CN107303286B

CN107303286B - Gelatin hybrid compound and preparation method and application thereof

Info

Publication number: CN107303286B
Application number: CN201610231684.5A
Authority: CN
Inventors: 刘昌胜; 李玉林; 王国营; 王金鹅; 袁媛; 孙懿
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2016-04-14
Filing date: 2016-04-14
Publication date: 2022-09-30
Anticipated expiration: 2036-04-14
Also published as: CN107303286A

Abstract

The invention relates to a gelatin hybrid compound and a preparation method and application thereof. Specifically, the invention discloses a compound, which comprises: nanoparticles and biodegradable gelatin, said gelatin being cross-linked to the surface of said nanoparticles; and the particle size of the compound is 20nm-500 mu m. The invention also discloses a preparation method and application of the compound. The compound has the excellent performances of high drug loading, high tumor permeability and gradual release in tumor cells. The preparation method has simple and controllable process and is convenient for large-scale industrialization. The compound can be widely applied to the diagnosis and treatment process of various diseases.

Description

Gelatin hybrid compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a gelatin hybrid compound and a preparation method and application thereof.

Background

With the increasing environmental pollution caused by modern industry, cancer has become a major problem to human health, and more patients die because of the incurability of cancer. Small molecule anticancer drugs are commonly used anticancer drugs, which usually have certain target sites (such as chromosome modification, heat shock proteins, molecular chaperones, protein kinases, etc.), and these target sites are more important for cancer cells than for normal cells. But has the disadvantages of faster metabolism in vivo, shorter effective blood concentration maintenance, low bioavailability and the like. Therefore, in order to improve the curative effect, a larger dosage or multiple administrations are often needed, and due to the lack of selectivity, the tumor cells are killed and simultaneously strong toxic and side effects are generated on normal cells.

The construction of nano-drugs by nanotechnology provides a suitable carrier for the management of small molecule anticancer drugs. In general, 1) long circulation, 2) tumor tissue enrichment and infiltration, 3) tumor cell entry and 4) controllable release of intracellular drugs of antitumor nano-drugs in vivo are 4 very critical links, and designing a nano-drug carrier capable of integrally overcoming the 4 links is very important for successfully treating tumors. However, the existing nano-drug carrier has poor tumor permeability due to too large size, or the drug is easily excreted out of the body by the kidney even if the size is too small, even if the obtained drug has good tumor permeability, or the drug loading capacity is limited, or the drug release process is not easy to control, so that the cancer cannot be effectively treated.

Therefore, in order to fully exert the therapeutic advantages of small molecule drugs, there is an urgent need in the art to develop a novel nano drug carrier that can satisfy the above 4 key requirements, has high drug loading, has excellent tumor permeability, and can realize the controlled release of drugs in cells.

Disclosure of Invention

The invention aims to provide a novel nano-drug carrier which can meet the 4 key requirements, has high drug loading, excellent tumor permeability and controllable release of drugs in cells.

In a first aspect of the invention, there is provided a complex comprising:

nanoparticles; and

biodegradable gelatin crosslinked to the nanoparticle surface;

and the particle size of the compound is 20nm-500 mu m.

In another preferred embodiment, the particle size of the complex is from 30nm to 300. mu.m, preferably from 50nm to 100. mu.m, more preferably from 80nm to 50 μm, most preferably from 100nm to 10 μm.

In another preferred embodiment, the particle size of the nano particles is 5-80 nm; and/or

The purity of the gelatin is more than or equal to 85 percent.

In another preferred embodiment, the particle size of the nanoparticles is 10-50nm, preferably 15-45nm, and more preferably 20-40 nm.

In another preferred embodiment, the purity of the gelatin is 90% or more, preferably 95% or more, more preferably 99% or more, preferably about 100%.

In another preferred embodiment, the molecular weight of the gelatin is 0.1 to 500 ten thousand, preferably 0.5 to 100 ten thousand, and more preferably 1 to 20 ten thousand.

In another preferred embodiment, the content of the nanoparticles is 1 to 30 wt%, preferably 2 to 25 wt%, more preferably 3 to 20 wt%, based on the total weight of the composite.

In another preferred example, in the compound, the coating rate of the gelatin on the nano particles is more than or equal to 95%.

In another preferred embodiment, in the compound, the coating rate of the gelatin on the nano-particles is more than or equal to 98%, preferably more than or equal to 99%, and preferably 100%.

