CN114404367B - Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof - Google Patents

Abstract

The invention discloses a nano-carrier distributed to lesion cells in a targeted manner, a preparation method and application thereof, wherein the nano-carrier comprises a shell and a core, and the shell is an enzyme substrate polypeptide-PEG modified lipid bilayer membrane disintegrated in a targeted manner under the action of an enzyme highly expressed by contacting interstitial fluid of placenta tissues; the inner core is a drug carrier modified by a marker antibody with high placenta trophoblast surface specificity expression; the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester; the drug carrier is loaded with superparamagnetic ferroferric oxide SPIO nano particles, micromolecule drugs for regulating and controlling the function of placenta trophoblasts, therapeutic genes or a combination thereof. The nano-carrier can effectively avoid the nonspecific drug absorption of other organs outside the maternal placenta and fetuses, thereby realizing the delivery and function regulation of the specific drug of the trophoblasts in the placenta.

Description

A nanocarrier that distributes by targeting diseased cells and its preparation method and application

technical field

The invention relates to the fields of chemistry and biomedical engineering, in particular to a nanometer carrier distributed through targeted distribution of diseased cells, a preparation method and application thereof.

Background technique

Placenta accreta is a group of diseases in which the placental villi invade the myometrium to varying degrees and is the main cause of perinatal hemorrhage, emergency hysterectomy and maternal death. The average blood loss in placenta accreta surgery is 3630±2216 ml, and it is prone to complications such as uterine rupture, neonatal death, infection, and ureteral and bladder injuries caused by difficult surgical management, resulting in fistula formation, all of which seriously threaten pregnant women and the life and health of newborns, the mortality rate of such mothers can reach 7-10%. Placenta accreta is related to common factors such as multiple induced abortions and curettage history, scarred uterine pregnancy after cesarean section, advanced age, placenta previa, and previous placental remnant. The number of patients is increasing day by day.

Although there are imaging methods such as MRI that can predict placenta accreta to a certain extent, there is still no reliable means to treat the disease mechanism and achieve placenta accreta in clinical practice. Even with early diagnosis, treatment cannot be achieved. From the perspective of pathological mechanism, placenta accreta is a pathological state characterized by the loss of part or all of the decidua basal layer and the abnormal invasion of part or even the whole placental villi into the myometrium. This abnormally invasive state causes the placenta to adhere to the uterine wall. Therefore, the origin of the disease is the invasion of placental trophoblast cells and the excessive activation of EMT function, which leads to the disorder of placental function. Only by regulating the placental function according to this mechanism can we hope to improve this important clinical problem.

Placental dysfunction is the root cause of many serious diseases in pregnant women, including placenta accreta. Most of this disorder is due to the most important cell in the placenta, placental trophoblast cell (trophoblast cell, TB) dysfunction. For example, excessive activation of EMT function and pro-angiogenic function of TB can trigger placenta accreta. It has been confirmed in previous studies that sulfasalazine is a compound that can precisely inhibit the NF-κB pathway in trophoblast cells [Gastroenterology.2000 Nov; 119(5):1209-18.] Inhibition of the -κB pathway can inhibit the EMT function of trophoblasts, thereby reducing the invasive ability of trophoblasts and improving placental function [Placenta.2010Nov; 31(11):997-1002]. CORO6 (Coronin 6) is an effective activation gene of the wnt pathway, which is closely related to cell ubiquitination [Front Cell Dev Biol.2021 Jun 22; 9:684643.], and its high expression can promote invasion and metastasis. wnt pathway activity [Front Cell Dev Biol. 2021 May 7;9:647301.d]. If siRNA technology can be used to achieve exact inhibition of CORO6 expression in trophoblast cells, the inhibition of EMT and invasion can be achieved through the inhibition of the wnt pathway. Studies have shown that if the inhibition of NF-κB and wnt pathways can be simultaneously achieved in EMT regulation, the EMT function of cells will be reliably downregulated [Food Chem Toxicol.2019 Feb; 124:219-230.]. Therefore, we expect to be able to realize the effective delivery of the above trophoblast function regulating drugs to trophoblasts in vivo.

However, at present, there is a lack of drug carrier means that can deliver drugs into the TB in the placenta, regulate its function, and then realize the treatment of related diseases. There are both maternal and fetal pathophysiological barriers to the development of drugs that modulate TB function. The use of drugs in pregnant women and the development of new drugs need to consider the distribution of drugs in both the mother and the fetus itself, as well as the toxicity of the two aspects. The vast majority of drugs can pass through the placenta, distribute into the fetal side, and affect fetal development. Therefore, there are many contraindications to medication for pregnant women, including emergency medicines. Medications for pregnant women are divided into 5 categories according to their teratogenic properties. Except for a very small number of drugs with the least toxicity that fall into categories a and b, most of the drugs in categories c, d and e have obvious damage to the fetus. During pregnancy, pregnant women have a heavy metabolic burden and complex immune changes. Therefore, even if the drug has no obvious toxicity during non-pregnancy, it may have significant side effects in pregnant women. Therefore, the current drugs that may be effective against TB in vitro experiments cannot achieve the regulation of TB function under the condition of ensuring the safety of the mother and fetus.

