KR20160105146A - Nanoformulated Anticancer Drug and Process for Preparing the Same - Google Patents

Abstract

The present invention relates to a nanoformulated anticancer drug and a manufacturing method thereof. More specifically, the present invention relates to a nanoformulated anticancer drug comprising a hydrophobic anticancer drug, pH-sensitive peptide, and a poloxamer-folate assembly, and relates to a manufacturing method thereof. The nanoformulated anticancer drug of the present invention can minimize side effects.

Description

[0001] The present invention relates to a nano-formulated anticancer agent and a method for preparing the same,

The present invention relates to a nano-anticancer drug type and a method for producing the same. More particularly, the present invention relates to a nano-anticancer agent type comprising a hydrophobic anticancer drug, a pH sensitive peptide, and a poloxamer-folate conjugate, and a method for producing the same.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and is the second most common cancer-related death worldwide. Clinical management of advanced cancer and metastatic cancer is difficult in many ways. Unfortunately, less than 30% of patients are considered to be able to undergo therapeutic procedures including surgical resection, liver transplantation, and chemoembolization.

Moreover, conventional chemotherapeutic agents such as doxorubicin do not appear to significantly prolong survival of non-operable HCC patients (Avila, MA; Berasain C .; Sangro, B .; Prieto, J. Oncogene 2006, 25, 3866-3884). Other conventional chemotherapeutic agents, such as epirubicin, cisplatin, 5-fluorouracil, etoposide, and combinations thereof, have been shown to exhibit far less efficacy than doxorubicin alone (Abdel-Rahman, O .; Fouad , M. Crit. Rev. Oncol / Hematol. Epub.doi.org/10.1016/j.critrevonc.2013.12.013). As a result, the overall prognosis for patients with advanced hepatocellular carcinoma is bleak.

Recently, customized target therapies have become an important strategy in the management of cancer patients at advanced stages, including HCC (Villanueva, A., J. Hepatol ., 2013, 59 , 392-395). Many ongoing clinical trials aim to improve treatment of HCC by targeting a subpopulation of patients.

In 2007, sorafenib, an oral multikinase inhibitor, was approved by the FDA as the first-line treatment for advanced HCC, but the progression-free survival rate progression-free survival increased by only two months.

However, over the last five years, more than 80% of the triple phase trials of targeted molecular therapies failed to prolong the survival rate of patients with advanced disease. Thus, the gloomy prognosis for most HCC patients further emphasizes the urgent need to find and develop more effective treatment strategies for HCC.

Tetriptyline, a diterpenoid triepoxide isolated from plant sterile glands (Tripterygium wilfordii), is a pancreatic cancer (a) Phillips, PA; Dudeja, V .; McCarroll, JA; Borja-Cacho, D .; Dawra, RK; Grizzle, WE; Vickers, SM; Saluja, AK Cancer Res . 2007, 67 , 9407-9416, b) Mujumdar, N .; Mackenzie, TN; Dudeja, V .; Chugh, R .; Antonoff, MB; Borja. Cacho, D .; Sangwan, V .; Dawra, R .; Vickers, SM; Saluja, AK Gastroenterology 2010, 139, 598-608), neuroblastoma (Antonoff, MB; Chugh, R .; Borja-Cacho, D .; Dudeja, V .; Clawson, KA; Skube, SJ; Sorenson, BS; Saltzman, DA; Vickers , SM; Saluja, AK Surgery Rachaphaew, N .; Reutrakul, V .; Sangsuwan, R .; Sirisinha, S. Cancer ( 2009), 146, 282-290) Lett . 1998, 133, 169-175) . & Lt; / RTI >

Though tryptolide was initially recognized as a promising chemotherapeutic drug by many people, its potential for clinical application has been limited due to its low solubility and very high toxicity (Zhou, ZL .; Yang, YX .; Ding, J Li, YC .; Miao, ZH, Nat . Prod ., Rep., 2012, 29, 457-475).

Recently, a synthetic prodrug of tryptolide, referred to as Mineralid, has been studied preclinically as a therapeutic agent for pancreatic cancer. Mineralide has a methyl phosphate group synthesized at the secondary alcohol moiety of the tripolyol that exhibits greater solubility and provides a site for rapid dephosphorylation by the common enzyme alkaline phosphatase. Although such prodrugs exhibit increased solubility and serum half-life, the prodrug is not specifically delivered to tumor cells and thus its efficacy is limited (Chugh, R .; Sangwan, V .; Patil, SP; Dudeja, V Dawra, RK; Banerjee, S .; Schumacher, RJ; Blazar, BR; Georg, GI; Vickers, SM Sci . Transl . Med ., 2012 , 156, 156ra13).

Nanotechnology-based drug delivery systems have been shown to increase drug accumulation and reduce toxicity in tumor cells. Multifunctional nanoparticles are emerging as next-generation anticancer drugs.

Due to excessive blood flow and fenestrated endothelia, generally intravenously injected nanoparticles are absorbed in the liver (Owens III, DE; Peppas, NA Int . J. Pharm . 2006, 307, 93-102). This process is more pronounced due to locally enhanced permeability and retention (EPR) effects in the case of liver cancer (Fang, J .; Nakamura, H.; Maeda, H. Adv Drug Delivery Rev. 2011, 63, 136-151).

Nanoparticles can also be designed to respond to tissue-specific interactions with selectively over-expressed receptors in a local tumor microenvironment or tumor. This may include "active targeting" of cells through surface receptors (e. G., Folate receptors) or drug release triggered by locally expressed enzymes.

In addition, the extracellular pH of solid tumors is stronger than that of normal tissue (about pH 6.0) and (a) Trdan, O .; Galmarini, CM; Patel, K .; Tannock, IF J. Natl. Cancer Inst . 2007 , b) Nichols, JW; Bae, YH Nano Today 2012, 7, 606-618; c) Gallagher, FA; Kettunen, MI; Day, SE; Hu, DE; Ardenkjær-Larsen, JH Nature 2008, 453, 940-943 99, 1441-1454), endo-lysosomes (endolysosome) of cancer cells is a much more strong acid (about pH 5.5) (Urano, Y .; Asanuma, D .; Hama, Y .; Koyama, Y., Barrett, T .; Kamiya, M .; Nagano, T .; Watanabe, T .; Hasegawa, A .; Choyke, PL Nat . Med ., 2008, 15, 104-109).

The present inventors have completed the present invention in view of the fact that drugs can be released only in a pH environment of a tumor tissue using a pH difference between a tumor tissue and a normal tissue, thereby minimizing side effects caused by drugs.

