Methods for treating cancer

Abstract

The present invention provides a conjugate comprising: (a) a cancer targeting ligand, (b) a hydrophilic polymer of polyethylene glycol (PEG), polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA), or glucan, and (c) a flavonoid. The present invention also provides a micellar nanoparticle composition comprising: (a) an outer shell comprising the conjugate, (b) an inner shell comprising oligomeric (-)-epigallocatechin gallate (OEGCG), and optionally (c) a cancer therapeutic molecule encapsulated in the inner shell. In one embodiment, the nanoparticle composition has at least 70% of nanoparticles having a diameter between 20 nm and 500 nm or between 50 nm and 300 nm, and has a single main peak in the size distribution. The invention also provides a method of treating cancer by administering to a subject an effective amount of a nanoparticle composition of the invention. The cancer targeting ligands target a tumor and deliver an active ingredient to the tumor to treat cancer.

Description

Technical Field

[0001] The present invention relates to a conjugate comprising: (a) a cancer targeting ligand, (b) a hydrophilic polymer of polyethylene glycol (PEG), polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA) or dextran, and (c) a flavonoid, wherein the hydrophilic polymer is covalently bound to the flavonoid and the cancer targeting ligand. The present invention relates to micellar nanoparticles comprising: (a) an inner shell comprising oligo(-)-epigallocatechin gallate (OEGCG), (b) an outer shell comprising a cancer targeting ligand-hydrophilic polymer-EGCG conjugate, and optionally (c) a cancer therapeutic agent encapsulated in the inner shell. Background Art

[0002] Green tea catechins have health benefits that prevent cancer. Among tea catechins, (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and plays an important role in the beneficial effects of green tea leaves. EGCG has antioxidant, anti-inflammatory and immunomodulatory effects. EGCG has also been shown to effectively inhibit tumor growth and metastasis by targeting multiple signal transduction pathways that are critical for cancer cell survival.

[0003] Despite these desirable activities, the clinical application of EGCG is limited by its poor stability and low oral bioavailability. For example, EGCG is unstable and easily decomposes under physiological conditions. Therefore, the plasma concentration of EGCG required to achieve the desired therapeutic effect cannot be achieved after oral administration.

[0004] Cancer is a complex disease with multiple signaling pathways, and its treatment faces three major challenges. First, cancer is caused by human immune dysfunction. Immunomodulation that restores host immune function is essential for long-term treatment solutions. Second, a single therapeutic agent can only change one disease pathway, with limited efficacy, drug resistance and no response. Cancer cells can escape single-agent treatment through alternative signaling pathways. Third, drug toxicity and ineffective administration to target tissues are common challenges faced by cancer treatment because the tumor size is only a small fraction of the body size. Only a small portion of the administered drug reaches the targeted tissue, and most of the drug enters non-targeted normal tissues, which leads to low efficacy to the targeted tissue and high toxicity to normal tissues.

[0005] There is a need for a pharmaceutical composition and drug delivery system that overcomes the above challenges and effectively enters the target tissue without potential toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1The micellar composition of the present invention is shown, wherein the drug molecule is encapsulated within the micelle, and the micelle comprises a ligand-PEG-EGCG conjugate in the outer shell and oligomeric EGCG (OEGCG) in the inner shell.

[0007] Figure 2 The micellar composition of the present invention is shown, wherein the drug molecule is encapsulated within the micelle, and the micelle comprises the ligand-PEG-EGCG conjugate plus naked PEG-EGCG in the outer shell and oligomeric EGCG (OEGCG) in the inner shell.

[0008] Figure 3 Shown is a scheme for the chemical synthesis of RGD-PEG-EGCG via conjugation of the N-terminus of the RGD peptide to HOOC-PEG-EGCG.

[0009] Figure 4 Shown is a scheme for the chemical synthesis of TP12-PEG-EGCG via conjugation of the N-terminus of the TP12 peptide to HOOC-PEG-EGCG.

[0010] Figure 5 The successful formulation of RGD-MINC-doxorubicin (A) and TP12-MINC-doxorubicin micelles (B) are shown.

[0011] Figure 6 Tumor cell uptake of MINC-doxorubicin, RGD-doxorubicin, or TP12(TfR)-MINC-doxorubicin was shown by measuring the fluorescence signal.

[0012] Figure 7 Tumor cell viability under treatment with MINC-doxorubicin, RGD-MINC-doxorubicin, or TP12(TfR)-MINC-doxorubicin is shown.

[0013] Figure 8 Shown is a scheme for the chemical synthesis of RGD-PEG-EGCG via conjugation of the C-terminus of the RGD peptide to HO-PEG-EGCG.

[0014] Fig. 9 Shown is a scheme for the chemical synthesis of TP12-PEG-EGCG via conjugation of the C-terminus of the TP12 peptide to HO-PEG-EGCG.

[0015] Fig.10 Shown is a scheme for the chemical synthesis of folate-PEG-EGCG via conjugation of the COOH group of folate to HO-PEG-EGCG.

[0016] Fig.11Shown is a scheme for the chemical synthesis of RGD-PLA-EGCG via conjugation of the N-terminus of the RGD peptide to HOOC-PLA-EGCG.

[0017] Fig.12 Shown is a scheme for the chemical synthesis of RGD-PLGA-EGCG via conjugation of the N-terminus of the RGD peptide to HOOC-PLGA-EGCG.

[0018] Fig.13 Shown is a scheme for the chemical synthesis of RGD-dextran-EGCG via conjugation of the C-terminus of the RGD peptide to HO-dextran-EGCG. DETAILED DESCRIPTION

[0019] definition

[0020] The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0021] As used herein, the term "cancer targeting ligand" refers to a molecule with a molecular weight of <10,000 Daltons, for example 300 to 3,500 Daltons, such as a peptide, acid or carbohydrate, which binds or targets a receptor on the surface of a cancer cell or in the tumor environment.

[0022] The term "cytokine" refers to proteins (about 5 kDa to 70 kDa) that are important in cell signaling. Cytokines have been shown to participate in autocrine, paracrine, and endocrine signaling as immunomodulators. Cytokines include interferons, interleukins, lymphokines, tumor necrosis factors, and chemokines.

[0023] The term "epigallocatechin gallate" refers to the ester of epigallocatechin and gallic acid, and is used interchangeably with "epigallocatechin-3-gallate" or EGCG.

[0024] The term "oligomeric EGCG" (OEGCG) refers to 3 to 20 covalently linked EGCG monomers. OEGCG preferably contains 4 to 12 EGCG monomers.

[0025] The term "nanoparticle" refers to particles having a diameter below 1 μm and between 1 nm and 999 nm.

[0026] The term "polyethylene glycol-epigallocatechin gallate conjugate" or "PEG-EGCG" refers to polyethylene glycol (PEG) conjugated to one or two EGCG molecules. The term "PEG-EGCG" refers to both PEG-mEGCG conjugate (monomeric EGCG) and PEG-dEGCG (dimeric EGCG) conjugates.

[0027] The term "MINC" (Multiple Pathway Immunomodulatory Nanocomplex Combination Therapy) is a platform technology. As used in this application, MINC utilizes the biological activity of PEG-flavonoid conjugates and oligomeric EGCG (OEGCG). MINC can encapsulate additional cancer therapeutics to form a MINC-agent.

[0028] As used in this application, the term "MINC-agent" is a micelle having a shell formed of a cancer targeting ligand-PEG-flavonoid conjugate and optionally an oligomeric flavonoid (such as OEGCG), and having an agent encapsulated within the shell.

[0029] Flavonoids

[0030] The flavonoids suitable for use in the present invention have the general structure of Formula I:

[0031]

[0032] in:

[0033] R1 is H or phenyl;

[0034] R2 is H, OH, gallate or phenyl; wherein the phenyl is optionally substituted with one or more (e.g., 2 to 3) hydroxyl groups;

[0035] R3 is H, OH or =O (oxo); or

[0036] R1 and R2 together form a closed loop structure; or

[0037] R2 and R3 together form a closed loop structure.

[0038] The 2-, 3-, 4-, 5-, 6-, 7- or 8-position of Formula I may be bonded to a group containing hydrocarbon, halogen, oxygen, nitrogen, sulfur, phosphorus, boron or a metal.

