CN111084759B

CN111084759B - Medicine carrying system for loading anticancer medicine and preparation and application thereof

Info

Publication number: CN111084759B
Application number: CN202010052562.6A
Authority: CN
Inventors: 吴允昆; 肖方南; 陈方满; 张维彬
Original assignee: Fujian Normal University
Current assignee: Fujian Normal University
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2021-10-08
Anticipated expiration: 2040-01-17
Also published as: CN111084759A

Abstract

The invention relates to the technical field of nano-medicine, in particular to a medicine carrying system for carrying an anti-cancer medicine, and preparation and application thereof. The drug-carrying particles formed by mesoporous silica nanospheres with good biocompatibility and large specific surface area and small molecular complexes are used as an inner core layer, the outer surface of the drug-carrying particles is coated with a liposome layer formed by specific target protein and a liposome membrane, and the specific target protein (formed by fusion construction and expression of H1299.2 target peptide, e-GFP and MscL) is fused with the liposome membrane, so that after the drug-carrying particles as an anti-cancer chemotherapeutic drug enter an organism, the drug-carrying particles are not accumulated in normal tissues without target protein binding sites, and the drug is bound on tumor cells with target sites and released at fixed points. Since the specific target protein contains the fluorescent protein e-GFP, the distribution of the e-GFP in the body can be determined by measuring the fluorescence value of the e-GFP, so that the chemotherapy can be guided more accurately.

Description

Medicine carrying system for loading anticancer medicine and preparation and application thereof

Technical Field

The invention relates to the technical field of nano-medicines, in particular to a protein liposome antitumor drug targeting carrier supported by mesoporous silica nanospheres, coupling of the carrier and an antitumor drug, a formed drug-loading system, and preparation and application thereof.

Background

Cancer is considered as the 'first killer' of human in the 21 st century by the world health organization, seriously threatens the survival of human, has high diagnosis difficulty and death rate, and the treatment of malignant tumor is mainly carried out by surgical excision, radiotherapy, immunotherapy and chemotherapy. The control of cancer has become a global health strategy focus. At present, China has become the first cancer kingdom in the world, and cancer not only seriously threatens the life and health of people. The chemotherapy method is still one of the main means for treating malignant tumors, the traditional chemotherapy method mainly uses chemical drugs to inhibit the proliferation of tumor cells and kill the tumor cells, but most of the chemotherapy drugs have no specificity and can damage normal tissues while inhibiting the tumor tissues, so the traditional chemotherapy method has non-negligible toxic and side effects, such as reduced immune function, bone marrow suppression, heart and liver toxicity and the like.

In order to overcome the defects in the aspect of chemical drug treatment, improve the targeting property of the chemical treatment drug, ensure that the added drug can be continuously accumulated in tumor tissues, reduce the harm of the drug to normal tissues, and is one of effective ways for improving the anti-tumor effect. The existing better method is to combine the antitumor drug with different drug carriers, and the drug carriers are modified to enable the drug carriers to have the targeted transportation capability, thereby achieving the purpose of targeted therapy.

Chinese patent application CN109364267A discloses a mesoporous silica nano drug-carrying particle with double targeting of tumor tissues and cells and a preparation method thereof, the drug-carrying particle is composed of a mesoporous silica nano particle with the surface modified by polyacrylic acid and polyethylene glycol and a drug, and the drug-carrying particle is enriched at a tumor part in a passive targeting manner through adaptive response of the drug-carrying particle to the pH value in an intestinal tract, particle size compatibility and prolongation of circulation time in blood, thereby achieving a treatment effect. However, the technique still cannot avoid the probability of misjudgment of the drug-loaded particles due to individual difference or dynamic change of body organs and body fluids, and the damage to body tissue cells is difficult to avoid. In other disclosed technologies, the targeting performance of the anti-cancer drug delivery system is not ideal enough, and the damage of chemotherapy to normal body cells cannot be reduced or avoided.

Disclosure of Invention

Therefore, a medicine carrying system for carrying an anticancer drug and preparation and application thereof are needed to be provided, so as to solve the problem that the anticancer drug or the medicine carrying system containing the anticancer drug in the prior art causes damage to normal body tissue cells of a patient in a chemotherapy process due to weak targeting performance. The medicine carrying system for carrying the anticancer medicine has the characteristics of strong targeting property, good biocompatibility, measurable distribution in a body and the like.

