KR101735467B1 - Nanoassembly for tumor-targeted drug delivery and cancer diagnosis comprising iodinated hyaluronic acid oligomer and hydrophobic drug - Google Patents

Abstract

The present invention relates to a nanoassembly for tumor-targeted drug delivery and cancer diagnosis comprising iodinated hyaluronic acid oligomer and hydrophobic drugs; a pharmaceutical composition for tumor-targeted drug delivery and cancer diagnosis comprising the same; and a method for manufacturing the nanoassembly. The nanoassembly, the pharmaceutical composition comprising the same and the manufacturing method can be used as a theranostic nanosystem to cancer.

Description

[0001] The present invention relates to a nanoassembly for tumor-targeted drug delivery and cancer diagnosis, including iodinated hyaluronic acid oligomers and hydrophobic drugs,

The present invention relates to a nanocomposite for tumor-targeted drug delivery and cancer diagnosis, and more particularly to a nanocomposite for tumor-targeted drug delivery and cancer diagnosis comprising iodinated hyaluronic acid oligomer and a hydrophobic drug.

A variety of approaches have been attempted so far to selectively deliver chemotherapeutic agents to tumor sites or to diagnose cancer [Non-Patent Documents 1-3]. In addition, tumor targeting strategies including passive and active targeting have been introduced into drug delivery systems to minimize the distribution of anticancer drugs to normal tissues and organs (Non-Patent Documents 4 and 5).

On the other hand, with respect to the distribution of drugs in tumors, nano-sized particles and macromolecules are retained in tumor sites due to angiogenesis of tumor tissue and abnormal vascular structure due to immature lymphatic system There is a tendency. This enhanced permeability and retention (EPR) effect is related to the physicochemical properties (eg size, shape, and surface charge) of the delivery vehicle and has been used as a strategy for passive tumor targeting [Non-Patent Document 6]. Such nonspecific properties can be complemented by an active targeting strategy using tumor targeting residues (e. G., Low molecular weight compounds, peptides, antibodies) as ligands to receptors overexpressed in cancer cells [Non-patent document 7 ,8]. That is, the combination of passive and active targeting strategies can increase tumor targeting capacity and chemotherapy effects.

Recently, a number of contrast agent compounds for X-ray computed tomography (CT), magnetic resonance (MR), positron emission tomography (PET) A technique of forming nano-sized particles or physically enclosing these particles has been reported [Non-Patent Documents 9 and 10]. This technique of performing cancer chemotherapy concurrently with cancer diagnosis can improve patient compliance and therapeutic efficacy.

Of the various nano-sized drug delivery systems currently available, self-assembling nanoparticles (NP) have been extensively studied for tumor-targeted drug delivery and cancer diagnosis [Non-Patent Documents 11, 15]. In addition, a technique of chemically covalently bonding a hydrophobic moiety having a specialized diagnostic and therapeutic function to a hydrophilic skeleton has been reported [Non-Patent Document 16].

However, to date, the extent to which the goal of tumor-targeted drug delivery and cancer diagnosis has been achieved has not been sufficient for clinical use, and the development of a delivery vehicle capable of achieving more effective tumor-targeting and diagnostic functions is required.

U.S. Patent Publication No. 2012-0308481A1 (2012.12.06) U.S. Patent Publication No. 2014-0056816A1 (Feb. Korean Patent Publication No. 10-2007-0104574 (October 26, 2007) U.S. Patent Publication No. 2009-0259297A1 (Oct. 15, 2009)

