CN108478528B - Targeting polymer drug-loaded micelle and preparation method thereof - Google Patents
Targeting polymer drug-loaded micelle and preparation method thereof Download PDFInfo
- Publication number
- CN108478528B CN108478528B CN201810362419.XA CN201810362419A CN108478528B CN 108478528 B CN108478528 B CN 108478528B CN 201810362419 A CN201810362419 A CN 201810362419A CN 108478528 B CN108478528 B CN 108478528B
- Authority
- CN
- China
- Prior art keywords
- solution
- micelle
- pmncf
- stirring
- loaded
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/545—Heterocyclic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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
- A61K47/6905—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 colloid or an emulsion
- A61K47/6907—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 colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
- A61K47/6909—Micelles formed by phospholipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention provides a targeting polymer drug-loaded micelle, which comprises an amphiphilic polymer and a hydrophobic drug, wherein the shell of the micelle is a zwitter-ionic group, the core of the micelle is the hydrophobic group and the hydrophobic drug, and the targeting groups are distributed on the outer surface of the micelle; the preparation method of the polymer drug-loaded micelle comprises the following steps: weighing amphiphilic polymer and hydrophobic drug, and dissolving in organic solvent to obtain solution A; dripping the solution A into a folic acid good solvent, and stirring to form a solution B; and adding a salt solution into the solution B, stirring for 6h, and purifying to obtain a micelle solution, wherein folic acid targeting groups in the polymer drug-loaded micelle are distributed on the outer surface of the micelle, so that the targeting capability of the targeting groups can be improved to the greatest extent, the targeting effect of the micelle on cancer cells is enhanced, the killing capability on the cancer cells is improved, and meanwhile, the toxic and side effects on normal tissue cells are reduced.
Description
Technical Field
The invention belongs to the field of high molecular biomedicine, and particularly relates to a targeting polymer drug-loaded micelle and a preparation method thereof.
Background
Cancer is a worldwide problem threatening human health, and methods for treating cancer include surgical treatment, radiotherapy and chemotherapy. Chemotherapy is the most common and common, but has serious side effects in clinical treatment because of its high lethality to normal cells of human body. In recent years, nano-drug carriers are widely used in cancer chemotherapy, and can reduce side effects of the chemotherapy by virtue of the high permeability of cancer blood vessels and the retention (EPR) effect of cancer tissues in a passive targeting manner. However, the traditional nano drug-loaded carrier is easily cleared by mononuclear phagocyte in blood in vivo, so that the circulation time in vivo is short, and the drug cannot be effectively transported to a tumor part.
Therefore, a great deal of research focuses on the modification of the surface of the nano-drug carrier to impart the ability of being invisible in blood, and polyethylene glycol (PEG) is widely used for the modification of the nano-drug carrier, and since PEG has good hydrophilicity, a hydration layer can be formed on the surface of the nano-drug carrier, so that the adhesion of proteins in blood and the clearance of mononuclear phagocyte can be reduced. However, clinical trials show that PEG is also easily oxidized in vivo, and repeated use of PEG can generate an effect of Accelerating Blood Clearance (ABC). To overcome the disadvantages of PEG, the biocompatibility of membrane-encapsulated nanoparticles, including nanoparticles derived from encapsulation of Red Blood Cells (RBCs), platelets, leukocytes, cancer cell membranes, etc., has been investigated. These membrane-coated nanopharmaceutical carriers showed significantly enhanced circulating half-life compared to nanoparticles coated by a layer of polyethylene glycol (PEG). Phosphorylcholine is a hydrophilic zwitterion with equal positive and negative charges in an outer cell membrane, can be combined with a large number of water molecules to form a hydration layer, and can be used as a hydrophilic chain segment of a polymer micelle to endow the polymer micelle with invisible capability in blood. Although the nano-drug carrier with the simulated cell membrane structure can circulate in vivo for a long time, the binding effect with cancer cells