Ultrahigh sensitivity carbon nanotube agents for photoacoustic molecular imaging in living mice.

Zhang Q., Iwakuma N., Sharma P., Moudgil B.M., Wu C., McNeill J., Jiang H., Grobmyer S.R.

Photoacoustic tomography (PAT) is a rapidly emerging non-invasive imaging technology that integrates the merits of high optical contrast with high ultrasound resolution. The ability to quantitatively and non-invasively image nanoparticles has important implications for the development of nanoparticles as in vivo cancer diagnostic and therapeutic agents. In this study, the ability of systemically administered poly(ethylene glycol)-coated (PEGylated) gold nanoparticles as a contrast agent for in vivo tumor imaging with PAT has been evaluated. We demonstrate that gold nanoparticles (20 and 50 nm) have high photoacoustic contrast as compared to mouse tissue ex vivo. Gold nanoparticles can be visualized in mice in vivo following subcutaneous administration using PAT. Following intravenous administration of PEGylated gold nanoparticles to tumor-bearing mice, accumulation of gold nanoparticles in tumors can be effectively imaged with PAT. With gold nanoparticles as a contrast agent, PAT has important potential applications in the image guided therapy of superficial tumors such as breast cancer, melanoma and Merkel cell carcinoma.

Kim J., Galanzha E.I., Shashkov E.V., Moon H., Zharov V.P.

Carbon nanotubes have shown promise as contrast agents for photoacoustic and photothermal imaging of tumours and infections because they offer high resolution and allow deep tissue imaging. However, in vivo applications have been limited by the relatively low absorption displayed by nanotubes at near-infrared wavelengths and concerns over toxicity. Here, we show that gold-plated carbon nanotubes—termed golden carbon nanotubes—can be used as photoacoustic and photothermal contrast agents with enhanced near-infrared contrast (∼102-fold) for targeting lymphatic vessels in mice using extremely low laser fluence levels of a few mJ cm−2. Antibody-conjugated golden carbon nanotubes were used to map the lymphatic endothelial receptor, and preliminary in vitro viability tests show golden carbon nanotubes have minimal toxicity. This new nanomaterial could be an effective alternative to existing nanoparticles and fluorescent labels for non-invasive targeted imaging of molecular structures in vivo. Carbon nanotubes coated with a thin layer of gold can be a good alternative to fluorescent labels and gold nanoparticles for non-invasive in vivo photoacoustic and photothermal imaging.

De La Zerda A., Zavaleta C., Keren S., Vaithilingam S., Bodapati S., Liu Z., Levi J., Smith B.R., Ma T., Oralkan O., Cheng Z., Chen X., Dai H., Khuri-Yakub B.T., Gambhir S.S.

Photoacoustic imaging of living subjects offers higher spatial resolution and allows deeper tissues to be imaged compared with most optical imaging techniques. As many diseases do not exhibit a natural photoacoustic contrast, especially in their early stages, it is necessary to administer a photoacoustic contrast agent. A number of contrast agents for photoacoustic imaging have been suggested previously, but most were not shown to target a diseased site in living subjects. Here we show that single-walled carbon nanotubes conjugated with cyclic Arg-Gly-Asp (RGD) peptides can be used as a contrast agent for photoacoustic imaging of tumours. Intravenous administration of these targeted nanotubes to mice bearing tumours showed eight times greater photoacoustic signal in the tumour than mice injected with non-targeted nanotubes. These results were verified ex vivo using Raman microscopy. Photoacoustic imaging of targeted single-walled carbon nanotubes may contribute to non-invasive cancer imaging and monitoring of nanotherapeutics in living subjects.

Maslov K., Zhang H.F., Hu S., Wang L.V.