[1] Ma X, Zhao Y, Liang XJ. Theranostic nanoparticles engineered for clinic and pharmaceutics. Acc Chem Res 2011; 44: 1114-1122. [2] Melancon MP, Stafford RJ, Li C. Challenges to effective cancer nanotheranostics. J Control Release 2012; 164: 177-182. [3] Park JH, Cho HJ, Yoon HY, Yoon IS, Ko SH, Shim JS, et al. Hyaluronic acid derivative-coated nanohybrid liposomes for cancer imaging and drug delivery. J Control Release 2014; 174: 98-108. [4] Lammers T, Kiessling F, Hennink WE, Storm G. Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress. J Control Release 2012; 161: 175-187. [5] Maeda H, Bharate GY, Daruwalla J. Polymeric drugs for efficient tumor-targeted drug delivery based on EPR-effect. Eur J Pharm Biopharm 2009; 71: 409-419. [6] Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm 2008; 5: 505-515. [7] Bertrand N, Wu J, Xu X, Kamaly N, Farokhzad OC. Cancer nanotechnology: the impact of passive and active targeting in the era of modern cancer biology. Adv Drug Deliv Rev 2014; 66: 2-25. [8] Zhong Y, Meng F, Deng C, Zhong Z. Ligand-directed active tumor-targeting polymeric nanoparticles for cancer chemotherapy. Biomacromolecules 2014; 15: 1955-1969. [9] Janib SM, Moses AS, MacKay JA. Imaging and drug delivery using theranostic nanoparticles. Adv Drug Deliv Rev 2010; 62: 1052-1063. [10] Xie J, Lee S, Chen X. Nanoparticle-based theranostic agents. Adv Drug Deliv Rev 2010; 62: 1064-1079. [11] Cho HJ, Yoon HY, Koo H, Ko SH, Shim JS, Lee JH, et al. Self-assembled nanoparticles based on hyaluronic acid-ceramide (HA-CE) and Pluronic® for tumor-targeted delivery of docetaxel. Biomaterials 2011; 32: 7181-7190. [12] Lee JY, Chung SJ, Cho HJ, Kim DD. Even acid-conjugated chondroitin sulfate A-based nanoparticles for tumor-targeted anticancer drug delivery. Eur J Pharm Biopharm 2015; 94: 532-541. [13] Lee JY, Chung SJ, Cho HJ, Kim DD. Phenylboronic acid-decorated chondroitin sulfate A-based theranostic nanoparticles for enhanced tumor targeting and penetration. Adv Funct Mater 2015; 25: 3705-3717. [14] Termsarasabi, Yoon IS, Park JH, Moon HT, Cho HJ, Kim DD. Polyethylene glycol-modified arachidyl chitosan-based nanoparticles for prolonged blood circulation of doxorubicin. Int J Pharm 2014; 464: 127-134. [15] Yhee JY, Son S, Kim SH, Park K, Choi K, Kwon IC. Self-assembled glycol chitosan nanoparticles for disease-specific theranostics. J Control Release 2014; 193: 202-213. [16] Shi J, Xiao Z, Kamaly N, Farokhzad OC. Self-assembled targeted nanoparticles: evolution of technologies and bench to bedside translation. Acc Chem Res 2011; 44: 1123-1134. [17] Criscione JM, Dobrucki LW, Zhuang ZW, Papademetris X, Simons M, Sinusas AJ, et al. Development and application of a multimodal contrast agent for SPECT / CT hybrid imaging. Bioconjug Chem 2011; 22: 1784-1792. [18] Lei K, Shen W, Cao L, Yu L, Ding J. An injectable thermogel with high radiopacity. Chem Commun 2015; 51: 6080-6083. [19] Negussie AH, Dreher MR, Johnson CG, Tang Y, Lewis AL, Storm G, et al. Synthesis and characterization of image-able polyvinyl alcohol microspheres for image-guided chemoembolization. J Mater Sci Mater Med 2015: 26: 198. [20] Singhana B, Chen A, Slattery P, Writing IK, Qiao Y, Tasciotti E, et al. Infusion of iodine-based contrast agents into poly (p-dioxanone) as a radiopaque resorbable IVC filter. J Mater Sci Mater Med 2015; 26: 124. [21] Cho HJ, Yoon IS, Yoon HY, Koo H, Jin YJ, Ko SH, et al. Polyethylene glycol-conjugated hyaluronic acid-ceramide self-assembled nanoparticles for targeted delivery of doxorubicin. Biomaterials 2012; 33: 1190-1200. [22] Ahrens T, Assmann V, Fieber C, Termeer C, Herrlich P, Hofmann M, et al. CD44 is the principal mediator of hyaluronic-acid-induced melanoma cell proliferation. J Invest Dermatol. 2001; 116: 93-101. [23] Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell 1990; 61: 1303-1313. [24] Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, et al. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 1999; 144: 789-801. [25] Turley EA, Austen L, Vandeligt K, Clary C. Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells. J Cell Biol 1991; 112: 1041-1047. [26] Ghosh SC, Alpay SN, Klostergaard J. CD44: a validated target for improved treatment of cancer therapeutics. Expert Opin Ther Targets 2012; 16: 635-650. [27] Jin YJ, Termsarasabi, Ko SH, Shim JS, Chong S, Chung SJ, et al. Hyaluronic acid derivative-based self-assembled nanoparticles for the treatment of melanoma. Pharm Res 2012; 29: 3443-3454. [28] Park JH, Lee JY, Termsarasabi, Yoon IS, Ko SH, Shim JS, et al. Development of poly (lactic-co-glycolic) acid nanoparticles-embedded hyaluronic acid-ceramide-based nanostructure for tumor-targeted drug delivery. Int J Pharm 2014; 473: 426-433. [29] Neises B, Steglich W. Simple method for the esterification of carboxylic acids. Angew Chem Int Ed Engl 1978; 17: 522-524. [30] Lee JY, Kim JS, Cho HJ, Kim DD. Poly (styrene) -b-poly (DL-lactide) copolymer-based nanoparticles for anticancer drug delivery. Int J Nanomed 2014; 9: 2803-2813. [31] Lee N, Choi SH, Hyeon T. Nano-sized CT contrast agents. Adv Mater 2013: 25: 2641-2660. [32] Termsarasabi, Cho HJ, Kim DH, Chong S, Chung SJ, Shim CK, et al. Chitosan oligosaccharide-arachidic acid-based nanoparticles for anti-cancer drug delivery. Int J Pharm 2013; 441: 373-380. [33] Phillips JN. The energetics of micelle formation. J N Trans Faraday Soc 1995; 51: 561-569. [34] Lee MJ, Oh NM, Oh KT, Youn YS, Lee ES. Functional poly (l-lysine) derivative nanogels with acidic pH-pulsed antitumor drug release properties. J Pharm Invest 2014; 44: 351-356.

The present inventors have been studying a carrier capable of simultaneously achieving tumor-targeting and diagnostic functions, and have found that an iodine-containing contrast agent, especially 2,3,5-triiodobenzoic acid (TIBA ) Was used as a functional unit for CT imaging and hydrophobic residues and hyaluronic acid (HA) oligomer was used as a hydrophilic skeleton for tumor targeting to prepare amphiphilic iodinated hyaluronic acid oligomer ; The nanocomposite comprising the iodinated hyaluronic acid oligomer and the hydrophobic anticancer drug (doxorubicin, DOX, etc.) is a nano-sized self-assembly exhibiting a drug releasing pattern favorable in the tumor environment, Not only increase the incorporation rate into the tumor, but also have excellent tumor targeting and cancer diagnostic efficacy, and are excellent in in vivo antitumor efficacy, and thus can be used as an effective theranostic nano-system for cancer.

Accordingly, the present invention relates to a nanocomposite for tumor-targeted drug delivery and cancer diagnosis comprising iodinated hyaluronic acid oligomer and a hydrophobic drug; A pharmaceutical composition for tumor-targeted drug delivery and cancer diagnosis comprising the same; And a method for producing the nanocomposite.

According to one aspect of the present invention, there is provided a nano-aggregate for tumor-targeted drug delivery and cancer diagnosis comprising iodinated hyaluronic acid oligomer and a hydrophobic drug.

In one embodiment, the iodinated hyaluronic acid oligomer is one or more selected from the group consisting of triiodobenzoic acid, diatrizoic acid, metrizoic acid, and iothalamate; And covalent conjugates of hyaluronic acid. The triiodobenzoic acid may be 2,3,5-triiodobenzoic acid, and the triiodobenzoic acid may be bound to 10 to 20 moles per 1 mole of hyaluronic acid.

In one embodiment, the iodinated hyaluronic acid oligomer may have an iodine content of 30 to 60% (w / w), and the iodinated hyaluronic acid oligomer and the hydrophobic drug are included in a weight ratio of 5: 1 to 40: 1 .

In one embodiment, the iodinated hyaluronic acid oligomer may be further combined with a dye for optical imaging.

According to another aspect of the present invention, there is provided a pharmaceutical composition for tumor-targeting drug delivery and cancer diagnosis comprising the above nanocomposite.

In one embodiment, the tumor is a carcinoma in which the CD44 receptor is expressed, and may be at least one selected from the group consisting of lung cancer, breast cancer, liver cancer, brain tumor, skin cancer and ovarian cancer.