[0039] Examples of flavonoids of Formula I include:

[0040]

[0041] Preferred flavonoid compounds of formula I include:

[0042] EGCG(CAS#989-51-5), EC(CAS#490-46-0), EGC(CAS#970-74-1) or ECG(CAS#1257-08-5)

[0043]

[0044] Conjugate

[0045] The present invention provides a conjugate comprising: (a) a cancer targeting ligand, (b) a hydrophilic polymer of polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA) or dextran, and (c) a flavonoid of formula I, wherein PEG is covalently bound to the flavonoid and the cancer targeting ligand.

[0046] The conjugate targets cancer through the cancer targeting ligand and delivers the active ingredient to the cancer tissue to treat the cancer.

[0047] The cancer targeting ligand is covalently linked to PEG, PLA, PLGA or dextran via its -COOH group or its -NH2 group by standard chemistry known to those skilled in the art. The molecular weight of the hydrophilic polymer in the conjugate is typically 1K to 100K, preferably 3K to 80K, and more preferably 5K to 40K.

[0048] The flavonoid in the conjugate has the general formula (I), and is preferably EGCG, EC, EGC or ECG. In one embodiment, the flavonoid is epigallocatechin gallate (EGCG).

[0049] In one embodiment, PEG contains an aldehyde group conjugated to position 5, 6, 7, or 8 (preferably position 6 or 8) of the A ring of the flavonoid compound.

[0050] In another embodiment, PEG contains a thiol group conjugated to R1 or R2 of the B ring of the flavonoid (when R1 or R2 is -OH).

[0051] In one embodiment, the conjugate comprises PEG-EGCG, which is PEG connected to one or two molecules of EGCG. It can be prepared by reacting the aldehyde-terminated PEG to EGCG via free aldehyde groups and 5, 6, 7 or 8 (preferably 6 or 8) of formula I, by connecting PEG, and conjugating the aldehyde-terminated PEG to EGCG. See WO2006 / 124000 and WO2009 / 054813. It can also be prepared by reacting the sulfo-terminated PEG to EGCG via free sulfo groups and R1 or R2 (wherein R1 or R2 is phenyl) of formula I, by connecting PEG, and conjugating the sulfo-terminated PEG to EGCG. See WO2015 / 171079.

[0052] In another embodiment, the conjugate comprises PEG-EC, PEG-EGC or PEG-ECG, and the conjugate can be prepared by conjugating the aldehyde-terminated PEG to EC, EGC or ECG via the reaction of the free aldehyde group with position 5, 6, 7 or 8 (preferably position 6 or 8) of Formula I, through the attachment of PEG.

[0053] HOOC-PEG-CHO and HO-PEG-CHO are commonly used. In one embodiment, according to WO2006 / 124000 and WO2009 / 054813, HOOC-PEG-CHO is conjugated to EGCG, EC, EGC or ECG. HOOC-PEG-flavonoids have a COOH group to react with the N-terminal of the cancer targeting peptide. Typically, the cancer targeting peptide is incubated together with HOOC-PEG-flavonoids, N, N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the C-terminal of the cancer targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions.

[0054] In another embodiment, according to WO2006 / 124000 and WO2009 / 054813, HO-PEG-CHO is conjugated to EGCG, EC, EGC or ECG. HO-PEG-flavonoids have an OH group to react with the C-terminus of the cancer targeting peptide. Typically, the peptide is incubated with HO-PEG-EGCG and N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the N-terminus of the cancer targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions. In this reaction, the COOH group on the peptide reacts selectively with the OH group on PEG because the primary OH group on PEG is more reactive than the tertiary OH group in the aromatic ring of the flavonoid.

[0055] HOOC-PLA-CHO, HOOC-PLGA-CHO, and HO-dextran-CHO are commercially available.

[0056] In one embodiment, according to WO2006 / 124000 and WO2009 / 054813, HOOC-PLA-CHO is conjugated to EGCG, EC, EGC or ECG. HOOC-PLA-flavonoids have a COOH group to react with the N-terminus of the cancer targeting peptide. Typically, the cancer targeting peptide is incubated with HOOC-PLA-flavonoids, N, N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the C-terminus of the cancer targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions.

[0057] In one embodiment, according to WO2006 / 124000 and WO2009 / 054813, HOOC-PLGA-CHO is conjugated to EGCG, EC, EGC or ECG. HOOC-PLGA-flavonoids have a COOH group to react with the N-terminus of the cancer targeting peptide. Typically, the cancer targeting peptide is incubated with HOOC-PLGA-flavonoids, N, N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the C-terminus of the cancer targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions.

[0058] In one embodiment, according to WO2006 / 124000 and WO2009 / 054813, HO-glucan-CHO is conjugated to EGCG, EC, EGC or ECG. HO-glucan-flavonoids have an OH group to react with the C-terminus of the cancer targeting peptide. Typically, the peptide is incubated with HO-glucan-EGCG and N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the N-terminus of the cancer targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions. In this reaction, the COOH group on the peptide reacts selectively with the OH in the CH2OH terminus of dextran because this is the only primary OH group in dextran, which is more reactive than other secondary OH in dextran and the tertiary OH in the aromatic ring of flavonoids.

[0059] The cancer targeting ligands in the present invention are ligands selected for targeting receptors on the surface of cancer cells or the tumor environment. The cancer targeting ligands of the present invention, for example, target the following receptors on cancer, including but not limited to integrin receptors (including α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, αLβ2, αMβ2, ICAM-1, αIIbβ3, αVβ1, αVβ3, αVβ3 αVβ5 αVβ5 αVβ3, αVβ5, αVβ5, αVβ6, αVβ8, α6β4), transferrin receptor, FGFR1, FGFR2, FGFR3, FGFR4, EGFR1 (HER1), EGFR2 (HER2), EGFR3 (HER3), EGFR 4 (HER4), TNFR1, TNFR2, c-MET (HGFR), NOTCH1, NOTCH2, NOTCH3, NOTCH4, IR, AR, ER, PRPTK7 receptor, TrkA, TrkB, TrkC, GPCR, Eph receptor, AXL receptor, Frizzled receptor, RET, ROS, folate receptor, IL-2R, IL-2RG, IL-2RB, IL-4R, IL-7R, IL-9R, CD133 receptor, LHRHR, LRP5, LRP6, CD44 receptor, CD47 receptor, CD20 receptor, Fas receptor, DR4, DR5, LEP-R, MUC1 receptor, adiponectin receptor, alpha-adrenergic receptor, nucleolin receptor, PD-L1, ASGPR, lectin receptor, annexin receptor, glycyrrhetinic acid receptor, cadherin, and EpCAM.The cancer targeting ligands of the present invention interact with, for example, the following targets in the tumor microenvironment, including but not limited to αlβ1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, αLβ2, αMβ2, ICAM-1, αIIbβ3, αVβ1, αVβ3, αVβ3αVβ5αVβ5 αVβ3, αVβ5, αVβ5, αVβ6, αVβ8, α6β4MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP1 7. MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, CCL2, CCL5, CXCL12, ICAM-1, VEGF-A, VEGF- B. VEGF-C, VEGF-D, PIGF, CCR2, CXCR4, CSF-1R, VEGFR1, VEGFR2, VEGFR3, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, WNT1, WNT2, WNT2B, WNT3, W NT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNTT0A, WNT10B, WNT11, WNT16, IL-1, IL-2, IL-3, I L-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-21, IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R , IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-15R, IL-17R, IL-21R, PDGF, PDGFR-α, PDGFR-β, IGF1, IGF2, TGF-β, EGF, HB-EGF, TNF-α, TNFβ, TNFγ, CD19, CD103, IDO, PD-1, CTLA-4, collagen, elastin, glycosaminoglycans, proteoglycans, cancer-associated fibroblasts, tumor-associated macrophages, T cells, B cells, and dendritic cells. .

[0060] In one embodiment, the cancer targeting ligand is RGD, which targets integrin αvβ3.

[0061] In one embodiment, the cancer targeting ligand is TP12 having the amino acid sequence of THRPPMWSPVWP (SEQ ID NO: 1), which targets the transferrin receptor on various cancers.

[0062] In one embodiment, the cancer targeting ligand is MC11 having an amino acid sequence of MQLPLATGGGC (SEQ ID NO: 2), which targets FGFR on various cancers.

[0063] In one embodiment, the cancer targeting ligand is FV12 having an amino acid sequence of FCDGFYACYMDV (SEQ ID NO: 3), which targets HER2 on various cancers.

