Trehalosomes: Colon targeting trehalose-based green nanocarriers for the maintenance of remission in inflammatory bowel diseases

Wessam H. Abd-Elsalam a,*, Mona M. Saber b, Samar M. Abouelatta c

Abstract

The use of non-steroidal anti-inflammatory drugs (NSAIDs) in inflammatory bowel diseases (IBDs) are contradictory between their beneficial effect in alleviating inflammation, and injurious outcomes in aggravating the symptoms of colitis. The study aimed to formulate trehalosomes (THs); innovative green trehalose-based nanocarriers, to alleviate the inflammation symptoms that might be provoked by NSAIDs in IBDs; as trehalose was proved to lighten the inflammation and the oxidative stress response, besides its resistance to the acidic conditions that rises its potentiality as a means for colon targeting. THs were fabricated using L-α-phosphatidylcholine (PL), trehalose, and transcutol, in a single step circumventing the incorporation of any organic solvent and loaded with Tenoxicam (TXM) as a model anti-inflammatory medication. A full 23 factorial design, using Design-Expert® software, was established to optimize the formulation variables. The optimized formulation composed of trehalose: PL at a weight ratio of 1:1, 377.72 mg transcutol, and sonicated for 4 min, possessed a spherical shape with a size of 268.61 nm and EE% of 97.83% and released 70.22% of its drug content over 24 h. The supreme protective action of TXM loaded THs compared to TXM suspension and drug-free THs was revealed by the suppression of the inflammatory biomarkers and the improved histopathology of the colonic tissue in male New Zealand rabbits. IL-1ß, IL-6, and TNF-alpha levels were notably dampened with TXM loaded THs, and oxidative stress markers, measured as GSH and MDA, were significantly altered. The study indicates the successful role of green THs in colon targeting and its anti-inflammatory characteristics in protecting against possible NSAIDs-driven exacerbation of colitis.

Keywords:
Trehalosomes Green
Phospholipids Trehalose
Colon targeting
Oxidative stress and inflammatory biomarkers

1. Introduction

Inflammatory bowel diseases (IBDs), namely Crohn’s disease and ulcerative colitis, are chronic and relapsing gastrointestinal inflammatory disorders that affect millions of people worldwide [21,34,5]. IBDs are proven to be triggered by a deregulated response of the immune system to the normal non-pathogenic gut microbiome that leads to an intestinal damage [18]. Besides, genetic tendency and environmental risk factors might contribute to the progression of the disease [7]. Ulcerative colitis primarily comprises diffused mucosal inflammation with the involvement of the rectum; due to the release of the inflammatory mediators and the progress of ulceration in the superficial mucosa. Alternatively, in Crohn’s disease, macrophages are aggregated mostly in the terminal ileum to form non-caseating granulomas. Peyer’s patches are the first position to bear mucosal lesions in Crohn’s disease. Crohn’s disease could be differentiated from ulcerative colitis through being patchy and segmental, where the inflammation is typically transmural [43].
Non-steroidal anti-inflammatory drugs (NSAIDs) are used in the treatment of various inflammatory conditions, however, their administration to patients with IBDs should be monitored cautiously to circumvent the probability of exacerbation of the intestinal inflammation. That highlights the need for advanced delivery systems that might decrease the possible undesirable effect of NSAIDs in worsening the intestinal inflammation in patients with IBDs. Nano-drug delivery systems targeting the colon are administered orally in most cases, and they are considered vital tools to cure IBDs [19]. These systems are supposed to get localized in the targeted parts, and hence the efficacy of the therapeutics is enhanced, and the systemic toxicity is minimized [33]. To deliver drugs specifically to the colon, formulations are designed to target pH, transit time, pressure, and microflora [6], where pH- dependent drug delivery is a unique strategy that utilizes acid- resistant polymers, such as Eudragit®, as coatings that are triggered only by colonic pH. Recently, Nutriose®, an indigestible dextrin water- soluble fiber that is stable at low pH, was employed in the fabrication of Nutriosomes [8]. In a parallel line, trehalose is a chemically-unique disaccharide, owing to its resistance to the acidic conditions and the lack of direct intramolecular hydrogen bonding [39]. In addition, trehalose was proved to alleviate inflammation and oxidative stress response, signifying its role in the treatment of chronic diseases that relate to oxidative stress and dysfunction of autophagy [32].
The originality of the present study can be summarized in the ability to formulate trehalosomes (THs) in a single step, adopting a green technique; without the use of any organic solvent, and the incorporation of trehalose for alleviating the possible deterioration that would be exacerbated by NSAIDs. THs are self-assembled nanovesicles fabricated using L-α-phosphatidylcholine (PL), trehalose, and transcutol, and loaded with Tenoxicam (TXM) as a model anti-inflammatory medication. The aqueous phase was charged with a considerable amount of trehalose, as a potential anti-inflammatory adjuvant, and a pH- dependent colon targeting agent. Design-Expert® software was utilized to determine the impact of the selected formulation variables on THs properties, and elect the optimized formulation. In addition, the advanced anti-colitis effect of THs was hypothesized, and to validate the proposed hypothesis, the in vivo outcome of the optimized TXM loaded THs was appraised against TXM suspension and drug-free THs in male New Zealand rabbits with induced colitis; to establish its prophylactic ability. The inflammatory biomarkers; IL-1ß, IL-6, and TNF-alpha, and the oxidative stress markers, GSH and MDA levels were measured to assess the anti-inflammatory response, in addition, a histopathological analysis of the colonic tissue was conducted.

