Lipid compositions, stock solutions, methods of preparation, systems of preparation, and applications for making broad-spectrum solvent-compatible liposomes

Abstract

This invention discloses a lipid composition, stock solution, preparation method, preparation system, and application for preparing broad-spectrum solvent-compatible liposomes. The lipid composition consists of HSPC, cholesterol, octadecylamine, and vitamin E polyethylene glycol succinate in specific proportions; it is dissolved in an aqueous organic solvent (such as ethanol) to form a lipid stock solution; the preparation system employs a three-channel intelligent control pump; in the preparation method, the above-mentioned lipid stock solution, an aqueous phase containing the target components (which can be pure water to 40% organic solvent-water solution), and a blank aqueous phase are injected into a microfluidic chip to generate a liposome dispersion, which is then purified. This invention overcomes the limitation of solvent polarity on liposome preparation, enabling the stable preparation of high-quality liposomes in a wide range of solvent systems. Experiments have shown that this liposome significantly enhances the stability and transdermal permeability of the encapsulated components, providing a universal, precise, and low-cost delivery platform for encapsulating various active ingredients.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical nanoparticles and microfluidic chips, and in particular to lipid compositions, stock solutions, preparation methods, preparation systems and applications for preparing broad-spectrum solvent-compatible liposomes. Background Technology

[0002] As a mature nanodelivery carrier, one of the core research objectives of liposomes is to solve two common problems in the application of various active ingredients (such as drugs and efficacy factors): low transdermal absorption efficiency and poor physicochemical stability. By encapsulating active ingredients in vesicles composed of a lipid bilayer, liposomes can effectively promote their penetration of the skin's stratum corneum barrier and improve bioavailability. At the same time, the lipid membrane can provide physical isolation between the encapsulated material and the external environment, significantly enhancing its stability to heat, light, oxygen, and hydrolysis, and extending its shelf life and application window.

[0003] However, the application efficiency of existing liposome preparation technologies is limited by a fundamental bottleneck: the compatibility of solvent systems. Traditional methods (such as membrane hydration) typically require the use of pure water or low-ionic-strength buffer solutions as hydration media. When the physicochemical properties of the target active ingredient (such as low water solubility or instability at a specific pH) require it to be dissolved or dispersed in an aqueous phase containing organic solvents (such as ethanol or propylene glycol), the introduced organic solvents severely damage the integrity of the lipid membrane, leading to liposome failure to form, extremely low encapsulation efficiency, or rapid product aggregation and precipitation. This greatly limits the universality of liposome technology in solving the aforementioned transdermal and stability problems, especially excluding many high-value active ingredients that require co-solvent solubilization or stabilization.

[0004] Microfluidic technology offers a novel approach to preparing uniform nanoparticles, but current research largely focuses on optimizing molding processes in specific two-phase (lipid-organic phase / pure aqueous phase) systems. The lipid formulations themselves lack tolerance to high proportions of organic solvents in the aqueous phase. Therefore, developing a universal lipid formulation with broad solvent compatibility, capable of adapting to preparation requirements ranging from pure water to aqueous phases containing organic solvents, and along with supporting precise preparation techniques, is of great significance for fully releasing the potential of liposomes in enhancing transdermal permeability and stability, and expanding their application to more diverse active ingredients. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing liposome preparation technologies in terms of poor compatibility with solvent systems, and to provide a lipid composition, stock solution, preparation method, preparation system and application for preparing broad-spectrum solvent-compatible liposomes. This enables the stable and efficient preparation of high-quality liposomes over a wide polarity range, from pure water to high-concentration organic solvent-water solutions (organic solvent content ≤40% v / v), providing a universal solution for encapsulating active ingredients that require special solvent treatment.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a lipid composition for preparing broad-spectrum solvent-compatible liposomes, comprising, by weight percentage: 50%-70% hydrogenated soybean phosphatidylcholine (HSPC), 20%-35% cholesterol, 5%-10% octadecylamine, and 1%-5% vitamin E polyethylene glycol succinate (TPGS). This specific combination imparts excellent stability to the liposomes in the presence of organic solvents.

[0008] Secondly, the present invention provides a lipid stock solution for preparing broad-spectrum solvent-compatible liposomes, obtained by dissolving the above-mentioned lipid composition in a water-soluble organic solvent.

[0009] Furthermore, the water-soluble organic solvent is anhydrous ethanol or isopropanol.

[0010] Furthermore, the concentration of the lipid composition in the solution is 8-12 mg / mL, preferably 10 mg / mL.

