Toward noncontact macroscopic imaging of multiple cancers using multi-spectral inelastic scattering detection.

Here we introduce a Raman spectroscopy approach combining multi-spectral imaging and a new fluorescence background subtraction technique to image individual Raman peaks in less than 5 seconds over a square field-of-view of 1-centimeter sides with 350 micrometers resolution. First, human data is presented supporting the feasibility of achieving cancer detection with high sensitivity and specificity - in brain, breast, lung, and ovarian/endometrium tissue - using no more than three biochemically interpretable biomarkers associated with the inelastic scattering signal from specific Raman peaks. Second, a proof-of-principle study in biological tissue is presented demonstrating the feasibility of detecting a single Raman band - here the CH2/CH3 deformation bands from proteins and lipids - using a conventional multi-spectral imaging system in combination with the new background removal method. This study paves the way for the development of a new Raman imaging technique that is rapid, label-free, and wide field.

Correlation of in vitro cell viability and cumulative singlet oxygen luminescence from protoporphyrin IX in mitochondria and plasma membrane.

SIGNIFICANCE: Photodynamic therapy (PDT) can be targeted toward different subcellular localizations, and it is proposed that different subcellular targets vary in their sensitivity to photobiological damage. Since singlet oxygen (1O2) has a very short lifetime with a limited diffusion length in cellular environments, measurement of cumulative 1O2 luminescence is the most direct approach to compare the PDT sensitivity of mitochondria and plasma membrane. APPROACH: PDT-generated near-infrared 1O2 luminescence at 1270 nm was measured together with cell viability for 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and exogenous PpIX, at different incubation times. Confocal fluorescence microscopy indicated that ALA-induced PpIX (2 h) localized in the mitochondria, whereas exogenous PpIX (1 h) mainly localized to the plasma membrane. Cell viability was determined at several time points during PDT treatments using colony-forming assays, and the surviving fraction correlated well with cumulative 1O2 luminescence counts from PpIX in mitochondria and plasmas membrane, respectively. RESULTS: The mitochondria are more sensitive than the plasma membrane by a factor of 1.7. CONCLUSIONS: Direct 1O2 luminescence dosimetry's potential value for comparing the PDT sensitivity of different subcellular organelles was demonstrated. This could be useful for developing subcellular targeted novel photosensitizers to enhance PDT efficiency.

Femtosecond pulsed laser photodynamic therapy activates melanin and eradicates malignant melanoma.

Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors.

Adaptation of a Clinical High-Frequency Transrectal Ultrasound System for Prostate Photoacoustic Imaging: Implementation and Pre-clinical Demonstration.

OBJECTIVE: High-frequency, high-resolution transrectal micro-ultrasound (micro-US: ≥15 MHz) imaging of the prostate is emerging as a beneficial tool for scoring disease risk and accurately targeting biopsies. Adding photoacoustic (PA) imaging to visualize abnormal vascularization and accumulation of contrast agents in tumors has potential for guiding focal therapies. In this work, we describe a new imaging platform that combines a transrectal micro-US system with transurethral light delivery for PA imaging. METHODS: A clinical transrectal micro-US system was adapted to acquire PA images synchronous to a tunable laser pulse. A transurethral side-firing optical fiber was developed for light delivery. A polyvinyl chloride (PVC)-plastisol phantom was developed and characterized to image PA contrast agents in wall-less channels. After resolution measurement in water, PA imaging was demonstrated in phantom channels with dyes and biodegradable nanoparticle contrast agents called porphysomes. In vivo imaging of a tumor model was performed, with porphysomes administered intravenously. RESULTS: Photoacoustic imaging data were acquired at 5 Hz, and image reconstruction was performed offline. PA image resolution at a 14-mm depth was 74 and 261 µm in the axial and lateral directions, respectively. The speed of sound in PVC-plastisol was 1383 m/s, and the attenuation was 4 dB/mm at 20 MHz. PA signal from porphysomes was spectrally unmixed from blood signals in the tumor, and a signal increase was observed 3 h after porphysome injection. CONCLUSION: A combined transrectal micro-US and PA imaging system was developed and characterized, and in vivo imaging demonstrated. High-resolution PA imaging may provide valuable additional information for diagnostic and therapeutic applications in the prostate.

