CN112957480B - Double-targeting polymer drug nano-carrier and preparation method and application thereof - Google Patents
Double-targeting polymer drug nano-carrier and preparation method and application thereof Download PDFInfo
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention provides a double-targeting polymer drug nano-carrier, belonging to the field of nano-biomedical materials, wherein the nano-carrier comprises a nano-material formed by polylactic-co-glycolic acid-polyethylene glycol block copolymer, the surface of the nano-material is modified with tumor targeting functional molecules and brain targeting peptide, the tumor targeting functional molecules are folic acid, and the brain targeting peptide is connected with the nano-material through sulfydryl-maleimide; the invention takes biodegradable polylactic acid-glycolic acid as a core, obtains the Angiopep-2/folic acid double-targeting polymer drug nano-carrier by surface modification of tumor targeting functional molecules and brain targeting peptides, can effectively overcome the barrier of BBB, targets tumor cells, and can be used as a nano preparation for encapsulating drugs to treat brain glioma in a targeting way.
Description
Technical Field
The invention relates to the field of nano biomedical materials, in particular to a double-targeting polymer drug nano-carrier and a preparation method and application thereof.
Background
Brain glioma is the tumor with the highest incidence rate in the central nervous system, accounts for about 46% of intracranial tumors, is considered to be one of the most destructive and fatal tumors, and has strong malignant proliferation capacity, high infiltration capacity and rapid recurrence capacity. Because the boundary between brain tumor tissue and normal tissue is not clear, the operation is difficult to completely remove the tumor, the success rate is low, the existence of blood-brain barrier (BBB), blood-cerebrospinal fluid barrier (BCB) and blood-tumor barrier (BTB) also makes most of chemotherapeutic drugs difficult to reach the brain tissue, and the drug enrichment concentration of the tumor part is low, which is not enough to kill tumor cells. Therefore, despite the combination of surgery, radiation therapy and chemotherapy, the median survival time of patients is less than 16 months. It has been proved that temozolomide, a novel alkylating agent, has a good anti-glioma effect, but its intracranial concentration is only 40% of blood. Therefore, the medicine is wrapped in the intelligent medicine carrying system which can penetrate through the barrier and has the brain tumor targeting capability, so that the toxic and side effects of the medicine are reduced while the medicine concentration of tumor tissues is improved, and the medicine can be used as one of important breakthrough points for treating the disease.
The Blood Brain Barrier (BBB) is a multicellular vascular structure that isolates the Central Nervous System (CNS) from peripheral blood circulation. The BBB is a highly selective, semi-permeable structural and chemical barrier, primarily through tight control of molecular and ionic pathways. On one hand, nutrition and oxygen are instantly provided according to the needs of neurons, on the other hand, more than 98% of small molecule candidate drugs and almost 100% of large molecule drugs are eliminated, so that the brain is protected from being damaged by toxins and pathogens, the microenvironment of the central nervous system is tightly controlled, and the normal functions of the neurons are ensured. BBB permeability is therefore the rate-limiting factor for drug permeation into the central nervous system. Overcoming the barrier effect of BBB, successfully delivering drugs, especially biotechnological drugs such as polypeptide proteins/genes, into the brain is a primary condition for the implementation of treatment of brain diseases; on the other hand, because the medicament lacks selectivity to the lesion site, presents whole brain distribution after entering the brain, and is difficult to concentrate to the lesion site, and the brain tissue is the center of the human body, the function is complex, and the neuron is very sensitive to the damage, so the whole brain distribution of the medicament not only reduces the concentration of the medicament reaching the lesion site, and weakens the treatment effect, but also may cause serious toxic and side effects to the normal center, and how to concentrate the medicament to the lesion site of the brain has very important significance for the treatment of the brain diseases.
Disclosure of Invention
Aiming at the problems, the invention provides a double-targeting polymer drug nano-carrier and a preparation method and application thereof.
The purpose of the invention is realized by adopting the following technical scheme:
a double-targeting polymer drug nano-carrier comprises a nano-material formed by polylactic glycolic acid-polyethylene glycol block copolymer, wherein the surface of the nano-material is modified with tumor targeting functional molecules and brain targeting peptide, the tumor targeting functional molecules are folic acid, and the brain targeting peptide is connected with the nano-material through sulfydryl-maleimide.
The invention also aims to provide a preparation method of the double-targeting polymer drug nano-carrier, which comprises the following steps:
s1, respectively weighing PLGA-PEG-MAL, PLGA-PEG-FA and PLGA-PEG-OMe, mixing to obtain a mixture, adding dichloromethane to dissolve the mixture, adding a first sodium cholate solution, performing pulse ultrasonic treatment to obtain a primary emulsion, dropwise adding the primary emulsion into a second sodium cholate solution, performing reduced pressure distillation to remove dichloromethane after dropwise adding is completed to obtain a nanoparticle colloidal solution, and performing ultrafiltration or centrifugal separation and precipitation on the nanoparticle colloidal solution to obtain the folic acid targeted nano micelle;
s2, re-dispersing the folic acid targeting nano micelle prepared by the S1 in deionized water, wherein the molar ratio of sulfydryl to maleimide is 2:1, adding brain targeting peptide Angiopep-2, then closing a reaction system, stirring for reaction at room temperature, performing ultrafiltration or centrifugal separation for precipitation after the reaction is finished, and washing to obtain the double-targeting polymer drug nano-carrier;
wherein the mass ratio of the PLGA-PEG-MAL to the PLGA-PEG-FA to the PLGA-PEG-OMe is 1:1: (1-9); the mass concentration of the first sodium cholate solution is 1-4%, and the mass concentration of the second sodium cholate solution is 0.1-0.4%; the mass-volume ratio of the mixture to the first sodium cholate solution is 1-6 g/L; the volume ratio of the first sodium cholate solution to the second sodium cholate solution is 2: 1.
preferably, the pulsed ultrasound conditions in step S1 are: the pulse ultrasonic time is 30-90s, the ultrasonic power is 100W, and the duty ratio is 5 s: and 5 s.
Preferably, the temperature of the reduced pressure distillation in the step S1 is 37 ℃, and the distillation time is 15 min.
