CN102068409A - Method for preparing mono-disperse microemulsion, liposome and microsphere based on microfluidic technology - Google Patents

Abstract

The invention discloses a method for preparing monodisperse microemulsion, liposome and microsphere based on microfluidic technology. The method comprises the following steps: using the aqueous solution (or oil solution) of hydrophilic drugs (or fat-soluble drugs) as a dispersed phase, the oil phase (or water phase) as a continuous phase, and transporting the dispersed phase and the continuous phase to the microfluidic The corresponding microchannels of the control chip device are sheared into monodisperse drug-encapsulated droplets, and then the droplets are solidified by a certain curing method to obtain drug-loaded liposome microspheres or biodegradable microspheres with uniform size and stable dispersion. ball. Under optimal conditions, the diameter distribution coefficient of microemulsions and microspheres can be less than 5%, and the diameters range from 10 to 500 microns. This technology solves the problems of drug-loaded microemulsions, liposomes, and microspheres with uneven size, low embedding rate, poor dispersion, poor targeting, and low bioavailability prepared by traditional ultrasound, stirring emulsification, and film hydration dispersion methods. Enzymes and cells have low biological activity, resulting in problems such as immunosuppression.

Description

A method for preparing monodisperse microemulsions, liposomes and microspheres based on microfluidic technology

technical field

The invention belongs to the field of pharmaceutical engineering drug preparations, and in particular relates to a method for preparing monodisperse microemulsions, liposomes and microspheres based on microfluidic technology.

Background technique

Microfluidic technology is an important science and technology, its main feature is integration and miniaturization, it will play a role in disease diagnosis, drug screening, environmental testing, food safety, judicial identification, sports competition, anti-terrorism, aerospace and other fields . Microfluidic chips are mainly characterized by the integration of microchannel networks and various functional units. They control and process micro-samples at the micro-scale, and realize the preparation, reaction, separation and detection of micro-samples. Droplets are a brand-new technology for manipulating tiny volumes of liquids developed on microfluidic chips in recent years. As a brand-new technology, the most common application of droplets is as a microreactor to study reactions and processes at the microscale. Among these applications, the preparation of uniform size-controlled microemulsions, liposomes, and biodegradable microspheres has important applications in drug delivery. At present, the methods for preparing microemulsions, liposomes and biodegradable microspheres mainly adopt the "top-down" method, that is, traditional ultrasound, mechanical stirring, etc. , the small emulsion will be absorbed by the large emulsion, and at the same time the large emulsion will be destroyed by the shear force; in the process of emulsion polymerization, many aggregates will be formed, and at the same time, in the process of merging and breaking of the emulsion, Targets such as drugs, enzymes, and cells in the emulsion are easy to escape to the surface of the emulsion, resulting in a low embedding rate of the target. Therefore, microemulsions, liposomes and biodegradable microspheres obtained by traditional methods have a wide particle size distribution, difficult shape control, and large deviations in the results of repeated preparations, giving microemulsions, liposomes and biodegradable microspheres Practical application brings many difficulties. For example, when liposomes are prepared by the commonly used film hydration dispersion method for hydrophilic drugs, the encapsulation efficiency is low, and the drug loading is difficult to meet the treatment needs. As a drug carrier, the size of the particle size determines its distribution in the body and affects its interaction with biological organs and cells. The small particle size carrier can minimize the potential irritation at the injection site; and if a monodisperse carrier is used, The drug release kinetics can be effectively controlled, making it easier to construct more complex drug release systems. In addition, as an enzyme immobilization and protein separation medium, due to the uneven particle size, small microspheres will gather in the gap between large microspheres, which will increase the separation pressure, and in severe cases, the separation will not be possible; as a cell culture As a carrier, uneven particle size will lead to uneven nutrient and cell density, resulting in uneven cell growth; as a drug sustained-release carrier, the embedding rate of the drug will be greatly reduced due to the instability of the emulsion. In addition, due to the uneven particle size , Poor drug targeting in systemic administration, leading to greatly reduced bioavailability of drugs in vivo, and differences between preparation batches, resulting in the inability to accurately investigate drug release patterns, greatly limiting its application. Therefore, new preparation methods are studied to prepare monodisperse microemulsions, liposomes and biodegradable microspheres in order to overcome the shortcomings of traditional preparation methods and the resulting application limitations.