is weak, and a targeting ligand needs to be added to increase the targeting effect on a tumor part. The Folate Receptor (FR) is tumor-related protein with very high affinity with folate, and enables the nano-drug carrier with the surface rich in folate to enter cancer cells through an endocytosis mechanism, thereby efficiently killing the cancer cells. However, a plurality of literature reports show that the nano-drug carrier modified by high content of folic acid (> 30%) has a targeting effect, while the nano-drug carrier modified by low content of folic acid (< 10%) does not show a targeting effect. In addition, most folate ligand-modified nanocarriers are not more than 3-fold selective for cancer cells. This is probably caused by the fact that folic acid targeting groups are easily embedded under the hydrophilic layer of the nano-drug carrier due to poor water solubility, and therefore, it is very important to research how to improve the density and the connection mode of the targeting groups on the surface of the nano-drug carrier.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a targeting polymer drug-loaded micelle which has higher cancer cell targeting performance;
the invention also aims to provide a preparation method of the targeting polymer drug-loaded micelle.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a targeting polymer drug-loaded micelle comprises an amphiphilic polymer and a hydrophobic drug, wherein the amphiphilic polymer has the following structural formula:
wherein R is1Is a zwitterionic group, n1Is a positive integer of 1 to 3, R2Is a hydrophobic group, n2Is a positive integer of 5 to 50, R3As a targeting group, n3Is a positive integer of 5 to 50; r is H or methyl, x/(x + y + z) is more than or equal to 0.5 and less than or equal to 0.8, y/(x + y + z) is more than or equal to 0.05 and less than or equal to 0.3, and z/(x + y + z) is more than 0 and less than or equal to 0.3, wherein the targeting group R is3Comprises a folate group; the shell of the micelle is a zwitter-ion group, the core of the micelle is a hydrophobic group and a hydrophobic drug, and the targeting group is distributed on the outer surface of the micelle.
Preferably, the zwitterion group is a phosphorylcholine, carboxylic acid betaine or sulfonic acid betaine group; the hydrophobic group is selected from one or more of cholesterol, alkyl with the carbon atom number more than 8 or heterocyclic hydrophobic group with the carbon atom number more than 8.
Preferably, the targeting group R3Also comprises one of cancer cell targeting peptide RGD, RGDm, cRGDyK or RGDFC.
A preparation method of the targeting polymer drug-loaded micelle comprises the following steps:
(1) weighing an amphiphilic polymer and a hydrophobic drug, and dissolving the amphiphilic polymer and the hydrophobic drug in an organic solvent to obtain a solution A, wherein the structural formula of the amphiphilic polymer is as follows:
wherein R is1Is a zwitterionic group, n1Is a positive integer of 1 to 3, R2Is a hydrophobic group, n2Is a positive integer of 5 to 50, R3As a targeting group, n3Is a positive integer of 5 to 50; r is H or methyl, x/(x + y + z) is more than or equal to 0.5 and less than or equal to 0.8, y/(x + y + z) is more than or equal to 0.05 and less than or equal to 0.3, and z/(x + y + z) is more than 0 and less than or equal to 0.3, wherein the targeting group R is3Comprises a folate group;
(2) dripping the solution A into a folic acid good solvent, and stirring for 1-10 h to form a solution B; the folic acid good solvent is an organic solvent/deionized water mixed solution or an alkaline aqueous solution;
(3) and adding a salt solution into the solution B, stirring for 1-8 h, and purifying to obtain the polymer drug-loaded micelle solution.
Preferably, the zwitterionic group in step (1) is a phosphorylcholine, carboxylic betaine or sulfonic betaine group; the hydrophobic group is selected from one or more of cholesterol, alkyl with the carbon atom number more than 8 or heterocyclic hydrophobic group with the carbon atom number more than 8; the targeting group further comprises one of cancer cell targeting peptides RGD, RGDm, cRGDyK or RGDFC.
Preferably, the organic solvent is dimethyl sulfoxide or N, N-dimethylformamide; the alkaline aqueous solution is aqueous solution of triethylamine, sodium hydroxide or potassium hydroxide.
Preferably, the temperature of the alkaline aqueous solution in the step (2) is 30-80 ℃, and the pH value is 8-10.
Preferably, the salt solution in step (3) is one or more selected from phosphate, carbonate, sodium chloride, potassium chloride or calcium chloride solution.
