CN114224870B - Placenta microenvironment targeted delivery probe and preparation method and application thereof - Google Patents
Placenta microenvironment targeted delivery probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a placenta microenvironment targeted delivery probe and a preparation method and application thereof, wherein the placenta microenvironment targeted delivery probe comprises the following components: a) The outer shell is an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is in targeted disintegration under the action of an enzyme which is in contact with the high expression of placenta interstitial fluid, and B) the inner core is a drug carrier modified by a marker antibody with the high expression of the surface specificity of placenta trophoblast cells; c) 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 microenvironment targeted delivery probe can effectively avoid the non-specific drug absorption of other organs outside the maternal placenta and the fetus, and further realize the delivery and function regulation of the nourishing cell specific drug in the placenta.
Description
Technical Field
The invention relates to the field of chemical and biomedical engineering, in particular to a placenta microenvironment targeted delivery probe and a preparation method and application thereof.
Background
Fetal Growth Restriction (FGR), refers to the inability of a fetus to reach its maximum growth potential as determined by its genetic predisposition in the uterus under the influence of various deleterious factors. It is mainly shown that fetal weight below the tenth percentile of the average body weight of its gestational age, or below two standard deviations of its average body weight, is one of the important causes of perinatal morbidity and mortality. FGR children have 4-6 times of perinatal death as compared with normal weight, and even if the FGR children survive, the physical constitution and the intelligence development of the FGR children are disturbed to different degrees. The causes of IUGR are many and complex, but in various models of pathogenesis, FGR development is considered to be closely related to maternal factors such as placental dysfunction [ Hum reproduced update.2020jun 18;26 (4): 501-513 ].
Most FGR pathogenesis is accompanied by placental trophoblast dysfunction [ placenta.2020jul;96 ] of the formula (I) 10-18 ]. Research currently shows that DNA methylation is crucial in the maintenance of normal placenta function and development of placenta-associated diseases [ hypertension.2020apr;75 (4): 1117-1124 ]. Our earlier studies found that the DNA methylation regulatory gene CTSG [ J Clin invest.2016jan;126 (1) 85-98 ] can realize the regulation and control of the methylation and proliferation invasion functions of the trophoblasts in vitro, and the previous research result proves that the function of the trophoblasts in the FGR environment is expected to be recovered by knocking CTSG out. On the other hand, maintenance and promotion of placental function requires efficient activation of PKC pathway functions [ Cell Biol int.2020dec;44 2409-2415. The product of Phorbol 12-myrristate 13-acetate (PMA, 12-O-Tetradecanoylphosphonol-13-acetate) is a potent PKC activator, which can widely regulate the phosphorylation of various protein sites in trophoblasts, and thus effectively regulate trophoblasts [ Biochem Biophys Res Commun.2012Jan27;417 (4): 1127-32. Activation of PKC pathway and DNA methylation regulation are expected to produce synergy in cells [ J Cell biochem.2011jul;112 1761-72, producing a functional regulatory effect on FGR placenta.
However, these drugs, which may achieve the regulation of TB function, present pathophysiological barriers both in the mother and in the fetus when applied in vivo. Drug use and new drug development in pregnant women requires consideration of both the distribution of the drug in the mother and in the fetus itself, as well as creating toxicity problems in both. Most of the drugs can pass through the placenta and distribute into the fetus, affecting the development of the fetus. Therefore, the medication of pregnant women including emergency drugs has many contraindications. The drugs for pregnant women are classified into 5 types according to teratogenicity, and except for a few drugs with the minimum toxicity which are classified into a and b types, most of the other drugs with c, d and e types have obvious damage to fetuses. The metabolism burden in the pregnant women is heavy during pregnancy, and the immune change is complex. Therefore, even if the drug is not significantly toxic during non-pregnancy, it may cause significant side effects to the pregnant woman. Therefore, the existing medicines which are possibly effective to TB in vitro experiments cannot realize the regulation and control of the TB function under the condition of ensuring the safety of a mother body and a fetus.
