Background
Cancer is an important health hazard for human life. In recent years, the incidence and mortality of cancer have become higher and higher, and the current treatment methods for cancer mainly include chemotherapy, radiotherapy and the like. The chemotherapy drugs are most widely used, such as clinically used adriamycin, cisplatin, paclitaxel and the like, however, the chemotherapy drugs can kill cancer cells and have higher toxicity to normal cells, so that adverse toxic and side effects are generated in the medication process. Therefore, how to reduce the toxicity of the chemotherapy drugs to normal cells is a technical problem which needs to be solved urgently when the anti-tumor drugs are used clinically.
To increase the targeting of drug molecules to tumor tissues is one approach to solve this problem, the following solutions are generally adopted: firstly, the structure of a drug molecule is modified by a target molecule, secondly, a targeting nano drug carrier is adopted to load the drug, and the drug molecule is released when the carrier reaches the vicinity of a tumor tissue, thereby achieving the effect of targeting and killing the tumor tissue. The chemical modification of the drug molecules may change the killing effect of the drug molecules on tumor tissues, so that the targeted drug delivery of most tumors is realized through a nano drug carrier clinically, and the targeted release of the drug molecules on the tumor tissues is ensured on the premise of not changing the drug efficacy of the drug.
The nano-drug carrier mainly utilizes the small-size effect and the high specific surface effect of nano-particles to improve the absorption of drugs and control the release of the drugs, and simultaneously utilizes the shielding effect of the carrier to realize the protection of the drugs, so the nano-drug carrier has obvious advantages compared with the traditional drug dosage form, and is mainly expressed in the following aspects: (1) the nano-drug carrier has small size and is easy to enter cells to realize high curative effect; (2) the nano-drug carrier has large specific surface area and strong adhesiveness, and is beneficial to the increase of retention during local administration, thereby prolonging the contact time of the drug and the digestive tract wall, increasing the contact area and finally improving the bioavailability of oral absorption of the drug; (3) the solubility of the drug with poor water solubility in the nano drug carrier is relatively enhanced, so that the use of the conventional emulsification solubilizer is avoided, and the toxic and side effects of the conventional emulsification solubilizer are avoided; (4) the nano-drug carrier generally has the structural characteristics of porosity, hollowness, multilayer and the like, and is easy to control the slow release of the drug; (5) the nanometer drug carrier can eliminate the limitation of special biological barrier to drug action.
Molecular Imprinting Technology (MIT) refers to a Technology for preparing a polymer selective for a specific target molecule (also called template molecule or imprinted molecule), which has the following advantages: (1) presetting: the template molecule and the functional monomer are selected before polymerization, so that the pre-determination determines that people can prepare different Molecularly Imprinted Polymers (MIP) according to own purposes so as to meet various different requirements; (2) specific recognition: MIPs are polymerized according to the configuration of the template molecule, so that the specific recognition site and the cavity of the MIP can specifically recognize the template molecule; (3) the practicability is as follows: the stability of MIP is good, and the MIP is not easily influenced by high temperature and acid and alkali; compared with natural biomolecules such as enzymes and substrates, antigens and antibodies, receptors and hormones, MIPs are resistant to more severe environmental influences, and are mild in storage conditions and capable of retaining recognition properties for a long time.
It is worth noting that the prepared nanoscale molecularly imprinted polymer has an "imprinted cavity" for template molecules, and when the molecularly imprinted polymer is applied to a system in which template molecules exist, the molecularly imprinted polymer can selectively adsorb the template molecules, thereby separating the template molecules (target molecules) from other substances.
In the prior art, the preparation of Molecularly Imprinted Polymers (MIPs) requires at least the following reagents: template molecules (templates); functional monomer (Functional monomer); porogens (solvents); a Crosslinking agent (Crosslinking); the function of the Initiator (Initiator) and each reactant is shown in table 1 below.
