Enhancing stability and anti-inflammatory properties of curcumin in ulcerative colitis therapy using liposomes mediated colon-specific drug delivery system

Chaofan Wang a, 1, Zhenlin Han b, 1, Yuhao Wu a, 1, Xiaoming Lu a, Xiaozhen Tang a,*, Jianbo Xiao c, d,**, Ningyang Li a,***


Curcumin liposomes (CUR-LPs) was identified by evaluating morphology, appearance, zeta potential, particle diameter, and drug encapsulation efficiency. The results indicated that particle diameter, surface charge and polydispersity index (PDI) of curcumin (CUR)-loaded anionic liposomes were 167 nm, —34 mV and 0.09, respectively. CUR-LPs is high stable pseudo-pH-sensitive nanoparticles system which has a favorable stability in simulated gastric fluid and slower degradation rate allowing CUR sustained release for prolonged times in simulated intestinal fluid. Within 1 h, the CUR consumption was 21.82% in simulated gastric fluid (SGF) and 27.32% in simulated intestinal fluid (SIF), respectively. CUR-LPs could attenuate clinical symptoms including weight loss, diarrhea and fecal bleeding. Especially, it could also prevent dextran sulfate sodium salt (DSS)- inducedcolon tissue damage and colon shortening, and reduce the production of malondialdehyde (MDA), colonic myeloperoXidase (MPO), Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in animal model. Our study illustrated that liposomes (LPs) was a potential carrier to develop the colon-specific drug delivery system incorporating CUR for treating ulcerative colitis.