Preferably, the purification in step (3) is performed by an ultrafiltration method.
The invention has the beneficial effects that: the folic acid targeting groups in the targeting polymer drug-loaded micelle are distributed on the outer surface of the micelle, so that the targeting capability of the targeting groups can be improved to the greatest extent, the targeting effect of the micelle on cancer cells is enhanced, the killing capability on the cancer cells is improved, and meanwhile, the toxic and side effects on normal tissue cells are reduced; the preparation method of the targeting polymer drug-loaded micelle provided by the invention regulates and fixes the orientation of the folate targeting groups in the self-assembly preparation process of the amphiphilic polymer to distribute the folate targeting groups on the outer surface of the micelle so as to improve the targeting performance of the polymer micelle on cancer cells.
Drawings
FIG. 1 is a scanning electron microscopy topography of an unloaded PMNCF-25 micelle;
FIGS. 2 and 3 are a fluorescence microscopic image and a fluorescence phagocytosis rate image of several coumarin-6-loaded polymer micelles after co-culturing with different cells for 6h, respectively;
FIG. 4 shows the cell viability of several doxorubicin-loaded polymer micelles after 48h of coculture with L929 cells (a) and Hela cells (b).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are described below clearly and completely, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The preparation method of the targeting polymer drug-loaded micelle regulates and fixes the orientation of the folic acid targeting groups in the process of preparing the micelle by self-assembling the amphiphilic polymer, so that the folic acid targeting groups are distributed on the outer surface of the micelle, and the targeting performance of the polymer micelle on cancer cells is improved. Firstly, an amphiphilic polymer solution is dripped into a folic acid good solvent to promote folic acid groups to migrate to the outer surface of a micelle in an oriented mode in the self-assembly process of a polymer, the folic acid groups are regulated and controlled to be more distributed on the outer surface of the micelle, then a salt solution is added to shrink and fix the core-shell structure of the polymer micelle, so that the micelle forms a compact zwitter-ion hydrophilic shell structure, the folic acid groups are not easy to migrate to hydrophobic cores of the micelle, the purpose of fixing the folic acid groups on the outer surface of the micelle is further achieved, and after organic solvents, small-molecule impurities and large-particle precipitates in the solution are removed through centrifugal ultrafiltration treatment, the drug-loaded polymer micelle solution.
The targeting polymer drug-loaded micelle is obtained by self-assembling an amphiphilic polymer, and the structural formula of the amphiphilic polymer is as follows:
wherein R is1Is a zwitterionic group, n1Is a positive integer of 1 to 3, R2Is a hydrophobic group, n2Is a positive integer of 5 to 50, R3As a targeting group, n3Is a positive integer of 5 to 50; r is H or methyl, x/(x + y + z) is more than or equal to 0.5 and less than or equal to 0.8, y/(x + y + z) is more than or equal to 0.05 and less than or equal to 0.3, and z/(x + y + z) is more than 0 and less than or equal to 0.3, wherein the targeting group R is3Including folic acid groups.
In order to further illustrate the present invention, the following examples are given to describe in detail a targeting polymer drug-loaded micelle and a preparation method thereof, wherein the following examples show the structural formula of the amphiphilic polymer PMNCF:
wherein the mol ratio of phosphorylcholine group, cholesterol group and folic acid group is 57: 13: 30.
example 1
Preparation of drug-free PMNCF-25 micelle
(1) Weighing 10mg of PMNCF, adding into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a PMNCF solution;
(2) slowly dropping the PMNCF solution into 10mL of deionized water containing 10% of dimethyl sulfoxide, and stirring for 6 h;
(3) after stirring was completed, 1mL of PBS solution was added and stirring was continued for 6 hours, the resulting reaction solution was transferred to an ultrafiltration centrifuge tube, centrifuged at 4000rpm for 20 minutes, the filtrate was discarded, and an equal amount of deionized water was added. Repeating the steps until the conductivity of the filtrate is close to that of deionized water, which shows that the ions and dimethyl sulfoxide in the micellar solution are almost completely removed, placing the micellar solution in a centrifugal tube and centrifuging the micellar solution at 6000rpm for 20 minutes to remove large-particle precipitates to prepare the micellar solution, wherein the SEM image of the micellar solution is shown in figure 1.