TABLE 1 Functions of the reagents
In addition, referring to fig. 1, the preparation process of the molecularly imprinted polymer in the prior art mainly includes: firstly, template molecules and functional monomers interact with each other in a covalent bond or non-covalent bond mode under the action of a pore-foaming agent to form a pre-polymerization compound; then, under the action of a cross-linking agent and an initiator, polymerizing around the template-functional monomer compound until the polymerization is finished to form a high molecular polymer; finally, the template molecule is eluted and removed, thus leaving a three-dimensional cavity in the high molecular weight polymer that is spatially matched to the template molecule and contains a recognition site specific to the template molecule. Due to the memory property of the three-dimensional cavity to the template molecules, the affinity of the MIP to the template molecules is greatly enhanced, and finally the molecular recognition capability is shown.
In addition, the Molecularly Imprinted Polymer (MIP) is also used for preparing a drug carrier, and is particularly mainly applied to two aspects of drug slow release and tumor targeting. In the aspect of drug slow release, generally, a drug molecule is taken as a template molecule, the template molecule and a functional monomer are connected through non-covalent bonds such as hydrogen bonds and hydrophobic acting forces to form a molecularly imprinted polymer, and the non-covalent bond effect between the drug molecule and the functional monomer is utilized to achieve the effect of drug slow release/controlled release. For example, there are reports in the literature that a graft copolymer formed from chitosan and methyl methacrylate can be imprinted with 5-fluorouracil using 5-fluorouracil as a template molecule; in addition, it has been shown that the drug release of the polymer is pH and time dependent in vitro drug release experiments. Furthermore, the molecularly imprinted polymer can be applied to targeting tumors; for example, a short peptide highly expressed on the surface of a tumor is selected as a template molecule, the prepared molecularly imprinted polymer can specifically recognize the template molecule, and the template molecule is the short peptide highly expressed on the surface of the tumor, so the prepared molecularly imprinted polymer has high affinity for the short peptide highly expressed on the surface of the tumor, and finally the targeting property on tumor tissues is realized; for example, the protein p32 with high expression on the surface of tumor is reported in literature and is modified to obtain short peptide with certain space structure as template molecule; in another example, the imprinted polymer shows significant targeting to tumor tissues in an in vivo experiment in animals by imprinting the epitope with a certain spatial conformation.
Disclosure of Invention
The invention aims to provide a molecularly imprinted polymer with a particle size reaching the nanometer level, and the molecularly imprinted polymer can further obtain a tumor targeted drug carrier, remarkably improve the dispersibility and stability of the drug, particularly has excellent selectivity/targeting property and is beneficial to improving the drug effect.
Specifically, the first aspect of the present invention provides a method for preparing a molecularly imprinted polymer, comprising the steps of:
s1: accurately weighing methacrylic acid, N-isopropyl acrylamide and N, N-dimethyl bisacrylamide, and dissolving in water by ultrasonic; then adding an absolute ethyl alcohol solution of N-tert-butyl acrylamide, and carrying out ultrasonic degassing;
s2: after the pH value is adjusted to be 5.8-6.2, a short peptide of yfqsmdk-C10 serving as a template molecule is added;
s3: slowly dripping an aqueous solution of an initiator, and carrying out polymerization reaction for 20-26 h at the temperature of 33-36 ℃;
s4: after the reaction is completed, ethanol-chloroform is used as an extracting agent, a Soxhlet extractor is adopted for refluxing overnight, so as to elute the template molecules and prepare a molecular imprinting polymer crude product;
s5: and dialyzing the crude product of the molecularly imprinted polymer to remove unpolymerized initiator or/and methacrylic acid or/and N-isopropyl acrylamide or/and N-tert-butyl acrylamide, thereby obtaining the molecularly imprinted polymer.
Preferably, in S1 of the above preparation method, the molar ratio of the methacrylic acid to the N-isopropylacrylamide is 1:2, the molar ratio of the methacrylic acid to the N, N-dimethylbisacrylamide is 1:10, and the molar ratio of the methacrylic acid to the N-tert-butylacrylamide is 1: 1.