2. Materials and methods

2.1. Materials

Tenoxicam (TXM) was supplied as a gift sample by Epico Co. (Cairo, Egypt). L-α-phosphatidylcholine (PL); extracted from egg-yolk, and trehalose were purchased from Sigma–Aldrich (MO, USA). Transcutol was donated by Gattefosse (Saint-Priest, France). Semi-permeable membrane tubing (Spectra Por©; MWCO 12,000–14,000) was procured from Spectrum Laboratories Inc., (Rancho Dominguez, CA). Phosphate salts; as potassium dihydrogen and disodium hydrogen, were obtained from Merck (Darmstadt, Germany). ELISA assay kit was acquired from Sunlong (Zhejiang, China).

2.2. Analysis of the formulation variables via a factorial design

A 23 factorial design was established and analyzed to enable the selection of an optimized formulation by means of Design-Expert® Software (Stat-Ease, Inc., Minneapolis, Minnesota, USA). The weight ratio of trehalose: PL (A), the amount of transcutol (B), and the sonication time (C), were set as independent variables, while the measured responses were particle size (PS), polydispersity index (PDI), zeta potential (ZP), entrapment efficiency percent (EE%), and the cumulative percent of TXM released after 2, 8 and 24 h; Q2, Q8, and Q24, Table 1. The optimized formulation was proposed to possess the highest EE %, ZP (as absolute value), and Q24, whilst PS, PDI, Q2, and Q8 were minimized.

2.3. Preparation of TXM loaded trehalosomes (THs)

Composition of the prepared TXM loaded THs (un-coded units) and their observed responses. TXM loaded trehalosomes (THs) were assembled in a single step, avoiding the incorporation of any organic solvent. During the preparation, PL, TXM (10 mg), and transcutol were mixed using a magnetic stirrer at 60 ◦C. An aqueous solution of trehalose (2 ml) was prepared and then added dropwise to the mixture. THs dispersions were formed spontaneously and were kept stirred for 30 min at 60 ◦C, then left at room temperature for cooling down. The prepared THs were subjected for different time intervals to ultrasonic waves (Crest Ultrasonics Corp., NJ, USA). As a step of purification, THs were separated from the dispersion by centrifugation of samples at 20,000 rpm for 1 h at 4 ◦C using cooling ultracentrifuge (Sigma 3-30 KS, Sigma Laborzentrifugen GmbH, Germany), the pellet was washed 3 times with bi-distilled water and THs were separated by ultracentrifugation and the supernatant was discarded. Table 2 represents the composition of the prepared TXM loaded THs. THs were kept in the refrigerator at 6 ± 2 ◦C till further characterization.

