TPZ / TPP-RBC-FA liposome and application thereof in oral cancer treatment

Abstract

The invention discloses a TPZ / TPP-RBC-FA liposome and an application thereof in oral cancer treatment, the brand new TPZ / TPP-RBC-FA liposome is creatively provided for the technical field of oral cancer treatment for the first time, and the effectiveness of the TPZ / TPP-RBC-FA liposome in oral cancer treatment is proved through an in-vitro cell experiment and an in-vivo experiment. The TPZ / TPP-RBC-FA liposome not only can effectively induce tumor cell apoptosis, but also can cause immunogenic cell death and immune cell activation, and has a wide application prospect in clinical treatment of oral cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to a TPZ / TPP@RBC-FA liposome and an application thereof in the treatment of oral cancer. Background Art

[0002] Oral cancer is one of the most common malignant tumors of the head and neck, of which squamous cell carcinoma accounts for more than 90%. There are more than 400,000 new cases of oral cancer each year worldwide, of which 2 / 3 are diagnosed in developing countries. Risk factors include betel nut chewing, smoking, human papillomavirus infection, and drinking. Most cases of oral cancer are diagnosed in the late stage, and the average 5-year survival rate is as low as 40%. It can be seen that oral cancer is an important public health issue and one of the most common malignant tumors in the world, with a high mortality rate. With the improvement of medical standards, the survival rate of oral cancer continues to increase, and patients have good functional recovery after surgery, but the non-specificity, non-selectivity, drug resistance and toxicity of traditional cancer treatments remain huge challenges.

[0003] In recent years, photodynamic therapy (PDT) has attracted increasing attention due to its low systemic toxicity and negligible drug resistance. In addition to surgery, PDT is regarded as another effective method for treating oral cancer. PDT is a new technology that uses the photodynamic effect for disease diagnosis and treatment. Its action is based on the photodynamic effect. This is a photosensitization reaction with biological effects involving oxygen molecules. The process is that laser irradiation of a specific wavelength excites the photosensitizer absorbed by the tissue, and the excited photosensitizer transfers energy to the surrounding oxygen to generate highly active singlet oxygen. The singlet oxygen undergoes an oxidation reaction with adjacent biomacromolecules, producing cytotoxic effects, which in turn leads to cell damage or even death.

[0004] However, the limited oxygen content in the tumor microenvironment reduces the efficiency of current photodynamic therapy. How to overcome the insufficient tumor killing caused by oxygen depletion in photodynamic therapy remains one of the technical problems that need to be urgently solved in this field. Summary of the invention

[0005] In view of this, the present invention has developed a complementary tumor killing system, which overcomes the technical problem of insufficient tumor killing caused by oxygen depletion in PDT in the current prior art by introducing hypoxia-activated chemotherapy drugs into tumor-targeted liposomes.

[0006] First, the present invention modifies tumor-targeting folic acid (FA) on the surface of liposomes based on red blood cell membranes, and loads photosensitizer TPP and hypoxia-activated drug TPZ into liposomes (TPZ / TPP@RBC-FA). After laser irradiation of the tumor site, the chemotherapy drug TPZ in the TPZ / TPP@RBC-FA liposomes is released due to the membrane rupture of reactive oxygen species (ROS) generated by photodynamic therapy, which is generated by oxygen during photodynamic therapy, leading to aggravated hypoxia in tumor tissues and further generation of TPZ carrying toxic free radicals. The combination of hypoxia-activated chemotherapy and photodynamic therapy can not only effectively induce cell apoptosis, but also cause immunogenic cell death and immune cell activation. The liposome drug loading strategy also shows good biosafety. It is believed that the strategy of combining photodynamic therapy and hypoxia-activated chemotherapy using liposomes can be applied to the treatment of clinical oral cancer in the near future.

[0007] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0008] The first aspect of the present invention provides a method for preparing TPZ / TPP@RBC-FA liposomes.

[0009] Further, the method comprises the following steps:

[0010] The tetracarboxylporphine solution and DSPE-PEG-FA powder were added to the red blood cell membrane suspension, and the mixture was shaken and mixed in the dark to obtain a mixed solution A;

[0011] Adding tirapazamine solution to the mixed solution A, shaking and mixing in the dark to obtain a mixed solution B;

[0012] The mixed solution B was ultrasonically disrupted on ice to obtain TPZ / TPP@RBC-FA liposomes.

