Carbon quantum dots and their uses

Abstract

The present invention relates to aromatic or partially aromatic polycyclic compounds substituted with one or more alkyl groups having amino and carboxyl functional groups, and includes carbon quantum dots (CQDs) containing these compounds. These compounds and CQDs have selective affinity for cells expressing LAT1 and tumor cells and can be internalized by these cells. These compounds and CQDs can be used to image such cells and deliver payloads to cells expressing LAT1 and tumor cells. Methods for preparing and using these compounds and CQDs for bioimaging and targeting certain tissues (including tumors) are disclosed.

Description

Field of the Invention

[0001] The field of the invention is compounds, compositions and methods related to tumor imaging and delivery of therapeutic agents to tumors, and methods of preparing novel compounds. The methods and compositions involve polycyclic aromatic structures commonly used as carbon quantum dots (CQDs). Background of the Invention

[0003] Cancer is one of the most devastating diseases, with more than 14 million new cases each year. The incidence of cancer is expected to continue to rise, with an estimated 21.7 million new cases per year by 2030. In current clinical practice, patients with cancer are typically treated with a combination of surgical resection (if applicable) and chemotherapy and radiation therapy. With the tremendous advances in surgery and medicine over the past few decades, this combined treatment approach has significantly increased patient survival time and quality of life. However, further improvements in these approaches have been hampered by the inability to selectively image and treat tumors. This challenge limits our ability to achieve maximal surgical resection or deliver chemotherapeutic agents and radiation without inducing significant side effects. 1-7

[0004] To enhance tumor-specific imaging and drug delivery, recent efforts have focused on designing a class of imaging or therapeutic agents that recognize ligands of "receptor" molecules expressed in cancer cells or in the tumor microenvironment. Increasing evidence shows that this approach has several major limitations. First, receptor molecules expressed only on the surface of cancer cells are rare, and most of them are also present in normal tissues. Therefore, the accumulation of cargo in normal tissues cannot be avoided. Second, cancer is a highly heterogeneous disease, and cancers of different tissue origins or different locations within tumors usually have different genotypes and phenotypes. Therefore, using only a single ligand cannot successfully target tumors. Due to these limitations, there is currently no strategy that can selectively image and deliver drugs to tumors regardless of their origin and location.

[0005] A promising approach for tumor-specific imaging and drug delivery is to exploit specific cargo transporters that are differentially upregulated in cancer cells. Examples of such transporters include large neutral amino acid transporter 1 (LAT1), alanine, serine, cysteine ​​preferential transporter 2 (ASCT2), and glucose transporters. 8-11 Among them, LAT1, which mediates large neutral amino acid transport, is particularly interesting. LAT1 has been shown to be highly expressed in a variety of tumors; in sharp contrast, according to the mRNA expression results, the distribution of LAT1 in normal tissues is limited to a few organs, including the placenta, blood-brain barrier, spleen, testis, and colon. 10LAT1 inhibitors have been used to target LAT1 for cancer chemotherapy, such as 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). 12 Unfortunately, BCH-mediated chemotherapy has largely failed due to lack of potency and specificity. BCH inhibits all four LATs, including LAT1, LAT2, LAT3, and LAT4, which have distinct physiological functions. The high doses of BCH required for treatment often induce significant toxicity. 12 LAT1 has also been used to enhance the targeted delivery of chemotherapeutic drugs to tumors. Results showed that binding to aspartic acid enabled LAT1-mediated delivery, increasing the accumulation of doxorubicin (DOX) in tumors by 3-6 times. 13

[0006] CQDs have emerged in recent years as a promising class of imaging agents and drug carriers for various biomedical applications due to their excellent biocompatibility, optical properties, drug loading capacity, and low toxicity. However, most previously reported CQDs interact non-selectively with both tumor and non-tumor cells and do not achieve a level of tumor specificity that would allow them to be used to selectively target tumor cells. 14,15 Summary of the invention

[0007] The present invention provides compounds with structures similar to large amino acids and carbon quantum dots (LAAM CQDs) containing these compounds, which can be used for selective imaging, drug delivery and / or detectable labeling of tissues expressing LAT1. Since many cancer cells express LAT1 at levels much higher than most human tissues, these compounds and CQDs can be used for tumor imaging and drug delivery to tumor cells, regardless of the source and location of the tumor, but rarely or almost never delivered to cells in normal tissues.

[0008] The LAAM compounds of the present invention are polycyclic compounds having a large aromatic or partially aromatic core substituted with an α-aminocarboxylic acid. The core of the compound is a polycyclic array consisting of 8 or more 6-membered rings, each of which is aromatic or partially aromatic or partially unsaturated. The polycyclic core is substituted with one or more, usually two or more, and in many embodiments three or four or more alkyl groups, each of which is substituted with a carboxylic acid and an amino group on the same carbon as the carboxylic acid, i.e., an α-aminocarboxylic acid moiety. The amino group may be -NH2 or a substituted form thereof. The alkyl group may also have one or more additional substituents.

[0009] The LAAM compounds of the present invention can be assembled into aggregates for use as carbon quantum dots (LAAM CQDs). LAAM CQDs have an α-aminocarboxylic acid portion that triggers multivalent interactions, thereby tightly binding to LAT1. Without being bound by theory, the high selective affinity exhibited by these compounds and LAAM CQDs for LAT1 explains their ability to selectively target tumor cells for tumor imaging and to deliver payloads (e.g., therapeutic agents and cytotoxic agents) to tumor cells. Some CQDs provided by the present invention, including the LAAM TC-CQDs described in the present invention, exhibit near-infrared (NIR) fluorescence (FL) and photoacoustic (PA) imaging capabilities, which allows them to image tumors. Because they are selectively internalized into cancer cells or tissues, such as by endocytosis associated with LAT1 or transported by LAT1 into cells expressing LAT1, the compounds and CQDs of the present invention can also be used as carriers for delivering chemotherapeutic drugs to tumors. The LAAM CQDs disclosed in the present invention, including LAAM CQDs synthesized using 1,4,5,8-tetraaminoanthraquinone (TAAQ) and citric acid (CA), provide new compositions and methods for selective imaging of tumors and certain other specific types of cells, and for selectively delivering therapeutic agents or labeling agents to these cells and tumors.

[0010] In one aspect, the present invention provides carbon quantum dots having useful fluorescent properties, being soluble in water and being biocompatible. The carbon quantum dots of the present invention can form organic (carbon-based) particles having a size of about 0.2-10 nm, and their carbon content is generally at least about 20% or 40% by weight, preferably at least about 50% by weight. Their fluorescent properties, water solubility and biocompatibility make them beneficial in vivo, and the LAAM CQDs selectively binding to LAT1 in the present invention are particularly useful in the methods described in the present invention for imaging tumors.

[0011] In another aspect, the present invention provides solvolysis products formed by the methods described herein, which can be effectively internalized into cells, for example, by endocytosis or transport by LAT1, and are selectively taken up by tumor cells, especially in tumors expressing LAT1. These solvolysis products contain polycyclic aromatic or partially aromatic nucleocyclic systems, and can be isolated and used as particles of about 0.2-10 nm in size that can act as carbon quantum dots. The use of solvolysis to prepare carbon quantum dots is known in the art. These compounds and particles prepared by the methods of the present invention can be used for tumor imaging or as a type of carrier that can deliver loads such as markers or therapeutic agents to the surface of tumor cells expressing LAT1, or to the interior of such cells. In some embodiments, the solvolysis products form particles containing one or more LAAM compounds disclosed in the present invention, which act as carbon quantum dots and can therefore be used for tumor imaging or tumor targeting that expresses the LAT1 transporter.

[0012] In another aspect, the present invention provides methods for preparing the above compounds and LAAM CQDs.

[0013] In another aspect, the present invention provides methods for imaging LAT1-expressing tissues, particularly LAT1-expressing tumors in vivo, using compounds and CQDs containing the same.

[0014] In another aspect, the present invention provides conjugates comprising the above compounds and CQDs, and methods of using these conjugates to selectively deliver imaging agents, therapeutic agents, and cytotoxic agents to cells expressing LAT1, including tumor cells.

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising the above compound and at least one pharmaceutically acceptable carrier or excipient.

[0016] In yet another aspect, the present invention provides a method of imaging a tumor or tissue expressing LAT1 using the LAAM compounds and LAAM CQDs of the present invention.

[0017] In yet another aspect, the present invention provides a use of the LAAM compounds and LAAM CQDs of the present invention for manufacturing a pharmaceutical product.

[0018] In another aspect, the present invention provides conjugates comprising the LAAM compounds or LAAM CQDs of the present invention, wherein the LAAM compounds or LAAM CQDs are covalently or non-covalently linked to a load and delivered to or into cells expressing LAT1. Since LAT1 is expressed in many tumors, these conjugates can be used to selectively deliver labeling agents or chemotherapeutic agents to tumor cells, with little or no delivery to normal cells.

[0019] In yet another aspect, the present invention provides methods of imaging and / or treating tumors expressing LAT1 using the conjugates of the present invention.

[0020] Other aspects and advantages of the products, compositions and methods of the present invention will be apparent to the skilled artisan in view of the following examples and further description.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 a Schematic diagram and hypothetical steps (dashed lines) for the synthesis of LAAM TC-CQDs.

[0023] Figure 1 b shows the TEM and HRTEM images of LAAM TC-CQDs (inset).

[0024] Figure 1 c shows the UV-visible absorption spectrum of LAAM TC-CQDs and a photograph of their appearance under sunlight (inset).

[0025] Figure 1 d shows the fluorescence emission spectrum and appearance photograph of LAAM TC-CQDs under 365 nm ultraviolet light (inset).

[0026] Figure 1 e shows the photoacoustic (PA) signal intensity and imaging of LAAM TC-CQDs at a concentration of 2 μg / mL (inset).

[0027] Figure 2 a shows laser confocal scanning microscopy (LCSM) images of representative cells, including HeLa cells and non-cancerous CCC-ESF-1 cells, after incubation with LAAM TC-CQDs. Scale bar is 100 μm.

[0028] Figure 2 b: Quantification of cellular uptake in cancer cells and non-cancerous cells by flow cytometry.

[0029] Figure 2 c provides near-infrared fluorescence (NIR FL) images of representative tumor-bearing mice after intravenous injection of LAAM TC-CQDs at the indicated time points.

[0030] Figure 2 d shows 8 hours after injection Figure 2 c 3D reconstruction of the distribution of LAAM TC-CQDs in mice.

[0031] Figure 2 e shows 8 hours after injection of LAAM TC-CQDs. Figure 2c Ex vivo NIRFL imaging of indicated organs and tumors in mice.

[0032] Figure 2 f provides cross-sectional photoacoustic (PA) imaging of the indicated tumors (circles) in representative tumor-bearing mice that received intravenous injection of LAAM TC-CQDs at the indicated time points.

[0033] Figure 3 a provides LCSM images of HeLa cells pretreated with Leu, Phe, Gly, or 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH, a known LAT1 inhibitor) and subsequently incubated with LAAM TC-CQDs. Scale bar is 25 μm.

[0034] Figure 3 bd show that knockdown of LAT1 expression by CRISPR / Cas9 reduced the cellular uptake of LAAM TC-CQDs (lower panels). Figure 3 The arrow in b indicates the sgRNA targeting sequence. Sanger sequencing (b, upper panel) and WB test (c) confirmed the successful targeting of LAT1. LCSM imaging of LAT1 downregulated cells is shown in Figure 3 d.

[0035] Figure 3 e shows the correlation between LAT1 expression and cellular uptake of LAAM TC-CQDs.

[0036] Figure 4 a: LCSM imaging of HeLa and CCC-ESF-1 cells after incubation with TPTC, LAAM TC-CQDs, or TPTC / LAAM TC-CQDs.

[0037] Figure 4 b: Cell survival rate of HeLa and CCC-ESF-1 cells after treatment with TPTC, LAAM TC-CQDs or TPTC / LAAM TC-CQDs.

[0038] Figure 4 c shows the serum concentration of TPTC over time when delivered as free drug or together with TPTC / LAAM TC-CQDs. Data are expressed as percentage of the total injected dose (%ID).

[0039] Figure 4 d shows the biodistribution of TPTC and TPTC / LAAM TC-CQDs in the indicated organs.

[0040] Figure 4 e shows representative images of tumor-bearing mice receiving indicated treatments.

[0041] Figure 4 f: Representative images of tumors obtained from mice receiving the indicated treatments on day 15 after treatment.

[0042] Figure 4 g: Changes in tumor volume over time in mice receiving the indicated treatments (n=5).

[0043] Figure 5 a: Molecular structures of selected compounds forming LAAM TC-CQDs, including NH2-free and COOH-free LAAMTC-CQDs, 2,6-CQDs, 1,4-CQDs, 1,5-CQDs, and Phe-CQDs.

[0044] Figure 5 b shows Figure 5 Imaging of cellular uptake / permeation of LAAM TC-CQDs shown in a.

[0045] Figure 5 c: Flow cytometric quantification of FL intensity in HeLa or CCC-ESF-1 cells after treatment with the indicated CQDs.

[0046] Figure 6 : Size distribution of TC-CQDs.

[0047] Figure 7 : Raman spectrum of TC-CQDs.

[0048] Figure 8 a: AFM image of TC-CQDs on silicon substrate.

[0049] Figure 8 b: Figure 8 Height profile along the indicated line in a.

[0050] Fig. 9 : XRD patterns of LAAM TC-CQDs.

[0051] Fig.10 : Full XPS spectrum of LAAM TC-CQDs.

[0052] Fig.11 : C1s spectrum of LAAM TC-CQDs.

[0053] Fig.12 : N1s spectrum of LAAM TC-CQDs.

[0054] Fig.13 : FT-IR spectrum of LAAM TC-CQDs.

[0055] Fig.14: LAAM TC-CQDs 13 C-NMR spectrum.

[0056] Fig.15 : Fluorescence emission spectrum of TC-CQDs aqueous solution at excitation wavelengths from 560-660nm.

[0057] Fig.16 : Main geometric parameters of the optimized ground state (a) and excited state (b) structures of a FL unit of LAAM TC-CQDs Optimized electron delocalized molecular orbital (MO) diagrams (c, d) and their energy band position diagram (e) of TC-CQDs obtained from density functional theory calculations (B3LYP / 6-31G(d, p)).

[0058] Fig.17 a: Under 650nm laser irradiation (0.5W / cm 2 ) were recorded every 30 seconds for TC-CQDs with different concentrations (0-10 μg / mL). The inset is the photothermal curves of 650 nm laser irradiation (0.5 W / cm 2 ) IR thermal imaging of TC-CQDs (0-10 μg / mL) after 5 minutes.

[0059] Fig.17 b: LAAM TC-CQDs (10 μg / mL) after 5 laser on / off cycles (650 nm laser irradiation, 0.5 W / cm 2 ) under temperature changes.

[0060] Fig.17 c: TC-CQDs aqueous solution was exposed to 650 nm laser (0.5 W / cm 2 ) Photothermal effect under irradiation. The laser was turned off after 5 minutes of irradiation.

[0061] Fig.17 d: Cooling time and cooling stage (such as Fig.17 c) The time constant of heat transfer in the system is determined to be τs = 83.86s.

[0062] Fig.18 : Photoacoustic (PA) signal intensity of LAAM TC-CQDs in the concentration range of 0-10 μg / mL.

[0063] Fig.19 : PA signal intensity and imaging of LAAM TC-CQDs in the concentration range of 2-10 μg / mL.

[0064] Fig. 20: Characterization of side population cells sorted from MDA-MB-231 cells. From cells stained with isotype control (a); from cells stained with anti-human CD44-FITC and CD24-PE antibodies (b). CSCs cells were analyzed by flow cytometry.

[0065] Fig.21 : LCSM imaging of different types of cancer cells after co-incubation with LAAM TC-CQDs. The scale bar is 100 μm.

[0066] Fig. 22 : LCSM images of different types of normal cells co-incubated with LAAM TC-CQDs. The scale bar is 100 μm.

[0067] Fig.23 : Uptake results of LAAM TC-CQDs in 20 cancer cells obtained by flow cytometry analysis.

[0068] Fig.24 : Uptake results of LAAM TC-CQDs in 18 normal cells obtained by flow cytometry analysis.

[0069] Fig.25 : LCSM images of HeLa cells treated with LAAM TC-CQDs for different time periods (1-10 h). Scale bar: 25 μm.

[0070] Fig.26 : LCSM images of CCC-ESF-1 cells treated with LAAM TC-CQDs for different time periods (1-48 h). Scale bar: 25 μm.

[0071] Fig. 27 : Semiquantitative biodistribution profiles of LAAM TC-CQDs in tumor-bearing mice determined by the average FL intensity of major organs and tumors.

[0072] Fig.28 : PA signal intensity of tumor area at different time.

[0073] Fig.29 : NIR FL imaging of subcutaneous tumor xenografts in nude mice derived from A549 (a), PANC-1 (b), MCF-7 (c), and MD-MBA-231 (d) cells within 10 h after intravenous injection of LAAM TC-CQDs.

[0074] Fig.30: Ex vivo NIR FL imaging of major organs (heart, liver, spleen, lung, and kidney) and tumors of nude mice with subcutaneous tumor xenografts derived from A549 (a), PANC-1 (b), MCF-7 (c), and MD-MBA-231 (d) cells 8 h after injection of LAAM TC-CQDs.

[0075] Fig.31 : NIR FL imaging of nude mice bearing A549 tumors (left) and HeLa tumors (right) 10 hours after intravenous injection of LAAM TC-CQDs.

[0076] Fig.32 : Ex vivo NIR FL imaging of major organs (heart, liver, spleen, lung, and kidney) and tumors of A549 tumor (left) and HeLa tumor (right) bearing nude mice 8 hours after injection of LAAM TC-CQDs.

[0077] Fig.33 : LCSM images of HeLa and CCC-ESF-1 cells treated with G-CQDs, Y-CQDs, B-CQDs, and B,S-CQDs, respectively. Scale bar is 100 μm. Note: G-CQDs refers to green fluorescent CQDs synthesized by thermal decomposition of citric acid; Y-CQDs refers to yellow fluorescent CQDs generated by electrolysis of graphite under alkaline conditions and reduction with hydrazine at room temperature; B-CQDs refers to boron (B)-doped CQDs synthesized by electrolysis of graphite in borax aqueous solution; B,S-CQDs refers to boron (B) and sulfur (S) co-doped CQDs synthesized by electrolysis of graphite rods in acetonitrile solution with sodium p-toluenesulfonate (TsONa).

[0078] Fig.34 : Uptake results of G-CQDs, Y-CQDs, B-CQDs and B,S-CQDs in HeLa and CCC-ESF-1 cells.

[0079] Fig.35 : Three-dimensional reconstruction of the distribution of G-CQDs in the tumor 8 hours after injection of G-CQDs.

[0080] Fig.36 : Ex vivo NIR FL imaging of major organs (heart, liver, spleen, lung, and kidney) and tumors 8 hours after G-CQDs injection (a). Semi-quantitative biodistribution of G-CQDs in tumor-bearing mice determined by the average FL intensity of major organs and tumors 8 hours after G-CQDs injection (b).

[0081] Fig.37: NIR FL imaging of nude mice bearing A549 tumors (left) and HeLa tumors (right) 10 hours after intravenous injection of FA-G-CQDs.

[0082] Fig.38 : Ex vivo NIR FL imaging of major organs (heart, liver, spleen, lung, and kidney) and tumors 8 hours after injection of FA-G-CQDs with surface-bound folate (FA).

[0083] Fig.39 : Uptake results of LAAM TC-CQDs (control), Leu, Phe, Gly and BCH pretreated LAAM TC-CQDs in HeLa, A549, PANC-1, MCF-7, MDA-MB-231 and CSCs cells.

[0084] Fig.40 : The uptake results of LAAM TC-CQDs in wild-type HeLa cells, sgLAT1-1 and sgLAT1-2 HeLa cells, respectively.

