Oral nano-drug delivery system for treating ulcerative colitis

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to an oral nano-drug delivery system for treating ulcerative colitis. The rosmarinic acid chondroitin sulfate A nanoparticles (RA-CSA nanoparticles) provided by the invention can effectively prevent the adsorption of plasma proteins and improve the stability of the nanoparticles in blood. In a hemolysis test, the RA-CSA nanoparticles have good biocompatibility in blood. A drug release mode of the RA-CSA nanoparticles shows explosive release within 24 hours at first and then enters a stable slow release stage, and the dynamic release mode is beneficial to long-term sustained release of the drug to an inflammatory site, ensures that the drug stays for a longer time in a body, stays for a longer time in a colon and acts for a longer time, and achieves a better anti-inflammatory effect. Meanwhile, the RA-CSA nanoparticles can effectively remove ROS (reactive oxygen species) and have strong antioxidant activity. In addition, compared with free RA, the RA-CSA nanoparticles have the advantages that inflammation activation of macrophages is remarkably reduced, colitis is effectively promoted to be relieved, and the RA-CSA nanoparticles have no obvious in-vivo toxicity.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to an oral nano drug delivery system for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD), a chronic inflammatory disease that affects the rectum and colon to varying degrees. In 2023, the global prevalence of ulcerative colitis is estimated to be 5 million cases and continues to grow at a high rate worldwide. UC is a complex, multifactorial, chronic disease with the main pathological features of inflammatory cell infiltration and glandular destruction. The pathogenesis of UC is related to changes in the colon environment in genetically susceptible individuals, which has a huge impact on the patient's life and can lead to chronic, lifelong, and recurrent complications, which brings great difficulties to treatment. Clinically, different types of drugs have been widely developed for the treatment of UC, mainly including the most commonly used first-line drug 5-aminosalicylic acid (5-ASA), immunosuppressive drugs (such as methotrexate), biological agents (such as anti-tumor necrosis factor (TNF), vedolizumab (anti-α4β7 integrin), ustekinumab) and small molecules (such as Janus kinase inhibitors, sphingosine-1 receptor modulators). However, the efficacy of these drugs is limited due to the lack of targeting, inevitable systemic side effects, high recurrence rate and strong drug resistance. Therefore, an effective therapy for the treatment of UC is urgently needed.

[0003] The active ingredients of natural medicines have the characteristics of multi-target, high efficacy and few side effects, and have become an important research object for the treatment of ulcerative colitis. Rosmaric acid (RA) is an active ingredient in the plant kingdom, such as rosemary and Prunella vulgaris, and is one of the most important polyphenol antioxidants. RA has a wide range of biological effects, including anti-inflammatory, anti-cancer, anti-ischemic and antibacterial activities. Data studies have shown that RA can block the production of PGE2 and TNF-α induced by LPS. In addition, rosemary extract can inhibit MAPKs phosphorylation, block NF-kB activation, lead to reduced iNOS and COX-2 expression, and have anti-inflammatory effects. Despite such high potential, RA also encounters the same problems as other small molecule bioactive compounds, namely poor water solubility, high instability, poor colon targeting ability, poor bioavailability, etc., which reduce its therapeutic effect. Recently, researchers have developed a delivery system for RA loading to improve stability and bioavailability and use it in biomedical applications. WO2021206428A1 discloses a PEG-modified RA aimed at improving the water solubility of RA.

