A TAT peptide-modified and membrane-coated lipid nanoparticle targeting pancreatic cancer and its preparation method and application

Abstract

The present invention relates to a TAT peptide-modified and CAF cell membrane-coated targeted lipid nanoparticle, which comprises phosphatidylcholine, cholesterol and DSPE-PEG. 2000 ‑TAT and drugs are prepared into TAT peptide-modified drug-loaded liposomes, and CAF cell membranes are coated on the surface of TAT peptide-modified drug-loaded liposomes to form TAT peptide-modified and CAF cell membrane-coated targeted lipid nanoparticles. The present invention coats the cell membrane of CAF cells on the surface of lipid nanoparticles, and at the same time modifies TAT ​​peptides to increase the number and aggregation of lipid nanoparticles at the cancer lesion site, thereby improving the efficacy.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutics, and in particular to a TAT peptide-modified and CAF cell membrane-coated targeted lipid nanoparticle, and a preparation method and application thereof. Background Art

[0002] Pancreatic cancer is a devastating malignancy characterized by high mortality, which is mainly attributed to the daunting challenges in early diagnosis. Despite the widespread use of surgical resection, chemotherapy and radiotherapy for treatment, the long-term prognosis of pancreatic cancer patients remains dismal. The rich extracellular matrix of pancreatic cancer changes the physical properties of the developing pancreatic tumor, leading to increased hardness and hydrostatic pressure, and forms a barrier to drug absorption, limiting clinical efficacy. Conventional nano-drug delivery systems that encapsulate free drugs can passively diffuse into pancreatic cancer tumor tissues through enhanced penetration and entrapment effects, thereby improving anti-tumor efficacy. Although nano-formulations can improve patient survival, the clinical treatment effect is not as optimistic as the results of preclinical experiments. Although TAT peptide-modified lipid nanoparticles can be used on the basis of conventional drug delivery systems to enhance penetration in pancreatic cancer tumors, their pancreatic cancer targeting ability is still unsatisfactory. The unsatisfactory targeting effect not only leads to drug accumulation in non-targeted locations, but also increases the risk of toxic side effects. Summary of the invention

[0003] The first object of the present invention is to provide a TAT peptide and tumor-associated fibroblast (CAF) cell membrane co-modified drug-loaded liposome that can actively target pancreatic cancer, improve the uptake of anti-tumor drugs by pancreatic cancer cells and increase the aggregation of drugs at the tumor site.

[0004] To achieve the above purpose, the technical solution provided by the present invention is:

[0005] A targeted lipid nanoparticle, characterized in that: CAF cell membrane extracted from CAF cells is coated on the surface of TAT peptide-modified drug-loaded liposomes to form TAT peptide-modified and CAF cell membrane-coated targeted lipid nanoparticles.

[0006] In some embodiments, the targeted lipid nanoparticles are composed of phosphatidylcholine, cholesterol and DSPE-PEG 2000 -TAT and drugs are prepared into TAT peptide-modified drug-loaded liposomes, and CAF cell membranes extracted from CAF cells are coated on the surface of TAT peptide-modified drug-loaded liposomes to form TAT peptide-modified and membrane-coated targeted lipid nanoparticles.

[0007] In some embodiments, the mass ratio of the CAF cell membrane extracted from the CAF cells and the TAT peptide-modified drug-loaded liposomes is 1:8-15 CAF cell membrane protein / phosphatidylcholine.

[0008] In some embodiments, the lipid nanoparticles contain phosphatidylcholine, cholesterol, and DSPE-PEG. 2000 -The mass ratio of TAT is 90-110:30-37:1.

[0009] In some embodiments, the drug is an anti-tumor drug.

