In vitro and Ex vivo Antioxidant Activity and Sustained Release Properties of Sinomenine-Loaded Liposomes-in-Hydrogel Biomaterials Simulating Cells-in-Extracellular Matrix

Abstract

Sinomenine (SIN), a natural product, has been used to treat rheumatoid arthritis (RA) in China for thousands of years. SIN has been developed for the treatment of RA by way of tablets and injections, but both dosage forms have been associated with severe adverse reactions. Making SIN into liposomes-in-hydrogel biomaterials for external use has a good slow-release effect and can play an important role in avoiding the first-pass effect, gastrointestinal reaction, and increasing the local action time of drugs. SIN-loaded liposomes were formed by the thin-film dispersion method, then SIN-loaded liposomes-in-hydrogels were prepared by combining the SIN-L with hyaluronic acid (HA) hydrogels. In this paper, the basic characteristics, In vitro and Ex vivo release, and antioxidant activity of SIN-loaded liposomes-in-hydrogels were studied. The results showed that SIN-loaded liposomes-in-hydrogels have good sustained-release and antioxidant effects, and the preparation is expected to be a good biomaterial.

Introduction

Sinomenine (SIN; 7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methylmorphinan-6-one, CAS Number: 115-53-7) is an isoquinoline alkaloid derived from Sinomenium acutum (Thunb.) Rehder & E.H. Wilson. SIN is a white powder, which is observed as needle-like crystals under the microscope. Clinical studies have shown that it has a variety of pharmacological effects, such as immunomodulation, anti-inflammation, anti-oxidation, and improving microcirculation.^1,2 SIN, as a natural good antioxidant, is easy to obtain with high safety and good stability. Therefore, it has been used to treat rheumatism in China for many years. ³ With regard to SIN, the existing preparations are mainly for administration orally and by injection. However, oral administration can stimulate the gastrointestinal tract, and long-term administration will cause gastric mucosal, liver, kidney, and heart damage. ⁴ In addition, SIN has problems of a short half-life and low bioavailability in vivo. ⁵ Therefore, it is of great significance to adopt some new methods, including nanoparticles, ⁵ transethosome, ⁶ and liposomes, ⁷ to change the administration mode of SIN, so that it can be better applied.

A drug carrier is a system that can change the way in which a drug can enter the body and its distribution in the body, controlling the drug release rate, and delivering the drug to the target organ. Inorganic nanoparticles (eg, cerium oxide nanoparticles ⁸ ), transethosome, and liposomes are all representative carriers, all of which are nano-scale and each has its own unique advantages. Cerium oxide nanoparticles are a strong antioxidant, which has the autocatalytic ability to scavenge reactive oxygen species (ROS), thus regulating the ROS level in the cell microenvironment, increasing the activity of antioxidant enzymes, and protecting cells from various harmful processes.^9–11 Transethosome contains a high content of ethanol and edge activator, and so it has a great capacity to carry hydrophilic drugs. ⁶ Liposomes have a lipid bilayer and can be loaded with either hydrophobic or hydrophilic drugs or both, ¹² which reduces the irritation and side effects of drugs and have good cell compatibility. At present, the technology of using liposomes to load drugs is well established and can release drugs continuously and stably, improve their bioavailability, and reduce toxicity.

Hydrogels are a kind of three-dimensional network polymer formed by physical and chemical cross-linking of monomers and hydrophilic groups. ¹³ They can be used for drug loading, tissue engineering, and biological research, and are a good biomedical material. ¹⁴ As a carrier, hydrogels have a good loading capacity for various drugs. Drug molecules such as growth factors or antibiotics can also be easily loaded into hydrogels. ¹⁵ Hyaluronic acid (HA) is a natural component of the extracellular matrix, and it is also a common hydrogel matrix. ¹⁶ It has a variety of physiological activities and is mainly used for wound repair, tissue regeneration, drug delivery, and the treatment of inflammatory diseases.^17,18 When HA is used in combination with drugs as a hydrogel matrix, it can significantly prolong the retention time of the drugs and improve their bioavailability.^19,20

Considering that SIN has a short half-life in vivo, rapid metabolism, and toxic effects caused by long-term oral administration, it was made into an external preparation. Liposomes have the advantages of controlling drug release, improving drug bioavailability, and reducing drug toxicity, while HA hydrogels have the advantage of prolonging drug retention time in the skin due to their biological adhesion. These advantages make SIN more widely applied. Therefore, it is a good choice to encapsule SIN into liposomes and then evenly disperse them in hydrogels to prepare sinomenine-loaded liposomes-in-hydrogels (SIN-L-H). In this study, liposomes were prepared by the thin-film dispersion method (SIN-L), then HA hydrogels were prepared by the swelling method (SIN-H), and the 2 methods were combined to prepare SIN-L-H. The appearance, particle size, Zeta potential, and other properties of SIN-L and the viscosity of SIN-H and SIN-L-H were investigated, and the release rate (In vitro and Ex vivo) and antioxidant activity (the scavenging rate of 1,1-diphenyl-2-picrylhydrazyl, H₂O_2, and mouse organ homogenates) of these 3 preparations were determined, which laid the foundation for a follow-up study of new dosage forms of SIN.

