CN114053428A - Nano-carrier for combined treatment of tumor chemotherapy and radiotherapy, preparation method and application - Google Patents
Nano-carrier for combined treatment of tumor chemotherapy and radiotherapy, preparation method and application Download PDFInfo
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- 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
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Abstract
The invention provides a nano-carrier for combined treatment of tumor chemotherapy and radiotherapy, a preparation method and application thereof. The particle size of the nanoparticle drug carrier for chemotherapy and radiotherapy combined treatment prepared based on the invention is 90-180nm, and the nanoparticle drug carrier can enter tumor cell cells through endocytosis, so that the rapid metabolism of small-molecule chemotherapy drugs in-vivo circulation is effectively reduced, AuNPs effectively enhances the intracellular ROS level under the irradiation of X-rays, so that the mitochondrial potential is unbalanced, the cell cycle is influenced, and the increase of cancer cells is inhibited by cooperating with the induction of apoptosis of chemotherapy drugs, thereby achieving the purpose of inhibiting tumors.
Description
Technical Field
The invention relates to the technical field of biomedical nano materials, in particular to a nano carrier for combined treatment of tumor chemotherapy and radiotherapy, a preparation method of the nano carrier and application of the nano carrier in combined treatment of chemotherapy and radiotherapy.
Background
Cancer is one of the major diseases that endanger human life and health. As shown by the world health organization data, the number of cancer cases and deaths is rapidly increasing, and cancer is expected to become the leading cause of death worldwide in the 21 st century. Therefore, the development of highly effective anticancer strategies is urgently needed. At present, chemotherapy is a treatment means widely applied in clinic, but the non-selectivity characteristic of small molecule chemotherapy drugs causes the small molecule chemotherapy drugs to kill normal tissues and cells, so that very obvious toxic and side effects are caused, such as broad-spectrum anticancer drug adriamycin (DOX), and very strong cardiotoxicity is caused. In such cases, chemotherapy may be combined with other treatments to reduce the chemotherapeutic dose. The combination therapy is a combination therapy system which is based on chemotherapy and other therapeutic means and mutually strengthened, is an effective way to overcome the adverse factors and can effectively improve the cure rate of the cancer. On the other hand, small molecule chemotherapy drugs have short half-life in vivo and are easy to be rapidly cleared, and in order to ensure the chemotherapy effect, the administration frequency is increased while the administration dosage is increased, which aggravates the systemic toxic and side effects.
The drug carriers to be studied at present mainly include liposomes, hydrogels, dendrimers, and the like. The peptide dendrimer is a polymer which is synthesized by taking natural amino acid as a raw material and has a highly branched structure, has the advantages of controllable molecular weight, high biocompatibility, biodegradability and the like, can be combined with the peptide dendrimer through physical embedding or environment-sensitive chemical bonds, further realizes effective delivery to a tumor part through a high-permeability long-retention effect (EPR effect) or by means of targeting factors, and finally realizes drug release through carrier swelling or breakage of sensitive chemical bonds. In order to achieve a more effective anti-tumor therapeutic effect, it is also a difficult point in the current research to design a peptide dendrimer drug delivery system that is more stable and accurate and has a combined therapeutic function.
Disclosure of Invention
The invention aims to provide a nano-drug carrier for combined treatment of tumor chemotherapy and radiotherapy, a preparation method and application thereof. AuNPs can affect the cell cycle after entering tumor cells, so that the AuNPs can be greatly arrested in a G2/M phase, and in the cell cycle, the tumor cells can be more sensitive to a radiotherapy medicament DOX, thereby achieving a synergistic effect. The nano-drug carrier can quickly release chemotherapeutic drugs in the application process, and realizes the combined treatment of chemotherapy and radiotherapy of tumors under the action of small-dose X rays.
In order to achieve the above object, a first aspect of the present invention provides a method for preparing a nanocarrier for a combination therapy of tumor chemotherapy and radiotherapy, comprising the following steps:
and 4, carrying out in-situ reduction on the ions coated in the cavity of the nano particles by adopting an in-situ compounding method to reduce the ions into nano silver-gold load, thereby obtaining the double-drug-loaded nano particles DOX-Hyd-PP @ AuNPs.
As an alternative embodiment, the preparation method specifically includes:
firstly, synthesizing G1-G3 generation peptide tree-shaped macromolecular material, mixing and dissolving carboxylated mPEG, EDC and NHS in an aqueous solution (the molar ratio of the carboxylated mPEG to the EDC to the NHS is 1:1.5:1.5), stirring for 1h, mixing with the deprotected G1-G3 generation peptide tree-shaped macromolecular aqueous solution, continuing stirring for 48h, dialyzing to remove unreacted substances (the cut-off molecular weight is 1000Da during dialysis), and freeze-drying to obtain a product G1-G3-mPEG;
then, reacting the deprotected G1-G3-mPEG peptide dendritic macromolecule with carboxylic acid tert-butyloxycarbonyl hydrazine to obtain a blank nano-carrier Hyd-G1-G3-mPEG with a rich internal cavity structure (the molar ratio of the deprotected G1-G3-mPEG peptide dendritic macromolecule to the carboxylic acid tert-butyloxycarbonyl hydrazine is 1:4), deprotecting the blank nano-carrier Hyd-G1-G3-mPEG, dissolving the deprotected blank nano-carrier Hyd-G1-G3-mPEG and DOX in DMF (pH5.4) (the molar ratio of the deprotected Hyd-G1-G3-mPEG to the DOX is 1:4), and reacting for 48 hours under the condition of avoiding light. Dialyzing at 4 deg.C for 24h (cut-off molecular weight is 1000Da during dialysis), and freeze drying to obtain medicine-carrying nanometer material DOX-Hyd-G1-G3-mPEG (abbreviation: DOX-Hyd-PP);
finally, the nano-gold can be loaded in the gaps of the drug carrier by adopting an in-situ compounding method. Dropwise adding chloroauric acid and NaOH solution into the drug-loaded nano material obtained in the step (the molar ratio of chloroauric acid to drug-loaded nano material is respectively 8: 1, 10:1 and 15: 1, NaOH mainly plays a role in neutralizing acidity of chloroauric acid), stirring for 30min, rapidly adding sodium borohydride solution (the molar ratio of chloroauric acid to sodium borohydride is 1:2.3), and continuously stirring for 3 h. Dialyzing to remove free substances, and freeze-drying to obtain the gold ion-loaded drug-loaded nano material DOX-Hyd-G1-G3-mPEG @ AuNPs (abbreviation: DOX-Hyd-PP @ AuNPs).
