CN113797353A - Radiotherapy and chemotherapy combined treatment reagent and preparation method thereof - Google Patents
Radiotherapy and chemotherapy combined treatment reagent and preparation method thereof Download PDFInfo
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- CN113797353A CN113797353A CN202010535662.4A CN202010535662A CN113797353A CN 113797353 A CN113797353 A CN 113797353A CN 202010535662 A CN202010535662 A CN 202010535662A CN 113797353 A CN113797353 A CN 113797353A
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- 238000011282 treatment Methods 0.000 title claims abstract description 24
- 238000002512 chemotherapy Methods 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 238000001959 radiotherapy Methods 0.000 title claims abstract description 20
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 63
- 238000003756 stirring Methods 0.000 claims description 55
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- 229910052757 nitrogen Inorganic materials 0.000 claims description 32
- 239000000243 solution Substances 0.000 claims description 26
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 24
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 23
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- 125000000304 alkynyl group Chemical group 0.000 claims description 3
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- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 3
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- 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
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/641—Branched, dendritic or hypercomb peptides
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/242—Gold; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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Abstract
The invention discloses a radiotherapy and chemotherapy combined treatment reagent and a preparation method thereof. The preparation method comprises the following steps: (1) preparing a peptide dendrimer carrier; (2) loading chemotherapeutic drug DOX and (3) loading radiosensitizer nanogold to obtain the chemoradiotherapy combined treatment reagent. The invention realizes the target delivery of the reagent to the tumor cells by loading the chemotherapeutic drug and the radiosensitization nano particles to the peptide dendrimer, achieves the combined treatment of radiotherapy and chemotherapy to the tumor cells, has excellent tumor cell targeting property and biocompatibility, simultaneously solves the problem of multidrug resistance of the tumor cells, and obviously enhances the treatment effect.
Description
Technical Field
The invention belongs to the technical field of nano-drugs, and relates to a radiotherapy and chemotherapy combined treatment reagent and a preparation method thereof.
Background
In order to improve the survival rate and the life quality of tumor patients and eradicate tumors caused by polygenic variation, the combined application of a plurality of treatment means becomes the trend of tumor treatment research. Currently, radiotherapy and chemotherapy combined treatment regimens are the preferred choice for clinical treatment of neoplastic diseases. In the cytodynamics cycle, the action points of cancer cell killing by radiotherapy are G2, M and G1 later stages, and have no effect on the S stage, while the main action point of chemotherapy is the S stage, so the radiotherapy can play a role in supplementing and killing cancer cells resistant to chemotherapy.
The generation of drug resistance is the most important problem in the clinical tumor chemotherapy process. The method finds a stable target which is not easy to mutate and has wide applicability in tumor pathology, and is an important way for overcoming multidrug resistance. Cathepsin B is a highly expressed lysosomal protease in tumor cells and tumor endothelial cells, and is often applied as a target for tumor therapy. In addition, the radiation therapy inevitably causes irreversible damage to normal tissues of a human body, and the application of the radiosensitizer can greatly reduce the irradiation dose, thereby reducing the damage to the surrounding normal tissues. The nano gold has good biocompatibility and surface modification, and is a sensitizer with wide application. The gold-sulfur bond is often selected as a chemical bond for modifying the nano-gold particle, and the tumor targeted delivery of the nano-gold particle can be realized by utilizing a hydrazone bond (Hyd) sensitive to acid.
Gu et al synthesized an arginine dendrimer and demonstrated that peptide dendrimers have drug-targeted delivery properties (Zhang X, Xu XH, Li YC, Hu C, Zhang ZJ, Gu ZW, Advanced materials, 2018). At present, many reports on the aspect of chemotherapy drug delivery of peptide dendrimers exist, however, simple chemotherapy is easy to generate multidrug resistance, and the treatment effect is limited. The combined therapeutic agent for radiotherapy and chemotherapy based on peptide dendrimers has not been reported.
Disclosure of Invention
The invention aims to provide a radiotherapy and chemotherapy combined treatment reagent and a preparation method thereof. The preparation method takes PEG-G1L-G3L peptide dendrimer as a carrier, and loads chemotherapeutic drugs and radiosensitizers in sequence based on cathepsin-sensitive linking peptide and acid-sensitive hydrazone bond, so that the prepared combined therapeutic agent has excellent tumor cell targeting property and biocompatibility, and solves the problem of multidrug resistance of tumor cells.