[0085] Fig.41 : WB test measured the relative expression of LAT1 in different cell types. For cancer cells, from left to right are HeLa, A549, PANC-1, MCF-7, MDA-MB-231, MKN-45, A498, HepG2, HTB-9, EC109, PC-3, SK-MEL-1, NCI-H1975, T84, CAL-27, SH-SY5Y, Hep-2, H460, Jurkat, and breast stem cells; for normal cells, from left to right are CCC-ESF-1, HUVEC, CCD-1095SK, HL-7702, RWPE-1, HaCaT, Hs578Bst, MCF 10A, BEAS-2B, MRC-5, WISH, HKC, 1301, HLF-a, CCC-HEK-1, CCC-HPE-2, CCC-HIE-2, and HBMSCs cells.

[0086] Fig.42 : UV-visible absorption spectra of LAAM TC-CQDs, TPTC and TPTC / LAAM TC-CQDs (a), TC-CQDs, DOX and DOX / LAAMTC-CQDs (b), and LAAM TC-CQDs, HCPT and HCPT / LAAM TC-CQDs aqueous solutions (c).

[0087] Fig.43 : FL emission spectrum of TPTC.

[0088] Fig.44 : Average FL intensity of TPTC and TPTC / LAAM TC-CQDs in the nucleus calculated using Image-Pro Plus 6.0 software.

[0089] Fig.45 : Cell viability of HeLa, A549, PANC-1, MCF-7, and MDA-MB-231 cells treated with LAAM TC-CQDs, TPTC, or TPTC / LAAM TC-CQDs.

[0090] Fig.46 : NIR FL imaging of HeLa tumor-bearing nude mice 10 hours after intravenous injection of TPTC or TPTC / LAAM TC-CQDs.

[0091] Fig.47 : Ex vivo NIR FL imaging of major organs (heart, liver, spleen, lung, and kidney) and tumors in HeLa tumor-bearing nude mice within 8 hours after intravenous injection of TPTC or TPTC / LAAM TC-CQDs.

[0092] Fig.48 : Relative survival rate of mice after treatment with different conditions.

[0093] Fig.49 : Representative images of nude mice bearing A549 tumors (left) and HeLa tumors (right) in different groups at different days after treatment.

[0094] Fig.50 : Growth curves (relative volume over days) of A549 tumors (a) and HeLa tumors (b) in mice treated with saline, TPTC, or TPTC / LAAM TC-CQDs.

[0095] Fig.51 : Body weights measured during the 15-day evaluation period of mice treated with saline, TPTC, or TPTC / LAAM TC-CQDs.

[0096] Fig.52 : Histological evaluation of major organs of mice treated with saline, TPTC, or TPTC / LAAM TC-CQDs. Each organ section has been stained with H&E. Scale bar is 200 μm.

[0097] Fig.53: Whole blood panel data from healthy controls and treated mice, including WBC (a), RBC (b), HGB (c), HCT (d), MCV (e), MCH (f), MCHC (g), PLT (h), RDW (i), PDW (j), MPV (k), and PCT (l). All indices of blood analysis were within normal ranges. No significant differences in all blood test data were noted between the control and treatment groups. Error bars are based on 5 mice per group.

[0098] Fig.54 : Serum biochemical parameters measurement. Three indices, including ALT, AST, and ALP for liver function (a), and two additional indices for renal function, UREA (b) and CREA (c), were evaluated by blood samples from healthy control and treated mice.

[0099] Fig.55 : Preparation process of NH2-free LAAM TC-CQDs and COOH-free LAAM TC-CQDs.

[0100] Fig.56 : FL emission spectra of LAAM TC-CQDs without NH2 (a) and LAAM TC-CQDs without COOH (b). Normalized FL emission spectra of LAAM TC-CQDs (λex = 600 nm), LAAM TC-CQDs without NH2 (ex = 580 nm), and LAAM TC-CQDs without COOH (ex = 600 nm) (c).

[0101] Fig.57 : FT-IR spectra of LAAM TC-CQDs without NH2 (a) and LAAM TC-CQDs without COOH (b).

[0102] Fig.58 : Uptake results of LAAM TC-CQDs, LAAM TC-CQDs without NH2, and LAAM TC-CQDs without COOH in HeLa and CCC-ESF-1 cells obtained by flow cytometry analysis.

[0103] Fig.59 : 1,4-CQDs (a), 1,5-CQDs (b), and 2,6-CQDs (c) prepared by hydrothermal treatment of CA and 1,4-DAAQ, 1,5-DAAQ, and 2,6-DAAQ, respectively.

[0104] Fig.60: TEM images of 1,4-CQDs (a), 1,5-CQDs (b), and 2,6-CQDs (c). AFM images of 1,4-CQDs (d), 1,5-CQDs (e), and 2,6-CQDs (f) on silicon substrates. The insets in Figures df are height profiles along the indicated lines.

[0105] Fig.61 : Raman (a), XRD (b) and FT-IR spectra of 1,4-CQDs, 1,5-CQDs and 2,6-CQDs.

[0106] Fig.62 : XPS full spectra (a), C1s (b, c, d) and N1s (e, f, g) spectra of 1,4-CQDs, 1,5-CQDs and 2,6-CQDs.

[0107] Fig.63 : UV-visible absorption spectra of 1,4-CQDs, 1,5-CQDs and 2,6-CQDs (a), and FL spectra of 1,4-CQDs (b), 1,5-CQDs (c) and 2,6-CQDs (d).

[0108] Fig.64 : Optimized electron delocalized molecular orbital (MO) maps of one fluorescent unit of 1,4-CQDs (a), 1,5-CQDs (b), and 2,6-CQDs (c) obtained from density functional theory calculations (B3LYP / 6-31G(d, p)).

[0109] Fig.65 : Uptake results of LAAM TC-CQDs, 1,4-CQDs, 1,5-CQDs, and 2,6-CQDs in HeLa and CCC-ESF-1 cells obtained by flow cytometry analysis.

[0110] Fig.66 : Phe-CQDs were prepared by a solvothermal method using Phe and ethanol as precursors.

[0111] Fig.67 : TEM (a) and HRTEM (b) images of Phe-CQDs.

[0112] Fig.68 : FT-IR (a), XPS full spectrum (b), C1s (c), N1s (c) and O1s (e) spectra of Phe-CQDs.

[0113] Fig.69 : UV-visible absorption (a) and FL emission (b) spectra of Phe-CQDs.

[0114] Fig.70 : The uptake results of LAAM TC-CQDs and Phe-CQDs in HeLa and CCC-ESF-1 cells were obtained by flow cytometry analysis.

[0115] Fig.71 a: Cell survival rate of HeLa and CCC-ESF-1 cells after treatment with LAAM TC-CQDs, TPTC, or TPTC / LAAM TC-CQDs.

[0116] Fig.71 b: Serum concentration of TPTC over time in HeLa tumor-bearing mice when delivered as free drug or together with TPTC / LAAM TC-CQDs (n=5). Data are expressed as percentage of the total injected dose (%ID).

[0117] Fig.71 c: Biodistribution of TPTC and TPTC / LAAM TC-CQDs in the indicated organs isolated from HeLa tumor-bearing mice (n=5).

[0118] Fig.71 d: Changes in tumor volume over time in HeLa tumor-bearing mice after receiving the indicated treatments (n=5).

[0119] Fig.71 e: Cell survival rate of A549 cells after treatment with LAAM TC-CQDs, TPTC, or TPTC / LAAM TC-CQDs.

[0120] Fig.71 f: Changes in serum concentrations of TPTC over time in A549 tumor-bearing mice when delivered as free drug or together with TPTC / LAAM TC-CQDs (n=5).

[0121] Fig.71 g: Biodistribution of TPTC and TPTC / LAAM TC-CQDs in the indicated organs isolated from A549 tumor-bearing mice (n=5).

[0122] Fig.71 h: Changes in tumor volume over time in A549 tumor-bearing mice receiving indicated treatments (n=5).

[0123] Fig.72 : FL emission spectra of LAAM TC-CQDs at selected pH values ​​(a). FL emission spectra of LAAM TC-CQDs in the presence of amino acids (Asp, Glu, Met, Phe, Tyr and Gly) or MMP-2 (b).

[0124] Fig.73 : Photostability of LAAM TC-CQDs after long-term storage or irradiation. PL spectra of freshly prepared LAAM TC-CQDs solution (black curve) and LAAM TC-CQDs stored at room temperature for three months (grey curve) (a). Ratio of PL intensity of LAAM TC-CQDs after irradiation at designated time points (F) to that of fresh LAAM TC-CQDs (F0) over time (b).

[0125] Fig.74 : Characterization of the effect of 365 nm UV irradiation for 5 h on LAAM TC-CQDs. FL emission spectra of LAAM TC-CQDs before (black curve) and after (grey curve) UV irradiation (a). UV-visible absorption spectra of LAAM TC-CQDs before (black curve) and after (grey curve) UV irradiation (b) after treatment with ninhydrin. Flow cytometric analysis of cellular uptake of control and UV-irradiated LAAM TC-CQDs in HeLa and CCC-ESF-1 cells (c).

[0126] Fig.75 : Size distribution of fresh LAAM TC-CQDs (a), and LAAM TC-CQDs after incubation in PBS buffer containing 10% FBS for 30 days (b). At the indicated time points (F), the PL intensity ratio of LAAM TC-CQDs after incubation in PBS buffer containing 10% FBS to the PL intensity of fresh LAAM TC-CQDs (F0) over time (c).

[0127] Detailed description

[0128] General Definition

[0129] Unless otherwise defined, the meaning of all technical and scientific terms used herein has the same meaning as that generally understood by those of ordinary skill in the art to which the present invention belongs. All patents, patent applications, published patent applications and other publications mentioned in the present invention are incorporated herein by reference in their entirety. If the definition set forth in this section is contrary to or inconsistent with the definition set forth in the patent, patent application or other publication incorporated herein by reference, the definition set forth in this section takes precedence over the definition incorporated herein by reference.

[0130] As used herein, "a" or "an" means "at least one" or "one or more".

[0131] As used herein, the term "alkyl" refers to a saturated hydrocarbon group in a linear, branched or cyclic configuration or any combination thereof, and specifically contemplates alkyl groups having ten or fewer carbon atoms, especially alkyl groups having 1-6 carbon atoms and lower alkyl groups having 1-4 carbon atoms. Exemplary alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, cyclopropylmethyl, and the like.

[0132] Alkyl groups may be unsubstituted or they may be substituted to such extent that it makes sense chemically. Typical substituents include, but are not limited to, halogen, =O, =N-CN, =N-OR a , =NR a 、-OR a 、-NR a 2. -SR a 、-SO2R a 、-SO2NR a 2. -NR a S02R a 、-NR a CONR a 2. -NR a COOR a 、-NR a COR a 、-CN、-COOR a 、-CONR a 2.-OOCR a 、-COR a and -NO2, where each R a are independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C3-C8 heterocyclyl, C4-C 10 Heterocycloalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C 10 Aryl or C5-C 10 heteroaryl, and each R a Optionally halogen, =O, =N-CN, =N-OR b , =NR b , OR b NR b 2. SR b 、SO2R b 、SO2NR b 2. NR b S02R b NR b CONR b 2. NR b COOR b NRb COR b 、CN、COOR b ,CONR b 2. OOCR b , COR b and NO2, where each R b are independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C3-C8 heterocyclyl, C4-C 10 Heterocycloalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl or C5-C 10 Heteroaryl. Alkyl, alkenyl and alkynyl may also be replaced by C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl or C5-C 10 Heteroaryl groups are substituted, each of which may be substituted with substituents appropriate for the particular group. If a substituent contains two R a or R b Groups (e.g., -NR b 2 or -NR b -C(O)R b ), then these two R a or R b The group may be optionally joined together with the atoms in the substituent to form a 5-8 membered ring, which may be a or R b The moiety itself is substituted where permitted and may contain one additional heteroatom (N, O or S) as a ring atom.

[0133] As used herein, the term "cycloalkyl" refers to a cyclic alkane (i.e., a chain of carbon atoms of a hydrocarbon forming a ring), preferably including 3-8 carbon atoms. Therefore, exemplary cycloalkanes include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. The cycloalkyl also includes one or two double bonds, thereby forming a "cycloalkenyl" group. The cycloalkyl can be optionally substituted with a group suitable for alkyl substitution as described herein.

[0134] As used herein, the term "aryl" or "aromatic moiety" refers to an aromatic ring system that may further include one or more non-carbon atoms. These are typically 5-6-membered isolated rings or 8-10-membered bicyclic groups and may be substituted. Thus, the aryl groups considered include (e.g., phenyl, naphthyl, etc.) and pyridyl, and the pyridone ring is considered to be aromatic as discussed herein. Further considered aryl groups may be fused to one or two 5- or 6-membered aryl or heterocyclic groups (i.e., covalently bonded to 2 atoms on the first aromatic ring), and are therefore referred to as "fused aryl" or "fused aromatic".

[0135] As used herein, the term "partially aromatic" refers to a ring that is fused to an aromatic ring and shares at least one bond. Partially aromatic fused ring systems are those in which each ring is aromatic or partially aromatic.

[0136] As used herein, the term "partially unsaturated" refers to a ring containing at least one double bond within the ring, but the ring is not aromatic. Note that a partially aromatic ring shares one aromatic bond and is considered partially unsaturated and also partially aromatic due to the aromatic ring.

[0137] Aryl groups containing one or more heteroatoms (usually N, O or S) as ring atoms may be referred to as heteroaryl or aromatic heterocyclic groups. Typical heteroaryl groups include monocyclic C5-C6 aryl groups such as pyridyl, pyrimidinyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl and imidazolyl, as well as fused bicyclic groups, which are formed by condensing one of these monocyclic groups with a benzene ring or any heterocyclic monocyclic group to form an 8-10 membered bicyclic group, such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridinyl, pyrazolopyrimidinyl, quinazolinyl, quinoxalinyl, cinnolinyl, etc. Any monocyclic or fused bicyclic ring system having aromatic characteristics in terms of the electron distribution of the entire ring system is included in this definition. It also includes bicyclic groups in which at least the ring directly attached to the rest of the molecule has aromatic character. Typically, the ring system contains 5-12 ring atoms.

[0138] As also used herein, the terms "heterocycle" and "heterocyclic" refer to any compound in which a plurality of atoms form a ring through a plurality of covalent bonds, wherein the ring includes at least one atom other than a carbon atom as a ring atom. Particularly contemplated heterocycles include 5- and 6-membered rings (e.g., imidazole, pyrrole, triazole, dihydropyrimidine, indole, pyridine, thiazole, tetrazole, etc.) with nitrogen, sulfur or oxygen as non-carbon atoms. Typically, these rings contain 0-1 oxygen or sulfur atoms, at least one and typically 2-3 carbon atoms, and up to 4 nitrogen atoms as ring atoms. Further contemplated heterocycles may be fused to one or two carbocyclic or heterocyclic rings (i.e., covalently bonded to two atoms on the first heterocyclic ring), and are therefore referred to as "fused heterocycles" or "fused heterocyclic" or "fused heterocyclic groups" as used herein. If the ring is aromatic, these may be referred to as "heteroaryls" or "aromatic heterocyclic groups" herein.

[0139] Non-aromatic heterocyclic groups may be substituted with the groups described herein as suitable for alkyl substitution.

[0140] Aryl and heteroaryl groups may be substituted where permitted. Suitable substituents include, but are not limited to, halogen, -OR a 、-NRa 2. -SR a 、-SO2R a 、-SO2NR a 2. -NR a S02R a 、-NR a CONR a 2. -NR a COOR a 、-NR a COR a 、-CN、-COOR a 、-CONR a 2.-OOCR a 、-COR a and -NO2, where each R a are independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C3-C8 heterocyclyl, C4-C 10 Heterocycloalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C 10 Aryl or C5-C 10 heteroaryl, and each R a Optionally halogen, =O, =N-CN, =N-OR b , =NR b , OR b NR b 2. SR b 、SO2R b 、SO2NR b 2. NR b S02R b NR b CONR b 2. NR b COOR b NR b COR b 、CN、COOR b ,CONR b 2. OOCR b , COR b and NO2, where each R b are independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C3-C8 heterocyclyl, C4-C 10 Heterocycloalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 Aryl or C5-C 10 Heteroaryl. Alkyl, alkenyl and alkynyl may also be replaced by C1-C8 acyl, C2-C8 heteroacyl, C6-C 10Aryl or C5-C 10 Heteroaryl groups are substituted, each of which may be substituted with substituents appropriate for the particular group. If a substituent contains two R a or R b Groups (e.g., -NR b 2 or -NR b -C(O)R b ), then these two R a or R b The group may be optionally joined together with the atoms in the substituent to form a 5-8 membered ring, which may be a or R b The moiety itself is substituted where permitted and may contain one additional heteroatom (N, O or S) as a ring atom.

[0141] As used herein, the term "alkoxy" refers to a hydrocarbon group connected by an oxygen atom, such as -O-Hc, wherein the hydrocarbon portion Hc may have any number of carbon atoms, typically 1-10 carbon atoms, may further include double or triple bonds, and may contain one or two oxygen, sulfur or nitrogen atoms in the alkyl chain, and may be substituted with aryl, heteroaryl, cycloalkyl and / or heterocyclic groups. For example, suitable alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, methoxyethoxy, benzyloxy, allyloxy, and the like. Similarly, the term "alkylthio" refers to an alkyl sulfide of the general formula -S-Hc, wherein the hydrocarbon portion Hc is as described for the alkoxy portion. For example, alkylthio groups contemplated include methylthio, ethylthio, isopropylthio, methoxyethylthio, benzylthio, allylthio, and the like.

[0142] As used herein, the term "amino" refers to an -NH2 group. The term "alkylamino" refers to an amino group in which one or two hydrogen atoms are replaced by a hydrocarbon group Hc as described above, wherein the amino nitrogen "N" may be substituted by one or two hydrocarbon groups Hc as described above for the alkoxy group. Exemplary alkylamino groups include methylamino, dimethylamino, ethylamino, diethylamino, and the like. In addition, the term "substituted amino" refers to an amino group in which one or two hydrogen atoms are replaced by a hydrocarbon group Hc as described above, wherein the amino nitrogen "N" may be substituted by one or two hydrocarbon groups Hc as described above for the alkoxy group.

[0143] As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine. When present as a substituent, halogen or halo typically refers to F or Cl or Br, more typically to F or Cl.

[0144] The term "haloalkyl" refers to an alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been replaced by a halo group. Examples of such groups include, but are not limited to, fluoroalkyl groups, such as fluoroethyl, trifluoromethyl, difluoromethyl, trifluoroethyl, etc.

[0145] The term "haloalkoxy" refers to an alkyl-O- group in which one or more hydrogen atoms on the alkyl group have been replaced by a halo group, including, for example, groups such as trifluoromethoxy and the like.

[0146] It should be further recognized that all the above defined groups may be further substituted by one or more substituents, which may be further substituted in turn by hydroxyl, amino, cyano, C1-C4 alkyl, halogen or C1-C4 haloalkyl. For example, the hydrogen atoms in alkyl or aryl may be substituted by amino, halogen or C1-C4 haloalkyl or alkyl.

[0147] As used herein, the term "substitution" refers to the replacement of a hydrogen atom on an unsubstituted group with a functional group, and functional groups specifically contemplated include nucleophilic groups (e.g., -NH2, -OH, -SH, -CN, etc.), electrophilic groups (e.g., C(O) or C(X)OH, etc.), polar groups (e.g., -OH), non-polar groups (e.g., heterocyclic, aryl, alkyl, alkenyl, alkynyl, etc.), ionic groups (e.g., -NH3 + ) and halogen (e.g., -F, -Cl), NHCOR, NHCONH2, OCH2COOH, OCH2CONH2, OCH2CONHR, NHCH2COOH, NHCH2CONH2, NHSO2R, OCH2-heterocycle, PO3H, SO3H, amino acids, and all chemically reasonable combinations thereof. In addition, the term "substituted" also includes multiple degrees of substitution, and in the case of disclosing or claiming multiple substituents, the substituted compounds can be independently substituted with one or more disclosed or claimed substituents.