[0013] In some embodiments, the order of adding the tetracarboxyporphine solution and the DSPE-PEG-FA powder to the red blood cell membrane suspension is not particularly limited, including but not limited to: first adding the tetracarboxyporphine solution to the red blood cell membrane suspension, and then adding the DSPE-PEG-FA powder; adding the tetracarboxyporphine solution and the DSPE-PEG-FA powder to the red blood cell membrane suspension at the same time; first adding the DSPE-PEG-FA powder to the red blood cell membrane suspension, and then adding the tetracarboxyporphine solution.

[0014] In the present invention, the tirapazamine (TPZ) is a hypoxia-selective cytotoxin, the corresponding CAS number is 27314-97-2, the molecular formula is C7H6N4O2, the molecular weight is 178.15, and the structural formula is shown in the following formula (I). In an oxygen-deficient environment, tirapazamine is reduced to a free radical, causing DNA double-strand breaks (DSBs), single-strand breaks and base damage. TPZ can inhibit DNA topoisomerase IIα and regulatory replication protein A (RPA), inhibiting DNA replication. In addition, TPZ can also strongly inhibit HIF-1α expression, and exerts its effect by regulating the phosphorylation of eukaryotic initiation factor eIF2α. TPZ can induce apoptosis of neuroblastoma cells, reduce mitochondrial membrane potential and glutathione levels, and this physiological effect may be p53-dependent. In the present invention, the tirapazamine can be used interchangeably with TPZ.

[0015]

[0016] In the present invention, the tetrakis(4-carboxyphenyl)porphyrin (TPP-4COOH) can be used as a photosensitizer to participate in photochemical reactions, and can also be used as an electron transport material in optoelectronic devices. Its corresponding CAS number is 14609-54-2, and its molecular formula is C 48 H 30 N4O8, molecular weight is 790.774, and the structural formula is shown in the following formula (II). In addition, the tetracarboxyporphine can also form a supramolecular structure with specific functions through interactions with other molecules, providing new ideas and methods for chemical synthesis and material preparation. Porphine is an organic compound with a large π conjugated system, and tetracarboxyporphine is a derivative obtained by introducing four 4-carboxyphenyl groups on the basis of porphine. This structure gives tetracarboxyporphine many unique properties, such as strong light absorption, good electron transport performance and high chemical stability. In the present invention, the tetracarboxyporphine and TPP-4COOH can be used interchangeably.

[0017]

[0018] In the present invention, the full name of DSPE-PEG-FA is distearoylphosphatidylethanolamine-polyethylene glycol-folic acid, which is a complex obtained by copolymerization of phosphatidylethanolamine (DSPE), polyethylene glycol (PEG) and folic acid (FA), wherein DSPE is a phospholipid having a hydrophobic phospholipid tail and a hydrophilic phosphatidylcholine head. PEG is a polymer with good water solubility. FA is a molecule that can bind to folate receptors and can achieve targeted delivery to specific cells. Its corresponding CAS number is 1236288-25-7, and its structural formula is shown in the following formula (III).

[0019]

[0020] Furthermore, the concentration of the tetracarboxyl porphine solution is 5-15 mg / mL, and the amount added is 7.5-17.5 μL;

[0021] The added amount of the DSPE-PEG-FA powder is 0.2-3.5 mg.

[0022] Furthermore, the concentration of the tetracarboxylporphine solution is 10 mg / mL, and the amount added is 12.5 μL

[0023] The added amount of the DSPE-PEG-FA powder is 1 mg.

[0024] Further, the amount of the red blood cell membrane is 0.5-3.5 mg;

[0025] Optionally, the amount of the red blood cell membrane is 2 mg;

[0026] Optionally, the light-proof oscillation time of the mixed solution A obtained after the light-proof oscillation mixing is 1-5h;

[0027] Optionally, the light-proof oscillation time of the mixed solution A obtained after the light-proof oscillation mixing is 2 hours.

[0028] Furthermore, the concentration of the tirapazamine solution is 5-15 mg / mL, and the amount added is 5-15 μL;

[0029] Optionally, the concentration of the tirapazamine solution is 10 mg / mL, and the amount added is 10 μL;

[0030] Optionally, the light-proof oscillation time of the mixed solution B obtained after the light-proof oscillation mixing is 10-60 min;

[0031] Optionally, the light-proof oscillation time of the mixed solution B obtained after the light-proof oscillation mixing is 30 minutes.

