CN109568598B

CN109568598B - Placenta-targeted nanoparticles for drug abortion and preparation method and application thereof

Info

Publication number: CN109568598B
Application number: CN201710905200.5A
Authority: CN
Inventors: 范秀军; 谭仑波; 张保珍; 程国钢; 汪宝蓓; 肖天霞; 张键
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2017-09-28
Filing date: 2017-09-28
Publication date: 2021-07-27
Anticipated expiration: 2037-09-28
Also published as: CN109568598A

Abstract

The invention provides a placenta-targeted nanoparticle which can be used for drug abortion and comprises a hydrophobic inner core, a monolayer lipid molecular layer wrapping the hydrophobic inner core and a hydrophilic outer shell, wherein the hydrophobic inner core comprises a hydrophobic polymer and a target delivery object loaded by the hydrophobic polymer, and the target delivery object comprises an abortion drug; the hydrophilic shell is an amphiphilic macromolecular compound grafted by polypeptide of targeted placenta-like chondroitin sulfate A, the hydrophobic end of the amphiphilic macromolecular compound is inserted into the single-layer lipid molecular layer in a penetrating manner, the hydrophilic end of the amphiphilic macromolecular compound is connected with the polypeptide through an amido bond, the polypeptide is exposed out of the single-layer lipid molecular layer, and the amino acid sequence of the polypeptide is selected from one or more of the amino acid sequences shown in SEQ ID NO 1-SEQ ID NO 3. The invention also provides a preparation method of the placenta-targeted nano-particles and application of the placenta-targeted nano-particles in preparation of a medicament for abortion of mammals.

Description

Placenta-targeted nanoparticles for drug abortion and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to placenta-targeted nanoparticles for medical abortion and a preparation method and application thereof.

Background

The world health organization estimates that 7500 million accidental pregnancies occur worldwide each year, and approximately 2600 to 5300 million cases end up with induced abortion. On the premise of such high artificial abortion rate, a safer and more effective artificial abortion technology is urgently needed to be developed. The existing artificial abortion techniques are mainly divided into two categories: drug abortion and negative pressure uterine aspiration operative abortion. Both of these techniques have major drawbacks. Although the surgical abortion has high success rate and short time, the surgical abortion has high surgical pain and is accompanied with surgical complications; although the medical abortion has the advantages of simple method, less pain and the like, the medical abortion has the risks of incomplete abortion and abortion failure and side effects of medicines in a certain proportion. Compared with the operation abortion which has more artificial uncertain factors, the innovation and promotion space of the medicine abortion is wider.

Recent research on targeting nanoparticles based on nanotechnology has brought new opportunities for drug abortion. The nano material can convey therapeutic drugs to a target organ to the maximum extent, has little influence on non-target organs, thereby achieving the high-efficiency and low-toxicity therapeutic effect, has the advantages of sustained and controlled release, transmucosal property, transdermal property and the like, and has great significance for drug abortion. The drug-loaded nanoparticle surface is connected with a ligand, such as an antibody, a peptide chain and the like, which can be specifically combined with a target cell (a brain microvascular endothelial cell constituting a blood brain barrier), so that the nanoparticle delivers the nano-drug to a specific part through receptor-mediated endocytosis transport.

Therefore, the development of a novel nano abortion medicine with high abortion success rate and low side effect is expected to break through the bottleneck of the existing artificial abortion. However, no nanoparticles with high targeting on placenta are found at present.

Disclosure of Invention

In view of the above, the invention provides a placenta-targeted nanoparticle, which is used for overcoming the defects of high failure rate and incomplete abortion of the traditional Chinese medicine in the prior art, and simultaneously improving the abortion efficiency and reducing the side effects of the traditional Chinese medicine.

In a first aspect, the invention provides a polypeptide targeting placenta-like chondroitin sulfate A (pl-CSA), wherein the amino acid sequence of the polypeptide is selected from one or more of the amino acid sequences shown in SEQ ID NO:1-SEQ ID NO: 3.

Wherein the amino acid sequence shown in SEQ ID NO.1 is LKPSHEKKNDDNGKKLCKAC.

The amino acid sequence shown in SEQ ID NO.2 is EDVKDINFDTKEKFLAGCLIVSFHEGKC.

The amino acid sequence shown in SEQ ID NO.3 is GKKTQELKNIRTNSELLKEWIIAAFHEGKC.

The polypeptide can be modified on common drug carriers or gene carriers such as polymers (such as polyethyleneimine, chitosan and the like), liposomes, gold nanoparticles, silicon dioxide, serum albumin and the like, and is used for targeting tissues expressing pl-CSA (platelet-derived antigen-binding protein) such as placental cells.