Since the droplets produced by microfluidic technology have the characteristics of no sample diffusion, stable reaction conditions, good monodispersity, no cross-contamination, and rapid mixing, microemulsions and lipids prepared by microfluidic technology based on the "bottom up" idea Plastids and biodegradable microspheres have the advantages of controllable shape, uniform size, and diversity of applicable materials. By introducing drugs, enzymes, and cells into droplets, monodisperse microemulsions, liposomes, and biodegradable microspheres are produced, which solves the problems of unbalanced size, low target encapsulation rate, and poor repeatability in traditional preparation methods, in order to construct One can control the release of drugs, improve the bioavailability of drugs, maintain the biological activity of enzymes and cells, improve the targeting of drug carriers, reduce the systemic toxicity of drugs, and immunosuppression and alloallergic reactions during cell transplantation.

Contents of the invention

The present invention prepares O/W, W/O and W/O/W microemulsions, liposomes and biodegradable microemulsions with uniform size and stable dispersion for various hydrophilic drugs and fat-soluble drugs, enzymes and cells. Spheres for drug delivery, control and release; enzyme immobilization and activity maintenance; cell culture, functional expression and targeted transplantation, etc.

To achieve the above object, the present invention adopts the following technical solutions:

A microfluidic chip device capable of preparing monodisperse microemulsions, liposomes, and biodegradable microspheres, including micropumps, microfluidic chips that generate droplets of different sizes, droplet receiving and storage containers; (1 ) According to the properties of the prepared microemulsion, the corresponding hydrophobic or hydrophilic modification is carried out on the microchannel of the chip; for example, when preparing a W/O microemulsion, a microchannel with a hydrophobic inner wall made of PDMS or glass can be used; When preparing O/W microemulsions, it is necessary to use polymers (such as polyvinyl alcohol, etc.) to modify the surface of PDMS microchannels to make their inner walls hydrophilic; in particular, through adsorption self-assembly-thermal curing method, using The mixed solution of 5% glycerol-2% polyvinyl alcohol is used to modify the PDMS microchannel, so that the surface of the modified PDMS has superhydrophilicity. (2) Dissolve the hydrophilic drug (or fat-soluble drug) in the water phase (or oil phase), then use the water phase (or oil phase) as the dispersed phase, and the oil phase (or water phase) as the continuous phase. (3) Prepare a corresponding solution according to the properties of the drug, pass it through the middle microchannel, and pass the solution of liposome or biocompatible polymer into the two side microchannel, and then use the continuous phase opposite to the above solution to shear the above solution into monodisperse drug-encapsulated droplets, and then use a certain curing method to solidify the droplets to obtain a drug-loaded lipid with uniform size and stable dispersion. Plastid microspheres or biodegradable microspheres. Under optimal conditions, the diameter distribution coefficient (CV value, definition sees the following formula) of microemulsion and microsphere is controlled within 5%, under the situation of microchannel size determination (the present invention is with 200 micron as example), by adjusting dispersed phase Concentration, flow rate of dispersed phase and continuous phase can be freely adjusted to achieve microemulsions, liposomes or microspheres with a diameter of 10-500 microns; when used as a drug carrier, the drug embedding rate is high, storage is stable, and embedding under optimized conditions The rate is close to 100%. When used to wrap enzymes and cells, the wrapping rate is high and the loss of activity is small.

The above diameter distribution coefficient (Coefficient of Variation, CV) = {[∑(d _i -d) ² /N] ^1/2 /d} × 100%

In the formula, d _i is the diameter of each microemulsion, liposome and polymer microsphere, d is the average diameter of the studied microemulsion, liposome and polymer microsphere, and N is the microemulsion used to study the diameter, The number of liposomes and polymer microspheres, N=100.

