CN104825490A - Hydroxyapatite nanoparticle with antitumor activity, preparation method and application thereof - Google Patents
Hydroxyapatite nanoparticle with antitumor activity, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a hydroxyapatite nanoparticle with antitumor activity, a preparation method and application thereof. The invention discloses the near-spherical nano-hydroxyapatite with a particle size of 10-100nm, amorphous state and uniform particle distribution. The nanoparticle has very excellent antitumor activity and a tumor cell inactivation rate up to about 80%. The hydroxyapatite nanoparticle is prepared by a liquid phase precipitation method. The invention also discloses the use of the nanoparticle, and a pharmaceutical composition and a medicine box containing the nanoparticle.
Description
Technical field
The invention belongs to inorganic material and field of medicaments, more specifically, the present invention relates to there is anti-tumor activity hydroxyapatite nano particle, its preparation method and application.
Background technology
Hydroxyapatite (Ca
10(PO
4)
6(OH)
2hydroxyapatite is called for short HAP) be main inorganic composition in skeleton and dental tissue, because of its have to most Protein nucleic acid molecules show adsorptivity, high associativity, outstanding biocompatibility, faint cytotoxicity, without challeng, without tumorigenicity and advantage (the Sergey V.Dorozhkin such as relatively good load transfection power and controllable release, Biomaterials., 2010,31:1465-1485; He LH, et al.ActaBiomaterials2008; 4:577-86), be extensively incorporated in biomedicine field, as organism embedded material, bone injury reparation, pharmaceutical carrier, anti-biotic material, immunosensor and gene therapy.Good and can the material of safe disposal as a kind of biocompatibility, in recent years, the interaction situation of hydroxyapatite and human body cell becomes study hotspot.
Existing research display hydroxyapatite nano particle (HAP nanoparticles, HAPN) presents certain inhibitory action to the growing multiplication of tumor cell.But the hydroxyapatite nano particle preparation method in currently available technology differs, and Morphological Characterization differs, and character differs, and antitumous effect is very different.Most of nanoparticles that in prior art, conventional method is produced do not have antitumous effect.
Therefore, this area needs the antitumor action exploring hydroxyapatite nano particle further, and optimizes its technology of preparing, to develop hydroxyapatite nano particle that is novel, excellent effect.
Summary of the invention
The object of the present invention is to provide hydroxyapatite nano particle with anti-tumor activity and its preparation method and application.
In a first aspect of the present invention, provide a kind of hydroxyapatite nano particle with antitumor cytotoxicity, this hydroxyapatite nano particle is near-spherical, particle diameter 20 ~ 100nm, and degree of crystallinity is low, and phase is amorphous state and distribution of particles is even.
In a preference, described hydroxyapatite particle to the suppression ratio more than 75% of tumor cell, preferably about 80%, there is high anti-tumor activity.
In another preference, the preparation method of described hydroxyapatite nano particle comprises:
(a) at 0 ~ 8 DEG C, preferably at 0 ~ 6 DEG C, by Ca (NO
3)
2aqueous solution and (NH
4)
2hPO
4aqueous solution, keep pH10 ~ 12, preferably pH10 ~ 11, stirring reaction 8 ~ 30 hours is preferably 20 ~ 30 hours;
B () reacts completely after, centrifugal collecting precipitate sample;
C sample that () washing (b) obtains;
The sample of (d) lyophilization process (c); Preferably, frozen dried 12 ~ 36 hours; More preferably, frozen dried 18 ~ 30 hours; With
E the sample of (d) is calcined by (), preferably calcine 1 ~ 5 hour, as 2, and 3,4 hours, the hydroxyapatite nano particle described in acquisition.
In another preference, in the step (a) of described method, Ca (NO
3)
2aqueous solution or (NH
4)
2hPO
4the concentration of aqueous solution is 0.1 ~ 0.8mol/L; Be preferably 0.15 ~ 0.6mol/L; As 0.2mol/L; 0.3mol/L; 0.4mol/L; 0.5mol/L; Or
In step (c), second alcohol and water replaces washing particles; Preferably, wash 1-5 time, as 2,3,4 times; Or
In step (e), the temperature of calcining is 600 ± 100 DEG C; Be preferably 600 ± 50 DEG C.
In another preference, in step (a), by reacting to make reaction temperature at 0 ~ 8 DEG C in ice-water bath.
