An oral nano-drug delivery system for treating ulcerative colitis

Abstract

The application 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 application can effectively prevent adsorption of plasma proteins and improve the stability of the RA-CSA nanoparticles in blood. In a hemolysis test, the RA-CSA nanoparticles have good biocompatibility in blood. The drug release mode of the RA-CSA nanoparticles shows an explosive release within the first 24 hours and then enters a stable slow-release stage. This dynamic release mode is beneficial to long-term sustained release of the drug to an inflammation site, ensures a longer in-vivo residence time of the drug, allows the drug to stay in the colon for a longer time, has a longer action time, and achieves a better anti-inflammatory effect. Meanwhile, the RA-CSA nanoparticles can effectively eliminate ROS and have strong antioxidant activity. In addition, compared with free RA, the RA-CSA nanoparticles significantly reduce the inflammatory activation of macrophages, effectively promote the relief of colonic inflammation, and have no obvious in-vivo toxicity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical preparations, and particularly relates to an oral nano-drug delivery system for treating ulcerative colitis. BACKGROUND

[0002] Ulcerative colitis (UC) is a kind of chronic inflammatory bowel disease (IBD), which is a chronic inflammatory disease affecting the rectum and colon to varying degrees. In 2023, the global prevalence of ulcerative colitis was estimated to be 5 million cases, and it continues to grow globally. UC is a complex, multifactorial chronic disease, and the main pathological features are inflammatory cell infiltration and gland destruction. The pathogenesis of UC is related to the changes in the colon environment of genetically susceptible individuals, which has a huge impact on the life of patients and can lead to chronic, lifelong, and recurrent complications, making treatment very difficult. In clinical practice, 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 antitumor necrosis factor (TNF), vedolizumab (anti-α4β7 integrin), ustekinumab), and small molecules (such as Janus kinase inhibitors, sphingosine-1 receptor modulators). However, due to the lack of targeting, inevitable systemic side effects, high recurrence rate, and strong drug resistance, the efficacy of these drugs is limited. Therefore, there is an urgent need for an effective treatment for UC.

[0003] The active ingredients of natural medicines have the characteristics of multi-target, high efficacy and less 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, which is one of the most important polyphenolic antioxidants. RA has a wide range of biological effects, including anti-inflammatory, anticancer, anti-ischemic and antibacterial activity. Data research shows that RA can block the production of PGE2 and TNF-a induced by LPS. In addition, rosemary extract can also inhibit the phosphorylation of MAPKs, block the activation of NF-kB, and lead to the decrease of iNOS and COX-2 expression, and has anti-inflammatory effect. Despite such high potential, RA has encountered the same problems as other small-molecule bioactive compounds, such as poor water solubility, high instability, poor colon targeting ability, and poor bioavailability, which reduces its therapeutic effect. Recently, researchers have developed RA-loaded delivery systems to improve stability and bioavailability, and used them for biomedical applications, WO2021206428A1 discloses a PEG-modified RA to improve the water solubility of RA.

[0004] There are few reports on chondroitin sulfate-derived RA-derived polymer prodrugs and achieving 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 belonging to the glycosaminoglycan (GAGs) family. The CS chain is composed of repeating disaccharide units of N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA), and is a component of the extracellular matrix in connective tissue. Due to its good biodegradability, high biocompatibility and chemical coupling, CS is widely used in many therapeutic methods. Previous studies have shown that CS can reduce the production of pro-inflammatory cytokines (phospholipase A2, cyclooxygenase 2, nitric oxide synthase-2), reduce C-reactive protein concentration, inhibit phagocytosis and intracellular oxidative burst of neutrophils. In addition, CS also shows significant binding affinity to transmembrane glycoprotein CD44. The CD44 receptor is overexpressed on the surface of macrophages in inflammatory colon and various malignant tumor cells, and the CD44 receptor in the lesion of many UC patients has the characteristics of high expression, 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, a new rosmarinic acid chondroitin sulfate A (RA-CSA) nanoparticle oral drug delivery system is developed for the treatment of UC. This strategy not only effectively improves the stability and bioavailability of RA, but also enhances the targeted drug accumulation at the UC lesion site. SUMMARY

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

[0006] The technical scheme of the present application 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 of 6-1300.