The method for preparing monodisperse drug-loaded microemulsion provided by the present invention comprises the steps of: using the aqueous solution (oil solution) containing water-soluble (fat-soluble) medicine as the dispersed phase; ) the oil solution (aqueous solution) of the emulsifier as the continuous phase; the dispersed phase and the continuous phase are delivered to the corresponding microchannels of the microfluidic chip device respectively, and the W/O (O/ W) microemulsion, the microemulsion is collected in the container that continuous phase or cross-linking agent solution is housed, obtains drug-loaded microemulsion.

The method for preparing monodisperse drug-loaded liposomes provided by the present invention comprises the steps of: using an aqueous solution (oil solution) containing water-soluble (fat-soluble) drugs and blank liposomes (that is, drug-free liposomes) As the dispersed phase; the oil solution (aqueous solution) containing W/O type (O/W type) emulsifier is used as the continuous phase; the dispersed phase and the continuous phase are respectively delivered to the corresponding microchannel of the microfluidic chip device, through The microfluidic chip device prepares W/O (O/W) microemulsions, and freeze-dries the generated microemulsions to obtain drug-loaded liposomes.

The method for preparing monodisperse drug-loaded microspheres provided by the present invention comprises the following steps: using an aqueous solution (oil solution) containing a water-soluble drug (fat-soluble) and a biodegradable polymer as a dispersed phase; The oil solution (aqueous solution) of the type (O/W type) emulsifier is used as the continuous phase 5; the dispersed phase and the continuous phase are respectively delivered to the corresponding microchannels of the microfluidic chip device, and prepared by the microfluidic chip device Obtain W/O (O/W) microemulsions, collect the microemulsions into a container with a continuous phase or a cross-linking agent solution, and adopt different solidification methods (such as: freeze-drying, heating to remove the solvent, adding a cross-linking agent to polymerize, etc.) for curing to obtain drug-loaded microspheres.

The method for preparing microemulsions, liposomes and microspheres of monodispersity encapsulating enzymes and cells provided by the present invention is the same as the above-mentioned method, but should pay attention to the use of biocompatible substances when selecting the dispersed phase and the continuous phase, Reduce the effect on the activity of enzymes and cells.

In the microemulsion prepared by the present invention, the concentration of encapsulated medicine (enzyme or cell) can be adjusted by adjusting the flow rate of the dispersed phase and the continuous phase in the microfluidic channel.

When preparing drug-loaded liposomes or drug-loaded microspheres, the size of liposomes or microspheres can be adjusted by the concentration of liposomes or biodegradable polymers in the dispersed phase (when other conditions are fixed, lipids The greater the concentration of liposomes or polymers, the larger the size of liposomes or microspheres produced), or through the flow adjustment of the dispersed phase and the continuous phase (when the flow rate of the dispersed phase is fixed, the flow rate of the continuous phase is smaller (larger) , the larger (smaller) the size of the microspheres will be produced; when the flow rate of the continuous phase is fixed, the larger (smaller) the flow rate of the dispersed phase will be, and the size of the microspheres will be larger (smaller)).

In the above method for preparing microemulsions, liposomes or microspheres loaded with drugs or encapsulating enzymes and cells, the mass concentration of the surfactant in the continuous phase can be 0.5%-10%, preferably in the range of 0.5%-4%. In the method of preparing microemulsions, liposomes or microspheres encapsulating enzymes and cells, surfactants with good biocompatibility should be selected.

In the present invention, the water-soluble drugs include water-soluble anticancer drugs, nucleic acid, RNA, interferon, various proteins, enzymes and polypeptide drugs, etc., which can be dissolved in the water phase together or separately according to needs; fat-soluble drugs include fat-soluble Anticancer drugs, hormones and various other fat-soluble drugs, etc. The emulsifiers used generally use nonionic surfactants.

The monodisperse microemulsion, liposome or microsphere drug carrier prepared by the present invention, and the liposome or microsphere of package enzyme and cell have the following advantages: (1) the method provided by the invention can be used for preparing various types of microemulsion Milk, liposomes and polymer microspheres can be used to embed a variety of hydrophilic and fat-soluble drugs, such as paclitaxel, pirarubicin, 5-fluorouracil, etc.; they can also be used for immobilized enzymes and cell culture , transplantation, etc., such as immobilized insulin enzyme for the treatment of diabetes, cells wrapped with recombinant urease gene are used to study the decomposition of harmful substances in the body, laying a certain foundation for the treatment of uremia; regulation of microemulsions, liposomes and microspheres It can be used for subcutaneous injection, intravenous injection, oral administration, arterial embolization, etc.