After micromolecule medicines or gene therapy medicines possibly having the TB function regulation and control function which are screened by in vitro experimental research enter the circulation of the pregnant woman through injection or oral administration, the medicines can take effect on the whole body cells of the pregnant woman outside the placenta to generate side effects; meanwhile, after the medicine enters the placenta, the medicine rapidly penetrates through a placenta barrier due to abundant blood supply to the side of the fetus, so that the fetus is damaged. Therefore, none of these drugs can be clinically applied. Therefore, at present, no exact pharmaceutical intervention means is provided for the diseases of restricted intrauterine growth of the fetus, placenta implantation and the like clinically. Doctors can only carry out passive symptomatic treatment on the symptoms caused by the placenta dysfunction disease. But not through TB function regulation, realize the real placenta function recovery. Therefore, how to avoid toxicity to the mother and fetus and realize effective delivery of TB function control drugs is the key to solving diseases caused by TB dysfunction.
The current macromolecule nano-carrier drug can realize the specific drug delivery to pathological target cells in various diseases. Development of TB-specific delivery vectors presents significant difficulties as these vectors fail to address the potential for toxic side effects on maternal and fetal non-specific distribution. 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, an excessively large particle size (> 100 nm) of the drug is disadvantageous for 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 everywhere in 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., epithelial cadherin, ecadherin) that distinguish it from other placental stromal cells in placental tissue, and from other cells in the placenta. However, analysis of the expression level of multi-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 Ecadrein 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 Ecadrein 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.
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 lacking at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a placenta microenvironment targeted delivery probe, which reduces the distribution of a medicament in maternal organ tissues before entering a placenta by utilizing the targeting of a placenta microenvironment and reduces the distribution of the medicament in fetal organ tissues after passing through the placenta by utilizing a trophoblast marker, so that the absorption of other organs outside the maternal placenta and fetal nonspecific medicaments can be effectively avoided, and the delivery and function regulation of a nourishing cell specific medicament in the placenta are further realized.
The invention also aims to provide a preparation method of the placenta microenvironment targeted delivery probe.
The invention is realized by the following technical scheme:
a placental microenvironment targeted delivery probe, comprising:
a) The shell is an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is disintegrated in a targeted manner under the action of an enzyme which is in contact with the high expression of placenta interstitial fluid, and the enzyme in high expression of placenta interstitial fluid is one or more of matrix metalloproteinase 1, matrix metalloproteinase 8, matrix metalloproteinase 14, lysozyme, kininase, histaminase or oxytocin;
b) The inner core is a drug carrier modified by a marker antibody with high surface specificity expression of the placenta trophoblast, the drug carrier is a copolymer formed by a polycation carrier modified by polyethylene glycol and hydrophobic degradable polyester, and the marker antibody with high surface specificity expression of the placenta trophoblast is a Fab segment of an epithelial cadherin Ecadherin antibody;
c) 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 pregnant women is rich in a plurality of enzymes for promoting placenta development and fetal nutrition, the enzymes highly expressed in placenta interstitial fluid are one or more of matrix metalloproteinase 1, matrix metalloproteinase 8, matrix metalloproteinase 14, lysozyme, kininase, histaminase, oxytocin or other matrix metalloproteinases, wherein Matrix Metalloproteinase (MMP) 1, matrix metalloproteinase 8 and matrix metalloproteinase 14 family have extremely high expression level in placenta interstitial fluid and hardly express in normal human blood and interstitial fluid, so that the mixture of one or more of matrix metalloproteinase 1, matrix metalloproteinase 8 or matrix metalloproteinase 14 is preferred.
Substrate polypeptides jointly suitable for the matrix metalloproteinase 1, the matrix metalloproteinase 8 and the matrix metalloproteinase 14 can be MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH2, and the molecular weight is as follows: 1221.32Da.
