Photo-thermal temperature-sensitive cell membrane drug delivery system capable of controlling drug release as well as preparation method and application of photo-thermal temperature-sensitive cell membrane drug delivery system

Abstract

The invention discloses a photo-thermal temperature-sensitive cell membrane drug delivery system capable of controlling drug release as well as a preparation method and application of the photo-thermal temperature-sensitive cell membrane drug delivery system. The delivery system comprises a cell membrane carrier, and temperature-sensitive lipid and a photosensitizer are doped in the cell membrane carrier. According to the scheme, the thermo-sensitive lipid and the photosensitizer are ingeniously introduced into a cell membrane delivery system at the same time, photo-thermal controllable drug depolymerization and release after target tissue arrives are achieved, then the drug is accurately delivered to heterotarget cells, and the treatment effects of disease tissue targeting and multi-cell regulation are achieved. The design obviously exceeds the limitation of a traditional cell membrane nano-drug delivery system, the traditional cell membrane nano-drug delivery system is only limited to targeting of a single cell type, and the action range is relatively limited.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a thermosensitive cell membrane drug delivery system with controllable photothermal drug release and a preparation method and application thereof. BACKGROUND

[0002] Traditional nanodrug delivery systems, mainly constructed by polymers, lipids and inorganic materials, have become the focus of extensive research due to their high drug loading capacity, targeted delivery and controllable drug release. However, such systems are often enriched in the liver, spleen and other non-target organs due to the capture of macrophages in the reticuloendothelial system, hindering the effective arrival of drugs at the lesion site. To solve this problem, in recent years, the introduction of cell-based carriers, especially nanodrug delivery systems based on cell membranes, has significantly improved the delivery efficiency through cell membrane camouflage, like the "Trojan horse" strategy. Such systems not only inherit the good biocompatibility and blood circulation stability of the source cells, effectively avoiding the recognition of the reticuloendothelial system and prolonging the in vivo circulation time, but also have tissue "homing" characteristics. The homologous cell targeting ability of the surface proteins of specific cell membranes ensures the precise delivery of drugs, reduces side effects and enhances therapeutic effects. Therefore, cell membrane nanodrug delivery systems have shown great application potential in anti-tumor, phototherapy, immunotherapy, inflammation control and bioimaging, among other fields. Among them, platelet membranes, tumor cell membranes, immune cell membranes and stem cell membranes are particularly noteworthy, and new modification methods such as extracellular vesicles and organelle membranes have also emerged. It is particularly worth mentioning that some immune cell membrane mimetic systems can also maintain their immune functions, such as M1 macrophage membranes that can polarize M2 macrophages, achieving the dual effects of anti-tumor and immune activation.

[0003] With the in-depth exploration of disease mechanisms, researchers have realized that the occurrence and development of various diseases involve complex interactions of multiple cell types and components, often requiring simultaneous administration of drugs targeting different cells or components to achieve synergistic therapeutic effects. For example, inflammation involves the combined action of local tissue cells, immune cells, blood vessels and various cytokines. The occurrence of tumors is even more complex, involving immune cells, stromal cells, regulatory factors and tumor extracellular matrix. Current cell membrane nanodrug delivery systems can achieve tissue-specific targeting through the modification of a single or several cell membranes, but after the drugs reach the target tissue, they are often taken up by a large number of the same cells or non-specifically taken up by multiple cells, which not only reduces drug efficiency and increases the risk of adverse reactions, but also cannot achieve precise drug delivery and regulation targeting different cells, especially in the treatment of diseases that require the simultaneous regulation of multiple cells.

[0004] In view of the above challenges, it is particularly urgent to develop a system that can precisely target disease sites and deliver drugs targeting different cell types. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes an improved cell membrane delivery system, which can realize light-controlled drug release and temperature-sensitive delivery, thereby realizing precise control of drug release time and location.

[0006] The present application also proposes a preparation method of the above delivery system.

[0007] The present application also proposes the application of the above delivery system.

[0008] According to the delivery system of the first aspect of the present application, the cell membrane carrier is doped with temperature-sensitive lipids and photosensitizers.

