CN112826938B - Intelligent nano-drug delivery system with cyclic gamma-polyglutamic acid coating and preparation method thereof - Google Patents
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Abstract
The invention relates to the field of nano biomaterials and nano medicines, in particular to an intelligent nano-drug delivery system with a ring gamma-polyglutamic acid (cyclo-gamma-PGA) coating and a preparation method thereof. The prepared cyclo-gamma-PGA coating intelligent nano-drug delivery system has good stability in high ion concentration and blood environment; the Cyclo-gamma-PGA coating can increase the uptake of tumor cells to nano micelles through an endocytosis pathway mediated by gamma-glutamyltranspeptidase, and can also increase the proportion of the medicine reaching the tumor part through an active targeting action mediated by GGT enzyme, thereby more effectively playing the anti-tumor effect. The cyclo-gamma-PGA biological coating can be applied to the fields of nano medicine, tumor treatment and the like.
Description
Technical Field
The invention relates to the field of nano biological materials and nano medicines, in particular to an intelligent nano drug delivery system with a ring gamma-polyglutamic acid coating and a preparation method thereof.
Background
Malignant tumors seriously threaten human health and are the second leading cause of death in the population (Siegel R L, miller K D, jemal A: cancer statistics,2019.CA Cancer J Clin,2019,69 (1): 7-34.). Chemotherapy is the main approach for tumor treatment, but the conventional tumor chemotherapy drugs have the problems of poor water solubility and stability, short circulation time in vivo, lack of tumor cell targeting, and the like, and often cannot achieve the expected treatment effect, even cause serious toxic and side effects (Montero J, sarosiek K A, deangelo J D, et al: drug-induced diagnosis therapy cancer response to chemotherapy. Cell 2015,160 (5): 977-989.). The polymer nano micelle is composed of a hydrophilic chain segment and a hydrophobic chain segment, can be self-assembled into an ordered aggregate with the grain diameter of 10-200 nm and the hydrophobic chain segment as a core and the hydrophilic chain segment as a shell in aqueous solution (Elsabayy M, wooley K L: design of polymeric nanoparticles for biological applications. Chem Soc Rev,2012,41 (7): 2545-2561.). In recent years, the polymer nano micelle as a nano drug delivery system has become one of effective ways to increase the targeting property of chemotherapeutic drugs, reduce toxic and side effects and improve the curative effect. The advantages of such drug carriers are: (1) the proportion of the drug reaching the tumor site can be increased by the Enhanced Permeability and Retention (EPR) effect of the tumor tissue; (2) the particle size is small, the distribution is narrow, and the specific targeting positioning can be carried out after the surface modification, so that the aim of targeted drug delivery is fulfilled; (3) the hydrophobic drug is wrapped in the hydrophobic core, so that the stability of the drug is improved; (4) slow release of the drug and prolonged duration of action (Kobayashi H, watanabe R, choyke P L: improving cosmetic enhanced performance and coverage (EPR) effects; what is the prepropriate target. However, the micelle carriers undergo a series of internal environmental changes after entering the body, including blood dilution effects, changes in pH and ion concentration in blood, and the like. In addition, for the cationic nano-micelle, cations on the surface of the cationic nano-micelle are easy to aggregate nonspecifically with electronegative erythrocytes and serum proteins, so that the stability of the micelle is influenced, and a loaded drug is released, thereby weakening the targeted antitumor effect and even causing serious toxic and side effects (Wang C, feng M, deng J, et al: poly (alpha-glutamic acid) combined with a nutrient as a nutrient for gene delivery. Inter J Pharmaceut,2010,398 (1-2): 237-245, qi N, tang B, liu G, et al.
At present, one of the methods for increasing the stability of cationic nanomicelles is to coat a surface of the nanomicelles with polyanion compounds as electronegative biomaterials. Gamma-polyglutamic acid (gamma-PGA) is a polyanion compound generated by bacillus fermentation, has the characteristics of strong water solubility, biodegradability, safety, no toxicity and the like, and is a commonly used polyanion biological coating material at present. For example, qi N. et al utilize electrostatic attraction to coat γ -PGA onto Doxorubicin-loaded liposome surfaces to enhance the stability of the liposomes in the blood circulation (Qi N, tang B, liu G, et al: poly (gamma-glutamic acid) -coated lipoplexes loaded with Doxorubicin for enhancing the activity of the antigen or activity against viral drugs in the liposomes Nanoscale Res Lett,2017,12 (1): 361.). Du X, et al, adsorbed gamma-PGA on the surface of mesoporous silica by continuous electrostatic interaction, can reduce nonspecific reaction between nanocarriers and erythrocytes and serum (Du X, xiong L, dai S, et al: gamma-PGA-coated mesoporous silica nanoparticles with a compatible adsorbed reagent for enhanced cellular uptake and intracellular GSH-reactive release. Adv healthcare Mater,2015,4 (5): 771-781). Research shows that the gamma-PGA coating reduces the inhibition effect of red blood cells and serum and improves the stability of a nano drug delivery system in blood circulation; on the other hand, γ -PGA can be transpeptidated by cell surface γ -glutamylThe enzyme (gamma-glutamyltreptdase, GGT) specifically recognizes, and GGT enzyme is overexpressed on the surface of various tumor cells, so that a γ -PGA-coated drug Delivery System can enter tumor cells through GGT enzyme-mediated endocytosis pathway, promote drug uptake, and enhance antitumor effect (Khalil I R, burns A T, radicka I, et al: bacterial-Derived Polymer Poly-y-glutaminic Acid (y-PGA) -Based Micro/nanoparticules as a Delivery System for antibacterial and Other biological applications. Int J. Of Mol Sci,2017,18 (2): 313.). However, gamma-PGA of natural origin has the following disadvantages: (1) molecular weight of 1X 10 5 ～8×10 6 The viscosity is too high, the rheological property is difficult to control, and chemical modification is difficult; (2) is a linear molecule, and a large number of amido bonds are arranged on the main chain, so that the polypeptide is easily degraded by aminopeptidase, carboxypeptidase and the like after entering a human body; (3) molecular flexibility of linear peptides results in conformational variability, and the strength and selectivity of binding to other molecules is affected. In contrast, the cyclic molecule has no free N-terminal and C-terminal, so that the sensitivity to aminopeptidase and carboxypeptidase is greatly reduced, and the cyclic molecule is more stable than a linear peptide; and cyclic molecules generally have a well-defined restricted conformation that can better fit into a receptor. Thus, cyclic molecules exhibit a greater functional diversity and application prospects.
