WO2016058447A1

WO2016058447A1 - Nano drug carrier and preparation method and use thereof

Info

Publication number: WO2016058447A1
Application number: PCT/CN2015/087714
Authority: WO
Inventors: 刘昌胜; 屈雪; 李金阳
Original assignee: 华东理工大学
Priority date: 2014-10-17
Filing date: 2015-08-20
Publication date: 2016-04-21
Also published as: CN104324380A; CN104324380B

Abstract

Disclosed are a nano drug carrier and a preparation method and use thereof. The nano drug carrier is mesoporous silicon dioxide composite particles, comprising an inner core, a middle layer and an outer layer, wherein the inner core is nano-mesoporous silicon dioxide particles; the middle layer is located on the surface of the inner core, and comprises at least one self-assembling layer, wherein the self-assembling layers comprise concanavalin A and glycogen that are mutually combined; and the outer layer is a transferrin layer disposed on the surface of the middle layer. The preparation method is: using layer self-assembling membrane manufacturing technology, forming at least one concanavalin A-glycogen self-assembling layer on the surface of the nano-mesoporous silicon dioxide particles, and finally forming a supramolecular layer membrane using transferrin as the outer layer. The nano drug carrier of the invention has the dual characteristics of being tumour-targeting and stimuli-responsive.

Description

Nano drug carrier and preparation method and application thereof

Technical field

The invention belongs to the field of biotechnology, and relates to a drug delivery system, in particular to a tumor targeting and stimuli-responsive nano drug carrier regulated by a functional protein membrane, a preparation method and application thereof.

Background technique

The idea of developing a drug that selectively destroys diseased cells without affecting healthy cells was originally proposed by Nobel Prize winner and chemotherapist Paul Ehrlich in the early 20th century, which Ehrlich called "magic bullets" ( Magic bullets). In the past few decades, researchers have made some progress in developing this ideal drug with specific targeting. In the field of pharmaceutical preparations, various targeted methods such as injection, oral administration, transdermal and ocular and pulmonary mucosal administration have been explored and practiced, and the targeted routes have been continuously expanded. In addition, novel targeted delivery vehicle liposomes

Equal target formulations have been marketed to benefit patients. The emergence of nanomedicine carriers and their in-depth research in cell and molecular biology have opened up new horizons. Nanoscale-based drug carriers are able to ooze out of tumor blood vessels by Enhanced Permeability and Retention, and the loaded drug is more highly available than free drug molecules. However, in order to achieve intelligent drug delivery, it is necessary to give the nanocarrier its targeting and responsiveness, so as to avoid degradation or early leakage of the drug during the delivery process, so that the drug is specifically delivered to the tumor site, and its special physiology The environment responds by regulating the timing and location of drug release.

However, with the gradual deepening of research, in view of the complexity of the physiological environment in the body, people are increasingly aware that the targeted nano drug delivery system still faces many challenges. In the past, the design of drug carriers was often simple, and it was difficult to cope with the complex physiological environment in the body. Only for a certain part of the body, the mechanism is relatively simple. At present, people have begun to design more complex adaptive drug delivery systems or intelligent drug delivery systems, that is, by multi-composite design of the drug delivery carrier, it can self-regulate with time or the environment, or External stimuli respond to smoothing through various physiological barriers in the body for better targeting.

The pH value of normal tissues is 7.4. Due to abnormal proliferation, tumor cells are in anoxic state for a long time, and anaerobic glycolysis produces lactic acid, and the pH around the tissue is about 6.8. In addition, the endosomes and lysosomes in tumor cells are more acidic. The pH of early endosomes in tumor cells measured by electron and chemical probes is about 6.0, and some even lower than 5.4. The pH of late endosomes is generally around 5.0, and some are even lower than 4.0. These acidic environments in tumors can be used as signals to trigger rapid drug release and organelle targeting.

Nano-mesoporous silica is an ideal intracellular drug transport carrier because of its good biocompatibility, particle size and pore size, nanometer scale, high specific surface area and good lysosomal escape ability. A large number of studies have been conducted to introduce functionalized "nano-caps" on the surface of nano-mesoporous silica (MSN), giving them better drug controlled release and targeted functions. Nano-mesoporous silica is used as a drug carrier according to its controlled release mechanism. It can be divided into two major categories by chemical modification. The MSN mesoporous channel is chemically modified by carboxyl group, amino group, etc., and the group is conjugated with the drug. Connection, using the sensitivity of the linking unit to the acidic or reducing microenvironment, thereby controlling drug release; second, loading the drug into the mesoporous channel, using quantum dots, nano-ferric oxide, polyelectrolyte, bovine serum white The particles or macromolecules of the protein block the mesoporous channels, and regulate the release or retention of the drug by regulating the switch of the blocking agent. The latter has no requirement for the chemical structure, solubility, and electronegativity of the drug molecule. The drug is stored in the mesoporous channel by the physical load, which can better preserve the chemical structure and activity of the drug, and thus has a broad Application prospects.

However, most of the nano-mesoporous silica drug-loading systems still have shortcomings, which limits their clinical application: 1) the response conditions do not meet the physiological environment of the tumor cells; 2) the normal physiological environment before the drug reaches the tumor site Lower premature dispersion; 3). The preparation process is cumbersome, usually requires complex chemical modification of the nanocaps, and then the target molecules are complexed to the surface of the MSN support.

Therefore, it is necessary to conceive a novel nanometer mesoporous silica drug controlled release carrier which is simpler to prepare, has a strong targeting effect, and responds to the actual physiological environment of the tumor cells in response to release conditions.

Summary of the invention

The object of the present invention is to provide a novel functional protein membrane-regulated tumor targeting and stimuli-responsive nano drug carrier in view of the deficiencies of the existing tumor-targeted drug delivery system.

According to a first aspect of the present invention, a mesoporous silica composite particle comprising a core, a middle layer and an outer layer, wherein

The inner core is a nanometer mesoporous silica particle;

The middle layer is disposed on a surface of the inner core, and includes at least one self-assembled layer, and the self-assembled layer comprises concanavalin A and a glycoside which are combined with each other;

The outer layer is a transferrin layer disposed on the surface of the intermediate layer.

In another preferred embodiment, a layer of concanavalin A is further disposed between the intermediate layer and the outer layer.

In another preferred embodiment, the transferrin is a human-derived iron-saturated transferrin.

In another preferred embodiment, the intermediate layer has 1-15 layers of self-assembled layers, preferably 2-10 layers of self-assembled layers.

In another preferred embodiment, the mesoporous silica composite particles dissociate at the intermediate layer and the outer layer at pH 4.0-6.0.

In another preferred embodiment, the mesoporous silica composite particles are pyrolyzed at a temperature higher than 200 ° C to 550 ° C, and the weight loss rate is 20-30%.