Preferably, the drug-loaded nanoparticles prepared in step 3 are DOX-Hyd-G1-G3-mPEG (DOX-Hyd-PP), and the particle size of the nanoparticles is kept between 120-130 nm.
Preferably, the step 4, finally freeze-drying, obtains double-drug-loaded nanoparticles DOX-Hyd-G1-G3-mPEG @ AuNPs (DOX-Hyd-PP @ AuNPs), and the particle size of the double-drug-loaded nanoparticles is kept between 90 nm and 100 nm.
The second aspect of the invention also provides a nano-carrier for combined treatment of tumor chemotherapy and radiotherapy, which is prepared according to the method, and takes the peptide dendrimer nanoparticles as a carrier, wherein the internal cavity of the carrier is loaded with radiotherapy sensitizer gold nanoparticles (AuNPs), the hydrophilic end of the peptide dendrimer nanoparticles as the carrier is composed of mPEG, and the hydrophobic end of the peptide dendrimer nanoparticles is composed of chemotherapeutic drug doxorubicin DOX.
The third aspect of the invention also provides application of the nano-carrier for the combined treatment of tumor chemotherapy and radiotherapy in preparing an oral medicament for the combined treatment of tumor chemotherapy and radiotherapy.
The particle size of the nanoparticle drug carrier for combined chemotherapy and radiotherapy prepared by the method is 90-100nm, the nanoparticle drug carrier can enter tumor cell cells through endocytosis, rapid metabolism of small molecule chemotherapy drugs in-vivo circulation is effectively reduced, and the physically-coated sensitizer AuNPs effectively enhances the intracellular ROS level under X-ray irradiation, so that mitochondrial potential is unbalanced, the cell cycle is influenced, and the growth of cancer cells is inhibited by cooperating with chemotherapy drugs to induce apoptosis, thereby achieving the purpose of inhibiting tumors.
Compared with the prior art, the invention has the following remarkable beneficial effects:
1) in the pH-sensitive amphiphilic peptide dendrimer nano drug-loading system for combined treatment of tumor chemotherapy and radiotherapy, the matrix material peptide dendrimer can be degraded by cells, so that the biological safety is good; the PEG is used for modifying the peptide dendrimer due to good hydrophilicity, so that not only can the basic skeleton of the dendrimer be not changed, but also the biocompatibility of the dendrimer be remarkably enhanced, the nano size of the dendrimer be changed, and the dendrimer be hydrophilic, and the PEG can be self-assembled to form a nano micelle subsequently, so that the carrier has abundant cavity physical embedding sensitizer, and the efficiency of loading nanogold in a cavity of the carrier is higher;
2) the nano-carrier in the pH-sensitive amphiphilic peptide dendrimer nano-drug-carrying system for combined treatment of tumor chemotherapy and radiotherapy can be delivered to tumor cells in a targeted manner through the EPR effect of tumor tissues, so that chemotherapeutic drugs are quickly released in the acidic environment of the tumor cells, and a better treatment effect is achieved under the action of radiosensitizer gold nanoparticles and X-rays under the condition of using smaller chemotherapeutic drugs;
3) the gold nanoparticles adopted by the invention provide a larger X-ray absorption cross section based on the nanoparticles with high atomic number, so that the gold nanoparticles are used as a radiosensitizer for enhancing radiotherapy, and can cause a large amount of ROS in cells after entering the cells, influence the mitochondrial potential of the cells, finally cause the mitochondria to release cytochrome c, and further induce apoptosis. And the cell cycle is blocked in the G2/M stage under the action of X rays, and the synergistic chemotherapeutic drug can kill tumor cells efficiently, thereby realizing the advantages of the combined treatment of tumor chemotherapy and radiotherapy. Meanwhile, the simultaneous combined delivery of multiple therapeutic agents using the advantages of AuNPs may not only improve the efficiency of each agent alone but also provide additional effects resulting from synergistic interactions occurring between the various modalities, thereby resulting in a stronger therapeutic effect. This approach to multimodal co-therapy helps avoid the associated side effects of high doses of drugs, as it allows for a reduction in the dose of each individual therapeutic agent administered.
It should be understood that all combinations of the foregoing concepts and additional concepts described in greater detail below can be considered as part of the inventive subject matter of this disclosure unless such concepts are mutually inconsistent. In addition, all combinations of claimed subject matter are considered a part of the presently disclosed subject matter.
The foregoing and other aspects, embodiments and features of the present teachings can be more fully understood from the following description taken in conjunction with the accompanying drawings. Additional aspects of the present invention, such as features and/or advantages of exemplary embodiments, will be apparent from the description which follows, or may be learned by practice of specific embodiments in accordance with the teachings of the present invention.
