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Chitosan (CS) is a substance abundant in nature. It is a biopolymer consisting of repetitive components of glucose and N-acetyl-glucose amine connected by (1,4)-glycosidic bonds. It has so many applications that are biodegradable, non-toxic, and biocompatible. The CS was loaded with 5-fluorouracil (5FU) via amide-mediated binding, and the resulting CSFUAC product was evaluated as a potential 5FU delivery agent. A new CS-Schiff base derivative was created using CS extracted from local fish scales by combining CS with another aromatic aldehyde. The antimicrobial effectiveness of the new product was evaluated. It includes two fungi and four strains of pathogenic bacteria. The MTT assay is employed to determine the cytotoxicity of the newly synthesized compounds. Finally, CS was used to synthesize a prodrug for colon cancer. As a colon cancer prodrug, methotrexate (MTX) was converted to the combined (methotrexate-imidazole) and linked with the CS to produce the CSMTX conjugate. Additionally, the compound’s hemolytic action and chemical stabilities were evaluated. In the MTT, three types of cancer cell lines (MDAMB231, MCF7, and MDAMB453) were utilized to test how toxic the compounds made in the lab were to cancer cells.
*Address all correspondence to: althker1@uokirkuk.edu.iq
1. Introduction
The chemical structure of chitin (SH)-derived chitosan (CS) has the chemical structure of “(1→ 4) 2-amino-2-deoxy—D-glucopyranose.” This is a typical co-biopolymer origin in the shells of cockroaches, the shells of crustaceans, and fungal cellular walls (Figure 1). The primary sources of CS and SH are crustaceans-like crabs, shrimp, and fish scales. CS is the most abundant and superior natural substance in nature, and second only to cellulose. Due to its excellent quality and adaptability, CS is in a league. In addition, they possess unique properties, for example, non-toxic, mucosal adhesion, biodegradability, biocompatibility, antimicrobial activities, and hydrophilicity. However, it is cholesterol lowering. These characteristics make CS useful in medicine, horticulture, stabilizers for staple foods, biocatalysts, and biology [2, 3, 4, 5, 6, 7].
Figure 1.
Chitin, chitosan, and cellulose’s molecular structures [1].
The beneficial biological effects of CS include antitumor, antibacterial, and hemostatic effects and wound healing. Applications include biomedicine design, pharmaceuticals, drug delivery, restoration materials, chelation of metal particles, water absorption processing, and plant safety [8, 9, 10]. From 1985 to June 2015, there was a significant increase in the research on chitosan and its derivatives; Figure 2 depicts the number of Scopus-indexed publications on chitosan and its derivatives [11].
Figure 2.
Publications indexed by Scopus concerning chitosan and its derivatives.
Figure 3 shows some chemical modifications of chitosan. Table 1 illustrates some applications of chitosan derivatives.
Figure 3.
Some chemical modifications of chitosan [12].
Fields
Applications
Descriptions/references
Food industry
Food packaging preservation
Film-forming property and antibacterial property [13].
Worldwide, the leading cause of death is cancer. In 2008, cancer was the cause of death for about 13% of all people who died, or 7.6 million people. Numerous kinds of cancer are known, such as “prostate, colon, lung, and breast cancers,” but colon cancer is the most lethal [30]. Numerous medications, such as Bevacizumab, (Avastin)Oxaliplatin, Folinic acid, 5-fluorouracil (5-FU), Methotrexate, and Celecoxib, are used to treat cancer and are used to treat colorectal cancer (Celecoxib). However, most drugs are consumed with food, restricting the higher gastrointestinal tract (GIT). They are not stable inside the body, which prevents the drug from concentrating effectively at the desired tumor site. Due to their lack of specificity, some medicines can also have the opposite effect [31]. 5-FU is a specific anticancer drug that is frequently employed in treatment. 5-FU has disadvantages, including rapid ingestion in the body, a small half-life, a propensity for activating cancer cell resistance and cytotoxicity. These facets necessitate higher medication dosages, which raises the danger of adverse properties. So, a perfect system for delivering 5-FU would send the drug in tiny amounts and let it out quickly at the target site [32, 33, 34]. A prodrug for colon cancer must meet several requirements, such as not being toxic, compatible with the body, and stable in the GIT [35, 36, 37, 38]. This section designates the synthesis of a prodrug for the colon by binding CS with 5-FU. The drug was transformed to “5-fluorouracil-1-acetic acid (FUAC),” which was then conjugated to CS to produce “chitosan-1-acetic acid-5-fluorouracil (CS-FUAC),” which has been validated for colon cancer treatment. The CS was extracted from fish scales using a chemical process. Infrared Fourier transform and ultraviolet spectrophotometry was used to characterize the product. It was looked at as a possible delivery agent after a covalent bond was made between CS-FUAC and 5-FU.