Capillaries, the smallest blood vessels, are the distal end of the vasculature where oxygen and nutrients are exchanged between blood and tissue. Hence, noninvasive imaging of capillaries and function in vivo has long been desired as a window to studying fundamental physiology, such as neurovascular coupling. Existing imaging modalities cannot provide the required sensitivity and spatial resolution. We present in vivo imaging of the microvasculature including single capillaries in mice using optical-resolution photoacoustic microscopy (OR-PAM) developed in our laboratory. OR-PAM provides a lateral resolution of 5 microm and an imaging depth >0.7 mm.

Schipper M.L., Nakayama-Ratchford N., Davis C.R., Kam N.W., Chu P., Liu Z., Sun X., Dai H., Gambhir S.S.

Single-walled carbon nanotubes are currently under evaluation in biomedical applications, including in vivo delivery1,2,3 of drugs4, proteins, peptides5,6,7 and nucleic acids8,9 (for gene transfer10 or gene silencing11), in vivo tumour imaging12 and tumour targeting of single-walled carbon nanotubes as an anti-neoplastic treatment5. However, concerns about the potential toxicity of single-walled carbon nanotubes have been raised13,14. Here we examine the acute and chronic toxicity of functionalized single-walled carbon nanotubes when injected into the bloodstream of mice. Survival, clinical and laboratory parameters reveal no evidence of toxicity over 4 months. Upon killing, careful necropsy and tissue histology show age-related changes only. Histology and Raman microscopic mapping demonstrate that functionalized single-walled carbon nanotubes persisted within liver and spleen macrophages for 4 months without apparent toxicity. Although this is a preliminary study with a small group of animals, our results encourage further confirmation studies with larger groups of animals.

Liu Z., Davis C., Cai W., He L., Chen X., Dai H.

Carbon nanotubes are promising new materials for molecular delivery in biological systems. The long-term fate of nanotubes intravenously injected into animals in vivo is currently unknown, an issue critical to potential clinical applications of these materials. Here, using the intrinsic Raman spectroscopic signatures of single-walled carbon nanotubes (SWNTs), we measured the blood circulation of intravenously injected SWNTs and detect SWNTs in various organs and tissues of mice ex vivo over a period of three months. Functionalization of SWNTs by branched polyethylene-glycol (PEG) chains was developed, enabling thus far the longest SWNT blood circulation up to 1 day, relatively low uptake in the reticuloendothelial system (RES), and near-complete clearance from the main organs in ≈2 months. Raman spectroscopy detected SWNT in the intestine, feces, kidney, and bladder of mice, suggesting excretion and clearance of SWNTs from mice via the biliary and renal pathways. No toxic side effect of SWNTs to mice was observed in necropsy, histology, and blood chemistry measurements. These findings pave the way to future biomedical applications of carbon nanotubes.

Yang X., Skrabalak S.E., Li Z., Xia Y., Wang L.V.

Poly(ethylene glycol)-coated Au nanocages have been evaluated as a potential near-infrared (NIR) contrast agent for photoacoustic tomography (PAT). Previously, Au nanoshells were found to be an effective NIR contrast agent for PAT; however, Au nanocages with their more compact sizes (100 nm for Au nanoshells) and larger optical absorption cross sections should be better suited for in vivo applications. We sequentially injected Au nanocages into the circulatory system of a rat in three administrations and in vivo PAT was conducted immediately prior to the first injection and continued until 5 h after the final injection. A gradual enhancement of the optical absorption in the cerebral cortex, by up to 81%, was observed over the course of the experiment.

Kim G., Huang S., Day K.C., O’Donnell M., Agayan R.R., Day M.A., Kopelman R., Ashkenazi S.