Small-molecule drugs or gene therapy drugs screened in previous in vitro experiments that may have regulatory effects on TB function are injected or taken orally. After the placenta, due to the rich blood supply to the fetal side, the drug quickly crosses the placental barrier and causes damage to the fetus. Therefore, none of these drugs can be used clinically. Therefore, clinically, there is still no definite drug intervention method for the aforementioned diseases such as placenta accreta. Doctors can only carry out passive symptomatic treatment for the symptoms caused by placental dysfunction diseases. Instead of adjusting the function of TB, the real recovery of placental function can be realized. Therefore, how to avoid the toxicity to the mother and fetus and realize the effective delivery of TB function-regulating drugs is the key to solving the disease caused by TB dysfunction.

The current polymer nano-carrier drugs may achieve specific drug delivery to diseased target cells in a variety of diseases. However, these vectors cannot solve the problem of possible toxic and side effects on maternal and fetal non-specific distribution. Therefore, there are great difficulties in the development of TB-specific delivery vectors. At present, researchers have tried two methods that may promote the delivery of TB-specific nano-drugs. One is to increase the particle size of nano-drugs so that they cannot pass through the fetal membrane barrier and remain in the placenta to produce drug delivery effects; It is aimed at the specific delivery of antibody-modified nanocarriers for TB cell membrane markers.

The principle of increasing the particle size of nano-drugs to promote drug distribution in the placenta is that experimental studies have found that nano-drugs <300nm cannot stay in the placenta and can easily enter the fetus through the placenta. Therefore, researchers try to synthesize nano-drugs with a particle size > 300nm, make them stay in the placenta, and regulate the functions of various cells including placental TB. However, too large drug particle size (>100nm) is not conducive to the distribution of the drug in vivo. Most of these nanomedicines >300nm are captured by the reticuloendothelial system in the maternal circulation, causing side effects throughout the body, and can reach the placenta, and the proportion of TB-specific distribution is also low. Therefore, other approaches are needed to achieve the retention of nanomedicine in the placenta and the targeting of TB cells.

Nano-drugs can use nano-drug-linked antibodies to target and recognize cell membrane markers of target cells to achieve specific delivery to target cells. TB has some definite surface markers (such as placental growth factor, Placental Growth Factor, PLGF for short) that distinguish it from other placental stromal cells in placental tissue, which can be used to distinguish it from other cells in the placenta. However, analysis of expression in multiple organs and tissues throughout the body found that this surface marker was also expressed in some cells in other parts of the placenta. The expression abundance on the surface of a small number of high-expressing cells was not significantly different from that of TB. If the antibody of PLGF is connected on the surface of the nanometer drug carrier and directly applied in vivo, it will cause side effects on other cells expressing the marker PLGF in the body. Therefore, only by shielding the TB cell recognition antibody of the nanocarrier before entering the placenta in the blood circulation, can it avoid its distribution in the cells outside the placenta and ensure its distribution in the TB cells in the placenta.

In summary, there is currently a lack of nanocarrier systems that can effectively avoid non-specific drug absorption by the mother and fetus, and then achieve specific drug delivery and functional regulation of TB cells in the placenta.

Contents of the invention

In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a nanocarrier that distributes diseased cells through targeted distribution. The nanocarrier utilizes the placental microenvironment to reduce the distribution of drugs in maternal organs and tissues before entering the placenta. Using trophoblast membrane markers to target and reduce the distribution of drugs in fetal organs and tissues after passing through the placenta can effectively avoid non-specific drug absorption in other organs outside the placenta of the mother and the fetus, and then realize the delivery and functional regulation of trophoblast-specific drugs in the placenta.

Another object of the present invention is to provide the above-mentioned preparation method of nanocarriers distributed through targeted distribution of diseased cells.

The present invention is achieved through the following technical solutions:

A nano-carrier distributed through targeted distribution of diseased cells, the nano-carrier includes an outer shell and an inner core, and the outer shell is an enzyme substrate polypeptide-PEG that is targeted for disintegration under the action of an enzyme highly expressed in the interstitial fluid of the placenta Modified lipid bimolecular membrane; the enzyme highly expressed in the interstitial fluid of the placenta is one or more of matrix metalloproteinase 9, lysozyme, kininase, histamine enzyme or oxytocinase;

The inner core is a drug carrier modified with marker antibodies specifically and highly expressed on the surface of placental trophoblast cells; the drug carrier is a copolymer formed of polyethylene glycol-modified polycation carrier and hydrophobic degradable polyester; The marker antibody specifically highly expressed on the surface of placental trophoblast cells is the Fab segment of the placental growth factor PLGF antibody;

The drug carrier is loaded with superparamagnetic iron ferric oxide SPIO nanoparticles, small molecule drugs that regulate the function of placental trophoblast cells, therapeutic genes or a combination thereof.

The placenta of pregnant women is rich in a variety of enzymes that promote placental development and fetal nutrition. The enzymes highly expressed in the placental interstitial fluid of the present invention are matrix metalloproteinase 9 (Matrix Metallopeptidase 9, MMP-9), lysozyme, and kininase , histamine, oxytocinase or one or more of the matrix metalloproteinase family, wherein matrix metalloproteinase 9 is highly expressed in placental tissue fluid, and is almost not expressed in normal human blood and tissue fluid. Therefore, matrix metalloproteinase 9 is preferably expressed Protease 9.

The matrix metalloproteinase 9 substrate polypeptide can be DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(N-Me-Abz)-NH2, molecular weight: 1077.22Da.

The drug carrier of the present invention is a copolymer formed of polyethylene glycol-modified polycation carrier and hydrophobic degradable polyester, and the copolymer is polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI- One or more of PCL, polyethylene glycol-polyethyleneimine-polylactic acid PEG-PEI-PLA or polyethylene glycol-polyethyleneimine-polylactic acid-glycolic acid PEG-PEI-PLGA, preferably poly Ethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL.