AAPM Task Group Report 311: Guidance for performance evaluation of fluorescence-guided surgery systems.

The last decade has seen a large growth in fluorescence-guided surgery (FGS) imaging and interventions. With the increasing number of clinical specialties implementing FGS, the range of systems with radically different physical designs, image processing approaches, and performance requirements is expanding. This variety of systems makes it nearly impossible to specify uniform performance goals, yet at the same time, utilization of different devices in new clinical procedures and trials indicates some need for common knowledge bases and a quality assessment paradigm to ensure that effective translation and use occurs. It is feasible to identify key fundamental image quality characteristics and corresponding objective test methods that should be determined such that there are consistent conventions across a variety of FGS devices. This report outlines test methods, tissue simulating phantoms and suggested guidelines, as well as personnel needs and professional knowledge bases that can be established. This report frames the issues with guidance and feedback from related societies and agencies having vested interest in the outcome, coming from an independent scientific group formed from academics and international federal agencies for the establishment of these professional guidelines.

Investigating the effects of a temperature dependent photodynamic dose: A numerical study.

SIGNIFICANCE: Photodynamic therapy (PDT) and photothermal therapy (PTT) show promise as cancer treatments, but challenges in generating large ablative volumes for deep-seated tumours persist. Using simulations, this study investigates combined PDT and PTT to increase treatment volumes, including the impact of a temperature-dependent PDT dose on the treatment volume radius. APPROACH: A finite-element model, using the open-source SfePy package, was developed to simulate combined interstitial photothermal and photodynamic treatments. Results compared an additive dose model to a temperature-dependent dose model with enhanced PDT dosimetry and examined typical clinical scenarios for possible synergistic effects. RESULTS: Findings revealed that the temperature-dependent dose model could significantly expand the damage radius compared to the additive model, depending on the tissue and drug properties. CONCLUSIONS: Characterizing synergistic effects of PDT and PTT could enhance treatment planning. Future work is ongoing to implement additional variables, such as photosensitizer photobleaching, and spatial and temporally varying oxygenation.

The use of nanomaterials in advancing photodynamic therapy (PDT) for deep-seated tumors and synergy with radiotherapy.

Photodynamic therapy (PDT) has been under development for at least 40 years. Multiple studies have demonstrated significant anti-tumor efficacy with limited toxicity concerns. PDT was expected to become a major new therapeutic option in treating localized cancer. However, despite a shifting focus in oncology to aggressive local therapies, PDT has not to date gained widespread acceptance as a standard-of-care option. A major factor is the technical challenge of treating deep-seated and large tumors, due to the limited penetration and variability of the activating light in tissue. Poor tumor selectivity of PDT sensitizers has been problematic for many applications. Attempts to mitigate these limitations with the use of multiple interstitial fiberoptic catheters to deliver the light, new generations of photosensitizer with longer-wavelength activation, oxygen independence and better tumor specificity, as well as improved dosimetry and treatment planning are starting to show encouraging results. Nanomaterials used either as photosensitizers per se or to improve delivery of molecular photosensitizers is an emerging area of research. PDT can also benefit radiotherapy patients due to its complementary and potentially synergistic mechanisms-of-action, ability to treat radioresistant tumors and upregulation of anti-tumoral immune effects. Furthermore, recent advances may allow ionizing radiation energy, including high-energy X-rays, to replace external light sources, opening a novel therapeutic strategy (radioPDT), which is facilitated by novel nanomaterials. This may provide the best of both worlds by combining the precise targeting and treatment depth/volume capabilities of radiation therapy with the high therapeutic index and biological advantages of PDT, without increasing toxicities. Achieving this, however, will require novel agents, primarily developed with nanomaterials. This is under active investigation by many research groups using different approaches.

Phonozen-mediated photodynamic therapy comparing two wavelengths in a mouse model of peritoneal carcinomatosis.