Materials and Methods

Materials

Sinomenine (≥ 98%, China), soy lecithin (99%, Shanghai Jinsong Industry Co., Ltd, China), cholesterols (99%, Shanghai Jinsong Industry Co., Ltd, China), absolute ethanol (AR, Shanghai RichJoint Chemical Reagents Co., Ltd, China), HA (99%, Shandong Xiya Reagents Co., Ltd, China), 1,1-diphenyl-2-picrylhydrazyl (DPPH, 98%, Shanghai Yuanye Biology Science and Technology Co., Ltd, China), H₂O₂ solution (AR, Shanghai SuYi Chemical Reagent Co., Ltd, China), FeSO₄·7H₂O (AR, Sinopharm Chemical Reagent Co., Ltd, China), thiobarbituric acid (TBA, 98%, Shanghai Yuanye Biology Science and Technology Co., Ltd, China), trichloroacetic acid (TCA, AR, Tianjin Damao Chemical Reagent Factory, China), phosphate buffered saline (PBS).

Preparation of SIN-L

Phospholipids (0.3 g) and 0.1 g of cholesterols (the ratio of phospholipids to cholesterols was adjusted based on our laboratory's preliminary test ²¹ ) were dissolved in absolute ethanol and placed in a rotary evaporator (RE-2000 E, Zhengzhou Yarong Experimental Instrument Co., Ltd, China) to form a film (70 °C, vacuum pressure 70 kPa, speed 100 r/min) until the absolute ethanol had completely volatilized. Then 10 mL of 2 mg/mL SIN solution was added to the membrane, rotated, and hydrated for 30 min. After ultrasonic treatment for 10 min, the suspension was filtered through a microporous membrane with a pore size of 0.22 μm, 3 times, to obtain SIN-L. In addition, PBS was used instead of the above SIN solution to carry out the same operation to obtain blank liposomes (B-L).

Preparation of SIN-H

HA (0.1 g) was added to 10 mL of a 2 mg/mL SIN solution and stirred with a magnetic stirrer (85-2, Changzhou Yineng Experimental Instrument Factory, China) at room temperature for 12 h until the HA had swollen sufficiently to form a transparent hydrogel. In addition, PBS was used instead of the above-mentioned SIN solution to perform the same operation to obtain blank hydrogels (B-H).

Preparation of SIN-L-H

The preparation of SIN-L-H was similar to the preparation of SIN-H. HA (0.1 g) was added to 10 mL of SIN-L suspension with a concentration of 2 mg/mL and stirred at room temperature with a magnetic stirrer for 12 h until HA was completely expanded to form hydrogels with uniform texture (Figure 1). In addition, B-L suspension was used instead of the above SIN-L suspension to carry out the same operation to obtain blank liposomes-in-hydrogels (B-L-H).

Figure 1.

SIN-L-H formed via SIN-L being uniformly located in the three-dimensional network structure of HA hydrogels.

Basic Characteristics of SIN-L, SIN-H, and SIN-L-H

Firstly, the appearance of the 3 kinds of preparations was observed. Then the microscopic features of SIN-L were observed under a cryo-EM (Glacios-200KV, Thermo Fisher Scientific, US), and the potential and particle size of SIN-L were measured using a Malvern Nanoparticle Potential Analyzer (ZEN3690, US Malvern Instrument Co., Ltd, UK).

The encapsulation efficiency (EE%) and loading efficiency (LE%) of SIN-L were determined by high-speed centrifugation. One mL of SIN-L suspension was placed in a centrifuge tube at a high speed of 12 500 r/min for 45 min to separate the supernatant and SIN-L precipitate. After the supernatant was absorbed, the content of free SIN (M_f) was measured at the characteristic wavelengths of SIN of 262 nm using an ultraviolet spectrophotometer (UV-1000, AoE Instrument Co., Ltd, China). The precipitate was freeze-dried and accurately weighed to obtain the total amount of liposomes (M_SIN-L). Another 1 mL of SIN-L suspension was added to 9 mL of absolute ethanol, and, after ultrasonic demulsification for 30 min, an appropriate amount of solution was taken to measure the content of total SIN (M_t) at 262 nm. The EE% was calculated according to formula (1) and the LE% was calculated according to formula (2), where “M_t” represents the total SIN content contained in the SIN-L suspension, and “M_f” represents the SIN content that was not encapsulated into the liposomes.

