A microfluidic acoustic cavitation chip and a method for preparing liposomes with different particle sizes by using the same

Abstract

The application provides a microfluidic acoustic cavitation chip and a method for preparing liposomes with different particle sizes by using the same. The microfluidic acoustic cavitation chip comprises a microfluidic mixing chip, a sealed soft film, a piezoelectric ceramic, an electrode and a clamping and fixing assembly. The microfluidic mixing chip comprises a buffer solution inlet, a lipid organic solution inlet, a serpentine mixing channel and a liposome solution outlet. The serpentine mixing channel is located on one side of the microfluidic mixing chip and is sealed by the sealed soft film. The clamping and fixing assembly comprises a perforated sheet, a matching sheet and a bolt and nut. The perforated sheet comprises a solution inlet and an outlet. The microfluidic acoustic cavitation chip can precisely control the particle size of liposomes without changing the flow rate ratio and the microchannel structure, can quickly prepare liposomes with different particle sizes, and can meet the diversified requirements of the biological and medical fields for the particle size distribution of liposome drugs.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microfluidics and liposome preparation, and particularly relates to a microfluidic acoustic cavitation chip and a method for preparing liposomes with different particle sizes by using the microfluidic acoustic cavitation chip. BACKGROUND

[0002] As one of the most promising drug carriers, liposomes can change the pharmacokinetic characteristics of drugs, prolong the circulation time of drugs, and reduce the toxic and side effects of drugs, and have been widely used in the fields of anti-tumor drug delivery, gene therapy, medical imaging, etc. It is worth noting that the particle size of liposomes plays an important role in the blood circulation, cell uptake and tissue penetration of liposomes in vivo, and has an important influence on the pharmacodynamics and pharmacokinetics of liposome drugs.

[0003] At present, common liposome preparation methods include the thin film hydration method, the reverse evaporation method, the ethanol injection method, etc. These methods are difficult to directly control the particle size of liposomes during the formation of liposomes. Traditional post-processing methods, such as liposome extrusion and ultrasonic oscillation, can reduce the particle size of liposomes, but have the disadvantages of time-consuming, low efficiency and poor repeatability. The microfluidic method can directly control the particle size of liposomes by adjusting the flow rate ratio of the buffer solution and the lipid solution during the formation of liposomes, but this method will undoubtedly change the concentration of liposomes, and thus affect the encapsulation efficiency and stability of liposomes. Therefore, it is of great significance to develop a method for accurately controlling the particle size of liposomes without changing the flow rate ratio, which can promote the production and application research of liposomes. SUMMARY

[0004] In view of the problems existing in the prior art, the present application provides a microfluidic acoustic cavitation chip and a method for preparing liposomes with different particle sizes by using the microfluidic acoustic cavitation chip. The microfluidic acoustic cavitation chip can directly control the particle size of liposomes during the synthesis of liposomes by using an acoustic-flow coupling field without changing the flow rate ratio of the buffer solution and the lipid solution.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] A microfluidic acoustic cavitation chip, comprising a microfluidic mixing chip, a sealed soft membrane, a piezoelectric ceramic, an electrode, and a clamping and fixing assembly; the microfluidic mixing chip contains a buffer solution inlet, a lipid organic solution inlet, a serpentine mixing channel, and a liposome solution outlet; the serpentine mixing channel is located on one side of the microfluidic mixing chip and is sealed by the sealed soft membrane; the clamping and fixing assembly comprises a perforated sheet, a matching sheet, and a bolt and nut; the perforated sheet contains a solution inlet and an outlet.

[0007] Preferably, the microfluidic mixing chip is made of photocured resin or polymethyl methacrylate (PMMA) or polydimethylsiloxane (PDMS).

[0008] Preferably, the piezoelectric ceramic has a resonance frequency range of 20-100 kHz.

[0009] Preferably, the inlet and outlet have an inner diameter range of 400-3000 μm and a depth range of 0.5-5 mm.

[0010] Preferably, the serpentine mixing channel has a width range of 50-1000 μm and a depth range of 50-1000 μm.

[0011] Preferably, the sealing soft film material is PDMS or polyurethane (PU) or silicone, and has a thickness range of 0.5-1.5 mm.