In another aspect of this invention, provide a kind of method preparing described hydroxyapatite nano particle, described method comprises:
(a) at 0 ~ 8 DEG C, preferably at 0 ~ 6 DEG C, by Ca (NO
3)
2aqueous solution and (NH
4)
2hPO
4aqueous solution, keep pH10 ~ 12, preferably pH10 ~ 11, stirring reaction 8 ~ 30 hours is preferably 20 ~ 30 hours;
B () reacts completely after, centrifugal collecting precipitate sample;
C sample that () washing (b) obtains;
The sample of (d) lyophilization process (c); Preferably frozen dried 12 ~ 36 hours; More preferably 18 ~ 30 hours; With
E the sample of (d) is calcined by (); Preferably calcine 1 ~ 5 hour, as 2,3,4 hours, the hydroxyapatite nano particle described in acquisition.
In a preference, in the step (a) of described method, Ca (NO
3)
2aqueous solution or (NH
4)
2hPO
4the concentration of aqueous solution is 0.1 ~ 0.8mol/L; Be preferably 0.15 ~ 0.6mol/L; As 0.2mol/L; 0.3mol/L; 0.4mol/L; 0.5mol/L; Or
In step (c), second alcohol and water replaces washing particles; Preferably, wash 1-5 time, as 2,3,4 times; Or
In step (e), the temperature of calcining is 600 ± 100 DEG C; Be preferably 600 ± 50 DEG C.
In another aspect of this invention, the purposes of arbitrary described hydroxyapatite nano particle is above provided, for the preparation of anti-tumor drug.
In a preference, described tumor comprises: pulmonary carcinoma, gastric cancer, hepatocarcinoma, melanoma, osteosarcoma, colon cancer.
In another aspect of this invention, provide a kind of for antitumor medicine composition, described pharmaceutical composition comprises: arbitrary described hydroxyapatite nano particle, and pharmaceutically acceptable carrier above.
In another aspect of this invention, provide a kind of for antineoplastic medicine box, described medicine box comprises described pharmaceutical composition.
In a preference, described tumor includes but not limited to: pulmonary carcinoma, gastric cancer, hepatocarcinoma, melanoma, osteosarcoma, colon cancer.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
The XRD spectra (A) of Fig. 1, hydroxyapatite nano particle-1 (HAPN-1), FTIR spectrogram (B) and TEM photo (C).
The TEM figure of Fig. 2, hydroxyapatite nano particle-2 (HAPN-2).
The TEM figure of Fig. 3, hydroxyapatite nano particle-3 (HAPN-3).
The TEM figure of Fig. 4, hydroxyapatite nano particle-4 (HAPN-4).
Fig. 5, hydroxyapatite nano particle-1,2, the XRD comparison diagram of 3 (HAPN-1, HAPN-2, HAPN-3).
Fig. 6, hydroxyapatite nano particle-1,2,3 (HAPN-1, HAPN-2, HAPN-3) to A549 cytotoxicity comparison diagram.
The XRD comparison diagram of Fig. 7, hydroxyapatite nano particle-1,4 (HAPN-1, HAPN-4).
Fig. 8, hydroxyapatite nano particle-1,4 (HAPN-1, HAPN-4) to A549 cytotoxicity comparison diagram.
The cytotoxicity comparison diagram of Fig. 9, hydroxyapatite nano particle-1 pair of A549 cell and 16HBE cell.
Figure 10, hydroxyapatite nano particle-1 (HAPN-1) are respectively to the cytotoxicity comparison diagram of A549, MGC803, Bel-7402, A857, U2OS cell.
Figure 11, mice with tumor carried out to tumor change photo after HAPN-1 vivo medicine-feeding.
Figure 12, mice with tumor carried out to gross tumor volume change curve after HAPN-1 vivo medicine-feeding.
Detailed description of the invention
The present inventor, through deep research, discloses a kind of novel hydroxyapatite nano particle, and it is active that it has extremely excellent antineoplastic, reaches about 80% to tumor cell inactivation ratio.Present invention further teaches the Preparation method and use of this nanoparticle, and contain pharmaceutical composition and the medicine box of this nanoparticle.
Nanoparticle and preparation thereof
The invention discloses a kind of hydroxyapatite nano particle with anti-tumor activity, it is near-spherical, particle diameter 20 ~ 100nm, and degree of crystallinity is low, and phase is amorphous state and distribution of particles is even.This hydroxyapatite nano particle is with Ca (NO
3)
2(NH
4)
2hPO
4for raw material, adopt liquid-phase precipitation method synthesis.This hydroxyapatite nano particle, compared with common hydroxyapatite nano particle, has high toxic effect to tumor cell.
