1-Laurin-3-Palmitin as a Novel Matrix of Solid Lipid Particles: Higher Loading Capacity of Thymol and Better Stability of Dispersions Than Those of Glyceryl Monostearate and Glyceryl Tripalmitate

To develop solid lipid nanoparticles (SLNs) with a new lipid matrix for delivery of hydrophobic bioactive molecules, high purity 1-laurin-3-palmitin (1,3-LP) was synthesized and the prepared 1,3-LP SLNs were compared with those of two common SLN matrices in glyceryl monostearate (GMS) and glyceryl tripalmitate (PPP). Conditions of preparing SLNs were first optimized by evaluating the particle size, polydispersity index (PDI), zeta-potential, and stability. Thereafter, the performance of SLN loading of a model compound in thymol was studied. The loading capacity of thymol in 1,3-LP SLNs was 16% of lipids and higher than 4% and 12% for GMS- and PPP-SLNs, respectively. The 1,3-LP SLNs also had the best efficiency to entrapment thymol during the prolonged storage. X-ray diffraction (XRD) analyses confirmed the excellent crystalline stability of 1,3-LP leading to the stable entrapment efficiency and better stability of thymol-loaded SLNs. Conversely, the polymorphic transformation of GMS and PPP resulted in the declined entrapment efficiency of thymol in the corresponding SLNs. This work indicated the 1,3-diacylglycerol (DAG) SLNs could be used as a promising delivery system for the encapsulation of hydrophobic bioactive molecules with high loading capacity and stability.


High Performance Liquid Chromatography Coupled with Evaporative Light Scattering Detection (HPLC-ELSD) Analysis of 1,3-LP
A Waters 1525 series HPLC system (Waters Corporation, Milford, MA, USA) and an Alltech 3300 ELSD detector (Alltech Associates, Inc., Deerfield, IL, USA) were used for the HPLC-ELSD analysis. The chromatography separation was performed on an Agilent C18 column (4.6 × 150 mm, 5 μm). An isocratic program was applied with acetonitrile and isopropanol (60:40, v/v) at a flow rate of 1.0 mL/min and a column temperature of 35 °C. The detection conditions of ELSD were as follows: drift tube temperature of 45 °C, nebulizer gas (N2) flow rate of 2.0 L/min, and gain of 4. The injection volume was 20 μL.

Preparation of SLNs
GMS-, 1,3-LP-, and PPP-SLN dispersions were prepared using a microemulsion method [26]. To illustrate the effects of lipid matrix on the thymol-loading performance, all formulations were prepared under the same formulation and conditions. Briefly, 80 mg lipid was melted at 70 °C under continuous stirring (500 rpm). The aqueous phase was prepared by adding soybean lecithin and T80 at a mass ratio of 1:3, 1:2, 1:1, 2:1, and 3:1 to double distilled water (2 g) which was also heated to 70 °C. The mass ratio of lipid to surfactant was fixed at 1:3. Then, the aqueous phase was added rapidly to the lipid phase at 70 °C, after which ethanol, about 0.5 mL, was added dropwise to the preemulsion and stirred for 5 min to form a transparent microemulsion. The hot microemulsion was subsequently dispersed in 5 parts of (v/v) of cold water (2-5 °C). All samples were stored at 25 °C before further analysis.

Determination of Z-Average Mean Diameter, Polydispersity Index (PDI), and Zeta-Potential
The Z-average mean diameter, PDI, and zeta-potential measurements of the prepared SLN dispersions were performed using a Malvern Zetasizer Nano ZS90 Particle Analyzer (Malvern The melting temperature (Tm) was measured on an X4 microscopic melting point apparatus (Gongyi City Kerui Instrument Co., Ltd., Henan, China) and was uncorrected. Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded at 400 MHz in CDCl 3 solution on a Varian Mercury Plus 400 spectrometer (Varian, Inc., Palo Alto, CA, USA) and chemical shifts were recorded in parts per million (ppm). The spectra were analyzed using these parameters: δ 0.88 (t, 6H, J = 8.0 Hz, CH 3 The Fourier transform infrared (FTIR) spectrum of 1,3-LP was collected on a Thermo Nicolet NEXUS 670 FTIR Raman spectrometer (Thermo Nicolet Corp., Madison, WI, USA). The wavenumber range recorded was from 4000 cm −1 to 500 cm −1 and 16 scans were collected for 1,3-LP. The mass spectrum (MS) of 1,3-LP was obtained on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Double distilled water was used throughout the study.

High Performance Liquid Chromatography Coupled with Evaporative Light Scattering Detection (HPLC-ELSD) Analysis of 1,3-LP
A Waters 1525 series HPLC system (Waters Corporation, Milford, MA, USA) and an Alltech 3300 ELSD detector (Alltech Associates, Inc., Deerfield, IL, USA) were used for the HPLC-ELSD analysis. The chromatography separation was performed on an Agilent C18 column (4.6 × 150 mm, 5 µm). An isocratic program was applied with acetonitrile and isopropanol (60:40, v/v) at a flow rate of 1.0 mL/min and a column temperature of 35 • C. The detection conditions of ELSD were as follows: drift tube temperature of 45 • C, nebulizer gas (N 2 ) flow rate of 2.0 L/min, and gain of 4. The injection volume was 20 µL.

Preparation of SLNs
GMS-, 1,3-LP-, and PPP-SLN dispersions were prepared using a microemulsion method [26]. To illustrate the effects of lipid matrix on the thymol-loading performance, all formulations were prepared under the same formulation and conditions. Briefly, 80 mg lipid was melted at 70 • C under continuous stirring (500 rpm). The aqueous phase was prepared by adding soybean lecithin and T80 at a mass ratio of 1:3, 1:2, 1:1, 2:1, and 3:1 to double distilled water (2 g) which was also heated to 70 • C. The mass ratio of lipid to surfactant was fixed at 1:3. Then, the aqueous phase was added rapidly to the lipid phase at 70 • C, after which ethanol, about 0.5 mL, was added dropwise to the pre-emulsion and stirred for 5 min to form a transparent microemulsion. The hot microemulsion was subsequently dispersed in 5 parts of (v/v) of cold water (2-5 • C). All samples were stored at 25 • C before further analysis.

Determination of Z-Average Mean Diameter, Polydispersity Index (PDI), and Zeta-Potential
The Z-average mean diameter, PDI, and zeta-potential measurements of the prepared SLN dispersions were performed using a Malvern Zetasizer Nano ZS90 Particle Analyzer (Malvern Instruments Ltd., Worcestershire, UK). The laser wavelength was 633 nm, and the material/dispersant reflective index was 1.590/1.330. Samples were prepared by diluting the SLN dispersions eighty-fold using double distilled water at pH 7. Each sample was repeated three times to obtain the mean value.

Comparison of the Loading Capacity and Entrapment Efficiency of SLNs
To verify the drug-loading performance of the three kinds of SLNs, thymol, a natural compound with antimicrobial and antifungal activities [23], was chosen as a model compound. Different amounts of thymol were used to prepare SLN dispersions based on the formula optimized in Section 2.4. The mass ratios of thymol to lipid were set as 4%, 8%, 12%, 16%, 20%, and 24%. The entrapment efficiency (EE) of samples was measured using a Waters 1525 series HPLC system (Waters Corporation, Milford, MA, USA) equipped with a UV-vis detector operating at 230 nm and an Agilent C18 column (4.5 × 250 mm, 5 µm) kept at 25 • C. After filtration of free thymol in SLN dispersions using an ultrafiltration membrane with a molecular weight cut-off of 10,000 Da (Vivaspin ® , Sartorius, Bagno a Ripoli, Florence, Italy), 20 µL of the permeate was injected to the HPLC. The mobile phase was composed of methanol and deionized water (75:27, v/v) and running at a flow rate of 1 mL/min during isocratic separation. The amount of encapsulated thymol was calculated based on the total thymol used in encapsulation (Wa) and the free thymol determined in the permeate (Ws). The EE of the SLN dispersions was calculated using the following equation [27]: (1)

X-ray Powder Diffraction (XRD) Spectroscopy Analysis
XRD was performed by following the literature method with slight modification [28]. In this study, only the spectra of bulk lipids were investigated to understand possible polymorphic structures of lipid matrix in the SLNs during storage. The powders of 1,3-LP, GMS, and PPP were obtained by melting the lipids at 70 • C and cooled immediately at 0-5 • C to simulate the thermal conditions during SLN preparation. The experiments were conducted using a Philips MPD-X'Pert diffractometer (Almelo, The Netherlands) equipped with Cu Kα radiation (λ = 1.54056 Å) and operating at a voltage of 45 kV, a current of 40 mA, and a step width of 0.039 • /s over the 2θ range of 5-75 • .

Statistical Analysis
The data reported in this paper were presented as mean ± standard deviation (SD). Significance of differences was evaluated using Student's t-test with p < 0.05.

Synthesis of 1,3-LP
In HPLC-ELSD, 1,3-LP and 1,2-LP had a retention time of 4.6 min and 5.0 min, respectively ( Figure 2), and the ratio of 1,2-LP to 1,3-LP was 7:1 after the ring opening reaction ( Figure 1). The purity of 1,3-LP was improved to 97% after isomerization at 55 • C for 72 h, and the total yield was 70%. The final 1,3-LP product appeared as a white solid and had a melting point of 60-62 • C. The present method gives an efficient approach to prepare unsymmetrical 1,3-DAG with good yield and high purity, which can be used as a lipid matrix for fabricating 1,3-DAG SLNs.
The structure of 1,3-LP was further confirmed by 1

Optimization of Formulations for the Preparation of SLNs
To fabricate stable SLN dispersions, the formulation was optimized firstly using the GMS that has the highest polarity among the three lipids. The surfactants soybean lecithin and T80 (widely utilized to prepare nanoscale lipid structures [29][30][31]) were studied for their mass ratios to prepare stable SLN dispersions. The Z-average mean diameter, PDI, zeta-potential, and visual stability of GMS-SLN dispersions prepared using different mass ratios of lecithin to T80 are summarized in Table  1. With the increase of mass ratio of lecithin/T80, the Z-average mean diameter increased significantly, which agreed with increase in visual turbidity ( Figure 6). As lecithin and T80 have an hydrophilic-lipophilic balance (HLB) value of 8.0 and 15.0, respectively, a higher lecithin/T80 mass ratio results in an overall more hydrophobic mixture that is closer to the HLB value of GMS (3.8) and therefore is more effective in reducing the interfacial tension to result in smaller particles [32]. On the contrary, the PDI decreased significantly from 0.39 ± 0.01 to 0.27 ± 0.01 as the lecithin/T80 mass ratio increased from 1:3 to 3:1. This again can be explained by the reduced interfacial tension making it easier to form more homogeneous droplets in microemulsions before quenching to form SLNs. PDI is an important indicator for the homogeneity of particle size distribution, and PDI values below 0.3 indicate the particles are in a narrow size range [33]. Therefore, the lecithin/T80 mass ratios of 2:1 and 3:1 were preferred to obtain relatively uniform GMS SLNs and were used to prepare SLNs using the other two lipids. Characteristics of the 1,3-LP and PPP SLNs are also shown in Table 1. Nanoscale particles and PDI values smaller than 0.3 were observed for both groups of SLNs. It was also observed that the particle dimension decreased with an increase in the number of fatty acid chains on lipids, likely due to the decreased viscosity. For all SLNs prepared at lecithin/T80 mass ratios of 2:1 and 3:1, dispersions were visually stable during two-month storage. These dispersions had a zeta-potential magnitude greater than 20 mV, which is considered to be sufficient to prevent the aggregation of colloidal particles by electrostatic repulsion [34]. The negative zeta-potential results from lecithin and the lecithin/T80 mass ratio of 3:1 were chosen as the optimized formulation for the rest of this study, so as to have a significantly higher magnitude of zeta-potential (Table 1) to stabilize the SLNs in dispersions. For dispersions prepared with a lecithin/T80 mass ratio of 3:1, both the Z-average mean diameter and PDI remained unchanged (p > 0.05) over 60-day storage at 25 °C (Figure 7).

Optimization of Formulations for the Preparation of SLNs
To fabricate stable SLN dispersions, the formulation was optimized firstly using the GMS that has the highest polarity among the three lipids. The surfactants soybean lecithin and T80 (widely utilized to prepare nanoscale lipid structures [29][30][31]) were studied for their mass ratios to prepare stable SLN dispersions. The Z-average mean diameter, PDI, zeta-potential, and visual stability of GMS-SLN dispersions prepared using different mass ratios of lecithin to T80 are summarized in Table 1. With the increase of mass ratio of lecithin/T80, the Z-average mean diameter increased significantly, which agreed with increase in visual turbidity ( Figure 6). As lecithin and T80 have an hydrophilic-lipophilic balance (HLB) value of 8.0 and 15.0, respectively, a higher lecithin/T80 mass ratio results in an overall more hydrophobic mixture that is closer to the HLB value of GMS (3.8) and therefore is more effective in reducing the interfacial tension to result in smaller particles [32]. On the contrary, the PDI decreased significantly from 0.39 ± 0.01 to 0.27 ± 0.01 as the lecithin/T80 mass ratio increased from 1:3 to 3:1. This again can be explained by the reduced interfacial tension making it easier to form more homogeneous droplets in microemulsions before quenching to form SLNs. PDI is an important indicator for the homogeneity of particle size distribution, and PDI values below 0.3 indicate the particles are in a narrow size range [33]. Therefore, the lecithin/T80 mass ratios of 2:1 and 3:1 were preferred to obtain relatively uniform GMS SLNs and were used to prepare SLNs using the other two lipids. Characteristics of the 1,3-LP and PPP SLNs are also shown in Table 1. Nanoscale particles and PDI values smaller than 0.3 were observed for both groups of SLNs. It was also observed that the particle dimension decreased with an increase in the number of fatty acid chains on lipids, likely due to the decreased viscosity. For all SLNs prepared at lecithin/T80 mass ratios of 2:1 and 3:1, dispersions were visually stable during two-month storage. These dispersions had a zeta-potential magnitude greater than 20 mV, which is considered to be sufficient to prevent the aggregation of colloidal particles by electrostatic repulsion [34]. The negative zeta-potential results from lecithin and the lecithin/T80 mass ratio of 3:1 were chosen as the optimized formulation for the rest of this study, so as to have a significantly higher magnitude of zeta-potential (Table 1) to stabilize the SLNs in dispersions. For dispersions prepared with a lecithin/T80 mass ratio of 3:1, both the Z-average mean diameter and PDI remained unchanged (p > 0.05) over 60-day storage at 25 • C (Figure 7). Table 1. The Z-average mean diameter (Z-average), PDI, zeta-potential (Zeta), and storage stability (at 25 • C for up to two months) of SLN dispersions prepared with three types of lipids using different mass ratios of lecithin and T80. All samples were prepared with 80 mg lipid, 240 mg surfactants, 2 g double distilled water, and 0.5 mL ethanol.      Table 1.

Properties of SLNs Loaded with Thymol
Different amounts of thymol were used to prepare SLN dispersions (based on the formulations optimized previously) at a lecithin/T80 mass ratio of 3:1. As the goal was to achieve stable dispersions with PDI smaller than 0.3 (Table 1), the loading capacity of SLNs was considered to be exceeded when dispersions showed visual instability. The loading capacity of thymol in SLNs was 4%, 16%, and 12% of the corresponding lipids when GMS, 1,3-LP, and PPP were studied (Table 2). Thymol is overall hydrophobic but has a polar hydroxyl group. The intermediate polarity of 1,3-LP when compared to GMS and PPP, as discussed previously, may have resulted in the best solubility for thymol to increase the loading capacity. For visually stable dispersions (fresh or after storage), the EE was mostly higher than 95%, with the exception of 85% for the GMS SLNs after storage ( Table 2). The data suggest most of the thymol was entrapped in these stable SLNs. Table 2. The Z-average mean diameter (Z-average), PDI, zeta-potential (Zeta), and encapsulation efficiency (EE%) for the thymol loaded GMS-, 1,3-LP-, and PPP-SLN. The Z-average mean diameter, PDI, and zeta-potential of treatments are also presented in Table 2. It is evident that the Z-average mean diameter of each group of SLN dispersions increased with the amount of thymol used in the preparation. The HLB of thymol calculated according to Equation (2) is 2.3, which makes the lipid phase more hydrophobic (with a HLB value even smaller than the mixture of lecithin and T80) and therefore results in bigger particles. The increase of the amount of thymol in dispersions also resulted in reduced zeta-potential magnitude and overall poorer stability initially and after storage ( Table 2)

