Preparation method and application of water-in-oil Pickering emulsion gel loaded with two-phase medicine

Abstract

The invention discloses a preparation method of water-in-oil Pickering emulsion gel loaded with a two-phase medicine and application of the water-in-oil Pickering emulsion gel loaded with the two-phase medicine. According to the method, an amino polymer with positive charges is dissolved in oil to serve as an oil phase, nano-particles with negative charges are dispersed in water to serve as a water phase, the oil phase and the water phase are mixed and emulsified to prepare the water-in-oil Pickering emulsion gel, and the water-in-oil Pickering emulsion gel has good stability and biocompatibility and can serve as an excellent drug delivery platform; the simultaneous loading of hydrophilic and hydrophobic active substances is realized. The degradation rate of the prepared Pickering emulsion gel can be controllably adjusted by adjusting the adding proportion of iodized oil and castor oil in the oil phase, release can be triggered under the ultrasonic action, and controlled release of the water-in-oil Pickering emulsion gel is achieved by adjusting and controlling different oil-water phase compositions, ultrasonic power and ultrasonic time. The prepared Pickering emulsion gel can be used for realizing efficient packaging of a hydrophobic sound sensitive agent, and can be used for realizing anti-tumor treatment through sonodynamic therapy.

Description

Technical Field

[0001] This invention relates to the field of drug delivery, and more specifically to a method for preparing a water-in-oil Pickering emulsion gel loaded with a biphasic drug and its application. Background Technology

[0002] Emulsion gels, as soft solid materials composed of aqueous and oil phases, possess both excellent stability and tunable viscoelasticity, enabling efficient encapsulation of hydrophilic or hydrophobic active substances. They are widely regarded as an excellent drug delivery platform. Encapsulating active substances through delivery systems can significantly enhance their stability, bioavailability, and functionality.

[0003] Most common emulsion gel preparation methods can only encapsulate single hydrophilic or hydrophobic active substances, making it difficult to achieve synergistic delivery of both. Conventional emulsion gels also exhibit poor stability, leading to leakage or inactivation of the encapsulated substance. Furthermore, the physical properties and network structure of emulsion gels may be disrupted by external stimuli such as pH, temperature, and enzymes, potentially releasing the encapsulated active substance and compromising its integrity, making precise control difficult. Hydrophobic sonosensitive agents suffer from poor water solubility, poor tumor aggregation, and strong phototoxicity to the skin, limiting their widespread clinical application. Currently, commonly used sonosensitive agent delivery methods mainly include liposomes and nanoparticles. However, liposomes and nanoparticles have low drug loading efficiency, poor long-term stability, and are prone to burst release.

[0004] Therefore, it is of great significance to develop a method that enables efficient loading and delivery of hydrophobic acoustic agents. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing a water-in-oil Pickering emulsion gel loaded with biphasic drugs and its applications. This method involves dissolving a positively charged amino polymer in oil as the oil phase and dispersing negatively charged nanoparticles in water as the aqueous phase. The oil and water phases are mixed and emulsified to prepare a water-in-oil Pickering emulsion gel, which exhibits good stability and biocompatibility, serving as an excellent drug delivery platform for the simultaneous loading of hydrophilic drugs and hydrophobic sonosensitive agents. By adjusting the ratio of iodized oil to castor oil in the oil phase, the degradation rate of the prepared Pickering emulsion gel can be controlled, and its release can be triggered by ultrasound. Controlled release of the water-in-oil Pickering emulsion gel can be achieved by regulating different oil-water phase compositions, ultrasonic power, and ultrasonic time. The prepared Pickering emulsion gel can achieve efficient encapsulation of hydrophobic sonosensitive agents and can be used for anti-tumor therapy via sonodynamic therapy.

