leaching out, is also demonstrated.

Modulation of Hydrogel Nanoparticle Intracellular Trafficking by Multivalent Surface Engineering with Tumor Targeting Peptide

Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NPs binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers.

Highly stable polymer coated nano-clustered silver plates: A multimodal optical contrast agent for biomedical imaging

Here, we present a new optical contrast agent based on silver nanoplate clusters embedded inside of a polymer nano matrix. Unlike nanosphere clusters, which have been well studied, nanoplate clusters have unique properties due to the different possible orientations of interaction between the individual plates, resulting in a significant broadening of the absorption spectra. These nanoclusters were immobilized inside of a polymer cladding so as to maintain their stability and optical properties under in vivo conditions. The polymer-coated silver nanoplate clusters show a lower toxicity compared to the uncoated nanoparticles. At high nanoparticle concentrations, cell death occurs mostly due to apoptosis. These nanoparticles were used for targeted fluorescence imaging in a rat glioma cell line by incorporating a fluorescent dye into the matrix, followed by conjugation of a tumor targeting an F3 peptide. We further used these nanoparticles as photoacoustic contrast agents in vivo to enhance the contrast of the vasculature structures in a rat ear model. We observed a contrast enhancement of over 90% following the nanoparticle injection. It is also shown that these NPs can serve as efficient contrast agents, with specific targeting abilities for broadband multimodal imaging that are usable for diagnostic applications and that extend into use as therapeutic agents as well.