A method for preparing ansamitocin P-3 loaded light-responsive thermosensitive liposomes using microfluidic technology and its application

Abstract

The present invention belongs to the field of nanomaterials and biomedical technology, and discloses a method and application of preparing light-responsive thermosensitive liposomes loaded with ansamitocin P‑3 by microfluidic technology. Ansamitocin P‑3 is combined with a nanocarrier for the first time, specifically relating to a method of preparing light-responsive thermosensitive liposomes loaded with ansamitocin P‑3 and a photosensitizer by microfluidic technology. In addition to ansamitocin P‑3, the liposomes also contain photosensitizers, phospholipids, cholesterol and PEGylated phospholipids. Through a vortex micromixer, stable and continuous production can be achieved with high repeatability. The prepared liposomes not only improve the stability of the drug, prolong the circulation time of the drug in the body, but also have the effect of light-controlled release. While improving the anti-tumor activity, the toxicity of the drug to normal tissues is also significantly reduced.

Description

Technical Field

[0001] The present invention belongs to the field of materials and biomedical technology, and relates to ansamitocin P-3 liposomes and preparation and application thereof, and specifically to a method for preparing light-responsive thermosensitive liposomes loaded with ansamitocin P-3 and a photosensitizer by using microfluidic technology. Background Art

[0002] Ansamitocin P-3 (AP-3) is an important fat-soluble anti-tumor drug with strong anti-tumor activity against a variety of tumor cell lines. The strong cytotoxicity and high price of AP-3 have limited its medicinal development. At the same time, since fat-soluble drugs are difficult to dissolve, a mature formulation process is urgently needed to solve these problems.

[0003] In the 20th century, Nils Rieberg-Finsen was one of the first scientists to use red light to treat smallpox and blue light to treat tuberculosis of the skin, and won the Nobel Prize in 1903. Since then, several studies have focused on using the energy (photons) radiated by light sources such as lasers for a variety of medical applications, including cancer treatment. Laser therapy for tumor ablation has several disadvantages, including non-selectivity for cancer cells and the high power lasers required to produce therapeutic effects, which have raised concerns about the convincingness and safety of this cancer treatment. To address these limitations, photosensitizers have been introduced to enhance the effects of light radiation and improve the efficiency of photodynamic therapy (PDT) and photothermal therapy (PTT). Indocyanine green (ICG), an FDA-approved drug for clinical use, is an ideal photosensitizer due to its significant near-infrared optical properties, but its disadvantages are a short plasma half-life and poor tissue permeability.

[0004] Liposomes are spherical structures with excellent biocompatibility. They can encapsulate amphiphilic drugs, overcome cell and tissue absorption barriers, increase drug stability, improve bioavailability, and reduce side effects. The temperature-sensitive liposomes with unique formulas can respond to the photothermal effect of photosensitizers. When the temperature is slightly higher than the human body temperature, the fluidity and permeability of the phospholipid bilayer will increase, thereby releasing a large amount of drugs, causing a large amount of drugs to accumulate in the target tissue. There are various methods for preparing liposomes. The traditional preparation method has low requirements for equipment precision and is mostly used for laboratory preparation. However, the disadvantage is that the size distribution of liposomes cannot be accurately controlled. The particle size needs to be reduced by ultrasound, extrusion and other methods, and the reproducibility between batches is poor, which cannot be achieved in industrial production. In order to overcome the defects of traditional methods, many modern methods for preparing liposomes have been reported in recent years, such as membrane contact method, supercritical fluid method, freeze drying method, microfluidics, etc. Among them, microfluidics technology uses micron-level or even millimeter-level micro-devices to manipulate tiny fluids. The devices used are also called microfluidic chips. Microfluidic liposome preparation technology can achieve continuous flow production, can well control the size uniformity of liposomes, and achieve industrial production. When preparing liposomes using microfluidic chips, cholesterol must be added to control the particle size by about 30% by weight. The addition of cholesterol will adjust the fluidity of the membrane and change the phase transition temperature of the membrane material. The addition of cholesterol will significantly affect the photothermal effect of liposomes. Therefore, most cases of preparing thermosensitive liposomes using traditional methods do not add cholesterol. 1-4 The present invention overcomes the disadvantage that a large amount of cholesterol must be added to prepare liposomes by microfluidics by adjusting the type and ratio of phospholipids, thereby combining thermosensitive liposomes with microfluidics for the first time, and successfully using microfluidics to embed Ansamitocin P-3 into nanocarriers, which not only improves the stability of the drug and prolongs the circulation time of the drug in the body, but also has the effect of light-controlled release.