In another preferred embodiment, the nanoparticles have a specific surface area of 10m ² /g－1500m ² (ii)/g; and/or

The nanoparticles have a mesoporous structure; and/or

The mesoporous aperture of the nano particles is 1 nm-50 nm; and/or

The nanoparticles are selected from the group consisting of: laponite, calcium phosphate microspheres/nanorods, carbon nanospheres, iron oxide, silica, ceramic, carbon nanospheres/nanotubes, titanium oxide, zinc oxide, magnesium oxide, zirconium oxide, or combinations thereof.

In another preferred embodiment, the nanoparticles have a specific surface area of 50m ² /g－1200m ² G, preferably 100m ² /g－1000m ² /g。

In another preferred embodiment, the mesoporous pore size of the nanoparticles is 2nm to 30nm, preferably 3nm to 20 nm.

In a second aspect of the present invention, there is provided a method for preparing the complex of the first aspect of the present invention, comprising the steps of:

1) providing a first solution, a first mixed solution, an interfacial agent and a crosslinking agent,

the first solution comprises a first solvent and gelatin dissolved in the first solvent;

the first mixed solution comprises a second solvent and nanoparticles;

2) mixing the first solution and the first mixed solution, and reacting to obtain a first reaction solution;

3) adding the interface forming agent into the first reaction liquid, and reacting to obtain a second reaction liquid; and

4) and adding the cross-linking agent into the second reaction solution, and reacting to obtain the compound of the first aspect of the invention.

In another preferred embodiment, the nanoparticles and the gelatin are as described in the first aspect of the invention.

In another preferred embodiment, the first solvent and the second solvent may be the same or different and are each independently selected from the group consisting of: water, methanol, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, dilute hydrochloric acid, or combinations thereof.

In another preferred embodiment, after step 4), the following steps are optionally included:

5) removing the interface forming agent in the product obtained in the previous step to obtain a third reaction liquid;

6) optionally centrifuging the third reaction solution obtained in the previous step, and removing the supernatant to obtain the complex of the first aspect of the present invention.

In another preferred example, in step 5), the removal is performed using a rotary evaporator or dialysis.

In another preferred embodiment, in step 6), the centrifugation rate of the centrifugation treatment is 5000-.

In another preferred example, in step 6), the treatment time of the centrifugation treatment is 10 to 120 minutes, preferably 30 to 80 minutes.

In another preferred embodiment, the method further comprises the following step after the step 6): removing aldehyde groups in the obtained product by using a substance selected from the following group: glycine, leucine, isoleucine, valine, methionine, phenylalanine, tryptophan, threonine, lysine, histidine, arginine, cystine, tyrosine, or combinations thereof.

In another preferred example, in the step 2), the mixing weight ratio of the nanoparticles to the gelatin in the first reaction solution is 1-50: 10-100.

In another preferred example, in the step 2), the mixing weight ratio of the nanoparticles to the gelatin in the first reaction solution is 1-30: 20-50, preferably 2-20: 20-40.

In another preferred embodiment, in step 2), the reaction temperature of the reaction is 40 to 80 ℃, preferably 50 to 70 ℃.

In another preferred example, in step 2), the reaction time of the reaction is 1 to 30 minutes, preferably 5 to 15 minutes.

In another preferred embodiment, the interfacial agent is selected from the group consisting of: acetone, ethanol, dimethyl sulfoxide, dichloromethane, methanol, diethyl ether, or a combination thereof; and/or

The crosslinking agent is selected from the group consisting of: glutaraldehyde, succinaldehyde, polyethylene glycol dialdehyde, polyaldehyde alginate, polyaldehyde hyaluronic acid, or combinations thereof.

In another preferred example, in the step 3), the volume ratio of the interfacial agent to the first reaction liquid is 1-10: 1-3, preferably 1-5: 1-2.

In another preferred embodiment, in step 3), the reaction is carried out at room temperature, preferably at 10-40 ℃.

In another preferred embodiment, in step 3), the reaction time of the reaction is 1 to 60 minutes, preferably 5 to 40 minutes, and more preferably 10 to 30 minutes.

In another preferred embodiment, in step 4), the ratio of the amount of the cross-linking agent to the amount of the gelatin in the second reaction solution is 0.1 to 50: 100-400, preferably 1-40: 100-200.

in another preferred embodiment, in step 4), the reaction is carried out at room temperature, preferably at 10 to 40 ℃.

