Evaluating the Mucoprotective Effects of Glycyrrhizic Acid Loaded Polymeric Nanoparticles Against 5-Fluorouracil Induced Intestinal Mucositis in Murine Model via Suppression of In ammatory Mediators and Oxidative Stress


 Objectives: 5-Flourouracil (5-FU), a chemotherapeutic drug, is linked with severe deteriorating effects on intestine leading to mucositis. Further, Glycyrrhizic acid is a renown herbal medicine with combined mucoprotective, antioxidant and anti-inflammatory actions, however associated with pharmacokinetics limitations. Owing to its remarkable therapeutic action in inflammatory bowel disease inside the polymeric nanocarriers, we have tried to explore its activity against 5-FU led intestinal mucositis. Polymeric nanocarriers proved to be efficient drug delivery vehicles for long-term remedy against inflammatory diseases, however, yet not explored for 5-FU induced mucositis. Therefore, the study aimed to produce Glycyrrhizic acid loaded poly lactic-co-glycolic acid (GA-PLGA) nanoparticles to evaluate its protective and therapeutic effects on intestinal mucosa against 5-FU mediated mucositis. Methods: For the said purpose, GA-PLGA nanoparticles were prepared using modified double emulsion method, physicochemically characterized and tested for invitro drug release. Thereafter, mucositis was induced by 5-FU (50 mg/kg; IP) administration to the mice for the first three days (day 0, 1, 2) and orally treated with GA-PLGA nanoparticles till seventh day (day 0-6). Results: GA-PLGA nanoparticles significantly reduced mucositis severity as manifested through recovered body weight, diarrhea score, distress, and anorexia. Further, 5-FU induced intestinal histopathological damage, altered villi-crypt length, low goblet cell count, elevated pro-inflammatory mediators and suppressed antioxidant enzymes were reversed by GA-PLGA nanoparticles’ sustained release therapeutic action. Conclusion: Morphological, behavioral, histological, and biochemical results suggested that GA-PLGA nanoparticles found to be efficient, biocompatible, targeted, sustained release drug delivery nano-vehicle for enhanced mucoprotective, anti-inflammatory and antioxidant effects in ameliorating 5-FU intestine mucositis.