Cyclic gamma-polyglutamic acid (cyclo-gamma-PGA) is a group of cyclic molecules consisting of 4 to 11 glutamic acid residues from medusa stinging capsule (applied national invention patent, application number: 201810295809.X, chinese patent document CN 105194670A). However, no report is found at present about the construction of a Cyclo-gamma-PGA coating intelligent nano-drug delivery system and a preparation method thereof.
Disclosure of Invention
The cyclic gamma-polyglutamic acid (cyclo-gamma-PGA) is a group of cyclic molecules consisting of 4 to 11 glutamic acid residues and derived from the medusa stinging capsule, is similar to the chain gamma-PGA derived from natural sources, is safe, nontoxic and good in biocompatibility, and also has the characteristics of high-quality biomaterials. The side chain of cyclo-gamma-PGA contains a large amount of electronegative free carboxyl, and after the side chain is adsorbed on the surface of the cation nano micelle by utilizing the electrostatic effect, the non-specific reaction of the nano micelle with negatively charged cell membranes and serum proteins can be effectively avoided, the aggregation of the micelle is prevented, and the stability of the micelle in blood circulation is improved. In addition, the Cyclo-gamma-PGA has small molecular weight, and the self structure is more stable than that of linear molecules, so that the effect of the Cyclo-gamma-PGA serving as a biological coating material is theoretically better than that of chain gamma-PGA.
The invention selects 9-element cyclo-gamma-PGA to be used as a biological coating material to be coated on the surface of the cation nano micelle NLS-LA-PpIX-DOX, and constructs a cyclo-gamma-PGA coating intelligent nano drug delivery system NLS-LA-PpIX-DOX @ cyclo-gamma-PGA. The system has the unique advantages that: (1) the cyclo-gamma-PGA coating can effectively increase the stability of the cationic nano-drug delivery system in high ion concentration and blood environment; (2) the cyclo-gamma-PGA coating can increase the uptake of tumor cells to nano-micelles through an GGT enzyme-mediated endocytosis pathway, and the effect is better than that of chain gamma-PGA; (3) the Cyclo-gamma-PGA coating can increase the proportion of the drug reaching tumor sites through GGT enzyme-mediated active targeting, and the effect is better than that of chain gamma-PGA, thereby better playing the anti-tumor capacity of the nano drug delivery system.
In order to solve the problem of poor stability of the cationic nano-micelle in blood circulation, the invention adopts cyclo-gamma-PGA to be adsorbed on the surface of the cationic nano-micelle to prepare a cyclo-gamma-PGA coating intelligent nano-drug delivery system, so that on one hand, the stability of the cationic nano-micelle in high ion concentration and blood environment is increased, on the other hand, the uptake of the drug by tumor cells is increased by utilizing GGT enzyme mediated endocytosis, and the proportion of the drug reaching tumor parts is increased by GGT enzyme mediated active targeting action, thereby enhancing the anti-tumor capability of the drug. The research of the invention discovers that a group of cyclic molecules consisting of 4-11 glutamic acid residues, namely cyclo-gamma-PGA (the patent of the invention takes 9-membered molecules as an example), has small molecular weight and stable self structure, and a large number of electronegative free carboxyl groups are adsorbed on the surface of a cation nano micelle, so that the nonspecific reaction of the nano micelle with red blood cells and serum proteins with negative charges can be effectively avoided, and the stability of the nano micelle in blood circulation is improved. Furthermore. The Cyclo-gamma-PGA coating can increase the medicine intake of tumor cells through an endocytosis way mediated by GGT enzyme, and can also increase the proportion of the medicine reaching the tumor part through an active targeting action mediated by the GGT enzyme, thereby better playing the anti-tumor capacity of a nano drug delivery system. Therefore, the invention establishes the Cyclo-gamma-PGA coating intelligent nano-drug delivery system and the preparation method thereof.
The invention aims to provide an intelligent nano drug delivery system with a ring gamma-polyglutamic acid (cyclo-gamma-PGA) coating and a preparation method thereof.
In order to achieve the above objects, in a first aspect of the present invention, a method for preparing a cyclic γ -polyglutamic acid (cyclo- γ -PGA) coating intelligent nano drug delivery system is provided, wherein a polymer NLS-LA-PpIX is used to prepare a cationic drug-loaded nano micelle, and the cyclic γ -polyglutamic acid is adsorbed on the surface of the cationic drug-loaded nano micelle through electrostatic interaction.
Further, the preparation method of the polymer NLS-LA-PpIX comprises the following steps:
(1) Swelling resin: 2-Chlorotrityl Chloride Resin was put into a reaction tube, DCM (15 ml/g) was added, and shaking was carried out for 30min.
(2) Grafting with the first amino acid: filtering off solvent by sand core, adding Fmoc-Gly-OH amino acid with 3 times molar excess, adding DMF to dissolve, adding DIEA with 10 times molar excess, and oscillating for 60min. Blocking with methanol.
(3) Deprotection: the DMF was removed, 20% piperidine DMF solution (15 ml/g) was added and the reaction was carried out for 5min, the DMF was removed and 20% piperidine DMF solution (15 ml/g) was added and the reaction was continued for 15min.
(4) And (3) detection: and (3) pumping out the piperidine solution, taking dozens of particles of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction.
(5) Washing: DMF (10 ml/g) was washed twice, DCM (10 ml/g) was washed twice, and DMF (10 ml/g) was washed twice.
(6) Condensation: and (3) dissolving protected amino acid with triple excess and HBTU with triple excess by using DMF as little as possible, adding into a reaction tube, immediately adding DIEA with ten-fold excess, and reacting for 30min.
