CN115449088B - High-pore COF nano-particle, preparation method thereof and application thereof as drug carrier - Google Patents
High-pore COF nano-particle, preparation method thereof and application thereof as drug carrier Download PDFInfo
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Abstract
The invention discloses a high-pore COF nano particle, a preparation method thereof and application thereof in a drug carrier, wherein the preparation method comprises the following steps: (1) Synthesis of COP nanoparticles: the method comprises the steps of using 4,4' -dithiodibenzoyl benzaldehyde and 1,3,5-tri (aminomethyl) benzene tri-hydrochloride as raw materials, using trifluoroacetic acid or acetic acid as a catalyst for Schiff base reaction, and synthesizing amorphous covalent organic polymer COP nano particles containing disulfide bonds in a Schiff base imine bond connection mode; (2) synthesizing the high-pore COF nano-particles by a recrystallization method: dispersing the prepared COP nano particles into etching liquid, fully reacting at 50-120 ℃, centrifugally collecting Gao Jiekong COF nano particles, and washing to remove residual reactants. The COF nano-carrier with high crystallinity and rich pores synthesized by the method can realize layered loading and cascade response release of Brig and Osi medicaments.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a high-pore COF nanoparticle, a preparation method thereof and application thereof as a drug carrier.
Background
Lung cancer is the leading cause of cancer-related death worldwide, with non-small cell lung cancer accounting for 85% of lung cancer, with a survival rate of only 19% in 5 years. Ornitinib (Osi) is approved by the FDA as a third generation EGFR-TKI for the first and second line treatment of EGFR mutant advanced NSCLC patients, and has remarkable curative effect. Patients receiving octenib treatment eventually develop resistance to disease progression. Previously reported, chemotherapeutic agents: cetuximab; ALK inhibitors: the combination of drugs such as the bujinib (Brigerinib, brig) and the like and the Ornithine can effectively overcome the acquired drug resistance of the Ornithine. However, tumor heterogeneity determines maldistribution of drug-resistant cells, and traditional administration methods limit local concentration of drug in drug-resistant cells, and increase drug dosage can increase toxic and side effects. How to increase the drug concentration of the drug-resistant tumor part is the key to enhance the anti-tumor curative effect.
Nanotechnology is a new strategy for delivering drugs. Covalent Organic Frameworks (COFs) are crystalline porous polymers linked by covalent bonds. Compared with the traditional high polymer material, the COF has the advantages of good chemical stability, abundant adjustable micropore structure, predictability control on composition, topology and porosity and the like, and can easily fix diagnostic and therapeutic drugs in the structure of the COF, thereby realizing effective loading of the drugs. Meanwhile, COF has better biocompatibility, more promising biodegradation and easier surface functionalization compared with emerging inorganic nanoparticles (metal, carbon, silica, etc.), and is widely used for drug delivery. Although current COF materials have the advantage of a rich, regular micro-channel structure. However, it has a low porosity and it is difficult to achieve independent loading and cascade response release of two or more drugs (Wang B, liu X, gong P, et al Fluorescent COFs with a highly conjugated structure for visual drug loading and responsive release [ J ]. Chemical Communications,2020,56 (4): 519-522.). Therefore, there is an urgent need to develop a COF nanodrug delivery system with high crystallization and multiple pores for multiple drug delivery.
Because the nano material has no targeting property, the nano material can only be passively enriched to the tumor part by means of the enhanced blocking and permeation effect (EPR effect). How to realize the active targeting of nano-carriers to tumor cells has been the direction of efforts. Cancer cell membranes are reported to have immune escape and homoadhesive capacity. Thus, a large number of researchers have employed cancer cell membranes to camouflage nanoparticles for specific binding to cognate cells. However, individual targeting of sensitive and drug resistant cells has not been considered. The current technical means still cannot realize the function of targeting the Ornitinib drug-resistant cells. Therefore, how to achieve active targeting of drug-loaded systems to drug-resistant cells, but not to sensitive cells, is yet another key challenge faced by people.
Disclosure of Invention
The inventor unexpectedly discovers that the drug-carrying system wrapped by the drug-resistant cell membrane of the Ornitinib has stronger targeting to drug-resistant tumors, but the nanometer drug-carrying system wrapped by the sensitive cell membrane cannot distinguish sensitive cells from drug-resistant cells. Aiming at the difficult problems of loading double drugs on a COF nano-carrier and responding to cascade response release, a recrystallization method is adopted to successfully synthesize the COF nano-carrier with high crystallinity and rich pores so as to realize layered loading and cascade response release of Brig and Osi drugs. Aiming at the difficult problem that the active targeting functionality of a COF nano drug-carrying system on drug-resistant tumors is poor, the targeted specificity on the drug-resistant tumors can be realized by adopting the drug-carrying system wrapped by the Ornitinib drug-resistant cell membrane.
