CN109568599B - Liposome-modified adriamycin and adriamycin-containing nanoparticles - Google Patents
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Abstract
The invention relates to liposome-modified adriamycin, nanoparticles containing the liposome-modified adriamycin and a preparation method thereof. The adriamycin modified by the liposome is prepared by connecting the adriamycin and the liposome through anhydride or dibasic organic acid, and is further wrapped by high molecular mPEG-DSPE to assemble the nano-drug. The nano-drug has the advantages of high entrapment rate, high tumor local drug concentration, low systemic toxicity, high drug effect, release of original drug after entering cells and the like.
Description
Technical Field
The invention relates to the field of drug modification, in particular to doxorubicin modification and preparation of doxorubicin nanoparticles, and more particularly relates to liposome-modified doxorubicin, nanoparticles containing liposome-modified doxorubicin and the like.
Background
In 2015, 4,292,000 new cases of cancer and 2,814,000 deaths were estimated. Over the past few decades, great efforts have been made to treat these serious diseases. Among the various therapies, chemotherapy is currently one of the most clinically effective means for treating cancer. Although various anticancer drugs have been developed and applied to chemotherapy, they often cause adverse side effects to patients, resulting in physical and mental pain, and the treatment efficiency is low. The chemotherapy drugs commonly used in clinic at present comprise adriamycin, paclitaxel, 10-hydroxycamptothecin, irinotecan and the like.
Doxorubicin (DOX) is an antitumor antibiotic, can inhibit the synthesis of RNA and DNA, has the strongest inhibition effect on RNA and wider antitumor spectrum, and is widely used for chemotherapy of various tumors. The traditional Chinese medicine composition is clinically used for treating acute lymphocytic leukemia, acute myelocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, breast cancer, lung cancer, ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, nephroblastoma, neuroblastoma, bladder tumor, thyroid tumor, chorioepithelial cancer, prostatic cancer, testicular cancer, gastric cancer, liver cancer and the like. However, the clinical application of the adriamycin is limited by two factors of the adriamycin, and on one hand, the adriamycin has toxic and side effects of bone marrow suppression, myocardial damage, myocardial toxicity and the like; on the other hand, doxorubicin resistance is an important factor.
Since most chemotherapy drugs achieve anti-tumor effects by interfering with the synthesis process of cancer cell DNA, they often have side effects such as myelosuppression, and there is no effective preventive measure for these accompanying side effects, which limits the application of tumor therapy. Therefore, the search for an anti-tumor related drug which can improve the sensitivity of chemotherapeutic drugs, enhance the inhibition effect on tumor cells, and reduce the toxic and side effects and drug resistance of the drugs becomes an urgent problem to be solved, and is also a hotspot of the research of modern oncologists.
Disclosure of Invention
To solve the above problems, the nano drug-loaded delivery strategy has received much attention. In vivo, the drug-loaded nanoparticles can be phagocytized by macrophages as foreign matters to reach target sites of liver, spleen and the like with concentrated distribution of a reticuloendothelial system and target sites connected with ligands, antibodies and enzyme substrates. The nano particles are highly dispersed, and the surface area is large, so that the contact area of the drug and the biomembrane of the absorption part is increased. The special surface property of the nano-particles greatly prolongs the retention time of the nano-particles in small intestines, and the nano-particles also have a protection effect on the loaded medicine, and the comprehensive effects can obviously improve the absorption and bioavailability of the medicine. Different from the transmembrane transport mechanism of common drugs, the nanoparticles enter cells through endocytosis and other mechanisms, so that the permeability of the drugs to biological membranes can be increased, and the transdermal absorption of the drugs and the exertion of intracellular drug effects are facilitated. The low molecular weight chemotherapeutic drug penetrates the capillary wall of healthy and tumor tissues through nonspecific diffusion, but the drug loaded by the nanoparticles can only penetrate into the tumor capillary bed with high permeability. The targeting property of the drug-loaded nanoparticles increases the local drug concentration and reduces the concentration of other parts of the whole body, thereby greatly reducing the systemic toxicity of the drug. In order to improve the curative effect of the nanoparticles and effectively reach tumor sites, Triphenylphosphine (TPP), folic acid, RGD, LHRH polypeptide, transferrin, aptamer and other common targeting elements with targeting effect are modified on the surfaces of the nanoparticles. Meanwhile, acid-sensitive groups are introduced into drug molecules, and after carboxylic acid and amino are condensed into ester, the toxic and side effects of the ester are reduced, so that the drug can be reduced into original drug after entering cells, and the drug effect is improved.