[0011] Thirdly, the present invention also provides a method for preparing broad-spectrum solvent-compatible liposomes, wherein the above-mentioned lipid stock solution, an aqueous solution containing the target active ingredient, and a blank aqueous solution are injected into a microfluidic chip for mixing to generate a liposome dispersion, which is then purified (e.g., by ultrafiltration replacement method, preferably by centrifugation with pure water three times for purification).

[0012] The aqueous solution containing the target active ingredient is pure water or an organic solvent-water mixture with an organic solvent volume fraction not exceeding 40%; the organic solvent is ethanol or isopropanol.

[0013] The target active ingredient is a substance that can dissolve in pure water or an organic solvent-water solution with an organic solvent volume fraction not exceeding 40%.

[0014] Furthermore, the lipid reserve solution, the aqueous solution containing the target active ingredient, and the blank aqueous solution are simultaneously injected into the microfluidic chip at a volumetric flow rate ratio of 1:3:7 for mixing.

[0015] Fourthly, the present invention also provides a preparation system for preparing broad-spectrum solvent-compatible liposomes, wherein the preparation system is an intelligent microfluidic system, which is applied in the above-mentioned preparation method, comprising:

[0016] Three-channel intelligent control pump;

[0017] Microfluidic chips;

[0018] Three independent fluid sources are connected to the three-channel intelligent control pump and the microfluidic chip, respectively; the three independent fluid sources are used to store: a) lipid stock solution; b) aqueous solution containing the target active ingredient; c) blank aqueous solution;

[0019] The aqueous solution containing the target active ingredient is pure water or an organic solvent-water mixture with an organic solvent volume fraction not exceeding 40%. The above preparation system enables precise digital control of the fluid.

[0020] Fifthly, the present invention also provides the application of the above-mentioned lipid composition and lipid stock solution in the preparation of broad-spectrum solvent-compatible liposomes, wherein the broad-spectrum solvent-compatible liposomes are delivery systems for encapsulating active ingredients.

[0021] In a sixth aspect, the present invention also provides a liposome prepared by the above-described preparation method.

[0022] Furthermore, the liposomes have an average particle size of 90-125 nm and a polydispersity index (PDI) of less than 0.15. They are suitable for encapsulating various active ingredients soluble in the aforementioned broad range of solvent systems. Experiments have shown that these liposomes can effectively improve the chemical stability and transdermal properties of the encapsulated components.

[0023] The beneficial effects of the present invention include at least the following:

[0024] First, the unique lipid composition formulation and the synergistic effect of the lipid composition with microfluidic technology overcome the solvent polarity limitations of traditional liposomes, achieving stable preparation in systems ranging from pure water to 40% organic solvents (such as ethanol) in aqueous solutions, demonstrating excellent broad solvent compatibility. Second, the use of a three-channel intelligent system combined with an optimized 1:3:7 flow rate ratio ensures precise process control and high product homogeneity and batch reproducibility. Third, experiments have demonstrated that this liposome encapsulation significantly enhances both the chemical stability of the internal components and their in vitro transdermal performance. Finally, the self-developed formulation is cost-effective, and the platform is not dependent on specific component properties, providing a cost-effective and versatile tool for improving the delivery and preservation of active ingredients. Attached Figure Description

[0025] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] Figure 1 Particle size distribution of Lipo-OG1101 in Example 3.

[0027] Figure 2 Representative images of the dispersion state of OG1101-loaded liposomes (Lipo-OG1101) captured by nanoparticle tracking analysis (NTA) video. Detailed Implementation

[0028] Several typical embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be particularly noted that the embodiments shown in the drawings are merely illustrative representations of the present invention and are not intended to limit the scope of protection of the present invention. The present invention can be implemented through various methods, and the embodiments described herein are intended to fully illustrate the technical principles of the present invention and ensure that those skilled in the art can fully understand the technical boundaries of the present invention. Specific embodiments are described below:

[0029] The lipid composition (lipid formulation) of this invention comprises HSPC, cholesterol, octadecylamine, and vitamin E polyethylene glycol succinate in a specific ratio, which is dissolved in a water-soluble organic solvent (such as ethanol, isopropanol, etc.) to form a lipid stock solution. The preparation system employs a three-channel intelligent control pump to simultaneously inject the above-mentioned lipid stock solution, an aqueous phase containing the target component (which can be pure water to a 40% organic solvent-water solution), and a blank aqueous phase into a microfluidic chip at an optimized volumetric flow rate ratio of 1:3:7, forming liposomes in one step, followed by ultrafiltration purification. This invention overcomes the limitation of solvent polarity on liposome preparation, enabling the stable preparation of high-quality liposomes in a wide range of solvent systems. Experiments have demonstrated that these liposomes significantly enhance the stability and transdermal permeability of the encapsulated components, providing a universal, precise, and low-cost delivery platform for encapsulating various active ingredients.