Lipid
where M Hydrophilic and M Lipophilic are the molecular mass of hydrophilic and lipophilic portions of a compound [35].
In addition, the Z-average mean diameter at the same amount of thymol used in preparations showed an identical trend to those of bare SLNs (GMS > 1,3-LP > PPP), which is expected due to the minor portion of thymol in the lipid phase. Overall, dispersions with a good initial EE and a PDI smaller than 0.3 maintained good visual stability, which is expected as big particles can phase separate especially after storage. It is worth noting the insignificant changes of Z-average mean diameter and PDI for the 1,3-LP SLN dispersion with thymol being 16% of lipid mass after storage at 25 • C for 90 days ( Figure 8B). The same results were found for both the GMS and PPP SLN dispersions with thymol being 4% and 12% of lipid mass, respectively ( Figure 8A,C). Considering most thymol remained entrapped (Table 2), SLNs prepared with 1,3-LP showed great promise as delivery systems. The release properties after applying the SLNs (e.g., after dilution with physiological fluid) are to be characterized in the future.

Polymorphic Structures of Lipids Studied with X-ray Powder Diffraction Spectroscopy
Polymorphic structures of the lipid phase play an important role in determining the stability of SLNs and the encapsulated compounds [6]. The XRD spectra of bulk GMS, 1,3-LP, and PPP following thermal treatments analogous to SLN preparation and storage at 25 °C were characterized, shown in Figure 9. According to the XRD American Oil Chemists' Society (AOCS) method [36], the α-form polymorph displays as one peak at a d-spacing of 4.1 Å, while the β-form displays as two peaks at a d-spacing of 4.6 Å and 3.8 Å. For the freshly prepared lipid crystals, the diffraction patterns in Figure  9A showed GMS and PPP both had one single high intensity peak near the d-spacing of 4.15 Å, which verified the dominant presence of α-form crystals in these two lipids. In contrast, the diffraction peaks of 1,3-LP were mainly located at d-spacings of 4.65 Å, 4.52 Å and 3.82 Å, indicating the formation of β-form polymorph crystals, and, a small peak at a d-spacing of 4.13 Å had a small portion of α-form crystals.

Polymorphic Structures of Lipids Studied with X-ray Powder Diffraction Spectroscopy
Polymorphic structures of the lipid phase play an important role in determining the stability of SLNs and the encapsulated compounds [6]. The XRD spectra of bulk GMS, 1,3-LP, and PPP following thermal treatments analogous to SLN preparation and storage at 25 • C were characterized, shown in Figure 9. According to the XRD American Oil Chemists' Society (AOCS) method [36], the α-form polymorph displays as one peak at a d-spacing of 4.1 Å, while the β-form displays as two peaks at a d-spacing of 4.6 Å and 3.8 Å. For the freshly prepared lipid crystals, the diffraction patterns in Figure 9A showed GMS and PPP both had one single high intensity peak near the d-spacing of 4.15 Å, which verified the dominant presence of α-form crystals in these two lipids. In contrast, the diffraction peaks of 1,3-LP were mainly located at d-spacings of 4.65 Å, 4.52 Å and 3.82 Å, indicating the formation of β-form polymorph crystals, and, a small peak at a d-spacing of 4.13 Å had a small portion of α-form crystals.

Polymorphic Structures of Lipids Studied with X-ray Powder Diffraction Spectroscopy
Polymorphic structures of the lipid phase play an important role in determining the stability of SLNs and the encapsulated compounds [6]. The XRD spectra of bulk GMS, 1,3-LP, and PPP following thermal treatments analogous to SLN preparation and storage at 25 °C were characterized, shown in Figure 9. According to the XRD American Oil Chemists' Society (AOCS) method [36], the α-form polymorph displays as one peak at a d-spacing of 4.1 Å, while the β-form displays as two peaks at a d-spacing of 4.6 Å and 3.8 Å. For the freshly prepared lipid crystals, the diffraction patterns in Figure  9A showed GMS and PPP both had one single high intensity peak near the d-spacing of 4.15 Å, which verified the dominant presence of α-form crystals in these two lipids. In contrast, the diffraction peaks of 1,3-LP were mainly located at d-spacings of 4.65 Å, 4.52 Å and 3.82 Å, indicating the formation of β-form polymorph crystals, and, a small peak at a d-spacing of 4.13 Å had a small portion of α-form crystals.