CN114272390B - Microenvironment targeted combined cell targeted tumor inhibition carrier and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a microenvironment targeted combined cell targeted tumor suppression carrier and a preparation method and application thereof, wherein the targeted tumor suppression carrier has a core-shell double-layer structure, an enzyme substrate polypeptide-PEG modified lipid bimolecular membrane which is targeted and disintegrated under the action of an enzyme which is contacted with interstitial fluid of placenta and highly expressed is taken as a shell, a drug carrier modified by a placenta trophoblast surface specificity high expression marker antibody is taken as an inner core, and the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester. The targeted tumor suppression carrier can effectively avoid the absorption of nonspecific drugs of other organs outside the placenta of the mother and the fetus, thereby realizing the delivery and function regulation of specific drugs of the nourishing cells in the placenta.
Description
Technical Field
The invention relates to the field of biomedical engineering, in particular to a microenvironment targeted combined cell targeted tumor inhibition carrier and a preparation method and application thereof.
Background
While malignant tumors during pregnancy have not been common in the past, the incidence of pregnancy complicated with malignant tumors has been increasing with the late average gestational age, the opening of "bikid" policy, and the development of assisted reproductive technologies in recent years. Reports show that in the death of pregnant and lying-in women caused by indirect obstetrical factors in Beijing city in 2010-2019, malignant tumors account for 7.14%. Pregnancy combined with malignant tumor enters five sites before the death cause of pregnant and lying-in women, and brings great influence on the safety of the mother and the child [ practical J. obstetrics and gynecology 2021,37(03): 178-. Among pregnancy complicated with malignant tumors, the most troublesome to handle is pregnancy complicated with trophoblastic tumors which are developed in the placenta of pregnant women. The pathological characteristics of these tumors are that the tumors occur in the placenta implantation site, and are solid tumors with different sizes, and the tumor bodies can be confined in the uterus, can protrude into the uterine cavity to form polyps, or invade the muscular layer and even break the serosa to form metastasis [ journal of Utility and obstetrics, 2006(09): 548-. The tumors are present in the placenta and are inseparable from the placenta tissue, the tumors cannot be treated by adopting an operation method, and if the tumors are treated by adopting medicaments, fetuses are often injured to cause abortion. Therefore, if the specific killing of trophoblast-derived tumor cells in placenta can be realized, the progress of diseases without effective treatment means can be effectively controlled.
The development of trophoblastic tumors is associated with excessive activation of the NF- κ B pathway [ Sci rep.2020 Aug 20; 14033 is used in 10 (1); the J of cell and molecular immunology 2012,28(10):1084-1087 ] Bortezomib (PS-341) is a reversible and selective proteasome inhibitor, has the functions of destroying cell cycle, inducing apoptosis and inhibiting nuclear factor NF-kB, and has anticancer activity [ blood.2018 Dec 13; 132(24) 2546 and 2554; protein cell.2018 Sep; 9(9), 770- > 784 ]. Therefore, we expect to achieve the inhibition of trophoblastic tumors with Bortezomib. DEFA1B (Defensin Alpha 1B) is a gene related to the response and resistance of therapeutic drugs such as paclitaxel [ Onco Targets Ther.2015 Jul 30; 1915-22. And, inhibition thereof can also effectively inhibit the activity of TB and TB-related tumors per se. In our previous experiments, it has been found that the combination of these two drugs can achieve better inhibition of trophoblastic tumors. Therefore, if a trophoblast tumor suppressor drug can be delivered to diseased cells in the placenta, a better therapeutic effect can be expected.
However, the existing drugs which may have a TB function modulating effect or a trophoblastic tumor suppressing effect are inevitably distributed in each organ of the mother and are distributed to the fetus through the placenta. These drugs, while modulating TB function, can produce maternal and fetal toxicity. The drug administration for pregnant women including emergency drugs at present has a lot of contraindications, and the problems of drug distribution and toxicity of the drugs in the mother and fetus need to be considered in the drug use and new drug development of the pregnant women are the first problems. Most of the drugs can pass through the placenta and distribute into the side of the fetus, affecting the development of the fetus. Therefore, the existing medicines for realizing the function regulation of TB such as EMT, angiogenesis promotion and the like in-vitro experiments cannot really realize the function regulation of the TB in the placenta on the premise of ensuring the safety of medication. Therefore, how to avoid toxicity to the mother and fetus and realize the accurate delivery of the TB function regulating drug to TB in the placenta is the key to solve the disease symptoms of pregnancy combined with trophoblastic tumor and the like caused by TB dysfunction.
The approaches that researchers try to promote the delivery of TB-specific nano-drugs include 2, one is to increase the particle size of nano-drugs, so that the nano-drugs cannot pass through a fetal membrane barrier and are retained in placenta to generate drug delivery effect; the other is specific delivery of antibody modified nano-carriers aiming at TB cell membrane markers.