[0010] A second object of the present invention is to provide a method for preparing the targeted lipid nanoparticles, comprising the following steps:

[0011] Step (1): Dissolve phosphatidylcholine in an appropriate amount of solvent, add cholesterol, DSPE-PEG 2000 -TAT and the drug are ultrasonically mixed in an ice bath to obtain a mixed solution; the mixed solution is injected into a PBS buffer (phosphate buffer) at a certain speed, and stirred while injecting, and continued to stir after injection until a liposome administration micelle solution is formed; the liposome administration micelle solution is dialyzed in a dialysis bag to obtain TAT peptide-modified drug-loaded liposomes;

[0012] Step (2): CAF cell membrane extracted from CAF cells and TAT peptide-modified drug-loaded liposomes are mixed at a CAF cell membrane protein / phosphatidylcholine mass ratio of 1:8-15, and extruded several times using a liposome extruder to obtain TAT peptide-modified and membrane-coated targeted lipid nanoparticles.

[0013] In some embodiments, in step (1), the mixed solution is injected into the PBS buffer at a speed of 0.4-0.6 ml / min, while magnetic stirring is performed at 800-1200 rpm.

[0014] In some embodiments, in step (2), the CAF cell membrane is extracted using a cell membrane extraction kit.

[0015] The third object of the present invention is to provide targeted lipid nanoparticles obtained by the above preparation method.

[0016] The fourth object of the present invention is to provide the use of the above-mentioned targeted lipid nanoparticles in the preparation of drugs for treating targeted tumors.

[0017] In some embodiments, the tumor is a pancreatic cancer tumor.

[0018] In some embodiments, the targeted lipid nanoparticles, relative to ordinary drug-loaded liposomes, in the in vitro uptake capacity evaluation experiment described in the present invention: the fluorescence intensity of coumarin 6 detected by flow cytometry after incubation for 6 hours increased by 50-70%; in the in vivo targeting capacity evaluation experiment described in the present invention: the DIR fluorescence intensity detected by a live animal imaging system in tumor tissue 24 hours after tail vein injection increased by 110-130%.

[0019] The targeted lipid nanoparticles can improve the uptake of drugs by tumor cells and increase the aggregation of drugs at the tumor site.

[0020] The targeted lipid nanoparticles of the present invention have specific targeting ability to pancreatic cancer.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention innovatively uses TAT ​​peptide and CAF cell membrane modified lipid nanoparticles at the same time, solving the difficult clinical problem in the prior art that lipid nanoparticles cannot aggregate more at pancreatic cancer lesions, greatly improving the efficacy and reducing toxic side effects.

[0023] At the same time, the immune response of liposomes to biological systems can be reduced, preventing them from being detected and cleared by the immune system in the blood, effectively introducing various nanoparticles into cells without affecting normal cell structure and function.

[0024] The present invention co-modifies the cell membrane of TAT peptide and CAF, and the two can synergistically significantly improve the uptake of anti-tumor drugs by pancreatic cancer cells and increase the aggregation of drugs in the tumor site, significantly improving the active targeting ability of liposomes to pancreatic tumor tissue. After encapsulating anti-tumor drugs by physical encapsulation, the clinical efficacy of tumor patients can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Schematic diagram of the structural composition of the targeted lipid nanoparticles of the present invention; wherein A is phosphatidylcholine, B is cholesterol, and C is DSPE-PEG 2000 -TAT, D is the drug, E is the drug-loaded liposome modified with TAT peptide, F is CAF cells, G is the cell membrane extracted from CAF cells, H is the drug-loaded liposome co-modified with synthetic TAT peptide and CAF cell membrane (targeted lipid nanoparticles), and I is a schematic diagram of in vivo targeting.

[0026] Figure 2 DSPE-PEG 2000 -NMR spectrum of TAT.

[0027] Figure 3 The particle size, potential and release curve of JQ1 in the release medium of ordinary drug-loaded liposomes, CAF cell membrane-modified drug-loaded liposomes, TAT peptide-modified drug-loaded liposomes, and TAT peptide and CAF cell membrane-co-modified drug-loaded liposomes.

[0028] Figure 4 Flow cytometry was used to detect the uptake results of ordinary drug-loaded liposomes, CAF cell-modified drug-loaded liposomes, TAT peptide-modified drug-loaded liposomes, and TAT peptide and CAF cell membrane co-modified drug-loaded liposomes on pancreatic cancer cells and CAF cells.

