CN112057630A

CN112057630A - Preparation method and application of targeted drug-loaded dumbbell-shaped Janus particles

Info

Publication number: CN112057630A
Application number: CN202010937214.7A
Authority: CN
Inventors: 郝红; 秦艳琼; 刘魏征; 高超权; 吴睿婕; 薛甲; 孙苗苗
Original assignee: Northwestern University
Current assignee: Northwestern University
Priority date: 2020-09-08
Filing date: 2020-09-08
Publication date: 2020-12-11
Anticipated expiration: 2040-09-08
Also published as: CN112057630B

Abstract

The invention discloses a preparation method and application of dumbbell-shaped Janus particles carrying targeted medicine, which is characterized by comprising the following steps of carrying out targeted medicine on dumbbell-shaped DOX @ (SiO)₂Adding NaOH solution into the-PS)/PMMA particle emulsion, hydrolyzing at 40-45 deg.C for 12-36h, washing to pH 7 to obtainDumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particles; mixing dumbbell-shaped DOX @ (SiO)₂dispersing-PS)/PMMA particles into deionized water, adding EDC, stirring uniformly, adding NHS and NH₂PEG-FA, stirring for 8-15h to obtain a solid, and washing the solid until no absorbance is detected at 270nm in the supernatant by UV-Vis spectrometry to obtain the dumbbell-shaped Janus particles carrying the drug. The preparation method of the particles is simple, the reaction conditions are mild, and the method is suitable for industrial production.

Description

Preparation method and application of targeted drug-loaded dumbbell-shaped Janus particles

Technical Field

The invention belongs to the field of medical high polymer materials, and particularly relates to a preparation method and application of a targeted drug-loaded dumbbell-shaped Janus particle.

Background

Nanoparticles play an important role in a variety of biomedical applications because they can be used as optically stable bioimaging agents and as solid supports for biomolecules in immunoassays and biosensor devices for early detection of disease. In addition, they also show excellent application prospects in vitro and in vivo diagnostic and therapeutic purposes. It follows that it is advantageous to synthesize nanoparticles expressing more than one functionality, which leads to the synthesis of multifunctional nanostructured particles that can combine various properties, such as magnetic, optical, good colloidal stability and biocompatibility.

Over the past two decades scientists have struggled to find new smart materials with the desired functionality, and Janus particles have gained attention due to their anisotropic nature with asymmetric geometry. Due to the non-centrosymmetric characteristic of the Janus particles, the Janus particles have wide application prospects in the fields of optical imaging, emulsion stabilizers, catalysts, drug delivery, highly specific sensors and the like. In the biomedical field, how to control the asymmetric structure of the Janus nanoparticles to maximize the effects of drugs, genes and immunotherapy has become a hot point for the research of the Janus particles in the biomedical field.

At present, the research on Janus particles has made a breakthrough progress, and the diversity of the preparation method thereof enables the Janus particles to have diverse shapes. The appearance of the Janus particles shown by SEM is divided into spherical, dumbbell-shaped, mushroom-shaped, raspberry-shaped and the like, and has asymmetry. Lv et al prepared PLGA/TTBO spherical composite particles by thermal calcination at 500 ℃ using an EHD co-spray composite solution process. Wang et al prepared anisotropic poly (tert-butyl acrylate)/polystyrene (PtBA/PS) composite particles with controllable morphology by soap-free seed emulsion polymerization, first synthesized non-crosslinked PtBA seeds with self-stabilizing polar functional groups, and then nucleated Polystyrene (PS) protrusions on the seeds, finally obtained PtBA/PS composite particles with the shapes of hamburger, litchi branch, mushroom, strawberry, bowl and snowman. In addition to the aforementioned Janus particles, rod-like, disk-like, snowman-like, and the like are common.

For many years, the preparation of dumbbell-shaped Janus particles with good application prospects still has certain challenges, and due to the limitations of low yield, large particle size, complicated synthesis steps and the like, the dumbbell-shaped Janus particles are difficult to realize application values. Janus particles are used in a wide range of applications, but there are still certain challenges to synthesizing particles of the desired morphology. Janus particles are composed of two or more parts with different properties, and are very suitable for carrying various medicines or modifying molecules with various functions. By loading functional monomers on Janus particles, the targeted release of the drug in a body can be realized. Targeted drug delivery is a major goal of inorganic nano-drug carrier research. The targeted drug delivery can keep relatively high drug concentration at the focus, prolong the action time of the drug and improve the lethality to tumor cells. Therefore, the Janus particles as the drug carrier show good application prospects in the aspects of realizing targeted drug delivery, controlled release and slow release of drugs, targeted cancer treatment and the like.

Conventional drug carriers have been greatly hindered in loading drugs of different chemical properties and controlling the independent release of each drug due to their properties, compositions and structural limitations, and the need to find a new material to serve as a drug carrier has been slow.

Disclosure of Invention

The invention aims to provide a preparation method and application of a targeted drug-loaded dumbbell-shaped Janus particle.

In order to achieve the above purpose, the invention is realized by the following technical scheme.

A preparation method of targeted drug-loaded dumbbell-shaped Janus particles comprises the following steps:

(1) dumbbell-shaped DOX @ (SiO)₂Adding NaOH solution into the emulsion of-PS)/PMMA particles, hydrolyzing at 40-45 deg.C for 12-36h, washing to pH 7, and drying to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particles;

(2) mixing dumbbell-shaped DOX @ (SiO)₂dispersing-PS)/PMMA particles into deionized water, adding EDC, stirring uniformly, adding NHS and NH₂And (3) PEG-FA, stirring for 8-15h to obtain a solid, washing the solid until no absorbance is detected at 270nm in the supernatant by UV-Vis spectrometry, and finally, drying in vacuum to obtain the targeted drug-loaded dumbbell-shaped Janus particles.

A further development of the invention is that, in step (1), DOX @ (SiO)₂-volume ratio of PS)/PMMA particle emulsion to NaOH solution (25-35): (3-5), the concentration of the sodium hydroxide solution is 2 mol/L.

The invention is further improved in that in the step (1), the dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion is prepared by the following procedure:

1.1) mixing 0.15-0.17g DOX @ SiO₂Adding into 70-100mL water to obtain suspension, introducing N₂Then adding a cross-linking agent, and uniformly stirring at 20-30 ℃ to obtain a mixed solution;

1.2) 23-25mg of NaSS was added to 8-15mL of H₂Obtaining NaSS aqueous solution in O;

dropwise adding NaSS aqueous solution and 2-3mL St into the mixed solution, uniformly stirring at 20-30 ℃, then raising the temperature to 55-80 ℃, adding KPS aqueous solution, and reacting for 1.5-3h to obtain DOX @ SiO₂-a PS particle emulsion;

1.3) mixing 15-30mL of water, 5-10mL of 0.1mol/L NaCl solution and DOX @ SiO₂-emulsion of PS particles, passing N₂Adding MMA, reacting at 20-30 deg.C for 1.5-3 hr, heating to 55-80 deg.C, adding KPS water solution, and reacting for 7-9 hr to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

In a further development of the invention, in step 1.1), the crosslinking agent is KH-570, KH-560 or DL-602; the volume of the crosslinking agent is 1.0% to 3.0% of the volume St.

The invention has the further improvement that in the step 1.2) and the step 1.3), the mass of KPS is 2-11% of that of MMA; DOX @ SiO₂Mass of the emulsion of PS particles is DOX @ SiO₂4.5-12.2% of the total mass of the PS particle emulsion and the MMA, and the mass of the MMA is DOX @ SiO₂87.8% -95.5% of the total mass of the PS particle emulsion and the MMA.

The invention is further improved in that DOX @ SiO₂Is prepared by the following steps: uniformly mixing ethanol and doxorubicin hydrochloride, dropwise adding 2-5g TEOS, 3-5mL ammonia water and 2-5mL water under stirring, and reacting at 25-35 ℃ for 2-4h to obtain DOX @ SiO₂(ii) a Wherein the mass ratio of the ethanol to the TEOS is 10:1-3: 5.

The invention has the further improvement that the mass ratio of the ethanol to the doxorubicin hydrochloride is 3 (1-8).

The invention is further improved in that in the step (2), the dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particles, EDC, NHS, NH₂-PEG-FA mass ratio (30-50): (75-85): (10-15): (10-20); dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particles to water ratio of (30-50) mg: (8-15) mL.

In a further development of the invention, NH₂-PEG-NH₂Is prepared by the following steps: adding diamino end-capped polyethylene glycol into dimethyl sulfoxide to obtain NH with concentration of 5-7mg/mL₂-PEG-NH₂A solution;

adding 20-25mg of FA, 45-50mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 10-20mg of N-hydroxysuccinimide into 5-15mL of DMSO, then adding 15-25uL of triethylamine, and uniformly stirring to obtain a yellow solution;

dropwise adding the yellow solution to NH with stirring₂-PEG-NH₂Stirring for 12-36h, dialyzing for 12-72h with dialysis bag, and vacuum drying to obtain NH₂-PEG-FA。

An application of dumbbell-shaped Janus carrying targeted drugs in a drug delivery carrier.

Compared with the prior art, the invention has the following beneficial effects: according to the invention, the synthesized drug-loaded and ester-group-containing Janus particles are hydrolyzed under alkaline conditions to hydrolyze ester groups into carboxyl groups, and functional monomers FA are successfully loaded on the ester ends of the Janus particles through an amide condensation reaction, so that the dumbbell-shaped Janus particles with targeted drug loading are synthesized, wherein the Janus particles are composed of two or more parts with different properties, so that the Janus particles are very suitable for multi-drug loading or modification of various functional molecules, are not interfered with each other when releasing drugs, and can be matched with drugs to achieve a good synergistic effect. According to the invention, the dumbbell-shaped Janus particles are prepared by soap-free seed emulsion polymerization, one end of the dumbbell-shaped Janus particles is coupled with folic acid, and the other end of the dumbbell-shaped Janus particles is loaded with hydrophilic drug doxorubicin hydrochloride, so that the dumbbell-shaped Janus particles can be used as a drug delivery carrier, targeted drug loading of the dumbbell-shaped Janus particles is realized, and the application potential of the Janus particles in the aspect of biomedicine is expanded. The preparation method is simple, mild in reaction conditions and suitable for industrial production.

Furthermore, the Janus particle containing ester groups in the invention has uniform, controllable and pure particle appearance.

Drawings

FIG. 1 is DOX @ SiO₂A schematic diagram of a particle synthesis mechanism;

FIG. 2 is DOX @ SiO₂-a schematic of the synthetic mechanism of PS particles;

FIG. 3 is DOX @ (SiO)₂-a schematic of the synthetic mechanism of PS)/PMAA particles;

FIG. 4 is DOX @ (SiO)₂-PS) PMAA map of the mechanism of action of targeted drug delivery by PMAA-FA particles;

FIG. 5 shows SiO in different KH-570₂-PS seed Pair DOX @ (SiO)₂-influence of PS)/PMAA-FA particle morphology; wherein (a) is 1 vol%, (b) is 1.5 vol%, (c) is 2 vol%, and (d) is 3 vol%.

FIG. 6 shows different SiO₂Mass ratio of PS to MMA DOX @ (SiO)₂-influence of PS)/PMAA-FA particle morphology; wherein (a) is 1:7, (b) is 1:13, (c) is 1:18, and (d) is 1: 21.

FIG. 7 is a graph of different KPS dosages vs. DOX @ (SiO)₂-influence of PS)/PMAA-FA particle morphology; it is composed ofWherein (a) is 2 wt%, (b) is 5 wt%, (c) is 8 wt%, and (d) is 11 wt%.

FIG. 8 is a graph of different reaction temperatures vs. DOX @ (SiO)₂-influence of PS)/PMAA-FA particle morphology; wherein (a) is 55 ℃, (b) is 65 ℃, (c) is 80 ℃.

FIG. 9 shows the targeting drug loading DOX @ (SiO) prepared in example 2₂-infrared spectrum of PS)/PMAA-FA particles;

FIG. 10 is the targeted drug loading DOX @ (SiO) prepared in example 2₂-uv spectrogram of PS)/PMAA-FA particles;

fig. 11 is an electron micrograph of targeted drug-loaded Janus particles prepared in example 2. Wherein, (a) is SEM spectrogram, (b) is TEM spectrogram;

FIG. 12 is a standard curve of DOX.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

The invention adopts an in-situ wrapping method to wrap the medicine in SiO₂Inside, dumbbell-shaped Janus particles which carry medicines and contain ester groups are synthesized by soap-free seed emulsion polymerization. And the dumbbell-shaped Janus particles are used as a matrix, and functional monomers are successfully loaded on the dumbbell-shaped Janus particles which are loaded with drugs and contain ester groups, so that the drug-loaded Janus particles are synthesized.

The invention relates to DOX @ (SiO) capable of carrying medicine in a targeted manner under physiological conditions₂-PS)/PMAA-FA particles and a preparation method thereof. DOX @ (SiO) is inspected by adopting an in-situ wrapping method and a posterior wrapping method₂The entrapment rate of the-PS)/PMAA-FA particles under different administration concentrations is finally encapsulated in SiO with the maximum drug loading entrapment rate by an in-situ encapsulation method with higher entrapment rate₂Inside, a dumbbell (SiO) carrying the drug and containing ester groups is synthesized by a soap-free seed emulsion polymerization method₂-PS)/PMMA particles. And in dumbbell Shape (SiO)₂The particles of-PS)/PMMA are taken as a matrix, functional monomers are successfully loaded on the particles, and DOX @ (SiO) of targeted drug loading is synthesized₂-PS)/PMAA-FA particles.

The invention discloses DOX @ (SiO)₂-PS)/PMAA-FA particles and a preparation method thereof. By in-situ wrappingEncapsulating drugs in SiO₂Inside, dumbbell-shaped Janus particles which carry medicines and contain ester groups are synthesized by soap-free seed emulsion polymerization. And the dumbbell-shaped Janus particles are used as a matrix, and the functional monomer is successfully loaded on the dumbbell-shaped Janus particles which are loaded with the medicine and contain ester groups, so that the particles have the capacity of targeted medicine loading. The addition amount of the cross-linking agent, the using amount of the initiator and the monomer ratio m (SiO) are considered₂And (3) m (MMA (methyl methacrylate)) and the influence of the reaction temperature on the particle morphology, wherein the crosslinking agent can be gamma- (methacryloyloxy) propyltrimethoxysilane (KH-570), gamma-glycidoxypropyltrimethylsilane (KH-560), N-beta- (aminoethyl) -gamma-aminopropylmethyldimethoxysilane (DL-602), and the like, and the initiator can be potassium persulfate (KPS), ammonium persulfate (NPS), and the like. Definitively synthesized dumbbell (SiO)₂The technique of the-PS)/PMMA particles is to add a cross-linking agent KH-570 which is SiO with the content of 1vol percent to 3vol percent of St of the monomer₂PS seed, initiator KPS 2-11 wt% of monomer MMA, and monomer ratio m (SiO)₂-PS) m (MMA (methyl methacrylate)) is 1:7 to 1:21, and the reaction temperature is controlled to be 55 ℃ to 80 ℃. Finally, the dumbbell-shaped Janus particles with the targeted drug loading capacity are successfully synthesized.

Referring to fig. 1, the invention provides dumbbell-shaped Janus particles (DOX @ (SiO) for targeting drug loading₂-PS)/PMAA-FA particles) as follows:

(1) referring to fig. 1, ethanol and a certain amount of doxorubicin hydrochloride (DOX) are uniformly mixed in a round bottom flask, 2-5g of TEOS, 3-5mL of ammonia water and 2-5mL of water (TEOS accounts for 28.57-33.33% of the total mass of TEOS, ammonia water and water, ammonia water accounts for 33.33-42.86% of the total mass of TEOS, ammonia water and water, and water accounts for 28.57-33.36% of the total mass of TEOS, ammonia water and water) are added dropwise by using a constant pressure funnel under stirring, the reaction temperature is 25-35 ℃, the reaction time is 2-4h, after the reaction is finished, the obtained product is centrifuged to remove impurities, filtrate is subjected to ultraviolet testing, and precipitate is dried to obtain pink particles, namely DOX @ SiO particles₂And then standby. Wherein the mass ratio of the ethanol to the doxorubicin hydrochloride (DOX) is 3:8-3:1, and the mass ratio of the ethanol to the TEOS is 10:1-3: 5.

(2) Referring to FIG. 2, the coating prepared in step (1) in situ is applied to 0.15-0.17g DOX @ SiO₂(DOX@SiO₂DOX @ SiO₂5.32-9% of the total mass of sodium p-styrene sulfonate (NaSS) and styrene (St) and 70-100mL of water are placed in a three-neck flask to prepare suspension, and N is introduced₂And (3) adding a crosslinking agent (the volume of the crosslinking agent is 1.0 vol% -3.0 vol% of St volume) into the mixture for 30min, wherein the crosslinking agent can be gamma- (methacryloyloxy) propyl trimethoxy silane (KH-570), gamma-glycidoxypropyltrimethylsilane (KH-560), N-beta- (aminoethyl) -gamma-aminopropylmethyldimethoxy silane (DL-602) or the like, and stirring the mixture for 1.5 to 3 hours at the temperature of between 20 and 30 ℃ to uniformly mix the mixture to obtain a mixed solution.

