CN112494660B

CN112494660B - Preparation method of nano targeting drug and application of nano targeting drug in treating gastric cancer

Info

Publication number: CN112494660B
Application number: CN202011525066.4A
Authority: CN
Inventors: 魏冬青; 莫达·沙扎德·洛迪; 阿里夫·马里克; 穆罕默德·塔希尔·汗; 塞拉·阿夫塔布; 扎胡尔·卡迪尔·萨姆拉; 王恒; 王艳菁
Original assignee: Haimen Zhiyi Pharmaceutical Technology Co ltd; Lahore Pakistan, University of; Yantai Intelligent Medical Technology Co ltd; Shanghai Jiaotong University; Panjab University
Current assignee: Haimen Zhiyi Pharmaceutical Technology Co ltd; Lahore Pakistan, University of; Yantai Intelligent Medical Technology Co ltd; Shanghai Jiaotong University; Panjab University
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2023-11-21
Anticipated expiration: 2040-12-22
Also published as: CN112494660A

Abstract

The invention relates to the technical field of biological medicines, in particular to a preparation method of a nano targeting drug and application thereof in treating gastric cancer, two different nano drugs are prepared, and main nano carrier Gold Nano Particles (GNP) and zinc sulfide quantum dots (ZnS QD) are coupled with doxorubicin and ligand transferrin to target protein tumor cells of transferrin receptors which are highly expressed on gastric cancer cells or stomach. Gold Nanoparticles (GNPs) are the most compatible nanostructures, have greater advantages over other metal nanocomposites, zinc sulfide quantum dots (ZnS QDs) are very important quantum dot nanoparticles, which are less toxic, human friendly, and spectrally narrow-band. The two therapeutic nano-drugs prepared are good in stability, high in drug loading, excellent in slow release property, capable of enhancing targeted concentration of drugs, enhancing the treatment effect of tumor killing, reducing non-specific toxic and side effects, and good in application prospect, and the design modes of the two therapeutic nano-drugs can be used for diagnosing, treating and prognosing the gastric cancer.

Description

Preparation method of nano targeting drug and application of nano targeting drug in treating gastric cancer

Technical Field

The invention relates to the technical field of biological medicines, in particular to a preparation method of a nano targeting drug and application thereof in treating gastric cancer.

Background

Among all terrible diseases, cancer is the leading cause of death worldwide, and their incidence is increasing worldwide. Among the different types of cancers, gastric cancer is one of the most common cancers with the highest mortality rate, and is probably caused by diet, organic solvents, pesticides and pesticides, and is also directly related to many factors, such as high salt intake in diet or microbial infection of helicobacter pylori (h.pyri) and the like, which is caused by cancer of the stomach wall infected with helicobacter pylori and the like or by some genetic alteration. Currently, various imaging techniques are being used for cancer diagnosis, such as single photon emission tomography (single photon emission tomography), magnetic Resonance Imaging (MRI), X-ray based computer-assisted tomography (CT) and Positron Emission Tomography (PET), all of which are non-invasive imaging methods for cancer detection. Pharmaceutical methods for cancer treatment include chemotherapy, radiation therapy and surgery, all of which unfortunately bring about a number of side effects, which not only cause diseases such as alopecia, nausea, fatigue, digestive system diseases, mouth ulcers and neurological diseases, but also destroy normal tissues and their eradication of cancer cells is incomplete.

In contrast to these therapies, nanobiotechnology promises to selectively deliver drugs targeting cancer cells with the help of targeted therapies to eliminate all of these side effects. The primary purpose of targeted drug delivery is to avoid toxic effects of the drug on normal cells, as another advantage of targeted drug delivery is the limited release of centrally controlled drug concentrations compared to the target cells. The controlled release of the drug not only expands the efficacy of the drug, but also avoids life loss caused by all adverse reactions related to the drug. Many successful approaches to targeted drug delivery have been disclosed involving different strategies for cells and organs, most of which have been reported as targeted delivery methods for cancer, targeting their cells using specific membrane-bound antigens for cancer.

The nano biotechnology brings new scientific prospect for targeted drugs and drug treatment. The participation of nanocarriers greatly changes the fate of targeted drug delivery, affecting not only the therapeutic field but also the diagnostic and prophylactic fields. Several decades of research have demonstrated that biodegradable nanoparticles are the best and friendly bioengineering for drug delivery or for target tissues. Researchers have focused on nanoparticles that are biodegradable and excreted from the body without any harm to the body, and by combining nanotechnology with drugs, called nanomedicines, the formed nanomedicines of interest are intended to provide maximum doses to the affected area and enhance their biodistribution to target sites, targeted delivery to cancer cells can minimize cytotoxicity of normal tissues where the anticancer agent acts.

The development of nano-drug formulations and the use of metal nanoparticles, such as iron, silver, gold or quantum dots, have advantages in delivery and imaging. The use of bioluminescent nanoparticles as ligands for drug-carrying and targeted nanocarrier targeting receptor sites can provide diagnostic and therapeutic effects, these diagnostic and therapeutic nanomedicines are termed therapeutic nanomedicines.

The main step in the selection of nanocarriers is to select targeted cancer cells for the appropriate ligand. Expression of many receptors increases many-fold on cancer cells compared to normal humans, such as folate and transferrin receptors. Elevated expression of transferrin receptor is observed on cancer cells, increasing iron uptake in cancer cells to promote biological activity, while expression of transferrin receptor is relatively limited on normal cells, so transferrin receptor can be used as a targeting site for nanomedicine for drug delivery targeting cancer.

Doxorubicin, although having high antitumor activity, cannot distinguish between normal cells and cancer cells. Doxorubicin (anticancer drug) has a low permeation molecular weight, is easily permeable to all cells, and is equally harmful to the health of normal cells. Adriamycin is used as an anticancer drug to overcome the obstacle, and the drug can be combined with the Adriamycin.

The therapeutic effect of doxorubicin at specific surface receptors can be enhanced by targeting cancer cells with ligands. Expression of transferrin receptors attracts researchers' attention to delivery on cells that target rapid drug growth. The main problem in determining therapeutic agents and targeting ligands is the choice of nanocarriers, gold Nanoparticles (GNPs) and zinc sulfide quantum dots (znqds) are chosen as nanocarriers for application in cancer diseases, which has not been reported in the open literature.

Disclosure of Invention

The invention aims to provide a preparation method of a nano targeting drug and application thereof in treating gastric cancer.

In order to achieve the above purpose, the technical scheme provided by the invention is as follows:

the nanometer targeting medicine includes GNP therapeutic nanometer medicine and ZnS QD therapeutic nanometer medicine.

The joint of the GNP therapeutic nano-drug is cysteine, the targeting ligand transferrin, the chemotherapeutic drug doxorubicin and the nano-carrier are GNP.

The joint of the ZnS QD therapeutic nano-drug is cysteine, the targeting ligand transferrin, the chemotherapeutic drug doxorubicin and the nano-carrier is ZnS QD.

The invention provides a preparation method of a nano targeting drug, which comprises preparation of a GNP therapeutic nano drug and preparation of a ZnS QD therapeutic nano drug.

Preparation of therapeutic nano-drugs for GNP, including synthesis of GNP, combination of cysteine and GNP, combination of doxorubicin and GNP-Cys, and combination of transferrin and GNP-Cys-Dox.

Preparation of ZnS QD therapeutic nano-drug, comprising synthesis of cysteine coated ZnS QD quantum dot, binding of doxorubicin to ZnS QD-Cys, and binding of transferrin to ZnS QD-Cys-Dox.

Cysteine-coated GNPs conjugated with transferrin can be used as targeted diagnostic probes.

The beneficial effects of the invention are as follows:

in the invention, two therapeutic nano-drugs are prepared, and gold nano-particles (GNP) serving as nano-carriers are the most compatible nano-structures, can be used for diagnosis, prognosis, imaging and treatment, and have greater advantages than other metal nano-composites. Zinc sulfide quantum dots (ZnS QDs) are very important quantum dot nanoparticles that are low in toxicity, narrow in spectrum, adjustable in emission profile, and high in emission quantum yield. The two therapeutic nano-drugs prepared are good in stability, high in drug loading, excellent in slow release property, capable of enhancing targeted concentration of drugs, enhancing the treatment effect of tumor killing, reducing non-specific toxic and side effects, and good in application prospect, and the design modes of the two therapeutic nano-drugs can be used for diagnosing, treating and prognosing the gastric cancer.