Example 2
Preparation of adriamycin-loaded PMNCF-25 micelle
(1) Weighing 10mg of PMNCF and 4mg of adriamycin, adding into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a solution containing PMNCF and adriamycin;
(2) slowly dripping the solution containing PMNCF and adriamycin into 10mL of deionized water containing 10% dimethyl sulfoxide, and stirring for 6 h;
(3) after stirring was completed, 1mL of PBS solution was added and stirring was continued for 6 hours, the resulting reaction solution was transferred to an ultrafiltration centrifuge tube, centrifuged at 4000rpm for 20 minutes, the filtrate was discarded, and an equal amount of deionized water was added. And repeating the steps until the conductivity of the filtrate is close to that of deionized water, which shows that the ions and the dimethyl sulfoxide in the micellar solution are almost completely removed, placing the micellar solution in a centrifugal tube and centrifuging at 6000rpm for 20 minutes, and removing large-particle precipitates to obtain the doxorubicin-loaded PMNCF-25 micellar solution.
Example 3
Preparation of adriamycin-loaded PMNCF micelle
(1) Weighing 10mg of PMNCF and 4mg of adriamycin, adding into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a solution containing PMNCF and adriamycin;
(2) slowly dripping the solution containing PMNCF and adriamycin into 10mL of deionized water with the temperature of 50 ℃ and the pH value of 9, and stirring for 6 hours;
(3) after stirring was completed, 1mL of a 0.3mg/mL sodium chloride solution was added and stirring was continued for 7 hours, and the resulting reaction solution was transferred to an ultrafiltration centrifuge tube, centrifuged at 4000rpm for 20 minutes, the filtrate was discarded, and an equal amount of deionized water was added. And repeating the steps until the conductivity of the filtrate is close to that of deionized water, which shows that the ions and the dimethyl sulfoxide in the micellar solution are almost completely removed, placing the micellar solution in a centrifugal tube and centrifuging at 6000rpm for 20 minutes, and removing large-particle precipitates to prepare the micellar solution.
Comparative example 1
Preparation of drug-free PMNCF-10 micelle
(1) Weighing 10mg of PMNCF, adding into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a PMNCF solution;
(2) the PMNCF solution was slowly dropped into 10mL of deionized water, stirred for 12 hours and then purified in an ultrafiltration centrifuge tube as described in example 1.
Comparative example 2
Preparation of drug-free PMNCF-18 micelle
(1) Weighing 10mg of PMNCF, adding into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a PMNCF solution;
(2) the PMNCF solution was slowly dropped into 10mL of deionized water containing 10% dimethyl sulfoxide, stirred for 12 hours, and then purified in an ultrafiltration centrifuge tube according to the method described in example 1.
Comparative example 3
Preparation of drug-free PMNCF-0 micelle
(1) Weighing 10mg of amphiphilic polymer, adding the amphiphilic polymer into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a polymer solution, wherein the amphiphilic polymer has the same structure as PMNCF except that no folic acid group is contained;
(2) slowly dropping the PMNCF solution into 10mL of deionized water containing 10% of dimethyl sulfoxide, and stirring for 6 h;
(3) after stirring was completed, 1mL of PBS solution was added and stirring was continued for 6 hours, and the obtained reaction solution was transferred to an ultrafiltration centrifuge tube and purified as described in example 1.
Test example 1
Measurement of particle size distribution and Zeta potential
The micellar solution prepared in example 1, comparative examples 1 and 2 was brought to a volume of 1mg mL-1The particle size distribution and the Zeta potential of the three polymer micelles are measured by dynamic light scattering by a Malvern Zetasizer Nano ZS90 Nano particle size analyzer, and the results show that the number average particle size of the three polymer micelles is between 57 and 73nm, the Zeta potential is respectively-24.7 +/-1.6 mV, -10.2 +/-0.4 mV and-18.0 +/-1.1 mV, and the test result of the Zeta potential shows that the orientation distribution of the folate groups on the surfaces of the micelles is remarkably changed, because only the folate groups in the amphiphilic polymer can be ionized, and phosphorylcholine and cholesterol groups are not charged under the experimental conditions, thereby showing that the addition of DMSO and PBS solutions in the process of preparing PMNCF micelles can remarkably improve the density of the folate groups on the surfaces of the Nano micelles.