[0029] Figure 5 The IndiGO live animal imager was used to detect the targeting ability of ordinary drug-loaded liposomes, CAF cell-modified drug-loaded liposomes, TAT peptide-modified drug-loaded liposomes, and TAT peptide and CAF cell membrane co-modified drug-loaded liposomes to mouse pancreatic cancer.

[0030] Figure 6 The therapeutic effect of TAT peptide and CAF cell membrane co-modified drug-loaded liposomes carrying the anti-tumor drug JQ1 on pancreatic cancer in mice.

[0031] Figure 7 The safety of the material was evaluated by HE staining of mouse heart, liver, kidney, spleen and lung tissues.

[0032] Figure 8 Effects of liposomes prepared with different mass ratios of cell membrane proteins and TAT on CAF cell uptake. DETAILED DESCRIPTION

[0033] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0034] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0035] The present invention is further described in detail below in conjunction with specific comparative examples and embodiments:

[0036] Comparative Example 1: Preparation of common drug-loaded liposomes (L)

[0037] Take 10 mg of phosphatidylcholine and dissolve it in 1 ml of anhydrous ethanol, add 3.3 mg of cholesterol and 0.1 mg of DSPE-PEG 2000 , 0.5-1 mg JQ1 was added, and then the probe was ultrasonicated in an ice bath for 30 times; then the ethanol mixed solution was injected into 10 ml of PBS buffer under magnetic stirring at 1000 rpm at a rate of 0.5 ml / min, and stirred for 10 minutes to form a common drug-loaded liposome dosing micelle solution, which was then dialyzed in a dialysis bag with MW=3500Da for 12 hours to separate the free JQ1 from the common drug-loaded liposome dosing micelle to obtain common drug-loaded liposomes.

[0038] Coumarin 6 labeled common drug-loaded liposomes: Dissolve 10 mg of phosphatidylcholine in 1 ml of anhydrous ethanol, add 3.3 mg of cholesterol, 0.1 mg of DSPE-PEG 2000, JQ1, and then add 10ug coumarin 6 (C6), and ultrasonicate with an ice bath probe 30 times; inject 10ml of PBS buffer under magnetic stirring at 1000rpm at a rate of 0.5ml / min to form a coumarin 6-labeled common drug-loaded liposome drug delivery micelle solution; add the coumarin 6-labeled common drug-loaded liposome drug delivery micelle solution into a dialysis bag, dialyze for 12 hours, centrifuge and take the supernatant to obtain coumarin 6-labeled common drug-loaded liposomes (C6@L).

[0039] DIR-labeled common drug-loaded liposomes: Dissolve 10 mg of phosphatidylcholine in 1 ml of anhydrous ethanol, add 3.3 mg of cholesterol, 0.1 mg of DSPE-PEG 2000 , JQ1, and then add 10ugDIR, ultrasonicate with ice bath probe 30 times; inject 10ml PBS buffer under magnetic stirring at 1000rpm at a speed of 0.5ml / min to form DIR-labeled common drug-loaded liposome drug delivery micelle solution; add the DIR-labeled common drug-loaded liposome drug delivery micelle solution into a dialysis bag, dialyze for 12 hours, centrifuge and take the supernatant to obtain DIR-labeled common drug-loaded liposomes (DIR@L).

[0040] Comparative Example 2: Preparation of CAF Cell Membrane Modified Drug-Loaded Liposomes (CL)

[0041] The cell membranes of 10 million NIH / 3T3 cells were extracted using a cell membrane extraction kit and then suspended in a PBS solution; the protein in the cell membrane was quantified using the BCA method, and then the cell membranes and the ordinary drug-loaded liposomes prepared in Comparative Example 1 were mixed at a cell membrane protein / phosphatidylcholine mass ratio of 1:10, and a liposome extruder was used to extrude them first with a pore size of 400 nm and then with a pore size of 200 nm, each for more than 11 times, to obtain CAF cell membrane-modified drug-loaded liposomes.