Curcumin liposomes Ulcerative colitis Stability
Colon-specific drug delivery

1. Introduction

Ulcerative colitis (UC) is a common inflammatory disease in the mucosa of the colon with rapidly increasing incidence worldwide (Imhann et al., 2018). It affects millions of individuals worldwide and persists in their lifetime because there is no permanent cure (Cosnes et al., 2011). Therefore, patients with UC have to take the medication throughout their whole life. The main drugs for clinical treatment of UC are highly depended on the application of aminosalicylates, corticoste- roids, anti-inflammatory molecules and immune modulatory agents with the goals of controlling inflammation and achieving mucosal healing (Gou et al., 2018; Sandborn et al., 2012; Farombi et al., 2013). However, these drugs are limited by low therapeutic efficiency and serious adverse effects caused by long-term utilizations, such as osteo- porosis, acute pancreatitis and infection as well as diarrhea (Dulbecco and Savarino, 2013; Farombi et al., 2013; Nidhi et al., 2016). Thus, development of alternative agents with high therapeutic efficacy and low side effects may be a promising therapeutic strategy.
CUR is a kind of functional ingredient extracted from ginger or turmeric, which has a wide variety of biological and pharmacological effects, including anti-inflammatory (Singla et al., 2014), antioXidant (Zhao et al., 2014), antitumor (Lim et al., 2014), and hepatoprotective activities (Nabavi et al., 2014). It has attracted increasing attention in UC treatment because it can scavenge oXygen radicals, reduce inflam- matory cytokine production and inhibit tumor growth (Chen et al., 2018). In addition, the distribution of CUR in human tissues suggests that it preferentially accumulates in the intestine, colon and liver compared to other organ systems (Li et al., 2015). This finding provides a theoretical basis for the positive effect of CUR on gastrointestinal diseases. It is worth noting that the standard therapy of CUR is relatively safe for humans and animals (Lao et al., 2006; Goel et al., 2008). However, its further application in clinics has been seriously restricted for its strong hydrophobicity, instability after light exposure, high in- testinal metabolic rate and rapid excretion from the body (Chen et al., 2009; Matloob et al., 2014; Kurien et al., 2007). Specially, CUR is easily esterified with carbohydrate, salts, etc., and metabolites are excreted with urine feces result in decreasing its absorption and narrowing its scope of application (Pan et al., 1999). Development of an appropriate delivery system to enhance CUR bioavailability is required for it to be a successful therapeutic agent.
In the past few years, nanoscale drug delivery systems, including LPs, nanoparticles (NPs) and micelles, have received significant attention due to their many advantageous properties (Chen et al., 2018; Xiao et al., 2016). LPs are spherical vesicles consisting of one or more phos- pholipid bilayers enclosing an aqueous core. It is formed through directed self-assembly of amphiphilic molecules such as phospholipids (Peng et al., 2018). LPs can encapsulate hydrophobic compounds such as CUR in their phospholipid bilayers (Fig. 1). Compared with conventional emulsions, the droplet of LPs is much smaller (typically<500 nm) (Yalçıno¨z and Erçelebi, 2018). The small size of a LPs provides advan- tages such as longterm stability, optical clarity, and increased bioavailability of the loaded lipophilic molecules (McClements, 2012). It has been recognized as a promising drug delivery system for UC therapy, which benefits from its sustained drug release capacity, colitis tissue-targeted ability based on the epithelial enhanced permeation and retention (eEPR) effect, and most importantly, high stability in gastric fluid due to its pseudo-pH-sensitivity (Taylor et al., 2005; Feng et al., 2014). From this perspective, the preparation of CUR-LPs for colon-targeted delivery could further optimize the clinical effects of UC. In present study, CUR-LPs was prepared by means of ethanol injec- tion method in order to enhance the poor aqueous solubility of CUR, to achieve sustained and prolonged drug release thereby extending the bioavailability. CUR without LPs was also prepared as control. Physi- cochemical properties of CUR-Lps were measured in terms of appear- ance, morphology, particle diameter, zeta potential, drug encapsulation efficiency and stability property in vitro. In this study, the applicability of the carriers was tested by means of an in-vitro digestion procedure allowing for measurement of the consumption of CUR. Meanwhile, anti- colitis activity of CUR-LPs was evaluated using DSS-induced colitis in mice model. 2. Materials and methods 2.1. Tested compounds and chemicals CUR was prepared in the laboratory. L-α-Phosphatidylcholine was purchased from Avanti Polar Lipids (America). Egg yolk was purchased from Beijing Solarbio Technology Co., Ltd (China). Other reagent chemicals were all of analytical grade and used without further purification. 