Nanoparticles 100 nm in diameter containing indocyanine green (ICG) have been developed as a contrast agent for photoacoustic (PA) imaging based on (photonic explorers for biomedical use by biologically localized embedding PEBBLE) technology using organically modified silicate (ormosil) as a matrix. ICG is an FDA-approved dye with strong optical absorption in the near-infrared (NIR) region, where light can penetrate deepest into biological tissue. A photoacoustic imaging system was used to study image contrast as a function of PEBBLE concentration in phantom objects. ICG-embedded ormosil PEBBLEs showed improved stability in aqueous solution compared with free ICG dye. The particles were conjugated with HER-2 antibody for breast cancer and prostate cancer cell targeting. Initial in vitro characterization shows high contrast and high efficiency for binding to prostate cancer cells. ICG can also be used as a photosensitizer (generating toxic oxygen by illumination) for photodynamic therapy. We have measured the photosensitization capability of ICG-embedded ormosil nanoparticles. This feature can be utilized to combine detection and therapeutic functions in a single agent.

Liu Z., Sun X., Nakayama-Ratchford N., Dai H.

We show that large surface areas exist for supramolecular chemistry on single-walled carbon nanotubes (SWNTs) prefunctionalized noncovalently or covalently by common surfactant or acid-oxidation routes. Water-soluble SWNTs with poly(ethylene glycol) (PEG) functionalization via these routes allow for surprisingly high degrees of pi-stacking of aromatic molecules, including a cancer drug (doxorubicin) with ultrahigh loading capacity, a widely used fluorescence molecule (fluorescein), and combinations of molecules. Binding of molecules to nanotubes and their release can be controlled by varying the pH. The strength of pi-stacking of aromatic molecules is dependent on nanotube diameter, leading to a method for controlling the release rate of molecules from SWNTs by using nanotube materials with suitable diameter. This work introduces the concept of "functionalization partitioning" of SWNTs, i.e., imparting multiple chemical species, such as PEG, drugs, and fluorescent tags, with different functionalities onto the surface of the same nanotube. Such chemical partitioning should open up new opportunities in chemical, biological, and medical applications of novel nanomaterials.

Eghtedari M., Oraevsky A., Copland J.A., Kotov N.A., Conjusteau A., Motamedi M.

The development of a contrast agent for a laser optoacoustic imaging system (LOIS) can significantly widen preclinical and clinical applications of this imaging modality for early detection of cancerous tumors. Gold nanorods were engineered to enhance the contrast for optoacoustic imaging. Under in vivo conditions, 25 microL of gold nanorods solution at a concentration of 1.25 pM were injected into nude mice and detected using a single-channel acoustic transducer. LOIS was used to visualize the distribution of gold nanoparticles at a concentration of 125 pM in vivo when 100 microL of solution of gold nanoparticles was delivered subcutaneously. Our results suggest that LOIS can be used for in vivo detection of gold nanorods at low concentrations and the nanoparticles can be engineered to enhance the diagnostic power of optoacoustic imaging.

Liu Z., Cai W., He L., Nakayama N., Chen K., Sun X., Chen X., Dai H.

Single-walled carbon nanotubes (SWNTs) exhibit unique size, shape and physical properties1,2,3 that make them promising candidates for biological applications. Here, we investigate the biodistribution of radio-labelled SWNTs in mice by in vivo positron emission tomography (PET), ex vivo biodistribution and Raman spectroscopy. It is found that SWNTs that are functionalized with phospholipids bearing polyethylene-glycol (PEG) are surprisingly stable in vivo. The effect of PEG chain length on the biodistribution and circulation of the SWNTs is studied. Effectively PEGylated SWNTs exhibit relatively long blood circulation times and low uptake by the reticuloendothelial system (RES). Efficient targeting of integrin positive tumour in mice is achieved with SWNTs coated with PEG chains linked to an arginine–glycine–aspartic acid (RGD) peptide. A high tumour accumulation is attributed to the multivalent effect of the SWNTs. The Raman signatures of SWNTs are used to directly probe the presence of nanotubes in mice tissues and confirm the radio-label-based results.

Zhang H.F., Maslov K., Stoica G., Wang L.V.