2.1 SH and CS extraction
In Kirkuk, Iraq, fish scales were collected at a local fish market. Based on a documented procedure, for demineralization and deproteinization, a 1% sodium hydroxide and hydrogen chloride solution were prepared with the receptivity of 40 g/mol and 36.5 g/mol [39].
2.2 Preparation of CS-FUAC
As published in the literature, FUAC was constructed with minor variations. 5-FU and aq. of KOH are reacted and heated at 100°C for 90 minutes. Then, for over 6 hours, chloroacetic acid was added regularly using a water bath at 60°C while stirring. The result was acetified to produce needle crystals of FUAC with a yield of 60% [40]. To achieve “1-acetic acid-5-fluorouracilimidazoline,” a solution of FUAC in DMSO and “1,1-carbonyldiimidazole” was combined with CS in a glacial acetic acid (GAA) aqueous solution (Figure 4). “FT-IR spectroscopy” was utilized to examine the CS conformation, and synthesized FUAC and the CS-FUAC. Using a UV spectrophotometer, the produced chemicals were analyzed. Experimentation was conducted at a wavelength of 273 nm. Using a “Bruker Avance (500) spectrometer”, 1H- and 13C-NMR spectra were collected from DMSO-d6 and 1% CF3COOD/D2O solutions (Figure 5).
Figure 4.
Method for preparing “CS-FUAC.”
Figure 5.
1H-NMR spectra of CS-FUAC.
2.3 The drug content percentage of CS-FUAC conjugates
UV-visible spectroscopy established the fraction of FUAC conjugated to CS. The amide link in the CS-FUAC conjugates was initially hydrolyzed in the primary medium. As predicted, the concentration of FUAC was raised by raising the molar fraction of CS to FUAC. Table 2 displays the FUAC concentration in conjugates where the molar ratios of “CS: FUAC were 1:1 and 1:2,” respectively.
During transit through GIT, the drug used in the treatment of colon cancer must be stable in various pH ranges. Therefore, two buffers with pH of 1.2 and 7.4 were used to examine the prodrug stability in acidic and basic environments to determine its performance in acidic and basic environments. Based on the way conjugates are released in an acidic buffer, only 3–5% could be released. In contrast, it was 3–4% in the primary buffer (Figure 6). In acidic environments, drug release is more significant than in neutral conditions. This could be because of the hydrolysis of an amide linkage in the acidic condition.
Figure 6.
Stability evaluation of CS-FUAC and 5-FU at pH 1.2 (A) and a pH of 7.4 (B).
2.5 Conclusion
CS-FUAC, a potential colon-prodrug, was synthesized. The created conjugates appeared more stable under the initial conditions. In addition, CS-FUAC was more durable than the original medication in different pH conditions. An in vitro cytotoxicity investigation revealed that those synthesized derivatives are more active than free drugs. The cytotoxicity test, in vitro, demonstrates that these prodrugs are significantly more effective against “human colorectal cancer cell lines (HT-29)” than free medicine. In addition, it was nearly twice as cytotoxic to colon cancer cells compared with normal cells. Considering these outcomes, CS-FUAC as a prodrug appears to be an excellent method for colon delivery.