The principle of increasing the particle size of the nano-drugs and promoting the distribution of the drugs in the placenta is that experimental research finds that the nano-drugs less than 300nm cannot be retained in the placenta and easily enter the fetus through the placenta. Researchers have therefore attempted to synthesize nanomedicines with particle sizes > 300nm, which are retained in the placenta, resulting in functional regulation of various cells including placental TB. However, too large a particle size (> 100nm) of the drug is detrimental to the in vivo distribution of the drug. Most of the nano-drugs with the particle size of more than 300nm are captured by a reticuloendothelial system in maternal circulation, generate side effects at all parts of the whole body, can reach the placenta and realize low proportion of specific distribution of TB. Therefore, other ways to achieve the retention of the nano-drug in the placenta and the targeting of TB cells are needed.
The nano-drug can adopt a nano-drug linked antibody to target and identify the cell membrane marker of the target cell, thereby realizing the specific delivery of the target cell. TB has some established surface markers (e.g., Cytokeratin 7) that are distinct from other placental stromal cells in placental tissue. Cytokeratin 7 is distinguishable from other cells in the placenta. However, analysis of the expression level of multiple organ tissues throughout the body revealed that some cells of the surface marker were expressed in other parts of the placenta. The expression abundance on the surface of a small number of high-expression cells is not significantly different from TB. If the antibody of the Cytokeratin 7 is connected to the surface of the nano-drug carrier, the direct in vivo application will cause side effects on other cells expressing the marker Cytokeratin 7 in vivo. Therefore, only before entering the placenta in 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 in cells outside the placenta, and the TB cell recognition antibody can be ensured to be distributed in the placenta.
It should be noted that Cytokeratin 7 is also highly expressed in the cell membranes of various tumors including trophoblastic tumors, and can be distinguished from other placental stromal cells in placental tissues other than TB.
In summary, a nano-carrier system capable of effectively avoiding the nonspecific drug absorption of the mother and the fetus and further realizing the specific drug delivery and function regulation of the TB cells in the placenta is absent at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a microenvironment targeted combined cell targeted tumor suppression carrier, the targeted tumor suppression carrier utilizes the placenta microenvironment targeted to reduce the distribution of the drug in maternal organ tissues before entering the placenta, and utilizes the nourishing cell membrane markers to reduce the distribution of the drug in fetal organ tissues after passing through the placenta in a targeted manner, so that the absorption of nonspecific drugs of other organs outside the maternal placenta and the fetus can be effectively avoided, and the delivery and the function regulation of the nourishing cell specific drug in the placenta can be further realized.
The invention also aims to provide a preparation method of the microenvironment targeted combined cell targeted tumor suppressor vector.
The invention is realized by the following technical scheme:
a microenvironment targeted combined cell targeted tumor suppression carrier 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 an enzyme which is contacted with high expression of placental interstitial fluid is used as a shell, a marker antibody modified drug carrier which is specifically and highly expressed on the surface of a placental trophoblast cell is used as an inner core, and the enzyme which is highly expressed in the placental interstitial fluid is one or more of matrix metalloproteinase 13 (MMP 13), lysozyme, histaminase or oxytocin; the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester; the marker antibody with high surface specificity expression of the placenta trophoblast is an Fab segment of endoglin Cytokeratin 7; 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 placenta of the pregnant woman is rich in a plurality of enzymes for promoting the development of the placenta and nutrition of a fetus, the enzyme highly expressed by the placenta interstitial fluid is one or more of matrix metalloproteinase 13, lysozyme, histaminase or oxytocin, wherein the expression level of the matrix metalloproteinase 13 in the placenta interstitial fluid is very high, and the matrix metalloproteinase 13 is hardly expressed in the blood and the interstitial fluid of a normal human body, so that the matrix metalloproteinase 13 is preferred.
The substrate polypeptide of the matrix metalloproteinase 13 can be MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH2, and the molecular weight is as follows: 1221.32 Da.
The drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester, 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 copolymer of the present invention can be synthesized by the prior art, for example, PEG is firstly reacted with polycation carrier to form copolymer, and then the active group of polycation is reacted with the activated polyester segment to form copolymer.
The copolymers of the present invention are also commercially available.
The drug carrier of the invention is loaded with superparamagnetic ferroferric oxide SPIO nano particles, micromolecule drugs for regulating and controlling the function of placenta trophoblasts, therapeutic genes or the combination thereof. The small molecule drug is Bortezomib, and the therapeutic gene is siRNA inhibiting DEFA1B (Defensin Alpha 1B) gene expression.
The average particle size of the targeted tumor inhibition carrier is 80nm-300nm, preferably 100nm-210nm, the particle size is too large to be beneficial to in vivo circulation, the particle size is too small to increase the preparation difficulty and to be beneficial to loading drugs and genes.
The invention also provides a preparation method of the microenvironment targeted and cell targeted tumor suppressor vector, which comprises the following steps:
s1, loading superparamagnetic ferroferric oxide (SPIO) nanoparticles, micromolecular drugs for regulating and controlling the function of placenta trophoblasts and/or genes to the copolymer to obtain composite nanoparticles;
s2, linking the placenta trophoblast surface marker antibody to the composite nanoparticle;
s3, linking enzyme substrate polypeptide for promoting placenta development and fetal nutrition 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 nanoparticles into a targeted tumor suppressor vector.
Preferably, in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15: 1.