As optimal way of the present invention, described hydroxyapatite nano particle adopts liquid phase method controllably to prepare; Preferably, the following method of described hydroxyapatite nano particle prepares:
(a) at 0 ~ 8 DEG C, by Ca (NO
3)
2aqueous solution and (NH
4)
2hPO
4aqueous solution, keep more than pH10, stirring reaction;
B () reacts completely after, centrifugal collecting precipitate sample;
C sample that () washing (b) obtains;
The sample of (d) lyophilization process (c); With
E the sample of (d) is calcined by (), the hydroxyapatite nano particle described in acquisition.
The present inventor find, in step (a), the anti-tumor activity of the nanoparticle prepared carry out hybrid reaction under cryogenic conditions (as ice-water bath) after is highly desirable, but not cryogenic conditions then anti-tumor activity is poor.Therefore, preferably at 0 ~ 8 DEG C, more preferably carry out Ca (NO at 0 ~ 6 DEG C
3)
2aqueous solution and (NH
4)
2hPO
4the hybrid reaction of aqueous solution; More preferably, lower temperature is kept by reaction in ice-water bath.
The present inventor also finds, the particle synthesized under same ice-water bath condition, when step (e) is calcined, the nanoparticle of 600 DEG C of sintering acquisitions has high anti-tumor activity, tumor cell inactivation ratio can reach 80%, but the nanoparticle of 800 DEG C of sintering acquisitions is almost complete absence of toxicity.Therefore, as optimal way of the present invention, the temperature of calcining is 600 ± 100 DEG C; Be preferably 600 ± 50 DEG C.
Hydroxyapatite nano particle of the present invention has high cytotoxicity to kinds of tumor cells, can be used as anti-tumor agent etc.
In the present invention, described tumor includes but not limited to: pulmonary carcinoma, gastric cancer, hepatocarcinoma, melanoma, osteosarcoma, colon cancer, breast carcinoma, carcinoma of prostate, nasopharyngeal carcinoma, the cerebral tumor, esophageal carcinoma, ovarian cancer, thyroid tumor, mediastinal tumor, intestinal tumor, tumor of kidney, adrenal gland neoplasms, tumor of bladder, tumor of testis, malignant lymphoma, multiple myeloma, nervous system neoplasms etc.Preferably, described tumor is pulmonary carcinoma, gastric cancer, hepatocarcinoma, melanoma, osteosarcoma, colon cancer.
Pharmaceutical composition
Present invention also offers a kind of pharmaceutical composition, it contains effective dose (as 0.000001-50wt%; Preferably 0.00001-20wt%; Better, 0.0001-10wt%) hydroxyapatite nano particle of the present invention, and pharmaceutically acceptable carrier.
Compositions of the present invention can be directly used in Tumor suppression.In addition, also can simultaneously with other therapeutic agent (as chemotherapeutics) or adjuvant conbined usage.
Usually, described hydroxyapatite nano particle can be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably, pH is about 6-8.As described in aqueous carrier medium be normal saline.
As used herein, term " contains " and represents that various composition can be applied in mixture of the present invention or compositions together.Therefore, term " primarily of ... composition " and " by ... form " be included in during term " contains ".As used herein, term " effective dose " or " effective dose " refer to can to people and/or animal produce function or activity and can by people and/or animal the amount that accepts.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or mammal and without excessive bad side reaction (as toxicity, stimulation and allergy), namely has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to the carrier being used for the treatment of agent administration, comprises various excipient and diluent.
Pharmaceutical composition of the present invention contains the described hydroxyapatite nano particle of safe and effective amount and pharmaceutically acceptable carrier.This kind of carrier comprises (but being not limited to): normal saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Usual pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by conventional method with normal saline or the aqueous solution containing glucose and other adjuvant.Described pharmaceutical composition should aseptically manufacture.The dosage of active component normally treats effective dose.Pharmaceutical preparation of the present invention also can be made into slow releasing preparation.
The effective dose of hydroxyapatite nano particle of the present invention can change with order of severity of the pattern of administration and disease to be treated etc.The selection of preferred effective dose can be determined (such as passing through clinical trial) according to various factors by those of ordinary skill in the art.Described factor includes but not limited to: pharmacokinetic parameter such as bioavailability, metabolism, the half-life etc. of described hydroxyapatite nano particle; The order of severity of the disease that patient will treat, the body weight of patient, the immune state of patient, the approach etc. of administration.Usually, when described hydroxyapatite nano particle every day is with about 0.00001mg-50mg/kg the weight of animals (preferably 0.0001mg-40mg/kg the weight of animals, as 20mg/kg the weight of animals, 10mg/kg the weight of animals, 5mg/kg the weight of animals, 3mg/kg the weight of animals) dosage give, gratifying effect can be obtained.By an urgent demand for the treatment of situation, the dosage that several times separate can be given every day, or dosage is reduced pari passu.
