Preparation method of hyaluronidase modified quercetin liposome

Abstract

The invention discloses a preparation method of hyaluronidase modified quercetin lipidosome, and relates to the technical field of natural plant preparations, and the preparation method comprises the following steps: step 1, weighing distearoyl phosphatidylacetamide-polyethylene glycol 2000-succinimide, dissolving in dimethylformamide, adding hyaluronidase, dialyzing in pure water, filtering, and drying to obtain a quercetin liposome solution; and carrying out freeze drying to obtain the DSPE-PEG2000-HAase. 2, adding quercetin, lecithin and cholesterol into a solvent, mixing, and carrying out reduced pressure rotary evaporation to obtain an oil phase; step 3, preparing a PBS (Phosphate Buffer Solution), adding distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-hyaluronidase, and uniformly mixing, so as to obtain a PBS-DSPE-PEG2000-HAase solution, namely a water phase; and 4, adding the water phase into the oil phase, heating in a water bath, and emulsifying to obtain the hyaluronidase modified quercetin liposome. The hyaluronidase modified quercetin liposome obtained by the invention is small in particle size, uniform in distribution and high in encapsulation efficiency, is beneficial to intravenous injection, effectively plays an anti-tumor role, and has a good market application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of natural plant preparations, and in particular to a method for preparing hyaluronidase-modified quercetin liposomes. Background Art

[0002] Quercetin is a widely existing natural plant flavonol with pharmacological effects such as anti-tumor, antioxidant, antibacterial, antiviral, anti-inflammatory and anti-allergic. According to current research, quercetin has low toxicity, and the LD50 of oral administration to mice is about 10-50g / kg, and no toxic reactions have been observed in long-term use. In view of the wide potential anti-tumor effect of quercetin, in recent years, many countries have used quercetin as a drug for the treatment of various tumors.

[0003] However, the pharmaceutical characteristics of quercetin are not ideal. The main problems include: poor in vitro stability of quercetin (light instability, acid-base instability), poor solubility and inability to be injected intravenously. Therefore, the development of a suitable quercetin carrier by using pharmaceutical methods to improve its stability in application has high potential and value.

[0004] As a new type of carrier, liposomes have the characteristic of improving the stability of the main drug. Although there has been some progress in the research on the preparation of quercetin liposomes (CN202010879360.9, CN201110453651.2, CN201210590103.9), the quercetin liposomes currently prepared cannot effectively penetrate the tumor microenvironment to play an effective therapeutic role.

[0005] Therefore, the occurrence and progression of malignant tumors are regulated by the tumor microenvironment. The inability of quercetin liposomes to penetrate the tumor microenvironment is due to the presence of hyaluronic acid. Studies have shown that the content of hyaluronic acid in the tumor microenvironment is higher than that in normal tissues. High content of hyaluronic acid is an independent factor affecting tumor prognosis. Hyaluronic acid has been proven to form a physical barrier that affects the penetration of chemotherapeutic drugs and treatment response. Therefore, there is an urgent need for a method to prepare hyaluronidase-modified quercetin liposomes with high encapsulation efficiency and good transdermal absorption. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing hyaluronidase-modified quercetin liposomes to solve the problems raised in the above background technology.

[0007] In order to achieve the above object, the present invention provides the following technical solution: a method for preparing hyaluronidase-modified quercetin liposomes, comprising the following steps:

[0008] Step 1: Distearoylphosphatidylacetamide-polyethylene glycol 2000-succinimide was weighed and dissolved in 1 mL of dimethylformamide, and 50 mg of hyaluronidase dissolved in PBS water was added. The reaction was allowed to react at room temperature for 0.5 h. The reaction solution was transferred to a dialysis bag and dialyzed in pure water for 24 h. The dialyzate was collected and freeze-dried to obtain DSPE-PEG2000-HAase.

[0009] Step 2: adding quercetin, lecithin and cholesterol into a solvent, mixing, and performing rotary evaporation under reduced pressure to obtain an oil phase;

[0010] Step 3: prepare PBS buffer, add distearoylphosphatidylethanolamine-polyethylene glycol 2000-hyaluronidase, mix well to obtain PBS-DSPE-PEG2000-HAase solution, i.e., aqueous phase;

[0011] Step 4: Add the aqueous phase to the oil phase, heat in a water bath, and then emulsify to obtain hyaluronidase-modified quercetin liposomes.

[0012] Preferably, in step 2, the mass ratio of quercetin, lecithin and cholesterol is 2:12:3.

[0013] Preferably, in step 2, the solvent is one or more of chloroform, ethanol, and ether, and the mass volume ratio of quercetin to the solvent is 1 g:200 mL.

