Magnetic smart drug delivery systems for theranostic using a personalized approach

Cancer is one of the most widespread group of diseases causing about 14.6% of all human deaths, representing a major public health issue worldwide. Standard treatment is usually causing side effects which can be reduced by targeting delivery with various nanostructures, including magnetic nanoparticles. Due to the fact that bare Fe3O4 nanoparticles are highly susceptible to degradation/dissolution in acidic and oxidative conditions as well as to the in vivo conditions, coating an outer protective layer to it is very important for maintaining the stability of the magnetic component until cellular internalization. Also, the proper choice of the shell will be essential in assisting the internalization of these nanostructures inside the tumor cells and further delivering the antitumoral agents directly inside the cells. The aim of the project is to improve medical strategies regarding the treatment of cancer, by developing new nanostructured systems for its targeted therapy, such as Fe3O4_dicarboxylic amino acids, hydroxy acids and keto acids, (as shell) and natural or synthetic antitumor compounds and thus to get improved cellular internalization and release of the antitumoral agents. The results obtained are promising, the Fe3O4@shell/cytostatic magnetic systems being potential solutions for the treatment of cancer, through the development of new nanostructured systems for its targeted therapy. The shell, natural products of catabolism (dicarboxylic amino acids, hydroxyacids and ketoacids) and polymers were used to assure stabilisation and/or internalization and natural or synthetic antitumor compounds were evaluated with the main aim to improve internalization into the tumoral cells and thus to reduce the harmful side effects (including systemic toxicity). The increase in internalization and targeted delivery of therapeutic agents was achieved by using amino acids, polyethylene glycol (PEG) and albumin as functionalization agent, with the role of increasing the stability of multifunctional magnetic systems and to control the release of embedded active substances in a triggered and targeted way. Polyethylene glycol (PEG) is frequently used as an agent to increase the stability of multifunctional magnetic systems in vitro and in vivo, due to its increased hydrophilicity in aqueous media, it can considerably reduce the resorption of magnetic nanoparticles by the reticuloendothelial system and, as a consequence, the circulation time compared to uncovered nanoparticles increases, nanoparticle aggregation decreases as a result of stabilization, and the association with non-targeted tissues and cells is reduced along with the systemic toxicity.

Also the “remote driving” of magnetic nanoparticles, under the influence of an external magnetic field, combined with the intrinsic penetrability of the magnetic field through the human body, magnetic nanoparticles are intended for targeted transport and triggered release of biologically active substances (transport agent in chemotherapy, antibiotics, analgesics, polyphenols, etc.) to a targeted region in the body. The advantage of the Fe3O4_CM type magnetic system using PEG, glutamic acid and aspartic acid as stabilizing and internalizing agents comes from the system’s multifunctionality, namely its role in treating cancer through the hyperthermia effect of magnetite as well as anti-inflammatory, antipyretic, antithermic, antiseptic and anti-infective thanks to the biologically active agents. The presence of these active biological agents will allow improved internalization and better stability at the cellular level because tumoral cells are more active compared to normal cells and must satisfy their greater need for specific compounds (vitamins, oligo-elements, …), nutrients and oxygen.

Based on these considerations, in stage I of the project, a series of multifunctional Fe3O4@amino acids, hydroxy acids and keto glutaric acid systems were synthesized and characterized. The magnetic systems were characterized from a structural and morphological point of view with the help of the physico-chemical methods characteristic for this type of materials. Depending on the nature of the CM, the change in the morphology of the mesoporous magnetic nanoparticles can be observed. Following the biological evaluation of the obtained Fe3O4@MC systems, most of the magnetic systems stabilized with the biologically active substances presented a better antimicrobial activity compared to the controls, and a synergistic effect was also observed. Among them, Fe3O4@ L-tryptophan determined the highest sensitivity of E. coli, P. aeruginosa and C. albicans, with MIC values of 0.001 mg/mL. Fe3O4@L-cysteine inhibited the adhesion of all strains studied. Cell viability, regardless of the nature of the stabilizer used (serine, tryptophan, cysteine, proline, glutaric, a-keto-glutaric, tartaric, usnic) is greater than 60%, which confirms good compatibility.