[0006] This invention first discloses a method for preparing a water-in-oil Pickering emulsion gel loaded with a biphasic drug, comprising the following steps:

[0007] (1) The hydrophobic acoustic sensitizer and the positively charged amino polymer are dissolved in oil as the oil phase, and the hydrophilic drug and the negatively charged nanoparticles are dispersed in water as the aqueous phase;

[0008] (2) The above oil phase and water phase are mixed, and the water phase is emulsified in the oil phase. During the emulsification process, the positively charged amino polymer and the negatively charged nanoparticles stabilize the oil-water interface through electrostatic interaction, forming a cross-linked network, so that the droplets are closely arranged and not easily flowed or deformed, thus obtaining a stable Pickering emulsion gel.

[0009] Preferably, in step (1), the oil is a mixture of iodized oil and castor oil, wherein the volume ratio of castor oil is 5-50%.

[0010] Preferably, in step (1), the concentration of the positively charged amino polymer in the oil phase is 8-30 mg / mL, and the concentration of the negatively charged nanoparticles in the aqueous phase is 0.83-10 mg / mL; the positively charged amino polymer is bis(amino) polystyrene; and the negatively charged nanoparticles are shellac nanoparticles.

[0011] Preferably, during step (2), the volume ratio of the oil phase is 20% to 50%.

[0012] Preferably, in step (2), the emulsification process can be formed by simple eddy current oscillation. In addition, high-energy emulsification methods such as high-speed homogenization and high-pressure homogenization can also be used.

[0013] Preferably, the hydrophobic acoustic sensitizer is one or more of hematoporphyrin, hematoporphyrin monomethyl ether (HMME), protoporphyrin, and IR780, and the concentration of the hydrophobic acoustic sensitizer in the oil phase is 0.1-5 mg / mL. More preferably, the hydrophobic acoustic sensitizer is hematoporphyrin monomethyl ether (HMME); the concentration of HMME is 1 mg / mL.

[0014] Preferably, the hydrophilic active substance is one or more of PD-1 antibody (αPD-1), T cell immunoglobulin, mucin domain protein 4 (Tim-4), and FITC, and the concentration of the hydrophilic active substance in the aqueous phase is 0.1-5 mg / mL.

[0015] The present invention also provides a water-in-oil Pickering emulsion gel loaded with a biphasic drug prepared by the method.

[0016] This invention also provides the application of the aforementioned water-in-oil Pickering emulsion gel in the preparation of tumor sonodynamic therapy drugs. The water-in-oil Pickering emulsion gel, in conjunction with ultrasound, achieves tumor treatment through sonodynamic therapy. Preferably, the tumor is pancreatic cancer.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) This invention proposes a method for preparing a water-in-oil Pickering emulsion gel loaded with biphasic drugs and its application. By adjusting the composition of the oil phase and the ratio of the oil and water phases, different emulsion gels can be prepared. Positively charged amino polymers and negatively charged nanoparticles stabilize the oil-water interface through electrostatic interaction, forming a cross-linked network, so that the droplets are tightly arranged and not easily flowed or deformed, thus obtaining a stable Pickering emulsion gel.

[0019] (2) The water-in-oil Pickering emulsion gel prepared by the present invention has strong stability and adjustable viscoelastic properties, and different emulsion gels can be prepared according to application requirements.

[0020] (3) The water-in-oil Pickering emulsion gel prepared by the present invention can efficiently encapsulate hydrophobic sonosensitive agents and hydrophilic drugs, and degrades slowly. It can be released by ultrasound triggering and has been widely used in the field of active substance delivery.

[0021] (4) Based on the preparation method of the Pickering emulsion gel of the present invention, the present invention also prepares a drug-loaded Pickering emulsion gel. The obtained drug-loaded Pickering emulsion gel can be directly used as a delivery system for drug administration. The drug is triggered and released by ultrasound, and tumor growth is effectively inhibited by sonodynamic therapy. Attached Figure Description

[0022] Figure 1 A schematic diagram illustrating the preparation of water-in-oil Pickering emulsion gel and its application in tumor sonodynamic therapy.