Preferably, the ultrafiltration separation precipitate in step S1 or step S2 is specifically obtained by centrifuging an ultrafiltration centrifuge tube with a molecular weight cut-off of 3kDa under the centrifugation condition of 5000rpm × 45 min.
Preferably, the centrifugation conditions for the centrifugation and precipitation in the step S1 or the step S2 are 12000rpm × 20 min.
Preferably, the mass concentration ratio of the first sodium cholate solution to the second sodium cholate solution is (5-20): 1.
still another object of the present invention is to provide a dual-targeting drug, comprising the dual-targeting polymer drug nanocarrier of claim 1 and an anti-tumor drug.
Preferably, the anti-tumor drug is paclitaxel.
The invention has the beneficial effects that:
the invention takes biodegradable polylactic acid-glycolic acid as a core, obtains the Angiopep-2/folic acid double-targeting polymer drug nano-carrier by modifying the surface of tumor targeting functional molecules and brain targeting peptides, can effectively overcome the barrier of BBB, targets tumor cells, and can be used as a nano preparation for encapsulating drugs to treat brain glioma in a targeting way.
Drawings
The invention is further illustrated by means of the attached drawings, but the embodiments in the drawings do not constitute any limitation to the invention, and for a person skilled in the art, other drawings can be obtained on the basis of the following drawings without inventive effort.
FIG. 1 is a schematic diagram of a process for preparing a drug-loaded nano-micelle according to an embodiment of the present invention;
FIG. 2 is a transmission electron microscope image of a double-targeting polymer drug nano-carrier according to an embodiment of the invention;
FIG. 3 is a BCA standard curve of example 16 of the present invention;
FIG. 4 is a DSC differential scanning calorimetry trace of example 16 of the present invention;
FIG. 5 is a standard curve of paclitaxel according to example 17 of the present invention;
FIG. 6 is a graph of sodium lauryl sulfate concentration versus percent paclitaxel dissolution;
FIG. 7 is a graph of the cumulative release of paclitaxel from example 17 of the present invention;
FIG. 8 is a serum and storage stability curve of drug-loaded nanomicelles of example 17 of the present invention;
FIG. 9 is a bar graph comparing the survival rates of U251 cells for 24h and 48h with dual targeting polymer drug nanocarriers;
FIG. 10 is a bar graph comparing the 24h, 48h survival rate of drug loaded nanomicelles against U251 cells;
FIG. 11 is a photograph of a scratch of cells collected at 0h, 12h, and 24h according to Experimental example 3 of the present invention;
FIG. 12 is a bar graph of the quantitative assessment of the wound healing rate of Experimental example 3 of the present invention;
FIG. 13 is a graph showing the effect of detecting U251 cells by flow cytometry in Experimental example 4 of the present invention;
FIG. 14 is a fluorescence micrograph of A549 cells after uptake of fluorescent nanoparticles;
FIG. 15 is a fluorescence micrograph of Hela cells after uptake of fluorescent nanoparticles;
FIG. 16 is a fluorescence micrograph of U251 cells after uptake of fluorescent nanoparticles;
figure 17 is the biodistribution of fluorescent agent nanomicelles in mice.
Detailed Description
The invention is further described with reference to the following examples.
The embodiment of the invention relates to a double-targeting polymer drug nano-carrier, which comprises a nano-material formed by polylactic glycolic acid-polyethylene glycol block copolymer, wherein the surface of the nano-material is modified with tumor targeting functional molecules and brain targeting peptide, the tumor targeting functional molecules are folic acid, and the brain targeting peptide is connected with the nano-material through sulfydryl-maleimide;
the Angiopep-2 is Kunitz type aprotinin, consists of 19 amino acids, has high affinity with Low-density lipoprotein receptor-related protein (LRP), has high expression in Brain Capillary Endothelial Cells (BCEC) and glioma cells, and can effectively improve the transport efficiency of the Angiopep-2 modified nano-carrier blood brain barrier and the blood tumor barrier. Experiments prove that compared with the common receptor-mediated brain targeting ligands of transferrin, lactoferrin and avidin, the Angiopep-2 has stronger capacity of crossing BCEC;
folic Acid (FA) is a ligand widely applied to active targeting of tumors, the activity and the number of folate receptors on the surfaces of tumor cells are obviously higher than those of normal cells, the folic acid and the folate receptors have extremely high affinity (Kd: 0.1-1 nmol/L), and even if the gamma-carboxyl of the folic acid is coupled with other small molecules, the affinity of the folic acid and the folate receptors is not influenced. Folic acid also has the advantages of low cost, small relative molecular mass, easy modification, low immunogenicity and the like.