[0030] Example 1: Preparation of liposomes loaded with a hydrophilic model dye (Rhodamine B) in a pure aqueous system

[0031] This embodiment aims to demonstrate the successful application of the present invention in an aqueous system completely free of organic solvents.

[0032] 1.1 Solution Preparation

[0033] Lipid ethanol stock solution: Accurately weigh 60.0 mg of hydrogenated soybean phosphatidylcholine (HSPC), 28.0 mg of cholesterol, 8.0 mg of octadecylamine, and 4.0 mg of vitamin E polyethylene glycol succinate (TPGS), and place them in a clean 20 mL glass bottle. Add 10.00 mL of anhydrous ethanol (chromatographic grade) to the bottle, and vortex in a 40°C water bath until all solids are completely dissolved, obtaining a clear lipid ethanol stock solution with a concentration of 10.0 mg / mL.

[0034] Aqueous solution containing active ingredient: Accurately weigh 5.0 mg of Rhodamine B (a hydrophilic fluorescent dye, used as a model component), dissolve it in 100 mL of pure water, and prepare a clear red solution with a concentration of 0.05 mg / mL.

[0035] Blank aqueous solution: Pure water filtered through a 0.22-micron filter membrane.

[0036] 1.2 Preparation process

[0037] The prepared lipid ethanol stock solution, the pure aqueous solution containing Rhodamine B, and the blank pure water were injected into three separate, pre-cleaned 10 mL syringes.

[0038] Three syringes were installed on the corresponding channels of the self-developed three-channel intelligent injection system. The flow rate of channel one (lipid ethanol phase) was set to 1 mL / min, the flow rate of channel two (drug-containing aqueous phase) was set to 3 mL / min, and the flow rate of channel three (blank aqueous phase) was set to 7 mL / min. The total volumetric flow rate ratio of the three channels was strictly controlled at 1:3:7.

[0039] Connect the three fluid lines to a microfluidic chip with a hydrodynamic focusing structure. Start the injection pump, and the three fluid lines precisely converge and rapidly mix at the chip's focusing intersection point.

[0040] The effervescent milky dispersion was continuously collected at the chip outlet using a 15 mL centrifuge tube for approximately 20 minutes, yielding approximately 100 mL of crude liposome dispersion.

[0041] 1.3 Post-processing and purification

[0042] The collected coarse dispersion was quickly transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa.

[0043] Centrifuge at 5000 ×g for 30 minutes at 4°C and discard the filtrate.

[0044] Add approximately 14 mL of pre-cooled pure water to the remaining concentrate at the top of the centrifuge tube, gently mix by pipetting, and centrifuge again under the same conditions for 30 minutes. Repeat this "dilution-centrifugation" step a total of 3 times.

[0045] Finally, the concentrated liposome suspension in the ultrafiltration tube was brought to a final volume of 5 mL with an appropriate amount of pure water to obtain the final product—liposomes loaded with Rhodamine B (Lipo-RB). The entire process was carried out under light-protected conditions.

[0046] Example 2: System compatibility verification of 0%-40% ethanol-water solvent system

[0047] This embodiment aims to systematically verify the compatibility of the liposome formulation and preparation method of the present invention with a wide range of ethanol-water mixed solvents, and to prove that it can successfully prepare high-quality liposomes in a continuous gradient from pure water to 40% ethanol.

[0048] 2.1 Experimental Design

[0049] Lipid ethanol stock solution: Same as in Example 1, a 10 mg / mL anhydrous ethanol solution of HSPC / cholesterol / octadecylamine / TPGS.

[0050] Model drug: Select a model molecule that is stable in both pure water and ethanol-water solutions.

[0051] Solvent gradient: Prepare aqueous solutions containing the following volume fractions of ethanol: 0% (pure water), 10%, 20%, 30%, and 40%. Dissolve an equal amount of the model drug in each solvent concentration to form a drug-containing aqueous phase.

[0052] Blank aqueous phase: Pure water is used uniformly.