[0042] Coumarin 6-labeled CAF cell membrane-modified drug-loaded liposomes: 10 million NIH / 3T3 cells were used to extract the cell membrane using a cell membrane extraction kit, and then suspended in a PBS solution; the BCA method (bicinchoninic acid method) was used to quantify the protein in the cell membrane, and then the cell membrane and the coumarin 6-labeled ordinary drug-loaded liposomes prepared in Comparative Example 1 were mixed at a cell membrane protein / phosphatidylcholine mass ratio of 1:10, and a liposome extruder was used to extrude first with a pore size of 400 nm, and then with a pore size of 200 nm, each for more than 11 times, to obtain coumarin 6-labeled CAF cell membrane-modified drug-loaded liposomes (C6@CL).

[0043] DIR-labeled CAF cell membrane modified drug-loaded liposomes: The cell membranes of 10 million NIH / 3T3 cells were extracted using a cell membrane extraction kit and then suspended in a PBS solution; the proteins in the cell membranes were quantified using the BCA method, and then the cell membranes and the DIR-labeled ordinary drug-loaded liposomes prepared in Comparative Example 1 were mixed at a cell membrane protein / phosphatidylcholine mass ratio of 1:10, and a liposome extruder was used to extrude first with a pore size of 400 nm and then with a pore size of 200 nm, each for more than 11 times, to obtain DIR-labeled CAF cell membrane modified drug-loaded liposomes (DIR@CL).

[0044] Comparative Example 3: Preparation of TAT peptide-modified drug-loaded liposomes (TL)

[0045] Take 10 mg of phosphatidylcholine and dissolve it in 1 ml of anhydrous ethanol, add 3.3 mg of cholesterol and 0.1 mg of DSPE-PEG 2000 -TAT, 0.5-1 mg JQ1, and then ultrasonicated with an ice bath probe 30 times; then the ethanol mixed solution was injected into 10 ml of PBS buffer under magnetic stirring at 1000 rpm at a rate of 0.5 ml / min, and stirred for 10 minutes to form a TAT peptide-modified drug-loaded liposome delivery micelle solution, which was then dialyzed in a dialysis bag with MW=3500Da for 12 hours to separate the free JQ1 from the TAT peptide-modified drug-loaded liposome delivery micelle to obtain TAT peptide-modified drug-loaded liposomes.

[0046] Coumarin 6-labeled TAT peptide-modified drug-loaded liposomes: 10 mg of phosphatidylcholine was dissolved in 1 ml of anhydrous ethanol, 3.3 mg of cholesterol, 0.1 mg of DSPE-PEG 2000 -TAT, JQ1, and then add 10ug coumarin 6, and ultrasonicate with an ice bath probe 30 times; inject 10ml of PBS buffer under magnetic stirring at 1000rpm at a rate of 0.5ml / min to form a coumarin 6-labeled TAT peptide-modified drug-loaded liposome delivery micelle solution; add the coumarin 6-labeled TAT peptide-modified drug-loaded liposome delivery micelle solution into a dialysis bag, dialyze for 12 hours, centrifuge and take the supernatant to obtain coumarin 6-labeled TAT peptide-modified drug-loaded liposomes (C6@TL).

[0047] DIR-labeled TAT peptide modified drug-loaded liposomes: 10 mg of phosphatidylcholine was dissolved in 1 ml of anhydrous ethanol, 3.3 mg of cholesterol, 0.1 mg of DSPE-PEG 2000-TAT, JQ1, then add 10ugDIR, ultrasonicate with ice bath probe 30 times; inject 10ml PBS buffer under magnetic stirring at 1000rpm at a speed of 0.5ml / min to form DIR-labeled TAT peptide-modified drug-loaded liposome delivery micelle solution; add the DIR-labeled TAT peptide-modified drug-loaded liposome delivery micelle solution into a dialysis bag, dialyze for 12 hours, centrifuge and take the supernatant to obtain DIR-labeled TAT peptide-modified drug-loaded liposomes (DIR@TL).