The technical scheme for realizing the purpose of the invention is as follows:
a preparation method of a combined treatment agent for radiotherapy and chemotherapy comprises the following steps:
s1, preparation of peptide dendrimers:
s11, synthesizing a generation of lysine peptide dendrimer G1L,
s12, synthesizing a second-generation lysine peptide dendrimer G1L-G1L,
s13, synthesizing a triline lysine peptide dendrimer G1L-G2L,
s14, synthesizing tetra-lysine peptide dendrimer G1L-G3L;
s2, preparation of mPEG-G1L-G3L-GFLG-DOX:
s21, dissolving Boc-GFLG (glycine-phenylalanine-leucine-glycine) -OMe in dichloromethane/trifluoroacetic acid (DCM/TFA) solution, stirring and reacting under nitrogen atmosphere, carrying out rotary evaporation on the product after the reaction is finished, adding anhydrous ether, collecting precipitate, carrying out centrifugal washing by using the anhydrous ether, dissolving in anhydrous Dimethylformamide (DMF) solution after vacuum drying, adding N, N-Diisopropylethylamine (DIPEA), 1-hydroxybenzotriazole (HOBt) and O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), stirring in ice bath under nitrogen atmosphere, stirring at room temperature, adding ethyl acetate (EtOAc) for dilution after the reaction is finished, and sequentially using NaHCO3、HCl、NaHCO3NaCl wash, organic phase over anhydrous MgSO4Drying, rotary evaporating, recrystallizing the residue at 4 deg.C to obtain N3-GFLG-OMe；
S22, adding N3Dissolving GFLG-OMe in MeOH and NaOH mixed solution, stirring at 4 deg.C, adjusting pH to 2-3, diluting with diethyl ether, washing with saturated NaCl three times, removing solvent to obtain N3-GFLG-OH；
S23，N3-GFLG-OH in Dichloromethane (DCM) and added thiazolidine and DIPEA, a solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) in DCM was added dropwise at 0 deg.C, stirred at 4 deg.C, diluted with EtOAc and successively diluted with NaHCO3、HCl、NaHCO3NaCl wash, organic phase over anhydrous MgSO4Drying and rotatingEvaporating, recrystallizing the residue at 4 deg.C to obtain N3-GFLG-Thiazolethione (N)3–GFLG–TT)；
S24, adding N3Dissolving GFLG-TT and doxorubicin hydrochloride (DOX. HCl) in anhydrous DMSO, adding distilled pyridine, stirring at room temperature in the dark, and adding ice EtOAc to obtain product N3–GFLG–DOX；
S25, dissolving the tetra-lysine peptide dendrimer G1L-G3L in a mixed solution of DCM/TFA under the protection of nitrogen, stirring and rotary evaporating at 0 ℃, adding anhydrous ether to form white precipitate, centrifugally washing with the anhydrous ether, drying in vacuum, dissolving the product in anhydrous DMF, adding DIPEA, HOBt, HBTU and 5-hexynic acid, stirring in an ice bath under the protection of nitrogen, stirring at room temperature, and stirring at 4 ℃ with NaHCO3Dialyzing for 24h, and finally freeze-drying to remove the solvent to obtain the alkynyl peptide dendrimer;
s26, under the protection of nitrogen, alkynylating the peptide dendrimer and CuSO4·5H2O、N3-GFLG-DOX and sodium ascorbate in dimethyl sulfoxide/water (DMSO/H)2O), stirring in a dark place, dialyzing in a dark place, and removing the solvent by freeze drying to obtain the peptide dendrimer-GFLG-DOX;
s3, synthesizing peptide dendrimer-GFLG-DOX-Hyd-Au:
s31, Hydroxypolyethyleneglyco-lamino (HO-PEG-NH)2) Generating LA-PEG-OH with dextro Lipoic Acid (LA), activating hydroxyl in the LA-PEG-OH by the hydroxyl of p-nitrobenzenechloroformate to generate LA-PEG-NPC, then reacting with hydrazine hydrate to generate LA-PEG-Hyd, and reacting with peptide dendrimer-GFLG-DOX to generate peptide dendrimer-GFLG-DOX-Hyd-PEG-LA;
s32, mixing the peptide dendrimer-GFLG-DOX-Hyd-PEG-LA with the nanogold stabilized by citric acid in water, adjusting the pH to 8.0, stirring at room temperature in the dark, and centrifuging to obtain the final product peptide dendrimer-GFLG-DOX-Hyd-Au.