$$\mathrm{EE}\text{\%}={\displaystyle \frac{{\mathrm{M}}_{\mathrm{t}}-{\mathrm{M}}_{\mathrm{f}}}{{\mathrm{M}}_{\mathrm{t}}}\times 100\text{\%}}$$

(1)

$$\mathrm{LE}\text{\%}={\displaystyle \frac{{\mathrm{M}}_{\mathrm{t}}-{\mathrm{M}}_{\mathrm{f}}}{{\mathrm{M}}_{\mathrm{SIN}-\mathrm{L}}}\times 100\text{\%}}$$

(2)

For SIN-H and SIN-L-H, an Ostwald viscometer (Shanghai Shenyi Glass Instrument Factory, China) was used to measure the viscosity of the 2 preparations. The viscosity of both was calculated according to formula (3). In this formula, “ŋ” is the viscosity of the liquid (mPa.s), “ρ” is the density of the liquid (g/mL), “t” is the time (s) for the liquid through the capillary tube of the austenite viscometer (s), “ρ₁” is the density of water, “t₁” is the time (s) for water to pass through the capillary test tube of the Ostwald viscometer, and “ŋ₁” is the viscosity of the water.

$$\eta ={\displaystyle \frac{\rho \mathrm{t}}{{\rho }_1{\mathrm{t}}_1}\times {\eta }_1}$$

(3)

Three batches of SIN-H were taken, where the prescription amount of SIN was 2 mg/mL. One mL of SIN-H and 1 mL of B-H, respectively, were placed in centrifuge tubes, to which absolute ethanol was added to fix the volume to 10 mL, and ultrasonically treated for 30 min. An appropriate amount of the above solution was centrifuged at 10 000 r/min for 10 min, and the absorbance of the supernatant was measured at 262 nm to calculate the drug content in SIN-H (M_measured, n  =  3).

In Vitro Release Across the Dialysis Membrane of SIN and its Preparations

A measured volume was added to prepared, numbered Franz diffusion cells. A stirring bar was added to each diffusion cell, which was filled with PBS. Dialysis membranes (MWCO: 8 000-14 000 Da) of appropriate sizes were cut out and placed on the lower cells, respectively. One mL of SIN, SIN-L, SIN-H, SIN-L-H, and their corresponding blank groups were added into the upper cells in turn. Two mL samples were removed after 5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 84 h, and replenished with 2 mL of PBS solution in the lower cell (Figure 2). An ultraviolet spectrophotometer was used to measure the absorbance at 262 nm. A standard curve was used to calculate the corresponding drug concentration, and the cumulative release rate was obtained according to formula (4), where “Q” is the cumulative drug release rate of SIN at the different sampling points, “C_n” is the mass concentration of SIN at the nth time point (μg/mL), “V_n” is the volume of the solution sampled at the nth time point (mL), “C_i” is the mass concentration of SIN in the receiving solution at the sampling point (i ≤ n−1) (μg/mL), “V” is the total volume of the lower cells (mL), and “Q_t” is the theoretical drug content (2000 μg/mL).

$$\mathrm{Q}(\text{\%})={\displaystyle \frac{{\mathrm{C}}_{\mathrm{n}}\times {\mathrm{V}}_{\mathrm{n}}+{\sum }_{\mathrm{i}=1}^{\mathrm{n}=1}{\mathrm{C}}_{\mathrm{i}}\times \mathrm{V}}{{\mathrm{Q}}_{\mathrm{t}}}}$$

(4)

Figure 2.

The schematic diagram of drug release across the dialysis membrane In vitro and mouse skin Ex vivo.

Ex Vivo Release Across Mouse Skin of SIN and its Preparations

Kunming mice were weighed and anesthetized. The hair on the back of the mice was removed by shaving, the skin cut to an appropriate size, cleaned of residual subcutaneous tissue and fat, and rinsed in PBS for later use (Figure 2). When required, it was used as described above.

The Scavenging Rate of SIN and its Preparations on DPPH Radicals

DPPH radicals are relatively stable, and deep purple when dissolved in absolute ethanol. The maximum absorption of DPPH is at 517 nm. When combined with antioxidant active substances, the color of DPPH will change from purple to yellow. ²² So, the scavenging ability of SIN and its preparations on DPPH radicals can be judged by color change. ²³ The lighter the color, the stronger the antioxidation ability of SIN and its preparations. The ability of SIN and its preparations to remove DPPH radicals is expressed by the IC₅₀ value, representing the concentration of an inhibitor required for 50% inhibition of DPPH radicals. The smaller the IC₅₀ value is, the stronger the antioxidant capacity of the material.

