Method for preparing polymer vesicles by using microfluidic microsphere template method

Abstract

The invention belongs to the field of preparation of polymer vesicles, and particularly relates to a method for preparing polymer vesicles by a microfluidic microsphere template method. Comprising the following steps: adjusting the material concentration of an oil phase solution and a continuous phase solution, dispersing the oil phase in the continuous phase to form an emulsion template, curing to form a microsphere template, collecting the microsphere template, carrying out vacuum drying for later use, and preparing the polymer vesicles by using the microsphere template through a rehydration method. According to the invention, the uniform microsphere template is creatively prepared by using a microfluidic technology, and then the polymer vesicles are hydrated, so that the problem of organic solvent residue in a single emulsion template method in the prior art is solved, and the problem of complex structure of microfluidic equipment in which double emulsion templates are used is solved. Meanwhile, the yield of the microsphere template prepared by the method disclosed by the invention is 4-20 times higher than that in the literature of preparing the high-molecular vesicles by a double-emulsion template method.

Description

Technical Field

[0001] The invention belongs to the field of polymer vesicle preparation, and in particular relates to a method for preparing polymer vesicles by a microfluidic microsphere template method. Background Art

[0002] Polymer vesicles are hollow spheres formed by self-assembly of amphiphilic block copolymers in aqueous solution. This assembly consists of a hydrophilic cavity and a thin film with hydrophilic-hydrophobic-hydrophilic layers. Polymer vesicles have been widely used in drug delivery, gene carriers, pesticide carriers, artificial cell membranes and microreactors. Traditional methods for preparing polymer vesicles, such as solvent exchange and membrane rehydration, have no selective mechanism for the size of polymer vesicles, which limits large-scale production and commercial applications. For example, the bimolecular membrane bending into spheres mechanism in the solvent exchange method has no selective mechanism for the size of polymer vesicles, which makes it difficult to accurately control the batch-to-batch quality and drug loading efficiency of the polymer vesicles obtained by this preparation method, and also brings about the problem of solvent residue; membrane rehydration can effectively avoid the problem of solvent residue, but the traditional membrane rehydration needs to undergo multiple phase changes from columnar phase to sponge phase, and is also affected by random mechanical stirring, so the particle size is also difficult to control.

[0003] In exploring ways to overcome the limitations of membrane rehydration, the conventional idea is to control the size of polymer vesicles by controlling the raw material supply of block copolymers. Some researchers have used photolithography to prepare silicon-based templates with uniform size, and then spin-coated the polymer body on the template to obtain a uniform polymer body template. After selecting a specific block copolymer for hydration, uniform micron-sized polymer vesicles can be spontaneously formed in aqueous solution. Other studies have shown that nanoparticle templates prepared by electrostatic spraying can rehydrate uniform nanoscale polymer vesicles based on the template.

[0004] In recent years, fluidic technology can create a stable fluid environment in microchannels, reduce the interference of uncontrollable factors, and provide technical support for the preparation of polymer vesicles with uniform size, such as emulsion phase transfer method, emulsion template method and nano-deposition method. A literature (Microparticle templating as a route to nanoscale polymer vesicles with controlled size distribution for anticancerdrug delivery) reported that the coefficient of variation CV of the double emulsion template based on microfluidic regulation is less than 5%, and micron-sized polymer vesicles can be prepared in a controllable manner by combining composition and concentration, but the particle generation rate in this literature is only 1 mL / h. In addition, there are still technical barriers to the large-scale preparation of uniform polymer templates and the preparation of polymer vesicles based on template rehydration. Although microfluidic technology has made it possible to prepare uniform emulsion templates and then prepare polymer vesicles with emulsion templates, there is no literature reporting the preparation of polymer vesicles by preparing microsphere templates using microfluidic technology. Summary of the invention

[0005] In order to solve the shortcomings and deficiencies of the prior art, the present invention provides a method for preparing polymer vesicles by a microfluidic microsphere template method. The method is based on the concentration-dependent characteristics of block copolymer assembly in aqueous solution, and prepares molecular vesicles by adjusting the concentration of the emulsion template, regulating the size of the microsphere template, and rehydrating the microspheres.