In another preferred embodiment, the mesoporous silica composite particles have one or more of the following characteristics:

(1) the nanometer mesoporous silica particles have a pore diameter of 1.5-30 nm;

(2) the nanometer mesoporous silica particles have a particle size of 50-300 nm;

(3) the mesoporous silica composite particles have an average particle diameter of 60-350 nm;

(4) The mesoporous silica composite particles are pyrolyzed at a temperature higher than 200 ° C to 550 ° C, and the weight loss rate is 20-30%.

In another preferred embodiment, the mesoporous silica composite particles have a typical amide group vibration peak at about 1532 cm ^-1 and about 1468 cm ^-1 with respect to the nano mesoporous silica particles.

According to a second aspect of the invention, there is provided a method for preparing a mesoporous silica composite particle according to the first aspect, comprising the steps of:

(a) providing nano-mesoporous silica particles as a core;

(b) the nano-mesoporous silica particles are positively charged and dispersed in a solution containing a concanavalin A solution, and then dispersed into a solution containing a glycogen to form a self-assembly on the surface of the nano-mesoporous silica particles. a layer, optionally repeating the above steps to form a plurality of layers of self-assembled layers;

(c) Dispersing the particles obtained in the step (b) into the solution containing the concanavalin A solution, and then dispersing into the transferrin solution to obtain the mesoporous silica composite particles.

The nano mesoporous silica particles used in the present invention can be produced by various methods known in the art, and are not particularly limited. In another preferred embodiment, the nanomesoporous silica particles are prepared by the following steps: preparing an aqueous solution of cetyltrimethylammonium bromide. Adding an appropriate amount of ammonia water, adjusting the pH of the solution to above 12; adding tetraethyl orthosilicate through a separatory funnel, after completion of the reaction, extracting by filtration to obtain a white solid product; and drying the solid product by high temperature calcination to remove the template , nano-mesoporous silica particles can be obtained.

In another preferred embodiment, the mesoporous nano silica is dispersed in a polyethyleneimine solution (0.1-5 mg/mL, preferably 0.5-4.5 mg/mL) to make the nanometer mesoporous silica particles. Positively charged.

In another preferred embodiment, the method of preparation comprises one or more of the following features:

(1) The concentration (or content) of the concanavalin A in the solution containing the concanavalin A in the step (b) or the step (c) is 0.2-5 mg/mL, preferably 0.5- 3 mg/mL, more preferably 0.8-2.5 mg/mL;

(2) the concentration (or content) of the glycogen in the glycogen-containing solution of the step (b) is 0.2-5 mg / mL, preferably 0.5-3 mg / mL;

(3) The volume ratio of the nano-mesoporous silica particles in the step (b) to the solution containing the concanavalin A solution is 1-60 mg: 1 ml, preferably 10-50 mg: 1 ml. More preferably 30-45 mg: 1 ml;

(4) The volume ratio of the mass of the nano-mesoporous silica particles to the solution containing the saccharide in the step (b) is 1-60 mg: 1 ml, preferably 10-50 mg: 1 ml, more Good land is 30-45mg: 1ml;

(5) the concentration (or content) of transferrin in the transferrin solution is 0.2-5 mg / mL, preferably 0.5-3 mg / mL;

(6) The volume ratio of the mass of the particles obtained in the step (b) to the solution containing the concanavalin A solution is 1-60 mg: 1 ml, preferably 10-50 mg: 1 ml, more preferably 30- 45mg: 1ml;

(7) The volume ratio of the mass of the particles obtained in the step (b) to the transferrin solution is 1-60 mg: 1 ml, preferably 10-50 mg: 1 ml, more preferably 30-45 mg: 1 ml. .

In a third aspect of the invention, the use of the mesoporous silica composite particles of the first aspect is provided for the preparation of a pharmaceutical carrier.

In another preferred embodiment, the pharmaceutical carrier is a stimuli-responsive pharmaceutical carrier.

In another preferred embodiment, the drug carrier is a tumor-targeting drug carrier

In another preferred embodiment, the pharmaceutical carrier is a tumor targeting and stimuli responsive pharmaceutical carrier.

In another preferred embodiment, the drug carrier is capable of inhibiting drug leakage under simulated extracellular pH conditions (pH 6.8-7.4), in the middle and outer layers of the pH of the simulated intracellular endosomes (pH 4.5-6.0). Dissociation occurs, thereby inducing drug release.

According to a fourth aspect of the invention, there is provided a composite comprising:

a mesoporous silica composite particle according to the first aspect; and

Anti-cancer drugs.

In another preferred embodiment, the anticancer drug is selected from the group consisting of: doxorubicin, 5-fluorouracil, busulfan, bleomycin, vinblastine, docetaxel, cyclophosphamide, gemcitabine, methotrexate, Carboplatin, capecitabine, lomustine, hydroxyurea, cisplatin, mitomycin, etoposide, paclitaxel, gefitinib.

In another preferred embodiment, the mesoporous silica composite particles are dispersed in an anticancer drug solution to obtain the complex.

According to a fifth aspect of the invention, a pharmaceutical composition comprising the complex of the fourth aspect and a pharmaceutically acceptable carrier is provided.

A sixth aspect of the invention provides the use of the complex of the fourth aspect or the pharmaceutical composition of the fifth aspect for the preparation of a medicament for preventing and/or treating a tumor.

In another preferred embodiment, the tumor includes, but is not limited to, liver cancer, lung cancer (including mediastinal cancer), oral cavity Skin cancer, nasopharyngeal cancer, thyroid cancer, esophageal cancer, lymphoma, chest cancer, digestive tract cancer, pancreatic cancer, intestinal cancer, breast cancer, ovarian cancer, uterine cancer, kidney cancer, gallbladder cancer, cholangiocarcinoma, central nervous system cancer , testicular cancer, bladder cancer, prostate cancer, skin cancer, melanoma, meat cancer, brain cancer, blood cancer (leukemia), cervical cancer, glioma, stomach cancer, or ascites.

The mesoporous silica composite particles of the present invention are tumor targeting and stimuli-responsive nano drug carriers regulated by functional protein membranes. The invention adopts nano mesoporous silica as a drug carrier, and utilizes the bioreversible bond between Concanavalin Con A and the sugar unit as a driving force, and the concanavalin A and the glycogen and transferrin are assembled units. A multi-layered protein membrane structure was constructed on the surface of nano-mesoporous silica by layer-by-layer self-assembly technology. After drug loading, a nano drug delivery system with tumor targeting and stimuli responsiveness was obtained.

The invention has the characteristics of simple and mild preparation method, good biocompatibility, physiological response condition and high tumor targeting efficiency, and is suitable for targeted therapy of various tumors.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 is a transmission electron micrograph.

Figure 2 is an infrared spectrum.

Figure 3 is a thermogravimetric map.

Figure 4 is a graph showing the release profile of the antitumor drug doxorubicin under different pH conditions.

Figure 5 is a graph showing the biospecific binding force of transferrin and concanavalin.

Figure 6 is a graph showing the results of specific uptake of human hepatoma cell line HepG2 against tumor targeting and stimuli-responsive nano drug carriers by confocal microscopy.