In a second aspect, the present invention provides a placenta-targeted nanoparticle for use in drug abortion, comprising a hydrophobic inner core comprising the hydrophobic polymer and its loaded target delivery comprising an abortion drug, a monolayer lipid molecular layer coating the hydrophobic inner core, and a placenta-targeted hydrophilic outer shell; the hydrophilic shell is composed of a polypeptide grafted amphiphilic macromolecular compound targeting pl-CSA, the hydrophobic end of the amphiphilic macromolecular compound is inserted into the single-layer lipid molecular layer, the hydrophilic end of the amphiphilic macromolecular compound is connected with the polypeptide through an amido bond, the polypeptide is exposed outside the single-layer lipid molecular layer, and the amino acid sequence of the polypeptide is selected from one or more of the amino acid sequences shown in SEQ ID NO 1-SEQ ID NO 3.

In the invention, the polypeptide can be one sequence shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3, or a plurality of sequences shown as SEQ ID NO. 1-SEQ ID NO. 3.

Preferably, the diameter of the placenta-targeted nanoparticle is 80-150 nm. The particle size is measured using a transmission electron microscope.

Preferably, the mass ratio of the hydrophobic polymer to the monolayer lipid molecule to the amphiphilic macromolecular compound is 1: (0.04-0.2): (0.1-0.4). Under the mass ratio, a structure with regular appearance, good dispersibility and uniform particle size distribution can be formed among the components of the placenta-targeted nanoparticles, the placenta-targeted nanoparticles have stable structure and are not easy to dilute and dissolve by body fluid of a human body to disintegrate, and the placenta-targeted nanoparticles are beneficial to targeting to main cells (such as placenta trophoblasts) forming the placenta.

Preferably, the monolayer lipid molecules are selected from at least one of lecithin and cephalin (phosphatidylethanolamine), the lecithin being selected from one or more of soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin and phosphatidylcholine. Further preferably, the monolayer lipid molecules have a hydrophobic portion facing the hydrophobic core and a hydrophilic portion facing the outside of the nanoparticle.

Preferably, the mass ratio of the amphiphilic macromolecular compound to the polypeptide is 1 (1-4). Under the mass ratio, the grafting rate of the polypeptide to the amphiphilic macromolecular compound is higher.

As described in the present invention, the polypeptide-grafted amphiphilic macromolecular compound layer includes an amphiphilic macromolecular compound having a hydrophobic end and a hydrophilic end connected to the lipid end, and a polypeptide. In the present invention, the hydrophobic end of the amphiphilic macromolecular compound may assist the insertion of the amphiphilic macromolecular compound into the monolayer lipid molecular layer, and the hydrophilic end is grafted with the polypeptide and extends outside the nanoparticle.

Preferably, the amphiphilic macromolecular compound is polyethylene glycol-derivatized phospholipid, and the polyethylene glycol-derivatized phospholipid is obtained by connecting polyethylene glycol and derivatives thereof with phospholipid substances through covalent bonds. In this case, the phospholipid material is present at the hydrophobic end of the amphiphilic macromolecular compound, and the carboxyl-or amino-modified polyethylene glycol or a polyethylene glycol derivative having another active functional group is present at the hydrophilic end.

Wherein the molecular weight of the polyethylene glycol is preferably 200-20000. Specifically, the molecular weight of the polyethylene glycol molecule may be 200, 500, 1000, 2000, 5000, 7000, 10000, 15000 or 20000. The phospholipid may be an artificially synthesized or naturally occurring phospholipid, and the phospholipid may be, but is not limited to, distearoyl phosphatidyl ethanolamine (DSPE), distearoyl phosphatidyl glycerol (DSPG), or cholesterol.

Further preferably, the amphiphilic macromolecular compound is distearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxylic acid copolymer (DSPE-PEG-COOH, also called phospholipid-PEG-carboxyl), distearoyl phosphatidyl ethanolamine-polyethylene glycol-amino copolymer (DSPE-PEG-NH)₂Also known as phospholipid-PEG-amino) or distearoylphosphatidylethanolamine-polyethylene glycol-maleamide.

Preferably, the hydrophobic polymer is selected from one or more of poly (lactic-co-glycolic acid) (also called polyglycolide-lactide, abbreviated as PLGA), polylactic acid and polycaprolactone, but is not limited thereto.

Further preferably, the hydrophobic polymer is polylactic-co-glycolic acid (abbreviated as PLGA), and the molecular weight of the PLGA is 7000-17000. Wherein the copolymerization ratio of the monomer lactic acid to the glycolic acid is 50: 50.

In the present application, the target delivery substance (containing the abortive drug) and the hydrophobic polymer together constitute the hydrophobic inner core. The hydrophobic polymer can adsorb or wrap the target delivery substance to form a hydrophobic inner core, so that the loaded target delivery substance can be effectively prevented from gathering or leaking before reaching placental cells, and the stability of the loaded delivery substance is ensured.