Weighing 23-25mg NaSS (NaSS accounts for DOX @ SiO)₂0.78-1.06% of the total mass of NaSS and St) in 8-15mL of H₂O, obtaining an aqueous NaSS solution, and passing 2-3mL of St (St stands for DOX @ SiO) through a constant pressure funnel₂92.04-93.9 percent of the total mass of NaSS and St) and NaSS water solution are added into the mixed solution drop by drop, and the mixture is stirred for 1.5-3h at 20-30 ℃ to be mixed evenly. Then the temperature is raised to 55-80 ℃, KPS (KPS accounts for 2 wt% -11 wt% of MMA) aqueous solution is slowly added to initiate polymerization, the solution color gradually changes from light white to milk white, the reaction lasts for 1.5-3h, and the emulsion is centrifugally washed to obtain the nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

Referring to FIG. 3, a three-necked flask was charged with 15-30mL of water, 5-10mL of NaCl solution (0.1mol/L), and an amount of DOX @ SiO₂-PS particle emulsion (DOX @ SiO)₂-PS particle emulsion DOX @ SiO₂4.5% -12.2% of total mass of PS particle emulsion and MMA, introducing N₂Adding MMA (MMA in DOX @ SiO) for 30min₂87.8 to 95.5 percent of the total mass of the PS particle emulsion and the MMA, and reacting for 1.5 to 3 hours at the temperature of between 20 and 30 ℃. Then raising the temperature to 55-80 ℃, slowly adding KPS aqueous solution (KPS accounts for 2 wt% -11 wt% of MMA), initiating polymerization, changing the color of the solution from light white to milky white, reacting for 7-9h, and centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

FIG. 2 is DOX @ SiO₂Schematic diagram of the mechanism of synthesis of PS particles. SiO prepared by Sol-Gel method₂The surface of the particles is free of groups polymerizable with styrene (St) and SiO is therefore reinforced₂Core andinteraction of PS shells, the requirement for SiO₂The surface is modified. SiO prepared by Sol-Gel method₂The surface contains hydroxyl, and can be subjected to condensation reaction with KH-570 to introduce C ═ C groups on the surface. SiO2₂The olefinic bond on the surface of the particle can be copolymerized with styrene under the initiation of KPS to form spherical inorganic solid particles as cores, and the SiO is coated with monomer liquid drops₂SiO with shell formed on surface₂-PS core-shell particles.

FIG. 3 is (DOX @ SiO)₂Schematic diagram of a synthetic mechanism of-PS)/PMAA particles, namely, SiO in a core-shell shape at 20-30 ℃ in the presence of MMA₂The PS cross-linked seed particle polymer chains are dissolved and relaxed (i.e. seed swelling) allowing MMA monomer to enter the interior of the seed. SiO2₂The PS seeds, when swelling reaches equilibrium or the temperature rises, lead to an increase in the elastic contractile force, SiO₂The PS seeds start to shrink, thereby reintroducing the monomer into the solution, forming the protrusion. After the initiator KPS is added, it will enter the seed particles and generate free radicals, which may be in SiO₂-transfer or transfer of PS seeds to MMA, KPS also in small amounts in the aqueous phase, by transfer together with MMA in the aqueous phase, MMA being in SiO phase under the action of initiator₂The protrusions formed outside the PS can undergo homogeneous or micellar nucleation, thus forming new (secondary) spherical particles.

(3) Taking 25-35mL dumbbell-shaped DOX @ (SiO)₂Adding 3-5mL of 2mol/L NaOH solution into the (-PS)/PMMA particle emulsion in a triangular flask, hydrolyzing for 12-36h in a shaking table at 40-45 ℃ and at the rotation speed of 100-₂-PS)/PMMA particles, ready for use.

(4) Capping diamino-terminated polyethylene glycol (NH)₂-PEG-NH₂) Dissolving in dimethyl sulfoxide (DMSO) to obtain 5-7mg/mL NH₂-PEG-NH₂A solution;

20-25mg of FA, 45-50mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 10-20mg of N-hydroxysuccinimide (NHS) (the weight of FA accounts for 26.32-26.67% of the total weight of FA, EDC and NHS, the weight of EDC accounts for 52.63-60% of the total weight of FA, EDC and NHS, and the weight of NHS accounts for FA, EDC21.05-13.33 percent of the total mass of NHS is dissolved in 5-15mL DMSO (DMSO accounts for 6.67-15.79 percent of the total mass of FA, EDC and NHS), then 15-25uL Triethylamine (TEA) (TEA accounts for 20-26.32 percent of the total mass of FA, EDC and NHS) is added, magnetic stirring is carried out for 0.5-1.5h, uniform mixing is carried out to obtain yellow solution, and the obtained yellow solution (one drop per 2 seconds) is added to NH dropwise under vigorous stirring (300-500r/min)₂-PEG-NH₂Dissolving in water, stirring the reaction mixture for 12-36h, dialyzing the reaction product with dialysis bag for 12-72h, and vacuum drying for 12-36h to obtain light yellow powder, i.e. NH₂-PEG-FA。

(5) Referring to FIG. 4, 30-50mg of dumbbell-shaped DOX @ (SiO) obtained in step (3)₂-PS)/PMMA particles (dumbbell DOX @ (SiO)₂-PS)/PMMA particle mass in dumbbell DOX @ (SiO)₂-PS)/PMMA particles, EDC, NHS and NH₂29.41-40% of the total mass of PEG-FA is dispersed in 8-15mL of deionized water (5.88-6.67% of the total mass of the reaction mass), then 75-85mg of EDC (33.34-50% of the total mass of the reaction mass) is dissolved to obtain a suspension, and the suspension is stirred for 10-30 min. Then, 10-15mg of NHS (mass accounting for 8.82-13.33% of the total reaction mass) and 10-20mg of NH obtained in the step (4) are added₂PEG-FA (11.77-13.33% by mass of the total reaction mass) was stirred for 8-15h to give a solid, which was washed magnetically until no absorbance was detected at 270nm in the washed supernatant by UV-Vis spectroscopy. Finally, the obtained product is dried in vacuum at 20-30 ℃ to obtain the targeted drug-loaded dumbbell-shaped Janus particle, namely DOX @ (SiO)₂-PS)/PMAA-FA particles. In this step, the total reactants include DOX @ (SiO) in dumbbell form₂-PS)/PMMA particles, EDC, NHS and NH₂-PEG-FA。

FIG. 4 is DOX @ (SiO)₂The action mechanism diagram of the targeting drug delivery of the-PS)/PMAA-FA particles. Encapsulating spectral antitumor drug doxorubicin hydrochloride (DOX) in (SiO)₂SiO of particles of-PS)/PMMA₂-a PS-terminus for drug release in tumor cells. Dumbbell Shape (SiO)₂The polymethyl methacrylate (PMMA) end of the-PS)/PMMA particles is hydrolyzed by ester group under alkaline environment, and is connected with the polymer FA-PEG-NH through amido bond₂Ligation, finally Folate (FA) conjugated to DOX @ (SiO)₂-PS)/PMAA particles. FA is a targeting recognition molecule, and because the activity and the number of FA receptors on the surface of a tumor cell membrane are obviously higher than those of normal cells, Janus particles modified by FA actively recognize FA receptors on tumor cells and enter the interior of the cells. After the Janus particles enter the interior of tumor cells, due to the concentration difference between the interior and exterior of the particles, DOX is released through diffusion, so that the purpose of treatment is achieved.

The performance of the particles prepared above was tested by the following steps:

(1) diluting a sample to be tested into a solution of 50 mu g/mL, taking water as a reference solution, measuring the maximum absorbance Abs under a UV-1801 type ultraviolet-visible spectrophotometer, and drawing a curve.

(2) Diluting a small amount of sample to a certain multiple, performing ultrasonic dispersion treatment for 10min, coating on a glass substrate, and drying. Spraying gold before testing, and adopting SIGMA field emission Scanning Electron Microscope (SEM) of ZEISS company in Germany to perform dumbbell-shaped DOX @ (SiO)₂Characterization of the-PS)/PMMA-FA particle morphology.

The meaning of the data in parentheses in example 1 is indicated in detail, and in the remaining examples, each substance in parentheses means the same as in example 1.

Example 1 KH-570 preparation of DOX @ (SiO) with a volume of 1.0 vol% of monomer St₂-PS)/PMAA-FA particles

(1) Uniformly mixing 39mL of ethanol and 33.6mg of DOX in a round-bottom flask, dropwise adding 3.0g of TEOS, 4.5mL of ammonia water (with the mass concentration of 28%) and 3.25mL of water by using a constant-pressure funnel under stirring, reacting at 30 ℃ for 3h, centrifugally removing impurities from the obtained product after the reaction is finished, and precipitating and drying to obtain pink particles for later use.

(2) Prepared by wrapping 0.16g DOX @ SiO in situ₂Placing the mixture and 80mL of water in a three-neck flask to prepare suspension, and introducing N₂After 30min, 25.5uL of KH-570 (the volume of KH-570 is 1.0 vol% based on the volume of the monomers St) and 20mL of water were added and stirred at 30 ℃ for 2 h.

24.25mg NaSS was weighed out and dissolved in 10mL H₂O, 2.55mL of St and NaSS aqueous solution were added dropwise through a constant pressure funnel, and stirred at 30 ℃ for 2 h. The temperature is raised to 65 ℃, 22.5mg KPS and10mL of water solution (KPS accounts for 5 wt% of monomer MMA) initiates polymerization, the solution color gradually changes from light white to milky white, the reaction lasts for 2h, and the emulsion is centrifugally washed to obtain nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.45g of MMA (DOX @ SiO) was added₂The mass ratio of PS to MMA is 1:13), and the reaction is carried out for 2h at the temperature of 20-30 ℃. The temperature is raised to 65 ℃, an aqueous solution prepared by 22.5mg KPS and 10mL water (KPS accounts for 5 wt% of monomer MMA) is slowly added to initiate polymerization, the color of the solution is changed from light white to milky white, after 8 hours of reaction, the emulsion is centrifugally washed to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

(3) Take 30mL dumbbell-shaped DOX @ (SiO)₂Putting the-PS)/PMMA particle emulsion into a triangular flask, adding 4mL of 2mol/L NaOH solution, hydrolyzing in a shaking table for 24h at 42 ℃ and at the rotating speed of 110r/min, repeatedly washing the mixed solution with distilled water to be neutral after the reaction is finished, and drying into powder for later use.

(4) 50mg of NH₂-PEG-NH₂Dissolved in 8mL DMSO, 22mg folic acid, 47.8mg EDC and 15mg NHS were dissolved in 10mL DMSO, then 20. mu.L TEA was added and the mixture was magnetically stirred for 1 hour. The resulting yellow solution (one drop per 2 seconds) was added dropwise to NH with vigorous stirring₂-PEG-NH₂In solution and the reaction mixture was stirred overnight, after which the reaction product was dialyzed against a dialysis bag and dried in vacuo to remove DMSO, obtaining NH as a pale yellow powder₂-PEG-FA。

(5) 40mg of dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particles were dispersed in 10mL of deionized water, then 80mg of EDC was dissolved. The suspension was stirred for 15 minutes. Then, 12mg of NHS and 15mg of NH obtained in step (4) were added₂-PEG-FA. The reaction was stirred overnight and washed magnetically until no absorbance was detected at 270nm in the supernatant by UV-Vis spectroscopy. Finally, the product obtained is dried under vacuum at 30 ℃.

Example 2 KH-570 preparation of DOX @ (SiO) at 1.5 vol% of monomer St₂-PS)/PMAA-FA particles

(1) Uniformly mixing 39mL of ethanol and 33.6mg of DOX in a round-bottom flask, dropwise adding 3.0g of TEOS, 4.5mL of ammonia water and 3.25mL of water by using a constant-pressure funnel under stirring, reacting at 30 ℃ for 3h, centrifuging and removing impurities from the obtained product after the reaction is finished, and precipitating and drying to obtain pink particles for later use.

(2) Prepared by wrapping 0.16g DOX @ SiO in situ₂Placing the mixture and 80mL of water in a three-neck flask to prepare suspension, and introducing N₂After 30min, 38.3uL KH-570(1.5 vol%) and 20mL of water were added and stirred at 20-30 ℃ for 2 h. 24.25mg NaSS was weighed out and dissolved in 10mL H₂O, 2.55mL of St and NaSS aqueous solution were added dropwise through a constant pressure funnel, and stirred at 20-30 ℃ for 2 h. The temperature is raised to 65 ℃, an aqueous solution (5 wt%) prepared by 22.5mg KPS and 10mL water is slowly added to initiate polymerization, the color of the solution is gradually changed from light white to milky white, the reaction is carried out for 2h, and the emulsion is centrifugally washed to obtain the nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.45g of MMA (DOX @ SiO) was added₂The mass ratio of PS to MMA is 1:13), and the reaction is carried out for 2h at the temperature of 20-30 ℃. Heating to 65 deg.C, slowly adding 22.5mg KPS and 10mL water solution (5 wt%), initiating polymerization, changing the color of solution from light white to milky white, reacting for 8h, centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

(4) 50mg of NH₂-PEG-NH₂Dissolved in 8mL DMSO, 22mg folic acid, 47.8mg EDC and 15mg NHS were dissolved in 10mL DMSO, then 20. mu.L TEA was added and the mixture was magnetically stirred for 1 hour. The resulting yellow solution (one drop per 2 seconds) was added dropwise to NH with vigorous stirring₂-PEG-NH₂In solution and the reaction mixture was stirred overnight, after which the reaction product was usedDialyzing with dialysis bag, vacuum drying to remove DMSO, and obtaining light yellow powder NH₂-PEG-FA。

(5) 40mg of dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particles were dispersed in 10mL of deionized water, then 80mg of EDC was dissolved. The suspension was stirred for 15 minutes. Then, 12mg of NHS and 15mg of NH obtained in step (4) were added₂-PEG-FA. The reaction was stirred overnight and washed magnetically until no absorbance was detected at 270nm in the supernatant by UV-Vis spectroscopy. Finally, the product obtained is dried under vacuum at 20-30 ℃.

FIG. 9 is DOX @ (SiO)₂-PS)/PMAA-FA particle infrared spectrum. As can be seen from FIG. 9, 3454cm^-1The absorption peak is due to the modification of the-OH bond under the stretching vibration of the N-H bond; 1750cm^-1-1500cm^-1The absorption peak at (a) is due to pterin and benzene ring backbone of FA; 1630cm^-1The absorption peak at (a) is due to stretching vibration of aromatic C ═ C bond and C ═ N bond and signal overlap of amide bond; 1731cm^-1The absorption peak at (a) is due to C ═ O stretching vibration of the quinone and ketone groups of DOX. Indicating the presence of DOX and FA.

FIG. 10 shows targeting drug-loaded DOX @ (SiO)₂UV spectrogram of-PS)/PMAA-FA particles. The UV-Vis spectrum shown in FIG. 10 shows that the UV characteristic absorption peak near λ 363nm confirms that FA is successfully coupled to (SiO) through carbodiimide mediation₂PMMA end of PS)/PMMA particles, and uv characteristic absorption peak at λ 480nm confirm that DOX is also successfully encapsulated in (SiO) as well₂-PS)/PMMA particles. Compared with ultraviolet characteristic absorption peaks of FA and DOX, the ultraviolet peak-emitting wavelengths of the two drugs are not changed, which shows that the structures and properties of the drugs are not affected in the loading process, so that the drugs are dumbbell-Shaped (SiO)₂-PS)/PMMA particles are preferred drug delivery vehicles.

Fig. 11 is an electron micrograph of targeted drug-loaded Janus particles. From the SEM images of (a) and (b) in FIG. 11, DOX @ SiO₂The dumbbell-shaped DOX @ (SiO) is successfully prepared by a soap-free seed emulsion method as seeds₂-PS)/PMMA particles. After the surface of the particle is enlarged, the PMMA end group of the Janus particle is rough in surface, probably because the carboxyl on the surface of the particle is grafted with the PMMA end groupFA-PEG-NH₂Thereby, the effect is achieved. The TEM spectrum shows that the crystal is dumbbell-shaped DOX @ (SiO)₂the-PS)/PMAA-FA particles have complete shapes and DOX is coated on the nuclear shell SiO₂-a PS terminal.

Example 3 KH-570 preparation of DOX @ (SiO) at 2.0 vol% of monomer St₂-PS)/PMAA-FA particles

(2) Prepared by wrapping 0.16g DOX @ SiO in situ₂Placing the mixture and 80mL of water in a three-neck flask to prepare suspension, and introducing N₂After 30min, 51uL KH-570(2.0 vol%) and 20mL of water were added and stirred at 20-30 ℃ for 2 h. 24.25mg NaSS was weighed out and dissolved in 10mL H₂O, 2.55mL of St and NaSS aqueous solution were added dropwise through a constant pressure funnel, and stirred at 20-30 ℃ for 2 h. The temperature is raised to 65 ℃, an aqueous solution (5 wt%) prepared by 22.5mg KPS and 10mL water is slowly added to initiate polymerization, the color of the solution is gradually changed from light white to milky white, the reaction is carried out for 2h, and the emulsion is centrifugally washed to obtain the nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.45g of MMA (DOX @ SiO) was added₂The mass ratio of PS to MMA is 1:13), and the reaction is carried out for 2h at the temperature of 20-30 ℃. The temperature is raised to 65 ℃, water solution (5 wt%) prepared by 22.5mg KPS and 10mL water is slowly added to initiate polymerization, the color of the solution is changed from light white to milky white, after 8 hours of reaction, the emulsion is centrifugally washed, and the emulsion of dumbbell-shaped DOX @ (SiO2-PS)/PMMA particles is obtained.

