CN101693111A - Method for increasing entrapment rate of polylactic acid microspheres to water soluble protein - Google Patents

Abstract

A method for increasing entrapment rate of polylactic acid microspheres to water soluble protein belongs to the field of medicinal preparation. On the basis of the existing multiple emulsion method (W/O/W), water soluble amphiphilic polymer carboxymethyl chitosan (O-CMC) of which the mass fraction ranges from 0.1% to 5% is added in internal water phase medicated solution used for forming multiple emulsion, wherein the molecular weight of the water soluble amphiphilic polymer carboxymethyl chitosan ranges from 15K to 30K, the degree of carboxymethylation ranges from 0.6 to 1, and the degree of deacetylation is 92%. Further, osmotically active substance NaCl or mannitol is added in external water phase solution used for forming multiple emulsions, wherein the mass fraction of NaCl ranges from 0.5% to 1.5%, and the mass fraction of mannitol ranges from 2% to 5%. The method reduces stability of protein during processes of preparation, storage and using of microspheres, and increases the entrapment rate of microspheres to protein and inhibits 'burst effect' of microspheres at initial stage of releasing.

Description

A method for improving the encapsulation efficiency of polylactic acid microspheres to water-soluble proteins

technical field

A method for improving the encapsulation rate of polylactic acid microspheres to water-soluble proteins, to be precise, it is a method to prepare polylactic acid microspheres by improving the double emulsion method (W/O/W) to improve the encapsulation rate of water-soluble proteins The method belongs to the field of pharmaceutical preparations.

Background technique

The drug-loaded microspheres of biodegradable materials can significantly improve the safety and effectiveness of clinical medication by controlling or targeting the drug release rate, and are suitable for a variety of drug delivery methods and drug delivery routes. Polylactic acid ball-forming materials, including polylactic acid PLA, lactic acid/glycolic acid copolymer PLGA and their derivatives, are by far the most researched and widely used biodegradable synthetic polymer materials at home and abroad. PLA, PLGA and other polylactic acid materials are currently recognized as good biodegradable controlled-release frameworks with good biocompatibility and will not cause obvious inflammatory reactions, immune reactions and cytotoxic reactions, and have been approved by the US FDA. Using polylactic acid materials such as PLA and PLGA as the carrier of drug-loaded microspheres and nanoparticles can protect drugs, solubilize them, and improve bioavailability, especially for peptides, vaccines, and hormones that are currently being researched and developed. and other biomacromolecular drugs. Such drugs are prepared into slow-release microsphere injections by embedding in PLA or PLGA, which can effectively broaden the route of administration and improve the bioavailability of drugs. In terms of drug release, polylactic acid materials with different degrees of polymerization and different hydrophilicity and hydrophobicity can be selected according to the properties of the drug, the route of administration, and the release time. Factors such as the amount and particle size can be used to control the drug to achieve different release characteristics.

However, since PLA and PLGA are hydrophobic materials, their encapsulation efficiency for water-soluble drugs is low; at the same time, the hydrophobic effect can easily cause damage to the protein structure, thereby inactivating or reducing the activity of the protein. Therefore, how to improve the encapsulation efficiency of the hydrophilic protein, maintain the activity of the protein in the microsphere, and improve the stability of the protein have become the key issues for the successful application of polylactic acid microspheres to the drug loading of the hydrophilic protein.

The double emulsion method (W/O/W) in the emulsification-solvent evaporation method is the most widely used method for encapsulating water-soluble drugs. The polylactic acid particles of water-soluble protein molecules are usually double-emulsion method (W/O/W) O/W) for preparation. This method avoids the destruction of relatively active drugs (such as genes and protein drugs) in the inner oil in the O/O method. However, the encapsulation efficiency of water-soluble protein by this method is generally low, which cannot meet the requirements of clinical application. At the same time, the protein is denatured during the preparation process, resulting in a decrease in the activity of the encapsulated drug. Most researchers have improved the encapsulation efficiency and activity of water-soluble proteins by synthesizing new materials, but it will take a long time for these newly synthesized materials to be applied clinically. Some researchers try to improve the encapsulation efficiency of drugs by improving the double emulsion method (W/O/W). For example, the 7th issue of volume 21 of journal J Micoencapsulation (p775-785) and Chinese invention patent CN1460468 have reported a kind of improved double emulsion method, this method adopts Tween to replace PVA as emulsifier, to contain or not contain water-soluble Salt or polyol (or sugar, polysaccharide) aqueous solution is external water phase, can improve the encapsulation efficiency of PLGA microsphere to bovine serum albumin, but this method can not improve the stability of protein; A method for improving the stability of proteins in the encapsulation process, characterized in that nonionic surfactant polyoxyethylene and polyoxypropylene copolymers are added to the internal aqueous phase, but this method does not propose a way to improve the encapsulation efficiency .

From the perspective of the process and principle of double emulsion preparation, the causes of protein inactivation and loss mainly include: 1) The compatibility between the material and the drug is poor, and the protein drug in the inner water phase is in the process of forming microspheres. The water phase diffuses, causing the loss and inactivation of the drug; if the solvent volatilizes slowly, the solidification time in the microspheres is prolonged, which will lead to a further increase in the diffusion of the drug to the outer water phase, and a decrease in the encapsulation efficiency; 2) protein drugs During the emulsification process, it contacts with the oil phase and aggregates at the oil-water interface of the emulsion droplets to cause denaturation, resulting in protein inactivation. In view of the above problems, the key ways to improve the encapsulation efficiency of water-soluble proteins by the double emulsion method are to reduce the diffusion of drugs from the inner water phase to the outer water phase, inhibit the hydrophilic-hydrophobic interaction between protein molecules and the oil-water interface of the emulsification system, and appropriately accelerate the solvent Evaporation speed.

Contents of the invention

The purpose of the present invention is to improve the encapsulation efficiency of polylactic acid microspheres to water-soluble protein and the stability of the protein by improving the technology of preparing polylactic acid microspheres by the existing double emulsion method (W/O/W).

The present invention is based on the double emulsion method (W/O/W) widely used in the art, and is realized through the following technical scheme: a method for improving the encapsulation efficiency of polylactic acid microspheres on water-soluble proteins, comprising the following steps :

Prepare the inner water phase: dissolve the drug in water to make a drug solution with a certain concentration, add a small amount of water-soluble amphiphilic polymer carboxymethyl chitosan in the solution; prepare the oil phase: make polylactic acid into a spherical material (poly Lactic acid PLA, lactic acid/glycolic acid copolymer PLGA and their derivatives) are dissolved in organic solvent methylene chloride or chloroform to obtain a certain concentration of oil phase solution; preparation of colostrum: add the inner water phase to the oil phase solution (both Volume ratio is 1:5-1:15), adding non-ionic surfactant Tween80 as emulsifier, ultrasonic or high-speed stirring to obtain colostrum; preparation of external water phase: at a concentration of 0.5% to 2.5% (w/v) PVA Add a small amount of osmotic active substances to the solution to obtain an external water phase; prepare double emulsion: add colostrum to the external water phase (volume ratio of colostrum and external water phase is 1:10 to 1:25), high-speed stirring or ultrasonic emulsification , to obtain W/O/W double emulsion; volatile solvent: add excess external aqueous phase solution, stir and evaporate the oil phase solvent, and precipitate to obtain microspheres; collect microspheres: collect microspheres from the microsphere suspension obtained by filtration, freeze-dry get the product.

Its main feature is that a small amount of water-soluble amphiphilic polymer carboxymethyl chitosan is added to the inner water phase forming the double emulsion, and a small amount of osmotic active substance is added to the outer water phase forming the double emulsion.

The water-soluble amphiphilic polymer hydroxymethyl chitosan added in the internal water phase has a molecular weight of 20K to 30K, a degree of carboxymethyl substitution of 0.6 to 1, and a degree of deacetylation of 92%. The mass in the internal water phase The fraction is 0.1% to 0.8%.

The osmotically active substance can be NaCl, with a mass fraction of 0.5%-1.2% in the external water phase; or mannitol, with a mass fraction of 2%-5% in the external water phase.

Principle of the present invention:

Adding hydroxymethyl chitosan, a hydrophilic polymer material, to the internal water phase can play two roles: on the one hand, hydroxymethyl chitosan replaces the hydration layer of the protein by hydrogen bonding, increasing the drug's Resistance to overflow from the inner aqueous phase; on the other hand, hydroxymethyl chitosan analysis preferentially adsorbed on the oil-water interface, reducing the interaction of the protein with the hydrophobic interface, thereby reducing the possibility of its inactivation. However, the hydroxymethyl chitosan accumulated at the oil-water interface increases the mass transfer resistance of water, so the microspheres have fewer pores after curing, so the release rate is slower, or the drug cannot be completely released.

Adding osmotically active substances to the outer water phase affects the osmotic pressure difference between the inner and outer water phases, and promotes the water in the outer water phase to flow into the inner water phase. On the one hand, it inhibits the diffusion of the outer water phase of the water-soluble protein phase; Diffusion also makes more pores formed inside the microspheres, avoiding the impact of hydroxymethyl chitosan on the release; adding osmotic active substances to the external aqueous phase can also increase the extraction rate of organic solvents, thereby improving the microspheres. curing rate.

The advantages and beneficial effects that the present invention has are:

1) Through the simple improvement of the existing double emulsion method, the inactivation of protein during the preparation of polylactic acid microspheres is reduced, and the encapsulation efficiency of the protein by the microspheres is improved;

2) In the prepared microspheres, a hydrophilic protective barrier is formed between the protein and the hydrophobic polylactic acid material, thereby improving the stability of the protein during the storage and use of the microspheres, and effectively inhibiting the microspheres. The "burst release" of the ball at the beginning of its release;

3) In the prepared microspheres, more pores are formed due to the diffusion of the external water phase into the interior of the microspheres, which is conducive to the diffusion and release of protein drugs inside the microspheres.

Description of drawings

Fig. 1 is the in vitro drug release curve of microspheres prepared by the method of the present invention.

Detailed ways

A method for improving the encapsulation efficiency of polylactic acid microspheres to water-soluble proteins of the present invention, the specific implementation method comprises the following steps:

1) Preparation of the inner water phase containing the drug: dissolve the protein in water to make a solution of a certain concentration, add a certain amount of carboxymethyl chitosan (O-CMC) to the solution, and the hydroxymethyl chitosan used The molecular weight of the sugar is 200K-300K, the degree of carboxymethyl substitution is 0.6-1, the degree of deacetylation is 92%, and the mass fraction of carboxymethyl chitosan in the internal water phase is 0.2%-0.8%;

2) Prepare the oil phase containing the ball-forming material: dissolve the polylactic acid ball-forming material (polylactic acid PLA, lactic acid/glycolic acid copolymer PLGA and their derivatives) in dichloromethane or chloroform to obtain a certain concentration of oil phase solution;

3) Preparation of colostrum: add the inner water phase to the oil phase solution (the volume of the two is 1:5-1:15), add non-ionic surfactant Tween 80 as emulsifier, ultrasonic or high-speed stirring, the prepared colostrum ;

4) Prepare the external water phase: in the PVA solution with a concentration of 0.5% to 2.5% (w/v), add one of NaCl or mannitol to obtain the external water phase; the mass fraction of NaCl in the external water phase is 0.5 %～1.5%, the mass fraction of mannitol in the external water phase is 2%～5%;

5) Preparation of double emulsion: add colostrum to the external water phase, the volume ratio of the two is 1:10-1:25, high-speed stirring or ultrasonic emulsification, to obtain W/O/W double emulsion;

6) Volatile solvent: Add excess external aqueous phase solution to the prepared W/O/W double emulsion, stir and evaporate the oil phase solvent at a rotation speed of 300-600 rpm until microspheres are precipitated;

7) Collect microspheres: filter the microsphere suspension obtained in step 6) to collect microspheres, and freeze-dry to obtain the product.

The specific implementation of the present invention can be illustrated by the following examples, but the protection scope of the present invention is not limited to the following examples.

Example 1

This embodiment is an experiment of the influence of O-CMC on the stability of protein lysozyme. Take 1 mL of 50 mg/mL (active concentration) lysozyme aqueous solution, add 0.2% and 0.4% O-CMC in mass fraction respectively, and take the solution without O-CMC as a comparison. In the above solution, add 3mL of dichloromethane, ultrasonically shake in a water bath for 1min, after standing for 5min, then ultrasonically shake for 1min, after standing for 5min, transfer the water phase, and extract the oil phase with aqueous solution for 5 times, and combine the extracts into the water phase , The active concentration of lysozyme was determined by the Schugach method, and the results are shown in Table 1. It can be seen that after adding O-CMC, the content of active lysozyme in the solution is higher.

Table 1 The active concentration of lysozyme

O-CMC/% 0.2 0.4 0 (control) Lysozyme/mg 44 42 32

Example 2

In this example, the method of the present invention was used to prepare microspheres whose carrier materials were PLGA-mPEG and the model drug lysozyme, and compared with the conventional method. The carrier material is PLGA (50:50, M _w =200k)-mPEG (M _w =5k), the mass ratio of drug and material is 1:6; the oil phase is dichloromethane, and the content of PLGA-mPEG is 0.1g /mL; the molecular weight of carboxymethyl chitosan is 250k, and the degree of carboxymethyl substitution is 0.8; the osmotic active substance is NaCl, the volume ratio of the external aqueous phase and colostrum is 1:20, and ultrasonic emulsification is used. The main properties of the lysozyme microspheres obtained under the three formulations with different mass fractions of NaCl and O-CMC are listed in Table 2. Other conditions are the same, but the main properties of lysozyme microspheres obtained under the condition of not adding O-CMC and NaCl are also listed in Table 2. It can be seen that the encapsulation efficiency and yield of the microspheres prepared by the method of the present invention are higher than those of the conventional method, and the average particle size is smaller.

Table 2 The main properties of lysozyme microspheres

serial number O-CMC/% NaCl/% Yield/% Encapsulation rate/% Average particle size/m Prescription A 0.1 0.9 81 80 80 Prescription B 0.2 1.2 86 85 85 Prescription C 0.4 0.5 92 82 65 Control I 0 0 68 65 190

Example 3

This implementation is the release characteristics of the microspheres prepared by the method of the present invention. The microspheres are: 1) the lysozyme microspheres prepared by the method in Example 1 according to prescription A, 2) the comparison microspheres in Example 1; the conditions for in vitro release are: release medium PBS, temperature 37°C, stirring speed 100rpm; the release rate is represented by the active concentration of lysozyme, and the activity of lysozyme is determined by the Schugard method. The cumulative drug release curve of the microspheres is shown in Figure 1. The initial "burst release" is suppressed to a certain extent, and the cumulative release time reaches more than 30 days, which can be used as long-acting release particles. At the same time, the microspheres prepared by the present invention can release more active lysozyme, and the cumulative amount of active lysozyme released is significantly higher than that of the control group.

Example 4

In this example, bovine serum albumin (BSA) microspheres whose carrier material is PLGA were prepared by the method of the present invention. The carrier material is PLGA (75:25, M _w =200k), and the mass ratio of drug and material is 1:10; the oil phase is dichloromethane, and the content of PLGA is 0.05g/mL; carboxymethyl chitosan The molecular weight is 250k, the carboxymethyl substitution degree is 0.6; mannitol is used as the osmotic active substance, and the volume ratio of the external aqueous phase and colostrum is 1:15; under the three prescriptions with different mass fractions of mannitol and O-CMC, the obtained BSA The main properties of the microspheres are listed in Table 3. Other conditions are the same, but the main properties of the BSA microspheres obtained without adding mannitol and O-CMC are also listed in Table 3.

Table 3 Main properties of BSA microspheres

serial number O-CMC/% Mannitol/% Yield/% Encapsulation rate/% Average particle size/m Prescription D 0.5 2 93 68 60 Prescription E 1 2 97 84 85 Prescription F 2 5 96 72 80 Control II 0 0 70 30 200

Example 5

In this example, the method of the present invention was used to prepare lysozyme microspheres whose carrier material was PLGA. The carrier material is PLGA (50:50, M _w =200k), the mass ratio of drug and material is 1:6; the oil phase is dichloromethane, and the content of PLGA is 0.06g/mL; the amphiphilic polymer is O -CMC, the molecular weight is 250k, the carboxymethyl substitution degree is 0.6-1, and the mass fraction in the inner aqueous phase solution is 0.2%; the osmotic active substance is NaCl, and the content in the inner aqueous phase is 0.9%; the outer aqueous phase and the initial The volume ratio of the milk is 1:10; the main properties of the obtained lysozyme microspheres are listed in Table 3. Other conditions are the same, but the main properties of the lysozyme microspheres obtained without adding O-CMC and NaCl are also listed in Table 4.

Table 4 The main properties of lysozyme microspheres

method Yield/% Encapsulation rate/% Average particle size/m this invention 84 80 60 routine 66 61 180

Claims (4)

1. method that improves polylactic acid microsphere to the water-solubility protein envelop rate may further comprise the steps:

1) water in the preparation: medicine is soluble in water, make drug solution, in solution, add water solublity amphipathic polymer carboxymethyl chitosan;

2) preparation oil phase: polylactic acid-based one-tenth ball material is dissolved in organic solvent dichloromethane or chloroform, obtains oil-phase solution;

3) preparation colostrum: interior water is added oil-phase solution (both volume ratios are 1: 5～1: 15), add non-ionic surface active agent Tween 80 and make emulsifying agent, stir and obtain colostrum;

4) the outer water of preparation: in concentration is in 0.5%～2.5% (w/v) PVA solution, adds osmo active substance, obtains outer water;

5) preparation emulsion: colostrum is added to outer water (colostrum and outer water volume ratio are 1: 10～1: 25), stirs or ultrasonic emulsification, obtain the W/O/W emulsion;

6) solvent flashing: add excessive outer aqueous phase solution again, stir evaporation oil phase solvent, precipitation obtains microsphere;

7) collect microsphere: filter the microsphere suspension liquid collection microsphere that step 6) obtains, lyophilizing gets product.

2. a kind of method that improves polylactic acid microsphere to the water-solubility protein envelop rate according to claim 1, it is characterized in that, described water solublity amphipathic polymer carboxymethyl chitosan, its molecular weight is 15K～30K, degree of substitution by carboxymethyl 0.6～1, deacetylation is 92%, and mass fraction is 0.1%～0.5% in interior aqueous phase solution.

3. a kind of method that improves polylactic acid microsphere to the water-solubility protein envelop rate according to claim 1 is characterized in that described osmo active substance is NaCl, and the mass fraction of aqueous phase is 0.5%～1.5% outside.

4. a kind of method that improves polylactic acid microsphere to the water-solubility protein envelop rate according to claim 1 is characterized in that described osmo active substance is a mannitol, and the mass fraction of aqueous phase is 2%～5% outside.