[0005] 1. Lu, T.; Lokerse, WJM; Seynhaeve, ALB; Koning, GA; Ten Hagen, TLM.

[0006] 2. Fu, X.; Lu, Y.; Guo, J.; Liu, H.; Deng, A.; Kuang, C.;

[0007] 3.Jose,A.;Ninave,KM;Karnam,S.;Venuganti,VVK,Temperature-sensitive liposomes for co-delivery of tamoxifen and imatinib for synergisticbreast cancer treatment.Journal of liposome research 2019,29(2),153-162.

[0008] 4. Du, C.; Li, S.; Li, Y.; Galons, H.; Guo, N.; Teng, Y.; Zhang, Y.; Li, M.; Yu, P., F7 and topotecan co-loaded thermosensitive liposome as a nano-drug delivery system for tumor hyperthermia. Drug delivery 2020, 27(1), 836-847. Summary of the invention

[0009] The purpose of the present invention is to provide ansamitocin P-3 liposomes and their preparation and application, and to provide a method for preparing light-responsive thermosensitive liposomes loaded with ansamitocin P-3 and photosensitizer based on microfluidic technology, which has a more stable and reliable process, improves the stability of the drug; prolongs the circulation time of the drug in the body; and reduces the toxicity of the drug to normal tissues. This method is closer to industrial production.

[0010] The present invention provides a microfluidic preparation method of drug-loaded thermosensitive liposomes, which adopts the following technical scheme:

[0011] (1) dissolving ansamitocin P-3 (AP-3), phospholipids, cholesterol, and PEGylated phospholipids in an organic solvent to obtain an organic phase;

[0012] (2) dissolving the photosensitizer in a buffer solution to obtain an aqueous phase;

[0013] (3) The aqueous phase and the organic phase are injected into the microfluidic chip from different channels at a certain total flow rate and flow rate ratio to form liposomes for self-assembly.

[0014] (4) Using different methods to remove the residual organic solvent and free substances in the liposomes obtained in step (3) and sterilize them.

[0015] Furthermore, in step (1), the mass ratio of phospholipid to cholesterol is 1:1 to 9:1, and preferably, the mass ratio of phospholipid to cholesterol is 2:1.

[0016] Further, the phospholipid is selected from one or a combination of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), egg yolk phosphatidylcholine (EPC), phosphatidylcholine (PC), hydrogenated soybean lecithin (HSPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylglycerol (DSPC), etc., preferably DPPC and DSPC; the mass ratio of DPPC to DSPC is 1:1 to 5:1, preferably, the mass ratio of DPPC to DSPC is 3:1 to 5:1.

[0017] Further, the PEGylated phospholipid is selected from DSPE-MPEG 1000 、DSPE-MPEG 2000 、DSPE-MPEG 5000 One or more of the above, preferably DSPE-MPEG 2000 .

[0018] Furthermore, the phospholipids, cholesterol, and PEGylated phospholipids of the present invention are preferably DPPC, DSPC, cholesterol, DSPE-MPEG 2000 The combination has a mass ratio of (4.5-5):(1-1.5):3:(0.4-1); further, the photosensitizer is indocyanine green (ICG).

[0019] Furthermore, AP-3, ICG and total lipid mass (phospholipids (DPPC / DSPC) + cholesterol + DSPE-MPEG 2000 The drug-to-lipid ratio of the total mass) is 1:1:20 to 1:1:25 (w / w).

[0020] Furthermore, the PEGylated phospholipid accounts for 5% to 25% of the total lipid mass. Preferably, the PEGylated phospholipid accounts for 5% to 10% of the total lipid mass.

[0021] Furthermore, the drug-lipid ratio of AP-3 to total lipid mass is 1:5 to 1:30 (w / w), preferably the drug-lipid ratio of AP-3 to total lipid mass is 1:20 to 1:30.

[0022] Furthermore, the organic solvent in step (1) is selected from any one or more of methanol, ethanol, acetone, acetonitrile, and ether, preferably ethanol; and the buffer described in step (2) is any one of PBS buffer, HEPES buffer, and TRIS buffer, preferably PBS buffer.

