CN106700098A - Preparation method of biodegradable supermolecule polylactic acid microspheres - Google Patents

Abstract

The invention relates to the field of biodegradable polymers and aims to provide a preparation method of biodegradable supramolecular polylactic acid microspheres. Dissolve polylactic acid modified with 2-ureido-4-[1H]-pyrimidinone (UPy) end group in a good solvent, and add the poor solvent drop by drop under stirring; after continuous stirring, centrifuge to wash and collect the solid precipitate ; After vacuum drying, biodegradable supramolecular polylactic acid microspheres were obtained. The preparation method of the invention is simple and easy, and has high implementability. The method of combining liquid-liquid phase separation and polymer crystallization is used to control the morphology of polymer microspheres, and at the same time control their structure and performance, and expand the scope of application. The raw materials of the prepared materials all come from renewable biomass resources, can be completely degraded after use, are green and environmentally friendly, and have good biocompatibility at the same time. The preparation of drug-loaded particles by co-precipitation of drugs and polymers can effectively avoid the use of emulsifiers, so that polymer nanoparticles or microspheres have wider application prospects.

Description

Preparation method of biodegradable supramolecular polylactic acid microspheres

technical field

The invention relates to the field of biodegradable polymers, in particular to a preparation method of biodegradable supramolecular polylactic acid microspheres.

Background technique

As a drug carrier material, nanoparticles or microspheres have been widely studied and applied in the field of controlled release of drugs. Microsphere carriers can overcome some disadvantages of existing pharmaceutical preparations, use their own small particle size and high dispersion of drugs, improve the water solubility and dissolution rate of insoluble drugs, and increase the bioavailability of drugs.

The traditional methods of preparing polymer micro-nanoparticles or microspheres include spray drying, emulsion solvent volatilization and coagulation methods. These methods have obvious deficiencies, such as the use of toxic solvents or additives, which are difficult to completely remove from the microspheres, affecting The biocompatibility of microspheres is difficult to control the size and size distribution of microspheres. In order to overcome the shortcomings of traditional methods, it has been reported in the literature that precipitation and solvent evaporation are used to prepare biodegradable polymer micro-nano microspheres. According to literature (Chen X et al., Biomacromolecules 2005, 6, 2843-2850) reports, stereo multi-block polylactic acid (PLA) was dissolved in a good solvent, and then replaced in a non-solvent to prepare flower and circle Disc-shaped polymer particles, the particle size distribution of the particles is relatively uniform, ranging from tens of nanometers to several microns. The paper (Zhou Z et al., Macromol. Mater. Eng. 2016, 301, 274-278) prepared petal-shaped PLA nanosheets by changing the type of solvent, the concentration of the polymer solution and the drying method. porosity and mechanical properties.

PLA can be prepared based on biomass resources and has many excellent properties, such as biodegradability, biocompatibility, and environmental friendliness. Due to the optical isomerism of PLA monomers, there are two enantiomers of PLA, namely poly-L-lactic acid (PLLA) and poly-D-lactic acid (PDLA), among which PLLA is more common. The melting point of PLLA is 170°C and the crystallization rate is slow. When PLLA and PDLA are blended, stereocomplex crystals can be formed with a melting point as high as 230°C, about 50°C higher than the homogeneous crystals of PLLA or PDLA alone. In addition, the functionalization of the PLA terminal can regulate the crystallization of the polymer, thereby improving the thermal properties and morphology of the polymer. Papers (Biela T et al., Macromolecules 2015,48,2994-3004) modified the end of PLA with 2-ureido-4[1H]-pyrimidinone (UPy) or uracil, blended PLLA and PDLA in equal proportions, and used N-methylpyrrolidone was dissolved and methanol precipitated to prepare spherical particles. When UPy double-terminal modification is used, it is dissolved with chloroform and precipitated with methanol to obtain a fibrous morphology. Therefore, the combination of stereocomplex crystallization and end group modification of PLA can obtain micro-nano materials with different structures and properties.

However, most traditional precipitation and solvent evaporation methods can only obtain spherical particles. When preparing drug-loaded microspheres, the traditional preparation method needs to add emulsifiers. Papers (Teng et al., J.Appl.Polym.Sci.2015,132,42213-42219) added 2.5% polyvinyl alcohol as an emulsifier when preparing star-shaped polylactic acid microspheres loaded with rifampicin drugs, and finally The polyvinyl alcohol emulsifier is removed by precipitation and washing. Papers (Zhang et al., Polym.Bull.2012,68,27-36) also used polyethylene Alcohol acts as an emulsifier. However, the use of emulsifiers makes it difficult to completely remove them from the microspheres, thus limiting the wide application of biodegradable particles.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a preparation method of biodegradable supramolecular polylactic acid microspheres.

In order to solve the problems of the technologies described above, the solution of the present invention is:

A method for preparing biodegradable supramolecular polylactic acid microspheres is provided, which comprises dissolving polylactic acid modified with 2-ureido-4-[1H]-pyrimidinone (UPy) end groups in a good solvent, and stirring Add a poor solvent drop by drop; after continuous stirring for 24 hours, centrifuge and wash to collect the solid precipitate; after vacuum drying, obtain biodegradable supramolecular polylactic acid microspheres; the good solvent is any one of dichloromethane, chloroform or tetrahydrofuran species; poor solvent is ethanol or methanol.

In the present invention, when 2-ureido-4-[1H]-pyrimidinone (UPy) end group-modified polylactic acid is dissolved in a good solvent, the concentration of the polylactic acid solution is 1 mg/mL.

In the present invention, the volume fraction of the poor solvent in the total amount of solvent used is 20%-90%.

In the present invention, the drying refers to drying at 60° C. for 6 hours.

In the present invention, the molecular form of the 2-ureido-4-[1H]-pyrimidinone (UPy) terminal modified polylactic acid is linear or three-arm star, and its specific structural formula is:

Linear PLA:

Three-arm star polylactic acid:

In the above formula,

Among them, n is 40-890.

In the present invention, the molecular weight of the polylactic acid is between 3-64kDa, which is poly-L-lactic acid, poly-D-lactic acid or a mixture of both.

Description of invention principle:

In solvent replacement, crystallization can induce the formation of different micro-nano assembly structures, and the structure of the micro-nano assembly is related to the nucleation form, crystallization rate, and crystal structure. PLA has two crystalline forms, and the ratio of the two crystalline forms can be regulated by the mixing ratio. Therefore, the different nucleation rates, crystallization growth rates, and chain packing forms of the two crystalline forms can be used to prepare biodegradable particles with different morphologies. In addition, when supramolecular groups that can form non-covalent bonds are introduced into the chain segments, the interchain interaction force and the degree of chain entanglement can be increased, thereby changing the size and morphology of self-assembled particles in solution crystallization.

The present invention prepares PLA microspheres by means of precipitation and solvent volatilization. In the process of solvent replacement, through the combination of liquid-liquid phase separation and polymer crystallization, polylactic acid microspheres with different shapes can be obtained. Moreover, by controlling The blending of different stereoisomeric polylactic acids can realize the transition between microspheres with different shapes, which is significantly different from the spherical particles obtained by traditional precipitation and solvent evaporation preparation methods.

Compared with the prior art, the present invention has the following advantages:

(1) The PLA microspheres of the present invention are prepared by precipitation and solvent volatilization, which is simple and easy to implement, and has high implementability.

(2) The present invention utilizes the method of combining liquid-liquid phase separation and polymer crystallization to control the morphology of polymer microspheres, and simultaneously control its structure and performance, thereby expanding the scope of application.

(3) The present invention uses the quadruple hydrogen bonds of UPy to construct supramolecular polymers. The quadruple hydrogen bonds have a strong force and can regulate the degree of chain entanglement of PLA with different chain topology structures, thereby obtaining PLA microspheres with different morphologies.

(3) The raw materials of the materials prepared in the present invention all come from renewable biomass resources, can be completely degraded after use, are green and environmentally friendly, and have good biocompatibility at the same time.

(4) The present invention adopts the co-precipitation method of drugs and polymers to prepare drug-loaded particles, which can effectively avoid the use of emulsifiers, so that polymer nanoparticles or microspheres have wider application prospects.

Description of drawings

Fig. 1 is the morphology characteristic of the PLA particle that embodiment 2 prepares.

Fig. 2 is the morphological characteristics of the PLA particles prepared in Example 3.

Fig. 3 is the morphological characteristics of the PLA particles prepared in Example 8.

Fig. 4 is the morphological characteristics of the PLA particles prepared in Example 9.

Figure 5 is the morphological characteristics of the PLA particles prepared in Comparative Example 1.

Figure 6 shows the morphology characteristics of the PLA particles prepared in Comparative Example 2.

Fig. 7 is the drug-loaded release curves of the supramolecular PLA particles of Examples 2, 3 and Comparative Example 1 in phosphate buffer.

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

The reagents and medicines used in the preparation of the present invention are as follows: L-lactide and D-lactide are purchased from Praque Company; L-lactide and D-lactide are recrystallized and purified in ethyl acetate for future use; 1 , 6-hexanediol, trimethylolpropane and stannous octoate were purchased from Sigma-Aldrich; 1,6-diisocyanate was purchased from Wanhua Chemical Company; 2-amino-4-hydroxyl-6-methylpyrimidine and Nitromethylpyrrolidone was purchased from J&K Company.

The structural formula of the isocyanate terminal functionalized 2-ureido-4[1H]pyrimidinone (UPy-NCO) of the present invention is:

Refer to the literature (Meijer E W et al., Science 1997,278,1601-1604) to prepare, the specific steps are as follows: 2-amino-4-hydroxyl-6-methylpyrimidine (10.0g) was added to a 500ml three-necked flask, 65°C Vacuum down for 0.5h, protect with argon, add 95.0g of hexamethylene diisocyanate and 3.2g of nitrogen methyl pyrrolidone as catalysts, wherein the number of moles of HDI is the number of moles of 2-amino-4-hydroxy-6-methylpyrimidine 7 times of that, the catalyst content is 3% of the total reactant mass. After reacting at 100°C for 16 hours, the product was dissolved in chloroform, dropped into a mixture of n-heptane and diisopropyl ether (total 700ml) with a volume ratio of 6:1, precipitated, and filtered. The white solid product was dried in a vacuum oven at 50° C. for 10 h, and set aside.

Linear and three-arm star-shaped PLLA and PDLA functionalized at the hydroxyl end were prepared by referring to the literature (Pan P et al., Cryst. Growth Des. 2016, 16, 1502-1511), and the three-terminal hydroxyl-terminated three-arm with a design molecular weight of 8 kg/mol The specific preparation steps of star-shaped PLLA are as follows: dry 20g of L-lactide, 0.335g of trimethylolpropane and 0.12g of stannous octoate into a flask, protect it with argon, and react at 130°C for 5h to obtain PLLA product. The obtained crude product was dissolved in chloroform, and unreacted lactide was removed by precipitation in a precipitant mixed with equal volumes of anhydrous ether and n-hexane, filtered, and dried to obtain a polymer. By changing the mass ratio of initiator to lactide, polymers with different molecular weights were prepared. The molecular weight of the polymer was determined by H NMR spectroscopy. Table 1 lists the preparation conditions and molecular weights of the two-terminal hydroxyl-terminated linear and three-terminal hydroxyl-terminated three-arm star-shaped PLLA and PDLA used in the present invention. Table 1: The preparation conditions and molecular weights of two-terminal hydroxyl-terminated linear and three-terminal hydroxyl-terminated three-arm star PLLA and PDLA

Note: In the name of the polymer, 2L, 2D, 3L, and 3D represent linear PLLA, PDLA terminated by two-terminal hydroxyl groups, and three-arm star-shaped PLLA, PDLA terminated by three-terminal hydroxyl groups, respectively, and the suffix numbers indicate the molecular weight of the polymer calculated from NMR .

1) Preparation of UPy-end functionalized PLLA and PDLA

Referring to (Pan P et al., Cryst.Growth Des.2016, 16, 1502-1511) method, further prepared linear and three-armed star-shaped PLLA and PDLA with UPy end group functionalization, the specific method is: functionalize the hydroxyl end Linear or three-arm star-shaped PLLA or PDLA, isocyanate-end functionalized UPy-NCO, stannous octoate and toluene were placed in a Hiddink tube and reacted at 110°C for 12 hours under the protection of argon; after the reaction, use The organic solvent was removed by a rotary evaporator, then added to dichloromethane to dissolve, and filtered. The solvent was evaporated at room temperature, and the obtained solid material was UPy-end-functionalized PLLA or PDLA. Among them, the molar amount of UPy-NCO is 3 times that of linear or three-arm star-shaped PLA functionalized at the hydroxyl end, stannous octoate accounts for 0.6% of the total mass, and the mass of toluene is 30 times that of PLLA or PDLA.

NMR test: nuclear magnetic resonance (Bruker, 400 MHz) was used to test the ¹ H NMR spectrum of the polymer, and then calculate its number average molecular weight (M _n ). The test temperature is room temperature, the solvent is deuterated chloroform, and the chemical shift (δ) is corrected by the solvent peak. Molecular weight calculation description: For PLA, the degree of polymerization and molecular weight are calculated by comparing the peak area ratio of the hydrogen on the tertiary carbon adjacent to the terminal hydroxyl group (δ=4.3ppm) and the hydrogen on the tertiary carbon in the main chain (δ=5.1ppm).

2) Preparation of supramolecular PLA microspheres

Examples 1-9

In Examples 1-9, linear or three-arm star-shaped PLLA and PDLA functionalized with UPy end groups are dissolved in good solvents such as dichloromethane, chloroform, tetrahydrofuran, etc. in a certain mass ratio, so that the concentration of the polymer solution is 1 mg /mL. After stirring and mixing for 2 hours, add dropwise into poor solvents such as ethanol and methanol, and stir until the volume fraction of poor solvents is 20% to 90%, and drop it off after 4 hours. Then after stirring for 24 h, it was centrifuged and washed to collect the solid precipitate. After the solid precipitate was vacuum-dried at 60° C. for 6 h, biodegradable supramolecular PLA microspheres were obtained.

In Comparative Examples 1 and 2, the same method was used to prepare PLA particles using hydroxyl end-functionalized linear or three-arm star-shaped PLLA, PDLA or a mixture of the two as raw materials. The proportion of PLLA and PDLA in Examples 1-9 and Comparative Examples 1-2, and the volume fraction of poor solvent in the solvent are listed in Table 2.

Morphological characterization: characterized by field emission scanning electron microscopy (FESEM). The dry polylactic acid particles adhered to the conductive carbon gel were observed using a CorlzeisD Utral55 FESEM with an accelerating voltage of 5keV.

Drug loading and drug release experiments on PLA particles Taking the anticancer drug rifampicin as an example, 10 mg of rifampicin, 100 mg of UPy terminal functionalized PLLA, and PDLA mixture were dissolved in dichloromethane, and the polymer The initial concentration is 1 mg/mL; after 2 hours, add absolute ethanol drop by drop, and stir; the volume fraction of ethanol is 70%, and drop it off after 4 hours. After stirring for 24 hours, centrifuging, washing and drying, biodegradable polylactic acid drug-loaded particles are obtained.

Dissolve 2.5 mg of PLA drug-loaded particles in 10 mL of N,N-dimethylformamide solvent, measure the absorbance of the solution at a wavelength of 340 nm with a UV-visible spectrophotometer, and calculate the drug-loaded amount of the particles based on the standard curve.

Enzymatic degradation experiment of PLA granules: 2.0 mg proteinase K, 1.0 mg sodium azide and a certain amount of PLA granules wrapped with filter paper were added to 10 mL of phosphate buffer solution (pH=7.4, 50 mM), and then placed in a 37°C Slowly degrade it on a constant temperature shaker. After a certain time interval, the PLA particles are freeze-dried, and the degradation rate is calculated according to the weight loss of the PLA particles.

Thermal performance test: use DSC test, nitrogen atmosphere. The sample was heated from room temperature to 180°C or 230°C at 10°C/min. The calculation method of thermal performance parameters is as follows: the endothermic peak between 120°C and 160°C is the melting peak of homogeneous crystals of PLLA and PDLA, the peak temperature is the melting point of homogeneous crystals, and the integral area is the melting enthalpy of homogeneous crystals (ΔH _{m, hc} ). The endothermic peak between 180°C and 220°C is the melting peak of PLLA/PDLA stereocomplex crystal, and the integrated area is the stereocomplex crystal enthalpy (ΔH _{m, sc} ). The relative fraction of the stereocomplex (f _SC ) is calculated by the formula f _SC =ΔH _m,sc /(ΔH _m,sc +ΔH _m,hc ).

Table 2 shows the preparation conditions, morphology, particle size, drug loading, degradation rate and thermal performance parameters of PLA particles in Examples 1-9 and Comparative Examples 1-2.

Table 2: Preparation conditions, morphology, particle size, drug loading, degradation rate and thermal performance parameters of PLA particles in Examples 1-9 and Comparative Examples 1-2

It can be seen from Table 2 and Figure 1 that in Example 2, the supramolecular PLA particles have a petal-shaped morphology. However, the morphology of the particles in Comparative Example 1 was irregular, indicating that the existence of supramolecular groups can promote chain entanglement, thereby forming a fluffy petal-shaped structure. Example 4 also cannot form a petal-shaped structure, mainly because the chain entanglement density of linear PLA is not as high as that of three-armed star-shaped PLA. When the blending mass ratio of PLLA and PDLA was 50/50 (such as Examples 3, 5 and Comparative Example 2), they all showed spherical characteristics, mainly due to the formation of stereocomplex crystals, which caused the crystallization rate to accelerate and the chains to pack tightly. Reduce the size of liquid-liquid microphase separation, resulting in the formation of smaller spherical particles. Comparing Examples 1, 2, and 6, changing the types of good solvents and poor solvents and the volume fraction of good solvents, all can obtain petal-shaped structures, indicating that different choices of good solvents and poor solvents will not affect the formation of particles.

Comparing Example 3 and Comparative Example 2, it can be seen from Figure 1 that the spherical particle size of Example 3 is larger and the surface smoothness is reduced, mainly because the existence of UPy supramolecular end groups increases the chain entanglement and easily forms larger particles. Example 4 exhibited a sheet-like structure and could not form a petal-like structure. The morphology characteristics of Example 6 show that high-molecular-weight PLA can also form a petal-shaped structure, but the results of Example 7 show that when high-molecular-weight PLLA and PDLA are blended in equal quantities, spherical particles cannot be formed.

As can be seen from Table 2, the blending ratio of supramolecular PLLA and PDLA was changed in Examples 8 and 9, and the comparison of Examples 2, 8 and 9 shows that by changing the mixing ratio of supramolecular PLLA and PDLA, polymer particles can be realized from The transition from petal-shaped to spherical morphology. It shows that the enhancement of stereocomplex crystallization, the packing of molecular chains is more compact, and the crystallization rate is accelerated, and it is easier to form spherical particles.

PLA particles have a slow-release effect on the drugs encapsulated in the particles. Take 2 mg of drug-loaded PLA particles and disperse them in 5 mL of PBS buffer solution (pH=7.4, 50 mM). ) for dialysis. The buffer solution outside the dialysis bag is replaced at certain time intervals, and the concentration of rifampicin in the buffer solution is tested by an ultraviolet spectrophotometer at the same time, and then the cumulative release amount is calculated. FIG. 2 is the drug release curves of PLA drug-loaded particles in Examples 2-3 and Comparative Example 1. FIG. As can be seen from Figure 2, comparing the results of Examples 2 and 3, as the blending ratio of PLLA and PDLA tends to 50/50, the content of stereocomplex crystals increases, the interaction between polymers and drug molecules is enhanced, and the chain The packing is more compact, so the drug loading of PLA particles gradually increases, and the release rate gradually slows down. At the same time, Table 3 also illustrates this result. Comparing Examples 2, 3, 8, and 9, it can be seen that as the blending ratio of PLLA and PDLA tends to 1/1, the drug loading of PLA particles increases from 0.34% to 4.11%. , the degradation rate dropped from 41% to 25%. Therefore, UPy-end-functionalized linear or three-arm star-shaped PLA particles can be used as carriers for drug release, and the release behavior of drug-loaded particles can be controlled by regulating the end-group modification of PLA and the crystalline form of PLA.

Finally, it should be noted that what is listed above are only specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (6)

1. a kind of preparation method of biodegradable supermolecule polylactic acid microsphere, it is characterised in that be by 2- urea groups -4- [1H] - After the PLA of pyrimidone end group modification is dissolved in good solvent, poor solvent is added dropwise under agitation；Lasting stirring After 24 hours, centrifuge washing, collection solids of sedimentation；After vacuum drying, biodegradable supermolecule polylactic acid microsphere is obtained；It is described Good solvent is any one in dichloromethane, chloroform or tetrahydrofuran；Poor solvent is ethanol or methyl alcohol.

2. method according to claim 1, it is characterised in that by the poly- breast of 2- urea groups -4- [1H]-pyrimidone end group modification When acid is dissolved in good solvent, the concentration for making PLA solution is 1mg/mL.

3. method according to claim 1, it is characterised in that the shared volume in the total consumption of solvent of the poor solvent Fraction is 20%~90%.

4. method according to claim 1, it is characterised in that the drying refers to dry 6h at 60 DEG C.

5. the method according to Claims 1-4 any one, it is characterised in that 2- urea groups -4- [the 1H]-pyrimidine The molecular forms of the PLA of ketone end group modification are linear or three-arm star-shaped, and its concrete structure formula is：

Linear polylactic acid：Or

Three-arm star-shaped PLA：

In above-mentioned formula,

Wherein, n is 40~890.

6. method according to claim 5, it is characterised in that the molecular weight of the PLA is between 3~64kDa The mixture of PLLA, poly- L-lactic acid or both.