The HemDelStim project aims as its main scientific objective to open a new approach and a new scientific direction for the treatment of patients with chronic renal dysfunction and liver cancer. Unlike the therapeutic approach so far, which involves performing the hemodialysis process (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 based on a stimulus – the tumor marker for this type of cancer (alpha fetoprotein). For this intelligent release, the concept of 'responsive drug delivery stimuli' will be used – supramolecular or polymeric architectures containing doxorubicin will contain monoclonal antibody for alpha fetoprotein. The presence of alpha fetoprotein in the blood undergoing dialysis will cause bursting of the encapsulars and release of doxorobicin.
The activities undertaken for phase III, according to the project financing contact, were as follows:
Activity 3.1. - Completion of activity - Cytotoxicity and hemotoxcity studies on synthesized membranes.
Activity 3.2. - Establishing parameters that can influence morphological properties.
Activity 3.3. - Studies of separation of compounds of interest in hemodialysis - creatinine, urea, uric acid and salts (Na+ and K+) from synthetic solutions. Separation of creatinine, urea, uric acid and salts (Na+ and K+) from synthetic solutions by UV-Viz and ICP-MS.
Activity 3.4. - Studies of controlled release of doxorubicin in the presence of separate compounds - creatinine, urea, uric acid and salts (Na+ and K+).
Activity 3.5. - Activity initiation - establishing optimal conditions for synthesis of membranes with dual role.
Activity 3.6. - Dissemination - scientific report, update of the project web page, three oral communications at international conferences.
All undertaken activities have been carried out, and below are presented some of the results obtained.
The parameters that influence morphological properties have been established to belong to two categories. The first category refers to the parameters of the polymer solution (concentration, coagulant, casting temperature), and the second to the parameters of functionalization reactions (degree of crosslinking and steric hindrance). In order to come up with additional data compared to the previous stages, an additional study was carried out on the morphology of functionalized membranes with crown ethers, with applications in both hemodialysis and osseointegration. The aim of this study was to develop a new generation of cellulose-based membranes with applications in the biomedical field, especially in osseointegration, by functionalizing the cellulose acetate surface with 4'-aminobenzo-15-crown-5-ether (AB15C5) using ethanolamine (EA) as modifying agent and glutaraldehyde (GA) as binding molecule. Crown ethers are macrocyclic polyethers containing a central cavity lined with oxygen atoms, where they can accommodate positive metal ions or a variety of neutral and ionic organic species. Metal cations are stabilized by interactions with lonely electron pairs on surrounding oxygen atoms, forming a host-guest complex. As mentioned before, ECM similarity is an essential aspect of material biocompatibility. Among the inorganic elements of bone ECM, calcium cations stand out because they play an important role in almost all stages of bone regeneration, starting from the formation of the temporary matrix to biomineralization and remodeling. Calcium cations (Ca2+) play a vital role in osseointegration, actively participating in plasma protein adsorption and intervening in intra and extracellular processes such as platelet activation, hemostasis and blood clotting. Ca2+ signaling stimulates osteoblast differentiation and osteogenesis by regulating osteocalcin, osteopontin, alkaline phosphatase, and bone morphogenetic protein expression in mesenchymal stem cells. Given the ability of crown ethers to retain calcium cations in their central cavity, functionalized cellulose acetate membranes AB15C5 were mineralized using the Taguchi method, resulting in biomimetic membranes containing bone-like apatite deposits in their structure, which enhance their bioactivity. It was postulated that the surface immobilized with Ca 2+ could enhance osteoblast proliferation, while the porous structure of CA facilitates osseointegration when these new materials are implanted at a site with bone defect.
The first stage consisted in partial deacetylation of cellulose acetate membranes using a 5% NaOH solution. The membranes were immersed in NaOH solution for 24 hours at room temperature. Then, the membrane surface was functionalized with ethanolamine (6 hours, 40°C) and glutaraldehyde (2 hours, 40°C); both reactions were performed under magnetic stirring in the NaOH medium. Functionalization with AB15C5 was performed in a slightly acidic ethanol environment, under magnetic stirring, for 2 hours at 40°C. After each step, the membranes were thoroughly rinsed with distilled water to remove any unresponsive compounds. The samples were dried for 72 hours at room temperature prior to characterization.
