CN105287431A - Polymer lipid sphere carrying active drugs and preparation method thereof - Google Patents

Abstract

The invention relates to a polymer lipid sphere loaded with active medicine and a preparation method thereof. The polymer lipid sphere comprises a hydrophobic skeleton composed of a polymer and a phospholipid layer coated outside the hydrophobic skeleton, the hydrophobic tails of the phospholipid molecules of the phospholipid layer are embedded in the hydrophobic skeleton, and The hydrophilic head group is exposed on the outer surface; active drugs are embedded inside the hydrophobic framework; the average particle diameter of the polymer lipid sphere is between 0.1-30 μm, and the dispersion coefficient is less than 0.1. The polymer lipid sphere of the present invention is uniform in size, controllable, high embedding rate, high activity, low burst release, stable drug release, long-acting sustained and controlled release of active drugs, degradable, multi-chamber or core The polymer lipid sphere with a shell structure can improve its uptake by M cells, thereby improving the transport volume and transport efficiency of drugs or vaccines through M cells.

Description

A kind of active drug-loaded polymer lipid sphere and its preparation method

technical field

The invention relates to the field of medical technology, in particular to a polymer lipid ball loaded with active drugs and a preparation method thereof, which can be used for loading active drugs such as proteins, polypeptides or vaccines.

Background technique

In recent years, with the rapid development of biotechnology and genetic engineering, a large number of biologically active drugs such as proteins, peptides, and vaccines have flourished, and more and more drugs have been used clinically. Bioactive drug molecules are relatively fragile and susceptible to various physical and chemical factors, resulting in short circulation half-life and low bioavailability. At present, most bioactive drugs are administered through parenteral routes, which usually require repeated injections to prolong the curative effect, resulting in Patient compliance is poor.

In order to prolong the circulating half-life of biologically active drugs in the body and reduce the number of administrations, active drugs such as proteins and peptides can be embedded in suitable carriers to make them release drugs continuously at the injection site or in vivo, and improve drug stability to achieve Efficient therapeutic effect. At present, a variety of microparticle sustained and controlled release systems based on synthetic polymers and natural component materials have been developed at home and abroad, including: oil-in-water emulsions, liposomes, microspheres and nanoparticles, etc. It is still a challenge to release and maintain the activity of the embedded protein.

Lipid globules are usually prepared by a method similar to liposomes, that is, after the lipid material is formed into a film, and then loaded with active drugs such as proteins during the hydration process, the lipoglobulins prepared by this method are easy to leak, and the protruding High release and fast release; later, the double emulsion method was developed, and the emulsion was prepared by traditional mechanical stirring, ultrasonic or homogeneous method. The prepared emulsion had a wide particle size distribution, resulting in a wide particle size distribution of the final microspheres. The repeatability of microsphere particle size and particle size distribution, embedding rate, and release behavior between batches is poor, making the reproducibility of the drug effect between batches poor. As a drug carrier, the repeatability of microsphere preparation between batches is crucial. In addition, the particle size and uniformity of the microspheres are also very important factors affecting the drug efficacy in vivo. However, the traditional methods cannot prepare microspheres with uniform particle size and controllability, and it is difficult to carry out the relationship between the particle size of the microspheres and the drug effect. It is difficult to screen the appropriate particle size requirements for different drugs and their different routes of administration.

Contents of the invention

Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a long-acting slow-controlled release degradable, active-loaded drug with uniform size, controllability, high embedding rate, high activity, low burst release and stable drug release. Polymeric lipid spheres with multi-chamber or core-shell structures and methods for their preparation.

In a first aspect, the present invention provides a polymer lipid sphere loaded with an active drug, the polymer lipid sphere comprises a hydrophobic skeleton composed of a polymer and a phospholipid layer coated on the outside of the hydrophobic skeleton, the The hydrophobic tail end of the phospholipid molecule of the phospholipid layer is embedded in the hydrophobic skeleton, while the hydrophilic head group is exposed on the outer surface; active drugs are embedded in the hydrophobic skeleton; the average particle size of the polymer lipid globule is The diameter is between 0.1-30μm, and the dispersion coefficient is less than 0.1.

The phospholipid layer on the surface of the polymer lipid sphere loaded with active drug provided by the invention has good affinity with the lipid on the cell membrane surface, and is easier to be absorbed by M compared with simple polymer (such as PLGA) microspheres. Cellular uptake, which facilitates drug transport through M cells, can be used as an oral drug carrier or an oral vaccine delivery system.

The polymer lipid globules loaded with active drugs provided by the present invention have a uniform and controllable size, and are degradable polymer lipid globules that can load various biologically active drugs such as proteins, polypeptides or vaccines. The lipid globules have Multi-chamber or core-shell structure, its particle size dispersion index (PDI) is less than 0.1, its particle size is controllable from nanometer to micrometer, the drug embedding rate of the lipid globule is above 80%, and the drug activity retention rate reaches 80% Above, the drug burst release rate is as low as 20%, and can be released continuously for 4 weeks or longer.