(7) And (3) detection: washing dozens of resin particles with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and taking colorless as a negative reaction.
(8) Washing: DMF (10 ml/g) was washed once, DCM (10 ml/g) was washed twice, and DMF (10 ml/g) was washed twice.
(9) Repeating the operation of 3 to 6 steps, and connecting the amino acids in the sequence from right to left. Wherein the first amino acid at the N terminal is Fmoc-Lys (Dde) -OH. After receiving the lipoic acid in the same way, dde is removed, and PpIX is continuously connected.
(10) The resin was drained and washed as follows: DMF (10 ml/g) was washed twice, methanol (10 ml/g) was washed twice, DMF (10 ml/g) was washed twice, DCM (10 ml/g) was washed twice, and suction-dried for 10min.
(11) Cutting the polymer from the resin: preparing cutting fluid: TFA 95%; 1% of water; EDT 2%; TIS 2%; cutting time: and (4) 120min.
(12) Drying and washing: the lysate is blown dry as much as possible with nitrogen, washed six times with ether and then evaporated to dryness at normal temperature.
(13) Analyzing and purifying: and purifying the crude product by using high performance liquid chromatography, and performing mass spectrum identification.
Chromatographic conditions are as follows: a chromatographic column: kromasil 100-5C18, 4.6mm. Times.250mm, 5micro; mobile phase Buffer a:0.1% TFA in Acetonitrile; buffer B:0.1% TFA in water; flow rate: 1.0ml/min; a gradient ratio of 0min 65% to 20min 0% to B; sample introduction amount: 10 mu L of the solution; column temperature: 25 ℃; detection wavelength: 220nm.
Mass spectrum conditions: an ion source: an ESI source; the detection mode is as follows: detecting positive ions; capillary voltage: 2.8KV
Taper hole voltage: 22V; ion source temperature: 120 ℃;
(14) Freeze-drying: the target polymer solution was collected and put into a freeze dryer for concentration and freeze-dried into white powder.
Further, the cation drug-carrying nano micelle is an adriamycin (DOX) -carrying nano micelle NLS-LA-PpIX-DOX.
Further, the preparation method of the adriamycin-loaded cationic nano micelle comprises the following steps: weighing 2mg of polymer NLS-LA-PpIX, dissolving in 800. Mu.l of dichloromethane, slowly dropwise adding the doxorubicin dichloromethane solution to the polymer solution under vigorous stirring, and then dropwise adding the resulting solution to pure water under vigorous stirring to give a final volume VR Methylene chloride/H2O ＝1:1, stirring and volatilizing while completely removing dichloromethane, transferring the solution into a dialysis bag (MWCO 1000 Da), dialyzing to remove unencapsulated DOX, and freeze-drying to obtain the adriamycin-loaded nano micelle NLS-LA-PpIX-DOX.
Furthermore, the adriamycin is fat-soluble adriamycin which is obtained by desalting DOX (DOX · HCl) Doxorubicin Hydrochloride, and the desalting method comprises the following steps: precisely weighing 1mg of DOX & HCl, dissolving in dichloromethane to prepare a DOX & HCl dichloromethane solution with the concentration of 1mg/ml, adding excessive triethylamine, and standing in the dark overnight to remove HCl, thereby obtaining fat-soluble adriamycin (DOX).
Further, the preparation method of the intelligent nano drug delivery system with the ring gamma-polyglutamic acid (cyclo-gamma-PGA) coating comprises the following steps: an aqueous solution of cyclic gamma-polyglutamic acid (cyclo-gamma-PGA) having a concentration of 0.25mg/ml was added in a volume ratio of 4:1, dropwise adding the solution into a cationic drug-loaded nano micelle aqueous solution with the concentration of 2mg/ml while stirring at room temperature, stirring at 1500rpm for 30min, putting the solution into a dialysis bag (MWCO 1500 Da), and dialyzing to remove free cyclo-gamma-PGA; and (4) freeze-drying the solution to obtain the compound.
Further, the cyclic gamma-polyglutamic acid (cyclic-gamma-PGA) is a cyclic polypeptide consisting of 9 glutamic acid residues, and the amino acid sequence (N → C) thereof is shown as follows, and the structural formula is shown in fig. 2:
cyclo[(γ-E)-(γ-E)-(γ-E)-(γ-E)-(γ-E)-(γ-E)-(γ-E)-(γ-E)-(γ-E)](SEQ ID NO:1)。
in a preferred embodiment of the present invention, the method for preparing the intelligent nano-drug delivery system with the cyclic gamma-polyglutamic acid (cyclo-gamma-PGA) coating comprises the following steps:
(A) Preparation of cation nano micelle NLS-LA-PpIX-DOX
a. Doxorubicin Hydrochloride (Doxorubicin Hydrochloride, DOX. HCl) for desalting: precisely weighing 1mg of DOX & HCl, dissolving in dichloromethane to prepare a DOX & HCl dichloromethane solution with the concentration of 1mg/ml, adding excessive triethylamine, standing in the dark overnight to remove HCl, and obtaining fat-soluble adriamycin (DOX);
b. preparation of the adriamycin-loaded nano micelle: precisely weighing 2mg NLS-LA-PpIX polymer, dissolving in 800 μ l dichloromethane, and removing a certain volume under strong stirringThe salted DOX dichloromethane solution was slowly added dropwise to the polymer solution, and then the resulting solution was added dropwise to pure water under vigorous stirring to give a final volume VR Methylene chloride/H2O =1:1, stirring and volatilizing while completely removing dichloromethane, transferring the solution into a dialysis bag (MWCO 1000 Da), dialyzing to remove unencapsulated DOX, and freeze-drying to obtain the adriamycin-loaded nano micelle NLS-LA-PpIX-DOX;
(B) Preparation of cyclo-gamma-PGA coating intelligent nano-drug delivery system NLS-LA-PpIX-DOX @ cyclo-gamma-PGA
A9-membered cyclo-gamma-PGA aqueous solution (0.25 mg/ml) was prepared by mixing a 9-membered cyclo-gamma-PGA aqueous solution at a volume ratio of 4:1 dropwise adding the cationic nano micelle aqueous solution NLS-LA-PpIX-DOX (2 mg/ml) into the solution at room temperature under stirring, stirring the solution at 1500rpm for 30min, putting the solution into a dialysis bag (MWCO 1500 Da), and dialyzing the solution in pure water for 24h to remove free cyclo-gamma-PGA; and (3) freeze-drying the solution to obtain the cyclo-gamma-PGA coating drug-loaded nano micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA.