[0009] According to the delivery system of the present application, at least the following beneficial effects are achieved: the present application ingeniously introduces temperature-sensitive lipids and photosensitizers into the cell membrane delivery system, realizes light-thermal controllable drug depolymerization and release after reaching the target tissue, and further precisely delivers the drug to the heterocellular target, achieving the therapeutic effect of disease tissue targeting and multiple cell regulation. This design significantly surpasses the limitations of traditional cell membrane nanodrug delivery systems, which are often limited to single cell type targeting and have a relatively limited range of action. Specifically, by introducing temperature-sensitive lipids into the cell membrane nanocarrier, the system is given sensitivity to temperature changes. This property allows the carrier to respond at a specific temperature threshold, causing structural depolymerization and thus effective release of the loaded drug. In addition, by ingeniously doping photosensitizers, the system further acquires light-thermal controllability. Under external laser irradiation, the photosensitizer can absorb light energy and convert it into heat energy, causing rapid local temperature changes. This light-heat conversion mechanism provides precise temporal and spatial control for drug release, making the delivery process more flexible and efficient.

[0010] In summary, the delivery system of the present application not only overcomes the limitation of traditional methods that can only target a single cell type, but also realizes dual control of the drug release process by introducing temperature-sensitive lipids and photosensitizers - temperature sensitivity and light-thermal controllability. This innovative design greatly broadens the scope of drug delivery, improves the precision and effectiveness of treatment, and provides new strategies and possibilities for the treatment of complex diseases.

[0011] According to some embodiments of the present application, the photosensitizer includes at least one of indocyanine green, hematoporphyrin and its derivatives, porphyrin compounds, phthalocyanine compounds, fused ring quinone compounds, and third-generation new photosensitizers (such as metal compounds, etc.). It can also be other newer photosensitizers.

[0012] According to some embodiments of the present application, the temperature-sensitive lipid comprises at least one of dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), and distearoyl phosphatidylglycerol (DSPG).

[0013] According to some embodiments of the present application, the concentration of the temperature-sensitive lipid in the delivery system is 5-15 mg / mL.

[0014] According to some embodiments of the present application, the mass ratio of the temperature-sensitive lipid to the non-temperature-sensitive lipid is 8:1. The controllable temperature-sensitive change can be achieved by adjusting the ratio between the temperature-sensitive lipid and the non-temperature-sensitive lipid.

[0015] According to some embodiments of the present application, the cell membrane comprises at least one of a tumor cell membrane, a stem cell membrane, a blood cell membrane, an immune cell membrane (such as a macrophage membrane, a neutrophil membrane, etc.), a stromal cell membrane (such as a fibroblast membrane, a muscle cell membrane, etc.), a cell vesicle or an exosome, a bacterial outer membrane or a bacterial vesicle, and a cell organelle membrane (such as a mitochondrial membrane, etc.).

[0016] According to some embodiments of the present application, the light-controlled depolymerization critical temperature of the delivery system is 20-70°C. The light-controlled depolymerization critical temperature of the delivery system can be adjusted by the phase transition temperature of the one or more temperature-sensitive lipids and the ratio thereof, the concentration of the photosensitizer, the light intensity, and the light time, which are flexible and variable.

[0017] According to some embodiments of the present application, the average hydration particle size of the delivery system is 100-300 nm.

[0018] According to some embodiments of the present application, the preparation method of the delivery system comprises the following steps: preparing the delivery system by a film dispersion-hydration method.

[0019] According to some embodiments of the present application, the raw materials for preparing the delivery system comprise a cell membrane, a temperature-sensitive lipid, cholesterol, and a photosensitizer; and the preparation method comprises the following steps:

[0020] The photosensitizer, the temperature-sensitive lipid, the cholesterol, and an organic solvent I are prepared into a mixed solution, the organic solvent I is removed, and a lipid membrane is obtained; a cell membrane solution is added to hydrate the lipid membrane, and the delivery system is obtained.

[0021] According to some embodiments of the present application, the organic solvent I comprises an alcohol solvent and chloroform, and the preparation process of the mixed solution comprises the following steps:

[0022] The photosensitizer is mixed with the alcohol solvent to prepare a solution I;

[0023] Mixing the chloroform solution containing the temperature-sensitive lipid and cholesterol with the solution I to obtain the mixed solution.

[0024] According to some embodiments of the present application, the alcohol solvent includes at least one of methanol or ethanol.

[0025] According to some embodiments of the present application, the concentration of the photosensitizer in the solution I is 0.1-1 mg / mL.

[0026] According to some embodiments of the present application, the concentration of the photosensitizer in the solution I is 0.5 mg / mL.

[0027] According to some embodiments of the present application, the mass ratio of the temperature-sensitive lipid to cholesterol is 6:1-10:1.

[0028] According to the application of the delivery system according to the third aspect of the present application, a drug delivery system includes a drug and the delivery system, and the drug is loaded in the delivery system.