Drawings
The drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures may be represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. Embodiments of various aspects of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which:
FIG. 1 is a graph of the particle size, potential and morphology of nanoparticles of the present invention.
FIG. 2 is a graphical representation of the in vitro cumulative release of DOX from DOX-Hyd-PP nanoparticles in phosphate buffered solutions at different pH's (pH 7.4, 6.8 and 5.0).
FIG. 3 is a schematic of cellular uptake of 4T1 cells incubated with free DOX and DOX-Hyd-PP @ AuNPs for 2h and 6h, respectively.
FIG. 4 is a graphical representation of the in vitro synergistic antitumor effect of 4T1 cells with free DOX and DOX-Hyd-PP @ AuNPs.
FIG. 5 is a schematic representation of intracellular ROS fluorescence images after co-incubation of blank, free DOX and DOX-Hyd-PP @ AuNPs groups with 4T1 cells, (+) representing the experimental group with added X-rays, with a scale of 25 μm.
FIG. 6 is a graph showing fluorescence images of mitochondrial membrane potential after incubation of blank, free DOX and DOX-Hyd-PP @ AuNPs groups with 4T1 cells, (+) represents the experimental group with X-ray addition, and the scale is 25 μm.
FIG. 7 is a graph showing cell cycle distribution after co-incubation of the blank group and the DOX-Hyd-PP @ AuNPs group with 4T1 cells, (+) representing the experimental group with the addition of X-rays.
Detailed Description
In order to better understand the technical content of the present invention, specific embodiments are described below with reference to the accompanying drawings.
In this disclosure, aspects of the present invention are described with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. Embodiments of the present disclosure are not necessarily intended to include all aspects of the invention. It should be appreciated that the various concepts and embodiments described above, as well as those described in greater detail below, may be implemented in any of numerous ways, as the disclosed concepts and embodiments are not limited to any one implementation. In addition, some aspects of the present disclosure may be used alone, or in any suitable combination with other aspects of the present disclosure.
The embodiment of the invention discloses a nano-drug carrier for combined treatment of tumor chemotherapy and radiotherapy, a preparation method and application thereof. After the radiosensitizer AuNPs enter tumor cells, the cell cycle is influenced, so that a large number of the AuNPs are arrested in the G2/M phase, and in the cell cycle, the tumor cells are more sensitive to a radiotherapy medicament DOX, thereby achieving a synergistic effect. The nano-drug carrier can quickly release chemotherapeutic drugs in the application process, and realizes the combined treatment of chemotherapy and radiotherapy of tumors under the action of small-dose X rays.
Meanwhile, since the environment in the body is very complicated, in order to achieve effective antitumor, it is necessary to ensure that the chemotherapeutic drug and the radiosensitizer can be effectively delivered to the desired site. Therefore, the peptide dendrimer is selected as the matrix material, and the purposes of effective circulation and high-efficiency delivery in vivo can be met by simple modification
Therefore, the invention provides a preparation method of a nano-carrier for combined treatment of tumor chemotherapy and radiotherapy, which comprises the following steps:
and 4, carrying out in-situ reduction on the ions coated in the cavity of the nano particles by adopting an in-situ compounding method to reduce the ions into nano silver-gold load, thereby obtaining the double-drug-loaded nano particles DOX-Hyd-PP @ AuNPs.
Wherein, the specific treatment of the step 1 comprises the following steps:
dissolving mono-Boc protected ethylenediamine, Z-Lys (Z) -OH, HBTU and HOBT in DMF under nitrogen atmosphere, adding DIEA, reacting in ice bath for 30min, and continuing to react for 24h after removing the ice bath; after the reaction is finished, the solvent is removed by rotary evaporation, and DCM is added for dissolutionDissolving with saturated NaHCO3Washing with 1mol/L HCl and saturated NaCl aqueous solution for three times, adding anhydrous magnesium sulfate after washing, drying overnight, filtering, performing rotary evaporation to 10mL, and further purifying by passing through a gel chromatographic column with a dichloromethane/methanol 10:1 mobile phase to obtain G1 generation peptide dendrimer;
dissolving the reaction product G1 in the last step in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, and continuing to react for 7h after removing the ice bath; after the reaction is finished, the solvent is rotationally evaporated, the solvent is precipitated by ethyl acetate, and white solid is collected; dissolving the collected white solid product and Boc-Lys (Boc) -OH, HBTU and HOBT in DMF under nitrogen atmosphere, adding DIEA, reacting in ice bath for 30min, and continuing to react for 24h after removing the ice bath; after the reaction is finished, performing rotary evaporation to remove the solvent, adding DCM for dissolution, washing with saturated sodium bicarbonate, 1mol/L hydrochloric acid and saturated NaCl aqueous solution for three times, adding anhydrous magnesium sulfate after washing for drying overnight, filtering, performing rotary evaporation to 15mL, and further purifying by passing a dichloromethane/methanol 10:1 mobile phase through a gel chromatographic column to obtain G1-G1 generation peptide dendrimer;
dissolving the reaction products G1-G1 in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, removing the ice bath, continuing to react for 7h, performing rotary evaporation to remove the solvent after the reaction is finished, precipitating with ethyl acetate, and collecting white solid; dissolving the white solid product and Boc-Lys (Boc) -OH, HBTU and HOBT in DMF under nitrogen atmosphere, adding DIEA, reacting in ice bath for 30min, and continuing to react for 48h after removing the ice bath; after the reaction is finished, the solvent is removed by rotary evaporation, DCM is added for dissolution, and saturated NaHCO is used3Washing with 1mol/LHCl and saturated NaCl aqueous solution for three times, adding anhydrous magnesium sulfate after washing, drying overnight, filtering, performing rotary evaporation to 20mL, and further purifying by passing through a gel chromatographic column with dichloromethane/methanol 10:1 mobile phase to obtain G1-G2 generation peptide dendrimer;
dissolving the reaction products G1-G2 in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, removing the ice bath, continuing to react for 7h, performing rotary evaporation to remove the solvent after the reaction is finished, precipitating with ethyl acetate, and collecting white solid; dissolving the white solid product and Boc-Lys (Boc) -OH, HBTU and HOBT in DMF under nitrogen atmosphere, adding DIEA, reacting in ice bath for 30min, removing the ice bath, and continuing to react for 72 h. And after the reaction is finished, performing rotary evaporation to remove the solvent, adding DCM for dissolution, washing with saturated sodium bicarbonate, 1mol/L hydrochloric acid and saturated NaCl aqueous solution for three times, adding anhydrous magnesium sulfate after washing, drying overnight, filtering, performing rotary evaporation to 25mL, and further purifying by passing through a gel chromatographic column by using a dichloromethane/methanol 10:1 mobile phase to obtain the G1-G3 generation peptide dendrimer.
