WO2020118787A1 - 一种磷基材料制剂及其制备方法和应用 - Google Patents

一种磷基材料制剂及其制备方法和应用 Download PDF

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WO2020118787A1
WO2020118787A1 PCT/CN2018/124634 CN2018124634W WO2020118787A1 WO 2020118787 A1 WO2020118787 A1 WO 2020118787A1 CN 2018124634 W CN2018124634 W CN 2018124634W WO 2020118787 A1 WO2020118787 A1 WO 2020118787A1
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phosphorus
based material
preparation
modified
cells
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French (fr)
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喻学锋
付文
潘婷
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中国科学院深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/42—Phosphorus; Compounds thereof
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/02—Preparation of phosphorus

Definitions

  • the invention relates to the technical field of medicine, in particular to a phosphorus-based material preparation and a preparation method and application thereof.
  • Cancers Malignant tumors (also called cancers) seriously endanger human health, and tumor treatment has become a major challenge facing the current medical research field.
  • the clinical treatment methods for tumors mainly include surgical treatment, radiotherapy and chemotherapy. Although these treatment methods have achieved certain effects in the treatment of tumors, they still have limitations, and a single treatment method cannot achieve good tumor treatment effects.
  • Traditional treatment methods are difficult to achieve complete removal of tumor cells and specific killing, leading to tumor cell metastasis, recurrence and side effects due to damage to normal cells.
  • a composite material of black phosphorus nanosheets-antitumor compounds and its preparation method and application discloses a black phosphorus nanosheets and antitumor composite materials with primary amino groups and/or phenolic hydroxyl groups And its preparation method and application, which constructs a two-dimensional black phosphorus loaded antitumor drug to achieve a synergistic anticancer effect.
  • the black phosphorus nanosheet in the composite material functions as an anti-cancer drug carrier. Black phosphorus itself is not used as an anti-cancer drug, and the drug has no targeting and specificity.
  • the present invention provides a phosphorus-based material preparation and a preparation method and application thereof.
  • the phosphorus-based material preparation has high stability in a neutral physiological environment, which can be applied to the preparation of drugs for treating tumors and can be effectively suppressed
  • the expansion and metastasis of tumor cells can prevent cancer cell metastasis and tumor recurrence more effectively, so as to improve the effect of treating tumors.
  • the preparation of phosphorus-based materials has little effect on normal tissues and cells, and is safe and reliable.
  • the present invention provides a phosphorus-based material preparation, which contains a phosphorus-based material and a calcium hydroxyphosphate modified on the surface of the phosphorus-based material, the phosphorus-based material is in In acidic environment can be converted to produce phosphate ion materials.
  • the phosphorus-based material is elemental phosphorus and/or phosphorus-containing compounds that can be converted into phosphate ions under an acidic environment.
  • Phosphorus-containing compounds include phosphorus-containing oxides, halides, and other phosphorus-containing compounds (such as phosphates) that can be converted into phosphate ions in an acidic environment.
  • Phosphorus-based materials specifically include but are not limited to black phosphorus, red phosphorus, white phosphorus, purple phosphorus and other phosphorus elements, phosphorus trioxide, phosphorus pentoxide and other phosphorus oxides, phosphorus pentahalide, phosphorus trihalide, phosphorus tetrahalide and other phosphorus halides, And one or more of phosphate compounds based on orthophosphoric acid, metaphosphoric acid, phosphorous acid, pyrophosphoric acid, triphosphoric acid, hypophosphorous acid, hypophosphoric acid, polyphosphoric acid and the like.
  • the surface of the phosphorus-based material can be oxidized, hydrolyzed or ionized to generate phosphate ions in situ.
  • concentration of phosphate ions in the local volume is much greater than the overall concentration of the solution, which is beneficial to the separation of phosphate ions from calcium sources and hydroxide
  • the sub-donor performs in-situ deposition reaction on the surface of the phosphorus-based material, thereby modifying calcium hydroxyphosphate on the surface of the phosphorus-based material.
  • the phosphorus-based material is a phosphorus-based micro-nano material, including nano- and micron-level materials.
  • nano-level materials are beneficial to intravenous administration and reduce the cumulative toxicity of organs; micron-level materials can be used for in situ administration, which has a stronger biological effect.
  • nanomaterials can achieve tumor cell targeting through surface modification of folic acid, antibodies and other targeted drugs while realizing drug delivery, modify fluorescent molecules or realize tumor cell imaging through its own optical effect, and realize the integration of tumor diagnosis and treatment treatment.
  • nanomaterials' huge specific surface area and long physiological cycle period and other characteristics ensure the drug delivery and drug delivery efficiency, which helps to achieve efficient and sustained killing of cancer cells.
  • the phosphorus-based material includes at least one of black phosphorus nanoplates, black phosphorus quantum dots, and surface modification materials thereof.
  • the surface of the surface modification material may be oxidized, hydrolyzed, or ionized to generate phosphate ions.
  • the black phosphorus nanoplatelets and/or black phosphorus quantum dots can also be surface modified, such as modifying rare earth metal ions, titanium ligands or other substances, as long as the surface modified black phosphorus nanoplatelets and/or black phosphorus quantum dot surfaces It can be oxidized, hydrolyzed or ionized to produce phosphate ions.
  • Such surface-modified black phosphorus nanosheets and/or black phosphorus quantum dots can also be used as phosphorus-based materials.
  • the degradation products of black phosphorus are phosphates, etc., which are necessary for human life activities and have good element biocompatibility. Its huge specific surface area is conducive to surface modification and drug delivery.
  • black phosphorus is rapidly degraded under the slightly acidic and high-pressure microenvironment inside and outside the cancer cells to produce a large amount of phosphate ions and active intermediate products (such as reactive oxygen species ROS, etc.), which itself has an anti-cancer effect.
  • the phosphorus-based material preparation is surface-modified for enhanced targeting.
  • the surface modification for enhancing targeting includes but is not limited to modification with compounds such as folic acid, modification of peptide substances such as transmembrane peptides, and modification of aptamers and antibodies targeting cancer cells.
  • Targeted modification of the surface of the phosphorus-based material preparation can more effectively inhibit the spread and metastasis of cancer cells or tumor cells, thereby more effectively prevent the recurrence of cancer cells or tumor cells, and further improve the therapeutic effect.
  • the black phosphorus-based material modified with calcium hydroxyphosphate surface has the function of loading functional molecules (such as targeted tumor marker molecules, fluorescent tracer molecules, combined drug molecules, genes, etc.), where the presence of hydroxyl groups is beneficial to the functional groups in the functional molecules
  • Stable grafting can be achieved by building a functional platform on the surface of the phosphor-based material to achieve functional modification of tumor targeting grafting, fluorescent molecule delivery, drug combination, gene delivery and so on.
  • the phosphorus-based material preparation is also loaded with functional molecules, including fluorescent tracer molecules (such as Ce6, Cy5.5, etc.), targeted tumor marker molecules, drug molecules, and biological activities Macromolecules (such as circular DNA, RNA, etc.), inorganic functional molecules such as ferric oxide and their arrangement and combination.
  • the functional molecule loaded with the phosphorus-based material preparation may specifically be a functional molecule that is adsorbed and/or embedded on the surface of the phosphorus-based material modified with calcium hydroxyphosphate.
  • the loaded functional molecules are released during the degradation process of calcium hydroxyphosphate to achieve the functionalization of the material preparation. This process is called "responsive co-release" Co-release)".
  • the above phosphorus-based material preparations can be used to prepare drugs for the treatment of tumors; the targeted tumor markers are loaded in the phosphorus-based material preparations, and the phosphorus-based material preparations can have both active and passive targeting during the tumor treatment process. Targeting and selective killing effects; loading fluorescent tracer molecules in phosphorus-based material preparations can achieve the purpose of integrating high-efficiency and low-toxicity tumor killing and fluorescent tracer diagnosis. This has multiple biological functions and is specific to tumor tissues.