[0009] The present application provides a preparation method of rosmarinic acid chondroitin sulfate A polymer prodrug, characterized in that it comprises the following steps:

[0010]

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

[0012] 2) after the reaction is completed, filtering the reaction solution with a 0.45μm organic filter membrane; dialyzing the filtrate in acidic Milli-Q water for 2 days; centrifuging the dialysate to collect the supernatant, freezing and lyophilizing to obtain the RA-CSA polymer prodrug.

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

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

[0015] A rosmarinic acid chondroitin sulfate A self-assembled nanoparticle, which is self-assembled from the rosmarinic acid chondroitin sulfate A polymer prodrug in an aqueous phase, has a hydrodynamic size of 247.3±2.99nm and a zeta potential of -19.6±1.23mV.

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

[0017] The rosmarinic acid chondroitin sulfate A polymer prodrug or rosmarinic acid chondroitin sulfate A self-assembled nanoparticle provided by the present application is used for preparing a drug for treating or preventing colon inflammation.

[0018] The rosmarinic acid chondroitin sulfate A polymer prodrug or rosmarinic acid chondroitin sulfate A self-assembled nanoparticle provided by the present application is used for preparing an injection, oral administration or topical administration system.

[0019] The present application provides rosmarinic acid chondroitin sulfate A nanoparticles (RA-CSA nanoparticles), the negative potential of the RA-CSA nanoparticles can effectively prevent the adsorption of plasma proteins, and improve the stability thereof in blood. In a hemolysis test, the RA-CSA nanoparticles have good biocompatibility in blood. The drug release mode of the RA-CSA nanoparticles shows an explosive release within the first 24 hours, and then enters a stable slow-release stage, and such a dynamic release mode is beneficial to the long-term sustained release of the drug to an inflammation site, ensures a longer in-vivo residence time of the drug, makes the drug stay in the colon for a longer time, has a longer action time, and achieves a better anti-inflammatory effect. Meanwhile, the RA-CSA nanoparticles can effectively scavenge ROS and have strong antioxidant activity. In addition, compared with free RA, the RA-CSA nanoparticles significantly reduce the inflammatory activation of macrophages, effectively promote the relief of colonic inflammation, and have no obvious in-vivo toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Synthetic 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 the RA-CSA nanoparticles. Release curve (D) of the RA-CSA nanoparticles in PBS at 37℃. Ultraviolet full-wavelength scanning (E) and hemolysis rate (F) of erythrocytes in different groups

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

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

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

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

[0026] The following examples can enable those skilled in the art to more completely understand the application, but are not intended to limit the application in any way.

[0027] Materials

[0028] RA was purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). CSA was from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Formamide was purchased from National Medicine Reagent Co., Ltd. (Shanghai, China). Carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) were from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Ethylenediamine and formamide were from Shanghai Aladdin Bio-Chem Technology Co., Ltd. 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and nitric oxide (NO) kits were purchased from Bioteke Biotech Co., Ltd.

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

[0030] Statistical analysis

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

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

[0033] RA and CSA were coupled via amide reaction using EDC and NHS as coupling agents. 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 continuously 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 ) were sonicated in an ultrasonic cell disruptor for 30 minutes (200 W, 3 s / 3 s per cycle). Unreacted material was subsequently removed and the mixture was concentrated by centrifugation (10,000 g, 15 min) to yield RA-CSA nanoparticles. Dynamic laser scattering (DLS) was used to measure the particle size and zeta potential of the nanoparticles on a Malvern Zeta slurry analyzer (Zetasizer nanoZS, Malvern, UK). Transmission electron microscopy was used to observe the morphology of the nanoparticles.

[0035] Results: The light yellow RA-CSA polymer prodrug ( Figure 1 A). FTIR( Figure 1 B) and 1 HNMR spectrum ( Figure 1 C) Confirm that RA is attached 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 All of these bands overlap with those of CSA, indicating that RA is bound to 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 HNMR spectrum showed a single peak at 6.25 ppm, which was attributed to H of carbon atom on ester bond in RA, thus it could be confirmed that RA-CSA polymer prodrug had been successfully synthesized.