The clinical significance of a burn depends on the percentage of total body involved and the depth of the burn. Hence a noninvasive method that is able to evaluate burn depth would be of great help in clinical evaluation. To this end, photoacoustic microscopy is used to determine the depth of acute thermal burns by imaging the total hemoglobin concentration in the blood that accumulates along the boundaries of injuries as a result of thermal damage to the vasculature. We induce acute thermal burns in vivo on pig skin with cautery. Photoacoustic images of the burns are acquired after skin excision. In a burn treated at 175 degrees C for 20 s, the maximum imaged burn depth is 1.73+/-0.07 mm. In burns treated at 150 degrees C for 5, 10, 20, and 30 s, respectively, the trend of increasing maximum burn depth with longer thermal exposure is demonstrated.

Zhang H.F., Maslov K., Stoica G., Wang L.V.

Although optical absorption is strongly associated with the physiological status of biological tissue, existing high-resolution optical imaging modalities, including confocal microscopy, two-photon microscopy and optical coherence tomography, do not sense optical absorption directly. Furthermore, optical scattering prevents these methods from imaging deeper than ∼1 mm below the tissue surface. Here we report functional photoacoustic microscopy (fPAM), which provides multiwavelength imaging of optical absorption and permits high spatial resolution beyond this depth limit with a ratio of maximum imaging depth to depth resolution greater than 100. Reflection mode, rather than orthogonal or transmission mode, is adopted because it is applicable to more anatomical sites than the others. fPAM is demonstrated with in vivo imaging of angiogenesis, melanoma, hemoglobin oxygen saturation (sO2) of single vessels in animals and total hemoglobin concentration in humans.

Barman S.N., Kumari K., Roy A.S.

Han X., Zhang X., Kang L., Feng S., Li Y., Zhao G.

Shaikh S.

Cancer imaging is challenging for various reasons, and one of the most important reasons is the inability to diagnose precisely using the available imaging modalities. The use of nanotechnology has evolved significantly due to the various nanoparticles used based on different components and properties. Additionally, the role of theranostics has also evolved recently, which will provide faster and more precise methods for the concept of personalized medicine.

Rong Q., Humayun L., Yao J.

Biomedical imaging allows us to explore the complex biology of living organisms and better understand the clinical progression of diseases through the visualization of dynamic, functional, and molecular events. Among the modern biomedical imaging technologies, photoacoustic microscopy (PAM) provides unique optical absorption contrast and high-spatial resolution at depths beyond the penetration limit of traditional optical microscopy. Over the last decades, PAM has become an increasingly popular anatomical, functional, and molecular information. In this book, we introduce the basic principles and typical system designs of PAM, including optical-resolution PAM and acoustic-resolution PAM. We also discuss the common characteristics of PAM, including spatial resolutions, penetration depth, PA signal detectors, and the scanning approach. Finally, we present the major biomedical applications of PAM, including functional measurement, anatomical imaging from cellular to organismal level, label-free functional imaging using endogenous biomolecules, molecular imaging using exogenous contrast agents, and preclinical and clinical applications.

Fatima S., Qamar F., Quadri S.N., Abdin M.Z., Ahmad F.J.

The complex process of visualizing internal structures for medical diagnosis and therapy has witnessed a transformative shift with inorganic nanoparticle-based bioimaging probes. As a potential substitute of traditional molecular imaging probes, inorganic nanoparticle-based bioimaging probes have been thoroughly explored over the past few decades. These probes, with their unique nanoscale attributes, customizable compositions, and adaptable functionalities, offer a versatile platform for stimuli-responsive, multimodal, and targeted imaging. Notably, the functionalization of these nanoparticles, through surface modification, has enriched their qualities, significantly impacting the bioimaging domain. Achieving high-quality, contrast-rich images is pivotal in bioimaging. This chapter outlines recent progress in enhancing inorganic nanoparticle-based bioimaging probes (e.g., gold, silica, magnetic nanoparticles, quantum dots, and carbon nanotubes) across multiple modalities (MRI, CT, optical imaging, and photoacoustic imaging) and their applications. The chapter also explores selective cell targeting using functionalized nanoparticles, minimizing background signals, and enabling extended multimodal imaging.