The copolymer described in the present invention can be synthesized by the prior art, such as firstly reacting PEG with a polycation carrier to form a copolymer, and then using the active group of the polycation to react with the activated polyester segment to form a copolymer.

The copolymers described in the present invention are also commercially available.

The drug carrier of the present invention is loaded with superparamagnetic iron ferric oxide SPIO nanoparticles, small molecule drugs regulating the function of placental trophoblast cells, therapeutic genes or combinations thereof. The small molecule drug is sulfasalazine, and the therapeutic gene is siRNA inhibiting the expression of CORO6 (Coronin 6) gene.

The average particle size of the nanocarrier of the present invention is 80nm-300nm, preferably 100nm-210nm. Too large particle size is not conducive to circulation in the body, and too small particle size increases the difficulty of preparation and is not conducive to loading drugs and genes.

The present invention also provides a method for preparing the above-mentioned nanocarriers distributed through targeted distribution of diseased cells, comprising the following steps:

S1. Load superparamagnetic iron ferric oxide SPIO nanoparticles, small molecule drugs and/or genes that regulate the function of placental trophoblast cells to the copolymer to obtain composite nanoparticles;

S2. Linking placental trophoblast cell surface marker antibodies to composite nanoparticles;

S3, linking the enzyme substrate polypeptide that promotes placental development and fetal nutrition to PEG to obtain polypeptide-PEG;

S4, mixing the polypeptide-PEG and liposomes to form a lipid bimolecular membrane modified by polypeptide-PEG;

S5. Assembling the lipid bimolecular membrane modified by the polypeptide-PEG and the composite nanoparticle into a nanocarrier for targeted distribution to diseased cells.

Preferably, in step S1, the mass ratio of the copolymer to the superparamagnetic ferric iron tetroxide SPIO nanoparticles is 5-15:1.

The present invention uses the lipid bimolecular membrane modified by the MMP-9 substrate polypeptide-PEG as the shell to ensure the stable distribution of the nano-transport system in the enzyme-free blood before entering the placental enzyme environment, reducing drug leakage, reducing or avoiding Phagocytosis by other cells outside the placenta. Thus ensuring the safety of other tissues and organs outside the placenta of the mother; the MMP-9 enzyme-sensitive shell disintegrates in the microenvironment containing enzymes on the mother's side of the placenta and releases the drug, which can ensure the efficient release and distribution of the drug in the placenta of the mother; The introduction of the enzyme-sensitive shell can ensure the distribution efficiency of the placenta without adopting a large-size nano-carrier structure, effectively reducing the particle size of the nano-carrier, ensuring the stable circulation distribution of the drug before entering the placenta, and ensuring the reticuloendothelial The system will not phagocytize a large number of vectors to cause reduced curative effect and increased side effects.

The present invention adopts the drug carrier modified by placental trophoblast cell surface marker antibody as the inner core, and the drug is modified by TB cell surface marker antibody, which can accurately anchor the TB cell membrane in the placenta after release, ensuring that in the complex placental environment, Specific drug administration to TB cells, while avoiding other cells in the placenta from taking drugs, resulting in unnecessary placental function damage; most drugs entering the placenta are targeted by antibodies and anchored to TB cells, ensuring minimal drug penetration Leakage passes through the placental barrier and enters the fetal side, ensuring the safety of the fetus; after the drug is anchored in the TB cell membrane, it promotes the endocytosis of therapeutic drugs and therapeutic genes into TB cells to achieve functional regulation and ensure accurate TB functional regulation.

The present invention also provides the application of the above-mentioned dual-target nano drug delivery system in the preparation of drugs for regulating the dysfunction of placental trophoblast cells, and the disease for regulating the dysfunction of placental trophoblast cells is placenta accreta.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention uses the lipid bimolecular membrane modified by the enzyme substrate polypeptide-PEG that can be disintegrated under the action of specific enzymes highly expressed in the interstitial fluid of the placenta as the shell; The drug carrier modified by the expressed marker antibody is used as the inner core; the nano-carrier with double-layer structure is synthesized. This double-layer structure can ensure that the nano-drugs in the blood circulation of pregnant women, the liposome shell structure is stable, maintain a stable circulation, and are not easily captured by other tissues and cells including the reticuloendothelial system, reducing the impact on the placenta in pregnant women distribution and release in other tissues, reducing toxic and side effects;

(2) After the transport system enters the placenta with the blood circulation, the enzyme substrate in the shell is decomposed by the corresponding enzyme highly expressed in the placental tissue, and the protective lipid bimolecular shell disintegrates rapidly in the placenta, releasing the anchoring Antibody-modified nanomedicine marked on the surface of TB cell membrane. The nano-medicine avoids being absorbed by other tissue cells of the mother, and is specifically anchored to the TB cell membrane in the placenta, and then is specifically endocytosed by the TB cells to produce functional regulation and ensure the exact treatment of TB cell diseases;

(3) Through the exact "antigen-antibody reaction", the drug is retained in the placenta rich in TB cells after the lipid bimolecular shell is disintegrated, reducing the leakage of the drug through the placental barrier and reducing the toxic and side effects on the fetus; Affects vascular endothelial cells, immune cells and other stromal cells in the placenta.

Description of drawings

FIG. 1 is a schematic structural view of the nanocarrier prepared in Example 1 of the present invention.

