KR102464624B1 - Photo-reactive drug delivery system for combination chemotherapy - Google Patents

Abstract

The present invention relates to a polymer drug delivery system containing a photosensitizer and an anticancer agent for photodynamic therapy. When irradiated with external light, the primary light stimulation local treatment and generated by reactive oxygen species generated from the photosensitizer inside the polymer drug delivery system. There is a synergistic effect of anticancer treatment that enables secondary chemotherapy by selectively releasing anticancer agents by means of functional groups having specific drug release properties by reactive oxygen species.

Description

Photo-reactive drug delivery system for combination chemotherapy

The present invention relates to a polymer drug carrier-photosensitizer complex capable of simultaneous photodynamic therapy and anticancer drug treatment. Specifically, the polymer drug carrier-photosensitizer complex of the present invention is a secondary chemotherapy by modifying an existing photosensitizer. It relates to possible anticancer drugs.

With the recent development of molecular biology technology, it is possible to find various targets applicable to cancer treatment, and based on this, various anticancer therapeutics such as new molecular target therapeutics, immunochemotherapy, and antibody therapy are being developed. However, these single chemotherapeutic agents do not show the excellent cure rate expected in the clinical field, but rather show problems such as drug resistance. to be.

Today, most cancer treatment drugs are designed based on molecular targets related to cancer growth and differentiation. These molecular targeting drugs are expected to be more effective than conventional therapies, but the efficacy of the drugs has not been successful so far due to problems such as genetic heterogeneity and signal complexity of cancer cells.

Recently, attention has been focused on combination therapy as a method for maximizing the therapeutic efficacy of molecularly targeted drugs, and photodynamic therapy (PDT) is being selected as a good candidate for combination therapy.

Photodynamic therapy refers to a technology that uses a photosensitizer to treat incurable diseases such as cancer without surgery. Such photodynamic therapy was tried from around 1400 BC, and active research has been conducted since the early 20th century, and up to now, it is being used in the diagnosis and treatment of cancer, treatment of inflammatory diseases, antibiotics, and skin transplantation. The scope is gradually expanding.

In photodynamic therapy, photosensitizers, which are substances sensitive to light, have almost no cytotoxicity when not exposed to light. Single oxygen or free radicals are generated by chemical reaction by

In addition, unlike anticancer drugs that show severe toxicity not only to cancer cells but also to normal cells, photosensitizers show little cytotoxicity when not exposed to light. It has the advantage that it can be operated with only simple local anesthesia.

Photosensitizers currently used in photodynamic therapy are known, such as porphyrin, bacteriochlorin, phthalocyanine, and 5-aminolevulinic acid derivatives. The pyrrole (cyclic tetrapyrrole) derivative not only selectively accumulates in cancer cells but also exhibits fluorescence or phosphorescence, so it can be used as a reagent for early diagnosis of tumors.

However, the currently used photodynamic therapy cannot be used for bulky tumors due to limited light transmission, phototoxic side effects appear due to slow metabolism of the photosensitizer in the human body, and it is difficult to accumulate in the tumor. There is a problem that the treatment effect cannot be seen.

In order to solve this problem, it is a polymer drug delivery system technology that can perform both photodynamic treatment function and anticancer drug treatment at the same time. Therefore, the technology to design a polymer by chemically modifying an anticancer agent so that the anticancer agent is released is considered the most promising in this field.

Free radicals generated from photosensitizers can be applied not only to cancer treatment but also to stimulatory drug delivery systems. Thio-ether, selenium, thioketal, aminoacrylate, boronic acid-ester, peroxalate-ester and polyproline A linker cleavable by various reactive oxygen species such as (polyproline) is applicable to an active oxygen reactive drug release system.

In general, cancer cells contain a higher concentration of reactive oxygen species than normal cells, and the linker is cleaved in response to cancer cell-specific reactive oxygen species, and thus can be designed to release the linked drug. The ability of cancer cells to scavenging free radicals is higher than that of normal cells, and free radicals are effectively eliminated at toxic concentrations for cell survival.

On the other hand, normal cells show a high concentration of free radicals under certain conditions, such as infection or diabetes. Therefore, the local photodynamic therapy, which can dramatically increase the local active oxygen concentration, is expected to enable effective cancer removal through reactive oxygen species reactivity and cancer-specific drug release.

Referring to the prior art,

A prior paper (Biomaterials., 34, 2013, 6454-6463) discloses a chitosan polymer bound with a photosensitizer, and this polymer has a chitosan moiety and pheophorbide A (PheoA) used as a photosensitizer part is disclosed.

Meanwhile, as the polymer drug delivery system of the present invention, chitosan-polyhydroxyl methacrylate particles containing a photosensitizer (Pheophorbide A; PheoA) and boronic acid-deoxyfluorosidine were used.

More specifically,

When near-infrared rays are irradiated using chitosan polymer as a basic skeleton and PheoA as a photosensitizer, it induces apoptosis through reactive oxygen species (ROS) activated in PheoA, thereby preventing and treating cancer. , There are some structural differences due to the inclusion of boronic acid-deoxyfluorocytidine conjugates,

The polymer of the present invention is characterized in that it has a synergistic effect through secondary chemotherapy by selectively releasing an anticancer agent as well as a primary light stimulation local treatment by reactive oxygen species generated from a photosensitizer.