(2) Microemulsions, liposomes or microspheres provided by the present invention are used as drug carriers, because the size is uniform and controllable, and the repeatability between batches is good. Therefore, in the study of drug release kinetics, and different drugs and their therapeutic effects The relationship between is more precise and simple. As a drug carrier, its size has a certain relationship with its distribution position and time in the body, and different drugs have different curative effects at different positions in the body; therefore, a series of monodisperse drug-encapsulated microemulsions are prepared for different drugs , liposomes or microspheres, and administer them separately, so that the drug effects of different drugs at different sizes can be found out, and the optimal size range required for different drugs can be found out. Research cannot be carried out effectively if microemulsions, liposomes or microspheres as drug carriers are not uniform in size.

(3) The preparation method provided by the present invention has mild conditions and is expected to maintain high activity of biologically active drugs such as polypeptides and proteins.

(4) The size of the microemulsion, liposome or microsphere provided by the present invention is uniform, and it is expected to realize stable and reproducible drug controlled release.

(5) The liposomes or microspheres provided by the present invention can be used for immobilization of enzymes, cell culture, and transplantation under mild conditions, which are expected to maintain the activity of enzymes and cells and realize the functionality of both.

The method of the present invention solves problems in the preparation of drug-loaded microemulsions, liposomes, and polymer microspheres by traditional techniques such as stirring emulsification and ultrasound, such as uneven particle size distribution, difficult shape control, low drug embedding rate, and batch-to-batch failure. There are large differences and other defects. Moreover, the traditional method of preparing microspheres affects the drug release behavior and reduces its targeting, and it is impossible to investigate the relationship between particle size and different diseases and therapeutic effects in clinical practice, which greatly limits its application. The uniform particle size of microemulsions, liposomes and microspheres prepared by microfluidics can be used to achieve stable and controllable drug release, and the drug release behavior, release mechanism and kinetic model can be accurately investigated to lay a certain foundation for clinical application. Base. At the same time, it is expected to solve the problem of large fluctuations in blood drug concentration, reduce the toxic and side effects of drugs, prolong the action time of drugs, and improve the utilization rate and therapeutic effect of drugs in the body. The preparation method has simple process, mild operating conditions, good repeatability and easy scale-up.

Description of drawings

Fig. 1 is the schematic diagram of the microfluidic chip that the present invention prepares monodisperse microemulsion, liposome or microsphere; And 3 introduce continuous phase, outlet 4 draws microemulsion or liposome, polymer droplet; Fig. 1B is a kind of microfluidic chip schematic diagram of preparing monodisperse multifunctional microemulsion, liposome or microsphere, import 1 introduces the continuous phase, and inlets 2-4 can respectively introduce multi-component drugs, magnetic nanoparticles, liposomes and multi-component polymers, etc., so as to realize multi-functional microemulsions, For liposomes or microspheres, the outlet 5 leads to droplets.

Fig. 2 is the microfluidic control preparation schematic diagram of embodiment 1 polylactic acid microsphere; Wherein, inlet 1 introduces the chloroform solution that is dissolved with polylactic acid, and inlet 2-3 introduces the aqueous solution of 2% polyvinyl alcohol (PVA), produces monodispersity droplet.

Fig. 3 is the optical micrograph that droplet produces in embodiment 1; Wherein, Fig. 3 (b) is the optical micrograph of droplet generation area; Fig. 3 (c) is the optical micrograph of droplet at microchannel tail Photos (Q ₁ =0.20mL/h, Q ₂ =Q ₃ 0.15mL/h; Q ₁ represents the flow rate of the dispersed phase; Q ₂ =Q ₃ represents the flow rate of the continuous phase, the same below).