[0148] In particular embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent, in addition to what is disclosed herein.

[0149] It is understood that, in all of the above-defined substituent groups, compounds obtained by defining a substituent having further substituents on itself (e.g., a substituted aryl having as a substituent a substituted aryl, which itself is substituted by a substituted aryl, which is further substituted by a substituted aryl, etc.) are not intended to be included herein. In such cases, the maximum number of such substituents is three. For example, serial substitutions of substituted aryl specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl.

[0150] Unless otherwise indicated, nomenclature of substituents not explicitly defined herein is by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to (aryl)-(alkyl)-OC(O)-.

[0151] For any group containing one or more substituents described herein, it is of course understood that such groups do not contain any substitutions or substitution patterns that are sterically impractical and / or synthetically unfeasible. In addition, the target compounds include all stereochemical isomers resulting from substitution of these compounds.

[0152] The term "pharmaceutically acceptable salt" refers to salts that are acceptable for administration to a patient (e.g., a mammal, such as a human) (counter ion salts with acceptable mammalian safety for a given dosage regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of a compound, which are derived from various organic and inorganic counterions well known in the art, including and by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formates, tartrates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, and the like.

[0153] The compounds and CQDs of the present invention have at least one α-aminocarboxylic acid moiety and are therefore capable of forming salts by protonation (acid addition) or deprotonation; in addition, they can exist as zwitterions, such as amino acid compounds known in the art. It should be understood that for convenience, unless otherwise specified, each time a compound of the present invention and a CQD of the present invention are mentioned, the substance can be prepared, isolated and used as a pharmaceutically acceptable salt or zwitterion in any suitable protonation state. In use or solution, the protonation state depends largely on the pH value of its environment, and the interconversion of protonation states is well understood. In addition, if the compounds or CQDs of the present invention have more than one α-amino acid carboxylic acid moiety, depending on the influence of the environment, each α-amino acid moiety can be any of these forms, so the compound can contain an internal mixture of salts and / or zwitterionic moieties.

[0154] The term "salt" refers to a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation, or a basic group, such as a nitrogen atom (especially sp 3 Hybridized nitrogen) accepts a proton to form a positively charged group (e.g., ammonium), in which case the positively charged compound is accompanied by an anionic counterion, such as a halide anion. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts of intermediate compounds that are not intended for administration to patients. For example, salts of compounds of the invention include portions in which the compound is protonated by an inorganic or organic acid to form a cation, and the conjugate base of the inorganic or organic acid is the anionic component of the salt.

[0155] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid polymers of any length, e.g., at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, or more amino acids. The polymer may be linear or branched, it may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component. Also included in the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.) and other modifications known in the art.

[0156] As used herein, the term "variant" is used to refer to a polypeptide having a certain degree of amino acid sequence identity with a parent polypeptide sequence. Variants are similar to the parent sequence, but have at least one substitution, deletion or insertion in their amino acid sequence, making them different in sequence from the parent polypeptide. In addition, the variant may retain the functional characteristics of the parent polypeptide, for example, maintaining at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the biological activity of the parent polypeptide.

[0157] "Antibody" is an immunoglobulin molecule that can specifically bind to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and can be any class of immunoglobulins, such as IgG, IgM, IgA, IgD and IgE. IgY is the main antibody type of poultry such as chickens and is also included in the definition. As used herein, the term includes not only complete polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chains (ScFv), mutants thereof, naturally occurring variants, fusion proteins comprising antibody portions having antigen recognition sites with desired specificity, humanized antibodies, chimeric antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites with desired specificity.

[0158] As used herein, the term "antigen" refers to a target molecule that specifically binds to an antibody through its antigen recognition site. An antigen may be monovalent or multivalent, i.e., it may have one or more epitopes recognized by one or more antibodies. Antigens that can be recognized by antibodies include polypeptides, oligosaccharides, glycoproteins, polynucleotides, lipids, and the like.

[0159] As used herein, the term "epitope" refers to a portion of an antigen, such as a peptide sequence of at least about 3 to 5, preferably about 5 to 10 or 15 and no more than about 1000 amino acids (or any integer therebetween), which defines a sequence alone or as part of a larger sequence, and an antibody produced in response to the sequence binds. There is no critical upper limit to the length of the fragment, for example, the fragment can almost contain the entire length of the antigen sequence, or even contain a fusion protein of two or more epitopes from the target antigen. The epitopes used in the present invention are not limited to peptides with the exact sequence derived from their parent protein portion, but also include sequences identical to the native sequence and modifications to the native sequence, such as deletions, additions and substitutions (conservative properties).

[0160] As used herein, the term "specific binding" refers to the binding specificity of a particular binding pair. The recognition of a specific target by an antibody in the presence of other potential targets is a characteristic of such binding. Specific binding involves two different molecules, one of which specifically binds to the second molecule by chemical or physical means. The connection between the two molecules is that the binding between them enables them to distinguish their binding partners from other components with similar characteristics. The members of the binding component pair are called ligands and receptors (counter-ligands), specific binding pairs (SBP) members, and SBP partners, etc. A molecule can also be a SBP member of a molecular aggregate; for example, an antibody produced against an immune complex of a second antibody and its corresponding antigen can be considered to be a SBP member of the immune complex.

[0161] "Polynucleotide" or "nucleic acid", used interchangeably herein, refers to nucleotide polymers of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogs. If present, the structure of the nucleotides may be modified before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by binding to a labeling component. Other types of modifications include, for example, "capping", replacement of one or more naturally occurring nucleotides with analogs, internucleotide modifications, for example, with uncharged bonds (e.g., methyl phosphoesters, phosphotriesters, phosphoamides, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), containing side chain moieties, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), with intercalating agents (e.g., acridine, psoralen, etc.), containing chelating agents (e.g., metals, radioactive metals, boron, oxidized metals, etc.), containing alkylating agents, with modified bonds (e.g., α-heteromeric nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any hydroxyl groups normally present in sugars can be replaced by, for example, phosphonic acid groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional links with additional nucleotides, or can be attached to a solid support. The 5' and 3' terminal OH can be phosphorylated or replaced by amines or by organic capping groups of 1 to 20 carbon atoms. Other hydroxyls can also be derived into standard protecting groups. Polynucleotides can also include ribose or deoxyribose sugars of similar forms generally known in the art, including, for example, 2'-O-methyl-2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-isopolysaccharides, epimeric sugars, such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs and abasic nucleoside analogs, such as methyl nucleosides. One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate group is replaced by P(O)S ("thioester"), P(S)S ("dithioester"), "(O)NR2 ("amide"), P(O)R, P(O)OR', CO or CH2 ("acetal"), wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether bond (-O-), aryl, alkenyl, cycloalkyl, cycloalkenyl or arylaldehyde. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0162] As used herein, "oligonucleotide" generally refers to a short, usually single-stranded, usually synthetic polynucleotide, which is usually, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is also fully applicable to oligonucleotides.

[0163] As used herein, the term "homolog" refers to a nucleic acid that differs from a naturally occurring nucleic acid (e.g., a "prototype" or "wild-type" nucleic acid) by making minor modifications to the naturally occurring nucleic acid, but retains the basic nucleotide structure of the naturally occurring form. Such changes include, but are not limited to, changes in one or several nucleotides, including deletions (e.g., truncations of nucleic acids), insertions and / or substitutions. Homologs may have enhanced, reduced, or substantially similar properties compared to naturally occurring nucleic acids. Homologs may be complementary or matching to naturally occurring nucleic acids. Homologs may be produced using techniques known in the art for producing nucleic acids, including, but not limited to, recombinant DNA technology, chemical synthesis, and the like.

[0164] As used herein, "substantially complementary or substantially matching" means that two nucleic acid sequences have at least 90% sequence identity. Preferably, two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Alternatively, "substantially complementary or substantially matching" means that two nucleic acid sequences can hybridize under high stringency conditions.

[0165] In general, the stability of the hybrid is a function of ion concentration and temperature. Typically, the hybridization reaction is carried out under conditions of lower stringency, followed by washing of different but higher stringency. Moderately stringent hybridization refers to conditions that allow nucleic acid molecules (such as probes) to bind to complementary nucleic acid molecules. Hybridized nucleic acid molecules typically have at least 60% identity, including, for example, at least any one of 70%, 75%, 80%, 85%, 90% or 95% identity. Moderately stringent conditions are equivalent to hybridization under conditions of 50% formamide, 5× Denhardt's solution, 5× SSPE, 0.2% sodium dodecyl sulfate at 42°C, followed by washing in 0.2× SSPE, 0.2% sodium dodecyl sulfate at 42°C. High stringency conditions can be provided, for example, by hybridization under conditions of 50% formamide, 5× Denhardt's solution, 5× SSPE, 0.2% sodium dodecyl sulfate at 42°C, followed by washing in 0.1× SSPE, 0.1% sodium dodecyl sulfate at 65°C. Low stringency hybridization refers to hybridization under conditions equivalent to 10% formamide, 5× Denhardt's solution, 6× SSPE, 0.2% sodium dodecyl sulfate at 22°C, followed by washing in 1× SSPE, 0.2% sodium dodecyl sulfate at 37°C. Denhardt's solution contains 1% Ficoll, 1% polyvinylpyrrolidone, and 1% bovine serum albumin (BSA). 20× SSPE (sodium chloride, sodium phosphate, ethylenediaminetetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M (EDTA). Other suitable moderate stringency and high stringency hybridization buffers and hybridization conditions are well known to those skilled in the art.

[0166] As used herein, the term "RNA interference" or "RNAi" generally refers to a process in which a double-stranded RNA molecule or a short hairpin RNA molecule reduces or inhibits the expression of a nucleic acid sequence, wherein the double-stranded or short hairpin RNA molecule has most or all homology with the nucleic acid sequence. The term "short interfering RNA" or "siRNA" or "RNAi agent" refers to an RNA (or RNA analog) sequence containing about 10-50 nucleotides (or nucleotide analogs) that triggers RNA interference. See Kreutzer et al., WO 00 / 44895; Zernicka-Goetz et al., WO 01 / 36646; Fire, WO 99 / 32619; Mello & Fire, WO 01 / 29058. As used herein, siRNA molecules include RNA molecules containing chemically modified nucleotides and non-nucleotides. The term "ddRNAi agent" refers to a DNA-guided RNAi agent transcribed from an exogenous vector. The term "short hairpin RNA" or "shRNA" refers to an RNA structure having a double-stranded region and a loop region. In certain embodiments, ddRNAi agents are initially expressed as shRNAs.

[0167] "Treatment" or "therapy" or "relieve" refers to a therapeutic treatment whose purpose is to slow down (reduce) if it cannot cure the target pathological condition or disorder or prevent recurrence of the disease. If the subject shows a reduction or absence of one or more observable and / or measurable signs and symptoms of a specific disease after receiving a therapeutic dose of a therapeutic agent or treatment, the subject is successfully "treated". The patient may also feel a reduction in disease signs or symptoms. If the patient's condition is stable, it may also be considered for treatment. In some embodiments, treatment with a therapeutic agent is effective in making the patient disease-free for 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years or more after treatment. These parameters for evaluating successful treatment and improvement of the disease are easily measured by routine procedures familiar to doctors with appropriate skills in the field. In some embodiments, "therapy" refers to any way to improve or otherwise beneficially change the symptoms of a condition, disorder or disease. Therapy also includes any pharmaceutical use of the compositions described herein. In some embodiments, "improvement" of a specific disorder by administering a specific pharmaceutical composition refers to any relief attributable to or associated with the administration of the composition, whether permanent or temporary, lasting or temporary.

[0168] As used herein, the term "prediction" or "prognosis" refers to the possibility that a patient will have a favorable or unfavorable reaction to a drug or a group of drugs, or the possible outcome of a disease. In one embodiment, the prediction is related to the degree of those responses or outcomes. In one embodiment, the prediction relates to whether the patient survives or improves after treatment (e.g., using a specific therapeutic agent for treatment), and there is no probability of disease recurrence within a certain period of time. The prediction method of the present invention can be used clinically to make treatment decisions by selecting the most appropriate treatment mode for any particular patient. The prediction method of the present invention is a valuable tool for predicting whether a patient may have a favorable response to a treatment regimen, such as a given treatment regimen, including, for example, the administration of a given therapeutic agent or combination, surgical intervention, steroid therapy, etc.

[0169] As used herein, "produced by recombinant means" refers to production methods using recombinant nucleic acid methods that rely on well-known molecular biology methods to express proteins encoded by cloned nucleic acids.

[0170] It should be understood that the aspects and embodiments of the present invention described herein include "comprising" and / or "consisting essentially of" aspects and embodiments.

[0171] Throughout the present invention, various aspects of the present invention are presented in range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be interpreted as an inflexible limitation to the scope of the present invention. Therefore, it should be considered that the description of the range has specifically disclosed all possible sub-ranges and the single numerical values ​​in the range. For example, the description of a range such as from 1 to 6 should be considered as having a specifically disclosed sub-range, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the single numerical values ​​in the range, for example, 1, 2, 3, 4, 5 and 6. This applies to the width of any range.

[0172] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. For example, see Remington, "Pharmacy Science and Practice", 20th edition, (Lippincott, Williams & Wilkins, 2003). Unless any conventional media or agents are incompatible with the active compound or substance, their use in the composition can be considered.

[0173] As used herein, the term "therapeutically effective dose" or "effective dose" refers to a therapeutic dose that is effective in preventing or ameliorating a disease or disorder in a subject when administered to a cell, tissue, or subject alone or in combination with an additional therapeutic agent. A therapeutically effective dose also refers to an amount of a therapeutic agent sufficient to cause symptomatic improvement, for example, treatment, healing, prevention, or improvement of a related medical condition, or to increase the rate of treatment, healing, prevention, or improvement of such a condition. When applied to the separate administration of a single active ingredient, a therapeutically effective dose refers to the administration of that ingredient alone. When applied to a composition, a therapeutically effective dose refers to the total amount of active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously. In some embodiments, an "effective dose of a compound or substance for treating a specific disease" refers to a dose that is sufficient to improve or in some way reduce the symptoms associated with the disease. The dose may be administered as a single dose, or may be administered according to a regimen by virtue of which it is effective. The dose may cure the disease, but is typically administered to improve the symptoms of the disease. Repeated administration may be required to achieve the desired symptom improvement.

[0174] As used herein, "prodrug" is a compound that is metabolized or otherwise converted into a biological, pharmacological or therapeutically active form of the compound after administration in vivo. In order to produce a prodrug, the pharmaceutically active compound is modified so that the active compound is regenerated by a metabolic process. A prodrug can be designed to change the metabolic stability or transport characteristics of a drug, mask side effects or toxicity, improve the taste of a drug, or change other characteristics or properties of a drug. With the knowledge of pharmacodynamic processes and drug metabolism in vivo, once a person skilled in the art knows a pharmaceutically active compound, a prodrug of the compound can be designed (e.g., see Nogrady (1985) "Medicinal Chemistry-Biochemical Methods", Oxford University Press, New York, pp. 388-392).

[0175] The compounds and compositions described herein can be administered to a subject in need of treatment for a cell proliferation disorder, such as cancer, particularly a cancer selected from leukemia, lymphoma, lung cancer, colon cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, head and neck cancer, and pancreatic cancer. The subject is typically a mammal diagnosed as needing treatment for one or more of such proliferative disorders, and the subject is typically a human. The method comprises administering an effective dose of at least one compound of the invention; optionally, the compound can be administered in combination with one or more additional therapeutic agents, particularly therapeutic agents known to be useful for treating cancer or proliferative disorders afflicting a particular subject.

[0176] The carbon quantum dots of the present invention are particles with suitable size and electronic properties used as quantum dots, and are composed of at least 20% by weight of carbon, preferably at least 50% by weight of carbon. When a CQDs is described as "comprising" a compound of a specific structure, it should be understood that these carbon quantum dots are not necessarily composed of a single pure compound. CQDs particles may contain other materials in the carbon core, and the carbon core may produce or affect the properties of the quantum dots (fluorescence, photoacoustics); but the particles still contain specific compounds, and their valuable biological properties (binding to LAT1, internalization into cells, selectivity for tumors or cells that specifically express LAT1 relative to other cells) can be attributed to the specified chemical structure, as shown in the data shown herein, which shows that modifications to the specific chemical structures of the CQDs of the embodiments and claims lead to reductions in these valuable biological properties. Therefore, for example, the CQDs described and claimed herein with respect to specific chemical structures are particles containing at least one biologically relevant amount of a specific chemical structure, and may contain a specific chemical structure of 5% or 10% by weight. Of course, CQDs may also include or substantially include the specified compounds in the specific embodiments or claims. Similarly, the methods described herein may produce compositions containing at least 5% or at least 10% (by weight) of the compounds contained in the products. These products may be obtained as particles or may be capable of forming particles having the properties of carbon quantum dots, but it is also understood that the compounds and compositions of the present invention, including products prepared by the methods described and claimed, and particles containing these compounds and compositions are expected to exhibit useful affinity for LAT1 and to be selectively internalized into LAT1-expressing cells by LAT1, even if their size is not sufficient to function as CQDs. Therefore, these compounds and compositions can be used to internalize markers or therapeutic agents into cells expressing LAT1, thereby showing valuable pharmacological, imaging and therapeutic uses; therefore, if the material herein is not explicitly described as a carbon quantum dot, the present invention includes the compounds, compositions and products described herein, whether or not they are in the form of functional particles having carbon quantum dots.

[0177] The following examples are representative of some aspects of the present invention.

[0178] 1. A carbon quantum dot, which:

[0179] a) comprising a core structure consisting of a fused polycyclic array of 6-membered rings, each of which is aromatic or unsaturated, wherein the polycyclic array of 6-membered rings is substituted with at least one C1-C3 alkyl substituted with a carboxyl group and an amino group; or

[0180] b) comprises a fused polycyclic ring system containing 6-membered rings, wherein each ring is aromatic or unsaturated, and the polycyclic ring system is substituted by at least one group of chemical formula A:

[0181]

[0182] Where R 1 is H or C1-C3 alkyl optionally substituted by one or two groups selected from halogen, -OH, -OMe, -NH2, -SMe, -COOH and -CONH2, and the dotted bond indicates the position at which the group of formula A is attached to the fused polycyclic system; or

[0183] c) a polycyclic aromatic or partially aromatic ring system comprising at least 10 fused 6-membered rings, wherein the polycyclic aromatic or partially aromatic ring system is fused to at least one subunit of formula B:

[0184]

[0185] in:

[0186] R 1 is H or C1-C3 alkyl optionally substituted by one or two groups selected from halogen, -OH, -OMe, -NH2, -SMe, -COOH and -CONH2,

[0187] Z 1 NR2 or C(R 2 )2, where each R 2 independently selected from H and C1-C3 alkyl, and wherein the dashed bond indicates the position where the chemical formula B is fused to the polycyclic aromatic or partially aromatic ring system; and / or

[0188] d) configured to selectively enter cells expressing the large neutral amino acid transporter (LAT1) or a subunit thereof; and / or

[0189] e) configured to selectively enter tumors or cancer cells, wherein the weight percentage of carbon atoms in the carbon quantum dots is 20% or higher. In some embodiments, the weight percentage of carbon atoms in the carbon quantum dots may be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or any sub-range thereof.

