Nanometer conjugate based on cuttlefish ink-porphyrin and used for treating tumor cells

Abstract

The nano material produced by organisms is more concerned in the field of tumor diagnosis and treatment due to the inherent biocompatibility of the nano material, the cuttlefish ink (M, extracted from ink sacs of cuttlefishes) and the meso-tetra (4-carboxyphenyl) porphin (TCPP) nano conjugate (M-TCPP) are prepared in the research, the nano size of the M-TCPP is about 120 nm, and the nano conjugate has the advantages that the nano conjugate can be applied to tumor diagnosis and treatment; the TCPP can be triggered by a 660 nm light source (visible light) to generate reactive oxygen species (ROS) for photodynamic therapy (PDT) of tumor cells; m is triggered by a 808 nm light source (near-infrared light) to perform effective photothermal conversion to generate heat for photothermal therapy (PTT) of tumor cells, and compared with single PDT or PTT, the synergistic PDT-PTT can kill the tumor cells to a greater extent. In addition, an in-vitro cell experiment shows that M-TCPP has no obvious toxic or side effect, and the research provides some inspiration for developing a natural multifunctional biological material and applying the natural multifunctional biological material to tumor treatment.

Description

Technical Field

[0001] The invention relates to application of a drug for combined in vitro photodynamic-photothermal treatment of cancer cells, and in particular to a cuttlefish juice-porphyrin nanoconjugate. Background Art

[0002] Malignant tumors seriously threaten human health. In order to treat tumors, in addition to traditional treatment methods, researchers have also explored a variety of new treatment technologies in recent years, mainly including photodynamic therapy (PDT), photothermal therapy (PTT), sonodynamic therapy (SDT) and chemodynamic therapy (CDT). However, the generation and development of tumors are very complicated. At present, there is no treatment method that can fully meet clinical needs. For this reason, researchers have tried to effectively combine different treatment methods in the hope of obtaining a "1+1>2" combined treatment strategy. So far, a variety of combined treatment strategies have emerged, such as PDT-PTT, SDT-PTT, SDT-PDT, PDT-CDT, PDT-chemotherapy and PTT-chemotherapy. It should be pointed out that in the phototherapy combination (PTT and PDT), PTT is considered to be an effective treatment method due to its non-oxygen dependence and high spatiotemporal accuracy. PTT usually uses photothermal agents to trigger local hyperthermia under near-infrared light (NIR) irradiation, resulting in irreversible ablation of tumor cells, with high treatment efficiency. During the DT process, laser irradiation of a specific wavelength excites the photosensitizer, and the excited photosensitizer transfers energy to the surrounding oxygen, generating highly cytotoxic reactive oxygen species (ROS), which in turn causes cell damage or even death. When PTT and PDT are used in combination, PTT can destroy the membrane structure of cancer cells and enhance the penetration of reactive oxygen species (ROS) in tumor cells, thereby improving the PDT ability. At the same time, the ROS generated during the PDT process can destroy heat shock proteins (HSP70 and HSP90), which usually play a role in preventing DNA denaturation and cell apoptosis. This destruction can promote the efficacy of PTT and supplement the effect of PDT in a synergistic manner. The PTT-PDT combined light therapy strategy has been proven to be an effective strategy because it can accelerate blood flow, increase tumor cell oxygen content, promote reactive oxygen generation, and increase the thermosensitivity of tumor cells. The key to synergistic PDT-PTT is to obtain new nanomaterials with significant photothermal effects and photodynamic properties. Up to now, several multifunctional nanomaterials have been constructed for combined PDT-PTT for tumor cell treatment.