  • the therapeutic method of sexual killing drugs is called "surface functionalized biologically active phosphorus-based diagnostic therapy (Functionalized Bioactive Phosphorus-based Diagnose & Therapy), referred to as "Functional Phosphorus-based Diagnose & Therapy" (FBPDT).
  • the present invention also provides a method for preparing more than one phosphorus-based material preparation, including the following steps:
  • the phosphorus-based raw material in an air-isolated environment, is ground and dispersed in a solvent to prepare a phosphorus-based raw material solution; and then the phosphorus-based raw material solution is subjected to ultrasonic peeling to obtain a phosphorus-based raw material solution;
  • the calcium source is a calcium ion-containing compound; the calcium source is preferably at least one of calcium gluconate, calcium chloride, and calcium citrate. Further preferably, the calcium source is added according to the molar ratio of P:Ca in the solution (the molar ratio of phosphorus and calcium) in the solution after the calcium source is added: 1:25 ⁇ 35.
  • the hydroxide ion donor is an alkaline compound that can be hydrolyzed or ionized to generate hydroxide ions; the hydroxide ion donor is preferably at least one of concentrated ammonia, sodium acetate, and sodium hydroxide . Further preferably, the amount of hydroxide ion donor added accounts for 5% to 10% of the total volume of the solution after the calcium source is added.
  • step S1 various solvents can be used, such as N-methylpyrrolidone (NMP), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), absolute ethanol, and isopropanol.
  • NMP N-methylpyrrolidone
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • absolute ethanol absolute ethanol
  • isopropanol various solvents can be used, such as N-methylpyrrolidone (NMP), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), absolute ethanol, and isopropanol.
  • NMP N-methylpyrrolidone
  • DMF dimethylformamide
  • DMSO dimethyl sulfoxide
  • absolute ethanol absolute ethanol
  • isopropanol isopropanol.
  • Ultrasonic peeling can use probe ultrasound, water bath ultrasound, or both in sequence. Specifically, the effect of peeling can be adjusted by changing the ultrasound mode and ultrasound frequency. It can also
  • step S2 after the hydroxide ion donor is added, the pH value is preferably adjusted to 10 to 11, and placed in a constant temperature water bath at 45 to 60°C to heat the reaction for 10 to 15 hours.
  • the present invention also provides the application of the above phosphorus-based material preparation in the preparation of a medicine for treating tumors, which inhibits the proliferation of tumor cells and/or induces apoptosis of tumor cells by changing the internal and external environment of the tumor cells.
  • the phosphorus-based material preparation can be engulfed by tumor cells, the surface of the phosphate-based material is degraded by calcium hydroxyphosphate, and the phosphorus-based material is converted into phosphate ions to change the internal and external environment of the tumor cells, thereby inhibiting tumor cell proliferation and And/or induce tumor cell apoptosis.
  • the phosphorus-based material preparation can be used as a single preparation; or the phosphorus-based material preparation can be used as an active ingredient to add a pharmaceutically acceptable adjuvant together to prepare a drug for treating tumors; in addition, other antitumor active ingredients can also be added To achieve synergy.
  • it can be made into clinically acceptable dosage forms according to conventional processes, including tablets, capsules, pills, granules, sustained-release preparations, controlled-release preparations, or injection preparations.
  • the proportion of the phosphorus-based material in the combined drug depends on the specific situation.
  • the specific addition amount of the phosphorus-based material can be 0.01wt% ⁇ 99.99 wt%, preferably 20 wt% to 99.99 wt%, further preferably 30 wt% to 80 wt%.
  • the above phosphorus-based material preparation is applied to the preparation of a medicine for treating tumors, and the administration method of the prepared medicine may be intravenous administration, or a method of placing it directly in and around the tumor.
  • the administration method of the prepared medicine may be intravenous administration, or a method of placing it directly in and around the tumor.
  • it can be used to prepare and treat brain, blood, breast, pancreas, uterus, endometrium, cervix, kidney, liver, gallbladder, head and neck, oral cavity, thyroid, skin, mucosa, glands, blood vessels originating in humans and animals.
  • the specific dosage in the course of treatment can be determined according to the type of phosphorus-based material contained in the drug, the type of tumor targeted, and the way of medication.
  • the present invention provides a phosphorus-based material preparation and its preparation method and application.
  • the surface-modified calcium hydroxyphosphate of the phosphorus-based material in the phosphorus-based material preparation can improve the neutrality of the phosphorus-based material Stability under physiological environment; in addition, after modification with calcium hydroxyphosphate, the surface roughness of the phosphorus-based material is significantly increased, which is beneficial to the endocytosis of the cell and improves the efficiency of endocytosis; and the modified layer on the surface of the phosphorus-based material has a tumor microenvironment response Degradation characteristics, when the material preparation acts on tumor cells, calcium hydroxyphosphate degrades in the acidic microenvironment inside and outside the tumor cells, exposing the activation site of the reaction between calcium hydroxyphosphate and phosphorus-based materials, and accelerates the degradation rate of phosphorus-based materials, A large amount of phosphate ions and other active products are generated instantaneously, destroying the ion balance of the tumor
  • phosphorus-based materials have a long retention effect due to the high penetration of tumor tissue (enhanced permeability and retention effect, EPR) and/or can accumulate in the tumor tissue microenvironment due to its surface targeting effect, etc., and/or after being taken up by tumor cells through endocytosis, due to the acidity of the tumor cells and extracellular
  • EPR enhanced permeability and retention effect
  • the microenvironment accelerates its conversion, and instantaneously generates a large amount of phosphate ions and other active products (that is, unstable intermediate products such as active free radicals, active oxygen, etc.) during its rapid conversion process, which can be called the "ion bomb effect" (Ionic Bomb Effect)", which can further induce changes in the microenvironment inside and outside the tumor cells, and promote non-specific phosphorylation of the protein, thereby disrupting the mitosis of the tumor cells, inhibiting the proliferation of the tumor cells, and ultimately inducing the death of the tumor cells.
  • Ionic Bomb Effect Ionic Bomb Effect
  • the phosphorous of the present invention In the material preparation, there are hydroxyl groups in the modified groups on the surface of the phosphorus-based material, and the electronegativity of the material surface is enhanced. Compared with the unmodified phosphorus-based material, it has more excellent functional molecular groups (such as tumor targeting molecules, Fluorescent tracer molecules, etc.) binding ability and adsorption capacity of positively charged molecules.
  • the surface active sites of phosphorus-based materials after degradation of calcium hydroxyphosphate can enhance the "ion bomb effect" of phosphorus-based materials, compared with unmodified phosphorus-based
  • the material can produce more phosphate ions and other active products per unit time, and has a better anti-tumor effect.
  • the solution of the present invention is simple and efficient.
  • the phosphorus-based material preparation of the present invention can be applied to the preparation of drugs for treating tumors, which are specific and targeted during the treatment of tumors.
  • Example 1 is a characterization diagram of the change in Zeta potential of black phosphorus nanosheets before and after modification with calcium hydroxyphosphate in Example 1;
  • FIG. 2 is the ultraviolet absorbance of black phosphate nanosheets modified with calcium hydroxyphosphate prepared in Example 1 in ultrapure water at pH 7.0 and pH 4.0 and naked black phosphorus nanosheets at 808 nm in ultrapure water at pH 7.0 Change curve graph;
  • FIG. 3 is a graph showing that the surface-modified black phosphorus-based nanosheet prepared in Example 1 inhibits breast cancer cell proliferation
  • Example 4 is a graph showing that the surface modified black phosphorus-based nanosheet prepared in Example 1 inhibits cervical cancer cell proliferation
  • Example 5 is a graph showing that the surface-modified black phosphorus-based nanosheet prepared in Example 1 inhibits proliferation of non-small lung cancer cells;
  • Example 6 is a graph showing that the surface modified black phosphorus-based nanosheet prepared in Example 1 inhibits normal cell proliferation
  • Example 7 is a diagram of breast cancer cell apoptosis induced by the surface modified black phosphorus-based nanosheet prepared in Example 1;
  • Example 8 is a diagram of cervical cancer cell apoptosis induced by the surface modified black phosphate-based nanosheet prepared in Example 1;
  • Example 9 is a graph of non-small lung cancer cell apoptosis induced by the surface modified black phosphorus-based nanosheet prepared in Example 1;
  • Example 10 is a graph of normal cell apoptosis induced by the surface modified black phosphorus-based nanosheet prepared in Example 1;
  • FIG. 11 is a comparison chart of the change in the fluorescence carrying efficiency of the black phosphorus-based nanosheets before and after surface modification of calcium hydroxyphosphate in Example 1;
  • FIG. 12 is a qualitative observation graph of gene delivery of the surface-modified black phosphorus-based nanosheets prepared in Example 1 with calcium hydroxyphosphate.
