CN112494660A - Preparation method of nano targeted drug and application of nano targeted drug in treatment of gastric cancer - Google Patents

Abstract

The invention relates to the technical field of biological medicines, in particular to a preparation method of a nano-targeting drug and application of the nano-targeting drug in treatment of gastric cancer. Gold Nanoparticles (GNPs) are the most compatible nanostructures, which have advantages over other metal nanocomposites, and zinc sulfide quantum dots (ZnS QDs) are very important quantum dot nanoparticles, which are low in toxicity, friendly to humans, and narrow in spectrum band. The two prepared therapeutic nano-drugs have good stability, high drug loading rate and excellent slow release property, can enhance the targeted concentration of the drugs, enhance the treatment effect of tumor killing and reduce the non-specific toxic and side effects, and the design modes of the two drugs can be used for diagnosis, treatment and prognosis of gastric cancer, thereby having good application prospect.

Description

Preparation method of nano targeted drug and application of nano targeted drug in treatment of gastric cancer

Technical Field

The invention relates to the technical field of biological medicines, in particular to a preparation method of a nano-targeting medicine and application of the nano-targeting medicine 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 cancer, stomach 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 associated with many factors, such as high salt intake in diet or microbial infection of helicobacter pylori (h. Currently, various imaging techniques are being used for cancer diagnosis, such as 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, radiotherapy and surgery, and unfortunately, all of these treatments bring about many side effects, and these treatments not only cause diseases such as alopecia, nausea, fatigue, digestive system diseases, canker sores and neurological diseases, but also destroy normal tissues and the cancer cells thereof are not completely eradicated.

In contrast to these therapies, nanotechnology promises the selective delivery of cancer cell-targeting drugs with the help of targeted therapies to eliminate all these side effects. The main objective of targeted drug delivery is to avoid the toxic effects of the drug on normal cells, since another advantage of targeted drug delivery compared to target cells is the limited, centrally controlled drug concentration released. 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 published involving different strategies against cells and organs, and most reports are targeted delivery approaches against cancer, targeting their cells using specific membrane-bound antigens of the cancer.

The nano biotechnology brings new scientific prospects for targeted drugs and drug therapy. The involvement of nanocarriers greatly changes the fate of targeted drug delivery, not only affecting the therapeutic field but also the diagnostic and prophylactic fields. Decades of research have demonstrated that biodegradable nanoparticles are the best and friendly bioengineering for drug delivery or for use in target tissues. Researchers have focused on those nanoparticles that are biodegradable and excreted from the body without any harm to the body, and by combining nanotechnology with drugs, called nanomedicines, targeted delivery to cancer cells can minimize cytotoxicity of normal tissues for the action of anticancer agents by aiming to provide maximum dose to the affected site and enhance their biodistribution to target sites.

With the development of nano-drug formulations and the use of metal nanoparticles, substances such as iron, silver, gold or quantum dots have advantages in delivery and imaging. The use of bioluminescent nanoparticles as drug-carrying and targeting nanocarriers to receptor site ligands can provide diagnostic and therapeutic effects, and these nanomedicines with diagnostic and therapeutic functions are called therapeutic nanomedicines.

The main step in the selection of nanocarriers is the selection of cancer cells targeted by appropriate ligands. Expression of many receptors, such as folate and transferrin receptors, is increased many-fold on cancer cells compared to normal humans. Increased expression of transferrin receptor is observed on cancer cells to increase iron uptake in cancer cells to facilitate biological activities for the following reasons, while expression of transferrin receptor is relatively limited on normal cells, and thus transferrin receptor can be used as a targeting site for nano-drugs for drug delivery targeting cancer.

Doxorubicin, although it has a high antitumor activity, cannot distinguish between normal cells and cancer cells. Adriamycin (an anticancer drug) has a low osmotic molecular weight, is easily permeated by all cells, and is as harmful to the health of normal cells. Overcoming the obstacles Adriamycin has a better effect as an anti-cancer agent, which can be combined with Adriamycin.

The therapeutic effect of doxorubicin at specific surface receptors can be enhanced by targeting cancer cells with ligands. The expression of transferrin receptor has attracted the interest of researchers' delivery on cells that target the rapid growth of drugs. Therapeutic agents and targeting ligands are determined, and the main problems are the selection of nano-carriers, and the selection of Gold Nanoparticles (GNPs) and zinc sulfide quantum dots (ZnS QDs) as nano-carriers to be applied to cancer diseases, which are not reported in the open literature.

Disclosure of Invention

The invention aims to provide a preparation method of a nano-targeted drug and application of the nano-targeted drug in treatment of gastric cancer.

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

a preparation method of a nano-targeted drug and application of the nano-targeted drug in the treatment of gastric cancer, wherein the nano-targeted drug comprises a GNP therapeutic nano-drug and a ZnS QD therapeutic nano-drug.

The linker of the GNP therapeutic nano-drug is cysteine, targeting ligand transferrin, chemotherapeutic drug adriamycin, and the nano-carrier is GNP.

The linker 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-targeted drug, which comprises the preparation of a GNP therapeutic nano-drug and the preparation of a ZnS QD therapeutic nano-drug.

The preparation of GNP therapeutic nano-medicine includes the synthesis of GNP, the combination of cysteine and GNP, the combination of adriamycin and GNP-Cys, and the combination of transferrin and GNP-Cys-Dox.

The preparation of the ZnS QD therapeutic nano-drug comprises the synthesis of ZnS QD quantum dots coated by cysteine, the combination of adriamycin and ZnS QD-Cys, and the combination of transferrin and ZnS QD-Cys-Dox.

Conjugation of cysteine-coated GNPs to transferrin can serve as targeted diagnostic probes.