[0190] In some examples of this embodiment, the carbon quantum dot comprises a core structure consisting of a 6-membered ring fused polycyclic array, each of which is aromatic or unsaturated (partially aromatic), wherein the 6-membered ring polycyclic array is substituted by at least one C1-C3 alkyl substituted by a carboxyl group and an amino group; and the 6-membered ring polycyclic array may be optionally substituted by one or more selected from C1-4 Alkyl, phenyl, C 1-4 Alkoxy, halogen, COOR*, -OH, CN and -NR*2, wherein each R* is independently selected from H and C 1-3 Alkyl, where each C 1-3 The alkyl group may be optionally substituted by one to three groups selected from -OH, -COOH, -NH2 and C 1-2 In addition, the C1-C3 alkyl group substituted with at least one carboxyl group and amino group may be optionally further substituted with a C1-C2 group, which may be optionally substituted with one to three selected from -OH, -COOH, -NH2 and C 1-2 Alkyl radicals are substituted.

[0191] In some examples of the first embodiment, the carbon quantum dot comprises a fused polycyclic ring system containing a 6-membered ring, wherein each ring is aromatic or unsaturated, and the polycyclic ring system is substituted by at least one group of chemical formula A:

[0192]

[0193] Where R 1 The C1-C3 alkyl group is H or optionally substituted by one or two groups selected from halogen, -OH, -OMe, -NH2, -SMe, -COOH and -CONH2, and the dotted bond indicates the position where the group of the chemical formula A is connected to the fused polycyclic system. In these compounds and CQDs, the polycyclic system may be optionally substituted by one or more groups selected from C 1-4 Alkyl, phenyl, C 1-4 Alkoxy, halogen, COOR*, -OH, CN and NR*2, wherein each R* is independently selected from H and C 1-3 Alkyl, each C 1-3 The alkyl group may be optionally substituted by one to three groups selected from -OH, -COOH, -NH2 and C 1-2 In addition, the C1-C3 alkyl group substituted with at least one carboxyl group and amino group may be optionally further substituted with a C1-C2 group, which may be optionally substituted with one to three selected from -OH, -COOH, -NH2 and C 1-2 In preferred embodiments of these compounds and CQDs, R 1 It is H, methyl or -CH2COOH.

[0194] In some examples of Example 1, the carbon quantum dot comprises a polycyclic aromatic or partially aromatic ring system of at least 10 fused 6-membered rings, wherein the polycyclic aromatic or partially aromatic ring system is fused to at least one subunit of Chemical Formula B:

[0195]

[0196] in:

[0197] R 1 is H or C1-C3 alkyl optionally substituted by one or two groups selected from halogen, -OH, -OMe, -SMe, -COOH and -CONH2,

[0198] Z 1 NR2 or C(R 2 )2, where each R 2 independently selected from H and C1-C3 alkyl, and wherein the dotted bond indicates the position where the chemical formula B is fused to the polycyclic aromatic system. In these compounds and CQDs, the polycyclic system may be further optionally replaced by one or more selected from C 1-4 Alkyl, phenyl, C 1-4 Alkoxy, halogen, COOR*, -OH, CN and -NR*2, wherein each R* is independently selected from H and C 1-3 Alkyl, where each C 1-3 The alkyl group may be optionally substituted by one to three groups selected from -OH, -COOH, -NH2 and C 1-2 In addition, the C1-C3 alkyl group substituted by at least one carboxyl group and amino group may be optionally further substituted by a C1-C2 group, which may be optionally substituted by one to three selected from -OH, -COOH, NH2 and C 1-2 Alkyl radicals are substituted.

[0199] In some examples of embodiment 1, including the foregoing examples of these embodiments, carbon quantum dots are configured to selectively enter cells expressing large neutral amino acid transporter (LAT1) or its subunits. In these examples, CQDs are preferentially adapted or configured to be selectively taken up into cells with an uptake rate of at least 90%, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0200] In some examples of embodiment 1, including the foregoing examples of these embodiments, the carbon quantum dots are configured to selectively enter tumor or cancer cells. In these examples, the tumor or cancer cells express a transporter that recognizes and binds to the carbon quantum dots.

[0201] 2. The carbon quantum dots according to Example 1 comprise a core structure consisting of a fused polycyclic array of 6-membered rings, each of which is aromatic or unsaturated, wherein the 6-membered polycyclic array is substituted by at least one C1-C3 alkyl substituted by a carboxyl group and an amino group.

[0202] 3. The carbon quantum dots according to embodiment 2, comprising at least 5 fused 6-membered aromatic rings or unsaturated nuclear rings.

[0203] 4. The carbon quantum dots according to Embodiment 2 or 3, wherein the polycyclic array of 6-membered rings is substituted by at least two C1-C3 alkyl groups, wherein each C1-C3 alkyl group is substituted by a carboxyl group and an amino group, and further optionally substituted by one or two groups selected from -OH, methyl, phenyl, -OMe, -NH2, -SMe, -COOH and -CONH2.

[0204] 5. The carbon quantum dot according to any one of embodiments 2-4, wherein the ratio of the number of 6-membered aromatic rings or unsaturated rings to the number of C1-C3 alkyl groups in the core structure is at least 2.

[0205] 6. The carbon quantum dots according to embodiment 5, wherein the ratio of the number of 6-membered aromatic rings or unsaturated rings to the number of C1-C3 alkyl groups in the core structure is about 2 to 70, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70, or any subrange thereof.

[0206] 7. The carbon quantum dot according to embodiment 1, comprising a fused polycyclic ring system containing a 6-membered ring, wherein each ring is aromatic or unsaturated, and the polycyclic ring system is substituted by at least one group of chemical formula A:

[0207]

[0208] Where R 1 is H or substituted by one or two C1-C3 alkyl groups selected from -OH, -OMe, -SMe, -COOH and -CONH2, and the dotted bond indicates the position at which the group of formula (A) is attached to the fused polycyclic system.

[0209] 8. The carbon quantum dots according to embodiment 7, wherein R 1 For H.

[0210] 9. The carbon quantum dots according to embodiment 7, wherein R 1 It is a C1-C3 alkyl group.

[0211] 10. The carbon quantum dot according to embodiment 9, wherein R 1 is a C1-C3 alkyl group optionally substituted by one or two groups selected from halogen, -OH, -OMe, -SMe, -COOH and -CONH2.

[0212] 11. The carbon quantum dot of embodiment 1, comprising a polycyclic aromatic or partially aromatic ring system of at least 10 fused 6-membered rings, wherein the polycyclic aromatic or partially aromatic ring system is fused to at least one subunit of chemical formula B:

[0213]

[0214] in:

[0215] R 1 is H or C1-C3 alkyl optionally substituted by one or two groups selected from halogen, -OH, -OMe, -SMe, -COOH and -CONH2,

[0216] Z 1 NR2 or C(R 2 )2, where each R 2 are independently selected from H and C1-C3 alkyl, and wherein the dashed bonds indicate the position where the chemical formula B is fused to the polycyclic aromatic system.

[0217] 12. The carbon quantum dot according to embodiment 11, wherein R 1 For H.

[0218] 13. The carbon quantum dot according to embodiment 11, wherein R 1 It is a C1-C3 alkyl group.

[0219] 14. The carbon quantum dot according to embodiment 11, wherein R 1 is a C1-C3 alkyl group optionally substituted by one or two groups selected from halogen, -OH, -OMe, -SMe, -COOH and -CONH2.

[0220] 15. The carbon quantum dot according to any one of embodiments 11-14, wherein Z 1 NR 2 ,, for example NH.

[0221] 16. The carbon quantum dot according to any one of embodiments 11-14, wherein Z 1 C(R 2 )2, for example CH2.

[0222] 17. The carbon quantum dot according to embodiment 15 or 16, wherein R 2 For H.

[0223] 18. The carbon quantum dot according to embodiment 15 or 16, wherein R 2 It is a C1-C3 alkyl group.

[0224] 19. The carbon quantum dot according to any one of embodiments 11-18, comprising at least two subunits of chemical formula B, for example, four subunits of chemical formula B fused to a polycyclic aromatic or partially aromatic ring system.

[0225] 20. The carbon quantum dots of any one of embodiments 11-19, having a molecular weight of about 500 to about 500,000, for example, about 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000 or any subrange thereof.

[0226] 21. The carbon quantum dots according to any one of embodiments 1-20 are formed by reacting at least two different precursors, at least one precursor containing carboxyl and hydroxyl groups and at least another precursor containing multiple 6-membered aromatic rings and at least two amino groups.

[0227] 22. The carbon quantum dots according to embodiment 21, wherein at least one precursor comprising carboxyl and hydroxyl groups is citric acid (CA).

[0228] 23. The carbon quantum dots according to embodiment 21 or 22, wherein the precursor comprising a plurality of 6-membered aromatic rings is 1,4,5,8-tetraaminoanthraquinone (TAAQ), 1,4-diaminoanthraquinone (1,4-DAAQ) or 1,5-diaminoanthraquinone (1,5-DAAQ).

[0229] 24. The carbon quantum dots according to any one of embodiments 1-20 are formed by reacting at least two different precursors, at least one precursor comprises an α-aminocarboxylic acid compound or an α-hydroxycarboxylic acid compound, and the second precursor comprises a C1-C8 alcohol.

[0230] 25. The carbon quantum dots according to embodiment 24, wherein the precursor comprising the α-aminocarboxylic acid compound is phenylalanine (Phe).

[0231] 26. The carbon quantum dots of embodiment 24 or 25, wherein the second precursor is ethanol.

[0232] 27. The carbon quantum dot according to any one of embodiments 1-26, which is configured to selectively enter cells expressing the large neutral amino acid transporter (LAT1) or a subunit thereof.

[0233] 28. The carbon quantum dots according to embodiment 27, wherein LAT1 comprises a 4F2hc / CD98 heavy subunit protein encoded by the SLC3A2 (solute carrier family 3 member 2) gene and a CD98 light subunit protein encoded by the SLC7A5 gene.

[0234] 29. The carbon quantum dots according to embodiment 27 or 28, wherein the relative uptake rate of the cell to large neutral amino acids is at least 90%, or the LAT1 expression level of the cell is at least 10 times higher than that of normal human bone marrow stromal cells. For example, the cell has a relative uptake rate of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more for large neutral amino acids, or any sub-range thereof. In another example, the LAT1 expression level of the cell is at least 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45× or 50× higher than that of normal human bone marrow stromal cells, or any sub-range thereof.

[0235] 30. The carbon quantum dot according to any one of embodiments 27-29, wherein the ratio between the expression level of the LAT1 gene of the cell and the expression level of another gene (e.g., a housekeeping gene such as GAPDH) is at least 0.5. For example, the ratio between the expression level of the LAT1 gene of the cell and the expression level of another gene (e.g., a housekeeping gene such as GAPDH) is at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 or higher, or any subrange thereof.

[0236] 31. The carbon quantum dot according to any one of embodiments 27-30, wherein the LAT1 preferentially internalizes branched-chain amino acids and / or aromatic amino acids.

[0237] 32. The carbon quantum dots according to embodiment 31, wherein the branched-chain amino acid is a proteinogenic branched-chain amino acid, such as leucine, isoleucine or valine.

[0238] 33. The carbon quantum dots according to embodiment 31, wherein the branched-chain amino acid is a non-proteinogenic branched-chain amino acid, such as 2-aminoisobutyric acid.

[0239] 34. The carbon quantum dots according to embodiment 31, wherein the aromatic amino acid is a proteinogenic amino acid, such as phenylalanine, tyrosine, arginine, tryptophan or histidine.

[0240] 35. The carbon quantum dots according to embodiment 31, wherein the aromatic amino acid is a non-proteinogenic aromatic amino acid, such as thyroxine, 5-hydroxytryptophan (5-HTP) or levodopa (L-DOPA).

[0241] 36. The carbon quantum dot according to any one of embodiments 27-35, wherein LAT1 is preferentially or highly expressed in the target organ relative to other tissues of a subject (eg, a mammal).

[0242] 37. The carbon quantum dot according to any one of embodiments 27-35, wherein LAT1 is preferentially or highly expressed in tumors or cancer cells relative to other tissues or cells of a subject (eg, a mammal).

[0243] 38. The carbon quantum dots according to any one of embodiments 1-37, doped with N, S, P, B and / or O.

[0244] 39. The carbon quantum dots according to any one of embodiments 1-38, having an average size or particle size range of about 0.2nm to about 10nm, such as about 0.2nm, 0.3nm, 0.4nm, 0.5nm, 0.6nm, 0.7nm, 0.8nm, 0.9nm, 1nm, 2nm, 3nm, 4nm, 5nm, 6nm, 7nm, 8nm or 9nm to about 10nm, or any sub-range thereof. In many embodiments, the carbon quantum dots have an average size between 1nm and 5nm or between 2nm and 4nm.

[0245] 40. The carbon quantum dots of any one of embodiments 1-39, having an excitation wavelength ranging from about 300 nm to about 900 nm, for example, about 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or any sub-range thereof.

[0246] 41. The carbon quantum dots of any one of embodiments 1-40, having an emission wavelength range of about 400 nm to about 1000 nm, for example, about 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, or any sub-range thereof.

[0247] 42. The carbon quantum dots according to embodiment 41, which emit near infrared (NIR) fluorescence (FL).

[0248] 43. The carbon quantum dot according to any one of embodiments 1-42, which is configured to perform photoacoustic (PA) imaging when irradiated.

[0249] 44. According to the carbon quantum dots described in embodiment 43, the carbon quantum dots generate ultrasonic waves under radiation with an excitation wavelength ranging from about 600nm to about 900nm (for example, about 600nm, 650nm, 700nm, 750nm, 800nm, 850nm or 900nm) or any sub-range thereof.

[0250] 45. The carbon quantum dots according to embodiment 44, wherein the ultrasound is configured to form a PA image.

[0251] 46. ​​The carbon quantum dots according to any one of embodiments 43-45, having a linear increasing relationship at a concentration of about 0.1 μg / mL to about 10 μg / mL, for example, about 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL or any sub-range thereof.

[0252] 47. The carbon quantum dot according to any one of embodiments 1-46, which emits near infrared (NIR) fluorescence (FL) and is configured to perform photoacoustic (PA) imaging upon irradiation.

[0253] 48. The carbon quantum dots of embodiment 47, configured for deep tissue, tumor or cancer imaging.

[0254] 49. The carbon quantum dot of any one of embodiments 1-48, configured to selectively enter tumor or cancer cells.

[0255] 50. The carbon quantum dots of embodiment 49, which are configured to selectively enter the nucleus of a tumor or cancer cell.

[0256] 51. Carbon quantum dots according to embodiment 49 or 50, wherein the tumor or cancer is selected from sarcoma, epidermoid carcinoma, fibrosarcoma, cervical cancer, gastric cancer, skin cancer, leukemia, lymphoma, lung cancer, non-small cell lung cancer, colon cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancer, pancreatic cancer, bladder cancer, esophageal cancer, tongue cancer and malignant melanoma of the skin.

[0257] 52. The carbon quantum dots of any one of embodiments 1-51, configured to selectively enter cells of one or more tumors or cancers selected from sarcoma, epidermoid carcinoma, fibrosarcoma, cervical cancer, gastric cancer, skin cancer, leukemia, lymphoma, lung cancer, non-small cell lung cancer, colon cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancer, pancreatic cancer, bladder cancer, esophageal cancer, tongue cancer, and malignant melanoma of the skin.

[0258] 53. The carbon quantum dot of any one of embodiments 1-52, configured to selectively enter a cell or cell nucleus of a subject.

[0259] 54. The carbon quantum dot of embodiment 53, wherein the subject is a mammal, such as a human or a non-human mammal.

[0260] 55. A method for preparing a polycyclic compound or a particle comprising a polycyclic compound (e.g., a carbon quantum dot according to any one of embodiments 1-54), the method comprising solvothermal synthesis using at least two different precursors, wherein:

[0261] 1) at least one precursor comprises an α-aminocarboxylic acid compound or an α-hydroxycarboxylic acid compound, and at least one other precursor comprising a plurality of 6-membered aromatic rings; or

[0262] 2) at least one precursor comprising phenylalanine or a phenylalanine analog, such as an analog of phenylalanine having a substituent on the phenyl ring selected from halogen, hydroxyl, methoxy, methyl and CF3, and C1-C8 alcohol.

[0263] In some embodiments, a fused polycyclic ring system containing a 6-membered ring, wherein each ring is aromatic or unsaturated (partially aromatic), and the polycyclic ring system is substituted with at least one group of formula A:

[0264]

[0265] Where R 1 The dotted bond indicates the position where the group of formula A is attached to the fused polycyclic ring system. In these compounds, the polycyclic ring system may be optionally substituted with one or more selected from C 1-4 Alkyl, phenyl, C 1-4 Alkoxy, halogen, COOR*, -OH, CN and NR*2, wherein each R* is independently selected from H and C 1-3 Alkyl, each C 1-3 The alkyl group may be optionally substituted by one to three groups selected from -OH, -COOH, -NH2 and C 1-2 In addition, the C1-C3 alkyl group substituted with at least one carboxyl group and amino group may be optionally further substituted with a C1-C2 group, which may be optionally substituted with one to three selected from -OH, -COOH, -NH2 and C 1-2 Alkyl radicals are substituted.

[0266] In some embodiments within Embodiment 55, the polycyclic compound comprises a polycyclic aromatic or partially aromatic ring system of at least 10 fused 6-membered rings, wherein the polycyclic aromatic or partially aromatic ring system is fused to at least one subunit of Formula B:

[0267]

[0268] in:

[0269] R 1 is H or C1-C3 alkyl optionally substituted by one or two groups selected from halogen, -OH, -OMe, -NH2, -SMe, -COOH and -CONH2.

[0270] Z 1 NR 2 or C(R 2 )2, where each R 2 independently selected from H and C1-C3 alkyl, and wherein the dotted bond indicates the position where the chemical formula B is fused to the polycyclic aromatic or partially aromatic ring system. In these compounds, the polycyclic system may be optionally replaced by one or more selected from C 1-4 Alkyl, phenyl, C 1-4 Alkoxy, halogen, COOR*, -OH, CN and NR*2, wherein each R* is independently selected from H and C 1-3 Alkyl, each C 1-3 The alkyl group may be optionally substituted by one to three groups selected from -OH, -COOH, -NH2 and C 1-2 In addition, the C1-C3 alkyl group substituted with at least one carboxyl group and amino group may be optionally further substituted with a C1-C2 group, which may be optionally substituted with one to three selected from -OH, -COOH, -NH2 and C 1-2 Alkyl radicals are substituted.

[0271] 56. The method according to embodiment 55, wherein the method comprises a solvothermal synthesis method using at least one α-aminocarboxylic acid compound or α-hydroxycarboxylic acid compound as a precursor and at least another precursor containing multiple 6-membered aromatic rings.

[0272] 57. The method of embodiment 56, wherein the precursor comprising the α-hydroxycarboxylic acid compound is citric acid (CA).

[0273] 58. The method of embodiment 56 or 57, wherein the precursor comprising multiple 6-membered aromatic rings is 1,4,5,8-tetraaminoanthraquinone (TAAQ), 1,4-diaminoanthraquinone (1,4-DAAQ), or 1,5-diaminoanthraquinone (1,5-DAAQ).

[0274] 59. The method of embodiment 55, wherein the method comprises using at least one solvothermal synthesis method comprising phenylalanine or a phenylalanine analog and a C1-C8 alcohol as precursors.

[0275] 60. The method of embodiment 59, wherein the precursor comprising phenylalanine or a phenylalanine analog is phenylalanine.

[0276] 61. The method of embodiment 59 or 60, wherein the C1-C8 alcohol is ethanol.

[0277] 62. The method according to any one of embodiments 55-61, comprising dissolving or dispersing the precursor molecules in a solvent to form a solution or mixture, and heating the solution or mixture at a temperature of about 100° C. to about 300° C. for about 10 minutes to about 72 hours, such as about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, 72 hours, or any sub-range thereof. In some embodiments, the temperature is between about 100° C., 150° C., 200° C., 250° C., 300° C., or any sub-range thereof.

