CN114748634A - Preparation and application of phenylboronic acid/folic acid dual-targeting nano delivery carrier - Google Patents
Preparation and application of phenylboronic acid/folic acid dual-targeting nano delivery carrier Download PDFInfo
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- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 title claims abstract description 121
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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Abstract
The invention relates to a nano delivery carrier with sialic acid and folic acid receptor dual targeting functions, which is prepared by taking dendritic polylysine (DGL), phenylboronic acid-polyethylene glycol (PBA-PEG-COOH) and folic acid-polyethylene glycol (FA-PEG-COOH) as main raw materials, and a preparation method and application thereof. Adding the activated PBA-PEG-COOH into the DGL solution, then adding the activated FA-PEG-COOH, stirring for reaction, dialyzing, and freeze-drying to obtain the PBA/FA-PEG-DGL with the surface modified by phenylboronic acid and folic acid targeting ligand. Adding tumor chemotherapy medicine into PBA/FA-PEG-DGL water solution by physical embedding method, stirring in dark place, dialyzing, and freeze drying to obtain final product. The invention adopts micromolecule targeting ligand, is cheap and easy to obtain, has high stability and simple preparation method, and reduces the cost of materials. Is suitable for delivering various tumor chemotherapy medicaments, realizes high-efficiency anti-tumor efficacy, reduces toxic and side effects, provides a new idea for clinical treatment of tumors, and brings new hopes.
Description
Technical Field
The invention relates to the field of nano biomedicine, in particular to a phenylboronic acid/folic acid dual-targeting nano delivery carrier; meanwhile, the preparation method of the phenylboronic acid/folic acid dual-targeting nano delivery carrier is simple and convenient and easy to operate; the double-targeting nano delivery carrier is suitable for specific targeting delivery of anti-tumor chemotherapeutic drugs, can prevent or treat malignant tumors with high sialic acid and folate receptors expression, such as liver cancer, cervical cancer, breast cancer, gastric cancer, kidney cancer and the like, and has potential application value in scientific research and clinical practice.
Background
The therapeutic effect of cancer depends mainly on the success of the drug delivery to the tumor tissue and uptake by the tumor cells. At present, chemotherapy is mainly used for treating cancer clinically, but chemotherapy drugs are easy to metabolize and clear in vivo due to small molecular weight, lack of targeting property and extremely easy to cause systemic toxic and side effects. Therefore, it is necessary to develop a specific drug which can prolong the blood circulation time and can be precisely targeted to the tumor site. In recent years, a drug targeted delivery system based on a nano-carrier is developed vigorously, so that on one hand, the abnormal condition of a blood vessel and a lymphatic reflux system at a solid tumor part is utilized, so that the local high permeability and retention effect (EPR effect) is enhanced, the nano-carrier is passively targeted and gathered to the solid tumor part, and on the other hand, the active targeting property of the nano-carrier can be endowed by means of the modification of a targeting ligand. The drug delivery system based on the nano-carrier can protect the loaded drug from being cleared and degraded, prolong the circulation time of the drug, thereby increasing the enrichment of the drug in tumor parts and the concentration in tumor cells and improving the clinical treatment effect.
Currently, active targeting drug delivery nanocarriers are mainly based on single ligand modification, but tumor cell uptake pathways and cell membrane receptor expression levels change with tumor types and growth stages, so the application of single ligand modified nanocarriers in different types of tumor treatment may be limited. After the uptake of a certain ligand in the cell is saturated, the further uptake of the ligand-modified nanocarrier will be inhibited, i.e., the extent of uptake of the nanocarrier modified with a single ligand by the cell is very limited. Therefore, further research into more effective strategies for targeted therapy of tumors is needed. The dual ligand targeting strategy has better cell selectivity and cellular uptake in cancer therapy than single ligand targeting, which may lead to uncontrollable and fluctuating targeting efficiencies, thus showing great potential in cancer therapy.
Dendritic polylysine (DGL) is a nano-scale macromolecular compound which has high water solubility and non-immunogenicity, can be completely degraded in vivo, and has the characteristics of high branching, controllable structure, monodispersity and the like; the DGL surface has a large number of modifiable amino groups, and the surface functional groups of the DGL surface exponentially increase with the increment of generation numbers, so that PEG, targeting molecules or imaging agents can be connected; the larger cavity inside can embed the drug molecules. However, high-generation DGL has some cytotoxicity due to its increased positive charge. It is reported that positive charges on the surface of the DGL nano-carrier can be shielded after PEG modification, but the internalization capability of the nano-carrier is reduced.
Phenylboronic acid is a non-toxic, non-immunogenic and low-cost small-molecule targeting ligand, Sialic Acid (SA) is an anionic monosaccharide, exists in an antigen approved as a tumor marker, and can form a stable complex with sialic acid to mediate internalization of cells. The folate receptor is highly expressed on the cell surface of most malignant tumors, such as liver cancer, cervical cancer, ovarian cancer and breast cancer, is a small molecular vitamin with good stability, is often used as a health product or a food additive, and has high specificity to the tumor cells. Therefore, we constructed a novel dual targeting nano delivery carrier PBA/FA-PEG-DGL with polyethylene glycol as a linking arm and phenylboronic acid and folic acid as targeting molecules. The polyethylene glycol on the connecting arm can reduce the cytotoxicity of the carrier, and simultaneously increase the particle size, so that the carrier can be passively enriched to a tumor part through a high permeability and retention effect (EPR effect); meanwhile, the internalization capability of the nanoparticles is improved by modification of two targeting ligands, namely phenylboronic acid and folic acid, and the triple effects of reducing the toxicity of the carrier and enhancing the targeting and internalization capability of the nano drug delivery system are finally achieved.
