NIRF Imaging Research Articles

Indocyanine green and methylene blue dye guided sentinel lymph node biopsy in patients with penile cancer (PeCa): results of 50 inguinal basins assessed at a single institution in India.

The primary aim of this study was to validate the reliability, sensitivity and safety profile of novel combination of ICG- methylene blue dye as an SN tracer for PeCa. This is a validation and non-randomised prospective observational study involving 25 patients (50 inguinal basins) who underwent SLNB where in ICG and methylene blue were used for localisation. The patients with clinically node negative groins were recruited in the study. SNs were identified intraoperatively using near infrared fluorescence imaging (NIRF Imaging system, SPY-PHI, Stryker, Sweden) and blue dye. The numbers of SNs identified by each tracer and the rates of complications and nodal recurrence during the followup. Overall 137 SNs were identified intraoperatively. Among the 137 SNs excised fluorescence, blue dye and Combined (blue + green) identified 57(41.6%), 27 (19.7%), and 51 (37.2%), respectively. The average number of SLNs removed per patient was 5 (range, 1-11) with sentinel lymph nodes detection rate at 94% (47/50). Seven patients had malignancy on SLNB and underwent ipsilateral radical inguinal lymphadenectomy. One patient had false negative SN and positive node in modified inguinal lymphadenectomy specimen. No adverse events were observed in all cases. The Novel combination of ICG fluorescence-Methylene blue dye technique is simple, reliable and safe. Moreover, it demonstrates a high SLN detection rate with a low false-negative rate, and it avoids radiation exposure.

The Feasibility of Assessing Perfusion of the Bone Using Quantitative ICG Fluorescence Imaging

Abstract Background Near-infrared fluorescence (NIRF) imaging using indocyanine green (ICG) allows perfusion to be visualized and objective perfusion parameters can be provided after additional measurements. Therefore, it has great potential in predicting adequate tissue perfusion. However, regarding bone tissue, evidence of the feasibility and usefulness of NIRF imaging using ICG is very limited. Methods A prospective monocentric pilot study was carried out at a tertiary hospital in the Netherlands. Patients undergoing autologous breast reconstruction from August 2021 to August 2022 were included. During surgery, ICG (0.1 mg/kg) was injected intravenously and a fluorescent angiogram of 4 minutes was made directly after injection. Post hoc time-intensity curves were generated for a 5-mm region of interest (ROI) positioned on the cross-sectional lateral surface of the rib. The first moment of increase of intensity was defined as t0. Fluorescent parameters included ingress and egress of ICG. Results Nine patients and 11 ribs were included for further analysis. Time-intensity curves were generated for endosteal measurement in 10 ribs. Three of the curves showed a steep and well-defined ingress and egress. In all other patients, the curves showed a much more flattened ingress and egress. Periosteal measurement was performed in nine ribs. No adverse events related to the ICG injection were observed intraoperatively. Conclusion This feasibility study suggests that quantitative NIRF imaging using ICG can provide objective parameters of endosteal rib perfusion. Larger prospective series are needed to investigate the value of NIRF imaging using ICG to assess bone perfusion intraoperatively and to establish cutoff values for adequate bone perfusion.

Exploring the anti-migraine effects of Tianshu capsule: chemical profile, metabolic behavior, and therapeutic mechanisms

