pH and redox dual response nano suppository for the treatment of ulcerative colitis

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Introduction
Ulcerative colitis (UC), one of the two main forms of inflammatory bowel disease (IBD), was first reported in 1859 1 .UC is characterized by mucosal in flammation that begins in the rectum and continues to spread to the proximal colon, the most common symptom is hemorrhagic diarrhea 2,3 .In recent years, t he incidence and prevalence of UC has increased worldwide, particularly in de veloping countries experiencing rapid industrialisation. 4.
The unique environment of the colon (pH, enzyme, redox potential, P-glyc oprotein efflux pump and microbiota, etc.) leads to multiple obstacles in the cu rrent delivery methods.Traditional carriers often release drugs prematurely, resu lting in UC therapeutics such as 5-ASA failing to achieve the desired therapeut ic effect. 5.For example, 5-ASA suppository, as a first-line clinical drug for the treatment of patients with mild to moderate ulcerative colitis (UC), can act on the lesion site through rectal administration, and have more advantages in the positioning treatment of local lesions than oral preparations 6 .However, the curr ent commercially available 5-ASA suppositories are mostly administered 3 time s/day, and the patient 's medication compliance is low.According to the physio logical conditions of the colon, researchers have developed a variety of new dr ug delivery systems including pH-responsive 7,8 and redox-responsive 9,10 systems using various functional biomaterials, such as polymers, lipids and inorganic m aterials 11,12 , to prolong the residence time of drugs at the site of inflammation and reduce the frequency of administration.
Hollow Mesoporous Silica Nanoparticles (HMSN), as an inorganic nanomat erial, are widely used in chemical catalysis, thermal insulation, functional coati ngs, and biomedical fields 13 .In brief, HMSN has both hollow and mesoporous advantages, with unique properties such as large specific surface area, easy fun ctionalization, high bioavailability and low toxicity (even non-toxic), which mak es it an ideal drug carrie 14 .Compared with traditional mesoporous silica, the or ganic groups in HMSN are uniformly and covalently bound in the pores at the molecular level, giving them multiple organic-inorganic hybrid frameworks, an d thus better biocompatibility, stability and higher drug loading [15][16][17] .As a redox-responsive group, disulfide bond is widely used in the constru ction of redox-responsive drug delivery systems because it can be reduced to s ulfhydryl (-SH) in a specific redox environment and trigger the drug release of the carrier 18 .The redox potential in the colon (-415 mV) is much lower than the standard reduction potential of the disulfide bond (-250 mV), so the disulfi de bond can be reduced and cracked in the colon 19 .Currently, researchers have developed a variety of novel nano-drug delivery systems based on disulfide re dox responsiveness for colon-specific drug delivery 20,21 .Polydopamine (PDA) and chitosan (CS) are both polymer biomaterials.Th ey have good biosafety and do not produce toxicity after entering the organism, and can be biodegraded into harmless metabolites.PDA also has good pH res ponsiveness, and its ability to construct pH-responsive nano-drug delivery syste ms has been demonstrated in many studies [22][23][24] .Since the pH-responsive charac teristics of PDA are in line with the low pH physiological environment caused by electrolyte imbalance, lactic acid production, microbial flora disorder and ab normal enzyme activity in the colorectal and rectal parts of UC patients, resear chers began to apply it to the development of new UC therapeutic drug delive ry systems 25 .CS has strong biological adhesion, which can prolong the retentio n time of drugs on the surface of intestinal mucosa 26 .At the same time, CS is easy to be chemically modified due to its rich hydroxyl and amino groups.A variety of new nano-drug delivery systems modified by CS have been widely used for UC drug delivery 27,28 .
In a word, this topic chooses the low pH and low redox potential environ ment of the colon of UC patients as the reaction conditions to construct a dua l-response suppository type nano drug delivery system that can be administered rectally and has strong mucosal adhesion, so that it can specifically aggregate i n the inflammatory colonic mucosa and achieve drug retention and slow releas e in the colon.
As shown in Scheme 1, 5-ASA was loaded into the pores of HMSN-SS, and then PDA and CS were modified on the surface of hollow mesoporous sili ca.Finally, the nano-suppository was prepared by using mixed fatty acid glycer ide as the matrix, which was named 5-ASA@HMSN-SS-PDA CS suppository (5-ASA@HSPC).The 5-ASA@HSPC nano drug delivery system adhered to the intestinal tract after rectal administration.In the intestinal environment of UC with low pH and low redox potential, PDA was protonated and dissociated fro m the surface of HMSN-SS, and at the same time the disulfide bond was bro ken, resulting in the slow release of encapsulated 5-ASA from the pores of H MSN-SS.In vivo imaging and in vivo pharmacokinetic experiments have show n that the HSPC suppository can complete the sustained release of 5-ASA.Su bsequent pharmacodynamic studies have shown that the HSPC suppository has good safety and the same therapeutic effect as the 5-ASA suppository group (3 times/day), showing great potential in the treatment of ulcerative colitis.DSS (molecular weight 36,000-50,000 kDa) was purchased from Dalian Meilun Biological T echnology Co.,Ltd.N-cetyltrimethylammonium bromide (CTAB), carbonate (Na2CO3, anhydrous, granular, ≥ 99.5%), hydrochloric acid (HCl, 36-38%), NaH2PO4•12H2O (99%), Na2HPO4•2H2O (99%), absolute ethanol, dimethyl sulfoxide (DMSO) were purchased from Tianjin Komiou Chemical Reagent Co., Ltd.(Tianjin, China).Dulbecco's modified eagle medium (DMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), trypsin, 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium-bromide (MTT) were purchased from Beijing Solarbio Technology Co., Ltd.(Solarbio, Beijing, China).Myeloperoxidase assay kit and Fecal occult blood test kit were purchased from Nanjing Jiancheng Bioengineering Institute.