The above-mentioned PDI is defined as: polydispersity coefficient, which represents the degree of uniform dispersion of particles, that is, particle size distribution coefficient. The smaller the value, the more uniform the particle distribution in the solution. Conversely, the larger the value, the broader the particle distribution in the solution. Emulsions with a PDI less than 0.1 are generally considered to be monodisperse.

The above-mentioned entrapment efficiency (EE) is defined as the percentage of the amount of drug embedded into lipid globules in the total amount of drug input, which is calculated according to the following formula.

$\mathrm{<span\; class="notranslate">\; EE</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; W</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}{\mathrm{<span\; class="notranslate">\; W</span>}}$

In the formula, EE represents the drug embedding rate, W1 represents the amount _of drug embedded into lipid globules, and W represents the total amount of drug input.

In the present invention, the average particle diameter of the polymer lipid globule is between 0.1-30 μm, such as 0.1 μm, 0.12 μm, 0.15 μm, 0.2 μm, 0.5 μm, 0.8 μm, 1.0 μm, 2.0 μm, 3.0 μm, 5.0 μm, 10.0 μm, 15.0 μm, 18.0 μm, 21.0 μm, 22.5 μm, 27 μm, 29 μm, 0.5-20 μm, 0.5-10 μm, 1-3 μm, 0.1-3 μm or 2-15 μm, preferably 0.1-3 μm.

In the present invention, active drug solutions such as proteins, polypeptides or vaccines of different concentrations can be embedded in the polymer lipid spheres, active drugs such as fat-soluble proteins, polypeptides or vaccines can also be embedded, and solid particle dispersions can also be embedded. Active drugs such as proteins, peptides or vaccines in the state. That is to say, the active drug can be a hydrophobic drug or a hydrophilic drug, and the hydrophilic drug can be in the form of an aqueous solution or a solid particle according to its existing form, and the corresponding active drug-loaded polymer lipid globules The structure is different, reflected in:

As a preferred solution of the present invention, when the active drug is a hydrophobic drug, the active drug is embedded in the hydrophobic framework, specifically: the hydrophobic drug is directly embedded in the hydrophobic framework. Wherein, the hydrophobic drugs include hydrophobic proteins, polypeptides or vaccines.

As a preferred solution of the present invention, when the active drug is a hydrophilic drug, the hydrophobic skeleton is embedded with an active drug, specifically: the hydrophobic skeleton is embedded with a cavity structure; The chamber structure includes a chamber surrounded by a phospholipid layer and an inner water phase inside the chamber and hydrophilic drugs in the inner water phase, or the chamber structure includes a chamber surrounded by a phospholipid layer and Hydrophilic drug particles inside the chamber. Wherein, the hydrophilic drugs include hydrophilic proteins, polypeptides or vaccines.

As a preferred solution of the present invention, the polymer lipid sphere adopts two materials of degradable polymer and phospholipid in the preparation process. Among them, phospholipid materials have the function of co-emulsifier, which is beneficial to improve the drug embedding rate, maintain biological activity, reduce burst release and stable release. The degradable polymer can be selected from polylactic acid, polyglycolic acid, polylactone, polyanhydride, polytrimethylene carbonate, polydioxanone or their copolymers; preferably, the copolymer The compound is selected from polylactic acid-polyglycolic acid copolymer or polylactic acid-polydioxanone. Preferably, the phospholipid molecules are selected from soybean lecithin, egg yolk lecithin, phospholipid derivatives, fatty acids, fatty amides, phosphatidylcholine or derivatives thereof, preferably hydrogenated soybean lecithin, phosphatidylcholine, egg yolk lecithin.

In a second aspect, the present invention provides a method for preparing the active drug-loaded polymer lipid sphere described in the first aspect. According to the different active drugs, the method can be any of the following three methods:

Where the active drug is a hydrophobic drug, the method comprises the steps of:

(1) Dissolving the degradable polymer and the phospholipid together in an organic solvent as the oil phase O;

(2) dissolving the hydrophobic drug in the oil phase to make the drug-containing oil phase;

(3) adding the drug-containing oil phase into the external water phase W containing a stabilizer, and mechanically stirring to form an O/W type pre-emulsion;

(4) Press the O/W type pre-emulsion through the microporous membrane to obtain the O/W type emulsion;

(5) Solidifying the O/W emulsion, removing residual organic solvent, and freeze-drying to obtain polymer lipid spheres loaded with active drugs in dry powder form.

In the case where the active drug is an aqueous solution of a hydrophilic drug, the method comprises the following steps:

(1) Dissolving the degradable polymer and the phospholipid together in an organic solvent as the oil phase O;

( ₂ ) dispersing the hydrophilic drug aqueous solution W1 in the oil phase O to make W1/O type colostrum _;

(3) adding the W ₁ /O type colostrum into the external water phase W ₂ containing a stabilizer, and mechanically stirring to form a W ₁ /O/W ₂ type pre-double emulsion;

(4) Press the W ₁ /O/W ₂ type pre-double emulsion through the microporous membrane to obtain the W ₁ /O/W ₂ type double emulsion;

(5) The W ₁ /O/W ₂ type double emulsion is solidified, the residual organic solvent is removed, and freeze-dried to obtain polymer lipid spheres loaded with active drugs in dry powder form.