[0048] Example 1: Preparation of TAT peptide and CAF cell membrane co-modified drug-loaded liposomes (CTL)

[0049] The cell membrane was extracted from 10 million NIH / 3T3 cells using a cell membrane extraction kit and then suspended in a PBS solution; the protein in the cell membrane was quantified using the BCA method, and then the cell membrane and the TAT peptide-modified drug-loaded liposomes prepared in Comparative Example 3 were mixed at a cell membrane protein / phosphatidylcholine mass ratio of 1:10, and a liposome extruder was used to extrude first with a pore size of 400 nm and then with a pore size of 200 nm, each for more than 11 times, to obtain TAT peptide and CAF cell membrane co-modified drug-loaded liposomes.

[0050] Coumarin 6-labeled TAT peptide and CAF cell membrane co-modified drug-loaded liposomes: 10 million NIH / 3T3 cells were extracted from the cell membrane using a cell membrane extraction kit and then suspended in a PBS solution; the protein in the cell membrane was quantified using the BCA method, and then the cell membrane and the coumarin 6-labeled TAT peptide-modified drug-loaded liposomes prepared in Comparative Example 3 were mixed at a cell membrane protein / phosphatidylcholine mass ratio of 1:10, and a liposome extruder was used to extrude first with a pore size of 400 nm, and then with a pore size of 200 nm, each for more than 11 times, to obtain coumarin 6-labeled TAT peptide and CAF cell membrane co-modified drug-loaded liposomes (C6@CTL).

[0051] DIR-labeled TAT peptide and CAF cell membrane co-modified drug-loaded liposomes: 10 million NIH / 3T3 cells were extracted from the cell membrane using a cell membrane extraction kit and then suspended in a PBS solution; the protein in the cell membrane was quantified using the BCA method, and then the cell membrane and the DIR-labeled TAT peptide-modified drug-loaded liposomes prepared in Comparative Example 3 were mixed at a cell membrane protein / phosphatidylcholine mass ratio of 1:10, and a liposome extruder was used to extrude first with a pore size of 400 nm and then with a pore size of 200 nm, each for more than 11 times, to obtain DIR-labeled TAT peptide and CAF cell membrane co-modified drug-loaded liposomes (DIR@CTL).

[0052] Determination of JQ1 content in drug-loaded liposomes by UV-Vis spectrophotometry

[0053] JQ1 was dissolved in anhydrous ethanol, and the anhydrous ethanol was diluted to prepare solutions of different concentrations to prepare a standard curve; a certain amount of ordinary drug-loaded liposomes, CAF cell-modified drug-loaded liposomes, TAT peptide-modified drug-loaded liposomes, and TAT peptide and CAF cell membrane co-modified drug-loaded liposomes were dissolved in anhydrous ethanol, the absorbance at 256 nm was measured, and the JQ1 content in the carrier was calculated using the standard curve.

[0054] The particle size and surface potential analyzer determined that the micelle particle sizes of ordinary drug-loaded liposomes, CAF cell-modified drug-loaded liposomes, TAT peptide-modified drug-loaded liposomes, and TAT peptide and CAF cell membrane co-modified drug-loaded liposomes were 115.2nm, 122.2nm, 139.0nm, and 155.5nm, and the Zeta potentials were -36.2mV, -39.6mV, -29.8mV, and -32.9mV, respectively.

[0055] 1 ml of common drug-loaded liposomes, CAF cell-modified drug-loaded liposomes, TAT peptide-modified drug-loaded liposomes, and TAT peptide and CAF cell membrane co-modified drug-loaded liposomes were placed in a dialysis bag, and the dialysis bag was placed in 50 ml of phosphate buffer at pH = 7.4. The dialysis bag was shaken in a shaker at 37°C, and samples were taken at different time points for 48 hours. The absorbance value at 256 nm was measured by UV-visible spectrophotometry, and the concentration of released JQ1 was calculated by the standard curve, as shown in FIG. Figure 3 shown.

[0056] The particle size and potential were maintained within an appropriate range, and the drug release rate of drug-loaded liposomes was lower than that of free drugs. No significant difference in drug release rate was observed among several groups of drug-loaded liposomes, indicating that they all had a good sustained-release effect in vivo and were able to release JQ1 after reaching the tumor tissue.