Introduction
Intestinal mucositis is clinically characterized through severe abdominal pain, nausea, vomiting, bloating, diarrhea, and abdominal cramps because of underlying mucosal in ammation and loss of epithelial lining that ends up in mucosal ulceration of the gastrointestinal tract (Blijlevens et al. 2000; Logan et al. 2009). It is one of the inevitable side effects associated with cancer chemotherapy (van Vliet et al. 2010), affecting almost 40% of the patients receiving standard doses and about 80-100% of the patients who received higher doses of chemotherapy (Keefe et al. 1997; Lalla et al. 2014). Chemotherapeutic agents, in general, interfere with rapidly dividing cells, therefore, fast proliferating mucosal cells are more prone to these agents (Duncan and Grant 2003). Intestinal mucositis halt chemotherapeutic regimen and alter the dosing schedule, thereby contributing towards higher mortality in the cancer patients (Naidu et al. 2004).
Recently, research paradigm concentrates on the resolution of intestinal injury caused by chemotherapeutic agents. 5-Fluorouracil (5-FU) is a renown anti-cancerous drug, used to treat various tumors such as head and neck, gastrointestinal and breast tumors. 5-FU, a uorinated pyrimidine analogue, exhibited its anti-metabolite chemotherapeutic action through inhibition of DNA synthesis, however, it can induce intestinal mucositis through intestinal epithelial disruptions and indiscriminate action against rapidly dividing, fast proliferating intestinal mucosal cells which have increased sensitivity to 5-FU (Soares et al. 2008; Wright et al. 2009). Although complete pathogenesis of 5-FU induced intestinal mucositis is not fully understood, however, main stages of mucosal barrier damage include initial in ammatory phase when DNA strands breakage occurs, characterized through production of reactive oxygen species (ROS) with minimal disruption to the epithelial lining. The second stage involves excessive burst of pro-in ammatory cytokines (IL-1, IL-6, TNF-α, IFN-γ) and transcription factors (NF-ĸB).
It leads to epithelial phase where cells proliferation terminates and cell death begins, then progressed towards ulcerative phase consisting of cell necrosis and ulceration which is followed by the nal healing phase that involves repair of damaged epithelial lining ( Unfortunately, the synthetic therapy available for 5-FU induced mucositis itself associated with deleterious side effects (Atiq et al. 2019). Currently, research has been directed towards exploring the medicinal uses of herbal products for a range of ailments to avoid unnecessary effects associated with synthetic compounds (Yuan et al. 2016). In this context, Glycyrrhizic acid, a triterpene glycoside, obtained from the roots of licorice plants, Glycyrrhiza glabra, holds a vital position because of its various medicinal bene ts including anti-in ammatory, anti-diabetic, antioxidant, anti-tumor, antimicrobial and anti-viral effects (Ming and Yin 2013). Recently, it has been explored that Glycyrrhizic acid possesses antiin ammatory activity through inhibition of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE-2) (Wang et al. 2011;Wang et al. 2017). Moreover, it can suppress in ammatory response through blocking NF-κB pathway, which is responsible for the production of pro-in ammatory cytokines and chemokines (Cherng et al. 2006;Wang et al. 2017). Further, it has been found to possess anti-cancerous properties enabling its dual usage including prevention of chemo-induced mucosal damage and proliferation of tumorous lesions (Khan et al. 2013;Su et al. 2017). The potential of Glycyrrhizic acid has been explored where it has reduced tumorous mass development and prevented in ltration of mast cells with attenuation of TNF-α level and prevent mucosal layer disruptions (Khan et al. 2013;Khan et al. 2018).
Furthermore, it was demonstrated that Glycyrrhizic acid owing to its anti-in ammatory and antioxidant properties have potential to serve as chemo-preventive agent (Bode and Dong 2015). Therefore, in the present study, Glycyrrhizic acid is explored to combat 5-FU induced intestinal mucositis and to highlight its anti-in ammatory, antioxidant, and muco-protective potential.
However, conventional delivery of Glycyrrhizic acid via oral route undergoes extensive rst-pass effect resulting in decreased bioavailability and reduced therapeutic e ciency in medical conditions like intestinal mucositis (Ploeger et al. 2000). Therefore, a drug-delivery system is required to deliver the therapeutic agent to the in amed intestines for an adequate duration without being metabolized in the gut. In this context, nanoparticle-based drug delivery system shows promising advantages to overcome conventional therapy limitations due to their nano-range size that ultimately results in improved accumulation and enhanced residence time at the in amed tissue via enhanced epithelial permeability and retention effect (eEPR) (Boisseau and Loubaton 2011;Zeeshan et al. 2019a). Previously, nanoparticles below 500 nm proved to be e ciently up taken by the in ltrated immune cells (Zeeshan et al. 2019a), which improves the time span for the drug release and therapeutic e cacy at the in amed tissues while overcomes diarrhea led clearance. For nanoparticle mediated drug delivery, a large range of polymers can be employed, however, polymers such as, poly lactic-co-glycolic acid (PLGA) hold a crucial role due to their biodegradability, biocompatibility and their ability to attain a sustained drug release over a longer period to achieve better therapeutic outcomes (Danhier et al. 2012). In our previously reported work, PLGA nanoparticles have been shown remarkable therapeutic e cacy in ulcerative colitis model (Ali et al. 2016;Zeeshan et al. 2019b).
To date, PLGA nanocarriers were employed only to cure oral mucositis (Takeuchi et al. 2018), however, no evidence is available to establish their role in 5-FU induced intestinal mucositis. In the present study, 5-FU induced intestinal mucositis is developed in BALB/c mice and treated with both Glycyrrhizic acid free drug and Glycyrrhizic acid loaded PLGA (GA-PLGA) nanoparticles separately through oral administration to demonstrate the therapeutic e cacy of the drug with or without its nanocarrier against 5-FU mediated intestinal toxicity through evaluation of multiple morphological, behavioral, biochemical, and histological manifestations.

