Synthesis and Characterization of Gold Nanostars Functionalized to Target Cancer Cells

Directly or indirectly, cancer affects the lives of most people, regardless of sex, social status or age. Each year millions of people die from this disease, in addition it causes pain and suffering, substantially affecting the quality of life of patients and their families, because of very long periods of hospitalization, recovery, and financial costs. Since first half of the twentieth century began to use different anti-cancer treatments that combine surgery with radiation and chemotherapy, achieving a tremendous progress in the fight against this disease. Anti-cancer agents can be as diverse as drugs, liposomes, natural products, therapeutic antibodies, cell growth inhibitors, angiogenesis inhibitors and apoptosis-inducing compounds. The introduction of new technologies has had a major impact on how cancer is treated and diagnosed. Specially with the identification of specific cancer biomarkers and the development of methods for early detection and monitoring, development of non-invasive diagnostic methods, alternative techniques to obtain and processing of biopsies, development of new contrast agents for imaging, revolutionary surgical procedures for removal of tumors and metastases, and the modification of compounds with anti-cancer activity to achieve tissue-specific delivery and reduce side effects. The recent advances in nanotechnology provide new alternatives to diagnose and treat cancer. It is possible to perform synthesis of inorganic materials with high precision and control at the nanoscale (1-100 nm), coupled with availability of functionalization/bioconjugation techniques to modify the surface of nanomaterials to achieve the development of smart devices based on functional nanoparticles for biomedical applications. Use of hybrid nanoparticles as nanomedicines requires a precise control of different properties like size, shape, morphology, charge, and physicochemical parameters to ensure biocompatibility and specificity for cancer cells. Obtaining smart nanodevices with advanced properties to be used for cancer therapy and diagnostics.

Metal nanoparticles have great potential as multifunctional therapeutic and diagnostic systems. The size, shape and chemical composition of nanoparticles determine the properties, functions and potential applications in nanomedicine. The movement, location and bio-distribution depend mainly on the size of the nanoparticles, that for biomedical applications ideally should be 10-100 nm to minimize accumulation and nonspecific interactions. Nanoparticles can be designed to target over-expressed receptors characteristic of cancer through functionalization with biomolecules (analogs, ligands, peptides, proteins or antibodies) to facilitate their adsorption and internalization. Nanotechnology-based systems have demonstrated their potential in diagnostic/treatment of cancer. Particularly in the early detection, as contrast agents in imaging techniques and tissue-specific administration of therapeutic agents. Nanotheranostics is a term derived from thera(py)+(diag)nostics that links properties of new advanced functional nanomaterials. This molecular-targeted nanomaterials have been developed to minimize the side effects of traditional cancer treatments as surgery, radiation and chemotherapy. Particularly, gold nanoparticles with anisotropic structures (star-shape, rods and core-shell) are of great interest as nanotheranostic devices, because they show properties of surface plasmon resonance with a maximum absorbance peak in the near infrared (NIR) spectra (650-900 nm). The exposure of nanoparticles to NIR light causes resonance and collective oscillations of free electrons converting electromagnetic energy into heat. This phenomena can cause controlled release of bioconjugated drugs or a direct photothermal-thermolytic effect on cancer cells by increasing the temperature at 42-47°C, inducing cell death, apoptosis and even necrosis. An important advantage is that NIR light can be effectively delivered to inaccessible tumors located several centimeters deep under skin, with no damage to healthy tissues.

Previous reports have observed that nanoparticles passively accumulate into tumor tissues, for, bioconjugation with different biomolecules including peptides, antibodies, analogs and drugs is a strategy to achieve specific attachment and internalization of nanoparticles on cancer cells. Folic acid (FA) have been used to target different anti-cancer compounds, taking into advantage that Folate receptor (FR) alpha is highly overexpressed on different cancer cells, including myelogenous leukemia, breast, lung, kidney, prostate, colon and brain. Another advantage of FR is that its expression in most normal cells is very low or even non-detectable, and possesses high affinity and specificity for drugs and particles bioconjugated with folic acid.

