CN103222961B - Injectable Cu(DDC)2 protein nanoparticle preparation for treating tumors and preparation method thereof - Google Patents

Abstract

The compound Cu(DDC) ₂ with high anti-tumor activity can treat melanoma, breast cancer, lung cancer, liver cancer and other cancers with a wide range of curative effects, and has a certain selective killing effect on tumor cells. The invention relates to Cu(DDC) ₂ protein nanoparticles for injection for treating tumors and a preparation method thereof, which solves the problem that Cu(DDC) ₂ has poor water solubility and is not easy to be administered intravenously. The present invention prepares Cu(DDC) protein nanoparticles for injection by ultrasonic method, high-pressure homogenization method and micro-jet method, and the Cu(DDC ₎ protein nanoparticles are prepared in proportion by the following components: (Cu(DDC ₎ ) ₂ + organic solvent): (protein + water) = 1:2 ~ 1:30, of which Cu(DDC) ₂ 0.01 ~ 5mg/mL, protein 0.05% ~ 25% (w/v), organic solvent: water ratio 1:2～1:30. Human serum albumin (HSA), bovine serum albumin (BSA), hydrophobin, glycoprotein, lipoprotein and other proteins or polypeptides are used as carriers and stabilizers to coat drug particles, which have good biocompatibility and high safety, improving the Cu(DDC) ₂ has antitumor efficacy and reduces adverse reactions.

Description

Injectable Cu(DDC)2 protein nanoparticle preparation for treating tumors and preparation method thereof

technical field

The invention belongs to the field of pharmaceutical preparations, and particularly provides a Cu(DDC) ₂ protein nanoparticle preparation for injection for treating tumors and a preparation method thereof.

Background technique

Cu(DDC) ₂ (Copper Diethyldithiocarbamate), the Chinese full name is N,N-diethyldithiocarbamate copper, which consists of two molecules of DDC (N,N-diethyldithiocarbamate) and one molecule of copper chelate synthesized (structure as formula I). DDC is the metabolite of the anti-alcoholic drug disulfiram (disulfiram, also known as disulfide tetraethylthiuram, disulfiram, structure such as formula II) in the body through the reduction of glutathione reductase system in red blood cells, and Both disulfiram and DDC are easily chelated with copper, eventually forming Cu(DDC) ₂ .

Formula Ⅰ

Formula II

Studies have found that Cu(DDC) ₂ has positive anticancer effects: nuclear transcription factor (NF-κB) is a factor that is closely related to anti-apoptosis and chemotherapy resistance. In normal cells, NF-κB combines with IκB to form an inactive NF-κB-IκB complex that exists in the cytoplasm. After being stimulated, IκB is phosphorylated and marked by ubiquitin protein, and the proteasome recognizes and degrades the ubiquitin-labeled IκB, releasing NF-κB from the NF-κB-IκB complex. Free NF-κB transfers to the nucleus, binds to DNA targets (κB), activates downstream genes and triggers a series of transcriptional reactions, and a large number of cell cycle proteins (cyclins D1, D2, D3, E and c- myc, etc.), adhesion molecules (ICAM-1 and E-selectin), angiogenic factors (VEGF), anti-apoptotic genes (Bcl-2, cIAPs and c-FLIP), and drug resistance proteins (MRP1, P-gp) and cyclooxygenase (COX). There are a large number of highly active NF-κB in drug-resistant tumor cell lines, and the abnormal overexpression of NF-κB blocks the apoptosis induced by cytotoxic drugs. Cu(DDC) ₂ indirectly inhibits the expression of NF-κB by inhibiting proteasome activity, thereby inhibiting the survival, proliferation and metastasis of tumor cells, and Cu(DDC) ₂ reduces P-glycoprotein (P-glycoprotein) through the NF-κB pathway gp), multidrug resistance-associated protein 1 (MRP1) and ATP efflux pump expression of mitoxantrone resistance-associated protein, reduce chemotherapeutic drug resistance and increase the sensitivity of drug-resistant tumor cells to chemotherapeutic drugs. Because tumor cells have a strong ability to proliferate, the activity of intracellular proteasome is much higher than that of normal cells, so Cu(DDC) ₂ has a selective killing effect on tumor cells, and has little killing effect on normal tissue cells. This is Cu(DDC) ₂ As a unique advantage of anti-tumor drugs. It has been reported in the literature that Cu(DDC) ₂ has a good curative effect on liver cancer, breast cancer, lung cancer, melanoma and other cancers.

Contents of the invention

The object of the present invention is to provide the injection Cu(DDC) _{2nanoparticle} preparation and preparation method thereof for the treatment of tumor, this preparation solves the problem of Cu(DDC) _2poor water solubility, takes albumen as the nanoscale intravenous injection Cu (DDC) ₂ preparation has the advantages of high drug efficacy, less toxic and side effects, long-lasting effect, and good stability. The pharmaceutical preparation of the present invention is powder for injection.

The invention provides a Cu(DDC) ₂ nanoparticle preparation for injection for treating tumors, which uses protein as a carrier to prepare protein nanoparticles, and is characterized in that: the preparation is prepared from the following components: Cu(DDC) _2. Organic solvent, protein and water, the volume ratio is (Cu(DDC) ₂ + organic solvent): (protein + water) = 1:2～1:30, and the concentration of Cu(DDC) ₂ is 0.01～5mg/ mL, the protein concentration is 0.05%-25% (w/v), and the percentages are weight-volume ratios unless otherwise specified.

Cu(DDC) ₂ is a brown-black powder with poor solubility in water, but good stability. In order to solve the problem of its poor water solubility and difficulty in intravenous administration, the present invention prepares it into nanoparticles for injection.

The Cu(DDC) ₂ nanoparticle preparation for injection for treating tumors of the present invention is characterized in that: the volume ratio of the components is preferably (Cu(DDC) ₂ + organic solvent): (protein + water) = 1:8 ～1:25, wherein, Cu(DDC) ₂ concentration is 0.1～2mg/mL, protein concentration is 0.5～5% (w/v). The optimal component volume ratio is (Cu(DDC) ₂ + organic solvent): (protein + water) = 1:12, where the concentration of Cu(DDC) ₂ is 1 mg/mL, and the concentration of protein is 1% (w/v ).

The Cu(DDC) ₂ nanoparticle preparation for injection for treating tumors of the present invention is characterized in that: the protein can be selected from human serum albumin (HSA, human serum albumin), bovine serum albumin (BSA, bovine serum albumin) , hydrophobin (hydrophobin), glycoprotein (glycoprotein) or lipoprotein (lipoprotein); the organic solvent is preferably dichloromethane, chloroform, acetone, dimethylformamide (DMF), dimethyl sulfoxide ( DMSO), dioxane, ethyl acetate, acetonitrile, methylpyrrolidone, methanol, ethanol, medium chain alcohol or one or more. Acid-base regulators, such as phosphoric acid, citric acid, hydrochloric acid, phosphoric acid, acetic acid, etc., can be added to the aqueous phase.

The present invention prepares and compares PLGA, PLA, chitosan nanoparticles and various protein nanoparticles through a large number of experimental studies. PLGA, PLA, and chitosan nanoparticles all have slow-release effects in the body, which is not conducive to Cu(DDC) ₂ reaching a therapeutic concentration in the blood to inhibit tumors; and its biocompatibility is not as good as that of HSA, BSA, hydrophobin, glycoprotein, lipid Protein, because protein can be metabolized into various amino acids in the body, which can be safely used by the body.

Due to the EPR effect (enhanced permeability and retention effect, high permeability and retention effect of macromolecular substances and particles in tumor tissue) of Cu(DDC) ₂ protein nanoparticles in tumor sites: the integrity of blood vessel walls in tumor sites is poor, and vascular endothelial cells The gap is large, and the pore size can reach 100-780nm, so macromolecular substances can enter the tumor tissue through the blood vessel wall, and the amount of retention in the tumor tissue increases; in addition, the lack of lymphatic clearance system in tumor tissue makes macromolecular substances, etc. Prolonged storage time in tumor tissue enables Cu(DDC) ₂ protein nanoparticles to passively target tumor cells, increase the concentration of Cu(DDC) ₂ in tumor cells, increase the anti-tumor effect, and reduce adverse reactions.