[0057] Evaluation of the in vitro uptake capacity of the conventional drug-loaded liposomes (L), CAF cell membrane-modified drug-loaded liposomes (CL), TAT peptide-modified drug-loaded liposomes (TL) of Comparative Examples 1-3 and the TAT peptide and CAF cell membrane co-modified drug-loaded liposomes (CTL) of Example 1:

[0058] In a 24-well cell culture plate, add 1 ml of 1 × 10 5 MT5 cells or NIH / 3T3 cells were placed at 37°C and 5% CO 2The cells were cultured in a box for 24 hours. After the cells were completely attached to the wall, 20ul of common drug-loaded liposomes labeled with coumarin 6, drug-loaded liposomes modified with coumarin 6-labeled CAF cells, drug-loaded liposomes modified with coumarin 6-labeled TAT peptide, and drug-loaded liposomes co-modified with coumarin 6-labeled TAT peptide and CAF cell membrane were added to each well. The mass of coumarin 6 contained in each well was 0.05ug. The cells were incubated for 3h or 6h, washed three times with isotonic phosphate buffer, digested with trypsin, centrifuged to obtain cell pellets, resuspended with 500ul buffer, and the fluorescence intensity of coumarin 6 was detected by flow cytometry. Figure 4 shown.

[0059] After 6 hours of co-incubation, the fluorescence signal of the CTL group in CAF cells was the strongest, which was 62.84% higher than that of the L group and 21.42% higher than that of the TL group. Compared with the L group, the fluorescence signals of the CL and TL groups were enhanced, indicating that the absorption of CTL by CAF cells was improved under the mediation of CAF cell membrane and TAT peptide.

[0060] In MT5 cells, only the fluorescence signals of TL and CTL increased, which increased by 31.85% and 32.53% respectively compared with L; however, there was no statistically significant difference between the L group and the CL group, and between the TL group and the CTL group. These results indicate that the liposomes encapsulating the CAF cell membrane have specific targeting ability for CAF cells, but not for MT5 cells.

[0061] Evaluation of the in vivo targeting ability of the common drug-loaded liposomes (L), CAF cell membrane-modified drug-loaded liposomes (CL), TAT peptide-modified drug-loaded liposomes (TL) of Comparative Examples 1-3 and the TAT peptide and CAF cell membrane co-modified drug-loaded liposomes (CTL) of Example 1:

[0062] MT5 cells and NIH / 3T3 cells were counted and suspended in a 1:1 ratio to form a mixed cell suspension. Then, an equal volume of matrix gel was added to the mixed cell suspension and thoroughly pipetted to mix. Subsequently, the mixed cell suspension was inoculated into the pancreas of mice at a concentration of 5×10 cells / 50 μL. 4 cells.

[0063] Two weeks later, a palpable round tumor tissue could be felt under the left rib. Subsequently, DIR-labeled common drug-loaded liposomes, DIR-labeled CAF cell-modified drug-loaded liposomes, DIR-labeled TAT peptide-modified drug-loaded liposomes, and DIR-labeled TAT peptide and CAF cell membrane-co-modified drug-loaded liposomes were injected into mice via the tail vein. The distribution of liposomes was then imaged using the IndiGO live animal imager 24 hours after injection. The mice were then dissected, and the fluorescence signal of each organ was quantified using the IndiGO live animal imager, as shown in Figure 2. Figure 5 shown.

[0064] In tumor tissue, the fluorescence intensity of the CTL group was the highest, increasing by 118.79% compared with the L group. In contrast, there was almost no observable fluorescence in the free DIR group; the fluorescence signals of the CL group and the TL group also increased, increasing by 47.94% and 63.72% respectively compared with the L group; then the tumor tissue was dissected and the main organs were imaged to monitor the distribution behavior of CTL in vivo. The fluorescence was mainly distributed in the tumor tissue, which was consistent with the results of in vivo live imaging. The fluorescence intensity of the CTL group was the highest, and the fluorescence signals of the CL group and the TL group also increased; CTL showed the strongest targeting and aggregation mechanism, which was mainly attributed to the wrapping effect of the CAF cell membrane, enabling CTL to more effectively aggregate in CAF-rich pancreatic cancer tissue through homologous targeting; after reaching the target tissue, CTL released the internalized TL under the mediation of TAT peptide, making it easier for lipid vesicles to penetrate the cell membrane and deliver drugs into the cells; in all groups, the fluorescence intensity in the liver and kidneys was relatively low, which may be due to the fact that these organs have metabolized most of their accumulated fluorescent liposomes within 24 hours.

