CN114377150B - MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, and preparation method and application thereof - Google Patents

MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, and preparation method and application thereof Download PDF

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CN114377150B
CN114377150B CN202111640369.5A CN202111640369A CN114377150B CN 114377150 B CN114377150 B CN 114377150B CN 202111640369 A CN202111640369 A CN 202111640369A CN 114377150 B CN114377150 B CN 114377150B
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placenta
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microenvironment
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CN114377150A (en
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郭宇
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Nanchang Research Institute Of Sun Yat Sen University
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    • A61K47/6835—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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    • 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
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    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • A61K47/6911—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Abstract

The invention discloses an MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, a preparation method and application thereof, wherein the targeting vector has a core-shell double-layer structure, takes an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is targeted to disintegrate under the action of enzyme which is in high expression in contact with placenta tissue interstitial fluid as a shell, takes a drug carrier modified by placenta trophoblast surface specificity high-expression marker antibody as an inner core, and is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester. The MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector can effectively avoid the absorption of other organs outside the mother placenta and fetal nonspecific drugs, thereby realizing the delivery and function regulation of the specific drugs of pathological change trophoblasts in the placenta.

Description

MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, and preparation method and application thereof
Technical Field
The invention relates to the field of biomedical engineering, in particular to an MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, and a preparation method and application thereof.
Background
Gestational trophoblastic tumors (gestational trophoblastic neoplasms, GTN) include aggressive grape embryo, choriocarcinoma, placental trophoblastic tumors, and epithelioid trophoblastic tumors, among others. Most of the GTNs with gestational onset react well to chemotherapy, and the effective rate of chemotherapy can reach more than 90%. However, among GTNs, the most difficult type of treatment is gestation-associated trophoblastic tumors that occur in the placental region of pregnant women. The tumor is developed in gestation period and coexists with fetus and placenta, and has the main pathological characteristics that the tumor occurs in placenta, the tumor body can be limited in uterus, and can also protrude to uterine cavity to form polyps, or invade muscular layer and even break through serosa to form metastasis [ J of Chinese gynaecology and obstetrics, 2003 (10): 4-5 ]. The tumors exist in the placenta, are indistinguishable from the placenta tissue, and cannot be treated by surgical means. If the common methods such as chemotherapy and the like are adopted, the fetus is damaged, and abortion is caused. Such cases are very difficult to handle clinically, often requiring multiple means of combination therapy after termination of pregnancy. Therefore, if means for delivering drugs to benign and malignant trophoblasts in placenta during gestation can be developed, specific killing of trophoblast-derived tumor cells in placenta can be realized, less drug distribution and lower toxicity to fetuses are ensured in the killing process, and the progress of diseases without effective treatment means can be effectively controlled.
Excessive activation of wnt/β -catenin pathway is a major cause of trophoblastic tumorigenesis and development [ plamenta.2009 Oct;30 (10) 876-83; mediators inflam.2020 Aug 12;2020:9578701; am J Physiol Cell Physiol.2020 Mar 1;318 (3) C664-C674 ] it is therefore desirable to achieve inhibition of trophoblastoma in the placenta with drugs that specifically inhibit the wnt pathway. Prodigiosin is a potent inhibitor of the Wnt/β -catenin pathway and has antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive and anticancer properties. [ Proc Natl Acad Sci U S A.2016 Nov 15;113 13150-13155 ], and it also has some inhibitory effect on tumor cell resistance [ Cancer Lett.2020 Jul 1;481:15-23 ]. Therefore, it is desirable to use Prodigiosin to achieve inhibition of trophoblastic tumors in the placenta. ISL1 (ISL LIM Homeobox 1) is an oncogene closely related to proliferation and metastasis and angiogenesis of various tumors [ Onco Targets Ther.2018 Feb 14;11:781-789 ], its carcinomatous activity is associated with AKT et al pathways [ Int J Mol med.2018 Nov;42 (5) 2343-2352 ]. We prove in the preliminary experiments that the proliferation and metastasis of the trophoblastic tumor can be effectively blocked by inhibiting the tumor. In our earlier experiments, it has been found that the combination of these two drugs can achieve a better inhibition of trophoblastic tumors. Therefore, if the aforementioned trophoblast tumor-inhibiting drug can be delivered to the diseased cells in the placenta, a better synergistic therapeutic effect can be expected.
However, the drugs that may have TB function controlling effects or trophoblast tumor inhibiting effects are inevitably distributed in various organs of the mother and distributed to the fetus through the placenta. These drugs, while regulating TB function, can produce toxicity to the mother and fetus. At present, the medication of pregnant women including emergency medicines has a great deal of contraindications, and the problems of medicine use of the pregnant women and development of new medicines are firstly faced with the problems of distribution and toxicity of the medicines in the mother and the fetus. Most drugs can pass through the placenta and be distributed into the fetal side, affecting fetal development. Therefore, the current drugs for realizing the function regulation of the EMT, angiogenesis promotion and the like of the TB in the in vitro experiments can not realize the function regulation of the TB in the placenta on the premise of guaranteeing the medication safety. Therefore, how to avoid toxicity to the mother and the fetus and realize accurate delivery of the TB function regulating medicine to the TB in the placenta is a key for solving the disease symptoms such as gestation complicated with trophoblastic tumor and the like caused by TB dysfunction.
There are 2 means that have been tried by researchers to potentially promote TB-specific nano-drug delivery, one is by enlarging the nano-drug particle size so that it cannot pass the membrane barrier and remain in the placenta to produce drug delivery effects; the other is specific delivery of antibody modified nanocarriers against TB cell membrane markers.