[0023] Figure 2 To prepare a water-in-oil Pickering emulsion gel simultaneously loaded with HMME and αPD-1;

[0024] Figure 3 Confocal images and 3D reconstructions of Pickering emulsion gels;

[0025] Figure 4 Rheological properties of Pickering emulsion gels prepared at different oil-water volume ratios;

[0026] Figure 5Centrifugal stability of water-in-oil Pickering emulsion gels simultaneously loaded with HMME and αPD-1;

[0027] Figure 6 To ensure the temperature stability of water-in-oil Pickering emulsion gels simultaneously loaded with HMME and αPD-1;

[0028] Figure 7 In vitro simulated release of Pickering emulsion gels prepared with different oil phase compositions (5%, 10%, 15% and 20% castor oil added to the oil phase, respectively); (A) FITC added to the aqueous phase; (B) Nile red added to the oil phase;

[0029] Figure 8 The release of Pickering emulsion gel under ultrasound;

[0030] Figure 9 In vivo degradation of Pickering emulsion gels prepared with different oil phase compositions;

[0031] Figure 10 To investigate the antitumor therapy of HMME-loaded Pickering emulsion gel in mice. Detailed Implementation

[0032] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the present disclosure and do not limit the scope of the invention. All raw materials used in the embodiments are commercially available.

[0033] like Figure 1 The diagram illustrates the preparation of the water-in-oil Pickering emulsion gel of this invention and its application in sonodynamic therapy for tumors. This invention prepares a water-in-oil Pickering emulsion gel by dissolving a hydrophobic sonosensitive agent and a positively charged amino polymer in oil as the oil phase, and dispersing a hydrophilic drug and negatively charged nanoparticles in water as the aqueous phase. The oil and water phases are mixed and emulsified. This process yields a water-in-oil Pickering emulsion gel with good stability and biocompatibility, serving as an excellent drug delivery platform for the simultaneous loading of hydrophilic drugs and hydrophobic sonosensitive agents. The degradation rate of the prepared Pickering emulsion gel can be controlled by adjusting the ratio of iodized oil to castor oil in the oil phase, and its release can be triggered by ultrasound. Controlled release of the water-in-oil Pickering emulsion gel can be achieved by regulating different oil-water phase compositions, ultrasonic power, and ultrasonic time. The prepared Pickering emulsion gel enables efficient encapsulation of hydrophobic sonosensitive agents and can achieve anti-tumor therapy through sonodynamic therapy. The invention is further described in detail below with reference to specific embodiments.

[0034] Example 1: Preparation of water-in-oil Pickering emulsion gel loaded with biphasic drugs

[0035] (1) The hydrophobic sound sensitizer HMME and diamino polystyrene were dissolved in the oil phase, wherein the concentration of the hydrophobic sound sensitizer was 1 mg / mL and the concentration of diamino polystyrene was 10 mg / mL, and the volume ratio of iodized oil to castor oil in the oil phase was 1:1; the hydrophilic drug αPD-1 and shellac nanoparticles were dispersed in water as the aqueous phase, wherein the concentration of the hydrophilic drug αPD-1 was 2 mg / mL and the concentration of shellac nanoparticles was 1.7 mg / mL;

[0036] (2) Weigh out the oil phase and water phase separately according to the oil phase volume ratio of 33% and add them to the glass bottle. Then, vortex the mixture until a viscous emulsion is formed. After standing for a few minutes, the emulsion forms a gel. When the glass bottle is inverted, the emulsion gel will not flow, as shown in the attached figure. Figure 2 As shown.

[0037] (3) Images of the Pickering emulsion gel loaded with HMME were taken using a laser confocal microscope under excitation light of 405 nm, as shown in the attached image. Figure 3 As shown, red fluorescence can be observed in the oil phase, verifying that the prepared emulsion gel is of the water-in-oil type.

[0038] Example 2: Properties determination of water-in-oil Pickering emulsion gels loaded with biphasic drugs