Figure 7 is a graph showing the selectivity of tumor targeting and stimuli-responsive nano drug carriers to human hepatoma cells HepG2 and human normal liver cells L02 by flow cytometry.

Figure 8 is a graph showing the results of dissociation behavior of tumor targeting and stimuli-responsive functional protein membranes in human hepatoma cells HepG2 and human normal liver cells L02 by confocal microscopy.

Figure 9 is a graph showing the results of drug release behavior of tumor targeting and stimuli-responsive nano drug delivery systems in human hepatoma cells HepG2 and human normal liver cells L02 by confocal microscopy.

Figure 10 is a MTT assay for tumor targeting and stimuli-responsive functional protein membrane anti-tumor drug doxorubicin on human hepatoma cells HepG2, human breast cancer cells MDA-MB-231, human gastric cancer cells MGC-803, human normal liver cells L02 The results of the toxicity regulation of mouse myoblast C2C12.

detailed description

The inventors of the present application have extensively and intensively studied to develop a novel drug carrier for the first time, which is a mesoporous silica composite particle, the core is a nano mesoporous silica particle, and the nano mesoporous silica The surface of the particle has one or more layers of self-assembled layers comprising Concanavalin A and a glycoside bonded to each other, and the surface of the outermost self-assembled layer has a transferrin layer. The drug carrier of the invention utilizes the targeting function of the outer layer transferrin on the tumor cells, and the response dissociation function of the inner layer concanavalin A-glycan element in the low pH environment of the tumor cells can realize the carrier anti-tumor drug Targeted transmission, fixed-point release. On the basis of this, the present invention has been completed.

the term

Concanavalin A is a globulin-type lectin extracted from the giant bean, which is tetramer at pH 6.8 or higher, and each subunit corresponds to a sugar binding site. Under acidic conditions, the protein depolymerizes to form a dimer, and the sugar binding site changes from four to two. A glycoside is a branched polysaccharide composed of glucose, which is capable of specifically binding to concanavalin A with pH-dependent properties.

The transferrin receptor (TfR, also known as CD71) is a type II transmembrane glycoprotein on the surface of the cell membrane. The expression of TfR in normal cells is low, while the rapidly proliferating tumor cells are due to iron. The increased demand has resulted in a receptor expression that is about 100 times that of normal cells.

Transferrin (Tf) is a single-chain glycoprotein containing two N-oligosaccharide chains with a molecular weight of 7.7 kDa and a sugar content of about 6%. It is also capable of undergoing sugar residue-based organisms with concanavalin A. Specific binding.

According to the International Association of Pure and Applied Chemistry (IUPAC), pores smaller than 2 nm are called micropores; those with pore diameters larger than 50 nm are called macropores; those with pore sizes between 2 and 50 nm are called mesopores. hole). The mesoporous material is a novel material having a large specific surface area and a three-dimensional pore structure between pores and macropores. Mesoporous silica has large encapsulation, large specific surface area (>900m ² /g), easy modification of inner and outer surfaces, orderly pores, adjustable pore size (2-10nm), non-toxic, good biocompatibility and thermodynamic stability. Highly characterized, it is an ideal nano-container storage and release carrier.

Mesoporous silica composite particles

The invention adopts concanavalin A, glycoside and transferrin as assembly units, and uses the biospecific binding force as a driving force to form a surface on the surface of the nanometer mesoporous silica particles by using a layer-by-layer self-assembly film forming technology. Layer or multi-layered concanavalin A-glycan self-assembled layer, and forms a supramolecular layered membrane with transferrin as the outer layer, using the outer transferrin to target the tumor cells, concanavalin The dissociation function of A-glycogen on the tumor cell internal environment enables the targeted delivery and targeted release of the anti-tumor drug.

Specifically, the mesoporous silica composite particles of the present invention comprise a core, a middle layer and an outer layer, wherein

The inner core is a nanometer mesoporous silica particle;

The object of the invention is achieved by the following technical solutions:

Preparation of Mesoporous Nanosilica: A certain amount of cetyltrimethylammonium bromide is dissolved in an appropriate amount of deionized water and heated to stir until completely dissolved. Add an appropriate amount of ammonia water and adjust the pH of the solution to above 12. Ethyl orthosilicate was added dropwise via a separatory funnel. After completion of the reaction, the mixture was filtered with suction to give a white solid. The dried solid product is subjected to high temperature calcination to remove the templating agent to obtain mesoporous nano silica.

Construction of multi-layer self-assembled protein multilayer film: Weigh a certain amount of mesoporous nano-silica and disperse it in a positively charged polyelectrolyte solution to make mesoporous nano-silica positively charged. After centrifugation washing, the nanoparticles were sequentially dispersed in the concanavalin A solution and the glycogen solution, and this step was repeated until a specified number of layered self-assembled protein multilayer films were obtained on the surface of the mesoporous nanosilica. Finally, the nanoparticles were sequentially redispersed in the concanavalin A solution and the transferrin solution, and the protein membrane was subjected to targeted modification. The present invention also replaces Concanavalin A with FITC-labeled Concanavalin A to prepare a layered self-assembled protein multilayer film having a fluorescent label to study the responsiveness of the multilayer film.

Construction of a tumor targeting and stimuli-responsive drug delivery system: the above-mentioned nanoparticles coated with a protein multilayer film (ie, The mesoporous silica composite particles were dispersed in a higher concentration of doxorubicin solution and shaken overnight. The unloaded free drug molecules are centrifuged and washed, and the tumor-targeted and stimuli-responsive drug delivery system is obtained after lyophilization.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an active ingredient in a safe and effective amount, together with a pharmaceutically acceptable carrier.

The "active ingredient" as used in the present invention means a complex according to the present invention, comprising the mesoporous silica composite particles of the present invention; and an anticancer drug.

The "active ingredient" and pharmaceutical composition of the present invention can be used for the preparation of a medicament for preventing and/or treating a tumor.

By "safe and effective amount" is meant that the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. In general, the pharmaceutical compositions contain from 1 to 2000 mg of active ingredient per dose, more preferably from 10 to 200 mg of active ingredient per dose. Preferably, the "one dose" is a tablet.

"Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By "compatibility" it is meant herein that the components of the composition are capable of intermingling with the active ingredients of the present invention and with respect to each other without significantly reducing the efficacy of the active ingredients. Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid). , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as

), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.

The administration form of the active ingredient or the pharmaceutical composition of the present invention is not particularly limited, and representative administration forms include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) a disintegrant such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent such as paraffin; (f) Absorbing accelerators, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, hard Calcium citrate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or a mixture thereof. In capsules, tablets and pills, the dosage form may also contain a buffer.

The solid dosage forms can also be prepared with coatings and shell materials, such as casings and other materials known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric and waxy materials.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances. In addition to these inert diluents, the compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.

In addition to the active ingredient, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.

Compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

The complex or pharmaceutical composition of the invention may be administered alone or in combination with other therapeutic agents such as chemotherapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the complex of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective effective administration dose, and for a person having a weight of 60 kg, The daily dose is usually from 1 to 2000 mg, preferably from 20 to 500 mg. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

The above-mentioned features mentioned in the present invention, or the features mentioned in the embodiments, may be arbitrarily combined. All of the features disclosed in the present specification can be used in combination with any of the compositions, and the various features disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Therefore, unless otherwise stated, the disclosed features are only general examples of equal or similar features.

The invention is advantageous in that:

(1) The present invention utilizes the biospecific binding force between concanavalin and a glycosyl group to construct a concanavalin A-glycan as a middle layer and a transferrin on the surface of mesoporous nanosilica by layer self-assembly. The outer layer of supramolecular layered membrane obtains a nanomedicine carrier with dual characteristics of tumor targeting and stimuli response.

(2) The nano drug carrier of the invention has good biocompatibility, simple preparation, targeted selection of a plurality of tumor cells, and the response condition conforms to the cell microenvironment.

(3) The preparation method of the invention is simple, and the drug or the carrier is not modified, and the protein supermolecular membrane having the dual characteristics of tumor targeting and stimulating response is prepared by using the sugar binding function of concanavalin A.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Percentages and parts are by weight unless otherwise stated.

Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described may be employed in the methods of the invention. The preferred embodiments and materials described herein are for illustrative purposes only.

In the following examples, Con A represents concanavalin A, Gly represents glycoside, Tf represents transferrin, MSN represents mesoporous nanosilica, and subscript n represents concanavalin A-glycan self-assembled multilayer. The number of layers of the membrane, the superscript D represents the antitumor drug doxorubicin loaded, and the structure of the nanoparticles is represented by sequentially arranging the constituent units from the outside to the inside, for example, targeting the nano drug-loading particles Tf/Con A (Gly /Con A) ₄ -MSN ^D refers to the concanavalin A-glycan self-assembled multilayer membrane with outer layer of transferrin and 4 layers of middle layer, and the core is nanometer loaded with antitumor drug doxorubicin. Particles.

Example 1

Preparation of tumor targeting and stimuli-responsive drug carriers (mesoporous silica composite particles and composites)

1.116 g of cetyltrimethylammonium bromide was dissolved in 480 mL of deionized water and stirred at 50 ° C until the end Fully dissolved. Add 52.8 mL of ammonia water and adjust the pH of the solution to above 12. 5.6 mL of tetraethyl orthosilicate was added dropwise through a separatory funnel. After completion of the reaction, the product was obtained as a white solid. The dried solid product is subjected to high temperature calcination to remove the templating agent to obtain mesoporous nano-silica MSN. The transmission electron micrograph is shown in Fig. 1, and the particle diameter is about 50-100 nm.

200 mg of mesoporous nano-silica was weighed and dispersed in a 5 mL polyethyleneimine solution (4 mg/mL, 0.5 M NaCl solution) to make the mesoporous nano-silica positively charged. After centrifugation, the nanoparticles were dispersed in 5 mL of concanavalin A solution (2 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), stirred slowly for 30 min, and then centrifuged and washed. A 5 mL saccharide solution (2 mg/mL, Tris-HCl buffer) was added, and the mixture was stirred slowly for 30 minutes, followed by centrifugation and washing. By repeating the assembly procedure of the above-mentioned concanavalin A and glycoside, two double-layered protein supramolecular membranes can be constructed on the surface of mesoporous nanosilica. Finally, the nanoparticles were sequentially redispersed in concanavalin A (2 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ) and transferrin solution (2 mg/mL, 10 mM Tris). -HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), slowly stirred for 30 min, centrifuged, and washed.

The above-mentioned protein-coated multilayered nanoparticles were dispersed in a 4 mg/mL doxorubicin solution and shaken overnight. The unloaded free drug molecules are centrifuged and washed, and the tumor-targeted and stimuli-responsive mesoporous silica composite particles are obtained after lyophilization.

Example 2

1.116 g of cetyltrimethylammonium bromide was dissolved in 480 mL of deionized water and stirred at 50 ° C until completely dissolved. Add 52.8 mL of ammonia water and adjust the pH of the solution to above 12. 5.6 mL of tetraethyl orthosilicate was added dropwise through a separatory funnel. After completion of the reaction, the product was obtained as a white solid. The dried solid product is subjected to high temperature calcination to remove the templating agent to obtain mesoporous nano silica.

150 mg of mesoporous nanosilica was weighed and dispersed in 5 mL of polyethyleneimine solution (4 mg/mL, 0.5 M NaCl solution) to make the mesoporous nanosilica positively charged. After centrifugation, the nanoparticles were dispersed in 5 mL of concanavalin A solution (1.5 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), slowly stirred for 30 min, centrifuged, and washed; 5 mL of glycoside solution (2.5 mg/mL, Tris-HCl buffer) was stirred slowly for 30 min, then centrifuged and washed. By repeating the assembly procedure of the above-mentioned concanavalin A and glycoside six times, seven double-layered protein supramolecular membranes can be constructed on the surface of mesoporous nanosilica. This sample is designated as (Gly/Con A) ₇ -MSN, and its transmission electron micrograph is shown in Fig. 1, and has a particle diameter of about 60 to 100 nm. Finally, the nanoparticles were sequentially redispersed in concanavalin A (2.5 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ) and transferrin solution (2.5 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), slowly stirred for 35 min, centrifuged, and washed.

The above-mentioned protein-coated multilayered nanoparticles were dispersed in a 4 mg/mL 5-fluorouracil solution and shaken overnight. The unloaded free drug molecules are centrifuged and washed, and the tumor-targeted and stimuli-responsive mesoporous silica composite particles are obtained after lyophilization.

Example 3

180 mg of mesoporous nanosilica was weighed and dispersed in 5 mL of polyethyleneimine solution (4 mg/mL, 0.5 M NaCl solution) to make the mesoporous nanosilica positively charged. After centrifugation, the nanoparticles were dispersed in 5 mL of concanavalin A solution (2 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), stirred slowly for 30 min, centrifuged, and washed; 5 mL was added. The glycogen solution (2 mg/mL, Tris-HCl buffer) was stirred slowly for 30 min, then centrifuged and washed. By repeating the assembly steps of the above-mentioned concanavalin A and glycoside nine times, ten double-layered protein supramolecular membranes can be constructed on the surface of mesoporous nanosilica. Finally, the nanoparticles were redispersed in concanavalin A solution (2.5 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), slowly stirred for 35 min, and then centrifuged and washed. This sample is referred to as Con A(Gly/Con A) ₁₀ -MSN, and its transmission electron micrograph is shown in Fig. 1, and has a particle diameter of about 60 to 100 nm.

