Dendrimer functionalized magnetic nanoparticles as promising platforms for cancer theranostics

2017-03-20T23:21:48Z (GMT) by Saumya Nigam
Cancer therapeutics deals with development of preclinical therapeutic drugs for cancer which are developed through innovative research and technologies. This itself is a very challenging task and it involves various approaches to prevent, diagnose and treat cancer. Various functional nanomaterials have been developed for improving the efficiency of therapeutic drugs against cancer. For use in biomedical applications, the nanomaterials must exhibit unique properties like reduced size, aqueous stability, biocompatibility, and interactive functional groups. The ‘active’ surfaces of nanoparticles could be modified by organic or inorganic materials, such as macromolecules, bio-molecules, drugs, etc. Amongst various functional materials, magnetic nanomaterials have emerged as versatile nanosystems promising for the detection, diagnosis and treatment of cancers. Superparamagnetic iron oxide (Fe<sub>3</sub>O<sub>4</sub>) nanoparticles have been thoroughly investigated as drug delivery vectors, magnetic drug targeting agents, contrast agents in magnetic resonance imaging (MRI) and hyperthermia treatment of cancer. Dendrimers are another emerging class of functional nanomaterials which are hyper branched, mostly symmetrical polymers with repetitive branching units. The presence of multiple functional groups makes them ideal candidates for anchoring guest molecules and therefore, they are assessed as delivery vectors, MR imaging agents, stabilizers of molecules, catalysis, sensing etc. Combining these two nanomaterials would contribute towards the development of ‘smart’ and versatile nanosystems with desired properties.<br><br>    Citric acid functionalized Fe<sub>3</sub>O<sub>4</sub> (CA–Fe<sub>3</sub>O<sub>4</sub>) and glutamic acid functionalized Fe<sub>3</sub>O<sub>4</sub> (Glu–Fe<sub>3</sub>O<sub>4</sub>) aqueous colloidal magnetic nanoparticles were synthesized. The surface engineering made the Fe<sub>3</sub>O<sub>4 </sub>hydrophilic and facilitated its aqueous suspensions. Their successful synthesis was confirmed by X-Ray diffraction (XRD) studies while infrared spectroscopy (FTIR) was used to confirm the surface modification. The electron micrographs showed that the nanoparticles were spherically-shaped, evenly dispersed and magnetometry confirmed their superparamagnetic nature (<i>M<sub>S</sub></i> = 57 emu/g at 20 kOe). Time-dependent calorimetric measurements determined the specific absorption rate (SAR) of CA–Fe<sub>3</sub>O<sub>4</sub> and Glu–Fe<sub>3</sub>O<sub>4</sub> which is an important parameter in evaluating their heating efficacy in the presence of alternating magnetic field (ACMF). The SAR of CA–Fe<sub>3</sub>O<sub>4</sub> was found to be 49.24 W/g at an applied field of 10 kA/m. On the other hand, SAR of Glu–Fe<sub>3</sub>O<sub>4</sub> nanoparticles was ~134 W/g. These values strongly suggested that these nanoparticles could also act as effective heating source for magnetic hyperthermia. Doxorubicin hydrochloride (DOX) was used as a model drug to evaluate their performance for drug delivery. The DOX molecules were released in substantial amounts in the mild acidic environments. These nanoparticles showed no loss of cell proliferation activity with the mouse fibroblast (L929), human cervical carcinoma (HeLa), oral carcinoma (KB), prostate cancer (PC-3) and human breast cancer (MCF-7) cells.<br><br>    A peptide dendrimer of second generation was synthesized by divergent method following the condensation reaction of L-lysine and L-arginine as monomeric units. Its structural characterization was carried out by various sophisticated techniques such as nuclear magnetic resonance (NMR), FTIR and X-ray photoelectron spectroscopy (XPS).<br><br>    The Glu–Fe<sub>3</sub>O<sub>4</sub> were functionalized by various generations of polyamidoamine (PAMAM) and as-prepared 2<sup>nd</sup> generation peptide dendrimer and their performances as delivery vectors for cationic (doxorubicin) and anionic (epigallocatechin gallate) drugs were assessed. It was seen that the drug loading and release efficiency increased with an increase in the dendrimer generation. Further, the peptide dendrimers exhibited similar drug loading efficiency as the PAMAM dendrimers, however, their drug release capacity was significantly improved. These nanosystems were also found to be potentially effective in magnetic hyperthermia and were thus used towards<i> in vitro </i>combinatorial chemo-thermo therapy of cancer using human cervical cancer (HeLa) cells as exemplary model. For the magnetic hyperthermia treatment, the exposure of these cells to ACMF for 10 min was successful in reducing the viable cell population by 50% (LD50). While exploring the combinatorial therapy, it was seen that DOX in synergism with magnetic hyperthermia, enhanced therapeutic effects and successfully reduced the viable cell population to ~2%.<br><br>    The <i>in vivo</i> studies investigated the performance of dendrimer functionalized Fe<sub>3</sub>O<sub>4</sub> nanoparticles in a subcutaneous syngeneic murine melanoma model. The systemic exposure of these nanoparticles (15 mg/kg body weight) caused changes in various blood and serum parameters. This assisted with the information about their toxicity and non-specific uptake by various organs. All the vital organs (heart, kidneys, lungs, liver, spleen, brain, stomach and thigh muscles) showed no loss of activity. The biochemical parameters also remained unaltered in the treated mice in comparison to the control mice population confirming a healthy liver and renal activity. The atomic emission spectroscopy (ICP-AES) of the organs showed that the magnetic nanoparticles were mainly accumulated in the liver, lung and spleen of the mice with a meagre amount also seen in heart and kidneys. The evaluation of efficient magnetic drug targeting (MDT) revealed ~6-fold accumulation of iron in the tumour region when compared to the tumour of mice which were not exposed to the magnetic field. This high localisation pattern led to high concentrations of DOX in the tumour and thus was effective in arresting the tumour growth significantly. It was seen that lower number of doses are sufficient to suppress the tumour growth in combination with magnetic field than that required without the magnetic field. By the end of 14<sup>th</sup> day, the average tumour volume was 55 ± 8.3 mm<sup>3</sup> as compared to the control animals in which the tumour volume was seen to be 4794 ± 844 mm<sup>3</sup> (~88-fold decrease).<br><br>    Furthermore, the dendrimer functionalized Fe<sub>3</sub>O<sub>4</sub> nanoparticles were also seen to be MR active and showed higher relaxivities when compared to the commercial magnetic contrast agent. Various physico-chemical parameters potentiate and affect the MR contrast properties. The relaxivities of the nanoparticles were evaluated under the varying parameters of iron concentration, buffer environments (ultrapure water, buffered saline and simulated body fluid) and temperatures (25, 37 and 45 °C). It was seen that under the conditions of simulated body fluid environment at 37 °C, the peptide dendrimer functionalized Fe<sub>3</sub>O<sub>4</sub> nanoparticles show higher <i>r<sub>2</sub></i> (spin-spin) relaxivity of 220 mM<sup>-1</sup>s<sup>-1</sup>. <i>In vitro T<sub>2</sub></i> weighted MR imaging of dendrimer functionalized Fe<sub>3</sub>O<sub>4</sub> treated (for various treatment times) HeLa cells showed increased contrast when compared to the untreated cells. Owing to the high magnetisation, high specific absorption rate and shorter transverse relaxation time, the dendrimer functionalized Fe<sub>3</sub>O<sub>4</sub> nanoparticles could be a suitable platform for MR imaging and multimodal cancer theranostics.<br>