İlaç-gen salımı ve optik görüntüleme ajanı olarak yakın kızılötesi ışıyan katyonik Ag2S kuantum noktacıklarının geliştirilmesi

Luminescent quantum dots (QDs) are widely studied in optical imaging, luminescent labelling, drug/gene delivery and for the combination of these. QDs are remarkable optical probes since they offer size tunable strong luminescence along with large absorption coefficients across a wide spectral range, long luminescence lifetime and superior stability against photo bleaching, allowing simultaneous detection and long working times. However, despite of such great promise, there is no clinical use of QDs, mostly due to the toxicity of the commercially available and most frequently used QDs and their emission in the visible range (400-700 nm). Visible light has low photon penetration depth, absorbed and scattered by some biological constituents. Besides, auto-fluorescence of collagen in the living tissue, decreases the contrast. On the other hand, emission in the near infrared I (NIR-I) (700-900 nm) offers high-resolution, high sensitivity, deeper penetration of light providing lower signal loss and thus greater signal/background ratio. Therefore, NIR emitting QDs have emerged as a new class of fluorescent probes for biological/medical use in the recent years. Ag2S QDs are the most promising NIR-emitting QDs with high cytocompatibility compared to heavy metal containing, more traditional QDs such as PbS, PbSe, CdHgTe. In this thesis work, we aimed to develop cytocompatable, cationic Ag2S NIRQDs with strong emission in the NIR I as theranostic nanoparticles to deliver genes or drugs to target cells and provide strong optical signal in the cells to detect the location of the delivery vehicle. Another objective of this work is to produce such QDs in a simple, green and economically viable way. Briefly, cationic Ag2S NIRQDs have been synthesized in a very simple one step aqueous synthetic route with a mixed coating of branched polyethyleneimine (PEI) and small capping agents at low temperatures. Molecular weight of the PEI (25kDa to 1.8 kDa), type of small molecule and PEI/small molecule ratio as well as the Ag/S, Ag/coating ratio, reaction duration, temperature and pH were all studied to tune emission wavelength and to maximize the colloidal stability, luminescence quantum yield while keeping the size small and transfection efficiency high. 2-mercaptopropionic acid (2MPA), l-cysteine (Cys), l-arginine, l-histidine and l-glutamic acid were tested as the small molecule component of the coating. These small molecules were chosen based on their functionalities, in case of the amino acids based on the side chain functionality. Low molecular weight PEI and small molecules without the thiol functionality have failed to produce colloidally stable and luminescent Ag2S. PEI coated Ag2S does not luminesce, but the ones with the mixed coating are strongly luminescent in the NIR I region. Best composition was determined as PEI/2MPA (80/20 mol/mol %) and PEI/Cys (60/40 mol/mol %) coatings, Ag/S ratio of 4 and coating/Ag of 5. Incorporation of 2MPA and Cys as a co-stabilizer reduced the surface defects on the crystal surface formed due to the inability of polymeric coating in providing a dense surface adsorption. This provided non-radiative events and hence provided strong luminescence. The most important results needed to be emphasized that the resulting quantum dots (1) have the highest quantum yield reported for Ag2S QDs until now (150 % - 157 % at pH 7.4 with respect to LDS 798 NIR dye), (2) are the first cationic ones of this class, (3) are smaller than 100 nm which is suitable for targeting and in vivo applications. The in vitro and in vivo optical imaging studies performed in cellular level, in worms and mice showed that Ag2S NIRQDs are very promising optical agents with efficient cell internalization and strong luminescence. Cationic nature of the particles was exploited for gene delivery using a standard gene, green fluorescent protein (GFP) and higher transfection efficiency was recorded especially with PEI(25kDa)/2MPA coated Ag2S QDs compared to commercial transfection reagents. The nanoparticles were also PEGylated to increase their biocompatibility further, and used for delivery of an anticancer drug, Doxorubicin, to cancer cells, in vitro. Selective delivery of nanoparticles via receptor-mediated endocytosis was also demonstrated via folic acid conjugation, which preferentially delivered DOX loaded Ag2S QDs to FR positive cell lines, inducing a strong apoptotic mechanism. In another study, the PEGylated nanoparticles were loaded with p53-GFP gene, a tumor suppressor gene, and used in the p53 -/- cancer cell lines. However, the transfection ability of the QDs dropped dramatically after PEGylation. Overall, within the scope of this thesis work, the very first and currently only cationic Ag2S NIRQDs was produced in an industrially feasible one step green synthesis, with the highest quantum yield ever reported for any Ag2S NIRQDs with excellent stability over 4 years now and in small sizes, allowing drug and gene delivery, molecular targeting and optical imaging. This work provides a practical QD composition which has high potential for clinical use.