[0029] According to some embodiments of the present application, the delivery system is applied in drug delivery.

[0030] According to some embodiments of the present application, the drug includes at least one of a small molecule drug, a gene complex or a nano drug; and the nano drug includes at least one of a polymer nanoparticle, a lipid carrier or an inorganic nanoparticle.

[0031] According to some embodiments of the present application, the drug includes at least one of a porphyrin or a PEI / siRNA gene complex.

[0032] According to some embodiments of the present application, the porphyrin is loaded in the delivery system in the form of a porphyrin-lipid nanodisc. The porphyrin is a lipid-soluble drug, and loading it in the nanodisc can make it better loaded in the delivery system, and improve its biocompatibility and targeting. According to some embodiments of the present application, the preparation process of the porphyrin-lipid nanodisc includes the following steps:

[0033] Dissolving a phospholipid in an organic solvent II, mixing the alcohol solution containing the porphyrin to obtain a mixed solution, removing the organic solvent II in the mixed solution, and hydrating with water to obtain the porphyrin-lipid nanodisc; wherein the phospholipid includes a long-chain lipid and a short-chain lipid.

[0034] According to some embodiments of the present application, the long-chain lipid includes at least one of dipalmitoyl phosphatidylcholine (DPPC) and distearoyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG).

[0035] According to some embodiments of the present application, the short-chain lipid comprises 1,2-dihexanoylphosphatidylcholine (DHPC).

[0036] According to some embodiments of the present application, the organic solvent II comprises at least one of chloroform, methanol or ethanol.

[0037] According to some embodiments of the present application, the preparation process of the drug delivery system comprises the following steps: mixing the drug to be loaded with the delivery system, extruding by ultrasound, high pressure homogenization or lipid extruder to obtain the drug delivery system.

[0038] Additional aspects and advantages of the present application will be partially given in the following description, partially will become obvious from the following description, or will be understood by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a particle size distribution graph of the delivery system prepared in Example 1 of the present application;

[0040] Figure 2 is a potential graph of the delivery system prepared in Example 1 of the present application;

[0041] Figure 3 is a TEM graph of the delivery system prepared in Example 1 of the present application;

[0042] Figure 4 is a SDS-PAGE electrophoresis strip of the delivery system prepared in Example 1 of the present application (in the figure, 1 is M1 macrophage, 2 is cell membrane extracted from M1 macrophage, 3 is protein sample extracted from the delivery system solution prepared in Example 1 of the present application);

[0043] Figure 5 is a flow graph of M1 macrophage and M2 macrophage after the drug delivery system prepared in Example 1 of the present application is co-incubated with M2 macrophage (in the figure, T-Lip is the drug delivery system without adding M1 macrophage membrane, TM is the drug delivery system prepared in Example 1, TM+L is the drug delivery system treated by 808 nm laser);

[0044] Figure 6 is a flow quantitative graph of M1 macrophage after the drug delivery system prepared in Example 1 of the present application is co-incubated with M2 macrophage (in the figure, T-Lip is the drug delivery system without adding M1 macrophage membrane, TM is the drug delivery system prepared in Example 1, TM+L is the drug delivery system treated by 808 nm laser);

[0045] Figure 7is a flow cytometry quantification chart of M2 macrophages after the drug delivery system prepared in Example 1 of the present application is co-incubated with the M2 macrophages (in the figure, T-Lip is the drug delivery system without adding the membrane of M1 macrophages, TM is the drug delivery system prepared in Example 1, TM+L is the drug delivery system treated by 808 nm laser);

[0046] Figure 8 is a temperature-time change curve of the drug delivery system prepared in Example 1 of the present application under 808 nm laser treatment;

[0047] Figure 9 is a particle size distribution chart of the drug delivery system prepared in Example 2 of the present application (in the figure, PEI / siRNA represents a PEI / siRNA gene complex, and TM@PEI / siRNA represents the drug delivery system of PEI / siRNA);

[0048] Figure 10 is a potential chart of the drug delivery system prepared in Example 2 of the present application (in the figure, PEI / siRNA represents a PEI / siRNA gene complex, and TM@PEI / siRNA represents the drug delivery system of PEI / siRNA);

[0049] Figure 11 is a particle size distribution chart of the drug delivery system prepared in Example 3 of the present application (in the figure, ErN represents a malarin-lipid nanodisc, and TM@ErN represents the drug delivery system carrying ErN);

[0050] Figure 12 is a potential chart of the drug delivery system prepared in Example 3 of the present application (in the figure, ErN represents a malarin-lipid nanodisc, and TM@ErN represents the drug delivery system carrying ErN).