[0032] Furthermore, the conditions for ultrasonic disruption on ice are to use a probe ultrasonic cell disruptor to insert the probe into the liquid surface of the mixed solution B for ultrasonication for 2-8 minutes;

[0033] Optionally, the ultrasonic disruption on ice is performed by inserting a probe into the liquid surface of the mixed solution B using a probe ultrasonic cell disruptor for 5 minutes;

[0034] Optionally, the erythrocyte membrane is a purified erythrocyte membrane;

[0035] Optionally, the method further comprises purifying the obtained TPZ / TPP@RBC-FA liposomes;

[0036] Optionally, the purification includes dialyzing the obtained TPZ / TPP@RBC-FA liposomes under magnetic stirring to obtain purified TPZ / TPP@RBC-FA liposomes;

[0037] Optionally, the magnetic stirring condition is 100-300 rpm, 1-3 days;

[0038] Optionally, the magnetic stirring condition is 200 rpm for 2 days.

[0039] In a specific embodiment of the present invention, the purified erythrocyte membrane is prepared by the following method: take 25 μL of EDTA solution and add it to 1 mL of PBS to prepare an anticoagulant. Then, collect mouse blood through the orbital sinus of Balb / c mice and add it to the anticoagulant prepared in advance. After centrifugation at 2500 rpm, discard the supernatant and residual blood cells, and the remaining precipitate is the purified erythrocytes. The purified erythrocytes are resuspended in 0.25×PBS, and the erythrocytes will be lysed under the action of osmotic pressure, lysed at 4°C for 25 minutes, and then centrifuged at 12000 rpm, and the supernatant is discarded to remove hemoglobin and impurities in the lysed erythrocytes. The remaining precipitate is the purified erythrocyte membrane. After repeating the above method for lysis and centrifugation three times, a purified erythrocyte membrane is finally obtained.

[0040] In some embodiments, the present invention has no particular restrictions on the source of the erythrocyte membrane, and is not limited to the purified erythrocyte membrane prepared by the specific method described in the specific embodiment of the present invention. The purified erythrocyte membrane prepared by any method known to those skilled in the art, or the purified erythrocyte membrane from any commercial source, falls within the protection scope of the present invention.

[0041] In a specific embodiment of the present invention, the tirapazamine solution is prepared by dissolving the above-mentioned specific amount of tirapazamine in a corresponding volume of DMSO solution. In a specific embodiment of the present invention, the tetracarboxyporphine solution is prepared by dissolving the above-mentioned specific amount of tetracarboxyporphine in a corresponding volume of DMSO solution.

[0042] In some embodiments, the solvent type described in the present invention is not limited to DMSO solution, and any solvent that can be used to dissolve the tirapazamine or tetracarboxylporphine described above in the present invention is within the protection scope of the present invention. In some embodiments, the solvent includes but is not limited to: DMSO solution, toluene, xylene, pentane, hexane, octane, cyclohexane, cyclohexanone, chlorobenzene, dichlorobenzene, methanol, ethanol, ether, methyl acetate, acetone, water, carbon tetrachloride, etc.

[0043] In a specific embodiment of the present invention, the obtained TPZ / TPP@RBC-FA liposomes are purified by the following method: a dialysis bag with a molecular weight pore size of 1 kDa is cut to a length of about 15 cm, one end is clamped with a special clamp, the above-mentioned mixed solution is added to the dialysis bag and the other end is clamped, and the bag is placed in a large beaker containing 1000 mL of PBS buffer solution. The solution is dialyzed at a stirring speed of 200 rpm for 2 days using magnetic stirring to remove unbound TPZ and TPP-4COOH molecules in the solution, and the substance remaining in the dialysis bag is the purified TPZ / TPP@RBC-FA liposome.

[0044] The second aspect of the present invention provides a TPZ / TPP@RBC-FA liposome.

[0045] Furthermore, the TPZ / TPP@RBC-FA liposomes are prepared by loading tirapazamine and tetracarboxylporphine into liposomes based on red blood cell membranes modified with folic acid;

[0046] Optionally, the TPZ / TPP@RBC-FA liposome is a TPZ / TPP@RBC-FA liposome prepared by the method described in the first aspect of the present invention.

[0047] The third aspect of the present invention provides a pharmaceutical composition.

[0048] Furthermore, the pharmaceutical composition comprises the TPZ / TPP@RBC-FA liposomes described in the second aspect of the present invention.