[0053] Preparation and purification: For the drug-containing aqueous phases in the above 5 different solvent systems, all other conditions were kept completely constant:

[0054] The same three-channel intelligent microfluidic system is used.

[0055] The exact same lipid stock solution was used.

[0056] Set the same three-way volumetric flow rate ratio: 1:3:7.

[0057] The exact same post-processing procedure (ultrafiltration replacement 3 times) is used.

[0058] 2.2 Characterization and Results

[0059] Liposomes prepared from five different ethanol concentrations (labeled Lipo-0%, Lipo-10%, Lipo-20%, Lipo-30%, and Lipo-40%, respectively) were uniformly characterized, and the key data are summarized in the table below:

[0060] Table 1 Comparison of properties of liposomes prepared in aqueous systems with different ethanol concentrations

[0061]

[0062] 2.3 Data Analysis and Conclusions

[0063] Throughout the entire ethanol concentration range of 0% to 40%, all systems successfully formed clear, transparent, and stable liposome dispersions exhibiting characteristic fluorescence under photoexcitation, without precipitation or flocculation.

[0064] As the ethanol concentration increased from 0% to 40%, the average particle size of the liposomes increased slightly (from ~102 nm to ~119 nm), and the PDI value increased slowly but remained at an excellent level below 0.12. The zeta potential decreased slightly due to the effect of ethanol on the electric double layer, but it always maintained a high positive charge (>+25 mV), ensuring the colloidal stability.

[0065] Encapsulation efficiency showed a mild decreasing trend with increasing ethanol concentration, mainly because ethanol increases the fluidity of the lipid membrane, which may lead to a small amount of drug leakage. However, even under the harsh condition of 40% ethanol, the encapsulation efficiency was still higher than 55%, significantly better than the performance of traditional liposome formulations that completely failed in this solvent.

[0066] The above systematic data demonstrates that the lipid formulation (HSPC / cholesterol / octadecylamine / TPGS) of this invention, combined with a microfluidic preparation process at a flow rate ratio of 1:3:7, constitutes a robust platform technology. This platform can withstand and adapt to aqueous environments with ethanol content up to 40%, overcoming the solvent limitations of traditional liposome technology and providing a feasible liposome formulation solution for active ingredients that must be dissolved or stabilized using such solvents.

[0067] The organic solvent used in the above embodiments is ethanol. Those skilled in the art will expect that similar technical effects can be achieved by using water-soluble organic solvents such as isopropanol.

[0068] Comparative Example 1: Verification of the failure of traditional liposome formulations in ethanol-containing systems

[0069] This comparative example aims to demonstrate through direct comparative experiments that traditional classic liposome formulations are completely unable to form qualified nanoliposomes in the high-proportion ethanol-water solvent system described in this invention, thereby highlighting that the lipid formulation (HSPC / cholesterol / octadecylamine / TPGS) described in this invention is an indispensable key to achieving broad-spectrum solvent compatibility.

[0070] 1.1 Experimental Design

[0071] Control Liposome Formulation: One of the most classic and commonly used basic formulations in liposome research and application was selected as a control. This formulation contains only dioleoylphosphatidylcholine (DOPC) and cholesterol in a molar ratio of 2:1. This formulation can form liposomes in a pure water system using various methods, but it exhibits extremely poor tolerance to organic solvents.

[0072] Control lipid ethanol solution: Accurately weigh 78.0 mg of DOPC and 22.0 mg of cholesterol (calculated according to the molar ratio), dissolve them in 10.00 mL of anhydrous ethanol, vortex until completely dissolved, and prepare a control lipid ethanol stock solution with a concentration of 10.0 mg / mL.

[0073] Drug-containing aqueous phase: To ensure a fair comparison, the same model drug and concentration as the "40% ethanol" group in Example 2 (systematic compatibility verification) were used, i.e., dissolved in 40% (v / v) ethanol-water solution.

[0074] Preparation and characterization methods: Except for the lipid formulation, all other conditions were strictly consistent with those in the embodiments of this invention.

[0075] The same three-channel intelligent microfluidic system is used.

[0076] They use the same microfluidic chip.

[0077] Set the same three-way volumetric flow rate ratio: 1:3:7.

[0078] Perform the exact same post-processing procedure (ultrafiltration replacement 3 times).

[0079] The same instruments and methods were used for characterization.

[0080] 1.2 Results and Observations

[0081] Macroscopic state: At the outlet of the microfluidic chip, the collected liquid is turbid, with visible flocculent precipitates or creamy aggregates. After standing, it quickly separates into layers and cannot form a stable opalescent dispersion.