Drawings

FIG. 1 is a UV-VIS spectrum, (a) a UV-VIS spectrum of GNP-Cys, and (b) a UV-VIS spectrum of transferrin-conjugated GNP-Cys.

FIG. 2 is an excitation and emission spectrum of a target diagnostic probe.

FIG. 3 is a TEM image of GNP-Cys and transferrin-bound GNP-Cys.

FIG. 4 is a whole tumor tissue examination and histological study of tumor tissue after (in vivo) binding of the targeted diagnostic probes. (a) bright field studies of tumor tissue revealed the morphology of the tissue; (b) The whole tissue examination under the uv excitation filter showed in vivo binding of the target probe to transferrin receptor on tumor cells; (c) Whole tissue examination under blue excitation filter; (d) And (e) histological study of tumor cryostat sections was identical to that observed in whole tissues.

FIG. 5 is a histological study of in vitro whole stomach tissue examination and in vivo tissue targeting diagnostic probe binding. (a) Bright field studies of gastric tissue show tissue morphology with tumor areas; (b) Throughout the tissue examination under the ultraviolet excitation filter, the target probe binds to transferrin receptor in the area of the clot in vivo; (c) The blue excitation filter under the tissue examination shows that the tissue emits more fluorescence in this region than ultraviolet light; (d) And (e) histological studies of gastric cancer tissue cryostat sections showed the same results as observed throughout the tissue examination.

FIG. 6 is an in vitro binding of a tumor section and gastric cancer tissue section histological study with a targeting diagnostic probe. (a) And (b) binding of the diagnostic probe to a tumor tissue section; (c) And (d) targeting the binding of the diagnostic probe to the gastric tissue section.

FIG. 7 is a cytotoxicity table of different GNP nanocomposites.

FIG. 8 is a graph of cytotoxicity of different GNP nanocomposites.

Figure 9 is a cytotoxic profile of GNP therapeutic nanomaterials at different concentrations.

Figure 10 is a graph of cytotoxicity of different concentrations of GNP therapeutic nanomaterials. (a) Cumulative effect of different concentrations of nanomedicine at 6 and 24 hours; (b) Cytotoxic effects at different concentrations at two time points 6 and 24 hours.

Figure 11 is a cytotoxicity table of GNP tumor treating nanomedicines at various time points.

FIG. 12 is a graph showing growth, cell morphology and fluorescence studies of GNP nanocomposites at the 6 hour time point.

FIG. 13 shows growth, cell morphology and fluorescence studies of GNP nanocomposites at the 12 hour time point.

FIG. 14 shows growth, cell morphology and fluorescence studies of GNP nanocomposites over a 24 hour time point.

FIG. 15 shows growth, cell morphology and fluorescence studies of GNP nanocomposites at the 48 hour time point.

Figure 16 is a graph showing survival and percent of live/dead cells for GNP nanocomposites at various time points. (a) The GNP tumor therapeutic nano-drug has cytotoxicity at 6, 12, 24, 48 and 72 hours; (b) The cytotoxic effects of GNP therapeutic nanomedicines caused a change in the proportion of viable and dead cells at 6, 12, 24, 48, 72 hours.

FIG. 17 is an intracellular trace of GNP nanocomposites in the form of GNP and doxorubicin at various time points.

Fig. 18 is a fluorescence intensity measurement of GNP therapeutic nanomaterials at different time points.

FIG. 19 is a graph showing the determination of the concentration of GNP synthesized nanocomposite at different time points based on fluorescence intensity.

Figure 20 is in vitro receptor binding of GNP therapeutic nanomaterials on gastric cancer tissue.

Figure 21 is a cell trace of GNP therapeutic nanomaterials at different time points.

Fig. 22 is fluorescence intensity of GNP therapeutic nanomaterials at different time points.

Fig. 23 is a comparison of fluorescence intensity (full window) at various time points after oral administration of GNP therapeutic nanomaterials.

Fig. 24 is the anti-gastric cancer efficacy of GNP tumor therapeutic nanomaterials during in vivo treatment.

Figure 25 shows survival of mice at various times during 40 days of GNP tumor therapeutic nanomedicine treatment.

Figure 26 is a graph of the change in body weight of living mice at various times during 40 days of GNP therapeutic nanomedicine treatment.

Fig. 27 is a cytotoxicity table of different ZnS nanocomposites.

Fig. 28 is a cytotoxicity table of ZnS QD therapeutic nanomaterials at different concentrations.

Fig. 29 is a graph of cytotoxicity of ZnS QD nanocomposites at different concentrations.

Figure 30 is a cytotoxicity table of ZnS QD therapeutic nanomedicine at various time points.

Fig. 31 is a ZnS QD nanocomposite growth at the time point of 6 hours, cell morphology and fluorescence study.

Fig. 32 is ZnS QD nanocomposite growth at the 12 hour time point, cell morphology and fluorescence studies.

Fig. 33 is ZnS QD nanocomposite growth at 24 hours time points, cell morphology and fluorescence studies.

Fig. 34 is a ZnS QD nanocomposite growth at the 48 hour time point, cell morphology and fluorescence study.

Fig. 35 is the viability of ZnS QD nanocomposites and the cytotoxic effect of surviving/dead cells at different time points.

Fig. 36 is an intracellular trace of ZnS QD choroidal nanocomposites in the form of ZnS QD and doxorubicin at different time points.

Fig. 37 is a graph of fluorescence intensity measurements of ZnS QD therapeutic nanomaterials at different time points.

Fig. 38 is fluorescence intensity for ZnS QD nanocomposites at different time points.

Fig. 39 is in vitro receptor binding of ZnS QD therapeutic nanomaterials in gastric cancer tissue.

Fig. 40 is a cell trace of ZnS QD therapeutic nanomedicine at various time points.

Fig. 41 is a comparison of fluorescence intensity at various time points after oral administration of ZnS QD venation nanomaterials.

Fig. 42 is the fluorescence intensity of ZnS QD therapeutic nanomedicine at different time points.

Fig. 43 is the anti-gastric cancer efficacy (in vivo) of ZnS QD therapeutic nanomedicines.

Fig. 44 is the survival of mice at different times during 40 days of treatment with ZnS QD therapeutic nanomedicine.

Fig. 45 shows the change in body weight of living mice at various times during the treatment period of 40 days with ZnS QD nanotherapeutics.

Fig. 46 is the anti-gastric cancer efficacy (in vivo) of ZnS QD nanotherapeutics.

Fig. 47 is a graph showing the release behavior of doxorubicin from GNP therapeutic nanomaterials at pH 5.

Fig. 48 is a graph showing the release behavior of doxorubicin from GNP therapeutic nanomaterials at pH 7.5.

Fig. 49 is a graph showing the release of doxorubicin from GNP therapeutic nanomaterials at various time points at pH 5.

Figure 50 is a graph showing the release of doxorubicin from GNP therapeutic nanomaterials at various time points at pH 7.5.

FIG. 51 is a chromatogram of doxorubicin.

Fig. 52 is doxorubicin concentration in GNP therapeutic nanomedicine targeted and non-targeted responses.

FIG. 53 is an analysis of doxorubicin concentration in dead cells and medium.

Figure 54 is the in vitro concentration of doxorubicin (GNP therapeutic nanomaterials) in the culture medium and dead cells after various time points.

Fig. 55 shows doxorubicin concentration (GNP therapeutic nanomaterials) in the culture medium incubated at various time points.

Fig. 56 is plasma drug activity profile data of GNP therapeutic nanomaterials analyzed by HPLC after intravenous administration (in vivo).

Fig. 57 is a graph showing pharmacokinetic parameters of GNP therapeutic nanomaterials.