Comprehensive analysis of MSN, (Gly/Con A) ₄ -MSN, (Gly / Con A) ₇ -MSN and Con A (Gly / Con A) ₁₀ -MSN transmission electron micrograph (as shown in Figure 1), visible coating After the multilayer film, the mesoporous pores of the nano-mesoporous silica are covered by the protein film, and as the number of layers of the multilayer film increases, the granular structure of the surface of the nano-particles becomes more apparent, and from Con A (Gly /Con A) The transmission electron micrograph of ₁₀ -MSN can directly observe the granular structure of the particle size corresponding to the size of Concanavalin A, which directly proves the successful formation of the multilayer film.

Example 4

1.116 g of cetyltrimethylammonium bromide was dissolved in 480 mL of deionized water and stirred at 50 ° C until completely dissolved. Add 52.8 mL of ammonia water and adjust the pH of the solution to above 12. 5.6 mL of tetraethyl orthosilicate was added dropwise through a separatory funnel. After completion of the reaction, the product was obtained as a white solid. The dried solid product is calcined at a high temperature to remove the templating agent to obtain mesoporous silica MSN, and the transmission electron micrograph is shown in FIG.

200 mg of mesoporous nano-silica was weighed and dispersed in a 5 mL polyethyleneimine solution (4 mg/mL, 0.5 M NaCl solution) to make the mesoporous nano-silica positively charged. After centrifugation, the nanoparticles were dispersed in 5 mL of concanavalin A solution (2 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), stirred slowly for 30 min, and then centrifuged and washed. A 5 mL saccharide solution (2 mg/mL, Tris-HCl buffer) was added, and the mixture was stirred slowly for 30 minutes, followed by centrifugation and washing. By repeating the assembly procedure of the above concanavalin A and glycoside three times, four bilayers of protein supramolecular membranes can be constructed on the surface of mesoporous nanosilica, and the sample is designated as (Gly/Con A) ₄ -MSN.

In order to directly study the dissociation behavior of the multilayer film, Concanavalin A was labeled with FITC fluorescence, and the rest of the operations were the same. The nanoparticles coated with four bilayer protein supramolecular membranes were prepared. The sample was recorded as (Gly). /Con A@FITC) ₄ -MSN.

(Gly/Con A) ₄ -MSN was dispersed in concanavalin A solution (2 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), slowly stirred for 30 min, centrifuged, and washed. This sample is referred to as Con A(Gly/Con A) ₄ -MSN.

Distribute Con A(Gly/Con A) ₄ -MSN into transferrin solution (2mg/mL, 10mM Tris-HCl buffer containing 1mM Ca ²⁺ , 1mM Mn ²⁺ ), slowly stir for 30min, then centrifuge and wash This sample is referred to as Tf/Con A(Gly/Con A) ₄ -MSN.

Tf/Con A(Gly/Con A) ₄ -MSN was dispersed in a 4 mg/mL doxorubicin solution and shaken overnight. The unloaded free drug molecules were centrifuged and washed, and after lyophilization, the nano drug-loaded particles (drug-loaded complex) were obtained, and the sample was recorded as Tf/Con A(Gly/Con A) ₄ -MSN ^D .

Example 5

200 mg of the mesoporous nanosilica prepared in Example 1 was weighed and dispersed in a 5 mL polyethyleneimine solution (1 mg/mL, 0.5 M NaCl solution) to bring a positive charge to the mesoporous nanosilica. After centrifugation, the nanoparticles were dispersed in 5 mL of concanavalin A solution (1 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), slowly stirred for 30 min, and then centrifuged and washed. A 5 mL saccharide solution (1 mg/mL, Tris-HCl buffer) was added, and the mixture was stirred slowly for 30 minutes, followed by centrifugation and washing. By repeating the assembly steps of the above concanavalin A and glycoside four times, five double-layered protein supramolecular membranes can be constructed on the surface of mesoporous nanosilica, and the sample is recorded as (Gly/Con A) ₅ -MSN .

(Gly/Con A) ₅ -MSN was dispersed in concanavalin A solution (1 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), stirred slowly for 40 min, then centrifuged and washed. This sample is referred to as Con A(Gly/Con A) ₅ -MSN.

Disperse Con A(Gly/Con A) ₅ -MSN into transferrin solution (1 mg/mL, 10 mM Tris-HCl buffer containing 1 mM Ca ²⁺ , 1 mM Mn ²⁺ ), slowly stir for 40 min, centrifuge, wash This sample is referred to as Tf/Con A(Gly/Con A) ₅ -MSN.

Tf/Con A(Gly/Con A) ₄ -MSN was dispersed in a 5 mg/mL docetaxel solution and shaken overnight. The unloaded free drug molecules are centrifuged and washed, and the drug-loaded complex is obtained after lyophilization.

Example 6

The properties of each of the particles prepared in Example 4 were exemplified.

Transmission electron micrographs of MSN and (Gly/Con A) ₄ -MSN are shown in Fig. 1. It is shown that a film-like structure is formed on the surface of mesoporous silica by layer-by-layer self-assembly.

The infrared analysis spectrum of MSN, (Gly/Con A) ₄ -MSN is shown in Figure 2. Compared with the nano-mesoporous silica particles, the nanomaterials coated with the protein film are at 1532 cm ^-1 and 1468 cm ^-1 . The typical amide group vibration peak further demonstrates the successful construction of the protein membrane.

Thermogravimetric analysis (shown in Figure 3) of MSN, (Gly/Con A) ₄ -MSN shows that the middle layer and the outer layer are pyrolyzed at a temperature higher than 200 ° C to 550 ° C, and the weight loss rate is reached. 20-30% (the specific gravity of the protein film is about 20%-30%).

Example 7

Drug carrier adsorption and multi-layer membrane regulation of drug release

Weigh 10 mg of fluorescently labeled mesoporous silica composite particles, namely (Gly/Con A@FITC) ₄ -MSN, and disperse them to 1 mL of different pH release media (pH 7.4 PBS, pH 5.5 acetic acid / sodium acetate buffer) In the pH 5.0 acetic acid/sodium acetate buffer solution, the sample was placed in a 37 ° C constant temperature shaking box. After centrifugation every 1 h, 500 μL of the release medium was taken to measure the fluorescence intensity, and 500 μL of the same fresh release medium was added. The fluorescence intensity was plotted on the ordinate to study the dissociation behavior of the multilayer film.

As shown in (A) of Figure 4, the multilayer film can be kept stable under neutral conditions; at pH 5.5, the multilayer film can be dissociated to a certain extent; at pH 5.0, the multilayer film is rapidly A response occurred, and the degree of dissociation in the first 2 hours reached about half of the first 12 hours. The layer-by-layer self-assembled multilayer film has the characteristics of maintaining structural stability in a neutral environment and different degrees of dissociation under weakly acidic conditions.

Ten mg of the nano drug-loaded particles (Gly/Con A) ₄ -MSN ^D prepared in Example 4 were weighed and dispersed in 1 mL of DMSO to destroy the surface protein supramolecular membrane and dissolve the drug molecules supported in the mesoporous channels. After centrifugation, the absorbance at the characteristic absorption peak in the supernatant is measured until no drug is released again, and the drug content in the material can be obtained by cumulatively calculating the drug release amount.