The principle of increasing the particle size of the nano-drug and promoting the drug distribution in the placenta is that experimental researches show that the nano-drug less than 300nm cannot be retained in the placenta and easily enters the fetus through the placenta. So researchers try to synthesize nano-drugs with particle size > 300nm, so that they remain in the placenta, and function regulation of various cells including placenta TB is generated. However, an excessively large drug particle size (> 100 nm) is detrimental to the in vivo distribution of the drug. Most of the nano-drugs with the wavelength of more than 300nm are captured by reticuloendothelial systems in the maternal circulation, side effects are generated everywhere in the whole body, the nano-drugs can reach the placenta, and the proportion of realizing the specific distribution of TB is low. Therefore, there is a need to achieve retention of nanomedicine in placenta and targeting of TB cells in other ways.
The nano-drug can adopt nano-drug linked antibodies to target and identify cell membrane markers of target cells, so that the specific delivery of the target cells is realized. TB and tumor cells have some established, high co-specificity expressed on the surface of these two cells, and are distinguishable from other placental stromal cells in placental tissue by surface markers (e.g., desmoplakin) that can be distinguished from other cells in the placenta. However, the whole body multi-organ tissue expression level analysis shows that the surface marker is expressed in partial cells at other parts outside the placenta. The difference between the expression abundance on the surface of a small amount of high-expression cells and TB is not obvious. If the antibody of desmoplakin is connected to the surface of the nano-drug carrier, the antibody can be directly applied in vivo, and the side effect on other cells expressing the marker desmoplakin in vivo can be caused. Therefore, only before entering the placenta in the blood circulation, the TB cell recognition antibody of the nano-carrier is shielded, so that the TB cell recognition antibody can be prevented from being distributed on the cells outside the placenta, and the TB cell distributed in the placenta is ensured.
More notably, desmoplakin is expressed in a variety of malignancies, including trophoblasts, and can be distinguished from other stromal cells and immune cells in the placenta, other than TB cells, to achieve specific drug delivery.
In view of the above, there is a lack of nanocarrier systems that can effectively avoid absorption of maternal and fetal nonspecific drugs, thereby achieving specific drug delivery and functional regulation of TB cells in the placenta.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary aim of the invention is to provide an MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, which utilizes the placenta microenvironment to target and reduce the distribution of drugs in maternal organs before entering the placenta, and utilizes a nourishing cell membrane marker to target and reduce the distribution of drugs in fetal organs after passing through the placenta, so that the absorption of other organs outside the maternal placenta and fetal nonspecific drugs can be effectively avoided, and the delivery and function regulation of the nourishing cell specific drugs in the placenta are realized.
The invention also aims to provide a preparation method of the MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector.
The invention is realized by the following technical scheme:
an MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector which has a core-shell double-layer structure, wherein an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is targeted to disintegrate under the action of enzymes which are in contact with placenta interstitial fluid and are expressed together with tumor interstitial fluid is taken as a shell, a drug carrier modified by a marker antibody which is expressed together with specificity and high on the surface of placenta trophoblast and tumor cells is taken as a kernel, and superparamagnetic ferroferric oxide SPIO nano particles, small molecular drugs for regulating functions of the placenta trophoblast, therapeutic genes or a combination thereof are loaded in the drug carrier;
the enzyme which is commonly and highly expressed by the placenta interstitial fluid and the tumor interstitial fluid is one or more of matrix metalloproteinase 9, lysozyme, kallikrein, histamine enzyme or oxytocin enzyme;
the marker antibody with common specificity and high expression on the surface of the placenta trophoblast and the surface of the tumor cell is a Fab segment of the desmoplakin antibody;
the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester.
The placenta of pregnant women is rich in a plurality of enzymes for promoting placenta development and fetal nutrition, and the enzyme with high expression of the placenta interstitial fluid is one or more of matrix metalloproteinase 9 (matrix metalloproteinase, MMP 9), lysozyme, kininase, histamine enzyme and oxytocin enzyme, wherein the matrix metalloproteinase 9 has extremely high expression level in placenta interstitial fluid and is hardly expressed in normal human blood and interstitial fluid. Also, matrix metalloproteinases (including MMP 9) are highly expressed in tumor tissues as well. Thus, matrix metalloproteinase 9 is preferred.
The matrix metalloproteinase 9 substrate polypeptide may be selected from DNP-Pro-Cha-Gly-Cys (Me) -His-Ala-Lys (N-Me-Abz) -NH2, molecular weight: 1077.22Da.
The drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester, wherein the copolymer is one or more of polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL, polyethylene glycol-polyethyleneimine-polylactic acid PEG-PEI-PLA or polyethylene glycol-polyethyleneimine-polylactic acid-glycolic acid PEG-PEI-PLGA, and preferably polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL.
The copolymers of the present invention can be synthesized by prior art techniques, such as first reacting PEG with a polycation carrier to form a copolymer, and then reacting the active groups of the polycation with the activated polyester segment to form a copolymer.
The copolymers according to the invention are also commercially available.
The superparamagnetic ferroferric oxide SPIO nano particles are loaded in the drug carrier, and small molecular drugs for regulating and controlling functions of placenta trophoblast cells, therapeutic genes or a combination of the therapeutic genes are used. The small molecule drug is Prodigiosin, and the therapeutic gene is siRNA for inhibiting ISL1 gene expression.
