WO2015188570A1 - 一种白蛋白吲哚菁绿紫杉醇复合物及其制备方法与应用 - Google Patents

一种白蛋白吲哚菁绿紫杉醇复合物及其制备方法与应用 Download PDF

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WO2015188570A1
WO2015188570A1 PCT/CN2014/089650 CN2014089650W WO2015188570A1 WO 2015188570 A1 WO2015188570 A1 WO 2015188570A1 CN 2014089650 W CN2014089650 W CN 2014089650W WO 2015188570 A1 WO2015188570 A1 WO 2015188570A1
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hsa
paclitaxel
icg
ptx
albumin
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PCT/CN2014/089650
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French (fr)
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刘庄
陈倩
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苏州大学张家港工业技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo

Definitions

  • the invention relates to the field of biomedicine, in particular to an albumin phthalocyanine green paclitaxel complex and a preparation method and application thereof.
  • Cancer is one of the major malignant diseases that threaten human health. Although a large amount of human, material and financial resources have been invested in the prevention and treatment of cancer since the 1950s, the progress made by humans in this respect is very limited.
  • Paclitaxel is a widely used anticancer drug after doxorubicin and cisplatin. It is mainly used for ovarian cancer and breast cancer. It is also suitable for lung cancer, colorectal cancer, melanoma, head and neck cancer, lymphoma and brain tumor. Both show a positive clinical effect. However, since paclitaxel is highly insoluble in water, polyoxyethylene castor oil and anhydrous ethanol are required as cosolvents, and polyoxyethylene castor oil causes various degrees of allergic reactions, neurotoxicity, hematological toxicity, etc., thereby causing paclitaxel. The dosage used is limited.
  • polyoxyethylene castor oil forms a large number of droplets in the blood circulation and encapsulates paclitaxel, which reduces paclitaxel leaving the blood circulation into the tissue, thereby reducing the dose-effect relationship of paclitaxel.
  • albumin-bound paclitaxel is a new type of white containing no polyoxyethylene castor oil and human albumin as a drug carrier and stabilizer. Protein-bound paclitaxel has the characteristics of no anti-allergic pretreatment, good curative effect and low toxicity.
  • albumin-bound paclitaxel can only be used as a chemotherapeutic agent for chemotherapy of tumors.
  • Chemotherapy is a systemic treatment of tumors that kills tumor cells at the cellular or molecular level, theoretically killing all tumor cells, but It is inevitable that it also has a killing effect on normal cells, so there are relatively large side effects in clinical use, which brings great pain to patients.
  • Photothermal therapy is a novel method for thermal ablation of tumor cells.
  • the main principle is to directly kill tumor cells by the thermal effect generated by photothermal conversion under excitation light, and whether it can produce strong light absorption on cancer cells.
  • high photothermal conversion efficiency is a key factor in the success of photothermal therapy.
  • Near-infrared light is a non-invasive new tool for tumor photothermal therapy that effectively penetrates normal tissues and reaches tumor sites, reducing damage to normal tissues.
  • Phthalocyanine green is a long-wavelength tricarbocyanine dye widely used in medical clinical diagnosis. ICG is essentially non-toxic and can be quickly removed from the body. It was initially used to examine choroidal tumors, central serous chorioretinopathy, various choroidal inflammations, degenerative diseases, vascular streaks, and vascular occlusion. Because ICG has good fluorescence and optical absorption properties in the near-infrared region, it has potential applications in biomedicine. In recent years, it has been used in fluorescence imaging and photothermal therapy. However, some physical characteristics, such as aggregation at early high concentrations, poor stability, non-specific adsorption and poor targeting ability limit the development and application of ICG in biomedicine.
  • the object of the present invention is to provide an albumin phthalocyanine green paclitaxel complex which has strong fluorescence and optical absorption properties in the near-infrared region, and can kill tumor cells, and can be in cells and living bodies. Combination therapy and imaging were performed on the upper level.
  • the present invention adopts the following technical solutions:
  • An albumin phthalocyanine green paclitaxel complex consisting of phthalocyanine, paclitaxel and human serum albumin, and phthalocyanine and paclitaxel are adsorbed onto human serum albumin by hydrophobic interaction.
  • Human Serum Albumin is a protein in human plasma, and its non-glycosylated single-chain polypeptide contains 585 amino acids with a molecular weight of 66 kD. Its concentration in plasma is 42 g / L, accounting for about 60% of total plasma protein.
  • human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions and many therapeutic molecules: while maintaining the normal osmotic pressure of the blood.
  • human serum albumin can be used to treat shock and burns, to supplement blood loss caused by surgery, accident or major bleeding, and also as a plasma compatibilizer.
  • Phthalocyanine green is commonly known as indigo green, diagnostic green needle, and indocyanine green. Its molecular weight is 774.9Da. It is a long-wavelength tricarbocyanine dye widely used in medical clinical diagnosis. At the same time, indocyanine green has good fluorescence and optical absorption properties in the near-infrared region, and has potential application in biomedicine, and can be used for fluorescence imaging and photothermal therapy.
  • Paclitaxel PTX
  • alias Taisu purple pigment
  • special element chemical name 5 ⁇ , 20-epoxy-1, 2 ⁇ , 4, 7 ⁇ , 10 ⁇ , 13 ⁇ -hexahydroxy taxane-11-ene-9- Keto-4,10-diacetate-2-benzoate-13[(2'R,3'S)-N-benzoyl-3-phenylisoserine] having a molecular weight of 853.9 Da.
  • the albumin phthalocyanine green paclitaxel complex of the present invention is composed of phthalocyanine derivative, paclitaxel and human serum albumin, and phthalocyanine and paclitaxel are adsorbed on human serum albumin by hydrophobic interaction to obtain the albumin.
  • Nanoparticles of phthalocyanine green paclitaxel complex HSA-ICG-PTX.
  • the indocyanine green in the albumin phthalocyanine green paclitaxel complex has good optical absorption in the near-infrared region, heat is generated under the irradiation of the laser, and more material can be promoted into the cell, and the Paclitaxel in the albumin phthalocyanine green paclitaxel complex is an ideal chemotherapeutic agent and can kill tumor cells. Therefore, the albumin phthalocyanine green paclitaxel complex of the present invention is an ideal combination therapeutic agent.
  • due to the indocyanine green in the albumin phthalocyanine green paclitaxel complex in the near infrared region It has strong fluorescence and is a good imaging agent for fluorescence imaging of living cells. Therefore, the biodistribution can be studied by fluorescence in vivo imaging technology, and the albumin phthalocyanine green paclitaxel complex can be monitored to reach the tumor site. Volume, guiding combination therapy in fluorescence imaging mode.
  • the invention determines the granules of the albumin phthalocyanine green paclitaxel complex of the invention by transmission electron microscopy and dynamic light scattering.
  • Transmission electron micrographs show that the particle size of HSA-ICG is about 7-8 nm, while the albumin phthalocyanine green paclitaxel complex (HSA-ICG-PTX) of the present invention has a particle size of about 50-60 nm. It indicated that the addition of paclitaxel could induce the self-assembly of HSA-ICG into large nanoparticles, which made the phthalocyanine green and paclitaxel adsorbed by hydrophobic interaction more stable.
  • the laser particle size distribution shows that the hydration diameter of HSA-ICG is about 10 nm, and the hydration diameter of HSA-PTX and HSA-ICG-PTX is about 100 nm. It is further proved that the addition of paclitaxel can cause albumin or HSA-ICG to self-assemble into large nanoparticles. It shows that paclitaxel not only acts as a chemotherapeutic drug, but also can self-assemble proteins into large nanoparticles like a cross-linking agent.
  • the albumin phthalocyanine green paclitaxel complex has a particle diameter of 50 nm to 60 nm.
  • the molar ratio of the human serum albumin to the phthalocyanine green is 1:2.
  • the loading amount of the paclitaxel is 5% to 8%.
  • the invention also provides a preparation method of the albumin phthalocyanine green paclitaxel complex, which comprises dissolving phthalocyanine green to obtain a phthalocyanine green solution, dissolving paclitaxel to obtain a paclitaxel solution, and dissolving human serum albumin in phosphate.
  • a phosphate buffer solution of human serum albumin is obtained in the buffer solution; then the phthalocyanine green solution and the paclitaxel solution are simultaneously added to the phosphate buffer solution of human serum albumin, and the mixture is stirred overnight in the dark.