Present invention also offers a kind of method of Tumor suppression, comprise the described hydroxyapatite nano particle giving experimenter's effective dose.
Medicine box
Present invention also offers a kind of medicine box for Tumor suppression, comprising: container, the hydroxyapatite nano particle of the present invention containing effective dose in this container, and pharmaceutically acceptable carrier.
Described medicine box is convenient to those skilled in the art's particularly clinician's application.As optimal way of the present invention, in described medicine box, also comprise operation instructions, take the method administration be applicable to instruct those skilled in the art.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.
The sign of material
Adopt X-Ray diffraction analysis (D/max 2550VB/PC polycrystalline diffractometer) respectively in the crystalline state of 10-80o analysis of material.Adopt the building stone of Fourier infrared spectrograph (the intelligent Fourier infrared spectrograph of Nicolet 5700) analysis of material.Transmission electron microscope (TEM 2100F type) is adopted to observe the microstructure of material.The micro-Electronic Speculum of transmission (JEM-1400 type) is adopted to observe surface topography and the microstructure of preparing material.
The preparation and characterization of embodiment 1, hydroxyapatite nano particle HAPN-1
Weigh 18.89 grams of Ca (NO respectively
3)
2with 6.34 grams of (NH
4)
2hPO
4, be dissolved in respectively in the high purity water of 400mL and 240mL, two kinds of raw materials be all mixed with the aqueous solution of 0.2M.The solution prepared is put into 4 DEG C of refrigerator pre-freezes, then solution complete for pre-freeze is placed in ice-water bath, under stirring condition, by above-mentioned (NH
4)
2hPO
4solution adds rapidly Ca (NO
3)
2mix in solution, and adopt ammonia that the pH of reaction system is adjusted to 10 ~ 12; Then in ice-water bath, stirring reaction spends the night, and notes keeping pH>10 in reaction.After question response is complete, centrifugal treating is carried out to reactant liquor and collects obtained particle, particle high purity water, ethanol are replaced cleaning 3 times; Then freeze dryer lyophilizing 24h is put into; Finally sample is put into Muffle furnace 600 DEG C calcining 2h, hydroxyapatite nano particle.
The hydroxyapatite nano particle (being designated as HAPN-1) of gained adopt the test of X-Ray diffraction analysis, Fourier transform infrared spectroscopy, transmission electron microscope, etc. measure microstructure, the pattern of material, result is as shown in Figure 1.
From Fig. 1 (A) XRD, synthesized material is occurred the diffraction maximum of typical hydroxyapatite between 20 ~ 50 ° at 2 θ, but peak shape imperfect, known HAPN-1 particle crystallization degree is low.
As can be seen from Fig. 1 (B) infrared spectrogram, at 3430cm
-1, 1630cm
-1, 1040cm
-1, 604cm
-1, 565cm
-1wave number place all has absworption peak to exist.By analysis: wherein 3430cm
-1the absworption peak at place is by the OH stretching vibration peak of hydrogen bond association; 565cm
-1, 604cm
-1, 1040cm
-1the strong absworption peak at place is PO
4 3-group vibration causes, and absorption peak strength is very strong; 1630cm
-1neighbouring absworption peak is because the water of HAPN surface adsorption causes.Known HAPN-1 is pure ha composition.
Be near-spherical by Fig. 1 (C) TEM visible material, particle diameter is about about 50nm.
Comprehensive above characterization result is known, and HAPN-1 particle is near-spherical, particle diameter is 20 ~ 100nm, and degree of crystallinity is low, and phase is amorphous state and distribution of particles is even.
The preparation and characterization of embodiment 2, hydroxyapatite nano particle HAPN-2
Weigh 18.89 grams of Ca (NO respectively
3)
2with 6.34 grams of (NH
4)
2hPO
4, be dissolved in respectively in the high purity water of 400mL and 240mL, be mixed with the aqueous solution of 0.2M.The solution prepared is put into 40 DEG C of water-bath preheatings, then solution complete for preheating is placed in 40 DEG C of water-baths, under stirring condition, by above-mentioned (NH
4)
2hPO
4solution adds rapidly Ca (NO
3)
2mix in solution, and adopt ammonia that the pH of reaction system is adjusted to 10 ~ 12; Then spend the night 40 DEG C of stirred in water bath reactions, note in reaction keeping pH>10.After question response is complete, centrifugal treating is carried out to reactant liquor and collects obtained particle, particle high purity water, ethanol are replaced cleaning 3 times; Then freeze dryer lyophilizing 24h is put into; Finally sample is put into Muffle furnace 600 DEG C calcining 2h, obtain hydroxyapatite nano particle (being designated as HAPN-2).