Two mL of SIN of various concentrations (0.025, 0.05, 0.075, 0.1, and 0.125 mg/mL) and 0.025 mg/mL of its preparations were added to 1 mL of DPPH solution, and reacted for 0.5 h in the dark. Then the absorbance was measured at 517 nm. According to the measured absorbance, the scavenging rate of SIN and its preparations on scavenging DPPH radicals was calculated according to the following formula (5), where “A₀” is the value of 2 mL of PBS and 1 mL of DPPH, “A_s” the value of 2 mL of sample and 1 mL of DPPH, and “A_c” the value of 2 mL sample and 1 mL of PBS.

$$\mathrm{E}\text{\%}={\displaystyle \frac{{\mathrm{A}}_0-({\mathrm{A}}_{\mathrm{s}}-{\mathrm{A}}_{\mathrm{c}})}{{\mathrm{A}}_0}\times 100\text{\%}}$$

(5)

The Scavenging Rate of SIN and its Preparations on H₂O₂

H₂O₂ is a source of toxic hydroxyl radicals, and it also oxidizes intracellular substances when it passes through the membrane, so the elimination of H₂O₂ and hydroxyl radicals is also an important part of antioxidant research. ²⁴ The operation of this experiment was the same as that of SIN and its preparation in scavenging DPPH radicals. A_s group was the sample group, each containing 0.6 mL of SIN with various concentrations of solution (0.2, 0.3, 0.4, 0.5, and 0.6 mg/mL) and 1.8 mL of H₂O₂. A_c was the control group, each containing 0.6 mL of SIN with various concentrations of solution (0.2, 0.3, 0.4, 0.5, and 0.6 mg/mL) and 1.8 mL of PBS. A₀ was the blank group, each containing 0.6 mL of PBS and 1.8 mL of H₂O₂. For each preparation group, 0.2 mg/mL was selected and the same operation was performed as above. All the solutions were mixed, and reacted for 10 min, and then the absorbance was measured at 230 nm to calculate the H₂O₂ scavenging rate and IC₅₀. The calculation formula was the same as formula (5).

Determination of Inhibitory Ability of SIN and its Preparations on Malondialdehyde (MDA) Production in Mouse Organs

The principle of the method is that TBA can combine with MDA to produce a strong red fluorescence. ²⁵ This method is widely used for the determination of MDA content. ²⁶ The mouse was put to death after the anesthesia, the livers and kidneys were taken out quickly, rinsed repeatedly to clean in 4 °C physiological salines, and weighed after removing surface water. Physiological saline was added (the ratio of organ mass to physiological saline volume was 1:9) to the weighed organs, and homogenized with a high-speed homogenizer (FSH-2A, Changzhou Putian Experimental Instrument Factory, China). The homogenized mixture was centrifuged (TG16-W, Hunan Xiangli Experimental Instrument Factory, China) at 4000 r/min for 15 min, and the supernatant was taken to obtain a 10% liver (or kidney) homogenate.

One mL of the 10% homogenate of the liver (or kidney) was added to test tubes and divided into a sample group, a model group, and a blank group. One hundred μL of a 0.85 mg/mL sample solution was added to the sample group and 100 μL of physiological saline to the blank and model groups. After mixing well, and being allowed to stand for 5 min, 100 μL of FeSO₄·7H₂O (10 mmol/L) was added to the sample and model groups, and 100 μL physiological saline to the blank group. After mixing, each group was incubated in a 37 °C water bath (HH-6, Changzhou Putian Experimental Instrument Factory, China) for 1.5 h. Three mL of TBA working solution (0.375% TBA solution and 5.6% TCA solution were mixed in a ratio of 2:1) was added and mixed well. The mixtures were then placed in a water bath at 95 °C for 40 min, cooled with running water, centrifuged at 4000 r/min for 8 min, and the supernatant was used for the determination. The ultraviolet spectrophotometer was adjusted to zero with physiological saline, the absorbance measured at 532 nm, and the inhibition rate of SIN, SIN-L, SIN-H, and SIN-L-H on MDA production was calculated by formula (6), in which “A_m” is the value of SIN, SIN-L, SIN-H, and SIN-L-H to the scavenging rate of MDA, “A_b” is the value of PBS, B-L, B-H, and B-L-H to the scavenging rate of MDA, “A_model” is the organ homogenate and FeSO₄·7H₂O, and “A_blank” is the value of organ homogenate and physiological saline.

$$\mathrm{E}\text{\%}={\displaystyle \frac{{\mathrm{A}}_{\mathrm{m}}-{\mathrm{A}}_{\mathrm{b}}}{{\mathrm{A}}_{\mathrm{model}}-{\mathrm{A}}_{\mathrm{blank}}}}$$

(6)

Statistical Analysis

All data were expressed as mean  ±  standard deviation. Differences in the experiments were considered statistically significant using the least significant differences at P < .05 and analyzed using the one-way analysis of variance test with SPSS 26.0 software.

