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BY 4.0 license Open Access Published by De Gruyter Open Access November 2, 2023

Important application value of injectable hydrogels loaded with omeprazole Schiff base complex in the treatment of pancreatitis

  • Jingrong Ma , Chaoqun Du , Yuehua Zhang , Jing Zhan , Yafang Lai and Mingwei Zhao EMAIL logo
From the journal Open Chemistry

Abstract

In this study, a novel Ho(iii) coordination complex with the chemical composition of [Ho6(acac)4L4(CH3O)6xCH3OH (1) has been prepared via using a polydentate Schiff base ligand N′-(2,3-dihydroxybenzylidene) picolinohydrazide (H2L) and a β-diketone (Hacac = acetylacetone) co-ligand, and then successfully loaded with drug omeprazole. Hyaluronic acid, a natural polysaccharide with good biocompatibility, was used as raw material to prepare drug carriers that can be injected into hydrogels based on chemical synthesis method. The internal microstructure of the hydrogel was observed by scanning electron microscope, which showed a good three-dimensional connected porous structure. After the synthesis of the hydrogel, the value of their application in the treatment of pancreatitis was evaluated and the related mechanisms were explored at the same time.

1 Introduction

As an acute inflammatory process of the pancreas, acute pancreatitis (AP) results in varying degrees of impact on neighboring tissues and other organ systems [1]. Research and practice have demonstrated that the pathogenesis of AP is a multifactorial, complicated pathophysiologic process. In the past several years, relevant research studies have progressed quickly, and put forward a number of new theories, which exert an essential role in the understanding of the developmental process of the disease and in the guidance of the clinical treatment [2,3]. Consequently, continued research into the pathogenesis of AP and an understanding of the diverse factors that participate in the complex pathologic process is a basic way to continually enhance the efficacy of disease treatment.

Over the decades, Schiff bases were considered as essential ligands because of their coordination chemistry; moreover, they could be easily produced and attached to a wide range of types of metal ions [4]. Because of the presence of amine groups, which resemble biological systems in nature, Schiff bases display a pivotal part in the observation of transformation mechanisms and racemization reactions within biological systems. Schiff bases have a wide range of essential bioactivities encompassing anti-bacterial, anti-HIV, anti-fertility, anti-tumor, anti-cancer, anti-inflammatory, and anti-mosquito larvae [511]. Among the various metal complexes, lanthanide metal complexes have been intensively investigated for their high bioactivity and low toxicity when bound to ligands. Lately, there has been a strong scientific interest in preparing lanthanide metal complexes as they are suitable for anticancer activities and DNA interactions [1215]. For instance, Wang et al. proved that the Schiff base La(iii) complex generated from diethylenetriamine and kaempferol interacts with DNA through an intercalation mode, and stronger cleavage of DNA and binding by the La(iii) complex than by the ligand was demonstrated by in vitro cytotoxicity behaviors on HL-60 cells (human leukocytomas) and on the HepG-2 cell line [16]. With the above considerations, in this work, a novel Ho(iii) coordination complex with the chemical composition of [Ho6(acac)4L4(CH3O)6xCH3OH (1) has been successfully prepared through applying a polydentate Schiff base ligand H2L (Scheme 1) together with a β-diketone (Hacac = acetylacetone) co-ligand. The CP 1’s structure was examined with IR spectroscopy, single crystal X-ray diffraction (SCXRD), elemental analysis (EA), thermogravimetric analysis (TGA), and powder X-ray diffraction (PXRD).

Scheme 1 
               Chemical drawings for the organic ligand.
Scheme 1

Chemical drawings for the organic ligand.

In recent years, with the rapid development of tissue engineering, injectable hydrogels have been widely used in the biomedical field [17,18]. Hydrogel has a 3D network structure consisting of polymer chains that can absorb and hold large amounts of water, giving it physical characteristics similar to those of extracellular matrices [19]. Hydrogels have great potential in the treatment of pancreatitis due to their advantages of simple preparation, adjustable mechanical properties, good biocompatibility, and biodegradation. Hyaluronic acid (HA) is a naturally occurring polysaccharide in human tissues, also known as HA, and is an important component of the extracellular matrix [20,21]. At the same time, as a natural polymer, HA has good biocompatibility and is increasingly important in the fields of biomedical engineering, pharmaceutical science, and medicine [22]. Omeprazole is an important hand subpump inhibitor, which is mainly used in the treatment of gastroesophageal reflux and peptic ulcer disease [23,24]. It has good acid inhibition effect, long duration of efficacy, and few side effects, and has been widely concerned by researchers.

