Translation of ionic liquids to be enteric nanoparticles for facilitating oral absorption of cyclosporine A

Abstract Ionic liquids (ILs) attract more and more interests in improving drug transport across membrane, including transdermal, nasal, and oral delivery. However, some drawbacks of ILs impede the application in oral drug delivery, such as rapid precipitation of poorly soluble drugs in stomach. This study aimed to employ enteric mesoporous silica nanoparticles (MSNs) to load ILs to overcome the shortcomings faced in oral administration. The choline sorbate ILs (SCILs) were synthesized by choline bicarbonate and sorbic acid and then adsorbed in mesopores of MSNs after dissolving cyclosporin A (CyA). MSNs loading SCILs and CyA were coated by Eudragit® L100 to form enteric nanoparticles. The in vitro release study showed that the CyA and SCILs released only 10% for 2 h in simulated gastric fluids but more than 90% in simulated intestinal fluid. In addition, SCILs and CyA were able to release from MSNs synchronously. After oral administration, enteric MSNs loading SCILs were capable of improving oral absorption of CyA significantly and the oral bioavailability of CyA was similar with that of oral Neoral®. In addition, the oral absorption of enteric MSNs was higher than that of nonenteric MSNs, which showed that enteric coating was necessary to ILs in oral delivery. These findings revealed great potential of translation of ILs to be enteric nanoparticles for facilitating oral absorption of CyA. It is predictable this delivery system is promising to be a platform for delivering poorly water‐soluble drugs and even biologics orally.

oral bioavailability of drugs, like insulin and sorafenib. 7,8 Unfortunately, ILs could dissociate in stomach once encountering enormous gastric fluids. 9 After dissociation, some poorly water-soluble drugs will quickly recrystallize and precipitate, while water-soluble drugs released in gastric fluid completely and could be degraded in harsh gastric environments. As a result, the effects of ILs are not able to exert perfectly, including increasing dissolution and enhancing permeation. Therefore, some studies adopted intrajejunal injection or enteric capsule for administration to animals. 10 Enteric capsule seems to be the feasible approach for clinical use; however, it could be broken by ILs during storage due to their strong solubilizing capacity and hygroscopicity. 11,12 Hence, it is urgent to develop a feasible carrier to protect ILs against dissociation in gastrointestinal tract for oral delivery.
Mesoporous silica nanoparticles (MSNs) have gained much attention for oral delivery due to many uniform mesopores, which are able to load drugs, and high dispersity in gastrointestinal tract that is able to inhibit drug recrystallization. 13 Besides, many reports have confirmed MSNs were capable of penetrating intestinal mucus layer. 14 Herein, cyclosporine A (CyA) was used as a model drug to verify the feasibility of the idea. CyA is a biopharmaceutics classification system (BCS) IV compound, whose bioavailability is limited by both poor water solubility and lower permeability across intestinal epithelia. 16 The commonly used product on the market is CyA self-emulsifying soft capsules (Neoral ® ), which are obtained by using high-dose surfactants (polyoxyethylene hydrogenated castor oil 40), corn oilmono-di-triglycerides and absolute ethanol. 17 Neoral ® can form a microemulsion in the gastrointestinal tract after oral administration.
However, long-term use of large dose surfactants could easily cause mucosal damage and even gastrointestinal bleeding. [18][19][20] This study fabricated enteric MSNs loaded with ILs and CyA to improve oral bioavailability without surfactants. This oral delivery system could further foster the application of ILs in pharmaceutical field.

| Preparation and characterization of MSNs
The preparation of MSNs was based on the template method reported previously. 21,22 Briefly, 960 ml of deionized water was added into a 2 L round-bottom flask and stirred mechanically at 200 rpm.
Then 2.0 g of CTAB was added into the flask, followed by the addition of 7.0 ml of 2 mol/L NaOH solution. The reaction mixture was heated to 80 C and kept at 80 C for 30 min. Next, 14.0 ml of TEOS was added dropwise and the stirring speed was increased to 300 rpm. The mixture was allowed to react for 2 h. After reaction was finished, the reaction solution was cooled to room temperature and centrifuged at 20,000 g for 10 min to settle down the particles. Deionized water and ethanol were used to wash precipitates alternately twice, after which the particles were placed in a vacuum oven at 60 C and dried overnight. Reflux of 2.0 g dried MSNs in a mixed solution of 400 ml ethanol and 20 ml hydrochloric acid at 80 C for 36 h was used to remove template. After reflux, the recovered particles were washed alternately with deionized water and ethanol twice. Finally, particles were placed in a vacuum oven and dried at 60 C overnight.
After MSNs were dispersed in pure water at 25 C, particle size, polydispersity index (PDI), and zeta potential were measured by a Malvern Zetasizer Nano ZS analyzer system (Malvern Instruments Ltd., Malvern, UK). MSNs were also characterized by a Nicolet Fourier transform infrared spectrometer (FTIR, Thermo Fisher Scientific Inc., Waltham, USA) and a Tecnai G2 transmission electron microscope (TEM, FEI, Netherlands).