Fig. 58 is plasma drug activity profile data of GNP therapeutic nanomaterials analyzed by HPLC after oral (in vivo).

Fig. 59 is a plasma drug activity profile of doxorubicin (GNP therapeutic nanopharmaceuticals) after oral administration.

Fig. 60 is the pharmacokinetic parameters after oral administration of GNP therapeutic nanomaterials.

Fig. 61 is a graph showing doxorubicin concentration in the liver following intravenous and oral administration of GNP therapeutic nanomaterials.

Fig. 62 is a comparison of the biodistribution of doxorubicin in the liver at various time points in GNP therapeutic nanomedicines following intravenous and oral administration.

Figure 63 shows doxorubicin concentration in hearts following intravenous and oral GNP therapeutic nanomaterials.

Figure 64 is a comparison of the biodistribution of doxorubicin in GNP therapeutic nanomedicines at different time points in the heart following intravenous and oral administration.

Figure 65 shows doxorubicin concentration in the kidneys after intravenous and oral GNP therapeutic nanomaterials.

Fig. 66 is a comparison of the biodistribution of doxorubicin in the kidney in GNP therapeutic nanomedicines at different time points following intravenous and oral administration.

Fig. 67 is a comparison of the biodistribution of GNP therapeutic nanodrug doxorubicin in gastric tumors at various time points following intravenous and oral administration.

FIG. 68 shows doxorubicin concentration in gastric tumors following intravenous and oral administration of GNP therapeutic nanomaterials.

Fig. 69 is the release behavior of doxorubicin from ZnS QD therapeutic nanopharmaceuticals at pH 5.

Fig. 70 is the release behavior of doxorubicin from ZnS QD therapeutic nanopharmaceuticals at pH 7.5.

Fig. 71 shows the release of doxorubicin at various time points for ZnS QD therapeutic nanomaterials at pH 5.

Fig. 72 is a graph showing the release of doxorubicin at various time points for ZnS QD therapeutic nanopharmaceuticals at pH 7.5.

Fig. 73 is doxorubicin concentration in ZnS QD therapeutic nanomedicine targeted and non-targeted reactions.

Figure 74 is an analysis of doxorubicin concentration in dead cells and medium (non-targeted and targeted delivery).

Fig. 75 shows the in vitro concentration of doxorubicin (znqd therapeutic nanomedicine) in culture medium and dead cells after various time points.

Fig. 76 shows doxorubicin concentration (ZnS QD therapeutic nanopharmaceuticals) in the culture medium incubated at various time points.

Fig. 77 is in vivo plasma drug activity profile data of nanomaterials measured by HPLC after ZnS QD intravenous administration.

Fig. 78 is a plasma drug activity profile of doxorubicin (ZnS QD therapeutic nanopharmaceuticals) after intravenous administration.

Fig. 79 is the pharmacokinetic parameters of ZnS QD therapeutic nanomaterials.

Fig. 80 is plasma drug activity profile data analyzed by HPLC after oral ZnS QD-based nanomaterials.

Fig. 81 is a plasma drug activity profile of doxorubicin (ZnS QD therapeutic nanopharmaceuticals) following oral administration.

Fig. 82 is the pharmacokinetic parameters of ZnS QD therapeutic nanomaterials after oral administration.

Fig. 83 is the concentration of doxorubicin in the liver following intravenous and oral administration of ZnS QD therapeutic nanomaterials.

Fig. 84 is a comparison of the biodistribution of doxorubicin in the liver at various time points ZnS QD therapeutic nanopharmaceuticals following intravenous and oral administration.

Fig. 85 is the concentration of doxorubicin in the heart following intravenous and oral administration of ZnS QD therapeutic nanomaterials.

Fig. 86 is a comparison of the biodistribution of doxorubicin in ZnS QD therapeutic nanopharmaceuticals at different time points in the heart following intravenous and oral administration.

Fig. 87 is the concentration of doxorubicin in the kidneys after intravenous and oral administration of ZnS QD therapeutic nanomaterials.

Fig. 88 is a comparison of the biodistribution of doxorubicin in the kidneys in ZnS QD therapeutic nanopharmaceuticals at different time points following intravenous and oral administration.

FIG. 89 comparison of biodistribution of ZnS QD therapeutic nanodrug doxorubicin in gastric tumors at different time points after intravenous and oral administration

Fig. 90 shows doxorubicin concentration in gastric tumors following intravenous and oral administration of ZnS QD therapeutic nanomaterials.

Detailed Description

The invention will be further described with reference to the drawings and specific examples, which do not limit the scope of the invention.

Example 1 preparation of nanotherapeutic drug

Two nanometer therapeutic drugs are prepared respectively. Both nanotherapeutic drugs have the same linker: cysteine, transferrin as targeting ligand, doxorubicin as chemotherapeutic drug, but the nanocarriers are different. Gold Nanoparticles (GNPs) are used in the first nanotherapeutic drug because of their unique physical properties, and zinc sulfide quantum dots (ZnS) are selected as the second nanotherapeutic drug nanocarriers.

The preparation of the nanometer therapeutic drug is as follows:

Preparation of GNP nanotherapeutics comprising synthesis of GNP, binding of cysteine to GNP, binding of doxorubicin to GNP-Cys, and binding of transferrin to GNP-Cys-Dox.

GNPs were synthesized using optimized citrate reduction, gold salts and sodium citrate at different concentrations were used to check for ideal concentrations, GNPs exhibited different optical properties, and 1% gold salt stock (chloric acid) was used to prepare HAuCl for optimal final reaction ₄ And the solution was boiled with continuous stirring on a hot plate maintained at a preset 100 c by diluting it to water in a 15mM working solution beaker in 400ml deionized water. A 1% trisodium citrate solution was prepared in deionized water and heated to 50 ℃, 12.5ml of preheated 1% trisodium citrate was quickly added to the boiling solution with vigorous stirring, continuous stirring and observation until the color of the reaction mixture became dark. The solution turned bright red, the reaction time was about 20 minutes, and the bright red showed a size of less than 15nm, completing the synthesis of GNP.

L-cysteine solution (0.2 mM) was prepared in deionized water, and 1ml was added to the prepared 400ml GNP solution. The solution was stirred at 25 ℃ for 2 hours and then shielded from interference for 12 hours. The cysteine-attached gold nanoparticles were centrifuged and run at 14,000rpm for 40 minutes. The precipitate was washed with deionized water.

Doxorubicin and cysteine attached gold nanoparticles by (1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide (EDC) method 50mg of cysteine coated 1ml gnp deionized water, 1ml EDC adjusted with 1M pH8 NaOH (20 mg/ml added deionized water) to activate carboxyl groups and pH adjusted with 1M HCl to pH6.4 the reaction mixture was incubated in the dark for 30 minutes with continuous shaking at 100rpm, after 30 minutes incubation 1ml carbodiimide (10 mg/ml deionized water) was added to the drug containing 50mg of doxorubicin and further incubated for 2 hours at 37 ℃ in dark, continuous shaking at 100rpm, centrifugation of conjugate particles at 14000rpm for 20 minutes and washing twice with deionized water and storage at 4 ℃.

Transferrin was conjugated to doxorubicin conjugated cysteine coated GNP nanocomposites by glutaraldehyde method. Briefly, the amine groups of cysteines on gold nanoparticles (GNP-Cys) were crosslinked with the amine groups of transferrin by glutaraldehyde crosslinking. 100mg of GNP-Cys-Dox was suspended in 100ml of coupling buffer (0.01M pyridine hydrochloride, pH6,0.1M NaCl) and sonicated for 10 min. After 10 minutes, the suspension was centrifuged at 14000rpm for 40 minutes, and the supernatant was then aspirated. The above procedure was repeated twice with coupling buffer and after the second wash. The nanocomposites were suspended in a ligation buffer (5% glutaraldehyde in coupling buffer), the suspension was shaken on an orbital shaker at 25℃for 3 hours at 100rpm, and then centrifuged again at 14000rpm for 20 minutes. The supernatant was aspirated and washed with the nanocomposite as before with coupling buffer to remove excess glutaraldehyde. 10mg of transferrin was dissolved in 100ml of coupling buffer and left to stand in 1ml of pre-coupling buffer. The remaining transferrin solution is mixed with 100mg of wash solution to form a nanocomposite. The mixture was shaken at 25℃for 24 hours and then centrifuged at 8000rpm at 4℃to obtain transferrin-coupled GNP-Cys-Dox nanocomposites (GNP therapeutic nanomedicine (GNP-Cys-Dox-transferrin)) the conjugated nanocomposites were suspended in storage buffer (0.01M Tris-Cl, pH7.4,0.1% sodium azide, 0.15M NaCl) in-20 ℃.