In this study, the injectable hydrogel was prepared by chemical synthesis method with natural polysaccharide HA and successfully loaded with omeprazole. The internal structure of the hydrogel showed a three-dimensional network structure with large pore characteristics by scanning electron microscope (SEM). In addition, a number of bioassays were carried out to assess its biological activity. Cholecystokinin (CCK)-8 assay presented that the hydrogel had a favorable protective effect on the pancreatitis cell viability. In addition, the levels of released inflammatory cytokines were also determined by ELISA.

2 Experimental

2.1 Chemicals and measurements

With all solvent and reagents being available in the market, they were not purified any further. EA of N, H, and C were implemented on the Perkin-Elmer 240C elemental analyzer. On a Bruker Vector 22 FT-IR spectrophotometer, FT-IR was recorded from 400 to 4,000 cm−1 utilizing KBr particles. On a Shimadzu XRD-6000 X-ray diffractometer with 1.54056 Å Cu Kα radiation at RT, the patterns of PXRD were obtained with 0.05° step size. TGA was conducted in air applying a Netzsch STA449 F1 thermal analyzer with 10°C/min heating rate between 25 and 800°C.

2.2 Preparation and characterization for [Ho6(acac)4L4(CH3O)6xCH3OH (1)

Ho(acac)3-2H2O of 0.03 mmol was added to 0.03 mmol of H2L solution in CH3OH/CH2Cl2 (12 mL, with a volume ratio of 3: 1) and mixed for 30 min at RT. The solution was subsequently sealed and placed in a glass vial (20 mL) and heated under self-pressurization at 80°C for 3 days. After that, colorless massive crystals were given that were appropriate for X-ray crystallography of 1. Yield: 52% (on the basis of Ho salt). Elemental analysis (%) calcd for C78H82Ho6N12O26: N, 6.48; H, 3.19; C, 36.13. Found: N, 6.46; H, 3.23; C, 35.96. The IR (KBr, cm−1, Figure 1) spectrum includes the following peaks: 685(m), 740(s), 768(s), 807(w), 853(s), 926(m), 1,018(s), 1,097(w), 1,156(w), 1,203(s), 1,265(s), 1,301(w), 1,345(m), 1,387(m), 1,438(s), 1,518(m), 1,551(s), 1,606(w), 2,354(w), 3,077(w), and 3,565(w). The absorption peak at 3,500 cm−1 is attributed to the stretching vibration of hydroxyl groups, the peaks at 1,620–1,400 cm−1 belong to the characteristic stretching vibrations of aromatic rings, and the peaks at 1,150–1,020 cm−1 correspond to the stretching vibrations of N–C bonds.

Figure 1 
                  IR spectrum of compound 1.
Figure 1

IR spectrum of compound 1.

X-ray data were taken by an Oxford Xcalibur E diffractometer. Optimization of the intensity data was carried out using CrysAlisPro software and transformed into HKL files. The generation and refinement of initial structure were conducted through the SHELXS program with direct method and SHELXL-2014 procedure based on least squares approach, separately [25,26]. The whole non-H-atoms were mixed utilizing the anisotropy parameter. Next, we utilized the AFIX command to fix the entire H-atoms geometrically to the C-atoms to which they are connected. Table 1 presents the crystallographic details and refinement particulars of CP 1.

Table 1

Crystallographic details and refinement particulars of CP 1

Empirical formula C78H82Ho6N12O26
Formula weight 2593.13
Temperature (K) 150.0
Crystal system Triclinic
Space group P-1
a (Å) 11.3690(2)
b (Å) 12.2267(3)
c (Å) 17.2362(11)
α (°) 104.259(3)
β (°) 98.7421(14)
γ (°) 109.529(2)
Volume (Å3) 2115.28(15)
Crystal size (mm3) 2.3 × 1.4 × 1.2
Z 1
ρ calc (g cm−3) 2.036
μ (mm−1) 5.627
Data/restraints/parameters 7,401/6/539
Goodness-of-fit on F 2 1.021
Final R indexes [I > = 2σ(I)] R 1 = 0.0491, ωR 2 = 0.1121
Final R indexes [all data] R 1 = 0.0847, ωR 2 = 0.1297
Largest diff. peak/hole/e Å−3 2.70/−1.60
CCDC 2089657

2.3 Synthesis of oxidized hyaluronic acid (OHA)

First, a certain amount of HA is dissolved in 100 mL deionized water and configured into an aqueous solution with 1 wt% concentration. The reaction was stopped by adding 0.5 mol/L sodium periodate solution (10 mL), protected from light for 2 h, and followed immediately by the addition of ethylene glycol (1.5 mL). After dialysis with DI water, the sample was freeze-dried and marked as OHA.

2.4 Preparation of carbonyl dihydrazin (CDH)-HA

First, a certain amount of HA is dissolved in 100 mL deionized water and configured into an aqueous solution with 1 wt% concentration. Later, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.3 g), 1-hydroxybenzotriazole (0.2 g), as well as CDH (0.5 g) were added to the HA solution overnight for amidation. After the reaction solution was dialyzed with DI water and lyophilized, the samples were marked as CDH-HA.

