Nanoparticle enabled diagnosis and combination therapy of cancer

Cancer causes approximately 10 million deaths yearly, with even more people diagnosed, comprising one-sixth of global mortality. It is both an economical and public health burden. Despite the developments in conventional therapies, which include surgery, chemotherapy and radiotherapy, the treatment outcome still falls behind the disease progression. As such, novel therapeutic approaches are needed to address the insufficiencies associated with conventional treatments, such as tumor specificity, poor prognosis, and side effects. Light-based therapies for different pathologies date back to ancient. Two main approaches, photodynamic therapy and photothermal therapy, have been highly popular in recent years. Photodynamic therapy mainly exploits the interaction between light, photosensitizer and oxygen, which produces reactive oxygen species killing the tumor cells via direct and indirect pathways. Photothermal therapy relies on localized temperature increase upon irradiation of a photosensitizer with light. Photothermal therapy does not require oxygen to be effective, thus, it has the potential to treat hypoxic tumors as well. For both modalities, photosensitizers that can work with near-infrared light is highly desired for safety and deep tissue penetration. Semiconducting nanocrystals, namely quantum dots (QDs), were initially designed as luminescent nanoprobes, and recently they are being investigated for theranostic approaches as drug delivery vehicles and photosensitizers for photothermal therapy. The first generation of QDs based on Cd, Hg or Pb suffered from toxicity. As a result of extensive research efforts, novel, heavy metal free Ag2S (AS) QDs emerged as a new class of theranostic QDs which may be tracked optically in NIR and induce local temperature increase upon irradiation in NIR. This thesis work was dedicated to exploit the theranostic applications of AS QDs in cancer treatment. In the first part, they were utilized as hydrophobic drug carriers, to efficiently deliver a commonly used chemotherapeutic agent, paclitaxel (PTX) and provide PTT at the same time. Since PTX suffers from low aqueous solubility, a novel design idea was proposed to produce small-sized, colloidally stable, NIR emitting AS QDs. Inspired by clinically approved albumin bound PTX structure, Abraxane®, bovine serum albumin (BSA) was covalently conjugated on the surface of the QDs and PTX was electrostatically loaded on the BSA. PTT/chemotherapy potential of this optically trackable nanoparticle (AS-BSA-PTX) was tested on SKBR3 and MDA-MB-231 breast cancer cells in vitro, utilizing a laser irradiation at 808 nm, 700 mW for 10 min, after 24 h incubation. For both cell lines, a significant difference between only PTT and combined treatment was observed at low doses. The IC50 value of PTX, 100 ng/mL, was reduced down 5-fold to 20 ng/mL with combination therapy. This design holds great potential as it can be adapted to deliver other hydrophobic species as well. In the second part of this thesis work, another chemotherapy drug, Cisplatin (CPt), was delivered to HER2 overexpressing breast cancer cells selectively via a targeted approach using Herceptin (Her) as a targeting molecule. Glutathione (GSH) coated AS QDs were utilized as imaging, drug delivery and PTT agents. Her was covalently conjugated on the QD surface and CPt was electrostatically loaded. AS-Her/CPt QDs were small-sized, colloidally stable, had strong NIR emission at 900 nm and provided sufficient temperature increase in the solution when irradiated with 808 nm (215 mW) laser. In vitro experiments showed 2-fold enhanced intracellular uptake of the QDs in HER2(+) SKBR3 cells compared to HER2(-) MDA-MB-231 cells due to receptor mediated internalization. The viability of the SKBR3 cells was reduced by 75% with PTT/chemotherapy combination at 100 μg/mL [Ag] concentration, with only 4 h of incubation and 808 nm (700 mW) laser irradiation for 10 min. Following up on the promising in vitro results, a safe dose of ASHer/CPt QDs was injected in vivo on tumor xenograft nude mice of SKBR3 cells. Selective accumulation on the tumor site was confirmed with IVIS imaging throughout the treatment period. Enhanced treatment efficiency was observed compared to mono therapies, chemotherapy and PTT with AS-Her QDs. Only chemotherapy treatment (only CPt at 0.7 mg/kg without laser irradiation) and only PTT treatment with AS-Her at 30 mg/kg dose (2.0 W/cm2, 10 min laser irradiation) resulted in 40% and 30% tumor volume decrease, respectively. When PTT/chemotherapy combination treatment was applied, complete tumor regression was observed at the same treatment parameters. Thus, AS-Her QDs were proposed as a novel, efficient PTT agents for the treatment of HER2 overexpressing tumors and delivering therapeutic cargo to tumor site. The third part of this thesis was focused on the delivery of a novel PDT agent, brominated hemicyanine (Hemi-Br) for the image-guided, combined treatment of folate receptor (FR) positive HeLa cells. For this purpose, AS-GSH QDs were decorated with folic acid (FA) and electrostatically loaded with Hemi-Br. AS-GSH-FA/Hemi-Br QDs were colloidally stable over one year, small-sized (<100 nm) and had dual emission at 705 and 900 nm from Hemi-Br and AS QDs, respectively. PTT and PDT potential of the final nanoparticle was first investigated in solution. With laser irradiation at 640 nm (215 mW),17.74oC temperature increase was observed with the combined nanoparticle compared to 6.67oC and 13.6oC with free Hemi-Br and AS-GSH-FA, respectively. This study signifies as the first study to investigate light-to-heat conversion efficiency of AS QDs at this wavelength. The singlet oxygen formation with laser irradiation of Hemi-Br was also confirmed in the solution. After promising results in the solution, particles were facilitated as FR targeted, NIR-emitting, PTT/PDT dual therapy agents in FR(+) HeLa and FR(-) A549 cells. No cytotoxic effect was observed in the absence of laser irradiation. On the other hand, complete cell death was observed for the targeted HeLa cell line at 10 μg/mL Hemi-Br (57 μg/mL [Ag]) dose when PDT/PTT combined therapy was applied. Whereas the viability was approximately 70% for untargeted A549 cells at the same concentration. The cell death mechanism was further investigated to show the effects of mono therapies. The fourth and the last chapter was aimed to enhance the PDT efficiency by improving the tumor hypoxia. For this purpose, oxygen and hydroxyl radical producing MnO2 was produced on the BSA coated AS QDs by the reduction of KMnO4, and a hybrid structure of AS-BSA-MnO2 was produced. To provide PDT, 5-aminolevulinic acid (ALA) was electrostatically loaded to these particles. Final nanoparticle had NIR emission and small hydrodynamic size with an ability to perform PDT and PTT in the solution. No significant cytotoxicity was observed on SKBR3 and MDA-MB-231 breast cancer cell lines without the laser irradiation, indicating the biocompatible nature of the nanoparticles. In the presence of both MnO2 and AS QDs, significant enhancement in the ALA-PDT treatment efficiency was observed for both cell lines with co-irradiation at 640 and 808 nm (300 and 700 mW) for 5 min. Additionally, the superiority of the enhanced combination treatment compared to mono therapies was shown. These findings demonstrated that AS-BSA-MnO2-ALA had excellent image-guided PTT/PDT potential against breast cancer cells.