Test example 2
Uptake assay for normal and tumor cells
Coumarin-6-loaded PMNCF-25, PMNCF-10, PMNCF-18 and PMNCF-0 micellar solutions were prepared using the same procedure as in example 1, comparative example 2 and comparative example 3, respectively, except that 0.02mg of coumarin-6 was added to dimethylsulfoxide at the same time in step (1).
The cellular uptake and targeting of PMNCF micelles were assessed using mouse fibroblasts L929 and human cervical carcinoma cells Hela, and qualitative analysis was performed using an inverted fluorescence microscope (Ti-U, Nikon). First, L929 and Hela cells were seeded into 24-well plates at about 1.0X 10 cells per well5Single cell, and placed in 5% CO2And culturing in a constant temperature incubator at 37 ℃ for 12 hours to allow the cells to adhere to the wall. Then, the stock culture was discarded, and 1mL of a culture containing coumarin-6-loaded PMNCF micelles at a micelle concentration of 0.2mgmL was added-1And culturing the cells together in a constant temperature incubator at 37 ℃ for 6 h. The culture containing the micellar solution was then aspirated and washed with sterile PBSThree times of washing, and observing by an inverted fluorescence microscope, the result is shown in FIG. 2. For quantitative analysis, the phagocytized culture broth was centrifuged at 1500rpm for 10 minutes to remove cells from the culture broth, the fluorescence intensity of the supernatant was measured with a fluorescence spectrophotometer (F-7000, Hitachi), and the uptake rate of L929, MCF-7 and Hela cells [ (I) was calculated using the following formula using the culture broth without phagocytosis as a controlcontrol-I)/Icontrol]X 100%, where I represents the fluorescence intensity of the medium after uptake by the cells, and the results are shown in FIG. 3. As can be seen from FIGS. 2 and 3, compared with PMNCF-0 micelles without folic acid, the coumarin-6-loaded PMNCF-25, PMNCF-10 and PMNCF-18 micelles have significant difference in the phagocytosis efficiency of several micelles by the cancer cells Hela and MCF-7 with high folate receptor protein expression, and the phagocytosis efficiency of the cells mediated by the folate receptor protein gradually increases with the increase of the folate group density on the surfaces of the micelles, while the phagocytosis of the several micelles by the normal cells L929 is not significantly different.
Test example 3
Toxicity test of adriamycin-loaded polymer micelle on normal cells and tumor cells
Adriamycin-loaded PMNCF-10, PMNCF-18 and PMNCF-0 micellar solutions were prepared using the same procedure as in comparative example 1, comparative example 2 and comparative example 3, respectively, except that 4mg of Adriamycin was added to the dimethylsulfoxide at the same time in step (1); freeze-drying the prepared adriamycin-loaded PMNCF-10 micelle solution to obtain adriamycin-loaded PMNCF-10 micelles, re-dispersing the PMNCF-10 micelles in water to ensure that the Zeta potential of the obtained water solution is about 1mV, and preparing the adriamycin-loaded PMNCF-1 micelle solution.