  • the white molar ratio of human serum albumin to phthalocyanine green or the loading of paclitaxel can be obtained.
  • Protein phthalocyanine green paclitaxel complex
  • human serum white can be obtained by controlling the amount of the phthalocyanine green solution or the paclitaxel solution added to the phosphate buffer solution of human serum albumin.
  • the albumin paclitaxel complex having a different molar ratio of protein to phthalocyanine green than the albumin phthalocyanine green complex or paclitaxel.
  • the method for preparing the albumin phthalocyanine green paclitaxel complex of the present invention comprises dissolving phthalocyanine green to obtain a 10 mg/ml phthalocyanine green solution, and dissolving paclitaxel to obtain a 20 mg/ml paclitaxel solution.
  • Human serum albumin was dissolved in phosphate buffer solution to obtain a phosphate buffer solution of 2 mg/ml human serum albumin; then 10 ⁇ l of phthalocyanine green solution and 6 ⁇ l of paclitaxel solution were added to 1 ml of phosphate buffer solution of human serum albumin. The mixture was stirred overnight in the dark to obtain an albumin phthalocyanine green paclitaxel complex having a molar ratio of human serum albumin to phthalocyanine green of 1:2 and a loading of paclitaxel of 5%.
  • the solvent for dissolving the phthalocyanine green is dimethyl sulfoxide.
  • the solvent for dissolving paclitaxel is ethanol.
  • the preparation method of the present invention further comprises the step of purifying the albumin phthalocyanine green paclitaxel complex.
  • the purification is specifically carried out by stirring the mixture to obtain a mixture, and the supernatant is collected by centrifugation to remove phthalocyanine green and paclitaxel which are not adsorbed to the protein.
  • the centrifugation is centrifuged at 14800 rpm for 5 min.
  • the present invention also provides an albumin phthalocyanine green paclitaxel complex prepared by the above preparation method.
  • the temperature change curve of the albumin phthalocyanine green paclitaxel complex of the present invention is tested by an infrared thermal imager, and the results show that the albumin phthalocyanine green paclitaxel of the present invention is within 5 minutes.
  • the temperature increase of the complex is remarkable, indicating that the albumin phthalocyanine green paclitaxel complex has relatively strong optical absorption properties and can be used as a material for photothermal therapy.
  • the combined therapeutic effect of HSA-ICG-PTX at the cellular level is examined, and the results show that the mild warming of the photothermal property of HSA-ICG-PTX can increase the permeability of the cell membrane under laser irradiation.
  • activity assays of 4T1 cells incubated with different paclitaxel concentrations of HSA-ICG-PTX are performed by standard MTT reagents, and the results show that when the concentration of paclitaxel is low, HSA-ICG-PTX is present on the cells. Very obvious chemotherapy effect.
  • HSA-ICG-PTX is injected into the mouse with 4T1 tumor through the tail vein, and a certain volume of blood is taken at different time points, and the change in the intensity of ICG fluorescence in the blood is analyzed. Behavior in the mouse blood circulatory system. The results showed that the amount of material retained in the blood of mice gradually decreased with time, but the amount of HSA-ICG-PTX retained in the blood of mice was significantly higher than the amount of HSA-ICG retained in the blood of mice. . It shows that the blood circulation time of HSA-ICG-PTX is significantly higher than that of HSA-ICG.
  • HSA-ICG-PTX is injected into the mouse with 4T1 tumor through the tail vein, and the tumor site is exposed to 808 nm laser for 10 min, and the tumor site temperature and laser irradiation time are counted. Relationship, changes in tumor volume, and survival rate in mice. The results showed that the temperature of the tumor site of mice injected with HSA-ICG-PTX could quickly rise to 48 ° C and maintain for 10 min, promoting more material into the cells, killing the tumor with the toxicity of PTX. The tumors of the mice were completely eliminated after two days under the combined treatment of photothermal and chemotherapy. The mice injected with HSA-ICG-PTX remained alive after 50 days, and the tumor sites were not regenerated. It shows that the combined treatment of HSA-ICG-PTX at the living level is remarkable.
  • the present invention also provides the use of the albumin phthalocyanine green paclitaxel complex for the preparation of a medicament for treating cancer.
  • albumin phthalocyanine green paclitaxel complex of the invention
  • the substance consists of phthalocyanine, paclitaxel and human serum albumin, and phthalocyanine and paclitaxel are adsorbed to human serum albumin by hydrophobic interaction.
  • the chemotherapeutic drug paclitaxel (PTX) is used as a crosslinking agent to combine a plurality of HSA-ICG into 50-60 nm nanoparticles, which not only improves
  • the stability of HSA-ICG also greatly improved the blood circulation time of HSA-ICG and greatly increased the enrichment of tumor parts.
  • the photothermal effect of ICG can promote the entry of HSA-ICG-PTX into cells, thereby improving the effect of combination therapy.
  • the albumin phthalocyanine green paclitaxel complex (HSA-ICG-PTX) of the invention has very good water solubility and biocompatibility, and has good dispersibility under water and physiological conditions, and there is no 14800 rpm. Significant precipitation; not only has very good optical absorption in the near-infrared region, it generates heat under the irradiation of laser light, promotes more materials into the cell, and can kill tumor cells, which is an ideal combination therapy reagent; It has strong fluorescence properties and can guide the combination of photothermal therapy and chemotherapy in fluorescence imaging mode.
  • the albumin phthalocyanine green paclitaxel complex of the present invention is injected into a tumor-bearing mouse through a tail vein, and is monitored by a fluorescence imaging technique, and the enrichment ratio of HSA-ICG-PTX at the tumor site is found.
  • HSA-ICG has been greatly improved, and then the laser is used for photothermal treatment. The effect is remarkable, and it will not cause damage to other parts, and will not recur after healing.
  • the preparation method of the albumin phthalocyanine green paclitaxel complex of the invention is simple in operation and easy to obtain raw materials, and can be used for mass preparation of albumin phthalocyanine green paclitaxel complex.
  • Figure 1 shows a transmission electron micrograph of HSA-ICG and HSA-ICG-PTX in Example 4; where a is a transmission electron micrograph of HSA-ICG and b is HSA-ICG Transmission electron micrograph;
  • FIG. 2 shows a laser particle size distribution map of HSA-ICG, HSA-PTX, and HSA-ICG-PTX in Example 4;
  • black line is HSA-ICG,
  • black dot dotted line is HSA-PTX,
  • grey line is HSA-ICG-PTX;
  • Figure 3 shows the loading curve of HSA-PTX obtained by high performance liquid chromatography in Example 5;
  • FIG. 4 is a view showing an ultraviolet absorption spectrum of HSA-ICG-PTX in different ratios of the same HSA concentration in Example 5; (black line) is HSA-ICG-PTX (1:1:10), (black dotted line) is HSA-ICG-PTX (1:2:10), (grey line) is HSA-ICG-PTX (1:4:10);
  • Figure 5 is a view showing the fluorescence spectrum of HSA-ICG-PTX in different ratios of the same HSA concentration in Example 5; (black line) is HSA-ICG-PTX (1:1:10), (black dotted line) is HSA-ICG-PTX (1:2:10), (grey line) is HSA-ICG-PTX (1:4:10);
  • Figure 6 is a graph showing the temperature rise of different materials in Example 6 under laser irradiation at 808 nm; Is water (aqueous solution); For HSA-PTX; For HSA-ICG; For HSA-ICG-PTX;
  • Figure 7 is a graph showing the cell survival rate of 4T1 cells after incubation of different concentrations of PTX with HSA-PTX and HSA-ICG-PTX in Example 7; (black line) is free PTX (solution of paclitaxel), (black dotted line) is HSA-PTX, (grey line) is HSA-ICG-PTX;
  • Figure 8 is a graph showing the effect of the combined treatment of HSA-ICG-PTX at the cellular level in Example 8; For HSA-ICG-PTX, For HSA-ICG+ laser; For HSA-ICG-PTX+ laser;
  • Figure 9 is a diagram showing the blood circulation of HSA-ICG and HSA-ICG-PTX in mice in Example 9, wherein For HSA-ICG-PTX, For HSA-ICG;
  • Figure 10 is a graph showing the enrichment of HSA-ICG and HSA-ICG-PTX in a tumor site of a mouse in Example 10; wherein, Figure a is a fluorescence imaging diagram of a living body of a mouse, and the upper panel of Figure a is an HSA-ICG -PTX, the lower part of the picture HSA-ICG in Figure a, from left to right, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h; Figure b is the fluorescence signal analysis diagram, Figure b For HSA-ICG-PTX, For HSA-ICG;
  • Figure 11 is a diagram showing the distribution of HSA-ICG and HSA-ICG-PTX in vivo in Example 11; wherein, Figure a is a fluorescence imaging diagram of different organs and tumor sites, and the upper panel of Figure a is HSA-ICG-PTX. Figure HSA-ICG in the lower part of Figure a; Figure b shows the fluorescence signal analysis of different organs and tumor sites, Figure b For HSA-ICG-PTX, For HSA-ICG;
  • Figure 12 is a graph showing the relationship between the temperature of the tumor site of the mouse and the laser irradiation time in Example 12; For HSA-ICG-PTX; For PBS;
  • Figure 13 is a graph showing changes in tumor volume of a mouse in Example 12; For PBS+ laser; For HSA-PTX; For HSA-ICG-PTX; For HSA-ICG-PTX+ laser;
  • Figure 14 is a graph showing the survival rate of mice in Example 12; For PBS+ laser; For HSA-PTX; For HSA-ICG-PTX; For the HSA-ICG-PTX+ laser.