The lipid bilayer membrane modified by MMP13 substrate polypeptide-PEG is used as a shell, so that the distribution of a nano transmission system in enzyme-free blood is stable before entering a placental enzyme environment, drug leakage is reduced, and other cells outside the placenta are reduced or avoided from phagocytosis. Thereby ensuring the safety of other tissues and organs outside the maternal placenta; the MMP13 enzyme sensitive shell is disintegrated in a microenvironment containing enzymes at the placenta matrix side to release the medicine, so that the high-efficiency release and distribution of the medicine in the matrix placenta can be ensured; due to the introduction of the enzyme sensitive shell, the distribution efficiency of the placenta can be ensured without adopting a large-particle-size nano-carrier structure, the particle size of the nano-carrier is effectively reduced, the stable circulation distribution of the medicine before entering the placenta is ensured, and the reticuloendothelial system is ensured not to phagocytize a large amount of carriers to cause the reduction of curative effect and the increase of side effect.
The invention adopts the medicine carrier modified by the placenta trophoblast surface marker antibody as the inner core, the medicine is modified by the TB cell surface marker antibody, and the TB cell membrane in the placenta can be anchored exactly after being released, thereby ensuring the specific administration of the TB cell in a complex placenta environment, and simultaneously avoiding unnecessary placenta function damage caused by the administration of other cells in the placenta; most of the medicines entering the placenta are targeted by the antibody and are exactly anchored in TB cells, so that the medicines are ensured to leak through a placenta barrier and enter the side of the fetus, and the safety of the fetus is ensured; after the medicine is anchored on the TB cell membrane, the therapeutic medicine and the therapeutic gene are promoted to be swallowed into the TB cell, so that the function regulation is realized, and the exact TB function regulation is ensured.
The invention also provides application of the double-targeting nano-drug delivery system in preparation of a drug for regulating and controlling the placenta trophoblastic dysfunction disease, wherein the regulating and controlling placenta trophoblastic dysfunction disease is pregnancy combined trophoblastic tumor.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention takes an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which can be targeted and disintegrated under the action of a specific enzyme highly expressed by contacting placenta interstitial fluid as a shell; the drug carrier modified by the marker antibody with high surface specificity expression of the placenta trophoblast is used as an inner core; synthesizing a targeting tumor inhibition carrier with a double-layer structure. The double-layer structure can ensure that the liposome shell structure is stable and keeps stable circulation in the blood circulation of the pregnant woman, so that the nano-drug is not easily captured by other tissues and cells including a reticuloendothelial system, the distribution and release of other tissues except a placenta which are influenced in the body of the pregnant woman are reduced, and the toxic and side effects are reduced;
(2) after the transmission system enters the placenta along with blood circulation, an enzyme substrate in the outer shell of the transmission system is decomposed by corresponding enzyme highly expressed in placenta tissues, and the protective lipid bimolecular outer shell is rapidly disintegrated in the placenta to release the antibody modified nano-drug capable of anchoring TB cell membrane surface marks. The nano-drug is prevented from being absorbed by other tissue cells of a parent body, is specifically anchored on a TB cell membrane in a placenta, is further specifically endocytosed by the TB cell, generates a function regulation and control effect, and ensures that TB cell diseases are treated exactly;
(3) through exact 'antigen-antibody reaction', the medicament is retained in the placenta rich in TB cells after the lipid bimolecular shells are disintegrated, so that the medicament leakage is reduced, the medicament passes through a placenta barrier, and the toxic and side effects on a fetus are reduced; and also avoids affecting vascular endothelial cells, immune cells and other stromal cells in the placenta.
Drawings
Fig. 1 is a schematic structural diagram of the microenvironment-targeted combined cell-targeted tumor suppressor vector prepared in example 1 of the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples, which are, however, not intended to limit the scope of the invention.
The raw materials of the invention are as follows:
the method for measuring the Fe content comprises the following steps:
the Fe content in the nano-drug system is measured by an atomic absorption spectrophotometer method and is used for measuring the dosage of the nano-drug. Weighing a certain amount of prepared drug solution (such as 1mL in step III), lyophilizing, and dissolving to 1mol L -1 The HCl solution is placed for 24 hours to ensure that Fe in SPIO is fully ionized, an atomic absorption spectrophotometer is used for detecting the absorbance of Fe atoms at the position of 248.3nm, the absorbance is substituted into a standard curve made by using a Fe standard solution to calculate the concentration of Fe, and then the content of Fe in the drug solution before freeze-drying is calculated in a reverse way.
The particle size test method comprises the following steps:
the particle size of the sample was measured with a Zeta-Plus potential particle size meter (Brooken Haven) at 25 ℃ at an incident laser wavelength λ of 532nm, an incident angle θ of 90 ° and a temperature of 532 ℃; the average of the three measurements was taken.
