CN107049951B

CN107049951B - Preparation and triple integrated application of thermosensitive liposome carrying hollow gold nanoparticles and tumor therapeutic agent together

Info

Publication number: CN107049951B
Application number: CN201710273592.8A
Authority: CN
Inventors: 沈雁; 尤晨; 王茹
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2017-04-19
Filing date: 2017-04-19
Publication date: 2021-06-29
Anticipated expiration: 2037-04-19
Also published as: CN107049951A

Abstract

The invention relates to preparation and 'triple integration' application of a thermosensitive liposome carrying hollow gold nanoparticles and a tumor therapeutic agent together. The preparation mainly comprises dipalmitoyl lecithin (DPPC), hollow gold nanoparticles and a therapeutic agent, wherein a thermosensitive liposome is prepared by a conventional method, the hollow gold nanoparticles are coated by an extrusion method, and the therapeutic agent is coated to obtain the preparation. The preparation not only reduces the toxic and side effects of whole body drug therapy, but also can achieve the triple integrated treatment effect of drug therapy and thermotherapy combined with radiotherapy, and improve the killing effect on tumors.

Description

Preparation and triple integrated application of thermosensitive liposome carrying hollow gold nanoparticles and tumor therapeutic agent together

Technical Field

The invention belongs to the technical field of medicines, and relates to a thermosensitive liposome carrying hollow gold nanoparticles and a tumor therapeutic agent together, a preparation method thereof and a triple-integrated anti-tumor application thereof.

Background

Thermosensitive liposomes (also known as temperature sensitive liposomes) are liposomes that release drugs under warm conditions. Under the heating temperature higher than the phase transition temperature of the liposome, the liposome membrane is converted from a colloid state to a liquid crystal structure, the permeability is increased, and the carried medicine is quickly released to a heating position to generate a heat target effect. The liposome preparation effectively utilizes the dual advantages of the liposome and the thermotherapy, further increases the targeting property of the liposome, reduces the uptake of a reticuloendothelial system to the liposome, improves the treatment effect and reduces the toxicity.

Hyperthermia (HT) is a method for treating tumors by artificially raising the temperature of human tissues, and the tumor treatment mechanism by hyperthermia is mainly as follows: the tumor cell protein is denatured at the temperature of more than 40 ℃, so that the tumor cell is killed, the tumor cell is stimulated to produce heat shock protein, and the tumor cell expressing the heat shock protein is particularly sensitive to natural killer cells and can be dissolved and damaged. Although the thermotherapy can not replace the operation, the radiotherapy or the chemotherapy as an independent tumor treatment scheme, the thermotherapy has obvious synergy and supplementary effects on tumor treatment means such as the chemotherapy, the radiotherapy and the operation.

Radiation Therapy (Radiation Therapy) is a process of causing damage to organism cells through ionization of rays, and is one of three important means for treating malignant tumors. Approximately 60% to 70% of patients with malignant tumors require radiation therapy. According to the biological behavior of tumors, the treatment of some tumors mainly adopts radiotherapy to achieve the purpose of radical treatment. For patients who are not suitable for operation, lose operation chance, have tumor residue or suspected tumor residue after operation, the radiation therapy is an important choice for local control of tumor. At present, increasing the radiation dose of a tumor target area, improving the local control rate of the tumor, reducing the radiation dose of normal tissues around the tumor, preserving the normal functions of important organs, improving the life quality of patients and becoming the main development trend of radiotherapy.

The hollow gold nanoparticles belong to one kind of gold nanoparticles, and have a higher photo-thermal conversion effect than solid gold nanoparticles due to a large absorption cross section formed by a cavity structure, and have the characteristics of low toxicity, small size (diameter of 30-60 nm), spherical shape, tunable (550-950 nm) absorption band and the like, so that the hollow gold nanoparticles can effectively replace the solid gold nanoparticles in photo-thermal treatment. In addition, the photothermal therapy of the gold nano material has fewer side reactions than chemotherapy and radiotherapy, does not damage normal tissues basically, is a novel tumor thermotherapy technology, and has wide application prospect in the field of biomedicine. On the other hand, after the gold nanoparticles are irradiated by X rays or gamma rays, strong photoelectric absorption effect and secondary electrons are generated, and the DNA chain breakage is accelerated, so that the gold nanoparticles can be used as a novel radiosensitizer in the field of radiotherapy.