Preparation of ZnS QD nanotherapeutics comprising synthesis of cysteine coated ZnS QD quantum dots, binding of doxorubicin to ZnS QD-Cys, binding of transferrin to ZnS QD-Cys-Dox.

In the reaction of zinc chloride with sodium sulfide in water in the presence of L-cysteine and taurine, cysteine coated zinc sulfide quantum dots (znqds) were synthesized, briefly, 0.01M pH8 zinc chloride solution, 0.01M pH8L-cysteine solution and 0.1M taurine were refluxed in 200ml total reaction volume for half an hour. Taurine is added as an antioxidant to prevent oxidation of the surface of the quantum dot synthesized in the air and improve the size and fluorescence quality of the quantum dot prepared in the air. After refluxing for half an hour, 0.01M sodium sulfide in 10ml deionized water was added dropwise to the reaction mixture, and further refluxed for 12 hours. The synthesized cysteine coated ZnS QD nanoparticles were centrifuged at 14000rpm for 20 minutes at 25 ℃. The precipitate was washed with deionized water.

Doxorubicin and cysteine coated ZnS QD 100mg of cysteine coated ZnS was substituted for GNP by (1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide (EDC) method.1 ml of EDC (20 mg/ml added deionized water) was adjusted with 1M pH8 NaOH to activate carboxyl groups and pH was adjusted to pH6.4 with 1M HCl. The reaction mixture was incubated in the dark for 30 minutes and continuously shaken at 100rpm, after incubation for 30 minutes 1ml of carbodiimide (10 mg/ml deionized water) was added to the drug containing 50mg of doxorubicin and further incubated for 2 hours at 37 ℃ in dark environment, the nanocomposite conjugated to doxorubicin was isolated by centrifugation at 14000rpm for 20 minutes and washed twice with deionized water, and the washed nanocomposite was stored at 4 ℃.

Glutaraldehyde method couples transferrin to doxorubicin conjugated cysteine coated ZnS QDs. 100mg of ZnS QD-Cys-Dox was suspended in 100ml of coupling buffer (0.01M pyridine hydrochloride, pH6,0.1M NaCl) and sonicated for 10 min. After 10 minutes, the suspension was centrifuged at 14000rpm for 40 minutes, and the supernatant was then aspirated. The above procedure was repeated twice with coupling buffer and after the second wash. The nanocomposites were suspended in a ligation buffer (5% glutaraldehyde in coupling buffer), the suspension was shaken on an orbital shaker at 25℃for 3 hours at 100rpm, and then centrifuged again at 14000rpm for 20 minutes. The supernatant was aspirated and washed with the nanocomposite as before with coupling buffer to remove excess glutaraldehyde. Transferrin (10 mg) is dissolved in 100ml coupling buffer and left to stand in 1ml pre-coupling buffer. The remaining transferrin solution is mixed with 100mg of wash solution to form a nanocomposite. The mixture was shaken at 25 ℃ for 24 hours and then centrifuged at 8000rpm at 4 ℃ to obtain transferrin-coupled ZnS QD-Cys-Dox nanocomposite (ZnS QD therapeutic nanopharmaceuticals (ZnS QD-Cys-Dox-transferrin)) the conjugated nanocomposite was added to a storage buffer (0.01M Tris-Cl, ph7.4,0.1% sodium azide, 0.15M NaCl) and stored at-20 ℃.

Example 2 preparation and characterization of diagnostic probes

Synthetic cysteine-coated GNPs were conjugated to transferrin for diagnostic detection. It was used to examine the efficiency of transferrin as a targeting ligand for cancer and hepatoma cells. Transferrin expression in induced gastric cancer tissues was also examined.

As shown in fig. 1 (b), UV-Vis spectra of the target diagnostic probe were compared with UV-Vis spectra of cysteine-coated GNPs, as shown in fig. 1 (a). The cysteine coated GNP spectrum showed a peak at 520nm, indicating a particle size less than 20nm, narrow size distribution. After transferrin binding, the new spectrum shows a shift between the inorganic peak at 525nm and the organic peak at 340nm, as shown in FIG. 1 (b). The peaks become broader, indicating that conjugation of the protein to the nanoparticle results in an increase in the size of the nanocomposite.

By immobilizing a drop of diagnostic probes on a slide and observing under a fluorescence microscope. The nanocomposites appear blue under uv excitation of the filters, and in the blue excitation filters they appear green. As shown in FIG. 2, excitation and emission spectra of the target diagnostic probe were obtained, which showed maximum excitation at 361.0nm and maximum emission at 362.4nm wavelength.

Hydrodynamic particle size distribution and target average diameter diagnostic probes were measured by DLS at 25 ℃. The particle size distribution is 100nm to 110nm in the solution, and the average diameter of the monodisperse particles is 100nm.

TEM micrographs of cysteine coated gold nanoparticles and targeted diagnostic probes show an increase in size after transferrin binding. The cysteine coated TEM micrograph GNP showed spherical nanoparticles with an average diameter of 10nm as shown in fig. 3 (a). Transferrin binding does not affect shape but results in a 25nm increase in size and an average diameter of 35nm as shown in figure 3 (b).

The receptor binding capacity of purified transferrin-conjugated cysteine-coated GNPs further demonstrates specific binding of probes and their potential use as targeted diagnostic probes in vivo and in vitro. Ex vivo and ex vivo histological studies of gastric cancer and tumor tissue showed that the probes bound to respective receptors on the cancer cells and produced fluorescent images under respective filters. Fig. 4 (a) shows the tumor tissue morphology under a field microscope. As shown in fig. 4 (b) and 4 (c), after in vivo administration, tumor tissue showed strong fluorescence under both filters, and binding of diagnostic probes to tumor cells was confirmed. As shown in fig. 4 (d) and 4 (e), histological studies of tumor cryostat sections showed the same results as observed in the whole tissue, which further confirmed the results of the histological examination. After one hour of oral administration, stomach tissue was examined under gastroscope. As shown in fig. 5 (b) and 5 (c), fluorescence microscopy confirmed binding of the probe to the infected cancer, and as shown in fig. 5 (d) and 5 (e), this section of histological study further confirmed the results. In vitro screening also showed that even after color fixation, probes could be imaged in vivo with receptor binding even after fixation.

It was further demonstrated that targeting the transferrin receptor of diagnostic probes on tissue, binding in vitro and fluorescence studies showed successful binding of the probes to immobilized tissue even at the receptor. As shown in fig. 6 (a) and 6 (b), tumor tissue showed slightly different fluorescence patterns ex vivo in vitro compared to in vitro. In vitro, binding of the targeted diagnostic probe to gastric tissue showed, as shown in fig. 6 (c) and 6 (d), a major gold fluorescence and blue and green. This further demonstrates the potential application of transferrin-conjugated cysteine coated GNPs as cancer in vitro targeting diagnostic probes.

EXAMPLE 3 therapeutic action of GNP and ZnS QDs

GNP tumor therapeutic nano-drug (in vitro)

Cellular uptake, therapeutic effect and cell targeting of therapeutic nanomedicines to colon cancer cells (HCT 116-Luc2And confirmed by different analyses. Techniques for studying cell viability intracellular nanomedicine tracking includes inverted microscopy, fluorescence microscopy, trypan blue assay, neutral red assay (metabolic activity), crystal violet staining assay @Cell adhesion studies) and fluorescence intensity measurements. And carrying out statistical comparison on all the results to obtain clear results.

