CN108324958B - Preparation method of purpurin 18-liposome nano-vesicles and application of purpurin 18-liposome nano-vesicles in preparation of drugs for treating tumors - Google Patents
Preparation method of purpurin 18-liposome nano-vesicles and application of purpurin 18-liposome nano-vesicles in preparation of drugs for treating tumors Download PDFInfo
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Abstract
A process for preparing the purpurin 18-liposome nano vesicles and its application in preparing the medicines for treating tumor includes such steps as covalent linking between purpurin 18 and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphorylcholine to obtain Pp18-lipid covalent conjugate, mixing Pp18-lipid covalent conjugate, distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethanediol 2000, filming, hydrating and extruding. The invention improves the loading rate of the purpurin 18 on the liposome; the water solubility of Pp18 is improved, the loading efficiency of Pp18 can be controlled independently, the particle size of the prepared liposome is about 100nm, the penetration retention effect of the liposome at a tumor part is enhanced, the drug utilization rate and the PDT treatment effect are improved, and the good photothermal conversion performance of high proportion Pp18-lipid is utilized, so that the liposome can be used for PTT treatment of tumors.
Description
Technical Field
The invention belongs to the field of nano-drug preparations, and relates to a preparation method of an purpurin 18-liposome nano-vesicle and application thereof in preparing drugs for treating tumors.
Background
Malignant tumors, one of the major public health problems worldwide, greatly endanger the health of human beings, and become the first killer of human beings in the new century. The current tumor treatment strategies mainly comprise surgical treatment, drug treatment, radiation treatment and biological treatment, and each method has certain defects: the operation treatment has high risk and is easy to relapse and transfer; the drug therapy can generate serious toxic and side effects due to non-tumor targeting; the biological treatment cost is high, and the recurrence is easy; the radiotherapy period is long, and radiation damage exists. Among them, Photodynamic Therapy (PDT) and Photothermal Therapy (PTT) generated by near-infrared laser irradiation can remove tumor tissues at a fixed point without affecting surrounding normal tissues due to the selectivity and controllability of laser, and are receiving more and more attention, and become a treatment mode with huge potential.
The key in the photodynamic and photothermal therapy of tumor application is the selection of photosensitizer and photothermal agent, and the chlorophyllin degradation product has the characteristics of ideal action spectrum, high near infrared region absorption coefficient, rapid elimination in vivo and the like, and can be used as PDT medicine and synthetic precursor with better application prospect.
The Purpurin 18(Purpurin 18, Pp18) is a chlorophyll derivative with strong hydrophobicity, has three characteristic absorption peaks which are respectively positioned at 410nm, 545nm and 705nm, wherein 705nm has strong absorption and longer absorption wavelength, belongs to a near infrared band, has good penetrability and can be used for PDT treatment of tumors. PDT treatment is to treat tumors by local irradiation, however, due to the strong hydrophobicity of Pp18, purpurin has poor solubility in vivo, is not easy to be taken up, and has low efficiency of reaching tumor sites, thereby affecting the effect of PDT treatment.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provides a preparation method of a nano vesicle and application of the nano vesicle in preparing a medicine for treating tumors.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing a purpurin 18-liposome nano vesicle comprises the steps of adopting organic acylation reaction to covalently connect the purpurin 18 and 1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphorylcholine to construct a Pp18-lipid covalent conjugate, mixing the Pp18-lipid covalent conjugate, distearoyl phosphatidylcholine, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol 2000, drying the mixture by using nitrogen to form a membrane, hydrating the membrane, and then adopting a liposome extrusion method to prepare the purpurin 18-liposome nano vesicle.
The invention is further improved in that the method specifically comprises the following steps:
(1) dissolving Pp18, P-lyso PC, EDCI and DMAP in chloroform, protecting from light and argon for acylation reaction, connecting Pp18 to a side chain of the P-lyso PC, and purifying to obtain Pp 18-lipid; wherein Pp18 is purpurin 18, P-lyso PC is 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine;
(2) dissolving distearoyl phosphatidylcholine, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and Pp18-lipid in chloroform, uniformly mixing, and blow-drying with nitrogen to form a film;
(3) oscillating and hydrating in PBS water bath, and repeatedly freezing and thawing to obtain a hydrated purpurin 18-lipid mixed solution;
(4) squeezing the hydrated purpurin 18-lipid mixed solution by using a squeezer to obtain the purpurin 18-liposome.
The invention further improves the method that in the step (1), the molar ratio of Pp18, P-lyso PC, EDCI and DMAP is (0.5-1): 1: (1-2): (1-2).
A further improvement of the invention is that Pp18 has a molecular weight of 564.631 and P-lyso PC has a molecular weight of 495.63.
The further improvement of the invention is that the specific purification process in the step (1) is as follows: performing column chromatography, wherein an eluent is a mixed solution of chloroform, methanol and acetic acid, and the polarity of the eluent is adjusted by adjusting the proportion of each solvent, wherein the volume ratio of the chloroform to the methanol to the acetic acid is (80-90): (10-15): (0-5).
In a further development of the invention, in step (2), the ratio of DSPC: chol: DSPE-PEG 2000: the quantity ratio of Pp18-lipid substance is (0-55): (30-40): 5: (1-65).
In a further improvement of the invention, the ratio of Pp18 to trichloromethane in step (1) is that the ratio of Pp18 to trichloromethane is 56.4 mg: 10 mL; the ratio of distearoyl phosphatidylcholine to chloroform in step (2) was 12.13 mg: 1 mL.
The invention is further improved in that the pH value of the PBS in the step (3) is 7.0-7.4, and the temperature of the water bath is 65 ℃.
The invention is further improved in that the extrusion temperature in the step (4) is 40-65 ℃.
An application of purpurin 18-liposome nanometer vesicle in preparing medicine for treating tumor is disclosed.
Compared with the prior art, the invention has the following beneficial technical effects:
the synthesized Pp18-lipos of the invention generates Pp 18-lipd through the organic reaction of P-lyso PC and Pp18, has an amphiphilic structure similar to common phospholipid, the lipid bilayer vesicle Pp18-lipos is formed by self-assembly in a water phase, and Pp18 is distributed among lipid bilayers, thereby effectively solving the characteristic of poor water solubility of Pp18, and the proportion of Pp18 can be adjusted independently, compared with the common liposome carrying Pp18, the loading capacity is obviously improved, because the phospholipid component is similar to the cell membrane component, the prepared nano-particles have good biocompatibility, the drug toxicity of Pp18 is reduced, meanwhile, the formed nano structure can be effectively and obviously enriched at a tumor part due to the enhanced penetration retention effect, and the prepared high-proportion Pp18-lipos can cause a fluorescence quenching phenomenon under the light activation due to the high-density arrangement of Pp18 between double-layer molecules, so that the nano structure has higher photo-thermal conversion efficiency. Pp18 used in the invention belongs to porphyrin derivatives, is hydrophobic molecules, has a carboxylic acid group on the porphyrin ring, and can be used for photodynamic therapy. The P-lyso PC used is one of phospholipid molecules, and a hydroxyl group is generated after one of two hydrophobic carbon chains of the phospholipid molecule is hydrolyzed, so that a hydrophilic head is reserved, and the P-lyso PC can also react with the carboxyl of the Pp 18. Compared with the traditional liposome system for loading porphyrin, the loading rate of the purpurin 18 on the liposome is greatly improved; improves the passive targeting performance of the purpurin 18 at the tumor part.
