阅读说明：本技术 纳米羟基磷灰石在制备预防或治疗基底细胞癌的药物中的用途 (Application of nano-hydroxyapatite in preparation of drugs for preventing or treating basal cell carcinoma ) 是由 解瑶 唐教清 王琳 于 2020-07-01 设计创作，主要内容包括：本发明属于生物医药技术领域,具体涉及一种纳米羟基磷灰石在制备预防或治疗基底细胞癌的药物中的用途。为了开发一种适用于预防或治疗基底细胞癌的药物,本发明提供了一种纳米羟基磷灰石在制备预防或治疗基底细胞癌的药物中的用途。本发明并具体给出了该纳米羟基磷灰石的粒径、形状等特征,通过构建小鼠基底细胞癌模型,摸索得出纳米羟基磷灰石在抑制基底细胞癌细胞中的使用剂量。本发明提供了一种纳米羟基磷灰石在制备预防或治疗基底细胞癌的药物中的用途,拓宽了纳米羟基磷灰石的应用领域,也为预防或治疗基底细胞癌的药物开发提供了基础和方向,具有重要的意义。(The invention belongs to the technical field of biological medicines, and particularly relates to application of nano-hydroxyapatite in preparation of a medicine for preventing or treating basal cell carcinoma. In order to develop a medicament suitable for preventing or treating basal cell carcinoma, the invention provides application of nano-hydroxyapatite in preparing a medicament for preventing or treating basal cell carcinoma. The invention also provides the characteristics of the nano hydroxyapatite such as particle size, shape and the like, and the dosage of the nano hydroxyapatite in the basal cell carcinoma inhibiting cell is obtained by constructing a mouse basal cell carcinoma model and searching. The invention provides the application of nano hydroxyapatite in preparing the drugs for preventing or treating basal cell carcinoma, widens the application field of nano hydroxyapatite, provides a basis and a direction for the development of the drugs for preventing or treating basal cell carcinoma, and has important significance.)

1. The application of the nano-hydroxyapatite in preparing the medicine for preventing or treating basal cell carcinoma.

2. Use according to claim 1, characterized in that: the particle diameter of the nano hydroxyapatite is 20-80 nm.

3. Use according to claim 1, characterized in that: the proportion of 30-60 nm in the particle diameter of the nano hydroxyapatite is more than or equal to 80 percent.

4. Use according to claim 1, characterized in that: the average particle size of the nano hydroxyapatite is 45.13 nm.

5. Use according to claim 1, characterized in that: the shape of the nano hydroxyapatite is as follows: granular, needle-like or columnar.

6. Use according to claim 1, characterized in that: the shape of the nano hydroxyapatite is needle-shaped.

7. Use according to claim 1, characterized in that: the in vitro dosage of the nano hydroxyapatite is 60-480 ug/ml.

8. Use according to claim 1, characterized in that: the in vitro dosage of the nano hydroxyapatite is 120 ug/ml.

9. Use according to claim 1, characterized in that: the in vivo dosage of the nano hydroxyapatite is more than or equal to 25 mg/kg.

10. Use according to claim 1, characterized in that: the nano hydroxyapatite consists of Ca (NO)₃)₂·4H₂O and (NH4)₂HPO₄Is prepared.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of nano-hydroxyapatite in preparation of a medicine for preventing or treating basal cell carcinoma.

Background

Basal cell carcinoma belongs to non-melanoma skin cancer, and has high incidence rate and heavy disease burden. Basal cell carcinoma often infiltrates, invades and destroys the skin and surrounding structures, including bone tissue, seriously affecting the aesthetic appearance of the patient. Currently, the clinical treatment of basal cell carcinoma is mainly surgical resection, but local recurrence may still exist after surgical resection, which affects prognosis. Therefore, it is extremely important to search for a drug suitable for the prevention or treatment of basal cell carcinoma.