[0049] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0050] In some embodiments, the pharmaceutically acceptable carrier and / or excipient is described in detail in Remington's Pharmaceutical Sciences (19th ed, 1995). These substances are used to help the stability of the drug or to help improve the activity of the active ingredient (the active ingredient is the TPZ / TPP@RBC-FA liposome of the present invention as described above). The substances include but are not limited to: diluents, surfactants, humectants, adhesives, fillers, disintegrants, adsorption carriers, lubricants, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents. The pharmaceutical composition prepared in this way can be administered by any appropriate administration method known to those skilled in the art as needed.

[0051] In some embodiments, the pharmaceutically acceptable carrier and / or excipient may additionally contain a liquid, such as water, saline, glycerol and / or ethanol, and the like.

[0052] A fourth aspect of the present invention provides a pharmaceutical preparation.

[0053] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the third aspect of the present invention.

[0054] In some embodiments, the dosage form of the pharmaceutical preparation includes, but is not limited to, suspensions, controlled release preparations, solutions, emulsions, tablets, pills, powders, granules, capsules, lozenges, syrups, aerosols, films, injections, intravenous drips, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories, nasal preparations, pulmonary preparations, etc., for patient ingestion.

[0055] In some embodiments, suitable modes of administration of the pharmaceutical composition or pharmaceutical formulation include any of a variety of methods and delivery systems known to those skilled in the art to physically introduce the pharmaceutical composition or pharmaceutical formulation of the present invention into a subject, including but not limited to: topical administration, rectal administration, nasal administration, oral administration, parenteral administration, administration by inhalation spray, buccal administration, or administration via an implanted reservoir.

[0056] The fifth aspect of the present invention provides the use of the TPZ / TPP@RBC-FA liposomes prepared by the method according to the first aspect of the present invention in the preparation of a drug for treating cancer.

[0057] Optionally, the cancer comprises oral cancer, nasopharyngeal cancer, gastric cancer, lung cancer, colorectal cancer, kidney cancer, ovarian cancer, liver cancer, breast cancer, cervical cancer, bladder cancer, colorectal cancer, soft tissue sarcoma, head and neck cancer, follicular lymphoma, B-cell lymphoma or pancreatic cancer.

[0058] In addition, the present invention also provides a method for treating cancer, which comprises administering a therapeutically effective amount of the TPZ / TPP@RBC-FA liposomes, pharmaceutical composition or pharmaceutical preparation of the present invention as described above to a subject in need thereof.

[0059] In some embodiments, the subject comprises one or more animals, including, for example, cattle, horses, sheep, primates, avian and rodent species. The subject can be a mammal, bird, fish, reptile or amphibian. The mammal includes a human or non-human mammal. In other embodiments, the subject can be a mouse, rat, hamster, ferret, gerbil, rabbit, monkey, chimpanzee, horse, pony, donkey, sheep, pig, chicken, goat, cat or dog. In a preferred embodiment, the subject is a human.

[0060] In some embodiments, the treatment generally involves treating humans or animals (e.g., used by veterinarians), where certain desired therapeutic effects can be achieved, such as inhibiting the development of the disease (including reducing the rate of development, stopping the development), improving the disease, and curing the disease. Treatment as a preventive measure (e.g., prevention) is also included. Use in patients who have not yet developed the disease but are at risk of developing the disease is also included in the term treatment.

[0061] In some embodiments, the therapeutically effective amount refers to an amount having a therapeutic effect or an amount required to produce a therapeutic effect in a subject. For example, a therapeutically effective amount refers to the amount of a drug required to produce a desired therapeutic effect, and the therapeutic effect can be reflected by the results of clinical trials, model animal studies, and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to: characteristic factors of the subject (such as height, weight, sex, age, and medication history), the severity of the disease, etc.

[0062] In some embodiments, the cancer includes, but is not limited to, oral cancer, nasopharyngeal cancer, gastric cancer, lung cancer, colorectal cancer, kidney cancer, ovarian cancer, liver cancer, breast cancer, cervical cancer, bladder cancer, colorectal cancer, soft tissue sarcoma, head and neck cancer, follicular lymphoma, B-cell lymphoma, or pancreatic cancer.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] The present invention creatively provides a new TPZ / TPP@RBC-FA liposome for the technical field of oral cancer treatment for the first time, and proves the effectiveness of the TPZ / TPP@RBC-FA liposome in the treatment of oral cancer through in vitro cell experiments and in vivo experiments. The TPZ / TPP@RBC-FA liposome can not only effectively induce tumor cell apoptosis, but also cause immunogenic cell death and immune cell activation, and has broad application prospects in the clinical treatment of oral cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 :Synthesis (a) and action mechanism (b) of TPZ / TPP@RBC-FA;