The invention has the beneficial effects that:

in the present invention, two therapeutic nano-drugs are prepared, and Gold Nanoparticles (GNPs) as nano-carriers are the most compatible nanostructures, which can be used for diagnosis, prognosis, imaging and therapy, with greater advantages than other metal nanocomposites. Zinc sulfide quantum dots (ZnS QDs) are very important quantum dot nanoparticles, and have the advantages of low toxicity, narrow spectrum band, adjustable emission profile size and high emission quantum yield. The two prepared therapeutic nano-drugs have good stability, high drug loading rate and excellent slow release property, can enhance the targeted concentration of the drugs, enhance the treatment effect of tumor killing and reduce the non-specific toxic and side effects, and the design modes of the two drugs can be used for diagnosis, treatment and prognosis of gastric cancer, thereby having good application prospect.

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.

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

FIG. 4 is a full tumor histological examination and histological study of tumor tissue after binding of a targeted diagnostic probe (in vivo). (a) Bright field studies of tumor tissue show the morphology of the tissue; (b) examination of the whole tissue under an ultraviolet-activated filter showed in vivo binding of the target probe to transferrin receptor on tumor cells; (c) whole tissue examination under blue excitation filters; (d) and (e) histological studies of tumor cryostat sections were identical to those observed in whole tissues.

FIG. 5 is a histological study of in vitro total stomach tissue examination and in vivo tissue-targeted diagnostic probe binding. (a) Bright field studies of stomach tissue have shown tissue morphology with regions of mass; (b) in the whole tissue examination under the ultraviolet ray excitation filter, the target probe is combined with transferrin receptor in a caking area in vivo; (c) the blue excitation filter under examination of the tissue shows that the tissue fluoresces more in this region than in the ultraviolet; (d) and (e) histological studies of gastric carcinoma tissue cryostat sections showed the same results as observed throughout the tissue examination.

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

Fig. 7 is a table of cytotoxicity of different GNP nanocomplexes.

Fig. 8 is a graph of the cytotoxicity effect of different GNP nanocomplexes.

Figure 9 is a table of cytotoxicity of different concentrations of GNP therapeutic nanomedicines.

Figure 10 is a graph of the cytotoxicity of GNP therapeutic nanomedicines at different concentrations. (a) The cumulative effect of the nano-drugs with different concentrations in 6 and 24 hours; (b) cytotoxic effects at different concentrations at 6 and 24 hours at the two time points.

Fig. 11 is a table of the cytotoxicity of GNP tumor treatment nano-drugs at different time points.

Figure 12 is a study of GNP nanocomposite growth, cell morphology and fluorescence at the 6 hour time point.

Figure 13 is a study of GNP nanocomposite growth, cell morphology and fluorescence at the 12 hour time point.

Figure 14 is a study of GNP nanocomposite growth, cell morphology and fluorescence at the 24 hour time point.

Fig. 15 is a study of GNP nanocomposite growth, cell morphology and fluorescence at the 48 hour time point.

Fig. 16 is a graph of the viability and percentage of live/dead cells of GNP nanocomplexes at different time points. (a) The GNP tumor treatment nano-drug has cytotoxic effect in 6, 12, 24, 48 and 72 hours; (b) the cytotoxic effect of GNP therapeutic nanomedicines causes a change in the proportion of live and dead cells at 6, 12, 24, 48, 72 hours.

Fig. 17 is an intracellular tracking of GNP nanocomplexes as GNP and doxorubicin at different time points.

Fig. 18 is a fluorescence intensity measurement of GNP therapeutic nanomedicines at various time points.

Fig. 19 is a graph of the concentration of GNP synthesized nanocomplex measured at various time points based on fluorescence intensity.

Figure 20 is in vitro receptor binding of GNP therapeutic nanomedicines on gastric cancer tissues.

Fig. 21 is a cell trace of GNP treatment nanomedicines at different time points.

Fig. 22 shows the fluorescence intensity of GNP therapeutic nanomedicines at different time points.

Figure 23 is a comparison of fluorescence intensity (full window) at different time points after oral administration of GNP therapeutic nanomedicines.

Fig. 24 shows the anti-gastric cancer effect of GNP tumor treatment nano-drug during in vivo treatment.

Figure 25 is the survival rate of mice at different times during 40 days of GNP tumor treatment with the nano-drug.

Figure 26 is a graph of the weight change of live mice over different times during 40 days of GNP therapeutic nano-drug treatment.

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

Fig. 28 is a table of cytotoxicity of various concentrations of ZnS QD for treatment of nano-drugs.

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

Fig. 30 is a table of cytotoxicity of ZnS QD therapeutic nanomedicines at different time points.

Fig. 31 shows ZnS QD nanocomplex growth at the 6 hour time point, cell morphology and fluorescence studies.

Fig. 32 shows ZnS QD nanocomplex growth at a 12 hour time point, cell morphology and fluorescence studies.

Fig. 33 shows ZnS QD nanocomplexes grown at 24 hour time point, cell morphology and fluorescence studies.

Fig. 34 shows ZnS QD nanocomplexes grown at 48 hour time point, cell morphology and fluorescence studies.

Fig. 35 is a graph of the survival and cytotoxic effects of viable/dead cells of ZnS QD nanocomplexes at different time points.

Fig. 36 is an intracellular tracking of ZnS QD choroidal nanocomplexes as ZnS QDs and doxorubicin at different time points.

Fig. 37 is a measurement of fluorescence intensity of ZnS QD therapeutic nano-drugs at different time points.

FIG. 38 shows fluorescence intensity of ZnS QD nano-compounds at different time points.

Figure 39 is the in vitro receptor binding of ZnS QD therapeutic nanomedicines in gastric cancer tissues.

Fig. 40 is a cell trace of ZnS QD therapeutic nano-drugs at different time points.

FIG. 41 is a comparison of fluorescence intensity at different time points after oral administration of ZnSQD choroidal Nanogen.

Fig. 42 shows the fluorescence intensity of ZnS QD therapeutic nanomedicines at different time points.

Fig. 43 shows the anti-gastric cancer efficacy of ZnS QD therapeutic nano-drugs (in vivo).

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

Figure 45 is the change in body weight of live mice for ZnS QD nanotherapeutic drugs at different times during 40 days of treatment.