Significance. It is necessary to develop new methods to treat and diagnose cancer by integrating recent advances in nanoscience and nanotechnology. Most cancer drugs are blunt instruments. We propose to engineer new chemotherapeutic agents at the molecular level to target prostate cancer cells and selectively destroy them through integration into a nanotechnology-based platform. The development of smart nanotheranostic devices require a multidisciplinary approach, integrating different areas of knowledge such as physics, material sciences, medicine and biotechnology, to perform the design, synthesis, characterization and functional evaluation of multifunctional hybrid nanoparticles with unique characteristics and potential in biomedical applications as therapeutic and diagnostic systems. The bioconjugation enables specific delivery of nanoparticles into malignant cells and avoiding healthy cells, making it feasible to achieve a non-invasive treatment of electromagnetic energy by exploiting the optical properties of nanoparticles.

Results and Discussion. The research objective of this project was to develop and evaluate novel nanotechnology-based devices with advanced properties to be used for cancer therapy and diagnostics. The aim is based on the hypothesis that folic acid allows the specific targeting of folate receptor alpha receptors over-expressed in cancer cells and mediate active transport of gold nanoparticles. Coupling with NIR radiation, nanotheranostic devices will induce cell death by apoptosis. To achieve the objective, our approach include: (1) design and synthesis of gold nanoparticles with star-shape, (2) bioconjugation of gold nanoparticles, (3) characterization of gold nanoparticles using advanced electron microscopy techniques, (4) characterization of properties of nanotheranostic devices, (5) in vitro assays of nanotheranostic devices. Analysis and characterization of nanomaterial bioconjugates used in biomedical applications require use of advanced techniques due to complexity of interactions of nanomaterials with living systems.

Synthesis of gold nanostars (GNS). Synthesis of anisotropic nanoparticles was obtained through a seed-mediated route using 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethane-sulfonic acid (HEPES) as a precise shape-directing agent and hydroxylamine as catalyst and reductant. This method allows controlling surface branching and nanostructuration, maintaining high level of monodispersion and controlled size.

Bioconjugation of GNS. Bioconjugation of folic acid to gold nanoparticle surface was through carbodiimide conjugation reactions to organic compounds used as capping agents during synthesis or gold nanoparticles. Raman spectroscopy revealed characteristic peaks corresponding to folic acid, confirming functionalization of gold nanoparticles. Gold nanoparticles conjugated were analyzed by advanced electron microscopy techniques to confirm nanostructuration, size distribution, shape, chemical composition, bonding and surface properties.

Characterization of GNS. UV-Vis spectroscopy determined that absorbance peak is centerd in 700 nm (surface plasmon resonance). Size distribution of nanoparticles in solution, charge and colloidal stability (zeta-potential) were determined by Dynamic Light Scattering. FE-SEM imaging (HITACHI FE-SEM S-5500) confirmed size distribution (75-85 nm) and monodispersity of gold nanostars. Aberration corrected HR-TEM (JEOL ARM 200F) revealed crystalline atomic structure of GNS, with the lattice spacing of 2.35 Å corresponding to fcc gold oriented over (111) face.

In vitro interaction of cells with GNS. In vitro assays with mouse macrophages revealed that GNS are biocompatible even at high concentrations. Cell viability remained at 97±1% after 4-24 h of incubation. We developed new advanced high resolution SEM techniques to analyze complete uncoated and unstained by SE (secondary electrons), BSE (Backscattered Electrons) and EDX microanalysis. Cells showed that GNS were efficiently adsorbed on cell membrane and internalized. Low voltage BF/DF-STEM of thin sections revealed that GNS are internalized in cytoplasm close to cell membrane and into lysosomes after 3 or 24 h of interaction. Chemical composition of internalized nanoparticles was confirmed by EDX and backscattered electron imaging.

As a perspective of this work, next step of the project include analysis of interaction of bioconjugated GNS with animal models for prostate cancer and use or NIR radiation for thermal-ablation of cancer cells.

Conclusion. In this work we obtained gold nanoparticles with star-shape through a seed-mediated method with high level of monodispersion and controlled size (75-85 nm). Surface of nanoparticles was functionalized with folic acid. Electron microscopy characterization revealed size distribution, morphology, chemical composition and atomic structure of gold nanostars. Biocompatibility and biodistribution using in vitro interaction of nanoparticles with cultured cells were analyzed by advanced electron microscopy techniques, including FE-SEM, Backscattered electron imaging, EDX microanalysis, low voltage BF/DF-STEM and HR-TEM confirming internalization of gold naoparticles.