[0278] 63. The method according to embodiment 62, wherein the solvent is water, C 1-10 alcohol (eg ethanol), amide (eg formamide, N,N-dimethylformamide, dimethylacetamide or N-methylpyrrolidone), ketone (eg acetone or 2-butanone) or sulfoxide (eg dimethyl sulfoxide).

[0279] 64. The method of any one of embodiments 55-63, further comprising separating or purifying carbon quantum dots. In some embodiments, the carbon quantum dots fluoresce in the near infrared region. Optionally, the CQDs have an average size between about 1 nm and 5 nm, such as about 1 nm, 2 nm, 3 nm, 4 nm, or 5 nm, or any sub-range thereof.

[0280] 65. The method according to embodiment 64, wherein the carbon quantum dots are separated or purified by filtration, centrifugation, chromatography (eg, column chromatography, gel electrophoresis, or dialysis).

[0281] 66. The method of any one of embodiments 55-65, comprising:

[0282] 1) mixing a precursor comprising an α-aminocarboxylic acid compound or an α-hydroxycarboxylic acid compound (e.g., citric acid (CA)) and another precursor comprising a plurality of 6-membered aromatic rings (e.g., TAAQ, 1,4-DAAQ or 1,5-DAAQ) in water to form a mixture;

[0283] 2) heating the mixture at about 180° C. for about 2 hours;

[0284] 3) cooling the mixture to a lower temperature, such as room temperature; and

[0285] 4) separating or purifying the carbon quantum dots from the mixture, for example by chromatography, solvent-solvent extraction and / or crystallization.

[0286] 67. The method according to embodiment 65 or 66 further comprises removing the solvent under vacuum drying to obtain isolated or purified carbon quantum dots.

[0287] 68. A carbon quantum dot prepared by the method described in any one of embodiments 55-67.

[0288] 69. The carbon quantum dots according to any one of embodiments 1-54 and 68, further comprising a releasable load, for example, the carbon quantum dots can be covalently or non-covalently linked to the releasable load. In some embodiments, the load is a therapeutic agent, a chemotherapeutic agent, or a labeling agent. In some embodiments, the load is an aromatic chemotherapeutic drug, such as topotecan (TPTC), doxorubicin (DOX), or hydroxycamptothecin (HCPT).

[0289] In some embodiments, the carbon quantum dots may be optionally conjugated to a therapeutic agent via a release linker. Any suitable therapeutic agent may be used in these compositions and conjugates. For example, the therapeutic agent may be a small molecule therapeutic agent, a macromolecular therapeutic agent, or a combination or complex thereof. Exemplary macromolecular therapeutic agents may be peptides, polypeptides, proteins, e.g., recombinant proteins, antibodies, antibody fragments or derivatives, polynucleotides or nucleic acids, e.g., DNA, RNA, PNA, antisense oligonucleotides or polynucleotides, or RNAi oligonucleotides or polynucleotides, lipids, sugars or carbohydrates, or a combination or complex thereof.

[0290] 70. The carbon quantum dots of embodiment 69, wherein the release of the releasable payload is triggered by contact between the carbon quantum dots and target cells, tissues, organs or subjects, or by enzymatic cleavage, or by changes in physical and / or chemical parameters surrounding the carbon quantum dots.

[0291] 71. The carbon quantum dots of embodiment 69 or 70, wherein the releasable payload is a therapeutic agent, a prophylactic agent, a diagnostic agent, a labeling agent or marker, a prognostic agent, an imaging agent, or a combination thereof.

[0292] 72. The carbon quantum dots according to embodiment 71, wherein the releasable cargo is a therapeutic agent. Suitable therapeutic drugs include topotecan (TPTC), doxorubicin (DOX) or hydroxycamptothecin (HCPT).

[0293] 73. Carbon quantum dots according to embodiment 72, wherein the therapeutic agent comprises a small molecule (e.g., an organic compound with a molecular weight between 200 and 2000), a macromolecule (e.g., a polypeptide or a polynucleotide), a cell therapeutic agent, a conjugate, or a combination thereof.

[0294] 74. The carbon quantum dots of Embodiment 72 or 73, wherein the therapeutic agent comprises an anti-tumor or anti-cancer agent.

[0295] 75. The carbon quantum dots according to embodiment 74, wherein the anticancer agent is a chemotherapy drug, such as topotecan hydrochloride (TPTC), doxorubicin (DOX) or hydroxycamptothecin (HCPT) or a kinase inhibitor.

[0296] 76. According to the carbon quantum dots described in any one of embodiments 69-75, the therapeutic agent is configured to treat cancer or tumors.

[0297] 77. Carbon quantum dots according to embodiment 76, wherein the therapeutic agent is configured to treat one or more tumors or cancers selected from sarcoma, epidermoid carcinoma, fibrosarcoma, cervical cancer, gastric cancer, skin cancer, leukemia, lymphoma, lung cancer, non-small cell lung cancer, colon cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancer, pancreatic cancer, bladder cancer, esophageal cancer, tongue cancer and malignant melanoma of the skin.

[0298] 78. A composition comprising the carbon quantum dots described in any one of Examples 1-54 and 68, which is an optional pharmaceutical composition formed by mixing the carbon quantum dots described in any one of Examples 1-54 and 68 and at least one pharmaceutically acceptable carrier or excipient.

[0299] 79. A composition according to embodiment 78, which is configured for use as a therapeutic agent, a prophylactic agent, a diagnostic agent, a sensor agent, a labeling agent, a prognostic agent, an imaging application or a combination thereof, for example by attaching or releasably attaching carbon quantum dots to a therapeutic agent (e.g., a therapeutic agent for treating cancer), such as topotecan hydrochloride (TPTC), doxorubicin (DOX) or hydroxycamptothecin (HCPT) or a kinase inhibitor.

[0300] 80. A kit comprising the carbon quantum dots of any one of embodiments 1-54 and 68-77 or the composition of embodiment 78 or 79, and instructions for using the carbon quantum dots or composition.

[0301] 81. A method for sensing, labeling or imaging a target cell, tissue or organ in a subject, the method comprising:

[0302] a) administering to a subject in need thereof an effective amount of the carbon quantum dots described in any one of Examples 1-54 and 68-77 or the composition described in Example 78 or 79; and

[0303] b) evaluating the carbon quantum dots for sensing, labeling or imaging target cells, tissues or organs in the subject.

[0304] In these embodiments, the step of evaluating the carbon quantum dots may include detecting near-infrared fluorescence generated by the carbon quantum dots.

[0305] 82. The method of embodiment 81, wherein the carbon quantum dots do not include a releasable payload.

[0306] 83. The method of embodiment 81, wherein the carbon quantum dots comprise a releasable payload.

[0307] 84. The method of any one of embodiments 81-83, for sensing, labeling or imaging a target cell, tissue or organ in a subject.

[0308] 85. The method of any one of embodiments 81-83, for sensing, labeling or imaging abnormal or diseased cells, tissues or organs in a subject.

[0309] 86. The method of embodiment 85, for sensing, labeling or imaging a tumor or cancer cell, tissue or organ in a subject.

[0310] 87. The method of embodiment 85, for sensing, labeling or imaging a tumor in a subject.

[0311] 88. The method of any one of embodiments 85-87, for diagnosing, prognosing, stratifying, risk assessing, or monitoring treatment of a disease or disorder in a subject.

[0312] 89. The method of embodiment 88, wherein the disease or disorder is one or more tumors or cancers selected from sarcoma, epidermoid carcinoma, fibrosarcoma, cervical cancer, gastric cancer, skin cancer, leukemia, lymphoma, lung cancer, non-small cell lung cancer, colon cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancer, pancreatic cancer, bladder cancer, esophageal cancer, tongue cancer and malignant melanoma of the skin.

[0313] 90. The method of any one of embodiments 85-87, for assisting or guiding the treatment or therapy of a disease or disorder in a subject.

[0314] 91. The method of embodiment 90, wherein the treatment or therapy comprises a course of therapy or surgery on the subject.

[0315] 92. The method of embodiment 91, for assisting or guiding a course of treatment or surgery for a tumor or cancer in a subject.

[0316] 93. The method of embodiment 91, used to assist or guide radiation therapy of a subject.

[0317] 94. Use an effective amount of the carbon quantum dots described in any one of Examples 1-54 and 68-77, or the composition described in Example 78 or 79 to manufacture a composition for sensing, labeling or imaging target cells, tissues or organs in a desired subject.

[0318] 95. A drug complex comprising an effective amount of the carbon quantum dots described in any one of embodiments 1-77, for example, the composition described in embodiments 72-77, or embodiments 78 or 79, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises a therapeutic agent for treating cancer, such as topotecan hydrochloride (TPTC), doxorubicin (DOX) or hydroxycamptothecin (HCPT) or a kinase inhibitor, wherein the therapeutic agent can be optionally releasably attached to the carbon quantum dots.

[0319] 96. A method for treating or preventing a disease or condition in a subject, comprising administering to a subject in need thereof an effective amount of the carbon quantum dots described in any one of Examples 1-77, such as the composition described in Examples 72-77, Example 78 or 79, or the pharmaceutical composition described in Example 95.

[0320] 97. The method of embodiment 96, for treating or preventing a tumor or cancer in a subject.

[0321] 98. The method of embodiment 96, for treating a tumor in a subject.

[0322] 99. The method of any one of embodiments 96-98, wherein the carbon quantum dots, composition or pharmaceutical composition is administered orally, nasally, by inhalation, parenterally, intravenously, intraperitoneally, subcutaneously, intramuscularly, intradermally, topically or rectly.

[0323] 100. The method of any one of embodiments 81-93 and 96-99, wherein the subject is a mammal.

[0324] 101. The method of embodiment 100, wherein the mammal is a non-human mammal.

[0325] 102. The method of embodiment 100, wherein the mammal is a human.

[0326] 103. Use an effective amount of the carbon quantum dots described in any one of Examples 1-54 and 68-77 or the composition described in Example 78 or 79 to manufacture a drug for treating or preventing a disease or condition in a desired subject, or a drug for detecting the presence or absence of cancer cells or tissues in a desired subject.

[0327] 104. The use according to embodiment 103, wherein the medicament is used to treat or prevent a tumor or cancer in a subject.

[0328] 105. The use according to embodiment 103, wherein the medicament is used to treat a tumor in a subject.

[0329] 106. A composition according to embodiment 78 or 79, wherein the carbon quantum dots are attached or fixed to a solid surface or support.

[0330] 107. The composition of embodiment 106, configured for detecting and / or isolating target cells from a sample.

[0331] 108. The composition of embodiment 107, wherein the sample is a biological sample, such as a blood or urine sample.

[0332] 109. The composition of embodiment 107 or 108, configured for detecting and / or isolating tumor or cancer cells from a sample.

[0333] 110. The composition of embodiment 109, configured for detecting and / or isolating circulating tumor or cancer cells from a biological sample (eg, a blood or urine sample).

[0334] 111. The composition of any one of embodiments 106-110, wherein the solid surface or support is a container, a tube, a plate, a portion of a microtiter plate, e.g., a well of a microtiter plate, a disc, a chip, a membrane, or a collection and / or analysis device.

[0335] 112. A composition according to any of embodiments 106-110, wherein the solid surface or support is part of an assay device selected from enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescence assay (IFA), nephelometry, flow cytometry, plasma resonance analysis, chemiluminescence analysis, lateral flow immunoassay, u-capture assay, inhibition assay, and affinity assay.

[0336] 113. A method for detecting and / or separating tumors or cancer cells from a sample, the method comprising contacting a sample containing or suspected of containing tumors or cancer cells with a composition according to any one of embodiments 106-110 under appropriate conditions to allow the tumor or cancer cells (if present in the sample) to bind to the carbon quantum dots attached or fixed on the solid surface or support.

[0337] 114. The composition according to embodiment 113, which is used for detecting and / or isolating circulating tumor or cancer cells from a biological sample (eg, a blood or urine sample).

[0338] 115. The method of any one of embodiments 81-90, for detecting and / or isolating circulating tumor or cancer cells from a biological sample (eg, a blood or urine sample).

[0339] 116. For use in treatment, the carbon quantum dots described in any one of Examples 1-54 and 68-77, or the composition described in Example 78 or 79.

[0340] 117. The carbon quantum dots of embodiment 116, wherein the therapeutic use is the treatment or detection of cancer or tumors.

[0341] 118. The carbon quantum dots according to embodiment 116, wherein the therapeutic use is to treat tumors.

[0342] The following exemplary compounds and carbon quantum dots or particles comprising these compounds are preferred embodiments of the present invention, and methods of preparing and using these exemplary compounds are preferred embodiments of the present invention.

[0343] Typical compounds

[0344]

[0345] The compounds, particles and compositions described herein can be used for any suitable purpose. In some embodiments, the compounds, particles and compositions described herein can be used for treatment, for example, for treating tumors expressing LAT1. The present invention also provides conjugates comprising the compounds and particles described herein, which are also suitable for treatment. In some embodiments, treatment refers to treating tumors, particularly tumors expressing LAT1.

[0346] In another aspect, the compounds can be used to image cells, tissues, organs or tumors, typically cells, tissues, organs or tumors expressing LAT1. Thus, the present invention includes methods of using the compounds, particles and compositions as imaging or labeling agents, and includes conjugates comprising the compounds and particles described herein suitable for use in these methods.

[0347] On the other hand, the compounds of the present invention include conjugates of carbon quantum dots and loads, which are typically therapeutic agents for treating or imaging cells, tissues, organs or tumors expressing LAT1. The load connected to the CQDs is releasable; in some embodiments, the load can be covalently connected to the CQDs via an amide or ester bond formed between the amino or carboxyl groups of the CQDs. In other embodiments, the load is physically adsorbed onto the particles or CQDs of the present invention, and the release of the load in the cell occurs passively. This method of carrying the load is particularly effective for polycyclic aromatic hydrocarbon loads, or loads with strong pi stacking ability (such as topotecan or irinotecan, doxorubicin and daunorubicin).

[0348] Disclosed herein are polycyclic compounds and particles containing these compounds, including some carbon quantum dots that selectively bind or internalize to tumor or cancer cells. Without being bound by theory, it is believed that their selectivity comes from their recognition by large amino acid transporters.

[0349] LAAM CQDs and demonstrated that LAAM CQDs allow imaging and drug delivery to tumors of different origins and locations with unprecedented specificity and efficiency ( Figure 2 , 4 By combining genetic and pharmacological approaches, we found that the tumor-specific interaction is mediated by LAT1 ( Figure 3 LAAM CQDs have three unique and significant advantages over previous imaging and delivery agents:

[0350] First, like previously reported CQDs, LAAM CQDs have intrinsic optical properties for imaging and can therefore be used for tumor imaging without further conjugation to imaging probes. We characterized LAAM TC-CQDs, one of the preferred LAAM CQDs, and found that it allows NIR FL / PA dual-modality imaging that is best suited for deep tissue imaging. Due to its high specificity for tumors, LAAM TC-CQDs have great potential in tumor imaging as well as imaging-guided surgery.

[0351] Second, LAAM TC-CQDs allow for targeted drug delivery to tumors with a high degree of specificity, which cannot be achieved by conventional approaches involving tumor-targeting ligand conjugation. A recent analysis showed that despite extensive efforts, conventional approaches have been unable to significantly improve tumor targeting efficiency. Consistent with this finding, we found that ligand conjugation enhanced the delivery of CQDs to some tumors but not others, and failed to significantly reduce their nonspecific accumulation in normal tissues ( Fig.37 , 38 ). Ligand binding methods have previously been tested for LAT1-mediated drug delivery to tumors via aspartate binding.13 . The study found that LAT1 targeting increased DOX accumulation in tumors by 3-6 fold. However, a considerable amount of DOX was also found in the liver. In contrast, we found that delivery of LAAM TC-CQDs increased TPTC accumulation in tumors by 16.7 fold, while TPTC accumulation in the liver was reduced to almost undetectable levels (1.8% in tumors) ( Figure 4 d). The observed differences between the two studies may be due to the different affinities of LAAM TC-CQDs and aspartic acid as drug carriers for LAT1. LAAM TC-CQDs contain four pairs of α-carboxyl and amino groups. We found that the specificity of LAAM CQDs correlated with the number of paired α-carboxyl and amino groups on the core ( Figure 5 ). It appears that the local high density of α-carboxyl and amino groups triggers a strong multivalent interaction with LAT1. Therefore, the penetration of TPTC into tumor cells was significantly enhanced by delivery via LAAM TC-CQDs. Since most normal tissues do not express LAT1, LAAM TC-CQDs do not easily penetrate normal cells and are preferentially eliminated from the circulation system in the absence of active transporters. Unlike LAAM CQDs, aspartic acid contains only one pair of α-carboxyl and amino groups. Due to the lack of multivalency, the interaction between aspartic acid-bound DOX and LAT1 is weak. Therefore, aspartic acid-bound DOX has a low affinity for tumor cells and can nonspecifically penetrate normal cells, leading to accumulation in normal organs 13 .

[0352] In summary, LAAM CQDs possess an ideal set of properties for tumor-specific imaging and drug delivery. Due to their ability to selectively target tumors regardless of their origin and location, minimal accumulation and toxicity in most normal tissues, inherent imaging capabilities, and the ability to load and deliver chemotherapeutic drugs, LAAM CQDs have great potential for clinical translational applications, such as imaging and drug delivery to various tumors.

[0353] Synthesis and characterization of LAAMTC-CQDs

[0354] LAAM TC-CQDs were synthesized by mixing 1,4,5,8-tetraaminoanthraquinone (TAAQ) with citric acid (CA) in aqueous solution followed by hydrothermal treatment at 180 °C for 2 h ( Figure 1 a). The reactants were purified by silica gel column chromatography to obtain a clear and transparent blue solution. Transmission electron microscopy (TEM) analysis showed that the LAAM TC-CQDs had good dispersion and an average diameter of 2.45 nm ( Figure 1 b and Figure 6). High-resolution TEM (HRTEM) analysis revealed a highly resolved lattice with a lattice spacing of 0.21 nm ( Figure 1 b inset), corresponding to the (100) lattice fringe of graphene 16 Raman spectroscopy confirmed the high degree of crystallinity, with 1605 cm -1 The crystalline G band at 1365 cm -1 The disordered D band at the position of G and D has an intensity ratio (I G / I D ) is 1.4( Figure 7 Atomic force microscopy (AFM) analysis found that the typical topological height is 0.943nm ( Figure 8 ), which indicates that most LAAMTC-CQDs are composed of 2-3 graphene layers. The X-ray powder diffraction (XRD) pattern shows a broad (002) peak at about 26° ( Fig. 9 ), confirming the graphene structure of LAAM TC-CQDs.

[0355] X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) and 13 The chemical composition and surface functional groups of LAAM TC-CQDs were characterized by C-nuclear magnetic resonance spectroscopy (NMR). XPS full spectrum analysis showed that the atomic percentages of C, N, and O in LAAM TC-CQDs were 72.43%, 12.25%, and 15.32%, respectively. Fig.10 The C1s deconvoluted spectrum shows three different types of carbon atoms, including graphitized / aliphatic (C=C / CC), oxygen-containing (CO, C=O, and OC=O), and N-containing (CN, C=N, and NC=O) carbons ( Fig.11 The N1s deconvoluted spectrum shows three peaks at 399.0, 399.9 and 400.8 eV ( Fig.12 ), corresponding to CN, NH and C=N, respectively. FT-IR spectroscopy analysis determined that NH (3298 and 3190 cm -1 )、C=O(1720cm -1 )、C=N(1624cm -1 )、CN(1387cm -1 ) and CO(1158cm -1 ) bond stretching vibration ( Fig.13 ), indicating the formation of polycyclic aromatic hydrocarbon structures and the presence of free carboxyl and amino groups at the edges of LAAM TC-CQDs. 13In the C-NMR spectrum, the peaks between 120.0 and 140.0 ppm can be attributed to aromatic functional groups and conjugated double bonds (C=C and C=N), while the peaks at 173.6, 170.2, and 53.3 ppm are related to OC=O, NC=O, and CN, respectively ( Fig.14 ). Taken together, the obtained structural information suggests that LAAM TC-CQDs may be formed via two steps. In the first step under hydrothermal conditions, one molecule of TAAQ reacts with two molecules of CA to produce a rigid carbon skeleton consisting of three benzene rings fused with four pyridine rings. Subsequently, the building block skeleton undergoes dehydration and decarboxylation processes, leading to the formation of LAAM TC-CQDs with high N-doping in a large rigid π-conjugated structure and free α-carboxyl and amino groups on the edges ( Figure 1 a).