The average particle size of the MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector is 80-300 nm, preferably 100-205 nm, and the particle size is too large to facilitate in vivo circulation, the preparation difficulty is increased when the particle size is too small, and the drug and gene loading is not facilitated.
The invention also provides a preparation method of the MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, which comprises the following steps:
s1, loading superparamagnetic ferroferric oxide SPIO nano particles, micromolecular drugs for regulating and controlling functions of placenta trophoblast and/or genes into a copolymer to obtain composite nano particles;
s2, linking an antibody or an antibody fragment of a marker which is commonly and highly expressed on the surface of placenta trophoblast and the surface of tumor cells to the composite nanoparticle;
s3, linking the enzyme substrate polypeptide which is commonly and highly expressed by placenta and tumor interstitial fluid with PEG to obtain polypeptide-PEG;
s4, mixing the polypeptide-PEG and the liposome to form a polypeptide-PEG modified lipid bilayer membrane;
s5, assembling the polypeptide-PEG modified lipid bilayer membrane and the composite nano particles into the MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector.
Preferably, in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15:1.
According to the invention, the lipid bilayer membrane modified by MMP9 substrate polypeptide-PEG is taken as a shell, so that the nano transmission system is ensured to be distributed stably in enzyme-free blood before entering placenta and tumor focus enzyme environment, drug leakage is reduced, and phagocytosis of other cells outside the placenta is reduced or avoided. Thereby ensuring the safety of other tissues and organs outside the mother placenta; MMP9 enzyme sensitive shell disintegrates in micro environment containing enzyme at placenta mother side to release medicine, which can ensure the high efficiency release and distribution of medicine in mother placenta and tumor focus; due to the introduction of the enzyme sensitive shell, the distribution efficiency of the placenta can be ensured without adopting a large-particle-diameter nano carrier structure, the particle diameter of the nano carrier is effectively reduced, the stability of the circulating distribution of the medicine before entering the placenta and tumor is ensured, and the reticuloendothelial system is ensured not to cause the reduction of curative effect and the increase of side effect due to a large amount of phagocytic carriers.
The invention adopts the drug carrier modified by the common marker antibody on the surface of the placenta trophoblast and the surface of the tumor cells as the inner core, and the drug is modified by the marker antibody on the surface of the TB cells, so that the TB cell membrane and the tumor cell membrane in the placenta can be anchored exactly after the drug carrier is released, the specific drug administration of the TB cells in a complex placenta environment is ensured, and meanwhile, the drug administration of other cells in the placenta is avoided, and unnecessary placenta function injury is generated;
most of medicaments entering the placenta are targeted by the antibody and are anchored to TB cells and tumor cells exactly, so that the medicaments are ensured to leak through placenta barriers very little and enter the fetal side, and the safety of the fetus is ensured; after being anchored to the TB cell membrane and the tumor cell membrane, the drug promotes the therapeutic drug and therapeutic gene to swallow the TB cell, realizes the function regulation and control, and ensures the exact regulation and control of the TB function and the inhibition of tumor focus in placenta.
The invention also provides application of the MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector in preparing a medicament for regulating and controlling placenta trophoblastic dysfunction, wherein the regulating and controlling placenta trophoblastic dysfunction is gestation combined trophoblastic tumor.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention takes the lipid bilayer membrane modified by the enzyme substrate polypeptide-PEG which can be targeted to disintegrate under the action of the specific enzyme which is commonly and highly expressed by the placenta tissue interstitial fluid and the tumor tissue interstitial fluid as the shell; the drug carrier modified by the marker antibody with common specificity and high expression on the surface of placenta trophoblast and tumor cells is used as the inner core; synthetic double-layer MMP enzyme sensitive placenta microenvironment and tumor microenvironment targeting vector. The bilayer structure can ensure that the nano-drug has stable liposome shell structure in the blood circulation of pregnant women, keeps stable circulation, is not easy to be captured by other tissues and cells including reticuloendothelial system, reduces the influence on the distribution and release of other tissues except placenta in the pregnant women, and reduces the toxic and side effects;
(2) After the transmission system enters the placenta along with blood circulation, enzyme substrates in the outer shell of the transmission system are decomposed by corresponding enzymes which are highly expressed in placenta tissues and tumor tissues, and the protective lipid bilayer outer shell is rapidly disintegrated in the placenta to release the nano-drug which can anchor the signs on the surfaces of TB cell membranes and tumor cell membranes and is modified by antibodies. The nano-drug is prevented from being absorbed by other tissue cells of a parent body, is specifically anchored to a TB cell membrane and a tumor cell membrane in the placenta, is further specifically endocytosed by the TB cell and the tumor cell, and has a function regulation and control function, so that the accurate treatment of the dysfunction and the tumor of the TB cell is ensured;
(3) Through definite antigen-antibody reaction, the medicine is remained in the placenta containing TB cell tumor cells after the lipid bilayer shell is disintegrated, so that the medicine leakage through placenta barrier is reduced, and the toxic and side effects on the fetus are reduced; also avoids influencing vascular endothelial cells, immune cells and other stromal cells in the placenta.
Drawings
FIG. 1 is a schematic structural diagram of MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector prepared in example 1 of the invention.
Detailed Description
The present invention will be further described by the following specific embodiments, which are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples.