Results

Basic Characteristics of SIN, SIN-L, SIN-H, and SIN-L-H

SIN solution was observed as a colorless and clear solution. “C” was used to perform linear regression on “A” to obtain the regression equation as A  =  0.0132 × C  +  0.0005 (R²  =  0.9995). According to the correlation coefficient R², SIN had a good linear relationship in the concentration range of 10 to 50 μg/mL. The accuracy and precision also met the requirements.

SIN-L was a viscous liquid with a certain consistency and a slight bean flavor. Observation under cryo-EM showed that SIN-L has a regular spherical structure, and there were obvious shadows in the sphere due to drug loading, with an EE% of 68.2  ±  7.6% and LE% of 3.25  ±  0.36%. The particle sizes of B-L and SIN-L were 121.6  ±  10.8 nm and 135.7  ±  11.4 nm, respectively. Zeta-potentials were all negative (−39.9  ±  3.1 mV and −38.5  ±  3.2 mV).

SIN-H was a transparent colorless liquid preparation with a certain viscosity, and SIN-L-H was an emulsion preparation with less fluidity than SIN-L, which was a milky white viscous fluid. Under the cryo-EM, it was found that the drug molecules in SIN-H and the spherical liposomes in SIN-H were uniformly dispersed. The viscosities of SIN-H and SIN-L-H were about 11.4615 and 12.6679 mPa·s after 5 dilutions.

Before determining the drug loading in SIN-H and SIN-L-H, the recovery rate of both formulations was determined; the recovery rate of both formulations was above 99.5%. The results of loading determination showed that the average contents of SIN-H and SIN-L-H samples were 1.99  ±  0.08 mg/mL and 1.98  ±  0.01 mg/mL, respectively, which met the quality requirements.

Release Tests of SIN and its Preparation In vitro

The release curves of SIN, SIN-L, SIN-H, and SIN-L-H In vitro are shown in Figure 3A. It can be seen that the samples from the upper cell gradually penetrate the lower cell with an increase in the release time. Within the first 6 h, the release rate of SIN was greater than that of SIN-L, SIN-H, and SIN-L-H. After 6 h, the release rate of SIN from SIN-H with a hydrophilic porous structure began to exceed that of the SIN group, which indicated that the hydrophilic structure of the hydrogels could make SIN penetrate across the dialysis membrane and enter the lower cell. With the increase of time, the release rate of SIN did not fluctuate greatly, and the release curve reached a stable state of about 68%. SIN-H and SIN-L reached the platform level at about 9 h and 24 h. It took a long time for the SIN-L-H to release quantities of SIN and reach the same platform level, which may be due to SIN needing to pass through the 2 barriers of phospholipid membrane and HA hydrogels with three-dimensional reticular structure.

Figure 3.

The release curves of SIN, SIN-L, SIN-H, and SIN-L-H across dialysis membrane In vitro and the mouse skin Ex vivo. (A) The release curves across the dialysis membrane of SIN, SIN-L, SIN-H, and SIN-L-H In vitro. (B) The release curves across the mouse skin of SIN, SIN-L, SIN-H, and SIN-L-H Ex vivo. (C) In vitro cumulative release rate curve fitted Weibull CDF model results. (D) Ex vivo cumulative release rate curve fitted Weibull CDF model results.

Release Tests Ex vivo of SIN and its Preparation

The release curves of SIN, SIN-L, SIN-H, and SIN-L-H indicated the level of penetration into mouse skin Ex vivo. As can be seen in Figure 3B, the amount of SIN passing through the mouse skin increased gradually with the increase in diffusion time. Because the liposomes contained phospholipids similar to those of the skin and had wide contact with the skin, they penetrated the skin the fastest. At 84 h, the permeability rate of SIN-L reached about 50%. SIN-L-H contained phospholipids and HA hydrogels similar to those of the extracellular matrix. The hydrogels have a three-dimensional structure that tightly binds the liposomes in its net, resulting in a lower skin rate absorption than SIN-L. SIN solution had poor compatibility with the skin. SIN was in the free state, so the amount and speed of entering the skin were small. The release rate from the SIN-H group was a little higher than that of the SIN group under the help of HA.

It was found that the difference in transdermal velocity of drugs in each group was obvious and their release was irregular. Mouse skin is thicker and more complex than that of the dialysis membrane, so drugs do not easily penetrate the skin into the diffusion fluid. In general, the rate of drug penetration through the skin was lower than that of the dialysis membrane. The amount of SIN through the dialysis membrane was about 3 times that through the mouse skin, and the amount of SIN-L-H through the dialysis membrane was about 1.5 times that of the skin. Considering the principle of similar compatibility between liposomes and cell membranes, we believe that drug-loaded liposomes are more easily deposited in the skin.