When human serum albumin (HSA, human serum albumin) is selected as the carrier and stabilizer, HSA-Cu(DDC) ₂ -NP can not only passively target tumor cells through EPR effect, but also endogenously target tumor cells through HSA-gp60-Caveolae-SPARC The pathway actively targets cancer cells, thereby enhancing the accumulation of Cu(DDC) ₂ into the tumor: albumin receptors gp60, gp30, gp18 and cysteine-rich acidic secretory protein (secreted protein acidic) present on the membrane of vascular endothelial cells and rich in cysteine, SPARC), in which gp60 is the main mediator of HSA adhesion to capillary endothelial cells, gp60-induced activation of Src kinase is a pre-signal for endothelial cells to achieve endocytosis of HSA. When Gp60 binds to HSA, gp60 binds to a scaffold protein caveolin-1 (Caveolin-1) on the cell membrane, and the morphology and function of Caveolin-1 will change, resulting in caveolae, forming A vesicle with a diameter of 50-100 nm, namely caveolae. The caveolus transcellularly transports the gp60-HSA conjugate, HSA-Cu(DDC) ₂ and free HSA into it, releasing them from the basolateral plasma membrane to the extracellular space. HSA and HSA-bound drugs in the tumor space are further mediated by SPARC. SPARC is an acidic secretory protein rich in cysteine, which can highly specifically bind to albumin, and SPARC is overexpressed in a variety of tumor cells, such as breast cancer, prostate cancer, esophageal cancer, colon cancer, lung cancer, melanoma wait. Therefore, SPARC can concentrate the drug that binds to HSA in the tumor cell interstitium and become a drug reservoir in tumor tissue, playing an extremely important role in increasing the accumulation of HSA-bound drugs in tumor tissue. In this way, a part of HSA-Cu(DDC) ₂ -NP penetrates into the tumor tissue through the gap of the tumor vascular endothelium, and most of the HSA-Cu(DDC) ₂ -NP makes the drug-carrying HSA transmute through the HSA-gp60-Caveolae-SPARC endogenous pathway. Actively targeting tumor tissue space through tumor vascular endothelial cells. The mechanism of action of bovine serum albumin (BSA, bovine serum albumin) is similar to that of HSA.

Hydrophobin (hydrophobin) is a protein secreted by filamentous fungi, which is divided into two types: class I and class II. Hydrophobin has hydrophilic and hydrophobic ends, and can be adsorbed on hydrophobic Cu(DDC) ₂ and arranged closely instead of in folded form. Cu(DDC) ₂ hydrophobin nanoparticles can significantly improve the water solubility of preparations, and have the advantages of high safety and good biocompatibility.

Glycoproteins and lipoproteins are human-derived proteins with good biocompatibility and high safety. In short, from the aspects of tumor targeting, biocompatibility and safety, human serum albumin, bovine serum albumin, hydrophobin, glycoprotein, lipoprotein and other proteins or polypeptides are Cu(DDC) ₂ nanoparticles The preferred carrier is human serum albumin, among which human serum albumin is the most preferred.

Proteins are added to the aqueous phase to act as carriers and stabilizers to form stable nano-droplets. Unlike conventional nanoparticle formation methods, no surfactant is added to the mixture.

Protein nanoparticles with solid or liquid cores of pharmacologically active substances can deliver high doses of pharmacologically active substances in a small volume. This minimizes patient discomfort and hospital stay when receiving large volumes of fluid. In addition, the carrier protein coated with the pharmacologically active substance can usually be completely degraded by proteolytic enzymes in vivo, therefore, the delivery system of the formulation of the present invention has high safety.

Those skilled in the art will recognize that many changes may be made within the scope and spirit of the invention. The organic medium within the nanocarriers can vary, a variety of different pharmacologically active substances can be used in their formation, and a variety of proteins or polypeptides and other natural or synthetic polymers can be used.

Another object of the present invention is to provide a preparation method of Cu(DDC) ₂ protein nanoparticle preparation for injection that is simple and easy to treat tumors with good stability, characterized in that the preparation adopts ultrasonic method, high-pressure homogenization Method or microfluidic preparation:

Ultrasonic preparation process: dissolve Cu(DDC) ₂ in an organic solvent, add it into a protein solution with a concentration of 0.05% to 25% while stirring in an ice bath at 0-10°C, perform ultrasonic emulsification treatment, and then evaporate under reduced pressure to remove Organic solvent, filtered to sterilize, and freeze-dried to obtain Cu(DDC) ₂ nanoparticles;

Preparation process by high-pressure homogenization method: Cu(DDC) ₂ is dissolved in organic solvent, added to 0.05%-25% protein aqueous solution under ice bath conditions at 0-10°C, transferred to high-pressure homogenizer after high-speed shearing internal circulation, and then evaporated under reduced pressure to remove the organic solvent, sterilized by filtration, and freeze-dried to obtain Cu(DDC) ₂ nanoparticles.

Preparation process by micro-jet method: Cu(DDC) ₂ is dissolved in organic solvent, added to 0.05% to 25% protein aqueous solution under ice bath conditions at 0-10°C, transferred to micro-jet internal circulation after high-speed shearing, Then, the organic solvent was removed by evaporation under reduced pressure, sterilized by filtration, and freeze-dried to obtain Cu(DDC) ₂ nanoparticles.

The organic solvent is evaporated under reduced pressure to form a colloid system composed of protein-coated pharmacologically active substance nanoparticles and protein. Acceptable methods of evaporation include the use of rotary evaporators, falling film evaporators, spray dryers, freeze dryers, and similar equipment.

The preparation method of Cu ₍ DDC) ₂ nanoparticle preparation for injection for treating tumors of the present invention is characterized in that, the specific process prepared by the ultrasonic method is: Cu(DDC) is dissolved in an organic solvent, and added to Protein double-distilled water solution, stirred in ice bath at 0-10°C for 1-20min, ultrasonicated for 3-30min under the condition of ultrasonic power 150-400W (3s/1s), and then decompressed and rotary evaporated to remove the organic solvent, the particle size range is 120nm- 160nm (the particles can be sterile filtered before use in liquid suspension, rather than using conventional methods such as autoclaving to denature the protein), to obtain Cu(DDC) ₂ nanoparticles, filter sterilized, frozen Vacuum dry for at least 24 hours.

The specific process of the high-pressure homogenization method is as follows: dissolve Cu(DDC) ₂ in an organic solvent, add it to the protein, and shear at a high speed for 3-5 minutes in an ice bath at 0-10°C, and then transfer it to a high-pressure homogenizer. Boost the pressure to 3000-6000psi, cycle 1-3 times, and then increase the pressure to 10000-35000psi, cycle 4-8 times; remove the organic solvent by rotary evaporation under reduced pressure to obtain Cu(DDC) ₂ nanoparticles with a particle size ranging from 100nm to 120nm (can be sterile-filtered before use in liquid suspension to avoid protein denaturation due to sterilization by conventional methods); freeze-dry in vacuum for at least 24 hours.

The specific process of preparation by micro-fluidic method is as follows: dissolve Cu(DDC) ₂ in organic solvent, add it into the protein with stirring in ice bath (0-10°C), high-speed shear for 3-5min in ice bath at 0-10°C, and then transfer Into the micro-jet, first increase the pressure to 3000-6000psi, cycle 1-2 times, then increase the pressure to 10000-36000psi, cycle 1-4 times; remove the organic solvent by rotary evaporation under reduced pressure to obtain Cu(DDC) ₂ nanoparticles, The particle size ranges from 90nm to 110nm (sterile filtration can be performed before use in liquid suspension to avoid protein denaturation due to conventional methods of sterilization); freeze-dry in vacuum for at least 24 hours.

The prepared freeze-dried preparation can be redispersed in a suitable aqueous medium at any suitable time to obtain a nanocolloid system that can be used for intravenous administration, and the particle size distribution is the same as before freeze-drying. Sealed and stored at low temperature. The aqueous medium includes one or more mixtures of physiological saline, buffered physiological saline, water, buffered aqueous medium, amino acid solution, vitamin solution or similar media.