The polymers of the preceding papers do not have these characteristics, and there is a difference in the effects of the invention.

An object of the present invention is to provide a complex having the function of simultaneously performing primary photodynamic therapy and secondary anticancer drug therapy.

In order to achieve the above object,

One aspect of the present invention is a main chain formed of a biocompatible polymer;

a photosensitizer bound to the main chain; and

It provides a complex for photodynamic (photodynamic) and chemical (chemo) treatment of cancer, including; an anticancer agent bound to the main chain.

The polymer drug delivery system containing the photosensitizer and anticancer agent for photodynamic therapy according to the present invention is primary light stimulation local treatment by active oxygen species generated from the photosensitizer inside the polymer drug delivery system when irradiated with external light and active oxygen generated There is a synergistic effect of anticancer treatment that enables secondary chemotherapy by selectively releasing anticancer agents by means of functional groups having specific drug release characteristics.

In FIG. 1, A is a scheme showing the process of synthesizing HEMA-PheoA, B is a scheme showing the process of synthesizing PBA/DFCR, and C is a scheme showing the process of synthesizing CSPM-PheoA-DFCR.
In FIG. 2, A is a result confirming that a broad absorption spectrum appears by the CSPM microgel when only CSPM is present in water, and B is a strong fluorescence emission signal in the range of 600-800 nm when CSPM-PheoA particles are present in water. This is the result of confirming that a specific absorption peak caused by PheoA occurs in the CSPM microgel.
3 is a TEM image of a CSPM-PheoA-DFCR microgel.
Figure 4 is the result of analyzing the green fluorescence emitted by the amine through a fluorescence microscope using a green fluorescent dye called NHS activated Oregon green to measure the amine residue of chitosan in CSPM-PheoA particles, and It is an image showing the result of analyzing the red fluorescence emitted by PheoA.
5A is a photograph and figure showing the average size of CSPM particles measured by a dynamic light scattering photometer (DLS) and an average dry diameter measured by TEM, and B is a CSPM-PheoA measured by a dynamic light scattering photometer (DLS). Photographs and drawings showing the average particle size and the average dry diameter measured by TEM, and C is a graph showing the size change of CSPM and CSPM-PheoA before and after microgel injection into the tumor.
6 is a graph showing the fluorescence spectrum of the DMA reacted by the generation of reactive oxygen species according to the near-infrared irradiation time.
7 is a graph comparing the fluorescence spectra of DMA with and without microgels.
8 is a graph comparing the amount of drug released from the CSPM-PheoA-DFCR microgel by active oxygen when irradiated with near-infrared rays and when not irradiated.
In FIG. 9, A is a graph comparing cell activity according to the concentration of CSPM and CSPM-PheoA microgels, and B is a graph comparing CSPM-PheoA and CSPM-PheoA-DFCR with or without near-infrared irradiation. It is a graph comparing cell activity.
In FIG. 10, A shows a microscope image of PANC-1 cells exposed to near-infrared rays after treatment with different microgels, and B is a graph comparing the relative fluorescence intensity in cells after treatment with different microgels.
In FIG. 11, A is a graph comparing the volume of the tumor with and without near-infrared irradiation when different microgels are administered to a CT-26 tumor of a mouse, B is a graph comparing the weight of the mouse, C is a photograph comparing H&E-stained tumor fragments, and D is a photograph comparing TUNEL-stained tumor fragments.
12 shows that when the CSPM-PheoA-DFCR microgel provided in one aspect of the present invention is used for cancer treatment, photodynamic, chemical, and local (locoregional) combination treatment is possible. It is a schematic diagram
13 is a schematic diagram illustrating a process in which an anticancer agent is released as light is irradiated to the CSPM-PheoA-DFCR microgel provided in one aspect of the present invention.

Hereinafter, the present invention will be described in detail.

On the other hand, the embodiment of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided in order to more completely explain the present invention to those of ordinary skill in the art.

Furthermore, "including" a certain element throughout the specification means that other elements may be further included, rather than excluding other elements, unless otherwise stated.

One aspect of the present invention is a main chain formed of a biocompatible polymer;

a photosensitizer bound to the main chain; and

It provides a complex for photodynamic (photodynamic) and chemical (chemo) treatment of cancer, including; an anticancer agent bound to the main chain.

At this time, the biocompatible polymer is, chitosan (chitosan), glycol chitosan (glycolchitosan), poly-L-lysine (poly-L-lysine), or poly (ethylene glycol) (poly (ethyleneglycol)) can be have.

In addition, the photosensitizer generates active oxygen when exposed to light,

Porphyrins, phthalocyanines, 5-aminolevulinic acid derivatives, cyclic tetrapyrrole derivatives, chlorins, bacteriochlorins, or It may be one selected from porphycenes.

Furthermore, the porphyrins photosensitizer,

One, or two or more selected from the group consisting of hematoporphyrin, protoporphyrin IX, talaporfin, pheophorbide A, and chlorin e6 It can be a combination.

Preferably, it may be a photosensitizer represented by the following formula (a).