[0004] There are few reports on chondroitin sulfate-derived RA-derived polymer prodrugs that achieve colon-active targeted therapy for UC. Polysaccharides can effectively protect small molecule compounds from the complex gastrointestinal environment after oral administration. Chondroitin sulfate (CS) is a naturally occurring negatively charged macromolecule that belongs to the glycosaminoglycans (GAGs) family. The CS chain is composed of repeating disaccharide units N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) and is a component of the extracellular matrix in connective tissue. CS is widely used in many therapeutic approaches due to its good biodegradability, high biocompatibility and chemical coupling. Previous studies have shown that CS can reduce the production of proinflammatory cytokines (phospholipase A2, cyclooxygenase 2, nitric oxide synthase-2), reduce C-reactive protein concentration, and inhibit phagocytosis and the intracellular oxidative burst of neutrophils. In addition, CS also exhibits significant binding affinity to the transmembrane glycoprotein CD44. The CD44 receptor is overexpressed on the surface of macrophages in the inflamed colon and various malignant tumor cells. The CD44 receptor is highly expressed in the lesions of many UC patients. Therefore, the CD44 receptor is considered to be a specific biomarker for UC. CS has been combined with nanoparticles to achieve efficient targeting, which enables it to promote its accumulation in the colon through CD44-mediated endocytosis. In addition, many researchers have designed CS-drug complexes to improve the solubility of poorly soluble drug molecules. Therefore, the present invention has developed a new oral delivery system of rosmarinic acid chondroitin sulfate A (RA-CSA) nanoparticles for the treatment of UC. This strategy not only effectively improves the stability and bioavailability of RA, but also enhances the targeted drug accumulation in UC lesions. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides an oral nano drug delivery system for treating ulcerative colitis.

[0006] The technical solution of the present invention is as follows: a rosmarinic acid chondroitin sulfate A polymer prodrug, the structural formula of which is as follows:

[0007]

[0008] Wherein n is an integer from 6 to 1300.

[0009] The present invention provides a method for preparing a rosmarinic acid chondroitin sulfate A polymer prodrug, which is characterized by comprising the following steps:

[0010]

[0011] 1) adding RA, CSA, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and n-hydroxysuccinimide in a molar ratio of 2:1:1:1:1, reacting in an ice bath for 2 h, storing at room temperature for 1 h, then adding a mixture of ethylenediamine and formamide to the reaction solution, and stirring continuously for 24 h;

[0012] 2) After the reaction is completed, the reaction solution is filtered with a 0.45 μm organic filter membrane. The filtrate is dialyzed in acidic Milli-Q water for 2 days; the dialyzate is centrifuged and the supernatant is collected and freeze-dried to obtain the RA-CSA polymer prodrug.

[0013] Preferably, in step 2), the dialysis membrane has a MWCO of 1.0 kDa.

[0014] Preferably, in step 2), the centrifugation condition is 2000 rpm, 10 min.

[0015] The invention discloses rosmarinic acid chondroitin sulfate A self-assembling nanoparticles, which are formed by self-assembling the rosmarinic acid chondroitin sulfate A polymer prodrug in an aqueous phase. The hydrodynamic size of the nanoparticles is 247.3±2.99nm and the zeta potential is -19.6±1.23mV.

[0016] A pharmaceutical composition comprises the rosmarinic acid chondroitin sulfate A polymer prodrug or the rosmarinic acid chondroitin sulfate A self-assembled nanoparticles and a pharmaceutically acceptable carrier or excipient.

[0017] The invention provides an application of the rosmarinic acid chondroitin sulfate A polymer prodrug or the rosmarinic acid chondroitin sulfate A self-assembled nanoparticles in the preparation of a drug for treating or preventing colon inflammation.

[0018] The present invention provides an application of the rosmarinic acid chondroitin sulfate A polymer prodrug or the rosmarinic acid chondroitin sulfate A self-assembled nanoparticles in the preparation of an injection, oral administration or local administration system.

[0019] Beneficial effects of the present invention: The present invention provides rosmarinic acid chondroitin sulfate A nanoparticles (RA-CSA nanoparticles). The negative potential of RA-CSA nanoparticles can effectively prevent the adsorption of plasma proteins and improve their stability in the blood. In the hemolysis test, RA-CSA nanoparticles have good biocompatibility in the blood. The drug release pattern of RA-CSA nanoparticles shows an explosive release within the first 24 hours, followed by a stable sustained release phase. This dynamic release pattern is conducive to the long-term continuous release of drugs to the site of inflammation, ensuring that the drug stays in the body longer, allowing the drug to stay in the colon longer, and the duration of action is longer, achieving better anti-inflammatory effects. At the same time, RA-CSA nanoparticles can effectively remove ROS and have strong antioxidant activity. In addition, compared with free RA, RA-CSA nanoparticles significantly reduce the inflammatory activation of macrophages, effectively promote the relief of colon inflammation, and have no obvious in vivo toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 .Synthesis route (A), FTIR spectrum (B) and 1 H NMR spectrum (C)