Example 1:
s1 synthesis of polyethylenimine grafted polyethylene glycol (PEG-PEI)
The method adopts a two-step method to synthesize polyethyleneimine grafted polyethylene glycol (PEG-PEI), firstly uses carbonyldiimidazole to activate the terminal hydroxyl of monomethyl ether polyglycol, and then reacts with the amino of polyethyleneimine to generate PEG-PEI. The specific operation is as follows: monomethyl ether glycol (8.0g, Mn ═ 2kDa) was weighed into a reaction flask, dried under vacuum at 80 ℃ for 6h, and dissolved by adding THF (60mL) under an argon atmosphere. Carbonyldiimidazole (CDI, 6.4g) was weighed into another reaction flask, and THF with mPEG-OH dissolved therein was slowly dropped into the CDI flask using an isopiestic dropping funnel, and the reaction was stirred at room temperature overnight. Distilled water (0.648mL) was added to inactivate excess CDI and stirring was continued for 30 min. Precipitating the solution into a large amount of cold ether, filtering, and drying in vacuum to obtain white powdery solid mPEG-CDI;
weighing PEI (4.4g, MW 1.8kDa) and adding into a two-mouth bottle (50mL), adding chloroform (20mL) to dissolve and add PEG-CDI (3.2g), stirring at room temperature for 24h, filling the solution into a dialysis bag (MWCO 3.5kDa), dialyzing with chloroform for 24h, concentrating the solution in the dialysis bag under reduced pressure, then precipitating in a large amount of cold ether, filtering and drying to obtain white powder packaged product mPEG-PEI;
s2 synthesis of poly (acetimide) grafted polyethylene glycol grafted polycaprolactone (PEG-PEI-PCL)
Firstly, synthesizing PCL-OH, adding 15g of dried dodecanol into a two-mouth bottle, vacuum-drying at 70 ℃ for 8h, adding 2ml of Sn (Oct) 2 Continuously drying for 0.5h, then adding 400mL of dried epsilon-caprolactone, and stirring and reacting for 24h at 105 ℃; 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 ether, filtering, and drying to obtain a white powdery product with the yield of 96%;
then PCL-CDI is synthesized, 10g of PCL-OH (Mn is 5000) is added into a two-mouth bottle, vacuum drying is carried out for 8h at the temperature of 50 ℃, 7.2g (10eq.) of Carbonyl Diimidazole (CDI) is added after the PCL-CDI is dissolved in 50mL of tetrahydrofuran, argon protection is carried out, room temperature reaction is carried out for 24h, precipitation is carried out in a large amount of anhydrous ether, filtration and vacuum drying at the room temperature are carried out, and a white powdery product is obtained, wherein the yield is 90%;
finally, reacting the PCL-CDI with PEG-PEI to prepare PEG-PEI-PCL, adding 1.6g of PEG-PEI into a 50mL two-mouth bottle, adding 30mL of trichloromethane to dissolve the PEG-PCL, slowly dropping 10mL of trichloromethane solution containing 200mg of PCL-CDI, stirring at room temperature to react for 24h, dialyzing in 1000mL of trichloromethane by using a dialysis bag (MWCO ═ 5kDa) for 24h, removing part of trichloromethane under reduced pressure, then precipitating in anhydrous ether, filtering and drying to obtain a white powder product, wherein the yield is 86%;
s3, preparation of polyethylene glycol-polyethyleneimine-polycaprolactone loaded SPIO nano-particles and drugs (PEG-PEI-PCL-SPIO/drug)
SPIO (superparamagnetic ferroferric oxide) according to the literature [ S.H.Sun, H.Zeng, D.B.Robinson, S.Raoux, P.M.Rice, S.X.Wang, G.X Li.Monodisperse MFe 2 O 4 (M ═ Fe, Co, Mn) nanoparticies.J.am.chem.Soc.2004, 126,273-279 ] iron acetylacetonate Fe (acac) 3 1.4126g (4mmol), 5.16g (20mmol) of 1, 2-hexadecanediol, 3.8ml (12mmol) of oleic acid and 3.8ml (12mmol) of oleylamine are added into a 200ml three-necked bottle, then 40ml of dibenzyl ether is added under the protection of nitrogen gas to be stirred and dissolved, the mixture is heated to 200 ℃ in a sand bath and stirred under reflux for 2h, then heated to 300 ℃ and refluxed for 1h, and the reaction system slowly turns from dark red to black; naturally cooling in air, precipitating in 150ml ethanol, centrifuging at 10000rpm for 5min, discarding the supernatant, dissolving the lower precipitate in 70ml n-hexane containing 4 drops of oleic acid and oleylamine, centrifuging at 10000rpm for 10min to remove insoluble part, precipitating the solution in 200ml ethanol, centrifuging at 10000rpm for 10min, dissolving the lower precipitate in 60ml n-hexane, introducing argon gas for protection, and storing at 4 deg.C;
drying and weighing the normal hexane solution of the SPIO, collecting 5mg of SPIO nano particles in a serum bottle (10mL), weighing 50mg of PEG-PEI-PCL polymer and 5mg of Bortezomib, dissolving and uniformly mixing the PEG-PEI-PCL polymer and the Bortezomib by using trichloromethane (3mL), dropwise adding the solution into 20mL of distilled water under ultrasonic dispersion, volatilizing to remove the trichloromethane, centrifuging at the rotating speed of 12000r/mim, collecting precipitates, and removing a supernatant. Dissolving the precipitate with water, ultrasonically dispersing, repeating centrifugal operation, ultrasonically dispersing the prepared PEG-PEI-PCL-SPIO/drug nanoparticles 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 nanoparticles to 0.145mg/mL with constant volume, and storing the product at 4 ℃ for later use;
