KR101809939B1 - Nano-drug delivery flatform for Sequential Release of Hydrophilic and Hydrophobic Drug - Google Patents

Abstract

The present invention provides a drug carrier which can carry hydrophilic and hydrophobic drugs, and sequentially release the drugs under acidic conditions according to pH conditions; and a method of preparing the same. The surface of the drug carrier of the present invention is coated with a biodegradable copolymer which is formed by polyethylene glycol (PEG), pyridyl disulfide (PDS), and 2-(diisopropylamino)ethyl methacrylate (DPAMA), wherein the biodegradable copolymer serves as a gate keeper layer which swells by an electrostatic interaction in acidic pH ranges. The biodegradable copolymer on the surface of the drug carrier of the present invention can be adjusted in size according to the surrounding pH conditions, and can regulate the release of drug by swelling in acidic environments similar to the pH of a primary tumor. Particularly, the drug carrier, when carrying a hydrophobic drug and a hydrophilic drug therein at the same time, can release the hydrophilic drug first prior to release of the hydrophobic drug, and thus may be effectively and beneficially used as a drug carrier for diseases such as multidrug resistant cancer that require treatments of multiple drugs.

Description

≪ Desc / Clms Page number 1 > Nano-drug delivery platform for sequential release of hydrophilic and hydrophobic drugs.

The present invention is based on the finding that a biodegradable copolymer crosslinked on the surface of a double-drug supported mesoporous silica nanoparticles (hollow Mesoporous Silica Nanoparticles (HMSN)) is swollen by electrostatic interactions in the acid pH range And has a gate keeper layer.

Multi drug resistance is a major cause of cancer deaths in more than 90% of patients with failed chemotherapy, especially in the absence of treatment effects of chemotherapy. Various attempts have been made to overcome MDR in chemotherapy, one of which is the co-administration of drugs in combination. However, because tumors vary in the tissue or cell environment depending on the cancer location or the timing of cancer progression, various drug doses and concentrations of the drug in such a tumor environment may limit the synergy in the two or more drugs administered . In addition, since two or more kinds of drugs must be administered in combination, there is a possibility that the side effects thereof are amplified. Therefore, sequentially delivering a complex drug to a target site at a cancer site can be an important method for improving the efficiency of chemotherapy through combination of drugs (Non-Patent Document 2 and Non-Patent Document 3).

Therefore, it is more preferable to administer the drug using the drug delivery system, which can deliver the drug to the target site and efficiently administer the drug. Selective targeting of tumor cells and release of drug molecules in the tumor microenvironment may be expected to reduce the side effects of traditional chemotherapy by reducing the dose. In targeted drug delivery that releases the drug in response to a particular stimulus, selective targeting of tumor cells and release of the drug molecule in the tumor microenvironment may reduce side effects of traditional chemotherapy by reducing the dose. This goal may be controlled using a targeted drug delivery system that releases the drug in response to a particular stimulus.

Nanoparticles, such as polymer nanoparticles or nanoparticles, can stably retain the drug before reaching the target cell during blood circulation, and can regulate the loading and emis- sion capacity of the drug to efficiently deliver the drug within the tumor tissue or tumor cells Is expected to be released. In order to prevent inaccurate release of the drug and ensure precise release at the target site, the delivery vehicle carrying the drug should have the property of controlling release in response to a particular stimulus.

Recently, it has been reported that core-shell nano- and microparticle-based drug delivery systems can sequentially release drugs through degradation of polymer-containing polymeric layers in the treatment of cancer. However, since it has not yet been specifically studied how to control the release of drugs appropriately during circulation through the blood in the body or to control the release pattern of two or more drugs in the encapsulated drug, To achieve effective and sequential treatment is still a limitation.

In this regard, the present inventors have developed a whole drug group capable of releasing a drug in response to a specific stimulus, and disclosed in Korean Patent Publication No. 10-2016-0089774 that polyethylene glycol (PEG) -pyridyl disulfide, PDS) on the surface of a drug-bearing MSN. It is known that the biodegradable copolymer can be coated as a non-covalent ligand on the surface of a drug-bearing MSN through cross-linking and released by GSH as a reducing agent. However, this method has a limitation in that it can release the drug effectively only through the reduction reaction with GSH or the like in the body.

Accordingly, the present inventors have made efforts to produce a drug delivery system capable of releasing a drug in a time-and-space-specific manner in a cancer tissue of multiple resistance. As a result, the present inventors have found that a combination of polyethylene glycol (PEG), pyridyl disulfide (PDS) ) Ethyl methyl acrylate (DPAMA) was coated on the surface of the double-drug-loaded HMSN and cross-linked to prepare a drug delivery vehicle (PMSN) carrying the drug. The biodegradable copolymer of the present invention can be controlled in size depending on the surrounding pH conditions and it is possible to control the drug release pattern in a carrier that detects the drug through electrostatic interaction in an acidic environment having a pH similar to that of the initial tumor Thus, the present invention has been completed upon confirming that the drug delivery system of the present invention can be used as a drug delivery system capable of simultaneously carrying a hydrophobic drug and a hydrophilic drug.

KR 10-2016-0089774 A (2016.07.28)

 Zhang, Kun, et al. &Quot; Facile large-scale synthesis of monodisperse mesoporous silica nanospheres with tunable pore structure. &Quot; Journal of the American Chemical Society 135.7 (2013): 2427-2430.  Cao, Yang, et al. "Polymer-controlled core? Shell nanoparticles: a novel strategy for sequential drug release." RSC Advances 4.57 (2014): 30430-30439.  Narayanan, Sreeja, et al. "Sequentially releasing dual-drug-loaded PLGA? Casein core / shell nanomedicine: Design, synthesis, biocompatibility and pharmacokinetics." Acta biomaterialia 10.5 (2014): 2112-2124.

Thus, the present inventors produced a drug delivery vehicle (PMSN) in which a biodegradable copolymer of PEG-PDS-DPAMA was coated on the surface of a double-drug-loaded HMSN and crosslinked to support a drug. The biodegradable copolymer of the present invention can be controlled in size depending on the surrounding pH conditions and it is possible to control the drug release pattern in a carrier that detects the drug through electrostatic interaction in an acidic environment having a pH similar to that of the initial tumor Thereby completing the present invention.

Accordingly, an object of the present invention is to provide a drug delivery system capable of carrying a double drug having hydrophilicity and hydrophobicity and sequentially releasing a drug under acidic conditions according to pH conditions, and a method for producing the drug delivery system.

It is still another object of the present invention to provide a pharmaceutical composition for preventing and treating cancer comprising the above drug delivery system as an active ingredient.

In order to achieve the above object, the present invention provides a method for producing a drug delivery vehicle, comprising the steps of:

i) preparing mesoporous silica nanoparticles (MSN);

ii) a biodegradable copolymer consisting of polyethylene glycol (PEG), pyridyl disulfide (PDS) and 2- (diisopropylamino) ethyl methacrylate (DPAMA) ;

iii) coating the biodegradable copolymer on the surface of HMSN by mixing the HMSN prepared in step i) and the biodegradable copolymer prepared in step ii);

iv) treating the coated HMSN in step iii) with dithiothreitol (DTT) to form a cross-linked copolymer-HMSN (PHMSN) of the biodegradable copolymer; And

v) obtaining the PHMSN formed in step iv) above.

The present invention also provides a drug delivery system manufactured by the above-described method.

In a preferred embodiment of the present invention, the mixing of step iii) may be a mixing of the HMSN and the biodegradable copolymer in a weight ratio of 1: 1 to 1: 4.

In a preferred embodiment of the present invention, the DTT of step iv) may be treated at a molar ratio of 20 to 50 mol% to the PDS in the biodegradable copolymer.

In a preferred embodiment of the present invention, the crosslinking in step iv) may be crosslinked at a crosslinking density of 30 to 60 mol%.

In a preferred embodiment of the invention, the drug delivery vehicle may comprise a drug or a biofunctional regulator within HMSN, the drug being selected from the group consisting of doxorubicin, verapamil, cisplatin, a hydrophilic drug selected from the group consisting of fluorouracil, oxaliplatin daunorubicin, irinotecan, topotecan and pharmaceutically acceptable salts thereof; A pharmaceutically acceptable salt thereof selected from the group consisting of paclitaxel, camptothecin, carboplatin, gemcitabine, methotrexalte, docetaxel and pharmaceutically acceptable salts thereof. drug; And a mixture thereof, and the biological function-controlling substance may be any one selected from the group consisting of DNA, RNA, polypeptides, vitamins, vitamin derivatives, amino acids and amino acid derivatives Or more.

