Emma Portnoy

Cetuximab-labeled liposomes containing near-infrared probe for in vivo imaging

UNLABELLED A new liposome-based near-infrared probe that combines both imaging and targeting abilities was developed for application in medical imaging. The near-infrared fluorescent molecule indocyanine green (ICG), and the cetuximab monoclonal antibody for epidermal growth factor receptor (EGFR) were attached to liposomes by passive adsorption. It was found that ICG molecules adsorbed to the liposomes are more fluorescent than free ICG and have a larger quantum yield. Cetuximab-adsorbed fluorescent liposomes preserved EGFR recognition, as is evident from internalization and selective binding to A431 colon carcinoma cells overexpressing EGFR. The binding of cetuximab-targeted fluorescent liposomes to A431 compared with IEC-6 cells (normal enterocytes expressing physiological EGFR levels) was greater by a factor of 3.5, ensuring imaging abilities with available fluorescent equipment. Due to relatively high quantum yield and specific tumor cell-recognizing ability, this technology deserves further in vivo evaluation for imaging and diagnostic purposes. FROM THE CLINICAL EDITOR A new liposome-based near-infrared probe combining both imaging and targeting abilities is reported. Due to relatively high quantum yield and EGFR-expressing tumor cell specificity, this technology deserves further in vivo evaluation for imaging and diagnostic purposes.

Evaluation of the near infrared compound indocyanine green as a probe substrate of p-glycoprotein.

The efflux transporter P-glycoprotein (P-gp) affects the pharmacokinetics of many drugs. Currently used methods for characterization of P-gps functional activity in vivo involve the use of radiolabeled substrates, are costly, and are technically demanding. Our objective was to evaluate whether the FDA-approved near-infrared compound indocyanine green (ICG) can be used as a probe substrate of P-gp. We also characterized the interaction of ICG with another efflux transporter, the breast cancer resistance protein (BCRP). We evaluated ICG accumulation and transport in MDCK cells overexpressing P-gp or BCRP (MDCK-MDR1 and MDCK-BCRP, respectively) compared to control MDCK cells, in the presence or the absence of transporter inhibitors. In vivo imaging of ICG biodistribution in mice was conducted over 3.5 h using valspodar as the P-gp inhibitor. The EC50 values for ICG accumulation in control MDCK and MDCK-MDR1 cells were 9.0 × 10(-6) ± 5.7 × 10(-7) M and 1.5 × 10(-5) ± 1.1 × 10(-6) M, respectively. The efflux ratio for ICG in MDCK-MDR1 cells was 6.8-fold greater than in control cells. P-gp inhibition attenuated ICG efflux from MDR1-MDCK cells, and their effects in those cells were greater than in control MDCK cells. In contrast, BCRP level of expression or pharmacological inhibition did not significantly affect ICG cellular accumulation. In vivo imaging indicated enhanced cerebral ICG distribution with valspodar (brain - foot area under the concentration-time curves of 3.0 × 10(10), 5.6 × 10(10) and 3.7 × 10(10) h·[p/s/sr]/μW in valspodar-treated mice vs 9.0 × 10(9) and 5.3 × 10(9) h·[p/s/sr]/μW in controls). The findings from this pilot study suggest that near-infrared imaging using ICG as the probe substrate should be further characterized as a methodology for in vivo evaluation of P-gp activity.

Tracking inflammation in the epileptic rat brain by bi-functional fluorescent and magnetic nanoparticles

Correct localization of epileptic foci can improve surgical outcome in patients with drug-resistant seizures. Our aim was to demonstrate that systemically injected nanoparticles identify activated immune cells, which have been reported to accumulate in epileptogenic brain tissue. Fluorescent and magnetite-labeled nanoparticles were injected intravenously to rats with lithium-pilocarpine-induced chronic epilepsy. Cerebral uptake was studied ex vivo by confocal microscopy and MRI. Cellular uptake and biological effects were characterized in vitro in murine monocytes and microglia cell lines. Microscopy confirmed that the nanoparticles selectively accumulate within myeloid cells in the hippocampus, in association with inflammation. The nanoparticle signal was also detectable by MRI. The in vitro studies demonstrate rapid nanoparticle uptake and good cellular tolerability. We show that nanoparticles can target myeloid cells in epileptogenic brain tissue. This system can contribute to pre-surgical and intra-surgical localization of epileptic foci, and assist in detecting immune system involvement in epilepsy.

Indocyanine Green Liposomes for Diagnosis and Therapeutic Monitoring of Cerebral Malaria.

