A response surface methodology to optimize the biocompatible vesicle loading of sinomenine hydrochloride

Abstract

The present invention discloses a method for optimizing the encapsulation of sinomenine hydrochloride (SH) in biocompatible vesicles using response surface analysis. The present invention uses SH as a model drug and screens out the delivery carrier with the best SH encapsulation rate: arginine decanoate vesicles [Arg][Dec]. Then, CCD response surface design is used to optimize the factors affecting the encapsulation of SH in [Arg][Dec] vesicles. The optimized [Arg][Dec] vesicle encapsulation rate is increased to 83.5%, and the centrifugal stability is increased to 90.4%. At the same time, the cumulative release rate of SH in [Arg][Dec] vesicles reaches more than 95% after 17 hours, and the cumulative skin penetration in [Arg][Dec] vesicles can reach 1665.59 μg / cm after 48 hours. 2 , with excellent in vitro skin penetration. This invention not only significantly improves the SH release rate and transdermal delivery effect, but also effectively saves experimental costs. It has potential application prospects in the treatment of local inflammation.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery, specifically relating to a method for encapsulating sinomenine hydrochloride in biocompatible vesicles. Background Technology

[0002] Sinomenine is an alkaloid extracted from the medicinal plant *Sinomenium acutum*. For centuries, it has been used in traditional Chinese medicine to treat rheumatoid arthritis. Modern pharmacological studies have shown that sinomenine hydrochloride (SH), a hydrochloride salt form of sinomenine, possesses a wide range of pharmacological effects, particularly anti-inflammatory, anti-immune, and anti-angiogenic effects. Therefore, SH is a promising and valuable drug. However, SH has low oral bioavailability (3-4%) and adverse effects on the digestive, blood, circulatory, and nervous systems. Furthermore, SH has a short half-life in the human body (0.791 hours), requiring frequent dosing and significantly reducing patient compliance.

[0003] Transdermal drug delivery systems (TDDS) are drug formulations that allow drugs to penetrate the skin at a controlled rate, be absorbed into the systemic circulation via capillaries, or be distributed in subcutaneous tissues to induce therapeutic effects. Compared to oral formulations, TDDS avoid gastrointestinal digestive enzyme metabolism and the first-pass effect of the liver. This minimizes the risk of pain and infection, significantly improving patient compliance. Despite these advantages, TDDS have not yet fully realized their potential, with the biggest challenge being overcoming the skin barrier effect. The stratum corneum (SC), the outermost layer of the skin, is the primary barrier to drug penetration. This route of administration offers convenient and painless delivery, sustained drug release, and reduced side effects, making it a hot topic in drug research in recent years. Extensive research focuses on using chemical penetration enhancers to circumvent the SC and improve transdermal penetration; these substances interact with the SC and induce increased skin permeability.

[0004] Certain fatty acids, such as oleic acid, enhance permeability because they disrupt the lamellar structure of the stratum corneum by inducing disorder and fluidization or lipid phase separation of natural lipids in the skin, thereby reducing the skin barrier function and allowing drugs to penetrate the skin layers more effectively. Oleic acid plays a crucial role in nanoparticle drug delivery systems, acting as a permeability enhancer and serving as a nanocarrier for transdermal drug delivery. It has been reported that oleic acid has successfully improved the transdermal therapeutic effects of various drugs, including celecoxib, flurbiprofen, piperine, and zinc phthalocyanine, for the treatment of various skin diseases. Therefore, fatty acids and arginine were selected to prepare vesicles for transdermal delivery of SH (skin dermal lesions). Summary of the Invention

[0005] To address the issues of low oral bioavailability of SH and skin barrier effects during transdermal delivery, this invention provides biocompatible vesicles encapsulating SH using fatty acid vesicles as delivery carriers and their preparation method.

[0006] The method for preparing SH-loaded biocompatible vesicles using a single-factor method provided by this invention includes the following steps:

[0007] (1) Preparation of [Arg][Ole] vesicles and [Arg][Dec] vesicles: fatty acids (oleic acid or decanoic acid) are added to an aqueous solution of arginine, and the solution is sonicated and magnetically stirred at room temperature until a homogeneous aqueous system is formed to obtain an aqueous solution of vesicles;

[0008] (2) Mix the SH solid with the vesicle aqueous solution described in step (1) and perform vortexing and ultrasonic treatment in sequence to obtain biocompatible vesicles with high SH encapsulation rate and centrifugal stability.

