A method for administering a nanomedicine co-loading glutamine and glutamine transporter inhibitor and its anti-tumor application

Abstract

The application belongs to the technical field of biological medicine materials, and provides a medicine application method of nanodrug co-loading glutamine and glutamine transporter inhibitors and anti-tumor application thereof. The application is directed to nanodrug containing glutamine, selects a series of glutamine transporter inhibitors, and prepares nanodrug through co-loading of glutamine and glutamine transporter inhibitors. After the nanodrug co-loading glutamine and glutamine transporter inhibitors is endocytosed by tumor cells, the nanodrug can further block the uptake of glutamine molecules by limiting the uptake of glutamine molecules due to the overexpression of glutamine transporters on the cell membrane of tumor cells, so that the metabolic pressure of glutamine forces the tumor cells to make adaptive metabolic conversion, promotes the endocytosis of nanodrug containing glutamine, and thus realizes the self-promoting uptake of nanodrug in tumor cells, and improves the tumor treatment effect of nanodrug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and in particular relates to a method of administering nanomedicine co-loaded with glutamine and glutamine transporter inhibitors and its anti-tumor application. Background Technology

[0002] Glutamine is a natural amino acid, a conditionally essential amino acid, and the most abundant free amino acid in the human body. It is inextricably linked to all aspects of cellular energy metabolism. Glutamine provides carbon and nitrogen sources for the synthesis of proteins, amino acids, and nucleic acids, participates in oxidative phosphorylation to release energy, and plays a crucial role in cellular nutrient signal transduction. Compared to normal cells, tumor cells exhibit significant heterogeneity in glutamine uptake, transformation, and metabolism. This is attributed to glutamine metabolic reprogramming caused by mutations in carcinogenic driver genes such as the Krölln rat sarcoma virus oncogene homolog (KRAS). Specific manifestations include increased glutamine uptake, upregulation of glutamine-based synthesis, upregulation of glutamine-related antioxidant metabolism, enhanced nutrient signal transduction in glutamine metabolic pathways, and the formation of a specific tumor microenvironment with the aid of glutamine. Therefore, glutamine metabolic rearrangement is a significant factor in the occurrence and development of tumors, influencing tumor spread, invasion, and metastasis. Furthermore, recent studies have shown that glutamine promotes apoptosis resistance and immune escape in tumor cells during clinical cancer treatment. Because of the important role of glutamine, the demand for glutamine in various tumor cells has been greatly increased. The demand for glutamine in tumor cells is dozens of times that of other amino acids, and many tumor cells exhibit glutamine addiction.

[0003] Tumor cells supply glutamine through two pathways: endogenous glutamine synthesis and exogenous glutamine uptake. Due to limited intracellular nutrients, exogenous glutamine uptake is the primary pathway, which can be further divided into the uptake of extracellular glutamine-containing substances via endocytosis and the uptake of molecular glutamine via amino acid transporters. To adapt to the variable nutritional environment of the tumor microenvironment, the two glutamine supply pathways of tumor cells are complementary. When one pathway cannot meet the metabolic demand for glutamine, the other pathway can be adaptively upregulated to increase glutamine uptake. Simultaneously, to meet the high glutamine supply demand, both pathways of exogenous glutamine uptake are significantly upregulated in tumor cells. In clinical treatment, the upregulated glutamine supply pathways of tumor cells are gradually becoming a drug target.

[0004] On the one hand, in the process of endocytosis to take up extracellular glutamine-containing substances, tumor cells exhibit enhanced endocytosis and specific macropinocytosis. This upregulation of endocytosis to take up nutrients such as glutamine has become a feasible drug delivery strategy in clinical treatment. Glutamine-based small molecule drugs, polyglutamines, glutamine assemblies, glutamine-containing peptides, and protein nanomedicines exhibit uptake preferences in tumor cells and are widely used in tumor diagnosis and treatment. However, in the study of nanomedicine delivery efficiency in tumor therapy, the delivery efficiency of most nanomedicines, including glutamine-based nanomedicines, remains extremely low, and the delivery efficiency of related nanomedicines still needs to be improved. Furthermore, considering various clinical drug delivery methods such as intravenous administration, oral administration, transdermal administration, topical application, and perfusion, tumor cell uptake of nanomedicines is an essential step. Improving the uptake efficiency of nanomedicines will greatly benefit the efficacy of nanomedicine treatment. Therefore, it is still necessary to develop a simple, universal, and efficient drug delivery strategy to improve the uptake efficiency of nanomedicines.