3. Synthesis and evaluation of Schiff base chitosan in biological systems
The Schiff base reactions produce the essential CS derivatives because of their application in the organic field. Furthermore, the response of CS with aromatic rings or heterocyclic aldehydes has led to the formation of stable Schiff bases (SBs), which are excellent molecules with uses in pharmacology, medicine, and other fields. Antimicrobial and cancer-prevention medications, for example [41, 42]. Interaction between CS amine sites and aldehydes or ketones, then removing water molecules, produces CSSBs [43]. Quinoline and quinazoline derivatives can also be found in various natural products, producing heterocyclic molecules with critical pharmacological applications. Quinoline and its results have antimalarial, antiviral, antibacterial, analgesic, anti-hepatoma, and anti-inflammatory properties. Oxazole derivatives are considered essential heterocyclic molecules in medicinal chemistry [44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56]. CH is extracted from a fish scale described previously and deacetylated to produce CS. This study’s objective is to estimate the DD percentage of CS, which is carried out by acid-base titration for samples of CS collected at various stages of the manufacturing process. In this investigation, FTIR and TGA will be used to characterize and validate the physicochemical parameters of the products. This investigation seeks to develop a synthetic method for three new CS Schiff bases (CSSB)s compounds by combining CS with “2-chloroquinoline-3-carbaldehyde, quinazoline-6-carbaldehyde, and oxazole-4-carbaldehyde.” Furthermore, the antibacterial possible of CS and its novel derivatives were investigated using two types of “fungi, C. Albicans and A. fumigate.” Utilizing FT-IR, (1H, and 13C) NMR spectroscopy, the structures of the manufactured products were established. The cytotoxicity of newly synthesized derivatives was determined using the MTT experiment.
3.1 Method of acid-base titration
The acid-base titration technique was used to calculate a CS sample’s degree of deacylation (DD). An indicator was used to control the endpoint, which turned to a blue-green color. The endpoint of the acid-base titration was used to calculate the DD percentage. The below equations were used to calculate the DD% of the deacetylated SH [57].
−NH2%=(0.016C1V1−C2V2Wx100E1
DD%=203(NH2%/16+42−NH2%x100E2
where the concentration and volume of HCl used are C1 and V1, the concentration and volume of NaOH used for titration are C2 and V2, and the weight of samples used for acid-base titration is W.
3.2 Synthesis of CSSBs
CSSBs were created by dissolving extracted CS in 2.0 percent aq. acetic acid, and carbonyl compounds dissolved in ethanol and added to the same amount of “CS. CS-P1, CSP2, and CS-P3” were the products of reactions between CS and “2-chloroquinoline-3-carbaldehyde, quinazoline-6-carbaldehyde, and oxazole-4-carbaldehyde”, respectively. Three CSSBs were synthesized, as exposed in Figure 7.
Figure 7.
Development of CSSB derivatives.
The configuration of the synthesized derivatives was established with “FT-IR spectroscopy, 1H NMR, and 13C NMR.” All FT-IR spectra of the newly prepared derivatives presented a band between 1633 and 1655 cm−1, related to the (-C=N) bond. Bands between 1400 and 1500 cm−1 and 1057 cm−1 correspond to (C-C) and in-plane (C-H) bonds. The absence of a band in the region between 1660 and 1730 cm−1 showed that the carbonyl group was absent, denoting that no free carbonyl remained. The (C-H) stretching of (CH3- and -CH2-) is represented by the vibrational bands at 2921 and 2883 cm−1. Bands at 1155 and 900 cm−1 were found in the glycosidic bonds. The glycosidic bonds and the (C-O, C-C, and C-O-C) stretching of the glycan ring were linked to the 1205–975 cm−1 bands [58, 59]. “Using 1H and 13C NMR, the structure of the prepared CSSBs was confirmed.” The 1H NMR spectra of the synthesized derivatives CS-P1and CS-P2 are depicted in Figures 8 and 9, respectively. The 13C NMR spectra for CSP1 is illustrated in Figure 10.
Figure 8.
1H NMR form CS-P1 compound.
Figure 9.
1H NMR spectra of CS-P2.
Figure 10.
13C NMR spectra CS-P1.
3.3 Examine the solubility
The solubility of the synthesized products was investigated using a variety of organic solvents. DMSO and mixtures of DMSO-CF3COOH in equal proportions were used. The results are shown in Table 3. Certain solvents, such as diluted HCl and CH3COOH, showed incomplete dissolution or swelling at 70°C. Most inorganic solvents are insoluble in the products.
Solvents
CH3COOH
CF3COOH
DMSO
HCl
NaOH
H2O
KOH
DMSO+ CF3COOH
Comp. codes
25°C
70°C
25°C
70°C
25°C
25°C
25°C
25°C
25°C
25°C
CS-P1
S+
S*
S**
S**
S
S*
S+
S+
S**
S
CS-P2
S+
S*
S**
S**
S
S*
S+
S+
S**
S
CS-P3
S+
S*
S**
S**
S
S*
S+
S+
S**
S
Table 3.