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 Phorbol 12-myristate 13-acetate (PMA), and the therapeutic gene is siRNA inhibiting CTSG (Cathepsin G) gene expression.
The placenta microenvironment targeted delivery probe has the average particle size of 80-300 nm, preferably 100-210 nm, is not favorable for in vivo circulation when too large, and is not favorable for loading drugs and genes when too small.
The invention also provides a preparation method of the placenta microenvironment targeted delivery probe, which comprises the following steps:
s1, loading superparamagnetic ferroferric oxide (SPIO) nano particles, micromolecular drugs for regulating and controlling functions of placenta trophoblasts and/or genes to a copolymer to obtain composite nano particles;
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;
and S5, assembling the polypeptide-PEG modified lipid bilayer membrane and the composite nanoparticles into a placenta microenvironment targeted delivery probe.
Preferably, in the step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15.
The MMP substrate polypeptide-PEG modified lipid bilayer membrane 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, the drug leakage is reduced, and the phagocytosis of other cells outside the placenta is reduced or avoided. Thereby ensuring the safety of other tissues and organs outside the maternal placenta; the MMP enzyme sensitive shell is disintegrated in a microenvironment containing enzymes at the placenta matrix side to release the medicament, so that the high-efficiency release and distribution of the medicament 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 an application of the double-targeting nano-drug delivery system in preparation of a drug for regulating and controlling the placenta trophoblast dysfunction disease, wherein the regulation and control of the placenta trophoblast dysfunction disease is that the intrauterine growth of a fetus is limited.
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 and expression of the placenta trophoblast is used as an inner core; synthesizing a placenta microenvironment targeted delivery probe with a double-layer structure. The double-layer structure can ensure that the liposome shell has stable structure and keeps stable circulation in the blood circulation of the pregnant woman, so that the liposome 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 by the body of the pregnant woman, 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 and is further specifically endocytosed by the TB cell to generate a function regulation and control effect, so that exact treatment on TB cell diseases is ensured;
(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 a placenta microenvironment targeted delivery probe prepared in example 1 of the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples, which are 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:
and measuring the Fe content in the nano-drug system by using an atomic absorption spectrophotometer method, wherein the Fe content is used for measuring the dosage of the nano-drug. Weighing a certain amount of prepared drug solution (such as 1mL of the solution in step three), lyophilizing, and dissolving to 1mol L -1 The HCl solution is placed for 24 hours to ensure that Fe in the SPIO is fully ionized, an atomic absorption spectrophotometer is used for detecting the absorbance of Fe atoms at 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 medicine solution before freeze-drying is calculated in a reverse mode.
The particle size test method comprises the following steps:
the particle size of the samples was measured with a Zeta-Plus potential particle sizer (Brooken Haven) with an incident laser wavelength λ =532nm, an incident angle θ =90 ° and a temperature of 25 ℃; the average of the three measurements was taken.