BackgroundMigraine is widely recognized as the third most prevalent medical condition globally. Tianshu capsule (TSC), derived from “Da Chuan Xiong Fang” of the Jin dynasty, is integral in the clinical treatment of migraine. However, the chemical properties and therapeutic mechanisms of TSC different portions remain unclear. PurposeThis study was designed to investigate the effects of TSC different portions (including small molecular TSCP-SM and polysaccharides TSC-P) on migraine and explore the underlying mechanisms. Study design and methodsFirst of all, migraine rats were established by nitroglycerin injection and treated with TSC, TSC-P, and TSC-SM. ELISA, qPCR, and immunofluorescence were used to evaluate the pharmacological effects on migraine rats. Secondly, UPLC-Q/TOF-MS and GC–-MS were employed to detect the components of TSC-SM. PMP-HPLC, NMR, FT-IR, UV–Vis, AFM, and SEM were used for the chemical profiling of polysaccharides. Thirdly, the metabolic behavior profile of TSC-P was characterized by oral administrated fluorescence-labeled TSC-P and detected by NIRF imaging. Finally, the anti-migraine mechanisms were explored by determining the composition of gut microbiota, analyzing colonic short-chain fatty acids (SCFAs), and examining serum tryptophan-related metabolites. ResultsBoth small molecules (45 volatiles and 114 small molecules) and polysaccharides (including Glc, Ara, Gal, and Gal A) have exhibited effectiveness in alleviating migraine, and this efficacy is associated with reduced CGRP and iNOS levels, along with increased β-EP expressions. Further mechanistic exploration revealed that small-molecules exhibited effectiveness in migraine treatment by exerting antioxidative actions, while polysaccharides demonstrated superior therapeutic effects in regulating 5-HT levels. By monitoring the metabolic behavior of polysaccharides with fluorescent labeling, it was observed that TSC-P exhibited poor absorption. Instead, TSC-P demonstrated its therapeutic effects by modulating the aberrations in gut microbiota (including Alloprevotella, Muribaculaceae_ge, and Ruminococcaceae_UCG-005), cecum short-chain fatty acids (such as isobutyric, isovaleric, and valeric acids), and serum tryptophan-related metabolites (including indole-3-acetamide, tryptophol, and indole-3-propionic acid). ConclusionThis research provides innovative insights into chemical composition, metabolic behavior, and proposed anti-migraine mechanisms of TSC from a polarity-based perspective, and pioneering an exploration focused on the polysaccharide components within TSC for the first time.

Fibronectin-Targeting Dual-Modal MR/NIRF Imaging Contrast Agents for Diagnosis of Gastric Cancer and Peritoneal Metastasis.

The prevalence and fatality rates of gastric cancer (GC) remain elevated, with advanced stages presenting a grim prognosis. Noninvasive diagnosis of GC cancer often proves challenging until the disease has progressed to an advanced stage or metastasized. Initially, the level of fibronectin (FN) in cancer-associated fibroblasts (CAFs) of GC was at least 3.7 times higher than that in normal fibroblasts. Herein, two FN-targeting magnetic resonance/near-infrared fluorescence (MR/NIRF) imaging contrast agents were developed to detect GC and peritoneal metastasis noninvasively. The probes CREKA-Cy7-(Gd-DOTA) and CREKA-Cy7-(Gd-DOTA)3 demonstrated significant FN-targeting capability (with dissociation constants of 1.0 and 2.1 mM) and effective MR imaging performance (with proton relaxivity values of 9.66 and 27.44 mM-1 s-1 at 9.4 T, 37 °C). In vivo imaging revealed a high signal-to-noise ratio and successful visualization of GC metastasis using NIRF imaging as well as successful tumor detection in MR imaging. Therefore, this study highlights the potential of FN-targeting probes for GC diagnosis and aids in the advancement of new diagnostic strategies for the clinical detection of GC.

Quantitative analysis of intestinal perfusion with indocyanine green (ICG) and methylene blue (MB) using a single clinically approved fluorescence imaging system: a demonstration in a porcine model

BackgroundNear-infrared fluorescence (NIRF) angiography with intraoperative administration of indocyanine green (ICG) has rapidly disseminated in clinical practice. Another clinically approved, and widely available dye, methylene blue (MB), has up to now not been used for this purpose. Recently, we demonstrated promising results for the real-time evaluation of intestinal perfusion using this dye. The primary aim of this study was to perform a quantitative analysis of bowel perfusion assessment for both ICG and MB.MethodsFour mature female Landrace pigs underwent laparotomy under general anesthesia. An ischemic bowel loop with five regions of interest (ROIs) with varying levels of perfusion was created in each animal. An intravenous (IV) injection of 0.25 mg/kg–0.50 mg/kg MB was administered after 10 min, followed by NIRF imaging in MB mode and measurement of local lactate levels in all corresponding ROIs. This procedure was repeated in ICG mode (IV dose of 0.2 mg/kg) after 60 min. The quest spectrum fluorescence camera (Quest Medical Imaging, Middenmeer, The Netherlands) was used for NIRF imaging of both MB and ICG.ResultsIntraoperative NIRF imaging of bowel perfusion assessment with MB and ICG was successful in all studied animals. Ingress (i/s) levels were calculated and correlated with local lactate levels. Both MB and ICG ingress values showed a significant negative correlation (r = − 0.7709; p = < 0.001; r = − 0.5367, p = 0.015, respectively) with local lactate levels. This correlation was stronger for MB compared to ICG, although ICG analysis showed higher absolute ingress values.ConclusionOur fluorescence quantification analysis validates the potential to use MB for bowel perfusion assessment besides the well-known and widely used ICG. Further human studies are necessary to translate our findings to clinical applications.

