5-FU, an anti-pyrimidine and antimetabolite drug, is widely used for chemotherapeutic purpose to treat malignant tumors of colorectal and breast regions (Chang et al. 2012; Chen et al. 2020). However, the continuous use of 5-FU is associated with intestinal mucositis leading to destruction of microstructure of intestinal like villi, crypts, epithelial cells and goblet cells and manifested through clinical symptoms like body weight loss, heavy diarrhea, anorexia and anxiety (Song et al. 2013). 5-FU led mucositis severely compromised patient’s compliance receiving anti-neoplastic chemotherapy and cancer treatment must be discontinued due to severe mucositis.
Glycyrrhizic acid is a marvelous herbal medicine, used to cure several ailments from centuries. Recently, it was found effective in the treatment of methotrexate induced enteritis (Wang and Du 2016). Owing to its anti-inflammatory, anti-ulcer, mucoprotective, immunomodulatory, anti-bacterial, antioxidant and anti-cancerous actions (Ming and Yin 2013), it is the suitable candidate to protect intestine from injury. However, its protective effect is limited in the intestine because of rapid elimination from the gut in response to heavy diarrhea and degradation by indigenous enzymes. Therefore, a sustained therapeutic effect can be achieved by encapsulating Glycyrrhizic acid inside a bio-compatible nanocarrier. In our previous research, Glycyrrhizic acid loaded polymeric nanocarriers proved to be efficient in ameliorating DSS induced ulcerative colitis (Zeeshan et al. 2019b). However, the scientific evidence of Glycyrrhizic acid and its nano carrier is not available to prove its efficacy against 5-FU induced intestinal mucositis. PLGA is widely used biocompatible, biodegradable, and harmless polymeric carrier inside the living systems. Therefore, PLGA was used to prepare Glycyrrhizic acid loaded PLGA nanoparticles using double emulsion method in this study. Previously, it was found to be a sustained drug release carrier in the treatment of oral mucositis (Takeuchi et al. 2018). Thus, PLGA would be an ideal carrier to be explored for 5-FU led mucositis of the intestines.
The synthesized nanoparticles have optimal size range < 200 nm with lesser PDI, indicating monodispersed system. The drug has suitable encapsulation efficiency and circular morphology (Table 1, Fig. 3). Particles with size lesser than 200 nm accumulate in the inflamed tissues through eEPR effect and prone to rapidly uptake by the immune cells recruited as a consequence of inflammation (Zeeshan et al. 2019a). Further, invitro drug release studies indicated initial burst release of drug, followed by sustained release drug behavior till 48 hours (Fig. 3). It was found to be consistent with previous findings (Zeeshan et al. 2019b).
To explore, preventive and therapeutic role of GA-PLGA nanoparticles against chemotherapeutic induced intestinal injury, 5-FU induced intestinal mucositis in the murine mice was developed via intraperitoneal injection according to the mentioned protocol (Fig. 1) (Atiq et al. 2019). The features of experimental animal model are nearly the same as found in the reported studies (Gelen et al. 2018; Zhang et al. 2017), explicated through diarrhea, loss in body weight, lower food intake, histological architecture destruction (villi and crypts lengths), mucin depletion and decreased goblet cell count. In addition to 5-FU IP injection, treatment groups received once daily intake of Glycyrrhizic acid free drug or GA-PLGA nanoparticles through oral route. Oral route is most preferable in this condition since it completely covers the natural way to intestines. Both, Glycyrrhizic acid and GA-PLGA nanoparticles alleviates the clinical symptoms and reduced the severity of diarrhea, weight loss, anorexia and distress level (Fig. 4, Fig. 5). Aggressive behavior was very prominent in the untreated mucositis mice. Similarly, the 5-FU induced mucositis highly increased the mortality rate, which was slow down by the Glycyrrhizic acid or GA-PLGA nanoparticles treatment (Fig. 4). Although free drug proved to be effective in remitting the symptoms and increases the survival chance, however, GA-PLGA nanoparticle always turned out to be more efficacious which paved a way for PLGA and other polymeric nanocarrier systems to be further explored in the management of intestinal mucositis.