According to still another aspect of the present invention, there is provided a method for preparing a pharmaceutical composition, comprising: (a) solubilizing an iodinated hyaluronic acid oligomer and a hydrophobic drug to obtain a solution; (b) removing the solvent from the solution of step (a) to obtain a solid; And (c) adding an aqueous solvent to the solids of step (b) to obtain a dispersion. The method includes the steps of: (a) preparing a nanocomposite for tumor-targeted drug delivery and cancer diagnosis;

According to the present invention, a nanocomposite comprising an iodinated hyaluronic acid oligomer and a hydrophobic drug has an average particle size of 207 nm and has been confirmed to be nano-sized, and the drug release pattern from the nanocomposite is pH dependent Free, and slow release, and was found to be suitable for the treatment of tumors. The interaction between hyaluronic acid and CD44 receptor not only increased the intracellular induction rate of the anti-cancer agent but also was excellent in tumor targeting and cancer diagnosis, Lt; RTI ID = 0.0 > in vivo < / RTI > antitumor efficacy.

Accordingly, the iodinated hyaluronic acid-based nanocomposite of the present invention and pharmaceutical compositions comprising it are useful as therapeutic diagnostic nanosystems for cancer-expressing drug delivery and cancer diagnosis, particularly for cancer expressing the CD44 receptor .

Figure 1 is a schematic diagram (A) of the synthesis of HA-TIBA covalent conjugate and a ¹ H-NMR spectrum (HA) of HA-TIBA (HA [a: 1.8 ppm] and TIBA [b and c: 7.8 and 8.2 ppm] And HA-TIBA was dissolved in DMSO-d ₆ for ¹ H-NMR (500 MHz) analysis).
Fig. 2 shows the particle size distribution (left panel) of the nanocomposite according to the average particle size, expressed as differential intensity, as a result of confirming the physicochemical properties of blank HA-TIBA and HA-TIBA / (Right panel) of the nanocomposite (the length of the reference in the TEM photograph is 200 nm).
Figure 3 is a graph showing the in vitro release (%) of DOX released from HA-TIBA / DOX nanocomposite at time of incubation time at pH 5.5, 6.8, and 7.4 (data are shown as mean ± SD, n = 3).
FIG. 4 shows the results of evaluating the extent of DOX incorporation into nano-aggregates in SCC7 cells, showing that DOX solution (green) and HA-TIBA / DOX (pink) corresponding to a DOX concentration of 50 μg / (A) and CLSM (B) (blue fluorescence intensity value [average ± standard deviation]), which was measured by flow cytometry after 1 hour of incubation And red represent DAPI and DOX, respectively, represented by 2D and 2.5D images of each test group).
FIG. 5 shows an in vivo scan of Cy5.5 and Cy5.5-HA-TIBA in SCC7 tumor-xenografted mouse models, and then real-time full-scan photographs at 0 (before treatment), 1, 3, 6, A graph showing the fluorescence intensities (average NC [normalized count], photon counts / mm ² ) in the in vivo NIRF imaging technique results (the circle indicated by the yellow dotted line corresponds to the tumor site) Data are presented as means ± SD, n = 3) (B).
FIG. 6 shows the results of evaluation of the biodistribution of the prepared nanoclusters by an in vitro NIRF imaging technique in an SCC7 tumor-xenografted mouse model. Cy5.5 and Cy5.5-HA-TIBA were administered to each mouse group (A), and fluorescence intensity (mean NC) in the extracted organs or tissues was measured by NIRF imaging technique after 24 hours of intravenous injection, and the liver, spleen, kidney, heart, lung, (Data are shown as mean ± standard deviation, n = 3, * p <0.05 [compared with Cy5.5 group]) (B).
Figure 7 shows in vivo CT images of SCC7 tumor-xenografted mouse models, in which SCC7 tumor-xenografted mouse models were injected intravenously with unconjugated TIBA and HA-TIBA followed by 0 (before treatment) and 24 hours (Data are mean ± SD, n = 3, * p <0.05 [non-binding TIBA (n = 3)], and the iodine concentration of the tumor tissues quantified by ICP-OES method 24 hours after administration (B).
Figure 8 shows the results of testing in vivo anti-tumor efficacy in a SCC7 tumor-xenografted mouse model, wherein blank HA-TIBA, DOX solution, and HA-TIBA / (A) tumor volume in mm ^{3 as a} function of time after intravenous injection on days 7, 9, 11, and 13; (P <0.05 compared with the control group), p <0.05 compared with the control group (p <0.05). Hour], + p &lt; 0.05 [compared to DOX solution group]); (C) showing the results of TUNEL analysis (left column) and H & E staining (right column) of heart tissues of the tumor taken from the mouse at day 16.

The present invention provides nano-aggregates for tumor-targeted drug delivery and cancer diagnosis comprising iodinated hyaluronic acid oligomers and hydrophobic drugs.

(I) passive tumor targeting by EPR effect and (ii) active tumor targeting by interaction between HA and CD44 receptors, and (iii) introducing a contrast agent for tumor diagnosis to deliver the drug to the cancer And provides a therapeutic diagnostic nano system for cancer diagnosing cancer and expressing CD44 receptor.

(I) Passive tumor targeting by EPR effect can be achieved by the above nanocomposite comprising iodinated hyaluronic acid oligomer and hydrophobic drug.

(Ii) Active tumor targeting can be achieved by using hyaluronic acid, known to bind to the CD44 receptor expressed on cancer cells, as a hydrophilic backbone.

(Iii) introduction of a contrast agent for tumor diagnosis can be achieved by the iodination moiety in the iodinated hyaluronic acid oligomer.

In the iodinated hyaluronic acid oligomer, the hyaluronic acid oligomer, which is part of it, corresponds to the hydrophilic backbone constituting the nanocomposite of (i) and the targeting ligand for (ii) CD44 receptor-expressing tumor. The iodine-containing substance, which is a part of the iodinated hyaluronic acid oligomer, corresponds to the hydrophobic moiety constituting the nanocomposite of (i) and the imaging function of (iii). That is, an iodinated hyaluronic acid oligomer is prepared by binding an iodine-containing substance, which is a hydrophobic moiety, to a hyaluronic acid (HA) oligomer, which is a hydrophilic skeleton, and a nano- - sized autologous aggregates, passive tumor targeting and active tumor targeting can be achieved while providing a therapeutic diagnostic nanosystem for cancer.

In one embodiment of the present invention, the iodinated hyaluronic acid oligomer is at least one selected from the group consisting of triiodobenzoic acid, diazotric acid, metrizoic acid, and iothalamate; And covalent conjugates of hyaluronic acid. In particular, 2,3,5-triiodobenzoic acid may be used as the triiodobenzoic acid.