[Formula a]

In addition, the active oxygen generated by exposure of the photosensitizer to light,

It may be to release the anticancer agent bound to the main chain formed of a biocompatible polymer, in which case the release of the anticancer agent is, as the anticancer agent bound to the main chain through a linker cleaved by active oxygen, the linker is cleaved by active oxygen It is preferred to release.

The linker cleaved by the active oxygen,

thio-ether, selenium, thioketal, aminoacrylate, boronic acid-ester, peroxalate-ester, Or it may be one selected from polyproline (polyproline).

In this case, the boronic acid in the boronic acid-ester is,

4-hydroxyphenylboronic acid, 4- (hydroxymethyl) phenylboronic acid (4- (hydroxymethyl) phenylboronic acid), 2-hydroxyphenylboronic acid (2-hydroxyphenylboronic acid), (3 -t-Butyl-5-methylphenyl) boronic acid ((3-t-butyl-5-methylphenyl) boronic acid), 2-hydroxy-3-methylphenylboronic acid (2-hydroxy-3-methylphenylboronic acid), 6- Hydroxypyridine-2-boronic acid (6-hydroxypyridine-2-boronic acid), 6- (hydroxymethyl) pyridine-3-boronic acid (6- (hydroxymethyl) pyridine-3-boronic acid), [3- ( 3-hydroxypropyl) phenyl] boronic acid ([3- (3-hydroxypropyl) phenyl] boronic acid), or 5- (hydroxymethyl) furan-2-boronic acid (5-hydroxymethyl) furan-2-boronic acid ) may be selected from

Preferably, it may be a boronic acid represented by the following formula (b).

[Formula b]

The anticancer agent may include a dihydroxyl functional group, and specifically, capecitabine, deoxyfluorocytidine (5'-deoxy-5-fluorocytidine; 5'-DFCR), and doxyfluridine (doxifluridine; 5-DFUR) may be one selected from the group consisting of, or a combination of two or more.

Preferably, it may be an anticancer agent (drug) represented by the following formula (c).

[Formula c]

The photosensitizer is preferably introduced into a main chain formed of a biocompatible polymer after a porphyrin-like compound having a carboxyl functional group and hydroxyethyl methacrylate (HEMA) are combined through an esterification reaction.

In the most preferred aspect,

The complex for photodynamic and chemical treatment of cancer is a high molecular compound represented by the following formula (A).

[Formula A]

In the above formula (A),

m and n are present in a molar ratio of 1:10 to 1000,

a + (bc) + (cd) + d is a + b, a + b is represented by the following formula B and is defined by chitosan having a weight average molecular weight (M _w ) in the range of 10,000 to 500,000:

[Formula B]

.

.

In another aspect, the weight average molecular weight (M _w ) of the chitosan may be in the range of 20,000 to 500,000, may be in the range of 30,000 to 500,000, may be in the range of 40,000 to 500,000, may be in the range of 45,000 to 500,000, and may be in the range of 50,000 to It can range from 500,000, it can be from 10,000 to 400,000, it can be from 10,000 to 300,000, it can be from 10,000 to 250,000, it can be from 10,000 to 200,000, it can be from 10,000 to 190,000, it can be from 20,000 to 400,000 may be in the range of 30,000 to 300,000, may be in the range of 40,000 to 200,000, and may be in the range of 50,000 to 190,000.

In the chitosan represented by Formula B,

The molar ratio of a and b may be in the range of 1:0.001 to 1000, but is not particularly limited thereto.

In addition, the complex for photodynamic and chemical treatment of cancer may be in the form of a spherical gel having an average diameter in the range of 100 to 2,000 nm, and the spherical gel is in the form of a core-shell type microgel, and the core has a light The sensitizer may be located, and the anticancer agent may be located in the shell. In this case, the average diameter may be in the range of 100 to 2,000 nm, may be in the range of 200 to 2,000 nm, may be in the range of 300 to 2,000 nm, may be in the range of 400 to 2,000 nm, and may be in the range of 500 to 2,000 nm. , 100 to 1800 nm, 100 to 1,600 nm, 100 to 1,400 nm, 100 to 1,300 nm, 200 to 1800 nm, 300 to 1,600 nm may be in the range, may be in the range of 400 to 1,400 nm, may be in the range of 500 to 1,300 nm.

The polymer drug delivery system containing the photosensitizer and anticancer agent for photodynamic therapy according to the present invention is primary light stimulation local treatment by active oxygen species generated from the photosensitizer inside the polymer drug delivery system when irradiated with external light and active oxygen generated There is a synergistic effect of anticancer treatment that enables secondary chemotherapy by selectively releasing an anticancer agent by a functional group having a specific drug release characteristic, which is directly supported by the experimental examples described below.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples.

However, the Examples and Experimental Examples described below are merely illustrative of the present invention in one aspect, and the present invention is not limited thereto.

<Experimental Example 1> Synthesis of CSPM-PheoA-DFCR microgel

The following experiments were performed for the synthesis of a polymer drug delivery system containing a photosensitizer and an anticancer agent for photodynamic therapy.