s4, preparation of antibody-targeted polyethylene glycol-polyethyleneimine-polycaprolactone-loaded SPIO nano-particles/drugs (Fab-PEG-PEI-PCL-SPIO/drug)
The Cytokeratin 7 antibody is first cleaved by methods known in the literature to obtain Fab fragments of Cytokeratin 7, which are then purified. Then, linking Cytokeratin 7-Fab to mal-PEG-COOH, and reacting the PEG connected with the antibody with amino on PEG-PEI-PCL-SPIO nano particles by amidation reaction to prepare Fab-PEG-PEI-PCL-SPIO;
the specific operation is as follows: 10mg of Cytokeratin 7 antibody was weighed out at 0.5 mg/ml -1 Papain, 10 mmol. multidot.L -1 Cysteine, 2 mmol. multidot.L -1 The enzyme is hydrolyzed for 4 hours under the condition of pH7.6. Separating the enzymolysis product by ProteinA affinity chromatography, further purifying the penetration peak by DEAE anion exchange chromatography, dialyzing, desalting and freeze-drying to obtain a Fab fragment of Cytokeratin 7 with higher purity;
1mg of the Fab fragment of Cytokeratin 7 (Mn. RTM.45 kDa) was weighed and pretreated with EDTA solution (500. mu.L 0.5M) for 15min at 4 ℃.5ml of PBS solution was added to dissolve the solution, 1mg of dithiothreitol was added thereto, and the reaction was carried out at 25 ℃ for 30 min. After removing dithiothreitol by centrifugation in a centrifugal ultrafiltration tube having a molecular weight cut-off of 1k, 5ml of a PBS solution was added to dissolve the dithiothreitol, and mal-PEG-COOH (2mg, Mn 4k) was added thereto and mixed well, followed by standing at 4 ℃ overnight. And then centrifuging by using a centrifugal ultrafiltration tube with the molecular weight cutoff of 5k to remove excessive mal-PEG-COOH. Activating carboxyl in Fab-PEG-COOH by using 500 mu g of EDC and NHS respectively for 15min, then adding 16mL of PEG-PEI-PCL-SPIO/drug prepared in the step 3, reacting overnight at 4 ℃, finally performing ultrafiltration and centrifugation to remove excessive small molecular impurities of EDC and NHS, performing centrifugation at 12000r/min to remove unconnected antibodies, collecting a solid solution, performing ultrasonic dispersion on the solid solution into distilled water, and performing constant volume adjustment on the concentration of Fab-PEG-PEI-PCL-SPIO/drug nanoparticles until the Fe content is 0.145mg/mL for later use;
s5 preparation of therapeutic gene composite nano particle
The PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle with positive electricity and DEFA1B-siRNA with negative electricity can be compounded through electrostatic interaction to prepare a nano compound. The specific operation is as follows: mu.g of DEFA1B-siRNA was diluted with PBS to a final volume of 1.5mL and shaken well. Taking 1.5mL of the PEG-PEI-SPIO prepared in the step (3) (or 1.6mL of the Fab-PEG-PEI-SPIO prepared in the step (4)) nanoparticles, ultrasonically dispersing the nanoparticles uniformly, mixing DEFA1B-siRNA diluted solution and PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle solution uniformly, fixing the volume of the composite system to 0.061mg/mL, blowing, uniformly mixing and standing for 30 minutes to prepare a uniform composite;
s6, synthesis of PEG-polypeptide
0.05mmol of matrix metalloproteinase 13-sensitive polypeptide (MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH2, molecular weight: 1221.32Da), 5mmol of EDC and 5mmol of DMAP were dissolved in 10mL of an aqueous acetonitrile solution (acetonitrile: water ═ 1:1), protected with N2 in an ice-water bath and magnetically stirred at 500rpm for 2h to activate Peptide. After 2h 0.5mmol PEG-NHS (molecular weight 3000Da) was added and the reaction was continued for 72 h. After the reaction is finished, putting the reaction solution into a dialysis bag (MWCO is 3.5kDa), dialyzing for 72h, and freeze-drying to obtain a product PEG-polypeptide;
s7, preparation of PEG-polypeptide modified liposome shell @ therapeutic gene composite nanoparticle
PEG-polypeptide and cholesterol (20 mg each) were dissolved in 5mL of methylene chloride and the methylene chloride was spun dry using a vacuum rotary vacuum to form a thin film of liposomes on the wall of the round bottom flask. 2mL of the therapeutic gene composite nanoparticle prepared in the step 5 is added dropwise into the liposome film formed by the PEG-polypeptide and cholesterol at the speed of 0.5mL/min under slow stirring. And (3) continuing stirring for 30min after the dropwise addition is finished, fully assembling the liposome and the therapeutic gene composite nanoparticles, and finally separating the liposome loaded with the therapeutic gene composite nanoparticles from the empty liposome by using strong magnets. And 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 syringe filter is constant volume until the Fe content is 0.061mg/mL, and the solution is stored at 4 ℃ for later use.