Doxorubicin (DOX) as the first generation of broad-spectrum anthracycline antibiotics can be used for treating various cancers, is one of the most common chemotherapeutic drugs in local treatment of liver cancer, however, free DOX has systemic toxicity, especially damage to myocardial cells seriously limits the clinical application of DOX, and after intravenous administration, the accumulation amount at the tumor part is difficult to reach the treatment amount. Therefore, how to effectively transmit DOX into tumor cells, improve the anti-tumor efficacy and reduce the toxic and side effects becomes an urgent problem to be solved. A phenylboronic acid-polyethylene glycol (PBA-PEG-COOH) and folic acid-polyethylene glycol (FA-PEG-COOH) co-modified nano delivery carrier (PBA/FA-PEG-DGL) is prepared, so that the problem of single-target ligand off-target effect possibly occurring in the drug delivery process is solved, and the enrichment of the drug at a tumor site and the concentration of the drug in tumor cells are increased.
Disclosure of Invention
The first purpose of the invention is to provide a phenylboronic acid/folic acid dual-targeting nano delivery carrier. Firstly, the nano delivery carrier has good biocompatibility and in vivo degradability. Secondly, the nano delivery carrier has better targeting property, so that the nano delivery carrier can be passively targeted to a tumor part through an EPR effect; on the other hand, the nano delivery carrier can mediate endocytosis through the interaction between two ligands and receptors to play an active targeting function.
The second purpose of the invention is to provide a method for preparing a phenylboronic acid/folic acid dual-targeting nano delivery carrier. The preparation method is simple, the materials are easy to obtain, the price is low, the reaction condition is mild, the operation is easy, and the method has the prospect of industrial implementation.
The third purpose of the invention is to provide an application of the phenylboronic acid/folic acid dual-targeting nano delivery carrier. The chemotherapy drug adriamycin is loaded by a physical embedding method, so that the enrichment of the loaded drug in tumor parts can be effectively increased, the distribution of the drug in the whole body is reduced while the high drug concentration in tumor cells is maintained, the anti-tumor effect is obviously improved, and the toxic and side effects of the whole body are reduced. This application has the advantage of bringing new hopes for clinical treatment of tumors.
In order to achieve the purpose, the invention adopts the following technical measures:
the technical conception is as follows: a phenylboronic acid/folic acid double-targeting drug-loaded nano system comprises dendritic polylysine (DGL), phenylboronic acid-polyethylene glycol (PBA-PEG-COOH) and folic acid-polyethylene glycol (FA-PEG-COOH), and the modification process comprises the following steps: EDC/NHS is used for activating carboxyl on the surface of PBA-PEG-COOH, the carboxyl reacts with amino on DGL to generate amido bond, and then EDC/NHS activated FA-PEG-COOH is added into the product, so that the nano delivery carrier (PBA/FA-PEG-DGL) with high biological safety and good targeting property is obtained.
The structural formula of each part is as follows:
dendritic polylysine nanoparticles (DGL): (the structure is schematically shown in figure 1)
PBA-PEG-COOH：
FA-PEG-COOH：
A phenylboronic acid/folate dual targeting nano delivery vehicle (PBA/FA-PEG-DGL): (the structural schematic diagram is shown in figure 2).
1. A preparation method of phenylboronic acid/folic acid dual-targeting nano delivery carrier comprises the following steps:
(1) synthesis of phenylboronic acid-polyethylene glycol-dendritic polylysine (PBA-PEG-DGL): dropwise adding a PBA-PEG-COOH solution activated by EDC/NHS into the DGL solution, carrying out shaking table reaction at room temperature for 20-30h, dialyzing, and freeze-drying to obtain PBA-PEG-DGL; wherein the molar ratio of EDC, NHS and PBA-PEG-COOH is 5-15: 1; the molar ratio of PBA-PEG-COOH to DGL is 25-35: 1.
(2) Synthesis of phenylboronic acid/folic acid-polyethylene glycol-dendritic polylysine (PBA/FA-PEG-DGL): adding EDC/NHS into FA-PEG-COOH solution drop by drop for activation, mixing the mixture into PBA-PEG-DGL solution, performing shake reaction at room temperature for 40-50h, dialyzing, and freeze-drying to obtain PBA/FA-PEG-DGL; wherein the molar ratio of EDC, NHS and FA-PEG-COOH is 5-15: 1; the molar ratio of FA-PEG-COOH to DGL is 25-35: 1.
The solvent of the solution in the steps (1) to (2) is dimethyl sulfoxide (DMSO).
The molecular weight of the dendritic polylysine (DGL) in the step (1) is 172300 g/mol; the molecular weight of PBA-PEG-COOH was 2050 g/mol.
The concentration of the DGL solution in the step (1) is 15-25mg/mL, the concentration of the PBA-PEG-COOH solution after EDC/NHS activation is 6-10mg/mL, the concentration of the EDC solution is 6-9mg/mL, and the concentration of the NHS solution is 4-7 mg/mL.
The molecular weight of FA-PEG-COOH in the step (2) is 2487 g/mol.