SIN, SIN-L, SIN-H, and SIN-L-H Release Test

We used Origin software to fit the cumulative release curve through zero-order, first-order, Higuchi, Hixson-Crowell, and Weibull CDF models. The Weibull CDF model (Figure 3C and D) had a good fit in the release curves (Tables 1 and 2). The SIN in SIN-L, SIN-H, and SIN-L-H had a good sustained-release effect.

Table 1.

Parameters for Fitting Curve Equations (Weibull CDF Model) In vitro and Their Correlation Coefficients.

Group	Fitting equation	R
SIN	$\mathrm{Q}=1.4873+68.1876\left(1-{\mathrm{e}}^{-{\left(\frac{\mathrm{t}}{120.4652}\right)}^{1.1949}}\right)$	0.99879
SIN-L	$\mathrm{Q}=-0.3587+70.9070\left(1-{\mathrm{e}}^{-{\left(\frac{\mathrm{t}}{270.5243}\right)}^{0.9614}}\right)$	0.99959
SIN-H	$\mathrm{Q}=0.9216+80.9263\left(1-{\mathrm{e}}^{-{\left(\frac{\mathrm{t}}{275.3529}\right)}^{1.0303}}\right)$	0.99957
SIN-L-H	$\mathrm{Q}=-2.5074+72.3691\left(1-{\mathrm{e}}^{-{\left(\frac{\mathrm{t}}{921.9931}\right)}^{0.6898}}\right)$	0.99903

Abbreviations: SIN, sinomenine; HA, hyaluronic acid; SIN-L-H, sinomenine-loaded liposomes-in-hydrogels; SIN-L, sinomenine-loaded liposomes.

Table 2.

Parameters for Fitting Curve Equations (Weibull CDF Model) Ex vivo and Their Correlation Coefficients.

Group	Fitting equation	R
SIN	$\mathrm{Q}=-2.9895+407.814\left(1-{\mathrm{e}}^{-{\left(\frac{\mathrm{t}}{1.1823}\right)}^{0.3097}}\right)$	0.99484
SIN-L	$\mathrm{Q}=1.8405+46.5235\left(1-{\mathrm{e}}^{-{\left(\frac{\mathrm{t}}{636.9335}\right)}^{0.8005}}\right)$	0.9843
SIN-H	$\mathrm{Q}=2.4867+30.3974\left(1-{\mathrm{e}}^{-{\left(\frac{\mathrm{t}}{633.0667}\right)}^{1.3759}}\right)$	0.99065
SIN-L-H	$\mathrm{Q}=2.3028+39.5569\left(1-{\mathrm{e}}^{-{\left(\frac{\mathrm{t}}{485.8491}\right)}^{0.8692}}\right)$	0.98225

Abbreviations: SIN, sinomenine; HA, hyaluronic acid; SIN-L-H, sinomenine-loaded liposomes-in-hydrogels; SIN-L, sinomenine-loaded liposomes.

Scavenging Rate of SIN and its Preparations on DPPH Radicals

The scavenging ability of SIN on DPPH radicals increased with the increase in concentration (Figure 4A), which indicates that SIN has a strong antioxidant capacity. The regression equation obtained of logE  =  1.76 × C−0.448 (R²  =  0.9918) showed that SIN concentration was linearly related to the logarithm of radicals scavenging in the range of 0.025 to 0.125 mg/mL. According to the regression equation, the IC₅₀ of SIN is about 0.084 mg/mL.

Figure 4.

The scavenging rate of different concentrations of SIN and its preparation on DPPH radicals. (A) The scavenging rate of different concentrations of SIN on DPPH radicals. The color changes of DPPH radicals scavenging by SIN solution with different concentrations are as follows from left to right: blank, 0.025, 0.05, 0.075, 0.1, and 0.125 mg/mL. (B) The scavenging rate of DPPH radicals by the same concentration of SIN, SIN-L, SIN-H, and SIN-L-H. The color changes of DPPH radicals scavenging by 3 preparations are as follows from left to right, from left to right: blank, SIN, SIN-L, SIN-H, and SIN-L-H. One-way ANOVA was used to determine statistical differences (*P < .05, **P < .01, ***P < .001).