Specifically, the invention provides liposome-modified adriamycin, nanoparticles containing the liposome-modified adriamycin and a preparation method thereof. The adriamycin modified by the liposome is prepared by connecting the adriamycin and the liposome through anhydride or dibasic organic acid, and is further wrapped by high molecular mPEG-DSPE to assemble the nano-drug. The nano-drug has the advantages of high entrapment rate, high tumor local drug concentration, low systemic toxicity, high drug effect, release of original drug after entering cells and the like, improves the inhibition effect of the drug on cancer cells, and reduces the toxic and side effects of the drug on normal cells.
In a first aspect, the present invention provides a liposome-modified drug, characterized by comprising a liposome molecule, a linker molecule, and a therapeutically active drug covalently linked in that order, the liposome molecule and the drug having a nucleophilic group, preferably the nucleophilic group is an amino group; the linker molecule has an amphiphilic group.
The liposome-modified drug of the present invention, wherein the liposome molecule comprises glutamic acid units and long-chain amine units, preferably the liposome molecule is N, N' -di-long-chain alkyl-L-glutamic acid diamide (LG).
The liposome modified drug is characterized in that the linker molecule is anhydride or dicarboxylic acid, preferably the linker molecule is cis-aconitic anhydride.
The liposome-modified drug of the present invention, wherein the therapeutically active drug is a chemotherapeutic with a primary amino group, preferably the chemotherapeutic is selected from the group consisting of Doxorubicin (Doxorubicin), Amrubicin (Amrubicin), Nimustine hydrochloride (Nimustine hydrochloride), Pirarubicin (Pirarubicin), Methotrexate (Methotrexate), Mitomycin (Mitomycin), vindesine (vindesine).
In a second aspect, the present invention also provides a method for preparing the liposome-modified drug, comprising:
(1) modifying the original drug with anhydride;
(2) subjecting an anhydride-modified prodrug (CAR) to a condensation reaction with a liposome molecule;
(3) washing, recrystallizing and freeze-drying to obtain the liposome modified drug.
The preparation method of the liposome modified drug comprises the following steps:
(1) preparing a cis-form aconitic anhydride modified parent drug CAR by taking triethylamine as a catalyst through a ring-opening reaction between the parent drug R and cis-form aconitic anhydride CA: dissolving the original drug R and CA in anhydrous DMF, adding triethylamine, and stirring for 24 hours at room temperature under the condition of keeping out of the sun and nitrogen; adding cold ethyl acetate, mixing and washing; drying the organic layer with anhydrous sodium sulfate for 12 hours, filtering and drying to obtain CAR;
(2) dissolving CAR with dichloromethane, activating with EDC and NHS, adding liposome molecule with amino group, and performing amide condensation reaction;
(3) after reacting for 48 hours, washing, recrystallizing and freeze-drying to obtain the liposome modified drug.
In a third aspect, the present invention provides a nanoparticle comprising the liposome-modified drug, wherein the drug-containing nanoparticle is formed by encapsulating the liposome-modified drug with a pharmaceutically acceptable polymer.
The nanoparticle is characterized in that the pharmaceutically acceptable high-molecular mPEG-DSPE is modified to have a targeting group.