1-1: Synthesis of poly-HEMA/chitosan microgel particle (CSPM) microgel

First, put distilled water containing 1 wt% acetic acid in a 100 ml three-necked round-bottom flask, and then put 5 mg/ml of 50 ml of chitosan (chitosan; molecular weight: 50000-190000, > 75% deacetylation) solution, then N ₂ , react vigorously for 30 minutes under a temperature of 80°C in an atmosphere. Add 1 ml of 2-hydroxyethyl methacrylate (HEMA) monomer and 1 ml of 5 mM tert-butyl hydroperoxide (TBHP) aqueous solution to the flask. The polymerization reaction proceeds for about 2 hours, and the synthesized CSPM microgel is purified by dialysis (MWCO 15K, Spectrum Lab, Rancho Dominguz) for 2 days, and freeze-dried.

CSPM: Chitosan Microgel

1-2: Synthesis step of CSPM-pheophrobide A (PheoA) microgel

To link CSPM and PheoA, HEMA was used as a core-forming and crosslinking agent. PheoA's carboxyl group is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; EDC) and 4-dimethylaminopyridine (4-dimethylaminopyridine; DMAP) Reacts with the hydroxyl group of the HEMA monomer for ester bonding in the presence of

50 mg of PheoA at a concentration of 84.4 μmol is dissolved in 10 ml of methanol, and 51 mg of EDC at a concentration of 270 μmol and 33 mg of DMAP at a concentration of 270 μmol are added to the PheoA solution at room temperature. Then, 1.4 mmol of HEMA monomer is added and reacted in the dark for 24 hours. After drying the above-reacted material in a vacuum, the HEMA monomer to which PheoA is connected is washed several times with distilled water, collected through centrifugation, and freeze-dried. The results are shown in Fig. 1.A.

Figure 1.A. is a diagram showing the process of synthesizing HEMA-PheoA.

The prepared HEMA-PheoA monomer is reacted with chitosan for the synthesis of CSPM-PheoA. HEMA monomer and HEMA-PheoA monomer mixture (HEMA 1 g, HEMA-PheoA 16 mg, HEMA : HEMA-PheoA = 1 : 0.002 M ratio) was added to 250 mg of chitosan solution, 80 °C in the presence of TBHP, N ₂ The reaction was carried out in the atmosphere for 2 hours. The synthesized CSPM-PheoA is purified by dialysis and freeze-dried. The PheoA content was found to be 0.14 wt% through UV-vis spectroscopy.

1-3: Synthesis step of CSPM-PheoA-5'-deoxy-5-fluorocytidine (5'-deoxy-5-fluorocytidine; DFCR) microgel

DFCR is mixed with phenylboronic acid for the synthesis of phenylboronic ester, which can be separated by active oxygen. Then, 735 mg of DFCR at a concentration of 3 mmol was reacted with 452 mg of 4-(hydroxymethyl)phenylboronic acid (PBA) at a concentration of 3 mmol in 100 ml of DMSO anhydrous. The prepared PBA/DFCR complex was ligated with the CSPM-PheoA microgel without further purification. To synthesize the CSPM-PheoA-DFCR complex, 8 mg of dried CSPM-PheoA was dissolved in 20 ml of DMSO anhydrous containing 73 mg of 450 μmol of carbonyldiimidazole (CDI), and 10 ml of PBA/DFCR is added slowly. After reaction in the dark, the synthesized mixture is purified through dialysis. The results are shown in FIGS. 1.B. and 1.C.

Figure 1.B. is a diagram showing the process of synthesizing PBA / DFCR,

Figure 1.C. is a diagram showing the process of synthesizing CSPM-PheoA-DFCR.

1-4: Structural analysis result of the synthesized CSPM-PheoA-DFCR microgel

¹ H NMR results of purified CSPM-PheoA-DFCR were measured.

The chitosan polymer alone produces a specific peak due to the acetyl group at 2 ppm (3H (1)). CSPM-PheoA produces specific peaks at 2.4 ppm (3H (2)), 1.1 ppm (3H (3)), and a small multiplet (3H (4,5)) due to the methyl group of PheoA linked to HEMA at 1.4-1.9 ppm. ) to make CSPM-PheoA-DFCR was 8.1 ppm (H(6)) due to the NH group of the carbamate group, 5.1 ppm (2H(7)) due to the methyl group of PBA, and 7.7 ppm (H(8)) due to the aromatic ring of PBA. )), which shows a specific peak at 7.5 ppm (H(9)) due to the DFCR moiety. The amount of DFCR linked to CSPM-PheoA-DFCR was measured by summing the peaks of 1.1 ppm (3H(3), PheoA) and 8.1 ppm (H(6), carbamate NH group) in ¹ H NMR spectrum, and CSPM-PheoA micro Calculated as approximately 33 mol% PheoA in the gel.

HEMA-PheoA was analyzed by FT-IR, and a specific peak was observed at 1735.8 cm ^-1 due to the C=O double bond and 1153.9 cm ^-1 due to the COC bond.

The absorption/emission spectra of CSPM-PheoA-DFCR microgels dispersed in water were measured by UV-vis spectroscopy. The results are shown in FIG. 2 .

As shown in Fig. 2.A., when only CSPM is present in water, a broad absorption spectrum generated by the CSPM microgel appears, but an emission spectrum does not appear,

As shown in FIG. 2.B., when CSPM-PheoA particles were present in water, a strong fluorescence emission signal was generated in the range of 600-800 nm, and a specific absorption peak caused by PheoA occurred in the CSPM microgel.

