ICG loaded antioxidant functionalized protein nanoparticles for targeted detection of cancer cells in sentinal lymph node.


Cancers of the oral cavity, oropharynx, supraglottis and hypopharynx are associated with a significant risk of metastatic cancer in the draining regional lymph nodes which is undetectable by standard clinical and radiological investigations. The current methodologies involve the injection of a radioactive substance, a blue dye, or both, near the tumor. it has however brought up two limitations- a) a suboptimal sensitivity with occasional false negative results; b) and toxicity and wastage problems associated with radioactive materials and the convetionally used blue dyes which may cause allergic reactions and skin necrosis. Due to Near infrared (NIR) fluorophoroe is being used widely as a contrast agent for optical imaging in angiography and guided biopsy as well as evaluating blood flow and hepatic function.Indocyanine green (ICG) is the only near-IR organic dye approved by the FDA for use in the human body. It is already in use for sentinel lymph node tracking and image guided surgery. Though it has numerous advantages over dye based and radio isotope based tracking systems, it also has some limitations. Low fluorescence quantum yield due to internal conversion and is prone to photobleaching, solvatochromic effects, and nonspecific quenching, all of which limit its utility in sensitive and prolonged in vivo imaging applications.Further, free-ICG molecules in the blood often bind with proteins, causing agglomeration leading to elimination from the body within minutes.Protein nanoparticles were used to enhance the stability of the fluorophore and extend its circulation life time. protein nanoparticles is the ease of synthesis, it exhibits biodegradability, weak antigenecity and superior biocompatibility compared with other natural polymers, such as albumin and gelatin. During 1970s and 1980s expanding medical applications of biomaterials and connective tissue research made laboratories to focus their studies on protein.Protein based nanoparticles or nanospheres are thermally stable and thus sterilization can easily be carried out. Moreover, nanoparticles can be taken up by the reticuloendothelial system, Due to a small size, a large surface area, high adsorptive capacity, and ability to disperse in water to form a clear colloidal solution, protein based nanoparticles have demonstrated their potential to be used as a sustained release formulation for anti-microbial agents or steroids Detecting optimistic tumor margins and local malignant masses during surgery is critical for long term patient survival. The use of image-guided surgery for tumor removal, particularly with fluorescent imaging, is a potential method to facilitate removing all neoplastic tissue at the surgical site. Herein I have synthesized nearly monodisperse spherical fluorescent antioxidant functionalized protein nanoparticles encapsulation covalently with indocyanine green dye molecules by chemical routes which are thoroughly characterized using analytical and spectroscopic techniques. TEM showed that the particles with diameters in the range of 70 nm–90 nm were obtained, with core and shell sizes controlled by varying component content. At the same time, the effect of ICG content on the fluorescent properties of these nanoparticles were studied, and the results indicated that fluorescence intensity of the fluorescent antioxidant functionalized nanoparticles could be precisely tuned by varying the doping amount of ICG dyes. Finally, the dye leakage was also tested; displaying that ICG molecules would not leak out from the protein nanoparticles after dispersing in aqueous media. As fluorescent intensity of surface functionalised nanoparticles was good and its fabrication procedure is much simpler, and nanoparticles obtained are very well formed with monodispersity, in the present work we are use new, economical and environment friendly methods of preparation of protein nanoparticles in which nanoparticle that retains much of the native properties of protein i.e. it is not hydrolysed or broken down; however, it has enhanced ability to increase half life of  ICG or become rapidly absorbed in vivo. And formulation is found non-toxic upto 1 mg/ml to human RBCs. The system exhibited 94% cell viability indicating that it is non-cytotoxic. I plan to proceed ahead with use of ICG loaded protein nanoparticles for detection of cancer cells in sentinal lymph node.

-Dr Mohammad Faiyaz Anwar 


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