CN111973576A

CN111973576A - Curcumin nano dispersion film and preparation method and application thereof

Info

Publication number: CN111973576A
Application number: CN202011004216.7A
Authority: CN
Inventors: 不公告发明人
Original assignee: Suzhou Quansuo Nanotechnology Co ltd
Current assignee: Suzhou Quansuo Nanotechnology Co ltd
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2020-11-24

Abstract

The invention provides a preparation method of a curcumin nano dispersion film, which comprises the following steps: dissolving curcumin active body and gelatin in a solvent; the curcumin active body is any one or more of curcumin, curcumin salt, curcumin co-crystal or curcumin derivatives; preparing a curcumin nano dispersion film intermediate by using an electrostatic spinning method; drying the intermediate of the curcumin nano dispersion membrane, incubating, washing and freeze-drying. The curcumin dispersion film prepared by the invention avoids the risk that the medicine carried by the dispersion film is damaged due to ultraviolet irradiation.

Description

Curcumin nano dispersion film and preparation method and application thereof

Technical Field

The invention relates to a chemical method, in particular to a curcumin nano dispersion film, a preparation method and application thereof.

Background

Tumors, especially malignant tumors, have become one of the most dangerous diseases threatening human health at present. Surgery to physically remove tumors remains the primary treatment for tumor-related diseases, especially benign tumors and early stage malignant tumors. However, residual tumor cells remain after surgical resection with a risk of recurrence and metastasis. The combination of surgery with chemotherapy, radiation therapy or immunotherapy can effectively reduce these hazards. For example, cytoreductive surgery (CRS) and intraperitoneal hyperthermia chemotherapy (HIPEC) are now considered standard treatments for certain appendiceal and peritoneal mesotheliomas, and have also shown success in pancreatic cancer treatment. However, radiation and side effects of radiotherapy and chemotherapy drugs (e.g. gastrointestinal problems, skin rashes, hair loss and lack of energy) cause non-specific damage, thereby reducing the quality of life of the patient and limiting the therapeutic dose. On the other hand, the high costs of chemotherapy and immunotherapy represent an unbearable economic burden for most patients, especially in developing countries.

Curcumin (chemical formula: C)₂₁H₂₀O₆(1E, 6E) -1, 7-bis (4-hydroxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione) has been identified as the main biologically active compound of turmeric. Curcumin has been shown to be effective in inducing apoptosis in various types of cancer cells. Curcumin, in addition to having excellent efficacy, is widely available and has been shown to have extremely low toxicity. In clinical trials, an adult oral safe dose of curcumin can be as high as 8000 mg per day. Although curcumin is safe, inexpensive and has a remarkable pharmacological action in vitro, it is not effective in the actual clinical treatment of tumors. This is mainly because curcumin is not easily absorbed by the human body, and is unstable itself and is easily and rapidly explained by the human body. Therefore, the curcumin bioavailability is effectively improved, an efficient, low-toxicity and low-cost tumor treatment means is provided, the survival rate and the life quality of patients are improved, and the economic burden of the patients and the society is reduced.

Currently, there is a limit to the use of curcumin for the preparation of formulations for the treatment of tumors. Patent CN110279677A discloses a method for preparing curcumin sustained release membrane, but this method needs ultraviolet light to irradiate the drug-loaded fiber membrane, and the irradiation of ultraviolet light increases the risk of damaging the loaded drug.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a method for preparing a curcumin nanodispersed film, which is used to solve the problems of easy degradation, poor solubility, low bioavailability, etc. in the prior art.

To achieve the above and other related objects, the present invention provides a method for preparing a curcumin nanodispersion film, comprising the steps of:

(1) dissolving curcumin active body and gelatin in a solvent; the curcumin active body is any one or more of curcumin, curcumin salt, curcumin co-crystal or curcumin derivatives;

(2) preparing a curcumin nano dispersion film intermediate by using an electrostatic spinning method;

(3) drying the intermediate of the curcumin nano dispersion membrane, incubating, washing and freeze-drying.

Naturally occurring curcuminoids include keto and enol forms of curcumin (1, 7-bis (4-hydroxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione), demethoxycurcumin (1- (4-hydroxyphenyl) -7- (4-hydroxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione), and bisdemethoxycurcumin (1, 7-bis (4-hydroxyphenyl) -1, 6-heptadiene-3, 5-dione), and any combination thereof.

