CN101721709B - Calcium phosphate and amphiphilic polymer composite drug-loaded nano-microspheres, preparation method and use - Google Patents

Abstract

The invention relates to a calcium phosphate and amphiphilic polymer composite medicament-carrying nano-microsphere, a preparation method and application in medicament carriers. Specifically, the calcium phosphate and amphiphilic polymer composite medicament-carrying nano-microsphere can carry different kinds of medicaments; the medicament-carrying nano-microsphere is modified by a modifier to have multi-functional characteristics such as tumor targeting function, reversing medicament-resistant function and medical diagnosis function; and the application of the composite medicament-carrying nano-microsphere comprises diagnosis and therapy for diseases. The calcium phosphate and amphiphilic polymer composite medicament-carrying nano-microsphere has the advantages of simple preparation, high bioavailability, strong sustained-release function, controllable degradation time, good biocompatibility of materials and the like, and has stronger medicinal therapy and diagnosis functions and can be used as a safe and effective clinical medicament.

Description

Calcium phosphate and amphiphilic polymer composite drug-loaded nano-microspheres, preparation method and use

technical field

The invention belongs to the application field of nanometer materials, in particular to calcium phosphate and amphiphilic polymer composite drug-carrying nano microspheres, a preparation method and its use in the preparation of carrier drugs.

Background technique

The route and method of administration of pharmaceutical preparations are crucial to the action of drugs, and improving the utilization and curative effect of drugs and reducing the side effects of drugs has always been an important research topic in the field of medicine. Nano-drug loading system is the main application form of nanotechnology in drug delivery and controlled release. It refers to the use of appropriate pharmaceutical techniques and methods to make drugs and pharmaceutical excipients into colloidal particles with a particle size in the range of 1-1000nm. Drug systems, including basic forms such as nanoparticles, nanospheres, and nanocapsules. Active components (drugs, biologically active substances, etc.) are distributed inside the particles through dissolution and encapsulation, or are located on the surface of the particles through adsorption. Nanocarriers have the advantages of high targeting, controlled drug release, and improved dissolution and absorption rates of insoluble drugs, which can increase the stability of drugs in vivo, improve drug efficacy, and reduce toxic and side effects. At the same time, specific targeting molecules, such as specific ligands, monoclonal antibodies, etc., can be coupled on the surface of nanoparticles, and the tertiary targeted delivery of genes/drugs can be realized by binding the targeting molecules to specific receptors on the cell surface.

Polymers and polymer micelle systems mainly include modified amphiphilic polymers and block copolymers. Supramolecular aggregates with a hydrophobic core and a hydrophilic shell formed by microphase separation of amphiphilic polymers in aqueous media. The nano-micelle is mainly used as a solubilizing carrier for insoluble drugs in the pharmaceutical field, avoiding adverse reactions of small molecule surfactant solubilizers (such as Tween, polyoxyethylene castor oil), and its hydrophobic inner core can also protect drugs. It does not degrade and controls the slow release of drugs, while the hydrophilic shell helps the long-term circulation of nanomicelles in the blood and the targeting of tumors, inflammation and other tissues. In recent years, many newly synthesized multifunctional The polymer micelle system, which can not only be used as a gene delivery system, but also realize the co-loading and co-therapy of insoluble water-soluble drugs and genes. The main physical methods for polymer micelles to entrap insoluble drugs in water are: direct dissolution method, dialysis method and emulsification method. The choice of method mainly depends on the solubility of the amphiphilic copolymer in water. If the water solubility is good, the direct dissolution method is used; otherwise, the dialysis method is used. Drugs can be loaded into the inner core of micelles by methods such as physical embedding and chemical binding.

