CN116283934B - Multifunctional unsaturated fatty acid-hemicyanine conjugate, preparation and application thereof - Google Patents
Multifunctional unsaturated fatty acid-hemicyanine conjugate, preparation and application thereof Download PDFInfo
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- CN116283934B CN116283934B CN202310281177.2A CN202310281177A CN116283934B CN 116283934 B CN116283934 B CN 116283934B CN 202310281177 A CN202310281177 A CN 202310281177A CN 116283934 B CN116283934 B CN 116283934B
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- hemicyanine
- fatty acid
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- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 1
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明属于医药技术领域，更具体地，涉及一种多功能不饱和脂肪酸‑半花菁偶联物、其制备和应用。本发明提供的一种不饱和脂肪酸‑半花菁偶联物，具有如式(一)所示的结构：其中，R1选自碳数为1‑18的烷基和碳数为1‑18的烷基磺酸；R2为不饱和脂肪链，所述不饱和脂肪链的碳数为6‑30。该偶联物在水中可自组装形成粒径均一，稳定性良好的纳米粒，实现构建100％载药量的单分子纳米药物递送系统；在660nm的激光照射下可以产生活性氧，实现抗肿瘤光动力治疗；其中阳离子型脂肪酸‑半花菁偶联物能够靶向线粒体，降低氧耗量功能，解决光动力治疗中氧供量不足的问题。
The present invention belongs to the field of medical technology, and more specifically, relates to a multifunctional unsaturated fatty acid-hemicyanine conjugate, its preparation and application. The present invention provides an unsaturated fatty acid-hemicyanine conjugate having a structure as shown in formula (1): Wherein, R1 is selected from an alkyl group with a carbon number of 1-18 and an alkyl sulfonic acid with a carbon number of 1-18; R2 is an unsaturated fatty chain, and the carbon number of the unsaturated fatty chain is 6-30. The conjugate can self-assemble in water to form nanoparticles with uniform particle size and good stability, thereby realizing the construction of a single-molecule nano drug delivery system with a 100% drug loading; reactive oxygen species can be generated under 660nm laser irradiation to achieve anti-tumor photodynamic therapy; wherein the cationic fatty acid-hemicyanine conjugate can target mitochondria, reduce oxygen consumption function, and solve the problem of insufficient oxygen supply in photodynamic therapy.
Description
Technical Field
The invention belongs to the technical field of medicines, and in particular relates to a multifunctional unsaturated fatty acid-hemicyanine conjugate, and preparation and application thereof.
Background
Photodynamic therapy (Photodynamic therapy, PDT) relies on photosensitizers to achieve explosive growth of ROS under light to achieve accurate treatment of diseased sites, which has been a relatively mature treatment modality. Oxygen molecules are a prerequisite for type II photodynamic therapy, however, hypoxia is one of the prominent features of solid tumors, limiting the clinical transformation of photodynamic therapy in the treatment of solid tumors.
In recent years, to solve the problem of insufficient oxygen supply in photodynamic therapy, the main strategy is covered by "open source throttling". Specifically, "open source" is to deliver oxygen to the tumor site with oxygen carrier material or to catalyze hydrogen peroxide at the tumor site to oxygen; "throttling" is the saving of intracellular oxygen consumption, such as co-delivering a photosensitizer and an oxidative phosphorylation inhibitor (e.g., atovaquone). However, the complex multi-drug delivery system manufacturing process limits its clinical transformation. Therefore, the photosensitizer with simple and high-efficiency development process solves the problem of insufficient oxygen supply in tumor photodynamic therapy and has wide application prospect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a multifunctional unsaturated fatty acid-hemicyanine conjugate, and preparation and application thereof, wherein single molecules of the conjugate can be self-assembled into nano particles, the nano particles are enriched in tumors at high concentration after systemic administration, active oxygen can be generated under 660nm laser irradiation, and the problem of insufficient oxygen supply in tumor photodynamic therapy can be solved by virtue of the biological activity of the nano particles.
To achieve the above object, the present invention provides an unsaturated fatty acid-hemicyanine conjugate having a structure as shown in formula (one):
Wherein R 1 is alkyl with 1-18 carbon atoms or alkyl sulfonic acid with 1-18 carbon atoms; r 2 is an unsaturated fatty chain, and the carbon number of the unsaturated fatty chain is 6-30.