DLS results show that the particle size of the nano-micelle after the coating of the cyclo-gamma-PGA is slightly increased compared with that of the nano-micelle without the coating, the particle size is 147.6nm, PDI is reduced to 0.043, TEM results show that a circle of light coating (red arrow) is arranged around the drug-loaded nano-micelle (yellow arrow) (FIG. 3A, B). The potential detection result shows that the potential of the nano micelle is reversed after the Cyclo-gamma-PGA coating (figure 3C), and the result shows that the Cyclo-gamma-PGA is successfully coated on the surface of the drug-loaded nano micelle.
In a second aspect of the present invention, an intelligent nano-drug delivery system with a cyclic gamma-polyglutamic acid (cyclo-gamma-PGA) coating is provided, which is prepared by the preparation method as described above.
In a third aspect of the present invention, an application of the intelligent nano-drug delivery system with a cyclic γ -polyglutamic acid (cyclo- γ -PGA) coating as described above in the preparation of anti-tumor drug carriers is provided.
Further, the anti-tumor drug is Doxorubicin (DOX).
The fourth aspect of the present invention provides an application of cyclic gamma-polyglutamic acid (cyclo-gamma-PGA) in preparing an antitumor drug carrier, wherein the cyclic gamma-polyglutamic acid is a cyclic polypeptide consisting of 9 glutamic acid residues, and the amino acid sequence of the cyclic gamma-polyglutamic acid is shown in SEQ ID NO 1; the drug carrier is an intelligent nano drug delivery system with a ring gamma-polyglutamic acid coating, and is obtained by preparing a cation drug-carrying nano micelle by using a polymer NLS-LA-PpIX and adsorbing the ring gamma-polyglutamic acid on the surface of the cation drug-carrying nano micelle through electrostatic interaction.
The invention has the advantages that:
the prepared cyclo-gamma-PGA coating intelligent nano-drug delivery system has good stability in high ion concentration and blood environment; the Cyclo-gamma-PGA coating can increase the uptake of tumor cells to nano-micelles through an endocytosis pathway mediated by gamma-glutamyltranspeptidase (GGT), and can also increase the proportion of the medicine reaching tumor parts through an active targeting action mediated by GGT enzyme, thereby more effectively playing an anti-tumor effect, effectively reducing the accumulation of DOX in other organs and further reducing the toxic and side effects of DOX. The cyclo-gamma-PGA biological coating can be applied to the fields of nano medicine, tumor treatment and the like.
Drawings
FIG. 1 is a particle size distribution (A) and a transmission electron micrograph (B) of an uncoated nanomicelle NLS-LA-PpIX-DOX with a scale bar of 200nm;
FIG. 2 is a 9-membered cyclic- γ -PGA 1 H-NMR spectrum and structural formula.
FIG. 3 shows the particle size distribution, electron microscope characterization and zeta potential change of nano-micelle before and after coating of Cyclo-gamma-PGA; the particle size distribution and the transmission electron microscope image of the uncoated nano micelle NLS-LA-PpIX-DOX (A) and the uncoated nano micelle NLS-LA-PpIX-DOX (A) coated with cyclo-gamma-PGA coated with the nano micelle (B); (C) The zeta potential detection result shows that the potential of the nano micelle is overturned after the cyclo-gamma-PGA coating;
FIG. 4 shows that the Cyclo-gamma-PGA coating increases the stability of nano-micelles in different media; (A) (B) comparing the hemolysis rate of the nano-micelle before and after the coating of cyclo-gamma-PGA; (C) The release conditions of DOX of the nano-micelle in 30ml of different media (PBS, PBS solution containing 2M NaCl, PBS solution containing 10 percent fetal calf serum) before and after the coating of the cyclo-gamma-PGA, * P<0.05， ** P<0.01， *** P<0.001。
FIG. 5 shows that Cyclo-gamma-PGA coating increases the uptake of nanomicelles by tumor cells via GGT enzyme-mediated endocytosis; (A) The laser confocal microscope image (blue fluorescence, DAPI; red fluorescence, adriamycin) of HCT-116 cells after being incubated with free DOX, NLS-LA-PpIX-DOX @ gamma-PGA and NLS-LA-PpIX-DOX @ cyclo-gamma-PGA for 4h is 20 mu m; (B) After the HCT-116 cells are incubated with free DOX, NLS-LA-PpIX-DOX @ gamma-PGA and NLS-LA-PpIX-DOX @ cyclo-gamma-PGA for 4 hours, detecting the drug uptake rate of the cells by a standard curve method; (C) (D) after the HCT-116 cells are incubated with free DOX, NLS-LA-PpIX-DOX @ gamma-PGA and NLS-LA-PpIX-DOX @ cyclo-gamma-PGA for 4 hours, detecting the fluorescence intensity of the average DOX in the cells by a flow cytometer; detecting the influence of GGT enzyme inhibitor GGsTop with different concentrations on the cellular uptake of cyclo-gamma-PGA coating nano micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA by a flow cytometer (E) (F) and a standard curve method (G); flow cytometry (H) and standard curve method (I) examined the effect of different concentrations of ggtase inhibitor ggsttop on cellular uptake of uncoated nanomicelles NLS-LA-PpIX-DOX, (. P. < 0.05), (. P. < 0.01), (. P. < 0.001).