The sources of the raw materials of the invention are as follows:
the Fe content measuring method comprises the following steps:
measuring Fe content in nano-drug system by atomic absorption spectrophotometryIs used for measuring the dosage of the nano-medicine. Taking a certain amount of prepared medicinal solution (for example, 1mL of the solution in the third step), freeze-drying and weighing, and dissolving to 1mol L -1 And (3) placing for 24 hours to fully ionize Fe in SPIO, detecting the absorbance of Fe atoms at 248.3nm by using an atomic absorption spectrophotometer, substituting the absorbance into a standard curve made by using a Fe standard solution to calculate the concentration of Fe, and then reversely calculating the Fe content in the drug solution before freeze-drying.
The particle size testing method comprises the following steps:
the particle size of the sample was measured with a Zeta-Plus potential particle sizer (Brooken Haven) with an incident laser wavelength λ=532 nm and an incident angle θ=90° at 25 ℃; the average of the three measurements was taken.
Example 1:
s1, synthesis of polyethylenimine grafted polyethylene glycol (PEG-PEI)
The polyethyleneimine grafted polyethylene glycol (PEG-PEI) is synthesized by a two-step method, the hydroxyl end groups of the monomethyl ether polyglycol are activated by carbonyl diimidazole, and then the hydroxyl end groups react with the amino groups of the polyethyleneimine to generate the PEG-PEI. The specific operation is as follows: monomethyl ether glycol (8.0 g, mn=2 kDa) was weighed into a reaction flask, dried under vacuum at 80 ℃ for 6h, and dissolved by adding THF (60 mL) under argon atmosphere. Carbonyl diimidazole (CDI, 6.4 g) was weighed into another reaction flask, and then THF with mPEG-OH dissolved therein was slowly added dropwise to the CDI flask using a constant pressure dropping funnel, and the reaction was stirred at room temperature overnight. Distilled water (0.648 mL) was added to deactivate excess CDI and stirring was continued for 30min. Precipitating the solution into a large amount of cold diethyl ether, filtering, and vacuum drying to obtain white powdery solid mPEG-CDI;
PEI (4.4 g, MW=1.8 kDa) is weighed and added into a two-mouth bottle (50 mL), chloroform (20 mL) is added to dissolve and add PEG-CDI (3.2 g), stirring is carried out at room temperature for 24h, the solution is filled into a dialysis bag (MWCO=3.5 kDa), dialysis is carried out for 24h by using chloroform, the solution in the dialysis bag is concentrated under reduced pressure, then the solution is precipitated into a large amount of cold diethyl ether, and the product mPEG-PEI is obtained by filtering and drying;
s2, synthesis of poly (acetylimide) -grafted polyethylene glycol-grafted polycaprolactone (PEG-PEI-PCL)
First synthesizePCL-OH,15g of dried dodecanol were added to a two-necked flask, dried in vacuo at 70℃for 8h, and added with 2ml of Sn (Oct) 2 Continuing to dry for 0.5h, then adding 400mL of dried epsilon-caprolactone, and stirring at 105 ℃ to react for 24h; cooling, adding 100mL of ethanol to dissolve unreacted epsilon-caprolactone, filtering, dissolving the crude product in 250mL of tetrahydrofuran, precipitating in a large amount of anhydrous diethyl ether, filtering, and drying to obtain a white powdery product, wherein the yield is 96%;
then synthesizing PCL-CDI, adding 10g PCL-OH (Mn=5000) into a two-mouth bottle, drying in vacuum at 50 ℃ for 8 hours, dissolving in 50mL tetrahydrofuran, adding 7.2g (10 eq.) of Carbonyl Diimidazole (CDI), protecting by argon, reacting at room temperature for 24 hours, precipitating in a large amount of anhydrous diethyl ether, filtering, and drying in vacuum at room temperature to obtain a white powdery product with the yield of 90%;
finally, the PCL-CDI and the PEG-PEI are reacted to prepare PEG-PEI-PCL,1.6g of PEG-PEI is added into a 50mL two-port bottle, 30mL of chloroform is added to dissolve the PEG-PEI, then 10mL of chloroform solution containing 200mg of PCL-CDI is slowly dripped into the PEG-PEI, the PCL-PEI and the PEG-PEI are stirred at room temperature for reaction for 24 hours, a dialysis bag (MWCO=5 kDa) is used for dialysis for 24 hours in 1000mL of chloroform, part of the chloroform is removed under reduced pressure, then the PEG-PEI is precipitated into anhydrous diethyl ether, and a white powder product is obtained through filtration and drying, and the yield is 86%;
s3, preparation of polyethylene glycol-polyethyleneimine-polycaprolactone-supported SPIO nano particles and medicines (PEG-PEI-PCL-SPIO/drugs)
SPIO (superparamagnetic ferroferric oxide) is described in literature [ S.H.Sun, H.Zeng, D.B.Robinson, S.Raoux, P.M.Rice, S.X.Wang, G.X Li. Monodiperse MFe 2 O 4 (m=fe, co, mn) nanoparticles.j.am.chem.soc.2004,126, 273-279) the synthesis of iron acetylacetonate Fe (acac) 3 1.4126g (4 mmol), 5.16g (20 mmol) of 1, 2-hexadecanediol, 3.8ml (12 mmol) of oleic acid, 3.8ml (12 mmol) of oleylamine, and then adding 40ml of dibenzyl ether under the protection of nitrogen to stir and dissolve, heating to 200 ℃ in a sand bath, refluxing and stirring for 2h, and then heating to 300 ℃ and refluxing for 1h, wherein the reaction system is changed from dark red to black slowly; naturally cooling in air, centrifuging the precipitate in 150ml ethanol at 10000rpm for 5min, removing supernatant, and dissolving the lower precipitate in water containing 4 drops70ml of oleic acid and oleylamine are added into 70ml of normal hexane, then the insoluble part is removed by centrifugation for 10min at 10000rpm, the solution is precipitated into 200ml of ethanol again, the solution is centrifuged for 10min at 10000rpm, the lower layer precipitate is dissolved into 60ml of normal hexane, argon is introduced for protection, and the solution is preserved at 4 ℃ for standby;