Fig. 46 shows the anti-gastric cancer efficacy of ZnS QD nano therapeutic drugs (in vivo).

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

Fig. 48 is a graph of doxorubicin release behavior from GNP therapeutic nano-drugs at pH 7.5.

Figure 49 is a graph of release of doxorubicin by GNP therapeutic nanomedicines at pH5 at various time points.

Figure 50 is a graph of release of doxorubicin by GNP therapeutic nanomedicines at pH7.5 at various time points.

FIG. 51 is a chromatogram of doxorubicin.

Fig. 52 is a graph of doxorubicin concentration in the GNP therapeutic nanomedicine targeting response versus non-targeting response.

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

Figure 54 is the in vitro concentration of doxorubicin in the culture medium and dead cells (GNP therapeutic nano-drug) after different time points.

Figure 55 is the concentration of doxorubicin in the medium (GNP therapeutic nanomedicine) at different time points of incubation.

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

Figure 57 is a pharmacokinetic parameter for GNP therapeutic nano-drugs.

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

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

Fig. 60 is a pharmacokinetic parameter after oral administration of GNP therapeutic nano-drug.

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

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

Figure 63 is the cardiac doxorubicin concentration after intravenous and oral administration of GNP therapeutic nano-drug.

Fig. 64 is a comparison of the biodistribution of doxorubicin in the GNP therapeutic nanomedicine in the heart at various time points after intravenous and oral administration.

Figure 65 is the adriamycin concentration in the kidney after intravenous and oral administration of GNP therapeutic nanomedicines.

Fig. 66 is a comparison of the biodistribution of doxorubicin in the GNP therapeutic nanomedicine in the kidney at various time points after intravenous and oral administration.

Fig. 67 is a comparison of the biodistribution of the GNP therapeutic nano-drug doxorubicin in gastric tumors at different time points after intravenous and oral administration.

Figure 68 is the concentration of doxorubicin in gastric tumors following intravenous and oral administration of GNP therapeutic nanomedicines.

Figure 69 is the release behavior of doxorubicin from ZnS QD therapeutic nano-drug at pH 5.

Fig. 70 is a graph of doxorubicin release behavior from ZnS QD therapeutic nano-drug at pH 7.5.

Fig. 71 shows release of doxorubicin from ZnS QD therapeutic nanomedicine at pH5 at different time points.

Fig. 72 shows release of doxorubicin from ZnS QD therapeutic nanomedicine at pH7.5 at different time points.

Figure 73 is the doxorubicin concentration in ZnS QD therapeutic nanomedicine targeting response versus non-targeting response.

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

Figure 75 is the in vitro concentration of doxorubicin in the culture medium and in dead cells (ZnS QD therapeutic nano-drug) after different time points.

Figure 76 is the concentration of doxorubicin in the medium (ZnS QD therapeutic nano-drug) at different time points of incubation.

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

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

Fig. 79 shows pharmacokinetic parameters of ZnS QD therapeutic nanomedicines.

Fig. 80 is data of plasma drug activity spectra analyzed by HPLC after oral administration of ZnS QD-based nano-drugs.

Figure 81 is a plasma drug activity profile of doxorubicin (ZnS QD therapeutic nano-drug) after oral administration.

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

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

Fig. 84 is a comparison of the biodistribution of doxorubicin in the liver in ZnS QD therapeutic nanomedicine at different time points after intravenous and oral administration.

Fig. 85 is a graph of doxorubicin concentration in the heart after intravenous and oral administration of ZnS QD therapeutic nanomedicine.

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

Figure 87 is the concentration of doxorubicin in the kidney following intravenous and oral administration of ZnS QD therapeutic nanomedicine.

Fig. 88 is a comparison of the biodistribution of doxorubicin in the kidney for ZnS QD therapeutic nanomedicine at different time points after intravenous and oral administration.

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

Fig. 90 is a graph of doxorubicin concentration in gastric tumors after intravenous injection and oral administration of ZnS QD therapeutic nano-drugs.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the accompanying drawings, which are not intended to limit the scope of the invention.

EXAMPLE 1 preparation of Nanotherapeutic drugs

Two kinds of nano medicine are prepared separately. Both nano-therapeutic drugs have the same linker: cysteine, the targeting ligand is transferrin, and the chemotherapeutic drug is adriamycin, but the nano-carriers are different. Gold Nanoparticles (GNPs) were used in the first nanotherapeutic drug due to their unique physical properties, zinc sulfide quantum dots (ZnS) were selected as the second nanotherapeutic drug nanocarriers.

The preparation of the nano therapeutic medicine is as follows:

preparation of GNP nano-therapeutic medicine, including synthesis of GNP, combination of cysteine and GNP, combination of adriamycin and GNP-Cys, and combination of transferrin and GNP-Cys-Dox.

GNPs were synthesized using an optimized citric acid reduction method, gold salts and sodium citrate at different concentrations were used to check the ideal concentrations, GNPs showed different optical properties, and HAuCl was prepared using a 1% gold salt stock solution (chlorous acid) in order to optimize the final reaction₄And diluted to water in a 15mM working solution beaker in 400ml of deionized water, the solution was continuously stirred and kept boiling on a hot plate preset at 100 ℃. Preparation of 1% lemon in deionized WaterThe trisodium citrate solution is heated to 50 ℃ and 12.5ml of preheated 1% trisodium citrate are rapidly added to the boiling solution with vigorous stirring, stirring is continued and observed until the color of the reaction mixture becomes dark. The solution turned bright red with a reaction time of about 20 minutes, and the bright red color showed a size of less than 15nm, completing the synthesis of GNP.

