Probiotics-loaded carrageenan microspheres for inflammatory bowel disease treatment

Nowadays, many studies have been established to develop strategies for inflammatory bowel disease (IBD) treatment. However, seldom of them explored the synergistic effects of materials and probiotics in IBD treatment. Herein, we prepared probiotics-loaded dietary fiber microspheres and studied their potential in IBD therapy. The carrageenan, a typical dietary fiber, was functionalized with polymerizable groups and employed to fabricate the methylacrylylated carrageenan hydrogel microspheres (CHMSs) by microfluidics. The CHMSs loaded with probiotics exhibited good monodispersity, biocompatibility, and the best effects in relieving the symptoms of IBD and body weight recovery than other groups, indicating the probiotics and carrageenan have synergistic effect for IBD treatment. These results show the huge potential of the probiotics-loaded CHMSs for IBD therapy.


Introduction
Inflammatory bowel disease (IBD) is a group of chronic intestinal inflammatory disorders and has caused huge burden on patients and medical institutions worldwide [1][2][3]. This disease results in many reactions, such as excessive inflammation and oxidative stress, and further leads to the imbalance or dysbiosis of the gut microbiota, which may cause more systematic diseases [4][5][6]. Recently, researchers have attempted to correct the dysbiosis of the gut microbiota, and found this strategy can help to relieve IBD in turn [7][8][9]. These results open the door of treating IBD by the probiotics. However, reliable and efficient strategies for constructing the probiotics-based IBD treatment system are still lacking.
In this work, we presented a novel probiotics-loaded carrageenan microsphere for IBD treatment. Carrageenan is a kind of edible dietary fiber extracted from marine red algae, and therefore it can be a good candidate as biocompatible polymers in biomedical fields [10][11][12][13][14][15][16]. Additionally, carrageenan has excellent bioactivity, such as inoxidizability, have been believed to play important roles in relieving IBD. Therefore, the joint usage of probiotics and carrageenan for IBD treatment is feasible, and the combination of them may obtain a more efficient effect. However, the joint of probiotics and carrageenan has not been developed, and the study in the joint effects is still lacking.
Herein, we prepared the probiotics-loaded carrageenan hydrogel microspheres (CHMSs) by microfluidics. Microfluidic technology can modulate fluid at submillimeter scale precisely and generate uniform droplets or fibers [17][18][19][20][21][22][23][24][25][26][27]. Therefore, the carrageenan solution could be cut into homogeneous droplets by a microfluidic device. It was worth mentioning that the carrageenan used in this work was functionalized by photocrosslinkable acrylate groups (CaMA), indicating they could be polymerized via ultraviolet (UV) light irradiation. Hence, the carrageenan droplets were polymerized by UV light and form hydrogel microspheres, which were then freeze-drying and incubated in the probiotics solution for probiotics uploading. The resultant CHMSs exhibited good monodispersity, biocompatibility, and the best effects in relieving the symptoms of IBD and body weight recovery than other groups, indicating the probiotics and carrageenan have synergistic effect for IBD treatment. This result indicates that the probiotics-loaded CHMSs are potential materials for IBD treatment.

Experimental section 2.1. Materials
Kappa carrageena was bought from purchased from Yuanye Bio-Technology Company (Shanghai, China). Lactobacillus were bought from China Center of Industrial Culture Collection. 2-hydroxy-2methylpropiophenone (HMPP), and Methacrylic anhydride were bought from Sigma Aldrich (St. Louis, MO, USA). Dextran sulphate sodium (DSS) was bought from Whiga Technology Company (Guangzhou, China). 6-8 weeks old C57BL/6 male mice were purchased from Jiake Biotechnology (Shanghai, China). BMSC of mice was bought from Cyagen Biosciences (Guangzhou, China). Calcein-AM was purchased from Thermo Fisher Scientific (USA). The ELISA kit was purchased from Neobioscience Technology Ltd. (Beijing, China). Microfluidic chip was purchased from Suzhou CChip scientific instrument Co., Ltd. The antibodies for ZO-1 and Occludin 3 were obtained from Boster Biological Technology co.ltd. (Nanjing, China).

