CN113713119A - Medicine for treating solid malignant tumor and preparation method thereof - Google Patents
Medicine for treating solid malignant tumor and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of chemotherapeutic drugs wrapped by nano-carriers, and discloses a drug for treating solid malignant tumors and a preparation method thereof. The nano-encapsulated chemotherapeutic drug is composed of polylactic acid-glycolic acid copolymer and dipalmitoyl phosphatidylcholine to form the shell of the nano-carrier; hyaluronic acidAcid for targeting tumor cytokine CD44, 1-butyl-3-methylimidazole-L-lactic acid as a thermal sensitizer and Ptx paclitaxel; according to the molar ratio, polylactic acid-glycolic acid copolymer: dipalmitoylphosphatidylcholine ═ 1: 3-1: 5; hyaluronic acid 1-5 x10‑4mmol/mL, 1-butyl-3-methylimidazole-L-lactic acid 2-10 x10‑5mmol/mLPtx 1-5 mg/mL. The invention effectively improves the microenvironment of the tumor, reduces the interstitial fluid pressure of the tumor tissue and reduces the drug resistance of the tumor tissue.
Description
Technical Field
The invention belongs to the technical field of chemotherapy drugs wrapped by nano-carriers, and particularly relates to a drug for treating solid malignant tumors and a preparation method thereof.
Background
Currently, solid tumors are poorly chemotherapeutically treated, for example, breast cancer. Breast cancer is the most common cancer in women worldwide and is one of the leading causes of cancer-related death. Despite early discovery and intervention, metastatic breast cancer remains largely incurable, particularly Triple Negative Breast Cancer (TNBC). TNBC is an aggressive subtype of breast cancer, accounting for 10-20% of all breast cancer cases. Due to the lack of specific targets and high probability of metastasis, currently available treatment regimens are very limited. Metastatic breast cancer is characterized by a unique Tumor Microenvironment (TME), distinct from other subtypes. Components of the TME include transformed extracellular matrix (ECM), soluble factors, immunosuppressive cells, epigenetic modifications and reprogrammed fibroblasts that collectively inhibit the anti-tumor response and aid in the progression and metastasis of TNBC. Another obstacle to breast cancer is its high heterogeneity, which complicates treatment. For example, a small piece of tumor tissue obtained from a biopsy does not necessarily represent all tumor components. In addition, the high Interstitial Fluid Pressure (IFP) generated by the tumor microenvironment also severely limits drug delivery to tumor cells, particularly in immunotherapy and nanocarrier-encapsulated chemotherapy drugs. The generation of high IFP in the tumor microenvironment may stress the blood vessels, leading to reduced intratumoral blood flow and reduced nanocarrier-encapsulated chemotherapeutic drug delivery. Because TME participates in proliferation, angiogenesis, apoptosis inhibition, immune system inhibition and drug resistance of metastatic breast cancer, TME becomes an important target for TNBC treatment.