Preferably, the unsaturated fatty chain is derived from an unsaturated fatty acid having the formula C nH2n-1COOH、CnH2n-3COOH、CnH2n-5COOH、CnH2n-7 COOH or C nH2n-9 COOH, wherein n is an integer from 8 to 29, more preferably n is an integer from 14 to 25.
Preferably, the conjugate is obtained by esterification reaction of the unsaturated fatty acid and the phenolic hydroxyl-containing hemicyanine.
According to another aspect of the present invention there is provided an unsaturated fatty acid-hemicyanine conjugate nano-formulation which is a self-assembled nanoparticle dispersion of said unsaturated fatty acid-hemicyanine conjugate.
According to another aspect of the present invention, there is provided a method for preparing the nano-preparation, comprising the steps of: and (3) dropwise adding an organic solution of the unsaturated fatty acid-hemicyanine conjugate dissolved in a benign organic solvent into deionized water in a stirring state, and finally dialyzing with the deionized water to obtain a self-assembled nanoparticle dispersion liquid, namely the unsaturated fatty acid-hemicyanine conjugate nano preparation.
According to another aspect of the present invention there is provided the use of said unsaturated fatty acid-hemicyanine conjugate or said unsaturated fatty acid-hemicyanine conjugate nano-formulation as a photosensitizer.
According to another aspect of the invention, the application of the unsaturated fatty acid-hemicyanine conjugate or the unsaturated fatty acid-hemicyanine conjugate nano-preparation in preparing anti-tumor drugs targeting mitochondria is provided.
According to another aspect of the invention, the application of the unsaturated fatty acid-hemicyanine conjugate or the unsaturated fatty acid-hemicyanine conjugate nano-preparation in preparing an oxygen-saving anti-tumor drug is provided.
In general, the above technical solutions conceived by the present invention have the following compared with the prior art
The beneficial effects are that:
(1) The unsaturated fatty acid-hemicyanine conjugate provided by the invention is obtained by coupling unsaturated fatty acid with hydrophobic hemicyanine dye, and a single-molecule self-assembled nano drug delivery system can be obtained by a nano precipitation method, and the preparation process is simple.
(2) According to the invention, the hydrophobic hemicyanine compound is used as a base for grafting modification to obtain the unsaturated fatty acid-hemicyanine conjugate, so that the unsaturated fatty acid-hemicyanine conjugate can be prepared into a nano preparation, and a single-molecule nano drug delivery system with 100% drug loading capacity can be constructed; and improves the in vivo pharmacokinetic characteristics of the hydrophobic hemicyanine compound and increases the concentration of the hydrophobic hemicyanine compound in tumor enrichment.
(3) The cationic unsaturated fatty acid-hemicyanine conjugate (CyOA) provided in the preferred embodiment of the invention can target mitochondria, reduce intracellular oxygen consumption, solve the problem of insufficient oxygen supply in photodynamic therapy, and remarkably improve photodynamic anti-tumor treatment effect.
Drawings
FIG. 1 is a synthetic route diagram of example 1 (CyOA) and example 2 (SO 3 -CyOA) of the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of example 1 (CyOA) of the present invention;
FIG. 3 is a hydrogen nuclear magnetic resonance spectrum of example 2 (SO 3 -CyOA) of the present invention;
FIG. 4 is a photograph of the dispersion of the nano-drug and hydrophobic hemicyanine material (CyOH) prepared in example 1 (CyOA) of the present invention in water;
FIG. 5 is a transmission electron microscope image of the nano-drug prepared in example 1 (CyOA) and example 2 (SO 3 -CyOA) of the present invention;
FIG. 6 is a Zeta potential diagram of the nano-drug prepared in example 1 (CyOA) and example 2 (SO 3 -CyOA) of the present invention;
FIG. 7 shows the in vitro active oxygen yields of the nanomedicines prepared in examples 1 (CyOA) and 2 (SO 3 -CyOA) of the present invention;