  • Example 1 Preparation of black phosphate nanosheets modified with calcium hydroxyphosphate
  • the liquid-phase stripping method is used to prepare biologically active black phosphorus nanosheets.
  • the specific steps include: in an air-isolated environment, a certain amount of black phosphorus crystals are ground and dispersed in the solvent N-methylpyrrolidone (NMP) and sealed; and then the probe ultrasound and water bath ultrasound are used in order
  • NMP solvent N-methylpyrrolidone
  • the black phosphorus solution is subjected to ultrasonic peeling to prepare a biologically active black phosphorus flake stock solution.
  • Ultrasonic peeling can also use the method of single probe ultrasound or water bath ultrasound, and the effect of peeling can be adjusted by changing the mode of ultrasound, ultrasound frequency, etc., and can also be combined with other peeling techniques such as thermal separation technology, ion intercalation technology, etc. Stripping efficiency and yield.
  • the black phosphorus flakes in the prepared black phosphorus flakes stock solution have biological activities that can be transformed into a large amount of phosphate ions after being engulfed by tumor cells, change the internal and external environment of the cells, and thereby inhibit tumor cell proliferation and induce tumor cell death.
  • the black phosphorus flakes are prepared, and the prepared black phosphorus flakes can also be surface coordinated or targeted modified to enhance the stability and targeting of the black phosphorus flakes, and the resulting two-dimensional black phosphorus ( Including exposed two-dimensional black phosphorus and modified two-dimensional black phosphorus) dispersed in a suitable solvent to facilitate long-term storage of the material.
  • Example 3 In vitro stability evaluation of black phosphorus nanosheets with surface-modified calcium hydroxyphosphate
  • the pH Under the neutral condition of 7.0 the stability of black phosphate nanosheets modified with calcium hydroxyphosphate is significantly higher than that of naked black phosphorus nanosheets, indicating that the surface modification of calcium hydroxyphosphate can improve the stability of black phosphorus nanosheets.
  • Example 4 Evaluation of the killing effect of black phosphorus nanosheets with surface-modified calcium hydroxyphosphate on tumor cells
  • the black phosphorus nanosheets with surface-modified calcium hydroxyphosphate prepared in Example 1 were applied to tumor cells and normal cells to perform cell proliferation detection experiments and apoptosis detection experiments. details as follows:
  • human cancer cells include cervical cancer cells (Hela), breast cancer cells (MCF-7) and non-small lung cancer cells (A549), and normal cells use human bone marrow mesenchyme Stem cells (Hmsc).
  • the cells can be planted in a 96-well plate at a density of 5000 cells/well, and each group is made into four multi-wells.
  • the medium for each well is 100 ⁇ L of DMEM containing 10% FBS.
  • the cells are placed in a 37°C incubator. 5 %CO 2 , cultured for 24 hours under saturated humidity.
  • the medium was replaced with a medium containing black phosphate nanosheets of surface-modified calcium hydroxyphosphate of appropriate size, and then cultured in a 37°C incubator, 5% CO 2 , and saturated humidity for 48 hours.
  • the size of the black phosphorus nanosheets with surface-modified calcium hydroxyphosphate can be 2-20 nm thick; 20-300 nm long and wide to facilitate endocytosis.
  • the final concentrations of black phosphorus nanosheets with surface-modified calcium hydroxyphosphate in culture wells were 0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4 and 8 ⁇ g/mL, respectively.
  • human cancer cells include cervical cancer cells (Hela), breast cancer cells (MCF-7) and non-small lung cancer cells (A549), and normal cells use human bone marrow mesenchyme Stem cells (Hmsc).
  • Hela cervical cancer cells
  • MCF-7 breast cancer cells
  • A549 non-small lung cancer cells
  • normal cells use human bone marrow mesenchyme Stem cells (Hmsc).
  • the cells were planted in a 24-well plate at a density of 5 ⁇ 104 cells/well. Three replicate wells were made in each group. The medium in each well was 1 mL of DMEM containing 10% FBS. The cells were placed in a 37°C incubator. Incubate at 5% CO 2 for 24 hours under saturated humidity.
  • the medium was replaced with a medium containing black phosphate nanosheets of surface-modified calcium hydroxyphosphate of appropriate size, and then cultured in a 37°C incubator, 5% CO 2 , and saturated humidity for 48 hours.
  • the size of the black phosphorus nanosheets with surface-modified calcium hydroxyphosphate can be 2-10 nm in thickness; 20-300 nm in length and width to facilitate endocytosis.
  • the final concentrations of black phosphorus nanosheets with surface-modified calcium hydroxyphosphate in the culture wells were 0, 0.25, 0.5, 1.0, and 2.0 ⁇ g/mL, respectively.
  • the cells were treated with trypsin and collected, centrifuged at 1000g for 5 min, the supernatant was discarded, the cells were collected, and the cells were gently resuspended in PBS and counted.
  • the cells were treated with trypsin and collected, centrifuged at 1000g for 5 min, the supernatant was discarded, the cells were collected, and the cells were gently resuspended in PBS and counted.
  • Take 50,000-100,000 resuspended cells centrifuge at 1000g for 5min, discard the supernatant, add 195 ⁇ L Annexin V-FITC binding solution to gently resuspend the cells, then add 5 ⁇ L Annexin V-FITC and 10 ⁇ L propidium iodide (PI) and mix gently Evenly stain for 15 min at room temperature in the dark, and use flow cytometry to test on the machine.
  • PI propidium iodide
  • FIG. 3 is a curve diagram of black phosphorus nanosheets with surface-modified calcium hydroxyphosphate inhibiting breast cancer cell proliferation in Example 1 of the present invention
  • FIG. 4 is a black phosphorus nanosheets with surface-modified calcium hydroxyphosphate inhibiting cervical Cancer cell proliferation curve diagram
  • FIG. 5 is a graph of surface modified calcium hydroxyphosphate black phosphorus nanosheets inhibiting proliferation of non-small lung cancer cells
  • FIG. 6 is a graph of surface modified calcium hydroxyphosphate black phosphorus nanosheets inhibiting human normal cell proliferation.
  • the abscissa is the concentration of black phosphorus nanosheets with surface-modified calcium hydroxyphosphate
  • the ordinate is the cell survival rate.
  • FIG. 7 is a diagram of black phosphorus nanosheets with surface modified calcium hydroxyphosphate inducing apoptosis of breast cancer cells in Example 1 of the present invention
  • FIG. 8 is a black phosphorus nanosheets with surface modified calcium hydroxyphosphate inducing cervical
  • Fig. 9 is a diagram of cancer cell apoptosis.
  • Fig. 9 is a graph of surface modified calcium hydroxyphosphate-based black phosphorus nanosheets inducing apoptosis of non-small lung cancer cells
  • Fig. 10 is a surface graph of surface-modified calcium hydroxyphosphate-based black phosphorus nanosheets.
  • the abscissa is Annexin V fluorescence intensity; ordinate is PI fluorescence intensity; Q4 area is normal active cells; Q3 is early apoptotic cells; Q2 is late apoptotic cells; Q1 is necrotic cells, which can be ignored.
  • FIGS. 7 to 10 further illustrate that at a lower dose of black phosphate nanosheets modified with calcium hydroxyphosphate, significant inhibition of cancer cell proliferation can be achieved, but the proliferation inhibitory effect on normal cells is significantly smaller.