After the solvent was evaporated, freeze-drying was used to obtain the powder of protein and Cu(DDC) ₂ . Before freeze-drying, it can be directly freeze-dried without adding a freeze-drying protection agent, because the protein itself has the effect of freeze-drying protection; it is also possible to add a freeze-drying protection agent to play an additional protective effect, and the freeze-drying protection agent is trehalose, mannose, Lactose, glucose, sucrose, aD-mannopyranose, cellobiose, maltose, inosose, raffinose, inulin, dextrose, maltodextrin, maltopolysaccharide, sucrose octasulfate, heparin, 2-hydroxy Propyl-beta Cyclodextrin, Glycerin, Mannitol, Inositol, Sorbitol, Thiol, Polyethylene Glycol, Sorbitol, Amino Acid, Polymer, Tween 80, Pluronic, Bridger, One or more of sodium dodecylsulfonate and ascorbic acid.

The innovation point of the present invention is:

1. Solve the problem that Cu(DDC) ₂ has poor water solubility and is difficult to administer intravenously. Cu(DDC) ₂ has very high antitumor activity on cancers such as liver cancer, breast cancer, lung cancer, and melanoma, and has a selective killing effect on cancer cells. The present invention prepares the pharmacologically active compound into an intravenous injection available for administration with preparations. Cu(DDC) ₂ indirectly inhibits the expression of nuclear transcription factor (NF-κB) by inhibiting proteasome activity, thereby inhibiting the survival, proliferation and metastasis of tumor cells, and Cu(DDC) ₂ reduces P-glycoprotein through the NF-κB pathway (P-gp), multidrug resistance-associated protein 1 (MRP1) and ATP efflux pump expression of mitoxantrone resistance-associated protein, reduce chemotherapeutic drug resistance, and increase drug-resistant tumor cell sensitivity to chemotherapeutic drugs . Because tumor cells have a strong ability to proliferate, the activity of intracellular proteasome is much higher than that of normal cells, so Cu(DDC) ₂ has a selective killing effect on tumor cells, and has little killing effect on normal tissue cells. This is Cu(DDC) ₂ As a unique advantage of anti-tumor drugs. The preparation method is used to prepare the nanoparticle with good water solubility, which solves the problem of clinical intravenous administration.

2. Human serum albumin, bovine serum albumin, hydrophobin, glycoprotein, lipoprotein and other proteins or polypeptides are used as stabilizers and carriers of Cu(DDC) ₂ nanoparticles, which are safe, non-toxic, non-immunogenic, and It has the advantages of biodegradation, good biocompatibility, etc., and retains the excellent biological activity of the protein in the process of preparing Pr-Cu(DDC) ₂ -NP and in the final preparation. At present, the common preparation methods of protein nanoparticles include emulsification and solidification method, solvent-non-solvent method, pH-coagulation method, etc., which involve chemical cross-linking or thermal denaturation. Chemical cross-linking is non-specific, and any nucleophile existing in the protein structure Groups such as amines and hydroxyls are reactive, and heat denaturation irreversibly alters protein structure. Residual formaldehyde, glutaraldehyde and other aldehydes will cause the inactivation of biologically active macromolecules, and the resulting protein nanoparticles have poor biodegradability and high toxicity. The invention adopts ultrasonic-emulsification method, high-pressure homogenization method and micro-jet method to prepare protein nanoparticles, fully retains the biological activity of protein itself, has high safety, and can be fully utilized by human body metabolism into amino acids.

3. When using human serum albumin (HSA) as a carrier and stabilizer, the combination of the passive targeting effect of Cu(DDC) ₂ -NP on tumors and the active targeting effect of HSA on tumor cells significantly improved tumor inhibition curative effect and reduce adverse reactions. The unique EPR effect of the tumor itself increases the retention of HSA-Cu(DDC) ₂ -NP in tumor tissue and prolongs the retention time in tumor tissue; HSA-Cu(DDC) ₂ -NP passes through HSA-gp60-Caveolae-SPARC The endogenous pathway crosses the tumor vascular endothelial cells to the tumor tissue space, and forms a drug reservoir in the rapidly growing tumor, thereby increasing the local concentration of Cu(DDC) ₂ in the tumor cells and prolonging the contact time of the drug with the tumor tissue. It also reduces the probability of the drug reaching normal cells and greatly improves the efficacy of the drug. The mechanism of action of bovine serum albumin (HSA) is similar to that of human serum albumin (HSA). From the aspects of cancer cell targeting, biocompatibility and biodegradability, human serum albumin, bovine serum albumin, hydrophobin, glycoprotein, lipoprotein and other proteins or polypeptides are all Cu(DDC) _2nm The preferred choice for particle carriers, among which human serum albumin is the most preferred.

Description of drawings

Fig. 1 Cu (DDC) ₂ human serum albumin nanoparticles transmission electron micrograph;

Fig. 2Cu(DDC) ₂ Degradation curves in 10% plasma, 50% plasma and whole blood;

Fig. 3 Cu (DDC) ₂ After acting for 12h, the inhibition curve to tumor cells and normal cells;

Fig. ₄ Cu (DDC) After 24h, the inhibition curve to tumor cells and normal cells;

Fig. 5 Cu (DDC) After ₂ acts on 48h, the inhibition curve to tumor cell and normal cell;

Fig. 6 Cu (DDC) ₂ After acting for 72h, the inhibition curve to tumor cells and normal cells;

Fig. 7 oral gavage Cu (DDC) ₂ suspension mean blood drug concentration-time curve;

Fig. 8 Average plasma concentration-time curve of Cu(DDC) ₂ albumin nanoparticles for intravenous injection.

Detailed ways

The present invention will be further described below in conjunction with the examples. The following examples are only several specific embodiments of the present invention, but the design concept of the present invention is not limited thereto, and any non-substantial changes to the present invention by using this concept should belong to the act of violating the protection scope of the present invention.

The methods in the following examples are conventional methods unless otherwise specified. The temperature range of the ice bath is 0-10°C. After obtaining the Cu(DDC) ₂ nanoparticles, unless otherwise specified, no lyoprotectant was added, and they were lyophilized and vacuum-dried for 24 hours. The obtained cake is quickly redissolved into the original suspension after adding sterile water or physiological saline, and the particle size distribution is the same as before freeze-drying. Sealed, 4 ℃ low temperature storage.

Example 1:

Dissolve 3mg Cu(DDC) ₂ in 3mL dichloromethane, add it into 36.0mL human serum albumin double distilled water solution (1%w/v) under stirring, stir in ice bath for 10min, and under the condition of ultrasonic power 300W (3s/1s) Sonicate for 9 minutes, and remove methylene chloride by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ albumin nanoparticles with a particle size ranging from 120nm to 160nm. The transmission electron microscope of the obtained Cu(DDC) ₂ nanoparticles is shown in FIG. 1 .

Example 2:

Dissolve 3mg Cu(DDC) ₂ in 3mL chloroform, add to 30.0mL human serum albumin double-distilled water solution (1%w/v) under stirring, stir in ice bath for 10min, and under the condition of ultrasonic power 300W (3s/1s) Sonicate for 9 minutes, remove chloroform by rotary evaporation under reduced pressure at 50°C to obtain Cu(DDC) ₂ albumin nanoparticles with a particle size ranging from 120nm to 160nm. Trehalose was added as a lyoprotectant.

Example 3:

Dissolve 3mg Cu(DDC) ₂ in 3mL acetone, add to 24.0mL human serum albumin double distilled water solution (1%w/v) under stirring, stir in ice bath for 10min, and ultrasonicate under the condition of ultrasonic power 300W (3s/1s) After 9 minutes, the acetone was removed by rotary evaporation under reduced pressure at 45°C to obtain Cu(DDC) ₂ albumin nanoparticles with a particle size ranging from 120nm to 160nm.