Hydroxyapatite is a calcium phosphate compound, is a main inorganic mineral component of human bones and teeth, has good biocompatibility, and is widely applied to the field of biological medicine materials. The nano-hydroxyapatite is a novel biological material, has unique biological activity, and gets rid of the characteristics of difficult plasticity, large brittleness and the like of the traditional hydroxyapatite. Research reports that the nano-hydroxyapatite can show special biological activity and has anti-tumor effect on osteosarcoma, gastric cancer, liver cancer and the like. However, the biological safety of nano-hydroxyapatite is still controversial, and most of the studies are limited to in vitro studies. Therefore, the anti-tumor effect of the nano-hydroxyapatite is yet to be further confirmed.

Currently, there are few in vivo studies on the treatment of tumors with nano-hydroxyapatite and animal models of basal cell carcinoma are lacking. The application of the nano-hydroxyapatite in preparing a medicament for preventing or treating basal cell carcinoma has not been reported yet, and needs to be researched.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: a drug suitable for the prevention or treatment of basal cell carcinoma is developed.

The invention provides application of nano hydroxyapatite in preparation of a medicine for preventing or treating basal cell carcinoma.

Wherein, in the application, the particle diameter of the nano hydroxyapatite is 20-80 nm.

Wherein, in the application, the proportion of 30-60 nm in the particle diameter of the nano hydroxyapatite is not less than 80%.

Preferably, in the above application, the nano hydroxyapatite has an average particle size of 45.13 nm.

Wherein, in the application, the shape of the nano hydroxyapatite is as follows: granular, needle-like or columnar.

Preferably, in the above use, the nano-hydroxyapatite is needle-shaped.

Wherein, in the application, the in vitro dosage of the nano-hydroxyapatite is 60-480 ug/ml.

Preferably, in the above application, the dosage of the nano-hydroxyapatite used in vitro is 120 ug/ml.

Wherein, in the application, the in vivo dosage of the nano hydroxyapatite is not less than 25 mg/kg.

In the application, the nano-hydroxyapatite is prepared from soluble calcium salt and soluble phosphate by a chemical precipitation method.

Further, in the above use, the nano-hydroxyapatite is composed of Ca (NO)₃)₂·4H₂O and (NH4)₂HPO₄Is prepared.

In particular, in the above application, the preparation method of the nano hydroxyapatite comprises the following steps:

a. 0.5ml/l Ca (NO) is prepared₃)₂·4H₂O160 ml and 0.5mol/l (NH4)₂HPO₄96ml of Ca (NO) was stirred continuously at 1000r/min with magnetic stirring₃)₂·4H₂O solution, at room temperature (NH4)₂HPO₄Slowly and uniformly dropping Ca (NO) into the solution at the speed of 4ml/min₃)₂·4H₂In O solution, ammonia NH is used at any time during the reaction₃·H₂Adjusting the pH value of the reaction system to 10;

b. treat (NH4)₂HPO₄After the solution is added dropwise, standing and aging for 24h at normal temperature, repeatedly centrifuging and washing the precipitate for 6 times by ultrapure water until the pH value of the slurry is washed to be neutral, and drying for 10h in a constant-temperature drying oven at 60 ℃ to prepare granular nano-hydroxyapatite;

or a, preparing 0.5ml/l Ca (NO)₃)₂·4H₂O160 ml and 0.5mol/l(NH4)₂HPO₄96ml of Ca (NO) was stirred continuously at 1000r/min with magnetic stirring₃)₂·4H₂O solution, at room temperature (NH4)₂HPO₄Slowly and uniformly dropping Ca (NO) into the solution at the speed of 4ml/min₃)₂·4H₂In O solution, ammonia NH is used at any time during the reaction₃·H₂Adjusting the pH value of the reaction system to 10;

b. treat (NH4)₂HPO₄After the solution is dropwise added, stirring and aging for 2h at normal temperature, transferring the obtained milky white slurry into a hydrothermal reaction kettle, carrying out hydrothermal reaction for 12h at 150 ℃, naturally cooling, then discarding supernatant, repeatedly carrying out centrifugal washing and precipitation for 6 times by using ultrapure water until the pH value of the slurry is washed to be neutral, and drying in a constant-temperature drying oven at 60 ℃ for 10h to prepare the needle-like nano-hydroxyapatite;