Preferably, in step S1, the specific steps for preparing the peptide dendrimer are as follows:
s11 Synthesis of a lysine peptideDendrimer G1L: adding 1.2 times of molar weight of Cbz amino-protected lysine into unilateral Boc-protected ethylenediamine, adding 1.5 times of molar weight of HOBT and HBTU, replacing nitrogen for three times, adding DMF, adding 5 times of molar weight of DIEA, reacting for 24h under the protection of nitrogen, distilling under reduced pressure to remove DMF solvent, adding DCM, sequentially adding 1mol/L HCl solution and saturated NaHCO3The solution was washed three times with saturated NaCl solution, dried over anhydrous sodium sulfate overnight, filtered and distilled under reduced pressure to dryness at VMethylene dichloride：VMethanolThe product is further purified by column chromatography with 10:1 as a mobile phase, and G1L is obtained after rotary evaporation;
in step S12, synthesizing a di-generation lysine peptide dendrimer G1L-G1L: adding G1L, replacing nitrogen, adding DCM and TFA, stirring for 30min under ice bath, reacting for 8h at room temperature under nitrogen protection, after the reaction is finished, performing rotary evaporation to remove the solvent, then dropwise adding the solvent into anhydrous ether, stirring overnight, precipitating for 4h, removing the upper solution, performing vacuum drying for 48h, adding lysine, HOBT and HBTU protected by Boc amino, replacing nitrogen, adding DIEA and DMF, stirring for 30min under ice bath, reacting for 24h at room temperature under nitrogen protection, performing rotary evaporation, washing, drying, filtering, performing reduced pressure distillation, and then using VMethylene dichloride：VMethanol20: 1, purifying, and then rotationally evaporating the solvent to obtain G1L-G1L;
in step S13, synthesizing a triline lysine peptide dendrimer G1L-G2L: step S12, column chromatography mobile phase is VMethylene dichloride：VMethanol＝25：1；
Step S14, synthesizing the tetra-lysine peptide dendrimer G1L-G3L: step S12, column chromatography mobile phase is VMethylene dichloride：VMethanol＝25：1。
Preferably, in step S21, the stirring reaction time under the nitrogen environment is 24 hours, the stirring time under ice bath is 1 hour, and the stirring time under room temperature is 48 hours; in the DCM/TFA solution, the volume ratio of DCM to TFA is 1: 1.
preferably, in step S22, the stirring time at 4 ℃ is 24 h.
Preferably, in step S23, the stirring time at 4 ℃ is 30 h.
Preferably, in step S24, the stirring time is 30h away from light.
Preferably, in step S25, the stirring time at 0 ℃ is 24h, the stirring time in ice bath is 1h, and the stirring time at room temperature is 72 h.
Preferably, in step S26, the stirring time is 3 days away from light; DMSO/H2In a mixed solution of O, DMSO and H2The volume ratio of O is 3: 1.
preferably, in step S32, the stirring time is 12h away from light.
The invention also provides a radiotherapy and chemotherapy combined treatment reagent prepared by the preparation method.
The chemotherapeutic drug is released in the tumor cell lysosome in a targeted manner due to the degradation of the cathepsin-sensitive interlinking peptide glycine-phenylalanine-leucine-glycine (GFLG) and promotes the apoptosis of tumor cells. The nanogold is released in the lysosome due to the damage of a hydrazone bond sensitive to the acidic environment of the tumor, then leaks from a lysosome membrane and is gathered in the mitochondria of the tumor cells, and the oxidative stress generated in the radiosensitization treatment process can make the mitochondria PTPC of the tumor cells in a high-permeability state. The peptide dendrimer has the characteristics of excellent biocompatibility and degradability, more functional groups, cavities in molecules and the like. The invention takes the peptide dendritic macromolecule as a carrier to load chemotherapeutic drugs and radiosensitization nano particles, after intravenous injection administration, a series of physiological obstacles such as degradation in blood circulation, elimination of an RES system and the like can be overcome, the peptide dendritic macromolecule is gathered on tumor cells due to enhanced osmotic retention Effect (EPR), the carried drugs also reach tumor cell targets, and the combination of the two combined targeted therapy modes can more effectively kill the tumor cells.
