Galactose targeted pH-responsive copolymer conjugated with near infrared fluorescence probe for imaging of intelligent drug delivery.

Sarcomas are cancers of the bones, muscles, nerves, and fat that require complete surgical removal for cure. The primary surgical goal therefore is to remove the tumor with a zone of normal, non-cancerous tissue surrounding the tumor, considered a ‘negative’ surgical margin. At present, surgeons rely on radiologic imaging and visual and tactile clues to gauge cancer depth and guide surgical excision. This can result in removal of too much or too little tissue, which can lead to unnecessary removal of vital structures or incomplete cancer removal, respectively. Both results can have negative effects on ultimate patient outcome, with positive margins reported in 23% of sarcoma surgeries. Near-infrared (NIR) fluorescence probes are molecules that when stimulated with specific, known frequencies of near-infrared light will emit light of another distinct frequency. NIR light penetrates human tissue reasonably well and therefore can be used to detect the presence and location of unseen structures labeled with NIR fluorescence probes through several centimeters of tissue. Intra-operative near-infrared (NIR) fluorescence probes have been effective for this purpose in brain tumor surgery and may be applicable to sarcoma surgery. Foundational research is needed to explore the potential of this affibody probe and perfusion probes to estimate margin thickness in sarcoma surgery. In this study we will determine if sarcoma labeling using NIR fluorescence probes is superior with perfusion probes or a novel affibody probe. We will also determine whether NIR fluorescence using perfusion probes or a novel affibody probe can be correlated accurately to margin thickness.

Near-infrared (NIR) fluorescence probes are especially useful for simple and noninvasive in vivo imaging inside the body because of low autofluorescence and high tissue transparency in the NIR region compared with other wavelength regions. However, existing NIR fluorescence probes for matrix metalloproteinases (MMPs), which are tumor, atherosclerosis, and inflammation markers, have various disadvantages, especially as regards sensitivity. Here, we report a novel design strategy to obtain a NIR fluorescence probe that is rapidly internalized by free diffusion and well retained intracellularly after activation by extracellular MMPs. We designed and synthesized four candidate probes, each consisting of a cell permeable or nonpermeable NIR fluorescent dye as a Förster resonance energy transfer (FRET) donor linked to the NIR dark quencher BHQ-3 as a FRET acceptor via a MMP substrate peptide. We applied these probes for detection of the MMP activity of cultured HT-1080 cells, which express MMP2 and MT1-MMP, by fluorescence microscopy. Among them, the probe incorporating BODIPY650/665, BODIPY-MMP, clearly visualized the MMP activity as an increment of fluorescence inside the cells. We then applied this probe to a mouse xenograft tumor model prepared with HT-1080 cells. Following intratumoral injection of the probe, MMP activity could be visualized for much longer with BODIPY-MMP than with the probe containing SulfoCy5, which is cell impermeable and consequently readily washed out of the tissue. This simple design strategy should be applicable to develop a range of sensitive, rapidly responsive NIR fluorescence probes not only for MMP activity, but also for other proteases.

A novel amphiphilic multi-block copolymer conjugated with both a near infrared fluorescence probe and drug has been designed and prepared by means of ring-opening polymerization (ROP) of N-Carboxy Anhydride (NCA) monomers following a Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization. At first, an amino group-containing RAFT agent was synthesized and it served as an initiator for the sequential ROP of aspartic acid β-benzyl ester N-carboxy anhydride (Asp-NCA) and e-carbobenzoxy-L-lysine NCA (ZLLys-NCA). Then the multi-block copolymer was prepared by a succeeding RAFT polymerization of poly(ethylene glycol) methyl ether acrylate (OGEA). At the end, both anticancer drug doxorubicin and hydrophobic aminocyanine dye were chemical conjugated to the block copolymer via a hydrazone or amide bond, respectively. The obtained NIRF copolymer and its micelles were characterized by nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), dynamic light scattering (DLS), and UV-vis and fluorescence spectrophotometry. The prodrug has strong fluorescence in the near infrared region and shows pH-responsive drug release behavior, and it has potential application in the theranostics of cancer.