2.2. Extraction and purification of CUR The CUR was extracted following the ultrasonic extraction method. The CUR was dissolved in ethanol and the supernatant was concentrated in a rotary evaporator under vacuum at 45 ◦C and filtered. Then, the supernatant was injected to a column (1.6 50 cm) of DM-301 mac- roporous resin. Eluate (5.0 mL/tube) was collected by automatic col- lector, and the absorbance at 425 nm was measured. The eluate was combined, concentrated, dialyzed, and then the supernatant was injec- ted to a column (1.6 × 50 cm) of silica gel with trichloromethane. After loading the sample, the column was eluted with CHCl3:CH3OH:HCOOH (99:0.6:0.4, 97:1.5:0.5, 75:24.5:0.5) for gradient elution. Eluate (4.0 mL/tube) was collected by automatic collector and three monomer components were obtained. The first monomer component was com- bined, concentrated, dialyzed, and determined by high performance liquid chromatography (HPLC) method and that was CUR. 2.3. Preparation of CUR-LPs CUR-LPs were prepared according to the ethanol injection method. Lecithin was miXed with cholesterol in a ratio of 1:3 (v/v) and dissolved in 1 mL of ethanol and then 0.5 mg of CUR was add, slowly inject into the phosphate buffer saline (PBS, pH 7.4) solution using a No.6 sy- ringe. And then incubated in water bath under stirring for 60 min at 45 ◦C. CUR-LPs suspension was sonicated in an ice bath at power of 100% for 60 s. 2.4. Characterization of CUR-LPs 2.4.1. Morphology The morphology of CUR-LPs was observed via transmission electron microscope (TEM, JEM-1200EX (120 KV) JEOL100 Ltd. Japan). The samples were stained with an aqueous solution of Phosphotungstic acid before observation. 2.4.2. Size distribution and zeta potential The average particle diameter, PDI and zeta potential of CUR-LPs were measured using dynamic light scattering (DLS) technique (Zeta- sizer ZEN3600, Malvern 95 Instruments Ltd., UK), and the sample was diluted 10-fold with PBS buffer before measurement, and the measurment temperature was 25 ◦C. Each measurement was made in triplicate. 2.4.3. Stability of CUR-LPs For testing the stability of CUR-LPs, the changes in mean particle diameter, PDI, and encapsulation rate were investigated over the storage periods. CUR-LPs samples were stored in an incubator at 4 ◦C and 25 ◦C for 28 days, and the particle diameter distribution, PDI, and encapsu- lation rate of different nanosystems was measured using DLS (Zetasizer ZEN3600, Malvern 95 Instruments Ltd., UK) every 7 days and the morphological changes were observed. 2.4.4. In vitro digestion of CUR from LPs and aqueous solution The release of CUR from nanoparticles was carried out in the pres- ence of gastrointestinal simulation solution. The samples were loaded into a dialysis bag and the dialysis bag was immersed in SGF (pH 1.2) for the first 2 h, followed by release in simulated duodenum fluid (pH 6.0, 2 h), simulated jejunum fluid (pH 7.0, 2 h) and simulated ileum fluid (pH 7.4, 2 h). The experiments were performed at 37 ◦C for 10 min in a shaking incubator at 100 rpm. At a scheduled time intervals, 3.0 mL of the solution was removed and the absorbance at 425 nm was measured. Then 3.0 mL of corresponding gastrointestinal simulation solution was added to the release medium to maintain a constant solution volume. According to the standard curve of CUR in the corresponding gastroin- testinal simulation solution, the cumulative amount of CUR release was calculated. All of the operations were carried out in triplicate. 2.5. Animal study 2.5.1. Animal care 7 to 8-week-old male C57BL/6 mice weighing 18–22 g were pur- chased from Peng Yue EXperimental Animal Breeding Co., Ltd.(Jinan, China). All animals were housed in individual cages. The mice were fed a standard rodent diet with free access to water, and were kept in rooms maintained at 21–23 ◦C and air humidity of 50% with 12 h light/dark cycle following international recommendations. All experimental procedures were consistent with the Directive 2010/63/EU and approved by the Ethic and Animal Shandong Agricultural University Animal Care and Use Committee (permit number: SDAUA-2017-016). 2.5.2. DSS-induced colitis in animal model experimental design Acute colitis was induced by oral administration of 5% (w/v) DSS dissolved in drinking water for 8 days (Li et al., 2015). Mice of each experimental group were monitored every day to confirm that they consumed equal volumes of DSS-containing water. Acute ulcerative colitis of the C57BL/6 mice was induced by oral administration of 5% (w/v) DSS ad libitum for 8 consecutive days. Fifty colitic mice were arbitrarily allocated in 5 groups: DSS model group, normal group, 5-Aminosalicylic acid (5-ASA)-treated group, CUR-treated group, CUR-LPs-treated group. The normal group was given autoclaved water for 9 days; the other groups were given water containing 5% DSS for 9 days. The 5-ASA-treated group was given 5-ASA for 8 days. The CUR-treated group was given CUR solution for 8 days, the CUR-LPs-treated group was given CUR-LPs for 8 days. A normal group with 10 normal mice received drinking water without DSS throughout the entire experimental period. CUR and CUR-LPs were administrated orally to colitic mice at doses of 100 mg/kg/day. 5-ASA was used as a positive reference agent and was given at 100 mg/kg/day. 2.5.3. Evaluation of the disease activity index (DAI) During the duration of the experiment, a DAI score was assessed to evaluate the clinical progression of colitis. The DAI was the combined score of weight loss, stool consistency, and bleeding (Kim et al., 2012). The DAI was determined by scoring the changes of body weight loss, diarrheal condition, and fecal bleeding. Diarrhea scoring and bleeding scoring were performed as modified criteria according to the method previously described (Shin et al., 2015). Every score was divided into 4 grades of body weight loss score: (0 means 0%; 1 means 1–5%; 2 means 5–10%; 3 means 10–15%; 4 means > 15%); diarrhea score: (0 means normal stool; 1 means mildly soft stool; 2 means soft stool; 3 means very soft stool; 4 means watery stool) and bloody feces score (0 means normal colored stool; 1 means brown stool; 2 means reddish stool; 3 means mildly bloody stool; 4 means bloody stool).