Calcium phosphate is widely used because of its good biocompatibility, degradability, bone conduction and bone integration ability, and safety and non-toxic properties. The performance of calcium phosphate depends largely on its morphology, phase, structure, crystallinity and other factors, and the composite of calcium phosphate and polymer can introduce the excellent characteristics of polymer into the calcium phosphate system to prepare the calcium phosphate system. Nano-microspheres with controllable appearance and powerful drug-loading function. Zhang et al. used polyvinylpyrrolidone (PVP) as a capping agent to prepare hydroxyapatite (HAP) nanorods with a higher specific surface area [Zhang YJ, Lu JJ.Crystal Growth & Design, 2008, 8 (7): 2101 -2107]; People such as Zhu prepared the flower-shaped calcium hydrogen phosphate in the water/ethylene glycol system with the small molecule surfactant sodium dodecyl sulfate (SDS) as the morphology regulator, and converted it into HAP keeps its morphology well [Ma MG, Zhu YJ and Chang J.J.Phys.Chem.B, 2006, 110: 14226-14230]; Mann et al. used polyacrylic acid (PAA ) as a template, polymer/calcium phosphate composite nanocapsules were prepared through biomineralization, and the material has the characteristics of pH response [Perkin KK, Turner JL, Wooley KL and MannS.Nano Letters, 2005, 5(7): 1457- 1461].

Contents of the invention

The present invention aims to provide a calcium phosphate and amphiphilic polymer composite drug-loaded nano-microsphere, a preparation method and the use of preparing carrier medicine, wherein the nano-particle is composed of an amphiphilic copolymer and calcium phosphate [Ca ₃ (PO4) ₂ ] Composites.

The composite drug-loaded nano-microspheres of calcium phosphate and amphiphilic polymer provided by the present invention, wherein the nano-microsphere is a complex composed of amphiphilic polymer and calcium phosphate, and contains drugs; the amphiphilic The percentage by weight of non-toxic polymer, calcium phosphate and the contained drug is 1%-60%, 0.1%-60% and 0.1%-50%; the calcium phosphate and amphiphilic polymer composite nano-microsphere The particle size is between 1-100000nm, wherein the particle size is preferably 5-5000nm.

The calcium phosphate and amphiphilic polymer composite drug-loaded nano-microspheres of the present invention are characterized in that the composition and preparation method of the nano-microspheres composed of amphiphilic polymers and calcium phosphate are preferably as follows:

Add the amphiphilic polymer to water at 0-60°C to prepare solution A with a mass fraction of 0.1-5%; add soluble calcium salt CaCl ₂ to solution A and stir to form a solution with a calcium ion concentration of 0.005-1M Solution B: Add dropwise or pour soluble phosphate solution (NH ₄ ) ₂ HPO ₄ with a concentration of 0.005-1M into solution B, so that the molar ratio of calcium ions and phosphate ions is 1:1-1.67:1, and the pH The value is controlled at 7-12; the stirring reaction is continued, and then the milky white reaction liquid is collected by centrifugation, repeatedly washed with deionized water and absolute ethanol, and vacuum-dried to obtain calcium phosphate/amphiphilic polymer composite porous nanospheres.

The calcium phosphate and amphiphilic polymer composite drug-carrying nano-microsphere of the present invention is characterized in that the drug-carrying method of calcium phosphate and amphiphilic polymer composite nano-microsphere not only includes adsorption method and solvent volatilization method, but also includes Dialysis method, pH gradient method, double emulsion method, complex coacervation method, etc., the drug loading sequence can be after the preparation of calcium phosphate and amphiphilic polymer composite nanospheres or during the preparation of nanospheres, such as The drug-loaded polymer micelles are prepared first, and then compounded with calcium phosphate to prepare drug-loaded micro-nanospheres.

The main methods are preferably as follows:

(1) Encapsulation of drugs during the preparation of nanospheres

Among them, the preferred method is to first configure solution A1 containing 0.1-5% amphiphilic polymer by weight and 0.1-5% drug by weight; or configure amphiphilic polymer nanoparticles containing drugs, Then prepare 0.1-5% drug loading and 0.1-5% amphiphilic polymer nanoparticle solution A2 by weight; then add soluble calcium salt CaCl ₂ to solution A1 or A2 respectively, and stir for 0.5-24h , to _{form a solution B} with _a calcium ion concentration of 0.005-1mol/L _; The ratio is 1/3-3/3, using ammonia water to adjust the pH value to 7-12, continuing to stir and react for 0-24 hours, collecting by centrifugation, washing, and drying to obtain composite drug-loaded nanospheres of calcium phosphate and amphiphilic polymer.