BACKGROUND: This study assessed the therapeutic efficacy of intraperitoneal photodynamic therapy (PDT) using photosensitizer activation at two different wavelengths, 405 and 664 nm, in a mouse model of peritoneal carcinomatosis. METHODS: The dark and light cytotoxicity of chlorin e6-polyvinylpyrrolidone (Phonozen) were measured in vitro under 402 ± 14 and 670 ± 18 nm LED activation in bioluminescent human gastric cancer cells, MKN45-luc. Cell viability was measured at 6 h after irradiation using the PrestoBlue assay. Corresponding in vivo studies were performed in athymic nude mice by intraperitoneal injection of 1 × 106 MKN45-luc cells. PDT was performed 10 d after tumor induction and comprised intraperitoneal injection of Phonozen followed by light irradiation at 3 h, delivered by a diffusing-tip optical fiber placed in the peritoneal cavity and coupled to a 405 or 664 nm diode laser to deliver a total energy of 50 J (20 mice per cohort). Whole-body bioluminescence imaging was used to track the tumor burden after PDT out to 130 days, and 5 mice in each cohort were sacrificed at 4 h post treatment to measure the acute tumor necrosis. RESULTS: Photosensitizer dose-dependent photocytotoxicity was higher in vitro at 405 than 664 nm. In vivo, PDT reduced the tumor growth rate at both wavelengths, with no statistically significant difference. There was substantial necrosis, and median survival was significantly prolonged at both wavelengths compared with controls (46 and 46 vs. 34 days). CONCLUSIONS: Phonozen-mediated PDT results in significant cytotoxicity in vitro as well as tumor necrosis and prolonged survival in vivo following intraperitoneal light irradiation. Blue light was more photocytotoxic than red in vitro and had marginally higher efficacy in vivo.

Exercise training augments brain function and reduces pain perception in adults with chronic pain: A systematic review of intervention studies.

Introduction: Chronic pain (CP) is a leading cause of disability worldwide. Pain may be measured using subjective questionnaires, but understanding the underlying physiology, such as brain function, could improve prognosis. Further, there has been a shift towards cost-effective lifestyle modification for the management of CP. Methods: We conducted a systematic review (Registration: #CRD42022331870) using articles retrieved from four databases (Pubmed, EMBASE, AMED, and CINAHL) to assess the effect of exercise on brain function and pain perception/quality of life in adults with CP. Results: Our search yielded 1879 articles; after exclusion, ten were included in the final review. Study participants were diagnosed with either osteoarthritis or fibromyalgia. However, two studies included "fibromyalgia and low back pain" or "fibromyalgia, back, and complex regional pain." Exercise interventions that were 12 weeks or longer (n = 8/10) altered brain function and improved pain and/or quality of life outcomes. The cortico-limbic pathway, default-mode network, and dorsolateral prefrontal cortex were key regions that experienced alterations post-intervention. All studies that reported an improvement in brain function also demonstrated an improvement in pain perception and/or quality of life. Discussion: Our review suggests that alterations in brain function, notably the cortico-limbic, default-mode and dorsolateral prefrontal cortex, may be responsible for the downstream improvements in the subjective experience of CP. Through appropriate programming (i.e., length of intervention), exercise may represent a viable option to manage CP via its positive influence on brain health.

Photodynamic and Photothermal Therapies: Synergy Opportunities for Nanomedicine.

Tumoricidal photodynamic (PDT) and photothermal (PTT) therapies harness light to eliminate cancer cells with spatiotemporal precision by either generating reactive oxygen species or increasing temperature. Great strides have been made in understanding biological effects of PDT and PTT at the cellular, vascular and tumor microenvironmental levels, as well as translating both modalities in the clinic. Emerging evidence suggests that PDT and PTT may synergize due to their different mechanisms of action, and their nonoverlapping toxicity profiles make such combination potentially efficacious. Moreover, PDT/PTT combinations have gained momentum in recent years due to the development of multimodal nanoplatforms that simultaneously incorporate photodynamically- and photothermally active agents. In this review, we discuss how combining PDT and PTT can address the limitations of each modality alone and enhance treatment safety and efficacy. We provide an overview of recent literature featuring dual PDT/PTT nanoparticles and analyze the strengths and limitations of various nanoparticle design strategies. We also detail how treatment sequence and dose may affect cellular states, tumor pathophysiology and drug delivery, ultimately shaping the treatment response. Lastly, we analyze common experimental design pitfalls that complicate preclinical assessment of PDT/PTT combinations and propose rational guidelines to elucidate the mechanisms underlying PDT/PTT interactions.