[0023] The total flow rate of step (3) is 12 to 200 ml / min, preferably 40 to 100 ml / min; the flow rate ratio of the aqueous phase to the organic phase is 1:1 to 29:1, preferably, the flow rate ratio of the aqueous phase to the organic phase is 2:1 to 4:1;

[0024] The microfluidic mixer consists of a vortex mixing unit, two inlet channels and one outlet channel.

[0025] The method for removing the residual organic solvent and free substances and sterilizing in step (4) is dialysis, low-speed centrifugation, ultrafiltration centrifugation and membrane method.

[0026] Specifically, the preferred solution is:

[0027] (1) Accurately weigh DPPC, DSPC, cholesterol, and DSPE-MPEG according to a certain ratio. 2000 and AP-3 were dissolved in ethanol and sonicated to prepare an organic phase.

[0028] (2) Weigh the photosensitizer and dissolve it in PBS. Ultrasonicate and dissolve it into the aqueous phase.

[0029] (3) After the solutions prepared in steps (1) and (2) are respectively filtered through a 0.22 μm filter membrane, the aqueous phase and the organic phase solution are injected into a microfluidic mixer through different inlet channels at a flow rate ratio of 3:1 to 4:1 and a total flow rate of 40 ml / min to 100 ml / min. The two-phase solution is briefly and fully mixed in the vortex mixing unit to self-assemble into liposomes.

[0030] (4) The liposome suspension obtained in step (3) is placed in a dialysis bag and dialyzed in PBS to remove residual ethanol.

[0031] (5) placing the dialyzed liposome suspension in step (4) into a centrifuge tube and centrifuging to precipitate free fat-soluble substances.

[0032] (6) Collect the supernatant after centrifugation in step (5), place it in an ultrafiltration centrifuge tube and centrifuge it to remove water-soluble free substances.

[0033] (7) Collect the liposome suspension in the ultrafiltration centrifuge tube in step (6), filter and sterilize it through a 0.22 μm mixed cellulose lipid membrane, and obtain liposomes with a particle size between 100 nm and 200 nm and a PDI of less than 0.1.

[0034] The prepared ansamitocin P-3 loaded light-responsive thermosensitive liposomes have a photothermal effect and realize drug release under light and heat stimulation; they can be used in the preparation of tumor treatment drugs and further prepared as anti-tumor photodynamic therapy drugs.

[0035] Compared with the existing technology, the present invention has the following beneficial effects:

[0036] The present invention successfully encapsulates ansamitocin P-3 into a nanocarrier for the first time and designs a long-circulation thermosensitive liposome through a unique lipid membrane material and ratio, which can be triggered in vitro with an infrared laser to achieve the effect of controlling drug release, reduce the damage of highly cytotoxic drugs to normal tissues, prolong the drug circulation time, and enhance the targeted treatment and inhibition effect on tumors.

[0037] Meanwhile, the microfluidic preparation process of the present invention is simple and stable, and can continuously produce liposomes with uniform particle size and good reproducibility, thus providing a new method for industrial production of anticancer targeted nanocarriers and expanding the application prospects of liposomes and ansamitocin P-3. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The morphology of ansamitocin P-3 photoresponsive thermosensitive liposomes in Example 1 was observed using an atomic force microscope.

[0039] Figure 2 It is the Zeta potential of ansamitocin P-3 photoresponsive thermosensitive liposomes in Example 1.

[0040] Figure 3 The results of the effect of the ratio of phospholipid to cholesterol on the liposome particle size and PDI in Example 2 are shown.

[0041] Figure 4 The results of the effect of total lipid concentration on liposome particle size and PDI in Example 2 are shown.

[0042] Figure 5 DSPE-MPEG in Example 2 2000 Results of the effect of dosage on liposome particle size and PDI.

[0043] Figure 6 The results of the effect of the flow rate ratio of the aqueous phase to the organic phase on the liposome particle size and PDI in Example 2 are shown.

[0044] Figure 7The results of the influence of the total flow rate of the two phases on the liposome particle size and PDI in Example 2 are shown.

[0045] Figure 8 The results of Example 2 are the effects of the Chinese medicine-lipid ratio on the liposome encapsulation efficiency.

[0046] Fig. 9 This is the effect of adding DSPC in Example 3 on the in vitro release of liposomes.

[0047] Fig.10 This is a thermal imaging image of the in vitro photothermal effect experiment in Example 4.

[0048] Fig.11 This is a temperature curve diagram of the in vitro photothermal effect experiment in Example 4.

