*3.1. Characteristics of the Liposomal System after Hydration of the Powder Formulations*

Double chain phospholipids with a phosphatidylcholine head group have a packing parameter (ratio between the cross-sectional area of the hydrophobic region and the hydrophilic head-group) close to 1. Therefore, these lipids are likely to form bilayer structures as compared to micellar assemblies [54–56].

*3.2. In Vitro Dissolution* 

3).

media, thus implying sink condition.

The DLS measurement, cryo-EM imaging, and fluorescent microscopy showed the size, morphology, and the formation dynamics of the liposomes after the hydration of the formulations in the acidic condition of SGF and the neutral condition of phosphate buffer (Figure 1, Figure S1).

The hydrodynamic diameter of the liposomes in SGF and phosphate buffer showed a population at 669 ± 169 nm and 490 ± 112 nm average size, respectively. However, in SGF, the liposomes additionally had a second population with an average size of 121 ± 36 nm (Figure 1, lower left panels). No difference was observed between the liposome sizes of different formulations (data not shown). Cumulant analysis of the samples obtained a z-average of 261 ± 83.07 (polydispersity index 0.56 ± 0.13) and 420 ± 86.86 nm (polydispersity index 0.60 ± 010) for the liposomes in SGF and phosphate buffer, respectively. *Pharmaceutics* **2020**, *12*, x doi: 7 of 18

The cryo-EM imaging of the liposomes corroborated the DLS results by showing many uni-lamellar and a small number of multi-lamellar liposomes after dissolving in SGF. On the other hand, the liposomes in the phosphate buffer had larger sizes and mostly multi-lamellar and multi-vesicular morphologies. the liposomes in the phosphate buffer had larger sizes and mostly multi-lamellar and multi-vesicular morphologies.

**Figure 1.** The morphology (i.e., uni-lamellar versus multi-lamellar) and size distribution of the liposomes after dispersing the powders in simulated gastric fluid without enzyme (SGF) (a and phosphate buffer (b). For each sample, the top panels show the cryo-EM images of the resulting liposomes. The bottom left panels show the average hydrodynamic diameter of the liposomes in each **Figure 1.** The morphology (i.e., uni-lamellar versus multi-lamellar) and size distribution of the liposomes after dispersing the powders in simulated gastric fluid without enzyme (SGF) (**a**) and phosphate buffer (**b**). For each sample, the top panels show the cryo-EM images of the resulting liposomes. The bottom left panels show the average hydrodynamic diameter of the liposomes in each medium, and the bottom right panels are the fluorescent microscopy images, showing the liposomes encapsulating a hydrophobic dye. In phosphate buffer, the liposomes can be seen emerging from the functionalized calcium carbonate (FCC) particles (white arrows).

encapsulating a hydrophobic dye. In phosphate buffer, the liposomes can be seen emerging from the functionalized calcium carbonate (FCC) particles (white arrows). Fluorescent imaging showed incorporation of the hydrophobic fluorescent dye (DiI) in the lipid bilayer of the liposomes. For samples prepared in SGF, almost no residuals of FCC particles could be observed. The supplementary optical microscopy video (Video S1) shows the dissolution process of FCC particles in SGF and formation of the liposomes. Formation of CO2 bubbles resulting from the Fluorescent imaging showed incorporation of the hydrophobic fluorescent dye (DiI) in the lipid bilayer of the liposomes. For samples prepared in SGF, almost no residuals of FCC particles could be observed. The supplementary optical microscopy video (Video S1) shows the dissolution process of FCC particles in SGF and formation of the liposomes. Formation of CO<sup>2</sup> bubbles resulting from the dissolution of calcium carbonate is clearly seen on the video. On the other hand, dispersion of the samples in phosphate buffer showed that FCC particles remain intact, while liposomes of various shapes and sizes slowly emerge out of the porous structure of the particles.

medium, and the bottom right panels are the fluorescent microscopy images, showing the liposomes

dissolution of calcium carbonate is clearly seen on the video. On the other hand, dispersion of the samples in phosphate buffer showed that FCC particles remain intact, while liposomes of various shapes and sizes slowly emerge out of the porous structure of the particles. The encapsulation efficiency of nifedipine (Figure 2b) was significantly greater in the liposomes created in SGF, as compared to phosphate buffer. The encapsulation efficiency was generally higher The encapsulation efficiency of nifedipine (Figure 2b) was significantly greater in the liposomes created in SGF, as compared to phosphate buffer. The encapsulation efficiency was generally higher in the formulations with lower phospholipid contents (5% and 10%) at a maximum average of 25% for D5N20. However, the encapsulation efficiency was not significantly different among the formulations in the same dissolution medium (*p*-value > 0.05).

Nifedipine equilibrium solubility in SGF, phosphate buffer, and FaSSIF were 17.94 ± 2.21 mg/L, 16.06 ± 1.71 mg/L, and 48.86 ± 25.26 mg/L, respectively. Therefore, the 5 mg/L drug concentration for in vitro dissolution tests was significantly below the saturation solubility of nifedipine in all three

For all media, the dissolution rate of nifedipine was faster for the formulations with FCC than for the reference formulation without FCC (Nif-DMPC), as well as for the physical mixture (Figure

in the formulations with lower phospholipid contents (5% and 10%) at a maximum average of 25%

The dissolution results in SGF showed that the rate of dissolution of nifedipine was inversely proportional to the phospholipid content. The changing ratio of drug to phospholipid did not have any influence on the rate of dissolution (Table S1). Therefore, all further analyses were carried out on the formulations with the highest drug load (20%) and with varying phospholipid content (i.e.,

**Figure 2.** Comparison of the dissolution rate and liposomal encapsulation efficiency of nifedipine for different formulations. (**a**) The dissolution rate of formulations obtained from fitting the dissolution **Figure 2.** Comparison of the dissolution rate and liposomal encapsulation efficiency of nifedipine for different formulations. (**a**) The dissolution rate of formulations obtained from fitting the dissolution data to Equation (5), and (**b**) encapsulation efficiency of the liposomes after the hydration of powders in different media. Increasing the phospholipid content inversely affected both the dissolution rate and encapsulation efficiency. Single asterisk indicates a *p*-value < 0.05 and the double asterisk indicates a *p*-value < 0.005.

#### data to Equation (5), and (**b**) encapsulation efficiency of the liposomes after the hydration of powders in different media. Increasing the phospholipid content inversely affected both the dissolution rate *3.2. In Vitro Dissolution*

and encapsulation efficiency. Single asterisk indicates a *p*-value < 0.05 and the double asterisk indicates a *p*-value < 0.005. Nifedipine equilibrium solubility in SGF, phosphate buffer, and FaSSIF were 17.94 ± 2.21 mg/L, 16.06 ± 1.71 mg/L, and 48.86 ± 25.26 mg/L, respectively. Therefore, the 5 mg/L drug concentration for in vitro dissolution tests was significantly below the saturation solubility of nifedipine in all three media, thus implying sink condition.

The 5% and 10% phospholipid formulations showed significantly faster drug release in SGF, compared to the formulations with 15% and 20% phospholipid content, and Nif-FCC (Figures 2a and For all media, the dissolution rate of nifedipine was faster for the formulations with FCC than for the reference formulation without FCC (Nif-DMPC), as well as for the physical mixture (Figure 3).

3a). All formulations with DMPC and FCC reached at least 85% drug dissolution. The dissolution results in SGF showed that the rate of dissolution of nifedipine was inversely proportional to the phospholipid content. The changing ratio of drug to phospholipid did not have any influence on the rate of dissolution (Table S1). Therefore, all further analyses were carried out on the formulations with the highest drug load (20%) and with varying phospholipid content (i.e., D5N20, D10N20, D15N20, and D20N20).

The 5% and 10% phospholipid formulations showed significantly faster drug release in SGF, compared to the formulations with 15% and 20% phospholipid content, and Nif-FCC (Figures 2a and 3a). All formulations with DMPC and FCC reached at least 85% drug dissolution.

None of the formulations reached 85% dissolution (Figure 3b) in FaSSIF. However, the rate of dissolution was significantly faster for the formulations with 5% and 10% phospholipid content (Figure 2a).

Similar results were obtained in the phosphate buffer, where none of the formulations reached 85% dissolution (Figure 3c). The rate of dissolution in the phosphate buffer was not significantly different within the formulations with FCC (*p*-value > 0.05).

The dissolution efficiency (DE) of formulations, as a measure of the dissolution behavior of the formulations, are shown in Table 2.

The results show a reduction in the DE of Nif-DMPC in all media. In SGF, a trend of reduction in DE was seen with increasing phospholipid contents.

**Figure 3.** Selected FCC formulations (see Table 1 1 ) tested for dissolution in three different dissolution media: (**a**) SGF without enzyme, (**b**) fasted state-simulated intestinal fluid (FaSSIF), and (**c**) phosphate buffer, pH 6.8. Solid symbols represent formulations containing an increasing amount of phospholipid. Open symbols represent control formulations. Values are mean ± SD of *n* = 2 or 3 tests.

formulations, are shown in Table 2.


**Table 2.** Dissolution efficiency of formulations calculated based on Equation (2). D20N20 77.45 ± 1.37 74.95 ± 6.76 80.34 ± 0.005

**Table 2.** Dissolution efficiency of formulations calculated based on Equation (2). **Formulation Code SGF Phosphate Buffer FaSSIF**  D5N20 91.53 ± 1.33 79.66 ± 4.33 86.40 ± 1.33 D10N20 89.74 ± 2.96 79.35 ± 1.31 84.88 ± 1.02 D15N20 84.23 ± 1.06 76.26 ± 2.19 78.09 ± 1.10

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Similar results were obtained in the phosphate buffer, where none of the formulations reached 85% dissolution (Figure 3c). The rate of dissolution in the phosphate buffer was not significantly

The dissolution efficiency (DE) of formulations, as a measure of the dissolution behavior of the

The results show a reduction in the DE of Nif-DMPC in all media. In SGF, a trend of reduction

#### *3.3. Characterization of the Dry Powder Formulations* (Figure 4a).

different within the formulations with FCC (*p*-value > 0.05).

in DE was seen with increasing phospholipid contents.

As an assessment of the loading efficiency, we visually investigated the appearance of the particles using SEM. The obtained images showed individual particles with neither agglomerates nor external drug crystallization (Figure 4b). There was no observable blocking or covering of the particle surfaces (Figure 4c). The images of the loaded samples were similar to the reference FCC material (Figure 4a). FIB-SEM images from the cross-section of the particles (Figure 5a) showed intra-particle larger pores evenly distributed throughout the entire FCC compact [40], while the drug-loaded formulations had smaller pores. The formulations Nif-FCC (Figure 5b) and D5N20 (Figure 5c) were not visually different, neither in pore sizes nor in pore shapes. On the contrary, the filled porous structure was seen for the formulation D20N20 (Figure 5d).

surfaces (Figure 4c). The images of the loaded samples were similar to the reference FCC material

**Figure 4.** SEM images used for visual analysis of drug-loaded FCC. (**a**) The surface lamella of the unloaded FCC particles can be seen. (**b**) Representative overview of the powder formulation with highest loaded material (D20N20), showing the absence of agglomerates and external crystals. (**c**) Formulation D20N20 showing preserved lamellar structure of FCC after loading. **Figure 4.** SEM images used for visual analysis of drug-loaded FCC. (**a**) The surface lamella of the unloaded FCC particles can be seen. (**b**) Representative overview of the powder formulation with highest loaded material (D20N20), showing the absence of agglomerates and external crystals. (**c**) Formulation D20N20 showing preserved lamellar structure of FCC after loading.

A loading efficiency of above 90% for the drug in all of the powder formulations was measured. On the basis of the results of BET surface area measurements, the specific surface area of the formulations decreased due to an increase in the phospholipid loading (Figure 6). FIB-SEM images from the cross-section of the particles (Figure 5a) showed intra-particle larger pores evenly distributed throughout the entire FCC compact [40], while the drug-loaded formulations had smaller pores. The formulations Nif-FCC (Figure 5b) and D5N20 (Figure 5c) were not visually different, neither in pore sizes nor in pore shapes. On the contrary, the filled porous structure was seen for the formulation D20N20 (Figure 5d).

pores.

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**Figure 5.** The focused ion beam scanning electron microscopy (FIB-SEM) images showed crosssections of the formulations after consolidating the powder. (**a**) Pure FCC, (**b**) Nif-FCC without phospholipid, (**c**) D5N20, and (**d**) D20N20. The red circle shows an example of a region with blocked **Figure 5.** The focused ion beam scanning electron microscopy (FIB-SEM) images showed cross-sections of the formulations after consolidating the powder. (**a**) Pure FCC, (**b**) Nif-FCC without phospholipid, (**c**) D5N20, and (**d**) D20N20. The red circle shows an example of a region with blocked pores.

The water sorption measurements showed an inverse correlation between the rate of water uptake and phospholipid content (Figure 6). A loading efficiency of above 90% for the drug in all of the powder formulations was measured. On the basis of the results of BET surface area measurements, the specific surface area of the formulations decreased due to an increase in the phospholipid loading (Figure 6). *Pharmaceutics* **2020**, *12*, x doi: 12 of 18

Figure 6 shows the correlation of the power law (represented as a semi-logarithmic plot) of the

**Figure 6.** The semi-logarithmic plot (base 7.66) of the correlation between the wetting constants versus **Figure 6.** The semi-logarithmic plot (base 7.66) of the correlation between the wetting constants versus specific surface area of the powder samples.

specific surface area of the powder samples. X-Ray powder diffraction was used to exclude an effect of complete drug amorphization. The The water sorption measurements showed an inverse correlation between the rate of water uptake and phospholipid content (Figure 6).

X-ray diffraction results of samples are presented in Figure 7. The control formulations in the absence of phospholipid or FCC both showed strong characteristic peaks of nifedipine at 8.11°, 11.76°, and

**Figure 7.** XRPD results of the formulations and physical mixture. The model drug was present in partial crystalline form in all formulations. The reference peaks of nifedipine (N) (8.11°, 11.76°, and 16.16°) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (D) (6.65° and 20.98°) are marked with dashed lines. A reference peak for FCC at 23.01° is also marked. The sample DMPC-Nif was

physically mixed with FCC to keep the ratio of the components constant for all samples.

16.16°. The intensity of these peaks was lower in the formulations with DMPC and FCC.

Figure 6 shows the correlation of the power law (represented as a semi-logarithmic plot) of the material wetting constant (obtained from Equation (4)) with the specific surface area of the formulations. For this correlation, the experimental data were fitted to Equation (5):

*Pharmaceutics* **2020**, *12*, x doi: 12 of 18

$$S = a\mathcal{C}^{\mathbb{I}} + b \tag{5}$$

where *S* is the specific surface area (*m*<sup>2</sup> ); *C* is the wetting constant obtained from Equation (4); *a* is the scaling parameter, intercept *b* → 0; and exponent *n* = 7.66 are the parameters obtained after fitting the experimental data to Equation (5). The two sets of results showed a strong positive power law correlation (adjusted *R* <sup>2</sup> = 0.99992).

X-Ray powder diffraction was used to exclude an effect of complete drug amorphization. The X-ray diffraction results of samples are presented in Figure 7. The control formulations in the absence of phospholipid or FCC both showed strong characteristic peaks of nifedipine at 8.11◦ , 11.76◦ , and 16.16◦ . The intensity of these peaks was lower in the formulations with DMPC and FCC. **Figure 6.** The semi-logarithmic plot (base 7.66) of the correlation between the wetting constants versus

The results of thermal analysis (Figure S2) showed the nifedipine melting peak at 172 ◦C. However, the samples with 5% phospholipid (D5N20) and without phospholipid (Nif-FCC) showed a reduced melting enthalpy for nifedipine. On the basis of calculations according to Equation (3), we detected crystallinity ratios of 60.76 ± 0.69% and 57 ± 9.91% for the D5N20 and Nif-FCC samples, respectively. The nifedipine melting peak was not present in the samples prepared with higher content of phospholipid. specific surface area of the powder samples. X-Ray powder diffraction was used to exclude an effect of complete drug amorphization. The X-ray diffraction results of samples are presented in Figure 7. The control formulations in the absence of phospholipid or FCC both showed strong characteristic peaks of nifedipine at 8.11°, 11.76°, and 16.16°. The intensity of these peaks was lower in the formulations with DMPC and FCC.

**Figure 7.** XRPD results of the formulations and physical mixture. The model drug was present in partial crystalline form in all formulations. The reference peaks of nifedipine (N) (8.11°, 11.76°, and 16.16°) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (D) (6.65° and 20.98°) are marked with dashed lines. A reference peak for FCC at 23.01° is also marked. The sample DMPC-Nif was **Figure 7.** XRPD results of the formulations and physical mixture. The model drug was present in partial crystalline form in all formulations. The reference peaks of nifedipine (N) (8.11◦ , 11.76◦ , and 16.16◦ ) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (D) (6.65◦ and 20.98◦ ) are marked with dashed lines. A reference peak for FCC at 23.01◦ is also marked. The sample DMPC-Nif was physically mixed with FCC to keep the ratio of the components constant for all samples.

physically mixed with FCC to keep the ratio of the components constant for all samples.