(2) Encapsulated drugs after preparation of nano-microspheres

The preferred method of loading water-soluble drugs is:

At room temperature, dry calcium phosphate and amphiphilic polymer composite nanospheres are mixed with an aqueous solution containing water-soluble drugs, and the weight percentage concentration of the calcium phosphate and amphiphilic polymer composite nanospheres and water-soluble drugs is 0.1-99% and 0.1-50% respectively, stirring for 0.5-24h, and centrifuging to collect the drug-encapsulated calcium phosphate and amphiphilic polymer composite nano-microspheres.

The preferred method for encapsulating fat-soluble drugs is:

First, the fat-soluble drug (0.1-30% by weight concentration) is dissolved in a mixed solvent composed of water and ethanol, and the volume ratio of the water and ethanol is 1/3-4/3; then dry calcium phosphate and Amphiphilic copolymer composite porous nano-microspheres, the calcium phosphate, amphiphilic polymer composite nano-microspheres and water-soluble drug have a weight percent concentration of 0.1-99% and 0.1-50% respectively, and are stirred for 0.5-24h , centrifuging to collect the drug-loaded calcium phosphate and amphiphilic polymer composite nanospheres.

Wherein the preferred scheme of solvent evaporation method is:

Dissolve fat-soluble drugs (0.1-30% by weight) in organic solvents such as chloroform to obtain an oil phase, then dissolve dry calcium phosphate and amphiphilic polymer composite nanospheres in an aqueous solution to obtain a water phase, and finally The water phase and the oil phase are mixed and stirred, the weight percent concentrations of the calcium phosphate and amphiphilic polymer composite nanospheres and the water-soluble drug are respectively 0.1-99% and 0.1-50%, and the oil phase is organic The volume ratio of the solvent to the aqueous solution is 1/3-5/3; after the solvent is volatilized, the calcium phosphate and the amphiphilic polymer composite nano-microspheres loaded with drugs are collected by centrifugation.

The calcium phosphate and amphiphilic polymer composite drug-loading nano microsphere of the present invention is characterized in that the used amphiphilic polymer includes polyester, polyamino acid, polylactic acid, phospholipid and copolymers thereof.

Among them, polycaprolactone (PCL), Pluronic (PEO-PPO-PEO), polyamino acid (PAGA), polyethylene glycol-polylactic acid (PEG-PLA), polyethylene glycol-(polylactic acid- co-polyglycolic acid) (PEG-PLGA), polyethylene glycol-polyaspartic acid (PEG-PASP), polyethylene glycol-polyglutamic acid (PEG-PGA), polyethylene glycol-poly Lysine (PEG-PLL), polyethylene glycol-polyethyleneimine (PEG-PEI), poly-L-lactic acid-b-polyethylene glycol-b-poly-L-lactic acid (PLLA-b-PEG-b -PLLA), polylactic acid-b-polyethylene glycol-b-polylactic acid (PLA-b-PEG-b-PLA), PEG-b-PLGA-b-PLL, PEG-b-PLGA-b-PEI, Any one of PEG-Pluronic, phospholipid-PLL or phospholipid-PEI, etc., the weight average molecular weight of the polymer is less than 1 million, preferably 0.2-200,000.

The calcium phosphate and amphiphilic polymer composite drug-loaded nano microspheres of the present invention can realize the controlled release of drugs by controlling the particle size and internal porous structure of the microspheres. The package of the drug through the composite nano-microspheres can form a relatively closed environment, which can enhance the stability of the drug to external factors; moreover, the nano-loaded microspheres can also increase the biological stability of the drug, so that the drug can maintain its stability before reaching the site of action. structural integrity.

The calcium phosphate and amphiphilic polymer composite drug-loaded nano-microspheres of the present invention can modify the calcium phosphate and amphiphilic copolymer composite drug-loaded nano-microspheres to make them targeted, and the modified preparations used include antibody Ligands (tumor-associated antigen markers), proteins and enzymes (enzyme markers), hormones, peptides, genes and small molecule preparations, etc.

Modified preparations may preferably be alpha-fetoprotein, carcinoembryonic antigen, tissue polypeptide antigen, amylase, lactate dehydrogenase, ribonuclease, 5-nucleotidase, adrenocorticotropic hormone, antidiuretic hormone, growth hormone, transformed growth factor, estrogen, progesterone, catecholamines and their derivatives, ras gene family and its expression products, myc gene family and its expression products, epidermal growth factor receptor, RB gene and p53 gene, folic acid, tamoxifen or magnetic particles.