[0009] Furthermore, in step (1), the concentration of the arginine aqueous solution is 100 mM.

[0010] Further, in step (1), the fatty acid is decanoic acid; in the aqueous homogeneous system, the concentration of arginine is 100 mM, and the concentration of decanoic acid is 150-350 mM.

[0011] Further, in step (1), the fatty acid is oleic acid; in the aqueous homogeneous system, the concentration of arginine is 100 mM, and the concentration of oleic acid is 50-250 mM.

[0012] Furthermore, in step (1), the ultrasonic time is 40 min and the magnetic stirring time is 20 min.

[0013] Furthermore, for the aqueous solution of vesicles formed by decanoic acid, in step (2), the concentration of SH is 5-45 mg / mL; the vortexing time is 0-40 min; and the ultrasonic treatment time is 0-40 min.

[0014] Preferably, the optimal preparation conditions for preparing loaded [Arg][Dec] vesicles are: arginine concentration of 100 mM, decanoic acid concentration of 250 mM, SH concentration of 5 mg / mL, vortexing time of 20 min, and sonication time of 20 min.

[0015] Furthermore, for the aqueous solution of vesicles formed by oleic acid, in step (2), the concentration of SH is 5-45 mg / mL; the vortexing time is 0-40 min; and the ultrasonic treatment time is 0-40 min.

[0016] Preferably, the optimal EE and CS conditions for preparing SH-loaded [Arg][Ole] vesicle aqueous solution are: arginine concentration of 100 mM, oleic acid concentration of 150 mM, SH concentration of 5 mg / mL, vortexing time of 10 min, and sonication time of 20 min.

[0017] The single-factor experiments revealed that, due to the high EE and CS of SH in [Arg][Ole] vesicles, only the concentration of oleic acid significantly affected EE and CS. Conversely, for [Arg][Dec] vesicles, multiple variables were at play. Therefore, further optimization experiments were conducted using CCD response surfaces to optimize the factors influencing the encapsulation of SH in [Arg][Dec] vesicles.

[0018] This invention optimizes the encapsulation efficiency and centrifugal stability of [Arg][Dec] vesicles for SH: Based on single-factor experiments, a CCD experimental design is used to optimize the process by considering four factors: decanoic acid concentration, SH concentration, sonication time, and vortexing time. The extreme values ​​(high and low) for each factor are +1.682 and -1.682, respectively, and bidirectional interaction is provided by setting each factor at four levels.

[0019] The encapsulation efficiency (EE) determination method used in this invention is as follows: 10 μL of [Arg][Dec] vesicle solution loaded with SH is taken, the vesicles are destroyed with methanol, and the solution is diluted to 1 mL. The total SH content in the sample is detected by HPLC. Separately, 200 μL of [Arg][Dec] vesicle solution loaded with SH is added to an ultrafiltration centrifuge tube and centrifuged. After centrifugation, 10 μL of the filtrate is collected and diluted to 100 μL with methanol. The free SH content in the sample is analyzed by HPLC.

[0020] Furthermore, the conditions for ultrafiltration centrifugation are: temperature: 277K, rotation speed: 13000rpm, centrifugation time: 90min.

[0021] The centrifugal stability (CS) determination method used in this invention involves placing the solution containing SH-loaded [Arg][Dec] vesicles in a centrifuge tube and centrifuging it. The SH content before and after centrifugation is determined by HPLC.

[0022] Furthermore, the centrifugation conditions for centrifugation stability are: temperature: 298K, rotation speed: 4000rpm, and centrifugation time: 20min.

[0023] The optimal preparation conditions for SH-loaded arginine-decanoic acid vesicles obtained by the above response surface methodology are: arginine concentration of 100 mM, decanoic acid concentration of 234 mM, sinomenine hydrochloride concentration of 18 mg / mL, vortexing time of 20 min, and sonication time of 20 min.

[0024] The biocompatible vesicles loaded with SH prepared by the above method are also within the scope of protection of this invention.

[0025] This invention also protects a transdermal drug delivery formulation of sinomenine hydrochloride.

[0026] The transdermal drug delivery formulation of sinomenine hydrochloride provided by the present invention comprises the biocompatible vesicles of sinomenine hydrochloride loaded as described above.