[0005] On the other hand, in the uptake of glutamine by transporters, tumor cells overexpress glutamine transporters on their cell membranes to take up small glutamine molecules from outside the cell, including alanine-serine-cysteine ​​transporter 2 (ASCT2), L-amino acid transporter 1 (LAT1), and Na+ transporter 2 (Na+). + - Coupled with neutral amino acid transporters (SNATs) and amino acid transporter B 0,+ (ATB 0,+ ASCT2 is a major glutamine transporter. Inhibitors of ASCT2, including V9302, L-γ-glutamyl-(p-nitroaniline) (GPNA), S-benzyl-L-cysteine ​​(BzlCys), and O-benzyl-L-serine (BzlSer), can pharmacologically block ASCT2, restricting glutamine uptake and thus inhibiting glutamine energy metabolism, increasing intracellular oxidative stress, and suppressing cancer cell growth and proliferation to achieve glutamine starvation therapy. However, to date, no glutamine transporter inhibitors have been approved for clinical use in cancer treatment. This is attributed to the sophisticated glutamine starvation adaptation mechanisms of tumor cells, including increasing glutamine synthesis, utilizing glucose and other nutrients as substitutes, and changing the way glutamine is obtained. Therefore, it is still necessary to optimize the use of glutamine transporter inhibitors to enhance their therapeutic effects on cancer.

[0006] To avoid the aforementioned technical problems, it is indeed necessary to provide a method of administration of nanomedicine co-loaded with glutamine and glutamine transporter inhibitor and its anti-tumor application to overcome the deficiencies in the prior art. Summary of the Invention

[0007] The purpose of this invention is to provide a method of administration of nanomedicine co-loaded with glutamine and glutamine transporter inhibitor and its anti-tumor application, aiming to solve the above-mentioned technical problems.

[0008] This invention selects a Fe / glutamine supramolecular self-assembled nanomedicine as a glutamine nanomedicine model. The raw materials for nanomedicine preparation are FeCl3·6H2O and glutamine. Further, a variety of glutamine transporter inhibitors are selected, including V-9302, L-γ-glutamyl-p-nitroaniline (GPNA), S-benzyl-L-cysteine ​​(BzlCys), O-benzyl-L-serine (BzlSer), raloxifene, tamoxifen, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), JPH203, and α-(methylamino)isobutyric acid (MeAIB). First, FeCl3·6H2O and glutamine were prepared into an aqueous solution of a certain concentration. Then, based on the water / oil solubility of the glutamine transporter inhibitor, a water / dimethyl sulfoxide solution of a certain concentration was prepared. Next, the FeCl3, glutamine, and glutamine transporter inhibitor solution were added to a certain volume of water and stirred at room temperature for a certain period of time. Fe underwent hydrolysis and self-assembly to form FeOOH nanoparticles. The carboxyl and amino groups in glutamine and the glutamine transporter inhibitor could react with Fe... 3+ Coordination and co-assembly were then employed to obtain nanomedicines co-loaded with glutamine and glutamine transporter inhibitors. These nanomedicines were spindle-shaped with a core-shell structure; the core was predominantly FeOOH, and the shell was composed of Fe... 3+ The main component is a coordination complex of glutamine / glutamine transporter inhibitors. The structural formula of V-9302 is shown in the figure below:

[0009]

[0010] The structural formula of GPNA is shown in the figure below:

[0011]

[0012] The structural formula of BzlCys is shown in the figure below:

[0013]

[0014] The structural formula of BzlSer is shown in the figure below:

[0015]

[0016] The structural formula of raloxifene is shown in the following figure:

[0017]

[0018] The structural formula of tamoxifen is shown in the following figure:

[0019]

[0020] The structural formula of BCH is shown in the figure below:

[0021]

[0022] The structural formula of JPH203 is shown in the figure below:

[0023]

[0024] The structural formula of MeAIB is shown in the figure below:

[0025]

[0026] Glutamine-containing nanomedicines can be recognized as a glutamine nutrient source by glutamine-addicted tumor cells, and subsequently internalized to supply glutamine metabolism. This allows glutamine-based nanomedicines to be specifically and efficiently taken up by tumor cells. Loading glutamine transporter inhibitors onto glutamine-containing nanomedicines represents a further improvement and enhancement of this strategy. After being taken up by tumor cells, the nanomedicine responsively releases glutamine transporter inhibitors to block overexpressed glutamine transporters on the cell membrane, thereby limiting glutamine molecule uptake and causing intracellular glutamine deficiency in tumor cells. The glutamine deficiency-induced metabolic stress guides the tumor cells to increase the uptake of glutamine-containing nanomedicines. Subsequent uptake of the nanomedicine further releases glutamine transporter inhibitors and promotes uptake again, thus achieving a self-promoting and efficient uptake of the nanomedicine in a cyclical manner.

[0027] This drug delivery strategy not only improves the uptake efficiency of glutamine-containing nanomedicines but also allows glutamine transporter inhibitors to be used as a commodity, achieving efficient accumulation in tumor cells through the aforementioned cyclical self-promoted uptake. This overcomes the disadvantage of low utilization rates caused by the poor water solubility of glutamine transporter inhibitors. Furthermore, this strategy is applicable to various glutamine-containing nanomedicines and glutamine transporter inhibitors, and shows significant efficacy in various glutamine-addicted tumors, providing a feasible method to improve the utilization rate of widely used nanomedicines in the biomedical field.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) The auto-uptake medication strategy proposed in this invention is applicable to the treatment of various cancers and tumors with glutamine addiction, including pancreatic cancer, lung cancer, colorectal cancer, liver cancer, and gastric cancer.

[0030] (2) The self-promoting uptake drug strategy proposed in this invention is applicable to a variety of glutamine-containing nanomedicines, including polyglutamine, glutamine assemblies, glutamine-containing peptides and proteins, etc.

[0031] (3) The self-promoted uptake drug strategy proposed in this invention significantly improves the uptake efficiency of glutamine-containing nanomedicines, and increases the uptake of nanomedicines by tumor cells by 2-10 times.

[0032] (4) The self-promoted uptake drug strategy proposed in this invention improves the tumor treatment effect of nanomedicine, reduces the half-inhibitory concentration of nanomedicine on tumor cell proliferation, and enhances the ability of nanomedicine to induce tumor cell apoptosis, necrosis, ferroptosis and pyroptosis. Attached Figure Description

[0033] Figure 1 Transmission electron microscopy image of the Fe / glutamine nanomedicine prepared in Example 1;

[0034] Figure 2 Transmission electron microscopy image of the Fe / glutamine / V-9302 nanomedicine prepared in Example 2;

[0035] Figure 3 Transmission electron microscopy image of the Fe / glutamine / GPNA nanomedicine prepared in Example 3;

[0036] Figure 4 Transmission electron microscopy image of the Fe / glutamine / BzlCys nanomedicine prepared in Example 4;

[0037] Figure 5 Transmission electron microscopy image of the Fe / glutamine / BzlSer nanomedicine prepared in Example 5;

[0038] Figure 6 Transmission electron microscopy image of the Fe / glutamine / raloxifene nanomedicine prepared in Example 6;

[0039] Figure 7 Transmission electron microscopy image of the Fe / glutamine / tamoxifen nanomedicine prepared in Example 7;