In a preferred embodiment of the present invention, the drug delivery vehicle may have a gate keeper layer in which the biodegradable copolymer cross-linked to the surface swells by electrostatic interaction in the acidic pH range, The pH range may be in the range of pH 4.0 to 6.5 of the initial tumor tissue. In addition, the electrostatic interaction may be such that the charge on the surface becomes zero or more positive.

The present invention also provides a pharmaceutical composition for preventing and treating cancer comprising the drug delivery system of the present invention as an active ingredient.

In one preferred embodiment of the present invention, the cancer may be at least one selected from the group consisting of multiple-resistant gastric cancer, breast cancer, lung cancer, liver cancer, esophageal cancer, and prostate cancer, each having resistance to an anticancer agent.

In one preferred embodiment of the present invention the anticancer agent is selected from the group consisting of doxorubicin, cisplatin, verapamil, fluorouracil, oxaliplatin, daunorubicin, irinotecan, may be any one selected from the group consisting of topotecan, paclitaxel, camptothecin, carboplatin, gemcitabine, methotrexalte, and docetaxel. have.

Accordingly, the present invention provides a drug delivery system capable of supporting dual drugs having hydrophilicity and hydrophobicity and sequentially releasing drugs under acidic conditions according to pH conditions, and a method for producing the same.

The drug delivery system of the present invention is formed by coating a biodegradable copolymer comprising polyethylene glycol (PEG), pyridyl disulfide (PDS) and 2- (diisopropylamino) ethyl methyl acrylate (DPAMA) There is a technical feature that the acidic copolymer serves as a gate keeper layer which swells by electrostatic interaction in an acidic pH range.

The biodegradable copolymer of the drug delivery surface of the present invention can be scaled according to ambient pH conditions and can be swollen in an acidic environment at pH similar to the initial tumor to control drug release. In particular, when a hydrophobic drug and a hydrophilic drug are simultaneously carried in the drug carrier, a hydrophilic drug may be released first and then a hydrophobic drug may be released. It can be effectively used as a drug delivery vehicle for diseases.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a process for preparing a dual-drug-supported PHMSN of the present invention:
FIG. 1A shows the swelling of the gatekeeper layer with PHMSN and pH control with dual drugs of verapamil hydrochloride (Ver) and doxorubicin (Dox);
Figure 1B shows the charge generation upon pH and structure of the PEG-PDS-PDAMA biodegradable copolymer coated on the surface of the PHMSN of the present invention; And
FIG. 1C shows the PHMSN of the present invention which is absorbed intracellularly in tumor cells to release the drug.
Figure 2 shows the characteristics of the prepared mesoporous silica nanoparticles (MSN)
2A shows the shape of the MSN determined by TEM image;
Figure 2b shows the result of confirming the shell thickness of the HMSN surface; And
Figure 2C shows the total pore volume and size of the HMSN surface.
Figure 3 shows the synthesis of a biodegradable copolymer for coating on a PHMSN surface:
3A shows the process of synthesizing the copolymer of PEG-PDS-PDPAMA of the present invention and the structure of the copolymer; And
Figure 3b shows the synthesis of the prior art PEG-PDS copolymer and the structure of the copolymer for use as a control.
Fig. 4 shows NMR analysis results for confirming the structure of the PEG-PDS-PDPAMA copolymer of the present invention.
Figure 5 shows the characteristics of the drug-supported PHMSN prepared in the present invention:
FIG. 5A is a TEM image analysis result showing the shape of a PMSN coated with a polymer layer; FIG.
Figure 5b shows the change in diameter size of the nanoparticles with or without coating of the polymer layer;
Figure 5c shows the surface charge change of the nanoparticles with or without coaching of the polymer layer;
FIG. 5D is a view showing changes in the surface charge of the dual drug-bearing carrier (PHMSN) of the present invention and the previously known drug delivery vehicle (PEG-PDS HMSN; crosslinking density 33 mol%) according to the change of the pH environment; And
FIG. 5E shows the change in diameter of Dox-CPT-PHMSN according to the pH environment change of the present invention.
Figure 6 shows the characterization of PHMSN following DTT treatment for cross-linking of a copolymer gatekeeper layer:
FIG. 6A is a view showing the difference in cross-linking of nanoparticle surfaces before and after the DTT treatment; FIG.
FIG. 6B is a graph showing that 33 mol% of crosslinking is formed when PDT in the copolymer is treated with 20 mol% of DTT;
FIG. 6C shows that 60 mol% of crosslinking is formed when PDT in the copolymer is treated with 50 mol% of DTT;
FIG. 6d is a view showing a change in diameter of Dox-CPT-PHMSN stored in a sodium acetate buffer solution at pH 5.5 over time; FIG.
FIG. 6E is a graph showing changes in diameter of Dox-CPT-PHMSN stored in pH 7.4 PBS according to time.
Figure 7 shows the drug release profile with pH change and GSH (reducing agent) treatment:
FIG. 7A is a graph showing the change of pH and the drug release pattern according to GSH treatment in the Dox-CPT-PHMSN of the present invention;
FIG. 7B is a view showing the pH change and the drug release pattern according to the GSH treatment in a dual drug-supported PHMSN coated with a PEG-PDS copolymer and crosslinked at a crosslinking density of 60 mol%; FIG. And
Figure 7c shows the change in surface charge as a function of pH of a dual drug-supported PHMSN (pink) coated with PEG-PDS-PHMSN (blue) and PEG-PDS copolymer of the present invention crosslinked at a crosslinking density of 60 mol% As shown in FIG.
Figure 8 shows the confirmation of whether the drug release pattern is modulated by various conditions in a dual drug or single drug loaded PHMSN:
8A is a diagram showing the drug release pattern of HMSN in the Dox-CPT carrier not coated with the copolymer;
8b is a diagram showing the drug release pattern of Dox-PHMSN at pH 7.5 depending on whether 5 mM GSH is present;
8c is a graph showing the drug release pattern of CPT-PHMSN at pH 7.5 depending on whether 5 mM GSH is present;
FIG. 8D is a graph showing drug release patterns of Dox-PHMSN or CPT-PHMSN at pH 6.5 medium treated with 5 mM GSH;
FIG. 8E is a diagram showing drug release patterns of Dox-PHMSN or CPT-PHMSN at pH 6.5 medium without 5 mM GSH; FIG. And
Figure 8f shows the drug release profile of CPT-Dox-PHMSN in PEG-PDS copolymer coating at pH 6.5 without GSH treatment.
Figure 9 shows the cytotoxic effect of the PHMSN of the present invention:
FIG. 9A is a graph showing the cytotoxic effect of drug-non-carrying HMSN and Dox-CTP-PHMSN on KB cells cultured for 24 hours; FIG.
Figure 9b shows the cell viability of KB cells cultured in a fresh medium after pretreatment of Dox-PHMSN in pH 6.5 or pH 7.4 medium for 30 minutes; And
Figure 9c shows the cell viability of KB cells cultured in fresh medium after pre-treatment with CPT-PHMSN in pH 6.5 or pH 7.4 medium for 30 minutes.
Figure 10 shows the intracellular influx of CPT-Dox-PHMSN:
10A is a fluorescence observation of KB cells treated with Dox-CPT-PHMSN for 1 hour in a pH 6.5 PBS buffer solution;
FIG. 10B is a fluorescence observation of KB cells treated with Dox-CPT-PHMSN for 1 hour in pH 7.4 PBS buffer solution;
Figure 10c shows the results of FACS analysis of KB cells treated with Dox-CPT-PHMSN for 2 hours at pH 7.4 (blue), pH 6.5 (red) and normal cell culture medium (green); And
FIG. 10d shows the cell viability of KB cells treated with Dox-CPT-PHMSN at pH 7.4 or pH 6.5 for 30 hours.
Figure 11 shows confirmation of the intracellular entry mechanism of CPT-Dox-PHMSN:
FIG. 11A is a view for confirming the intracellular influx of Doc-PHMSN in a medium pretreated with sucrose;
FIG. 11B is a view showing the intracellular influx of Doc-PHMSN in a medium pretreated with amiloride;
FIG. 11C is a graph showing the intracellular inflow pattern of Doc-PHMSN in the medium pretreated with M? CD (methyl-β-cyclodextrin); And
FIG. 11D is a FACS result showing the intracellular inflow pattern of Doc-PHMSN in a medium pretreated with sucrose (blue), amiloride (red), or MβCD (green).
Figure 12 shows confirmation of the release mechanism of CPT-Dox-PHMSN into the cytoplasm:
FIG. 12A is a fluorescence imaging analysis of endosomes and drugs in KB cells cultured by treatment with Dox-PHMSN or CPT-PHMSN for 1 hour in pH 6.5 buffer solution and exchanging with fresh medium; And
FIG. 12B is a fluorescence imaging analysis of lysosomes and drugs in KB cells cultured in DOC-PHMSN or CPT-PHMSN treated with pH 6.5 buffer solution for 1 hour and exchanged with fresh medium.
Figure 13 shows confirmation of the release mechanism of CPT-Dox-PHMSN into the cytoplasm:
FIG. 13A is a fluorescence imaging analysis of KB cells cultured for 4 hours in Dox-CPT-PHMSN treated with pH 6.5 buffer solution for 1 hour, exchanged with fresh medium;
FIG. 13B is a fluorescence imaging analysis of KB cells cultured for 6 hours in Dox-CPT-PHMSN treated with pH 6.5 buffer for 1 hour and exchanged with fresh medium.
Figure 14 shows confirmation of the synergistic cell killing effect of Dox-CPT-PHMSN on Dox resistant gastric cancer cell lines:
FIG. 14A is a graph showing the cell death effect of cells cultured for 48 hours in Free-Dox, Dox-PHMSN, or Dox-CPT-PHMSN treated with pH 6.5 solution for 6 hours and then replaced with fresh medium;
FIG. 14B shows the appearance of a drug introduced from a cell cultured for 48 hours after Dox-PHMSN or Dox-CPT-PHMSN was treated with pH 6.5 solution for 2 hours or 6 hours and then replaced with fresh medium.
Figure 15 shows confirmation of the synergistic cell killing effect of Dox-Ver-PHMSN on Dox resistant gastric cancer cell lines:
FIG. 15A is a graph showing the cell death effect of cells cultured for 48 hours after free-Dox or Dox-Ver-PHMSN was treated with pH 6.5 solution for 6 hours and then replaced with fresh medium; And
FIG. 15B is a view showing the appearance of a drug introduced from a cell cultured for 2 hours after free-Dox or Dox-Ver-PHMSN was treated in a pH 6.5 solution for 2 hours and then replaced with a fresh medium.
16 shows the influx of the intracellular drug into the multidrug resistant cancer cell line according to the drug delivery treatment of the present invention:
FIG. 16A is a view showing the influx of the intracellular drug into cells treated with Dox-PHMSN and Free Dox; FIG. And
FIG. 16B is a view showing the flow of the drug in the cancer cell line treated with Dox-PHMSN after the pretreatment of Free-Ver for 30 minutes.