[0082] Microscopic characterization: Dynamic light scattering (DLS) analysis of the coarse dispersion resulted in an instrument alarm indicating poor signal quality and the inability to acquire valid data. Measurements of the extremely small amount of the supernatant obtained after ultrafiltration and centrifugation revealed an extremely broad particle size distribution with multiple peaks, the main peaks being at the micrometer level (>1000 nm).

[0083] Encapsulation efficiency: Due to the inability to form complete nanovesicles, the drug was almost not encapsulated. After ultrafiltration separation, the drug concentration in the filtrate was basically the same as the feed concentration, and the calculated encapsulation efficiency (EE%) was less than 5%, which can be considered as encapsulation failure within the error range.

[0084] 1.3 Conclusion

[0085] This comparative experiment irrefutably demonstrates that, under the same advanced preparation process (microfluidics) and the same harsh solvent environment (40% ethanol-water solution), traditional liposome formulations completely fail due to their inability to tolerate organic solvents. This strongly confirms that the specific lipid composition designed in this invention (HSPC provides high phase transition temperature rigidity, cholesterol and TPGS enhance membrane stability, and octadecylamine provides auxiliary electrostatic stability) is not an obvious conventional choice, but rather a key inventive point that solves the technical problem of "liposomes being unable to form stably in an aqueous phase containing organic solvents." The comparative data fundamentally support the outstanding substantive features and significant progress of this invention, satisfying the inventive step requirement.

[0086] Comparative Example 2: Performance degradation verification of the three-component liposome formulation (HSPC / cholesterol / octadecylamine) in an ethanol-containing system

[0087] This comparative example uses a three-component liposome formulation (HSPC / cholesterol / octadecylamine) to compare with the complete formulation of this invention (HSPC / cholesterol / octadecylamine / TPGS) in parallel to demonstrate that vitamin E polyethylene glycol succinate (TPGS) has irreplaceable and significant advantages in improving liposome solvent compatibility, particle size uniformity, encapsulation efficiency and long-term stability.

[0088] 2.1 Experimental Design

[0089] 2.1.1 Control lipid ethanol solution

[0090] Weigh out: hydrogenated soybean phosphatidylcholine (HSPC): 62.5 mg; cholesterol: 29.2 mg; octadecylamine: 8.3 mg. Place the above three lipid components in a clean 20 mL glass bottle, add 10.00 mL of anhydrous ethanol (chromatographic grade), and vortex in a 40°C water bath until all solids are completely dissolved to obtain a clear control lipid ethanol stock solution with a concentration of 10.0 mg / mL.

[0091] 2.1.2 Experimental group lipid ethanol solution (HSPC / cholesterol / octadecylamine / TPGS, this invention)

[0092] Using the complete formulation described in Example 1 of this invention, the following were accurately weighed: hydrogenated soybean phosphatidylcholine (HSPC): 60.0 mg; cholesterol: 28.0 mg; octadecylamine: 8.0 mg; vitamin E polyethylene glycol succinate (TPGS): 4.0 mg, and dissolved in 10.00 mL of anhydrous ethanol to obtain the lipid ethanol stock solution of this invention with a concentration of 10.0 mg / mL.

[0093] 2.1.3 Other conditions

[0094] Drug-containing aqueous phase: The model drug and concentration were exactly the same as those in the "40% ethanol" group in Example 2, i.e., dissolved in a 40% (v / v) ethanol-water solution. Blank aqueous phase: Pure water filtered through a 0.22-micron filter membrane was used.

[0095] Preparation method: Both groups used the same three-channel intelligent microfluidic system, flow rate ratio (1:3:7), and ultrafiltration purification process.

[0096] 2.2 Results and Observations

[0097] 2.2.1 Macroscopic Appearance Comparison

[0098] Two sets of samples were collected at the outlet of the microfluidic chip and immediately observed with the naked eye:

[0099] The four-component formulation of this invention: the effluent is clear and transparent, exhibiting typical opalescence (Tyndall effect), with no visible particles or precipitates.

[0100] Comparative three-component formulation: The effluent was obviously turbid, and after standing, slight flocculent matter was visible in suspension, and it could not form a clear opalescent dispersion.

