There are some evidence showing that the optimal treatment effects of 5-ASA are dose-dependent, and its mucosal concentrations in the colon and rectum region are limited in response to the oral dosages (Naganuma et al., 2001, Binienda et al., 2020). Hence, this reasoned hypothesis is reinforced by the discovery that increased mucosal concentrations of 5-ASA correspond to enhanced clinical response rates. Notably, the utilization of a gastric mucoadhesive drug delivery system holds great promise for facilitating particulate uptake. This report identifies a transcytosis-based mechanism to achieve efficient delivery of 5-ASA by HbNPs to the colonic tissue.
The effect of 5-ASA-HbNPs on the severity of DSS-induced colitis
After consuming DSS in drinking water, mice showed severe clinical symptoms of colitis such as gross bleeding, diarrhea, and weight loss. As shown in Fig. 1a-c, there were obvious differences in the stool consistency, body weight loss, and bleeding between healthy and DSS groups. In the treatment groups, it was significantly decreased and the improvement of all parameters compared with the DSS mice were observed on the fifth days of observation. Furthermore, the control group (DSS+) had a higher DAI value than the healthy group (DSS−), which indicates the development of acute colitis in the mice, Fig. 1d. While, starting treatments, all treatment groups showed the lower levels of DAI scores than the DSS group. The group treated with 5-ASA demonstrated a low DAI value (3.0) attributed to the substantial quantity of 5-ASA administered (1 mg/ 20g) that can suppress the acute colitis as described previously (Iwao et al., 2018). At the same time, different dose of 5-ASA-HbNPs was administered to find the minimal effective administration dose and the results show that at the high amount of administered 5-ASA-HbNPs (200 − 100 µg/20g) mice had intestinal constipation and in some case eventually death. This might be explained by iron (HbNPs) that can often result from digestive disorders (Tolkien et al., 2015).
Notably, the 5-ASA-HbNPs containing 5% of the dose of the 5-ASA group (50 µg/20g), represented a similar low DAI value (3.5), suggesting that the anti-inflammatory effect of 5-ASA -HbNPs could be dose dependent (Naganuma et al., 2001). In addition, HbNPs groups containing equivalent of the dose of the 5-ASA-HbNPs group (2.5 mg/20g), also showed a similar trend like healthy group without specific side-effects. Whereas, compared with the DSS (+) group, a significant suppression of effects (DAI scores) was observed in the HbNPs/DSS (+) group.
Therapeutic effect of 5-ASA-HbNPs on macroscopic changes of the colon
In order to ascertain whether 5-ASA-HbNPs could exhibit a therapeutic impact on acute colitis, an assessment of colon weight/length ratios was conducted. As shown in Fig. 2, the severity of acute colitis was assessed by the reduced colon weight and the shortening of colon length (Oh et al., 2014, Huynh et al., 2019).
Among all the experimental groups, the shortest colon lengths and the lowest colon weight belongs to DSS-treated mice, as expected (Fig. S1). Furthermore, the 5-ASA-HbNPs and the 5-ASA groups exhibited markedly reduced colon weight/length ratios than the (DSS+) group. Notably, the 5-ASA-HbNPs recovered the colon shortening and colon weight loss induced by DSS administration (P < 0.01) and exhibited better outcomes. Treatment with HbNPs in the present of (DSS+) also resulted in a relatively lower ratio that explained by the pharmacological effects of Hb. The nano-sized formulation and The ability of free radical scavenging of Hb allowed significant accumulation of the particles at the site of inflammation (Gorudko et al., 2009). It seems that, HbNPs alone can suppress acute colonic inflammation and show a therapeutic effect in DSS-induced UC mouse model.
Release of 5-ASA-HbNPs in cecum and rectum
Consistent with the previous findings, 5-ASA formulations (gels, foams and suppositories) have the same effect in the treatment of proctitis and due to the limited access of suppositories to the rectum, the utility of that is limited (Harris and Lichtenstein, 2011). The gradient of 5-ASA concentrations across left and right colon with oral therapy depends on a number of factors such as proximal absorption (Sandborn and Hanauer, 2003). The more proximal absorption the higher rate of 5-ASA inactivation, the lower concentration of active 5-ASA are available to exert distal effects. Once the 5-ASA is absorbed, it may also be inactivated by acetylation in the gut epithelium, therefore, the lower concentrations of active 5-ASA are available to the distal colon (Vree et al., 2000, Zou et al., 2005). Our data demonstrate that, 5-ASA conjugated HbNPs was chemically and enzymatically stable in the upper intestine and the systemic absorption of that was negligible, while it was de-conjugated to release 5-ASA in the lumen of the colon and rectum (Vaezi et al., 2022). To assess the absorption and systemic concentration of 5-ASA-HbNPs, which is related to efficient colonic delivery of 5-ASA, the absorption of 5-ASA in the cecum and rectum was measured and compared with free 5-ASA after 4 h of oral administration. As shown in Fig. 3A (a, b), free 5-ASA accumulated about 10% of the dose, while the conjugated one greatly increased the accumulated amount of 5-ASA up to near 2 folds. On the other side, HbNPs greatly reduced the acetylation form of 5-ASA, a process that renders it inactive. As a result of proximal acetylation, about 40% of the contents remained unchanged, implying that over 80% of the orally administered substances successfully reached the colon and rectum. The similar result has been reported about 5-aminosalicyltaurine prodrug (Jung et al., 2006, Beiranvand, 2021).