Biomineralization studies were conducted using the alternative soaking method described by Taguchi et al. The samples were first incubated in a 200 mM CaCl solution at 37 °C for 24 hours. The pH of the solution was adjusted to 7.4 using the base HCl and Tris. The membranes were then rinsed briefly with distilled water and incubated for a further 24 hours in a 120 mM Na2HPO4 solution at 37°C. The cycle was repeated twice. Finally, the membranes were rinsed with distilled water and dried for 72 hours at 37°C prior to characterisation.
In carrying out this activity, we started from the results reported in the previous stage regarding the synthesis of functionalized membranes with anticoagulant molecules. The elegant solution chosen to perform these reactions was to coat the polysulfone membranes with cellulose acetate using an electrospinning gun, followed by modifying the cellulose acetate surface, which has a much higher versatility. Vitamin E was dissolved in an ethanol/water mixture under magnetic stirring for 24 hours at 35°C. The Berzelius glass was covered throughout the process. After 24 hours, a small amount of acetic acid was added to the vitamin E solution and left to stir for 5 minutes at 300 rpm. This step was necessary because an acidic medium is needed for the partial diacetylation reaction of the membrane to have several vitamin E binding sites. After homogenizing the vitamin E and acetic acid solution, the membranes were placed in a petri dish, over which the previously obtained solution was added.
Similar to the tests performed on these membranes, retention tests were performed on polysulfone and polysulfone functionalized membranes with supramolecular architectures based on cyclodextrins, the results being presented in the graph, at 4 hours of recirculation through the tested membranes. Na+ and K+ retention was performed from NaCl and KCl salts, respectively, added at an initial concentration of 0.5 g/L and analyzed by ICP-MS technique. It can be seen from the data obtained that in the case of the functionalized membrane the retentions were higher, this representing an advantage in terms of improving the retention efficiency and possibly decreasing the time of performing the medical procedure. This increase in retention can be explained by steric tripping effects occurring at the active surface of the membrane. This is an advantage only until the moment when supramolecular architectures have to release doxorubicin, precisely this very good retention, fulfilling the process of releasing the active substance and implicitly decreasing the yield for the ultimate goal of the synthesized membranes – to release doxorubicin while retaining the species of interest.
For the doxorubicin controlled release study, initial diclofenac release studies were conducted on a chitosan control membrane to develop the release method. A chitosan (CS) solution was prepared by dissolving 2 g of chitosan powder in 18 ml of acetic acid for 30 min. at room temperature. Then add 180 ml of distilled water and allow to homogenize for 2 hours at room temperature. The concentration of acetic acid solution used was 10 weight % and the concentration of CS solution was 1 %. A volume of 10 ml of solution was poured into a petri dish with a diameter of 10 cm and left to dry for 72 hours at room temperature. To obtain the DCF-loaded CS-based membrane, we added diclofenac to a CS:DCF mass ratio of 1:0.05 to the previously obtained CS solution and let it homogenize for 2 hours at room temperature. The solution obtained was poured into a petri dish and left to dry for 72 hours at room temperature. 0.5 g LDH was dispersed in 200 ml distilled water for 30 min. at room temperature. After homogenisation, 0,1 g diclofenac was added and allowed to homogenize for a further 2 hours, also at room temperature. The suspension obtained was placed in petri dishes and left to dry for 72 hours at room temperature. To obtain the composite membrane based on CS/LDH/DCF, LDH/DCF was added to the CS solution obtained at a mass ratio CS:LDH:DCF of 1:0.25:0.05 and homogenized for 2 hours at room temperature. A volume of 10 ml of solution was poured into the Petri dish with a diameter of 10 cm and left to dry for 72 hours at room temperature. The in vitro release of diclofenac was studied by a UV-Vis spectrophotometer (Shimadzu UV-3600 UV-VIS). The samples were placed in a dialysis membrane bag with 4 ml phosphate buffer solution (PBS) and then the samples were immersed in 200 ml PBS and spun for 72 hours at 100 rpm at room temperature. The spectra were recorded at a maximum absorption wavelength of 276 nm, and the standard curve was determined for drug concentrations ranging from 2 to 10 μg/ml. The membrane samples used to release diclofenac were 2 × 2 cm and weighed 23 ± 3 mg. The measured standard curve is shown in the figure. The morphological tests of the obtained membranes were performed using scanning electron microscopy (SEM). Observations were carried out on the surface of CS-LDH membranes. The figure shows SEM micrographs of the film surface at various magnifications (25× and 500×). In Fig. A, B it can be noted that clean chitosan has a smooth surface, without porosity or roughness. After adding diclofenac, it can be seen that diclofenac is dispersed unevenly, slightly agglomerated in the polymer matrix, possibly due to poor magnetic mixing (Fig C,D). This could indicate poor dispersion of diclofenac in the chitosan solution. In figure 2 E,F a significant change in membrane morphology can be observed, with the introduction of LDH, so that a slight stratification of the composite membrane compared to the clean CS membrane is observed (Figure 2 A,B ) and an increased dispersion of diclofenac in the polymer matrix. The addition of LDH provides additional protection against external degradation factors, but also for a more effective, gradual release of the drug.