2.4. Characterization of TXM loaded THs

2.4.1. Particle size (PS), polydispersity index (PDI) and zeta potential (ZP) determinations

Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) was used to record the average PS, PDI, and ZP of TXM loaded THs. Before carrying out the measurements, THs were appropriately diluted with bi-distilled water. Measurements were conducted for three different batches [1].

2.4.2. TXM entrapment efficiency percent (EE%) determination and calculation

To separate THs from the dispersion, centrifugation of samples at 20,000 rpm for 1 h at 4 ◦C was conducted using a cooling ultracentrifuge (Sigma 3-30 KS, Sigma Laborzentrifugen GmbH, Germany). The pellet was washed three times with bi-distilled water, and the washes were added to the supernatant. The supernatant was collected and its TXM concentration was determined spectrophotometrically at λmax 368 nm (Shimadzu, model UV-1601 PC, Kyoto, Japan). The calculation of EE% was as follows: Total amount of TXM − Unentrapped TXM EE% = Total amount of TXM × 100 (1)

2.4.3. In vitro drug release experimentation

The in vitro release experimentation of TXM from THs and the aqueous suspension of TXM were conducted within a Type II-USP Dissolution Testing Station at 37 ± 0.5 ◦C (VK 7000, Vankel Industries, Inc., NJ, USA). A sample of each THs was packed in a semi- permeable membrane tubing and then placed into the dissolution vessel. For 2 h, 900 ml of 0.1 N HCl (pH 1.2) were added to the vessel, followed by phosphate buffer (pH 6.8) for 6 h, then finally phosphate buffer (pH 7.4) for 16 h. Aliquot samples were collected at certain time intervals and replaced with a fresh media, and TXM content was analyzed spectrophotometrically at λmax 368 nm [12].

2.4.4. THs behaviour at gastrointestinal pH

Before the experiment, the pH value of the optimized TXM loaded THs formulation was determined via a digital pH meter (Jenway, UK) at 25 ◦C. To study THs behaviour at the gastrointestinal pH, 1 ml of the optimized THs was diluted with 9 ml of 0.1 N HCl solution (pH 1.2) or phosphate buffer solution (pH 7.4), then loaded into Type II-USP Dissolution Testing Station (VK 7000, Vankel Industries, Inc., NJ, USA), thermostatted at 37 ± 0.5 ◦C, maintained for 2 h at pH 1.2, or for 6 h at pH 7.4. After the dilution and at the termination of the experiment, particle measurements (PS, PDI, and ZP) of the optimized THs were determined [8].

2.4.5. Transmission electron microscopy (TEM)

Micrographs; showing the shape and the topography of the optimized THs, were captured via TEM (Joel JEM 1230, Tokyo, Japan). The optimized THs formulation was properly diluted and adsorbed on a copper grid. The grid was then left to dry at room temperature before being examined under a transmission electron microscope [2].

2.4.6. Stability studies

The optimized THs was charged in a tightly closed vial and kept in the refrigerator (6 ± 2 ◦C) for 3 months. The scrutiny of the deviations in the physical properties of THs (e.g., visual appearance, PS, PDI, and ZP), and TXM EE% were assessed to judge the formulation instability [3]. The formulation was prepared and reported in triplicates. Results are expressed as mean ± standard deviation. The statistical significance of the difference between the results was compared using Student’s t-test. The level of the statistical significance was considered at P < 0.05.

2.5. In vivo studies

2.5.1. Study design and samples collection

The experimental procedures in this study were conducted as per the approval of the ethics committee of the Faculty of Pharmacy, Cairo University, Egypt (PI 2609). The purpose of the in vivo study was to verify the boosted protective effect of TXM loaded THs compared to that of TXM aqueous suspension and drug-free THs on the colonic tissue. A randomized parallel design was applied, where twenty-five male New Zealand rabbits; weighing 2–2.5 kg, were randomly assigned to five groups, with five rabbits per each. Group I: Negative control; where rabbits did not receive any treatment, Group II: Positive control, rabbits with induced colitis, Group III: rabbits with induced colitis pre-treated with TXM suspension, Group IV: rabbits with induced colitis pre- treated with the optimized drug-free THs. Group V: rabbits with induced colitis pre-treated with the optimized TXM loaded THs. Prior to the experimentation, animals were kept fasted for 24 h but allowed free access to water. Groups III, IV, and V were administered orally and once daily for 1 week, before the induction of colitis, TXM suspension, the optimized drug-free THs, and the optimized TXM loaded THs dispersion (containing 10 mg TXM), respectively. Induction of colitis was done following the method described by [12] via the rectal administration of acetic acid (2%, v/v). An hour later, rabbits were then permitted food and water without treatments. On the next day, rabbits were sacrificed, and 8 cm of the distal colon was collected, and longitudinally cut along the mesenteric edge. The colonic segments were rinsed with a normal saline solution, and preserved in formalin (10%, v/v) for further investigations.