The purpurin 18-liposome nano vesicle prepared by the invention is applied to the preparation of tumor treatment drugs, so that the property of the purpurin 18 which is easy to aggregate in aqueous solution is improved, the histocompatibility is improved, and the toxicity is reduced; due to the constructed Pp18-lipos nano vesicles, a large amount of purpurin 18 is regularly stacked between lipid bilayers to generate a fluorescence self-quenching phenomenon, and under the irradiation of specific wavelength, originally excited fluorescence is converted into heat energy which can be used as a photo-thermal agent to realize photo-thermal conversion and be used for tumor photo-thermal therapy (PTT); by reducing the proportion of Pp18-lipid, in a low proportion of Pp18-lipid, porphyrin is not quenched under the irradiation of light with specific wavelength, and singlet oxygen is generated and used as a photosensitizer for photodynamic therapy (PDT) tumor; tumor diagnosis and imaging: the low proportion of Pp18-lipos can be used for in vivo tumor near infrared fluorescence imaging because the fluorescence is not quenched. The constructed porphyrin liposome is passively targeted to a tumor part, namely, the porphyrin liposome is used as a photosensitizer/photothermal agent for tumor treatment and can also be used for tumor diagnosis imaging, so that the defects of the traditional medicine are overcome, and the tumor cure rate is improved. The Purpurin 18-liposome (Pp18-lipos) nano vesicle constructed by the invention not only improves the water solubility of Pp18, can independently control the load efficiency of Pp18, has the particle size of about 100nm after being extruded, enhances the permeation retention effect, improves the drug utilization rate and the PDT treatment effect, utilizes the controllable load rate, and after the load rate is improved, because the high-density Pp18 is regularly arranged between Pp18-lipos double-layer molecules, the fluorescence quenching phenomenon occurs, so that the energy conversion is changed from light energy to heat energy, thereby introducing the PTT treatment.
Drawings
FIG. 1 shows the reaction structure of Pp18-lipid covalent conjugate.
FIG. 2 is a thin layer chromatography assay of Pp18-lipid covalent conjugate, wherein, a is before reaction and b is after reaction.
FIG. 3 is a high performance liquid assay of Pp18-lipid covalent conjugates, wherein a is before reaction and b is after reaction.
FIG. 4 is a high resolution mass spectrometric detection of Pp18-lipid covalent conjugates.
FIG. 5 is Pp18-lipid covalent conjugate column chromatography purification thin layer chromatography detection, wherein, a is before purification, b is after purification and not color, c is after purification and color.
FIG. 6 is an electron microscope image of Pp18-lipos nanovesicles, wherein a is the product prepared in example 6 and b is the product prepared in example 11.
FIG. 7 is a particle size distribution diagram of Pp18-lipos nanovesicles, wherein a is the product prepared in example 6 and b is the product prepared in example 11.
FIG. 8 shows the singlet oxygen yield of Pp18-lipos nanovesicles.
FIG. 9 shows the in vitro photothermal temperature change of Pp18-lipos nanovesicles.
FIG. 10 shows the cytotoxicity test of Pp18-lipos nanovesicles, wherein a is Pp18-lipos toxicity, and b is Pp18 toxicity.
FIG. 11 is 2% Pp18-lipos nanovesicle-mediated killing of breast cancer cells by PDT.
FIG. 12 is the killing of breast cancer cells by 65% Pp18-lipos nanovesicle mediated PTT.
FIG. 13 shows in vitro and in vivo near infrared fluorescence imaging of Pp18-lipos nanovesicles.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Adopting a catalyst to catalyze organic acylation reaction to covalently connect Pp18 with 1-Palmitoyl-2-hydroxy-sn-glycerol-3-phosphorylcholine (1-Palmitoyl-sn-glycero-3-phosphorylcholine, P-lysoPC, lipid) to construct Pp 18-lipid-covalent conjugate, using Pp 18-lipid-covalent conjugate to mix distearoyl phosphatidylcholine (DSPC), cholesterol (Chol), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) according to a certain proportion, adopting nitrogen to blow and dry the membrane, then hydrating the membrane, and adopting a liposome extrusion method to prepare the purpurin 18-liposome (Pp 18-lipids) nano-vesicle. Wherein 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (1-Palmitoyl-sn-glycero-3-phosphoricholine, P-lyso PC, lipid) is a phospholipid main structure, porphyrin photosensitizer purpurin 18(Pp18) is a photoactivatable element, and two molecules are covalently connected together as shown in fig. 1. The method specifically comprises the following steps:
the invention provides an in-vitro singlet oxygen detection method of Pp18-lipos nano vesicles, which comprises the following specific steps of:
and irradiating the Pp18-lipos nano vesicle with 705nm light to activate the Pp18-lipos nano vesicle to generate singlet oxygen, and detecting the singlet oxygen by a fluorescence spectrophotometer by using a singlet oxygen probe.
The invention provides a method for detecting in-vitro photothermal conversion capacity of Pp18-lipos nano vesicles, wherein the percentage of the content of Pp18-lipid substances is 1-65%, and the method comprises the following steps:
irradiating Pp18-lipos at 705nm, and detecting the temperature rise of the Pp18-lipos nano vesicle by using an infrared thermal imager.
The invention provides a method for applying nano vesicles in preparing a photodynamic tumor cell treatment drug, which comprises the following steps:
administering Pp18-lipos nanovesicles having an amount of Pp18-lipid of 2% in percentage to MDA-MB-231 cells;
irradiating Pp18-lipos in MDA-MB-231 cells by using 705nm wavelength light to activate Pp18-lipos to generate active oxygen to kill tumor cells, and detecting by using an MTT method.
The invention provides an application method of nano vesicles in preparation of tumor cell photothermal treatment medicines, which comprises the following steps:
administering Pp18-lipos nanovesicles having an amount of substance of Pp18-lipid percentage of 65% to MDA-MB-231 cells;
irradiating Pp18-lipos in MDA-MB-231 cells by using 705nm light to activate Pp18-lipos to increase temperature and kill tumor cells, and detecting by using an MTT method.
The invention provides a method for detecting in-vivo drug distribution by nano vesicles in near-infrared fluorescence imaging, which comprises the following steps:
administering Pp18-lipos nanovesicles having an amount of Pp18-lipid of 2% in percentage to 4T1 transplanted tumor mice;
irradiating the Pp18-lipos nano-vesicles with the percentage of 2% of the substance in the laser target region Pp 18-lipd by using 705nm wavelength light to activate the Pp18-lipos nano-vesicles in the target region to generate fluorescence; the intensity of the generated fluorescent signal is detected using small animal live body imaging.
The purpurin 18-liposome nano vesicle prepared by the method is applied to preparing medicaments for treating tumors.