[0006] The purpose of the present invention is achieved through the following technical solutions: A method for preparing polymer vesicles by a microfluidic microsphere template method comprises the following steps: (1) Adjusting the material concentration of the oil phase and the continuous phase solution, and injecting the oil phase and the continuous phase into the microfluidic device; (2) controlling the injection flow rates of the oil phase and the continuous phase solution to disperse the oil phase in the continuous phase to form an oil-in-water emulsion template, evaporating the solvent of the obtained emulsion, filtering and washing with water, and then vacuum drying to obtain a microsphere template for use; (3) Using the microsphere template obtained in step (2), a rehydration method is used to prepare polymer vesicles.

[0007] Preferably, the microfluidic device in step (1) is a fluid focusing microfluidic device, a coaxial co-flow microfluidic device or a cross-flow interlaced microfluidic device.

[0008] More preferably, the microfluidic device is a fluid focusing type microfluidic device, including a liquid inlet tube composed of a metal capillary or a glass tube, a glass liquid outlet tube composed of a glass capillary, and a glass flow chamber.

[0009] Preferably, the solvent in the oil phase in step (1) may be at least one of chloroform, dichloromethane, toluene, dioxane, n-hexane, tetrahydrofuran, ethyl acetate and n-butanol.

[0010] Preferably, the oil phase in step (1) contains at least one of an amphiphilic block copolymer, a drug molecule or a fluorescent dye, etc., for constructing a bimolecular membrane of a nanoscale polymer vesicle.

[0011] More preferably, the amphiphilic block copolymer is at least one of PEG-PDLLA, PEG-PS, PEG-PLA, PEG-PCL, PEG-PLGA, PS-PEG, PS-PAA, PLA-PEG-PLA and PCL-PEG-PCL.

[0012] More preferably, the concentration of the amphiphilic block copolymer in the oil phase is 0.03 wt% to 20 wt%, preferably 2 to 10 wt%, and most preferably 2 to 5 wt%.

[0013] More preferably, the drug molecule may be at least one of a biomolecule, a nanoparticle or a lipophilic drug, and the fluorescent dye may be at least one of rhodamine, Nile red, curcumin, paclitaxel and coumarin-6.

[0014] More preferably, the biomolecules include at least one of proteins, gene fragments, polypeptides and growth factors; and the nanoparticles include but are not limited to at least one of gold rods, manganese dioxide and iron tetroxide.

[0015] Preferably, the continuous phase in step (1) contains a surfactant and / or a hydrophilic drug to prevent emulsion fusion or aggregation at the water-oil interface. The surfactant may be an anionic surfactant, a cationic surfactant, a zwitterionic surfactant or a nonionic surfactant.

[0016] More preferably, the surfactant in the continuous phase is polyvinyl alcohol; the concentration of the surfactant in the continuous phase ranges from 0.05 wt% to 10 wt%, preferably, the concentration is from 0.05 wt% to 2 wt%.

[0017] More preferably, the hydrophilic drug includes but is not limited to one of doxorubicin, daunorubicin, cytarabine, and bupivacaine.

[0018] Preferably, in step (2), the injection rate ratio of the oil phase to the continuous phase is 1:2-1:20; the flow rate of the oil phase is 0.05 mL / h-10 mL / h; and the flow rate of the continuous phase is 1 mL / h-60 mL / h.

[0019] Preferably, in step (2), the receiving distance between the oil phase outlet of the microfluidic device and the receiving tube is 0-500 μm, preferably 0-200 μm.

[0020] Preferably, the solvent evaporation in step (2) refers to placing the emulsion in an open container at room temperature and pressure to allow the solvent to evaporate naturally.

[0021] Preferably, the vacuum drying temperature in step (2) is -50 to 60°C, and the vacuum drying time is more than 24 hours.

[0022] Preferably, the rehydration method in step (3) is carried out at 25-60°C, more preferably at 25-45°C.

[0023] Preferably, in the rehydration method in step (3), the stirring speed is 100 rpm to 500 rpm, and the stirring time is more than 24 hours.

[0024] Preferably, the aqueous solution used in the rehydration method in step (3) includes, but is not limited to, one of deionized water, PBS buffer, and Tris buffer.

[0025] Preferably, in the rehydration method of step (3), the concentration of the microsphere template in the aqueous solution is 0.5wt%-10wt%.

[0026] The polymer vesicles obtained by the present invention can be used in various applications, such as drug delivery (such as nanoparticles, biomolecules, etc.), nanoreactors, material preparation, biological cell modeling, etc.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention innovatively uses microfluidic technology to prepare a uniform emulsion template. On this basis, after solidification (i.e., evaporation of the solvent), the microspheres are vacuum dried at room temperature and then hydrated to obtain polymer vesicles. This method solves the problem of organic solvent residue in the single emulsion template method in the disclosed technology and the problem of complex structure of microfluidic devices in double emulsion templates.

[0028] (2) Compared with the traditional double emulsion template method, the method of the present invention breaks through the limitation that the original double emulsion template method can only prepare micron-sized polymer vesicles, and realizes the preparation of nano-sized polymer vesicles by the microsphere template method based on the single emulsion, thereby being able to better meet the needs of different practical applications.

[0029] (3) The yield of microsphere templates prepared by the method of the present invention is 4-20 times greater than that of the double emulsion template method for preparing polymer vesicles in the literature. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1Schematic diagram of a microfluidic device used in an embodiment of the present invention.

[0031] Figure 2 Optical microscopy images of different microsphere templates prepared by adjusting the concentration of amphiphilic block copolymers in the oil phase at a fixed continuous phase concentration. The scale bar is 300 μm.

[0032] Figure 3 This is the distribution diagram of the microsphere template prepared in Example 1.

[0033] Figure 4 This is a diagram showing the dynamic light scattering results of the polymer vesicles prepared in Example 1.

[0034] Figure 5 This is a morphological structure diagram of the polymer vesicles prepared in Example 1.

[0035] Figure 6 This is a transmission electron microscopy image of the worm-like micelle assembly prepared in Comparative Example 1.

[0036] Figure 7 This is a laser confocal image of the micron-sized polymer vesicles prepared in Example 2.

[0037] Figure 8 This is a scanning electron microscope image of the micron-sized polymer vesicles prepared in Example 2. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. The raw materials involved in the present invention can be purchased directly from the market. For process parameters not particularly noted, conventional techniques can be used.

[0039] Example 1 (1) Adjusting the material concentration of the oil phase and the continuous phase solution, and injecting the oil phase and the continuous phase into the microfluidic device; The microfluidic device described in this embodiment is a coaxial fluid confocal microfluidic device, and the structural schematic diagram is shown in FIG. Figure 1 As shown, it includes a base, a metal liquid inlet tube, a glass liquid outlet tube, a glass flow chamber, and several adapters and gaskets; the inner diameter of the metal liquid inlet tube is 100 μm, the inner diameter of the glass liquid outlet tube is 300 μm, and the inner diameter of the glass flow chamber is 500 μm; the central axes of the glass flow chamber, the metal nozzle and the glass liquid outlet tube are in the same straight line, and the metal nozzle, the liquid outlet tube and the flow chamber are connected by adapters and gasket parts; the glass liquid outlet tube is connected to a liquid collecting device; the receiving distance between the oil phase outlet of the microfluidic device and the receiving tube is 0 μm (the needle and the receiving tube are tangent).

[0040] In this embodiment, the continuous phase is 0.5 wt% polyvinyl alcohol solution and the oil phase is 10 wt% PEG5000 -PDLLA 12000 Ethyl acetate solution.

[0041] (2) Control the injection flow rate of the oil phase and the continuous phase solution to disperse the oil phase in the continuous phase to form an oil-in-water emulsion template. After evaporating the solvent of the obtained emulsion, filter it and wash it with deionized water for 3 times. Place it in a vacuum drying oven and dry it at room temperature for 48 h to obtain a microsphere template, such as Figure 2 As shown; In this embodiment, the flow rate of the oil phase is 7 mL / h, and the flow rate of the continuous phase is 30 mL / h.

[0042] (3) Weigh an appropriate amount of the microsphere template prepared in step (2), place it in deionized water, prepare a 10 mg / mL vesicle solution (i.e., the concentration of the microspheres in deionized water is 10 mg / mL), stir at room temperature for 24 h at a stirring speed of 100 rpm. After stirring, centrifuge the solution and take the supernatant to obtain nanopolymer vesicles.

[0043] The average particle size of the nano-scale polymer vesicles is 121±2 nm, and the particle size distribution is as follows: Figure 4 As shown, the morphology of the nanoscale polymer vesicle is as follows Figure 5 As shown, it can be seen that the particle size of the polymer vesicles is relatively uniform. The PDI of the polymer vesicles prepared by this method is 0.16, which is smaller than the PDI of the traditional membrane rehydration method of 0.28. It has certain advantages over traditional methods. And it is 6.7 times faster than the fastest speed of template preparation in the literature. The yield of polymer vesicles in Example 1 is 630 μg / mL, and the maximum yield in the literature (Microparticle templating as a route to nanoscale polymer vesicles with controlled size distribution for anticancer drug delivery) is 143μg / mL. The yield of Example 1 is 4.4 times greater than that in the literature.

[0044] The calculation method of the microsphere template yield is as follows: microsphere template yield = mass per milliliter of oil phase solution × mass fraction of oil phase × oil phase flow rate, the mass per milliliter of solution is obtained by the density of the solvent. Because the amphiphilic block copolymer generally accounts for a very small proportion, the density increase caused by it can be ignored.

[0045] Referring to the above steps (1) and (2), the mass concentration of the amphiphilic block copolymer in the oil phase was changed to 0.008 wt%, 0.025 wt%, 0.125 wt%, 0.25 wt%, 0.5 wt%, 1 wt%, 2 wt%, and 5 wt%, respectively, to prepare microsphere templates. The results are as follows: Figure 2 As shown in the figure, it can be seen that microsphere templates of different sizes can be obtained by adjusting the concentration of the amphiphilic block copolymer.

[0046] Comparative Example 1 In the same coaxial fluid confocal microfluidic device as in Example 1, the material concentrations of the oil phase and the continuous phase solution and the flow rate of the microfluidic device were changed: 1) The continuous phase is 0.5 wt% polyvinyl alcohol solution, and the oil phase is 0.25 wt% PEG 5000 -PDLLA 12000 Dichloromethane solution.

[0047] 2) The oil phase flow rate is 1 mL / h, and the continuous phase flow rate is 20 mL / h to obtain a microsphere template.

[0048] Based on this template, only worm-like micelle assemblies were obtained, and the transmission electron micrograph is shown in Figure 6. This indicates that the method of the present invention requires strict concentration screening, rather than simply improving the equipment to easily prepare polymer vesicles. Referring to the calculation method of Example 1, the yield of Comparative Example 1 is 3.3 μg / mL.

[0049] Example 2 (1) Adjusting the material concentration of the oil phase and the continuous phase solution, and injecting the oil phase and the continuous phase into the microfluidic device; The microfluidic device described in this embodiment is the same as that in embodiment 1; In this embodiment, the continuous phase is a 0.5 wt % polyvinyl alcohol solution.

[0050] In this example, 50 μg of Nile red was added to 2 mL of 4 wt% PEG 2500 -PCL 7500 The mixture was mechanically stirred in ethyl acetate solvent for 120 minutes to obtain an oil phase.

[0051] (2) controlling the injection flow rate of the oil phase and the continuous phase solution to disperse the oil phase in the continuous phase to form an oil-in-water emulsion template, evaporating the solvent of the obtained emulsion for 12 hours, filtering and washing the microspheres with water for 5 times to remove the residual polyvinyl alcohol in the solution, and then vacuum drying at room temperature for 36 hours to obtain a microsphere template for use; In this embodiment, the oil phase flow rate is 2 mL / h, and the continuous phase flow rate is 60 mL / h.

[0052] (3) The microsphere template prepared in step (2) was stirred in deionized water for 24 h at room temperature in a light-proof manner. The concentration of the microsphere template in the deionized water was 5 wt %, and the stirring speed was 200 rpm. After the stirring was completed, the solution was dialyzed for 12 h, and micron-sized polymer vesicles were obtained. The laser confocal microscopy image is shown in FIG. Figure 7As shown in the scanning electron microscope image Figure 8 shown.

[0053] Referring to the calculation method of Example 1, the microsphere template output of Example 2 is 72 μg / h.

[0054] In summary, the present invention provides a method for preparing polymer vesicles by a microfluidic microsphere template method, which creatively uses microfluidic technology to prepare microsphere templates, and then hydrates the polymer vesicles, solving the problem of organic solvent residue in the single emulsion template method in the disclosed technology, and solving the problem of complex structure of microfluidic devices with double emulsion templates. At the same time, the yield of the microsphere template prepared by the method of the present invention is 4-20 times greater than that in the literature on the preparation of polymer vesicles by the double emulsion template method, and has great commercial value.

[0055] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing polymer vesicles by microfluidic microsphere template method, characterized in that: The following steps are involved: (1) Adjusting the material concentration of the oil phase and the continuous phase solution, and injecting the oil phase and the continuous phase into the microfluidic device; (2) controlling the injection flow rates of the oil phase and the continuous phase solution to disperse the oil phase in the continuous phase to form an oil-in-water emulsion template, evaporating the solvent of the obtained emulsion, filtering and washing with water, and then vacuum drying to obtain a microsphere template for use; (3) Using the microsphere template obtained in step (2), a rehydration method is used to prepare polymer vesicles.

2. The method for preparing polymer vesicles by microfluidic microsphere template method according to claim 1, characterized in that: The microfluidic device in step (1) is a fluid focusing microfluidic device, a coaxial co-flow microfluidic device or a cross-flow interlaced microfluidic device.

3. The method for preparing polymer vesicles by microfluidic microsphere template method according to claim 2, characterized in that: The microfluidic device is a fluid focusing type microfluidic device, comprising a liquid inlet tube composed of a metal capillary or a glass tube, a glass liquid outlet tube composed of a glass capillary, and a glass flow chamber.

4. The method for preparing polymer vesicles by microfluidic microsphere template method according to claim 1, characterized in that: The solvent in the oil phase in step (1) is at least one of chloroform, dichloromethane, toluene, dioxane, n-hexane, tetrahydrofuran, ethyl acetate and n-butanol; and the oil phase contains at least one of an amphiphilic block copolymer, a drug molecule or a fluorescent dye.

5. The method for preparing polymer vesicles by microfluidic microsphere template method according to claim 4, characterized in that: The amphiphilic block copolymer is at least one of PEG-PLA, PEG-PS, PEG-PCL, PEG-PLGA, PS-PEG, PS-PAA, PLA-PEG-PLA and PCL-PEG-PCL; the concentration of the amphiphilic block copolymer in the oil phase is 0.03 wt% to 20 wt%; The drug molecule is at least one of a biomolecule, a nanoparticle or a fat-soluble drug; The fluorescent dye is at least one of rhodamine, Nile red, curcumin, paclitaxel and coumarin-6.

6. The method for preparing polymer vesicles by microfluidic microsphere template method according to claim 1, characterized in that: In step (1), the continuous phase contains a surfactant and / or a hydrophilic drug; the surfactant is one or more of anionic surfactant, cationic surfactant, zwitterionic surfactant or non-ionic surfactant.

7. The method for preparing polymer vesicles by microfluidic microsphere template method according to claim 6, characterized in that: The surfactant in the continuous phase is polyvinyl alcohol; the concentration range of polyvinyl alcohol in the continuous phase is 0.05 wt%~10wt%; The hydrophilic drug includes at least one of doxorubicin, daunorubicin, cytarabine, bupivacaine and sunitinib.

8. The method for preparing polymer vesicles by microfluidic microsphere template method according to claim 1, characterized in that: In step (2), the injection rate ratio of the oil phase to the continuous phase is 1:2 to 1:20; the flow rate of the oil phase is 0.05 mL / h to 10 mL / h; the flow rate of the continuous phase is 1 mL / h to 60 mL / h; and the receiving distance between the oil phase outlet of the microfluidic device and the receiving tube is 0-500 μm.

9. The method for preparing polymer vesicles by microfluidic microsphere template method according to claim 1, characterized in that: The solvent evaporation in step (2) refers to placing the emulsion in an open container at normal temperature and pressure to allow the solvent to evaporate naturally; The vacuum drying temperature in step (2) is -50~60°C, and the vacuum drying time is more than 24 hours.

10. The method for preparing polymer vesicles by microfluidic microsphere template method according to claim 1, characterized in that: The rehydration method in step (3) is carried out at 25-60°C, more preferably at 25-45°C; In the rehydration method described in step (3), the stirring speed is 100 rpm to 500 rpm, and the stirring time is more than 24 hours; The aqueous solution used in the rehydration method in step (3) is one of deionized water, PBS buffer and Tris buffer; In the rehydration method of step (3), the concentration of the microsphere template in the aqueous solution is 0.5 wt%-10 wt%.

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