2.5.2. Measurement of oxidative stress

The colonic tissue was homogenized in a normal saline solution and then centrifuged. The supernatant was used to determine the content of Malondialdehyde (MDA) and Glutathione (GSH). The determination of MDA in the distal colonic specimen was performed according to the method described by Draper and Hadley [10]. For the determination of GSH content, a method previously reported by Ellman was applied [13].

2.5.3. Biochemical analysis

An enzyme-linked immunosorbent assay (ELISA) kit was utilized to measure the levels of Interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alfa (TNF-α). Sandwich ELISA kits involve the use of a pre-coated 96 well plate with antibodies specific to IL-1β, IL-6, and TNF-α to detect the correspondent soluble cytokines in the samples. The amount of the cytokine produced is proportional to the intensity of the yellow colour developed, which was measured at 450 nm.

2.5.4. Statistical analysis

All data are stated as mean ± SD (n = 5). The data were analyzed by one-way analysis of variance (ANOVA), then Tukey-Kramer multiple comparison test using Graphpad InStat, version 7.01 (Graphpad, USA). The level of the statistical significance was considered at P < 0.05.

2.5.5. Histopathological evaluation

The preserved specimens were dehydrated, then entrenched in paraffin slabs. Afterward, sections of 4–6 µm thick films were cut, de- paraffinized using xylol, and stained with hematoxylin and eosin [22], the sonication time (C) on, A) PS, B) PDI, C) ZP, and D) EE%. and then examined under a light microscope fitted with a digital camera (Leica Microsystems, Germany).

3. Results and discussion

3.1. Preparation of TXM loaded THs by green technique and analysis of factorial design

THs were formulated through a single-step method, adopting no organic solvent incorporation, where both PL and trehalose were locked in the aqueous compartment. In addition, transcutol was introduced as a non-toxic and biocompatible penetration enhancer, which is known to be soluble in both water and oils. Moreover, transcutol functions as an intestinal absorption enhancer via the inhibition of multidrug resistance protein 2, known as ATP-binding cassette subfamily C member 2, an efflux transporter protein mainly expressed in the liver, kidney, and intestine [4]. Upon applying ultrasound waves, the dispersion was capable of forming vesicles with uniform size and homogeneity. During the analysis of the full 23 factorial design, values above 4 for the adequate precision ratio, and a difference less than 0.2 between the predicted R2 values and the adjusted R2 values, were observed in all responses.

3.1.1. Effect of formulation variables on PS

The reduction of the size of drug delivery carriers to the nanoscale could offer better IBDs therapy [20]. The PS of THs, depicted in Table 2, ranged from 296.85 ± 4.80 to 730.8 ± 15.56 nm. The normal plot in Fig. 1(A) and 3D surface plots Fig. 2(A) displayed the effect of the studied factors on the PS. Upon the statistical analysis, it was observed that the weight ratio of trehalose: PL (A) was insignificant, while the amount of transcutol and sonication time (B and C) have a negative significant effect (P < 0.05) on the PS of the prepared THs. Increasing the amount of trancsutol resulted in the formation of smaller vesicles. This could be attributed to its hydrophobicity (HLB = 4) which decreased water imbibition as well as reducing the surface energy, thus forming smaller vesicles. Aboelwafa et al. reported similar results on comparing the PS achieved after the addition of transcutol to that of PEG 400 (HLB = 11.5) [16]. Furthermore, it was observed that increasing the sonication time decreased the PS of THs. This is due to exposure of THs to the ultrasonic vibrations with higher energy for a longer period [36]. These findings comply with the results reported by Farid Badria et al. [5], where sonication of AKBA nanospanlastics for 5 min caused a significant decrease in the size of the vesicles.

