Temperature response type essential oil liposome based on amide molecule conformation transformation and preparation method thereof

Abstract

The invention discloses a temperature response type essential oil liposome based on amide molecule conformation transformation and a preparation method thereof, and belongs to the technical field of liposome embedding essential oil. Oleamide is introduced into a lipid bilayer, an embedded structure construction method based on pH-induced conformation transformation is adopted, oleamide forms a relatively loose conformation and is dispersed in a lipid bilayer interface area under a weak alkaline condition, then the pH of a system is slowly adjusted to a weak acid interval, and the lipid bilayer is prepared. Hydrogen bonds among amide molecules are reestablished and conformational contraction occurs, so that'conformational locking type embedding 'of the amide molecules in a lipid double-layer hydrophobic region is realized. According to the invention, not simple physical mixing is adopted, but pH is adopted to drive molecular rearrangement, so that oleamide is stably positioned in a lipid membrane, hydrogen bond network rupture and conformation expansion occur in a specific temperature interval, local space occupation mutation and structural defect formation in the membrane are triggered, and rapid release is realized.

Description

Technical Field

[0001] This invention relates to a temperature-responsive essential oil liposome based on amide molecular conformational change and its preparation method, belonging to the field of liposome-encapsulated essential oil technology. Background Technology

[0002] Liposomes, as nanoscale vesicles composed of a lipid bilayer, have become an ideal carrier for the encapsulation and delivery of essential oils due to their excellent biocompatibility, low toxicity, and broad-spectrum encapsulation ability for hydrophobic / hydrophilic substances. Essential oils, as natural plant extracts, possess various biological activities such as antibacterial, anti-inflammatory, and aromatherapy properties, and are widely used in daily chemical products, textile finishing, and environmental fragrance materials. However, they inherently suffer from high volatility, poor stability, susceptibility to oxidative degradation, low bioavailability, and difficulty in achieving long-lasting and controlled release. Encapsulation with liposomes can effectively isolate essential oils from external environmental interference, prolong their duration of action, reduce irritation, and achieve long-lasting and controllable delivery.

[0003] Currently, some studies attempt to induce a temperature-responsive release of lipids by modulating their composition, causing the lipid membrane to transition from a gel state to a liquid crystal state within a specific temperature range. However, this type of technology still has significant limitations: (1) The lipid bilayer has limited ability to block small molecule essential oils. It still has a continuous chronic leakage phenomenon at room temperature, resulting in a large loss before the temperature is triggered, which weakens the temperature-sensitive response difference. (2) The permeability change brought about by the improvement of membrane fluidity is small, and the release behavior is mostly gradual rather than obvious "threshold" change, making it difficult to form a clear temperature triggering effect; (3) Liposomes are usually in liquid dispersion system, which limits their storage stability. They are prone to vesicle fusion, membrane structure collapse or rupture during spray drying or freeze drying. After rehydration, the particle size increases and the encapsulation efficiency decreases, making it difficult to maintain the temperature response function. (4) The release behavior of thermosensitive liposomes is mostly gradual rather than sudden; the phase transition temperature is greatly affected by components such as cholesterol, and the regulation window is limited; it is difficult to form sufficient membrane structure defects by simply relying on phase transition; the system usually needs to introduce outer layer modification or polymer response structure to increase complexity.

[0004] Therefore, there is an urgent need to develop a new liposome structure regulation strategy that enables liposomes to form stronger diffusion restriction on essential oil molecules at low temperatures, and to achieve temperature-triggered rapid release of essential oils without the need for additional polymerization structures, but only through the rearrangement of small molecular structures within the membrane. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a temperature-responsive essential oil liposome based on the conformational change of amide molecules and its preparation method. Specifically, this invention introduces oleic acid amide into a lipid bilayer and employs an embedded structure construction method based on pH-induced conformational change. Under weakly alkaline conditions, oleic acid amide forms a relatively loose conformation and disperses in the lipid bilayer interface region. Subsequently, the pH of the system is slowly adjusted to a weakly acidic range, causing the hydrogen bonds between amide molecules to re-establish and undergo conformational contraction, thereby achieving "conformation-locked embedding" in the hydrophobic region of the lipid bilayer. This invention is not a simple physical mixing process, but rather uses pH-driven molecular rearrangement to stably position oleic acid amide within the lipid membrane, causing hydrogen bond network rupture and conformational unfolding within a specific temperature range. This induces abrupt changes in local space occupancy and the formation of structural defects within the membrane, achieving rapid release. This invention does not rely on an outer polymer-responsive structure, does not involve complex chemical modifications, and achieves temperature-sensitive burst release solely through the conformational change of amide molecules within the membrane. It has advantages such as simple structure, convenient preparation, and ease of industrialization.