Preferably, the target delivery further comprises one or more of a contrast agent and a fluorescent tracking agent.

Preferably, the mass ratio of the hydrophobic polymer to the target delivery substance is 1 (0.1-0.8). Further preferably 1 (0.1-0.6), more preferably 1 (0.1-0.2). Most preferably 1 (0.1-0.16).

Further preferably, the mass ratio of the abortive drug to the fluorescent tracking agent and/or the contrast agent in the target delivery is 1 (0.1-0.8).

Preferably, the abortive drug is selected from one or more of mifepristone, misoprostol, prostamethyl ester, sulprostone, tamoxifen, letrozole, and methotrexate, but is not limited thereto.

Preferably, the fluorescent tracer is selected from one or more of indocyanine green, evans blue, isothio blue, patent blue, methylene blue, coumarin 6, IR780 iodide (11-chloro-1, 1' -di-n-propyl-3, 3,3',3' -tetramethyl-10, 12-trimethyleneindole tricarbocyanine iodide) and DiR iodide, but is not limited thereto.

Preferably, the contrast agent is selected from one or more of iohexol, iopromide, lopiramide, iodophenyl ester and barium sulfate, but is not limited thereto.

The placenta-targeted nanoparticle provided by the second aspect of the invention has the advantages that the single-layer lipid molecules can be self-assembled into the single-layer lipid molecule layer in the preparation process and wrap the hydrophobic inner core, the hydrophobic end of the amphiphilic macromolecular compound is combined with the lipid molecules in the single-layer lipid molecule layer through physical action so as to be inserted into the single-layer lipid molecule layer, the polypeptide is covalently connected with the hydrophilic end of the amphiphilic macromolecular compound and extends outside the targeted nanoparticle, the amphiphilic macromolecular compound grafted by the polypeptide provides a hydrophilic outer layer and a receptor of the targeted placenta for the targeted nanoparticle, so that the placenta-targeted nanoparticle has good targeting property for placenta trophoblast cells, can well carry target delivery substances into the placenta trophoblast cells, and is helpful for ensuring the sufficient administration concentration of the targeted parts, the abortion efficiency is improved.

In a third aspect, the present invention provides a preparation method of placenta-targeted nanoparticles, comprising the following steps:

(1) dissolving hydrophobic polymer in organic solvent to obtain hydrophobic polymer solution;

(2) dissolving monolayer lipid molecules, amphiphilic macromolecular compounds and target delivery materials into a first solvent to obtain a first mixed solution, wherein the target delivery materials comprise abortion medicines;

(3) adding the hydrophobic polymer solution into the first mixed solution, carrying out ultrasonic treatment for 4-6min to obtain a second mixed solution, carrying out centrifugal treatment on the second mixed solution, and collecting supernatant to obtain a targeting nanoparticle precursor;

(4) taking the targeting nanoparticle precursor, and carrying out an amide reaction on the targeting nanoparticle precursor, a polypeptide of targeting placenta-like chondroitin sulfate A, a catalyst and a dehydrating agent in a second solvent to graft the polypeptide onto an amphiphilic macromolecular compound; collecting reaction liquid, and separating and purifying the reaction liquid to obtain the placenta-targeted nanoparticles. The resulting placenta-targeted nanoparticles are as described in the first aspect of the invention.

Preferably, in the step (1), the organic solvent includes one or more of acetonitrile, acetone, diethyl ether, chloroform, dichloromethane and n-hexane, but is not limited thereto. The organic solvent is preferably a volatile solvent capable of dissolving the hydrophobic polymer.

Wherein the first solvent comprises at least one hydrophilic solvent or a mixed solvent formed by water and at least one hydrophilic solvent. Wherein the hydrophilic solvent is selected from the group consisting of ethanol, methanol, 1-octanol, acetonitrile, acetone, Dimethylformamide (DMF) and Dimethylsulfoxide (DMSO), but is not limited thereto. The first solvent is required to dissolve the amphiphilic macromolecular compound, the monolayer lipid molecules and the target delivery.

Preferably, the first solvent is a mixed solvent of water and at least one hydrophilic solvent, such as ethanol aqueous solution with various concentrations and methanol aqueous solution with various concentrations. Further preferably, the volume fraction of water in the first solvent is 3 to 8%.

In one embodiment of the present invention, the first solvent is a 4% volume fraction ethanol aqueous solution or a 4% volume fraction methanol aqueous solution.

Wherein, the first solvent contains water, and can reduce the solubility of the hydrophobic polymer when being mixed with the organic solvent solution of the hydrophobic polymer at the later stage, thereby facilitating the later-stage ultrasonic and emulsification balling.

Preferably, in the first mixed solution, the concentration of the monolayer lipid molecules is 10-300 mug/mL, and the concentration of the amphiphilic macromolecular compound is 30-600 mug/mL.