[0040] Figure 8 Transmission electron microscopy image of the Fe / glutamine / BCH nanomedicine prepared in Example 8;

[0041] Figure 9 Transmission electron microscopy image of the Fe / glutamine / JPH2O3 nanomedicine prepared in Example 9;

[0042] Figure 10 Transmission electron microscopy image of the Fe / glutamine / MeAIB nanomedicine prepared in Example 10;

[0043] Figure 11 The difference in cellular uptake of Fe / glutamine nanomedicine and Fe / glutamine / V-9302 nanomedicine in pancreatic cancer cells MiaPaCa2 cells was measured in Example 11;

[0044] Figure 12 The difference in cytotoxicity between Fe / glutamine nanomedicine and Fe / glutamine / V-9302 nanomedicine in pancreatic cancer cells MiaPaCa2 cells was measured in Example 12. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0046] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0047] Example 1

[0048] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, and 10 mL of glutamine aqueous solution were mixed under stirring at room temperature and stirred for 48 hours. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine nanodrug suspension.

[0049] Example 2

[0050] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; the glutamine transporter inhibitor V-9302 was dissolved in dimethyl sulfoxide to prepare a 10 mM V-9302 dimethyl sulfoxide solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, 9.5 mL of glutamine aqueous solution, and 0.5 mL of V-9302 dimethyl sulfoxide solution were mixed under stirring at room temperature for 48 hours. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine / V-9302 nanodrug suspension.

[0051] Example 3

[0052] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; the glutamine transporter inhibitor GPNA was dissolved in dimethyl sulfoxide to prepare a 10 mM GPNA dimethyl sulfoxide solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, 9.5 mL of glutamine aqueous solution, and 0.5 mL of GPNA dimethyl sulfoxide solution were mixed under stirring at room temperature for 48 hours. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine / GPNA nanodrug suspension.

[0053] Example 4

[0054] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; the glutamine transporter inhibitor BzlCys was dissolved in dimethyl sulfoxide to prepare a 10 mM BzlCys dimethyl sulfoxide solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, 9.5 mL of glutamine aqueous solution, and 0.5 mL of BzlCys dimethyl sulfoxide solution were mixed under stirring at room temperature for 48 hours. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine / BzlCys nanomedicine suspension.

[0055] Example 5

[0056] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; the glutamine transporter inhibitor BzlSer was dissolved in dimethyl sulfoxide to prepare a 10 mM BzlSer dimethyl sulfoxide solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, 9.5 mL of glutamine aqueous solution, and 0.5 mL of BzlSer dimethyl sulfoxide solution were mixed under stirring at room temperature and stirred for 48 hours at room temperature. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine / BzlSer nanomedicine suspension.

[0057] Example 6

[0058] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; the glutamine transporter inhibitor raloxifene was dissolved in dimethyl sulfoxide to prepare a 10 mM raloxifene dimethyl sulfoxide solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, 9.5 mL of glutamine aqueous solution, and 0.5 mL of raloxifene dimethyl sulfoxide solution were mixed under stirring at room temperature for 48 hours. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine / raloxifene nanoparticle suspension.

[0059] Example 7

[0060] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; the glutamine transporter inhibitor tamoxifen was dissolved in dimethyl sulfoxide to prepare a 10 mM tamoxifen dimethyl sulfoxide solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, 9.5 mL of glutamine aqueous solution, and 0.5 mL of tamoxifen dimethyl sulfoxide solution were mixed under stirring at room temperature for 48 hours. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine / tamoxifen nanoparticle suspension.

[0061] Example 8

[0062] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; the glutamine transporter inhibitor BCH was dissolved in water to prepare a 10 mM BCH aqueous solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, 9.5 mL of glutamine aqueous solution, and 0.5 mL of BCH aqueous solution were mixed under stirring at room temperature for 48 hours. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine / BCH nanodrug suspension.

[0063] Example 9

[0064] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; the glutamine transporter inhibitor JPH203 was dissolved in dimethyl sulfoxide to prepare a 10 mM JPH203 dimethyl sulfoxide solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, 9.5 mL of glutamine aqueous solution, and 0.5 mL of JPH203 dimethyl sulfoxide solution were mixed under stirring at room temperature for 48 hours. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine / JPH203 nanodrug suspension.

[0065] Example 10

[0066] FeCl3·6H2O was dissolved in water to prepare a 100 mM FeCl3 aqueous solution; glutamine was dissolved in water to prepare a 10 mM glutamine aqueous solution; the glutamine transporter inhibitor MeAIB was dissolved in water to prepare a 10 mM MeAIB aqueous solution; 80 mL of deionized water, 10 mL of FeCl3 aqueous solution, 9.5 mL of glutamine aqueous solution, and 0.5 mL of MeAIB aqueous solution were mixed under stirring at room temperature for 48 hours. The resulting product was centrifuged at 8000 r / min for 10 minutes and washed with deionized water to obtain an iron / glutamine / MeAIB nanodrug suspension.

[0067] Example 11

[0068] Imaging flow cytometry was used to determine the self-promoted uptake behavior of nanomedicines in cancer cells. The specific steps were as follows:

[0069] Control group: 300,000 MiaPaCa2 cells were seeded into 12-well cell culture plates and cultured for 24 hours without nanomedicine. The cells were then cultured for another 6 hours without nanomedicine. Afterwards, the culture medium was removed, adherent cells were washed three times with PBS, and cells were collected by trypsin digestion. Finally, the uptake of nanomedicine was measured by imaging flow cytometry.

[0070] Experimental group (iron / glutamine nanomedicine group): 300,000 MiaPaCa2 cells were seeded into 12-well cell culture plates and cultured for 24 hours. Iron / glutamine nanomedicine (prepared in Example 1) was then added to achieve a nanomedicine concentration of 100 μg / mL in the culture medium, and the cells were cultured for another 6 hours. Afterwards, the culture medium was removed, adherent cells were washed three times with PBS, and cells were collected by trypsin digestion. Finally, the uptake of the nanomedicine was measured by imaging flow cytometry.

[0071] Experimental group (iron / glutamine / V-9302 nanomedicine): 300,000 MiaPaCa2 cells were seeded into 12-well cell culture plates and cultured for 24 hours. Iron / glutamine nanomedicine (prepared in Example 2) was then added to bring the nanomedicine concentration in the culture medium to 100 μg / mL, and the cells were cultured for another 6 hours. Afterward, the culture medium was removed, adherent cells were washed three times with PBS, and cells were collected by trypsin digestion. Finally, the uptake of the nanomedicine was measured by imaging flow cytometry.

[0072] Example 12

[0073] The antitumor activity of nanomedicines was determined using the CCK-8 assay. First, 8000 MiaPaCa2 cells were seeded into 96-well cell culture plates and cultured for 12 hours. Then, different concentrations of nanomedicines were added and cultured for another 48 hours. The relative cell viability was calculated by measuring the absorbance of each well at 450 nm and subtracting the background absorbance.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0075] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. The application of a nanomedicine co-loaded with glutamine and a glutamine transporter inhibitor in the preparation of drugs that increase the endocytic uptake of glutamine-containing nanomedicines by cancer cells, characterized in that, Introducing glutamine transporter inhibitors into glutamine-containing nanomedicines artificially creates glutamine deficiency in cancer cells by blocking the uptake of glutamine molecules by transporters, thereby guiding the adaptive metabolism of cancer cells to change, thus increasing the endocytic uptake of glutamine-containing nanomedicines and enhancing the therapeutic effect of the drug on tumors; the cancer cells are pancreatic cancer cells. The glutamine-containing nanomedicine is an Fe / glutamine supramolecular nanomedicine; The glutamine transporter inhibitor is V-9302.

2. The application according to claim 1, characterized in that, The structural formula of V-9302 is shown in Formula I: Formula I.

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