Based on the above, the invention protects the following technical scheme:
a method for preparing high pore COF nanoparticles, comprising the steps of:
(1) Synthesis of COP nanoparticles: the method comprises the steps of using 4,4' -dithiodibenzoyl benzaldehyde and 1,3,5-tri (aminomethyl) benzene tri-hydrochloride as raw materials, using trifluoroacetic acid or acetic acid as a catalyst for Schiff base reaction, and synthesizing amorphous covalent organic polymer COP nano particles containing disulfide bonds in a Schiff base imine bond connection mode;
(2) High pore COF nano-particles are synthesized by a recrystallization method: dispersing the prepared COP nano particles into etching liquid, fully reacting at 50-120 ℃, centrifugally collecting Gao Jiekong COF nano particles, and washing to remove residual reactants to obtain the nano-particles.
In the step (1), the solvent of 4,4 '-dithiodibenzoyl aldehyde and 1,3,5-tri (aminomethyl) benzene tri-hydrochloride is acetonitrile, and trifluoroacetic acid is added into the acetonitrile dispersion solution of 4,4' -dithiodibenzoyl aldehyde, and stirring and fully reacting are carried out; and adding acetonitrile solution of 1,3,5-tri (aminomethyl) benzene tri hydrochloride, fully reacting at room temperature, centrifugally collecting a product, washing with ethanol and N, N-dimethylacetamide to remove residual reactants, and obtaining COP nano particles.
The catalyst is trifluoroacetic acid, the amount of the trifluoroacetic acid is 400-600 mu L of the trifluoroacetic acid used for each 0.01 mol of 4,4 '-dithiodibenzoyl benzaldehyde, and preferably 450-550 mu L or 500 mu L of the trifluoroacetic acid is used for each 0.01 mol of 4,4' -dithiodibenzoyl benzaldehyde;
preferably, the concentration of the 4,4' -dithiodibenzoyl benzaldehyde solution is 0.35-0.55mM or 0.4-0.5mM, and the concentration of the 1,3, 5-tris (aminomethyl) benzene tri-hydrochloride solution is 0.2-0.4mM or 0.25-0.35mM;
preferably, the ratio of the amount of 4,4' -dithiodibenzoyl aldehyde to the amount of 1,3, 5-tris (aminomethyl) benzene tri-hydrochloride is 3:2.
Dispersing the prepared COP nano particles into etching liquid in the step (2), and reacting for 12-60 hours, preferably 42-54 hours, 44-52 hours or 48 hours at 50-100 ℃ or 50-90 ℃ or 60-80 ℃; centrifugally collecting mesoporous COF nano particles, and washing the mesoporous COF nano particles with ethanol to remove residual reactants;
the etching liquid comprises: 10-40 ml of N, N-dimethylacetamide, 10-40 ml of 1,3, 5-trimethylbenzene, 0.5-3ml of water and 0.5-2.5ml of trifluoroacetic acid, wherein the volume ratio of the N, N-dimethylacetamide to the 1,3, 5-trimethylbenzene is as follows: 1 to 4:1 to 4, preferably 4:1; the volume of water is preferably 2ml, and the volume of trifluoroacetic acid is preferably 2ml.
The ratio of the COP nanoparticles to the etching solution is 1 mg/40-60 ml, preferably 1 mg/45-55 ml or 50ml, based on the mass of the COP nanoparticles to the volume of the etching solution.
The invention also protects the high pore COF nano particles prepared by the preparation method.
The invention also protects the application of the high-pore COF nano-particles in serving as a drug carrier.
According to the application technical scheme, the application is that a bionic drug-carrying system for targeting lung cancer drug-resistant tumors is prepared by taking COF nano-particles as drug carriers, the bionic drug-carrying system is obtained by anchoring a buntinib drug to the COF nano-particles to obtain a COF-Brig nano-material, embedding an Ornitinib drug into a mesoporous structure of the COF-Brig nano-material to obtain the COF-Brig@Osi nano-material, and then wrapping the COF-Brig@Osi nano-material by adopting cell membranes of lung cancer Ornitinib drug-resistant cells.
In the application technical scheme, the COF-Brig nano material is synthesized by anchoring a buntinib drug molecule through a hydrogen bond by utilizing a lone pair electron on imine nitrogen in the COF nano particles; the preparation of the COF-Brig@Osi nanomaterial is to embed the Ornitinib drug into the mesoporous structure of the COFs material by a BSA embedding method.
In the application technical scheme, a physical extrusion method is adopted to wrap the cell membrane of the lung cancer octenib drug-resistant cell on the surface of the COF-Brig@Osi nanomaterial.