The invention aims to combine a tumor therapeutic agent with hollow gold nanoparticles to prepare a preparation with photosensitivity and temperature sensitivity, effectively combines a triple-integrated treatment means of drug therapy, thermotherapy and radiotherapy, improves the tumor targeting treatment effect and reduces the toxic and side effects of single-means treatment.

Disclosure of Invention

One of the purposes of the invention is to provide a thermosensitive liposome carrying hollow gold nanoparticles and a tumor therapeutic agent, which can be used as a good antitumor drug carrier, enables drugs to be efficiently released at tumor positions through NIR, combines the functions of thermal therapy and radiotherapy, and improves the treatment effect.

The second purpose of the invention is to provide a preparation method of the thermosensitive liposome preparation carrying the hollow gold nanoparticles and the tumor therapeutic agent together.

The invention also aims to provide the application of the thermosensitive liposome preparation carrying the hollow gold nanoparticles and the tumor therapeutic agent in tumor treatment.

The invention provides the following technical scheme

The thermosensitive liposome carrying the hollow gold nanoparticles and the tumor therapeutic agent is characterized in that the preparation is prepared by wrapping the hollow gold nanoparticles with the thermosensitive liposome through an extrusion method and then wrapping the tumor therapeutic agent, the phase transition temperature of the liposome is 39.0-45.0 ℃, and the liposome contains dipalmitoyl lecithin (DPPC).

Further, the thermosensitive liposome formulation is characterized in that: the weight ratio of the hollow gold nanoparticles to dipalmitoyl lecithin (DPPC) is 1-4: 60, the particle size of the liposome is 150-250nm, and the potential is-15 to-30 mV.

Further, the thermosensitive liposome preparation is characterized in that the particle size of the hollow gold nanoparticles is 30-60nm, and the maximum absorption wavelength is 650-850 nm.

Further, the thermosensitive liposome preparation, wherein the tumor therapeutic agent is selected from a small molecule chemotherapeutic drug, a bioprotein drug, or a gene therapy-related drug.

Further, the thermosensitive liposome preparation is characterized in that the small molecule chemotherapeutic drug is selected from paclitaxel, doxorubicin hydrochloride, mitoxantrone, 5-fluorouracil, cisplatin, daunorubicin, hormonal drugs or traditional Chinese medicine monomers; the biological protein drug is selected from rituximab, bevacizumab or trastuzumab; the gene medicine is selected from plasmid DNA, oligonucleotide or small interfering RNA containing reporter gene, anticancer gene and cytokine gene which can be recombined and expressed in eukaryotic cells.

Further, the thermosensitive liposome formulation is characterized in that: the prescription of the liposome preparation is as follows:

DPPC 60mg

DSPE-PEG2000 12mg

DOX.HCl 10mg

hollow gold nanoparticles 100mL

The preparation method comprises the following steps: weighing DPPC and DSPE-PEG2000 according to the prescription amount, adding an organic solvent for dissolving after uniformly mixing, placing on a rotary evaporator to remove the organic solvent for film formation, adding a concentrated hollow gold nanoparticle solution for hydration, and filtering for dozens of times through a 0.2 mu m filter membrane at 60 ℃ to prepare the thermosensitive liposome carrying the hollow gold nanoparticles; adding the adriamycin powder with the prescription amount, dissolving at 60 ℃, stirring, incubating for 4 hours, cooling, dialyzing to remove the unencapsulated adriamycin, and obtaining the thermosensitive liposome preparation containing the adriamycin and the hollow gold nanoparticles.