Weigh 10 mg of the above-mentioned nano drug-loaded particles (Gly/Con A) ₄ -MSN ^D and disperse them into 1 mL of different pH release media (pH 7.4 PBS, pH 6.8 PBS, pH 6.0 PBS, pH 5.5 acetic acid / sodium acetate buffer). In the solution, pH 5.0 acetic acid/sodium acetate buffer, pH 4.0 acetic acid/sodium acetate buffer, the sample was placed in a constant temperature shaking box at 37 °C. After centrifugation at 1 h, 500 μL of the release medium was taken to measure the absorbance at the characteristic absorption peak. At the same time, add 500 μL of the same fresh release medium to make a cumulative release curve.

As shown in (B) and (C) of Fig. 4, in the pH 7.4 simulating normal human body fluid, the drug was almost not released within 12 hours, which is consistent with the experimental results that the multilayer film remained structurally stable at pH 7.4. It is indicated that the self-assembled layer is firmly fixed on the surface of the MSN, thereby ensuring that the nano drug-loaded particles remain stable during the circulation in the body, and avoids side effects caused by early escape of the drug.

The release behavior of the nano drug-loaded particles in the simulated intracellular weakly acidic conditions is basically consistent with the dissociation behavior of the multilayer film. Under the pH 5.5 of the simulated weak body environment, the degree of dissociation of the multilayer membrane is not high, but the degree of cross-linking is decreased, thus “opening” the mesoporous channel and inducing drug release. The release rate after about 12 h is about 45%; at pH 5.0 Under the simulated lysosome pH environment, the multilayer film rapidly dissociated. After 7h, the release rate reached 50%, and the 12h release rate was about 70%.

In summary, under the regulation of the protein membrane, the stimuli-responsive mesoporous silica composite particles of the present invention achieve controlled release of the drug, and the response condition is in accordance with the physiological environment, and is an ideal intracellular drug transport carrier.

Example 8

Biospecific binding between concanavalin A and transferrin

When both ligand A and ligand B are capable of binding to the receptor R and the affinity of molecule A is higher, the high affinity ligand A is first premixed with the acceptor, and then the low affinity ligand B is added. The body will preferentially interact with ligand A, and only a small fraction of ligand A is replaced by ligand B from the binding site, so there will be only a small apparent heat change.

Based on the above theory, the present invention firstly studied the thermodynamic binding of transferrin and concanavalin A using an isothermal titration calorimeter (ITC 200, Micarcal, Inc.) and has been conjugated to the sugar binding site of concanavalin A. When the high affinity ligand methyl-α-D-mannopyranoside is occupied, the competitive thermodynamic binding of transferrin to it is demonstrated, and the interaction between transferrin and Con A is based on transferrin Tf. Biospecific binding between the sugar chain and Con A.

The specific operations are as follows:

First, transferrin (denoted as Tf) and concanavalin A were combined into a solution, which was degassed by high speed centrifugation (8000 rpm, 3 min) after passing through a 220 nm filter. Set the experimental temperature to 25 ° C, the reference power 5 μcal / sec, the stirring speed of 1500 rpm; add 200 μL of Concanavalin A solution in the sample cell, 40 μL of transferrin solution in a syringe, drop one drop every 120s, 1.5 drops per drop μL, using the Origin plug provided by Microcal, Inc. for data processing, using a single binding model to calculate the thermodynamic parameters such as binding constant, number of binding sites, and molar binding enthalpy. In order to further study the biospecific binding between the two, pre-mixed concanavalin A and methyl-α-D-mannopyranoside (molar ratio = 1:100), and stored at 4 ° C for 2 h to fully After the bond and the load, 200 μL was loaded into the sample cell, and the transferrin solution was loaded into the syringe, and the concanavalin A concentration and the transferrin concentration were consistent with the previous experiment, and titration was performed.

The photograph of A in Fig. 5 shows that transferrin is cross-linked with concanavalin A at pH 7.4 to form a precipitate, and the process is high-affinity ligand methyl-α-D-pyridine of concanavalin A. Inhibition by melanoside (Me-α-man) initially demonstrated the existence of a biospecific binding interaction between transferrin and concanavalin A. C in Figure 5 indicates that methyl-α-D-mannopyranoside (Me-α-man) is a high-affinity ligand for concanavalin A.

Further study on the affinity between the two by isothermal titration calorimetry (B, D in Fig. 5) showed that the binding constant between concanavalin A and transferrin was 5.71×10 ⁶ M ^-1 , which was a single The sugar Me-α-man is nearly 800 times; the number of binding sites between the two is about 0.188, that is, there are 5.3 sugar binding sites on each transferrin that can bind to the Con A monomer.

In Figure 5, D shows that the presence of me-α-man greatly reduces the amount of heat released by transferrin to the concanavalin A titration process, demonstrating that the binding of transferrin to concanavalin A is related to the sugar recognition process. Although according to the calculation results, the binding constant of transferrin-concanavalin A is greater than that of me-α-man-concanavalin A, transferrin competitively replaces me-α-man with concanavalin A. may. Due to the large amount of me-α-man in the concanavalin A solution, it will actually inhibit the substitution of transferrin, which leads to a significant reduction in the amount of heat released during the titration process, which strongly proves Concanavalin A and transfer iron. Biospecific binding between proteins.

Example 9

Tumor targeting

Using human hepatoma cell line HepG2 as a cell model, mesoporous silica was fluorescently labeled with FITC, mesoporous silica (MSN) and unmodified transferrin mesoporous silica composite particles (Con A(Gly) /Con A) ₄ -MSN) is a control group, and pre-incubation by adding free transferrin to the culture medium to occupy the cell surface transferrin receptor binding site by confocal microscopy and flow cytometry Semi-quantitative and quantitative analysis were performed separately to further investigate the specific recognition of tumor-targeted mesoporous silica composite particles by transferrin receptor.

The specific operation was as follows: HepG2 cells were seeded at a density of 2 × 10 ⁴ /well in a NEST laser confocal special glass bottom petri dish to grow adherently. For the free transferrin competition experiment, one group (named Tf/Con A(Gly/Con A) ₄ -MSN+ competition factor Tf group) was removed after co-culture for 22 h, and the addition of 200 μg/mL was added. The cell culture medium of ferritin is pre-incubated. After 2 h, the wells were removed and cell culture media containing 50 μg/mL of different functionalized nano drug carriers were added. After co-culture for 4 h, the cell culture medium containing nanoparticles was removed, washed 3 times with PBS, the unintaked nanoparticles were removed, fixed in 1% glutaraldehyde solution for 15 min, labeled with DAPI, and passed through a confocal microscope. (Nikon A1R) Observed the intracellular distribution of nanoparticles and drugs. The wavelength is set as follows: DAPI channel excitation at 404.3 nm, reception at 450.0 nm; FITC channel (ie MSN) excitation at 488.0 nm, reception at 525.0 nm, observation under 60 times oil mirror, as shown in Figure 6, respectively, from left to right The MSN group, the Con A (Gly/Con A) ₄ -MSN group, the Tf/Con A (Gly/Con A) ₄ -MSN group, and the Tf/Con A (Gly/Con A) ₄ -MSN+ competition factor Tf group.

