The main scientific objective of the HemDelStim project is to open a new approach and a new scientific direction for the treatment of patients with chronic kidney dysfunction and liver cancer. Unlike the therapeutic approach so far, which involves performing the process of hemodialysis (to replace kidney function) and performing chemotherapy in parallel with this procedure, the project aims to combine the two, in an intelligent way, with minimal impact on the body. The drug used to treat liver cancer (doxorubicin) will be encapsulated in supramolecular or polymeric architectures, the release being made strictly on the basis of a stimulus - the tumor marker for this type of cancer (alpha fetoprotein). The concept of ‘responsive drug delivery stimuli’ will be used for this intelligent release - supramolecular or polymeric architectures containing doxorubicin will contain a monoclonal antibody for alpha fetoprotein. The presence of alpha fetoprotein in dialysis blood will cause encapsulation to break down and doxorubicin to be released. Supramolecular or polymeric architectures containing alpha fetoprotein monoclonal antibody, with encapsulated doxorubicin on the surface, will be immobilized on polysulfone membranes for hemodialysis (membranes containing anticoagulants on the surface). The combination of the two therapeutic procedures - hemodialysis and responsive drug delivery stimuli, is the main novelty of the project. It should be noted that so far there is no reference in the literature to combine hemodialysis with controlled release, the research conducted so far in the field of hemodialysis referring strictly to the development of membranes for this field.
The activities undertaken for stage I of the project were the following:
1.1. Molecular modeling of reactions to optimize working conditions by computational chemistry.
1.2. Synthesis of monoclonal antibody-functionalized supramolecular architectures for encapsulated drug AFP.
1.3. Synthesis of polymeric architectures based on polyethylene glycol (PEG) or polycarbonate (PC) for encapsulation of doxorubicin.
1.4. Morphological characterization (SEM and TEM), structural (FT-IR, Raman, XPS, MRI), cytotoxicity studies and assessment of doxorubicin release capacity.
1.5. Dissemination - scientific report, project web page, two oral communications at international conferences, and two ISI articles sent for evaluation.
All the assumed activities were carried out, below being presented some of the results obtained. The results regarding the synthesis of supramolecular architectures, as well as the synthesis of membranes for dual role hemodialysis are being patented.
Hepatocellular carcinoma is the most common type of abdominal cancer and ranks 3rd in the world in cancer mortality. Unfortunately, the treatment of liver cancer is limited due to the lack of adequate treatment options and the side effects caused by commonly used treatment methods (e.g. chemotherapy). Among the drugs used for chemotherapy, doxorubicin (DOX) is one of the most effective, but when DOX is given intravenously, about half the dose is eliminated through bile secretions. Moreover, although DOX generates cancer cell apoptosis and an antitumor immune response during chemotherapy, it also induces severe side effects such as systemic immunosuppression, which limits its clinical applications [1]. To maximize the antitumor efficacy of doxorubicin, it is desirable to develop controlled release systems for this drug. By integrating DOX into a controlled release system, the degradation of the drug in the circulatory system is reduced, thus minimizing cytotoxicity to normal cells and improving the half-life and pharmacokinetic profile. Over time, a number of controlled release systems for doxorubicin have been developed based on nanoparticles, metal-organic structures or polymers. Organic controlled release systems such as polymers or liposomes are generally more biocompatible but have low drug loading capacity while inorganic systems (eg nanoparticles of silica, iron, gold, etc.) have a high drugs retention capacity but may have biocompatibility issues [2].
By integrating cancer cell-specific recognition molecules into the structure of controlled delivery systems, targeted drug delivery to the tumor can be achieved [3]. For example, galactose is one of the most effective targeting molecules for liver cancer because it has the ability to recognize asialoglycoprotein receptors, overexpressed in hepatomas [4]. In a recent study, Pooresmaeil et al. developed such a targeted release system based on photoluminescent glycodendrimer synthesized by increasing the polyamidoamine dendrimer on zirconium-based metal-organic structures. The system was subsequently functionalized with D-galactose to increase biocompatibility and induce the targeted release of doxorubicin in hepatomas. Controlled release tests were performed at pH 5 and 7.4 and a temperature of 37 °C to accurately simulate the biological environment of tumor cells and normal cells. In vitro cell uptake, DAPI staining, MTT test and cell cycle analysis showed that the synthesized material has a high biocompatibility when not loaded with drug (80% cell viability) and has the ability to release both hydrophilic and hydrophobic drugs with a acidic pH-sensitive release profile characteristic of tumor cell environment [2]. Yang and co-workers combined doxorubicin with a cleavable peptide (B-cathepsin) and coated the system with Pluronic F68, resulting in stable prodrug-stable self-assembled nanoparticles. In vitro tests showed that the nanoparticles were cleaved and released cytotoxic molecules of DOX only in the presence of tumor cells that overexpressed cathepsin B, this behavior suggesting a tendency for selective accumulation of drug in the tumor and a significant reduction in systemic side effects of chemotherapy. [1]. Phenylboronic acid is also known for its ability to form reversible complexes with overexpressed sialic acids found on the terminal monosaccharides of glycans on the surface of tumor cells. Self-assembled nanocomplexes of doxorubicin with polymerized phenylboronic acid have shown high antitumor efficacy due to both targeted release of the drug to tumor tissues and accumulation of complexes in cells, resulting in sustained long-term release [5]. Another strategy is to target lysosomes in tumor cells because increasing the permeability of the lysosomal membrane leads to the release of enzymes inside the cell, thus causing its apoptosis. GLPG tetra-peptide and 4- (2-aminoethyl) morpholine are examples of molecules that have shown excellent lysosome targeting abilities and enhanced the cytotoxic effect of doxorubicin on tumor cells [6].