Further, the preparation method of the thermosensitive liposome preparation carrying the hollow gold nanoparticles and the tumor therapeutic agent comprises the following steps:

(1) adding 0.5-1mL of 0.05M sodium citrate solution and 100 mu L of 0.4M cobalt chloride solution into 100mL of ultrapure water, and protecting with nitrogen; adding 0.5-1mL of 0.1M sodium borohydride solution, stirring for 15-30min, adding 500 mu L of 1% chloroauric acid, introducing air, and stirring for 30min to obtain hollow gold nanoparticles;

(2) respectively weighing 30-60mg of DPPC and 6-15mg of DSPE-mPEG, dissolving in 5-10mL of chloroform, and performing rotary evaporation at 30-40 ℃ to remove the organic solvent to obtain a phospholipid membrane;

(3) centrifuging the hollow gold nanoparticles prepared in the step (1) at a high speed according to the prescription amount, removing supernatant, redispersing the precipitated hollow gold nanoparticles by using ultrapure water, hydrating the phospholipid membrane prepared in the step (2), and filtering the phospholipid membrane with the diameter of 0.2 mu m for ten times at the temperature of 50-60 ℃; adding tumor therapeutic agent, dissolving, stirring at 50-60 deg.C for 2-6h, cooling, dialyzing or centrifuging to remove the unencapsulated tumor therapeutic agent to obtain thermosensitive liposome preparation carrying hollow gold nanoparticles and tumor therapeutic agent.

Further, the invention provides application of the co-carried hollow gold nanoparticles and the tumor therapeutic agent thermosensitive liposome in preparation of an anti-tumor carrier. The administration route of the anti-tumor carrier is injection, oral administration or mucosa administration, and the anti-tumor carrier is applied to 'triple integrated' cooperative treatment of drug therapy of tumors and thermotherapy combined radiotherapy.

Furthermore, the invention provides an application of the thermosensitive liposome carrying the hollow gold nanoparticles and the tumor therapeutic agent together in 'triple integration' treatment, and the dosage of the thermosensitive liposome is 1-5w/cm after the preparation is given²The NIR of (3) irradiates the tumor part for 2-15min, and the process is repeated for 2-4 rounds; irradiating the tumor part with 2-12Gy power ray for 3-15 min.

Features of the invention

The invention has the advantages that the thermosensitive liposome is combined with the hollow gold nanoparticles with better photothermal conversion capability and radiotherapy sensitization effect, only no more than 20 percent of medicine is released at the normal body temperature of 37 ℃, the tumor part is irradiated by NIR, the hollow gold nanoparticles are subjected to photothermal conversion, the temperature of the liposome rapidly exceeds the phase transition point, the medicine is rapidly and completely released to kill tumor cells, and the purpose of thermotherapy is achieved by the heat production of the hollow gold nanoparticles. Meanwhile, the strong photoelectric absorption effect of the hollow gold nanoparticles on X-rays enhances the sensitivity of tumors to radiotherapy, realizes a triple-integration cooperative treatment strategy and improves the tumor treatment effect.

The thermosensitive liposome carrying the hollow gold nanoparticles prepared by the invention can release drugs at tumor parts through NIR irradiation to achieve a targeting effect, so that the toxic and side effects of drug treatment are reduced, and the treatment effect is improved through the further killing capacity of thermotherapy and radiotherapy, and the combined treatment mode is one of the main trends of future tumor treatment.

Drawings

FIG. 1 is a transmission electron microscope image of the hollow gold nanoparticles of the present invention.

FIG. 2 is a UV full-wavelength scanning diagram of the hollow gold nanoparticles of the present invention.

FIG. 3 is a temperature rise curve of photothermal conversion of several gold nanomaterials.

FIG. 4 is a transmission electron microscope image of the thermosensitive liposome carrying the hollow gold nanoparticles.