The thermotherapy combines the nano particles and the chemotherapeutic drugs and has great prospect for treating cancers. However, its efficacy in the treatment of solid malignancies has not been clinically proven. For example, since 2006, a number of clinical trials have been conducted using ThermoDox, Adriamycin loaded cryo-sensitive liposomes (LTSLs) for the treatment of liver, colorectal, prostate and breast cancers. Hyperthermia can be achieved by different heating techniques, such as Radio Frequency (RF), Focused Ultrasound (FUS), and Microwave (MW), among others. A recent phase I study (TARDOX) showed that the combination therapy of LTSLs and non-invasive FUS hyperthermia appears clinically feasible, safe, and capable of increasing intratumoral dosing concentrations. Although increased intratumoral drug delivery has been demonstrated in preclinical studies, most phase II and phase III clinical trials fail to demonstrate that combination therapy is superior to simple chemotherapy or hyperthermia. Therefore, preclinical studies and clinical transformations of thermosensitive nanocarrier-encapsulated chemotherapeutic drugs still face enormous needs and challenges. The high permeability and long retention effect (abbreviated as EPR) refers to the phenomenon that some macromolecular substances with specific sizes (such as liposome, nanoparticle and some macromolecular drugs) are easier to permeate into tumor tissues and retain for a long time (compared with normal tissues). This is commonly explained by the fact that tumor cells, in order to grow rapidly, require more nutrients and oxygen and therefore secrete growth factors related to tumor angiogenesis, such as vascular endothelial growth factor. Especially when tumors reach 150-. The newly formed tumor vessels are structurally and morphologically very different from normal vessels. The endothelial cell space is larger, the smooth muscle layer of the vascular wall is lacked, and the function of the angiotensin receptor is lacked. In addition, the lack of lymphatic vessels in the tumor tissue may prevent lymphatic return. Both of these results in the fact that macromolecular substances can easily pass through the vascular wall to be enriched in tumor tissues, and are not carried away by lymph return and can be stored in tumor tissues for a long time, so the high permeability and long retention Effect (EPR) of solid tumors is called. The EPR effect can be further enhanced by several pathophysiological factors, such as bradykinin, nitric oxide, peroxynitrite ion, prostaglandins, vascular endothelial growth factor, tumor necrosis factor, etc., which stimulate tumor vasodilation. In addition, lymphopenia at the tumor site also increases the retention effect of macromolecular substances therein. The EPR effect was originally thought to be important for nanoparticle and liposome delivery to tumor sites; clinical trial results showed that the EPR effect was effective only in some animal tumor models, but not in solid tumors in clinical patients. See: journal of Controlled Release 244(2016) 108-. "To extension the needle microorganisms, silicon the EPR effect factors in the clinical, what is the future of the nanomedicine? "
Meanwhile, microwave, light and ultrasonic induced thermotherapy is a promising adjuvant therapy method, and can induce tumor cell apoptosis and destroy tumor cells and microenvironment (peripheral tissues). For example, focused ultrasound and microwaves can penetrate into most solid tumors of patients, and the drugs resistance of the tumors are not generated, so that the traditional Chinese medicine composition has no systemic side effect. The mild heat treatment can also induce the apoptosis of tumor cells at the temperature of 39-45 ℃. Therefore, a new method for treating solid malignant tumor based on mild hyperthermia and a chemotherapeutic drug encapsulated by a thermosensitive nano-carrier is needed.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) due to the lack of specific targets and high probability of metastasis, currently available treatment regimens are very limited.
(2) The curative effect in treating solid malignant tumor by combining thermotherapy with nano particle and chemotherapy medicine has not been proved clinically.
(3) Most of the existing phase II and phase III clinical tests fail to prove that the combined nano-drug therapy is superior to the single chemotherapy or thermotherapy, so the preclinical research and clinical transformation of the chemotherapy drugs wrapped by the thermosensitive nano-carrier still face huge requirements and challenges.
The difficulty in solving the above problems and defects is: (1) the carrier-wrapped chemotherapeutic drug is difficult to reach local tissues of the tumor (< 1%), and the nano-drug cannot generate EPR effect in solid tumor (Enhanced Permation and Retention effect); (2) solid malignant tumor microenvironment (high interstitial fluid pressure, reduced blood perfusion, immunosuppression); (3) the natural immune system responds to the elimination of the nano-drug (opsonin action, complement reaction, immune cell phagocytosis, etc.).
The significance of solving the problems and the defects is as follows: improving or ameliorating the therapeutic effect on malignant solid tumors. Revealing the importance of thermal treatment to alter the tumor microenvironment. Explore a new path for treating solid tumors by the novel nano-drug.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a medicine for treating solid malignant tumor and a preparation method thereof.
The invention is realized in such a way that the medicine for treating the solid malignant tumor is a chemotherapeutic medicine wrapped by a nano-carrier;
the nano-encapsulated chemotherapeutic drug is composed of polylactic acid-glycolic acid copolymer and dipalmitoyl phosphatidylcholine to form the shell of the nano-carrier; hyaluronic acid, used for targeting tumor cell factor CD44, 1-butyl-3-methylimidazole-L-lactic acid as a thermal sensitizer and Ptx paclitaxel;
according to the molar ratio, polylactic acid-glycolic acid copolymer: dipalmitoylphosphatidylcholine ═ 1: 3-1: 5; hyaluronic acid 1-5 x10-4mmol/mL, 1-butyl-3-methylimidazole-L-lactic acid 2-10 x10-5mmol/mLPtx 1～5mg/mL。
Further, the nano-carrier at least comprises phospholipid, polylactic acid-glycollic acid copolymer, tumor cell targeting molecules, a thermal effect sensitizer and anti-malignant tumor drugs.
