The permeation profile, as the cumulative amount permeated through AHS over 25 h, is outlined in Figure 4. It shows that the liposome POPC:CHOL exhibited the highest permeation, with liposome POPC:CHOL:CER following behind, and liposome POPC showing the lowest permeation of the three liposomes tested. The permeation parameters calculated are presented in Table 3. The permeation profile, as the cumulative amount permeated through AHS over 25 h, is outlined in Figure 4. It shows that the liposome POPC:CHOL exhibited the highestpermeation, with liposome POPC:CHOL:CER following behind, and liposome POPCshowing the lowest permeation of the three liposomes tested. The permeation parameterscalculated are presented in Table 3. *Pharmaceuticals* **2023**, *16*, 894 6 of 17

**Figure 4.** BNB permeation profile: cumulative amount of BNB permeated (µg) vs. time (h). The results are presented as mean ± SD (*n* = 5). **Figure 4.** BNB permeation profile: cumulative amount of BNB permeated (µg) vs. time (h). The results are presented as mean ± SD (*n* = 5).

**Table 3.** Permeation parameters of three liposomes: cumulative amount of BNB permeated at 25 h (*AP25h*, µg), flux (*Jss*, µg/h), permeability coefficient (*Kp*, cm/h) and predicted steady-state plasma concentration in human steady state on a 10 cm2 surface application (*Css*, ng/mL). **Table 3.** Permeation parameters of three liposomes: cumulative amount of BNB permeated at 25 h (*AP25h*, µg), flux (*Jss*, µg/h), permeability coefficient (*Kp*, cm/h) and predicted steady-state plasma concentration in human steady state on a 10 cm2 surface application (*Css*, ng/mL).

POPC:CHOL:CER 14.36 ± 1.40 0.77 ± 0.07 4.01 ± 0.37 1.28 ± 0.12 ANOVA test analysis of variance, followed by Tukey's multiple comparison test, were performed, ANOVA test analysis of variance, followed by Tukey's multiple comparison test, were performed, and all were statistically different from each other (*p* < 0.001). Results are expressed by mean ± SD (*n* = 5).

and all were statistically different from each other (*p* < 0.001). Results are expressed by mean ± SD

when compared to liposome POPC:CHOL and liposome POPC:CHOL:CER. However, no significant differences were observed between liposome POPC:CHOL and liposome

**Figure 5.** Retained amount (µg/cm2) of BNB at 25 h in the AHS. Results are expressed as mean ± SD (*n* = 5). *ANOVA* test analysis of variance, followed by Tukey's multiple comparison test, were performed. Statistically significant difference: \*\*\* = *p* < 0.0001; ns = no statistically significant difference.

The tolerability of the liposomal formulations on altered skin (previously subjected to microneedle punches) was evaluated on New Zealand rabbits. Figure 6 shows the progression of altered rabbit's skin (ARS) monitored for 3 h 30 min after applying xylol so as to induce erythema and edema. This was intended to stimulate AD flare-ups. All three

ns

(*n* = 5).

POPC:CHOL:CER.

150 \*\*\*

POPC

2.4.1. In Vivo Tolerance Study

0

50

100

**Retained amount of**

**baricitinib (**μ**g/cm2**

**)**

POPC:CHOL

*2.4. Tolerance Study and Histological Analysis*

POPC:CHOL:CER

Figure 5 illustrates the quantity of BNB retained in the tissues and the corresponding statistical outcomes. Liposome POPC demonstrated significant statistical differences when compared to liposome POPC:CHOL and liposome POPC:CHOL:CER. However, no significant differences were observed between liposome POPC:CHOL and liposome POPC:CHOL:CER. Figure 5 illustrates the quantity of BNB retained in the tissues and the corresponding statistical outcomes. Liposome POPC demonstrated significant statistical differences when compared to liposome POPC:CHOL and liposome POPC:CHOL:CER. However, no significant differences were observed between liposome POPC:CHOL and liposome POPC:CHOL:CER.

**Figure 4.** BNB permeation profile: cumulative amount of BNB permeated (µg) vs. time (h). The re-

**Table 3.** Permeation parameters of three liposomes: cumulative amount of BNB permeated at 25 h (*AP25h*, µg), flux (*Jss*, µg/h), permeability coefficient (*Kp*, cm/h) and predicted steady-state plasma

*AP25h (***µg)** *Jss* **(µg/h)** *Kp* **(10−4 cm/h)** *Css* **(ng/mL)**

ANOVA test analysis of variance, followed by Tukey's multiple comparison test, were performed, and all were statistically different from each other (*p* < 0.001). Results are expressed by mean ± SD

concentration in human steady state on a 10 cm2 surface application (*Css*, ng/mL).

POPC 5.13 ± 0.52 0.22 ± 0.03 1.14 ± 0.16 0.36 ± 0.05 POPC:CHOL 21.93 ± 2.20 0.29 ± 0.03 1.52 ± 0.16 0.48 ± 0.05 POPC:CHOL:CER 14.36 ± 1.40 0.77 ± 0.07 4.01 ± 0.37 1.28 ± 0.12

POPC:CHOL POPC:CHOL:CER

POPC

*Pharmaceuticals* **2023**, *16*, 894 6 of 17

0 10 20 30

**Time (h)**

sults are presented as mean ± SD (*n* = 5).

0

10

20

**Cum amount baricitinib**

**permeated(**

(*n* = 5).

μ**g)**

30

**Figure 5.** Retained amount (µg/cm2) of BNB at 25 h in the AHS. Results are expressed as mean ± SD (*n* = 5). *ANOVA* test analysis of variance, followed by Tukey's multiple comparison test, were performed. Statistically significant difference: \*\*\* = *p* < 0.0001; ns = no statistically significant difference. **Figure 5.** Retained amount (µg/cm2 ) of BNB at 25 h in the AHS. Results are expressed as mean ± SD (*n* = 5). *ANOVA* test analysis of variance, followed by Tukey's multiple comparison test, were performed. Statistically significant difference: \*\*\* = *p* < 0.0001; ns = no statistically significant difference.

#### *2.4. Tolerance Study and Histological Analysis 2.4. Tolerance Study and Histological Analysis*

2.4.1. In Vivo Tolerance Study 2.4.1. In Vivo Tolerance Study

The tolerability of the liposomal formulations on altered skin (previously subjected to microneedle punches) was evaluated on New Zealand rabbits. Figure 6 shows the progression of altered rabbit's skin (ARS) monitored for 3 h 30 min after applying xylol so as to induce erythema and edema. This was intended to stimulate AD flare-ups. All three The tolerability of the liposomal formulations on altered skin (previously subjected to microneedle punches) was evaluated on New Zealand rabbits. Figure 6 shows the progression of altered rabbit's skin (ARS) monitored for 3 h 30 min after applying xylol so as to induce erythema and edema. This was intended to stimulate AD flare-ups. All three liposomes appear to delay the onset of edema and erythema caused by xylol. The use of POPC liposomes was found to reduce the damage caused by xylol. The negative control of ARS was not treated with any substance or formulation. *Pharmaceuticals* **2023**, *16*, 894 7 of 17 liposomes appear to delay the onset of edema and erythema caused by xylol. The use of POPC liposomes was found to reduce the damage caused by xylol. The negative control of ARS was not treated with any substance or formulation.

**Figure 6.** Pictures of 5 groups of rabbits used for the modified Draize test: before the application of xylol and liposomes, at 15 min, 45 min and 3 and a half hours after the respective applications. **Figure 6.** Pictures of 5 groups of rabbits used for the modified Draize test: before the application of xylol and liposomes, at 15 min, 45 min and 3 and a half hours after the respective applications.

xylol exposure, both with and without subsequent application of the liposomes (mean ± SD, *n* = 5).

**Ed 15 min 45 min 3 h**

 **Erythema Edema Erythema Edema Erythema Edema Erythema Edema**

Negative control 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Liposome POPC 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

POPC:CHOL 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1.00 ± 0.53 0.00 ± 0.00 0.65 ± 0.40 0.00 ± 0.00

(xylol) 0.00 ± 0.00 0.00 ± 0.00 3.54 ± 0.20 1.62 ± 0.41 3.50 ± 0.40 1.84 ± 0.13 2.40 ± 0.30 0.50 ± 0.02

POPC:CHOL:CER 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1.45 ± 0.42 0.00 ± 0.00 0.37 ± 0.03 0.00 ± 0.00

scoring system (see Table 4).

Er = erythema; Ed = Edema.

dration, of rabbits with altered skin.

**Chemicals Before Induced Er and**

Liposome

Liposome

Positive control

The erythema and edema presented on the rabbits' backs are classified by the view

The pictures of Figure 6 are supported by the graphs shown in Figure 7, which show the evolution of the transepidermal water loss (TEWL), and the evolution of the skin hy-

The erythema and edema presented on the rabbits' backs are classified by the view scoring system (see Table 4).

**Table 4.** A modified Draize score was employed to evaluate erythema and edema resulting from xylol exposure, both with and without subsequent application of the liposomes (mean ± SD, *n* = 5).

Er = erythema; Ed = Edema.

The pictures of Figure 6 are supported by the graphs shown in Figure 7, which show the evolution of the transepidermal water loss (TEWL), and the evolution of the skin hydration, of rabbits with altered skin. *Pharmaceuticals* **2023**, *16*, 894 8 of 17

**Figure 7.** Results of TEWL and skin hydration during 210 min before and after formulations application on rabbits with induced dermatitis by xylol, except for the negative control. (**a**) TEWL; (**b**) Skin hydronation (AU). Results are expressed as mean ± SD (*n* = 5). **Figure 7.** Results of TEWL and skin hydration during 210 min before and after formulations application on rabbits with induced dermatitis by xylol, except for the negative control. (**a**) TEWL; (**b**) Skin hydronation (AU). Results are expressed as mean ± SD (*n* = 5).

#### 2.4.2. Histological Analysis

2.4.2. Histological Analysis Once the modified Draize test had been finished, a histological study was conducted to evaluate whether any structural change had occurred to the altered skin due to the application of the formulations. The histology results of the different liposomes, as well as the positive and negative controls, are set out in Figure 8. The skin treated with liposome POPC:CHOL:CER presented stratum corneum loss (Figure 8e) indicating some dis-Once the modified Draize test had been finished, a histological study was conducted to evaluate whether any structural change had occurred to the altered skin due to the application of the formulations. The histology results of the different liposomes, as well as the positive and negative controls, are set out in Figure 8. The skin treated with liposome POPC:CHOL:CER presented stratum corneum loss (Figure 8e) indicating some disruptive effect. This is in contrast to the skins treated with either liposomes POPC or POPC:CHOL, which do not demonstrate any alteration to the skin.

ruptive effect. This is in contrast to the skins treated with either liposomes POPC or

**Figure 8.** Skin sections colored with eosin and hematoxylin. (**a**) Control negative skin; (**b**) positive control; (**c**) treated with POPC; (**d**) treated with POPC:CHOL; and (**e**) treated with POPC:CHOL:CER. sc = stratum corneum, d = dermis, \* shows loss of stratum corneum, ∆ arrowhead

AD can also be present on facial skin, with symptoms such as dryness, redness, sen-

sitivity and itching, and facial skin might need the application of soothing formulations. This is why the potential tolerance of the liposomal formulations on the eye, after a periocular application on the eye, was assessed by the in vitro technique HET-CAM of fertilized chicken eggs. Table 5 shows the irritation score (IS) estimated for the liposomal formulations. The three liposomes resulted in non-irritant formulations exhibiting IS values similar to the negative control, whereas the positive control resulted in higher IS values

2.4.3. In Vitro Tolerance of the Liposomal Formulations for Periocular Application

indicates leukocyte infiltrate. Magnification = 200×, scale bar = 100 µm.

corresponding to a severely irritant substance.

POPC:CHOL, which do not demonstrate any alteration to the skin.

**0**

**10**

**20**

**g/h·m2**

**30**

**40**

**0 50 100 150 200 250**

2.4.2. Histological Analysis

**Time (min)**

Control positive Liposome POPC Liposome POPC:CHOL Liposome POPC:CHOL:CER

Control negative

**0 50 100 150 200 250**

**Time (min)**

**0**

**Figure 7.** Results of TEWL and skin hydration during 210 min before and after formulations application on rabbits with induced dermatitis by xylol, except for the negative control. (**a**) TEWL; (**b**)

Once the modified Draize test had been finished, a histological study was conducted to evaluate whether any structural change had occurred to the altered skin due to the application of the formulations. The histology results of the different liposomes, as well as the positive and negative controls, are set out in Figure 8. The skin treated with liposome POPC:CHOL:CER presented stratum corneum loss (Figure 8e) indicating some disruptive effect. This is in contrast to the skins treated with either liposomes POPC or

**5**

**10**

**Skin hydratation (AU)**

(**a**) (**b**)

Skin hydronation (AU). Results are expressed as mean ± SD (*n* = 5).

POPC:CHOL, which do not demonstrate any alteration to the skin.

**15**

**Figure 8.** Skin sections colored with eosin and hematoxylin. (**a**) Control negative skin; (**b**) positive control; (**c**) treated with POPC; (**d**) treated with POPC:CHOL; and (**e**) treated with POPC:CHOL:CER. sc = stratum corneum, d = dermis, \* shows loss of stratum corneum, ∆ arrowhead indicates leukocyte infiltrate. Magnification = 200×, scale bar = 100 µm. **Figure 8.** Skin sections colored with eosin and hematoxylin. (**a**) Control negative skin; (**b**) positive control; (**c**) treated with POPC; (**d**) treated with POPC:CHOL; and (**e**) treated with POPC:CHOL:CER. sc = stratum corneum, d = dermis, \* shows loss of stratum corneum, ∆ arrowhead indicates leukocyte infiltrate. Magnification = 200×, scale bar = 100 µm.

#### 2.4.3. In Vitro Tolerance of the Liposomal Formulations for Periocular Application

2.4.3. In Vitro Tolerance of the Liposomal Formulations for Periocular Application AD can also be present on facial skin, with symptoms such as dryness, redness, sensitivity and itching, and facial skin might need the application of soothing formulations. This is why the potential tolerance of the liposomal formulations on the eye, after a periocular application on the eye, was assessed by the in vitro technique HET-CAM of fertilized chicken eggs. Table 5 shows the irritation score (IS) estimated for the liposomal formulations. The three liposomes resulted in non-irritant formulations exhibiting IS values AD can also be present on facial skin, with symptoms such as dryness, redness, sensitivity and itching, and facial skin might need the application of soothing formulations. This is why the potential tolerance of the liposomal formulations on the eye, after a periocular application on the eye, was assessed by the in vitro technique HET-CAM of fertilized chicken eggs. Table 5 shows the irritation score (IS) estimated for the liposomal formulations. The three liposomes resulted in non-irritant formulations exhibiting IS values similar to the negative control, whereas the positive control resulted in higher IS values corresponding to a severely irritant substance.

8.9 < IS ≤ 21, severely irritating [18].

Figure 9 shows the results from the HET-CAM test. They revealed no irritant effect of any kind; no hemorrhage, coagulation or lysis vessel were observed 5 min after 300 µL of the respective formulations had been applied. This is opposite to the positive control, in which lysis vessel and coagulation appeared (Figure 9b).

in which lysis vessel and coagulation appeared (Figure 9b).

obtained for the positive and negative controls are also reported.

