WO2018095017A1 - 一种靶向光热黑磷纳米制剂及其制备方法和应用 - Google Patents

一种靶向光热黑磷纳米制剂及其制备方法和应用 Download PDF

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WO2018095017A1
WO2018095017A1 PCT/CN2017/088021 CN2017088021W WO2018095017A1 WO 2018095017 A1 WO2018095017 A1 WO 2018095017A1 CN 2017088021 W CN2017088021 W CN 2017088021W WO 2018095017 A1 WO2018095017 A1 WO 2018095017A1
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black phosphorus
preparation
photothermal
folic acid
nano
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French (fr)
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张晗
陶伟
张家宜
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Shenzhen University
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Shenzhen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy

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  • the invention relates to the field of biomedical nano material technology, in particular to a photothermal black phosphorus nano preparation and a preparation method and application thereof.
  • photothermal therapy uses the thermal effect of photothermal conversion to kill cancer cells and perform thermal ablation treatment on tumor sites.
  • the excitation light for illumination is generally NIR, which is a non-invasive method.
  • Sexual cancer treatment can effectively penetrate normal human tissues to reach cancer sites, greatly reducing damage to normal tissues.
  • black phosphorus two-dimensional materials have great potential for application in biomedical fields, especially in photothermal therapy of cancer, due to their low toxicity, high extinction coefficient and high photothermal conversion efficiency.
  • the simple black phosphorus nanomaterial prepared by the prior art has poor stability under physiological conditions when it is directly applied to photothermal treatment of cancer, and the blood circulation time is not long, and the enrichment effect at the cancer site needs to be improved.
  • the first aspect of the present invention provides a photothermal black phosphorus nano preparation, which has high stability, good target recognition ability, excellent photothermal performance, and can effectively kill cancer. Cells that target photothermal therapy of cancer at the cellular level.
  • the present invention provides a photothermophilic black phosphorus nano-preparation comprising black phosphorus nanoflakes, polyethylene glycol adsorbed on the surface of the black phosphorus nanosheet by electrostatic attraction, and an amide bond a folic acid on polyethylene glycol, wherein one end of the polyethylene glycol is an amino group, and the other end is an imino group, and a nitrogen atom of the imino group is bonded to a carbonyl carbon atom in the folic acid to form the amide bond.
  • the folic acid is exposed to the outermost layer of the nanoformulation.
  • the invention adopts polyethylene glycol to modify black phosphorus nanosheets and simultaneously adopts folic acid for targeted modification, wherein polyethylene glycol can effectively avoid aggregation of black phosphorus nanosheets, and polyethylene glycol has hydrophilicity. Characteristics and polarity can significantly inhibit the recognition of the immune system, reduce the phagocytosis of the mononuclear-phagocytic system, and effectively prolong the long blood circulation time of the nanoflakes, thereby enhancing the bioavailability of nano-formulations and enrichment of cancer sites.
  • the folic acid is exposed to the outermost layer of the nano preparation to provide a targeting ligand, which can specifically bind to the cancer cell, improve the targeting specificity of the nano preparation to the cancer cell, and further enhance the nano preparation in the cancer site.
  • the aggregation significantly improves the efficacy of photothermal therapy.
  • the targeted photothermal black phosphorus nano-preparation is obtained by mass ratio (1 ⁇ 3):(5 ⁇ 12):(2-6) of black phosphorus nanosheet, bis-aminopolyethylene glycol, and folic acid. Proportioned.
  • the black phosphorus nanoflakes have a size of 50 nm to 200 nm.
  • the black phosphorus nanoflakes have a thickness of 2 nm to 3 nm.
  • the bis-aminopolyethylene glycol has a weight average molecular weight of 550 to 30,000.
  • the targeted photothermal black phosphorus nano preparation provided by the first aspect of the invention has high stability, good target recognition ability, excellent photothermal performance, can effectively kill cancer cells, and can perform cancer target light at the cell level.
  • Thermal treatment which can significantly reduce the in vivo clearance of black phosphorus nanosheets; and the targeted photothermal black phosphorus nano preparation has good biocompatibility, can be excreted by biodegradation or normal physiological route, and is non-toxic to organisms. Side effects and high biosecurity.
  • the present invention provides a method for preparing a photothermal black phosphorus nano preparation, comprising the following steps:
  • the hydroxysuccinimide-activated folic acid is added to a dimethyl sulfoxide solution of bis-aminopolyethylene glycol, and an appropriate amount of a catalyst is added to obtain a mixed solution, and the mixed solution is stirred at room temperature and protected from light. -32 hours, after completion of the reaction, filtration, to obtain a folic acid-modified polyethylene glycol, which is attached to polyethylene glycol by an amide bond, one end of which is an amino group and the other end is an imino group. The nitrogen atom of the imino group is bonded to the carbonyl carbon atom in the folic acid to form the amide bond;
  • the surface of the black phosphorus nanoflake is coated with folic acid modified polyethylene glycol by probe ultrasonication and magnetic stirring to obtain a photothermal black phosphorus nano preparation, and the folic acid modified polyethylene glycol is passed. Electrostatic attraction is adsorbed on the surface of the black phosphorus nanoflakes, and the folic acid is exposed to the outermost layer of the targeted photothermal black phosphorus nanoformulation.
  • the black phosphorus nanoflakes, the bisaminopolyethylene glycol, and the folic acid are fed in a mass ratio (1 to 3): (5 to 12): (2 to 6).
  • the catalyst may be triethylamine.
  • the volume ratio of the catalyst to the reaction system is 1:75-85.
  • the probe is ultrasonically subjected to a time of 20-40 minutes, and during the ultrasonic process of the probe, the ultrasonic is continuously performed for 45 seconds to 1 hour, and waiting for 15 seconds to 1 hour is a cycle, and the power amplifier is 12%-20%.
  • the magnetic stirring may be rotated at a speed of 800 rpm to 1200 rpm for a duration of 3-4 hours.
  • the black phosphorus nanoflakes are prepared by solution stripping and probe ultrasonication, and are collected and purified by centrifugal tube centrifugation.
  • the ultrasonic time of the probe is 12-18 hours, and the probe ultrasonic process Medium, continuous ultrasound for 45 seconds - 1 hour, and waiting for 15 seconds - 1 hour for a cycle, the power amplifier is 20% - 30%, the rate of centrifugation is 1000 rpm - 2000 rpm, time is 6-15 minutes, temperature is 4 ° C .
  • the hydroxysuccinimide-activated folic acid is prepared by dissolving folic acid in anhydrous dimethyl sulfoxide (DMSO), and then adding hydroxysuccinimide and N,N'-dicyclohexyl. A mixed solution of the carbon imide is obtained, and the mixture is stirred and reacted at room temperature, protected from light and triethylamine for 16-32 hours, and after completion of the reaction, filtration is carried out to obtain the hydroxysuccinimide-activated folic acid.
  • DMSO dimethyl sulfoxide
  • the method for preparing the photothermophilic black phosphorus nano preparation further comprises at least one purification and screening of the obtained photothermal black phosphorus nano preparation by ultrafiltration membrane centrifugation to obtain the purity of the optimized particle size.
  • the photothermal black phosphorus nanoformulation is targeted, the ultrafiltration membrane is centrifuged at a rate of 2000-4000 rpm, the time is 30-60 minutes, and the temperature is 4-8 °C.
  • the preparation method of the photothermal black phosphorus nano preparation provided by the second aspect of the invention is characterized in that the black phosphorus nanosheet and the folic acid modified polyethylene glycol are coated by electrostatic adsorption to obtain a targeted photothermal black phosphorus.
  • Nano preparation the preparation process is simple and easy to operate.
  • the present invention provides the use of the above-mentioned photothermophilic black phosphorus nano-preparation in the preparation of a medicament for the treatment of cancer photothermal ablation.
  • the targeted photothermal black phosphorus nano-preparation is applied to targeted photothermal ablation therapy of cancer.
  • the photothermal ablation treatment is laser excited by a 808 nm near-infrared band.
  • the 808 nm near-infrared laser has a strong skin penetration ability and can effectively reach deep tissues and cancer sites.
  • the targeted photothermal black phosphorus nano preparation has good biocompatibility and no toxic side effects.
  • the targeted photothermal black phosphorus nano preparation according to the invention can effectively prevent the aggregation of the black phosphorus nanosheet, improve the stability of the black phosphorus nanosheet, prolong the blood circulation time, and enhance the targeting effect of the black phosphorus nanosheet;
  • the targeted photothermal black phosphorus nano preparation prepared by the present invention can be applied to targeted photothermal therapy of cancer.
  • Example 1 is a schematic view showing the structure of a photothermophilic black phosphorus nano preparation (BP-PEG-FA) prepared in Example 1 of the present invention
  • Example 2 is a scanning transmission electron microscope (STEM) and an X-ray energy dispersive spectrum (EDS mapping) of a photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention;
  • FIG. 3 is a particle size distribution diagram of a photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention
  • FIG. 4 is a diagram showing changes in zeta potential of a photothermophilic black phosphorus nano-preparation BP-PEG-FA in the preparation process according to Example 1 of the present invention
  • FT-IR Fourier infrared spectrum
  • Example 6 is a photothermal curve of time-dependent changes of BP-PEG-FA targeting photothermophilic black phosphorus nano-preparation prepared by Example 2 of the present invention under 808 nm near-infrared laser irradiation;
  • Example 7 is a photothermal stability of a photothermophilic black phosphorus nano-prepared BP-PEG-FA prepared in Example 2 of the present invention under multiple 808 nm near-infrared laser irradiation, wherein the black phosphorus concentration is 100 ⁇ g/mL;
  • Example 8 is a photothermal curve of the photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 2 of the present invention at different preparation times, wherein the black phosphorus concentration is 100 ⁇ g/mL;
  • CLSM laser confocal scanning electron microscope
  • Example 10 is a result of toxicity of a photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention in different cancer cells;
  • Example 11 is an in vitro targeted photothermotherapy effect of the photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention in HeLa cells.