In an embodiment of the present invention, a hydrophobic residue, 2,3,5-triiodobenzoic acid (TIBA), was bonded to a hydrophilic skeleton, hyaluronic acid (HA) oligomer Reference). Specifically, by covalently bonding 2,3,5-triiodobenzoic acid to the hyaluronic acid oligomer by esterification between the carboxylic acid group of 2,3,5-triiodobenzoic acid (TIBA) and the hydroxy group of HA, 3,5-triiodobenzoic acid-bonded hyaluronic acid oligomer (HA-TIBA) can be synthesized.

In the present invention, hyaluronic acid used as a hydrophilic skeleton is a component existing in an extracellular matrix (ECM) and has been proved to be related to cancer proliferation, metastasis, and migration (Non-Patent Document 22). On the other hand, CD44 is a receptor for hyaluronan-mediated motility (RHAMM) / intracellular hyaluronic acid binding protein (IHABP), and a lymphatic endothelial hyaluronan receptor-1 (lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1), which is known as a receptor for HA [Non-Patent Documents 23-25]. Such CD44 receptors have been expressed in a large number of cancer cells and thus have been reported to be used as targeting receptors for HA [Non-Patent Document 26]. HA derivative-based autologous conjugates using tumor targeting capabilities of HA to the CD44 receptor have been reported and their use in chemotherapy has been disclosed [Non-Patent Literatures 11, 21, 27, 28].

TIBA is insoluble in water but is soluble in alkaline solutions (NaOH, KOH) and in some organic solvents (ethanol, dimethyl sulfoxide, DMSO, and ethers). In addition, hydrophobic TIBA, including iodine atoms, can be used as a CT contrast agent. TIBA is structurally similar to the commercial contrast agent iohexol (Omnipaque, GE Healthcare), which has been approved for clinical use by the United States Food and Drug Administration (FDA) [Non-patent document 17]. It has been reported that TIBA can be covalently bound to the polymer backbone to prepare the delivery vehicle and can be encapsulated in a variety of formulations such as microspheres and NP as CT contrast agents [Non-Patent Literatures 18-20]. It is believed that, rather than physically introducing the diagnostic contrast agent (i. E., TIBA) into the pharmaceutical formulation, the nano-sized system is prepared by covalently binding TIBA to the polymer backbone as in the present invention, Tracking effect can be achieved.

The triiodobenzoic acid may be bonded at 10 to 20 moles per 1 mole of hyaluronic acid, preferably at 12 to 18 moles, more preferably at 14 to 16 moles, but is not limited thereto.

In one embodiment, the iodide content of the iodinated hyaluronic acid oligomer may be 30 to 60% (w / w), preferably 35 to 50% (w / w), more preferably 40 to 45% w / w). &lt; / RTI &gt;

In one embodiment, the iodinated hyaluronic acid oligomer and the hydrophobic drug may be included in a weight ratio of 5: 1 to 40: 1, preferably 10: 1 to 35: 1, more preferably 20: : 1, but is not limited thereto.

In one embodiment, the iodinated hyaluronic acid oligomer may be further combined with a dye for optical imaging. The optical image dye may be a polymethine dye, and commercially available Cy5 and Cy7 may be used.

The optical image dyes can be used for NIRF imaging and have been reported to be used in near infrared ray fluorescence (NIRF) imaging techniques in the prior art [Non-Patent Documents 11 and 21] Cy5.5 was covalently linked to HA-TIBA. Dual (optical / CT) imaging moieties can synergistically improve diagnostic capabilities by combining the advantages of each.

The nanocomposite of the present invention had an average particle size of 207 nm and was found to be nano-sized (see FIG. 2), and the drug release pattern from the nanocomposite exhibited a higher drug release at acidic pH than at neutral pH, , Indicating a drug release pattern favorable to the tumor environment (see FIG. 3). In addition, not only did the induction rate of DOX (anticancer drug) from the nanocomposite into the cell increased by the introduction into the cell based on the interaction between HA and CD44 receptor in CD44 receptor positive squamous cell carcinoma cells (see FIG. 4) 6), in vivo CT imaging assessment (see FIG. 7), in vivo NIRF imaging evaluation (see FIG. 5), in vivo in SCC7 tumor-xenografted mouse models, As a result, it was confirmed that HA-TIBA nanocomposite exhibited excellent tumor targeting and cancer diagnostic efficacy. In vivo anti-tumor efficacy test results (see FIG. 8) in HA-TIBA / DOX Nanocomposites have been shown to significantly reduce tumor volume growth and thus exhibit superior in vivo antitumor efficacy.

The present invention also provides a pharmaceutical composition for tumor-targeting drug delivery and cancer diagnosis comprising the above nanocomposite.

In one embodiment, the tumor is a carcinoma in which the CD44 receptor is expressed, for example, one or more selected from the group consisting of lung cancer, breast cancer, liver cancer, brain tumor, skin cancer and ovarian cancer.

The present invention also provides a method for preparing a pharmaceutical composition comprising: (a) solubilizing an iodinated hyaluronic acid oligomer and a hydrophobic drug to obtain a solution; (b) removing the solvent from the solution of step (a) to obtain a solid; And (c) adding a water-soluble solvent to the solid of step (b) to obtain a dispersion. The present invention also provides a method of producing a nanocomposite for tumor-targeted drug delivery and cancer diagnosis.

Step (a) comprises reacting iodinated hyaluronic acid oligomer (e.g., HA-TIBA) and a hydrophobic drug with a surfactant (e.g., DMSO) and a suitable solvent (e. G., DMSO) in order to prepare a nanocomposite comprising a hydrophobic drug For example, water).

Step (b) may be carried out by removing the solvent from the solution in which the iodinated hyaluronic acid oligomer and the hydrophobic drug have been solubilized using an appropriate method (e.g. solvent evaporation method) to obtain a solid (e.g. in the form of a film) .

Step (c) is a step of adding a water-soluble solvent (for example, water) to the solid to obtain a dispersion of the nanocomposite.

Hereinafter, the present invention will be described in more detail through examples and test examples. However, the following examples and test examples are provided for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example  1. HA- TIBA  Synthesis and identification of covalent conjugates

(1) Material

HA oligomer (average molecular weight: 4.7 kDa) was purchased from Bioland Co., Ltd., Cheonan, Korea. 2,3,5-Triiodobenzoic acid (TIBA) was obtained from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan. Doxorubicin hydrochloride (DOX HCl) was purchased from Boryung Pharmaceutical Co., Ltd., Seoul, Korea. N, N'- dicyclohexylcarbodiimide (N, N'-dicyclohexylcarbodiimide, DCC) , 4- ( dimethylamino) pyridine [4- (dimethylamino) pyridine, DMAP ], and triethylamine (trimethylamine, TEA) is Sigma-Aldrich Co., St. Louis, Mo., USA. Cy5.5 (Amine-functionalized Cy5.5, FCR-675 amine) with amine functional groups was purchased from BioActs, Incheon, Korea. RPMI 1640 (developed by Roswell Park Memorial Institute), penicillin, streptomycin, and heat-inactivated fetal bovine serum (FBS) were purchased from Gibco Life Technologies , Inc., Grand Island, NY, USA). Other reagents were purchased at analytical grade.

