(M1030-03-17) Enhance Transport of Adenosine Integrated in Lipid Nanoparticles Crossing Reconstructed Human Epidermis

Purpose: Adenosine is one of the four nucleosides with extensive of pharmacological effects, including antitumor, anti-inflammatory, antioxidant, and antiwrinkle effects. Upon skin permeation, adenosine promotes the growth of fibroblasts and collagen synthesis. accordingly, skin wrinkles reduce, while skin elasticity increases. In this study, we used free fatty acids to prepare liquid nanoparticles to enhance skin permeation of the low permeability drug adenosine and conducted skin permeation studies using rat skin.

Methods: First, a first-emulsion (W₁/O) was prepared using Span^® 80, a hydrophobic surfactant having a hydrophilic-lipophilic balance value of 4.3. Second, poloxamer 188 was finally choosed as the surfactant used to stabilize W₁/O/W₂ 4 g of lipid (stearic acid) was dissolved in each 10 mL glass vial. After dissolving the lipids, 1, 2, 4, 8, or 16 mg of Adenosine was added to the lipid, and the compound was observed with the visually. A first emulsion (W₁/O) was prepared by homogenizing the dissolved oil phase containing adenosine and the internal aqueous phase at 5000 rpm at a lipid melting point of 10 ℃ or higher using an Ultra- Turrax T18 (IKA, Staufen, Germany). The first-emulsion and external aqueous phase (W₂) were mixed using an Ultra-Turrax T18 at 1000 rpm and then homogenized for 3 cycles at 1300 bar in a NanoDeBEE homogenizer (BEE International, South Easton, MA). The nano-sized W₁/O/W₂ emulsion was preserved at 4 ℃. The prepared lipids were measured for particle size, polydispersity index and zeta potential of the nanoparticles at 25° C. using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK). The samples were dispersed 10 times using deionized water before the measurements. The Lipid Nanoparticles were dispersed 10 times using deionized water and smoothly vortexed. The distributed Lipid Nanoparticles were then centrifuged at 14000 rpm and 4 ℃ for 2 h. After 48 h incubation of the SkinEthic RHE tissues, the culture inserts were located into a new sterile 6-well plate containing 3 mL of receptor medium, and 0.3 mL of each formulation was placed on the epidermal surface. The 6-well plate with the RHE tissues was placed on a shaker (NB-101SRC, N-BIOTEK, Korea) in an incubator at a temperature of 37 ℃ under an atmosphere of 5 % CO with a speed of 100 rpm. At specific time points at 0.5, 1, 2, 4 and 8 hours during incubation, 1 mL of receptor solution was removed from the wells of the 6-well plate

Results: 1. Average particle size and polydispersity characteristics of the adenosine-contained lipid nanoparticles. Figure 1) The particle size of lipid nanoparticles ranges from 82.22 nm (F3) to 177.7 nm (F1). It was explained that the particle size increases as the concentration of Stearic acid increases. Additionally, it was observed that the particle size increases as the concentration of Span® 80 is reduced.The polydispersity index of all the lipid nanoparticles ranged from 0.19 to 0.41, indicating that the lipid nanoparticles were prepared homogeneously. 2. Entrapment efficiency and loading amount of the adenosine-contained Lipid Nanoparticles. Figure 2) As shown in Figure 2, the encapsulation efficiency and loading amount of adenosine-containing lipid nanoparticles were detected as 55.96~66.79% and 2.72~9.09%, respectively. 3. Zeta potential value of the adenosine-contained lipid nanoparticles. Figure 3) Formulations were detected to range from -21.87 (F1) to -23.08 mV (F3). As a result, more stable lipid nanoparticle formulations appear to be formed when the concentration of stearic acid or Span^® 80 increases encapsulation efficiency and loading. Encapsulation efficiency and loading amount a little increased as the concentrations of the surfactants used in the formulations increased. In the case of lipids, increasing the lipid concentration resulted in higher encapsulation efficiency but lower loading. Figure 4) As a result of the cumulative percentage of adenosine release from the lipid nanoparticles in the medium, F1 and F2 were all released within about 12 hours, and F3 was all released within 24 hours.

Conclusion: In this study, we prepared adenosine-contained lipid nanoparticles to improve permeation of adenosine into skin. The size of particle for F3 formulation was the smallest, which induces high permeation of adenosine due to its lipophilic and nano properties. Lipid nanoparticle is one of the pharmaceutical formulation that sustained release property for encapsulated drug. In our result of release, we confirmed that adenosine from lipid nanoparticles showed sustained release profile. Thus, our adenosine->contained lipid nanoparticle is a promising to improve skin permeation and long acting anti-wrinkle effect.

Acknowledgements: This work was supported by the Mid-Career Researcher Program (No. NRF-2021R1A2C2008834), the Basic Research Infrastructure Support Program (University-Centered Labs-2018R1A6A1A03023718), and the Bio & Medical Technology Development Program (NRF-2022M3A9G8016257) through the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT).

Table 1. Adenosine-contained Lipid Nanoparticles were prepared via the modified double emulsion method (W₁/O/W₂) with diverse furtherance.

Figure 1. Average particle size and polydispersity index of the adenosine-contained lipid nanoparticles. The results are represented as the mean ± SD of three separate experiments (n = 3).

Figure 2. (A) Entrapment efficiency and (B) loading amount of the adenosine-contained Lipid Nanoparticles. The results are represented as the mean ± SD of three separate experiments (n = 3).

Figure 3. Zeta potential value of the adenosine-contained lipid nanoparticles. The results are represented as the mean ± SD of three separate experiments (n = 3).

Figure 4. Cumulative percentage of adenosine release from Lipid Nanoparticle in the meduim. The results are represented as the mean ± SD of three separate experiments (n = 3).