[0064] In one embodiment, the cancer targeting ligand is YI12 having an amino acid sequence of YHWYGYTPQNVI (SEQ ID NO: 4), which targets EGFR.

[0065] In one embodiment, the cancer targeting ligand is CTT having an amino acid sequence of CTTHWGFTLC (SEQ ID NO: 5), which targets MMP2 / MMP9 in the tumor microenvironment.

[0066] In one embodiment, the cancer targeting ligand is the H2009.1 peptide having the amino acid sequence of RGDLATLRQLAQEDGVVGVR (SEQ ID NO: 6), which targets the integrin αvβ6 receptor.

[0067] In one embodiment, the cancer targeting ligand is an IL-13 peptide having the amino acid sequence of GSETWKTIITKN (SEQ ID NO: 7), which targets the IL-13Rα2 receptor.

[0068] In one embodiment, the cancer targeting ligand is an AP-1 peptide having the amino acid sequence of RKRLDRN (SEQ ID NO: 8), which targets the IL-4 receptor.

[0069] In one embodiment, the cancer targeting ligand is CVKTPAQSC (SEQ ID NO: 9), which targets the CD133+ receptor.

[0070] In one embodiment, the cancer targeting ligand is a CC9 peptide having an amino acid sequence of CDCRGDCFC (SEQ ID NO: 10), which targets integrins in cancer and tumor environments, including αvβ3, αvβ5, αvβ6, αvβ8, αIIbβ3, α8β1, and α5β1.

[0071] In one embodiment, the cancer targeting ligand is an RGDS peptide having the amino acid sequence of RGDS (SEQ ID NO: 11), which targets the integrin αvβ3 receptor.

[0072] In one embodiment, the cancer targeting ligand is an NR7 peptide having an amino acid sequence of NSVRGSR (SEQ ID NO: 12), which targets

[0073] In one embodiment, the cancer targeting ligand is a LHRH peptide having the amino acid sequence of EHWSYGLRPG (SEQ ID NO: 13), which targets LHRHR.

[0074] In one embodiment, the cancer targeting ligand is angiopep-2 peptide having the amino acid sequence of TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 14), which targets LRP.

[0075] In one embodiment, the cancer targeting ligand is a TbFGF peptide having an amino acid sequence of KRTGQYKLC (SEQ ID NO: 15), which targets EGFR.

[0076] In one embodiment, the cancer targeting ligand is an EGF peptide having an amino acid sequence of YHWYGYTPQNVI (SEQ ID NO: 16), which targets EGFR.

[0077] In one embodiment, the cancer targeting ligand is folic acid or folate (C 19 H 19 N7O6), which targets folate receptors in cancer.

[0078] In one embodiment, the cancer targeting ligand is hyaluronic acid, which targets the CD44 receptor.

[0079] In one embodiment, the cancer targeting ligand is lactose or galactosamine, which targets ASGPR.

[0080] In one embodiment, the cancer targeting ligand is galactose, which targets ASGPR and lectin receptors.

[0081] In one embodiment, the cancer targeting ligand is glycyrrhetinic acid, which targets the glycyrrhetinic acid receptor.

[0082] Nanoparticle composition

[0083] The term "MINC" (Multiple Immunomodulatory Nanocomplex Combination Therapy) is a platform technology. The present invention provides nanoparticle micelle (MINC) compositions. The micelles contain a cancer targeting ligand-PEG-flavonoid conjugate in an outer shell and oligomeric EGCG (OEGCG) in an inner shell (see Figure 1 ). Cancer targeting ligands allow the nanoparticle compositions to be specifically targeted to cancer tissues.

[0084] In one embodiment, the nanoparticle micelle composition has a defined and narrow size distribution, wherein at least 70% of the nanoparticles have a diameter between 20nm and 500nm or 50nm and 300nm, and the size distribution of the nanoparticles has only one main peak containing more than 90% of all nanoparticles.

[0085] The micelles optionally comprise a cancer therapeutic molecule (agent) encapsulated within the micelle (MINC-agent).

[0086] The MINC-pharmaceutical composition of the present invention comprises three active ingredients that are functionally complementary to address both immune responses and signaling pathways through its backbone components (PEG-flavonoids / OEGCG), as well as additional signaling pathways through selected drug molecules for treating cancer. Each nanoparticle is a fixed-dose combination drug with three active ingredients in a fixed molar ratio.

[0087] The present invention delivers MINC-agents to targeted cancer tissues by actively delivering micelles to tumors with specific receptors via cancer targeting ligands. In addition, the present invention delivers MICN-agents to tumors by passive delivery relying on nanoparticle size.

[0088] The size of the drug product determines how much drug selectively enters the tumor relative to other unexpected tissues. Normal, unexpected healthy tissues usually have vascular openings less than 10nm. Nanoparticles with a single size distribution of about 100nm can be more effectively taken up by target cells. It has been shown that nanoparticles with a single peak and a limited size of about 100nm can deliver more therapeutic agents to target cells. Most of the particles of the nanoparticle micelle composition of the present invention have a size of 20nm to 500nm or 50nm to 300nm. If the particle size is less than 50nm, the risk that the particles cannot be well taken up by cells is higher. If the particle size is greater than 300nm, it may cause excessive intake by the reticuloendothelial (RE) system and cause side effects. More than 70% of the particles in the composition of the present invention are within the size range of 20nm to 500nm, which ensures that the therapeutic agent enters the disease lesion in preference to normal tissues and the RE system. In addition, the limited nanoparticle size of 20nm to 500nm can be selectively taken up by target cells to increase the efficacy of drugs (pharmaceuticals).

[0089] The nanoparticle micellar composition of the present invention has a narrow particle size distribution because it has only one main peak containing more than 90% of all nanoparticles. It is important to have a therapeutic composition with only one particle size distribution peak, rather than several peaks or multiple peaks. If the therapeutic composition has more than one molecular size, it may cause serious variations in therapeutic efficacy and patient response rate.

[0090] The nanocomposite of the present invention contains the first two active ingredients, namely OEGCG and PEG-flavonoids, such as PEG-EGCG, in the skeleton of the micellar composition. They are derivatives of EGCG, which is a strong immunomodulator and regulates a variety of disease signal transduction pathways. EGCG regulates both innate immunity and adaptive immunity. However, the bioavailability of EGCG is low and EGCG is unstable. The nanocomposite composition of the present invention overcomes the bioavailability problem of EGCG by forming a nanocarrier that carries EGCG to the target position of treatment, and overcomes the stability problem of EGCG by forming an OEGCG and PEG-EGCG complex, which effectively enables EGCG to become an efficient therapeutic agent.

[0091] The nanocomplex of the present invention optionally contains a third active ingredient, which is a drug molecule encapsulated in the nanoparticles for treating cancer.

[0092] Nanoparticles with a size of 20 nm to 500 nm preferentially reach the intended treatment site. Large nanoparticles (500 nm to 999 nm) or micron-sized (1000 nm to 5000 nm) particles can cause toxicity due to the aggregation of smaller nanoparticles, because large nanoparticles are usually efficiently taken up by the reticuloendothelial system (RES) (also known as the mononuclear phagocytic system (MPS)) located in the liver, lungs, and bone marrow.

[0093] The inventors have found a nanoparticle micelle composition of the present invention for targeted delivery to a target tissue, wherein at least 70% of the nanoparticles have a diameter between 20nm and 500nm or 50nm and 300nm, and the size distribution of the nanoparticles has only one main peak containing more than 90% of all particles. The inventors have also found a method for preparing such a nanoparticle composition.

[0094] The present invention relates to a nanoparticle composition comprising nanoparticles, wherein the nanoparticles have: (a) an inner core comprising oligomeric (-)-epigallocatechin gallate (OEGCG); (b) an outer core comprising a cancer targeting ligand-PEG-flavonoid conjugate; and (c) a drug molecule encapsulated in the inner core. In one embodiment, at least 70% of the nanoparticles have a diameter between 20nm and 500nm or 50nm and 300nm, and the size distribution of the nanoparticles has only one main peak comprising more than 90% of all particles. The flavonoid in the conjugate is preferably EGCG, EC, EGC or ECG, more preferably EGCG. Figure 1 The structure of the nanoparticles of the present invention is shown in FIG.