Further, the curcumin active is selected from compounds of 1, 7-bis (4-hydroxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione or 1, 7-bis (4-hydroxyphenyl) hept-4-en-3-one backbone, wherein the phenyl groups may independently carry one or more alkoxy residues, especially one methoxy residue in position 3.

A curcumin salt, a curcumin co-crystal, or a curcumin derivative. The curcumin derivative can be curcumin copper salt dimer, demethoxycurcumin, bisdemethoxycurcumin and other chemical substances with therapeutic effect.

Further, the mass ratio of the curcumin active body to the gelatin in the step (1) is 1: 10-15.

Further, the solvent is an organic solvent, such as trifluoroethanol.

Further, the final concentration of curcumin in the solvent is 9-11% wt/v.

Furthermore, the curcumin and the gelatin are dissolved in the solvent and can be stirred, so that the mixed solution is more uniformly mixed.

Further, the technological parameters of the electrostatic spinning method are as follows: the voltage is 15-17kV, the speed of the injection pump is 1-2.5ml/h, and the distance between the needle tip and the receiver is 10-20 cm.

Further, the drying method comprises the step of placing the curcumin nano dispersion film intermediate in a vacuum drying oven at the temperature of 3-4 ℃ for 20-28 hours.

Further, the incubation conditions are: placing the curcumin nano dispersion membrane intermediate in an organic solvent, and incubating for 20-28 hours at 20-28 ℃.

Further, the freeze-drying refers to that the curcumin nano dispersion film intermediate is placed at the temperature of-35 to-45 ℃ for 20 to 28 hours.

Another aspect of the present invention provides a curcumin nanodispersion film prepared by the above method.

The invention also provides application of the curcumin nano dispersion film in preparing a tumor treatment medicament.

Further, the tumor is selected from any one of nasopharyngeal tumor, oral tumor, tongue tumor, throat tumor, esophageal tumor, lung tumor, liver tumor, kidney tumor, Gongmen tumor, stomach tumor, pylorus tumor, membrane gland tumor, pancreas tumor, intestine tumor, Guardian tumor, prostate tumor, cervix tumor, uterus tumor, ovary tumor, lip tumor, skin tumor, breast tumor, bone tumor, sarcoma, malignant brain glioma, Ewing's tumor, Hodgkin's disease, non-Hodgkin's disease.

As described above, the method for preparing the curcumin nano-dispersion film of the present invention has the following beneficial effects:

(1) the preparation method is simple and rapid, the flexibility of technological parameters is higher, and the nano-fiber membrane loaded with the curcumin dispersion can be produced in a low-cost large-scale manner;

(2) the prepared nano-fiber is swelled but not dissolved after contacting with water, and drug molecules can be slowly released from the nano-fiber;

(3) the cross-linked drug-loaded membrane has certain elasticity, and the prepared gelatin nanofiber membrane has no biotoxicity and antigenicity and is degradable in organisms, so that the gelatin nanofiber membrane is a good in-situ drug-delivery matrix material;

(4) the method avoids ultraviolet irradiation, and increases the risk of damage to the loaded medicine.

Drawings

Fig. 1 shows a process for preparing a curcumin dispersion film.

Fig. 2 shows a picture of a curcumin dispersion film as a real object.

Fig. 3 curcumin nanodispersed film scanning electron microscope image.

Fig. 4 curcumin nanodispersion film fluorescence microscopy images.

FIG. 5X-ray crystal diffraction pattern of curcumin nanodispersion film.

FIG. 6 is the time-varying curcumin concentration released from the curcumin nano-dispersion film in vitro.

Fig. 7 effect of curcumin nanodispersion membrane on normal stem cells.

Figure 8 the effect of curcumin nanodisperse membrane leachate on the expression of proteins associated with pathways.

Fig. 9 shows the killing effect of the curcumin nano dispersion membrane leachate on pancreatic cancer cells.

FIG. 10 shows the effect of curcumin nanodispersion membrane leachate on tumor cell clusters under fluorescence microscope observation (right side is bright field, left side is green fluorescence living cell tracer to observe the growth morphology of tumor mass under fluorescence microscope; upper row is control, and lower row is treatment group with flavin-loaded nanodispersion membrane).