[0356] The UV-vis absorption spectrum of LAAM TC-CQDs shows two π-π* transitions at 230 and 280 nm, which are assigned to aromatic C=C bonds and aromatic sp 2 In addition to the typical absorption peak of CQDs converted by n-π*, the system exhibits a strong characteristic absorption band at about 650nm ( Figure 1 c) 14,17-19 . Characterize the fluorescence (FL) spectra at different excitation wavelengths ( Figure 1 d, Fig.15 ). The aqueous solution of LAAM TC-CQDs exhibits a near-infrared fluorescence (NIRFL) emission peak at 700nm, and the FL emission wavelength is almost excitation-independent. The absolute FL quantum yield of LAAM TC-CQDs was determined to be 6.8% by the integrating sphere method.

[0357] Density functional theory (DFT) simulations using the B3LYP / 6-31G(d,p) basis set confirmed that LAAM TC-CQDs have a local structure that generates near-NIR FL. Fig.16 a, b and Table 1 show the optimized Cartesian coordinates and main geometric parameters of the ground and excited states of LAAM TC-CQDs. The electron delocalized molecular orbital (MO) diagram of one FL structural unit obtained from theoretical calculations shows that the energy gap (Eg) between the highest occupied molecular orbital (HOMO) position and the lowest unoccupied molecular orbital (LUMO) position is 2.14 eV ( Fig.16 ce). The simulation predicted an excitation wavelength of 580 nm and an oscillator strength of 0.1092, and the corresponding fluorescence wavelength was 692 nm and an oscillator strength of 0.1003; these values ​​are very close to the experimentally measured excitation / emission wavelengths (λex=600nm,λem=700nm, Fig.15 ).

[0358] Table 1. Optimized Cartesian coordinates of the ground and excited states of the FL building block

[0359]

[0360]

[0361]

[0362] In addition to the FL property, LAAM TC-CQDs were also found to possess the capability of photoacoustic (PA) imaging. Fig.17 The results in Table 2 show that at 650 nm laser (0.5 W / cm 2 ) irradiation, LAAM TC-CQDs absorb NIR light, resulting in a thermally induced pressure transition. The resulting ultrasonic wave can be received by an acoustic detector to form a PA image. 20 Monitoring the PA spectra of the agarose gel model filled with LAAM TC-CQDs (0-10 μg / mL) at different excitation wavelengths. Figure 1 e and Fig.18 The peak at about 650 nm corresponds to Figure 1 c shows the observed NIR absorption band of LAAM TC-CQDs. In the concentration range of 2–10 μg / mL, the PA signal increases linearly with the increase of LAAM TC-CQDs concentration (R 2 =0.9989)( Fig.19 ).

[0363] Table 2. Examples of experimentally determined photothermal conversion efficiencies (η) of different PTCAs arranged from high to low.

[0364]

[0365] NIR has the advantage of penetrating biological tissues more efficiently than visible light. Ultrasound waves scatter to a lesser extent than photons; therefore, PA imaging can overcome the penetration limitation of optical imaging to a few centimeters. 21 With this unique NIR FL / PA dual-modality imaging capability that combines the high contrast of optical imaging with the high spatial resolution of ultrasound, LAAM TC-CQDs represent a promising probe for deep tissue imaging.

[0366] LAAMTC-CQDs selectively target cancer cells in vitro and in vivo

[0367] We investigated the interaction between LAAM TC-CQDs and a broad panel of cell cultures, including 27 cancer cell lines of different origin, a cancer stem cell-like side population (SP) isolated from MDA-MB-231 cells ( Fig. 20 ) 22,12 and 18 non-cancerous cell lines. Table 3 includes a detailed list of cells used in this study. Using confocal laser scanning microscopy (LCSM), we found that LAAM TC-CQDs permeated all tested cancer cells, regardless of their origin or stemness. In contrast, they had limited ability to permeate non-cancerous cells, as confirmed by flow cytometric analysis ( Figure 2 a, Figure 21-24 Quantitative analysis showed that approximately 99% of cancer cells (except for one BCSC line with a rate of 95%) were positive for LAAM TC-CQDs, while the positivity of non-cancerous cells was limited to <30% (average 13%) ( Figure 2 b and Table 3). Further LCSM analysis revealed that a considerable portion of LAAM TC-CQDs entered the cell nucleus, as can be seen from the overlap of the FL of LAAM TC-CQDs and the 4,6-diamidino-2-phenylindole (DAPI) FL that stains the cell nucleus. To determine the dynamics of cell penetration and nuclear localization, we incubated HeLa cells with LAAM TC-CQDs and monitored the location of LAAM TC-CQDs within the cell over time ( Fig.25 ). At the 1-hour time point, most LAAM TC-CQDs were located on the cell membrane. Subsequently, LAAM TC-CQDs penetrated into the cell and localized in the cytoplasm. After 6 hours, a significant portion of LAAM TC-CQDs entered the nucleus. By 8 hours, the concentration of LAAM TC-CQDs in the nucleus exceeded that in the cytoplasm. We used the same procedure to monitor the interaction of LAAM TC-CQDs with noncancerous CCC-ESF-1 cells ( Fig.26 ). Consistent with the observations from LCSM and flow cytometry analyses, we did not detect any interaction between LAAM TC-CQDs and representative non-cancerous CCC-ESF-1 cells throughout the 48 h time window.

[0368] Table 3. Flow cytometric analysis of TC-CQDs uptake results in the indicated cells.

[0369]

[0370]

[0371] We evaluated whether LAAM TC-CQDs maintain the observed high tumor specificity in vivo. LAAM TC-CQDs were intravenously injected into HeLa tumor-bearing mice at a dose of 5 mg / kg. FL / PA imaging was performed on the mice at 0, 2, 4, 6, 8, and 10 hours after injection. Figure 2 c shows that FL in the tumor area gradually increased over time and reached a peak at 8 h, while no FL was observed in other areas. The three-dimensional (3D) reconstruction of FL imaging confirmed that LAAM TC-CQDs selectively imaged tumors rather than normal tissues ( Figure 2 d). This high selectivity was validated by ex vivo imaging of isolated organs and tumors, which showed that the FL signal in tumors was significantly greater than that in normal organs ( Figure 2 e and Fig. 27 ). Consistent with the findings from FL imaging, the average PA intensity in the tumor continued to increase over time until 8 h after injection, when the PA signal was primarily concentrated in the tumor ( Figure 2 f and Fig.28 ).

[0372] To exclude the possibility that the observed tumor specificity was unique to HeLa tumors, we evaluated LAAM TC-CQDs in four additional tumor xenografts derived from A549 cells, PANC-1 cells, MCF-7 cells, and MD-MBA-231 cells. We found that LAAM TC-CQDs imaged tumors in all models tested with kinetics and specificity similar to that observed in the HeLa model ( Fig.29 and 30 To further validate tumor specificity, we evaluated LAAM TC-CQDs in mice bearing A549 lung cancer in the left flank and HeLa ovarian tumors in the right flank. Fig.31 and 32 As shown, LAAMTC-CQDs penetrated both tumors with similar specificity.

[0373] We investigated whether the observed high tumor specificity could be achieved using conventional CQDs. 17 、Y-CQDs 19 , B-CQDs 23 and B,S-CQDs 14 As an example, these four previously reported CQDs were incubated with a panel of cancer cells and non-cancer cells and their specificity was determined using flow cytometric analysis. Fig.33 and 34As shown, all tested CQDs were able to non-selectively permeate cancer cells and non-cancerous cells. After permeating cells, most of the CQDs were localized in the cytoplasm. G-CQDs were selected and further evaluated in HeLa tumor-bearing mice. The experiments were performed following the same procedures used in the LAAM TC-CQDs study. 3D reconstructions of FL imaging showed that FL signals were also present in the liver, lungs, and kidneys in addition to the tumors ( Fig.35 ). Ex vivo imaging showed that the FL intensity in the lungs and kidneys was significantly greater than that in the tumor ( Fig.36 We evaluated whether conventional CQDs could be linked to tumor-targeting ligands to achieve the same degree of tumor specificity observed in LAAM TC-CQDs. We followed our recently published procedure 18 synthesized G-CQDs with folic acid (FA) attached to their surface. The resulting CQDs, called FA-G-CQDs, were evaluated in mice bearing HeLa and A549 tumors. HeLa tumors, but not A549 tumors, are known to overexpress FA receptors. 21,24 Sure enough, we found that FA attachment enhanced the accumulation of FA-G-CQDs in HeLa tumors, but not in A549 tumors ( Fig.37 ), indicating that the ligand-linked approach mainly enhances the targeting efficiency of CQDs to receptor-expressing tumors. Although the FL signal in HeLa tumors is larger than that in other organs, our ex vivo imaging confirmed that a large amount of FL signal can be observed in the liver, lungs, and kidneys ( Fig.38 ).

[0374] Taken together, these data suggest that LAAM TC-CQDs have a unique ability to selectively interact with cancer cells regardless of origin, but have limited interaction with non-cancerous cells. Due to their unique NIR FL / PA dual-modality imaging capability, LAAM TC-CQDs enable in vivo imaging of tumors with minimal background.

[0375] LAT1 mediates the internalization of LAAMTC-CQDs in cancer cells

[0376] To investigate the cellular uptake mechanism of LAAM TC-CQDs, we treated HeLa cells with excess leucine (Leu), phenylalanine (Phe), or glycine (Gly) before adding LAAM TC-CQDs. In addition to Gly, Leu and Phe are both known high-affinity substrates for LAT1. 27 .like Figure 3As shown in Figure 1a, Leu and Phe significantly inhibited the cellular uptake of LAAM TC-CQDs, whereas Gly did not. This observation led to the hypothesis that LAAM TC-CQDs penetrate into cancer cells at least in part through interaction with LAT1. This hypothesized mechanism is supported by several lines of evidence. First, pretreatment with the LAT1 inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) significantly reduced the uptake of LAAM TC-CQDs by HeLa cells ( Figure 3 a). Quantitative results by flow cytometry showed that pretreatment with BCH reduced the cellular uptake rate from ∼99% to ∼20% in all cell lines tested, including HeLa, A549, PANC-1, MCF-7, MDA-MB-231, and MDA-MB-231SP cells ( Fig.39 ). Second, using CRISPR / Cas9 technology to generate LAT1 gene knockout and verified by sequencing and western blotting (WB), the uptake of LAAM TC-CQDs by HeLa cells was significantly reduced by about 80% ( Figure 3 bd and Fig.40 ). Third, in various cells, the expression level of LAT1 correlated with the number of LAAM TC-CQDs that permeated the cells ( Figure 3 e. Fig.41 and Table 4). Consistent with previous reports, the expression level of LAT1 in cancer cells was significantly higher than that in non-cancerous cells. 8,10,11 .

[0377] Table 4. Relative LAT1 expression and TC-CQDs uptake in various cancer cells and normal cells tested. (Note: The relative LAT1 expression of different cell types is obtained by Fig.37 The WB test measured,

[0378] The uptake of TC-CQDs was determined by Fig.17 and 18 obtained).

[0379]

[0380]

[0381] Taken together, our results suggest that the interaction between LAAM TC-CQDs and cancer cells is mediated by LAT1.

[0382] LAAM TC-CQDs for tumor-specific drug delivery

[0383] Major limitations associated with conventional chemotherapy include significant adverse toxicity to normal tissues and intrinsic or acquired drug resistance due to overexpression of multidrug resistance ATP-binding cassette (ABC) transporters. The efficacy of DNA-damaging chemotherapeutic agents that exert their effects by interacting with DNA within the nucleus is often further reduced by their limited ability to penetrate the nucleus. 28 . Using LAAM TC-CQDs as drug carriers can overcome these limitations. First, LAAM TC-CQDs have great tumor-specific targeting and can avoid systemic toxicity. Second, LAAM TC-CQDs will not be removed by ABC transporters due to their nanosize. Third, LAAM TC-CQDs can effectively penetrate the cell nucleus, making it possible to maximize the efficacy of DNA-damaging chemotherapeutic drugs.

[0384] We evaluated LAAM TC-CQDs as drug carriers for the delivery of DNA-damaging chemotherapeutic drugs. LAAM TC-CQDs have a large π-conjugated structure and can load aromatic chemotherapeutic drugs such as topotecan hydrochloride (TPTC), doxorubicin (DOX), and hydroxycamptothecin (HCPT) ( Fig.42 ). We further evaluated TPTC-loaded LAAMTC-CQDs, named TPTC / TC-CQDs, which were synthesized by mixing LAAM TC-CQDs with TPTC overnight followed by extensive dialysis. Evidence for successful loading of TPTC was the appearance of its characteristic UV-vis absorption peak at 390 nm, which was superimposed on the absorption spectrum of LAAM TC-CQDs ( Fig.42 a). The absorption peak of TPTC has a slight red shift after loading, which is attributed to the π-π stacking effect between TPTC and LAAM TC-CQDs. TPTC / TC-CQDs have dual FL emission corresponding to TPTC (yellow channel, Fig.43 ) and LAAM TC-CQDs (red channel, Figure 1 d) We characterized the interactions between TPTC / TC-CQDs and a panel of cancer cells and non-cancer cells. Figure 4 As shown in a, TPTC / TC-CQDs maintained the selective permeability to cancer cells, while the delivery of LAAM TC-CQDs abolished the penetration of TPTC into non-cancerous cells. In particular, the delivery of LAAM TC-CQDs increased the accumulation of TPTC in the nucleus by 4.5 times ( Fig.44). Delivery of TC-CQDs via LAAM significantly enhanced the toxicity of TPTC to cancer cells but reduced its toxicity to non-cancerous cells. At a concentration equivalent to 5 μg / mL TPTC, TPTC / TC-CQDs killed all HeLa cells but showed no toxicity to non-cancerous CCC-ESF-1 cells ( Figure 4 b). In contrast, the same concentration of free TPTC had comparable inhibitory efficiency against both cell types. The apparent enhancement of toxicity to cancer cells is not unique to HeLa cells, and similar enhancements were also observed in other cancer cells, including A549, PANC-1, MCF-7, and MDA-MB-231 cells ( Fig.45 ). This clearly demonstrates that the LAAM CQDs of the present invention can be used to selectively carry chemotherapeutic drugs into cancer cells, greatly enhancing the efficacy and safety of cytotoxic therapeutic agents.

[0385] We determined the pharmacokinetics of TPTC / TC-CQDs in HeLa tumor-bearing mice. TPTC or TPTC / TC-CQDs were injected intravenously at a dose equivalent to 10 mg / kg TPTC, and blood was collected at different time points. TPTC in plasma was quantified by high-performance liquid chromatography (HPLC). Figure 4 As shown in c, delivery via LAAM TC-CQDs significantly enhanced the blood circulation and bioavailability of TPTC, with the half-life of TPTC increasing from 5 to 40 minutes and the area under the curve (AUC) increasing from 13.4 to 138.1. The second group of mice that received the same treatment had their biodistribution of TPTC characterized based on TPTC fluorescence. As expected, free TPTC was not specifically and widely distributed throughout the body. In contrast, TPTC / TC-CQDs exhibited similar kinetic properties to LAAM TC-CQDs ( Fig.46 ). By 8 hours, TPTC could only be detected in tumors. Ex vivo imaging of isolated organs confirmed that LAAM TC-CQDs were able to deliver TPTC to tumors with high specificity ( Fig.47 ). Quantitative results based on TPTC FL intensity showed that the concentration of TPTC delivered via LAAM TC-CQDs increased by 13.8 times in tumors, while significantly reducing the accumulation of TPTC in other organs ( Figure 4 d).

[0386] We evaluated TPTC / TC-CQDs for cancer therapy using HeLa tumor-bearing mice. 3Mice were randomized and received intravenous injections of saline, TPTC (10 mg / kg), or TPTC / TC-CQDs (equivalent to 10 mg / kg TPTC) on days 1, 4, 8, and 12. After 15 days, tumors in mice treated with TPTC / TC-CQDs shrank to undetectable levels; in contrast, tumors in mice receiving free TPTC continued to grow, albeit at a slower rate than in control mice ( Figure 4 All mice treated with TPTC / TC-CQDs survived for more than 60 days; in contrast, mice treated with TPTC and saline survived for 40 and 20 days, respectively ( Fig.48 The significant antitumor effect of TPTC / TC-CQDs was not unique to HeLa tumors; we treated mice with left-flank A549 tumors and right-flank HeLa tumors in the same manner. After 20 days of treatment, both tumors were eradicated ( Fig.49 and 50 ).

[0387] TPTC / TC-CQDs have little toxicity to mice. Fig.51 As shown, treatment with TPTC / TC-CQDs did not induce significant weight loss throughout the study. In contrast, weight loss was observed in the group treated with free TPTC. Consistent with this finding, H&E analysis of major organs isolated from mice treated with TPTC / TC-CQDs did not detect inflammatory infiltration or pathological lesions ( Fig.52 ). However, free TPTC-treated mice developed significant myocardial fiber disruption, diffuse ballooning degeneration of hepatocytes, pulmonary congestion, and focal tubular necrosis. Complete blood count analysis showed that standard parameters, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), red blood cell distribution width (RDW), platelet distribution width (PDW), platelet volume (MPV), and platelet hematocrit (PCT), were within the normal range in mice treated with TPTC / TC-CQDs compared with saline control mice ( Fig.53 ), indicating that TPTC / TC-CQDs are blood compatible. Serum biochemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) to detect liver function, and urea nitrogen (UREA) and creatinine (CREA) to detect renal function, did not detect abnormalities in TPTC / TC-CQDs-treated mice ( Fig.54 ).

[0388] Overall, these data suggest that LAAM CQDs can be used as a safe carrier for the delivery of tumor-specific chemotherapeutic drugs, with the delivery of LAAM CQDs significantly enhancing the therapeutic efficacy while reducing the systemic toxicity of the loaded therapeutic agents.