Preparation of GA-PLGA nanoparticles
GA-PLGA nanoparticles were prepared by modi ed double W/O/W emulsi cation-evaporation method, as reported previously (Zeeshan et al. 2019b). Glycyrrhizic acid (monoammonium salt), purchased from Sigma Aldrich, Germany, was dissolved in 5mL of distilled water and added dropwise in 5mL of organic solution, containing 100 mg PLGA (Resomer RG502, 50:50, Evonik, Germany) in 5 mL ethyl acetate. The resultant primary emulsion was probe sonicated and added into external aqueous phase, comprising 5mL of 2% poly vinyl alcohol (PVA) solution (Sigma Aldrich. Germany). The obtained W/O/W emulsion was stirred to evaporate organic solvent, centrifuged, and freeze-dried to get lyophilized Glycyrrhizic acid loaded PLGA nanoparticles.

Physicochemical characterization
Average particle diameter was determined through dynamic light scattering technique (Brookhaven 90Plus Instrument, USA) in triplicate and results are expressed in mean ± standard deviation (S.D.). Zeta potential of nanoparticles was analyzed in triplicate using Malvern zeta sizer 2000 HS (Malvern instrument, UK) and stated as mean ± S.D. The percentage yield of the freeze-dried nanoparticles was obtained by weighing freeze-dried nanoparticles and calculated by the formula: The surface morphology of GA-PLGA nanoparticles was analyzed under scanning electron microscope (SEM) (Vega 3 TESCAN, Czech Republic).

Encapsulation e ciency (EE)
Drug entrapment within PLGA nanoparticles was determined through the spectroscopic estimation of free drug content in the supernatant collected after centrifugation of the nano-formulation at a wavelength (λ max ) of 252 nm (Ultraviolet-Visible Spectrophotometer, Agilent Technologies, US). %EE was measured through the formula: Afterwards, loaded drug content was determined spectroscopically at 252 nm by the method previously described (Zeeshan et al. 2019b). % Drug content was calculated from the formula:

Fourier transformed infrared spectroscopy (FTIR)
FTIR analysis of Glycyrrhizic acid drug, PLGA, and GA-PLGA nanoparticles was performed using potassium bromide (KBr) disc technique. FTIR spectra were observed in the region of 400-4000 cm − 1 wave number against % transmittance using Perkin-Elmer100 FTIR spectrometer (US).

In-vitro drug release studies
In-vitro drug release was tested at pH 1.2 and 6.8 that corresponds to the physiological pH values of gastrointestinal tract (GIT) organs. 50 mg of the GA-PLGA nanoparticles were suspended in simulated gastric uid (SGF), prepared according to United States Pharmacopeia. The release pro le was observed for 2 hours, afterward salts Na 2 HPO 4 .2H 2 O and KH 2 PO 4 were added and pH was adjusted to 6.8 with 0.1N NaOH solution. The release pro le was continued for 48 hours and the samples were collected at the pre-determined intervals from the buffers and analyzed under UV-Vis spectrophotometer for the drug release estimation.
2.6 Animal studies 2.6.1 Experimental Animals Experiments were performed on 3-4 weeks old BALB/c mice obtained from National Institute of Health (NIH, Islamabad, Pakistan) and kept under standard laboratory condition (20-25 C, 55 ± 5% humidity) in stainless steel cages. Mice were acclimatized in the same environment for a period of one week before experimentation.
All animal experiments were conducted according to the bioethical committee protocols of Quaid-i-Azam University, Islamabad for the care and use of lab animals (Approval no. BES-fbs-QAU2018-75). Mice were given ad libitum access to food and drinking water.