Wide-field Stokes polarimetric microscopy for second harmonic generation imaging.

We employ wide-field second harmonic generation (SHG) microscopy together with nonlinear Stokes polarimetry for quick ultrastructural investigation of large sample areas (700 µm × 700 µm) in thin histology sections. The Stokes vector components for SHG are obtained from the polarimetric measurements with incident and outgoing linear and circular polarization states. The Stokes components are used to construct the images of polarimetric parameters and deduce the maps of ultrastructural parameters of achiral and chiral nonlinear susceptibility tensor components ratios and cylindrical axis orientation in fibrillar materials. The large area imaging was employed for lung tumor margin investigations. The imaging shows reduced SHG intensity, increased achiral susceptibility ratio values, and preferential orientation of collagen strands along the boarder of tumor margin. The wide-field Stokes polarimetric SHG microscopy opens a possibility of quick large area imaging of ultrastructural parameters of tissue collagen, which can be used for nonlinear histopathology investigations.

Multispectral singlet oxygen luminescent dosimetry (MSOLD) for Photofrin-mediated Photodynamic Therapy.

Direct detection of singlet-state oxygen ([1O2]) constitutes the holy grail dosimetric method for type II PDT, a goal that can be quantified using multispectral singlet oxygen dosimetry (MSOLD). However, the short lifetime and extremely weak nature of the singlet oxygen signal produced has given rise to a need to improve MSOLD signal-to-noise ratio. This study examines methods for optimizing MSOLD signal acquisition, specifically employing an orthogonal arrangement between detection and PDT treatment light, consisting of two fiber optics - connected to a 632-nm laser and an InGaAs detector respectively. Light collected by the InGaAs detector is then passed through a filter wheel, where spectral emission measurements are taken at 1200 nm, 1240 nm, 1250 nm, 1270 nm, and 1300 nm. The data, after fitting to the fluorescence background and a gaussian-fit for the singlet oxygen peak, is established for the background-subtracted singlet oxygen emission signal. The MSOLD signal is then compared with the singlet oxygen explicit dosimetry (SOED) results, based on direct measurements of in-vivo light fluence (rate), in-vivo Photofrin concentration, and tissue oxygenation concentration. This study focuses on validating the sensitivity and minimum detectability of MSOLD signal in various in-vitro conditions. Finally, the MSOLD device will be tested in Photofrin-mediated PDT for mice bearing Radiation-Induced Fibrosarcoma (RIF) tumors.

Unsupervised determination of lung tumor margin with widefield polarimetric second-harmonic generation microscopy.

The extracellular matrix (ECM) is amongst many tissue components affected by cancer, however, morphological changes of the ECM are not well-understood and thus, often omitted from diagnostic considerations. Polarimetric second-harmonic generation (P-SHG) microscopy allows for visualization and characterization of collagen ultrastructure in the ECM, aiding in better understanding of the changes induced by cancer throughout the tissue. In this paper, a large region of hematoxylin and eosin (H&E) stained human lung section, encompassing a tumor margin, connecting a significant tumor portion to normal tissue was imaged with P-SHG microscopy. The resulting polarimetric parameters were utilized in principal components analysis and unsupervised K-Means clustering to separate normal- and tumor-like tissue. Consequently, a pseudo-color map of the clustered tissue regions is generated to highlight the irregularity of the ECM collagen structure throughout the region of interest and to identify the tumor margin, in the absence of morphological characteristics of the cells.

Picosecond Infrared Laser Mass Spectrometry Identifies a Metabolite Array for 10 s Diagnosis of Select Skin Cancer Types: A Proof-of-Concept Feasibility Study.