[0027] The present invention has the following beneficial effects:

[0028] (1) The raw materials used in this invention are fatty acids and arginine, which have the advantages of readily available raw materials and low cost.

[0029] (2) In this invention, after CCD optimization, the encapsulation efficiency of decanoic acid-arginine loaded with sinomenine hydrochloride was increased to 83.5%, the centrifugal stability was increased to 90.4%, and the release time of SH was extended to 17h.

[0030] (3) In this invention, the cumulative skin penetration of the optimized decanoic acid-arginine can reach 1665.59 μg / cm³ after 48 hours. 2 It has excellent in vitro skin penetration effect, which can provide support and ideas for the transdermal delivery of sinomenine hydrochloride, and has certain technical reference value and application promotion value. Attached Figure Description

[0031] Figure 1 The images show Cryo-TEM patterns and physical images of two types of vesicles; where AB: Cryo-TEM image of aqueous solution with a molar ratio of [Arg]:[Ole] = 100:250; CD: Cryo-TEM image of aqueous solution with a molar ratio of [Arg]:[Dec] = 100:250; EF: Cryo-TEM images of [Arg][Ole] and [Arg][Dec] at different concentrations.

[0032] Figure 2 The graph shows the effect of different fatty acid concentrations on the encapsulation of SH by two types of vesicles and their influence on centrifugal stability; where A: [Arg][Ole] concentration, B: [Arg][Dec] concentration.

[0033] Figure 3 EE and CS plots of SH encapsulation in [Arg][Ole] vesicles and [Arg][Dec] vesicles with different SH concentrations; where A: SH concentration in [Arg][Ole] and B: SH concentration in [Arg][Dec].

[0034] Figure 4 The EE and CS plots show the effects of different vortex times on the encapsulation of two types of SH vesicles; where A: vortex time for [Arg][Ole] and B: vortex time for [Arg][Dec].

[0035] Figure 5The EE and CS plots show the effects of different centrifugation times on the encapsulation of two types of vesicles (SH). A: sonication time for [Arg][Ole], B: sonication time for [Arg][Dec].

[0036] Figure 6 The response surface and contour plot for the [Arg][Dec] vesicle encapsulation efficiency.

[0037] Figure 7 This is a graph showing the cumulative release rate of SH in the [Arg][Ole] and [Arg][Dec] vesicles.

[0038] Figure 8 This is a cumulative permeation curve of SH for two types of vesicles, [Arg][Ole] and [Arg][Dec].

[0039] Figure 9 Cytotoxicity of two types of vesicles: [Arg][Ole] and [Arg][Dec]. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] Example 1

[0043] Preparation of oleic acid arginine [Arg][Ole] vesicles and decanoic acid arginine [Arg][Dec] vesicles

[0044] Add different proportions of oleic acid (to a final concentration of 50 mM–250 mM) or decanoic acid (to a final concentration of 150 mM–350 mM) to a 100 mM aqueous solution of arginine, sonicate for 40 min, and magnetically stir at room temperature for 20 min until a homogeneous liquid is formed (see [link to article]). Figure 1 ).

[0045] Example 2

[0046] Different concentrations of SH were added to two types of vesicles (oleic arginine [Arg][Ole] and decanoic arginine [Arg][Dec]) prepared in Example 1, and the vesicles were subjected to vortexing and sonication for different durations to obtain SH-loaded vesicles.

[0047] Single-factor experiment

[0048] 1. Effects of fatty acid concentration on EE and CS of SH encapsulation by [Arg][Ole] vesicles and [Arg][Dec] vesicles.

[0049] SH was loaded into two types of vesicles at different concentrations ([Arg][Dec] vesicles with decanoic acid concentrations of 100, 125, 150, 175, and 200 mM, respectively, or [Arg][Ole] vesicles with oleic acid concentrations of 50, 75, 100, 125, and 150 mM, respectively). The specific method is as follows: A certain amount of solid SH was added to aqueous solutions of vesicles of different concentrations to achieve an SH concentration of 5 mg / mL. The solutions were then subjected to vortexing for 3 min and sonication for 10 min to obtain SH-loaded vesicle solutions. 10 μL of the SH-loaded vesicle solution was diluted to 1 mL with methanol to break down the vesicles, and the total SH content (W) in the sample was determined by HPLC. t ). Take another 200 μL of the SH-loaded vesicle solution and add it to an ultrafiltration centrifuge tube. Centrifuge at 277 K and 13000 rpm for 90 min. After centrifugation, collect 10 μL of the filtrate and dilute it to 100 μL with methanol. Determine the content of unencapsulated SH in the sample (W) using HPLC. f ).