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

[0037] 2. Drug release behavior

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

[0039]

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

[0041] Results: RA-CSA nanoparticles were then analyzed for in vitro release of RA in PBS (pH = 7.4) at 37°C. Figure 2 ​​​D shows the release curve of RA-CSA nanoparticles at different time points. There is a rapid release phase at the beginning, followed by a platform period, and the cumulative release amount is 38.33% after 24 hours. RA-CSA nanoparticles continue to slowly release RA in the next 7 days, and the final release amount reaches 43.03%. This release mode shows an explosive release in the first 24 hours, followed by a stable slow-release phase. This dynamic release mode is beneficial for the long-term sustained release of drugs to the inflammation site, ensuring longer drug residence time in the body, longer drug residence time in the colon, longer action time, and better anti-inflammatory effect.

[0042] 3. Hemolysis analysis

[0043] The mouse blood was taken, centrifuged to obtain red blood cells, and then diluted with physiological saline to 2%. RA-CSA nanoparticles were prepared into 1000 μg mL -1 samples, mixed with pure water or PBS and red blood cell solution. After vortexing, the mixed solution was stored at 37°C for 1 h, centrifuged, and the supernatant absorbance was detected by UV. The hemolysis rate was calculated according to the following formula:

[0044]

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

[0046] Results: The hemolysis of RA-CSA nanoparticles is shown in Figure 2 E, the supernatant of the H2O group is red, and there is obvious hemolysis phenomenon, while the supernatant of the PBS group and the RA-CSA group is colorless and no hemolysis occurs. The full wavelength scanning result shows that the supernatant of the H2O group has the strongest absorption value at 540 and 579 nm, while the PBS group and the RA-CSA group have no absorption value at the same position. The hemolysis rate of each group is calculated as shown in Figure 2 F, the hemolysis rate of RA-CSA at a concentration of 1000 μg mL -1 is only 0.76%, indicating that it has good biocompatibility in blood.

[0047] 4. In vitro antioxidant study

[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 bleaching effect of DPPH on its unpaired electron. Briefly, 2 mL of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) ethanol solution (0.1 mM) was added to 200 μL of corresponding free RA, CSA or RA-CSA nanoparticles (1.0 mg mL"1). The mixture was stirred for 30 min in the dark at room temperature. Then, the absorbance was measured at 515 nm with a Microplate Reader. The smaller the absorbance of the mixture, the higher the free radical scavenging activity of the sample tested. RSA was expressed as a percentage and the results were expressed as mean ± SD (n = 3).

[0050]

[0051] where A0is the blank absorbance and A1is the sample absorbance.

[0052] 4.2 Hydrogen peroxide scavenging ability

[0053] The scavenging activity of hydrogen peroxide (H2O2) on RA, CSA and RA-CSA nanoparticles was evaluated by measuring the bleaching degree of titanium sulfate. Briefly, 2 mL of H2O2solution (10 mM) was added to 200 μL of corresponding free RA, CSA or RA-CSA nanoparticles (1.0 mg mL"1). The mixture was stirred for 10 min at room temperature in the dark. The supernatant was reacted with 1.3 mL of titanium sulfate. Finally, the absorbance was measured at 405 nm with a Microplate Reader. -1

[0054]

[0055] where A0is the blank absorbance and A1is the sample absorbance.

[0056] Results: The free radical scavenging activity of antioxidants against DPPH and H2O2, among others, is commonly used to study the ability of molecules as free radical terminators. Thus, the antioxidant capacity of free RA, CSA and RA-CSA nanoparticles was studied using this method. As shown in Figure A, CSA exhibited very low antioxidant capacity, while free RA and RA-CSA had a strong antioxidant capacity. It was then checked whether the RA-CSA nanoparticles retained the ability of RA to scavenge ROS. H2O2is one of the most common types of endogenous ROS. Compared to free RA and CSA, RA-CSA nanoparticles had a stronger ability to scavenge ROS (Figure B). These results indicate that RA-CSA has a good antioxidant capacity. Figure 3 Figure 3

[0057] 5. Nitric oxide and TNF-α release test in vitro​​​

[0058] RAW264.7 cells were seeded at a density of 3 x 105cells / well in 96-well microplates and incubated at 37 °C in 5% CO2for 24 h. Then, 500 ng mL-1LPS was added to induce Ml macrophages. After removing LPS, the cells were further incubated with free RA, CSA and RA-CSA NPs. After 24 h, the secretion of NO and TNF-a in the culture medium was detected by ELISA. 5 -1 LPS polarized the cells to Ml type macrophages. After removing LPS, the cells were further incubated with free RA, CSA and RA-CSA NPs. After 24 h, the secretion of NO and TNF-a in the culture medium was detected by ELISA.