Cerebral malaria (CM) is a major cause of death of Plasmodium falciparum infection. Misdiagnosis of CM often leads to treatment delay and mortality. Conventional brain imaging technologies are rarely applicable in endemic areas. Here we address the unmet need for a simple, non-invasive imaging methodology for early diagnosis of CM. This study presents the diagnostic and therapeutic monitoring using liposomes containing the FDA-approved fluorescent dye indocyanine green (ICG) in a CM murine model. Increased emission intensity of liposomal ICG was demonstrated in comparison with free ICG. The Liposomal ICGs emission was greater in the brains of the infected mice compared to naïve mice and drug treated mice (where CM was prevented). Histological analyses suggest that the accumulation of liposomal ICG in the cerebral vasculature is due to extensive uptake mediated by activated phagocytes. Overall, liposomal ICG offers a valuable diagnostic tool and a biomarker for effectiveness of CM treatment, as well as other diseases that involve inflammation and blood vessel occlusion.

Imaging the urinary pathways in mice by liposomal indocyanine green

UNLABELLED Intraoperative ureter identification can assist in the prevention of ureteral injury and consequently improve surgery outcomes. Our aim was to take advantage of the altered pharmacokinetics of liposomal indocyanine green (ICG), the only FDA-approved near-infrared (NIR) dye, for imaging of ureters during surgeries. ICG was passively adsorbed to liposomes. NIR whole mice body and isolated tissue imaging were used to study liposomal ICG properties vs. free ICG. In vivo, the urinary bladder could be clearly observed in most of the liposome-treated mice. Liposomal encapsulation of ICG enhanced ureteral emission up to 1.9 fold compared to free ICG (P<0.01). Increase in liposomal micropolarity and microviscosity and differential scanning calorimetry supported ICG localization within the liposomal bilayer. Our findings suggest that liposomal ICG could be utilized for ureteral imaging intra-operatively, thus potentially improving surgical outcomes. FROM THE CLINICAL EDITOR Iatrogenic ureteral injury is a serious complication of abdominal surgery and intra-operative recognition of the ureters is usually the best method of injury prevention. In this article, the authors developed liposomal indocyanine green, which could be excreted via the urinary system and investigated its in-vivo use in mice.

Near Infrared Imaging of Indocyanine Green Distribution in Pregnant Mice and Effects of Concomitant Medications

The transfer of indocyanine green (ICG) across the placenta is considered to be very low based on measurements in fetal blood. The goal of this study was to evaluate in mice ICGs distribution within fetuses themselves and effects of concomitant medications on fetal exposure. Mid-gestational (day 12.5) and late-gestational (day 17.5) age mice were imaged after administration of ICG (0.167 mg), in the presence and the absence of the organic anion transporting polypeptide (OATP) inhibitor rifampin (10 mg/kg, n = 11, or 20 mg/kg, n = 1) or the P-glycoprotein inhibitor valspodar (12.5 mg/kg). In vivo ICG emission intensity was followed by ex vivo analysis of blood and tissue emission. Both valspodar and rifampin increased ICGs emission intensity within maternal tissues. In addition, valspodar enhanced the ex vivo signal in mid-pregnancy placentae (2.1-fold; p < 0.01) and fetuses (2.4-fold; p < 0.01), and reduced late-pregnancy placenta:blood and fetus:blood ratios. Rifampin increased placental (1.4-fold, p < 0.05, and 2.3-fold, p < 0.01, in mid- and late-pregnancy, respectively) and fetal (2.2-fold, p < 0.01, and 3.2-fold, p < 0.01, in mid- and late-pregnancy) ICG signal. Similarly to valspodar, late-pregnancy placenta:blood and fetus:blood ratios were reduced by rifampin. Both inhibitors enhanced ICGs emission in fetal leg, liver, and brain. In conclusion, ICG distribution into the mouse fetus can be enhanced when used concomitantly with OATP or P-glycoprotein inhibitors. The greater distribution within individual fetal tissues is likely related to ICGs greater transplacental transfer. Until further data are available on ICGs safety when combined with medications that affect its maternal handling, such combinations should be used with caution.

Effects of valproic acid on the placental barrier in the pregnant mouse: Optical imaging and transporter expression studies.