In previous animal experimental models, 5-FU significantly affect weight of spleen, kidney and to lesser extent liver (Gelen et al. 2018; Whittaker et al. 2016; Yang et al. 2017). In the current experiment, visceral organs weight assessment indicated that 5-FU decline splenic weight about 66% and kidney weight about 32%, as compared to the normal control (Fig. 5). Although, 5-FU did not significantly alter liver weight, but accounting for 22% decline. The weight decline possibly driven because of 5-FU induced excessive inflammatory response or immunosuppressive action of 5-FU that decreases splenic weight (Whittaker et al. 2016). Further, 5-FU shorten length of small intestine and large intestine (colon) (Fig. 5). Glycyrrhizic acid plain drug restore colon to an appropriate length (p < 0.01), but lesser lengthening effect on small intestine (Fig. 5). Whereas, GA-PLGA nanoparticles endure greater protection as manifested from significant increase in both small intestine (p < 0.05) and colonic length (p < 0.001).
Moreover, intestinal mucosal and epithelial layer destruction by 5-FU were efficiently restored through Glycyrrhizic acid or GA-PLGA nanoparticles administration. Our findings indicated that 5-FU induced histomorphological damage as manifested through shortened villi and crypt lengths, disintegrated epithelium, immune cells intrusion and goblet cells loss has been alleviated through Glycyrrhizic acid or GA-PLGA nanoparticles treatment. Constantly, GA-PLGA nanoparticles have superior mucoprotective effects as compared to free drug alone (Fig. 6). The results suggest that recovery of intestinal epithelia and mucosal damage could be the possible reason behind reduction in diarrhea (Zhang et al. 2019). Additionally, the histopathological findings were supported through investigating goblet cells count and mucin content. Consistent with previous data, 5-FU distort barrier function through loss of goblet cells number and disintegration of its structure leading to mucin discharge and depletion (Ali et al. 2019; Stringer et al. 2009). Again, the treatment elevated goblet cell count and MUC-2 protein containing mucin content in the intestine, with more notable improvement in GA-PLGA nanoparticles group (Fig. 7).
To date, pathogenesis of 5-FU mediated intestinal mucositis is not completely elucidated. However, several underlying elements that are responsible for mucositis damages DNA and cellular and sub-cellular structures because of overproduction of ROS, uncontrolled inflammation that destroy intestinal homeostasis and natural microbial flora alterations (Yan et al. 2020). In fact, ROS excessive production trigger the inflammatory pathways and pro-inflammatory cytokines production (Gelen et al. 2018; Yan et al. 2020). Among many others, some basic transcription factors activated by ROS are NF-ĸB and MAPK proteins that increase the production of pro-inflammatory cytokines including TNF-α, IL-6 and IL-1β (Chang et al. 2012; Song et al. 2013; Zhang et al. 2017). Elevation of pro-inflammatory cytokines activates a series of acute reactions to damage cells and tissues, thus causing mucosal injury and mucositis. Interestingly, Glycyrrhizic acid has been explored to mediate its action through inhibition of NF-ĸB and MAPK pathways (Wang and Du 2016), thus reducing pro-inflammatory cytokines production and resulting inflammation. In this study, Glycyrrhizic acid as a free drug or encapsulated within nanoparticles reduced the expression of TNF-α, IL-6 and IL-1β cytokines, thus protecting intestinal mucosa from 5-FU induced damage. More significant reduction was found to be with the encapsulated drug within nanoparticles (Fig. 8).
Furthermore, 5-FU led inflammation and ROS overproduction has weakened body’s defensive antioxidant system (Yan et al. 2020). Thus, the antioxidant protective enzymes could not be able to capture excessive ROS, leading to increased oxidative stress and cellular damage. In the present study, 5-FU depleted antioxidant enzymes including GSH, GST and catalase (Fig. 8) and causing mucosal damage. Glycyrrhizic acid proved to be an anti-inflammatory and antioxidant drug (Ming and Yin 2013); and reduces oxidative stress in DSS-induced colitis through elevation of antioxidant protective enzymes (Zeeshan et al. 2019b). Consistent with the previous findings, both free drug and encapsulated drug increased mucosal protection through rise in GSH, GST and catalase levels. While, GA-PLGA nanoparticles exhibited more pronounced effect (Fig. 8), because of sustained drug release at the targeted intestinal tissues with more localized therapeutic effect.
Hence, Glycyrrhizic acid proved to be an ideal candidate in alleviating intestinal mucositis, whereby, its pharmacokinetics limitation and prolonged protective and therapeutic effect is achieved through encapsulating it in PLGA polymeric nanocarrier. PLGA nanoparticles with their inert nature successfully deliver the drug to the inflamed intestinal tissues and enhanced therapeutic action through accumulation of nanoparticles at the site and minimizing drug clearance due to diarrhea.