[0059] Results: The anti-inflammatory ability of RA-CSA NPs was evaluated by NO release assay and ELISA assay in the macrophage RAW 264.7 cell line. The LPS-induced levels of NO and TNF-a were evaluated and used as a positive control. Figure 3 C shows the total amount of NO released after treatment with RA, CSA and RA-CSA NPs. Almost no NO release was observed in unstimulated cells (negative control). RA, CSA and RA-CSA NPs reduced the LPS-induced NO level. More importantly, RA-CSA NPs can reduce the NO production by half compared to the positive control (control + LPS), and are significantly lower than free RA and CSA. This can be because RA and CSA have a synergistic effect, producing a stronger anti-inflammatory effect. Similarly, the TNF-a level also shows the same trend Figure 3 D).

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

[0061] Female C57BL / 6 mice (6 weeks, 18-20 g) were from Beijing Sipper-Bio Technology Co., Ltd. The handling and care of all experimental animals were reviewed and 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 changed to regular drinking water. The mice were monitored daily, and their body weight and diarrhea symptoms were recorded.

[0062] The mice were divided into 6 groups (n = 8) for each experiment: healthy control group (normal water), PBS-treated dss-induced colitis group, free RA, CSA, RA-CSA NPs and 5-ASA (RA 20 mg kg-1, 5-ASA 150 mg kg-1, 5-ASA 300 mg kg-1, RA-CSA NPs 20 mg kg-1) treated dss-induced colitis group. The mice were given free access to drinking water containing 3% (w / v) DSS for 7 consecutive days to induce acute colitis, and then changed to regular drinking water. -1 , 5-ASA 150 mg kg-1, 5-ASA 300 mg kg-1, RA-CSA NPs 20 mg kg-1) treated dss-induced colitis group. The mice were given free access to drinking water containing 3% (w / v) DSS for 7 consecutive days to induce acute colitis, and then changed to regular drinking water. -1 ​) Treatment of DSS-induced colitis group. Mice were treated with different formulations orally with DSS, with one day interval for 9 days. Body weight was monitored daily for 9 days of observation period. Mice were sacrificed on day 9 and colon length was measured. To determine the pro-inflammatory cytokine levels, colon tissues were weighed and homogenized. Homogenized tissue suspension was centrifuged at 20000g (4°C) for 15 min and the levels of TNF-a and IL-6 in mouse tissues and serum were measured using ELISA kits. In vivo toxicity evaluation was performed in mice after 9 days of treatment. Body weight changes after treatment with different formulations were recorded daily and colon H&E staining was performed for histological evaluation. Untreated mice were used as control group.

[0063] Since RA-CSA showed good antioxidant and anti-inflammatory activities in vitro, the therapeutic effect of RA-CSA in a mouse UC model was evaluated. The colon length change on day 9 was measured as a key indicator to assess the severity of DSS-induced acute colitis. As shown in Figure 4 A-C, PBS-treated mice exposed to DSS showed the shortest colon length and the lowest body weight. After oral treatment with RA or CSA, the colon length shortening was improved and the body weight loss was significantly inhibited. More importantly, RA-CSA treatment significantly slowed down the colon length shortening (4.20 ± 0.15 cm), indicating that RA-CSA improved the colonic 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 degree of colonic inflammation. The colon tissue of the control group (healthy control mice) was structurally intact without inflammatory infiltration. In comparison with the histology of a representative colon, PBS-treated colitic colon showed disorganized colon structure, irregular morphology, and strong inflammatory cell infiltration Figure 4 D). In contrast, mice treated with 5-ASA and RA-CSA showed a relatively intact surface epithelium, similar to the healthy control colon, indicating epithelial repair and reduced inflammation. At the same time, Tunel staining showed a significant increase in DSS-induced apoptosis. However, both 5-ASA and RA-CSA showed an improvement in DSS-induced colon tissue apoptosis. 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 assess the effect of RA-CSA nanoparticles on UC, the effect of RA-CSA nanoparticles on the production of TNF-alpha and IL-6, which are well known as markers of inflammation, was studied. Findings indicate that RA-CSA nanoparticles have the potential to effectively inhibit the progression of UC. The results show that treatment with RA-CSA nanoparticles significantly reduced the production of TNF-alpha Figure 5 (A and 5C) and IL-6 Figure 5 (B and 5D) in DSS-induced colitis colon and serum, similar to the effect observed in the 5-ASA group. These findings indicate that RA-CSA nanoparticles have the potential to effectively inhibit the progression of UC.