An L-cysteine solution (0.2mM) 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 for 12 hours without interference. 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 were purified by the (1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide (EDC) method, 50mg cysteine coated 1ml gnp deionized water, adjusted with 1M NaOH at pH 81 ml EDC (20mg/ml added deionized water) to activate the carboxyl group, and adjusted to pH6.4 with 1M HCl, the reaction mixture was incubated in the dark for 30 minutes with continuous shaking at 100rpm, after 30 minutes of incubation, 1ml carbodiimide (10mg/ml deionized water) was added to the drug containing 50mg doxorubicin and incubated for a further 2 hours in a dark environment at 37 ℃, continuously shaken at 100rpm, centrifuged to separate the conjugate particles at 14000rpm for 20 minutes, and washed twice with deionized water and stored at 4 ℃.

Transferrin is conjugated to doxorubicin conjugated cysteine coated GNP nanocomplexes by the glutaraldehyde method. Briefly, the amine group of cysteine on gold nanoparticles (GNP-Cys) was crosslinked with the amine group 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 then the supernatant was aspirated. The above procedure was repeated twice with coupling buffer and was performed after the second wash. The nanocomposite was suspended in 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 nanocomplexes with coupling buffer as described previously to remove excess glutaraldehyde. 10mg transferrin was dissolved in 100ml coupling buffer and left to stand in 1ml pre-coupling buffer. The remaining transferrin solution was mixed with 100mg of washing solution to mix the nanocomposite. The mixture was shaken at 25 ℃ for 24 hours and then centrifuged at 8000rpm at 4 ℃ to obtain transferrin-coupled GNP-Cys-Dox nanocomplexes (GNP therapeutic nanomedicines (GNP-Cys-Dox-transferrin) — the conjugated nanocomplexes were suspended in storage buffer (0.01M Tris-Cl, ph7.4, 0.1% sodium azide, 0.15M NaCl) and stored at-20 ℃.

The preparation of ZnS QD nano therapeutic drug comprises the synthesis of ZnS QD quantum dots coated by cysteine, the combination of adriamycin and ZnS QD-Cys, and the combination of transferrin and ZnS QD-Cys-Dox.

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

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

The glutaraldehyde method couples transferrin to doxorubicin-coupled cysteine-coated ZnS QDs. 100mg ZnSQD-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 then the supernatant was aspirated. The above procedure was repeated twice with coupling buffer and was performed after the second wash. The nanocomposite was suspended in 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 nanocomplexes with coupling buffer as described previously to remove excess glutaraldehyde. Transferrin (10mg) was dissolved in 100ml of coupling buffer and left to stand in a 1ml pre-coupling buffer. The remaining transferrin solution was mixed with 100mg of washing solution to mix the 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 nanocomplex (ZnS QD-Cys-Dox-transferrin) the conjugated nanocomplex was added to 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 are conjugated to transferrin for diagnostic detection. It is used to examine the efficiency of transferrin as a targeting ligand on cancer cells and hepatoma cells. Transferrin was also examined for expression in tissues that induce gastric cancer.

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

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

Hydrodynamic particle size distribution and target mean 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 100 nm.

TEM micrographs of cysteine coated gold nanoparticles and targeted diagnostic probes showed an increase in size upon transferrin binding. Cysteine-coated TEM micrographs GNPs exhibited 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 with an average diameter of 35nm, as shown in figure 3 (b).

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

The transferrin receptor on tissue targeted diagnostic probes was further confirmed, binding in vitro and fluorescence studies showed successful binding to fixed tissue even with probes on 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, the binding of the targeted diagnostic probe to stomach tissue showed mainly gold fluorescence and blue and green color as shown in FIGS. 6(c) and 6 (d). This further confirms the potential use of transferrin conjugated cysteine coated GNPs as in vitro targeted diagnostic probes for cancer.

Example 3 therapeutic Effect of GNP and ZnS QDs

GNP tumor treatment nano-drugs (in vitro)

Cellular uptake, therapeutic efficacy and cellular targeting of therapeutic Nanoparticulates to colon cancer cells (HCT 116-Luc 2)

And confirmed by different analyses. Techniques for studying cell viability intracellular nano-drug tracking include inverted microscopy, fluorescence microscopy, trypan blue assay, neutral red assay (metabolic activity), crystal violet staining assay (cell adhesion studies) and fluorescence intensity measurements. All results were statistically compared to give clear results.

Human colon cancer cell lines: HCT-116 (male patient-derived cell line) was used to examine the effect of therapeutic nano-drugs in vitro. The cell line specification involved p53 wild-type gene and K-Ras gene mutation mismatch defect repair system. The morphology of the cells is spindle-shaped in appearance. The remaining vial was removed from the-80 ℃ freezer and thawed prior to use. Memory (

High glucose) Medium with streptomycin and pencillin 100mg/ml solution

And 10% heat-inactivated fetal bovine serum

HCT-116 cells in the presence of 5% CO₂Growth was carried out in a humidified chamber with air and 87% humidity at 37 ℃. Throughout the drug analysis, the culture should be routinely performed. The medium was changed every 2 days and subcultured after 5 days.

Three GNP nanocomposites, lower concentrations of 5 μ Ι (5 μ g/μ Ι), GNP-Cys-Dox and GNP-Cys-Dox-transferrin, were tested against colon cancer cell lines and compared by statistical analysis (ANOVA) to observe the therapeutic effect of the nanocomposites, as shown in figure 7. GNP values have a toxic effect on cancer cells, but only 8% of cells have 92% viability. GNP-Cys-Dox demonstrated higher toxicity due to doxorubicin binding and cell viability, 83%, as shown in fig. 8, which shows cell viability within 6 hours for the first complex (GNP-Cys), the second complex (GNP-Cys-Dox) and the third complex (final therapeutic nanomedicine (GNP-Cys-Dox-Trans)). Mean (n-3) ± SD, p <0.0001 for all groups analyzed, and p <0.001 between the second and third synthesis. After transferrin binding, minor differences in cell viability were observed. GNP tumor treatment nano-drugs showed 80% cell viability and observation of cell morphology results showed that even adherent cells were not in good condition due to the longer residence time of the nano-drug. The data are within the confidence interval, showing the significance of the comparison.