[0012] Preferably, the opening thin plate and the matching thin plate in the clamping and fixing assembly have a thickness range of 3-15 mm.

[0013] Another aspect of the present application is to protect the use method of the device described above, which comprises the following steps:

[0014] (1) input the resonance frequency of the piezoelectric ceramic into a signal generator, set the excitation voltage; connect the positive and negative electrodes of the power amplifier to the positive and negative electrodes of the piezoelectric ceramic in sequence after setting the amplification multiple of the power amplifier after connecting the signal generator signal to the power amplifier;

[0015] (2) prepare the corresponding solution; dissolve the liposome adjuvant in an organic solvent to form a lipid organic solution; according to the solubility of the encapsulated drug, dissolve the water-soluble drug in a buffer or dissolve the fat-soluble drug in the lipid organic solution;

[0016] (3) set the flow rate ratio and total flow rate of the buffer and the lipid organic solution, use a multi-channel microsyringe pump to inject the buffer and the lipid organic solution into the buffer inlet and the lipid organic solution inlet respectively through a syringe and a catheter, and turn on the signal generator and the power amplifier to generate acoustic cavitation effect in the channel of the microfluidic mixing chip;

[0017] (4) as the buffer and the lipid organic solution are mixed in the microfluidic mixing chip, the lipid organic solution is quickly dissolved in the buffer, so that the lipid molecules quickly self-assemble to form liposomes in the buffer; under the condition of not changing the flow rate ratio of the buffer and the lipid organic solution, according to the required particle size of the liposomes, the excitation voltage of the piezoelectric ceramic is adjusted to quickly produce liposomes of different particle sizes.

[0018] Preferably, the excitation voltage amplification multiple of the power amplifier is 50 times.

[0019] Preferably, the main excipients in the liposome excipients include phospholipids, cholesterol and N-(carbonyl-methoxypolyethylene glycol 2000)-1, 2-distearoyl-SN-glycero-3-phosphoethanolamine (DSPE-MPEG2000); the phospholipids include one or more of dipalmitoyl phosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoyl lecithin (DMPC), dioleoyl lecithin (DOPC), egg yolk lecithin or soybean lecithin (Lecithin).

[0020] Preferably, the organic solvent is ethanol or isopropanol.

[0021] Preferably, the buffer is PBS buffer or HEPES buffer or Tris buffer

[0022] The microfluidic acoustic cavitation chip and the method for preparing liposomes with different particle sizes provided by the application have the following beneficial effects:

[0023] (1) Compared with other microfluidic mixing chips for synthesizing liposomes, the microfluidic acoustic cavitation chip can precisely control the particle size of the liposomes without changing the flow rate ratio and the microchannel structure.

[0024] (2) The microfluidic acoustic cavitation chip can quickly prepare liposomes with different particle sizes, which can meet the diversified needs of the biological and medical fields for the particle size distribution of liposome drugs. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a schematic diagram of the overall structure of the microfluidic acoustic cavitation chip of the embodiment of the application;

[0026] Figure 2 is a schematic diagram of the structure of the microfluidic mixing chip of the embodiment of the application;

[0027] Figure 3 is a schematic diagram of the system for preparing liposomes with different particle sizes using the microfluidic acoustic cavitation chip of the embodiment of the application;

[0028] Figure 4 is a photo of the acoustic cavitation effect in the microfluidic mixing chip of the embodiment of the application;

[0029] Figure 5 is a comparison diagram of the particle sizes of blank liposomes prepared under different excitation voltages of the embodiment of the application;

[0030] Figure 6 is a comparison diagram of the particle sizes of blank liposomes prepared with different components of the embodiment of the application;

[0031] Figure 7A comparison chart of particle sizes of various drug-loaded liposomes prepared in the embodiment of the present application;

[0032] Figure 8 A freeze transmission electron microscope chart of various drug-loaded liposomes of different particle sizes prepared in the embodiment of the present application;

[0033] Wherein: 1 is a microfluidic mixing chip, 2 is a sealed soft film, 3 is an electrode, 4 is a piezoelectric ceramic, 5 is a clamping and fixing assembly, 6 is a signal generator, 7 is an information transmission line, 8 is a power amplifier, 9 is a lead, 10 is a micro-injection pump, 11 is a syringe, 12 is a catheter, 101 is a buffer inlet, 102 is a lipid organic solution inlet, 103 is a liposome solution outlet, and 104 is a serpentine mixing channel. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiment of the present application clearer, the technical scheme in the embodiment of the present application will be described clearly and completely below in combination with the drawings in the embodiment of the present application. Obviously, the described embodiment is a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0035] The embodiment provides a microfluidic acoustic cavitation chip and a method for preparing liposomes of different particle sizes by using the same.