Controlled release systems based on nanostructures (eg nanoparticles, nanocapsules, liposomes) have been developed due to the need for appropriate transport vehicles to overcome the pharmacokinetic limitations associated with conventional drug formulations [7]. Below are some such systems that have been shown to increase the antitumor efficacy of doxorubicin in the treatment of hepatocellular carcinoma. Mdlovu and colleagues synthesized mesoporous nanocomposites based on iron oxide magnetic nanoparticles coated with SBA-15 silica and Pluronic F127 polymer, with applications in pH and thermosensitive release of doxorubicin. The nanocomposites were loaded with drug by impregnation in solutions with different concentrations of DOX, followed by ultrasound for DOX retention in the porous structure of SBA-15. The physico-chemical characterization showed that the nanocomposites are superparamagnetic, mesoporous and sensitive to pH and temperature, characteristics that recommend them for the targeted release of the antitumor drug, which can be directed to the site of interest through an external magnetic field. They also induced apoptosis and prevented the proliferation of Hep G2 tumor cells in vitro [3]. The same study group also developed controlled release systems based on dextran-coated magnetite nanoparticles and conjugated with DOX for the treatment of liver cancer. Dextran coating increases the systemic circulation time of nanoparticles, improves biocompatibility and gives the ability to target tumor tissues as they overexpress asialoglycoprotein receptors with high affinity for the carbohydrate structure of dextran [8]. Another type of mesoporous composite based on superparamagnetic iron oxide nanoparticles was developed by Shu et al. The superparamagnetic particles were coated with mesoporous polydopamine, modified with sialic acid and chelated with Fe3+ ions, after which they were charged with doxorubicin, thus obtaining a theranostic agent for chemo-photothermal therapy guided by MRI of liver cancer. In vitro and in vivo studies have shown that nanoparticles can effectively target tumor liver tissue due to specific interactions between surface sialic acid molecules and E-selectins overexpressed by cancer cells. Moreover, due to the combination of chemotherapy with chemo-photothermal therapy, the therapeutic effect has been greatly improved compared to conventional DOX treatment [9]. Calcium phosphate nanoparticles have also been used as multifunctional therapeutic agents for the localization and treatment of liver tumors. The nanoparticles were synthesized by the hydrothermal method, functionalization with A54 peptide and loading with DOX for targeting and treatment of hepatomas being performed during the synthesis. Biological tests have shown that the drug is released after absorption into cells due to the dissolution of Ca phosphate in an acidic medium [10]. Another study focused on the functionalization of the surface of mesoporous silica nanoparticles with long chains of polyethylene glycol (PEG) or polypropylene glycol (PPG) terminated with lactose-like targeting molecules through amide bonds. The systems were then loaded with doxorubicin, which was retained mainly in the pores of the nanoparticles, and evaluated for in vitro cytotoxicity on Hep G2 tumor cells. Studies have shown that nanoparticles decorated with lactose-anchored polypropylene glycol are the best combination for the treatment of tumors because the lipophilic nature of PPG facilitates interactions with DOX thus ensuring increased retention in silica pores, while lactose confers system selectivity for liver tumor cells that present receptors for asialoglycoprotein on the surface [11].
Liposomes have been used over time as carriers for drugs in both biomedical research and clinical applications, but their poor ability to target tissues of interest leads to inefficient eradication of tumor cells and unnecessary damage to normal cells. To overcome this inconvenience, the studies aimed to modify the surface of liposomes with specific molecules for targeting tumor tissues or to develop thermo-sensitive formulations that allow the drug to be released only in mild conditions of hyperthermia obtained by thermal ablation, microwave hyperthermia or focused high intensity ultrasounds [12]. Glycyrrhetinic acid (GA) and hazelnut agglutinin (PNA) were covalently crosslinked with DSPE-PEG 2000 for the preparation of liposomes for the delivery of doxorubicin to malignant liver tumors. Modification with GA and PNA has improved the ability of liposomes to bind to liver tumor cells, as can be seen during in vivo tests, where the volume and weight of DOX-encapsulated BALB/C mice tumors encapsulated in liposomes showed a marked decrease in compared to those treated with simple DOX [13]. Zhang and colleagues developed a similar system based on poly (ethylene glycol) -b-poly (trimethylene carbonate-co-dithiolane trimethylene carbonate), a biodegradable polymer decorated with SP94 peptide with high specificity for hepatocellular carcinoma [14].