Detailed ways

The present invention will be further described below through specific embodiments. The following examples are preferred implementation forms of the present invention, but the implementation manners of the present invention are not limited by the following examples.

The raw material source of the present invention is as follows:

C57BL/6J-Marveld1 knockout mice (strain number: KOCMP-277010-Marveld1-B6J-VA) Saiye (Suzhou) Biotechnology Co., Ltd.

Fe content determination method:

The Fe content in the nano-medicine system was measured by atomic absorption spectrophotometer, which was used to measure the dose of nano-medicine. Take a certain amount of the prepared drug solution (for example, 1 mL of the solution in step 3) is lyophilized and weighed, and then dissolved in a 1 molL ^-1 HCl solution, left for 24 hours to fully ionize Fe in SPIO, and then analyzed by atomic absorption spectrometry Photometer detects the absorbance of Fe atom at 248.3nm, substitutes it into the standard curve made with Fe standard solution to calculate the concentration of Fe, and then back calculates the Fe content in the drug solution before lyophilization.

Particle size test method:

The particle size of the sample was measured with a Zeta-Plus potential particle sizer (Brooken Haven), the incident laser wavelength λ=532nm, the incident angle θ=90°, and the temperature was 25°C; the average value of three measurements was taken.

Example 1:

Synthesis of S1, polyacetylimide grafted polyethylene glycol (PEG-PEI)

Polyethyleneimine-grafted polyethylene glycol (PEG-PEI) was synthesized by a two-step method. First, the terminal hydroxyl groups of monomethyl ether polyglycol were activated with carbonyldiimidazole, and then reacted with the amino groups of polyethyleneimine to generate PEG. -PEI. The specific operation is as follows: weigh monomethyl ether glycol (8.0 g, Mn=2 kDa) into a reaction flask, dry it in vacuum at 80° C. for 6 h, and add THF (60 mL) under an argon atmosphere to dissolve it. Weigh carbonyldiimidazole (CDI, 6.4g) and another reaction bottle, then slowly drop THF dissolved in mPEG-OH into the CDI bottle with a constant pressure dropping funnel, and stir the reaction overnight at room temperature. Distilled water (0.648 mL) was added to inactivate excess CDI, and stirring was continued for 30 min. Precipitate the solution into a large amount of cold ether, filter and dry in vacuo to obtain mPEG-CDI as a white powdery solid;

Weigh PEI (4.4g, MW=1.8kDa) into a two-necked bottle (50mL), add chloroform (20mL) to dissolve it, add PEG-CDI (3.2g), stir and react at room temperature for 24h, and put the solution in In a dialysis bag (MWCO=3.5kDa), dialyze with chloroform for 24h, concentrate the solution in the dialysis bag under reduced pressure, then precipitate in a large amount of cold ether, filter and dry to obtain the product mPEG-PEI as a white powder;

S2, Synthesis of polyacetylimide grafted polyethylene glycol grafted polycaprolactone (PEG-PEI-PCL)

First synthesize PCL-OH, add 15g of dry dodecanol into a two-neck bottle, dry in vacuum at 70°C for 8h, add 2ml of Sn(Oct) ₂ to continue drying for 0.5h, then add 400mL of dry ε-caprolactone, and dry at 105°C Stir and react for 24 hours; after cooling, add 100 mL of ethanol to dissolve the unreacted ε-caprolactone, filter, dissolve the crude product in 250 mL of tetrahydrofuran, precipitate in a large amount of anhydrous ether, filter and dry to obtain a white powder product with a yield of 96%;

Then synthesize PCL-CDI, add 10g PCL-OH (Mn=5000) into a two-necked flask, dry it under vacuum at 50°C for 8h, dissolve it in 50mL tetrahydrofuran, add 7.2g (10eq.) of carbonyldiimidazole (CDI), and protect it under argon , reacted at room temperature for 24 hours, precipitated in a large amount of anhydrous ether, filtered, and dried in vacuum at room temperature to obtain a white powder product with a yield of 90%;

Finally, react PCL-CDI with PEG-PEI to prepare PEG-PEI-PCL, add 1.6g PEG-PEI into a 50mL two-necked bottle, add 30mL chloroform to dissolve it, and then slowly drop into 10mL of 200mg PCL-CDI Chloroform solution, stirred and reacted at room temperature for 24h, dialyzed in 1000mL chloroform with a dialysis bag (MWCO=5kDa) for 24h, removed part of chloroform under reduced pressure, then precipitated in anhydrous ether, filtered and dried to obtain a white powder Product, yield 86%;

S3, Preparation of polyethylene glycol-polyethyleneimine-polycaprolactone-loaded SPIO nanoparticles and drugs (PEG-PEI-PCL-SPIO/drug)

SPIO (superparamagnetic iron tetroxide) according to the literature [SH Sun, H. Zeng, DB Robinson, S. Raoux, PMRice, SX Wang, GX Li. Monodisperse MFe ₂ O ₄ (M=Fe, Co, Mn) Nanoparticles.J. Synthesized by the method reported in Am.Chem.Soc.2004,126,273-279], iron acetylacetonate Fe(acac) ₃ 1.4126g (4mmol), 1,2-hexadecanediol 5.16g (20mmol), oleic acid 3.8ml (12mmol) and 3.8ml (12mmol) of oleylamine were added to a 200ml three-necked flask, and then 40ml of dibenzyl ether was added to stir and dissolve under the protection of nitrogen, heated to 200°C in a sand bath and refluxed for 2h, then heated to 300°C to reflux After 1h, the reaction system slowly changed from dark red to black; naturally cooled in air, precipitated in 150ml of ethanol, centrifuged at 10000rpm for 5 minutes, discarded the upper clear night, and dissolved the lower layer in 4 drops of oleic acid and oleylamine in 70ml of n-hexane, then centrifuged at 10000rpm for 10min to remove the insoluble part, the solution was reprecipitated in 200ml of ethanol, and then centrifuged at 10000rpm for 10min, the lower precipitate was dissolved in 60ml of n-hexane, and argon was introduced Store under gas protection at 4°C for later use;