[0006] The first objective of this invention is to provide a method for preparing temperature-responsive essential oil liposomes, comprising the following steps: (1) Lipid membrane formation: Phospholipids and cholesterol are dissolved in an organic solvent to obtain a mixed solution; then essential oils are added to the mixed solution, stirred evenly, and then the solvent is removed by evaporation to form a uniform lipid film; (2) Alkaline hydration: The lipid membrane was hydrated at 45-55℃ for 10-40 min using a buffer solution to form a primary liposome dispersion system. (3) Introduction of amides: Slowly add the oleamide solution to the primary liposome dispersion system and stir at 45-50℃ for 20-40 minutes to form a mixed system; (4) pH-induced embedding: The pH of the mixture was adjusted to 6.2-6.8, and the mixture was kept at 45-50℃ for 15-30 minutes. The mixture was then extruded or ultrasonically treated to obtain temperature-responsive essential oil liposomes.

[0007] In one embodiment of the present invention, the mass ratio of phospholipids, cholesterol and oleamide in the temperature-responsive essential oil liposomes is 75:5-20:3-15.

[0008] In one embodiment of the present invention, the mass ratio of the sum of the masses of phospholipids, cholesterol, and oleamide to the mass of the essential oil in step (1) is 1:0.05-0.2.

[0009] In one embodiment of the present invention, the organic solvent in step (1) is one or more of chloroform, ethanol, diethyl ether, and methanol.

[0010] In one embodiment of the present invention, the mass concentration of phospholipid in the organic solvent in step (1) is 60-80%.

[0011] In one embodiment of the present invention, the essential oil in step (1) is lavender essential oil.

[0012] In one embodiment of the present invention, the stirring in step (1) is carried out at 15-35°C and 400-600 rpm for 20-40 minutes.

[0013] In one embodiment of the present invention, the evaporation in step (1) is rotary evaporation, specifically evaporation at 40-60°C for 15-30 minutes.

[0014] In one embodiment of the present invention, the buffer solution in step (2) is a phosphate buffer with a pH of 8.0-8.5.

[0015] In one embodiment of the present invention, the mass ratio of lipid membrane to buffer solution in step (2) is 1:1-3.

[0016] In one embodiment of the present invention, the oleamide solution in step (3) is an ethanol solution of oleamide with a mass concentration of 5-10%.

[0017] In one embodiment of the present invention, the addition rate in step (3) is 2-5 mL / min.

[0018] In one embodiment of the present invention, the stirring speed in step (3) is 200-600 rpm, the temperature is 45-50℃, and the time is 10-30 min.

[0019] In one embodiment of the present invention, step (3) causes oleic amide to be dispersed in the membrane interface region.

[0020] In one embodiment of the present invention, the pH adjustment in step (4) is performed using a phosphate buffer solution with a pH of 6-7.

[0021] In one embodiment of the present invention, step (4) causes the oleamide molecules to undergo conformational contraction and rearrange through hydrogen bonds to embed into the lipid bilayer.

[0022] In one embodiment of the present invention, the heat preservation process in step (4) is carried out by magnetic stirring at a speed of 100-300 rpm.

[0023] In one embodiment of the present invention, in step (4), the extrusion is performed using a filter membrane with a pore size of 50-150 nm; the ultrasonic treatment is performed at 150-300 W for 3-10 min.

[0024] In one embodiment of the present invention, after extrusion or ultrasonic treatment in step (4), impurity removal can be performed, specifically by dialysis to remove non-embedded components.

[0025] The second objective of this invention is to prepare temperature-responsive essential oil liposomes using the method described herein.

[0026] In one embodiment of the present invention, the particle size of the temperature-responsive essential oil liposomes is 80-200 nm; more preferably 100-150 nm.

[0027] The third objective of this invention is the application of the temperature-responsive essential oil liposomes described herein in the preparation of daily chemical products, textile finishing, or environmental fragrance materials.

[0028] A fourth objective of this invention is to provide a method for improving the temperature response performance of essential oil liposomes, wherein the temperature-responsive essential oil liposomes described in this invention are used.

[0029] The fifth objective of this invention is to provide a method for enhancing the rapid release of essential oils at 38-42°C, wherein the temperature-responsive essential oil liposomes described in this invention are employed.

[0030] The technical effects of this invention are as follows: (1) Oleamide is a molecule containing long-chain hydrophobic alkyl groups and amide functional groups. At lower temperatures, it can form a locally ordered aggregated state through intermolecular hydrogen bonding, making its conformation relatively compact and forming a relatively stable mixed structure with phospholipid fatty chains. When the temperature rises to the range of 38-42℃, the hydrogen bonds between amides gradually break, and the molecular conformation changes from a relatively coiled or aggregated state to a more unfolded state, resulting in an increase in local volume occupation and an increase in the degree of disorder in the arrangement. This conformational transformation process is different from simple thermal expansion, but belongs to the nonlinear change of the structural level, which generates local spatial mismatch and lateral stress concentration in the membrane, forming nanoscale structural defects, which cause the encapsulated contents to be released rapidly.

[0031] (2) This invention does not rely on the outer polymer response structure, does not involve complex chemical modifications, and achieves thermosensitive burst release only through the conformational change of the amide molecule in the membrane. It has the advantages of simple structure, clear mechanism and easy scale-up of preparation process.

[0032] (3) The temperature-responsive essential oil liposomes of the present invention have an encapsulation rate of over 80% for essential oils and a phase transition temperature of 38-41℃; and can achieve rapid release of large quantities at 42℃. Attached Figure Description

[0033] Figure 1 The images show particle size test results for the examples and comparative examples.

[0034] Figure 2Viscosity test graphs for the examples and comparative examples. Detailed Implementation

[0035] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0036] Test method: 1. Release performance test; Accurately weigh 1 mL of the essential oil liposome sample and place it in a stoppered conical flask. Add 50 mL of a 30% (v / v) ethanol aqueous solution as the release medium. Place the conical flask in a constant-temperature shaking water bath at a shaking speed of 100 rpm to ensure uniform dispersion of the system and avoid microcapsule sedimentation affecting release stability. Release the system at the target temperature for 60 min. After release, measure the absorbance of the solution and calculate the corresponding release amount based on a pre-established standard curve.

[0037] 2. Particle size test: Essential oil liposome samples were taken and appropriately diluted with pH 7.4 phosphate buffer to a concentration of 0.1 mg / mL to avoid the influence of multiple scattering on the test results. The diluted samples were then transferred to disposable plastic cuvettes and measured using a Dynamic Light Scattering (DLS) analyzer. The test temperature was set to 25℃, the equilibration time to 120 s, and the detection angle to 90°. Each sample was measured three times consecutively, with each measurement lasting 60 s, to obtain the average hydrated particle size (Z-average) and polydispersity index (PDI) of the essential oil liposomes.

[0038] 3. Embedding rate test: The determination was performed using ultrafiltration centrifugation.

[0039] Take 1 mL of essential oil liposomes and place them in a 10 kDa ultrafiltration centrifuge tube. Centrifuge at 10,000 rpm for 20 min to separate the free essential oil.

[0040] The free essential oil content in the filtrate and the total essential oil content after liposome perforation were determined, and the encapsulation efficiency was calculated. Encapsulation efficiency (EE, %) = (Total essential oil volume) (Free essential oil content) / Total essential oil content × 100% Each sample group has three parallel samples.

[0041] 4. Phase transition temperature test: The prepared essential oil liposomes were centrifuged at 200,000 g for 20 min to remove the supernatant and collect the lower liposomes.

[0042] Weigh 10 mg of liposomes and spread them on the bottom of a sealed aluminum crucible. Scan the sample under a heating range of 20℃ to 60℃, with a heating rate of 5℃ / min. Repeat twice and measure the DSC spectrum. Use a blank crucible as a control.

[0043] 5. Lipid bilayer flowability and microviscosity test: Take an appropriate amount of the prepared liposomes and dilute them with pH 7.4 phosphate buffer to a phospholipid concentration of 0.1 mg / mL. Add DPH probe stock solution (dissolved in tetrahydrofuran, concentration 2.0 × 10⁻⁶). -3 The DPH was diluted to a 1:200 molar ratio with 1 mol / L, and incubated at 25°C for 30 min in the dark to allow the probe to fully embed into the hydrophobic region of the lipid bilayer. The sample was then transferred to a fluorescence spectrophotometer with a temperature control and equilibrated at set temperatures (e.g., 32°C and 42°C) for 10 min. The fluorescence intensity in the parallel direction (I∥) and perpendicular direction (I⊥) was measured using an excitation wavelength of 360 nm and an emission wavelength of 430 nm. The fluorescence anisotropy value r was calculated using the following formula: r = (I ∥ G·I ⊥ ) / (I ∥ + 2G·I ⊥ ) Among them, I ∥ I represents the fluorescence intensity in the parallel polarization direction. ⊥ G represents the fluorescence intensity in the vertical polarization direction, and G is the instrument correction factor (G = IHV / IHH, which is automatically determined by the instrument).

[0044] The lipid membrane microviscosity η is further calculated based on the fluorescence anisotropy value, using the following formula: η = (2r / (0.46 r)) × η0 Where η is the lipid bilayer microviscosity (mPa·s), r is the fluorescence anisotropy value, 0.46 is the theoretical limit of anisotropy of the DPH probe in a rigid medium, and η0 is the viscosity constant of DPH in a standard reference system (taken as 1 mPa·s, used for relative microviscosity calculation).

[0045] Each sample was measured in triplicate, and the average value was taken as the final result. By comparing the changes in the microviscosity of the liposome membrane at different temperatures, the fluidity and structural order of the lipid bilayer can be reflected, thereby evaluating the effect of oleamide intercalation on the structural stability and temperature response characteristics of the lipid membrane.

[0046] Raw materials used in the examples: Phospholipids: 99.9%; Cholesterol: 99.9%; Oleamide: 99%; Lavender essential oil: Purity > 90%; All other raw materials are sourced from commercially available sources.

[0047] Example 1 A method for preparing temperature-responsive essential oil liposomes includes the following steps: (1) Lipid membrane formation: Phospholipids and cholesterol were dissolved in chloroform to obtain a mixed solution; then lavender essential oil was added to the mixed solution and stirred at 25°C and 500 rpm for 30 min. The solvent was then removed by rotary evaporation (evaporation at 50°C for 15 min) to form a uniform lipid film. The phospholipid concentration in chloroform was 75%. (2) Alkaline hydration: The lipid membrane and buffer solution were hydrated at 48°C for 30 min with a mass ratio of 1:2 and a phosphate buffer solution at pH 8.2 to form a primary liposome dispersion system. (3) Introduction of amides: An 8% (w / w) oleamide ethanol solution was slowly added to the primary liposome dispersion system and stirred at 48°C and 400 rpm for 20 min to form a mixed system. The addition rate was 5 mL / min. (4) pH-induced embedding: The pH of the mixture was adjusted to 6.5 using an acidic buffer (phosphate buffer, pH 6.4), and the mixture was kept at 48℃ and 200 rpm for 20 min. The mixture was then extruded through a 100 nm filter membrane to obtain temperature-responsive essential oil liposomes (particle size approximately 130 nm). The mass ratio of phospholipids, cholesterol, and oleamide in the essential oil liposomes is 75:15:10, and the sum of the masses of phospholipids, cholesterol, and oleamide is 1:0.1 with the mass of the essential oil.