[0051] Figure 13 is a particle size chart of the drug delivery system prepared in Example 4 of the present application after 808 nm laser treatment;

[0052] Figure 14 is a potential chart of the drug delivery system prepared in Example 4 of the present application after 808 nm laser treatment;

[0053] Figure 15 is a TEM chart of the drug delivery system prepared in Example 4 of the present application after 808 nm laser treatment;

[0054] Figure 16The immunofluorescence section pictures of the tumor of the drug delivery system prepared in Example 4 of the present application injected into tumor-bearing mice through the tail vein for 8 hours and after 8 hours of laser treatment at 808 nm, the scale length is 100 μm (in the figure, DAPI marks the cell nucleus, CD31, α-SMA marks the blood vessels, Cy5-N represents Cy5-lipid nanodisc, and PEI / FAM-siN represents PEI / FAM-siN gene complex). DETAILED DESCRIPTION

[0055] The concept and technical effects of the present application will be described below in combination with examples, so as to fully understand the purpose, features and effects of the present application. Obviously, the described examples are only some of the examples of the present application, but not all the examples, and other examples obtained by those skilled in the art based on the examples of the present application without creative labor are within the protection scope of the present application. The test methods used in the examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified. The same parameters are used in the same way in each example unless otherwise specified. The examples described below are exemplary and are used to explain the present application, and cannot be understood as limiting the present application.

[0056] In the description of the present application, the description of the terms "some embodiments" and the like means that the specific features, structures, materials or characteristics described in combination with the embodiments or examples are contained in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0057] In the description of the present application, if the first, second, etc. are described, it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features or the sequence of indicated technical features.

[0058] Example 1

[0059] This example provides a delivery system, which is prepared into a cell membrane carrier by using a macrophage membrane, and the cell membrane carrier is doped with a temperature-sensitive lipid DPPC and a photosensitizer indocyanine green (ICG). The delivery system can release the drug under the control of temperature and photothermal at 43°C. The preparation process is as follows:

[0060] 1. Extraction of macrophage membrane

[0061] RAW 264.7 macrophage cells (Shanghai Cell Bank of Chinese Academy of Sciences) were cultured at 37°C in 5% CO2. Before collection, the cells were induced to differentiate for 24 h by adding LPS (lipopolysaccharide, 100 ng / mL) to the culture medium. The induced M1 macrophage cells were collected using a cell scraper and centrifuged at 1000 rpm for 5 min to remove the supernatant, leaving the cell pellet for use. When extracting the membranes of other types of cells, a digestion solution containing EDTA but not trypsin was used, and trypsin digestion was avoided as much as possible to prevent damage to the membrane proteins.

[0062] According to the instructions of the Cell Membrane Protein and Cytosol Protein Extraction Kit (Bi Yun Tian, P0033), cell membrane extraction solution containing 1 mM of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added to the cell pellet. The cells were gently blown and fully suspended, incubated on ice for 10-15 min, and then repeatedly frozen and thawed 3-5 times under liquid nitrogen and room temperature. The frozen and thawed solution was observed under a microscope to check the cell lysis. If 70-80% of the cells had no perinuclear halo and intact cell morphology, it indicated that the cells had been fully broken.

[0063] The frozen and thawed solution was centrifuged at 4°C and 700 g for 10 min to remove the snot-like viscous solid (nuclei and unbroken cells) in the solution, and the supernatant was carefully collected into a new centrifuge tube. The supernatant was centrifuged at 4°C and 14000 g for 30 min to obtain the precipitated cell membrane fragments. PBS (pH 7.4) was added to fully resuspend the membrane fragments, and the membrane proteins were quantified using the BCA Protein Concentration Assay Kit (Bi Yun Tian, P0010). The extracted membrane protein solution was stored in a -80°C refrigerator.

[0064] 2. Preparation of the delivery system

[0065] ICG solids were weighed and dissolved in anhydrous ethanol to prepare an ICG stock solution at 0.5 mg / mL. 20.0 mg of DPPC and 2.5 mg of cholesterol were weighed into a 20 mL pear-shaped flask, 4 mL of chloroform and 1 mL of the ICG stock solution were added, and the mixture was ultrasonically dissolved in a water bath for more than 10 min to fully dissolve and mix.

[0066] The delivery system was prepared by the thin film dispersion-hydration method: the organic solvent was removed by rotary evaporation at about 42°C to form a uniform lipid film on the wall of the flask; 2 mL of cell membrane solution (about 1 mg of cell membrane) was added for hydration, and the lipid film was fully hydrated by rotating and oscillating at 45°C to obtain a solution containing the delivery system.