The cytotoxicity of GNP at different concentrations, therapeutic nano-drug of GNP at different concentrations, is equivalent to examining the effect of doxorubicin at different concentrations on colon cancer cells at 6 hours and 24 hours. The present invention is used to examine the optimal time and optimal time concentration of the GNP therapeutic nanomedicine effect for further determination. This assay is also used to obtain IC 50 values and the value of lethal doses of GNP therapeutic nanopharmaceuticals. The average cell viability values, neutral red assay, absorbance at different concentrations and percentage viability are summarized in figure 9, and compared statistically (n-3), the lethal dose 50(IC 50) for GNP is 10 μ l (50 μ g) for nanopharmaceuticals and 50 μ l for lethal dose. Fig. 10(a) shows that the different concentrations and increasing drug effect concentrations show a decrease in cell viability, as shown in fig. 10(b), accumulated at the two time points 6 and 24 hours. Mean and variance analysis and data lie within a confidence interval of p < 0.0001.

Cytotoxicity and drug binding at different times, cytotoxicity was observed at different time points according to cell viability and live-dead cell comparison, as shown in figure 11. After each time point, the plates were viewed 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 compared to the control, and the same pattern was that the last time point was observed. The cells appeared round, wrinkled, pale-bordered, indicating that the drug was present only on the cells and not in the blank. After 6 hours, fluorescence observation confirmed the presence of the nanomedicine, particularly doxorubicin, in the cells indicating its presence in the nucleus, as shown in fig. 12. After 6 hours, the cells began to clamp and showed GNPs and doxorubicin concentration in the cells as shown in fig. 13. The fluorescence enhancement dots and magnified images after 12 hours show the separation of the nanoparticles from the 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 point, as shown in fig. 15, the cells began to lose fluorescence and at 72 hours the cells appeared to darken under the fluorescence microscope. Cytotoxicity studies have shown that GNPs have therapeutic effect nano-drugs are effective even at 72 hours due to the slow release of doxorubicin. Feasibility studies show different effects of the drug at different times. The drug was most effective within 12 hours and 24 hours. The viability was only 83% at 6 hours and 43% at 12 hours. Comparison of mean cell viability, as shown in fig. 16(a), the different time points indicated that the data were within the effective region (p < 0.0001). Live and dead cells were counted and their mean was compared by ANOVA. Studies showed that the data lie within the valid interval, with p < 0.0001. Comparison of live and dead cells also confirmed that the feasibility study for a potent drug effect remained 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, and fluorescence intensity measurements at different time points were subtracted using Image J software to subtract the anti-fluorescence and study the independence of GNP and doxorubicin fluorescence. Subtracting the red from the green and going from the red to the green fluorescence plot, GNP and doxorubicin were traced inside the cell as shown in fig. 17, which shows receptor binding and intracellular localization of GNP nmr nano-drugs with binding and fluorescence intensity changes correlated with nanoparticle and doxorubicin concentrations at 6, 12, 24, 48, 72 hours. Most of the nano-drugs bound to the cells after 6 hours and showed fluorescence on the whole cells. After 12 hours, the cells emitted clear combined fluorescence, but after subtraction of the anti-fluorescence, the decrease indicated that most doxorubicin was still associated with GNP. Doxorubicin is therefore traced not only in the nucleus, but also in the cytoplasm with GNPs after 12 hours. Within 24 and 48 hours, most of the doxorubicin was localized in the nucleus and GNPs in the cytoplasm, and within 72 hours, the intracellular concentration began to disappear. The green fluorescence intensity is in direct proportion to the amount of GNP nanoparticles, and the red fluorescence intensity is proportional to the amount of doxorubicin. Twenty cells per group were used to calculate intensities and compare their means by analysis of variance as shown in figure 18. As shown in fig. 19, the points showing higher intensity were 6 hours, and the time at which the intensity started to decrease after 24 hours was 12 hours, respectively. Data were located at time intervals of p < 0.0001.

GNP tumor therapy nanomedicines bind to receptors on gastric cancer tissue (in vitro), and frozen sections of gastric tissue are incubated with GNP nanomedicines to examine the binding capacity of the nanomedicines to target tissue in vitro. As shown in fig. 20, successful binding of GNP tumor treating nano-drugs to transferrin receptor on gastric tissue was demonstrated. Fluorescent blue and green filters showed the presence of GNP nanoparticles in the tissue, while red fluorescence confirmed doxorubicin in the tissue. GNP tumor treatment nano-drugs showed successful and robust binding to transferrin receptor on gastric cancer tissues. The combination also proves that the preparation does not influence the combination of transferrin and transferrin receptor, and can be used for in vivo examination of the curative effect of the medicine.

Therapeutic efficacy (in vivo) of GNP therapeutic Nanoparticulates

GNP treatment the therapeutic effect and intracellular drug management were examined by oral administration of nano-drugs equivalent to 5mg/kg doxorubicin.

Receptor binding, intracellular tracking, drug release and fluorescence intensity at different time points, immunohistochemical study of nano-therapeutic drug oral dose in vitro tissue for receptor binding activity and intracellular tracking of gastric tissues collected after 3, 6, 12, 24, 48 and 72 hours of GNP. The nanomedicine binds to the receptor and begins to move the compartment inward into contact with the surface immediately thereafter. Figure 21 shows the binding and localization of the nanomedicine within the cell after 3, 6, and 12 hours. This was observed in tissue studies 12 hours after oral administration of the maximum drug. Doxorubicin fluoresces red and GNP fluoresces blue and green at different intensities. After 12 hours, the drug and nanoparticles began to clear from the cells, and fluorescence intensity decreased at 48 and 72 hours. As shown in fig. 22, the fluorescence intensity in the tissue was measured at the above time points using image J software. Among three different fluorescence filters, at 67.17cm each²The intensities of the triplet were measured and their average values were statistically compared, and the results are shown in fig. 23. GNP tumor treatment nano-drugs show maximal binding to the receptor within 6 hours and start to clear from the tissue after their removal. After 48 hours, most of the drug was approximately 30-50nm red from the nanoparticle size (green fluorescence decreased), while nanoparticles with a size less than this were still shown by the blue emission filter intensity. However, gold nanoparticles of various sizes and doxorubicin showed the same pattern of tissue localization and removal from the tissue.