2.5 Preparation of the injectable HA hydrogel

The freeze-dried samples of 200 mg OHA and 400 mg CDH-HA were added to 10 mL PBS until completely dissolved, and the reaction solution was prepared. It was moved into a double-barreled syringe and rapidly squeezed into the mold to create a hydrogel.

2.6 Morphology observation

Internal microstructure of the injectable hydrogel was visualized by SEM. Sample was sprayed with gold prior to testing.

2.7 CCK-8

To characterize the protective effect of hydrogels on the pancreatitis cells, the effect of the hydrogels on cellular activity was first examined using the CCK-8 assay. Pancreatitis cells were inoculated into cell culture plates (96-well) at a terminal number of 104 per well. After incubation in an incubator at 37°C with 5% CO2 for 12 h, the cells were treated with hydrogels of various dilutions and incubation was continued for 48 h. Next, discard the cell culture medium and add fresh medium with CCK-8 reagent. Finally, at 450 mm, the absorbance of each well was determined. The assay was replicated a minimum of three times.

2.8 ELISA assay

The hydrogels were evaluated for their inhibitory activity on the tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels released into plasma by ELISA. Cells were treated with various concentrations of hydrogels for 48 h, and subsequently the TNF-α and IL-1β levels in the cell supernatant were assayed by ELISA.

3 Results and discussion

3.1 Structural characterization

Structure solving and refinement according to gathered single-crystal data under environmental circumstances showed that complex 1 crystallizes in a three-oblique space group P-1 with Z = 1. A molecular configuration of 1, illustrated in Figure 2(a), is composed primarily of four L2−, six Ho(iii) ions, four acac, and six CH3O. In the asymmetric group, Ho2 and Ho1 ions are eight-coordinated. Ho1 ion is attached to N1 atom and O1, O2, O2a, O3, O4, O5, and O12 atoms, the Ho2 ion is attached to N4 and N3 atoms and O1, O3, O5, O6, O11, and O13 atoms, and the Ho3 ion at the center is a hepta-coordinated NO6 ligand. The coordination polyhedra of central Ho1 and Ho2 atoms could be characterized as twisted bipyramidal trigonal geometries, and the coordination polyhedra of central Ho3 atoms could be described as twisted pentagonal bipyramidal geometries, which were achieved via SHAPE 2.0 software. In 1, H2L is linked to the central Ho1 ion employing two distinct coordination modes. Six central Ho ions are connected to 12 μ2-O atoms derived from two CH3O and four L2 ligands, forming an approximately linear Ho6 arrangement (Figure 2(b)). In Ho6 core, the distances of Ho1⋯Ho2, Ho2⋯Ho3, and Ho1⋯Ho1a are 3.5064, 3.7515, and 3.6402 Å, the angles of Ho1–Ho2–Ho3 and Ho1a–Ho1–Ho2 are 148.792° and 146.694°. In addition, the distances of Ho–O bond of CP 1 are between 2.238(8)–2.389(6) Å, while the lengths of the Ho–N bonds are 2.417(8), 2.518(8), and 2.540(8) Å, separately. The O–Ho–O bonds have angles ranging between 66.2(2)° and 169.6(3)°. These bond angles and lengths are comparable to Ln6 clusters investigated earlier in the literature.

Figure 2 
                  (a) Asymmetrical unit of 1 and (b) Ho6 cluster-based structure of 1.
Figure 2

(a) Asymmetrical unit of 1 and (b) Ho6 cluster-based structure of 1.

In order to examine the purity of the product’s phases, PXRD experiments were performed on the prepared crystals of 1 (Figure 3(a)). The positions of the peaks of the experiment and the simulated PXRD pattern coincide with each other, suggesting that the crystal structure genuinely represents the bulk crystal product. The difference in intensity may be caused by the favorable orientation of the crystal samples. To assess the complexes’ thermal stability, we conducted a TGA of the crystal samples of CP 1 in air at 10°C min−1 heating rate. As illustrated in Figure 3(b), the TGA curve showed a 4.82% weight loss (with a calculated value of 4.91%) between 40 and 320°C, as a result of the loss of four ligand methanol molecules. This was followed by a weight loss of 16.12% from 320 to 400°C, which correlates with the shedding of four acetylacetonate molecules (15.34% calculated value). Thereafter, cluster 1 progressively decomposed between 400 and 800°C.

Figure 3 
                  (a) 1’s PXRD pattern and (b) its TGA curve.
Figure 3

(a) 1’s PXRD pattern and (b) its TGA curve.