[0389] LAAM CQDs as a new class of CQDs for tumor-specific imaging and drug delivery

[0390] To identify the molecular structure that explains the tumor specificity observed in LAAM TC-CQDs, we systematically analyzed a series of TC-CQDs analogs. First, we determined whether the carboxyl group, the amino group, or both were required. We reduced the TC-CQDs to NH2-NH2 groups by hydrazine hydrate (NH2-NH2). 19 Coupling reaction of EDC / NHS with acetic acid 18 , removing the carboxyl and amino groups at the edge of LAAM TC-CQDs ( Figure 55-56 The synthesized NH2- and COOH-free LAAM TC-CQDs were evaluated in HeLa and CCC-ESF-1 cells. Figure 5 As shown in a, b, the removal of the carboxyl or amino groups abolished the tumor-specific permeability of LAAM TC-CQDs, and the resulting CQDs non-selectively permeated cancer cells and non-cancerous cells. Flow cytometry analysis ( Fig.58 ) showed that compared with unmodified LAAM TC-CQDs, the uptake of LAAM TC-CQDs without NH2 groups and COOH groups was reduced by 46% and 48%, respectively, in HeLa cells, and the uptake was increased by 414% and 613% in non-cancerous CCC-ESF-1 cells ( Figure 5 c). Next, we tested whether the presence of carboxyl and amino groups at the same α position is essential. 2,6-CQDs with the same number of carboxyl and amino groups were synthesized using 2,6-diaminoanthraquinone (2,6-DAAQ) and CA and their structures and optical properties were characterized ( Figure 59-64 and Table 5). All carboxyl and amino groups of 2,6-CQDs were located at different positions in LAAM TC-CQDs. LCSM and flow cytometry analysis showed that the altered carboxyl and amino positions resulted in a complete loss of tumor-specific permeability; 2,6-CQDs permeated HeLa and CCC-ESF-1 cells with comparable efficiency ( Figure 5 ac and Fig.65). We evaluated whether the number of paired α-carboxyl and amino groups correlates with tumor-specific permeability. 1,4-CQDs and 1,5-CQDs were synthesized under the same conditions as LAAMTC-CQDs, whereas TAAQ was replaced by 1,4-diaminoanthraquinone (1,4-DAAQ) or 1,5-diaminoanthraquinone (1,5-DAAQ). The structural and optical properties of both CQDs were characterized ( Figure 59-64 and Table 5). 1,4-CQDs are structurally similar to LAAM TC-CQDs but have half the pairing groups to maintain tumor-specific permeability ( Figure 5 a). However, their permeation efficiency is significantly lower, only 61% of that of LAAM TC-CQDs ( Figure 5 b, c). 1,5-CQDs, which are also structurally similar to LAAM TC-CQDs and have the same number of pairing groups, permeate HeLa cells with comparable efficiency to LAAM TC-CQDs ( Figure 5 ac). Finally, we determined whether the backbone structure of LAAM TC-CQDs is important. Phe-CQDs were synthesized using Phe, which carries a paired α-carboxyl and amino group at the α position but has a significantly different backbone from LAAM TC-CQDs, and their structures and optical properties were characterized ( Figure 65-69 ). Evaluation of HeLa and CCC-ESF-1 cells found that Phe-CQDs maintained tumor-specific permeability, although the uptake of Phe-CQDs in HeLa cells was 36% of that of LAAM TC-CQDs ( Figure 5 ac and Fig.70 The lower permeability of Phe-CQDs may be attributed to the poor water solubility of Phe-CQDs, which cannot be well dispersed in the solution, thereby reducing cellular uptake.

[0391] Table 5. Optimized Cartesian coordinates of a FL structural unit of 1,4-CQDs, 1,5-CQDs and 2,6-CQDs in the ground state and excited state

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400] In conclusion, we found that the observed high tumor specificity is not unique to LAAM TC-CQDs, but rather to a class of CQDs bearing paired α-carboxyl and amino groups. It appears that the anchoring of the α-carboxyl and amino groups disguises the CQDs as large amino acids, thereby triggering LAT1-mediated tumor-specific interactions. We designate CQDs bearing paired α-carboxyl and amino groups as “large amino acid mimicking CQDs” or LAAM CQDs. Structurally, the position and number of carboxyl and amino groups determine the tumor specificity and cell penetration efficiency of LAAMTC-CQDs.

[0401] Pharmaceutical compositions, compositions and other related uses

[0402] The compounds and CQDs disclosed herein can be used as drugs for imaging or treatment. When used in this way, they can be mixed with one or more pharmaceutically acceptable carriers or excipients.

[0403] Thus, the present invention provides a pharmaceutical composition comprising a compound, particle or CQD(s) as described herein, mixed with at least one pharmaceutically acceptable carrier or excipient. Optionally, the pharmaceutical composition comprises at least two pharmaceutically acceptable carriers or excipients. Suitable excipients and carriers for pharmaceutical compositions of these compounds for therapeutic or imaging purposes are known in the art. Since the compounds and CQDs of the present invention are generally water-soluble, they are generally formulated into aqueous solutions / suspensions, for example, in sterile water, or isotonic aqueous solutions containing saline, glucose, lactose, etc. For imaging purposes, they are generally administered parenterally and formulated accordingly.

[0404] In yet another aspect, the present invention provides the use of the compounds and particles as described above for the manufacture of a medicament.

[0405] In one aspect, the present invention provides pharmaceutical compositions comprising compounds described herein or CQDs for use in treatment. In some embodiments, the use in treatment is for the treatment of one or more tumors or cancers selected from sarcoma, epidermal cancer, fibrosarcoma, cervical cancer, gastric cancer, skin cancer, leukemia, lymphoma, lung cancer, non-small cell lung cancer, colon cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancer, pancreatic cancer, bladder cancer, esophageal cancer, tongue cancer and skin malignant melanoma.

[0406] formula

[0407] Any suitable formulation of the compounds of the present invention can be prepared. In general, see Remington's Pharmaceutical Sciences, (2000) Hoover, JE editor, 20th edition, Lippincott Williams and Wilkins Publishing Company, Easton, Pa., pages 780-857. Select a formulation suitable for the appropriate route of administration. In the case where the alkalinity or acidity of the compound is sufficient to form a stable non-toxic acid or base salt, it may be appropriate to administer the compound in the form of a salt. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form physiologically acceptable anions, such as toluenesulfonates, mesylates, acetates, citrates, malonates, tartrates, succinates, benzoates, ascorbates, α-ketoglutarates, and α-glycerophosphates. Suitable inorganic salts can also be formed, including hydrochlorides, sulfates, nitrates, bicarbonates, and carbonates. Pharmaceutically acceptable salts are obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids are also prepared.

[0408] When the compound under consideration is administered in a pharmaceutical composition, it is contemplated that the compound can be formulated in admixture with pharmaceutically acceptable excipients and / or carriers. For example, the compound under consideration can be administered orally as a neutral compound or a pharmaceutically acceptable salt, or intravenously in normal saline. Conventional buffers such as phosphates, bicarbonates or citrates can be used for this purpose. Of course, a person of ordinary skill in the art can modify the formulation within the teachings of the specification to provide a variety of formulations for a specific route of administration. In particular, the compound under consideration can be modified to make it more soluble in water or other media, for example, which can be easily achieved by subtle modifications (salt preparations, esterification, etc.) by a person of ordinary skill in the art. It is also common technology in the art to modify the route of administration and dosage regimen of a particular compound to manage the pharmacokinetics of the current compound so that it has the greatest beneficial effect on the patient.

[0409] A person skilled in the art can modify the formulation within the scope of the instructions to provide multiple formulations for specific routes of administration. In particular, the compound can be modified to make it more soluble in water or other media. It is also within the ordinary skill of the art to modify the route of administration and dosage regimen of a specific compound to manage the pharmacokinetics of the current compound so that it produces the greatest beneficial effect on the patient.

[0410] Drug combinations

[0411] The methods of embodiments of the present invention comprise administering an effective amount of at least one exemplary compound disclosed herein; optionally, the compound may be administered in combination with one or more additional therapeutic agents, particularly known therapeutic agents useful for treating the condition or disease afflicting the subject by the methods of the present invention.

[0412] The additional active ingredients can be administered in separate pharmaceutical compositions from at least one exemplary compound of the present invention, or can be included in a single pharmaceutical composition with at least one exemplary compound of the present invention. The additional active ingredients can be administered simultaneously, before or after at least one exemplary compound of the present invention.

[0413] Methods of using exemplary compounds and pharmaceutical compositions thereof

[0414] To practice the methods of the present invention, the compounds and pharmaceutical compositions thereof with formulations can be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally, vaginally, by implanted reservoirs or other administration methods. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrathecal, intracerebral, intrathecal and intracranial injection or infusion techniques.

[0415] When the carbon quantum dots of the present invention are used to image tumors or tissues in a subject, they are usually administered parenterally, and often by intravenous injection or infusion. Suitable formulations and methods for such administration are known in the art.

[0416] Sterile injection compositions, such as sterile injection water or oily suspensions, can be prepared according to known techniques in the art using appropriate dispersants or wetting agents and suspending agents (if necessary). Sterile injection preparations can also be sterile injection solutions or suspensions in non-toxic parenteral acceptable diluents or solvents. The preferred carrier of the CQDs of the present invention is sterile water, which can be changed by adding appropriate buffers and other excipients. Acceptable carriers that can be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. Suitable carriers and other pharmaceutical composition components are usually sterile.

[0417] In addition, sterile fixed oils are often used as solvents or suspension media (e.g., synthetic mono- or di-glycerides). Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injections, as are pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylene forms. These oil solutions or suspensions may also contain long-chain ethanol diluents or dispersants, or carboxymethyl cellulose or similar dispersants. Various emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms may also be used in the formulation.

[0418] The composition for oral administration can be any oral acceptable dosage form, including but not limited to tablets, capsules, emulsifiers and aqueous suspensions, dispersants and solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate can also be added. For oral capsule forms, useful diluents include lactose and dried corn starch. When oral aqueous suspensions or emulsifiers, the active ingredient can be suspended or dissolved in an oily phase together with an emulsifier or suspending agent. If necessary, certain sweeteners, flavorings or coloring agents can be added. Nasal aerosols or inhalation compositions can be prepared according to well-known techniques in pharmaceutical formulation technology, and can be prepared into solutions in, for example, physiological saline, using appropriate preservatives (e.g., benzyl alcohol), absorption promoters to improve bioavailability, and / or other solubilizers or dispersants known in the art.

[0419] In addition, the compounds and compositions of the present invention can be administered alone or in combination with other therapeutic agents such as anticancer agents to treat various cancers or tumors that can be treated with the compounds and compositions of the present invention. The combined therapy of the present invention includes the administration of at least one exemplary compound of the present invention and at least one other pharmaceutically active ingredient. The compounds of the present invention and other pharmaceutically active ingredients can be administered alone or together. The amount of active ingredients and pharmaceutically active agents and the relative administration time will be selected to achieve the desired combined treatment effect.

[0420] example

[0421] Materials and reagents

[0422] 1,4-Diaminoanthraquinone (1,4-DAAQ, 90%), 1,5-diaminoanthraquinone (1,5-DAAQ, 90%), 2,6-diaminoanthraquinone (2,6-DAAQ, 90%), 1,4,5,8-tetraaminoanthraquinone (TAAQ, 90%), and citric acid (CA, 99.8%) were purchased from Sigma-Aldrich. Dichloromethane (99.5%) and methanol (99.5%) were provided by Beijing Chemical Reagent Co., Ltd. Cell Counting Kit-8 (CCK-8) was provided by Dojindo Laboratory (Japan). All water used in the experiments was purified by Millipore system (ULUPURE, Chengdu, China). All BALB / c female mice were purchased from Beijing Laboratory Animal Research Center, and the weight of each mouse was 18-20 g.

[0423] Synthesis of TC-CQDs

[0424] First, 0.04g CA and 0.03g TAAQ were mixed in 10mL pure water, and then the solution was transferred to a polytetrafluoroethylene-lined reactor (25mL). After heating in an oven at 180°C for 2 hours, it was naturally cooled to room temperature to obtain a blue suspension. The crude product was then purified by silica gel column chromatography using a mixture of dichloromethane and methanol (10:1) as an eluent. After removing the solvent and further drying under vacuum, purified TC-CQDs were finally obtained with a yield of 25wt%.

[0425] Characterization

[0426] Transmission electron microscopy images (TEM) were taken on a JEOL JEM 2100 transmission electron microscope (FEI). Atomic force microscopy (AFM) images were obtained by MultiMode V SPM (VEECO). X-ray diffraction (XRD) patterns were performed using X-ray diffraction with Cu-Kα radiation (XRD, PANalytical X'Pert Pro MPD). Raman spectra were measured using a laser confocal micro-Raman spectrometer (LabRAM Aramis). X-ray photoelectron spectroscopy (XPS) was measured using an ESCALab 250Xi electron spectrometer from VG Scientific with 300W Al Kα radiation. UV-visible absorption and fluorescence spectra were recorded on a UV-2600 spectrophotometer and a PerkinElmer-LS55 fluorescence spectrometer, respectively. Fourier transform infrared spectroscopy (FT-IR) was measured using a Nicolet 380 spectrometer. 13 C NMR spectra were recorded on a 400 MHz Bruker Advance III spectrometer using CD3OD as solvent, and chemical shift values ​​are reported in parts per million.

[0427] Calculation method

[0428] The ground state and the first excited state of a fluorescent structural unit of CQDs were obtained by theoretical calculations using density functional theory (B3LYP / 6-31G(d)). The geometric parameters of the ground state were optimized and verified at the B3LYP / 6-31G(d) level, and the geometric parameters of the first excited state were optimized using TD-B3LYP / 6-31G(d).

[0429] Cell culture and materials. Cells other than BCSCs were cultured in a 10% fetal bovine serum (Invitrogen), 100 unit mL -1 Penicillin and 100 μg mL -1The cells were cultured in DMEM or RPMI medium (Invitrogen) containing streptomycin (Invitrogen) at 37°C in an incubator containing 5% carbon dioxide. Human BCSCs were enriched and cultured as previously reported. 34 Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich. LAT1 antibody was purchased from Novus Biologicals.

[0430] Synthesis of CQDs. LAAM TC-CQDs were synthesized by treating CA with TAAQ in aqueous solution at 180 °C for 2 h, and the obtained LAAM TC-CQDs were purified by silica gel column chromatography.

[0431] Photothermal effect of LAAM TC-CQDs aqueous solution. To evaluate the photothermal effect in aqueous solution, aqueous solutions of LAAM TC-CQDs with different concentrations (0-10 μg / mL) were exposed to 650 nm laser irradiation (0.5 W / cm 2 ), the irradiation direction was from the top to the bottom of the cuvette, and it lasted for 5 minutes. An equal amount of pure water was used as a control under the same laser irradiation. The real-time temperature was recorded every 30 seconds by the infrared thermal imager.

[0432] In view of its effective NIR absorption properties, the photothermal properties of TC-CQDs aqueous solution were investigated. 2 ) to monitor the temperature of TC-CQDs aqueous solution with different concentrations (0-10 μg / mL), such as Fig.17 As shown in a. No obvious temperature increase was observed in the control sample of pure water, while TC-CQDs (10 μg / mL) could rapidly trigger a temperature increase under short-term (5 minutes) light irradiation and exhibit concentration-dependent hyperthermia (above 42°C), leading to irreversible damage to tumor cells. 7,8 To further investigate the photothermal stability and transduction efficiency of TC-CQDs, an aqueous solution of TC-CQDs (10 μg / mL, 3 mL) was illuminated by a 650 nm laser (0.5 W / cm 2 ) was continuously irradiated until the steady-state temperature was reached, at which point the laser irradiation was stopped and the suspension was allowed to cool naturally. The temperature change during the heating-cooling process was monitored for 5 cycles, and the following results were obtained: Fig.17 Heat generation and heat dissipation curves shown in b. During each laser on / off cycle, the temperature rise is almost equal to 25.6 °C, indicating that TC-CQDs have better photothermal stability than widely used organic dyes and inorganic nanomaterials with surface plasmon resonance. 3-15 The photothermal conversion efficiency (η) of TC-CQDs was calculated using the following equation: 16 :

[0433]

[0434] Where h is the heat transfer coefficient, S is the surface area of ​​the container, T max is the maximum system temperature, T surr is the ambient temperature, Q Dis is the baseline energy input to the sample cell, I is the incident laser power and A 650 is the absorbance of TC-CQDs at a wavelength of 650nm. Fig.17 c,(T max -T surr ) is 25.6°C, I is 0.5W / cm 2 And A 650 It is 1.4908. Dis The heat dissipated by the light absorbed by the quartz sample cell itself was measured to be 37.2 mW using a quartz cuvette containing pure water and no TC-CQDs. Therefore, only hS is unknown in the calculation of η.

[0435] In order to obtain hS, the dimensionless driving force temperature (θ) and the sample system time constant τ are introduced s .

[0436]

[0437]

[0438] Where m and C p are the mass and heat capacity of water respectively. When θ and τ s When substituting into the total energy balance equation (4) of the following system, equation (5) is obtained.

[0439]

[0440] Where Q NC is the energy input to TC-CQDs, and Q Surr It is the conduction of heat away from the system surface through the air.

[0441]

[0442] During the cooling stage of the aqueous dispersion of TC-CQDs, the light source was turned off and Q NC +Q Dis =0.

[0443]

[0444] t=-τ s lnθ (7)

[0445] Therefore, by applying the linear time data of the cooling period (after 300 s) and the negative natural logarithm of the driving force temperature, the time constant of the system heat transfer is determined to be τ s =83.86s( Fig.17 d). In addition, m is 0.3g, C p is 4.2 J / g. Therefore, according to equation (3), hS is derived to be 16.1 mW / ℃. Substituting hS of 16.1 mW / ℃ into equation (1), the η of TC-CQDs can be calculated to be 77.4%, which is comparable to previously reported photothermal conversion agents (PTCA), such as golden bell flower (74%), gold nanocages (64%), gold nanorods (55%), dopamine-melanin nanospheres (40%), carbon dots (38.5%), Cu9S5 nanocrystals, and Cu 2-x Se nanocrystals (22%), etc. s3-s15 .

[0446] Flow cytometry. According to our previously reported method 22 Sorting of MDA-MB-231SP cells 22 Briefly, cells harvested at approximately 85% confluence were plated at 1 × 10 6 The cells were resuspended at a density of 1×10 cells / mL in RPMI-1640 supplemented with 2% fetal bovine serum (FBS) and incubated at 37°C with 5 μg / mL Hoechst 33342. After 90 minutes, the cells were plated at 1×10 6 The cells were suspended in cold PBS at a concentration of 10 cells / mL, filtered through a 40 μm cell strainer to remove cell aggregates, stained with 1 μg / mL of propidium iodide (PI), and analyzed and sorted using FACSDiva (Becton Dickinson, USA). To characterize SP cells, freshly sorted SP cells were suspended in cold PBS and stained with anti-human CD44-FITC and CD24-PE or their appropriate isotype controls on ice for 30 minutes. The cells were washed three times with cold PBS, resuspended in 400 mL of cold PBS, and analyzed using a FACScan flow cytometer (BD, Ann Arbor, MI). To characterize the interaction between CQDs and cells, selected cells were placed on 6-well plates and treated with 10 μg / mL of LAAM TC-CQDs. Cells without CQDs treatment served as controls. After 12 hours, the cells were washed with fresh medium, trypsinized, resuspended in PBS containing 0.5% FBS, and analyzed using BD FACSCalibur (BD Biosciences, USA). Data analysis was performed using FlowJo7.6.

[0447] Confocal laser scanning microscopy imaging.To determine the uptake of CQDs, cells were placed on glass slides and treated with 10 μg / mL of LAAM TC-CQDs. After 8 hours, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes at room temperature, and then DAPI was added for nucleus staining. Finally, the slides were washed three times, fixed, sealed with coverslips, and imaged with a laser confocal scanning microscope (Leica TCS-SP8, Germany) with an excitation / emission of: 561 / 700. To determine the effect of Leu, Phe, Gly, or BCH on the uptake of CQDs, HeLa cells were placed and treated with Leu, Phe, Gly, or BCH. After 4 hours, LAAM TC-CQDs were added to the cells. After another 8 hours, the cells were washed, fixed, and imaged.

[0448] PA imaging of LAAM TC-CQDs. LAAM TC-CQDs at different concentrations (0, 2, 4, 6, 8 and 10 μg / mL) were added to agarose tubes (37°C) and scanned using a PA imager (mode: iTheraMedical Co. MOST in Vision128; excitation wavelength range 640-840 nm, interval 5 nm). PA signals were recorded.

[0449] Cytotoxicity assay. Cells were plated at 1×10 4 The cells were placed in a 96-well plate at a density of 100 cells / mL and treated with different concentrations of LAAM TC-CQDs, TPTC, or TPTC / TC-CQDs. After 12 hours, the culture medium was removed and washed with PBS. 100 μL of fresh culture medium containing 10 μL of CCK-8 (Sigma) was added to each well. After culturing at 37°C for 2 hours, the absorbance at 450 nm was measured using a microplate reader.