  • Example 5 Evaluation of small molecule drug delivery capacity of black phosphorus nanosheets with surface-modified calcium hydroxyphosphate
  • the fluorescent small molecule Ce6 was added during the deposition of calcium hydroxyphosphate on the surface of the black phosphorus nanosheets. After the reaction was completed, the supernatant was centrifuged to absorb the supernatant. Supernatant; In addition, according to a similar method, the fluorescent small molecule Ce6 was added during the deposition of calcium hydroxyphosphate on the surface of the black phosphorus nanosheets, and the supernatant was immediately centrifuged to obtain the solution before being carried. The fluorescence intensity of the supernatant after transportation and the fluorescence intensity of the solution before transportation were measured separately. The results are shown in Figure 11.
  • Example 5 Evaluation of bioactive macromolecule drug delivery capacity of black phosphorus nanosheets with surface-modified calcium hydroxyphosphate
  • a small molecule circular DNA labeled with a fluorescent molecule Cy-5.5 was used for gene delivery experiments. Specifically, according to the preparation method of the black phosphorus nanosheet modified with calcium hydroxyphosphate in Example 1, the calcium hydroxyphosphate was added during the deposition of the surface of the black phosphorus nanosheet Cy-5.5 labeled small circular DNA, co-localized tracing of fluorescent molecules and DNA, 24 hours later using a fluorescence microscope to observe, the results are shown in Figure 12. Results in Figure 12 show 24 After h, the fluorescence area is consistent with the cell contour, indicating that the calcium hydroxyphosphate-modified black phosphorus nanosheet can carry genes and achieve its release in the cell.
  • the two-dimensional black phosphorus nanosheets modified with calcium hydroxyphosphate can effectively inhibit the proliferation of cancer cells and induce their apoptosis, while having low cytotoxicity to normal cells.
  • the phosphorus-based material preparation is very suitable for the development of new multifunctional anticancer drugs.
  • the black phosphorus nanosheets modified with calcium hydroxyphosphate surface have the potential for biological activity and multifunctional modification.
  • the mechanism of action mainly includes the physical embedding of functional molecules and the release of molecules under environmental response, which produces corresponding functions and functions inside and outside tumor cells.
  • the binding site of black phosphorus modified with calcium hydroxyphosphate is exposed, and the surface of black phosphorus is in an activated state, which is oxidized to produce a large amount of phosphate ions and other active intermediate products (such as ROS, etc.). It has huge application potential in cancer treatment, tumor tracing, gene combination therapy and drug delivery.
  • the surface of the black phosphorus quantum dots can be oxidized to generate phosphate ions like the nanosheets, and combined with calcium ions and hydroxide ions to form calcium hydroxyphosphate deposited on the surface of the material, which is based on the similarity to the black phosphorus nanosheets modified with calcium hydroxyphosphate
  • the mechanism can be applied to the preparation of anti-tumor drugs. Specifically, black phosphorus can be carefully ground into powder in a small amount of N-methylpyrrolidone (NMP), and then appropriate amount of N-methylpyrrolidone can be added and the probe of the cell disrupter can be used to sonicate for 3 hours. The resulting dispersion is then bathed in an ultrasonic cleaner Ultrasound 10 h.
  • the black phosphorus quantum dots are prepared; and then the preparation method of the black phosphorus nanosheets modified with calcium hydroxyphosphate is modified on the surface of the black phosphorus quantum dots to prepare black phosphorus quantum dots with surface-modified calcium hydroxyphosphate.
  • calcium hydroxyphosphate can also be modified on the surface of the black phosphor nanosheets or black phosphor quantum dots in other ways, which can enhance the stability of the black phosphor nanosheets and black phosphor quantum dots in a neutral environment and facilitate endocytosis. Improve internal rate.
  • black phosphorus nanosheets have biological activity, and can be engulfed by tumor cells after contact with tumor cells, and converted into phosphate ions in tumor cells, due to the acidity in tumor cells and extracellular
  • the microenvironment can accelerate its transformation, and during the rapid transformation process, it can generate a large amount of phosphate ions and other active products (that is, unstable intermediate products), which can further induce changes in the microenvironment of the tumor cells and destroy the microenvironment of the tumor cells. Ion balance, and promote non-specific phosphorylation of tumor cell proteins, thereby disrupting tumor cell mitosis, inhibiting tumor cell proliferation, and ultimately inducing tumor cell death.

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Abstract

一种磷基材料制剂及其制备方法和应用，其中磷基材料制剂包含磷基材料和修饰于磷基材料表面的羟基磷酸钙，磷基材料为在酸性环境下可转化产生磷酸根离子的材料。该磷基材料制剂在中性生理环境下稳定性高，其可应用于制备治疗肿瘤的药物，可有效抑制肿瘤细胞的扩增和转移，从而更有效防止癌症细胞的转移和肿瘤的复发，以提高治疗肿瘤效果，且在整个治疗过程中，磷基材料制剂对正常组织和细胞影响较小，安全可靠。

Description

一种磷基材料制剂及其制备方法和应用
技术领域
本发明涉及医药技术领域，具体涉及一种磷基材料制剂及其制备方法和应用。
背景技术
恶性肿瘤（也被称为癌症）严重危害人类健康，肿瘤治疗已经成为当前医学研究领域所面临的一个重大挑战。目前临床上肿瘤的治疗方法主要包括手术治疗、放射治疗及化学治疗等方法。这些治疗方法在肿瘤治疗过程中虽然取得了一定效果，但它们依然存在局限性，并且单一的治疗方法无法实现良好的肿瘤治疗效果。传统的治疗方式难以实现肿瘤细胞的完全清除以及特异性杀伤，导致肿瘤细胞转移、复发以及由于对正常细胞的损害产生的副作用。
专利“一种黑磷纳米片-抗肿瘤化合物的复合材料及其制备方法和应用”（申请号CN106267204A），披露了一种黑磷纳米片和带伯氨基和/或酚羟基的抗肿瘤复合材料及其制备方法和应用，其构建二维黑磷负载抗肿瘤药物，实现协同抗癌效果。该复合材料中黑磷纳米片起到的是抗癌药物载体的作用，黑磷本身不作为一种抗癌药物，且药物无靶向性和特异性。
发明内容
为了解决上述技术问题，本发明提供一种磷基材料制剂及其制备方法和应用，该磷基材料制剂在中性生理环境下稳定性高，其可应用于制备治疗肿瘤的药物，可有效抑制肿瘤细胞的扩增和转移，从而更有效防止癌症细胞的转移和肿瘤的复发，以提高治疗肿瘤效果，且在整个治疗过程中，磷基材料制剂对正常组织和细胞影响较小，安全可靠。
本发明所采用的技术方案是：本发明提供了一种磷基材料制剂，该磷基材料制剂包含磷基材料和修饰于所述磷基材料表面的羟基磷酸钙，所述磷基材料为在酸性环境下可转化产生磷酸根离子的材料。
优选地，所述磷基材料为在酸性环境下可转化产生磷酸根离子的单质磷和/或含磷化合物。含磷化合物包括含磷氧化物、卤化物及其他在酸性环境下可转化产生磷酸根离子的含磷化合物（如磷酸盐等）。磷基材料具体包括但不限于黑磷、红磷、白磷、紫磷等磷单质，三氧化磷、五氧化磷等磷氧化物，五卤化磷、三卤化磷、四卤化磷等磷卤化物，以及基于正磷酸、偏磷酸、亚磷酸、焦磷酸、三磷酸、次磷酸、连二磷酸、聚磷酸等磷酸盐化合物中的一种或多种。
优选地，所述磷基材料的表面可被氧化、水解或电离原位产生磷酸根离子，局部体积内磷酸根离子浓度远大于溶液整体浓度，有利于磷酸根离子与钙源、氢氧根离子供体在磷基材料表面原位沉积反应的进行，从而在磷基材料的表面修饰羟基磷酸钙。
优选地，所述磷基材料为磷基微纳材料，包括纳米和微米级别的材料。其中，纳米级别材料利于静脉给药，减少器官累计毒性；微米级别材料可用于原位给药，具有更强的生物学效应。另外，纳米材料能够在实现药物运载的同时，通过表面修饰叶酸、抗体等靶向药物获得肿瘤细胞靶向性，修饰荧光分子或通过其本身的光学效应实现肿瘤细胞成像，实现肿瘤的诊疗一体化治疗。另外，纳米材料巨大的比表面积以及较长的生理循环周期等特征，保证了药物运载及给药效率，有助于实现癌细胞的高效持续杀伤。
优选地，所述磷基材料包括黑磷纳米片、黑磷量子点及其表面修饰材料中的至少一种，所述表面修饰材料的表面可被氧化、水解或电离产生磷酸根离子。也就是，黑磷纳米片和/或黑磷量子点也可经过表面修饰，如修饰稀土金属离子、钛配体或其他物质，只要经表面修饰的黑磷纳米片和/或黑磷量子点表面可被氧化、水解或电离产生磷酸根离子，这类经表面修饰的黑磷纳米片和/或黑磷量子点也可作为磷基材料。以上这些磷基材料中，黑磷的降解产物为磷酸根等，为人体生命活动所必需，具有良好的元素生物相容性。其巨大的比表面积有利于表面修饰及药物运载。另外，黑磷在癌细胞内外微酸性及高压的微环境下迅速降解产生大量磷酸根离子和活性中间产物（如：活性氧ROS等），本身具有抗癌作用效果。
优选地，所述磷基材料制剂经过表面修饰以用于增强靶向性。所述用于增强靶向性的表面修饰包括但不限于利用叶酸等化合物修饰、穿膜肽等肽类物质修饰、以及靶向癌细胞的适配体、抗体等的修饰。通过对磷基材料制剂的表面进行靶向性修饰，可更有效地抑制癌细胞或肿瘤细胞的扩散和转移，从而更有效地防止癌细胞或肿瘤细胞的复发，以更进一步提高治疗效果。
经羟基磷酸钙表面修饰的黑磷基材料具有负载功能分子（如靶向肿瘤标记物分子、荧光示踪分子、联用药物分子、基因等）的功能，其中羟基的存在有利于功能分子中官能团的稳定接枝，故可通过在磷基材料表面构建功能化平台，可实现肿瘤靶向接枝、荧光分子运载、药物联用、基因运载等功能化改性。因而，进一步优选地，所述磷基材料制剂内还负载有功能分子，所述功能分子包括荧光示踪分子（如Ce6、Cy5.5等）、靶向肿瘤标记物分子、药物分子和生物活性大分子（如环状DNA、RNA等）、四氧化三铁等无机功能分子及排列组合。磷基材料制剂负载功能分子具体可为表面修饰羟基磷酸钙的磷基材料上以吸附和/或包埋的方式负载功能分子。负载的功能分子在羟基磷酸钙降解过程中得以释放，以实现材料制剂的功能化，该过程称为“响应性共释放（Responsive Co-release）”。以上磷基材料制剂可应用于制备治疗肿瘤的药物；在磷基材料制剂内负载靶向肿瘤标记物，在肿瘤治疗过程中磷基材料制剂可同时具备主动靶向、被动靶向以及选择性杀伤等效果；在磷基材料制剂内负载荧光示踪分子，可达到高效低毒杀伤肿瘤与荧光示踪诊断一体化的目的。这一具有多重生物学功能的针对肿瘤组织特异性杀伤药物治疗方法称为“表面功能化生物活性磷基诊疗法（Functionalized Bioactive Phosphorus-based Diagnose & Therapy）”，简称为“功能化磷基诊疗(FBPDT)”。
本发明还提供了一种以上磷基材料制剂的制备方法，包括以下步骤：
S1、在隔绝空气的环境，将磷基原材料研磨后分散于溶剂中，制得磷基原材料溶液；而后对所述磷基原材料溶液进行超声剥离，得磷基材料原液；
S2、对所述磷基材料原液进行离心分离，取上清液；向所述上清液中加入钙源，混匀后加入氢氧根离子供体进行反应，再进行纯化。
优选地，所述钙源为含钙离子化合物；所述钙源优选为葡萄糖酸钙、氯化钙、柠檬酸钙中的至少一种。进一步优选地，按照加入钙源后溶液中P:Ca摩尔浓度比（磷和钙摩尔浓度比）为1:25~35添加钙源。
优选地，所述氢氧根离子供体为可水解或电离产生氢氧根离子的碱性化合物；所述氢氧根离子供体优选为浓氨水、醋酸钠、氢氧化钠中的至少一种。进一步优选地，氢氧根离子供体的添加量占加入钙源后溶液总体积的5%~10%。
另外，步骤S1中，溶剂可采用各种溶剂，如N-甲基吡咯烷酮（NMP）、二甲基甲酰胺（DMF）、二甲基亚砜（DMSO）、无水乙醇、异丙醇等。超声剥离可采用探头超声、水浴超声或者两者依次作用的方式，具体可通过改变超声的作用方式、超声频率等调节剥离的效果，也可结合其他的剥离技术如热分离技术、离子插层技术等，提高剥离的效率和产率。此外，还可对超声剥离所得的磷基材料原液进行表面配位或靶向修饰，以增强磷基材料的稳定性和靶向性。
步骤S2中，加入氢氧根离子供体后，优选调节pH值为10~11，并置于45~60℃恒温水浴加热反应10~15h。
另外，本发明还提供了以上磷基材料制剂在制备治疗肿瘤的药物中的应用，所述药物通过改变所述肿瘤细胞的内外环境，来抑制肿瘤细胞增殖和/或诱导肿瘤细胞凋亡。具体可通过其中的磷基材料制剂被肿瘤细胞吞噬，磷基材料表面修饰的羟基磷酸钙降解，磷基材料转化产生磷酸根离子，以改变所述肿瘤细胞的内外环境，进而抑制肿瘤细胞增殖和/或诱导肿瘤细胞凋亡。
优选地，所述磷基材料制剂可作为单一制剂；或者以所述磷基材料制剂作为活性成分，加入药学可接受的助剂一同制备治疗肿瘤的药物；此外，还可加入其他抗肿瘤活性成分，以达到协同作用。具体可按照常规工艺，制成临床接受的剂型，包括片剂、胶囊剂、丸剂、颗粒剂、缓释制剂、控释制剂或注射制剂等，应用于临床。其中，磷基材料在组合药物中所占的比例因具体情况而定，基于药物组合物，磷基材料的具体添加量可为0.01wt%~99.99 wt%，优选20wt%~99.99wt%，进一步优选30wt%~80wt%。
将以上磷基材料制剂应用于制备治疗肿瘤的药物中，所制得药物的给药方式可通过静脉给药，或肿瘤内和肿瘤周围直接放置的方式。具体可用于制备治疗起源于人及动物的大脑、血液、乳腺、胰腺、子宫、子宫内膜、子宫颈、肾脏、肝、胆囊、头颈部、口腔、甲状腺、皮肤、黏膜、腺体、血管、肝脏、肺脏、食管、卵巢、前列腺、骨组织、淋巴结、膀胱、结肠或直肠的原发或继发的癌、肉瘤或癌肉瘤的药物。治疗过程中的具体用药量可根据药物中所含磷基材料的类型、所针对的肿瘤类型及用药方式等情况进行确定。
本发明的有益技术效果是：本发明提供了一种磷基材料制剂及其制备方法和应用，该磷基材料制剂中磷基材料的表面修饰性羟基磷酸钙，可提高磷基材料在中性生理环境下的稳定性；另外，羟基磷酸钙修饰后，磷基材料的表面粗糙度显著增加，有利于细胞的内吞，提高内吞效率；并且磷基材料表面的修饰层具有肿瘤微环境响应降解特性，当该材料制剂作用于肿瘤细胞时，羟基磷酸钙在肿瘤细胞内外偏酸性的微环境下降解，暴露羟基磷酸钙与磷基材料反应的活化位点，加快磷基材料的降解速率，瞬时产生大量磷酸根离子及其它活性产物，破坏肿瘤微环境的离子平衡，诱发肿瘤细胞的蛋白发生磷酸化，从而实现抑制肿瘤细胞增殖、诱导其死亡的作用。具体地，磷基材料由于肿瘤组织的高渗透长滞留效应（enhanced permeability and retention effect，EPR）和/或由于其表面的靶向作用等原因可在肿瘤组织微环境中积累，和/或被肿瘤细胞通过内吞作用摄取后，由于肿瘤细胞内及胞外所具有的偏酸性微环境加速其转化，并在其快速转化过程中瞬时产生大量磷酸根离子及其他活性产物（即不稳定的中间产物，如活性自由基、活性氧等），该过程可称为“离子炸弹效应（Ionic Bomb Effect）”，进而可进一步诱导肿瘤细胞内外微环境的改变，并促使蛋白发生非特异性磷酸化作用，从而扰乱肿瘤细胞的有丝分裂，抑制肿瘤细胞增殖，并最终诱导肿瘤细胞死亡。由于本发明磷基材料制剂中，磷基材料表面修饰的基团中存在羟基，材料表面的电负性增强，相比于未经修饰的磷基材料具有更为优异的功能分子基团（如肿瘤靶向分子、荧光示踪分子等）结合能力以及正电荷分子吸附能力。另外，羟基磷酸钙降解后磷基材料表面活性位点可增强磷基材料的“离子炸弹效应”，相比于未经修饰的磷基材料，能够在单位时间内产生数量更多的磷酸根离子及其它活性产物，具有更为优良的抗肿瘤效果。而对于正常细胞而言，由于其较慢的分裂活性以及偏中性的胞内外微环境，磷基材料制剂在正常组织及细胞中的转化降解较为缓慢，在这一温和转化过程中被缓慢地释放的磷酸根离子具有极高的生物相容性，因此，对正常组织细胞的影响非常小。
综上，本发明的方案简单、高效，本发明的磷基材料制剂可应用于制备治疗肿瘤的药物，其在治疗肿瘤过程中具有特异性和靶向性。
附图说明
为了更清楚的说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图做简单说明。
图1是实施例1中羟基磷酸钙修饰前后黑磷纳米片Zeta电位变化情况的表征图；
图2是实施例1所制得羟基磷酸钙修饰的黑磷纳米片分别在pH7.0和pH4.0超纯水中和裸黑磷纳米片在pH7.0超纯水中808nm处紫外吸光度的变化曲线图；
图3是实施例1所制得羟基磷酸钙表面修饰的黑磷基纳米片抑制乳腺癌细胞增殖曲线图；
图4是实施例1所制得羟基磷酸钙表面修饰的黑磷基纳米片抑制宫颈癌细胞增殖曲线图；
图5是实施例1所制得羟基磷酸钙表面修饰的黑磷基纳米片抑制非小肺癌细胞增殖曲线图；
图6是实施例1所制得羟基磷酸钙表面修饰的黑磷基纳米片抑制正常细胞增殖曲线图；
图7是实施例1所制得羟基磷酸钙表面修饰的黑磷基纳米片诱导乳腺癌细胞凋亡图；
图8是实施例1所制得羟基磷酸钙表面修饰的黑磷基纳米片诱导宫颈癌细胞凋亡图；
图9是实施例1所制得羟基磷酸钙表面修饰的黑磷基纳米片诱导非小肺癌细胞凋亡图；
图10是实施例1所制得羟基磷酸钙表面修饰的黑磷基纳米片诱导正常细胞凋亡图；
图11是实施例1中羟基磷酸钙表面修饰前后黑磷基纳米片的荧光运载效率变化对比图；
图12是实施例1所制得羟基磷酸钙表面修饰的黑磷基纳米片基因运载的定性观察结果图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1：制备羟基磷酸钙修饰的黑磷纳米片
先通过液相剥离方法制备具有生物活性的黑磷纳米薄片。具体步骤包括：在隔绝空气的环境下，将一定量的黑磷晶体研磨后分散于溶剂N-甲基吡咯烷酮（NMP）中并密封；而后采用探头超声和水浴超声两者依次作用的方式，对黑磷溶液进行超声剥离，以制备具有生物活性的黑磷薄片原液。超声剥离也可采用单独探头超声或水浴超声的方式，并且可通过改变超声的作用方式、超声频率等调节剥离的效果，也可结合其他的剥离技术如热分离技术、离子插层技术等，提高剥离的效率和产率。所制得黑磷薄片原液中的黑磷薄片具有在被肿瘤细胞吞噬后可转化产生大量磷酸根离子，改变细胞内外环境，进而抑制肿瘤细胞增殖及诱导肿瘤细胞死亡的生物活性。
按如上液相剥离方法制得黑磷薄片，还可对所制得黑磷薄片进行表面配位或靶向修饰，以增强黑磷薄片的稳定性和靶向性，得到的二维黑磷（包括裸露的二维黑磷以及经修饰的二维黑磷）分散于适当溶剂中，以便于材料的长期保存。
取适量黑磷原液（溶剂为NMP），在7000 rpm下离心10 min，测量上清液黑磷浓度，按照P:Ca的摩尔浓度比为1:30加入葡萄糖酸钙，摇匀后超声15 min使其分散均匀。在超声后的溶液中按照占溶液体积的10%加入浓氨水，调节反应pH为10~11，并置于50℃恒温水浴加热12 h；而后在15000 rpm下离心纯化，并用超纯水洗涤沉淀两次，离心纯化后制得表面修饰羟基磷酸钙的黑磷纳米片。
实施例2：Zeta电位测量实验
对实施例1中经羟基磷酸钙修饰前后的黑磷纳米片进行Zeta电位测量实验，所得结果如图1所示。如图1可知，经羟基磷酸钙修饰前，黑磷纳米片的Zeta电位为-21.0mV，而经羟基磷酸钙修饰后，黑磷纳米片的Zeta电位为-29.7mV。这表明经羟基磷酸钙表面修饰的黑磷纳米片表面电负性相对裸黑磷（即未经羟基磷酸钙修饰的黑磷）来说增强了。
实施例3：对表面修饰羟基磷酸钙的黑磷纳米片的体外稳定性评价
通过分别对裸黑磷纳米片和实施例1所制得表面修饰羟基磷酸钙的黑磷纳米片进行体外降解实验，以对其体外稳定性进行评价。具体如下：
分别取等量实施例1所制得的表面修饰羟基磷酸钙的黑磷纳米片溶解于pH 7.0以及pH 4.0的超纯水中（超纯水的pH由稀盐酸调节）。每隔24 h测量各样品的300~900 nm紫外吸光度，根据吸光度值与初始值的比值计算降解率，直至黑磷完全降解。另外，取等量的裸黑磷纳米片溶于pH 7.0的超纯水中，按以上的方法进行体外降解实验。
具体实验结果如图2所示。结果表明4天后表面修饰羟基磷酸钙的黑磷纳米片在pH4.0的超纯水中的降解率为62.0%，明显高于pH 7.0时所对应的31.0%降解率，证明该经羟基磷酸钙表面修饰的黑磷纳米片具有一定的pH响应性。
与对照组裸黑磷纳米片相比，在pH 7.0中性条件下，羟基磷酸钙修饰的黑磷纳米片稳定性明显高于裸黑磷纳米片，表明羟基磷酸钙的表面修饰能够提高黑磷纳米片的稳定性。
实施例4：表面修饰羟基磷酸钙的黑磷纳米片对肿瘤细胞的杀伤效果评价
将实施例1所制得表面修饰羟基磷酸钙的黑磷纳米薄片作用于肿瘤细胞和正常细胞，进行细胞增殖检测实验和细胞凋亡检测实验。具体如下：
（一）细胞增殖检测实验方法
预先培养不同类型的人癌细胞和正常细胞，其中，人癌细胞包括宫颈癌细胞（Hela）、乳腺癌细胞（MCF-7）和非小肺癌细胞（A549），正常细胞选用人骨髓间充质干细胞(Hmsc)。具体地，可将细胞按5000个/孔的密度种植于96孔板，每组做四个复孔，每孔培养基为100μL包含有10% FBS的DMEM，细胞置于37℃培养箱，5%CO2，饱和湿度条件下培养24h。
然后，将培养基替换为含适宜尺寸的表面修饰羟基磷酸钙的黑磷纳米薄片的培养基，再在37℃培养箱，5%CO2，饱和湿度条件下培养48h。其中，表面修饰羟基磷酸钙的黑磷纳米薄片的尺寸可以为厚度2-20nm；长宽尺寸20-300nm，以利于细胞内吞。另外，培养孔中表面修饰羟基磷酸钙的黑磷纳米薄片的终浓度分别为0、0.0625、0.125、0.25、0.5、1、2、4和8μg/mL。培养后，弃去原培养基，加入100μL CCK-8工作液孵育1h后，检测A450nm处的吸光度（OD）值，通过各孔OD值计算细胞存活率。
（二）细胞凋亡检测实验方法
预先培养不同类型的人癌细胞和正常细胞，其中，人癌细胞包括宫颈癌细胞（Hela）、乳腺癌细胞（MCF-7）和非小肺癌细胞（A549），正常细胞选用人骨髓间充质干细胞（Hmsc）。具体地，将细胞按5×104个/孔的密度种植于24孔板，每组做三个复孔，每孔培养基为1mL包含有10% FBS的DMEM，细胞置于37℃培养箱，5%CO2，饱和湿度条件下培养24h。
然后，将培养基替换为含适宜尺寸的表面修饰羟基磷酸钙的黑磷纳米薄片的培养基，再在37℃培养箱，5%CO2，饱和湿度条件下培养48h。其中，表面修饰羟基磷酸钙的黑磷纳米薄片的尺寸可以为厚度2-10nm；长宽尺寸20-300nm，以利于细胞内吞。另外，培养孔中表面修饰羟基磷酸钙的黑磷纳米薄片的终浓度分别为0、0.25、0.5、1.0和2.0μg/mL。培养结束后，用胰蛋白酶处理并收集细胞，1000g离心5min，弃上清，收集细胞，用PBS轻轻重悬细胞并计数。取5-10万重悬的细胞，1000g离心5min，弃上清，加入195μL Annexin V-FITC结合液轻轻重悬细胞，再加入5μL Annexin V-FITC和10μL碘化丙啶（PI）轻轻混匀，室温避光染色15min，用流式细胞仪上机检测。
对表面修饰羟基磷酸钙的黑磷纳米薄片进行如上体外应用研究，所得结果如图3-10所示。
请参阅图3至图6，图3是本发明实施例1中表面修饰羟基磷酸钙的黑磷纳米薄片抑制乳腺癌细胞增殖曲线图，图4是表面修饰羟基磷酸钙的黑磷纳米薄片抑制宫颈癌细胞增殖曲线图，图5是表面修饰羟基磷酸钙的黑磷纳米薄片抑制非小肺癌细胞增殖曲线图，图6是表面修饰羟基磷酸钙的黑磷纳米薄片抑制人正常细胞增殖曲线图。在以上细胞增殖曲线图中，横坐标为表面修饰羟基磷酸钙的黑磷纳米薄片的浓度，纵坐标为细胞存活率。
如图3至图6所示，在细胞增殖检测实验中，一定浓度梯度的表面修饰羟基磷酸钙的黑磷纳米薄片作用于乳腺癌细胞（MCF-7）、宫颈癌细胞（Hela）和非小肺癌细胞（A549）三种癌细胞48h后，该表面修饰羟基磷酸钙的黑磷纳米薄片对三种癌细胞呈现显著的增殖抑制作用。分析结果发现：培养48h后，当羟基磷酸钙修饰的黑磷纳米薄片的浓度为0.5μg/mL时，乳腺癌细胞的增殖率被抑制了50%左右（如图3所示）；当羟基磷酸钙修饰的黑磷纳米薄片的浓度为0.5μg/mL左右时，宫颈癌细胞的增殖抑制率达到了80%（如图4所示）；当羟基磷酸钙修饰的黑磷纳米薄片浓度为0.5μg/mL左右时，非小肺癌细胞增殖抑制率达到90%左右（如图5所示）。而对于正常细胞人骨髓间充质干细胞，在培养48h后，各浓度羟基磷酸钙修饰的黑磷纳米薄片处理的细胞其细胞存活率均在40%以上（如图6所示）。且由图3至图6可知，羟基磷酸钙修饰的黑磷纳米薄片在较低的剂量下便可显著抑制癌细胞的增殖；在相同剂量下黑磷纳米薄片对正常细胞的增殖抑制作用远远小于癌细胞。
请参阅图7至图10，图7是本发明实施例1中表面修饰羟基磷酸钙的黑磷纳米薄片诱导乳腺癌细胞凋亡图，图8是表面修饰羟基磷酸钙的黑磷纳米薄片诱导宫颈癌细胞凋亡图，图9是表面修饰羟基磷酸钙的黑磷纳米薄片诱导非小肺癌细胞凋亡图，图10是表面修饰羟基磷酸钙的黑磷纳米薄片诱导人正常细胞凋亡图。在以上细胞凋亡图中，横坐标为Annexin V荧光强度；纵坐标为PI荧光强度；Q4区域为活性正常的细胞；Q3为早期凋亡细胞；Q2为晚期凋亡细胞；Q1为坏死细胞，可忽略不计。