Statistical analysis of the data was performed using two-tailed t-test and ANOVA. All examples and test examples were performed at least 3 times and the data are expressed as mean ± standard deviation (SD).

(2) HA- TIBA  synthesis

HA-TIBA was synthesized as follows by using DCC as a coupling reagent and DMAP as a catalyst, by esterification between a carboxylic acid group of TIBA and a hydroxy group of HA [Non-Patent Document 29]. TIBA (1.00 mmol), DCC (2.00 mmol) and DMAP (0.20 mmol) were dissolved in 40 mL of N, N -dimethylformamide (DMF) And stirred for 15 minutes. The resulting TIBA / DCC / DMAP solution was then slowly added to the HA solution and the mixture was stirred at room temperature for 20 hours. The obtained solution was put on a dialysis membrane (molecular weight cut-off: 3.5 kDa) and then dialyzed in methanol for 2 days, in a methanol / secondary distilled water (DDW) mixture (1: 1, v / v) . After lyophilization, the resultant HA-TIBA was stored at 2-8 ° C and used for subsequent testing.

^For analysis, HA-TIBA was dissolved in hexadeuterodimethyl sulfoxide (DMSO-d ₆ ) for ¹ H-NMR (Varian FT-500 MHz, Varian Inc., Palo Alto, The iodine content of HA-TIBA was measured by inductively coupled plasma optical emission spectrometry (ICP-OES, Optima-4300 DV, PerkinElmer Inc., Waltham, The degree of substitution (DS) of TIBA was determined.

(3) HA- TIBA  Identify characteristics

As described above, an iodine-containing compound, TIBA (as a hydrophobic moiety), was linked to the HA oligomer (as the hydrophilic backbone) to synthesize the amphiphilic HA-TIBA covalent conjugate. HA and TIBA have been used as tumor targeting ligands and CT contrast agents for the CD44 receptor, respectively. Although iodine is used as a CT contrast agent, it is nonspecifically distributed in intravascular and extravascular spaces and rapidly released through the kidney [Non-Patent Document 31]. Thus, unmodified iodinated molecules provide unascertained CT images with short imaging time after administration. HA-TIBA nanocomposites were prepared by synthesizing HA-TIBA to overcome these limitations and improve tumor targeting ability. Due to the amphipathic nature of HA-TIBA, HA-TIBA can exist as a nano-sized self-aggregate in the aqueous phase at conditions above the critical aggregation concentration (CAC). DOX (the hydrophobic drug used in the present invention) can enter the internal hydrophobic space of the HA-TIBA nanocomposite.

HA-TIBA was synthesized by covalently bonding the carboxylic acid group of TIBA to the hydroxyl group of the HA oligomer (see Fig. 1 (A)). One-step synthesis via DCC and DMAP-mediated esterification between -COOH and -OH groups was used to attach TIBA to the HA backbone. The synthesis of HA-TIBA was confirmed by ¹ H-NMR analysis, and the results are shown in Fig. 1 (B). Representative signals (b and c: 7.8 and 8.2 ppm [2H at positions 4 and 6 of the phenyl ring]) of TIBA in the ¹ H-NMR spectrum of the final product (HA-TIBA) - Indicates that the TIBA has been synthesized successfully. The extent of TIBA substitution in HA was measured by ICP-OES. The average molar substitution (molar substitution, MS) of the introduced TIBA per mole of HA was 14.9 and the iodine content of HA-TIBA was 42.8% (w / w).

The autocomber in the aqueous solution of the prepared HA-TIBA covalent bond was identified by particle size analysis. Unlike the method of non-patent documents 11 and 32 in which pyrene is used as a fluorescence probe, in the examples of the present invention, the change of the average particle size of the HA-TIBA dispersion in DW As a function. The average particle size of the HA-TIBA dispersion in the DW was almost zero up to a concentration of 2.5 μg / mL. However, at a concentration of 5 μg / mL, the particle size increased to about 198 nm. The average particle size was measured at 155 to 198 nm in the range of 5-500 μg / mL of HA-TIBA. Therefore, it is assumed that the critical association concentration of HA-TIBA is in the range of 2.5-5 μg / mL. This value is lower than the value obtained for other amphiphilic HA derivatives (for example, hyaluronic acid-ceramide) reported in the prior art [non-patent document 11]. It can be deduced that the nanocomposite of the present invention will maintain the structure of the nanocomposite even after dilution in body fluids when the nanocomposite of the present invention is injected intravenously in view of having a relatively low CAC value compared to a low molecular weight surfactant Patent Documents 11 and 33].

Example  2. DOX -include Nano-aggregate  Manufacturing and characterization

(One) DOX -include Nano-aggregate  Produce

In the examples of the present invention, a DOX base was used as a hydrophobic model drug. To prepare the DOX base, DOX HCl (100 mg) was dissolved in 10 mL of DMSO and 0.12 mL of TEA was added [Non-Patent Document 21]. The resulting solution was stirred at room temperature for 12 hours, freeze-dried and stored in a dry state. HA-TIBA (30 mg) and DOX (1 mg) were solubilized in 1 mL of DMSO / DDW (1: 1, v / v) to prepare DOX-containing nanocomposites. The solvent was removed under a weak stream of nitrogen gas at 70 DEG C for 8 hours. To prepare NP, DDW (1 mL) was added to HA-TIBA / DOX film and the resulting dispersion was applied to a probe sonicator (VC-750, Sonics & Materials, Inc., Newtown, CT , USA) for 1 minute.

Morphological shapes of the drug-containing nanocomposites were observed by transmission electron microscopy (TEM). The nanocomposite dispersion was dyed with 2% (w / v) phosphotungstic acid, placed on a copper grid along with the film, and dried for 10 minutes to prepare a TEM (JEM 1010; JEOL, Tokyo , Japan).

(2) DOX -include Nano-aggregate  Identification

As described above, DOX was introduced into the HA-TIBA nanocomposite as a model drug of a hydrophobic drug. In the present invention, a solvent evaporation method was used to prepare DOX-containing HA-TIBA nanocomposite [Non-Patent Documents 11-13, 21, 27, 32]. Physical and chemical properties of HA-TIBA / DOX were confirmed and are shown in FIG. 2 and Table 1 below.