[0065] Evaluation of the antitumor efficacy and safety of the drug-loaded liposomes co-modified with TAT peptide and CAF cell membrane in Example 1:

[0066] Using pancreatic orthotopic transplanted tumor mice as a model, the inhibitory effects of free drug (JQ1) and drug (JQ1) encapsulated in drug-loaded liposomes co-modified with TAT peptide and CAF cell membrane on pancreatic tumors were evaluated.

[0067] Mouse pancreatic cancer cell line MT5-luc cells and embryonic fibroblast NIH / 3T3 cells were mixed in a ratio of 1:1 to prepare a cell suspension; then an equal volume of matrix gel was added to the cell mixture and thoroughly pipetted and mixed; then, the mixed cell suspension was inoculated into the pancreatic tissue of mice to simulate the formal pancreatic cancer environment, with a concentration of 5×10 per 50 μL. 4 cells; after 5 days, a round tumor tissue could be felt under the left rib of the mouse.

[0068] The mice were randomly divided into three groups (control group, free drug group, CTL drug group), and tumor growth was monitored by luciferase imaging; subsequently, the mice were injected with phosphate buffer (control group), free JQ1 (free drug group), and TAT peptide and CAF cell membrane co-modified liposomes (CTL drug group) through the tail vein every two days, and the drugs were given every other day, for a total of 7 times; the concentration of JQ1 in the free drug group and the CTL drug group was 10 mg / kg; the body weight of the mice was recorded every three days; at the end of the treatment period, all mice were euthanized, and tumors and organs, including heart, liver, kidney, spleen and lung, were harvested and analyzed.

[0069] like Figure 6 As shown in the figure, the free JQ1 group did not significantly inhibit tumor growth compared with the PBS group. However, the CTL drug group could significantly inhibit tumor growth, indicating that CTL could target JQ1 to the tumor, thereby enhancing tumor perfusion, while only a small amount of JQ1 in the free JQ1 group was distributed to the tumor tissue, so its tumor inhibition effect was poor.

[0070] There was no significant change in body weight among the three groups of mice. The collected major organs were fixed and embedded, and HE staining was performed after paraffin sectioning. The results showed that the cell morphology and tissue structure of the organs in each group did not change significantly, indicating that the CTL of the present invention has good in vivo safety.

[0071] Effects of C6@CTL with different cell membrane protein / TAT peptide / phosphatidylcholine mass ratios on cell uptake in Example 1:

[0072] Dissolve 10 mg of phosphatidylcholine in 1 ml of anhydrous ethanol, add 3.3 mg of cholesterol, and add 0.033 mg, 0.05 mg, 0.10 mg, and 0.20 mg of DSPE-PEG, respectively. 2000 -TAT, then add 10ug coumarin 6, and ultrasonicate 30 times with an ice bath probe; inject 10ml of PBS buffer under magnetic stirring at 1000rpm at a rate of 0.5ml / min to form a dosing micelle solution, which is then added to a dialysis bag and dialyzed for 12 hours. The supernatant is centrifuged to obtain C6@TL with different TAT peptide / phosphatidylcholine mass ratios.

[0073] The cell membranes of 10 million NIH / 3T3 cells were extracted using a cell membrane extraction kit and then suspended in a PBS solution. The proteins in the cell membranes were quantified using the BCA method. The cell membranes were then mixed with C6@TL with different TAT peptide / phosphatidylcholine mass ratios at a cell membrane protein / phosphatidylcholine mass ratio of 1:50, 1:20, 1:10, and 1:5, respectively. The mixture was extruded using a liposome extruder with a pore size of 400 nm and then with a pore size of 200 nm for more than 11 times each to obtain C6@CTL with different cell membrane protein / TAT peptide / phosphatidylcholine mass ratios.

