3.4.1. Aqueous Solubilization

The partitioning of FEN between the aqueous phase and the pellet was measured over 90 min during two-phase gastrointestinal lipolysis. The total percentage of FEN recovered ranged between 70 to 98% for all formulations (Figures S2 and S3). Difficulty in isolating the pellet and possible pellet loss during the separation of the aqueous phase and pellet have contributed to having less than 100% total recovered FEN. This issue has been previously reported and was attributed to either drug loss during the separation process or inability to analyze drug retained in the undigested oil phase [28].

The aqueous concentration-time profiles of solubilized FEN during in vitro gastrointestinal lipolysis are displayed in Figure 6A,B. Crystalline FEN displayed negligible solubilization during the gastric phase, while achieving only 3.5% solubilization after complete intestinal lipolysis. For APO-fenofibrate, once re-dispersed in simulated intestinal digesting media, 5% of total FEN was immediately solubilized with no further changes in solubilization over the entirety of the lipolysis period. The characteristic precipitation of FEN from LBFs caused difficulties in data interpretation and distinguishing enabling formulations. However, the extent and occurrence of FEN precipitation was influenced by the silica nanostructure, type of lipid and drug load evident in patterns observed from the aqueous solubilization-time profiles and area under the intestinal solubilization-time curve (AUC) reported in Figure 6. It is important to note that SLH formulations prepared at 400% and 600% Seq did not improve FEN dissolution (Figure 5); therefore, these formulations were not pursued further in aqueous solubilization studies.

When comparing solidified formulations to 80% liquid lipid, enhanced performance was observed with 80% liquid PG8 during the gastric step, achieving the highest FEN solubilization of 25%, after 30 min (*p* < 0.05). Despite FEN precipitation during the intestinal phase between 1–5 min, FEN solubilization gradually increased to reach 18%, after 90 min, which was significantly higher than all SLH PG8 formulations (*p* < 0.05). However, 80% liquid C300 formulation displayed an enhanced FEN aqueous solubilization profile during the intestinal phase compared to corresponding solidified 80% FS C300 but lower performance than corresponding 80% MPS C300 (*p* < 0.05) (Figure 6A,C).

For FEN solubilization from SLH formulations prepared at 80% Seq, FEN precipitation was triggered by the transition into the intestinal phase at time points ranging between 1–5 min and was followed by a period of slightly enhanced solubilization prior to another decrease in solubilization after 15 min. During the gastric step, 80% FS PG8, 80% MPS PG8, 80% FS C300, and 80% MPS C300 formulations displayed enhanced FEN solubilization of 15%, 16%, 19%, and 12.4%, after 30 min, respectively (*p* > 0.05). This corresponds to 3.5- to 5-fold and 3- to 3.7-fold significantly enhanced solubilization compared to crystalline FEN and APO-fenofibrate, respectively (*p* < 0.05) (Figure 6A,B). Transition into the intestinal phase triggered FEN precipitation where the percentage FEN solubilized decreased to 13% and 6.7% from 80% MPS PG8 and 80% MPS C300, respectively. However, a more dramatic drop in FEN solubilization to 9% and 5.9% was observed from 80% FS PG8 and 80% FS C300 formulations, respectively, at 1 min of the intestinal phase. Furthermore, 80% MPS C300 was able to gradually enhance FEN solubilization and prevent its precipitation, achieving 16% FEN solubilization, after 90 min which was significantly greater than FEN solubilization achieved by corresponding 80% FS C300 formulation (*p* < 0.05).

**Figure 6.** FEN aqueous concentration-time profiles from crystalline FEN, APO-fenofibrate, and FENloaded SLH formulations, in fasted state simulated gastric fluid (FaSSGF) (30 min) and fed state simulated intestinal fluid (FaSSIF) (60 min) under digesting conditions, dosed at 3 mg of FEN (mean ± SD, *n* = 3). (**A**) SLH C300 formulations and (**B**) SLH PG8 formulations. Dotted line represents the FEN Seq in biorelevant media. The area under the curve (AUC) of FEN aqueous solubilization-time profile measured during the intestinal phase from (**C**) crystalline FEN, APO-fenofibrate, and SLH C300 formulations and (**D**) crystalline FEN, APO-fenofibrate, and SLH PG8 formulations. **Figure 6.** FEN aqueous concentration-time profiles from crystalline FEN, APO-fenofibrate, and FEN-loaded SLH formulations, in fasted state simulated gastric fluid (FaSSGF) (30 min) and fed state simulated intestinal fluid (FaSSIF) (60 min) under digesting conditions, dosed at 3 mg of FEN (mean ± SD, *n* = 3). (**A**) SLH C300 formulations and (**B**) SLH PG8 formulations. Dotted line represents the FEN Seq in biorelevant media. The area under the curve (AUC) of FEN aqueous solubilization-time profile measured during the intestinal phase from (**C**) crystalline FEN, APO-fenofibrate, and SLH C300 formulations and (**D**) crystalline FEN, APO-fenofibrate, and SLH PG8 formulations.