2.5.4. Histological analysis

At the end of experiment, the mice were sacrificed by cervical dislocation, the abdominal cavity was dissected, the colon was separated from ileo-cecal junction and anal verge, and then take pictures, measure and record colon length. Colon tissues were harvested and fiXed in 10% neutral formalin solution. Tissue sections were prepared by conven- tional tissue processing methods, stained with hematoXylin and eosin (H&E), and examined under the light microscope.

2.5.5. Determination of colonic MPO activity

One unit of MPO activity was defined as the amount of enzyme present that produced a change in optical density of 1.0 U/min at 25 ◦C in the final reaction volume. The results were normalized to equal protein levels and quantified as units/mg protein.

2.5.6. Determination of MDA content

MDA can be condensed with thibabituric acid under acidic (pH 3) conditions to produce a red product. The red product has the largest absorption peak at 523 nm. MDA was determined in colon tissue by measuring the absorbance at a wavelength of 523 nm and quantified as nmol/mg.

2.5.7. Enzyme-linked immunosorbent assay (ELISA) analysis

The cytokines TNF-α and IL-6 in the culture supernatants of colonic tissues were measured with ELISA kits (eBioscience, San Diego, CA, U.S. A.) according to the manufacture’s protocols, respectively.

2.6. Statistical analysis

All data analysis were presented as mean S.D. Data were evaluated by one-way ANOVA using IBM SPSS statistical software version 24.0 (IBM Corporation, Chicago, IL, USA), the differences between means were assessed by analysis of variance (ANOVA) with Duncan’s test using a significant difference level of p < 0.05. 3. Results 3.1. Characteristics of CUR-LPs CUR-LPs was prepared through ethanol injection method. CUR-LPs were well distributed and spherical, with smooth surfaces (Fig. 1). The average diameter of CUR-LPs was around 167.34 9.42 nm and carried a charge of 34.11 0.24 mV. In addition, PDI of CUR-LPs was 0.09 0.01 indicating that particle diameter was well controlled with a narrow dispersity. 3.2. Stabitity of CUR-LPs It is well known that the stability of nanoparticles is critical to establish their safe and effective use. To assess the stability of CUR-LPs over the storage periods at 4 ◦C and 25 ◦C, the changes in particle diameter and morphology were investigated (Table 1 and Fig. 1). The particle diameter of CUR-LPs increased from 168.04 1.78 nm to 268.77 3.36 nm and without obvious precipitation appeared at 4 ◦C, while the particle diameter increased from 184.13 2.04 nm to 345.43 1.96 nm and obvious precipitation appeared at 25 ◦C. On the 28th day, the encapsulation rate of CUR-LPs at 4 ◦C (59.14%) was higher than that at 25 ◦C (27.62%). The PDI of CUR-LPs at 4 ◦C (0.31) was lower than that at 25 ◦C (0.67) (Table 1). It can be clearly seen from Fig. 1 that the color of CUR-LPs is darker at 4 ◦C and lighter at 25 ◦C, which is caused by curcumin degradation. It is concluded that 4 ◦C is beneficial to the preservation of CUR-LPs and makes the nanoparticles system more stable. High storage temperature resulted in higher permeability of the CUR-LPs membrane, leakage and degradation of the CUR. Meanwhile, CUR-LPs membrane presents in colloidal state at low temperature which prevented leakage of CUR (Gonzalez-Perez et al., 2004). Generally, lower loss of CUR occurred during storage in a refrigerator compared with at room temperature. 3.3. In vitro digestion of CUR from LPs and aqueous solution The consumption profile of CUR and CUR-LPs from nanoparticles was studied in vitro in the presence of gastrointestinal simulation solu- tion (Fig. 2). In SGF, a burst consumption within the first 0.5 h was observed at pH 1.2 from CUR, approXimately 69.17%. Then CUR appeared a slow consume during the next 2.5 h. While CUR-LPs dis- played the slow consumption behavior during the 1 h, and appeared a slightly rapid release during the next 2 h, approXimately 17.53%. 3 h later, the finally consumption of CUR solution and CUR-LPs were 75.52% and 21.82%, respectively. CUR solution and CUR-LPs displayed a sustaining consumption behavior by a similar consumption tendency in SIF. 3 h later, the cumulative amount of CUR consumption from CUR solution and CUR-LPs were 44.78% and 27.32%, respectively. 3.4. Anti-colitis activity of CUR-LPs 3.4.1. Effects on clinical symptoms Starting from day 4, In this study, we found that C57BL/6 mice subjected to the oral administration of 5% DSS regularly developed ul- cerative colitis with weight loss, severe diarrhea and rectal prolapse accompanied by extensive wasting disease in untreated mice. In severe cases, gross blood adhering to the anus was noted. The DAI, an indicator of the severity of intestinal inflammation, was used to analyze the therapeutic benefit of CUR-LPs treatment. In this experiment, CUR-LPs- treated group showed remarkable improvements in body weight loss, intestinal bleeding and diarrhea, resulting in significant amelioration of UC as assessed by DAI when compared with the DSS of group and CUR- treated group. Fig. 3 show the time course of the DAI scores after treatment with DSS. DAI scores of mice were increased in a time-dependent fashion. Compared with the normal group, the DAI scores were markedly increased in DSS model group (P < 0.01). By contrast, treatment with CUR-LPs-treated group significantly reduced the DAI scores in mice induced by DSS (P < 0.01). We also found that CUR-LPs- treated group exhibited the similar improvements to positive medicine group of 5-ASA (P > 0.05). These results suggest that oral administration of CUR-LPs have inhibitory activity on body weight loss, diarrhea and fecal bleeding in DSS-induced colitis.