A basic object of the present invention is to provide a nano-anticancer agent type comprising a hydrophobic anticancer drug, a pH sensitive peptide, and a poloxamer-folate conjugate.

Another object of the present invention is to provide a pharmaceutical composition comprising (i) a hydrophobic anticancer drug, a pH sensitive peptide, and a poloxamer-folate conjugate in a solvent; (ii) drying the solution obtained in step (i); And (iii) hydrating the dried material obtained in the step (ii).

The basic object of the present invention described above can be achieved by providing a nano-anticancer agent type comprising a hydrophobic anti-cancer drug, a pH sensitive peptide, and a poloxamer-folate conjugate.

The hydrophobic anticancer drug, pH sensitive peptide, and poloxamer-folate conjugate spontaneously form micellar nanogels in water. More specifically, the micelle-structured nanogel is characterized in that the hydrophobic portion of the hydrophobic anticancer drug, the pH sensitive peptide and the poloxamer-folate conjugate forms a core and the hydrophilic portion of the poloxamer- And is exposed to the outside of the core.

The pH sensitive peptide is degraded in tumor cells that are more acidic than normal tissue, thereby allowing the hydrophobic anti-cancer drug to be released. As a result, the hydrophobic hydrophobic anti-cancer drug acts only on the tumor cells.

The hydrophilic portion of the poloxamer-folate conjugate is exposed to the outside of the core, and the folate moiety acts as a targeting ligand for the tumor cells.

That is, when the folate moiety induces the nano-anticancer drug type of the present invention to a tumor cell, the pH-sensitive peptide is degraded in response to the acidic pH environment of the tumor cell, whereby the hydrophobic anti-cancer drug is released to act on the tumor cell do.

The hydrophobic anticancer drugs included in the nano-anticancer drug type of the present invention include Bendamustine, Busulfan, Carmustine, Chlorambucil, Cyclophosphamide, An alkylating agent such as Dacarbazine, Ifosfamide, Melphalan, Procarbazine, Streptozocin or Temozolomide; or an alkylating agent such as Dacarbazine, Ifosfamide, Melphalan, Procarbazine, Streptozocin or Temozolomide; Asparaginase, Capecitabine, Cytarabine, 5-Fluorouracil, Fludarabine, Gemcitabine, Methotrexate, Femetrexed, Antimetabolites such as Pemetrexed or Raltitrexed; But are not limited to, Actinomycin D (Dactinomycin), Bleomycin, Daunorubicin, Doxorubicin, Pegylated liposomal Doxorubicin, Epirubicin, Anti-Tumor Antibiotics such as Idarubicin, Mitomycin or Mitoxantrone; A plant alkaloid such as Etoposide, Docetaxel, Irinotecan, Paclitaxel, Topotecan, Vinblastine, Vincristine or Vinorelbine, / Plant alkaloids / microtubule inhibitors; DNA binding agents such as Carboplatin, Cisplatin or Oxaliplatin; Alemtuzamab, BCG (bacille de Calmette-Guerin vaccine), Bevacizumab, Cetuximab, Denosumab, Erlotinib, The compounds of the present invention may be selected from the group consisting of Gefitinib, Imatinib, Interferon, Ipilimumab, Lapatinib, Panitumumab, Rituximab, Sunitinib, Biological agents such as Sorafenib, Temsirolimus or Trastuzumab; Or bisphosphonates such as Clodronate, Ibandronic acid, Pamidronate, or Zolendronic acid.

Also, the pH sensitive peptide contained in the nano-anticancer drug type of the present invention is octadecylamine-p (API-Asp) ₁₀ ; Fusogenic GALA peptides; Or pHLIP peptides such as H ₂ N- (AALEALAEALEALAEALEALAEAAAAGGC) -CO ₂ H, H ₂ N- (AALAEALAEALAEALAEALAAAAAGGC) -CO ₂ H or H ₂ N- (ALEALAEALEALAEA) -CONH ₂ .

In the nano-anticancer drug type of the present invention, the pH-sensitive peptide is decomposed under an acidic environment to release the hydrophobic anti-cancer drug. That is, in the weakly alkaline normal cells or tissues, the nano-anticancer drug type remains unchanged, but the pH sensitive peptide is degraded in the tumor cells or tissues in the acidic environment, thereby releasing the hydrophobic anti-cancer drug.

The poloxamer-folate conjugate included in the nano-anticancer drug form of the present invention may be pluronic F127-folate.

In the present specification, Pluronic F127 refers to a compound having the structure of formula (I) below.

(I)

In the formula (I), a is an integer of 80 to 150, and b is an integer of 50 to 100.

The nano-anticancer drug type of the present invention may be a therapeutic agent for liver cancer. The hydrophobic anticancer drug acts on liver cancer cell lines such as ZNF114, BHLHE40, CKS2, RND3, SLC38A2, TXNIP, ZC3H11A, TLE1, HIST1H1E, MKI67IP, ANKRD1, LEO1, AURKA, PLIN2, WDR82, SETX, ARRDC3, PUM2, WDR43 or SETD5 .

The nano-anticancer drug type of the present invention may be a therapeutic agent for liver cancer. The hydrophobic anticancer drugs include ZNF114, BHLHE40, CKS2, RND3, SLC38A2, TXNIP, ZC3H11A, TLE1, HIST1H1E, MKI67IP, ANKRD1, LEO1, AURKA, PLIN2, WDR82, SETX, ARRDC3, PUM2, WDR43, SETD5, MH- H1, HF1-5, HF1, C2-Rev7, C2, H5, HTC, Hepa 1-6, SK-HEP-1, MCA-RH 7777, N1-S1, H4S, Fao, ARL-6, H-4 May act on liver cancer cell lines such as Hep-2-E, Hep 3B, MH1C1, PLC / PRF / 5, H4-II-E-C3, HTC (BUdR), Hep G2 or Huh-7D12.

Another object of the present invention described above is to provide a method for preparing a pharmaceutical composition comprising the steps of (i) dissolving a hydrophobic anti-cancer drug, a pH sensitive peptide, and a poloxamer-folate conjugate in a solvent; (ii) drying the solution obtained in step (i); And (iii) hydrating the dried product obtained in the step (ii).

In the nano-anticancer drug form produced by the method of the present invention, the pH-sensitive peptide is degraded under an acidic environment to release the hydrophobic anti-cancer drug. That is, in the weakly alkaline normal cells or tissues, the nano-anticancer drug type remains unchanged, but the pH sensitive peptide is degraded in the tumor cells or tissues in the acidic environment, thereby releasing the hydrophobic anti-cancer drug.

The hydrophobic anti-cancer drug used in the method of the present invention may be tryptolide, sorapenib, doxorubicin or daunorubicin.