blowing the SPIO normal hexane solution to dry, weighing and collecting 5mg of SPIO nano particles in a serum bottle (10 mL), weighing 50mg of PEG-PEI-PCL polymer and 5mg of Prodigiosin, dissolving and uniformly mixing the PEG-PEI-PCL polymer and the Prodigiosin with chloroform (3 mL), dropwise adding the solution into 20mL of distilled water under ultrasonic dispersion, volatilizing to remove the chloroform, centrifuging at a rotating speed of 12000 r/mm, collecting precipitate, and discarding supernatant. Dissolving the precipitate with water, performing ultrasonic dispersion, repeating centrifugal operation, finally performing ultrasonic dispersion on the prepared PEG-PEI-PCL-SPIO/drug nano particles into water, filtering with a needle filter with the aperture of 220nm, adding purified water, adjusting the concentration of the PEG-PEI-PCL-SPIO/drug nano particles to reach the Fe content of 0.145mg/mL at constant volume, and preserving the product at 4 ℃ for later use;
s4, preparation of antibody-targeted polyethylene glycol-polyethyleneimine-polycaprolactone-loaded SPIO nano particle/drug (Fab-PEG-PEI-PCL-SPIO/drug)
The desmoplakin antibody is first cleaved by the method in the prior literature to obtain the Fab fragment of desmoplakin, and then purified. Then the desmoplakin-Fab is linked to mal-PEG-COOH, and then the PEG connected with the antibody is reacted with the amino on the PEG-PEI-PCL-SPIO nano particle by amidation reaction to prepare Fab-PEG-PEI-PCL-SPIO;
the specific operation is as follows: 10mg of desmoplakin antibody was weighed out at 0.5 mg.ml -1 Papain of (10 mmol.L) -1 Cysteine of (2 mmol.L) -1 Is subjected to enzymolysis for 4 hours at pH 7.6. Separating the enzymolysis product by protein A affinity chromatography, purifying the penetration peak by DEAE anion exchange chromatography, dialyzing to remove salt, and lyophilizing to obtain Fab fragment of desmoplakin with higher purity;
1mg of the Fab fragment of desmoplakin (Mn=45 kDa) was weighed and pretreated with EDTA solution (500. Mu.L 0.5M) at 4℃for 15min. 5ml of PBS solution was added for dissolution, 1mg of dithiothreitol was added and the mixture was reacted at 25℃for 30 minutes. After removal of dithiothreitol by centrifugation through a centrifuge ultrafiltration tube having a molecular weight cut-off of 1k, 5ml of PBS solution was added for dissolution, and mal-PEG-COOH (2 mg, mn=4k) was added for uniform mixing and left at 4℃overnight. The excess mal-PEG-COOH was removed by centrifugation through a 5k cutoff centrifuge tube. Activating carboxyl in Fab-PEG-COOH with 500 mug of EDC and NHS for 15min, adding 16mL of PEG-PEI-PCL-SPIO/drug prepared in the step 3, reacting at 4 ℃ overnight, finally removing excessive EDC and NHS small molecular impurities by ultrafiltration and centrifugation, removing unconnected antibodies by centrifugation at 12000r/min, collecting solid solution, ultrasonically dispersing into distilled water, and adjusting the concentration of Fab-PEI-SPIO/drug nano particles to 0.145mg/mL for standby;
s5, preparation of therapeutic gene composite nano particles
The positively charged PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticles and negatively charged ISL1-siRNA can be compounded into a nano-composite through electrostatic action. The specific operation is as follows: 400 μg of ISL1-siRNA was diluted with PBS to a final volume of 1.5mL and shaken well. Uniformly dispersing 1.5mL of PEG-PEI-SPIO prepared in the step 3 (or 1.6mL of Fab-PEG-PEI-SPIO prepared in the step 4) by ultrasonic waves, uniformly mixing an ISL1-siRNA diluted solution with PEG-PEI-SPIO (or Fab-PEI-SPIO) nanoparticle solution, fixing the volume of a compound system to the Fe content of 0.061mg/mL, blowing and uniformly mixing, and standing for 30 minutes to prepare a uniform compound;
s6, synthesis of PEG-polypeptide
0.05mmol of matrix metalloproteinase 9 (matrix metalloproteinase, MMP 9) -sensitive polypeptide (DNP-Pro-Cha-Gly-Cys (Me) -His-Ala-Lys (N-Me-Abz) -NH2, molecular weight: 1077.22 Da), 5mmol of EDC and 5mmol of DMAP were dissolved in 10mL of acetonitrile in water (acetonitrile: water=1:1), and magnetically stirred for 2h under N2 protection on an ice-water bath at 500rpm to activate Peptide. After 2h, 0.5mmol of PEG-NHS (molecular weight 3000 Da) was added and the reaction continued for 72h. After the reaction is finished, placing the reaction solution into a dialysis bag (MWCO=3.5 kDa), dialyzing for 72 hours, and freeze-drying to obtain a product PEG-polypeptide;
preparation of S7, PEG-polypeptide modified liposome shell @ therapeutic gene composite nano-particles
PEG-polypeptide and cholesterol (each 20mg in weight) were dissolved in 5mL of dichloromethane, and the dichloromethane was spin-dried using a vacuum spin-on apparatus to form a thin film of liposomes on the walls of the round-bottomed flask. 2mL of the therapeutic gene composite nanoparticle prepared in the step 5 is dropwise added into a liposome film formed by the PEG-polypeptide and cholesterol at a speed of 0.5mL/min under slow stirring. And (3) continuing stirring for 30min after the dripping is finished, fully assembling the liposome and the therapeutic gene composite nano particles, and finally separating the liposome loaded with the therapeutic gene composite nano particles from the empty liposome by using a strong magnet. Finally, adding 2mL of physiological saline (0.9% NaCl) solution to dissolve the PEG-polypeptide modified liposome shell@therapeutic gene composite nano particles, wherein the aperture is 220nm, the filtration rate of a needle filter is increased, the volume is fixed until the Fe content is 0.061mg/mL, and the mixture is preserved at 4 ℃ for later use.