Example 4:

Dissolve 3mg Cu(DDC) ₂ in 3mL dichloromethane: acetone (1:5, v/v), add to 30.0mL human serum albumin double distilled water solution (1%w/v) under stirring, and stir in ice bath for 10min , under the condition of ultrasonic power 300W (3s/1s) for 9 minutes, the mixed solvent was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ albumin nanoparticles with a particle size range of 120nm-160nm.

Example 5:

Dissolve 3mg Cu(DDC) ₂ in 3mL dichloromethane: chloroform (1:5, v/v), add to 30.0mL human serum albumin double distilled water solution (1%w/v) under stirring, ice bath Stir for 10 minutes, sonicate for 9 minutes under the condition of ultrasonic power 300W (3s/1s), and remove the mixed solvent by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ albumin nanoparticles with a particle size ranging from 120nm to 150nm.

Embodiment 6:

The difference from Example 5 is that 3 mg of Cu(DDC) ₂ was dissolved in 3 mL of dichloromethane: acetone: chloroform (1:0.5:3, v/v), and added to 24.0 mL of human serum albumin under stirring Double distilled water (1% w/v).

Embodiment 7:

The difference from Example 6 is that dichloromethane: acetone: chloroform = 1:3:2 (v/v).

Embodiment 8:

Dissolve 3mg Cu(DDC) ₂ in 3mL DMF, add to 36.0mL human serum albumin double distilled water solution (1% w/v) under stirring, stir in ice bath for 10min, and ultrasonicate under the condition of ultrasonic power 300W (3s/1s) After 9 minutes, dialyze in double distilled water for 12 hours to remove DMF to obtain Cu(DDC) ₂ albumin nanoparticles with a particle size ranging from 130nm to 170nm.

Embodiment 9:

The difference from Example 8 is that Cu(DDC) ₂ is dissolved in DMSO to obtain Cu(DDC) ₂ albumin nanoparticles with a particle size ranging from 140nm to 170nm.

Example 10:

The difference from Example 8 is that 3 mg Cu(DDC) ₂ was dissolved in 3 mL DMF:acetone (1:1.5), and added to 30.0 mL human serum albumin double distilled water solution (1% w/v) under stirring to obtain Cu(DDC) ₂ albumin nanoparticles, the particle size range is 140nm-170nm.

The methods in the following examples 11 to 53, unless otherwise specified, are conventional methods of ultrasonic method. Stir in an ice bath for 10 minutes, the ultrasonic power is 300W (3s/1s), and the ultrasonic time is 9 minutes.

The difference between Embodiment 11～53 and Embodiment 10 is shown in Table 1:

Table 1 embodiment 11～53 data parameters

Example 54:

Dissolve 3mg Cu(DDC) ₂ in 3mL dichloromethane, add to 33.0mL bovine serum albumin double-distilled water solution (1% w/v) under stirring, stir in ice bath for 10min, and under the condition of ultrasonic power 300W (3s/1s) Sonicate for 9 minutes, remove dichloromethane by rotary evaporation under reduced pressure at 25°C, and obtain Cu(DDC) ₂ bovine serum albumin nanoparticles with a particle size ranging from 120nm to 160nm.

Example 55:

Dissolve 3mg Cu(DDC) ₂ in 3mL DMF, add to 30.0mL hydrophobin double-distilled water solution (0.2% w/v) under stirring, stir in ice bath for 10min, and ultrasonicate for 9 minutes under the condition of ultrasonic power 300W (3s/1s) , DMF was removed by dialysis in double distilled water for 12 hours to obtain Cu(DDC) ₂ hydrophobin nanoparticles with a particle size ranging from 130nm to 160nm.

Example 56:

Dissolve 3mg Cu(DDC) ₂ in 3mL dichloromethane, add to 30.0mL glycoprotein double-distilled water solution (0.5%w/v) under stirring, stir in ice bath for 3min, and sonicate under the condition of ultrasonic power 300W (3s/1s) After 12 minutes, dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ nanoparticles with a particle size ranging from 140nm to 180nm.

Example 57:

Dissolve 3mg Cu(DDC) ₂ in 3mL dichloromethane, add to 30.0mL lipoprotein double-distilled water solution (0.1%w/v) under stirring, stir in ice bath for 12min, and sonicate under the condition of ultrasonic power 300W (3s/1s) After 12 minutes, dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ nanoparticles with a particle size ranging from 170nm to 210nm.

Example 58:

Dissolve 10mg Cu(DDC) ₂ in 10mL dichloromethane, add it into 120.0mL human serum albumin (1%w/v) under stirring, shear at high speed in ice bath for 3-5min, then transfer to a high-pressure homogenizer , first boost to 2000psi, cycle 2 times, then boost to 10000psi, cycle at least 5 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ nanoparticles with a particle size ranging from 120nm to 160nm. Trehalose was added as a lyoprotectant.

Example 59:

The difference from Example 58 is that in the high-pressure homogenizer, the pressure is first increased to 3000 psi and circulated for 2 times, and then the pressure is increased to 18000 psi for at least 5 cycles. Cu(DDC) ₂ nanoparticles were obtained with a particle size ranging from 100nm to 120nm.

Example 60:

The difference from Example 58 is that in the high-pressure homogenizer, the pressure is first increased to 6000 psi and circulated for 2 times, and then the pressure is increased to 35000 psi for at least 5 cycles. Cu(DDC) ₂ nanoparticles were obtained with a particle size ranging from 110nm to 150nm.

Example 61:

The difference from Example 58 is that in the high-pressure homogenizer, the pressure is first increased to 3000 psi and circulated for 2 times, and then the pressure is increased to 18000 psi and circulated for 4 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ nanoparticles with a particle size ranging from 100nm to 140nm.

Example 62:

The difference from Example 58 is that in the high-pressure homogenizer, the pressure is first increased to 3000 psi and circulated for 2 times, and then the pressure is increased to 18000 psi and circulated for 8 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ nanoparticles with a particle size ranging from 100nm to 150nm.

Example 63:

Dissolve 10mg Cu(DDC) ₂ in 10mL chloroform, add it into 100.0mL human serum albumin (1%w/v) under stirring, shear at high speed in ice bath for 3-5min, then transfer to high pressure homogenizer , first boost to 3000psi, cycle 2 times, then boost to 18000psi, cycle 7 times. The chloroform was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ nanoparticles with a particle size ranging from 100nm to 120nm.

Example 64:

Dissolve 10mg Cu(DDC) ₂ in 10mL acetone, add it into 80.0mL human serum albumin (1%w/v) under stirring, shear at high speed in ice bath for 3-5min, then transfer to a high-pressure homogenizer, first Boost to 3000psi, cycle 2 times, then boost to 18000psi, cycle 7 times. Acetone was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ nanoparticles with a particle size ranging from 120nm to 140nm.

The methods in the following examples 65-105, unless otherwise specified, are conventional methods of high-pressure homogenization. First boost to 3000psi, cycle 2 times, then boost to 18000psi, cycle 7 times.

The difference between Embodiment 65～105 and Embodiment 64 is shown in Table 2:

Table 2 embodiment 65～105 data parameters

Example 106:

Dissolve 10mg Cu(DDC) ₂ in 10mL dichloromethane, add it into 120.0mL bovine serum albumin (1%w/v) under stirring, shear at high speed in ice bath for 3-5min, then transfer to a high-pressure homogenizer , first boost to 3000psi, cycle 2 times, then boost to 18000psi, cycle 7 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ bovine serum albumin nanoparticles with a particle size ranging from 100nm to 120nm.

Example 107:

Dissolve 10mg Cu(DDC) ₂ in 10mL DMF, add it into 100.0mL hydrophobin (0.2%w/v) under stirring, shear at high speed in ice bath for 3-5min, then transfer to a high-pressure homogenizer, first increase the pressure To 3000psi, cycle 2 times, then boost to 18000psi, cycle 8 times. DMF was removed by dialysis in double distilled water for 16 hours to obtain Cu(DDC) ₂ hydrophobin nanoparticles with a particle size ranging from 100nm to 130nm. Trehalose was added as a lyoprotectant.