CSSB solubility characteristics in a range of solvents.
S = soluble, S+ = insoluble, S* = partially soluble and swelling, S** = partially soluble.
3.4 In vitro cytotoxicity examination
The MTT test is a colorimetric test used to measure cytotoxicity and cell viability. Based on MTT, the cytotoxicity of the synthesized derivatives was assessed, as depicted in Table 4.
Comp. conc. (mg)
Viable cells in the presence of CS
Viable cells in the presence of CS-P1
Viable cells in the presence of CS-P2
Viable cells in the presence of CS-P3
25
99 ± 0.83
99 ± 0.73
99 ± 0.60
99 ± 0.75
50
97 ± 0.73
99 ± 0.91
98 ± 1.3
99 ± 0.89
100
94 ± 0.63
98 ± 0.74
98 ± 1.2
98 ± 0.50
150
93 ± 0.88
97 ± 0.65
98 ± 0.62
96 ± 1.2
200
89 ± 0.53
90 ± 0.72
90 ± 0.74
91 ± 1.2
Table 4.
The CS and SB derivative cytoxicity assessment.
The experiment was repeated three times and mean was calculated.
Compared to the control, the outcome of the verified derivatives ‘CS-P1, CS-P2, and CS-P3’ exhibit the minimal difference between them. Several previous types of research have confirmed that CS and CSSB derivatives are non-toxic to cells. Consequently, CS has numerous medical applications [60, 61, 62].
3.5 Antimicrobial assessment
The CSSB derivatives’ antibacterial activity was assessed using the inhibition zone technique. Table 5 illustrates the results. The outcomes show that CS and all CSSBs affect E. coli and K. pneumonia strains as CS. According to the study, CS can prevent S. aureus and E. coli from forming new cells [63]. CSSBs derivatives presented antimicrobial activity against S. aureus, with inhibition zones of “22±0.3, 20±1.2, and 19±0.62 mm” for “CS-P1, CS-P2, and CS-P3,” respectively. The synthesized compounds also have antibacterial activity against S. mutans, with inhibition zones of “15±0.89, 17±0.50, and 18±1.20 mm,” respectively. Two fungal strains were used to test the CSSBs’ antifungal activity, and all the CSSBs tested showed positive results.
Gram-negative bacteria
Gram-positive bacteria
Fungi
Comp. codes
E. coli
K. pneumonia
S. aureus
S. mutans
C. albicans
Ammophilus fumigatus
CS
24 ± 0.63
26 ± 0.73
NA
NA
26 ± 0.79
16 ± 0.83
CS-P1
22 ± 0.73
28 ± 0.91
22 ± 0.3
15 ± 0.89
34 ± 0.99
26 ± 0.91
CS-P2
27 ± 0.83
27 ± 0.72
20 ± 1.2
17 ± 0.5
31 ± 1.29
25 ± 0.72
CS-P3
22 ± 0.98
26 ± 0.65
19 ± 0.62
18 ± 1.2
26 ± 0.49
21 ± 0.65
Table 5.
Results of antibacterial and antifungal action of CS and CSSBs.
NA means not detected.
According to the published studies, numerous mechanisms are expected to explain how CS acts on bacteria, which vary depending on the metabolic process, the type of microorganism, and the cell wall composition. The initial recommendation is to disrupt the organism’s cell wall electrostatic attraction between the positively charged amine in CS and the negatively charged residue group in bacterial nucleic acid cellular components such as COO− or PO4−2. The interface of bacterial DNA with CS is proposed as the second procedure. Protein and messenger RNA perversion into bacterial cells is induced by CS, followed by nuclei. Another theory relies on the ability of CS to form metal complexes. Metal complexes such as Zn2+, Mg2+, and Ca2+ are examples. Metals are required for bacterial metabolic and growth processes [63].
