Paola Luciani

Quantitative Imaging of Lymphatic Function with Liposomal Indocyanine Green

Lymphatic vessels play a major role in cancer progression and in postsurgical lymphedema, and several new therapeutic approaches targeting lymphatics are currently being developed. Thus, there is a critical need for quantitative imaging methods to measure lymphatic flow. Indocyanine green (ICG) has been used for optical imaging of the lymphatic system, but it is unstable in solution and may rapidly enter venous capillaries after local injection. We developed a novel liposomal formulation of ICG (LP-ICG), resulting in vastly improved stability in solution and an increased fluorescence signal with a shift toward longer wavelength absorption and emission. When injected intradermally to mice, LP-ICG was specifically taken up by lymphatic vessels and allowed improved visualization of deep lymph nodes. In a genetic mouse model of lymphatic dysfunction, injection of LP-ICG showed no enhancement of draining lymph nodes and slower clearance from the injection site. In mice bearing B16 luciferase-expressing melanomas expressing vascular endothelial growth factor-C (VEGF-C), sequential near-IR imaging of intradermally injected LP-ICG enabled quantification of lymphatic flow. Increased flow through draining lymph nodes was observed in mice bearing VEGF-C-expressing tumors without metastases, whereas a decreased flow pattern was seen in mice with a higher lymph node tumor burden. This new method will likely facilitate quantitative studies of lymphatic function in preclinical investigations and may also have potential for imaging of lymphedema or improved sentinel lymph detection in cancer.

Use of a PEG-conjugated bright near-infrared dye for functional imaging of rerouting of tumor lymphatic drainage after sentinel lymph node metastasis

Tumor lymphangiogenesis promotes metastatic cancer spread to lymph nodes and beyond. However, the potential remodeling and functionality of tumor-draining lymphatic vessels has remained unclear. Thus, we aimed to develop non-invasive imaging methods for repeated quantitative imaging of lymphatic drainage and of contractile collecting lymphatic vessel function in mice, with colloidal near-infrared (NIR) tracers and a custom fluorescence stereomicroscope specially adapted for NIR sensitive imaging. Using these tools, we quantitatively determined pulse rates and valvular function of collecting lymphatic vessels with high resolution. Unexpectedly, we found that tumor-draining lymphatic vessels in a melanoma footpad model initially were dilated but remained functional, despite lower pulse rates. In two independent tumor models, impaired lymphatic function was detected once metastases were present in draining lymph nodes. Importantly, we found that lymphatic dysfunction, induced by metastatic tumor spread to sentinel lymph nodes, can lead to a rerouting of lymphatic flow away from the metastatic lymph node, via collateral lymphatic vessels, to alternate lymph nodes. These findings might have important clinical implications for the procedure of sentinel lymph node mapping that represents the standard of care for determining prognosis and treatment of melanoma and breast cancer patients.

Sustained gastrointestinal activity of dendronized polymer–enzyme conjugates

Methods to stabilize and retain enzyme activity in the gastrointestinal tract are investigated rarely because of the difficulty of protecting proteins from an environment that has evolved to promote their digestion. Preventing the degradation of enzymes under these conditions, however, is critical for the development of new protein-based oral therapies. Here we show that covalent conjugation to polymers can stabilize orally administered therapeutic enzymes at different locations in the gastrointestinal tract. Architecturally and functionally diverse polymers are used to protect enzymes sterically from inactivation and to promote interactions with mucin on the stomach wall. Using this approach the in vivo activity of enzymes can be sustained for several hours in the stomach and/or in the small intestine. These findings provide new insight and a firm basis for the development of new therapeutic and imaging strategies based on orally administered proteins using a simple and accessible technology.

Breakthrough discoveries in drug delivery technologies: the next 30 years.

What if we could open the 2044 special issue of the Journal of Controlled Release? Which drug delivery technologies will have led the field? Which ones will have successfully reached the marketplace? In attempting to answer these questions, we selected a few recent technologies and concepts that could, in our opinion, play a crucial role in coming years. In each case, emblematic papers are cited to introduce and discuss the selected topic.

Chronic High-Fat Diet Impairs Collecting Lymphatic Vessel Function in Mice

Lymphatic vessels play an essential role in intestinal lipid uptake, and impairment of lymphatic vessel function leads to enhanced adipose tissue accumulation in patients with lymphedema and in genetic mouse models of lymphatic dysfunction. However, the effects of obesity on lymphatic function have been poorly studied. We investigated if and how adipose tissue accumulation influences lymphatic function. Using a lymphatic specific tracer, we performed in vivo near-infrared (NIR) imaging to assess the function of collecting lymphatic vessels in mice fed normal chow or high-fat diet (HFD). Histological and whole mount analyses were performed to investigate the morphological changes in initial and the collecting lymphatic vessels. HFD was associated with impaired collecting lymphatic vessel function, as evidenced by reduced frequency of contractions and diminished response to mechanostimulation. Moreover, we found a significant negative correlation between collecting lymphatic vessel function and body weight. Whole mount analyses showed an enlargement of contractile collecting lymphatic vessels of the hind limb. In K14-VEGF-C mice, HFD resulted in a reduced spreading of the tracer within dermal lymphatic vessels. These findings indicate that adipose tissue expansion due to HFD leads to a functional impairment of the lymphatic vasculature, predominantly in collecting lymphatic vessels.