[0066] Figure 2 : Zeta potential diagram of TPZ / TPP@RBC-FA;

[0067] Figure 3 : TEM image and particle size distribution of TPZ / TPP@RBC-FA;

[0068] Figure 4 : Dynamic light scattering (DLS) particle size distribution of TPZ / TPP@RBC-FA;

[0069] Figure 5 :In vitro killing effect of TPZ / TPP@RBC-FA;

[0070] Figure 6 : Live and dead staining results of tumor cells after different treatments;

[0071] Figure 7 :Flow cytometry results of cell apoptosis after different treatments;

[0072] Figure 8 : Results of the release of cell immunogenic death (ICD)-related markers (ATP, HMGB1, CRT) after different treatments;

[0073] Fig. 9 : Growth curves of mouse tumors after different treatments;

[0074] Fig.10 : Evaluation of in vivo immune efficacy, testing of DC cell (ab) maturation and T lymphocyte (cd) activation levels in primary and secondary tumors of different groups of mice. DETAILED DESCRIPTION

[0075] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0076] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0077] Example 1 Preparation and detection of TPZ / TPP@RBC-FA liposomes

[0078] 1. Specific preparation methods of TPZ / TPP@RBC-FA liposomes in the experimental group and TPZ@RBC, TPP@RBC, TPZ / TPP@RBC and other control groups

[0079] First, take 25 μL of EDTA solution and add it to 1 mL of PBS to prepare an anticoagulant. Then, collect mouse blood through the orbital sinus of Balb / c mice and add it to the anticoagulant prepared in advance. After centrifugation at 2500 rpm, discard the supernatant and residual blood cells, and the remaining precipitate is the purified red blood cells. Resuspend the purified red blood cells in 0.25×PBS, and the red blood cells will be lysed under the action of osmotic pressure. Lyse at 4°C for 25 minutes, then centrifuge at 12000 rpm, and discard the supernatant to remove hemoglobin and impurities in the lysed red blood cells. The remaining precipitate is the purified red blood cell membrane. After repeating the above method for lysis and centrifugation three times, the purified red blood cell membrane is finally obtained.

[0080] Next, weigh 10 mg of TPZ (tirapazamine, CAS number: 27314-97-2) and 10 mg of TPP-4COOH (tetracarboxylporphine, CAS number: 14609-54-2) powder, dissolve them in 1 mL of DMSO, and prepare 10 mg / mL TPZ storage solution and 10 mg / mL TPP-4COOH storage solution. Resuspend 2 mg of purified erythrocyte membrane in 1 mL of PBS, add 12.5 μL of the above-mentioned TPP-4COOH storage solution, and weigh 1 mg of DSPE-PEG-FA powder (CAS number: 1236288-25-7) and dissolve it in the above solution, and place it on a shaker to oscillate and mix for 2 hours in the dark. Subsequently, add 10 μL of the above-mentioned TPZ storage solution and continue to place it on a shaker to oscillate and mix for 30 minutes in the dark. The above solution was then placed on ice, and a probe ultrasonic cell disruptor was used to insert the probe into the liquid surface for ultrasonication for 5 minutes to prepare TPZ / TPP@RBC-FA lipid nanoparticles.

[0081] Finally, a dialysis bag with a molecular weight pore size of 1 kDa was cut into a length of about 15 cm, one end of which was clamped with a special clamp, and the above-mentioned mixture was added to the dialysis bag and the other end was clamped. It was placed in a large beaker containing 1000 mL of PBS buffer and dialyzed for 2 days at a stirring speed of 200 rpm using magnetic stirring to remove unbound TPZ and TPP-4COOH molecules in the solution. The substance left in the dialysis bag is the prepared TPZ / TPP@RBC-FA liposome.

[0082] Similarly, TPZ@RBC, TPP@RBC and TPP / TPZ@RBC were prepared by methods similar to those described above, except that during the preparation process, only the above-mentioned TPZ storage solution was added to prepare TPZ@RBC liposomes, only the above-mentioned TPP-4COOH storage solution was added to prepare TPP@RBC liposomes, and the above-mentioned TPZ and TPP-4COOH storage solutions were added to prepare TPZ / TPP@RBC liposomes.

[0083] The synthesis and mechanism of action of the TPZ / TPP@RBC-FA liposomes are shown in the figure Figure 1 shown.