The effect of GNP therapeutic nano-drugs on body weight and survival rate of gastric cancer-treated mice, the anticancer efficacy of GNP tumor-treating nano-drugs on induced cancer mice (third group induced cancer) was examined. Three groups: the first saline, second doxorubicin, and third GNP therapeutic nano-drug treatment group was observed for 40 days. The experiment was scheduled for two months but terminated at 40 days due to death of the last mouse in the second group. In contrast to treatment with doxorubicin, saline and GNP nano-therapeutic drug treated mice were active all the time. Although the group was dizzy hours after the nano-drug was taken, it started to be active after a while. Mice in the second group of mice became lethargic and weak daily, while mice in the first group were still 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 survival rate was zero at 40 days in the second group of mice, and the third group of mice showed 80% survival rate and possessed 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, treated with GNP therapeutic nanomedicines, not only had an extended lifespan, but also were living healthily, and had a smooth weight gain.

ZnS QD nano therapeutic drug (in vitro)

Statistical comparisons (ANOVA) of the three ZnSQD nanocomplexes, lower concentrations of 5 μ l (5 μ g/μ l) (ZnS-Cys, ZnS-Cys-Dox and ZnS-Cys-Dox-transferrin) on colon cancer cell lines were performed to observe the therapeutic effects of the nanocomplexes, as shown in FIG. 27. ZnS-Cys has a toxic effect on cancer cells, but only 2% have 98% cell viability. ZnS-Cys-Dox is more toxic due to the binding of the chemotherapeutic drug doxorubicin and the cell viability is 85%. After transferrin binding, cell viability showed a difference of 81%. Data are within the confidence interval, indicating comparative significance of p < 0.0001.

After 6 and 24 hours of incubation, different concentrations of ZnS QD therapeutic nano-drug (equivalent to different concentrations of doxorubicin) were examined for colon cancer cells. The present invention was used to examine the optimum time and optimum time concentration for ZnS nanomedicine action for further assays. This assay was also used to obtain IC 50 values and the value of lethal dose ZnS QD for tumor treatment nano-drugs. Fig. 28 and 29 summarize the average viability percentages for different concentrations and make a statistical comparison (n-3). The lethal dose 50(IC 50) of ZnS QD nano-therapeutic drug was 15. mu.l (57. mu.g) and the lethal dose was 50. mu.l. The cumulative effect of different concentrations of drug at the two time points 6 and 24 hours indicates a decrease in cell viability. The mean was compared to ANOVA and data was placed with p-median <0.0001 at confidence intervals.

Cytotoxicity and drug binding of ZnS QD therapeutic nano-drugs at different time points cytotoxicity was observed at different time points as shown in figure 30 based on cell viability and comparison of live and dead cells. After each time point, the plates were viewed upside down. Cell morphology and drug fluorescence were observed with a fluorescence microscope. Cells initially showed a change in cell morphology in the test medium compared to the control, and the same pattern was observed until the last time point. The cellular appearance in the presence of ZnS QD nanotherapeutic drugs is different from the cellular morphology previously observed in GNP nanotherapeutic drug samples and from the control. The cell plate showed that the drug was only present on the cells and not in the blank. After 6 hours, fluorescence observation confirmed the presence of the nanomedicine, particularly doxorubicin, in the cells indicating its presence in the nucleus, as shown in fig. 31. After 6 hours, cells began to clamp and showed an increase in ZnS QD and doxorubicin concentrations. After the 12 hour time point, fluorescence increased as shown in fig. 32, and maximum fluorescence was observed at the 24 hour point. ZnS QD nanoparticles were observed in the cytoplasm, while most doxorubicin was in the nucleus, as shown in figure 33. After 24 hours, doxorubicin was used to kill and release from the cells. After 48 hours the nuclei were quiescently filled with doxorubicin as shown in FIG. 34. At 72 hours the cells died and the cells began to discolour and deform. Cytotoxicity studies showed that ZnS QD treatment of nanomedicines was effective even within 72 hours. Feasibility studies confirmed that previously observed nano-drugs affecting GNP tumor therapy showed the greatest efficacy within 12 hours. As shown in fig. 35, the ZnS QD therapeutic nano-drug 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 at 12 hours 44%, which was still close to this level at 24 hours and 48 hours. Statistical comparison of cell viability averages at different time points indicated that the data were located in the important region (p < 0.0001). Live and dead cells were counted and their mean values were compared by analysis of variance. Studies indicate that data exists between significant regions of p < 0.0001. Live and dead cell comparison also verifies that viability studies indicate that the ZnS QD therapeutic nano-drug has a prolonged effect on cells.

Intracellular ZnS quantum dots and doxorubicin tracking and fluorescence intensity measurements at different time points, back-fluorescence was subtracted at different time points using Image J software, and ZnS QD and doxorubicin fluorescence were studied independently. The red was subtracted from the green and shifted from the red to the green fluorescence plot, and ZnS QD and doxorubicin were traced inside the cells as shown in figure 36. Most of the nano-drugs were incorporated in the cells after 6 hours and showed fluorescence throughout the cells. At 12 hours, the cells fluoresced clearly combined, but the decrease after subtraction of the back-fluorescence indicates that most doxorubicin was still present with ZnS QDs. Therefore ZnS QD traced doxorubicin not only in the nucleus, but also in the cytoplasm at 12 hours. After 24 and 48 hours, most of the doxorubicin was localized in the nucleus, while ZnS QD was localized 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 anti-fluorescence was analyzed by image J. Twenty cells per group were used to calculate intensities and their means compared by analysis of variance are shown in figure 37. Fig. 38 shows that the higher intensity points were 6 hours and 12 hours, respectively, and the intensity started to decrease after 24 hours. Data are in the significant interval with p < 0.0001.

ZnS QD therapeutic nano-drugs bind to receptors on gastric cancer tissues (in vitro), and frozen sections of gastric tissues are incubated with ZnS QD nano-therapeutic drugs to examine the ability of the nano-drugs to bind to target tissues in vitro. Figure 39 shows successful binding of ZnS QD tumor therapy nanomedicine to transferrin receptor on gastric tissue. Fluorescence in 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 confirmed that ZnS QD tumor treatment nanomedicines do not affect the receptor binding site of transferrin. The transferrin in the therapeutic nano-drug is completely activated and can be combined with transferrin to a receptor.

Therapeutic efficacy (in vivo) of ZnS QD therapeutic Nanoparticulates

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

Receptor binding, intracellular tracking, drug release and fluorescence intensity of ZnS QD therapeutic nanomedicines at different time points, receptor binding activity of tissues and intracellular cell tracking were observed by ex vivo immunohistochemical studies of gastric tissues collected 3, 6, 12, 24, 48 and 48 hours after oral administration of ZnS QD nanomedicines. The nanomedicine receptor binding and localization pattern mimics the GNP therapeutic nanomedicine pattern with no change in pattern. FIG. 40 shows binding and localization of intracellular nano-drugs after 3, 6 and 12 hours. The largest drug was observed in the tissue study after 12 hours, and also after 12 hours in the GNP therapeutic nano-drug study. The decrease in fluorescence was observed by confirming the gradual elimination behavior after 12 hours. The size range of ZnS QD nanoparticles is lower than that of GNP therapeutic nanomedicines, so in the case of quantum dots, blue fluorescence is clearer and more intense than green fluorescence. The fluorescence intensity was measured in a 67.17cm2 area 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 started to leave the tissue. After 48 hours, most of the doxorubicin was cleared from the tissue as shown by the decrease in red fluorescence. ZnS QDs are smaller in size than GNPs, but they escape from tissue more efficiently at the 72 hour time point.

The influence of ZnS QD nano therapeutic drugs on mouse body weight and survival rate during gastric cancer treatment was also examined for the anticancer effect of ZnS QD therapeutic nano drugs, three groups: the first group of saline, the second group of doxorubicin and the third group of GNP therapeutic nano-drugs re-experiment was terminated at day 40 because the survival rate of the group 2 mice at day 40 was zero. Throughout the treatment, the group treated with ZnS QD therapeutic nanomedicine remained active 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, rather than the 15-day group observed in GNP animal treatment. The effect of treatment on body weight was observed in the first 15 days and the results are shown in fig. 45 and 46, where ZnS QD treatment nano-drugs not only can prolong life but also have a healthy life and have a smooth weight gain pattern.

Example 4 pharmacokinetic and biodistribution Studies

Pharmacokinetics (in vitro) of GNP tumor treatment nanomedicines

The pH-dependent drug release of GNP therapeutic nano-drugs was performed 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 the physiological pH is almost 7.5 for plasma and organs. The pH of the medium containing the nano-drug was also 7.5, and therefore, the drug release behavior was examined to see the stability of the drug and the release at the desired site. The drug release behavior of GNP tumor treatment nano-drugs at pH5 and pH7.5 is shown in fig. 47 and 48. Drug release behavior demonstrates the stability of the therapeutic nano-drug at pH7.5 and dominant release behavior at acidic pH 5. The drug release behavior indicates that doxorubicin is released, the free form and the drug release rate are greater at pH5, and the drug is stable at pH 7.5. These results also predict the in vivo stability of the drug and its release at the tumor site and in the stomach due to pH-dependent release. At pH5, half of the doxorubicin was released in 85 hours, as shown in fig. 49, while at pH7.5, it took 850 minutes to release half of the conjugated doxorubicin, as shown in fig. 50. Data points are presented as mean values of three in figure 49 and figure 50 as ± SD.

The GNP nano-therapeutic drug released doxorubicin in cell culture, and doxorubicin release was measured in two sets of reactions in cell culture. Measurements were made between the first target concentration and the non-target concentration, and at the second time, the concentration of doxorubicin in dead cells and the culture medium was determined by HPLC. FIG. 51 shows chromatograms of pure doxorubicin (a), standard doxorubicin (b), doxorubicin (c) extracted from dead cells and culture medium, and doxorubicin (d) extracted from plasma.

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

The doxorubicin concentration in GNP tumor treatment nano-drugs was analyzed by HPLC at different time points, under different conditions in media with dead cells and compared by analysis of variance. 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 data expressed as standard deviation and significance (p <0.005) as mean values, as shown in FIG. 55.

Pharmacokinetics (in vivo) of GNP tumor treatment nanomedicines

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

Pharmacokinetics of GNP nmr nano-drugs, intravenous administration, and following intravenous administration of GNP therapeutic nano-drugs, drug distribution in plasma was characterized by HPLC analysis. Figure 56 summarizes the data collected for HPLC analysis of doxorubicin release in plasma at different time points and for statistical comparison (analysis of variance). The analysis showed that the data are within a significant time interval (p)<0.001) and all pharmacokinetic parameters were calculated and are summarized in figure 57. The clearance rate of GNP-Cys-Dox-transferrin is 0.002L/h, and the plasma half-life period (t)_1/2) It was 12.70 hours. Over time, plasma drug concentration decreased for the time and small amounts of doxorubicin were observed at 48 and 72 hours.

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

Biodistribution of GNP tumor treatment nanomedicines, and also after detection of doxorubicin concentrations in liver, heart, kidney and stomach tumors with 485 excitation and 525 emission fluorescence scanners for IV and PO dosing (luciferase reader).

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

Figure 63 summarizes doxorubicin concentrations and PO doses in the heart at various time points after intravenous infusion. Data represent mean (n-3) with standard deviation, significance (p <0.005) was calculated by ANOVA and independent T-test. Figure 64 shows doxorubicin concentrations at various time points, demonstrating rapid elimination of doxorubicin from the heart, with orally administered doxorubicin concentrations lower than intravenous injection.