Example 1:
s1, synthesis of polyethyleneimine 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 (8.0 g, mn = 2kDa) was weighed into a reaction flask, dried under vacuum at 80 ℃ for 6 hours, and dissolved by adding THF (60 mL) under an argon atmosphere. Carbonyldiimidazole (CDI, 6.4 g) 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 (0.648 mL) was added to inactivate the 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;
weighing PEI (4.4 g, MW = 1.8kDa) and adding the PEI into a two-mouth bottle (50 mL), adding trichloromethane (20 mL) to dissolve the PEI and adding PEG-CDI (3.2 g), stirring at room temperature for reaction for 24h, filling the solution into a dialysis bag (MWCO =3.5 kDa), dialyzing the solution in the trichloromethane for 24h, concentrating the solution in the dialysis bag under reduced pressure, precipitating the solution 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, drying at 70 ℃ for 8 hours in vacuum, adding 2ml of Sn (Oct) 2 Continuing to dry for 0.5h, then adding 400mL of dried epsilon-caprolactone, and stirring and reacting for 24h at 105 ℃; after 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 = 5000) is added into a two-mouth bottle, vacuum drying is carried out for 8h at the temperature of 50 ℃, 7.2g (10 eq.) 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, then slowly dripping 10mL of trichloromethane solution containing 200mg of PCL-CDI, stirring and reacting for 24h at room temperature, dialyzing for 24h in 1000mL of trichloromethane by using a dialysis bag (MWCO =5 kDa), decompressing to remove part of trichloromethane, then precipitating in anhydrous ether, filtering and drying to obtain a white powder product with the yield of 86%;
s3, preparation of polyethylene glycol-polyethyleneimine-polycaprolactone loaded SPIO nano particles and drugs (PEG-PEI-PCL-SPIO/drug)
SPIO (superparamagnetic ferroferric oxide) is 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) nanoparticles.J.am.chem.Soc.2004,126,273-279 ] iron acetylacetonate Fe (acac) 3 1.4126g (4 mmol), 5.16g (20 mmol) of 1, 2-hexadecanediol, 3.8ml (12 mmol) of oleic acid and 3.8ml (12 mmol) of oleylamine are added into a 200ml three-necked bottle, then 40ml of dibenzyl ether is added under the protection of nitrogen and stirred for dissolution, 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 changes 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 (10 mL), weighing 50mg of PEG-PEI-PCL polymer and 5mg of Phorbol 12-myritate 13-acetate (PMA), dissolving and uniformly mixing the PEG-PEI-PCL polymer and the Phorbol 12-myritate 13-acetate (PMA) by using trichloromethane (3 mL), 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 discarding 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 particle/drug (Fab-PEG-PEI-PCL-SPIO/drug)
The Ecadrein antibody is firstly cracked by adopting the method in the prior literature to obtain the Fab segment of Ecadrein, and then the Fab segment is purified. Then linking Ecadrein-Fab to mal-PEG-COOH, and reacting 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 Ecadrein antibody was weighed out at 0.5 mg. Multidot.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 Ecadrein with high purity;
1mg of Fab fragment of Ecadherin (Mn =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, and 1mg of dithiothreitol was added thereto to react at 25 ℃ for 30min. After removing dithiothreitol by centrifugation with a centrifugal ultrafiltration tube with a molecular weight cutoff of 1k, 5ml of PBS solution was added for dissolution, mal-PEG-COOH (2mg, mn = 4k) was added and mixed uniformly, and the mixture was left overnight at 4 ℃. 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 500 micrograms of EDC and NHS respectively for 15min, then adding the PEG-PEI-PCL-SPIO/drug 1695l prepared in the step 3, reacting overnight at 4 ℃, finally removing excessive small molecular impurities of EDC and NHS by ultrafiltration and centrifugation, removing unconnected antibodies by centrifugation at 12000r/min, collecting solid solution, ultrasonically dispersing the solid solution into distilled water, and regulating the concentration of Fab-PEG-PEI-PCL-SPIO/drug nanoparticles to a constant volume 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 the CTSG-siRNA with negative electricity can be compounded through electrostatic interaction to prepare a nano compound. The specific operation is as follows: mu.g of CTSG-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 uniformly, uniformly mixing a CTSG-siRNA diluted solution and a PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle solution, 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, PEG-polypeptide synthesis