or a, preparing 0.5ml/l Ca (NO)₃)₂·4H₂O160 ml and 0.5mol/l (NH4)₂HPO₄96ml of Ca (NO) was stirred continuously at 1000r/min with magnetic stirring₃)₂·4H₂O solution, at room temperature (NH4)₂HPO₄Slowly and uniformly dropping Ca (NO) into the solution at the speed of 4ml/min₃)₂·4H₂In O solution, ammonia NH is used at any time during the reaction₃·H₂Adjusting the pH value of the reaction system to 10;

b. treat (NH4)₂HPO₄After the solution is dropwise added, stirring and aging are carried out for 24h at normal temperature, ultrapure water is repeatedly centrifuged, washed and precipitated for 6 times until the pH value of the slurry is washed to be neutral, hydrothermal reaction is carried out for 12h at 150 ℃, and then drying is carried out in a constant-temperature drying oven for 10h at 60 ℃ to prepare the columnar nano-hydroxyapatite.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a new application of nano-hydroxyapatite in basal cell carcinoma, the invention discovers for the first time that the nano-hydroxyapatite with a specific form can inhibit the basal cell carcinoma, and the effect is verified by normal human fibroblasts and human keratinocyte cell strains. The invention also establishes a basal cell carcinoma animal model for the first time, researches the influence of the nano-hydroxyapatite on the important organs of the mouse model in vivo for the first time, and determines the safety of the nano-hydroxyapatite in the application field of the basal cell carcinoma. The invention provides the application of nano hydroxyapatite in preparing the drugs for preventing or treating basal cell carcinoma, widens the application field of nano hydroxyapatite, provides a basis and a direction for the development of the drugs for preventing or treating basal cell carcinoma, and has important significance.

Drawings

Fig. 1 shows nano-hydroxyapatite with different forms prepared by different methods. A is granular, B is needle-shaped, and C is columnar.

FIG. 2 shows the cell growth morphology (200) observed under an inverted microscope. The morphology of TE354.T, HaCat and HSF cells is observed under an inverted phase contrast microscope, the TE354.T, HSF cells all grow adherent to the wall in a single layer, the TE354.T cells grow in a polygonal shape and in a cluster shape under the normal condition, the HaCat cells grow in an epithelial shape under the normal condition, the HSF cells grow in a fusiform shape under the normal condition, and 2-4 bulges with different lengths extending outwards can be seen. (a) TE354.T cells; (b) HaCat cells; (c) HSF cells.

FIG. 3 shows the effect of different concentrations of nHAP on the proliferative capacity of basal cell carcinoma cell line TE354.T tumor cells. Compared with the control group, higher concentrations of nHAP material (60, 120, 240 and 480 mu g/mL) in the experimental group have the effect of inhibiting proliferation of T cells after being acted on TE354, 48 hours, and the difference has statistical significance (P is less than 0.05). And there is a gradually increasing tendency for TE354.T cell proliferation inhibition as the material concentration increases. The above results indicate that higher concentrations of nHAP have the effect of inhibiting TE354. The lower concentration (30. mu.g/mL) was not significantly different (P >0.05) compared to the control group, and had no significant effect on cell proliferation.

FIG. 4 shows the effect of different concentrations of nHAP on the proliferative capacity of normal human keratinocytes. Compared with a control group, the experimental groups with the concentration of nHAP materials (30, 60, 120, 240 and 480 mu g/mL) do not obviously inhibit or promote cell proliferation after acting on HaCat cells for 48h, and the difference is not statistically significant (P is greater than 0.05). Indicating that the nHAP material at each concentration had no significant effect on the proliferation of HaCat cells.

FIG. 5 shows the effect of different concentrations of nHAP on the proliferative capacity of normal human fibroblasts. Compared with the control group, the nHAP materials (30, 60, 240 and 480 mu g/mL) in most experimental groups have the effect of promoting the proliferation of the HSF cells after being acted on the HSF cells for 48 hours, and the difference has statistical significance (P < 0.05). The individual concentration (120 mug/mL) in the experimental group has no significant difference (P >0.05) compared with the control group, and the concentration does not obviously promote or inhibit the proliferation of the HSF cells.