In the step (2), the concentration of the PBA-PEG-DGL solution is 7-10mg/mL, and the concentration of the FA-PEG-COOH solution after EDC/NHS activation is 6-10 mg/mL. The concentration of EDC solution is 6-9mg/mL, and the concentration of NHS solution is 4-7 mg/mL.
The dialysis conditions in the steps (1) to (2) are as follows: the dialysis membrane is a biotechnology regenerated fiber (RC) membrane, the molecular weight cut-off of the RC membrane is 8000-14000 Da, the RC membrane is dialyzed in deionized water for 2-4 days, and dialysate is replaced every 10-15 hours.
2. And (3) performing transmission electron microscope observation and dynamic light scattering analysis on the phenylboronic acid/folic acid dual-targeting nano delivery carrier prepared in the step (2), and finding that compared with the unmodified ligand nano delivery carrier, the modified nano delivery carrier is regular and spherical in shape, obviously increased in particle size and reduced in surface charge, and is the dual-targeting nano delivery carrier with excellent physicochemical properties.
3. Respectively co-culturing unmodified dendritic polylysine (DGL), polyethylene glycol modified dendritic polylysine (PEG-DGL), phenylboronic acid-polyethylene glycol modified dendritic polylysine (PBA-PEG-DGL), folic acid-polyethylene glycol modified dendritic polylysine (FA-PEG-DGL) and the phenylboronic acid/folic acid dual-target nano delivery carrier prepared in the step (2) with HepG2 tumor cells, and detecting the influence of the four carriers with different concentrations on the cell activity. The four modified carriers are found to have no obvious toxicity to tumor cells, and the cell survival rate is high; as for the phenylboronic acid/folic acid ligand modified dual-targeting nano delivery carrier, even if the phenylboronic acid/folic acid ligand modified dual-targeting nano delivery carrier is incubated at the concentration of 200 mu g/mL for 24 hours, the cell survival rate is still higher than 85%, and the dual-targeting nano delivery carrier is proved to have excellent biological safety.
The application of the phenylboronic acid/folic acid dual-targeting nano delivery carrier in preparing the local chemotherapeutic medicine for treating or preventing tumors comprises the following steps:
1. synthesis of phenylboronic acid-polyethylene glycol-dendritic polylysine:
weighing 35.7mg of PBA-PEG-COOH, putting the PBA-PEG-COOH into a eggplant-shaped bottle, dissolving the PBA-PEG-COOH in 3mL of DMSO, performing ultrasonic treatment for 30min to uniformly disperse the PBA-PEG-COOH, then weighing 33.4mg of EDC, dissolving the EDC in 3.5mL of DMSO, adding the solution into the solution, performing shaking table reaction at room temperature for 2h, after the reaction is finished, weighing 20.1mg of NHS, dissolving the NHS in 3.5mL of DMSO, dropwise adding the NHS into the reaction system, and performing shaking table reaction at room temperature for 2h to activate carboxyl on the PBA-PEG-COOH. Meanwhile, 100mg of DGL is weighed and dissolved in 5mL of DMSO, and the mixture is subjected to ultrasonic treatment for 30min to be uniformly dispersed. Finally, the activated PBA-PEG-COOH solution is added into the DGL solution drop by drop, and the shaking table reaction is carried out for 24 hours at room temperature. After the reaction is finished, the mixture is transferred into a dialysis bag (the cut-off molecular weight of the dialysis bag is 8000-14000 Da), and is dialyzed in a 500mL deionized water system for three days, and the dialysate is replaced every 12 h. And (5) after dialysis, freeze drying to obtain a white solid product PBA-PEG-DGL.
2. Synthesis of phenylboronic acid/folic acid-polyethylene glycol-dendritic polylysine (PBA/FA-PEG-DGL):
weighing 43.3mg of FA-PEG-COOH, dissolving in 4.5mL of DMSO, performing ultrasonic treatment for 30min to uniformly disperse, then weighing 33.4mg of EDC, dissolving in 3.5mL of DMSO, adding into the solution, performing shaking table reaction at room temperature for 2h, after the reaction is finished, weighing 20.1mg of NHS, dissolving in 3.5mL of DMSO, dropwise adding into the reaction system, and performing shaking table reaction at room temperature for 2h to activate the carboxyl on the FA-PEG-COOH. The freeze-dried product PBA-PEG-DGL preserved in the step 1 is re-dissolved in 8mL of DMSO and then is subjected to ultrasonic treatment for 30min to be uniformly dispersed. And (3) dropwise adding the activated FA-PEG-COOH into the PBA-PEG-DGL solution, carrying out shaking table reaction at room temperature for 24 hours, transferring the mixture into a dialysis bag (the cut-off molecular weight of the dialysis bag is 8000-14000 Da) after the reaction is finished, dialyzing for three days in a 500mL deionized water system, and replacing the dialysate every 12 hours. After dialysis, the solid product PBA/FA-PEG-DGL is obtained by freeze drying.
3. The synthesis of the phenylboronic acid/folic acid double-targeting drug-loaded nano system loaded with the chemotherapeutic drug Doxorubicin (DOX):
weighing 10mg DOX, dissolving in 5mL deionized water, transferring to 2mL deionized water solution containing 20mg PBA/FA-PEG-DGL, and carrying out shake reaction at room temperature for 24h in a dark place. And (3) after the reaction is finished, transferring the product into a dialysis bag (the cut-off molecular weight of the dialysis bag is 8000-14000 Da), dialyzing for 12 hours in a dark place, freeze-drying to obtain the phenylboronic acid/folic acid dual-target drug-loaded nano system loaded with the chemotherapeutic drug adriamycin, and weighing.