Adopt transmission electron microscope etc. to measure microstructure, the pattern of material, result as shown in Figure 2.
Can obtain from Fig. 2 analysis, compared with HAPN-1, HAPN-2 draw ratio adds, and be ellipsoid shape, diameter and length are respectively about 20nm and 50nm.
The preparation and characterization of embodiment 3, base phosphorite nano particle HAPN-3
Weigh 18.89 grams of Ca (NO respectively
3)
2with 6.34 grams of (NH
4)
2hPO
4, be dissolved in respectively in the high purity water of 400mL and 240mL, be mixed with the aqueous solution of 0.2M.The solution prepared is put into 60 DEG C of water-bath preheatings, then solution complete for preheating is placed in 60 DEG C of water-baths, under stirring condition, by above-mentioned (NH
4)
2hPO
4solution adds rapidly Ca (NO
3)
2mix in solution, and adopt ammonia that the pH of reaction system is adjusted to more than 10; Then spend the night 60 DEG C of stirred in water bath reactions, note in reaction keeping pH>10.After question response is complete, centrifugal treating is carried out to reactant liquor and collects obtained particle, particle high purity water, ethanol are replaced cleaning 3 times; Then freeze dryer lyophilizing 24h is put into; Finally sample is put into Muffle furnace 600 DEG C calcining 2h, obtain hydroxyapatite nano particle, be designated as HAPN-3.
Adopt transmission electron microscope etc. to measure microstructure, the pattern of material, result as shown in Figure 3.
Can obtain from Fig. 3 analysis, along with the rising of reaction temperature, length of particle increases than continuing, and HAPN-3 particle is corynebacterium substantially, and diameter and length are respectively about 20nm and 60nm.
The preparation and characterization of embodiment 4, base phosphorite nano particle HAPN-4
Weigh 18.89 grams of Ca (NO respectively
3)
2with 6.34 grams of (NH
4)
2hPO
4, be dissolved in respectively in the high purity water of 400mL and 240mL, be mixed with the aqueous solution of 0.2M.The solution prepared is put into 4 DEG C of refrigerator pre-freezes, then solution complete for pre-freeze is placed in ice-water bath, under stirring condition, by above-mentioned (NH
4)
2hPO
4solution adds rapidly Ca (NO
3)
2mix in solution, and adopt ammonia that the pH of reaction system is adjusted to more than 10; Then in ice-water bath, stirring reaction spends the night, and notes keeping pH>10 in reaction.After question response is complete, centrifugal treating is carried out to reactant liquor and collects obtained particle, particle high purity water, ethanol are replaced cleaning 3 times; Then freeze dryer lyophilizing 24h is put into; Finally sample is put into Muffle furnace 800 DEG C calcining 2h, hydroxyapatite nano particle.The particle of gained is designated as HAPN-4.
Adopt transmission electron microscope etc. to measure microstructure, the pattern of material, result as shown in Figure 4.
From the TEM of Fig. 4, compared with particle above, HAPN-4 particle diameter is obviously comparatively large, size heterogeneity, and this is due under high-temperature process, particle sufficient crystallising, and together, particle diameter is about about 200-500nm in some particle growths of reuniting together.
The anti-tumor activity of embodiment 5, differential responses temperature gained particle
For embodiment 1,2,3 gained particle (HAPN-1, HAPN-2, HAPN-3), carry out X-Ray diffraction analysis to it, result as shown in Figure 5.
The analysis of mtt assay cytotoxicity is carried out to embodiment 1,2,3 gained particle.MTT cell toxicity test process is as follows: with human lung cancer cell A549's cell for model, is inoculated in by A549 cell (every porocyte number is 3000-5000) in 96 well culture plates, in 37 DEG C of constant temperature, 5%CO
2middle cultivation 12 hours, after cell is completely adherent, abandon liquid and the new culture medium of displacement containing variable concentrations hydroxyapatite nano particle (autoclaving process 30min, ensures integral asepsis at high-pressure sterilizing pot 121 DEG C), continue at 37 DEG C of constant temperature, 5%CO
2middle cultivation 72h.After cultivation terminates, every hole adds 30 μ L MTT reagent, 37 DEG C continue to hatch 4h after, sop up upper liquid, every hole adds 200 μ LDMSO, after room temperature concussion 10min, every hole is taken out 150 μ L and is put into 96 new hole ELISA Plate detect every hole absorbance at 492nm wavelength place, hatches the ratio of group data and matched group data as particle toxic effect to cell under incubation conditions.Result as shown in Figure 6.
