The HemDelStim project aims to open as a main scientific objective the opening of 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 the function of the kidneys) and performing chemotherapy in parallel with this procedure, the project aims to combine the two, in an intelligent way, with a minimal impact on the body. The drug used for the treatment of hepatic canecr (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). For this intelligent release, the concept of 'responsive drug delivery stimuli' will be used – supramolecular or polymeric architectures containing doxorubicin will have in their monoclonal antibody component for alpha fetoprotein. The presence of fetoprotein alpha in the blood subjected to dialysis will cause the rupture of the incapsulars and the release of doxorobicin.
The activities assumed for phase II, according to the project financing contact, were the following:
A.2.1. Establishing the optimal working conditions for the encapsulation of medicine. Following the analyzes performed, the optimal synthesis method for each category of architecture will be established;
A.2.2. Derivatization of polysulphonous membranes with vitamin E using formilated PSf (by the Vilsmeyer-Haak method) to induce the anticoagulant character.
A.2.3. Covalent immobilization of supramolecular architectures with encapsulated drug and monoclonal antibody for immobilized or polymeric AFP with encapsulated drug and monoclonal antibody for immobilized AFP.
A.2.4. Morphological (SEM, TEM, CT) and structural -FT-IR, Raman, XPS (survey and hi-resolution) and NMR characterization of the synthesized membranes.
A.2.5. Initiation of activity - Cytotoxicity and hemotoxity studies on synthesized membranes.
A.2.6. Dissemination – scientific report, project web page update, three oral communications at international conferences, two ISI articles.
All the assumed activities were carried out, further being presented some of the results obtained. The results regarding the synthesis of supramolecular architectures, as well as the synthesis of membranes for hemodialysis with a double role are patented.
To obtain cyclodextrin (CD) it was necessary to use 0.5g of CD powder, 100 ml of dimethylformamide (DMF), 1 ml of aminopropyltriethoxysilane (APTES) and 0.2g sodium hydroxide (NaOH) powder. APTES, NaOH and DMF were put under magnetic stirring for 1h at a temperature of 40°C on a magnetic stirring hob, in a Berzelius glass, and after about 1h the cyclodextrin was added, and the solution will be allowed to solubilize for another 2h at a temperature of 70°C. At this stage, the same substances that we used to obtain the previous sample were used. Aptes, DMF and NaOH were put and 0.02g of doxorubicin powder were put in another Berzelius beaker, left on the hob for 1h at 70°C, then the 0.5g of cyclodextrin were added, and the solution obtained was left on the magnetic stirring hob for 2h at 70°C. The obtained sample was dried in the oven at 150°C for about 4 days.
The elegant solution chosen to perform these reactions was to cover the polysulphon membranes with cellulose acetate with the help of an electro-filtering gun, a coating followed by the modification of the cellulose acetate surface, which has a much higher versatility. Vitamin E was dissolved in a mixture of ethanol/water under magnetic stirring for 24 hours at 35°C. The Berzelius glass was covered throughout the process. After the 24 hours over the vitamin E solution, a small amount of acetic acid was added and the solution was left to shake for 5 minutes at 300 rpm. This step was necessary because it takes an acidic medium for the reaction of partial diacetillary of the membrane to have several sites of binding vitamin E. After homogenizing the solution of vitamin E and acetic acid, the membranes were placed in a Petri dish, over which the previously obtained solution was added. In Figure 3.1.1.3. the reaction between vitamin E and cellulose acetate is represented. The membranes immersed in the solution were maintained at a temperature of 40°C for 48 hours in the laboratory oven. After the 48 hours the membranes were rinsed with distilled water and left to dry at room temperature for 48 hours. The next step consisted in the physico-chemical characterization of the functionalized membranes. To begin with, acetylsalicylic acid was dissolved in distilled water under magnetic stirring for 15 minutes at 40°C. Cellulose acetate membranes were placed in a Petri dish and acetylsalicylic acid solution was added over them. After 15 minutes of immersion, a low concentration of NaOH solution was added over the membranes to facilitate functionalization. The NaOH solution has a role in the deacetylation of the membrane, thus helping to form several ether-type bonds between it and acetylsalicylc acid. In Figure 3.1.2.2. the reaction between acetylsalicylic acid and cellulose acetate is represented. The membranes were left in the solution for 8 hours at 40°C. After the 8 hours the membranes were washed with distilled water and left to dry for 48 hours at room temperature. After the membranes were dried, they were subjected to physico-chemical characterization.
Figure 4 shows the SEM images of the cellulose acetate (CA) membrane, before (pictures a-c) and after (d-f images) functionalization with vitamin E and after (g-i) acetylsalicylic acid (AA) functionalization. In Image 3.4.1.1.b. the structure of the membrane of the strati dedicated, porous CA can be seen. The pores (Figure 3.4.1.1.c.) are uniform, have xvasispheric shapes, interconnected, with micrometric dimensions. Towards the depth (Figure 3.4.1.1.a.) a fibrillate structure can be observed. This structure of the CA membrane is specific to an asymmetrical membrane. The porous structure of the membrane provides effective microfiltration and favors the infiltration of vitamin E solution. All six images show dimensional homogeneity. In image 3.4.1.1.d. you can see the darker areas where the vitamin E solution was captured. In image 3.4.1.1.e. the deeper fibrilla area is no longer observed, and in figure 3.4.1.1.f. it is observed that the vitamin E solution was retained on the surface of the membrane in the form of aggregates, the CA membrane being hydrophilic, vitamin E oily solution shows an uneven distribution due to its hydrophobicity.
In picture 4. g. the dark areas where acetylsalicylic acid has been fixed can be observed. Unlike vtamine E it is represented punctately. Figure 3.4.1.1.1.h. is the same as that of vitamin E, the fibrillar structure is no longer observed. In image 3.4.1.1.i. you can see agglomerations of AA, but the agglomerates are significantly fewer as opposed to those of vitamin E and with a tendency to low agglomeration. This suggests a uniform distribution in the membrane pores of AA versus vitamin E.
From the six sem images presented it can be concluded that vitamin E is not evenly distributed in the pores of the membrane, compared to AA which has a uniform distribution.
Figure 5 and 6 shows the FT-IR spectrum of non-functional CA membranes, CA-Vitamin E and CA-AA membranes. The simple membranes in CA have 3 peaks: at 1720, 1376 and 1245 cm-1, these being generated by the stretching of the C=O, C-H and C-O bonds in the acetil grouping. The tip with the value of 1053 cm-1 corresponds to the stretching vibration of the O-C-O of the pyranosis ring. The C1-H, O-H curvature vibrations corresponding to the β-glycosidic glucose bonds have the characteristic peak at the value of 842 cm-1 . The presence of aromatic rings in the structure of vitamin E and acetylsalicylic acid is signaled by the presence of a small pick at around 1508 cm-1, associated with π-π interactions and C=C vibrations in the benzene ring. The increase in the intensity of the peaks corresponding to the C-O groups (1245 cm-1 ) is due to the formation of new etheric bonds, between cellulose acetate and vitamin E, respectively, cellulose acetate and acetylsalicylic acid.
The functionalization reaction of cellulose acetate membranes with acetylsalicylic acid is confirmed by the presence of peaks from 1454 cm-1 and 1735–1750 cm-1 in the spectrum of functionalized membranes. These absorption maxima are corresponding to benzene rings and ester groups in the structure of acetylsalicylic acid. As for the reaction of functionalization with vitamin E, it is confirmed by the presence of peaks in the range of 1210 and 1260 cm-1, in the spectrum of functionalized membranes. At the value of 1210 cm-1, the loss of the phenol group in the structure of vitamin E is signalled in order to form the etheric group between vitamin E and cellulose acetate.
The flow of water through the membranes was evaluated using 500 ml of deionized water in continuous recirculation for 4 hours under simulated conditions comparableto the medical procedure a.
The simple CA membranes had a continuous decrease in flow over the course of the 4 hours. Ca membranes functionalized with acetylsalicylic acid and vitamin E had a slight decrease after the first 2 hours. This decrease is due to hydrodynamic stabilization of membranes. After the 2 hours the functionalized membranes kept their flow constant. Functionalized membranes with acetylsalicylic acid have a higher, constant flow, unlike those functionalized with vitamin E. This is due to the uniform capture in the membrane pores of acetylsalicylic acid as opposed to vitamin E.
To evaluate the performance of membranes for hemodialysis, 250 ml of synthetic solutions of urea (0,1 mg/ml) and creatinine (0,1 mg/ml) were used to evaluate the clearance of these two uremic toxinse (Figure 8).
After the 4 hours of hemodialysis, under simulated conditions comparable to the medical procedure, an increase in urea retention is observed, from 21% for the CA membrane, to 29% for CA-acetylsalicylic acid and to 31% for CA-vitamin E. An increase in creatinine retention from 18% for CA, to 23% for CA-acetylsalicylic acid is also observed, At 26% for CA-vitamin E. From the results obtained, the more pronounced improvement of the retention of uremic toxins for the functionalized membrane with vitamin E. is observed, which recommends it for use in hemodialysis.
Fig.10 represents the scanning electron microscopy of the polysulphonous membrane obtained. Scanning electron microscopy is a modern technique of visualization and characterization of materials, used to evaluate the morphological and structural changes that occurred following the addition of cyclodextrin architectures with the drug. As can be seen in the images A and D that are representative of the simple polysulphonous membrane, over which cyclodextrin or medicine has not been added, its morphology is a microporous one, in which the pores have various sizes and irregular shapes, but with a uniform distribution on their membrane surface. With the addition of cyclodextrin (images B and E), the microporosity decreases, the pores still remain varying in shape and size, while it can be seen on the surface of the membrane the presence of cyclodextrin that appears in the form of agglomerates, with varying sizes and irregular shape that would lead us to think of a polyhedral shape of it.
From these images we can state that the functionalization of the polysulphon membrane with cyclodextrin architectures took place. In the C and F images characteristic of the polysulphonous membrane over which the architectures of cyclodextrin with medicine were added, we can see that the pores of the membrane are reduced in number, they have acquired a circular shape with various sizes. The architectures of cyclodextrin and active substance can still be noticed on the surface of the membrane, but comparing the images with the membrane made of simple polysulphon and this last image, we can state that both the drug has been successfully incorporated into the architecture of cyclodextin, but also all this complex has been integrated into the pores of the functionalized membrane.
The absorption strips for the polysulphon are found at 1000 cm-1- 1300 cm-1, being specific to the bond -O=S=O-, another peak characteristic of the polysulfone is found at 1716 cm-1, we also meet the specific absorption band for -CH3 at 2854 cm-1.
Samples containing cyclodextrin can be identified by the presence of the -OH-link band in cyclodextrin from 2936 cm-1, but the peaks of 1663 cm-1 are also visible, this being characteristic of the benzene deformation strip, and at 1579 cm-1 the strip characteristic of the connection between the polysulphon and the cyclodextrin is observed.
In order to confirm also the existence of doxorubicin in cyclodextrin architectures, we can observe the peak at 2931 cm-1 and the band specific to the vibration of the stretching of the C-H bond from 2829 cm-1, and to state that doxorubicin is also found in the membrane made of polysulphone with cyclodextrin and DOX architectures, we have the presence of the characteristic N-H band in pure doxorubicin. XPS analysis was used to observe compositional changes in the surface of the samples. The data can be found in Table 2 and fig. 12th
As can be seen in the membrane made of polysulphon, commercial, there are elements C, O2, N2 and S2, the presence of nitrogen being due to the presence of amino groups in the structure of the commercial membrane. It can also be noted the presence of Si2 in all samples other than the commercial membrane, since for the functionalization of cyclodextrin it was necessary to add APTES, and the presence of Na is also due to the functionalization of cyclodextrin.
For the polysulphonous membrane sample and the CD and drug architectures, the presence of all the elements found in the rest of the samples is observed, indicating that the functionalization of cyclodextrin has successfully occurred and cyclodextrin and active substance architectures have been incorporated into the membrane.
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