0.05mmol of matrix metalloproteinase 1/8/14 sensitive polypeptide (MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH 2, molecular weight: 1221.32 Da), 5mmol of EDC and 5mmol of DMAP were dissolved in 10mL of acetonitrile in water (acetonitrile: water = 1), and the mixture was magnetically stirred at 500rpm for 2h on an ice-water bath under protection of N2 to activate the Peptide. After 2h 0.5mmol PEG-NHS (molecular weight 3000 Da) was added and the reaction was continued for 72h. After the reaction is finished, putting the reaction solution into a dialysis bag (MWCO =3.5 kDa), dialyzing for 72h, and freeze-drying to obtain a product PEG-polypeptide;
preparation of S7 and 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 spinner to form a thin film of liposomes on the wall of the round bottom flask. 2mL of the therapeutic gene composite nanoparticle prepared in step 5 was added dropwise to the liposome film formed of the above PEG-polypeptide and cholesterol at a rate of 0.5mL/min with 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. Finally, 2mL of physiological saline (0.9 percent NaCl) solution is added to dissolve the PEG-polypeptide modified liposome shell @ therapeutic gene composite nano particles, the filtration rate of a syringe filter with the aperture of 220nm is determined, the volume is fixed 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 dual-targeting nano-drug delivery system 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 amounts of polymer, drug and SPIO fed in step S3 or omitting one of steps S3, S4, S5, S6, S7, as shown in table 1 below:
table 1: examples and comparative examples
Function evaluation test
1. Magnetic Resonance Imaging (MRI) assay to evaluate the placental-specific delivery function of drugs
Establishing a model:
SPF grade C57BL/6 mice (purchased from guangdong provincial medical laboratory animal center) 8 weeks old, female and male mice 2:1, mating in estrus cages, smearing the vaginal secretion of the female mouse with Papanicolaou staining on the vaginal secretion smear on the next day, observing a sample under an optical microscope, and marking the vaginal sperm-positive person as pregnancy as the pregnancy day 0 (D0).
At the time of induction of FGR formation, mice were given a tobacco-wine treated group starting 7 days before mating until parturition, and a fetal intrauterine growth restricted model was established. The tobacco and wine treatment method comprises passively smoking for 2h (9 cigarettes per hour; brand of cigarette is Liqun (soft blue) purchased from Hangzhou cigarette factory, tar amount: 11mg, nicotine amount: 1.0mg, carbon monoxide amount: 11 mg) in a closed container with volume of 5L per day, and orally administering 45% ethanol 2ml (ethanol purchased from Sigma) per mouse per day by intragastric gavage. The control mice were administered to a smokeless closed container for 2 hours a day and then to the stomach with an equal volume of distilled water. The pregnant mice were placed in a closed container with a volume of 5L daily, and the pregnant mice without passive smoking treatment served as normal control group.
MRI imaging to detect placental distribution of drugs:
on day 11, after chloral hydrate anesthesia, the fetal intrauterine growth restricted model animal was subjected to MRIT2 sequence scanning at time points before (0 h) and 2h (2 h) after drug injection, and the in vivo distribution of the nano-drug containing SPIO was observed. The dosage of the tail vein injection nano-medicament is as follows: (therapeutic dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline);
MRI imaging of the uterus of C57BL/6j mice was performed using a Philips Intera 1.5T MRI scanner, with its animal specific coils. The evolution of signal intensity in the uterus and embryonic regions in mice was observed on the MRIbTFE sequence and the T2 relaxation time changes due to SPIO distribution in drugs 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 rate of increase of R2 (RSI (Rela) at 2h after drug injection was calculatedtive Signal Intensity)％＝R2 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 | 23.23 | 1.10 | 3.27 |
| Example 2 | 21.96 | 3.22 | 2.00 |
| Example 3 | 20.01 | 1.41 | 2.95 |
| Example 4 | 24.20 | 1.09 | 3.73 |
| Comparative example 1 | 11.14 | 16.96 | 5.70 |
| Comparative example 2 | 7.87 | 11.30 | 8.04 |
| Comparative example 3 | 3.84 | 4.13 | 8.06 |
| Comparative example 4 | 3.53 | 3.36 | 10.40 |
| Comparative example 5 | 14.74 | 1.05 | 17.31 |
| Comparative example 6 | 10.08 | 1.03 | 26.23 |
From the above results, in comparative example 1, the placental trophoblast cell 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 placental retention, a large amount of drug leaks through the placental barrier, and low placental RSI is detected; the drugs are accumulated in the embryo, which results in high embryo RSI; the drug can not be anchored in TB cells to obtain placenta retention, and part of the drug is separated from the placenta and distributed systemically, so that the liver RSI is high.