如图7至图10所示，在细胞凋亡检测实验中，发现三种癌细胞的凋亡细胞（Q2+Q3）比例随浓度的增加而显著增大，表面修饰羟基磷酸钙的黑磷纳米薄片对三种癌细胞具有一定的浓度依赖性，即表面修饰羟基磷酸钙的黑磷纳米薄片浓度增加，其对细胞抑制程度相应增大。而正常细胞的凋亡细胞（Q2+Q3）比例较小，且随材料浓度增大凋亡细胞比例增加不明显。当羟基磷酸钙修饰的黑磷纳米片浓度为1μg/mL左右时，凋亡的MCF-7、Hela和A549细胞所占比例分别为50.05 %、34.4 %和37.02 %，相比之下，凋亡的Hmsc细胞所占比例仅为10.88 %。这表明羟基磷酸钙修饰的黑磷纳米片能够实现对肿瘤细胞和正常细胞的差异化作用，即选择性杀伤肿瘤细胞。
图7至图10的结果进一步说明了，在较低剂量的羟基磷酸钙修饰的黑磷纳米片便可实现癌细胞增殖的显著抑制，但对正常细胞的增殖抑制作用明显较小。
以上结果证明该羟基磷酸钙修饰的黑磷纳米薄片可显著有效地抑制癌细胞的增殖，并诱导其凋亡，但在相同剂量下对正常细胞的杀伤作用远小于癌细胞。
实施例5：表面修饰羟基磷酸钙的黑磷纳米片对小分子药物运载能力评价
以荧光小分子Ce6为模板，检测表面修饰羟基磷酸钙的黑磷纳米片的小分子药物运载能力。
按照实施例1中羟基磷酸钙修饰的黑磷纳米片的制备方法，在羟基磷酸钙沉积于黑磷纳米片表面的过程中加入荧光小分子Ce6，反应结束后离心吸取上清液，得运载后上清液；另外，按照类似的方法，在羟基磷酸钙沉积于黑磷纳米片表面的过程中加入荧光小分子Ce6，立即离心取上清液，得运载前溶液。分别检测运载后上清液的荧光强度和运载前溶液的荧光强度，所得结果如图11所示，按照药物运载率η=I/I0(I和I0分别为运载后上清液的荧光强度以及运载前溶液荧光强度)，由图11结果计算运载率，得荧光小分子的运载量大约为90.17%。
实施例5：表面修饰羟基磷酸钙的黑磷纳米片对生物活性大分子药物运载能力评价
以荧光分子Cy-5.5标记小型环状DNA进行基因运载实验，具体按照实施例1中羟基磷酸钙修饰的黑磷纳米片的制备方法，在羟基磷酸钙沉积于黑磷纳米片表面的过程中加入Cy-5.5标记小型环状DNA，进行荧光分子与DNA的共定位示踪，24h后采用荧光显微镜进行观察，观察结果如图12所示。图12中结果显示24 h后荧光区域与细胞轮廓相一致，表明该羟基磷酸钙修饰的黑磷纳米片能够运载基因，并实现其在细胞内的释放。
由上可知，羟基磷酸钙修饰的二维黑磷纳米片能够在显著有效抑制癌细胞的增殖，并诱导其凋亡的同时，对正常细胞存在较低的细胞毒性。另外，羟基磷酸钙形成过程中能够成功运载多种功能分子，实现抗癌药物的多功能化。因此，该磷基材料制剂非常适用于作为新型多功能化抗癌药物的开发。
综上，经羟基磷酸钙表面修饰的黑磷纳米薄片具有生物活性及多功能修饰潜力，其作用机制主要包括功能分子的物理包埋及环境响应下的分子释放，产生相应功能以及在肿瘤细胞内外微酸性环境下，羟基磷酸钙修饰的黑磷结合位点暴露，黑磷表面呈活化状态，被氧化产生大量磷酸根离子及其他活性中间产物（如：ROS等）。在癌症治疗、肿瘤示踪以及基因联合治疗、药物运载方面具有巨大应用潜力。
黑磷量子点的表面能够与纳米片一样被氧化产生磷酸根离子，并与钙离子、氢氧根离子结合形成羟基磷酸钙沉积于材料表面，进而基于与羟基磷酸钙修饰的黑磷纳米片相似的机理，可应用于制备抗肿瘤药物。具体可通过将黑磷在少量N-甲基吡咯烷酮（NMP）中仔细研磨成粉，再加入适量N-甲基吡咯烷酮并使用细胞破碎仪探头超声3h，得到的分散液再在超声波清洗器中水浴超声10 h，制备得到黑磷量子点；再采用类似以上羟基磷酸钙修饰的黑磷纳米薄片的制备方法在黑磷量子点的表面修饰羟基磷酸钙，以制备表面修饰羟基磷酸钙的黑磷量子点。
当然，羟基磷酸钙也可以其他方式修饰于黑磷纳米片或黑磷量子点的表面，从而可增强黑磷纳米片和黑磷量子点在中性环境下的稳定性，以及利于细胞内吞，提高内吞率。
另外，由上可知，黑磷纳米薄片具有生物活性，在与肿瘤细胞接触后，可被肿瘤细胞吞噬，并在肿瘤细胞内转化产生磷酸根离子，由于肿瘤细胞内及胞外所具有的偏酸性微环境可加速其转化，在其快速转化过程中瞬时产生大量磷酸根离子及其他活性产物（即不稳定的中间产物），可进一步诱导肿瘤细胞内外微环境的改变，破坏肿瘤细胞内外微环境的离子平衡，并促使肿瘤细胞的蛋白发生非特异性磷酸化作用，从而扰乱肿瘤细胞的有丝分裂，抑制肿瘤细胞增殖，并最终诱导肿瘤细胞死亡。根据以上黑磷纳米薄片针对肿瘤细胞的作用机理，可推断其他在酸性环境下可转化产生磷酸根离子的磷基材料，包括其他单质磷和/或含磷化合物，也可应用于制备治疗肿瘤的药物，以通过以上类似的作用机理作用于肿瘤细胞，抑制肿瘤细胞的增殖及诱导肿瘤细胞死亡，实现对肿瘤的治疗。并且可通过羟基磷酸钙的表面修饰增强磷基材料在中性环境下的稳定性，以及利于细胞内吞，提高内吞效率；甚至可赋予其在肿瘤微环境的pH响应功能，加速在酸性环境下的降解，快速有效抑制肿瘤细胞的扩增和转移，从而更有效防止癌症细胞的转移和肿瘤的复发，以提高治疗肿瘤效果。
尽管结合优选实施方案具体展示和介绍了本发明，但所属领域的技术人员应该明白，在不脱离所述权利要求书所限定的本发明的精神和范围内，在形式上和细节上可以对本发明做出各种变化，均为本发明的保护范围。

Claims (14)

  1. 一种磷基材料制剂，其特征在于，所述磷基材料制剂包含磷基材料和修饰于所述磷基材料表面的羟基磷酸钙，所述磷基材料为在酸性环境下可转化产生磷酸根离子的材料。
  2. 根据权利要求1所述的磷基材料制剂，其特征在于，所述磷基材料为在酸性环境下可转化产生磷酸根离子的单质磷和/或含磷化合物。
  3. 根据权利要求1所述的磷基材料制剂，其特征在于，所述磷基材料的表面可被氧化、水解或电离产生磷酸根离子。
  4. 根据权利要求1-3中任一项所述的磷基材料制剂，其特征在于，所述磷基材料为磷基微纳材料；优选地，所述磷基材料包括黑磷纳米片、黑磷量子点以及经表面修饰的黑磷纳米片和黑磷量子点中的至少一种，所述经表面修饰的黑磷纳米片和黑磷量子点的表面可被氧化、水解或电离产生磷酸根离子。
  5. 根据权利要求1-3中任一项所述的磷基材料制剂，其特征在于，所述磷基材料制剂经过表面修饰以用于增强靶向性；所述用于增强靶向性的表面修饰包括肽类物质修饰和靶向癌细胞的适配体或抗体修饰中的至少一种。
  6. 根据权利要求1-3中任一项所述的磷基材料制剂，其特征在于，所述磷基材料制剂内还负载有功能分子，所述功能分子包括荧光示踪分子、靶向肿瘤标记物分子、药物分子和生物活性大分子中的至少一种。
  7. 一种权利要求1-6中任一项所述的磷基材料制剂的制备方法，其特征在于，包括以下步骤：
    S1、在隔绝空气的环境，将磷基原材料研磨后分散于溶剂中，制得磷基原材料溶液；而后对所述磷基原材料溶液进行超声剥离，得磷基材料原液；
    S2、对所述磷基材料原液进行离心分离，取上清液；向所述上清液中加入钙源，混匀后加入氢氧根离子供体进行反应，再进行纯化。
  8. 根据权利要求7所述的制备方法，其特征在于，所述钙源为含钙离子化合物。
  9. 根据权利要求7所述的制备方法，其特征在于，所述氢氧根离子供体为可水解或电离产生氢氧根离子的碱性化合物。
  10. 权利要求1-6中任一项所述的磷基材料制剂在制备治疗肿瘤的药物中的应用，其特征在于，所述药物通过改变所述肿瘤细胞的内外环境，来抑制肿瘤细胞增殖和/或诱导肿瘤细胞凋亡。
  11. 根据权利要求10所述的应用，其特征在于，基于所述治疗肿瘤的药物，所述磷基材料制剂的添加量为1wt%~99.99wt%。
  12. 根据权利要求10所述的应用，其特征在于，所述肿瘤包括人及动物大脑、血液、乳腺、胰腺、子宫、子宫内膜、子宫颈、肾脏、肝、胆囊、头颈部、口腔、甲状腺、皮肤、粘膜、腺体、血管、肝脏、肺脏、食管、卵巢、前列腺、骨组织、淋巴结、前列腺、膀胱、结肠或直肠的原发或继发的癌、肉瘤或癌肉瘤。
  13. 根据权利要求10所述的应用，其特征在于，所述治疗肿瘤的药物还包括药学可接受的助剂。
  14. 根据权利要求13所述的应用，其特征在于，所述治疗肿瘤的药物为片剂、胶囊剂、丸剂、颗粒剂、缓释制剂、控释制剂、注射制剂中的任一种。
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