FIG. 5 is a particle size distribution diagram of the thermosensitive liposome with encapsulated hollow gold nanoparticles.

FIG. 6 is a photo-thermal temperature rise curve of the thermosensitive liposome carrying the hollow gold nanoparticles and the solid gold nanoparticles.

FIG. 7 is an in vitro release curve of the encapsulated hollow gold nanoparticle thermosensitive liposome at the temperatures of 37 ℃ and 42 ℃.

FIG. 8 shows the cell survival rates of the encapsulated hollow gold nanoparticle thermosensitive liposome, the encapsulated solid gold nanoparticle thermosensitive liposome, the encapsulated adriamycin thermosensitive liposome and free adriamycin in MCF-7 cells.

FIG. 9 shows the survival rates of MCF-7 cells after laser treatment of the encapsulated hollow gold nanoparticle thermosensitive liposome, the encapsulated solid gold nanoparticle thermosensitive liposome, the encapsulated adriamycin thermosensitive liposome and free adriamycin of the invention.

FIG. 10 shows the cell survival rate of the encapsulated solid gold nanoparticle thermosensitive liposome after X-ray irradiation.

Detailed Description

The following are examples of this patent, but the following examples do not limit the scope of the patent claims.

Example 1:

synthesis and characterization of hollow gold nanoparticles

Adding 1mL of 0.05M sodium citrate solution and 100 mu L of 0.4M cobalt chloride solution into 100mL of ultrapure water, and protecting with nitrogen; adding 1mL of 0.1M sodium borohydride solution, stirring for 15min, adding 200 mu L of 1% chloroauric acid, introducing air, stirring for 30min, centrifuging for 15min at 15000 rpm, and re-dispersing and precipitating with ultrapure water to obtain hollow gold nanoparticles; the product was examined using a transmission electron microscope and a full-wavelength ultraviolet scanner, and the results are shown in fig. 1 and 2.

Example 2:

comparison of photo-thermal conversion capability of several gold nano materials

Preparing solid gold nanoparticles, gold nanostars and hollow gold nanoparticles according to related literature methods, and performing centrifugal concentration to adjust the gold content of the solid gold nanoparticles, the gold nanostars and the hollow gold nanoparticles to be the same; respectively taking 1mL of the solution from the above solution, placing the solution in a quartz dish, and performing 808nm laser at 3w/cm²The irradiation time is 10min, the temperature is measured every 1min by an infrared thermometer, and the result is shown in figure 3 by using physiological saline as a control.

Example 3:

DPPC 60mg

DSPE-PEG2000 12mg

DOX.HCl 10mg

hollow gold nanoparticles 100mL

Weighing DPPC and DSPE-PEG2000 according to the prescription amount, adding an organic solvent for dissolving after uniformly mixing, placing on a rotary evaporator to remove the organic solvent for film formation, adding a concentrated hollow gold nanoparticle solution for hydration, and filtering for dozens of times through a 0.2 mu m filter membrane at 60 ℃ to prepare the thermosensitive liposome carrying the hollow gold nanoparticles; adding the adriamycin powder with the prescription amount, dissolving at 60 ℃, stirring, incubating for 4 hours, cooling, dialyzing to remove the unencapsulated adriamycin, and obtaining the thermosensitive liposome preparation containing the adriamycin and the hollow gold nanoparticles.

Example 4:

DPPC 100mg

MPPC 15mg

cisplatin 20mg

Hollow gold nanoparticles 150mL

Weighing DPPC and MPPC according to the prescription amount, adding an organic solvent for dissolving after uniformly mixing, placing on a rotary evaporator to remove the organic solvent for film formation, adding a concentrated hollow gold nanoparticle solution for hydration, and filtering through a 0.2 mu m filter membrane for dozens of times at 50 ℃ to prepare the thermosensitive liposome carrying the hollow gold nanoparticles; adding cisplatin powder with the prescription amount, dissolving at 50 ℃, stirring, incubating for 3 hours, cooling, dialyzing to remove the unencapsulated cisplatin, and obtaining the heat-sensitive liposome preparation containing the cisplatin and the hollow gold nanoparticles.