[0014] Preferably, in step three, the PBS buffer concentration is 20-30 mmol / L and the pH value is 7.4.

[0015] Preferably, in step 4, the water bath heating temperature is 45° C. and the heating time is 30 min.

[0016] Preferably, in step 4, the emulsification speed is 800 r / min and the emulsification time is 30 min.

[0017] Preferably, in step 4, the emulsified solution is stable at 4° C. for 15 days.

[0018] In the above technical solution, the technical effects and advantages provided by the present invention are:

[0019] The quercetin liposome modified by hyaluronidase obtained by the present invention has a small particle size and uniform distribution, a high encapsulation rate, is conducive to intravenous injection, effectively exerts an anti-tumor effect, and has a good market application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0021] Figure 1 This is an appearance diagram of the quercetin liposome modified with hyaluronidase of the present invention;

[0022] Figure 2 is a particle size distribution diagram of the hyaluronidase-modified quercetin liposome of the present invention;

[0023] Figure 3 It is a transmission electron microscopy image of the quercetin liposome modified with hyaluronidase of the present invention. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0025] The present invention provides Figures 1 to 3 The method for preparing a hyaluronidase-modified quercetin liposome comprises the following steps:

[0026] (1) Preparation of the oil phase:

[0027] 1. Weigh 2g quercetin, 12g lecithin and 3g cholesterol into a beaker, add about 10mL anhydrous ethanol, stir and ultrasonically dissolve until no obvious solids are left;

[0028] 2. Place the beaker on a magnetic stirrer and heat it in water at 45°C. Spin dry the anhydrous ethanol in the solution. At this time, there will be a uniform film layer on the inner wall of the beaker, which is the oil phase.

[0029] (2) Preparation of the aqueous phase:

[0030] Weigh distearoylphosphatidylacetamide-polyethylene glycol 2000-succinimide and dissolve it in 1 mL of dimethylformamide, add 50 mg of hyaluronidase dissolved in PBS water (8 mL pH = 7.4), react at room temperature for 0.5 h, transfer the reaction solution to a dialysis bag (molecular weight cutoff 3500 Da), dialyze in pure water for 24 h, collect the dialysate and freeze-dry to obtain DSPE-PEG2000-HAase. Prepare 10 mL of PBS buffer, add 0.25 g of DSPE-PEG2000-HAase, mix well to obtain PBS-DSPE-PEG2000-HAase solution, i.e., the aqueous phase;

[0031] (3) Emulsification:

[0032] The above 10 mL of aqueous phase was added to the oil phase, and a magnetic stirrer was used at a speed of 7000 r / min, and heated in a water bath at 45°C for 30 min, and finally stabilized at 4°C for 2 hours to obtain quercetin liposomes. Figure 1 Store at 4°C overnight.

[0033] (4) Characterization:

[0034] 1. Use AVESTIN liposome extruder LF1 to filter the prepared quercetin liposome samples, use 0.4um, 0.2um, and 0.1um filter membranes to filter, sonicate the samples for 30 minutes, and measure the particle size distribution. Most of them are around 130nm, such as Figure 2 shown.

[0035] 2. Spread the sealing film flat on the glass slide, use tweezers to place the copper mesh clip on the sealing film, use a liquid gun to take 10u l of sample and drop it on the copper mesh, time it for 10 minutes, and use a small piece of filter paper to absorb the excess liquid. Use a liquid gun to take 10u l of 3% uranyl acetate and drop it on the copper mesh, time it for 1-3 minutes, and then use a small piece of filter paper to absorb the excess staining liquid. Time it for 10 minutes and let the copper mesh dry naturally. Use a JEM-1200EX transmission electron microscope from JEOL Ltd. to take pictures, and you can clearly see the phospholipid bilayer, such as Figure 3 shown.

[0036] Test Example 1: Hyaluronidase-modified Quercetin Liposome Encapsulation Efficiency Test

[0037] The overall concentration of quercetin in the hyaluronidase-modified quercetin liposomes obtained in Example 1 was measured by ultraviolet spectrophotometry, and the encapsulation efficiency of the hyaluronidase-modified quercetin liposomes was calculated.

[0038] The experimental conditions are as follows

[0039] Wavelength: 200-600nm;

[0040] Blank solvent: methanol;

[0041] Control solution: weigh 10 mg of sample, add 10 ml of methanol, shake to dissolve, and filter (0.22 um filter head).

[0042] Sample solution (quercetin liposome modified with hyaluronidase): 100uL of quercetin liposome modified with hyaluronidase was taken and diluted 10 times with methanol, then centrifuged at 12000r / min for 10min and the supernatant was taken.