In another preferred embodiment, in step 4), the reaction time of the reaction is 1 to 24 hours, preferably 3 to 20 hours, and more preferably 5 to 15 hours.

In a third aspect of the present invention, there is provided a composition comprising:

a complex according to the first aspect of the invention; and

one or more substances selected from the group consisting of: antineoplastic medicine, contrast agent and antiphlogistic medicine.

In another preferred embodiment, the anti-tumor drug is selected from the group consisting of: doxorubicin (DOX), methotrexate, paclitaxel, daunorubicin, irinotecan, topotecan, vinblastine, or a combination thereof.

In another preferred embodiment, the anti-inflammatory drug is selected from the group consisting of: dexamethasone, hydrocortisone, aspirin, amoxicillin, penicillin, enzyme-resistant penicillin, ampicillin, cephalexin, cephradine, cephalexin, cefaclor, or a combination thereof.

In another preferred embodiment, the contrast agent is selected from the group consisting of: fluorescein isothiocyanate, an integral imaging agent (e.g., gold particles, magnetic particles, etc.), an iodine preparation, iohexol, or a combination thereof.

In a fourth aspect of the invention, there is provided a use of a complex according to the first aspect of the invention or a composition according to the third aspect of the invention for 1) the manufacture of a medicament for the treatment and/or diagnosis of cancer; and/or 2) for the preparation of a medicament for the treatment of inflammation.

In a fifth aspect of the present invention, there is provided a medicament comprising:

a complex according to the first aspect of the invention;

one or more substances selected from the group consisting of: antineoplastic drugs, contrast agents, anti-inflammatory drugs; and

a pharmaceutically acceptable carrier.

In another preferred embodiment, the drug is preferably administered by intravenous injection.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a microscopic morphology change diagram of GLD nanoparticles in the presence and absence of gelatinase (MMP 2), respectively.

FIG. 2 shows the kinetics of drug release of GLD composition in phosphate buffered saline at various pH and temperature.

FIG. 3 shows the results of the tumor depth permeability of Doxorubicin DOX, Doxorubicin-carrying laponite microsphere LD, Doxorubicin-carrying gelatin/laponite microsphere pharmaceutical composition GLD.

FIG. 4 shows the results of cellular uptake and intracellular drug distribution of doxorubicin DOX, doxorubicin-loaded gelatin/laponite microsphere pharmaceutical composition GLD.

FIG. 5 shows the antitumor activity of Doxorubicin DOX, gelatin/laponite microspheres GL, LD, gelatin/laponite microsphere pharmaceutical composition GLD carrying Doxorubicin.

Detailed Description

Through long-term and intensive research, the inventor prepares a compound which has high drug loading, excellent tumor permeability and can realize graded drug release in cells by crosslinking and modifying a gelatin layer on the surface of a nanoparticle with a specific surface structure and a specific particle size. Specifically, the inventor prepares a compound capable of releasing the nano-drug carrier in a grading manner in vivo by using nano-particles with strong interaction with small-molecule drugs as a core drug carrier and cross-linking and hybridizing gelatin which is easy to react with collagenase widely existing in vivo on the surface of the core drug carrier to serve as a coating layer. By loading an antitumor drug, a contrast agent, or other drugs, etc. in the complex, highly effective treatment of the corresponding disease can be achieved. On this basis, the inventors have completed the present invention.

Composite material

The inventor discovers that the following components are contained in the traditional medicine carrier through research: the nano-drugs with the size of about 100nm can increase long circulation time and improve the aggregation of the drugs around the tumor, but the higher osmotic pressure in the tumor limits the deep permeability of the tumor; particles smaller than 10nm are easily excreted by the kidney despite their high tissue permeability. In particular, the current liposome-loaded anticancer drugs in clinical use, although the aggregation of the drugs around the tumor is improved by increasing the long circulation period of the drugs, and the toxic and side effects are reduced to some extent, the large size (100nm) of the drugs causes poor tumor permeability, which greatly reduces the drug efficacy to some extent.

In order to solve the above problems, the present invention provides a complex comprising:

nanoparticles; and

biodegradable gelatin crosslinked to the nanoparticle surface;

and the particle size of the compound is 20nm-500 mu m.

In the invention, the particle size of the nano particles is 5-80 nm; and/or

The purity of the gelatin is more than or equal to 85 percent.