FIG. 6 is a graph showing that Cyclo-gamma-PGA coating increases the ability of nanomicelle to target tumors through GGT enzyme-mediated active targeting; (A) After HCT-116 tumor-bearing nude mice are injected with physiological saline, free DOX, NLS-LA-PpIX-DOX @ gamma-PGA or NLS-LA-PpIX-DOX @ cyclo-gamma-PGA for 12h, the fluorescence distribution of DOX in main organs and tumors is obtained; (B) Quantitatively analyzing the fluorescence intensity of DOX in main organs and tumors, *** P<0.001。
Detailed Description
The following examples are carried out on the premise of the technical scheme of the present invention, and give detailed embodiments and specific operation procedures, but the scope of the present invention is not limited to the following examples. The experimental procedures in the following examples are all conventional ones unless otherwise specified.
Example 1 preparation of cationic drug-loaded nanomicelle NLS-LA-PpIX-DOX
(1) Preparing blank nano-micelles: adopting a solvent co-volatilization method to accurately weigh the polymer NLS-LA-PpIX according to massProduct ratio of 1:1 is dissolved in dichloromethane and added dropwise to pure water (VR) under vigorous stirring Methylene dichloride/pure water = 1:1), volatilizing while stirring to remove dichloromethane, centrifuging the obtained solution at 3000rpm for 10min, and filtering through a 0.45 μm filter membrane to obtain a blank nano micelle solution. And (3) freeze-drying the solution to obtain blank nano-micelle NLS-LA-PpIX.
(2) Doxorubicin Hydrochloride (DOX. HCl) was desalted: precisely weighing 1mg of DOX & HCl, dissolving in dichloromethane to prepare 1mg/ml DOX & HCl dichloromethane solution, adding excessive triethylamine, and standing overnight in a dark place to remove HCl to obtain fat-soluble Doxorubicin (DOX).
(3) Preparation of the adriamycin-loaded nano micelle: precisely weighing 2mg NLS-LA-PpIX polymer, dissolving the NLS-LA-PpIX polymer in 800 mul dichloromethane, slowly dripping a certain volume of desalted DOX dichloromethane solution into the polymer solution under the condition of strong stirring, and then dripping the obtained solution into pure water under the condition of strong stirring to ensure that the final volume VR is Methylene chloride/H2O =1:1, stirring and volatilizing while completely removing dichloromethane, transferring the solution into a dialysis bag (MWCO 1000 Da), dialyzing to remove unencapsulated DOX, and freeze-drying to obtain the adriamycin-loaded nano micelle NLS-LA-PpIX-DOX. The particle size of the nano-micelle is 121.3nm and the PDI is 0.201 (figure 1A) detected by Dynamic Light Scattering (DLS), and the micelle has uniform and spherical particle size and is similar to the DLS measurement result (figure 1B) under a transmission electron microscope.
Example 2: preparation of cyclo-gamma-PGA coating intelligent nano-drug delivery system NLS-LA-PpIX-DOX @ cyclo-gamma-PGA
An aqueous solution (0.25 mg/ml) of 9-membered cyclo-gamma-PGA (structural formula shown in FIG. 2) was prepared in a volume ratio of 4:1, dropwise adding the cationic nano micelle aqueous solution NLS-LA-PpIX-DOX (2 mg/ml) into the solution at room temperature under stirring, stirring the solution at the rotating speed of 1500rpm for 30min, putting the solution into a dialysis bag (MWCO 1500 Da), and dialyzing the solution in pure water for 24h to remove free cyclo-gamma-PGA to obtain a uniform and transparent aqueous solution; and (3) freeze-drying the solution to obtain the cyclo-gamma-PGA coating drug-loaded nano micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA. DLS results show that the particle size of the nano-micelle after the coating of the cyclo-gamma-PGA is slightly increased compared with that of the nano-micelle without the coating, the particle size is 147.6nm, PDI is reduced to 0.043, TEM results show that a circle of light coating (red arrow) is arranged around the drug-loaded nano-micelle (yellow arrow) (FIG. 3A, B). The potential detection result shows that the potential of the nano micelle is reversed after the Cyclo-gamma-PGA coating (figure 3C), and the result shows that the Cyclo-gamma-PGA is successfully coated on the surface of the drug-loaded nano micelle.
Example 3: cyclo-gamma-PGA coating for increasing nano micelle stability
(1) Hemolytic activity of nano-micelle before and after coating of cyclo-gamma-PGA
(1) Preparation of the erythrocyte suspension: the mouse pericardial puncture method comprises the steps of taking 1ml of fresh blood, adding the fresh blood into a 15ml centrifuge tube containing an anticoagulant, adding physiological saline for washing, centrifuging at 1000rpm for 5min to remove upper serum and protein, continuously adding the physiological saline, repeating the washing step and centrifuging for several times until a supernatant is clear and transparent.
(2) Determination of hemolytic Activity: 500 mul of uncoated drug-loaded nano-micelle NLS-LA-PpIX-DOX solution or cyclo-gamma-PGA coated drug-loaded nano-micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA solution with different concentrations (31 mug/ml, 62 mug/ml, 125 mug/ml, 250 mug/ml, 500 mug/ml) is added into a 1.5ml centrifuge tube, and then 500 mul of red blood cell stock solution is added. Placing the centrifuge tube into a constant temperature shaker at 37 deg.C, shaking and incubating for 4h, and centrifuging at 1500rpm for 5min to remove precipitate. The supernatant 200. Mu.l was pipetted and transferred to a 96-well plate, and the absorbance (OD) at 545nm was measured using a microplate reader. Physiological saline was used as a negative control and saponin (4 mg/ml) was used as a positive control during the experiment. The results show that the hemolysis rate of erythrocytes of the Cyclo-gamma-PGA coated nano-micelle is obviously lower than that of uncoated nano-micelle, when the micelle concentration is 500 mug/ml, the hemolysis rate of the uncoated nano-micelle reaches about 18 percent, and the hemolysis rate of the Cyclo-gamma-PGA coated nano-micelle is maintained below 1 percent (FIG. 4A, B). The results prove that after the nano micelle of the cyclo-gamma-PGA coating is finished, on one hand, the cyclo-gamma-PGA has good blood compatibility, and on the other hand, the cyclo-gamma-PGA coating reduces the interaction between the cationic nano-carrier and red blood cells, thereby reducing the hemolytic effect and enhancing the stability of the nano micelle in blood.