Fig. 11 is a fluorescent microscope to observe the effect of curcumin nanodispersion membrane leachate on tumor cell clusters (staining of cell clusters after administration with two dyes, red for dead cells and green for live cells, upper row for control group with increased cell clusters (number of cells) before and after administration and substantially all live cells (green), lower row for administration with decreased cell clusters (number of cells) after administration and substantially all dead cells (red)).

Fig. 12a is a photograph showing mouse tumor tissue real images after 14 days of curcumin nanodispersion membrane treatment.

Figure 12b in vivo inhibitory effect of curcumin nanodispersion membrane on tumor growth in mice (left panel is tumor tissue volume change over time, right panel is tumor volume statistics on day 28 of tumor growth (day 14 of treatment)).

Fig. 13 shows BrdU immunohistochemical staining results of mouse tumor tissue sections after curcumin nanodispersion membrane treatment (the upper row is blank gelatin membrane group, the lower row is curcumin nanodispersion membrane treatment group, the right panel is partial area enlargement of the left panel, the stronger BrdU staining indicates stronger cell proliferation activity, and the weaker BrdU staining signal in sections after curcumin nanodispersion membrane treatment indicates decreased tumor cell proliferation activity).

FIG. 14 shows the p-STAT3 immunohistochemical staining results of mouse tumor tissue slices after curcumin nanodispersion membrane treatment (the upper row is a blank gelatin membrane group, the lower row is a curcumin nanodispersion membrane treatment group, and the p-STAT3 staining signals in the slices after curcumin nanodispersion membrane treatment are weaker, which indicates that the p-STAT3 expression level in tumor cells is reduced).

Fig. 15 shows the result of BIP immunohistochemical staining of mouse tumor tissue sections after curcumin nanodispersion membrane treatment (the upper row is blank gelatin membrane group, the lower row is curcumin nanodispersion membrane treatment group, and BIP staining signals in sections after curcumin nanodispersion membrane treatment are stronger, which indicates that BIP expression level of tumor cells is increased).

Detailed Description

In order to facilitate understanding of the present invention, the raw material drugs of the present invention will now be further described.

Curcumin:

the name of Chinese: curcumin (curcumin)

The foreign language name: curcumin

CAS number: 458377

The chemical formula is as follows: c₂₁H₂₀O₆(1E, 6E) -1, 7-bis (4-hydroxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione

Molecular weight: 368.39

Density l.307g/cm³

Melting Point 183 deg.C

Boiling point 593.2 deg.C (760 mmHg lower)

Chemical structural formula:

gelatin:

gelatin is a partially hydrolyzed product of collagen, and is colorless to pale yellow solid, in the form of powder, tablet or block. Has luster, no smell and no taste. The relative molecular mass is about 50000-100000. The relative density is 1.3 to 1.4. Gelatin is widely used in tissue engineering and drug delivery systems because of its high biocompatibility, biodegradability, no generation of other by-products after in vivo degradation, no immunogenicity, blood compatibility, and the same composition and biological properties as collagen.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art.

Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

Experimental materials:

(1) human mesenchymal stem cells (cell bank of chinese academy of sciences);

(2) DMEM high glucose medium (seimer feishell science (china) ltd);

(3) mouse pancreatic cancer cells 399 (boo Kong doctor heft);

(4) human pancreatic cancer cell PANC-1 (Chinese academy of sciences cell bank);

(5) human pancreatic cancer cell T3M4 (shanghai source, biotechnology limited);

(6) human pancreatic cancer cell MIA PaCa-2 (cell bank of Chinese academy of sciences).