FIG. 8 is a graph showing the intracellular distribution of the nanomedicines prepared in examples 1 (CyOA) and 2 (SO 3 -CyOA) of the present invention;
FIG. 9 is a graph showing confocal imaging detection of intracellular hypoxia levels following treatment of the nanomedicine prepared in examples 1 (CyOA) and 2 (SO 3 -CyOA) of the present invention;
FIG. 10 is a graph showing intracellular hypoxia level detected by flow cytometry after treatment of the nanomedicine prepared in examples 1 (CyOA) and 2 (SO 3 -CyOA) of the present invention;
FIG. 11 is a graph showing the oxygen consumption of tumor cells detected by the oxygen measuring microelectrode after the nano-drugs prepared in example 1 (CyOA) and example 2 (SO 3 -CyOA) of the present invention are treated;
FIG. 12 is a graph showing intracellular ROS content in hypoxic and normoxic conditions following treatment of the nanomedicine prepared in examples 1 (CyOA) and 2 (SO 3 -CyOA) of the present invention;
FIG. 13 is a graph showing photosensitizer dose-cell viability curves under hypoxic and normoxic conditions after treatment with the nanomedicine prepared in examples 1 (CyOA) and 2 (SO 3 -CyOA) of the present invention;
FIG. 14 shows tumor volume-time curves (content a) and tumor photographs (content b) of the nano-drug prepared in example 1 (CyOA) of the present invention and clinical camptotheca treatment.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The unsaturated fatty acid-hemicyanine conjugate provided by the invention has a general formula shown in a formula (I):
in the formula (I), R 1 is selected from alkyl with 1-18 carbon atoms and alkyl sulfonic acid with 1-18 carbon atoms; r 2 is an unsaturated fatty chain, and the carbon number of the unsaturated fatty chain is 6-30.
In a preferred embodiment, in formula (one), R 1 is selected from one of alkyl having 1-10 carbons, alkyl sulfonic acid having 1-10 carbons; more preferably one of alkyl groups having 1 to 6 carbons and alkylsulfonic acids having 1 to 6 carbons.
In some embodiments, the unsaturated fatty chain is derived from an unsaturated fatty acid having the formula C nH2n-1COOH、CnH2n-3COOH、CnH2n-5COOH、CnH2n-7 COOH or C nH2n-9 COOH, wherein n is an integer from 8 to 29, preferably n is an integer from 14 to 25. The unsaturated fatty acids include, but are not limited to, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid (Eicosapentaenoic Acid, EPA) or docosahexaenoic acid (Docosahexaenoic Acid, DHA).
In some embodiments, the multifunctional unsaturated fatty acid-hemicyanine conjugate of the present invention may be obtained by esterification of an unsaturated fatty acid with a hemicyanine containing a phenolic hydroxyl group.
The invention also provides the unsaturated fatty acid-hemicyanine conjugate nano preparation which is the self-assembled nanoparticle dispersion liquid of the unsaturated fatty acid-hemicyanine conjugate.
In some embodiments, the preparation method of the unsaturated fatty acid-hemicyanine conjugate nano preparation comprises the following steps of dissolving the unsaturated fatty acid-hemicyanine conjugate in a benign organic solvent to obtain a conjugate solution, then dropwise adding the conjugate solution into deionized water in a stirring state, and finally dialyzing by adopting the deionized water to remove the organic solvent and unsaturated fatty acid-hemicyanine conjugate small molecules which do not participate in assembly, thereby obtaining a self-assembled nano particle dispersion liquid, namely the unsaturated fatty acid-hemicyanine conjugate nano preparation.
In some embodiments, the benign organic solvent is one or more of dimethyl sulfoxide, methanol, ethanol, acetonitrile, and tetrahydrofuran. The stirring speed of the deionized water in the stirring state is not lower than 200rpm, preferably 200-2000rpm. The concentration of conjugate in the conjugate solution is 0.1-50mM, more preferably 0.1-20mM. The cut-off molecular weight of the dialysis bag is 1kDa-14kDa, and the dialysis is carried out for 12-36 hours.
