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AZERBAIJAN CHEMICAL JOURNAL № 1 2022
ISSN 2522-1841 (Online) ISSN 0005-2531 (Print)
UDC: 544.23.02/.03
IMMOBILIZATION OF LEVOTHYROXINE IN QUATERNIZED N,N-DIETYL N-METHYL CHITOSAN HYDROGEL AND CHEMICAL NATURE OF THE
INTERACTION
S.F.Safaraliyeva1, A.F.Hummetov2, Sh.Z.Tapdiqov3, S.S.Fatullayeva1, M.Raucci4,
D.B.Tagiyev1, N.A.Zeynalov1
1M.Nagiyev Institute of Catalysis and Inorganic Chemistry, NAS of Azerbaijan
[email protected] Azerbaijan Medical University, Educational-Surgical Clinic, Department of Surgical Diseases
SOCAR Oilgasresearchproject Institute 4Institute of Polymers, Composites and Biomaterials, National Research Council of Italy
Received 26.11.2021 Accepted 24.12.2021
Immobilization of a thyroid hormone substitute of L-thyroxine on chitosan-based smart hydrogel structure was performed to reduce the side effects of the required daily dose. The hydrogel was obtained by the Schiff reaction of chitosan with acetaldehyde and quaternization with methyl iodide then by ion exchange with NaCl salt. Finally, a hydrogel was obtained from the treatment of chitosan-based salt at -200C. The amount of levothyroxine immobilized by physical sorption on the hydrogel was determined by molecular electron spectroscopy on basis especially absorption peak at 227 nm. Results of IR- and UV-Vis spectroscopic analyses revealed that the interaction between levothyroxine and hydrogel occurs mainly due to hydrogen bonding and orientation forces of intermolecular interaction.
Keywords: chitosan, quaternized, hydrogel, levothyroxine, immobilized, chemical nature of the interaction.
doi.org/10.32737/0005-2531-2022-1-6-12
Introduction
Hydrogels can be formed by the cross-linking reaction of linear or graft copolymers with small-molecules reagents by covalent chemical bonds. Hydrogels can also be created by the join of macromolecules with each other due to the forces of physical-electrostatic interaction. Such materials have the property of biocompatibility by expanding by containing a certain amount of water in their volumes or collapsing by desorption. This property allows them to be widely used in medicine and biotechnology, as drug carriers, capsules and wound dressings [1].
Hydrogels demonstrate phase transition by being responsive to external factors such as pH, temperature, ionic strength, and physical and mechanical effects. Due to these properties, they are also called "stimuli responsive" or "smart" gels [2].
In general, gels used in the delivery of drugs are characterized by the size of the matrixes. Nanogels - are hydrogel structures that are around 10-200 nm in size. They are
able to carry and protect the active molecules which are loaded into the gel network and respond to changes as a result of physical and chemical factors. The highly regulable property of the chemical composition in this form has led to the widespread use of nanogels in the delivery of biologically active compounds to target address [3].
In recent years, the Nagiyev Institute of Catalysis and Inorganic Chemistry of ANAS carries out a lot of research work on the synthesis and investigation of polymer nanogels for immobilization and targeted delivery of biologically active substances and drugs [4-13]. Among these of particular importance is the work on the synthesis and application of polymer vectors to maintain the long-term biological activity of the thyroid hormone substitute levothyroxine in the organism.
It is known that L-thyroxine, a hormone of the thyroid gland, stimulates growth and vegetation, increases the oxygen demand of tissues, provides the metabolism of proteins, fats and carbohydrates, regulates the activity of the
cardiovascular and central nervous systems. Due to hypothyroidism caused by the removal of part or all of the gland due to the dysfunction, patients receive a salt form of the hormone substitute - levothyroxine-Na pentahydrate throughout life. Side effects of the drug, such as weight gain, hair loss, impaired renal function, tachycardia, arrhythmias, sleep disorders, unexplained anxiety and etc., are observed due to increased appetite during long-term use [14-16].
In recent years, levothyroxine has been immobilized on natural and synthetic polymer carriers, as in antibiotics and proteins, to avoid overdose. As a result, the period of activity of the drug increases in terms of fulfilling its function by prolonging its circulation in the bloodstream. Among such polymer carriers, chitosan, a natural polyaminosaccharide, is of particular importance and is considered to be a more favorable matrix in terms of biocompatibility [17-19].
OH OH
Fig. 1. Chemical structure of levothyroxine-Na pentahydrate and chitosan macromolecule.