Another object of the present invention is to provide a method for preparing the drug for treating solid malignant tumor, the method comprising:
firstly, completely dissolving DPPC, amino NH2-PLGA-NH2 and paclitaxel in dichloromethane DCM, and then adding a thermal sensitizer BML dissolved in deionized water into the mixed solution;
and (4) performing acoustic vibration emulsification on the mixed solution on an ice bath by using an ultrasonic oscillator. Adding a polymer polyvinyl alcohol (PVA) aqueous solution with the concentration of 2% w/w during emulsification, and emulsifying until the nano emulsion reaches the required diameter;
and (2) completely evaporating DCM in a fume hood under stirring, washing and collecting the DCM by ultra-high-speed centrifugation, covalently coupling the collected nano liquid particles with hyaluronic acid through amino groups on PLGA, and washing and removing unreacted hyaluronic acid by centrifugation and deionized water to obtain the PLGA-DPPC nano particles which have a targeting function and are loaded with anticancer drugs and thermal sensitizers.
Further, the emulsion is filtered by a polyether sulfone membrane to reduce the diameter of the nanoparticles, and a sterilization method can be adopted for sterilization; and finally, placing the prepared targeting nano emulsion into a refrigerator at 4 ℃ for storage.
Further, 10mg of DPPC, 20mg of NH2-PLGA-NH2 and 2mg of Ptx were completely dissolved in 2ml of dichloromethane DCM.
Further, 400 μ L of BML was added to DCM solution, 0.5mg/mL in BML deionized water, and the solution containing Ptx and BML was emulsified on ice bath for 2 minutes using an ultrasonic shaker with a 5s on-off duty cycle.
Further, 8mL of PVA solution was added to the emulsion and emulsified as before until the desired nanoparticle diameter was obtained; the DCM was then evaporated thoroughly in a fume hood for 3h and finally the nanoemulsion was collected by centrifugation at 10000g for 10 min and washed twice with deionized water.
Further, the thermosensitive nanoparticle targeting CD44 is prepared by covalently coupling hyaluronic acid through an amino group on PLGA; the coupling reaction is as follows: 2mg of HA was dissolved in 195mg MES buffer pH 5.5.
Further, 27mg EDC and 8.6mg NHS were added to the HA hyaluronic acid solution; subsequently, the mixture was incubated with 1 ml of the solution in the nanoemulsion for 24 hours with continuous stirring on an ice bath.
Further, collected by centrifugation at 10000g for 10 minutes for secondary washing; and (3) sterilizing and filtering the collected nano emulsion by adopting a polyether sulfone membrane, and storing in a refrigerator at 4 ℃.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention provides a medicine for treating solid malignant tumor, in particular to a method for treating solid tumor by adopting twice thermotherapy combined with a chemotherapeutic medicine wrapped by a nano carrier. The first thermal therapy aims at pretreating tumor tissues, and the temperature of the thermal therapy is 39-49 ℃, so that the microenvironment of the tumor is effectively improved, interstitial fluid pressure of the tumor tissues is reduced, drug resistance of the tumor tissues is reduced, and the ingestion and storage of chemotherapeutic drugs by the tumor and the tissues around the tumor (microenvironment) are improved; the second heat treatment effectively activates the sensitizer wrapped by the nano-carrier, so that the temperature of the tumor and the surrounding tissues is rapidly increased (more than or equal to 50 ℃), and the effects of local rapid release of the medicine and heat treatment ablation are achieved. The purpose of the second heat treatment is to directly destroy the tumor tissue, resulting in apoptosis and necrosis of the tumor cells.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flowchart of a method for determining the efficacy of a drug for treating solid malignant tumors according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a medicament for treating solid malignant tumor and a preparation method thereof, and the invention is described in detail with reference to the accompanying drawings.