As seen from Figure 5, after 600 DEG C of calcining (sintering) process, the crystallization degree of particle is roughly the same.As shown in Figure 6, the HAPN-1 synthesized under ice-water bath condition has high anti-tumor activity, and tumor cell inactivation ratio can reach 80%, but the particle anti-tumor activity synthesized under 40 DEG C and 60 DEG C of conditions is poor.
From above experimental result, the anti-tumor activity impact of change on particle of reaction temperature is very large, HAPN-1 is character optimal particle, this particle is near-spherical, particle diameter is 20 ~ 100nm, degree of crystallinity is low, phase is amorphous state and distribution of particles is even, and subsequent experimental proves that this particle has high cytotoxicity to kinds of tumor cells, can be used as anti-tumor agent etc.
The anti-tumor activity of embodiment 6, different sintering temperature gained particle
For embodiment 1,4 gained particle (HAPN-1, HAPN-4), carry out X-Ray diffraction analysis to it, result as shown in Figure 7.
The analysis of mtt assay cytotoxicity is carried out to embodiment 1,4 gained particle.MTT cell toxicity test process is as follows: with human lung cancer cell A549's cell for model, is inoculated in by A549 cell (every porocyte number is 3000-5000) in 96 well culture plates, in 37 DEG C of constant temperature, 5%CO
2middle cultivation 12 hours, after cell is completely adherent, abandon liquid and the new culture medium of displacement containing variable concentrations hydroxyapatite nano particle (autoclaving process 30min, ensures integral asepsis at high-pressure sterilizing pot 121 DEG C), continue at 37 DEG C of constant temperature, 5%CO
2middle cultivation 72h.After cultivation terminates, every hole adds 30 μ L MTT reagent, 37 DEG C continue to hatch 4h after, sop up upper liquid, every hole adds 200 μ LDMSO, after room temperature concussion 10min, every hole is taken out 150 μ L and is put into 96 new hole ELISA Plate detect every hole absorbance at 492nm wavelength place, hatches the ratio of group data and matched group data as particle toxic effect to cell under incubation conditions.Result as shown in Figure 8.
As seen from Figure 7, after 600 DEG C of sintering processes, HAPN-1 particle occurs characteristic peak, but peak shape is imperfect, not crystallization completely.And after 800 DEG C of sintering processes, HAPN-4 characteristic peak is complete, peak shape is sharp-pointed, intensity is high, and in typical hydroxyapatite feature peak shape, after known 800 DEG C of heat treatments, particle can reach complete crystalline state.As shown in Figure 8, the particle synthesized under similarity condition, the HAPN-1 of 600 DEG C of sintering has high anti-tumor activity, and tumor cell inactivation ratio can reach 80%, but the HAPN-4 of 800 DEG C of sintering processes is almost complete absence of toxicity.Visible, the particle anti-tumor activity synthesized under 800 DEG C of sintering conditions is poor.
From above experimental result, the change of sintering temperature is very large on the pattern of particle, degree of crystallinity and anti-tumor activity impact, the anti-tumor activity of HAPN-1 is the highest, this particle is near-spherical, particle diameter is 20 ~ 100nm, degree of crystallinity is low, phase is amorphous state and distribution of particles is even, and subsequent experimental proves that this particle all has high cytotoxicity to kinds of tumor cells, can be used as anti-tumor agent etc.
The selective toxicity difference of embodiment 7, HAPN-1
Select people's lung normal cell 16HBE and human lung cancer cell A549 to be model, A549 and 16HBE cell is inoculated in (every porocyte number is 3000-5000) in 96 well culture plates, in 37 DEG C of constant temperature, 5%CO
2middle cultivation 12 hours, after cell is completely adherent, abandons liquid and the new culture medium of displacement containing variable concentrations HAPN-1 particle (autoclaving process 30min, ensures integral asepsis at high-pressure sterilizing pot 121 DEG C), continues at 37 DEG C of constant temperature, 5%CO
2middle cultivation 72h.After cultivation terminates, every hole adds 30 μ L MTT reagent, 37 DEG C continue to hatch 4h after, sop up upper liquid, every hole adds 200 μ LDMSO, after room temperature concussion 10min, every hole is taken out 150 μ L and is put into 96 new hole ELISA Plate detect every hole absorbance at 492nm wavelength place, hatches the ratio of group data and matched group data as particle toxic effect to cell under incubation conditions.
As shown in Figure 9, HAPN-1 has obvious Difference In Toxicity to A549 and 16HBE to result, and when administration concentration reaches 1000 μ g/ml, the cytoactive of 16HBE is about 100%, and the activity of A549 only has about 15%.
Selective toxicity difference is provable, and HAPN-1 and human normal cell's compatibility well, have extremely strong killing action to tumor cell simultaneously.