4. And (3) detecting the drug loading rate and the encapsulation rate of the drug by using an ultraviolet spectrophotometry for the phenylboronic acid/folic acid double-targeting drug loading nano system loaded with the chemotherapeutic drug adriamycin obtained in the step (3).
5. Co-culturing the phenylboronic acid/folic acid double-targeting drug-loaded nano system loaded with the chemotherapeutic drug adriamycin obtained in the step 3 with tumor cells, detecting the uptake efficiency of the tumor cells by means of fluorescence microscopy, flow cytometry and the like, and finding that the drug-loaded nano system can be effectively endocytosed by the tumor cells.
6. Injecting free DOX, DGL loaded with adriamycin, a polyethylene glycol modified drug-loaded nano system loaded with DOX, a phenylboronic acid-polyethylene glycol modified single-target drug-loaded nano system loaded with DOX, a folic acid-polyethylene glycol modified single-target drug-loaded nano system loaded with DOX and a phenylboronic acid/folic acid double-target drug-loaded nano system loaded with chemotherapeutic drug adriamycin obtained in the step 3 into a transplanted tumor mouse model body through tail veins respectively, and monitoring the weight and tumor growth change of the mouse every day. After the treatment was completed, the mice were euthanized, tumors were removed and weighed, and the dual-targeted drug-loaded nanosystems were found to have the highest tumor inhibition rate in each of the groups.
The invention has the beneficial effects that:
1. the invention adopts dendritic polylysine (DGL) which has no immunogenicity, can be completely degraded in vivo, contains a cavity inside and is easy to modify and carry medicaments as a framework, and can effectively protect the stability of chemotherapeutic medicaments in vivo.
2. The phenylboronic acid/folic acid dual-targeting nano delivery carrier prepared by the invention has excellent biocompatibility and degradability in vivo, the raw materials are cheap and easily available, the synthesis process is simple and easy to operate, and the synthesis condition is mild, so that the phenylboronic acid/folic acid dual-targeting nano delivery carrier is an excellent dual-targeting nano delivery carrier.
3. The phenylboronic acid/folic acid dual-targeting nano delivery carrier prepared by the invention is spherical, is uniformly dispersed, has no obvious aggregation phenomenon, and has higher drug loading rate and encapsulation efficiency.
4. The surface of the double-targeting drug-loaded nano system prepared by the invention is modified with phenylboronic acid and folic acid ligands, so that the drug-loaded nano system has double targeting characteristics on sialic acid and folic acid receptor high-expression tumor cells, the intracellular drug concentration is realized, and a better drug targeting delivery effect and lower toxic and side effects are achieved. Reasonable design and simple operation.
In conclusion, the invention takes the dendritic polylysine as a framework material, and phenylboronic acid-polyethylene glycol (PBA-PEG-COOH) and folic acid-polyethylene glycol (FA-PEG-COOH) are modified to prepare the nano delivery carrier with the double-targeting function. The phenylboronic acid/folic acid modified nano-carrier has excellent biocompatibility and degradability. The prepared phenylboronic acid/folic acid dual-targeting nano delivery carrier is uniform and spherical, is good in dispersion, free of obvious aggregation phenomenon, and high in encapsulation efficiency and drug loading. Can enhance the aggregation of the medicine at the tumor part and the concentration in tumor cells, enhance the anti-tumor effect, reduce the non-specific toxic and side effects of the whole body and have good application prospect.
Drawings
FIG. 1 is a schematic diagram of the structure of a dendritic polylysine nanocarrier (DGL). Wherein: NH (NH)2Representing amino groups, 963 representing 963 ammonia on the surface of the nanoparticleAnd (4) a base.
FIG. 2 is a schematic structural diagram of a phenylboronic acid/folic acid dual-targeting nano-delivery carrier (PBA/FA-PEG-DGL). The exterior of the carrier is that the phenylboronic acid and the folic acid are connected to the surface of the DGL through an amide bond formed by PEG-COOH and amino on the DGL.
FIG. 3 is a structural schematic diagram of a phenylboronic acid/folic acid dual-targeting drug-loaded nano-system loaded with chemotherapeutic drug doxorubicin, the phenylboronic acid and folic acid are connected to the surface of DGL through an amide bond formed by PEG-COOH and amino groups on DGL, and the chemotherapeutic drug Doxorubicin (DOX) is loaded inside the carrier.
FIG. 4 is a particle size distribution diagram of the nano delivery carrier before and after the phenylboronic acid and folic acid dual ligand is modified on DGL, A: DGL, B: PBA/FA-PEG-DGL.
Respectively carrying out dynamic light scattering experiments on unmodified dendritic polylysine nanoparticles (DGL) and phenylboronic acid/folic acid modified double-targeting nano delivery carriers (PBA/FA-PEG-DGL), and detecting the particle size change of the nano delivery carriers before and after modification. The result shows that the phenylboronic acid/folic acid modified dual-targeting nano delivery carrier (PBA/FA-PEG-DGL) has a larger particle size (42.4nm), and is beneficial to passively accumulating at a tumor site through an EPR effect in a circulation process, so that the accumulation of the drug at the tumor site is increased.
FIG. 5 is transmission electron micrograph of pre-and post-modified nano delivery vehicle A DGL, B: PBA/FA-PEG-DGL.
Respectively carrying out transmission electron microscope experiments on unmodified dendritic polylysine nanoparticles (DGL) and phenylboronic acid/folic acid modified dual-targeting nano delivery carriers (PBA/FA-PEG-DGL), and detecting the morphology, particle size and dispersion condition of the nanoparticles before and after dual-ligand modification. The result shows that the nano particles before and after modification are spherical, uniformly dispersed and free of aggregation; and the phenylboronic acid/folic acid modified double-targeting nano delivery carrier (PBA/FA-PEG-DGL) has larger particle size, which indicates that the nano delivery carrier has better passive targeting capability, and the detection result is consistent with the detection result of dynamic light scattering.
Fig. 6 is the effect of the nano delivery vector on HepG2 cell activity under no-load and drug-load conditions.
And (3) respectively incubating each no-load nano delivery carrier and each drug-loaded nano system loaded with the drug DOX with the liver cancer HepG2 cells for 24h, and detecting the light absorption value (OD value) of each hole at 490nm by using an enzyme-labeling instrument after the incubation is finished. The results show that in each group without drug loading, the four nano delivery carriers after ligand modification have no obvious influence on the survival of tumor cells. In each group after drug loading, the phenylboronic acid/folic acid modified double-targeting nano drug carrier (PBA/FA-PEG-DGL/DOX) has the highest cytotoxicity. Combining the above results, the phenylboronic acid/folic acid modified dual-targeting nano delivery carrier (PBA/FA-PEG-DGL) has excellent biological safety. Meanwhile, after the drug is loaded, the drug-loaded nano system has higher cytotoxicity and can effectively inhibit the growth of tumor cells.
Fig. 7 is the uptake capacity of HepG2 cells for drug-loaded nanosystems modifying different ligands.
And (3) incubating each drug-loaded nano system with liver cancer HepG2 cells for 4h, staining the incubated cell nucleuses by using DAPI dye, and observing the uptake condition of the HepG2 cells to each drug-loaded nano system by using a fluorescence microscope. The results show that compared with a drug-loaded nano system without a modified targeting ligand and a drug-loaded nano system with a modified single targeting ligand, the uptake of the double-targeting drug-loaded nano system by the cells is the largest, and the uptake of the double-targeting drug-loaded nano system by the tumor cells is promoted under the synergetic mediation of the two ligands. Notably, the uptake of free DOX by cells is higher than that of the dual-targeting drug-loaded nanosystems, mainly due to the fact that small-molecule DOX enters cells by means of free diffusion.
Figure 8 is the in vivo anti-tumor efficacy of each drug-loaded nanosystem.
Respectively injecting Phosphate Buffer Solution (PBS), free DOX, DGL (DGL/DOX) loaded with adriamycin, a drug-loaded nano system (PEG-DGL/DOX) modified with polyethylene glycol, a drug-loaded nano system (PBA-PEG-DGL/DOX, FA-PEG-DGL/DOX) modified with a single targeting ligand and a drug-loaded nano system (PBA/FA-PEG-DGL/DOX) modified with two targeting ligands into a mouse model body of liver cancer transplantation tumor through tail veins, injecting for 1 time every other day, injecting for 7 times in total, and treating for 14 days. Mice were measured daily for weight and tumor size (tumor size) during the treatment periodMajor diameter x shortest tumor meridian2/2), after the treatment is over, the mice are euthanized, the tumors are removed, photographed and weighed. The result shows that the drug-loaded nano system for modifying the two targeting ligands has the best anti-tumor growth effect and the highest tumor inhibition rate, and the body weight of the mouse always shows the trend of slow increase.
Detailed Description
The present invention will be further described with reference to examples, but the following description is only for the purpose of explaining the present invention and does not limit the contents thereof.
Example 1:
synthesis of a phenylboronic acid/folate dual targeting nano delivery vehicle (PBA/FA-PEG-DGL):
1. synthesis of phenylboronic acid-polyethylene glycol-dendritic polylysine (PBA-PEG-DGL):
weighing 35.7mg of PBA-PEG-COOH, putting the PBA-PEG-COOH into a eggplant-shaped bottle, dissolving the PBA-PEG-COOH in 3mL of DMSO, performing ultrasonic treatment for 30min to uniformly disperse the PBA-PEG-COOH, then weighing 33.4mg of EDC to dissolve in 3.5mL of DMSO, adding the solution into the solution, performing shaking table reaction at room temperature for 2h, after the reaction is finished, weighing 20.1mg of NHS to dissolve in 3.5mL of DMSO, dropwise adding the reaction system into the DMSO, and performing shaking table reaction at room temperature for 2h to activate carboxyl on the PBA-PEG-COOH. Meanwhile, 100mg of DGL is weighed and dissolved in 5mL of DMSO, and ultrasonic uniform dispersion is carried out. Finally, the activated PBA-PEG-COOH solution is added into the DGL solution drop by drop, and the shaking table reaction is carried out for 24 hours at room temperature. After the reaction is finished, the mixture is transferred into a dialysis bag (the cut-off molecular weight of the dialysis bag is 8000-14000 Da), and is dialyzed in a 500mL deionized water system for three days, and the dialysate is replaced every 12 h. And after dialysis, freeze drying is carried out, and a white solid product PBA-PEG-DGL is obtained.
2. Synthesis of phenylboronic acid/folic acid-polyethylene glycol-dendritic polylysine (PBA/FA-PEG-DGL):
weighing 43.3mg of FA-PEG-COOH, dissolving in 4.5mL of DMSO, performing ultrasonic treatment for 30min to uniformly disperse, then weighing 33.4mg of EDC, dissolving in 3.5mL of DMSO, adding into the solution, performing shaking table reaction at room temperature for 2h, after the reaction is finished, weighing 20.1mg of NHS, dissolving in 3.5mL of DMSO, dropwise adding into the reaction system, and performing shaking table reaction at room temperature for 2h to activate the carboxyl on the FA-PEG-COOH. The freeze-dried product PBA-PEG-DGL preserved in the step 1 is re-dissolved in 8mL of DMSO and then is subjected to ultrasonic treatment for 30min to be uniformly dispersed. And (2) dropwise adding the activated FA-PEG-COOH into the PBA-PEG-DGL solution, reacting for 24 hours in a shaking table at room temperature, transferring the mixture into a dialysis bag after the reaction is finished (the cut-off molecular weight of the dialysis bag is 8000-14000 Da), dialyzing for three days in a 500mL deionized water system, and replacing the dialysate every 12 hours. After dialysis, the solid product PBA/FA-PEG-DGL is obtained by freeze drying.
Example 2:
a bio-safety test of a phenylboronic acid/folic acid dual-targeting nano delivery carrier comprises the following steps:
1. synthesis of phenylboronic acid-polyethylene glycol-dendritic polylysine:
weighing 35.7mg of PBA-PEG-COOH, putting the PBA-PEG-COOH into a eggplant-shaped bottle, dissolving the PBA-PEG-COOH in 3mL of DMSO, performing ultrasonic treatment for 30min to uniformly disperse the PBA-PEG-COOH, then weighing 33.4mg of EDC, dissolving the EDC in 3.5mL of DMSO, adding the DMSO into the solution, performing shaking table reaction at room temperature for 2h, weighing 20.1mg of NHS after the reaction is finished, dissolving the NHS in 3.5mL of DMSO, dropwise adding the DMSO into the reaction system, and performing shaking table reaction at room temperature for 2h to activate carboxyl on the PBA-PEG-COOH. Meanwhile, 100mg of DGL was weighed out and dissolved in 5mL of DMSO, and uniformly dispersed by ultrasonic. Finally, the activated PBA-PEG-COOH solution is added into the DGL solution drop by drop, and the shaking table reaction is carried out for 24 hours at room temperature. After the reaction is finished, the mixture is transferred into a dialysis bag (the cut-off molecular weight of the dialysis bag is 8000-14000 Da), and is dialyzed in a 500mL deionized water system for three days, and the dialysate is replaced every 12 h. After dialysis, white solid product PBA-PEG-DGL is obtained by freeze drying.
2. Synthesis of phenylboronic acid/folic acid-polyethylene glycol-dendritic polylysine (PBA/FA-PEG-DGL):
weighing 43.3mg of FA-PEG-COOH, dissolving in 4.5mL of DMSO, performing ultrasonic treatment for 30min to uniformly disperse, then weighing 33.4mg of EDC, dissolving in 3.5mL of DMSO, adding into the solution, performing shaking table reaction at room temperature for 2h, after the reaction is finished, weighing 20.1mg of NHS, dissolving in 3.5mL of DMSO, dropwise adding into the reaction system, and performing shaking table reaction at room temperature for 2h to activate the carboxyl on the FA-PEG-COOH. The freeze-dried product PBA-PEG-DGL preserved in the step 1 is re-dissolved in 8mL DMSO and then is subjected to ultrasonic treatment for 30min to be uniformly dispersed. And (3) dropwise adding the activated FA-PEG-COOH into the PBA-PEG-DGL solution, carrying out shaking table reaction at room temperature for 24 hours, transferring the mixture into a dialysis bag (the cut-off molecular weight of the dialysis bag is 8000-14000 Da) after the reaction is finished, dialyzing for three days in a 500mL deionized water system, and replacing the dialysate every 12 hours. After dialysis, the solid product PBA/FA-PEG-DGL is obtained after freeze drying.
3. And (3) performing transmission electron microscope observation and dynamic light scattering analysis on the phenylboronic acid/folic acid double-targeting nano delivery carrier prepared in the step (2), and finding that compared with unmodified nano particles of a control group, the modified nano particles have obviously increased particle size, meet the particle size range (20-200nm) with an ERP effect function, and have good physicochemical properties. (FIG. 4 and FIG. 5)
4. Co-culturing unmodified nanoparticles (DGL) with different concentrations (3.125-200 mug/mL), polyethylene glycol modified nano delivery vectors (PEG-DGL), phenylboronic acid modified single-target nano delivery vectors (PBA-PEG-DGL), folic acid modified single-target nano delivery vectors (FA-PEG-DGL) and the phenylboronic acid/folic acid dual-target nano delivery vectors prepared in the step 2 with liver cancer HepG2 cells respectively, and detecting proliferation and activity change of the cells by using a microplate reader, wherein the four modified vectors have no obvious toxicity to the tumor cells and high cell survival rate; as for the phenylboronic acid/folic acid dual-targeting nano delivery carrier, even if the phenylboronic acid/folic acid dual-targeting nano delivery carrier is incubated at the concentration of 200 mu g/mL for 24 hours, the cell survival rate is still higher than 85%, and the dual-targeting nano delivery carrier is proved to have excellent biological safety. (FIG. 6)
Example 3: the preparation method of the phenylboronic acid/folic acid double-targeting drug-loading nano system loaded with the chemotherapeutic drug adriamycin comprises the following steps:
1. synthesis of phenylboronic acid-polyethylene glycol-dendritic polylysine:
weighing 35.7mg of PBA-PEG-COOH, putting the PBA-PEG-COOH into a eggplant-shaped bottle, dissolving the PBA-PEG-COOH in 3mL of DMSO, performing ultrasonic treatment for 30min to uniformly disperse the PBA-PEG-COOH, then weighing 33.4mg of EDC, dissolving the EDC in 3.5mL of DMSO, adding the DMSO into the solution, performing shaking table reaction at room temperature for 2h, weighing 20.1mg of NHS after the reaction is finished, dissolving the NHS in 3.5mL of DMSO, dropwise adding the DMSO into the reaction system, and performing shaking table reaction at room temperature for 2h to activate carboxyl on the PBA-PEG-COOH. Meanwhile, 100mg of DGL was weighed out and dissolved in 5mL of DMSO, and uniformly dispersed by ultrasonic. Finally, the activated PBA-PEG-COOH solution is added into the DGL solution drop by drop, and the shaking table reaction is carried out for 24 hours at room temperature. After the reaction is finished, the mixture is transferred into a dialysis bag (the cut-off molecular weight of the dialysis bag is 8000-14000 Da), and is dialyzed in a 500mL deionized water system for three days, and the dialysate is replaced every 12 h. After dialysis, white solid product PBA-PEG-DGL is obtained by freeze drying.
2. Synthesis of phenylboronic acid/folic acid-polyethylene glycol-dendritic polylysine (PBA/FA-PEG-DGL):
weighing 43.3mg of FA-PEG-COOH, dissolving in 4.5mL of DMSO, performing ultrasonic treatment for 30min to uniformly disperse, then weighing 33.4mg of EDC, dissolving in 3.5mL of DMSO, adding into the solution, performing shaking table reaction at room temperature for 2h, after the reaction is finished, weighing 20.1mg of NHS, dissolving in 3.5mL of DMSO, dropwise adding into the reaction system, and performing shaking table reaction at room temperature for 2h to activate the carboxyl on the FA-PEG-COOH. The freeze-dried product PBA-PEG-DGL preserved in the step 1 is re-dissolved in 8mL DMSO and then is subjected to ultrasonic treatment for 30min to be uniformly dispersed. And (2) dropwise adding the activated FA-PEG-COOH into the PBA-PEG-DGL solution, reacting for 24 hours in a shaking table at room temperature, transferring the mixture into a dialysis bag after the reaction is finished (the cut-off molecular weight of the dialysis bag is 8000-14000 Da), dialyzing for three days in a 500mL deionized water system, and replacing the dialysate every 12 hours. After dialysis, the solid product PBA/FA-PEG-DGL is obtained after freeze drying.
3. Synthesis of a phenylboronic acid/folic acid double-targeting drug-loaded nano system loaded with chemotherapeutic drug adriamycin:
10mg of DOX is weighed, dissolved in 5mL of deionized water, transferred to 2mL of deionized water solution containing 20mg of PBA/FA-PEG-DGL, and subjected to shaking table reaction at room temperature in the absence of light for 24 hours. And after the reaction is finished, transferring the product solution into a dialysis bag (the cut-off molecular weight of the dialysis bag is 8000-14000 Da), dialyzing for 12 hours in a dark place, freeze-drying to obtain the phenylboronic acid/folic acid double-target drug-carrying nano system carrying the chemotherapeutic drug adriamycin, and weighing.
4. Respectively co-culturing free DOX, DGL (DGL/DOX) loaded with adriamycin, a drug-loaded nano system (PEG-DGL/DOX) modified with polyethylene glycol, a drug-loaded nano system (PBA-PEG-DGL/DOX, FA-PEG-DGL/DOX) modified with single-targeting ligand and a phenylboronic acid/folic acid double-targeting drug-loaded nano system loaded with chemotherapeutic drug adriamycin prepared in the step 3 with liver cancer HepG2 cells for 4 hours, removing incubation drugs, washing with PBS for 3-5 times, fixing for 15min with 4% paraformaldehyde, performing DAPI staining on cell nuclei, and observing the fluorescence signal density of DOX in the cells under a fluorescence microscope. The results show that compared with a drug-loaded nano system without a modified targeting ligand and a drug-loaded nano system with a modified single targeting ligand, the uptake of the double-targeting drug-loaded nano system by the cells is the largest, and the uptake of the double-targeting drug-loaded nano system by the tumor cells is promoted under the synergetic mediation of the two ligands. Notably, the uptake of free DOX by cells is higher than that of the dual-targeting drug-loaded nanosystems, mainly due to the fact that small-molecule DOX enters cells by means of free diffusion. (FIG. 7)
Example 4:
the application of the phenylboronic acid/folic acid double-targeting drug-loading nano system loaded with the chemotherapeutic drug adriamycin in preparing the local chemotherapeutic drug for treating or preventing liver cancer comprises the following steps:
h22 cells adjusted to 2X 107Inoculating cell suspension (0.15 mL/mouse) subcutaneously into BALB/c male mouse (weight 18-22g) to construct mouse liver cancer transplantation tumor model, and observing and measuring tumor volume (tumor longest diameter × tumor shortest diameter)22) until the tumor volume reaches 150mm3The mice were then randomly assigned to 7 groups (n-7) for subsequent experiments.
2. Injecting 7 groups of mice with PBS, free DOX, DGL (DGL/DOX) loaded with adriamycin, a drug-loaded nano system (PEG-DGL/DOX) modified with polyethylene glycol, a drug-loaded nano system (PBA-PEG-DGL/DOX, FA-PEG-DGL/DOX) modified with a single-target ligand and a drug-loaded nano system (PBA/FA-PEG-DGL/DOX) modified with two target ligands respectively through tail veins, wherein the dosage of the DOX is 5mg/kg, 0.2mL is injected into each mouse, the mice are placed back into cages to be raised after the injection is finished, 1 time of injection is performed every other day for 7 times, the treatment time is totally 14 days, and the weight and the tumor volume of the mice are recorded every day during the treatment period.
3. After treatment, mice were euthanized, tumor tissue was taken, photographed and tumor weights were weighed.
4. Compared with other groups, the phenylboronic acid/folic acid double-targeting drug-loaded nano system loaded with the DOX exerts the optimal anti-tumor effect, the tumor volume is increased slowly, the tumor inhibition rate is highest, the weight of a mouse shows a slow rising trend, and no obvious toxic or side effect exists. (FIG. 8)
Claims (9)
1. A phenylboronic acid/folate dual targeted nano delivery vehicle, comprising:
consists of dendritic polylysine (DGL), phenylboronic acid-polyethylene glycol (PBA-PEG-COOH) and folic acid-polyethylene glycol (FA-PEG-COOH);
the dendritic polylysine (DGL) is a nano-carrier with a large number of amino groups on the surface and the particle size of the nano-carrier is between 10 and 20nm, has high biocompatibility, and has no toxic or side effect, and the in-vivo degradation product is amino acid necessary for human body;
phenylboronic acid-polyethylene glycol (PBA-PEG-COOH)), having the structural formula:
folic acid-polyethylene glycol (FA-PEG-COOH), whose structural formula is:
2. the method for preparing the phenylboronic acid/folic acid dual-targeting nano delivery carrier according to claim 1, comprising the following steps of:
(1) synthesis of phenylboronic acid-polyethylene glycol-dendritic polylysine (PBA-PEG-DGL): dropwise adding a PBA-PEG-COOH solution activated by EDC/NHS into the DGL solution, carrying out shaking table reaction at room temperature for 40-50h, dialyzing, and freeze-drying to obtain PBA-PEG-DGL; wherein the molar ratio of EDC, NHS and PBA-PEG-COOH is 5-15: 1; the molar ratio of PBA-PEG-COOH to DGL is 25-35: 1;
(2) synthesis of phenylboronic acid/folic acid-polyethylene glycol-dendritic polylysine (PBA/FA-PEG-DGL): adding EDC/NHS into FA-PEG-COOH solution drop by drop for activation, mixing the mixture into PBA-PEG-DGL solution, performing shake reaction at room temperature for 40-50h, dialyzing, and freeze-drying to obtain PBA/FA-PEG-DGL; wherein the molar ratio of EDC, NHS and FA-PEG-COOH is 5-15: 1; the molar ratio of FA-PEG-COOH to DGL is 25-35: 1.
3. The method for preparing phenylboronic acid/folic acid dual-targeting nano delivery carrier according to claim 2, wherein the method comprises the following steps: the solvent of the solution in the steps (1) to (2) is dimethyl sulfoxide (DMSO).
4. The method for preparing phenylboronic acid/folic acid dual-targeting nano delivery carrier according to claim 1, wherein the method comprises the following steps: the molecular weight of the dendritic polylysine (DGL) in the step (1) is 172300 g/mol; the molecular weight of PBA-PEG-COOH was 2050 g/mol.
5. The method for preparing phenylboronic acid/folic acid dual-targeting nano delivery carrier according to claim 1, wherein the method comprises the following steps: the concentration of the DGL solution in the step (1) is 15-25mg/mL, the concentration of the PBA-PEG-COOH solution after EDC/NHS activation is 6-10mg/mL, the concentration of the EDC solution is 6-9mg/mL, and the concentration of the NHS solution is 4-7 mg/mL.
6. The method for preparing phenylboronic acid/folic acid dual-targeting nano delivery carrier according to claim 1, wherein the method comprises the following steps: the molecular weight of FA-PEG-COOH in the step (2) is 2487 g/mol.
7. The method for preparing phenylboronic acid/folic acid dual-targeting nano delivery carrier according to claim 1, wherein the method comprises the following steps: in the step (2), the concentration of the PBA-PEG-DGL solution is 7-10mg/mL, the concentration of the FA-PEG-COOH solution after EDC/NHS activation is 6-10mg/mL, the concentration of the EDC solution is 6-9mg/mL, and the concentration of the NHS solution is 4-7 mg/mL.
8. The method for preparing phenylboronic acid/folic acid dual-targeting nano delivery carrier according to claim 1, wherein the method comprises the following steps: the dialysis conditions in the steps (1) to (2) are as follows: the dialysis membrane is a biotechnology regenerated fiber (RC) membrane, the molecular weight cut-off of the RC membrane is 8000-14000 Da, the RC membrane is dialyzed in deionized water for 2-4 days, and dialysate is replaced every 10-15 hours.
9. The use of the phenylboronic acid/folic acid dual-targeted nano-delivery vector according to claim 1 for treating tumors, wherein the tumors are liver cancer, cervical cancer, breast cancer, gastric cancer and renal cancer.
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