Example 1: preparation of curcumin nano dispersion film

1.5g of gelatin and 0.1g of curcumin are dissolved in 10ml of trifluoroethanol at normal temperature, and stirred for 2 hours to ensure that the curcumin and the gelatin are completely and uniformly mixed and dissolved in the trifluoroethanol. The solution was filled into a 10ml syringe and electrospun using a 0.4 mm-diameter injection head, a feed rate of 1.5ml/h, and a direct current voltage of 15kV to obtain a nanofiber membrane containing curcumin. And (2) placing the prepared membrane in a vacuum drying oven at 4 ℃ for 24 hours, drying to remove residual organic solvent, then placing the prepared nanofiber membrane in a mixed solution of 25% glutaraldehyde and 1% ethanol, incubating for 28 hours at 20 ℃, washing the treated curcumin nano dispersion membrane with ultrapure water for 2 times, and freeze-drying for 24 hours at-40 ℃ to prepare the finished curcumin nano dispersion membrane (as shown in figure 1).

A common camera shoots the curcumin dispersion film as shown in figure 2, and a Scanning Electron Microscope (SEM) proves that the fiber is smooth and has no visible crystal, which indicates that curcumin is converted from a crystal into a solid dispersion and is uniformly distributed in the gelatin nanofiber filament (as shown in figure 3). The electrostatic spinning was spun on a glass slide and examined with a fluorescence microscope, and since curcumin itself has fluorescence (green), it can exhibit green fluorescence under the fluorescence microscope, confirming that curcumin is present in the nanofiber (see fig. 4). X-ray crystal diffraction (XRD) confirmed that the characteristic peak of curcumin bulk drug (crystal) disappeared in the drug-loaded nanofiber membrane, confirming that curcumin had been converted from crystal to amorphous state and existed in the nanofiber membrane (see fig. 5).

Example 2: in vitro release experiment of curcumin nano dispersion film

1.5 cm × 1.5 cm curcumin nanodispersion membrane was immersed in 50 ml phosphate buffered saline (PBS, pH 7.4, 37 ℃). Curcumin content was determined using high performance liquid chromatography with 200 microliters of solution at intervals, while an equal volume of PBS was added back to the original system to maintain a total volume of 50 milliliters.

The detection result is shown in fig. 6, which shows that curcumin can be dissolved into the solution from the nano-dispersion film, and the curcumin concentration in the buffer solution can still reach more than about 50 μ M after the curcumin nano-dispersion film is immersed for 120 hours.

Example 3: biocompatibility of curcumin nano dispersion film

We further evaluated the biocompatibility of the drug loaded material. The human mesenchymal stem cells and the drug-loaded material are co-cultured in vitro for 7 days. The drug-loaded material was found to have no toxic effect on normal stem cells by LDH (see figure 7); the result shows that the curcumin dispersion film has good compatibility with human tissues and meets the medicinal standard.

Example 4: killing effect of curcumin nano dispersion film on in-vitro tumor cells

a) Mouse pancreatic cancer cells 399 were co-cultured with either blank gelatin membrane or curcumin nanodispersion membrane. After 2 days, total protein was prepared by adding protease inhibitor (Shanghai Bin Yun Tian Biotechnology Co., Ltd.) and phosphatase inhibitor (Shanghai Bin Yun Tian Biotechnology Co., Ltd.) to the diluted cell lysis buffer (Shanghai Bin Yun Tian Biotechnology Co., Ltd.). The expression levels of BiP, p-PERK, p-STAT3, p-elF2a and cleared Caspase-3 were measured using antibodies (anti-BiP antibody (3177), anti-p-PERK (Thr 980) antibody (3179), anti-p-eIF 2a (Ser 51) antibody (9721) and anti-p-STAT 3 antibody (9145)) of Cell Signaling Technology (Seikagaku Bioagent Ltd.) and GAPDH was used as an internal reference protein.

The results are shown in figure 8, and show that the expression of clear Caspase-3, p-PERK, BIP and p-elF2a is up-regulated and the expression of p-STAT3 is reduced in mouse pancreatic cancer cells after the co-culture of the curcumin nano-disperse membrane, thereby indicating that the curcumin nano-disperse membrane can trigger endoplasmic reticulum stress response, namely a Bip/p-PERK/p-elF2a pathway and inhibit the phosphorylation of STAT3 by increasing the generation of active oxygen in pancreatic cancer cells, so as to induce the apoptosis of pancreatic cancer cells.

b) Immersing the curcumin nano dispersion membrane into a DMEM high glucose medium containing 10% fetal calf serum, 100U/ml penicillin and 100 mug/ml streptomycin, and obtaining a culture medium containing 5 muM curcumin after 72 hours. Mouse pancreatic cancer cell 399, human pancreatic cancer cell PANC-1, T3M4 and MIA PaCa-2 were cultured in the curcumin-containing medium at 37 ℃ and 5% CO, respectively₂The culture was carried out under an atmosphere. After 7 days the cells were fixed using methanol, stained with 10% crystal violet and cell clone counts were performed under the microscope.