[0074] In a 24-well cell culture plate, add 1 ml of 1 × 10 5 NIH / 3T3 cells were placed at 37°C and 5% CO 2 The cells were cultured in an incubator for 24 hours. After the cells were completely attached, 20ul of C6@CTL with different cell membrane protein / TAT peptide / phosphatidylcholine mass ratios were added to each well, and the mass of coumarin 6 contained in each well was 0.05ug. The cells were incubated for 6 hours, washed three times with isotonic phosphate buffer, digested with trypsin, and the cell pellet was obtained by centrifugation. The cell pellet was resuspended with 500ul buffer and the fluorescence intensity of coumarin 6 was detected by flow cytometry.

[0075] After 6 hours of co-incubation, the results showed that when the mass ratio of cell membrane protein / phosphatidylcholine was less than 1:10, the cell uptake of C6@CTL increased with the increase of cell membrane protein, and when the mass ratio of cell membrane protein / phosphatidylcholine was greater than 1:10, the cell uptake of C6@CTL did not change. When the mass ratio of TAT peptide / phosphatidylcholine was less than 1:100, the cell uptake of C6@CTL increased with the increase of TAT peptide, and when the mass ratio of TAT peptide / / phosphatidylcholine was greater than 1:10, the cell uptake of C6@CTL did not change.

[0076] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A targeted lipid nanoparticle, characterized in that: The CAF cell membrane extracted from CAF cells is coated on the surface of the TAT peptide-modified drug-loaded liposomes to form TAT peptide-modified and CAF cell membrane-coated targeted lipid nanoparticles; The TAT peptide-modified drug-loaded liposomes are prepared using phosphatidylcholine, cholesterol, DSPE-PEG2000-TAT and drugs; The method for preparing the targeted lipid nanoparticles comprises the following steps: Step (1): dissolving phosphatidylcholine in an appropriate amount of solvent, adding cholesterol, DSPE-PEG2000-TAT and the drug, and ultrasonically mixing in an ice bath to obtain a mixed solution; injecting the mixed solution into a PBS buffer at a certain speed, stirring while injecting, and continuing to stir after injection until a liposome administration micelle solution is formed; dialyzing the liposome administration micelle solution in a dialysis bag to obtain TAT peptide-modified drug-loaded liposomes; Step (2): CAF cell membrane extracted from CAF cells and TAT peptide-modified drug-loaded liposomes are mixed at a CAF cell membrane protein / phosphatidylcholine mass ratio of 1:8-15, and extruded several times using a liposome extruder to obtain TAT peptide-modified and membrane-coated targeted lipid nanoparticles; The targeted lipid nanoparticles are used in preparing medicine for treating targeted tumors, and the tumor is a pancreatic cancer tumor.

2. The targeted lipid nanoparticle according to claim 1, characterized in that: The phosphatidylcholine, cholesterol and DSPE-PEG 2000 -The mass ratio of TAT is 90-110:30-37:

1.

3. The targeted lipid nanoparticle according to claim 1, characterized in that: The medicine is an anti-tumor medicine.

4. The targeted lipid nanoparticle according to claim 1, characterized in that: In the step (1), the mixed solution is injected into the PBS buffer at a speed of 0.4-0.6 ml / min, and magnetic stirring is performed at 800-1200 rpm. In the step (2), the CAF cell membrane is extracted using a cell membrane extraction kit.

5. The targeted lipid nanoparticle according to claim 1, characterized in that: When the targeted lipid nanoparticles are used to prepare drugs for treating targeted tumors, compared with common drug-loaded liposomes, in an in vitro uptake capacity evaluation experiment of the present invention: after incubation for 6 hours, the fluorescence intensity of coumarin 6 detected by flow cytometer increases by 50-70%; in an in vivo targeting capacity evaluation experiment of the present invention: 24 hours after tail vein injection, the DIR fluorescence intensity detected by live animal imaging device in tumor tissue increases by 110-130%.

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