Constituent HA-TIBA HA-TIBA / DOX ^c Average particle diameter (nm) 156.0 ± 2.6 206.6 ± 5.0 Polydispersity Index 0.13 + 0.03 0.10 0.02 Zeta potential (mV) -14.89 ± 2.59 -9.73 + -0.62 Inclusion rate (%) ^a - 91.30 ± 1.00 Drug content (%) ^b - 3.04 + 0.03

c: The weight ratio between HA-TIBA and DOX is 30: 1.

Data are expressed as means ± SD (n ≥ 3).

As shown in Table 1 above, the coarse (drug-free) HA-TIBA exhibited an average particle size of 156 nm, a narrow particle size distribution (< 0.2 polydispersity index), and negative zeta potential values. After incorporation of DOX in HA-TIBA, the mean particle size slightly increased to 207 nm compared to HA-TIBA, but the narrow particle size distribution and negative zeta potential remained unchanged. It also showed a high drug loading rate (> 90%) in the prepared HA-TIBA / DOX. The particle size and spherical shape of the HA-TIBA nanocomposite were observed by TEM and are shown in Fig. The particle size range of the prepared DOX-containing HA-TIBA nanocomposite was measured to be about 200 nm, confirming that the HA-TIBA nanocomposite was suitable for passive tumor targeting (based on the EPR effect) during intravenous injection.

Test Example  One. In vitro DOX  Emission evaluation

To evaluate the drug release pattern from the nanocomposite formulation, a dispersion (75 μL) of HA-TIBA / DOX nanocomposite (HA-TIBA: DOX = 30: 1) was transferred to a Mini- GeBAflex tube Molecular weight cut-off: 14 kDa, Gene Bio-Application Ltd., Kfar Hanagide, Israel). Each tube was then placed in a 10 mL dissolution test solution (phosphate buffered saline, adjusted to pH 5.5, 6.8 and 7.4 with PBS, phosphate buffered saline) in a shaking bath at 37 ° C and 50 rpm. A predetermined amount (200 μL) of the elution test solution was collected at a fixed time ((2, 4, 6, 8, 10, 24, 48, 72, 96, 120 and 144 hours) was supplemented. emissions of DOX is high performance liquid chromatography (HPLC) was measured by using the method, the reverse phase C-18 column ^{(Xbridge ® RP-18, 250} × 4.6 mm, 5 μm, Waters Co.), the separation module ( (Waters HPLC system, Waters Co., Milford, Mass., USA) equipped with a separation module (Waters e2695) and a fluorescence detector (Waters 2475) (71:29, v / v) at a flow rate of 1.0 mL / min, excitation and emission of 470 and 565 nm, respectively, consisting of M sodium acetate buffer (adjusted to pH 4.0 with acetic acid) and acetonitrile The injection volume of the drug analysis was 20 μL and the lower limit of quantification (LLOQ) was 50 ng / mL. Day group (interday) and days (intraday) deviation (variance) of the HPLC method, was within the allowable range.

As above, DOX release from the HA-TIBA nanocomposite was evaluated at multiple pH values (pH 5.5, 6.8, and 7.4) and the results are shown in FIG. In the development of intravenous injection formulations of anticancer drugs, sustained release and pH-dependent drug release of nanotransporters is preferred. The sustained drug release pattern in intravenous formulations for cancer treatment may reduce the in vivo loss of the drug, improve the residence time in the systemic circulation, and thus increase the accumulation of drug at the tumor site. Decreased frequency of drug administration can also contribute to improve patient compliance. Since the pH values of 5.5, 6.8 and 7.4 are pH values indicating endosomes and lysosomes, tumor microenvironment, and normal physiological environment [Non-Patent Document 34], as shown in Fig. 3, Identification of improvements in drug release has the additional advantage of increasing the anticancer activity of anticancer drugs and reducing unwanted effects. In the present invention, it was confirmed that the release of DOX from HA-TIBA / DOX was slow and pH-dependent, as shown in the results of Fig. Especially at pH 5.5, drug release from HA-TIBA / DOX was maintained for 4 days. At 6 days, the release of DOX from nanocomposites at pH 5.5 and 6.8 was 2.2- and 1.3-fold higher, respectively, than pH 7.4 (p <0.05). This pH-dependent release of DOX is associated with weak interactions between drug and nanocomposite structures; And increased DOX solubility at acidic pH [Non-Patent Document 21].

Test Example  2. Intracellular Introduction rate  evaluation

SCC7 (squamous cell carcinoma) cells were obtained from ATCC (American Type Culture Collection, Rockville, Md., USA). The incorporation rate of DOX-containing nanocomposite into cells into SCC7 cells was evaluated by flow cytometry as follows. SCC7 cells were seeded in a 6-well plate at a density of 6.0 × 10 ⁵ cells per well and cultured at 37 ° C. for 24 hours. Then, the cell culture medium was inoculated into a DOX solution or a dispersion of a DOX-containing nanocomposite μg / mL DOX) and incubated for 1 hour. The cells were then washed with PBS (pH 7.4) and collected. The collected cell pellet was resuspended in PBS containing FBS (2%, v / v). Using FACSCalibur fluorescence-activated cell sorter (FACS ^TM ) equipped with a measurement program (CellQuest software, Becton Dickinson Biosciences, San Jose, Calif., USA), the introduction rate of DOX labeled with fluorescence intensity was measured .

Confocal laser scanning microscopy (CLSM) was used to evaluate the intracellular distribution of DOX-containing nanocomposites. After reaching 70-80% of the cell density (confluency), the cells inoculated per well at a density of 1.0 × 10 ⁵ cells (per well surface area 1.7 cm ²⁾ on the trypsinized culture slide (BD Falcon, Bedford, MA, USA) And seeded at 37 ° C for 24 hours. DOX (50 μg / mL) alone or in the state of HA-TIBA nanocomposite was incubated at 37 ° C for 1 hour, washed three times with PBS (pH 7.4), and 10 times with 4% formaldehyde solution Min. The cells on the culture slides were dried and incubated with 4 ', 6-diamidino-2-phenylindole, DAPI, H-1200, Vector laboratories, Inc., Burlingame, VECTASHIELD mounting medium was added to inhibit fading and to stain the cell nuclei. DOX fluorescence was detected with CLSM (LSM 710, Carl-Zeiss, Thornwood, NY, USA).