(2) And (3) measuring the drug release rate of the nano-micelle in a high ion concentration and serum-containing culture medium before and after the coating of the cyclo-gamma-PGA.
Precisely measuring 1ml of uncoated drug-loaded nano-micelle solution NLS-LA-PpIX-DOX and cyclo-gamma-PGA coated drug-loaded nano-micelle solution NLS-LA-PpIX-DOX @ cyclo-gamma-PGA (the final concentration is 1 mg/ml) and respectively placing the solutions in a dialysis tube, and slightly and flexibly oscillating the solutions in a constant-temperature water bath oscillation device at 37 ℃ in 30ml of different media (PBS, PBS solution containing 2M NaCl and PBS solution containing 10% fetal calf serum) to inspect the DOX release condition. 3ml of the medium was sampled (simultaneously, the same medium was supplemented in the same volume) at 1, 3, 6, 9, 12, and 24 hours, respectively, and the fluorescence intensity of DOX in the samples taken at different time points was measured based on the DOX standard curve to calculate the released amount. The result shows that the release rate of DOX in 24h of the uncoated nano micelle NLS-LA-PpIX-DOX in the PBS solution is less than 20 percent; under the condition of high ion concentration, the release rate of DOX reaches 20% within about 6h, and the accumulative release rate reaches about 30% within 24h, which shows that the stability of micelle particles is reduced by the high ion concentration; while NLS-LA-PpIX-DOX @ cyclo-gamma-PGA micelle still maintains the accumulated release rate of DOX below 20% within 24h in a high-concentration NaCl environment, which indicates that the cyclo-gamma-PGA coating can increase the stability of the micelle in a high-ion concentration environment (FIG. 4C); similarly, NLS-LA-PpIX-DOX and NLS-LA-PpIX-DOX @ cyclo-gamma-PGA micelles were mixed with the serum-containing medium, and as a result, it was found that the release of DOX in uncoated nanobelts was very rapid, and the 24h cumulative release rate exceeded 40%; while the DOX release of the micelle of the cyclo- γ -PGA coating was hardly affected (fig. 4C), which shows that the polymer micelle can effectively reduce the reaction with serum after being coated with cyclo- γ -PGA. The above results suggest that the Cyclo-gamma-PGA coating can increase the stability of nano-micelle in different media.
Example 4: effect of Cyclo-gamma-PGA coating on cellular uptake of nanomicelles
(1) Confocal microscopy of the Effect of Cyclo-gamma-PGA coating on cellular uptake
HCT-116 cells were plated at 1X 10 5 Cells/well were seeded in confocal culture dishes until cells attached. Preparing uncoated nano micelle solution NLS-LA-PpIX-DOX, cyclo-gamma-PGA coating nano micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA and gamma-PGA coating nano micelle NLS-LA-PpIX-DOX @ gamma-PGA solution (DOX)The concentration was 10. Mu.g/ml). Adding the naked drug DOX and the 3 kinds of drug-loaded micelle solutions into a culture dish respectively, incubating for 4h, and then absorbing the culture medium. Cells were fixed for 20min by adding 4% paraformaldehyde. In a dark room, a small amount of DAPI solution is added into a culture dish, after 3min of staining, the culture dish is washed by PBS for a plurality of times, and the uptake condition of HCT-116 cells to 3 kinds of drug-loaded nano-micelles is observed under a confocal microscope. The results show that the fluorescence intensity in cells incubated by naked drug DOX solution is weaker, and the fluorescence intensity of the uncoated nano-micelle NLS-LA-PpIX-DOX group is slightly stronger than that of the naked drug DOX group, but is weaker than that of the NLS-LA-PpIX-DOX @ gamma-PGA and NLS-LA-PpIX-DOX @ cyclo-gamma-PGA nano-micelle groups. Meanwhile, the uptake of the Cyclo-gamma-PGA coating nano micelle in cells is higher than that of NLS-LA-PpIX-DOX @ gamma-PGA, which shows that the Cyclo-gamma-PGA coating nano micelle can increase the uptake of DOX by cells and has better effect than that of chain gamma-PGA (figure 5A).
(2) Standard curve method and flow cytometry for detecting influence of cyclo-gamma-PGA coating on cell uptake
Human colon cancer cell HCT-116 cell is 2 x 10 5 Cells/well were seeded in 6-well plates and cultured for 24h before changing the medium. Preparing uncoated nano-micelle solution NLS-LA-PpIX-DOX, cyclo-gamma-PGA coating nano-micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA and gamma-PGA coating nano-micelle NLS-LA-PpIX-DOX @ gamma-PGA solution (the DOX concentration is 10 mu g/ml). Adding naked drug DOX and 3 kinds of drug-loaded micelle solutions into a culture plate respectively, incubating for 4h, then absorbing the culture medium, and washing twice with PBS. Adding a proper amount of DMSO to dissolve DOX taken up by cells. Under the observation of a microscope, after the cells are completely detached from the wall, the solution dissolved with DOX and cell debris is sucked out to determine the fluorescence intensity, and the content of DOX entering the cells is calculated according to a DOX standard curve. As a result, the content of uptake of the Cyclo-gamma-PGA coated nano-micelle group DOX by cells is higher than that of the naked drug DOX, the uncoated nano-micelle NLS-LA-PpIX-DOX and the chain gamma-PGA coated nano-micelle NLS-LA-PpIX-DOX @ gamma-PGA group, which shows that the Cyclo-gamma-PGA coating can enhance the uptake of the nano-micelle by the cells (FIG. 5B).