In another preferred embodiment, the particle size of the nanoparticles is 10-50nm, preferably 15-45nm, more preferably 20-40 nm.

In another preferred embodiment, the gelatin has a molecular weight of 0.1 to 500, preferably 0.5 to 100, more preferably 1 to 20 ten thousand.

In another preferred embodiment, the content of the nanoparticles is 1-30 wt%, preferably 2-25 wt%, more preferably 3-20 wt%, based on the total weight of the composite.

In the compound, the coating rate of the gelatin on the nano particles is more than or equal to 95%.

In another preferred example, in the compound, the coating rate of the gelatin on the nano particles is more than or equal to 98%, preferably more than or equal to 99%, and preferably 100%.

In the present invention, the nanoparticles have a specific surface area of 10m ² /g－1500m ² (iv) g; and/or

The nanoparticles have a mesoporous structure; and/or

The mesoporous aperture of the nano particles is 1 nm-50 nm; and/or

The nanoparticles include (but are not limited to): laponite, calcium phosphate microspheres/nanorods, carbon nanospheres, iron oxide, silica, ceramic, carbon nanospheres/nanotubes, titanium oxide, zinc oxide, magnesium oxide, zirconium oxide, or combinations thereof.

In another preferred embodiment, the nanoparticles have a specific surface area of 50m ² /g－1200m ² In g, preferably 100m ² /g－1000m ² /g。

In another preferred embodiment, the mesoporous pore size of the nanoparticle is 2nm to 30nm, preferably 3nm to 20 nm.

In the invention, the inventor takes small-scale nanoparticles (with the particle size of 5-80 nm) capable of carrying drugs as drug carrier subunits, and prepares the nanometer hybrid microspheres (namely the compound) with the graded release function through the self-assembly and in-situ crosslinking technology of the nanoparticles and degradable functional polymers. The microspheres can be controllably degraded under the stimulation of a tumor microenvironment signal (such as high-concentration collagenase MMP2, a lower pH value or a reductive microenvironment) to release the internally coated small-scale nano carrier subunits, the specific particle size of the nano carrier subunits can obviously enhance the permeability of tumor tissues to the nano carrier subunits and the uptake of drugs by tumor cells, and the drugs loaded by the nano carrier subunits can be sustained and released in the tumor cells for a long time under the stimulation of the microenvironment in the tumor or an external heating signal, so that the disease treatment effect is enhanced.

In the invention, the nanoparticles have large specific surface area, so that the nanoparticles and small-molecule drugs have strong intermolecular interaction, the drug loading rate can be obviously improved, and the drug stability is further prolonged; the nanoparticles have a specific particle size, so that the obtained medicine can permeate into tumor cells in a high permeability manner, and is not easy to be discharged out of the body by the kidney, thereby improving the utilization rate of the medicine.

In the present invention, in the complex, the gelatin layer on the surface of the nanoparticle can be degraded under the stimulation of one or more biological signals (enzyme, pH, redox, etc. signals) or external signals (light, heat, electric, magnetic, etc. signals) and release the small-scale nanoparticle subunits carrying the drug for subsequent infiltration into tumor cells.

In the invention, the nano particles entering the tumor cells can realize enhanced and long-acting drug release under the stimulation of microenvironment signals (such as pH and strong reduction biological signals) in the tumor cells or external light, heat, magnetism, electricity and other signals, thereby improving the drug effect and effectively curing diseases.

In addition, the hybrid microsphere can be labeled by a fluorescent reagent or coated with an imaging/developing agent such as gold particles, magnetic particles and the like in the interior for in vivo biological imaging, so that the hybrid microsphere can be used as a new technology for further microscopic evaluation of curative effect to detect precancerous lesion molecular abnormality, cell growth kinetics, tumor cell markers, gene changes and the like. The integration of disease treatment and dynamic evaluation can be effectively realized by the combination of the imaging technology, the drug treatment technology and the like.

Preparation method

The invention also provides a preparation method of the compound, which comprises the following steps:

the first mixed solution comprises a second solvent and nanoparticles;

3) adding the interfacial agent into the first reaction solution, and reacting to obtain a second reaction solution; and

4) and adding the cross-linking agent into the second reaction solution, and reacting to obtain the compound.

In another preferred embodiment, the nanoparticles and the gelatin are as described herein.