The specific structural schematic diagrams of the prepared MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector are shown in figure 1.
Examples 2-4, comparative examples 1-6:
examples 2-4 or comparative examples 1-6 can be prepared by varying the amounts of polymer, drug and SPIO dosed in step S3 or omitting any of steps S3, S4, S5, S6, S7 as compared to example 1, see table 1 below:
table 1: examples and comparative examples
Functional evaluation experiment
1. Nuclear Magnetic Resonance (MRI) experiments to evaluate placenta-specific delivery function of drugs in pregnancy-associated trophoblast tumor models
Model establishment and weight detection:
8 week old SPF-class immunodeficiency model BALB/c-nu mice (purchased from medical laboratory animal center, guangdong province) were placed in SPF light-controlled feeding environment and subjected to light/dark cycle at constant temperature of 22+ -2deg.C and humidity of 60% for 12 hours, and food and drinking water were freely obtained. Body weight was monitored daily.
Building tumor blocks: 10 7 The JEG-3 trophoblastic cancer cells are inoculated under the skin of the right rib of a mouse, 14d of tumor growth after the tumor formation is performed, the tumor is cut, necrotic tissues are removed, and good growth tissues are cut to form 0.5mm 3 Is used for planting tumors.
Tumor inoculation of pregnant mice: female mice and male mice 2:1 mating in estrus, the next day female vaginal secretion smear on Papannicola Wu Ranse, and the person observing sample vaginal sperm positivity under light microscope is diagnosed as pregnant, marked as day 0 of gestation (D0). On the 8 th day of model establishment, after anesthetizing pregnant mice, opening the abdomen, selecting 1 embryo, puncturing the uterine wall with ophthalmic forceps on the placenta side of uterus, feeding fresh tumor into placenta, suturing the rupture, closing the abdomen after hemostasis by compression, and establishing a gestation combined trophoblast tumor model.
Detection of placenta distribution of drug by MRI imaging:
gestation-combined trophoblastic tumor model animals were scanned on day 11 after chloral hydrate anesthesia at time points of pre-drug injection (0 h) and 2 hours post-injection (2 h) for MRIT2 sequences and SPIO-containing nano-drug in vivo distribution was observed. The dose of the tail vein injection nano-drug is as follows: (therapeutic dose 0.31mg/Kg iron equivalent drug, or equivalent volume of physiological saline);
c57BL/6j mice uterine MRI imaging was performed using a Philips Intra 1.5T MRI scanner, and its animal-specific coils. Signal intensity evolution of uterus and embryo regions in mice is observed on MRIbTFE sequence, T2map imaging technology is adopted to measure T2 relaxation time changes caused by SPIO in the drug in vivo uterus, placenta, embryo and other organ distribution, and relaxation rates R2 at 0h and 2h are calculated. Calculate the relative increase ratio of R2 at 2h post drug injection (RSI (Relative Signal Intensity)% = R2) 2h /R2 0h ) The results are shown in Table 2.
TABLE 2 placenta-specific delivery function evaluation results
From the above results, it is clear that, in comparative example 1, the placenta trophoblast and tumor cell surface marker antibody are not linked, and after the polypeptide-PEG modified lipid bilayer disintegrates, the content of the drug cannot be anchored to TB cells to obtain placenta retention, and a large amount of the drug leaks through placenta barriers, so that lower placenta RSI is detected; the drug gathers in the embryo, resulting in higher embryo RSI; the inability of the drug to anchor to TB cells and tumor cells to obtain placental retention also results in partial drug release from the placenta and systemic distribution leading to higher liver RSI.
The delivery system of comparative example 2 does not contain polypeptide-PEG modified lipid bilayer membranes as the outer shell, and cannot achieve targeted release against the placental microenvironment; in addition, desmoplakin antibodies target a variety of cells expressing desmoplakin in vivo, including TB, and cell membrane targeting is not strong; therefore, lower placenta RSI is detected and lower liver RSI; the drug without lipid membrane had smaller particle size and entered the placenta, and a higher RSI in the embryo was detected by passing the placenta barrier in a larger proportion.