Flow cytometry was used to examine the effect of cyclo-gamma-PGA coating on cellular uptake: adding naked drug DOX and 3 drug-loaded micelle solutions respectively, incubating for 4h, adding pancreatin to digest cells, centrifuging at 1000rpm for 3min, collecting cell sediment suspended in 1ml PBS, filtering with a filter screen, detecting the fluorescence content of DOX in cells with a flow cytometer, and comparing and analyzing the influence of the cyclo-gamma-PGA coating on the uptake of drug-loaded micelles by cells. Results show that compared with naked drug DOX and uncoated nano micelle groups, the intracellular DOX fluorescence content after treatment of the Cyclo-gamma-PGA coating nano micelle is remarkably increased, and the results show that the Cyclo-gamma-PGA coating nano micelle can increase the cellular uptake of DOX and the uptake rate is greater than that of the chain gamma-PGA coating nano micelle (figure 5C, D). The detection result of the flow cytometer is consistent with that of the standard curve method.
(3) Effect of GGT enzyme inhibitors on cellular uptake
Measuring the content of intracellular DOX: HCT-116 cells were plated at 2X 10 5 Cells/well were seeded in 6-well plates and the medium was changed after 24h of culture. After cells were pretreated with GGTenzyme inhibitor GGsTOP (0,0.05. Mu.g/ml, 0.5. Mu.g/ml, 1. Mu.g/ml) of different concentrations for 1h, naked drug DOX, uncoated nano-micelle solution NLS-LA-PpIX-DOX, cyclo-gamma-PGA coated nano-micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA, and gamma-PGA coated nano-micelle NLS-LA-PpIX-DOX @ gamma-PGA solution (DOX concentration of 10. Mu.g/ml) were added to each well, incubated for 4h, the medium was aspirated, and PBS was washed twice. Adding a proper amount of DMSO to dissolve DOX taken up by cells. And calculating and comparing the content of DOX entering cells before and after GGsTOP treatment according to a DOX standard curve, and calculating the cell uptake rate.
Flow cytometry detection: HCT-116 cells were seeded in 6-well plates and cultured for 24h before changing the medium. After cells are pretreated by GGsTOP (0,0.05 mug/ml, 0.5 mug/ml and 1 mug/ml) with GGT enzyme inhibitors with different concentrations for 1h, naked drugs DOX, uncoated nano-micelle solution NLS-LA-PpIX-DOX, cyclo-gamma-PGA coated nano-micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA and gamma-PGA coated nano-micelle NLS-LA-PpIX-DOX @ gamma-PGA solution (the DOX concentration is 10 mug/ml) are added. After continuously culturing for 4h, removing the culture medium, washing with PBS for three times, adding pancreatin to digest the cells, centrifuging for 3min at 1000rpm, collecting cell precipitates and suspending the cell precipitates in 1ml of PBS, filtering by using a filter screen, detecting the fluorescence content of intracellular DOX by using a flow cytometer, and comparing and analyzing the influence of GGsTOP treatment on the uptake of drug-loaded micelles by the cells.
Flow cytometry detection results show that after HCT-116 cells are pretreated with GGsTop of GGT enzyme inhibitor with different concentrations for 1h, the fluorescence intensity of intracellular DOX of the cyclo-gamma-PGA coating nano micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA group is obviously reduced, which indicates that DOX entering the cells is reduced, namely the cell uptake of the nano micelles is reduced, and the DOX fluorescence intensity is lower along with the increase of the concentration of GGsTop (figure 5E, F). The results were consistent with the results of cellular uptake measured by the standard curve method, and when the concentration of GGT enzyme inhibitor reached 1. Mu.g/ml, the cellular uptake rate was reduced from 90% to about 50% (FIG. 5G). In contrast, GGsTop did not affect the cellular uptake rate of uncoated nanomicelle NLS-LA-PpIX-DOX (FIG. 5H, I), and there was no significant change in intracellular DOX fluorescence intensity and content. The results prove that the Cyclo-gamma-PGA coating increases the uptake of the nano-micelle by the cells through GGT enzyme-mediated endocytosis pathway.
Example 5: cyclo-gamma-PGA coating nano micelle is distributed in vivo of tumor-bearing mice
HCT-116 cells (5X 10) 6 cells in 120. Mu.l DMEM) were subcutaneously inoculated into BALB/c nude mice to establish a tumor-bearing model of mice. When the tumor volume reaches 50mm 3 According to the dose of 2mg/kg DOX, the naked drug DOX, the uncoated nano-micelle solution NLS-LA-PpIX-DOX, the cyclo-gamma-PGA coated nano-micelle NLS-LA-PpIX-DOX @ cyclo-gamma-PGA and the gamma-PGA coated nano-micelle NLS-LA-PpIX-DOX @ gamma-PGA solution are respectively injected into a mouse body through the tail vein, and PBS is used as a negative control. Mice were sacrificed 12h after administration, and the inoculated tumor bodies and major organs (heart, liver, spleen, lung, kidney) were collected, observed with a small animal in vivo imaging system (emission wavelength 500-750 nm) and the fluorescence content of DOX in the tumor bodies and major organs of the mice was calculated (fluorescence of all administration groups was subtracted from that of PBS group). The results show that the naked drug DOX group can realize drug accumulation in various organs, and although the uncoated nano-micelle NLS-LA-PpIX-DOX and the gamma-PGA coated nano-micelle NLS-LA-PpIX-DOX @ gamma-PGA tumor tissues have DOX distribution, the fluorescence intensity is obviously lower than that of the NLS-LA-PpIX-DOX @ cyclo-gamma-PGA group, and the DOX fluorescence of the NLS-LA-PpIX-DOX @ cyclo-gamma-PGA group can be observed only in tumor bodies, so that the naked drug DOX group has better tumor targeting capability (figure 6). The above results indicate that Cyclo-gamma-PGA can be increased by GGT enzyme-mediated active targetingThe drug is added to reach the tumor part, so that the accumulation of DOX in other organs is effectively reduced, and the toxic and side effects of DOX are reduced.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Sequence listing
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Claims (8)