The analysis shows that there are limited references on the process of release of levothy-roxine by loading it into chitosan-based matrixes. Only a small number of studies have been performed to obtain the biological activity of levothyroxine by immobilization of chitosan-based hydrogels in vitro or in vivo. Some of these are listed below.
In the study [20], levothyroxine was loaded onto a hydrogel based on chitosan/col-lagen and the release kinetics was studied in phosphate buffer saline solution, hydrogen peroxide and lysozyme medium. The angiogenic potential of the hydrogel-drug complex containing 1 -10 mcg of levothyroxine was found to be high for 8-14 days. However, the study undis-
closed a chemical description of the interaction between hydrogel and levothyroxine.
In another study [21], levothyroxine was loaded on chitosan/carboxymethylcellulose/hy-droxyapatite hydrogel and angiogenic experiments were studied ex-ovo. Hydrogel containing 0.1 mcg of levothyroxine was non-toxic and had maximum neovascularization potential, with good bending, folding, spreading and stretching properties. In this case, the authors did not touch on the bonds that bind levothyroxine to the hydrogel too.
Lubna and others [22] developed chitosan-based wound dressings immobilized with levothyroxine and conducted clinical experiments in mice. Chitosan-based hydrogel obtained from the treatment of alkali with an alcohol solution at low temperatures is 90% cumulative release of levothyroxine into the medium within 4 days. As in the above studies, the nature of the chemical interaction that causes immobilization between levothyroxine and the matrix has remained unclear.
In general, the reference analysis shows that after loading levothyroxine on the chitosan-based hydrogels, the main focus is on its biological research and the study of the degree of release of the active drug into the medium. However, the nature of the bond between levothyroxine and the polymer chain has not been studied. However, this directly affects the dose of immobilization drug molecules and the degree of activity of the drug, as well as the controlled release. In this regard, the Schiffs base of chitosan with acetaldehyde initially was synthesized, then quaternized with methyl iodide, and NaCl ion exchange reaction, then freeze modification, at the end levothyroxine immobilized into chitosan based gel, and the chemical nature of the interaction finally studied by spectroscopic methods.
Experimental part
Chitosan average molecular weight 35 kDa (deacetylation degree 85-87%), acetaldehyde (>99.0%), NaBH4 (chemically pure >96%), acetic acid (Glacial), ethanol (95%), acetone (>99.9%), diethyl ether (1 ppm inhibitor, anhydrous, >99.7%), NaCl (BioXtra, >99.5%),
acetonitrile (99.8% anhydrous), and NaOH from Sigma Aldrich. Methyl iodide stabilized with metallic copper (99% c.p.) is from Acros Organics. Levothyroxine-Na pentahydrate (CAS 6106-07-6) was also obtained from Sigma Aldrich.
Synthesis of N, N-diethyl, N-methyl chi-tosan chloride (DEMX) was synthesized according to the methodology [23]. The synthesis of DEMX-based hydrogel was performed according to the appropriate methodology. 5 g of DEMX is dispersed in 20 ml of 0.5 M acetic acid and mixed continuously until completely dissolved. The solution is shed into a Petri dish and frozen at -200C for 15 hours. The sample is then immersed in a 3M cold ethanol solution of NaOH and restored at -200C for 24 h. The hydrogel is first washed with a 50% ethanol solution and then with distilled water until a neutral pH medium is obtained. Finally, after washing with absolute ethanol, it is dried at room temperature. For levothyroxine intercorporation, a sample of hydrogel with a diameter of 15-20 mm pieces is added to 10 ml of 1 Mg/ml levothyroxine solution and incubated for 24 hours. After physical absorption of the drug, DEMX-levothyroxine hydrogel separates and is dried at 30-350C temperature for 2 days.
The amount of levothyroxine in solution was determined by electron spectroscopy based on the absorption spectrum in the range of 180600 nm in UV-Vis. Thus, at 227 nm, a calibration curves with the function A=(C) of drug
solutions of different concentrations was established according to the optical density of the characteristic peak. IR spectra of chitosan, levothyroxine and DEMX-levothyroxine hydrogel were recorded in the range of 4000-400 cm-1 and absorption bands characteristic of functional groups were observed.
Result and discussion
In order to increase the hydrophilicity of chitosan, its N,N-diethyl derivative was synthesized and then quaternized with CH3I. By changing the iodine ions with chlorine, a DEMX salt of quaternary chitosan, which is better soluble in alkali medium, was obtained. The synthesis process and the chemical structure of the final product can be shown as follows.
The interaction of functional groups that caused immobilization between the hydrogel and the levothyroxine molecule obtained after structuring DEMX at freeze temperature was identified by spectroscopy. For this purpose, IR spectra of chitosan, its quaternized DEMX salt, levothyroxine and levothyroxine complex samples with hydrogel obtained after immobilization were recorded (Figures 4, 5).