[0049] Fig.12 This is a fluorescence image of the cell live-death staining experiment in Example 5.

[0050] Fig.13 The cell morphology of each group in Example 6 was observed using an atomic force microscope after administration. DETAILED DESCRIPTION

[0051] The present invention is not limited to the following specific embodiments. A person skilled in the art can implement the present invention in various other specific embodiments according to the contents disclosed in the present invention, or any simple changes or modifications made to the design structure and ideas of the present invention fall within the protection scope of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0052] The microfluidic chip described in the present invention is a vortex structure; a vortex structure chip refers to a chip with a cylindrical vortex structure unit. After multiple liquids converge in the upper mixing chamber, a vortex will be formed and rotated downward, which can more quickly change the fluid state from laminar flow to turbulent flow, so that the liquid can be fully mixed in a short time. The present invention has no restriction on the number of vortex structure units, and two can be preferably used. The present invention has no restriction on the size of the chip.

[0053] The vortex structure chip belongs to the prior art. However, the key innovation is to prepare the ansamitocin P-3 light-responsive thermosensitive liposome using a unique material ratio and parameters, which is reported for the first time in the present invention.

[0054] Example 1: Preparation of Ansamitocin P-3 Photoresponsive Thermosensitive Liposomes

[0055] Prepare PBS buffer (10mM, PH 7.4): weigh 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4, add 800ml deionized water, dissolve under ultrasonication, and then make up to volume with a 1L volumetric flask.

[0056] Prepare the organic phase: weigh DPPC, DSPC, cholesterol, DSPE-MPEG 2000 and ansamitocin P-3 (4.5:1.5:3:0.5:0.5, w / w) were placed in a small glass bottle, and a certain amount of anhydrous ethanol was added to make the total lipid concentration reach 6 mg / ml. Ultrasonication was performed for 30 min to completely dissolve the mixture, and the mixture was filtered through a 0.22 μm filter membrane for use.

[0057] Prepare the aqueous phase: Weigh an amount of ICG equal to that of AP-3 and dissolve it in PBS three times the volume of ethanol, then sonicate to completely dissolve it and filter it through a 0.22 μm filter membrane for later use.

[0058] Preparation of liposomes: The organic phase and the aqueous phase were sucked into syringes respectively. Two syringes were connected to the two inlets of the microfluidic device and fixed on the syringe pump. The organic phase and the aqueous phase were pumped into the microfluidic chip at a speed of 20 ml / min and 60 ml / min respectively. After the first 5 ml of liquid was discharged from the outlet, the liposome suspension was collected in a container.

[0059] Removal of organic solvent: The liposome suspension was placed in a 3000 molecular weight dialysis bag, which was placed in pre-cooled PBS. The dialysis solution was dialyzed at 4°C using a magnetic stirrer. The dialysate was replaced every 6 hours for a total of 3 times to completely remove the ethanol.

[0060] Removal of free substances: Place the dialyzed liposome suspension in a centrifuge tube and centrifuge it at 3000 rpm / min for 10 min at 4°C using a low-speed centrifuge to remove free fat-soluble precipitates. Then place the liposomes in a 3KD molecular weight ultrafiltration centrifuge tube and centrifuge it at 4000 rpm / min for 20 min to remove free water-soluble substances.

[0061] Sterilization (for cell experiments): The liposome suspension in the ultrafiltration tube was sterilized by filtration through a 0.22 μm mixed cellulose lipid membrane.

[0062] 2. Characterization

[0063] The particle size, PDI and Zeta potential of liposomes were determined using a Malvern Zetasizer particle size analyzer. The surface morphology of liposomes was observed using an atomic force microscope (AFM). Figure 1 The Zeta potential results are as follows: Figure 2 3. Determination of AP-3 and ICG Content

[0064] The content of AP-3 was determined by high performance liquid chromatography (HPLC). The column model used was Eclipse XDB-C18 4.6 mm × 150 mm. The mobile phase was 70% methanol + 30% ddH2O. The flow rate of the mobile phase pump was 0.8 mL / min, and the detection wavelength was 254 nm. ICG was quantitatively determined by UV-visible spectrophotometry, and the detection wavelength was 800 nm.