Figure 4 is a photo of the contact angle of PDMS modified by the adsorption self-assembly-thermal curing method (the contact angle in the figure is the measured value of the PDMS sheet modified by the same modification method as the microfluidic channel), wherein, Figure 4(a) is the contact angle of PDMS (120°, hydrophobic); Fig. 4(b) is the contact angle (52°, hydrophilic) of PVA-modified PDMS; Fig. 4(c) is the PDMS modified by PVA/glycerol (Gly) contact angle (5°, superhydrophilic).

Figure 5 is an optical micrograph; among them, picture (ac) is the array of polylactic acid droplets on the glass slide; picture (d) is the solidification process of polylactic acid droplets in the container, the darker color indicates that it has been cured, and the lighter color The indications are droplets or semi-cured (monochrome image); image (ef) is cured polylactic acid microspheres. Flow rate conditions: (a) Q ₁ =0.15mL/h; Q ₂ =Q ₃ =0.35mL/h; (b) Q ₁ =0.20mL/h; Q ₂ =Q ₃ =0.15mL/h; (c) Q ₁ =0.25mL/h; Q ₂ =Q ₃ =0.45mL/h; (d)-(f)Q ₁ =0.10mL/h, Q ₂ =Q ₃ =0.15mL/h. The scale in the figure is 100 Micron.

6 is an optical micrograph (ab) and a size distribution diagram (c) of drug-loaded polylactic acid microspheres with different particle sizes prepared in Example 2. Flow rate conditions: (a) Q ₁ =0.15mL/h, Q ₂ =Q ₃ =0.35mL/h; (b) Q ₁ =0.20mL/h, Q ₂ =Q ₃ =0.15mL/h, the scale is 50 Micrometers, scale bar for interpolated pictures is 25 micrometers.

Fig. 7 is the in vitro drug release diagram of paclitaxel-polylactic acid microspheres of different sizes and different drug loadings prepared in Example 2, wherein, a) is a cumulative release diagram of paclitaxel, and b) is a logarithmic diagram of release amount and release time; S a-b description: S represents the sample, a represents the particle size (1 represents 31 microns, 2- represents 50 microns), and b represents the theoretical drug loading (1 represents 1 mg, 2 represents 2 mg).

Figure 8 is an optical micrograph of polylactic acid droplets coated with pirarubicin prepared in Example 3.

9 is an optical micrograph of polylactic acid microspheres coated with pirarubicin prepared in Example 4.

Fig. 10 is the in vitro drug release diagram of pirarubicin-polylactic acid microspheres prepared in Example 4 with different sizes and different drug loadings.

11 is an optical micrograph of droplets and microspheres of polylactic acid-glycolic acid prepared in Example 5.

FIG. 12 is an optical micrograph of chitosan microspheres prepared in Example 6 with different magnifications.

13 is an optical micrograph of calcium alginate microspheres prepared in Example 7 with different magnifications.

Fig. 14 is a schematic diagram of a microfluidic device for mass production.

Detailed ways

The method of the present invention will be described below through specific examples, but the present invention is not limited thereto.

The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources.

The invention includes a microfluidic preparation method of microemulsions, liposomes or microspheres monodispersely encapsulating medicines, enzymes or cells. This method can prepare O/W, W/O, O/W/O and other microemulsions, liposomes and microspheres that embed water-soluble and fat-soluble drugs; liposomes or liposomes that encapsulate functional enzymes can also be prepared. Microspheres; the preparation of cell-encapsulated microspheres is similar to the preparation of hydrophilic drug microspheres.

The preparation of microspheres of fat-soluble drugs is prepared according to the steps shown in Figure 2, and the specific methods and steps are as follows:

(1) Modification of the channel of the microfluidic chip: After the PDMS chip was plasma-treated for 90 seconds, the mixed solution of mass concentration 5% glycerin-2% polyvinyl alcohol was filled, and left at room temperature for 20 minutes; then the excess solution was used Vacuum pump, place at 60°C for 2 hours to heat-cure the coating, repeat the above steps to obtain multi-layer modified channels, then cure at 110°C for 20 minutes, and cool naturally to room temperature for later use.