As a preferred solution of the present invention, the drug concentration in the hydrophilic drug aqueous solution W1 is _1-100 mg/mL.

As a preferred version of the present invention, the volume ratio of the hydrophilic drug aqueous solution W1 to the oil phase O is ₁ :1 to 1:10, preferably 1:10 _; the volume ratio of the oil phase O to the external water phase W2 The volume ratio is 1:1～1:100.

In the case where the active drug is a hydrophilic drug particle, the method comprises the following steps:

(1) Dissolving the degradable polymer and the phospholipid together in an organic solvent as the oil phase O;

(2) dispersing the hydrophilic drug particles S in the oil phase O to make S/O type colostrum;

(3) adding the S/O type colostrum into the external water phase W containing a stabilizer, and mechanically stirring to form an S/O/W type pre-double emulsion;

(4) the S/O/W type pre-multiplex emulsion is pressed through the microporous membrane to obtain the S/O/W type double emulsion;

(5) The S/O/W type double emulsion is solidified, the residual organic solvent is removed, and freeze-dried to obtain dry powder polymer lipid spheres loaded with active drugs.

As a preferred version of the present invention, in the above three methods, the stabilizer in the external water phase is selected from polyvinyl alcohol, polyglycerol fatty acid ester, polyoxyethylene sorbitan monooleate (Tween80), polyoxyethylene One or a combination of at least two of ethylene sorbitan laurate (Tween20) or sodium dodecyl sulfonate (SDS), preferably polyvinyl alcohol, polyoxyethylene sorbitan monooleate or sodium dodecyl sulfonate;

As a preferred solution of the present invention, in the above three methods, the concentration of the stabilizer is 0.1-5 wt%, preferably 1-5 wt%.

As a preferred solution of the present invention, in the above three methods, the organic solvent is an organic solvent in which both the degradable polymer and the phospholipid are soluble and easily volatilized, preferably dichloromethane or chloroform.

As a preferred version of the present invention, in the above three methods, the mass percentage range of the phospholipid and the polymer is 1-100%, which means that the quality of the phospholipid is 1-100% of the polymer mass, preferably 1 -30%.

As a preferred solution of the present invention, in the above three methods, the pore diameter of the microporous membrane in the step (4) is 0.5-50 μm, preferably 0.8-9.2 μm.

As a preferred solution of the present invention, in the above three methods, the pressure of the microporous membrane in the step (4) is 0.1-2 MPa.

Compared with the prior art, the method of the present invention has the following characteristics:

(1) In the degradable polymer lipid globules carrying proteins, polypeptides, vaccines and other active drugs provided by the present invention, the phospholipids of the lipid globules are embedded on the surface of the polymer to form a multi-chamber or core-shell structure, which is coated on the polymer surface. The phospholipids in the outer layer have a good affinity with the cell membrane. Compared with PLGA microspheres, lipid globules are easier to be taken up by M cells, which is conducive to the transport of drugs through M cells, increasing the amount of drugs or vaccines transported through M cells and Transport efficiency, can be used as oral drug carrier and oral vaccine delivery system.

(2) The degradable polymer lipid spheres loaded with proteins, polypeptides, vaccines and other active drugs provided by the present invention have uniform and controllable size, and the dispersion index (PDI) is less than 0.1, which ensures the repeatability of the experiment, and the drug The reproducibility of the in vivo and in vitro behavior of the carrier; the drug embedding rate is above 80%, and the burst release rate is as low as 20%, which overcomes the problem of high burst release rate of traditional sustained-release drugs, and is stable during embedding, release and storage. Both can effectively maintain the drug activity, and can release continuously for 4 weeks or longer.

(3) The method provided by the present invention for rapidly preparing active drug-degradable polymer lipid spheres with uniform size, such as proteins, polypeptides, and vaccines, can control the product by controlling the pore size and operating pressure of the microporous membrane during the preparation process. particle size and uniformity.

(4) The present invention does not need additional stabilizers and emulsifiers in the internal water phase or solid particles and oil phases to achieve the effects of stable emulsion droplets and sustained release, eliminating the difficulties caused by the determination of residues in the later stage, and at the same time Help reduce costs.

(5) The preparation conditions of the method of the present invention are low in energy consumption and mild; the preparation parameters are highly controllable and reproducible; the prepared lipid spheres have a uniform and controllable particle size, which is beneficial to large-scale industrial production.

In a word, the beneficial effect of the present invention is reflected in: for active drugs such as therapeutic proteins, polypeptides, and vaccines, the carrier can protect the drugs from acid and enzymatic hydrolysis, prolong the therapeutic effect, and will not cause unnecessary damage to the body. At the same time, the lipid sphere with multi-chamber or core-shell structure provided by the present invention can improve its uptake by M cells, thereby improving the transport volume and transport efficiency of drugs or vaccines through M cells. Therefore, the protein, polypeptide, vaccine and other active drug delivery systems can be used in a wide range of applications, and can be administered in a variety of ways to reasonably exert the drug effect and relieve pain for patients.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Figure 1 is a schematic flow chart of the preparation of drug-loaded degradable polymer lipid spheres by the rapid membrane emulsification method.