[0067] The delivery system prepared by the above operation was characterized as follows:

[0068] 1) Particle size and potential determination

[0069] The solution containing the delivery system prepared in the above operation was diluted 20 times with ultrapure water, and particle size and potential were determined using a Marven particle size analyzer. The results are shown in Figure 1 and 2 As can be seen from the figure, the particle size of the system is uniform, about 162.20±0.54 nm; the Zeta potential is negative, about -24.89±1.26 mV.

[0070] 2) Morphological characterization

[0071] The solution containing the delivery system prepared in the above operation was diluted 10 times with ultrapure water, and copper mesh was added and soaked for 5 min, then the copper mesh was taken out and soaked in 2 wt% phosphotungstic acid for 30 s, and then dried and photographed using a 120 kV transmission electron microscope. The results are shown in Figure 3 As can be seen from the figure, the system presents a spherical nanoparticle morphology with uniform particle size.

[0072] 3) Macrophage membrane protein characterization

[0073] The solution containing the delivery system, the macrophage precipitate collected by step 1, and the membrane protein solution extracted by step 1 were centrifuged. 10 times the volume of cell lysis solution containing 1 mM PMSF was added to the three different precipitates, and lysis was performed on ice for 30 min, with shaking every 10 min to ensure complete lysis. Centrifugation was performed at 4°C, 12000 rpm for 10-15 min, and the supernatant was used to measure the protein concentration by BCA method. After determining the concentration, 5x SDS solution was added, and protein denaturation was performed in a special metal bath at 99°C for 7 min. 10 wt% separating gel and 5 wt% concentrated gel were prepared, and after the instrument was assembled, 20 μg of denatured protein was added to each well, and electrophoresis was performed according to the steps of 75 V, 30 min and 150 V, 60 min. When the blue dye reached the bottom of the gel, the electrophoresis was stopped. The gel was removed and stained with Coomassie brilliant blue, washed and developed for observation. The results are shown in Figure 4 As can be seen from the figure, the system and the macrophage have similar protein bands, indicating that the M1 type macrophage membrane surface related proteins in the system are still retained, and do not affect the biological functions of tumor tissue targeting and macrophage polarization.

[0074] 4) Verification of the biological function of macrophage membrane

[0075] To verify the biological function of macrophage membrane inducing M2 type macrophages to repolarize into M1 type macrophages, bone marrow-derived macrophages (BMDM) were first extracted from BALB / c mice. After cervical dislocation of healthy mice, the body surface was disinfected with 75% ethanol, and then the hind limb bones were aseptically isolated in a clean bench. The femur and tibia were completely removed after the muscle and ligament tissues were completely removed, and then the bone surface was disinfected with 75% ethanol and stored in DMEM medium containing double antibiotics (penicillin 100 U / mL, streptomycin 100 μg / mL). When the bone marrow was extracted, the bone marrow cavity was repeatedly flushed 3 times by drilling holes at both ends of the bone with pre-cooled DMEM medium. The collected cell suspension was centrifuged at 1500 rpm for 5 minutes, and then lysed with 3 times the volume of red blood cell lysis solution for 2 minutes. The finally obtained bone marrow cells were washed with PBS, and the density was adjusted to 1×10 5 cells / well with complete DMEM medium containing 20 ng / mL M-CSF, and then inoculated in a 6-well plate in a 37°C, 5% CO2 incubator for primary culture. Half of the medium was changed on the 2nd, 4th, and 6th days, and after the cells were stable, the culture medium was changed to contain 40 ng / mL IL-4 to continue culturing the cells for 24 hours, i.e. to induce mouse M2 type BMDM.

[0076] The culture medium of M2 type BMDM was changed to complete medium containing different components (PBS, T-Lipo, TM, TM+L), wherein T-Lip is a drug delivery system without adding M1 type macrophage membrane, TM is a drug delivery system with added M1 type macrophage membrane, and TM+L is TM treated with 808 nm laser. After continuing to culture for 24 hours, the cells were collected after digestion, centrifuged at 500g for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in flow cytometry staining buffer. CD16 / 32 antibody was added at 4°C for 10 minutes, and then antibody was added for labeling (PE-labeled anti-CD11b antibody, FITC-labeled anti-CD86 antibody), and incubated at 4°C for 1 hour in the dark. After washing twice with PBS, the cells were broken and fixed by adding BD Cytofix / Cytofix, and then CD16 / 32 antibody prepared with BD Perm / wash TM buffer was added for blocking at 4°C for 10 minutes, and finally APC-labeled anti-CD206 antibody prepared with BD Perm / wash TM buffer was added, and incubated at 4°C for 1 hour in the dark. After washing twice with BD Perm / wash TM buffer, the cells were resuspended with BD Perm / wash TM buffer.