The delivery systems of comparative examples 3 and 4, without desmoplakin antibody, failed to target anchoring of the drug into the placenta to TB cells and tumor cells, failed to obtain placenta retention, leaked across the placenta barrier in large amounts, detected lower placenta RSI; the drug aggregates in the embryo, resulting in a higher RSI in the embryo. Meanwhile, the lipid bilayer membrane shell of comparative example 3 is not modified by enzyme-sensitive polypeptide, and the distribution of placenta is reduced, which also results in lower placenta RSI and higher liver RSI. Comparative example 4 has no lipid bilayer membrane envelope, the RSI of placenta is lower and the RSI of liver is higher than comparative example 3.
The particle size of comparative example 5 is too large, so that the internal circulation distribution effect is extremely poor, the medicine is mainly phagocytized by reticuloendothelial system of the liver, so that the RSI of the liver is obviously higher, and the placenta RSI is obviously lower; but its large particle size retards its leakage across the maternal-fetal barrier, so embryo RSI is low. Comparative example 6 has a much larger particle size than comparative example 5 and a worse circulation, so that its liver RSI is higher than comparative example 5; the particle size was larger and less likely to leak across the maternal-fetal barrier, so that placenta RSI was lower than comparative example 5.
In examples 1-4, a lipid bilayer membrane modified by a substrate polypeptide of MMP 9-PEG is taken as a shell, a drug carrier modified by a placenta trophoblast cell surface marker antibody is taken as a kernel, and an MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting carrier with a double-layer structure is synthesized, wherein the particle size range is 80-205nm. The particle size of about 100nm and the outer negative lipid bilayer membrane are convenient for avoiding being phagocytized by reticuloendothelial system in a large amount, so that the internal circulation time is prolonged, and the effective internal circulation is realized. The substrate polypeptide-PEG modified lipid bilayer membrane shell is stable in the circulation of other tissues and organs in the body, reaches the placenta and tumor microenvironment of the specific high-expression MMP9, disintegrates along with the degradation of the polypeptide, and realizes the specific distribution of the drug in placenta tissues. The drug shell revealed a drug core containing desmoplakin antibody fragments after disintegration of the placenta and tumor microenvironment. The desmoplakin antibody fragment can be anchored in TB cells and tumor cells with high cell membrane specificity and high expression desmoplakin in placenta, promotes the drug to realize the regulation and control of the functions of the TB and the tumor cells after being specifically endocytosed by the TB cells and the tumor cells, reduces the distribution in other cells of the placenta, and reduces the influence on the functions of the placenta. The desmoplakin antibody enables the drug in the placenta to be anchored to TB cells and tumor cells, and also effectively reduces the leakage of the drug through the maternal-fetal barrier and reduces the arrival of the drug at the embryo.
2. Establishing gestation-combined trophoblastic tumor animal model to evaluate treatment effect
Drug treatment (therapeutic dose 0.31mg/Kg iron equivalent drug, or equivalent volume of physiological saline) was injected at D3, D6, D9, D12, D15, and serial measurements were performed at D17, with the results shown in table 3:
placenta and miscarriage examination: placental tissue, namely, killing pregnant mice, opening abdominal cavity, dissecting uterus, taking out the fetuses and placenta in sequence, and recording the number of surviving fetuses. Removing placenta membrane and umbilical cord, cutting off umbilical cord at the fetal head along umbilical root, placing placenta and fetal head on sterile gauze, sucking surface amniotic fluid, and weighing placenta and fetal head with analytical balance. Placenta of the planted tumor is cut, the weight of the tumor is weighed, and the size of the tumor is detected. Tumor volumes were calculated using the following formula:
tumor volume (mm) 3 ) =0.5× (long diameter×short diameter 2 )。
TABLE 3 evaluation of therapeutic Effect by gestation-associated trophoblastic tumor animal models
From the above results, it can be seen that in comparative example 1, the placenta trophoblast and tumor cell surface marker antibody are not linked, and after the polypeptide-PEG modified lipid bilayer disintegrates, the drug contained therein cannot be anchored to the TB cells and tumor cells to obtain placenta retention, and a large amount of drug leaks across the placenta barrier, so that poor therapeutic effect, larger tumor volume in the placenta, lower weight of the fetus and lower litter size are detected; meanwhile, the medicine gathers in embryo, which leads to embryo toxicity, and also leads to lower weight and lower litter size.
The delivery system of comparative example 2 does not contain polypeptide-PEG modified lipid bilayer membranes as the outer shell, targeting release against placenta and tumor microenvironment is not achieved; the desmoplakin antibody targets desmoplakin-expressing cells in vivo including TB and tumors, has weak cell targeting, poor therapeutic effect detected, larger tumor volume in placenta, lower weight of embryo and lower litter size. Meanwhile, the medicine without lipid membrane has smaller particle size, enters the placenta, passes through the placenta barrier in a larger proportion, and also has lower weight and lower litter size.
The delivery systems of comparative examples 3 and 4, which did not contain desmoplakin antibody, failed to target anchoring of the drug into the placenta to TB and tumor cells, failed to obtain placenta retention, resulted in massive leakage across the placenta barrier, detected poor therapeutic effect, large tumor volume in the placenta, low weight of the embryo, and low litter size. Meanwhile, the lipid bilayer membrane shell of comparative example 3 is not modified by enzyme-sensitive polypeptide, the distribution of placenta is reduced, the treatment effect is detected to be poor, the tumor volume in the placenta is large, the weight of the embryo is low, and the number of born is low. Comparative example 4 has no lipid bilayer membrane envelope and has a poorer therapeutic effect than comparative example 3.