As shown in FIG. 9, it was revealed that the number of cell clones formed by the mouse pancreatic cancer cells 399 in the culture medium containing curcumin nanodispersion membrane-leached curcumin was reduced by about 60% as compared with the normal culture medium (cell clone number: 75.2. + -. 7.7 (DMEM), 29.6. + -. 6.5 (curcumin nanodispersion membrane leachate)). Similar results are shown on human pancreatic cancer cells T3M4, MIA PaCa-2 and PANC-1 by the curcumin nano dispersion membrane leachate, which indicates that the curcumin nano dispersion membrane leachate can effectively kill mouse and human pancreatic cancer cells.

c) 2 x 10 to⁵The cultured human pancreatic cancer cells T3M4, MIA PaCa-2 and PANC-1 and murine pancreatic cancer cells were inoculated into 10ml of pre-cooled Matrigel solution, and gently pipetted in an ice bath until the cells were well mixed with the Matrigel solution. Then one drop of 1.5ml containing-3 x 10⁴Matrigel solution of cells was added to 6-well cell culture plates. After the Matrigel was solidified at 37 ℃ for 1 hour, 3 ml of DMEM was added to each well and cultured at 37 ℃ for 24 hours, and then a blank gelatin film or a curcumin nanodispersion film was immersed in the medium. After 14 days, the cell clusters were stained with LIVE/DEAD kit (seimer feishell technologies (china) ltd) and imaged by fluorescence microscope to observe DEAD and LIVE cells.

The results are shown in fig. 10 and 11, compared with a blank gelatin film, the volume of the pancreatic cancer cell cluster in the curcumin nano dispersion film administration group is obviously reduced, and most of cells in the cell cluster are dead cells, which indicates that the curcumin nano dispersion film not only has a killing effect on cultured adherent pancreatic cancer cells, but also has an effective proliferation inhibition effect on clustered pancreatic cancer cell clusters.

Example 5: killing effect of curcumin nano dispersion film on tumor cells in mice

a) Will be 1X 10 in total⁶A number of 399 mouse pancreatic cancer cells were subcutaneously transplanted to both sides of the back of 8-week-old wild-type mice, after which tumor volumes were measured weekly. After 14 days, surgery was performed with curcumin nanodispersion membrane and blank material placed between the subcutaneously inoculated tumor and muscle, tightly against the tumor.

The results are shown in fig. 12a and 12b, which show that the tumor volume growth rate is obviously reduced after adding the curcumin nano-dispersion film compared with that after adding the gelatin film without curcumin, the tumor volume is only about one fifth of that of the blank gelatin film control group after adding the curcumin nano-dispersion film for 14 days (28 days for tumor inoculation), and the curcumin nano-dispersion film effectively inhibits the further growth of the tumor in the mouse.

b) The tumor tissue sections were deparaffinized and rehydrated and antigen retrieval was performed using citrate buffer or proteinase K (shanghai bi yunnan biotechnology limited). After blocking endogenous peroxidase and non-specific binding, sections were incubated with anti-BiP, anti-BrdU, anti-p-STAT 3 antibodies, respectively. And labeled with secondary antibody labeled with HRP, and developed with DAB staining working solution. Then counterstained with hematoxylin, dehydrated and fixed. The result shows that BrdU staining signals in mouse tumor tissue slices are obviously weakened after curcumin nano dispersion membrane treatment, which indicates that the tumor cell proliferation activity is reduced; meanwhile, the staining signal of p-STAT3 is weaker, and the expression level of p-STAT3 in tumor cells is reduced; the BIP staining signal is strong, the BIP expression level of the tumor cells is increased, and the result shows that the curcumin nano dispersion membrane can effectively inhibit the tumor proliferation activity by triggering the endoplasmic reticulum stress response in the tumor tissues, namely BIP/p-PERK/p-elF2a pathway and inhibiting the phosphorylation of STAT 3.