3.6 Conclusion
The hunt for new antibiotics has risen in tandem with the rise in antibiotic-resistant microorganisms. CS may be good material in this field. Cyprinus scales extracted 89% of the CS from local market-purchased Carpio fish. Acid-base titration was used to determine the DD % and describe how CS is put together. Three new CSSB derivatives that included the branches’ distinct parts were synthesized, and their formations were confirmed using “FT-IR and 1H and 13C NMR spectroscopy.” The new SB configuration was antibacterial against many bacteria and fungi tested. CSSBs had practically no effect on cytotoxic mouse fibroblast cell lines after being prepared. As a result of the findings above, it is reasonable to believe that the prepared CSSBs could be utilized with high efficiency, care, and performance in numerous biomedical fields.
4. A novel prodrug of methotrexate based on chitosan and evaluation of their bioactivity
Methotrexate (MTX) is the most effective medicine for treating several types of cancer, including colon cancer. Nevertheless, this medication can reduce the bioavailability of the goal material. It is administered orally and rapidly digested. MTX is an antimetabolic agent that inhibits folic acid metabolism. 1948 marked the beginning of clinical use of the drug as an anticancer agent, following the finding of its abnormal effects on DNA combination [64]. Due to the drug’s physical and chemical characteristics, oral administration results in sedate retention at the beginning of the gastrointestinal tract. The drug’s numerous significant limitations and disadvantages were demonstrated once aimed directly at a specific position of absorption or a particular portion of the alimentary channel, for instance, the colon. As a result, it is critical to explain how the medicine arrived at a specific treatment location [65]. Recent preparation of the prodrug involved covalently attaching the colon drug to the carrier. These prodrugs frequently alter their physical and chemical characteristics to improve infatuation at the activity site, increase the duration of action, and reduce toxicity and side effects [66]. This project is designed to develop a prodrug for colon cancer treatment by incorporating MTX into a biopolymer. The MTX was then converted to “methotrexate – imidazole” and loaded into CS to create colon cancer prodrugs containing CS-MTX conjugates. The structure of the synthesized derivatives was confirmed using spectroscopic analysis. The compound’s chemical stability and hemolytic activity were also investigated. The drug concentration percentages were calculated. The “MDA-MB-231, MCF-7, and MDA-MB-453” cell lines were used to test the cytotoxicity of the prepared derivatives in a dish with MTT.
4.1 Synthesis of CS-MTX
The CS-MTX compound was developed as a possible treatment for MTX-induced colon cancer. As previously described, the CS was extracted and used to prepare the CS-MTX. As shown in Figure 11, methotrexate and imidazole were used to kickstart the process. To make MTX-imidazole in situ, equal quantities of “MTX and imidazole” were added to the mixture. A small quantity of “N, N-carbonyl diimidazole (CDI)” was added. At a concentration of 2%, CS was dissolved in GAA. The catalyst, triethylamine (TEA), was added in a few drops [67].
Figure 11.
Synthesis of CS-MTX.
The confirmation of the CS-MTX compounds was established by “NMR and FTIR spectroscopy.” A downfield signal at 6.02 Hz on the 1HNMR chart indicated the proton was linked to C1 of the sugar (Figure 12). The proton signal was observed at 3.60, which is connected to C1. Since the two protons were in diverse environments, the methylene group C5 showed signals at 2.53, and C6 led signals at 2.08 and 2.28. The carbonyl signals were shown in the 13C NMR spectrum (Figure 13) at 173.2 and 173.4. The value of the anomeric carbon was 101.5. The IR spectrum of the conjugate (Figure 14) revealed the CS and MTX vibrations. The N-H group had peaks at 3064 cm−1, while the C-H group had 3032–2834 cm−1. At 1660 and 1720 cm−1, COOH group C-O vibration-related signals were detected. The (C-C, C-N, and CO) vibrations were found in signals between 1499 and 1183 cm−1. A new peak at 1700 cm−1 confirmed the development of an amide bond between MTX and CS. This bond was vulnerable to acid hydrolysis or the amidase enzyme, allowing the drug to be released.
Figure 12.
The 1H NMR of “CS-MTX.”
Figure 13.
The 13C NMR of “CS-MTX.”
Figure 14.
The FT-IR of the “CS-MTX.”