(4) 50mg of NH₂-PEG-NH₂Dissolved in 8mIn L of DMSO, 22mg folic acid, 47.8mg EDC and 15mg NHS were dissolved in 10mL DMSO, then 20. mu.L TEA was added and the mixture was magnetically stirred for 1 hour. The resulting yellow solution (one drop per 2 seconds) was added dropwise to NH with vigorous stirring₂-PEG-NH₂In solution and the reaction mixture was stirred overnight, after which the reaction product was dialyzed against a dialysis bag and dried in vacuo to remove DMSO, obtaining NH as a pale yellow powder₂-PEG-FA。

Example 4: KH-570 being 3.0 vol% of monomer St DOX @ (SiO2-PS)/PMAA-FA particles were prepared

(2) Prepared by wrapping 0.16g DOX @ SiO in situ₂Placing the mixture and 80mL of water in a three-neck flask to prepare suspension, and introducing N₂After 30min, 76.5uL KH-570(3.0 vol%) and 20mL of water were added and stirred at 20-30 ℃ for 2 h. 24.25mg NaSS was weighed out and dissolved in 10mL H₂O, 2.55mL of St and NaSS aqueous solution were added dropwise through a constant pressure funnel, and stirred at 20-30 ℃ for 2 h. The temperature is raised to 65 ℃, an aqueous solution (5 wt%) prepared by 22.5mg KPS and 10mL water is slowly added to initiate polymerization, the color of the solution is gradually changed from light white to milky white, the reaction is carried out for 2h, and the emulsion is centrifugally washed to obtain the nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.45g of MMA (DOX @ SiO) was added₂Mass ratio of PS to MMA 1:13) And reacting for 2 hours at the temperature of 20-30 ℃. Heating to 65 deg.C, slowly adding 22.5mg KPS and 10mL water solution (5 wt%), initiating polymerization, changing the color of solution from light white to milky white, reacting for 8h, centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

Example 5: SiO2₂Preparation of DOX @ (SiO) with a mass ratio of PS to MMA of 1:7₂-PS)/PMAA-FA particles

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, add 0.24g of MMA (DOX @ SiO)₂The mass ratio of PS to MMA is 1:7), and the reaction is carried out for 2h at the temperature of 20-30 ℃. Heating to 65 deg.C, slowly adding water solution (5 wt%) prepared from 12mg KPS and 10mL water, initiating polymerization, changing the color of the solution from light white to milky white, reacting for 8h, centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

(5) 40mg of dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particles were dispersed in 10mL of deionized water, then 80mg of EDC was dissolved. The suspension was stirred for 15 minutes. Then, 12mg of NHS and 15mg of NH obtained in step (4) were added₂-PEG-FA. The reaction was stirred overnight and washed magnetically until no detection at 270nm in the supernatant by UV-Vis spectroscopyTo an absorbance. Finally, the product obtained is dried under vacuum at 20-30 ℃.

Example 6: SiO2₂Preparation of DOX @ (SiO) with a mass ratio of PS to MMA of 1:18₂-PS)/PMAA-FA particles

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.62g of MMA (DOX @ SiO) was added₂The mass ratio of PS to MMA is 1:18), and the reaction is carried out for 2h at the temperature of 20-30 ℃. Heating to 65 deg.C, slowly adding water solution (5 wt%) prepared from 31mg KPS and 10mL water, initiating polymerization, changing the color of the solution from light white to milky white, reacting for 8h, centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

(4) 50mg of NH₂-PEG-NH₂Dissolved in 8mL DMSO, 22mg folic acid, 47.8mg EDC and 15mg NHS were dissolved in 10mL DMSO, then20 μ L of TEA was added and the mixture was magnetically stirred for 1 hour. The resulting yellow solution (one drop per 2 seconds) was added dropwise to NH with vigorous stirring₂-PEG-NH₂In solution and the reaction mixture was stirred overnight, after which the reaction product was dialyzed against a dialysis bag and dried in vacuo to remove DMSO, obtaining NH as a pale yellow powder₂-PEG-FA。

Example 7: SiO2₂Preparation of DOX @ (SiO) with a mass ratio of PS to MMA of 1:21₂-PS)/PMAA-FA particles

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.73g of MMA (DOX @ SiO) are added₂The mass ratio of PS to MMA is 1:21), and the reaction is carried out for 2h at the temperature of 20-30 ℃. The temperature is raised to 65 ℃, and 3 is slowly added6.5mg KPS and 10mL water prepared aqueous solution (5 wt%), initiate polymerization, the solution color changed from light white to milk white, after 8h reaction, the emulsion centrifugal washing, get dumbbell shape DOX @ (SiO)₂-PS)/PMMA particle emulsion.

Example 8: preparation of DOX @ (SiO) with KPS in an amount of 2 wt.% of monomeric MMA₂-PS)/PMAA-FA particles

(2) Prepared by wrapping 0.16g DOX @ SiO in situ₂Placing the mixture and 80mL of water in a three-neck flask to prepare suspension, and introducing N₂30min, 38.3uL KH-570(1.5 vol%) and 20mL of water, 20-30 deg.CStirring for 2 h. 24.25mg NaSS was weighed out and dissolved in 10mL H₂O, 2.55mL of St and NaSS aqueous solution were added dropwise through a constant pressure funnel, and stirred at 20-30 ℃ for 2 h. The temperature is raised to 65 ℃, an aqueous solution (2 wt%) prepared by 9mg KPS and 10mL water is slowly added to initiate polymerization, the color of the solution is gradually changed from light white to milky white, the reaction is carried out for 2h, and the emulsion is centrifugally washed to obtain the nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.45g of MMA (DOX @ SiO) was added₂The mass ratio of PS to MMA is 1:13), and the reaction is carried out for 2h at the temperature of 20-30 ℃. Heating to 65 deg.C, slowly adding water solution (2 wt%) prepared from 9mg KPS and 10mL water, initiating polymerization, changing the color of the solution from light white to milky white, reacting for 8h, centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

Example 9: preparation of DOX @ (SiO) with KPS at 8 wt.% of monomeric MMA₂-PS)/PMAA-FA particles

(2) Prepared by wrapping 0.16g DOX @ SiO in situ₂Placing the mixture and 80mL of water in a three-neck flask to prepare suspension, and introducing N₂After 30min, 38.3uL KH-570(1.5 vol%) and 20mL of water were added and stirred at 20-30 ℃ for 2 h. 24.25mg NaSS was weighed out and dissolved in 10mL H₂O, 2.55mL of St and NaSS aqueous solution were added dropwise through a constant pressure funnel, and stirred at 20-30 ℃ for 2 h. The temperature is raised to 65 ℃, an aqueous solution (8 wt%) prepared by 36mg KPS and 10mL water is slowly added to initiate polymerization, the color of the solution is gradually changed from light white to milky white, the reaction is carried out for 2 hours, and the emulsion is centrifugally washed to obtain the nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.45g of MMA (DOX @ SiO) was added₂The mass ratio of PS to MMA is 1:13), and the reaction is carried out for 2h at the temperature of 20-30 ℃. Heating to 65 deg.C, slowly adding water solution (8 wt%) prepared from 36mg KPS and 10mL water, initiating polymerization, changing the color of the solution from light white to milky white, reacting for 8h, centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

(4) 50mg of NH₂-PEG-NH₂Dissolved in 8mL DMSO, 22mg folic acid, 47.8mg EDC and 15mg NHS were dissolved in 10mL DMSO, then 20. mu.L TEA was added and the mixture was magnetically stirred for 1 hour. Under the condition of vigorous stirringTo obtain a yellow solution (one drop per 2 seconds) is added dropwise to the NH₂-PEG-NH₂In solution and the reaction mixture was stirred overnight, after which the reaction product was dialyzed against a dialysis bag and dried in vacuo to remove DMSO, obtaining NH as a pale yellow powder₂-PEG-FA。

Example 10: preparation of DOX @ (SiO) with KPS at 11 wt.% of monomeric MMA₂-PS)/PMAA-FA particles

(2) Prepared by wrapping 0.16g DOX @ SiO in situ₂Placing the mixture and 80mL of water in a three-neck flask to prepare suspension, and introducing N₂After 30min, 38.3uL KH-570(1.5 vol%) and 20mL of water were added and stirred at 20-30 ℃ for 2 h. 24.25mg NaSS was weighed out and dissolved in 10mL H₂O, 2.55mL of St and NaSS aqueous solution were added dropwise through a constant pressure funnel, and stirred at 20-30 ℃ for 2 h. The temperature is raised to 65 ℃, aqueous solution (11 wt%) prepared by 49.5mg KPS and 10mL water is slowly added to initiate polymerization, the color of the solution is gradually changed from light white to milk white, the reaction is carried out for 2h, and the emulsion is centrifugally washed to obtain nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.45g of MMA (DOX @ SiO) was added₂The mass ratio of PS to MMA is 1:13), and the reaction is carried out for 2h at the temperature of 20-30 ℃. The temperature is raised to 65 ℃, an aqueous solution (11 wt%) prepared by 49.5mg KPS and 10mL water is slowly added to initiate polymerization, and the color of the solution is changed from lightChanging white into milky white, reacting for 8h, and centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

Example 11: preparation of DOX @ (SiO) at a reaction temperature of 55 deg.C₂-PS)/PMAA-FA particles

(2) Prepared by wrapping 0.16g DOX @ SiO in situ₂Placing the mixture and 80mL of water in a three-neck flask to prepare suspension, and introducing N₂After 30min, 38.3uL KH-570(1.5 vol%) and 20mL of water were added and stirred at 20-30 ℃ for 2 h. 24.25mg NaSS was weighed out and dissolved in 10mL H₂O, 2.55mL of St was mixed with a constant pressure funnelNaSS aqueous solution is added dropwise and stirred for 2h at the temperature of 20-30 ℃. The temperature is raised to 55 ℃, an aqueous solution (5 wt%) prepared by 22.5mg KPS and 10mL water is slowly added to initiate polymerization, the color of the solution is gradually changed from light white to milky white, the reaction is carried out for 2h, and the emulsion is centrifugally washed to obtain the nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.45g of MMA (DOX @ SiO) was added₂The mass ratio of PS to MMA is 1:13), and the reaction is carried out for 2h at the temperature of 20-30 ℃. Heating to 55 deg.C, slowly adding 22.5mg KPS and 10mL water solution (5 wt%), initiating polymerization, changing the color of solution from light white to milky white, reacting for 8h, centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

Example 12: preparation of DOX @ at a reaction temperature of 80 ℃ @ ((SiO₂-PS)/PMAA-FA particles

(2) Prepared by wrapping 0.16g DOX @ SiO in situ₂Placing the mixture and 80mL of water in a three-neck flask to prepare suspension, and introducing N₂After 30min, 38.3uL KH-570(1.5 vol%) and 20mL of water were added and stirred at 20-30 ℃ for 2 h. 24.25mg NaSS was weighed out and dissolved in 10mL H₂O, 2.55mL of St and NaSS aqueous solution were added dropwise through a constant pressure funnel, and stirred at 20-30 ℃ for 2 h. The temperature is raised to 80 ℃, an aqueous solution (5 wt%) prepared by 22.5mg KPS and 10mL water is slowly added to initiate polymerization, the color of the solution is gradually changed from light white to milky white, the reaction is carried out for 2h, and the emulsion is centrifugally washed to obtain the nuclear shell-shaped DOX @ SiO₂-a PS particle emulsion.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-emulsion of PS particles, passing N₂30min, 0.45g of MMA (DOX @ SiO) was added₂The mass ratio of PS to MMA is 1:13), and the reaction is carried out for 2h at the temperature of 20-30 ℃. Heating to 80 deg.C, slowly adding water solution (5 wt%) prepared from 22.5mg KPS and 10mL water, initiating polymerization, changing the color of the solution from light white to milky white, reacting for 8h, centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

(4) 50mg of NH₂-PEG-NH₂Dissolved in 8mL DMSO, 22mg folic acid, 47.8mg EDC and 15mg NHS were dissolved in 10mL DMSO, then 20. mu.L TEA was added and the mixture was magnetically stirred for 1 hour. The resulting yellow solution (one drop per 2 seconds) was added dropwise to NH with vigorous stirring₂-PEG-NH₂In solution, andthe reaction mixture was stirred overnight, after which the reaction product was dialyzed against a dialysis bag and dried in vacuo to remove DMSO, obtaining NH as a pale yellow powder₂-PEG-FA。

Example 13: DOX @ (SiO)₂Preparation of-PS)/PMAA particles

(1) Uniformly mixing 39mL of ethanol and 33.6 mL of DOX in a round-bottom flask, dropwise adding 3.0g of TEOS, 4.5mL of ammonia water and 3.25mL of water by using a constant-pressure funnel under stirring, reacting at 30 ℃ for 3h, centrifugally removing impurities from the obtained product after the reaction is finished, and precipitating and drying to obtain pink particles for later use.

A three-necked flask was charged with 21mL of water, 5.6mL of NaCl solution (0.1mol/L), and 4g of DOX @ SiO₂-PS particle emulsion through N₂After 30min, 0.45g of MMA (1:13) was added and the reaction was carried out at 20-30 ℃ for 2 h. Heating to 65 deg.C, slowly adding 22.5mg KPS and 10mL water solution (5 wt%), initiating polymerization, changing the color of solution from light white to milky white, reacting for 8h, centrifuging and washing the emulsion to obtain dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particle emulsion.

(3) Get30mL dumbbell DOX @ (SiO)₂Putting the-PS)/PMMA particle emulsion into a triangular flask, adding 4mL of 2mol/L NaOH solution, hydrolyzing in a shaking table for 24h at 42 ℃ and at the rotating speed of 110r/min, repeatedly washing the mixed solution with distilled water to be neutral after the reaction is finished, and finally drying the product into powder.

Preparation of DOX @ (SiO) with different KH-570 contents₂Performance testing of the-PS)/PMAA-FA particles

The samples obtained in examples 1, 2, 3, 4 were characterized by comparison with the difference in the content of KH-570 vs. DOX @ (SiO)₂-influence of PS)/PMAA-FA particle morphology. And (3) taking a 15uL sample to dilute to 3mL, performing ultrasonic dispersion treatment for 10min, coating on a glass substrate, and drying. Spraying gold before testing, using SIGMA field emission Scanning Electron Microscope (SEM) of ZEISS, Germany, for DOX @ (SiO)₂Characterization of the particle morphology of-PS)/PMAA-FA.

The results are shown in FIG. 5 (a), (b), (c) and (d), in which the KH-570 content was (a)1 vol%; (b)1.5 vol%; (c)2 vol%; (d)3 vol%. As can be seen, 1.5 vol% KH-570 in SiO₂As a seed, PS was successfully synthesized at DOX @ (SiO)₂-PS)/PMAA-FA particles having a particle size of about 340nm at one end and about 310nm at the other end, wherein the particles are uniformly distributed in the system and substantially free of irregular particles. When SiO is present₂When the amount of KH-570 in the PS particles is small, the crosslinked network is not dense enough, the seeds swell first in the presence of MMA to allow the monomer to diffuse into the seeds, the diffusion rate is relatively fast, but the seeds have poor crosslinkability during shrinkage, and provide a small elastic shrinkage force, resulting in a slow shrinkage rate of the seeds, thereby not leaving MMA droplets on the surface thereof, and the monomer remaining inside the seeds cannot cause phase separation under the squeezing force of the shrinkage force, thereby obtaining spherical particles larger than the seeds, and having a particle size of about 370 nm. With increasing KH-570, the seed crosslinked network becomes increasingly dense, and SiO when the elastic contractive force provided by seed contraction is large enough₂The PS seeds form a new phase outside them, resulting in dumbbell-shaped particles. When SiO is present₂At higher amounts of KH-570 in the PS particles, the seed crosslinked network is extremely dense, the elastic shrinkage force is higher at higher temperature, and the shrinkage speed is faster, which allows the seeds to leave MMA droplets on different surfaces or on different surfacesSiO under extrusion of shrink force₂Monomers in PS tend to phase separate in all directions, and upon addition of initiator, MMA will participate in the polymerization in different directions, forming topographically irregular particles (see fig. 5(c), (d)).

Illustrating the synthesized DOX @ (SiO) when the KH-570 content is 1.5 vol%₂The particle shape of the-PS)/PMAA-FA particles is optimal, the particles are uniformly distributed, and irregular particles are basically absent.

Different SiO₂Performance test for preparing DOX @ (SiO2-PS)/PMAA-FA particles by mass ratio of PS to MMA

The samples obtained in examples 2, 5, 6 and 7 were characterized in comparison with SiO₂Different mass ratios of PS to MMA vs DOX @ (SiO)₂-influence of PS)/PMAA-FA particle morphology. And (3) taking a 15uL sample to dilute to 3mL, performing ultrasonic dispersion treatment for 10min, coating on a glass substrate, and drying. Spraying gold before testing, using SIGMA field emission Scanning Electron Microscope (SEM) of ZEISS, Germany, for DOX @ (SiO)₂Characterization of the particle morphology of-PS)/PMAA-FA.