[0003] There are three main strategies for constructing PDT-PTT combinations. The first is to construct inorganic photosensitizers and inorganic photothermal nanomaterials for PDT-PTT, among which gold, iron, copper, tungsten and molybdenum-based nanomaterials are the mainstream photothermal and photodynamic nanoagents. However, one of the main challenges of inorganic nanoagents is their potential toxicity. Over time, inorganic nanoagents will accumulate in the body. Since inorganic nanoagents are difficult to degrade / metabolize in a short period of time, they may cause unnecessary side effects / toxicity. The second strategy is to encapsulate organic photosensitizers in inorganic photothermal shells, and vice versa. For example, H-TiO2@PDA@ICG (indocyanine green) and Fe3O4 / SiO2@CUR (curcumin) composite nanoagents exhibit significant photothermal effects and photodynamic properties. However, the inorganic material part is difficult to effectively decompose and metabolize from the body, and the organic part is difficult to effectively / quickly release from the inorganic part, resulting in low utilization efficiency. In order to solve these problems, a series of organic photothermal-organic photosensitive nanocomposites have been derived. However, the loading efficiency of organic small molecules is not ideal, and the drug release is spontaneous and uncontrollable. In addition, the ROS generated by photosensitizers has a short lifetime (200 ns) and a limited diffusion distance (<20 nm), which directly leads to poor utilization of ROS inside the composite nanoagents. Therefore, it is necessary to develop new organic PDT-PTT nanoagents with ideal ROS diffusion distance, biocompatibility and biodegradability. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a cuttlefish juice (M, extracted from the ink sac of cuttlefish) and meso-tetra(4-carboxyphenyl)porphine (TCPP) nanoconjugate (M-TCPP). The TCPP can be triggered by a 660 nm light source (visible light) to produce reactive oxygen species (ROS) for photodynamic therapy (PDT) of tumor cells; the M can be triggered by near-infrared light to perform effective photothermal conversion to generate heat for photothermal therapy (PTT) of tumor cells. Compared with single PDT or PTT, synergistic PDT-PTT can kill tumor cells to a greater extent. In addition, in vitro cell experiments show that M-TCPP has no obvious toxic side effects.

[0005] The invention provides a nano conjugate based on cuttlefish juice-porphyrin, which is a nano conjugate M-TCPP of cuttlefish juice and meso-tetra(4-carboxyphenyl)porphine. The M-TCPP is a nano microsphere with an average particle size of about 120 nm and a Zeta potential of -15 to -18 mV.

[0006] Another object of the present invention is to provide a method for preparing a nano-conjugate based on cuttlefish juice-porphyrin, comprising the following steps: N -Hydroxysulfosuccinimide is dissolved in N,N-dimethylformamide solution, after stirring in the dark, cuttlefish juice is added to the mixed solution, magnetically stirred overnight, centrifuged and washed to obtain the cuttlefish juice-porphyrin nanoconjugate.

[0007] TCPP, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N The mass / volume ratio of -hydroxysulfosuccinimide and cuttlefish ink is 1: 3-10: 1-4 (mg: μL: mg: mg).

[0008] Another object of the present invention is to provide a product 1 O2 reagent, the reagent is the cuttlefish juice-porphyrin nanoconjugate or the cuttlefish juice-porphyrin nanoconjugate prepared by the method.

[0009] The cuttlefish juice-porphyrin nanoconjugate is produced under the condition of 600-850nm. 1 Application of O2.

[0010] The present invention also provides a photothermal material, wherein the reagent is the cuttlefish juice-porphyrin nanoconjugate or the cuttlefish juice-porphyrin nanoconjugate prepared by the method.

[0011] The cuttlefish juice-porphyrin nanoconjugate achieves a temperature increase effect under 800-850nm near-infrared laser irradiation, and the concentration of the cuttlefish juice-porphyrin nanoconjugate is 0.025-0.2 mg mL -1 .

[0012] The invention discloses an application of a nanoconjugate based on cuttlefish juice-porphyrin in preparing a drug for treating anti-tumor cells, wherein the tumor cells include cancer cells 4T1.

[0013] The cuttlefish juice-porphyrin nanoconjugate is produced by 1 O2 and / or photothermal effect realizes the use of in vitro photodynamic-photothermal combined treatment of tumor cells.

[0014] In the technical scheme of the present invention, cuttlefish juice (M) is an excellent photothermal agent in PTT due to its inherent biocompatibility and degradability. Cuttlefish juice contains abundant amine-active chemical units and is expected to undergo amidation reaction with photosensitizers containing carboxyl groups to prepare multifunctional nanoagents. TCPP (m-tetrakis (4-carboxyphenyl) porphine) is an organic molecule containing carboxyl groups and has been considered as an effective photosensitizer in clinical trials for many years. In addition, considering that the diffusion distance of ROS is short, TCPP is evenly distributed on the surface of M, which can maximize the utilization rate of ROS. For this reason, this study uses M and TCPP to undergo amidation reaction to construct M-TCPP nanoconjugates. M-TCPP has high PDT efficiency and high photothermal effect, and can be used as a good PDT-PTT nanoagent for synergistic treatment of tumor cells ( Figure 1 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 . Schematic diagram of the design of M-TCPP and its application in photodynamic-photothermal (PDT-PTT) synergistic treatment of tumor cells.

