Periodic Reporting for period 1 - MANTARGET (Super resolution imaging of nanoPMOs for cancer drug delivery)
Reporting period: 2021-01-04 to 2023-01-03
Prostate cancer is one of the leading causes of deaths in Europe and US and is becoming a dramatically increasing malignancy in Asian countries. An important increase in the detection of tumors of small size has also been observed, estimated to at least 450,000 new cases of prostate cancer in 2018 in Europe. Prostatectomy, radiotherapy, or hormono therapy are currently the standard treatments of prostate cancer. However, all these therapies are very invasive and often not effective. Nanotechnology can offer new opportunities for overcoming the drawbacks of conventional therapy providing innovative solutions for a selective and side-effect free therapy for prostate cancer. Therefore the development of novel nanosystems and their biological evaluation is a topic of great importance in the European strategy for improving health. This is an ambitious challenge that requires a multidisciplinary approach (ranging from synthetic chemistry to biology) and a strong technical expertise. In this project, we proposed to combine the state-of-the-art technologies towards the treatment of prostate cancer: nanoPMOs (Periodic Mesoporous Organosilica Nanoparticles) and super resolution microscopy. NanoPMOs are a recent family of nanoparticles (NP) with promising properties for drug delivery. The full potential of this novel platform has still to be explored. In order to study and evaluate the behaviour of nanoPMOs in cells, we proposed to use an innovative microscopy technique: super resolution Stochastic Optical Reconstruction Microscopy (STORM). STORM allows for imaging with 20 nm resolution and therefore was ideal for tracking nanoPMOs. Thanks to this the dynamic of internalization of the nanoPMOs in prostate cancer cells through the newly identified over-expressed mannose-6-phosphate (M6P) receptor, and their degradability behaviour in cells was investigated. This project allowed a better understanding of the nanoPMOs-prostate cancer cell interactions through a potent imaging technique and provided the necessary information for the further application of nanoPMOs in the field of nanomedicine for cancer theranostics.
i) The development of fluorescently-labeled mannose-6-phosphate targeted and biodegradable periodic mesoporous organosilica nanoparticles (nanoPMOs) for the treatment of prostate cancer was successfully performed. (Milestone 1)
We successfully synthesized biodegradable bistriethoxysilylpropyl disulfide (BTEDS) and tretrasulfide, bis(triethoxysilyl)benzene (BTEB) and bis(triethoxysilyl)ethylene (BTEE)-based nanoPMOs. Those nanoPMOs were labelled with Cy3 dye and after optimization of the amount of the dye inside the nanoparticles, super-resolution microscopy of the nanoparticles was successfully achieved. After the synthesis of the nanoparticles, their functionalization with Cy5-labeled antibody was carried out. Two ways of functionalization were studied: An oriented manner through oxidation of the carbohydrate chains and immobilisation of the antibody through the semicarbazide, oxo-semicarbazone function and a random immobilization through carboxylate-amide EDC NHS coupling. The number of oriented grafted antibodies on the nanoparticles was evaluated through STORM imaging depending on the conditions of grafting.
ii) The use of STORM super-resolution microscopy to study the interactions of nanoPMOs with cancer cells was successfully carried out. (Milestone 2)
The antibody-functionalized nanoparticles were incubated with prostate cancer cells and normal cells. The oriented antibody was much more efficient than the randomly attached antibody for the targeting of LNCaP prostate cancer cells.
iii) The biological evaluation of drug-loaded nanoPMOs in models of prostate cancer was carried out. (Milestone 3)
The antibody-functionalized nanoparticles were loaded with doxorubicin and incubated with prostate cancer cells. The oriented antibody was much more efficient than the nanoparticles without antibody for killing LNCaP prostate cancer cells. Very few amount of nanoparticles were used.
We successfully synthesized biodegradable bistriethoxysilylpropyl disulfide (BTEDS) and tretrasulfide, bis(triethoxysilyl)benzene (BTEB) and bis(triethoxysilyl)ethylene (BTEE)-based nanoPMOs. Those nanoPMOs were labelled with Cy3 dye and after optimization of the amount of the dye inside the nanoparticles, super-resolution microscopy of the nanoparticles was successfully achieved. After the synthesis of the nanoparticles, their functionalization with Cy5-labeled antibody was carried out. Two ways of functionalization were studied: An oriented manner through oxidation of the carbohydrate chains and immobilisation of the antibody through the semicarbazide, oxo-semicarbazone function and a random immobilization through carboxylate-amide EDC NHS coupling. The number of oriented grafted antibodies on the nanoparticles was evaluated through STORM imaging depending on the conditions of grafting.
ii) The use of STORM super-resolution microscopy to study the interactions of nanoPMOs with cancer cells was successfully carried out. (Milestone 2)
The antibody-functionalized nanoparticles were incubated with prostate cancer cells and normal cells. The oriented antibody was much more efficient than the randomly attached antibody for the targeting of LNCaP prostate cancer cells.
iii) The biological evaluation of drug-loaded nanoPMOs in models of prostate cancer was carried out. (Milestone 3)
The antibody-functionalized nanoparticles were loaded with doxorubicin and incubated with prostate cancer cells. The oriented antibody was much more efficient than the nanoparticles without antibody for killing LNCaP prostate cancer cells. Very few amount of nanoparticles were used.
In this work, we have synthesized and well-characterized a library of anti-M6PR antibody conjugated fluorescent and biodegradable nanoPMOs with varied sizes, shapes, compositions, orientations, and multivalency in order to investigate their physicochemical properties predominantly biodegradability and surface functionality by super-resolution dSTORM imaging modality for effective stimuli-responsive drug delivery for targeted chemotherapy of prostate cancer. In dSTORM imaging with nanometric spatial resolution (around 20 nm), we have quantified the biodegradation of various nanoPMOs, which was unprecedented and found that their excellent degradation behavior depends on their structural characteristics. A single particle level analysis of nanoPMOs conjugated with an anti-M6PR antibody has revealed that one of the most important parameters, namely the surface functionality of nanoPMOs, such as the number, distribution, and density of antibody per nanoparticle, shows a significant impact on prostate cancer cell targeting using dSTORM imaging. Aside from the surface functionality of nanoPMOs, the size, and shape of nanoPMOs also significantly influence their interaction with living cells, leading to variations in cellular uptake by healthy and cancerous prostate cells. According to the results, small-sized nanorods functionalized with oriented antibody have higher M6PR overexpressed prostate cancer cell labeling capabilities when they are functionalized at high density, whereas heterogeneous surface functionality reduces the ability of nanoPMOs with low multivalency to bind to cancer cells. The excellent hydrophobic drug loading capacity of nanoPMOs into the porous structure, and glutathione-triggered controlled drug release properties could improve the effectiveness of highly biodegradable nanoPMOs, which exhibit better cancer cell targeting ability in targeted chemotherapy of prostate cancer, resulting in the potent anticancer activity of EAB4H nanorods with low lethal concentrations (LC50).