  • the invention discloses an albumin phthalocyanine green paclitaxel complex and a preparation method and application thereof. Those skilled in the art can learn from the contents of this document and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the products and methods and applications of the present invention have been described in terms of the preferred embodiments, and it is obvious that the products and methods and applications described herein may be modified or appropriately modified and combined without departing from the spirit, scope and scope of the invention. To implement and apply the techniques of the present invention.
  • albumin Based on albumin (HAS), the phthalocyanine green and paclitaxel are adsorbed by hydrophobic interaction. Paclitaxel can act as a cross-linking agent to make albumin self-assembled into large nanoparticles, forming albumin phthalocyanine green paclitaxel complex (HSA- ICG-PTX).
  • HAS albumin phthalocyanine green paclitaxel complex
  • the phthalocyanine green (ICG) was dissolved in a dimethyl sulfoxide solution to prepare a 10 mg/mL phthalocyanine green solution, and paclitaxel (PTX) was dissolved in ethanol to prepare a 10 mg/mL paclitaxel solution;
  • 2 mg of human serum albumin (HSA) was dissolved in 2 mL of phosphate buffer solution to prepare a phosphate buffer solution of 1 mg/mL human serum albumin;
  • the phthalocyanine green solution and the paclitaxel solution are added to the phosphate buffer solution of human serum albumin in different proportions to obtain albumin phthalocyanine having different molar ratio of human serum albumin to phthalocyanine green or different loading of paclitaxel. Green paclitaxel complex.
  • the phthalocyanine green (ICG) was dissolved in a dimethyl sulfoxide solution to prepare a 10 mg/mL phthalocyanine green solution; 2 mg of human serum albumin (HSA) was weighed and dissolved in 2 mL of a phosphate buffer solution. Further, 6 ⁇ L of the phthalocyanine green solution was added, and the mixture was stirred overnight (12 h) in the dark; finally, the mixture was stirred overnight to obtain a mixture of the ICG which was not reacted by centrifugation at 14800 rpm for 5 minutes, and the supernatant was an albumin phthalocyanine green complex ( HSA-ICG).
  • HSA-ICG albumin phthalocyanine green complex
  • the paclitaxel solution was added to the phosphate buffer solution of human serum albumin in different proportions to obtain different ratios of albumin paclitaxel complex.
  • paclitaxel paclitaxel
  • ethanol a 10 mg/mL paclitaxel solution
  • HSA human serum albumin
  • phosphate buffer solution 2 mg
  • paclitaxel solution 25 ⁇ L
  • HSA-ICG Albumin phthalocyanine green complex
  • HSA-ICG-PTX albumin phthalocyanine green paclitaxel complex
  • the albumin phthalocyanine green complex (HSA-ICG) and the albumin phthalocyanine green paclitaxel complex (HSA-ICG-PTX) (wherein the concentration of HSA is 1 mg/ml) Dynamic scatterometry measures the hydration radius.
  • Figure 1 is a transmission electron micrograph of albumin phthalocyanine green complex (HSA-ICG) and albumin phthalocyanine green paclitaxel complex (HSA-ICG-PTX). It can be seen from Fig. 1 that the particle size of HSA-ICG is about 7-8 nm, and the particle size of HSA-ICG-PTX is about 50-60 nm. It indicated that the addition of paclitaxel could induce the self-assembly of HSA-ICG into large nanoparticles, which made the phthalocyanine green and paclitaxel adsorbed by hydrophobic interaction more stable.
  • HSA-ICG albumin phthalocyanine green complex
  • HSA-ICG-PTX albumin phthalocyanine green paclitaxel complex
  • Figure 2 is a laser particle size distribution of albumin phthalocyanine green complex (HSA-ICG), albumin paclitaxel complex (HSA-PTX) and albumin phthalocyanine green paclitaxel complex (HSA-ICG-PTX) .
  • HSA-ICG albumin phthalocyanine green complex
  • HSA-PTX albumin paclitaxel complex
  • HSA-ICG-PTX albumin phthalocyanine green paclitaxel complex
  • the hydration diameter of HSA-ICG is about 10 nm
  • the hydration diameter of HSA-PTX and HSA-ICG-PTX is about 100 nm.
  • the addition of paclitaxel can cause albumin or HSA-ICG to self-assemble into large nanoparticles. It shows that paclitaxel not only acts as a chemotherapeutic drug, but also can self-assemble proteins into large nanoparticles like a cross-linking agent.
  • the amount of PTX loaded on HSA was measured by high performance liquid chromatography (HPLC) to investigate the effect of loading of paclitaxel.
  • HSA-PTX synthesized according to the method described in Example 3 were separately dissolved in chromatographically pure methanol, incubated at room temperature for 12 h, centrifuged at 14800 rpm for 10 minutes to remove the precipitate, and the needle was tested.
  • the amount of paclitaxel was measured by high performance liquid chromatography (detection wavelength: 227 nm) loaded with a UV detector, and a mixture of methanol and water in a volume ratio of 1:1 was used as a mobile phase, and the elution time of paclitaxel was 7.8 min.
  • the loading of the ICG was studied using an ultraviolet-visible absorption spectrometer and a fluorescence spectrometer. Results are shown in Figure 3-5.
  • Figure 3 is the loading curve of HSA-PTX.
  • the amount of PTX loaded onto the HAS increases with the amount of added PTX, and the PTX is selected from the loading curve.
  • the concentration was 0.2 mg/ml, that is, 5% of the loading was used in the following experiment.
  • Figure 4 is a graph showing the ultraviolet absorption spectrum of HSA-ICG-PTX in different ratios (molar ratio of human serum albumin to phthalocyanine green) of the same HSA concentration (0.2 mg/ml). It can be clearly seen from the figure that HSA-ICG-PTX has very good optical absorption in the near-infrared region of 700 nm to 850 nm, and is a very good photothermal therapeutic reagent. As the ICG loading ratio increases, the absorption peak of ICG at around 800 nm increases significantly.
  • Figure 5 is a fluorescence spectrum of HSA-ICG-PTX in different ratios (molar ratio of human serum albumin to phthalocyanine green) of the same HSA concentration (0.2 mg/ml). It can be clearly seen from the figure that when ICG is adsorbed to HSA, the fluorescence in the near-infrared region is obviously enhanced. When the ratio of HSA to ICG is 1:2, the fluorescence is the strongest, and when ICG continues to increase. The amount of fluorescence will be quenched. Therefore, the molar ratio of human serum albumin to phthalocyanine green was selected to be 1:2 in subsequent experiments.
  • HSA-PTX, HSA-ICG and HSA-ICG-PTX and an aqueous solution prepared in Example 1-3 of the same HSA concentration (2 mg/ml) of 2 ml were placed in a cuvette, and a laser of 808 nm (power of 0.5 W/) was used. Cm 2 ) directly irradiated on the sample, and the temperature change curve was tested by an infrared thermal imager. The result is shown in Fig. 6.
  • HSA-PTX and HSA-ICG-PTX were incubated with 4T1 cells for 72 h, respectively, using standard MTT reagents. Cell viability assays were performed and the results are shown in FIG.
  • Example 8 combination therapy of HSA-ICG-PTX at the cellular level
  • HSA-ICG-PTX has a certain influence on cell activity under no laser irradiation and HSA-ICG under laser irradiation.
