Indocyanine Green (herein also named ICG) is an FDA-approved fluorescent probe, but suffers of a fast metabolic clearance because it is rapidly eliminated by the liver. It is known that after intravenous injection, ICG is bound to albumin and subsequently taken up almost exclusively by the hepatic parenchymal cells. When ICG is administered at the human recommended dose of 0.5 mg/kg a normal blood half-time is around 3.0 minutes (Rosenthal E, Zinn K R, editors. Optical Imaging of Cancer: Clinical Applications. New York, N.Y., USA: Springer; 2009. pp 72). This efficient hepatobiliary excretion prevents the selective accumulation of ICG at specific pathological sites, limiting its possible clinical uses, which actually are mainly confined to optical examinations of blood flow with applications in ocular angiography, hepatic function characterization, or in the measurement of cardiac output.
Indocyanine Green is considered a promising candidate for high sensitive tumor detection and lymph nodes mapping in the clinical fluorescence imaging applications. Particularly wished is a formulation of ICG for use in the real-time visualization of cancerous lesions and sentinel lymph node detection during surgery lesions removal or endoscopic surgical treatments.
However, as said above, due to its very low residence time in human blood, ICG does not have any significant targeting property at the tumor tissue after intravenous administration, when used at the clinical recommended dose of 0.5 mg/kg.
Moreover, ICG is unstable in aqueous solution (already at μM concentration) and must be used within 6 hours due to its tendency to aggregate. The dye-dye interactions have adverse effects on the optical properties of ICG as the decreasing of the extinction coefficient and the fluorescence self-quenching effect after the dye excitation (Landsman, M. L.; Kwant, G.; Mook, G. A.; Zijlstra, W. G. Light absorbing properties, stability, and spectral stabilization of indocyanine green. J. Appl. Physiol. 1976, 40, 575-83).
Saxena V. et al. (Journal of Photochemistry and Photobiology B: Biology 74 (2004) 29-39) disclose poly(DL-lactic-co-glycolic acid) and polyvinyl alcohol polymeric nanoparticles for improving aqueous-stability, photo-stability and thermal stability to ICG. In this work, even if the stability of ICG loaded nanoparticles was improved due to the entrapment of ICG in the polymeric envelop (showing half-life in aqueous solution of 2.5-3 days), ICG in nanoparticles shows a decrease in its peak fluorescence intensity with respect to the free ICG solution. This finding together with high particle size distribution (mean diameter around 350 nm) could result in low efficiency in the in vivo imaging of tumor targeting.
WO2010/018216 discloses a fluorescent nanoemulsion of ICG Indocyanine green, comprising in the oily phase ICG, at least one amphiphilic lipid and at least one solubilising lipid which is solid at 25° C. According to this reference, the droplets in the nanoemulsion should have an amorphous core, because crystallinity is deemed detrimental to the stability of the nanoemulsion favouring the expulsion of the encapsulated molecules to the outside of the droplets or their aggregation.
In spite of the apparent progress provided by the above nanoparticles and nanoemulsion, ICG use in near-infrared imaging still encounters problems. Altinoglu and Adair (WIREs Nanomedicine and Nanobiotechnology, Volume 2, September/October 2010, 461-477) confirm superior optical and stability properties of Quantum Dots (QDs) in NIR imaging. However, toxicity problems hinder their use and NIR dyes are still proposed. Encapsulation of ICG in nanoparticles synthesized from calcium phosphate is reviewed and in spite of the lower performance with respect to QDs, their clinical application is proposed.
Zheng et al. (Mol. Pharmaceutics 2011, 8, 447-456), in order to overcome the problems of poor aqueous stability of ICG, its nonspecific binding to proteins and lack of target specificity, disclose an ICG-containing nanostructure exploiting the non-covalent self-assembly chemistry between phospholipid-polyethylene glycol (PL-PEG) and ICG. The dual functionality of this nanostructure for targeted optical imaging and photothermal therapy is proposed. Their use in in vivo photothermal therapy has been recently described (Zheng et al. Mol. Pharm, 2012, 9(3):514-522).
Navarro et al. (Journal of Biomedical Nanotechnology, Vol. 8, 594-604 and 730-741, 2012) disclose lipid nanoparticle vectorization (LNP) of ICG as beneficial for intra-operative fluorescence. In the work, it is quantified for up to two days the improvement on in vivo tumor/skin and ex vivo tumor/muscle fluorescence ratio of the ICG-LNP in comparison to the free dye injection (by a factor of 2 between 24 and 48 h).
US2006/0083781 discloses solid lipid nanoparticles which are functionalized in view of their use in tumor targeting therapeutic systems, thermoresponsive payload delivery systems, magnetic-driven targeting systems, therapeutic diagnostic systems, stabilized ink compositions and cosmetic formulations. Furthermore, the developed process is amenable to encapsulation of the quantum dots in a lipid environment diminishing their accessibility to oxidative species and Cd-associated toxicity.
A review of the state of the art dealing with all methods for solid lipid nanoparticles preparation is provided in Sawant and Dodiya (Recent Patents on Drug Delivery & Formulation 2008, 2, 120-135). In order to optimize the delivery properties of a nanoparticle a particle size lower than 100 nm is preferably required. On the contrary, many of the methods exploited in the literature provide SLNs with an average particle size in the micrometer range (Cortesi et al., Biomaterials 2002; 23:2283-2294) or not lower than 200 nm (Garcia-Fuentes et al. Colloids and Surfaces B: Biointerfaces, 2003, 27: 159-168; Morel et al. European Journal of Pharmaceutics and Biopharmaceutics 45 (1998) 157-163), i.e. well above the preferred nano-size range.
Advantageously, in the present invention the formulation of nanosuspensions shows particle size lower than 100 nm and shows a prolonged blood circulation half-life and an improved photostability and fluorescence signal.
These unexpected results were achieved by the optimization of the amphiphilic components and the preparation method.