Biopharmaceutical profiling of cancer targeted gold nanoparticles for doxorubicin delivery

This thesis project is focused on the development of folate-targeted gold nanoparticles for the delivery and controlled release of doxorubicin to the tumour. Moreover, this DDS can be exploited for both ultrasounds cancer treatment and in vivo imaging. GNPs were produced by the Turkevich method, according to an adapted protocol to obtain particles with a diameter of 15 nm at high concentration. The particles were decorated with different functional agents: folic acid as the targeting agent, Bodipy (BDP) as the fluorescent probe, doxorubicin as the therapeutic drug. mPEG2kDa-SH was used to saturate the particles’ surface to guarantee stealth properties to the system. In this project, folate-PEG3.5kDa-SH (FA-PEG3.5kDa-SH) was conjugated to particles’ surface at 1: 50 GNP/FA-PEG3.5kDa-SH molar ratio to selectively target folate receptor-expressing tumour cells. The particle surface was saturated with an excess of hydrophilic polymer (mPEG2kDa-SH) to increase GNPs colloidal stability by limiting aggregation phenomena; also, PEGylation was used to provide stealth features to the drug delivery system and prevent the opsonization process that could occur upon intravenous administration. Doxorubicin (DOXO) was selected as the therapeutic model for its anticancer activity. DOXO was conjugated to α-lipoic acid through a hydrazone bond in order to be released under acidic pH. Indeed, this well-known pH-sensitive bond can be exploited to trigger the drug release under the acidic environment of the endosomal cellular compartment, while preventing the undesired release of DOXO in the bloodstream. Nanoparticles were decorated with increasing [DOXO-hydrazyl-lipoic acid]:[GNPs] molar ratio to evaluate the effect of drug densities on the particles stability. After testing 200:1 400:1 500:1 700:1 1000:1 and 1500:1 [ DOXO-hydrazyl-lipoic acid]:[GNPs] molar ratios, the formulation with 1000:1 molar ratio was chosen due to the high stability and high degree of loading. The pharmaceutical properties of the DDS was investigated by, a fluorescence study and exploiting the nanomaterial surface energy transfer (NSET) effect. At pH 5 the release of DOXO by decorated GNP is significantly faster than at pH 7.4. Subsequently, an additional dialysis study showed that at pH 5 all doxorubicin is released by the GNP within 5 days. On the other hand, at pH 7.4, less than 30% of doxorubicin is released in the same time range. Stability of the decorated GNP formulation over time was investigated over ten weeks in PBS pH 7.4. Size analysis by DLS showed that the samples remained colloidal stable without sign of aggregation. However, after five weeks particles tend to adsorb on glass vials. In order to develop decorated GNP formulations for long storage, a systematic study on lyophilization process was conducted. Several cryoprotectants have been tested, such as mannitol, sucrose, trehalose, PVP, and Tween 20. The combination of PVP (0.5% w/v) and Tween 20 (0.5% w/v) allowed to liophilize the decorated GNP yielding formulations after redispersion with comparable colloidal features, namelly size and the z-potential, with respect to freshly prepared formulations. Moreover, complete recovery of decorated GNP was achieved and no release of DOXO was detected by fluorometric studies which implies no hydrolysis of the hydrazone bond. Biological studies were conducted on KB cells (human cervical carcinoma cell line), which overexpress the folate receptor (FR). The lyophilized particles incubated after redispersion with KB cells showed comparable cytotoxicity to the one of the freshly prepared formulations. Particles’ association with KB cells was investigated by flow cytometry to confirm the selectivity of the decorated GNPs after the lyophilization process. The association of the lyophilized version of the GNPs to KB cells was maintained also confirming the selectivity provided by the folate on the particles’ surface with respect to the control