[0065] Example 2: Preparation of thermosensitive liposomes by vortex micromixer and screening of parameters

[0066] 1. Effect of the ratio of phospholipids to cholesterol on liposome particle size and PDI

[0067] Blank liposomes were prepared according to Example 1. The preparation method was the same as Example 1. The difference was that due to the high price of the drugs, AP-3 and ICG were not added in the early process screening, and the mass ratios of DSPC+DPPC to cholesterol were 1:1, 1.5:1, 2:1, 4:1 and 9:1 respectively. Other conditions remained unchanged. The changes in liposome particle size and PDI were measured. The results are shown in Figure 3 The more cholesterol is added, the smaller the particle size of the liposome is, and it has no effect on PDI. The smaller the particle size and PDI, the better. Considering that cholesterol will occupy the space of the phospholipid bilayer and affect the embedding of fat-soluble drugs, the mass ratio of phospholipid to cholesterol is preferably 2:1.

[0068] 2. Effect of total lipid concentration on liposome particle size and PDI

[0069] Blank liposomes were prepared according to Example 1. The preparation method was the same as Example 1. The difference was that due to the high price of the drugs, AP-3 and ICG were not added in the early process screening, and the total lipid concentrations were 2 mg / ml, 4 mg / ml, 6 mg / ml, 8 mg / ml and 10 mg / ml respectively. Other conditions remained unchanged. The changes in liposome particle size and PDI were measured. The results are shown in Figure 4 When the total lipid concentration is greater than or equal to 4 mg / ml, there is no effect on the particle size and PDI of the liposomes. The amount of lipid added can be changed according to the actual demand for the liposome concentration.

[0070] 3. DSPE-MPEG 2000 Effect of dosage on liposome particle size and PDI Blank liposomes were prepared according to Example 1. The preparation method was the same as Example 1, except that AP-3 and ICG were not added in the early process screening due to the high price of the drug. 2000 The dosage of liposomes was 5%, 10%, 15%, 20% and 25% of the total lipid mass, and other conditions remained unchanged. The changes in liposome particle size and PDI were measured. The results are shown in Figure 5 DSPE-MPEG2000 The less the dosage, the smaller the particle size, and it has little effect on PDI. The addition of long-chain PEG is mainly to prolong the circulation time of liposomes. The dosage does not need to be large, 5% to 10% is optimal.

[0071] 4. Effect of the flow rate ratio of aqueous phase to organic phase on liposome particle size and PDI

[0072] Blank liposomes were prepared according to Example 1. The preparation method was the same as Example 1, except that AP-3 and ICG were not added in the early process screening due to the high price of the drugs. The flow rate ratios of the aqueous phase to the organic phase were 1:1, 2:1, 3:1, 4:1, 5:1, 9:1, 19:1 and 29:1, respectively. Other conditions remained unchanged. The changes in liposome particle size and PDI were measured. The results are shown in Figure 6 It can be seen that the flow rate ratio can greatly change the particle size and PDI of liposomes. When the flow rate ratio is between 2:1 and 4:1, the particle size and PDI are optimal.

[0073] 5. Effect of total two-phase flow rate on liposome particle size and PDI

[0074] Blank liposomes were prepared according to Example 1. The preparation method was the same as Example 1, except that AP-3 and ICG were not added in the early process screening due to the high price of the drugs. The total flow rates of the two phases were 4 ml / min, 12 ml / min, 24 ml / min, 40 ml / min, 60 ml / min, 80 ml / min, 100 ml / min, 120 ml / min and 140 ml / min, respectively. Other conditions remained unchanged. The changes in liposome particle size and PDI were measured. The results are shown in FIG. Figure 7 It can be seen that when the total flow rate is greater than 12 ml / min, the liposome particle size has little effect on PDI.

[0075] VI. Effect of drug-lipid ratio on liposome encapsulation efficiency

[0076] Liposomes encapsulating AP-3 were prepared according to Example 1. The preparation method was the same as Example 1, except that ICG was not added and the drug-lipid ratio of AP-3 to total lipids was 1:5, 1:10, 1:15, 1:20 and 1:30, respectively. Other conditions remained unchanged, and the changes in encapsulation efficiency were measured. The results are shown in FIG. Figure 8 It can be estimated that when the drug-lipid ratio is between 1:20 and 1:25, the encapsulation efficiency is higher.

[0077] Example 3: In vitro release experiment

[0078] Ansamitocin P-3 thermosensitive liposomes were prepared according to Example 1. The preparation method was the same as Example 1, except that ICG was not added to prepare AP3-TSL.