Compared with the prior art, the invention has the following advantages:
the chemotherapeutics and the radiosensitization nanoparticles are loaded on the peptide dendrimer matrix, and the prepared chemoradiotherapy combined treatment reagent, namely the peptide dendrimer-GFLG-DOX-Hyd-Au reagent has excellent tumor cell targeting property and biocompatibility, overcomes the multidrug resistance of tumor cells, and obviously enhances the treatment effect.
Drawings
FIG. 1 is a schematic diagram of a multifunctional peptide dendrimer targeted nano-delivery system and a tumor radiotherapy and chemotherapy combination therapy based on the radiotherapy and chemotherapy combination therapy reagent of the invention;
FIG. 2 is a nuclear magnetic hydrogen spectrum of peptide dendrimers;
FIG. 3 is a TEM image and a particle size analysis image of the gold nanoparticles;
table 1 shows the reagent cytotoxicity assay results;
FIG. 4 is a graph of the results of plate colony formation experiments under different irradiation intensities.
Detailed Description
The invention will be further described in the following by means of specific embodiments and the accompanying drawings.
In The present invention, reagents to be used are commercially available unless otherwise specified, and The peptide dendrimers are described in The references of The patent of self-assembled, pH-responsive nanoparticles of mEGlated peptide polymers for cancer therapy, Biomaterials,2013,34,1603 1623.
Example 1
The preparation method of the radiotherapy and chemotherapy combined treatment reagent specifically comprises the following steps:
(1) preparation of peptide dendrimers
S11, synthesis G1L: adding 1.2 times of molar amount of Cbz amino-protected lysine (lys (Z)) into ethylene diamine (2.79g,17.4mmol) protected by unilateral Boc, adding 1.5 times of molar amount of HOBT and HBTU, replacing nitrogen for three times, adding DMF and 5 times of molar amount of DIEA, reacting for 24h under nitrogen protection, distilling under reduced pressure to remove DMF solvent, adding 200ml of DCM, sequentially adding 1mol/L HCl solution and saturated NaHCO3Washing the solution with saturated NaCl solution for three times, drying with anhydrous sodium sulfate overnight, filtering, distilling under reduced pressure to 15ml, and purifying by column chromatography (column chromatography mobile phase V)Methylene dichloride：VMethanol10:1) to yield G1L as a white powder.
S12, Synthesis of G1L-G1L: G1L (6.5G,11.7mmol) was added, nitrogen was replaced, DCM and TFA (9.0ml,117mmol) were added, and the mixture was stirred for 30min under ice bath with nitrogen blanketedAnd (3) reacting at room temperature for 8 hours, after the reaction is finished, removing the solvent by rotary evaporation, then dropwise adding the solvent into anhydrous ether, stirring the mixture overnight, precipitating the mixture for 4 hours, removing the upper solution, and performing vacuum drying for 48 hours. Adding Boc amino protected lysine (4.9g,14.1mmol), HOBT (5.4g,4.1mmol), HBTU (1.9g,14.1mmol), replacing nitrogen, adding 50ml DIEA, DMF (11.7ml,70.5mmol), stirring in ice bath for 30min, reacting at room temperature under nitrogen protection for 24h, rotary steaming, washing, drying, filtering, distilling under reduced pressure, and using VMethylene dichloride：VMethanol20: 1, purifying, and then rotationally evaporating the solvent to obtain white powder G1L-G1L.