In the second stage of the project, series of multifunctional Fe3O4@PEG_CM magnetic systems was obtained using PEG, glutamic acid and aspartic acid as stabilizing and internalizing agents. The presence of these active biological agents will allow improved internalization and better stability at the cellular level because tumor cells are more active compared to normal cells and must satisfy their greater need for nutrients and oxygen. These nanoparticle systems were compositionally and morphologically characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM-HRTEM), as well as chemical stability in specific environments. Detailed structural characterization using the specified techniques confirmed the successful formation of Fe3O4/amino acid (AA) and Fe3O4/PEG/amino acid (AA) core/shell hybrid structure. The X-ray diffractograms obtained on the magnetic nanopowders coated with PEG and/or amino acids indicate the presence of the magnetite core and thus the specific peaks of crystalline magnetite can be observed. TEM and DLS analyses showed good stability and polydispersity of these systems with an almost spherical shape.

These Fe3O4@MC/PEG nanostructures are able to adsorb cytostatic molecules and target them to the diseased organ/tissue followed by targeted drug release. This innovative and targeted drug delivery strategy involves coupling the drug to magnetic nanoparticles (NPs) that can be guided to the target using external magnetic fields. Once they reach the target organ/tissue, the nanoparticles release the drugs under the influence of the alternating magnetic field that can generate hypothermia and implicitly increase the release rate of cytostatics. At the present stage, the internalization capacity of the proposed and realized magnetic structures has been proven, and the studies will continue with a view to their optimization and access to higher levels of technological development

Also in the second stage of the project, a series of drug delivery systems based on citrate-stabilized MNPs, which act as nanocarriers for polyphenolic compounds (PCs) from extracts (BBE and BPE) with 5-fluorouracil. This study presents the development of drug delivery nanocarriers based on magnetite and trisodium citrate to improve the antitumor activity of 5-FU and mitigate the adverse effects on gut microbiota. MNPs were synthesized by a spray assisted method and loaded with bee pollen extracts (BPE) or bee bread extracts (BBE) and 5-FU. BPEs and BBEs have been characterized in previous studies. First, the loaded MNPs were morphologically and structurally characterized by physical analysis. The release behaviour of the bioactive agents was evaluated for 5-FU and PC, and their antioxidant activity was evaluated by three assays. The purpose of this research is based on the use of low concentrations of extracts and 5-FU to establish the influence of each bioactive compound and the effects on a selected bacteria with probiotic potential. In addition, the novelty of this study involves the increase of the inhibitory activity of the bacterial adhesion capacity to the inert substrate induced by the synthesized nanocarriers.

The results demonstrated that MNPs with narrow size distributions and an average NP size of ~10 nm were obtained. Moreover, it was shown that the antitumoral drug and PCs had fast release rates in the first 24 h from MNPs. Furthermore, the PCs exhibited prolonged release behaviour (up to 96 h), and loading MNPs with extracts enhanced their antioxidant and antibacterial properties. Additionally, the non-toxic properties of magnetite and the prebiotic potential of the developed DDS on L. rhamnosus MF9 were demonstrated, underline the role of the antioxidants (PCs) in supporting gut microbiota which is essential in the cancer treatment, microbiota being able to control the healing capacity of the body, in general via different mechanisms. The moderate antiproliferative activity on a colorectal tumoral cell line at low concentrations of MNPs and 5-FU suggests the potential of DDS to alleviate the adverse effects of anticancer drugs and improve gut balance.

In the third stage of the project the antimicrobial, antitumoral and internalization capacity of the obtained systems in the first and second stage was evaluated. These magnetic nanoparticles were developed, these being able to function as Trojan Horses and to carry cytostatic drugs into the tumoral cells. This is important because the antitumoral activity is correlated with the cytostatic level from the cell and this is why the internalization is so important and many research teams are looking for solutions to improve the internalization. In the literature, most of the internalization studies are done using folic acid and here we proved that Aspartic acid could also improve the internalization capacity and thus improved antitumoral activity is obtained. Based on the high resolution images, it can see that PEG-ylated nanoparticles are quite stable and can reach and enter the mitochondria and organize around lipid vesicles in quite a high concentration. Best results, according to this study are obtained for Fe₃O₄_PEG_Asp_CisPt and Fe₃O₄-Asp-CisPt at both 100 and 500 µg/ml. Further research is needed to study the complex mechanism occurring inside the cells, which involves internalization and resorption and to correlate the ratio between Fe₃O₄, PEG and Aspartic acid, with the synthesis procedure and the type of the cancer. It is important to mention that a proper carrier have to be stable enough to assure internalization and, once these are inside, they can be destroyed and release the cargo (cytostatic drugs). Certainly, magnetic triggering could be also expected to be a solution to achieve a personalized therapy with external control of the delivery rate.