As could be seen from Figure 4B, when the concentration was 0.025 mg/mL, SIN-L, SIN-H, and SIN-L-H had lower scavenging rates on DPPH radicals than SIN, which indicated that all 3 preparations slow down the radical scavenging rate of SIN due to the sustained-release effect. The color of the SIN-L, SIN-H, and SIN-L-H groups changed from purple to yellow, which indicated that all 3 preparations had good antioxidant activity. In the solution, the diffusion rate of SIN was the fastest, the contact area with DPPH radicals was large, and the scavenging rate of DPPH radicals was the highest. SIN-L-H contained 2 barriers, liposomes, and hydrogels. In addition, its contact time and area with DPPH radicals were less, so its scavenging rate of DPPH radicals was the lowest. The phospholipids in SIN-L have some antioxidant capacity, and the interaction between phospholipids and DPPH radicals will change the structure of phospholipids, causing damage to the liposome's structure and releasing SIN to act with the remaining DPPH. In SIN-H, SIN was wrapped in hydrogel structures, there was a smaller barrier than in SIN-L-H, and the scavenging rate of DPPH radicals was faster than that of SIN-L-H. At the same time, the scavenging rate of phospholipids on DPPH radicals was better than HA, so the scavenging rate of SIN-H for DPPH radicals was slower than that of SIN-L. Therefore, the order of scavenging rate (E) of DPPH radicals by SIN and 3 preparations is E_SIN > E_SIN-L > E_SIN-H >E_SIN-L-H.

Scavenging Rate of SIN and its Preparations on H₂O₂

Using different SIN concentrations (C) to perform linear regression on the scavenging rate (E%) of H₂O₂, the regression equation E% = 168.18 × C−13.428 (R² = 0.9912) was obtained. The experiment showed that SIN concentration in the range of 0.2 to 0.6 mg/mL was linearly correlated with the H₂O₂ scavenging rate (Figure 5A). According to the regression equation, the IC₅₀ of SIN on H₂O₂ was 0.3771 mg/mL. Figure 5B shows the scavenging rate of SIN and preparations of each group on H₂O₂ after deducting the blank group. From the data in the figure, we can see that when the concentration was 0.02 mg/mL, the scavenging rate on H₂O₂ by the 3 groups of preparations was approximately the same, and all of them were lower than SIN, which indicated that SIN-L, SIN-H, and SIN-L-H can release the drug slowly.

Figure 5.

Scavenging rate of SIN and its preparations on the H₂O₂. (A) Scavenging rate of different concentrations of SIN on H₂O₂. (B) Scavenging rate of 0.02 mg/mL SIN, SIN-L, SIN-H, and SIN-L-H on H₂O₂. One-way ANOVA was used to determine statistical differences (*P < .05, **P < .01, ***P < .001).

Determination of Inhibitory Ability of SIN and its Preparations on MDA Production in Mouse Organs

When the drug concentration was 1.65 mg/mL, SIN and its 3 preparations inhibited MDA production in organ homogenates (Figure 6), which shows that SIN, SIN-L, SIN-H, and SIN-L-H have good antioxidant properties. By comparing the difference in inhibition ability of SIN and its preparations on MDA production in liver and kidney homogenates (removing the inhibition ability of blank preparations corresponding to each preparation), it was found that SIN and its preparations had an inhibition effect on MDA in liver and kidney homogenates, but they were more sensitive to MDA in the liver, and the inhibition rate was higher. SIN inhibited MDA in liver homogenates about 2 times as much as SIN-L and about 3 times as much as SIN-H and SIN-L-H. Compared with SIN, the effects of 3 preparations on MDA in liver homogenates were significantly different (**P ＜ .01). Compared with SIN, SIN-L, SIN-H, and SIN-L-H had lower inhibition rates on MDA in kidney homogenates, but there were also significant differences (**P ＜ .01). In short, the experimental results showed that the antioxidant activity of SIN was extended in the preparation.

Figure 6.

Inhibitory effect of SIN and its 3 preparations on malondialdehyde (MDA) produced by mouse liver and kidney. One-way ANOVA was used to determine statistical differences (*P < .05, **P < .01, ***P < .001).

Discussion

As a biodegradable material, liposomes are de-formable, flexible, and controllable in size. ²⁷ Phospholipids are biodegradable and biocompatible, so the lipid bilayers in liposomes have good compatibility with the cell membrane. ²⁸ Among the new pharmaceutical technologies, liposomes are preferred because of the way they bind to cells. In this experiment, SIN-L prepared by a thin-film dispersion method loaded SIN in a lipid bilayer, so that SIN could penetrate the skin into the body better with improved bioavailability. The particle size of SIN-L was 135.7  ±  11.4 nm, slightly larger than that of the B-L group. The charge on the surface of SIN-L makes liposomes disperse more evenly in the system, preventing aggregation and flocculation and thus forming a more stable state.