Abstract 4656: Novel mitochondria-based targeting overcomes resistance in prostate cancer

Abstract Background: Prostate cancer (PC) is a major contributor to cancer-related mortality in men, with metastatic castration-resistant PC (mCRPC) presenting a significant obstacle due to its resistance to standard treatments. Current therapies, including nonsteroidal antiandrogens and chemotherapy drugs, provide limited effectiveness and are often accompanied by severe side effects. Recently, statins have gained attention as antitumor agents, demonstrating chemopreventive effects on a range of human cancers while maintaining a well-established safety profile. However, the use of statins as standalone treatment is limited by insufficient cancer cell targeting. Methods: We synthesized a DZ-simvastatin (DZ-SIM) conjugate that combines a near-infrared heptamethine carbocyanine dye (DZ) with simvastatin for selective tumor cell targeting. Cancer cell survival was measured by crystal violet and MTT assay. Co-localization of DZ-SIM with mitochondria and lysosome in cells was detected under confocal microscope using Mitotracker and Lysotracker. Cellular and mitochondrial cholesterol levels were detected by LC-MS. Male nude mice were inoculated subcutaneously with 22Rv1-Luc cells as a prostate cancer xenograft mouse model. We used Swiss-PDB and Rosetta algorithms to model the structure of the OATP1B3 channel. Oxygen consumption rate and extracellular acidification rate were measured by Seahorse assay. After treatment with DZ-SIM, differentially expressed genes profiling of prostate cancer cells was performed using RNAseq. Results: Our research demonstrates the conjugate's ability to effectively kill androgen-independent and metastatic PC cells without harming normal cells. Furthermore, DZ-SIM has been shown to kill multiple human PC cell lines, regardless of androgen receptor status or therapeutic resistance. DZ-SIM has been shown to inhibit growth in human prostate cancer cell line xenograft models. NIR fluorescence imaging showed specific localization of DZ-SIM to cancer, but not to normal cells and tissues. It could localize into cancer cells by binding the transmembrane transporter OATP to primarily target subcellular organelle mitochondria, decreasing the OCR and ECAR of cancer cells. DZ-SIM can bind to multiple cytosolic and mitochondrial proteins as detected by NIRF imaging. Cholesterol levels also decrease in whole cells and mitochondria. Ingenuity Pathway Analysis (IPA) of RNAseq showed that the four signaling pathways in the DZ-SIM treatment group are significantly enriched in genes related to cholesterol synthesis. Conclusion: DZ-SIM specifically targets PC cells and their mitochondria, resulting in loss of mitochondrial function and consequent cell death. With a unique subcellular targeting strategy, the conjugate holds the potential to outperform existing chemotherapeutic drugs. Citation Format: Yan Ou, Adrian Lim, Ning Zhai, Mouad Edderkaoui, Stephen Pandol, Ruoxiang Wang, Yi Zhang. Novel mitochondria-based targeting overcomes resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4656.

Comparison of Postoperative Outcomes Between Near-Infrared Fluorescent Imaging-Guided Mediastinal Lymphadenectomy and Conventional Surgery for Esophageal Cancer.