[0016] Figure 2 (a, b) Scanning electron microscope (SEM) images of M-TCPP. (c) UV-visible absorption spectra of M-TCPP, TCPP, and cuttlefish ink. (d) Fluorescence spectra of M-TCPP, TCPP, and cuttlefish ink. (e) Dynamic light scattering particle size of M-TCPP. (f) Zeta potential of TCPP and M-TCPP.

[0017] Figure 3 . (a) M-TCPP produces singlet oxygen under light irradiation ( 1 Schematic diagram of the oxidation of DPBF by adding O2. (b) UV-visible absorption spectrum of M-TCPP + DPBF + light over time. (c) Changes in absorbance of different test groups under the same conditions.

[0018] Figure 4 (a) UV-visible absorption spectra of M-TCPP at different concentrations. (b) Temperature rise curves of M-TCPP at different concentrations. (c) Photothermal imaging of M-TCPP. (d) Temperature variation of M-TCPP during four irradiation cycles. (e) Temperature variation curve of M-TCPP aqueous dispersion after 808 nm laser on / off and linear time data -ln(θ) after near-infrared laser off.

[0019] Figure 5 (a, b) Cytotoxicity of different concentrations of M-TCPP on HUVEC and 4T1 cells. (c) Fluorescence images of 4T1 cells after incubation with M-TCPP or M for 4 h.

[0020] Figure 6 (a) Schematic diagram of in vitro photothermal-photodynamic (PTT-PDT) combined therapy. (b) Relative survival rate of 4T1 cells after treatment in each group. (c) Fluorescence images of 4T1 cells after treatment in each group. DETAILED DESCRIPTION

[0021] Experimental Section Instruments and reagents Meso-tetrakis(4-carboxyphenyl)porphine (TCPP), purity 97%, N-hydroxysuccinimide (NHS), purity 98%, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), purity 95%, were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; N, N -Dimethylformamide (DMF), analytical grade, and 1,3-diphenylisobenzofuran (DPBF), purity 97%, were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0022] JSM-7500F scanning electron microscope (JEOL, SEM, 10 kV); Shimadzu UV-2600 ultraviolet-visible spectrophotometer (UV-Vis), Shimadzu Co., Japan; Hitachi F-4600 fluorescence spectrometer, Hitachi Co., Japan; NANOTRAC WAVE II nanoparticle size and zeta potential analyzer, Microtrac Co., USA.

[0023] Experimental procedures Example 1 Preparation of M-TCPP TCPP (5 mg), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 40 μL) and N -Hydroxysulfosuccinimide (NHS, 20 mg) was dissolved in N,N -dimethylformamide (DMF, 5 mL) solution, stirred in the dark for 2 h. Then, cuttlefish juice (10 mg) was added to the mixed solution, magnetically stirred overnight, and finally, the mixture was centrifuged (10000 rpm, 5 min) and washed three times with water / ethanol (1 / 4 mL).

[0024] Synthesis and characterization of M-TCPP like Figure 1As shown, TCPP is an organic molecule containing a carboxyl group and is commonly used as a photosensitizer for PDT. Cuttlefish juice (M) is a natural biopolymer that contains abundant amine groups and can be used as a photothermal agent for PTT. The M-TCPP composite nanoagent is obtained by coupling the carboxyl group of TCPP with the amine group of cuttlefish juice and can be used for PDT-PTT combined therapy. Subsequently, the morphology of M-TCPP was studied using scanning electron microscopy (SEM). The results showed that M-TCPP was spherical in structure with a particle size of about 120 nm ( Figure 2 a, 2b). Subsequently, the optical properties of M-TCPP were measured by UV-visible spectrophotometer. Cuttlefish ink M exhibited strong light absorption at 300-800 nm, and TCPP exhibited a characteristic absorption peak at 410 nm. Compared with the absorption spectra of M or TCPP, the absorption spectrum of M-TCPP showed all the characteristic absorption peaks of TCPP and M. It is worth noting that compared with pure TCPP, M-TCPP showed a slight peak shift due to the energy transfer between TCPP and cuttlefish ink ( Figure 2 c). In addition, under 560 nm excitation, the main emission peak of TCPP in DMF solution is 660 nm; while the fluorescence intensity of M-TCPP is significantly lower than that of TCPP, indicating that energy transfer from TCPP to M occurs ( Figure 2 d), indicating the successful complexation of TCPP on the surface of squid ink. In addition, the average hydrodynamic size of M-TCPP is ~165 nm ( Figure 2 e). Zeta potential changes from -13.7 mV for TCPP to -17.9 mV for M-TCPP ( Figure 2 f), further confirming the successful compounding of TCPP on the surface of cuttlefish ink.