The preparation method of the bionic drug-carrying system for targeting lung cancer drug-resistant tumors comprises the following steps:
(1) The method comprises the steps of dissolving a buntinib drug into THF or absolute ethanol solution, adding the COFs nano-particles into the buntinib solution, uniformly dispersing, stirring, fully reacting, centrifuging and washing after the reaction is finished, and removing residual buntinib drug to obtain COF-Brig nano-particles;
the molar mass ratio of the buntinib drug to the COFs nano-particles is 0.01-10 mmol/1 mg, preferably 0.01-0.1 mmol/1 mg or 0.01-0.05 mmol/1 mg or 0.02-0.05 mmol/1 mg;
adding the COFs nano particles into a bunatinib solution, carrying out ultrasonic oscillation until the COFs nano particles are uniformly dispersed, stirring overnight for reaction, and washing with ethanol after centrifugation.
(2) Loading an osiertinib drug: the Ornitinib drug is captured by BSA or polyethylene glycol and embedded into the COFs mesoporous: adding COF-Brig nano particles into the Ornitinib solution, dispersing uniformly, dripping the dispersion liquid into the BSA solution, stirring, fully reacting, centrifuging and washing to remove residual Ornitinib drug after the reaction is finished, and obtaining the COF-Brig@Osi nano material;
the solvent of the solution of the Ornitinib is ethanol, the molar mass ratio of the Ornitinib to the COF-Brig nano particles is 0.001-10 mmol/1 mg, preferably 0.004-10 mmol/1 mg or 0.004-1 mmol/1 mg or 0.004-0.1 mmol/1 mg or 0.004-0.05 mmol/1 mg or 0.004-0.02 mmol/1 mg, the COF-Brig nano particles are added into the solution of the Ornitinib, the dispersion is uniformly dispersed by adopting ultrasonic oscillation, the dispersion is dripped into the BSA solution, the stirring reaction is carried out for 2-10 hours, the reaction is finished, and the residual Ornitinib medicine is removed by washing with deionized water;
the BSA solution preparation method comprises the following steps: 10mgBSA was dispersed in 1ml of 2.5mM NaCl solution, and 200. Mu.L of 0.1mM NaCl solution was added thereto after complete dissolution.
(3) Wrapping cell membranes of the lung cancer octenib drug-resistant cells on the surface of a COF-Brig@Osi nano material by adopting a physical extrusion method to obtain a drug-carrying system COF-Brig@Osi-M nano particle;
uniformly mixing COF-Brig@Osi nano particles and cell membranes of lung cancer octenib drug-resistant cells, repeatedly extruding the mixture through a nanoscale polycarbonate filter membrane by using an extruder, and then centrifuging at a high speed to obtain COF-Brig@Osi-M nano particles;
the mass ratio of the COF-Brig@Osi nano particles to the cell membrane of the lung cancer octenib resistant cells is 1:2-7, preferably 1:3-7 or 1:4-6 or 1:5;
preferably, the pore diameter of the nano-grade polycarbonate filter membrane is 200nm; the high-speed centrifugation was performed at 15000rpm for 10min.
The bionic drug-carrying system for targeting lung cancer drug-resistant tumors can be applied to delivering the anti-tumor drugs of the bujiatinib and the Oritinib or applied to preparing tumor treatment drugs. Wherein the tumor is lung cancer, preferably non-small cell lung cancer; preferably, the tumor is of the type of acquired resistance to octreotide.
The beneficial effects of the invention are as follows:
aiming at the difficult problems of loading double drugs on a COF nano-carrier and responding to cascade response release, a recrystallization method is adopted to successfully synthesize the COF nano-carrier with high crystallinity and rich pores so as to realize layered loading and cascade response release of Brig and Osi drugs.
Aiming at the problem that the active targeting function of a COF nano drug-carrying system on drug-resistant tumors is poor, the drug-carrying system wrapped by the drug-resistant cell membrane of the Ornitinib (the synthetic process of the drug-carrying system is shown in figure 15) can realize the specific targeting on the drug-resistant tumors.
The drug carrying system can realize the on-demand release of the anti-tumor drug, greatly improve the concentration of the anti-tumor drug at the drug-resistant tumor part, and further achieve the effect of enhancing the anti-tumor.
The high-pore COF nano drug-carrying system wrapped by the oritinib resistant cell membrane is expected to be an effective means for overcoming the oritinib resistance. On one hand, the system can realize active targeted delivery of drug-resistant tumor cells; on the other hand, the cascade responsive release of the anti-tumor drug in the drug-resistant cells can be realized, so that the anti-tumor effect is enhanced.
Drawings
Fig. 1 is a TEM image of the synthesized COP.
Fig. 2 is a TEM and SEM image of high pore COF nanoparticles.
FIG. 3 is a TEM structure diagram of COF-Brig.
FIG. 4 is a TEM structure diagram of COF-Brig@Osi.
Fig. 5 is a loading optimization and encapsulation of high pore COF nanoparticle loaded brigerinib and osimerinib drugs.