[0021] Figure 2 .TEM (A), particle size distribution (B) and serum stability (C) of RA-CSA nanoparticles. Release curve of RA-CSA nanoparticles in PBS at 37°C (D). UV full wavelength scan (E) and hemolysis rate of red blood cells in different groups (F)

[0022] Figure 3 .DPPH (A) and H2O2 (B) scavenging abilities of RA, CSA, and RA-CSA nanoparticles. NO release (C) and TNF-α levels (D) in LPS-induced RAW 264.7 macrophages after treatment with RA, CSA, and RA-CSA nanoparticles.

[0023] Figure 4 Effects of RA-CSA on colon length in a mouse model of DSS-induced colitis. Colon length (A) and images (B). Daily changes in body weight in different groups (C). Histopathological examination of colon using H&E and Tunel staining (D)

[0024] Figure 5 In vivo levels of proinflammatory cytokines TNF-α (A, C) and IL-6 (B, D) in colon and serum

[0025] Figure 6 . Targeted uptake rate of RA-CSA in macrophages. . DETAILED DESCRIPTION

[0026] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0027] Material

[0028] RA was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). CSA was from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Formamide was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were from Shanghai Aladdin Biochemical Technology Co., Ltd. Ethylenediamine and formamide were from Shanghai Aladdin Biochemical Technology Co., Ltd., and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and nitric oxide (NO) kits were purchased from Bio-Tech Biotechnology.

[0029] Enzyme-linked immunosorbent assay (ELISA) kit was from ThermoFisher Scientific, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from ThermoFisher Scientific Co., Ltd (China), and DMEM was from Gibco Invitrogen (USA).

[0030] Statistical analysis

[0031] All measurements were performed at least three times, and the data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance or t-test (GraphPad Prism). The significance of the difference was indicated by *p<0.05, **p<0.01, and ***p<0.001, respectively, indicating significant, moderately significant, and highly significant.

[0032] 1. Synthesis and characterization of RA-CSA polymer prodrug

[0033] Using EDC and NHS as coupling agents, RA and CSA were coupled through amide reaction. The reaction was carried out in an ice bath for 2 hours, stored at room temperature for 1 hour, and then a mixture of ethylenediamine and formamide was added to the reaction solution and stirred for 24 hours. At the end of the reaction, the reaction solution was filtered with a 0.45 μm organic filter membrane. The filtrate (dialysis membrane MWCO 1.0 kDa) was dialyzed in acidic Milli-Q water for 2 days. After centrifugation of the dialysate (2000 rpm, 10 min), the supernatant was collected and freeze-dried to obtain RA-CSA polymer prodrug. The freeze-dried RA-CSA polymer prodrug has a high absorption wavelength in the mid-infrared absorption region (400-3500 cm -1 ) FTIR spectra were recorded using a spectrometer equipped with Nicolet (Nicolet, UK). 1 H NMR spectra were recorded on a Varian Mercury NMR spectrometer at 400 MHz and 45° C. RA-CSA polymer prodrugs were dissolved in deuterium oxide (D 2 O).

[0034] 20mL RA-CSA polymer prodrug (20mg mL -1 ) was sonicated in an ultrasonic cell disruptor for 30 minutes (200 W, 3s / 3s per cycle). Subsequently, the unreacted materials were removed and centrifuged (10000g, 15min) to obtain RA-CSA nanoparticles. The particle size and zeta potential of the nanoparticles were measured on a Malvern zeta slurry machine (Zetasizer nanoZS, Malvern, UK) using dynamic laser scattering (DLS). The morphology of the nanoparticles was observed by transmission electron microscopy.

[0035] Results: The light yellow RA-CSA polymer prodrug ( Figure 1 A). FTIR( Figure 1 B) and 1 HNMR spectroscopy ( Figure 1 C) Confirm that RA is connected to the CSA backbone. Figure 1 As shown in B, at 1030cm -1 The C-OH stretching vibration involved in the CSA main chain vibration was observed at 1245 cm -1 In addition to the peak, all these bands overlap with the band of CSA, which indicates that RA is bound in the RA-CSA sample. To further confirm the structure of the RA-CSA polymer prodrug, 1 The H NMR spectrum showed characteristic peaks of RA and CSA: the singlet at δ4.3 ppm corresponded to the H atom of CSA, δ3.5 ppm corresponded to the H atom of ethylenediamine, and δ5.3 ppm corresponded to the H atom of the phenolic hydroxyl group in RA.1 The H NMR spectrum showed a single peak at 6.25 ppm, which was attributed to the H of the carbon atom on the ester bond in RA, thus confirming that the RA-CSA polymer prodrug had been successfully synthesized.

[0036] RA-CSA polymer prodrug has amphiphilic properties and can self-assemble into nanoparticles in an aqueous environment. TEM observation of RA-CSA nanoparticles is spherical ( Figure 2 A), its hydrodynamic size is (247.3±2.99)nm, and its zeta potential is (-19.6±1.23)mV( Figure 2 B). The negative potential of the nanoparticles can effectively prevent the adsorption of plasma proteins and improve their stability in the blood. Using FBS as the experimental object, the stability of RA-CSA under simulated physiological conditions was investigated. After incubation with PBS containing 10% FBS for 12 hours, the particle size of RA-CSA increased slightly, indicating that the nanoparticles have good particle size stability ( Figure 2 C).

[0037] 2. Drug release behavior

[0038] To study the release of RA from RA-CSA nanoparticles, 2 mL of sample (dialysis membrane MWCO 1.0 kDa) was placed in a 50 mL centrifuge tube containing 10 mL of PBS (pH 7.4) and shaken at 50 rpm at 37 °C in a shaker. At pre-set time intervals, 1 mL of supernatant was taken from each tube and the same volume of fresh PBS was added. The RA released from the supernatant was quantitatively analyzed by HPLC. 2 =0.9998(Abs(330nm)=11936×[RA](μg mL -1 The release amount of RA in each sample was determined by using a calibration curve of 43887). Each experiment was repeated 3 times and the results were expressed as mean ± standard deviation (SD).

[0039]

[0040] Where Er (%) is the cumulative release of the drug, V0 is the total volume of PBS, C n is the concentration of RA at the nth sampling time, V e The volume of each sample, m RA is the total mass of RA in the nanoparticles.

[0041] Results: The in vitro release of RA from RA-CSA nanoparticles was then analyzed in PBS (pH=7.4) at 37°C. Figure 2D shows the release curves of RA-CSA nanoparticles at different time points. There was an initial rapid release phase, followed by a plateau phase, with a cumulative release of 38.33% after 24 hours. RA-CSA nanoparticles continued to slowly release RA over the next 7 days, with a final release of 43.03%. This release pattern showed an explosive release within the first 24 hours, followed by a stable sustained release phase. This dynamic release pattern is conducive to the long-term and continuous release of drugs to the site of inflammation, ensuring that the drug stays longer in the body, allowing the drug to stay longer in the colon and act longer, achieving better anti-inflammatory effects.

[0042] 3. Hemolysis Analysis

[0043] Blood was collected from mice, centrifuged to obtain red blood cells, and then diluted to 2% with saline. RA-CSA nanoparticles were prepared to 1000 μg mL -1 The sample was mixed with red blood cell solution using purified water or PBS. The mixed solution was vortexed and stored at 37°C for 1 hour. After centrifugation, the absorbance of the supernatant was detected by UV. The hemolysis rate was calculated as follows:

[0044]

[0045] Among them, A sample , A PBS and Represent the absorbance of sample, PBS and water, respectively.

[0046] Results: The hemolysis of RA-CSA nanoparticles was as follows Figure 2 As shown in E, the supernatant of the H2O group was red, with obvious hemolysis, while the supernatants of the PBS and RA-CSA groups were colorless, without hemolysis. The full wavelength scanning results showed that the supernatant of the H2O group had the strongest absorption values ​​at 540 and 579 nm, while the PBS and RA-CSA groups had no absorption values ​​at the same position. The hemolysis rate of each group was calculated as shown in Figure 2 F, RA-CSA at 1000 μg mL -1 The hemolysis rate was only 0.76% at this concentration, indicating that it has good biocompatibility in the blood.

[0047] 4. In vitro antioxidant studies

[0048] 4.1 Free radical scavenging activity

[0049] The free radical scavenging activity of free RA, CSA and RA-CSA nanoparticles was evaluated by measuring the decolorization effect of DPPH on their unpaired electrons. Briefly, 2 mL of 1,1-diphenyl-2-pyridinium hydrazine radical (DPPH) ethanol solution (0.1 mM) was added to 200 μL of the corresponding free RA, CSA or RA-CSA nanoparticles (1.0 mg mL-1). The mixture was stirred for 30 min in the dark and at room temperature. Then, the absorbance was measured at 515 nm using a Microplate Reader. The lower the absorbance of the mixture, the higher the free radical scavenging activity of the tested sample. RSA was expressed as a percentage and the results were expressed as mean ± SD (n = 3).

[0050]

[0051] Where A0 is the blank absorbance and A1 is the sample absorbance.

[0052] 4.2 Hydrogen peroxide removal capacity

[0053] The scavenging activity of hydrogen peroxide (H2O2) on RA, CSA and RA-CSA nanoparticles was evaluated by measuring the decolorization degree of titanium sulfate. Briefly, 2 mL of H2O2 solution (10 mM) was added to 200 μL of the corresponding free RA, CSA or RA-CSA nanoparticles (1.0 mg mL -1 The mixture was stirred and reacted for 10 minutes in the dark at room temperature. The supernatant was reacted with 1.3 mL of titanium sulfate. Finally, the absorbance was measured at 405 nm using a Microplate Reader.

[0054]

[0055] Where A0 is the blank absorbance and A1 is the sample absorbance.

[0056] Results: The free radical scavenging activity of antioxidants against substances such as DPPH and H2O2 is usually used to study the ability of molecules to act as free radical terminators. Therefore, this method was used to study the antioxidant capacity of free RA, CSA, and RA-CSA nanoparticles. Figure 3 As shown in A, CSA exhibited very low antioxidant capacity, while free RA and RA-CSA had strong antioxidant capacity. We then examined whether RA-CSA nanoparticles retained the ability of RA to scavenge ROS. H2O2 is one of the most common types of endogenous ROS. RA-CSA nanoparticles had a stronger ability to scavenge ROS than free RA and CSA ( Figure 3 B). These results indicate that RA-CSA has good antioxidant capacity.

[0057] 5. In vitro nitric oxide and TNF-α release assay

[0058] RAW264.7 cells were cultured at 3 × 10 5 The cells were seeded at a density of 100 cells / well in a 96-well microplate and incubated at 37°C in 5% CO2 for 24 h. Then 500 ng mL -1 LPS polarized the cells into M1 macrophages. After LPS removal, the cells were further incubated with free RA, CSA and RA-CSA nanoparticles. After 24 h, the secretion of NO and TNF-α in the culture medium was detected by ELISA.

[0059] Results: The anti-inflammatory capacity of RA-CSA nanoparticles was evaluated by NO release assay and ELISA assay in macrophage RAW264.7 cell line. LPS-induced NO and TNF-α levels were evaluated and used as positive controls. Figure 3 C shows the total amount of NO released after treatment with RA, CSA, and RA-CSA nanoparticles. Almost no NO release was observed in unstimulated cells (negative control). RA, CSA, and RA-CSA nanoparticles reduced LPS-induced NO levels. More importantly, RA-CSA nanoparticles could reduce NO production by half compared to the positive control (control + LPS) and were significantly lower than free RA and CSA. This may be because RA and CSA have a synergistic effect, producing a stronger anti-inflammatory effect. Similarly, TNF-α levels showed the same trend ( Figure 3 D).

[0060] 6. In vivo therapeutic effect of RA-CSA nanoparticles on UC

[0061] Female C57BL / 6 mice (6 weeks, 18-20 g) were obtained from Spectro (Beijing) Biotechnology Co., Ltd. All experimental animal handling and care were approved by the Experimental Animal Center of Jiangnan University. From day 0, mice were given free access to drinking water containing 3% (w / v) dextran sulfate sodium salt (DSS, MW 36000-50000, colitis grade, MP Biomedicals, Santa Ana, CA) for 7 consecutive days to induce acute colitis, and then switched to regular drinking water. The mice were monitored daily, and their body weight and diarrhea symptoms were recorded.

[0062] In each experiment, mice were divided into 6 groups (n = 8): healthy control group (normal water), PBS-treated DSS-induced colitis group, free RA, CSA, RA-CSA nanoparticles and 5-ASA (RA 20 mg kg -1 ,5-ASA 150mg kg -1) treated with DSS-induced colitis group. Mice were treated with different formulations orally for DSS, with administration every other day for 9 days. Body weight was monitored daily during the 9-day observation period. Mice were killed on the 9th day and the colon length was measured. In order to determine the levels of proinflammatory cytokines, the colon tissue was weighed and homogenized. The homogenized tissue suspension was centrifuged at 20,000 g (4°C) for 15 minutes, and the levels of TNF-α and IL-6 in mouse tissues and serum were detected using ELISA kits. In vivo toxicity evaluation was performed on mice 9 days after administration. The changes in body weight after treatment with different formulations were recorded every day, and histological evaluation was performed by H&E staining of the colon. Untreated mice served as the control group.

[0063] Because RA-CSA has good antioxidant and anti-inflammatory activities in vitro, the therapeutic effect of RA-CSA was evaluated in a mouse UC model. The change in colon length on day 9 was measured as a key indicator for evaluating the severity of DSS-induced acute colitis. Figure 4 As shown in Figures AC, PBS-treated mice exposed to DSS exhibited the shortest colon length and lowest body weight. After oral RA or CSA treatment, colon length shortening was ameliorated and body weight loss was significantly inhibited. More importantly, RA-CSA treatment significantly slowed colon length shortening (4.20 ± 0.15 cm), indicating that RA-CSA improved colon inflammation. These results were similar to those obtained with 5-ASA (positive control group).

[0064] In addition, histopathological scoring takes into account the degree of loss of structural integrity and the degree of inflammatory infiltration, which is a way to understand the extent of colonic inflammation. The colonic tissue of the control group (healthy control mice) was intact and had no inflammatory infiltration. Compared with the histology of representative colons, the PBS-treated colitis colon showed disorganized colonic architecture, irregular morphology, and strong inflammatory cell infiltration ( Figure 4 D). In contrast, mice treated with 5-ASA and RA-CSA exhibited relatively intact surface epithelium, similar to healthy control colons, indicating epithelial repair and reduced inflammation. Meanwhile, Tunel staining showed a significant increase in DSS-induced apoptosis. However, both 5-ASA and RA-CSA showed improved DSS-induced apoptosis in colon tissue. In short, RA-CSA nanoparticles can improve inflammation in DSS-induced colitis.

[0065] Inflammatory cytokines play a crucial role in the development and progression of ulcerative colitis (UC). Therefore, to evaluate the effect of RA-CSA nanoparticles on UC, their effects on the production of TNF-α and IL-6, which are well-known markers of inflammation, were investigated. The findings suggest that RA-CSA nanoparticles have the potential to effectively inhibit the progression of UC. The results showed that treatment with RA-CSA nanoparticles significantly reduced TNF-α ( Figure 5 A and 5C) and IL-6 ( Figure 5 B and 5D), similar to the effect observed in the 5-ASA group. These findings suggest that RA-CSA nanoparticles have the potential to effectively inhibit the progression of UC.

