CN118252817A - Composite nanoparticle and large-scale preparation method thereof - Google Patents
Composite nanoparticle and large-scale preparation method thereof Download PDFInfo
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- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
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Abstract
The invention relates to a large-scale preparation method of compound nanoparticles, which is prepared from human serum albumin and polylactic acid by an emulsification-solvent volatilization method, and comprises the following steps: (1) high speed shear mixing; (2) high-pressure homogenization; (3) removing the organic solvent under reduced pressure; (4) tangential flow purification; (5) And (3) membrane filtration sterilization, wherein the mass ratio of the human serum albumin to the polylactic acid is 1-12:1, a step of; the polylactic acid is selected from the group consisting of: ester-terminated levorotatory polylactic acid, carboxyl-terminated levorotatory polylactic acid, ester-terminated racemic polylactic acid. The composite nanoparticle obtained by the preparation method has the advantages of smaller than 130nm in particle size, good uniformity, higher protein load and stability, and solves the problems that the laboratory ultrasonic preparation method is poor in repeatability, poor in emulsification effect, incapable of realizing large-scale preparation and the like.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a human serum albumin/polylactic acid compound nanoparticle and a large-scale preparation method thereof.
Background
Immunotherapy is a new therapeutic approach that has emerged in recent years to perform tumor killing by activating or restoring the immune system of the human body. Currently, most antibodies approved for marketing and clinical trial studies worldwide are monospecific and interact only with a single target. However, cancer is often induced by multiple factors as a complex disease, and cross-talk between multiple ligands, receptors and signaling pathways is involved, so that it is difficult to completely destroy tumor cells by using a monospecific antibody therapy directed against only a single antigen, and thus, a low tumor immune response rate of a monoclonal antibody drug becomes a pain spot problem to be solved in clinical urgent need.
Along with the continuous and deep development of antibody structure research and bioengineering technology, the development of industrialization of the double-antibody medicament is realized, and meanwhile, a plurality of research institutions develop multi-antibody medicament research and development of three-antibody, four-antibody and the like. Compared with monoclonal antibodies, the double/multiple antibodies increase antigen binding sites, obviously improve the response rate of patients, and become a new trend of current drug development. It has been counted that more than 30 platforms have been developed for bispecific antibodies to ameliorate structural-induced manufacturing and clinical defects, such as the ease of heavy-light chain mismatch during fc fragment antibody manufacture, and the low molecular weight of the fragment antibodies results in short half-life in vivo. The construction technology basically adopts a bioengineering technology to reform molecules, the development difficulty is high, and currently, only 6 types of double-antibody medicines are sold in the market in batches, however, the clinical curative effect and the expected market performance are very considerable, and a high market value-added space still exists in the future. The technical threshold of trispecific antibodies is higher, no product is currently marketed, and most of the trispecific antibodies are in preclinical studies. The invention adopts the gene recombination technology in the earlier stage, prepares the fragment antibody and albumin into fusion protein, and prepares the single/double/multi-specificity nano antibody drug by the physical mixing mode of the fusion protein and the high polymer material. The method is a revolutionary strategy for constructing a single/double/multi-specificity antibody drug platform, has strong universality and has been applied for Chinese patent application (CN 114516921A). In order to realize the clinical transformation and marketization application of the technical platform, the development of the production process aiming at the technical platform is particularly important. Although the ultrasonic emulsification-solvent volatilization method in the laboratory can realize the simple preparation of the mono/bi/multi-specificity nano antibody, the preparation process has certain defects, such as poor repeatability, poor emulsification effect, poor particle uniformity, incapability of realizing large-scale preparation and the like.
Disclosure of Invention
Based on the above, the invention aims to provide a preparation method of human serum albumin/polylactic acid composite nanoparticles, and can realize large-scale preparation of fusion protein/polylactic acid composite nanoparticles recombined by fragment antibodies and albumin, and the obtained composite nanoparticles have higher protein load and stability, have a particle size of less than 130nm and good uniformity, and lay a foundation for providing single/double/multi-specific antibody medicaments with controllable quality for clinical anti-tumor treatment.
In a first aspect, the invention provides a preparation process of human serum albumin/polylactic acid composite nanoparticles.
A preparation method of composite nanoparticles, wherein the composite nanoparticles are prepared from serum albumin and polylactic acid, and the preparation method comprises the following steps:
(1) High-speed dispersion and mixing: mixing aqueous phase solution of albumin and organic phase solution of polylactic acid by a high-speed dispersing machine or a dispersing machine to prepare colostrum; the mass ratio of the albumin to the polylactic acid is 1-12:1, a step of;
(2) Homogenizing under high pressure: homogenizing the colostrum by a micro-jet high-pressure homogenizer to obtain uniform O/W nano emulsion;
(3) Drying under reduced pressure to remove the organic solvent to obtain a stable nanoparticle solution;
(4) Purifying: removing large particles or impurities in the nanoparticle solution by utilizing a microfiltration membrane, and then performing ultrafiltration to remove free proteins, and simultaneously performing solvent replacement;
(5) Filtering with membrane, and sterilizing.
The second object of the present invention is to provide serum albumin/polylactic acid composite nanoparticles obtained by the above preparation method.
The third object of the invention is to provide the application of the prepared composite nanoparticle in preparing specific antibody medicines.
According to the invention, the preparation process is optimized, and particularly the dosage ratio of the human serum albumin/polylactic acid and the volume ratio during preparation are controlled, so that the human serum albumin/polylactic acid composite nanoparticles are small and uniform in particle size, have higher protein load and stability, and solve the problems of poor repeatability, poor emulsification effect, poor particle uniformity, incapability of realizing large-scale preparation and the like of a laboratory ultrasonic preparation method.
Drawings
FIG. 1 is a graph showing the effect of different protein to polylactic acid mass ratios, oil-water volume ratios and albumin concentrations on the protein assembly rate and protein loading of human serum albumin/polylactic acid composite nanoparticles examined in examples 7 to 9.
FIG. 2 is a graph showing the effect of different protein concentrations, mass ratios of protein to polylactic acid and volume ratios of oil to water on the particle size and distribution of the human serum albumin/polylactic acid composite nanoparticles examined in examples 7 to 9.
FIG. 3 is a graph showing the results of the influence of the rotational speeds of the batch-type and pipeline-type online dispersing machines on the protein assembly rate and the protein load of the homogenized human serum albumin/polylactic acid composite nanoparticles, respectively.
FIG. 4 is a graph showing the effect of the rotational speed of the batch-type and pipeline-type online dispersing machines on the particle size and distribution of the homogenized human serum albumin/polylactic acid composite nanoparticles.
FIG. 5 is a graph showing the effect of the high pressure homogenization pressure and the number of homogenization cycles of the microfluidics on the protein assembly rate and protein loading of the human serum albumin/polylactic acid composite nanoparticles.
FIG. 6 is a graph showing the effect of the high pressure homogenization pressure and the number of homogenization cycles of the microfluidics on the size and distribution of the particle size of the human serum albumin/polylactic acid composite nanoparticle.
FIG. 7 is a particle size distribution and electron microscopy image of microparticles and nanoparticles prepared in example 2.
FIG. 8 is a circular dichroism spectrum of free protein in the microparticles and nanoparticles prepared in example 2.
FIG. 9 is a graph showing the stability of the human serum albumin/polylactic acid composite nanoparticle prepared by the particle 1 in example 1 in PBS buffer.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental methods, which are not specified in the following examples, are generally carried out according to conventional conditions, such as Green and Sambrook-s.A.fourth edition, molecular cloning, instruction manual (Molecular Cloning: ALaboratory Manual), published in 2013, or according to the conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
In some embodiments, the present invention relates to a method for preparing composite nanoparticles prepared from serum albumin and polylactic acid by an emulsion-solvent evaporation method, the method comprising the steps of:
(1) High-speed dispersion and mixing: mixing aqueous phase solution of albumin with organic phase solution of polylactic acid to obtain colostrum by a high-speed dispersing machine or a dispersing machine; the mass ratio of the albumin to the polylactic acid is 1-12:1, a step of;
(2) Homogenizing under high pressure: homogenizing the primary emulsion by a micro-jet high-pressure homogenizer to obtain uniform O/W nano emulsion;
(3) The organic solvent was removed under reduced pressure: the nano emulsion is rapidly evaporated by a parallel evaporator or a thin film evaporator to remove the organic solvent, so as to obtain a stable nano particle solution;
(4) Purifying: removing large particles or impurities in the nanoparticle solution by utilizing a microfiltration membrane, and then ultrafiltering the nanoparticle solution to remove free proteins by utilizing a hollow fiber tangential flow system or a membrane-coated tangential flow system, and simultaneously performing solvent replacement;
(5) Membrane filtration sterilization: and (3) performing membrane filtration sterilization on the purified nanoparticle solution by using a capsule filter.
In some of these embodiments, the albumin is serum albumin, which is bovine serum albumin, mouse serum albumin, or human serum albumin, and in actual clinical use, human serum albumin is selected for use if used in human therapy.
In some embodiments, the mass ratio of albumin to polylactic acid is 2.5-10:1, preferably 4-10:1,4-6:1, further preferably the mass ratio of albumin to polylactic acid is 4.5-5.5:1.
In some of these embodiments, the albumin concentration in the aqueous solution is 2.5-40mg/mL,2.5-20mg/mL, preferably 2.5-10mg/mL, more preferably 3-6 mg/mL; the concentration of the polylactic acid in the organic phase solution is 2.5-40mg/mL,2.5-20mg/mL, preferably 2.5-10mg/mL, more preferably 3-6 mg/mL.
In some of these embodiments, the volume ratio of the aqueous phase solution of albumin to the organic phase solution of polylactic acid is from 5 to 20:1, preferably 5-10:1, more preferably 5-7.5:1.
The above preferred parameters are favorable for improving the serum albumin encapsulation efficiency and the loading capacity of the nanoparticles, and have better dispersion coefficient.
In some embodiments, the polylactic acid is levorotatory polylactic acid (PLLA) or the polylactic acid is racemic polylactic acid (PDLLA).
In some of these embodiments, the l-polylactic acid (PLLA) is ester-terminated or carboxyl-terminated.
In some of these embodiments, the racemic polylactic acid (PDLLA) is ester-terminated.
In some of these embodiments, the molecular weight of the ester-terminated levorotatory polylactic acid (PLLA) is 10000-250000 Da, preferably 15000-240000 Da, more preferably 19000-140000 Da.
In some embodiments, the organic solvent in the organic phase solution is selected from one or more of chloroform, dichloromethane and ethyl acetate, preferably a mixed solvent of one or two of chloroform and dichloromethane.
In some of these embodiments, the mixing means is batch high speed disperser mixing.
In some of these embodiments, the batch high speed disperser is used to mix at a speed of 6000 to 22000rpm, preferably 14000 to 18000rpm; the dispersing time is 30s-5min, preferably 1-2min.
In some of these embodiments, the mixing means is a pipeline online disperser mixing.
In some of these embodiments, the in-line pipeline disperser is used for mixing at a linear speed of 5 to 40m/s, preferably 10 to 40m/s, more preferably 20 to 40m/s, and even more preferably 20 to 30m/s.
In some of these embodiments, the microfluidic high pressure homogenization has a homogenization pressure of 6000 to 25000psi, preferably 10000 to 22000psi, more preferably 10000 to 18000psi, and even more preferably 10000 to 15000psi.
The homogenization pressure has great influence on the stability of the nano particles and the encapsulation rate and the loading capacity of the albumin, and the proper homogenization pressure can promote the gradual increase of the energy input in the emulsification process, so that the interaction between the polylactic acid and the albumin is enhanced, and the encapsulation rate and the loading capacity of the albumin are improved.
In some of these embodiments, the number of homogenizations of the microfluidic high pressure homogenizations is 1-9, preferably 3-7.
In some of these embodiments, the method of removing the organic solvent is drying under reduced pressure.
In some of these embodiments, the reduced pressure drying is at a temperature of 20 to 40℃and a pressure of 10 to 50mbar. Preferably, the temperature of the reduced pressure drying is 20-30 ℃ and the pressure is 10-30 mbar.
In some embodiments, the micro-filtration membrane is one or more of Polyethersulfone (PES), polyvinylidene fluoride (PVDF), and Cellulose Acetate (CA), preferably one or more of PES and PVDF.
In some of these embodiments, the microfiltration membrane has a pore size of 0.22 μm or 0.45 μm.
In some of these embodiments, the microfiltration membrane is a two-stage membrane with a pore size of 0.45/0.22 μm.
In some embodiments, the ultrafiltration purification is performed by using hollow fiber membranes and membrane packages, preferably hollow fiber membranes. The membrane pore size is 300kD and 500kD, preferably 500kD.
In some embodiments of the present invention, it also relates to human serum albumin/polylactic acid complex nanoparticles obtained by the above preparation method.
In some of these embodiments, the average particle size of the human serum albumin/polylactic acid complex nanoparticle is 50 to 300nm, preferably 50 to 180nm, more preferably 70 to 130nm.
In some of these embodiments, the human serum albumin/polylactic acid complex nanoparticles have a particle size polydispersity index (PDI) of less than 0.3, preferably less than 0.2, more preferably less than 0.15.
The raw materials and the auxiliary materials used by the human serum albumin/polylactic acid compound nanoparticle are approved by FDA, so that the source is wide, and the quality is controllable; the preparation method of the human serum albumin/polylactic acid composite nanoparticle is simple, industrial production can be realized by high-speed dispersion and high-pressure homogenization and reduced-pressure drying, and products with stable quality can be produced with high efficiency; the method can provide process reference for large-scale preparation of fusion protein/polyester composite nanoparticles recombined by the fragment antibody and albumin, is suitable for providing quality-controllable preparation of single/double/multi-specificity antibody medicaments, and lays a foundation for clinical anti-tumor treatment.
The sources of raw materials and the treatment methods used in the following examples:
Human Serum Albumin (HSA), purchased from SIGMA ALDRICH;
polylactic acids with different molecular weights and structures are purchased from Jinan Dai, biological technology Co., ltd, and specific information is as follows:
organic solvents such as chloroform, dichloromethane and ethyl acetate are purchased from national pharmaceutical group chemical reagent company, ltd;
Instrument model and brand used in the examples:
Batch type high-speed disperser: model T18 digital (IKA, germany);
pipeline type online dispersing machine: model magic LAB (IKA, germany);
Microfluidic high-pressure homogenizer: model Genizer K (us Genizer);
Parallel evaporator: model SyncorePlus (Germany Bucchi);
Ultra-high speed centrifuge: model Optima XPN (Beckman, usa);
tangential flow ultrafiltration system: model number KR2i (Repligen, usa);
nanometer particle size analyzer: model Nano ZSE (Malvern, uk);
Scanning electron microscope: model Merlin (zeiss, germany);
high performance liquid chromatography: model ARC PREMIER (Waters, USA)
Kjeldahl apparatus: model HANON K1160 (sea energy instrument)
Round two chromatograph: model CHIRASCAN (British Applied PhotoPhysics)
The present invention will be described in further detail with reference to specific examples.
Example 1: preparation of human serum albumin/polylactic acid composite nanoparticle
Preparing 5mL of chloroform solution of ester-terminated L-polylactic acid (OH-PLLA-COOR-130K) with the concentration of 5mg/mL and 25mL of human serum albumin water solution with the concentration of 5mg/mL respectively, adding the human serum albumin water solution into the chloroform solution of ester-terminated L-polylactic acid, and mixing in an ice-water bath by using an IKA T18 digital batch type dispersing machine to prepare colostrum, wherein the dispersing speed is 10000rpm, and the dispersing time is 1min. Immediately transferring the primary emulsion after obtaining the primary emulsion which is uniformly mixed into a NanoGenizer K micro-jet high-pressure homogenizer for high-pressure homogenization, wherein the homogenization pressure is 10000psi, and the homogenization times are 3 times; immediately after the homogenization, the homogenized emulsion was transferred to a parallel evaporator, and the mixture was sequentially maintained at room temperature for 5 minutes according to vacuum levels of 400, 300, 200, 100, 50, 30, 20, and 10mbar to sufficiently remove chloroform, thereby obtaining a human serum albumin/polylactic acid complex nanoparticle solution. This particle was designated particle 1.
The preparation method of the nano-particles prepared by assembling the ester-terminated L-polylactic acid, carboxyl-terminated L-polylactic acid and ester-terminated racemized polylactic acid with human serum albumin with other different molecular weights refers to the preparation of the particles 1.
Example 2: preparation of human serum albumin/polylactic acid composite nanoparticle
100ML of chloroform solution of ester-terminated L-polylactic acid (OH-PLLA-COOR-130K) with the concentration of 5mg/mL and 500mL of human serum albumin water solution with the concentration of 5mg/mL are respectively prepared, the human serum albumin water solution is added into the ester-terminated L-polylactic acid chloroform solution, and an IKA Magic lab pipeline type online dispersing machine is used for mixing to prepare colostrum, wherein the linear speed of the dispersing machine is 20m/s. After obtaining uniformly mixed colostrum, transferring 50ml of the colostrum solution into a parallel evaporator, and sequentially maintaining at room temperature for 5 minutes according to vacuum degrees of 400, 300, 200, 100, 50, 30, 20 and 10mbar to fully remove chloroform so as to obtain a human serum albumin/polylactic acid compound particle solution; in addition, the rest colostrum solution is immediately transferred into a NanoGenizer K micro-jet high-pressure homogenizer for high-pressure homogenization, the homogenization pressure is 10000psi, and the homogenization cycle times are 3 times; immediately after the homogenization, the homogenized emulsion was transferred to a parallel evaporator, and the mixture was sequentially maintained at room temperature for 5 minutes according to vacuum levels of 400, 300, 200, 100, 50, 30, 20, and 10mbar to sufficiently remove chloroform, thereby obtaining a human serum albumin/polylactic acid complex nanoparticle solution.
Example 3: albumin encapsulation efficiency of human serum albumin/polylactic acid composite nanoparticle
The particle 1 prepared in example 1 was filtered using a 0.22 μm polyethersulfone filter to remove large particles, a proper amount of filtrate was diluted by a certain multiple (n), and the ultraviolet absorbance at 280nm was detected using UHPLC (Waters, ARC PREMIER), and pure HSA was subjected to gradient dilution as a standard, to obtain the concentration of free protein C1 in the particle. Taking the particle 1 to perform Kjeldahl nitrogen determination to measure that the total protein concentration in the particle is C0, the albumin encapsulation efficiency isEncapsulation efficiency results of nanoparticles prepared by assembling polylactic acid and human serum albumin with different molecular weights and structures are shown in table 1.
The liquid phase conditions are as follows: ① The chromatographic column model isBEH SEC，3.5 Μm,7.8 mm. Times.150 mm; ② mobile phase: 1 x PBS buffer salt (10 mm, ph=7.4); ③ Mobile phase flow rate: 0.8ml/min; sample injection volume: 10. Mu.L; ④ Sample cell temperature: 8+ -5deg.C, column temperature: 30.+ -. 5 ℃.
Example 4: purification of human serum albumin/polylactic acid complex nanoparticles
Taking the particles 1 prepared in example 1, pre-filtering to remove large particles and other impurities by using a polytetrafluoroethylene filter membrane with a membrane pore diameter of 0.45 μm, transferring the filtered solution into a clean sample bottle, performing ultrafiltration to remove unassembled free albumin by using a tangential flow ultrafiltration system (C02-E500-05-N hollow fiber column with a membrane pore diameter of 500 KD), controlling the shearing force of tangential flow to be 3000/s, controlling the transmembrane pressure TMP to be 3psi, washing and filtering 15 times, and collecting a reflux end for purifying the sample A.
Example 5: protein load analysis of human serum albumin/polylactic acid complex nanoparticles
Taking sample A in example 4, carrying out Kjeldahl nitrogen determination to determine that the protein content in the particles is M1, simultaneously taking a corresponding amount of sample, freeze-drying, weighing to obtain the total mass of the particles is M2, and then obtaining the protein load in the particles as followsThe albumin loading results of the nanoparticles prepared by assembling polylactic acid and human serum albumin with different molecular weights and structures are shown in table 1.
Example 6: characterization of particle size of human serum albumin/polylactic acid composite nanoparticles
The sample A purified in example 4 was diluted to a protein concentration of 0.1mg/mL, and the hydration diameter of the nanoparticles was measured by a nanoparticle analyzer, and the average particle size and the particle size dispersion coefficient were shown in Table 1. As can be seen from Table 1, when the molecular weight of the ester-terminated L-polylactic acid (PLLA-COOR) is 19kD-240kD, the particle size of the prepared composite nano particles is about 100nm, PDI is less than 0.2, and the size uniformity is good; the encapsulation efficiency and the loading capacity of the protein are relatively high when the molecular weight of PLLA-COOR is 19kD-140kD, and are respectively more than 20% and 50%. In addition, the carboxyl end-capped L-polylactic acid (PLLA-COOH) has no significant difference in various performance indexes when the molecular weight of the carboxyl end-capped L-polylactic acid is about 160 kD. The size and uniformity of the particle diameter of the nano-particles prepared by the ester-terminated racemized polylactic acid (PDLLA-COOR) with the molecular weight of 150kD are good, and the protein encapsulation efficiency and the loading capacity are similar to those of PLLA-COOR, so that the optical activity of PLA has no obvious influence on the performance of the nano-particles.
TABLE 1 physicochemical characterization of nanoparticles assembled from polylactic acids and human serum albumin of different molecular weights and structures
Example 7: influence of mass ratio of human serum albumin to polylactic acid on nanoparticle performance
125Mg of human serum albumin is weighed and dissolved in 25mL of ultrapure water, and simultaneously, 50 mg, 25mg, 16.67 mg and 12.5mg of ester-terminated L-polylactic acid (OH-PLLA-COOR-130K) are respectively weighed and dissolved in 5mL of chloroform, and the polylactic acid and the albumin are respectively weighed according to the mass ratio of 1:2.5,1:5, a step of; 1:7.5,1:10, human serum albumin/polylactic acid composite nanoparticles were prepared according to the method of example 1, and purified according to the tangential flow ultrafiltration method of example 4. Protein encapsulation efficiency LE and protein loading LC were measured as in example 2 and example 4, respectively, and as shown in fig. 1a, the protein encapsulation efficiency gradually decreased with increasing albumin mass ratio, while the protein loading gradually increased, probably due to the fact that as the concentration of polylactic acid decreases, more hydrophobic domains of albumin in the oil-in-water emulsion can enter the oil phase to bind with polylactic acid through hydrophobic forces, resulting in an increase in protein content on the particles; but the protein encapsulation efficiency is generally low due to the higher overall protein content. The particle size and its dispersion coefficient were measured as in example 5, and as shown in fig. 2a, the particle size of the nanoparticles decreased with increasing albumin mass ratio, probably due to the decrease in polylactic acid concentration, resulting in a smaller hydrophobic core size.
Example 8: influence of volume ratio of human serum albumin to polylactic acid on nanoparticle performance
125Mg of human serum albumin is weighed and dissolved in 25mL of ultrapure water, meanwhile 25mg of ester-terminated L-polylactic acid (OH-PLLA-COOR-130K) is weighed and dissolved in 5,3.33,2.5 mL of chloroform solution and 1.25mL of chloroform solution respectively, and the polylactic acid and the albumin are mixed according to the volume ratio of 1:5,1:7.5,1:10,1:20, human serum albumin/polylactic acid complex nanoparticles were prepared according to the method of example 1 and purified according to the tangential flow ultrafiltration method of example 4. Protein encapsulation efficiency LE and protein loading LC were measured as in example 3 and example 5, respectively, and as shown in fig. 1b, as the aqueous phase volume ratio of albumin increases, both the encapsulation efficiency and loading of albumin tended to decrease, probably as the volume of the oil phase decreased and the concentration of polylactic acid increased, the number of albumin molecules receivable by the oil phase in the oil-in-water emulsion formed during emulsification decreased, resulting in a simultaneous decrease in protein encapsulation efficiency and loading. The particle size and its dispersion coefficient were measured as in example 6, and as shown in fig. 2b, the particle size of the nanoparticles did not change much with decreasing volume ratio of the oil phase, but the dispersion coefficient tended to increase, probably due to uneven distribution of oil droplets during emulsification when the volume of the oil phase was small.
Example 8: influence of the concentration of human serum albumin and polylactic acid on the nanoparticle Properties
Preparing chloroform solution of ester-terminated L-polylactic acid (OH-PLLA-COOR-130K) with concentration of 2.5,5, 10, 20 and 40mg/mL and aqueous solution of human serum albumin with equal concentration respectively, and mixing the polylactic acid and the albumin according to volume ratio of 1:5, human serum albumin/polylactic acid complex nanoparticles were prepared according to the method of example 1 and purified according to the tangential flow ultrafiltration method of example 3. Protein encapsulation efficiency LE and protein loading LC were measured as in example 3 and example 5, respectively, and as shown in fig. 1c, as the concentration of the system increases, both the encapsulation efficiency and loading of albumin tended to decrease, probably as the concentration of the oil phase polylactic acid increases, and the number of albumin molecules that the oil phase can hold in the oil-in-water emulsion formed during the emulsification process continuously decreased, resulting in a simultaneous decrease in protein encapsulation efficiency and loading. The particle size and the dispersion coefficient thereof were measured as in example 6, and as a result, as shown in fig. 2c, the particle size and the dispersion coefficient of the nanoparticles did not significantly change as the concentration of the system increased.
Example 9: influence of the rotational speed of the batch disperser on the size of the primary emulsion particles
Preparing 5mL of chloroform solution of ester-terminated L-polylactic acid (OH-PLLA-COOR-134K) with the concentration of 5mg/mL and 25mL of human serum albumin water solution with the concentration of 5mg/mL respectively, adding the human serum albumin water solution into the chloroform solution of ester-terminated L-polylactic acid, preparing primary emulsion in ice water bath by using an IKA T18 digital batch type dispersing machine, setting the dispersing rotating speed of the dispersing machine to 6000, 10000, 14000, 18000 and 22000rpm, and the dispersing time to 1min. After obtaining the uniformly mixed colostrum, the human serum albumin/polylactic acid composite nanoparticle was prepared according to the method of example 1, and purified according to the tangential flow ultrafiltration method of example 3. Protein encapsulation efficiency LE and protein load LC were measured as in example 3 and example 5, respectively. The results are shown in FIG. 3a, where protein encapsulation efficiency and loading increased and then decreased with increasing dispersion speed, reaching a maximum at 14000 rpm. The particle size and the dispersion coefficient thereof were measured as in example 6, and as shown in fig. 4a, the particle size of the homogenized nanoparticles gradually decreased with increasing dispersion speed of the disperser, and the particle sizes were substantially uniform at 14000rpm and above, and the dispersion coefficients of the nanoparticles prepared at different disperser speeds were not significantly different, and the average PDI was within 0.2, indicating that the particle size distribution was more uniform by further homogenizing the primary emulsion.
Example 10: influence of line speed of pipeline type on-line disperser on size of primary emulsion particles
100ML of chloroform solution of ester-terminated L-polylactic acid (OH-PLLA-COOR-134K) with the concentration of 5mg/mL and 500mL of human serum albumin water solution with the concentration of 5mg/mL are respectively prepared, the human serum albumin water solution and the ester-terminated L-polylactic acid chloroform solution are conveyed into an IKA Magic lab disperser according to the volume ratio of 5:1 by a peristaltic pump to be mixed, and the dispersion linear speeds of the disperser are respectively set to be 13.3, 20, 30 and 40m/s to prepare the colostrum. After obtaining the uniformly mixed colostrum, the human serum albumin/polylactic acid composite nanoparticle was prepared according to the method of example 1, and purified according to the tangential flow ultrafiltration method of example 3. Protein encapsulation efficiency LE and protein load LC were measured as in example 3 and example 5, respectively. As shown in fig. 3b, when the linear velocity of the online disperser reaches 20m/s or more, the protein encapsulation efficiency and the loading capacity are obviously increased, and the emulsification is more sufficient when the linear velocity is increased, so that the interaction between the polylactic acid and the albumin is enhanced, and the encapsulation efficiency and the loading capacity of the albumin are improved. As a result of measuring the particle size and the dispersion coefficient thereof in the method of example 6, as shown in FIG. 4b, the particle size of the nanoparticle increased to some extent with the increase in the linear velocity of the in-line disperser, and the particle size increased most when the linear velocity reached 30m/s or more, but was substantially within 120nm, and the increase in the particle size was likely related to the increase in the protein load. In addition, the PDI of the nano particles prepared by different linear speeds of the dispersing machine is not significantly different, and the average PDI is within 0.2, which indicates that the primary emulsion can make the particle size distribution more uniform through further homogenization.
Example 11: influence of the homogenization pressure of the microfluidic homogenizer on the nanoparticle properties
Preparing 5mL of chloroform solution of ester-terminated L-polylactic acid (OH-PLLA-COOR-130K) with the concentration of 5mg/mL and 25mL of human serum albumin water solution with the concentration of 5mg/mL respectively, adding the human serum albumin water solution into the chloroform solution of ester-terminated L-polylactic acid, and mixing in an ice-water bath by using an IKA T18 digital batch type dispersing machine to prepare colostrum, wherein the dispersing speed is 10000rpm, and the dispersing time is 1min. Immediately transferring the primary emulsion after obtaining the primary emulsion which is uniformly mixed into a NanoGenizer K micro-jet high-pressure homogenizer for high-pressure homogenization, setting the homogenization pressure to 6000 and 10000, 14000, 18000, 22000psi, and setting the homogenization times to 3 times; immediately after the homogenization, the homogenized emulsion was transferred to a parallel evaporator, and the mixture was sequentially maintained at room temperature for 5 minutes according to vacuum levels of 400, 300, 200, 100, 50, 30, 20, and 10mbar to sufficiently remove chloroform, thereby obtaining a human serum albumin/polylactic acid complex nanoparticle solution. Purification was performed according to the tangential flow ultrafiltration method in example 4. The protein encapsulation efficiency LE and the protein loading LC were measured according to the methods of example 3 and example 5, respectively, and the results are shown in fig. 5a, and the encapsulation efficiency and the loading of albumin increase to different extents with increasing homogenizing pressure, which means that the energy input in the emulsification process gradually increases with increasing homogenizing pressure, thereby enhancing the interaction between polylactic acid and albumin and improving the encapsulation efficiency and the loading of albumin. The particle size and its dispersion coefficient PDI were measured as in example 6, and as shown in FIG. 6a, the particle size of the nanoparticles decreased first and then increased with increasing homogenization pressure, but the particle size was below 120nm. In addition, when the homogenization pressure is greater than 14000psi, the dispersion coefficient PDI increases somewhat, indicating that higher pressures may deteriorate the uniformity of the particles.
Example 12: influence of the number of homogenization cycles of a microfluidic homogenizer on the nanoparticle Performance
Preparing 5mL of chloroform solution of ester-terminated L-polylactic acid (OH-PLLA-COOR-130K) with the concentration of 5mg/mL and 25mL of human serum albumin water solution with the concentration of 5mg/mL respectively, adding the human serum albumin water solution into the chloroform solution of ester-terminated L-polylactic acid, and mixing in an ice-water bath by using an IKA T18 digital batch type dispersing machine to prepare colostrum, wherein the dispersing speed is 10000rpm, and the dispersing time is 1min. Immediately transferring the primary emulsion after obtaining the primary emulsion which is uniformly mixed into a NanoGenizer K micro-jet high-pressure homogenizer for high-pressure homogenization, setting the homogenization pressure to 10000psi, and setting the homogenization cycle times to 1-9 times; immediately after the homogenization, the homogenized emulsion was transferred to a parallel evaporator, and the mixture was sequentially maintained at room temperature for 5 minutes according to vacuum levels of 400, 300, 200, 100, 50, 30, 20, and 10mbar to sufficiently remove chloroform, thereby obtaining a human serum albumin/polylactic acid complex nanoparticle solution. Purification was performed according to the tangential flow ultrafiltration method in example 4. The protein encapsulation efficiency LE and the protein loading LC were measured according to the methods of example 3 and example 5, respectively, and as shown in fig. 5b, the encapsulation efficiency and the loading of albumin increased to different extents with the increase of the number of homogenization, which means that the emulsification was more sufficient with the increase of the number of homogenization, thereby enhancing the interaction between polylactic acid and albumin and improving the encapsulation efficiency and the loading of albumin. As a result of measuring the particle diameter and the dispersion coefficient PDI thereof in the method of example 6, as shown in FIG. 6b, when the homogenization pressure was fixed at 10000psi, the particle diameter remained substantially at about 115nm after the number of homogenization was increased to 3 times, and the PDI was gradually decreased with the increase of the number of homogenization, but the PDI was increased after the number of homogenization was 9 times, indicating that the number of homogenization was not too large and was kept below 9 times.
Example 13: observing the surface morphology of the purified human serum albumin/polylactic acid compound nano particles by a scanning electron microscope
According to the preparation method of example 2, the human serum albumin/polylactic acid composite particles and the human serum albumin/polylactic acid composite nanoparticles are prepared, after the particles are purified according to the method of example 4, the concentrations of the particles and the nanoparticles are diluted to 0.1mg/ml HSA, 10 mu L of the particles and the nanoparticles are dripped on a silicon wafer, and the particles and the nanoparticles are observed under a scanning electron microscope after naturally airing at room temperature. As shown in fig. 7a, the human serum albumin/polylactic acid composite particles prepared by using an online dispersing machine are spherical and have good uniformity; the nano particles prepared by further high-pressure homogenization are also spherical, uniform and regular in size, and about 100nm in size (fig. 7 b), which shows that the high-speed dispersion and high-pressure homogenization emulsification enable the protein and polylactic acid to be well assembled into the nano particles through hydrophobic acting force.
Example 14: round dichromatic analysis of changes in secondary structure of albumin in preparation process of human serum albumin/polylactic acid composite nanoparticles
The preparation method of example 2 is used for preparing the human serum albumin/polylactic acid composite particles and the human serum albumin/polylactic acid composite nanoparticle solution. Taking a proper amount of microparticle and nanoparticle solution, performing high-speed centrifugation (70000 g,4 ℃) for 1 hour by using a super-high-speed centrifuge, taking supernatant solution, determining the protein content by using the method in the example 3, diluting a proper amount of sample to 0.2mg/ml, placing the sample in a cuvette for circular dichromatic analysis, and obtaining the result, as shown in figure 8, wherein a circular dichromatic spectrogram of free albumin is basically overlapped with a circular dichromatic spectrogram of a human serum albumin reference substance in the preparation process of the human serum albumin/polylactic acid composite microparticle and nanoparticle, which indicates that the secondary structure of the protein is not changed remarkably in the preparation process of high-speed dispersion and high-pressure homogenization.
Example 15: influence of microfiltration membrane material and aperture of microfiltration membrane on physical and chemical properties of nanoparticles
A human serum albumin/polylactic acid composite nanoparticle solution was prepared as in example 1, then prefiltered with microfiltration membranes (polyethersulfone, polyvinylidene fluoride, cellulose acetate) of different materials having a membrane pore size of 0.45 μm or 0.22 μm to remove large particles and other impurities, the filtrate was transferred to a clean sample bottle, ultrafiltration was performed as in example 4 to remove unassembled free albumin, and the reflux end was collected to purify sample A for analysis of protein loading in the particles. The total protein contents M0 and M1 in the nanoparticle solutions before and after prefiltering were measured by Kjeldahl nitrogen, respectively, as in example 3, and the recovery rate of albumin in the nanoparticle solutions before and after prefiltering wasThe recovery rate of albumin in the nanoparticle solution before and after prefiltering is shown in table 2, and when microfiltration membranes made of two materials, namely polyethersulfone and polyvinylidene fluoride, are used for prefiltering and impurity removal, the recovery rate of albumin in the particle solution is higher than that when prefiltering is carried out by adopting a cellulose acetate microfiltration membrane; and the recovery rate was lower with PES filter membrane with membrane pore size of 0.22 μm than with 0.45 μm, indicating that more particles were trapped. The protein loading was measured as in example 5, and it can be seen that the highest protein loading was achieved with PVDF membranes and the lowest loading was achieved with CA membranes, and that the smaller pore size membranes also reduced protein loading, indicating more significant nanoparticle retention by CA and lower pore size membranes. The particle size and the dispersion coefficient PDI thereof were measured as in example 6, and the results showed that the filters of different materials had little effect on the particle size of the nanoparticles, while the effect on the pore size of the membranes was larger, and the particle size and PDI were smaller after filtration with the filters of smaller pore size.
TABLE 2 influence of different prefiltered microfiltration membrane materials and pore sizes on physicochemical Properties of purified nanoparticles and recovery of albumin in the particles
Example 16: stability of human serum albumin/polylactic acid composite nanoparticles in PBS buffer particle 1 prepared in example 1 was purified by tangential flow ultrafiltration according to example 4, and solvent displacement was performed using 1 x PBS buffer (ph=7.4, 10 mM), and ultrafiltration to obtain a nanoparticle sample dispersed in PBS. The nanoparticle samples were stored at 4 ℃ and sampled at 1d,3d,6d and 15d, respectively, and the particle size analysis was performed as in example 6, and the results were shown in fig. 9, in which no significant particle size change was observed during incubation, the nanoparticle was maintained substantially at about 115nm, and the PDI was also very stable, indicating that the nanoparticle had better stability.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (18)
1. A method for preparing composite nanoparticles prepared from albumin and degradable polylactic acid, wherein the method comprises the following steps:
(1) High-speed dispersion and mixing: mixing aqueous phase solution of albumin and organic phase solution of polylactic acid by a high-speed dispersing machine or a dispersing machine to prepare colostrum; the mass ratio of the albumin to the polylactic acid is 1-12:1, a step of;
(2) Homogenizing under high pressure: homogenizing the colostrum by a micro-jet high-pressure homogenizer to obtain uniform nano emulsion;
(3) Drying under reduced pressure to remove the organic solvent to obtain a stable nanoparticle solution;
(4) Purifying: removing large particles or impurities in the nanoparticle solution by utilizing a microfiltration membrane, and then performing ultrafiltration to remove free proteins, and simultaneously performing solvent replacement;
(5) Filtering with membrane, and sterilizing.
2. The preparation method of claim, wherein the mass ratio of the albumin to the polylactic acid is 2.5-10:1, preferably 4-6:1, more preferably 4.5-5.5:1.
3. The preparation method according to claim 1, wherein the albumin concentration in the aqueous phase solution is 2.5mg/mL-20mg/mL, preferably 2.5mg/mL-10mg/mL; more preferably 3mg/mL-6mg/mL;
The concentration of the polylactic acid in the organic phase solution is 2.5-20mg/mL, preferably 2.5-10mg/mL, more preferably 3-6 mg/mL;
The volume ratio of the aqueous phase solution of albumin to the organic phase solution of polylactic acid is 5-20:1, preferably 5-10:1, more preferably 5-7.5:1.
4. The method of preparation according to claim 1, wherein the albumin is serum albumin, preferably the serum albumin is bovine serum albumin, mouse serum albumin or human serum albumin.
5. The preparation method according to claim 1, wherein the polylactic acid is an ester-terminated or carboxyl-terminated levorotatory polylactic acid, preferably the ester-terminated levorotatory polylactic acid has a molecular weight of 10000Da to 250000Da, preferably 15000Da to 240000Da, more preferably 19000Da to 140000Da;
Or the polylactic acid is racemized polylactic acid with end capped by ester.
6. The preparation method according to claim 1, wherein the organic solvent is selected from one or more of chloroform, dichloromethane and ethyl acetate, preferably a mixed solvent of one or both of chloroform and dichloromethane.
7. The production method according to claim 1, wherein the mixing is performed by a batch type high-speed disperser; the rotation speed of the batch type high-speed dispersing machine for mixing is 6000rpm-22000rpm, preferably 14000rpm-20000rpm; the dispersing time is 30s-5min, preferably 1min-2min.
8. The production method according to claim 1, wherein the mixing means is a pipe-type in-line disperser, and the pipe-type in-line disperser is used for mixing at a linear speed of 10m/s to 40m/s, more preferably 20m/s to 40m/s, still more preferably 20m/s to 30m/s.
9. The method of any one of claims 1-8, wherein the high pressure homogenization of the microfluidics has a homogenization pressure of 10000psi-22000psi, preferably 10000psi-18000psi, more preferably 10000psi-15000psi.
10. The preparation method according to claim 9, wherein the number of homogenizations of the microfluidic high pressure homogenizations is 1-9, preferably 3-7.
11. The production method according to any one of claims 1 to 8, wherein the drying under reduced pressure removes the organic solvent: the nano emulsion is rapidly evaporated to remove the organic solvent by a parallel evaporator or a thin film evaporator, the temperature of the decompression drying is 20-40 ℃, and the pressure is 10-50 mbar; preferably, the temperature of the reduced pressure drying is 20-30 ℃ and the pressure is 10-30 mbar.
12. The method according to claim 1, wherein the microfiltration membrane has a pore size of 0.22 μm or 0.45 μm and/or the microfiltration membrane is a two-stage membrane having a pore size of 0.45/0.22 μm.
13. The method of claim 1, wherein the ultrafiltration is performed on the nanoparticle solution by a hollow fiber tangential flow system or a membrane-enclosed tangential flow system; preferably hollow fibre membranes with a membrane pore size of 300kD and 500kD, more preferably 500kD.
14. The preparation method of claim 1, wherein the purified nanoparticle solution is subjected to membrane filtration sterilization using a capsule filter.
15. Composite nanoparticles obtainable by the method according to any one of claims 1 to 14.
16. The composite nanoparticle according to claim 15, wherein the composite nanoparticle has an average particle size of 50 to 300nm, preferably 50 to 180nm, more preferably 70 to 130nm.
17. The composite nanoparticle according to claim 15, wherein the composite nanoparticle has a polydispersity of less than 0.3, preferably a PDI of less than 0.2, more preferably a PDI of less than 0.15.
18. Use of a complex nanoparticle according to any one of claims 15 to 17 for the preparation of a specific antibody drug.
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