3.4.2. Effects on histological examination

As shown in Fig. 4(A) and (B), 5% DSS-induced mice developed serious mucosal damage, bloody and watery stool, and significant colon length shortening (P < 0.01) compared with normal group. However, the length of the shortened colon was significantly increased after the treatment of agents in mice with DSS-induced colitis (P < 0.05). CUR- LPs-treated group and 5-ASA-treated group displayed the strongest ef- ficacies to increase the length of the shortened colon induced by DSS, and the therapeutic effects were similar (P > 0.05). Compared with DSS model group and CUR-treated group, CUR-LPs-treated group significantly inhibited the mucosal damage and improve diarrhea, fecal bleeding and colon shortening in mice.
As shown in Fig. 4 (C), we characterized the histologic features of UC in C57BL/6 mice subjected to 5% DSS. The severity of UC-like lesions was most marked in the colon of DSS-induced mice. Compared with normal mice, the distal colon of UC mice exhibited marked mucosal inflammation in all layers of the bowel wall, including severe submu- cosal edema, depletion of goblet cells, inflammatory cells infiltration and crypt abscesses. CUR-LPs protected against both the infiltration of inflammatory cells and the mucosal damage, particularly, significantly suppressed lymphocyte infiltration, resulting in a significant reduction of histopathology damage. However, compared with CUR-LPs-treated group, CUR-treated group produced more obvious inflammatory cells infiltration and depletion of goblet cells. These findings indicate that oral administration of CUR-LPs improved the colon disease symptoms by recovering severity of inflammation, inflammatory cell infiltration to colonic mucosa and extent of epithelia damage.

3.4.3. Effects on MPO activity and MDA content

Consistent with the histological scores, Fig. 5 (A) showed that colonic MPO activity was greatly increased in the DSS model group (0.96 ± 0.03 U/mg) than normal group (0.25 ± 0.03 U/mg), whereas the MPO ac- tivities in the CUR-LPs-treated group (0.43 ± 0.02 U/mg) and CUR- treated group (0.58 0.08 U/mg) were significantly reduced. As shown in Fig. 5 B, CUR-LPs and 5-ASA substantially decreased the colonic content of MDA by about 65.75% and 68.61% respectively, compared to DSS model group, and there was no significant difference between the two groups (P > 0.05).