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

Biodistribution experiments were performed in mice that induced gastric cancer (group 3). Post-intravenous administration, only a small amount could reach the target site, but still rarely showed an effect until 48 hours. FIG. 67 shows that most drugs administered orally bind to the target site and show higher concentrations in the gastric tumor fraction at 48 hours, and the data in FIG. 68 are summarized as mean (n-3) with standard deviation and significance (p < 0.0001).

Pharmacokinetics (in vitro) of ZnS QD therapeutic Nanoparticulates

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

Doxorubicin release from ZnS QD therapeutic nano-drug, nano-drug in cell culture (in vitro) in cell culture, doxorubicin release was measured in two sets of reactions. Dead cells and media were measured by HPLC at different time points at the first target and non-target concentrations and at the second doxorubicin concentration.

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

The concentration of doxorubicin in ZnS QD was analyzed by HPLC for the concentration of doxorubicin in media with dead cells 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 data expressed as mean values. Standard deviation and significance (p <0.005) as shown in figure 76.

ZnS QD tumor treatment NanoTaharmaceutical pharmacokinetics (in vivo)

The temporal profile of doxorubicin blood activity of the ZnS QD therapeutic nano-drug was divided into two groups of studies. One group received the nano-drug via intravenous route and the other group via oral route. The biodistribution of doxorubicin was also characterized after intravenous and oral administration.

Following intravenous injection of ZnS QD, plasma drug profiles were characterized by HPLC analysis. Figure 77 summarizes data collected from HPLC of doxorubicin in plasma at different time points and performs statistical comparisons (ANOVA). Analysis showed data at 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 plasma decreased with small amounts of doxorubicin observed at 48 and 72 hours.

Pharmacokinetics of ZnS QD therapeutic nanomedicines after oral administration, plasma concentrations of doxorubicin were analyzed by HPLC at various times after oral administration. Data are shown in fig. 80 as mean values with standard deviation (n-3) and intervals with p <0.001 as the significand. The drug concentration in the plasma increased after oral administration, and the drug was administered to reach the maximum concentration in 48 hours. After 48 to 72 hours, the concentration lines showed a sharp drop and elimination of drug from the plasma, fig. 81. Figure 82 summarizes pharmacokinetic parameters after oral dosing.

Biodistribution of nano-drugs for ZnS QD tumor therapy, and IV and PO doses (luciferase labeling) with 485 excitation and 525 emission by a fluorescence scanner for postoperative detection of doxorubicin concentrations in liver, heart, kidney and stomach tumors.

Figure 83 shows doxorubicin concentration and PO dose in the liver at different time points after intravenous injection. As shown in fig. 84, the results confirmed the reduction of doxorubicin in liver tissue over time. The doxorubicin concentration in the liver tissue after oral administration was less than the blood concentration after intravenous injection at all time points and showed rapid clearance from the liver tissue.

Figure 85 summarizes doxorubicin concentrations and PO doses in the heart at various time points after intravenous injection. Data have mean values of standard deviation (n-3), significance (p <0.005) calculated by ANOVA and independent T-test. Figure 86 shows doxorubicin concentrations at different time points. The doxorubicin concentrations tracked in the heart were extremely low, and the results confirmed the rapid elimination of doxorubicin from the heart. Oral administration of doxorubicin is less concentrated than intravenous injection.

FIG. 87 shows the elimination of doxorubicin from the kidney at various time points and the data obtained from the analysis represent the deviation and significance (p <0.001) as the mean value of the standard (n-3). Doxorubicin concentrations decreased over time, with no significant doxorubicin concentrations after intravenous and oral administration at the 6 hour time point, as shown in fig. 88. The kidney was orally taken at higher concentrations for 24 hours, but most doxorubicin was cleared from the body at 48 hours.

Biodistribution experiments were performed in mice that induced gastric cancer (group 3). 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, with a redistribution pattern 89 after 48 hours at higher concentrations in gastric tumors. The data in figure 90 are summarized as mean (n-3) with standard deviation and significance (p < 0.0001).

Claims (7)

1. The preparation method of the nano-targeted drug and the application of the nano-targeted drug in the treatment of gastric cancer are characterized in that the nano-targeted drug comprises GNP therapeutic nano-drug and ZnS QD therapeutic nano-drug.

2. The preparation method of the nano-targeting drug and the application of the nano-targeting drug in the treatment of gastric cancer according to claim 1, wherein the linker of the GNP therapeutic nano-drug is cysteine, the targeting ligand is transferrin, the chemotherapeutic drug is doxorubicin, and the nano-carrier is GNP.

3. The preparation method of the nano-targeted drug and the application thereof in the treatment of gastric cancer according to claim 1, wherein the linker 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.

4. The preparation method of the nano-targeted drug and the application of the nano-targeted drug in the treatment of gastric cancer according to claim 1, wherein the preparation method of the nano-targeted drug comprises the preparation of GNP therapeutic nano-drugs and the preparation of ZnS QD therapeutic nano-drugs.

5. The method for preparing nano-targeted medicament according to claim 4, wherein the preparation of GNP therapeutic nano-medicament comprises the synthesis of GNP, the combination of cysteine and GNP, the combination of adriamycin and GNP-Cys, and the combination of transferrin and GNP-Cys-Dox.

6. The method for preparing nano targeted drug according to claim 4, characterized in that the preparation of ZnS QD therapeutic nano drug comprises the synthesis of ZnS QD quantum dots coated with cysteine, the combination of adriamycin and ZnS QD-Cys, and the combination of transferrin and ZnS QD-Cys-Dox.

7. The preparation method of the nano-targeted drug and the application of the nano-targeted drug in the treatment of gastric cancer according to claim 1, wherein cysteine-coated GNP and transferrin are conjugated to serve as a targeted diagnostic probe.

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