In contrast, FEN solubilization behavior from super-SLH (200% Seq) was dependent on the type of lipid utilized, where a decrease in FEN solubilization was attained in the gastric phase for 200% FS PG8 and 200% MPS PG8 followed by enhanced solubilization in the intestinal phase achieving 15% and 10% solubilization, respectively (Figure 6C). FEN solubilization was maintained at 4–5% during the gastric phase by 200% FS C300 and 200% MPS C300 formulations followed by enhanced FEN solubilization in the intestinal phase, achieving 15.3% and 10.4%, after 5 min, respectively, prior to FEN precipitation by only 200% FS C300, at 15 min, dropping FEN solubilization to less than 10%. In contrast, FEN solubilization from 200% MPS C300 gradually increased during the intestinal phase In contrast, FEN solubilization behavior from super-SLH (200% Seq) was dependent on the type of lipid utilized, where a decrease in FEN solubilization was attained in the gastric phase for 200% FS PG8 and 200% MPS PG8 followed by enhanced solubilization in the intestinal phase achieving 15% and 10% solubilization, respectively (Figure 6C). FEN solubilization was maintained at 4–5% during the gastric phase by 200% FS C300 and 200% MPS C300 formulations followed by enhanced FEN solubilization in the intestinal phase, achieving 15.3% and 10.4%, after 5 min, respectively, prior to FEN precipitation by only 200% FS C300, at 15 min, dropping FEN solubilization to less than 10%. In contrast, FEN solubilization from 200% MPS C300 gradually increased during the intestinal phase

achieving 12.7%, after 90 min. This corresponds to 3.6- and 2-fold enhanced solubilization compared

to crystalline FEN and APO-fenofibrate, respectively.

achieving 12.7%, after 90 min. This corresponds to 3.6- and 2-fold enhanced solubilization compared to crystalline FEN and APO-fenofibrate, respectively. *Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 15 of 22 All SLH formulations achieved FEN solubilization above FEN equilibrium solubility in FaSSGF

All SLH formulations achieved FEN solubilization above FEN equilibrium solubility in FaSSGF (1.1 ± 0.4 µg/mL) during the gastric phase and below its equilibrium solubility in FaSSIF during the intestinal phase (18.1 ± 0.8 µg/mL). Regardless of lipid type, a pattern of enhanced FEN solubilization with increased drug load in FS formulations was clearly evident in data obtained from AUC of FEN solubilization-time profile during the intestinal phase (Figure 6C,D), whereas a decrease in performance with supersaturation was observed for MPS formulations. It was evident that 80% MPS C300 improved the aqueous solubilization of FEN when compared to the 80% liquid C300. In contrast, for PG8 formulations, the presence of both silica types reduced FEN aqueous solubilization when compared to 80% liquid PG8. (1.1 ± 0.4 µg/mL) during the gastric phase and below its equilibrium solubility in FaSSIF during the intestinal phase (18.1 ± 0.8 µg/mL). Regardless of lipid type, a pattern of enhanced FEN solubilization with increased drug load in FS formulations was clearly evident in data obtained from AUC of FEN solubilization-time profile during the intestinal phase (Figure 6C,D), whereas a decrease in performance with supersaturation was observed for MPS formulations. It was evident that 80% MPS C300 improved the aqueous solubilization of FEN when compared to the 80% liquid C300. In contrast, for PG8 formulations, the presence of both silica types reduced FEN aqueous solubilization when compared to 80% liquid PG8.