Figure 6 shows the effect of nHAP on TE354.T cell apoptosis (total apoptosis). Compared with the control group, after the nHAP material in each concentration nHAP experimental group acts on TE354.T cells for 48 hours, the proportion of the cells in total apoptosis (early and late apoptosis) is obviously increased, and the difference has statistical significance (P)<0.01). Shows that higher concentration nHAP (60, 120, 240 and 480 mu g/mL) can promote apoptosis of basal cell carcinoma cell strain TE354.T tumor cell, (b), (c)^**P<0.01，^***P<0.001，^****P<0.0001)。

FIG. 7 shows nHAP vs. TE 354T cell cycle (G)₂Period) of the vehicle. Higher concentrations of nHAP (60, 120, 240 and 480. mu.g/mL) in the experimental groups compared to the control group, were exposed to TE354.T cells for 48h, at cell cycle G₂The proportion of cells in the phase is significantly reduced, and the difference is statistically significant (P)<0.05). Indicating that higher concentrations of nHAP may inhibit cell proliferation by affecting the cell cycle of te354.t tumor cells. (^*P<0.05，^**P<0.01)。

FIG. 8 shows a comparison between the tumor growth of the nude mice in the control group. It can be seen that the tumor size of the mice bearing tumors of basal cell carcinoma which had not been treated with nHAP was significantly increased at the end of the observation period.

FIG. 9 shows the comparison between the tumor growth of the nude mice in the experimental group. After the observation period, the tumor growth of the mice with the basal cell carcinoma negative tumor treated by 25mg/kg nHAP is obviously inhibited.

FIG. 10 shows the comparison of tumor tissues after sacrifice of control and experimental nude mice. Therefore, the tumor volume of the experimental group is obviously smaller than that of the control group, and the in vivo experiment further proves that the nHAP has obvious inhibition effect on the basal cell carcinoma.

Detailed Description

The invention provides application of nano hydroxyapatite in preparation of a medicine for preventing or treating basal cell carcinoma.

Particularly, the invention also determines that the nano hydroxyapatite is the nano hydroxyapatite with the particle size of 20-80 nm. The inventor finds that when the particle size of the nano-hydroxyapatite is too small, the nano-hydroxyapatite can be quickly eliminated in blood circulation and tumor tissues or reach peripheral tissues, and the effect and the safety are poor; when the particle size is too large, the permeability in tumor tissues is poor, and the tumor-inhibiting effect is affected. According to the invention, the nano-hydroxyapatite with the particle size of 20-80 nm is adopted, so that the penetration depth of the material in a tumor is ensured, the endocytosis of the material by tumor cells is facilitated, meanwhile, the nano-particles with the particle size have a tumor tissue targeting effect through an EPR effect, the deposition of the nano-hydroxyapatite in a non-tumor tissue is avoided, and the biological safety is ensured.

In order to obtain suitable nano-hydroxyapatite, the inventor adopts Ca (NO)₃)₂·4H₂O and (NH)₄)₂HPO₄The preparation method of the nano hydroxyapatite comprises the following steps:

in particular, in the above application, the preparation method of the nano hydroxyapatite comprises the following steps:

a. 0.5ml/l Ca (NO) is prepared₃)₂·4H₂O160 ml and 0.5mol/l (NH4)₂HPO₄96ml of Ca (NO) was stirred continuously at 1000r/min with magnetic stirring₃)₂·4H₂O solution, at room temperature (NH4)₂HPO₄Slowly and uniformly dropping Ca (NO) into the solution at the speed of 4ml/min₃)₂·4H₂In O solution, ammonia NH is used at any time during the reaction₃·H₂Adjusting the pH value of the reaction system to 10;