3.1.2. Effect of formulation variables on PDI

The homogeneity of the particle size distribution is expressed by the values of PDI, and its value is designated between 0 and 1. Smaller values indicate uniformity of PS [14]. In this study, PDI values of 0.22 ± 0.01 to 0.83 ± 0.17 were detected, as shown in Table 2. The normal plot and 3D surface plots demonstrated the factors affecting PDI, Fig. 1(B) and 2(B), respectively. Sonication time (C) showed a negative significant effect (P < 0.05) on PDI, whereas the other studied factors were insignificant. PDI decreased with increasing the sonication time. This observation correlate size and PDI; where sonication time antagonizes both responses significantly. This could be attributed to the continuous supply of energy during a longer sonication period; yielding a narrower size distribution through the disruption of the vesicles, as previously described by Emami et al. [15].

3.1.3. Effect of formulation variables on ZP

ZP is considered the main parameter in determining the stability of a colloidal dispersion. Absolute large values of ZP confirm the stability of the colloidal dispersions through the repulsion between particles, thus avoiding aggregation and consenting to the formation of highly dispersed systems [1]. ZP of THs with values between − 36.20 ± 0.28 and − 41.95 ± 1.34 mV are recorded in Table 2. As per Fig. 1(C) and 2 (C), ZP was found to be synergistically and significantly (P < 0.05) affected by the amount of transcutol (B), while trehalose: PL weight ratio and sonication time (A and C) were insignificant. This could be referred to the ability of transcutol to interpenetrate the phospholipid bilayer, and consequently modifying the lipid bilayer packing [28]. These results follow that of Manca et al., who designated that increasing the amount of transcutol increased the stability of the colloidal dispersions [27].

3.1.4. Effect of formulation variables on EE %

The percent of TXM, entrapped inside THs, is of great importance for its success as a new drug delivery system. The EE % ranged from 98.60 ± 0.16 to 99.07 ± 0.02% as presented in Table 2. According to Fig. 1(D) and 2(D), it is relevant that the amount of transcutol (B) and sonication time (C) were antagonistically affecting the amount of the drug entrapped, whereas the trehalose: PL weight ratio (A) was insignificant. Increasing transcutol amount resulted in a decrease in EE %, which might be referred to the partitioning of TXM from the internal to the external phase, and thus causing a drug leakage and reducing its entrapment. Besides, a longer sonication time led to a decrease in EE % due to the breaking and the reformation of the vesicles during ultrasonication [14]. The harmonious effect of the higher amounts of transcutol and the longer sonication time can be explained in light of the interfacial tension reduction, thus facilitating TXM partition during a longer sonication time and a higher shear mixing [38]. 3.1.5. Effect of formulation variables on Q2, Q8, and Q24
The cumulative percent of TXM released over 24 h in buffers simulating the gastrointestinal fluids was studied as shown in Fig. 3(A). Regarding colon targeting behaviour of THs, the model was found to be significant during the studied time intervals; where most of TXM dose was released at pH 7.4. In the case of the percent of drug released after 2 h (Q2), the maximal amount of the drug released did not exceed 7.99 ± 0.19% over 2 h. As displayed in Figs. 4 and 5, trehalose: PL weight ratio (A) has a significantly (P < 0.05) negative effect on the percent of the drug released. As previously reported, PL is known to be unstable in aggravated acidic conditions of the stomach, and thus failed to delay the drug release in the stomach [44]; this is contradictory in this model as a minimal amount of the drug was released, which should be solely attributed to the presence of trehalose which is known for its acid resistance characteristics. This might be referred to its polar nature which facilitates the formation of a hydration layer via water replacement, and finally stabilizes the PL bilayers under vigorous acidic conditions, and minimizing the drug release in the gastric pH [41]. The percent of drug released after 8 h and 24 h (Q8 and Q24) was found to range from 8.20 ± 0.81 to 33.95 ± 1.11 % and from 32.33 ± 2.48 to 81.88 ± 0.11 %, respectively. Trehalose: PL weight ratio (X1) showed a synergistic significant effect (P < 0.05) on TXM release in the neutral pH. Phospholipids are well known for their degradation in the gastrointestinal fluids, and thus increased the percent of the drug released.
These findings are in agreement with the results previously reported by Coma-Cros et al. [30] and Liu et al. [24]. Moreover, trehalose is known for its acid resistance characteristics due to its 1–1 alpha bond, which decreased the release of the drug in the acidic pH, however, higher amounts of trehalose enhanced the release of TXM in both buffers; pH = 6.8 and 7.4. These results are in line with the findings achieved by Dolinina et al. [9] and Li et al. [23]. As displayed in Figs. 4 and 5, the amount of transcutol (B) and Sonication time (C) were non-significant at all studied time intervals (P > 0.05).