  • the mild warming of the photothermal properties of HSA-ICG-PTX can increase the permeability of the cell membrane, thereby promoting the ability of HSA-ICG-PTX to enter cells, and then killing cells with PTX. It shows that the combined treatment of HSA-ICG-PTX at the cellular level is remarkable.
  • Blood the behavior of the above materials in the blood circulation system of mice was analyzed by measuring changes in the intensity of ICG fluorescence in the blood.
  • the specific measurement method is as follows: the collected blood is weighed, and the taken blood is dissolved by a solution (1% SDS, 1% Triton-100, 40 mM Tris buffer solution), and the cell debris is removed by centrifugation at a low rotation speed, and the supernatant is determined.
  • the fluorescence intensity of ICG wherein ICG has an excitation wavelength of 730 nm, an emission peak of about 810 nm, and a receiving spectrum of 750 nm to 900 nm.
  • the statistical results are shown in Figure 9.
  • Figure 9 is a blood circulation diagram of HSA-ICG and HSA-ICG-PTX in mice. As shown in Figure 9, the amount of material retained in the blood of mice gradually decreases with time, but HSA-ICG - The amount of PTX retained in the blood of mice is significantly higher than the amount of HSA-ICG retained in the blood of mice. It is indicated that the particle size of the nanoparticles is significantly increased due to the cross-linking of PTX. The blood circulation time of HSA-ICG-PTX is significantly higher than that of HSA-ICG, which can be used for the enrichment of the tumor site.
  • the HSA-ICG prepared in Example 2 and the HSA-ICG-PTX prepared in Example 1 were injected into the mouse through the tail vein, and the pictures were collected in real time at different time points on the Small Animal Imaging System (CRI). The amount of enrichment of the material at the tumor site was observed, and the results are shown in Fig. 10. Among them, the selected excitation light source is 730 nm, and the exposure time is 100 ms.
  • Figure 10 is a graph showing the enrichment of materials in tumor sites in mice. As shown in Figure 10(a), the enrichment of HSA-ICG-PTX at the tumor site was significantly higher than that of HSA-ICG, consistent with blood circulation data. Over time, HSA-ICG is quickly metabolized due to its small particle size.
  • Figure 10 (b) is a plot of fluorescence signal values over time at tumor sites showing that the enrichment of HSA-ICG-PTX at the tumor site was significantly higher than HSA-ICG and peaked at 8 h.
  • the HSA-ICG prepared in Example 2 and the HSA-ICG-PTX prepared in Example 1 were injected into the back of a mouse bearing 4T1 tumor through the tail vein. After 24 hours, the experimental mice sacrificed and the tumor was removed. Important organs were placed in a watch glass, and fluorescent photographs were taken on a small animal imaging system to observe the enrichment of materials in various organs and tumor sites. The results are shown in Fig. 11.
  • Figure 11 is a graph showing the enrichment of materials in mice. As shown in Fig. 11(a), at 24h, the enrichment of HSA-ICG-PTX in the tumor site was the highest, and the kidney site also had a certain enrichment, indicating that HSA-ICG-PTX has some depolymerization in the body. Slowly, some materials are also metabolized from the kidney; while HSA-ICG is low in various parts, indicating that HSA-ICG is easily metabolized due to its small particle size. This result is consistent with the results of live imaging. .
  • Figure 11 (b) shows the fluorescence signal values of different organs and tumor sites obtained semi-quantitatively by the small animal imaging system. The experimental data further indicates that the blood circulation time of HSA-ICG-PTX is significantly higher than that of HSA-ICG.
  • HSA-ICG-PTX prepared in Example 1 was injected from the tail vein. After 4 hours, the tumor site was exposed to 808 nm laser for 10 min. The power was 0.3 W/cm 2 and the temperature of the tumor site was controlled at 48 °C. The other three groups of mice with tumors on the back (5 in each group) were used as the control group. The following treatments were performed (1) injection of phosphate buffer (PBS) and irradiation for 10 min under the same power laser; (2) The same dose of HSA-PTX prepared in Example 3 was injected; (3) The same dose of HSA-ICG-PTX prepared in Example 1 was injected, but no laser irradiation was applied.
  • PBS phosphate buffer
  • Figure 12 is a graph showing the relationship between the temperature of a mouse tumor site and the laser irradiation time. As shown in Figure 12, the temperature of the tumor site of mice injected with HSA-ICG-PTX can quickly rise to 48 ° C and maintain for 10 min, promoting more material into the cells, killing the tumor with the toxicity of PTX; The temperature of the mouse tumor is almost unchanged and does not affect the growth of the mouse tumor.
  • Figure 13 is a graph showing changes in tumor volume. As shown in Fig. 13, the tumors of the mice in the experimental group were completely eliminated after two days under the combined treatment of photothermal and chemotherapy, and the photothermal and chemotherapy alone could only inhibit the growth of the tumor at an early stage.
  • Figure 14 is a graph showing the survival rate of each group of mice. It can be seen from Fig. 14 that all of the control mice died at 18 to 22 days, while the experimental mice were still alive after 50 days, and the tumor sites were not regenerated.

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Abstract

一种白蛋白吲哚菁绿紫衫醇复合物，由吲哚菁绿、紫衫醇和人血清白蛋白组成。制备方法为将吲哚菁绿和紫衫醇通过疏水作用力吸附于人血清白蛋白上。用于制备治疗癌症的药物，在荧光成像模式下，进行光热治疗和化疗。

Description

一种白蛋白吲哚菁绿紫杉醇复合物及其制备方法与应用
本申请要求于2014年6月13日提交中国专利局、申请号为201410265653.2、发明名称为“一种白蛋白吲哚菁绿紫杉醇复合物及其制备方法与应用”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
本发明涉及生物医药领域，具体的说是涉及一种白蛋白吲哚菁绿紫杉醇复合物及其制备方法与应用。
背景技术
癌症是威胁人类健康的重大恶性疾病之一。虽然从上世纪50年代起，数十年中大量的人力、物力、财力被投入到癌症的预防和治疗中，但在这方面人类所取得的进展十分有限。
紫杉醇是继阿霉素和顺铂之后的一种广范使用的抗癌药物，主要适用于卵巢癌和乳腺癌，对肺癌、大肠癌、黑色素瘤、头颈部癌、淋巴瘤、脑瘤也都显示出较肯定的临床疗效。但由于紫杉醇高度不溶于水，需要聚氧乙烯蓖麻油和无水乙醇作为助溶剂，而聚氧乙烯蓖麻油在体内会导致不同程度的过敏反应、神经毒性、血液学毒性等，从而造成紫杉醇的使用剂量受到限制。此外，聚氧乙烯蓖麻油在血循环中形成大量微滴并将紫杉醇包裹，减少了离开血循环进入组织的紫杉醇，从而降低了紫杉醇的量效关系。为了降低毒性,提高其疗效，近年来临床上陆续开发了紫杉醇的新剂型，其中白蛋白结合型紫杉醇是一种不含聚氧乙烯蓖麻油、以人血白蛋白作为药物载体与稳定剂的新型白蛋白结合型紫杉醇，其具有不用抗过敏预处理、疗效较好、毒性较低等特点。但白蛋白结合型紫杉醇仅可作为化疗剂，对肿瘤进行化学治疗。化学治疗属于全身效应的肿瘤治疗方法，它可在细胞水平乃至分子水平上杀死肿瘤细胞，理论上来讲可以杀死全部肿瘤细胞，但 是不可避免的对正常细胞也有杀伤作用，因此临床使用中有比较大的毒副作用，给病人带来极大的痛苦。
光热治疗是一种新颖的肿瘤细胞热消融方法，其主要原理为在激发光的照射下，利用光热转换产生的热效应来直接杀死肿瘤细胞，能否在癌细胞上产生强的光照吸收以及高的光热转换效率是光热疗法是否成功的关键因素。近红外光是一种非侵入性的用于肿瘤光热治疗的新工具，能够有效的穿透正常组织到达肿瘤部位，可以减少对正常组织的伤害。
吲哚菁绿(简称ICG)，是一种长波长的三碳菁的染料，广泛地用于医学临床诊断。ICG基本没有毒并且可以迅速的从体内清除出去，起初主要用于检查脉络膜肿瘤、中心性浆液性脉络膜视网膜病变、各种脉络膜炎症、变性性疾病、血管样条纹和血管阻塞病等。由于ICG在近红外区具有很好的荧光及光学吸收性质在生物医学中有应用潜力，最近几年人们利用它可以用于荧光成像及光热治疗中。然而一些物理的特征，如早高浓度下聚集，稳定性差，非特异性吸附和靶向能力差等限制了ICG在生物医学中的发展和应用。目前已有文献报道将ICG吸附到白蛋白上，可以增强它的荧光及稳定性。然而ICG与白蛋白的结合稳定性不是很好，并且血液循环时间也不是很好，在肿瘤部位的富集量不高。
目前对于肿瘤的治疗，学者们一致认为多种治疗方法联合应用是目前较为有效且切实可行的，选用几种作用机制、作用时象和作用途径不同的疗法联合使用治疗肿瘤。
发明内容
有鉴于此，本发明目的是提供一种白蛋白吲哚菁绿紫杉醇复合物，该复合物在近红外区具有较强的荧光及光学吸收性能，而且可以杀死肿瘤细胞，可在细胞和活体上平上实现联合治疗与成像。
为实现本发明的目的，本发明采用如下技术方案：
一种白蛋白吲哚菁绿紫杉醇复合物，由吲哚菁衍、紫杉醇和人血清白蛋白组成，吲哚菁衍和紫杉醇通过疏水作用力吸附于人血清白蛋白上。
其中，人血清白蛋白(Human Serum Albumin，简称HSA)是人血浆中的蛋白质，其非糖基化的单链多肽包含585个氨基酸，分子量为66kD。在血浆中其浓度为42g/L，约占血浆总蛋白的60％。在体液中人血清白蛋白可以运输脂肪酸、胆色素、氨基酸、类固醇激素、金属离子和许多治疗分子等：同时维持血液正常的渗透压。在临床上人血清白蛋白可用于治疗休克与烧伤，用于补充因手术、意外事故或大出血所致的血液丢失，也可以作为血浆增容剂。
吲哚菁绿俗称靛氰绿、诊断用绿针、吲哚花青绿，其分子量为774.9Da，是一种长波长的三碳菁的染料，广泛地用于医学临床诊断。同时吲哚菁绿在近红外区具有很好的荧光及光学吸收性质，在生物医学中有应用潜力，可以用于荧光成像及光热治疗。
紫杉醇(Paclitaxel，PTX)，别名泰素，紫素，特素，化学名称5β，20-环氧-1，2α，4，7β，10β，13α-六羟基紫杉烷-11-烯-9-酮-4，10-二乙酸酯-2-苯甲酸酯-13[(2’R，3’S)-N-苯甲酰-3-苯基异丝氨酸酯]，其分子量为853.9 Da。
本发明所述白蛋白吲哚菁绿紫杉醇复合物由吲哚菁衍、紫杉醇和人血清白蛋白组成，吲哚菁衍和紫杉醇通过疏水作用力吸附于人血清白蛋白上，得到所述白蛋白吲哚菁绿紫杉醇复合物的纳米颗粒(HSA-ICG-PTX)。由于所述白蛋白吲哚菁绿紫杉醇复合物中的吲哚菁绿在近红外区具有很好的光学吸收，在激光的照射下会产生热量，可以促进更多的材料进入细胞，而所述白蛋白吲哚菁绿紫杉醇复合物中的紫杉醇又是一种理想的化疗试剂，可以杀死肿瘤细胞，因此本发明所述白蛋白吲哚菁绿紫杉醇复合物是理想的联合治疗试剂。另一方面，由于所述白蛋白吲哚菁绿紫杉醇复合物中的吲哚菁绿在近红外区 具有很强的荧光，是一种很好的成像剂，可用于活体细胞的荧光成像，因而可以通过荧光活体成像技术研究生物分布，监测所述白蛋白吲哚菁绿紫杉醇复合物达到肿瘤部位的量，在荧光成像模式下指导联合治疗。
在具体实施方案中本发明通过透射电镜和动态光散射确定本发明所述白蛋白吲哚菁绿紫杉醇复合物的粒剂。透射电镜照片显示HSA-ICG的粒径大约为7～8nm，而本发明所述白蛋白吲哚菁绿紫杉醇复合物(HSA-ICG-PTX)的粒径大约为50～60nm。表明紫杉醇的加入可以诱导HSA-ICG进行自组装成大的纳米粒子，使得疏水作用吸附上的吲哚菁绿与紫杉醇更加稳定。而激光粒径分布图显示HSA-ICG的水化直径约为10nm左右，HSA-PTX与HSA-ICG-PTX的水化直径约为100nm左右。进一步证明了紫杉醇的加入可以引起白蛋白或者HSA-ICG自组装成大的纳米粒子。表明紫杉醇不仅起到化疗药物的作用，还可以像交联剂一样使蛋白自组装成大的纳米粒子。
因此，作为优选，所述白蛋白吲哚菁绿紫杉醇复合物的粒径为50nm～60nm。
进一步的，作为优选，所述人血清白蛋白与吲哚菁绿的摩尔比为1∶2。
进一步的，作为优选，所述紫杉醇的装载量为5％～8％。
本发明还提供了所述白蛋白吲哚菁绿紫杉醇复合物的制备方法，为将吲哚菁绿溶解得到吲哚菁绿溶液，将紫杉醇溶解得到紫杉醇溶液，将人血清白蛋白溶于磷酸盐缓冲溶液中得到人血清白蛋白的磷酸盐缓冲溶液；然后将吲哚菁绿溶液与紫杉醇溶液同时加入到人血清白蛋白的磷酸盐缓冲溶液，避光搅拌过夜即得。
其中，通过控制加入到人血清白蛋白的磷酸盐缓冲溶液中吲哚菁绿溶液与紫杉醇溶液的量即可得到人血清白蛋白与吲哚菁绿的摩尔比不同或紫杉醇的装载量不同的白蛋白吲哚菁绿紫杉醇复合物。
同样，在制备白蛋白吲哚菁绿复合物和白蛋白紫杉醇复合物时，通过控制加入到人血清白蛋白的磷酸盐缓冲溶液中吲哚菁绿溶液或紫杉醇溶液的量即可得到人血清白蛋白与吲哚菁绿的摩尔比不同白蛋白吲哚菁绿复合物或紫杉醇的装载量不同的白蛋白紫杉醇复合物。
在一些实施方案中，本发明所述白蛋白吲哚菁绿紫杉醇复合物的制备方法，将吲哚菁绿溶解得到10mg/ml吲哚菁绿溶液，将紫杉醇溶解得到20mg/ml紫杉醇溶液，将人血清白蛋白溶于磷酸盐缓冲溶液中得到2mg/ml人血清白蛋白的磷酸盐缓冲溶液；然后将10μl吲哚菁绿溶液与6μl紫杉醇溶液加入到1ml人血清白蛋白的磷酸盐缓冲溶液，并避光搅拌过夜得到人血清白蛋白与吲哚菁绿的摩尔比为1:2、紫杉醇的装载量为5％的白蛋白吲哚菁绿紫杉醇复合物。
其中，优选的，所述溶解吲哚菁绿的溶剂为二甲基亚砜。
优选的，所述溶解紫杉醇的溶剂为乙醇。
进一步的，在一些实施方案中，本发明所述制备方法还包括对白蛋白吲哚菁绿紫杉醇复合物进行纯化的步骤。
作为优选，所述纯化具体为将搅拌过夜得到混合物离心收集上清，以除去未吸附到蛋白上的吲哚菁绿和紫杉醇。
进一步的，作为优选，所述离心为14800rpm离心5min。
本发明还提供了上述制备方法制备得到的白蛋白吲哚菁绿紫杉醇复合物。
在一个具体实施方案中，通过红外热成像仪测试本发明所述白蛋白吲哚菁绿紫杉醇复合物温度变化的曲线，结果显示，在5min之内，本发明所述白蛋白吲哚菁绿紫杉醇复合物的温度升高显著，表明所述白蛋白吲哚菁绿紫杉醇复合物具有比较强的光学吸收性质，可以作为光热治疗的材料。
在一个具体实施方案中，考察HSA-ICG-PTX在细胞水平的联合治疗效果，结果显示在激光照射情况下，HSA-ICG-PTX的光热性质产生的温和升温可以增加细胞膜的通透性，从而 促进HSA-ICG-PTX进细胞的能力，进而利用PTX杀死细胞。表明HSA-ICG-PTX在细胞水平的联合治疗效果显著。
在一个具体实施方案中，通过标准的MTT试剂对经不同紫杉醇浓度的HSA-ICG-PTX孵育的4T1细胞进行活性检测，结果显示当紫杉醇的浓度很低时，HSA-ICG-PTX对细胞就有非常明显的化疗作用。
在一个具体实施方案中，将HSA-ICG-PTX通过尾静脉注射到带有4T1肿瘤的小鼠体内，在不同时间点取出一定体积的血液，通过测量血液中ICG荧光强度的变化来分析其在小鼠血液循环系统中的行为。结果显示，随着时间的增加，滞留在小鼠血液中的材料的量逐渐衰减，但HSA-ICG-PTX滞留在小鼠血液中的量明显高于HSA-ICG滞留在小鼠血液中的量。表明HSA-ICG-PTX血液循环时间比HSA-ICG有明显地提高。
在另一个具体实施方案中，将HSA-ICG-PTX通过尾静脉注射到带有4T1肿瘤的小鼠体内，将肿瘤部位暴露在808纳米激光下照射10min，统计小鼠肿瘤部位温度与激光照射时间的关系、肿瘤体积的变化以及小鼠的存活率。结果显示注射HSA-ICG-PTX的小鼠肿瘤部位的温度很快可以升到48℃并维持10min，促进更多的材料进入细胞，利用PTX的毒性杀死肿瘤。小鼠的肿瘤在光热与化疗的联合治疗作用下，两天后全部消除，注射HSA-ICG-PTX的小鼠在50天后仍然全部活着，并且肿瘤部位没有重新再生。表明HSA-ICG-PTX在活体水平的联合治疗效果显著。
因此，本发明还提供了所述白蛋白吲哚菁绿紫杉醇复合物在制备治疗癌症的药物中的应用。
由上述技术方案可知，本发明提供了一种白蛋白吲哚菁绿紫杉醇复合物及其制备方法与应用。本发明所述白蛋白吲哚菁绿紫杉醇复合 物由吲哚菁衍、紫杉醇和人血清白蛋白组成，吲哚菁衍和紫杉醇通过疏水作用力吸附于人血清白蛋白上。本发明所述白蛋白吲哚菁绿紫杉醇复合物(HSA-ICG-PTX)中，化疗药物紫杉醇(PTX)作为交联剂，使多个HSA-ICG组合成50-60nm的纳米粒子，不仅提高了HSA-ICG的稳定性，还大幅度地提高了HSA-ICG的血液循环时间，极大地提高了肿瘤部分的富集量。另一方面，ICG的光热效果可以促进HSA-ICG-PTX进入细胞，从而提高联合治疗的效果。
本发明所述白蛋白吲哚菁绿紫杉醇复合物(HSA-ICG-PTX)具有非常好的水溶性和生物相容性，在水和生理条件下都具有很好的分散性，14800rpm不会有明显沉淀；不仅在近红外光区具有非常好的光学吸收，在激光的照射下会产生热量，促性更多的材料进细胞，而且可以杀死肿瘤细胞，是理想的联合治疗试剂；此外还具有很强的荧光性能，可以在荧光成像模式下指导光热治疗与化疗治疗的联合治疗。
将本发明所述白蛋白吲哚菁绿紫杉醇复合物通过尾静脉注射到带有肿瘤的小鼠体内，并由荧光成像技术对其进行监测，发现HSA-ICG-PTX在肿瘤部位的富集比HSA-ICG有很大的提高，然后用激光进行光热治疗，效果显著，并且不会对其他的部位造成损伤，治愈后不复发。
而本发明所述白蛋白吲哚菁绿紫杉醇复合物的制备方法，操作简单，原料易得，可用于白蛋白吲哚菁绿紫杉醇复合物的大量制备。
附图说明
图1示实施例4中HSA-ICG和HSA-ICG-PTX的透射电镜照片；其中a为HSA-ICG的透射电镜照片，b为HSA-ICG 的透射电镜照片；
图2示实施例4中HSA-ICG、HSA-PTX及HSA-ICG-PTX的激光粒径分布图；其中
Figure PCTCN2014089650-appb-000001
(黑色直线)为HSA-ICG，
Figure PCTCN2014089650-appb-000002
(黑色点虚线)为HSA-PTX，
Figure PCTCN2014089650-appb-000003
(灰色直线)为HSA-ICG-PTX；
图3示实施例5中由高效液相色谱得到的HSA-PTX的装载曲线；
图4示实施例5中相同HSA浓度不同比例的HSA-ICG-PTX的紫外吸收光谱图；其中，
Figure PCTCN2014089650-appb-000004
(黑色直线)为HSA-ICG-PTX(1:1:10)，
Figure PCTCN2014089650-appb-000005
(黑色虚线)为HSA-ICG-PTX(1:2:10)，
Figure PCTCN2014089650-appb-000006
(灰色直线)为HSA-ICG-PTX(1:4:10)；
图5示实施例5中相同HSA浓度不同比例的HSA-ICG-PTX的荧光光谱图；其中，
Figure PCTCN2014089650-appb-000007
(黑色直线)为HSA-ICG-PTX(1:1:10)，
Figure PCTCN2014089650-appb-000008
(黑色虚线)为HSA-ICG-PTX(1:2:10)，
Figure PCTCN2014089650-appb-000009
(灰色直线)为HSA-ICG-PTX(1:4:10)；
图6示实施例6中不同材料在808nm的激光照射下的升温曲线；其中，
Figure PCTCN2014089650-appb-000010
为water(水溶液)；
Figure PCTCN2014089650-appb-000011
为HSA-PTX；
Figure PCTCN2014089650-appb-000012
为HSA-ICG；
Figure PCTCN2014089650-appb-000013
为HSA-ICG-PTX；
图7示实施例7中不同浓度的PTX与HSA-PTX和HSA-ICG-PTX孵育4T1细胞后的细胞存活率曲线；其中，
Figure PCTCN2014089650-appb-000014
(黑色直线)为free PTX(紫杉醇的溶液)，
Figure PCTCN2014089650-appb-000015
(黑色虚线)为HSA-PTX，
Figure PCTCN2014089650-appb-000016
(灰色直线)为HSA-ICG-PTX；
图8示实施例8中HSA-ICG-PTX在细胞水平的联合治疗效果图；其中，
Figure PCTCN2014089650-appb-000017
为HSA-ICG-PTX，
Figure PCTCN2014089650-appb-000018
为HSA-ICG+激光；
Figure PCTCN2014089650-appb-000019
为HSA-ICG-PTX+激光；
图9示实施例9中为HSA-ICG及HSA-ICG-PTX在小鼠体内的血液循环图，其中，
Figure PCTCN2014089650-appb-000020
为HSA-ICG-PTX，
Figure PCTCN2014089650-appb-000021
为HSA-ICG；
图10示实施例10中HSA-ICG及HSA-ICG-PTX在小鼠肿瘤部位的富集情况图；其中，图a为小鼠活体的荧光成像图，图a中上部的图为HSA-ICG-PTX，图a中下部的图HSA-ICG，从左至右依次为0.5h、1h、2h、4h、8h、12h、24h；图b为荧光信号分析图，图b中
Figure PCTCN2014089650-appb-000022
为HSA-ICG-PTX，
Figure PCTCN2014089650-appb-000023
为HSA-ICG；
图11示实施例11中HSA-ICG及HSA-ICG-PTX在生物体内的分布图；其中，图a为不同器官及肿瘤部位的荧光成像图，图a中上部的图为HSA-ICG-PTX，图a中下部的图HSA-ICG；图b为不同器官及肿瘤部位的荧光信号分析图，图b中
Figure PCTCN2014089650-appb-000024
为HSA-ICG-PTX，
Figure PCTCN2014089650-appb-000025
为HSA-ICG；
图12示实施例12中小鼠肿瘤部位温度与激光照射时间的关系图；其中，
Figure PCTCN2014089650-appb-000026
为HSA-ICG-PTX；
Figure PCTCN2014089650-appb-000027
为PBS；
图13示实施例12中小鼠肿瘤体积的变化图；其中，
Figure PCTCN2014089650-appb-000028
为PBS+激光；
Figure PCTCN2014089650-appb-000029
为HSA-PTX；
Figure PCTCN2014089650-appb-000030
为HSA-ICG-PTX；
Figure PCTCN2014089650-appb-000031
为HSA-ICG-PTX+激光；
图14示实施例12中小鼠成活率统计图；其中，
Figure PCTCN2014089650-appb-000032
为PBS+激光；
Figure PCTCN2014089650-appb-000033
为HSA-PTX；
Figure PCTCN2014089650-appb-000034
为HSA-ICG-PTX；
Figure PCTCN2014089650-appb-000035
为HSA-ICG-PTX+激光。
具体实施方式
本发明实施例公开了一种白蛋白吲哚菁绿紫杉醇复合物及其制备方法与应用。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的产品和方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的产品和方法及应用进行改动或适当变更与组合，来实现和应用本发明技术。
为了进一步理解本发明，下面结合实施例对本发明进行详细说明。其中，说明书和权利要求书中所使用的缩写的含义列于下表中：
HSA 人血清白蛋白
ICG 吲哚菁绿
PTX 紫杉醇
HSA-ICG 白蛋白吲哚菁绿复合物
HSA-PTX 白蛋白紫杉醇复合物
HSA-ICG-PTX 白蛋白吲哚菁绿紫杉醇复合物
PBS 磷酸盐缓冲液
实施例1：白蛋白吲哚菁绿紫杉醇复合物的制备：
以白蛋白(HAS)为基底，通过疏水作用吸附吲哚菁绿与紫杉醇，紫杉醇可以作为交联剂使白蛋白自主装成大的纳米粒子，形成白蛋白吲哚菁绿紫杉醇复合物(HSA-ICG-PTX)。具体制备方法如下：
将吲哚菁绿(ICG)溶于二甲亚砜溶液中，配成10mg/mL的吲哚菁绿溶液，同时将紫杉醇(PTX)溶于乙醇中，配成10mg/mL的紫杉醇溶液；称取2mg的人血清蛋白(HSA)，溶于2mL的磷酸盐缓冲溶液中，配成1mg/mL的人血清白蛋白的磷酸盐缓冲溶液；
吲哚菁绿溶液与紫杉醇溶液按不同比例加入到人血清白蛋白的磷酸盐缓冲溶液即可得到人血清白蛋白与吲哚菁绿的摩尔比不同或紫杉醇的装载量不同的白蛋白吲哚菁绿紫杉醇复合物。
其中取6μL的吲哚菁绿溶液和25μL的紫杉醇溶液加入到1ml人血清白蛋白的磷酸盐缓冲溶液中，避光搅拌过夜(12h)；最后将搅拌过夜得到混合物经14800rpm离心5min，除去没有反应上的PTX和ICG，取上清即得人血清白蛋白与吲哚菁绿的摩尔比为1:2、紫杉醇的装载量为5％的白蛋白吲哚菁绿紫杉醇复合物 (HSA-ICG-PTX)。
实施例2、白蛋白吲哚菁绿复合物的制备
将吲哚菁绿(ICG)溶于二甲亚砜溶液中，配成10mg/mL的吲哚菁绿溶液；称取2mg的人血清蛋白(HSA)，溶于2mL的磷酸盐缓冲溶液中，再加入6μL的吲哚菁绿溶液，避光搅拌过夜(12h)；最后将搅拌过夜得到混合物经14800rpm离心5min，除去没有反应上的ICG，取上清即为白蛋白吲哚菁绿复合物(HSA-ICG)。
实施例3、白蛋白紫杉醇复合物的制备
将紫杉醇溶液按不同比例加入到人血清白蛋白的磷酸盐缓冲溶液即可得到不同比例的白蛋白紫杉醇复合物。
其中，将紫杉醇(PTX)溶于乙醇中，配成10mg/mL的紫杉醇溶液；称取2mg的人血清蛋白(HSA)，溶于2mL的磷酸盐缓冲溶液中，再加入25μL的紫杉醇溶液，搅拌过夜(12h)；最后将搅拌过夜得到混合物经14800rpm离心5min，除去没有反应上的PTX，取上清即为0.2mg/ml白蛋白紫杉醇复合物(HSA-PTX)。
实施例4、白蛋白吲哚菁绿紫杉醇复合物的表征
分别采用透射电镜和动态光散射观察实施例1-3制得的各复合物的粒径。结果见图1和图2。
白蛋白吲哚菁绿复合物(HSA-ICG)和白蛋白吲哚菁绿紫杉醇复合物(HSA-ICG-PTX)滴到铜网上后，由磷钨酸染色以提高蛋白的衬度，透射电镜(FEI Tecnai F20)进行表征。
将白蛋白吲哚菁绿复合物(HSA-ICG)和白蛋白吲哚菁绿紫杉醇复合物(HSA-ICG-PTX)(其中HSA的浓度为1mg/ml)用 动态光散射仪测其水合半径。
图1为白蛋白吲哚菁绿复合物(HSA-ICG)和白蛋白吲哚菁绿紫杉醇复合物(HSA-ICG-PTX)的透射电镜照片。由图1可以看到HSA-ICG的粒径大约为7～8nm，而HSA-ICG-PTX的粒径大约为50～60nm。表明紫杉醇的加入可以诱导HSA-ICG进行自组装成大的纳米粒子，使得疏水作用吸附上的吲哚菁绿与紫杉醇更加稳定。
图2是白蛋白吲哚菁绿复合物(HSA-ICG)、白蛋白紫杉醇复合物(HSA-PTX)及白蛋白吲哚菁绿紫杉醇复合物(HSA-ICG-PTX)的激光粒径分布图。如图2所示，HSA-ICG的水化直径约为10nm左右，HSA-PTX与HSA-ICG-PTX的水化直径约为100nm左右。进一步证明了紫杉醇的加入可以引起白蛋白或者HSA-ICG自组装成大的纳米粒子。表明紫杉醇不仅起到化疗药物的作用，还可以像交联剂一样使蛋白自组装成大的纳米粒子。
实施例5、吲哚菁绿、紫杉醇装载量的确定
采用高效液相色谱(HPLC)测量装载在HSA上的PTX的量，以研究紫杉醇的装载量的影响。
按照实施例3所述的方法合成的不同比例的HSA-PTX，分别溶在色谱纯的甲醇中，在室温下孵育12h，14800rpm离心10分钟除去沉淀，过针头后待测。紫杉醇的量由装载着紫外检测器的高效液相色谱检测(检测波长227nm)，以体积比为1:1的甲醇和水的混合物作为流动相，紫杉醇的流出时间为7.8min。再用紫外-可见光吸收光谱仪和荧光光谱仪来研究ICG的装载。结果见图3-5。
图3是HSA-PTX的装载曲线，装载到HAS上PTX的量随着加入的PTX量的增加而增加，由装载曲线选取了PTX 的浓度为0.2mg/ml，即装载量5％用于下面的实验中。