Hereinafter, the present invention will be described in detail.

As described above, a complex anticancer treatment method is required for obtaining a therapeutic effect in a multi-resistant cancer. Therefore, it is required to develop a drug delivery system capable of controlling the release of such a complex drug. However, in the encapsulated drug delivery system, Achieving effective and sequential therapies using nanomaterial transporters remains a limitation, since control of the release profile of more than one drug has not yet been specifically studied.

The biodegradable copolymer of the drug delivery surface of the present invention can be scaled according to ambient pH conditions and can be swollen in an acidic environment at pH similar to the initial tumor to control drug release. In particular, when a hydrophobic drug and a hydrophilic drug are simultaneously carried in the drug carrier, a hydrophilic drug may be released first and then a hydrophobic drug may be released. And can be usefully used as a drug delivery vehicle for diseases.

Accordingly, the present invention provides a biodegradable copolymer crosslinked on the surface of a dual-drug supported mesoporous silica nanoparticles (hollow Mesoporous Silica Nanoparticles (HMSN)) by electrostatic interactions in the acid pH range A drug carrier having a swollen gate keeper layer is provided.

The " drug delivery vehicle " of the present invention preferably comprises a drug or a biofunctional regulator within the HMSN.

The medicament may be selected from the group consisting of doxorubicin, verapamil, cisplatin, fluorouracil, oxaliplatin, daunorubicin, irinotecan, topotecan, Lt; / RTI > acceptable salt; A pharmaceutically acceptable salt thereof selected from the group consisting of paclitaxel, camptothecin, carboplatin, gemcitabine, methotrexalte, docetaxel and pharmaceutically acceptable salts thereof. drug; And mixtures thereof. More preferably, the drug is one or more selected from the group consisting of doxorubicin, verapamil and camptothecin, but is not limited thereto. Any combination of drugs known in the art to be synergistic when used in combination in the treatment of resistant cancers can be used.

More preferably, the drug that can be carried on the drug delivery system of the present invention may be a combination of a silver hydrophobic drug and a hydrophilic drug. In the drug delivery system of the present invention, the drug is carried or captured in the hollow heterogeneous silica nanoparticles (HMSN), wherein the hydrophilic drug can be injected into the HMSN inner space having a high injection capacity, Lt; RTI ID = 0.0 > HMSN < / RTI >

The biological function-controlling substance of the " drug delivery vehicle " of the present invention may be any one or more selected from the group consisting of NA, RNA, polypeptides, vitamins, vitamin derivatives, amino acids and amino acid derivatives. The biofunction regulating material may also be carried or trapped by a combination of a hydrophobic substance and a hydrophilic substance. Among them, the hydrophilic substance may be supported in the HMSN and the hydrophobic substance may be physically adsorbed on the surface of the HMSN.

The drug or the biofunctional substance to be supported on the " drug delivery vehicle " of the present invention can be effectively supported on the HMSN by simple mixing and stirring in a solution containing HMSN in the course of production. Therefore, the drug can be effectively carried on the carrier without requiring a chemical coupling reaction or a complex purification separation process, so that a drug carrier can be manufactured through a simple process at a low cost.

The term "drug delivery vehicle" of the present invention refers to a drug delivery system comprising polyethylene glycol (PEG), pyridyl disulfide (PDS) and 2- (diisopropylamino) ethyl methacrylate, DPAMA) is preferably coated by cross-linking. The crosslinked biodegradable copolymer can protect or release the drug into a gate keeper layer that is swelled by electrostatic interactions in the acid pH range.

The acidic pH range is preferably pH environment of the initial tumor tissue. Specifically, the acidic pH range is preferably in the range of 4.0 to 7.0, more preferably in the range of 4.5 to 6.5, but is not limited thereto. The electrostatic interaction may occur as the surface charge of the polymer-coated drug-bearing HMSN (PHMSN) becomes zero or more positive.

In a preferred embodiment of the present invention, the present inventors prepared a mesoporous silica nanoparticle (MSN) first to prepare a drug delivery system capable of regulating drug release by changing the pH and inducing reduction (FIG. 1) 2). In addition, a PEG-PDS-PDAMA copolymer was prepared to produce a positive charge polymer (FIGS. 3 and 4).

In addition, the present inventors carried out a method of coating DMSO (Dox) and camptothecin (CPT) in the HMSN thus prepared and coating the HMSN with the PEG-PDS-PDAMS copolymer (Table 1 and FIGS. 5A to 5C) The copolymer layer was treated with DTT and crosslinked to prepare Dox-CPT-PHMSN (Figs. 6A to 6C).

In order to examine the sequential release effect of the drug according to the pH change in the PHMSN carrying the double drug, the present inventors dispersed Dox-CPT-PHMSN in various pH environments to examine the drug release pattern. As a result, CPT-PHMSN has stability against long-term storage (Fig. 6D and Fig. 6E) and increases in diameter due to electrostatic interactions created by gradually changing the surface charge to a positive charge as the pH decreases and becomes an acidic environment (Fig. 5D, Fig. 5E and Fig. 7C).

The inventors of the present invention found that when GSH as a disulfide bonded reducing agent is treated, the release of the drug is significantly increased because the characteristic of the Dox-CPT-PHMSN is the effect of the biodegradable copolymer coated on the surface of the nanoparticles by cross- (Figs. 7A, 7B and 8).

Therefore, the drug delivery system of the present invention can be adjusted in size depending on the surrounding pH conditions due to the characteristics of the cross-linked biodegradable copolymer coated on the surface and swelled in an acidic environment at a pH similar to that of the initial tumor to control the release of the drug The drug delivery system of the present invention is an environment that can control the release of a drug through pH control and can be controlled by controlling the amount of drug The nanoparticles can be used as nanoparticles.

The present invention also provides a process for preparing the drug delivery vehicle comprising the steps of i) to v):

i) preparing mesoporous silica nanoparticles (MSN);

ii) a biodegradable copolymer consisting of polyethylene glycol (PEG), pyridyl disulfide (PDS) and 2- (diisopropylamino) ethyl methacrylate (DPAMA) ;

iii) coating the biodegradable copolymer on the surface of HMSN by mixing the HMSN prepared in step i) and the biodegradable copolymer prepared in step ii);

iv) treating the coated HMSN in step iii) with dithiothreitol (DTT) to form a cross-linked copolymer-HMSN (PHMSN) of the biodegradable copolymer; And

v) obtaining the PHMSN formed in step iv) above.