3.4.4. Effects on TNF-α and IL-6 levels

DSS-induced UC was accompanied by the release of pro- inflammatory cytokines including TNF-α and IL-6. The levels of TNF-α and IL-6 in the supernatant of colon tissue homogenate from each group were measured to determine whether CUR-LPs inhibits the production of pro-inflammatory cytokines, therefore protecting the colon mucosa. As shown in Fig. 5 C and D, the increase in the amount of TNF-α and IL-6 expression was significantly reduced after the treatment of agents in mice with DSS-induced colitis (P < 0.05). We could find that CUR-LPs- treated group displayed strong efficacies to inhibit the production of TNF-α and IL-6 induced by DSS, and the therapeutic effects were better than CUR-treated group (P < 0.05). 4. Discussion In recent years, drug delivery system for UC therapy has been described in numerous reports (Tian et al., 2018; Li et al., 2015). However, two major drawbacks of colonic delivery systems are limited mucosal diffusion and pre-systemic degradation. One approach to improve these situation is to use nanoparticle drug carriers, which possess sustained drug release capacity, colitis tissue-targeted ability As a relatively non-toXic functional ingredient combined with based on the epithelial enhanced permeation and retention (eEPR) ef- fect, owing to their large interfacial surface area and minute dimension (McClements, 2012). It is well documented that intestinal permeability is increased during inflammation, allowing permeation of the particles to the desired colon lesion site in a preferable manner when compared to the normal intestine (McGuckin et al., 2009). Naeem et al. (2020) have already shown that fluorescent nanoparticles exhibited high fluores- cence intensities in the mucosa of DSS-induced rats after systemic administration, probably due to the retention effect of the inflamed tissue. In the current study, CUR-LPs is high stable pseudo-pH-sensitive nanoparticles system which has favorable stability in SGF and slower degradation rate allowing CUR sustained release for prolonged times in SIF. In the intestine, lipase efficiently digests LPs to release free fatty acids and monoglycerides (Golding and Wooster, 2010). The nano- particles system is thus disintegrated to release the loaded lipophilic molecules, namely, CUR. The released lipophilic molecules can form micelles with endogenous surfactants (e.g. bile salts and phospholipids) in the intestine where the micelles can be absorbed by the epithelial cells, and further relieve UC. Because the pH-sensitivity is due to the action of lipase enzymes, this nanoparticles system is pseudo-pH-sensitive (Xiong et al., 2020). excellent anti-inflammatory and antioXidant properties, CUR has been attracted increasing attention in UC treatment (Chen et al., 2018). Un- fortunately, dosing CUR alone, researchers would face the dilemma of poor absorption for a few drawbacks of CUR such as its strong hydro- phobicity, instability after light exposure, high intestinal metabolic rate and rapid excretion from the body, it is thus very vital for clinical application to improve absorption of CUR in the further studies (Mat- loob et al., 2014). In the present study, the CUR-LPs formulation with negative charge was prepared to improve the stability, solubility and anti-colitis activity of CUR. CUR encapsulation may cause significant changes in the LPs surface structure, resulting in the change in orien- tation of the phosphatidylcholine head groups at the surface of LPs, thus contributing to the negative zeta potential measured (Jin et al., 2016). Tirosh et al. (2009) have recently shown that targeting the inflamed mucosa could be accomplished with negatively charged dosage forms for the topical treatment of UC. Therefore, CUR and piperine (PIP) were delivered via anionic self-microemulsifying drug delivery system (SMEDDS) to the inflamed mucosa of experimental colitis-induced mice were more effective in ameliorating the induced inflammation compared with their aqueous suspensions. CUR-LPs has small particle diameter (<500 nm) and great zeta potentials values (>30 mV) which can increase electrostatic repulsion between adjacent LPs and prevent LPs from aggregating, and thus, indicating high stability (Liu et al., 2016). The rapid consumption of CUR was probably caused by the gastric acid condition (Anand et al., 2007). CUR-LPs is quite stable in the gastric acid condition due to its pseudo-pH-sensitive, which enables the nanoparticles system to inhibit the consumption of CUR to some extent (Xiong et al., 2020). In addition, LPs unique structure of vesicular phospholipid membranes offers the opportunity to encapsulate hydro- phobic CUR in their phospholipid bilayer, which could reduce the and finally, evaluated the therapeutic effects of CUR-LPs treatment. Our findings demonstrated that oral administration of CUR-LPs have inhib- itory activity on body weight loss, diarrhea and fecal bleeding in DSS- induced colitis. In addition, the DSS-induced colitis exhibited marked colon length shortening and mucosal inflammation in all layers of the bowel wall, including severe submucosal edema, depletion of goblet cells, inflammatory cells infiltration and crypt abscesses. CUR-LPs pro- tected against the colon shortening, the infiltration of inflammatory cells and the mucosal damage, particularly, significantly suppressed interaction between oXygen with CUR, and slow the diffusion of lymphocyte infiltration, resulting in a significant reduction of histopa- gastrointestinal simulation solution into the internal aqueous phase of the phospholipid bilayer, thereby slowing the pH change of the internal aqueous phase of the liposome (Peng et al., 2018). Hence, LPs can be advantageous to protect CUR from degradation processes in gastric juice.
In order to investigate the topical therapeutic effects of CUR-LPs on UC, we selected a model of colitis induced by DSS in mice. The model exhibits many symptoms and signs similar to those seen in human UC, such as diarrhea, bloody feces, body weight loss, mucosal ulceration and shortening of colon length (Xiao et al., 2015). In addition, the DSS-induced UC animal model has a variety of advantages over others, such as simple experimental methods, reproducibility of the time-course of development as well as colitis severity among individual mice, and relative uniformity of the induced lesions (Camuesco et al., 2005). Therefore, this model is thought to be reliable for testing drug formu- lations or phytochemicals for UC treatment.
In the present study, we measured the severity of clinical symptoms by assessing the body weight loss, stool consistency and fecal bleeding, thology damage. Studies have demonstrated that treatment with CUR on UC could significantly lower MPO activity and MDA content (Arafa et al., 2009). Inflammation is characterized by various prooXidative/antioXidative processes. One of the hallmarks of oXidative stress is the elevated level of MPO. This enzyme catalyses the production of potent oXygen radicals and is increased in the inflamed colon in patients with UC (Chami et al., 2018). In vivo, the role of reactive oXygen radicals produce peroXidation, the end product of lipid oXidation is MDA. MDA can oXidize cell mem- brane, resulting in the damage of organic structure. Therefore, MDA is often used to measure the level of oXygen radical, the degree of lipid peroXidation and the degree of tissue damage (Li et al., 2015). The suppression of MPO activity and MDA production by CUR-LPs treatment in the DSS-induced colitis model is effective for the inhibition of UC progression.
It is well known that there is an inflammatory cascade within the gut tissues of UC that is characterized by the recruitment of circulating leukocytes into the gut tissues and the aberrant expression of pro inflammatory cytokines such as TNF-α and IL-6 (Pedersen et al., 2014; Takac et al., 2014). In the present study, we have also shown the development of such a cascade of inflammatory events in colitis induced by DSS. Analysis of inflammatory cytokine production in colon tissue homogenate revealed a significant reduction in the levels of TNF-α and IL-6 in mice treated with CUR-LPs, compared to the DSS-induced colitis model. Based on these results, the reduced TNF-α and IL-6 production in the colonic tissues represents a possible means for decreasing the severity of UC. Indeed, we found that the administration of CUR-LPs not only reduced DAI and histopathological lesion, but also downregulated TNF-α and IL-6 production, limited the inflammatory response, and thereby significantly ameliorated the severity of DSS-induced colitis.