More preferably integrin, transmembrane peptide, transferrin, alpha-fetoprotein, transforming growth factor, estrogen, progesterone, epidermal growth factor receptor, p53 gene, folic acid, tamoxifen, magnetic particles, rhodamine and quantum dots.

The calcium phosphate and amphiphilic polymer composite drug-carrying nano microspheres of the present invention, the drugs can be organic drugs, water-soluble drugs, water-insoluble drugs, genes, probes and diagnostic reagents.

The drugs are preferably paclitaxel, indomethacin, antifolates (such as methotrexate), antipurines (such as mercaptopurine), antipyrimidines (such as fluorouracil, tegafur), nucleotide reductase inhibitors (such as Hydroxyurea), deoxyribonucleotide polymerase inhibitors (such as cyclocitidine), drugs that directly affect and damage DNA structure and function (such as nitrogen mustard, cyclophosphamide, nitrogen methyl, cisplatin, mitomycin, Camptothecin), drugs that inhibit protein synthesis (such as doxorubicin, L-asparaginase, daunorubicin, daunorubicin), drugs that affect microtubule protein assembly and spindle filament formation (vinblastine, etopol glycosides), quantum dots, fluorescent dyes or therapeutic genes.

More preferably paclitaxel, indomethacin, methotrexate, mercaptopurine, fluorouracil, tegafur, hydroxyurea, cyclocytidine, nitrogen mustard, cyclophosphamide, azamethanone, cisplatin, mitomycin, camptothecin , doxorubicin, L-asparaginase, daunorubicin, mithromycin, vinblastine, etoposide, p53 gene.

The calcium phosphate and amphiphilic polymer composite drug-loaded nano-microspheres of the present invention are characterized in that their applications in drug carriers include delivery of drugs, slow and controlled release, reversal of drug resistance characteristics of tumor cells, and diagnosis and treatment of diseases. treat.

Composite drug-loaded nanospheres can change the route of drug administration and diversify the route and mode of administration; for example, composite nanospheres of calcium phosphate and amphiphilic polymers can protect drugs such as peptides, proteins or antisense nucleic acids. Not being enzymatically or hydrolyzed, the drug can be taken orally, and the dosage and frequency of medication can be reduced. At the same time, the targeting of the composite drug-loaded nanospheres can increase the local drug concentration while reducing the drug concentration in other parts of the body, thereby reducing the systemic toxicity of the drug. Furthermore, after the drug is nanosized, the increase in the absolute absorption can reduce the dosage, and also achieve the purpose of reducing the toxic and side effects of the drug; Concentration fluctuations are reduced, thereby improving drug safety and bioavailability.

The calcium phosphate and amphiphilic polymer composite drug-loaded nano-microspheres of the present invention and their application in drug carriers are characterized in that in the diagnosis of diseases, the drug-loaded nano-microspheres carry or surface-modify antibody ligands and proteins , peptides, short peptides, magnetic nanoparticles, quantum dots or fluorescent probes and other diagnostic reagents.

The calcium phosphate and amphiphilic polymer composite drug-loading nano-microspheres of the present invention can not only realize the entrapment of drugs, but also realize the co-encapsulation and delivery of drugs and genes to achieve co-therapy of drugs and genes.

Compared with existing materials, the calcium phosphate and amphiphilic polymer composite drug-loaded nano-microspheres of the present invention have the following advantages:

(1) Composite drug-loaded nano-microspheres combine the characteristics of calcium phosphate and amphiphilic copolymers, and have the characteristics of simple preparation, convenient drug loading, strong slow and controlled release of drugs, and good biocompatibility.

(2) Composite drug-loaded nano-microspheres can carry water-soluble drugs or fat-soluble drugs separately or simultaneously, which overcomes the shortcoming that amphiphilic copolymers are generally only suitable for carrying fat-soluble drugs, and expands the scope of application of amphiphilic copolymers. At the same time, the scope of use of calcium phosphate has also been expanded.

(3) The method of loading water-soluble drugs by composite drug-loaded nanospheres is simple. When the adsorption method is used, only the nanospheres and the drug-containing solution are mixed and oscillated, which can reduce the loss of drug activity and help maintain the drug. curative effect.