Human colon cancer cell line: HCT-116 (male patient derived cell line) was used to examine the effect of therapeutic nanomedicines in vitro. The cell line involved is a mismatch defect maintenance system for p53 wild-type gene and K-Ras gene mutation. The morphology of the cells was spindle-shaped in appearance. The remaining vials were removed from the freezer at-80 ℃ and the colon cancer cell line was thawed prior to use. Memory body [ ]High glucose) medium with streptomycin and pencilin 100mg/ml +.>And 10% heat-inactivated fetal bovine serum +>HCT-116 cells in the presence of 5% CO ₂ The growth was carried out in a humidity chamber at 37℃with gas and 87% humidity. Throughout the course of the drug analysis, culture should be routinely performed. The medium was changed every 2 days and subcultured after 5 days.

Three GNP nanocomposites, 5 μl (5 μg/μl), GNP-Cys-Dox and GNP-Cys-Dox-transferrin at lower concentrations were tested against colon cancer cell lines and compared for statistical analysis (ANOVA) to observe the therapeutic effect of the nanocomposites, as shown in fig. 7. GNP values have a toxic effect on cancer cells, but only 8% of cells have 92% viability. GNP-Cys-Dox demonstrated 83% higher toxicity due to doxorubicin binding and cell viability, as shown in fig. 8, which shows cell viability of the first complex (GNP-Cys), the second complex (GNP-Cys-Dox) and the third complex (final therapeutic nanomedicine (GNP-Cys-Dox-Trans)) within 6 hours. Average (n=3) ±sd, p <0.0001 for all groups analyzed, p <0.001 between second and third syntheses. After transferrin binding, small differences in cell viability were observed. The GNP tumor therapeutic nanomaterials showed 80% cell viability and the observed cell morphology results showed that even adherent cells were not in good condition due to the longer lifetime of the nanomaterials. The data lie within the confidence interval, showing the meaning of the comparison.

Cytotoxicity of GNPs at different concentrations, GNP therapeutic nanomaterials at different concentrations, corresponds to examining the effects of doxorubicin at different concentrations on colon cancer cells at 6 hours and 24 hours for incubation. The present invention is useful for examining the optimal time and optimal time concentration of GNP therapeutic nanomedicine effects for further determination. This assay was also used to obtain IC 50 values and the value of the lethal dose GNP therapeutic nanomedicine. Average cell viability values, neutral red analysis, absorbance at different concentrations and percent viability are summarized in fig. 9, statistical comparison (n=3), with a lethal dose of 50 (IC 50) nanomedicine for GNP of 10 μl (50 μg) and a lethal dose of 50 μl. Fig. 10 (a) shows that the different concentrations and increasing concentrations of drug effect, accumulated at two time points 6 and 24 hours, show a decrease in cell viability, as shown in fig. 10 (b). The mean and analysis of variance and data lie within the confidence interval of p <0.0001.

Cytotoxicity and drug binding at different times cytotoxicity was observed at different time points based on cell viability and live dead cell comparison, as shown in fig. 11. After each time point, the plates were observed upside down. Cell morphology and drug fluorescence were observed with a fluorescence microscope. Cells began to show a change in cell morphology in the test medium as compared to the control, and the same pattern was that the last time point was observed. The appearance of the cells was round, shrunken, pale borderline, showing that the drug was only present on the cells and not in the blank. After 6 hours, fluorescence observations confirm that the presence of the nanomedicine, especially doxorubicin, in the cells indicates its presence in the nuclei, as shown in fig. 12. After 6 hours, the cells began to clamp and showed GNPs and doxorubicin concentrations in the cells, as shown in fig. 13. The fluorescence enhancement spot and magnified image after 12 hours showed separation of the nanoparticle from doxorubicin. Nanoparticles were observed in the cytoplasm, while most doxorubicin was in the nucleus, as shown in fig. 14. After 24 hours, the cells were killed with doxorubicin and then released from the cells. At this time, as shown in fig. 15, the cells began to lose fluorescence, and at 72 hours, the cells appeared to darken under a fluorescence microscope. Cytotoxicity studies indicate that GNPs have therapeutic effect nano-drugs are effective even at 72 hours due to slow release of doxorubicin. Feasibility studies showed different effects of drugs at different times. The drug was most effective within 12 hours and 24 hours. The 6 hour viability was only 83% and 43% at 12 hours. Comparison of average cell viability, as shown in fig. 16 (a), the various time points indicate that the data are within the effective region (p < 0.0001). Live dead cells were counted and their mean values were compared by ANOVA. Studies have shown that data lie within the effective interval, p <0.0001. Live cell comparison also demonstrated that the feasibility study of the drug effect remained at 72 hours after 12 hours, as shown in fig. 16 (b).

Intracellular GNP and doxorubicin tracking and fluorescence intensity measurements were performed at different time points, fluorescence intensity measurements at different time points were used to subtract the anti-fluorescence using Image J software and to study GNP and doxorubicin fluorescence independence. Subtracting red from green and going from red into green fluorescence plots, GNP and doxorubicin were traced inside the cell, as shown in fig. 17, which shows receptor binding and intracellular localization of GNP nuclear magnetic resonance nanomaterials with the associated binding and fluorescence intensity changes at 6, 12, 24, 48, 72 hours with nanoparticle and doxorubicin concentrations. Most nanomedicines bind to cells after 6 hours and exhibit fluorescence on whole cells. After 12 hours, the cells fluoresce clearly in combination, but after subtraction of the anti-fluorescence, the decrease indicates that most of the doxorubicin is still related to GNP. Doxorubicin was therefore not only traced inside the nucleus but also in the cytoplasm with GNPs after 12 hours. Within 24 and 48 hours, most of the doxorubicin was located in the nucleus and GNP in the cytoplasm, and within 72 hours, the intracellular concentration began to disappear. The green fluorescence intensity is proportional to the number of GNP nanoparticles, and the red fluorescence intensity is proportional to the amount of doxorubicin. Twenty cells per group were used to calculate intensity and to compare their mean value by analysis of variance as shown in figure 18. As shown in fig. 19, the points showing higher intensities were each 6 hours, and the time after 24 hours at which the intensity began to decrease was 12 hours. The data is at a time interval of p < 0.0001.

GNP tumor therapeutic nanomaterials bind to receptors on gastric cancer tissue (in vitro), and frozen sections of gastric tissue are incubated with GNP nanomaterials to examine the binding capacity of the nanomaterials to target tissues in vitro. As shown in fig. 20, successful binding of GNP tumor therapeutic nanomaterials to transferrin receptor on gastric tissue is shown. Fluorescent blue and green filters showed the presence of GNP nanoparticles in the tissue, while red fluorescence confirmed doxorubicin in the tissue. The GNP tumor therapeutic nanomedicine showed successful and firm binding to transferrin receptor on gastric cancer tissue. Binding also demonstrates that the formulation does not affect transferrin binding to transferrin receptor and can be used to examine drug efficacy in vivo.

Therapeutic efficacy (in vivo) of gnp therapeutic nanomedicines

GNP treatment was examined for therapeutic effect and intracellular drug treatment methods by orally administering a nano-drug equivalent to 5mg/kg doxorubicin.

Receptor binding, intracellular tracking, drug release and fluorescence intensity at various time points, receptor binding activity of tissues was observed ex vivo and immunohistochemical study of gastric tissues collected after intracellular tracking of GNPs 3, 6, 12, 24, 48 and 72 hours was performed at oral doses of nanotherapeutic drug. The nano-drug binds to the receptor and begins immediately after the inward movement of the compartment into contact with the surface. Figure 21 shows the binding and localization of nanomedicine within cells after 3, 6 and 12 hours. This phenomenon was observed in tissue studies 12 hours after maximum drug administration. Doxorubicin fluoresces in red and GNPs fluoresce in blue and green of different intensities. After 12 hours, the drug and nanoparticle began to clear from the cells, with a decrease in fluorescence intensity at 48 and 72 hours. As shown in fig. 22, fluorescence intensity in the tissue was measured at the above time points using image J software. In three different fluorescence filters, at 67.17cm respectively ² The intensities of the triplet were measured and their averages were statistically compared and the results are shown in figure 23. GNP tumor therapeutic nanomedicines showed maximum binding to the receptor within 6 hours and began to clear from the tissue after their removal.After 48 hours, most of the drug was approximately 30-50nm (green fluorescence reduction) from the nanoparticle size, while nanoparticles with a size smaller than this were still shown by the blue emission filter intensity. But gold nanoparticles of various sizes and doxorubicin showed the same pattern of tissue localization and removal from the tissue.