Figure 2 is a schematic diagram of the prepared lipid spheres with a core-shell (left) or multi-chamber (right) structure. Among them, 1 represents the phospholipid layer, 2 represents the hydrophobic skeleton composed of polymers, 3 represents the hydrophobic drug molecule, 4 represents the hydrophilic drug molecule, 5 represents the internal aqueous solution, and 6 represents the phospholipid layer of the cavity structure.

3 is a scanning electron micrograph of PLGA microspheres loaded with hydrophilic protein in Example 1.

Fig. 4 is the scanning electron micrograph of the lipid sphere loaded with hydrophilic protein polymer in Example 2.

Figure 5 is a scanning electron micrograph of the hydrophilic protein polymer-loaded lipid sphere in Example 3.

Figure 6 is a scanning electron micrograph of the hydrophobic protein polymer-loaded lipid sphere in Example 4.

7 is a transmission electron micrograph of the core-shell structure of the polymer lipid sphere in Example 5.

Fig. 8 is a laser confocal micrograph of the core-shell structure of polymer lipid spheres in Example 5 (scale bar 10 μm).

9 is a scanning electron micrograph of the drug-loaded polymer lipid sphere in Example 6.

10 is a scanning electron micrograph of the drug-loaded polymer lipid sphere in Example 7.

Fig. 11 is a scanning electron micrograph of the drug-loaded polymer lipid sphere in Example 8.

Fig. 12 is a scanning electron micrograph of the drug-loaded polymer lipid sphere in Example 9.

Fig. 13 is a graph showing the release behavior of drug-loaded microspheres (Example 1 and Example 2) in Example 10.

Fig. 14 is a comparison chart of the transport amount of the drug-loaded microspheres (Example 1 and Example 2) through M cells in Example 11.

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only preferred examples of the present invention, so as to better understand the present invention, and thus should not be considered as limiting the scope of the present invention.

The experimental methods in the following examples, unless otherwise specified, are conventional methods; the experimental materials used, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.

Example 1

A microporous membrane with a pore size of 9.2 μm was used to prepare PLGA microspheres loaded with hydrophilic proteins. The specific implementation method is shown in Figure 1: 10 mg bovine serum albumin was accurately weighed and dissolved in 500 μL deionized water as the inner water phase (W ₁ ); 160 mg of PLGA was added to 5 mL of dichloromethane solution to fully dissolve it as the oil phase (O); 50 mL of aqueous solution containing 1% PVA was taken as the external water phase (W ₂ ). Add the inner water phase to the oil phase, and undergo homogeneous emulsification (20000rpm, 1min) to form water-in-oil (W ₁ /O) type colostrum, then add the colostrum to the outer water phase, and mechanically stir (300rpm, 1min) , forming W ₁ /O/W ₂ type double emulsion. The double emulsion was poured into a rapid membrane emulsification reaction tank, and passed through the membrane 5 times under the action of nitrogen pressure of 0.1MPa to form an emulsion with uniform particle size. It was mechanically stirred and solidified overnight in a ventilated environment at room temperature, and evaporated to dryness. of organic solvents. Then, centrifuge wash 5 times to remove unloaded drug and residual stabilizer. Finally, the dispersed suspension is freeze-dried, and the dry powder is collected as drug-loaded PLGA microspheres. Its average particle size and particle size distribution are measured by a British Malvern nanometer particle size analyzer (ZetasizerNanoZS90), and the average particle size in water is 2893nm, and the particle size distribution coefficient PDI is 0.031. The particle size of the drug-loaded PLGA microspheres is uniform. Among them, the drug loading rate of the drug carrier detected by a microplate reader was 4.15%, and the embedding rate was 83.68%.

Example 2

A microporous membrane with a pore size of 9.2 μm was used to prepare polymer lipid spheres loaded with hydrophilic proteins. The specific implementation method is as shown in Figure 1: 10 mg of transferrin was accurately weighed and dissolved in 500 μL of deionized water as the inner water phase (W ₁ ); 20mgHSPC (hydrogenated soybean lecithin) and 140mgPLGA are added to 5mL methylene chloride solution to make it fully dissolved, as the oil phase (O); get a volume of 50mL containing 1% PVA aqueous solution as the external water phase (W ₂ ). Add the inner water phase to the oil phase, and undergo homogeneous emulsification (20000rpm, 1min) to form water-in-oil (W ₁ /O) type colostrum, then add the colostrum to the outer water phase, and mechanically stir (300rpm, 1min) , forming W ₁ /O/W ₂ type double emulsion. The double emulsion was poured into a rapid membrane emulsification reaction tank, and passed through the membrane 5 times under the action of nitrogen pressure of 0.1MPa to form an emulsion with uniform particle size. It was mechanically stirred and solidified overnight in a ventilated environment at room temperature, and evaporated to dryness. of organic solvents. Then, centrifuge wash 5 times to remove unloaded drug and residual stabilizer. Finally, the dispersed suspension is freeze-dried, and the dry powder is collected as the drug-loaded polymer lipid sphere. Its average particle size and particle size distribution are measured by a British Malvern nanometer particle size analyzer (ZetasizerNanoZS90), and the average particle size in water is 2831nm, and the particle size distribution coefficient PDI is 0.051. The particle size of the drug-loaded polymer lipid sphere is uniform. Among them, the drug loading rate of the drug carrier detected by a microplate reader was 6.07%, and the embedding rate was 95.45%.