In the scintigraphic studies, Basit et al. demonstrated that oral administration of simple 5-ASA tablets is not sufficient because the therapeutic concentrations are not reached beyond the sigmoid or descending colon and 5-ASA is effective if the combination therapy was applied, topically in the form of an enema (Varum et al., 2020). On the other hand, significant loss of prodrugs and also side effects due to insufficient time for activation can be considered as disadvantages of administrating these 5-ASA prodrugs. Colonic nano-delivery can be used to delay absorption to achieve optimal time-concentration for treatment, and the reduction of particle size with increased residence time in the colon is likely, even for patients with diarrhea (Hua et al., 2015). As illustrated in Fig. 3B (a, b), the highest mucosal concentration of free 5-ASA was achieved in the right colon, whereas the concentration in the rectum were negligible. While, an acceptable percentage of the administered 5-ASA-HbNPs reached to the left colon and rectum that can measure through the absorption spectra and image by in-vivo imaging. According to the white arrow in the fluorescence image Fig. 3B a, the suitability of HbNPs with mucoadhesive properties was confirmed by the accumulation of nanoparticles in the colon with enhanced permeability and retention (EPR) effect. This leads to releases 5-ASA gradually from the stomach to small intestine, passing through them without significant degradation, followed by the site-specifically degradation in the colon compared to the free 5-ASA.
The effect of 5-ASA-HbNPs on microscopic changes of colon
It is necessary to support the results of macroscopic observations with histological evaluation. The extracted tissue of colon from UC model mice were histologically analyzed and shown in Fig. 4A. The H&E staining revealed structural changes of colon tissues and the DSS challenge showed considerable inflammatory cell infiltration, hyperplasia, goblet cell loss, crypt loss, villus atrophy, and ulceration (Abbasi-Kenarsari et al., 2020). Also, the HbNPs/ DSS (+) group displayed damage to the epithelial cells of the mucosa. Some of the inflammatory damages were decreased in the colon in the 5-ASA and the 5-ASA-HbNPs groups, and the degree of damage was mild compared to the DSS (+) group. This indicates that mucosal repair and replacement with fibrous granulation tissue has occurred.
Moreover, the structural changes of colon tissues in red square with magnification 400X (Fig. 4 (f) 5-ASA-HbNPs) compared with the (a) healthy group. In the mucosa of (f) group due to DSS-induced acute colitis, the inflammatory cells have infiltrated into the stroma, the goblet cell architecture of the colon is damaged, and the structure of the gland has been destroyed (Xu et al., 2020). Interestingly, the better mucosal healing was occurred after 5-ASA-HbNPs treatment than free 5-ASA. In the submucosa, the present of edema decreased, which was mainly due to inflammatory cell infiltration. Within the muscularis propria layer, changes were noted in the smooth muscle cell structure, with nuclei adopting a more rounded form and cell boundaries becoming less distinct. Nonetheless, no inflammatory cells were detected. Furthermore, no observable distinction was evident in the serosa between the healthy and 5-ASA-HbNPs groups. Furthermore, the lower histological scores were observed in the mice treated with 5-ASA-HbNPs compared to 5-ASA alone (Fig. 4B). Therefore, these results indicated that although the colon lesion was significant in DSS-induced UC mice, the 5-ASA-HbNP formulation can suppress the colon inflammation with better structurally recovered after treatment.
The effect of 5-ASA-HbNPs on histological changes of other tissue
As shown in Fig. 5, the H&E staining in the cardiac histopathology shows a purplish blue color for cell nuclei, and cytoplasm and the extracellular matrix are stained pink, indicating normal histological structure in the heart tissues of the healthy group. Moreover, a healthy morphology without cardio toxicity was observed in the cardiac tissue of the 5-ASA-HbNPs group in where neutrophils and macrophages did not accumulate.
The healthy portal tract of liver shows the bile duct cuboidal epithelium without lymphocytic infiltration (Blanco et al., 2004). In the case of 5-ASA-HbNPs group, proliferation of the bile ductules and infiltration with inflammatory cells are not evident. For the normal kidney, a thin connective tissue capsule was observed with underlying renal cortex (at the top of the figure), which contains a glomerulus and surrounding tubules with cuboidal epithelium. Inflammation and toxicity in the kidney can occur in the renal tubule, interstitium, and glomerulus. The 5-ASA-HbNPs group had similar results compared to the healthy group, and inflammatory cell congestion and infiltration was not observed. The staining of the spleen structures for both groups depict normal white pulp (purple) and red pulp (pink) without significant structural disturbances.
Quantitation of gene expression using real-time PCR
One of the main pro-inflammatory cytokines including tumour necrosis factor-α (TNF-α) plays an important role in the severity of IBD (Gareb et al., 2020). Since, TNF-α can cause damage gut wall tissue and increase luminal antigens has led to intense activation of the innate and adaptive immune system (Geremia et al., 2014, Friedrich et al., 2019). Therefore, the evaluation of TNF-α tissue levels in the lamina propria and colon of IBD patients result in better screen regulation in immune system response and decrease inflammation situation. An effective treatment correlates with reduce localized TNF-α in the gastrointestinal tract (GIT). As a result, low level of TNF-α production led to regulate activity, relapse and type of IBD (Olsen et al., 2007, Hozumi et al., 2013).
Figure 6 shows the TNF-α production regulation during treatment on the 5-ASA and the 5-ASA-HbNPs, which it is the main inflammation cytokine in IBD. For this reason, a practical and efficient treatment can decrease secretion TNF-α. In this study we were able to show the mice that have taken the 5-ASA-HbNPs have significantly lower TNF-α production than DSS mice (P < 0.001) 95% CI: 0.5139, SEM: (0.6000 ± 0.02000). However, the mice were treated by common 5-ASA did not show significantly reduce TNF-α production (P < 0.46) 95% CI: 46.81, SEM: (8.130 ± 8.990).