Example 108:

Dissolve 10mg Cu(DDC) ₂ in 10mL dichloromethane, add it into 100.0mL glycoprotein (0.5%w/v) under stirring, shear at high speed in ice bath for 3-5min, then transfer to a high-pressure homogenizer, first Boost to 3000psi, cycle 2 times, then boost to 18000psi, cycle 9 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ glycoprotein nanoparticles with a particle size ranging from 130nm to 160nm. Lactose was added as a lyoprotectant.

Example 109:

Dissolve 10mg Cu(DDC) ₂ in 10mL dichloromethane, add it into 100.0mL lipoprotein (0.5%w/v) under stirring, shear at high speed in ice bath for 3-5min, then transfer to a high-pressure homogenizer, first Boost to 3000psi, cycle 2 times, then boost to 18000psi, cycle 9 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ lipoprotein nanoparticles with a particle size ranging from 150nm to 190nm. Trehalose was added as a lyoprotectant.

Example 110:

Dissolve 10 mg of Cu(DDC) in 10 mL of dichloromethane, add to 120.0 mL of human _serum albumin (HSA) (1% w/v) with stirring, high-speed shear in ice bath for 3-5 min, and then transfer to microfluidizer Inside, first boost to 2000psi, cycle 1 time, then boost to 10000psi, cycle 3 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ nanoparticles with a particle size range of 110-140nm.

Example 111:

The difference from Example 110 is that in the micro jet, the pressure is first increased to 3000psi, and the cycle is 1 time, and then the pressure is increased to 20000psi, and the cycle is 3 times. Cu(DDC) ₂ nanoparticles range in size from 90-110nm.

Example 112:

The difference from Example 110 is that in the micro jet, the pressure is first increased to 6000psi, and the cycle is 1 time, and then the pressure is increased to 36000psi, and the cycle is 3 times. Cu(DDC) ₂ nanoparticles range in size from 110-130nm. Mannitol was added as a lyoprotectant.

Example 113:

The difference from Example 110 is that in the micro jet, the pressure is first increased to 3000psi, and the cycle is 1 time, and then the pressure is increased to 20000psi, and the cycle is 2 times. The particle size range is 110-140nm.

Example 114:

The difference from Example 110 is that in the micro jet, the pressure is first increased to 3000psi, and the cycle is 1 time, and then the pressure is increased to 20000psi, and the cycle is 4 times. Cu(DDC) ₂ nanoparticles range in size from 100-140nm.

The methods in the following examples 115-157, unless otherwise specified, are conventional methods of microfluidics. First boost to 3000psi, cycle 1 time, then boost to 20000psi, cycle 3 times.

The difference between Embodiment 115～157 and Embodiment 114 is shown in Table 3:

Table 3 embodiment 115～157 data parameters

Example 158:

Dissolve 10 mg of Cu(DDC) ₂ in 10 mL of dichloromethane, add it into 120.0 mL of bovine serum albumin (1% w/v) under stirring, shear at high speed in an ice bath for 3-5 min, and then transfer it into the microjet, first Boost to 3000psi, cycle 1 time, then boost to 20000psi, cycle 3 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ nanoparticles with a particle size ranging from 100-120nm.

Example 159:

Dissolve 10mg Cu(DDC) ₂ in 10mL DMF, add it into 100.0mL hydrophobin (0.2%w/v) under stirring, high-speed shear in ice bath for 3-5min, then transfer to micro jet, first increase the pressure to 3000psi , cycle 1 time, then increase the pressure to 20000psi, cycle 3 times. DMF was removed by dialysis in double distilled water for 16 hours to obtain Cu(DDC) ₂ hydrophobin nanoparticles with a particle size ranging from 100nm to 120nm. Trehalose was added as a lyoprotectant.

Example 160:

Dissolve 10mg Cu(DDC) ₂ in 10mL dichloromethane, add it into 100.0mL glycoprotein (0.5%w/v) under stirring, shear at high speed in ice bath for 3-5min, then transfer to the micro jet, first increase the pressure To 3000psi, cycle 1 time, then boost to 20000psi, cycle 3 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ glycoprotein nanoparticles with a particle size ranging from 120nm to 160nm. Lactose was added as a lyoprotectant.

Example 161:

Dissolve 10mg Cu(DDC) ₂ in 10mL dichloromethane, add it into 100.0mL lipoprotein (0.5%w/v) under stirring, shear at high speed in ice bath for 3-5min, then transfer to micro jet, first increase the pressure To 3000psi, cycle 1 time, then boost to 20000psi, cycle 3 times. Dichloromethane was removed by rotary evaporation under reduced pressure at 25°C to obtain Cu(DDC) ₂ glycoprotein nanoparticles with a particle size ranging from 140nm to 170nm. Mannitol was added as a lyoprotectant.

Comparative Example 1: Preparation of Cu(DDC) ₂ PLGA nanoparticles

2mgCu(DDC) ₂ and 10mgPLGA were dissolved in 2mL dichloromethane solution, added to 20ml F68 aqueous solution (0.5%, w/v), after high-speed shear (15000rpm, 3min), under the condition of ultrasonic power 300W (3s/1s) Sonicate for 9 minutes to form an milky dispersion system, remove dichloromethane by low-pressure rotary evaporation at 25°C, and obtain Cu(DDC) ₂ nanoparticles with a particle size range of 180nm-210nm. Trehalose was added as a freeze-drying protective agent, and freeze-dried in vacuum for 24 hours. The obtained cake is quickly redissolved into the original suspension after adding sterile water or physiological saline, and the particle size distribution is the same as before freeze-drying. Sealed and stored at low temperature.

Comparative Example 2: Preparation of Cu(DDC) ₂ PLA nanoparticles

3 mg Cu(DDC) ₂ and 8 mg PLA (molecular weight 10,000) were dissolved in 3 ml of dichloromethane, 20 mg of F68 was dissolved in 6 ml of double distilled water, the organic phase was slowly dropped into the water phase under stirring, and the dichloromethane was removed by rotary evaporation at 25°C to obtain Cu(DDC) ₂ nanoparticles, the particle size range is 200nm-230nm. Trehalose was added as a freeze-drying protective agent, and freeze-dried in vacuum for 24 hours. The obtained cake is quickly redissolved into the original suspension after adding sterile water or physiological saline, and the particle size distribution is the same as before freeze-drying. Sealed, 4 ℃ low temperature storage.

Comparative Example 3: Preparation of Cu(DDC) ₂ Chitosan Nanoparticles

3 mg Cu(DDC) ₂ and 3.5 mg lecithin were dissolved in 3 mL of dichloromethane and then mixed with 1 mL of acetone. 2.4 mg of F68 was dissolved in 60 mL of chitosan (0.5%, w/w) hydrochloric acid solution, and the oil phase was injected into the water phase under stirring. Transfer to the high-pressure homogenizer, boost the pressure to 3000psi at first, then boost to 15000psi, and cycle 5 times. The dichloromethane was removed by rotary evaporation at 25°C to obtain Cu(DDC)2 nanoparticles with a particle size ranging from 160nm to 190nm. Trehalose was added as a freeze-drying protective agent, and freeze-dried in vacuum for 24 hours. The obtained cake is quickly redissolved into the original suspension after adding sterile water or physiological saline, and the particle size distribution is the same as before freeze-drying. Sealed, 4 ℃ low temperature storage.

Comparative example 4: select other organic solvents for use

Since Cu(DDC) _has poor solubility in organic solvents such as methanol, ethanol and ethyl acetate, it is not suitable for separate application in the present invention, but it can be used as a mixed solvent in a small proportion. However, Cu(DDC) ₂ will appear green precipitate in tetrahydrofuran. It is speculated that tetrahydrofuran will compete with Cu(DDC) ₂ for copper, thereby chelating with copper, making Cu(DDC) ₂ unstable, so this organic solvent cannot be used.

Comparative Example 5: Prepared by Ultrasonic Method Using Traditional Stabilizer

Dissolve 1mg Cu(DDC) ₂ in 1mL dichloromethane, add to 20.0mL F68 double distilled water solution (0.5%w/v) under stirring, stir in ice bath for 10min, and ultrasonicate 9 under the condition of ultrasonic power 300W (3s/1s) minutes, dichloromethane was removed by rotary evaporation under reduced pressure at 25°C. The obtained preparation has large particles visible to the naked eye, and is stratified upon standing.