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 Ecadrein antibody targets other cells with various cell membranes expressing Ecadrein in vivo including TB, and the cell membrane targeting property is not strong; therefore, low placenta RSI and low liver RSI were detected; the drug without lipid membrane had a smaller particle size and entered the placenta, and passed the placental barrier in a larger proportion, and a higher RSI of the embryo was detected.
The delivery systems of comparative examples 3 and 4, which did not contain the Ecadherin antibody, failed to target and anchor the drug into the placenta to TB cells, failed to obtain placental retention, leaked a significant amount of placenta 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 is not modified by enzyme-sensitive polypeptide, so that the distribution of the lipid bilayer membrane outer shell in the placenta is reduced, and the RSI of the placenta is lower and the RSI of the liver is higher. Comparative example 4, without the lipid bilayer envelope, had lower RSI for placenta and higher RSI for liver than comparative example 3.
The comparative example 5 has an excessively large particle size, so that the in vivo circulation distribution effect is extremely poor, the medicament is mainly phagocytosed by a 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 has poorer circulation, so its liver RSI is higher than that of comparative example 5; the particle size was larger and less prone to leakage through the maternal-fetal barrier, so placenta RSI was lower than comparative example 5.
In examples 1-4, a substrate polypeptide-PEG modified lipid bilayer membrane of MMP 1/8/14 is used as an outer shell, a drug carrier modified by a placenta trophoblast surface marker antibody is used as an inner core, and a placenta microenvironment targeted delivery probe with a double-layer structure is synthesized, wherein the particle size range is 80-210nm. 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 MMP, disintegrates along with degradation of the polypeptide, and realizes drug specificity distribution in the placenta tissue. After the drug shell is disintegrated in the placenta microenvironment, the drug inner core containing the Ecadherin antibody fragment is revealed. The Ecadirin antibody fragment can be anchored in a cell membrane specific high-expression Ecadirin TB cell in a placenta, so that the drug is promoted to be specifically endocytosed by the TB cell to realize TB function regulation and control, the distribution of the Ecadirin antibody fragment in other cells of the placenta is reduced, and the influence on the placenta function is reduced. The Ecadrein antibody enables the drug in the placenta to be anchored in the TB cell, so that the drug leakage through the maternal-fetal barrier is effectively reduced, and the drug reaching the embryo is reduced.
2. Establishing animal model with restricted intrauterine growth of fetus for evaluating treatment effect
Drugs (therapeutic dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline) are injected into D3, D6, D9, D12 and D15, and serial detection is performed in D17, and the detection 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 order, and the number of surviving litter 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. Shearing placenta tissue, and storing in liquid nitrogen at-80 deg.C.
And counting the body weights of all the fetal rats in the normal control group, and setting the fetal rats with the weight less than the tenth percentile of all the fetal rats as FGR. The FGR incidence rate of the fetal mice in each treatment group = (FGR amount/total animal number in this group) × 100% was calculated
TABLE 3 evaluation of therapeutic Effect in animal models with fetal intrauterine growth restriction
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 contained drug cannot be anchored in TB cells to obtain placenta retention, a large amount of drug leaks through the placenta barrier, and the therapeutic effect is poor, the number of dead fetus + absorbed fetus is high, the FGR occurrence rate is high, the weight of the fetus is low, and the number of live fetus is low; meanwhile, the drug is accumulated in the embryo, which causes embryo toxicity, lower weight of the fetus and lower number of live fetus.