Induction and evaluation of mucositis
Mice weighing 25-30 g were randomly divided into four groups (n = 5/group). Number of mice in each group was decided while keeping in mind the ethical condition of minimal harm and applying resource equation to generate sound statistical data. Group one received vehicle only (drinking water) and served as a normal control group. Group 2, 3 and 4 received 5-FU (Sigma Aldrich, Germany) through intraperitoneal injection at a dose of 50 mg/kg for three consecutive days to induce experimental intestinal mucositis (Atiq et al. 2019). In addition, group 3 and 4 were treated with Glycyrrhizic acid free drug and GA-PLGA nanoparticles respectively through oral gavage, at a drug dose equivalent to 10mg/kg body weight for 7 days. The dose was optimized for anti-in ammatory activity in ulcerative colitis model in our previous study (Zeeshan et al. 2019b). For the rst three days, treatment either in the form of free drugs or nanoparticles was given thirty minutes before 5-FU IP administration. At the 7th day, mice were euthanized, and tissue samples were removed for further testing (Fig. 1).

Assessment of intestinal mucositis severity through morphological and behavioral parameters
Mice were monitored to assess 5-FU induced mucositis severity on daily basis. It was manifested through body weight loss, stool consistency, stress scores, anorexia and mortality rate (Basile et al. 2019). Therefore, mice from all four groups were observed daily for the body weight loss. The results were expressed as percent average body weight loss ± standard deviations (SD). Similarly, stools were examined for diarrheal indication and scored using Bowen's score system (Leocádio et  Likewise, daily distress scores were calculated based on cumulative average scores of fur/coat condition, change in temperament and reluctance to movement. Fur/coat was characterized as 0, shiny and smooth; 1, coat raised around neck; 2, coat raised around neck and belly; 3, coat raised around whole body with or without coat loss. Change in temperament was noticed and scored as 0, normal; 1, agitated behavior; 2, stress marks on body parts; 3, stress marks with hunching. Movement reluctance of the mice was scored as 0, normal activity; 1, movement on the placement of near object; 2, movement only after lifting mice up; 3, no movement even on handling (Atiq et al. 2019).

Evaluation of food consumption and survival analysis
Consumption of food on each day was recorded to assess food intake pattern of the mice in different groups (n = 5). Anorexia is a common symptom of chemotherapy induced mucositis (Basile et al. 2019). Therefore, daily food intake demonstrated development of FU-induced mucositis and the effect of therapy. Moreover, survival analysis was performed by observing the death rate of mice in each group daily.

Effect of 5-FU induced mucositis on organs
At the end of experiment, mice were euthanized, and entire small intestine and colon were excised. The weight of small intestine and colon length were recorded after removal of feces and adjacent fats. The tissues were xed in 10% formalin and frozen at -80ºC for further analysis. The weight of spleen was evaluated as an in ammation index. In addition, liver and kidney were resected and weigh to evaluate the effect of chemotherapy and the treatment on these vital organs.

Histopathological evaluation
For histological analysis, a 1-cm excised tissue sample of small intestine (jejunum) and large intestine from each mice group was xed in 10% buffered formalin and embedded in a para n wax. Afterwards, thin section of 5 µm thickness were incised from each sample, mounted on a glass slides and stained with hematoxylin and eosin (H & E). The prepared samples were observed microscopically at different magni cations (4x, 10x and 40x) to assess severity of intestinal damage. The histo-morphometric parameters for small intestine include measurement of small intestinal villus height (top of villus to villuscrypt junction; n = 20 villi), crypt depth (adjacent villi intussusceptions; n = 20) and calculating the index to estimate villi-crypt damage using TCapture imaging software (Tucsen Photonics Co. Ltd.) and a light microscope equipped with high resolution camera (ISH 500, Tuscen CMOS USB 2.0). The villi-crypts measurements were performed using ImageJ software (NIH, USA). While, large intestine histological score based on in ammation indexes including sum scores of surface epithelial loss, crypt destruction and in ammatory cells in ltration according to pre-determined scale (Erben et al. 2014).