The pH-sensitive peptide used in the method of the present invention may be octadecylamine-p (API-Asp) ₁₀ . In addition, the poloxamer-folate conjugate may be pluronic F127-folate.

The nano-anticancer drug type may be a therapeutic agent for liver cancer. In addition, the hydrophobic anticancer drug may be selected from the group consisting of ZNF114, BHLHE40, CKS2, RND3, SLC38A2, TXNIP, ZC3H11A, TLE1, HIST1H1E, MKI67IP, ANKRD1, LEO1, AURKA, PLIN2, WDR82, SETX, ARRDC3, PUM2, WDR43, It acts on the cell line.

In step (iii) of the method of the present invention, phosphate buffered saline (PBS) can be used and ultrasonication can be performed.

In the step (iii) of the method of the present invention, the hydrophobic anticancer drug, the pH sensitive peptide, and the poloxamer-folate conjugate spontaneously form a micellar nanogel, and the nanogel of the micellar structure, Wherein the hydrophobic portion of the pH sensitive peptide and the poloxamer-folate conjugate forms a core, and the hydrophilic portion of the poloxamer-folate conjugate is exposed to the outside of the core.

In accordance with one embodiment of the present invention, low-solubility and highly toxic tryptolide is administered to a follicular-coated tumor pH that is capable of targeting HCC-subpopulations that overexpress a folate receptor - It can be formulated with a tumor pH-sensitive nanogel.

A nanoformulated triptolide (Nf-Trip) is composed of two polymers synthesized from natural tryptolide and FDA-approved materials. One polymer is pluronic F127 esterified with folic acid 3a and 3b), and the other polymer is a synthesized 10-mer of aspartic acid having an octadecylamine tail and an ionizable amidazole side chain (see FIGS. 3A and 3C) .

The pH sensitive peptide of the present invention has the following two advantages. First, the pH-sensitive polymer is prepared by (1) ring-opening polymerization (ROP) of an α-amino acid N-carboxyanhydride (NCA) using octadecylamine as an initiator and (2) is synthesized through a two-step reaction of introducing a pH-sensitive ionizable functional group (e.g., an imidazole group); This simple process facilitates the application of cancer nanotechnology to clinical practice.

Second, the use of octadecylamine as an initiator for ROP provides a superhydrophobic moiety in addition to the resulting pH-sensitive polypeptide, which enhances the charging efficiency of hydrophobic drugs such as tryptolide.

The two polymers spontaneously form micelle structured nanogels in water (see Figure 3d). The folate exposed on the surface acts as a cancer cell-targeting ligand, and the internally densified imidazole provides an ionizable moiety for pH-sensitive ionisation and dispersion (see Figures 3e-3g).

The tryptolide is filled into the hydrophobic core of the nanogel by thin-film hydration. With this Nf-Trip, hydrophobic tryptolide can be administered intravenously. The acidic environment of the tumor provides prodrugs to the imadazole compounds therein, leading to tumor-specific drug release when the micelles collapse. The folate (targeting ligand) can promote receptor-mediated intracellular entry into tumor cells that overexpress the folate receptor (FR), and that during the endolysosomal acidification process, the tryptolide is very < RTI ID = And is efficiently released.

Since the nano-anticancer drug of the present invention delivers anticancer drugs to tumor cells or tissues and reaches the tumor and releases the anticancer drugs, anticancer drugs with high toxicity can also be formulated. Therefore, the use of the nano-anticancer agent of the present invention minimizes or eliminates side effects.

Figure 1 a shows in vitro compound screening for the identification of tryptolide using the MTS assay and Figures 1b to 1d show the results of the in vitro screening of tryptolide, sorapenib, and daunorhodide in liver cancer cell lines, as analyzed by MTS, Dose-dependent inhibitory effects of < RTI ID = 0.0 >
Figure 2 shows the increase in Annexin-V + and 7-AAD + cells observed by flow cytometry for both Bel-7404 and HCCLM3 cells treated with 10 ng / mL tryptolide.
FIG. 3A shows the synthesis process of a pluronic F127-folate conjugate and octadecylamine-p (API-Asp) ₁₀ , FIG. 3B shows the synthesis of a folate-pluronic F127 conjugate synthesized in Example 2 of the present invention FIG. 3C is a molecular formula and ¹ H-NMR analysis result of octadecylamine-p (API-Asp) ₁₀ synthesized in Example 3 of the present invention, and FIG. 3D is a result of molecular formula and ¹ H- 3e is a SEM image of Nf-Trip at pH 7.4 and pH 6.0, and Fig. 3f is a DLS size measurement of Nf-Trip (0.1 mg / ml) as a function of pH , And Fig. 3g is the percent transmittance of the Nf-Trip suspension as a function of pH.
Figure 4a is a conceptual diagram of tumor pH-responsive drug release of Nf-Trip in tumor microenvironment, Figure 4b shows the pH dependent drug release of tryptolide from Nf-Trip, Figure 4c shows FR + 4d and 4e show the inhibition effect of Nf-Trip on Bel-7404 and FR-MIHA cells, respectively. Fig. 4d and Fig. 4f shows the effect of Nf-Trip on the inhibition of the Bel-7404 cell cycle in the G2 / M phase analyzed by flow cytometry, FIG. 4g shows the inhibitory effect of the free tryptolide on TUNEL (Scale bar = 50 μm) in the analyzed Bel-7404 and MIHA cells, and FIG. 4h shows DNA induced by Nf-Trip, analyzed by P-H2AX staining This is a picture of the damage (scale bar = 50 μm).
5a and 5b show cell cycle arrest and apoptosis-related cyclin kinase subunit-2 (CKS2) (Fig. 5a) by Nf-Trip treatment as assessed by RT-qPCR. And Aurora A kinase (AURKA) (Fig. 5B), and Figs. 5C and 5D show the expression of CKS2 and AURKA in tissues of HCC patients significantly compared with corresponding adjacent non-tumor liver tissues by RT- (T: tumorous liver, MN: corresponding adjacent non-tumor liver; NN: histologically normal liver tissue), FIGS. 5e and 5f show that CKS2 and AURKA (Fisher ' s exact test and Kaplan-Meier analysis results, respectively, showing expression.
Figure 6a shows the results of Nf-Trip (10 ng / ml) in FR ⁺ Bel-7404 liver cancer cells and FR ^- liver parenchymal cells (MIHA) obtained using Nikon's bio- 6B is a photograph (scale bar = 50 μm) of Nf-Trip cell uptake in FR ⁺ Bel-7404 and FR ^- MIHA cells using CLSM (scale bar = 50 μm) Is an in vivo photograph of a mouse containing luciferase expressing an isoHCC tumor derived from FR ⁺ Bel-7404 cells after intravenous injection of Nf-Trip (containing RITC), obtained using the Xenogen IVIS Lumina system.
Figure 7a is a photograph of a weekly interval for an islet tumor xenotransplantation during treatment, Figure 7b is a tumor-containing liver cut from the mouse at the end of treatment of the other therapies, Figure 7c is a week FIG. 7D shows the results of quantitative analysis on the bioluminescence signal of the intervals, FIG. 7D shows the average mouse body weight in the weekly intervals for the three treatment groups, FIG. 7E shows Fisher's exact test for the mouse survival rate over time in the above three groups (Scale bar = 50 μm). Fig. 7 (g) is a photograph of H & E staining obtained from the above three groups and IHC staining of tumor tissue against CKS2 and AURKA, A TUNEL analysis of the tumor from the three groups (scale bar = 50 μm).