Kumari P., Arora S., Pan Y., Ahmed I., Kumar S., Parshad B.

Huang H., Zheng Y., Chang M., Song J., Xia L., Wu C., Jia W., Ren H., Feng W., Chen Y.

Taylan U., Akçimen S., Eş I., Küçük B.N., Kendir Tekgül E., Çelebi Ç., Kumru Y., Köymen H., Inci F., Ortaç B.

AbstractCopper sulphide nanoparticles are synthesized by laser ablation of a copper target in DMSO by a 527 nm nanosecond pulsed laser. These nanoparticles have double stoichiometry (CuS and Cu1.8S) and crystalline structures, sizes under 30 nm, and they present substantial absorbance in the second near‐infrared window and photoluminescence in the visible region. The nanoparticles are used as photothermal and photoacoustic agents at 1080 nm and 1064 nm, respectively. Utilizing as a photothermal agent, these nanoparticles rapidly exhibit local heating, photothermal stability, and a temperature change of 52.2 °C within 300 s at 1 mg mL−1 concentration and 3.23 W cm−2 laser intensity. On the other hand, while employed as a photoacoustic agent, they enhanced the contrast significantly and increased the brightness proportional to their concentrations when compared to ultrasound imaging. Additionally, the biocompatibility properties of these nanoparticles were tested with cancer cells, and they were subjected to laser ablation to assess their photothermal effects. In this article, we demonstrate that copper sulphide nanoparticles synthesized by pulsed laser ablation hold great promise for photothermal and photoacoustic applications, especially in biomedical applications.

Kalathil J., Antony A., P S., Sekhar P.M., Christopher S., Perumal D., Paul S., Samanta D., Singh M.S., Varghese R.

Gao R., Liu Y., Qi S., Song L., Meng J., Liu C.

Yusufbeyoğlu S., Cinar V., Ildiz N., Hamurcu Z., Ocsoy İ., Kilic A.B.

AbstractMethicillin‐resistant Staphylococcus aureus (MRSA) is one of the deadliest pathogenic bacteria. Using photothermal therapy (PTT) to eradicate MRSA bacteria easily and effectively, it has directed this bacterium to be destroyed. Gold nanorods (AuNRs), which are nanoparticles that provide PPT, were synthesized and removed from the CTAB molecule reduce the toxic effect caused by CTAB. Subsequently, mercaptophenylboronic acid (MFBA) coated AuNRs were synthesized and used in photothermal therapy to develop a targeting agent to selectively eliminate MRSA. A decrease in cytotoxic effect of CTAB@AuNRs after conjugation with MFBA was also demonstrated by the MTS cell viability test. We found that at the end of 48 hours and 72 hours of interaction, IC50 values of MFBA@ AuNR increased by approximately 50 % compared to CTAB@AuNR. Also, it shows that the cytotoxicity of AuNRs conjugated with MFBA was reduced. Herein, photothermal efficiency was achieved with MFBA@AuNRs targeting MRSA. The purpose of using and modifying gold nanorods is to reduce the toxicity of AuNRs and to examine their efficacy on resistant pathogenic bacteria strains by taking advantage of photothermal therapy properties.

Hirasawa T., Tachi K., Ishikawa T., Miyashita M., Ito K., Ishihara M.

Wang K., Wada Y., Nakamura K.

Abstract Photoacoustic imaging is considered useful for evaluating the effects of treatment because it has a good resolution to capture minute vascular lesions and changes in the progression of atherosclerosis, which is difficult to detect with conventional imaging methods. In this study, the authors prepared a thin silicone tube filled with a mixture of red ink and olive oil as a model that mimics arteriosclerosis. The tube was embedded in a soft phantom. Photoacoustic measurements were performed using 405 nm and 520 nm laser diodes. As a result, the 405 nm laser produced a higher photoacoustic signal as the oil concentration in the mixture increased, whereas the 520 nm laser produced lower photoacoustic signals as the oil concentration increased. By focusing on the difference in the optical absorption at different wavelengths between the red ink and oil, it was shown that there was a possibility of estimating the oil concentration from the ratio of photoacoustic signals between different wavelengths.

Nguyen P., Zhe J., Hu J., Ahmed U., Paulus Y.

The application of molecular and cellular imaging in ophthalmology has numerous benefits. It can enable the early detection and diagnosis of ocular diseases, facilitating timely intervention and improved patient outcomes. Molecular imaging techniques can help identify disease biomarkers, monitor disease progression, and evaluate treatment responses. Furthermore, these techniques allow researchers to gain insights into the pathogenesis of ocular diseases and develop novel therapeutic strategies. Molecular and cellular imaging can also allow basic research to elucidate the normal physiological processes occurring within the eye, such as cell signaling, tissue remodeling, and immune responses. By providing detailed visualization at the molecular and cellular level, these imaging techniques contribute to a comprehensive understanding of ocular biology. Current clinically available imaging often relies on confocal microscopy, multi-photon microscopy, PET (positron emission tomography) or SPECT (single-photon emission computed tomography) techniques, optical coherence tomography (OCT), and fluorescence imaging. Preclinical research focuses on the identification of novel molecular targets for various diseases. The aim is to discover specific biomarkers or molecular pathways associated with diseases, allowing for targeted imaging and precise disease characterization. In parallel, efforts are being made to develop sophisticated and multifunctional contrast agents that can selectively bind to these identified molecular targets. These contrast agents can enhance the imaging signal and improve the sensitivity and specificity of molecular imaging by carrying various imaging labels, including radionuclides for PET or SPECT, fluorescent dyes for optical imaging, or nanoparticles for multimodal imaging. Furthermore, advancements in technology and instrumentation are being pursued to enable multimodality molecular imaging. Integrating different imaging modalities, such as PET/MRI (magnetic resonance imaging) or PET/CT (computed tomography), allows for the complementary strengths of each modality to be combined, providing comprehensive molecular and anatomical information in a single examination. Recently, photoacoustic microscopy (PAM) has been explored as a novel imaging technology for visualization of different retinal diseases. PAM is a non-invasive, non-ionizing radiation, and hybrid imaging modality that combines the optical excitation of contrast agents with ultrasound detection. It offers a unique approach to imaging by providing both anatomical and functional information. Its ability to utilize molecularly targeted contrast agents holds great promise for molecular imaging applications in ophthalmology. In this review, we will summarize the application of multimodality molecular imaging for tracking chorioretinal angiogenesis along with the migration of stem cells after subretinal transplantation in vivo.

Jagdale S.C., Chandorkar P.U., Podutwar A.A., Polshettiwar S.A., Chabukswar A.R.

In the last two decades, application of nanomaterials as a tool for imaging and diagnosis has gained the attention of many researchers, as they have showed promising results in every aspect of disease management right from the diagnosis, delivering and real time tracking of the drug and monitoring the diseased condition to the in-vivo imaging. In-vivo imaging helps in early diagnosis of lethal diseases like, cancer, cardiovascular diseases, and renal failure, which can improve patient's lifestyle. Nanomaterials offer properties like enhanced diagnostic specificity, increased blood circulation time, and organ specific delivery just with the help of functionalization and conjugation with desired materials. Nanomaterials like dendrimer, silica nanoparticles, and some magnetic nanoparticles are used as contrast agents in imaging technology such as magnetic resonance imaging (MRI), computed tomography (CT) and radiopharmaceuticals. This chapter covers nanomaterials (NPs) as a biomedical tool for imaging and diagnosis of various diseases.