Our aim was to evaluate the effects of valproic acid (VPA) on the function of the placental barrier in vivo, in pregnant mice. Studies were conducted on gestational days 12.5 (mid‐gestation) or 17.5 (late gestation), following intraperitoneal treatment with 200 mg/kg VPA or the vehicle. Indocyanine green (ICG; 0.167 mg, i.v.) was used as a marker for the placental barrier permeability. Transporter expression was evaluated by quantitative ‐PCR. VPA treatment was associated with a 40% increase (p < 0.05) in accumulation of ICG in maternal liver in mid‐pregnancy and a decrease by one fifth (p < 0.05) in late pregnancy. Ex vivo, VPA treatment led to a 20% increase (p < 0.05) in fetal ICG emission in mid‐pregnancy. Also in mid‐pregnancy, the placental expression of the L‐type amino acid transporter, the organic anion–transporting polypeptide (Oatp)4a1 (thyroid hormone transporter), and the reduced folate carrier was lower in VPA‐treated mice (p < 0.05). In late pregnancy, hepatic Oatp4a1 levels were 40% less than in controls (p > 0.05). The observed changes in placental transporter expression and function support further research into the potential role of the placenta in the adverse pregnancy outcomes of VPA. Near‐infrared imaging provides a noninvasive, nonradioactive tool for future studies on the effects of epilepsy and antiepileptic drugs on tissue transport functions.

Therapeutic efficacy of combined PEGylated liposomal doxorubicin and radiofrequency ablation: Comparing single and combined therapy in young and old mice

ABSTRACT Antitumor therapy in the elderly is particularly challenging due to multiple, often chronic diseases, poly‐therapy, and age‐related physiological changes that affect drug efficacy and safety. Furthermore, tumors may become more aggressive and drug‐resistant with advanced age, leading to poor patient prognosis. In this study, we evaluated in mice bearing medulloblastoma xenografts the effect of age on tumor progression and tumor therapy. We focused on therapeutic efficacy of two treatment modalities alone radiofrequency ablation therapy (RFA), PEGylated liposomal doxorubicin (PLD) equivalent to Doxil, and their combination. We demonstrated that tumor growth rate was higher and survival was lower in old versus young mice (p < 0.05). Likewise, tumors in old mice were less susceptible to either PLD or RFA monotherapy. However, combined therapy of PLD and RFA succeeded to eliminate the age‐related differences in anti‐cancer treatment efficacy (p > 0.05) by the two monotherapies. The results on PLD therapy are supported by preferable PEGylated nano‐liposomes accumulation in tumors of young mice compared to old mice, as determined by near‐infrared imaging with indocyanine green (ICG)‐labeled PEGylated nano‐liposomes. Taken together, our findings suggest that age effects on tumor progression and tumor monotherapy outcome may potentially be related to changes in tumor microenvironment, and that these changes can be overcome by RFA as this technique abolishes these differences and significantly improves success of PLD treatment.

Evaluation of Near Infrared Dyes as Markers of P-Glycoprotein Activity in Tumors.

Aim: The multidrug resistance protein 1 (MDR1; P-glycoprotein) has been associated with efflux of chemotherapeutic agents from tumor cells and with poor patient prognosis. This study evaluated the feasibility of non-invasive, non-radioactive near infrared (NIR) imaging methodology for detection of MDR1 functional activity in tumors. Methods: Initial accumulation assays were conducted in MDR1-overexpressing MDCK cells (MDCK-MDR1) and control MDCK cells (MDCK-CT) using the NIR dyes indocyanine green (ICG), IR-783, IR-775, rhodamine 800, XenoLight DiR, and Genhance 750, at 0.4 μM-100 μM. ICG and IR-783 were also evaluated in HT-29 cells in which MDR1 overexpression was induced by colchicine (HT-29-MDR1) and their controls (HT-29-CT). In vivo optical imaging studies were conducted using immunodeficient mice bearing HT-29-CT and HT-29-MDR1 xenografts. Results: ICGs emission intensity was 2.0- and 2.2-fold higher in control versus MDR1-overexpressing cells, in MDCK and HT-29 cell lines, respectively. The respective IR-783 control:MDR1 ratio was 1.4 in both MDCK and HT-29 cells. Optical imaging of mice bearing HT-29-CT and HT-29-MDR1 xenografts revealed a statistically non-significant, 1.7-fold difference (p > 0.05) in ICG emission intensity between control and MDR1 tumors. No such differences were observed with IR-783. Conclusion: ICG and IR-783 appear to be weak MDR1 substrates. In vivo, low sensitivity and high between-subject variability impair the ability to use the currently studied probes as markers of tumor MDR1 activity. The results suggest that, for future use of this technology, additional NIR probes should be screened as MDR1 substrates.