In a fourth aspect, the present invention provides a method for preparing the nanoparticle, comprising: mixing mPEG-DSPE and liposome modified drug, dissolving in anhydrous DMF, magnetically stirring, slowly dropwise adding secondary water, dialyzing overnight, centrifuging, and collecting supernatant to obtain nanoparticle containing liposome modified drug.
Optionally, mPEG-DSPE modified by a targeting molecule is further added in the mixing process of the mPEG-DSPE and the liposome modified drug, and the preferred targeting molecule is selected from the group consisting of Triphenylphosphine (TPP), folic acid, RGD, LHRH polypeptide, transferrin, and aptamer.
In a fifth aspect, the invention provides the liposome modified drug and the use of the nanoparticle in the following aspects:
(1) preparing a medicament for treating cancer;
(2) preparing an intracellular targeting drug;
(3) prodrugs with reduced toxic side effects are prepared.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) the introduction of liposome molecule N, N' -di-long chain alkyl-L-glutamic acid diamide (LG) improves the entrapment rate of drug molecules and the bioavailability of drugs.
(2) Modification of NH with Triphenylphosphine (TPP)2The mPEG-DSPE is mixed with the mPEG-DSPE to prepare the nano-drug with mitochondrion targeting, and the anti-cancer effect of the drug is improved. The concentration of the local medicine is increased and the concentration of other parts of the whole body is reduced, thereby greatly reducing the systemic toxicity of the medicine.
(3) Acid-sensitive groups are introduced into drug molecules, and after carboxylic acid and amino are condensed into ester, the toxic and side effects of the ester are reduced, so that the drug can be reduced into original drug after entering cells, and the drug effect is improved.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:
FIG. 1: nuclear magnetic characterization of Liposomal N, N' -didodecyl-L-glutamic acid diamide (LGC12)
FIG. 2: MALDI-TOF-MS mass spectrometry characterization of the liposome LGC 12;
a: boc-protected LGC12 mass spectrum; b: LGC12 mass spectrum of Boc deprotection
FIG. 3: nuclear magnetic characterization of LGC 12-modified Doxorubicin (DOX-LGC12)
FIG. 4: MALDI-TOF-MS mass spectrometry characterization of DOX-LGC 12;
FIG. 5: preparation condition optimization of nanoparticles containing DOX-LGC12
The X axis is the mass ratio of DOX-LGC12 to mPEG-DSPE; y-axis is diameter (nm)/PDI
FIG. 6A: diameter distribution of DOX-LGC 12-containing nanoparticles
FIG. 6B: the diameter distribution of the nanoparticles containing DOX-LGC 12-TPP;
FIG. 7: TEM scanning electron micrograph of DOX-LGC12 nanoparticle
FIG. 8: relation curve between DOX mass concentration and ultraviolet absorption in DOX-LGC 12-containing nanoparticles
FIG. 9: cytotoxicity test of nanoparticles containing DOX-LGC12 and DOX-LGC12-TPP
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
The invention provides liposome-modified adriamycin, nanoparticles containing the liposome-modified adriamycin and a preparation method thereof. The adriamycin modified by the liposome is prepared by connecting the adriamycin and the liposome through anhydride or dibasic organic acid, and is further wrapped by high molecular mPEG-DSPE to assemble the nano-drug. The nano-drug has the advantages of high entrapment rate, high tumor local drug concentration, low systemic toxicity, high drug effect, release of original drug after entering cells and the like.
Example 1: preparation and characterization of Liposomal N, N' -Di-Long-chain alkane-L-glutamic acid diamide (LG)
Adding Boc-glutamic acid and two times of equivalent of long-chain amine (octa, dodeca and octadecamine) into a reaction bottle, adding dichloromethane for dissolving, adding 1.1 times of equivalent of EDC and HOBt for amide condensation, then filtering, washing and recrystallizing to obtain N, N '-di-long-chain alkyl-L-Boc-glutamic acid diamide, then removing Boc protection by trifluoroacetic acid, washing and drying to obtain white powder N, N' -di-long-chain alkyl-L-glutamic acid diamide (LG). The reaction steps are as follows:
the prepared LGC12 was subjected to nuclear magnetic hydrogen spectrum characterization, and the results are shown in FIG. 1. Peaks in the nuclear magnetic hydrogen spectrum, all successfully assigned, indicate successful synthesis of LGC 12.