The unsaturated fatty acid-hemicyanine conjugate provided by the invention is a multifunctional conjugate, and the multifunctional conjugate is characterized in that: (1) The nanometer particles with uniform particle size and good stability can be formed by self-assembly in water, so that a single-molecule nanometer drug delivery system with 100% drug loading capacity is constructed; (2) Active oxygen can be generated under 660nm laser irradiation, so that anti-tumor photodynamic therapy is realized; (3) The cationic fatty acid-hemicyanine conjugate can target mitochondria, and experiments prove that the cationic fatty acid-hemicyanine conjugate can reduce intracellular oxygen consumption of tumors, improve hypoxia level of tumor cells, and solve the problem of insufficient oxygen supply in photodynamic therapy. Therefore, the unsaturated fatty acid-hemicyanine conjugate or the unsaturated fatty acid-hemicyanine conjugate nano preparation provided by the invention can be used as a photosensitizer for photodynamic therapy, can be used for preparing anti-tumor drugs targeting mitochondria, and can also be used for preparing oxygen-saving anti-tumor drugs so as to reduce intracellular oxygen consumption of tumors and improve hypoxic level of tumor cells. The tumors of the present invention include, but are not limited to, breast cancer, liver cancer, colon cancer, ovarian cancer, melanoma, pancreatic cancer, etc.
The following are examples:
Example 1
The unsaturated fatty chain with R 1 as C 3H7,R2 is derived from oleic acid, and has a structure shown in a formula (II) and is named as CyOA.
As shown in fig. 1, the synthesis steps include:
(1) Resorcinol (220 mg,2 mmol) and K 2CO3 (276 mg,2 mmol) were dissolved in 5mL anhydrous N ', N' -dimethylformamide and stirred at room temperature for 30min, then a solution of IR780 (667 mg,1 mmol) in 2mL N ', N' -dimethylformamide was added to the above reaction system and reacted at 60 ℃ for 5h under N 2 protection. The post-treatment adopts dichloromethane extraction, saturated saline water is used for washing, anhydrous sodium sulfate is used for drying, concentration and silica gel sample mixing, and dichloromethane is used for: methanol=10: and 1 is eluent column chromatography separation and purification to obtain an intermediate CyOH.
(2) CyOH (4395 mg,0.8 mmol) was dissolved in 10mL of dichloromethane and a mixed solution of oleic acid (282 mg,1 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (192 mg,1 mmol) and 4-dimethylaminopyridine (24.3 mg,0.2 mmol) in 10mL of dichloromethane was added dropwise in a round bottom flask. The reaction was stirred at room temperature for 8h. After the completion of the reaction, the post-treatment was extracted with methylene chloride, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the concentrate was purified by silica gel column chromatography (methylene chloride: methanol=10:1) to give a dark blue viscous oily substance (yield: 71%). FIG. 2 shows the nuclear magnetic resonance hydrogen spectrum and high resolution mass spectrum of the compound prepared in example 1 .1H NMR(600MHz,CDCl3)δ8.62(d,J＝15.0Hz,1H),7.52–7.48(m,3H),7.44–7.36(m,2H),7.11(s,1H),7.07–7.03(m,1H),7.00–6.96(m,1H),6.87(d,J＝15.0Hz,1H),5.37–5.32(m,2H),4.75–4.61(m,2H),2.88–2.82(m,2H),2.76–2.70(m,2H),2.61(t,J＝7.6Hz,2H),2.10–1.92(m,10H),1.79(s,6H),1.31–1.22(m,21H),1.12–1.06(m,3H),0.88–0.85(m,3H).HRMS(ESI):m/z calcd for C46H62NO3 +[M]+676.4724,found 676.47155.
Example 2
The unsaturated fatty chain with R 1 as C 4H8O3S-,R2 is derived from oleic acid, and has a structure shown in a formula (II) and is named as SO 3 -CyOA.
The synthetic route is the same as CyOA (fig. 1), the starting material is replaced by IR780 to IR783, and the specific steps include:
(1) Resorcinol (220 mg,2 mmol) and K 2CO3 (276 mg,2 mmol) were dissolved in 5mL anhydrous N ', N' -dimethylformamide and stirred at room temperature for 30min, then a solution of IR783 (749.3 mg,1 mmol) in 2mL N ', N' -dimethylformamide was added to the above reaction system and reacted at 60 ℃ for 5h under N 2 protection. The post-treatment adopts dichloromethane extraction, saturated saline water is used for washing, anhydrous sodium sulfate is used for drying, concentration and silica gel sample mixing, and dichloromethane is used for: methanol=10: and 1 is eluent column chromatography separation and purification to obtain an intermediate SO 3 -CyOH.