Wherein, the specific treatment of the step 2 comprises the following steps:
dissolving mPEG-OH500, succinic anhydride, DMAP and triethylamine in tetrahydrofuran, and reacting for 24 hours under rapid stirring; and after the reaction is finished, removing the solvent by rotary evaporation, adding a small amount of DCM for dissolution, adding glacial ethyl ether for precipitation under the condition of vigorous stirring, and drying in vacuum to obtain a white solid product mPEG-COOH, wherein mPEG-OH 500: succinic anhydride: DMAP: the molar ratio of triethylamine is 1:1.5:1.05: 1.05;
weighing G1-G3 peptide dendrimer, dissolving in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, removing the ice bath, continuing to react for 7h, performing rotary evaporation to remove the solvent after the reaction is finished, precipitating with diethyl ether, and collecting white solid; dissolving white solid products mPEG-COOH, EDC and NHS in 10mL of water, stirring and activating for 1h, dripping the activated product into the water solution of the deprotected product at a constant speed after activation, reacting for 48h at normal temperature, dialyzing for 3 times by using PBS (for example, 0.2M) after the reaction is finished, dialyzing for three times by using deionized water, and freeze-drying to obtain white solid G1-G3-mPEG; wherein G1-G3 peptide dendrimers: mPEG-COOH: EDC: the molar ratio of NHS is 1:16:24: 24;
dissolving succinic anhydride and DMAP in DCM, and slowly dropwise adding tert-butoxycarbonylhydrazine dissolved in DCM under the condition of vigorous stirring; and (3) after the reaction is finished, evaporating to remove the solvent, and vacuumizing and drying for 24 hours to obtain the carboxylated tert-butyloxycarbonyl hydrazine, wherein the weight ratio of succinic anhydride: the molar ratio of tert-butyloxycarbonyl hydrazine is 1.5: 1;
dissolving G1-G3-mPEG500 in anhydrous methanol, adding Pd/C (46.3mg) and reacting for 48 hours in a hydrogen atmosphere to remove Cbz protection; after the reaction is finished, removing palladium carbon by suction filtration, and removing the solvent by rotary evaporation; dissolving carboxylated tert-butyloxycarbonyl hydrazine, EDC and NHS in 5mL of water, and carrying out activation reaction for 1 h; dissolving G1-G3-mPEG500 without Cbz protection in 5mL of water, slowly dropwise adding activated carboxylic tert-butyloxycarbonyl hydrazine, and reacting for 48 h; after the reaction is finished, dialyzing the mixture for 3 days by deionized water, and freeze-drying the mixture to obtain Boc-Hyd-G1-G3-mPEG, wherein G1-G3-mPEG 500: carboxylated t-butyloxycarbonylhydrazine: EDC: the molar ratio of NHS was 1:4:8: 8.
Wherein, the specific treatment of the step 3 comprises the following steps:
dissolving Boc-Hyd-G1-G3-mPEG in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, removing the ice bath, continuing to react for 7h, removing the solvent by rotary evaporation after the reaction is finished, precipitating with ethyl acetate, and collecting white solid; the collected white solid was dissolved in DMF with DOX. HCl, and a drop of TFA was added to catalyze the reaction for 48h in the absence of light. After the reaction is finished, the solvent is removed by rotary evaporation, a small amount of deionized water is added for dissolution, deionized water dialysis is carried out for 24h at the temperature of 4 ℃, the freeze-dried product is the drug-loaded nanoparticle DOX-Hyd-G1-G3-mPEG (DOX-Hyd-PP), and the particle size of the nanoparticle is kept between 120 and 130 nm. Wherein Boc-Hyd-G1-G3-mPEG: the molar ratio of hcl is 1: 4.
Wherein, the specific processing of the step 4 comprises the following steps:
adding 1 w% of chloroauric acid into 20mL of water, adjusting the pH value to be neutral by using 0.1mol/L of NaOH, dissolving DOX-hyd-G1-G3-mPEG in the chloroauric acid solution, stirring for 30min, quickly adding the sodium borohydride solution, and continuously stirring for 3 h;
and dialyzing with deionized water for 24h after stirring, and freeze-drying to obtain double-drug-loaded nanoparticles DOX-Hyd-G1-G3-mPEG @ AuNPs (DOX-Hyd-PP @ AuNPs), wherein the particle size of the double-drug-loaded nanoparticles is kept between 90 nm and 100 nm. Wherein, DOX-hyd-G1-G3-mPEG: the molar ratio of AuNPs is 1: 8.