The TEM image of the CSPM-PheoA-DFCR microgel dispersed in water was observed, and the results are shown in FIG. 3 .

As shown in FIG. 3 , the TEM image of the CSPM-PheoA-DFCR microgel clearly shows the poly-HEMA core structure of the particle and the chitosan shell structure stained black at ~50 nm.

In order to measure the amine residues of chitosan in CSPM-PheoA particles, green fluorescence emitted by amines was analyzed through a fluorescence microscope using a green fluorescent dye called NHS activated Oregon green. In addition, the red fluorescence emitted by PheoA linked to poly-HEMA was analyzed. The results are shown in FIG. 4 .

As shown in FIG. 4 , particles of about 1 μm in size were seen by Oregon green and Red PheoA dyes in the same area through the fluorescence microscope image.

<Experimental Example 2> Colloidal stability of CSPM microgel

In order to evaluate the colloidal stability of CSPM, the hydrodynamic size of CSPM changing with time in 10% FBS containing water, PBS, and DMEM medium was measured using a dynamic light scattering spectrophotometer (DLS). And the shape of the particles incubated for 24 hours was confirmed through TEM.

There was no change in the hydrodynamic size of CSPM in water and PBS, but the particle size in DMEM medium increased with time.

In water, CSPM was observed to be simply dispersed in a spherical shape. On the other hand, phosphate in PBS was absorbed on the particle surface, which was observed only in a dry state through low-magnification TEM, but no aggregation or precipitation of microgels was found. In addition, in the case of DMEM, CSPM particles showed aggregation with time due to the interaction of CSPM with various proteins.

In water, CSPM and CSPM-PheoA microgels form a stable turbid colloidal suspension without aggregation, and CSPM-PheoA exhibits red fluorescence when exposed to ultraviolet (wavelength = 365 nm). The water absorption and swelling characteristics suggest that CSPM microgels can be injected directly into the tumor and can take advantage of the drug release generated in part by PDT.

<Experimental Example 3> Expansion of CSPM-PheoA-DFCR microgel

The average size of the CSPM particles measured by dynamic light scattering spectroscopy (DLS) was 1309±306 nm, and the average dry diameter measured by TEM was 511±59 nm. The average particle size of CSPM-PheoA was 1214±320 nm (DLS) and 332±30 nm (TEM), which was not changed by binding between CSPM-PheoA and DFCR. These results show that the particle size is highly dependent on the water content. The results are shown in FIG. 5 .

Figure 5.A. is a photograph and figure showing the average size of CSPM particles measured by dynamic light scattering spectroscopy (DLS) and the average dry diameter measured by TEM.

Figure 5.B. is a photograph and figure showing the average size of CSPM-PheoA particles measured by dynamic light scattering photometer (DLS) and the average dry diameter measured by TEM.

The volumetric expansion rate of the particles was calculated using the following equation.

Volumetric expansion rate = V _wet /V _dry = r _wet ³ /r _dry ³

(V _wet : wet sample volume, V _dry : dry sample volume, r _wet : wet particle radius as determined by DLS, r _dry : dried particle radius as determined by TEM)

It can be seen that the volumetric expansion rate of CSPM-PheoA is much larger than that of CSPM (48.9 vs 16.8). Furthermore, we observed that the particle size of CSPM-PheoA was smaller than that of CSPM because of the hydrophobicity of PheoA. The results are shown in Fig. 5.C.

Figure 5.C. is a graph showing the size change of CSPM and CSPM-PheoA before and after the microgel is injected into the tumor.

The effectiveness of local cancer treatment using drug-eluting hydrogels is affected by the physical properties of hydrogels. In general, swelling of the microgel after topical injection promotes the release of the drug and raises the concentration of the drug.

<Experimental Example 4> Generation of active oxygen generated by near-infrared irradiation

1-1: Experimental method

In order to measure singlet oxygen generated by CSPM-PheoA due to near-infrared irradiation, 9,10-dimethylanthracene (DMA) emitting fluorescence was used. DMA is a well-known blue fluorescent dye sensitive to singlet oxygen, which can be used for the detection of singlet oxygen through fluorescence quenching by the formation of endoperoxide anthracene. DMA is added to 4.5 mg/ml of CSPM-PheoA in DMSO and immediately exposed to a near-infrared laser (λ = 671 nm, 167 mW/cm ² ).

After irradiating near infrared rays using a microplate reader (Infinite 200 pro, Tecan, Mannedorf, Switzerland) for different times (t = 0, 1, 2, 3, 4, 5, 10, 30 minutes), λ _max = 420 nm The degree of fluorescence of DMA was measured.

1-2: CSPM-PheoA-DFCR Microgel generates reactive oxygen species generated by near-infrared rays

For the measurement of singlet oxygen generated by the synthesized CSPM-PheoA-DFCR complex, DMA with a final concentration of 100 μM was added to the sample, and a near-infrared laser (671 nm, 167 mW/cm ² ) was applied to the sample for 1 to 30 minutes. After irradiation, DMA fluorescence intensity was measured using a fluorescence analyzer. In DMA (380-600 nm) and CSPM-PheoA-DFCR microgels, the fluorescence intensity of PheoA (600-800 nm) gradually decreased with increasing exposure time. The results are shown in FIG. 6 .