The specific structural schematic diagram of the prepared microenvironment targeting combined cell targeting tumor suppressor vector is shown in fig. 1.
Examples 2-4, comparative examples 1-6:
compared with example 1, examples 2-4 or comparative examples 1-6 can be prepared by changing the dosage of the polymer, the drug and the SPIO in step S3 or omitting one of steps S3, S4, S5, S6 and S7, and the following table 1 specifically shows:
table 1: examples and comparative examples
Function evaluation test
1. Magnetic Resonance Imaging (MRI) assay to evaluate the placenta-specific delivery function of drugs in a tumor model of gestational merged trophoblastic tumors
Model establishment and weight detection:
an 8-week-old SPF-grade immunodeficient model BALB/c-nu mouse (purchased from the center of medical laboratory animals, Guangdong province) was housed in an SPF light-controlled rearing environment, and subjected to light/dark cycles of 12 hours at a constant temperature of 22. + -. 2 ℃ and a humidity of 60% to freely obtain food and drinking water. Body weight was monitored daily.
Tumor mass establishment: 10 7 Inoculating JEG-3 trophoblastic cancer cells under the skin of right costal region of mouse, growing after forming tumor for 14d, cutting tumor, removing necrotic tissue, and cutting well-grown tissue to 0.5mm 3 The fresh tumor mass of (2) is used for tumor implantation.
Tumor inoculation of pregnant mice: female and male mice 2: 1 mating in estrus coops, carrying out Papanicolaou staining on vaginal secretion smears of female mice on the next day, and marking the vaginal sperm-positive person of the sample as pregnancy when the diagnosis is observed under an optical microscope as pregnancy (D0). On the 8 th day of model establishment, the abdomen of the pregnant mouse is opened after anesthesia, 1 embryo is selected, after the placenta side of the uterus punctures the uterine wall by using an ophthalmic forceps, a fresh tumor mass is sent into the placenta, an incision is closed, the abdomen is closed after hemostasis by compression, and a pregnancy merged trophoblastic tumor model is established.
MRI imaging to detect placental distribution of drugs:
pregnancy merged trophoblast tumor model animals were scanned on day 11, after chloral hydrate anesthesia, for the MRIT2 sequence at time points before (0h) and 2h (2h) after drug injection to observe the in vivo distribution of SPIO-containing nano-drugs. The dosage of the tail vein injection nano-drug is as follows: (therapeutic dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline);
c57BL/6j mouse uterus MRI imaging was performed using a Philips Intera 1.5T MRI scanner, with its animal specific coils. The evolution of signal intensity in the uterine and embryonic regions in mice was observed on the MRIbTFE sequence and the relaxation time changes of T2 with SPIO in the drug distributed in the uterus, placenta, embryo and other organs in vivo were measured using T2map imaging technique, calculating the relaxation rates R2 at 0h and 2h, respectively. The relative increase rate of R2 (rsi (relative Signal intensity)% -, R2) at 2h after drug injection was calculated 2h /R2 0h ) The results are shown in Table 2.
Table 2 evaluation results of placenta-specific delivery function
| Group of | Placenta RSI (relative signal multiple) | Embryo RSI (relative signal multiple) | Liver RSI (relative multiple of signal) |
| Physiological saline group | 1.00 | 1.00 | 1.00 |
| Example 1 | 27.42 | 1.06 | 3.37 |
| Example 2 | 26.67 | 2.23 | 2.07 |
| Example 3 | 22.94 | 1.38 | 3.09 |
| Example 4 | 29.66 | 1.02 | 3.79 |
| Comparative example 1 | 10.24 | 18.28 | 5.78 |
| Comparative example 2 | 7.83 | 11.11 | 9.24 |
| Comparative example 3 | 3.51 | 2.85 | 9.07 |
| Comparative example 4 | 3.14 | 2.61 | 11.51 |
| Comparative example 5 | 12.67 | 1.07 | 18.20 |
| Comparative example 6 | 9.31 | 1.01 | 29.92 |
From the above results, in comparative example 1, the placenta trophoblast surface marker antibody is not linked, and after the polypeptide-PEG modified lipid bilayer is disintegrated, the drug in the content cannot be anchored in TB cells to obtain placenta retention, a large amount of drug leaks through the placenta barrier, and low placenta RSI is detected; the drug is gathered in the embryo, which results in high embryo RSI; the failure of the drug to anchor to TB cells to achieve placental retention also results in partial drug detachment from the placenta and systemic distribution, resulting in higher liver RSI.
The delivery system of comparative example 2 does not contain a polypeptide-PEG modified lipid bilayer membrane as a shell, and cannot achieve targeted release for the placenta microenvironment; in addition, the Cytokeratin 7 antibody targets other various cells expressing Cytokeratin 7 in vivo including TB, and the cell membrane targeting property is not strong; therefore, lower placental RSI and lower liver RSI were detected; the drug without lipid membrane has smaller particle size, enters the placenta, passes the placenta barrier in larger proportion, and the embryo RSI is detected to be higher.