The n-hexane solution of SPIO was blown dry and weighed to collect 5 mg of SPIO nanoparticles in a serum bottle (10 mL), weighed 50 mg of PEG-PEI-PCL polymer, 5 mg of sulfasalazine, and washed with dimethyl sulfoxide (3 mL) Dissolve and mix them evenly, add the above solution dropwise to 20mL distilled water under ultrasonic dispersion, place the reaction solution in a dialysis bag (MWCO=3.5kDa) and dialyze for 24h to remove dimethyl sulfoxide, and centrifuge at a speed of 12000r/min , collected the precipitate, and discarded the supernatant. Then dissolve the precipitate with water, ultrasonically disperse, and repeat the centrifugation operation. Finally, ultrasonically disperse the prepared PEG-PEI-PCL-SPIO/drug nanoparticles into water, use a needle filter with a pore size of 220nm, add pure water, and adjust the volume PEG-PEI-PCL-SPIO/drug nanoparticles concentration to Fe content of 0.145mg/mL, the product was stored at 4°C for later use; S4, antibody-targeted polyethylene glycol-polyethyleneimine-polycaprolactone Preparation of loaded SPIO nanoparticles/drug (Fab-PEG-PEI-PCL-SPIO/drug)

Firstly, the PLGF antibody is cracked by the method in the existing literature, and the Fab fragment of PLGF is obtained and purified. Then PLGF-Fab is linked to mal-PEG-COOH, and then the PEG linked to the antibody is reacted with the amino groups on the PEG-PEI-PCL-SPIO nanoparticles by amidation reaction to prepare Fab-PEG-PEI-PCL-SPIO ;

The specific operation is as follows: Weigh 10 mg of PLGF antibody and digest it with 0.5 mg·ml ^-1 papain, 10 mmol·L ^- 1 cysteine, 2 mmol·L ^- 1 EDTA, pH 7.6 for 4 hours . The enzymatic hydrolysis product was separated by Protein A affinity chromatography, and the breakthrough peak was further purified by DEAE anion exchange chromatography, and the Fab fragment of PLGF with high purity was obtained after dialysis, desalination and lyophilization;

1 mg of PLGF Fab fragment (Mn=45kDa) was weighed and pretreated with EDTA solution (500 μL 0.5M) at 4° C. for 15 min. Add 5ml of PBS solution to dissolve, add 1mg of dithiothreitol, and react at 25°C for 30min. After removing dithiothreitol by centrifugation with a centrifugal ultrafiltration tube with a molecular weight cut-off of 1k, add 5ml of PBS solution to dissolve, then add mal-PEG-COOH (2mg, Mn=4k) to mix well, and place at 4°C overnight. The excess mal-PEG-COOH was removed by centrifugation with a centrifugal ultrafiltration tube with a molecular weight cut-off of 5k. Activate the carboxyl group in Fab-PEG-COOH with 500 μg each of EDC and NHS for 15 minutes, then add 16 mL of PEG-PEI-PCL-SPIO/drug prepared in step 3, react overnight at 4°C, and finally remove excess EDC and NHS by ultrafiltration and centrifugation small molecule impurities, centrifuge at 12000r/min to remove the unlinked antibody, collect the solid solution, ultrasonically disperse it into distilled water, adjust the concentration of Fab-PEG-PEI-PCL-SPIO/drug nanoparticles to a Fe content of 0.145mg/ mL spare;

S5. Preparation of therapeutic gene composite nanoparticles

Positively charged PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticles and negatively charged CORO6-siRNA can be complexed by electrostatic interaction to form a nanocomposite. The specific operation is as follows: Dilute 400 μg of CORO6-siRNA with PBS to a final volume of 1.5 mL, and shake evenly. Take 1.5mL of PEG-PEI-SPIO prepared in step 3, (or Fab-PEG-PEI-SPIO net weight 1.6mL prepared in step 4) and ultrasonically disperse the nanoparticles evenly, mix CORO6-siRNA dilution solution with PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle solution was mixed evenly, and the composite system was fixed to a Fe content of 0.061 mg/mL, mixed by pipetting and allowed to stand for 30 minutes to obtain a uniform composite;

S6, PEG-polypeptide synthesis

0.05mmol matrix metalloproteinase 9 sensitive polypeptide (DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(N-Me-Abz)-NH2, molecular weight: 1077.22Da), 5mmol EDC and 5mmol The DMAP was dissolved in 10 mL of acetonitrile aqueous solution (acetonitrile: water = 1:1), placed on an ice-water bath under the protection of N2, and magnetically stirred at 500 rpm for 2 h to activate Peptide. After 2h, 0.5mmol of PEG-NHS (molecular weight: 3000Da) was added, and the reaction was continued for 72h. After the reaction, the reaction solution was placed in a dialysis bag (MWCO=3.5kDa), dialyzed for 72 hours, and freeze-dried to obtain the product PEG-polypeptide;

S7. Preparation of PEG-polypeptide modified liposome shell@therapeutic gene composite nanoparticles