Preferably, the concentration of the abortive drug in the first mixed solution is 33-750 μ g/mL.

Preferably, in the first mixed solution, the mass ratio of the abortive drug to the amphiphilic macromolecular compound is (1-5): 1. more preferably (1.9 to 3.5): 1 or (1-1.5): 1. more preferably (1-1.5): 1.

preferably, in step (1), the concentration of the solution of the hydrophobic polymer is 1 to 4 mg/ml.

Preferably, in the step (3), the volume ratio of the hydrophobic polymer solution to the first mixed solution is 1: 3.

preferably, in the step (3), the hydrophobic polymer solution is mixed with the first mixed solution in a dropwise manner, and further preferably, the dropwise addition rate of the hydrophobic polymer solution is 0.2 to 0.5 mL/min. Thus, the hydrophobic polymer can be fully complexed with the target delivery substance, and the target delivery substance is wrapped in the shell and matched with ultrasound to form the drug-loaded nano-particles.

Preferably, after the ultrasonication, the second mixed solution is gently stirred at 40 to 80 ℃. And (3) mild stirring is carried out, a proper solvent volatilization condition is provided, the interaction of the hydrophobic polymer, the target delivery substance, the monolayer lipid molecules and the amphipathic molecules is promoted, and the finally obtained nanoparticle precursor has good dispersibility and uniform particle size. Compared with the finally obtained placenta-targeted nanoparticles, the nanoparticle precursor is different only in that the surface of the nanoparticle precursor is not modified with placenta-targeted polypeptides.

Preferably, in the step (3), the centrifugal treatment is carried out for 2-5 times in an ultrafiltration centrifugal tube with the molecular weight cutoff of 5-10kDa, and washing is carried out by water.

Preferably, in the step (3), the centrifugation treatment is performed for 3-6min at a centrifugation rotation speed of 3000-.

Preferably, in the step (3), the sonication is performed with an ultrasonic cell disruptor at a frequency of 20kHz and a power of 80-160W.

In the step (3), the hydrophobic polymer, the target delivery substance, the monolayer lipid molecules and the amphiphilic macromolecular compound form the targeting nanoparticle precursor (i.e., the targeting-free nanoparticle) through a self-assembly process, no chemical reaction is needed, the preparation process is environment-friendly and nontoxic, and the method is simple and easy to operate.

Preferably, in step (4), the amide reaction is carried out at room temperature. Optionally, the time of the amide reaction is 15-24 h.

The second solvent may be water or other hydrophilic solvent. Preferably, in the step (4), the second solvent includes water, 2- (N-morpholine) ethanesulfonic acid buffer (abbreviated as "MES buffer solution") having a pH of 5.5 to 6.7, Phosphate (PBS) buffer having a pH of 7.0 to 7.9, and the like, but is not limited thereto.

In step (4), the method of the amidation reaction is well known to those skilled in the art. The catalyst, which may also be referred to as an activator, is often used in conjunction with a condensing agent for the amidation reaction.

Preferably, in the step (4), the condensing agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC).

Preferably, in the step (4), the catalyst comprises any one of N-hydroxysuccinimide (NHS) and N-hydroxythiosuccinimide sodium salt (sulfo-NHS).

Preferably, in the step (4), the mass ratio of the condensing agent, the catalyst and the amphiphilic macromolecular compound is (0.2-0.4): (0.05-0.3): 1.

more preferably, the mass ratio of EDC, NHS and DSPE-PEG-COOH is 1:0.4: 5.

Preferably, in the step (4), the separation and purification is performed by using an ultrafiltration centrifugal tube with molecular weight cut-off of 5-10kDa, and the supernatant obtained after centrifugation is collected to obtain the placenta-targeted nanoparticles. Preferably, the ultrafiltration centrifugation is performed 2-5 times. Water or PBS wash was used after each centrifugation except the last ultrafiltration centrifugation.

In another embodiment of the present invention, the polypeptide may be grafted to the amphiphilic macromolecular compound, and then the hydrophobic polymer, the target delivery substance, the polypeptide-grafted amphiphilic macromolecular compound, and the monolayer lipid molecule are processed according to the steps (1) to (3) to form the placenta-targeted nanoparticle.

The preparation method of the placenta-targeted nano-particles provided by the invention is simple and easy to implement and convenient to operate. The prepared placenta-targeted nanoparticles have strong targeting property and high enrichment degree on placenta trophoblast cells, high abortion success rate and lower side effect.

In a fourth aspect, the present invention provides a polypeptide for targeting the placenta according to the first aspect of the present invention or a placenta-targeting nanoparticle according to the second aspect of the present invention for use in the preparation of a medicament for abortion in a mammal. Preferably, the application in preparing the medicament for early pregnancy abortion of mammals.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising the placenta-targeted nanoparticles of the second aspect of the invention. The pharmaceutical composition is used for abortion in mammals.