The delivery systems of comparative examples 3 and 4, which did not contain the Cytokeratin 7 antibody, failed to target and anchor the drug into the placenta to TB cells, failed to achieve placental retention, leaked a significant amount of the placental barrier, and detected low placental RSI; the drug is accumulated in the embryo, resulting in high RSI of the embryo. Meanwhile, the lipid bilayer membrane outer shell of the comparative example 3 has no enzyme-sensitive polypeptide modification, and the distribution in the placenta is reduced, so that the placenta RSI is lower, and the liver RSI is higher. Comparative example 4, which has no lipid bilayer envelope, has a lower RSI for placenta and a higher RSI for liver than comparative example 3.
The comparative example 5 has extremely poor in vivo circulation distribution effect due to excessively large particle size, and the medicines are mainly phagocytosed by the reticuloendothelial system of the liver in a large amount, so that the RSI of the liver is obviously higher, and the RSI of the placenta is obviously lower; but its large particle size retards its leakage across the maternal-fetal barrier, so the embryo RSI is low. Comparative example 6 has a much larger particle size than comparative example 5 and a much poorer circulation, so its liver RSI is higher than that of comparative example 5; the placenta has a larger particle size and is less likely to leak through the maternal-fetal barrier, so placenta RSI is lower than comparative example 5.
In examples 1-4, a substrate polypeptide-PEG modified lipid bilayer membrane of MMP13 was used as a shell, a drug carrier modified by a placental trophoblast surface marker antibody was used as a core, and a microenvironment targeted combined cell targeted tumor suppressor carrier with a bilayer structure was synthesized, with a particle size range of 80-210 nm. The particle size of the liposome is about 100nm, and the outer negative electricity lipid bilayer membrane is convenient to avoid being phagocytized by a reticuloendothelial system in a large amount, so that the in vivo circulation time is prolonged, and the in vivo effective circulation is realized. The substrate polypeptide-PEG modified lipid bilayer membrane shell is stable in circulation of other tissues and organs in vivo, reaches a placenta microenvironment with high specificity expression MMP13, disintegrates along with degradation of the polypeptide, and realizes drug specificity distribution in placenta tissues. The drug shell disintegrates in the placental microenvironment, revealing the inner drug core containing the Cytokeratin 7 antibody fragment. The antibody fragment of the Cytokeratin 7 can be anchored in a placenta to a TB cell with a cell membrane specificity and high expression of the Cytokeratin 7, so that the regulation of the TB function is realized after the drug is specifically endocytosed by the TB cell, the distribution in other cells of the placenta is reduced, and the influence on the function of the placenta is reduced. The Cytokeratin 7 antibody enables the drug in the placenta to be anchored in TB cells, thereby effectively reducing the drug from leaking through the maternal-fetal barrier and reducing the drug from reaching the embryo.
2. Establishing animal model of gestational combined trophoblastic tumor for evaluating treatment effect
The injection of drugs for treatment (treatment dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline) into D3, D6, D9, D12, and D15, and the serial tests in D17, the test results are shown in table 3:
placenta and litter examination: placenta tissue, pregnant mice were sacrificed, the abdominal cavity was opened, the uterus was dissected open, the litter and placenta were removed in sequence, and the number of surviving litters was recorded. Removing the placenta and umbilical cord from placenta, cutting umbilical cord from the fetus end along the root of umbilical cord, placing placenta and fetus on sterile gauze, sucking out amniotic fluid on the surface, and weighing placenta and fetus with analytical balance. And cutting placenta for planting tumor, and detecting the size of the tumor. Tumor volume was calculated using the following formula:
tumor volume (mm) 3 ) 0.5 × (major axis × minor axis) 2 )。
TABLE 3 animal model evaluation of treatment effect of gestational merged trophoblastic tumors
From the above results, it can be seen that, in comparative example 1, the placenta trophoblast surface marker antibody is not linked, and after the polypeptide-PEG modified lipid bilayer is disintegrated, the drug in the content cannot be anchored to the TB cell and the tumor cell to obtain placenta retention, and a large amount of drug leaks through the placenta barrier, so that the therapeutic effect is poor, the tumor volume in the placenta is large, the weight of the fetus is low, and the litter size is low; meanwhile, the drug is accumulated in the embryo, which causes embryo toxicity, lower weight of the fetus and lower litter size.
The delivery system of comparative example 2 does not contain a polypeptide-PEG-modified lipid bilayer membrane as a shell, and targeted release of the placenta microenvironment cannot be achieved; the Cytokeratin 7 antibody targets in vivo expression Cytokeratin 7 cells including TB, the cell targeting is weak, the detection result is poor, the tumor volume in the placenta is large, the litter weight is low, and the litter size is low. Meanwhile, the medicine without lipid membrane has smaller particle size, and the medicine enters the placenta and passes through the placenta barrier in a larger proportion, so that the weight of the fetus is lower, and the litter size is lower.