#### 3.4.2. Lipid Digestion 3.4.2. Lipid Digestion

The lipid digestion profiles during the intestinal phase of the gastrointestinal lipolysis of all investigated LBF are displayed in Figure 7. Crystalline FEN and APO-fenofibrate did not display any lipid digestion as they did not contain lipid (data not shown). The fabricated formulations differed in their lipid content per dose, as dosing was based on the amount of formulation equivalent to 3 mg of FEN. The amount of lipid dosed ranged from 19.5–48.7 mg (Table S2). The 80% liquid PG8 formulation exhibited the lowest extent of digestion compared to its corresponding solidified SLH formulation (FS or MPS). In contrast, 80% liquid C300 displayed a slow gradual increase in lipolysis over 60 min, but the final extent of lipolysis was lower than corresponding SLH formulations. The amount of digestion decreased as a function of increasing drug load, since all 80% SLH formulations displayed significantly higher extent of digestion when compared to their corresponding 200% formulations, regardless of the type of lipid (*p* < 0.05). FA released from C300 SLH formulations were 5-fold greater than corresponding SLH PG8 formulations, regardless of the type of silica or drug load. At the same saturation level and lipid type, FS facilitated greater extent of lipolysis when compared to corresponding MPS formulations; however, the difference was not statistically significant (*p* > 0.05). The lipid digestion profiles during the intestinal phase of the gastrointestinal lipolysis of all investigated LBF are displayed in Figure 7. Crystalline FEN and APO-fenofibrate did not display any lipid digestion as they did not contain lipid (data not shown). The fabricated formulations differed in their lipid content per dose, as dosing was based on the amount of formulation equivalent to 3 mg of FEN. The amount of lipid dosed ranged from 19.5–48.7 mg (Table S2). The 80% liquid PG8 formulation exhibited the lowest extent of digestion compared to its corresponding solidified SLH formulation (FS or MPS). In contrast, 80% liquid C300 displayed a slow gradual increase in lipolysis over 60 min, but the final extent of lipolysis was lower than corresponding SLH formulations. The amount of digestion decreased as a function of increasing drug load, since all 80% SLH formulations displayed significantly higher extent of digestion when compared to their corresponding 200% formulations, regardless of the type of lipid (*p* < 0.05). FA released from C300 SLH formulations were 5-fold greater than corresponding SLH PG8 formulations, regardless of the type of silica or drug load. At the same saturation level and lipid type, FS facilitated greater extent of lipolysis when compared to corresponding MPS formulations; however, the difference was not statistically significant (*p* > 0.05).

**Figure 7.** Fatty acid (FA) release-time profile from: (**A**) C300 formulations and (**B**) PG8 formulations, during the intestinal phase of in vitro gastrointestinal (GI) lipolysis in FaSSIF for 60 min, dosed at 3 mg of FEN (mean − SD, *n* = 3). **Figure 7.** Fatty acid (FA) release-time profile from: (**A**) C300 formulations and (**B**) PG8 formulations, during the intestinal phase of in vitro gastrointestinal (GI) lipolysis in FaSSIF for 60 min, dosed at 3 mg of FEN (mean − SD, *n* = 3).

#### **4. Discussion 4. Discussion**

formulation.

Numerous studies have shown that small changes in the nanostructure, surface chemistry and composition of SLH particles can significantly impact lipid digestion dynamics, which in turn influences drug solubilization and release kinetics [23,40]. Therefore, the aim here was to examine Numerous studies have shown that small changes in the nanostructure, surface chemistry and composition of SLH particles can significantly impact lipid digestion dynamics, which in turn influences drug solubilization and release kinetics [23,40]. Therefore, the aim here was to examine the influence of (i) silica nanostructure, (ii) type of lipid, and (iii) drug load on the in vitro solubilization and solid-state stability of the model drug FEN when incorporated into an SLH formulation.

It is well known that spray drying is amongst the techniques commonly employed to solidify liquid-state LBFs and is recognized to generate highly porous three-dimensional nanostructured matrices with superior physicochemical properties [14]. Here, spray drying was employed to generate porous silica microparticles (FS) from precursor fumed silica nanoparticles suspensions, prior to drug/lipid encapsulation via adapting the simple mixing technique [29]. Supersaturation was achieved by adding drug above its equilibrium saturation level in either of two lipids (PG8 or C300) using heat (≤60 ◦C) [29]. The generated, thermodynamically unstable, supersaturated liquid lipids were adsorbed into the nanopores or onto the surface of FS or MPS to allow for direct comparison.