Example 3

A microporous membrane with a pore size of 9.2 μm was used to prepare polymer lipid spheres loaded with hydrophilic proteins. The specific implementation method is shown in Figure 1: 10 mg ovalbumin was accurately weighed as the solid phase (S); 40 mg HSPC and 120 mg PLGA were added together. Dissolve it fully in 5 mL of dichloromethane solution, and use it as the oil phase (O); take a 100 mL aqueous solution containing 0.8% PVA as the external water phase (W ₂ ). Disperse the protein solid phase S in the oil phase, and homogeneously emulsify (20000rpm, 1min) to form a solid-in-oil (S/O) emulsion system, then add it to the external water phase, and mechanically stir (300rpm, 1min) , forming S/O/W2 double emulsion. The double emulsion was poured into a rapid membrane emulsification reaction tank, and passed through the membrane 4 times under a nitrogen pressure of 0.05MPa to form an emulsion with uniform particle size, which was mechanically stirred and solidified overnight in a ventilated environment at room temperature, and evaporated to dryness. of organic solvents. Then, centrifuge wash 5 times to remove unloaded drug and residual stabilizer. Finally, the dispersed suspension is freeze-dried, and the dry powder is collected as the drug-loaded polymer lipid sphere. Its average particle size and particle size distribution are measured by a British Malvern nanometer particle size analyzer (ZetasizerNanoZS90), and the average particle size in water is 2789nm, and the particle size distribution coefficient PDI is 0.061. The particle size of the drug-loaded polymer lipid sphere is uniform. Among them, the drug loading rate of the drug carrier detected by a microplate reader was 7.12%, and the embedding rate was 98.24%.

Example 4

A microporous membrane with a pore size of 9.2 μm was used to prepare polymer lipid spheres loaded with hydrophobic proteins. The specific implementation method is shown in Figure 1: Accurately weigh 10 mg of apolipoprotein, 60 mg of HSPC and 100 mg of PLGA and add them together to 5 mL of dichloromethane solution Dissolve it fully and use it as the oil phase (O); take a 200 mL aqueous solution containing 0.5% PVA as the water phase (W ₂ ). Add the oil phase to the water phase, and use mechanical stirring (300rpm, 1min) to form an oil-in-water (O/W) type pre-emulsion. After 3 passes through the membrane, an emulsion with uniform particle size was formed, which was solidified overnight with mechanical stirring in a ventilated environment at room temperature, and the residual organic solvent was evaporated to dryness. Then, centrifuge wash 5 times to remove unloaded drug and residual stabilizer. Finally, the dispersed suspension is freeze-dried, and the dry powder is collected as the drug-loaded polymer lipid sphere. Its average particle size and particle size distribution are measured by a British Malvern nanometer particle size analyzer (ZetasizerNanoZS90), and the average particle size in water is 2699nm, and the particle size distribution coefficient PDI is 0.059. The particle size of the drug-loaded polymer lipid sphere is uniform. Among them, the drug loading rate of the drug carrier detected by a microplate reader was 8.12%, and the embedding rate was 99.65%.

Example 5

The preparation of lipid spheres was carried out using FITC fluorescently labeled phospholipids with a hydrophilic head group. The preparation process and conditions were consistent with those in Example 2, and the prepared PLGA microspheres and PLGA/HSPC lipid spheres were transmitted The characterization of the electron microscope and the laser confocal microscope, the transmission electron microscope photos and the laser confocal microscope photos are shown in Figure 7 and Figure 8, respectively. It can be seen from the results in the figure that the prepared PLGA microspheres are solid and uniform microspheres. The transmission electron microscope photos show that there is a dark ring on the surface of the PLGA/HSPC lipid spheres, and the confocal microscope photos show that the lipid spheres have a circle of fluorescence. ring, indicating that the prepared lipid spheres have a core-shell structure.