In the step of the production method of the present invention, the surface charge of MSN produced in step i) preferably represents negative charge, and the biodegradable copolymer produced in step ii) preferably exhibits a positive charge, ) And coating the biodegradable copolymer on the surface of HMSN can be coated with a positive charge copolymer on the surface of the negatively charged nanoparticles due to the electrostatic interaction.

In the production method of the present invention, the mixing of the step iii) is preferably, but not limited to, mixing the HMSN and the biodegradable copolymer in a weight ratio of 1: 1 to 1: 4. More preferably, the copolymer is added in such a range that the surface charge after coating of the positive charge copolymer on the surface of the negatively charged HMSN can be a negative charge of zero or less.

In the production process of the present invention, the DTT of step iv) is preferably treated in a molar ratio of 20 to 50 mol% with respect to PDS in the biodegradable copolymer, wherein the copolymer is crosslinked by DTT , And the crosslinking can be crosslinked with a crosslinking density of 30 to 60 mol%. Since the cross-linking density may increase in proportion to the treatment concentration of DTT, a drug and / or a drug delivery vehicle carried in the drug delivery vehicle can be appropriately selected and adjusted according to the pH environment of the target organ to be used.

The drug carrier prepared according to the preparation method of the present invention can be sized according to the surrounding pH condition due to the characteristics of the cross-linked biodegradable copolymer coated on the surface and is swollen in an acidic environment similar to the initial tumor, And the release of the drug can be controlled to be increased as compared with the case where the disulfide bond of the crosslinking between the copolymers is reduced through the reducing agent. Therefore, the production method of the present invention can control the release of the drug through pH control And can be usefully used as a method for producing a drug delivery vehicle for delivering a drug to an environment in which the drug is delivered.

The present invention also provides a pharmaceutical composition for preventing and treating cancer, which comprises the above drug delivery vehicle as an active ingredient.

In the "pharmaceutical composition" of the present invention, it is preferable that the cancer is any one or more selected from the group consisting of multiple cancer resistant gastric cancer, breast cancer, lung cancer, liver cancer, esophageal cancer and prostate cancer. Gastric cancer or breast cancer, but is not limited thereto.

The anticancer agent may be selected from the group consisting of doxorubicin, cisplatin, verapamil, fluorouracil, oxaliplatin, daunorubicin, irinotecan, topotecan, paclitaxel, But is not limited to, any one selected from the group consisting of camptothecin, carboplatin, gemcitabine, methotrexalte, and docetaxel.

In another specific example of the present invention, the inventors of the present invention compared the degree of intracellular uptake of Dox-CPT-PHMSN according to the pH environment of the culture medium, And FIG. 9b), it was confirmed that cancer cells could be killed because the Dox was first released to the cytoplasm and then the CPT was released and the drug was sequentially released (FIG. 9). In addition, it was confirmed that Dox-CPT-PHMSN, Dox-PHMSN and CPT-PHMSN of the present invention showed apoptotic effects depending on the treatment concentration, and after the drug carriers were treated for the first 30 minutes, , Indicating a significant cancer killing effect (FIG. 10).

In addition, the inventors of the present invention confirmed that the drug delivery system of the present invention can be introduced into cells through clathrin-dependent inclusion and ovarian cell action (FIG. 11), and they are introduced into cells in the form of endosomes and lysosomes, and could be released into the cytoplasm by pH change (FIGS. 12 and 13).

The present inventors also confirmed the synergistic effect of Dox and CPT drugs delivered into cells by Dox-CPT-PHMSN in drug-resistant cancer cells. As a result, in the case of Dox-CPT-PHMSN treatment, the cell survival rate was decreased in a concentration- (Fig. 14a to Fig. 14c). This shows that the Dox is released into the cytoplasm at the initial stage of intracellular inflow, and then the CPT is released sequentially, so that the synergistic effect of the cytotoxic effect may increase the cytotoxic effect (Fig. 14D).

In addition, the inventors of the present invention carried out, in a cancer cell line showing resistance to a versatile drug, the Verapamil hydrochloride (Ver), which is a calcium channel antagonist capable of inhibiting the activity of p-glycoprotein, Ver-Dox-PHMSN can be absorbed well in Dox-resistant breast cancer cells, and the concentration of Dox in the cytoplasm gradually increases with time, It was confirmed that the cancer cell killing effect was exhibited (FIGS. 15 and 16).

Therefore, when the drug carrier of the present invention is loaded with a hydrophobic drug and a hydrophilic drug, the drug carrier can be effectively absorbed into the tumor cells showing an acidic pH, thereby releasing the drug sequentially. In addition, synergistic effects are exhibited in cancer cells having resistance to anticancer drugs through sequential release of double-drugs, and the killing effect on cancer cells can be significantly increased while overcoming the drug resistance, Can be effectively used as an active ingredient of a pharmaceutical composition for the treatment and prevention of a multi-resistant cancer having resistance to an anticancer drug.

The double-drug-carrying drug delivery system of the present invention can be administered parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. Parenteral administration may refer to administration via routes other than oral, such as rectal, intravenous, peritoneal, muscular, arterial, percutaneous, nasal, inhalation, ocular and subcutaneous. When the drug delivery vehicle carrying the double-drug of the present invention is used as a medicine, it may further contain one or more active ingredients exhibiting the same or similar functions.

That is, the drug delivery system carrying the double-drug of the present invention can be administered in various forms of parenteral administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, Is prepared using an excipient. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Witepsol, macrogol, Tween 61, cacao bean, Liu lingzhi, glycerogelatin and the like can be used as the base of the suppository.

In addition, the drug delivery system carrying the double-drug of the present invention can be used in combination with various carriers permitted as medicines such as physiological saline or an organic solvent, and can be used in combination with glucose, sucrose or dex- Antioxidants such as carbohydrates such as tran, ascorbic acid or glutathione, chelating agents, low molecular proteins or other stabilizers can be used as pharmaceuticals.

The effective dose of the drug delivery system carrying the double-drug of the present invention is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and can be administered once to three times a day.

In a pharmaceutical composition of the present invention, the total effective amount of the drug delivery vehicle carrying the double-drug of the present invention is increased by a single dose (bolus) or a single dose, for example, by infusion for a relatively short period of time And may be administered by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. Since the concentration of the effective dose of the patient is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient, in view of this point, Lt; / RTI > as a pharmaceutical composition of a < RTI ID = 0.0 > double-drug-carrying < / RTI > drug delivery system.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Mesoporous  Silica nanoparticles ( Mesoporous  Silica Nanoparticles , MSN)

<1-1> Hollow MSN (hollow Mesoporous  Silica Nanoparticles, HMSN )

In the present invention, HMSN was prepared for use as a drug delivery system carrying a drug. HMSN was prepared according to the previously known method (Non-Patent Document 1, Patent Document 1).

Concretely, 3.5 ml of 2 M NaOH aqueous solution was mixed with 480 ml of purified water, and then hexadecyltrimethyl-ammonium bromide (CTAB) was added and heated to 80 캜 with stirring. When the CTAB was completely dissolved, 4.67 g of TEOS (tetraethyl orthosilicate) was stirred at 80 DEG C for 2 hours while being slowly added. After stirring, the resulting precipitate was filtered, washed with purified water three times, and then dried at 60 DEG C overnight. A dry sample was obtained and calcined at 550 &lt; 0 &gt; C for 5 hours in air to give HMSN.

In order to analyze the structural characteristics of the obtained HMSN, analysis was performed using a transmission electron microscope (TEM) (JEOL-2100, JEOL) at an acceleration voltage of 200 kV to confirm the distribution of pore sizes. Nitrogen adsorption-desorption isotherm measurements were carried out at -196 ° C using a BELZORP-Max system to determine the total pore volume and size of HMSN.

As a result, it was confirmed that the MSN prepared as shown in Fig. 2 was HMSN in the form of a hollow and well knit mesoporous shell (Fig. 2A). It was confirmed that the shell thickness of the HMSN was 15 nm and the average diameter was 100 nm (Figs. 2A and 2B). The Bruaner-Emmett-Teller (BET) surface area was also analyzed to confirm that the HMSN had a total pore volume of 1.56 cm ³ / g and a pore size of 2.4 nm (Figure 2c) .

&Lt; 1-2 > Preparation of positive charge polymer

In order to make a form of polymer HMSN (PHMSN) bound to the polymer in the present invention, it was desired to construct a gatekeeper of polymer on the surface of HMSN. Since the surface of HMSN has a negative charge, a positive charge copolymer that can be cross-linked non-covalently through electrostatic interactions was prepared (Figure 3) .