[0048] Example 2 The mass ratio of phospholipids, cholesterol, and oleamide in the essential oil liposomes of Example 1 was adjusted to 75:10:10, while other aspects remained the same as in Example 1, to obtain the essential oil liposomes.

[0049] Example 3 The mass ratio of phospholipids, cholesterol, and oleamide in the essential oil liposomes of Example 1 was adjusted to 75:15:5, while other aspects remained the same as in Example 1, thus obtaining the essential oil liposomes.

[0050] Example 4 The mass ratio of phospholipids, cholesterol, and oleamide in the essential oil liposomes of Example 1 was adjusted to 75:20:5, while other aspects remained the same as in Example 1, thus obtaining the essential oil liposomes.

[0051] Example 5 In step (4) of Example 1, the forming process was adjusted to use ultrasonic treatment at 300W for 5 minutes, while other steps remained the same as in Example 1, to obtain essential oil liposomes.

[0052] Example 6 The pH of the mixture in step (4) of Example 1 was adjusted to 6.2, while other parameters remained the same as in Example 1, to obtain essential oil liposomes.

[0053] Example 7 In Example 1, the ratio of the sum of the masses of phospholipids, cholesterol, and oleamide to the mass of the essential oil was adjusted to 1:0.2, while other aspects remained the same as in Example 1, to obtain essential oil liposomes.

[0054] Comparative Example 1 The step of adjusting pH in step (4) of Example 1 is omitted, and the rest is kept the same as in Example 1 to obtain essential oil liposomes.

[0055] Comparative Example 2 Oleamide was omitted in Example 1, and everything else remained the same as in Example 1 to obtain essential oil liposomes.

[0056] Comparative Example 3 In Example 1, oleamide was replaced with stearamide, while other aspects remained the same as in Example 1, resulting in essential oil liposomes.

[0057] Comparative Example 4 The mass ratio of phospholipids, cholesterol, and oleamide in the essential oil liposomes of Example 1 was adjusted to 75:15:20, while other aspects remained the same as in Example 1, to obtain the essential oil liposomes.

[0058] Comparative Example 5 The mass ratio of phospholipids, cholesterol, and oleamide in the essential oil liposomes of Example 1 was adjusted to 75:15:1, while other aspects remained the same as in Example 1, thus obtaining the essential oil liposomes.

[0059] Comparative Example 6 Add the oleamide ethanol solution from step (3) of Example 1 to step (1), that is: mix the oleamide ethanol solution, phospholipids and cholesterol in step (1) evenly; only stir in step (3); keep everything else the same as in Example 1 to obtain essential oil liposomes.

[0060] The obtained essential oil liposomes were subjected to performance testing, and the results are as follows: Table 1

[0061] Note: Response amplitude = 42℃ release rate / 32℃ release rate.

[0062] As shown in Table 1, embedding oleamide into the liposome membrane structure via pH induction significantly improves the structural stability and temperature-responsive release performance of liposomes. In Examples 1-7, using appropriate ratios of phospholipids, cholesterol, and oleamide, and inducing embedding via pH gradient, the resulting liposomes exhibited uniform particle size distribution (approximately 118-133 nm), high embedding efficiency (greater than 80.9%), and phase transition temperatures concentrated in the range of 38.2-40.7℃. The release rate remained low at 32℃ (14.9%-21.6%), while significantly increasing at 42℃. The temperature-sensitive response amplitude was greater than 4.0, demonstrating obvious temperature-triggered release characteristics. Ultrasonic treatment can further reduce particle size and improve embedding efficiency, while appropriately lowering the embedding pH can improve lipid membrane stability and reduce low-temperature leakage, but may slightly reduce high-temperature release efficiency. Increasing the essential oil loading can increase high-temperature release, but it reduces membrane stability and increases low-temperature release rate.