Cytotoxicity of several doxorubicin-loaded polymer micelles was evaluated by tetrazolium salt colorimetry (MTT) using mouse fibroblast cells L929, human cervical carcinoma cells Hela, and Free doxorubicin solution (Free DOX) at the same concentration was used as a control. L929 cells were cultured in DMEM high-sugar medium, Hela cells were cultured in RPMI-1640 medium, and both cells were cultured in saturated humidity medium containing 5% CO2Culturing in a constant temperature incubator at 37 ℃. All cytotoxicity assays were performed in 96-well plates, approximately 1X 10 per well4Culturing the individual cells in a pore plate for 12 hours until the cells are completely attachedAfter the cell wall was removed, the culture medium was aspirated, 200. mu.L of doxorubicin-loaded polymer micelle solutions of different concentrations were added, and the mixture was dispersed in DMEM or RPMI-1640 medium without folic acid, wherein the concentrations of doxorubicin were 0.002, 0.005, 0.01, and 0.02mg mL, respectively-1. After an additional 48 hours of incubation, the medium was aspirated, adherent cells were washed twice with sterile PBS and 20. mu.L of 5mg mL were added to each well in the dark-1The MTT solution was further cultured at 37 ℃ for 4 hours, then the MTT solution was aspirated, 200. mu.L of formazan dissolved in chromatographically pure dimethyl sulfoxide was added thereto, the resulting mixture was shaken in a constant temperature shaker at 37 ℃ for 15 minutes and mixed uniformly, and then the Optical Density (OD) of each well was measured using a microplate readersampleValue, untreated cells were taken as control group (OD)controlCalculating the relative cell survival rate, which is expressed by the following formula:
Cell viability(％)＝(OD)sample/(OD)control×100％
as shown in FIG. 4, the toxicity test results of doxorubicin-loaded polymeric micelles and free doxorubicin on normal cells L929 and cancer cells HeLa are shown in FIG. 4, and it can be seen from FIG. 4 that the toxicity of doxorubicin-loaded PMNCF-25, PMNCF-10, PMNCF-18, PMNCF-1 and PMNCF-0 micelles on normal cells L929 is significantly lower than that of free doxorubicin, while the toxicity of doxorubicin-loaded PMNCF-25 micelles prepared in example 2 on folate receptor protein-highly-expressed Hela cells is significantly higher than that of PMNCF-10, PMNCF-18, PMNCF-1 and PMNCF-0 micelles and free doxorubicin.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (4)
1. A targeting polymer drug-loaded micelle is characterized in that the drug-loaded micelle is an unloaded PMNCF-25 micelle, and is prepared by the following method:
(1) weighing 10mg of PMNCF, adding into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a PMNCF solution;
(2) slowly dropping the PMNCF solution into 10mL of deionized water containing 10% of dimethyl sulfoxide, and stirring for 6 h;
(3) after stirring, adding 1mL of PBS solution, continuously stirring for 6h, transferring the obtained reaction solution into an ultrafiltration centrifugal tube, centrifuging for 20 minutes at 4000rpm, discarding the filtrate, adding an equal amount of deionized water, repeating the steps until the conductivity of the filtrate is close to that of the deionized water, indicating that the ions and dimethyl sulfoxide in the micelle solution are almost completely removed, placing the micelle solution into the centrifugal tube, centrifuging for 20 minutes at 6000rpm, removing large-particle precipitates, and preparing the micelle solution, wherein the Zeta potential of the micelle is-24.7 +/-1.6 mV;
the temperature of the PBS solution is 30-80 ℃, and the pH value is 8-10;
the structure of the PMNCF is as follows:
wherein the mol ratio of phosphorylcholine group, cholesterol group and folic acid group is 57: 13: 30.
2. a method for preparing the targeting polymer drug-loaded micelle of claim 1, comprising the following steps:
(1) weighing 10mg of PMNCF, adding into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a PMNCF solution;
(2) slowly dropping the PMNCF solution into 10mL of deionized water containing 10% of dimethyl sulfoxide, and stirring for 6 h;
(3) after stirring, adding 1mL of PBS solution, continuously stirring for 6h, transferring the obtained reaction solution into an ultrafiltration centrifugal tube, centrifuging for 20 minutes at 4000rpm, discarding the filtrate, adding an equal amount of deionized water, repeating the steps until the conductivity of the filtrate is close to that of the deionized water, indicating that the ions and dimethyl sulfoxide in the micelle solution are almost completely removed, placing the micelle solution into the centrifugal tube, centrifuging for 20 minutes at 6000rpm, and removing large-particle precipitates to obtain the micelle solution;
the temperature of the PBS solution is 30-80 ℃, and the pH value is 8-10.