The medicine for treating solid malignant tumor provided by the invention is a chemotherapeutic medicine wrapped by a nano-carrier. The drug-loaded nano-composition comprises: PLGA (polylactic-co-glycolic acid), DPPC (dipalmitoylphosphatidylcholine) make up the outer shell of the nanocarrier. HA hyaluronic acid, used for targeting tumor cell factor CD44, BML (1-butyl-3-methylimidazole-L-lactic acid) as a thermal sensitizer and Ptx paclitaxel, a natural anticancer drug. PLGA: DPPC 1: 3-1: 5 (mol: mol), HA 1-5 x10-4mmol/mL,BML 2～10x10-5mmol/mLPtx 1-5 mg/mL. Milliliter refers to the volume of the nanosuspension.
The nano-carrier for injection of the invention at least comprises the following 5 raw materials: (1) phospholipid, (2) PLGA, (3) tumor cell targeting molecules, (4) thermal effect sensitizer, and (5) anti-malignant tumor drugs.
As shown in fig. 1, the preparation method of the drug for treating solid malignant tumor provided by the embodiment of the present invention adopts a double-emulsion method to prepare DPPC-PLGA targeted nanoparticles loaded with paclitaxel Ptx and a thermal sensitizer BML, and specifically includes the following steps:
s101: firstly, completely dissolving DPPC, amino NH2-PLGA-NH2 and paclitaxel in Dichloromethane (DCM), and then adding a thermal sensitizer BML dissolved in deionized water into a mixed solution;
s102: the mixed solution was subjected to acoustic emulsification with an ultrasonic oscillator (cell disruptor) on an ice bath. Adding a polymer polyvinyl alcohol PVA aqueous solution (2% w/w) during emulsification, and further emulsifying until the nano emulsion reaches the required diameter;
s103: and (2) completely evaporating DCM in a fume hood under stirring, washing and collecting (at least twice) through ultra-high-speed centrifugation, covalently coupling the collected nano liquid particles with hyaluronic acid through amino groups on PLGA, and washing and removing unreacted hyaluronic acid through centrifugation and deionized water to obtain the PLGA-DPPC nano particles with the targeting function and loaded with the anti-cancer drugs and the thermal sensitizer.
The emulsion can be filtered by a polyether sulfone membrane (with the aperture of 0.45 and the diameter of 0.2 micron) to further reduce the diameter of the nanoparticles, and can be sterilized by adopting a proper sterilization method. And finally, placing the prepared targeting nano emulsion into a refrigerator at 4 ℃ for storage.
The preparation method of the medicine for treating solid malignant tumor provided by the embodiment of the invention specifically comprises the step of completely dissolving 10mg of DPPC, 20mg of NH2-PLGA-NH2 and 2mg of Ptx in 2ml of Dichloromethane (DCM). 400 μ L of BML (0.5 mg/mL in deionized water) was added to the DCM solution. The solution containing Ptx and BML was emulsified on an ice bath using an ultrasonic oscillator (frequency 24kHz, 600W, 12% output) (2 minutes, 5s on-off duty cycle). 8mL of PVA (2% w/w, aq) solution was added to the emulsion and emulsified as before until the desired nanoparticle diameter was obtained. The DCM was then evaporated thoroughly in a fume hood (3 h). Finally the nanoemulsion was collected by centrifugation at 10000g for 10 min and washed twice with deionized water. Thermosensitive nanoparticles targeting CD44 were prepared by covalently coupling hyaluronic acid through an amino group on PLGA. The coupling reaction is as follows: 2mg of HA was dissolved in 195mg MES buffer (pH 5.5). 27mg EDC and 8.6mg NHS were then added to the HA hyaluronic acid solution. Subsequently, the mixture was incubated with 1 ml of the solution in the nanoemulsion for 24 hours with continuous stirring on an ice bath. Finally collected by centrifugation at 10000g for 10 min with a second wash. The collected nano emulsion is sterilized and filtered by a polyether sulfone membrane (with the aperture of 0.45 and the diameter of 0.2 micron), and is stored in a refrigerator at 4 ℃.