Synthesis of HMSN-SS
HMSN carrier was prepared by selective etching method.75ml ethanol, 10ml water, and 1.6 mL NH3•H2O were mixed evenly, 2ml TEOS was added dropwise under stirring conditions, reacted at 30 °C for 2h, and the obtained solution was centrifuged for 10min to obtain solid silica nanoparticles.The precipitate was ultrasonically treated in 20ml of water to disperse evenly and 46.8ml of water, 7ml of ethanol, 0.056ml of triethylamine, and 0.116g of CTAB were added.The mixture of 188μl TEOS and 80μl bis [ 3-(triethoxysilyl) propyl] tetrasulfide was added dropwise after stirring in a water bath at 80 °C for 50 min.The reaction was continued for 4h and centrifuged to obtain nanoparticles with a solid silica core and mesoporous silica shell.
It was re-dispersed in 40ml water, added 3.18g Na2CO3, stirred in 50 °C water bath for 10h, and centrifuged to collect the precipitate.The precipitate was re-dispersed in 60ml of methanol, added to 2ml of concentrated hydrochloric acid, and stirred at room temperature for 12h.Samples were collected by centrifugation, washed with 100 mL of absolute ethanol and 10 mL of HCl, centrifuged 3 times, and dried to obtain HMSN.

Synthesis of HMSN-SS-PDA
100 mg HMSN-SS was placed in a 250 mL round bottom flask, 100 mL Tris-HCL (10 mM, pH = 8.5) buffer was added, stirred at 600 rpm, and 50 mg dopamine hydrochloride was added.The speed was increased to 900 rpm, and the reaction was carried out at room temperature for 24 h.Finally, the precipitations followed by extensive washing with deionized water, and lyophilized to obtain HMSN-SS-PDA.

Synthesis of HMSN-SS-PDA-CS
200 mg of HMSN-SS-PDA was dispersed in Tris buffer solution (10 mM, pH = 8.5). 100 mg of chitosan was then added into the solution and stirred for 24 h at room temperature.These as-synthesized HMSN-SS-PDA-CS (HSPC)nanocomposites were collected and stored in vials for further characterization and experiments.