[0063] In contrast, comparative results showed that both insufficient and excessive oleamide content affected the thermosensitive regulation of the lipid membrane structure. Specifically, low oleamide content led to a decrease in the liposome phase transition temperature and high-temperature release rate, resulting in a weakened thermosensitive response. Conversely, excessive oleamide content or direct addition before hydration without pH-induced intercalation disrupted the ordered arrangement of the lipid membrane, leading to increased particle size, a lower phase transition temperature, and a significant increase in low-temperature leakage. Simultaneously, the improvement in high-temperature release rate was limited, and the thermosensitive response performance significantly decreased. These results clearly demonstrate that oleamide, through pH-induced intercalation, can effectively regulate the conformational stability and phase transition behavior of the lipid bilayer. Within a suitable ratio range, thermosensitive liposome systems with well-defined phase transition temperatures and good temperature-responsive release performance can be constructed, thereby achieving controlled release of essential oils. This verifies the effectiveness and superiority of this method in the construction of thermosensitive controlled-release liposomes.

[0064] Figure 2 Viscosity test graphs for the examples and comparative examples. From Figure 2It can be seen that at 32℃, the microviscosity of the liposome membrane in Example 1 reached 972 mPa·s, significantly higher than that of the liposomes without oleamide (892 mPa·s) and those with oleamide added before hydration (845 mPa·s), indicating that oleamide successfully intercalated into the lipid bilayer and enhanced the membrane's structural order. At 42℃, the microviscosity of Example 1 significantly decreased to 398 mPa·s, a much larger decrease than the comparative example, indicating that oleamide underwent a conformational change during heating and significantly improved the lipid membrane's fluidity. These results demonstrate that oleamide intercalated via pH-induced induction can effectively regulate the microstructure and fluidity changes of the lipid bilayer, directly proving at the microscopic level that oleamide has successfully intercalated into the liposome membrane structure and participated in temperature-response regulation.

[0065] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing temperature-responsive essential oil liposomes, characterized in that, Includes the following steps: (1) Lipid membrane formation: Phospholipids and cholesterol are dissolved in an organic solvent to obtain a mixed solution; then essential oils are added to the mixed solution, stirred evenly, and then the solvent is removed by evaporation to form a uniform lipid film; (2) Alkaline hydration: The lipid membrane was hydrated at 45-55℃ for 10-40 min using a buffer solution to form a primary liposome dispersion system. (3) Introduction of amides: Slowly add the oleamide solution to the primary liposome dispersion system and stir at 45-50℃ for 20-40 minutes to form a mixed system; (4) pH-induced embedding: The pH of the mixture was adjusted to 6.2-6.8, and the mixture was kept at 45-50℃ for 15-30 minutes. The mixture was then extruded or ultrasonically treated to obtain temperature-responsive essential oil liposomes.

2. The method according to claim 1, characterized in that, The mass ratio of phospholipids, cholesterol, and oleamide in temperature-responsive essential oil liposomes is 75:5-20:3-15.

3. The method according to claim 1, characterized in that, In step (3), the oleamide solution is an ethanol solution of oleamide with a mass concentration of 5-10%.

4. The method according to claim 1, characterized in that, In step (1), the sum of the masses of phospholipids, cholesterol, and oleamide is in a mass ratio of 1:0.05-0.2 to the mass of the essential oil.

5. The method according to claim 1, characterized in that, The essential oil used in step (1) is lavender essential oil.

6. Temperature-responsive essential oil liposomes prepared by the method according to any one of claims 1-5.

7. The temperature-responsive essential oil liposome according to claim 6, characterized in that, The particle size of temperature-responsive essential oil liposomes is 80-200 nm.

8. The application of the temperature-responsive essential oil liposomes as described in claim 6 in the preparation of daily chemical products, textile finishing, or environmental fragrance materials.

9. A method for improving the temperature response performance of essential oil liposomes, characterized in that, The temperature-responsive essential oil liposomes described in claim 6 were used.

10. A method for enhancing the rapid release of essential oils at 38-42℃, characterized in that, The temperature-responsive essential oil liposomes described in claim 6 were used.

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