Drawings

Fig. 1 is a schematic structural diagram of placenta-targeted nanoparticles prepared in example 1 of the present invention;

fig. 2 is the results of ultrasonography of abortion experiments in pregnant mice of placenta-targeted nanoparticles prepared in example 1 of the present invention and other experimental groups;

fig. 3 is a result of influence of placenta-targeted nanoparticles prepared in example 1 of the present invention and other experimental groups on the body weight of an embryo.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

In the present invention, the sequence of the polypeptide for targeting placenta-like chondroitin sulfate A (pl-CSA) is shown in SEQ ID NO:1-SEQ ID NO: 3. Specifically, LKPSHEKKNDDNGKKLCKAC is shown as SEQUENCE No. 1.

EDVKDINFDTKEKFLAGCLIVSFHEGKC is shown as SEQUENCE No. 2.

GKKTQELKNIRTNSELLKEWIIAAFHEGKC is shown as SEQUENCE No. 3.

The polypeptides are synthesized according to a conventional polypeptide synthesis process, wherein the leftmost end of each sequence is an N-terminal, the rightmost end is a C-terminal of the polypeptide, and the C-terminal or the N-terminal can be covalently linked with the amphiphilic polymer compound according to the properties of the amphiphilic polymer compound. Wherein, when the amphiphilic polymer compound has-COOH, the carboxyl group thereof can be utilized to perform an amide reaction with an amino group at the C-terminal of the polypeptide (i.e., an amino group at cysteine C). When the amphiphilic polymer compound has an amino group, the amino group on the amphiphilic polymer compound can be used for an amide reaction with a carboxyl group on the N-terminus of the polypeptide.

Example 1

A preparation method of placenta targeted nanoparticles comprises the following steps:

(1) dissolving polylactic acid-glycolic acid copolymer (PLGA, the molecular weight is 15000, and the copolymerization ratio of monomer lactic acid and glycolic acid is 50: 50) in acetonitrile to obtain an acetonitrile solution of PLGA, wherein the concentration is 2 mg/mL;

(2) dissolving 90 μ g of soybean lecithin, 210 μ g of DSPE-PEG-COOH (molecular weight of PEG is 2000), 315 μ g of methotrexate in 3mL of 4% ethanol aqueous solution by volume fraction to obtain a first mixed solution;

(3) dropwise adding 1mL of PLGA acetonitrile solution into 3mL of the first mixed solution at the speed of 0.3mL/min, and carrying out ultrasonic treatment by adopting an ultrasonic cell disruption instrument at the frequency of 20KHz and the power of 130W, wherein the ultrasonic time is 5 min;

carrying out ultrafiltration centrifugation on the solution after ultrasonic treatment in an ultrafiltration centrifugal tube with the molecular weight cutoff of 10kDa, washing with water, repeating for 4 times, wherein the centrifugal speed is 4000rpm, centrifuging for 4min each time, and collecting supernatant to obtain a target nanoparticle precursor;

(4) dissolving the targeting nanoparticle precursor in water, adding 42 mu g of EDC and 17 mu g of NHS for surface activation for 2h, then adding 0.5mg of polypeptide with the SEQUENCE of LKPSHEKKNDDNGKKLCKAC (shown as SEQUENCE NO. 1), and performing amidation reaction for 16h at room temperature to obtain a reaction solution;

and (3) carrying out ultrafiltration centrifugation on the reaction solution by using an ultrafiltration tube with the molecular weight cutoff of 10kDa, washing with water, repeating for 4 times, wherein the centrifugation speed is 3500rpm, centrifuging for 4min each time, and collecting supernatant to obtain the placenta-targeted nanoparticles.

Fig. 1 is a schematic structural diagram of placenta-targeted nanoparticles prepared in example 1 of the present invention. The placenta-targeted nanoparticle comprises a hydrophobic inner core 1, a single-layer lipid molecular layer 2 wrapping the hydrophobic inner core and a placenta-targeted hydrophilic outer shell 3, wherein the single-layer lipid molecular layer 2 is composed of soybean lecithin, the hydrophilic outer shell 3 is composed of polypeptide 32-grafted DSPE-PEG31, 11 is methotrexate, 12 is hydrophobic polymer PLGA, and 1 is a hydrophobic inner core composed of 11 and 12; in the polypeptide grafted DSPE-PEG, the lipid end DSPE of the DSPE-PEG31 is inserted into the soybean lecithin layer 22, the hydrophilic end PEG is connected with the polypeptide through an amido bond, and the polypeptide is exposed outside the single-layer lipid molecular layer.

The entrapment rate of methotrexate is calculated by the following formula: and EN% (1-Cf/Ct) × 100%, wherein Cf is the amount of free drug, and Ct is the total amount of drug, so that the EN% encapsulation rate of the targeted nanoparticles to methotrexate is 52.3 +/-4.4%. In addition, the linkage rate of the polypeptide measured by the BCA method was: 53.4 +/-3.5 percent.

Example 2

(1) dissolving polycaprolactone (molecular weight is 10000) in acetone to obtain polycaprolactone solution with the concentration of 1 mg/mL;

(2) mixing 40 μ g of egg yolk lecithin and 100 μ g of DSPE-PEG-NH₂(the molecular weight of PEG is 3000), 50 mu g of mifepristone and 50 mu g of misoprostol are dissolved in 3mL of methanol aqueous solution with the volume fraction of 4% to obtain a first mixed solution;

(3) dropwise adding 1mL of polycaprolactone solution into 3mL of the first mixed solution at the speed of 0.2mL/min, and carrying out ultrasonic treatment by adopting an ultrasonic cell disruption instrument at the frequency of 20KHz and the power of 80W for 6 min;

carrying out ultrafiltration centrifugation on the solution after ultrasonic treatment in an ultrafiltration centrifugal tube with molecular weight cutoff of 5kDa, washing with water, repeating for 4 times, wherein the centrifugation speed is 3000rpm, each time is 6min, and collecting supernatant to obtain a target nanoparticle precursor;

(4) dissolving the targeting nanoparticle precursor in water, adding 20 mu g of EDC and 5 mu g of NHS for surface activation for 2h, then adding 0.4mg of polypeptide with the SEQUENCE of LKPSHEKKNDDNGKKLCKAC (shown as SEQUENCE NO. 1), and performing amidation reaction for 15h at room temperature to obtain a reaction solution;

and (3) carrying out ultrafiltration centrifugation on the reaction solution by using an ultrafiltration tube with the molecular weight cutoff of 5kDa, washing with water, repeating for 4 times, wherein the centrifugation speed is 3000rpm, each time is 6min, and collecting supernatant to obtain the placenta-targeted nanoparticles.

Example 3

(1) dissolving polylactic acid (with a molecular weight of 21800) in DMF to obtain a polylactic acid solution with a concentration of 4 mg/mL;

(2) dissolving 800 μ g of cephalin, 1600 μ g of DSPE-PEG-NH2 (molecular weight of PEG is 2000), 2250 μ g of letrozole in 3mL of acetone to obtain a first mixed solution;

(3) dropwise adding 1mL of polylactic acid solution into 3mL of the first mixed solution at the speed of 0.4mL/min, and carrying out ultrasonic treatment by adopting an ultrasonic cell disruption instrument at the frequency of 20KHz and the power of 160W, wherein the ultrasonic time is 4 min;

carrying out ultrafiltration centrifugation on the solution after ultrasonic treatment in an ultrafiltration centrifugal tube with the molecular weight cutoff of 10kDa, washing with water, repeating for 4 times, wherein the centrifugation speed is 5000rpm, each time is 3min, and collecting supernatant to obtain a target nanoparticle precursor;

(4) dissolving the targeting nanoparticle precursor in water, adding 640 mu g of EDC and 80 mu g of NHS for surface activation for 4h, then adding 1.6mg of polypeptide with the SEQUENCE of EDVKDINFDTKEKFLAGCLIVSFHEGKC (shown as SEQUENCE NO. 2), and performing amidation reaction for 18h at room temperature to obtain a reaction solution;

and (3) carrying out ultrafiltration centrifugation on the reaction solution by using an ultrafiltration tube with the molecular weight cutoff of 10kDa, washing with water, repeating for 4 times, wherein the centrifugation speed is 5000rpm, each time is 3min, and collecting supernatant to obtain the placenta-targeted nanoparticles.

The TEM particle size of the placenta-targeted nanoparticle prepared by the embodiment is 100-120 nm; the linkage rate of the polypeptide measured by the BCA method is as follows: 55.4 +/-2.5 percent.

Example 4

(1) dissolving polylactic acid-glycolic acid copolymer (PLGA, molecular weight is 10000, copolymerization ratio of monomer lactic acid and glycolic acid is 50: 50) in acetonitrile to obtain an acetonitrile solution of PLGA, wherein the concentration is 2 mg/mL;

(2) mixing 120 μ g of phosphatidyl choline, 250 μ g of DSPE-PEG-NH₂(molecular weight of PEG is 3000), 300. mu.g of prostacyclin is dissolved in 3mL of methanol to obtain a first mixed solution;

(3) 1mL of PLGA acetonitrile solution is added into 3mL of the first mixed solution drop by drop at the speed of 0.5mL/min, and ultrasonic treatment is carried out by an ultrasonic cell disruptor at the frequency of 20KHz and the power of 120W, and the ultrasonic time is 5 min;

carrying out ultrafiltration centrifugation on the solution after ultrasonic treatment in an ultrafiltration centrifugal tube with molecular weight cutoff of 5kDa, washing with water, repeating for 4 times, wherein the centrifugation speed is 3500rpm, centrifuging for 4min each time, and collecting supernatant to obtain a target nanoparticle precursor;

(4) dissolving the targeting nanoparticle precursor in water, adding 75 mu g of EDC and 50 mu g of NHS for surface activation for 3h, then adding 0.75mg of polypeptide with the SEQUENCE of GKKTQELKNIRTNSELLKEWIIAAFHEGKC (shown as SEQUENCE NO. 3), and performing amidation reaction for 17h at room temperature to obtain a reaction solution;

and (3) carrying out ultrafiltration centrifugation on the reaction solution by using an ultrafiltration tube with the molecular weight cutoff of 5kDa, washing with water, repeating for 4 times, wherein the centrifugation speed is 3000rpm, each time is 3min, and collecting supernatant to obtain the placenta-targeted nanoparticles.

The TEM particle size of the placenta-targeted nanoparticle prepared by the embodiment is 90-130 nm; the linkage rate of the polypeptide was 54% as determined by BCA method.

Application examples

The effect test of pregnant mouse abortion was performed on the methotrexate-coated placenta-targeted nanoparticles (CSA-MNPs for short) prepared in example 1 of the present invention, and PBS group, free methotrexate group, common nanoparticle group, and disordered polypeptide group were used as controls, wherein the sequence of the polypeptide modified by SCR-MNPs in disordered polypeptide group was: PNNKCESDKLAKHKKLGDKC (shown as SEQUENCE No. 4), the polypeptide has no targeting effect on placenta. The specific operation is as follows:

female CD-1 mice with the age of 6 weeks and the weight of 15-20g are adopted as test animals, the female mice and the male mice are combined in a ratio of 1:2, the female mice are examined for vaginal embolus the next day, and the day of vaginal embolus is 0.5 day of pregnancy. Starting from 5.5 days of gestation in pregnant mice, every other day, pregnant mice were injected with different drugs via tail vein (equivalent of methotrexate is 1 μ g/g body weight) and divided into the following groups: PBS group, Free methotrexate group (Free DOX), general nanoparticle group (MNPs, unmodified placenta-targeted polypeptide, i.e., nanoparticle precursor in the present invention), disordered polypeptide group (SCR-MNPs), placenta-targeted nanoparticles encapsulating methotrexate (CSA-MNPs). Meanwhile, the size and growth of the embryo are observed and recorded by using a vevo2100 ultra-high resolution small animal ultrasonic real-time molecular imaging system every day, and the test result of ultrasonic development is shown in figure 2. Meanwhile, the embryos were taken out every day, the fetal weights were weighed and recorded, and the experimental results are shown in fig. 3.

In fig. 2, the last 2 figures of each row in the ultrasonic imaging results of day 10 and day 12 are the experimental results of CSA-MNPs, which show two different states of the pregnant mouse embryo after drug administration, the last 2 figure shows the pregnant mouse embryo development retardation, and the last 1 figure shows that the pregnant mouse embryo is nearly dead. As can be seen from FIG. 3, the CSA-MNPs group can see that the embryo has a large abortion (miscarriage rate is 80%) on the 14 th day of pregnancy, and the embryo development condition is slower than that of the PBS group between the 9 th day and the 12 th day of pregnancy, which indicates that the placenta-targeted nano-drug particles have already produced obstruction to the embryo growth and further prevent pregnancy, the Free DOX group can also see abortion, but the abortion quantity is not much on the 14 th day of pregnancy, and the MNPs group and the SCR-MNPs group have no abortion, and the fetus growth condition is better.

As can be seen from fig. 3, the weight of the fetus did not increase with the increase of the pregnancy time in the CSA group, i.e., the fetus died, compared to the other groups.

The test results show that the placenta-targeted nanoparticles provided by the invention can obviously inhibit pregnancy and can achieve a good abortion effect.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute of Chinese academy of sciences

<120> placenta targeted nano-particles for drug abortion and preparation method and application thereof

<130> 2017

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> PRT

<213> Artificial sequence

<400> 1

Leu Lys Pro Ser His Glu Lys Lys Asn Asp Asp Asn Gly Lys Lys Leu

1 5 10 15

Cys Lys Ala Cys

20

<210> 2

<211> 28

<212> PRT

<213> Artificial sequence

<400> 2

Glu Asp Val Lys Asp Ile Asn Phe Asp Thr Lys Glu Lys Phe Leu Ala

1 5 10 15

Gly Cys Leu Ile Val Ser Phe His Glu Gly Lys Cys

20 25

<210> 3

<211> 30

<212> PRT

<213> Artificial sequence

<400> 3

Gly Lys Lys Thr Gln Glu Leu Lys Asn Ile Arg Thr Asn Ser Glu Leu

1 5 10 15

Leu Lys Glu Trp Ile Ile Ala Ala Phe His Glu Gly Lys Cys

20 25 30

<210> 4

<211> 20

<212> PRT

<213> Artificial sequence

<400> 4

Pro Asn Asn Lys Cys Glu Ser Asp Lys Leu Ala Lys His Lys Lys Leu

1 5 10 15

Gly Asp Lys Cys

20

Claims

1. A placenta-targeted nanoparticle for drug abortion, comprising a hydrophobic inner core comprising the hydrophobic polymer and its loaded target delivery comprising an abortive drug, a monolayer lipid molecular layer encapsulating the hydrophobic inner core, and a placenta-targeted hydrophilic outer shell; the hydrophilic shell is composed of an amphiphilic macromolecular compound grafted by a polypeptide targeting placenta-like chondroitin sulfate A, wherein the hydrophobic end of the amphiphilic macromolecular compound is inserted into the single-layer lipid molecular layer in a penetrating way, the hydrophilic end of the amphiphilic macromolecular compound is connected with the polypeptide through an amido bond, the polypeptide is exposed outside the single-layer lipid molecular layer, the amino acid sequence of the polypeptide is shown as SEQ ID NO:1, the amphiphilic macromolecular compound is polyethylene glycol-derivatized phospholipid, and the single-layer lipid molecule is selected from at least one of lecithin and cephalin; the mass ratio of the hydrophobic polymer to the monolayer lipid molecule to the amphiphilic macromolecular compound is 1: (0.04-0.2): (0.1-0.4); the mass ratio of the amphiphilic macromolecular compound to the polypeptide is 1 (1-4).

2. The placenta-targeted nanoparticle of claim 1, wherein the abortive drug is selected from one or more of mifepristone, misoprostol, prostamethyl ester, sulprostone, tamoxifen, letrozole, and methotrexate.

3. The placenta-targeted nanoparticle of claim 1, wherein the mass ratio of the abortive drug to the amphiphilic macromolecular compound is (1-5): 1.

4. the placenta-targeted nanoparticle of claim 1, wherein the target delivery substance further comprises one or more of a contrast agent and a fluorescent tracer; in the target delivery object, the mass ratio of the abortion medicine to the fluorescence tracking agent and/or the contrast agent is 1 (0.1-0.8).

5. The placenta-targeted nanoparticle of any one of claims 1-4, wherein the mass ratio of the hydrophobic polymer to the target delivery substance is 1 (0.1-0.6).

6. A preparation method of placenta targeted nanoparticles is characterized by comprising the following steps:

(4) taking the targeting nanoparticle precursor, and carrying out an amide reaction on the targeting nanoparticle precursor, a polypeptide of targeting placenta-like chondroitin sulfate A, a catalyst and a dehydrating agent in a second solvent to graft the polypeptide onto an amphiphilic macromolecular compound; collecting reaction liquid, and separating and purifying the reaction liquid to obtain the placenta-targeted nanoparticles;

the placenta-targeted nanoparticle comprises a hydrophobic inner core, a single lipid molecular layer wrapping the hydrophobic inner core and a placenta-targeted hydrophilic outer shell, the hydrophobic inner core comprises the hydrophobic polymer and target delivery materials loaded by the hydrophobic polymer, the target delivery materials comprise abortion medicines, the hydrophilic shell is composed of placenta-like chondroitin sulfate A polypeptide grafted amphiphilic macromolecular compound, the hydrophobic end of the amphiphilic macromolecular compound is inserted into the single-layer lipid molecular layer in a penetrating way, the hydrophilic end of the amphiphilic macromolecular compound is connected with the polypeptide through amido bond, the polypeptide is exposed out of the single-layer lipid molecular layer, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO:1, the amphiphilic macromolecular compound is polyethylene glycol-derivatized phospholipid, and the monolayer lipid molecules are selected from at least one of lecithin and cephalin; the mass ratio of the hydrophobic polymer to the monolayer lipid molecule to the amphiphilic macromolecular compound is 1: (0.04-0.2): (0.1-0.4); the mass ratio of the amphiphilic macromolecular compound to the polypeptide is 1 (1-4).

7. The production method according to claim 6, wherein the mass ratio of the abortive drug to the amphiphilic macromolecular compound in the first mixed solution is (1-5): 1.

8. use of the placenta-targeted nanoparticles according to any one of claims 1 to 5 or prepared by the preparation method according to claims 6 to 7 for the preparation of a medicament for abortion in a mammal.

9. A pharmaceutical composition comprising the placenta-targeted nanoparticles of any one of claims 1-5.