The results showed that HepG2 cells were very limited in the uptake of MSN without transferrin modification. After the transfer of transferrin on the outermost layer of the protein membrane, a large number of MSN particles appeared around the nucleus, indicating that the MSN was transferred. Ferritin modification greatly improved the uptake efficiency of nanoparticles by HepG2 cells. Competition experiments have further shown that this uptake is a specific uptake mediated by transferrin receptors.

Example 10

Tumor targeting

In this embodiment, a human liver cancer cell line HepG2 and a human normal liver cell L02 are used as a cell model.

HepG2 cells and L02 cells were seeded in a 6-well plate at a density of 40 × 10 ⁴ /well to grow adherently. For the free transferrin competition experiment, one group of HepG2 cells (designated Tf/Con A (Gly/Con A) ₄ -MSN+ competition factor Tf group) was removed from the cell culture medium after co-culture for 22 h, and the addition was 200 μg/ The mL culture medium of transferrin is pre-incubated. After 2 h, the wells were removed and cell culture media containing 50 μg/mL of different functionalized nano drug carriers were added. After co-culture for 4 h, the cell culture medium containing the nanoparticles was removed, washed twice with PBS, digested with trypsin for 1-2 minutes, and the cells were collected into a centrifuge tube, centrifuged at 1000 rpm/5 min, and washed twice with PBS. In order to prevent the nanoparticles adhering to the cell surface from interfering with the experimental results, extracellular fluorescence was quenched by adding 1 mL of 0.4% trypan blue solution, and the residual trypan blue flow cytometry was washed away with PBS. The results are shown in Fig. 7. Show.

The results showed that the uptake rate of unmodified MSN by HepG2 cells and L02 cells was about 20%; after MSN was modified by (Con A/Gly) ₄ multilayer membrane (self-assembled layer), it may be due to Con A and cell membrane surface. The specific binding of some glycoproteins mediated a part of endocytosis, and the uptake rate of HepG2 cells and L02 cells increased to about 30%. After the outermost layer of the multilayer film (self-assembled layer) was connected to Tf, the system was given The excellent targeting performance, the surface of HepG2 cells due to the presence of a large number of TfR1 and TfR2, and the specific recognition of Tf on the surface of nanoparticles mediated a large number of endocytosis, the uptake rate reached 60%. At the same time, since the sugar binding site of Con A on the surface of the nanoparticle is mostly occupied by Tf, the specific binding of the particle to the glycoprotein on the cell membrane surface no longer exists, and the uptake rate of L02 cells is reduced to about 25%.

The above results indicate that the mesoporous silica composite particles of the present invention have excellent tumor targeting properties.

Example 11

Dissociation behavior of nano drug carriers in tumor cells and normal cells

The mesoporous silica was labeled with a red fluorescent probe, Texas Red, and the concanavalin A was labeled with a green fluorescent probe FITC to synthesize a dual fluorescently labeled nano drug carrier, and the nanoparticles were co-cultured with the cells. Confocal fluorescence microscopy was used to study the uptake of nano drug carriers and the dissociation behavior of multilayer films.

The specific operation was as follows: HepG2 and L02 cells were seeded at a density of 2×10 ⁴ /well in a special glass-bottomed Petri dish of NEST laser confocal, and the cell culture medium was removed after adherent growth for 24 hours, and a double fluorescence of 20 μg/mL was added. The cell culture medium of the labeled nano drug carrier was incubated for 3 h, 8 h, and 24 h, respectively, and washed with PBS three times to remove the unintaked nanoparticles, and fixed in a 1% glutaraldehyde solution for 15 min, and the nuclei were labeled with DAPI. Laser confocal microscopy (Nikon A1R) was used to observe the intracellular distribution of nanoparticles and drugs.

The wavelength settings are as follows: DAPI channel excitation at 404.3 nm, reception at 450.0 nm; FITC channel (ie Con A) excitation at 488.0 nm, reception at 525.0 nm; Texas Red channel (ie MSN) excitation at 561.0 nm, reception at 595.0 nm, Observed under a 60-fold oil microscope, the results are shown in Fig. 8.

The results showed that the uptake of the targeted drug carrier by HepG2 cells was significantly higher than that of L02 cells, and scattered green fluorescence was observed in the cytoplasm of HepG2 cells, indicating that the multi-layer membrane was weakly acidic in the cell after targeting the nano drug carrier into the cell. Dissociation occurs under the stimulation of the environment, releasing Con A-FITC.

Example 12

Drug release behavior of nano drug-loading systems in tumor cells and normal cells

Using the autofluorescence characteristics of doxorubicin itself, mesoporous silica was labeled with the red fluorescent probe Texas Red, and a multi-layered targeting protein membrane was constructed on the surface (MSN drug-loaded with uncoated protein membrane) The particles, MSN ^{D, were used} as the control group. After the drug was loaded, the cells were co-cultured, and the uptake and drug release behavior of the drug-loaded nanoparticles were observed under a confocal fluorescence microscope.

The specific operations are as follows:

HepG2 and L02 cells were inoculated into the NEST laser confocal special glass bottom culture dish at a density of 2×10 ⁴ /well, and the cell culture medium was removed after adherent growth for 24 hours, and the doxorubicin content was 0.5 μg/mL. The cells of the above-mentioned fluorescently labeled drug-loaded nanoparticles were incubated for 3 or 8 hours respectively, and then washed three times with PBS to remove the unintaked nanoparticles, and fixed in a 1% glutaraldehyde solution for 15 minutes, and the nuclei were labeled with DAPI. The intracellular distribution of nanoparticles and drugs was observed by laser confocal microscopy (Nikon A1R).

The wavelength settings are as follows: DAPI channel excitation at 404.3 nm, reception at 450.0 nm; DOX channel excitation at 488.0 nm, reception at 525.0 nm; Texas Red channel (ie MSN) excitation at 561.0 nm, reception at 595.0 nm, 60 times under oil mirror Observed, the results are shown in Figure 9.

HepG2 cells were co-cultured with the material for 3 h, and it was found that the MSN drug-loaded particles were significantly higher in the uptake amount and intracellular drug level of the targeted nano drug-loaded particles than the unmodified protein film. After the cells were co-cultured with the targeted nano drug-loaded particles, the drug was uniformly dispersed in the cytoplasm at 3 h, and the cells showed green fluorescence; and at this time, since the MSN particles still coexisted with the drug, they exhibited superimposed yellow fluorescence.