(4) The encapsulation efficiency and drug loading rate of the composite drug-loaded nano-microspheres for water-soluble drugs or fat-soluble drugs are high, and the encapsulation rate can reach more than 95%, so the loss of drugs can be reduced; the composite drug-loaded nano-microspheres The ball has a powerful function of slow and controlled release of drugs, and its release of drugs can be controlled by adjusting the particle size of the nano-microspheres, adjusting the size of the internal pores of the microspheres, and adjusting the molecular weight of the polymer.

(5) Composite drug-loaded nano-microspheres have a wide range of applications as drug carriers, which is reflected in the fact that they can carry different drugs for disease treatment, and can carry or surface-modified diagnostic reagents for disease diagnosis. diagnosis and treatment.

(6) The composite drug-loaded nano-microsphere can be used as a drug or gene carrier at the same time.

Description of drawings

Fig. 1. TEM photo of calcium phosphate and PEG-PLA composite nanospheres loaded with mitoxantrone;

Figure 2. Calcium phosphate and PEG-PLA composite nanospheres loaded with mitoxantrone release curve of mitoxantrone;

Figure 3. Accumulation of mitoxantrone calcium phosphate and PEG-PLA composite nanospheres in breast cancer cell MCF-7 and breast cancer drug-resistant cell MDR/RES MCF-7;

Specific implementation method

The following examples of the present invention are given to further illustrate the present invention, rather than limit the scope of the present invention.

Example 1:

Add 0.2g of polyethylene glycol-polylactic acid and glycolic acid copolymer (mPEG-b-PLGA) (molecular weight is 10000, mPEG segment molecular weight is 2000, LA/GA=5:5) to 20ml of water, 37°C Stir for 1 hour to dissolve the polymer and obtain solution A; add 10ml of 0.5M CaCl2 solution to A and stir for 0.5 hours to obtain solution B; add 10ml of 0.3M (NH4)2HPO4 aqueous solution to solution B dropwise, during The pH value of the reaction solution was controlled at 10 with ammonia water, and the temperature was 37°C; after the dropwise addition, the reaction solution was centrifuged, washed three times with deionized water and once with absolute ethanol, and then vacuum-dried at 37°C for 24h , to obtain calcium phosphate/mPEG-b-PLGA composite nanoporous spheres.

Example 2:

Encapsulation of water-soluble drugs by adsorption method:

At room temperature, 100 mg of dry calcium phosphate and polyethylene glycol-polylactic acid (PEG-PLA) composite nanospheres are mixed with 15 ml of aqueous solution containing water-soluble drug mitoxantrone (drug content 10 mg/ml), stirred for 0.5- At 24 hours, the calcium phosphate and PEG-PLA composite nanospheres loaded with mitoxantrone were collected by centrifugation. Then, calcium phosphate and PEG-PLA composite nano-microspheres were used to measure drug encapsulation efficiency and drug-loading rate, analyze nano-microsphere particle size and detect drug activity. The particle size of the calcium phosphate and PEG-PLA composite nano microsphere is about 200nm, and its encapsulation rate for mitoxantrone is 90%.

Example 3:

Encapsulation of fat-soluble drugs by adsorption method:

Dissolve the fat-soluble drug paclitaxel in a mixed solvent of water and ethanol (volume ratio 1:1) (drug concentration 1mg/ml); take dry calcium phosphate and polyethylene glycol-polyaspartic acid (PEG) at room temperature - 100 mg of PASP) composite nanospheres were mixed with 15 ml of the paclitaxel-containing solution, stirred for 0.5-24 hours, and the paclitaxel-encapsulated calcium phosphate and PEG-PASP composite nanospheres were collected by centrifugation. Then, calcium phosphate and PEG-PASP composite nano-microspheres were used to measure drug encapsulation efficiency and drug-loading rate, analyze nano-microsphere particle size and detect drug activity. The particle size of the calcium phosphate and PEG-PASP composite nano microsphere is about 30nm, and the encapsulation efficiency of paclitaxel is 85%.