Hereinafter, the present invention will be described in more detail with reference to the following examples or drawings. It is to be understood, however, that the following description of the embodiments or drawings is intended to illustrate specific embodiments of the invention and is not intended to be exhaustive or to limit the scope of the invention to the precise forms disclosed.

Example  1. Toxicity In vitro  Screening

Triptolide was chosen because of its efficacy on various cell lines (Figure 1a). In contrast to the more commonly used clinical chemotherapeutic agents (sorafenib, doxorubicin and daunorubicin) for HCC patients, tryptolide resulted in a much greater cytotoxic effect on human liver cancer cell lines (Fig. 1B 1E). Two liver cancer cell lines, Bel-7404 and HCCLM3, were treated with tryptolide at a concentration of 10 ng / mL to assess the effect of tryptolide on cell death in hepatoma cell lines. As expected, tryptolide treatment resulted in a significant effect on apoptosis in both liver cancer cell lines. The cell lines were analyzed by Annexin-V and 7-AAD using flow cytometry. Both Annexin-V positive cells and 7-AAD positive cells were significantly increased in both Bel-7404 and HCCLM3 cells within 24 hours after treatment with 10 ng / mL tryptolide, indicating significant apoptosis and necrosis (Fig. 2).

Example  2. Folate- Pluronic  F127 junction ( Folate - pluronic  F127 ( FA - PF127 ) conjugate

100 mg of the pluronic F127 powder was dissolved in anhydrous DMSO. 5 mg of folic acid (1.5 eq) was dissolved in 10 mL of DMSO and 1.5 molar equivalents of DCC (N, N'-dicyclohexylcarbodiimide) and DMAP (4-dimethylaminopyridine) were added. Pluronic F127 was dissolved in dimethyl sulfoxide (DMSO) with vigorous stirring and then slowly mixed with activated folic acid. The reaction mixture was then stirred at room temperature for 24 hours. The reaction mixture was dialyzed against distilled water for 2 days (MWCO: 1000 Da) to remove unfused folate and DMSO. The final solution was frozen for an instant and lyophilized. The folate-pluronic F127 conjugate thus obtained was purified using Sephadex LH-20 (Sigma-Aldrich, Co.). The molecular formula and the ¹ H-NMR analysis of the folate-pluronic F127 conjugate are shown in FIG.

Folic ^acid-1 conjugate on Nick F127 Pluronic H NMR (500 MHz, DMSO- d 6): σ = 8.6

ppm (1H, d, folic acid of _{-N = C H 1 CH 1 =} ), 7.6 ppm (t, 1H, folic acid of -C H = CH-), 6.6 ppm (t, 1H, -CH = C H of folic acid -), 3.4 - 3.6 ppm ( m, 848H, Nick, -C H ₂ C H ₂ O-) of Pluronic F127, and 1.0 ppm (d, 210H, Pluronic F127 of -CH ₂ CH- (C H ₃ ) O-).

Example  3. Octadecylamine -p ( API - Asp ) ₁₀ Synthesis of

3 g (12 mmol) of β- benzyl -L- aspartate N- carboxy anhydride (BLA-NCA) a, 20 mL of DMF (dimethylformamide) and 50 mL of CH ₂ Cl ₂ mixtures in octadecylamine (M _W = 269.51 g / mol, 0.3 g, 1.2 mmol) and polymerized at 40 < 0 > C. The reaction mixture was stirred for 2 days. The reaction mixture was rotary evaporated under high vacuum to remove the CH ₂ Cl ₂ . 20 mL of 0.1 N HCl was slowly added to precipitate the polymer, followed by centrifugation (3000 rpm) for 5 minutes. The supernatant was discarded and the remaining residue was lyophilized to remove residual water. PH-sensitive octadecylamine-p (API-Asp) ₁₀ was synthesized by aminolysis of the octadecylamine-PBLA octadecylamine-PBLA using 1- (3-aminopropyl) imidazole . Octadecylamine-p (API-Asp) ₁₀ (0.2, 74.8 μmol) was dissolved in 5 mL of DMSO and reacted with API (1 g, 7.9 mmol) at 25 ° C. under nitrogen and stirred for 12 hours. The reaction mixture was added dropwise to 20 mL of 1 N cold HCl aqueous solution and dialyzed three times against 0.01 N HCl solution (Spectra / Por; MWCO: 1,000 Da). The final solution was lyophilized to give octadecylamine-p (API-Asp) ₁₀ as a white solid.

1 H NMR (500 MHz, DMSO-d ₆ ) for octadecylamine-p (API-Asp) ₁₀ :? = 7.8

ppm (1H, s, -NC H = N-) of the imidazole ring, 7.7 ppm (1H, s, -NC H = CH- of the imidazole ring), 7.3 ppm = C H -N-), 4.5 ppm (1H, m, -NHCHC = O-), 4.0 ppm (2H, s, = NCH2CH2), 3.0 ppm (2H, s, -NHC H 2 CH 2 -), 2.6 ppm (2H, m, -C ₂ H C = ONH-), ppm 1.8 (2H, s, C H ₂ -CH ₂ CH ₂ -), ppm 1.2 (17H, s, of octadecylamine -CH ₂ -) , And 0.8 ppm (3H, t, CH ₃ of octadecylamine).

Example  4. With fluorescein (RITC) Labeled Octadecylamine -p ( API - Asp ) ₁₀ Synthesis of

100 mg of octadecylamine-p (API-Asp) ₁₀ in 20 mL of DMSO was reacted with 30 mg of RITC for 12 hours at room temperature in the dark. The reaction mixture was dialyzed against distilled water for 2 days (MWCO: 1000 Da) to remove unlabeled RITC and DMSO.