S13, Synthesis of G1L-G2L: step S12, column chromatography mobile phase is VMethylene dichloride：VMethanol＝25：1。
S14, Synthesis of G1L-G3L: step S12, column chromatography mobile phase is VMethylene dichloride：VMethanol＝25：1。
(2) Preparation of mPEG-G1L-G3L-GFLG-DOX
S21, Boc-GFLG-OMe (2.0g,3.94mmol) was dissolved in 10mL DCM/TFA (1:1, v/v) solution, stirred under nitrogen for 24h and the temperature was kept constant at 0 ℃. The product was rotary evaporated and anhydrous ether was added to give a white precipitate. The precipitate was collected and washed 3 times with anhydrous ether. Dried under vacuum for half an hour, dissolved in 30mL anhydrous DMF and added DIPEA (4.2mL,23.69mmol), HOBt (0.798g,5.91mmol) and HBTU (2.24g,5.91 mmol). The mixture was stirred in an ice bath for 1h under nitrogen and then at room temperature for 48 h. After the reaction was complete, 500ml of LEtOAc was added for dilution, and 1mol/L NaHCO was used successively3、HCl、NaHCO3NaCl wash, organic phase over anhydrous MgSO4Drying, rotary evaporating to remove solvent, and recrystallizing the residue at 4 deg.C to obtain N3-GFLG-OMe。
S22, adding N3-GFLG-OMe (1.0g,1.81mmol) was dissolved in 2mL MeOH and 9.0mL of 1.0M NaOH solution. Stirring at 4 ℃ for 24 h. Adjusting the pH value to 2-3. Diluted with 500mL of diethyl ether and washed three times with saturated NaCl and the solvent removed to give N as a white solid3-GFLG-OH。
S23，N3-GFLG-OH (924.37mg,1.72mmol) dissolved in 10mL of DCM solution and added thiazolidine (307.5mg,2.58mmol) and DIPEA (1.8mL,10.32 mmol). A solution of 5mL EDC (494.6mg,2.58mmol) in DCM was added dropwise at 0 ℃. Stirred at 4 ℃ for 30h, diluted with 500mL EtOAc and sequentially with NaHCO3、HCl、NaHCO3NaCl wash, organic phase over anhydrous MgSO4Drying, rotary evaporating to remove solvent, and recrystallizing the residue at 4 deg.C to obtain N3-GFLG-TT。
S24, adding N3-GFLG-TT (500mg,0.93mmol) and DOX & HCl (592.8mg,1.02mmol) were dissolved in 10mL of anhydrous DMSO, and 5mL of distilled pyridine was added, and stirred at room temperature in the dark for 30 h. Final addition of ice EtOAc produced red solid product (N)3-GFLG-DOX)。
S25, under the protection of nitrogen, the peptide dendrimer (3.0g,0.11mmol) prepared in step (1) was dissolved in a mixed solution of 10mL CDM/TFA (1/1, v/v). The solution was stirred at 0 ℃ for 24h and the solvent was removed by rotary evaporation and a white precipitate was formed by addition of anhydrous ether. After washing with anhydrous ether by centrifugation for 3 times, vacuum drying is carried out for 0.5 h. The product was dissolved in 10mL anhydrous DMF and DIPEA (1.0g,7.92mmol), HOBt (303mg,1.98mmol), HBTU (750mg,1.98mmol) and 5-hexynoic acid (222mg,1.98mmol) were added. The mixture was stirred in an ice bath for 1h under nitrogen and at room temperature for 72 h. 0.1M NaHCO at 4 deg.C3Dialyzing for 24 h. And finally, freeze drying to remove the solvent to obtain the alkynyl peptide dendrimer.
S26, alkynylating peptide dendrimer (150mg,29.02mmol), CuSO under nitrogen protection4·5H2O(87.5mg,0.35mmol),N3-GFLG-DOX (1.06g,0.52mmol), sodium ascorbate (69.03mg,0.35mmol) was dissolved in 20mL DMSO/H2Mixed solution of O (3:1, V/V). Stirring in dark for 3 days. And dialyzed against light. Freeze drying to remove solvent to obtain red product (peptide dendrimer-GFLG-DOX).
(3) Synthesis of peptide dendrimer-GFLG-DOX-Hyd-Au
S31, (0.16g,0.04mmol) hydroxypolyethyleneglycoamido (HO-PEG-NH)2) Mixing with 8ml of dextro Lipoic Acid (LA) at room temperature, and stirring for 2h to obtain LA-PEG-OH.
S32, 5ml of LA-PEG-OH is mixed with 10ml of p-nitrobenzoic chloroformic acid at room temperature to generate LA-PEG-NPC. Then reacts with 5ml of 80% hydrazine hydrate to generate LA-PEG-Hyd.
S33, taking 5ml LA-PEG-Hyd to react with the peptide dendrimer-GFLG-DOX (0.1g,0.04mmol) prepared in the step (2), and freeze-drying to remove the solvent to obtain the peptide dendrimer-GFLG-DOX-Hyd-PEG-LA.