PEG-polypeptide and cholesterol (20 mg each) were dissolved in 5 mL of dichloromethane, and the dichloromethane was spin-dried with a vacuum rotary device, so that PEG-polypeptide and cholesterol formed a layer of liposomes on the wall of the round-bottomed flask film. Under slow stirring, 2 mL of therapeutic gene composite nanoparticles prepared in step 5 was added dropwise at a rate of 0.5 mL/min into the liposome film formed by the above-mentioned PEG-polypeptide and cholesterol. Continue to stir for 30 minutes after the dropwise addition is completed, so that the liposomes and the therapeutic gene composite nanoparticles are fully assembled, and finally the liposomes loaded with the therapeutic gene composite nanoparticles and the empty liposomes are separated with a strong magnet. Finally, 2 mL of normal saline (0.9% NaCl) was added to dissolve the PEG-polypeptide-modified liposome shell@therapeutic gene composite nanoparticle with a pore size of 220 nm and a syringe filter filtration rate of 220 nm. Set the volume to Fe content of 0.061 mg/mL, 4 Store at ℃ for later use.

The schematic diagram of the specific structure of the prepared nanocarrier is shown in Fig. 1 .

Embodiment 2-4, comparative example 1-6:

Compared with Example 1, Example 2-4 or Comparative Example 1-6 can be prepared by changing the dosage of polymer, drug and SPIO in Step S3 or omitting a certain step in Steps S3, S4, S5, S6, and S7 , see Table 1 below for details:

Table 1: Examples and comparative examples

Functional evaluation experiment

1. Nuclear magnetic resonance (MRI) experiments to evaluate the placenta-specific delivery function of drugs

Marveld1 (MARVEL Domain Containing 1) is a gene with EMT suppression function. Previous studies have confirmed that Marveld1 knockout mice will experience uterine bleeding and dystocia. Anatomy found that this is because the placental trophoblast cells of such animals invaded into the uterine wall, causing the placenta to adhere tightly to the uterine wall, causing dystocia. Even if some fetuses are delivered, the placenta cannot be separated spontaneously, and the placenta pulls on the uterus, causing uterine deformation, uterine bleeding, and birth canal blockage. Marveld1 knockout mice can be used to conceive, to simulate the pathophysiological process of placenta accreta, and to detect the distribution and functional regulation of the carrier.

Model building:

8-week-old C57BL/6J-Marveld1 knockout mice and control group SPF-grade C57BL/6 mice (purchased from Guangdong Provincial Medical Experimental Animal Center), female mice and male mice 2:1 mated in the same cage during estrus On the second day, the vaginal secretion smears of the female rats were stained with Papanicolaou, and those with positive vaginal spermatozoa observed under an optical microscope were diagnosed as pregnant, which was marked as the 0th day of pregnancy (D0). Pregnant C57BL/6J-Marveld1 knockout mice were used as the placenta accreta model, and pregnant C57BL/6 mice were used as the normal control group.

Placental distribution of drugs detected by MRI imaging:

Placenta accreta model animals were anesthetized with chloral hydrate on the 11th day, and MRIT2 sequence was scanned before drug injection (0h) and 2 hours after injection (2h) to observe the distribution of nanomedicine containing SPIO in vivo. The dose of the tail vein injection of nano-medicine is: (therapeutic dose 0.31mg/Kg iron equivalent medicine, or equal volume of normal saline);

Uterine MRI imaging of C57BL/6j mice was performed using a Philips Intera 1.5T MRI scanner and its animal-specific coils. Observe the evolution of the signal intensity of the uterus and embryos in the mouse on the MRIbTFE sequence, and use T2map imaging technology to measure the T2 relaxation time changes caused by the distribution of SPIO in the drug in the uterus, placenta, embryos and other organs in the body, and calculate 0h The relaxation rate R2 at time and 2h respectively. The relative increase ratio of R2 (RSI (Relative Signal Intensity)%=R2 _2h /R2 _0h ) was calculated 2 hours after drug injection, and the results are shown in Table 2.

Table 2 Evaluation results of placenta-specific delivery function

From the above results, it can be seen that in Comparative Example 1, after the peptide-PEG-modified lipid bilayer was disintegrated without linking the placental trophoblast cell surface marker antibody, the drug contained in it could not be anchored in TB cells to obtain placental retention, and a large number of drugs were leaked. Placental barrier, the placental RSI is lower; the drug accumulates in the embryo, resulting in a higher embryo RSI; the drug cannot be anchored to TB cells to obtain placental retention, which also causes some drugs to leave the placenta and distribute throughout the body, resulting in a higher liver RSI.

The delivery system in Comparative Example 2 does not contain a polypeptide-PEG modified lipid bimolecular membrane as a shell, and cannot achieve targeted release for the placental microenvironment; in addition, the PLGF antibody targets other cell membranes in the body including TB to express PLGF cells, the cell membrane targeting is not strong; therefore, the detected RSI of the placenta is lower, and the RSI of the liver is lower; the particle size of the drug without lipid membrane is smaller, and those who enter the placenta pass through the placental barrier in a larger proportion, and the embryos are detected The RSI is higher.