[0079] Ansamitocin P-3 thermosensitive liposomes were prepared according to Example 1. The preparation method was the same as Example 1, wherein AP-3 and ICG were loaded at the same time to prepare AI-TSL.

[0080] According to Example 1, liposomes without DSPC were prepared, that is, DSPC was replaced with an equal amount of DPPC, and other conditions remained unchanged to prepare AI-TSL without DSPC. Further, ICG was not added to prepare AP3-TSL without DSPC.

[0081] To evaluate the controlled release of drugs, the aqueous dispersions of thermosensitive liposomes (AP3-TSL) containing the same amount of AP-3 and thermosensitive liposomes (AI-TSL) loaded with AP-3 and ICG were placed in dialysis bags and placed in 100 mL PBS (containing 1% SDS) at 37°C. The dialysis bags were heated at 808 nm, 1 W / cm 2 The dissolution medium was collected after 5 min of laser irradiation; AP3-TSL(+) and AI-TSL(+) refer to those after laser irradiation.

[0082] The liposomes without DSPC were operated in the same manner as above. Using a magnetic stirrer at 200 rpm / min, 1 mL of release medium was obtained at different time points, and 1 mL of fresh release medium was added. The content of AP-3 was determined by HPLC. The results are shown in Fig. 9 .

[0083] Depend on Fig. 9 It can be seen that the release of AP-3 in vitro by AP3-TSL(+) and AI-TSL is low. Since AP3-TSL(+) is not loaded with ICG, although it is illuminated, there is no photothermal effect, and the release of AP-3 is low; although AI-TSL is loaded with ICG, it will not generate heat without laser irradiation, resulting in low release efficiency. However, AI-TSL(+) has a higher efficiency in releasing AP-3. The release of AP-3 at 12h is 89%±7%, which is about 44% higher than AI-TSL (P<0.05). It can be seen that laser irradiation and ICG synergistically produce photothermal effect.

[0084] At the same time, it can be found that the amount of release after illumination does not change much regardless of whether DSPC is added or not, probably because the temperature exceeds the phase transition temperature of the liposome after laser irradiation. In the absence of laser irradiation, the release of liposomes without DSPC increases significantly, probably because the addition of cholesterol changes the fluidity of the membrane, lowers the phase transition temperature of the liposome, and increases the release under body temperature. It can be seen that the addition of DSPC in the prescription increases the phase transition temperature of the liposome and significantly enhances the effect of temperature-controlled release.

[0085] Example 4: In vitro photothermal effect experiment

[0086] PBS, ICG solution (25 μg / mL) and AI-TSL (ICG = 25 μg / mL) were placed in 24-well plates to observe the photothermal effect in vitro. Each sample was heated at 1 W / cm 2 The samples were exposed to 808 nm laser for 5 minutes. Then a thermal imaging camera was used to take pictures every half a minute (see Fig.10 ) and record the temperature (see Fig.11 ).from Fig.10 and Fig.11 It can be seen that after 5 minutes of laser irradiation, the temperature of AI-TSL is 18°C ​​higher than that of PBS. This shows that AI-TSL has a good light response effect, which can achieve the effect of controlled release in tumor tissue and reduce the toxicity of drugs to healthy tissues.

[0087] Example 5: Cell live and dead staining experiment

[0088] The live-dead staining method was used to investigate the cytotoxicity and light-controlled release effect of the ansamitocin P-3 photoresponsive thermosensitive liposomes prepared in Example 1 on human breast cancer cells (MCF-7). 5 cells / mL) were inoculated in 6-well plates and cultured overnight, and then replaced with normal culture medium (control), free AP-3, AI-TSL or AI-TSL(+), where the concentration of AP-3 was the same, all 30μM. Then, the wells of the AI-TSL(+) group were irradiated with 808nm laser (1W / cm 2 , for 5 minutes). After culturing for 12 hours, the liquid in the 6-well plate was aspirated and the cells were gently washed with PBS. For qualitative analysis of cell apoptosis, staining with calcein-AM (2 μM) and PI (4.5 μM) was performed at 37°C for 30 minutes. Staining with DAPI (10 μg / mL) was performed at 37°C for 20 minutes. After washing with PBS, the cells were placed under a fluorescence microscope for observation.