  • Embodiments of the present invention provide a photothermal black phosphorus nano-preparation, comprising black phosphorus nano-flakes, polyethylene glycol adsorbed on the surface of the black phosphorus nano-sheet by electrostatic attraction, and the aggregation by the amide bond.
  • a folic acid on ethylene glycol the polyethylene glycol having an amino group at one end and an imino group at the other end, the nitrogen atom of the imino group being bonded to a carbonyl carbon atom in the folic acid to form the amide bond, the folic acid Exposure to the outermost layer of the nanoformulation.
  • the targeted photothermal black phosphorus nano-preparation is obtained by mass ratio (1 ⁇ 3):(5 ⁇ 12):(2-6) of black phosphorus nanosheet, bis-aminopolyethylene glycol, and folic acid. Proportioned. Further optionally, the targeted photothermal black phosphorus nano-preparation is obtained by mass ratio of black phosphorus nano-flakes, bis-amino-terminated polyethylene glycol, and folic acid. Prepared by 1:4.2:2.1 ratio.
  • Suitable black phosphorus nanosheets, bis-amino-terminated polyethylene glycol, and folic acid mass ratio are beneficial to form a structurally stable targeted photothermal black phosphorus nano-preparation, which can effectively avoid the aggregation of black phosphorus nano-flakes and improve the nano-photothermal preparation. Stability and targeting effects.
  • the surface of the black phosphorus nanosheet is negatively charged, so that it can be combined with the amino terminated polyethylene glycol by electrostatic attraction, and the polyethylene glycol provides a hydrophilic surface for the photothermal black phosphorus nano preparation.
  • the black phosphorus nanoflakes have a size of 50 nm to 200 nm.
  • the suitable size of the black phosphorus nanosheet can effectively enhance the EPR effect of the nano preparation, increase the enrichment of the nano preparation in the cancer site, and ensure that it is not easily removed by the human reticuloendothelial tissue during blood circulation.
  • the black phosphorus nanoflakes have a size of 50 nm to 150 nm, 100 nm to 150 nm, 120 nm to 180 nm, and 160 nm to 200 nm.
  • the black phosphorus nanoflakes have a thickness of 2 nm to 3 nm.
  • the suitable thickness of the black phosphorus nanosheet can significantly increase the surface area-mass ratio of the nano preparation and enhance the photothermal treatment effect of the nano preparation. The thinner the thickness, the larger the surface area to mass ratio. The same quality of the black phosphorus nanosheet preparation, the larger the specific surface, the better the photothermal effect; on the contrary, the photothermal effect is reduced.
  • the black phosphorus nanosheet has a very high surface area-mass ratio, on the one hand, it can absorb the near-infrared excitation light to the utmost, and the excellent photothermal property is generated; on the other hand, the surface of the black phosphorus nanosheet is filled with a negative charge. It can enhance the electrostatic adsorption capacity with polyethylene glycol, enhance the surface loading of folic acid modified polyethylene glycol, and enhance the stability and targeting effect of the targeted photothermal black phosphorus nano preparation.
  • the bis-aminopolyethylene glycol has a weight average molecular weight of 550 to 30,000.
  • the targeted photothermal black phosphorus nano preparation provided by the first aspect of the invention has high stability, good target recognition ability, excellent photothermal performance, can effectively kill cancer cells, and can perform cancer target light at the cell level.
  • Thermal treatment which can significantly reduce the in vivo clearance of black phosphorus nanosheets; and the targeted photothermal black phosphorus nano preparation has good biocompatibility and can be excreted by biodegradation or normal physiological pathways, The organism has no toxic side effects and high biosafety.
  • the embodiment of the invention further provides a preparation method for targeting a photothermal black phosphorus nano preparation, comprising the following steps:
  • the black phosphorus nanoflakes have a size of 50 nm to 200 nm.
  • the suitable size of the black phosphorus nanosheet can effectively enhance the EPR effect of the nano preparation, increase the enrichment of the nano preparation in the cancer site, and ensure that it is not easily removed by the human reticuloendothelial tissue during blood circulation.
  • the black phosphorus nanoflakes have a size of 50 nm to 150 nm, 100 nm to 150 nm, 120 nm to 180 nm, and 160 nm to 200 nm.
  • the black phosphorus nanoflakes have a thickness of 2 nm to 3 nm. The suitable thickness of the black phosphorus nanosheet can significantly increase the surface area-mass ratio of the nano preparation and enhance the photothermal treatment effect of the nano preparation.
  • the black phosphorus nanoflakes can be prepared by solution stripping and probe ultrasonic method, and collected and purified by centrifugal tube centrifugation, and the ultrasonic time of the probe is 12-18 hours.
  • the ultrasonic process of the probe the ultrasonic is continuously performed for 45 seconds to 1 hour, and waiting for 15 seconds to 1 hour is one.
  • the cycle is 20%-30% for the power amplifier, the centrifugation rate is 1000 rpm-2000 rpm, the time is 6-15 minutes, and the temperature is 4 °C.
  • the centrifugation operation is to precipitate and separate the un-stripped bulk black phosphorus to purify the separated black phosphorus nanoflakes.
  • the bis-aminopolyethylene glycol has a weight average molecular weight of 550-30000.
  • the catalyst may be triethylamine.
  • the volume ratio of the catalyst to the reaction system is 1:75-85.
  • the stirring speed during the stirring reaction is 300-600 rpm.
  • the hydroxysuccinimide-activated folic acid can be prepared by dissolving folic acid in anhydrous dimethyl sulfoxide (DMSO) and then adding hydroxysuccinimide (NHS). a mixture of triethylamine and N,N'-dicyclohexylcarbimide (DCC), the mixture is stirred at room temperature and protected from light for 16-32 hours, and filtered after completion of the reaction. The hydroxysuccinimide activates folic acid.
  • DMSO dimethyl sulfoxide
  • NHS hydroxysuccinimide
  • DCC N,N'-dicyclohexylcarbimide
  • the filtration operation after completion of the reaction may specifically be: dialysis using a dialysis bag (MWCO: 3000) to remove unreacted hydroxysuccinimide-activated folic acid.
  • the folic acid-modified polyethylene glycol obtained in the embodiment of the invention has strong affinity for the cell membrane, can promote the nano-formation into the cell, and is beneficial to improving the subsequent photothermal treatment effect of the targeted photothermal black phosphorus nano preparation. .
  • the black phosphorus nanoflakes, the bisaminopolyethylene glycol, and the folic acid are fed in a mass ratio (1 to 3): (5 to 12): (2 to 6). Further optionally, the black phosphorus nanoflakes, bis-aminopolyethylene glycol, and folic acid are fed at a mass ratio of 1:4.2:2.1.
  • the black phosphorus nanosheet is pre-coated by probe ultrasonic method, and further coated by magnetic stirring.
  • the probe is ultrasonic for 20-40 minutes, and the probe is ultrasonic for 45 seconds to 1 hour, and waits for 15 seconds to 1 hour for one cycle, and the power amplifier is 12%-20 %.
  • the magnetic stirring speed can be 800 rpm - 1200 rpm, continuous The time is 3-4 hours.
  • the obtained photothermal black phosphorus nano preparation is purified and screened at least once by ultrafiltration membrane centrifugation to obtain a pure target photothermal black phosphorus nano preparation with optimized particle size.
  • the ultrafiltration membrane is centrifuged at a rate of 2000-4000 rpm for a period of 30-60 minutes and a temperature of 4-8 °C.
  • the method for preparing the photothermophilic black phosphorus nano preparation comprises repeating the ultrafiltration membrane centrifugation operation a plurality of times, specifically: resuspending the super saline solution after each ultrafiltration membrane centrifugation operation ends.
  • the photothermal black phosphorus nanoformulation on the filter is then subjected to the next ultrafiltration membrane centrifugation operation.
  • the parameter setting of each index of the ultrasonic and centrifugation duration, period, rotation speed, and power amplifier in the above respective steps of the present invention is not limited to the above limitation.
  • the preparation method of the photothermal black phosphorus nano preparation provided by the second aspect of the invention is characterized in that the black phosphorus nanosheet and the folic acid modified polyethylene glycol are coated by electrostatic adsorption to obtain a targeted photothermal black phosphorus.
  • Nano preparation the preparation process is simple and easy to operate.
  • the present invention provides the use of the above-mentioned photothermophilic black phosphorus nano-preparation in the preparation of a medicament for the treatment of cancer photothermal ablation.
  • the targeted photothermal black phosphorus nano-preparation is applied to targeted photothermal ablation therapy of cancer.
  • the photothermal ablation treatment is laser excited by a 808 nm near-infrared band.
  • the 808 nm near-infrared laser has a strong skin penetration ability and can effectively reach deep tissues and cancer sites.
  • the targeted photothermal black phosphorus nano preparation has good biocompatibility and no toxic side effects.
  • a method for preparing a photothermal black phosphorus nano preparation comprises the following steps:
  • Triethylamine 0.5 mL
  • NHS 0.52 g, 5.00 mmol
  • DCC 0.50 g, 2.50 mmol
  • the mixture was magnetically stirred at room temperature under light-shielding conditions for 24 hours, and then filtered to remove insoluble by-products and unreacted raw materials to obtain NHS-activated folic acid.