In order to achieve the above object, in a first aspect of the present invention, the inventors provide a drug-loading system for loading an anticancer drug, comprising an inner core layer and an outer shell layer, wherein the outer shell layer is coated on the outer surface of the inner core layer, the inner core layer comprises a carrier and a small molecule complex, the carrier is a mesoporous silica nanosphere, the outer shell layer comprises a targeting protein and a liposome membrane, and the targeting protein is fused to the liposome membrane.

Further, the targeted protein includes a protein expressed by the fusion construct of the H1299.2 target peptide, eGFP and MscL.

Further, the particle size of the mesoporous silica nanosphere is as follows: 85.6-99.2nm, the pore diameter is: 7.2-10.0 nm. Compared with the pore diameters in other ranges, the mesoporous silica nanospheres in the pore diameters in the range are beneficial to large-scale loading of the medicament.

Further, the mass ratio of the liposome membrane to the targeting protein is (19-22): (1-2), preferably 20: (1-2). The target protein-liposome membrane fusion membrane prepared in the mass ratio range is more stable.

Further, the small molecular complex is a ruthenium metal complex with an anticancer effect, and the molecular weight is 662 g/mol.

The mesoporous silica nanosphere/particle (MSN) has the advantages of biocompatibility, good water solubility, large pore volume, large surface area and the like. The invention provides a preparation method of a novel targeted protein liposome (Lip) system, which is designed and synthesized, and can stably reach a tumor part in a targeted manner by combining with mesoporous silica nanospheres/particles (MSN) to realize effective accumulation of a medicament at the tumor part, so that a protein liposome antitumor medicament targeted carrier supported by silicon mesopores is provided.

The anti-cancer drug is a DNA targeting drug. The medicine is enriched in tumor cells H1299 under the action of the target protein and inserts and destroys nuclear DNA, resulting in tumor cell apoptosis. And according to the ultraviolet absorbance value obtained by measurement, the amount of the released ruthenium complex anticancer drug RuDPNI is obtained by conversion through a standard curve.

In a second aspect of the present invention, the inventors provide a method for preparing a drug-loaded system for loading anticancer drugs, comprising the following steps:

preparing mesoporous silica sphere drug-loaded particles,

dispersing mesoporous silica nanospheres in a small molecular complex emulsion, and preparing mesoporous silica drug-loaded particles through first self-assembly; and

preparing a medicine carrying system for carrying the anti-cancer medicine,

adding DSPE, cholesterol and DSPE-PEG2000 into a trichloromethane solution, evaporating a solvent to obtain a liposome membrane, adding a targeting protein to perform hydration reaction, combining the liposome membrane with the targeting protein to obtain a liposome layer containing the targeting protein, mixing the liposome layer with the mesoporous silica sphere drug-loaded particle solution, and performing a second self-assembly action to obtain the drug-loaded system loaded with the anticancer drug;

the drug-loading system for loading the drug-resistant drug comprises: the drug-loaded mesoporous silica gel comprises an inner core layer and an outer shell layer, wherein the inner core layer is mesoporous silica drug-loaded particles formed by mesoporous silica nanospheres and small molecule complexes, and the outer shell layer is formed by targeted protein and liposome membranes and is coated on the outer surface of the inner core layer.

In the above step of preparing the anticancer drug-loaded drug delivery system, preferably, the solvent is removed by slow reduced pressure evaporation at 40 ℃. Targeted liposomes (Lip) are also preferably formed by extrusion using a liposome extruder with a 0.1 μm filter.

As a preferable technical scheme, the mass ratio of the mesoporous silica nanospheres to the small molecular complex is 10:3-1: 1.

As a preferable technical scheme of the invention, the ratio of the DSPE, cholesterol, DSPE-PEG2000 and chloroform is 10:2:0.63: 10.

As a preferable technical scheme, the addition amount of the targeting protein is 0.5mg, and the proportioning relation of the liposome layer and the mesoporous silica sphere drug-loaded particle solution is 800 muL to 100 muL.

In a third aspect of the present invention, the inventors provide the use of the drug delivery system according to the first aspect of the present invention for carrying an oral anticancer drug.

Since mesoporous silica nanospheres/particles (MSN) have the advantages of biocompatibility, good water solubility, large pore volume, large surface area, etc., there have been a large number of literature reports on the use of MSN for drug delivery. The invention provides a preparation method of a novel targeted protein liposome (Lip) system, which is designed and synthesized, and can stably reach a tumor part in a targeted manner by combining with mesoporous silica nanospheres/particles (MSN) to realize effective accumulation of a medicament at the tumor part, so that a protein liposome antitumor medicament targeted carrier supported by silicon mesopores is provided.