[0025] Example 2 Singlet oxygen ( 1 O2) Experiment First, DPBF (1,3-diphenylisobenzofuran, 20 μL, 2.0 mg mL -1 ) was added to a DMF solution containing M-TCPP (3 mL, 50 mg mL -1 ) to form a uniform mixed solution, and then the mixed solution was exposed to a light source (660 nm, 100 mW• cm −2 ) was irradiated and labeled (M-TCPP + DPBF + Light). The absorption spectrum was recorded with a UV-visible spectrophotometer to detect the effect of M-TCPP photodynamic production of ROS. At the same time, the absorbance changes of (DPBF +Light) and (M-TCPP + Light) under the same conditions were recorded.

[0026] The M-TCPP nanocomplex is made of TCPP and cuttlefish juice, in which the TCPP part can be excited by a 660 nm light source to convert O2 into cytotoxic 1 O2, 1 O2 can be detected using 1,3-diphenylisobenzofuran (DPBF) ( Figure 3 a). When the mixed solution (M-TCPP + DPBF) was excited by the light source for 5 min, the characteristic absorption peak of DPBF (at 415 nm) decreased rapidly with the increase of illumination time, indicating that (M-TCPP + DPBF + Light) produced 1 O2( Figure 3 b). In addition, to exclude other influences, we also tested the absorbance changes of the pure DPBF group and the M-TCPP group under the same conditions. The study found that 34.1% of DPBF was oxidized by the M-TCPP + Light (5 min) group, while the absorption intensity of the pure DPBF group or the M-TCPP group did not decrease significantly ( Figure 3 c). Therefore, the above results show that M-TCPP can produce 1 O2 has potential application prospects in PDT.

[0027] Example 3 Photothermal effect experiment M-TCPP aqueous dispersion (100 μL) was diluted with 0, 0.025, 0.05, 0.1 and 0.2 mg mL -1 The concentration of 1.0 W cm-1 was added to plastic tubes (150 μL), and the tubes were then exposed to near-infrared laser light (808 nm, 1.0 W cm-1). -2 ) for 10 min and record the temperature changes with an infrared thermal imager.

[0028] With the increase of M-TCPP concentration (0.025~0.2 mg mL -1 ), the light absorption of M-TCPP dispersion was significantly enhanced. When exposed to 808 nm laser (1.0 W cm -2 ), these dispersions exhibited a rapid temperature rise response to laser irradiation and a strong concentration-dependent photothermal effect. -1 The temperature increases of M-TCPP dispersion were 5.8, 12.5, 19.5, 30.8 and 47.5 °C, respectively. -1) showed that with the increase of 808 nm laser irradiation time, the intensity of thermal imaging signal became higher and higher, indicating that M-TCPP has a good photothermal conversion effect. -1 ) dispersion after 4 photothermal cycles, the photothermal conversion effect was well maintained ( Figure 4 d), indicating that M-TCPP has good photothermal cycle stability. After calculation, the photothermal conversion efficiency of M-TCPP is 31.5%. In summary, the M-TCPP composite nanoreagent has a good photothermal conversion effect, which makes it have broad application prospects in PTT.

[0029] Example 4 In vitro cell experiments Human umbilical vein endothelial cells (HUVEC) and mouse breast cancer cells (4T1) were cultured in 96-well plates under standard conditions. After 24 hours, the cell culture medium was removed and new culture medium containing different concentrations of M-TCPP was added. After 24 hours, the cells were washed with PBS and the cell survival rate was detected by standard tetrazolium salt (MTT) colorimetric method.

[0030] To verify the biosafety of M-TCPP, M-TCPP was co-incubated with HUVEC and 4T1 cells for the same time (12 / 24 h), and the cell survival rate was tested by tetrazolium salt (MTT) colorimetry. The results showed that after 24 h of co-incubation, these cells still maintained high activity, with an average survival rate of more than 88% (400 μg•mL −1 ), verifying that M-TCPP has good biosafety (Figure 5a, 5b). Subsequently, the effect of M-TCPP (200 μg•mL -1 ) under the same test conditions, using non-fluorescent squid juice as a comparison, it was found that after M-TCPP was co-incubated with 4T1 cells for 4 h, CT26 cells showed strong red fluorescence, indicating that M-TCPP was effectively taken up / endocytosed by 4T1 cells ( Figure 5 c).