3. A targeting polymer drug-loaded micelle is characterized in that the drug-loaded micelle is an adriamycin-loaded PMNCF-25 micelle, and is prepared by the following method:
(1) weighing 10mg of PMNCF and 4mg of adriamycin, adding into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a solution containing PMNCF and adriamycin;
(2) slowly dripping the solution containing PMNCF and adriamycin into 10mL of deionized water containing 10% dimethyl sulfoxide, and stirring for 6 h;
(3) after stirring, adding 1mL of PBS solution, continuing stirring for 6h, transferring the obtained reaction solution into an ultrafiltration centrifugal tube, centrifuging for 20 minutes at 4000rpm, discarding the filtrate, adding an equal amount of deionized water, repeating the steps until the conductivity of the filtrate is close to that of the deionized water, indicating that the ions and dimethyl sulfoxide in the micelle solution are almost completely removed, placing the micelle solution into the centrifugal tube, centrifuging for 20 minutes at 6000rpm, and removing large-particle precipitates to obtain the adriamycin-loaded PMNCF-25 micelle solution;
the temperature of the PBS solution is 30-80 ℃, and the pH value is 8-10;
the structure of the PMNCF is as follows:
wherein the mol ratio of phosphorylcholine group, cholesterol group and folic acid group is 57: 13: 30.
4. a method for preparing the targeting polymer drug-loaded micelle of claim 3, comprising the following steps:
(1) weighing 10mg of PMNCF and 4mg of adriamycin, adding into 1mL of dimethyl sulfoxide, and stirring overnight to obtain a solution containing PMNCF and adriamycin;
(2) slowly dripping the solution containing PMNCF and adriamycin into 10mL of deionized water containing 10% dimethyl sulfoxide, and stirring for 6 h;
(3) after stirring, adding 1mL of PBS solution, continuing stirring for 6h, transferring the obtained reaction solution into an ultrafiltration centrifugal tube, centrifuging for 20 minutes at 4000rpm, discarding the filtrate, adding an equal amount of deionized water, repeating the steps until the conductivity of the filtrate is close to that of the deionized water, indicating that the ions and dimethyl sulfoxide in the micelle solution are almost completely removed, placing the micelle solution into the centrifugal tube, centrifuging for 20 minutes at 6000rpm, and removing large-particle precipitates to obtain the adriamycin-loaded PMNCF-25 micelle solution;
the temperature of the PBS solution is 30-80 ℃, and the pH value is 8-10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810362419.XA CN108478528B (en) | 2018-04-20 | 2018-04-20 | Targeting polymer drug-loaded micelle and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810362419.XA CN108478528B (en) | 2018-04-20 | 2018-04-20 | Targeting polymer drug-loaded micelle and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108478528A CN108478528A (en) | 2018-09-04 |
| CN108478528B true CN108478528B (en) | 2020-11-10 |
Family
ID=63312864
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810362419.XA Active CN108478528B (en) | 2018-04-20 | 2018-04-20 | Targeting polymer drug-loaded micelle and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108478528B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103788211A (en) * | 2012-11-01 | 2014-05-14 | 中国科学院上海药物研究所 | Bifunctional peptide, composite formed by the bifunctional peptide and nucleic acid molecule, and pharmaceutical composite used for treating tumor |
| CN104804195A (en) * | 2015-01-13 | 2015-07-29 | 西北大学 | Mussel-adhered and cytomembrane antifouling double-bionic multi-armed PEG and preparation method thereof |
| CN106146761A (en) * | 2016-06-03 | 2016-11-23 | 西北大学 | The nano-micelle of a kind of amphipathic multifunctional polymer and preparation thereof and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6679593B2 (en) * | 2014-09-03 | 2020-04-15 | ジーンセグエス，インコーポレイテッド | Therapeutic nanoparticles and related compositions, methods and systems |
| CN105902518B (en) * | 2016-06-02 | 2019-03-26 | 北京林业大学 | A kind of preparation method of water soluble nanometer particles |
-
2018