Different from the prior art, the technical scheme has the following advantages:

the drug-carrying particles formed by mesoporous silica nanospheres with good biocompatibility and large specific surface area and small molecular complexes are used as an inner core layer, the outer surface of the drug-carrying particles is coated with a liposome layer formed by specific target protein and a liposome membrane, and the specific target protein (formed by fusion construction and expression of H1299.2 target peptide, eGFP and MscL) is fused with the liposome membrane, so that after the drug-carrying particles are taken as an anti-cancer chemotherapeutic drug and enter an organism orally, the drug-carrying particles are not accumulated in normal tissues without target protein binding sites, and the drug is bound on tumor cells with target sites and released at fixed points. As the specific target protein contains the fluorescent protein eGFP, the distribution of the e-GFP in vivo can be determined by measuring the fluorescence value of the e-GFP, so that the chemotherapy can be more accurately guided.

Drawings

FIG. 1 shows N of mesoporous silica nanoparticles before and after loading RuDPNI in an embodiment of the present invention₂Sucking and removing the attached drawings;

FIG. 2 is a table of the pore diameter, pore volume and specific surface area of mesoporous silica nano before and after loading RuDPNI in the embodiment of the present invention;

FIG. 3 is a transmission electron microscope image of a targeted silicon mesoporous liposome nano-drug (Ru-MSN-Lip) according to an embodiment of the present invention;

FIG. 4 is a RuDPNI release profile of Ru-MSN-Lip drug of an embodiment of the invention in PBS at pH5.0(a) and pH7.4 (b);

FIG. 5 is a table showing the toxicity of Ru-MSN-Lip on HELF, H1299, HepG2 and HeLa cells detected by MTT method according to an embodiment of the invention;

FIG. 6 is a graph showing the toxicity of different concentrations of Ru-MSN-Lip on HELF, H1299, HepG2 and HeLa cells in accordance with one embodiment of the present invention.

Detailed Description

To explain technical contents of technical solutions and achieved objects and effects in detail, the following detailed description is given with reference to specific embodiments and accompanying drawings.

The medicines, specifications, properties and sources used by the invention are explained as follows:

CTATos, hexadecyltrimethyl para-toluenesulfonate ammonium salt: 5g, national drug group chemical reagents, Inc.;

TEA, triethanolamine: 500ml, national pharmaceutical group chemical reagents Limited;

TEOS, tetraethoxysilane: 500ml, national pharmaceutical group chemical reagents Limited;

DSPE-PEG2000, distearoylphosphatidylethanolamine-polyethylene glycol: 500mg, Shanghai Aladdin Biotechnology Ltd;

PBS, phosphate buffer: 500ml, bio-engineering (Shanghai) Ltd;

medium DMEM, Dulbecco's modified eaglemedia: 500ml, Saimer Feishale science and technology (China) Co., Ltd.;

MTT, thiazole blue tetrazolium bromide: 1g, bio-engineering (Shanghai) Ltd;

RuDPNI: the laboratory is self-made, and the specific preparation method can refer to the related content in the Chinese invention patent application 201910375826.9.

The specification and source of the instrument used in the invention are as follows:

liposome extruder: LiposoEasyLE-1, Merge mechanical (Shanghai) Co., Ltd;

TEM transmission electron microscope: TecnaiF20FETEM, seimer feishel technologies (china) ltd;

a dialysis bag: 14kd, bio-engineering (Shanghai) Ltd;

ultraviolet spectrophotometer: lambda950, perkin elmer instruments (shanghai) ltd;

microplate detector: synergy2, beton BioTek instruments ltd, usa;

inductively coupled plasma optical emission instrument: ultima2, horiba jobinbyvon, france, for determining the loading of RuDPNI;

example 1 preparation of mesoporous silica nanospheres MSN

0.9614g CTATos and 0.14mL of LTEA were added to 50mL of deionized water, heated to 60-80 ℃ and stirred for 1 h. TEOS (2-2.5mL) was added and stirring continued for 2 h. Then standing and cooling to room temperature, and centrifugally collecting the mesoporous silica nanospheres, namely the MSN. And centrifugally washing the mixture for several times by using absolute ethyl alcohol. The precipitate was dispersed in 100mL of 0.6% wt ammonium nitrate ethanol solution and extracted at 60 ℃ for 24h to remove the template. Centrifuging at 11500rpm, washing precipitate with water and anhydrous ethanol alternately for several times, repeating the extraction for 3 times, and storing the obtained MSN in anhydrous ethanol.