3.2. Optimization of the design

THs were optimized to obtain vesicles possessing minimum PS and PDI, maximum ZP to enhance the stability of vesicles, and showing maximum TXM EE%, along with a controlled-release profile with a maximum drug release percentage after 24 h. THs optimized formulation composed of trehalose: PL weight ratio of 1:1, 377.72 mg transcutol, and sonicated for 4 min, where desirability equal to 0.706 was achieved. The validity of the design was checked by calculating the residual as shown in Table 3. In addition, the release profile of TXM from the aqueous suspension and the optimized THs is displayed in Fig. 3(B).

3.3. Characterization of the optimized formulation

3.3.1. THs behaviour at gastrointestinal pH

The pH value for the formulation was found to be 6.84, which falls almost in the neutral range; excluding pH effects on the zeta potential measurements. Particle characteristics (PS, PDI, and ZP) of the optimized formulation were determined after 2 h in 0.1 N HCl (pH = 1.2), and after 6 h in phosphate buffer (pH = 7.4). After 2 h in pH = 1.2, it was observed that the PS and PDI showed non-significant change (P > 0.05) as shown in Table 4. This indicates the stability of THs in the acidic medium which might be due to the strong acid resistance character of trehalose, thus prevented the erosion or the change in the vesicular structure [29]. ZP showed a marked change from a highly negative to a slightly positive charge because of the protons found in the acidic medium. At the neutral media, a significant decrease in PS was detected (P < 0.05), also ZP decreased without changing to a positive sign as observed in the acidic medium [8].

3.3.2. TEM and stability studies

TEM micrographs of the optimized THs, Fig. 6, showed spherically shaped, non-aggregating vesicles. Regarding the stability results, the optimized THs showed a non-significant change (P > 0.05) in PS, PDI, or zeta potential excluding aggregation, Table 5. Furthermore, no significant change (P > 0.05) in drug EE % was revealed; confirming the stability of the optimized formulation upon storage at 6 ± 2 ◦C.

3.4. In vivo study

Results of the studies investigating the effect of NSAIDs on IBDs were opposing; relating its valuable effect in relieving the inflammation, and its potential to aggravate the symptoms and precipitate colitis [25]. Apart from their possible use as anti-inflammatory agents against IBDs, their use is vital in numerous inflammatory conditions putting their administration even in patients with colitis inevitable [40]. TXM is used in the treatment of various cases of chronic inflammation; including rheumatoid arthritis and osteoarthritis, and although its gastrointestinal effects in patients with IBDs have not been fully elucidated, it is used with caution in patients suffering from ulcerative colitis or Crohn’s disease due to its association with exacerbations. On the other hand, trehalose was proved to alleviate inflammation and oxidative stress. Its usefulness was reported in improving the status of many diseases involving chronic inflammatory response; thus trehalose could be beneficial in alleviating the chronic diseases linked to the oxidative stress [32]. To overcome the limitations that NSAIDs have in their use in conditions involving gastrointestinal inflammation, nanovesicles incorporating TXM and trehalose were formulated in an attempt to decrease the intestinal damage caused by provoking the inflammation.