As described above, the results of evaluating the introduction rate and intracellular distribution of DOX into HA-TIBA / DOX by flow cytometry and CLSM analysis are shown in FIG. As reported in the prior art [Non-patent document 21], SCC7 cells were selected as CD44 receptor-positive cells to evaluate the tumor targeting ability of HA-based nanoparticles. Utilization of an active tumor targeting strategy based on HA and CD44 receptors has been evaluated in previous literature using HA-based nanoparticles of the inventors of the present invention [Non-Patent Documents 3, 11, 21, 27]. As shown in Fig. 4 (A), after the incubation of the nanoclusters and the DOX solution with SCC7 cells for 1 hour, the rate of introduction of DOX from the nanoclusters was 2.3-fold higher than that from the DOX solution (p < 0.05). As reported in the prior art [Non-patent Document 27], DOX-containing nanoparticles and DOX solutions have different mechanisms of introducing into cells, namely, receptor-mediated endocytosis and passive diffusion. Notably, the interaction between HA and CD44 receptors leads to an increase in the rate of HA-TIBA / DOX incorporation into cells compared to the drug solution group. Also, in the CLSM image (see Fig. 4 (B)), it can be confirmed that the fluorescence of DOX in the nano-association group is stronger than that in the DOX solution group. Increased intracellular uptake of the drug in the nanoclase group can contribute to increased toxicity to cancer cells after reaching the tumor site.

Test Example  3. In vivo NIRF  Imaging assessment

In order to observe the in vivo distribution of the HA-TIBA nanocomposite prepared, Cy5.5-covalently bound HA-TIBA was synthesized as described below and used for real-time NIRF imaging evaluation in a tumor-xenografted mouse model [Non-Patent Documents 11, 21]. The Cy5.5 (Cy5.5-NH ₂₎ with an amine functional group carboxylic acid groups of HA were connected via the amide bond formation. To synthesize Cy5.5-HA-TIBA, the carboxylic acid groups of HA were activated by dissolving HA-TIBA (80 mg), EDC (9.9 mg), and NHS (5.9 mg) in DMSO (20 mL). Adding the Cy5.5-NH ₂ (0.1 mg) solubilized in DMSO (0.1 mL) to the solution and stirred for 16 hours. Thereafter, the obtained mixture was dialyzed against DW for 2 days and lyophilized.

The fluorescence intensities of Cy5.5 solution (control) and Cy5.5-HA-TIBA / DOX were measured using an imaging observation system (Fig. 4B) before intravenous injection of Cy5.5 solution (control) and Cy5.5-HA- TIBA / Optix MX3, ART Advanced Research Technologies Inc., Saint-Laurent, QC, Canada).

Female BALB / c nude mice (5 weeks old, Charles River, Wilmington, Mass., USA) were used to generate SCC7 tumor-xenografted mouse models. SCC7 cells (2 x 10 &lt; ⁶ &gt; cells in 0.1 mL) were administered by subcutaneous injection of the mice. Tumor volume (V, mm ³ ) was calculated using the following equation: V = 0.5 × maximum diameter × (minimum diameter) ² . When the tumor volume exceeds 200 mm ³ , Cy5.5 solution or Cy5.5-HA-TIBA nanocomposite was administered to the tail vein of mice at a dose of 60 μg / kg. For excitation of Cy5.5 dye, 670 nm wavelength laser was used and whole body NIRF images were scanned at 1, 3, 6, and 24 hours after administration. At 24 hours after administration, tissues and organs including the tumor were extracted to obtain in vitro NIRF images. The tumor targeting potency was evaluated using the nanocomposites prepared by comparing fluorescence intensities at the tumor site with in vivo and in vitro imaging.

The in vivo distribution of the prepared HA-TIBA-based nanocomposite was observed by real-time NIRF imaging as described above, and the results are shown in FIG. 5 and FIG. For the NIRF imaging, Cy5.5-NH ₂ (as the NIRF dye) was covalently attached to an amide bond forming groups -COOH of HA oligomer. Cy5.5-HA-TIBA nanoclusters were intravenously injected into SCC7 tumor-xenografted mice and the whole body of the mice was scanned for up to 24 hours (see FIG. 5). The fluorescence intensity of tumor sites in the nano-aggregate-treated group was stronger than the fluorescence intensity of Cy5.5 group at all measurement times for 24 hours. At 24 hours after administration, the fluorescence intensity of the nano-associate group was 1.8-fold higher than that of the Cy5.5 group (p <0.05). In the Cy5.5-HA-TIBA nanoassociative group, fluorescence signals were detected in other tissues and organs (ie, liver and spleen) 3 hours after administration, but the nanocomplexes disappeared in the body over time And accumulate in the tumor area. In addition, in vitro imaging results support in vivo imaging data. That is, as shown in FIG. 6, the HA-TIBA nanocomposite was found to accumulate mainly in tumor tissues rather than other normal tissues and organs. In addition, the fluorescence intensity of Cy5.5-HA-TIBA nanocomposite in tumor tissues was 2.4-fold higher than that of Cy5.5 (p <0.05). From the results of the NIRF imaging technique, it can be seen that the tumor targeting ability of the HA-TIBA nanocomposite is excellent.

Test Example  4. In vivo CT imaging evaluation

In vivo μCT scanning was performed with an imaging instrument (IVIS ^® Spectrum CT, PerkinElmer, Waltham, Mass., USA). The test X-ray conditions are as follows (voltage: 50 kVp, anodic current: 1 mA, resolution: 425 μm, filtration: aluminum). Treated with 1.77 mg / mL of control TIBA solution or HA-TIBA nanocomposite (30 mg / mL polymer concentration) in DMSO / polyethylene glycol 400 (PEG400) / DDW (1: 1: 1, v / A scanning procedure was performed on four pairs of control and test subjects consisting of one SCC7 tumor-xenografted female BALB / c nude mice (5 weeks old, Charles River, Wilmington, MA, USA;> 200 mm ³ tumor volume) . Mice were anesthetized with isoflurane and intravenously injected with TIBA solution or HA-TIBA nanocomposite at an iodine dose of 125 mg / kg. Systemic images were measured at 0 hour (untreated) and 24 hours after administration. ΜCT images were obtained using measurement software (Living Image ^® Software, ver. 4.3.1). At 24 hours after administration, tumor tissues from all groups were extracted and quantitatively analyzed for iodine content with a measuring instrument (ICP-OES, Optima-4300 DV, PerkinElmer Inc.).