Example 4:

Encapsulation of fat-soluble drugs by solvent evaporation method:

First fat-soluble drug indomethacin (mass percentage concentration 2%) is dissolved in 25ml of chloroform to obtain an oily phase, then dry calcium phosphate/polyethylene glycol-polylysine (PEG-PLL) composite nanospheres 100mg Dissolve in 50ml of phosphate buffer solution to obtain the water phase, and finally mix and stir the water phase and the oil phase, evaporate the solvent, and centrifuge to collect the calcium phosphate/PEG-PLL composite nanospheres loaded with indomethacin. Then, calcium phosphate/PEG-PLL composite nano-microspheres were used to measure the drug encapsulation efficiency and drug loading rate, analyze the particle size of the nano-microspheres and detect the drug activity. The particle diameter of the calcium phosphate/PEG-PLL composite nano microsphere is about 30nm, and its encapsulation rate for indomethacin is 90%.

Example 5:

Encapsulation of drugs before the preparation of composite nanospheres:

Dissolve 5 mg of fat-soluble drug paclitaxel and 100 mg of PEG-PLGA (weight average molecular weight 10,000) in 3 ml of dimethyl sulfoxide, add 10 ml of water to stir and mix, and then put it into a dialysis bag with a molecular weight cut-off of 12,000 for dialysis for 5-12 hours. The final preparation is a drug-loaded polymer micelle solution A with a mass fraction of 0.1-5%; then add a soluble calcium salt to the solution A and stir for 0.5-24 hours to form a solution B with a calcium ion concentration of 0.005-1M; A soluble phosphate solution with a concentration of 0.005-1M was added to B, and the pH value was controlled to be 7-12; finally, soluble phosphate was added, and the stirring reaction was continued for 0-24h, collected by centrifugation, washed, and dried to obtain calcium phosphate/PEG-PLGA composite carrier Drug Nanospheres.

Embodiment 6:

Encapsulation of water-soluble drugs by pH gradient method:

Draw an appropriate amount of 9g/L sodium chloride injection and add it to the mitoxantrone hydrochloride bottle for injection and shake to completely dissolve the mitoxantrone hydrochloride; put the dissolved mitoxantrone hydrochloride aqueous solution in a water bath (55°C～ 60°C) for 10 min; draw 2 mL of calcium phosphate/polyethylene glycol-polylysine (PEG-PLL) composite nanosphere suspension (50 mg/ml) preheated in a water bath and add it to hydrochloric acid mitoxantrone solution, then add sodium carbonate solution, shake well and heat in a water bath (55°C-60°C); after the mixed solution is equilibrated at 55°C-60°C for 10 minutes, fill it with nitrogen and stir for 10 minutes, then centrifuge to collect and pack Calcium phosphate/PEG-PLL composite nanospheres of mitoxantrone. Then, calcium phosphate/PEG-PLL composite nano-microspheres were used to measure the drug encapsulation efficiency and drug loading rate, analyze the particle size of the nano-microspheres and detect the drug activity. The particle size of the calcium phosphate/PEG-PLL composite nano-microsphere is about 300nm, and its encapsulation efficiency for mitoxantrone hydrochloride is 96%.

Embodiment 7:

Use modifiers such as transferrin Tf to modify the surface of calcium phosphate/amphiphilic polymer composite drug-loaded nanospheres to make them targeted:

Take 1ml of calcium phosphate/polyethylene glycol-polyethyleneimine (PEG-PEI) composite nanospheres (50mg/ml) loaded with methotrexate, add 15ml of phosphate buffer solution (pH=7.4), and 1mg The tumor targeting agent transferrin Tf was mixed, 2ml of 1-ethyl-3(3-dimethylpropyl)carbodiimide hydrochloride (EDC) was added dropwise, and reacted at 4°C for 4h. The Tf-modified calcium phosphate/PEG-PEI composite nanospheres loaded with methotrexate were collected by centrifugation. The modification of transferrin Tf can increase the targeting of calcium phosphate/PEG-PEI composite drug-loaded nanospheres to most tumor cells, increase the ability of the nanospheres to cross the cell membrane, and make the loaded drugs play a greater role .