[0039] (1) Rheological properties: According to the method in Example 1, in step (2), water-in-oil Pickering emulsion gels loaded with biphasic drugs were prepared with oil-water volume ratios of 1:1, 1:1.5, and 1:2, respectively. The rheological properties of the obtained gels were measured using a plate system of a rotational rheometer. The strain scanning results are attached. Figure 4 As shown in Figure A, the emulsion exhibits shear-thinning properties. When the strain is 0.1%, the elastic modulus G′ is greater than the viscous modulus G″, indicating that the emulsion gel is non-flowable. As the strain gradually increases, the elastic modulus G′ gradually decreases, while the viscous modulus G″ gradually increases. When the strain exceeds the yield point (10%), the elastic modulus G′ becomes less than the viscous modulus G″, and the emulsion gel begins to flow. The frequency scan results are attached. Figure 4 As shown in Figure B, the elastic modulus G′ is always greater than the viscous modulus G″, indicating that the emulsion hydrogel is non-flowable. This further proves that the obtained water-in-oil Pickering emulsion gel is in a gel state, and within this frequency scanning range, the storage modulus and loss modulus do not change significantly with increasing frequency. The viscosity curves of the emulsion gels prepared at different oil-to-water ratios exhibit shear thinning characteristics; as the amount of water phase increases, the viscosity of the emulsion gel increases, such as... Figure 4 As shown in C.

[0040] (2) Centrifugal stability: Following the method in Example 1, a water-in-oil Pickering emulsion gel loaded with HMME and αPD-1 was prepared using an oil-to-water volume ratio of 1:2. The prepared gel was poured into centrifuge tubes and centrifuged at speeds of 800 rpm, 1000 rpm, 1200 rpm, 1500 rpm, and 2000 rpm for 10 min, respectively. The stability of the emulsion gel was then observed. When the centrifugation speed was less than or equal to 1000 rpm, the aqueous and oil phases in the gel did not separate into layers, indicating good stability, as shown in the attached figure. Figure 5 As shown.

[0041] (3) Temperature stability: Following the method in Example 1, a water-in-oil Pickering emulsion gel loaded with HMME and αPD-1 was prepared using an oil-to-water volume ratio of 1:2. The prepared gels were placed at -20℃, 4℃, and 50℃ for 12 h, respectively. The stability of the gels was then observed, as shown in the attached figure. Figure 6 As shown. The emulsion gel is stable when the temperature is between 4℃ and 50℃; when the temperature is below the freezing point, the emulsion gel is destroyed due to water crystallization, however, a stable Pickering emulsion gel can be obtained again by shaking.

[0042] Example 3: In vitro simulated release and in vivo degradation of water-in-oil Pickering emulsion gel

[0043] The effect of different oil phase compositions on the in vitro release of water-in-oil Pickering emulsion gels was further investigated at 37℃. 0.5 mL of emulsion gel was added to the bottom of a 3 mL glass bottle, and 1 mL of PBS buffer was added to the top of the gel. The top buffer was collected every other day to determine the dye concentration, and fresh buffer was used after each measurement.

[0044] FITC was loaded into the aqueous phase and Nile red into the oil phase, respectively. The release of FITC and Nile red was determined by measuring their concentrations. Castor oil at concentrations of 5 vol%, 10 vol%, 15 vol%, and 20 vol% was added to iodized oil, and the corresponding release curves for FITC and Nile red are shown below. Figure 7 As shown in Figures A and B, the release rate of the water-in-oil Pickering emulsion gel decreases with increasing castor oil content.

[0045] Furthermore, the release of water-in-oil Pickering emulsion gels can be controlled by ultrasound. When the volume ratio of iodized oil to castor oil in the oil phase is 1:1, and hydrophobic HMME is loaded into the oil phase, the resulting release curve is shown in the figure. Figure 8 As shown, with the addition of ultrasound, the release of water-in-oil Pickering emulsion gel can be controlled and adjusted.

[0046] The degradation of Pickering emulsion gels in vivo was compared between Pickering emulsion gels prepared with iodized oil and castor oil in a 1:1 volume ratio. The two oil phase compositions were injected into mice, and the gel residues in the mice were analyzed. (See attached image.) Figure 9 As shown, adding castor oil to the oil phase can slow down the in vivo degradation rate of Pickering emulsion gel.

[0047] Example 4: Drug-loaded oil-in-water Pickering emulsion gel combined with sonodynamic therapy can effectively inhibit tumor growth.