[0077] The flow cytometry was used to detect the re-polarization of M2 type BMDM cells to M1 type macrophages. The results are shown in Figs. 6, 7 and 8. Figure 5 、 6 As shown in Figs. 6, 7 and 8, the T-Lip group had no significant effect on the content of M2 type macrophages and could not induce the re-polarization of M2 type macrophages. The TM group could significantly induce the re-polarization of M2 type macrophages to M1 type macrophages, proving that the M1 type macrophage membrane in the drug delivery system could still exert its biological function. In addition, compared with the TM group, the TM+L group further enhanced the induction effect of the re-polarization of M2 type macrophages, indicating that after laser treatment, the drug delivery system could respond to laser to depolymerize and generate more M1 type macrophage membrane fragments, ultimately amplifying the effect of inducing the re-polarization of M2 type macrophages.

[0078] 5) Photothermal controllable temperature-sensitive characterization

[0079] The prepared solution was taken into a sharp-bottom centrifuge tube, and the tube bottom was irradiated with an 808 nm laser to cover the solution with laser. A thermal infrared imager was used for temperature monitoring, and the solution temperature was maintained at a mild photothermal temperature (above 43℃, 43-47℃) for at least 5 min. The temperature monitoring results are shown in Fig. 3. As shown in the figure, by controlling the laser treatment time and range, the solution temperature can be stably maintained in the mild temperature range of 37.0-45.0℃, which can not only ensure the stability of the loaded temperature-sensitive drugs, but also cause the increase of the fluidity of the temperature-sensitive lipids and the explosion of the system to expose the loaded drugs. Figure 8

[0080] Example 2

[0081] This example provides an application of a photothermal controllable drug release temperature-sensitive cell membrane drug delivery system in delivering PEI / siRNA gene complexes.

[0082] Specifically, a drug delivery system is provided, which is composed of a delivery system loaded with PEI / siRNA gene complexes, wherein the preparation process of the delivery system is the same as that of Example 1. The preparation process of the drug delivery system is specifically as follows:

[0083] 2.4 μg of siRNA powder (the sequence is shown in Seq No. 1, specifically 5'-CTATAAAGCTTCCAAAGGA-3' (sense)) was taken and dissolved in 200 μL of sterile enzyme-free water; similarly, 8.6 μg of PEI complex (molecular weight of ~25000 kDa, BCBZ4957, Guangzhou Ribo Bio-technology Co., Ltd.) was taken and dissolved in 200 μL of sterile enzyme-free water. Under vortex conditions, the siRNA solution was slowly dropped into the PEI solution, and incubated at room temperature for more than 30 min in the dark to obtain PEI / siRNA gene complexes. ​

[0084] The solution containing the delivery system prepared by the same operation as in Example 1 was mixed with 200 μL of the PEI / siRNA gene complex solution, and extruded using a lipid extruder and a 200 nm filter membrane, and the extrusion was repeated 10 times to obtain a solution of the delivery system loaded with the PEI / siRNA gene complex.

[0085] The particle size and potential were determined after the solution was diluted 10 times. The results are shown in Figure 9 and 10 As can be seen from the figure, the particle size of the delivery system after co-extrusion increased from 98.57 ± 1.47 nm to 146.13 ± 1.76 nm, and the potential changed from 43.69 ± 2.67 mV of the PEI / siRNA gene complex to -28.64 ± 0.18 mV. The increase in particle size and the reversal of potential indicate that the system can successfully load the PEI / siRNA gene complex.

[0086] Example 3

[0087] This example provides an application of a thermosensitive cell membrane drug delivery system with photo-thermal controlled drug release in delivering malarin-lipid nanodiscs.