b. treat (NH4)₂HPO₄After the solution is added dropwise, standing and aging for 24h at normal temperature, repeatedly centrifuging and washing the precipitate for 6 times by ultrapure water until the pH value of the slurry is washed to be neutral, and drying for 10h in a constant-temperature drying oven at 60 ℃ to prepare granular nano-hydroxyapatite;

or a, preparing 0.5ml/l Ca (NO)₃)₂·4H₂O160 ml and 0.5mol/l (NH4)₂HPO₄96ml of Ca (NO) was stirred continuously at 1000r/min with magnetic stirring₃)₂·4H₂O solution, at room temperature (NH4)₂HPO₄Slowly and uniformly dropping Ca (NO) into the solution at the speed of 4ml/min₃)₂·4H₂In O solution, ammonia NH is used at any time during the reaction₃·H₂Adjusting the pH value of the reaction system to 10;

b. treat (NH4)₂HPO₄After the solution is dropwise added, stirring and aging for 2h at normal temperature, transferring the obtained milky white slurry into a hydrothermal reaction kettle, carrying out hydrothermal reaction for 12h at 150 ℃, naturally cooling, then discarding supernatant, repeatedly carrying out centrifugal washing and precipitation for 6 times by using ultrapure water until the pH value of the slurry is washed to be neutral, and drying in a constant-temperature drying oven at 60 ℃ for 10h to prepare the needle-like nano-hydroxyapatite;

or a, preparing 0.5ml/l Ca (NO)₃)₂·4H₂O160 ml and 0.5mol/l (NH4)₂HPO₄96ml of Ca (NO) was stirred continuously at 1000r/min with magnetic stirring₃)₂·4H₂O solution, at room temperature (NH4)₂HPO₄Slowly and uniformly dropping Ca (NO) into the solution at the speed of 4ml/min₃)₂·4H₂In O solution, ammonia NH is used at any time during the reaction₃·H₂Adjusting the pH value of the reaction system to 10;

b. treat (NH4)₂HPO₄After the solution is dropwise added, stirring and aging are carried out for 24h at normal temperature, ultrapure water is repeatedly centrifuged, washed and precipitated for 6 times until the pH value of the slurry is washed to be neutral, hydrothermal reaction is carried out for 12h at 150 ℃, and then drying is carried out in a constant-temperature drying oven for 10h at 60 ℃ to prepare the columnar nano-hydroxyapatite.

The basal cell carcinoma cell line adopted by the in vivo and in vitro experiments is a mature and stable basal cell carcinoma cell line TE354. T.

The invention adopts experiments of cell proliferation, cell cycle, apoptosis and the like to evaluate the influence of the nano-hydroxyapatite on the phenotype of the basal cell carcinoma cell strain, and the evaluation index is relatively comprehensive.

The invention successfully establishes the basal cell carcinoma animal model, and further verifies the feasibility of the application of the nano-hydroxyapatite in the research field of basal cell carcinoma through animal experiments. The invention establishes different nano-hydroxyapatite concentrations (30, 60, 120, 240 and 480 mu g/mL) in a body experiment, finds out that when the optimal application concentration of the nano-hydroxyapatite in the application field of basal cell carcinoma is 120 mu g/mL, the effects of inhibiting the proliferation of basal cell carcinoma tumor cells and promoting the apoptosis of the tumor cells are strongest, and has the most obvious influence on the basal cell carcinoma tumor cell cycle; the in vivo application dose of 25mg/kg body weight shows significant anti-tumor effect.

The invention also solves the safety problem of the application of the nano-hydroxyapatite in basal cell carcinoma, and finds that the nano-material can promote the proliferation of the human normal fibroblasts while having no obvious influence on the human keratinocytes through in vitro experiments on the human normal fibroblasts and the application of the nano-hydroxyapatite in the human keratinocytes. The nano material of the invention has biological safety and potential repairable effect on tissues. And further verifying that the deposition of the nano material does not occur in the important visceral organs of the mouse after the treatment is finished and each important visceral organ does not have obvious damage through in vivo animal experiments, and further verifying the targeting property and the safety of the nano hydroxyapatite disclosed by the invention.

The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.