Comparative Example 6: Prepared by homogeneous method using traditional stabilizer

Dissolve 10mg of Cu(DDC) ₂ in 10mL of dichloromethane: acetone (1:10), add it into 90.0mL of F68 double distilled water solution (0.5%w/v) under stirring, shear at high speed in ice bath for 3-5min, then Transfer to a high-pressure homogenizer, boost the pressure to 3000psi at first, then boost to 18000psi, and cycle 7 times. Dichloromethane and acetone were removed by rotary evaporation under reduced pressure at 25°C, and the particle size of the obtained preparation was 190nm-230nm, but precipitation occurred after several days of storage, indicating poor physical stability.

Example 162:

IC ₅₀ of copper gluconate, disulfiram and Cu(DDC) ₂ on the proliferation inhibition of human tumor cell lines:

1. Materials: 6 tumor cell lines: lung cancer A549, α-2; liver cancer HepG2, Hep3B; breast cancer MDA-MB-435s, MCF-7;

2. Method: Take human tumor cells in the logarithmic growth phase, adjust the cell density to 5×10 ⁴ cells/ml with fresh medium, inoculate in 96-well plates, 100 μl/well, at 37°C, 5% Culture in a CO ₂ incubator. After 12-24 hours of adherent culture, the cells were replaced with fresh culture medium containing drugs. Continue to culture the cells for 72 hours after adding the drug, then add MTT solution to the cell solution, 10 μl/well, incubate the cells with 0.25 mg/ml MTT at 37°C for 4 hours, aspirate the culture medium, add 100 μl DMSO, and shake well , measure its optical density OD value.

For data processing, use the corresponding software of the microplate reader for data processing, calculate the average value of OD values of 3 to 5 wells for each sample, and use the average value to calculate the cell viability (CV%) according to the following formula.

Cell survival rate% = average value of OD value of sample group / average value of OD value of blank control group × 100% (CV% = OD _sample / OD _control × 100%)

3. Results: as shown in Table 4.

Table 4 Proliferation inhibition IC ₅₀ of human tumor cell lines

4. Conclusion: As a complex of Cu ions and DSF, Cu(DDC) ₂ has significant antitumor effects on human liver cancer, lung cancer and breast cancer in vitro, and is significantly better than copper gluconate and disulfiram.

Example 163: Inhibitory effect of Cu(DDC) ₂ protein nanoparticle preparation and Cu(DDC) ₂ common solution on tumor cells.

1. Materials: human breast cancer cell MCF7, Cu(DDC) ₂ human serum albumin nanoparticle preparation (prepared according to the method in Example 1), Cu(DDC) ₂ bovine serum albumin nanoparticle preparation (according to this embodiment 54 method), Cu(DDC) ₂ hydrophobin nanoparticle preparation (prepared according to the method of this embodiment 55), Cu(DDC) ₂ glycoprotein nanoparticle preparation (prepared according to the method of this embodiment 56), Cu( DDC) ₂ lipoprotein nanoparticle preparation (prepared according to the method of Example 57), Cu(DDC) ₂ common solution.

2. Methods: Human breast cancer cells MCF7 were cultured in six culture media containing Cu(DDC) ₂ with the same content, in which the culture media contained Cu(DDC) ₂ human serum albumin nanoparticle preparations, Cu(DDC) ) ₂ bovine serum albumin nanoparticle preparation, Cu(DDC) ₂ hydrophobin nanoparticle preparation, Cu(DDC) ₂ glycoprotein nanoparticle preparation, Cu(DDC) ₂ lipoprotein nanoparticle preparation, Cu(DDC) ₂ common solution agent, and the rest of the medium was the same. After the breast cancer cells were cultured at 37° C. for 72 hours, the survival rate of the breast cancer cells in the culture medium was determined.

3. Results: Cu(DDC) ₂ human serum albumin nanoparticle preparation, Cu(DDC) ₂ bovine serum albumin nanoparticle preparation, Cu(DDC) ₂ hydrophobin nanoparticle preparation, Cu(DDC) ₂ glycoprotein nanoparticle preparation In the culture medium of granule preparation and Cu(DDC) ₂ lipoprotein nanoparticle preparation, the survival rates of MCF7 cells were 43.72%, 46.81%, 47.23%, 50.74% and 53.33% _respectively ; In the culture medium, the survival rate of MCF7 cells was 56.40%.

4. Conclusion: Compared with Cu(DDC) ₂ common solution, Cu(DDC) ₂ protein nanoparticle preparation has a higher effect of inhibiting tumor cell activity, and improves the anti-tumor efficacy of Cu(DDC) ₂ .

Example 164: Selective killing effect of Cu(DDC) ₂ human serum albumin nanoparticles on six kinds of human cell lines:

Take 6 tumor cell lines in logarithmic growth phase: liver cancer Hep3B; leukemia cell line Jurkat, human normal cell line L-02, MCF-10A, HUVEC, VE, and adjust the cell density to 5×10 ⁴ with fresh medium cells/ml, seeded in 96-well plates, 100 μl/well, and cultured in an incubator at 37°C and 5% CO ₂ . After 12-24 hours of adherent culture, the cells were replaced with fresh culture medium containing drugs. Continue to culture the cells for 12h, 24h, 48h, 72h after dosing, then add MTT solution to the cell solution, 10μl/well, incubate the cells with 0.25mg/ml MTT at 37°C for 4h, suck out the culture solution, and then add 100μl DMSO, after shaking well, measure its optical density OD value.

Data processing, use the corresponding software of microplate reader to carry out data processing, calculate the average value of OD value of 3～5 wells of each sample, use the average value to calculate cell survival rate according to the following formula (CV%=OD _sample /OD _control ×100 %)

The selective killing effect of Cu(DDC) ₂ on six human cell lines at different time points, 12h, 24h, 48h, and 72h, is shown in Figures 3-6.

The results showed that Cu(DDC) _2, a complex with copper ions, had in vitro cytotoxic effects on a variety of human tumor cells. It was found that at 48h and 72h, the drug had little effect on normal cells at the level of 0.1-1uM L-02 , the survival rate of MCF-10A and HUVEC, but the effect on the tumor lines Hep3B and Jurkat was obvious.

Example 165: Biological Evaluation of Cu(DDC) _Protein Nanoparticles for Injection

1. Safety study of Cu(DDC) ₂ protein nanoparticles for injection:

1.1 Allergy test: observe the allergic reaction caused by Cu(DDC) ₂ protein nanoparticles for injection to animals after systemic administration.

Dose grouping:

Cu(DDC) ₂ human serum albumin nanoparticle preparation (prepared according to the method of Example 1): sensitization dose 2ml (40mg)/time, a total of three times (ip); challenge dose 2ml (40mg)/time, a total of one time ( iv);

Cu(DDC) ₂ bovine serum albumin nanoparticle preparation (prepared according to the method of Example 54): sensitization dose 2ml (40mg)/time, a total of three times (ip); challenge dose 2ml (40mg)/time, a total of one time ( iv);

Cu(DDC) ₂ hydrophobin nanoparticle preparation (prepared according to the method of Example 55): sensitization dose 2ml (40mg)/time, a total of three times (ip); attack dose 2ml (40mg)/time, a total of one time (iv) ;

Cu(DDC) ₂ glycoprotein nanoparticle preparation (prepared according to the method in Example 56): sensitization dose 2ml (40mg)/time, three times (ip); challenge dose 2ml (40mg)/time, once (iv) ;

Cu(DDC) ₂ lipoprotein nanoparticle preparation (prepared according to the method of Example 57): sensitization dose 2ml (40mg)/time, a total of three times (ip); challenge dose 2ml (40mg)/time, a total of one time (iv) ;

Cu(DDC) ₂ solution: sensitization dose 2ml (40mg)/time, three times (ip); attack dose 2ml (40mg)/time, once (iv);

Egg white intravenous injection: sensitization dose 2ml/time, three times (i.p.); challenge dose 2ml/time, once (i.v.);