Example 6

A microporous membrane with a pore size of 0.8 μm was used to prepare drug-loaded polymer lipid spheres. The specific implementation method is shown in Figure 1: 10 mg of insulin was accurately weighed and dissolved in 500 μL of deionized water as the inner water phase (W ₁ ); 5 mg of HSPC and 150 mg of PLGA Add them together to 5 mL of dichloromethane solution to make them fully dissolve, as the oil phase (O); take a volume of 300 mL of 0.1% PVA aqueous solution as the external water phase (W ₂ ). Add the inner water phase to the oil phase, and undergo homogeneous emulsification (24000rpm, 1min) to form water-in-oil (W ₁ /O) type colostrum, then add the colostrum to the outer water phase, and mechanically stir (300rpm, 1min) , forming W ₁ /O/W ₂ type double emulsion. The double emulsion was poured into a rapid membrane emulsification reaction tank, and passed through the membrane 5 times under the action of nitrogen pressure of 2 MPa to form an emulsion with uniform particle size, which was mechanically stirred and solidified overnight in a ventilated environment at room temperature, and the residual Organic solvents. Then, centrifuge wash 5 times to remove unloaded drug and residual stabilizer. Finally, the dispersed suspension is freeze-dried, and the dry powder is collected as the drug-loaded polymer lipid sphere. The scanning electron micrograph is shown in Figure 9, and the particle size and particle size distribution are measured by a British Malvern nanometer particle size analyzer (ZetasizerNanoZS90). The diameter distribution coefficient PDI is 0.081. Among them, the drug loading rate of the drug carrier detected by a microplate reader was 4.82%, and the embedding rate was 80.16%.

Example 7

A microporous membrane with a pore size of 1.4 μm was used to prepare drug-loaded polymer lipid spheres. The specific implementation method is shown in Figure 1: 100 mg of exenatide was accurately weighed and dissolved in 2500 μL of deionized water as the internal water phase (W ₁ ); 80mg of phosphatidylcholine and 80mg of PLA are added to 5mL of dichloromethane solution to make it fully dissolved, as the oil phase (O); get a volume of 400mL containing 2% sodium dodecylsulfonate aqueous solution as the external water phase (W ₂ ). Add the inner water phase to the oil phase, and homogeneously emulsify (10000rpm, 1min) to form water-in-oil (W ₁ /O) type colostrum, then add the colostrum to the outer water phase, and mechanically stir (300rpm, 1min) , forming W ₁ /O/W ₂ type double emulsion. The double emulsion was poured into a rapid membrane emulsification reaction tank, and passed through the membrane 5 times under the action of nitrogen pressure of 0.5MPa to form an emulsion with uniform particle size. It was mechanically stirred and solidified overnight in a ventilated environment at room temperature, and evaporated to dryness. of organic solvents. Then, centrifuge wash 5 times to remove unloaded drug and residual stabilizer. Finally, the dispersed suspension is freeze-dried, and the dry powder is collected as the drug-loaded polymer lipid sphere. Scanning electron micrograph is shown in Figure 10, and particle size and particle size distribution are measured by British Malvern nanometer (ZetasizerNanoZS90), and the results show that the prepared drug-loaded polymer lipid spheres have uniform particle size, and the average particle size is 240nm. The diameter distribution coefficient PDI is 0.071. Among them, the drug loading rate of the drug carrier detected by a microplate reader was 5.28%, and the embedding rate was 85.88%.

Example 8

A microporous membrane with a pore size of 2.8 μm was used to prepare drug-loaded polymer lipid spheres. The specific implementation method is shown in Figure 1: Accurately weigh 50 mg model vaccine chicken serum albumin and dissolve it in 3000 μL deionized water as the inner water phase (W ₁ ); 40mg egg yolk lecithin and 120mgPLA were added to 5mL chloroform solution to make it fully dissolved, as the oil phase (O); the volume was 500mL containing 5% polyoxyethylene sorbitan monooleate aqueous solution as the external water phase (W ₂ ). Add the inner water phase to the oil phase, and homogeneously emulsify (6000rpm, 1min) to form water-in-oil (W ₁ /O) type colostrum, then add the colostrum to the outer water phase, and mechanically stir (300rpm, 1min) , forming W ₁ /O/W ₂ type double emulsion. The double emulsion was poured into a rapid membrane emulsification reaction tank, and passed through the membrane 5 times under a nitrogen pressure of 1 MPa to form an emulsion with uniform particle size. It was solidified overnight with mechanical stirring in a ventilated environment at room temperature, and the residual organic solvent was evaporated. Then, centrifuge wash 5 times to remove unloaded drug and residual stabilizer. Finally, the dispersed suspension is freeze-dried, and the dry powder is collected as the drug-loaded polymer lipid sphere. The scanning electron microscope photo is shown in Figure 11, and the particle size and particle size distribution are measured by a British Malvern nanometer particle size analyzer (ZetasizerNanoZS90). The diameter distribution coefficient PDI is 0.051. Among them, the drug loading rate of the drug carrier detected by a microplate reader was 5.49%, and the embedding rate was 89.22%.