The results are shown in FIG. 6 (a), (b), (c) and (d), in which SiO is present₂-the mass ratio of PS to MMA is (a)1:7, respectively; (b)1: 13; (c)1: 18; (d)1: 21. As can be seen, SiO₂The mass ratio of PS seed to MMA is 1:13 and 1:18, and DOX @ (SiO)₂The particle sizes of one end of the-PS)/PMAA-FA particles are all about 310nm, the particle sizes of the other end of the-PS)/PMAA-FA particles are respectively 280nm and 300nm, the particles in the system are uniformly distributed, and irregular particles are not generated. When the amount of MMA is small, it swells into SiO₂Less MMA of the PS crosslinked network, insufficient to reach the swelling equilibrium of the seed particles, elevated temperature SiO₂The PS crosslinked network shrinks so that MMA polymerizes inside the seed, giving spherical particles with a particle size of about 370 nm. As the MMA addition was increased, it swelled into SiO₂Increased MMA of PS cross-linked network, equilibrium of seed particle swelling, elevated temperature SiO₂The PS crosslinked network shrinks, causing phase separation of the monomers. When the amount of MMA is too large, MMA is in SiO₂After the phase separation is formed outside the PS seeds, excessive monomers in the system continue to participate in polymerization, and irregular particles are formed.

When SiO is used₂Masses of PS and MMADOX @ (SiO) synthesized at a ratio of 1:13 to 1:18₂The particle shape of the-PS)/PMAA-FA particles is optimal, the particles are uniformly distributed, and irregular particles are basically absent.

Preparation of DOX @ (SiO) with different KPS dosages₂Performance testing of the-PS)/PMAA-FA particles

The samples obtained in examples 2, 8, 9 and 10 were characterized by comparing the different KPS amounts at DOX @ (SiO)₂-influence of PS)/PMAA-FA particle morphology. And (3) taking a 15uL sample to dilute to 3mL, performing ultrasonic dispersion treatment for 10min, coating on a glass substrate, and drying. Spraying gold before testing, using SIGMA field emission Scanning Electron Microscope (SEM) of ZEISS, Germany, for DOX @ (SiO)₂Characterization of the particle morphology of-PS)/PMAA-FA.

The results are shown in FIG. 7 (a), (b), (c) and (d), and FIG. 7 shows the results of different KPS dosages vs. DOX @ (SiO)₂-PS)/PMAA-FA particles. Wherein the KPS is used in an amount of (a)2 wt%, (b)5 wt%, (c)8 wt%, and (d)11 wt%, respectively. As is clear from the figure, when KPS is 5 wt% of the monomer, DOX @ (SiO) is synthesized₂The particle size of one end of the-PS)/PMAA-FA particle is about 310nm, the particle size of the other end of the-PS)/PMAA-FA particle is about 310nm, and the particles in the system are uniformly distributed and have no irregular particles. When the KPS is used in a small amount, the free radicals in the system are not enough to form bulges outside the seeds, but participate in polymerization inside the seeds to form spherical particles with the particle size of about 375 nm. As the amount of KPS is increased, a part of KPS enters the interior of the seed to generate free radicals which can be in SiO₂-diffusing or transferring the PS seeds into MMA to initiate MMA polymerization, and forming protrusions on the surfaces of the seeds; the other part, in aqueous phase, diffuses with the MMA onto the surface of the projections and polymerizes with the inner extruded projections, forming new (secondary) spherical particles. When the amount of KPS is increased to 8 wt% and 10 wt%, too many radicals are generated in the system, and they are vigorously polymerized with MMA of the system, forming irregular PMMA particles.

Illustrating the synthesized DOX @ (SiO) when KPS is used in an amount of 5 wt% based on the monomer₂The particle shape of the-PS)/PMAA-FA particles is optimal, the particles are uniformly distributed, and irregular particles are basically absent.

Preparation of DOX @ (SiO) at different reaction temperatures₂Performance testing of the-PS)/PMAA-FA particles

The samples obtained in examples 2, 11 and 12 were characterized by comparison of the difference in reaction temperature with respect to DOX @ (SiO)₂-influence of PS)/PMAA-FA particle morphology. And (3) taking a 15uL sample to dilute to 3mL, performing ultrasonic dispersion treatment for 10min, coating on a glass substrate, and drying. Spraying gold before testing, using SIGMA field emission Scanning Electron Microscope (SEM) of ZEISS, Germany, for DOX @ (SiO)₂Characterization of the particle morphology of-PS)/PMAA-FA.

The results are shown in FIG. 8 (a), (b) and (c), and FIG. 8 shows the reaction temperature vs. DOX @ (SiO)₂-influence of PS)/PMAA-FA particle morphology. Wherein (a) is 55 ℃, (b) is 65 ℃, (c) is 80 ℃. As is clear from FIG. 8, DOX @ (SiO) was synthesized at a reaction temperature of 65 ℃₂-PS)/PMAA-FA particles, wherein the particle size of one end is about 310nm, the particle size of the other end is about 300nm, and the particles in the system are uniformly distributed and have no irregular particles. When the reaction temperature is low, SiO₂The PS seeds have weak elastic contractive force and slow contraction speed, can not form bulges on the surfaces of the seeds, and generate less free radicals by KPS decomposition in a system at lower temperature, so spherical particles with the particle size of about 370nm are obtained. With the temperature rise, the seed shrinkage speed becomes fast, and the KPS half-life temperature is reached, and the dumbbell-shaped particles are obtained. When the reaction temperature continues to rise to 80 ℃, SiO₂The PS has strong elastic contractive force and high contraction speed, and generates a plurality of bulges on the surface of the seed, and the bulges form irregular particles after the MMA is rapidly polymerized.

Illustrating the DOX @ (SiO) synthesized when the reaction temperature is 65 deg.C₂The particle shape of the-PS)/PMAA-FA particles is optimal, the particles are uniformly distributed, and irregular particles are basically absent.

The following examples examined DOX @ (SiO) using in-situ wrapping and post-wrapping₂The encapsulation rate of the-PS)/PMAA-FA particles to the medicine, and finally, an in-situ encapsulation method is selected to encapsulate the medicine in SiO₂Inside with SiO loaded with drug₂The particles are seeds, the surfaces of which are modified, and C ═ C groups are introduced.

Example 14: SiO when the dosage DOX is 16.8mg/5mg₂Preparation of microspheres

An in-situ wrapping method: uniformly mixing 39mL of ethanol and 16.8mg of DOX in a round-bottom flask, dropwise adding 3.00g of TEOS, 4.5mL of ammonia water and 3.25mL of water by using a constant-pressure funnel under stirring, reacting at 30 ℃ for 3h, centrifuging and removing impurities from the obtained product after the reaction is finished, taking the filtrate to test the ultraviolet of the filtrate, and precipitating and drying to obtain pink particles for later use.

Or a posterior wrapping method: preparing 5mg/mL solution of 25mg DOX and 5mL ethanol, taking 1mL mixed solution in a single-neck flask by a pipette, and taking 10mg SiO₂Adding the particles into a flask, performing ultrasonic dispersion at the reaction temperature of 30 ℃ for 24h, centrifuging after the reaction is finished, testing the ultraviolet of the filtrate, and drying the precipitate to obtain pink particles for later use.

Example 15: SiO when the dosage DOX is 25mg/10mg₂Preparation of microspheres

An in-situ wrapping method: uniformly mixing 39mL of ethanol and 25mg of DOX in a round-bottom flask, dropwise adding 3.00g of TEOS, 4.5mL of ammonia water and 3.25mL of water by using a constant-pressure funnel under stirring, reacting at 30 ℃ for 3h, centrifuging and removing impurities from the obtained product after the reaction is finished, taking the filtrate to test the ultraviolet of the filtrate, and precipitating and drying to obtain pink particles for later use.

Or a posterior wrapping method: preparing 5mg/mL solution of 25mg DOX and 5mL ethanol, taking 2mL mixed solution by a pipette, and taking 10mg SiO₂Adding the particles into a flask, performing ultrasonic dispersion at the reaction temperature of 30 ℃ for 24h, centrifuging after the reaction is finished, testing the ultraviolet of the filtrate, and drying the precipitate to obtain pink particles for later use.

Example 16: SiO when the dosage DOX is 33.6mg/20mg₂Preparation of microspheres

An in-situ wrapping method: uniformly mixing 39mL of ethanol and 33.6mg of DOX in a round-bottom flask, dropwise adding 3.00g of TEOS, 4.5mL of ammonia water and 3.25mL of water by using a constant-pressure funnel under stirring, reacting at 30 ℃ for 3h, centrifuging and removing impurities from the obtained product after the reaction is finished, taking the filtrate to test the ultraviolet of the filtrate, and precipitating and drying to obtain pink particles for later use.