[0048] Following the method in Example 1, the hydrophobic sonosensitive agent HMME was dissolved in the oil phase to prepare a water-in-oil Pickering emulsion gel (Gel@HMME) loaded with HMME. A low-immunogenic KPC pancreatic tumor model was used to verify the effect of Gel@HMME combined with sonodynamic therapy on inhibiting tumor growth in mice. After establishing a pancreatic tumor model in mice, different groups were set up: control group (PBS), ultrasound group (US), HMME group, Gel@HMME group, and Gel@HMME + US group. Each group was injected with 100 μL of the sample once. 24 h after injection, US treatment (1.0 MHz, 5 min) was performed for two consecutive days. The tumor volume was measured every 3-4 days. After treatment on day 15, the mice were sacrificed, and tumor mass was analyzed, as shown in the attached figure. Figure 10 As shown in Figure A. After 15 days of treatment, in terms of tumor volume among different treatment groups, the Gel@HMME + US group showed the highest tumor suppression effect, as shown in the attached figure. Figure 10 As shown in Figure B. Based on the tumor weight of mice in each group, the combined treatment of Gel@HMME + US resulted in the greatest reduction in tumor weight, as shown in the attached figure. Figure 10 As shown in C and D, drug-loaded oil-in-water Pickering emulsion gels combined with sonodynamic therapy can effectively inhibit tumor growth.

[0049] The above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a water-in-oil Pickering emulsion gel loaded with a biphasic drug, characterized in that, Includes the following steps: (1) The hydrophobic acoustic sensitizer and the positively charged amino polymer are dissolved in oil as the oil phase, and the hydrophilic drug and the negatively charged nanoparticles are dispersed in water as the aqueous phase; (2) The above oil phase and water phase are mixed, and the water phase is emulsified in the oil phase. During the emulsification process, the positively charged amino polymer and the negatively charged nanoparticles stabilize the oil-water interface through electrostatic interaction, forming a cross-linked network, so that the droplets are closely arranged and not easily flowed or deformed, thus obtaining a stable Pickering emulsion gel.

2. The method for preparing water-in-oil Pickering emulsion gel according to claim 1, characterized in that, In step (1), the oil is a mixture of iodized oil and castor oil, wherein the volume ratio of castor oil is 5-50%.

3. The method for preparing the water-in-oil Pickering emulsion gel according to claim 1, characterized in that, In step (1), the concentration of the positively charged amino polymer in the oil phase is 8-30 mg / mL, and the concentration of the negatively charged nanoparticles in the aqueous phase is 0.83-10 mg / mL; the positively charged amino polymer is bisamino polystyrene; and the negatively charged nanoparticles are shellac nanoparticles.

4. The method for preparing water-in-oil Pickering emulsion gel according to claim 1, characterized in that, During step (2), the volume ratio of the oil phase is 20% to 50%.

5. The method for preparing water-in-oil Pickering emulsion gel according to claim 1, characterized in that, The emulsification process is achieved through eddy current oscillation or high-energy emulsification.

6. The method for preparing the water-in-oil Pickering emulsion gel according to claim 1, characterized in that, The hydrophobic acoustic sensitizer is one or more of hematoporphyrin, hematoporphyrin monomethyl ether (HMME), protoporphyrin and IR780, and the concentration of the hydrophobic acoustic sensitizer in the oil phase is 0.1-5 mg / mL.

7. The method for preparing the water-in-oil Pickering emulsion gel according to claim 1, characterized in that, The hydrophilic active substance is one or more of PD-1 antibody (αPD-1), T cell immunoglobulin, mucin domain protein 4 (Tim-4), and FITC, and the concentration of the hydrophilic active substance in the aqueous phase is 0.1-5 mg / mL.

8. The method for preparing the water-in-oil Pickering emulsion gel according to claim 6, characterized in that, The preferred hydrophobic sonosensitive agent is hematoporphyrin monomethyl ether (HMME); the concentration of HMME is 1 mg / mL.

9. A water-in-oil Pickering emulsion gel loaded with a biphasic drug, prepared by the method according to any one of claims 1-8.

10. The use of the water-in-oil Pickering emulsion gel according to claim 9 in the preparation of tumor sonodynamic therapy drugs.

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