The preparation method of the double-targeting polymer drug nano-carrier comprises the following steps:
s1, respectively weighing PLGA-PEG-MAL, PLGA-PEG-FA and PLGA-PEG-OMe, mixing to obtain a mixture, adding dichloromethane to dissolve the mixture, adding a first sodium cholate solution, performing pulse ultrasonic treatment to obtain a primary emulsion, dropwise adding the primary emulsion into a second sodium cholate solution, performing reduced pressure distillation to remove dichloromethane after dropwise adding is completed to obtain a nanoparticle colloidal solution, and performing ultrafiltration or centrifugal separation and precipitation on the nanoparticle colloidal solution to obtain the folic acid targeted nano micelle;
s2, re-dispersing the folic acid targeted nano micelle prepared by the S1 in deionized water, wherein the molar ratio of sulfydryl to maleimide is 2:1, adding brain targeting peptide Angiopep-2, then closing a reaction system, stirring for reaction at room temperature, performing ultrafiltration or centrifugal separation for precipitation after the reaction is finished, and washing to obtain the double-targeting polymer drug nano-carrier;
wherein the mass ratio of the PLGA-PEG-MAL to the PLGA-PEG-FA to the PLGA-PEG-OMe is 1:1: (1-9); the mass concentration of the first sodium cholate solution is 1-4%, and the mass concentration of the second sodium cholate solution is 0.1-0.4%; the mass-volume ratio of the mixture to the first sodium cholate solution is 1-6 g/L; the volume ratio of the first sodium cholate solution to the second sodium cholate solution is 2: 1;
wherein, the PLGA-PEG-OMe is a poly (lactic-co-glycolic acid) -polyethylene glycol block copolymer modified by a terminal methoxy group, LA/GA is 50:50, Mw is 45000, provided by the biotechnology limited of Peng in shanghai; the PLGA-PEG-MAL is a poly (lactic-co-glycolic acid) -polyethylene glycol block copolymer modified by maleimide at the end group, wherein LA/GA is 50:50, Mw is 45000, and is provided by Peng Shuo Biotechnology Co., Ltd in Shanghai; the PLGA-PEG-FA is a polylactic glycolic acid-polyethylene glycol block copolymer modified by end-group folic acid, LA/GA is 50:50, Mw is 45000, and is provided by Peng Shuo Biotech limited in Shanghai;
examples 1 to 3
The preparation method comprises the steps of precisely weighing PLGA-PEG-OMe, PLGA-PEG-MAL and PLGA-PEG-FA by an emulsion solvent volatilization method, weighing PLGA-PEG-OMe, PLGA-PEG-MAL and PLGA-PEG-FA at a ratio of 1:1:1, 4:1:1 and 9:1:1, weighing total amounts of 3mg, 6mg and 11mg respectively, adding 0.5ml of dichloromethane for dissolution, adding 2ml of a first sodium cholate solution with a mass concentration of 1%, performing pulse ultrasonic treatment (ultrasonic treatment for 5s and interruption for 5s) by using an ultrasonic cell disruptor, obtaining a primary emulsion with the ultrasonic power of 100W and the pulse ultrasonic treatment time of 60s, dropwise adding the primary emulsion into 1ml of a second sodium cholate solution with the mass concentration of 0.1%, performing rotary evaporation for 15min at the temperature of 37 ℃ to remove dichloromethane to obtain a nanoparticle colloidal solution, placing the nanoparticle colloidal solution into an ultrafiltration centrifugal tube with the molecular weight cutoff of 3kDa, centrifuging for 45min at 5000rpm, collecting precipitates, preparing the folic acid targeted nano micelle, and then re-dispersing the folic acid targeted nano micelle in deionized water; the particle size parameters of the micelles were measured by a Zetasizer Nano-S90 laser particle size analyzer, and the results are shown in Table 1.
By measuring the particle sizes of the nano-micelles prepared from the three PLGA materials in different concentrations and ratios, the particle sizes are less influenced by the ratios of 1:1:1 and 4:1:1, while the particle sizes of the nano-particles obtained from 9:1:1 can be enlarged without significant difference, PLGA-PEG-OMe is a main carrier component of a nano-particle structure, PLGA-PEG-FA and PLGA-PEG-Mal are targeting molecules, so that the total amount of the materials is preferably 6mg, and the ratio of 4:1: 1.
Examples 4 to 6
The folic acid targeting nano micelle is prepared by adopting an emulsifying solvent volatilization method, PLGA-PEG-OMe, PLGA-PEG-MAL and PLGA-PEG-FA are precisely weighed respectively, the total weight is 6mg, the weighing ratio is 4:1:1, 0.5ml of dichloromethane is added for dissolving, 2ml of a first sodium cholate solution with the mass concentration of 1% is added, pulse ultrasonic treatment (5 seconds of ultrasonic treatment and 5 seconds of interruption) is carried out by an ultrasonic cell crushing instrument, the ultrasonic power is 100W, the pulse ultrasonic treatment time is respectively 30 seconds, 60 seconds and 90 seconds, a primary emulsion is obtained, the primary emulsion is dropwise added into 1ml of a second sodium cholate solution with the mass concentration of 0.1%, the dichloromethane is removed by rotary evaporation at the temperature of 37 ℃ for 15min, a nano particle colloidal solution is obtained, the nano particle colloidal solution is placed in an ultrafiltration centrifugal tube with the molecular weight cutoff of 3kDa, centrifugation is carried out at the speed of 5000rpm for 45min, precipitation is collected, and the folic acid targeting nano micelle is prepared, re-dispersing the folic acid targeted nano micelle in deionized water; the particle size parameters of the micelles were measured by a Zetasizer Nano-S90 laser particle size analyzer, and the results are shown in Table 1.
When the ultrasonic treatment is carried out for 60s, the particle size of the obtained nanoparticles is about 165.9nm at minimum. The ultrasonic time is too short, the emulsification is incomplete, the obtained nanoparticles are large, the ultrasonic time is too long, the PDI is obviously large, the aggregation of molecules can occur, and the ultrasonic time is preferably 60 s.
Examples 7 to 12
The preparation method is characterized by adopting an emulsion solvent volatilization method, accurately weighing PLGA-PEG-OMe, PLGA-PEG-MAL and PLGA-PEG-FA respectively, weighing 6mg of total amount, weighing 4:1:1, adding 0.5ml of dichloromethane for dissolution, adding 2ml of first sodium cholate solution, performing pulse ultrasonic treatment (5 s of ultrasound and 5s of interruption) by using an ultrasonic cell crushing instrument, obtaining primary emulsion with the ultrasonic power of 100W and the pulse ultrasonic treatment time of 60s, dropwise adding the primary emulsion into 1ml of second sodium cholate solution, rotary evaporating at 37 deg.C for 15min to remove dichloromethane to obtain nanoparticle colloidal solution, placing in ultrafiltration centrifuge tube with molecular weight cutoff of 3kDa, centrifuging at 5000rpm for 45min to obtain the folic acid targeted nano micelle, and re-dispersing the folic acid targeted nano micelle in deionized water; wherein, the concentration ratio of the sodium cholate of the fixed inner phase to the fixed outer phase is 10:1, and the concentrations are respectively 1-0.1%, 1.5-0.15%, 2-0.2%, 2.5-0.25%, 3-0.3% and 4-0.4%; the Zetasizer Nano-S90 laser particle size analyzer measures the particle size parameters of the micelles, and the measurement results are shown in Table 1.
The particle size of the nanoparticles obtained by the sodium cholate of the internal phase and the external phase is the minimum between 2 percent and 0.2 percent; too low a concentration may not be effective in emulsification, resulting in an increase in the particle size of the nanoparticles; when the concentration is too high, sodium cholate excessively reduces interfacial energy on the surface of the nanoparticles, so that the nanoparticles are unstable, and the particle size of the obtained nanoparticles is increased.
Examples 13 to 15
The preparation method is characterized by adopting an emulsion solvent volatilization method, accurately weighing PLGA-PEG-OMe, PLGA-PEG-MAL and PLGA-PEG-FA respectively, weighing 6mg of total amount, weighing 4:1:1, adding 0.5ml of dichloromethane for dissolution, adding 2ml of first sodium cholate solution, performing pulse ultrasonic treatment (5 s of ultrasound and 5s of interruption) by using an ultrasonic cell crushing instrument, obtaining primary emulsion with the ultrasonic power of 100W and the pulse ultrasonic treatment time of 60s, dropwise adding the primary emulsion into 1ml of second sodium cholate solution, rotary evaporating at 37 deg.C for 15min to remove dichloromethane to obtain nanoparticle colloidal solution, placing in ultrafiltration centrifuge tube with molecular weight cutoff of 3kDa, centrifuging at 5000rpm for 45min, collecting precipitate to obtain the folic acid targeted nano micelle, and re-dispersing the folic acid targeted nano micelle in deionized water; wherein the concentration of the fixed inner phase sodium cholate is 2%, the concentration ratios of the inner phase sodium cholate and the outer phase sodium cholate are 20:1, 10:1 and 5:1, and the concentrations are respectively 2% -0.1%, 2% -0.2% and 2% -0.4%; the ZetaSizer Nano-S90 laser particle size analyzer measures the particle size parameters of the micelle, and the measurement results are shown in Table 1.
The particle size of the obtained nanoparticles is minimum when the ratio of the sodium cholate in the inner phase to the sodium cholate in the outer phase is 2 percent and 0.1 percent. The concentration gradient of the sodium cholate is large, which is beneficial to the dispersion of the emulsion, when the sodium cholate of the external water phase is distilled water, the formed nanoparticle solution contains partial precipitate, and the pure distilled water is possibly not beneficial to the dispersion of the emulsion.
TABLE 1 Effect of preparation conditions on particle size parameters of folate-targeted nanomicelles
Example 16
Accurately weighing 4.0mg of PLGA-PEG-OMe, 1.0mg of PLGA-PEG-Fa and 1.0mg of PLGA-PEG-Mal, dissolving with 0.5ml of dichloromethane, adding 1ml of sodium cholate solution with the concentration of 2%, and performing intermittent ultrasound (ultrasound for 5s and intermittent 5s) for 60s at the power of 100W; dropwise adding the obtained primary emulsion into 1ml of 0.1% sodium cholate aqueous solution, carrying out rotary evaporation for 15min at 37 ℃ by using a rotary evaporator to remove dichloromethane to obtain a nanoparticle colloidal solution, placing the nanoparticle colloidal solution into an ultrafiltration centrifugal tube with the molecular weight cutoff of 3kDa, centrifuging for 45min at 5000rpm, and collecting precipitates to obtain the folic acid targeted nano-gel;
re-dispersing the folic acid targeting nano micelle in deionized water according to the ratio of sulfydryl: adding brain-targeted peptide Angiopep-2 into the maleimide group-2: 1, sealing a reaction system, stirring and reacting for 6 hours at room temperature, after the reaction is finished, performing ultrafiltration or centrifugal separation and precipitation, washing to obtain the double-targeted polymer drug nano-carrier, and dispersing the double-targeted polymer drug nano-carrier in deionized water.
Characterization of the double targeting polymer drug nanocarriers:
(1) appearance form
And observing the appearance of the double-target polymer drug nano-carrier by using a Transmission Electron Microscope (TEM), dropwise adding 5 mu L of 1mg/mL solution of the double-target polymer drug nano-carrier onto a copper screen, drying at room temperature, immersing the copper screen into phosphotungstic acid, carrying out negative dyeing for 2min, drying, and observing the appearance under the TEM, wherein the result is shown in an attached figure 2.
(2) Particle size and zeta potential measurements
Diluting 50 mu L of the solution of the double-targeting polymer drug nano-carrier with 1mg/mL to 1.5mL, placing the solution in a cuvette, measuring the particle size and zeta potential by using a laser particle size analyzer, and comparing the particle size and the zeta potential with the solution before modification of the brain-targeting peptide Angiopep-2; the results are shown in Table 2.
TABLE 2 particle size and zeta potential measurements before and after modification of brain-targeting peptides
| Whether to modify a polypeptide | Z-Ave(nm) | PDI | ZP(mV) |
| Unmodified | 127.9±3.8 | 0.109±0.15 | -22.8±5.4 |
| Decoration | 157.9±3.1 | 0.183±0.052 | -7.35±3.8 |
When the nanoparticles are modified with polypeptide, the particle size is obviously increased, and the potential is relatively increased.
(3) Determination of graft ratio
Detecting the content of the residual brain targeting peptide in the supernatant by using a BCA protein concentration determination kit, and calculating the grafting ratio of the brain targeting peptide, wherein the steps are as follows:
s1, preparing working solution: adding 1 volume of Cu reagent (50:1) into 50 volumes of BCA reagent according to the specification to prepare BCA working solution, and uniformly mixing;
s2, diluting a standard product: diluting 10 μ L BSA standard substance to 100 μ L with PBS to a final concentration of 0.5mg/ml, adding the standard substance to the protein standard substance well of 96-well plate at a ratio of 0, 2, 4, 6, 8, 12, 16, 20 μ L, and adding PBS to make up to 20 μ L;
s3, adding 20 mu L of the supernatant after the nano micelle centrifugation into sample wells of a 96-well plate, adding 200 mu L of BCA working solution into each well, standing at 37 ℃ for 30min, measuring by using an enzyme-labeling instrument at a wavelength of 562nm, and calculating the protein concentration according to a standard curve.
The BCA standard curve is shown in figure 3, and according to the standard curve, the grafting rate of the brain targeting peptide is calculated to be 63.9%.
(4) DSC differential scanning calorimetry characterization
Respectively measuring brain targeting peptide Angiopep-2(ANG), folic acid targeting nano micelle (FA-PLGA-NPs) and double targeting polymer drug nano carrier (ANG/FA-PLGA-NPs), taking 1mL of solution with the concentration of 5mg/mL, and measuring the glass transition temperature by using a DSC differential scanning calorimeter; the initial temperature is 10 ℃, the final temperature is 130 ℃, the pressure disturbance (PPC) is controlled to 3 atmospheric pressures, the temperature rise speed is 1 ℃/min, and finally, a thermal analysis chart is drawn, and the result is shown in the attached figure 4.
According to the spectrogram, the method comprises the following steps: the glass transition temperature of the brain targeting peptide Angiopep-2(ANG) is 29.84 ℃, the glass transition temperature of the folic acid targeting nano-micelle (FA-PLGA-NPs) is 34.16 ℃, and the double targeting polymer drug nano-carrier (ANG/FA-PLGA-NPs) shows two peaks in the graph, and the glass transition temperature is ANG: 33.14 ℃; FA-PLGA: 36.89 ℃. The chemical reaction between the brain targeting peptide and PLGA is obtained according to the differential thermal results, and the connection of the brain targeting peptide and the PLGA is further proved.
Example 17
Accurately weighing 4.0mg of PLGA-PEG-OMe, 1.0mg of PLGA-PEG-MAL, 1.0mg of PLGA-PEG-FA and 0.6mg of paclitaxel, adding 0.5ml of dichloromethane for dissolution, adding 2ml of sodium cholate solution with the mass concentration of 1%, performing pulse ultrasonic treatment (5 s of ultrasound and 5s of interruption) by using an ultrasonic cell crushing instrument, performing ultrasonic power of 100W for 90s of pulse ultrasonic treatment to obtain a primary emulsion, dropwise adding the primary emulsion into 1ml of sodium cholate solution with the mass concentration of 0.1%, performing rotary evaporation for 15min at the temperature of 37 ℃ to remove dichloromethane to obtain a nanoparticle colloidal solution, placing the nanoparticle colloidal solution into an ultrafiltration centrifugal tube with the molecular weight cutoff of 3kDa, centrifuging at 5000rpm for 45min, collecting precipitates, and preparing the targeted drug-loaded nano-micelle;
adding 1ml of deionized water into the prepared folic acid targeting nano micelle, dispersing, transferring into a penicillin bottle, and treating according to the ratio of sulfydryl: adding brain targeting peptide Angiopep-2 into the maleimide group-2: 1, sealing a reaction system, stirring and reacting for 6 hours at room temperature, placing the solution into an ultrafiltration centrifugal tube with the molecular weight cutoff of 3kDa after the reaction is finished, centrifuging for 45 minutes at 5000rpm, collecting precipitates, washing, preparing the double-targeting drug-loaded nano micelle, and dispersing in deionized water.
The characterization of the double-targeting drug-loaded nano-micelle is as follows:
(1) determination of paclitaxel content
Measured by a UPLC method, the chromatographic conditions are as follows: xue Yue C 18 The chromatographic column adopts methanol-acetonitrile-water as a mobile phase (V: V: V: 40: 30: 30), and the isocratic elution is carried out at the flow rate of 0.4 mL/min -1 (ii) a The column temperature is 30 ℃; the detection wavelength is 228 nm; the sample size was 1. mu.L.
Accurately weighing appropriate amount of PTX (paclitaxel) in 10mL measuring flask, adding methanol to dissolve, and metering to desired volume to obtain 1mg/mL -1 The stock solution of the standard substance (1), 2, 5, 10, 20, 50, 100. mu.g.mL respectively -1 The standard solution of (2) is plotted with the peak area as the ordinate (Y) and the mass concentration as the abscissa (X), and a regression equation is calculated. The standard curve is shown in figure 5, and the result shows that the linear regression equation of PTX is Y0.491X +0.0556, and the correlation coefficient R 2 1.000. It is found that the concentration of PTX is 1 to 100. mu.g/mL -1 The time and the peak area show good linear relation.
(1-1) precision
Taking appropriate amount of paclitaxel reference substance, and making into low, medium and high (17.3, 57.7, 164.8 mg. L) -1 ) The reference substance solutions with three mass concentrations are filtered by a 0.22 mu m microporous filter membrane, the subsequent filtrate is measured according to the chromatographic condition, the sample injection is carried out for 6 times, the RSD is respectively calculated to be 0.78%, 0.25% and 0.07%, and the precision of the instrument is good.
(1-2) recovery rate
Taking blank nano-carrier 2mg, adding appropriate amount of paclitaxel reference substance into 2mL volumetric flask, diluting with methanol to scale, and making into low, medium and high (17.3, 57.7, 164.8 mg. L -1 ) Three mass concentrations of sample solution. Filtering with 0.22 μm microporous membrane, collecting filtrate, and measuring by chromatographyThe calculated average recovery rates were 101.4. + -. 0.34%, 101.3. + -. 1.33%, 101.3. + -. 0.14% respectively, and RSD were 0.34%, 1.31% and 0.14% respectively, all in accordance with the methodological requirements.
(2) Encapsulation efficiency and drug loading
Precisely weighing 2mg of the double-target drug-loaded nano micelle, transferring the double-target drug-loaded nano micelle into a 2mL volumetric flask, adding acetonitrile for dissolving, carrying out ultrasonic treatment for 15min, fixing the volume to a scale by using the acetonitrile, and passing through a 0.22 mu m microporous filter membrane to be used as a sample to be detected. The paclitaxel content of the sample is determined according to the chromatographic conditions, and the Encapsulation Efficiency (EE) and Drug Loading (DL) are calculated.
The encapsulation rate (%) is PTX content/drug dosage in the nano micelle multiplied by 100%
The drug loading capacity (%) is PTX content in the nano-micelle/total mass of the nano-micelle multiplied by 100%
The drug loading rate and the encapsulation rate of the double-targeting drug-loaded nano-micelle with different proportions are measured by UPLC, and the drug loading rate is measured to be 7.2% +/-0.75%, and the encapsulation rate is measured to be 50.7% +/-1.0%.
(3) Stability of
Precisely weighing 2mg of double-target drug-loaded nano micelle, placing the double-target drug-loaded nano micelle in a 2mL conical flask with a plug, precisely adding 2mL of acetonitrile, carrying out ultrasonic treatment for 15min, cooling to room temperature, filtering by a 0.22 mu m microporous membrane, respectively carrying out sample injection after preparation for 0, 2, 4, 8, 12, 16 and 24h, and measuring according to chromatographic conditions to obtain the RSD of 0.93 percent by calculation. From the results, it was found that the stability of the sample was good.
(4) In vitro drug release test
The conditions of the leakage groove of the paclitaxel are as follows:
preparation of phosphate buffer solution with pH 7.4: weighing 1.36g of monopotassium phosphate, adding 79ml of 0.lmol/L sodium hydroxide solution, adding 0.5% lauryl sodium sulfate, and diluting with water to 200ml to obtain the potassium phosphate.
preparation of phosphate buffer solution with pH 6.8: 250mL of 0.2mol/L potassium dihydrogen phosphate solution is taken, 118mL of 0.2mol/L sodium hydroxide solution is added, 0.5% sodium dodecyl sulfate is added, and the mixture is diluted to 1000mL by water and shaken up to obtain the potassium dihydrogen phosphate.
preparation of phosphate buffer solution with pH 5.0: weighing potassium dihydrogen phosphate 8.34g and dipotassium hydrogen phosphate 0.87g, respectively, adding 0.5% sodium dodecyl sulfate, and adding water to dissolve into 1000ml to obtain the final product.
Taking 6 parts of phosphate buffer solution with the pH value of 7.4, respectively adding sodium dodecyl sulfate with the proportion of 0.05%, 0.1%, 0.3%, 0.5%, 0.7% and 1.0%, adding 1.0mg of paclitaxel, performing ultrasonic treatment for 15min, and detecting the content of the paclitaxel by using UPLC; the measurement results are shown in FIG. 6.
After adding 1.0% sodium dodecyl sulfate, the dissolution ratio reaches 99.0%, and when 0.5% sodium dodecyl sulfate is added, the dissolution of the loaded paclitaxel nanoparticles is satisfied (about 4 times).
An in-vitro drug release test for researching the drug-loaded nano-micelle by adopting a dialysis method comprises the following steps: putting the drug-loaded nano micelle solution into a dialysis bag, respectively adding 20mL of phosphate buffer solution with pH5.0, pH6.8 and pH7.4 as an external dialysis solution, putting the dialysis system into a constant-temperature shaking box at 37 ℃, respectively collecting 1mL of external dialysis solution for 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 24h and 48h, and replacing with PBS with the same volume; the collected concentration of the dialysate was measured by UPLC, and the cumulative release curve of paclitaxel was plotted with time as abscissa and cumulative release rate as ordinate, and the measurement results are shown in fig. 7.
The in vitro drug release result shows that the accumulative release amount of the drug-loaded nano-micelle at pH7.4 and pH6.8 is about 35%, the accumulative release amount at pH5.0 is 43%, and the release amounts of the micelle at different pH values are slightly different; the nano-micelle has certain pH responsiveness.
(5) Serum and storage stability
Taking a proper amount of the drug-loaded nano-micelle solution, dispersing the drug-loaded nano-micelle solution into a phosphate buffer solution (pH7.4), storing the solution for 7 days at the temperature of 4 ℃ in the dark, measuring the particle size of the nano-micelle by using a laser particle size analyzer, and observing the change condition of the particle size of the nano-micelle along with time.
Taking a proper amount of the drug-loaded nano-micelle solution, dispersing the drug-loaded nano-micelle solution into phosphate buffer (pH7.4) containing 10% FBS, keeping the drug-loaded nano-micelle solution in the dark at 4 ℃ for 7 days, measuring the particle size of the nano-micelle by using a laser particle size analyzer, and observing the change condition of the particle size of the nano-micelle along with time.
The stability determination result is shown in figure 8, and the result shows that the nano micelle has good stability within seven days.
Examples of the experiments
1. Cytotoxicity of dual targeting polymeric drug nanocarriers
Preparation of a test solution: ANG/FA-PLGA-NPs was diluted to 1mg/mL of a mother liquor, filtered through a 0.22 μm needle-type sterile filter, and diluted to different concentrations of 0, 0.01, 0.1, 1, 10, 100, 500 μ g/mL in MEM/EBSS medium containing 10% fetal bovine serum for use.
Well-digested log phase U251 cells were diluted to 5X 10 with MEM/EBSS medium containing 10% fetal bovine serum 4 each.mL -1 100 μ L of cell suspension was plated onto 96-well plates and incubated for 24 h. After the cells adhered to the wall, the old culture medium was discarded, 200. mu.L of ANG/FA-PLGA-NPs solutions of different concentrations were added, and 5% CO was added at 37 deg.C 2 The treated cells were incubated in a humid environment for 24h or 48 h. Adding 20 mu L of CCK-8 solution into each hole, continuously incubating for 2h in a constant-temperature incubator, measuring the A value by using a microplate reader at the wavelength of 450nm, and calculating the cell survival rate.
The determination results are shown in the table 3 and the attached figure 9, and show that the double-targeting polymer drug nano-carrier has almost no inhibition effect on the proliferation and the growth of cells and basically has no toxicity.
TABLE 3 toxicity of Dual Targeted Polymer drug nanocarriers on U251 cells
2. Cytotoxicity of drug-loaded nanomicelles
Preparation of a test solution: dissolving PTX in DMSO to 20mg/mL of mother liquor, and diluting with MEM/EBSS medium containing 10% fetal calf serum to different concentrations of 0, 0.001, 0.01, 0.1, 0.2, 1, 2, 5, 10 μ g/mL; drug-loaded nanomicelles (PTX @ ANG/FA-PLGA-NPs) were diluted to the same concentration with the medium.
By containing10% fetal bovine serum MEM/EBSS Medium well-digested log phase U251 cells were diluted to 5X 10 4 each.mL -1 100 μ L of cell suspension was plated onto 96-well plates and incubated for 24 h. After the cells were attached to the wall, the old medium was discarded, 200. mu.L of different concentrations of free PTX and PTX @ ANG/FA-PLGA-NPs (PTX equi-concentration) were added, and 5% CO was added at 37 ℃ to 2 Incubated the treated cells for 24h in a humid environment. Adding 20 mu LCCK-8 solution into each hole, continuously incubating for 2h in a constant-temperature incubator, measuring the A value by using a microplate reader at the wavelength of 450nm, and calculating the survival rate of cells.
The determination results are shown in Table 4 and figure 10, and show that the half inhibition concentration IC50 of PTX @ ANG/FA-PLGA-NPs is 1.07 mu g/mL, the half inhibition concentration IC50 of PTX is 1.27 mu g/mL, the PTX @ ANG/FA-PLGA-NPs form a dose-dependent trend, the drug-loaded nano-micelle and the free PTX have obvious inhibition effect on the proliferation and growth of tumor cells, and the PTX @ ANG/FA-PLGA-NPs has better effect.
TABLE 4 toxicity of drug-loaded nanomicelles on U251 cells
3. Cell scratch test
Removing the protective adhesive tape from the bottom of the culture dish of the wound healing insert, and adding 5X 10-containing solution into the cell insert 3 The cell suspension per well was incubated for 24h at 37 ℃ in an incubator. The cells were cultured in serum-free DMEM medium for 4 hours, the insert was removed with forceps, the cells were washed, and then an appropriate amount of free PTX (PTX), ANG/FA-PLGA-NPs (ANG/FA-NPs) and PTX @ ANG/FA-PLGA-NPs (PTX @ ANG/FA-NPs) was added for culture. The plates were photographed under a microscope at 0, 12, 24h and the area of the scratched area covered by cells was analyzed for each image.
The experimental results are shown in figures 11-12, and show that the ANG/FA-PLGA-NPs group has little inhibition effect on U251 cells, and PTX @ ANG/FA-PLGA-NPs have the effect of inhibiting the healing of tumor cells compared with the Control group. Compared with a control group, the cell healing rates of PTX and PTX @ ANG/FA-PLGA-NPs at 24h are respectively 26.1% and 22.7%, the two groups can obviously reduce the migration capacity of the glioma cells U251, and the brain targeting peptide folic acid double-modified nano drug delivery system has the best effect of inhibiting the migration of the U251 cells. Probably due to the sustained release effect of nanomicelle drug release.
4. Apoptosis assay
The apoptosis detection is analyzed by a flow cytometer. Taking U251 cells in logarithmic growth phase for 3X 10 cells per well 5 The individual cells were inoculated uniformly and cultured in 6-well plates for 48 h. Cells were treated with free PTX, PTX @ ANG/FA-PLGA-NPs and ANG/FA-PLGA-NPs, incubated for 24h, cells were collected and washed twice with PBS, then stained with the FITC annexin apoptosis detection kit according to the manufacturer's instructions, and finally, the apoptosis was detected with a flow cytometer.
The experimental results are shown in figure 13; after the U251 cells are treated by the drug for 48 hours, compared with a control group, the drug-free nano-micelle ANG/A-NPs basically have no influence on apoptosis, free drugs PTX and PTX @ ANG/FA-NPs mainly undergo early apoptosis, the sum of the early apoptosis and the late apoptosis can respectively reach 28.8 percent and 33.8 percent, and the PTX @ ANG/FA-NPs nano-micelle has better apoptosis effect on the brain glioma U251 cells than the free drugs.
5. Cell uptake assay
U251, HeLa, A549 cells (3X 10 per well) 5 Cell/cell) were seeded in 6-well plates and cultured for 24 h. 1mL of complete medium containing 20. mu.g of fluorescent nanoparticles (RHB @ PLGA-NPs, RHB @ ANG-PLGA-NPs, RHB @ FA-PLGA-NPs, RHB @ ANG/FA-PLGA-NPs) was added and incubated with the cells for 4h, and then the cells were washed completely with PBS and collected. Adding 4% paraformaldehyde to fix cell morphology, and washing with PBS phosphate buffer solution after 3-5 min. The DAPI staining solution and the Lysotracker fluorescent probe staining solution stain the cell nucleus and the cell cytoplasm respectively. After staining was completed, the cells were washed three times with PBS and observed for cell uptake under a fluorescent microscope.
The preparation method of the RHB @ PLGA-NPs comprises the following steps:
accurately weighing a proper amount of PLGA-PEG-OMe and RHB, dissolving with a proper amount of dichloromethane, adding 1ml of sodium cholate solution with the concentration of 2%, and performing intermittent ultrasound (ultrasound for 5s and intermittent 5s) for 60s at the power of 100W; dropwise adding the obtained primary emulsion into 1ml of 0.1% sodium cholate aqueous solution, carrying out rotary evaporation for 15min at 37 ℃ by a rotary evaporator to remove dichloromethane to obtain a nanoparticle colloidal solution, placing the nanoparticle colloidal solution into an ultrafiltration centrifugal tube with the molecular weight cutoff of 3kDa, centrifuging for 45min at 5000rpm, collecting precipitates, adding distilled water solution to re-disperse to obtain rhodamine B-loaded nano micelles, namely RHB @ PLGA-NPs, and placing the rhodamine B-loaded nano micelles into a penicillin bottle for storage.
The preparation method of the RHB @ ANG-PLGA-NPs comprises the following steps:
accurately weighing appropriate amount of PLGA-PEG-OMe, PLGA-PEG-MAL and RHB, dissolving with dichloromethane, adding 1ml of 2% sodium cholate solution, and performing intermittent ultrasound (ultrasound for 5s, intermittent 5s) with power of 100W for 60 s; dropwise adding the obtained primary emulsion into 1ml of 0.1% sodium cholate aqueous solution, carrying out rotary evaporation for 15min at 37 ℃ by using a rotary evaporator to remove dichloromethane to obtain a nanoparticle colloidal solution, placing the nanoparticle colloidal solution into an ultrafiltration centrifugal tube with the molecular weight cutoff of 3kDa, centrifuging for 45min at 5000rpm, collecting precipitate, adding distilled water solution to re-disperse, transferring the precipitate into a penicillin bottle, and carrying out the steps of: adding brain-targeted peptide Angiopep-2 into the maleimide group-2: 1, sealing a reaction system, stirring and reacting for 6 hours at room temperature, after the reaction is finished, performing ultrafiltration or centrifugal separation on the precipitate, washing and re-dispersing to obtain the rhodamine B-loaded targeted nano micelle, namely RHB @ ANG-PLGA-NPs, and placing the rhodamine B-loaded targeted nano micelle in a penicillin bottle for storage.
The preparation method of the RHB @ FA-PLGA-NPs comprises the following steps:
accurately weighing a proper amount of PLGA-PEG-OMe, PLGA-PEG-FA and RHB, dissolving with a proper amount of dichloromethane, adding 1ml of sodium cholate solution with the concentration of 2%, and performing intermittent ultrasound (ultrasound for 5s and intermittent 5s) for 60s at the power of 100W; dropwise adding the obtained primary emulsion into 1ml of 0.1% sodium cholate aqueous solution, carrying out rotary evaporation for 15min at 37 ℃ by a rotary evaporator to remove dichloromethane to obtain a nanoparticle colloidal solution, placing the nanoparticle colloidal solution into an ultrafiltration centrifugal tube with the molecular weight cutoff of 3kDa, centrifuging for 45min at 5000rpm, collecting precipitates, adding distilled water solution to re-disperse to prepare rhodamine B folic acid-loaded targeting nano micelles, namely RHB @ FA-PLGA-NPs, and placing the rhodamine B folic acid-loaded targeting nano micelles into a penicillin bottle for storage.
The experimental results are shown in figures 14-16, the human lung cancer A549 cells do not take in three nano-micelles of RHB @ FA-PLGA-NPs, RHB @ ANG-PLGA-NPs and RHB @ ANG/FA-PLGA-NPs, the human cervical carcinoma Hela cells take in the three nano-micelles of RHB @ FA-PLGA-NPs and RHB @ ANG/FA-PLGA-NPs well, the nano-micelles of RHB @ ANG-PLGA-NPs are less, and the human brain glioma U251 cells take in the three nano-micelles well.
6. Biodistribution
And observing the biological distribution of the fluorescent agent-loaded nano micelle in the mouse by adopting a living body imaging method. After 10 mg.kg-1 doses of nano-micelle solutions of free DiR (DiR), DiR @ FA-PLGA-NPs (DiR @ FA-NPs) and DiR @ ANG/FA-PLGA-NPs (DiR @ ANG/FA-NPs) were injected via tail vein, the whole body fluorescence distribution patterns were collected at time points of 0h, 1h, 4h, 8h and 12h, respectively, using in vivo bio-optical imaging technique.
The experimental result is shown in figure 17, and the fluorescence result shows that ANG/FA-PLGA-NPs can penetrate through a blood brain barrier to be more accumulated at a tumor part based on targeting.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A double-targeting polymer drug nano-carrier is characterized by comprising a nano-material formed by polylactic glycolic acid-polyethylene glycol block copolymer, wherein the surface of the nano-material is modified with tumor targeting functional molecules and brain targeting peptide, the tumor targeting functional molecules are folic acid, and the brain targeting peptide is connected with the nano-material through sulfydryl-maleimide;
the preparation method of the double-targeting polymer drug nano-carrier comprises the following steps:
s1, respectively weighing PLGA-PEG-MAL, PLGA-PEG-FA and PLGA-PEG-OMe, mixing to obtain a mixture, adding dichloromethane to dissolve the mixture, adding a first sodium cholate solution, performing pulse ultrasonic treatment to obtain a primary emulsion, dropwise adding the primary emulsion into a second sodium cholate solution, performing reduced pressure distillation to remove dichloromethane after dropwise adding is completed to obtain a nanoparticle colloidal solution, and performing ultrafiltration or centrifugal separation and precipitation on the nanoparticle colloidal solution to obtain the folic acid targeted nano micelle;
s2, re-dispersing the folic acid targeting nano micelle prepared by the S1 in deionized water, wherein the molar ratio of sulfydryl to maleimide is 2:1, adding brain targeting peptide Angiopep-2, then closing a reaction system, stirring for reaction at room temperature, performing ultrafiltration or centrifugal separation for precipitation after the reaction is finished, and washing to obtain the double-targeting polymer drug nano-carrier;
wherein the mass ratio of the PLGA-PEG-MAL to the PLGA-PEG-FA to the PLGA-PEG-OMe is 1:1: 4; the mass concentration of the first sodium cholate solution is 2%, and the mass concentration of the second sodium cholate solution is 0.1%; the mass-volume ratio of the mixture to the first sodium cholate solution is 1-6 g/L; the volume ratio of the first sodium cholate solution to the second sodium cholate solution is 2: 1.
2. the double-targeting polymer drug nanocarrier of claim 1, wherein the pulsed ultrasound conditions in step S1 are: the pulse ultrasonic time is 30-90s, the ultrasonic power is 100W, and the duty ratio is 5 s: and 5 s.
3. The nanocarrier of claim 1, wherein the temperature of the vacuum distillation in step S1 is 37 ℃ and the distillation time is 15 min.
4. The dual-targeting polymer drug nanocarrier of claim 1, wherein the ultrafiltration separation pellet of step S1 or step S2 is obtained by centrifuging an ultrafiltration centrifuge tube with a molecular weight cut-off of 3kDa under the centrifugation condition of 5000rpm x 45 min.
5. The dual targeting polymer drug nanocarrier of claim 1, wherein the centrifugation conditions of the centrifugation and precipitation in step S1 or step S2 are 12000rpm x 20 min.
6. A dual-targeting drug, comprising the dual-targeting polymer drug nanocarrier of claim 1 and an anti-tumor drug.
7. The dual targeting drug of claim 6 wherein said antineoplastic drug is paclitaxel.
8. Use of a dual targeting agent according to claim 6 for the manufacture of a medicament for the treatment of brain glioma.
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