Or a posterior wrapping method: preparing 5mg/mL solution of 25mg DOX and 5mL ethanol, taking 4mL mixed solution in a single-neck flask by a pipette, and taking 10mg SiO₂Adding the particles into a flask, performing ultrasonic dispersion at the reaction temperature of 30 ℃ for 24h, centrifuging after the reaction is finished, testing the ultraviolet of the filtrate, and drying the precipitate to obtain pink particles for later use.

Example 17: SiO when the dosage DOX is 42mg/30mg₂Preparation of microspheres

An in-situ wrapping method: uniformly mixing 39mL of ethanol and 42mg of DOX in a round-bottom flask, dropwise adding 3.00g of TEOS, 4.5mL of ammonia water and 3.25mL of water by using a constant-pressure funnel under stirring, reacting at 30 ℃ for 3h, centrifuging and removing impurities from the obtained product after the reaction is finished, taking the filtrate to test the ultraviolet of the filtrate, and precipitating and drying to obtain pink particles for later use.

Or a posterior wrapping method: preparing 5mg/mL solution of 25mg DOX and 5mL ethanol, taking 6mL mixed solution in a single-neck flask by using a pipette, and taking 10mg SiO₂Adding the particles into a flask, performing ultrasonic dispersion at the reaction temperature of 30 ℃ for 24h, centrifuging after the reaction is finished, testing the ultraviolet of the filtrate, and drying the precipitate to obtain pink particles for later use.

Example 18: SiO when the dosage DOX is 50.4mg/40mg₂Preparation of microspheres

An in-situ wrapping method: uniformly mixing 39mL of ethanol and 50.4mg of DOX in a round-bottom flask, dropwise adding 3.00g of TEOS, 4.5mL of ammonia water and 3.25mL of water by using a constant-pressure funnel under stirring, reacting at 30 ℃ for 3h, centrifuging and removing impurities from the obtained product after the reaction is finished, taking the filtrate to test the ultraviolet of the filtrate, and precipitating and drying to obtain pink particles for later use.

Or a posterior wrapping method: preparing 5mg/mL solution of 25mg DOX and 5mL ethanol, taking 8mL mixed solution in a single-neck flask by using a pipette, and taking 10mg SiO₂Adding the particles into a flask, performing ultrasonic dispersion at the reaction temperature of 30 ℃ for 24h, centrifuging after the reaction is finished, testing the ultraviolet of the filtrate, and drying the precipitate to obtain pink particles for later use.

SiO₂Determination of encapsulation efficiency

25mgDOX was weighed and dissolved in a 100mL volumetric flask to prepare a 250. mu.g/mL aqueous solution of DOX. 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL of the standard solution was pipetted precisely and made to a volume of 25mL to prepare DOX standard solutions having concentrations of 2. mu.g/mL, 4. mu.g/mL, 6. mu.g/mL, 8. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 30. mu.g/mL, 40. mu.g/mL, 50. mu.g/mL. The maximum absorption wavelength was measured at a DOX standard solution of 50. mu.g/mL in the 200nm-700nm band. The absorbance of the above standard solutions was measured at the maximum absorption wavelength using water as a blank and a linear regression method was used to fit the standard curve, see fig. 12.

Loading 10mg of SiO into the mixture₂The ball was diluted with deionized water to a dose concentration within the range of the standard curve concentration, and then centrifuged to take 5mL of supernatant for testing. And (4) obtaining the concentration of the solution according to a standard curve fitting equation, and further calculating the corresponding encapsulation efficiency.

The effects of different dosages on encapsulation efficiency obtained for examples 14, 15, 16, 17, 18 are shown in table 1.

TABLE 1 DOX dosing vs. SiO₂Effect of encapsulation efficiency

As can be seen from table 1, with increasing dose of DOX, the encapsulation efficiency of both the post-wrap and in-situ wrap increased and then decreased. The reason for this is that SiO increases when the dose of DOX is increased₂The amount of drug encapsulated in the spheres will increase, but SiO will increase₂The drug loading capacity of the ball is not infinite, and after the dosage concentration of DOX is increased to a certain value, SiO is added₂The ball will reach saturation state, and DOX is increased at this time, so the encapsulation efficiency is not increased or decreased. DOX-loaded SiO obtained by in-situ wrapping method₂The ball encapsulation efficiency is higher because the in-situ wrapping method is adopted to prepare the product by forming SiO₂Directly encapsulating DOX in SiO during ball₂Internally without regard to concentration gradients and SiO₂Pore size, but the post-packing method causes the drug to enter SiO from the pore canal by diffusion₂In the ball, there is a possibility that DOX is adsorbed on the surface of the ball, pores of the ball and SiO₂In the ball, SiO is reduced₂The encapsulation efficiency of (2).

Using the highest envelope delivery ratePreparation of drug-loaded SiO by drug concentration₂And using it as seed to synthesize dumbbell-shaped DOX @ (SiO)₂-PS)/PMAA-FA particles.

Combined with single factor method to DOX @ (SiO)₂The preparation process of the-PS)/PMAA-FA particle is optimized, and the functional monomer FA is used for preparing dumbbell-Shaped (SiO)₂-PS)/PMMA particles. The following conclusions were reached:

(1) synthesis of DOX @ (SiO)₂The optimal process for the-PS)/PMAA-FA particles is as follows: KH-570 is 1.5 vol% SiO₂PS seeds, reaction temperature 65 ℃ C, m (SiO)₂PS) m (MMA) is 1:13 to 1:18, and KPS is used in an amount of 5 wt% based on the monomer.

(2) Loading functional monomer FA on dumbbell-shaped DOX @ (SiO)₂-PS)/PMAA end of PMAA particles, to obtain DOX @ (SiO)₂-PS)/PMAA-FA particles, rendering them targeting.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A preparation method of targeted drug-loaded dumbbell-shaped Janus particles is characterized by comprising the following steps:

2. The preparation method of the dumbbell-shaped Janus particle with targeted drug loading according to claim 1, wherein the preparation method is characterized in thatIn step (1), DOX @ (SiO)₂-volume ratio of PS)/PMMA particle emulsion to NaOH solution (25-35): (3-5), the concentration of the sodium hydroxide solution is 2 mol/L.

3. The preparation method of the dumbbell Janus particle with targeted drug loading according to claim 1, wherein in step (1), the dumbbell DOX @ (SiO) is adopted₂-PS)/PMMA particle emulsion is prepared by the following procedure:

4. The method for preparing dumbbell Janus particles with targeted medicine loading according to claim 3, wherein in step 1.1), the cross-linking agent is KH-570, KH-560 or DL-602; the volume of the crosslinking agent is 1.0% to 3.0% of the volume St.

5. The preparation method of the dumbbell-shaped Janus particle with the targeted medicine carrying function according to claim 3, wherein in the step 1.2) and the step 1.3), the mass of KPS is 2% -11% of that of MMA; DOX @ SiO₂Mass of the emulsion of PS particles is DOX @ SiO₂4.5-12.2% of the total mass of the PS particle emulsion and the MMA, and the mass of the MMA is DOX @ SiO₂87.8% of the total mass of PS particle emulsion and MMA95.5％。

6. The preparation method of the dumbbell-shaped Janus particle with targeted drug loading according to claim 3, wherein the preparation method is characterized in that DOX @ SiO₂Is prepared by the following steps: uniformly mixing ethanol and doxorubicin hydrochloride, dropwise adding 2-5g TEOS, 3-5mL ammonia water and 2-5mL water under stirring, and reacting at 25-35 ℃ for 2-4h to obtain DOX @ SiO₂(ii) a Wherein the mass ratio of the ethanol to the TEOS is 10:1-3: 5.

7. The preparation method of the dumbbell-shaped Janus particle with the targeted medicine carrying function according to claim 6, wherein the mass ratio of ethanol to doxorubicin hydrochloride is 3 (1-8).

8. The preparation method of the dumbbell Janus particle with targeted drug loading according to claim 3, wherein in the step (2), the dumbbell DOX @ (SiO) is adopted₂-PS)/PMMA particles, EDC, NHS, NH₂-PEG-FA mass ratio (30-50): (75-85): (10-15): (10-20); dumbbell-shaped DOX @ (SiO)₂-PS)/PMMA particles to water ratio of (30-50) mg: (8-15) mL.

9. The method for preparing dumbbell-shaped Janus particles carrying medicine in targeted mode according to claim 8, wherein NH is adopted₂-PEG-NH₂Is prepared by the following steps: adding diamino end-capped polyethylene glycol into dimethyl sulfoxide to obtain NH with concentration of 5-7mg/mL₂-PEG-NH₂A solution;

10. Use of drug-loaded dumbbell Janus prepared according to the method of any one of claims 1-9 in a drug delivery vehicle.