Currently, a large number of skin biopsies are taken for each true skin cancer case detected, creating a need for a rapid, high sensitivity, and specificity skin cancer detection tool to reduce the number of unnecessary biopsies taken from benign tissue. Picosecond infrared laser mass spectrometry (PIRL-MS) using a hand-held sampling probe is reported to detect and classify melanoma, squamous cell carcinoma, and normal skin with average sensitivity and specificity values of 86-95% and 91-98%, respectively (at a 95% confidence level) solely requiring 10 s or less of total data collection and analysis time. Classifications are not adversely affected by specimen's quantity of melanin pigments and are mediated by a number of metabolic lipids, further identified herein as potential biomarkers for skin cancer-type differentiation, 19 of which were sufficient here (as a fully characterized metabolite array) to provide high specificity and sensitivity classification of skin cancer types. In situ detection was demonstrated in an intradermal melanoma mouse model wherein in vivo sampling did not cause significant discomfort. PIRL-MS sampling is further shown to be compatible with downstream gross histopathologic evaluations despite loss of tissue from the immediate laser sampling site(s) and can be configured using selective laser pulses to avoid thermal damage to normal skin. Therefore, PIRL-MS may be employed as a decision-support tool to reduce both the subjectivity of clinical diagnosis and the number of unnecessary biopsies currently required for skin cancer screening.

Translational feasibility and efficacy of nasal photodynamic disinfection of SARS-CoV-2.

The lack of therapeutic options to fight Covid-19 has contributed to the current global pandemic. Despite the emergence of effective vaccines, development of broad-spectrum antiviral treatment remains a significant challenge, in which antimicrobial photodynamic therapy (aPDT) may play a role, especially at early stages of infection. aPDT of the nares with methylene blue (MB) and non-thermal light has been successfully utilized to inactivate both bacterial and viral pathogens in the perioperative setting. Here, we investigated the effect of MB-aPDT to inactivate human betacoronavirus OC43 and SARS-CoV-2 in vitro and in a proof-of-principle COVID-19 clinical trial to test, in a variety of settings, the practicality, technical feasibility, and short-term efficacy of the method. aPDT yielded inactivation of up to 6-Logs in vitro, as measured by RT-qPCR and infectivity assay. From a photo-physics perspective, the in vitro results suggest that the response is not dependent on the virus itself, motivating potential use of aPDT for local destruction of SARS-CoV-2 and its variants. In the clinical trial we observed variable effects on viral RNA in nasal-swab samples as assessed by RT-qPCR attributed to aPDT-induced RNA fragmentation causing falsely-elevated counts. However, the viral infectivity in clinical nares swabs was reduced in 90% of samples and undetectable in 70% of samples. This is the first demonstration based on quantitative clinical viral infectivity measurements that MB-aPDT is a safe, easily delivered and effective front-line technique that can reduce local SARS-CoV-2 viral load.

Machine learning-enabled cancer diagnostics with widefield polarimetric second-harmonic generation microscopy.

The extracellular matrix (ECM) collagen undergoes major remodeling during tumorigenesis. However, alterations to the ECM are not widely considered in cancer diagnostics, due to mostly uniform appearance of collagen fibers in white light images of hematoxylin and eosin-stained (H&E) tissue sections. Polarimetric second-harmonic generation (P-SHG) microscopy enables label-free visualization and ultrastructural investigation of non-centrosymmetric molecules, which, when combined with texture analysis, provides multiparameter characterization of tissue collagen. This paper demonstrates whole slide imaging of breast tissue microarrays using high-throughput widefield P-SHG microscopy. The resulting P-SHG parameters are used in classification to differentiate tumor from normal tissue, resulting in 94.2% for both accuracy and F1-score, and 6.3% false discovery rate. Subsequently, the trained classifier is employed to predict tumor tissue with 91.3% accuracy, 90.7% F1-score, and 13.8% false omission rate. As such, we show that widefield P-SHG microscopy reveals collagen ultrastructure over large tissue regions and can be utilized as a sensitive biomarker for cancer diagnostics and prognostics studies.

Preclinical evaluation of a clinical prototype transrectal diffuse optical tomography system for monitoring photothermal therapy of focal prostate cancer.