(2) Preparation of dispersed phase and continuous phase: dissolve fat-soluble drug paclitaxel and polylactic acid in chloroform (oil phase) as dispersed phase; dissolve polyvinyl alcohol or other water-soluble emulsifiers in water (water phase) as Continuous phase; in the microfluidic chip device shown in Figure 1 (the channel is modified by hydrophilicity), the dispersed phase is passed into channel 1 through the pump, and the continuous phase is passed into channels 2 and 3, passing through the fluid focusing area to obtain monodispersity O/W microemulsion (or droplet); when water-soluble drugs are to be embedded, the water-soluble drug solution can be used as dispersed phase 1, and the chloroform solution of polylactic acid can be used as dispersed phase 2, and dispersed phase 1 can be sheared by dispersed phase 2. W/O microemulsion, and then the continuous phase in which the hydrophilic emulsifier is dissolved is cut into the dispersed phase 2 again to form a W/O/W microemulsion.

(3) Preparation of polylactic acid microspheres

The microemulsion obtained in the above step (2) is placed in a water bath at room temperature, stirred gently to remove chloroform; the solidified drug-coated microspheres are filtered, washed with water, and vacuum-dried or freeze-dried at room temperature.

Embodiment 1, preparation polylactic acid microsphere

The microfluidic chip device was assembled as shown in Figure 1. A chloroform solution of 30 mg/mL polylactic acid (molecular weight 10000) was prepared; a solution of PVA (model: average polymerization degree 1750±50) with a mass concentration of 2% was prepared as the continuous phase. The dispersed phase and the continuous phase are transported to the microfluidic chip through a micropump (see Figure 2), and the dispersed phase forms monodisperse droplets through the shearing action of the continuous phase in the chip (see Figure 3); chip channel modification The hydrophilicity characterization is shown in Figure 4.

The collected droplets are placed in a water bath at room temperature to remove the organic solvent, and the pelletized material is precipitated and solidified into microspheres; the solidified drug-loaded microspheres are filtered, washed with water, and vacuum-dried or freeze-dried at room temperature. The size of droplets and microspheres can be adjusted by the flow rate of dispersed phase and mobile phase, and the size of microspheres can also be adjusted by the concentration of polylactic acid. Take 100 droplets or microspheres to measure the size and take the average value. Accompanying drawing 5 (ac) is the array optical micrograph of the polylactic acid droplet that different flow rates produce, and its CV value is all less than 1.5%, shows that particle size is very uniform; Figure (d) is the solidification process of polylactic acid droplet in container , Figure (ef) is the cured polylactic acid microspheres. Flow rate conditions: (a) Q ₁ =0.15mL/h; Q ₂ =Q ₃ =0.35mL/h; (b) Q ₁ =0.20mL/h; Q ₂ =Q ₃ =0.15mL/h; (c) Q ₁ =0.25 mL/h; Q ₂ =Q ₃ =0.45 mL/h; (d)-(f) Q ₁ =0.10 mL/h, Q ₂ =Q ₃ =0.15 mL/h.

Example 2, preparation of polylactic acid microspheres wrapped with paclitaxel

The microfluidic chip device was assembled as shown in Figure 1. Prepare a chloroform solution of 30 mg/mL polylactic acid (molecular weight 10000), weigh a certain amount of paclitaxel and dissolve it in the above solution, and prepare solutions containing paclitaxel 1 mg/mL and 2 mg/mL respectively as the dispersed phase; prepare a mass concentration of 2 % PVA solution as the continuous phase. The dispersed phase and the continuous phase are transported to the microfluidic chip by a micropump to form monodisperse droplets; the collected droplets are placed in a 40°C water bath to remove the organic solvent; the solidified drug-loaded microspheres are filtered and washed with water , vacuum-dried or freeze-dried at room temperature.

Accompanying drawing 6 (ab) is the optical micrograph of the prepared drug-loaded polylactic acid microspheres with different particle sizes, flow rate conditions: (a) Q ₁ =0.15mL/h, Q ₂ =Q ₃ =0.35mL/h; (b) Q ₁ =0.20mL/h, Q ₂ =Q ₃ =0.15mL/h; Figure 6(c) is the size distribution. Take 100 microspheres to measure the size, and take the average value. The particle diameters of the two microspheres are about 31 and 50 microns respectively, and the CV values are both less than 4.0%, indicating that the particle diameters are very uniform.