[0084] 2. Specific detection steps of TPZ / TPP@RBC-FA

[0085] (1) The prepared TPZ / TPP@RBC-FA lipid nanoparticles were diluted and the particle size of the nanoparticles in the solution was measured using a dynamic light scattering (DLS) instrument. The lipid nanoparticles composed of TPZ, TPP and DSPE-PEG-FA were prepared by ultrasound.

[0086] (2) After the prepared TPZ / TPP@RBC-FA lipid nanoparticles were diluted, transmission electron microscopy (TEM) was used to observe and count the particle size.

[0087] (3) After diluting the prepared TPZ / TPP@RBC-FA lipid nanoparticles, a Zeta potential analyzer was used to detect the Zeta potential of the TPZ / TPP@RBC-FA lipid nanoparticles and the red blood cell membrane itself (RBCm).

[0088] 3. Test results

[0089] Zeta potential diagram of TPZ / TPP@RBC-FA is shown in Figure 2 As shown, the potential change shown in the potential diagram of TPZ / TPP@RBC-FA proves that the membrane surface has been successfully covalently modified with the target molecular groups.

[0090] TEM image and particle size distribution of TPZ / TPP@RBC-FA are shown in Figure 3 As shown, the transmission electron microscopy results intuitively prove the successful synthesis of TPZ / TPP@RBC-FA liposomes and statistically calculate their particle size distribution.

[0091] The dynamic light scattering (DLS) particle size distribution of TPZ / TPP@RBC-FA is shown in Figure 4 As shown, the results prove that the present invention successfully synthesized nanoliposomes with relatively uniform particle size.

[0092] Example 2 Verification of the in vitro and in vivo tumor killing effect of TPZ / TPP@RBC-FA liposomes 1. Verification of the in vitro tumor killing effect of TPZ / TPP@RBC-FA liposomes

[0093] (1) Experimental methods

[0094] SCC7 cells (oral cancer cell line) were cultured at 1×10 5 / mL was inoculated in a 96-well plate and irradiated with near-infrared laser (655nm, 0.05W / cm 2 , 5min), and then the cell viability was detected by MTT method. 20μL MTT solution (5mg / mL) was added to each well. After incubation for 4 hours, DMSO was used to dissolve the formazan crystals formed in each well, and the absorbance of each well was measured by microplate reader. The same method was used to determine the cytotoxicity of TPZ@RBC, TPP@RBC, TPZ / TPP@RBC, and TPZ / TPP@RBC-FA.

[0095] SCC7 cells were cultured at 2×10 4 / well were seeded in a 96-well plate and incubated for 24 hours. Then SCC7 cells were divided into 10 groups and treated as follows: (I) PBS (control), (II) TPZ@RBC, (III) TPP@RBC, (IV) TPZ / TPP@RBC, (V) TPZ / TPP@RBC-FA, (VI) laser, (VII) TPZ@RBC+laser, (VIII) TPP@RBC+laser, (IX) TPZ / TPP@RBC+laser and (X) TPZ / TPP@RBC-FA+laser. The untreated group I was used as the control group, and groups II and VII were treated with TPZ@RBC (10μg / mL). Groups III and VIII were treated with TPP@RBC (10μg / mL). Groups IV and IX were treated with TPZ / TPP@RBC (10μg / mL). Groups V and X were treated with TPZ / TPP@RBC-FA (10 μg / mL). After 24 h of co-incubation, SCC7 cells were washed three times with PBS and then added with fresh culture medium. Groups VI, VII, VIII, IX, and X were irradiated with 655 nm laser (0.05 W / cm 2 , 5min). Finally, 1μM calcein-am (Ex / Em: 495 / 515nm) and 2μM PI (Ex / Em: 535 / 617nm) were added to stain the different groups of SCC7 cells at 25°C. After washing with PBS three times, SCC7 cells were imaged using a confocal microscope.

[0096] The SCC7 cells after the ten different treatments were collected, washed with PBS three times, stained with V-FITC / PI apoptosis detection kit, and detected by flow cytometry.

[0097] SCC7 cells were plated at 2 × 10 4 The cells were inoculated in a 24-well plate at a density of 100 cells and cultured overnight. After the above 10 different treatments, the supernatant was collected and the concentrations of ATP and HMGB1 were detected using an ELISA kit. Finally, the corresponding absorbance was measured using an enzyme-labeled instrument.