6 is a graph showing the fluorescence spectrum of the DMA reacted by the generation of reactive oxygen species according to the near-infrared irradiation time.

When only the DMA solution without microgel was exposed to near-infrared light for the same time, there was no change in fluorescence intensity. Looking at the graph showing the fluorescence quenching of DMA with respect to the near-infrared irradiation time, when the microgel was exposed to near-infrared light at a constant light intensity, the amount of singlet oxygen produced increased with time, and when the greatest amount of singlet oxygen was produced, 10 minutes had elapsed since saturation began. In the absence of microgel, fluorescence quenching of DMA did not appear even when irradiated with near-infrared rays, indicating that singlet oxygen was generated by PheoA in the microgel, and the singlet oxygen generation efficiency of CSPM-PheoA-DFCR was 100 J/ It shows the highest when the energy is larger than cm ² . The results are shown in FIG. 7 .

7 is a graph comparing the fluorescence spectra of DMA with and without microgels.

<Experimental Example 5> DFCR emitted by near-infrared irradiation

1-1: Experimental method

In order to confirm the release characteristics of the drug by reactive oxygen species in the microgel, PBA (phenylboronic acid) was used to connect the microgel and DFCR. The complex is synthesized by the boronic acid ester bond between PBA and DFCR, and the amine group of D-glucosamine in chitosan and 4-hydroxymethyl in the PBA-DFCR complex to form an ester bond. ) to form a CSPM-PheoA complex through the reaction between the groups. CSPM-PheoA-DFCR was purified by dialysis in the presence of DMSO and used for drug release experiments by light.

The amount of DFCR released from CSPM-PheoA-DFCR by active oxygen was measured through HPLC. 5 mg/ml of microgel samples were exposed to near-infrared (λ = 671 nm, 167 mW/cm ² , ) for different times (t = 0, 1, 2, 3, 4, 5, 10, 30 min), It was precipitated by centrifugation at 13000 rpm for 30 minutes. DFCR floating on the upper layer had a column size of 4.6 X 160 mm, a particle size of 5 μm (GL Science, Japan), an injection volume of 10 μl, a distilled water/methanol buffer solution (8:2, v/v), and an absorption wavelength of 220 nm. It was measured by HPLC (Alliance 2695 equipped with a 2487 detector; Waters, MA, USA).

1-2: Release of DFCR by reactive oxygen species in CSPM-PheoA-DFCR microgels

Near infrared rays initiated DFCR emission in the CSPM-PheoA-DFCR microgel, and the emission of DFCR was maximized when near infrared rays were irradiated for 10 minutes. In the case of not irradiating near-infrared rays, no trace of DFCR emission could be seen. These results suggest that the release of DFCR from CSPM-PheoA-DFCR under near-infrared irradiation coincides with the generation of singlet oxygen. The results are shown in FIG. 8 .

8 is a graph comparing the amount of drug released from the CSPM-PheoA-DFCR microgel by active oxygen when irradiated with near-infrared rays and when not irradiated.

To measure the effects of reactive oxygen species and H ₂ O ₂ generated in the microgel under near-infrared irradiation, cell imaging studies were conducted in the presence of DCFDA (2',7'-dichlorofluorescine diacetate). DCFDA is a fluorescent indicator and shows strong fluorescence in the oxidized state. In the case of CT-26 cells themselves and cells treated with CSPM-PheoA-DFCR without near-infrared irradiation, cytotoxicity and DCFDA fluorescence were not observed. However, in the case of cells treated with CSPM-PheoA-DFCR exposed to near-infrared or H ₂ O ₂ , the cell population decreased, and DFCR fluorescence increased in the cells. These results indicate that cytotoxicity is caused by reactive oxygen species or H ₂ O ₂ under near-infrared irradiation.

<Experimental Example 6> Cytotoxicity evaluation

1-1: Cell Conditions

The human pancreatic cancer cell line PANC-1 and the murine colon cancer cell line CT-26 are cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS, 1% penicillin, and 1% streptomycin, which are fetal bovine serum.

1-2: Cytotoxicity of CSPM-PheoA-DFCR microgels when active oxygen and DFCR are combined

To investigate the cytotoxicity of microgels, a water-soluble tetrazolium (WST) assay is used. Cells (4 x 10 ⁴ per well) are cultured in a 94-well plate, incubated at 37 ° C., 5% CO ₂ in an incubator for 24 hours, and then DFCR, CSPM, or CSPM-PheoA is added at different concentrations. . After incubation for 24 hours, 48 hours, and 72 hours, WST-1 is added to each well and incubated for 4 hours. Cell viability was analyzed by measuring the degree of light absorption using a microplate reader (Infinite 200 pro, Tecan, Mannedorf, Switzerland). To measure the PDT effect, cells in 5% DMSO medium were reacted with CSPM-PheoA or CSPM-PheoA-DFCR in DMSO with a final concentration of 65 μg/ml, followed by a near-infrared laser (λ=671 nm, 100 J/ml). cm ² ) after irradiating for 2 hours, the viability of cells was evaluated using WST-1.