Probiotics microspheres preparation
Photocrosslinkable Carrageenan (CaMA) was synthesized from Kappa carrageena and Methacrylic anhydride as previous reports [10][11][12]. Then 5% CaMA solution containing 2% HMPP were used for microspheres generation using a microfluidic chip. The CaMA solution containing photoinitiator was pumped into a microfluidic chip as the inner phase while the HFE7500 oil was employed as the outer phase. The generated drops were collected in HFE7500 oil and then crosslinked under ultraviolet light for 30 s to form the crosslinked CaMA microspheres. After evaporating the oil by an oven and drying the microspheres with a freezing-dryer, the dried microspheres are soaked in PBS containing 6 × 10 7 /ml Lactobacillus for the probiotics loading.

Characterization and biocompatibility of the microspheres
The morphology of microspheres was observed by microscopy with a CCD camera (Media Cybernetics EvolutionMP 5.0) to characterize the size. At the same time, the collected microspheres cleaned with ultrapure water and placed in vacuum to remove excess water. Then dried samples were observed by a scanning electron microscopy. Bone marrow stromal cells (BMSCs) of mice was used for testing the biocompatibility of the CHMSs. The BMSCs were divided into two groups including the control group and CHMSs. After 5 days' coculture, the cells were stained by Calcein-AM and the cell status was observed under fluorescence microscope. Control group: Free water without special treatment, 10 ml kg −1 PBS for gavage treatment daily. DSS group: The drinking water was added with 4% DSS and treated with 10 ml kg −1 PBS daily. Prob group: The drinking water was added with 4% DSS and treated with 10 ml kg −1 PBS containing 1.5 × 10 7 CFU probiotics daily. CHMS: The drinking water was added with 4% DSS and treated with 10 ml kg −1 PBS containing 0.1 g CHMSs daily. CHMS + Prob: The drinking water was added with 4% DSS and treated with 10 ml kg −1 PBS containing 0.1 g CHMSs loaded with 1.5 × 10 7 CFU probiotics daily. The weight of the mice was recorded every day. After 8 days treatment, mice were executed and colonic tissue was collected and colonic length was measured. In addition, we collected blood for further analysis.

Therapeutic evaluation
The collected intestinal tissues were fixed in 4% paraformaldehyde solution for 48 h. The intestinal tissues were cleaned, dehydrated in a gradient, and embedded in paraffin. Subsequently, the paraffin-embedded intestinal tissues were processed by tissue sectioning on slides of 5 μm thickness, and the effect of intestinal quality was examined by H&E staining, and immunofluorescence staining including ZO-1 and Occludin 3. Next, we also measured the expression of IL-10, IL-6, IL-β, TNF-α in serum samples by ELISA.

Statistical analysis
All the results were expressed as the mean ± SD. Student's T-tests were used to determine the significance between control and different experimental group. Statistical significance was defined as * p < 0.05, ** p<0.01, and *** p < 0.001.

Fabrication of the probiotics-loaded CHMSs
In a typical experiment, the CHMSs were generated from a microfluidic system [28][29][30][31][32]. As shown in figure 1(a), the CaMA solution containing photoinitiator was pumped into a microfluidic chip as the inner phase while the oil was employed as the outer phase. In the chip, the outer phase cut the inner phase into droplets by shearing force, and the resultant droplets were collected in a container containing oil. Followed with UV light irradiation to form CHMSs. The generated CHMSs were then washed and freeze-drying, and immersed in probiotic solution. After probiotic uploading, the probiotics-loaded CHMSs were prepared ( figure 1(b)).

Characterization of the CHMSs
The generated CHMSs were observed under an optical microscope firstly. As shown in figure 2(a), the CaMA droplets were polymerized into CHMSs and collected in a container, and exhibited spherical shapes. Owing to the precisely control of microfluidic system, the generated CHMSs had a relative uniform size distribution (figure 2(b)), and they can maintain the shape within 3 weeks (figure S1).
The microstructure of CHMSs was characterized by a scanning electron microscope (SEM). As shown in figures 2(c) and (d), the CHMS maintained the spherical shape after drying and had macroporous and rough microstructure. The dried CHMS showed a smaller size than that before drying due to the water loss and shrinking of hydrogel. Meanwhile, the macroporous and rough microstructure made it possible to uploading probiotics.
Since the CHMSs were employed to load probiotics and delivered into bowel for treatment, the biocompatibility of them should be concerned. Herein, we cocultured the CHMSs with BMSCs for the evaluation of the biocompatibility of the CHMSs, and the culture of BMSCs without CHMSs was set as the control group. As the results shown in figures 2(e) and (f), the BMSCs in the both groups exhibited similar growth situation, indicating the CHMSs had good biocompatibility and thus, they could be reliable materials for probiotics loading and the further in vivo usage.