[0095] In one embodiment, the micellar shell further comprises a naked PEG-flavonoid conjugate, such as PEG-EGCG, which has no cancer targeting ligand attached to the PEG-flavonoid. In such micellar shells, the ratio of ligand-PEG-EGCG to ligand-PEG-EGCG plus PEG-EGCG is typically greater than 10%, or greater than 20%, or greater than 30%, or greater than 50%, and up to 100%. See Figure 2 .

[0096] In one embodiment, at least 80% or at least 85% or at least 90% or at least 95% of the nanoparticles have a diameter between 20 nm and 500 nm or between 50 nm and 300 nm.

[0097] In one embodiment, the median nanoparticle diameter in the nanoparticle composition is between 50 nm and 250 nm, 50 nm and 200 nm, 80 nm and 200 nm, 100 nm and 200 nm, or 50 nm and 150 nm.

[0098] In one embodiment, the size distribution of the nanoparticles shows only one major (and narrow) peak containing more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of all the particles.

[0099] In one embodiment, the cancer therapeutic drug molecule in the MINC-agent is a cytokine, an antibody, a chemotherapeutic agent, or a small compound inhibitor.

[0100] Suitable cancer therapeutic cytokines for use in the present invention include, but are not limited to, IL-2, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, TARIL, IGF1, GLP-1, IFN-α, IFN-β, IFN-γ, CCL5, CXCL9, CXCL10, CXCL11, CX3CL1, and recombinant cytokine products.

[0101] Suitable cancer treatment antibodies for use in the present invention include, but are not limited to, monoclonal antibodies, polyclonal antibodies, antibody-drug-conjugates, and bispecific antibodies. Preferred antibodies of the present invention are monoclonal antibodies. Antibodies suitable for use in the present invention include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-LAG3 antibodies, anti-TIGIT antibodies, anti-TIM3 antibodies, anti-HER2 antibodies, anti-HER3 antibodies, anti-HGFR antibodies, anti-EGFR antibodies, anti-EpCAM, anti-FOLR1 antibodies, anti-c-Met antibodies, anti-GD2 ganglioside antibodies, anti-GD3 gangliosides, anti-VEGFR1 antibodies, anti-VEGF antibodies, anti-TGF-β antibodies, anti-TNF-α antibodies, anti-IGF-1R antibodies, anti-IL-4 antibodies, anti-IL-10 antibodies, anti-IL-13 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD Anti-CD40 antibody, anti-CD40L antibody, anti-CD43 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD28 antibody, anti-CD38 antibody, anti-CD20 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-DR5 antibody, anti-MUC1 antibody, anti-Tau antibody, anti-β-amyloid antibody, abagovomab, abituzumab, adalimumab, aducanumab, alemtuzumab, amatuxi mab), amivantamab, anifrolumab, atezolizumab, avelumab, bapineuzumab, basiliximab, belimumab, benralizumab, besilesomab, bevacizumab, bezlotoxumab, blinatumomab ab), brazikumab, brontictuzumab, cabiralizumab, camrelizumab, carlumab, carotuximab, catumaxomab, cedelizumab, cetrelimab, cetuximab, cibisatamab, crenezumab,Cusatuzumab, daclizumab, dalotuzumab, daratumumab, detumomab, dinutuximab, drozitumab, duligotuzumab, dupilumab, durvalumab, ecoromeximab, emibetuzumab, epcori tamab), epratuzumab, eptinezumab, erenumab, ertumaxomab, etaracizumab, etesevimab, farletuzumab, fezakinumab, ficlatuzumab, figitumumab, fletikumab, foralumab, fresolumab imumab), futuximab, ganitumab, gantenerumab, gatipotuzumab, gevokizumab, golimumab, guselkumab, icrucumab, igovomab, imalumab, imgatuzumab, inebilizumab, infliximab, intetumumab, ipilimumab, istiratumab, ixekizumab, 1etolizumab, 1exatumumab, lintuzumab, mapatumumab, matuzumab, mavrilimumab, mepolizumab, mogamulizumab, monalizumab,mosunetuzumab, natalizumab, naxitamab, necitumumab, nimotuzumab, nivolumab, ocaratuzumab, ocrelizumab, ofatumumab, olaratumab, olaratumab icinumab), panitumumab, pembrolizumab, pertuzumab, ponezumab, ramucirumab, ranibizumab, rituximab, samalizumab, sarilumab, secukinumab, sintilimab, solanezumab, teprotumumab, tigatuzumab, tildrakizumab, timigutuzumab, tocilizumab, tomucoximab, trastuzumab, ustekinumab, vanucizumab, varisacumab, variruzumab varlilumab, vedolizumab, vepalimomab, vesencumab, visilizumab, vonlerolizumab, zanolimumab, zatuximab, zenocutuzumab, zolbetuximab, ado-trastuzumab emtansine, anetumab ravtansine, brentuximab vedotin, cantuzumab mertansine, certolizumab pegol, coltuximab ravtansine,depatuxizumab mafodotin, enapotamab vedotin, gemtuzumab ozogamicin, glembatumumab vedotin, iladatuzumab vedotin, inatuzumab vedotin, indatuximab ravtansine, indusatumab vedotin, lifastuzumab vedotin, lilotomab satetraxetan, lorvotuzumab mertansine), losatuxizumab vedotin, lulizumab pegol, mirvetuximabsoravtansine, naratuximab emtansine, notuzumab ozogamicin, polatuzumab vedotin-piiq, rovalvpituzumab tesirine, sacituzumab govitecan, samrotamab vedotin, telisotuzumab vedotin, trastuzumab deruxtecan) and tucotuzumabcelmoleukin. Antibodies also include antibody fragments that are capable of binding to their corresponding antigens, such as Fab, (Fab)2 or single-chain antibodies.

[0102] Suitable cancer treatment chemotherapeutic agents for use in the present invention include, but are not limited to, altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, trabectedin, 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, topotecan, daunorubicin, doxorubicin, epirubicin, idarubicin, and valrubicin.

[0103] Suitable cancer therapeutic small compound inhibitors for use in the present invention include, but are not limited to, imatinib, gefitinib, erlotinib, sunitinib, lapatinib, nilotinib, sorafenib, temsirolimus, everolimus, pazopanib, crizotinib, ruxolitinib, vandetenib, axitinib, bosutinib, cabozantinib, ponatinib, regorafenib, ibrutinib, trametinib, or perifosine for targeting tyrosine and serine / threonine kinases.

[0104] Suitable cancer therapeutic small compound inhibitors for use in the present invention include, but are not limited to, bortezomib, carfilzomib, or marizomib for targeting the proteasome.

[0105] Suitable cancer therapeutic small compound inhibitors for use in the present invention include, but are not limited to, batimastat, neovastat, prinomastat, rebimastat, marimastat, ganetespib, or NVP-AUY922 for targeting MMPs and HSPs.

[0106] Suitable cancer therapeutic small compound inhibitors for use in the present invention include, but are not limited to, Obatoclax or Navittoclax for inducing apoptosis.

[0107] The nanoparticle composition of the present invention has a majority particle size of 20nm to 500nm or 50nm to 300nm in diameter, wherein OEGCG, PEG-EGCG and drug molecules are held together by hydrophobic interactions. The nanoparticle composition is stable in a hydrophilic environment (such as blood circulation) and dissociates in a hydrophobic environment (such as tumor tissue). Due to inflammation and other excessive activities (such as rapid, uncontrolled tumor growth), the nanoparticle composition can selectively diffuse from blood vessels to surrounding tissues with vascular leakage. Due to the size of the nanoparticle composition, it is restricted from entering normal tissues with less vascular leakage. Once the nanoparticle complex enters the hydrophobic tissue, it dissociates and releases its active components OEGCG, PEG-flavonoids (such as PEG-EGCG) and drug molecules in the nanocomposite. The free active components restore their biological activity in cancer delay. The active components in the nanoparticles have a longer circulation half-life and act as a sustained release mechanism, which further reduces the drug dosage requirements. Therefore, any adverse effects on normal tissues are further reduced.

[0108] MINC-pharmaceutical composition also has the following advantages:

[0109] MINC is stable in blood circulation for >15 days. The encapsulated drug molecules are protected within the MINC nanoparticle shell during circulation.

[0110] MINC nanoparticles use the enhanced permeability and retention (EPR) effect and deliver most of the drug molecules to the target cells.