Effect of GNP therapeutic nanomaterials on body weight and survival of gastric cancer treated mice the anticancer efficacy of GNP tumor therapeutic nanomaterials on induced cancer mice (third group of induced cancers) was examined. Three groups: the first group of physiological saline, the second group of doxorubicin and the third group of GNP therapeutic nanomedicine treatments were observed for 40 days. The experiment was scheduled for two months but ended up with 40 days due to death of the last rat in the second group. Treatment of doxorubicin groups, saline and GNP nanotherapeutic drug treated mice were active at all times. Although the group dizziness a few hours after the administration of the nano-drug, it started to be active after a period of time. Mice in the second group became drowsy and weak daily, while mice in the first group remained active. After a period of time, as shown in fig. 24 and 25, the survival rate of the first group began to decrease, 60% at the end of the study. The second group of mice had zero survival at 40 days, and the third group of mice exhibited 80% survival and had an active lifestyle. The effect of treatment on body weight is summarized in fig. 26, and the results show that the third group of mice was treated with GNP therapeutic nanomedicine, not only for prolonged life, but also for healthy life with smooth weight gain.

ZnS QD nanotherapeutic drug (in vitro)

Three ZnS QD nanocomposites, 5 μl (5 μg/μl) at lower concentrations (ZnS-Cys, znS-Cys-Dox and ZnS-Cys-Dox-transferrin) were compared statistically (ANOVA) to colon cancer cell lines to observe the therapeutic effect of the nanocomposites, as shown in fig. 27. ZnS-Cys has toxic effects on cancer cells, but only 2% have 98% cell viability. ZnS-Cys-Dox was more toxic due to the binding of the chemotherapeutic drug doxorubicin with 85% cell viability. After transferrin binding, cell viability showed a different value of 81%. The data lie within the confidence interval, indicating the significance of the comparison of p <0.0001.

After 6 hours and 24 hours incubation, different concentrations of ZnS QD therapeutic nanomaterials (corresponding to different concentrations of doxorubicin) were examined against colon cancer cells. The present invention is used to examine the optimal time and optimal time concentration of ZnS nanomedicine effects for further determination. This assay was also used to obtain IC 50 values and the value of lethal dose ZnS QD tumor therapeutic nanomedicines. Fig. 28 and 29 summarize the mean percent viability for the different concentrations and make a statistical comparison (n=3). The lethal dose 50 (IC 50) of ZnS QD nanotherapeutic was 15. Mu.l (57. Mu.g) and the lethal dose was 50. Mu.l. The cumulative effect of different concentrations of drug at two time points 6 and 24 hours indicates a decrease in cell viability. The mean was compared to ANOVA and the data placed in the p-median of confidence interval <0.0001.

Cytotoxicity and drug binding of ZnS QD therapeutic nanomedicine at different time points cytotoxicity was observed at different time points based on cell viability and live-dead cell comparison, as shown in fig. 30. After each time point, the plates were observed upside down. Cell morphology and drug fluorescence were observed with a fluorescence microscope. Cells started to show changes in cell morphology in the test medium compared to the control, and the same pattern was observed until the last time point. The appearance of cells in the presence of ZnS QD nanotherapeutics is different from the cell morphology previously observed in GNP nanotherapeutics samples and from controls. The cell plate showed that the drug was only present on the cells and not in the blank. After 6 hours, fluorescence observations confirm that the presence of the nanopharmaceuticals, in particular doxorubicin, in the cells indicates its presence in the nucleus, as shown in fig. 31. After 6 hours, the cells began to clamp and showed an increase in ZnS QD and doxorubicin concentrations. After the 12 hour time point, fluorescence was enhanced as shown in fig. 32, and maximum fluorescence was observed at the 24 hour time point. ZnS QD nanoparticles were observed in the cytoplasm, while most doxorubicin was in the nucleus, as shown in fig. 33. After 24 hours, doxorubicin was used to kill and release from the cells. After 48 hours the nuclei were quiescent filled with doxorubicin as shown in figure 34. At 72 hours the cells die and the cells begin to fade and deform. Cytotoxicity studies indicate that ZnS QD therapeutic nanomedicines remain effective even within 72 hours. Feasibility studies demonstrated that the previously observed nanomedicines affecting GNP tumor therapy showed the greatest efficacy within 12 hours. As shown in fig. 35, znS QD therapeutic nanomaterials still continued to have a severe effect on cells after 12 hours until the last examination time point was 72 hours. The survival rate at 6 hours was only 84% and 44% at 12 hours, which was still close to this level at 24 hours and 48 hours. Statistical comparisons of cell viability averages showed that the data was located in the important areas (p < 0.0001) at different time points. Live dead cells were counted and their mean values were compared by analysis of variance. Studies indicate that the data is present in the salient region of p < 0.0001. Live-dead cell comparison also verifies that viability studies indicate that ZnS QD therapeutic nanomaterials have prolonged effects on cells.

Intracellular ZnS quantum dots and doxorubicin tracking and fluorescence intensity measurements at different time points, anti-fluorescence was subtracted at different time points using Image J software and ZnS QD and doxorubicin fluorescence were studied independently. Subtracting red from green and going from red into green fluorescence plot ZnS QD and doxorubicin were traced inside the cell as shown in fig. 36. Most nano-drugs bind in cells after 6 hours and exhibit fluorescence throughout the cells. At 12 hours, the cells emitted clear combined fluorescence, but the decrease after subtraction of the anti-fluorescence indicated that most of the doxorubicin was still present with ZnS QD. So ZnS QD tracks not only doxorubicin in the nucleus but also in the cytoplasm at 12 hours. After 24 and 48 hours, most of the doxorubicin was located in the nucleus, while ZnS QD was located in the cytoplasm, and after 72 hours, the intracellular concentration began to disappear. The green fluorescence intensity is proportional to the number of ZnS QD nanoparticles, and the red fluorescence intensity is proportional to the amount of doxorubicin. After subtraction, the fluorescence intensity was analyzed for anti-fluorescence with image J. Twenty cells per group were used to calculate intensity and their mean values were compared by analysis of variance as shown in figure 37. Fig. 38 shows that the higher intensity points are 6 hours and 12 hours, respectively, and that the intensity begins to decrease after 24 hours. The data lie in the significant interval of p < 0.0001.

ZnS QD therapeutic nanomaterials bind to receptors on gastric cancer tissue (in vitro), frozen sections of gastric tissue were incubated with ZnS QD therapeutic nanomaterials to examine the ability of the nanomaterials to bind to target tissue in vitro. Fig. 39 shows successful binding of ZnS QD tumor therapeutic nanomedicine to transferrin receptor on gastric tissue. Fluorescence in the blue and green filters indicated the presence of ZnS QD nanoparticles in the tissue, while red fluorescence confirmed the presence of doxorubicin in the tissue. In vitro studies confirm that ZnS QD tumor therapeutic nanomedicines do not affect the receptor binding sites of transferrin. Transferrin in therapeutic nanomedicines has been fully activated and can bind to transferrin receptors.

Therapeutic efficacy (in vivo) of zns QD therapeutic nanomedicine

ZnS QD nanotherapeutics corresponding to 5mg/kg doxorubicin were orally administered to examine the therapeutic effect and intracellular drug treatment method. The results of ZnS QD therapeutic nanomaterials were used to confirm and support the behavior and effects of GNP therapeutic nanomaterials.