[0043] The ultraviolet spectrophotometer was used to detect at 200-600 nm, and the absorbance value was obtained and then brought into the standard curve of quercetin methanol solution. Under the above conditions, the free concentration of quercetin was 0.19 mg / ml; the concentration of quercetin dosage was 0.2 mg / ml.

[0044] The formula for calculating the liposome encapsulation efficiency is EE% = (Wa / Wb) × 100%; Wa is the total amount of the drug in the liposome suspension; Wb is the amount of drug delivered.

[0045] The quercetin liposome encapsulation efficiency was found to be 95%.

[0046] Test Example 2: Hyaluronidase Activity Test of Quercetin Liposomes Modified with Hyaluronidase

[0047] The overall concentration of hyaluronidase in the hyaluronidase-modified quercetin liposomes obtained in Example 1 was detected by ultraviolet spectrophotometry, and the enzyme activity of the hyaluronidase-modified quercetin liposomes was calculated.

[0048] The experimental conditions are as follows

[0049] Wavelength: 200-600nm;

[0050] Blank solvent: 1 mL of 0.01% (w / v) bovine serum albumin solution mixed with 1 mL of 0.3 mg / mL hyaluronic acid solution;

[0051] Control solution: 0.5 mL 300 U / mL hyaluronidase solution, 0.5 mL 0.01% (w / v) bovine serum albumin solution, and 1 mL 0.3 mg / mL hyaluronic acid solution mixed;

[0052] Sample solution (quercetin liposome modified with hyaluronidase): 0.5 mL of quercetin liposome modified with hyaluronidase, 0.5 mL of 0.01% (w / v) bovine serum albumin solution and 1 mL of 0.3 mg / mL hyaluronic acid solution were mixed;

[0053] The ultraviolet spectrophotometer was used to detect at 200-600 nm, and the absorbance value was obtained and then brought into the standard curve of the bovine serum albumin hyaluronic acid mixed solution. Under the above conditions, the hyaluronidase-modified quercetin liposomes contained 201 U / mg of hyaluronidase; the control solution contained 266 U / mg of hyaluronidase.

[0054] The hyaluronidase activity calculation formula is: enzyme activity % = (T1 / T2) × 100%; T1 is the total amount of hyaluronidase contained in the quercetin liposome modified with hyaluronidase; T2 is the total amount of hyaluronidase.

[0055] The results showed that the enzymatic activity of hyaluronidase-modified quercetin liposomes was 93.55% compared with the pure enzyme.

[0056] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing hyaluronidase-modified quercetin liposomes, characterized in that: The steps include: Step 1: Distearoylphosphatidylacetamide-polyethylene glycol 2000-succinimide was weighed and dissolved in 1 mL of dimethylformamide, and 50 mg of hyaluronidase dissolved in PBS water was added. The reaction was allowed to react at room temperature for 0.5 h. The reaction solution was transferred to a dialysis bag and dialyzed in pure water for 24 h. The dialyzate was collected and freeze-dried to obtain DSPE-PEG2000-HAase. Step 2: adding quercetin, lecithin and cholesterol into a solvent, mixing, and performing rotary evaporation under reduced pressure to obtain an oil phase; Step 3: prepare PBS buffer, add distearoylphosphatidylethanolamine-polyethylene glycol 2000-hyaluronidase, mix well to obtain PBS-DSPE-PEG2000-HAase solution, i.e., aqueous phase; Step 4: Add the aqueous phase to the oil phase, heat in a water bath, and then emulsify to obtain hyaluronidase-modified quercetin liposomes.

2. The method for preparing a hyaluronidase-modified quercetin liposome according to claim 1, characterized in that: In the step 2, the mass ratio of quercetin, lecithin and cholesterol is 2:12:

3.

3. The method for preparing a hyaluronidase-modified quercetin liposome according to claim 1, characterized in that: In the step 2, the solvent is one or more of chloroform, ethanol, and ether, and the mass volume ratio of quercetin to the solvent is 1g:200mL.

4. The method for preparing a hyaluronidase-modified quercetin liposome according to claim 1, characterized in that: In step three, the concentration of the PBS buffer is 20-30 mmol / L, and the pH value is 7.

4.

5. The method for preparing a hyaluronidase-modified quercetin liposome according to claim 1, characterized in that: In step 4, the water bath is heated at 45° C. for 30 minutes.

6. The method for preparing a hyaluronidase-modified quercetin liposome according to claim 1, characterized in that: In the step 4, the emulsification speed is 800 r / min and the emulsification time is 30 min.

7. The method for preparing a hyaluronidase-modified quercetin liposome according to claim 1, characterized in that: In the step 4, the emulsified solution is stable at 4° C. for 15 days.

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