| Preparation and characterization of choline sorbate ILs
Sorbate ILs (SCILs) were synthesized by a salt metathesis reaction. In

| Solubilization of CyA in SCILs
The saturated solubility of CyA was measured by dissolving excess CyA in solvent. In brief, excess CyA and 10 ml of methanol were dissolved in SCILs, then dried by rotary evaporation at 60 C for 30 min and underwent vacuum drying for 12 h to remove methanol. Subsequently, the solution was vortexed for 5 min and kept at 37 C ± 0.5 C for 2 h.
Afterward, the mixture was subject to centrifugation two times for sampling. The first round of centrifugation at 4000 g, 10 min was intended to take samples from the bottom layer due to larger density of SCILs than CyA. The second round of centrifugation at 13,000 rpm for 10 min aimed at taking samples from supernatant to ensure no CyA precipitate contamination. The supernatant was followed by appropriate dilution with methanol. Twenty microliters were analyzed by high performance liquid chromatography (HPLC) to determine the saturated solubility of CyA in SCILs. Detail quantification method of CyA in methanol by HPLC was described in the supporting material S1.
Briefly, MSNs, SCILs, and CyA were weighed according to the formulation ratios. Ultrasonication for 30 min was employed to promote the dissolution of CyA and SCILs. Then, the suspension was magnetically stirred at room temperature for 24 h to allow the CyA and SCILs fully to enter the MSNs channels. Subsequently, methanol was removed by rotary evaporation at 60 C for 30 min. The samples were dried in a vacuum oven at 60 C for 12 h to remove the residual solvents and stored in a desiccator for use. For enteric coating, the dried nanoparticles were coated by spraying 10% Eudragit ® L100 solution (in methanol), then dried in a well-ventilated place at room temperature for 4 h, and subsequently transferred to a vacuum oven at 60 C for 12 h to remove the residual solvents. The final products were ground until they could pass across 80-mesh sieve. MSNs loading CyA was denoted as CyA@MSNs and used as control group. Enteric CyA@MSNs were prepared via the same method for enteric CyA@S-CIL@MSNs but without the addition of SCILs.

| In vitro release profiles
The in vitro release was evaluated using a 708-DS dissolution tester 1% SDS) and pH 6.8 phosphate buffer (containing 0.1% SDS) was investigated using the same dissolution method as above. The concentration of SCILs was represented by determination of sorbic acid, whose HPLC analysis method and validation were described in supporting material (Table S1-S7).

| Pharmacokinetic study
In vivo pharmacokinetic studies were conducted in male Sprague- and analysis method of CyA in rat whole blood was described in supporting material S1.

| Data analysis
The data were expressed as mean ± standard deviation (SD

| Preparation and characterization of SCILs
We successfully prepared SCILs with sorbic acid and choline bicarbonate using a conventional metathesis reaction (Figure 2a) affected by newly-generated ionic bonds moved to the upfield. Notably, the chemical shift of 14-H moved from δ 7.15 to δ 6.65 ppm, exhibiting a change of 0.5 ppm, which is also a strong proof of the successful synthesis of SCIL. In addition, the water content of neat IL determined by Karl Fischer titration was 1.73% ± 0.26% (see supporting material S1).   What is more, after 10 months storage, the CyA content in enteric CyA@SCIL@MSNs-4 and the dissolution curves of CyA from enteric CyA@SCIL@MSNs-4 were close to those at Day 0, demonstrating the robust stability after long-term storage ( Figure S3).

| In vitro release profiles
The release profiles of CyA and SCILs (Figure 5a) showed that only

| Pharmacokinetic study
After oral administration, the concentration-time curves of CyA are shown in Figure 6. It could be concluded that the concentration of CyA in Neoral ® group reached the peak at the most rapid rate and the peak concentration was the highest among five groups. There was a However, the AUC 0-24 h of other three control formulations was significantly lower than Neoral ® . This is also true for C max . Conversely, the T max of enteric CyA@SCIL@MSNs-4 was not significantly different from other three control formulations except Neoral ® . Yet, there was no significant difference of T max between other three control formulations and Neoral ® . The T max of Neoral ® was basically the same as reported in the relevant literature. 23 Release site of enteric CyA@SCIL@MSNs-4 was at the small intestine, thus the T max was relatively longer. We also found that compared with CyA@SCIL, the enteric CyA@MSNs could also improve the oral bioavailability of CyA.