Receptor binding, intracellular tracking, drug release and fluorescence intensity of ZnS QD therapeutic nanomedicine at various time points the receptor binding activity and intracellular cell tracking of tissues was observed by ex vivo immunohistochemical study of gastric tissues collected at 3, 6, 12, 24, 48 and 48 hours with oral ZnS QD therapeutic nanomedicine. The pattern of nano-drug receptor binding and localization mimics the pattern of GNP therapeutic nano-drugs, with no change in pattern. Figure 40 shows the binding and localization of the nanomaterials within the cells after 3, 6 and 12 hours. The largest drug was observed in the tissue study after 12 hours and also in the GNP therapeutic nanomedicine study after 12 hours. The decrease in fluorescence was observed by confirming the gradual elimination behavior after 12 hours. The ZnS QD nanoparticles range in size below GNP therapeutic nanomaterials, so in the case of quantum dots, blue fluorescence is clearer than green fluorescence and higher in intensity. Fluorescence intensity was in the 67.17cm2 region with image J and compared using SPSS 26, as shown in FIG. 41. The results are summarized in fig. 42, znS QD therapeutic nanomedicine showed maximum binding to the receptor at 6 hours and gradually began to leave the tissue. After 48 hours, most of the doxorubicin was cleared from the tissue as indicated by the decrease in red fluorescence. ZnS QDs are smaller in size than GNPs, but they escape from the tissue more efficiently at the 72 hour time point.

The effect of ZnS QD nanotherapeutics on mouse body weight and survival during gastric cancer treatment, the anticancer effect of ZnS QD therapeutic nanotherapeutics was also examined, three groups: the first group of saline, the second group of doxorubicin and the third group of GNP therapeutic nanomedicine again experiments terminated at day 40, as the survival rate of group 2 mice was zero at day 40. The group treated with ZnS QD therapeutic nanomedicines remained active throughout the treatment compared to the doxorubicin group. As shown in fig. 43 and 44, the survival rate of ZnS QD-treated group was 80%, but the first mice died after 18 days, instead of the 15 day group observed in GNP animal treatment. The effect of treatment on body weight was observed over the first 15 days, and the results are shown in fig. 45 and 46, zns QD-treated nanomedicines not only can extend life, but also have a healthy life and have a smooth body weight gain pattern.

Example 4 pharmacokinetic and biodistribution studies

Pharmacokinetics (in vitro) of gnp tumor therapeutic nanomedicines

The GNP therapeutic nanomaterials were subjected to pH dependent drug release experiments at acidic pH5 and neutral pH7.5 (in vitro). The purpose of the experiment was to examine the kinetic release behavior conditions of the drug at different pH. Acidic conditions are prevalent in the vicinity of tumors and in the stomach, while physiological pH is almost 7.5 for plasma and organs. The pH of the culture medium containing the nano-drug was also 7.5, and thus, drug release behavior was checked to see the stability of the drug and release at the desired site. The drug release behavior of GNP tumor therapeutic nanomaterials at pH5 and pH7.5 is shown in fig. 47 and 48. Drug release behavior demonstrates the stability of therapeutic nanomaterials at pH7.5 and dominant release behavior at acidic pH 5. The drug release behavior indicated that doxorubicin was released, with greater free form and drug release rate at pH5 and drug stability at pH 7.5. These results also predict the in vivo stability of the drug and release in tumor sites and stomach due to pH dependent release. At pH5, half of the doxorubicin was released within 85 hours as shown in fig. 49, while at pH7.5, half of the conjugated doxorubicin was released over 850 minutes as shown in fig. 50. Data points are represented in figures 49 and 50 as the mean of three in ± SD.

The GNP nanotherapeutic released doxorubicin in cell culture, in which the release of doxorubicin was measured in both sets of reactions. The measurement was performed between the first target concentration and the non-target concentration, and at the second time, the concentration of doxorubicin in dead cells and medium was determined by HPLC. FIG. 51 shows chromatograms of pure doxorubicin (a), standard doxorubicin (b), doxorubicin (c) extracted from dead cells and medium, and doxorubicin (d) extracted from plasma.

The release of doxorubicin from targeted and non-targeted GNPs, nanocomposite formulations, concentration of doxorubicin in dead cells incubated with GNP-Cys-Dox and GNP-Cys-Dox was calculated at 6 hours and 24 hours for the time of reaction with GNP-Cys-Dox-Trans, values compared by analysis of variance for p <0.005 for the non-target population over 24 hours, as shown in figure 52. Figure 53 shows that targeted GNP nanocomposites showed higher doxorubicin concentrations than non-targeted GNP nanocomposites at 24 hours.

The concentration of doxorubicin in the GNP tumor therapeutic nanomedicine was analyzed by HPLC under different conditions at different time points and compared by analysis of variance in the medium with dead cells. The maximum concentration of doxorubicin was found to be 72 hours, then 12 hours. As shown in fig. 54, the lowest concentration was recorded at 6 hours. All reactions were performed in triplicate and the data were expressed as mean as standard deviation and significance (p < 0.005) as shown in FIG. 55.

Pharmacokinetics (in vivo) of gnp tumor therapeutic nanomedicines

The blood activity time profile of GNP therapeutic nanomaterials to release doxorubicin was studied in two groups. One group receives the nano-drug via intravenous route (IV route) and the other group via oral route.

GNP nuclear magnetic resonance nano-drugPharmacokinetic, intravenous administration, and after intravenous administration of GNP therapeutic nanomaterials, the drug was analyzed by HPLC to characterize the distribution in plasma. Figure 56 summarizes the data collected for HPLC analysis of doxorubicin release in plasma at various time points and for statistical comparison (analysis of variance). Analysis showed that the data were within a significant time interval (p<0.001 And all pharmacokinetic parameters were calculated and summarized in figure 57. The clearance of GNP-Cys-Dox-transferrin was 0.002L/h, plasma half-life (t _1/2 ) For 12.70 hours. Small amounts of doxorubicin were observed over time, at decreasing plasma drug concentrations and for 48 and 72 hours.

Plasma concentration management of doxorubicin at various times after oral administration was analyzed by HPLC, and the data shown in fig. 58 are expressed as mean values with standard deviation (n=3) and in significant intervals of p < 0.001. The plasma concentration of drug in figure 59 was elevated for oral administration and reached maximum concentration at 48 hours. After 48 hours to 72 hours, the concentration line showed a sharp drop and the drug was eliminated from the plasma. Figure 60 summarizes the pharmacokinetic parameters after oral doses.

Biodistribution of the GNP tumor therapeutic nanomedicine was also detected after IV and PO doses were performed with a fluorescence scanner (fluorescence microplate reader) with 485 excitation and 525 emission at doxorubicin concentration in liver, heart, kidney and stomach tumors.

Figure 61 shows doxorubicin concentration and PO dose in liver at various time points following intravenous infusion. The results shown in fig. 62 demonstrate that doxorubicin decreased in liver tissue over time. The doxorubicin concentration in liver tissue following oral administration was less than the increase in blood concentration following intravenous infusion at all time points and showed rapid clearance from liver tissue.

Figure 63 summarizes doxorubicin concentration and PO dose in hearts at different time points following intravenous infusion. Data represent mean values (n=3), with standard deviation, significance (p < 0.005) calculated by ANOVA and independent T-test. Figure 64 shows doxorubicin concentrations at various time points, which confirm rapid elimination of doxorubicin from the heart, with oral administration having a lower doxorubicin concentration than intravenous injection.

FIG. 65 shows the elimination of doxorubicin from the kidneys at various time points, with the mean value (n=3) representing the deviation and the significant value (p < 0.05). Doxorubicin concentration decreased in time and showed rapid elimination from the body, as shown in figure 66. Most doxorubicin is found in the kidneys after oral administration and cleared from the body.

Biodistribution experiments were performed in mice (group 3) that induced gastric cancer. Post intravenous administration only a small amount can reach the target site, but still little will show effect until 48 hours. Figure 67 shows that most of the drug orally taken was bound to the target site and higher concentrations appeared in the gastric tumor portion at 48 hours, and the data of figure 68 were summarized as mean (n=3) with standard deviation and significance values (p < 0.0001).