Embodiment 8:

Using modifiers such as transforming growth factor to modify the surface of calcium phosphate/amphiphilic polymer composite drug-loaded nanospheres to make them targeted:

Take 1ml of calcium phosphate/polyethylene glycol-polyethyleneimine (PEG-PEI) composite nanospheres (50mg/ml) loaded with cyclocytidine, add 15ml of phosphate buffer solution (pH=7.4), and mix with 1mg of tumor The targeted preparation was mixed with transforming growth factor, 2ml of 1-ethyl-3(3-dimethylpropyl)carbodiimide hydrochloride (EDC) was added dropwise, and reacted at 4°C for 4h. The calcium phosphate/PEG-PEI composite nanospheres modified by transforming growth factor and loaded with methotrexate were collected by centrifugation. The modification of transforming growth factor can increase the targeting of calcium phosphate/PEG-PEI composite drug-loaded nanospheres to most tumor cells, increase the ability of the nanospheres to cross the cell membrane, and make the loaded drugs play a greater role.

Embodiment 9:

Preparation of calcium phosphate/amphiphilic polymer composite drug-loaded nanospheres with medical diagnostic and therapeutic functions:

Take the dry calcium phosphate/polyethylene glycol-(polylactic acid-co-polyglycolic acid)-polylysine triblock copolymer (PEG-b-PLGA-b-PLL) composite nanoparticle at room temperature Mix 100mg of balls with 15ml of aqueous solution containing water-soluble drug doxorubicin and magnetic nanoparticles (drug content: 10mg/ml, magnetic nanoparticle content: 1mg/ml), stir for 0.5-24h, centrifuge to collect the loaded doxorubicin and have NMR. Functional calcium phosphate/PEG-b-PLGA-b-PLL composite nanospheres. Then, the calcium phosphate/PEG-b-PLGA-b-PLL composite nanometer microspheres were tested for drug encapsulation efficiency, drug loading rate, particle size analysis and drug activity. The particle size of the calcium phosphate/PEG-b-PLGA-b-PLL composite nano-microsphere is about 200nm, and its encapsulation efficiency for doxorubicin is 90%.

Calcium phosphate/PEG-b-PLGA-b-PLL composite magnetic drug-loaded micro-nanospheres not only have the function of anti-tumor drugs to treat tumors, but also have the function of nuclear magnetic imaging of magnetic nanoparticles, and can be used as MRI diagnostic reagents.

Example 10:

Take the dry calcium phosphate/polyethylene glycol-(polylactic acid-co-polyglycolic acid)-polylysine triblock copolymer (PEG-b-PLGA-b-PLL) composite nanoparticle at room temperature Mix 100mg of balls with 15ml of aqueous solution containing water-soluble drug fluorouracil and water-soluble quantum dots (drug content 10mg/ml, quantum dot content 1mg/ml), stir for 0.5-24h, and centrifuge to collect fluorouracil-encapsulated calcium phosphate with fluorescence imaging function /PEG-b-PLGA-b-PLL composite nanospheres. Then, the calcium phosphate/PEG-b-PLGA-b-PLL composite nanometer microspheres were tested for drug encapsulation efficiency, drug loading rate, particle size analysis and drug activity. The particle size of the calcium phosphate/PEG-b-PLGA-b-PLL composite nano microsphere is about 200nm, and its encapsulation efficiency for fluorouracil is 90%.

Calcium phosphate/PEG-b-PLGA-b-PLL composite magnetic drug-loaded micro-nanospheres not only have the function of anti-tumor drugs to treat tumors, but also have the function of fluorescence imaging of quantum dots.

Example 11:

Application of calcium phosphate/amphiphilic polymer composite nanospheres as drug and gene carriers.

The prepared calcium phosphate/PEG-PLA composite nanospheres loaded with mitoxantrone can reverse drug resistance to drug-resistant breast cancer cells (MCF-7/MIT, BCAP37/MDR). Calcium phosphate/PEG-PLA composite nanospheres can increase the accumulation of anticancer drugs in tumor cells, see Figure 3, improve the toxicity of anticancer drugs to non-drug-resistant and drug-resistant tumor cells, and to a certain extent can reverse the tumor The drug resistance of cells can increase the fluidity of cell membrane, improve the permeability of cell membrane of drugs, and inhibit or reduce the efflux of P-gp protein to the substrate, inhibit the level of MDRI, MRP and GST-pmRNA of drug-resistant cells and reduce ATP levels required for efflux of the P-gp protein, ABCG2. Calcium phosphate/PEG-PLA composite nanospheres can change the in vivo pharmacokinetics and tissue distribution parameters of the model drug, significantly prolong the blood circulation time of the model drug in rats and mice, and change the drug distribution in various organs in the body.