(2) A mixed solution of SO 3 -CyOH (404.5 mg,0.8 mmol) in 10mL of dichloromethane, oleic acid (282 mg,1 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (192 mg,1 mmol) and 4-dimethylaminopyridine (24.3 mg,0.2 mmol) in 10mL of dichloromethane was added dropwise in a round bottom flask. The reaction was stirred at room temperature for 8h. After the completion of the reaction, the post-treatment was extracted with methylene chloride, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated, and the concentrate was purified by silica gel column chromatography (methylene chloride: methanol=10:1) to give a dark blue viscous oily substance (yield: 64%). FIG. 3 shows the NMR hydrogen spectrum and high resolution mass spectrum of the compound prepared in comparative example 1 .1H NMR(600MHz,DMSO-d6)δ8.59(d,J＝15.2Hz,1H),7.83–7.75(m,2H),7.60–7.37(m,5H),7.12–7.08(m,1H),6.78–6.69(m,1H),5.34–5.30(m,2H),4.55–4.42(m,2H),2.78–2.66(m,6H),2.64–2.60(m,2H),2.03–1.96(m,6H),1.92–1.88(m,2H),1.84–1.78(m,3H),1.75(s,6H),1.40–1.25(m,17H),1.04–1.02(m,2H),0.87–0.82(m,3H).HRMS(ESI):m/z calcd for C47H63NO6S[M+H]+770.4449,found 770.44373.
When R in the molecule (a) in FIG. 1 is CH 3, the molecule is IR780; when R is CH 2CH2SO3 -, the molecule is IR783; when R in the molecule (b) is CH 3, the molecule is CyOH; when R is CH 2CH2SO3 -, the molecule is SO 3 -CyOH; when R in the molecule (c) is CH 3, the molecule is CyOA; when R is CH 2CH2SO3 -, the molecule is SO 3 -CyOA.
Example 3
Preparation and characterization of self-assembled nanoparticles of example 1 (CyOA) and example 2 (SO 3 -CyOA) self-assembly of CyOA NPs and SO 3 -CyOA NPs was performed spontaneously and was prepared using a nano-precipitation method. Specifically, 10mM CyOA or SO 3 -CyOA dissolved in 200 μl of methanol is added dropwise to deionized water (4 mL) with a magnetic stirring speed of 500rpm, and after the addition, dialysis is carried out for 12 hours by using a dialysis bag with molecular weight cutoff of 3500Da, the organic solvent is removed, and the obtained nanoparticles are blue solution without visible precipitation.
Fig. 4 is a photograph of intermediate CyOH and CyOA NPs suspended in water. As can be seen from the figure, the hydrophobicity CyOH is poorly soluble in water, while the prepared CyOA NPs is clear and transparent.
FIG. 5 is a transmission electron microscope image of CyOA NPs and SO 3 -CyOA NPs prepared in example 3, and the CyOA NPs and SO 3 -CyOA NPs nano-drugs can be obtained with uniform particle size distribution, and the average diameters are about 60nm and 120nm, respectively.
FIG. 6 shows the zeta potential measurement result, cyOA NPs positive charges on the surface, about +12 mv; in SO 3 -CyOA NPs, the sulfonate group is modified to neutralize the positive charge of the cation on the indole ring, SO that SO 3 -CyOA NPs has weak negative charge, about-8 mv.
Example 4
In vitro ROS (reactive oxygen species) production capability assays of example 1 (CyOA) and example 2 (SO 3 -CyOA).
After co-incubation with ROS, the absorbance value of 1, 3-Diphenylisobenzofuran (DPBF) decreases and is inversely related to ROS amount. Therefore, we used this reagent to quantify the ROS-producing capacity of CyOA NPs and SO 3 -CyOA NPs prepared in example 3 in vitro. Specifically, 1mg of DPBF was dissolved in 1mL of ethanol to obtain a stock solution. Then, 120. Mu.L of the stock solution was added to 3880. Mu. L CyOH (2.5. Mu.M) or 3880. Mu.L of SO3-CyOA NPs (2.5. Mu.M) solution, and mixed well. The mixture was irradiated with a laser at 660nm (200 mW/cm 2), 100. Mu.L of the above mixed solution was taken out at each time point designed, mixed with 100. Mu.L of ethanol, and the absorbance at 406nm was recorded on-line.
FIG. 7 is a graph of DPBF absorbance versus time, showing that CyOA NPs has no significant difference in the ability to generate ROS in vitro from SO 3 -CyOANPs.
Example 5
Example 1 (CyOA) was examined for intracellular co-localization with example 2 (SO 3 -CyOA).
Due to the negative potential of the inner mitochondrial membrane, cationic compounds may be efficiently enriched in mitochondria in an inverse concentration gradient. We examined the intracellular locations of CyOA NPs and SO 3 -CyOA NPs prepared in example 3 using confocal microscopy. Specifically, 20 ten thousand 4T1 cells were seeded in 20mm copoly Jiao Min overnight, incubated with 1 μ M CyOA NPs or 1 μΜ SO 3 -CyOA NPs for 8h after cell attachment, then rinsed 3 times with phosphate buffer (ph=7.4), then lysosomes and mitochondria were labeled with lysosome probes and mitochondrial probes, respectively, and then intracellular locations of the nanoparticles were visualized on the machine.
FIG. 8 is a photograph of the intracellular co-localization of CyOA NPs and SO 3 -CyOA NPs with mitochondria and lysosomes. It can be seen from the figure that CyOA NPs is polydisperse in mitochondria, whereas SO 3 -CyOA NPs sits in lysosomes. The difference in intra-cellular co-localization of the nanoparticles prepared in example 1 and example 2 is likely to be caused by the difference in their surface charges.
Example 6
Example 1 (CyOA) and example 2 (SO 3 -CyOA) were examined for the effect of oxygen consumption on cells.
Intracellular hypoxia levels were detected by hypoxia/oxidative stress detection kit ROS-ID. Specifically, after seeding 4T1 cells in a petri dish for 12h, incubation with 2 μ M CyOA NPs or 20 μΜ SO 3 -CyOA NPs for 4h followed by washing 3 times with phosphate buffer (ph=7.4) and staining with a hypoxic probe, confocal microscopy imaging. In addition, intracellular hypoxia levels were also analyzed by flow cytometry under the same treatment.
FIG. 9 is a confocal microscopy image, wherein CTR indicates that tumor cells were not treated with drug, and SO 3 -CyOA NPs and CyOA NPs indicate that tumor cells were pretreated with 20. Mu.M SO 3 -CyOA NPs or 2. Mu. M CyOA NPs, respectively. As can be seen from the figure, the fluorescence of the intracellular hypoxia probes of group CyOA NPs was significantly darker than that of the CTR and SO 3 -CyOA NPs groups, indicating that CyOA NPs improved the intracellular hypoxia levels of tumor cells.
FIG. 10 is fluorescence intensity and quantitative data for each group of hypoxia probes analyzed using flow cytometry. Also, as can be seen from the figure, the fluorescence intensity of the CyOA NPs group intracellular hypoxia probes was lower by about 60% compared to the CTR and SO 3 -CyOA NPs groups. The data indicate that CyOA NPs can significantly improve the intracellular hypoxia level of tumor cells and increase intracellular oxygen content compared to SO 3 -CyOA NPs without affecting the intracellular oxygen content of tumor cells.
Next, we examined the oxygen consumption of tumor cells after treatment with different groups using oxygen microelectrodes. Specifically, 100 ten thousand 4T1 cells were seeded in 6-well plates and treated with SO 3 -CyOA NPs (20. Mu.M) or CyOA NPs (2. Mu.M) for 6 hours. Then, 1X 10 6 cells were resuspended in 1mL Hank's balanced salt solution, the medium was sealed with liquid paraffin to avoid oxygen exchange, and oxygen probes extended into the lower layer broth, recording dissolved oxygen every 1 second for a total of 2 minutes.
FIG. 11 is a graph showing the concentration of dissolved oxygen versus time, wherein Blank represents the change in oxygen concentration in Hank's balanced salt solution without cells, CTR represents tumor cells without drug treatment, and SO 3 -CyOA NPs and CyOA NPs represent tumor cells pretreated with 20. Mu.M SO 3 -CyOA NPs or 2. Mu. M CyOA NPs, respectively. From the figure, cyOA NPs can significantly reduce the oxygen consumption of tumor cells compared to SO 3 -CyOA NPs, which does not affect the oxygen demand of the cells.
Example 6
Intracellular ROS production capability assays of example 1 (CyOA) and comparative example 1 (SO 3 -CyOA) under normoxic and hypoxic conditions.
2',7' -Dichlorofluorescein diacetate (DCFDA), which is cell membrane permeable and non-fluorescent in itself, is hydrolyzed by cellular esterases to produce 2',7' -dichlorofluorescein and further oxidized to produce strongly fluorescent 2',7' -Dichlorofluorescein (DHF) once it enters cells, and is a versatile ROS indicator. Normoxic conditions (21% o 2): firstly, 50 ten thousand 4T1 cells are planted in a 6-hole plate, after the cells are attached, the cells are respectively incubated for 4 hours in an normoxic incubator by SO 3 -CyOA NPs (20 mu M) or CyOA NPs (2 mu M), then 10 mu M DCFDA is added for incubation for 30 minutes, the non-cytoblast medicines are washed and removed by phosphoric acid buffer solution, and then, each hole is irradiated with 200mW/cm 2 660nm laser for 5 minutes. Fluorescence intensity was quantified for each group using flow cytometry. Hypoxia condition (1%O 2): drug incubation concentrations under hypoxic conditions were consistent with time and normoxic, but cells were always cultured in a hypoxic incubator (1%O 2).
The left side of fig. 12 shows the flow patterns after illumination of each group, and the right side shows the quantification of fluorescence intensity. From the figure, it can be derived that: 1. under normoxic conditions, the treatment of groups SO 3 -CyOA NPs and CyOA NPs can significantly increase intracellular ROS, and the increase amplitude of group CyOA NPs is higher than that of SO 3 -CyOA NPs; 2. following treatment in the SO 3 -CyOA NPs group, intracellular ROS rise was significantly reduced under hypoxic conditions, in contrast to CyOA NPs treated cells which exhibited the same ability to produce high concentrations of ROS under both normoxic and hypoxic conditions. These results demonstrate CyOA NPs that endogenous O 2 can be preserved for subsequent PDT, solving the problem of insufficient oxygenation in tumor photodynamic.
Example 7
Cytotoxicity assays under normoxic and hypoxic conditions example 1 (CyOA) and comparative example 1 (SO 3 -CyOA).
Normoxic conditions: 1 ten thousand 4T1 cells are inoculated in a 96-well plate, cultured in a constant-temperature constant-oxygen incubator with 5% CO 2 at 37 ℃, after the cells are attached to the wall, the culture medium is sucked, 100 mu L of RPMI 1640 culture solution containing CyOA NPs and SO 3 -CyOA NPs with different concentrations is respectively added, the mixture is incubated for 6 hours, and each well is irradiated with 200mW/cm 2 660nm laser for 4 minutes. After further culturing for 24 hours, the cell viability was calculated using the azobromate (MTT) method. Hypoxia conditions: the parameters such as the drug incubation concentration, the illumination time and the like under the hypoxia condition are consistent with normoxic, but the cells are always cultured in a hypoxia incubator (1%O 2).
The left side of FIG. 13 shows the change in cell viability after treatment with the different concentrations of SO 3 -CyOA NPs, and the right side shows the change in cell viability after treatment with the different concentrations of CyOA NPs. It can be seen that: 1. the phototoxicity of CyOA NPs, whether under normoxic or hypoxic conditions, is significantly greater than the IC 50 value (79.3. Mu.M) of SO 3-CyOA NPs;2、SO3 -CyOA NPs under hypoxic conditions than IC 50 (45.7. Mu.M) under normoxic conditions, while CyOA NPs shows similar phototoxicity under normoxic (IC 50: 1.9. Mu.M) and hypoxic (IC 50: 2.1. Mu.M) conditions. Thus, these results demonstrate that the use of self-assembled CyOA NPs achieves both endogenous O 2 and PDT conservation, addressing the inherent shortcomings of traditional PDT, showing great potential for clinical use.
Example 8
Example 1 (CyOA NPs) examination of antitumor efficacy against subcutaneous tumors of breast cancer in mice.