In another preferred embodiment, the first solvent and the second solvent may be the same or different and are each independently selected from the group consisting of (but not limited to): water, methanol, ethanol, acetone, tetrahydrofuran, dimethyl sulfoxide, dilute hydrochloric acid, or a combination thereof.

6) optionally centrifuging the third reaction solution obtained in the previous step, and removing the supernatant to obtain the compound.

In another preferred example, in step 6), the centrifugation rate of the centrifugation treatment is 5000-.

In another preferred embodiment, the method further comprises the following step after the step 6): removing aldehyde groups in the resulting product using a material selected from the group consisting of: glycine, leucine, isoleucine, valine, methionine, phenylalanine, tryptophan, threonine, lysine, histidine, arginine, cystine, tyrosine, or combinations thereof.

In the present invention, in step 2), the mixing weight ratio of the nanoparticles and the gelatin in the first reaction solution is 1 to 50: 10-100.

In another preferred embodiment, in step 2), the reaction time of the reaction is 1 to 30 minutes, preferably 5 to 15 minutes.

In the present invention, the interfacial agent includes (but is not limited to): acetone, ethanol, dimethyl sulfoxide, dichloromethane, methanol, diethyl ether, or combinations thereof; and/or

The crosslinking agent includes (but is not limited to): glutaraldehyde, succinaldehyde, polyethylene glycol dialdehyde, alginic acid polyaldehyde, hyaluronic acid polyaldehyde, or a combination thereof.

In another preferred example, in the step 4), the ratio of the amount of the cross-linking agent to the amount of the gelatin in the second reaction solution is 0.1-50: 100-400, preferably 1-40: 100-200.

In the present invention, the crosslinking reaction of the nanoparticles and the gelatin is performed at the interface of two phases of water and an interfacial agent (e.g., acetone). Firstly, the horizontal dispersion of natural macromolecular gelatin in water molecules is beneficial to improving the interaction between the natural macromolecular gelatin and the surfaces of the nanoparticles, so that the nanoparticles can be uniformly dispersed in the aqueous solution of the gelatin; the addition of the non-solvent acetone can reduce the solubility of the gelatin in water, induce the gelatin to gradually aggregate from molecular level dispersion and form a dispersed phase; the ratio of water to acetone can be used to adjust the size of the dispersed phase, the nanoparticles can be wrapped by gelatin in situ during the formation of the gelatin dispersed phase, and the hybrid microspheres with good colloidal stability are formed by the cross-linking treatment of the cross-linking agent.

Applications of

The present invention also provides a composition comprising:

the complex as described; and

In another preferred embodiment, the anti-neoplastic agents include (but are not limited to): doxorubicin (DOX), methotrexate, paclitaxel, daunorubicin, irinotecan, topotecan, vinblastine, or a combination thereof.

In another preferred embodiment, the anti-inflammatory agent includes (but is not limited to): dexamethasone, hydrocortisone, aspirin, amoxicillin, penicillin, enzyme-resistant penicillin, ampicillin, cephalexin, cephradine, cephalexin, cefaclor, or a combination thereof.

In another preferred embodiment, the contrast agents include (but are not limited to): fluorescein isothiocyanate, an integral imaging agent (e.g., gold particles, magnetic particles, etc.), an iodine preparation, iohexol, or a combination thereof.

The invention also provides a use of said complex or said composition for 1) the preparation of a medicament for the treatment and/or diagnosis of cancer; and/or 2) for the preparation of a medicament for the treatment of inflammation.

The present invention also provides a medicament comprising:

the complex as described;

a pharmaceutically acceptable carrier.

Compared with the prior art, the invention has the following main advantages:

(1) the compound has the characteristics of high drug loading, excellent tumor permeability and capability of releasing the drug in a grading manner;

(2) the preparation method of the compound is simple, controllable in conditions, low in cost and easy for large-scale industrialization;

(3) the compound has good biocompatibility and intelligent responsiveness;

(4) in the compound, the existence of gelatin can effectively improve the colloid stability of the microsphere (namely the compound of the invention), and amino and carboxyl functional groups in gelatin molecules can be used for surface modification of hybrid microspheres, such as the modification of RGD polypeptide, folic acid and other tumor targeting groups, or fluorescent agents (such as fluorescein isothiocyanate, erythrosin B, rhodamine) or imaging/developing agents (such as iodotaltamamine, iodixanoic acid, iohexol, iosimidyl alcohol, iopromide, ioversol, Gd-DTPA and linear and cyclic polyamine polycarboxylic chelates thereof), and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are exemplary only.