Negative control group: give the same volume of 5% glucose injection;

Take 48 healthy and uninjured all-white guinea pigs, and divide them into 8 groups according to body weight and gender, namely, Cu(DDC) ₂ human serum albumin nanoparticles for injection, Cu(DDC) ₂ bovine serum albumin nanoparticles for injection, and Cu(DDC) 2 bovine serum albumin nanoparticles for injection. Cu(DDC) ₂ hydrophobin nanoparticles, Cu(DDC) ₂ glycoprotein nanoparticles for injection, Cu(DDC) ₂ lipoprotein nanoparticles for injection, Cu(DDC) ₂ solution, egg white intravenous injection and 5% Glucose injection control group, 6 rats in each group, half male and half male. Each guinea pig was injected ip with Cu(DDC) ₂ protein nanoparticles, Cu(DDC) ₂ solution, egg white intravenous injection and 5% glucose injection 2ml every other day, a total of three consecutive times, and the reaction of the guinea pigs was observed after each injection Condition. After the ip is over, each group is divided into two groups, with three dogs in each group. On the 14th day after the first injection of the test substance in one group, and in the 21st day after the first injection of the other group, each mouse was challenged with 2 ml of the corresponding test substance above iv respectively, and observed and recorded whether the animals scratched their noses, Allergic symptoms such as shrugging, dyspnea, convulsions, shock, and death were observed continuously for 15 minutes. The test results were comprehensively evaluated by the degree of allergic reaction, occurrence rate, death situation and the grade of allergic reaction in guinea pigs.

Test results: After each administration, all guinea pigs did not show any abnormal reaction, and their activities, food intake, and drinking water were all normal, which was the same as the negative control (5% glucose). 14 days and 21 days after the first administration, a single intravenous injection (iv) of Cu(DDC) ₂ protein nanoparticles for injection, Cu(DDC) ₂ solution, egg white intravenous injection and 5% glucose injection 2ml/rat . After the injection, Cu(DDC) ₂ protein nanoparticles for injection 2ml (40mg)/time/mouse and Cu(DDC) ₂ solution 2ml(30mg)/time/mouse dose group had no obvious reaction in guinea pigs, and the degree of allergic reaction was 0. However, the guinea pigs in the egg white group had serious and obvious symptoms of anaphylaxis, mainly manifested as dyspnea, convulsions, urinary incontinence, and shock death. The death time was all within 2 minutes, and the mortality rate reached 100%. class. There was no abnormal reaction in the 5% glucose injection control group.

The results showed that under the dosage conditions of this test, Cu(DDC) ₂ protein nanoparticles for injection were allergy test-qualified drugs, while egg white intravenous injection had severe allergic reactions on guinea pigs.

1.2 In vitro hemolysis experiment: observe whether Cu(DDC) ₂ protein nanoparticles for injection directly contact with blood, whether there is hemolysis effect.

About 10ml of blood was collected from the rabbit's ear vein, stirred with a glass rod (with absorbent cotton on the top) for 10min to remove fibrin, centrifuged (1000rpm × 5min), removed the supernatant, and washed with 10 times the amount of normal saline for three times (both were centrifuged) Then discard the supernatant) until the supernatant has no red color. Then prepare 2% erythrocyte suspension with normal saline for later use.

Take 8 10ml test tubes and add various solutions according to Table 5. Among them, the sixth tube is the blank control of 5% glucose injection, and the seventh tube is the positive control (distilled water). The 8th tube is Cu(DDC) ₂ solution. After gently shaking each tube, place it in a constant temperature water bath at 37°C and incubate for 4 hours, observe the degree of hemolysis of each tube within 0.5 to 4 hours, and judge according to the standards in Table 6. The test results are shown in Table 7.

Table 5 Sample addition table for in vitro hemolysis test

Table 6 Judgment of hemolysis results

Table 7 Cu(DDC) ₂ protein nanoparticles for injection in vitro hemolysis test results

It can be seen from this test that under the test conditions, Cu(DDC) ₂ protein nanoparticles for injection have no hemolysis and erythrocyte aggregation.

1.3 Vascular stimulation experiment: Observe the stimulating effect of Cu(DDC) ₂ protein nanoparticles for injection on the rabbit auricular vein (vessel) after continuous injection of Cu(DDC) 2 protein nanoparticles into the same (relatively concentrated) segment of blood vessel.

The first group of Cu(DDC) ₂ human serum albumin nanoparticle preparation (prepared according to the method of Example 1), 2ml (40mg)/piece/time×1 time×1d;

The second group of Cu(DDC) ₂ bovine serum albumin nanoparticle preparations (prepared according to the method of Example 54)

The third group of Cu(DDC) ₂ hydrophobin nanoparticle preparations (prepared according to the method of Example 55)

The fourth group of Cu(DDC) ₂ glycoprotein nanoparticle preparations (prepared according to the method of Example 56)

The fifth group of Cu(DDC) ₂ lipoprotein nanoparticle preparations (prepared according to the method of Example 57)

The sixth group Cu(DDC) ₂ solution 2ml (40mg)/piece/time×1 time×1d;

The seventh group 5% glucose injection 2ml/piece/time×1 time×1d;

Take 42 rabbits and divide them into seven groups according to their body weight and sex. They were given Cu(DDC) ₂ protein nanoparticle injection 2ml (40mg)/kg/time intravenously after alcohol disinfection of the left ear vein according to the above grouping respectively. Cu(DDC) ₂ solution 2ml (40mg)/kg/time; the seventh group was given an equal volume of 5% glucose injection as a contrast, administered once, and killed the animal 24 hours after the administration, and placed it 1cm below the needle site and At 5cm, the rabbit ears were cut, and redness, swelling, papules, etc. were visible to the naked eye. And fixed with 10% formaldehyde, paraffin section, HE staining, vascular endothelium, subcutaneous tissue and thrombus formation were observed under light microscope.

As a result of the experiment, there was no obvious abnormality in the rabbits of the Cu(DDC) ₂ protein nanoparticle injection group during the administration. However, during the administration of Cu(DDC) ₂ solution, the rabbits had reactions such as screaming and head shaking.

Visual observation: No obvious congestion, exudation, edema and necrosis were observed in the ear veins of animals in the Cu(DDC) ₂ protein nanoparticle injection group.

Microscopic findings: Cu(DDC) ₂ protein nanoparticle injection 2ml (40mg)/kg/time group In the five groups, the amount of red blood cells in the blood vessel at 1cm below the needle insertion site varies, and there is no thrombus formation in the blood vessel Among them, there were two cases of mild swelling of vascular endothelial cells in the hydrophobin nanoparticle injection group, and one case of mild swelling of vascular endothelial cells in the lipoprotein nanoparticle injection group. Cell infiltration; no thrombus, no swelling and hyperplasia of endothelial cells, no hemorrhage outside the blood vessel and other pathological changes were seen in the blood vessel 5 cm below the needle insertion site; no blood vessels were seen in the blood vessel 1 cm below the injection site of Cu(DDC) ₂ solution Thrombus, endothelial cells in all 3 cases were swollen and proliferated to varying degrees, no hemorrhage, edema and necrosis were found outside the blood vessel. No thrombus was seen at 5 cm, no hemorrhage, edema, necrosis and inflammatory cell infiltration around the blood vessel, but mild swelling of vascular endothelial cells was also seen. 5% glucose injection group: No obvious abnormalities were found in the blood vessels and surrounding tissues at 1cm and 5cm.

Test conclusion: Cu(DDC) ₂ human serum albumin nanoparticles, bovine serum albumin nanoparticles, and glycoprotein nanoparticles for injection are basically non-irritating to rabbit ear veins at 1 cm below the injection site; Cu(DDC) ₂ hydrophobin nanoparticles, the clinical dosage of Cu(DDC) ₂ lipoprotein nanoparticles for injection are slightly irritating to rabbit ear veins at 1cm below the injection site; while Cu(DDC) ₂ solution It is irritating to rabbit ear veins at 1cm and 5cm below the injection site.