In the spectrum, characteristic of chitosan the peak characteristic of the axial stretching of the chemical bond >C=O in I amide in the acet-amide group in the region 1657 cm-1, angular deformation of the -NH2 group in 1571 cm-1 and vibrational bands characteristic of the C-N group in 1260 cm-1 were observed.
_H+
H 2 C ^C H 2
C H 3 C H 3
Fig. 2. Synthesis reaction of N,N-diethyl chitosan from chitosan.
HC
O
+ H3
30 min, T=20C, Str
H
HJC / CH3J, Asetonitril r h,C 1 /
35 0C, 30 saat / \
HO-^^I--- HC CH n . >r-CHs . H2C CH2 J0 n
CH3 CH3 CH3 CH3
Fig. 3. Quaternization of N,N-diethyl chitosan and chloride salt synthesis reactions of DEMX. AZERBAIJAN CHEMICAL JOURNAL № 1 2022
Fig.4. IR spectra of chitosan and levothyroxine.
____DEMX
\ jS2938 lessl/^W 13S2 L
DEMX/L-tiroksin
11082
3ZO<5 1 J HOS \
U i«3 \
16-50
33 00 3000 2 J00 2000 1300 1000 300 WuvenumbcT (cm
Fig. 5. IR spectrum of DEMX and levothyroxine immobilized hydrogel
The wide band observed in the area of 3478 cm-1 belongs to the stretching vibrations of -OH and -NH2 groups. It was found that during alkylation and after quaternization, the adsorption band characteristic of the N-H bond in the region of 1592-1598 cm-1 loses and this proves that the substitution of protons with ethyl groups during alkylation. In addition, the low-intensity peaks formed in the 1465-1365 cm-1 region for —CH2— and -CH3 in the ethyl group, prove that the incorporation of alkyl groups in the chitosan structure. In addition, the formation of salts by the quaternization of nitrogen atoms results in the formation of a new peak in the spectrum around 1480 cm-1, which is characteristic of the >N+< group. Peaks for the hydroxyl and carbonyl groups and C-I bonds of Ar-OH, -NH2, -COOH, which are characteristic of levothyroxine, are observed around 1465, 16043250, 3500-1640 and 490 cm-1, respectively.
After obtaining the levothyroxine complex with DEMX hydrogel, the characteristic bands of the main matrix in the IR spectrum of the biomaterial were controlled. After immo-
bilization of levothyroxine into the hydrogel, certain imprints of the drug are observed in the IR spectrum. This is due to the fact that its content in the hydrogel is 2-5 mcg for every 50 mg of gel matrix. However, it is possible to observe certain chemical shifts in the absorption bands characteristic of both hydrogel and levothyroxine. Thus, for DEMX, the chemical shift of the absorption bands characteristic of single amine, carbonyl, C-N chemical bonds shift to the region of 1585, 1643, 1268 cm-1, as well as the formation of a broad band around 3470 cm-1 is a manifestation of drug interaction with the polymer structure. Thus, the hydrogen bond between functional groups causes the bonds to stretch and vibrate. The slight changes in the bands around 1390 and 1470 cm-1, which are characteristic of methyl, methylene, aromatic cycle, also prove the existence of hydrophobic interactions between the drug and the hydrogel. These absorption bands belong to hydrophobic groups and are mainly characteristic of the C=C bond in the benzene group. It also appears with low intensity in the area of 1470-1590 cm-1.
Fig. 6. UV-Vis spectra of chitosan, levothyroxine and DEMX/levothyroxine complex solution.
These results suggest that the interaction between the chitosan macromolecule and the levothyroxine molecule is mainly due to hydrogen bonds and hydrophobic forces. The formation of immobilization of drugs due to this type of interaction is found in some references [24].