The synthetic method of the purpurin 18-phospholipid comprises the following steps:
(1) pp18, lipid, EDCI (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) and DMAP (4- (dimethylamino) pyridine) in a molar ratio (0.5-1): 1: (1-2): (1-2) mixing in 10mL of dehydrated chloroform, mixing, keeping out of the sun, protecting with argon, and stirring for 24 hours to form a mixed solution I. Wherein, Pp18 has a molecular weight of 564.631 and P-lyso PC has a molecular weight of 495.63. The ratio of Pp18 to chloroform was 56.4 mg: 10 mL.
(2) Placing the dried three-mouth bottle on a magnetic stirrer, connecting argon gas for protection, adding the mixed solution I into the three-mouth bottle, opening the magnetic stirrer, stirring for 24 hours in a dark place to form a reaction mixed solution II, and carrying out thin-layer chromatography, high-resolution mass spectrometry and high-performance liquid chromatography detection on the reaction mixed solution II, as shown in fig. 2, 3 and 4. As can be seen from FIGS. 2, 3 and 4, purpurin 18-phospholipid (Pp18-lipid) was produced.
(3) In order to purify the obtained Pp18-lipid by adopting column chromatography and gradient elution, the used eluent is a mixed solution of chloroform, methanol and acetic acid, the polarity of the eluent is adjusted by adjusting the proportion of each solvent, and the volume ratio of the chloroform, the methanol and the acetic acid is (80-90): (10-15): (0-5). The specific process is as follows: mixing the reaction mixed solution II with silica gel powder, carrying out rotary evaporation at 37 ℃ to remove trichloromethane, adsorbing a sample on the silica gel, loading the silica gel column, loading the sample, adding an eluent, carrying out column chromatography purification on the mixed sample, and carrying out gradient elution on the mixed sample, wherein the polarity of the eluent is (r) trichloromethane sequentially from high to low: methanol volume ratio of 9:1, chloroform: the volume ratio of methanol is 8.5: 1.5, trichloromethane: methanol: the volume ratio of acetic acid is 8: 1.5: 0.5, the purified product Pp18-lipid was detected by thin layer chromatography.
(4) Removing chloroform and methanol at 37 ℃ by using a rotary evaporator, removing acetic acid at 65 ℃, primarily drying, transferring and subpackaging into a test tube, further drying by using nitrogen, placing in a vacuum drying tank for vacuum drying for 3h, sealing by using a sealing film, and storing in a refrigerator at-20 ℃ for later use.
The preparation method of the purpurin 18-liposome nano vesicle comprises the following steps:
(1) distearoylphosphatidylcholine, cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (calculated as purpurin 18 by mass), were dissolved in chloroform, respectively, and the ratio of the total amount of distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and Pp18-lipid was 100, DSPC: chol: DSPE-PEG 2000: the molar ratio of Pp18-lipid is (0-55): (30-40): 5: (1-65) mixing, sequentially adding into a test tube to form a phospholipid mixed solution with the Pp18-lipid content of 1-65%, and heating in a 65 ℃ water bath for 5 min;
wherein the molar ratio of distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and Pp18-lipid is (0-55) based on the total amount of distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and Pp18-lipid being 100: (30-40): 5: (1-65) the sum of the amounts of distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and Pp18-lipid is 100. For example, when the amount of the distearoylphosphatidylcholine species is 30, the amount of the cholesterol species is 40, the amount of the distearoylphosphatidylethanolamine-polyethylene glycol 2000 species is 5, the amount of the Pp18-lipid species is 25, and the total amount of the distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and Pp18-lipid is 100.
(3) Fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating chloroform along with the blowing of the nitrogen, and closing the nitrogen after a uniform lipid film is formed on the wall of the test tube and no liquid is at the bottom of the test tube;
(4) sealing the opening of the test tube by using a sealing film, pricking a small hole on the sealing film to communicate the sealing film with the outside, putting the sealing film into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 h to fully volatilize and clean residual solvent;
(5) oscillating a lipid film in a hydration test tube in 65 ℃ water by using 1mL of 0.01mol/L PBS (phosphate buffered saline) with the pH value of 7.2-7.4 for 2min, vortexing on a vortexing instrument for 20s (after 5 cycles), allowing the film on the wall of the test tube to fall off, freezing by using liquid nitrogen, rapidly melting and vortexing at 65 ℃, and after 5 cycles, causing no macroscopic aggregated particles in the liquid to obtain a hydrated purpurin lipid mixed solution;
(6) preparing a liposome squeezer, heating the squeezer to 40-65 ℃, squeezing the hydrated purpurin 18-liposome by using the squeezer, sequentially passing through a polycarbonate membrane with the particle size of 200nm and 100nm, and after 10 cycles, changing a purpurin 18-liposome suspension with the content of the prepared Pp18-lipid accounting for 1-65% into a transparent liquid to obtain the purpurin 18-liposome, adding a freeze-drying protective agent for freeze-drying, and storing by using a purpurin 18-liposome freeze-drying agent; wherein the freeze-drying protective agent is one or a mixture of more than two of mannose, lactose, glucose, amino acid, sucrose and polyethylene glycol, and is placed in a refrigerator at-80 deg.C for pre-freezing for 12h, and then is placed in a freeze-drying machine for freeze-drying for 24h to obtain the dried purpurin 18-liposome freeze-drying agent.
Pp18 used in the invention belongs to porphyrin derivatives, is a hydrophobic molecule, has a carboxylic acid group molecular weight of 564.631 on the porphyrin ring, and can be used for photodynamic therapy. The P-lyso PC adopted by the invention is one of phospholipid molecules, one of the two hydrophobic carbon chains of the phospholipid molecule is hydrolyzed to generate a hydroxyl group, a hydrophilic head is reserved, and the molecular weight of the P-lyso PC is 495.63 and can also react with Pp18 carboxyl.
The synthesized Pp18-lipos of the invention generates Pp 18-lipd through the organic reaction of P-lyso PC and Pp18, has an amphiphilic structure similar to common phospholipid, the lipid bilayer vesicle Pp18-lipos is formed by self-assembly in a water phase, and Pp18 is distributed among lipid bilayers, thereby effectively solving the characteristic of poor water solubility of Pp18, and the proportion of Pp18 can be adjusted independently, compared with the common liposome carrying Pp18, the loading capacity is obviously improved, because the phospholipid component is similar to the cell membrane component, the prepared nano-particles have good biocompatibility, the drug toxicity of Pp18 is reduced, meanwhile, the formed nano structure can be effectively and obviously enriched at a tumor part due to the enhanced penetration retention effect, and the prepared high-proportion Pp18-lipos can cause a fluorescence quenching phenomenon under the light activation due to the high-density arrangement of Pp18 between double-layer molecules, so that the nano structure has higher photo-thermal conversion efficiency.