Preparation of 5-ASA@HMSN-SS-PDA-CS suppository
According to the dosage, the required 5-ASA@HSPC freeze-dried powder was weighed, and the appropriate amount of mixed fatty acid glycerides was melted in a water bath at 40 °C, added, so that the matrix and 5-ASA@HSPC nanoparticles were mixed evenly, and the medium speed was filled into the bullet-shaped plug mold, cooled at 4 °C for 15 min, and the excess part was removed.The 5-ASA@HSPC suppository nano-drug delivery system was obtained by opening the mold.

Characterizations
The morphologies of HMSN-SS and HMSN-SS-PDA-CS were observed by a transmission electron microscope (JEM-F200, JEOL).Data on particle sizes and zetapotentials were obtained by a Zetasizer Nano ZS90 analyzer (Malvern, UK).Fourier transform infrared (FT-IR) spectra of all the samples were recorded on a Nicolet iS10 spectrometer (Thermo Electron, USA).Raman spectroscopy was performed on a RM5 Raman spectrometer using an argon ion laser at an excitation wavelength of 532 nm(Edinburgh Instruments, UK).Specific surface area, pore volume and pore size of all the samples were determined by nitrogen gas adsorption/desorption isotherm calculated through the Brunauer-Emmett-Teller (BET) method and Barrett-Joyner-Halenda (BJH) approach (ASAP 2020M+C, Micromeritics Instrument Co., USA).Thermogravimetric analysis (TGA) was performed from 25 °C to 600 °C at a heating rate of 10 °C/min under N2 atmosphere with a DTG-60AH (Shimadzu, JPN).

Investigation of redox responsiveness of HMSN-SS nanoparticles
The particle size distribution of HMSN-SS nanoparticles was determined b y dynamic light scattering method to investigate their redox sensitivity 29 .Appro priate amounts of HMSN-SS were weighed and sonicated and dispersed in PB S buffer containing 10 mmol/L and 10 µmol/L GSH, respectively, while approp riate amounts of HMSN without disulfide bonds were sonicated and dispersed i n the same medium as a control group.The experimental and control groups were incubated in a constant temperature oscillator at 37°C and 100 rpm, and 1 mL of samples were taken at 0 h and 24 h to measure the particle size dist ribution of the nanoparticles in a laser particle sizer, respectively.

5-ASA loading
200 mg of HMSN-SS and 200 mg of 5-ASA were dispersed in 100 mL of PBS (pH 7.4).The mixture was stirred vigorously at room temperature for 24 h.After the reaction was stopped, the supernatant was collected, washed wi th PBS several times, and freeze-dried to obtain 5-ASA@HMSN-SS.PDA and CS were modified to their surfaces to obtain 5-ASA@HMSN-SS-PDA-CS (5-A SA@HSPC).The content of 5-ASA encapsulated in the carriers was determined by UV-vis spectrometer.The loading capacity (LC) was measured through a UV-vis spectrometer (UV-2600, Shimadzu, JPN) at 330 nm.The LC was calcul ated by the following equations:

In vitro drug release studies
In different environments, the in vitro release test of the drug was carried out, the content of 5-ASA was measured by UV, the cumulative release rate ( CRP ) was calculated and the cumulative release curve was drawn.
The cumulative drug release rate Ve-displacement volume of release solution; V0-volume of release liquid in release system Ci-drug concentration in release solution during the i-th displacement sa mpling

Study on pH-responsive drug release ability
To study the in vitro release properties, 10 mg of 5-ASA@HSPC was dis persed in 5 mL of the buffer; and 1 mL of the dispersion was transferred into a dialysis bag whose molecular weight cut-off is 14000 Da.Then, the dialysis bag was placed in 30 mL of three different pH buffer solutions (pH=5.5, 6.8, and 7.4) in a shaking table at 37 °C.In the time interval required, 3 mL of the solution was collected and 3 mL of fresh buffer was added.All samples w ere measured by a UV-vis spectroscopy at the wavelength of 330 nm.