To determine the potential therapeutic effect of CSPM-PheoA-DFCR, PANC-1 cancer cells were used to investigate cytotoxicity. When cells were incubated with CSPM or CSPM-PheoA microgels in the concentration range of 0.35-700 μg/ml for 3 days, no specific cytotoxicity was observed. Similarly, no toxicity was observed in CSPM-PheoA-DFCR as well.

To investigate the cytotoxicity of microgels exposed to near-infrared rays, PANC-1 cells were treated with CSPM-PheoA or CSPM-PheoA-DFCR, and then light was irradiated. In the absence of microgels, cells were not affected by near-infrared radiation even when irradiated with near-infrared rays, but when treated with CSPM-PheoA or CSPM-PheoA-DFCR, a sharp decrease in cell activity was observed (< 20%). According to these results, it can be seen that the cytotoxic effect of CSPM-PheoA-DFCR is slightly greater than that of CSPM-PheoA. The synthesized CSPM microgel was expected to have a very large size (~1 μm in water) and to have low cell uptake and active oxygen diffusion efficiency. However, the PDT effect of CSPM-PheoA and CSPM-PheoA-DFCR was >80% in cytotoxicity. These results suggest that the reactive oxygen species generated by PheoA in the microgel affect cells despite limitations such as short half-life and short diffusion distance. These results also suggest that the amine-rich chitosan in microgels exhibits a positive charge and can bind more effectively with the negatively charged cell membrane through the results of increased diffusion and toxicity of active oxygen in the cell. The results are shown in FIG. 9 .

9.A. is a graph comparing cell activity according to the concentration of CSPM and CSPM-PheoA microgel.

Figure 9.B. is a graph comparing the cell activity according to the presence or absence of CSPM-PheoA and CSPM-PheoA-DFCR when irradiated with near-infrared rays and when not irradiated.

1-3: Cell Imaging

We measure the cytotoxic effect of reactive oxygen species generated from microgels by near-infrared rays through cell imaging. In 0.5 ml DNEM, PANC-1 cells (50000 cells/well) are cultured, and incubated for 24 hours at a temperature of 37° C. and 5% CO ₂ atmosphere. Add the same amount of CSPM, CSPM-PheoA, or CSPM-PheoA-DFCR microgel to DMSO (25 μl, 4.6 mg/ml), and incubate for 2 hours after adding this to the cells. Then, after exposure to near-infrared rays for 10 minutes, incubation is performed for another 2 hours. Then, it is stained with nuclear staining (ReadyProbes cell viability imaging kit, Blue/Green, Thermo Fisher), and washed 3 times with PBS buffer. It was fixed with 4% paraformaldehyde, and images of cells were acquired using a confocal microscope (FV1000, Olympus).

Untreated and CSPM microgel-treated PANC-1 cells showed weak green nuclear fluorescence after near-infrared irradiation, but cells treated with CSPM-PheoA and CSPM-PheoA-DFCR showed strong green fluorescence. Fewer cells could be seen in the region of interest (512x512 pixel) due to the separation of dead cells from the glass bottom. As a result of measuring the fluorescence image using ImageJ, it was found that the degree of green fluorescence in CSPM-PheoA-DFCR was statistically larger than in CSPM-PheoA. These results were shown to be consistent with those in cytotoxicity experiments. The results are shown in FIG. 10 .

Figure 10.A. shows a microscopic image of PANC-1 cells exposed to near-infrared light after treatment with different microgels.

10.B. shows a graph comparing the relative fluorescence intensities in cells after treatment with different microgels.

<Experimental Example 7> IN VIVO experiment

1-1: Experimental method

15-20 g of BALB/c mice with free access to water and food were placed at 25°C, followed by a 12-hour day and night cycle. Experiments using all animals were conducted in accordance with the guidelines for animal experiments made by the Institute of Life Sciences, Inha University.

Murine colon cancer CT-26 cells (2.5x10 ⁶ ) were injected into the back of BALB/c mice. When the initial tumor size reached 100 mm ³ , the mice were separated into 4 groups, and an amount of 3 microgels (CSPM, CSPM-PheoA, CSPM-PheoA-DFCR) or PBS 10 mg/kg was applied directly to the tumor. injected. And the tumor on the left side was irradiated with a near-infrared laser (λ = 671 nm, 100 J/cm ² ) once a day for 3 days. Initial tumor size and weight were measured daily for 2 weeks. Resected tumors were analyzed using hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated neck end label (TUNEL) staining.

1-2: Inhibition of colorectal cancer growth through cancer therapy

When NIR was exposed to CSPM-PheoA and CSPM-PheoA-DFCR, tumor growth was effectively inhibited, and no change in body weight was observed. In particular, when the tumor was irradiated with CSPM-PheoA-DFCR and near-infrared rays, it was found to be more effective in inhibiting tumor growth after 2 weeks compared to CSPM-PheoA.

Additionally, hematoxylin-eosin (H&E) staining of tumor slices and terminal deoxynucleotidyl transferase-mediated neck end label (TUNEL) staining were performed. This shows that when CSPM-PheoA or CSPM-PheoA-DFCR was irradiated with near-infrared rays, higher cancer cell apoptosis and necrosis were seen compared to other groups such as PBS or CSPM. The results are shown in FIG. 11 .