[0036] A microfluidic acoustic cavitation chip, as shown in Figure 1 The microfluidic mixing device is composed of a microfluidic mixing chip 1, a sealed soft film 2, an electrode 3, a piezoelectric ceramic 4, and a clamping and fixing assembly 5; as shown in Figure 2 The microfluidic mixing chip 1 is provided with a buffer inlet 101, a lipid organic solution inlet 102, a liposome solution outlet 103, and a serpentine mixing channel 104; the serpentine mixing channel 104 is located on one side of the microfluidic mixing chip 1 and is sealed by the sealed soft film 2; the clamping and fixing assembly 5 includes an open sheet, a matching sheet, and a bolt and nut; and the open sheet contains solution inlets and outlets.

[0037] Preferably, the microfluidic mixing chip 1 is made of photocurable resin or PMMA or PDMS.

[0038] Preferably, the piezoelectric ceramic 4 has a resonance frequency range of 20-100 kHz.

[0039] Preferably, the inlets 101 and 102 and the outlet 103 have an inner diameter range of 400-3000 μm and a depth range of 0.5-5 mm.

[0040] Preferably, the width of the serpentine mixing channel 104 ranges from 50 to 1000 μm, and the depth ranges from 50 to 1000 μm.

[0041] Preferably, the material of the sealing soft film 3 is PDMS or PU or silica gel, and the thickness ranges from 0.5 to 1.5 mm.

[0042] Preferably, the thickness of the opening sheet and the matching sheet in the clamping and fixing assembly 5 ranges from 3 to 15 mm.

[0043] A method for preparing liposomes with different particle sizes,

[0044] As shown in Figure 3 The method for preparing liposomes with different particle sizes comprises the following steps:

[0045] (1) input the resonant frequency of the piezoelectric ceramic 4 into the signal generator 6, set the excitation voltage, input the signal of the signal generator into the power amplifier 8 through the signal transmission line 7, set the amplification multiple of the power amplifier, and connect the positive and negative leads 9 of the power amplifier to the positive and negative poles of the piezoelectric ceramic 4 in sequence;

[0046] (2) prepare the corresponding solution; dissolve the liposome auxiliary materials in an organic solvent to form a lipid organic solution; according to the solubility of the encapsulated drug, dissolve the water-soluble drug in a buffer or dissolve the fat-soluble drug in the lipid organic solution;

[0047] (3) set the flow rate ratio and total flow rate of the buffer and the lipid organic solution, use the multi-channel micro-injection pump 10 to inject the buffer and the lipid organic solution into the buffer inlet 101 and the lipid organic solution inlet 102 respectively through the syringe 11 and the catheter 12, and turn on the signal generator 6 and the power amplifier 8 to generate acoustic cavitation effect in the channel of the microfluidic mixing chip 1;

[0048] (4) as the buffer and the lipid organic solution are mixed in the microfluidic mixing chip 1, the lipid organic solution is quickly dissolved in the buffer, so that the lipid molecules are quickly self-assembled to form liposomes in the buffer; under the condition that the flow rate ratio of the buffer and the lipid organic solution is not changed, according to the particle size of the required liposomes, the excitation voltage of the piezoelectric ceramic 4 is adjusted, and liposomes with multiple different particle sizes can be quickly produced.

[0049] Preferably, the amplification multiple of the excitation voltage of the power amplifier is 50 times.

[0050] Preferably, the main auxiliary materials in the liposome auxiliary materials include phospholipids, cholesterol and DSPE-MPEG2000; the phospholipids include one or more of DPPC, DSPC, DMPC, DOPC, egg yolk lecithin or soybean lecithin.

[0051] Preferably, the organic solvent is ethanol or isopropanol.

[0052] Preferably, the buffer is PBS buffer or HEPES buffer or Tris buffer.