Embodiment 8, HAPN-1 are to the antitumous effect of kinds of tumor cells
Select human lung cancer cell A549, gastric carcinoma cells MGC803, Human hepatoma cell line Bel-7402, people's malignant melanoma cell A857, human osteosarcoma cell U2OS to be model, tumor cell is inoculated in respectively in 96 well culture plates, in 37 DEG C of constant temperature, 5%CO
2middle cultivation 12 hours, after cell is completely adherent, abandon liquid and the new culture medium of displacement containing variable concentrations hydroxyapatite nano particle (autoclaving process 30min, ensures integral asepsis at high-pressure sterilizing pot 121 DEG C), continue at 37 DEG C of constant temperature, 5%CO
2middle cultivation 72h.After cultivation terminates, every hole adds 30 μ L MTT reagent, 37 DEG C continue to hatch 4h after, sop up upper liquid, every hole adds 200 μ LDMSO, after room temperature concussion 10min, every hole is taken out 150 μ L and is put into 96 new hole ELISA Plate detect every hole absorbance at 492nm wavelength place, hatches the ratio of group data and matched group data as particle toxic effect to cell under incubation conditions.
As shown in Figure 10, HAPN-1 all has obvious toxic effect to A549, MGC803, Bel-7402, A857, U2OS to result.
Provable, HAPN-1 has high cytotoxicity to kinds of tumor cells, and repeatability is good, can be used as anti-tumor agent etc.
Embodiment 9, animal experiment in vivo
Select 8 female tumor Mus transplanting A549 tumor cell, matched group and each random selecting of administration group 4, the average body weight average of each group is at about 20g, and average tumor diameter is about 9mm.Monday weekly, Wednesday, Friday are at the HAPN-1 normal saline suspension of the nude mice intratumor injection 200 μ L of administration group, administration concentration is 40mg/kg, the normal saline of matched group injection same volume, administration 6 weeks altogether, during administration, timing detects the change in volume situation of tumor.
Figure 11 is after administration in 6 weeks terminates, mice with tumor in-vivo tumour photo.During Figure 12 is administration, the gross tumor volume change curve of normal saline group (saline) and administration group (HAPN-1,40mg/kg).From Figure 11, after administration terminates, administration group tumor is obviously less.From Figure 12, administration is after 3 weeks, the tumor not regrowth substantially of administration group.
Mice with tumor zoopery is provable, and the growth of HAPN-1 to tumor has obvious inhibitory action.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. have a hydroxyapatite nano particle for antitumor cytotoxicity, it is characterized in that, this hydroxyapatite nano particle is near-spherical, particle diameter 20 ~ 100nm, and degree of crystallinity is low, and phase is amorphous state and distribution of particles is even.
2. hydroxyapatite nano particle as claimed in claim 1, it is characterized in that, the preparation method of this hydroxyapatite nano particle comprises:
(a) at 0 ~ 8 DEG C, by Ca (NO
3)
2aqueous solution and (NH
4)
2hPO
4aqueous solution, keep pH10 ~ 12, stirring reaction 8 ~ 30 hours;
B () reacts completely after, centrifugal collecting precipitate sample;
C sample that () washing (b) obtains;
The sample of (d) lyophilization process (c); With
E the sample of (d) is calcined by (), the hydroxyapatite nano particle described in acquisition.
3. hydroxyapatite nano particle as claimed in claim 2, is characterized in that, in step (a), and Ca (NO
3)
2aqueous solution or (NH
4)
2hPO
4the concentration of aqueous solution is 0.1 ~ 0.8mol/L; Or
In step (c), second alcohol and water replaces washing particles; Or
In step (e), the temperature of calcining is 600 ± 100 DEG C; Be preferably 600 ± 50 DEG C.
4. prepare a method for hydroxyapatite nano particle according to claim 1, it is characterized in that, described method comprises:
(a) at 0 ~ 8 DEG C, by Ca (NO
3)
2aqueous solution and (NH
4)
2hPO
4aqueous solution, keep pH10 ~ 12, stirring reaction 8 ~ 30 hours;
B () reacts completely after, centrifugal collecting precipitate sample;
C sample that () washing (b) obtains;
The sample of (d) lyophilization process (c); With
E the sample of (d) is calcined by (), the hydroxyapatite nano particle described in acquisition.
5. method as claimed in claim 4, is characterized in that, in step (a), and Ca (NO
3)
2aqueous solution or (NH
4)
2hPO
4the concentration of aqueous solution is 0.1 ~ 0.8mol/L; Or
In step (c), second alcohol and water replaces washing particles; Or
In step (e), the temperature of calcining is 600 ± 100 DEG C; Be preferably 600 ± 50 DEG C.