3.4.1. MDA and GSH contents

As shown in Fig. 7(A), after pre-treatment with TXM suspension oxidative stress increased in the colonic tissue as evident by the increase in MDA levels by 4.4 folds compared to the respective untreated negative control group. Administration of TXM loaded THs dispersion resulted in a 3.3folds increase in MDA which was statistically significant compared to a 4.4folds increase in the case of TXM suspension, and 3.7folds for drug-free THs dispersion. Similarly, and as exhibited in Fig. 7B, GSH levels showed the lowest suppression; of only 35%, after TXM loaded THs pre-treatment compared to the untreated negative control group. While, TXM suspension and drug-free THs dispersion decreased GSH by 65% and 56%, respectively. In line with these results, Echigo et al. have reported that the presence of trehalose in the formulation decreased the oxidative stress significantly in the colon [11].

3.4.2. Inflammatory markers levels

As represented in Fig. 7 (C, D, and E) and according to the results obtained from the measurement of the inflammatory markers; IL-1β, IL- 6, and TNF-α levels, group III that received TXM suspension showed that the drug aggravated the inflammatory reactions in the colon resulting from the induction of colitis by the acetic acid. Pontes-Quero et al. demonstrated an increase in IL-1β and IL-6 with tenoxicam treatment in vitro in human articular chondrocytes and murine RAW264.7 macrophages [35]. Moreover, exacerbation of inflammatory bowel disease has been linked to NSAIDs use [25]. Despite the useful role of TXM in several musculoskeletal disorders and inflammatory conditions, its use is limited in patients suffering from IBDs. In the current study, incorporation of TXM into THs resulted in attenuation of the inflammatory response compared to the positive control, and this was proved by the decrease in IL-1β, IL-6, and TNF-α by 7.8, 1.8, and 2folds, respectively, while the inflammatory markers decreased by 3.5, 2.5, and 4folds on comparing TXM loaded THs to TXM suspension. In addition, TXM loaded THs decreased IL-1β, IL-6, and TNF-α by 1.9, 1.4, and 2.6folds, respectively, compared to drug-free THs dispersion. Several studies investigated the effect of trehalose on inflammation, including Taya et al., who showed that trehalose treatment suppressed IL-1β and TNF-α production [37]. Other studies showed that trehalose significantly blunted IL-1beta, and TNF-alpha gene expression, and markedly reduced the release of inflammatory cytokines [31,11,42]. Several formulations containing trehalose demonstrated a significant effect in reducing the inflammatory cytokines; namely IL-1β and IL-6 [17]. Therefore, amelioration of the inflammation through formulations or drugs that decrease pro-inflammatory cytokines is an important approach to minimize the side effects and the damage to the colonic tissue that may result from NSAIDs use, and this was achieved in the current study by the formulated TXM loaded THs.

3.4.3. Histopathological analysis

The micrographs of the colonic tissue of the negative control group revealed an intact mucosal lining with unremarkable inflammation, Fig. 8A. Untreated rabbits of the positive control group showed a widely ulcerated mucosal lining and a dense inflammation compared to the normal colonic tissue, Fig. 8B. A similar observation was detected in group III; received TXM suspension, where in addition to the severe inflammation, a severe congestion was observed, Fig. 8C. Group IV; received drug-free THs, showed an ulcerated mucosal lining and a pseudomembranous inflammation, and a severe congestion, Fig. 8D. Group V; received TXM loaded THs, showed a significant reduction in the inflammation signs that could be supported by the presence of a relatively preserved mucosal lining and a mild inflammation, Fig. 8E. These verdicts were in reliable agreement with the observed levels of the investigated biomarkers.

4. Conclusion

Colon targeting THs were successfully formulated through a green, single-step method, avoiding the usage of organic solvents and incorporating trehalose. A 23 full factorial design was exploited for the formulation of THs. The optimized TXM loaded THs composed of trehalose: PL weight ratio of 1:1, 377.72 mg transcutol, and subjected to 4 min sonication. The formulation showed spherical nanovesicles with high EE%, and was capable of sustainig the release of the drug over 24 h.
The results of the biomarkers analysis and the improved inflammatory response to acetic acid-induced colitis in the histopathology of the colonic tissue indicated the efficacious role of trehalose-based nanovesicles in the protection against the possible NSAIDs provoked exacerbation of colitis.

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