A controllable liposome synthesis microfluidic chip and synthesis method based on electrodynamic mixing technology

Abstract

The present invention discloses a controllable liposome synthesis microfluidic chip and synthesis method based on electrodynamic mixing technology, relating to the field of liposome micro-nano biomedical material synthesis technology, and designs a microfluidic chip comprising a chip body, a microchannel, and a metal electrode. The prepared phospholipid solution and deionized water are injected into the microfluidic chip according to a preset ratio and flow rate, and an electrical signal of preset voltage and frequency is provided to the two electrodes of the microfluidic chip at the same time, so that the phospholipid solution and deionized water are rapidly mixed under the action of electrodynamic force, and the electric field changes in which the phospholipid solution and deionized water are located when synthesizing liposomes are controlled, thereby obtaining liposome particle size data corresponding to different flow rates, voltages, and frequencies through experiments, and then determining the synthesis conditions for synthesizing liposomes of different particle sizes to synthesize liposomes. The microfluidic chip of the present invention has the characteristics of low production cost, simple structure, small volume, and low experimental cost. The liposome synthesis process has the advantages of high degree of automation and continuous controllable synthesis process.

Description

Technical Field

[0001] The present invention relates to the technical field of liposome micro-nano biomedical material synthesis, and more particularly to a controllable liposome synthesis microfluidic chip and a synthesis method based on electrodynamic mixing technology. Background Art

[0002] Drug delivery systems refer to biocompatible transport vehicles used in pharmaceutical, cosmetic, and nutritional applications. Among drug delivery systems, liposomes have attracted widespread attention due to their advantages such as size, amphiphilicity, and biocompatibility.

[0003] Microfluidics is a multidisciplinary technology that integrates nanotechnology, biotechnology, chemistry, physics, and engineering. It processes microdevices with microchannels and microchambers ranging in size from tens to hundreds of micrometers. Samples are processed and analyzed by controlling the flow behavior of small volumes of fluid in these microchannels and microchambers. Currently, there are several microfluidics-based liposome synthesis technologies, including thin film hydration, extrusion, emulsification, and hydrodynamic focusing. However, in microchannels, the viscous force between the two phases of liquid is greater than the inertial force, and the two phases flow in a laminar manner. Due to the laminar flow of the two phases of liquid, the two phases of liquid mix by molecular diffusion. This mixing method often has poor mixing effects and cannot control the degree of mixing, which cannot meet the actual needs of users.

[0004] Therefore, how to control the mixing mode in the microchannel to achieve controllable, efficient and rapid synthesis of liposomes is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a controllable liposome synthesis microfluidic chip and synthesis method based on electrodynamic mixing technology. The chip uses electrodynamic mixing technology to mix reagents, and based on the influence of flow rate, reagent flow ratio, voltage, and electric field frequency in the chip on the particle size of synthesized liposomes, realizes controllable, efficient and rapid synthesis of nanoliposomes.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention discloses a controllable liposome synthesis microfluidic chip based on electrodynamic mixing technology, comprising: a chip body, a microchannel, and metal electrodes. The microchannel is located in the chip body, and two sample inlets and one sample outlet of the microchannel are Y-shaped. The metal electrodes are located on both sides of the chip body, and liquid mixing is promoted by applying an electric field to the liquid in the microchannel.

[0008] Preferably, the pole piece of the metal electrode is parallel and closely attached to the mixing part of the microchannel, and the gap between the pole piece and the microchannel is less than or equal to 100 μm.

[0009] Preferably, the microfluidic chip is manufactured by the following method:

[0010] S1: Pour the premixed liquid into the mold and defoam it;

[0011] S2: After the premixed liquid solidifies, the microchannel mold and metal electrodes are placed in the mold;

[0012] S3: Pour the premixed liquid again for defoaming treatment, solidify and demould, and cut according to the designed size;

[0013] S4: dissolving the microchannel mold using acetone solution to obtain a microfluidic chip.

[0014] Preferably, the premixed liquid is obtained by mixing polydimethylsiloxane and a cross-linking agent in a fixed ratio and stirring them uniformly; and the microchannel mold is a Y-shaped mold formed by bonding ABS plastic.