The technical solution of the present invention is further described below with reference to specific examples.
1. The embodiment of the invention uses mild thermotherapy to improve the tumor microenvironment so as to reduce the hydraulic pressure of tumor tissues, increase the uptake of chemotherapeutic drugs wrapped by nano-carriers by malignant tumors and quickly release the chemotherapeutic drugs wrapped by the nano-carriers.
2. Content providing method and apparatus
(1) Before injecting the chemotherapeutic drug wrapped by the nano-carrier, irradiating the tumor by adopting an external heating source to enable the temperature of the tumor area to reach 39-49 ℃;
(2) the purpose of radiation is to change the microenvironment of the tumor, change the blood circulation inside the tumor, reduce the fluid pressure in the tumor and the tissues around the tumor or the permeability of tumor blood vessels, and increase the uptake and accumulation of the chemotherapeutic drugs wrapped by the nano-carrier;
(3) the external heat source comprises microwave, focused ultrasound, light, radio frequency and other methods for heating and warming local tissues of the tumor;
(4) rapidly injecting chemotherapy drug wrapped by nano-carrier intravenously after irradiation;
(5) performing second irradiation on the tumor by adopting corresponding thermotherapy technology at the time point (which can be displayed by imaging or biomarker method) of the highest uptake and accumulation concentration of the chemotherapy drug wrapped by the nano-carrier at the tumor part to achieve the rapid release of the drug at the tumor part;
(6) the particle size of the drug-loaded nano particles is 10-500 nm;
(7) the nano drug-carrying system can comprise a sensitizer (such as hematoporphyrin derivative), so that the heat effect of light, ultrasound or microwave can be increased;
(8) the material for preparing the nano carrier also has heat-sensitive performance, for example, the material (phospholipid or high polymer, biomolecule) can generate phase change at about 40 ℃, so that the permeability of the nano carrier is changed, and the release of therapeutic drugs is facilitated;
(9) the nano-carrier can be linked with biomolecules with a targeting tumor function, such as antibodies, polypeptides and high-specificity targeting small molecules;
(10) tumor chemotherapeutic drugs include, but are not limited to, the following chemotherapeutic drugs, biologicals. The chemotherapeutic drugs are divided into:
A. medicine for interfering nucleic acid biosynthesis
1. Dihydrofolate reductase inhibitors: methotrexate (MTX)
2. Thymidylate synthase suppressants: fluorouracil (5-FU)
3. Purine nucleotide tautomerism inhibitors: mercaptopurine (6-MP)
4. Ribonucleotide reductase inhibitors: hydroxyurea (HU)
DNA polymerase inhibitory drugs: cytarabine (Ara-C)