The results are shown in fig. 13-15, and immunohistochemistry also verifies that the proliferative cell ginger-yellow material group is obviously reduced, endoplasmic reticulum stress is obviously increased, and p-STAT3 is obviously inhibited.

Example 6: preparation of curcumin nano dispersion film and in-vitro release and activity experiment thereof

1.1g of gelatin and 0.11g of curcumin are dissolved in 10ml of trifluoroethanol at normal temperature, and stirred for 2 hours to ensure that the curcumin and the gelatin are completely and uniformly mixed and dissolved in the trifluoroethanol. The solution is filled into a 10ml syringe, and electrostatic spinning is carried out by using an injection head with the caliber of 0.4mm, the feeding rate of 1ml/h and the direct current voltage of 16kV to obtain the nano-fiber membrane containing curcumin. And (3) placing the prepared membrane in a vacuum drying oven at 4 ℃ for 28 hours, drying to remove residual organic solvent, then placing the prepared nanofiber membrane in a mixed solution of 25% glutaraldehyde and 1% ethanol, incubating for 24 hours at 24 ℃, washing the treated curcumin nano dispersion membrane with ultrapure water for 2 times, and freeze-drying for 20 hours at-45 ℃ to prepare the curcumin nano dispersion membrane.

A 1.5 cm x 1.5 cm film of the finished curcumin nanodispersion prepared was cut out and immersed in 50 ml of phosphate buffered saline (PBS, pH 7.4, 37 ℃). Curcumin content was determined using high performance liquid chromatography with 200 microliters of solution at intervals, while an equal volume of PBS was added back to the original system to maintain a total volume of 50 milliliters. The determination result shows that the curcumin can be dissolved into the solution from the nano-dispersion film, and the curcumin concentration in the buffer solution can still reach more than about 50 mu M after the curcumin nano-dispersion film is immersed for 120 hours. Referring to the method in example 3, the curcumin nanodispersion membrane obtained in this example was examined for its killing effect on tumor cells in vitro. As a result, similar to FIG. 8, the expression of clear Caspase-3, p-PERK, BIP and p-elF2a in mouse pancreatic cancer cells was up-regulated, while the expression of p-STAT3 was reduced after the co-culture of curcumin nanodispersed membranes. Thus, the curcumin nanodispersion membrane can trigger endoplasmic reticulum stress response, namely BIP/p-PERK/p-elF2a pathway and inhibit phosphorylation of STAT3 by increasing the generation of active oxygen in pancreatic cancer cells, thereby inducing pancreatic cancer cell apoptosis. Similar to fig. 9, the curcumin nanodispersion membrane leachate of the present example can effectively kill pancreatic cancer cells of mice and humans. Similar to fig. 10 and 11, the curcumin nanodispersion membrane of the present example has not only a killing effect on cultured adherent pancreatic cancer cells but also an effective proliferation-inhibiting effect on clustered pancreatic cancer cell clusters. In addition, the curcumin nanodispersion membrane of this example was examined for its killing effect on tumor cells in mice by referring to the method in example 5. The results show that the curcumin nanodispersion film of the present example effectively inhibited further tumor growth in mice.

Example 7: preparation of curcumin nano dispersion film and in-vitro release and activity experiment thereof

1.35g of gelatin and 0.09g of curcumin were dissolved in 10ml of trifluoroethanol at normal temperature, and stirred for 2 hours to completely and uniformly mix and dissolve the curcumin and the gelatin in the trifluoroethanol. The solution was filled into a 10ml syringe and subjected to electrostatic spinning using a 0.4 mm-diameter injection head, a 2.5ml/h feed rate, 17kV dc voltage to obtain a nanofiber membrane containing curcumin. And (2) placing the prepared membrane in a vacuum drying oven at 4 ℃ for 20 hours, drying to remove residual organic solvent, then placing the prepared nanofiber membrane in a mixed solution of 25% glutaraldehyde and 1% ethanol, incubating for 20 hours at 28 ℃, washing the treated curcumin nano dispersion membrane with ultrapure water for 2 times, and freeze-drying for 20 hours at-35 ℃ to prepare the curcumin nano dispersion membrane.