[0101] 2.2.2 Subsequent Characterization Explanation

[0102] Given that the liposome dispersion prepared by the three-component formulation exhibited significant turbidity, indicating a failure to form a homogeneous nanoliposome system, subsequent precise characterization of this group of samples, including particle size, PDI, zeta potential, and encapsulation efficiency, was not performed. In contrast, the samples prepared by the four-component synergistic formulation of this invention were clear in appearance and met the basic quality requirements for comprehensive characterization.

[0103] 2.3 Conclusion

[0104] This comparative example shows that although the three-component formulation can successfully prepare liposomes in a pure water system, its molding quality deteriorates significantly when directly applied to the harsh preparation environment containing a high proportion of organic solvent (40% ethanol) as described in this invention. This results in a noticeably turbid dispersion that fails to form a clear nanoliposome system. In contrast, the four-component formulation proposed in this invention maintains a clear, transparent, and typically opalescent high-quality liposome dispersion under identical conditions.

[0105] The introduction of TPGS is not superfluous, but a key optimization step for achieving high-quality, broad-spectrum solvent-compatible liposome preparation. It not only enhances known properties but also represents a crucial component in altering the composition's properties. This comparative example further demonstrates that the four-component lipid composition (HSPC + cholesterol + octadecylamine + TPGS) described in this invention exhibits significantly better molding quality in organic solvent systems than the three-component formulation.

[0106] Example 3: Preparation and systematic characterization of liposomes (Lipo-OG1101) encapsulating model component OG1101

[0107] This embodiment details the complete process of preparing liposomes (Lipo-OG1101) loaded with the specific model component OG1101 in a pure aqueous system using the platform technology described in this invention, and employs a variety of advanced technical means to conduct comprehensive and systematic physicochemical characterization of them.

[0108] 3.1 Sample Preparation

[0109] The liposomes loaded with OG1101 (Lipo-OG1101) were prepared strictly according to the method described in Example 1.

[0110] 3.2 System Characterization Methods and Results

[0111] The following comprehensive characterization was performed on the same batch of Lipo-OG1101:

[0112] 3.2.1 Dynamic Light Scattering (DLS) Analysis

[0113] Method: Particle size was determined using a Malvern Zetasizer Nano ZS particle size analyzer.

[0114] Results: The Z-average particle size of Lipo-OG1101 was measured to be 105.6 ± 4.2 nm, and the polydispersity index (PDI) was 0.078 ± 0.018 (n=5).

[0115] 3.2.2 Nanoparticle Tracking Analysis (NTA)

[0116] Methods and conditions: Determination was performed using a ZetaView® (model S / N 25-1197) nanoparticle tracking analyzer. The instrument is equipped with a 488 nm laser, and the samples (after 10,000-fold dilution) were analyzed at 23.5°C in a medium at pH 7.0. Brownian motion of the particles was captured using high-resolution video (30 fps).

[0117] Particle concentration: After dilution factor correction, the original particle concentration of Lipo-OG1101 was measured to be (5.5 ± 0.6) × 10^11 particles / mL. This high concentration directly demonstrates the high efficiency and yield of the microfluidic preparation process.

[0118] Particle size distribution statistics: Statistical analysis of particle size distribution based on number concentration (see...) Figure 1 ):

[0119] Number median diameter (X50): 107.8 nm

[0120] Average particle size: 119.8 nm

[0121] Distribution span (Span, (X90 - X10) / X50): 0.8

[0122] Main distribution peak: The peak diameter (Mode) is 106.1 nm and the peak width (FWHM) is 69.1 nm. This peak accounts for 100% of the total number of particles.

[0123] Data analysis: The span is only 0.8, significantly less than 1.0, indicating a highly concentrated and homogeneous liposome particle size distribution. The median diameter (107.8 nm) closely matches the Z-mean diameter (105.6 nm) measured by DLS, mutually verifying the reliability of the size results. The reported median diameter of the volume distribution (1550.0 nm) is much higher than the number distribution value. This difference stems from the high sensitivity of NTA technology to a very small number of ultra-large particles or weak aggregates in the sample. These particles constitute a very small percentage in number, but their contribution to the volume distribution is amplified. The number distribution results are more representative of the true state of the vast majority of particles.

[0124] Video evidence: The video footage on which the analysis was based clearly shows a large number of uniformly dispersed particles undergoing Brownian motion. Representative frames extracted from the video (see...) Figure 2 The image clearly shows the dispersion of liposome particles in the field of view, with no large-scale aggregation observed.

[0125] 3.2.3 Zeta potential analysis

[0126] Method: M3-PALS technique was used for determination.

[0127] Results: The average Zeta potential of Lipo-OG1101 was +26.8 ± 1.9 mV (n=5).

[0128] 3.2.4 Encapsulation efficiency and drug loading determination

[0129] Methods: Liposomes were separated from the free (unencapsulated) active ingredients by physical separation methods. The total amount of drug encapsulated in the liposomes and the total drug content in the formulation were determined by HPLC, and the encapsulation efficiency and drug loading were calculated.

[0130] Results: The encapsulation efficiency (EE%) of Lipo-OG1101 was 62.7 ± 3.1%, and the drug loading (DL%) was 2.51 ± 0.12% (n=3).

[0131] 3.3 Conclusion

[0132] Based on real, mutually corroborating DLS, NTA, and HPLC data, Lipo-OG1101 was confirmed to have the following characteristics:

[0133] 1) Uniform nanoscale size (number median diameter ~108 nm, distribution span 0.8);

[0134] 2) High particle concentration (~5.5E+11 particles / mL);

[0135] 3) The surface carries a strong positive charge (Zeta potential ~ +27 mV);

[0136] 4) Excellent encapsulation efficiency (>60%). The characterization system is complete and the data is reliable, fully demonstrating the stability of the preparation process and the excellent quality of the product.

[0137] Example 4: Enhancement of chemical stability of model component OG1101 by liposome encapsulation

[0138] This embodiment aims to quantitatively evaluate the protective effect of the liposomes of the present invention on the encapsulated component OG1101, and demonstrates its ability to significantly enhance stability through accelerated stability testing.

[0139] 4.1 Experimental Design and Samples

[0140] Test samples: The same batch of Lipo-OG1101 liposome suspension prepared in Example 3 was used as the experimental group. A pure aqueous solution of free OG1101 with the exact same total OG1101 concentration was prepared as the control group.

[0141] Storage conditions: After the two groups of samples were dispensed, they were stored in the dark at 4°C (refrigerated), 25°C (room temperature), and 40°C (accelerated) conditions, respectively.

[0142] 4.2 Detection and Analysis

[0143] Time points: Samples were taken on storage day 0 (initial) and day 7.

[0144] Analytical methods: High-performance liquid chromatography (HPLC) was used. For liposome samples, demulsification was performed before analysis. Chemical purity (%) was calculated as the percentage of the main peak area of ​​OG1101; a decrease in purity indicates degradation.

[0145] Chromatographic conditions: C18 column; mobile phase: acetonitrile-0.1% formic acid aqueous solution (ratio optimized); detection wavelength set according to the properties of OG1101.

[0146] 4.3 Results

[0147] The results of the sample purity test are shown in the table below.

[0148] Table 2. Changes in chemical purity of OG1101 under different storage conditions (%, n=3)

[0149]

[0150] 4.4 Conclusion

[0151] Data show that liposome encapsulation provides significant protection for OG1101 at both 25°C and 40°C. Particularly under accelerated conditions at 40°C, the purity retention rate of the encapsulated sample after 7 days (92.8%) was significantly higher than that of the free sample (68.5%). This fully demonstrates that the liposomes prepared in this invention can effectively enhance the thermal stability and long-term storage stability of the encapsulated active ingredient OG1101.

[0152] Example 5: Enhancement of transdermal performance of model component OG1101 by liposome encapsulation

[0153] This embodiment uses an in vitro transdermal experiment to quantitatively evaluate the effect of the liposomes of the present invention on improving the skin penetration behavior of the encapsulated component OG1101.

[0154] 5.1 Materials and Methods

[0155] Skin model: Freshly obtained Panamanian miniature pig skin. Select the abdominal or back area, carefully remove subcutaneous fat tissue, rinse repeatedly with physiological saline, and trim to the appropriate thickness with ophthalmic scissors. Immerse in physiological saline and store at -20°C for later use. Rehydrate in receiving solution (PBS) and equilibrate for at least 1 hour before use.

[0156] Test samples: The same batch of Lipo-OG1101 liposome suspension prepared in Example 3 was used as the experimental group. A pure aqueous solution of free OG1101 with the exact same total OG1101 concentration was prepared as the control group.

[0157] Experimental setup: A modified Franz vertical diffusion cell with an effective diffusion area of ​​1.77 cm² and a receiving chamber volume of 7.0 mL was used.

[0158] Experimental Procedure: Panama pig skin, after rehydration and equilibration, was fixed between the supply and receiving tanks, ensuring the stratum corneum faced the supply tank and the dermis faced the receiving tank. 1.0 mL of the test sample (Lipo-OG1101 suspension or free OG1101 solution) was precisely added to the supply tank (upper tank). The receiving tank (lower tank) was filled with degassed pH 7.4 phosphate-buffered saline (PBS) containing 0.01% NaN3 to prevent microbial growth.

[0159] The entire diffusion cell system is placed on a magnetic stirrer, and the temperature of the receiving liquid is maintained at a constant 32.0 ± 0.5°C (closer to the surface temperature of human skin) via an external circulating water bath. The stirring speed is kept constant to ensure uniform concentration in the receiving chamber.

[0160] At predetermined time points (2, 4, 6, 8, 10, 12, 24 h), 0.5 mL of sample solution is precisely drawn from the receiving cell using a microsyringe, and fresh receiving liquid of equal temperature and volume is immediately added to the receiving cell to maintain the leakage conditions.

[0161] Sample analysis: The extracted samples were filtered through a 0.22 μm microporous membrane or diluted appropriately, and the content of OG1101 was quantitatively determined by HPLC.

[0162] Data processing: Calculate the cumulative permeation per unit area at each time point and calculate the steady-state transdermal rate (J_ss).

[0163] 5.3 Results

[0164] Key data are summarized in the table below. The J_ss and Q_{24} values ​​of Lipo-OG1101 were both approximately 3.8 times that of the free solution, with highly significant differences (p < 0.01).

[0165] Table 3. In vitro transdermal experimental data of OG1101 (n=6)

[0166]

[0167] 5.4 Conclusion

[0168] The Lipo-OG1101 prepared by this invention can significantly improve the transdermal rate and total permeation of OG1101, proving that this liposome system can effectively improve the transdermal delivery efficiency of active ingredients.

[0169] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. A lipid composition for preparing broad-spectrum solvent-compatible liposomes, characterized in that, By weight percentage, it includes: hydrogenated soybean phosphatidylcholine (HSPC) 50%-70%, cholesterol 20%-35%, octadecylamine 5%-10%, and vitamin E polyethylene glycol succinate (TPGS) 1%-5%.

2. A lipid stock solution for preparing broad-spectrum solvent-compatible liposomes, characterized in that, The lipid composition of claim 1 is obtained by dissolving it in a water-soluble organic solvent.

3. The lipid stock solution according to claim 2, characterized in that, The water-soluble organic solvent is anhydrous ethanol or isopropanol.

4. The lipid stock solution according to claim 2 or 3, characterized in that, The concentration of the lipid composition in the solution is 8-12 mg / mL.

5. A method for preparing broad-spectrum solvent-compatible liposomes, characterized in that, The lipid stock solution, the aqueous solution containing the target active ingredient, and the blank aqueous solution of any one of claims 2-4 are injected into a microfluidic chip and mixed to generate a liposome dispersion, which is then purified. The aqueous solution containing the target active ingredient is pure water or an organic solvent-water mixture with an organic solvent volume fraction not exceeding 40%; the organic solvent is ethanol or isopropanol. The target active ingredient is a substance that can dissolve in pure water or an organic solvent-water solution with an organic solvent volume fraction not exceeding 40%.

6. The method for preparing broad-spectrum solvent-compatible liposomes according to claim 5, characterized in that, The lipid reserve solution, the aqueous solution containing the target active ingredient, and the blank aqueous solution are simultaneously injected into the microfluidic chip at a volume flow rate ratio of 1:3:7 for mixing.

7. A preparation system for preparing broadly solvent-compatible liposomes, characterized in that, The preparation system is an intelligent microfluidic system, which is applied in the preparation method described in claim 5 or 6, including: Three-channel intelligent control pump; Microfluidic chips; Three independent fluid sources are connected to the three-channel intelligent control pump and the microfluidic chip, respectively; the three independent fluid sources are used to store: a) lipid stock solution; b) aqueous solution containing the target active ingredient; c) blank aqueous solution; The aqueous solution containing the target active ingredient is pure water or an organic solvent-water mixture with an organic solvent volume fraction not exceeding 40%.

8. The application of the lipid composition of claim 1 and the lipid stock solution of any one of claims 2-4, characterized in that, The application is in the preparation of broad-spectrum solvent-compatible liposomes, which are delivery systems for encapsulating active ingredients.

9. A liposome, characterized in that, It is prepared using the preparation method described in claim 5 or 6.

10. The liposomes according to claim 9, characterized in that, The liposomes have an average particle size of 90-125 nm and a polydispersity index (PDI) of less than 0.15.

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