The present invention will be described in further detail by way of specific examples
EXAMPLE 1 Synthesis of purpurin 18-Phospholipids
Weighing Pp 18: 56.4mg, P-lyso PC: 98.7mg, EDCI: 38.35mg, DMAP: 24.35mg, dissolving in 10mL anhydrous chloroform, adding into a reaction bottle filled with argon in sequence, stirring at room temperature in a dark place for 24h, ending the reaction, mixing the reaction mixture with silica gel powder, evaporating the solvent to 37 ℃ in a rotating way, purifying by using silica gel column chromatography, performing gradient elution, and sequentially adding an eluent with the polarity of (i) chloroform from high to low: methanol volume ratio of 9:1, chloroform: the volume ratio of methanol is 8.5: 1.5, trichloromethane: methanol: the volume ratio of acetic acid is 8: 1.5: 0.5, collecting Pp18-lipid, primarily evaporating the solvent by using a rotary evaporator, drying by blowing nitrogen, drying for 3h in vacuum, and storing in a refrigerator at the temperature of minus 20 ℃.
EXAMPLE 2 Synthesis of purpurin 18-Phospholipids
Weighing Pp 18: 112.8mg, P-lyso PC: 98.7mg, EDCI: 38.35mg, DMAP: 24.35mg, dissolving in 10mL anhydrous chloroform, adding into a reaction bottle filled with argon in sequence, stirring at room temperature in a dark place for 24h, ending the reaction, mixing the reaction mixture with silica gel powder, evaporating the solvent to 37 ℃ in a rotating way, purifying by using silica gel column chromatography, performing gradient elution, and sequentially adding an eluent with the polarity of (i) chloroform from high to low: methanol volume ratio of 9:1, chloroform: the volume ratio of methanol is 8.5: 1.5, trichloromethane: methanol: the volume ratio of acetic acid is 8: 1.5: 0.5, collecting Pp18-lipid, primarily evaporating the solvent by using a rotary evaporator, drying by blowing nitrogen, drying for 3h in vacuum, and storing in a refrigerator at the temperature of minus 20 ℃.
EXAMPLE 3 Synthesis of purpurin 18-Phospholipids
Weighing Pp 18: 112.8mg, P-lyso PC: 98.7mg, EDCI: 76.7mg, DMAP: 48.7mg, dissolve in 10mL anhydrous chloroform, add in proper order in the reaction flask that leads to the argon gas, the room temperature is protected from the sun and is stirred 24h, the reaction is ended, mix reaction mixture and silica gel powder, rotatory evaporation to dryness solvent uses silica gel column chromatography purification, gradient elution, the eluant polarity is from high to low is (r) chloroform in proper order: methanol volume ratio of 9:1, chloroform: the volume ratio of methanol is 8.5: 1.5, trichloromethane: methanol: the volume ratio of acetic acid is 8: 1.5: 0.5, collecting Pp18-lipid, primarily evaporating the solvent by using a rotary evaporator, drying by blowing nitrogen, drying for 3h in vacuum, and storing in a refrigerator at the temperature of minus 20 ℃.
By varying the ratio of reactant Pp18, P-lyso PC to both catalysts (EDCI and DMAP are both catalysts), when Pp 18: a lipid: EDCI: DMAP molar ratio 1: 1: 2: 2, the reaction yield is relatively high, the HPLC is shown in FIG. 2, and the purification components are single, as shown in FIG. 5.
Example 4 preparation of purpurin 18-Liposome nanovesicles
(1) Drying by nitrogen to form a film: in terms of a molar ratio of Distearoylphosphatidylcholine (DSPC), cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (Pp18-lipid prepared in example 1, 2 or 3, calculated on the mass of purpurin 18) of 55: 40: 5: 0 weighing the four substances, respectively dissolving 12.13mg, 4.31mg, 3.92mg and 0mg in chloroform, mixing in a test tube of 2 × 18cm, keeping the final volume at 1mL, placing in a 65 deg.C water bath, mixing, and heating; fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating trichloromethane along with the blowing of the nitrogen, sealing the opening of the test tube by using a sealing film after a uniform lipid thin film is formed on the wall of the test tube and no liquid is left at the bottom of the test tube, poking a small hole on the sealing film to communicate the small hole with the outside, putting the small hole into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 hours to fully volatilize residual solvent;
(2) film hydration: oscillating a lipid film in a hydration test tube in water at 65 ℃ by using 1mL of 0.01mol/L PBS (phosphate buffered saline) with the pH value of 7.2-7.4 for 2min, vortexing on a vortexing instrument for 20s (after 5 cycles), falling off the film on the wall of the test tube, freezing by using liquid nitrogen, rapidly melting and vortexing at 65 ℃, and after 5 cycles, obtaining a liposome mixed solution without macroscopic aggregated particles in the liquid;
(3) and (3) liposome extrusion: preparing a liposome squeezer, heating the squeezer to 40 ℃, squeezing the hydrated purpurin 18-liposome by the squeezer, sequentially passing through a polycarbonate membrane with the wavelength of 200nm and 100nm for 10 cycles, then preparing 0% Pp18-lipos suspension to be transparent liquid, cooling to room temperature and storing in a refrigerator with the temperature of 4 ℃.
Example 5 preparation of purpurin 18-Liposome nanovesicles
(1) Drying by nitrogen to form a film: according to the molar ratio of Distearoylphosphatidylcholine (DSPC), cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (calculated by the weight of purpurin 18, Pp18-lipid prepared in example 1, 2 or 3) of 54: 40: 5: 1 weighing 11.91mg, 4.31mg, 3.92mg and 0.157mg of the four substances respectively, dissolving in trichloromethane, mixing in a test tube of 2 x 18cm, keeping the final volume at 1mL, placing in a 65 ℃ water bath, mixing uniformly, and heating; fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating trichloromethane along with the blowing of the nitrogen, sealing the opening of the test tube by using a sealing film after a uniform lipid thin film is formed on the wall of the test tube and no liquid is left at the bottom of the test tube, poking a small hole on the sealing film to communicate the small hole with the outside, putting the small hole into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 hours to fully volatilize residual solvent;
(2) film hydration: oscillating a lipid film in a hydration test tube in water at 65 ℃ by using 1mL of 0.01mol/L PBS (phosphate buffered saline) with the pH value of 7.2-7.4 for 2min, vortexing on a vortexing instrument for 20s (after 5 cycles), falling off the film on the wall of the test tube, freezing by using liquid nitrogen, rapidly melting and vortexing at 65 ℃, and after 5 cycles, obtaining a liposome mixed solution without macroscopic aggregated particles in the liquid;
(3) and (3) liposome extrusion: preparing a liposome squeezer, heating the squeezer to 40 ℃, squeezing the hydrated purpurin 18-liposome by the squeezer, sequentially passing through a polycarbonate membrane with the wavelength of 200nm and 100nm for 10 cycles, then changing the prepared suspension of 1% Pp18-lipos into transparent liquid, cooling to room temperature and storing in a refrigerator with the temperature of 4 ℃.
