CN109464402B

CN109464402B - Multifunctional nano-medicine composition and preparation method thereof

Info

Publication number: CN109464402B
Application number: CN201811360486.4A
Authority: CN
Inventors: 聂广军; 吴雁; 陈龙; 朱瑾; 覃好; 覃钰婷
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2018-11-15
Filing date: 2018-11-15
Publication date: 2021-03-19
Anticipated expiration: 2038-11-15
Also published as: CN109464402A

Abstract

The invention belongs to the field of nano biomedicine, and particularly relates to a multifunctional nano pharmaceutical composition and a preparation method thereof. The pharmaceutical composition comprises a carrier and an active ingredient loaded on the carrier, wherein the carrier is an HPMA-NAS-PLA polymer, and the active ingredient is a hydrophobic drug; the carrier is obtained by connecting poly N-2-Hydroxypropyl Methacrylamide (HMPA) and polylactic acid (PLA) by acrylic acid N-hydroxysuccinimide ester (NAS). The multifunctional nano-drug composition provided by the invention can carry hydrophilic and hydrophobic drugs, and particularly has high drug loading capacity on the hydrophobic drugs. The size of the obtained medicinal composition is nano-scale and the distribution is narrow by a nano-precipitation method. In addition, the N-hydroxysuccinimide group contained in the carrier copolymer can be covalently linked with a material containing an amino group, and has the potential of carrying gene drugs. Therefore, the compound has good combined application prospect of chemotherapy and gene therapy.

Description

Multifunctional nano-medicine composition and preparation method thereof

Technical Field

The invention belongs to the field of nano biomedicine, and particularly relates to a multifunctional nano pharmaceutical composition and a preparation method thereof.

Background

The nano medicine carrying system uses nano particles with the particle size of 1-1000nm as a carrier to realize the common delivery, slow release and controlled release of different kinds of (hydrophilic and hydrophobic) medicines. Due to the characteristics of the nano drug delivery system, such as improvement of the defects of the traditional drugs in the aspects of solubility, pharmacokinetics, targeting property and the like, passive or active targeted delivery of the drugs is realized, and the release of the drugs is triggered under various sensitive conditions, so that the treatment effect is greatly enhanced. Therefore, the method has wide application prospect. The nano drug-carrying system can utilize the special structure and performance characteristics of the nano drug-carrying system, and overcome many problems which are difficult to solve in the traditional chemotherapy drugs.

Compared with other drug carriers, the nano drug delivery system has the following advantages: (1) overcoming multidrug resistance: particles with a particle size of more than 500nm enter cells mainly through energy-dependent endocytosis; particles with a particle size between 200-500nm enter cells primarily by virtue of caveolae (fossa, fovea, etc.) mediated endocytosis; nanoparticles with a particle size between 100-200nm mainly rely on clathron-mediated phagocytosis into cells. The entry mode can overcome the efflux action of a cell membrane surface protein (such as P-glycoprotein) pump on intracellular drugs, and reduce the MDR effect of tumor cells; (2) the targeting property of the medicine is improved: when the chemotherapeutic drug is distributed in normal tissues, toxic and side effects are generated on the tissues, and the administration dosage is limited; however, the low dosage of the drug can lead to the drug concentration in the tumor tissue to be too low to achieve the expected therapeutic effect. The nano carrier system has large specific surface area and more functional groups, and the surface modification or modification of the nano carrier system can improve the targeting property to tumor tissues or cells and efficiently deliver the medicament to the focus part to be treated; (3) improving the biocompatibility: after the medicine enters the body through blood circulation, part of the medicine is discharged out of the body due to the exosmosis or the kidney clearance effect, the medicine concentration is reduced, and the treatment effect is weakened; for the purpose of treatment, the dosage is usually increased or continuous instillation is adopted, which greatly increases the treatment cost. The nano drug-loaded system can reduce the clearing effect of the organism on the drug as much as possible by adjusting the physical and chemical characteristics (such as material structure, particle size, charge intensity and the like) of the carrier, prolong the stabilization time of the drug in a circulatory system and improve the drug effect; (4) improving the solubility of hydrophobic drugs: some drugs (such as paclitaxel and curcumin) contain a large amount of lipophilic groups, and have low solubility in water, so that a proper administration form is difficult to prepare. The nano particles prepared by using amphiphilic material polyethylene glycol polylactic acid (PEG-PLA) block copolymer as a carrier can provide both hydrophilic environment and hydrophobic environment, and after being combined with drug molecules, the solubility of the hydrophobic drug in water and the in-vivo drug concentration can be improved to a great extent, and the treatment effect of the chemotherapeutic drug is greatly enhanced.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a multifunctional nano-drug composition and a preparation method thereof.

The technical scheme for realizing the aim of the invention is as follows:

a multifunctional nano-drug composition comprises a carrier and an active ingredient loaded on the carrier, wherein the carrier is a HPMA-NAS-PLA polymer, and the active ingredient is a hydrophobic drug; the carrier and the active ingredient loaded on the carrier form nanoparticles;

wherein the carrier is obtained by connecting poly N-2-Hydroxypropyl Methacrylamide (HMPA) and polylactic acid (PLA) by acrylic acid N-hydroxysuccinimide ester (NAS).

Preferably, the nanoparticles are 50-1000nm in size; preferably, the nanoparticle size is 50-300 nm.

In the nano-drug composition, the mass ratio of the carrier to the hydrophobic drug is (20-150) to 1; preferably (30-60): 1; the optimal mass ratio is 50: 1.

In the nano-drug composition, the hydrophobic drug is paclitaxel.

With respect to the vector: specifically, the poly-HPMA-NAS-PLA amphiphilic block copolymer is obtained by connecting poly-N-2-Hydroxypropyl Methacrylamide (HMPA) and polylactic acid (PLA) by acrylic acid N-hydroxysuccinimide ester (NAS).

Specifically, in the polymer structure: the NAS and the HPMA are connected by a carbon-carbon double bond, and PLA is grafted and copolymerized on the HPMA by utilizing-OH thereof.

Wherein, in the HPMA-NAS-PLA polymer, the molar ratio of N-2-hydroxypropyl methacrylamide to lactide is (4-8): (1-2);

preferably, the ratio of m to n is (4-5): 1; more preferably 4: 1. At the above ratios, the resulting product stability and yield are optimal, significantly superior to other ratios.

The weight-average molecular weight of the polymer disclosed by the invention is 8000-10000Da, and the polymer has good solubility and stability.

Preferably, in the polymer, the mass ratio of the N-hydroxysuccinimide acrylate-linked poly (N-2-hydroxypropyl methacryloyl) (HPMA-NAS) to the Lactide (LA) is 1 (20-80).

Preferably, the carrier of the invention is prepared by the following method: polymerizing N-2-hydroxypropyl methacrylamide and N-hydroxysuccinimide acrylate to obtain an HPMA-NAS polymer, and polymerizing LA (lactide) and the HPMA-NAS polymer to obtain the HPMA-NAS-PLA polymer.

Preferably, the molar ratio of the N-2-hydroxypropyl methacrylamide to the N-hydroxysuccinimide acrylate NAS is (16-32): 1; and/or the molar ratio of lactide to HPMA-NAS polymer is (4-8): 1.

or, the mass ratio of the N-2-hydroxypropyl methacrylamide to the N-hydroxysuccinimide acrylate NAS is (260-300): (4-5); and/or the mass ratio of the lactide to the HPMA-NAS polymer is (50-70): 1; more preferably, the mass ratio of lactide to HPMA-NAS polymer is 60: 1.

in the system, the proper concentration ratio of the reactants is needed to be maintained, so that the reaction is facilitated to occur and proceed forwards. More preferably, the concentration of the N-2-hydroxypropyl methacrylamide in the reaction system is 0.1-0.2 g/mL; the concentration of the lactide in the reaction system is 1-2 g/mL.

Specifically, the method comprises the following steps:

(1) carrying out polymerization reaction on N-2-hydroxypropyl methacrylamide and NAS under the action of a chain initiator, and precipitating to obtain an HPMA-NAS polymer;

(2) and (2) carrying out polymerization reaction on LA and the HPMA-NAS polymer obtained in the step (1) under the action of organic amine to obtain the HPMA-NAS-PLA polymer.

In the step (1):

the chain initiator is preferably selected from one of azobisisobutyric acid dimethyl ester AIBME, azobisisobutyronitrile AIBN, more preferably azobisisobutyric acid dimethyl ester. Dimethyl azodiisobutyrate is selected as a chain initiator, so that the reaction can be efficiently initiated, and the method is non-toxic and more suitable for industrial production.

More preferably, the chain initiator is used in an amount of 5-10 mM.

Carrying out the precipitation by using a precipitator, wherein the precipitator is an alcohol ether mixture; preferably a mixture of methanol and diethyl ether (preferably methanol/diethyl ether-1/8). The inventors have tried a plurality of different conventional precipitating agents, which have not achieved good technical effects (for example, the product obtained by precipitation with an ice-water mixture is an oily liquid, which cannot meet the production requirements); a number of studies have surprisingly found that the precipitant is a mixture of alcohol ethers can have unexpectedly superior technical effects, especially when methanol/diethyl ether is 1/8.

The reaction temperature of the polymerization reaction is 50-60 ℃, and the reaction time is 20-25 hours.

In the step (2): the organic amine is preferably triethylamine.

Preferably, the mass to volume ratio of the HPMA-NAS polymer to the organic amine is (0.2-1) g/1 mL; more preferably (0.2-0.5) g/1 mL.

In the step, a precipitator is used for precipitation, and the precipitator is tertiary water with the temperature of 0 ℃. The tertiary water at 0 ℃ is used as a precipitator, so that the reaction product at high temperature (95 ℃) can reach the instant temperature reduction, no new impurities are introduced, and the purity of the obtained reaction product is improved.

In the step, the reaction temperature of the reaction is 90-98 ℃ (preferably 95 ℃), and the reaction time is 12 hours; preferably, the reaction is carried out under nitrogen.

In the preparation method of the invention, the step (a) and the step (b) are both carried out in an organic solvent system, and the organic solvent is preferably anhydrous dimethyl sulfoxide. Anhydrous dimethyl sulfoxide is used as a solvent, so that the chemical reaction can be carried out under strict anhydrous conditions; the solubility is higher, and the reaction is more thorough; high boiling point and good stability. And the forward progress of the reaction can be facilitated by aiming at other reaction conditions such as reaction temperature and the like.

It will be understood by those skilled in the art that the above preparation process may further comprise the step of washing the precipitated product with three times of water (for example, three times), and after the washing, drying the product in a vacuum drying oven.

A multifunctional nano-drug composition comprises a carrier and an active ingredient loaded on the carrier, wherein the carrier is poly N-2-hydroxypropyl methacrylamide and polylactic acid block copolymer connected with acrylic acid N-hydroxysuccinimide ester; the active ingredient is hydrophobic drug paclitaxel, and the carrier and the active ingredient loaded on the carrier form nanoparticles. Preferably, the nanoparticle size is 50-1000 nm. Further preferably, the nanoparticle size is 50-300 nm.

The invention further provides a preparation method of the nano-drug composition according to any one of the technical schemes, which comprises the following steps:

dissolving the HPMA-NAS-PLA polymer and the hydrophobic drug, uniformly mixing to obtain a mixed solution, dropwise adding the mixed solution into water for three times, dialyzing, and freeze-drying to obtain the HPMA-NAS-PLA polymer.

Specifically, the following steps are adopted:

(a) dissolving the HPMA-NAS-PLA polymer in a water-immiscible organic solvent to obtain a carrier solution;

(b) dissolving the hydrophobic drug in a water-immiscible organic solvent to obtain a drug solution;

(c) slowly adding the carrier solution dropwise to the drug solution;

(d) dropwise adding the carrier solution and the hydrophobic drug solution into the water for three times to obtain a mixed solution;

(e) and dialyzing the mixed solution to remove the organic solvent, washing and freeze-drying to obtain the compound.

In the preparation method of the present invention, in step (a), the concentration of the HPMA-NAS-PLA polymer in the carrier solution is 15 to 25mg/mL, preferably 20 mg/mL.

Step (b), in the drug solution, the concentration of the hydrophobic drug is 1-3 mg/ml;

a step (c) of slowly adding the carrier solution dropwise to the drug solution; slowly dropwise adding the mixture to form the nano-particles with the best particle size uniformity. Increasing the dropping speed or continuously dropping easily causes the nanoparticles to aggregate, which affects the formation of the obtained nanoparticles.

Step (d), also includes the process of magnetic stirring for 20-40min after obtaining the mixed solution;

and (e) the molecular weight of a dialysis bag used for dialysis is 3000-3500Da, and the dialysis time is 65-75 h.

In the preparation method of the present invention, the speed of the "dropwise addition" in the step (d) can be operated according to the common knowledge of those skilled in the art, and the preferable scheme here is: approximately at a rate of 100 uL/min.

The multifunctional nano-drug composition provided by the invention can carry hydrophilic and hydrophobic drugs, and particularly has high drug loading capacity on the hydrophobic drugs. The medicinal composition is prepared by a nano precipitation method, and the obtained medicinal composition has a nano-scale size and narrow distribution. In addition, the N-hydroxysuccinimide group contained in the carrier copolymer can be covalently linked with materials containing amino groups (such as protamine, polyethyleneimine and the like), and has the potential of carrying gene drugs. Therefore, the compound has good combined application prospect of chemotherapy and gene therapy.

The nano-medicine of the invention has the advantages that: (1) HPMA (hydrophilic material) has good biocompatibility and good biodegradability. It can form hard sphere by connecting PLA through hydroxyl (-OH), and can also be connected with FITC (fluorescein isothiocyanate, a fluorescent dye mainly used in a fluorescent antibody technology, can be combined with various antibody proteins, the combined antibody does not lose the specificity of combining with certain antigen, and has strong green fluorescence in an alkaline solution); and its double bond may be directly linked to other double bonds. Its double bond can also form a gel. (2) Can carry hydrophilic and hydrophobic drugs, and particularly has high drug loading capacity to hydrophobic drugs. The size of the obtained medicinal composition is nano-scale and the distribution is narrow by a nano-precipitation method. In addition, the N-hydroxysuccinimide group contained in the carrier copolymer can be covalently linked with a material containing an amino group, and has the potential of carrying gene drugs. Therefore, the compound has good combined application prospect of chemotherapy and gene therapy.

Drawings

FIG. 1 is a transmission electron microscope image of nanoparticles formed by the N-hydroxysuccinimide acrylate-linked N-2-hydroxypropyl methacrylamide amphiphilic block copolymer of polylactic acid in example 2 without loading with water;

FIG. 2 is a graph showing the particle size distribution of N-hydroxysuccinimide acrylate-linked N-2-hydroxypropyl methacrylamide amphiphilic block copolymer of polylactic acid formed by dynamic light scattering of nanoparticles in example 2 with no loading of tertiary water.

FIG. 3 is a transmission electron microscope image of nanoparticles formed by amphiphilic block copolymer of N-hydroxysuccinimide acrylate-linked polylactic acid N-2-hydroxypropyl methacrylamide amphiphilic block copolymer of example 7, which entraps hydrophobic drug paclitaxel;

FIG. 4 is a schematic view of example 7, in which an N-hydroxysuccinimide acrylate-linked amphiphilic block copolymer of polylactic acid N-2-hydroxypropyl methacrylamide entraps hydrophobic drug paclitaxel to form nanoparticles, and dynamic light scattering is shown.

Detailed Description

The multifunctional nano-drug composition and carrier obtained in the following examples were subjected to dynamic light scattering (Zetasizer NanoZS), transmission electron microscopy (FEI, Tecnai G220S-TWIN, 200KV) characterization, and fluorescence analysis (Perkin Elmer, LS-55, USA).

Example 1

This example provides a HPMA-NAS-PLA polymer and a method of making the same:

2.8636g of N-2-hydroxypropyl methacrylamide was dissolved with 0.43g of N-hydroxysuccinimide acrylate in 20ml of anhydrous DMSO, and 10mM dimethyl azodiisobutyrate was added. After stirring at 60 ℃ for 20 hours, the mixture was immediately poured into a precipitant to precipitate the polymer, thereby obtaining a HPMA-NAS polymer.

After completion of step (a), 0.5g of the HPMA-NAS polymer obtained in step (a) was dissolved with 30g of lactide in 25ml of anhydrous DMSO and 1ml of triethylamine was added. Stirring at 95 deg.C under nitrogen for 12 hr, and immediately pouring into precipitant for precipitation. Washing the obtained precipitation product with water for three times, and drying in a vacuum drying oven; thus obtaining the amphiphilic block copolymer (HPMA-NAS-PLA polymer) of polylactic acid N-2-hydroxypropyl methacrylamide connected with acrylic acid N-hydroxysuccinimide ester.