Dissolve 1 mg of the dried microspheres in 1 mL of chloroform, sonicate for 2 minutes, add 2 mL of acetonitrile/water (1:1, v/v), vortex for 2 minutes, let stand for 15 minutes, and pass through nitrogen to remove the phases. Chloroform; then add acetonitrile/water (1:1, v/v) to keep the total volume at 2 mL; after ultrasonication for 5 minutes, centrifugation at 4000 rpm for 10 minutes, the supernatant was analyzed by high performance liquid chromatography. Chromatographic conditions: 25 cm long _C18 chromatographic column, mobile phase acetonitrile/water (1:1, v/v), flow rate 1 mL/min, detection wavelength 227 nm, injection volume 20 microliters. The measured embedding rates were all higher than 93%.

Figure 7 is the in vitro drug release diagram of microspheres with four different particle sizes and theoretical drug loadings. It can be seen from the figure that the release behavior of paclitaxel from polylactic acid microspheres is divided into two steps: first, the rapid burst in the first three hours. release phase followed by a gradual and sustained release process. And with the increase of microsphere size and theoretical drug loading, the burst release phenomenon increased slightly.

Embodiment 3, preparation wraps the polylactic acid microemulsion of pirarubicin

As in Example 1, there is no solidification step, only the dispersed phase is changed to polylactic acid chloroform solution containing pirarubicin, and the optical micrograph of the collected microemulsion is shown in Figure 8.

Embodiment 4, preparation wraps the polylactic acid microsphere of pirarubicin

As in Example 2, only the dispersed phase was changed to a polylactic acid chloroform solution containing 1 mg/mL pirarubicin, and the collected optical micrographs of polylactic acid microspheres coated with pirarubicin are shown in FIG. 9 . The in vitro release profiles of pirarubicin-PLA microspheres with different sizes are shown in Figure 10. It can be seen from the figure that the small-sized microspheres are released slowly during the whole release process, and there is no obvious burst release phenomenon. The large-sized microspheres showed a certain burst release phenomenon, while the later stage also showed slow release.

Example 5

As in Example 1, only the dispersed phase was changed to a chloroform solution of PLGA (poly(lactic acid-glycolic acid)), and the optical micrographs of the droplets and microspheres are shown in FIG. 11 .

Embodiment 6, preparation hydrophilic medicine chitosan microsphere

Prepare the mass concentration of 1mg/mL hydrophilic drug 5-fluorouracil as 2% chitosan acetic acid aqueous solution as dispersed phase, mineral oil (or soybean oil) containing mass concentration 5% Span80 as continuous phase, at a certain flow rate Produce monodisperse droplet under the following conditions, put droplet into the container that fills mass concentration and be 10% sodium polyphosphate (containing a small amount of isopropanol), solidify after 2 hours, filter, wash with secondary water, lyophilize or Tumble dry low. The average particle size of the microspheres is 44 microns, and the optical micrograph is shown in Figure 12, and the particle size is uniform.

Embodiment 7, preparation calcium alginate microsphere

As in Example 6, the dispersed phase was changed to a 2% sodium alginate aqueous solution, and a 10% calcium chloride solution was used as the curing agent. After curing for 1 hour, gently filter, wash with water, and dry in the air. Optical micrographs of the droplets and microspheres are shown in Figure 13.

Embodiment 8 mass production design

Various microspheres can be mass-produced by adopting the design of Fig. 14 .