11.A. is a graph comparing the volume of tumors when different microgels were administered to CT-26 tumors of mice, when irradiated with near-infrared rays and when not irradiated.

11.B. is a graph comparing the body weight of mice when different microgels were administered to CT-26 tumors in mice, and when near-infrared rays were irradiated and when not irradiated.

11.C. is a photograph comparing H&E-stained tumor fragments when different microgels were administered to mouse CT-26 tumors and irradiated with near-infrared rays.

11.D. is a photograph comparing TUNEL-stained tumor fragments when different microgels were administered to CT-26 tumors of mice and irradiated with near-infrared rays.

In general, the synergistic effect of combination therapy results from multiple inhibition of cancer molecules or oncogenes. In this study, we investigated the combination of the effects of two functional drugs (DFCR for inhibition of DNA synthesis and PheoA for free radical-induced cancer cell apoptosis) using a microgel delivery system with a mechanism emitted by near-infrared rays. did. And, when CSPM-PheoA-DFCR was exposed to near-infrared rays, it showed strong cancer cell inhibition both in vivo and in vitro. In all cases, a synergistic effect of PDT and chemotherapy could be demonstrated.

Claims (15)

a main chain formed of a biocompatible polymer;
a photosensitizer bound to the main chain; and
an anticancer agent bound to the main chain; A polymer complex for cancer treatment comprising a
The biocompatible polymer is chitosan (chitosan), glycol chitosan (glycolchitosan), poly-L-lysine (poly-L-lysine), or poly (ethylene glycol) (poly (ethyleneglycol)),
The photosensitizer generates active oxygen when exposed to light, and the photosensitizer is a porphyrin-like compound having a carboxyl functional group after hydroxyethyl methacrylate (HEMA) is combined through an esterification reaction, It is introduced into the main chain formed of a biocompatible polymer,
The anticancer agent includes a dihydroxyl functional group, and the anticancer agent is a polymer for cancer treatment, characterized in that the anticancer agent is released only under light exposure by binding to the main chain through a linker cleaved by active oxygen generated by the photosensitizer. complex.
According to claim 1,
The porphyrins compound is selected from the group consisting of hematoporphyrin, protoporphyrin IX, talaporfin, pheophorbide A and chlorin e6. A polymer complex for cancer treatment, characterized in that it is one type, or a combination of two or more types.
According to claim 1,
Active oxygen generated by exposure of the photosensitizer to light is a polymer complex for cancer treatment, characterized in that it releases the anticancer agent bound to the main chain formed of the biocompatible polymer.
According to claim 1,
The anticancer agent is a polymer complex for cancer treatment, characterized in that it is released as the linker is cleaved by active oxygen.
According to claim 1,
The linker cleaved by the active oxygen is thio-ether, selenium, thioketal, aminoacrylate, boronic acid-ester, peroxal Late-ester (peroxalate-ester), or polyproline (polyproline), characterized in that selected from, cancer treatment polymer complex.
6. The method of claim 5,
In the boronic acid-ester, boronic acid is 4-hydroxyphenylboronic acid, 4-(hydroxymethyl)phenylboronic acid, 2-hydroxyphenylboronic acid ( 2-hydroxyphenylboronic acid), (3-t-butyl-5-methylphenyl)boronic acid ((3-t-butyl-5-methylphenyl)boronic acid), 2-hydroxy-3-methylphenylboronic acid (2-hydroxy- 3-methylphenylboronic acid), 6-hydroxypyridine-2-boronic acid, 6-(hydroxymethyl)pyridine-3-boronic acid (6-(hydroxymethyl)pyridine-3- boronic acid), [3- (3-hydroxypropyl) phenyl] boronic acid ([3- (3-hydroxypropyl) phenyl] boronic acid), or 5- (hydroxymethyl) furan-2-boronic acid (5- hydroxymethyl) furan-2-boronic acid), characterized in that selected from, cancer treatment polymer complex.
According to claim 1,
1 selected from the group consisting of capecitabine, deoxyfluorocytidine (5'-deoxy-5-fluorocytidine;5'-DFCR) and doxifluridine (5-DFUR) A polymer complex for cancer treatment, characterized in that it is a species, or a combination of two or more.
According to claim 1,
The complex for photodynamic and chemical treatment of cancer is a polymer complex for cancer treatment, characterized in that it comprises a polymer compound represented by the following formula (A):
[Formula A]

In the above formula (A),
m and n are present in a molar ratio of 1:10 to 1000,
a + (bc) + (cd) + d is a + b, a + b is represented by the following formula B and is defined by chitosan having a weight average molecular weight (M _w ) in the range of 10,000 to 500,000:
[Formula B]

.
9. The method of claim 8,
In the chitosan represented by Formula B,
The molar ratio of a and b is 1:0.001 to 1000, characterized in that the range, cancer treatment polymer complex.
According to claim 1,
The polymer complex for cancer treatment is characterized in that it is in the form of a spherical gel having an average diameter in the range of 100 to 2,000 nm, the polymer complex for cancer treatment.
11. The method of claim 10,
The spherical gel form is a core-shell type microgel form,
A polymer complex for cancer treatment, characterized in that the photosensitizer is located in the core, and the anticancer agent is located in the shell. delete delete delete delete

Images