The preparation of the calcium phosphate/amphiphilic polymer composite drug-loaded nano-microspheres disclosed and disclosed in the present invention and its application in drug carriers can be referred to in this disclosure. Although the preparation of the calcium phosphate/amphiphilic polymer composite drug-loaded nano-microspheres of the present invention and the application in drug carriers have been described through preferred embodiments, those skilled in the art can obviously , within the spirit and scope of the method changes described herein, more specifically, all similar substitutions and changes will be obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention .

Claims (6)

1. the composite medicament-carrying nano-microsphere of a calcium phosphate and amphipathic nature polyalcohol, the wherein complex that formed by amphipathic nature polyalcohol and calcium phosphate of Nano microsphere, and bag medicine carrying thing; The percentage by weight of described amphipathic nature polyalcohol, calcium phosphate and the medicine carrying thing that wraps is followed successively by 1%-60%, 0.1%-60% and 0.1%-50%; The particle diameter of described calcium phosphate and amphipathic nature polyalcohol composite nano-microsphere is 1-100000nm; Described amphipathic nature polyalcohol is polyethylene glycol-polylactic acid.

2. calcium phosphate as claimed in claim 1 and amphiphilic polymer composite medicament-carrying nano-microsphere, the particle diameter that it is characterized in that described calcium phosphate and amphipathic nature polyalcohol composite nano-microsphere is 5-5000nm.

3. calcium phosphate as claimed in claim 1 and amphiphilic polymer composite medicament-carrying nano-microsphere, it is characterized in that the described Nano microsphere that is formed by amphipathic nature polyalcohol and calcium phosphate form and preparation method is preferably as follows:

Under 0～60 ℃, amphipathic nature polyalcohol is added in the water, is mixed with mass fraction and is 0.1～5% solution A; In solution A, add soluble calcium salt CaCl ₂, stirring and forming calcium ion concentration is the solution B of 0.005～1M; In solution B, drip or pour into the soluble phosphoric acid saline solution (NH that concentration is 0.005～1M ₄) ₂HPO ₄, making calcium ion and phosphate anion mol ratio is 1: 1～1.67: 1, pH value is controlled at 7～12 simultaneously; Continue stirring reaction, then with the centrifugal collection of milky reactant liquor, deionized water and dehydrated alcohol cyclic washing, vacuum drying obtains calcium phosphate/amphiphilic polymer composite medicament-carrying nano-microsphere.

4. the preparation method of a calcium phosphate as claimed in claim 1 and amphiphilic polymer composite medicament-carrying nano-microsphere, it is as follows to it is characterized in that wrapping support method:

(1) bag medicine carrying thing in the Nano microsphere preparation process

First configuration contains the solution A 1 of the medicine that amphipathic nature polyalcohol that percentage by weight is 0.1-5% and percentage by weight be 0.1-5%; In solution A 1, add soluble calcium salt CaCl respectively again ₂, stir 0.5-24h, forming calcium ion concentration is the solution B of 0.005-1mol/L; Then adding concentration in the solution B is the (NH of 0.005-1mol/L ₄) ₂HPO ₄Solution, (NH ₄) ₂HPO ₄Solution and solution B volume ratio are 1/3-4/3, use ammonia to regulate pH value and are 7-12, continue stirring reaction 0-24h, centrifugal collection, and washing, drying obtains calcium phosphate and amphiphilic polymer composite medicament-carrying nano-microsphere;

(2) bag medicine carrying thing after the Nano microsphere preparation

At room temperature, calcium phosphate and amphipathic nature polyalcohol composite nano-microsphere and the aqueous solution that contains medicine with drying, the weight percent concentration of described calcium phosphate and amphipathic nature polyalcohol composite nano-microsphere and medicine is respectively 0.1-99% and 0.1-50%, stir 0.5-24h, calcium phosphate and the amphipathic nature polyalcohol composite nano-microsphere of centrifugal collection bag medicine carrying thing.

5. a calcium phosphate as claimed in claim 1 and the amphiphilic polymer composite medicament-carrying nano-microsphere purposes in the preparation carrier medicament.

6. purposes as claimed in claim 5 is characterized in that described carrier medicament is the resistant characterization medicine of reversing tumor cell or to the medicine of disease treatment.

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