Confirmation of the interaction between DEMX-based hydrogel and levothyroxine was also studied by comparative study of molecular electron spectra of aqueous solutions of samples with UV-Vis spectroscopy (Figure 6). As can be seen, an intense peak around 250 nm, characteristic of the chitosan macromolecule, is observed. This peak represents the n^n transition in the double bond of the carbonyl group in the acetoamide group, which is characteristic of non-deacetylated fragments. The inclusion of alkyl or diethyl groups to the macromolecular chain causes structural changes in the polymer content, resulting in changes in the electron density in the links. This leads to a change in the line of the spectrum. Thus, it causes the formation of a wide absorption band in the of 200-300 nm region. If we look at the UV-Vis spectrum of levothyroxine, a characteristic low-intensity band is observed in the range of 208265 nm, which belongs to the aromatic cycle or phenyl group. In addition, the absorption bands of the C-I group at 260 nm and the absorption bands of the other groups -COOH and >C=O observed together in the same region, form a broad spectrum, indicating both immobilization and chemical interaction. Also, the formation of a broad absorption band around 300 nm in the UV-Vis spectrum of DEMX-levothyroxine, which is not observed in the chitosan or DEMX
spectrum, indicates a strong interaction of functional groups between the drug and DEMX. Such chemical shifts in the UV-Vis spectrum with bathochromic and hypsochromic effects are due to the occurrence of electrostatic interaction as well as hydrogen bonds between drugs and polymer macromolecules [25].
Acknowledgments
The presented research work was carried out with the financial support of the Science Development Foundation under the President of the Republic of Azerbaijan "Grant No SDF-GAT-5-2020-3 (37)-12/04/4-M-04".
The research also was carried out in collaboration with the Azerbaijan National Academy of Sciences and the Italian National Research Council under the project "In vivo biological study of the encapsulation of L-thyroxine in chitosan N-trimethyl iodine derivative and its long-term controlled release' according to the order of the Presidium of ANAS No 7/5 March 14, 2018.
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KVATERNÍZO OLUNMUS N,N-DÍETÍL N-METÍL XÍTOZAN HÍDROGELÍNO LEVOTÍROKSÍNÍN ÍMMOBÍLÍZO OLUNMASI VO QAR§ILIQLI OLAQONÍN KÍMYOVÍ TOBÍOTÍ
S.F.Safaraliyeva, A.F.Hümmatov, §.Z.Tapdiqov, S.S.Fatullayeva, M. Raucci, D.B.Tagiyev, N.A.Zeynalov
Tiroid hormon avazedicisi olan levotiroksinin gündalik talab olunan dozasinin yan tasirlarini azaltmaq maqsadi ila xitozan asasli agilli hidrogel strukturuna immobilizasiyasi hayata kegirilmi§dir. Hidrogel xitozanin asetat aldehidi ila §iff reaksiyasi va sonuncunun metil yodidla kvaterniza va NaCl duzu ila ion mübadila reaksiyasindan alinmi§dir. Sonda xitozan asasli duzun -20 °C-da i§lanilmasindan hidrogel alinmi§dir. Hidrogela fiziki sorbsiya üsulu ila immobiliza olunmu§ levotiroksinin miqdari molekulyar elektron spektroskopiya üsulu ila 227 nm-daki xarakterik udma pikina göra tayin edilmi§dir. ÍQ va UV-Vis spektroskopik analiz üsullarinin naticalari ila müayyan olunmu§dur ki, levotiroksin ila hidrogel arasindaki alaqa asasan hidrogen rabitasi va molekullararasi qar§iliqli tasirin oriyentasiya qüvvalari hesabina ba§ verir.
Agar sözlar: xitozan, kvaterniza, hidrogel,levotiroksin,immobiliz3, qar§iliqli slaqsnin kimyavi tabiati.
ИММОБИЛИЗАЦИЯ ЛЕВОТИРОКСИНА В КВАТЕРНИЗОВАННЫЙ NjN-ДИЭТИЛ N-МЕТИЛ ХИТОЗАН ГИДРОГЕЛЬ И ХИМИЧЕСКАЯ ПРИРОДА ВЗАИМОДЕЙСТВИЯ
С.Ф.Сафаралиева, А.Ф.Гумматов, Ш.З.Тапдыгов, С.С.Фатуллаева, М.Рауччи, Д.Б.Тагиев, Н.А.Зейналов
Для снижения побочных эффектов необходимой суточной дозы заменителя тиреоидного гормона L-тироксина была проведена его иммобилизация на «умную» гидрогелевую структуру на основе хитозана. Гидрогель получали реакцией Шиффа хитозана с ацетальдегидом и последующей кватернизацией метилиодидом, затем ионным обменом с NaCl. Наконец, при обработке соль на основе хитозана при температуре -20°С был получен гидрогель. Количество левотироксина, иммобилизованного физической сорбцией на гидрогеле было определено методом молекулярной электронной спектроскопии на основе особенностей пика поглощения в области 227 нм. Результаты анализов ИК- и УФ-видимой спектроскопии показали, что взаимодействие между левотироксином и гидрогелем происходит, в основном, за счет водородных связей и ориентационных сил межмолекулярного взаимодействия.
Ключевые слова: хитозан, кватернизация, гидрогель, левотироксин, иммобилизация, химическая природа взаимодействия.