[0098] (2) Experimental results

[0099] The in vitro killing effect of TPZ / TPP@RBC-FA is as follows Figure 5 As shown, the results showed that at the concentrations of TPZ: 50 μg / mL and TPP: 40 μg / mL, the prepared TPZ / TPP@RBC-FA liposomes had basically achieved a strong killing effect on the target cells.

[0100] The results of live and dead staining of tumor cells after different treatments are shown in Figure 2. Figure 6As shown, the results showed that the experimental group (TPZ / TPP@RBC-FA liposome treatment group) had achieved a strong killing effect on target cells.

[0101] The results of flow cytometry detection of cell apoptosis after different treatments are as follows Figure 7 As shown, the flow cytometry results after staining the target cells after different treatments using the cell apoptosis kit showed that the prepared TPZ / TPP@RBC-FA liposomes had a strong effect on inducing apoptosis of target cells.

[0102] The release detection results of cell immunogenic death (ICD) related markers (ATP, HMGB1, CRT) after different treatments are shown in Figure 2. Figure 8 As shown, the release detection results of cell immunogenic death-related markers proved that the experimental group (TPZ / TPP@RBC-FA liposome treatment group) had the strongest ability to induce cell immunogenic death on target cells.

[0103] Among them, 1: control group (PBS treatment), 2: TPZ@RBC treatment group, 3: TPP@RBC treatment group, 4: TPZ / TPP@RBC treatment group, 5: TPZ / TPP@RBC-FA treatment group, 6: 655nm wavelength laser irradiation for 5 minutes, PBS treatment group, 7: 655nm wavelength laser irradiation for 5 minutes, TPZ@RBC treatment group, 8: 655nm wavelength laser irradiation for 5 minutes, TPP@RBC treatment group, 9: 655nm wavelength laser irradiation for 5 minutes, TPZ / TPP@RBC treatment group, 10: 655nm wavelength laser irradiation for 5 minutes, TPZ / TPP@RBC-FA treatment group.

[0104] 2. Verification of the in vivo tumor killing effect of TPZ / TPP@RBC-FA liposomes

[0105] (1) Experimental methods

[0106] Each mouse was subcutaneously injected with 150 μL of 2×10 7 SCC7 cells were injected subcutaneously on the right back with 1×10 cells suspended in 150 μL PBS. 7 SCC7 cells were used to establish SCC7 xenograft tumor models. One week later, the cells were divided into 10 groups and treated with different treatments: 1: control group (100 μL PBS treatment), 2: TPZ@RBC (0.2 g / kg, RBC: 5×10 6 ) treatment group, 3: TPP@RBC (0.2 g / kg, RBC: 5×10 6 ) treatment group, 4: TPZ / TPP@RBC (0.2 g / kg, RBC: 5×10 6) treatment group, 5: TPZ / TPP@RBC-FA (0.2 g / kg, RBC: 5×10 6 ) Treatment group, 6: 100 μL PBS, 655 nm wavelength, 0.1 W / cm 2 Laser irradiation 5 minutes treatment group, 7: TPZ@RBC (0.2g / kg, RBC: 5×10 6 ), 655nm wavelength, 0.1W / cm 2 Laser irradiation 5 minutes treatment group, 8: TPP@RBC (0.2g / kg, RBC: 5×10 6 ), 655nm wavelength, 0.1W / cm 2 Laser irradiation 5 minutes treatment group, 9: TPZ / TPP@RBC (0.2g / kg, RBC: 5×10 6 )655nm wavelength, 0.1W / cm 2 Laser irradiation 5 minutes treatment group, 10: TPZ / TPP@RBC-FA (0.2g / kg, RBC: 5×10 6 )655nm wavelength, 0.1W / cm 2 The laser was irradiated for 5 minutes in each treatment group. The size of the subcutaneous tumor was measured with a vernier caliper every other day.

[0107] All mice were killed after 18 days of different treatments, and the tumor tissue of each mouse was removed and ground. The cells obtained from the ground tumor tissue were stained with CD45, CD80, CD86, CD3, CD4 and CD8, and then detected by flow cytometry to evaluate the in vivo immune efficacy.

[0108] (2) Experimental results

[0109] The growth curve of mouse tumors after different treatments is shown in the figure below. Fig. 9 As shown, in the in vivo experiment, the tumor growth of mice treated with the experimental group (TPZ / TPP@RBC-FA liposome treatment group) was the slowest, and it had a significant tumor inhibitory effect compared with other control groups, verifying the in vivo anti-tumor ability of liposomes under light treatment.