[0066] 7. Cell Targeted Uptake Experiments

[0067] Polyethylene glycol-rosemary acid (PEG-RA) was selected for targeted uptake studies with RA-CSA. RAW 264.7 were routinely cultured in DMEM medium containing 10% fetal bovine serum. Cells were seeded in 12-well microplates at a density of 2 x 10 5 cells / well and incubated at 37°C in a 5% CO2 gas environment for 24 h. Then, FITC fluorescently labeled RA-CSA and PEG-RA nanoparticles 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 that have taken up nanoparticles and the total number of cells, respectively.

[0070] Results: The cell uptake rate of RA-CSA nanoparticles was higher than that of PEG-RA nanoparticles, indicating that they have a higher uptake ability into macrophages Figure 6 . After pre-treatment of cells with free CS, the uptake of RA-CSA nanoparticles was significantly reduced, as the occupation of the CD44 receptor by CS led to a decrease in nanoparticle uptake. Therefore, RA-CSA nanoparticles can be efficiently taken up by macrophages through CD44 receptor-mediated endocytosis to exert their effects.

[0071] Conclusion: The present invention developed a highly efficient drug delivery system based on CSA and RA natural products for the treatment of UC. RA-CSA polymeric prodrugs were successfully synthesized, and their structures were confirmed by FTIR and 1H NMR analysis was confirmed. The resulting RA-CSA prodrugs can self-assemble into nanoparticles under aqueous conditions. RA-CSA can effectively scavenge ROS and have strong antioxidant activity. In addition, they reduce the inflammatory activation of macrophages compared with free RA. Importantly, RA-CSA nanoparticles promote colonic inflammation relief without obvious in vivo toxicity. Therefore, RA-CSA nanoparticles can be considered as a potential anti-inflammatory therapy for further treatment of UC.

Claims

1. A rosmarinic acid-chondroitin sulfate A polymer prodrug, having the following structure: wherein n is an integer from 6 to 1300.

2. A method for preparing the rosmarinic acid-chondroitin sulfate A polymer prodrug of claim 1, comprising the following steps:

2. A process for the preparation of the rosmarinic acid chondroitin sulfate A polymer prodrug of claim 1, characterized in that, 1) adding rosmarinic acid, chondroitin sulfate A, hydrochloric acid (1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and n-hydroxysuccinimide in a molar ratio of 2:1:1:1, reacting for 2 h in an ice bath, storing for 1 h at room temperature, then adding a mixture of ethylenediamine and formamide to the reaction solution, and continuously stirring for 24 h; 2) after the reaction is completed, filtering the reaction solution with a 0.45 pm organic filter membrane, dialyzing the filtrate in acidic Milli-Q water for 2 days; centrifuging the dialyzed solution, collecting the supernatant, freezing and lyophilizing to obtain the rosmarinic acid-chondroitin sulfate A polymer prodrug. In step 2), the dialysis membrane has a MWCO of 1.0 kDa.

3. The preparation method according to claim 2, characterized in that In step 2), the centrifugation conditions are 2000 rpm for 10 min.

4. The production method according to claim 2, characterized by, 5. A rosmarinic acid-chondroitin sulfate A self-assembled nanoparticle formed by self-assembly of the rosmarinic acid-chondroitin sulfate A polymer prodrug of claim 1 in an aqueous phase, having a hydrodynamic size of 247.3 ± 2.99 nm and a zeta potential of -19.6 ± 1.23 mV.

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

7. Use of the rosmarinic acid-chondroitin sulfate A polymer prodrug of claim 1 or the rosmarinic acid-chondroitin sulfate A self-assembled nanoparticle of claim 5 in the preparation of a medicament for treating or preventing colonic inflammation.

8. Use of the rosmarinic acid-chondroitin sulfate A polymer prodrug of claim 1 or the rosmarinic acid-chondroitin sulfate A self-assembled nanoparticle of claim 5 in the preparation of an injection, oral administration or topical administration system. ​

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