Goblet cells evaluation
Similarly, 1 cm thin sections from each harvested small intestine (jejunum) were xed in 10% formalin and embedded in para n wax. Then sections of 5 µm thickness were stained with periodic acid schiffacian blue (PAS-acian blue) stain to visualize goblet cells and mucin content in the jejunum samples of each group (Ali et al. 2019;Stringer et al. 2009). The stained images were captured as mentioned above and processed to calculate number of intact goblet cells per villus, mucin-stained area containing MUC-2 protein and uorescence intensity of the blue colored stain using ImageJ software. Results are expressed as mean ± SD.

Enzyme linked immunosorbent assay (ELISA) for cytokines pro ling
The severity of intestinal in ammation was determination through expression of pro-in ammatory cytokines (IL-1β, IL-6 and TNF-α) in the jejunum sections excised from each mice group (n = 5 mice/group) using ELIZA kits (eBioscience, Inc., San Diego, CA, USA).
All assays were conducted according to the manufacturer's instructions.

Biochemical assays to determine intestinal antioxidant protection level
To assess antioxidant enzymatic activity in the intestine, 1 cm jejunum tissue was excised at the end of experiment from all groups (n = 5). The tissue was immediately immersed in phosphate buffer saline (PBS) (1 mL) and homogenized at 10,000 rpm until single cell suspension was obtained, then centrifuged (489g x 10 min) to get clear supernatant sample. Further, biochemical assays were performed to estimate antioxidant enzymatic levels of GSH, GST and catalase.
Reduced Glutathione (GSH) levels were determined in each mice group using Ellman's regent method (Arruda et al. 2013; Moron et al. 1979) under UV/Vis spectrophotometer at 412 nm. PBS was used as a blank. The obtained GSH values were expressed as nmol/g tissue sample ± SD.
Afterwards, Glutathione sulfotransferase (GST) assay was conducted to determine intestinal tissue detoxi cation level against oxidative damage. It was determined spectroscopically at 340 nm using potassium phosphate buffer as a blank. Assay mixture without supernatant was used as a control to determine non-speci c binding of the substrates (GSH and 1-chloro-2,4-dinitrobenzene (CDNB)) (Arruda et al. 2013;Habig et al. 1974). GST activity was expressed as nmol of CDNB conjugated/g tissue weight.
Furthermore, catalase activity was determined to nd its protective effects by mixing 3 ml of H 2 O 2potassium phosphate buffer (0.6 M) and 40ul of supernatant. Then decrease in absorbance was recorded spectrophotometrically at 240 nm. The same assay mixture without H 2 O 2 was used as a blank.
Results were stated as enzyme units/min/mg. One enzymatic unit is the time required to reduce the absorbance by 0.05 units at 240 nm (Hadwan and Abed 2016).

Physicochemical characterization of the prepared GA-PLGA nanoparticles
GA-PLGA nanoparticles were successfully prepared with an average particle size of 190.46 ± 7.49 nm, zeta potential of -11.67 ± 2.5mV and PDI lesser than 0.3 (0.123 ± 0.112). Particle size below 200 nm can easily accumulate and reside at the targeted site (Collnot et al. 2012;Zeeshan et al. 2019a). Moreover, nanoparticles were fabricated with good percentage yield (78 ± 2.51%) and drug entrapment (67.66 ± 2.5%) ( Table 1).  vibrations at 980 cm -1 , C-C at 702 cm -1 and NH out of plane at 617 cm -1 . The ngerprint regions of both drug and polymer were retained in the spectra which con rmed compatibility and exclude possibility of chemical interaction or bond formation. A relative change in the intensity of transmittance showed an overlap of the same functional group of both drug and the polymer which con rmed drug entrapment (Fig. 2c).

Morphological analysis
SEM analysis con rmed the particle size within required size range (< 200 nm). Further, SEM revealed nanoparticles morphological features as spherical in shape with smooth surface (Fig. 3).

