Significance: Photodynamic therapy (PDT) involves complex light-drug-pathophysiology interactions that can be affected by multiple parameters and often leads to large variations in treatment outcome from patient to patient. Direct PDT dosimetry technologies have been sought to optimize the control variables (e.g., light dose, drug administration, tissue oxygenation, and patient conditioning) for best patient outcomes. In comparison, singlet oxygen (O21) dosimetry has been tested in various forms to provide an accurate and perhaps comprehensive prediction of the treatment efficacy.
Aim: We discuss an advanced version of this approach provided by a noninvasive, continuous wave dosimeter that can measure near-infrared spectrally resolved luminescence of both photosensitizer (PS) and O21 generated during PDT cancer treatment.
Approach: This dosimetry technology uses an amplified, high quantum efficiency InGaAs detector with spectroscopic decomposition during the light treatment to continuously extract the maximum signal of O21 phosphorescence while suppressing the strong PS luminescence background by spectrally fitting the data points across nine narrow band wavelengths. O21 and PS luminescence signals were measured in vivo in FaDu xenograft tumors grown in mice during PDT treatment using Verteporfin as the PS and a continuous laser treatment at 690 nm wavelength.
Results: A cohort of 19 mice was used and observations indicate that the tumor growth rate inhibition showed a stronger correlation with O21 than with just the PS signal.
Conclusions: These results suggest that O21 measurement may be a more direct dosimeter of PDT damage, and it has potential value as a definitive diagnostic for PDT treatment, especially with spectral separation of the background luminescence and online estimation of the PS concentration.
Light-responsive compounds can be utilized to control spatially and temporally the initiation of biochemical processes. This has the potential to improve the treatment of diseases, such as cancers, by controlling the location of drug activity at the site of disease rather than the drug acting systemically. This would reduce side-effects as well as the overall systemic dose of anticancer agents. Until recently light-responsive molecules that released active compounds in a stoichiometric manner required the use of short wavelength, high energy ultraviolet light (UV). These compounds cannot be used to treat disease because the required light wavelengths are readily absorbed by biological molecules preventing the light from penetrating tissue to an appreciable extent. Recently, we have a developed a platform technology that combines Vitamin B12 and a near infrared (NIR) absorbing fluorophore that converts an inactivated drug into an active form when exposed to NIR. In contrast to UV, NIR is poorly absorbed by biological tissues. Therefore, NIR penetrates tissue and can be used for photochemotherapeutic treatment of disease. In addition to targeting diseased tissue based on controlling drug activity by regulating light exposure, these compounds target cancer cells due to the Vitamin B12 moiety because rapidly dividing cancer cells have an increased demand for Vitamin B12 in comparison to normal, healthy cells. The reported technology could improve treatment of certain by diseases by affording effective treatment while reducing side effects.
X-Ray activated pharmaceutical therapy is highly sought after as it provides deep tissue, synergistic method of treating cancers in which the standard method of care involves radiotherapy. Traditional drugs utilized as neoadjuvant chemotherapy have significant side effects and a lack of selectivity, leaving a dire need for targeted drug delivery methods. We have recently developed a unique delivery platform whereby the drug is conjugated to an alkylcobalamin vitamin B12 scaffold, and these alkylcobalamins are actively transported into cells by transcobalamin receptors (TCblR). A large number of cancer types have enhanced expression of these receptors; therefore, the drug-cobalamin conjugate could be effectively ferried into the tumor selectively via the TCblR pathway. This delivery system provides light-activatable release of chemotherapeutics. Due to the drug becoming active at the specific site that it is needed, such as a tumor, the potential side effects of that drug in organs at risk are mitigated. As a proof of concept, we have found that a fluorescent cobalamin derivative localized within xenograft tumors in mice, demonstrating the effectiveness of the vitamin B12 scaffold as a theranostic targeting agent. In addition, this derivative is also activated with clinical X-ray doses from a linear accelerator. We explored the ability of a variety of cobalamin drug conjugates to be used in combination with radiotherapy to elicit an enhanced reduction in tumor margins in pancreatic adenocarcinoma models.
Luminescence molecular tomography with Cherenkov excitation offers the ability to non-invasively image and quantify temporal changes in fluorescence throughout the body, and then further realize tumor localization. This can be done in radiotherapy to determine the response to treatment in fractionated therapy. To obtain high signal-to-background or signal-to-noise ratio measurement, it is critical to know the best post time point of in-vivo agent-based molecular imaging, which could account on a high signal ratio of target to skin (TSR). For this purpose, ex-vivo murine experiments were performed to quantify the biokinetics and biodistribution of the major organs, plasma, tumor, and skin.
Cherenkov emission, which is generated during radiation therapy, can be utilized for imaging that is synergistic with radiation therapy. Cherenkov light can be utilized to excite phosphors, which then can be imaged utilizing Cherenkov excited luminescence scanned imaging (CELSI).
Europium chelate microspheres, which exhibit bright luminescence with long luminescent lifetime, were appended with multiple copies of cetuximab. This will allow for selective imaging of EGFR overexpressing tumors during the course of radiation therapy via CELSI. We have characterized the functionality of the cetuximab loaded microspheres in vitro via ELISA, as well as via fluorescence microscopy in EGFR overexpressing A431 cells. These microspheres were intravenously injected into athymic nude mice bearing A431 flank tumors and allowed to incubate for a series of time points. They were then imaged first via standard fluorescence imaging to determine the ideal time point for visualizing tumors via CELSI. After demonstrating selective accumulation in tumors, imaging was then undertaken in vivo via CELSI. These antibody conjugated europium microspheres provide promise to image tumors selectively with CELSI. Future studies involve conjugating other antibodies to the europium microspheres to utilize in CELSI.
The discovery of new tumor targeting agents is desirable to expand imaging and drug delivery platforms. Cobalamins, vitamin B12 derivatives, selectively accumulate in tumor versus benign tissue due to overexpression of transcobalamin receptors in a variety of cancer types. Multiple forms of this vitamin are taken into cells via transport through transcobalamin receptors on the cell surface. Alkylcobalamins are light-activatable, and we have discovered that the wavelength of this light activation is tunable via appendage of a fluorophore. We have been able to harness this cobalamin platform to release drugs with a variety of wavelengths of light, including those within the optical window of tissue. This cobalamin drug delivery platform provides selective spatiotemporal activation of drug only where needed, thereby diminishing side effects of traditional chemotherapy.
A Bodipy650-cobalamin was synthesized and utilized to study the tumor targeting ability of cobalamin derivatives in athymic nude mice with subcutaneous MCF-7 and MIA PaCa-2 tumors, which have been demonstrated to overexpress transcobalamin receptors. The fluorescently labeled cobalamin was injected intravenously into the mice and allowed to incubate for a series of time points. Fluorescence imaging revealed that this cobalamin conjugate selectively accumulated in both tumor types. We utilized this cobalamin platform for tumor selective, light-activated delivery of the pancreatic cancer drugs erlotinib and SN38. We determined light-induced apoptosis in MIA PaCa-2 cells in vitro and explored the reduction of MIA PaCa-2 tumors in vivo utilizing these cobalamin drug conjugates. This cobalamin platform provides potential for development of new theranostic tools for drug delivery.
Solid tumors often exhibit abnormal morphology which can be characterized by increased permeability and low perfusion. The resulting tumor hypoxia has been correlated with poor prognosis, which may be due to ineffective therapy or survival of more aggressive phenotypes. External beam radiation therapy (EBRT) is often used to treat such tumors, where radiation dose is delivered on a daily fractionated basis over the course of weeks. A non-contact optical method for measuring in vivo oxygen levels during EBRT treatments has been developed to provide early indications of hypoxic tumor environments. This method uses a time-gated intensified imaging device to measure both Cherenkov emissions, which are generated in tissue by high energy electrons traveling faster than the phase-velocity of the medium, and Cherenkov-excited luminescence generated by the oxygen-sensitive phosphorescent compound, PtG4. Murine models have shown the ability to discriminate phosphorescence lifetime changes before and after animal sacrifice. Pixel-maps of the estimated pO2 can be generated from this data to show high spatial variability within a region of interest. By further camera optimization, this method can be expanded to show pO2 distributions for other physiological conditions in near real-time. Our imaging method has the unique ability to be integrated within existing clinical applications while providing a wide-field mapping of oxygen saturation, which is currently unavailable with existing point probes.
Cherenkov emission generated in tissue during radiotherapy can be harnessed for the imaging biochemistry of tissue microenvironments. Cherenkov-excited luminescence scanned imaging (CELSI) provides a way to optically and noninvasively map oxygen-related signals, which is known to correlate to outcomes in radiotherapy. Four candidate phosphorescent reagents PtG4, MM2, Ir(btb)2 ( acac ) , and MitoID were studied for oxygen sensing, testing in a progressive series of (a) in solution, (b) in vitro, and (c) in subcutaneous tumors. In each test, the signal strength and response to oxygen were assessed by phosphorescence intensity and decay lifetime measurement. MM2 showed the most robust response to oxygen changes in solution, followed by PtG4, Ir(btb)2 ( acac ) , and MitoID. However, in PANC-1 cells, their oxygen responses differed with Ir(btb)2 ( acac ) exhibiting the largest phosphorescent intensity change in response to changes in oxygenation, followed by PtG4, MM2, and MitoID. In vivo, it was only possible to utilize Ir(btb)2 ( acac ) and PtG4, with each being used at nanomole levels, to determine signal strength, lifetime, and pO2. Oxygen sensing with CELSI during radiotherapy is feasible and can estimate values from 1 mm regions of tissue when used in the configuration of this study. PtG4 was the most amenable to in vivo sensing on the timescale of external beam LINAC x-rays.
Cherenkov-excited luminescence scanned imaging (CELSI) has been proposed for radiation-dose determination in medical physics due to its high spatial-resolution over centimeters of tissue. However, dense line-scanning illumination in typical CELSI is time-cost owing to the mechanical movement of the leaves in multi leaf collimator (MLC), resulting into increased radiation exposure. As a result, a scanningless Cherenkov luminescence imaging modality is herein proposed through structuring epi-illumination with MLC-based Hadamard-patterns, which utilizes a reduced radiation does by limiting illumination patterns, extremely shortening the sampling process. In order to effectively reconstruct unknowns from the resultant underdetermined linear system with sparse samplings, a compressed sensing-based reconstruction methodology with l1-norm regularization is adopted. Numerical and phantom experiments show that the proposed methodology achieves the same image quality as the traditional CELSI does.
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