Example 6 preparation of purpurin 18-Liposome nanovesicles
(1) Drying by nitrogen to form a film: according to the mol ratio of Distearoylphosphatidylcholine (DSPC), cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (calculated by the weight of the purpurin 18) of 53: 40: 5: 2 weighing 11.69mg, 4.31mg, 3.92mg and 0.314mg of the four substances respectively, dissolving in trichloromethane, mixing in a test tube of 2 x 18cm, keeping the final volume at 1mL, placing in a 65 ℃ water bath, mixing uniformly, and heating; fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating trichloromethane along with the blowing of the nitrogen, sealing the opening of the test tube by using a sealing film after a uniform lipid thin film is formed on the wall of the test tube and no liquid is left at the bottom of the test tube, poking a small hole on the sealing film to communicate the small hole with the outside, putting the small hole into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 hours to fully volatilize residual solvent;
(2) film hydration: oscillating a lipid film in a hydration test tube in 65 ℃ water by using 1mL of 0.01M PBS (with the pH value of 7.2-7.4), oscillating for 2min, carrying out vortex on a vortex instrument for 20s (after 5 cycles), enabling the film on the wall of the test tube to fall off, freezing by using liquid nitrogen, rapidly melting and vortex at 65 ℃, and after 5 cycles, enabling no macroscopic aggregated particles to exist in the liquid to obtain a liposome mixed solution;
(3) and (3) liposome extrusion: preparing a liposome squeezer, heating the squeezer to 40 ℃, squeezing the hydrated purpurin 18-liposome by using the squeezer, sequentially passing through a polycarbonate membrane with the wavelength of 200nm and 100nm for 10 cycles, then preparing 2% Pp18-lipos suspension to be transparent liquid, cooling to room temperature and storing in a refrigerator with the temperature of 4 ℃.
Example 7 preparation of purpurin 18-Liposome nanovesicles
(1) Drying by nitrogen to form a film: according to the mol ratio of distearoyl phosphatidylcholine (DSPC), cholesterol (Chol), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (calculated by the weight of the purpurin 18) of 50: 40: 5: 5 weighing 11.03mg, 4.31mg, 3.92mg and 0.785mg of the four substances respectively, dissolving in trichloromethane, mixing in a test tube of 2 x 18cm, keeping the final volume at 1mL, placing in a 65 ℃ water bath, mixing uniformly, and heating; fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating trichloromethane along with the blowing of the nitrogen, sealing the opening of the test tube by using a sealing film after a uniform lipid thin film is formed on the wall of the test tube and no liquid is left at the bottom of the test tube, poking a small hole on the sealing film to communicate the small hole with the outside, putting the small hole into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 hours to fully volatilize residual solvent;
(2) film hydration: oscillating a lipid film in a hydration test tube in 65 ℃ water by using 1mL of 0.01M PBS (with the pH value of 7.2-7.4), oscillating for 2min, carrying out vortex on a vortex instrument for 20s (after 5 cycles), enabling the film on the wall of the test tube to fall off, freezing by using liquid nitrogen, rapidly melting and vortex at 65 ℃, and after 5 cycles, enabling no macroscopic aggregated particles to exist in the liquid to obtain a liposome mixed solution;
(3) and (3) liposome extrusion: preparing a liposome squeezer, heating the squeezer to 40 ℃, squeezing the hydrated purpurin 18-liposome by using the squeezer, sequentially passing through a polycarbonate membrane with the wavelength of 200nm and 100nm for 10 cycles, then preparing 5% Pp18-lipos suspension to be transparent liquid, cooling to room temperature and storing in a refrigerator with the temperature of 4 ℃.
Example 8 preparation of purpurin 18-Liposome nanovesicles
(1) Drying by nitrogen to form a film: according to the mol ratio of distearoyl phosphatidylcholine (DSPC), cholesterol (Chol), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (calculated by the weight of the purpurin 18) of 45: 40: 5: 10 weighing the four substances, respectively dissolving 9.93mg, 4.31mg, 3.92mg and 1.57mg in chloroform, mixing in a test tube of 2 × 18cm, keeping the final volume at 1mL, placing in a 65 deg.C water bath, mixing, and heating; fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating trichloromethane along with the blowing of the nitrogen, sealing the opening of the test tube by using a sealing film after a uniform lipid thin film is formed on the wall of the test tube and no liquid is left at the bottom of the test tube, poking a small hole on the sealing film to communicate the small hole with the outside, putting the small hole into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 hours to fully volatilize residual solvent;
(2) film hydration: oscillating a lipid film in a hydration test tube in 65 ℃ water by using 1mL of 0.01M PBS (with the pH value of 7.2-7.4), oscillating for 2min, carrying out vortex on a vortex instrument for 20s (after 5 cycles), enabling the film on the wall of the test tube to fall off, freezing by using liquid nitrogen, rapidly melting and vortex at 65 ℃, and after 5 cycles, enabling no macroscopic aggregated particles to exist in the liquid to obtain a liposome mixed solution;
(3) and (3) liposome extrusion: preparing a liposome squeezer, heating the squeezer to 65 ℃, squeezing the hydrated purpurin 18-liposome by using the squeezer, sequentially passing through a polycarbonate membrane with the wavelength of 200nm and 100nm for 10 cycles, then preparing 10% Pp18-lipos suspension to be transparent liquid, cooling to room temperature and storing in a refrigerator with the temperature of 4 ℃.
Example 9 preparation of purpurin 18-Liposome nanovesicles
(1) Drying by nitrogen to form a film: according to the mol ratio of distearoyl phosphatidylcholine (DSPC), cholesterol (Chol), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (calculated by the weight of the purpurin 18) of 40: 40: 5: 15 weighing the four substances, respectively dissolving 8.83mg, 4.31mg, 3.92mg and 2.355mg in chloroform, mixing in a test tube of 2 × 18cm, keeping the final volume at 1mL, placing in a 65 deg.C water bath, mixing, and heating; fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating trichloromethane along with the blowing of the nitrogen, sealing the opening of the test tube by using a sealing film after a uniform lipid thin film is formed on the wall of the test tube and no liquid is left at the bottom of the test tube, poking a small hole on the sealing film to communicate the small hole with the outside, putting the small hole into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 hours to fully volatilize residual solvent;
(2) film hydration: oscillating a lipid film in a hydration test tube in 65 ℃ water by using 1mL of 0.01M PBS (with the pH value of 7.2-7.4), oscillating for 2min, carrying out vortex on a vortex instrument for 20s (after 5 cycles), enabling the film on the wall of the test tube to fall off, freezing by using liquid nitrogen, rapidly melting and vortex at 65 ℃, and after 5 cycles, enabling no macroscopic aggregated particles to exist in the liquid to obtain a liposome mixed solution;
(3) and (3) liposome extrusion: preparing a liposome squeezer, heating the squeezer to 65 ℃, squeezing the hydrated purpurin 18-liposome by using the squeezer, sequentially passing through a polycarbonate membrane with the wavelength of 200nm and 100nm for 10 cycles, then preparing 15 percent Pp18-lipos suspension to be transparent liquid, cooling to room temperature and storing in a refrigerator with the temperature of 4 ℃.