[0450] NIRFL imaging of LAAMTC-CQDs in vivo. Female BALB / c nude mice were maintained in a sterile environment and used for in vivo imaging of LAAMTC-CQDs. This project was approved by the Animal Use Committee of Beijing Normal University and the IACUC of Yale University. To establish HeLa tumor-bearing mice, we prepared 2 × 10 6 HeLa cells were subcutaneously inoculated into female BALB / c mice. When the tumor volume reached about 100 mm 3 LAAM TC-CQDs (5 mg / kg) were intravenously injected into mice. NTR FL images were collected at 1, 2, 4, 6, 8, and 10 h using an animal optical imaging system (IVIS Lumina III, Caliper Life Sciences).

[0451] In vivo PA imaging of LAAM TC-CQDs.Pre-contrast data with excitation wavelengths of 640 to 840 nm were obtained before intravenous injection of LAAM TC-CQDs. Tumor-bearing mice were treated with LAAM TC-CQDs at a dose of 5 mg / kg. Post-contrast data were obtained at 2, 4, 6, 8, and 10 hours after injection. PA images were reconstructed using data obtained from all 128 sensors in each view using a modified back-projection algorithm.

[0452] Therapeutic evaluation in tumor-bearing mice. HeLa cells were subcutaneously inoculated into female BALB / c mice. When the tumor volume reached approximately 100 mm 3 At 1 d, mice were intravenously injected with saline, TPTC, or TPTC / TC-CQDs (n=5). Changes in tumor volume and body weight were monitored daily. The tumor volume was calculated according to the following formula: V=D×d 2 / 2 (where D and d are the longest and shortest diameters of the tumor, respectively, measured with a vernier caliper). The relative tumor volume was calculated as V / V0 (V0 is the initial tumor volume at the start of treatment).

[0453] Toxicity Evaluation in Mice. Female BALB / c mice were intravenously injected with TPTC (10 mg / kg, 0.05 mL per mouse) or TPTC / TC-CQDs (10 mg / kg TPTC, 0.05 mL per mouse) (n=5). Mice treated with normal saline were used as controls. At selected time points, blood samples were collected in heparinized microhematocrit tubes and centrifuged at 3000 rpm for 10 minutes. At the end of the study, mice were euthanized. Major organs were removed, fixed in formalin and analyzed, including heart, liver, spleen, kidney and lung.

[0454] TPTC / LAAMTC-CQDs were used to treat HeLa tumors. The delivery of TC-CQDs via LAAM significantly enhanced the toxicity of TPTC to HeLa cells, but reduced its toxicity to non-cancerous cells (see Fig.71 a). At a concentration equivalent to 5 μg / mL TPTC, TPTC / LAAM TC-CQDs killed all HeLa cells but showed no toxicity to noncancerous CCC-ESF-1 cells. We determined the pharmacokinetics of TPTC / LAAM TC-CQDs in HeLa tumor-bearing mice. For comparison, TPTC-loaded liposomes were synthesized using HSPC:cholesterol:PEG 2000-DSPE (molar ratio 56:39:5) and designated as TPTC / liposomes. The same lipid composition was used in several FDA-approved liposome formulations, including TPTC / LAAM TC-CQDs with a dose equivalent to 2 mg / kg TPTC and free TPTC or TPTC / liposomes in the control group were injected intravenously. Blood was collected at different time points. TPTC in plasma was quantified by high performance liquid chromatography (HPLC). Fig.71 b, Delivery by LAAMTC-CQDs significantly enhanced the blood circulation time and bioavailability of TPTC, with the half-life of TPTC increasing from 5.4 to 40.1 minutes and the area under the curve (AUC) increasing from 13.4 to 138.1. In contrast, the half-life and AUC of TPTC / liposomes were 11.8 minutes and 55.1 ( Fig.71 b).

[0455] Next, we examined the biodistribution of TPTC in a second group of mice that received the same treatment. As expected, free TPTC was widely distributed in the body without specificity. In contrast, TPTC / LAAM TC-CQDs exhibited similar kinetic properties as LAAM TC-CQDs. By 8 hours, TPTC could only be detected in tumors. Quantification based on the FL intensity of TPTC showed that TPTC delivered via LAAM TC-CQDs increased the concentration of TPTC in tumors by 16.7-fold, while significantly reducing the accumulation of TPTC in other organs. The amount of LAAM TC-CQDs accumulated in tumors accounted for 15.3% of the total injected dose. In contrast, a large amount of TPTC / liposomes was found in the liver. Liposomal delivery increased the concentration of TPTC in tumors by 6.7-fold ( Fig.71 c).

[0456] We also evaluated the treatment of HeLa tumors with TPTC / LAAM TC-CQDs. When the tumor volume reached approximately 100 mm 3 Mice were randomized and received intravenous injections of saline, TPTC (2 mg / kg), TPTC / liposomes, or TPTC / LAAM TC-CQDs (all at a dose equivalent to 2 mg / kg TPTC) on days 1, 4, 8, and 12. After 15 days, tumors in mice treated with TPTC / LAAM TC-CQDs shrank to undetectable levels. In contrast, tumors in mice treated with free TPTC or TPTC / liposomes continued to grow, although at a slower rate than in control mice ( Fig.71 d). All mice treated with TPTC / LAAM TC-CQDs survived for more than 50 days. However, the median survival of mice treated with saline, TPTC, and TPTC / liposomes was limited to 25, 30, and 35 days, respectively.

[0457] We repeated the characterization study using A549 tumors. The experiments were performed following the same procedures as described above. Similar to the results in HeLa tumors, we found that the delivery of LAAM TC-CQDs significantly enhanced the toxicity of TPTC to A549 cells ( Fig.71 e), increasing the half-life and AUC of TPTC in the blood circulation by 5.5 times (half-life from 5.9 minutes to 32.5 minutes) and 13.9 times (AUC from 15.9 to 221.5) respectively. Fig.71 f). It also increased the accumulation of TPTC in tumors by 10.3-fold, while reducing the accumulation of TPTC in other organs ( Fig.71 h), and significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice ( Fig.71 h). The overall delivery efficiency to the tumor was 15.8%. Consistently, treatment with TPTC / liposomes failed to achieve an inhibitory or therapeutic effect comparable to that achieved with the addition of LAAM TC-CQDs ( Fig.71 fh).

[0458] Characterization of the stability of LAAMTC-CQDs for cancer imaging. The tumor microenvironment is acidic (pH between 6.2 and 6.9) and is saturated with proteases and amino acids. 28,29 Therefore, we characterized the FL stability of LAAM TC-CQDs at pH 6-8 in the presence of amino acids or matrix metalloproteinase-2 (MMP-2). We found that the FL emission spectra of LAAM TC-CQDs remained unchanged under all tested conditions ( Fig.72 ). Next, we characterized the photostability of LAAM TC-CQDs after long-term storage or irradiation. Fig.75 The results in showed that all tested conditions, including storage at room temperature for 3 months, continuous irradiation with a 500W xenon lamp for 12 hours, and continuous UV irradiation at 365nm for 5 hours, did not significantly change the FL emission spectrum. Finally, we evaluated the effect of prolonged incubation in serum on the size and FL emission of LAAM TC-CQDs. We found that the diameter and FL emission intensity of LAAM TC-CQDs did not change after 30 days of incubation in serum ( Fig.75 ).

[0459] Statistical analysis

[0460] All data were collected in triplicate and reported as mean and standard deviation. Comparisons between two conditions were assessed using unpaired t-tests. One-way ANOVA was performed to determine the statistical significance of treatments associated with survival. p<0.05 (*), 0.01 (**), and 0.0001 (****) were considered significant.

[0461] The above detailed description is provided to assist those skilled in the art in implementing the present invention. However, the invention described and claimed herein is not limited in scope by the specific embodiments disclosed herein, as these embodiments are intended to illustrate several aspects of the present invention. Any equivalent embodiments are intended to be within the scope of the present invention. In fact, various modifications of the present invention, other than those shown and described herein, will make the above description apparent to those skilled in the art, and these modifications do not depart from the spirit or scope of the present invention. Such modifications are also intended to fall within the scope of the appended claims.

[0462] All publications, patents, patent applications, and other references cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that it is prior art to the present invention.

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Claims

1. A carbon quantum dot, which: Include A fused polycyclic ring system containing 8 or more 6-membered rings, each of which is aromatic or partially aromatic, and the polycyclic ring system is substituted with at least one group of formula A: Where R 1 is H or C1-C3 alkyl optionally substituted by one or two groups selected from halogen, -OH, -OMe, -NH2, -SMe, -COOH and -CONH2, and the dotted bond indicates the position at which the group of the chemical formula A is attached to the fused polycyclic system; And the carbon quantum dots: d) can selectively enter cells that express the large neutral amino acid transporter LAT1 or LAT1 subunit; wherein the LAT1 subunit refers to the 4F2hc / CD98 heavy subunit protein encoded by the SLC3A2 gene and the CD98 light subunit protein encoded by the SLC7A5 gene; and e) can selectively enter tumor or cancer cells, wherein the weight percentage of carbon atoms in the carbon quantum dots is 20% or higher.

2. The carbon quantum dot according to claim 1, wherein R 1 is C1-C3 alkyl which is optionally substituted by one or two groups selected from halogen, -OH, -OMe, -SMe, -COOH and -CONH2.

3. The carbon quantum dots according to claim 1 or 2, having a molecular weight of 500 to 500,000.

4. The carbon quantum dots according to claim 1 or 2, which are formed by reacting at least two different precursors, at least one precursor containing carboxyl and hydroxyl groups and at least another precursor containing multiple 6-membered aromatic rings and at least two amino groups. The carbon quantum dots according to claim 4 , wherein at least one precursor containing carboxyl groups and hydroxyl groups is citric acid CA.

6. The carbon quantum dots according to claim 4, wherein the precursor comprising a plurality of 6-membered aromatic rings and at least two amino groups is 1,4,5,8-tetraaminoanthraquinone TAAQ, 1,4-diaminoanthraquinone 1,4-DAAQ or 1,5-diaminoanthraquinone 1,5-DAAQ.

7. The carbon quantum dots according to claim 1 are formed by reacting at least two different precursors, at least one precursor comprises an α-aminocarboxylic acid compound, and the second precursor comprises a C1-C8 alcohol. 8 . The carbon quantum dot according to claim 7 , wherein the precursor comprising the α-aminocarboxylic acid compound is phenylalanine (Phe).

9. The carbon quantum dots according to claim 7 or 8, wherein the second precursor is ethanol.

10. The carbon quantum dots according to claim 1, wherein the relative uptake rate of large neutral amino acids by cells is at least 90%, or the LAT1 expression level of cells is at least 10 times higher than that of normal human bone marrow stromal cells. The carbon quantum dots according to claim 1 , wherein the LAT1 preferentially internalizes branched-chain amino acids and / or aromatic amino acids. 12 . The carbon quantum dots according to claim 11 , wherein the branched-chain amino acid is a proteinogenic branched-chain amino acid or a non-proteinogenic branched-chain amino acid, and the aromatic amino acid is a proteinogenic amino acid or a non-proteinogenic aromatic amino acid.

13. The carbon quantum dots according to claim 12, wherein the branched-chain amino acid is leucine, isoleucine, valine or 2-aminoisobutyric acid, and the aromatic amino acid is phenylalanine, tyrosine, arginine, tryptophan, histidine, thyroxine, 5-hydroxytryptophan or levodopa. The carbon quantum dots according to claim 1 , which are doped with N, S, P, B and / or O.

15. The carbon quantum dots according to claim 1, having a size or particle diameter ranging from 0.2 nm to 10 nm. The carbon quantum dots according to claim 1 , have an excitation wavelength ranging from 300 nm to 900 nm. The carbon quantum dots according to claim 1 , have an emission wavelength ranging from 400 nm to 1000 nm.

18. The carbon quantum dots of claim 1, configured to perform photoacoustic imaging upon irradiation.

19. The carbon quantum dots according to claim 18, which generate ultrasonic waves under the radiation of an excitation wavelength range of 600nm to 900nm.

20. The carbon quantum dots of claim 19, wherein the ultrasound wave is configured to form a photoacoustic image.

21. The carbon quantum dots of claim 1, emitting near-infrared fluorescence and configured to be photoacoustically imaged upon irradiation.

22. The carbon quantum dots of claim 21, configured for tumor or cancer imaging.

23. The carbon quantum dot of claim 1 or 2, which is configured to selectively enter cancer cells.

24. The carbon quantum dots of claim 23, configured to selectively enter the nucleus of a cancer cell.

25. The carbon quantum dots of claim 22, wherein the tumor or cancer is selected from sarcoma, epidermoid carcinoma, fibrosarcoma, cervical cancer, gastric cancer, skin cancer, leukemia, lymphoma, lung cancer, colon cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancer, pancreatic cancer, bladder cancer, esophageal cancer and tongue cancer.

26. The carbon quantum dots according to claim 1 or 2, which are configured to be able to selectively enter tumor cells.

27. The carbon quantum dots of claim 26, configured to selectively enter the nucleus of a tumor cell in a subject.

28. The carbon quantum dot of claim 27, wherein the subject is a mammal.

29. The carbon quantum dot according to claim 1 or 2, further comprising a releasable loading substance, wherein the carbon quantum dot can be covalently or non-covalently linked to the releasable loading substance.

30. The carbon quantum dots of claim 29, wherein the release of the releasable cargo is triggered by contact between the carbon quantum dots and target cells, tissues, organs or subjects, or by enzymatic cleavage, or by changes in physical and / or chemical parameters surrounding the carbon quantum dots.

31. The carbon quantum dot of claim 29, wherein the releasable cargo is a therapeutic agent, a prophylactic agent, a diagnostic agent, a labeling agent, a prognostic agent, an imaging agent, or a combination thereof.

32. The carbon quantum dot of claim 31, wherein the therapeutic agent comprises a small molecule, a macromolecular cell therapeutic agent, a conjugate, or a combination thereof.

33. The carbon quantum dots according to claim 32, wherein the small molecule refers to an organic compound with a molecular weight between 200 and 2000, and the macromolecule refers to a polypeptide or a polynucleotide.

34. The carbon quantum dots of claim 31 or 32, wherein the therapeutic agent comprises an anti-tumor or anti-cancer agent.

35. The carbon quantum dots according to claim 34, wherein the anticancer agent is a chemotherapeutic drug.

36. According to the carbon quantum dots of claim 35, the chemotherapy drug refers to topotecan hydrochloride TPTC, doxorubicin DOX or hydroxycamptothecin HCPT or a kinase inhibitor.

37. The carbon quantum dots according to claim 31 or 32, wherein the therapeutic agent is configured so as to be used to treat a disease or disorder in a subject.

38. The carbon quantum dots of claim 31, wherein the therapeutic agent is configured to be useful for treating a tumor or cancer.

39. A method for preparing the carbon quantum dots according to any one of claims 1 to 38, The method comprises a solvothermal synthesis using at least two different precursors, wherein at least one precursor comprises an α-hydroxycarboxylic acid compound, and at least one other precursor comprises a plurality of 6-membered aromatic rings and at least two amino groups; or The method comprises using at least one solvothermal synthesis method comprising phenylalanine or a phenylalanine analog and a C1-C8 alcohol as precursors.

40. The method of claim 39, wherein the precursor comprising the alpha-hydroxycarboxylic acid compound is citric acid.

41. The method of claim 39, wherein the other precursor is 1,4,5,8-tetraaminoanthraquinone TAAQ, 1,4-diaminoanthraquinone 1,4-DAAQ or 1,5-diaminoanthraquinone 1,5-DAAQ.

42. The method of claim 39, wherein the precursor comprising phenylalanine or a phenylalanine analog is phenylalanine.

43. The method of claim 39, wherein C 1- The C8 alcohol is ethanol.

44. The method of claim 39, comprising dissolving or dispersing the precursor molecules in a solvent to form a solution or a mixture, and heating the solution or the mixture at a temperature of 100°C to 300°C for 10 minutes to 72 hours.

45. The method of claim 44, wherein the solvent is water, C 1-10 alcohol, amide, ketone or sulfoxide.

46. ​​The method of claim 45, wherein the C 1-10 The alcohol refers to ethanol; the amide refers to formamide, N,N-dimethylformamide, dimethylacetamide or N-methylpyrrolidone; the ketone refers to acetone or 2-butanone; and the sulfoxide refers to dimethyl sulfoxide.

47. The method of any one of claims 39-46, further comprising separating or purifying the carbon quantum dots.

48. The method of claim 47, wherein the carbon quantum dots are separated or purified by filtration, centrifugation, or chromatography.

49. The method of claim 39, comprising: 1) mixing the precursor citric acid CA containing an α-hydroxycarboxylic acid compound and the other precursor TAAQ in water to form a mixture; 2) heating the mixture at 180° C. for 2 hours; 3) cooling the mixture to a lower temperature; and 4) Separating or purifying the carbon quantum dots from the mixture.

50. The method of claim 49, further comprising removing the solvent by drying under vacuum to obtain isolated or purified carbon quantum dots.

51. A composition comprising an optional pharmaceutical composition formed by mixing the carbon quantum dots described in any one of claims 1 to 38 and at least one pharmaceutically acceptable carrier or excipient.

52. The composition of claim 51, which is formulated for use as a therapeutic agent, a prophylactic agent, a diagnostic agent, a sensing agent, a labeling agent, a prognostic agent, an imaging application, or a combination thereof.

53. The composition according to claim 52, wherein the carbon quantum dots are linked or releasably linked to a cancer therapeutic agent, wherein the cancer therapeutic agent is topotecan hydrochloride TPTC, doxorubicin DOX or hydroxycamptothecin HCPT or a kinase inhibitor.

54. A kit comprising the carbon quantum dots described in any one of claims 1-38 or the composition described in any one of claims 51-53, and instructions for use of the carbon quantum dots or the composition.

55. The kit of claim 54, which can be used for sensing, labeling or imaging target cells, tissues or organs in a subject.

56. The kit of claim 54, which can be used for diagnosis, prognosis, stratification, risk assessment or treatment monitoring of a disease or disorder in a subject.

57. A pharmaceutical composition comprising an effective amount of the carbon quantum dots described in any one of claims 1-38, or the composition described in any one of claims 51-53, and a pharmaceutically acceptable carrier or excipient, and further comprising a therapeutic agent for treating cancer, wherein the therapeutic agent is releasably attached to the carbon quantum dots.

58. Use of an effective amount of the carbon quantum dots described in any one of claims 1-38 or the composition described in any one of claims 51-53 to manufacture a drug for treating or preventing a disease or condition in a desired subject, or for detecting the presence or absence of cancer cells or tissues in a desired subject.

59. The use according to claim 58, wherein the medicament is for treating or preventing a tumor or cancer in a subject.

60. The carbon quantum dots according to any one of claims 1-38, which can be used for therapeutic purposes.

61. A composition according to any one of claims 51-53 for therapeutic use.

62. The composition of claim 61, wherein the therapeutic use is the treatment or detection of cancer or tumor.

63. according to the composition described in any one of claims 51-53, wherein this carbon quantum dot is attached or fixed on solid surface or support.

64. The composition of claim 63, which is configured for detecting and / or isolating target cells from a sample.

65. The composition of claim 64, wherein the sample is a biological sample.

66. The composition of claim 63, configured for detecting and / or isolating tumor or cancer cells from a sample.

67. The composition of claim 63, configured for detecting and / or isolating circulating tumor or cancer cells from a biological sample.

68. The composition of claim 63, wherein the solid surface or support is a portion of a container, a tube, a plate, a microtiter plate.

69. The composition of claim 63, wherein the solid surface or support is part of an assay device selected from enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescence assay (IFA), nephelometry, flow cytometry, plasma resonance assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, inhibition assay, and affinity assay.

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