- 2018-04-20 CN CN201810362419.XA patent/CN108478528B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103788211A (en) * | 2012-11-01 | 2014-05-14 | 中国科学院上海药物研究所 | Bifunctional peptide, composite formed by the bifunctional peptide and nucleic acid molecule, and pharmaceutical composite used for treating tumor |
| CN104804195A (en) * | 2015-01-13 | 2015-07-29 | 西北大学 | Mussel-adhered and cytomembrane antifouling double-bionic multi-armed PEG and preparation method thereof |
| CN106146761A (en) * | 2016-06-03 | 2016-11-23 | 西北大学 | The nano-micelle of a kind of amphipathic multifunctional polymer and preparation thereof and application |
Non-Patent Citations (2)
| Title |
|---|
| pH and redox dual-responsive biodegradable polymeric micelles with high drug loading for effective anticancer drug delivery;Jye Yng Teo,etal;《Nanomedicine: Nanotechnology, Biology, and Medicine》;20171231(第13期);431-442 * |
| 两亲性多功能聚合物合成及其仿细胞膜结构纳米胶束的构建;姜海涛 等;《2015年全国高分子学术论文报告会论文摘要集-主题F-生物医用高分子》;20151017;785 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108478528A (en) | 2018-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sun et al. | Bone microenvironment regulative hydrogels with ROS scavenging and prolonged oxygen-generating for enhancing bone repair | |
| CN105727307B (en) | Lipoic acid modified nano-polypeptide carrier and preparation method and application thereof | |
| CN113633625B (en) | Nano-drug of hybrid membrane loaded oxidative phosphorylation inhibitor and preparation method thereof | |
| CN107184987B (en) | Lipoic acid modified targeted integrin alpha v beta 3 nano-polypeptide carrier and preparation method and application thereof | |
| CN112999153B (en) | Nano micelle carrying chemotherapeutic drug/photosensitizer and preparation method and application thereof | |
| Li et al. | Dual pH-responsive micelles with both charge-conversional property and hydrophobic–hydrophilic transition for effective cellular uptake and intracellular drug release | |
| Pan et al. | Synthesis and characterization of biodegradable polyurethanes with folate side chains conjugated to hard segments | |
| EP3995152A1 (en) | Heparin nano drug carrier system for loading amino anti-tumor drug, and preparation method therefor | |
| US20200392296A1 (en) | Nano coordination polymer and preparation method and application thereof | |
| Biosca et al. | Zwitterionic self-assembled nanoparticles as carriers for Plasmodium targeting in malaria oral treatment | |
| CN110403917A (en) | A kind of artificial excretion body, preparation method and application | |
| An et al. | Hyaluronic acid-coated poly (β-amino) ester nanoparticles as carrier of doxorubicin for overcoming drug resistance in breast cancer cells | |
| CN114224838B (en) | Tumor microenvironment activated fusion membrane wrapped bionic nano delivery system and preparation method and application thereof | |
| Zhao et al. | Construction of functional targeting daunorubicin liposomes used for eliminating brain glioma and glioma stem cells | |
| CN113648401B (en) | Hybrid nano-assembly for proteasome inhibition sensitization photodynamic therapy and preparation and application thereof | |
| CN114224839A (en) | Method for modifying liposome by cell membrane | |
| CN108478528B (en) | Targeting polymer drug-loaded micelle and preparation method thereof | |
| CN104434792A (en) | Polymer micelle, preparation method thereof, antitumor pharmaceutical composition, preparation and preparation method thereof | |
| WO2023193389A1 (en) | Resveratrol-lecithin nanoparticle, method for preparing same, and use thereof | |
| CN110859820A (en) | Biocompatible alkaline earth metal peroxide nano preparation, preparation method and application thereof | |
| CN114642736B (en) | Blood-brain-penetrating barrier drug delivery system and preparation method and application thereof | |
| CN112426537B (en) | Polypeptide nano micelle and preparation method and application thereof | |
| CN115006367A (en) | Antibiotic-carrying bacterial outer membrane vesicle and preparation method and application thereof | |
| CN114246843A (en) | Preparation method of drug-loaded nanoparticles embedded with hypocrellin and cisplatin simultaneously | |
| CN116262138A (en) | PH responsive exosome carrier with core-shell structure and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |