CN116425728A - Carboxylesterase-responsive diagnosis and treatment integrated probe and preparation method and application thereof - Google Patents
Carboxylesterase-responsive diagnosis and treatment integrated probe and preparation method and application thereof Download PDFInfo
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- CN116425728A CN116425728A CN202310363045.4A CN202310363045A CN116425728A CN 116425728 A CN116425728 A CN 116425728A CN 202310363045 A CN202310363045 A CN 202310363045A CN 116425728 A CN116425728 A CN 116425728A
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- 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
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- 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
- A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
- A61K41/0033—Sonodynamic cancer therapy with sonochemically active agents or sonosensitizers, having their cytotoxic effects enhanced through application of ultrasounds
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- 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
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- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
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Abstract
The invention belongs to the field of biological medicine, and in particular relates to a carboxylesterase response diagnosis and treatment integrated probe as well as a preparation method and application thereof. The invention discloses a small molecular diagnosis and treatment probe shown in the following structural general formula,
the small molecular diagnosis and treatment probe is prepared by adding near infrared fluorescent dye, chlorambucil, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 4-Dimethylaminopyridine (DMAP) into an organic solvent for reaction. The invention encapsulates the micromolecule diagnosis and treatment probe by using poly (lactic acid-co-glycolic acid) -polyethylene glycol (PLGA-PEG) to obtain the nanometer diagnosis and treatment integrated probe, and the fluorescence wavelength of the obtained nanometer diagnosis and treatment integrated probe is in a near infrared region, so that the near infrared fluorescence imaging diagnosis of tumors can be realized, and the nanometer diagnosis and treatment integrated probe has the combined treatment performance of chemistry, photodynamic and sonodynamic and can obviously kill tumor cells.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a carboxylesterase response diagnosis and treatment integrated probe as well as a preparation method and application thereof.
Background
The incidence and death rate of malignant tumors are continuously increased, the life and health of human beings are seriously threatened, and a heavy economic burden is brought to families and society. Therefore, there is a need for developing means for early detection of tumors and for effective new methods of treatment. The diagnosis and treatment integrated probe based on the molecular image integrates the molecular image module and the tumor treatment module simultaneously, not only can simultaneously meet the requirements of diagnosis and treatment, but also can visualize the in-vivo distribution and release process of the medicine, and the treatment mode of the imaging guidance is favorable for real-time monitoring of the treatment effect and timely optimization of the treatment scheme, and is hopeful to provide a new scheme for realizing accurate treatment and personalized treatment of tumors. Carboxylesterase is overexpressed in a variety of malignancies and is a potential tumor marker. Therefore, the diagnosis and treatment integrated probe for developing carboxylesterase response has great significance for early diagnosis and effective treatment of malignant tumors.
In recent years, near infrared fluorescence imaging based on a near infrared fluorescence probe can effectively avoid background fluorescence interference from organisms, has strong tissue penetrating capacity and small light damage to living bodies, and is widely used for research of early diagnosis of malignant tumors. Because of the inherent shortages of chemical, photodynamic and sonodynamic treatment means, the multi-means combined treatment can achieve better tumor inhibition effect. Therefore, the development of the molecular probe with near infrared fluorescence imaging and multi-means combined treatment provides a new technical means for the diagnosis and treatment integration of tumors.
However, near infrared fluorescent probes for carboxylesterase response and integrated application thereof in diagnosis and treatment of tumors are rarely reported at present, and are technical problems to be solved urgently.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides the carboxylesterase-responsive nano diagnosis and treatment integrated probe, the fluorescence wavelength of the probe is in a near infrared region, near infrared fluorescence imaging diagnosis of tumors can be realized, and the probe has the combined treatment performance of chemistry, photodynamic and sonodynamic and can obviously kill tumor cells.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
in a first aspect, the invention provides a carboxylesterase-responsive small molecule diagnosis and treatment probe, which has a structural general formula as follows:
wherein R is any one of methyl, ethyl and propyl.
In a second aspect, the invention provides a method for preparing a carboxylesterase-responsive small molecule diagnostic probe, the method comprising the steps of:
step 1: adding near infrared fluorescent dye, chlorambucil, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 4-Dimethylaminopyridine (DMAP) into an organic solvent for reaction;
step 2: and after the reaction is finished, separating and purifying to obtain the small molecular diagnosis and treatment probe.
Further, in the step 1, the organic solvent is at least one of tetrahydrofuran, N-dimethylformamide, dichloromethane, chloroform, acetonitrile and methanol; the structural formula of the near infrared fluorescent dye is as follows:
wherein R is any one of methyl, ethyl and propyl.
Further, in the step 1, the volume of the organic solvent is 1-20 mL; the molar ratio of the near infrared fluorescent dye to chlorambucil to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to 4-dimethylaminopyridine is 1 (1-5) (0.005-0.05); the reaction time is 10-200 min, and the reaction temperature is-10-50 ℃.
In a third aspect, the invention provides a carboxylesterase-responsive nano diagnosis and treatment integrated probe, which is obtained by encapsulating the small molecule diagnosis and treatment probe by poly (lactic-co-glycolic acid) -polyethylene glycol (PLGA-PEG) through a microemulsion method.
In a fourth aspect, the invention provides a method for preparing a carboxylesterase-responsive nano diagnosis and treatment integrated probe, which comprises the following steps:
step 1: adding the small molecular diagnosis and treatment probe and poly (lactic acid-co-glycolic acid) -polyethylene glycol into an organic solvent, performing ultrasonic treatment, adding an aqueous solution of polyvinyl alcohol, and continuing to react;
step 2: and after the reaction is finished, carrying out centrifugal treatment to obtain the nano diagnosis and treatment integrated probe.
Further, in the step 1, the organic solvent is at least one of tetrahydrofuran, dichloromethane, chloroform and cyclohexane.
Further, in the step 1, the volume of the organic solvent is 1-20 mL; the mass ratio of the small molecular diagnosis and treatment probe to the poly (lactic acid-co-glycolic acid) -polyethylene glycol is 1 (1-15); the aqueous solution of the polyvinyl alcohol is formed by dissolving 100-500 mg of polyvinyl alcohol in 5-50 mL of water; the ultrasonic time is 2-30 min, and the reaction time is 10-48 h.
In a fifth aspect, the invention provides an application of the carboxylesterase-responsive nano diagnosis and treatment integrated probe in preparation of a reagent for tumor diagnosis and/or tumor treatment.
Further, the tumor diagnosis is tumor near infrared fluorescence imaging diagnosis; the tumor treatment is combined treatment of chemistry, photodynamic and sonodynamic.
Compared with the prior art, the invention has the following beneficial effects:
1. the analysis wavelength of the invention is in the near infrared region, can effectively avoid the interference of organism background fluorescence, has strong tissue penetrating power and small damage to living body light.
2. The carboxylesterase-responsive nano diagnosis and treatment integrated probe prepared by the invention can show near infrared fluorescence enhancement response to carboxylesterase highly expressed by tumor cells, and can realize near infrared fluorescence imaging diagnosis of tumors.
3. The carboxylesterase-responsive nanometer diagnosis and treatment integrated probe prepared by the invention has the combined treatment performance of chemistry, photodynamic and sonodynamic, can obviously kill tumor cells and inhibit the growth of nude mice tumor, and is an excellent diagnosis and treatment reagent.
4. The carboxylesterase-responsive nano diagnosis and treatment integrated probe prepared by the invention has determined composition, good biological safety, no obvious toxicity to nude mice and meets the basic requirement of clinical medication.
5. The method has the advantages of simple operation, low cost, rapidness and sensitivity, and is easy to popularize and apply.
Drawings
FIG. 1 shows a small molecule diagnostic probe HC synthesized in example 1 1 H NMR chart.
FIG. 2 shows HC synthesized in example 1 13 C NMR chart.
Fig. 3 is a high resolution mass spectrum of HC synthesized in example 1.
Fig. 4 is a transmission electron microscope image of the encapsulated nano diagnosis and treatment integrated probe PLGA-peg@hc of example 1.
Fig. 5 is a particle size distribution diagram of the encapsulated nano-diagnosis and treatment integrated probe PLGA-peg@hc of example 1.
FIG. 6 is a graph showing the absorption spectrum response of PLGA-PEG@HC to carboxylesterase in the integrated probe for nano diagnosis and treatment in example 2, wherein curve a is the absorption spectrum of PLGA-PEG@HC (0.2 mg/mL), and curve b is the absorption spectrum after reaction of PLGA-PEG@HC (0.2 mg/mL) with carboxylesterase (100U/L).