At 8h, the nano drug-loaded particles showed red fluorescence due to the large amount of drug escaping; and the behavior of nuclear concentration and cell shrinkage and rounding was observed, indicating that the cells undergo apoptosis under the action of drugs.

For L02 cells, the uptake of the two nanoparticles was small, and the intake did not increase with time. However, some of the nanoparticles are still taken up by the clathrin-mediated pathway, and drug leakage occurs under the stimulation of intracellular lysosomes.

Overall, the accumulation of drugs in L02 cells was much lower than that of HepG2, and the cell morphology was maintained in a normal state after co-culture.

Example 13

The role of functional protein membranes in regulating the cytotoxicity of anti-doxorubicin against different tumor cell lines and normal cell lines

Targeted drug-loaded nanoparticles (Tf/Con A(Gly/Con A) ₄ -MSN ^D ) and different tumor cell lines (human hepatoma cell HepG2, human breast cancer cell MDA-MB-231, human gastric cancer cell MGC-) 803) Co-cultured with normal cell line (human normal liver cell L02, mouse myoblast C2C12), using mesoporous silica drug-loaded particles (MSN ^D ) without uncoated protein membrane and particle-free blank as control group The targeting and inhibition of tumor cells were evaluated by MTT assay.

The specific operations are as follows:

The cells were seeded at a density of 2 × 10 ⁴ /well in 24-well plates (6 parallel experiments in each set of experiments), and after 24 hours of adherent growth, one of them (Tf/Con A(Gly/Con A) ₄ -MSN ^D + competition factor Tf) was preincubated with 1 mL of cell culture medium containing 200 μg/mL transferrin for 30 min to occupy the TfR1 and TfR2 binding sites on the cell membrane surface. Cell culture medium was removed from each well, and mesoporous silica drug-loaded nanoparticles (MSN ^D ) and targeted drug-loaded nanoparticles (Tf/Con A(Gly/Con A) ₄ -MSN ^D with different doxorubicin contents were added ^. 1 mL of the suspension culture solution was cultured for 4 hours, and the cell culture solution was removed, and washed with PBS three times to remove the nanoparticles not taken up by the cells, and the culture was continued for 44 hours. The cell viability was evaluated by the MTT method, and the results are shown in Fig. 10.

Compared with the MSN drug delivery system MSN ^D without the multi-layer membrane structure, the targeting drug-loaded nanocarrier Tf/Con A(Gly/Con A) ₄ -MSN ^D can increase the inhibition rate of tumor cells, which is normal. The toxicity of the cells is greatly reduced. This is because the mesoporous silica composite particles are rapidly endocytosed by the tumor cells mediated by the transferrin receptor, and rapidly release the drug under the stimulation of the intracellular microenvironment of the tumor, so that the inhibition of the tumor cells is stronger; However, normal cells can only target drug-loaded nanoparticles through clathrin-mediated non-specific endocytic uptake, and the toxicity of the drug-loading system is greatly reduced. After the addition of the competitive factor Tf, the toxicity of the targeted drug-loaded nanoparticles on tumor cells decreased, and it was confirmed that the inhibition rate of the tumor cells was based on the transferrin receptor-mediated active targeting.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

a mesoporous silica composite particle, comprising: an inner core, a middle layer and an outer layer, wherein

The inner core is a nanometer mesoporous silica particle;

The middle layer is disposed on a surface of the inner core, and includes at least one self-assembled layer, and the self-assembled layer comprises concanavalin A and a glycoside which are combined with each other;

The outer layer is a transferrin layer disposed on the surface of the intermediate layer.
The mesoporous silica composite particle according to claim 1, wherein the intermediate layer has 1 to 15 self-assembled layers, preferably 2 to 10 self-assembled layers.
The mesoporous silica composite particle according to claim 1, wherein the mesoporous silica composite particles are dissociated from the intermediate layer and the outer layer at pH 4.0 to 6.0.
The mesoporous silica composite particle according to claim 1, wherein the mesoporous silica composite particle has one or more of the following characteristics:

(1) the nanometer mesoporous silica particles have a pore diameter of 1.5-30 nm;

(2) the nanometer mesoporous silica particles have a particle size of 50-300 nm;

(3) the mesoporous silica composite particles have an average particle diameter of 60-350 nm;

(4) The mesoporous silica composite particles are pyrolyzed at a temperature higher than 200 ° C to 550 ° C, and the weight loss rate is 20-30%.
A method for preparing mesoporous silica composite particles according to claim 1, wherein the preparation method comprises the following steps:

(a) providing nano-mesoporous silica particles as a core;

(b) the nano-mesoporous silica particles are positively charged and dispersed in a solution containing a concanavalin A solution, and then dispersed into a solution containing a glycogen to form a self-assembly on the surface of the nano-mesoporous silica particles. a layer, optionally repeating the above steps to form a plurality of layers of self-assembled layers;

(c) Dispersing the particles obtained in the step (b) into the solution containing the concanavalin A solution, and then dispersing into the transferrin solution to obtain the mesoporous silica composite particles.
The preparation method according to claim 5, wherein the preparation method comprises one or more of the following features:

(1) the concentration of the concanavalin A in the solution containing the concanavalin A in the step (b) or the step (c) is 0.1-5 mg/mL, preferably 0.2-3 mg/mL;

(2) the concentration of the glycogen in the glycogen-containing solution of the step (b) is 0.2-5 mg / mL, preferably 0.5-3 mg / mL;

(3) The volume ratio of the nano-mesoporous silica particles in the step (b) to the solution containing the concanavalin A solution is 1-60 mg: 1 ml, preferably 10-50 mg: 1 ml. More preferably 30-45 mg: 1 ml;

(4) The volume ratio of the mass of the nano-mesoporous silica particles to the solution containing the saccharide in the step (b) is 1-60 mg: 1 ml, preferably 10-50 mg: 1 ml, more Good land is 30-45mg: 1ml;

(5) the concentration of transferrin in the transferrin solution is 0.2-5 mg / mL, preferably 0.5-3 mg / mL;

(6) The volume ratio of the mass of the particles obtained in the step (b) to the solution containing the concanavalin A solution is 1-60 mg: 1 ml, preferably 10-50 mg: 1 ml, more preferably 30- 45mg: 1ml;

(7) The volume ratio of the mass of the particles obtained in the step (b) to the transferrin solution is 1-60 mg: 1 ml, preferably 10-50 mg: 1 ml, more preferably 30-45 mg: 1 ml. .
Use of the mesoporous silica composite particles according to any one of claims 1 to 3, which is for use in the preparation of a pharmaceutical carrier.
A composite characterized in that the composite comprises:

The mesoporous silica composite particle according to any one of claims 1 to 3;

Anti-cancer drugs.
A pharmaceutical composition comprising the complex of claim 8 and a pharmaceutically acceptable carrier.
Use of the complex according to claim 8 or the pharmaceutical composition according to claim 9, for the preparation of a medicament for the prevention and/or treatment of a tumor.