Example  5. Nanotriptolide Formulation ( nanoformulated triptolide  ( Nf - Trip ))

20 mg of octadecylamine-p (API-Asp) ₁₀ , 20 mg of FA-F127 and 4 mg of tryptolide were dissolved in 6 mL of methanol and added to a wall of a 25 mL round bottom flask at 60 & The film was dried to form. Then, 5 mL of PBS (pH 7.4) was added to the flask to hydrate the film. After sonication for 5 minutes using a probe sonicator (Sonics Vibra cell, Sonics & Material Inc., Newtown, CT, USA), the solution was stirred continuously at 60 ° C for 6 hours. To remove uncharged tryptolide, the sample was dialyzed against PBS (pH 7.4) for 12 hours.

The Nf-Trip thus obtained showed a drug-to-carrier ratio of about 9% and a high charging efficiency of about 94%. The hydrodynamic radius of the Nf-Trip at physiological pH was measured to be about 120 nm (Figure 3f). First, the acid-sensitivity of the Nf-Trip was examined by scanning electron microscopy (SEM) and dynamic light scattering (DLS). At physiological pH, pronounced nanoparticles were observed, but upon acidification at pH 6.0, the nanoparticles completely disintegrated (FIGS. 3e and 3f). Also showed a stepwise decrease in% transmittance at a critical pH range of about 6.5 by acid-base titration (Figure 3g).

Example  6. Cell line and cell culture

Human liver cancer cells (Bel-7404 or HCCKM3) or immortalized normal hepatocytes (MIHA) were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin- Lt; / RTI > The cells were maintained at 37 ℃ under 5% CO ₂ present.

Example  7. In vitro cell uptake

After confluence, the cells were removed, counted, and inoculated overnight in an 8-well coverglass chamber (LAB-TEK, Nagle Nunc, IL, USA). The medium was then replaced with Nf-Trip spiked medium at different concentrations and incubated for different times. The cell uptake of Nf-Trip was observed using a Nikon live cell imaging system. The cell uptake behavior and intracellular distribution of the Nf-Trip were analyzed using a confocal laser scanning microscope (CLSM, Carl Zeiss). Nf-Trip treated cells were washed twice with prewarmed 1X PBS and fixed with 4% paraformaldehyde for 15 minutes. The cells were then washed twice with PBS, stained for nuclei using Hoechst 33342 and imaged with CLSM. For analysis, data was processed using Adobe Photoshop 7.0 software.

Example  8. In vitro  toxicity

Liver cancer cells or immortalized normal hepatocytes (MIHA) were inoculated into 96-well plates at a density of 5 x 10 ³ cells per well (100 μL). The medium was replaced with a medium in which Nf-Trip was mixed at different concentrations. For MTS analysis, CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, Wis., USA) was used according to manufacturer's instructions. Two hours before each desired time point, 20 [mu] L of MTS reagent was added to each well and the cells were incubated at 37 [deg.] C for 2 hours. Absorbance was detected at 490 nm using a Wallac Victor 1420 Multilabel plate reader. All experiments were repeated 3 times.

Cytotoxicity can be effected by folate receptor-mediated cell endocytosis following pH-dependent extracellular release or intercellular release, thus testing the in vitro drug release and efficacy of Nf-Trip. Release by pH from the Nf-Trip was faster at about pH 6 (tumor environment) compared to pH 7.4 (systemic circulation), but full drug release took several days (Figs. 4a and 4b). Nf-Trip inhibited the survival of FR ⁺ Bel-7404 cells more effectively than FR ^- MIHA cells (Fig. 4C). In addition, the Nf-Trip more strongly inhibited the FR ^- MIHA cell growth at pH 6.0 (Fig. 4d) and at the same time showed reduced cytotoxicity to normal FR ^- MIHA cells at pH 7.4 (Fig. 4e). Moreover, the extracellularly released tryptolide showed a much reduced effect compared to Nf-Trip at lower concentrations regardless of cell type or pH, as seen in vitro (Fig. 4d and 4e). Thus, the main driving force for cytotoxicity is due to folate-mediated intracellular release, which is more pronounced in the FR ⁺ Bel-7404 cells compared to the FR ^- MIHA cells (Figures 4c-4e). Due to the high cancer-selective nature of the Nf-Trip nanoparticles observed in vitro (Figures 4c-4e), the pH-dependent intracellular drug release can significantly reduce toxicity to non-tumor tissues and cells.

Example  9. Apoptosis  analysis( TUNEL ) And DNA  Damage analysis

To mark the nuclei of apoptotic cells, Nf-Trip treated HCC cells were placed on glass coverslips in 24-well plates and fixed with 4% paraformaldehyde 24 hours after Nf-Trip treatment Respectively. Terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining was performed using the DeadEnd fluorometric TUNEL system (Promega) according to the manufacturer's protocol. The percentage of apoptotic cells was determined by dividing the number of TUNEL-positive cells by the number of Hoechst 33342-stained cells. In two 40x objective fields containing about 100 cells, each was counted per cover slip, and three cover slips per condition were analyzed.

The HCC cells were inoculated into BD Falcon ^TM 8-well culture slides (CultureSlide) and treated with Nf-Trip for 24 hours. After incubation the cells were fixed to the handle by using the ilchang antibody to p-H2AX (primary antibodies), Alexa Fluor ® 488 goat anti-incubated with the rabbit IgG (Invitrogen). The slides were contrast dyed using Hoechst 33342 and imaged using CLSM.

The effect of Nf-Trip on apoptosis and DNA damage in FR ⁺ Bel-7404 and FR ^- MIHA cells at pH 7.4 was evaluated by TUNEL (DeadEnd fluorometric TUNEL system from Promega) and DNA damage marker P-H2AX According to the results of the above investigation, apoptosis and DNA damage were seriously induced in Nf-Trip treated FR ⁺ HCC cells, but they were minimized in FR ^- MIHA cells (FIGS. 4g and 4h) , Which implies a specific targeting and inhibitory effect of Nf-Trip on FR ⁺ HCC cells in vitro.

Example  10. Flow cytometry

The cell cycle was analyzed by flow cytometry (FACSCanto II, BD Biosciences) using PI staining (BD Biosciences) according to standard protocols. Briefly, the same number of cells (2.0 x 10 ⁵ ) were inoculated on a 6-well tissue culture plate one day prior to treatment. The cells were then treated with Nf-Trip (0.01 [mu] L / mL) or control. After 24 hours, the cells were fixed in ice-frozen 70% ethanol and stained using a Coulter DNA-Prep reagent kit (Beckman Coulter, Fullerton, Calif.). From each sample, cellular DNA content corresponding to 5 × 10 ⁵ cells was determined by flow cytometry (FACSCanto II, BD Biosciences). The distribution of the cell cycle phase was analyzed using FlowJo software, which uses data from two separate experiments performed three times each time.