Preferably, in S1 of the above preparation method, the duration of the ultrasonic degassing is 5 to 15 min.
Preferably, in the preparation method, the molar ratio of the methacrylic acid to the short peptide of yfqsmdk-C10 is 1: 2-1: 16.
Further preferably, in the above production method, the molar ratio of the methacrylic acid to the short peptide of yfqsmdk-C10 is 1: 8.
Preferably, in the above preparation method, the initiator comprises: ammonium persulfate, sodium bisulfite and sodium lauryl sulfate.
Further preferably, in the above preparation method, the mass of the ammonium persulfate is 3 times that of the yfqsmdk-C10 short peptide, the mass of the sodium bisulfite is 1 time that of the yfqsmdk-C10 short peptide, and the mass of the sodium dodecyl sulfate is 2 times that of the yfqsmdk-C10 short peptide.
Meanwhile, the second aspect of the invention provides a molecularly imprinted polymer, which is prepared by the preparation method of the first aspect of the invention.
In addition, the third aspect of the invention provides an application of the molecularly imprinted polymer of the second aspect in preparing tumor-targeted drug carriers. For example, the molecularly imprinted polymer can be combined with other small molecule compounds or polymers to prepare various drug carriers without changing the three-dimensional cavity containing the specific recognition site, so that the drug carriers can show tumor targeting.
It should be noted that, in this context, the water used in each step is distilled water.
Compared with the prior art, the molecularly imprinted polymer provided by the invention is suitable for preparing tumor-targeted drug carriers, and mainly has the following beneficial effects: according to the technical scheme provided by the invention, firstly, short peptide molecules, methacrylic acid, N-isopropyl acrylamide, N-dimethyl bisacrylamide and N-tert-butyl acrylamide interact through non-covalent bonds (hydrogen bonds, Van der Waals force and the like), then a proper amount of initiator is added for polymerization reaction, so that the short peptide molecules are imprinted on the surface of a polymer, and after the polymerization reaction is finished, the short peptide molecules imprinted on the surface are eluted by a proper solvent (namely an extracting agent), so that a cavity capable of specifically identifying the short peptide molecules is left; because the short peptide molecule is a section of short peptide highly expressed on the surface of the tumor, after the short peptide is eluted, the formed cavity can specifically identify the short peptide molecule highly expressed on the surface of the tumor, thereby finally realizing the selectivity and the targeting of the molecularly imprinted polymer to the tumor cells.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the following embodiments.
Example 1: preparation of Polymer NIP-1
Accurately weighing methacrylic acid (32.0 mu L), N-isopropylacrylamide (73.2mg) and N, N-dimethylbisacrylamide (4.2mg), and ultrasonically dissolving in 80ml of water; then adding a solution of N-tert-butyl acrylamide (31.1mg) in absolute ethanol (1ml), and ultrasonically degassing for 10 min; adjusting the pH value to 5.8-6.2; slowly dropwise adding an initiator (ammonium persulfate (60.0mg), sodium bisulfite (20.0mg) and sodium dodecyl sulfate (40.0mg) dissolved in 20mL of distilled water), and carrying out polymerization reaction for 20-26 h at 33-36 ℃; and dialyzing the prepared crude polymer to remove unpolymerized initiator or/and methacrylic acid or/and N-isopropyl acrylamide or/and N-tert-butyl acrylamide, thus obtaining the polymer NIP-1.
Example 2: preparation of Polymer NIP-2
Accurately weighing methacrylic acid (27.0 mu L), N-isopropylacrylamide (73.9mg) and N, N-dimethylbisacrylamide (3.9mg), and dissolving in 80ml of water by ultrasonic waves; then adding a solution of N-tert-butylacrylamide (39.2mg) in absolute ethanol (1ml), and ultrasonically degassing for 10 min; adjusting the pH value to 5.8-6.2; slowly dropwise adding an initiator (ammonium persulfate (60.0mg), sodium bisulfite (20.0mg) and sodium dodecyl sulfate (40.0mg) dissolved in 20mL of distilled water), and carrying out polymerization reaction for 20-26 h at 33-36 ℃; and dialyzing the prepared crude polymer to remove unpolymerized initiator or/and methacrylic acid or/and N-isopropyl acrylamide or/and N-tert-butyl acrylamide, thus obtaining the polymer NIP-2.
Example 3: preparation of Polymer NIP-3
Accurately weighing methacrylic acid (21.0 mu L), N-isopropylacrylamide (73.9mg) and N, N-dimethylbisacrylamide (3.8mg), and ultrasonically dissolving in 80ml of water; then adding a solution of N-tert-butyl acrylamide (47.5mg) in absolute ethanol (1ml), and ultrasonically degassing for 10 min; adjusting the pH value to 5.8-6.2; slowly dropwise adding an initiator (ammonium persulfate (60.0mg), sodium bisulfite (20.0mg) and sodium dodecyl sulfate (40.0mg) dissolved in 20mL of distilled water), and carrying out polymerization reaction for 20-26 h at 33-36 ℃; and dialyzing the prepared crude polymer to remove unpolymerized initiator or/and methacrylic acid or/and N-isopropyl acrylamide or/and N-tert-butyl acrylamide, thus obtaining the polymer NIP-3.
Example 4: preparation of Polymer NIP-4
Accurately weighing methacrylic acid (30.3 mu L), N-isopropylacrylamide (80.4mg) and N, N-dimethyl bisacrylamide (3.8mg), and ultrasonically dissolving in 80ml of water; then adding a solution of N-tert-butyl acrylamide (26.2mg) in absolute ethanol (1ml), and ultrasonically degassing for 10 min; adjusting the pH value to 5.8-6.2; slowly dropwise adding an initiator (ammonium persulfate (60.0mg), sodium bisulfite (20.0mg) and sodium dodecyl sulfate (40.0mg) dissolved in 20mL of distilled water), and carrying out polymerization reaction for 20-26 h at 33-36 ℃; and dialyzing the prepared crude polymer to remove unpolymerized initiator or/and methacrylic acid or/and N-isopropyl acrylamide or/and N-tert-butyl acrylamide, thus obtaining the polymer NIP-4.
Example 5: preparation of Polymer NIP-5
Accurately weighing methacrylic acid (18.0 mu L), N-isopropylacrylamide (80.4mg) and N, N-dimethylbisacrylamide (3.8mg), and ultrasonically dissolving in 80ml of water; then adding a solution of N-tert-butyl acrylamide (44.3mg) in absolute ethanol (1ml), and ultrasonically degassing for 10 min; adjusting the pH value to 5.8-6.2; slowly dropwise adding an initiator (ammonium persulfate (60.0mg), sodium bisulfite (20.0mg) and sodium dodecyl sulfate (40.0mg) dissolved in 20mL of distilled water), and carrying out polymerization reaction for 20-26 h at 33-36 ℃; and dialyzing the prepared crude polymer to remove unpolymerized initiator or/and methacrylic acid or/and N-isopropyl acrylamide or/and N-tert-butyl acrylamide, thus obtaining the polymer NIP-5.
Example 6: preparation of Polymer NIP-6
Accurately weighing methacrylic acid (10.0 mu L), N-isopropylacrylamide (88.6mg) and N, N-dimethylbisacrylamide (4.1mg), and ultrasonically dissolving in 80ml of water; then adding a solution of N-tert-butyl acrylamide (47.2mg) in absolute ethanol (1ml), and ultrasonically degassing for 10 min; adjusting the pH value to 5.8-6.2; slowly dropwise adding an initiator (ammonium persulfate (60.0mg), sodium bisulfite (20.0mg) and sodium dodecyl sulfate (40.0mg) dissolved in 20mL of distilled water), and carrying out polymerization reaction for 20-26 h at 33-36 ℃; and dialyzing the prepared crude polymer to remove unpolymerized initiator or/and methacrylic acid or/and N-isopropyl acrylamide or/and N-tert-butyl acrylamide, thus obtaining the polymer NIP-6.
Example 7:
calculate Q value (mg/g): taking 1mL of short peptide solution of 3mg of each of the polymers NIP-1 to NIP-6 and yfqsmdk-C10 as a template molecule, placing the solution on a shaking table overnight, centrifuging (12000rpm, 30min), taking the supernatant, measuring the concentration of the template molecule in the supernatant by HPLC, and determining the concentration of the template molecule according to the formula Q ═ [ (C)0-Ct)×V]/M(C0Concentration of template molecule solution before adsorption (mg/mL), Ct-the concentration of template molecule in the supernatant after adsorption (mg/mL), V-the volume of template molecule solution added (mL), M-the amount of polymer substance added (g)) and the calculated Q-values (i.e. the amount of adsorption on the template molecule) are respectively: 53.11(NIP-1), 53.16(NIP-2), 52.94(NIP-3), 50.36(NIP-4), 42.39(NIP-5), 40.96 (NIP-6). It can be seen that the adsorption amount of the polymer NIP-2 to the template molecule was the greatest.
The preparation method of the molecularly imprinted polymer according to the first aspect of the invention comprises the following steps:
s1: accurately weighing methacrylic acid (0.2-0.3mmol), N-isopropylacrylamide and N, N-dimethyl bisacrylamide, and ultrasonically dissolving in 80mL of water; then adding an absolute ethyl alcohol (1mL) solution of N-tert-butyl acrylamide, and ultrasonically degassing for 10 min;
s2: after adjusting the pH to 6.0, a short peptide of yfqsmdk-C10 was added as a template molecule; wherein the sequence of the short peptide of yfqsmdk-C10 is as follows: Tyr-Phe-Gln-Ser-Met-Asp-Lys-c10, the structural formula is shown in figure 1;
wherein, template molecules are respectively added according to the following molar ratios:
MIP-1: (methacrylic acid: template molecule ═ 1:2)
MIP-2: (methacrylic acid: template molecule ═ 1:4)
MIP-3: (methacrylic acid: template molecule ═ 1:8)
MIP-4: (methacrylic acid: template molecule ═ 1:16)
S3: slowly dripping an aqueous solution of an initiator, and carrying out polymerization reaction for 20-26 h at the temperature of 33-36 ℃;
s4: after the reaction is completed, ethanol-chloroform is used as an extracting agent, a Soxhlet extractor is adopted for refluxing overnight, so as to elute the template molecules and prepare a molecular imprinting polymer crude product;
s5: and dialyzing the crude product of the molecularly imprinted polymer to remove unpolymerized initiator or/and methacrylic acid or/and N-isopropyl acrylamide or/and N-tert-butyl acrylamide, thereby obtaining the molecularly imprinted polymer.
In addition, the adsorption capacity of each molecularly imprinted polymer to the short peptide of yfqsmdk-C10 is detected by an HPLC method, and the molecularly imprinted polymer with the largest adsorption amount is determined, so that the MIP-3 has the largest adsorption amount (see FIG. 3); characterizing the morphology of the sample by adopting a TEM and an AFM; the adsorption equilibrium curve of the molecularly imprinted polymer to the template molecule is determined by the HPLC method.
The molecularly imprinted polymer according to the second aspect of the present invention is produced by the production method according to the first aspect of the present invention.
The molecularly imprinted polymer according to the third aspect of the invention is applied to the preparation of tumor-targeted drug carriers.