[0110] The results of in vivo immune efficacy evaluation of DC cell maturation and T lymphocyte activation levels in primary tumors and secondary tumors of different groups of mice are as follows Fig.10 As shown, the results of flow cytometry detection of immune cells in tumor tissues proved that the activation and maturation levels of intratumoral immune cells (DC cells and T cells) in mice treated with the experimental group (TPZ / TPP@RBC-FA liposome treatment group) were the highest, proving that the TPZ / TPP@RBC-FA liposomes prepared by the present invention have a strong ability to activate the immune system of mice in vivo under light conditions.

Claims

1. A method for preparing TPZ / TPP@RBC-FA liposomes, characterized in that: The method comprises the following steps: The tetracarboxylporphine solution and DSPE-PEG-FA powder were added to the red blood cell membrane suspension, and the mixture was shaken and mixed in the dark to obtain a mixed solution A; Adding tirapazamine solution to the mixed solution A, shaking and mixing in the dark to obtain a mixed solution B; The mixed solution B was ultrasonically disrupted on ice to obtain TPZ / TPP@RBC-FA liposomes.

2. The method according to claim 1, characterized in that The concentration of the tetracarboxyl porphine solution is 5-15 mg / mL, and the amount added is 7.5-17.5 μL; The added amount of the DSPE-PEG-FA powder is 0.2-3.5 mg.

3. The method according to claim 2, characterized in that The concentration of the tetracarboxylporphine solution is 10 mg / mL, and the amount added is 12.5 μL The added amount of the DSPE-PEG-FA powder is 1 mg.

4. The method according to claim 1, characterized in that The amount of the red blood cell membrane is 0.5-3.5 mg; Optionally, the amount of the red blood cell membrane is 2 mg; Optionally, the light-proof oscillation time of the mixed solution A obtained after the light-proof oscillation mixing is 1-5h; Optionally, the light-proof oscillation time of the mixed solution A obtained after the light-proof oscillation mixing is 2 hours.

5. The method according to claim 1, characterized in that The concentration of the tirapazamine solution is 5-15 mg / mL, and the amount added is 5-15 μL; Optionally, the concentration of the tirapazamine solution is 10 mg / mL, and the amount added is 10 μL; Optionally, the light-proof oscillation time of the mixed solution B obtained after the light-proof oscillation mixing is 10-60 min; Optionally, the light-proof oscillation time of the mixed solution B obtained after the light-proof oscillation mixing is 30 minutes.

6. The method according to claim 1, characterized in that The conditions for ultrasonic disruption on ice are to use a probe ultrasonic cell disruptor to insert the probe into the liquid surface of the mixed solution B for ultrasonication for 2-8 minutes; Optionally, the ultrasonic disruption on ice is performed by inserting a probe into the liquid surface of the mixed solution B using a probe ultrasonic cell disruptor for 5 minutes; Optionally, the erythrocyte membrane is a purified erythrocyte membrane; Optionally, the method further comprises purifying the obtained TPZ / TPP@RBC-FA liposomes; Optionally, the purification includes dialyzing the obtained TPZ / TPP@RBC-FA liposomes under magnetic stirring to obtain purified TPZ / TPP@RBC-FA liposomes; Optionally, the magnetic stirring condition is 100-300 rpm, 1-3 days; Optionally, the magnetic stirring condition is 200 rpm for 2 days.

7. A TPZ / TPP@RBC-FA liposome, characterized in that: The TPZ / TPP@RBC-FA liposomes are prepared by loading tirapazamine and tetracarboxylporphine into liposomes based on red blood cell membranes modified with folic acid; Optionally, the TPZ / TPP@RBC-FA liposome is a TPZ / TPP@RBC-FA liposome prepared by the method according to any one of claims 1-6.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the TPZ / TPP@RBC-FA liposomes described in claim 7.

9. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the pharmaceutical composition according to claim 8.

10. Use of the TPZ / TPP@RBC-FA liposomes prepared according to any one of claims 1 to 6 in the preparation of drugs for treating cancer; Optionally, the cancer comprises oral cancer, nasopharyngeal cancer, gastric cancer, lung cancer, colorectal cancer, kidney cancer, ovarian cancer, liver cancer, breast cancer, cervical cancer, bladder cancer, colorectal cancer, soft tissue sarcoma, head and neck cancer, follicular lymphoma, B-cell lymphoma or pancreatic cancer.

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