2. Degradation kinetics of Cu(DDC) ₂ albumin nanoparticles in plasma and whole blood.

Take 15 μL of Cu(DDC) ₂ human serum albumin nanoparticle injection (5 mg/mL), add 0.5, 2.5, and 5 mL of fresh rat blank plasma preheated at 37 °C respectively, and add normal saline to each tube to make the final volume of 5mL, shake well and place in a 37°C water bath, sample 100μL at 0, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24h, add 900μL acetonitrile to precipitate protein, vortex for 10min, and centrifuge at 13000rpm for 10min. The supernatant was taken for HPLC analysis. See Figure 2.

Experimental conclusion: Cu(DDC) ₂ albumin nanoparticles for injection can significantly improve the stability of Cu(DDC) ₂ in the blood, prolong the residence time of Cu(DDC) ₂ in the body, act on the tumor site for a long time, and play an anti-inflammatory role. tumor activity.

3. Study on plasma pharmacokinetics of Cu(DDC) ₂ albumin nanoparticles for injection in rats.

3.1 Materials and methods

3.1.1 Drugs and reagents: Cu(DDC) ₂ albumin nanoparticles for injection (prepared according to the method in Example 1), specification 10ml: 8mg, self-made; Cu(DDC) ₂ suspension, specification 10ml: 24mg, Self-made; chromatographically pure methanol, chloroform, ether, Shandong Yuwang Chemical Factory; diphenhydramine methanol solution, specification 1ml: 200ng, self-made; experimental water, double distilled water.

3.1.2 Animals: SD rats, body weight (250±30) g, 12, male and female, provided by Experimental Animal Center of Shenyang Pharmaceutical University. They were randomly divided into two groups, 6 rats in each group.

3.2 Dosing regimen and sample collection: SD male rats were fasted for 12 hours before the experiment, and Cu(DDC) ₂ albumin nanoparticles were injected intravenously into the tail vein according to the doses of Cu(DDC) ₂ 4mg/kg and 12mg/kg respectively. solution, intragastric Cu(DDC) ₂ suspension, 0.0833, 0.1667, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12h after administration for the intravenous injection group and 0.5 , 1, 2, 4, 8, 10, 12, 24 and 36 hours, about 0.5ml of blood was taken from the orbital vein, placed in a heparinized test tube, centrifuged at 6000rpm for 10min, separated from the plasma, and stored at -20°C for testing.

3.3 Plasma sample processing:

Precisely draw 100 μl of plasma sample, accurately add 20 μl of diphenhydramine internal standard solution, shake well, add 4ml of dichloromethane, vortex extract for 10 minutes, centrifuge at 4000rpm for 10 minutes, take the dichloromethane layer, and dry it with nitrogen in an air bath at 30°C , the residue was redissolved in 200 μl of acetonitrile, 20 μl was taken for HPLC measurement, the peak areas of Cu(DDC) ₂ and the internal standard were recorded, the ratio of the two was calculated, and the internal standard method was used for quantification.

3.4 Average blood drug concentration-time curve: the average blood drug concentration-time curve of oral gavage Cu(DDC) ₂ suspension is shown in Figure 7; the average blood drug concentration-time of Cu(DDC) ₂ albumin nanoparticles for intravenous injection The curve is shown in Figure 8.

Claims (7)

1. treat the injection Cu (DDC) of tumor ₂nano particle preparations, selects albumen to prepare nanoparticle as carrier, it is characterized in that: described preparation is prepared from by following component: Cu (DDC) ₂, albumen, organic solvent and water, its volume ratio is (Cu (DDC) ₂+ organic solvent): (albumen+water)=1:2 ~ 1:30, wherein Cu (DDC) ₂concentration is 0.01 ~ 5mg/mL, and protein concentration is 0.05% ~ 25% (w/v); During preparation, first by Cu (DDC) ₂be dissolved in organic solvent, join in the protein solution of 0.05% ~ 25% concentration under condition of ice bath while stirring, described albumen is bovine serum albumin, hydrophobin, glycoprotein or lipoprotein; Described organic solvent be dichloromethane, chloroform, acetone, dimethyl formamide, dimethyl sulfoxide, dioxane, ethyl acetate, acetonitrile, methyl pyrrolidone, methanol, ethanol, medium chain alcohol one or more; Aqueous phase adds acid-base modifier.

2. according to the injection Cu (DDC) treating tumor described in claim 1 ₂nano particle preparations, is characterized in that: described volume ratio is (Cu (DDC) ₂+ organic solvent): (albumen+water)=1:8 ~ 1:25, wherein, Cu (DDC) ₂concentration is 0.1 ~ 2mg/mL, protein concentration is 0.5 ~ 5%.

3. according to the injection Cu (DDC) treating tumor described in claim 2 ₂nano particle preparations, is characterized in that: described volume ratio is (Cu (DDC) ₂+ organic solvent): (albumen+water)=1:12, wherein, Cu (DDC) ₂concentration is 1mg/mL, protein concentration is 1%.

4. treat the injection Cu (DDC) of tumor as claimed in claim 1 for one kind ₂the preparation method of nano particle preparations, is characterized in that, described preparation adopts ultrasonic method or high pressure homogenization method preparation:

Adopt ultrasonic method preparation process: by Cu (DDC) ₂be dissolved in organic solvent, join in the protein solution of 0.05% ~ 25% concentration under condition of ice bath while stirring, ultrasonic, reduction vaporization removing organic solvent, filtration sterilization, lyophilizing, obtains Cu (DDC) ₂nanoparticle;

Adopt high pressure homogenization method preparation process: Cu (DDC) ₂be dissolved in organic solvent, join in the protein solution of 0.05% ~ 25% concentration under condition of ice bath, after high speed shear, transfer to high pressure homogenizer internal recycle, then reduction vaporization removing organic solvent, filtration sterilization, lyophilizing, obtains Cu (DDC) ₂nanoparticle;

The temperature of described condition of ice bath is 0-10 DEG C.

5. according to the injection Cu (DDC) treating tumor described in claim 4 ₂the preparation method of nano particle preparations, is characterized in that, detailed process prepared by described ultrasonic method is: by Cu (DDC) ₂be dissolved in organic solvent, join in albumen double steaming solution under stirring, 1 ~ 20min is stirred under 0-10 DEG C of condition of ice bath, ultrasonic 3 ~ 30min under ultrasonic power 150 ~ 400W condition, then reduce pressure rotary evaporation removing organic solvent, particle size range is at 120nm ~ 160nm, and filtration sterilization, obtains Cu (DDC) ₂nanoparticle, lyophilisation at least 24 hours.

6. according to the injection Cu (DDC) treating tumor described in claim 4 ₂the preparation method of nano particle preparations, is characterized in that, detailed process prepared by described high pressure homogenization method is: by Cu (DDC) ₂be dissolved in organic solvent, join while stirring in protein solution under 0-10 DEG C of condition of ice bath, high speed shear 3 ~ 5min, then transfer in high pressure homogenizer, first boost to 3000 ~ 6000psi, circulate 1 ~ 3 time, after boost to 10000 ~ 35000psi, circulate 4 ~ 8 times; Decompression rotary evaporation removing organic solvent, filtration sterilization obtains Cu (DDC) ₂nanoparticle, particle size range is at 100nm ~ 120nm; Lyophilisation at least 24 hours.

7. according to the injection Cu (DDC) of the arbitrary described treatment tumor of claim 4 ~ 6 ₂the preparation method of nano particle preparations, is characterized in that: Cu (DDC) before lyophilization ₂nanoparticle adds freeze drying protectant, described freeze drying protectant is that trehalose, mannose, lactose, glucose, sucrose, a-D-mannopyranose, cellobiose, maltose, inose, Raffinose, inulin, dextrose are liquor-saturated, maltodextrin, Fructus Hordei Germinatus polysaccharide, eight sulphuric acid sucrose, heparin, 2-hydroxy propyl-Beta cyclodextrin, glycerol, mannitol, inositol, sorbitol, mercaptan, Polyethylene Glycol, adonite, aminoacid, polymer, Tween 80, pluronic, background of cloth outstanding person, dodecyl sodium sulfate, ascorbic acid one or more.