In vitro drug release study
Drug release from the GA-PLGA nanoparticles was conducted in SGF (pH 1.2) for the rst 2 hours, afterwards conducted in PBS (pH 6.8) at 37ºC up to 48 hours. The pattern obtained for the drug release at pre-determined time points indicated initial burst release, followed by sustained release pattern up till 48 hours, accounting 77.67 ± 1.1547 % of drug release (Fig. 3). The drug release behavior is the typical pattern of drug release from PLGA nanoparticles (Ali et al. 2016).
3.5 Assessment of 5-FU mucositis induction and treatment through morphological and behavioral parameters 5-FU induction declines mice body weight, as manifested from daily weight assessment. It is one of the symptoms associated with intestinal in ammation. Treatment either with Glycyrrhizic acid (drug) or GA-PLGA nanoparticles restore mice body weight (Fig. 4a). However, the highest recovery in % body weight was noticed with GA-PLGA nanoparticles administration (p < 0.001). The two treatment groups differ by the factor 4, in terms of %BW (Fig. 4a). Furthermore, mice from all groups were assessed for stool consistency; initially softened stools were observed in all groups. After 3rd day, mice injected with 5-FU have suffered from pronounced diarrhea. Treatment groups relieved diarrheal symptoms to much extent, although not completely; GA-PLGA nanoparticles have shown signi cant improvement (p < 0.001) (Fig. 4b).
Induction of mucositis by 5-FU causes mice distress, as manifested from behavioral signs like di culty to move, anxious behavior and fur condition (score = 3; p < 0.001 ### ).), when compared with normal healthy mice. Glycyrrhizic acid and GA-PLGA nanoparticles showed signi cant stress reduction in the 5-FU induced in amed mice. At the end of experiment, Glycyrrhizic acid have decreased stress score up to 1.67 ± 0.57 (p < 0.05) and GA-PLGA reduced stress up to 1.25 ± 0.5 points (p < 0.001), when compared to 5-FU group (Fig. 4c).

Evaluation of daily food consumption and survival analysis
Mortality rate was assessed in all groups. Percentage survival rate was lowest for the 5-FU group; it differs widely from the normal mice group. While, free Glycyrrhizic drug improved survival rate up to 40%, when compare with 5-FU induced group. Whereas, GA-PLGA nanoparticles have greater survival rate (60%), in comparison to both 5-FU and Glycyrrhizic acid treated mice (Fig. 4d).
Anorexia is most commonly linked with chemotherapy (Basile et al. 2019); therefore, food intake was assessed in the mice of all four groups on daily basis. After 4th day of 5-FU intraperitoneal injection, a sharp decline in food consumption was observed (p < 0.001), as compared to the normal healthy mice. At the end of experiment, anorexia aggravates, and food intake left up to 31% of the initial food intake in the 5-FU group. Treatment groups (Glycyrrhizic acid or GA-PLGA nanoparticles) eradicate anorexia to a great extent (p < 0.001) in the 5-FU induced mice. Improvement in the nutritional intake was about 59% of the initial food intake in the mice treated with Glycyrrhizic acid, while it was up to 78% in the GA-PLGA nanoparticles administered mice (Fig. 5a).

Effect of 5-FU induced mucositis and GA-PLGA nanoparticles treatment on the organs
Effect of 5-FU on major organs like spleen, liver, kidney, small intestine and large intestine (colon) were investigated and compared with the treatment groups (Song et al. 2013). 5-FU signi cantly decreased weight of spleen (p < 0.01) and kidney (p < 0.0.5), whereas did not affect liver profoundly (Fig. 5b). Likewise, shortening of small intestine (p < 0.05) and colon (p < 0.001) was observed to a signi cant extent (Fig. 5c, d). Treatment groups, either Glycyrrhizic acid or GA-PLGA nanoparticles, retard the negative effects of 5-FU on organs, meanwhile improves splenic weight signi cantly (p < 0.01). Moreover, Glycyrrhizic acid restored kidney weight (p < 0.05) and GA-PLGA nanoparticles signi cantly normalizes the kidney weight (p < 0.01). Further, Glycyrrhizic acid recovered the length of small intestine and colon to a little extent, whereas, GA-PLGA nanoparticles have aptly lengthen the 5-FU a icted small intestine (p < 0.05) and colon (p < 0.001) (Fig. 5c, d).