Example 5

DPPC 80mg

DSPE-PEG2000 15mg

Rituximab 10mg

Hollow gold nanoparticles 120mL

Weighing DPPC and DSPE-PEG2000 according to the prescription amount, adding an organic solvent for dissolving after uniformly mixing, placing on a rotary evaporator to remove the organic solvent for film formation, adding a PBS solution (0.5-2 mg/mL) of rituximab for hydration, adding concentrated hollow gold nanoparticles, and filtering through a 0.2 mu m filter membrane for dozens of times at 50 ℃ to prepare the thermosensitive liposome preparation carrying the rituximab and the hollow gold nanoparticles.

Example 6: detection of a transmission electron microscope and measurement of particle size distribution of the encapsulated hollow gold nanoparticle thermosensitive liposome: the morphology of the thermosensitive liposome was examined by JEM transmission electron microscope, and the results are shown in FIG. 4; the liposome size distribution was measured by Malvern laser granulometer, and the average liposome size was 200nm, and the particle size distribution is shown in FIG. 5.

Example 7: the photo-thermal conversion capability of the encapsulated hollow gold nanoparticles and the solid gold nanoparticle thermosensitive liposome is compared

Preparing the thermosensitive liposome encapsulating the hollow gold nanoparticles and the solid gold nanoparticles according to the process, and adjusting the concentration to ensure that the gold contents are the same; respectively taking 1mL of the solution from the above solution, placing the solution in a quartz dish, and performing 808nm laser at 3w/cm²The irradiation was carried out for 10min, and the temperature was measured every 1min with an infrared thermometer and PBS (7.4) as a control, and the results are shown in FIG. 6.

Example 8: evaluation of in vitro release of thermosensitive liposome co-loaded with hollow gold nanoparticles and adriamycin

Taking 2mL of the doxorubicin-loaded preparation in a dialysis bag (molecular weight cut-off =7000 Da), fastening both ends with cotton threads, placing the preparation in a 50mL iodine vial, adding 20mL of release medium (pH 7.4) respectively, placing the preparation in constant-temperature water bath oscillators at 37 ℃ and 42 ℃ respectively, rotating at 100r/min for 0.5h, 1h, 2h, 4h, 6h, 8h, 12h and 24h, sampling 1mL each time, supplementing 1mL of the constant-temperature release medium, detecting the sample in a fluorescence spectrophotometer, and calculating the drug release amount, wherein the result is shown in FIG. 7.

Example 9: evaluation of in vitro antitumor Activity of thermosensitive liposomes co-loaded with hollow gold nanoparticles and tumor therapeutic agent

(1) Single chemotherapy effect

MCF-7 cells were seeded at 5000 cells/well in 96-well plates and after 24h incubation, the formulations were diluted to different concentrations and added to 96-well plates with free doxorubicin solution as a control. After 48h incubation, 20 μ L of tetramethylazodicarbonyl blue (MTT, 5 mg/mL) was added into each well, incubation was continued for 4h, the liquid in the well was discarded, 150 μ L of DMSO was added, the crystals were sufficiently dissolved by shaking for 10min, the absorbance (ODsample) of each sample was measured at a wavelength of 570 nm with a microplate reader, the OD (ODcontrol) of the blank group was also measured, and the cell viability was calculated, the results are shown in FIG. 8.