The copolymers prepared in the present invention are two copolymers of PEG-PDS-DPAMA and PEG-PDS. First, PEG-PDS-DPAMA was prepared. 2-cyano-2-propyl benzodithioate (RAFT reagent, 4.72 L, 0.025 mmol), poly (ethylene glycol) methacrylate (1.02 g, 2.15 mmol), PDSEMA (549 mg, 2.15 mmol) 849.5 mg, 4.26 mmol) and AIBN (7 mg, 0.043 mmol) were dissolved in L (-) - 5,6,7,8-tetrahydrofolic acid (THF) solution and degassed with argon gas. The reaction vessel was sealed and then heated in an oil bath preheated to 70 ° C. for 12 hours. To remove the unreacted monomer and purify the polymer, the reaction mixture was precipitated from diethyl ether to give a random copolymer. The structure of the obtained PEG-PDS-DPAMA copolymer was confirmed by ¹ H NMR analysis (FIG. 4): ¹ H NMR (600 MHz, CDCl ₃ ): δ 8.46, 7.68, 7.11, 4.21, -3.52, 3.37, 2.97, 2.61, 1.85-1.86, 1.81. GPC (PMMA standard): Mn 78 kDa, Mw 132 kDa and PDI 1.69.

Next, a PEG-PDS copolymer was prepared. (1 g, 3.92 mmol), poly (ethylene glycol) methacrylate (931 mg, 0.98 mmol) and AIBN (2.4 mg, 0.0146 mmol) were added to a solution of 2-cyano-2-propyl benzodithioate (RAFT reagent, And then a random copolymer was obtained in the same manner as above. ¹ H NMR analysis of the obtained PEG-PDS copolymer confirmed the structure: ¹ H NMR (600 MHz, CDCl ₃ ):? 8.46, 7.68, 7.11, 4.50-4.01, 3.72-3.58, 3.38, 3.03, 2.15 -1.71, 1.13-0.79. GPC (PMMA standard): Mn 9.8 kD, Mw 14.4 kD and PDI 1.47.

Drug Bearing (loaded) PHMSN  Produce

&Lt; 2-1 > Bearing PHMSN  Produce

In the present invention, a PHMSN capable of carrying a double-acting drug effective for the purpose of killing cancer cells resistant to an anticancer agent was prepared.

Specifically, 5 mg of the HMSN prepared in Example <1-1> was added to 1 ml of purified water and ultrasonically pulverized to disperse the particles until a uniform colloidal solution was obtained. DMSO in which 2.5 mg, 5.0 mg or 7.5 mg of camptothecin (CPT) was dissolved was dissolved in the colloidal solution and stirred at room temperature for 24 hours. The stirred suspension was then centrifuged to obtain CPT-loaded nanoparticles and the concentration of the remaining drug remaining in the supernatant was determined. To remove DMSO from the nanoparticles, the CPT-loaded nanoparticles were vacuum dried, washed three times with purified water, and stored freeze-dried. The molecular weight of the CPT-loaded nanoparticles was calculated using the supernatant and wash solution. To calculate the amount of supported CPT, spectrum analysis was performed using a UV-Vis spectrophotometer set at a molecular absorption coefficient of? Max = 365 nm and 42,282 M-1 cm-1. Then, the nanoparticles were suspended in 1 ml of purified water and added to the reaction solution suspended in 2.5 mg, 5.0 mg or 7.5 mg of doxorubicin (Dox), and the mixture was stirred at room temperature for 24 hours.

Then, 10 mg of the PEG-PDS-DPA copolymer prepared in the above Example <1-2> was added to the polymer gatekeeper to coat the obtained double drug-carrying HMSN with the polymer gatekeeper, and further stirred overnight. To cross-link the surface-wrapped copolymer, dithiothreitol (DTT) was added to the stirred reaction solution and stirred at room temperature for 3 hours. The DTT was added in a molar ratio of 20 mol% or 50 mol% to the PDS group constituting the copolymer. The PHMSN carrying the double drug was collected by centrifugation and then washed with phosphate buffer solution of pH 7.4 and distilled water to finally obtain PHMSN (Dox-CPT-PHMSN) carrying CPT and Dox.

The density of cross-linking in the Dox-CPT-PHMSN obtained was calculated by confirming the concentration of pyridothione produced as a byproduct during crosslinking. The molar extinction coefficient of pyridothione is known to be 8.08 × 10 ³ M ^-1 ㎝ ^-2 at 343 ㎚. The amount (% by weight) of Dox carried in Dox-CPT-PHMSN was calculated from the amount of residual Dox in the final reaction solution from the total amount of drug added to the initial solution. The amount of Doc in solution was analyzed using a UV-Vis absorption spectrophotometer using a molar extinction coefficient of 11,500 M ^-1 cm ^-1 . The loading capacity and entrapment efficiency of the drug were calculated using the following equations (1) and (2).

[Equation 1]

Drug loading capacity (%) = Mass of the drug in MSN / Mass of MSN x 100

&Quot; (2) &quot;

Entrapment efficiency (%) = Mass of the loaded drug / Initial mass of the drug x 100

As a result, as shown in FIG. 5, it was confirmed that the HMSN was formed into a nanoparticle which was not formed as a composite through a polymer coating on a TEM image, and that the average diameter increased from 100 nm to 130 nm, , It was confirmed that even when Dox-CPT-PHMSN of the present invention was administered into the body, it could be decomposed and removed in the body after long-term circulation (FIGS. 5A and 5B). As shown in Table 1 below, Dox-CPT-PHMSN loaded with dual drugs had different loading efficiency and trapping efficiency depending on the concentration of CPT and Dox, and the loading efficiency of Dox increased to 36 wt% The loading efficiency of Dox and CPT was about 1: 2 (Table 1). The surface transfer of Dox-CPT-PHMSN was confirmed to show a high negative charge of -40 이전 before the polymer coating, but it was confirmed that the surface charge was neutralized to -2 한 after coating the polymer gatekeeper (Fig. 5C).

Comparison of loading efficiency and trapping efficiency of drugs to support dual drugs of Dox and CPT in PHMSN Drug concentration Dox loading efficiency%
(Capture efficiency%) CPT carrying efficiency (%)
Capture efficiency (%) 2.5 mg / ml 15% (30%) 23% (46%) 5.0 mg / ml 32% (32%) 60% (60%) 7.5 mg / ml 36% (24%) 80% (54%)

&Lt; 2-2 > Cross-linking agent  Depending on treatment concentration PHMSN  Identify features

In the Dox-CPT-PHMSN preparation process of the present invention, since the drug efflux can be controlled through the cross-linking of the surface copolymer gatekeepers, the concentration of the cross-linking inducing agent (DTT) We confirmed whether the characteristics of PHMSN were changed. The copolymer was crosslinked in a molar ratio of 20 mol% or 50 mol% with respect to the PDS group constituting the copolymer in Example <2-1> to induce copolymer cross-linking, thereby confirming the surface cross-linking density.

As a result, as shown in FIG. 6, the final density when cross-linked with 20 mol% of DTT was about 33 mol% for the PDS, and the final density when crosslinked with 50 mol% Was about 60 mol% (Figs. 6A to 6C).

<2-3> Single drug Bearing Dox - PHMSN  Produce

For comparison with the CPT-Dox-PHMSM prepared in the present invention, Dox-PHMSN was prepared as PHMSN carrying a single drug.

Specifically, 5 mg of the HMSN prepared in Example <1-1> was added to 1 ml of purified water and ultrasonically pulverized to disperse the particles until a uniform colloidal solution was obtained. Then, 2.5 mg, 5.0 mg or 7.5 mg of Dox was dissolved in the colloidal solution and stirred at room temperature for 24 hours. The stirred suspension was then centrifuged to obtain Dox-loaded nanoparticles and the concentration of the remaining drug remaining in the supernatant was determined. To remove DMSO from the nanoparticles, it was washed with purified water and a phosphate buffer solution of pH 7.4 and distilled water and centrifuged to obtain PHMSN (Dox-PHMSN) carrying a single drug, Dox. In the above procedure, all the remaining supernatants were collected into one, and the concentration of residual Dox was calculated, and the collection efficiency and the supporting efficiency were calculated using this. The amount (% by weight) of Dox carried in Dox-PHMSN was calculated from the amount of residual Dox in the final reaction solution from the total amount of drug added to the initial solution.