The invention utilizes the mouse 4T1 breast cancer subcutaneous tumor model to examine the antineoplastic effect of CyOA NPs under the illumination and non-illumination conditions, and compares the antineoplastic effect with the camptotheca of the commercial positive medicine. The method comprises the following specific steps:
At 6 weeks of age, 16-18g female BALB/c mice were inoculated subcutaneously with a 4T1 cell suspension of mouse breast cancer, about 1X 10 6 cells, and a 4T1 subcutaneous tumor mouse model of mouse breast cancer was established. When the tumor volume in situ was about 80mm 3, mice were randomly divided into 4 groups of 6, physiological Saline (Saline), cyOANPs-light, cyOA NPs + light and camptothecins+light, respectively. The dosage of CyOA NPs and camptotheca is equal to 10 mu mol/kg. The administration time on the first day is recorded as 1 day, and the above doses are administered on the 4 th, 7 th and 10 th days respectively, and after 12 hours of administration, the illumination group is illuminated. Illumination parameters: 200mW/cm 2, 660nm,10min. From day 1, the mice body weight and in situ tumor volume were measured once a day and tumor volume-time curves were plotted. Mice were sacrificed on day 12 and photographs taken of the peeled subcutaneous tumors.
Fig. 14, content (a) is a mouse tumor volume-time curve, and content (b) is a tumor photograph after exfoliation. It can be seen that CyOA NPs + light group can significantly inhibit tumor growth, the tumor inhibition rate reaches 67%, and the tumor inhibition rates of CyOA NPs-light group and camptotheca+ light group are 42% and 53% respectively. These results indicate that CyOA NPs has optimal in vivo photodynamic effects for tumor treatment, even above clinical camptothecins. Thus, the results further demonstrate that CyOA NPs, which is excellent in therapeutic effect but simple in process, shows great potential for clinical application.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. An unsaturated fatty acid-hemicyanine conjugate for use in the preparation of a photosensitizer, characterized by having a structure represented by formula (one):
wherein R 1 is alkyl with 1-10 carbon atoms;
r 2 is an unsaturated fatty chain derived from an unsaturated fatty acid having the formula C nH2n-1COOH、CnH2n-3COOH、CnH2n-5COOH、CnH2n-7 COOH or C nH2n-9 COOH, wherein n is an integer from 8 to 29.
2. The conjugate of claim 1, wherein n is an integer from 14 to 25.
3. The conjugate according to claim 1, characterized in that it is obtained by esterification of said unsaturated fatty acid with a phenolic hydroxyl group-containing hemicyanine.
4. An unsaturated fatty acid-hemicyanine conjugate nano-formulation, characterized in that it is a self-assembled nanoparticle dispersion of the unsaturated fatty acid-hemicyanine conjugate as claimed in any of claims 1 to 3.
5. The method of preparing a nano-formulation according to claim 4, comprising the steps of: and (3) dropwise adding an organic solution of the unsaturated fatty acid-hemicyanine conjugate dissolved in a benign organic solvent into deionized water in a stirring state, and finally dialyzing with the deionized water to obtain a self-assembled nanoparticle dispersion liquid, namely the unsaturated fatty acid-hemicyanine conjugate nano preparation.
6. The method of claim 5, wherein the benign organic solvent is one or more of dimethyl sulfoxide, methanol, ethanol, acetonitrile, and tetrahydrofuran.
7. Use of an unsaturated fatty acid-hemicyanine conjugate as claimed in any of claims 1 to 3 or an unsaturated fatty acid-hemicyanine conjugate nano-formulation as claimed in claim 4 for the preparation of a photosensitizer.
8. Use of an unsaturated fatty acid-hemicyanine conjugate as claimed in any of claims 1 to 3 or an unsaturated fatty acid-hemicyanine conjugate nano-formulation as claimed in claim 4 for the preparation of a mitochondria-targeted antitumor drug.
9. Use of an unsaturated fatty acid-hemicyanine conjugate as claimed in any of claims 1 to 3 or an unsaturated fatty acid-hemicyanine conjugate nano-formulation as claimed in claim 4 for the preparation of an oxygen-saving antitumor drug.
10. The use according to claim 8 or 9, wherein the tumour is breast cancer, liver cancer, colon cancer, ovarian cancer, melanoma or pancreatic cancer.
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| CN114748639B (en) * | 2022-03-28 | 2024-02-09 | 华中科技大学 | A photosensitizer-hydroxyalkyl starch-polypeptide coupled amphiphilic macromolecular compound, nano drug delivery system and preparation method thereof |
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| CN105617394A (en) * | 2016-01-26 | 2016-06-01 | 北京大学 | Self-assembled nano-system of unsaturated fatty acid-anti-tumor drug conjugates as well as preparation method and application thereof |
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