The cytotoxic effect on liver cancer cells may be related to the effect on cell cycle regulation. The effect of Nf-Trip on FR ⁺ HCC cell cycle arrest was analyzed using PI staining and flow cytometry. In contrast to the control group, cells that underwent G2 / M cell cycle suppression in FR ⁺ HCC cells treated with Nf-Trip were doubled (G2 / M group: 25.29% in Nf-Trip vs. 11.44 in control group %) (Figure 4f).

Example  11. Microarray  And real-time quantitative RT -PCR ( qRT - PCR )

Total RNA (total RNA) was extracted from tissue samples or Bel-7404 cells treated with Nf-Trip or a control using TRIzol reagent (Invitroge). The quality and quantity of isolated total RNA was analyzed using an Agilent 2100 Bioanalyzer and a NanoDrop ND-1000 Spectrophotometer (Agilent, Santa Clara, Calif., USA). Microarray analysis was performed according to manufacturer's instructions using GeneChip ^® Human Gene ST Arrays (Affymetrix, USA). Affymetrix GeneChip Fluidics Station 450 was washed after hybridization. The array was scanned over an Affymetrix GeneChip Scanner 3000. Partek software ((Partek Incorporated. St. Louis, were normalized to the scanned array using the GCRMA MO). Quality control was an array using the Affymetrix Expression Console ^{® TM.} Was converted to a signal strength log2 Partek Gemonics Software Suite (Partek Inc., St. Louis, Mo.) and subjected to statistical analysis. The microarray data were stored in the European Bioinformatics Institute database of the European Molecular Biology Laboratory http://www.ebi.ac.uk/array express /), access numbers E-MEXP-84 and E-TABM-292 through the ArrayExpress public database. For mRNA detection, the total RNA was reversely transcribed using SuperScript III First-Strand Synthesis System (Invitrogen, CA) for RT-PCR. Hypoxanthin phospholiposyl transfer Claim was used for 1 (HPRT1), and performing qPCR using ^{SsoFast TM EvaGreen ® Supermix (Bio-} Rad), and calculated the difference in concentration over a relatively Assay (2 ^{-Δ Ct)} Kaplan-Meier survival using the method Survival curves were generated and statistically compared using a log-rank test.

In addition to cytotoxicity, mechanisms of action at the gene level are considered. To identify potential downstream targets, gene expression profiles were generated for Bel-7404 treated with 10 ng / mL tryptolide or DMSO (dimethyl sulfoxide). The top 20 genes with reduced expression (Table 1) were further examined. The microarray data was further confirmed by real-time RT-PCR. According to the results, both CKS2 and AURKA were downregulated in HCC cells after treatment with Nf-Trip (Figures 5a and 5b).

The top 20 genes with reduced expression by tryptolide in HCC cells Transit cluster ID
(Transcript Cluster ID) Gene symbol
(Gene Symbol) Fold Change
(Tryptolide / control (Control)) 8030002 ZNF114 -25.3734 8077441 BHLHE40 -17.2767 8156290 CKS2 -13.5553 8055688 RND3 -12.9608 7962537 SLC38A2 -12.0721 7904726 TXNIP -11.3302 7908978 ZC3H11A -11.2426 8161919 TLE1 -10.1573 8117377 HIST1H1E -10.0923 8054930 MKI67IP -9.83051 7934979 ANKRD1 -9.82121 7988838 LEO1 -9.72329 8067167 AURKA -9.25794 8160297 PLIN2 -9.05077 8087874 WDR82 -9.02924 8164701 SETX -8.88432 8113073 ARRDC3 -8.389 8050565 PUM2 -8.33998 8041149 WDR43 -8.17517 8077528 SETD5 -8.02955

Because preclinical data for HCC differ from model to model, it is beneficial to examine these results in the context of more reliable and relevant clinical data sets. We established a global gene profile database for HCC tumor tissues and histologically normal liver tissues using Affymetrix Human Genome U133 plus 2.0 Arrays (Affymetrix, Santa Clara, Calif., USA) , H. Ooi, LLP & Hui, KM MicroRNA-216a / 217.induced epithelial.Mesenchymal transition targets PTEN and SMAD7 to promote drug resistance and recurrence of liver cancer. Hepatology 58 , 629-641 (2013); Wang, SM, Ooi, LLPJ & Hui, KM Upregulation of Rac GTPase-activating protein 1 is significantly associated with early recurrence of human hepatocellular carcinoma. Clin . Cancer Res . 17, 6040-6051 (2011); Xia, H., Ooi, LLPJ & Hui, KM MiR-214 targets β-Catenin pathway to suppress invasion, stem-like traits and recurrence of human hepatocellular carcinoma. PLoS ONE 7, e44206 (2012)). Using this, we analyzed the expression levels of CKS2 and AURKA in HCC tissue samples of patients and found that both CKS2 and AURKA were significantly upregulated in the human HCC samples, as compared to the corresponding peripheral or histologically normal liver tissue ) (Figs. 5C and 5D). The median value of expression of CKS2 and AURKA in all 76 HCC samples was chosen as the cut-off point. Fisher's exact test and Kaplan-Meier analysis showed that high levels of expression of CKS2 and AURKA were closely related to patient survival (Figures 5e and 5f). This result implies that Nf-Trip can be an important therapeutic target for HCC because CKS2 and AURKA actually play an important role in hepatocarcinogenesis.

Example  12. Immunohistochemistry and Dynamic HCC  Model

Paraffin-embedded tissue samples from patients were cut into 5 μm slices, placed on polylysine-coated slides, paraffin removed in xylene, rehydrated using graded alcohols. Antigen was recovered by heat mediation in citrate buffer (pH 6, Dako). Samples were blocked using 10% goat serum prior to incubation with the primary antibody. The sample was incubated overnight with a primary antibody that is anti-CKS2 and AURKA (1: 100) (Abcam) or isotype-matched IgG as negative control in a wet vessel at 4 째 C Lt; / RTI > Immunohistochemical staining was performed using Dako Envision Plus System (Dako, Carpinteria, Calif.) According to manufacturer's instructions. Staining intensity was evaluated on a scale of 0 to 4 according to the percentage of benign tumors.