EXAMPLE 1 purification of gelatin

2G of commercially available unpurified gelatin (dark yellow from Sigma, type G-9382) were weighed out, 50mL of ultrapure water was added thereto, stirred in a water bath at 60 ℃ for 2 hours, 50mL of acetone as a precipitant was added thereto, stirred at room temperature for 1 hour, the supernatant was removed, 50mL of ultrapure water was added thereto again, and dissolved by heating in a water bath at 60 ℃. After complete dissolution, the purified Gelatin (Gelatin) is obtained by subpackaging and freeze-drying. The Gelatin used for the construction of the microspheres is not specifically indicated, but is purified Gelatin (Gelatin).

The purified gelatin was tested to be up to 90% pure.

EXAMPLE 2 preparation of Complex GL

Weighing 50mg of purified gelatin, dissolving the purified gelatin in 5mL of ultrapure water, and stirring and dissolving the gelatin at the temperature of 60 ℃;

② 20mg of Laponite (LP) with the grain diameter of 10-30 nm (the specific surface area is 100 m) ² /g-900m ² (g)/g) dispersed in 2mL of ultrapure water, and sonicated for 30 minutes to uniformly disperse;

thirdly, quickly adding 0.5-1mL of the solution of Gelatin prepared in the step II into the solution of LP prepared in the step II, and reacting for 10 minutes at 60 ℃;

dripping 20mL of interfacial agent acetone into the mixed solution prepared in the step (III), and reacting for 20 minutes at room temperature after dripping;

fifthly, dropwise adding 50 mu L of glutaraldehyde solutions (1-25 wt%) with different concentrations into the solution prepared in the step (iv), and reacting for 12 hours at room temperature;

sixthly, evaporating the acetone in the solution obtained in the step (fifth) by using a rotary evaporator;

seventhly, centrifuging the milky white liquid obtained in the step (c), wherein the centrifugation speed is 12000rpm, and the time is 60 minutes;

removing the supernatant obtained in the step (sixthly), dissolving the precipitate again by using 5mL of ultrapure water, and storing the precipitate in a refrigerator at 4 ℃ for later use.

Description of the invention: in the case of the material used for cell experiments, the excess aldehyde groups can be removed by adding glycine solution to GL and stirring at room temperature for 4h, followed by centrifugal purification.

Results

The particle size and coating rate of the composite GL obtained in example 2 are tested, and the results show that the particle size of the composite GL is (105 +/-10) nm, and the coating rate of the gelatin layer on the laponite nano particles is about 90%.

EXAMPLE 3 preparation of composition GLD

To 5mL of GL microsphere dispersion prepared in example 2, 1mL of doxorubicin aqueous solution (1mg/mL) was added, the mixture was stirred for 24 hours in the absence of light, centrifuged at 12000rpm for 40 minutes, the supernatant (for drug encapsulation efficiency) was removed, and the resulting precipitate was dispersed in 5mL of ultrapure water to prepare a microsphere drug assembly GLD.

Description of the drawings: the gelatin/laponite microspheres can be used for loading various medicaments including anticancer medicaments and anti-inflammatory medicaments, and the prepared medicament combination can be used for treating cancers, skin diseases and other related diseases.

EXAMPLE 4 degradation responsiveness of composition GLD to gelatinase

250ng of gelatinase (MMP 2) was dissolved in 1mL of the HEPES solution, 1mL of GLD nanoparticles was added to the solution, the solution was cultured at 37 ℃ for 24 hours, and changes in microscopic morphology of GLD nanoparticles with and without MMP2 treatment were observed by transmission electron microscopy, respectively.

As can be seen from FIG. 1, the GLD microspheres without MMP2 enzyme appeared spherical, whereas the GLD microspheres with MMP2 enzyme showed much more fragments. The reason is that MMP2 enzyme can degrade gelatin on the surface of GLD microspheres under the action of enzyme, smaller fibrous nanoparticles with the size of 40-50nm are released, and the aim of gradual release is fulfilled.