Example 10 preparation of purpurin 18-Liposome nanovesicles
(1) Drying by nitrogen to form a film: according to the mol ratio of distearoyl phosphatidylcholine (DSPC), cholesterol (Chol), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (calculated by the weight of the purpurin 18) of 35: 40: 5: 20 weighing the four substances, respectively dissolving 7.73mg, 4.31mg, 3.92mg and 3.14mg in chloroform, mixing in a test tube of 2 × 18cm, keeping the final volume at 1mL, placing in a 65 deg.C water bath, mixing, and heating; fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating trichloromethane along with the blowing of the nitrogen, sealing the opening of the test tube by using a sealing film after a uniform lipid thin film is formed on the wall of the test tube and no liquid is left at the bottom of the test tube, poking a small hole on the sealing film to communicate the small hole with the outside, putting the small hole into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 hours to fully volatilize residual solvent;
(2) film hydration: oscillating a lipid film in a hydration test tube in 65 ℃ water by using 1mL of 0.01M PBS (with the pH value of 7.2-7.4), oscillating for 2min, carrying out vortex on a vortex instrument for 20s (after 5 cycles), enabling the film on the wall of the test tube to fall off, freezing by using liquid nitrogen, rapidly melting and vortex at 65 ℃, and after 5 cycles, enabling no macroscopic aggregated particles to exist in the liquid to obtain a liposome mixed solution;
(3) and (3) liposome extrusion: preparing a liposome squeezer, heating the squeezer to 65 ℃, squeezing the hydrated purpurin 18-liposome by using the squeezer, sequentially passing through a polycarbonate membrane with the wavelength of 200nm and 100nm for 10 cycles, then preparing 20 percent Pp18-lipos suspension into transparent liquid, cooling to room temperature and storing in a refrigerator with the temperature of 4 ℃.
Example 11 preparation of purpurin 18-Liposome nanovesicles
(1) Drying by nitrogen to form a film: according to the mol ratio of distearoyl phosphatidylcholine (DSPC), cholesterol (Chol), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (calculated by the weight of the purpurin 18) of 0: 30: 5: 65 weighing 0mg, 4.31mg, 3.92mg and 10.205mg of the four substances respectively, dissolving in chloroform, mixing in a test tube of 2 x 18cm, keeping the final volume at 1mL, placing in a 65 ℃ water bath kettle, mixing uniformly, and heating; fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating trichloromethane along with the blowing of the nitrogen, sealing the opening of the test tube by using a sealing film after a uniform lipid thin film is formed on the wall of the test tube and no liquid is left at the bottom of the test tube, poking a small hole on the sealing film to communicate the small hole with the outside, putting the small hole into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 hours to fully volatilize residual solvent;
(2) film hydration: oscillating a lipid film in a hydration test tube in 65 ℃ water by using 1mL of 0.01M PBS (with the pH value of 7.2-7.4), oscillating for 2min, carrying out vortex on a vortex instrument for 20s (after 5 cycles), enabling the film on the wall of the test tube to fall off, freezing by using liquid nitrogen, rapidly melting and vortex at 65 ℃, and after 5 cycles, enabling no macroscopic aggregated particles to exist in the liquid to obtain a liposome mixed solution;
(3) and (3) liposome extrusion: preparing a liposome squeezer, heating the squeezer to 65 ℃, squeezing the hydrated purpurin 18-liposome by using the squeezer, sequentially passing through a polycarbonate membrane with the wavelength of 200nm and 100nm for 10 cycles, then preparing 65% Pp18-lipos suspension to be transparent liquid, cooling to room temperature and storing in a refrigerator with the temperature of 4 ℃.
By observation, the prepared Pp18-lipos solution was clear and had no aggregated particles visible to the naked eye.
By changing the temperature during extrusion by spinning, the obtained Pp18-lipos was found to have good stability during extrusion at 65 ℃.
The ratio of Pp18-lipid was varied to prepare 0-65% Pp18-lipid, i.e., the percentage of Pp18-lipid to the total amount of Distearoylphosphatidylcholine (DSPC), cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid was 1-65%, and the products prepared in example 6 and example 11 were observed by transmission electron microscopy to have a spherical vesicular shape and uniform size, and were distributed at about 100nm, as shown in FIG. 6.
The particle sizes of the products prepared in examples 7-11 were all around 100 nm. The particle size of 1-65% Pp18-lipos prepared by dynamic light scattering detection is consistent through electron microscope observation, the dispersion coefficient is small, and the dispersibility is good, as shown in FIG. 7.
Example 12
The singlet oxygen yield was measured using a fluorospectrophotometer with a singlet oxygen probe.
Taking prepared purpurin 18-liposome with different proportions, diluting the purpurin 18-liposome to 10 mug/mL (calculated by equivalent Pp18 concentration) by using 0.2 muM SOSG, taking a PBS group as a control group, wherein four light doses in each proportion are respectively 0J/cm2, 5J/cm2, 10J/cm2 and 15J/cm2, three times of each light dose are repeated, respectively placing the control group in a 96-well plate, 200 muL of each hole, turning on a laser, adjusting the laser power to be 50mW, adjusting the light spot diameter to be 1cm (the light density to be 63.66mW/cm2), preheating for 30min, vertically irradiating each group of samples, and adjusting the light dose by adjusting the irradiation time (the time corresponding to the light dose is respectively 0s, 78s, 156s and 234 s); and (3) collecting the buffer solution after treatment, detecting the fluorescence intensity at the wave band of 510-540 nm (excitation wavelength of 504nm) by using a fluorescence spectrophotometer, and counting the fluorescence intensity at 522.5 nm.
The 2% Pp18-lipos was determined to have significantly higher singlet oxygen yield than the other ratios of Pp18-lipos, as shown in FIG. 8.
Example 13
Detecting the photo-thermal conversion performance of the purpurin 18-liposome with different proportions by using a thermal infrared camera, diluting the purpurin 18-liposome with different proportions by a certain multiple to ensure that the molar contents of the liposome are the same, adding 10 mu l of diluted purpurin 18-liposome to the bottom of an EP tube with the diameter of 0.35cm, opening a laser with the wavelength of 705nm, adjusting the power to be 75mW, adjusting the diameter of a light spot to be 0.35cm (the optical density is 750mW/cm2), preheating for 30min until the output power is stable, vertically irradiating a sample in the EP tube, simultaneously monitoring the temperature change of the sample by using the thermal infrared camera until the temperature is not obviously increased, photographing, counting data, and drawing a change curve of the temperature of the purpurin 18-liposome with different proportions along with the time under illumination.
Data analysis shows that 65% Pp18-lipos has a significantly faster temperature rise and the highest temperature than other ratios of Pp18-lipos, as shown in FIG. 9.
Example 14
MTT is adopted to detect the cytotoxicity of Pp18-lipos prepared in the embodiment 11 of the invention to human breast cancer MDA-MB-231.
(1) Plate distribution: MDA-MB-231 cells were trypsinized to a density of 2X 105cells/mL are inoculated into a 96-well plate, each well is 100 mu l, each group is cultured for 12-16 h at 37 ℃ in a 5% CO2 incubator, and the cells are fully attached to the wall and spread at the bottom of the well plate.
(2) Adding medicine: when the cells are completely stretched, the cells are randomly divided into a Control group (Control), a Pp18-lipos single drug group and a Pp18 single drug group.
(3) Adding MTT: and at 0h, 24h and 48h, adding 20 mu l/well of MTT, incubating in an incubator for 4h in a dark place, and detecting the OD value of each well by using a microplate reader.
The toxicity of Pp18-lipos is obviously reduced compared with that of Pp18 by detection, as shown in figure 10.
Example 15
MTT is adopted to detect Pp18-lipos prepared in example 6 of the invention to treat human breast cancer MDA-MB-231 mediated Photodynamic therapy (PDT).