Example 2 preparation of mesoporous silica nanosphere MSN/drug nanoparticles

Adding 30mg/mL RuDPNI dimethyl sulfoxide solution into the MSN prepared by the method in example 1, wherein the mass ratio of the added MSN to the small molecular complex RuDPNI is 1:1, stirring for 72h, centrifuging at 5000rpm for 3min, then re-dispersing with 25mL of water, and centrifuging again to obtain the drug-loaded silicon mesoporous nanoparticles (Ru-MSN).

Example 3 preparation of mesoporous silica nanosphere MSN/drug nanoparticles

The difference from the example 2 is that the mass ratio of the added MSN to the small molecule complex RuDPNI is 1.5: 1.

Example 4 preparation of mesoporous silica nanosphere MSN/drug nanoparticles

The difference from the example 2 is that the mass ratio of the added MSN to the small molecule complex RuDPNI is 1.75: 1.

Example 5 preparation of mesoporous silica nanosphere MSN/drug nanoparticles

The difference from the example 2 is that the mass ratio of the added MSN to the small molecule complex RuDPNI is 2: 1.

Example 6 preparation of mesoporous silica nanosphere MSN/drug nanoparticles

The difference from the example 2 is that the mass ratio of the added MSN to the small molecule complex RuDPNI is 2.5: 1.

Example 7 preparation of mesoporous silica nanosphere MSN/drug nanoparticles

The difference from the example 2 is that the mass ratio of the added MSN to the small molecule complex RuDPNI is 3: 1.

Example 8 preparation of mesoporous silica nanosphere MSN/drug nanoparticles

The difference from the example 2 is that the mass ratio of the added MSN to the small molecule complex RuDPNI is 10: 3.

The mesoporous silica nanosphere MSN prepared in the examples 2-8 and the drug-loaded (small molecule complex RuDPNI) nanoparticles thereof were subjected to pore diameter, pore volume and specific surface area and N₂The adsorption and desorption experiments show that the experimental results are shown in figure 1 and figure 2.

Referring to fig. 1 and 2, it can be seen from the experimental results of fig. 2 that the pore diameter and the specific surface area of the mesoporous silica nano after the RuDPNI loading are both reduced, and the pore diameter and the specific surface area are respectively 8.565nm and 1.156m³The/g is reduced to 6.435nm and 0.660m³The/g shows that the mesoporous silica nano MSN loads the RuDPNI drug; the loading of RuDPNI was 14.0% as measured by inductively coupled plasma optical emission spectroscopy.

Example 9 Synthesis of Targeted liposomes (Lip)

In a clean 50mL round bottom flask, 10mL chloroform was added to dissolve 10mg DSPC, 2mg cholesterol, 0.63mg DSPE-PEG 2000. The solvent was evaporated off slowly at 40 ℃ under reduced pressure to form a thin, uniform white liposome film on the wall of the flask, which was dried overnight under vacuum. Adding a liposome membrane into 10ml of Phosphate Buffered Saline (PBS) with pH7.4, and adding 0.5-1.0mg of targeting protein and 9.5-22mg of liposome membrane, wherein the mass ratio of the liposome membrane to the targeting protein is (19-22): (1-2). Rehydrating at 35 deg.C for 30min, and slowly washing liposome membrane. Ice water bath, and intermittent ultrasonic treatment for 30 min. The targeted liposomes (Lip) were obtained by repeated squeezing about 20 times rapidly with a liposome squeezer with a 0.1 μm filter.

EXAMPLE 10 preparation of Targeted Liposome-MSN-drug combination System

Taking 100 mu L (16mg/mL) of the mesoporous silica nanosphere MSN/drug nanoparticle prepared in the embodiment 2-8, adding 800 mu L of the embodiment 9, fusing silicon mesopores with a liposome membrane when intermittently shaking for 1h to obtain targeted mesoporous liposome nanoparticles, centrifuging for 2min at 6000rpm, removing redundant Lip to obtain targeted silicon mesoporous liposome nanoparticles (Ru-MSN-Lip), and dispersing in 10mL of 0.5 xPBS. The morphology and size were characterized by TEM and the detailed results are shown in fig. 3.