[0089] Results Fig.12

[0090] exist Fig.12 In the figure, calcein-AM dyes live cells green, PI dyes dead cells red, and DAPI dyes live and dead cell nuclei blue. It can be inferred from the figure that AI-TSL shows good stability at body temperature, with less drug leakage and lower toxicity than free drugs within 12 hours. After laser irradiation, the photoresponse of ICG increases the internal temperature of the liposomes, increases the permeability of the liposome membrane, and makes it easy for the drug to be released through the membrane, further confirming the anti-tumor effect and photoresponse effect of AI-TSL.

[0091] Example 6: Cell morphology analysis

[0092] MCF-7 cells (1×10 5 cells / mL) were inoculated in a 6-well plate containing a cell slide and cultured overnight, and then replaced with normal culture medium (control), free AP-3, AI-TSL or AI-TSL(+), respectively, where the concentration of AP-3 was the same, all 30μM. The wells in the AI-TSL(+) group were then irradiated with 808nm laser (1W / cm2, for 5 minutes). After continuing to culture for 12 hours, the liquid in the 6-well plate was aspirated, the cells were gently washed 3 times with PBS, incubated with glutaraldehyde for 45 seconds, and then fixed with paraformaldehyde for 1 hour. After removing the solution, gently wash 5 times with PBS, place the cell slide on a slide, blow dry the surface with nitrogen, and observe the cell morphology of each group using an atomic force microscope (AFM). The results are shown in Fig.13 .

[0093] like Fig.13 As shown, the cell morphology of the untreated control group was intact, the cell membrane did not change, and the nucleus and flagella were clearly visible. After treatment with free AP-3, the peripheral cell membrane ruptured and the cell surface was uneven, indicating that the cytoskeleton was collapsing. After treatment with AI-TSL, the cells swelled and there was no obvious damage to the membrane, indicating that AI-TSL was taken up by the cells, but the drug was not released through the phospholipid layer. The cell morphology of the AI-TSL (+) group collapsed completely, indicating that after laser irradiation, ICG responded to release heat, AP-3 penetrated the phospholipid bilayer, inhibited the aggregation of tubulin, and caused the cytoskeleton to collapse. This further confirms the anti-tumor effect and photoresponsive effect of AI-TSL.

Claims

1. A method for preparing ansamitocin P-3 loaded light-responsive thermosensitive liposomes using microfluidic technology, characterized in that: The following steps are involved: (1) dissolving ansamitocin P-3, phospholipids, cholesterol, and PEGylated phospholipids in an organic solvent to obtain an organic phase; wherein the mass ratio of phospholipids to cholesterol is 1:1 to 9:1; The phospholipids are dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine; the PEGylated phospholipids are selected from DSPE-MPEG 2000 ; DPPC, DSPC, cholesterol and DSPE-MPEG 2000 The mass ratio is (4.5~5):(1~1.5):3:(0.4~1); (2) dissolving a photosensitizer in a buffer solution to obtain an aqueous phase; the photosensitizer is indocyanine green; (3) The flow rate ratio of the aqueous phase to the organic phase is 1:1 to 29:1, and the total flow rate is greater than 12 ml / min. The two-phase solutions are injected into the microfluidic chip from different channels for self-assembly to form liposomes; The organic solvent and free substances remaining in the liposomes are removed and sterilized to obtain ansamitocin P-3 thermosensitive liposomes.

2. The method according to claim 1, characterized in that The mass ratio of the phospholipid to cholesterol is 2:

1.

3. The method according to claim 1, characterized in that The organic solvent described in step (1) is selected from any one or more of methanol, ethanol, acetone, acetonitrile and ether; the buffer described in step (2) is any one of PBS buffer, HEPES buffer and TRIS buffer.

4. The method according to claim 1, characterized in that The total flow rate of the aqueous phase and the organic phase in step (3) is 40 ml / min to 100 ml / min; the flow rate ratio of the aqueous phase to the organic phase is 2:1 to 4:

1.

5. The method according to claim 1, characterized in that AP-3, photosensitizer and phospholipids + cholesterol + DSPE-MPEG in Ansamitocin P-3 thermosensitive liposomes 2000 The total lipid mass ratio is 1:1:20~1:1:25; PEGylated phospholipids account for 5%~10% of the total lipid mass.

6. The method according to claim 1, characterized in that The method for removing the residual organic solvent and free substances in the liposomes and sterilizing in step (3) is one or more of dialysis, low-speed centrifugation, ultrafiltration centrifugation and membrane permeation.

7. Use of ansamitocin P-3 loaded light-responsive thermosensitive liposomes prepared by the method according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.

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