4.2 Determination of drug contains
The percentage of MTX-CS was calculated using UV-visible spectroscopy. The initial conditions for this experiment were based on the amide bond hydrolysis between MTX and CS. As expected, increasing the amounts of CS and MTX in the conjugates improved the results. The CS-MTX (1:1) contained 0.60% w MTX, and the yield was 65.0%.CS-MTX (1:2), on the other hand, had a content of 0.72% w and produced 70%. All reactions were carried out at 50°C for 20 hours. The proportion of medicine left was determined using this equation [68]:
The percentage of hemolysis produced by CS-MTX derivatives was investigated at various concentrations. The proportion of hemolysis increased as the concentration of CS-MTXs increased, as shown in Figure 15. According to the test results, the CSMTXs’ hemolysis rate was less than 4.5 percent, below the international standard of less than 5% [69]. This assessment was conducted depending on the method described [70]. The rate of hemolysis increases as the level of CS-MTX rises. The hemolysis rate in the CSMTXs was less than 4.5 percent, less than the international standard of less than 5 percent. White rabbit red blood cell (RBC) samples were used in this experiment. A 0.9 percent of NaCl solution was added after 3 mL of blood was extracted and centrifuged for 20 minutes at 4000 rpm. The controls were made by mixing 1 mL of red blood cells and distilled water with 5 mL of normal saline. A sequence of solutions containing both derivatives was carried out by adding 6,2,0.6,0.3,0.7 g of MTX and the prepared combination to tubes, followed by 2 mL of normal saline and 1 mL of red blood cells. The resulting solutions were kept for 2 hours at 37°C in a water bath. Separately, the tube was centrifuged at 4000 rpm for 20 minutes. At a wavelength of 541 nm, UV absorbance was measured. The following equation used to calculate the percentage of hemolysis was as follows:
Three human breast cancer cell lines were used in this project “MDA-MB-231, MCF-7, and MDA-MB-453.” The cell lines’ viability was decreased by CSMTX conjugates in a dose-dependent manner, according to MTT analyses. CS-MTX cytotoxicity was variable across all cell lines tested. The IC50 values for “MCF-7, MDA-MB-231, and MDA-MB-453 were 363.5±31.2, 198.8±20.4, and 163.4±10.8 g of CS-MTX/mL,” respectively, over 24 hours. However, the cell “MDA-MB-453” used CS-MTX more precisely than the other cell lines in Figure 16. The cytotoxicity of CS MTX and the free medicine MTX was studied using “MCF-7, MDA-MB-231, and MDA-MB-453 cells,” and the effect was calculated using the MTT assay [71]. A 96-well plate was used to test a range of CS-MTX and MTX concentrations (1–10 M). These concentrations were added before coating the plate with roughly 3 x 103 cells. After that, the plate was kept for 12, 24, and 48 hours. After removing the previous media, new media were added, including (DMEM/F12) supplemented with 15 L of (MTT, concentration—500 g/mL). The cells were then grown at 37°C for 3–4 hours. The dark-blue formazan crystals were dissolved in DMSO. A Cytation three multimode plate reader manufactured in the United States was used to calculate their absorptivity at 574 nm for each well. The equation below was used to convert the obtained absorbance values into applied rate cells for unprocessed control cells:
Figure 16.
MTT viability tests cell lines after mixing 200 g/mL in DMSO as a control for 24 hours. “Data were calculated as mean SD (n = 3) with a P-value of 0.01 compared to the control.” Each solution’s final exhibition was equal to 0.5 percent.
Relative cell viabillity=Absorbance of the sampleAbsorbance of the control×100E5
4.5 Conclusion
The synthesized CS-MTX derivatives are most likely human colon cancer prodrugs. Under acidic conditions, the prepared derivatives were satisfactorily stable at a pH of 1.2. The synthesized compounds had a long half-life value of 4.52 in acidic conditions and 16.01 in basic media than the original drug. The CS-MTX conjugate produced significant results in the MTT assay. The three “cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-453” showed a dose-dependent reduction in viability compared to the origin medicine. The IC50 values were “363.53±1.2, 198.82±0.4, and 163.41±0.8 g of CS-MTX/mL after 24 hours.” These findings suggest that CS-MTX as a prodrug could be helpful in the treatment of colon cancer. Additional tests are being conducted to estimate the synthesized prodrug’s various biological activities.
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Written By
Mohsin Mohammed and Nadia Haj
Submitted: 17 May 2022Reviewed: 10 June 2022Published: 09 November 2022
Open access peer-reviewed chapter