EXAMPLE 11 measurement of Effect of sustained Release of Carrier

Taking RuDPNI of 0.5-10 mug/mL, measuring ultraviolet absorbance (Abs ═ 264nm), and fitting a standard curve according to the absorbance.

5mL of Ru-MSN-Lip (containing 2.8mg of RuDPNI) was added to the dialysis bag and placed in 100mL of PBS at pH7.4 and 5.0, respectively.

Stirring at 37 deg.C, collecting dialysate at different time points, measuring ultraviolet absorbance, and quantifying the amount of RuDPNI released by standard curve, as shown in FIG. 4.

Example 12 cytotoxicity testing of Lip/MSN/drug System

The toxicity of the material was analyzed by the international MTT method. The subject HELF, H1299, Hela, HepG2 cells.

HELF, H1299, Hela and HepG2 cells were co-cultured with the Ru-MSN-Lip prepared from the different precursor ratios in example 3 for 48H in DMEM medium. Then MTT (5mg/mL, 20. mu.L) was added to each well and co-incubated at 37 ℃ for 4 h. The supernatant was carefully removed, 150. mu.L of DMSO was added and the mixture was shaken gently on a shaker at 37 ℃ for 10 min. A50. mu.L sample supernatant was placed in a microplate detector (Bio-Rad550) and read at a wavelength of 570nm to determine the OD. Cell viability was calculated using the following formula:

cell viability ═ 100% (OD treatment-OD blank)/(OD control-OD blank) X%

Wherein the OD treatment is obtained from material-treated cells, the OD blank is obtained from the culture medium by the same MTT treatment step, and the OD control is obtained from untreated cells.

Referring to fig. 5 and fig. 6, experimental results show that the target protein labeled Lip-encapsulated Ru-MSN has an obvious targeting effect on H1299 tumor, cytotoxicity is obviously increased along with increase of concentration, anti-tumor activity on H1299 cell is 4.18 times of normal cytotoxicity (HELF cell), and the target protein labeled Lip-encapsulated Ru-MSN has a targeting effect on tumor and can reduce side effects.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

Claims

1. The drug loading system for loading the anti-cancer drugs is characterized by comprising an inner core layer and an outer shell layer, wherein the outer shell layer is coated on the outer surface of the inner core layer, the inner core layer comprises a carrier and a small molecule complex, the carrier is a mesoporous silica nanosphere, the outer shell layer comprises a targeting protein and a liposome membrane, the targeting protein is fused with the liposome membrane, and the targeting protein comprises a protein which is constructed and expressed by fusion of H1299.2 target peptide, eGFP and MscL.

2. The drug delivery system of claim 1, wherein the mesoporous silica nanospheres have a particle size of: 85.6-99.2nm, the pore diameter is: 7.2-10.0 nm.

3. The drug delivery system of claim 1, wherein the mass ratio of liposome membrane to targeting protein is (19-22): (1-2).

4. The drug delivery system of claim 3, wherein the mass ratio of liposome membrane to targeting protein is 20 (1-2).

5. The drug delivery system of claim 1, wherein the small molecule complex is a ruthenium metal complex with anticancer effect having a molecular weight of 662.

6. The preparation method of the medicine carrying system for carrying the anticancer medicine is characterized by comprising the following steps:

preparing mesoporous silica sphere drug-loaded particles,

preparing a medicine carrying system for carrying the anti-cancer medicine,

the drug-loading system for loading the anticancer drug comprises: the drug-loaded mesoporous silica nanoparticle comprises an inner core layer and an outer shell layer, wherein the inner core layer is mesoporous silica drug-loaded particles formed by mesoporous silica nanospheres and small-molecule complexes, the outer shell layer is formed by a targeting protein and a liposome membrane and is coated on the outer surface of the inner core layer, and the targeting protein comprises a protein expressed by fusion construction of H1299.2 target peptide, eGFP and MscL.

7. The preparation method of claim 6, wherein the mass ratio of the mesoporous silica nanospheres to the small molecule complex is 10:3-1: 1.

8. The method of claim 6, wherein the ratio of DSPE, cholesterol, and DSPE-PEG2000 to chloroform is 10:2:0.63: 10.

9. The preparation method of claim 6, wherein the addition amount of the targeting protein is 0.5mg, and the ratio of the liposome layer to the mesoporous silica sphere drug-loaded particle solution is 800 μ L to 100 μ L.

10. Use of a pharmaceutical carrier system according to any one of claims 1 to 5 for the preparation of an oral anti-cancer medicament.