1) Plate distribution: MDA-MB-231 cells were trypsinized to adjust the density to 2X 105cells/mL were seeded in 96-well plates at 100. mu.l per well, 6 replicates per group, and cultured at 37 ℃ in a 5% CO2 cell incubator for 12-16 h.
2) Adding medicine: randomly divided into a Control group (Control), an individual illumination group (Laser), a single drug group, a treatment group: setting 3 light dose gradients 2.5, 5, 7.5J/cm 2; the drug was dosed in groups, MDA-MB-231, at a concentration of 2.5. mu.g/mL.
3) And (3) treatment: after 24h incubation, the supernatant was discarded, 100. mu.l of fresh complete medium was added, the laser was turned on, the laser power was adjusted to 50mW, the spot diameter was adjusted to 1cm (optical density 63.66mW/cm2), and the group samples were treated by vertical irradiation.
4) Adding MTT: after the treatment for 6h and 24h by illumination, 20 mul/well of MTT solution is added, and after the incubation for 4h in an incubator in the dark, the OD value of each well is detected by a microplate reader.
The Pp18-lipos is detected to have obvious killing effect on human breast cancer MDA-MB-231 mediated PDT, as shown in figure 11.
Example 16
MTT is adopted to detect Pp18-lipos prepared in example 11 of the invention for MDA-MB-231 mediated Photothermal (PTT) treatment of human breast cancer
1) Plate distribution: MDA-MB-231 cells were trypsinized to adjust the density to 2X 105cells/mL were seeded in 384-well plates at 20. mu.l/well in 6 replicates per set for 6 sets, cultured at 37 ℃ in a 5% CO2 cell incubator for 12-16 h.
2) Adding medicine: randomized into Control (Control) and treatment groups: setting 6 concentration gradients, adding medicine 0.01825, 0.0375, 0.075, 0.15, 0.3, 0.5 μ l/mL for MDA-MB-231 cells according to groups, and continuing culturing at 20 μ l per well.
3) And (3) treatment: and (3) incubating for 24h, turning on a laser, adjusting the work of the laser to 80mW, adjusting the diameter of a light spot to 0.35cm (the optical density is 800mW/cm2), preheating for 30min, vertically irradiating the treatment group sample, and adjusting the irradiation time.
4) Adding MTT: after the light treatment for 4h, adding 10 mul/well MTT solution, incubating for 4h in dark, and detecting the OD value of each well by a multifunctional microplate reader.
The detection result shows that Pp18-lipos has obvious effect on human breast cancer MDA-MB-231 mediated PTT killing, as shown in figure 12.
Example 17
The Pp18-lipos prepared in example 6 of the invention was evaluated by in vivo imaging of mice by near infrared fluorescence and the accumulation of Pp18-lipos in tumors and tissues was observed.
3 BABL/c mice weighing 18 g-20 g were inoculated with 1X 10 cells of 4T1 on the lower right of the back6When the tumor volume of the mice is measured by a vernier caliper, the tumor volume (mm)3)＝(a×b2) A is the longest diameter of the tumor, b is the shortest diameter of the tumor, and the average volume reaches 50-100 mm3Three mice were selected and injected with 200. mu.l of Pp18-lipos (Pp18 concentration 3.14mg/kg) at 0.5h, 1h, 3h, 6h, 12h, 24h, 36h, 48h, respectivelyh, carrying out isoflurane gas anesthesia on the mouse, and placing the mouse in a living body imaging system for photographing.
In order to examine the distribution of Pp18-lipos in mouse tumors and organs, 200. mu.l of Pp18-lipos (Pp18 concentration of 3.14mg/kg) was injected into the tail vein of a mouse tumor model 3 as an object to be examined, and the time at which the fluorescence intensity of the tumor site was the maximum was selected as a reference, and mice at that time were sacrificed to rapidly and accurately obtain tumors, hearts, livers, spleens, lungs, and kidneys, and imaged using a live mouse imaging system.
Fluorescence intensity was counted, and as shown in FIG. 13, the tumor site of the mouse was enriched more than that of other organs.
EXAMPLE 18 Synthesis of purpurin 18-Phospholipids
(1) Pp18, lipid, EDCI (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) and DMAP (4- (dimethylamino) pyridine) were mixed in a molar ratio of 0.5: 1: 1: 2, mixing in 10mL of dehydrated trichloromethane, mixing, keeping out of the sun, protecting with argon, and stirring for 24 hours to form a mixed solution I. Wherein, Pp18 has a molecular weight of 564.631 and P-lyso PC has a molecular weight of 495.63. The ratio of Pp18 to chloroform was 56.4 mg: 10 mL.
(2) And (3) placing the dried three-necked bottle on a magnetic stirrer, connecting argon gas for protection, adding the mixed solution I into the three-necked bottle, opening the magnetic stirrer, and stirring for 24 hours in a dark place to form a reaction mixed solution II.
(3) Mixing the reaction mixed solution II with silica gel powder, carrying out rotary evaporation at 37 ℃ to remove trichloromethane, adsorbing a sample on the silica gel, loading the silica gel column, loading the sample, adding an eluent, carrying out column chromatography purification on the mixed sample, and carrying out gradient elution on the mixed sample, wherein the polarity of the eluent is (r) trichloromethane sequentially from high to low: methanol volume ratio of 9:1, chloroform: the volume ratio of methanol is 8.5: 1.5, trichloromethane: methanol: the volume ratio of acetic acid is 8: 1.5: 0.5, the purified product Pp18-lipid was detected by thin layer chromatography.
(4) Removing chloroform and methanol at 37 ℃ by using a rotary evaporator, removing acetic acid at 65 ℃, primarily drying, transferring and subpackaging into a test tube, further drying by using nitrogen, placing in a vacuum drying tank for vacuum drying for 3h, sealing by using a sealing film, and storing in a refrigerator at-20 ℃ for later use.
EXAMPLE 19 Synthesis of purpurin 18-Phospholipids
In contrast to example 18, the molar ratio of Pp18, lipid, EDCI to DMAP (4- (dimethylamino) pyridine) was 0.8: 1: 2: 1.
EXAMPLE 20 Synthesis of purpurin 18-Phospholipids
In contrast to example 18, the molar ratio of Pp18, lipid, EDCI (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) to DMAP (4- (dimethylamino) pyridine) was 1: 1: 1: 1.5.
EXAMPLE 21 Synthesis of purpurin 18-Phospholipids
In contrast to example 18, the molar ratio of Pp18, lipid, EDCI (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) to DMAP (4- (dimethylamino) pyridine) was 0.7: 1: 1.5: 1.2.
example 22 preparation of purpurin 18-Liposome nanovesicles
(1) Distearoylphosphatidylcholine, cholesterol (Chol), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000) and Pp18-lipid (calculated as purpurin 18 mass) were dissolved in chloroform, respectively, and distearoylphosphatidylcholine: chol: DSPE-PEG 2000: the molar ratio of Pp18-lipid is 47: 40: 5: 8, sequentially adding the materials into a test tube to form a phospholipid mixed solution with the Pp18-lipid content of 1-65%, and heating in a 65 ℃ water bath for 5 min.