The tests for the release of diclofenac from the composite membrane were performed by UV-Vis technique according to the procedure developed in a previous research at pH 7.4, and the data are shown in Fig. The tests were conducted for 72 hours; In the first 6 hours, 26% of the amount of the drug was released. In the first 24 hours, 50% was released; within 48 hours, 88% were released; and in 72 h 90% was released for the CS-DCF system. In the first 6 hours, 16% were released; in the first 24 hours, 31% were released; within 48 hours, 46% were released; and within 72 hours, 65% was released for the CS-LDH-DCF system. There are several mechanisms that drive drug delivery from a polymer matrix, the most important being related to diffusion-controlled release, controlled swelling release, controlled erosion and degradation release, and controlled stimulus release.
Diffusion controlled release occurs in the case of the porous polymer matrix and is governed by the diffusion mechanism. Controlled swelling release occurs when polymer chains break down under the interactions that occur between the polymer and the environment, a process that is preceded by swelling. Polymer erosion involves swelling, diffusion and dissolution, the release of the drug is regulated by the type of polymer and the internal bond of other species present in the system. Controlled stimulus release occurs when the shape, size, porosity or other properties of the polymer matrix are affected by an external stimulus, such as pH, temperature or the presence of biological markers, and the drug is released based on these interactions, which is the most advantageous strategy in terms of delivery to the destination and minimizing side effects. In the case of chitosan-based drug delivery systems, the release mechanism is a combination of all these mechanisms, depending on the method of synthesis and the environment of the polymer system. Compact chitosan films synthesized for drug administration in strongly acidic pH (1.5 for gastric juice) or moderately acidic pH (5.5-6.5 for cancer tumors) are systems with rapid release ability due to the polymer structure. In such systems, especially those intended for release into strongly acidic environments, the entire amount of the active pharmaceutical substance is released very quickly, within a few hours. Due to the insolubility of chitosan at normal extracellular pH (6.8-7.4), the release is much slower and occurs due to the formation of a hydrogel. In our case, the greatest release for the CS-DCF system is based on this formation of the gel structure, while in the case of the CS-LDH-DCF system, despite the fact that the film forms the same gel structure, the release occurs from the LDH layers by extracting diclofenac into the environment, a process that takes longer. Similar results were obtained for the release of diclofenac sodium from the Chitosan-Oxidized Konjac Glucomannan system by Korkiatithaweechai et al. The data obtained show ideal efficiency for the first 48 hours after a potential implantation, and the release profile shows that the membrane obtained can provide an effective anti-inflammatory response for the critical period, with applications in dentistry and orthopedics.
Doxorubicin release studies were conducted under the same conditions, being evaluated by UV-Viz under conditions similar to those presented above. From the data obtained, a smaller amount of active substance is observed to be released from the membrane functionalized under complex environmental conditions containing both urea and creatinine, as well as sodium and potassium salts, this can be explained by steric obstructions that occur in solution due to the even volume that urea and creatinine have, but also due to the influence of sodium and potassium salts that interact with hydroxyl groups on the surface of supramolecular architectures from cyclodextrin molecules, preventing the breaking of these architectures and implicitly the release of doxorubicin.
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