A 1.5 cm x 1.5 cm film of the finished curcumin nanodispersion prepared was cut out and immersed in 50 ml of phosphate buffered saline (PBS, pH 7.4, 37 ℃). Curcumin content was determined using high performance liquid chromatography with 200 microliters of solution at intervals, while an equal volume of PBS was added back to the original system to maintain a total volume of 50 milliliters. The determination result shows that the curcumin can be dissolved into the solution from the nano-dispersion film, and the curcumin concentration in the buffer solution can still reach more than about 50 mu M after the curcumin nano-dispersion film is immersed for 120 hours. Referring to the method in example 3, the curcumin nanodispersion membrane obtained in this example was examined for its killing effect on tumor cells in vitro. The results show that similar to FIG. 8, after the co-culture of the curcumin nanodispersion membrane, the expression of cleared Caspase-3, p-PERK, BIP and p-elF2a in mouse pancreatic cancer cells is up-regulated, and the expression of p-STAT3 is reduced, thereby indicating that the curcumin nanodispersion membrane can trigger endoplasmic reticulum stress response, namely BIP/p-PERK/p-elF2a pathway and inhibit the phosphorylation of STAT3 by increasing the generation of active oxygen in pancreatic cancer cells, so as to induce pancreatic cancer cell apoptosis. Similar to fig. 9, the curcumin nanodispersion membrane leachate of the present example can effectively kill pancreatic cancer cells of mice and humans. Similar to fig. 10 and 11, the curcumin nanodispersion membrane of the present example has not only a killing effect on cultured adherent pancreatic cancer cells but also an effective proliferation-inhibiting effect on clustered pancreatic cancer cell clusters. In addition, the curcumin nanodispersion membrane of this example was examined for its killing effect on tumor cells in mice by referring to the method in example 5. The results show that the curcumin nanodispersion film of the present example effectively inhibited further tumor growth in mice.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims

1. A method for preparing a curcumin nanodispersed film, comprising the steps of:

2. The method according to claim 1, wherein the mass ratio of the curcumin active substance to the gelatin in the step (1) is 1:10 to 1: 15.

3. The method as claimed in claim 1, wherein the curcumin active agent is selected from compounds of 1, 7-bis (4-hydroxy-3-methoxyphenyl) -1, 6-heptadiene-3, 5-dione or 1, 7-bis (4-hydroxyphenyl) hept-4-en-3-one backbone, wherein the phenyl groups may independently carry one or more alkoxy residues.

4. The method as claimed in claim 1, wherein the final concentration of curcumin in the solvent is 9 to 11% wt/v.

5. The method according to claim 1, wherein the process parameters of the electrospinning method are: the voltage is 15 to 17kV, the speed of the syringe pump is 1 to 2.5ml/h, and the distance between the needle tip and the receptor is 10 to 20 cm.

6. The method according to claim 1, characterized in that it further comprises any one or several of the following technical features:

a) the drying method comprises the steps of placing the curcumin nano dispersion film intermediate in a vacuum drying oven at 3-4 ℃ for 20-28 hours;

b) the incubation conditions were: placing the curcumin nano dispersion film intermediate in an organic solvent, and incubating for 20-28 hours at 20-28 ℃.

7. The method as claimed in claim 1, wherein the freeze-drying is performed by placing the curcumin nanodispersed membrane intermediate at a temperature of-35 to-45 ℃ for 20 to 28 hours.

8. A curcumin nanodispersed film prepared by the preparation method as claimed in any one of claims 1 to 7.

9. The use of curcumin nanodispersion membrane as claimed in claim 8 in the preparation of a medicament for the treatment of tumor.

10. Use according to claim 9, wherein the tumor is selected from any of nasopharyngeal tumors, oral tumors, tongue tumors, laryngeal tumors, esophageal tumors, lung tumors, liver tumors, kidney tumors, gongmen tumors, stomach tumors, pyloric tumors, membranous tumors, pancreatic tumors, intestinal tumors, breast tumors, prostate tumors, cervical tumors, uterine tumors, ovarian tumors, lip tumors, skin tumors, breast tumors, bone tumors, sarcomas, malignant brain gliomas, Ewing's tumors, Hodgkin's disease, non-Hodgkin's disease.