The delivery systems of Comparative Examples 3 and 4 do not contain PLGF antibody, the drug entering the placenta cannot be targeted and anchored to TB cells, cannot obtain placental retention, a large number of placental barriers are leaked, and the placental RSI is detected to be low; the drug accumulates in the embryo, resulting in a higher embryonic RSI. At the same time, the lipid bimolecular membrane shell of Comparative Example 3 is not modified by the enzyme-sensitive polypeptide, and the distribution in the placenta is reduced, which also leads to lower RSI of the placenta and higher RSI of the liver. Comparative Example 4 has no lipid bimolecular membrane shell, and compared with Comparative Example 3, the RSI of the placenta is lower, and the RSI of the liver is higher.

The particle size of Comparative Example 5 was too large, resulting in extremely poor circulation distribution in the body, and the drug was mainly swallowed by the reticuloendothelial system of the liver, resulting in a significantly higher RSI of the liver and a significantly lower RSI of the placenta; It leaks through the maternal-fetal barrier, so the embryo RSI is lower. The particle size of Comparative Example 6 is much larger than that of Comparative Example 5, and the circulation is worse, so its liver RSI is higher than that of Comparative Example 5; its particle size is larger, and it is less likely to leak through the maternal-fetal barrier, so the placental RSI is lower than Comparative Example 5.

In Example 1-4, the lipid bimolecular membrane modified by the substrate polypeptide-PEG of MMP-9 is used as the outer shell, and the drug carrier modified by the surface marker antibody of the placental trophoblast cell is used as the inner core to synthesize a double-layer nanocarrier. The diameter range is 80-210nm. Its particle size of about 100nm and the outer layer of negatively charged lipid bimolecular membrane are convenient to avoid being swallowed by the reticuloendothelial system in large quantities, so as to prolong the circulation time in the body and realize the effective circulation in the body. The lipid bimolecular membrane shell modified by its substrate polypeptide-PEG is stable in the circulation of other tissues and organs in the body, and reaches the placental microenvironment with specific high expression of MMP-9. Drug-specific distribution in tissues. After the drug shell disintegrates in the placental microenvironment, a drug core containing PLGF antibody fragments is revealed. PLGF antibody fragments in the placenta can be anchored to TB cells with high expression of PLGF in the cell membrane, which can promote the regulation of TB function after the drug is specifically internalized by TB cells, reduce the distribution in other cells of the placenta, and reduce the impact on placental function. Influence. The PLGF antibody anchors the drug in the placenta to TB cells, and also effectively reduces the drug leakage through the maternal-fetal barrier and reduces the drug reaching the embryo.

2. Establish a placenta accreta animal model to evaluate the therapeutic effect

Drugs were injected on D3, D6, D9, D12, and D15 (a therapeutic dose of 0.31 mg/Kg iron-equivalent drug, or an equal volume of normal saline), and a series of tests were carried out during production. The test results are shown in Table 3:

Placenta and fetus inspection: record the number of normal births after delivery, open the abdominal cavity of pregnant mice, open the uterus, take out the remaining fetuses and placenta in turn, and record the number of dystocia fetuses. The fetal membranes and umbilical cord on the placenta were removed, and the umbilical cord of the fetus was cut off along the root of the umbilical cord. The placenta and fetus were placed on sterile gauze to blot the surface amniotic fluid, and the placenta and fetus were weighed with an analytical balance. Cut the placental tissue into liquid nitrogen and store at -80°C.

Judgment of dystocia caused by placenta accreta: Anatomy found that the placenta was tightly adhered to the uterine wall during delivery and could not be peeled off from the implantation site during delivery, and then the placenta pulled the uterus toward the cervix, resulting in cervical Blockage, eventually increased uterine bleeding, mothers and fetuses died were recorded as dystocia. Dystocia rate of pregnant mice in each group=(number of cases of dystocia/total number of cases in this group)*100%.

Table 3 Placenta accreta animal model to evaluate the therapeutic effect

group Dystocia rate (%) litter size normal control group 2.52 8.59 Example 1 11.27 8.32 Example 2 11.86 5.94 Example 3 11.53 8.17 Example 4 12.28 8.11 Comparative example 1 62.75 3.98 Comparative example 2 64.67 3.83 Comparative example 3 64.61 3.52 Comparative example 4 65.17 3.21 Comparative example 5 65.94 3.21 Comparative example 6 66.35 3.06

From the above results, it can be known that in Comparative Example 1, after the antibody of the surface marker of the placental trophoblast cell was not linked, the lipid bilayer modified by the polypeptide---PEG was disintegrated, and the drug contained in it could not be anchored in the TB cells to obtain placental retention, and a large amount of leakage occurred. After passing the placental barrier, it was detected that the treatment effect was poor, the dystocia rate was high, and the litter size was low; at the same time, the drug accumulated in the embryo, resulting in embryotoxicity and low litter size.

The delivery system of Comparative Example 2 does not contain the lipid bimolecular membrane modified by polypeptide-PEG as the shell, and the targeted release for the placental microenvironment cannot be realized; the PLGF antibody targets the cells expressing PLGF in vivo including TB, and the cell targeting Not strong, it was detected that the treatment effect was poor, the dystocia rate was high, and the litter size was low. At the same time, the particle size of the drug without lipid membrane is small, and those who enter the placenta pass through the placental barrier in a larger proportion, resulting in embryotoxicity and lower litter size.

The delivery systems of Comparative Examples 3 and 4 do not contain PLGF antibody, and the drugs that enter the placenta cannot be targeted and anchored to TB cells, and the placental retention cannot be obtained, resulting in a large number of leakage through the placental barrier, poor therapeutic effect and high dystocia rate , lower litter size. At the same time, the lipid bimolecular membrane shell of Comparative Example 3 is not modified by enzyme-sensitive polypeptides, and its distribution in the placenta is reduced. It is detected that the treatment effect is poor, the dystocia rate is high, and the litter size is low. Comparative example 4 has no lipid bimolecular membrane shell, and compared with comparative example 3, the therapeutic effect is worse.