The comparative examples 5 and 6 have too large particle size, which results in poor in-vivo circulation distribution effect, and the drug is largely phagocytized by reticuloendothelial system of liver, resulting in insufficient distribution of placenta drug, poor therapeutic effect, large tumor volume in placenta, low weight of embryo, and low litter size. The particle size of comparative example 6 is much larger than that of comparative example 5, and the circulation distribution is worse, so that the therapeutic effect is worse than that of comparative example 5.
In examples 1-4, a lipid bilayer membrane modified by a substrate polypeptide-PEG of MMP9 is used as a shell, a drug carrier modified by a placenta trophoblast and tumor cell surface marker antibody is used as a kernel, and an MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting carrier with a bilayer structure is synthesized, wherein the particle size range is 80-205nm. The particle size of about 100nm and the outer negative lipid bilayer membrane are convenient for avoiding being phagocytized by reticuloendothelial system in a large amount, so that the internal circulation time is prolonged, and the effective internal circulation is realized. The substrate polypeptide-PEG modified lipid bilayer membrane shell is stable in the circulation of other tissues and organs in the body, reaches the placenta and tumor microenvironment of the specific high-expression MMP9, disintegrates along with the degradation of the polypeptide, and realizes the specific distribution of the drug in the placenta and tumor tissues. The drug shell revealed a drug core containing desmoplakin antibody fragments after disintegration of the placenta and tumor microenvironment. The desmoplakin antibody fragment can be anchored in a TB and a tumor cell which are in cell membrane specificity and highly express desmoplakin in the placenta, can promote the drug to realize the regulation and control of the TB function after being specifically endocytosed by the TB and the tumor cell, can reduce the distribution in other cells of the placenta, can reduce the influence on the placenta function, and can realize better treatment effect through the effective regulation and control of the TB and the tumor function. The desmoplakin antibody enables the drug in the placenta to be anchored to TB and tumor cells, effectively reduces the leakage of the drug through the maternal-fetal barrier, reduces the arrival of the drug at the embryo, and has less toxicity to the embryo.
3. Toxicity evaluation of drugs for animal models
Mice in the control group were bled from the tail vein 72 hours after drug injection, and were tested for the liver function index glutamic pyruvic transaminase (alanine transaminase, ALT), total bilirubin (TBil), and the kidney function index blood urea nitrogen (blood urea nitrogen, BUN) and serum creatinine (sCr). The detection instrument is a Hitachi 7600 type full-automatic biochemical analyzer, and the detection result is shown in Table 4.
TABLE 4 toxicity evaluation results
From the results, the MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector prepared by the invention has no obvious toxic or side effect on a mother and a fetus.

Claims (5)

1. An MMP enzyme sensitive placenta microenvironment and tumor microenvironment targeting vector, characterized in that the targeting vector comprises
A core-shell bilayer structure wherein the target is obtained by contacting an enzyme highly expressed in the placental interstitial fluid and the tumor interstitial fluid
The lipid bilayer membrane modified by the polypeptide-PEG of the enzyme substrate which disintegrates is taken as an outer shell, and placenta trophoblast and tumor cell surfaces are taken as the outer shell
Drug carrier modified by co-specificity high-expression marker antibody is used as inner core, and superparamagnetic tetraoxide is loaded in the drug carrier
Three-iron SPIO nano particles, small molecule drugs for regulating and controlling placenta trophoblast functions, therapeutic genes or a combination thereof;
the small molecule drug is Prodigiosin, and the therapeutic gene is siRNA for inhibiting ISL1 gene expression;
the enzyme substrate polypeptide is DNP-Pro-Cha-Gly-Cys (Me) -His-Ala-Lys (N-Me-Abz) -NH2;
the enzyme which is commonly and highly expressed by the placenta interstitial fluid and the tumor interstitial fluid is matrix metalloproteinase 9;
the marker antibody with common specificity and high expression on the surface of the placenta trophoblast and the surface of the tumor cell is a Fab segment of the desmoplakin antibody;
the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester, and the copolymer is polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL;
the average particle size of the MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector is 80-300 nm.
2. The MMP enzyme-sensitive placental microenvironment and tumor microenvironment targeting vector according to claim 1, wherein the MMP enzyme-sensitive placental microenvironment and tumor microenvironment targeting vector has an average particle size of 100nm to 205nm.
3. The method for preparing the MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector according to any one of claims 1-2, comprising the following steps:
s1, loading superparamagnetic ferroferric oxide SPIO nano particles, micromolecular drugs for regulating and controlling functions of placenta trophoblast and/or genes into a copolymer to obtain composite nano particles;
s2, linking a placenta trophoblast cell surface and a tumor cell surface common marker antibody or antibody fragment to the composite nanoparticle to obtain an antibody composite nanoparticle;
s3, linking the enzyme substrate polypeptide which is commonly and highly expressed by the placenta tissue interstitial fluid and the tumor tissue interstitial fluid with PEG to obtain polypeptide-PEG;
s4, mixing the polypeptide-PEG and the liposome to form a polypeptide-PEG modified lipid bilayer membrane;
s5, assembling the polypeptide-PEG modified lipid bilayer membrane and the antibody composite nano particles into an MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector.
4. The preparation method of the MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector according to claim 3, wherein in the step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15:1.
5. Use of the MMP enzyme-sensitive placental microenvironment and tumor microenvironment targeting vector according to any one of claims 1-2 in the preparation of a medicament for modulating placental trophoblastic dysfunction, even leading to a malignant disease, said modulating placental trophoblastic dysfunction even leading to a malignant disease being a gestational combined trophoblastic tumor.
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