Fig. 6 is a graph showing the change in specific surface area of the high pore COF nanocarriers before and after loading the dual drug.
FIG. 7 is a TEM image of synthesized COF-Brig@Osi-M nanoparticles.
FIG. 8 shows the change in particle size of Gao Jiekong COF nanodrug delivery system before and after encapsulation of cell membranes.
FIG. 9 shows the protein profile of the cell membrane (SDS-PAGE) of the material after encapsulation of the cell membrane by the Gao Jiekong COF nanodrug delivery system.
FIG. 10 shows the inhibitory effect of COF-Brig@Osi-M nanoparticles on the activity of PC-9OR resistant cells and the IC50 value thereof.
FIG. 11 is a COF-Brig@Osi-M nanoparticle inhibition PC-9OR drug resistant cell clone formation assay.
FIG. 12 is a nude mouse injected COF Cy5 -Brig@Osi-M PC-9 Fluorescence intensity of the nanoparticle group main organ and tumor.
FIG. 13 is a nude mouse injected COF Cy5 -Brig@Osi-M PC-9OR1 Fluorescence intensity of the nanoparticle group main organ and tumor.
FIG. 14 shows the expression of N-cadherin, EPCAM and Galectin-3 proteins in sensitive and drug resistant cell membranes.
Fig. 15 is a schematic representation of the synthetic process of the drug delivery system of the present invention.
Detailed Description
The invention is further illustrated, but is not limited, by the following examples.
The experimental methods in the following examples are conventional methods unless otherwise specified; the chemical reagents and materials used, unless otherwise specified, are all conventional in the art and are commercially available.
Major reagents and material sources:
4,4' -dithiodibenzoyl benzaldehyde: CAS:100538-31-6,
1,3, 5-tris (aminomethyl) benzenetricarboxylic acid hydrochloride: CAS number: 69146-57-2,
PC-9OR cell line: the human non-small cell lung cancer Ornitinib drug resistant cell strain is obtained by the establishment of the laboratory.
PC-9 cells: human lung adenocarcinoma cells, commercially available.
Example 1 preparation of COF-Brig@Osi
1. Synthesis of COP nanoparticles:
amorphous covalent organic polymer (covalent organic polymers, COP) nanoparticles containing disulfide bonds are synthesized by adopting a Schiff base type imine bond connection mode:
4,4 '-Dithiodibenzoyl (4, 4' -disulanediyldibenzoaldehyde, 0.45 mM) was dissolved in acetonitrile, and after complete dispersion, 500. Mu.L of trifluoroacetic acid was rapidly added to 25ml of the dispersion solution, followed by magnetic stirring for 10 minutes. 25ml of a solution of 1,3, 5-tris (aminomethyl) Benzene tri-hydrochloride (Benzene-1, 3,5-triyltrimethanamine,0.3 mM) in acetonitrile was slowly added to the above solution, reacted at room temperature for 0.5 hours, and the product was collected by centrifugation, washed with ethanol and N, N-dimethylacetamide several times to remove the residual reactants, and COP nanoparticles were obtained, and a TEM image of the synthesized COP was shown in FIG. 1. 2. Synthesis of high pore COF nanoparticles (COFs) by recrystallization
The prepared 1mg COP nanoparticles were dispersed in 50ml of an etching solution (etching solution component: 40ml of N, N-dimethylacetamide, 10ml of 1,3, 5-trimethylbenzene, 2ml of water and 2ml of trifluoroacetic acid) prepared in advance, reacted at 70℃for 48 hours, and then the mesoporous COF nanoparticles were collected by centrifugation and washed with ethanol multiple times. TEM and SEM images of the formed high pore COF nanoparticles are shown in FIG. 2, and the particle size of the COF nanoparticles synthesized by recrystallization is not changed much from that of the COP nanoparticles to about 160-170nm, but the COFs nanoparticles exhibit a significant macroporosity structure, which is very advantageous for loading drugs.
3. The high pore COF nanoparticle is loaded with brigerinib and Osimertinib antitumor drugs:
(1) The high pore COF nano material is loaded with Brigerinib drug (namely COF-Brig):
the bunatinib molecule is anchored by a lone pair of electrons on the imine nitrogen in COFs: the bunatinib drug was dissolved in anhydrous Tetrahydrofuran (THF) solution, and 1mg COFs nanoparticles were dispersed in bunatinib THF solution (10 ml) of different concentrations (1, 2,5,10 mm), sonicated for 10min, and stirred magnetically overnight. After the reaction is completed, the mixture is centrifuged and washed with ethanol for a plurality of times to remove the residual buntinib drug.
Optimizing the loading and encapsulation rate of brigerinib drug: as a result of detection, the 1mg COFs showed that the loading amounts of 1mg COFs to 1,2,5,10mM of bujitinib were 0.05, 0.13, 0.33, and 0.41mM, respectively, and the encapsulation rates were 21.4, 24.0, 28.3, and 19.6%, respectively. Thus, the optimal bunatinib loading conditions were determined as: 5mM buntinib solution, its corresponding loading and encapsulation rates were: 0.33mM and 28.3%.
The TEM structure of the COF-Brig nanoparticle after being loaded with the buntinib is shown in FIG. 3, and the corresponding BET specific surface area is shown in FIG. 6. Comparing TEM results of COF nanoparticles before loading, it can be seen that part of mesoporous structure of COF after loading is occupied by drug, and BET results further reveal that specific surface area of COF-Brig nanoparticles is reduced. FIG. 8 shows a graph of nanoparticle hydrated particle size distribution, with particle size of COF-Brig comparable to COF after Brigerinib loading, of about 170nm.
(2) The high pore COF nano material is loaded with Brigerinib and then reloaded with Osimertinib medicine (namely COF-Brig@Osi):
ornitinib drug was captured with Bovine Serum Albumin (BSA) and embedded into COFs mesopores: 10mg of BSA was dispersed in 1ml of 2.5mM NaCl solution, and 200. Mu.L of 0.1mM NaOH solution was added after complete dissolution. Simultaneously, 4ml of 5mM of the Ornitinib ethanol solution is prepared, 1mg of the COF-Brig nano particles are added, ultrasonic oscillation is carried out for 10min, and magnetic stirring is used for completely dispersing the COF-Brig nano particles. Finally, the COF-Brig ethanol solution containing the Ornitinib drug is dripped into 1ml of the previously prepared BSA solution and stirred for 6 hours. After the reaction is completed, the solution is centrifuged and washed with deionized water for a plurality of times to remove the residual octtinib drug. The TEM structure after loading the dual drug is shown in fig. 4.
Optimizing the loading capacity and encapsulation efficiency of the Osimertinib drug: the buntinib-loaded COF-bright nanoparticles were used to load the octtinib drug. The different concentrations set by Osimertinib were 1,2,5,10mM, respectively. The loading and encapsulation efficiency of Osimertinib are shown in FIG. 5. The loading amounts of the 1,2,5,10mM Osimertinib drugs after 1mg COFs loaded with Brigerinib drugs were 0.05, 0.11, 0.26, 0.39mM, respectively, and the encapsulation rates were 24.3, 23.9, 26.1, 20.0% respectively. The optimal octreotide loading conditions are determined as follows: 5mM of Ornitinib solution, its corresponding loading and encapsulation rates were: 0.26mM and 26.1%.
TEM images before and after loading the high pore COF nano carrier with double drugs show that the mesoporous structure of the COF is almost completely occupied by the drugs. The specific surface area as shown in fig. 6 is significantly reduced. The hydrated particle size results in FIG. 8 reveal that the particle size of the COF-Brig@Osi nanoparticles is about 180nm.
Example 2 preparation of Ornitinib drug resistant cell membrane coated COF-Brig@Osi nanomaterial
(1) Extracting the cell membrane of the lung cancer octenib drug resistance:
PC-9OR resistant cells were first dispersed in hypotonic lysis buffer (hypotonic lysis buffer: 20mM Tris HCl, 10mM KCl, 2mM MgCl) 2 And 1 tablet of protease inhibitor without EDTA) and broken with a homogenizer. The cell solution was centrifuged (3200 g,5 min) and the supernatant was collected. The supernatant was centrifuged at 20000g for 30min, the pellet discarded, and centrifuged again at 80000g using a super high speed centrifuge for 1.5h, removing the supernatant. PC-9OR resistant cell membranes were collected and PC-9OR resistant cell membrane vesicles were then physically extruded through a 400nm polycarbonate porous membrane on a micro-extruder. The extraction method of the PC-9 sensitive cell membrane is the same as that of PC-9OR.
(2) Wrapping the COF-Brig@Osi nano material by adopting a physical extrusion method:
100. Mu.g/ml of PBS solution of COF-Brig@Osi nanoparticles and 0.5ml of PBS solution of PC-9OR drug-resistant cell membrane vesicles (concentration 500 ug/ml) were mixed, and after 10min of ultrasound, the mixture was repeatedly extruded through a polycarbonate film containing a 200nm pore size with a small extruder, and then centrifuged at 15000rpm for 10min to obtain COF-Brig@Osi-M nanoparticles. The method for wrapping the PC-9 sensitive cell membrane is the same as that of PC-9OR. The synthesized COF-Brig@Osi-M nanoparticle structure is shown in FIG. 7: TEM results revealed that the nanoparticle surface is coated with a layer of cell membrane approximately 8nm thick, consistent with the monolayer cell membrane thickness reported in the literature. The SDS-PAGE result shown in FIG. 9 shows the retention of protein spectrum after the nanomaterial wraps the cell membrane, and the successful transfer of the protein on the cell membrane to the surface of the nanomaterial is found, so that the homologous targeting adhesion function of the material is realized. The change of particle size before and after the Gao Jiekong COF nano drug-carrying system wraps the cell membrane is shown in fig. 8: the particle size of the COF-Brig@Osi-M nanoparticles is about 200nm.
Example 3 evaluation of anti-tumor Effect of COF-Brig@Osi-M nanodrug delivery System in vitro
(1) Killing effect of COF-Brig@Osi-M nanoparticles on PC-9OR drug-resistant cells
By CCK-8 experimentsThe method tests the toxic and side effects of COF-Brig@Osi-M nanoparticles on PC-9OR drug-resistant cells. PC-9OR cells were plated at 5X10 3 The number of individual wells/well was plated in 96-well plates and incubated for 12h. Different concentrations of COF-M, COF-Brig-M, COF-Osi-M and COF-Brig@Osi-M nanoparticles (concentration settings: 0, 0.1, 0.2, 0.5, 1,2,5,10 mg/ml) were then added. Incubation was carried out at 37℃for 72h, medium was discarded, 10% CCK-8 solution was added, and after incubation for 2h the absorbance at 450nm was measured. And cell viability was calculated as shown in fig. 10: COF-M, COF-Brig-M nanoparticles have poor inhibitory effect on the activity of PC-9OR cells, COF-Osi-M has a certain inhibitory effect, but the IC50 is relatively large (about 2.2 mg/ml). The COF-Brig@Osi-M nanoparticle has a remarkable inhibition effect on the activity of PC-9OR cells, and the IC50 of the nanoparticle is about 0.45mg/ml.
(2) COF-Brig@Osi-M nanoparticles inhibit PC-9OR drug-resistant cell clone formation
The ability of COF-Brig@Osi-M nanoparticles to inhibit proliferation of PC-9OR resistant cells was evaluated by a colony formation assay. PC-9OR cells were plated at 1X10 3 The number of individual wells/well was plated in 6-well plates and cultured for 24 hours. COF-M, COF-Brig-M, COF-Osi-M and COF-Brig@Osi-M nanoparticles were then added at a concentration of 0.2mg/ml (IC 25), respectively. After incubation at 37℃for 15 days, the medium was discarded, washed 3 times with PBS and dried at room temperature. 0.5ml of crystal violet is added to each hole, after 1h of dyeing, the crystal violet is sucked out, dried at room temperature, washed 1 time with PBS and finally photographed.
The experimental results are shown in fig. 11: the COF-M, COF-Osi-M has no obvious inhibition effect on the clone formation of PC-9OR cells, and the COF-Brig-M has a certain inhibition effect. But the COF-Brig@Osi-M nano particles have remarkable inhibition effect on the clone formation of PC-9OR cells.
The experimental results show that the Gao Jiekong COF loaded with the Brig and the Osi double drugs has remarkable inhibition effect on the activity and proliferation of PC-9OR drug-resistant cells.
Example 4 in vivo evaluation of targeting of COF-Brig@Osi-M nanodrug delivery System
(1) In order to observe the in vivo targeting of COF-Brig@Osi-M nano particles in a lung cancer transplantation tumor nude mouse animal model, the COF-Brig@Osi is marked by fluorescein Cy5 and wrapped by sensitive cell membrane MPC-9OR drug resistant cell membrane MPC-9OR1, namely COFCy5-Brig@Osi-MPC-9 and COFCy5-Brig@Osi-MPC-9OR.
(2) Constructing a nude mice lung cancer transplantation tumor model: PC-9 sensitive cells and PC-9OR drug-resistant cell suspensions with the density of 1x107/mL are respectively inoculated on the left armpit and the right armpit of a nude mouse subcutaneously, and tumor masses of about 100mm3 appear after one week.
(3) Targeting of COFCy5-Brig@Osi-MPC-9 and COFCy5-Brig@Osi-MPC-9OR nanoparticles in different tumors was observed: nanoparticles were injected into mice via the tail vein. After 8 hours of administration, mice were sacrificed, organs (heart, liver, spleen, lung, kidney) and transplants were removed, and the distribution of nanoparticles in the organs and transplants was observed by a fluorescence imager.
The fluorescence intensities of the main organs and tumors of the COFCY5-Brig@Osi-MPC-9 nanoparticle group injected are shown in FIG. 12: the fluorescence intensity is strongest in liver, stronger in tumor, lung and kidney, and weaker in heart and spleen. The nano material is obviously enriched in the liver and is more enriched in the tumor. Comparison of PC-9 and PC-9OR tumors revealed that the fluorescence intensities of the sensitive cell membrane PC-9 encapsulation material after PC-9 and PC-9OR tumors were substantially very similar. The targeting effect of the sensitive cell membrane wrapping nano material on sensitive and drug-resistant tumors is equivalent, and the sensitive and drug-resistant tumors cannot be distinguished.
The fluorescence intensities of the main organs and tumors of the COFCY5-Brig@Osi-MPC-9OR1 nanoparticle group injected are shown in FIG. 13: the fluorescence intensity is strongest at the tumor site, stronger in liver, lung and kidney, weaker in heart and spleen. The nano material is obviously enriched in the tumor part. Comparing PC-9 and PC-9OR tumors, the fluorescence intensity of the drug-resistant cell membrane PC-9OR wrapping material in the drug-resistant tumor is very strong, but the fluorescence intensity in the sensitive tumor is weaker. Therefore, the targeting effect on drug-resistant tumors is obvious after the drug-resistant cell membrane wraps the nano material.
From the results, the targeting effect of the sensitive cell membrane on PC-9 and PC-9OR1 tumors after the sensitive cell membrane wraps the nano material is equivalent. The targeting effect of the drug-resistant cell membrane coated with the nano material on PC-9OR tumor is obviously better than that of PC-9 sensitive tumor.
EXAMPLE 5 mechanism analysis of drug-resistant cell membrane targeting
SDS-PAGE experiment is adopted to detect the total protein spectrum of PC-9 sensitive cells and PC-9OR drug-resistant cell membranes and the expression condition of target related adhesion proteins N-cadherin, EPCAM and Galectin-3 in sensitive and drug-resistant cell membranes.
The experimental method comprises the following steps: the sensitive and resistant cell membranes extracted in example 2 were used for SDS-PAGE and WesternBlot experiments, and compared with the sensitive and resistant cells, the SDS-PAGE and Western Blot experiments were performed using conventional methods.
The experimental results are shown in fig. 14: as can be seen from SDS-PAGE protein patterns, the difference of the expression quantity of various proteins exists in the sensitive cell membrane and the drug-resistant cell membrane, and a large amount of proteins in the drug-resistant cell membrane are remarkably and highly expressed. The expression of cell adhesion related protein N-cadherin, EPCAM and Galectin-3 was further analyzed by Western Blot. The contrast sensitive cell membrane shows that the N-cadherein and Galectin-3 proteins in the drug-resistant cell membrane are remarkably high-expressed, and the EPCAM protein is not remarkably changed.
From the above results, it can be seen that: the remarkable targeting of drug-resistant cell membranes to drug-resistant tumors is attributed to the high expression of N-cadherein and Galectin-3 proteins.
Claims (10)
1. The preparation method of the high-pore COF nano-particles is characterized by comprising the following steps of:
(1) Synthesis of COP nanoparticles: using 4,4' -dithiodibenzoyl aldehyde and 1,3,5-tri (aminomethyl) benzene tri-hydrochloride as raw materials, using trifluoroacetic acid as a catalyst of Schiff base reaction, and adopting Schiff base type imine bond connection mode to synthesize amorphous covalent organic polymer COP nano particles containing disulfide bonds, wherein the use amount of the trifluoroacetic acid is 400-600 mu L of trifluoroacetic acid for every 0.01 mol of 4,4' -dithiodibenzoyl aldehyde, and the concentration of 4,4' -dithiodibenzoyl benzaldehyde solution is 0.35-0.55mM;
(2) High pore COF nano-particles are synthesized by a recrystallization method: dispersing the prepared COP nano particles into etching liquid, fully reacting at 50-120 ℃, centrifugally collecting Gao Jiekong COF nano particles, and washing to remove residual reactants to obtain the nano-particles; the etching liquid comprises: 10 to 40ml of N, N-dimethylacetamide, 10 to 40ml of 1,3, 5-trimethylbenzene, 0.5 to 3ml of water and 0.5 to 2.5ml of trifluoroacetic acid.
2. The method of manufacturing according to claim 1, characterized in that:
in the step (1), the solvent of 4,4 '-dithiodibenzoyl aldehyde and 1,3,5-tri (aminomethyl) benzene tri-hydrochloride is acetonitrile, and trifluoroacetic acid is added into the acetonitrile dispersion solution of 4,4' -dithiodibenzoyl aldehyde, and stirring and fully reacting are carried out; and adding acetonitrile solution of 1,3,5-tri (aminomethyl) benzene tri hydrochloride, fully reacting at room temperature, centrifugally collecting a product, washing with ethanol and N, N-dimethylacetamide to remove residual reactants, and obtaining COP nano particles.
3. The preparation method according to claim 2, characterized in that: 450-550. Mu.L of trifluoroacetic acid per 0.01 mol of 4,4' -dithiodibenzoyl;
the concentration of the 4,4' -dithiodibenzoyl benzaldehyde solution is 0.4-0.5mM, and the concentration of the 1,3, 5-tris (aminomethyl) benzene tri-hydrochloride solution is 0.2-0.4mM;
the ratio of the use amount of the 4,4' -dithiodibenzoyl benzaldehyde to the use amount of the 1,3, 5-tris (aminomethyl) benzene tri-hydrochloride is 3:2.
4. The method of manufacturing according to claim 1, characterized in that:
dispersing the prepared COP nano particles into etching liquid in the step (2), and reacting for 12-60 hours at 50-100 ℃; centrifugally collecting mesoporous COF nano particles, and washing the mesoporous COF nano particles with ethanol to remove residual reactants;
the volume ratio of the N, N-dimethylacetamide to the 1,3, 5-trimethylbenzene in the etching liquid is as follows: 1 to 4:1 to 4.
5. The method of manufacturing according to claim 4, wherein: the using amount ratio of the COP nano particles to the etching liquid is 1 mg/40-60 ml.
6. The high-pore COF nanoparticle produced by the production process of any one of claims 1 to 5.
7. Use of the high pore COF nanoparticle of claim 6 as a pharmaceutical carrier.
8. The use according to claim 7, characterized in that: the application is that a bionic drug-carrying system for targeting lung cancer drug-resistant tumors is prepared by taking COF nano-particles as a drug carrier, wherein the bionic drug-carrying system is obtained by anchoring a buntinib drug to the COF nano-particles in claim 6 to obtain a COF-Brig nano-material, embedding an octtinib drug into a mesoporous structure of the COF-Brig nano-material to obtain the COF-Brig@Osi nano-material, and then wrapping the COF-Brig@Osi nano-material by adopting a cell membrane of lung cancer octtinib drug-resistant cells.
9. The use according to claim 8, characterized in that: the COF-Brig nano material is synthesized by anchoring a buntinib drug molecule through hydrogen bonds by utilizing lone pair electrons on imine nitrogen in COF nano particles; the preparation of the COF-Brig@Osi nanomaterial is to embed the Ornitinib drug into the mesoporous structure of the COFs material by a BSA embedding method.
10. The use according to claim 8, characterized in that: and wrapping the cell membrane of the lung cancer octenib drug-resistant cell on the surface of the COF-Brig@Osi nano material by adopting a physical extrusion method.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2015271991A1 (en) * | 2010-09-29 | 2016-01-21 | Philogen S.P.A. | Thiazolidine linker for the conjugation of drugs to antibodies |
| CN108421047A (en) * | 2018-03-31 | 2018-08-21 | 湖北大学 | A kind of composite Nano medicament carrier system for having tumour and causing targeting ability |
| CN110859826A (en) * | 2019-12-09 | 2020-03-06 | 深圳先进技术研究院 | Brain tumor targeted bionic drug-loaded nanoparticle and preparation method and application thereof |
| CN114010783A (en) * | 2021-11-03 | 2022-02-08 | 大连理工大学 | Multifunctional boron-rich nano targeting preparation based on covalent organic framework material, and preparation method and application thereof |
| CN114377149A (en) * | 2021-12-14 | 2022-04-22 | 西北工业大学 | Mn-based degradable MOF nano reactor and preparation method and application thereof |
| CN115192708A (en) * | 2022-07-07 | 2022-10-18 | 安徽医科大学 | Nano composite material loaded with anti-tumor drug, nano drug-loaded system, preparation and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6590802B2 (en) * | 2013-11-06 | 2019-10-16 | ザ ユニバーシティ オブ シカゴＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｃｈｉｃａｇｏ | Nanoscale transporter for delivery or co-delivery of chemotherapeutic drugs, nucleic acids and photosensitizers |
| WO2018218052A1 (en) * | 2017-05-24 | 2018-11-29 | Northwestern University | Nanoparticle-lipid composite carriers and uses thereof |
-
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2015271991A1 (en) * | 2010-09-29 | 2016-01-21 | Philogen S.P.A. | Thiazolidine linker for the conjugation of drugs to antibodies |
| CN108421047A (en) * | 2018-03-31 | 2018-08-21 | 湖北大学 | A kind of composite Nano medicament carrier system for having tumour and causing targeting ability |
| CN110859826A (en) * | 2019-12-09 | 2020-03-06 | 深圳先进技术研究院 | Brain tumor targeted bionic drug-loaded nanoparticle and preparation method and application thereof |
| CN114010783A (en) * | 2021-11-03 | 2022-02-08 | 大连理工大学 | Multifunctional boron-rich nano targeting preparation based on covalent organic framework material, and preparation method and application thereof |
| CN114377149A (en) * | 2021-12-14 | 2022-04-22 | 西北工业大学 | Mn-based degradable MOF nano reactor and preparation method and application thereof |
| CN115192708A (en) * | 2022-07-07 | 2022-10-18 | 安徽医科大学 | Nano composite material loaded with anti-tumor drug, nano drug-loaded system, preparation and application |
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