[0088] Specifically, a delivery system is provided, which is composed of a delivery system loaded with malarin-lipid nanodiscs, wherein the preparation process of the delivery system is the same as in Example 1. The preparation process of the delivery system is as follows:

[0089] 38.4 mg of DPPC, 14.8 mg of DSPE-PEG, and 7.8 mg of DHPC were weighed, and 4 mL of chloroform and 1 mL of an ethanol solution of malarin were added, and ultrasonic treatment was performed in a water bath for more than 10 min to fully dissolve them. A thin film dispersion-hydration method was used to prepare: the organic solvent was removed by rotary evaporation at about 42°C, and a uniform lipid film was formed on the wall of the flask; 1 mL of ultrapure water was added for hydration, and the lipid film was fully hydrated by rotary oscillation at 45°C to obtain a solution after hydration, which was subjected to ultrasonic treatment to obtain a malarin-lipid nanodisc solution.

[0090] The solution containing the delivery system prepared by the same operation as in Example 1 was mixed with 200 μL of the PEI / siRNA gene complex solution, and extruded using a lipid extruder and a 200 nm filter membrane, and the extrusion was repeated 10 times to obtain a solution of the delivery system loaded with the PEI / siRNA gene complex.

[0091] The particle size and potential were determined after the solution was diluted 10 times. The results are shown in Figure 11 and 12As shown in FIG. 1 and FIG. 2, after co-extrusion, the particle size of the drug delivery system increased from 15.07 ± 7.24 nm to 127.06 ± 1.23 nm, and the electric potential slightly decreased from -25.91 ± 1.22 mV to -23.85 ± 0.61 mV, indicating that the system successfully loaded the heparin-lipid nanodisks.

[0092] Example 4

[0093] This example provides an application of the thermosensitive cell membrane drug delivery system of photothermal controlled drug release in simultaneously delivering PEI / siRNA gene complexes and heparin-lipid nanodisks.

[0094] Specifically, this example provides a drug delivery system composed of PEI / siRNA gene complexes and heparin-lipid nanodisks, wherein the preparation process of the drug delivery system is the same as that of Example 1, the preparation process of the PEI / siRNA gene complexes is the same as that of Example 2, and the preparation process of the heparin-lipid nanodisks is the same as that of Example 3. The preparation process of the drug delivery system is as follows:

[0095] Take 1 mL of the solution containing the drug delivery system prepared in the same manner as in Example 1 and 200 μL of the solution containing the PEI / siRNA gene complexes prepared in the same manner as in Example 2, and 200 μL of the solution containing the heparin-lipid nanodisks prepared in the same manner as in Example 3, and mix them. Use a lipid extruder and a 200 nm filter membrane to perform extrusion, and repeat the extrusion for 10 times, to obtain a solution of the thermosensitive cell membrane drug delivery system simultaneously loaded with PEI / siRNA gene complexes and heparin-lipid nanodisks.

[0096] Characterize the drug delivery system prepared in the above operation:

[0097] 1) In vitro photothermal controlled drug release characterization

[0098] Take the prepared solution containing the drug delivery system, and perform laser treatment according to the operation of Example 1. Dilute the solution after irradiation with ultrapure water by 20 times, use a Marven particle size analyzer to determine the particle size and electric potential, and the results are shown in FIG. 3 and FIG. 4. Figure 13 and 14 As can be seen from the figures, after laser treatment, the particle size graph shows multiple particle size peaks, and the negative potential also slightly decreases, indicating that the system has exploded after laser treatment and exposed the contents loaded therein.

[0099] Take the solution after irradiation treatment, dilute it with ultrapure water by 10 times, add copper mesh and soak for 5 min, then take out the copper mesh and soak it in 2 wt% phosphotungstic acid for 30 s, and then dry it thoroughly and take a photo using a 120 kV transmission electron microscope. The results are shown in FIG. 5 and FIG. 6. Figure 15The results are shown in FIG. 6. As can be seen from the figure, after laser treatment, a variety of nanoparticles with different particle sizes and morphologies appeared in the system, indicating that the system exploded after laser treatment and exposed the contents loaded therein.

[0100] 2) In vivo multi-target cell drug delivery characterization

[0101] 4-6 week old female BALB / c mice were purchased from the Guangdong Medical Laboratory Animal Center (Foshan, China). The experiments were strictly in accordance with the relevant provisions of the guidelines of the Sun Yat-sen University Animal Care and Use Committee.

