Multifunctional croconaine nanoparticles for efficient optoacoustic imaging of deep tumors and photothermal therapy

The proper design of near-infrared light-Absorbing agents enables efficient optoacoustic imaging-guided phototherapy. In particular, several croconaine-based organic agents with excellent optical properties have been recently reported for this purpose. However, most of them absorb light below 800 nm, limiting deep-Tissue imaging applications. To this end, we utilized a recently described novel croconaine derivative (CR880) to develop CR880-based nanoparticles (CR880-NPs) for effective in vivo delivery, deep tissue optoacoustic imaging and photothermal therapy applications. Radicals and strong π-πstacking in CR880 result in an 880 nm absorption peak with no blue-shift upon condensing to the solid phase. DSPE-PEG2000-formulated CR880-NPs exhibited high optoacoustic generation efficiency and photostability, and could be visualized in the tumors of three different mouse tumor models (breast, brain, and colon tumor) with high image contrast. The high photothermal conversion efficiency of CR880-NPs (∼58%) subsequently enabled efficient in vivo tumor elimination using a low energy laser, while remaining biocompatible and well-Tolerated. This work introduces a promising novel agent for cancer theranostics of challenging deep-seated tumors

Facile Synthesis of a Croconaine-Based Nanoformulation for Optoacoustic Imaging and Photothermal Therapy

Near-infrared (NIR) light absorbing theranostic agents can integrate optoacoustic imaging and photothermal therapy for effective personalized precision medicine. However, most of these agents face the challenges of unstable optical properties, material-associated toxicity, and nonbiodegradability, all of which limit their biomedical application. Several croconaine-based organic agents able to overcome some of these limitations have been recently reported, but these suffer from complicated multistep synthesis protocols. Herein, the use of CR760, a croconaine dye with excellent optical properties, is reported for nanoparticle formulation and subsequent optoacoustic imaging and photothermal therapy. Importantly, CR760 can be conveniently prepared in a single step from commercially available materials. Furthermore, CR760 can be covalently attached, via a polyethylene glycol linker, to the αvβ3 integrin ligand c(RGDyC), resulting in self-assembled nanoparticles (NPs) with cancer-targeting capability. Such CR760RGD-NPs exhibit strong NIR absorption, high photostability, high optoacoustic generation efficiency, and active tumor-targeting, making them ideal candidates for optoacoustic imaging. Due to favorable electron transfer, CR760RGD-NPs display a 45.37% photothermal conversion efficiency thereby rendering them additionally useful for photothermal therapy. Targeted tumor elimination, biosafety, and biocompatibility are demonstrated in a 4T1 murine breast tumor model. This work points to the use of CR760RGD-NPs as a promising nanoagent for NIR-based cancer phototheranostics

Concurrent fluorescence and volumetric optoacoustic tomography of nanoagent perfusion and bio-distribution in solid tumors

Intravenously administered liposomes and other nano-sized particles are known to passively accumulate in solid tumors via the enhanced permeability and retention (EPR) effect, which is extensively explored toward the improvement of diagnosis and drug delivery in oncology. Agent extravasation into tumors is often hampered by the mononuclear phagocytic and renal systems, which sequester and/or eliminate most of the nanoparticles from the body. Dynamic imaging of the tumor microcirculation and bolus perfusion can thus facilitate optimization of the nanoparticle delivery. When it comes to non-invasive visualization of rapid biological dynamics in whole tumors, the currently available pre-clinical imaging modalities are commonly limited by shallow penetration, lack of suitable contrast or otherwise insufficient spatial or temporal resolution. Herein, we demonstrate the unique capabilities of a combined epi-fluorescence and optoacoustic tomography (FLOT) system for characterizing contrast agent dynamics in orthotopic breast tumors in mice. A liposomal indocyanine green (Lipo-ICG) preparation was administered intravenously with the time-lapse data continuously acquired during and after the injection procedure. In addition to the highly sensitive detection of the fluorescence agent by the epi-fluorescence modality, the volumetric multi-spectral optoacoustic tomography readings further enabled resolving deep-seated vascular structures with high spatial resolution and hence provided accurate readings of the dynamic bio-distribution of nanoparticles in the entire tumor in 3D. The synergetic combination of the two modalities can become a powerful tool in cancer research and potentially aid the diagnosis, staging and treatment guidance of certain types of cancer in the clinical setting

Croconaine-based nanoparticles enable efficient optoacoustic imaging of murine brain tumors

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors

Hepatoprotective activity of alcoholic and aqueous extracts of leaves of Tylophora indica (Linn.) in rats

Objective: To investigate the hepatoprotective activity of alcoholic (ALLT) and aqueous (AQLT) extracts of leaves of Tylophora indica (asclepiadaceae) against ethanol-induced hepatotoxicity. Materials and Methods: Leaf powder of Tylophora indica was successively extracted with alcohol and water. Preliminary phytochemical tests were done and the LD50 values for both extracts determined. The hepatoprotective activity of the ALLT and AQLT were assessed in ethanol-induced hepatotoxic rats. Results: The ALLT showed presence of alkaloids, carbohydrates, steroids, saponins and triterpenes, while alkaloids, carbohydrates and saponins were present with AQLT. The ALLT did not produce any mortality even at 5000 mg/kg while LD50 of AQLT was found to be 3162 mg/kg. Ethanol produced significant changes in physical (increased liver weight and volume), biochemical (increase in serum alanine transaminase, aspartate transaminase, alkaline phosphatase, direct bilirubin, total bilirubin, cholesterol, triglycerides and decrease in total protein and albumin level), histological (damage to hepatocytes) and functional (thiopentone-induced sleeping time) liver parameters. Pretreatment with ALLT or AQLT extract significantly prevented the physical, biochemical, histological and functional changes induced by ethanol in the liver. Conclusion: The present study indicates that ALLT and AQLT extracts possessed hepatoprotective activity. The alcoholic extract was found to exhibit greater hepatoprotective activity than the aqueous extract

Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging

Bacterial outer membrane vesicles (OMVs) are increasingly used as carriers for drug delivery. Here the authors encapsulate biopolymer melanin into OMVs, extending their use to optoacoustic imaging both in vitro and in vivo, and demonstrate the potential of this tool for photothermal therapy applications

Merged molecular switches excel as optoacoustic dyes: azobenzene-cyanines are loud and photostable NIR imaging agents

Optoacoustic imaging, also known as photoacoustic imaging, promises micron-resolution noninvasive imaging in biology at much deeper penetration (>cm) depths than e.g. fluorescence. However, the loud, photostable, NIR-absorbing molecular contrast agents which would be needed for optoacoustic imaging of enzyme activity remain unknown: most organic molecular contrast agents are simply repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging conditions, which are consequences of their slow S1→S0 electronic relaxation rates. We now disclose that known fluorophores can be rationally modified to reach ultrafast S1→S0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches to cyanine dyes to give ultrafast relaxation (100-fold faster). Even without adapting instrument settings, these azohemicyanine optoacoustic imaging agents deliver outstanding improvements in signal longevity (>1000-fold increase of photostability) and signal loudness (here: >3-fold even at time zero). We show why this still-unexplored design strategy can offer even stronger performance in the future, as a simple method that will also increase the spatial resolution and the quantitative linearity of photoacoustic response even over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on unsolved problems that have faced optoacoustic agents, this practical strategy may be a crucial step towards unleashing the full potential, in fundamental studies and in translational uses, of optoacoustic imaging

Bacterial outer membrane vesicles as cationic dye carriers for optoacoustics-guided phototherapy of cancer

Abstract Background Cationic dyes are widely used as biomarkers for optical imaging. However, most of these are hydrophobic and cannot be employed in vivo without chemical conjugation or modification. Herein, we report for the first time the use of bacterial outer membrane vesicles (OMVs) as nanocarriers of cationic dyes for cancer theranostics. Results We demonstrate that cationic dyes (IR780, Cy7, and Cy7.5) form stable complexes with negatively charged bacterial-OMVs, improving the dyes’ in vivo circulation and optoacoustic properties. Such OMV-Dye complexes are biodegradable and safe for in vivo applications. Importantly, this method of cationic dye loading is faster and easier than synthetic chemistry approaches, and the efficient tumor accumulation of OMV-Dyes enables sensitive tumor detection using optoacoustic technology. As a proof-of-concept, we generated OMV-IR780 for optoacoustics-guided in vivo tumor phototherapy in a mouse model. Conclusions Our results demonstrate cationic dye-bound OMVs as promising novel nanoagents for tumor theranostics

Bioengineered Bacterial Outer Membrane Vesicles as Cell-Specific Drug-Delivery Vehicles for Cancer Therapy

Advances in genetic engineering tools have contributed to the development of strategies for utilizing biologically derived vesicles as nanomedicines for achieving cell-specific drug delivery. Here, we describe bioengineered bacterial outer membrane vesicles (OMVs) with low immunogenicity that can target and kill cancer cells in a cell-specific manner by delivering small interfering RNA (siRNA) targeting kinesin spindle protein (KSP). A mutant <i>Escherichia coli</i> strain that exhibits reduced endotoxicity toward human cells was engineered to generate OMVs displaying a human epidermal growth factor receptor 2 (HER2)-specific affibody in the membrane as a targeting ligand. Systemic injection of siRNA-packaged OMVs caused targeted gene silencing and induced highly significant tumor growth regression in an animal model. Importantly, the modified OMVs were well tolerated and showed no evidence of nonspecific side effects. We propose that bioengineered OMVs have great potential as cell-specific drug-delivery vehicles for treating various cancers