[0053] The specific operation process of this embodiment is as follows: the microArch TM The microfluidic mixing chip 1 is made by S1403D printer, and the photocuring resin used for printing is GR type resin produced by Chongqing Mofang Precision Technology Co., Ltd. The overall structure diagram of the microfluidic acoustic cavitation chip is shown in Figure 1 The microfluidic mixing chip 1, the sealing soft film 2, the electrode 3, the piezoelectric ceramic 4 and the clamping and fixing assembly 5 are assembled into the microfluidic acoustic cavitation chip. As shown in Figure 2 The microfluidic mixing chip 1 is ring-shaped, with an outer diameter of 30 mm, an inner diameter of 12 mm and a thickness of 3 mm; the inner diameters of the inlets 101 and 102 and the outlet 103 are 2 mm, and the depth is 3 mm; the serpentine mixing channel 104 is made on one side of the microfluidic mixing chip 1, with a width of 0.2 mm and a depth of 0.2 mm. The sealing soft film 2 is made of PU, with a thickness of 0.5 mm. The electrode 3 is made of phosphor copper, with a thickness of 0.2 mm. The piezoelectric ceramic is ring-shaped, with an outer diameter of 30 mm, an inner diameter of 12 mm, a thickness of 5 mm and a resonant frequency of 51.1 kHz. The clamping and fixing assembly 5 is circular, wherein the diameter of the perforated plate is 56 mm, the thickness is 12 mm, the diameter of the bolt hole is 5 mm, the diameters of the solution inlet and outlet holes are 6 mm; the diameter of the matching plate is 56 mm, the thickness is 5 mm, the diameter of the bolt hole is 5 mm; the specification of the bolt and nut is M5.

[0054] The system diagram for preparing liposomes with different particle sizes using the microfluidic acoustic cavitation chip is shown in Figure 3 The resonant frequency 51.1 kHz of the piezoelectric ceramic 4 is input into the signal generator 6, and the excitation voltage is set to 6V pp; the signal generator signal is connected to the power amplifier 8 through the signal transmission line 7, and the amplification multiple of the power amplifier is set to 50 times; the positive and negative electrodes of the power amplifier are connected to the positive and negative electrodes of the piezoelectric ceramic 4 in sequence; a corresponding solution is prepared, DPPC, DSPE-MPEG2000 and cholesterol are dissolved in ethanol according to a molar ratio of 70:5:25 to form a lipid organic solution, and the concentration of DPPC in ethanol is 20 mmol / L; the flow rate ratio of PBS buffer (pH 7.4, 0.01 mol / L) to the lipid organic solution is set to 4, and the total flow rate is 60 mL / h; a multi-channel micro-injection pump 10 is used in combination with a syringe 11 and a catheter 12 to inject the PBS buffer and the lipid organic solution into the buffer inlet 101 and the lipid organic solution inlet 102 respectively, and the signal generator 6 and the power amplifier 8 are turned on to generate acoustic cavitation effect in the channel of the microfluidic mixing chip 1; as the PBS buffer and the lipid organic solution are mixed in the microfluidic mixing chip 1, the lipid organic solution is quickly dissolved in the PBS buffer, so that the lipid molecules are quickly self-assembled to form liposomes in the buffer. Without changing the flow rate ratio of the buffer to the lipid organic solution, the excitation voltage (100 V PP , 200 V PP , 300 V PP ) of the piezoelectric ceramic 4 is adjusted according to the required particle size of the liposomes, different intensities of microfluidic acoustic cavitation effect (as shown in Figure 4 ) can be quickly generated in the serpentine mixing channel 104, thereby changing the acoustic-flow coupling field and generating liposomes with multiple different particle sizes, and the experimental results are shown in Figure 5 . In order to verify the applicability of the microfluidic acoustic cavitation chip, without changing other conditions, the main auxiliary material DPPC of the liposome auxiliary material is replaced by DMPC or DSPC or soybean lecithin in sequence, and it is found that using the microfluidic acoustic cavitation chip can effectively reduce the particle size of liposomes with different components, and the experimental results are shown in Figure 6 . Further, under the conditions of a flow rate ratio of 4 of the buffer to the lipid organic solution, a total flow rate of 60 mL / h and an excitation voltage of 300 V PP of the piezoelectric ceramic, drug-loaded liposomes with different particle sizes are prepared, and the model drugs selected are isothiocyanate fluorescein (FITC), indocyanine green (ICG), paclitaxel (PTX)-celastrol (CEL) and doxorubicin (DOX), and the particle size comparison and cryogenic transmission electron microscope pictures of the prepared drug-loaded liposomes are shown in Figure 7 , Figure 8 . The results show that under the condition of not changing the flow rate ratio of the buffer to the lipid organic solution, different particle sizes of drug-loaded liposomes can be obtained by using the microfluidic acoustic cavitation chip.