Study on redox-responsive drug release ability
Studies have shown that 10 mmol/L GSH concentration can simulate the r edox potential environment of the colon, so dialysis is often used to simulate t he redox release behavior of the drug delivery system at this concentration 30 .20 mg of 5-ASA@HMSN-PDA-CS (5-ASA@HPC) and 20 mg of 5-ASA@HSPC were accurately weighed and dispersed in 10 mL of pure water to form a solu tion of 2 mg/mL.1 mL solution was transferred to a dialysis bag with a molec ular weight cut-off of 14000 Da.The dialysis bags were placed in 30 mL of t hree PBS simulation media (pH 5.5), in which the GSH content of 5-ASA@H PC was 10 mmol/L, and the GSH content of 5-ASA@HSPC was 10 mmol/L, 10 µmol/L, and the release experiment was carried out in a constant temperatu re oscillator at 37 °C and 120 rpm.

In vitro cytotoxicity assay
The cytotoxicity of nanoparticles was evaluated using the MTT assay.HL-7702 cells were seeded respectively at a density of 8000 cells per well in 96-well plates and cultured for 24 h.Then the media were replaced with fresh media containing the free drug and the different formulation of AHPA.After incubation for 48 h, the media were replaced with fresh media.Next, 10 µL of the MTT reagent (5 mg/mL in PBS) was added and incubated for another 4 h at 37 °C.Then, the formed formazan crystals were dissolved in 100 µL of DMSO and the absorbance was measured using a microplate reader (CLARIOstar, BD, Germany) at 570 nm.The cell viability was calculated using the optical density (OD) with this formula: Cytotoxicities of blank and HSPC were also determined on HL-7702 cells to evaluate the biosafety of the nanoparticles.

In vivo biodistribution and pharmacokinetic
Mice were randomly divided into four groups, three in each group.Cy7 fluorescent dye ( Cy7 dose of 4 mg / kg ) was prepared into Cy7@ordinary suppository, Cy7@HMSN-SS suppository, Cy7@HMSN-PDA-CS suppository, Cy7@HMSN-SS-PDA-CS suppository.The fine silicone tube was fully lubricated with liquid paraffin.The suppositories in each group were plugged into the anus 2 cm away from the anal orifice of the mice, and the anus was blocked with a self-made rubber plug.After 20 min, the suppositories were removed.After 2,4,8,12 and 24 h of administration, the mice were gas anesthetized with isoflurane for small animal fluorescence imaging.

Scheme 2 C57BL/6 mice modeling and administration time diagram
The experimental protocol is shown in Scheme 2. The DSS model group, 5-ASA ordinary suppository group (1 time/day), 5-ASA ordinary suppository gr oup (3 times/day), 5-ASA @ HMSN-SS suppository group, 5-ASA @ HPC su ppository group and 5-ASA @ HSPC suppository group were given drinking w ater containing 3 % DSS, and the normal control group was given sterile drin king water without DSS for 10 days.Except for the DSS model group, the ot her groups were given rectal administration from the third day of drinking wat er containing 3 % DSS for 7 days.All mice were sacrificed 10 days later and the colon was dissected.The body weight, fecal traits and fecal occult blood of mice were observe d daily to evaluate the disease progression of UC.At the same time, the disea se activity index (DAI) was scored.On day 10, animals were sacrificed and th eir colon lengths measured.A small section of colon was used for myeloperoxi dase (MPO) analysis.The remainder of the colon was used for histopathologic al analysis.

Statistical Analysis
Statistical significance in difference was analysed using student's T-Test: * p < 0.05, **p < 0.01 and ***p < 0.001.All data were exhibited via mean ± SD. Figure 1 showed the transmission electron microscope (TEM), the Zeta pot ential diagrams and particle size distribution of the prepared samples.It can be seen from the TEM diagram (Fig. 1a) that HMSN-SS was spherical nanoparti cles with regular morphology，Clear hollow structure, good dispersion and narr ow particle size distribution.When the HMSN-SS were modified with PDA an d CS, the cladding layer is clearly visible (Fig. 1b).The average particle sizes of HMSN-SS, HMSN-SS-PDA and HMSN-SS-PDA-CS (Fig. 1d.e.f) were ~180.4 nm, ~200.1 nm, and ~220.9 nm, respectively.When the nanoparticles were modified with PDA and CS, the particle size obviously increased, indicating su ccessful grafting of functionalized groups.The Zeta potentials of HMSN-SS (Fi g. 1c) was -19.8 eV because of hydroxyl group on the surface.After the surfa ce was modified with PDA, the potential value decreased to -26.5 mV, which was attributed to the deprotonation of the phenolic hydroxyl group of PDA at neutral pH.CS contains a large number of free amino groups, so it is positive ly charged.When HSP is modified by CS, some amino groups are consumed, and the Zeta potential of HSPC increases to -15.0 mV.The change of Zeta po tential also indicated that HSPC was successfully prepared.The nitrogen adsorption-desorption isotherm was carried out for the core-sh ell nanoparticles HMSN-SS to evaluate the porosity (Fig. 2a).HMSN-SS exhibi ted a typically reversible type IV isotherm, confirming that HMSN-SS had a w ell-defined mesoporous structure.The BET surface areas (SBET) of MSN-COO H was ~754.991m 2 /g, with the BJH pore volume (Vp) and BJH pore diamete r (DBJH) of ~1.879 cm 3 /g and ~9.954 nm, respectively.HMSN-SS has a H2-t ype hysteresis loop, and the reaction pore structure is more complex.

Preparation and characterization of 5-ASA@HMSN-PDA-CS
The FT-IR spectra were shown in Figure 2b.The strong absorption peak a t 1080 cm -1 is attributed to the asymmetric stretching vibration of Si-O-Si.The absorption peak at 801 cm -1 is attributed to the symmetric stretching vibration of Si-O-Si.The weak absorption peak at 954 cm -1 is attributed to the bendin g vibration of Si-OH.The wide peak at 3419 cm -1 is the stretching vibration of H-O-H.The absorption peaks at 568 cm -1 and 694 cm -1 correspond to the vibration of S-S and C-S, respectively, indicating that the preparation of HMS N-SS is successful.The two characteristic peaks of 1623 cm -1 and 1501 cm -1 after PDA modification were assigned to N-H stretching vibration and aromatic ring stretching vibration.For the infrared spectrum of HSPC, the peak at 1647 cm -1 in the infrared spectrum of the final system was attributed to the characte ristic peak of the carbon-nitrogen double bond produced by the Schiff base rea ction, and the absorption peak at 894 cm -1 was the characteristic peak of the β -glycosidic bond, indicating that CS has been successfully modified to HMSN-SS-PDA.
The absorption peak of S-S bond in HMSN-SS infrared results is weak, s o it is further characterized by Raman spectroscopy.The peaks at Figure 2c,43 8 and 487 cm -1 are attributed to the stretching vibration of the S-S bond, and the peak at 630 cm -1 is attributed to the stretching vibration of the S-C bond 31 .
To characterize the intermediate product from multi-step surface modificati on of the nanoplatform, TGA was used to measure the weight loss of all sam ples in the range of 30℃ to 800℃ (Fig. 2d).The weight loss of HMSN-SS a t 800 °C is only 15.66 %, which may be due to the fact that the nanoparticle s contain few organic components.Compared with HMSN-SS, the weight loss of HMSN-SS-PDA was about 24.73 %, which was due to the introduction of PDA to coat the nanoparticles.For HMSN-SS-PDA-CS, the weight loss at 800 °C was further increased to 46.69 %, indicating that CS was successfully mo dified on HMSN-SS-PDA.Six capsules each of 5-ASA suppositories for mice, 5-ASA@HSPC suppos itory for mice, 5-ASA suppositories for rats and 5-ASA@HSPC suppository for rats were randomly taken to evaluate their appearance.The results of Fig. 3 sh ow that the prepared suppository has uniform color, smooth section and no ob vious bubbles.The results in Fig. 4 show that the average particle size of HMSN-SS na noparticles changed significantly with time under the simulated low redox pote ntial environment of colon (10 mmol/L GSH ).At 0 h, the average particle si ze was 171.2 nm, the PDI was 0.126, the particle size was small and the distr ibution was narrow.After 24 h, the average particle size changed to 742.5 nm, and more distribution appeared around 1000 nm, and the PDI was 0.854.It i s indicated that the disulfide bond in HMSN-SS nanoparticles is broken under the action of 10 mmol/L GSH, the shell of HMSN-SS is destroyed, and the di ssociated part is irregularly aggregated to form larger nanoparticles, resulting in uneven particle size distribution.As a control group, the particle size of HMS N nanoparticles without disulfide bond did not change significantly with time i n 10 mmol/L GSH buffer.At the same time, the average particle size and PDI of HMSN-SS nanoparticles were almost unchanged within 24 h in a buffer co ntaining 10 μmol/L GSH (simulating a normal redox environment).It shows th at HMSN-SS nanoparticles have good stability under normal redox potential.Before the in vitro drug release study, the drug loading of 5-ASA@HSPC was determined to be 34.6 %.The cumulative release curve of 5-ASA is show n in Fig. 5a.The release of 5-ASA@HSPC in PBS buffer with different pH valu es was significantly different.In PBS buffer with pH 7.4, the release rate of 5 -ASA was slow, and the release amount accumulated to 48 h was only 51.93 %.In PBS buffer with pH 6.8, although the release of 5-ASA increased, the cumulative release amount only increased to 58.06 %.In pH 5.5 PBS buffer, t he cumulative release of 5-ASA reached 68.79 %.The results showed that the drug release amount of each experimental group entered the platform period aft er 24 h, and the release amount of 5-ASA gradually increased with the decrea se of pH value, and had a higher cumulative release amount under acidic cond itions, indicating that the nanoparticles had the characteristics of sustained relea se and pH-responsive release.