The delivery systems of comparative examples 3 and 4, which did not contain the antibody to Cytokeratin 7, failed to target and anchor the drug into the placenta to TB cells, failed to achieve placental retention, resulted in a large number of leaks across the placental barrier, and were detected to be less effective, larger tumor volumes in the placenta, lower litter weights, and lower litter sizes. Meanwhile, the lipid bilayer membrane outer shell of the comparative example 3 is not modified by enzyme-sensitive polypeptide, the distribution in the placenta is reduced, the detected treatment effect is poor, the tumor volume in the placenta is large, the litter weight is low, and the litter size is low. Comparative example 4, without the lipid bilayer membrane shell, was less effective than comparative example 3.
Comparative examples 5 and 6 have an excessively large particle size, resulting in extremely poor in vivo circulation distribution effect, and the drug is mainly phagocytosed by the reticuloendothelial system of the liver in a large amount, resulting in insufficient distribution of the placenta drug, poor therapeutic effect, large tumor volume in the placenta, low litter weight, and low litter size. Comparative example 6, which has a particle size much larger than comparative example 5, has a poorer circulation distribution, and thus has a poorer therapeutic effect than comparative example 5.
In examples 1-4, a substrate polypeptide-PEG modified lipid bilayer membrane of MMP13 was used as a shell, a drug carrier modified by a placental trophoblast surface marker antibody was used as a core, and a microenvironment targeted combined cell targeted tumor suppressor carrier with a bilayer structure was synthesized, with a particle size range of 80-210 nm. The particle size of the liposome is about 100nm, and the outer negative electricity lipid bilayer membrane is convenient to avoid being phagocytized by a reticuloendothelial system in a large amount, so that the in vivo circulation time is prolonged, and the in vivo effective circulation is realized. The substrate polypeptide-PEG modified lipid bilayer membrane shell is stable in circulation of other tissues and organs in vivo, reaches a placenta microenvironment with high specificity expression MMP13, disintegrates along with degradation of the polypeptide, and realizes drug specificity distribution in placenta tissues. The drug shell disintegrates in the placental microenvironment, revealing the inner drug core containing the Cytokeratin 7 antibody fragment. The Cytokeratin 7 antibody fragment can be anchored in the TB cell and the tumor cell of the cell membrane specificity high expression Cytokeratin 7 in the placenta, promote the medicine to realize the regulation and control of the TB function after being specifically endocytosed by the TB cell and the tumor cell, reduce the distribution in other cells of the placenta, reduce the influence on the placenta function, and realize better treatment effect through the effective regulation and control of the TB function. The Cytokeratin 7 antibody enables the drug in the placenta to be anchored in TB cells, thereby effectively reducing the drug leakage through maternal-fetal barriers, reducing the drug reaching embryos and having less toxicity to the fetus.
3. Drug for toxicity evaluation of animal models
At 72 hours after the injection of the drug, the mice in the normal control group were bled from the tail vein, and liver function indices glutamic-pyruvic transaminase (ALT), total bilirubin (TBil), and kidney function indices Blood Urea Nitrogen (BUN) and serum creatinine (sCr) were measured. 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
According to the results, the microenvironment targeted combined cell targeted tumor inhibition vector prepared by the invention has no obvious toxic or side effect on the mother and the fetus.
Claims (5)
1. A microenvironment targeted combined cell targeted tumor suppression carrier is characterized by having 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 contacting with a placenta interstitial fluid highly-expressed enzyme is used as a shell, a placenta trophoblast cell surface specificity highly-expressed marker antibody modified drug carrier is used as a core, and the placenta interstitial fluid highly-expressed enzyme is matrix metalloproteinase 13; the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester; the copolymer is polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL; the marker antibody with high surface specificity expression of the placenta trophoblast is a Fab segment of Cytokeratin 7; superparamagnetic ferroferric oxide SPIO nano particles, micromolecule medicines for regulating and controlling the function of placenta trophoblasts and therapeutic genes are loaded in the medicine carrier; the small molecule drug is Bortezomib, and the therapeutic gene is siRNA inhibiting DEFA1B gene expression;
the average grain size of the targeting tumor inhibition carrier is 80nm-210 nm;
the enzyme substrate polypeptide is MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH 2.
2. The microenvironment-targeted combined cell-targeted tumor suppressor vector of claim 1, wherein the mean particle size of the targeted tumor suppressor vector is in the range of 100nm to 210 nm.
3. The preparation method of the microenvironment targeted combination cell-targeted tumor suppressor vector of any one of claims 1 to 2, comprising the following steps:
s1, loading superparamagnetic ferroferric oxide (SPIO) nanoparticles, micromolecular drugs for regulating and controlling functions of placenta trophoblasts and therapeutic genes to the copolymer to obtain composite nanoparticles;
s2, linking the placenta trophoblast surface marker antibody to the composite nanoparticles to obtain antibody composite nanoparticles;
s3, linking the substrate polypeptide of the matrix metalloproteinase 13 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 nanoparticle into a targeted tumor suppressor vector.
4. The preparation method of the microenvironment targeted combination cell targeted tumor suppressor vector of claim 3, wherein in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15: 1.
5. Use of the nano-drug delivery system of any one of claims 1-2 in the preparation of a medicament for modulating placental trophoblast dysfunction disease, which is gestational merged trophoblastic tumor.
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