Therapeutic effects of the probiotics-loaded CHMSs
To investigate the therapeutic effects on IBD of the probiotics-loaded CHMSs, we employed mice as the animal model and randomly divided them into five groups. Dextran sulphate sodium (DSS) is widely used for inducing IBD model. Therefore, in this work, the groups were treated with PBS (Control), DSS (DSS), DSS and probiotics (Prob), DSS and CHMSs (CHMS), DSS and probiotics-loaded CHMSs (CHMS + Prob), respectively, and the body weight of the mice was recorded every day. As shown in figure 3(a), the mice in the groups treated with DSS all showed weight loss, while the mice in the Control group displayed a little increase. This can be attributed to the DSS induced IBD. The DSS group displayed the most body weight loss. When the mice were treated with Prob or CHMSs, the IBD was relieved. It was worth mentioning that the body weight of the mice in CHMS + Prob group was slight lower that of the Control group and better that those of the DSS, Prob, and CHMS group, indicating the CHMS + Prob group had the best therapeutic efficiency, and the CHMSs and probiotics had the synergistic therapeutic effects. The colon lengths of the mice in the five groups were shown in figure 3(b). Similar to the results of the body weight loss, the mice in the DSS group had the shortest colon, and the CHMS + Prob group displayed the best therapeutic effects.
Inflammatory factors level within body can be employed evaluate the inflammatory conditions. Herein, the levels of IL-10, IL-6, IL-β, and TNF-α were measured for the evaluation.
As illustrated in figure 4, the levels of them were significantly raised when the mice were treated with DSS only, indicating the mice in the DSS group suffered strong inflammatory response. When the mice were treated with Prob or CHMSs simultaneously, the inflammatory factors were decreased, indicating the Prob or CHMSs are efficient in relieving IBD. The most marked results could be found in the CHMS + Prob group, which displayed the lowest inflammatory factors level among the group treated by DSS. These results were consistent with the results of the body weight and colon length, confirming that the probiotics and CHMSs both could play roles in treating IBD, and the probiotics-loaded CHMSs had the best therapeutic effect.  The intestinal epithelial barrier is crucial for protecting the body from bacteria translocation. However, when the body suffered IBD, the bacteria translocation would be occurred because of the disruption tight junctions or apoptosis. To study the effect of probiotics-loaded CHMSs on intestinal epithelial barrier, the bowels were stained with hematoxylin and eosin (H&E). As shown in figure 5(a), the bowels of the mice in  Control group had complete morphologies, while intestinal epithelial barrier of the mice in DSS group was broken, suggesting that the DSS could induce the damage of intestinal epithelial barrier. In contrast, the intestinal epithelial barrier of the mice in Prob and CHMSs groups were similar and better than that of DSS group, meaning the probiotics and CHMSs played protection roles. Similar to the results above, the CHMS + Prob group displayed the best morphologies among the groups treated with DSS, and the morphologies were close to that of the Control group, indicating the probiotics and CHMSs had synergistic effect on protecting intestinal epithelial barrier and IBD treatment.
The morphology changes of the intestinal epithelial barrier were further studied by fluorescent staining. Herein, we stained the Occludin 3 (OCC) and ZO-1, the two main protein components of tight junctions of the barrier, respectively, and the results was shown in figures 5(b)-(d). It could be found that both the OCC and ZO-1 were at a low level in the DSS group, indicating the large amount of tight junctions loss and increased permeability. The Prob and CHMSs groups displayed a relative higher level on the expression of ZO-1 and OCC, respectively, while the other protein was at a low level. The fluorescent straining of the CHMS + Prob group illustrated the both high levels of the OCC and ZO-1, confirming that the probiotics-loaded CHMSs had the best therapeutic effect.

Conclusion
In this work, we prepared novel probiotics-loaded CHMSs by a microfluidic device. The CHMSs had relatively uniform spherical shape and good biocompatibility. We dosed mice treated by DSS up with the probioticsloaded CHMSs, and found that both probiotics and CHMSs are helpful for IBD relieving and recovery, and the probiotics-loaded CHMSs are the most efficient due to the synergistic effect. These results indicated the probiotics-loaded CHMSs can be used as a new type of materials for IBD treatment.