EXAMPLE 5 drug Release study of GLD composition

Study of GLD drug Release Performance Using stimulated emission Spectroscopy

Composition GLD containing 30. mu.g of doxorubicin was dispersed in 2mL of Phosphate Buffered Saline (PBS) and transferred to a dialysis bag (molecular weight cut-off: 14kDa), which was then immersed in 8mL of PBS solution (pH 7.4, 6.5 or 5.0, respectively, at 25 or 37 ℃ C.). After a certain time interval, 100. mu.L of the solution was taken from the release system for fluorometric analysis (lambda.) _ex ＝480nm,λ _em 580nm) and 100 μ L of fresh PBS solution was refreshed. Cumulative release at different times (C) _r ) Doxorubicin (iv) can be calculated using the following formula:

C _r ＝100×F _t /F _tot

wherein F _t And F _tot Respectively represents the fluorescence of the solution at the time t and the fluorescence of the total adriamycin contained in the hybrid microspheres for release.

As can be seen from FIG. 2A, the cumulative release of DOX increases as the pH decreases from 7.4 to 6.5, 5.0. For example, the amount released at pH 7.4 was (30. + -. 2)%, the amount increased to (41. + -. 3)%, at pH 6.5 and the amount increased to (55. + -. 3)%, at pH 5.0, at 120 hours.

As can be seen from FIG. 2B, the cumulative DOX release was also improved as the temperature was increased (25 ℃,37 ℃, 42 ℃). For example, the DOX release amount increases from (20. + -.2)% at 25 ℃ to (30. + -.2)% at 37 ℃ and then to (36. + -.2)% at 42 ℃ over 120 hours.

This shows that GLD nanospheres can achieve gradual release of DOX in an acidic environment or at an elevated temperature, thereby achieving an enhanced antitumor effect, and on the other hand, the pH and thermal sensitivity of GLD nanospheres are fully demonstrated.

Example 6 tumor sphere depth penetration study

The shape and size of the tumor sphere and the tumor depth permeability of GLD were observed by laser confocal: a549 tumor sphere model was grown in 96-well plates coated with agarose on the bottom: coating a thin layer of 2% agarose solution on a 96-well plate, sterilizing at high temperature and high pressure, adding DMEM culture solution containing 2000/2000-density A549 cell strain, culturing for a week until the diameter of the tumor ball is about 300 μm, adding DOX, LD and GLD, incubating for 6 days, and observing the shape and size and the tumor ball depth penetration ability of the adriamycin drug under laser confocal condition.

The abscissa in fig. 3 is the depth of the tumor microsphere, and it can be seen from fig. 3 that the gelatin/laponite microsphere drug composition GLD carrying doxorubicin delivers the DOX drug to the middle of the tumor microsphere (the microsphere diameter is 330 μm, so the 160 μm photograph reflects the drug distribution of the drug in the middle of the microsphere), while the fluorescence intensity of the doxorubicin DOX of the control group and the laponite microsphere LD carrying doxorubicin in the middle of the microsphere is significantly reduced, indicating that they are not favorable for the penetration of the drug in the deep tumor.

EXAMPLE 7 cellular uptake and intracellular accumulation Studies of drugs

And (3) observing the distribution of the drug of the sample in the A549 cells by a laser confocal microscope: a549 cells were first seeded on a plastic culture dish, and after one day of incubation, DOX and GLD were added thereto, respectively, and incubated for 2 hours and 48 hours. After the indicated time, the culture medium was removed, washed 3 times with PBS, fixed with 2.5% glutaraldehyde for 15 minutes, the glutaraldehyde solution was removed, washed 3 times with PBS, stained with 4', 6-diamidino-2-phenylindole (DAPI) for 15 minutes, washed 3 times with PBS, added 1mL of PBS, and finally observed in 3 dimensions under laser confocal.

As can be seen in fig. 4, the first column is the bright field effect of DOX and GLD microspheres in the cells for 2 and 48 hours, the second column is the red fluorescence effect of DOX, the third column is DAPI staining the nuclei blue, and the last column is the combination of the first two columns. From these figures, it can be seen that DOX and GLD have been taken up into the cytoplasm and the interior of the nucleus, with more GLD entering the nucleus and less DOX entering the interior of the nucleus; GLD has a higher accumulation of DOX than DOX, mainly because GLD can release smaller nanoparticles inside the cell, which promotes GLD to release more DOX to kill cancer cells under acidic conditions, as can be seen by the number of cells at 48 hours being significantly less than 2 hours.

Example 8 antitumor Activity study

Anti-tumor method using human lung cancer cell A549 cell as tumor model research drugIs as follows. The cell culture comprises the following specific steps: the cells were cultured in DMEM medium containing 10% calf serum, 100U/mL penicillin, and 100U/mL streptomycin at 37 ℃ with 5% CO ₂ Subculturing in an incubator under the atmosphere. A549 cells are inoculated into a 96-well plate, culture medium solutions of GL, DOX, LD, GLD and a blank control group are added according to corresponding concentrations (DOX concentration) after one day of incubation, the culture is continued for 48 hours, and the cell activity and the cell proliferation are detected by using a 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide (MTT) assay method: after further incubation for 4 hours with 30. mu.L of tetramethylazozolium salt reagent, the culture medium was removed, 200. mu.L of dimethyl sulfoxide (DMSO) was added, the mixture was shaken at 37 ℃ for 10 minutes to dissolve the crystals, and the light absorption value of the solution was measured at 490nm using a continuous spectrum microplate reader.

As can be seen from FIG. 5, gelatin/laponite microspheres GL had no significant cytotoxicity, DOX, LD and GLD all showed cancer cytotoxicity, and GLD showed better anticancer activity than the control group. For example, IC of GLD ₅₀ The sample concentration at 50% cell survival rate is 0.25 μm, while DOX and LD are 1.57 μm and 0.56 μm, respectively, which is probably because GLD nanospheres with graded drug release function promote DOX tumor permeability and drug uptake rate of cancer cells, thereby improving killing effect on tumor cells.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims

1. A composite, comprising:

nanoparticles, the nanoparticles are laponite nanoparticles, and the nanoparticlesThe specific surface area of the rice grains is 50m ² /g－1200m ² (iv) g; and the nanoparticles have a mesoporous structure and the mesoporous aperture of the nanoparticles is 1nm to 50 nm; and

biodegradable gelatin crosslinked to the nanoparticle surface;

the particle size of the compound is 30nm-10 mu m;

the particle size of the nano particles is 5-80 nm;

the purity of the gelatin is more than or equal to 90 percent;

in the compound, the coating rate of the gelatin on the nano particles is more than or equal to 95 percent;

the content of the nano particles is 1-30 wt% based on the total weight of the compound;

and the compound is prepared by the following method:

the first mixed solution comprises a second solvent and nanoparticles;

4) adding the cross-linking agent into the second reaction solution, and reacting to obtain the compound;

wherein the interfacial agent is selected from the group consisting of: acetone, ethanol, dimethyl sulfoxide, dichloromethane, methanol, diethyl ether, or a combination thereof.

2. The composite of claim 1, wherein the nanoparticles have a particle size of 10-50 nm; and/or

The purity of the gelatin is more than or equal to 95 percent; or

The specific surface area of the nano particles is 100m ² /g－1000m ² In each case of

The mesoporous aperture of the nano particles is 2 nm-30 nm.

3. The compound of claim 1, wherein the nanoparticle is coated with the gelatin at a rate of 98% or more.

4. A method of preparing the compound of claim 1, comprising the steps of:

the first mixed solution comprises a second solvent and nanoparticles;

4) adding the cross-linking agent into the second reaction solution to react to obtain the compound of claim 1;

wherein the interfacial agent is selected from the group consisting of: acetone, ethanol, dimethyl sulfoxide, dichloromethane, methanol, diethyl ether, or combinations thereof.

5. The method according to claim 4, wherein in the step 2), the mixing weight ratio of the nanoparticles and the gelatin in the first reaction solution is 1 to 50: 10-100.

6. The method of claim 4, wherein the interfacial agent is selected from the group consisting of: acetone, ethanol, dimethyl sulfoxide, dichloromethane, methanol, diethyl ether, or combinations thereof; and/or

7. A composition, characterized in that the composition comprises:

the complex of claim 1; and

one or more substances selected from the group consisting of: antineoplastic medicine, contrast agent, and anti-inflammatory medicine.

8. The composition of claim 7, wherein said antineoplastic agent is Doxorubicin (Doxorubicin).

9. Use of a complex according to claim 1 or a composition according to claim 7 for 1) the preparation of a medicament for the treatment and/or diagnosis of cancer; and/or 2) for the preparation of a medicament for the treatment of inflammation.

10. A medicament, comprising:

the complex of claim 1;

a pharmaceutically acceptable carrier.