[0102] 4T1 cells grown to the logarithmic growth phase were collected, added to 50% Matrigel matrix gel to prepare a cell suspension, and injected subcutaneously into the left abdominal region of each mouse at a concentration of 1 x 10 7 After the unilateral 4T1 tumor model was established, the mice were continued to be fed and observed for tumor growth, and after the tumor volume reached ~ 100 mm 3 After the unilateral 4T1 tumor model was established, the mice were continued to be fed and observed for tumor growth, and after the tumor volume reached ~ 100 mm 2 The tumor volume (V) was calculated according to the formula V = L x W

[0103] The PEI / FAM-siN gene complex was prepared according to the operation of Example 2 using FAM-siN instead of siRNA, the Cy5-lipid nanodisc was prepared according to the operation of Example 3 using Cy5 instead of malachite green, and the drug delivery system simultaneously loaded with the PEI / FAM-siN gene complex and the Cy5-lipid nanodisc was prepared according to the operation of Example 1. After the tumor-bearing mice were injected via the tail vein (FAM-siN: 1 mg / kg, Cy5: 0.5 mg / kg), the mice were sacrificed at 8 hours after administration, and the tumors were collected. A parallel group was set up, and after administration in the same way for 8 hours, the tumor site was irradiated with an 808 nm laser for 5 cycles, and then the mice were sacrificed and the tumors were collected. The tumors treated in different ways were washed with PBS and then frozen sectioned, and then the cell nuclei were stained with DAPI, the blood vessels in the tumors were stained with Alexa 594-labeled anti-CD31 antibody and IF 546-labeled anti-a-SMA antibody. The diffusion of the drug delivery system in the tumor tissue after laser irradiation treatment was observed by two-photon confocal microscopy, and the results are shown in FIG. 7. Figure 16It can be seen from the figure that the fluorescence of FAM-siN and Cy5 mostly coincides after 8 hours of injection, and also basically coincides with the fluorescence of the antibody labeled blood vessels, indicating that both PEI / FAM-siN gene complex and Cy5-lipid nanodisc are loaded in the drug delivery system, and mainly exist around blood vessels, and the traditional cell membrane carrier cannot target the delivery of different nanoparticles due to the lack of photothermal depolymerization effect; in the group treated by laser after 8 hours of injection, the fluorescence of FAM-siN and Cy5 only partially overlaps, and is mostly dispersed in different cells and enters the tumor tissue from the blood vessels, indicating that the drug delivery system can depolymerize and expose the different nanoparticles loaded therein after laser treatment, and can achieve precise drug delivery and regulation for different cells, and has great application prospects in the treatment of diseases requiring the synchronous regulation of multiple cells.

[0104] The above has described the embodiments of the present application in detail, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application.

Claims

1. A delivery system, characterized in that: The invention comprises a cell membrane carrier doped with a temperature-sensitive lipid and a photosensitizer.

2. The delivery system according to claim 1, wherein: The photosensitizer includes at least one of indocyanine green, hematoporphyrin and its derivatives, porphyrin compounds, phthalocyanine compounds, condensed ring quinone compounds and third generation novel photosensitizers.

3. The delivery system according to claim 1, wherein: The thermosensitive lipid comprises at least one of dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine or distearoylphosphatidylglycerol; and / or, in the delivery system, the concentration of the thermosensitive lipid is 5-15 mg / mL.

4. The delivery system according to claim 1, wherein: The cell membrane includes at least one of a tumor cell membrane, a stem cell membrane, a blood cell membrane, an immune cell membrane, a matrix cell membrane, a cell vesicle or an exosome, a bacterial outer membrane or a bacterial vesicle, and an organelle membrane.

5. The delivery system according to any one of claims 1 to 4, characterized in that: The photo-controlled depolymerization critical temperature of the delivery system is 20-70° C.; and / or the average hydrated particle size of the delivery system is 100-300 nm.

6. A method for preparing the delivery system according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: preparing the delivery system by a thin film dispersion-hydration method.

7. The method for preparing the delivery system according to claim 6, wherein: The raw materials for preparing the delivery system include cell membrane, temperature-sensitive lipid, cholesterol and photosensitizer; the preparation method includes the following steps: A photosensitizer, a temperature-sensitive lipid, cholesterol and an organic solvent are prepared into a mixed solution, and the organic solvent is removed to obtain a lipid membrane; a cell membrane solution is added to hydrate the lipid membrane to obtain the delivery system.

8. The method for preparing the delivery system according to claim 7, wherein: The concentration range of the photosensitizer is 0.1-1 mg / mL, and the mass ratio of the temperature-sensitive lipid to cholesterol is 6:1-10:

1.

9. A drug delivery system, characterized in that: The method comprises a drug and the delivery system according to any one of claims 1 to 5, wherein the drug is loaded in the delivery system.

10. The drug delivery system according to claim 9, wherein: The drug includes at least one of small molecule drugs, gene complexes or nano drugs; the nano drugs include at least one of polymer nanoparticles, lipid carriers or inorganic nanoparticles.

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