[0050] Encapsulation efficiency EE is calculated using the following formula: EE(%) = (W t -W f ) / W t ×100%

[0051] The solution containing SH was placed in a centrifuge tube and centrifuged at 298 K and 4000 rpm for 20 min. The SH (W) content before centrifugation was determined by HPLC. b ) content and SH content after centrifugation (W a Centrifugal stability is calculated using the following formula:

[0052] CS(%) = W a / W b ×100%.

[0053] Figure 2 The graph shows the effect of different fatty acid concentrations on the EE and CS of SH encapsulation in two types of vesicles. Figure 2It can be seen that for [Arg][Ole], increasing the oleic acid concentration slightly decreases the EE, but it remains above 90%. When the oleic acid concentration is 150 mM, the CS reaches an equilibrium value of approximately 96.48%. For [Arg][Dec], increasing the decanoic acid concentration leads to an initial increase in EE, followed by a minimum EE. When the decanoic acid concentration is 300 mM, the EE reaches its maximum value of 88.32%, and the CS initially shows a stable trend, followed by a decrease. When the decanoic acid concentration is 250 mM, both EE and CS remain at relatively high levels. Therefore, the optimal concentrations for adding oleic acid and decanoic acid to a 100 mM arginine aqueous solution are 150 mM and 250 mM, respectively.

[0054] 2. Effects of different SH concentrations on the EE and CS of [Arg][Ole] and [Arg][Dec] vesicles.

[0055] When the concentrations of SH solid in the two types of vesicles were 5 mg / mL, 15 mg / mL, 25 mg / mL, 35 mg / mL, and 45 mg / mL, respectively, and the concentration of oleic acid in the oleic acid-arginine [Arg][Ole] vesicles was 150 mM, and the concentration of decanoic acid in the decanoic acid-arginine [Arg][Dec] vesicles was 250 mM, the vesicles were subjected to vortexing for 3 min and sonication for 10 min to obtain SH-loaded vesicle solutions, and then EE and CS were measured.

[0056] Figure 3 The diagram shows the effect of different SH concentrations on the EE and CS of [Arg][Ole] and [Arg][Dec] vesicle encapsulation. Figure 3 It was found that increasing the SH concentration in [Arg][Ole] slightly decreased the EE, but still maintained it above 91.81% and the CS above 83.18%. Conversely, with increasing the SH concentration in [Arg][Dec] vesicles, the EE value decreased significantly, while the CS value first decreased and then increased. Therefore, the optimal SH concentration for both [Arg][Ole] and [Arg][Dec] is 5 mg / mL.

[0057] 3. The effect of vortex time on the EE and CS of [Arg][Ole] and [Arg][Dec] vesicle encapsulation of SH.

[0058] Two fatty acid vesicles of certain concentrations were prepared (oleic acid-arginine [Arg][Ole] vesicles with oleic acid concentration of 150 mM and arginine concentration of 100 mM; decanoic acid-arginine [Arg][Dec] vesicles with decanoic acid concentration of 250 mM and arginine concentration of 100 mM). A certain amount of SH solid was added to each vesicle (to a concentration of 15 mg / mL in the vesicle aqueous solution). The vesicles were subjected to vortexing for different durations (0 min, 10 min, 20 min, 30 min, 40 min) and ultrasonic treatment for 20 min to obtain SH-containing vesicles. Then, EE and CS were measured.

[0059] Figure 4 The graph shows the effect of different vortex times on the EE and CS of two types of vesicle encapsulation SH. Figure 4 It can be seen that for [Arg][Ole] vesicles, changing the vortex time results in both EE and CS around 90%. A vortex time of 10 min shows good effects on EE and CS, reaching 91.48% and 89.95%, respectively. For [Arg][Dec] vesicles, EE gradually decreases with increasing vortex time, while CS shows an initial increase followed by a decrease. Therefore, the optimal vortex times for SH in [Arg][Ole] and [Arg][Dec] are 10 min and 20 min, respectively.