1. The preparation method of the intelligent nano-drug delivery system with the ring gamma-polyglutamic acid coating is characterized in that the intelligent nano-drug delivery system is obtained by preparing a cationic drug-loaded nano-micelle by using a polymer NLS-LA-PpIX and adsorbing the ring gamma-polyglutamic acid on the surface of the cationic drug-loaded nano-micelle through electrostatic action; the cyclic gamma-polyglutamic acid is a cyclic polypeptide consisting of 9 glutamic acid residues, and the amino acid sequence of the cyclic gamma-polyglutamic acid is shown as SEQ ID NO. 1; the preparation method of the polymer NLS-LA-PpIX comprises the following steps:
(1) Swelling resin: placing 2-Chlorotrityl Chloride Resin in a reaction tube, adding 15ml/g DCM, and oscillating for 30min;
(2) Grafting with the first amino acid: filtering off solvent by sand core, adding Fmoc-Gly-OH amino acid with molar excess of 3 times, adding DMF for dissolving, adding DIEA with molar excess of 10 times, and oscillating for 60min; blocking with methanol;
(3) Deprotection: removing DMF, adding 15ml/g 20% piperidine DMF solution, reacting for 5min, removing DMF, adding 15ml/g 20% piperidine DMF solution, and continuing to react for 15min;
(4) And (3) detection: pumping out the piperidine solution, taking dozens of resin, washing with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and taking a positive reaction when the resin turns dark blue;
(5) Washing: twice washing with 10ml/g DMF, twice washing with 10ml/g DCM and twice washing with 10ml/g DMF;
(6) Condensation: protecting amino acid, dissolving HBTU with DMF as little as possible, adding into a reaction tube, adding DIEA for over ten times, and reacting for 30min;
(7) And (3) detection: washing dozens of resin particles with ethanol for three times, adding a detection reagent for detection, heating at 105-110 ℃ for 5min, and taking colorless as a negative reaction;
(8) Washing: washing once with 10ml/g DMF, twice with 10ml/g DCM and twice with 10ml/g DMF;
(9) Repeating the operation of 3 to 6 steps, and sequentially connecting the amino acids in the sequence from right to left; wherein the first amino acid at the N terminal is Fmoc-Lys (Dde) -OH; after the lipoic acid is grafted in the same way, dde is removed, and PpIX is continuously connected;
(10) The resin was drained and washed as follows: washing twice with 10ml/g DMF, twice with 10ml/g methanol, twice with 10ml/g DMF, twice with 10ml/g DCM, and draining for 10min;
(11) Cutting the polymer from the resin: preparing cutting fluid: TFA 95%; 1% of water; 2% of EDT; TIS 2%; cutting time: 120min;
(12) Drying and washing: drying the lysate with nitrogen as much as possible, washing with diethyl ether for six times, and then volatilizing at normal temperature;
(13) Analyzing and purifying: purifying the crude product by high performance liquid chromatography, and performing mass spectrum identification;
chromatographic conditions are as follows: a chromatographic column: kromasil 100-5C18, 4.6mm. Times.250mm, 5micro; mobile phase Buffer a:0.1% TFA in Acetonitrile; buffer B:0.1% TFA in water; flow rate: 1.0ml/min; a gradient ratio of 0min 65% to 20min 0% to B; sample introduction amount: 10 mu L of the solution; column temperature: 25 ℃; detection wavelength: 220nm;
mass spectrum conditions: an ion source: an ESI source; the detection mode is as follows: detecting positive ions; capillary voltage: 2.8KV;
taper hole voltage: 22V; ion source temperature: 120 ℃;
(14) Freeze-drying: the target polymer solution was collected and put into a freeze dryer for concentration and freeze-dried into white powder.
2. The method for preparing a cyclogamma-polyglutamic acid coated intelligent nano-drug delivery system according to claim 1, comprising the steps of: mixing a 0.25mg/ml aqueous solution of cyclic gamma-polyglutamic acid according to a volume ratio of 4:1, dropwise adding the solution into a cationic nano micelle aqueous solution with the concentration of 2mg/ml while stirring at room temperature, stirring at 1500rpm for 30min, putting the solution into a dialysis bag, and dialyzing to remove free cyclic gamma-polyglutamic acid; and (4) freeze-drying the solution to obtain the compound.
3. The method for preparing the intelligent nano-drug delivery system with the cyclo-gamma-polyglutamic acid coating according to claim 1, wherein the cationic drug-loaded nano-micelle is an adriamycin-loaded nano-micelle NLS-LA-PpIX-DOX.
4. The preparation method of the intelligent nano-drug delivery system with the cyclic gamma-polyglutamic acid coating according to claim 3, wherein the preparation method of the adriamycin-loaded cationic nano-micelle comprises the following steps: weighing 2mg of polymer NLS-LA-PpIX, dissolving in 800. Mu.l of dichloromethane, slowly dropwise adding the doxorubicin dichloromethane solution to the polymer solution under vigorous stirring, and then dropwise adding the resulting solution to pure water under vigorous stirring to give a final volume VR Methylene chloride/H2O =1:1, stirring and volatilizing while completely removing dichloromethane, transferring the solution into a dialysis bag, dialyzing to remove the unencapsulated DOX, and freeze-drying to obtain the adriamycin-loaded nano micelle.
5. The method for preparing the intelligent nano-drug delivery system with the cyclic gamma-polyglutamic acid coating according to claim 4, wherein the adriamycin is fat-soluble adriamycin obtained by desalting doxorubicin hydrochloride, and the desalting method comprises the following steps: weighing 1mg of doxorubicin hydrochloride, dissolving the doxorubicin hydrochloride in dichloromethane to prepare 1mg/ml doxorubicin hydrochloride dichloromethane solution, adding excessive triethylamine, standing in the dark overnight to remove HCl, and obtaining the fat-soluble doxorubicin.
6. An intelligent nano-drug delivery system with a cyclic gamma-polyglutamic acid coating, which is prepared by the preparation method of any one of claims 1 to 5.
7. The application of the intelligent nano-drug delivery system with the coating of cyclic gamma-polyglutamic acid as claimed in claim 6 in preparing anti-tumor drug carriers.
8. The use according to claim 7, wherein the anti-neoplastic drug is doxorubicin.
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