| DISCUSSION
Oral delivery is a common administration route with high patient compliance. However, a lot of drug molecules are not suitable to be delivered by oral route due to their low aqueous solubility and membrane permeability, 24 such as BCS II and IV drugs. Hence, more and more novel technologies are emerging for improving oral bioavailability of these drugs, including solid dispersion, 25 self-microemulsion, 26 nanoparticles, 27 and permeation enhancers. 28 These technologies must be combined with traditional formulations to meet requirements of clinical administration. For instance, self-microemulsions are filled in soft capsules 29 and solid dispersions are prepared to be tablet after mixing with other excipients. 30 ILs have been widely used as solvents or permeation enhancers to dissolve poorly soluble drugs 31 or enhance transmembrane ability of drug molecules. 32 In addition, they have been reported to enhance oral absorption of insulin 33 and poorly soluble drugs. 34 ILs can be tai-  is not easy to separate out in mucus due to the presence of SCILs.
In addition, SCILs were distributed in large area by transportation of MSNs, which would not lead to local high concentration and severe mucus toxicity.
The oral bioavailability of enteric nanoparticles has no statistically difference with Neoral ® after oral administration to rats, which demonstrated that the combination of SCILs and MSNs were able to improve the oral absorption of CyA, because Neoral ® is the most successful formulation of CyA which employs self-microemulsion to facilitate oral absorption of CyA. 41 The bioavailability of nonenteric nanoparticles (CyA@SCIL@MSNs-4) is lower than that of enteric nanoparticles significantly (Figure 6c). Although nonenteric nanoparticles also improved oral absorption of CyA to a certain extent, their effects were similar with the group without SCILs (enteric CyA@MSNs), which implies SCILs could release and dissociate in stomach immediately if nanoparticles were not protected by enteric materials. The in vivo study also confirmed that ILs could be dissociated in stomach and lost their capability of improving absorption, as supported by low oral bioavailability of CyA@SCIL group. The oral bioavailability of nanoparticles without SCILs (enteric CyA@MSNs) is also lower than that of enteric CyA@SCIL@MSNs significantly, implying the permeation-enhanced absorption effect exerted by SCILs.
Although the onset of therapeutic action of enteric CyA@SCIL@MSNs was slower than Neoral ® due to enteric coating (Figure 6e), the bioavailability and C max (Figure 6d) were not changed significantly.
Consequently, ILs must avoid the dilution of gastric fluids as oral carriers or permeation enhancers. And ILs can be adsorbed into MSNs to achieve higher level of enhancing oral absorption. In addition, we evaluated the in vivo toxicity of Neoral ® and enteric CyA@S-CIL@MSNs by 7 days of once-a-day repeat oral administration to rats. The results showed though the rats body weight steadily increased in both groups, the weight gain of rats in the group of enteric CyA@SCIL@MSNs-4 was significantly more than that of Neoral group ( Figure S4), implying enteric CyA@SCIL@MSNs-4 reduced irritation to intestinal tracts compared to Neoral ® .
Meanwhile, the extent of tissue inflammation induced by enteric CyA@SCIL@MSNs was lower than Neoral ® ( Figure S5). Besides, we only observed the irritation to GI tract after 7 days consecutive administration of two formulations, the difference of in vivo toxicity between two groups was not very large. It might be more evident if they are administered for long periods of time in clinical use.

| CONCLUSION
Although ILs could be good permeation enhancer or solubilizer, some evident shortcomings limited their use in oral delivery, such as rapid drug precipitation when exposed to enormous gastric fluids This study employed MSNs to load ILs, which dissolved CyA and avoided leakage in stomach by enteric coating. This delivery system is able to protect ILs against dissociation in gastric fluids and ensure synchronous release of SCILs and CyA in small intestine. In addition, the enteric nanoparticles loading SCILs can improve the oral absorption of CyA significantly and achieve similar oral bioavailability with Neoral ® .
Except for BCS IV drugs, this system has great potential to be a platform for delivering other poorly soluble drugs and even biologics orally.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available within the paper and its supplementary information. Any other data are available from the corresponding authors upon request.