[0015] The present invention also discloses a controllable liposome synthesis method based on electrodynamic mixing technology, the specific steps of which are as follows:

[0016] Step 1: Prepare phospholipid solution and pretreat;

[0017] Step 2: The pretreated phospholipid solution and deionized water are injected into the corresponding inlets of the microfluidic chip at a preset ratio and flow rate, and an electrical signal of a set voltage and frequency is applied to the microfluidic chip to cause the phospholipid solution and deionized water to mix rapidly under the action of the electric force to synthesize liposomes;

[0018] Step 3: collecting the synthesized liposomes, and detecting and recording the particle size and potential of the synthesized liposomes;

[0019] Step 4: changing the voltage and frequency of the electrical signal and the gradient value of the flow rate within a set range to synthesize liposomes and detect the liposome particle size and potential;

[0020] Step 5: Analyze the relationship between different voltages, frequencies, and flow rates and the size of the synthesized liposomes to determine the synthesis conditions for synthesizing liposomes of different particle sizes;

[0021] Step 6: Synthesize liposomes according to the determined synthesis conditions.

[0022] Preferably, the step 1 comprises: adding lecithin to anhydrous ethanol, and placing in a 60° C. water bath for 10 minutes to fully dissolve the lecithin; and then filtering using a filter membrane to obtain a 10 mM phospholipid solution.

[0023] Preferably, in step 2, the phospholipid solution syringe and the deionized water syringe are fixed by a syringe pump and the injection speed is adjusted, thereby adjusting the flow rate of the phospholipid solution and deionized water injected into the microfluidic chip.

[0024] Preferably, step 2 further comprises: controlling the flow ratio of the phospholipid solution and the deionized water by changing the specifications of the phospholipid solution syringe and the deionized water syringe, thereby analyzing the relationship between the flow ratio and the particle size of the synthesized liposomes.

[0025] Preferably, in step 3, after 800 μL of ×PBS reagent is added to the collected 200 μL reaction solution for dilution, the liposome suspension is immediately placed in a Malvern particle size analyzer to measure the liposome particle size and potential.

[0026] Preferably, the electrical signal is provided by an arbitrary function generator, the output waveforms of the two output channels of the arbitrary function generator are square waves, and the phase difference between the output channels is 180°.

[0027] Preferably, the analysis of the relationship between different voltages, frequencies and flow rates and the synthesized liposome particle size specifically includes: establishing a linear regression equation between the microfluidic chip voltage, frequency and flow rate and the liposome particle size; solving the linear regression equation based on the microfluidic chip voltage, frequency, flow rate and liposome particle size data obtained in steps 1-6.

[0028] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a controllable liposome synthesis microfluidic chip and synthesis method based on electrodynamic mixing technology, wherein the microfluidic chip is cast from PDMS material and metal electrodes, and has the characteristics of low production cost; and the microfluidic chip has a simple structure and a small size, which can significantly simplify the experimental process, reduce the consumption of experimental reagents and samples, and reduce experimental costs; at the same time, the liposome synthesis process has a high degree of automation, does not require manual operation, and the synthesis process is continuously controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0030] Figure 1 Schematic diagram of the structure of the microfluidic chip according to an embodiment of the present invention.

[0031] Figure 2 Schematic diagram of the process of liposome synthesis according to an embodiment of the present invention.

[0032] Figure 3 Schematic diagram of the relationship between liposome particle size and voltage in an embodiment of the present invention.

[0033] Figure 4 Schematic diagram of the relationship between liposome particle size and frequency in an embodiment of the present invention.

[0034] Figure 5 Schematic diagram of the relationship between liposome particle size and flow rate in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] The embodiment of the present invention discloses a controllable liposome synthesis microfluidic chip based on electrokinetic mixing technology, such as Figure 1 As shown, it includes: a chip body, a microchannel, and metal electrodes. The microchannel is located in the chip body. The two sample inlets and one sample outlet of the microchannel are Y-shaped. The metal electrodes are located on both sides of the chip body. Liquid mixing is promoted by applying an electric field to the liquid in the microchannel.

[0037] In a specific embodiment, the pole piece of the metal electrode is parallel and closely attached to the mixing part of the microchannel, and the gap between the pole piece and the microchannel is less than or equal to 100 μm.

[0038] In a specific embodiment, the microchannel has a diameter of 1 mm and the chip size is 2x2 cm;

[0039] In a specific embodiment, the microfluidic chip is manufactured by the following method:

[0040] S1: Pour the premixed liquid into the mold and defoam it;

[0041] S2: After the premixed liquid solidifies, the microchannel mold and metal electrodes are placed in the mold;

[0042] S3: Pour the premixed liquid again for defoaming treatment, solidify and demould, and cut according to the designed size;

[0043] S4: Dissolve the microchannel mold using acetone solution to obtain a microfluidic chip.

[0044] In a specific embodiment, the premixed liquid is obtained by mixing polydimethylsiloxane and a cross-linking agent in a fixed ratio and stirring them uniformly; the microchannel mold is a Y-shaped mold made of ABS plastic.