Example 9

A microporous membrane with a pore size of 5.3 μm was used to prepare drug-loaded polymer lipid spheres. The specific implementation method is shown in Figure 1: 50 mg growth hormone was accurately weighed and dissolved in 1000 μL deionized water as the inner water phase (W ₁ ); 30 mg HSPC and 130mg of polydioxanone was added to 5mL of dichloromethane solution to make it fully dissolved, as the oil phase (O); the volume was 100mL containing 5% sodium dodecylsulfonate aqueous solution as the external water phase ( W ₂ ). Add the inner water phase to the oil phase, and undergo homogeneous emulsification (14000rpm, 1min) to form a water-in-oil (W/O) type colostrum, then add the colostrum to the outer water phase, and mechanically stir (300rpm, 1min), Form W ₁ /O/W ₂ double emulsion. The double emulsion was poured into a rapid membrane emulsification reaction tank, and passed through the membrane 5 times under a nitrogen pressure of 0.5 MPa to form an emulsion with uniform particle size. It was solidified overnight with mechanical stirring in a ventilated environment at room temperature, and the residual organic solvent was evaporated. Then, centrifuge wash 5 times to remove unloaded drug and residual stabilizer. Finally, the dispersed suspension is freeze-dried, and the dry powder is collected as the drug-loaded polymer lipid sphere. The scanning electron microscope photo is shown in Figure 12, and the particle size and particle size distribution are measured by a British Malvern nanometer particle size analyzer (ZetasizerNanoZS90). The diameter distribution coefficient PDI is 0.041. Among them, the drug loading rate of the drug carrier detected by a microplate reader was 5.82%, and the embedding rate was 92.36%.

Example 10

The cumulative release behavior in vitro was tested for the drug-loaded PLGA microspheres in Example 1 and the drug-loaded polymer lipid spheres in Example 2. The release is carried out in a constant temperature oscillator in a water bath with a constant temperature of 37.0±0.5°C and an oscillation rate of 100 times/min. 10mg microspheres were dissolved in 1.0mL pH7.4 phosphate buffer solution (PBS), samples were taken at corresponding time points, and the samples were centrifuged (5000rpm, 5min), and the supernatant was taken to detect the protein concentration by micro-BCA method. The results are shown in Figure 13. The protein drugs in the two carriers completed burst release within 24 hours. The burst release rate of the drug in Example 1 was about 40%, while that in Example 2 was only about 20%. Thereafter, the remaining protein in the microspheres continues to be released at a slow rate, and the release of the protein at this time depends largely on the degradation rate of the carrier. Correspondingly, the release rate of the polymer lipid sphere is relatively slower, and when reaching one month, the cumulative release rate of BSA in Example 1 is about 55%, while that in Example 2 is 35%, that is, the polymer with more stable structure Lipid spheres will produce lower drug burst release and slower release behavior when embedding hydrophilic proteins.

Example 11

Caco-2 cells were co-incubated with Raji cells to construct M cells, and the prepared PLGA microspheres and PLGA/HSPC lipid spheres with a particle size of 200nm and fluorescence were added to the upper layer of Transwell, and co-incubated with M cells at 37°C. After incubation, after a certain period of time, the solution was collected from the lower layer of the Transwell, and the fluorescence intensity value was read to quantitatively characterize its M cell translocation. The results are shown in Figure 14. The results showed that M cells transported PLGA/HSPC lipid spheres significantly stronger than PLGA microspheres.

The invention adopts rapid membrane emulsification technology to prepare protein-loaded polymer lipid microspheres with uniform particle size and controllability. When using membrane emulsification technology to prepare uniform lipid spheres, compared with the preparation of polymer microspheres, its process parameters and formula parameters need to be adjusted according to the system requirements, and the phospholipids act as co-emulsifiers during the preparation process, so that the external water phase The formula is significantly different from that of polymer microspheres. Phospholipids can be distributed on the oil-water interface or the oil phase and the surface of hydrophilic solid particles, which can improve drug embedding rate, protect drug activity, and reduce burst release; at the same time, its operating parameters are also affected by phospholipids. There are also significant differences in addition, which need to be optimized and adjusted according to the needs of the system. In addition, due to the addition of phospholipids, not only can the embedding rate of active substances such as proteins, polypeptides, and vaccines be greatly improved by lipid globules, but also because of its role as a co-emulsifier, it can protect active drugs such as proteins from contacting with the hydrophobic oil-water interface. Greatly improve the activity retention of drugs; at the same time, based on the surface activity of phospholipids, active drugs such as proteins can be embedded in lipid spheres to the greatest extent. While improving the drug embedding rate, the burst release of drugs is greatly reduced, and drug stable release.

The applicant states that the present invention illustrates the detailed features and detailed methods of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed features and detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed features and detailed methods. implement. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of selected components of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (10)

1. one kind carries the polvmeric lipid ball of active medicine, it is characterized in that, described polvmeric lipid bag draws together the hydrophobic skeleton that polymer is formed and the phospholipid layer be coated on outside described hydrophobic skeleton, the hydrophobic tail end of the phospholipid molecule of described phospholipid layer is embedded in described hydrophobic skeleton inside, and hydrophilic head base is exposed to outer surface; Described hydrophobic skeleton inside is embedded with active medicine; The mean diameter of described polvmeric lipid ball is between 0.1-30 μm, and the coefficient of dispersion is less than 0.1.

2. polvmeric lipid ball according to claim 1, is characterized in that, described hydrophobic skeleton inside is embedded with active medicine, is specially: described hydrophobic skeleton inside directly embeds hydrophobic drug;

Preferably, described hydrophobic drug comprises hydrophobic protein, polypeptide or vaccine.

3. polvmeric lipid ball according to claim 1, is characterized in that, described hydrophobic skeleton inside is embedded with active medicine, is specially: described hydrophobic skeleton inside is embedded with chamber structure; Described chamber structure comprises the hydrophilic medicament in the interior aqueous phase of chamber that phospholipid layer surrounds and described chamber interior and described interior aqueous phase, or described chamber structure comprises the hydrophilic medicament granule of chamber that phospholipid layer surrounds and described chamber interior;

Preferably, described hydrophilic medicament comprises hydrophilic protein matter, polypeptide or vaccine.

4. the polvmeric lipid ball according to any one of claim 1-3, is characterized in that, described polymer is degradable polymer;

Preferably, described polymer is selected from polylactic acid, polyglycolic acid, polylactone, condensing model, PTMC, PPDO or their copolymer;

Preferably, described copolymer is selected from PLGA or polylactic acid-PPDO;

Preferably, described phospholipid molecule is selected from soybean lecithin, Ovum Gallus domesticus Flavus lecithin, phospholipid derivative, fatty acid, fatty acid amide, phosphatidylcholine or derivatives thereof, preferred hydrogenated soy phosphatidyl choline, phosphatidylcholine, Ovum Gallus domesticus Flavus lecithin.

5. prepare a method for the polvmeric lipid ball of according to claim 1 year active medicine, comprise the following steps:

(1) degradable polymer and phospholipid are dissolved in organic solvent jointly as oil phase O;

(2) hydrophobic drug is dissolved in described oil phase makes pastille oil phase;

(3) added by described pastille oil phase in the outer aqueous phase W containing stabilizing agent, mechanical agitation forms O/W type pre-emulsion;

(4) described O/W type pre-emulsion is pressed through microporous membrane, obtain O/W type emulsion;

(5) by described O/W type emulsion solidification, removing residual organic solvent, lyophilization, obtains the polvmeric lipid ball that powdered carries active medicine.

6. prepare a method for the polvmeric lipid ball of according to claim 1 year active medicine, comprise the following steps:

(1) degradable polymer and phospholipid are dissolved in organic solvent jointly as oil phase O;

(2) by hydrophilic medicament aqueous solution W ₁be scattered in described oil phase O and make W ₁/ O type colostrum;

(3) by described W ₁/ O type colostrum adds the outer aqueous phase W containing stabilizing agent ₂in, mechanical agitation forms W ₁/ O/W ₂the pre-emulsion of type;

(4) by described W ₁/ O/W ₂the pre-emulsion of type presses through microporous membrane, obtains W ₁/ O/W ₂type emulsion;

(5) by described W ₁/ O/W ₂type emulsion solidifies, and removing residual organic solvent, lyophilization, obtains the polvmeric lipid ball that powdered carries active medicine.

7. method according to claim 6, is characterized in that, described hydrophilic medicament aqueous solution W ₁drug concentration is 1-100mg/mL;

Preferably, described hydrophilic medicament aqueous solution W ₁be 1:1 ~ 1:10 with the volume ratio of oil phase O, described oil phase O and outer aqueous phase W ₂volume ratio be 1:1 ~ 1:100.

8. prepare a method for the polvmeric lipid ball of according to claim 1 year active medicine, comprise the following steps:

(1) degradable polymer and phospholipid are dissolved in organic solvent jointly as oil phase O;

(2) hydrophilic medicament granule S is scattered in described oil phase O makes S/O type colostrum;

(3) add in the outer aqueous phase W containing stabilizing agent by described S/O type colostrum, mechanical agitation forms the pre-emulsion of S/O/W type;

(4) the pre-emulsion of described S/O/W type is pressed through microporous membrane, obtain S/O/W type emulsion;

(5) by described S/O/W type emulsion solidification, removing residual organic solvent, lyophilization, obtains the polvmeric lipid ball that powdered carries active medicine.

9. the method according to any one of claim 5-8, it is characterized in that, in described outer aqueous phase, stabilizing agent is selected from the combination of in polyvinyl alcohol, polyglyceryl fatty acid ester, Tween-81, polyoxyethylene sorbitol acid anhydride laurate or dodecyl sodium sulfate a kind or at least 2 kinds, is preferably polyvinyl alcohol, Tween-81 or dodecyl sodium sulfate;

Preferably, the concentration of described stabilizing agent is 0.1-5wt%;

Preferably, described organic solvent is that degradable polymer and phospholipid are all solvable wherein and hold volatile organic solvent, is preferably dichloromethane or chloroform;

Preferably, the mass percent scope of described phospholipid and described polymer is 1-100%.

10. the method according to any one of claim 5-9, is characterized in that, in described step (4), the aperture of microporous membrane is 0.5-50 μm, is preferably 0.8-9.2 μm;

Preferably, the pressure crossing microporous membrane in described step (4) is 0.1 ~ 2MPa.