  • the surface of the black phosphorus nanosheet is coated with folic acid modified polyethylene glycol to prepare a targeted photothermal black phosphorus nano preparation; wherein the black phosphorus nanoflake and folic acid modification
  • the mass ratio of polyethylene glycol is 1:6.3.
  • the black phosphorus nanosheets were pre-coated with folic acid modified polyethylene glycol by probe ultrasonic method, and the probe ultrasonic process was carried out in both suspensions.
  • the probe ultrasonic time was 40 minutes, and the process was continuous. Ultrasound for 45 seconds, wait 15 seconds for a cycle, and the amplifier is 15%.
  • the black phosphorus nanosheets were further coated with folic acid modified polyethylene glycol by magnetic stirring at room temperature, and the magnetic stirring speed was 800 rpm for 4 hours; the polyethylene glycol coated black phosphorus nanometer light was used.
  • the hot preparation (without folic acid modification) can be prepared in the same manner, and the raw material is prepared from folic acid.
  • the modified amino-terminated polyethylene glycol is replaced by an unmodified ammonia-terminated polyethylene glycol;
  • step (3) Purifying and screening the photothermal black phosphorus nano-preparation obtained by the step (3) by ultrafiltration membrane centrifugation to obtain a pure target photothermal black phosphorus nano preparation with optimized particle size: taking the reaction in step (3) The mixture was added to an ultrafiltration tube (MWCO 100 kDa; Millipore) for centrifugation to separate the uncoated folic acid-modified polyethylene glycol and the prepared targeted photothermal black phosphorus nano preparation; the ultrafiltration membrane was centrifuged at a rate of 4000 rpm, time. For 30 minutes, the temperature was 4 °C.
  • MWCO 100 kDa; Millipore millipore
  • the targeted photothermal black phosphorus nano preparation on the filter membrane was resuspended in physiological saline, and the ultrafiltration membrane was centrifuged and resuspended in physiological saline for 3 times to obtain a pure targeted photothermal black phosphorus nano preparation.
  • BP-PEG-FA BP-PEG-FA
  • FIG. 1 is a schematic view showing the structure of a photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention, comprising black phosphorus nanosheets 10, and polyethylene glycol adsorbed on the surface of black phosphorus nanosheets 10 by electrostatic attraction.
  • a folic acid 30 attached to the polyethylene glycol 20 via an amide bond; the polyethylene glycol 20 has an amino group at one end and an imino group at the other end, and the nitrogen atom of the imino group is bonded to a carbonyl carbon atom in the folic acid 30.
  • the amide bond, the folic acid 30 is exposed to the outermost layer of the nanoformulation.
  • 2(a) and 2(b) are scanning transmission electron microscopy (STEM) and X-ray energy dispersive spectroscopy (EDS mapping) of the photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention, respectively.
  • STEM scanning transmission electron microscopy
  • EDS mapping X-ray energy dispersive spectroscopy
  • the particle size of the BP-PEG-FA prepared in this embodiment is about 100 nm, the particle size range is suitable for producing a good EPR effect, and the BP-PEG prepared in this embodiment can be known.
  • the elements of -FA are composed of carbon, nitrogen, oxygen and phosphorus. It is preliminarily verified that the target photothermal black phosphorus nano-preparation BP-PEG-FA is successfully prepared.
  • Example 3 is a particle size distribution diagram of the photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention, which has good consistency with the results in FIG.
  • FIG. 4 is a graph showing changes in zeta potential of a photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention during preparation.
  • the zeta potential of black phosphorus nanosheets BP NSs is around -14mV, The absolute value of the negative side is relatively high, and it can effectively interact with the ammonia-terminated polyethylene glycol to carry out polyethylene glycol coating.
  • the black phosphorescent photothermal nano-preparation (including folate-free modified BP-PEG NSs and folate-modified BP-PEG-FA NSs) with polyethylene glycol has a zeta potential between -10mV and -18mV, which is maintained at a high level. Absolute values, nano-formulations have a strong mutual repulsion and are therefore highly stable in the dispersed phase.
  • FT-IR Fourier infrared spectrum
  • a method for preparing a photothermal black phosphorus nano preparation comprises the following steps:
  • Triethylamine 0.5 mL
  • NHS 0.52 g, 5.00 mmol
  • DCC 0.50 g, 2.50 mmol
  • the mixture was magnetically stirred at room temperature under light-shielding conditions for 24 hours, and then filtered to remove insoluble by-products and unreacted raw materials to obtain NHS-activated folic acid.
  • the power amplifier is 12%; further, the black phosphorus nano-flakes are further coated with folic acid-modified polyethylene glycol by magnetic stirring at room temperature, the magnetic stirring speed is 1000 rpm, and the duration is 4 hours;
  • the coated black phosphorus nanophotothermal preparation (no folic acid modification) can be prepared in the same manner, and the preparation raw material is replaced by folic acid modified amino terminal polyethylene glycol to unmodified ammonia terminal polyethylene glycol;
  • step (3) Purifying and screening the photothermal black phosphorus nano-preparation obtained by the step (3) by ultrafiltration membrane centrifugation to obtain a pure target photothermal black phosphorus nano preparation with optimized particle size: taking the reaction in step (3) The mixture was added to an ultrafiltration tube (MWCO 100 kDa; Millipore) for centrifugation to separate the uncoated folic acid-modified polyethylene glycol and the prepared targeted photothermal black phosphorus nano preparation; the ultrafiltration membrane was centrifuged at a rate of 4000 rpm, time. For 30 minutes, the temperature was 4 °C.
  • MWCO 100 kDa; Millipore millipore
  • the targeted photothermal black phosphorus nano preparation on the filter membrane was resuspended in physiological saline, and the ultrafiltration membrane was centrifuged and resuspended in physiological saline for 4 times to obtain a pure targeted photothermal black phosphorus nano preparation.
  • BP-PEG-FA BP-PEG-FA
  • FIG. 6 is a photothermal curve of the photothermal black phosphorus nano-prepared BP-PEG-FA prepared according to Example 2 of the present invention under 808 nm near-infrared laser irradiation with time.
  • the photothermal black phosphorus nano-preparation BP-PEG-FA of the embodiment of the invention has good photothermal characteristics, and the concentration is 100 ⁇ g/at a 808 nm laser irradiation intensity of 1.0 W/cm 2 .
  • the temperature of the mL-targeted photothermal black phosphorus nanoformer increased by about 26 °C in 10 minutes.
  • Example 7 is a photothermal stability of a photothermophilic black phosphorus nano-prepared BP-PEG-FA prepared in Example 2 of the present invention under multiple 808 nm near-infrared laser irradiation. It can be seen from the figure that the temperature-dependent change of the target photothermal black phosphorus nano-preparation BP-PEG-FA under the 808 nm near-infrared laser irradiation is stable, and the light stability of the near-infrared laser excitation is good.
  • Example 8 is a photothermal curve of the photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 2 of the present invention at different preparation times. It can be seen from the figure that after one week of preparation, the temperature change after the near-infrared laser excitation under the same conditions as the newly prepared target photothermal black phosphorus nano-preparation BP-PEG-FA is consistent, thus indicating the preparation of the examples of the present invention.
  • the targeted photothermal black phosphorus nanoformulation BP-PEG-FA can be used after storage.
  • a method for preparing a photothermal black phosphorus nano preparation comprises the following steps:
  • Triethylamine 0.5 mL
  • NHS 0.52 g, 5.00 mmol
  • DCC 0.50 g, 2.50 mmol
  • the mixture was magnetically stirred at room temperature under light-shielding conditions for 24 hours, and then filtered to remove insoluble by-products and unreacted raw materials to obtain NHS-activated folic acid.
  • the power amplifier is 15%; further, the black phosphorus nano-flakes are further coated with folic acid-modified polyethylene glycol by magnetic stirring at room temperature, the magnetic stirring speed is 850 rpm, and the duration is 4 hours;
  • the coated black phosphorus nanophotothermal preparation (no folic acid modification) can be prepared in the same manner, and the preparation raw material is replaced by folic acid modified amino terminal polyethylene glycol with no folic acid modified ammonia terminal polyethylene glycol;
  • step (3) Purifying and screening the photothermal black phosphorus nano-preparation obtained by the step (3) by ultrafiltration membrane centrifugation to obtain a pure target photothermal black phosphorus nano preparation with optimized particle size: taking the reaction in step (3) The mixture was added to an ultrafiltration tube (MWCO 100 kDa; Millipore) for centrifugation to separate the uncoated folic acid-modified polyethylene glycol and the prepared targeted photothermal black phosphorus nano preparation; the ultrafiltration membrane was centrifuged at a rate of 4000 rpm, time. For 40 minutes, the temperature was 4 °C.
  • MWCO 100 kDa; Millipore millipore
  • the targeted photothermal black phosphorus nano preparation on the filter was resuspended in physiological saline, and the ultrafiltration membrane was centrifuged and resuspended in physiological saline for 5 times to obtain a pure targeted photothermal black phosphorus nano preparation.
  • BP-PEG-FA BP-PEG-FA
  • a method for preparing a photothermal black phosphorus nano preparation comprises the following steps:
  • Triethylamine 0.5 mL
  • NHS 0.52 g, 5.00 mmol
  • DCC 0.50 g, 2.50 mmol
  • the mixture was magnetically stirred at room temperature under light-shielding conditions for 24 hours, and then filtered to remove insoluble by-products and unreacted raw materials to obtain NHS-activated folic acid.