[0031] The PDT and PTT effects of M-TCPP on 4T1 tumor cells in vitro were then analyzed. -1 ) cell culture medium for 12 h, some groups were irradiated with 808 nm laser / 660 nm laser ( Figure 6a). These cells were divided into 7 groups, including: (1) Control group, (2) M-TCPP group, (3) near-infrared light (NIR, 808 nm) irradiation group, (4) light (Light, 660 nm) irradiation group, (5) M-TCPP + NIR group, (6) M-TCPP + Light group, and (7) M-TCPP + NIR + Light group. The cell viability of these groups was detected by MTT method and the cell fluorescence staining photos were observed by fluorescence microscopy ( Figure 6 ). In groups (1-4), the cell survival rates were all over 85%, indicating that M-TCPP, NIR or Light alone had no killing effect on cancer cell 4T1. However, the cell survival rate of group (5) cells was 44.3%, indicating that M-TCPP had a good PTT effect under NIR light irradiation. In addition, the cell survival rate of group (6) cells decreased to 53.8%, confirming a good PDT effect. More importantly, group (7) had the lowest cell survival rate of only 12.2%, which was attributed to the good synergistic PTT-PDT treatment effect ( Figure 6 b). At the same time, the fluorescence photos showed that the cells in group (1-4) were almost all green fluorescent (living cells). Compared with group (1-4), the green fluorescent area of ​​cells in group (5-7) decreased, while the red fluorescent area (dead cells) increased rapidly, indicating that the survival rate of cancer cells decreased. In particular, the M-TCPP + NIR + Light group (7) showed red fluorescence of most dead cells (Figure 8b). In summary, M-TCPP combined with 808 nm laser and 660 nm laser has excellent photothermal-photodynamic combined therapeutic efficacy and can efficiently kill tumor cells.

[0032] In summary, the biomimetic M-TCPP nanoreagent was successfully prepared by coupling TCPP with cuttlefish juice. M-TCPP can efficiently generate reactive oxygen species under 660 nm laser irradiation and quickly generate heat under 808 nm laser irradiation, showing excellent photodynamic-photothermal effects.

[0033] In addition, M-TCPP showed good biosafety, and compared with the therapeutic effect of PDT or PTT alone, the ability of PDT-PTT combination therapy to significantly kill tumor cells was greatly improved, providing an effective new strategy for biosafe material treatment of tumor cells.

Claims

1. A nanoconjugate based on cuttlefish juice-porphyrin, characterized in that: It is a nanoconjugate of cuttlefish juice and meso-tetra(4-carboxyphenyl)porphine, M-TCPP. M-TCPP is a nano-microsphere with an average particle size of ~120 nm and a Zeta potential of -15~-18mV.

2. A method for preparing a nanoconjugate based on cuttlefish juice-porphyrin, characterized in that: The method comprises the following steps: preparing TCPP, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N -Hydroxysulfosuccinimide is dissolved in N,N -dimethylformamide solution, after stirring in a dark environment, cuttlefish juice is added to the mixed solution, magnetically stirred overnight, centrifuged and washed to obtain a cuttlefish juice-porphyrin nanoconjugate.

3. The method for preparing the nanoconjugate based on cuttlefish juice-porphyrin according to claim 2, characterized in that: TCPP, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N The mass / volume ratio of -hydroxysulfosuccinimide and cuttlefish ink is 1: 3-10: 1-4 (mg: μL: mg: mg).

4. A product 1 O2 reagent, characterized in that The reagent is the cuttlefish juice-porphyrin nanoconjugate according to claim 1 or the cuttlefish juice-porphyrin nanoconjugate prepared by the method according to claim 2 or 3.

5. The product according to claim 4 1 O2 reagent, characterized in that The cuttlefish juice-porphyrin nanoconjugate is produced under the condition of 600-850nm. 1 Application of O2.

6. A photothermal material, characterized in that: The photothermal material is the cuttlefish juice-porphyrin nanoconjugate according to claim 1 or the cuttlefish juice-porphyrin nanoconjugate prepared by the method according to claim 2 or 3.

7. The photothermal material according to claim 6, characterized in that: The cuttlefish juice-porphyrin nanoconjugate achieves a temperature increase effect under 800-850nm near-infrared laser irradiation, and the concentration of the cuttlefish juice-porphyrin nanoconjugate is 0.025-0.2 mg mL -1 .

8. Use of a nanoconjugate based on cuttlefish juice-porphyrin in the preparation of a drug for treating anti-tumor cells, characterized in that: The tumor cells include cancer cell 4T1.

9. The use according to claim 8, characterized in that: The cuttlefish juice-porphyrin nanoconjugate is produced by 1 O2 and / or photothermal effect realizes the use of in vitro photodynamic-photothermal combined treatment of tumor cells.

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