[0111] MINC nanoparticles did not aggregate during freeze-thaw cycles.

[0112] ·MINC drug retains its original biological activity after lyophilization.

[0113] MINC is stable at 2°C to 8°C.

[0114] Methods for preparing nanoparticle compositions

[0115] The present invention provides a method for preparing a nanoparticle composition of a fixed-dose combination drug. The method is optimized so that only nanoscale particles are produced, wherein at least 70% of the particles have a diameter between 20nm and 500nm or 50nm and 300nm and a main peak.

[0116] The method comprises the following steps: (a) mixing drug molecules with OEGCG and a cancer targeting ligand-PEG-EGCG conjugate in an aqueous solution; and (b) filtering the mixture through a membrane with a molecular weight cutoff of 8,000 to 300,000 Daltons to remove small molecular weight molecules and retain large molecular weight molecules.

[0117] In a preferred embodiment, the method further comprises step (c), filtering the large molecular weight molecules through a 0.2 μm to 0.3 μm membrane and collecting the filtrate.

[0118] The method of the present invention optionally further comprises a freeze-drying step (d) after step (c). Step (d): freeze-drying the filtrate by stepwise freezing at (i) about 0°C to 5°C, (ii) about -20°C to -30°C and (iii) about -60°C to -100°C and then drying.

[0119] In step (a), the drug molecule is dissolved in an aqueous solvent such as phosphate buffered saline, saline, water, bicarbonate buffer, oxygenated hemoglobin buffer, bis-tris paraffin, Tris-HCl, HEPES, histidine buffer, NP-40, RIPA (radioimmunoprecipitation assay buffer), tricine, TES, TAPS, TAPSO, Bicine, MOPS, PIPES, cacodylate or MES. Preferred solvents are phosphate buffered saline, saline or water. The protein drug concentration is generally 0.01 mg / ml to 50 mg / ml, preferably 0.05 mg / ml to 10 mg / ml, more preferably 0.1 mg / ml to 5 mg / ml.

[0120] OEGCG, PEG-EGCG and optional EGCG are dissolved in ketone, acetonitrile, alcohol, aldehyde, ether, acetate, sulfoxide, benzene, organic acid, amide, aqueous buffer and any combination thereof. Preferred solvents are alcohol, acetonitrile, sulfoxide, amide and any combination thereof. OEGCG / EGCG and PEG-EGCG concentrations are usually independently 0.001 mg / ml to 10 mg / ml, preferably 0.005 mg / ml to 1 mg / ml or 0.1 mg / ml to 5 mg / ml.

[0121] It is important that OEGCG is in molar excess relative to the drug agent. Generally speaking, the molar ratio of EGCG to drug molecules in OEGCG is between 1-500:1, 2-500:1, 3-500:1 or 5-500:1, preferably 3-100:1, 5-100:1 or 10-50:1. The molar ratio is calculated by the number of moles of monomeric EGCG in OEGCG to the number of moles of drug molecules. The molar excess of EGCG ensures that most or all of the drug agent is encapsulated by the OEGCG molecules. In the method of the present invention, unencapsulated drug agents are avoided by controlling the molar ratio of OEGCG to protein, which will not be selectively distributed to the target tissue and will lead to lower efficacy and safety issues.

[0122] The pharmaceutical agent, OEGCG and PEG-EGCG are mixed at a temperature between about 0°C to 60°C, preferably 0°C to 45°C or 0°C to 37°C for 1 minute to 2 days, preferably 1 minute to 12 hours.

[0123] In step (b), the mixture is filtered through a membrane having a molecular weight cut-off of 8,000 to 300,000 daltons, preferably 8,000 to 200,000 daltons, 8,000 to 150,000 daltons or 8,000 to 12,000 daltons to remove small molecular weight molecules and retain large molecular weight molecules. The ultrafiltration membrane material is selected from the group consisting of: cellulose (and derivatives thereof), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, polyvinylidene fluoride or polyvinylidene fluoride (PVDF) and polypropylene (PP); preferably cellulose (and derivatives thereof), PTFE and PVDF.

[0124] Prior to ultrafiltration, the mixture is optionally diluted in an aqueous solvent, such as those described above in step (a).

[0125] Ultrafiltration step (b) removes unwanted small molecular weight impurities, such as unreacted OEGCG or EGCG, or reaction byproducts. These impurities can reduce drug efficacy and safety. Excessive unreacted OEGCG or EGCG can also cause single nanoparticles to aggregate into particles of about 1000nm size, which will reduce efficacy and cause potential toxicity.

[0126] In step (c), the retained large molecular weight molecules are filtered through a membrane having a pore size of about 0.2 μm to 0.3 μm (such as 0.22 μm), and the filtrate is collected. This is to remove unwanted large molecular impurities, such as giant aggregates. These aggregates can be discharged from the tissues into which they enter due to their huge size. These aggregates reduce the overall efficacy / safety and have a higher chance of inducing immunogenicity in patients. Large size nanoparticles are also more likely to be taken up by the RES in the liver, lungs, and more undesirable organs.

[0127] The membrane material of step (c) is selected from the group consisting of cellulose (and its derivatives), PES, PTFE, nylon, PVDF and PP; preferably cellulose (and its derivatives), PES and PP.

[0128] In one embodiment, steps (b) and (c) are repeated at least once, for example 1, 2, 3 or 4 times, before step (d) to effectively remove unwanted small molecule impurities and large aggregates.

[0129] After step (c), the filtrate is stored at 2°C to 8°C and is stable for at least 100 days.

[0130] The method of the present invention optionally further comprises a freeze-drying step (d) after step (c) to provide long-term stability of the nanoparticle composition.

[0131] In step (d), the filtrate collected after filtering through a 0.2 μm to 0.3 μm membrane is first freeze-dried by stepwise freezing under the following conditions: (i) freezing at about 0°C to 5°C, e.g., for about 1 hour to 3 hours; (ii) freezing at about -25°C to -30°C, e.g., for about 1 hour to 3 hours; and then (iii) freezing at -60°C to -100°C or -70°C to -100°C, e.g., for at least 8 hours.

[0132] After freezing, the material was lyophilized for 1 to 7 days.

[0133] Freezing and lyophilization often cause nanoparticles to form complexes and aggregates. These large particles may be too large to penetrate blood vessels and enter the tissue environment. Therefore, efficacy and safety are lower, and immunogenicity can be increased. In order to avoid these changes caused by lyophilization, the method of the present invention uses a stepwise freezing method, rather than a continuous freezing method (gradually and continuously reducing the temperature during freezing), to maintain the nanoparticle size during lyophilization.

[0134] Pharmaceutical composition

[0135] The present invention provides a pharmaceutical composition comprising a nanoparticle composition of the present invention and optionally one or more pharmaceutically acceptable carriers. For tablets, powders or parenteral preparations, the nanoparticle composition in the pharmaceutical composition is generally about 1% to 90%, preferably 20% to 90% or 30% to 80%. For capsule preparations, the nanoparticle composition in the pharmaceutical composition is generally 1% to 100%, preferably 20% to 100%, 50% to 100% or 70% to 100%. For liquid suspension preparations, the nanoparticle composition in the pharmaceutical composition is generally 1% to 50%, 5% to 50% or 10% to 40%.

[0136] In one embodiment, the pharmaceutical composition may be in the form of tablets, capsules, granules, fine granules, powders, suspensions, patches, parenteral preparations, injectable preparations, etc. The pharmaceutical composition may be prepared by conventional methods.

[0137] Pharmaceutically acceptable carriers are inactive ingredients that can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers can contain a variety of ingredients, including but not limited to saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol and glucose; pH adjusters and buffers such as salts of hydroxides, phosphates, citrates, acetates, borates and triethanolamine; antioxidants such as salts, acids and / or bases of bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol and ascorbyl palmitate; surfactants such as lecithin and phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidylcholine. alcohols; poloxamers and poloxamines; polysorbates such as polysorbate 80, polysorbate 60 and polysorbate 20; polyethers such as polyethylene glycol and polypropylene glycol; polyethylenes such as polyvinyl alcohol and polyvinyl pyrrolidone (PVP, povidone); cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose and hydroxypropylmethylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di- and triglycerides; polysaccharides such as dextran; and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved to prevent bacterial contamination with well-known preservatives including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as non-preserved preparations for single or multiple use.