S34, mixing 1mg of peptide dendrimer-GFLG-DOX-Hyd-PEG-LA and 1mg of citric acid stable nanogold in 10mL of ultrapure water, and adjusting the pH value to 8.0. Stirring at room temperature for 12h in the dark. Centrifuging (14000rpm for 15min), and extracting the final product peptide dendrimer-GFLG-DOX-Hyd-Au, namely the radiotherapy and chemotherapy combined treatment reagent.
As shown in FIG. 2, there are G1L peptide type dendrimer nuclear magnetic hydrogen spectrum, G1L-G1L peptide type dendrimer nuclear magnetic hydrogen spectrum, G1L-G2L peptide type dendrimer nuclear magnetic hydrogen spectrum and G1L-G3L peptide type dendrimer nuclear magnetic hydrogen spectrum. The structure diagram of the peptide dendrimer can be obtained by nuclear magnetic hydrogen spectrum.
As shown in FIG. 3, the left image is a TEM image of the loaded gold nanoparticles, and the right image is a histogram of the nanoparticle size distribution. The average grain diameter of the nano gold particles obtained by analysis is 10.8nm, and the standard deviation is 5.8.
Example 2
Cytotoxicity test of peptide dendrimer-GFLG-DOX-Hyd-Au reagent:
the invention tests the cell activity of the cells treated by the peptide dendrimer-GFLG-DOX-Hyd-Au reagent by an MTT method. L929 mouse fibroblast cells are seeded into a 96-well plate, the density of each well is 5000, the cells are cultured for 24 hours, then reagents with different dilution times are added, 20 mu L of MTT is added after the cells are continuously cultured for 24 hours, the incubation is continuously carried out for 4 hours at 37 ℃, the culture solution is sucked out, 150 mu L of DMSO is added into each well for dissolution, and the absorption at 490nm is measured by a microplate reader.
Table 1 shows the effect of the reagent on cell viability, and the analysis shows that the reagent has good biocompatibility.
Example 3
Evaluating the radiosensitization performance and the combined treatment performance of the peptide dendrimer-GFLG-DOX-Hyd-Au reagent:
the effect of radiosensitization of the samples was assessed by colony survival analysis. The scattered single U251/ADM suspension cells were plated in known numbers. 1 day after attachment, cells were treated with the peptide dendrimer-GFLG-DOX-Hyd-Au reagent for 24 hours and then irradiated with 0, 2, 4, 6, 8Gy of radiation, respectively, with untreated controls. After 10 days of incubation, colonies were washed with cold PBS, stained with Giemsa dye, and then counted by counting colonies >50 cells to confirm viable fractions. The survival cell rate was compared with the survival number of the control cells. The irradiation survival curve is obtained by comparing and comprehensively processing the number of the surviving flora and the number of the surviving colonies after sample treatment.
As can be seen from FIG. 4, for the U251/ADM cell line which already has multidrug resistance, compared with the control group without the added reagent, the growth of the cells treated by the reagent is inhibited correspondingly before irradiation, the growth of the cells treated by the reagent is inhibited more obviously after irradiation of a certain dose, and the two groups with 6 Gy and 8Gy radiation intensity hardly see colony generation. The chemoradiotherapy combined treatment reagent, namely the peptide dendrimer-GFLG-DOX-Hyd-Au reagent, really plays a sensitization effect, can overcome the multidrug resistance of tumor cells, and shows a more excellent treatment effect compared with single chemoradiotherapy and radiotherapy.
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, it is intended that all equivalent alterations and modifications be included within the scope of the invention, without departing from the spirit and scope of the invention.