Irritation score (IS) 0.07 ± 0.00 16.10 ± 0.08 0.07 ± 0.00 0.07 ± 0.00 0.07 ± 0.00

 **Formulation**

Positive control (0.1 N NaOH)

and 8.9 < IS ≤ 21, severely irritating [18].

Negative control

**Figure 9.** The irritant effect of the formulations was evaluated using the HET-CAM method, with (**a**) a negative control using saline solution, and (**b**) a positive control using 0.1 N sodium hydroxide solution. The other three images show the evolution of the liposomal formulations: (**c**) POPC liposome, (**d**) POPC:CHOL liposome and (**e**) POPC:CHOL:CER liposome. **Figure 9.** The irritant effect of the formulations was evaluated using the HET-CAM method, with (**a**) a negative control using saline solution, and (**b**) a positive control using 0.1 N sodium hydroxide solution. The other three images show the evolution of the liposomal formulations: (**c**) POPC liposome, (**d**) POPC:CHOL liposome and (**e**) POPC:CHOL:CER liposome.

**Table 5.** Irritation score calculated for the liposomal formulations (mean ± SD of *n* = 3). The scores

IS ≤ 0.9, non-irritating/slightly irritating; 0.9 < IS ≤ 4.9, moderately irritating; 4.9 < IS ≤ 8.9, irritating;

Figure 9 shows the results from the HET-CAM test. They revealed no irritant effect of any kind; no hemorrhage, coagulation or lysis vessel were observed 5 min after 300 µL of the respective formulations had been applied. This is opposite to the positive control,

POPC POPC:CHOL POPC:CHOL:CER

#### **3. Discussion 3. Discussion**

All physicochemical characteristics, such as pH, vesicle size and ZP, influence the liposome interaction with the skin [19]. Liposome POPC was the largest vesicle, followed by POPC:CHOL, and the smallest was POPC:CHOL:CER. All of them had a PDI below 0.2, which is indicative of the uniformity of the particle size, and their small size and the low PDI mean the formulations tend to pass more easily through the sterilizing filters, which, in turn, renders them suitable for skins that are grazed or scratched and could become infected. Instead, the ZP helps to predict the stability of particles. A high ZP indicates a stable formulation, so the liposome POPC:CHOL:CER is the most stable of the tested liposomes [20]. Differences in the lipid used for each liposome elaboration may lead to different release profiles described by different kinetic models [21]. In our work, the two liposomes containing CHOL displayed the same kinetic model suggesting that the presence of CHOL in the liposome strongly impacts on their drug release characteristics. The fastest BNB release over a short period of time was presented only by liposome POPC:CHOL:CER but not by liposome POPC:CHOL (Figure 2). Although liposomes POPC and POPC:CHOL exhibited similar BNB released at 54 h, POPC:CHOL showed a higher AUC than POPC:CHOL:CER, indicating a superior performance by POPC:CHOL. All physicochemical characteristics, such as pH, vesicle size and ZP, influence the liposome interaction with the skin [19]. Liposome POPC was the largest vesicle, followed by POPC:CHOL, and the smallest was POPC:CHOL:CER. All of them had a PDI below 0.2, which is indicative of the uniformity of the particle size, and their small size and the low PDI mean the formulations tend to pass more easily through the sterilizing filters, which, in turn, renders them suitable for skins that are grazed or scratched and could become infected. Instead, the ZP helps to predict the stability of particles. A high ZP indicates a stable formulation, so the liposome POPC:CHOL:CER is the most stable of the tested liposomes [20]. Differences in the lipid used for each liposome elaboration may lead to different release profiles described by different kinetic models [21]. In our work, the two liposomes containing CHOL displayed the same kinetic model suggesting that the presence of CHOL in the liposome strongly impacts on their drug release characteristics. The fastest BNB release over a short period of time was presented only by liposome POPC:CHOL:CER but not by liposome POPC:CHOL (Figure 2). Although liposomes POPC and POPC:CHOL exhibited similar BNB released at 54 h, POPC:CHOL showed a higher AUC than POPC:CHOL:CER, indicating a superior performance by POPC:CHOL. The mathematical modelling in the release studies is an excellent tool to evaluate differences between formulations [22–24]. Furthermore, the non-modelistic parameters are useful to compare formulations which exhibit different kinetic models. In this work, the nonmodelistic parameters showed a relation between the amount of BNB that is released from the formulation and the release efficiency, with the cumulative permeated amount throughout the 25 h period being the liposome POPC:CHOL, the formulation with the highest values. Baricitnib is a molecule with poor water solubility under physiological conditions, as observed from its structure. It does not present charged structures at pH 7.40, suggesting that it could be found deep within the lipid bilayer. Moreover, it presents several groups capable of stabilizing the molecule in hydrophobic regions through hydrogen bonds. These facts point to baricitinib incorporating into the liposome lipid membrane, remaining there and not being released to permeate through the skin. To overcome this problem, liposomes containing baricitinib were supplemented with 5% Transcutol. The structure of Transcutol allows it to be incorporated in parallel to hydrocarbon chains with its OH group near the headgroup of the phospholipids, distorting the phospholipid bilayer. Then, when liposomes containing baricitinib interact with the skin surface, baricitinib can easily

be released from the liposome, and Transcutol molecules can increase the water solubility of baricitinib and facilitate its distribution towards the surface of the skin.

Different authors have demonstrated that, if the lipids are like those in the stratum corneum, more drugs will penetrate the skin [19]. It is worth mentioning that human skin contains CHOL and CER and disturbances in their levels cause disruption of the skin barrier function. Yet, harmonizing the CHOL and CER levels in the skin restores its barrier function [25]. Sinico and Fadda explained that a greater permeation and release could be produced when the liposomes are composed of lipids that are more similar to those of the skin [26]. This would explain the higher permeation of POPC:CHOL and POPC:CHOL:CER compared with liposome POPC without CHOL. The liposome POPC:CHOL:CER triplicated the results values of flux, permeability coefficient and theoretical plasma concentration in human steady state compared with the other studied liposomes (POPC and POPC:CHOL, see Table 3). Therefore, CER could be acting like a permeation enhancer. CERs have been considered the principal factor in the control of the skin barrier as the enhancer effect of CER analogues has been demonstrated in diverse studies [27]. Since the predicted plasma concentration at steady state did not reach therapeutic levels, it is likely that BNB only exerts a local effect on the skin [14].

Retention of BNB in the skin was not statistically different between POPC:CHOL and POPC:CHOL:CER, so both liposomes would be a good option in order to achieve a local effect of BNB because both liposomes reached values a hundred times higher than liposome POPC (Figure 5). Liposomes with CHOL and CER—which are the lipids found in the skin—were able to diffuse through the skin, thus creating a reservoir effect in the BNB. This achieved concentrations inside the skin higher than those obtained in the blood in oral therapy for AD treatment [28], considering the human skin density to be 1 g/mL [29].

The HET-CAM test showed no irritation effect caused by any of the three liposomes and, in reference to the modified Draize test, we observed a good Draize score for erythema and edema (Table 4). Liposome POPC effectively counteracted the impact of xylol on the skin and neither erythema nor edema were observed over the 3 h of the study. Xylol caused both reactions, erythema and edema, which were observed from the first assessment (after 15 min) and up to the end of the tolerance study, which was after 3 h. Liposomes POPC:CHOL and POP:CHOL:CER also effectively counteracted the edema caused by xylol. However, after 45 min of application, a slight erythema appeared in the areas where liposomes POPC:CHOL and POPC:CHOL:CER were tested. Nevertheless, this effect was more than 2-fold and 3-fold lower than the erythema caused by xylol. TEWL values were constant for all the liposomes with similar values to the negative control (within the range of 8–15 g/h·m2 , which means a healthy value for rabbits), whereas the positive control showed a significant increase. The results are in accordance with those obtained in control rabbits, as observed by Babu M. Medi and Angela Anigbogu [30,31]. The skin hydration values were constant and followed the trend of the negative control, while the positive control showed a steady increase during the tolerance study. Finally, the histological study demonstrated that liposome POPC and POPC:CHOL avoided any damage to the tissue structures. Based on these promising results, further biochemical studies should be carried out to test the efficacy of these liposomal formulations on animal models of the disease, including mutant animals, before proceeding to clinical trials.

#### **4. Materials and Methods**

#### *4.1. Materials*

BNB, Ammonium Formate and POPC were purchased from Sigma–Aldrich (Madrid, Spain). Transcutol® P [Diethylene glycol monoethyl ether] was bought at Gattefossé (Barcelona, Spain). Fisher Chemical (Loughborough, UK) supplied the Acetonitrile. Finally, CHOL (ovine wool > 98%) and CER (bovine spinal cord ≥ 98%) were acquired at Avanti Polar Lipid Inc. (Alabaster, AL, USA). A microneedle roller (Currentbody, Barcelona, Spain) contains 540 titanium needles that are 0.25 mm long, corresponding to 72.3 microneedles/cm2 .

BNB (C16H17N7O2S) is a pyrrolopyrimidine, the chemical structure of which is shown in the figure below, Figure 10, which has been obtained from PubChem, an open chemistry database at the National Institute of Health (NIH). BNB (C16H17N7O2S) is a pyrrolopyrimidine, the chemical structure of which is shown in the figure below, Figure 10, which has been obtained from PubChem, an open chemistry database at the National Institute of Health (NIH).

BNB, Ammonium Formate and POPC were purchased from Sigma–Aldrich (Madrid, Spain). Transcutol® P [Diethylene glycol monoethyl ether] was bought at Gattefossé (Barcelona, Spain). Fisher Chemical (Loughborough, UK) supplied the Acetonitrile. Finally, CHOL (ovine wool > 98%) and CER (bovine spinal cord ≥ 98%) were acquired at Avanti Polar Lipid Inc. (Alabaster, AL, USA). A microneedle roller (Currentbody, Barcelona, Spain) contains 540 titanium needles that are 0.25 mm long, corresponding to 72.3 mi-

in control rabbits, as observed by Babu M. Medi and Angela Anigbogu [30,31]. The skin hydration values were constant and followed the trend of the negative control, while the positive control showed a steady increase during the tolerance study. Finally, the histological study demonstrated that liposome POPC and POPC:CHOL avoided any damage to the tissue structures**.** Based on these promising results, further biochemical studies should be carried out to test the efficacy of these liposomal formulations on animal models

of the disease, including mutant animals, before proceeding to clinical trials.

*Pharmaceuticals* **2023**, *16*, 894 11 of 17

**4. Materials and Methods**

*4.1. Materials*

croneedles/cm2.

**Figure 10.** Chemical structure of BNB. **Figure 10.** Chemical structure of BNB.

#### *4.2. Biological Materials 4.2. Biological Materials*

The abdominal human skin (protocol code 93-01162 02/18 approved on 17 January 2020 by the 99 Bioethics Committee of SCIAS Hospital de Barcelona), was dermatomed to 400 µm thickness (Aesculap GA 630, Aesculap, Tuttligen, Germany). All the skin was subjected to a physical alteration with microneedles in order to simulate an AD skin by piercing the epidermis. Since the needles do not come into contact with the nerves or blood vessels, this technique is painless when performed on live animals. The abdominal human skin (protocol code 93-01162 02/18 approved on 17 January 2020 by the 99 Bioethics Committee of SCIAS Hospital de Barcelona), was dermatomed to 400 µm thickness (Aesculap GA 630, Aesculap, Tuttligen, Germany). All the skin was subjected to a physical alteration with microneedles in order to simulate an AD skin by piercing the epidermis. Since the needles do not come into contact with the nerves or blood vessels, this technique is painless when performed on live animals.

#### *4.3. Methods 4.3. Methods*

#### 4.3.1. Preparation of the Liposomes

4.3.1. Preparation of the Liposomes Three different liposome formulations were prepared: (i) pure POPC liposomes, (ii) POPC:CHOL (0.8:0.2, mol/mol) liposomes and (iii) POPC:CHOL:CER (0.36:0.24:0.40, Three different liposome formulations were prepared: (i) pure POPC liposomes, (ii) POPC:CHOL (0.8:0.2, mol/mol) liposomes and (iii) POPC:CHOL:CER (0.36:0.24:0.40, mol/mol/mol) liposomes.

mol/mol/mol) liposomes. All liposomes samples were elaborated in accordance with the methods published in other articles [32]. In short, to elaborate each liposome sample, we added 500 mg of BNB in a round bottom flask to the corresponding chloroform–methanol (2:1, *v*/*v*) lipid solutions to achieve the desired molar lipid concentration for each composition. Then, the mixture was sonicated for 10–15 s to make sure all BNB was dissolved. Following the removal All liposomes samples were elaborated in accordance with the methods published in other articles [32]. In short, to elaborate each liposome sample, we added 500 mg of BNB in a round bottom flask to the corresponding chloroform–methanol (2:1, *v*/*v*) lipid solutions to achieve the desired molar lipid concentration for each composition. Then, the mixture was sonicated for 10–15 s to make sure all BNB was dissolved. Following the removal of the solvent using a rotary evaporator, the thin lipid film was kept in a high vacuum overnight in the absence of light to ensure the absence of organic solvent traces. The thin films were rehydrated using a solution containing 10 mM TRIS·HCl ([tris(hydroxymethyl)aminomethane] 150 mM NaCl), and 5% (*v*/*v*) of Transcutol® P pH 7.40. Vigorous vortexing was performed for 5 cycles, at a temperature above that of the lipid mixture's transition, so as to obtain large multilamellar vesicles. The liposome size was homogenized using an ultrasound bath with temperature control for 15 min. Finally, the liposomes were passed through a Sephadex® G50 column mounted in a 5 mL syringe and centrifuged at 1000 rpm for 10 s using a Rotanta 460R centrifuge (Andreas Hettich GmbH & Co. KG, DE, Tuttlingen, Germany) to eliminate non-encapsulated BNB.

#### 4.3.2. Liposomes' Physicochemical Characterization

The physicochemical properties of the liposomes were analyzed using various parameters, including pH, vesicle size, polydispersity index, ZP and encapsulation efficiency. pH measurement was carried out, using a pH-meter micro pH 2001 (Crison Instruments SA, Alella, Spain), in triplicate. The Zetasizer Nano S (Malvern Instrument, Malvern, UK) was used to determine the liposomes size, PDI and ZP. All the physicochemical characteristics were determined in triplicate [33]. All the measures were analyzed by one-way ANOVA

test followed by a Tukey's multiple comparison test. These measurements were repeated on samples stored for up to 1 month at 4 ◦C to assess the stability of the liposomes.

To study the EE percentage (EE%), the liposomes were broken down with Transcutol® P: Triton at 10% (8:1), so that they released the BNB, and quantified using HPLC (see Section 4.3.6). The comparison between the initial amount and the extracted amount gave the *EE*% (Equation (1)) [32]:

$$EE\% = \frac{Q\_f}{Q\_0} \times 100\tag{1}$$

where *Qf* is the total mass of BNB retained inside the liposomes and *Q*0 is the total mass of BNB initially used to prepare the liposomes. Both quantities are expressed in mg.

#### 4.3.3. In Vitro Drug Release Study

In order to investigate the release of the drug, we employed a dialysis membrane that had a molecular cut-off weight of 14,000 Da (Sigma–Aldrich, Madrid, Spain) in Franz-type diffusion cells. These cells had a diffusion area of 0.64 cm2 and a receptor chamber of 4.9 mL (Crown Glass Company, Inc., Jersey City, NJ, USA) [34,35]. Prior to use, the membrane was hydrated in methanol:water (1:1) for 24 h and then washed before being set in the Franz diffusion cells. The receptor medium used was Transcutol® P which was stirred at 500 r.p.m. to keep sink conditions. The experiment was conducted at 32 ◦C by means of a thermostatic water bath. An amount of 500 µL of formulation was added to the donor compartment; five replicates per each liposome were included. During the experiment, samples of 200 µL were collected at specific time intervals and, in order to maintain a constant volume in the cells, Transcutol® P was added after each sample collection. The collected samples were analyzed by a validated HPLC-fluorescence method, as described in Section 4.3.6. The cumulative amounts of BNB released from each liposomal formulation were plotted over time, and the data were analyzed by different kinetic models to describe the drug release profile. The determination coefficient (r2 ) was used to assess the goodness of fit [36].

Moreover, some non-modelestic parameters were calculated to compare different release profiles. These estimated parameters were the MRT of BNB from the formulation, the AUC representing the amount of BNB that was released from the formulation [37] and the efficiency (E) as the percentage of BNB released from the initial formulation.

#### 4.3.4. Ex Vivo Permeation Study

The permeation studies were conducted using Franz diffusion cells with a surface area measuring 0.64 cm2 and a receptor chamber of 4.9 mL. The altered abdominal human skin was placed between the donor and the receptor compartments [34,35]. The receptor medium was Transcutol® P. Amounts of 500 µL of each liposome was put on the donor compartment. Samples of 200 µL were taken over 25 h and replaced by Transctuol® P after every sampling time. We analyzed the samples using the validated HPLC-fluorescence method (see Section 4.3.6).

Three permeation parameters of each liposome were calculated: flux (*Jss*, µg/h), permeability coefficient (*Kp*, cm/h) and theoretical predicted plasma concentration in human steady state applied to a 10 cm2 surface (*Css*, ng/mL). Flux is the slope calculated with the aligned points in the permeation profile [35]. The permeability coefficient was obtained by the following equation:

$$Kp = \frac{Jss}{\mathbb{C}\_0 \cdot A} \tag{2}$$

where *C*0 (µg/mL) is the initial concentration of BNB in the liposome and *A* is the surface of the diffusion area (cm2 ) [34].

*Css* was calculated by the equation detailed below:

$$\mathcal{C}\_{ss} = \frac{Jss \cdot TSA}{\mathcal{C}lp \cdot A} \tag{3}$$

where *Jss* (µg/h) is the flux, *TSA* (cm2 ) is the theoretical surface area of application, *Clp* (ml/min) is the human plasma clearance of BNB and *A* (cm2 ) is the diffusion area of the Franz cells [34]. The area of application considered was 10 cm2 .

After completing the permeation study, we removed all tissues from the diffusion cells and washed away any liposomes remaining on the surface with distilled water. Then, we extracted the BNB that was retained in the permeation area of the tissue, cutting it, weighing it and immersing it in 1 mL of Transcutol® P. The sample was then sonicated for 10 min using an ultrasonic water bath [33]. The final step was to analyze the Transcutol® P with the extracted BNB using HPLC. We calculated the retained BNB in the tissues (*Qret*) using Equation (4) and the results were stated, normalized by the weight of the tissue and the diffusion area (0.64 cm2 ) and multiplied by the recuperation of the drug:

$$Q\_{rtl} = \frac{Q\_{ext}}{W \times A} \times \frac{100}{R} \tag{4}$$

In this equation, *Qext* represents the quantity of drug extracted from the tissue and is measured in µg, *W* denotes the weight of the tissue in grams, while *A* is the diffusion area in cm2 . Finally, *R* represents the proportion of BNB that is recovered in each tissue [35].

#### 4.3.5. Baricitinib Determination by HPLC

The measurement of BNB in each sample was carried out using high-performance liquid chromatography (HPLC) equipped with a fluorescence detector. The HPLC system consisted of a Chromatograph Waters Alliance 2695 and a Fluorescence Jasco FP-1520 detector that operated at an excitation wavelength of 310 nm and an emission wavelength of 390 nm. The chromatographic column was a Symmetry C18 (4.6 × 75 mm, 3.5 µm) and the mobile phase was Ammonium Formate 10 mM pH 7.4 (75:25 *v*/*v*); the flux was 1 mL/min and the volume of injection was 10 µL. The validated range for the quantification of BNB was from 0.03 to 1 µg/mL.

#### 4.3.6. Tolerance Study and Histological Analysis

Liposomes were also evaluated by a modified Draize skin test to study the effect on induced erythema and edema with xylol to simulate atopic skin. We used non-anesthetized New Zealand healthy rabbits (Harlan, Barcelona, Spain). They were cared for in accordance with the standard conditions, receiving food and water ad libitum. The objective was to detect the possible signs of damage on altered skin as indicated by the level of erythema and edema [38,39]. The studies were conducted under a protocol in accordance with the Mexican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999).

To obtain an ARS, we used microneedles so as to compromise the skin barrier function on the rabbits' backs. This was carried out one day after having measured their basal values and having shaved them. The animals were divided into different groups: the negative control group which only underwent microneedles; the positive control group which, in addition to the microneedles, received xylol to induce skin irritation; 3 other groups which received microneedles, plus xylol, plus one of the liposomes. Next, we applied 0.5 mL of xylol to all the rabbits minus the negative control (which also did not have liposomes). The positive control received microneedles plus xylol.

The responses were recorded at 15 min, 45 min and 3 h 30 min. At the same points in time, the transepidermal water loss (TEWL) and skin hydration were measured. For the positive control, 0.5 mL of xylol was applied and evaluated at the same times as the liposomes. A visual scoring system was used to evaluate inflammation; this system classifies from 1 to 4 according to whether the erythema and edema were practically imperceptible or had reached a large area of exposure [40]: 0 denotes no erythema and no edema, 1 denotes a little sign of erythema or edema, 2 denotes explicit erythema or slight edema, 3 denotes a moderate to severe erythema or edema and 4 refers to serious erythema (beet redness) through to the first signs of depth injuries or severe edema [41].

Xylol was used as an irritant component to induce allergic contact dermatitis (ACD), characterized by manifestations such as skin redness, swelling, warmth and itching and accompanied by dryness and flaking. Other articles, like those of Patricia L Nadworny et al. [42] or Liu Tang et al. [43], used dinitrochlorobenzene, or Paul L. Stanley et al. [44], who used 12-O-tetradecanoylphorbol-13-acetate (TPA).

TEWL is the amount of water that can diffuse through the skin stratum corneum per unit of time. The appropriate structure of intracellular lipids in the stratum corneum is one of the key factors in retaining transdermal water [45,46]. TEWL was measured by Tewameter (TEWL-Dermalab, Agaram Industries, India). An increment of this value means there is damage on the skin barrier [47]. The skin hydration was measured by a Corneometer (EnviroDerm, Ireland), which can quantify the amount of water within the corneum.

After monitoring the mentioned parameters for 3 h, the treated animal parts were histologically evaluated to assess the effect of the liposomes. To ensure this, the animals were euthanized with xylazine (Rompun® 20 mg/mL, Bayer Hispania, Sant Joan Despí, Spain) and ketamine (Imalgene® 100 mg/mL, Boehringer Ingelheim Animal Health España, Sant Cugat del Vallès, Spain). The mixture was injected via the right ear vein at 4 mg/kg [48]. After clinical death, the back skin was immediately excised, rinsed with PBS pH 7.4, and set overnight at room temperature in 4% buffered formaldehyde and subsequently embedded in paraffin wax. Transverse sections measuring 5 µm were stained with hematoxylin and eosin and then examined using a light microscope (Olympus BX41 and camera Olympus XC50) on blinded-coded samples to assess the histological structure [49].

Finally, a HET-CAM test was conducted to evaluate the potential risk of causing irritation to the eye after a periocular application. The HET-CAM test assesses the potential toxicity of formulations when applied to the CAM of a 10-day embryonated hen's egg (supplied by the G.A.L.L.S.A. farm, Tarragona, Spain). The CAM was observed for five minutes following application, with any reactions, such as bleeding (hemorrhage), blood vessel disintegration (coagulation) and protein denaturation (intra- and extra-vascular vessel lysis coagulation), being noted [18]. To this end, we applied 300µL of liposomes to the CAM and we waited 5 min to see if there had been any reaction working with the INVITTOX protocol [50]. The positive control used was a 0.1 N solution of NaOH, while the negative control was a solution containing 0.9% NaCl [50]. The IS was calculated, as described by Garrós et al., including 3 replicates per formulation [49].

#### **5. Conclusions**

Three liposomal formulations containing BNB, a Janus kinase inhibitor, were developed for the topical treatment of complement flare-ups in AD. The liposomes were prepared using three different lipids: POPC, CHOL and CER. The POPC:CHOL:CER formulation showed higher flux and permeation compared to the other formulations. However, there were no statistically significant differences in the retention concentration in the skin between POPC:CHOL and POPC:CHOL:CER. These formulations also showed that they retained higher amounts of BNB than liposome POPC; this may be due to them having a longer effect. However, further biomedical investigations should be carried out. The formulations did not demonstrate any irritant effect in the HET-CAM test, and the liposomes POPC and POPC:CHOL did not cause any structural alteration according to the histological analysis.

**Author Contributions:** Conceptualization, Ò.D., M.M. and A.C.C.; methodology, Ò.D., M.M. and H.C.; validation, M.M. and H.C.; formal analysis, N.G.; investigation, N.G., M.J.R.-L., N.B. and R.M.-M.; resources, Ò.D., M.M., A.C.C. and M.J.R.-L.; data curation, P.B.-S., Ò.D. and M.M.; writing original draft preparation, N.G. and P.B.-S.; writing—review and editing, P.B.-S. and M.M.; visualization, M.M.; supervision, A.C.C., M.M. and H.C.; project administration, M.M., A.C.C. and H.C.;

funding acquisition, A.C.C., M.M. and H.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The protocol used to carry out the studies was in accordance with the Mexican Official Norm for Animal Care and Handing (NOM-062-ZOO-1999) and with the approval of the Academic Committee of Ethics of the Vivarium of the Autonomous University of the Morelos State of Mexico, number 0122013. Moreover, the experiments were carried out in agreement with the guidelines stated in the protocol "Principles of Laboratory Animal Care" (publication 214/97 of 30 July).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors would like to thank Pol Alegret and Alexander Parra-Coca for their excellent technical support. We also thank Harry Paul for revising the English language.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

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## *Article* **Preparation, Characterization, and In Vivo Evaluation of Gentiopicroside‑Phospholipid Complex (GTP‑PC) and Its Self‑Nanoemulsion Drug Delivery System (GTP‑PC‑SNEDDS)**

**Yingpeng Tong 1,2,† , Wen Shi 2,† , Qin Zhang 2 and Jianxin Wang 1,2,3,\***

**Abstract:** The objective of the present study was to develop a gentiopicroside‑phospholipid com‑ plex (GTP‑PC) and its self‑nanoemulsion drug delivery system (GTP‑PC‑SNEDDS) to increase the oral bioavailability of gentiopicroside (GTP). The factors affecting the formation of GTP‑PC were studied with the complexation efficiency and dissociation rate. The properties of the complex were investigated by means of differential scanning calorimetry (DSC), X‑ray diffraction (XRD), Fourier transform infrared spectra (FT‑IR), dissolution, etc. Then, GTP‑PC was loaded into SNEDDS by in‑ vestigating the effects of weight ratios of GTP‑PC to blank SNEDDS, preparation technology, dilution media, and dilution multi, based on the screening results of oils, surfactants, and cosurfactants. In rats, GTP, GTP‑PC, and GTP‑PC‑SNEDDS were orally administered at different times, and GTP con‑ centrations were determined using RP‑HPLC. The optimal GTP‑PC was prepared with tetrahydrofu‑ ran as the reaction solvent, GTP:phospholipid = 1:2, and stirring for 4 h. The optimal prescription for GTP‑PC‑SNEDDS was as follows: Maisin 35‑1:Miglycol = 30%, Labrasol:Cremophor EL = 1:4 = 40%, Transcutol P = 30%; Maisin 35‑1:Miglycol = 12, and the ratio of GTP‑PC to blank was 1:10—then the mixture was stirred at 37 ◦C for 1 d and then placed for 2 d to form stable GTP‑PC‑SNEDDS. After oral administration of GTP, GTP‑PC and GTP‑PC‑SNEDDS, and mean plasma GTP concentration– time curves were all in accordance with the single‑compartment model. The Cmax, AUC0–∞, and Fr of the three formulations were significantly higher than that of GTP, demonstrating that GTP was metabolized rapidly, and its higher bioavailability could be achieved by the formation of GTP‑PC and GTP‑PC‑SNEDDS. Among the three formations, the bioavailability of GTP‑PC‑SNEDDS was highest, with approximately 2.6‑fold and 1.3‑fold of Fr value, compared with GTP‑PC (suspension) and GTP‑PC (oil solution), respectively. Compared with GTP, GTP‑PC and GTP‑PC‑SNEDDS en‑ hanced the bioavailability of GTP significantly. In the future, this study could serve as a reference for clinical trials using GTP‑PC and GTP‑PC‑SNEDDS.

**Keywords:** gentiopicroside; phospholipid complex; self‑nanoemulsion drug delivery system; oral bioavailability; pharmacokinetics

#### **1. Introduction**

Gentiopicroside (GTP, Figure 1A), a kind of iridoid glycoside, is one of the key ac‑ tive components from Gentiana species [1–3]. It exhibits many activities, including anti‑ inflammatory, analgesic, antioxidant, and anti‑hepatotoxic activities, which imply its ben‑ efit in the treatment of rheumatoid arthritis, liver illness (hepatitis), fever, digestive, intesti‑ nal disorders, and so on [4–9].

**Citation:** Tong, Y.; Shi, W.; Zhang, Q.; Wang, J. Preparation, Characterization, and In Vivo Evaluation of Gentiopicroside‑ Phospholipid Complex (GTP‑PC) and Its Self‑Nanoemulsion Drug Delivery System (GTP‑PC‑SNEDDS). *Pharmaceuticals* **2023**, *16*, 99. https://doi.org/10.3390/ph16010099

Academic Editors: Ana Catarina Silva, João Nuno Moreira and José Manuel Sousa Lobo

Received: 9 October 2022 Revised: 1 January 2023 Accepted: 5 January 2023 Published: 9 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

intestinal disorders, and so on [4–9].

**Figure 1.** The structure of GTP and its influencing factors on the formation of phospholipid complexes. (**A**): The structure of GTP. (**B**): Cumulative Dissolution of phytosome of different type of solvent in phosphate buffer saline (pH 6.8). (**C**): Cumulative Dissolution of phytosome of different molar ratios of GTP and phospholipids. **Figure 1.** The structure of GTP and its influencing factors on the formation of phospholipid com‑ plexes. (**A**): The structure of GTP. (**B**): Cumulative Dissolution of phytosome of different type of solvent in phosphate buffer saline (pH 6.8). (**C**): Cumulative Dissolution of phytosome of different molar ratios of GTP and phospholipids.

benefit in the treatment of rheumatoid arthritis, liver illness (hepatitis), fever, digestive,

Unfortunately, the clinical efficacy was limited by the low bioavailability of oral GTP. Several studies attributed the low bioavailability of oral GTP to the first-pass metabolism, bacterial metabolic processes, or decomposition in the intestine, as well as to poor absorption from the gastrointestinal tract [10–16]. To overcome the problem of the slight clinical efficacy of GTP, it is important to improve its bioavailability. Some reports had exposed that the bioavailability of GTP could be significantly improved by the interaction of the compound–compound in an herb extract [11] or the herb–herb in a formulae decoction [12,16]. Additionally, no data about pharmaceutical approaches have been published, in relation to improving the bioavailability of GTP after oral administration. Unfortunately, the clinical efficacy was limited by the low bioavailability of oral GTP. Several studies attributed the low bioavailability of oral GTP to the first‑pass metabolism, bacterial metabolic processes, or decomposition in the intestine, as well as to poor absorp‑ tion from the gastrointestinal tract [10–16]. To overcome the problem of the slight clin‑ ical efficacy of GTP, it is important to improve its bioavailability. Some reports had ex‑ posed that the bioavailability of GTP could be significantly improved by the interaction of the compound–compound in an herb extract [11] or the herb–herb in a formulae decoc‑ tion [12,16]. Additionally, no data about pharmaceutical approaches have been published, in relation to improving the bioavailability of GTP after oral administration.

The formation of a drug–phospholipid complex is an important, and the most common, way to improve bioavailability. Up until now, several drug–phospholipid complexes have been approved to use in clinical treatment, such as Meriva® (curcumin-PC) [17], Siliphos® (Silybin-PC) [17]. SNEDDS, which consists of a drug, oil, surfactant, and cosurfactant, can produce an oil-in-water (O/W) nanoemulsion with a droplet size of less than 100 nm by gently mixing with water [18–21]. It is another pharmaceutical means to enhance the drug's bioavailability. More importantly, the combination of PC and SNEDDS can overcome the same limitations of drugs with low oral bioavailability. It has been proposed that combining PC with SNEDDS can enhance the oral bioavailability of bioactive compounds or biomacromolecules, such as morin [22–25], akebia saponin D [26,27], rosuvastatin calcium [28], paclitaxel [29], curcumin [30], ellagic acid [31], baicalin [32], and matrine [33]. The formation of a drug–phospholipid complex is an important, and the most com‑ mon, way to improve bioavailability. Up until now, several drug–phospholipid complexes have been approved to use in clinical treatment, such as Meriva® (curcumin‑PC) [17], Siliphos® (Silybin‑PC) [17]. SNEDDS, which consists of a drug, oil, surfactant, and co‑ surfactant, can produce an oil‑in‑water (O/W) nanoemulsion with a droplet size of less than 100 nm by gently mixing with water [18–21]. It is another pharmaceutical means to enhance the drug's bioavailability. More importantly, the combination of PC and SNEDDS can overcome the same limitations of drugs with low oral bioavailability. It has been pro‑ posed that combining PC with SNEDDS can enhance the oral bioavailability of bioactive compounds or biomacromolecules, such as morin [22–25], akebia saponin D [26,27], ro‑ suvastatin calcium [28], paclitaxel [29], curcumin [30], ellagic acid [31], baicalin [32], and matrine [33].

In the above experiments, the tested compounds are water insoluble and belong to classes 2 or classes 4 of the biopharmaceutical classification system. However, there are no related studies on class 3 compounds. Classified as class 3 drugs, they are highly soluble in gastrointestinal fluids, but possess low absorption membrane permeability, often resulting in low bioavailability [34]. GTP was a typical class 3 compound with high water solubility (7.65 g/100 g at 23 °C) and low membrane permeability [35]. So, GTP was adopted in this study, and then GTP-PC and GTP-PC-SNEDDS were optimized to test their advantages on enhancing GTP's oral bioavailability. In the above experiments, the tested compounds are water insoluble and belong to classes 2 or classes 4 of the biopharmaceutical classification system. However, there are no related studies on class 3 compounds. Classified as class 3 drugs, they are highly soluble in gastrointestinal fluids, but possess low absorption membrane permeability, often resulting in low bioavailability [34]. GTP was a typical class 3 compound with high water solubility (7.65 g/100 g at 23 ◦C) and low membrane permeability [35]. So, GTP was adopted in this study, and then GTP‑PC and GTP‑PC‑SNEDDS were optimized to test their advantages on enhancing GTP's oral bioavailability.

ids.

#### **2. Results and Discussion** *2.1. Factors affecting the Formation of GTP-Phospholipid Complex*

**2. Results and Discussion**

#### *2.1. Factors Affecting the Formation of GTP‑Phospholipid Complex* 2.1.1. Different Type of Phospholipids 2.1.1. Different Type of Phospholipids **2. Results and Discussion**

2.1.1. Different Type of Phospholipids The complexation efficiency and dissociation rate of GTP-PC of different type of The complexation efficiency and dissociation rate of GTP-PC of different type of *2.1. Factors affecting the Formation of GTP-Phospholipid Complex*

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 3 of 20

The complexation efficiency and dissociation rate of GTP‑PC of different type of phos‑ pholipids, including hydrogenated phospholipids, soybean phospholipids, and egg yolk phospholipids, are shown in Table 1. The results display that the complexation efficiency of the GTP‑PC prepared by the soybean phospholipid and egg yolk phospholipid exceeded 95%. The dissociation rate of GTP‑PC in pH 6.8 PBS was hydrogenated phospholipid > egg yolk phospholipid > soybean phospholipid, indicating that the stability of hydrogenated phospholipids prepared by hydrogenated phospholipid was poor, and it was easy to dis‑ sociate in aqueous environment. The cost of egg yolk phospholipid is higher than that of soybean phospholipid. Considering the complexation efficiency, dissociation rate, and cost factors, soybean phospholipid was selected for preparing GTP‑PC. phospholipids, including hydrogenated phospholipids, soybean phospholipids, and egg yolk phospholipids, are shown in Table 1. The results display that the complexation efficiency of the GTP-PC prepared by the soybean phospholipid and egg yolk phospholipid exceeded 95%. The dissociation rate of GTP-PC in pH6.8 PBS was hydrogenated phospholipid > egg yolk phospholipid > soybean phospholipid, indicating that the stability of hydrogenated phospholipids prepared by hydrogenated phospholipid was poor, and it was easy to dissociate in aqueous environment. The cost of egg yolk phospholipid is higher than that of soybean phospholipid. Considering the complexation efficiency, dissociation rate, and cost factors, soybean phospholipid was selected for preparing GTP-PC. phospholipids, including hydrogenated phospholipids, soybean phospholipids, and egg yolk phospholipids, are shown in Table 1. The results display that the complexation efficiency of the GTP-PC prepared by the soybean phospholipid and egg yolk phospholipid exceeded 95%. The dissociation rate of GTP-PC in pH6.8 PBS was hydrogenated phospholipid > egg yolk phospholipid > soybean phospholipid, indicating that the stability of hydrogenated phospholipids prepared by hydrogenated phospholipid was poor, and it was easy to dissociate in aqueous environment. The cost of egg yolk phospholipid is higher than that of soybean phospholipid. Considering the complexation efficiency, dissociation rate, and cost factors, soybean phospholipid was selected for preparing GTP-PC. 2.1.1. Different Type of Phospholipids The complexation efficiency and dissociation rate of GTP-PC of different type of phospholipids, including hydrogenated phospholipids, soybean phospholipids, and egg yolk phospholipids, are shown in Table 1. The results display that the complexation efficiency of the GTP-PC prepared by the soybean phospholipid and egg yolk phospholipid exceeded 95%. The dissociation rate of GTP-PC in pH6.8 PBS was hydrogenated phospholipid > egg yolk phospholipid > soybean phospholipid, indicating that the stability of hydrogenated phospholipids prepared by hydrogenated phospholipid was poor, and it was easy to dissociate in aqueous environment. The cost of egg yolk phospholipid is higher than that of soybean phospholipid. Considering the complexation efficiency, dissociation *Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 3 of 20 **2. Results and Discussion** *2.1. Factors affecting the Formation of GTP-Phospholipid Complex* 2.1.1. Different Type of Phospholipids The complexation efficiency and dissociation rate of GTP-PC of different type of

**2. Results and Discussion**

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 3 of 20

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 3 of 20

*2.1. Factors affecting the Formation of GTP-Phospholipid Complex*

**Table 1.** Complexation efficiency and dissociation rate of GTP-PC of different types of phospholip-

hydrogenated phospholipid 88.20 ± 4.74 8.52

highest complexation efficiency and slowest dissociation. Therefore, tetrahydrofuran was

rate, and cost factors, soybean phospholipid was selected for preparing GTP-PC.

phospholipids, including hydrogenated phospholipids, soybean phospholipids, and egg

the solvent within 24 h; ++ indicates substance is easily dissoluble in the solvent within 8~12 h; ※

Ethyl Ether - - n-hexane ++ - - - indicates substance is not soluble in the solvent within 48 h; + indicates substance is soluble in the solvent within 24 h; ++ indicates substance is easily dissoluble in the solvent within 8~12 h; ※

n-hexane ++ - -

**)**

**Table 1.** Complexation efficiency and dissociation rate of GTP‑PC of different types of phospholipids. ids. yolk phospholipids, are shown in Table 1. The results display that the complexation effi-

**Table 1.** Complexation efficiency and dissociation rate of GTP-PC of different types of phospholip-

2.1.2. Dissolving Solvents

#### 2.1.2. Dissolving Solvents 2.1.2. Dissolving Solvents than that of soybean phospholipid. Considering the complexation efficiency, dissociation rate, and cost factors, soybean phospholipid was selected for preparing GTP-PC.

The solubility of GTP and GTP‑PC is shown in Table 2. The results show that GTP is in‑ soluble in dichloromethane, ethyl acetate, and tetrahydrofuran, but GTP‑PC and soybean phospholipid are easily soluble. In this case, the formation of GTP‑PC could be judged ac‑ cording to the change of solubility. Then, the most suitable solvent was chosen further by complexation efficiency and dissociation. The results are shown in Table 3 and Figure 1B, indicating that the phospholipid complex prepared in tetrahydrofuran had the highest complexation efficiency and slowest dissociation. Therefore, tetrahydrofuran was identi‑ fied as the dissolving solvent. The solubility of GTP and GTP-PC is shown in Table 2. The results show that GTP is insoluble in dichloromethane, ethyl acetate, and tetrahydrofuran, but GTP-PC and soybean phospholipid are easily soluble. In this case, the formation of GTP-PC could be judged according to the change of solubility. Then, the most suitable solvent was chosen further by complexation efficiency and dissociation. The results are shown in Table 3 and Figure 1B, indicating that the phospholipid complex prepared in tetrahydrofuran had the highest complexation efficiency and slowest dissociation. Therefore, tetrahydrofuran was identified as the dissolving solvent. The solubility of GTP and GTP-PC is shown in Table 2. The results show that GTP is insoluble in dichloromethane, ethyl acetate, and tetrahydrofuran, but GTP-PC and soybean phospholipid are easily soluble. In this case, the formation of GTP-PC could be judged according to the change of solubility. Then, the most suitable solvent was chosen further by complexation efficiency and dissociation. The results are shown in Table 3 and Figure 1B, indicating that the phospholipid complex prepared in tetrahydrofuran had the highest complexation efficiency and slowest dissociation. Therefore, tetrahydrofuran was identified as the dissolving solvent. 2.1.2. Dissolving Solvents The solubility of GTP and GTP-PC is shown in Table 2. The results show that GTP is insoluble in dichloromethane, ethyl acetate, and tetrahydrofuran, but GTP-PC and soybean phospholipid are easily soluble. In this case, the formation of GTP-PC could be judged according to the change of solubility. Then, the most suitable solvent was chosen further by complexation efficiency and dissociation. The results are shown in Table 3 and Figure 1B, indicating that the phospholipid complex prepared in tetrahydrofuran had the **Table 1.** Complexation efficiency and dissociation rate of GTP-PC of different types of phospholipids. **Type of Phospholipids Complexation Efficiency (%) K (h−1** soybean phospholipid 97.15 ± 3.27 1.60 egg yolk phospholipid 100 ± 4.92 2.49 hydrogenated phospholipid 88.20 ± 4.74 8.52

**Table 2.** Dissolubility of GTP, GTP‑PC, and phospholipids in different solvents (mg/mL). **Table 2.** Dissolubility of GTP, GTP-PC, and phospholipids in different solvents (mg/mL). **Table 2.** Dissolubility of GTP, GTP-PC, and phospholipids in different solvents (mg/mL). identified as the dissolving solvent. 2.1.2. Dissolving Solvents

Ethyl Ether - - n-hexane ++ - - - indicates substance is not soluble in the solvent within 48 h; + indicates substance is soluble in Ethyl Ether - - n-hexane ++ - - - indicates substance is not soluble in the solvent within 48 h; + indicates substance is soluble in Trichloromethane ++ + ++ Ethyl Ether - - - - indicates substance is not soluble in the solvent within 48 h; + indicates substance is soluble in the solvent within 24 h; ++ indicates substance is easily dissoluble in the solvent within 8~12 h; **Solvents Soybean Phospholipid GTP GTP-PC** Water ※ ++ ※ Methanol + ++ + indicates substance is dispersed in the solvent within 48 h.

indicates substance is dispersed in the solvent within 48 h. indicates substance is dispersed in the solvent within 48 h. - indicates substance is not soluble in the solvent within 48 h; + indicates substance is soluble in Ethanol - - ※ Acetone ++ - - **Table 3.** Complexation efficiency and dissociation rate of GTP‑PC of different types of solvent.

the solvent within 24 h; ++ indicates substance is easily dissoluble in the solvent within 8~12 h; ※

### 2.1.3. Molar Ratio of GTP to Phospholipid

Complexation efficiency and dissociation of the GTP‑PC of different molar ratios of GTP and phospholipid are shown in Table 4 and Figure 1C. The data indicated that the com‑ plexation efficiency of phospholipid complex was close to 100% for the GTP of GTP:phospholipid ≤ 1:1. The dissociation rate increased with the ratio of GTP‑to‑phosp‑ holipid increase, suggesting that the greater the amount of phospholipid, the higher the stability of GTP‑PC. However, the content of phospholipid could not be too much, due to the poor dispersion and fluidity of the GTP‑PC of the drug‑to‑lipid ratio ≥ 1:3. Addition‑ ally, according to the results of the bioavailability, the molar ratio of GTP and phospholipid was identified as 1:2 [36].

**Table 4.** Complexation efficiency and dissociation rate of phytosome of different molar ratios of GTP and phospholipid.

#### 2.1.4. Stirring Time

It was found that, with the stirring time increasing from 0.5 to 4 h, the complexation efficiency increased from 83.57% to 99.50%, and at 4 h reached the peak. When stirring for 5 h and 6 h, the complexation efficiency declined to 92.63% and 88.63%. This may be due to the instability of GTP. Therefore, the optimal stirring time was 4 h.

## *2.2. Characterization of GTP‑PC*

#### 2.2.1. Differential Scanning Calorimetry (DSC)

In DSC, an interaction can be detected by eliminating endothermic peaks, showing new peaks, changing peak shape, and changing the peak temperature and relative peak area or enthalpy [37]. The DSC curves of the GTP, phospholipids, physical mixture, and GTP‑PC are shown in Figure 2A. The GTP displayed an abroad endothermal peak at 260.5 ◦C (Figure 2Aα). The phospholipid had a peak at 100.6 ◦C (Figure 2Aβ). The DSC of the phospholipid complex showed that the phospholipid peak vanished, and the onset temperature was lower than GTP's, which was at 245.08 ◦C (Figure 2Aδ). The GTP and phospholipids were thought to interact through hydrogen bonds or van der Waals forces. As the GTP and phospholipid polarity parts were combined, the carbon–hydrogen chain of phospholipids was free to turn and enclose the polarity parts and GTP of the phospho‑ lipids. After that, the sequence between the aliphatic hydrocarbon chains of phospholipids decreased and the peak of phospholipids disappeared, lowering the onset temperature of GTP [38]. The physical mixture of the GTP and phospholipid showed two peaks. The for‑ mer was 60.5 ◦C, lower than the onset temperature of phospholipid; the other was 245.8 ◦C, the same with the onset temperature of GTP‑PC (Figure 2Aχ). It was thought that, as the temperature increased, the phospholipid melted, and the drugs dissolved into it, partially forming phospholipid complexes.

#### 2.2.2. X‑ray Diffractometry (XRD)

The powder X‑ray diffraction patterns of the GTP, phospholipids, physical mixture, and complex are shown in Figure 2B. The GTP displayed sharp crystalline peaks. In con‑ trast, phospholipids were amorphous and lacking crystalline peaks. Despite the physical mixtures of the GTP and phospholipid, some drug signals could still be detected. How‑ ever, the crystalline peaks had disappeared in the GTP‑PC. The combination of the polar

ends of phospholipid with GTP led to their being highly dispersed, thus inhibiting their crystalline characteristics. *Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 5 of 20

> **Figure 2.** Characterization of GTP-PC. (**A**): Differential scanning calorimetry (DSC). (**B**): X-ray diffractometry (XRD). (**C**): Thin-layer chromatography (TLC). (**D**): Ultraviolet (UV) spectra. (**E**): Degradation of GTP-PC in phosphate buffer solutions and diluted hydrochloric acid at different pH (n = 3). GTP (α), phospholipids (β), physical mixture (χ), GTP-PC (δ), and standard GTP (ε). **Figure 2.** Characterization of GTP‑PC. (**A**): Differential scanning calorimetry (DSC). (**B**): X‑ray diffrac‑ tometry (XRD). (**C**): Thin‑layer chromatography (TLC). (**D**): Ultraviolet (UV) spectra. (**E**): Degrada‑ tion of GTP‑PC in phosphate buffer solutions and diluted hydrochloric acid at different pH (n = 3). GTP (α), phospholipids (β), physical mixture (χ), GTP‑PC (δ), and standard GTP (ε).

#### 2.2.2. X-ray Diffractometry (XRD) 2.2.3. Fourier Transform Infrared Spectra (FT‑IR)

The powder X-ray diffraction patterns of the GTP, phospholipids, physical mixture, and complex are shown in Figure 2B. The GTP displayed sharp crystalline peaks. In contrast, phospholipids were amorphous and lacking crystalline peaks. Despite the physical mixtures of the GTP and phospholipid, some drug signals could still be detected. However, the crystalline peaks had disappeared in the GTP-PC. The combination of the polar ends of phospholipid with GTP led to their being highly dispersed, thus inhibiting their crystalline characteristics. 2.2.3. Fourier Transform Infrared Spectra (FT-IR) The FT-IR of the GTP, phospholipids, physical mixture, and GTP-PC are shown in Figure 3A–D, respectively. The FT-IR spectrum of GTP was shown in Figure 3A, disclosing a broad band of 3600–3000 cm−1 for hydrogen-bonded O-H stretching vibrations. The absorption bands at 1713.35, 1609.84 cm−1 related to the C=O and C=C stretching. The signal at 1273.48 and 1007.06 cm−1 were assigned to C–O–C vibration. This FT-IR spectrum was consistent with the previously published reports. The stretching vibration of hydroxyl in GTP (υOH, 3420.86) and phospholipids (υOH, 3389.75) were superimposed in the physical mixture, forming a large, wide peak. The vibration of the hydrocarbon of saturated long-chain fatty (υCH, 2924.56, 2853.51), carbonyl of fatty acid ester (υC=O, 1737.68), phosphorus oxygen double bond (υP=O, 1241.04), and phosphorus oxygen single bond (υP-O-C, 1089.90) in phospholipid can be found, indicating little interaction between the GTP and phospholipid in the physical mixture (Figure 3C). In the phospholipid (Figure 3B), there were strong hydrogen bonds, so the vibration of hydroxyl was very The FT‑IR of the GTP, phospholipids, physical mixture, and GTP‑PC are shown in Figure 3A–D, respectively. The FT‑IR spectrum of GTP was shown in Figure 3A, disclos‑ ing a broad band of 3600–3000 cm−1 for hydrogen‑bonded O‑H stretching vibrations. The absorption bands at 1713.35, 1609.84 cm−1 related to the C=O and C=C stretching. The signal at 1273.48 and 1007.06 cm−1 were assigned to C–O–C vibration. This FT‑IR spec‑ trum was consistent with the previously published reports. The stretching vibration of hydroxyl in GTP (υOH, 3420.86) and phospholipids (υOH, 3389.75) were superimposed in the physical mixture, forming a large, wide peak. The vibration of the hydrocarbon of satu‑ rated long‑chain fatty (υCH, 2924.56, 2853.51), carbonyl of fatty acid ester (υC=O, 1737.68), phosphorus oxygen double bond (υP=O, 1241.04), and phosphorus oxygen single bond (υP‑O‑C, 1089.90) in phospholipid can be found, indicating little interaction between the GTP and phospholipid in the physical mixture (Figure 3C). In the phospholipid (Figure 3B), there were strong hydrogen bonds, so the vibration of hydroxyl was very strong and had a relatively low wavenumber (3389.75). This was due to the fact that the phospholipid phosphate and quaternary ammonium in the phospholipids had a strong ability to ion‑ ize, and hydrogen bonds formed by positive and negative ions were significantly strong, possibly with ionic character. However, the combination of phospholipid and GTP made new hydrogen bonds, formed between the two, and also destroyed part of the hydrogen bonds between the phospholipids (Figure 3D), resulting in the vibration of the hydroxyl moving to a higher wavenumber (υOH, 3396.32). Moreover, the υC=O and υP‑O‑C in phospholipids moved to a lower wavenumber (υC=O, 1737.68 to 1735.78; υP‑O‑C, 1089.90 to 1085.39), indicating that new hydrogen bonds may have been formed.

strong and had a relatively low wavenumber (3389.75). This was due to the fact that the phospholipid phosphate and quaternary ammonium in the phospholipids had a strong ability to ionize, and hydrogen bonds formed by positive and negative ions were significantly strong, possibly with ionic character. However, the combination of phospholipid and GTP made new hydrogen bonds, formed between the two, and also destroyed part of the hydrogen bonds between the phospholipids (Figure 3D), resulting in the vibration

and υP-O-C in phospholipids moved to a lower wavenumber (υC=O, 1737.68 to 1735.78; υP-O-C, 1089.90 to 1085.39), indicating that new hydrogen bonds may have been formed.

**Figure 3.** Fourier Transform Infrared spectra (FT-IR) of GTP-PC. GTP (**A**), phospholipids (**B**), physical mixture (**C**), GTP-PC (**D**). **Figure 3.** Fourier Transform Infrared spectra (FT‑IR) of GTP‑PC. GTP (**A**), phospholipids (**B**), physi‑ cal mixture (**C**), GTP‑PC (**D**).

The FT-IR of the GTP, phospholipids, physical mixture, and GTP-PC is shown in Figure 3A–D, respectively. The GTP spectrum (Figure 3A) disclosed the characteristic hydrogen-bonded O-H stretching vibration at 3600–3000 cm−1, C=O stretching vibration at 1713.35 cm−1, C=C stretching vibration at 1609.84 cm−1, and C–O stretching vibrations at 1273.48, 1083.96, and 1007.06 cm−1. The characteristic C-H stretching and bending vibrations of the long fatty acid chain of PC shown in Figure 3B were at 2924.56, 2853.51, and 1466.19 cm−1. Carbonyl of fatty acid ester (υC=O) at 1737.68 cm−1, phosphorus oxygen double bond (υP=O) at 1241.04 cm−1, and phosphorus oxygen single bond (υP-O-C) at 1089.90 cm−1 in phospholipid can also be found. In the spectrum of the physical mixture of GTP and PC, the peaks of O-H stretching vibrations of GTP and PC were superposed to form a wide peak at 3409.70 cm−1, with the other main peaks from GTP and PC still existing in the physical mixture of GTP and PC, but the intensity of some peaks from GTP were significantly weakened, such as the C=C stretching vibration at 1609.84 cm−1 and C–O stretching vibration at 1007.06 cm−1. However, the combination of the phospholipid and GTP may form new hydrogen bonds between them and destroy part of the hydrogen bonds between the phospho-lipids (Figure 3D), resulting in the hydroxyl vibration of PC moving to higher wavenumber at 3396.32 cm−1. The intensity of the peaks from GTP at 1609.84 cm−1 and 1007.06 cm−1 were also weakened, but they were relatively stronger than those from the physical mixture of GTP and PC. The FT‑IR of the GTP, phospholipids, physical mixture, and GTP‑PC is shown in Figure 3A–D, respectively. The GTP spectrum (Figure 3A) disclosed the characteristic hydrogen‑bonded O‑H stretching vibration at 3600–3000 cm−1 , C=O stretching vibration at 1713.35 cm−1 , C=C stretching vibration at 1609.84 cm−1 , and C–O stretching vibrations at 1273.48, 1083.96, and 1007.06 cm−1 . The characteristic C‑H stretching and bending vi‑ brations of the long fatty acid chain of PC shown in Figure 3B were at 2924.56, 2853.51, and 1466.19 cm−1 . Carbonyl of fatty acid ester (υC=O) at 1737.68 cm−1 , phosphorus oxygen double bond (υP=O) at 1241.04 cm−1 , and phosphorus oxygen single bond (υP‑O‑C) at 1089.90 cm−1 in phospholipid can also be found. In the spectrum of the physical mixture of GTP and PC, the peaks of O‑H stretching vibrations of GTP and PC were superposed to form a wide peak at 3409.70 cm−1 , with the other main peaks from GTP and PC still existing in the physical mixture of GTP and PC, but the intensity of some peaks from GTP were significantly weakened, such as the C=C stretching vibration at 1609.84 cm−1 and C–O stretching vibration at 1007.06 cm−1 . However, the combination of the phospholipid and GTP may form new hydrogen bonds between them and destroy part of the hydrogen bonds between the phospho‑lipids (Figure 3D), resulting in the hydroxyl vibration of PC moving to higher wavenumber at 3396.32 cm−1 . The intensity of the peaks from GTP at 1609.84 cm−1 and 1007.06 cm−1 were also weakened, but they were relatively stronger than those from the physical mixture of GTP and PC.

#### 2.2.4. Thin-Layer Chromatography (TLC) 2.2.4. Thin‑Layer Chromatography (TLC)

Only one spot with the same Rf as GTP was observed in the chromatogram of GTP-PC, similar to that of the physical mixture (Figure 2C). As the GTP and phospholipid have no chemical groups that can react with each other under our preparation conditions, they were unable to form new compounds. Therefore, the TLC did not reveal any new spots, and the UV confirmed the result. Only one spot with the same Rf as GTP was observed in the chromatogram of GTP‑ PC, similar to that of the physical mixture (Figure 2C). As the GTP and phospholipid have no chemical groups that can react with each other under our preparation conditions, they were unable to form new compounds. Therefore, the TLC did not reveal any new spots, and the UV confirmed the result.

#### 2.2.5. Ultraviolet (UV) Spectra

2.2.5. Ultraviolet (UV) Spectra As can be seen in Figure 2D, the phospholipid exhibited only end absorptions close to 200 nm, while GTP, the physical mixture, and GTP-PC showed nearly identical absorption curves. All of them exhibited two characteristic absorption bands at 240 and 270 nm. As can be seen in Figure 2D, the phospholipid exhibited only end absorptions close to 200 nm, while GTP, the physical mixture, and GTP‑PC showed nearly identical absorption curves. All of them exhibited two characteristic absorption bands at 240 and 270 nm. There‑ fore, the UV spectra of GTP was not changed during the combination with the phospholipid, indicating that no chemical groups could react between the GTP and the phospholipid.

#### 2.2.6. Dissolution Studies in Phosphate Buffer Saline (pH 6.8)

Figure 2E shows the dissolution profile of GTP from the GTP‑PC and GTP in phos‑ phate buffer saline (pH 6.8). The dissolution of GTP was complete in 30 min, while the GTP from GTP‑PC had a fast dissolution in the 0–2 h (about 50%); then, the dissolution tended to increase slowly in 2–12 h, and eventually, the phospholipid complex almost completely dissolved in 12 h. Therefore, the GTP and GTP‑PC had significantly different dissolution characteristics, in that the dissolution of GTP in GTP‑PC was delayed, compared with the GTP material.

#### *2.3. Preparation Process of GTP‑PC‑SNEDDS*

#### 2.3.1. Screening of Oils, Surfactants and Cosurfactant

Among the oils surfactants and cosurfactants that have been experimentally inves‑ tigated and shown in Table 5, imwitor 742, labrasol, and transcutol P had the highest solubility, which were 0.146 ± 0.028, 0.107 ± 0.011, and 0.315 ± 0.012 g/g, respectively. Additionally, the mixtures of Maisin 35‑1/Miglycol 812N, Imwitor742/Miglycol 812N, and Labrasol/Cremophor EL with different ratios also had a relatively high solubility.

In previous preliminary studies on the blank formulation of SNEDDS, the SNEDDS could not have been formed by choosing Imwitor 742 or maisin 35‑1 as the oil, Labrasol as the surfactant, or Transcutol P as the cosurfactant. Only when the oil loading was less than 10% could the microemulsion be infinitely diluted, but layered after 30 min, which might be related to the weak emulsifying ability of Labrasol. It was reported that the combination of Labrasol and Cremophor EL in the formulation could easily form self‑microemulsion because of the high emulsifying ability of Cremophor EL and the excellent dissolving ca‑ pacity of Labrasol. When the ratio of Labrasol:Cremophor EL = 1:4, the microemulsion area was larger. When the surfactant was changed to Labrasol:Cremophor EL = 1:4, the formed SNEDDS would not layer after 24 h, while the oil loading was still less than 10%, which might have been related with the weak polarity of Imwitor 742. However, the mixture

of Maisin 35‑1:Miglyol 812N and Imwitor742:Miglyol 812N would significantly improve the oil loading of SNEDDS, which is consistent with the previous reports. After the Maisin 35‑1 was mixed with Miglyol 812N, the mixture would change the polarity of the oil phase, improve the ability of oil phase molecule penetrating into surfactant, and reduce the inter‑ facial tension between oil and water, which would result in the formulation of stable mi‑ croemulsion droplets. In the mixture, the combination of the long‑chain oils solubilizing in microemulsions and short‑chain oils acting as cosurfactants would help the hydrophilic groups of oil molecules to disperse in polar cephalic groups of surfactants form chelating effect, which would increase the formation area of microemulsions. ports. After the Maisin 35-1 was mixed with Miglyol 812N, the mixture would change the polarity of the oil phase, improve the ability of oil phase molecule penetrating into surfactant, and reduce the interfacial tension between oil and water, which would result in the formulation of stable microemulsion droplets. In the mixture, the combination of the longchain oils solubilizing in microemulsions and short-chain oils acting as cosurfactants would help the hydrophilic groups of oil molecules to disperse in polar cephalic groups of surfactants form chelating effect, which would increase the formation area of microemulsions. Based on previous results shown above, the pseudo-ternary phase diagrams were

as the surfactant, or Transcutol P as the cosurfactant. Only when the oil loading was less than 10% could the microemulsion be infinitely diluted, but layered after 30 min, which might be related to the weak emulsifying ability of Labrasol. It was reported that the combination of Labrasol and Cremophor EL in the formulation could easily form self-microemulsion because of the high emulsifying ability of Cremophor EL and the excellent dissolving capacity of Labrasol. When the ratio of Labrasol:Cremophor EL = 1:4, the microemulsion area was larger. When the surfactant was changed to Labrasol:Cremophor EL = 1:4, the formed SNEDDS would not layer after 24 h, while the oil loading was still less than 10%, which might have been related with the weak polarity of Imwitor 742. However, the mixture of Maisin 35-1:Miglyol 812N and Imwitor742:Miglyol 812N would significantly improve the oil loading of SNEDDS, which is consistent with the previous re-

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 8 of 20

Based on previous results shown above, the pseudo‑ternary phase diagrams were con‑ structed by selecting Imwitor742:Miglyol 812N = 1:1 or Maisin 35‑1:Miglyol 812N = 1:1 as the oil phase, Labrasol:Cremophor EL = 1:4 as the surfactant, and Transcutol P as the cosurfactant. It can be seen that the self‑microemulsifying region of Maisin 35‑1:Miglyol 812N = 1:1 was big‑ ger than that of Imwitor742: Miglyol = 1:1 (Figure 4A,B). The effects of different weight ratios of Maisin 35‑1 and Miglyol 812N on the self‑microemulsifying region was shown in Figure 4B,C, and Maisin 35‑1:Miglyol 812N = 1:2 was chosen as oil phase because of its lager self‑microemulsifying region and higher oil loading. Labrasol:Cremophor EL = 1:4 would also been selected by comparing the effects of different weight ratios on the self‑ microemulsifying region and oil loading, which are presented in Figure 4C–F. constructed by selecting Imwitor742:Miglyol 812N = 1:1 or Maisin 35-1:Miglyol 812N = 1:1 as the oil phase, Labrasol:Cremophor EL = 1:4 as the surfactant, and Transcutol P as the cosurfactant. It can be seen that the self-microemulsifying region of Maisin 35-1:Miglyol 812N = 1:1 was bigger than that of Imwitor742: Miglyol = 1:1 (Figure 4A,B). The effects of different weight ratios of Maisin 35-1 and Miglyol 812N on the self-microemulsifying region was shown in Figure 4B,C, and Maisin 35-1:Miglyol 812N = 1:2 was chosen as oil phase because of its lager self-microemulsifying region and higher oil loading. Labrasol:Cremophor EL = 1:4 would also been selected by comparing the effects of different weight ratios on the self-microemulsifying region and oil loading, which are presented in Figure 4C–F.

**Figure 4.** Pseudo‑ternary phase diagrams of different compositions of oil, surfactant, and co‑ surfactant. The region of self‑microemulsifaction is in black. (**A**,**B**) indicates effects of different oil on the efficient self‑microemulsifacation. The oil phase was as follows: (**A**) as Imwitor742:Miglyol 812N = 1:1; (**B**) as Maisin 35‑1:Miglyol 812N = 1:1. (**C**–**F**) indicates effects of different ratios between Labrasol and Cremophor EL from 1:1 to 1:4 on the efficient self‑microemulsifacation, respectively.

#### 2.3.2. Factors Affecting the Formation of GTP‑PC‑SNEDDS

In the present work, the effect factors for preparation of GTP‑PC‑SNEDDS, including the weight ratios of GTP‑PC to blank SNEDDS, preparation technology, dilution media, and dilution multi, were further optimized, and the results are presented in Tables 6–9.

**Table 6.** The particle size of GTP‑PC‑SNEDDS with different ratios of drug/blank formula (*w*/*w*) in 0.1 M HCl, pH 6.8 PBS, and water (nm).

**Table 7.** The effect of preparation technology on the efficiency of self‑microemulsifacation of GTP‑ PC‑SNEDDS.

**Table 8.** The effects of different dilution media on the efficiency of self‑microemulsifacation of GTP‑ PC‑SNEDDS.

**Table 9.** The effects of different dilution multi on the efficiency of self‑microemulsifacation of GTP‑ PC‑SNEDDS.

As described in Table 6, the formulation with the weight ratio of 1:10 (GTP‑PC:blank SNEDDS) could form a stable self‑microemulsion in both the PBS and HCl. This might be related to the solubility of GTP‑PC. When the weight ratio was 1:10, the most stable self‑microemulsion with the smallest particle size could be formed. With the increase of drug concentration, resulting in the increase of phospholipids, the particle size of self‑ microemulsion became bigger, which would affect the efficiency of the self‑microem‑ ulsification. In addition, the undissolved drugs also had some influence on the particle size and stability of the system. Therefore, the ratio of drug‑to‑blank medium was 1:10 (*w*/*w*).

As shown in Table 7, the GTP‑PC‑SNEDDS prepared by ultrasonic method and vo‑ lution would become turbid after dilution, and then be clear after standing for 30 min. Therefore, the excessively vigorous preparation process had a certain influence on the self‑microemulsifying drug delivery system. Prepared by placing at different tempera‑ ture, the self‑microemulsifying drug delivery system would become layered, implying that it was an unstable system. The GTP‑PC‑SNEDDS, prepared by stirring at room tem‑ perature, was the most stable system with the smallest particle size and the shortest self‑ microemulsification time.

The effects of different dilution media on the efficiency of self‑microemulsifacation is shown in Table 8. After being diluted 250 times by different media, the particle diameters of GTP‑PC‑SNEDDS, from large to small, were: pH 6.8 PBS > 0.1 M HCl ≈ distilled water. It might be due to the NaCl in the PBS, which can cause salting out to lower the surfactant content and reduce the solubility of the surfactant, resulting in an increase in particle size. After repeated verification, the particle size in PBS was about two times larger than that in HCl, and the system was relatively stable. Considering the cost and ease of operation, the 0.1 M HCl was used to replace pH 6.8 PBS for the following investigation.

As described in Table 9, there was not a significant effect on the particle size of GTP‑ PC‑SNEDDS when it was diluted 100–500 times by 0.1 M HCl. However, the particle size was remarkably reduced when it was diluted 1000 times, which might be due to the fact that SNEDDS was in a solution state after being infinitely diluted. In keeping with the previous investigations, the dilution factor was chosen to be 250 times.

In summary, the preparation process with the following parameters had the highest efficiency of self‑microemulsifacation of GTP‑PC‑SNEDDS: the ratio of GTP‑PC to blank was 1:10, and then the mixture was stirred at room temperature to form GTP‑PC‑SNEDDS, which was diluted 250 times by 0.1 M HCl, subsequently.

After many validation tests, the formulation with the most stable system was deter‑ mined, and it had the highest solubility, a suitable particle size (<100 nm), and a shorter emulsion time (<2 min). Therefore, the optimal prescription was as follows: Maisin 35‑ 1:Miglycol = 30%, Labrasol: Cremophor EL = 14 = 40%, Transcutol P = 30%, and Maisin 35‑1:Miglycol = 12, and the ratio of GTP‑PC to blank was 1:10; then, the mixture was stirred at 37 ◦C for 1 d and placed for 2 d to form stable GTP‑PC‑SNEDDS.

## *2.4. Characterization of GTP‑PC‑SNEDDS*

### 2.4.1. Transmittance Electron Microscope

The diluted GTP‑PC‑SNEDDS was examined by a transmission electron microscope. Because the particle size of GTP‑PC‑SNEDDS was only about 20 nm, which could eas‑ ily form a relatively uniform spherical emulsion, the emulsion droplets were small and evenly dispersed under transmission electron microscope after negative staining with 1% phosphotungstic acid.

#### 2.4.2. Droplet Size and Zeta Potential

As presented in Figure 5A,B, the average droplet sizes of GTP‑PC‑SNEDDS diluted by pH 1.0 HCl or pH 6.8 PBS were (25.2 ± 5.3) nm and (46.7 ± 8.2) nm, respectively, showing Gaussian distribution. The Zeta potentials of the GTP‑PC‑SNEDDS diluted by pH 1.0 HCl or pH 6.8 PBS were (−19.23 ± 5.84) mV and (−23.43 ± 3.93) mV, respectively, showing no significant difference (*p* > 0.05).

#### 2.4.3. Stability of Microemulsion Particle Size after Microemulsification

As described in Table 10, the particle size of the self‑microemulsifying solution was not significantly changed within 8 h after dilution with 0.1 M HCl and distilled water (*p* > 0.05), and there was no drug for crystallization. However, the particle size of GTP‑PC‑ SNEDDS diluted in pH 6.8 PBS significantly increased with time, and this might be due to the present of salt in PBS, which can reduce the surfactant content by salting out. The de‑ crease of the surfactant content will reduce the surfactant solubilizing ability and weaken the self‑microemulsifying ability, resulting in an increase in particle size. The larger par‑ ticle size may be also due to the easily aggregation of the particles when dispersed in pH 6.8 PBS, with time increasing [39].

**Figure 5.** Characterization of GTP-PC-SNEDDS. (**A**): Particle Size distribution in dynamic light scattering (DLS) of SNEDDS at the dilution of 1:250 by pH 6.8 PBS. (**B**): Particle Size distribution DLS of SNEDDS at the dilution of 1:250 by pH 1.0 HCl. **Figure 5.** Characterization of GTP‑PC‑SNEDDS. (**A**): Particle Size distribution in dynamic light scat‑ tering (DLS) of SNEDDS at the dilution of 1:250 by pH 6.8 PBS. (**B**): Particle Size distribution DLS of SNEDDS at the dilution of 1:250 by pH 1.0 HCl.

2.4.3. Stability of Microemulsion Particle Size after Microemulsification As described in Table 10, the particle size of the self-microemulsifying solution was **Table 10.** Stability of particle size after self‑microemulsifying in 0.1 M HCl, pH 6.8 PBS, and distilled water at 37 ◦C.

persed in pH 6.8 PBS, with time increasing [39].

**Table 10.** Stability of particle size after self-microemulsifying in 0.1M HCl, pH6.8 PBS, and distilled water at 37 °C. Therefore, since various salts are present in the intestinal tract, the particle size of the self‑microemulsifying liquid in the intestinal tract easily became larger.

#### *2.5. Bioavailability Experiments in Rats*

**Time (h) HCl Particle Size (nm) pH6.8 PBS Particle Size (nm) Distilled Water Particle Size (nm)**  0 27.8 ± 3.9 46.7 ± 2.1 24.6 ± 3.4 1 30.5 ± 5.0 56.7 ± 8.5 28.2 ± 4.1 2 31.5 ± 4.4 62.7 ± 8.4 30.8 ± 4.8 4 32.7 ± 4.3 61.6 ± 5.7 31.4 ± 3.9 8 35.4 ± 5.7 65.3 ± 6.2 34.6 ± 2.5 Therefore, since various salts are present in the intestinal tract, the particle size of the self-microemulsifying liquid in the intestinal tract easily became larger. Figure 6 shows the complete separation of the GTP in plasma under analytical condi‑ tions (A = 0.0133 C + 0.004, r = 0.9996), where C is the concentration ratio of GTP to theo‑ phylline, and A is the corresponding peak‑area ratio of GTP/theophylline. The results at‑ tained from the RSD inter‑days of low, middle, and high concentrations were 8.45%, 2.39%, and 3.78%, respectively. The corresponding RSD intra‑days were 7.99%, 2.59%, and 5.42%, and the recoveries were (96.67 ± 6.22)%, (102.24 ± 2.71)%, and (102.40 ± 5.58)%, which indicated recoveries and RSD inter‑days, and the RSD intra‑days were satisfying, with the lowest detection limit at 50 ng·mL−1 .

Figure 6 shows the complete separation of the GTP in plasma under analytical conditions (A = 0.0133 C + 0.004, r = 0.9996), where C is the concentration ratio of GTP to theophylline, and A is the corresponding peak-area ratio of GTP/theophylline. The results attained from the RSD inter-days of low, middle, and high concentrations were 8.45%, 2.39%, and 3.78%, respectively. The corresponding RSD intra-days were 7.99%, 2.59%, and 5.42%, and the recoveries were (96.67 ± 6.22)%, (102.24 ± 2.71)%, and (102.40 ± 5.58)%, which indicated recoveries and RSD inter-days, and the RSD intra-days were satisfying,

*2.5. Bioavailability Experiments in Rats*

with the lowest detection limit at 50 ng·mL−1.

**Figure 6.**  **Figure 6.**

Figure 7 and Table 11 illustrate the mean plasma concentration–time curve of GTP in rats after oral administration of GTP, GTP-PC, and GTP-PC-SNEDDS (equivalent to 80 mg/kg of GTP). All of them followed a single-compartment absorption model with first order. The main pharmacokinetic parameters of GTP and the other three formulations are presented in Table 11. As shown in Table 11, the Cmax, AUC0-∞, and Fr of three formulations were significantly higher than that of GTP, demonstrating that GTP was metabolized rapidly, and its higher bioavailability could be achieved by the formation of GTP-PC and GTP-PC-SNEDDS. Among the three formations, the bioavailability of GTP-PC-SNEDDS was highest, with approximately 2.6-fold and 1.3-fold of Fr value, compared with GTP-PC (suspension) and GTP-PC (oil solution), respectively. The improved bioavailability by GTP-PC and GTP-PC-SNEDDS might be attributed to the following reasons: (1) phospholipids are an important component of cell membrane, having the effects of keeping the cell membrane fluidity. Moreover, phosphatidylcholine and its digestion product lysophosphatidylcholine could promote lymphatic transport efficiently [40–42]. Figure 7 and Table 11 illustrate the mean plasma concentration–time curve of GTP in rats after oral administration of GTP, GTP‑PC, and GTP‑PC‑SNEDDS (equivalent to 80 mg/kg of GTP). All of them followed a single‑compartment absorption model with first order. The main pharmacokinetic parameters of GTP and the other three formulations are presented in Table 11. As shown in Table 11, the Cmax, AUC0–∞, and Fr of three formula‑ tions were significantly higher than that of GTP, demonstrating that GTP was metabolized rapidly, and its higher bioavailability could be achieved by the formation of GTP‑PC and GTP‑PC‑SNEDDS. Among the three formations, the bioavailability of GTP‑PC‑SNEDDS was highest, with approximately 2.6‑fold and 1.3‑fold of Fr value, compared with GTP‑ PC (suspension) and GTP‑PC (oil solution), respectively. The improved bioavailability by GTP‑PC and GTP‑PC‑SNEDDS might be attributed to the following reasons: (1) phospho‑ lipids are an important component of cell membrane, having the effects of keeping the cell membrane fluidity. Moreover, phosphatidylcholine and its digestion product lysophos‑ phatidylcholine could promote lymphatic transport efficiently [40–42]. (2) Compared with that of GTP, the lipophilicity of GTP‑PC was effectively increased, and the dissolution rate of GTP in GTP‑PC was reduced. Therefore, improved bioavailability could be achieved by the use of delivery systems, which could enhance the rate and/or the extent of drug ab‑ sorbing into intestinal mucosa [43]. (3) The instability of GTP in GI tract might decrease the bioavailability and activity of the GTP; however, the GTP‑PC and GTP‑PC‑SNEDDS might protect it from metabolization by gastric secretions and gut bacteria [44–46] and improve the absorption of GTP. (4) By enhancing the solubility of bile to GTP, liver targeting may be facilitated, and phosphatidylcholine also acts as a hepatoprotective, hence giving the syn‑ ergistic effect. Additionally, compared with GTP‑PC, these fine droplets, with nano‑size, produced by GTP‑PC‑SNEDDS could further enhance the dispersion of drug dissolved in‑

side the oil phase into gastrointestinal fluid, resulting in the significant improvement of absorption in gastrointestinal (GI) tract. of drug dissolved inside the oil phase into gastrointestinal fluid, resulting in the significant improvement of absorption in gastrointestinal (GI) tract.

(2) Compared with that of GTP, the lipophilicity of GTP-PC was effectively increased, and the dissolution rate of GTP in GTP-PC was reduced. Therefore, improved bioavailability could be achieved by the use of delivery systems, which could enhance the rate and/or the extent of drug absorbing into intestinal mucosa [43]. (3) The instability of GTP in GI tract might decrease the bioavailability and activity of the GTP; however, the GTP-PC and GTP-PC-SNEDDS might protect it from metabolization by gastric secretions and gut bacteria [44–46] and improve the absorption of GTP. (4) By enhancing the solubility of bile to GTP, liver targeting may be facilitated, and phosphatidylcholine also acts as a hepatoprotective, hence giving the synergistic effect. Additionally, compared with GTP-PC, these fine droplets, with nano-size, produced by GTP-PC-SNEDDS could further enhance the dispersion

*Pharmaceuticals* **2023**, *16*, x FOR PEER REVIEW 13 of 20

**Figure 7.** Mean plasma concentration–time curve of GTP, GTP-PC (suspension), GTP-PC (oil) solution, and GTP-PC-SNEDDS after single po dose of 80 mg/kg GTP in SD rats, n = 6. **Figure 7.** Mean plasma concentration–time curve of GTP, GTP‑PC (suspension), GTP‑PC (oil) solu‑ tion, and GTP‑PC‑SNEDDS after single po dose of 80 mg/kg GTP in SD rats, n = 6.

AUC0-∞/μg·mL−1·h 6.273 ± 2.123 23.375 ± 10.665 \*\* 47.009 ± 20.532 \*\* 60.749 ± 33.759 \*\* Fr (%) - 372.65 749.45 968.49 \* *p* < 0.05 and \*\* *p* < 0.01 indicate a statistically significant difference, when compared with GTP \* *p* < 0.05 and \*\* *p* < 0.01 indicate a statistically significant difference, when compared with GTP concentration used Student's *t* test; Ka: rate constant of absorption; Ke: rate constant of elimination; T1/2Ka: absorption half‑life; T1/2Ke: elimination half‑life; Tmax: time to reach peak concentration; Cmax: the maximum concentration; AUC0–∞: the area under the plasma concentration–time curve; Fr: relative bioavailability.

concentration used Student's t test; Ka: rate constant of absorption; Ke: rate constant of elimination;

#### T1/2Ka: absorption half-life; T1/2Ke: elimination half-life; Tmax: time to reach peak concentration; Cmax: **3. Materials and Methods**

#### the maximum concentration; AUC0-∞: the area under the plasma concentration–time curve; Fr: rela-*3.1. Material and Animals*

tive bioavailability. **3. Materials and Methods**  *3.1. Material and Animals*  National Institute for Control of Pharmaceuticals supplied the reference substances of GTP with purity of 98%. GTP extract with a purity of 85% (*w*/*w*) was isolated in our laboratory and used for preparation of GTP-PC and GTP-PC-SNEDDS. Soybean National Institute for Control of Pharmaceuticals supplied the reference substances of GTP with purity of 98%. GTP extract with a purity of 85% (*w*/*w*) was isolated in our laboratory and used for preparation of GTP‑PC and GTP‑PC‑SNEDDS. Soybean phospho‑ lipids were obtained from Shanghai Tai‑Wei pharmaceutical Co. Ltd. (Shanghai, China), and the phosphatidylcholine content was about 98% (*w/w*). Egg yolk phospholipid and hydrogenated phospholipid were presented by Lipoid Company (Ludwigshafen, LS, Ger‑ many). Imwitor 742 and Migyol 812N were presented by SASOL Company (Johannesburg, JB, South Africa). Maisin 35‑1, Labrasol, Plurol Qleique CC 497, and Transcutol P were pre‑ sented by GATTEFOSSE SAS Company (SAINT‑PRIEST, SP, France). Cremophor EL was purchased from Shanghai Licheng Food Industry Co., Ltd (Shanghai, China). HPLC‑grade methanol was purchased from TEDIA company, Inc. (Fairfield, CA, USA). A Milli‑Q water purification system (Millipore, Bedford, MA, USA) was used to purify the water. All other chemicals were of analytical grade.

Male SD rats (180 ± 20 g) were supplied by Experimental Animal Center of Chinese Academy of Sciences. The rats were housed in an environmentally controlled room. Un‑ less otherwise indicated, standard laboratory food and water were given. The Animal Experimentation Ethics Committee of Fudan University recommended all animal experi‑ mentation procedures (2019‑03‑YJ‑WJX‑01).

#### *3.2. Chromatography*

The GTP in GTP‑PC was analyzed by LC‑20AB HPLC system with SPD‑20A UV de‑ tector (SHIMADZU, Kyoto, Japan) at 270 nm. The chromatography was performed on a Venusil MP C18 column 300A (150 mm × 4.6 mm, 5 µm) by using a mixture of methanol– water (30:70, *v/v*) as mobile phase at 1 mL/min and 30 ◦C. The GTP concentration in plasma samples were also detected following this method, except for the mobile phase, which was changed to a mixture of methanol–water (24:76, *v/v*).

The GTP content in GTP‑PC was determined according to the external standard method. The peak area of GTP had a good linear relationship, with a concentration ranging from 19.84 to 744 µg/mL. The standard curve equation between peak area of GTP (A) and its concentration (C, µg/mL) was A = 6808.5C + (r = 0.9999). While the GTP was quantitatively determined by internal standard method, theophylline was used as internal standard. The linear regression curve between GTP concentration (C, µg/mL) and peak radio between GTP and theophylline (A) was calculated. The r value was 0.9996, indicating this curve had a good linear relationship (A = 0.033 C + 0.004).

## *3.3. Preparation of GTP‑PC*

#### 3.3.1. Complexation Efficiency of GTP‑PC

The complexation efficiency of GTP‑PC was measured as previously reported [36]. Briefly, methanol and dichloromethane were used to dissolve two samples of GTP‑PC with approximately the same amount. After the dichloromethane solution was filtered, the GTP contents in both the methanol and dichloromethane solutions were determined by HPLC, as described in Section 3.2. The GTP in dichloromethane and methnol was regarded as complexed GTP and total GTP in GTP‑PC (complexed and uncomplexed), respectively, because methanol can easily dissolve complexed and uncomplexed GTP in GTP‑PC, while only complexed GTP in GTP‑PC is dissolvable in dichloromethane. Therefore, the com‑ plexation efficiency was calculated as follows:

Complexation efficiency % = Wc/Wt × 100%

where Wc is the GTP content dissolved in dichloromethane, and Wt is the GTP content in methanol.

### 3.3.2. Measurement of Dissociation Rate of GTP‑PC

GTP was easily dissolved in water, but GTP‑PC was practically insoluble. GTP dis‑ solved in water was considered to be from the dissociation of GTP‑PC. The dissolution studies could be carried out using the paddle method to assess the dissociation of GTP‑ PC [47]. At 37 ◦C, 200 mL of pH 6.8 phosphate buffer saline (PBS) was continually stirred at 50 rpm in dissolution flasks. The stirred medium was first containing GTP‑PC (0.2 g). At 10, 20, 30, 45, 60, and 120 min, 5 mL samples were withdrawn and filtrated with 0.45 µm cellulose nitrate membranes, and then 5 mL fresh mediums were added into flask. The GTP concentration in the resulting solution was determined by HPLC by mixing 0.4 mL filtrate with 4 mL PBS. The dissociation rate was calculated according to the following equation [48]:

$$\text{Log C} = \text{Log C}\_0 - \text{Kt/2.303}$$

where C0 is the initial concentration of GTP, K is the first order rate constant, and t is the time in hours.

#### 3.3.3. Investigations of GTP‑PC

GTP and phospholipid were placed in a round‑bottom flask and suspended in differ‑ ent solvents (trichloromethane, ethyl acetate, and tetrahydrofuran) with a certain molar ratio (GTP: phospholipids = 2:1, 1:1, 1:2, 1:3, and 1:4). Then, the mixture was stirred at 40 ◦C for different times (0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h). The dried residues were col‑ lected as GTP‑PC after the solvent was evaporated off under vacuum at 40 ◦C. By measur‑

ing complexation efficiency and dissociation, the optimal preparation process for GTP‑PC was determined.

## *3.4. Characterization of GTP‑PC*

#### 3.4.1. Differential Scanning Calorimetry (DSC)

In a nitrogen atmosphere, samples sealed in aluminum crimp cells were heated at 10 ◦C/min from 0 ◦C to 300 ◦C (PERKIN‑ELMER 7, Waltham, MA, USA). The peak transi‑ tion onset temperatures of four types of samples were compared, including phospholipid, GTP, the mixture of phospholipid and GTP, and the GTP‑PC.

#### 3.4.2. X‑ray Diffractometry (XRD)

Using a graphite monochromator with Cu/Ka radiation with a voltage window of 40 kV and current density of 60 mA with a scanning rate of 4 ◦C/min ranging from 5 ◦C to 45 ◦C, the X‑ray diffraction (D/MAXX Rigaku, Tokyo, Japan) was performed.

#### 3.4.3. Fourier Transform Infrared Spectra (FT‑IR)

Samples were compressed into a KBr pellet, and their FT‑IR spectra were recorded by FT‑IR spectrometer (Avatar TM 360E.S.P.TM, Avatar).

#### 3.4.4. Thin‑Layer Chromatography (TLC)

Sample was prepared by dissolving standard GTP, GTP‑PC, the physical mixture, and GTP‑PC in methanol. The experiment was carried out according to a TLC test of China pharmacopoeia. A total of 5 µL solution was spotted onto a silica gel GF254 plate with ethyl acetate‑menthol‑water (20:2:1, *v/v/v*) as developing solvent. The resulting plate had a picture taken under ultraviolet lamp (254 nm).

#### 3.4.5. Ultraviolet (UV) Spectra

Sample methanol solutions were scanned by a UV spectrometer over the wavenumber range of 200–400 nm.

#### 3.4.6. Dissolution Studies in Phosphate Buffer Saline (pH 6.8)

The dissolution studies were carried out according to paddle method demonstrated in Section 3.3.2. Additionally, samples were withdrawn at 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h.

#### *3.5. Preparation of GTP‑PC‑SNEDDS*

## 3.5.1. Solubility Studies

Suitable excipients for preparation of GTP‑PC were screened by solubility studies. Briefly, excess GTP‑PC was added into about 1 g of various oils, surfactants, or co‑surfactants, respec‑ tively. Then, the mixture was shaken at 37 ◦C for 48 h; after centrifuging at rpm for 10 min, the content of GTP was assayed by HPLC, and the GTP solubility of GTP‑PC in each oily medium was calculated.

#### 3.5.2. Construction of Pseudo‑Ternary Phase Diagrams

Pseudo‑ternary phase diagrams can be used for preliminary screening of self‑micro‑ emulsifying systems. The points in the triangle represent the different composition ratios of ternary systems, such as oil phase, surfactant, and cosurfactant. In this work, about 1 g of blank formulation (only including different ratios of oil phase, surfactant, and cosurfactant) was first weighed accurately, and then about 10% (*w/w*) of GTP‑PC was added. The mixture was stirred at 37 ◦C for 24 h and followed to keep for 48 h to observe whether all the drugs were dissolved and formed a uniform and transparent solution. If so, part of formulation (equivalent to 4 mg of GTP) was diluted 250 times with 0.1 M HCl and then stirred at 37 ◦C and 50 rpm. If a clear and transparent solution could be formed, it is believed that

the formulation, including this ratio of oil phase, surfactant, and cosurfactant, could been used to form microemulsion and draw pseudo‑ternary phase diagrams, in turn.

#### 3.5.3. Investigations of GTP‑PC‑SNEDDS

In order to optimize the preparation process of GTP‑PC‑SNEDDS, the effects of weight ratios of GTP‑PC to blank SNEDDS, preparation technology, dilution media, and dilution multi were further investigated.

#### 3.5.4. Characterization of GTP‑PC‑SNEDDS

#### Morphological Characterization

Transmission electron microscope (TEM) (PHILIPS CM‑120, Eindhoven, EIN, Nether‑ lands) was used to observe the morphology of SMEDDS. SMEDDS was diluted with dis‑ tilled water 1:25 and gently shaken to mix. Afterwards, a drop of the diluted sample was placed on copper grids, and the excess was rubbed off with filter paper. The grids were then stained with 1% phosphor‑tungstic acid solution for 30 s.

## Droplet Size and Zeta Potential

Droplet size distribution and zeta potential of GTP‑PC‑SNEDDS were determined using NICOMP 380 ZLS Zeta Potential/Particle Sizer (PSS‑380, NICOMP, Santa Barbara, CA, USA).

#### Solubility

Excess GTP‑PC was added into about 0.5 g of blank formulation, which was shaken at 37 ◦C for 48 h and then centrifuged at 15,000 rpm for 10 min. The solubility of GTP in blank formulation was evaluated by the content measurement of GTP in supernatant by HPLC.

#### Self‑Microemulsifying Time

The self‑microemulsifying time of GTP‑PC‑SNEDDS was analyzed according to Chi‑ nese pharmacopoeia (2015 edition). Certain amount of formulation (equivalent to 4 mg of GTP) was diluted 250 times with 0.1 M HCl, and then stirred at 37 ◦C and 50 rpm. Self‑ microemulsifying time was recorded, since the droplets contacted with the liquid level to a clear and transparent solution was formed. According to the literature, the time of self‑microemulsification should not exceed 2 min.

#### *3.6. Bioavailability Experiments in Rats*

#### 3.6.1. Plasma Sample Preparation and Validity

A total of 250 µL internal standard solution (4.031 µg/mL theophylline in methanol solution) was added to 100 µL plasma and agitated for 30 s. Then, the solution was cen‑ trifuged (15 min). Aliquots (20 µL) of the supernatant were injected for HPLC analysis.

Rat plasma blanks were supplemented with different amounts of GTP for valida‑ tion of the method. The resulting concentrations of GTP were 0.13, 0.53, 5.3, 21.2, and 42.4 µg/mL. For testing the method's precision, accuracy, and detection limit, the calibra‑ tions were subjected to the above analytical procedure.

#### 3.6.2. Pharmacokinetic Study

Twelve four male rats (150–200 g) were divided randomly into four groups, and each group had six animals. They were fasted for 24 h, but allowed to take water freely. They were orally administered GTP solution in water (equivalent to 80 mg/kg of GTP), GTP‑PC suspension in oil (equivalent to 80 mg/kg of GTP), GTP‑PC suspension in water (equivalent to 80 mg/kg of GTP), and GTP‑PC–SNEDDS (equivalent to 80 mg/kg of GTP), respectively. About 0.4 mL blood samples were collected from the tail vein into tubes containing heparin at 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h. Plasma was separated by centrifugation (5000 rpm, 10 min) and stored at −20 ◦C until analysis.

Peak concentration (Cmax) and peak times (tmax) were derived directly from the exper‑ iment points, and AUC0–∞ was calculated by trapezoidal method. A computer program called 3p87 was used to compute the rest of the pharmacokinetic parameters.

#### **4. Conclusions**

In this study, we firstly prepared GTP‑PC by studying the factors affecting the forma‑ tion of GTP‑PC with complexation efficiency and a dissociation experiment. DSC, XRD, FT‑IR, TLC, and UV measurements confirmed that GTP is bonded to phospholipids only through hydrogen bonds and/or van der Waals forces. Then, GTP‑PC was loaded into SNEDDS by investigating the effects of the weight ratios of GTP‑PC to blank SNEDDS, preparation technology, dilution media, and dilution multi based on the screening results of the oils, surfactants, and cosurfactants. As a result, the dissolution of the GTP in GTP‑ PC was delayed, compared with GTP material. In rats, GTP‑PC and GTP‑SNEDDS could significantly enhance GTP bioavailability, compared with GTP. Our study may serve as a basis for the clinical applications of GTP‑PC and GTP‑PC‑SNEDDS.

There are also the same limits for this work, for example, the morphology and sizes of aggregates of GTP‑PC were not characterized. The characterization of SNEDDS with‑ out GTP‑PC, including size, Z‑potential, and PDI, was not clarified. Furthermore, several studies need to be further executed, such as regarding the absorbed mechanism of GTP‑PC and GTP‑PC‑SNEDDS, an experiment with the delivery of GTP to show that the GTP‑PC and GTP‑PC‑SNEDDS are really able to work in vivo.

**Author Contributions:** Conceptualization, J.W.; data curation, W.S. and Y.T.; writing—original draft preparation, Y.T. and W.S.; writing—review and editing, Y.T., J.W. and Q.Z.; methodology, W.S. and Q.Z., formal analysis, Y.T., J.W. and Q.Z.; supervision, J.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China (No. 81573616 and No. 81773911) and the Development Project of Shanghai Peak Disciplines‑Integrative Medicine (20180101).

**Institutional Review Board Statement:** The animal study protocol was approved by The Animal Experimentation Ethics Committee of Fudan University (2019‑03‑YJ‑WJX‑01).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **Quality by Design of Pranoprofen Loaded Nanostructured Lipid Carriers and Their Ex Vivo Evaluation in Different Mucosae and Ocular Tissues**

**María Rincón 1 , Lupe Carolina Espinoza 2 , Marcelle Silva-Abreu 3,4,\* , Lilian Sosa 5 , Jessica Pesantez-Narvaez 6 , Guadalupe Abrego 7 , Ana Cristina Calpena 3,4 and Mireia Mallandrich 3,4**

**Abstract:** Transmucosal delivery is commonly used to prevent or treat local diseases. Pranoprofen is an anti-inflammatory drug prescribed in postoperative cataract surgery, intraocular lens implantation, chorioretinopathy, uveitis, age-related macular degeneration or cystoid macular edema. Pranoprofen can also be used for acute and chronic management of osteoarthritis and rheumatoid arthritis. Quality by Design (QbD) provides a systematic approach to drug development and maps the influence of the formulation components. The aim of this work was to develop and optimize a nanostructured lipid carrier by means of the QbD and factorial design suitable for the topical management of inflammatory processes on mucosal tissues. To this end, the nanoparticles loading pranoprofen were prepared by a high-pressure homogenization technique with Tween 80 as stabilizer and Lanette® 18 as the solid lipid. From, the factorial design results, the PF-NLCs-N6 formulation showed the most suitable characteristics, which was selected for further studies. The permeability capacity of pranoprofen loaded in the lipid-based nanoparticles was evaluated by ex vivo transmucosal permeation tests, including buccal, sublingual, nasal, vaginal, corneal and scleral mucosae. The results revealed high permeation and retention of pranoprofen in all the tissues tested. According to the predicted plasma concentration at the steady-state, no systemic effects would be expected, any neither were any signs of ocular irritancy observed from the optimized formulation when tested by the HET-CAM technique. Hence, the optimized formulation (PF-NLCs-N6) may offer a safe and attractive nanotechnological tool in topical treatment of local inflammation on mucosal diseases.

**Keywords:** design of experiment; porcine mucous membrane; ophthalmic tissues; permeation; nanostructured lipid carriers

## **1. Introduction**

Pranoprofen (PF) or 2-(5H-chromeno[2,3-b]pyridin-7-yl)propanoic acid is a non-steroidal anti-inflammatory drug (NSAID), which can be used as an effective and safe anti-inflammatory treatment option in ocular therapy [1–3]. It is usually indicated in chronic but non-bacterial inflammatory processes affecting the anterior segment of the eye and in the treatment of postoperative pain of cataract or strabismus surgery [4]. PF may also be used to control the

**Citation:** Rincón, M.; Espinoza, L.C.; Silva-Abreu, M.; Sosa, L.; Pesantez-Narvaez, J.; Abrego, G.; Calpena, A.C.; Mallandrich, M. Quality by Design of Pranoprofen Loaded Nanostructured Lipid Carriers and Their Ex Vivo Evaluation in Different Mucosae and Ocular Tissues. *Pharmaceuticals* **2022**, *15*, 1185. https://doi.org/10.3390/ ph15101185

Academic Editors: Ana Catarina Silva, João Nuno Moreira and José Manuel Sousa Lobo

Received: 20 August 2022 Accepted: 19 September 2022 Published: 24 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

inflammation and pain in the posterior segment, such as in the case of posterior chamber intraocular lens implantation, acute central serous chorioretinopathy, uveitis age-related macular degeneration or cystoid macular edema [5]. PF can also be used for acute and chronic management of osteoarthritis and rheumatoid arthritis [6,7]. PF inhibit COX-1 and COX-2 enzymes, and thus it blocks arachidonic acid being converted to eicosanoids and reduces prostaglandins synthesis. Although this drug has shown high anti-inflammatory and analgesic efficiency, and its side effects on the gastrointestinal tract are minimal, the pharmaceutical use of PF is limited due to its inadequate biopharmaceutical profile. PF has a short plasmatic half-life, low water solubility and is unstable in aqueous solution, particularly when exposed to light [6,8].

The application of NSAIDs through the mucosal tissues offers a set of advantages, e.g., it avoids the first pass metabolism, averts the risk of gastrointestinal disturbance, targeting only the areas of disease. The use of ophthalmic NSAIDs is the usual treatment to prevent and treat common ocular disorders inflammatory [9]. However, this conventional dosage form cannot be considered optimal in the treatment of ocular diseases due to the fact that most of the drugs is removed from the surface of the eye, following the instillation, by various mechanisms (tear dilution and tear turn over). Moreover, the relatively impermeable corneal barrier restricts the entry of foreign substances. As a result, less than 5% of the administered drug penetrates the cornea and reaches intraocular tissues [10].

Intranasal drug application is currently most used for the treatment of local inflammations such as common rhinitis, allergic rhinitis or for easing nasal congestion. The main advantage of nasal administration is the rapid drug absorption through this membrane due to the prevailing physical conditions of the nose, such as good blood circulation of the nasal mucosa, resulting in a quick local effect, and the rapid onset of action. Due to the fast local absorption, an unintentional systemic distribution of the active ingredient is prevented, eliminating the first pass metabolism and its side effects are avoided [11,12]. Other advantages are the high permeability of some drugs in the nasal epithelium and better compliance with the recommended treatment, improved comfort for the patient, and a sustained and prolonged effect compared to other delivery systems such as oral drug delivery systems [11].

The administration of drugs through the oral mucosa, particularly the buccal and sublingual mucosa, has been attracting great interest. The main advantage of using the buccal route is the direct access of drugs to the systemic circulation by the internal jugular vein, eliminating the hepatic first-pass metabolism and mitigating possible side effects [13,14]. Nevertheless, buccal drug delivery suffers from some disadvantages such as low permeability and a smaller absorptive surface area, in contrast to the high absorptive surface area of the small intestine [15,16].

Vaginal drug delivery treats or prevents diseases and allows a controlled or enhanced drug absorption with the advantage of a systemic circulation delivery of drugs avoiding the hepatic first-pass effect and gastrointestinal interferences while assuring, in accordance with the selected formulation, stable and/or high local concentrations and reduced, enhanced and/or controlled systemic absorption [17].

In an attempt to overcome, the biopharmaceutical profile and improve the permeability of drugs through the tissues, nanostructured Lipid Carriers (NLCs) are one of the colloidal systems that have been most widely studied due to their characteristics such as small particle size, biocompatibility, prolonged release, drug protection, and incorporation of both hydrophilic and lipophilic drugs [18]. In ocular delivery, conventional ophthalmic formulations are effortlessly drained through the nasolacrimal pathway, while nanoparticles are cleared more slowly and therefore can release the drug upon interaction with the cornea over a longer time period [10].

Taking into account all these considerations, the main purpose of the work in question here was to target PF-loaded NLCs (PF-NLCs) in mucosal tissues (buccal, sublingual nasal, vaginal, cornea and sclera) with controlled release effect, enhancing the contact of the PF

and improving its mucosal tissues retention, thus to increase the anti-inflammatory and analgesic. For this purpose, a factorial design and detailed statistical studies shed light on the most adequate lipid and led to the optimized formulation with physicochemical properties to permeate the mucosae in a competent way. Taking into consideration that factorial designs provide the maximum information from the least number of experiments [19], a 23 +star central composite factorial design was applied to study the main effects and interactions of three factors: PF concentration (cPF), the concentration of the solid lipid in relation to the liquid lipid (cSL/cLL) and the concentration of Tween® 80 (cTW), on dependent variables: average particle size (Z-Ave), zeta potential (ZP) and polydispersity index (PI), and encapsulation efficacy (EE). From a total of 16 formulations obtained by factorial design, an optimized formulation was selected to carry out additional studies. These formulations were characterized for their morphology rheological and extensibility. The physical stability of the optimized formulation was also evaluated, as well as the ex vivo permeation profile through porcine mucosal tissues (buccal, sublingual nasal, vaginal, cornea and sclera) and the in vitro tolerance study were assessed.

Design of Experiments is extensively used for the implementation of Quality by Design (QbD) in research and industrial settings. In QbD, formula (product) and process understanding are the key enablers of assuring quality in the final product [20]. QbD is not only a guide, but it also provides a systematic approach to drug development, focusing on quality through the application of analytical and risk management methodologies for the design, development and manufacture of new drugs. Likewise, it maps the influence and interaction of various formulations and operating parameters on process performance [21].

#### **2. Results**

#### *2.1. Lipid Screening*

The PF was dissolved in different solid lipids (SL) and liquid lipids (LL) in order to determine the components of the lipid phase before producing the NLCs containing PF. Therefore, components of the lipid phase were determined by evaluating the solubility of PF in 14 different lipids as shown in Table 1. Selected liquid lipids included LAS (PEG-8 Caprylic/Capric Glycerides), Castor oil, rose mosqueta oil, plantacare oil, Jojoba oil and Miglyol® 812, and isopropyl myristate. The solubility of PF was also evaluated in solid lipids such as stearic acid, Precifac® ATO, Compritol® ATO 888, Precirol® ATO 5, Lanette® 18, Geleol® and Gelucire®. Following on from this, PF (0.1 to 1% of PF was dissolved in different SL and LL or physical mixtures of them (PF, 1.5 to 3%, with regard to total lipid). PF in either physical mixtures or lipids was heated at about 10 ◦C above the melting point of the SL. The samples obtained were then observed to verify the presence/absence of insoluble drug crystals. The next step was that the mixtures were cooled down to room temperature (RT) for solidification, and this was followed by the analysis of the lipids and PF by Differential Scanning Calorimetry (DSC) to evaluate non-solubilized PF. The analysis was carried out under the conditions described below in the assessment of the lipid matrix crystallinity. Another condition that we imposed is that any resulting solid lipids that were "soft" or "semi-solid" at room temperature were not considered for the design of the NPs (Tables 1 and 2).

+ pranoprofen is soluble; − pronoprofen is insoluble.

+ pranoprofen is soluble; − pronoprofen is insoluble.

Based on the results from the lipid screening, the selected lipids for the preparation of the PF-NLC formulations were Castor oil, LAS and Lanette® 18. In previous studies, we formulated the NLCs with Castor oil, LAS and Precirol® ATO 5, which were chosen and studied [8,18].

#### *2.2. Design of Experiments*

The PF-NLC formulations were optimized by means of a 23 + star central composite rotatable design (Tables 3 and 4). The main effects and interactions of the independent variables, such as concentration of solid lipid with regard to liquid lipid (cSL/LL), concentration of PF, and concentration of Tween 80® (cTW) were investigated on the mean particle size (Z-Ave), polydispersity index (PI), zeta potential (ZP) and encapsulation efficiency (%EE). A total of 16 experiments are summarized in Table 4.

**Table 3.** Factors and their corresponding coded levels of experimental design.

cSL/LL: concentration of solid lipid concerning liquid lipid; cPF: concentration of PF, cTW: concentration of TW 80.

**Table 4.** Independent and dependent variables of the 23 start central composite rotatable factorial design factors.

cSL/LL: concentration of solid lipid concerning liquid lipid; cPF: concentration of PF; cTW: concentration of TW 80; Z-Ave: mean particle size; PI: poly dispersity index; ZP: zeta potential and EE: encapsulation efficiency.