SIGNIFICANCE: Our work demonstrates in preclinical models that continuous-wave transrectal diffuse optical tomography (TRDOT) can be used to accurately monitor photothermal therapy (PTT) and, in particular, the progression of the photocoagulation boundary toward the rectum. When used in patients, this should prevent rectal damage during PTT, thereby achieving maximum treatment efficacy while ensuring safety, using a technology platform suitable for wide dissemination. AIM: We aim to validate that TRDOT measurements analyzed using a shape-based image-reconstruction algorithm (SBDOT) allow localization of the photocoagulation boundary during PTT within ±1 mm toward the rectum in the transverse plane. APPROACH: TRDOT measurements were performed in tissue-simulating phantoms, ex vivo tissues, and an in vivo canine prostate model. The accuracy and sensitivity of reconstructing the size and location of the coagulation zone were determined, based on changes in the tissue absorption and reduced scattering coefficients upon photocoagulation. The reconstruction also yields the native and coagulated tissue optical properties. RESULTS: The TRDOT measurements and SBDOT reconstruction algorithm were confirmed to perform sufficiently well for clinical translation in PTT monitoring, recovering the location of the coagulation boundary within ±1 mm compared to the true value as determined by direct visualization postexcision and/or MRI. CONCLUSIONS: Implementing previously described TRDOT instrumentation and SBDOT image reconstruction in different tissue models confirms the potential for clinincal translation, including required refinements of the system and reconstruction algorithm.

Perspective on the integration of optical sensing into orthopedic surgical devices.

SIGNIFICANCE: Orthopedic surgery currently comprises over 1.5 million cases annually in the United States alone and is growing rapidly with aging populations. Emerging optical sensing techniques promise fewer side effects with new, more effective approaches aimed at improving patient outcomes following orthopedic surgery. AIM: The aim of this perspective paper is to outline potential applications where fiberoptic-based approaches can complement ongoing development of minimally invasive surgical procedures for use in orthopedic applications. APPROACH: Several procedures involving orthopedic and spinal surgery, along with the clinical challenge associated with each, are considered. The current and potential applications of optical sensing within these procedures are discussed and future opportunities, challenges, and competing technologies are presented for each surgical application. RESULTS: Strong research efforts involving sensor miniaturization and integration of optics into existing surgical devices, including K-wires and cranial perforators, provided the impetus for this perspective analysis. These advances have made it possible to envision a next-generation set of devices that can be rigorously evaluated in controlled clinical trials to become routine tools for orthopedic surgery. CONCLUSIONS: Integration of optical devices into surgical drills and burrs to discern bone/tissue interfaces could be used to reduce complication rates across a spectrum of orthopedic surgery procedures or to aid less-experienced surgeons in complex techniques, such as laminoplasty or osteotomy. These developments present both opportunities and challenges for the biomedical optics community.

Multispectral label-free Raman spectroscopy can detect ovarian and endometrial cancer with high accuracy.

Up to 70% of ovarian cancer patients are diagnosed with advanced-stage disease and the degree of cytoreduction is an important survival prognostic factor. The aim of this study was to evaluate if Raman spectroscopy could detect cancer from different organs within the abdominopelvic region, including the ovaries. A Raman spectroscopy probe was used to interrogate specimens from a cohort of nine patients undergoing cytoreductive surgery, including four ovarian cancer patients and three patients with endometrial cancer. A feature-selection algorithm was developed to determine which spectral bands contributed to cancer detection and a machine-learning model was trained. The model could detect cancer using only eight spectral bands. The receiver-operating-characteristic curve had an area-under-the-curve of 0.96, corresponding to an accuracy, a sensitivity and a specificity of 90%, 93% and 88%, respectively. These results provide evidence multispectral Raman spectroscopy could be developed to detect ovarian cancer intraoperatively.

Radiodynamic Therapy Using TAT Peptide-Targeted Verteporfin-Encapsulated PLGA Nanoparticles.

Radiodynamic therapy (RDT) is a recent extension of conventional photodynamic therapy, in which visible/near infrared light irradiation is replaced by a well-tolerated dose of high-energy X-rays. This enables greater tissue penetration to allow non-invasive treatment of large, deep-seated tumors. We report here the design and testing of a drug delivery system for RDT that is intended to enhance intra- or peri-nuclear localization of the photosensitizer, leading to DNA damage and resulting clonogenic cell kill. This comprises a photosensitizer (Verteporfin, VP) incorporated into poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) that are surface-functionalized with a cell-penetrating HIV trans-activator of transcription (TAT) peptide. In addition to a series of physical and photophysical characterization studies, cytotoxicity tests in pancreatic (PANC-1) cancer cells in vitro under 4 Gy X-ray exposure from a clinical 6 MV linear accelerator (LINAC) showed that TAT targeting of the nanoparticles markedly enhances the effectiveness of RDT treatment, particularly when assessed by a clonogenic, i.e., DNA damage-mediated, cell kill.