The study aimed to evaluate the efficacy of using near-infrared fluorescent imaging (NIRF) imaging with indocyanine green as an intraoperative tool for achieving complete mediastinal lymph node (LN) resection. Between September 2019 and July 2021, patients with potential for esophagectomy due to middle and lower thoracic esophageal cancer were enrolled in this study. All patients were scheduled for NIRF-guided mediastinal lymphadenectomy during esophageal cancer surgery and were appropriately assigned to the NIRF group. Patients who underwent esophagectomy between September 2017 and September 2019 were assigned to the historical control group upon satisfying the inclusion/exclusion criteria. Surgical outcomes and the number of removed LNs were compared between the two groups using 1:1 propensity score matching. Of 67 eligible patients, 59 patients were included in the NIRF group after postsurgical exclusions. The operative time was significantly shorter in the NIRF group than in the historical control group [180 (140-420)min versus 202 (137-338)min; P < 0.001]. The incidence of postoperative chylothorax and hoarseness were significantly lower in the NIRF group than in the historical control group (0% versus 10.2 %; P = 0.036, 3.4% versus 13.6%; P = 0.047). The number of dissected total LNs, mediastinal LNs, and negative LNs was significantly larger in the NIRF group than in the historical control group. The number of overall metastatic LNs and abdominal LNs was comparable between the two groups. NIRF imaging can assist in the thorough and complete mediastinal LNs dissections without increasing complications in patients undergoing esophagectomy.

Modular Self-Assembling Dendrimer Nanosystems for Magnetic Resonance and Multimodality Imaging of Tumors.

Bioimaging is a powerful tool for diagnosing tumors but remains limited in terms of sensitivity and specificity. Nanotechnology-based imaging probes able to accommodate abundant imaging units with different imaging modalities are particularly promising for overcoming these limitations. In addition, the nanosized imaging agents can specifically increase the contrast of tumors by exploiting the enhanced permeability and retention effect. A proof-of-concept study is performed on pancreatic cancer to demonstrate the use of modular amphiphilic dendrimer-based nanoprobes for magnetic resonance (MR) imaging (MRI) or MR/near-infrared fluorescence (NIRF) multimodality imaging. Specifically, the self-assembly of an amphiphilic dendrimer bearing multiple Gd3+ units at its terminals, generates a nanomicellar agent exhibiting favorable relaxivity for MRI with a good safety profile. MRI reveals an up to two-fold higher contrast enhancement in tumors than in normal muscle. Encapsulating the NIRF dye within the core of the nanoprobe yields an MR/NIRF bimodal imaging agent for tumor detection that is efficient both for MRI, at Gd3+ concentrations 1/10 the standard clinical dose, and for NIRF imaging, allowing over two-fold stronger fluorescence intensities. These self-assembling dendrimer nanosystems thus constitute effective probes for MRI and MR/NIRF multimodality imaging, offering a promising nanotechnology platform for elaborating multimodality imaging probes in biomedical applications.

A SPECT/NIR Fluorescence Dual-Modality Imaging Agent Composed of Drugs and Hospital Available Isotope for Preoperative Sentinel Lymph Node Mapping and Intraoperative Biopsy.

Sentinel lymph node (SLN) mapping-guided biopsy is crucial for cancer staging and treatment. Optical/nuclide dual-modality imaging agents for mapping SLN are ideal for preoperative planning and intraoperative biopsy, which are enabled by penetration-depth unlimited nuclide imaging and dynamic real-time optical imaging, respectively. However, commonly reported dual-modality imaging agents are composed of novel but safety-unproven materials, making their quick clinical translation challenging. Herein, we report a novel nanoparticle composed of facile hospital-available drugs and isotope for single-photon emission computed tomography (SPECT)/near-infrared (NIR) fluorescence imaging to detect SLNs. Indocyanine green-human serum albumin (ICG-HSA) nanoparticles (NPs) were synthesized by ICG-induced HSA self-assembly and further 99mTc-labeling via a one-step, facile hospital-available method. After injecting 99mTc-ICG-HSA into the rats' forepaw pads, the rats' draining axillary lymph nodes were visualized by preoperative mapping with SPECT/CT and intraoperative biopsy with NIR fluorescence. The axillary lymph nodes of rats were identified by pathology and fluorescent staining after execution. Additionally, its toxicity testing and comparison with 99mTc-sulfur colloid imaging were also explored. The study reported a self-assembled 99mTc-ICG-HSA with a high radiochemical yield (85.6 ± 3.8%). Compared with conventional 99mTc-sulfur colloid, 99mTc-ICG-HSA NPs showed faster SLN identification, higher renal clearance, and lower hepatic retention. Furthermore, NIRF imaging allowed for the accurate visualization of the SLN and guided SLN biopsy intraoperatively. Notably, the 99mTc-ICG-HSA NPs were composed of hospital-available drugs and isotope, which are safe for acute toxicity evaluation by a certified institute. The proposed 99mTc-ICG-HSA NPs are safe and capable of noninvasive SLN identification and biopsy guidance with multi-modal imaging strategies and could be a promising tool for clinically assisted SLN biopsy.

P-150 - Folate receptor targeted 64Cu/ICG-labeled layered double hydroxide nanoprobe for PET/CT and NIRF imaging

P-150 - Folate receptor targeted 64Cu/ICG-labeled layered double hydroxide nanoprobe for PET/CT and NIRF imaging

Efficacy and outcome of indocyanine green-based intraoperative cholangiography using near-infrared fluorescence imaging: A prospective study.

The most dreaded complication during laparoscopic cholecystectomy still remains to be injury to the common bile duct. The primary cause for bile duct injury during LC is misinterpretation of the biliary anatomy. Intra-operative cholangiography was introduced as a means of reducing the chances of biliary injury, done using Fluoroscopic imaging or Near-infrared fluorescence imaging method. NIRF is one of the most popular imaging methods in biomedical sciences. Indocyanine Green is sterile and water soluble which completely binds to albumin and is excreted in bile. This prospective study was conducted among 70 patients between July 2020 and December 2021. Subjects were administered 5mg of ICG dye pre-operatively and procedure performed using Karl Storz HD image S1 system with a D-light P light source for NIRF imaging. The average duration of surgery was 58.10 minutes. After calot's dissection, the CBD was visualized in 88.71 % patients, with a mean time to visualization at 26.33 minutes. The cystic duct was visualized in 87.3% cases with a mean time of visualization of 32.10 minutes. The hepatic duct was visualized in 28.57% and the hepatic duct-CBD confluence was visualized in 34.28% patients. Near infrared imaging based intra-operative cholangiography, using Indocyanine Green dye, during Lap. Cholecystectomy is an easy, useful and inexpensive method of visualizing the biliary ductal anatomy.

Imaging Investigation of Hepatocellular Carcinoma Progress via Monitoring γ-Glutamyltranspeptidase Level with a Near-Infrared Fluorescence/Photoacoustic Bimodal Probe.

Hepatocellular carcinoma (HCC) is one of the main principal causes of cancer death, and the late definite diagnosis limits therapeutic approaches in time. The early diagnosis of HCC is essential, and the previous investigations on the biomarkers inferred that the γ-glutamyltranspeptidase (GGT) level could indicate the HCC process. Herein, a near-infrared fluorescence/photoacoustic (NIRF/PA) bimodal probe, CySO3-GGT, was developed for monitoring the GGT level and thus to image the HCC process. After the in-solution tests, the bimodal response was convinced. The various HCC processes were imaged by CySO3-GGT at the cellular level. Then, the CCl4-induced HCC (both induction and treatment) and the subcutaneous and orthotopic xenograft mice models were selected. All throughout the tests, CySO3-GGT achieved NIRF and PA bimodal imaging of the HCC process. In particular, CySO3-GGT could effectively realize 3D imaging of the HCC nodule by visualizing the boundary between the tumor and the normal tissue. The information here might offer significant guidance for the dynamic monitoring of HCC in the near future.

NIRF/PET imaging of γδ T cells via metabolic glycoengineering and bioorthogonal labeling

Non-invasive tracking of γδ T cells may help to predict patient responsiveness and therapeutic efficacy. In this study, we developed an efficient γδ T cell NIRF/PET imaging strategy by combining metabolic glycoengineering and bioorthogonal click chemistry. Initially, γδ T cells were incubated with N-azidoacetyl-D-mannosamine-tetraacylated, and azide (N3) groups were then incorporated onto the surface of γδ T cells via metabolic glycoengineering to obtain N3-γδ T cells. Next, Cy5.5 dibenzylcyclooctyne (DBCO-Cy5.5) and the short half-life 68Ga-labeled chelator 68Ga-NETA-DBCO were conjugated with N3-γδ T cells via in vitro or in vivo bioorthogonal metal-free click chemistry for NIRF and PET imaging, respectively. In Daudi tumor-bearing mice, Cy5.5-labeled γδ T cells migrated specifically to tumor sites, and adoptive cells were observed clearly via non-invasive NIRF imaging over 10 days. Furthermore, in the pre-targeted PET images, high tumor uptake of 68Ga-NETA-DBCO was observed in mice three days post-injection of N3-γδ T cells, which was 1.5-fold higher than that of control mice. Interestingly, dead-Cy5.5-γδ T cells also showed high tumor targeting and good tumor retention, peaking at day 1 and retaining NIRF signal up to 10 days, indicating a high potential of γδ T cells as a drug delivery system. These findings demonstrated that a bioorthogonal labeling method followed by NIRF/PET imaging maps the biodistribution, migration, and tumor-homing efficiency of adoptive γδ T cells in vivo, representing a new potential γδ T cell imaging technology.

Simultaneous fluorescence imaging of bowel perfusion and ureter delineation using methylene blue: a demonstration in a porcine model

BackgroundIntraoperative near-infrared fluorescence imaging (NIRF) with preoperative optical dye administration is a promising technique for quick and easy intraoperative visualization of the ureter and for an improved, real-time assessment of intestinal perfusion. During colorectal surgery, there is a need for simultaneous non-invasive ureteral imaging and bowel perfusion assessment, using one single camera system. The purpose of this study is to investigate the feasibility of simultaneous intestinal perfusion and ureteral imaging using a single commercially available NIRF imaging system.MethodsSix Landrace pigs underwent laparotomy under general anesthesia in this experiment. An intravenous (IV) dose of 0.2 mg/kg indocyanine green (ICG) was given to assess bowel perfusion. Two pairs received a methylene blue (MB) iv injection of 0.75, 0.50 or 0.25 mg/kg respectively to investigate ureteral visualization. Quest Spectrum Fluorescence Camera (Quest Medical Imaging, Middenmeer, The Netherlands) was used for NIRF imaging.ResultsUreter visualization and bowel perfusion under NIRF imaging was achieved in all animals. All ureters were visible after five to ten minutes and remained clearly visible until the end of every experiment (120–420 min). A mixed model analysis did not show any significant differences neither between the three groups nor over time. Importantly, we demonstrated that bowel perfusion could be visualized with methylene blue (MB) as well. We observed no interference between ICG and MB and a faster washout of MB.ConclusionWe successfully demonstrated simultaneous fluorescence angiography with ICG and ureteral imaging with MB in the same surgical procedure, with the same commercially available NIRF imaging equipment. More importantly, we showed that the use MB is adequate for bowel perfusion assessment and ureter visualization with this NIRF imaging system. Besides, MB showed an earlier washout time, which can be clinical beneficial as a repeated dye injection may be necessary during a surgical procedure.

Visual Investigation of Tumor-Promoting Fibronectin Potentiated by Obesity in Pancreatic Ductal Adenocarcinoma Using an MR/NIRF Dual-Modality Dendrimer Nanoprobe.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease characterized by dense stroma. Obesity is an important metabolic factor that greatly increases PDAC risk and mortality, worsens progression and leads to poor chemotherapeutic outcomes. With omics analysis, magnetic resonance and near-infrared fluorescence (MR/NIRF) dual-modality imaging and molecular functional verification, obesity as an important risk factor is proved to modulate the extracellular matrix (ECM) components and enhance Fibronectin (FN) infiltration in the PDAC stroma, that promotes tumor progression and worsens response to chemotherapy by reducing drug delivery. In the study, to visually evaluate FN in vivo and guide PDAC therapy, an FN-targeted nanoprobe, NP-CREKA, is synthesized by conjugating gadolinium chelates, NIR797 and fluorescein isothiocyanate to a polyamidoamine dendrimer functionalized with targeting peptides. A dual-modality strategy combining MR and NIRF imaging is applied, allowing effective visualization of FN in orthotopic PDAC with high spatial resolution, ideal sensitivity and excellent penetrability, especially in obese mice. In conclusion, the findings provide new insights into the potential of FN as an ideal target for therapeutic evaluation and improving treatment efficacy in PDAC, hopefully improving the specific management of PDAC in lean and obese hosts.

Electrochemotherapy with Bleomycin Supported by NIRF Imaging with Indocyanine Green (ICG)—In Vitro and In Vivo Case Study

Electrochemotherapy (ECT) with bleomycin has been effectively used in recent years to treat various skin tumors. Microsecond electric pulses significantly improve bleomycin (BLM) delivery and its anticancer potential. Up to now, we can determine electric field distribution in the targeted tissue, however, the distribution of the injected drug is still not well known. In this study, we propose the combination of indocyanine green (ICG) with bleomycin as a practical approach for ECT, enabling drug distribution control and detection. Normal skeletal muscle (L6) and fibrosarcoma (WEHI-164) cells were used for the viability evaluation by MTT assay after 24 and 72 h. Cells were exposed to the ESOPE protocol alone and in combination with drugs. Additionally, visualization of the uptake of ICG and ICG + BLM supported by electroporation was performed by confocal microscopy. The mast cell tumor (MCTs) was diagnosed in the feline case. The mixture of ICG + BLM was injected into the tumor, and ECT was performed under near-infrared fluorescence imaging (NIRF). The obtained results indicate the safety of the used procedure in vitro and in vivo. ICG does not affect ECT protocols in vitro. No significant cell viability decrease was noted only in the case of WEHI-164 cells post-ECT. Moreover, it does not adversely affect the procedure; in the case of in vivo surgery, it helps to control the drug distribution before and after ECT and identify the sentinel lymph node.

A Novel PSMA-Targeted Probe for NIRF-Guided Surgery and Photodynamic Therapy: Synthesis and Preclinical Validation.

A total of 20% to 50% of prostate cancer (PCa) patients leave the surgery room with positive tumour margins. The intraoperative combination of fluorescence guided surgery (FGS) and photodynamic therapy (PDT) may be very helpful for improving tumour margin delineation and cancer therapy. PSMA is a transmembrane protein overexpressed in 90-100% of PCa cells. The goal of this work is the development of a PSMA-targeted Near InfraRed Fluorescent probe to offer the surgeon a valuable intraoperative tool for allowing a complete tumour removal, implemented with the possibility of using PDT to kill the eventual not resected cancer cells. PSMA-617 binding motif was conjugated to IRDye700DX-NHS and the conjugation did not affect the photophysical characteristics of the fluorophore. The affinity of IRDye700DX-PSMA-617 towards PCa cells followed the order of their PSMA expression, i.e., PC3-PIP &gt; LNCaP &gt; PC3, PC3-FLU. NIRF imaging showed a significant PC3-PIP tumour uptake after the injection of 1 or 5 nmol with a maximum tumour-to-muscle ratio (ca. 60) observed for both doses 24 h post-injection. Importantly, urine, healthy prostate, and the bladder were not fluorescent at 24 h post-injection. Flow cytometry and confocal images highlighted a co-localization of PSMA+ cells with IRDye700DX-PSMA uptake. Very interestingly, ex vivo analysis on a tumour specimen highlighted a significant PSMA expression by tumour-associated macrophages, likely attributable to extracellular vesicles secreted by the PSMA(+) tumour cells. FGS proved that IRDye700DX-PSMA was able to easily delineate tumour margins. PDT experiments showed a concentration-dependent decrease in cell viability (from 75% at 10 nM to 12% at 500 nM), whereas controls did not show any cytotoxicity. PC3-PIP tumour-bearing mice subjected to photodynamic therapy showed a delayed tumour growth. In conclusion, a novel PSMA-targeted NIRF dye with dual imaging-PDT capabilities was synthesized and displayed superior specificity compared to other small PSMA targeted molecules.

P-216 - Development of a dual imaging probe for bimodal PET/ two-photon NIRF imaging

P-216 - Development of a dual imaging probe for bimodal PET/ two-photon NIRF imaging

Targeting Contrast Agents With Peak Near-Infrared-II (NIR-II) Fluorescence Emission for Non-invasive Real-Time Direct Visualization of Thrombosis.

Thrombosis within the vasculature arises when pathological factors compromise normal hemostasis. On doing so, arterial thrombosis (AT) and venous thrombosis (VT) can lead to life-threatening cardio-cerebrovascular complications. Unfortunately, the therapeutic window following the onset of AT and VT is insufficient for effective treatment. As such, acute AT is the leading cause of heart attacks and constitutes ∼80% of stroke incidences, while acute VT can lead to fatal therapy complications. Early lesion detection, their accurate identification, and the subsequent appropriate treatment of thrombi can reduce the risk of thrombosis as well as its sequelae. As the success rate of therapy of fresh thrombi is higher than that of old thrombi, detection of the former and accurate identification of lesions as thrombi are of paramount importance. Magnetic resonance imaging, x-ray computed tomography (CT), and ultrasound (US) are the conventional non-invasive imaging modalities used for the detection and identification of AT and VT, but these modalities have the drawback of providing only image-delayed indirect visualization of only late stages of thrombi development. To overcome such limitations, near-infrared (NIR, ca. 700–1,700 nm) fluorescence (NIRF) imaging has been implemented due to its capability of providing non-invasive real-time direct visualization of biological structures and processes. Contrast agents designed for providing real-time direct or indirect visualization of thrombi using NIRF imaging primarily provide peak NIR-I fluorescence emission (ca. 700–1,000 nm), which affords limited tissue penetration depth and suboptimal spatiotemporal resolution. To facilitate the enhancement of the visualization of thrombosis via providing detection of smaller, fresh, and/or deep-seated thrombi in real time, the development of contrast agents with peak NIR-II fluorescence emission (ca. 1000–1,700 nm) has been recently underway. Currently, however, most contrast agents that provide peak NIR-II fluorescence emissions that are purportedly capable of providing direct visualization of thrombi or their resultant occlusions actually afford only the indirect visualization of such because they only provide for the (i) measuring of the surrounding vascular blood flow and/or (ii) simple tracing of the vasculature. These contrast agents do not target thrombi or occlusions. As such, this mini review summarizes the extremely limited number of targeting contrast agents with peak NIR-II fluorescence emission developed for non-invasive real-time direct visualization of thrombosis that have been recently reported.

Acidic tumor microenvironment-sensitive liposomes enhance colorectal cancer therapy by acting on both tumor cells and cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) play a crucial role in facilitating tumor invasion and metastasis, which act as the "soil" in the tumor microenvironment (TME). Accordingly, it would be a promising strategy to enhance the antitumor effect by killing both tumor cells and CAFs simultaneously. Herein, novel TME acid-responsive liposomes for co-delivery of IRI and 398 (IRI&398-s-LPs) were developed, in which the rapid release of both drugs could be triggered under acidic conditions. Notably, a CT-26/3T3 cell co-culture system was used to mimic the real TME both in vitro and in vivo. Cellular immunofluorescence revealed that IRI&398-s-LPs could efficiently decrease the activation of CAFs. In vitro cytotoxicity evaluation demonstrated that IRI&398-s-LPs exhibited higher cytotoxicity than the other liposomal formulations in the CT-26 and CT-26/3T3 cell co-culture system. In vivo NIRF imaging showed that the IRI&398-s-LPs could increase drug accumulation in the tumor sites. Furthermore, IRI&398-s-LPs not only presented superior in vivo anti-tumor activity in CT-26 bearing BALB/c mice, but also enhanced the effect in CT-26/3T3 cell bearing mice with decreased collagen and CAF biomarker expression. Furthermore, IRI&398-s-LPs also presented superior anti-metastatic efficiency in a lung metastasis model. These results indicated that this combinational strategy for eliminating both tumor cells and CAFs provides a new approach for cancer therapy, and the prepared TME-responsive liposomes for co-delivery of drugs hold promising clinical application prospects.