Genetic Ablation of SOX18 Function Suppresses Tumor Lymphangiogenesis and Metastasis of Melanoma in Mice

The lymphatic vasculature provides a major route for tumor metastasis and inhibiting neolymphangiogenesis induced by tumors can reduce metastasis in animal models. Developmental biology studies have identified the transcription factor SOX18 as a critical switch for lymphangiogenesis in the mouse embryo. Here, we show that SOX18 is also critical for tumor-induced lymphangiogenesis, and we show that suppressing SOX18 function is sufficient to impede tumor metastasis. Immunofluorescence analysis of murine tumor xenografts showed that SOX18 is reexpressed during tumor-induced neolymphangiogenesis. Tumors generated by implantation of firefly luciferase-expressing B16-F10 melanoma cells exhibited a reduced rate of metastasis to the regional draining lymph node in Sox18-deficient mice, as assessed by live bioluminescence imaging. Lower metastatic rates correlated with reduced tumoral lymphatic vessel density and diameter and with impaired drainage of peritumoral injected liposomes specific for lymph vessels from the sentinel lymph nodes. Overall, our findings suggested that SOX18 induction is a key step in mediating tumor lymphangiogenesis and metastasis, and they identify SOX18 as a potential therapeutic target for metastatic blockade.

Expansion of the lymphatic vasculature in cancer and inflammation: New opportunities for in vivo imaging and drug delivery

Over the last 15 years, discovery of key growth factors and specific molecular markers for lymphatic vessels has enabled a new era of molecular research on the lymphatic vascular system. As a result, it has been found that lymphangiogenesis, the expansion of existing lymphatic vessels, plays an important role in tumor progression and in the control of chronic inflammation. At the same time, technical advancements have been made to improve the visualization of the lymphatic system. We have recently developed liposomal and polymer-based formulations of near-infrared lymphatic-specific imaging tracers for the non-invasive quantitative in vivo imaging of lymphatic vessel function. Using these tracers, a near-infrared stereomicroscope system allows imaging of initial and collecting lymphatic vessels with high spatial and temporal resolution in mice. In addition, we have developed a new method, using antibodies to a lymphatic specific marker and positron emission tomography, to sensitively detect lymphatic expansion in lymph nodes as the earliest sign of cancer metastasis. These imaging methods have great potential to provide non-invasive measures to assess the functionality of the lymphatic system and to assess the efficiency of lymphatic drug delivery.

Non-invasive dynamic near-infrared imaging and quantification of vascular leakage in vivo

Preclinical vascular research has been hindered by a lack of methods that can sensitively image and quantify vascular perfusion and leakage in vivo. In this study, we have developed dynamic near-infrared imaging methods to repeatedly visualize and quantify vascular leakage in mouse skin in vivo, and we have applied these methods to transgenic mice with overexpression of vascular endothelial growth factors VEGF-A or -C. Near-infrared dye conjugates were developed to identify a suitable vascular tracer that had a prolonged circulation lifetime and slow leakage into normal tissue after intravenous injection. Dynamic simultaneous imaging of ear skin and a large blood vessel in the leg enabled determination of the intravascular signal (blood volume fraction) from the tissue signal shortly after injection and quantifications of vascular leakage into the extravascular tissue over time. This method allowed for the sensitive detection of increased blood vascularity and leakage rates in K14-VEGF-A transgenic mice and also reliably measured inflammation-induced changes of vascularity and leakage over time in the same mice. Measurements after injection of recombinant VEGF-A surprisingly revealed increased blood vascular leakage and lymphatic clearance in K14-VEGF-C transgenic mice which have an expanded cutaneous lymphatic vessel network, potentially indicating unanticipated effects of lymphatic drainage on vascular leakage. Increased vascular leakage was also detected in subcutaneous tumors, confirming that the method can also be applied to deeper tissues. This new imaging method might facilitate longitudinal investigations of the in vivo effects of drug candidates, including angiogenesis inhibitors, in preclinical disease models.

Treatment of calcium channel blocker-induced cardiovascular toxicity with drug scavenging liposomes

Calcium channel blocker (CCB) overdose is potentially lethal. Verapamil and diltiazem are particularly prone to acute toxicity due to their dual effect on cardiac and vascular tissues. Unfortunately, conventional decontamination measures are ineffective in accelerating blood clearance and, to date, few efforts have been made to develop antidotes. To address the issue, injectable long-circulating liposomes bearing a transmembrane pH-gradient are proposed as efficient detoxifying agents of CCB poisoning. By scavenging the drug in situ, these circulating nanocarriers can restrict its distribution in tissues and hinder its pharmacological effect. In vitro, we showed that liposomes stability in serum and their ability to sequester CCBs could be finely-tuned by modulating their internal pH, surface charge, and lipid bilayer structure. Subsequently, we verified their efficacy in reversing the cardiovascular effects of verapamil in rats implanted with telemetric pressure/biopotential transmitters. In animals orally intoxicated to verapamil, an intravenous injection of the liposomal antidote rapidly attenuated the reduction in blood pressure. Areas under diastolic, systolic, and mean pressures curves were significantly reduced by up to 60% and the time to hemodynamic recovery was shortened from 19 to only 11 h. These findings confirm the protective effect of pH-gradient liposomes against cardiovascular failure after CBB intoxication, and endorse their potential as efficient, versatile antidotes.

Ratiometric Fluorescent Probes for the Detection of Reactive Oxygen Species

Reactive oxygen species (ROS) are endogenously produced oxidants with various functions ranging from host defense to signaling. These transient species can cause severe damage to the body when their production is dysregulated or when environmental factors elevate their concentrations. To study their effects and prevent oxidative harm, tools capable of monitoring ROS in cells and tissue in a sensitive and selective fashion are required. In this Review, a summary of existing ratiometric probes is provided, together with a critical discussion of selected examples.