(2) Chemotherapy combined with thermotherapy

MCF-7 cells were seeded at 5000 cells/well in 96-well plates and after 24h incubation, the formulations were diluted to different concentrations and added to 96-well plates with free doxorubicin solution as a control. After 6h incubation, the mixture is treated with 808nm laser at 2w/cm²Irradiating for 5min, treating for 4 times in 2h, continuing to incubate for 42h, adding 20 μ L of tetramethylazodicarbonazole blue (MTT, 5 mg/mL) into each well, continuing to incubate for 4h, discarding the liquid in the well, adding 150 μ L of DMSO, shaking for 10min to fully dissolve the crystals, measuring the absorbance (ODsample) of each sample by a microplate reader at a wavelength of 570 nm, measuring the OD (ODcontrol) of a blank group, and calculating the cell survival rate, wherein the result is shown in FIG. 9.

(3) Radiotherapy sensitization

MCF-7 cells were seeded at 5000 cells/well in 96-well plates and after 24h incubation, the therapeutic-free formulations were diluted to different concentrations (in terms of Au content) and added to the 96-well plates, with PBS solution as a control. After 6h incubation, irradiating with 6 Gay radiation for 5min, continuing to incubate for 42h, adding 20 μ L of tetramethylazodicarbonazole blue (MTT, 5 mg/mL) into each well, continuing to incubate for 4h, discarding the liquid in the well, adding 150 μ L of DMSO, shaking for 10min to fully dissolve the crystals, measuring the absorbance (ODsample) of each sample with a microplate reader at a wavelength of 570 nm, measuring the OD (ODcontrol) of a blank group, and calculating the cell survival rate, wherein the result is shown in FIG. 10.

Claims

1. A thermosensitive liposome preparation carrying hollow gold nanoparticles and a tumor therapeutic agent together is characterized in that the prescription of the thermosensitive liposome preparation is as follows:

DPPC 60mg

DSPE-PEG2000 12mg

DOX.HCl 10mg

hollow gold nanoparticles 100mL

The thermosensitive liposome preparation is mainly prepared by the following method:

(1) adding 0.5-1mL of 0.05M sodium citrate solution and 100 mu L of 0.4M cobalt chloride solution into 100mL of ultrapure water, and protecting with nitrogen; adding 0.5-1mL of 0.1M sodium borohydride solution, stirring for 15-30min, adding 200 mu L of 1% chloroauric acid, introducing air, and stirring for 30min to obtain hollow gold nanoparticles;

(2) respectively weighing 60mg of DPPC and DSPE-PEG 200012 mg, dissolving in 5-10mL of chloroform, and rotary evaporating at 30-40 deg.C to remove organic solvent to obtain phospholipid membrane;

(3) centrifuging the hollow gold nanoparticles prepared in the step (1) at a high speed according to the prescription amount, removing supernatant, redispersing the precipitated hollow gold nanoparticles by using ultrapure water, hydrating the phospholipid membrane prepared in the step (2), and filtering the phospholipid membrane with the diameter of 0.2 mu m for ten times at the temperature of 50-60 ℃; adding and dissolving the tumor therapeutic agent, stirring for 2-6h at 50-60 ℃, cooling, dialyzing or centrifuging to remove the unencapsulated tumor therapeutic agent to obtain the thermosensitive liposome preparation carrying the hollow gold nanoparticles and the tumor therapeutic agent together;

wherein the particle diameter of the liposome is 150-250nm, and the potential is-15 to-30 mV.

2. The thermosensitive liposome formulation according to claim 1, wherein the NIR irradiation power is 1-5w/cm in photothermal therapy²The irradiation time is 2-15 min; the radiation power is 2-12Gy and the irradiation time is 3-15 min.

3. The thermosensitive liposome preparation according to claim 1, wherein the hollow gold nanoparticles have a particle size of 30-60nm and a maximum absorption wavelength of 650-850 nm.

4. The use of the thermosensitive liposome preparation co-carrying hollow gold nanoparticles and a tumor therapeutic agent according to claim 1 in the preparation of an antitumor carrier.

5. The use of claim 4, wherein the anti-tumor carrier is administered by injection, oral administration or mucosa administration, and is used for triple integrated synergistic therapy of tumor drug therapy and thermotherapy combined with radiotherapy.