Therefore, according to the embodiment, the nano-carrier DOX-Hyd-PP @ AuNPs for combined treatment of tumor chemotherapy and radiotherapy is prepared, peptide dendrimer nanoparticles are used as a carrier, gold nanoparticles (AuNPs) serving as radiotherapy sensitizers are loaded in an inner cavity of the carrier, hydrophilic ends of the peptide dendrimer nanoparticles serving as the carrier are composed of mPEG, and hydrophobic ends of the peptide dendrimer nanoparticles are composed of DOX serving as a chemotherapeutic drug. The particle size of the nano-particle drug carrier is 90-100nm, the nano-particle drug carrier can enter tumor cell cells through endocytosis, rapid metabolism of small-molecule chemotherapeutic drugs in vivo circulation is effectively reduced, the physically-coated sensitizer AuNPs effectively enhances the intracellular ROS level under X-ray irradiation, mitochondrial potential is unbalanced, the cell cycle is influenced, and then the chemotherapeutics are cooperated to induce apoptosis to inhibit growth of cancer cells, so that the purpose of inhibiting tumors is achieved.
Therefore, the DOX-Hyd-PP @ AuNPs double-drug-loaded nanoparticles can be used for preparing an internal medicine for combined treatment of tumor chemotherapy and radiotherapy, and enter a human body through oral administration as described above, wherein the physically-coated sensitizer AuNPs effectively enhance the intracellular ROS level under X-ray irradiation, so that the mitochondrial potential is unbalanced, the cell cycle is influenced, and then the chemotherapeutics are induced by the chemotherapeutics to apoptosis to inhibit the growth of cancer cells in coordination, thereby achieving the purpose of tumor inhibition, and simultaneously realizing the combined treatment effect of chemotherapy and radiotherapy by combining with the chemotherapeutics doxorubicin DOX.
The prepared nano particles are characterized by combining the characteristics shown in figure 1, the results are shown in figure 1, the three nano particles Hyd-G1-G3-mPEG, DOX-Hyd-PP and DOX-Hyd-PP @ AuNPs have good dispersion indexes, the particle size is kept between 90 nm and 150nm, the particle size of the finally obtained double drug-carrying particles is 90 nm to 100nm, and the drug-carrying particles can be successfully phagocytized by cells after entering a human body.
Example 1
To determine the release of DOX from drug loaded materials in vitro, we performed drug release experiments simulating in vivo conditions. 4mg of DOX-Hyd-PP was weighed out and dissolved in 1mL of PBS buffer solution of different pH values (pH5.4, 6.8, 7.4), the solution was transferred into a dialysis bag of cut-off volume 1000, and the dialysis bag was clamped and put into a centrifuge tube containing 25mL of PBS buffer solution (pH5.4, 6.8, 7.4). The centrifuge tube was placed in a constant temperature shaker at 37 ℃ and 120r/min to start timing. 1mL of PBS buffer at corresponding pH was removed at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, 24, 36, 48, 60, 72, 84, and 96 h. And measuring the fluorescence absorption value of DOX at 480nm by using an ultraviolet spectrophotometer, calculating the DOX released at different time points according to a DOX concentration-absorption intensity standard curve, and calculating the accumulated release amount by taking an average value (n is 3).
As shown in FIG. 2, DOX release is related to pH conditions, and the lower the pH, the greater the drug release, and the more stable the release over time.
Example 2
To track phagocytosis of nanogels within cells, 4T1 cells were plated at 4 × 10 per well4The cells were seeded at a density in confocal laser glass and incubated for 24 h. After the cells grow adherently, the culture medium is discarded, and DOX.HCl and DOX-Hyd-PP @ AuNPs (DOX content is 5 mu g/mL) fresh culture media are added for incubation for 2h and 6h respectively. After incubation, the medium was removed, washed three times with PBS, cells were fixed with 4% paraformaldehyde for 15min, and washed three times with PBS. Adding instant DAPI staining agent for staining for 8min, washing with PBS for three times, adding anti-fluorescence quenching sealing liquid for sealing, and observing fluorescence intensity in cells by laser confocal method.
4T1 cells were then plated at 4X 10 per well5The cells were inoculated in 6-well plates at density, 3 multiple wells were set for each group, 2mL of complete medium containing 10% fetal bovine serum was added to each well, and placed in a cell incubator for incubation for 24 h. After the cells grow adherently, the culture medium is discarded, and DOX.HCl and DOX-Hyd-PP @ AuNPs (DOX content is 5 mu g/mL) fresh culture media are added for incubation for 2h and 6h respectively. After the incubation was completed, the medium was removed, washed three times with PBS, 1mL of pancreatin was added to each well and placed in an incubator for digestion for 4min, then 2mL of complete medium was added to stop digestion, cells were collected by centrifugation at 1000rpm for 5min, washed three times with PBS, finally cells were resuspended with 0.5mL PBS, and the cell suspension was filtered through a 40 μm nylon mesh into a flow tube, set to an excitation wavelength of 480nm, the fluorescence intensity of each group of DOX was measured using a flow meter, and plotted using Flowjo software.
As shown in FIG. 3, DOX-Hyd-PP @ AuNPs gradually accumulate in cytoplasm with the lapse of time, free drugs without being coated directly enter cell nucleus, and flow cytometry quantitatively shows that the uptake of the nano drug-loaded material DOX-Hyd-PP @ AuNPs is increased by 1.69 times and 1.25 times at 2h and 6h compared with the free DOX group. These results are consistent with fluorescence imaging, and confirm that the nano drug-loaded material DOX-Hyd-PP @ AuNPs can enhance the uptake and accumulation of DOX.
Example 3
To characterize the nanometerThe synergistic antitumor effect of the medicine carrying material in cell is 1 × 10 of 4T14The number of individual cell densities was seeded in 96-well plates and incubated for 24 h. After the cells have grown adherent, the original medium is discarded, fresh medium containing DOX.HCl, DOX-Hyd-PP @ AuNPs (DOX concentrations of 0. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL, 1.5. mu.g/mL, 2. mu.g/mL and 2.5. mu.g/mL, corresponding to AuNPs concentrations of 0mM, 0.5mM, 1mM, 1.5mM, 2mM and 2.5mM) is added and placed in an incubator for 12 h. The cells were irradiated with 4Gy of X-ray and incubated in an incubator for 12 hours. And after the incubation is finished, removing the supernatant, washing with PBS for three times, adding a serum-free culture medium containing 10 mu LCCK-8 into each hole, continuously putting the holes into an incubator for culturing for 2h, taking untreated cells as a control group, taking a blank plate of the serum-free culture medium only containing CCK-8 as a background absorbance group, and setting 4 parallel samples in each group.
As shown in FIG. 4, DOX-Hyd-PP @ AuNPs showed more significant antitumor effect than free DOX. Then, the CCK-8 method is used for researching the chemotherapy/radiotherapy synergistic antitumor effect of DOX-Hyd-PP @ AuNPs in vitro. Under the sensitization of the AuNPs radiosensitizer, the combined inhibition effect of radiotherapy and chemotherapy of the DOX-Hyd-PP @ AuNPs (+) group is obviously higher than that of chemotherapy of the DOX-Hyd-PP @ AuNPs (+) group. And the cell survival rate becomes smaller as the concentration of the drug increases. Under the action of X rays, the DOX-Hyd-PP @ AuNPs (+) group realizes the synergistic inhibition of tumor cell growth by AuNPs and DOX.
Example 4
In order to determine the mechanism problem of the nano drug-loaded material in the chemotherapy and radiotherapy combined treatment process, the generation of active oxygen in cells is evaluated by DCFH-DA staining. Mitochondrial membrane potential was detected by mitochondrial membrane potential dye (MitoView-633) probe and cell cycle distribution was detected by flow cytometry for the different treatment groups.
As shown in FIG. 5, after treatment, AuNPs generated a large amount of active oxygen under the irradiation of X-rays after the DOX-Hyd-PP @ AuNPs group, and both the blank group and free DOX generated almost no active oxygen under the irradiation of no rays. Excess intracellular ROS can alter mitochondrial membrane permeability, leading to mitochondrial membrane potential collapse.
As shown in FIG. 6, the fluorescence intensity of the control group was the highest, and the mitochondrial membrane potential of the control group + the X-ray group was decreased, because the X-ray radiation decomposed water molecules to generate active oxygen, which caused the decrease of the mitochondrial membrane potential. The fluorescence intensity of the free DOX group is lower than that of the control group, and the mitochondrial membrane potential of the control group and the ray group is reduced, which is caused by the apoptosis caused by the drug action and is reduced. The DOX-Hyd-PP @ AuNPs group has the lowest fluorescence intensity, and mitochondrial transmembrane potential depolarization under triple actions of DOX, AuNPs and X-ray radiation causes mitochondrial outer membrane pores to be opened to release factors such as cytochrome c and the like, thereby accelerating apoptosis.
As shown in FIG. 7 and the following table, the cells were affected by X-ray irradiation, and it can be seen from the data of Control and Control + that X-ray decreased the G0/G1 phase and increased the G2/M phase of the cells (the Control with + sign indicating the application of X-ray). After the DOX-Hyd-PP @ AuNPs material group is used for treating the cells, the G0/G1 phase of the cells is reduced, and the G2/M phase of the cells is increased. Similarly, after the cells are irradiated by X-rays, the G0/G1 phase of the cells is reduced, and the G2/M phase of the cells is increased. Under the influence of AuNPs and X rays in the nano drug-loaded material, the cell cycle is blocked in S and G2/M phases, and tumor cells are more sensitive to chemotherapeutic drugs in the cell cycle, so that the dosage of the chemotherapeutic drugs is reduced, and the synergistic effect of the chemotherapeutic and radiotherapy combined treatment is achieved. As shown in the table below, the band + sign refers to the add ray group. The cell cycle distribution is shown in FIG. 7, in which (a) represents a control group, (b) represents a control group (+), (c) represents DOX-Hyd-PP @ AuNPs, and (d) represents DOX-Hyd-PP @ AuNPs (+).
| Control | Control+ | DOX-Hyd-pp@AuNPs | DOX-Hyd-pp@AuNPs+ | |
| G0/G1 | 40.47％ | 12.13％ | 33.77％ | 5.92％ |
| S | 46.01％ | 47.50％ | 48.70％ | 47.23％ |
| G2/M | 13.52％ | 40.36％ | 17.53％ | 46.85％ |
In conclusion, the nano-drug carrier prepared by the invention takes the peptide dendrimer as a matrix material carrier, chemotherapeutics are chemically grafted by modifying mPEG and pH sensitive hydrazone bonds, and the radiation sensitizer AuNPs is physically wrapped, so that the pH sensitive amphipathic peptide dendrimer nano-drug carrier for the combined treatment of tumor chemotherapy and radiotherapy is constructed, the mPEG provides a hydrophilic end, the biocompatibility of the mPEG is remarkably enhanced, the nano size of the mPEG is changed, and the mPEG has hydrophilicity, so that the peptide dendrimer can be self-assembled into micelles, and the sensitizer with abundant spatial physical embedding is available; the hydrophobic end (oleophilic end) is the drug DOX, and the connection of the DOX is mainly connected with the peptide dendrimer through a pH sensitive hydrazone bond, so that the drug delivery of the combined treatment of tumor chemotherapy and radiotherapy is realized.
Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, the protection scope of the present invention should be determined by the appended claims.
Claims (9)
1. A preparation method of a nano-carrier for combined treatment of tumor chemotherapy and radiotherapy is characterized by comprising the following steps:
step 1, selecting lysine as a raw material, taking ethylenediamine as a core, and synthesizing G1-G3 generation peptide dendrimer by a divergent method;
step 2, modifying mPEG on the surface of G1-G3 generation peptide tree-shaped macromolecules to be used as an acid sensitive bond, and synthesizing to obtain Boc-Hyd-G1-G3-mPEG (Boc-Hyd-PP) nanoparticles as a carrier;
step 3, linking the chemotherapeutic drug DOX with the obtained nano-particles through chemical bonds to obtain DOX-Hyd-PP;
and 4, carrying out in-situ reduction on the ions coated in the cavity of the nano particles by adopting an in-situ compounding method to reduce the ions into nano silver-gold load, thereby obtaining the double-drug-loaded nano particles DOX-Hyd-PP @ AuNPs.
2. The method for preparing the nanocarrier for the combination therapy of tumor chemotherapy and radiotherapy according to claim 1, wherein the specific treatment of step 1 comprises the following steps:
dissolving mono-Boc protected ethylenediamine, Z-Lys (Z) -OH, HBTU and HOBT in DMF under nitrogen atmosphere, adding DIEA, reacting in ice bath for 30min, and continuing to react for 24h after removing the ice bath; after the reaction is finished, the solvent is removed by rotary evaporation, DCM is added for dissolution, and saturated NaHCO is used3Washing with 1mol/L HCl and saturated NaCl aqueous solution for three times, adding anhydrous magnesium sulfate after washing, drying overnight, filtering, performing rotary evaporation to 10mL, and further purifying by passing through a gel chromatographic column with a dichloromethane/methanol 10:1 mobile phase to obtain G1 generation peptide dendrimer;
dissolving the reaction product G1 in the last step in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, and continuing to react for 7h after removing the ice bath; after the reaction is finished, the solvent is rotationally evaporated, the solvent is precipitated by ethyl acetate, and white solid is collected; dissolving the collected white solid product and Boc-Lys (Boc) -OH, HBTU and HOBT in DMF under nitrogen atmosphere, adding DIEA, reacting in ice bath for 30min, and continuing to react for 24h after removing the ice bath; after the reaction is finished, performing rotary evaporation to remove the solvent, adding DCM for dissolution, washing with saturated sodium bicarbonate, 1mol/L hydrochloric acid and saturated NaCl aqueous solution for three times, adding anhydrous magnesium sulfate after washing for drying overnight, filtering, performing rotary evaporation to 15mL, and further purifying by passing a dichloromethane/methanol 10:1 mobile phase through a gel chromatographic column to obtain G1-G1 generation peptide dendrimer;
dissolving the reaction products G1-G1 in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, removing the ice bath, continuing to react for 7h, performing rotary evaporation to remove the solvent after the reaction is finished, precipitating with ethyl acetate, and collecting white solid; dissolving the white solid product and Boc-Lys (Boc) -OH, HBTU and HOBT in DMF under nitrogen atmosphere, adding DIEA, reacting in ice bath for 30min, and continuing to react for 48h after removing the ice bath; after the reaction is finished, the solvent is removed by rotary evaporation, DCM is added for dissolution, and saturated NaHCO is used3Washing with 1mol/LHCl and saturated NaCl aqueous solution for three times, adding anhydrous magnesium sulfate after washing, drying overnight, filtering, performing rotary evaporation to 20mL, and further purifying by passing through a gel chromatographic column with dichloromethane/methanol 10:1 mobile phase to obtain G1-G2 generation peptide dendrimer;
dissolving the reaction products G1-G2 in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, removing the ice bath, continuing to react for 7h, performing rotary evaporation to remove the solvent after the reaction is finished, precipitating with ethyl acetate, and collecting white solid; dissolving the white solid product and Boc-Lys (Boc) -OH, HBTU and HOBT in DMF under nitrogen atmosphere, adding DIEA, reacting in ice bath for 30min, removing the ice bath, and continuing to react for 72 h. And after the reaction is finished, performing rotary evaporation to remove the solvent, adding DCM for dissolution, washing with saturated sodium bicarbonate, 1mol/L hydrochloric acid and saturated NaCl aqueous solution for three times, adding anhydrous magnesium sulfate after washing, drying overnight, filtering, performing rotary evaporation to 25mL, and further purifying by passing through a gel chromatographic column by using a dichloromethane/methanol 10:1 mobile phase to obtain the G1-G3 generation peptide dendrimer.
3. The method for preparing the nanocarrier for the combination therapy of tumor chemotherapy and radiotherapy according to claim 2, wherein the step 2 comprises the following steps:
dissolving mPEG-OH500, succinic anhydride, DMAP and triethylamine in tetrahydrofuran, and reacting for 24 hours under rapid stirring; and after the reaction is finished, removing the solvent by rotary evaporation, adding a small amount of DCM for dissolution, adding glacial ethyl ether for precipitation under the condition of vigorous stirring, and drying in vacuum to obtain a white solid product mPEG-COOH, wherein mPEG-OH 500: succinic anhydride: DMAP: the molar ratio of triethylamine is 1:1.5:1.05: 1.05;
weighing G1-G3 peptide dendrimer, dissolving in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, removing the ice bath, continuing to react for 7h, performing rotary evaporation to remove the solvent after the reaction is finished, precipitating with diethyl ether, and collecting white solid; dissolving white solid products mPEG-COOH, EDC and NHS in 10mL of water, stirring and activating for 1h, dripping the activated product into the water solution of the deprotected product at a constant speed after activation, reacting for 48h at normal temperature, dialyzing for 3 times by using PBS buffer solution after the reaction is finished, dialyzing for three times by using deionized water, and freeze-drying to obtain white solid G1-G3-mPEG; wherein G1-G3 peptide dendrimers: mPEG-COOH: EDC: the molar ratio of NHS is 1:16:24: 24;
dissolving succinic anhydride and DMAP in DCM, and slowly dropwise adding tert-butoxycarbonylhydrazine dissolved in DCM under the condition of vigorous stirring; and (3) after the reaction is finished, evaporating to remove the solvent, and vacuumizing and drying for 24 hours to obtain the carboxylated tert-butyloxycarbonyl hydrazine, wherein the weight ratio of succinic anhydride: the molar ratio of tert-butyloxycarbonyl hydrazine is 1.5: 1;
dissolving G1-G3-mPEG500 in anhydrous methanol, adding Pd/C (46.3mg) and reacting for 48 hours in a hydrogen atmosphere to remove Cbz protection; after the reaction is finished, removing palladium carbon by suction filtration, and removing the solvent by rotary evaporation; dissolving carboxylated tert-butyloxycarbonyl hydrazine, EDC and NHS in 5mL of water, and carrying out activation reaction for 1 h; dissolving G1-G3-mPEG500 without Cbz protection in 5mL of water, slowly dropwise adding activated carboxylic tert-butyloxycarbonyl hydrazine, and reacting for 48 h; after the reaction is finished, dialyzing the mixture for 3 days by deionized water, and freeze-drying the mixture to obtain Boc-Hyd-G1-G3-mPEG, wherein G1-G3-mPEG 500: carboxylated t-butyloxycarbonylhydrazine: EDC: the molar ratio of NHS was 1:4:8: 8.
4. The method for preparing the nanocarrier for the combination therapy of tumor chemotherapy and radiotherapy according to claim 3, wherein the step 3 comprises the following steps:
dissolving Boc-Hyd-G1-G3-mPEG in DCM under nitrogen atmosphere, adding TFA, reacting in ice bath for 30min, removing the ice bath, continuing to react for 7h, removing the solvent by rotary evaporation after the reaction is finished, precipitating with ethyl acetate, and collecting white solid; the collected white solid was dissolved in DMF with DOX. HCl, and a drop of TFA was added to catalyze the reaction for 48h in the absence of light. After the reaction is finished, removing the solvent by rotary evaporation, adding a small amount of deionized water for dissolution, dialyzing with the deionized water at 4 ℃ for 24h, and freeze-drying to obtain a product which is a drug-loaded nanoparticle DOX-Hyd-G1-G3-mPEG (DOX-Hyd-PP), wherein the Boc-Hyd-G1-G3-mPEG: the molar ratio of hcl is 1: 4.
5. The method for preparing the nanocarrier for the combination therapy of tumor chemotherapy and radiotherapy according to claim 4, wherein the specific treatment of step 4 comprises the following steps:
adding 1 w% of chloroauric acid into 20mL of water, adjusting the pH value to be neutral by using 0.1mol/L of NaOH, dissolving DOX-hyd-G1-G3-mPEG in the chloroauric acid solution, stirring for 30min, quickly adding the sodium borohydride solution, and continuously stirring for 3 h;
and dialyzing with deionized water for 24 hours after stirring, and freeze-drying to obtain double-drug-loaded nanoparticles DOX-Hyd-G1-G3-mPEG @ AuNPs (DOX-Hyd-PP @ AuNPs), wherein DOX-Hyd-G1-G3-mPEG: the molar ratio of AuNPs is 1: 8.
6. The method for preparing the nanocarrier for the combination of tumor chemotherapy and radiotherapy in the step 3, wherein the particle size of the nanoparticle DOX-Hyd-G1-G3-mPEG is maintained between 120 and 130nm after the nanoparticle is lyophilized;
in the step 4, the particle size of the freeze-dried double-drug-loading nanoparticle DOX-hyd-G1-G3-mPEG @ AuNPs is kept between 90 nm and 100 nm.
7. The method for preparing the nanocarrier for the combination of tumor chemotherapy and radiotherapy as claimed in claim 4, wherein the cut-off molecular weight of the dialysis bag used in the dialysis with deionized water in the steps 2 and 3 is 1000 Da.
8. The pH-sensitive amphiphilic peptide dendrimer nano-carrier for combined tumor chemotherapy and radiotherapy, prepared according to the preparation method of the nano-carrier for combined tumor chemotherapy and radiotherapy, disclosed by any one of claims 1 to 7, takes peptide dendrimer nanoparticles as a carrier, and radiotherapy sensitizer gold nanoparticles (AuNPs) are loaded in an inner cavity of the carrier, wherein a hydrophilic end of the peptide dendrimer nanoparticles as the carrier is composed of mPEG, and a hydrophobic end of the peptide dendrimer nanoparticles as the carrier is composed of doxorubicin DOX serving as a chemotherapy drug.
9. The application of the pH-sensitive amphiphilic peptide dendrimer nano-carrier for combined treatment of tumor chemotherapy and radiotherapy, according to claim 8, in preparation of internal medicines for combined treatment of tumor chemotherapy and radiotherapy.
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