CN111374945A - Platelet-like liposome drug delivery system and preparation method and application thereof - Google Patents
Platelet-like liposome drug delivery system and preparation method and application thereof Download PDFInfo
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- CN111374945A CN111374945A CN201811610714.9A CN201811610714A CN111374945A CN 111374945 A CN111374945 A CN 111374945A CN 201811610714 A CN201811610714 A CN 201811610714A CN 111374945 A CN111374945 A CN 111374945A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Abstract
The invention belongs to the technical field of pharmaceutical preparations, relates to a targeted drug delivery system, and particularly relates to a platelet-like liposome drug delivery system, and a preparation method and application thereof. The drug delivery system is made by fusing a platelet membrane with a traditional liposome extruded membrane. The drug delivery system has the advantages of platelets in both natural targeting and long circulation of liposomes in vivo, and in vitro adhesion experiments prove that the platelet-like liposome has the adhesion capacity to collagen and fibrinogen similar to pure platelet vesicles; in vitro targeting experiments prove that the platelet-like liposome has good targeting property of the approximately pure platelet vesicles to tumor cells; pharmacokinetic experiments prove that the platelet-like liposome has longer in vivo circulation time close to that of the traditional liposome; in vivo targeting experiments prove that the platelet liposome has a targeting effect on residual tumor and metastatic tumor which is obviously higher than that of the traditional liposome and pure platelet vesicle.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, relates to a targeted drug delivery system, and particularly relates to a platelet-like liposome drug delivery system, and a preparation method and application thereof.
Background
The prior art discloses that the main treatment means for solid tumors in clinical practice is still surgical treatment, and practice proves that residual tumor tissues and circulating tumor cells caused by surgical excision are the key points for seriously influencing the prognosis of tumor treatment. Research shows that death caused by tumor recurrence and metastasis is a very serious challenge for tumor therapy, and therefore, it is of great clinical significance to develop a tumor therapy scheme aiming at postoperative residual tumor and circulating tumor cells to reduce or prevent tumor recurrence and metastasis.
Clinical practice shows that most of the clinical chemotherapeutic drugs do not achieve the ideal chemotherapeutic effect due to the non-specificity and poor bioavailability. In the last decades, the nano drug delivery system has been developed rapidly in terms of solving the problems of fast clearance in vivo, non-specific toxicity, etc. of small molecule drugs due to the characteristics of adjustable size, potential, surface modification, etc., wherein in recent years, the bionic nano drug delivery system represented by the membrane-coated nanoparticles of cell membranes has received much attention due to the functional diversity and many significant advantages endowed by natural cell membranes. Research shows that the blood platelet participates in physiological and pathological processes such as hemostasis, wound healing, inflammatory reaction, thrombosis and the like, and is easy to actively enrich in the wound part; recent studies reveal that platelets also participate in the metastatic process of tumors, playing an important role in identifying and combining Circulating Tumor Cells (CTCs), assisting CTCs in evading immune clearance, promoting CTCs to adhere to vascular endothelium and forming metastases; therefore, according to the natural tendency effect of the platelets on wound sites, the specific combination of CTC and the natural accumulation characteristics of the platelets on metastasis, the platelet-like liposome with platelet functions is designed, so that natural targeting on residual tumors, CTC and tumor metastasis can be realized, the targeted delivery of chemotherapeutic drugs to the residual tumors and the metastasis is expected to be increased, and the treatment effect of recurrence and metastasis of tumor after operation is further improved.
At present, in the existing research of utilizing platelets to carry out targeted delivery on tumors, platelet membrane-coated nanoparticles, namely PLGA nanoparticles and platelet vesicles coated by platelet membranes, show a tumor targeting effect, but have the defect of short in vivo circulation time, which seriously influences the exertion of the in vivo targeting effect of the platelets, so that the problem of prolonging the in vivo circulation time can be solved on the premise of keeping the platelet targeting function by constructing a platelet-like liposome drug delivery system with the in vivo long circulation capability.
Meanwhile, the traditional platelet membrane coated PLGA nanoparticles have the defect of low drug loading rate which can not meet the dosage requirement of clinical medication; the platelet-like liposome has the drug loading capacity of the traditional liposome, and taking adriamycin as an example, the drug loading capacity of the traditional liposome can reach 90 percent, and the drug loading capacity of PLGA does not exceed 10 percent.
In addition, the current situation of shortage of platelet resources exists in clinic, so that the platelet-like liposome drug delivery system can be constructed to greatly reduce the dosage of platelets on the premise of ensuring the functions of the platelets, save the cost of a preparation and be more beneficial to further clinical transformation of the platelet-like liposome drug delivery system.
Based on the current situation of the prior art, the inventor of the application intends to provide a novel targeted drug delivery system, and particularly relates to a platelet-like liposome drug delivery system and a preparation method and application thereof.
Disclosure of Invention
The invention aims to provide a novel targeted drug delivery system based on the current situation of the prior art, and particularly relates to a platelet-like liposome drug delivery system and a preparation method and application thereof.
In particular, the invention provides a platelet-like liposome drug delivery system;
the invention also provides a preparation method for constructing the platelet-like liposome drug delivery system;
the invention further provides application of the constructed platelet-like liposome drug delivery system.
The purpose of the invention is realized by the following technical scheme:
1) the platelet-imitated liposome drug delivery system is characterized by comprising three components of a platelet membrane, phospholipid and distearoyl phosphatidyl ethanolamine-polyethylene glycol DSPE-PEG, wherein the platelet membrane is an activated platelet membrane, the phospholipid is egg yolk lecithin, and the molecular weight of the DSPE-PEG is 2000; the drug delivery system is formed by fusing a platelet membrane and a traditional liposome, wherein the mass ratio of the platelet membrane to phospholipid is 1: 25-1: 50, and the mass ratio of the phospholipid to distearoyl phosphatidyl ethanolamine-polyethylene glycol is 10: 1; the particle size of the drug delivery system is 80-120 nm.
2) The preparation method of the platelet-like liposome drug delivery system is characterized by comprising the following specific steps of:
the method comprises the following steps: preparing traditional liposome by a thin film dispersion method;
step two: adding the activated platelet membrane into the hydration solution of the liposome, and uniformly mixing by ultrasonic;
step three: the platelet-like liposome is prepared by an extrusion and membrane-passing method.
3) The application of the platelet-like liposome drug delivery system is characterized in that the drug delivery system can utilize the natural targeting effect of platelets on wounds, circulating tumor cells and metastasis sites, and is used for targeted therapy of residual tumors after breast cancer operation and lung metastasis thereof.
The invention carries out experiments, and the results show that:
the platelet-like liposome delivery system of the present invention functions by:
1) the platelet-like liposome simulates the natural tendency action of platelets to wound sites, specific combination to circulating tumor cells and natural accumulation characteristic in metastatic foci through platelet membrane components in the preparation, thereby improving the targeting action of a drug delivery system to postoperative residual tumors and metastatic foci thereof.
2) The platelet-like liposome enables the drug delivery system to obtain the in vivo long circulation capacity similar to that of the traditional liposome through the distearoyl phosphatidyl ethanolamine-polyethylene glycol component in the preparation.
Compared with the prior art, the platelet-like liposome has the following beneficial effects:
1) the platelet-like liposome drug delivery system has a natural targeting function of platelets, and has a natural targeting effect on postoperative residual tumors and a metastasis stove thereof compared with the traditional liposome.
2) The platelet-like liposome drug delivery system has the in-vivo long circulation capacity of the traditional liposome, and compared with the existing platelet membrane-coated nanoparticle and platelet vesicle preparation, the drug delivery system has the advantages that the in-vivo circulation time is remarkably prolonged, and the platelet membrane targeting effect in the drug delivery system can be more efficiently exerted in vivo.
3) Compared with the PLGA nanoparticle preparation coated by the platelet membrane in the prior art, the platelet-like liposome drug delivery system has higher drug loading capacity and is closer to the dosage requirement of clinical medication.
4) Compared with the platelet membrane-coated nanoparticle and platelet vesicle preparations in the prior art, the platelet membrane-simulated liposome drug delivery system can greatly reduce the dosage of the platelet membrane, save the preparation cost, facilitate the expanded production and be beneficial to the clinical transformation of the platelet membrane-coated nanoparticle and platelet vesicle preparations.
Drawings
FIG. 1 is a schematic diagram of a preparation process of a platelet-like liposome.
FIG. 2 is an electron microscope representation of a platelet-mimicking liposome,
it is shown that the platelet-like liposomes PL are round in shape and uniform in size, close to the electron microscope morphology of LI, and have slightly larger PV size.
FIG. 3 particle size characterization of the platelet-like liposomes
It is shown that the particle size of the platelet-like liposomes PL is about 100 nm.
FIG. 4 potential characterization of the platelet-mimetic liposomes
It is shown that the potential of the platelet-like liposomes PL is about-32 mV, between the potentials of LI and PV, i.e., -22 to-37 mV.
FIG. 5 in vitro stability assay of the platelet-mimetic liposomes
It is shown that the particle size of the platelet-like liposome PL does not change much within one week, demonstrating its good in vitro stability.
FIG. 6 measurement of in vitro adhesion of platelet-like liposomes to collagen
It is shown that the adhesion of the platelet-like liposomes PL on collagen whiteboards is 20.22 times that of the conventional liposomes LI, and that the adhesion is approximately half of that of pure platelet vesicles PV.
FIG. 7 measurement of the in vitro adhesion of platelet-like liposomes to fibrinogen
It is shown that the amount of adhesion of the platelet-like liposomes PL to the fibrinogen plate is 11.4 times that of the conventional liposomes LI and exceeds half of that of the pure platelet vesicles PV.
FIG. 8 pharmacokinetic results of platelet-like liposomes
The half-life and area under the curve of the platelet-like liposome PL are shown to be significantly higher than that of the pure platelet vesicle PV, and the pharmacokinetic behavior in vivo of the platelet-like liposome PL is close to that of the traditional liposome LI.
FIG. 9 qualitative test of targeted binding of platelet-like liposomes to 4T1 cells
It is shown that the fluorescence intensity of the simulated platelet liposome PL group is similar to that of the pure platelet vesicle PV group and is significantly higher than that of the conventional liposome LI.
FIG. 10 Targeted binding quantification assay of platelet-mimetic liposomes to 4T1 cells
It is shown that 4T1 tumor cells bind platelet-like liposome PL in an amount 82% of pure platelet vesicles PV, 18.7 times that of conventional liposome LI.
FIG. 11 in vivo targeted bimodal qualitative validation of platelet-like liposomes against 4T1 breast carcinoma in situ residual tumor
It is shown that NIR signals of residual tumors in the platelet-mimicked liposome PL group are significantly higher than those of the conventional liposome LI group and the pure platelet vesicle PV group.
FIG. 12 in vivo targeted bimodal semi-quantitative validation of platelet-like liposomes against 4T1 breast carcinoma in situ residual tumor
It is shown that the NIR signal intensity of the residual tumors in the platelet-mimicked liposome PL group is 6.17 and 16.04 times that of the conventional liposome LI group and the pure platelet vesicle PV group, respectively.
FIG. 13 in vivo targeted in vivo bimodal qualitative validation of platelet-like liposomes against lung metastasis of 4T1 breast cancer
It is shown that NIR signals of lung tissues in the platelet-mimicked liposome PL group are significantly higher than those of the conventional liposome LI group and the pure platelet vesicle PV group.
FIG. 14 in vivo targeted in vivo bimodal semi-quantitative validation of platelet-mimetic liposomes on lung metastasis of 4T1 breast cancer
It is shown that the NIR signal intensity of lung tissue in the simulated platelet liposome PL group is 2.50 and 3.03 times higher than that of the conventional liposome LI group and the pure platelet vesicle PV group, respectively.
Detailed Description
For a better understanding of the present invention, reference will now be made to the following examples, which are to be considered in all respects to be illustrative and not restrictive.
EXAMPLE 1 preparation of the platelet-mimicking liposomes
1) Preparation of conventional liposomes: accurately weighing 4mg of yolk lecithin and 0.4mg of DSPE-PEG2000Dissolving in 10mL of dichloromethane, performing rotary evaporation under reduced pressure at normal temperature to remove dichloromethane, forming a film, and adding 1.6mL of distilled water for hydration after 30 min;
2) preparation of the platelet-like liposome: adding 0.4mL of activated human platelet membrane suspension into the hydration solution in the step 1), ultrasonically mixing the mixture for 1min, sequentially passing through polycarbonate membranes with the sizes of 800 microns, 400 microns, 200 microns and 100 microns to obtain the platelet-like liposome PL,
in addition, the liposome suspension obtained by the method in the step 1) is subjected to ultrasonic treatment and extrusion treatment under the same conditions in the step 2) to obtain the traditional liposome LI, and 1mL of the activated human platelet membrane suspension is subjected to ultrasonic treatment and extrusion treatment under the same conditions in the step 2) to obtain the pure platelet vesicles PV. The unactivated Platelet also served as a characterization control for this example;
the preparation process of the platelet-like liposome is schematically shown in figure 1.
Example 2 characterization of the platelet-mimicking liposomes
1) And (3) observing the surface morphology of the platelet-like liposome: LI, PL and PV in example 1 were diluted to 0.1mg/mL, stained with 2% phosphotungstic acid, and observed under a transmission electron microscope, and the experimental results are shown in FIG. 2;
2) particle size and potential investigation of the platelet-like liposome: LI, PL, PV and Platlet in example 1 were each diluted to 0.2mg/mL, and the particle size and surface charge were measured by a dynamic light scattering apparatus. The experimental results are shown in fig. 3 and 4;
3) in vitro stability study of the platelet-like liposomes: LI, PL and PV were allowed to stand at 0.2mg/mL for one week at 4 ℃ and the change in particle size was monitored by a dynamic light scattering instrument, and the results of the experiment are shown in FIG. 5.
Example 3 in vitro adhesion assay of platelets in the form of platelet-like liposomes
1) In vitro adhesion of the platelet-like liposome to collagen was verified: DiD-labeled LI, PL and PV samples were prepared as described in example 1, with the mass ratio of DiD to phospholipid being 1: 1000. 96-well plates were coated with 2mg/mL of human collagen IV and air dried overnight at 4 ℃. Then 100. mu.L of either LI, PL or PV labeled with DiD was added to each well, PBS was washed 3 times after 5min, 100. mu.L of DMSO was added to dissolve DiD, and the amount of each sample adhered to the collagen white plate was quantified by a microplate reader, wherein the excitation wavelength/emission wavelength was 643/665nm, respectively. The experimental results are shown in fig. 6;
2) in vitro adhesion of the platelet-like liposome to fibrinogen was verified: DiD-labeled LI, PL and PV samples were prepared as described in example 1, with the mass ratio of DiD to phospholipid being 1: 1000. 100 mu L2.0mg/mL fibrinogen and 10 mu L0.4M CaCl were added to a 96-well plate in this order 210 μ L of 0.1U/mL thrombin, incubated at 37 ℃ for 90 min. The dosing and treatment conditions were the same as 1) in example 3, and the results are shown in FIG. 7.
Example 4 pharmacokinetic study of the platelet-mimicking liposomes
15 BALB/c mice were randomized into 3 groups and received tail vein injections of 200 μ L of 2.0mg/mL DiD-labeled LI, PL or PV, respectively. Collecting medicated blood samples after 1min, 10min, 20min, 40min, 1h, 2h, 4h, 8h, 12h and 24h of administration, collecting buccal blood samples, measuring the drug concentration at each time point by a microplate reader, and drawing a concentration-time curve, wherein the excitation wavelength/emission wavelength is 643/665nm respectively, and the experimental result is shown in fig. 8.
Example 5
In-vitro targeting verification of platelet-simulated liposome on 4T1 breast cancer cells
DiO-labeled LI, PL and PV samples were prepared as described in example 1, with the mass ratio of DiO to phospholipid being 1: 1000.4 for T1 cells at 2 × 105The density of each well was plated on 12-well plates, and after 24h the medium was replaced with serum-free medicated medium, wherein the concentrations of LI, PL and PV labeled with DiO were 100. mu.g/mL. And after adding the drugs, incubating for 15min at 4 ℃ in the dark, washing for 3 times by PBS, and observing the binding condition of each sample to 4T1 cells under a fluorescence microscope after fixed dyeing. In parallel experiments under the same administration conditions, the cells were finally digested and the binding of each sample to 4T1 cells was quantified by flow cytometry, and the results are shown in fig. 9 and 10.
Example 6
In-vivo targeting verification of platelet-simulated liposome on 4T1 breast cancer in-situ residual tumor and lung metastasis thereof
1) In vivo targeting verification of 4T1 breast cancer residual tumor in situ by platelet-simulated liposome preparation of DiR-labeled LI, PL and PV samples in the manner described in example 1, wherein the mass ratio of DiR to phospholipid was 1: 1000.12 BALB/c mice were randomly divided into 3 groups and inoculated with 1 × 10 breast fat pad64T1-luc cells. The tumor to be in situ grows to 300mm390% of the tumor tissue was excised to obtain a post-operative residual tumor animal model. The tail vein was then injected with 200 μ L of 2mg/mL DiR-labeled LI, PL or PV, respectively. Injecting D-fluorescein sodium salt with a dose of 150mg/kg into the abdominal cavity of each mouse after 24h, killing and collecting main organs and residual tumors after 10min, and performing bioluminescence/near infrared fluorescence bimodal qualitative and semi-quantitative analysis, wherein the excitation wavelength/emission wavelength is 745/780nm respectively, and the experimental results are shown in fig. 11 and 12;
2) in-vivo targeting verification of 4T1 breast cancer lung metastasis by platelet-simulated liposome, wherein 12 BALB/c mice are randomly divided into 3 groups and respectively subjected to tail intravenous injection of 5 × 1054T1-luc cells are used for constructing an animal model of lung metastasis of breast cancer. The tail vein was then re-injected with 200 μ L of 2mg/mL DiR-labeled LI, PL or PV, respectively. Injecting D-fluorescein sodium salt with dosage of 150mg/kg into abdominal cavity of each mouse after 24h, killing and collecting main viscera after 10min, and performing bioluminescence/near infrared fluorescence bimodal qualitative and semi-quantitative analysis, wherein excitation wavelength/emission wavelength is 745/780 nm. The experimental results are shown in fig. 13 and 14.
Claims (4)
1. A platelet-like liposome drug delivery system is characterized in that the drug delivery system is composed of a platelet membrane, phospholipid, distearoylphosphatidylethanolamine-polyethylene glycol DSPE-PEG;
the platelet membrane is activated platelet membrane;
the phospholipid is egg yolk lecithin;
the molecular weight of the DSPE-PEG is 2000;
the mass ratio of the platelet membrane to the phospholipid is 1: 25-1: 50, and the mass ratio of the phospholipid to the distearoyl phosphatidyl ethanolamine-polyethylene glycol is 10: 1; the particle size of the drug delivery system is 80-120 nm.
2. A method of preparing the platelet-mimetic liposome delivery system of claim 1, comprising the steps of:
the method comprises the following steps: preparing traditional liposome by a thin film dispersion method;
step two: adding the activated platelet membrane into the hydration solution of the liposome, and uniformly mixing by ultrasonic;
step three: the platelet-like liposome is prepared by an extrusion and membrane-passing method.
3. Use of the platelet-mimetic liposome delivery system of claim 1 for the preparation of a targeted therapeutic for post-operative residual tumors of breast cancer and lung metastases thereof.
4. Use according to claim 3, characterized in that the delivery system utilizes the natural targeting of platelets to wounds and circulating tumor cells and metastases for the targeted treatment of residual tumors after breast cancer surgery and their lung metastases.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113769118A (en) * | 2021-06-28 | 2021-12-10 | 中山大学附属第三医院（中山大学肝脏病医院） | Platelet membrane bionic ultrasonic microbubble and preparation method and application thereof |
| CN113952461A (en) * | 2021-09-07 | 2022-01-21 | 浙江大学 | Neutrophil-imitating nano drug delivery system and preparation method and application thereof |
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| CN113769118A (en) * | 2021-06-28 | 2021-12-10 | 中山大学附属第三医院（中山大学肝脏病医院） | Platelet membrane bionic ultrasonic microbubble and preparation method and application thereof |
| CN113952461A (en) * | 2021-09-07 | 2022-01-21 | 浙江大学 | Neutrophil-imitating nano drug delivery system and preparation method and application thereof |
| CN113952461B (en) * | 2021-09-07 | 2024-03-22 | 浙江大学 | Neutrophil-imitating nano drug delivery system and preparation method and application thereof |
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Application publication date: 20200707 |