Example 8:
s1: accurately weighing methacrylic acid (27.0 mu L), N-isopropylacrylamide (73.9mg), N, N-dimethylbisacrylamide (3.9mg) dissolved in 80mL of distilled water, N-tert-butylacrylamide (39.2mg) dissolved in 1mL of absolute ethanol, and performing ultrasonic treatment for 10 min;
s2: pH was adjusted to 6.0 with NaOH and 20.0mg template molecule (yfqsmdk-C10 short peptide) (dissolved in distilled water) was added;
s3: an aqueous solution of an initiator (ammonium persulfate (60.0mg), sodium bisulfite (20.0mg), sodium lauryl sulfate (40.0mg) dissolved in 20mL of distilled water) was slowly added dropwise, and polymerization was carried out at 35 ℃ for 24 hours;
s4: after the reaction is completed, ethanol-chloroform is used as an extracting agent, a Soxhlet extractor is adopted for refluxing overnight, so as to elute the template molecules and prepare a molecular imprinting polymer crude product;
s5: and dialyzing the crude product of the molecularly imprinted polymer to remove unpolymerized initiator or/and methacrylic acid or/and N-isopropyl acrylamide or/and N-tert-butyl acrylamide, thus obtaining the molecularly imprinted polymer MIP-3.
The particle size of the molecularly imprinted polymer MIP-3 is measured by adopting a nanometer particle size analyzer, the morphology of the MIP-3 is observed by a TEM and an AFM, and the prepared MIP-3 is found to be nanospheres with the particle size of 200 nm. The electron microscope image of the molecularly imprinted polymer MIP-3 is shown in FIG. 4, and the atomic force microscope image is shown in FIG. 5.
Example 9:
preparing template molecule solutions (0.2,0.5,1.0,2.0,3.0,6.0,12.0mg/ml) with distilled water as solvent, taking 1ml of the template molecule solutions with different concentrations to respectively interact with MIP-3(3ml, 1mg/ml) and NIP-2(3ml, 1mg/ml), centrifuging (12000rpm, 30min), taking supernatant, measuring template molecule concentration in the supernatant by HPLC, and obtaining the template molecule according to the formula Q [ (C ═ C-0-Ct)×V]/M(C0Concentration of template molecule solution before adsorption (mg/mL), CtQ value was calculated from the concentration (mg/mL) of the template molecule in the supernatant after centrifugation after adsorption, V, the volume (mL) of the template molecule solution added, and M, the amount (g) of the polymer added, and a graph showing the isothermal adsorption equilibrium of NIP-2 and MIP-3 was prepared (FIG. 6).
Example 10: targeting of molecularly imprinted polymer of imprinted short peptide to tumor
Method for co-culturing tumor cell MDA-MB-231 through normal cell HaCaTThe method proves the selectivity/targeting of the molecularly imprinted polymer MIP-3 to tumor cells. Firstly, carrying out covalent bonding on a molecularly imprinted polymer MIP-3 and a red dye rhodamine, and removing the rhodamine dye which is not bonded by a dialysis method, so that the MIP-3 has red fluorescence; then, tumor cells in exponential growth phase were taken, the medium was discarded, washed 2 times with PBS, and then digested into single cell suspension with 1mL of trypsin, the cells were counted, and the cell concentration was diluted to 2.5X 10 by adding the medium4One cell/mL, and then inoculated into a 3cm glass dish containing 1mL of the cell per well, and the cells were placed in 5% CO at 37 ℃2Incubating in a saturated humidity incubator; after the tumor cells adhered (24h), 1mL of GFP-labeled normal cells (2.5X 10/mL) were added4Cell suspension/mL, placing the cells in 5% CO at 37 ℃2After incubation in a saturated humidity incubator and cell adherence, supernatant was aspirated, MIP-3 (dissolved in sterile PBS, 5, 10 and 20 μ M) diluted with medium at different concentrations was added for 24h, cells were fixed with 4% formaldehyde for 20min, DAPI stained for 10min, and observed with confocal microscopy. The results are shown in FIG. 7, where the blue color is labeled with breast cancer cells MDA-MB-231, the green color is labeled with normal cells HaCaT, and the red color is labeled with MIP-3. As can be seen, red marked MIP-3 is mainly distributed in blue marked tumor cells through laser confocal microscope observation, MIP-3 is few in normal cells, and therefore, MIP-3 prepared by the inventor can be used for identifying tumor cells in a targeted mode.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.