Cathepsin activity as a biomarker for human pancreatic dysplasia and carcinoma

Activatable near-infrared fluorescent (NIRF) probes have been used for ex vivo and in vivo detection of intestinal tumors in animal models. We hypothesized that NIRF probes activatable by cathepsins or metalloproteinases will detect and quantify dextran sulphate sodium (DSS)-induced acute colonic inflammation in wild type mice or chronic colitis in interleukin-10 (IL-10)-null mice ex vivo or in vivo. Wild type mice given DSS, water controls, and IL-10-null mice with chronic colitis were administered probes by retro-orbital injection. FMT2500 LX system imaged fresh and fixed intestine ex vivo and mice in vivo. Inflammation detected by probes was verified by histology and colitis scoring. NIRF signal intensity was quantified using 2-dimensional region of interest ex vivo or 3-dimensional region of interest analysis in vivo. Ex vivo, 7 probes tested yielded significant higher NIRF signals in colon of DSS-treated mice versus controls. A subset of probes was tested in IL-10-null mice and yielded strong ex vivo signals. Ex vivo fluorescence signal with 680 series probes was preserved after formalin fixation. In DSS and IL-10-null models, ex vivo NIRF signal strongly and significantly correlated with colitis scores. In vivo, ProSense680, CatK680FAST, and MMPsense680 yielded significantly higher NIRF signals in DSS-treated mice than controls, but background was high in controls. Both cathepsin or metalloproteinase-activated NIRF probes can detect and quantify colonic inflammation ex vivo. ProSense680 yielded the strongest signals in DSS colitis ex vivo and in vivo, but background remains a problem for in vivo quantification of colitis.

Introduction and Hypothesis: The prevalence of myocardial infarction increases with the average age of the population and is currently the leading cause of death worldwide. In order to define anatomical changes and biomarkers related to cardiac infarction, we tested the use of combined computed tomography (CT) and near infrared fluorescent (NIRF) probes to facilitate non-invasive imaging of processes concerned with tissue degeneration/regeneration. This development of new non-invasive diagnostic methods will lead to better treatment options in the future. Methods: Mice were subjected to left anterior descending artery (LAD) ligation inducing an acute myocardial infarction and subsequently imaged in vivo using fast and low dose microCT scanning (QuantumFX, Perkin Elmer) and near infrared (NIRF) probes to monitor MMP activity (MMPsense680). Immediate contrast enhanced CT imaging using eXIA160 during its blood-pool phase allowed registration of changes in ventricular anatomy and function of important global parameters, like end-diastolic volume (EDV), end-systolic volume (ESV). These were used to calculate stroke volume (SV), and left ventricular ejection fraction (LVEF). Delayed eXIA160 contrast uptake during its myocardial phase, subsequently allowed analysis of the infarct size and infarct healing. In addition, in vivo molecular MMPsense localization was combined with micro-CT imaging for accurate 3D co-registration. Results: Changes in ventricular anatomy and myocardial viability were assessed 3 hours and 2 months post LAD occlusion in the same animal, demonstrating the feasibility of monitoring myocard viability over time. The decreased uptake of eXIA160 in the myocardium was subsequently quantitated. A concomitant increase in MMP activity, as determined by fluorescence mediated tomography using MMPsense680, could be localized to the infarcted site. Conclusions: Non-invasive imaging, using NIRF probes, enables longitudinal imaging of processes concerned with myocard infarction. Consequently, disease progression can be monitored over time and the effect of (new) pharmacotherapy can be studied.

Green Stereoregular Polymerization of Poly(methyl methacrylate)s Through Vesicular Catalysis

A facile pH near-infrared fluorescence probe for the diagnosis of cancer in vivo

We used a molecular probe activated by protease cleavage to image expression of matrix metalloproteinases (MMPs) in the heart after myocardial infarction. We synthesized and characterized a near-infrared fluorescent (NIRF) probe that is activated by proteolytic cleavage by MMP2 and MMP9. The NIRF probe was injected into mice at various time points up to 4 weeks after myocardial infarction induced by ligation of the left anterior descending coronary artery. NIRF imaging of MMP activity increased in the infarct region, with maximal expression at 1 to 2 weeks, persisting to 4 weeks. Zymography and real-time polymerase chain reaction analysis showed that MMP9 expression is increased at 2 to 4 days, and MMP2 expression is increased at 1 to 2 weeks. Dual-label confocal microscopy showed colocalization of NIRF imaging with neutrophils on day 2, and flow cytometric analysis confirmed that NIRF signal is associated with leukocytes in the infarct zone. This study demonstrates that the activity of MMPs in the myocardium may be imaged by use of specific activity-dependent molecular probes.

Bioluminescence imaging (BLI) has shown its appeal as a sensitive technique for in vivo whole body optical imaging. However, the development of injectable tumor-specific near-infrared fluorescent (NIRF) probes makes fluorescence imaging (FLI) a promising alternative to BLI in situations where BLI cannot be used or is unwanted (e.g., spontaneous transgenic tumor models, or syngeneic mice to study immune effects).In this study, we addressed the questions whether it is possible to detect tumor progression using FLI with appropriate sensitivity and how FLI correlates with BLI measurements. In addition, we explored the possibility to simultaneously detect multiple tumor characteristics by dual-wavelength FLI (∼700 and ∼800 nm) in combination with spectral unmixing. Using a luciferase-expressing 4T1-luc2 mouse breast cancer model and combinations of activatable and targeting NIRF probes, we showed that the activatable NIRF probes (ProSense680 and MMPSense680) and the targeting NIRF probes (IRDye 800CW 2-DG and IRDye 800CW EGF) were either activated by or bound to 4T1-luc2 cells. In vivo, we implanted 4T1-luc2 cells orthotopically in nude mice and were able to follow tumor progression longitudinally both by BLI and dual-wavelength FLI. We were able to reveal different probe signals within the tumor, which co-localized with immuno-staining. Moreover, we observed a linear correlation between the internal BLI signals and the FLI signals obtained from the NIRF probes. Finally, we could detect pulmonary metastases both by BLI and FLI and confirmed their presence histologically.Taken together, these data suggest that dual-wavelength FLI is a feasible approach to simultaneously detect different features of one tumor and to follow tumor progression with appropriate specificity and sensitivity. This study may open up new perspectives for the detection of tumors and metastases in various experimental models and could also have clinical applications, such as image-guided surgery.

Alzheimer's disease (AD) is a common progressive neurodegenerative disease characterized by abnormal deposition of amyloid-β protein (Aβ) and the formation of neurofibrillary tangles (NFT). Although there are drugs available for clinical treatment, they do not fundamentally cure AD, so early diagnosis and intervention for AD are essential. In recent years, near-infrared fluorescent (NIRF) probes have been widely used in diagnosing AD due to their advantages of high tissue penetration, low autofluorescence interference, and non-invasiveness. This paper reviewed the recent progress of NIRF probes in AD. We described the effects of NIRF probes on AD biomarkers, including Aβ protein, Tau tangles, mitochondrial viscosity, reactive oxygen species, copper ions, zinc ions, and so on. In detail, we also described the optical properties, in vitro detection, and in vivo imaging of NIRF probes based on different dyes and their derivatives, such as curcumin, Boron dipyrromethene, Quinoline, and Donor-Acceptor. Despite significant progress, NIRF probes remain challenging in imaging and treating AD in vivo. Their selectivity and sensitivity need to be improved due to the complex structure of brain tissue and interfering factors. In conclusion, studying NIRF probes provides us with new ideas. Through further optimization, these probes may be used for early diagnosis and treatment detection of AD to better cope with the challenges brought by AD.

A TPP-OH-based near-infrared fluorescent nanoprobe for cellular and in vivo H2S detection and imaging.

A BTK-activatable targeted covalent NIR fluorescent probe for kinase monitoring and B-cell lymphoma imaging.

We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.

A series of diblock copolymers, poly(ethylene oxide)-block-poly{6-(4-(4-ethoxyphenylazo) phenoxy) hexyl methacrylate} (PEO-b-PMA6AB2), with different hydrophilic/hydrophobic ratios were synthesized by reversible addi- tion-fragmentation chain transfer (RAFT) polymerization. The structures and properties of diblock copolymers were charac- terized by 1 H NMR and gel permeation chromatography (GPC). The self-assembly method was as follows: block copolymers were dissolved in tetrahydrofuran (THF) at first, and then by adding water at the rate of 2.5 μL/s, the aggregates self-assembled when the critical micelle concentration (CMC) was reached. Field emission transmission electron microscope (FETEM), scanning electron microscope (SEM), field emission scanning electron microscope (FESEM), optical microscope, particle size analyzer, UV-Vis absorption spectroscope and fluorescence emission spectroscope were used to study the mor- phology and size of the aggregates and their stimuli-responsive behavior. The SEM and FETEM results showed that poly- meric micelles and large compound micelles (LCM) coexisted in the aqueous solution. Furthermore, it was found that the size of the self-assembly reduced with the decrease of initial concentration and the increase of stirring rate, and the distribution tended to be uniform. Meanwhile, the morphology of large compound micelles turned to be different, changing from common sphere to peanut shape or rod-like shape when the hydrophilic/hydrophobic ratio was 2.3. By encapsulating Nile Red into the aggregates, the peanut shape and rod-like shape were captured by fluorescence microscope. UV-Vis absorption spectra of the self-assembly revealed that the photo-responsive behavior of azobenzene moieties in the aggregates was reversible but re- stricted to some extent. The change on fluorescence intensity of Nile Red encapsulated in the diblock copolymer aggregates indicated that the alternative irradiation of UV and visible light induced the release of Nile Red resulted from the photoisom- erization of the azobenzene moieties. In addition, the photo-responsive study showed that large compound micelles self-assembled from BCP-S3-0.82 could dissociate under the irradiation of UV light. Other large compound micelles assem- bled from the rest of block copolymers also dissociated, but the extent of the dissociation was not as remarkable as BCP-S3-0.82, which had the maximal hydrophobic ratio. Keywords RAFT; amphiphilic block copolymer; azobenzene moiety; hydrophilic/hydrophobic ratio; self-assembly; mi- celles