[0066] 7. Cellular Targeted Uptake Experiment

[0067] Polyethylene glycol-rosmarinic acid (PEG-RA) and RA-CSA were selected for targeted uptake studies. RAW 264.7 cells were routinely cultured in DMEM medium containing 10% fetal bovine serum. The cells were seeded in a 12-well microplate at a density of 2×10 5 cells / well, and incubated at 37°C for 24 h in a 5% CO2 atmosphere. Then, RA-CSA and PEG-RA nanoparticles labeled with FITC were added and incubated for 4 h, and the cell uptake rate was determined by flow cytometry.

[0068]

[0069] Where N S and N T represent the number of cells taking up nanoparticles and the total number of cells, respectively.

[0070] Results: The cellular uptake rate of RA-CSA nanoparticles was higher than that of PEG-RA nanoparticles, indicating that they have a higher uptake capacity into macrophages ( Figure 6 ). However, the uptake of RA-CSA nanoparticles decreased significantly after the cells were pre-treated with free CS. This is because CS occupied the CD44 receptor, which led to a decrease in the entry of nanoparticles into the cells. Therefore, RA-CSA nanoparticles can be efficiently taken up by macrophages through CD44 receptor-mediated endocytosis and exert their effects.

[0071] Conclusion: This paper developed an efficient drug delivery system based on CSA and RA natural products for the treatment of UC. RA-CSA polymer prodrug was successfully synthesized and characterized by FTIR and 1H NMR analysis confirmed that the obtained RA-CSA polymer prodrugs can self-assemble into nanoparticles under aqueous conditions. RA-CSA can effectively scavenge ROS and has strong antioxidant activity. In addition, they reduced the inflammatory activation of macrophages compared with free RA. Importantly, RA-CSA nanoparticles promoted the remission of colonic inflammation without obvious in vivo toxicity. Therefore, RA-CSA nanoparticles can be considered as a potential anti-inflammatory therapy for the further treatment of UC.

Claims

1. A rosmarinic acid chondroitin sulfate A polymer prodrug, the structural formula of which is as follows: Wherein n is an integer from 6 to 1300.

2. The method for preparing the rosmarinic acid chondroitin sulfate A polymer prodrug according to claim 1, characterized in that: The steps include: 1) adding RA, CSA, (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and n-hydroxysuccinimide in a molar ratio of 2:1:1:1:1, reacting in an ice bath for 2 h, storing at room temperature for 1 h, then adding a mixture of ethylenediamine and formamide to the reaction solution, and stirring continuously for 24 h; 2) After the reaction is completed, the reaction solution is filtered through a 0.45 μm organic filter membrane, and the filtrate is dialyzed in acidic Milli-Q water for 2 days; the dialyzate is centrifuged and the supernatant is collected and freeze-dried to obtain the RA-CSA polymer prodrug.

3. The preparation method according to claim 2, characterized in that: In step 2), the dialysis membrane has a MWCO of 1.0 kDa.

4. The preparation method according to claim 2, characterized in that: In step 2), the centrifugation condition is 2000 rpm, 10 min.

5. A rosmarinic acid chondroitin sulfate A self-assembled nanoparticle, which is formed by self-assembly of the rosmarinic acid chondroitin sulfate A polymer prodrug according to claim 1 in an aqueous phase, wherein the hydrodynamic size of the nanoparticle is 247.3±2.99 nm and the zeta potential is -19.6±1.23 mV.

6. A pharmaceutical composition comprising the rosmarinic acid chondroitin sulfate A polymer prodrug according to claim 1 or the rosmarinic acid chondroitin sulfate A self-assembled nanoparticles according to claim 5 and a pharmaceutically acceptable carrier or excipient.

7. Use of the rosmarinic acid chondroitin sulfate A polymer prodrug according to claim 1 or the rosmarinic acid chondroitin sulfate A self-assembled nanoparticles according to claim 5 in the preparation of a drug for treating or preventing colon inflammation.

8. Use of the rosmarinic acid chondroitin sulfate A polymer prodrug according to claim 1 or the rosmarinic acid chondroitin sulfate A self-assembled nanoparticles according to claim 5 in the preparation of an injection, oral administration or local administration system.

Images