Example 2

This example provides a nano-drug composition, which uses the polymer described in example 1 as a carrier, paclitaxel as an active ingredient, and the carrier encapsulating the active ingredient.

The mass ratio of the carrier to the active ingredient is 50: 1.

Example 3

This example provides a nano-drug composition, differing from example 2 only in that the mass ratio of the carrier and the active ingredient is 20: 1.

Example 4

This example provides a nano-drug composition, differing from example 2 only in that the mass ratio of the carrier and the active ingredient is 150: 1.

Example 5

This example provides a nano-drug composition, differing from example 2 only in that the mass ratio of the carrier and the active ingredient is 100: 1.

Example 6

This example provides a nano-drug composition, differing from example 2 only in that the mass ratio of the carrier and the active ingredient is 30: 1.

Example 7

This example provides a method for preparing the nano-drug composition described in example 2, including the following steps:

dissolving 20mg of N-hydroxysuccinimide acrylate-linked poly (N-2-hydroxypropyl methacrylamide) and polylactic acid block copolymer in 1ml of DMSO to obtain a polymer solution;

0.4mg of paclitaxel was dissolved in 0.4ml of DMSO to obtain a drug solution.

The two were mixed well and then slowly added dropwise to 5ml of three times of water with constant magnetic stirring at room temperature for 30 min.

Then dialyzing with 3500Da molecular weight dialysis bag for 72 h.

And finally, washing with water for three times, and freeze-drying to obtain the multifunctional nano-drug composition.

Through detection, the nano particles formed by the polylactic acid N-2-hydroxypropyl methacrylamide amphiphilic block copolymer (HPMA-NAS-PLA polymer) connected with the acrylic acid N-hydroxysuccinimide ester and carrying the hydrophobic drug paclitaxel have a regular spherical structure; the particle size is 166.4 +/-3.3 nm, and the dispersion coefficient is 0.129 +/-0.019.

FIGS. 1 and 2 are graphs showing the particle size distribution of nanoparticles formed by water-unloaded triple time of N-hydroxysuccinimide acrylate-linked polylactic acid N-2-hydroxypropyl methacrylamide amphiphilic block copolymer (HPMA-NAS-PLA polymer) in example 2, respectively.

Fig. 3 and fig. 4 are a transmission electron microscope image and a dynamic light scattering schematic diagram of nanoparticles formed by the amphiphilic block copolymer of N-hydroxysuccinimide acrylate-linked polylactic acid N-2-hydroxypropyl methacrylamide coated with hydrophobic drug paclitaxel prepared in this example, respectively.

The methods of example 3-6 provide the nano-drug compositions by adjusting the amounts of the raw materials/reagents according to the method of example 7.

Test example 1

This test example provides a comparison of the encapsulation efficiency and drug loading of the nano-drug compositions provided in examples 2-5, as shown in table 1.

TABLE 1

Mass ratio of	20:1	50:1	100:1	150:1
					Drug loading (%)	5.14±0.23	4.67±0.13	2.03±0.12	1.21±0.18
Encapsulation efficiency (%)	43.16±1.32	57.37±1.76	63.29±1.83	79.88±2.16

The mass ratio in table 1 refers to the mass ratio of HPMA-NAS-PLA vector to the hydrophobic drug paclitaxel.

Test example 2

Particle size and distribution the particle size distribution of the nano-carrier prepared in example 2 was measured using a malvern laser particle size meter (zetasizer nanozs90), and the result showed that the particle size distribution of the nano-carrier prepared was as shown in fig. 2.

An appropriate amount of the nanocarrier of example 2 was diluted with distilled water, and a drop of the nanocarrier was dropped on a copper mesh coated with a carbon support film, and after standing for a while, a drop of 2% Pigeon Phosphoric acid was further dropped for negative staining, and the particle morphology was observed by a transmission electron microscope (FEI, Tecnai G220S-TWIN, 200KV), and the observation results of example 1 are shown in FIG. 1. As can be seen from FIG. 1, the particles are distributed uniformly and in the shape of regular spheres.

Test example 3

This test example provides stability testing of the nano-pharmaceutical compositions described in examples 2-6.

The nanoparticle solutions prepared in examples 2 to 6 were placed in a refrigerator at 4 ℃ for 72 hours, and visual observation revealed that the solutions were almost unchanged without the occurrence of the precipitation of nanoparticles, the precipitation of drugs, and the like, and the particle size was measured by a malvern laser particle size analyzer, and it was found that the particle size did not significantly change after the nanoparticle solutions were placed for 72 hours.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A nano-drug composition comprises a carrier and nano-particles consisting of an active ingredient loaded on the carrier, and is characterized in that the carrier is a HPMA-NAS-PLA polymer, and the active ingredient is a hydrophobic drug; the carrier is obtained by connecting poly N-2-Hydroxypropyl Methacrylamide (HMPA) and Polylactide (PLA) by acrylic acid N-hydroxysuccinimide ester (NAS);

in the HPMA-NAS-PLA polymer, the molar ratio of N-2-hydroxypropyl methacrylamide to lactide is (4-8): (1-2); the mol ratio of the N-2-hydroxypropyl methacrylamide to the N-hydroxysuccinimide acrylate is (16-32): 1;

the weight average molecular weight of the HPMA-NAS-PLA polymer is 8000-10000 Da.

2. The nano-pharmaceutical composition of claim 1, wherein the nano-particles are 50-1000nm in size.

3. The nano-pharmaceutical composition of claim 2, wherein the nano-particles are 50-300nm in size.

4. The nano-drug composition according to claim 1, wherein the mass ratio of the carrier to the hydrophobic drug is (20-150): 1.

5. The nano-drug composition according to claim 4, wherein the mass ratio of the carrier to the hydrophobic drug is (30-60): 1.

6. the nano-drug composition according to claim 5, wherein the mass ratio of the carrier to the hydrophobic drug is 50: 1.

7. The nano-drug composition of claim 1, wherein the hydrophobic drug is paclitaxel.

8. The nano-drug composition of claim 1, wherein the mass ratio of the N-hydroxysuccinimide acrylate-linked poly (N-2-hydroxypropyl methacryloyl) to the lactide in the polymer is 1 (20-80).

9. The nano-pharmaceutical composition according to claim 8, wherein the mass ratio of the N-hydroxysuccinimide acrylate-linked poly (N-2-hydroxypropyl methacryloyl) to lactide in the polymer is 1: (50-70).

10. The nano-pharmaceutical composition according to claim 9, wherein the mass ratio of the N-hydroxysuccinimide acrylate-linked poly-N-2-hydroxypropyl methacryloyl to lactide in the polymer is 1: 60.

11. The nano-drug composition according to any one of claims 1 to 10, wherein the polymer is prepared by the following method:

(1) carrying out polymerization reaction on N-2-hydroxypropyl methacrylamide and acrylic acid N-hydroxysuccinimide ester under the action of a chain initiator, and precipitating to obtain an HPMA-NAS polymer;

(2) and (2) under the action of organic amine, carrying out polymerization reaction on lactide and the HPMA-NAS polymer obtained in the step (1) to obtain the HPMA-NAS-PLA polymer.

12. The nano-drug composition of claim 11, wherein in step (1): the chain initiator is selected from one of dimethyl azodiisobutyrate AIBME and azodiisobutyronitrile AIBN; and/or, in step (2): the organic amine is triethylamine.

13. The nano-drug composition according to claim 12, wherein in step (1): the chain initiator is dimethyl azodiisobutyrate.

14. The nano-pharmaceutical composition according to claim 12 or 13, wherein the mass to volume ratio of the HPMA-NAS polymer to the organic amine is (0.2-1) g/1 mL.

15. A method for preparing the nano-drug composition of any one of claims 1 to 14, wherein the HPMA-NAS-PLA polymer and the hydrophobic drug are dissolved and uniformly mixed to obtain a mixed solution, the mixed solution is added dropwise to water three times, dialyzed and lyophilized to obtain the nano-drug composition.

16. The method of claim 15, comprising the steps of:

(c) slowly adding the carrier solution dropwise to the drug solution;

17. The method according to claim 15 or 16,

step (a), the concentration of the HPMA-NAS-PLA polymer in the carrier solution is 15-25 mg/mL;

and/or the presence of a gas in the gas,