In vitro drug release studies
The in vitro release of 5-ASA@HPC and 5-ASA@HSPC in buffers with t he same pH concentration (pH5.5) and different GSH concentrations was invest igated.As shown in Figure 5b, the cumulative release rate of 5-ASA@HPC na noparticles in 10 mmol/L GSH buffer for 48 h was 69.12 %, while that of 5-ASA@HSPC nanoparticles was as high as 84.73 %.In 10 μmol/L GSH buffer (simulated normal redox environment), the cumulative release rate of 5-ASA@ HSPC nanoparticles for 48 h was 72.29 %, which was similar to that of HPC nanoparticles without disulfide bond.These results indicated that 5-ASA@HSPC nanoparticles had the characteristics of redox-responsive release and could be released slowly in simulated colon environment.Excellent biocompatibility is the prerequisite for nanoparticles to be used i n vivo.To assess the biocompatibility of blank nanoparticles HSPC, the cytoto xicity of HSPC against HL7702 cells was tested.As shown in Figure 6, HL-7 702 cells did not show significant cytotoxicity after incubation with different c oncentrations of HSPC for 48 hours, and the cell viability of each experimenta l group was above 90 %.The results showed that HSPC had low cytotoxicity and good biocompatibility.Figure 7 In vivo imaging of Cy7 @ suppository, Cy7 @ HMSN-SS suppository, Cy7 @ HPC suppository and Cy7 @ HSPC suppository at 2,4,8,12,24 h, respectively.

In vivo biodistribution and pharmacokinetic
The imaging of Cy7ordinary suppository group, Cy7@HMSN-SS suppository group, Cy7@HPC suppository group and Cy7@HSPC suppository group in mice after rectal administration is shown in Figure 7.The Cy7 ordinary suppository group showed strong fluorescence intensity at 2 hours after administration, and the fluorescence intensity peaked at about 4 hours.At 24 hours, there was almost no fluorescence in mice, indicating that Cy7 had been basically digested and excreted.In contrast, the fluorescence intensity of Cy7@HMSN-SS suppository group reached the peak at 4 h after administration, and still had a low fluorescence intensity after 12 h, which was attributed to the fact that although the outer layer of HMSN-SS was not modified and had no adhesion, hollow mesoporous silica had a certain sustained release effect.Cy7@HPC suppository group and Cy7@HSPC suppository group had good sustained release effect, and there was still fluorescence at 24 h, indicating that the modified nanodrug delivery system had significant adhesion and retention in the colon.The fluorescence intensity of Cy7@HPC suppository group without disulfide bond reached the peak at about 8 h, while the fluorescence intensity of Cy7@HSPC suppository group reached the peak at about 12 h, and the fluorescence intensity at 24 h was significantly higher than that of Cy7@HPC suppository group, which was attributed to the continuous cleavage of disulfide bond in the low redox potential environment of colon, and the shell of nanoparticles was gradually broken, so that the loaded Cy7 was continuously released and the release amount was higher.The above experimental results showed that Cy7@HSPC suppository group had better sustained release effect.

Table 1 Plasma pharmacokinetic parameters(n=6)
The results of the drug-time curve of figure 8 show that the blood concentration of 5-ASA@HMSN-SS suppository group, 5-ASA@HPC suppository group and 5-ASA@HSPC suppository group in rats is relatively stable, indicating that the 5-ASA of these experimental groups is slowly absorbed in the body, and the dosage form has a sustained release effect.The results of pharmacokinetic experiments in Table 1 showed that the Tmax of the ordinary suppository group was 2.0 h, and the half-life t1/2 was 4.6 h.Compared with 5-ASA ordinary suppository group, 5-ASA@HMSN-SS suppository group, 5-ASA@HPC suppository group and 5-ASA@HSPC suppository group can effectively prolong the peak time and half-life of 5-ASA, but the effect of 5-