Claims (5)

1. A method for preparing monodisperse drug-loaded microemulsions, according to the difference in drug properties, is divided into following a) and b) two methods; The a) method comprises the following steps: using the aqueous solution containing water-soluble medicine as the dispersed phase, which is denoted as dispersed phase 1; using the oil solution containing W/O emulsifier as the continuous phase, denoted as continuous phase 1; The dispersed phase 1 and the continuous phase 1 are transported to the corresponding microchannels of the microfluidic chip through the micropump, and the W/O microemulsion is prepared through the microfluidic chip, and the W/O microemulsion is collected in the continuous phase 1 Or in the container of cross-linking agent solution, obtain carrying water-soluble drug microemulsion; Wherein, the microchannel inner wall surface of described microfluidic chip device has hydrophobicity; The b) method comprises the steps of: using the oil solution containing fat-soluble drugs as the dispersed phase 2; using the aqueous solution containing the O/W emulsifier as the continuous phase 2; transporting the dispersed phase 2 and the continuous phase 2 respectively To the corresponding microchannel of the microfluidic chip device, the O/W microemulsion is prepared by the microfluidic chip device, and the O/W microemulsion is collected into the continuous phase 2 solution to obtain the fat-soluble drug microemulsion ; Wherein, the microchannel inner wall surface of the microfluidic chip device has hydrophilicity. 2. A method for preparing monodisperse drug-loaded liposomes or enzyme-loaded liposomes is divided into following c) and d) methods according to the difference in drug properties; The c) method comprises the following steps: using the aqueous solution containing blank liposomes and water-soluble drugs as the dispersed phase 3, or using the aqueous solution containing blank liposomes and enzymes as the dispersed phase 3; The oil solution of the agent is used as the continuous phase 3; the dispersed phase 3 and the continuous phase 3 are respectively transported to the corresponding microchannels of the microfluidic chip device, and the W/O microemulsion is prepared by the microfluidic chip device, which will produce The W/O microemulsion is freeze-dried to obtain drug-loaded liposomes or enzyme-loaded liposomes; wherein, the microchannel inner wall surface of the microfluidic chip device has hydrophobicity; Described d) method comprises the following steps: use the oil solution containing fat-soluble medicine and blank liposome as dispersed phase 4; Use the aqueous solution containing O/W type emulsifier as continuous phase 4; Describe dispersed phase 4 and The continuous phase 4 is respectively transported to the corresponding microchannels of the microfluidic chip device, the O/W microemulsion is prepared through the microfluidic chip device, and the generated W/O microemulsion is freeze-dried to obtain drug-loaded liposomes; Wherein, the surface of the inner wall of the microchannel of the microfluidic chip device is hydrophilic. 3. A method for preparing monodisperse drug-loaded microspheres or enzyme-loaded microspheres or encapsulated cell microspheres, which can be divided into the following e) and f) methods according to the different properties of the drug; The e) method includes the following steps: using an aqueous solution containing a biodegradable polymer and any one of the following substances as the dispersed phase 5: water-soluble drugs, enzymes and cells; using an oil solution containing a W/O emulsifier As the continuous phase 5; the dispersed phase 5 and the continuous phase 5 are respectively transported to the corresponding microchannels of the microfluidic chip device, and the W/O microemulsion is prepared by the microfluidic chip device, and the W/O microemulsion Collect in the container that continuous phase 5 or cross-linking agent solution is housed, solidify, obtain the microsphere of drug-loaded microsphere or enzyme-loaded microsphere or wrapping cell; Wherein, the microchannel inner wall surface of described microfluidic chip device has Hydrophobic; The f) method comprises the steps of: using the oil solution containing fat-soluble drugs and biodegradable polymers as the dispersed phase 6; using the aqueous solution containing the O/W emulsifier as the continuous phase 6; using the dispersed phase 6 and the continuous phase 6 are delivered to the corresponding microchannels of the microfluidic chip device, and the O/W microemulsion is prepared by the microfluidic chip device, and the O/W microemulsion is collected into the continuous phase 6 or the crosslinking agent The container of the solution is solidified to obtain drug-loaded microspheres; wherein, the surface of the inner wall of the microchannel of the microfluidic chip device has hydrophilicity. 4. The method according to any one of claims 1-3, characterized in that: the diameter distribution coefficients of the microemulsions, liposomes and microspheres are all less than or equal to 5%. 5. The method according to any one of claims 1-4, characterized in that: the mass concentration of the surfactant in the continuous phase is 0.5%-10%, preferably 0.5%-4%.

Images