PROJECT | PN-iii-TE20-Iacovita

SUMMARY

The aim of this project is to further extend the very recent advancements in both preparation and application of thermosensitive magneto-liposomes (ThMLs) to cancer treatment with the precise goal of elaborating a new class of biofunctionalized hybrid ThMLs capable to efficiently induce in vitro apoptosis of cancer cells. This involves the realization of the following objectives:

Objective 1. Synthesis and size sorting of hydrophilic cubic SPIONs exhibiting enhanced heating power:

We propose to synthesize different series of superparamgentic nanoparticles (SPIONs) exhibiting cubic shape. The side of the cubic SPIONs will be varied in the range going from 10 nm up to a certain value (estimated to be around 35 nm) where the MNPs are not anymore single magnetic domain passing from the superparamagnetic regime to the ferromagnetic one. The cubic SPIONs that will heat better will be selected as heating sources for the proposed hybrid nano-system. We believe that the high heating capabilities of cubic shape SPIONs in comparison to the previous used spherical SPIONs will induce the disruption of the lipidic bilayer as well as they will sensitize the cancer cell to the action of the anticancer therapeutic drug loaded in the aqueous lumen of the magneto-liposomes.

Objective 2. Preparation of thermosensitive liposomes (ThLs) with transition temperature slightly above physiological conditions:

Within this second objective, our strategy is to design ThLs which will undergo a phase transition from the crystalline form to liquid within the temperature window above physiological temperature but in a range attainable by mild local hyperthermia - which is typically around 42°C. A reasonable choice for the primary liposomal lipid is the di-palmitoyl phosphati-dylcholine (DPPC) which displays a Tc of 41°C . The addition of various proportions of distearoyl phosphatidylcholine (DSPC) with a Tc of 54°C in the mixture will allow us to ThLs with Tc in the desired range between 42-43°C. In case of any leakage of test molecules from ThLs, cholesterol will be introduced in the composition in order to tighten the bilayer. As a long as these ThLs are intended for medical usage, we envisage to add a small percentage of Polyethylene glycol (PEG) derivatized lipids in the membrane, as an option to confer them a stealth character. We strongly believe that by carefully adjusting the amount of DPPC, DSPC, PEG-derivatized lipids and cholesterol in the composition of the lipid bilayer, we will prepare stealth and integer ThLs with a transition temperature from the crystalline form to liquid between 42-43°C.

Objective 3. Elaboration of thermosensitive magneto-liposomes loaded with anticancer drug and assessment of triggered drug-releasing features:

Different preparation methods of ThMLs will be elaborated as reversed-phase evaporation, double emulsion methods and dry film hydration in order to obtain high encapsulation efficiencies of cubic SPIONs. At this stage of the project, the cubic SPIONs will be encapsulated into ThLs together with a water-soluble fluorophore as carboxyfluorescein or rhodamine 6G. By means of self-quenching of their fluorescence, it can be established a solute release index from the liposomes lumen as a function of the temperature rise upon exposure of the ThMLs to external AC magnetic field. At sufficiently high concentration, due to the interaction between neighboring fluorophore molecules inside the liposomes lumen, their fluorescence is entirely self-quenched. The release of fluorophore molecules, as a consequence of membrane disruption at Tc due to the local heating of cubic SPIONs within magneto-liposomes in AC magnetic field, is then related directly to the progressive increase in fluorescence. A correlation between the index of release, the amplitude of the AC magnetic field and the time exposure will be done. Once all mentioned parameters are well established, the fluorophore molecules will be replaced in the preparation method of ThMLs by anticancer therapeutically agents as doxorubicin.

Objective 4. Toxicity, cellular uptake and magneto-chemotherapy capabilities assessment of the anticancer drug-loaded magneto-liposomes:

We propose within this project to compare the mechanisms involved in the internalization of as-synthesized cubic SPIONs and drug-loaded ThMLs in order to reveal the role of protective liposomal membrane in the internalization process and integrity of ThMLs inside intracellular compartment. The intrinsic and comparative cytotoxic properties of cubic SPIONs and drug-loaded ThMLs will also be assessed by using 4 standardized tests: the membrane integrity test by measuring the release of lactate dehydrogenase (LDH), the metabolic activity by the Alamar blue test, the quantitation of the ATP, and the Trypan Blue test for the viability assay. The optimal anticancer drug and cubic SPIONs loading of ThMLs will be thus evaluated on different normal and cancer cell lines. Furthermore, the hyperthermia effects on the cellular viability when either cubic SPIONs or liposomes loaded cubic SPIONs are internalized will be assessed respecting the safety limit of MH. Upon cellular internalization of drug-loaded ThMLs, the efficiency of magnetically triggered drug release will be evaluated. The major goal is to correlate the hyperthermia and chemotherapy effects with the differential internalization, biocompatibility and cytotoxic properties of the payload (cubic SPIONs and anticancer drug). The final outcome of these studies is to find the optimal preparation of the anticancer drug-loaded ThMLs in order to achieve cell apoptosis at concentrations bellow the intrinsic toxicity of payload.