Parameters
Unit 5-ASA suppository 5-ASA@HMSN-SS suppository 5-ASA@HPC suppository 5-ASA@HSPC suppository ASA@HSPC suppository group is the best.At the same time, the AUC(0-t) of 5-ASA@HSPC suppository group was greater than that of 5-ASA@HMSN-SS suppository group and 5-ASA@HPC suppository group, indicating that it had better bioavailability.The Tmax of the HSPC suppository group was 12.0 h, the half-life t1/2 was 14.1 h, the peak time and half-life were 6 times and 3.1 times longer than those of the 5-ASA ordinary suppository group, and the peak time and half-life were significantly delayed, which proved that the drug delivery system could release the drug slowly for a long time.The body weight of mice in the normal control group increased slightly, and the feces were normal and dry.Except for the normal control group, the mice in the other experimental groups began to lose weight and loose stools and bloody stools after 3 days of drinking water containing 3 % DSS.As shown in Figure 9a, on the 10 th day of the experiment, the average body weight of the DSS model group was significantly lower than that of the normal control group (p<0.001).The experimental groups treated with 5-ASA can alleviate the weight loss trend to a certain extent.Among the experimental groups, the weight loss of mice in the 5-ASA ordinary suppository group (3 times/day) and the 5-ASA@HSPC suppository group (1 time/day) was the least.
From Figure 9b, it can be seen that the DAI value of the normal control group is basically 0, while the DAI value of the DSS group continues to rise, which is significantly different from the normal control group (P<0.001), which proves that the UC model has been successfully modeled.The results showed that 6 days after the start of the experiment, the disease performance of mice in the DSS group gradually increased, and the DAI score continued to rise.There was a significant difference in the scores of mice in the treatment groups (P<0.05),indicating that the disease development of mice in each experimental group treated with 5-ASA was alleviated.The DAI scores of 5-ASA ordinary suppository group (3 times/day) and 5-ASA@HSPC suppository group (1 time/day) were lower than those of the other experimental groups, and there was no significant difference between the groups, indicating that the therapeutic effect of 5-ASA ordinary suppository group (3 times/day) and 5-ASA@HSPC suppository group (1 time/day) was more obvious, and the efficacy was similar.The experimental results in Fig. 10 showed that the colon length of the normal control group was usually between 9-10 cm, and the intestinal mucosa was clear and complete without congestion and swelling.After 10 days of drinking water with 3 % DSS concentration, the intestinal wall of the model group was congested and swollen, and the length was shortened to 5-6 cm.As shown in Figure 5-4, compared with the DSS group, the other experimental groups given drug treatment inhibited colon shortening to varying degrees, showing different therapeutic effects.The colon length of 5-ASA ordinary suppository group (1 time/day), 5-ASA ordinary suppository group (3 times/day), 5-ASA @ HMSN-SS suppository group, 5-ASA@HPC suppository group and 5-ASA @ HSPC suppository group was longer than that of DSS group, and there was significant difference compared with DSS group (P<0.05).Among them, the colon length of the 5-ASA ordinary suppository group (3 times/day) and the 5-ASA @ HSPC suppository group (1 time/day) is the closest to the normal group, and there is no significant difference between the two groups, indicating that the 5-ASA ordinary suppository group (3 times/day) and the 5-ASA @ HSPC suppository group (1 time/day) can effectively inhibit colon shortening, and both have similar inhibitory effects.MPO activity is an indicator for quantitative evaluation of inflammatory re sponse and is widely used to evaluate the degree of intestinal inflammatory res ponse.The results of Fig. 11 showed that the MPO activity in the colon of the model group was significantly higher than that of the normal control group (P <0.01), indicating that the inflammatory response in the colon tissue was relati vely serious.In the other experimental groups treated with drugs, the MPO act ivity of mice decreased to a certain extent, and there was a significant statistic al difference compared with the DSS model group (P<0.05), which proved the inhibitory effect of drug treatment on inflammation.The expression levels of MPO in the 5-ASA ordinary suppository group (3 times/day) and the 5-ASA@ HSPC suppository group were the lowest, and there was no significant differen ce between the groups, indicating that the 5-ASA ordinary suppository group (3 times/day) and the 5-ASA@HSPC suppository group (1 time/day) had simila r inhibitory effects on inflammation and were superior to the other administrati on groups.The pathological sections of Figure 12 showed that the epithelial cells and crypts of the colon mucosa in the normal control group were intact, and the goblet cells were not reduced.The morphological changes of epithelial cells, cr ypt cells and goblet cells in the intestinal tract of mice in the model group gi ven 3 % DSS drinking water were extremely significant, and the structure of e pithelial cells, crypts and goblet cells in the colon tissue almost completely dis appeared.The colon injury of the other experimental groups (c-g) treated with drugs was significantly reduced compared with the DSS model group, the rang e of colon injury was reduced, and the damaged area of crypt structure was re duced.In each administration test group, the damage area of the crypt structur e and the degree of pathological damage in the 5-ASA ordinary suppository gr oup (3 times/day) and the 5-ASA@HSPC suppository group were significantly reduced compared with the 5-ASA ordinary suppository group (1 time/day), 5-ASA@HMSN-SS suppository group and 5-ASA@HPC suppository group, indica ting that the 5-ASA@HSPC suppository group (1 time/day) and the 5-ASA ord inary suppository group (3 times/day) have significant therapeutic effects in mai ntaining colon tissue morphology and inhibiting inflammation, and the effects a re similar.

Fig. 4
Fig. 4 The particle size data of HMSN-SS at 0 h (a) and 24 h (b) in 10 mmol/L GSH P BS The particle size data of HMSN at 0 h (c) and 24 h (d) in 10 mmol/L GSH PBS.The particle size data of HMSN-SS at 0 h (e) and 24 h (f) in 10 μmol/L GSH PBS.

Figure 5
Figure 5 Cumulative release curves of 5-ASA@HSPC in different pH values (a), Cumulati ve release curves of 5-ASA@HSPC in different GSH at the same pH (b)

Figure 6
Figure 6 Cell viabilities of HL-7702 cells co-incubated with different concentrations of HS PC.