  • the power amplifier is 20%; further, the black phosphorus nano-flakes are further coated with folic acid-modified polyethylene glycol by magnetic stirring at room temperature, the magnetic stirring speed is 1200 rpm, and the duration is 3 hours;
  • the coated black phosphorus nanophotothermal preparation (no folic acid modification) can be prepared in the same manner, and the preparation raw material is replaced by folic acid modified amino terminal polyethylene glycol to unmodified ammonia terminal polyethylene glycol;
  • step (3) Purifying and screening the photothermal black phosphorus nano-preparation obtained by the step (3) by ultrafiltration membrane centrifugation to obtain a pure target photothermal black phosphorus nano preparation with optimized particle size: taking the reaction in step (3) The mixture was added to an ultrafiltration tube (MWCO 100 kDa; Millipore) for centrifugation to separate the uncoated folic acid-modified polyethylene glycol and the prepared targeted photothermal black phosphorus nano preparation; the ultrafiltration membrane was centrifuged at a rate of 4000 rpm, time. For 40 minutes, the temperature was 4 °C.
  • MWCO 100 kDa; Millipore millipore
  • the targeted photothermal black phosphorus nano preparation on the filter membrane was resuspended in physiological saline, and the ultrafiltration membrane was centrifuged and resuspended in physiological saline for 4 times to obtain a pure targeted photothermal black phosphorus nano preparation.
  • BP-PEG-FA BP-PEG-FA
  • HeLa cells were cultured in a DMEM high glucose medium containing 10% fetal bovine serum in a 37 ° C, 5% CO 2 incubator. After the cells are covered with the bottom of the cell culture flask, discard the old medium, rinse once with PBS, add 1 mL of 0.25% trypsin, digest for 1-2 minutes at 37 ° C, add a small amount of complete medium containing serum, and use a straw. Gently blow to separate the cells from the wall into a single cell suspension. This single cell suspension was uniformly inoculated into a 6-well cell culture plate, and 1 mL of the medium was added thereto, and cultured in a 37 ° C, 5% CO 2 incubator for 24 hours.
  • the targeted photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention was added to the cells, and the culture was continued for 4 hours.
  • the cells were washed three times with ice-cold PBS, fixed cells were added with methanol for 20 min, methanol was discarded, DAPI staining solution was added for 5 min, and then washed three times with PBS, and the cell uptake was observed under a FV-1000 laser confocal microscope.
  • the wavelength of the excitation light used for DAPI was observed to be 340 nm, and the uptake of the targeted photothermal black phosphorus nano-preparation BP-PEG-FA was observed through the Bright channel.
  • the old medium was discarded, washed once with PBS, and 1 mL of 0.25% trypsin was added, and the mixture was digested at 37 °C. - 2 minutes, add a small amount of complete medium containing serum, gently blow to separate the cells from the wall into a single cell suspension.
  • the suspension was subjected to cell counting, diluted, and uniformly seeded in a 96-well cell culture plate at a density of 5000 cells/well, and culture was continued for 24 hours at 37 ° C in a 5% CO 2 incubator to adhere the cells.
  • the target photothermal black phosphorus nano-preparation BP-PEG-FA (concentration is 10, 25, 50 ⁇ g/mL) prepared in Example 1 of the present invention was dispersed as a sample to be tested; Hela cells cultured in complete medium of the nanoformulation BP-PEG-FA served as a control group.
  • Figure 10 is a graph showing the toxicity results of the photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention in different cancer cells (Hela, MCF-7, HepG2, and PC3). It can be seen from the figure that the targeted photothermal black phosphorus nano-preparation BP-PEG-FA of the embodiment of the invention has no cytotoxicity under the condition of no laser irradiation, and has good biosafety and no toxic and side effects.
  • FIG. 11 is an in vitro targeted photothermotherapy effect of different concentrations of the photothermophilic black phosphorus nano-preparation BP-PEG-FA prepared in Example 1 of the present invention in HeLa cells.