Bel-7404 Luciferase-expressing cells were subcutaneously injected into a 5-week to 6-week-old athymic mouse (BioLASCO Taiwan Co., Ltd) to generate tumors. The mice were then anesthetized using hip / midazolam and the tumors were dissected and sectioned. An orthotopic xenograft liver tumor model was established by transplanting a similar sized tumoroid into a new mouse liver. One week after the surgery, the mice were photographed and grouped based on uniform signal intensity. The mice were then treated with Nf-Trip, free triptolide or vehicle intravenously twice weekly for 7 weeks. Tumor growth was measured weekly by bioluminescence imaging using the Xenogen IVIS Lumina system (Xenogen Corporation, Hopkinton, Mass.). Briefly, D-luciferin (150 mg / kg; Caliper Life Sciences, Inc., Hopkinton, Mass.) Was intraperitoneally injected into the mice after anesthesia with 2% isoflurane, For 1 min.

In vitro cell uptake efficiency of Nf-Trip over folate receptor overexpression was examined by comparing FR ⁺ HCC cell line (Bel-7404) in comparison with FR ^- normal hepatocyte (MIHA). When cultured with FR ^- MIHA cells, Nf-Trip had little cellular uptake, whereas the uptake into FR ⁺ Bel-7404 was considerably more efficient, thus imaging living cells with differential interference contrast (DIC) , Exhibited excellent cell-specific efficacy (Fig. 6A). Next, confocal with the laser scanning microscope (CLSM) were investigated in addition to the absorption of a Nf-Trip of FR ⁺ HCC vertical, FR ^- liver within MIHA cells only dispersed cells with a minimum amount of intracellular fluorescence (Fig. 6B).

To analyze in vivo tumor targeting, a HCC isoform transplantation model developed from luciferase expressing FR ⁺ HCC was used. All experiments with mice were approved by the SingHealth Institutional Animal Care and Use Committee (IACUC). FR ⁺ Bel-7404 cells transfected with the luciferase vector were subcutaneously transplanted and grown in nude mice (BioLASCO Taiwan Co., Ltd). These ectopic tumors were excised and small tumoroids transplanted orthotopically into the liver of new mice. When the xenogeneic xenograft tumor was established, treatment was initiated by intravenous administration of Nf-Trip (1 mgeq / kg) by a tail vein injection twice a week. After the tail vein injection, the systemic distribution of Nf-Trip was imaged using RITC by the Xenogen IVIS Lumina System. After 20 hours, the Xenogen system was used to confirm that the Nf-Trip was accumulated in liver tumor tissue, which was co-localized with the luciferase signal from the transplanted tumor cells (FIG. 6c).

To examine the efficacy of the nanotriptolide formulations according to the invention, an orthotopic HCC model expressing luciferase was used. Because the tumor cells grow in the liver, it is a better model for natural tumors, but it is difficult to examine changes in size of deep tissue tumors. Luciferase in these tumors allows imaging through the Xenogen IVIS lumina system, which makes significant advances in terms of clinical relevance. Once the iso xenograft tumor is established, the free tryptolide and diluted DMSO vehicle in Nf-Trip (1 mgeq / kg), dimethylsulfoxide (DMSO, 1 mg / kg) are injected intravenously Lt; / RTI > The therapeutic efficacy was determined by mouse survival as well as tumor size measurements estimated from bioluminescence signals after administration of luciferin. Both groups showed efficacy as compared to the untreated control group and the Nf-Trip showed greater efficacy than the drug liberated by both the luciferase signal and physical examination of the excised organ (Figs. 7a-c). In addition, the untreated control and Nf-Trip treated mice maintained their body weight stable, whereas the mice treated with the free tryptolide significantly reduced body weight (FIG. 7d), indicating that the high toxicity of tryptolide And was mitigated by targeted formulation. In terms of survival rate, mice treated with Nf-Trip showed a survival rate of about 80% after 3 months, survival rate of about 50% for mice treated with free tryptolide, and about 50% 30% survival rate (Fig. 7E).

To assess cell-level efficacy, tumors were collected and histochemically examined after the pharmacodynamic study. The Nf-Trip treated tumors by hematoxylin and eosin (H & E) staining were much necrotic as compared to the free tryptolide treated tumors. In the DMSO control, necrosis necrosis) was not observed (Fig. 7f). Consistent with the in vitro test results, the TUNEL staining showed that the population of cells suffering apoptosis in Nf-Triop treated tumor tissue was much higher than the free tryptolized tumor and the DMSO control (Fig. 7G). Finally, immunohistochemical (IHC) staining significantly reduced the expression of CKS2 and AURKA in Nf-Trip treated tumor tissue compared to the free tryptolide treated tumor tissue or the DMSO treated tumor tissue (Fig. 7F).

Example  14. Survival rate  And statistical analysis

Experimental data are expressed as mean ± standard deviation. All statistical analyzes were performed using ANOVA or two-tailed Student's t test (GraphPad Prism 5). Survival curves were generated using the Kaplan-Meier method and statistically compared using the log-rank test. P values less than 0.05 were considered statistically significant.

Example  7. No toxicity in equine mouse models Nf - Trip Tumor burden by Survival rate  increase

To examine the efficacy of the nanotriptolide formulations according to the invention, an orthotopic HCC model expressing luciferase was used. Because the tumor cells grow in the liver, it is a better model for natural tumors, but it is difficult to examine changes in size of deep tissue tumors. Luciferase in these tumors allows imaging through the Xenogen IVIS lumina system, which makes significant advances in terms of clinical relevance. Once the iso xenograft tumor is established, the free tryptolide and diluted DMSO vehicle in Nf-Trip (1 mgeq / kg), dimethylsulfoxide (DMSO, 1 mg / kg) are injected intravenously Lt; / RTI > The therapeutic efficacy was determined by mouse survival as well as tumor size measurements estimated from bioluminescence signals after administration of luciferin. Both groups were efficacious compared to the untreated control group and the Nf-Trip showed greater efficacy than the drug liberated by both the luciferase signal and physical examination of the excised organ (Figs. 9a-9c). In addition, the untreated control and Nf-Trip treated mice maintained their body weight stably, whereas the mice treated with free tryptolide significantly reduced body weight (FIG. 9d), indicating that the high toxicity of tryptolide And was mitigated by targeted formulation. In terms of survival rate, mice treated with Nf-Trip showed a survival rate of about 80% after 3 months, survival rate of about 50% for mice treated with free tryptolide, and about 50% 30% survival rate (Fig. 9E).

To assess cell-level efficacy, tumors were collected and histochemically examined after the pharmacodynamic study. The Nf-Trip treated tumors by hematoxylin and eosin (H & E) staining were much necrotic as compared to the free tryptolide treated tumors. In the DMSO control, necrosis necrosis) was not observed (Fig. 9f). Consistent with the in vitro test results, the TUNEL staining showed that the population of cells suffering apoptosis in Nf-Triop treated tumor tissue was much higher than the free tryptolized tumor and the DMSO control (Fig. 9G). Finally, immunohistochemical (IHC) staining significantly reduced the expression of CKS2 and AURKA in Nf-Trip treated tumor tissue compared to the free tryptolide treated tumor tissue or the DMSO treated tumor tissue (Fig. 9f).