Then, the dried Dox-HMSN was suspended in 1 ml of distilled water, and 10 mg of the PEG-PDS-DPA copolymer prepared in Example <1-2> was added thereto, followed by further stirring overnight. To cross-link the surface-wrapped copolymer, dithiothreitol (DTT) was added to the stirred reaction solution and stirred at room temperature for 3 hours. The DTT was added in a molar ratio of 20 mol% to the PDS group constituting the copolymer. After stirring, Dox-HPMSN was collected by centrifugation and then washed with phosphate buffer solution of pH 7.4 and distilled water to obtain the final Dox-PHMSN.

<2-4> Single drug Bearing  CPT- PHMSN  Produce

For comparison with the CPT-Dox-PHMSM prepared in the present invention, CPT-PHMSN was prepared as PHMSN carrying a single drug.

Specifically, 5 mg of the HMSN prepared in Example <1-1> was added to 1 ml of purified water and ultrasonically pulverized to disperse the particles until a uniform colloidal solution was obtained. Then, 2.5 mg, 5.0 mg or 7.5 mg of Dox was dissolved in the colloidal solution and stirred at room temperature for 24 hours. Then, the same procedure as in Example <2-3> was conducted to obtain a final CPT-PHMSN.

<2-5> Preparation of PEG-PDS copolymer-coated drug delivery vehicle for use as a control group

For use as a control for the present invention, PHMSNs of biodegradable copolymer coatings known in the prior art were prepared (Non-Patent Document 1).

Specifically, as the biodegradable copolymer, the PEG-PDS prepared in Example <1-2> was used, and the PHMSN carrying the double drug of CPT and Dox was performed in the same manner as in Example <2-1> To prepare a control-dual drug-PHMSN. DTT for copolymer crosslinking was added in a molar ratio of 50 mol% with respect to the PDS period.

Double drugs Bearing From PHMSN  Sequential release effect of drugs according to pH change

<3-1> Depending on the pH environment PHMSN  Identify drug release effects

It was confirmed that the PHMSN carrying the dual drug prepared in the present invention could release the drug by the surrounding pH environment.

Specifically, 5 mg of Dox-CPT-PHMSN prepared in Example <2-1> was suspended in a buffer solution having various pH conditions of pH 4 to pH 8.5, and then stored for 72 hours. Five hours after the initiation of storage, the surface charge and the size of the nanoparticles of Dox-CPT-PHMSN were measured. After 24, 48, and 72 hours from the start of storage, the size of the nanoparticles was measured to determine whether the size of the nanoparticles varied with time.

5 mg of Dox-CPT-PHMSN was stored in phosphate-buffered saline (pH 7.4) for 5 hours in order to determine whether the same Dox-CPT- , And then transferred to a sodium acetate buffer solution at pH 5.5 for further storage for 10 hours, after which 1 mM glutathione (GSH) was added to the buffer solution and further stored for 17 hours. The drug release patterns over time were determined for both Dox and CPT and absorbance was measured at 480 ㎚ and 360 ㎚ using a Shimadzu RFPC 5430 spectrophotometer. As a control, the control group-dual drug-PHMSN prepared in Example <2-5> was stored under the same conditions as in Example <3-1> to measure the charge on the surface of the nanoparticles.

As a result, when the Dox-CPT-PHMSN was stored for 72 hours as shown in FIG. 6D and FIG. 6E, the particle size did not change in both the pH 5.5 and pH 7.4 environments, and Dox-CPT-PHMSN was used (Fig. 6 (d) and Fig. 6 (e)).

As for the size change of Dox-CPT-PHMSN according to various pH conditions, as shown in FIG. 5D and FIG. 5E, the pH of the Dox-CPT- It was confirmed that the charge was changed to positive charge. It can be predicted that this is a phenomenon in which the di-isopropyl group of the PEG-PDA-DPA copolymer protonates as the pH is lowered and acidified (Fig. 2B). In PDA among PEG-PDA-DPA copolymers coated with a gatekeeper, it is presumed that the amine group is positive as the pH is lowered, and electrostatic repulsion is generated between the generated quaternary amine groups to induce expansion of the coating layer of the gatekeeper have. As a result, it was confirmed that the size of Dox-CPT-PHMSN increased in an acidic environment having a low pH, and nanoparticles having a diameter of 130 nm at pH 7.5 increased to 300 nm in diameter at pH 4.5 (FIGS. 5D and 5E) .

As a result, it can be expected that the Dox-CPT-PHMSN of the present invention can increase the size of the polymer coating the surface of the Dox-CPT-PHMSN as the pH condition of the surrounding environment becomes acidic environment. FIGS. 7A and 7B When the cross-linked copolymer gate-keeper coating layer was decomposed through treatment with disulfide-bonded GSH as shown in FIG. 7A, the pattern of drug release was significantly increased (FIG. 7A). In Dox-CPT-PHMSN with a crosslinking density of 33 mol%, it was confirmed that the release rate of hydrophilic Dox among the two drugs appeared faster than the hydrophobic CPT. When Dox-CPT-PHMSN was dispersed in a pH 5.5 buffer solution, the release rate of Dox was more than 70%, whereas the release of CPT was not significant. Thereafter, it was confirmed that when GSH was treated, the release rate of CTP was increased, and finally, more than 40% of CTP was released. This pattern of drug release shows a lower level of drug release (Fig. 7b) when it has a crosslinking density of 60 mol% than that with a crosslinking density of 33 mol% (Fig. 7b) Was affected by the cross-linked copolymer gatekeepers on the PHMSN surface.

In addition, as shown in Figs. 5D and 7C, it was confirmed that all of the crosslinking densities of 33 mol% and 60 mol% produced high positive charges of +45 ㎷ and +43 에서 at the acidic pH, it was confirmed that it had a negative charge of -5 ㎷ and -14 pH under pH conditions (Figs. 5D and 7C). This indicates that the surface charge of Dox-CPT-PHMSN of the present invention is affected by the surrounding pH environment, and as a result, the drug carried by the swelling can be released. On the other hand, it was confirmed that the surface charge was not significantly changed even in the case of the control-double drug-PHMSN coated with the gatekeeper, the PEG-PDA copolymer used as the control group, despite the change of the pH.

&Lt; 3-2 > When a double drug or a single drug Supported From PHMSN  Determination of whether drug release pattern is controlled by various conditions

In the PHMSN drug delivery system of the present invention using PEG-PDA-DPA as a gatekeeper copolymer, the possibility of controlling the drug release pattern through other conditions was confirmed.

Specifically, PHMSN carrying the double drug prepared in Example 2 or PHMSN carrying a single drug was prepared. Then, PHMSN was dispersed under the conditions shown in [Table 2] below to confirm differences in drug release patterns.

Identification of changes in drug release pattern of PHMSN under various conditions Sample number Whether gatekeeper Carrier drug GSH treatment PH of medium One none Dox or CPT × pH 7.5 2 PEG-PDA-DPA Dox 5 mM GSH treatment
And GSH untreated pH 7.5 3 CPT 5 mM GSH treatment
And GSH untreated pH 7.5 4 Dox 5 mM GSH treatment pH 6.5 5 CPT × pH 6.5 6 PEG-PDA Dox or CPT × pH 6.5

As a result, it was confirmed that dispersing PHMSN under various conditions as shown in FIG. 8 can control the drug release pattern supported on PHMSN. First, when the copolymer gatekeeper was not present, it was confirmed that the release of Dox and CPT carried on the HMSN appeared at a high rate (FIG. 8A). In addition, Dox-PHMSN or CPT-PHMSN was dispersed in a pH 7.4 and pH 6.5 environment similar to that of Dox-CPT-PHMSN loaded with dual drugs. As a result, drug release was higher in pH 6.5 than pH 7.5 , And the effect of drug release was remarkably increased when 5 mM GSH as a reducing agent for crosslinking was treated (FIG. 8B to FIG. 8E). As a control, PHMSN using a PEG-PDA copolymer as a gatekeeper was used, and it was confirmed that no significant drug release was observed at pH 6.5 as compared with the Dox-CPT-PHMSN of the present invention (FIG.

Dox- CPT - PHMSN  Identification of intracellular absorption

&Lt; 4-1 > Dox- CPT - PHMSN  Comparison of intracellular absorption

Since it was confirmed that Dox-CPT-PHMSN prepared in the present invention can control the drug release by the surrounding pH, the efficiency of Dox-CPT-PHMSN in the cells was confirmed.