  • the black phosphorus concentration on the abscissa in Fig. 11 refers to the concentration of black phosphorus contained in the black phosphorus-targeted photothermal preparation. Control means that no cells have been treated as a blank reference. It can be seen from the figure that compared with the black phosphorus nanosheets (BP NSs) and the black phosphorescent thermal nano preparations (BP-PEG NSs) which are not targeted modified at 808 nm laser irradiation with an intensity of 1.0 W/cm 2 .
  • BP NSs black phosphorus nanosheets
  • BP-PEG NSs black phosphorescent thermal nano preparations
  • the targeted photothermal black phosphorus nano-preparation BP-PEG-FA has better in vitro targeted photothermal therapy effect, can effectively kill cancer cells, and only produces therapeutic effects in the irradiation site of the near-infrared laser, thereby greatly To a lesser extent, it damages normal cells, tissues and organs and is targeted.

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Abstract

一种靶向光热黑磷纳米制剂，包括黑磷纳米薄片、通过静电引力吸附在所述黑磷纳米薄片表面的聚乙二醇、以及通过酰胺键连接在所述聚乙二醇上的叶酸，所述聚乙二醇的一端为氨基，另一端为亚氨基，所述亚氨基的氮原子与所述叶酸中的羰基碳原子相连构成所述酰胺键，所述叶酸暴露在所述纳米制剂的最外层。该靶向光热黑磷纳米制剂的制备方法和其用于制备在细胞层面进行癌症的靶向光热治疗的药物中的应用。

Description

一种靶向光热黑磷纳米制剂及其制备方法和应用
本申请要求了2016年11月25日提交中国专利局的，申请号201611055858.3，发明名称为“一种靶向光热黑磷纳米制剂及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医用纳米材料技术领域，特别是涉及一种靶向光热黑磷纳米制剂及其制备方法和应用。
背景技术
作为威胁人类健康的重大恶性疾病之一，癌症的治疗无论是化学治疗、手术治疗或放射治疗都是对身体有极大副作用的，并且在发生恶性转移以后，无论采用上述何种方式都难以彻底治愈。虽然已有大量的人力、物力、财力已经投入到癌症的研究，但进展十分有限、并且其有效治疗仍然是人类面对的极大考验。
有研究表明，将纳米技术应用到癌症诊断治疗领域，具有广阔的前景及临床价值。光热治疗作为一种新型的癌症治疗方式，利用光热转换产生的热效应以杀死癌细胞、对肿瘤部位进行热消融治疗，其照射用的激发光一般采用近红外光，是一种非侵入性的癌症治疗方式，能够有效穿透人体正常组织到达癌症部位，极大程度地减少对正常组织的损害。
近期研究发现，黑磷二维材料由于具有低毒性、高消光系数和高光热转换效率，在生物医用领域，尤其是癌症的光热治疗方面具有极大的应用潜力。然而， 现有方式制备的单纯黑磷纳米材料在直接应用于癌症的光热治疗时，其在生理状态下稳定性较差，并且血液循环时间不长，且在癌症部位的富集效果有待改善。
发明内容
鉴于此，本发明第一方面提供了一种靶向光热黑磷纳米制剂，该靶向光热黑磷纳米制剂稳定性高、靶向识别能力好、光热性能优异、能够有效杀死癌细胞，可在细胞层面进行癌症的靶向光热治疗。
第一方面，本发明提供了一种靶向光热黑磷纳米制剂，包括黑磷纳米薄片、通过静电引力吸附在所述黑磷纳米薄片表面的聚乙二醇、以及通过酰胺键连接在所述聚乙二醇上的叶酸，所述聚乙二醇的一端为氨基，另一端为亚氨基，所述亚氨基的氮原子与所述叶酸中的羰基碳原子相连构成所述酰胺键，所述叶酸暴露在所述纳米制剂的最外层。
本发明通过采用聚乙二醇对黑磷纳米薄片进行修饰，并同时采用叶酸进行靶向修饰，其中，聚乙二醇可以有效避免黑磷纳米薄片发生聚集，且聚乙二醇兼具亲水特性和极性，可以显著抑制免疫系统的识别，减少单核-吞噬细胞系统的吞噬，并有效延长纳米薄片的血液长循环时间，从而增强纳米制剂的生物利用度和癌症部位的富集。而叶酸暴露在所述纳米制剂最外层提供靶向配体，所述靶向配体可以和癌细胞特异性结合，提高纳米制剂对癌细胞的靶向特异性，进一步增强纳米制剂在癌症部位的聚集，显著提高光热治疗效果。
可选地，所述靶向光热黑磷纳米制剂通过将黑磷纳米薄片、双氨端聚乙二醇、叶酸按质量比(1～3)∶(5～12)∶(2～6)配比制得。
本发明中，所述黑磷纳米薄片的尺寸为50nm-200nm。
本发明中，所述黑磷纳米薄片的厚度为2nm-3nm。
本发明中，所述双氨端聚乙二醇的重均分子量为550-30000。
本发明第一方面提供的所述靶向光热黑磷纳米制剂，稳定性高、靶向识别能力好、光热性能优异、能够有效杀死癌细胞，可在细胞层面进行癌症的靶向光热治疗，从而可显著减少黑磷纳米薄片的体内清除；且该靶向光热黑磷纳米制剂具有良好的生物相容性，可通过生物降解或者正常的生理途径排出体外，对生物体无毒副作用、生物安全性高。
第二方面，本发明提供了一种靶向光热黑磷纳米制剂的制备方法，包括以下步骤：
提供黑磷纳米薄片；
将羟基琥珀酰亚胺活化叶酸加入到双氨端聚乙二醇的二甲基亚砜溶液中，并加入适量催化剂，得到混合溶液，所述混合溶液在室温、避光条件下进行搅拌反应16-32小时，反应完成后过滤，得到叶酸修饰的聚乙二醇，所述叶酸通过酰胺键连接在聚乙二醇上，所述聚乙二醇的一端为氨基，另一端为亚氨基，所述亚氨基的氮原子与所述叶酸中的羰基碳原子相连构成所述酰胺键；
依次采用探针超声、磁力搅拌的方式，在所述黑磷纳米薄片的表面包覆叶酸修饰的聚乙二醇，得到靶向光热黑磷纳米制剂，所述叶酸修饰的聚乙二醇通过静电引力吸附在所述黑磷纳米薄片表面，所述叶酸暴露在所述靶向光热黑磷纳米制剂的最外层。
本发明中，可选地，所述黑磷纳米薄片、双氨端聚乙二醇、叶酸按质量比(1～3)∶(5～12)∶(2～6)投料。
本发明中，所述催化剂可以是三乙胺。所述催化剂与反应体系的体积比为1:75-85。
本发明中，可选地，所述探针超声的时间为20-40分钟，所述探针超声过程中，持续超声45秒-1小时，及等待15秒-1小时为一个周期，功放为12％-20％。
本发明中，可选地，所述磁力搅拌的转速可以为800rpm-1200rpm，持续时间为3-4小时。
本发明中，所述黑磷纳米薄片采用溶液剥离结合探针超声法制备，并采用离心管离心方式收集、纯化，所述探针超声的超声时间为12-18小时，所述探针超声过程中，持续超声45秒-1小时，及等待15秒-1小时为一个周期，功放为20％-30％，所述离心的速率为1000rpm-2000rpm，时间为6-15分钟，温度为4℃。
本发明中，所述羟基琥珀酰亚胺活化叶酸采用如下方式制备：将叶酸溶解于无水二甲基亚砜(DMSO)中，再加入羟基琥珀酰亚胺和N,N'-二环己基碳酰亚胺得到混合液，所述混合液在室温、避光和三乙胺催化条件下进行搅拌反应16-32小时，反应完成后过滤，得到所述羟基琥珀酰亚胺活化叶酸。
本发明中，所述靶向光热黑磷纳米制剂的制备方法进一步包括采用超滤膜离心的方式对所得靶向光热黑磷纳米制剂进行至少一次纯化、筛选，得到优化粒径大小的纯净靶向光热黑磷纳米制剂，所述超滤膜离心的速率为2000-4000rpm，时间为30-60分钟，温度为4-8℃。
本发明第二方面提供的靶向光热黑磷纳米制剂的制备方法，通过将黑磷纳米薄片与叶酸修饰的聚乙二醇通过静电吸附的方式包覆结合，制得靶向光热黑磷纳米制剂，制备过程简单易操作。
第三方面，本发明提供了一种上述的靶向光热黑磷纳米制剂在制备癌症光热消融治疗药物中的应用。所述靶向光热黑磷纳米制剂应用于癌症的靶向光热消融治疗。所述光热消融治疗通过808nm近红外波段激光激发。所述808nm近红外波段激光具有很强的皮肤穿透能力，能够有效到达深层组织及癌症部位。 同时，所述靶向光热黑磷纳米制剂具有很好的生物相容性、无毒副作用。
综上所述，本发明有益效果包括以下几个方面：
(1)本发明所述的靶向光热黑磷纳米制剂可以有效防止黑磷纳米薄片的聚集，提高黑磷纳米薄片的稳定性，延长血液循环时间，增强黑磷纳米薄片的靶向效果；
(2)本发明方法制备过程简单易操作，制备的靶向光热黑磷纳米制剂具备优异生物相容性、无毒副作用；
(3)本发明制备的所述靶向光热黑磷纳米制剂可以应用于癌症的靶向光热治疗。
附图说明
图1为本发明实施例1制备的靶向光热黑磷纳米制剂(BP-PEG-FA)的结构示意图；
图2为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA的扫描透射电子显微镜(STEM)和X射线能量色散谱(EDS mapping)；
图3为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA的粒径分布图；
图4为本发明实施例1靶向光热黑磷纳米制剂BP-PEG-FA在制备过程中Zeta电位的变化图；
图5为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA的傅里叶红外光谱图(FT-IR)；
图6为不同浓度的本发明实施例2制备的靶向光热黑磷纳米制剂BP-PEG-FA在808nm近红外激光照射下随时间变化的光热曲线图谱；
图7为本发明实施例2制备的靶向光热黑磷纳米制剂BP-PEG-FA在多次808nm近红外激光照射下的光热稳定性，其中黑磷浓度为100μg/mL；
图8为本发明实施例2制备的靶向光热黑磷纳米制剂BP-PEG-FA在不同制备时间下的光热曲线图谱，其中黑磷浓度为100μg/mL；
图9为激光共聚焦扫描电子显微镜(CLSM)观察本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA的Hela细胞摄取图片；
图10为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA在不同癌症细胞中的毒性结果；
图11为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA在Hela细胞中的体外靶向光热治疗效果。
具体实施方式
以下所述是本发明实施例的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明实施例原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明实施例的保护范围。
本发明实施例提供了一种靶向光热黑磷纳米制剂，包括黑磷纳米薄片、通过静电引力吸附在所述黑磷纳米薄片表面的聚乙二醇、以及通过酰胺键连接在所述聚乙二醇上的叶酸，所述聚乙二醇的一端为氨基，另一端为亚氨基，所述亚氨基的氮原子与所述叶酸中的羰基碳原子相连构成所述酰胺键，所述叶酸暴露在所述纳米制剂的最外层。
可选地，所述靶向光热黑磷纳米制剂通过将黑磷纳米薄片、双氨端聚乙二醇、叶酸按质量比(1～3)∶(5～12)∶(2～6)配比制得。进一步可选地，所述靶向光热黑磷纳米制剂通过将黑磷纳米薄片、双氨端聚乙二醇、叶酸按质量比 1∶4.2∶2.1配比制得。适合的黑磷纳米薄片、双氨端聚乙二醇、叶酸质量比，有利于形成结构稳定的靶向光热黑磷纳米制剂，可有效避免黑磷纳米薄片的聚集，提高纳米光热制剂的稳定性和靶向效果。
本发明中，所述黑磷纳米薄片表面带负电，因此可通过静电引力与氨基封端的聚乙二醇相结合，聚乙二醇为靶向光热黑磷纳米制剂提供了亲水性表面。
本发明中，可选地，所述黑磷纳米薄片的尺寸为50nm-200nm。适合的黑磷纳米薄片尺寸，可以有效增强纳米制剂的EPR效应，增加纳米制剂在癌症部位的富集，同时保证其在血液循环过程中不易被人体网状内皮组织清除。可选地，黑磷纳米薄片的尺寸为50nm-150nm、100nm-150nm、120nm-180nm、160nm-200nm。
本发明中，所述黑磷纳米薄片的厚度为2nm-3nm。黑磷纳米薄片适合的厚度能够显著提高纳米制剂的表面积-质量比，强化纳米制剂的光热治疗效果。厚度越薄，表面积-质量比比值越大。相同质量的黑磷纳米薄片制剂，比表面越大，光热效果越好；反之，光热效果下降。
本发明中，所述黑磷纳米薄片具有非常高的表面积-质量比，一方面能够最大限度地吸收近红外激发光，产生优异的光热性能；另一方面黑磷纳米薄片表面充满负电荷，能够增强与聚乙二醇之间的静电吸附能力，增强叶酸修饰的聚乙二醇表面负载量，加强靶向光热黑磷纳米制剂的稳定性和靶向效果。
本发明中，所述双氨端聚乙二醇的重均分子量为550-30000。
本发明第一方面提供的所述靶向光热黑磷纳米制剂，稳定性高、靶向识别能力好、光热性能优异、能够有效杀死癌细胞，可在细胞层面进行癌症的靶向光热治疗，从而可显著减少黑磷纳米薄片的体内清除；且该靶向光热黑磷纳米制剂具有良好的生物相容性，可通过生物降解或者正常的生理途径排出体外，对 生物体无毒副作用、生物安全性高。
相应地，本发明实施例还提供了一种靶向光热黑磷纳米制剂的制备方法，包括以下步骤：
(1)提供黑磷纳米薄片；
(2)将羟基琥珀酰亚胺活化叶酸加入到双氨端聚乙二醇的二甲基亚砜溶液中，并加入适量催化剂，得到混合溶液，所述混合溶液在室温、避光条件下进行搅拌反应16-32小时，反应完成后过滤，得到叶酸修饰的聚乙二醇，所述叶酸通过酰胺键连接在聚乙二醇上，所述聚乙二醇的一端为氨基，另一端为亚氨基，所述亚氨基的氮原子与所述叶酸中的羰基碳原子相连构成所述酰胺键；
(3)依次采用探针超声、磁力搅拌的方式，在所述黑磷纳米薄片的表面包覆叶酸修饰的聚乙二醇，得到靶向光热黑磷纳米制剂，所述叶酸修饰的聚乙二醇通过静电引力吸附在所述黑磷纳米薄片表面，所述叶酸暴露在所述靶向光热黑磷纳米制剂的最外层。
本发明实施例步骤(1)中，可选地，所述黑磷纳米薄片的尺寸为50nm-200nm。适合的黑磷纳米薄片尺寸，可以有效增强纳米制剂的EPR效应，增加纳米制剂在癌症部位的富集，同时保证其在血液循环过程中不易被人体网状内皮组织清除。可选地，黑磷纳米薄片的尺寸为50nm-150nm、100nm-150nm、120nm-180nm、160nm-200nm。可选地，所述黑磷纳米薄片的厚度为2nm-3nm。黑磷纳米薄片适合的厚度能够显著提高纳米制剂的表面积-质量比，强化纳米制剂的光热治疗效果。
本发明实施例步骤(1)中，所述黑磷纳米薄片可采用溶液剥离结合探针超声法制备，并采用离心管离心方式收集、纯化，所述探针超声的超声时间为12-18小时，所述探针超声过程中，持续超声45秒-1小时，及等待15秒-1小时为一 个周期，功放为20％-30％，所述离心的速率为1000rpm-2000rpm，时间为6-15分钟，温度为4℃。离心操作是为了沉淀分离未被剥离的块状黑磷，以提纯分离黑磷纳米薄片。
本发明实施例步骤(2)中，可选地，所述双氨端聚乙二醇的重均分子量为550-30000。可选地，所述催化剂可以是三乙胺。所述催化剂与反应体系的体积比为1:75-85。所述搅拌反应过程中的搅拌速度为300-600rpm。
本发明实施例步骤(2)中，所述羟基琥珀酰亚胺活化叶酸可采用如下方式制备：将叶酸溶解于无水二甲基亚砜(DMSO)中，再加入羟基琥珀酰亚胺(NHS)、三乙胺和N,N'-二环己基碳酰亚胺(DCC)得到混合液，所述混合液在室温、避光条件下进行搅拌反应16-32小时，反应完成后过滤，得到所述羟基琥珀酰亚胺活化叶酸。
本发明步骤(2)中，反应完成后的过滤操作具体可以是：采用透析袋(MWCO:3000)透析除去未反应的羟基琥珀酰亚胺活化叶酸。
本发明实施例中所得叶酸修饰的聚乙二醇对细胞膜有较强的亲和力，可以很好地促进纳米制剂进入细胞内，有利于提高靶向光热黑磷纳米制剂的后续癌症光热治疗效果。
本发明中，可选地，所述黑磷纳米薄片、双氨端聚乙二醇、叶酸按质量比(1～3)∶(5～12)∶(2～6)投料。进一步可选地，所述黑磷纳米薄片、双氨端聚乙二醇、叶酸按质量比1∶4.2∶2.1投料。
本发明步骤(3)中，先采用探针超声方式对所述黑磷纳米薄片进行预包覆，再采用磁力搅拌方式进行进一步包覆。可选地，所述探针超声的时间为20-40分钟，所述探针超声过程中，持续超声45秒-1小时，及等待15秒-1小时为一个周期，功放为12％-20％。所述磁力搅拌的转速可以为800rpm-1200rpm，持续 时间为3-4小时。
本发明步骤(3)中，进一步包括采用超滤膜离心的方式对所得靶向光热黑磷纳米制剂进行至少一次纯化、筛选，得到优化粒径大小的纯净靶向光热黑磷纳米制剂，所述超滤膜离心的速率为2000-4000rpm，时间为30-60分钟，温度为4-8℃。
进一步可选地，所述靶向光热黑磷纳米制剂的制备方法包括重复多次的所述超滤膜离心操作，具体为：每次超滤膜离心操作结束后，用生理盐水重悬超滤膜上的靶向光热黑磷纳米制剂，然后进行下一次所述超滤膜离心操作。
本发明上述各个步骤中超声及离心持续时间、周期、转速及功放等各指标的参数设置不局限于上述的限定。
本发明第二方面提供的靶向光热黑磷纳米制剂的制备方法，通过将黑磷纳米薄片与叶酸修饰的聚乙二醇通过静电吸附的方式包覆结合，制得靶向光热黑磷纳米制剂，制备过程简单易操作。
第三方面，本发明提供了一种上述的靶向光热黑磷纳米制剂在制备癌症光热消融治疗药物中的应用。所述靶向光热黑磷纳米制剂应用于癌症的靶向光热消融治疗。所述光热消融治疗通过808nm近红外波段激光激发。所述808nm近红外波段激光具有很强的皮肤穿透能力，能够有效到达深层组织及癌症部位。同时，所述靶向光热黑磷纳米制剂具有很好的生物相容性、无毒副作用。
下面分多个实施例对本发明实施例进行进一步的说明。其中，本发明实施例不限定于以下的具体实施例。在不变主权利的范围内，可以适当的进行变更实施。
实施例1
一种靶向光热黑磷纳米制剂的制备方法，包括以下步骤：
(1)采用溶液剥离结合探针超声法制备黑磷纳米薄片：取50mg块状黑磷材料，置于100mL超纯水中，进行探针超声，所述探针超声法的超声时间为12小时，过程为持续超声45秒，等待15秒为一个周期，功放为20％；超声结束后，取棕黑色剥离液置于离心管中，1000rpm离心10分钟，温度为4℃，沉淀未被剥离的块状黑磷，并小心分离沉淀的块状黑磷和上清液中的黑磷纳米薄片，以进一步提纯分离黑磷纳米薄片；
(2)在溶解有叶酸(1.0g,2.27mmol)的40mL的无水DMSO中加入三乙胺(0.5mL)，NHS(0.52g,5.00mmol)以及DCC(0.50g,2.50mmol)，得到混合液，该混合液在室温、避光条件下磁力搅拌反应24h后，过滤除去不溶的副产物和未反应的原料，得到NHS活化叶酸。在三乙胺存在的前提下，将2.5mL的NHS活化叶酸加入到双氨端聚乙二醇(分子量为3000)的无水DMSO溶液中，两者(NHS活化后叶酸：双端氨聚乙二醇)摩尔比为1:2，得到混合溶液。该混合溶液在室温、避光条件下磁力搅拌反应24h，通过耦合氨端共聚物，合成叶酸修饰的聚乙二醇FA-PEG-NH2，在蒸馏水中透析(MWCO:1000)，以除去未反应的叶酸，对FA-PEG-NH2进行纯化；
(3)采用探针超声与磁力搅拌的相结合的方式，在黑磷纳米薄片的表面包覆叶酸修饰的聚乙二醇制备靶向光热黑磷纳米制剂；其中黑磷纳米薄片和叶酸修饰的聚乙二醇的质量比为1:6.3。具体地，先采用探针超声方式对黑磷纳米薄片进行叶酸修饰的聚乙二醇预包覆，探针超声过程在二者悬液中进行，探针超声的时间为40分钟，过程为持续超声45秒，等待15秒为一个周期，功放为15％。然后采用在室温下磁力搅拌的方式对黑磷纳米薄片进行叶酸修饰的聚乙二醇进一步包覆，磁力搅拌转速为800rpm，持续时间为4小时；单纯聚乙二醇包覆的黑磷纳米光热制剂(无叶酸修饰)可以通过相同的方式制备，制备原料由叶酸 修饰的氨端聚乙二醇替换为无修饰的氨端聚乙二醇；
(4)采用超滤膜离心的方式纯化、筛选步骤(3)所得靶向光热黑磷纳米制剂，得到优化粒径大小的纯净靶向光热黑磷纳米制剂：取步骤(3)中反应混合液加入到超滤管(MWCO 100kDa；Millipore)中离心，分离未包覆的叶酸修饰的聚乙二醇和制备得到的靶向光热黑磷纳米制剂；超滤膜离心的速率为4000rpm，时间为30分钟，温度为4℃。超滤膜离心后，用生理盐水重悬滤膜上的靶向光热黑磷纳米制剂，重复超滤膜离心和生理盐水重悬3次，即可得到纯净的靶向光热黑磷纳米制剂BP-PEG-FA。
图1为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA的结构示意图，包括黑磷纳米薄片10、通过静电引力吸附在黑磷纳米薄片10表面的聚乙二醇20和通过酰胺键连接在聚乙二醇20上的叶酸30；聚乙二醇20的一端为氨基，另一端为亚氨基，所述亚氨基的氮原子与叶酸30中的羰基碳原子相连构成所述酰胺键，所述叶酸30暴露在纳米制剂的最外层。
图2(a)和图2(b)分别为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA的扫描透射电子显微镜(STEM)和X射线能量色散谱(EDS mapping)，从图中可看出，本实施例制得的BP-PEG-FA粒径大约为100nm左右，粒径范围适合产生较好的EPR效应，并且可以获知本实施例制得的BP-PEG-FA的元素构成为碳、氮、氧、磷四种元素，初步验证靶向光热黑磷纳米制剂BP-PEG-FA制备成功。
图3为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA的粒径分布图，与图2中结果具有很好的一致性。
图4为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA在制备过程中Zeta电位的变化图。黑磷纳米薄片BP NSs的Zeta电位在-14mV左右，表 面负电绝对值较高，能够有效与氨端聚乙二醇发生相互作用，进行聚乙二醇包覆。聚乙二醇包覆后的黑磷光热纳米制剂(包括无叶酸修饰BP-PEG NSs和有叶酸修饰BP-PEG-FA NSs)的Zeta电位在-10mV～-18mV之间，维持了较高的绝对值，纳米制剂之间相互排斥作用较强，因而在分散相中高度稳定。
图5为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA的傅里叶红外光谱图(FT-IR)。从图中可以看出，在～2900cm-1吸收峰来自靶向光热黑磷纳米制剂BP-PEG-FA中聚乙二醇部分的CH振动峰和C-O振动峰，在～1637到～1653cm-1的吸收峰来自叶酸部分的酰胺结合，进一步验证了靶向光热黑磷纳米制剂BP-PEG-FA制备成功。
实施例2
一种靶向光热黑磷纳米制剂的制备方法，包括以下步骤：
(1)采用溶液剥离结合探针超声法制备黑磷纳米薄片：取50mg块状黑磷材料，置于100mL超纯水中，进行探针超声，所述探针超声法的超声时间为18小时，过程为持续超声1小时，等待1小时为一个周期，功放为20％。超声结束后，取棕黑色剥离液置于离心管中，2000rpm离心6分钟，温度为4℃，沉淀未被剥离的块状黑磷，并小心分离沉淀的块状黑磷和上清液中的黑磷纳米薄片，以进一步提纯分离黑磷纳米薄片；
(2)在溶解有叶酸(1.0g,2.27mmol)的40mL的无水DMSO中加入三乙胺(0.5mL)，NHS(0.52g,5.00mmol)以及DCC(0.50g,2.50mmol)，得到混合液，该混合液在室温、避光条件下磁力搅拌反应24h后，过滤除去不溶的副产物和未反应的原料，得到NHS活化叶酸。在三乙胺存在的前提下，将2.5mL的NHS活化叶酸加入到双氨端聚乙二醇(分子量为5000)的DMSO溶液中， 两者等摩尔比，得到混合溶液。该混合溶液在室温、避光条件下磁力搅拌反应24h，通过耦合氨端共聚物，合成叶酸修饰的聚乙二醇FA-PEG-NH2，在蒸馏水中透析(MWCO:3000)，以除去未反应的叶酸，对FA-PEG-NH2进行纯化；
(3)采用探针超声与磁力搅拌的相结合的方式，在黑磷纳米薄片的表面包覆叶酸修饰的聚乙二醇制备靶向光热黑磷纳米制剂，其中黑磷纳米薄片和叶酸修饰的聚乙二醇的质量比为1:5。具体地，先采用探针超声方式对黑磷纳米薄片进行叶酸修饰的氨端聚乙二醇进行预包覆，探针超声过程在二者悬液中进行，探针超声的时间为持续30分钟，功放为12％；再在室温下采用磁力搅拌的方式对黑磷纳米薄片进行叶酸修饰的聚乙二醇的进一步包覆，磁力搅拌转速为1000rpm，持续时间为4小时；单纯聚乙二醇包覆的黑磷纳米光热制剂(无叶酸修饰)可以通过相同的方式制备，制备原料由叶酸修饰的氨端聚乙二醇替换为无修饰的氨端聚乙二醇；
(4)采用超滤膜离心的方式纯化、筛选步骤(3)所得靶向光热黑磷纳米制剂，得到优化粒径大小的纯净靶向光热黑磷纳米制剂：取步骤(3)中反应混合液加入到超滤管(MWCO 100kDa；Millipore)中离心，分离未包覆的叶酸修饰的聚乙二醇和制备得到的靶向光热黑磷纳米制剂；超滤膜离心的速率为4000rpm，时间为30分钟，温度为4℃。超滤膜离心后，用生理盐水重悬滤膜上的靶向光热黑磷纳米制剂，重复超滤膜离心和生理盐水重悬4次，即可得到纯净的靶向光热黑磷纳米制剂BP-PEG-FA。
图6为本发明实施例2制备的靶向光热黑磷纳米制剂BP-PEG-FA在808nm近红外激光照射下随时间变化的光热曲线图谱。从图中可以看出，本发明实施例靶向光热黑磷纳米制剂BP-PEG-FA具有很好的光热特性，在强度为1.0W/cm2的808nm激光照射下，浓度为100μg/mL的靶向光热黑磷纳米制剂的温度在10 分钟内上升了约26℃。
图7为本发明实施例2制备的靶向光热黑磷纳米制剂BP-PEG-FA在多次808nm近红外激光照射下的光热稳定性。从图中可以看出，本发明实施例靶向光热黑磷纳米制剂BP-PEG-FA在多次808nm近红外激光照射下温度变化稳定，近红外激光激发的光稳定性好。
图8为本发明实施例2制备的靶向光热黑磷纳米制剂BP-PEG-FA在不同制备时间下的光热曲线图谱。从图中可以看出，制备一周后与刚刚制备的靶向光热黑磷纳米制剂BP-PEG-FA在相同条件下的近红外激光激发后温度变化一致，因此，表明本发明实施例制备的靶向光热黑磷纳米制剂BP-PEG-FA可以储备放置后使用。
实施例3
一种靶向光热黑磷纳米制剂的制备方法，包括以下步骤：
(1)采用溶液剥离结合探针超声法制备黑磷纳米薄片，取50mg块状黑磷材料，置于100mL超纯水中，进行探针超声，所述探针超声法的超声时间为16小时，过程为持续超声1小时，等待1小时为一个周期，功放为25％。超声结束后，取棕黑色剥离液置于离心管中，1000rpm离心15分钟，温度为4℃，沉淀未被剥离的块状黑磷，并小心分离沉淀的块状黑磷和上清液中的黑磷纳米薄片，以进一步提纯分离黑磷纳米薄片；
(2)在溶解有叶酸(1.0g,2.27mmol)的40mL的无水DMSO中加入三乙胺(0.5mL)，NHS(0.52g,5.00mmol)以及DCC(0.50g,2.50mmol)，得到混合液，该混合液在室温、避光条件下磁力搅拌反应24h后，过滤除去不溶的副产物和未反应的原料，得到NHS活化叶酸。在三乙胺存在的前提下，将2.5mL 的NHS活化叶酸加入到双氨端聚乙二醇(分子量为550)的DMSO溶液中，两者等摩尔比，得到混合溶液。该混合溶液在室温、避光条件下磁力搅拌反应24h，通过耦合氨端共聚物，合成叶酸修饰的聚乙二醇FA-PEG-NH2，在蒸馏水中透析(MWCO:500)，以除去未反应的叶酸，对FA-PEG-NH2进行纯化；
(3)采用探针超声与磁力搅拌的相结合的方式，在黑磷纳米薄片的表面包覆叶酸修饰的聚乙二醇制备靶向光热黑磷纳米制剂，其中黑磷纳米薄片和叶酸修饰的聚乙二醇的质量比为1:8。具体地，先采用探针超声方式对黑磷纳米薄片进行叶酸修饰的氨端聚乙二醇进行预包覆，探针超声过程在二者悬液中进行，探针超声的时间为持续30分钟，功放为15％；再在室温下采用磁力搅拌的方式对黑磷纳米薄片进行叶酸修饰的聚乙二醇的进一步包覆，磁力搅拌转速为850rpm，持续时间为4小时；单纯聚乙二醇包覆的黑磷纳米光热制剂(无叶酸修饰)可以通过相同的方式制备，制备原料由叶酸修饰的氨端聚乙二醇替换为无叶酸修饰的氨端聚乙二醇；
(4)采用超滤膜离心的方式纯化、筛选步骤(3)所得靶向光热黑磷纳米制剂，得到优化粒径大小的纯净靶向光热黑磷纳米制剂：取步骤(3)中反应混合液加入到超滤管(MWCO 100kDa；Millipore)中离心，分离未包覆的叶酸修饰的聚乙二醇和制备得到的靶向光热黑磷纳米制剂；超滤膜离心的速率为4000rpm，时间为40分钟，温度为4℃。超滤膜离心后，用生理盐水重悬滤膜上的靶向光热黑磷纳米制剂，重复超滤膜离心和生理盐水重悬5次，即可得到纯净的靶向光热黑磷纳米制剂BP-PEG-FA。
实施例4
一种靶向光热黑磷纳米制剂的制备方法，包括以下步骤：
(1)采用溶液剥离结合探针超声法制备黑磷纳米薄片，取50mg块状黑磷材料，置于100mL超纯水中，进行探针超声，所述探针超声法的超声时间为14小时，过程为持续超声0.5小时，等待0.5小时为一个周期，功放为30％。超声结束后，取棕黑色剥离液置于离心管中，2000rpm离心10分钟，温度为4℃，沉淀未被剥离的块状黑磷，并小心分离沉淀的块状黑磷和上清液中的黑磷纳米薄片，以进一步提纯分离黑磷纳米薄片；