(3) Fixing the test tube on a nitrogen blow-drying device, keeping out of the sun, opening nitrogen until airflow is stable and uniform, blowing the nitrogen into the test tube, evaporating chloroform along with the blowing of the nitrogen, and closing the nitrogen after a uniform lipid film is formed on the wall of the test tube and no liquid is at the bottom of the test tube;
(4) sealing the opening of the test tube by using a sealing film, pricking a small hole on the sealing film to communicate the sealing film with the outside, putting the sealing film into a vacuum drying tank, keeping out of the sun, opening a vacuum pump, and vacuumizing for 3-4 h to fully volatilize and clean residual solvent;
(5) oscillating a lipid film in a hydration test tube in 65 ℃ water by using 1mL of 0.01mol/L PBS (phosphate buffered saline) with the pH value of 7.2-7.4 for 2min, vortexing on a vortexing instrument for 20s (after 5 cycles), allowing the film on the wall of the test tube to fall off, freezing by using liquid nitrogen, rapidly melting and vortexing at 65 ℃, and after 5 cycles, causing no macroscopic aggregated particles in the liquid to obtain a hydrated purpurin lipid mixed solution;
(6) preparing a liposome extruder, heating the extruder to 40-65 ℃, extruding the hydrated purpurin 18-liposome by the extruder, sequentially passing through a polycarbonate membrane with the wavelength of 200nm and 100nm, changing suspension into transparent liquid after 10 cycles to obtain the purpurin 18-liposome, adding a freeze-drying protective agent for freeze-drying, and storing by using a purpurin 18-liposome freeze-drying agent; wherein the freeze-drying protective agent is one or a mixture of more than two of mannose, lactose, glucose, amino acid, sucrose and polyethylene glycol, and is placed in a refrigerator at-80 deg.C for pre-freezing for 12h, and then is placed in a freeze-drying machine for freeze-drying for 24h to obtain the dried purpurin 18-liposome freeze-drying agent.
Example 23 preparation of purpurin 18-Liposome nanovesicles
The difference from example 22 is that DSPC: chol: DSPE-PEG 2000: the molar ratio of Pp18-lipid is 35: 30: 5: 30.
EXAMPLE 24 preparation of purpurin 18-Liposome nanovesicles
The difference from example 22 is that DSPC: chol: DSPE-PEG 2000: the molar ratio of Pp18-lipid is 20: 35: 5: 40.
example 25 preparation of purpurin 18-Liposome nanovesicles
The difference from example 22 is that DSPC: chol: DSPE-PEG 2000: the molar ratio of Pp18-lipid is 13: 32: 5: 50.
the invention adopts acylation reaction to organically synthesize Pp18-lipid by DSPC: chol: DSPE-PEG 2000: pp18-lipid is mixed in a certain proportion, and Pp18-lipid nano-vesicles with the particle size of 100nm are synthesized by an extrusion method, so that the method has the following advantages: (1) compared with the traditional liposome system for loading porphyrin, the loading rate of the purpurin 18 on the liposome is greatly improved; (2) the passive targeting performance of the purpurin 18 at the tumor part is improved; (3) the property of the purpurin 18 which is easy to aggregate in the water solution is improved, the histocompatibility is improved, and the toxicity is reduced; (4) the medicine for preparing the tumor treatment comprises the following components: due to the constructed Pp18-lipos nano vesicles, a large amount of purpurin 18 is regularly stacked between lipid bilayers to generate a fluorescence self-quenching phenomenon, and under the irradiation of specific wavelength, originally excited fluorescence is converted into heat energy which can be used as a photo-thermal agent to realize photo-thermal conversion and be used for tumor photo-thermal therapy (PTT); (5) by reducing the proportion of Pp18-lipid, in a low proportion of Pp18-lipid, porphyrin is not quenched under the irradiation of light with specific wavelength, and singlet oxygen is generated and used as a photosensitizer for photodynamic therapy (PDT) tumor; (6) tumor diagnosis and imaging: the low proportion of Pp18-lipos can be used for in vivo tumor near infrared fluorescence imaging because the fluorescence is not quenched. The constructed porphyrin liposome is passively targeted to a tumor part, namely, the porphyrin liposome is used as a photosensitizer/photothermal agent for tumor treatment and can also be used for tumor diagnosis imaging, so that the defects of the traditional medicine are overcome, and the tumor cure rate is improved.
Claims (5)
1. A preparation method of a purpurin 18-liposome nano vesicle is characterized in that purpurin 18 and 1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphorylcholine are covalently connected by adopting organic acylation reaction to construct Pp18-lipid covalent conjugate, Pp18-lipid covalent conjugate, distearoyl phosphatidylcholine, cholesterol and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 are mixed, nitrogen is adopted to blow dry to form a membrane, then the membrane is hydrated, and then a liposome extrusion method is adopted to prepare the purpurin 18-liposome nano vesicle;
the method specifically comprises the following steps:
(1) dissolving Pp18, P-lyso PC, EDCI and DMAP in chloroform, protecting from light and argon for acylation reaction, connecting Pp18 to a side chain of the P-lyso PC, and purifying to obtain Pp 18-lipid; wherein Pp18 is purpurin 18, P-lyso PC is 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine; wherein in the step (1), the molar ratio of Pp18, P-lyso PC, EDCI and DMAP is (0.5-1): 1: (1-2): (1-2);
(2) dissolving distearoyl phosphatidylcholine, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and Pp18-lipid in chloroform, uniformly mixing, and blow-drying with nitrogen to form a film; wherein, based on the total amount of distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylethanolamine-polyethylene glycol 2000 and Pp18-lipid as 100, DSPC: chol: DSPE-PEG 2000: the quantity ratio of Pp18-lipid substance is (0-55): (30-40): 5: (1-65);
(3) oscillating and hydrating in PBS water bath, and repeatedly freezing and thawing to obtain a hydrated purpurin 18-lipid mixed solution;
(4) squeezing the hydrated purpurin 18-lipid mixed solution by using a squeezer to prepare the purpurin 18-liposome; wherein the extrusion temperature is 40-65 ℃.
2. The method for preparing the purpurin 18-liposome nano-vesicle according to claim 1, wherein the purification process in the step (1) is as follows: performing column chromatography, wherein an eluent is a mixed solution of chloroform, methanol and acetic acid, and the polarity of the eluent is adjusted by adjusting the proportion of each solvent, wherein the volume ratio of the chloroform to the methanol to the acetic acid is (80-90): (10-15): (0-5).
3. The method for preparing the purpurin 18-liposome nanovesicle according to claim 1, wherein the ratio of Pp18 to chloroform in the step (1) is 56.4 mg: 10 mL; the ratio of distearoyl phosphatidylcholine to chloroform in step (2) was 12.13 mg: 1 mL.
4. The method for preparing the purpurin 18-liposome nanovesicles according to claim 1, wherein the pH value of PBS in the step (3) is 7.0-7.4, and the temperature of the water bath is 65 ℃.
5. Use of the purpurin 18-liposome nanovesicles prepared according to the method of any one of claims 1 to 4 in the preparation of a medicament for the treatment of tumors.
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