FIG. 7 shows the in vivo tumor targeting ability of HA-TIBA nanocomposite by CT imaging in SCC7 tumor-xenografted mouse model as described above. TIBA, which is a hydrophobic moiety for the preparation of nano-sized self-assemblies and also used as CT contrast agent due to the contained iodine molecules, is covalently bound to the HA oligomer. TIBA covalently bound to HA was expected to form a self-assembled complex after intravenous administration, resulting in a slower disappearance in the body and a longer circulation in blood than unconjugated TIBA in solution. As shown in Fig. 7 (A), the HA-TIBA nanocomposite was found to exist mainly in tumor tissues rather than normal tissues and organs at 24 hours after administration. On the other hand, unconjugated TIBA was not detected in tumor tissue and other parts of the body at 24 hours after administration. HA-TIBA nanocomposites can be more efficiently accumulated in tumor tissues because of their low blood loss and long half-life in the blood. The results are also presumably due to an active targeting strategy based on HA and CD44 receptor interactions. Tumor tissues were extracted 24 hours after administration and the iodine content in tumor tissues was quantitatively analyzed by ICP-OES. As shown in FIG. 7 (B), the iodine content in the tumor tissues of the HA-TIBA-treated group was 10.1-fold higher than that in the non-conjugated TIBA group (p <0.05). These results demonstrate the high targeting potential of HA-TIBA nanocomposites based on passive and active tumor targeting strategies.

Test Example  5. Evaluation of anti-tumor efficacy in vivo

For in vivo anticancer activity studies, SCC7 tumor-xenografted mouse models were prepared using female BALB / c nude mice (5 weeks old, Charles River). The mice were raised at a temperature of 22 ± 2 ° C and a relative humidity of 55 ± 5%. The experimental procedure was carried out with the approval of the relevant institution (Animal Care and Use Committee of the College of Pharmacy, Seoul National University). SCC7 cell suspension (2 x 10 &lt; ⁶ &gt; cells in 0.1 mL cell culture medium) was subcutaneously injected into the mice. After the tumor volume reached 50-100 mm ³ , tumor size and body weight were measured. Tumor volume (mm ³ ) was calculated using the following equation: V = 0.5 × maximum diameter × (minimum diameter) ² . Test groups consisted of control, blank HA-TIBA, DOX, and HA-TIBA / DOX groups. DOX solution and HA-TIBA / DOX corresponding to a dose of 5 mg / kg were injected into the tail vein of each group on days 5, 7, 9, 11, and 13. The same HA-TIBA / DOX HA-TIBA amount of HA-TIBA was administered by the same method. Tumor volume and body weight of mice were measured for 16 days after administration, and tumors and hearts were harvested for histological staining 16 days after administration. The extracted specimens were fixed in 4% (v / v) formaldehyde for one day, and 6-μm sections were then deparaffinized and hydrated with ethanol. Tumor tissues were used for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis using standard procedures and heart tissue was treated with hematoxylin and eosin ) &Lt; / RTI &gt; (H &amp; E). Chromogen diaminobenzene (DAB) was used for TUNEL analysis and the samples were incubated and developed to detect DNA fragmentation caused by the apoptotic signaling cascade.

The in vivo antitumor activity of HA-TIBA / DOX in the SCC7 tumor-xenografted mouse model was evaluated, and the results are shown in FIG. Each of the formulations prepared was administered to mice in multiple doses to evaluate tumor growth inhibition, weight change, apoptosis in tumor tissue, and cardiac toxicity. There was no significant difference in tumor growth pattern between the control and co-HA-TIBA groups during the entire test period (see Fig. 8 (A)). In addition, there was no significant difference in body weight between the two groups (see Fig. 8 (B)). This means that the HA-TIBA covalent bond itself did not exhibit severe anti-tumor effects or systemic toxicity at the concentration range tested. The mean tumor volume of the DOX solution group was lower than that of the control group, but there was no significant difference, but the tumor growth inhibitory effect of the HA-TIBA / DOX group was significantly different from that of the other groups (p <0.05 ). The tumor volume of the HA-TIBA / DOX group measured at 16 days was 41.1% of that of the control group. The cell suicide effect of HA-TIBA / DOX was evaluated by TUNEL analysis, and as a result, high cell suicide rate was observed in the HA-TIBA / DOX group compared to the other groups (see left column of FIG. The H & E staining results of the control and HA-TIBA / DOX groups showed that cardiac toxicity, known as the major side effect of DOX, was not severe (see right column of FIG. 8 (C)). From the above results, it can be seen that the nanocomposite of the present invention exhibits increased antitumor efficacy and minimized side effects.

Claims (11)

delete An ester covalent bond of a hyaluronic acid oligomer in which a carboxylic acid group of a compound selected from the group consisting of triiodobenzoic acid, diazotric acid, metrizoic acid, and iothalamate is ester-covalently bonded to a hydroxy group of hyaluronic acid; And nano-aggregates for tumor-targeted drug delivery and cancer diagnosis, including hydrophobic anticancer agents. 3. The composition of claim 2, wherein the ester covalent bond of a hyaluronic acid oligomer in which a carboxylic acid group of triiodobenzoic acid and a hydroxy group of hyaluronic acid are ester covalently bonded; And nano-aggregates for tumor-targeted drug delivery and cancer diagnosis, including hydrophobic anticancer agents. 4. The nanocomposite according to claim 3, wherein the triiodobenzoic acid is bonded at 10 to 20 moles per mole of hyaluronic acid. 4. The nanocomposite according to claim 3, wherein the iodine content of the ester covalent bond of the hyaluronic acid oligomer is 30 to 60% (w / w). 4. The nanocomposite according to claim 3, wherein the ester covalent bond of the hyaluronic acid oligomer and the hydrophobic anticancer agent are contained in a weight ratio of 5: 1 to 40: 1. The nanocomposite according to claim 2, wherein a polymethine dye is further bound to the ester covalent bond of the hyaluronic acid oligomer. 7. A pharmaceutical composition for tumor-targeted drug delivery and cancer diagnosis comprising the nanocoating complex of any one of claims 2-7. 9. The pharmaceutical composition according to claim 8, wherein the tumor is a carcinoma in which the CD44 receptor is expressed. [Claim 9] The pharmaceutical composition according to claim 8, wherein the tumor is at least one selected from the group consisting of lung cancer, breast cancer, liver cancer, brain tumor, skin cancer and ovarian cancer. (a) an ester covalent bond of a hyaluronic acid oligomer in which a carboxylic acid group of a compound selected from the group consisting of triiodobenzoic acid, diazotric acid, metrizoic acid and iothalamate is ester-covalently bonded with a hydroxy group of hyaluronic acid, and Solubilizing the hydrophobic anticancer agent to obtain a solution;
(b) removing the solvent from the solution of step (a) to obtain a solid; And
(c) adding an aqueous solvent to the solids of step (b) to obtain a dispersion
Lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; tumor-targeted drug delivery and cancer diagnosis.

Images