Claims (10)
1. A preparation method of a combined treatment reagent for radiotherapy and chemotherapy is characterized by comprising the following steps:
s1, preparation of peptide dendrimers:
s11, synthesizing a generation of lysine peptide dendrimer G1L,
s12, synthesizing a second-generation lysine peptide dendrimer G1L-G1L,
s13, synthesizing a triline lysine peptide dendrimer G1L-G2L,
s14, synthesizing tetra-lysine peptide dendrimer G1L-G3L;
s2, preparation of mPEG-G1L-G3L-GFLG-DOX:
s21, dissolving Boc-GFLG-OMe in DCM/TFA solution, stirring and reacting under nitrogen environment, carrying out rotary evaporation on the product after the reaction is finished, adding anhydrous ether, collecting precipitate, centrifugally washing with the anhydrous ether, dissolving in the anhydrous DMF solution after vacuum drying, adding DIPEA, HOBt and HBTU, stirring under nitrogen environment in ice bath, stirring at room temperature, adding EtOAc for dilution after the reaction is finished, and sequentially using NaHCO3、HCl、NaHCO3NaCl wash, organic phase over anhydrous MgSO4Drying, rotary evaporating, recrystallizing the residue at 4 deg.C to obtain N3-GFLG-OMe；
S22, adding N3Dissolving GFLG-OMe in MeOH and NaOH mixed solution, stirring at 4 deg.C, adjusting pH to 2-3, diluting with diethyl ether, washing with saturated NaCl three times, removing solvent to obtain N3-GFLG-OH；
S23，N3-GFLG-OH was dissolved in DCM and thiazolidine and DIPEA were added, EDC in DCM solution was added dropwise at 0 deg.C, stirred at 4 deg.C, EtOAc was diluted with NaHCO in turn3、HCl、NaHCO3NaCl wash, organic phase over anhydrous MgSO4Drying, rotary evaporating, recrystallizing the residue at 4 deg.C to obtain N3–GFLG–TT；
S24, adding N3dissolving-GFLG-TT and DOX & HCl in anhydrous DMSO, adding distilled pyridine, stirring at room temperature in dark, and adding ice EtOAc to obtain product N3–GFLG–DOX；
S25, dissolving the tetra-lysine peptide dendrimer G1L-G3L in a mixed solution of DCM/TFA under the protection of nitrogen, stirring and rotary evaporating at 0 ℃, adding anhydrous ether to form a white precipitate, centrifuging and cleaning with the anhydrous ether, drying in vacuum, dissolving the product in anhydrous DMF, adding DIPEA, HOBt, HBTU and 5-hexynoic acid, under the protection of nitrogen,stirring in ice bath, stirring at room temperature, NaHCO at 4 deg.C3Dialyzing for 24h, and finally freeze-drying to remove the solvent to obtain the alkynyl peptide dendrimer;
s26, under the protection of nitrogen, alkynylating the peptide dendrimer and CuSO4·5H2O、N3-GFLG-DOX and sodium ascorbate in DMSO/H2Stirring in dark place in the O mixed solution, dialyzing in dark place, and removing the solvent by freeze drying to obtain peptide dendrimer-GFLG-DOX;
s3, synthesizing peptide dendrimer-GFLG-DOX-Hyd-Au:
S31，HO-PEG-NH2generating LA-PEG-OH with LA, activating hydroxyl in the LA-PEG-OH by chloroformic acid hydroxyl of p-nitrobenzene to generate LA-PEG-NPC, then reacting with hydrazine hydrate to generate LA-PEG-Hyd, and reacting with peptide dendrimer-GFLG-DOX to generate peptide dendrimer-GFLG-DOX-Hyd-PEG-LA;
s32, mixing the peptide dendrimer-GFLG-DOX-Hyd-PEG-LA with the nanogold stabilized by citric acid in water, adjusting the pH to 8.0, stirring at room temperature in the dark, and centrifuging to obtain the final product peptide dendrimer-GFLG-DOX-Hyd-Au.