Claims (18)

A nano-anticancer drug type comprising a hydrophobic anticancer drug, a pH sensitive peptide, and a poloxamer-folate conjugate. The method of claim 1, wherein the hydrophobic anti-cancer drug is selected from the group consisting of vendamustine, ruby, camustine, chlorambucil, cyclophosphamide, dacarbazine, ipospamide, melphalan, procarbazine, streptozocin, temozolomide Diclofenacin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, But are not limited to, pegylated liposomal doxorubicin, epirubicin, dirubicin, mitomycin, mitoxantrone, etoposide, docetaxel, irinotecan, paclitaxel, topotecan, vinblastine, vincristine, vinorelbine, carboplatin, cisplatin, , Interferon, eicilimumab, lapatinib, panituumatum, rituximab, sutinitinib, soracin, erythromycin, eicosapentaenoic acid, Fenip, Wherein the anticancer agent is selected from the group consisting of temairolimus, trastuzumab, claudronate, ibandronic acid, pamidronate, and zoledronic acid. The method of claim 1, wherein the pH sensitive peptides octadecylamine -p (API-Asp) _10, generating a fusion peptide _{GALA, H 2 N- (AALEALAEALEALAEALEALAEAAAAGGC) -CO} 2 H, H 2 N- (AALAEALAEALAEALAEALAEALAAAAGGC) -CO 2 H and H ₂ N- (ALEALAEALEALAEA) -CONH ₂ . The nano-anticancer drug type according to claim 1, wherein the pH-sensitive peptide is decomposed under an acidic environment to release the hydrophobic anti-cancer drug. The nano-cancer drug formulation according to claim 1, wherein the poloxamer-folate conjugate is pluronic F127-folate. The nano-anticancer drug formulation according to claim 1, wherein the nano-anticancer drug is a drug for treating liver cancer. The method of claim 1, wherein the hydrophobic anti-cancer drug is selected from the group consisting of ZNF114, BHLHE40, CKS2, RND3, SLC38A2, TXNIP, ZC3H11A, TLE1, HIST1H1E, MKI67IP, ANKRD1, LEO1, AURKA, PLIN2, WDR82, SETX, ARRDC3, PUM2, WDR43, , MH-22A, Hepa-1c1c7, HF1-5, HF1, C2-Rev7, C2, H5, HTC, Hepa 1-6, SK-HEP-1, MCA-RH 7777, N1-S1, H4S, 6, H-4-II-E, Hep 3B, MH1C1, PLC / PRF / 5, H4-II-E-C3, HTC (BUdR), Hep G2 and Huh-7D12 Wherein the nano-anticancer agent is an anticancer agent. The method of claim 1, wherein the hydrophobic anticancer drug, the pH sensitive peptide, and the poloxamer-folate conjugate spontaneously form micellar nanogels in water,
Wherein the hydrophobic portion of the hydrophobic anticancer drug, the pH sensitive peptide and the poloxamer-folate conjugate forms a core, and the hydrophilic portion of the poloxamer-folate conjugate is bonded to the outside of the core Wherein the nanotubes are exposed. (i) dissolving a hydrophobic anti-cancer drug, a pH sensitive peptide, and a poloxamer-folate conjugate in a solvent;
(ii) drying the solution obtained in step (i); And
(iii) hydrating the dried material obtained in the step (ii). 10. The method of claim 9, wherein the hydrophobic anti-cancer drug is selected from the group consisting of vendamustine, ruby, camustine, chlorambucil, cyclophosphamide, dacarbazine, ipospamide, melphalan, procarbazine, streptozocin, temozolomide Diclofenacin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, doxorubicin, But are not limited to, pegylated liposomal doxorubicin, epirubicin, dirubicin, mitomycin, mitoxantrone, etoposide, docetaxel, irinotecan, paclitaxel, topotecan, vinblastine, vincristine, vinorelbine, carboplatin, cisplatin, , Interferon, eicilimumab, lapatinib, panituumatum, rituximab, sutinitinib, soracin, erythromycin, eicosapentaenoic acid, Fenip, Wherein the anticancer agent is selected from the group consisting of temairolimus, trastuzumab, claudronate, ibandronic acid, pamidronate, and zoledronic acid. 10. The method of claim 9, wherein the pH sensitive peptides octadecylamine -p (API-Asp) _10, generating a fusion peptide _{GALA, H 2 N- (AALEALAEALEALAEALEALAEAAAAGGC) -CO} 2 H, H 2 N- (AALAEALAEALAEALAEALAEALAAAAGGC) -CO 2 H and H ₂ N- (ALEALAEALEALAEA) -CONH ₂ . 10. The method according to claim 9, wherein the pH sensitive peptide is degraded under an acidic environment to release the hydrophobic anti-cancer drug from the nano-anticancer drug type. 10. The method according to claim 9, wherein the poloxamer-folate conjugate is pluronic F127-folate. [Claim 11] The method according to claim 9, wherein the nano-anticancer agent is a therapeutic agent for liver cancer. 11. The method of claim 9, wherein the hydrophobic anti-cancer drug is selected from the group consisting of ZNF114, BHLHE40, CKS2, RND3, SLC38A2, TXNIP, ZC3H11A, TLE1, HIST1H1E, MKI67IP, ANKRD1, LEO1, AURKA, PLIN2, WDR82, SETX, ARRDC3, PUM2, WDR43, , MH-22A, Hepa-1c1c7, HF1-5, HF1, C2-Rev7, C2, H5, HTC, Hepa 1-6, SK-HEP-1, MCA-RH 7777, N1-S1, H4S, 6, H-4-II-E, Hep 3B, MH1C1, PLC / PRF / 5, H4-II-E-C3, HTC (BUdR), Hep G2 and Huh-7D12 Wherein the method comprises the steps of: The method according to claim 9, wherein the phosphate buffered saline (PBS) is used in step (iii). The method according to claim 9, wherein the ultrasonic treatment is performed in step (iii). 10. The method of claim 9, wherein in step (iii), the hydrophobic anticancer drug, the pH sensitive peptide, and the poloxamer-folate conjugate spontaneously form a micellar nanogel, Wherein the hydrophobic portion of the anticancer drug, the pH sensitive peptide and the poloxamer-folate conjugate forms a core, and the hydrophilic portion of the poloxamer-folate conjugate is exposed to the outside of the core. (Method for manufacturing anticancer agent type).

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