图4是相同HSA浓度(0.2mg/ml)不同比例(人血清白蛋白与吲哚菁绿的摩尔比)的HSA-ICG-PTX的紫外吸收光谱图。从图中可以明显地看出HSA-ICG-PTX在700nm～850nm的近红外区具有非常好的光学吸收，是非常好的光热治疗试剂。随着ICG装载比例的增加，ICG在800nm左右的吸收峰明显增加。
图5是相同HSA浓度(0.2mg/ml)不同比例(人血清白蛋白与吲哚菁绿的摩尔比)的HSA-ICG-PTX的荧光光谱图。从图中可以明显地看出，当ICG吸附到HSA后，在近红外区的荧光有明显的增强，当HSA与ICG的比例为1:2时，荧光是最强的，当继续增加ICG的量，荧光会有一定的淬灭。因此，在后续的实验中选择人血清白蛋白与吲哚菁绿的摩尔比为1:2。
实施例6、HSA-ICG-PTX在激光照射下的升温曲线的测试
将2ml相同HSA浓度(2mg/ml)的实施例1-3制得HSA-PTX、HSA-ICG及HSA-ICG-PTX及水溶液置于比色皿中，将808nm的激光(功率为0.5W/cm2)直接照射在样品上，利用红外热成像仪测试其温度变化的曲线，结果如图6。
由图6结果可见，在5min之内，HSA-ICG和HSA-ICG-PTX溶液的温度升高显著，说明吸附有ICG的纳米粒子具有很高的光热转换率；而对照组HSA-PTX及水在同样的激光照射下基本上没有发生变化。表明HSA-ICG-PTX复合物具有比较强的光学吸收性质，可以作为光热治疗的材料。
实施例7、HSA-ICG-PTX纳米粒子在细胞水平上的毒性研究
取不同紫杉醇浓度的PTX的溶液(PTX溶在体积比为1:1的乙醇与蓖麻油的溶液中)、HSA-PTX和HSA-ICG-PTX，分别与4T1细胞孵育72h，采用标准的MTT试剂进行细胞活性检测，结果如图7所示。
由图7结果可见，当紫杉醇的浓度很低时，PTX的溶液(PTX溶在体积比为1:1的乙醇与蓖麻油的溶液中)、HSA-PTX和HSA-ICG-PTX对细胞均有非常明显的化疗作用，且HSA-ICG-PTX的化疗作用强于PTX的溶液和HSA-PTX。
实施例8、HSA-ICG-PTX在细胞水平的联合治疗
将25μL不同浓度的实施例2制得的HSA-ICG及实施例1制得的HSA-ICG-PTX材料分别加入到含有100μL 4T1细胞液的96孔板中，用808nm、0.4W/cm2的激光器照射30min并监测温度不超过45℃，继续培养1h后，在洗去多余的材料。继续培养24h后，用MTT方法检测细胞活性，以考察HSA-ICG-PTX在细胞水平的联合治疗效果，结果见图8。
从图8可以看出，HSA-ICG-PTX在没有激光照射情况下及HSA-ICG在激光照射下，对细胞活性有一定的影响。而在激光照射情况下，HSA-ICG-PTX的光热性质产生的温和升温可以增加细胞膜的通透性，从而促性HSA-ICG-PTX进细胞的能力，进而利用PTX杀死细胞。表明HSA-ICG-PTX在细胞水平的联合治疗效果显著。
实施例9、HSA-ICG及HSA-ICG-PTX的体内血液循环分析
将200μL实施例2制得的HSA-ICG及实施例1制得的HSA-ICG-PTX(CHSA＝5mg/mL)的水溶液通过尾静脉注射到小鼠体内，在不同时间点取出一定体积的血液，通过测量血 液中ICG荧光强度的变化来分析上述材料在小鼠血液循环系统中的行为。具体测定方法为：取采集的血液称重后用溶解液(1％SDS，1％曲拉通-100，40mMTris缓冲溶液)把取出的血液溶解，低转速离心去除细胞碎片，测定上清液中ICG的荧光强度；其中ICG的激发波长为730nm，发射峰在810nm左右，接收光谱范围为750nm到900nm。统计结果见图9。
图9为HSA-ICG及HSA-ICG-PTX在小鼠体内的血液循环图，如图9所示，随着时间的增加，滞留在小鼠血液中的材料的量逐渐衰减，但HSA-ICG-PTX滞留在小鼠血液中的量明显高于HSA-ICG滞留在小鼠血液中的量。表明由于PTX的交联使得纳米粒子的粒径有明显的增大，HSA-ICG-PTX的血液循环时间比HSA-ICG有了明显的提高，可以为材料肿瘤部位的富集争取时间。
实施例10、HSA-ICG及HSA-ICG-PTX的活体成像
将实施例2制得的HSA-ICG及实施例1制得的HSA-ICG-PTX通过尾静脉注射到小鼠体内，在小动物成像系统(CRI)上在不同的时间点进行实时采集图片，观察材料在肿瘤部位的富集量，结果见图10。其中，选用的激发光源是730nm，曝光时间是100ms。
图10为材料在小鼠肿瘤部位的富集情况图。如图10(a)所示，HSA-ICG-PTX在肿瘤部位的富集量明显高于HSA-ICG的肿瘤富集量，与血液循环的数据相一致。随着时间的推移，HSA-ICG由于粒径较小，很快被代谢出去。图10(b)是肿瘤部位的荧光信号值随时间变化的曲线，其中显示HSA-ICG-PTX在肿瘤部位的富集量明显高于HSA-ICG，并且在8h达到高峰。
实施例11、HSA-ICG及HSA-ICG-PTX在生物体内的分布
分别将实施例2制得的HSA-ICG及实施例1制得的HSA-ICG-PTX通过尾静脉注射到背部带有4T1肿瘤的小鼠体内，24小时后，实验小鼠牺牲，取出肿瘤及重要的器官置于表面皿中，在小动物成像系统上拍摄荧光照片，观察各个器官及肿瘤部位材料的富集量，结果见图11。
图11为材料在小鼠体内的富集情况图。如图11(a)所示，在24h时，HSA-ICG-PTX在肿瘤部位的富集量还是最高，肾部位也有一定的富集，表明HSA-ICG-PTX在体内会有一定的解聚，慢慢地也有部分材料从肾代谢出去；而HSA-ICG在各个部位的富集都很低，说明HSA-ICG由于粒径很小很容易被代谢出去，这一结果与活体成像的结果相符。图11(b)为由小动物成像系统半定量地得到的不同器官及肿瘤部位的荧光信号值，实验数据进一步说明HSA-ICG-PTX的血液循环时间比HSA-ICG有明显地提高。
实施例12、HSA-ICG-PTX在活体水平的联合治疗
选取5只背部带有4T1肿瘤的小鼠作为实验组，从尾静脉注射实施例1制得的HSA-ICG-PTX，经过4小时后，然后将肿瘤部位暴露在808纳米激光下照射10min，激光功率为0.3W/cm2，将肿瘤部位的温度控制在48℃。另外三组背部带有肿瘤的小鼠(每组5只)做为对照组试验，分别做如下处理(1)注射磷酸盐缓冲液(PBS)并在相同功率的激光下照射10min；(2)注射同样剂量的实施例3制得的HSA-PTX；(3)注射同样剂量的实施例1制得的HSA-ICG-PTX，但是没有加激光照射。当处理完每组小鼠之后，小鼠背部的肿瘤体积每隔二天测量一次,体积的计算方法是：长*宽2/2，当肿瘤的体积超过1cm3时，认为小鼠死亡。统计小鼠肿瘤部位 温度与激光照射时间的关系、肿瘤体积的变化以及各组小鼠的存活率，结果见图12-图14。
图12为小鼠肿瘤部位温度与激光照射时间的关系。如图12所示，注射HSA-ICG-PTX的小鼠肿瘤部位的温度很快可以升到48℃并维持10min，促进更多的材料进入细胞，利用PTX的毒性杀死肿瘤；而对照组的小鼠肿瘤的温度几乎没有改变，不会影响小鼠肿瘤的生长。
图13为肿瘤体积的变化图。如图13所示，实验组小鼠的肿瘤在光热与化疗的联合治疗作用下，两天后全部消除，而单独的光热及化疗只能在初期对肿瘤的生长有一定的抑制作用。
图14为各组小鼠的存活率统计图。由图14可以看出在18～22天时对照组小鼠全部死亡，而实验组小鼠在50天后仍然全部活着，并且肿瘤部位没有重新再生。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 一种白蛋白吲哚菁绿紫杉醇复合物，其特征在于，由吲哚菁绿、紫杉醇和人血清白蛋白组成，吲哚菁绿和紫杉醇通过疏水作用力吸附于人血清白蛋白上。
  2. 根据权利要求1所述白蛋白吲哚菁绿紫杉醇复合物，其特征在于，所述白蛋白吲哚菁绿紫杉醇复合物的粒径为50nm～60nm。
  3. 根据权利要求1所述白蛋白吲哚菁绿紫杉醇复合物，其特征在于，所述人血清白蛋白与吲哚菁绿的摩尔比为1∶2。
  4. 根据权利要求1所述白蛋白吲哚菁绿紫杉醇复合物，其特征在于，所述紫杉醇的装载量为5％～8％。
  5. 一种权利要求1所述的白蛋白吲哚菁绿紫杉醇复合物的制备方法，其特征在于，将吲哚菁绿溶解得到吲哚菁绿溶液，将紫杉醇溶解得到紫杉醇溶液，将人血清白蛋白溶于磷酸盐缓冲溶液中得到人血清白蛋白的磷酸盐缓冲溶液；然后将吲哚菁绿溶液与紫杉醇溶液加入到人血清白蛋白的磷酸盐缓冲溶液中，避光搅拌过夜即得。
  6. 根据权利要求5所述的制备方法，其特征在于，所述溶解吲哚菁绿的溶剂为二甲基亚砜。
  7. 根据权利要求5所述的制备方法，其特征在于，所述溶解紫杉醇的溶剂为乙醇。
  8. 根据权利要求5所述的制备方法，其特征在于，还包括对白蛋白吲哚菁绿紫杉醇复合物进行纯化的步骤。
  9. 权利要求4-8所述制备方法制备得到的白蛋白吲哚菁绿紫杉醇复合物。
  10. 权利要求1、2、3、4和9所述白蛋白吲哚菁绿紫杉醇复合物在制备治疗癌症的药物中的应用。
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