5. Conclusion

In this study, CUR-LPs was successfully prepared by ethanol injection method. The results indicate that CUR-LPs possessed spherical morphology. Particle diameter, polydispersity index (PDI) and surface charge of CUR-loaded anionic liposomes were 167 nm, 0.09 and 34 mV, respectively. CUR-LPs is high stable pseudo-pH-sensitive nano- particles system which inhibits the consumption of CUR to some extent in gastrointestinal simulation solution. Within 3 h, the CUR consump- tion is 21.82% in SGF and 27.32% in SIF, respectively. In addition, low temperature is beneficial to maintain the colloidal stability of nano- particles system. As a conclusion of DSS-induced colitis in animal model assay, CUR-LPs could attenuate clinical symptoms including weight loss, stool consistency and bleeding. It could also prevent colon shortening and colon tissue damage induced by DSS, and attenuate the production of colonic MPO activity, MDA, TNF-α, IL-6 in animal model. The CUR- Lps developed in this study was a potential carrier for developing colon-specific drug delivery system of CUR for ulcerative colitis treat- ment. It has good potential to formulate more efficacious colloidal de- livery systems for application in the food, dietary supplement, and pharmaceutical industries, and further significantly improve the bioavailability of functional food ingredients. Further research is needed to reveal the effects of other gastrointestinal components such as ions and enzymes on the stability and toXicity of CUR-LPs. To solve these problems needing prompt solution is the common goal of our team. Combinations of in vitro and in vivo experiments would help to accom- plish this objective and accelerate the commercialization of CUR-LPs.


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