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Multi-Functional Nano-Entities for Seamless Breast Cancer Detection and and Tumor Specific Treatment

Hanzhu Jin1, Bin Hong1, Sham Kakar2, and Kyung A. Kang1. (1) Chemical Engineering Department, University of Louisville, Ernst Hall 106, Speed School of Engineering, University of Louisville, Louisville, KY 40292, (2) School of Medicine: Oncology, University of Louisville, Baxter BioMedical Research Bldg., Rm. 204C, University of Louisville, Louisville, KY 40292

X-ray mammography, the method currently used for breast cancer detection, has low sensitivity for younger women (<40 years). Tumors have unorganized vasculature, and therefore, tend to accumulate hemoglobin – one of the strongest, natural chromophores in tissue. Therefore, optical mammography by near infrared (NIR) light, is can be effective detection approach for these women. Some metal nanoparticles can be used as NIR contrast agents for the optical mammography because of their high NIR absorption for detecting small and deeply seated tumors. Fluorophores can be also linked to the metal nanoparticles to add fluorescence contrast. Low heat (45oC) hyperthermia can induce slow death of the tumor by deactivating some enzymes in the tumor with little side effects. Nanoparticles with magnetic properties can also be non-invasively heated by alternating electromagnetic (AEM) field. These nanoparticles can target tumors specifically if they are linked with tumor specific peptides or antibodies. Therefore, combining all these features, a multifunctional nano-entity can be developed for the seamless breast cancer detection and treatment.

Magnetic nanoparticles, such as Fe3O4, are heated by a well selected AEM frequency. At a frequency of 450 KHz or lower, Fe3O4 nanoparticles at a size of 10~30 nm were heated effectively, without heating the tissue components, such as water, hemoglobin, 0.9% of NaCl solution, and tissue (ground beef). An empty vitamin E capsule (1.5x1x1 cm, tumor model) filled with 1% Fe3O4 nanoparticles in agar was placed at 1 and 2.5 cm deep in an experimental breast model made by agar and water. When a pancake shaped, AEM heater coil (diameter of 3 cm) was placed on the surface of the breast model, after only 2 minutes, the tumor model became heated by 17 and 1oC, for 1, and 2.5 cm deep, respectively. The breast model was not heated, showing the feasibility of tumor specific hyperthermia by AEM field.

Gold nanoparticles are known as strong near infrared (NIR) absorbers. Gold is inert to human body and also can be easily linked with tumor specific antibodies or peptides. We have studied the photon absorption properties of various sized nanogold (5~250 nm) particles. Nanogold particles at 150 nm showed an optical density of 0.3 at concentration of 0.01 wt% at 780 nm, which is the wavelength frequently used for optical mammography. Nanogold particles were added to a tumor model, and the tumor model was placed in an experimental breast model at various depths between 1 and 2.5 cm. The surface of the breast model was scanned with NIR at 780 nm. For 1 cm deep tumor, 150 nm sizes of gold particles at a concentration of 0.01%, showed 3.5 dB, greater than that by a usual tumor model (2.5 dB). Therefore, if the Fe3O4 nanoparticle is coated by gold, it can be an optical/thermal marker because of its high NIR absorption by gold layer and its heating by AEM.

Fluorophores can be linked to the nanoparticles to add fluorescence contrast for more accurate detection of tumors. Indocyanine green (ICG) is one of a few, FDA approved, NIR fluorophore, with a very low quantum yield (0.012 in whole blood). Hong and Kang showed that the fluorescence signal of low quantum yield fluorophors can be increased as much as 10 times by linking the fluorephores to nanogold particles at proper distance. ICG linked nanogold via streptavidin spacer showed 100% higher fluorescence contrast than ICG alone.

Luteinizing hormone releasing hormone (LHRH) is a hormone with a peptide chain of 10 amino acids. A number of human breast cancer cells over express LHRH receptors and therefore, the cancer cells can be targeted using LHRH. Among many tumor specific biomarkers, LHRH was selected because of its cost-effectiveness and high affinity. Also, LHRH can easily be linked to the gold surfaces. The binding affinity of LHRH linked nanogold particles was examined in the mouse gonadotrope cell line (LâT2) that express high levels of LHRH receptors. The result showed that the binding affinity of LHRH linked nanogold was similar to LHRH alone.

Therefore, LHRH and ICG linked, gold-coated Fe3O4 nanoparticles can be NIR absorbing, fluorescence contrast agents for optical mammography as well as thermal agents for tumor specific hyperthermic treatment.

US Army, Breast Cancer Research Program (BCRP) [DAMD 17-03-1-0572] and Kentucky Science Engineering Foundation (KSEF-148-502-03-55) had partially supported this project.