Specifically, the cancer cell KB cells derived from non-papillary epidermal cancer tissue were cultured in RPM 1640 medium. KB cells were inoculated into a sterilized single well glass plate (Lab Tek II, Thermo Scientific) at a concentration of 2 × 10 ⁵ cells / well and then cultured in a 37 ° C. incubator with 5% CO ₂ for 24 hours. To confirm the cell survival rate, the culture medium of the cultured cells was replaced with PBS medium of pH 6.5 or pH 7.5, and Dox-CPT-PHMSN was treated so that the final concentration of Dox and CPT became 10 g / Lt; / RTI &gt; After incubation, the PBS medium was replaced with fresh medium, the cells were covered with a cover glass (LabTeil II glass chamber coverglass, Thermo Fisher Scientific Inc), and the cells were observed with an Olympus FV1000 fluorescence microscope connected to a CO ₂ incubator. The red fluorescence signal represents the distribution of Dox and the blue fluorescence signal represents the distribution of CPT.

Further, to perform flow cytometry (FACS), the KB cells were seeded into 5 × 10 ⁴ concentration of cells / well in 24-well plates containing a medium of pH 6.5 or PBS pH 7.4. After preincubation for 24 hours, Dox-CPT-PHMSN was treated and cultured for 2 hours. The inflow of intracellular Dox was then analyzed using a FASC analyzer (FACS caliber, BD Bioscience).

As a result, it was confirmed that Dox-CPT-PHMSN was treated at pH 6.5 medium rather than pH 7.4 medium, as shown in FIGS. 9A and 9B, showing a higher level of inflow (FIGS. 9A and 9B) . In particular, among Dox-CPT-PHMSN absorbed into the cells in pH 6.5 medium, Dox that develops red fluorescence is widely distributed in cytoplasm, and CPT that develops blue fluorescence during 1 hour treatment is found to be aggregated in PHMSN . From this, it was confirmed that Dox was released from PHMSN in a pH dependent manner at the initial stage of intracellular entry. The Dox-CPT-PHMSN of the present invention was internalized into the cells, and then the Dox-CPT-PHMSN of the present invention was sequentially internalized And that the drug could be released to kill cancer cells.

In addition, flow cytometry analysis again confirmed the difference in the manner in which nanoparticles were absorbed into KB cells cultured at different pH conditions (FIG. 9c). This indicates that pH-dependent absorption occurs and that the same PHMSN nanoparticles can increase cell entry at pH 6.5 over pH 7.5.

&Lt; 4-2 > PHMSN  Biocompatibility check

It was confirmed that Dox-CPT-PHMSN of the present invention can effectively control drug release according to pH conditions and can be effectively introduced into cells. To confirm the biocompatibility of PHMSN of the present invention, cytotoxicity against KB cells was confirmed Respectively.

Specifically, KB cells were inoculated to a microplate of sterilized 96-well Nunc (Thermo Fisher Scientific Inc.) at a concentration of 5 × 10 ³ cells / well and incubated in a 37 ° C. incubator with 5% CO ₂ for 24 hours Lt; / RTI &gt; After the culture, the medium was replaced with PBS medium of pH 6.5 or pH 7.5, and then the HMSN prepared in Example <1-1>, Dox-CPT-PHMSN, Dox-PHMSN or the like prepared in <Example 2> CPT-PHMSN was treated in each well at various concentrations. After 30 minutes of treatment, the PBS medium was removed, and the medium was replaced with RPM 1640 medium and further cultured for 48 hours. Finally, after the completion of the incubation, the ratio of viable cells to the total inoculated cells was determined by Alamar Blue assay and expressed as the cell viability (%). Fluorescence intensity of each well was measured using a plate reader (Tecan Infinite Series, Germany) with excitation wavelength of 565 nm and emission wavelength of 590 nm.

As a result, as shown in FIG. 10, in the case of the Dox-CPT-PHMSN, Dox-PHMSN and CPT-PHMS treated groups carrying the drug, cell death was increased depending on the PHMSN treatment concentration (FIGS. 10d). On the other hand, in the case of HMSN not carrying the drug, it was confirmed that even if the treatment concentration was increased, there was no significant cell death, and it was confirmed that HMSN had cell compatibility that can be used as a carrier for drug delivery 10a). In addition, even after exchanging fresh media for 30 minutes after the PHMSN carrying the drug, the difference in cell viability after the final (after 48 hours) due to the pH difference of the initial 30 minutes, It was confirmed that the drug-bearing PHMSN of the present invention can exhibit high cytotoxicity at the pH of the tumor (FIGS. 10B to 10D).

PHMSN  Intracellular entry and cytoplasmic release Mechanistic  Confirm

&Lt; 5-1 > Dox- CPT - PHMSN  Intracellular entry Mechanistic  Confirm

Since it was confirmed that the PHMSN of the present invention exhibits effective biocompatibility and can be rapidly introduced into cells, it was attempted to confirm the absorption mechanism of nanoparticles at the cell and intracellular level. After the condition that the surface charge changes according to the pH of the surrounding environment and can be absorbed into the cell, the mechanism of the introduction of the PHMSN into the cell of the present invention is confirmed.

More specifically, the KB cells were seeded at a concentration of 2 × 10 ⁵ cells / well in a cover glass for a single well. Endocytosis dependent on caveolin, a membrane protein involved in the transport of clathrin or protein, a protein involved in water-soluble dependent intracellular uptake; In order to inhibit macropinocytosis, inoculated KB cells were pretreated with 450 mM sucrose or 10 mM methyl-β-cyclodextrin for 30 minutes or treated with 3 mM amiloride for 15 minutes Lt; / RTI &gt; After pre-incubation, KB cells in PBS medium at pH 6.5 were treated with Dox-CPT-PHMSN and cultured for 2 hours in the same manner as in Example <4-1>, and the distribution of Dox was confirmed by fluorescence microscopy Respectively. Alternatively, KB cells were cultured in a PBS medium at pH 6.5 for 1 hour, followed by further culture with fresh medium, followed by further incubation for 2 hours (total of 3 hours), and then fluorescence-labeled cells were quantitated by FACS analysis.

As a result, as shown in FIG. 11, intracellular influx of Dox-CPT-PHMSN was inhibited in KB cells pretreated with sucrose or amiloride, whereas in KB cells pretreated with MβCD (methyl-β-cyclodextrin) It was confirmed that intracellular inflow was maintained (Figs. 11A to 11C). Flow cytometry revealed that the intracellular inflow of Dox was inhibited by sucrose (blue) and amilorin (red) in the presence of the inhibitor, and that Dox was introduced into the cells in the MβCD (green) treatment (Fig. Since sucrose is an inhibitor of clathrin, amiloride is an inhibitor of ovarian cell action, and M? CD is known as a caveolin inhibitor, the PHMSN of the present invention thereby provides clathrin- It was confirmed that it could be introduced into cells through the action of

&Lt; 5-2 > Dox- CPT - PHMSN  Release into the cytoplasm Mechanism  Confirm

The mechanism by which clathrin-dependent intracellular inclusion and domesticated cell action release PHMSN into the cytoplasm was investigated.

Specifically, KB cells were inoculated into sterilized 96-well Nunc microplates at a concentration of 5 × 10 ³ cells / well, and then cultured in a 37 ° C. incubator with 5% CO ₂ for 24 hours. After the culture, the medium was replaced with PBS medium of pH 6.5, and then the HMSN prepared in Example <1-1>, CPT-PHMSN, Dox-PHMSN or Dox-CPT-PHMSN prepared in <Example 2> Were incubated for 1 hour, replaced with fresh medium, and cultured for a total of 20 hours to observe cells with a Confocal Laser Scanning Microscope. Appropriate trackers were added to observe the late endosomes or lysosomes and identified as green fluorescence wavelengths. CPT was identified as the blue fluorescence wavelength and Dox was identified as the red fluorescence wavelength.

As a result, as shown in FIG. 12, the cells treated with CPT-PHMSN and Dox-PHMSN containing the drug alone did not coincide with the position of the green fluorescence signal of the late endosomes and the Dox and red fluorescence, Dox was initially able to escape from the endosomes and released into the cytoplasm. CPT was also released after 8 hours of incubation and secreted into the cytoplasm (Fig. 12A). In addition, similar to endosomes in the lysosomes, the release of Dox appeared in the early stage of culture and CPT was also released from the lysosome after 6 hours of incubation and secreted into the cytoplasm (Fig. 12B).

Also, as shown in Fig. 13, it was confirmed that Dox was released from lysosomes and that CPT was released after 6 hours from the initiation of culture even in the cells treated and cultured with Dox-CPT-PHMSN containing double-drug ). That is, it was confirmed that PHMSN could be introduced into endosomes and lysosomes in the cells and released into the cytoplasm by pH change.

For drug resistant cancer cells, PHMSN  Identify synergy

<6-1> Dox  Resistant to gastric cancer cell lines Dox -CPT- PHMSN  Identify synergy

In cancer cells, cells over-expressing the MDK (midkine) protein show resistance to drugs such as Dox, 5-flurouracil and cisplatin. Therefore, in order to overcome resistance to Dox in MDC overexpressing gastric cancer cells, PHMSN carrying double-drug of Dox-HCl and CPT can be used to effectively exhibit anticancer activity in drug-resistant gastric cancer cells Respectively.

Specifically, a microplate of 96-well Nunc (Thermo Fisher Scientific Inc.) sterilized SNU-620-ADR / 300 cells, which is a gastric cancer cell line resistant to Dox, was inoculated at a concentration of 5 × 10 ³ cells / And then cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After the culture, the medium was replaced with PBS medium of pH 6.5, and free-Dox-HCl, Dox-CPT-PHMSN or Dox-PHMSN prepared in Example 2 was treated in each well. After 6 hours of treatment, the PBS medium was removed, and the medium was replaced with fresh RPM 1640 medium and further cultured for 48 hours. Finally, after the completion of the incubation, the ratio of viable cells to the total inoculated cells was determined by Alamar Blue assay and expressed as the cell viability (%). Fluorescence intensity of each well was measured using a plate reader (Tecan Infinite Series, Germany) with excitation wavelength of 565 nm and emission wavelength of 590 nm.

As a result, as shown in FIG. 14, Dox not carried on drug carriers and Dox carried on PHMSN showed low toxicity to SNU-620-ADR / 300 cells due to drug resistance due to MDK even after culturing for 72 hours In contrast, the PHMSN-treated group carrying the double drug of CPT-Dox showed a significant toxic effect because the cell survival rate was decreased depending on the treatment concentration (FIG. 14A). After 72 hours of incubation, Dox-CPT-PHMSN elicited an IC ₅₀ value of 1.2 μg / ml for the SNU-620-ADR / 300 cell line. This value is lower than the respective IC ₅₀ values of CPT-PHMSN and Dox-PHMSN of 4.2 and 2.1 占 퐂 / ml, so that Dox-CPT-PHMSN shows a significantly increased apoptotic effect on Dox-resistant gastric cancer cells Respectively.

In addition, fluorescence microscopy of the drug delivery system into the cells revealed that the fluorescence intensity of the Dox signal in the cytoplasm gradually increased with the passage of time from 1 hour to 6 hours, and the intensity of the CPT was kept constant (Fig. 14B). These results indicate that Dox - resistant gastric cancer cell lines show significant synergistic effects through dual drug administration of CPT and Dox.

<6-2> P- glycoprotein  Dependent drug resistance In Cancer Cells  Double-drug PHMSN Of cancer cell death by

In cancer cells, it is well known that proteins such as P-glycopeotein or topoisomerase II are overexpressed to show resistance to drugs. Overexpression of P-glycoprotein in cancer cells can release drug to the outside of the cell membrane, thereby reducing drug concentration in the cytoplasm, thereby reducing the anti-tumor effect. Therefore, in a cancer cell line showing tolerance to a multidrug drug, when verapamil hydrochloride (Ver), which is a calcium channel antagonist capable of inhibiting the activity of p-glycoprotein, was administered to cells in the PHMSN of the present invention , And it was confirmed whether or not it could exhibit significant anticancer effect.

Specifically, first, PHMSN carrying Ver and Dox double drug was prepared. 5 mg of the HMSN prepared in Example <1-1> was dispersed in a hydrophobic Dox solution (2.5 mg dissolved in DCM) and stirred at room temperature for 24 hours. After 24 hours, the nanoparticles carrying the drug were obtained by centrifugation, and the supernatant was separately stored and used to confirm drug loading efficiency. The nanoparticles loaded with the drug were vacuum dried, then washed with purified water and stored by freeze drying. The nanoparticles were again suspended in 1 ml of purified water and 2.5 mg of verapamil hydrochloride (Ver) was added to the suspended solution, followed by stirring at room temperature for 24 hours.

Then, 10 mg of the PEG-PDS-DPA copolymer prepared in the above Example <1-2> was added to the polymer gatekeeper to coat the obtained double drug-carrying HMSN with the polymer gatekeeper, and further stirred overnight. To cross-link the surface-wrapped copolymer, dithiothreitol (DTT) was added to the stirred reaction solution and stirred at room temperature for 3 hours. The DTT was added in a molar ratio of 20 mol% to the PDS group constituting the copolymer. The PHMSN carrying the double drug was collected by centrifugation and then washed with phosphate buffer solution of pH 7.4 and distilled water to finally obtain Ver-Dox-PHMSN carrying Ver and Dox.

Then, microflora of 96-well Nunc (Thermo Fisher Scientific Inc.) sterilized cancer cell host MCF-7 / ADR cells having resistance to an anticancer drug was inoculated at a concentration of 5 × 10 ³ cells / well , And cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After the culture, the medium was replaced with PBS medium of pH 6.5, and Ver-CPT-PHMSN; Or Dox-CPT-PHMSN and 5 μM free Ver and cultured for 48 hours. The proportion of viable cells to total inoculated cells was determined by Alamar Blue assay and expressed as the cell viability (%). Fluorescence intensity of each well was measured using a plate reader (Tecan Infinite Series, Germany) with excitation wavelength of 565 nm and emission wavelength of 590 nm.

As a result, as shown in FIG. 15, the loading capacity of Dox and the loading capacity of Ver-Dox-PHMSN were set to 21 wt% and 9 wt%, respectively, so that the loading ratio of Ver and Dox was 1: 2 Respectively. Ver and Dox exhibited high cytotoxicity against MCF7 / ADR cells, and it was confirmed that PHMSN exhibited a killing effect on cancer cells remarkably when compared with that treated with Free Dox (FIG. 15C). In addition, Ver-Dox-PHMSN can be well absorbed in MCF-7 / ADR cells, and Dox concentration in the cytoplasm gradually increased with time (Figs. 15B and 16).

Claims (14)

i) preparing hollow Mesoporous Silica Nanoparticles (HMSN);
ii) a biodegradable copolymer consisting of polyethylene glycol (PEG), pyridyl disulfide (PDS) and 2- (diisopropylamino) ethyl methacrylate (DPAMA) ;
iii) coating the biodegradable copolymer on the surface of HMSN by mixing the HMSN prepared in step i) and the biodegradable copolymer prepared in step ii);
iv) treating the coated HMSN in step iii) with dithiothreitol (DTT) to form a cross-linked copolymer-HMSN (PHMSN) of the biodegradable copolymer; And
v) obtaining the PHMSN formed in step iv).
The method of claim 1, wherein the mixing of step iii) comprises mixing the HMSN and the biodegradable copolymer in a weight ratio of 1: 1 to 1: 4.
The method according to claim 1, wherein the DTT of step iv) is treated in a molar ratio of 20 to 50 mol% with respect to PDS in the biodegradable copolymer.
The method of claim 1, wherein the cross-linking in step iv) is crosslinked at a cross-linking density of 30 to 60 mol%.
A drug delivery vehicle produced by the method of claim 1,
Said drug delivery system comprising a hydrophilic drug selected from the group consisting of doxorubicin, verapamil and pharmaceutically acceptable salts thereof in HMSN; A hydrophobic drug selected from the group consisting of camptothecin and a pharmaceutically acceptable salt thereof; &Lt; / RTI &gt; and mixtures thereof.
delete delete delete 6. The drug delivery vehicle of claim 5, wherein the drug delivery vehicle has a gate keeper layer wherein the biodegradable copolymer cross-linked to the surface swells by electrostatic interaction in the acidic pH range.
10. The drug delivery system according to claim 9, wherein the acidic pH range is in the range of pH 4.0 to 6.5 of the initial tumor tissue.
10. A drug delivery system according to claim 9, wherein the electrostatic interaction occurs as the surface charge becomes zero or more positive.
A pharmaceutical composition for preventing and treating cancer, which comprises the drug carrier of claim 5 as an active ingredient.
The pharmaceutical composition for cancer prevention and treatment according to claim 12, wherein the cancer is any one or more selected from the group consisting of gastric cancer, breast cancer, lung cancer, liver cancer, esophageal cancer and prostate cancer which have resistance to an anticancer agent. Gt;
14. The method of claim 13, wherein the anticancer agent is selected from the group consisting of doxorubicin, cisplatin, verapamil, fluorouracil, oxaliplatin, daunorubicin, irinotecan, topotecan, Wherein the composition is any one selected from the group consisting of paclitaxel, camptothecin, carboplatin, gemcitabine, methotrexalte, docetaxel, Pharmaceutical composition for prevention and treatment of cancer

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