FIG. 7 is a graph showing fluorescence response of PLGA-PEG@HC of the integrated probe for nano diagnosis and treatment to carboxylesterase with different concentrations, wherein the concentrations of carboxylesterase are 0,2,5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100U/L in sequence from bottom to top in the graph of example 2.
FIG. 8 is a confocal imaging of the nano-diagnostic integrated probe PLGA-PEG@HC of example 3 on HeLa and HcerEpic cells, wherein the first column is a white light channel and the second column is a fluorescent channel.
FIG. 9 is a confocal imaging of the nano-diagnostic integrated probe PLGA-PEG@HC of example 3 for carboxylesterase inhibitor triphenyl phosphate pretreatment of HeLa cells, wherein the first behavior is a white light channel and the second behavior is a fluorescence channel.
FIG. 10 is a graph of in vivo fluorescence imaging of different time nodes after intravenous injection of PLGA-PEG@HC into the tail of a tumor in example 4, with the control group on the left side and the experimental group on the right side of each graph.
FIG. 11 shows the change in fluorescence intensity at 525nm of DCFH-DA after 660nm laser irradiation in PBS (curve a) and PLGA-PEG@HC (0.2 mg/mL) with carboxylesterase (100U/L) reaction solution (curve b) in example 5.
FIG. 12 shows the change in absorbance at 425nm after ultrasonic stimulation in PBS (curve a) and PLGA-PEG@HC (0.2 mg/mL) and carboxylesterase (100U/L) reaction solution (curve b) for DPBF in example 5.
FIG. 13 shows the viability of HeLa cells (Panel A) and HcerEpic cells (Panel B) treated in the different ways described in example 6.
FIG. 14 is a graph showing the tumor volume change in different treatment groups of nude mice in example 7.
Fig. 15 is a photograph of tumors of the different treatment groups of nude mice of example 7 after 14 days of treatment.
FIG. 16 is a graph showing the change in body weight of nude mice in different treatment groups in example 8.
FIG. 17 shows the primary serum biochemical markers of the nude mice of the different treatment groups of example 8 after 14 days of treatment.
Fig. 18 is a pathological section analysis of the major viscera of the nude mice of the different treatment groups of example 8 after 14 days of treatment.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1 preparation of carboxylesterase-responsive nanometer diagnosis and treatment Integrated Probe PLGA-PEG@HC
The small molecule diagnosis and treatment probe HC is synthesized according to the following route:
the method comprises the following specific steps: near infrared fluorescent dye (410 mg,1 mmol), chlorambucil (450 mg,1.5 mmol), EDC (230 mg,1.2 mmol), DMAP (1.8 mg,0.01 mmol) were mixed well in dichloromethane (5 mL) and reacted at room temperature for 90min. Concentrating the mixed solution under reduced pressure, and purifying by column chromatography to obtain small molecular probe HC as purple powder.
Encapsulation of small molecule diagnostic probes HC:
1mg of HC and 5mg of PLGA-PEG are weighed, dissolved in 1mL of dichloromethane respectively, and after 5min of ultrasound at room temperature, the two are mixed, dichloromethane is added to 4mL, and ultrasound is continued for 5min. Polyvinyl alcohol (200 mg) is dissolved in 10mL of water, the organic phase is added, stirring is carried out for 24h at room temperature, centrifugal treatment is carried out, and the supernatant fluid is the encapsulated nanometer diagnosis and treatment integrated probe PLGA-PEG@HC.
The structure of the small molecule probe HC is confirmed as follows:
1 h NMR 13 C NMR diagrams are shown in FIGS. 1 and 2. 1 H NMR(400MHz,298K,CDCl 3 )：δ8.63(d,1H),7.49(m,2H),7.40(d,2H),7.27(s,1H),7.12(d,3H),7.08(s,1H),6.98(m,1H),6.77(d,1H),6.66(d,2H),4.57(t,2H),3.72(t,4H),3.64(t,4H),2.76(d,4H),2.65(m,4H),2.07(t,4H),1.98(t,2H),1.80(s,6H),1.08(t,3H). 13 C NMR(100MHz,298K,CDCl 3 ):δ178.8,171.7,160.0,153.1,152.8,146.4,144.7,142.2,141.5,131.1,130.3,130.2,129.9,129.5,128.3,128.0,122.6,119.8,119.1,115.6,113.5,112.4,112.4,109.5,106.5,53.7,51.1,47.7,40.7,34.0,33.8,29.6,28.3,28.3,26.7,24.3,21.7,20.3,11.6.
High resolution mass spectrometry: c (C) 42 H 47 Cl 2 N 2 O 3+ ，M + The method comprises the steps of carrying out a first treatment on the surface of the Calculated values: 697.2958; measurement value: 697.2954 (FIG. 3).
The transmission electron microscope (figure 4) and dynamic light scattering (figure 5) prove that the small molecular probe is successfully encapsulated, and the obtained nanometer diagnosis and treatment integrated probe PLGA-PEG@HC is spherical with uniform size, has the particle size of about 100nm and has good dispersibility.
Example 2 optical response of nanodiagnostic Integrated Probe PLGA-PEG@HC to carboxylesterase
Weighing 0.2g of nanometer diagnosis and treatment integrated probe PLGA-PEG@HC, dissolving in 10mL of ultrapure water, and preparing a probe stock solution (also called mother solution, the concentration is 20 mg/mL); mu.L of probe stock solution was added to a certain amount of phosphate buffer solution (PBS, 0.01M), then carboxylesterase solutions of different concentrations were added, and the volume was fixed to 5mL with PBS. After reaction at 37 ℃ for 10 minutes, the absorption spectrum and fluorescence emission spectrum were tested. Excitation was carried out at 670nm and excitation and emission slit widths were 10nm and 5nm, respectively, when fluorescence emission spectrum was measured.
FIG. 6 shows the absorption spectrum response of the nano diagnosis and treatment integrated probe PLGA-PEG@HC to carboxylesterase, wherein a is the absorption spectrum of PLGA-PEG@HC (0.2 mg/mL), and b is the absorption spectrum after the reaction of PLGA-PEG@HC (0.2 mg/mL) and carboxylesterase (100U/L). FIG. 7 shows fluorescence response of nanometer diagnosis and treatment integrated probe PLGA-PEG@HC to carboxylesterase, and the concentration range of carboxylesterase is 0-100U/L. The carboxylesterase concentrations in FIG. 7 were 0,2,5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100U/L in order from bottom to top. The experiment shows that the nanometer diagnosis and treatment integrated probe PLGA-PEG@HC shows remarkable fluorescence enhancement response to carboxylesterase.
Example 3 nanometer diagnosis and treatment Integrated Probe PLGA-PEG@HC for tumor cell imaging
(1) At 37 DEG CAnd 5% CO 2 Human cervical cancer cells HeLa and human normal cervical epithelial cells HcerEpic were cultured in DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, 100. Mu.g/mL streptomycin under conditions.
(2) HeLa and HcerEpic cells in logarithmic growth phase are inoculated into a laser confocal dish, the culture solution is discarded after 24 hours of culture, the culture solution is washed three times by PBS (pH 7.4), and then the culture solution is incubated with PLGA-PEG@HC (1 mg/mL) prepared by the invention for 30 minutes and then is imaged by a laser confocal microscope. The imaging excitation wavelength is 635nm, and the collection range of the emission wavelength is 650-750nm.
FIG. 8 shows confocal imaging of HeLa and HcerEpic cells, which can be seen that HeLa cells showed bright red fluorescence, while normal cells had weak HcerEpic fluorescence, indicating that PLGA-PEG@HC can selectively image tumor cells.
FIG. 9 shows confocal imaging of PLGA-PEG@HC for HeLa cells pre-incubated with the carboxylesterase inhibitor triphenyl phosphate. The fluorescence of the cells pretreated with triphenyl phosphate in the inhibitor group is significantly reduced, which indicates that the fluorescence enhancement response of PLGA-PEG@HC in tumor cells is caused by carboxylesterase over-expressed by tumor cells.
Example 4 nanometer diagnosis and treatment Integrated Probe PLGA-PEG@HC for in vivo tumor imaging of nude mice
(1) The experiment is approved by the ethical and use committee of animals of Shanxi medical university, the experimental animals are BALB/c nude mice of 5 weeks old, and 15 g+/-1 g/animal weight are all fed in a sterile environment. Tumor model was established using HeLa cells, which were grown at 5X 10 6 Dilution was performed at a density of one/mL, placed on ice, 200 μl of cell suspension was taken after anesthetizing a nude mouse with 6% chloral hydrate, subcutaneously injected into the right side of the nude mouse, the weight of the nude mouse was weighed every other day and the tumor size was measured, and the formula v=a×b was calculated 2 Calculating tumor volume (a is long diameter, b is short diameter) until the volume exceeds 50mm 3 Imaging experiments were performed at that time.
(2) Taking a bare mouse which is successfully modeled, injecting PLGA-PEG@HC (final concentration is 10 mg/mL) into a tail vein, performing fluorescence imaging on the bare mouse at different time nodes, and observing the distribution of fluorescence in vivo. The excitation wavelength is 670nm during imaging, and the emission wavelength is 750nm.
Fig. 10 shows in vivo fluorescence imaging of nude mice at different time nodes. The control group (PBS injected into tail vein) has no fluorescence all the time, and the tumor of the nude mice injected with PLGA-PEG@HC emits fluorescence in 2 hours, and reaches the maximum in 8 hours, and the fluorescence of the tumor part is far higher than that of other parts. The PLGA-PEG@HC can reach the tumor site of the nude mice through passive targeting and specifically turn on fluorescent signals, and has potential application value in tumor imaging diagnosis.
Example 5 in vitro photodynamic and sonodynamic effects of nanometer diagnosis and treatment Integrated Probe PLGA-PEG@HC
To evaluate the photodynamic and photodynamic effects of PLGA-peg@hc, the performance of the system in generating active oxygen under laser irradiation and ultrasonic stimulation was evaluated using commercial active oxygen probes 2',7' -dichlorofluorescein diacetate (DCFH-DA) and 1, 3-diphenyl isochroman (DPBF), respectively. The fluorescence intensity of DCFH-DA at 525nm is positively correlated with the active oxygen concentration, and the absorbance of DPBF at 425nm is negatively correlated with the singlet oxygen concentration. PLGA-PEG@HC (0.2 mg/mL) and carboxylesterase (100U/L) are reacted for 20min in a shaking table at 37 ℃, activated DCFH-DA (5 nM) is added into the reaction system, and then the reaction solution is irradiated with 660nM laser for a certain time to test fluorescence spectrum to evaluate the photodynamic performance, and the fluorescence excitation wavelength is 490nM. DPBF (20. Mu.g/mL) was added to the reaction system, followed by sonication (1W/cm) 2 ) After various times, absorbance at 425nm was recorded to evaluate its sonodynamic performance.
FIG. 11 shows the fluorescence at 525nm as a function of the laser irradiation time for different systems (a is DCFH-DA, b is the reaction solution of DCFH-DA and PLGA-PEG@HC and carboxylesterase). It can be seen that along with the extension of the laser irradiation time, the fluorescence of DCFH-DA at 525nm in the reaction system of PLGA-PEG@HC and carboxylesterase is gradually enhanced, and the laser irradiation hardly influences the fluorescence of DCFH-DA, which indicates that the reaction system of PLGA-PEG@HC and carboxylesterase can generate active oxygen under the irradiation of 660nm laser, and the photodynamic therapy potential is provided.
FIG. 12 is a graph showing the absorbance at 425nm versus ultrasound time for different systems (a is DPBF and b is the reaction solution of DPBF with PLGA-PEG@HC and carboxylesterase). It can be seen that the absorbance of DPBF at 425nm in the reaction system of PLGA-PEG@HC and carboxylesterase gradually decreases along with the extension of ultrasonic time, and the absorbance of DPBF is hardly affected by ultrasonic, which indicates that the reaction system of PLGA-PEG@HC and carboxylesterase has the capability of generating singlet oxygen under ultrasonic stimulation and can be used for sonodynamic therapy.
Example 6 nanometer diagnosis and treatment Integrated Probe PLGA-PEG@HC for selectively killing tumor cells
The killing properties of PLGA-PEG@HC on different cells were evaluated by MTT method. HeLa and HcerEpic cells in logarithmic growth phase are inoculated in a 96-well plate, PLGA-PEG@HC with different concentrations is added after wall-attaching culture for 24 hours, and incubated for 24 hours, the culture solution is discarded, and after 3 times of washing with PBS, a laser group is irradiated with 660nm laser (0.25W/cm) 2 ) Irradiating for 10min, and intermittently ultrasonic (0.75W/cm 2 ) Treatment is carried out for 5min (namely, ultrasonic treatment is carried out for 15s and intermittent treatment is carried out for 15s, and total time is 5 min), the compound group is processed by superposition of laser and ultrasonic treatment, and the control group is placed in a dark environment. After 3 washes of cells with PBS, 100. Mu.L of MTT solution (0.5 mg/mL) was added to each well, the MTT was discarded after further incubation for 4 hours, and 150. Mu.L of DMSO was added after PBS wash. And finally, measuring absorbance at 490nm by using an enzyme-labeled instrument, and calculating the cell survival rate.
FIG. 13 shows the viability of HeLa (Panel A) and HcerEpic (Panel B) cells after treatment in different ways. It can be seen that the viability of HeLa cells gradually decreased with increasing PLGA-PEG@HC concentration, with HeLa cells viability only 66% at a concentration of 2.0mg/mL, while the viability of HcerEpic cells was still as high as 86%. This is due to the fact that carboxylesterase overexpressed in HeLa cells initiates hydrolysis of the probe, and the released chemotherapeutic agent chlorambucil has a killing effect on the cells. In addition, in the presence of 660nm laser or ultrasonic stimulation alone, the survival rate of HeLa cells incubated by PLGA-PEG@HC is further reduced, which indicates that PLGA-PEG@HC has the capacity of killing by sound power and light power on the cell level; the survival rate of HeLa cells is the lowest when ultrasound and laser are simultaneously present, only 18% remains, while the survival rate of HcerEpic cells under the same conditions is still above 60%. These results indicate that the probe PLGA-PEG@HC can kill tumor cells in a combined way through chemotherapy, photodynamic therapy and sonodynamic therapy, and has lower toxic and side effects on normal cells.
Example 7 nanometer diagnosis and treatment Integrated Probe PLGA-PEG@HC for anti-tumor treatment of nude mice
The tumor model nude mice were established as in example 4 until the tumor volume exceeded 100mm 3 Treatment was started at that time. Nude mice were randomly divided into 7 groups (n=3) and the corresponding treatment regimen was taken. The grouping situation is as follows: PBS group, PBS+laser (L) +ultrasonic (US) group, HC+L+US group, PLGA-PEG@HC group, PLGA-PEG@HC+US group, PLGA-PEG@HC+L group and PLGA-PEG@HC+L+US group. The probe was injected into nude mice by tail vein at the time of treatment (100. Mu.L, wherein PLGA-PEG@HC concentration is 10mg/mL, HC concentration is 13.95. Mu.g/mL), and 660nm laser (0.75W/cm) was used for 8h post injection 2 ) An ultrasonic instrument for ultrasonic group (1.5W/cm) 2 ) And carrying out ultrasonic treatment for 5min, wherein the composite group is subjected to laser and ultrasonic superposition treatment. The treatment is carried out once every two days for 3 times. During treatment, tumor length and diameter were measured every other day, according to formula v=a×b 2 The tumor volume was calculated by 2 (a is the long diameter and b is the short diameter). After the treatment is completed, the tumor is dissected and photographed.
Fig. 14 shows tumor volume change curves of different groups of nude mice, and fig. 15 shows tumor pictures of different groups of nude mice after 14 days of treatment. These results indicate that PLGA-PEG@HC can exert antitumor effect in combination with chemotherapy, photodynamic therapy and sonodynamic therapy.
Example 8 biological safety of nanodiagnostic Integrated Probe PLGA-PEG@HC
The body weight of the nude mice was recorded every other day during the 14 day treatment period in example 7. After the treatment period of 14 days is finished, the nude mice are anesthetized, the eyesockets are sampled, the collected blood samples are centrifuged (3000 rpm/min) for 10min, and upper serum is taken and respectively detected by a biochemical analyzer to obtain liver function indexes alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) and kidney function indexes urea nitrogen (BUN) and Creatinine (CRE); after the above procedure, nude mice were sacrificed, hearts, livers, spleens, lungs, kidneys were removed, fixed with 4% paraformaldehyde, paraffin-embedded sections were stained with hematoxylin and eosin, and observed under a fluorescence microscope.
FIG. 16 is a graph showing the weight change curves of different groups of nude mice, each group of nude mice having slightly increased weight; FIG. 17 shows the main serum biochemical indicators of different groups of nude mice after 14 days of treatment, and it can be seen that the liver function and kidney function indicators of the nude mice in each treatment group have no significant difference compared with the control group; fig. 18 shows pathological section analysis of main organs of different groups of nude mice after 14 days of treatment, and the results show that the main organs are not obviously abnormal, and the results all show that the nanometer diagnosis and treatment integrated probe PLGA-PEG@HC has good biological safety, and no obvious side effect is caused in tumor treatment based on the probe.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The carboxylesterase-responsive small molecule diagnosis and treatment probe is characterized by having a structural general formula:
wherein R is any one of methyl, ethyl and propyl.
2. A method for preparing a carboxylesterase-responsive small molecule diagnostic probe of claim 1, comprising the steps of:
step 1: adding near infrared fluorescent dye, chlorambucil, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 4-dimethylaminopyridine into an organic solvent for reaction;
step 2: and after the reaction is finished, separating and purifying to obtain the small molecular diagnosis and treatment probe.
3. The method for preparing a carboxylesterase-responsive small molecule diagnosis and treatment probe according to claim 2, wherein in the step 1, the organic solvent is at least one of tetrahydrofuran, N-dimethylformamide, dichloromethane, chloroform, acetonitrile and methanol; the structural formula of the near infrared fluorescent dye is as follows:
wherein R is any one of methyl, ethyl and propyl.
4. The method for preparing a carboxylesterase-responsive small molecule diagnosis and treatment probe according to claim 2, wherein in the step 1, the volume of the organic solvent is 1-20 mL; the molar ratio of the near infrared fluorescent dye to chlorambucil to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to 4-dimethylaminopyridine is 1 (1-5) (0.005-0.05); the reaction time is 10-200 min, and the reaction temperature is-10-50 ℃.
5. The carboxylesterase-responsive nano diagnosis and treatment integrated probe is characterized in that the nano diagnosis and treatment integrated probe is obtained by encapsulating the small molecule diagnosis and treatment probe in claim 1 through poly (lactic acid-co-glycolic acid) -polyethylene glycol by a microemulsion method.
6. A method for preparing the carboxylesterase-responsive nano-diagnosis and treatment integrated probe as claimed in claim 5, which is characterized by comprising the following steps:
step 1: adding the small molecular diagnosis and treatment probe and the poly (lactic acid-co-glycolic acid) -polyethylene glycol according to claim 1 into an organic solvent, performing ultrasonic treatment, adding an aqueous solution of polyvinyl alcohol, and continuing to react;
step 2: and after the reaction is finished, carrying out centrifugal treatment to obtain the nano diagnosis and treatment integrated probe.
7. The method for preparing carboxylesterase-responsive integrated probe for nano diagnosis and treatment according to claim 6, wherein in the step 1, the organic solvent is at least one of tetrahydrofuran, dichloromethane, chloroform and cyclohexane.
8. The method for preparing carboxylesterase-responsive integrated probe for nano diagnosis and treatment according to claim 6, wherein in the step 1, the volume of the organic solvent is 1-20 mL; the mass ratio of the small molecular diagnosis and treatment probe to the poly (lactic acid-co-glycolic acid) -polyethylene glycol is 1 (1-15); the aqueous solution of the polyvinyl alcohol is formed by dissolving 100-500 mg of polyvinyl alcohol in 5-50 mL of water; the ultrasonic time is 2-30 min, and the reaction time is 10-48 h.
9. Use of the carboxylesterase-responsive nano-diagnostic integrated probe of claim 5 in the preparation of a reagent for tumor diagnosis and/or tumor treatment.
10. The use of a carboxylesterase-responsive integrated nanodiagnostic probe as claimed in claim 9, wherein said tumor diagnosis is a near infrared fluorescence imaging diagnosis of a tumor; the tumor treatment is combined treatment of chemistry, photodynamic and sonodynamic.
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| CN117731664B (en) * | 2023-12-20 | 2024-05-10 | 山东第一医科大学(山东省医学科学院) | Preparation and application of tumor MRI diagnosis and treatment integrated nano probe for acidosis monitoring |
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