[0138] For example, the tablet, capsule or parenteral preparation of the active compound may contain other excipients that are not biologically active and do not react with the active compound. The excipients of tablets or capsules may include fillers, binders, lubricants and glidants, disintegrants, wetting agents and release rate regulators. Examples of excipients of tablets or capsules include but are not limited to carboxymethyl cellulose, cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly (acrylic acid) and polyvinyl pyrrolidone.

[0139] For example, tablet formulations may contain inactive ingredients such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and titanium dioxide. Capsule formulations may contain inactive ingredients such as gelatin, magnesium stearate, and titanium dioxide. Powder oral formulations may contain inactive ingredients such as silica gel, sodium benzoate, sodium citrate, sucrose, and xanthan gum.

[0140] Treatment

[0141] The present invention relates to a method for treating cancer, comprising the step of administering an effective amount of the nanoparticle composition of the present invention to a subject in need thereof.

[0142] As used herein, an "effective amount" is an amount effective for treating a disease by ameliorating the pathological condition or reducing disease symptoms.

[0143] Suitable cancers to be treated by the present invention include adrenocortical tumors, AIDS-related lymphomas, anal cancer, appendix cancer, grade I (anaplastic) astrocyte tumors, grade II astrocyte tumors, grade III astrocyte tumors, grade IV astrocyte tumors, atypical teratoma / rhabdomyosarcoma of the central nervous system, basal cell carcinoma, bladder cancer, breast cancer, bronchial carcinoma, bronchioloalveolar carcinoma, Burkitt's lymphoma, lymphoma, cervical cancer, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, uterine cancer, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, olfactory neuroblastoma, Ewing's sarcoma, ectocranial ectodermal tumor, gonadal ectodermal tumor, extrahepatic bile duct cancer, fibrous histiocytoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic tumor, glioma, head and neck cancer, heart cancer, hepatocellular carcinoma, hilar cholangiocarcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, Langerhans cell histiocytosis histiocytosis), laryngeal cancer, lip cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, endocrine tumors, multiple myeloma, mycosis fungoides, myelodysplasia, myelodysplasia / myeloproliferative neoplasms, myeloproliferative disorders, nasal cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma lymphoma), oral cancer, oropharyngeal cancer, osteosarcoma, ovarian clear cell carcinoma, ovarian epithelial cancer, ovarian germ cell tumor, papillomatosis, paranasal sinus cancer, parathyroid cancer, penile cancer, nasopharyngeal cancer, pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasmacytoma, plasmacytoma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, respiratory cancer with chromosome 15 alteration, retinoblastoma, rhabdomyosarcoma, salivary gland cancer adenocarcinoma, Sezary syndrome, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymic cancer, thymoma, thyroid cancer, renal pelvic cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

[0144] The ligand-PEG-EGGC conjugate is typically administered at 0.01 mg / kg to 1200 mg / kg (total weight of the conjugate / body weight of the subject) or 0.1 mg / kg to 1000 mg / kg.

[0145] The dosing of the MINC-agent is based on the known doses of the agent used to treat the specific disease and the condition of the subject. The dose can be a dose approved by the Food and Drug Administration (FDA) or a dose used in a clinical trial.

[0146] In the MINC-agent, the weight of the ligand-PEG-EGCG is close to that of the encapsulated drug agent. Typically, the dose of PEG-EGCG combined with OEGCG is between 10 ug / kg and 100 mg / kg.

[0147] The dosing of the nanoparticle composition is based on the known doses of protein drugs used to treat specific diseases and the subject's condition. For example, for the treatment of breast cancer in adults, trastuzumab is administered via IV infusion at 4 mg / kg to 8 mg / kg once a week for 52 weeks. The effective dose of ligand-MINC-trastuzumab is within the same dose range, with a lower dosing frequency of once every 12 to 16 weeks for 52 weeks.

[0148] For example, for the treatment of melanoma, interferon-α is induced by IV infusion of 20 million IU / m 2 , 5 consecutive days per week for 4 weeks. The effective dose of ligand-MINC-interferon-α is within the same dose range, administered 1 day per week for 2 weeks to achieve the same efficacy with reduced toxicity.

[0149] For example, for the treatment of renal cancer, 600,000 IU / kg (0.037 mg / kg) of IL-12 is administered three times daily for a maximum of 14 doses. After a 9-day rest, the schedule is repeated for 14 doses as tolerated. The effective dose of ligand-MINC-IL-12 in the same dose range is 1 dose per day for a total of 9 doses over 3 days.

[0150] The present invention can be used to treat humans and non-human animals. For example, the present invention can be used to treat mammalian subjects, such as humans, horses, pigs, cats and dogs.

[0151] The following examples further illustrate the present invention. These examples are intended only to illustrate the present invention and should not be construed as limiting.

[0152] Example

[0153] Example 1: Conjugation of RGD peptide with HOOC-PEG-EGCG

[0154] Material

[0155] RGD peptide was purchased from BIOTOOLS.

[0156] HOOC-PEG-CHO was purchased from NBC Chemicals.

[0157] method

[0158] According to WO2006 / 124000 and WO2009 / 054813, HOOC-PEG-CHO was conjugated to EGCG; RGD peptide was conjugated to HOOC-PEG-EGCG via conjugation between the COOH group on PEG and the NH2 group on RGD to form RGD-PEG-EGCG (N′-linked) ( Figure 3 ).

[0159] Specifically, 1 mg to 1000 mg RGD was PEGylated by incubation with 1 mg to 1000 mg HOOC-PEG-EGCG, 1 mg to 1000 mg N, N'-dicyclohexylcarbodiimide (DCC) and 1 mg to 1000 mg N-hydroxysuccinimide (NHS) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane molecular weight cutoff = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0160] result

[0161] HPLC was used to confirm the preparation of RGD-PEG-EGCG. HPLC was performed under the following conditions: column: C18, 4.6 mm×150 mm, 4 μm; eluent: A=0.1% TFA / H2O, B=0.1% TFA / ACN; oven temperature: 40°C; flow rate: 1 ml / min; autosampler temperature: 15°C; measurement: UV280.

[0162] The retention time of HOOC-PEG-EGCG was 5.95 minutes, and after RGD conjugation, there was a new peak with a retention time of 6.38 minutes. The HPLC results indicated the successful conjugation of RGD-PEG-EGCG.

[0163] Example 2: Conjugation of transferrin peptide (TP12) with HOOC-PEG-EGCG

[0164] Material

[0165] TP12 peptide was purchased from BIOTOOLS.

[0166] HOOC-PEG-CHO was purchased from NBC Chemicals.

[0167] method

[0168] For TP12-PEG-EGCG formulations, HOOC-PEG-CHO was conjugated to EGCG according to WO2006 / 124000 and WO2009 / 054813; TP12 peptide was conjugated to HOOC-PEG-EGCG via conjugation between the COOH group on PEG and the NH2 group on TP12 to form TP12-PEG-EGCG (N′-linked) ( Figure 4 ).

[0169] Specifically, 1 mg to 1000 mg TP12 was PEGylated by incubating with 1 mg to 1000 mg HOOC-PEG-EGCG, 1 mg to 1000 mg N, N'-dicyclohexylcarbodiimide (DCC) and 1 mg to 1000 mg N-hydroxysuccinimide (NHS) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0170] result

[0171] HPLC was used to confirm the preparation of TP12-PEG-EGCG. HPLC was performed under the following conditions: column: C18, 4.6 mm×150 mm, 4 μm; eluent: A=0.1% TFA / H2O, B=0.1% TFA / ACN; oven temperature: 40°C; flow rate: 1 ml / min; autosampler temperature: 15°C; measurement: ELSD.

[0172] The retention time of HOOC-PEG-EGCG was 6.04 minutes. After TP12 conjugation, there was a new peak with a retention time of 6.28 minutes. The results indicated the successful conjugation of TP12-PEG-EGCG.

[0173] Example 3: Preparation of RGD-MINC-doxorubicin and TP12-MINC-doxorubicin

[0174] Material

[0175] RGD-PEG-EGCG was prepared according to Example 1.

[0176] TP12-PEG-EGCG was prepared according to Example 2.

[0177] Doxorubicin was purchased from Sigma-Aldrich or other suppliers.

[0178] method

[0179] RGD-MINC-doxorubicin nanoparticles and TP12-MINC-doxorubicin nanoparticles were prepared according to the following protocol:

[0180] 1. Incubate 5 μg to 500 μg of doxorubicin in 1 mL of DMSO for 15 minutes to 1 hour.

[0181] 2. Add 1 μg to 100 μg of OEGCG and 1 μg to 10,000 μg of RGD-PEG-EGCG or TP12-PEG-EGCG. Incubate the mixture at 25°C for 3 hours.

[0182] 3. Filter out the liquid using a 10KMWCO filter unit. Wash the filter 3 times with 0.9% NaCl.

[0183] 4. Freeze-dried into dry powder.

[0184] result

[0185] Nanoparticle size was measured by DLS (Anton Paar, Litesizer 500). Figure 5 The successful formulation of RGD-MINC-doxorubicin (A) and TP12-MINC-doxorubicin (B) is shown.

[0186] Example 4: Different ligands for drug delivery into cancer cells - MINC-doxorubicin

[0187] Material

[0188] RGD-MINC-doxorubicin, TfR-MINC-doxorubicin (TP12-MINC-doxorubicin) or MINC-doxorubicin were formulated according to Example 3.

[0189] method

[0190] The breast cancer cell line MDA-MB-231 was cultured at 1×10 5 Cells / well were seeded in 12-well plates with coverslips and incubated overnight. On the second day, cells were treated with 2.5 μM of MINC-doxorubicin, RGD-MINC-doxorubicin or TfR-MINC-doxorubicin for 2 hours and 24 hours. After treatment, cells were fixed with ice-cold methanol. Fluorescence images were taken by fluorescence microscopy to evaluate delivery efficiency. Fluorescence intensity was measured by Image J using parameter fixed area: 10.309. Higher fluorescence signals mean more doxorubicin is taken up by cells.

[0191] result

[0192] Doxorubicin is a red fluorescent compound, and its delivery into cells was visualized using fluorescence microscopy. Higher fluorescence intensity means more doxorubicin was delivered into the cells. Figure 6 In the Figure 2, the fluorescence signal of RGD-MINC-doxorubicin or TfR-MINC-doxorubicin was stronger in MDA-MB-231 cancer cells compared with MINC-doxorubicin. These results demonstrate that tumor targeting peptides increase specific drug delivery into cancer cells.

[0193] Example 5: Study on the efficacy of different ligands-MINC-doxorubicin on cancer cells

[0194] Material

[0195] RGD-MINC-doxorubicin, TfR-MINC-doxorubicin (TP12-MINC-doxorubicin) or MINC-doxorubicin were formulated according to Example 3.

[0196] Acid phosphatase (ACP) assay kit was purchased from LSBio.

[0197] MDA-MB-231 cell line was purchased from ATCC (HTB26).

[0198] method

[0199] Breast cancer MDA-MB-231 cells were cultured at 1×10 4 Cells / well were seeded in 96-well plates and incubated overnight. On the second day, cells were treated with different concentrations of MINC-doxorubicin, RGD-MINC-doxorubicin or TfR-MINC-doxorubicin for 72 hours. After treatment, cell viability was assessed with an ACP kit according to the manufacturer's instructions. Optical density (OD) values ​​were measured by a spectrophotometer. Acid phosphatase is an enzyme present in cancer cells. Enzyme activity is positively correlated with cell viability and can be measured by OD values. Cell viability (%) was calculated by [(OD value of treatment group-blank) / (OD value of untreated group-blank)] × 100%.

[0200] result

[0201] exist Figure 7 In the experiment, compared with naked MINC-doxorubicin, at the same concentration of doxorubicin, the RGD-MINC-doxorubicin and TfR-MINC-doxorubicin treated groups had lower cell viability. The calculated 50% inhibition concentration (IC50) was much lower in the RGD-MINC-doxorubicin and TfR-MINC-doxorubicin treated groups than in the MINC-doxorubicin treated group. The results demonstrated that tumor targeting ligands improved the anticancer efficacy of MINC-doxorubicin.

[0202] Example 6: Conjugation of RGD peptide with HO-PEG-EGCG (prophetic example)

[0203] Purpose

[0204] This experiment was designed to demonstrate the conjugation of RGD to HO-PEG-EGCG. HPLC was used to detect the formation of a new product (RGD-PEG-EGCG) with a different retention time than HO-PEG-EGCG. NMR was used to confirm the structure.

[0205] Material

[0206] RGD peptide was purchased from BIOTOOLS.

[0207] HO-PEG-CHO was purchased from Huanteng Pharmaceutical.

[0208] method

[0209] According to WO2006 / 124000 and WO2009 / 054813, HO-PEG-CHO was conjugated to EGCG to prepare HO-PEG-EGCG.

[0210] RGD was conjugated to HO-PEG-EGCG via conjugation between the OH group on PEG and the COOH group on RGD to form RGD-PEG-EGCG (C' linked). Figure 8 .

[0211] Specifically, 1 mg to 1000 mg of RGD was PEGylated by incubation with 1 mg to 1000 mg of HO-PEG-EGCG and 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0212] Example 7: Conjugation of transferrin peptide (TP12) with HO-PEG-EGCG (prophetic example)

[0213] Purpose

[0214] This experiment was designed to demonstrate the conjugation of TP12 to HO-PEG-EGCG. HPLC was used to detect the formation of a new product (TP12-PEG-EGCG) with a different retention time than HO-PEG-EGCG. NMR was used to confirm the structure.

[0215] Material

[0216] TP12 peptide was purchased from BIOTOOLS.

[0217] HO-PEG-CHO was purchased from Huanteng Pharmaceutical.

[0218] method

[0219] For TP12-PEG-EGCG formulation, HO-PEG-CHO was conjugated to EGCG according to WO2006 / 124000 and WO2009 / 054813.

[0220] TP12 peptide was conjugated to HO-PEG-EGCG via conjugation between the OH group on PEG and the COOH group on TP12 to form TP12-PEG-EGCG (C′ linked). Fig. 9 .

[0221] Specifically, 1 mg to 1000 mg of TP12 was PEGylated by incubation with 1 mg to 1000 mg of HO-PEG-EGCG and 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0222] Example 8: Conjugation of folate to HO-PEG-EGCG (prophetic example)

[0223] Purpose

[0224] This experiment was designed to demonstrate the conjugation of folate to HO-PEG-EGCG. HPLC was used to detect the formation of a new product (folate-PEG-EGCG) with a different retention time than HO-PEG-EGCG. NMR was used to confirm the structure.

[0225] Material

[0226] Folate was purchased from TCI Chemicals.

[0227] HO-PEG-CHO was purchased from Huanteng Pharmaceutical.

[0228] method

[0229] According to WO2006 / 124000 and WO2009 / 054813, HO-PEG-CHO was conjugated to EGCG.

[0230] Folic acid or folate is conjugated to HO-PEG-EGCG via conjugation between the OH group on PEG and the COOH or COOR group on folate to form folate-PEG-EGCG. Fig.10 .

[0231] Specifically, 1 mg to 1000 mg of folate was PEGylated by incubation with 1 mg to 1000 mg of HO-PEG-EGCG and 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0232] Example 9: Conjugation of RGD peptide to HOOC-PLA-EGCG (prophetic example)

[0233] Purpose

[0234] This experiment was designed to demonstrate the conjugation of RGD peptide to HOOC-PLA-EGCG. HPLC was used to detect the formation of a new product (RGD-PLA-EGCG) with a different retention time than HOOC-PLA-EGCG. NMR was used to confirm the structure.

[0235] Material

[0236] RGD peptide was purchased from BIOTOOLS.

[0237] HOOC-PLA-CHO was purchased from Merck (Sigma-Aldrich).

[0238] method

[0239] According to WO2006 / 124000 and WO2009 / 054813, HOOC-PLA-CHO was conjugated to EGCG; RGD peptide was conjugated to HOOC-PLA-EGCG via conjugation between the COOH group on PLA and the NH2 group on RGD to form RGD-PLA-EGCG (N′ linked). Fig.11 .

[0240] Specifically, 1 mg to 1000 mg of RGD was incubated with 1 mg to 1000 mg of HOOC-PLA-EGCG, 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) and 1 mg to 1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light and nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane molecular weight cutoff = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0241] Example 10: Conjugation of RGD peptide to HOOC-PLGA-EGCG (Prophetic Example)

[0242] Purpose

[0243] This experiment was designed to demonstrate the conjugation of RGD peptide to HOOC-PLGA-EGCG. HPLC was used to detect the formation of a new product (RGD-PLGA-EGCG) with a different retention time than HOOC-PLGA-EGCG. NMR was used to confirm the structure.

[0244] Material

[0245] RGD peptide was purchased from BIOTOOLS.

[0246] HOOC-PLGA-CHO was purchased from Merck (Sigma-Aldrich).

[0247] method

[0248] According to WO2006 / 124000 and WO2009 / 054813, HOOC-PLGA-CHO was conjugated to EGCG; RGD peptide was conjugated to HOOC-PLGA-EGCG via conjugation between COOH groups on PLGA and NH2 groups on RGD to form RGD-PLGA-EGCG (N′ linked). Fig.12 .

[0249] Specifically, 1 mg to 1000 mg of RGD was incubated with 1 mg to 1000 mg of HOOC-PLGA-EGCG, 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) and 1 mg to 1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane molecular weight cutoff = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0250] Example 11: Conjugation of RGD peptide with HO-glucan-EGCG (prophetic example)

[0251] Purpose

[0252] This experiment was designed to demonstrate the conjugation of RGD to HO-Glucan-EGCG. HPLC was used to detect the formation of a new product (RGD-Glucan-EGCG) with a different retention time than HO-Glucan-EGCG. NMR was used to confirm the structure.

[0253] Material

[0254] RGD peptide was purchased from BIOTOOLS.

[0255] HO-dextran-CHO was purchased from Merck (Sigma-Aldrich).

[0256] method

[0257] According to WO2006 / 124000 and WO2009 / 054813, HO-dextran-CHO was conjugated to EGCG to prepare HO-dextran-EGCG.

[0258] RGD was conjugated to HO-dextran-EGCG via conjugation between the OH groups on PEG and the COOH groups on dextran to form RGD-dextran-EGCG (C′ linked). Fig.13 .

[0259] Specifically, 1 mg to 1000 mg of RGD was incubated with 1 mg to 1000 mg of HO-glucan-EGCG and 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0260] Example 12: Formulation of Folate-MINC-Doxorubicin (Prophetic Example)

[0261] Purpose

[0262] This experiment was designed to demonstrate the formulation of folate-MINC-doxorubicin. DLS was used to measure the size of the nanoparticles.

[0263] Material

[0264] Folate-PEG-EGCG was formulated according to Example 8.

[0265] Doxorubicin was purchased from Sigma-Aldrich or other suppliers.

[0266] method

[0267] Folate-MINC-doxorubicin nanoparticles were prepared according to the following protocol:

[0268] 1. Incubate 5 μg to 500 μg of doxorubicin in 1 mL of DMSO for 15 minutes to 1 hour.

[0269] 2. Add 1 μg to 100 μg OEGCG and 1 μg to 10,000 μg folate-PEG-EGCG. Incubate the mixture at 25°C for 3 hours.

[0270] 3. Filter out the liquid using a 10KMWCO filter unit. Wash the filter 3 times with 0.9% NaCl.

[0271] 4. Freeze-dried into dry powder.

[0272] List of abbreviations

[0273] AP-1 activator protein-1

[0274] AR androgen receptor

[0275] ASGPR Sialylglycoprotein receptor

[0276] CCL2 Chemokine (CC motif) ligand 2

[0277] CCR2 CC chemokine receptor type 2

[0278] CD20 Cluster of differentiation 20

[0279] CD44 Cluster of differentiation 44

[0280] c-MET Mesenchymal-epithelial transition factor

[0281] DR4 Death receptor 4

[0282] EGal Extracellular domain of glycoprotein A1

[0283] EGF Epidermal Growth Factor

[0284] EGFR Epidermal Growth Factor Receptor

[0285] EpCAM epithelial cell adhesion molecule

[0286] ER estrogen receptor

[0287] Fas Fas cell surface death receptor

[0288] IL-13

[0289] IL-4R Interleukin-4 receptor

[0290] IR Insulin receptor

[0291] LHRH Luteinizing hormone releasing hormone

[0292] LRP5 Low-density lipoprotein receptor-related protein 5

[0293] MMP1 matrix metalloproteinase 1

[0294] MUC1 Mucin 1

[0295] NOTCH1 Neurogenic locus Notch homologous protein 1

[0296] PDGF Platelet-derived growth factor

[0297] PD-L1 Programmed death ligand 1

[0298] PEG Polyethylene glycol

[0299] PIGF Placental Growth Factor

[0300] PLA Polylactic acid

[0301] PLGA Polylactic-co-glycolic acid

[0302] PRPTK7 Protein tyrosine phosphatase receptor type K7

[0303] TbFGF truncated basic fibroblast growth factor

[0304] TNFR1 Tumor necrosis factor receptor 1

[0305] WNT1 Wingless-related integration site 1

Claims

1. A conjugate comprising: (a) a cancer targeting ligand, (b) a hydrophilic polymer of polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA) or dextran, and (c) a flavonoid of EGCG, EC, EGC or ECG, as shown in the following structures: wherein the hydrophilic polymer is covalently bound to the flavonoid and the cancer targeting ligand, Wherein the cancer targeting ligand is selected from the group consisting of: RGD, TP12 having an amino acid sequence of THRPPMWSPVWP (SEQ ID NO: 1), MC11 having an amino acid sequence of MQLPLATGGGC (SEQ ID NO: 2), FV12 having an amino acid sequence of FCDGFYACYMDV (SEQ ID NO: 3), YI12 having an amino acid sequence of YHWYGYTPQNVI (SEQ ID NO: 4), CTT having an amino acid sequence of CTTHWGFTLC (SEQ ID NO: 5), The H2009.1 peptide having the amino acid sequence of RGDLATLRQLAQEDGVVGVR (SEQ ID NO: 6), an IL-13 peptide having an amino acid sequence of GSETWKTIITKN (SEQ ID NO: 7), AP-1 peptide having the amino acid sequence of RKRLDRN (SEQ ID NO: 8), CVKTPAQSC (SEQ ID NO: 9) a CC9 peptide having an amino acid sequence of CDCRGDCFC (SEQ ID NO: 10), an RGDS peptide having the amino acid sequence of RGDS (SEQ ID NO: 11), an NR7 peptide having an amino acid sequence of NSVRGSR (SEQ ID NO: 12), an LHRH peptide having the amino acid sequence of EHWSYGLRPG (SEQ ID NO: 13), angiopep-2 peptide having an amino acid sequence of TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 14), a TbFGF peptide having an amino acid sequence of KRTGQYKLC (SEQ ID NO: 15), an EGF peptide having an amino acid sequence of YHWYGYTPQNVI (SEQ ID NO: 16), Folic acid or folate, hyaluronic acid, lactose, galactose, galactosamine and glycyrrhetinic acid.

2. The conjugate according to claim 1, wherein the flavonoid is EGCG.

3. The conjugate of claim 1, wherein the hydrophilic polymer is PEG.

4. A nanoparticle composition comprising nanoparticles, the nanoparticles having: (a) an outer shell comprising the conjugate according to claim 1, (b) an inner shell comprising oligo(-)-epigallocatechin gallate (OEGCG), and (c) cancer therapeutic drug molecules of cytokines, antibodies or chemotherapeutic agents encapsulated in the inner shell; wherein at least 70% of the nanoparticles have a diameter between 20 nm and 500 nm, and the size distribution of the nanoparticles has only one main peak containing more than 90% of all particles.

5. The nanoparticle composition of claim 3, wherein the shell further comprises a naked hydrophilic polymer-flavonoid conjugate that is not covalently bound to the cancer targeting ligand.

6. The nanoparticle composition of claim 4 or 5, wherein at least 80% of the nanoparticles have a diameter between 50 nm and 300 nm.

7. The nanoparticle composition of claim 4 or 5, wherein at least 90% of the nanoparticles have a diameter between 50 nm and 300 nm.

8. The nanoparticle composition of claim 4 or 5, wherein the nanoparticles have a median diameter of about 50 nm to 250 nm.

9. The nanoparticle composition according to claim 4 or 5, wherein the size distribution of the nanoparticles has only one main peak containing more than 95% of all particles.

10. A method for treating cancer, comprising the step of administering an effective amount of the nanoparticle composition according to any one of claims 1 to 9 to a subject in need thereof.

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