Histopathological evaluation
Histological microscopy revealed intact epithelial integrity, well oriented villi, and aligned crypt cells, lack of in ammatory in ltrates in normal small intestine (jejunum) and large intestine (colon). Whereas, 5-FU mediate marked injury to both small and large intestine, but more severely affect small intestine, as shown in Fig. 6a-e. 5-FU induced small intestine histomorphological damage is evident from disrupted epithelium, shortened and distorted villi, vacuolated crypt cells, necrosis and immune cells in ltrations.
Marked decrease in villi length (p < 0.001) was noticed with slightly enlarged crypts (p < 0.01), which altered villi to crypt ratio (p < 0.001), as compared to normal intestine (Fig. 6a,c-e). Similarly, large intestine has pronounced deformation with epithelium loss, villi-crypt morphological destruction and immune cells intrusion (Fig. 6a,b). Treatment with Glycyrrhizic acid or GA-PLGA nanoparticles restored the histomorphological features to a wide extent. Small intestine villi-crypt length and ratio and large intestine histopathological scores were improved through Glycyrrhizic acid treatment of 5-FU mice.
3.9 Exploration of goblet cell count and mucin content 5-FU mediated in ammation resulted in the goblet cells decline and distortion in the jejunum, thus reducing the mucin content in the intestinal tissue. Treatment with Glycyrrhizic acid restore the goblet cell count to some extent (p < 0.01), while GA-PLGA nanoparticles increase the goblet cells number and architecture in the tissues predominantly (p < 0.001) (Fig. 7a,b). Furthermore, MUC-2 protein is mainly responsible for the mucin formation in the intestine (Tadesse et al. 2017), it was indirectly assessed through PAS-acian blue stained mucin area and resulting uorescence intensity within goblet cells using ImageJ software. Mucin stained area (p < 0.01) and uorescence intensity (p < 0.001) was found to be signi cantly altered on 5-FU induction as compared to normal control (Fig. 7c,d). Glycyrrhizic acid have a limited increase in mucin overall area, however, enhanced uorescence intensity profoundly. While, GA-PLGA nanoparticle markedly elevated the stained area (p < 0.05) and uorescence (p < 0.001), as compared to 5-FU group (Fig. 7c,d).

Discussion
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 e cient in ameliorating DSS induced ulcerative colitis (Zeeshan et al. 2019b). However, the scienti c evidence of Glycyrrhizic acid and its nano carrier is not available to prove its e cacy 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 e ciency and circular morphology ( Table 1, Fig. 3). Particles with size lesser than 200 nm accumulate in the in amed tissues through eEPR effect and prone to rapidly uptake by the immune cells recruited as a consequence of in ammation (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 ndings 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 e cacious 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 signi cantly 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 signi cantly alter liver weight, but accounting for 22% decline. The weight decline possibly driven because of 5-FU induced excessive in ammatory 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 signi cant 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 e ciently restored through 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-in ammatory cytokines production and resulting in ammation. 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 signi cant reduction was found to be with the encapsulated drug within nanoparticles (Fig. 8).
Furthermore, 5-FU led in ammation 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-in ammatory 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 ndings, 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 in amed intestinal tissues and enhanced therapeutic action through accumulation of nanoparticles at the site and minimizing drug clearance due to diarrhea.

Conclusion
The present work concluded that GA-PLGA nanoparticles with appropriate physicochemical properties and drug release behavior could be a promising approach to protect and cure small and large intestine Disclosure statement: