Colon-targeted protein delivery by using solid lipid nanoparticles

Materials

BSA (Sigma-Aldrich, Saint Louis, MO), glyceryl monostearate (Chemical Point, Oberhaching, Germany), Tween 80 (Brataco, Jakarta, Indonesia), soy lecithin (Unitechem Co., Ltd., Suqian, China), inulin (BENEO-Orafti SA, Oreye, Belgium), polyethylene glycol (PEG) 6000 (NOF Corporation, Tokyo, Japan), Eudragit S100 (Evonik, Bekasi, Indonesia), Lowry reagent, trichloroacetic acid (Merck, Darmstadt, Germany), and Aquadest were acquired.

Colyophilization BSA with inulin

An inulin solution (5 mg/ml) was added to the BSA solution (2 mg/ml) and stirred homogeneously. The mixed solution was placed in a vial and frozen in a freezer at −80°C and then freeze-dried at 0.2 mBar.

Preparation of BSA–SLN

Table 1 shows the formula of BSA–SLN. The method of SLN preparation used in this study was double emulsion. Glyceryl monostearate was melted at 55°C. Then, soy lecithin, 20 ml of BSA solution (2 mg/ml), and PEG 6000 were added. The solution was stirred using a T25 digital ULTRA-TURRAX homogenizer (IKA, Kuala Lumpur, Malaysia) for 10 minutes at 10,000 rpm. The emulsion formed was added to the surfactant solution (Tween 80) and sonicated for 25 minutes.

BSA–SLN cCoating with Eudragit S100

An Eudragit S100 solution with a concentration of 5% w/v was prepared using deionized water. The optimum BSA–SLN formula solution was then added to the Eudragit S100 solution with the same volume. The addition of SLN to the polymer solution was carried out drop by drop with continuous agitation at room temperature (20^oC) for 10 minutes to produce the desired coating (Zhang et al., 2014). The SLN coating results were immediately frozen at −85^oC and lyophilized using a freeze-dryer at −65^oC condenser temperature and 0.211 mBar pressure for 24 hours.

Characterizations of BSA–SLN

Particle Size Distribution

The measures of the mean intensity (Dp) and polydispersity index (PDI) were taken by photon correlation spectroscopy using Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK). Measurements were made on the SLN dispersion in 1:1,000 Aquadest at 25°C with a scattering angle of 90^o.

Zeta Potential

Zeta potential from the SLN was measured by electrophoretic light scattering using the Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK). Zeta potential measurements were used to determine the physical stability of the SLN and the electric charge on the SLN’s surface. Measurements were made on the SLN dispersion in a 1 :1,000 Aquadest at 25°C with a scattering angle of 90^o.

Particle Morphology

The surface morphology of the SLN-Eudragit S100 was seen using a transmission electron microscope (FEI Tecnai G2 20 S-TWIN, Hillsboro, OR). SLN-Eudragit S100 powder was dispersed in distilled water. As many as one drop of the dispersion was dropped on the carbon-coated copper network to form a thin liquid layer (Elmowafy et al., 2017). The layers on the grid were allowed to dry at room temperature. Next, the grid was attached to the instrument, and photos were taken at various magnifications with an Eagle^TM CCD Camera (Shah et al., 2016).

Entrapment Efficiency of SLN

The entrapment efficiency of BSA in the SLN is determined indirectly by calculating the BSA that is not trapped. The final SLN preparation results were centrifuged at a speed of 12,000 rpm. Protein is calculated from the free BSA in the supernatant from the centrifugation result. 1 ml of supernatant was taken, and then the BSA that is not trapped was precipitated using a 10% TCA solution. The precipitate was then dissolved in 1 ml deionized water. The BSA in the solution was tested using the Lowry assay. The absorbance was measured at a wavelength of 750 nm using a microplate reader (VersaMax, Chattanooga, TN). The BSA concentration was calculated by comparing it against a standard curve of known standard BSA concentrations. The percentage of entrapment efficiency was calculated using the following formula:

Table 1. Formulae of SLN containing BSA. [Click here to view]

$\mathrm{Entrapment}\mathrm{efficiency}\mathrm{(\%)}=\frac{\mathrm{Total}\mathrm{amount}\mathrm{of}\mathrm{drug}\mathrm{loaded}\mathrm{-}\mathrm{free}\mathrm{drug}\mathrm{in}\sup \mathrm{erna}\tan \mathrm{t}}{\mathrm{Total}\mathrm{amount}\mathrm{of}\mathrm{drug}\mathrm{loaded}}\times 100$

In Vitro Drug Release Studies

The in vitro release studies were carried out by a modified dissolution method using a dialysis membrane. Before use, dialysis bags with a molecular weight cut-off of 100 kDa were immersed in distilled water for 12 hours. Furthermore, 100 mg of optimal SLN formula was suspended in distilled water. One milliliter of the suspension was filled in a dialysis bag. Then, the dialysis bag was put in a beaker glass containing 250 ml of release media operated at 37^oC using a magnetic stirrer at a speed of 100 rpm. The in vitro release study was carried out in hydrochloric acid media of 0.1 N pH 1.2, phosphate buffer pH 7.4, and phosphate buffer pH 6.8 that simulate the stomach, small intestine, and colon, respectively (Iswandana et al., 2018b). The time of drug release in the hydrochloric acid medium of 0.1 N pH 1.2 was observed for 2 hours, in the phosphate buffer media pH 7.4 for 3 hours, and in the phosphate buffer media pH 6.8 for 3 hours.

A total of 1 ml of the sample was taken at 15, 30, 45, 60, 90, 120, and 180 minutes of each medium and then replaced immediately with new media at the same volume to maintain sink conditions. The BSA content in the dissolution medium was analyzed using the Lowry protein assay. The absorbance was measured at a wavelength of 750 nm using a microplate reader (VersaMax, Chattanooga, TN). The BSA concentration was calculated using a standard calibration curve.