The particle size of Comparative Examples 5 and 6 was too large, resulting in extremely poor circulation distribution in the body, and the drug was mainly swallowed by the reticuloendothelial system of the liver, resulting in insufficient drug distribution in the placenta, poor therapeutic effect, high dystocia rate, and poor delivery rate. The number is low. The particle size of Comparative Example 6 is much larger than that of Comparative Example 5, and the circulation distribution is worse, so its therapeutic effect is worse than that of Comparative Example 5.

In Example 1-4, the lipid bimolecular membrane modified by the substrate polypeptide-PEG of MMP-9 is used as the outer shell, and the drug carrier modified by the surface marker antibody of the placental trophoblast cell is used as the inner core to synthesize a double-layer nanocarrier. The diameter range is 80-210nm. Its particle size of about 100nm and the outer layer of negatively charged lipid bimolecular membrane are convenient to avoid being swallowed by the reticuloendothelial system in large quantities, so as to prolong the circulation time in the body and realize the effective circulation in the body. The lipid bimolecular membrane shell modified by its substrate polypeptide-PEG is stable in the circulation of other tissues and organs in the body, and reaches the placental microenvironment with specific high expression of MMP-9. Drug-specific distribution in tissues. After the drug shell disintegrates in the placental microenvironment, a drug core containing PLGF antibody fragments is revealed. PLGF antibody fragments in the placenta can be anchored to TB cells with high expression of PLGF in the cell membrane, which can promote the regulation of TB function after the drug is specifically internalized by TB cells, reduce the distribution in other cells of the placenta, and reduce the impact on placental function. Through the effective regulation of TB function, a better therapeutic effect has been achieved. The PLGF antibody anchors the drug in the placenta to TB cells, effectively reduces the drug leakage through the maternal-fetal barrier, reduces the drug reaching the embryo, and has less toxicity to the fetus.

3. Toxicity evaluation of drugs in animal models

72 hours after injection of the drug in the mice in the normal control group, blood was taken from the tail vein to detect liver function indicators alanine transaminase (ALT), total bilirubin (total bilirubin, TBil) and kidney function indicators blood urea nitrogen (blood urea nitrogen). nitrogen, BUN) and serum creatinine (serum creatinine, sCr). The testing instrument is a Hitachi 7600 automatic biochemical analyzer, and the testing results are shown in Table 4.

Table 4 Toxicity evaluation results

From the above results, it can be seen that the nanocarrier prepared by the present invention has no obvious toxic and side effects on the mother and fetus.

Claims (5)

1. a kind of nano-carrier by targeted distribution to pathological cells, it is characterized in that, described nano-carrier comprises shell and core, The shell is an enzyme substrate polypeptide-PEG modified lipid bimolecular membrane that is targeted for disintegration under the action of an enzyme highly expressed in the interstitial fluid of the placenta; the enzyme highly expressed in the interstitial fluid of the placenta is a matrix metalloproteinase 9; the enzyme substrate polypeptide is DNP-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys(N-Me-Abz)-NH2; The inner core is a drug carrier modified with marker antibodies specifically and highly expressed on the surface of placental trophoblast cells; The drug carrier is a copolymer formed of polyethylene glycol-modified polycation carrier and hydrophobic degradable polyester; the copolymer is polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL ; The marker antibody specifically highly expressed on the surface of the placental trophoblast cell is the Fab segment of the placental growth factor PLGF antibody; The drug carrier is loaded with superparamagnetic iron ferric oxide SPIO nanoparticles, small-molecule drugs and therapeutic genes that regulate the function of placental trophoblast cells; the small-molecule drug is sulfasalazine, and the therapeutic gene is siRNA that inhibits the expression of CORO6 gene ; The average particle diameter of the nano-carrier is 80nm-300nm. 2. A nano-carrier distributed through targeted distribution of diseased cells according to claim 1, characterized in that the average particle diameter of the nano-carrier is 100nm-210nm. 3. A method for preparing nanocarriers distributed by diseased cells according to any one of claims 1-2, characterized in that it comprises the following steps: S1. Load superparamagnetic iron ferric oxide SPIO nanoparticles, small molecule drugs that regulate the function of placental trophoblast cells, and therapeutic genes to the copolymer to obtain composite nanoparticles; S2. Linking the placental trophoblast cell surface marker antibody to the composite nanoparticle to obtain the antibody composite nanoparticle; S3, linking the enzyme substrate polypeptide that promotes placental development and fetal nutrition to PEG to obtain polypeptide-PEG; S4, mixing the polypeptide-PEG and liposomes to form a lipid bimolecular membrane modified by polypeptide-PEG; S5. Assembling the polypeptide-PEG modified lipid bimolecular membrane and the antibody composite nanoparticle into a nanocarrier for targeted distribution to diseased cells. 4. a kind of preparation method by the nano-carrier of pathological cell targeted distribution according to claim 3, is characterized in that, in step S1, the mass ratio of copolymer and superparamagnetic iron tetraoxide SPIO nanoparticle is 5 -15:1. 5. The application of a kind of nano-carrier according to any one of claims 1-2 in the preparation and regulation of placental trophoblast dysfunction disease medicine by targeted distribution of diseased cells, and the regulation and control of placental trophoblast dysfunction disease is placenta implant.

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