B. Drugs directly influencing DNA structure and function
1. Alkylating agent
2. Platinum compounds for DNA disruption
3. DNA damaging antibiotics
4. Topoisomerase inhibitors
C. Agents interfering with the transcription process and preventing RNA synthesis
Actinomycin D (DACT), doxorubicin, daunorubicin
D. Medicinal use as tubulin activity inhibitor for inhibiting protein synthesis and function
1. Vinblastine and taxol
2. Drugs that interfere with the function of the nucleoprotein: cephalotaxus fortunei alkaloids
3. Drugs that affect the supply of amino acids: l-asparaginase.
The invention adopts the twice thermotherapy combined with the chemotherapy drug wrapped by the nano carrier to treat the solid tumor. The first heat treatment aims at preprocessing the tumor tissue, the temperature of the heat treatment is 39-49 ℃, so that the microenvironment of the tumor is effectively improved, interstitial fluid pressure of the tumor tissue is reduced, drug resistance of the tumor tissue is reduced, and the absorption and storage of chemotherapy drugs by the tumor and the tissues (microenvironment) around the tumor are improved. The second heat treatment effectively activates the sensitizer wrapped by the nano-carrier, so that the temperature of the tumor and the surrounding tissues is rapidly increased (more than or equal to 50 ℃), and the effects of local rapid release of the medicine and heat treatment ablation are achieved. The purpose of the second heat treatment is to directly destroy the tumor tissue, resulting in apoptosis and necrosis of the tumor cells.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The medicine for treating the solid malignant tumor is characterized in that the medicine for treating the solid malignant tumor is a chemotherapeutic medicine wrapped by a nano-carrier;
the nano-encapsulated chemotherapeutic drug is composed of polylactic acid-glycolic acid copolymer and dipalmitoyl phosphatidylcholine to form the shell of the nano-carrier; hyaluronic acid, used for targeting tumor cell factor CD44, 1-butyl-3-methylimidazole-L-lactic acid as a thermal sensitizer and Ptx paclitaxel;
according to the molar ratio, polylactic acid-glycolic acid copolymer: dipalmitoylphosphatidylcholine ═ 1: 3-1: 5; hyaluronic acid 1-5 x10-4mmol/mL, 1-butyl-3-methylimidazole-L-lactic acid 2-10 x10-5mmol/mLPtx 1～5mg/mL。
2. The drug for treating solid malignant tumor of claim 1, wherein the nanocarrier comprises at least phospholipid, poly (lactic-co-glycolic acid), tumor cell targeting molecule, thermoeffect sensitizer, and anti-malignant tumor drug.
3. A method of preparing a medicament for the treatment of solid malignancies as claimed in any one of claims 1 to 2, comprising:
firstly, completely dissolving DPPC, amino NH2-PLGA-NH2 and paclitaxel in dichloromethane DCM, and then adding a thermal sensitizer BML dissolved in deionized water into the mixed solution;
performing acoustic vibration emulsification on the mixed solution on an ice bath by using an ultrasonic oscillator; adding a polymer polyvinyl alcohol (PVA) aqueous solution with the concentration of 2% w/w during emulsification, and emulsifying until the nano emulsion reaches the required diameter;
and (2) completely evaporating DCM in a fume hood under stirring, washing and collecting the DCM by ultra-high-speed centrifugation, covalently coupling the collected nano liquid particles with hyaluronic acid through amino groups on PLGA, and washing and removing unreacted hyaluronic acid by centrifugation and deionized water to obtain the PLGA-DPPC nano particles which have a targeting function and are loaded with anticancer drugs and thermal sensitizers.
4. The preparation method of claim 3, wherein the emulsion is filtered by a polyethersulfone membrane to reduce the diameter of the nanoparticles, and can be sterilized by a sterilization method; and finally, placing the prepared targeting nano emulsion into a refrigerator at 4 ℃ for storage.
5. The process according to claim 3, wherein 10mg DPPC, 20mg NH2-PLGA-NH2 and 2mg Ptx are completely dissolved in 2ml dichloromethane DCM.
6. The method of claim 5, wherein 400 μ L of BML is added to a DCM solution, 0.5mg/mL of BML in deionized water, and the Ptx and BML containing solution is emulsified on an ice bath for 2 minutes using an ultrasonic shaker with a 5s on-off duty cycle.
7. The method of claim 6, wherein 8mL of PVA solution is added to the emulsion and emulsified as before until the desired nanoparticle diameter is obtained; the DCM was then evaporated thoroughly in a fume hood for 3h and finally the nanoemulsion was collected by centrifugation at 10000g for 10 min and washed twice with deionized water.
8. The method of claim 7, wherein the thermosensitive nanoparticle targeting CD44 is prepared by covalently coupling hyaluronic acid through an amino group on PLGA; the coupling reaction is as follows: 2mg of HA was dissolved in 195mg MES buffer pH 5.5.
9. The method of claim 8, wherein 27mg EDC and 8.6mg NHS are added to the HA hyaluronic acid solution; subsequently, the mixture was incubated with 1 ml of the solution in the nanoemulsion for 24 hours with continuous stirring on an ice bath.
10. The method of claim 9, wherein the collection is performed by centrifugation at 10000g for 10 minutes and a second wash; and (3) sterilizing and filtering the collected nano emulsion by adopting a polyether sulfone membrane, and storing in a refrigerator at 4 ℃.
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