[0055] The above embodiments only express the implementation ways of the present application, but cannot be understood as a limitation to the scope of the patent of the present application. It should be pointed out that, for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection of the present application.

Claims

1. A method for preparing liposomes of different particle sizes, characterized in that, This method utilizes a microfluidic acoustic cavitation chip, which includes a microfluidic mixing chip, a sealing membrane, piezoelectric ceramics, electrodes, and a clamping and fixing assembly. The microfluidic mixing chip contains a buffer solution inlet, a lipid organic solution inlet, a serpentine mixing channel, and a liposome solution outlet. The serpentine mixing channel is located on one side of the microfluidic mixing chip and is sealed by the sealing membrane. The resonant frequency range of the piezoelectric ceramics is 20-100kHz. The clamping and fixing assembly includes an open-ended thin plate, a mating thin plate, and bolts and nuts. The perforated thin plate has a solution inlet and an outlet; The method also includes the following steps: (1) Input the resonant frequency of the piezoelectric ceramic into the signal generator and set the excitation voltage; after connecting the signal generator signal to the power amplifier, set the amplification factor of the power amplifier; connect the positive and negative terminals of the power amplifier to the positive and negative terminals of the piezoelectric ceramic in sequence. (2) Prepare the appropriate solutions; dissolve the liposome excipients in an organic solvent to form a lipid organic solution; according to the solubility of the encapsulated drug, dissolve the water-soluble drug in a buffer solution or the lipid-soluble drug in a lipid organic solution; (3) Set the flow rate ratio and total flow rate of the buffer solution and the lipid organic solution. Use a multi-channel microinjection pump combined with a syringe and catheter to inject the buffer solution and the lipid organic solution into the buffer solution inlet and the lipid organic solution inlet, respectively. Turn on the signal generator and power amplifier to generate acoustic cavitation effect in the channel of the microfluidic mixing chip. (4) As the buffer solution and the lipid organic solution are mixed in the microfluidic mixing chip, the lipid organic solution dissolves rapidly in the buffer solution, allowing lipid molecules to quickly self-assemble into liposomes in the buffer solution. Without changing the flow rate ratio of the buffer solution to the lipid organic solution, the excitation voltage of the piezoelectric ceramic can be adjusted according to the required liposome particle size to quickly generate liposomes of various particle sizes.

2. The method for preparing liposomes of different particle sizes according to claim 1, characterized in that, The microfluidic hybrid chip is made of photocurable resin, polymethyl methacrylate (PMMA), or polydimethylsiloxane (PDMS); the inner diameter of the inlet and outlet ranges from 400 to 3000 μm, and the depth ranges from 0.5 to 5 mm; the width of the serpentine hybrid channel ranges from 50 to 1000 μm, and the depth ranges from 50 to 1000 μm.

3. The method for preparing liposomes of different particle sizes according to claim 1, characterized in that, The thickness range of the perforated thin plate and the mating thin plate in the clamping and fixing assembly is 3-15mm.

4. The method for preparing liposomes of different particle sizes according to claim 1, characterized in that, The main excipients in the liposome excipients include phospholipids, cholesterol, and N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-distearyl-SN-glycerol-3-phosphoethanolamine (DSPE-MPEG2000). The phospholipids include one or more of dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), myristoyl lecithin (DMPC), dioleoyl lecithin (DOPC), egg yolk lecithin, or soybean lecithin.

5. The method for preparing liposomes of different particle sizes according to claim 1, characterized in that, The organic solvent is ethanol or isopropanol, and the buffer solution is PBS buffer, HEPES buffer, or Tris buffer.

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