Colyophilization BSA with inulin

Colyophilization is a process of protecting the BSA from conformational changes and denaturation during storage and passing through the GI tract. Colyophilization is carried out by coating the BSA protein with inulin. The ratio of the concentration of BSA and inulin is 1 :2.5. From the experimental results, we obtained 32 mg of BSA-inulin containing 10 mg of BSA. The addition of inulin to BSA by colyophilization can increase protein stability and decrease denaturation caused by conformational change. Inulin as a protein protectant can prevent the unfolding of proteins (Furlán et al., 2010).

Preparation of BSA–SLN

The preparation of BSA–SLN uses the double-emulsion method. From the experimental results, it was found that the appearance of the SLN is a white-yellow, odorless, suspension-like solution, as shown in Figure 1.

Figure 1. BSA–-SLN preparation from formulations 1–8. [Click here to view]

Table 2. Particle size, PDI, zeta potential, and entrapment efficiency of BSA–SLN. [Click here to view]

Preliminary characterizations of BSA–SLN

Particle Size Distribution

The particle size distribution is measured by a particle size analyzer and seen at the Z-Ave value. The Z-Ave value is the average value of the particle size in the sample. The distribution of particle sizes ranges from 94.24 nm to 186.8 nm as can be seen in Table 2. This result proves that, from the F1 to F8 formulae, all have the ideal size for the SLN, which is in the range of 50–1,000 nm. The increase of Tween 80 as a surfactant leads to a decrease in particle size and PDI due to reduced interfacial tension by a higher concentration of surfactant. The addition of soy lecithin as a cosurfactant can also decrease the mean particle size by stabilizing emulsion (Niculae et al., 2013). Formula 4 (F4) shows the best result of particle size measurement with 1.5% Tween 80 and 0.25% soy lecithin. Previous research showed that a smaller PDI (less than 0.7) indicates that the SLN has homogenous particle distribution (Trinovita et al., 2019). All the formulae tested in this research proved to achieve the ideal PDI for the SLN and therefore show that the SLN is distributed homogeneously.

Zeta Potential

Zeta potential is the electrical potential between the medium and interface of the fluid attached to the particle. Suppose the absolute value of the zeta potential is too high. In that case, the system deflocculates due to increased repulsion and cake dispersion. Suppose the zeta potential decreases below a particular value. In that case, attractive forces exceed the repulsive forces so that the particles will unite. In general, the zeta potential value of an emulsion is +/− 30 mV. Based on Table 2, it is found that the zeta potential value of the formula varies from −22.8 mV to −34.3 mV. According to previous research, a zeta potential greater than + 30 mV or −30 mV stipulates the excellent stability of the dispersion. This strongly indicates that all formulae of the SLN could be more stable (Chandran et al., 2018).

Entrapment Efficiency of SLN

The entrapment efficiency value is the ability of the matrix to trap the protein. The measurement of entrapment efficiency is carried out indirectly by calculating the amount of BSA that is not trapped. The value of entrapment efficiency can be seen in Table 2. From the observations, the most considerable SLN’s entrapment efficiency is F4, which is 78.69%. Greater entrapment efficiency can be obtained by increasing the concentration of surfactant to improve BSA’s stability and emulsifying capacity in the SLN system (Trinovita et al., 2019). Double-emulsification methods were found to increase drug solubility and entrapment efficiency (Ngwuluka et al., 2017). This research proved that the F4 SLN provides high entrapment efficiency, which can load a higher protein concentration.

Figure 2. TEM of the optimized formula (F4 SLN). [Click here to view]

Final characterizations of BSA–SLN

After conducting preliminary characterizations of BSA–SLN, the optimum formula was obtained based on the best initial descriptions, namely, F4. Furthermore, F4 preparation was evaluated for further tests, such as particle morphology and in vitro drug release studies.

Particle morphology

The particle morphology of the SLN was obtained from transmission electron microscopy (TEM). The results of the TEM of F4 can be seen in Figure 2. The SLN particles consist of a hydrophobic core with a surfactant monolayer layer based on the test results. The inside of the core contains proteins that are dispersed in a lipid matrix. The SLN’s morphological characteristic, almost spherical with a smooth surface, was observed using a TEM photograph. Further, no precipitation of BSA was observed, indicating the SLN was stable (Kesharwani et al., 2016).

Table 3. Cumulative drug release of SLN in HCl, PBS 7.4, and PBS 6.8 (n = 3). [Click here to view]

Figure 3. Cumulative drug release profile of F4 SLN. Data are expressed as mean ± SD (n = 3). HCl media pH 1.2 (0–120 minutes); phosphate buffer pH 7.2 (120–300 minutes); and phosphate buffer pH 6.8 (300–480 minutes). [Click here to view]

In Vitro Drug Release Studies

Based on the research results shown in Table 3 and Figure 3, the SLN F4 formulation could deliver a maximum drug to the colon. 102.93% ± 3.19% BSA was released from the system after 8 hours of release study in three different media. Therefore, the F4 SLN had a potential delivery system in enhancing protein absorption in the colon. However, drug release had begun to occur in the simulated gastric and small intestine fluids. The release of BSA in the SLN system of F4 showed initial burst release followed by a slow controlled release of proteins. The drug release study indicated that, using F4 SLN, 25.97% ± 4.91% BSA was released in the HCl solution pH 1.2 in the first 2 hours. These results can be caused by the imperfect coating of BSA–-SLN with Eudragit S100. The addition of excipients such as sustained-release polymers or polysaccharides can be used to prevent premature release.