[0045] The present invention also discloses a controllable liposome synthesis method based on electrokinetic mixing technology. Figure 2 The specific steps are as follows:

[0046] Step 1: Prepare phospholipid solution and pretreat;

[0047] Step 2: The pretreated phospholipid solution and deionized water are injected into the corresponding inlets of the microfluidic chip at a preset ratio and flow rate. At the same time, an electrical signal of a set voltage and frequency is applied to the microfluidic chip to cause the phospholipid solution and deionized water to mix rapidly under the action of electrodynamics to synthesize liposomes;

[0048] Step 3: collecting the synthesized liposomes, and detecting and recording the particle size and potential of the synthesized liposomes;

[0049] Step 4: Changing the voltage, frequency, and flow rate gradient of the electrical signal within a set range to synthesize liposomes and detect liposome particle size and potential;

[0050] Step 5: Analyze the relationship between different voltages, frequencies, and flow rates and the size of the synthesized liposomes to determine the synthesis conditions for synthesizing liposomes of different particle sizes;

[0051] Step 6: Synthesize liposomes according to the determined synthesis conditions.

[0052] In a specific embodiment, step 1 comprises: adding lecithin to anhydrous ethanol, immersing in a 60°C water bath for 10 minutes to fully dissolve the lecithin; then filtering using a filter membrane to obtain a 10 mM phospholipid solution. For example, 0.06 g of lecithin is added to 8 mL of anhydrous ethanol, immersing in a 60°C water bath for 10 minutes to fully dissolve the lecithin, and then filtering using a 20 μm filter membrane to obtain a phospholipid solution. The phospholipid solution can also be obtained by dissolving and filtering other phospholipids, such as synthetic anionic liposomes (phosphoserine, PSs) or cationic liposomes DMG-PEG2000.

[0053] In a specific embodiment, in step 2, the phospholipid solution syringe and the deionized water syringe are fixed by a syringe pump and the injection speed is adjusted, thereby adjusting the flow rate of the phospholipid solution and deionized water injected into the microfluidic chip.

[0054] In a specific embodiment, step 2 further includes: controlling the flow ratio of the phospholipid solution and the deionized water by changing the specifications of the phospholipid solution syringe and the deionized water syringe, thereby analyzing the relationship between the flow ratio and the particle size of the synthesized liposomes.

[0055] In a specific embodiment, in step 3, after adding 800 μL of ×PBS reagent to the collected 200 μL reaction solution for dilution, the liposome suspension is immediately placed in a Malvern particle size analyzer to measure the liposome particle size and potential, and the particle size of the synthesized liposome is characterized by the peak value of the particle size distribution of the test results. The charge on the surface of the liposome will affect its interaction with other particles in the surrounding environment. Measuring the zeta potential can help determine the charge properties of the liposome surface, such as positive or negative charge, thereby affecting its distribution and interaction in the body. Zeta potential is also an indicator for evaluating the stability of liposomes. Liposomes with a larger zeta potential generally have better stability because the electrostatic repulsion between them prevents them from aggregating together to form larger clusters. In the process of preparing liposomes, adjusting the preparation conditions can change the zeta potential of the liposomes. Therefore, measuring the zeta potential can help optimize the preparation process to obtain liposomes with desired properties.

[0056] In a specific embodiment, the electrical signal is provided by an arbitrary function generator. The output waveforms of the two output channels of the arbitrary function generator are square waves, and the phase difference between the output channels is 180°.

[0057] In a specific embodiment, the relationship between different voltages, frequencies and flow rates and the size of the synthesized liposomes was analyzed, wherein the voltage gradient range was 0-10V. p-p , the frequency gradient range is 10kHz-40MHz, and the flow rate gradient range is 10-50μL / min. Figure 3 As shown, when the frequency and injection flow rate are fixed, the voltage of the microfluidic chip is 2V. p-p , 4V p-p , 6V p-p , 8V p-p , 10V p-p (V p-p represents the voltage between the two output channels of the arbitrary function generator), it can be seen that the particle size of the liposome decreases with the increase of voltage; Figure 4 As shown in FIG, when the voltage and injection flow rate are fixed, by detecting the synthetic liposome particle size when the microfluidic chip electrical signal frequency is 100kHz, 1MHz, 10MHz, and 40MHz, it can be seen that the liposome particle size increases with the increase of frequency; Figure 5As shown in the figure, when the voltage and frequency are fixed, by detecting the liposome particle size when the injection flow rate of the phospholipid solution and deionized water is 10μL / min, 20μL / min, 30μL / min, and 40μL / min, it can be seen that the liposome particle size generally decreases with the increase of the injection flow rate. Therefore, the regression equation of different liposome particle sizes can be obtained by the linear regression method, and based on this equation, liposomes of different particle sizes can be accurately synthesized according to the needs of liposome synthesis. The linear regression method specifically includes:

[0058] Establish a linear regression equation between the voltage, frequency, and flow rate of the microfluidic chip and the liposome particle size;

[0059] According to the microfluidic chip voltage, frequency, flow rate and liposome particle size data obtained in steps 1-6, the linear regression equation is solved. The linear regression equation is:

[0060] Y=k1X f +k2X T +k3X V +μ;

[0061] Where, Y is the particle size, in nm; X f is the frequency in MHz; X T is the flow rate, in μL / min; X V is the voltage, the unit is V p-p ; k1, k2, k3, and μ are linear regression coefficients; k1, k2, k3, and μ obtained through multiple experimental fitting calculations are 1.164, -1.73, -8.55, and 618.5, respectively.

[0062] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. Reference can be made to the common and similar parts between the various embodiments. For the devices disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the method description.

[0063] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A controllable liposome synthesis method based on electrokinetic mixing technology, characterized in that: The specific steps are as follows: Step 1: Prepare phospholipid solution and pretreat; Step 2: Injecting the pretreated phospholipid solution and deionized water into the corresponding inlets of the microfluidic chip at a preset ratio and flow rate, and applying an electrical signal of a set voltage and frequency to the microfluidic chip to synthesize liposomes; Step 3: collecting the synthesized liposomes, and detecting and recording the particle size and potential of the synthesized liposomes; Step 4: changing the voltage and frequency of the electrical signal and the gradient value of the flow rate within a set range to synthesize liposomes and detect the liposome particle size and potential; Step 5: Analyze the relationship between different voltages, frequencies, and flow rates and the size of the synthesized liposomes to determine the synthesis conditions for synthesizing liposomes of different particle sizes; Step 6: synthesizing liposomes according to the determined synthesis conditions; The analyzing the relationship between different voltages, frequencies, and flow rates and the synthesized liposome particle size specifically includes: establishing a linear regression equation between the microfluidic chip voltage, frequency, and flow rate and the liposome particle size; solving the linear regression equation based on the microfluidic chip voltage, frequency, flow rate, and liposome particle size data obtained in steps 1-6; The microfluidic chip comprises: a chip body, a microchannel, and metal electrodes. The microchannel is located in the chip body. Two sample inlets and one sample outlet of the microchannel are in a Y shape. The metal electrodes are located on both sides of the chip body and promote liquid mixing by applying an electric field to the liquid in the microchannel. The pole piece of the metal electrode is parallel and closely attached to the mixing part of the microchannel, and the gap between the pole piece and the microchannel is less than or equal to 100 μm.

2. The method for synthesizing controllable liposomes based on electrokinetic mixing technology according to claim 1, wherein: In step 2, the phospholipid solution syringe and the deionized water syringe are fixed by a syringe pump and the injection speed is adjusted, thereby adjusting the flow rate of the phospholipid solution and deionized water injected into the microfluidic chip.

3. The method for synthesizing controllable liposomes based on electrokinetic mixing technology according to claim 2, wherein: The step 2 further includes: controlling the flow ratio of the phospholipid solution and the deionized water by changing the specifications of the phospholipid solution syringe and the deionized water syringe, thereby analyzing the relationship between the flow ratio and the particle size of the synthesized liposomes.

4. The method for synthesizing controllable liposomes based on electrokinetic mixing technology according to claim 1, wherein: The electrical signal is provided by an arbitrary function generator. The output waveforms of the two output channels of the arbitrary function generator are square waves, and the phase difference between the output channels is 180°.

5. The method for synthesizing controllable liposomes based on electrokinetic mixing technology according to claim 1, wherein: The microfluidic chip is manufactured in the following manner: S1: Pour the premixed liquid into the mold and defoam it; S2: After the premixed liquid solidifies, the microchannel mold and metal electrodes are placed in the mold; S3: Pour the premixed liquid again for defoaming treatment, solidify and demould, and cut according to the designed size; S4: dissolving the microchannel mold using acetone solution to obtain a microfluidic chip.

6. The method for synthesizing controllable liposomes based on electrokinetic mixing technology according to claim 5, characterized in that: The premixed liquid is obtained by mixing polydimethylsiloxane and a cross-linking agent in a fixed ratio and stirring them uniformly; the microchannel mold is a Y-shaped mold formed by bonding ABS plastic.

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