Imaging of Vascular-Injured Tissue by Liposomal Quantum-Dots

SUMMARY This work examined a novel liposomal Quantum-Dots (LipQD) formulation to effectively accumulate, after been phagocytized by circulating monocytes, at the inflamed restenotic region. Negatively charged QDs were encapsulated in positively charged liposomes, and their uptake, stability and cytotoxicity were examined in murine monocyte/macrophage cell line, and in a rat model of vascular injury. High fluorescent intensities were observed, for at least 24 hrs, after local incubation of the carotid artery lumen of Sabra male rats with LipQD after arterial injury. In contrast, accumulation of QDs following incubation with free DQs suspension was not detected, at all time points, in the injured artery. In a preliminary study, a high fluorescent signal was seen in the injured artery of restenotic rats after intravenous administration of LipQD. The novel LipQD formulation offers a promising and attractive approach for inflammation imaging by exploiting the innate-immunity cells. INTRODUCTION Inflammation characterizes several cardiovascular pathological disorders including restenosis, reobstruction of the artery following endovascular procedures. The inflammatory cascade involves significant infiltration of phagocytic cells 1 , which can be exploited for therapeutic as well as diagnostic purposes when the cargo is non-cytotoxic to the cell carrier. Liposomal delivery systems, negativelyand positively-charged vesicles in particular, are phagocytized by monocytes in the blood stream 2 , and the latter cells infiltrate into the inflammation site as part of innate immune response. QDs, also known as semiconductor nano-crystals, have a number of advantages in comparison to more conventional dyes, and are of great interest as novel fluorescence markers in biological and medical research. However, a major concern is their biocompatibility and cytotoxicity impeding their use in biomedical applications. In order to effectively target QDs to inflamed regions enabling efficient fluorescent signal, and at the same time avoiding cytotoxicity, a suitable delivery system is required. Liposomal formulations of QDs is a formidable task 3 . A liposomal delivery system containing QDs could address these objectives. EXPERIMENTAL METHODS QDs Preparation: CdSe/ZnS hydrophobic nanocrystals were synthesized and water solubility was rendered by glutathione via a thiol exchange reaction. Red emitting negatively charged QDs (655 nm emission maxima) were utilized in these experiments with an average size of 10 nm. Preparation and Characterization of LipQD: Liposomes, composed from a mixture of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl3-trimethylammonium-propane (DOTAP) and cholesterol, were prepared by a modified thin film hydration method 4 . The lipids mixture was dissolved in t-butanol and lyophilized overnight, and the lyophilized cake was hydrated with the QDs suspension (125 nM). The liposomes were homogenized by means of tip sonication yielding small unilamellar vesicles characterized by size and zeta potential. QDs concentration was determined spectrophotometrically after solubilizing the liposomes with octyl β-D glucopyranoside (OGP) 200 mM solution. Monocyte/macrophage uptake of LipQD and fluorescent stability: RAW 264 cells were grown in supplemented DMEM, and 6×10 4 cells were plated and allowed to adhere overnight. Uptake of LipQD by the cells was assessed 1 and 4 hrs after treatment following washing, fixation and mounting. The slides were examined for the fluorescent signal by means of a confocal microscopy. Quantitative determination of uptake extent was measured by means of flow cytometry (FACS). Fluorescent stability was assessed in cells incubated with LipQD, washed and incubated in treatment-free media for additional 24 hrs. Formulation toxicity: Cytotoxicity of RAW 264 cell line by LipQD and free QDs at different concentrations was determined 24 and 48 hrs post treatment. Cells were plated at 4×10 4 cells per well and allowed to adhere overnight. The viability was assessed using 3-(4, 5-dimethylthiazolyl 2)-2,5 diphenyltetrazoliumbromide (MTT) assay. Fluorescent intensity in vivo (restenosis model): Male Sabra rats (n=32), weighing 350-400g, underwent a carotid injury 5 . The left common carotid artery was denuded of endothelium by the intraluminal passage of balloon catheter introduced through the external carotid artery. Immediately after injury, a 0.1 mL of 125 nM LipQD was instilled in the isolated balloon-injured segment of the carotid artery. After a 15-minute incubation period the remainder of the suspension was evacuated and blood circulation through the injured segment was restored. The animals were euthanized 4 and 24 hrs after blood flow restoration, arteries were harvested, and scanned by means of Typhoon Scanner for fluorescent signal analyses. In a preliminary experiment the fluorescent signal was measured following IV administration of LipQD in restenostic animals. RESULTS AND DISCUSSION Typical LipQD batches were characterized as containing 100-150 nM, ranging diameter size of 100-150 nm, and a zeta potential of 25-35 mV. Uptake of LipQD and fluorescent stability: Confocal microscopy and FACS analysis of cells incubated with LipQD revealed a marked and dose dependent intracellular fluorescent signal, but to a lesser extent than free QDs treated cells. The fluorescent stability of phagocytized LipQD in macrophages, incubated for 1 hr, was stable for at least 24 hours (Fig. 1). Formulation toxicity: No cytotoxic effects were noted for both free and liposomal QDs 24 hrs after treatment, at all concentrations, in contrast to liposomal alendronate (LipALN, a positive control, data not shown). Nevertheless, LipQD were found less toxic to the RAW264 cells 48 hrs after treatment in comparison to free QDs and LipALN (Fig. 2). Fluorescent intensity in restenotic rats: In contrast to free QDs treated rats, a significant accumulation and retention of LipQD, for up to 24 hrs, was observed in restenotic and intact rats 15 minutes after local incubation (Fig. 3). CONCLUSION A liposomal QDs formulation was successfully prepared and exhibited marked accumulation at the arterial injured site, stability and negligible cytotoxicity. These favorable results are in contrast to free QDs treatment, which exhibited cytotoxicity in vitro, and no accumulation at the injured tissue in restenotic animals. LipQD were retained both in the injured and intact tissue, after a short incubation time, for at least 24 hrs. Moreover, in-vivo preliminary results indicated a marked accumulation of LipQD at the injured artery following systemic intravenous administration. It is concluded that encapsulating QDs in liposomal system is a promising approach in order to avoid free QDs toxicity and to achieving effective fluorescent signal in the inflamed tissue. REFERENCES 1. Toutouzas, K.; Colombo, A.; Stefanadis, C., Eur Heart J 2004, 25, (19), 1679-87. 2. Afergan, E.; Danenberg, H. D.; Golomb, G., J Control Release 2008, 132, (2), 84-90. 3. Hansen, M.; Storm, G.; Löwik, D. P. M., J Nanopart Res 2012, 15, (1), 1-9. 4. Epstein, H.; Danenberg, H. D.; Golomb, G., AAPS J 2008, 10, (4), 505-15. 5. Danenberg, H. D.; Monkkonen, J.; Golomb, G., J Cardiovasc Pharmacol 2003, 42, (5), 671-9. 0 50 100 150 200 % c e lls v ia b ili ty Treatment 0 100 200 300 400 500 600 700 injured artery intact artery % f lu o re sc e n t in te n si ty LipQD