(2)在溶解有叶酸(1.0g,2.27mmol)的40mL的无水DMSO中加入三乙胺(0.5mL)，NHS(0.52g,5.00mmol)以及DCC(0.50g,2.50mmol)，得到混合液，该混合液在室温、避光条件下磁力搅拌反应24h后，过滤除去不溶的副产物和未反应的原料，得到NHS活化叶酸。在三乙胺存在的前提下，将2.5mL的NHS活化叶酸加入到双氨端聚乙二醇(分子量为30000)的DMSO溶液中，两者等摩尔比，得到混合溶液。该混合溶液在室温、避光条件下磁力搅拌反应24h，通过耦合氨端共聚物，合成叶酸修饰的聚乙二醇FA-PEG-NH2，在蒸馏水中透析(MWCO:3000)，以除去未反应的叶酸，对FA-PEG-NH2进行纯化；
(3)采用探针超声与磁力搅拌的相结合的方式，在黑磷纳米薄片的表面包覆叶酸修饰的聚乙二醇制备靶向光热黑磷纳米制剂，其中黑磷纳米薄片和叶酸修饰的聚乙二醇的质量比为1:12。具体地，先采用探针超声方式对黑磷纳米薄片进行叶酸修饰的氨端聚乙二醇进行预包覆，探针超声过程在二者悬液中进行，探针超声的时间为持续20分钟，功放为20％；再在室温下采用磁力搅拌的方式对黑磷纳米薄片进行叶酸修饰的聚乙二醇的进一步包覆，磁力搅拌转速为1200rpm，持续时间为3小时；单纯聚乙二醇包覆的黑磷纳米光热制剂(无叶酸修饰)可以通过相同的方式制备，制备原料由叶酸修饰的氨端聚乙二醇替换为无修饰的氨端聚乙二醇；
(4)采用超滤膜离心的方式纯化、筛选步骤(3)所得靶向光热黑磷纳米制剂，得到优化粒径大小的纯净靶向光热黑磷纳米制剂：取步骤(3)中反应混合液加入到超滤管(MWCO 100kDa；Millipore)中离心，分离未包覆的叶酸修饰的聚乙二醇和制备得到的靶向光热黑磷纳米制剂；超滤膜离心的速率为4000rpm，时间为40分钟，温度为4℃。超滤膜离心后，用生理盐水重悬滤膜上的靶向光热黑磷纳米制剂，重复超滤膜离心和生理盐水重悬4次，即可得到纯净的靶向光热黑磷纳米制剂BP-PEG-FA。
应用实施例
使用含有10％胎牛血清的DMEM高糖培养液培养Hela细胞于37℃，5％CO2的培养箱中。待细胞铺满细胞培养瓶底部后，弃去陈旧培养基，用PBS冲洗一次，加入1mL 0.25％的胰蛋白酶，37℃消化1-2分钟，加入少量含有血清的完全培养基，用弯头吸管轻轻吹打使细胞脱离瓶壁成为单细胞悬液。将此单细胞悬液均匀接种于6孔细胞培养板中，再加入1mL培养基，37℃、5％CO2孵箱中培养24小时。于细胞中加入本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA，继续培养4小时。用冰冷的PBS冲洗三次，加入甲醇固定细胞20min，弃去甲醇，加入DAPI染液孵育5min，再用PBS冲洗三次，于FV-1000激光共聚焦显微镜下观察细胞摄取情况。观察DAPI所用激发光波长为340nm，通过Bright通道观察靶向光热黑磷纳米制剂BP-PEG-FA的摄取情况。
待正常培养于10％胎牛血清的DMEM高糖培养液中的Hela细胞铺满细胞培养瓶底部后，弃去陈旧培养基，用PBS冲洗一次，加入1mL 0.25％的胰蛋白酶，37℃消化1-2分钟，加入少量含有血清的完全培养基，轻轻吹打使细胞脱离瓶壁成为单细胞悬液。将此悬液进行细胞计数、稀释后按5000个细胞/孔的密度 均匀接种于96孔细胞培养板中，于37℃，5％CO2的培养箱中继续培养24小时使细胞贴壁。用完全培养基分散本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA(浓度分别是10，25，50μg/mL)作为待测样品；不含靶向光热黑磷纳米制剂BP-PEG-FA的完全培养基培养的Hela细胞作为对照组。待接种于96孔细胞培养板中的Hela细胞培养24h贴壁后，弃去陈旧培养基，用PBS冲洗一次，加入不同浓度的实验组和对照组分别培养4小时，随后在强度为1.0W/cm2的808nm激光照射10分钟，弃去陈旧培养基，用PBS冲洗一次后，加入完全培养基，37℃孵育过夜后，通过MTT法(每个组均设置5个复孔)测定不同组的细胞活性以测定靶向光热黑磷纳米制剂BP-PEG-FA体外癌症光热治疗效果。
图9为激光共聚焦扫描电子显微镜(CLSM)观察本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA的Hela细胞摄取图片。具体地图9(a)、图9(b)、图9(c)分别为细胞核用DAPI染成蓝色，依次通过DAPI通道、Bright通道和混合通道观察的图像。从图中可以看出，本发明实施例的靶向光热黑磷纳米制剂BP-PEG-FA能够有效进入癌症细胞，进而能够在细胞内部发生光热治疗效果。
图10为本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA在不同癌症细胞(Hela、MCF-7、HepG2和PC3)中的毒性结果。从图中可以看出，本发明实施例的靶向光热黑磷纳米制剂BP-PEG-FA在无激光照射情况下不具备细胞毒性，生物安全性好、无毒副作用。
图11为不同浓度的本发明实施例1制备的靶向光热黑磷纳米制剂BP-PEG-FA在Hela细胞中的体外靶向光热治疗效果。图11中横坐标黑磷浓度是指黑磷靶向光热制剂中含有黑磷的浓度。Control指未做任何处理细胞，作为 空白参照。从图中可以看出，在强度为1.0W/cm2的808nm激光照射下，与未经靶向修饰的黑磷纳米薄片(BP NSs)和黑磷光热纳米制剂(BP-PEG NSs)相比，靶向光热黑磷纳米制剂BP-PEG-FA具有更为优异的体外靶向光热治疗效果，能够有效杀死癌细胞，并且只在近红外激光的照射部位产生治疗效果，从而极大程度上减少了对正常细胞、组织及器官的伤害，具有针对性。
需要说明的是，根据上述说明书的揭示和和阐述，本发明所属领域的技术人员还可以对上述实施方式进行变更和修改。因此，本发明并不局限于上面揭示和描述的具体实施方式，对本发明的一些等同修改和变更也应当在本发明的权利要求的保护范围之内。此外，尽管本说明书中使用了一些特定的术语，但这些术语只是为了方便说明，并不对本发明构成任何限制。

Claims (13)

  1. 一种靶向光热黑磷纳米制剂，其特征在于，包括黑磷纳米薄片、通过静电引力吸附在所述黑磷纳米薄片表面的聚乙二醇、以及通过酰胺键连接在所述聚乙二醇上的叶酸，所述聚乙二醇的一端为氨基，另一端为亚氨基，所述亚氨基的氮原子与所述叶酸中的羰基碳原子相连构成所述酰胺键，所述叶酸暴露在所述纳米制剂的最外层。
  2. 如权利要求1所述的靶向光热黑磷纳米制剂，其特征在于，所述靶向光热黑磷纳米制剂通过将黑磷纳米薄片、双氨端聚乙二醇、叶酸按质量比(1～3)∶(5～12)∶(2～6)配比制得。
  3. 如权利要求1所述的靶向光热黑磷纳米制剂，其特征在于，所述黑磷纳米薄片的长宽尺寸为100nm-200nm；所述黑磷纳米薄片的厚度为2nm-3nm。
  4. 如权利要求2所述的靶向光热黑磷纳米制剂，其特征在于，所述双氨端聚乙二醇的重均分子量为550-30000。
  5. 一种靶向光热黑磷纳米制剂的制备方法，其特征在于，包括以下步骤：
    提供黑磷纳米薄片；
    将羟基琥珀酰亚胺活化叶酸加入到双氨端聚乙二醇的二甲基亚砜溶液中，并加入适量催化剂，得到混合溶液，所述混合溶液在室温、避光条件下进行搅拌反应16-32小时，反应完成后过滤，得到叶酸修饰的聚乙二醇，所述叶酸通过酰胺键连接在聚乙二醇上，所述聚乙二醇的一端为氨基，另一端为亚氨基，所述亚氨基的氮原子与所述叶酸中的羰基碳原子相连构成所述酰胺键；
    依次采用探针超声、磁力搅拌的方式，在所述黑磷纳米薄片的表面包覆叶酸修饰的聚乙二醇，得到靶向光热黑磷纳米制剂，所述叶酸修饰的聚乙二醇通 过静电引力吸附在所述黑磷纳米薄片表面，所述叶酸暴露在所述靶向光热黑磷纳米制剂的最外层。
  6. 如权利要求5所述的靶向光热黑磷纳米制剂的制备方法，其特征在于，所述黑磷纳米薄片、双氨端聚乙二醇、叶酸按质量比(1～3)∶(5～12)∶(2～6)投料。
  7. 如权利要求5所述的靶向光热黑磷纳米制剂的制备方法，其特征在于，所述催化剂包括三乙胺，所述催化剂与反应体系的体积比为1:75-85。
  8. 如权利要求5所述的靶向光热黑磷纳米制剂的制备方法，其特征在于，所述探针超声的时间为20-40分钟，所述探针超声过程中，持续超声45秒-1小时，及等待15秒-1小时为一个周期，功放为12％-20％。
  9. 如权利要求5所述的靶向光热黑磷纳米制剂的制备方法，其特征在于，所述磁力搅拌的转速为800rpm-1200rpm，持续时间为3-4小时。
  10. 如权利要求5所述的靶向光热黑磷纳米制剂的制备方法，其特征在于，所述黑磷纳米薄片采用溶液剥离结合探针超声法制备，并采用离心收集、纯化，所述探针超声的超声时间为12-18小时，所述探针超声过程中，持续超声45秒-1小时，及等待15秒-1小时为一个周期，功放为20％-30％，所述离心的速率为1000rpm-2000rpm，时间为6-15分钟，温度为4℃。
  11. 如权利要求5所述的靶向光热黑磷纳米制剂的制备方法，所述羟基琥珀酰亚胺活化叶酸采用如下方式制备：将叶酸溶解于无水二甲基亚砜中，再加入羟基琥珀酰亚胺和N,N'-二环己基碳酰亚胺得到混合液，所述混合液在室温、避光和三乙胺催化条件下进行搅拌反应16-32小时，反应完成后过滤，得到所述羟基琥珀酰亚胺活化叶酸。
  12. 如权利要求5所述的靶向光热黑磷纳米制剂的制备方法，其特征在于， 进一步包括采用超滤膜离心的方式对所得靶向光热黑磷纳米制剂进行至少一次纯化、筛选，得到优化粒径大小的纯净靶向光热黑磷纳米制剂，所述超滤膜离心的速率为2000-4000rpm，时间为30-60分钟，温度为4-8℃。
  13. 如权利要求1-4任一项所述的靶向光热黑磷纳米制剂在制备癌症光热消融治疗药物中的应用。
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