Pharmaceutical Nanotechnology
Polymeric nanoparticulate delivery system for Indocyanine green: Biodistribution in healthy mice

https://doi.org/10.1016/j.ijpharm.2005.11.003Get rights and content

Abstract

The objective of this study is to investigate the biodistribution of Indocyanine green (ICG) in healthy mice, when delivered through polymeric nanoparticles. The poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticles entrapping ICG were engineered and characterized. The extraction method for ICG recovery from biological samples was developed. The biodistribution of ICG was determined in healthy C57BL/6 mice (female, 10-week old) when delivered through PLGA nanoparticles in comparison to free ICG solution, using a fluorometric assay method. The extraction method for ICG shows efficiency above 80% for various organs and plasma. When nanoparticles were used to deliver ICG, 2–8 times higher concentrations of ICG was deposited in various organs, with 5–10 times higher plasma levels till 4 h, after an i.v. dose as compared to free ICG solution. In conclusion, the nanoparticle formulation significantly increased the ICG concentration and circulation time in plasma as well as the ICG uptake, accumulation and retention in various organs. Overall, this study represents the first step in exploring and establishing the potential of nanoparticles as an ICG-delivery system for use in tumor-diagnosis and photodynamic therapy.

Introduction

Indocyanine green (ICG) is a water-soluble tricarbocyanine dye, which has been approved by the United States Food and Drug Administration for various medical diagnostic applications (Philip et al., 1996, Maarek et al., 2001, Saxena et al., 2003). Recently, much attention has been focused on the potential of ICG as fluorescence contrast agent in diagnostic imaging for early detection of superficial tumors such as of breast and skin cancer (Saxena et al., 2004a, Saxena et al., 2004b). Moreover photodynamic therapy using ICG is also a promising method, which is now being evaluated for superficial tumor destruction (Fickweiler et al., 1997, Abels et al., 2000). It has been shown that incubation of tumor cells with ICG and then subsequent irradiation with a diode laser leads to cell killing by photo-oxidation (Fickweiler et al., 1997, Abels et al., 2000). An important motivation for using ICG in above-mentioned studies is its strongest absorption band around 800 nm and its most intense emission around 820 nm. These are the wavelengths for which the blood and other tissues are relatively transparent and the penetration depth of light in biological tissue is the highest (Saxena et al., 2003).

For both tumor-imaging and photodynamic anticancer therapy applications, the delivery of ICG to the tumor site, intracellular uptake of ICG and accumulation and retention in the tumor are the crucial steps. Once injected in blood circulation, all these crucial steps mainly depend upon the protein binding and blood circulation time of ICG. Now, ICG has a very high protein binding in the blood and shows rapid elimination from the body (plasma t1/2 = 2–4 min) (Mordon et al., 1998, Desmettre et al., 2000). This leads to low intracellular uptake and minimal accumulation of ICG, at the tumor site, respectively. Thus, these characteristics of ICG are the major limitations in its above-mentioned novel applications in tumor-imaging and anticancer therapy.

Thus, efficient delivery of ICG to the tumor site and its retention at tumor site is required for application of ICG in tumor-diagnosis and destruction. For this purpose, an intravenously administrable product/system for ICG having ability for tumor targeting and efficient intracellular uptake will be of great interest. One of the approaches can be ICG delivery to the tumor site and subsequently into the tumor cells through a polymeric nanoparticulate system (Leroux et al., 1996). Now, nanoparticulate delivery systems are associated with enhanced permeation and retention (EPR) effect, which is filtering out of the nanoparticle from the blood stream (in the blood vessel) into the tumor site (due to the leaky vasculature at the tumor site) and subsequently accumulating at that site (Monsky et al., 1999). Thus, the EPR effect provides passive targeting to the tumor site for the nanoparticulate delivery systems. The polymeric nanoparticles containing ICG will thus provide passive tumor targeting of ICG to the tumor site.

In our earlier studies, we have engineered a nanoparticulate formulation for ICG by entrapping ICG within poly(dl-lactic-co-glycolic acid) (PLGA) polymeric matrix (Saxena et al., 2004a, Saxena et al., 2004b, Saxena et al., 2004c). Also, we have established that these nanoparticles provided efficient aqueous-stability, photo-stability and thermal-stability to ICG (Saxena et al., 2004a). Moreover we have proved that these nanoparticles enhanced the in vitro intracellular uptake of ICG in the tumor cells (Saxena et al., 2005). Now, the main focus of our present study is to characterize the in vivo biodistribution of ICG in healthy animal model, when delivered through PLGA nanoparticles, to access the merit of PLGA nanoparticles as ICG carriers and delivery systems over free ICG solution, for tumor-diagnosis and anticancer therapy.

Thus, studies on development of extraction method of ICG from biological samples and determination of the biodistribution of ICG using PLGA nanoparticles were performed on healthy mice. Also for comparison ICG biodistribution was also determined using free ICG solution. This knowledge of in vivo biodistribution of ICG through PLGA nanoparticles will help to optimize the ICG delivery to tumors. Moreover, this study will also form the reference for studies involving ICG delivery to tumors using delivery systems such as nanoparticles. Overall this research will help in the efficient use ICG for tumor-diagnosis and anticancer therapy applications.

Section snippets

Materials

ICG (free of sodium iodide) was obtained from Fisher Scientific (Fisher Scientific Inc., Pittsburgh, PA). Poly(dl-lactic-co-glycolic acid) 50:50 and polyvinyl alcohol (PVA) (88–89% hydrolyzed) were purchased from Sigma (Sigma Chemical Co., St. Louis, MO). All organic chemicals and solvents used were of reagent grade.

Animals

Pathogen free (healthy) C57BL/6 mice (female, 10-week old) were purchased from Taconic, Germantown, NY, housed and used according to the protocol approved by the university animal

Characterization of nanoparticles

The following characterization of the nanoparticles was carried out according to our previous studies (Saxena et al., 2004a, Saxena et al., 2004c). The entrapment efficiency of ICG in PLGA nanoparticles was about 74.5 ± 2.2%, the ICG content of nanoparticles was 0.20 ± 0.01% (w/w) and nanoparticle recovery was about 45%. Mean diameter of the nanoparticles was 300 ± 10 nm with polydispersity index of 0.06. The nanoparticles obtained were nearly spherical in shape. The AFM image shows the numerous pores

Conclusions

A PLGA nanoparticulate delivery system for ICG was developed and characterized. The biodistribution of ICG when delivered through nanoparticles in healthy mice were determined using a fluorometric assay method. Compared to free solution, nanoparticles led to higher ICG deposit in organs (2–8 times) as well as in blood (5–10 times), reflecting the enormous potential of PLGA nanoparticles as delivery systems for ICG for its use in tumor-diagnosis and photodynamic therapy.

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