The prepared LGC12 was characterized by MALDI-TOF-MS, and the results are shown in FIG. 2. FIG. 2A is a mass spectrum of Boc-protected LGC12, wherein 604.1 is Boc-LGC12+ Na+620.1 Boc-LGC12+ K+(ii) a FIG. 2B is a mass spectrum of Boc deprotection with 481.5 LGC12,503.6 LGC12+ Na+All successfully assigned, the MALDI-TOF-MS mass spectrometry results also indicated successful synthesis of LGC 12.
Example 2: preparation method of LGC12 modified adriamycin (Doxorubicin)
R (R ═ doxorubicin DOX), hereinafter referred to as CAR, was modified with cis-aconitic anhydride (CA), and synthesized by a ring-opening reaction between R and CA using triethylamine as a catalyst. Bulk R (0.4mmol) and CA (0.44mmol) were dissolved in 20.0mL of anhydrous DMF in a completely dry flask, followed by the addition of 67.0. mu.L of triethylamine. Placing the mixture at room temperatureIn the dark and under nitrogen (N)2) Stirred under atmosphere for 24 hours. Next, the solution was mixed with 200.0mL of cold ethyl acetate, washed first with a saturated sodium chloride solution at pH 2-3 and then with a saturated solution at pH 7.4. The obtained organic layer was dried over anhydrous sodium sulfate for 12 hours. Finally, the solid CAR was isolated by filtration and dried under vacuum at room temperature to give the product.
② adding the drug CAR (R ═ adriamycin (DOX)) into a reaction bottle, adding dichloromethane to dissolve, adding 2-3 times of equivalent of EDC and NHS to activate for 2-4 hours, adding 1 time of equivalent of N, N' -didodecyl-L-glutamic acid diamide (LGC12) to carry out amide condensation reaction for 48 hours, then washing, recrystallizing and freeze-drying to obtain the LG modified drug (CAR-LGC 12). The reaction steps are as follows:
wherein R is DOX, and the structural formula is as follows:
example 3: structural characterization of LGC 12-modified DOX
1. Nuclear magnetic resonance spectrum (NMR)
N, N' -didodecyl-glutamic acid diamide (LGC12) modified drug R prepared in example 2 was characterized with Tetramethylsilane (TMS) as internal standard and deuterated chloroform (CDCl)3) As solvent, a 400MHZ nuclear magnetic resonance instrument is adopted to carry out the reaction1H NMR was scanned.
The nuclear magnetic hydrogen spectrum of DOX-LGC12 is shown in FIG. 3, and the peaks in the nuclear magnetic hydrogen spectrum of DOX-LGC12 were all successfully assigned.
2. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
To further confirm the synthesized CAR-LG (DOX-LGC12) of example 2, its mass spectrum was tested by MALDI-TOF-MS, matrix-selective gentisic acid (DHB).
The MALDI-TOF-MS mass spectra of DOX-LGC12 are shown in FIG. 4. 1141.6 in FIG. 4 is DOX-LGC12+ Na+。
The experimental results of nuclear magnetic hydrogen spectrum and mass spectrum prove that the LGC12 modified drug DOX-LGC12 is successfully synthesized.
Example 4: preparation and characterization of nanoparticles containing DOX-LGC12
Nanoparticles were prepared by loading the DOX-LGC12 prepared in example 2 with a polymer. Selecting mPEG-DSPE or mPEG-DSPE modified by mitochondrion targeting group Triphenylphosphine (TPP) as an entrapped molecule, dissolving DOX-LGC12 and a macromolecule in a mass ratio of 1:1,1:2,1:3,1:4,1:5 and 1:6 in 1mL of anhydrous DMF, magnetically stirring, slowly dropwise adding 5mL of secondary water, dialyzing overnight (molecular weight cut-off 3500) after half an hour, and centrifuging to obtain a supernatant to obtain the nanoparticles. Changes in particle size and zeta potential were observed by Dynamic Light Scattering (DLS). The results of optimizing the conditions for nanoparticle formation by DOX-LGC12 are shown in FIG. 5.
According to FIG. 5, DOX-LGC 12: the optimal ratio of mPEG-DSPE is 1:3
Preparing corresponding nano particles according to the selected optimal conditions (the optimal ratio of the LGC12 modified drug to the polymer), detecting the particle size distribution of the nano particles, and carrying out TEM morphology observation.
The particle size distribution diagram of DOX-LGC12 nanoparticles formed under the optimal conditions is shown in FIG. 6, wherein FIG. 6A is DOX-LGC12 nanoparticles prepared by using mPEG-DSPE as an entrapment molecule, and FIG. 6B is DOX-LGC12-TPP nanoparticles prepared by using TPP modified mPEG-DSPE as an entrapment molecule; the electron microscope image is shown in FIG. 7. The results show that the nanoparticles prepared by the method are stable in structure and uniform in size.
Example 5: the DOX-LGC 12-containing nanoparticles have the advantages of traditional Chinese medicine content, loading rate and packaging efficiency
1. Method of producing a composite material
The content of DOX in the nanoparticles prepared in example 4 was examined. The ultraviolet absorbance value of the solution at 480nm (DOX) is measured by ultraviolet visible spectrum (UV-Vis), and then the linear relation between the DOX content and the ultraviolet absorbance value is calculated. Measuring the ultraviolet absorbance value of the nano-drug solution at the characteristic ultraviolet absorption position to calculate the content of the drug DOX;
the loading rate and the wrapping efficiency of the drug R are calculated by the following formulas:
loading rate (%) ([ content of drug R in nanoparticle/mass of total nanoparticle ] × 100
Encapsulation efficiency (%) ([ content of drug R in nanoparticle/mass of drug R administered ] × 100
2. Results
The relationship curve between the mass concentration of doxorubicin in the DOX-LGC12 nanoparticles and the uv absorption is shown in fig. 8. The calculation formula is that Y is 8.959 XX + 0.03118; r20.9996. Measuring ultraviolet absorbance at 480nm to calculate to obtain drug content of 388 μ M; the loading rate is 7.5%; the wrapping efficiency is 22.5 percent
Example 6: cytotoxicity of nanoparticles containing LGC 12-modified drug
The DOX-LGC 12-containing nanoparticles prepared in example 4 were tested for their toxicity to cancer cells.
1. Method of producing a composite material
A549 (human lung cancer cells), A549DDP (human lung cancer platinum-resistant cells), MCF7 (human breast cancer cells) and 4T1 (murine breast cancer cells) are selected for researching the toxicity problem of the medicine. Four cells were cultured in DMEM (GIBCO) medium. DMEM medium contains 10% fetal bovine serum and 1% penicillin streptomycin mixture (100X).
The cytotoxicity is detected by an MTT method, and the specific steps are as follows:
(1) after a549, a549DDP, MCF7 and 4T1 cells were cultured to log phase, digested with pancreatin and counted. The cell solution was diluted to 5X 104cells/mL；
(2) Inoculating the pre-diluted cells into a 96-well plate, wherein each well is 100 mu L, and then placing the plate in an incubator for overnight culture;
(3) respectively diluting DOX, NP and NP-TPP according to a certain multiple, adding into a 96-well plate, adding 10 mu L into each well, and enabling the final DOX concentration of the medicine to be 100,50,25,12.5,1.25,0.125 and 0.0125 mu M in sequence. Setting four multiple holes for each concentration, and culturing for 72 h;
(4) diluting 10 times of pre-configured 10% MTT solution by using a phenol-free red culture medium, adding the diluted solution into 96-well plates with different culture time, adding 100 mu L of MTT solution into each well, continuously placing the plates in an incubator for culture for 4 hours, then adding 100 mu L of SDS solution into each well, keeping the plates away from light, and placing the plates in a constant-temperature incubator at 37 ℃ for 12 hours;
(5) measuring the absorbance OD value of each hole of the 96-hole plate at 570nm by using a microplate reader, selecting the background wavelength to be 650nm, taking the average value of the OD values of the three multiple holes as the OD value of a target sample, and calculating the cell survival rate:
cell viability ═ sample OD/blank OD
2. Results
2.1 cytotoxicity assays with Dox nanoparticles
The cytotoxicity of NPS (DOX-LGC12), NPS-TPP (DOX-LGC12-TPP) and DOX was measured by MTT method, and the time taken for examination was 72 hours, and the results are shown in FIG. 9, IC50The values are listed in table 2.
Table 2: NPS (DOX-LGC12), NPS (DOX-LGC12) -TPP, IC of DOX on four cells50 (μM)
According to the toxic effects of the three drugs NPS (DOX-LGC12), NPS-TPP (DOX-LGC12-TPP) and DOX on four cell lines, the fact that the cytotoxicity is NPS-TPP (DOX-LGC12-TPP) > NPS (DOX-LGC12) > DOX in sequence from strong to weak indicates that the polymer is used as a carrier, the endocytosis of the drug by cells can be enhanced, and the nanoparticle with the mitochondrion targeting can improve the toxic effect of the drug on cancer cells.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (6)
1. A lipid-modified drug characterized by comprising a lipid molecule, a linker molecule, and a therapeutically active drug covalently linked in that order; the lipid molecule is N, N' -di-long-chain alkyl-L-glutamic acid diamide, and the structural formula is shown as the formula (I); the therapeutically active drug is doxorubicin; the linker molecule is cis-aconitic anhydride;
wherein n is 7, 11 or 17.
2. The process for preparing a lipid-modified pharmaceutical according to claim 1, comprising:
(1) modifying the original drug with anhydride;
(2) carrying out condensation reaction on the anhydride modified technical product and lipid molecules;
(3) washing, recrystallizing and freeze-drying to obtain the lipid modified drug.
3. The method of claim 2, comprising:
(1) preparing a cis-form aconitic anhydride modified parent drug CAR by taking triethylamine as a catalyst through a ring-opening reaction between the parent drug R and cis-form aconitic anhydride CA: dissolving the original drug R and CA in anhydrous DMF, adding triethylamine, and stirring for 24 hours at room temperature under the condition of keeping out of the sun and nitrogen; adding cold ethyl acetate, mixing and washing; drying the organic layer with anhydrous sodium sulfate for 12 hours, filtering and drying to obtain CAR;
(2) dissolving CAR with dichloromethane, activating with EDC and NHS, and adding lipid molecule with amino group to perform amide condensation reaction;
(3) after reacting for 48 hours, washing, recrystallizing and freeze-drying to obtain the lipid modified drug.
4. A nanoparticle comprising the lipid-modified drug prepared according to claim 2 or 3, characterized in that the drug-containing nanoparticle is formed by assembly using a pharmaceutically acceptable polymer to encapsulate the lipid-modified drug.
5. A method of preparing nanoparticles as claimed in claim 4, comprising: mixing mPEG-DSPE and lipid modifying drug, dissolving in anhydrous DMF, magnetically stirring, slowly dropwise adding secondary water, dialyzing overnight, centrifuging, and collecting supernatant to obtain nanoparticles containing lipid modifying drug.
6. Use of the lipid-modified drug of claim 1 or the nanoparticle of claim 4 for:
(1) preparing a medicament for treating cancer;
(2) preparing a medicament with a targeting effect;
(3) prodrugs with reduced toxic side effects are prepared.
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