2. The method of claim 1, wherein the step of preparing the peptide dendrimer in step S1 comprises the following steps:
s11, synthesizing a generation of lysine peptide dendrimer G1L: adding 1.2 times of molar weight of Cbz amino-protected lysine into unilateral Boc-protected ethylenediamine, adding 1.5 times of molar weight of HOBT and HBTU, replacing nitrogen for three times, adding DMF, adding 5 times of molar weight of DIEA, reacting for 24h under the protection of nitrogen, distilling under reduced pressure to remove DMF solvent, adding DCM, sequentially adding 1mol/L HCl solution and saturated NaHCO3The solution was washed three times with saturated NaCl solution, dried over anhydrous sodium sulfate overnight, filtered and distilled under reduced pressure to dryness at VMethylene dichloride：VMethanolThe product is further purified by column chromatography with 10:1 as a mobile phase, and G1L is obtained after rotary evaporation;
in step S12, synthesizing a di-generation lysine peptide dendrimer G1L-G1L: adding G1L, replacing nitrogen, beatingAdding DCM and TFA, stirring for 30min under ice bath, reacting for 8h at room temperature under nitrogen protection, performing rotary evaporation after the reaction is finished to remove the solvent, then dropwise adding the solvent into anhydrous ether, stirring overnight, precipitating for 4h, removing the upper solution, performing vacuum drying for 48h, adding lysine, HOBT and HBTU protected by Boc amino, replacing nitrogen, adding DIEA and DMF, stirring for 30min under ice bath, reacting for 24h at room temperature under nitrogen protection, performing rotary evaporation, washing, drying, filtering, performing reduced pressure distillation, and then using VMethylene dichloride：VMethanol20: 1, purifying, and then rotationally evaporating the solvent to obtain G1L-G1L;
in step S13, synthesizing a triline lysine peptide dendrimer G1L-G2L: step S12, column chromatography mobile phase is VMethylene dichloride：VMethanol＝25：1；
Step S14, synthesizing the tetra-lysine peptide dendrimer G1L-G3L: step S12, column chromatography mobile phase is VMethylene dichloride：VMethanol＝25：1。
3. The preparation method according to claim 1, wherein in step S21, the stirring reaction time under nitrogen atmosphere is 24h, the stirring time in ice bath is 1h, and the stirring time at room temperature is 48 h; in the DCM/TFA solution, the volume ratio of DCM to TFA is 1: 1.
4. the method according to claim 1, wherein the stirring time at 4 ℃ in step S22 is 24 hours.
5. The method according to claim 1, wherein the stirring time at 4 ℃ in step S23 is 30 hours.
6. The method according to claim 1, wherein the stirring in step S24 is carried out for 30 hours without light.
7. The preparation method according to claim 1, wherein in step S25, the stirring time at 0 ℃ is 24h, the stirring time in ice bath is 1h, and the stirring time at room temperature is 72 h.
8. The method according to claim 1, wherein in step S26, the stirring is performed away from light for 3 days; DMSO/H2In a mixed solution of O, DMSO and H2The volume ratio of O is 3: 1.
9. the method according to claim 1, wherein the stirring time in step S32 is 12 hours under dark conditions.
10. A combined therapeutic agent for radiotherapy and chemotherapy prepared by the preparation method according to any one of claims 1 to 9.
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| US20020123609A1 (en) * | 2000-09-29 | 2002-09-05 | The Regents Of The University Of California | Dendrimeric support or carrier macromolecule |
| US20130004427A1 (en) * | 2009-12-11 | 2013-01-03 | The Regents Of The University Of Michigan | Targeted dendrimer-drug conjugates |
| CN103599070A (en) * | 2013-11-26 | 2014-02-26 | 上海交通大学 | Preparation method of temperature and fluorescence probe of lipidosome loaded with gold nanocluster and anti-cancer drug |
| CN104258391A (en) * | 2014-08-08 | 2015-01-07 | 中山大学 | Multi-functional stimuli sensitive polymer-gold nanocage carrier and preparation method thereof |
| US20210015944A1 (en) * | 2017-01-09 | 2021-01-21 | The Curators Of The University Of Missouri | Targeted Doxorubicin-Gold Nanocojugates for Tumor Therapy |
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| US20020123609A1 (en) * | 2000-09-29 | 2002-09-05 | The Regents Of The University Of California | Dendrimeric support or carrier macromolecule |
| US20130004427A1 (en) * | 2009-12-11 | 2013-01-03 | The Regents Of The University Of Michigan | Targeted dendrimer-drug conjugates |
| CN103599070A (en) * | 2013-11-26 | 2014-02-26 | 上海交通大学 | Preparation method of temperature and fluorescence probe of lipidosome loaded with gold nanocluster and anti-cancer drug |
| CN104258391A (en) * | 2014-08-08 | 2015-01-07 | 中山大学 | Multi-functional stimuli sensitive polymer-gold nanocage carrier and preparation method thereof |
| US20210015944A1 (en) * | 2017-01-09 | 2021-01-21 | The Curators Of The University Of Missouri | Targeted Doxorubicin-Gold Nanocojugates for Tumor Therapy |
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