Hydrogels have high permeability and hydration, because they have a three-dimensional network structure, and can absorb up to 90% of water, providing an environment similar to the extracellular matrix for drugs. ²⁹ Its loose and porous structure can maintain optimal drug concentration, improve the stability and adhesion of drugs, and achieve the effect of sustained release of drugs. ³⁰ In HA hydrogels, the drugs are evenly distributed in the network through an intermolecular force bond with HA, so HA hydrogels are an ideal drug carrier for external use. ³¹ SIN-L-H has a milky white texture with a certain viscosity, which can quickly moisturize the skin surface when it comes in contact with the skin. The adhesion of hydrogels improves the poor adhesion of liposome suspension on the skin surface, increases the retention time of drugs on the skin surface, and improves the bioavailability of drugs. ³² The cumulative release curves of drugs in the dialysis membrane diffusion experiment In vitro and transdermal diffusion experiment Ex vivo showed that SIN encapsulated in SIN-L-H had sustained-release characteristics. The curve fitting showed that the release characteristics of SIN in SIN-L, SIN-H, and SIN-L-H were in accordance with the Weibull CDF model. The presence of a hydrogel matrix makes the distribution of liposomes more uniform and prevents them from aggregating and fusing, which results in a more uniform distribution of SIN and a slower release on the basis of improved SIN stability.

In the metabolism process, mitochondria, endoplasmic reticulum, and various enzymes will all produce a large amount of ROS. ³³ Daily skin contact with ultraviolet light-B can also promote the increase of cell oxidation reaction and the production of ROS in the cell microenvironment. ³⁴ Studies have shown that the inflammatory process will produce a large number of ROS radicals. When the production of ROS radicals exceeds the endogenous scavenging capacity of cellular antioxidants, the body will enter into an oxidative stress state. ³⁵ Oxidative stress reflects the disorder in the balance of oxygen-promoting/anti-oxidant reactions in the body, which ultimately need to be adjusted through redox mechanisms. ³⁶ The presence of ROS can oxidize macromolecules such as phospholipids, enzymes, and membrane receptors on the surface of the cell membrane, resulting in the formation of peroxides such as MDA and 4-hydroxynonenal. So, the content of these aldehydes is often used to judge the degree of lipid peroxidation.^37,38 Aldehydes, as electrophilic substances, can directly react with DNA bases or generate DNA adducts to damage DNA, causing irreversible damage to the organism. In addition, the high level of ROS in the body promotes the release of various pro-inflammatory cytokines, which leads to some diseases, such as acute lung injury, diabetes, and diabetes wounds.^39–41 There are systems for scavenging ROS in the organism, such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px). These enzymes will neutralize or remove active substances or interrupt the chain reaction to resist oxidative stress. ⁴² However, the increase in ROS production is accompanied by a decrease in antioxidant production, which further leads to the decrease in ROS scavenging ability and the increase in oxidative stress. Therefore, using antioxidants to inhibit the production of ROS can reduce the development of diseases from the source. In vivo, SIN can increase the levels of GSH-Px and SOD, reduce the production of MDA, enhance the resistance to oxidative stress and the defense function of cell membranes.^43,44

According to the experimental results, SIN had a good scavenging rate on DPPH radicals in the concentration range of 0.025 to 0.125 mg/mL, and with an IC₅₀ of 0.084 mg/mL; 0.2 to 0.6 mg/mL of SIN had a good scavenging rate for H₂O₂, and the IC₅₀ was 0.3771 mg/mL. The ability of antioxidants to scavenge radicals depends on the ability of hydroxyl groups to provide hydrogen ions (H⁺). H⁺ provided by antioxidants can combine with the radicals produced by oxidation to stop or interrupt the chain reaction, thus preventing the continuation of the oxidation process. The more H  +  the antioxidants provide, the stronger its antioxidant activity. ⁴⁵ The C-4 of SIN is connected to the hydroxyl group, and the hydroxyl group, as an electron donor, can interact with radicals in the body to prevent oxidation. Compounds such as VC (IC₅₀  =  21 μg/mL ⁴⁶ ) and resveratrol (IC₅₀  =  12.8 μg/mL ⁴⁷ ) have more hydroxy groups, so they have stronger antioxidant activity than SIN. Natural phospholipids, such as soybean lecithin, can produce certain antioxidant effects under proper conditions. ⁴⁸ HA, as a macromolecular polysaccharide, also has the antioxidant capacity and has a role in scavenging ROS in vivo. ⁴⁹ According to our experimental results, SIN-L-H had a good scavenging ability on DPPH and H₂O₂ In vitro, and it also had an obvious inhibitory effect on MDA in organs. Therefore, in the treatment of oxidative stress, the combination of SIN, phospholipids, and HA will be an excellent choice, and SIN-L-H might be a good antioxidant for the clinic in the future.

Conclusion

As a natural antioxidant, SIN can scavenge ROS radicals and inhibit peroxide damage. It was shown that SIN-L, SIN-H, and SIN-L-H have significant slow-release effects. SIN and its preparations could scavenge DPPH radicals and H₂O₂ In vitro, and also had a certain scavenging effect on MDA in isolated organ homogenate, which indicated that they all have a good antioxidant effect. The pharmacodynamics and mechanism of action need further study.