6. the purposes of the arbitrary described hydroxyapatite nano particle of claims 1 to 3, is characterized in that, for the preparation of anti-tumor drug.
7. purposes as claimed in claim 6, it is characterized in that, described tumor comprises: pulmonary carcinoma, gastric cancer, hepatocarcinoma, melanoma, osteosarcoma, colon cancer.
8. for an antitumor medicine composition, it is characterized in that, described pharmaceutical composition comprises: the arbitrary described hydroxyapatite nano particle of claims 1 to 3, and pharmaceutically acceptable carrier.
9. for an antineoplastic medicine box, it is characterized in that, described medicine box comprises pharmaceutical composition according to claim 8.
10. pharmaceutical composition as claimed in claim 8 or medicine box according to claim 9, it is characterized in that, described tumor comprises: pulmonary carcinoma, gastric cancer, hepatocarcinoma, melanoma, osteosarcoma, colon cancer.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018052825A (en) * | 2016-09-26 | 2018-04-05 | 秀希 青木 | Therapeutic agent of cell growth disorders using hydroxyapatite sol |
| CN108371668A (en) * | 2018-02-25 | 2018-08-07 | 四川大学 | Nanometer hydroxyapatite particle and preparation method with antitumor action and purposes |
| CN110078880A (en) * | 2018-01-26 | 2019-08-02 | 华东理工大学 | Isocyanate-crosslinked polyethylene glycol decanedioic acid glyceride bioelastomer and its preparation method and application |
| CN111573647A (en) * | 2020-06-16 | 2020-08-25 | 北京市创伤骨科研究所 | Application of nano-hydroxyapatite in preventing or inhibiting metastasis and recurrence of osteosarcoma |
| CN111643522A (en) * | 2020-07-01 | 2020-09-11 | 四川大学华西医院 | Application of nano-hydroxyapatite in preparation of drugs for preventing or treating basal cell carcinoma |
| CN112843090A (en) * | 2019-11-28 | 2021-05-28 | 华东理工大学 | Preparation method and application of hydroxyapatite nanoparticles for reversing tumor drug resistance |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110135577A1 (en) * | 2009-12-03 | 2011-06-09 | National Taiwan University | Superparamagnetic nanoparticles IN MEDICAL THERAPEUTICS and manufacturing method THEREOF |
-
2015
- 2015-04-23 CN CN201510197642.XA patent/CN104825490A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110135577A1 (en) * | 2009-12-03 | 2011-06-09 | National Taiwan University | Superparamagnetic nanoparticles IN MEDICAL THERAPEUTICS and manufacturing method THEREOF |
Non-Patent Citations (3)
| Title |
|---|
| E. BOANINI ET AL: "Ionic substitutions in calcium phosphates synthesized at low temperature", 《ACTA BIOMATERIALIA》 * |
| 汤薇: "羟基磷灰石纳米粒子抗肿瘤活性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 王志强 等: "湿法合成纳米羟基磷灰石粉末的研究", 《无机盐工业》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018052825A (en) * | 2016-09-26 | 2018-04-05 | 秀希 青木 | Therapeutic agent of cell growth disorders using hydroxyapatite sol |
| CN110078880A (en) * | 2018-01-26 | 2019-08-02 | 华东理工大学 | Isocyanate-crosslinked polyethylene glycol decanedioic acid glyceride bioelastomer and its preparation method and application |
| CN110078880B (en) * | 2018-01-26 | 2022-06-28 | 华东理工大学 | Isocyanate cross-linked polyethylene glycol-polysebacic acid glyceride biological elastomer and preparation method and application thereof |
| CN108371668A (en) * | 2018-02-25 | 2018-08-07 | 四川大学 | Nanometer hydroxyapatite particle and preparation method with antitumor action and purposes |
| CN108371668B (en) * | 2018-02-25 | 2020-11-24 | 四川大学 | Nano-hydroxyapatite particles with anti-tumor effect, preparation method and application |
| CN112843090A (en) * | 2019-11-28 | 2021-05-28 | 华东理工大学 | Preparation method and application of hydroxyapatite nanoparticles for reversing tumor drug resistance |
| CN111573647A (en) * | 2020-06-16 | 2020-08-25 | 北京市创伤骨科研究所 | Application of nano-hydroxyapatite in preventing or inhibiting metastasis and recurrence of osteosarcoma |
| CN111643522A (en) * | 2020-07-01 | 2020-09-11 | 四川大学华西医院 | Application of nano-hydroxyapatite in preparation of drugs for preventing or treating basal cell carcinoma |
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