[0060] 4. Effects of ultrasound time on EE and CS of [Arg][Ole] and [Arg][Dec] vesicle encapsulation of SH

[0061] Two fatty acid vesicles of certain concentrations were prepared (oleic acid-arginine [Arg][Ole] vesicles, with oleic acid concentration of 150 mM and arginine concentration of 100 mM; and decanoic acid-arginine [Arg][Dec] vesicles, with decanoic acid concentration of 250 mM and arginine concentration of 100 mM). A certain amount of SH solid was added to each vesicle (to a concentration of 5 mg / mL in the vesicle aqueous solution), and the vesicles were subjected to vortexing for 3 min and sonication for different times (0 min, 10 min, 20 min, 30 min, 40 min) to obtain SH-containing vesicles. Then, EE and CS were measured.

[0062] Figure 5 This image shows the effects of different ultrasound techniques on the electrical activity (EE) and scleresy (CS) of two types of vesicle-encapsulated SH. Figure 5 It can be seen that for [Arg][Ole] and [Arg][Dec] vesicles, changing the ultrasound time has little effect on EE, and the effect on CS shows an initial increase followed by a decrease, reaching a maximum value of 95.31% when the ultrasound time is 20 min. Therefore, the optimal ultrasound time for SH in both [Arg][Ole] and [Arg][Dec] vesicles is 20 min.

[0063] Through the above single-factor experiments, the optimal process conditions for EE and CS of SH-loaded [Arg][Ole] vesicles were determined to be: oleic acid concentration of 150 mM, SH concentration of 5 mg / mL, vortexing time of 10 min, and sonication time of 20 min. SH has a relatively high EE and CS in [Arg][Ole] vesicles, and only the concentration of oleic acid has a significant impact on EE and CS. Conversely, for [Arg][Dec] vesicles, multiple variables are at play. Therefore, further optimization experiments were conducted on the EE of [Arg][Dec] vesicles using CCD response surface, as shown in Example 3.

[0064] Example 3: Optimization of vesicle encapsulation process using CCD response surface methodology

[0065] Based on single-factor experiments, a four-factor, four-level response surface optimization experiment was conducted using the central composite design principle of CCD for four factors: decanoic acid concentration, SH concentration, vortex time, and ultrasonic time. The extreme values ​​(high and low) for each factor were +1.682 and -1.682, respectively. Only the maximum and minimum values ​​were required for each factor, and the system automatically set four levels to provide bidirectional interaction. Specific factors and levels are shown in Table 1.

[0066] Table 1. CCD Response Surface Factors and Levels

[0067]

[0068] Example 4: CCD Response Surface Experiment

[0069] The experimental design included 30 test points, as shown in Table 2 and the variance in Table 4.

[0070] Table 2. Experimental design and results of the four-factor CCD response surface methodology.

[0071]

[0072] Example 5: Establishment of CCD Response Surface Model

[0073] Analysis of variance was performed on the experiment using Design Expert 13.0.1.0 software.

[0074] Based on the variance analysis results of the regression models in Table 3, it was found that the p-values ​​for both response value models were less than 0.05, and the coefficient of determination R0 was... 2The coefficients of performance (COPs) were 0.9547 and 0.9916, respectively, indicating a good model fit. The lack-fit terms were both greater than 0.05, meaning the model's lack of fit was not significant, indicating that the model could describe the actual relationship between the dependent variable and the response values ​​well. Furthermore, the exact coefficients for both response values ​​were greater than 4, at 20.4566 and 46.9243, respectively. The coefficients of variation were both less than 10%, at 6.36% and 1.85%, respectively, indicating that the model was highly reliable. In short, the model has high reliability within the scope of this study and can be used to predict the EE and CS of [Arg][Dec]-encapsulated SH.

[0075] Based on the significance test results of the regression models shown in Table 4, both EE and CS exhibit quaternary regression models, indicating an interaction between the four factors and the response value. In the regression models, a positive coefficient of the dependent variable indicates a promoting effect on the response value, while a negative coefficient indicates an inhibiting effect. The absolute value of the coefficient represents the degree of influence on the response value; the larger the absolute value, the greater the influence. According to the analysis of variance results, the coefficients for A-decanoic acid concentration and B-SH concentration are negative in both models. The coefficients for the dependent variables C-vortex time and D-ultrasound time are positive in the EE model but negative in the CS model. The coefficients for A-decanoic acid concentration and B-SH concentration are relatively large in both models, indicating a significant impact on the response value.

[0076] Table 3. Significance Tests of the Regression Model

[0077]

[0078] Table 4. Analysis of Variance for Regression Models

[0079]

[0080]

[0081] Example 6: CCD Response Surface Analysis

[0082] To determine the effects of the four variables and their interactions on encapsulation efficiency and centrifugal stability, surface plots and contour plots of the response surface results were generated, such as... Figure 6 As shown.

[0083] Taking EE as an example, the response surface methodology was analyzed, such as... Figure 6 (As shown in A, B, C, D, E, F).

[0084] Figure 6A describes the effects of decanoic acid concentration and SH concentration on EE. With increasing SH concentration, EE decreased from 78.39% to 70.34%. Decanoic acid concentration had a dual effect on EE: when the decanoic acid concentration increased from 150 mM to 250 mM, EE increased from 78.42% to 86.88%; however, when the decanoic acid concentration further increased from 250 mM to 350 mM, EE decreased again to 66.80%. Figure 6 B describes the effects of decanoic acid concentration and vortexing time on EE. The results show that vortexing time has no significant effect on EE; when the vortexing time increases from 15 min to 25 min, the EE only increases from 75.31% to 77.85%. The effect of decanoic acid concentration on EE is related to... Figure 6 The concentration of decanoic acid in A has the same effect on EE. Figure 6 C describes the effects of decanoic acid concentration and sonication time on EE. EE increased with increasing sonication time, reaching 61.35% after 15 minutes of sonication and further increasing to 64.68% after 40 minutes. The effect of decanoic acid concentration on EE is compared with... Figure 6 The concentration of decanoic acid in A has the same effect on EE. Figure 6 The impact of the four variables (D, E, F) on EE, and... Figure 6 The situations described in (A, B, C) are consistent.

[0085] Taking EE as an example, the contour plot of the response surface projection was analyzed, such as... Figure 6 As shown in (a, b, c, d, e, f), the contour lines in the figure are all elliptical, indicating interactions between the variables. The interactions between variables BC and CD are relatively significant, while the interactions between variables AD and BD are relatively weak. This conclusion can also be drawn by comparing the absolute values ​​and p-values ​​of the interaction coefficients in the two model equations in Tables 3 and 4.

[0086] The smaller the distance between contour lines in a contour map, the greater the impact of that variable on EE. For example, Figure 6 In section a, using decanoic acid concentration and SH concentration as references, the differences between the 85-80% range of the EE contour lines were compared. It was found that when decanoic acid concentration was used as a reference, the difference between the two contour lines was smaller than when SH concentration was used as a reference. Therefore, decanoic acid concentration has a greater impact on EE. Comprehensive analysis of other contour maps revealed that A—decanoic acid concentration—has the greatest impact on EE, followed by B—SH concentration and D—ultrasound time, while C—vortex time—has the least impact.

[0087] Example 7: Optimization Experiment Verification

[0088] Response surface methodology analysis revealed the optimal encapsulation (EE) and saturation (CS) conditions for SH in [Arg][Dec] vesicles as follows: decanoic acid concentration of 234 mM, SH concentration of 18 mg / mL, vortexing time of 20 min, and sonication time of 20 min. The predicted average encapsulation efficiency of SH by decanoic acid-arginine was 87.95%, while the experimental value was 83.45%. The predicted centrifugal stability of SH after loading with decanoic acid-arginine was 92.23%, while the experimental value was 90.38%. The experimental and predicted values ​​for both response methods are very close, with an error of less than 5%, which is within the allowable error range. This indicates that the response surface methodology is reliable for optimizing SH encapsulation.

[0089] Example 8: In vitro release experiment

[0090] Fatty acid vesicles [Arg][Dec] and [Arg][Ole] with the best encapsulation efficiency and centrifugal stability were selected as research subjects (molar ratios of [Arg]:[Ole] = 100:150 and [Arg]:[Dec] = 100:234, respectively). 2 mL of sample was added to each dialysis bag and then immersed in a storage bottle containing 150 mL of phosphate buffer (pH = 7.45). The dialysis system was placed in an air-controlled shaker at 298 K and 120 rpm. At selected time intervals (3 h, 4 h, 6 h, 8 h, 10 h, 17 h), 1 mL of sample was aspirated and an equal volume of dialysis buffer was added. The aspirated samples were filtered through a membrane and analyzed by HPLC. Each sample was analyzed in triplicate.

[0091] Figure 7 This represents the cumulative release rate of SH in [Arg][Ole] and [Arg][Dec] vesicles. Figure 7 It can be seen that the release rate of SH in [Arg][Ole] and [Arg][Dec] vesicles is similar, but slightly lower than the release rate in aqueous solution.

[0092] Example 9: In vitro transdermal experiment

[0093] Abdominal skin from male Wistar rats (180-200g) was used. After hair removal, the entire skin layer was soaked in physiological saline, and subcutaneous fat and connective tissue were removed with cotton swabs. The skin was then thoroughly rinsed with physiological saline, and its integrity was checked. The excised skin was sandwiched between the upper and lower chambers, with the epidermis facing upwards. The lower chamber was filled with 0.9% physiological saline:ethanol = 80:20 (v / v) at 37℃ and magnetically stirred at 400 rpm. After equilibration for 30 min, the sample was uniformly injected into the upper chamber. At 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, 14h, 18h, 20h, 24h, 28h, 32h, 39h, and 48h, 200 μL of sample was taken from the lower chamber, and an equal volume of physiological saline:ethanol = 80:20 (v / v) was quickly added. The concentration of SH in the sample was determined by high performance liquid chromatography (HPLC). Each sample was repeated three times. Osmosis curves were plotted based on the cumulative amount (Qn) passing through a unit area of ​​skin over different time intervals. Relevant osmosis parameters are shown in Table 5.

[0094] Two types of vesicle samples were used in this experiment, the same as those used in Example 8.

[0095] Table 5. Correlation fitting parameters of different models for the transdermal behavior of sinomenine hydrochloride.

[0096]

[0097] Figure 8 This is a cumulative permeation curve of SH for the two types of vesicles. (From...) Figure 8 As shown in Table 5, the cumulative permeation of SH-carrying materials is [Arg][Dec] > [Arg][Ole].

[0098] Example 10, In vitro toxicity test

[0099] Count 500,000 cells in each well of a 96-well plate. Remove the culture medium, wash the wells twice with PBS, add 100 μL of medium containing 10% CCK-8 and 5% CO2, and incubate at 310 K for 1 hour. Measure the absorbance at 450 nm using a microplate reader and calculate the relative cell viability using the formula.

[0100] RCV = (As - Ab) / (Ac - Ab)

[0101] Where Ab is the absorbance of the multi-well plate itself (i.e., the blank well), As is the absorbance of the experimental group, and Ac is the absorbance of the control group.

[0102] Figure 9 Cytotoxicity of two vectors, [Arg][Dec] and [Arg][Ole]. (From...) Figure 9It was found that after 48 hours, the survival rates of HaCaT cell lines in [Arg][Dec] and [Arg][Ole] were 64.42% and 66.99%, respectively. Analysis of the experimental results showed that the toxicity of [Arg][Dec] was similar to that of [Arg][Ole], both exceeding 60%. Combined with the EE value, CS value, and cumulative SH penetration in the above examples, [Arg][Dec] is the optimal carrier for delivering SH.

[0103] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing sinomenine hydrochloride-loaded biocompatible vesicles using response surface methodology, comprising the following steps: (1) Add fatty acids to an aqueous solution of arginine, sonicate and magnetically stir at room temperature until the liquid forms a homogeneous aqueous system to obtain an aqueous vesicle solution; (2) Mix the solid sinomenine hydrochloride with the vesicle aqueous solution described in step (1) to obtain a mixture, and then subject the mixture to vortexing and sonication treatment in sequence to obtain a biocompatible vesicle solution loaded with sinomenine hydrochloride. In step (1), the fatty acid is decanoic acid, and the preparation conditions for preparing arginine-decanoic acid vesicles loaded with sinomenine hydrochloride are: arginine concentration of 100 mM, decanoic acid concentration of 234 mM, sinomenine hydrochloride concentration of 18 mg / mL, vortexing time of 20 min, and sonication time of 20 min.

2. The biocompatible vesicles loaded with sinomenine hydrochloride prepared by the method of claim 1.

3. A transdermal drug delivery formulation of sinomenine hydrochloride, comprising the biocompatible vesicles of sinomenine hydrochloride as described in claim 2.

Images