3.2 Synthesis and micromorphology of injectable hydrogels

Natural polysaccharide HA possesses excellent biocompatibility and is widely used in tissue engineering. In this experiment, aldehyaluronic acid was prepared by sodium periodate oxidation, while starch-based HA was produced by a dicarbonate trap replacing HA. Finally, the injectable hydrogel was created in situ through the nature of the Schiff base between the amino group and aldehyde group, which was safe and reliable without toxic crosslinking agent in the crosslinking process. We further examined the in situ cross-linking capacity of the hydrogels. The injection physical drawing of the hydrogel is shown in Figure 4(a). Different shapes of the hydrogel could be prepared according to different molds, and no blockage or fracture occurred during the extrusion process, indicating that the injectable hydrogel had good in situ molding ability (Figure 4(b)). The diagram shows the microscopic morphology inside the hydrogel. Hydrogels have a typical macroporous structure and are interconnected in three dimensions, which is of great significance as drug carriers (Figure 4(c)).

Figure 4 
                  (a) Preparation process of injectable hydrogel, (b) molding of injectable hydrogel, and (c) morphology of injectable hydrogel.
Figure 4

(a) Preparation process of injectable hydrogel, (b) molding of injectable hydrogel, and (c) morphology of injectable hydrogel.

3.3 Remarkable protective effect of hydrogels on the viability of pancreatitis cells

The effect of the new hydrogels on cellular activity was first evaluated. CCK-8 assay was implemented to evaluate the cellular activity of pancreatitis cells after treating with different concentrations of hydrogels. As shown in Figure 5, the hydrogels had a good enhancement impact on the viability of pancreatitis cells compared to the control group, and this effect was more pronounced as the concentration of the hydrogels increased. This result indicates that the hydrogels have a good application in pancreatitis treatment.

Figure 5 
                  Significant protective effect of hydrogels on cell viability in pancreatitis. Cell activity was detected using CCK8 assay after treatment of cells with different concentrations of compounds for 48 h; * and ** indicate P < 0.05 and 0.01, respectively.
Figure 5

Significant protective effect of hydrogels on cell viability in pancreatitis. Cell activity was detected using CCK8 assay after treatment of cells with different concentrations of compounds for 48 h; * and ** indicate P < 0.05 and 0.01, respectively.

3.4 Hydrogels inhibit the release of inflammatory cytokines

In the above experiments, we demonstrated a significant protective effect of the new hydrogels on the viability of pancreatitis cells. Inflammatory cytokine levels are usually elevated during the development of pancreatitis. Therefore, in the current work, real-time ELISA was exploited to determine the levels of inflammatory cytokines in the supernatants of cells treated with different concentrations of hydrogels. The results in Figure 6 show that the hydrogels were able to significantly reduce the release of inflammatory cytokine levels compared to the control.

Figure 6 
                  Effect of hydrogels on inflammatory cytokines. The amount of inflammatory cytokines released from the cells was determined by ELISA; * and ** indicate P < 0.05 and 0.01, respectively.
Figure 6

Effect of hydrogels on inflammatory cytokines. The amount of inflammatory cytokines released from the cells was determined by ELISA; * and ** indicate P < 0.05 and 0.01, respectively.

4 Conclusion

In conclusion, we have synthesized a novel Ho(iii) CP via employing a polydentate Schiff base ligand N′-(2,3-dihydroxybenzylidene)picolinohydrazide (H2L) and a β-diketone (Hacac = acetylacetone) co-ligand. The CP 1’s structure was examined with IR spectra, SCXRD, EA, TGA, and PXRD. Using HA with good biocompatibility as raw material, the injectable hydrogel was prepared by Schiff base reaction of amino group and aldehyde group, and the loading of omeprazole was realized successfully. We further observed the internal microstructure of the hydrogel and conducted a series of experiments to evaluate its biological activity. SEM results showed that the hydrogel presented a three-dimensional network structure with good connectivity between pores. CCK-8 exhibited that the hydrogel had a good protective effect on the cellular activity of pancreatitis cells. In addition, the impact of the hydrogel on the inflammatory cytokines release was determined by ELISA. The results surfaced that the hydrogel was able to inhibit the release of inflammatory cytokines, which predicts that the hydrogel has a good application value in the treatment of pancreatitis.

  1. Funding information: The authors state no funding information.

  2. Author contributions: Jingrong Ma, Chaoqun Du, and Yuehua hang synthesized and characterized the compounds; Jing Zhan, Yafang Lai, and Mingwei Zhao performed other experiments.

  3. Conflict of interest: The authors state that no conflict of interest exists in the publication of this article.

  4. Ethical approval: Research experiments performed in this article using humans or animals have been authorized by the Ethical Committee and the competent authorities of research organization(s), and all guidelines, legal, regulations, and ethical standards for humans or animals have been followed.

  5. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-08-16
Revised: 2023-10-04
Accepted: 2023-10-07
Published Online: 2023-11-02

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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