Transcatheter arterial chemoembolization is a method of treatment used for inoperable liver cancer that involves combining chemotherapy with obstruction of blood vessels that vascularize tumor tissue. Emulsions (e.g. Lipiodol) were initially used for embolization and drug delivery, but studies have shown that there are no significant differences between pharmacokinetic parameters and systemic toxicity of doxorubicin administered by this method vs. that injected intravenously [15, 16]. Recently, drug-loaded microcapsules have shown promising results during clinical trials for this type of therapy. The "layer by layer" method was used to prepare structures from anionic alginate and chitosan, with doxorubicin being encapsulated inside them. When the capsules were irradiated with a light wavelength of 365 nm, the electrostatic interactions between the layers were weakened and the drug released. Cellular experiments showed that the materials were incorporated into Hep G2 cells by endocytosis and laser scanning confocal microscopy tests showed that the intensity of DOX fluorescence increased in proportion to the duration of UV irradiation, suggesting that the intracellular release of the drug can be controlled by irradiation. UV [17]. Kim and colleagues used a microfluidic system based on borosilicate glass capillaries to make glutaraldehyde-crosslinked albumin microcapsules loaded with doxorubicin. Controlled-release studies have shown that DOX was released from microcapsules gradually over 30 days and in vivo tests determined that the antitumor effect of DOX administered by this method is superior to conventional intravenous injection chemotherapy [18].
In recent years, membrane technology has gained major importance in the biomedical field, with polymeric membranes being used in various medical procedures, such as controlled drug release, tissue regeneration, biosensors, and bioseparation [19, 20]. The market for medical membranes is projected to reach $ 3.31 billion by 2022, and demand for these membranes will steadily increase in the coming years due to the growing global prevalence of diseases such as end-stage renal disease (ESRD), which requires a membrane-based treatment technique. Most of the medical market is made up of membranes for hemodialysis, tissue engineering and controlled release of drugs, and in all these applications biocompatible and sometimes biodegradable materials are needed for membrane preparation [21]. Cellulose, an abundant natural polymer, is a promising material for the biomedical field due to its biocompatibility, biodegradability, low toxicity and excellent physical, chemical and mechanical properties [22]. However, strong intra- and intermolecular bonds make pure cellulose insoluble or partially soluble in common organic solvents, which limits its processing into fibers, films and membranes [23, 24]. Cellulose acetate (CA) is a cellulose derivative obtained by replacing hydroxyl groups in anhydroglucose units with acetyl groups by ester substitution [25, 26]. Compared to pure cellulose, CA has a good solubility in organic solvents, which gives it a high processability and a wide range of applications. Due to its high biocompatibility, low cost, environmental friendliness, high desalination capacity, chlorine resistance and good mechanical strength, one of the most common uses of cellulose acetate is the preparation of polymeric membranes for industrial or biomedical applications [23, 26].
To give cellulose acetate the properties needed for a particular application, surface modification techniques (e.g., polymer blending, physical adsorption, covalent immobilization, etc.) and composite membrane synthesis are considered the most effective methods [27, 28]. For example, a trend toward the development of new hemodialysis membranes is to mix cellulose acetate with other polymers, such as polyethyleneimine [29], polyvinyl alcohol / polyethylene glycol [30], or quaternized polysulfone [31] to obtain mixed matrix membranes with superior selectivity and hemocompatibility. Another method involves the incorporation of biological macromolecules, such as sericin [32] or inorganic compounds such as zeolite [33, 34], hydroxyapatite [35] and the fraxiparinized nanotubes of titanium dioxide [36] into the polymer matrix, resulting in composite membranes with improved properties in terms of protein rejection, toxin clearance and clogging susceptibility. Bone tissue engineering is another area in which cellulose acetate membranes have been extensively studied. The main goal in bone engineering is to design biomaterials that have a structure similar to the extracellular matrix (ECM) for better cytocompatibility and biomineralization [37]. For this purpose, nanofiber membranes were prepared by electrospinning from mixtures of cellulose acetate with lactic acid [38], iron acetate [39], graphene oxide/reduced cobalt [40], graphene oxide / carbon nanotubes [41] ], nanosilica [42] or hydroxyapatite [43, 44]. Membranes with antibacterial properties were also prepared by electrospinning using silver nanoparticles as a functional filler for cellulose acetate [45, 46]. Composite membranes showed improved thermal and mechanical properties, higher apatite formation potential, higher rates of cell proliferation and an ideal structure for osseointegration.
Of all the existing membrane modification techniques, the functionalization of the surface by covalent immobilization is the most convenient because it allows an extremely selective binding and prevents the leeching of the immobilized compound in the surrounding aqueous medium. During covalent immobilization, stable bonds are formed between the functional groups of the substrate and the functional groups of the active compound. Functionalization can be performed directly on the substrate or may include initial activation of the surface by binding agents [47]. Previous studies have reported successful covalent immobilization of sericin [48] and resveratrol [49] cellulose acetate membrane surfaces using aminopropyl triethoxy silane (APTES) as a modifying agent and glutaraldehyde (GA) as a linker molecule. APTES ensured the presence of highly reactive amino groups on the membrane surface, while GA was used as a spacer to prevent steric agglomeration of active compounds that were further immobilized.
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