Pharmacokinetics (in vitro) of zns QD therapeutic nanomedicines

pH dependent drug release of ZnS QD therapeutic nanomaterials the drug release behavior of ZnS QD therapeutic nanomaterials at pH5 and pH7.5 is as shown in fig. 69 and 70. The drug release behavior demonstrates the stability of the nano-drug at pH7.5 and the significant release behavior at acidic pH 5. Fig. 69 is a plot of percent drug release at different time intervals. Doxorubicin was released in a pH5 medium at a higher rate of pH7.5 release than at pH 7.5. These results enhance the previously observed release pattern of doxorubicin from GNP tumor therapeutic nanomaterials. In both drugs doxorubicin is coupled to the carboxyl group of cysteine and thus released in a similar manner. At pH5, half of the doxorubicin was released within 140 hours, while at pH7.5, it took 800 minutes to release half of the bound doxorubicin. The data points are shown in fig. 71 and 72.

Doxorubicin release from ZnS QD therapeutic nanomaterials in cell culture (in vitro) in cell culture, doxorubicin release was measured in both sets of reactions. Dead cells and medium were measured by HPLC at different time points at a first target versus non-target concentration and at a second doxorubicin concentration.

Doxorubicin release from targeted and non-targeted ZnS quantum dots the doxorubicin concentration in the therapeutic nanocomposite in the dead cells incubated with ZnS-Cys-Dox and ZnS and the reaction time of ZnS-Cys-Dox-transferrin were calculated at 6 hours and 24 hours, respectively. The values were compared by analysis of variance as shown in figure 73. Meaning that 24 hours localized to the non-localized group p <0.01. The targeted ZnS QD nanocomposites showed higher doxorubicin concentrations, shown in figure 74, over 24 hours, than the untargeted ZnS QD nanocomposites.

Concentration of doxorubicin in ZnS QDs the concentration of doxorubicin in the medium with dead cells was analyzed by HPLC at different time points and compared by ANOVA. The maximum concentration of doxorubicin was found to be 72 hours, then 12 hours. After 12 hours, the next two time points remained almost stable, as shown in fig. 75. All reactions were performed in triplicate and the data are presented as average values. Standard deviation and significance (p < 0.005), as shown in figure 76.

Nano-drug pharmacokinetics (in vivo) of zns QD tumor treatment

Doxorubicin blood activity time spectrum of ZnS QD therapeutic nanomaterials was divided into two groups of studies. One group receives the nano-drug by intravenous route and the other group by oral route. The biodistribution of doxorubicin after intravenous injection and oral administration was also characterized.

After intravenous injection of ZnS QD, the plasma drug profile was characterized by HPLC analysis. Figure 77 summarizes the data collected from HPLC of doxorubicin in plasma at various time points and makes statistical comparisons (ANOVA). Analysis showed that the data was located within significant time intervals (p < 0.001), as shown in FIG. 78. FIG. 79 shows that the plasma half-life of ZnS-Cys-Dox-transferrin is 16.91h at a rate of 0.001L/h. Over time, the drug concentration in the plasma decreased and small amounts of doxorubicin were observed at 48 and 72 hours.

After pharmacokinetic oral administration of ZnS QD therapeutic nanomaterials, plasma concentrations of doxorubicin were analyzed by HPLC at various times after oral administration. The data are represented as average values with standard deviation (n=3) in fig. 80, and the interval of p <0.001 is represented by the significant bit number. The concentration of drug in plasma increased after oral administration and reached maximum concentration at 48 hours. After 48 to 72 hours, the concentration line showed a sharp drop and drug was eliminated from the plasma, fig. 81. Figure 82 summarizes the pharmacokinetic parameters after oral dosing.

Biodistribution of ZnS QD tumor therapeutic nanomaterials, IV and PO doses (fluorescence microplate reader) at 485 excitation and 525 emission were measured by fluorescence scanner for post-operative detection of doxorubicin concentration in liver, heart, kidney and stomach tumors.

Figure 83 shows doxorubicin concentration and PO dose in liver at various time points following intravenous injection. As shown in fig. 84, the results demonstrate that doxorubicin decreased in liver tissue over time. Doxorubicin concentrations in liver tissues following oral administration were less than blood concentrations following intravenous injection at all time points and showed rapid clearance from liver tissues.

Figure 85 summarizes doxorubicin concentration and PO dose in hearts at different time points after intravenous injection. The data had an average of standard deviations (n=3) with significance (p < 0.005) calculated by ANOVA and independent T-test. Figure 86 shows doxorubicin concentrations at various time points. The very low concentration of doxorubicin tracked in the heart, and the results confirm rapid elimination of doxorubicin from the heart. The concentration of orally administered doxorubicin is lower than that of intravenous injection.

FIG. 87 shows the elimination of doxorubicin from the kidneys at various time points, the data obtained by analysis represent the deviation and the significant value (p < 0.001) as the mean value of the standard (n=3). The doxorubicin concentration decreased with time, and at the 6 hour time point, the doxorubicin concentration was not significant after intravenous injection and oral administration, as shown in FIG. 88. The concentration was higher when the kidney was orally taken for 24 hours, but most doxorubicin was cleared from the body at 48 hours.

Biodistribution experiments were performed in mice (group 3) that induced gastric cancer. Post intravenous administration only small amounts of doxorubicin could reach the target site, but at 48 hours, very small amounts were also tracked in the stomach. Oral administration showed that most of the drug bound to the target site and that the concentration in gastric tumors was reassigned after 48 hours more figure 89. The data in figure 90 are summarized as mean (n=3) with standard deviation and significance values (p < 0.0001).

Claims

1. The application of the nano targeting drug in preparing the drug for treating gastric cancer is characterized in that the nano targeting drug is ZnS QD therapeutic nano drug;

the joint of the ZnS QD therapeutic nano-drug is cysteine, the targeting ligand is transferrin, the chemotherapeutic drug is doxorubicin, and the nano-carrier is ZnS QD;

the preparation method of the ZnS QD therapeutic nano-drug comprises the steps of synthesizing a cysteine coated ZnS QD quantum dot ZnS QD-Cys, preparing ZnS QD-Cys-Dox by combining doxorubicin and ZnS QD-Cys, and combining transferrin and ZnS QD-Cys-Dox;

the synthesis method of the cysteine coated ZnS QD quantum dot comprises the step of synthesizing the cysteine coated ZnS QD quantum dot in the water reaction of zinc chloride and sodium sulfide in the presence of L-cysteine and taurine, wherein the synthesis method specifically comprises the following steps:

After refluxing 0.01M solution of pH8 zinc chloride, 0.01M solution of L-cysteine and 0.1M taurine in 200ml total reaction volume for half an hour, 0.01M sodium sulfide in 10ml deionized water was added dropwise to the reaction mixture, and further refluxed for 12 hours; centrifuging the synthesized cysteine coated ZnS QD nano particles ZnS QD-Cys at 25 ℃ for 20 minutes at 14000rpm, and washing the precipitate with deionized water;

the binding method of the doxorubicin and ZnS QD-Cys is characterized in that the doxorubicin and the ZnS QD coated by cysteine are synthesized by a 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide EDC method, and specifically comprises the following steps:

100mg of cysteine coated ZnS QDs were adjusted in 1ml deionized water with 1M NaOH pH8; 1ml of EDC to activate carboxyl groups and adjust pH to pH6.4 with 1M HCl, incubating the reaction mixture in the dark for 30 minutes and continuously shaking at 100rpm, after incubation for 30 minutes, adding 1ml of carbodiimide to the drug containing 50mg of doxorubicin and further incubating for 2 hours in a dark environment at 37℃and continuously shaking at 100 rpm; the nanocomposite conjugated with doxorubicin was isolated by centrifugation at 14000rpm for 20 minutes and washed twice with deionized water, and the washed nanocomposite znqd-Cys-Dox was stored at 4 ℃;

The binding method of transferrin and ZnS QD-Cys-Dox is that the glutaraldehyde method couples transferrin to doxorubicin-coupled cysteine coated ZnS QD.