The delivery system of comparative example 2 does not contain a polypeptide-PEG modified lipid bilayer membrane as a shell, and targeted release aiming at the placenta microenvironment cannot be realized; the Ecadrein antibody targets Ecadrein cells expressed in vivo including TB, the cell targeting is weak, the detected treatment effect is poor, the number of dead fetus and absorbed fetus is high, the FGR incidence is high, the weight of the fetus is low, and the number of live fetus 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 number of the live fetus is lower.
The delivery systems of comparative examples 3 and 4, which do not contain Ecadherin antibody, do not target and anchor the drug entering the placenta to TB cells, and do not obtain placenta retention, resulting in a large amount of leakage across the placenta barrier, and detected as poor therapeutic effect, high number of dead and absorbed fetuses, high FGR incidence, low weight of the fetuses, and low number of live fetuses. Meanwhile, the lipid bilayer membrane outer shell of the comparative example 3 is not modified by enzyme-sensitive polypeptide, the distribution of the lipid bilayer membrane outer shell in placenta is reduced, the detected treatment effect is poor, the number of dead fetus and absorbed fetus is high, the FGR incidence rate is high, the weight of fetus is low, and the number of live fetus is low. Comparative example 4, which has no lipid bilayer membrane shell, is less effective than comparative example 3.
The particle sizes of comparative examples 5 and 6 were too large, resulting in poor in vivo circulation distribution, poor in treatment effect, high in dead fetus + absorbed fetus number, high in FGR incidence, low in litter weight, and low in viable fetus number, and the drugs were mainly phagocytosed by the reticuloendothelial system of the liver, resulting in insufficient distribution of the placenta drugs. 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 MMP 1/8/14 is used as a shell, a drug carrier modified by a placenta trophoblast surface marker antibody is used as an inner core, and a placenta microenvironment targeted delivery probe with a double-layer structure is synthesized, wherein the particle size range is 80-210nm. 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 MMP, disintegrates along with degradation of the polypeptide, and realizes drug specificity distribution in the placenta tissue. The drug shell disintegrates in the placental microenvironment, revealing the inner drug core containing the Ecadherin antibody fragment. The Ecadirin antibody fragment can be anchored in a TB cell with cell membrane specificity and high expression in the placenta, promote the drug to be specifically endocytosed by the TB cell to realize TB function regulation and control, reduce the distribution in other cells in the placenta, reduce the influence on the placenta function, and realize better treatment effect through effective TB function regulation and control. The Ecadrein antibody enables the drug in the placenta to be anchored in TB cells, effectively reduces the drug leakage through a maternal-fetal barrier, reduces the drug reaching the embryo, and has low 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 index glutamic pyruvic transaminase (ALT), total bilirubin (TBil) and kidney function index 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 a table 4.
TABLE 4 toxicity evaluation results
According to the results, the placenta microenvironment targeted delivery probe prepared by the invention has no obvious toxic or side effect on the mother and the fetus.
Claims (5)
1. A placental microenvironment targeted delivery probe, comprising:
a) The outer shell is an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is disintegrated in a targeted manner under the action of an enzyme which is in contact with the high expression of the placenta interstitial fluid, and the enzyme in the high expression of the placenta interstitial fluid is one or more of matrix metalloproteinase 1, matrix metalloproteinase 8 or matrix metalloproteinase 14;
the enzyme substrate polypeptide is MCA-Lys-Pro-Leu-Gly-Leu-DNP-Dpa-Ala-Arg-NH2;
b) The inner core is a drug carrier modified by a marker antibody with high surface specificity expression of the placental trophoblasts, the drug carrier is a copolymer formed by a polycation carrier modified by polyethylene glycol and hydrophobic degradable polyester, and the marker antibody with high surface specificity expression of the placental trophoblasts is a Fab segment of an epithelial cadherin Ecadherin antibody;
the copolymer is polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL;
c) 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 small molecule drug is Phorbol 12-myrristate 13-acetate, and the therapeutic gene is siRNA for inhibiting CTSG gene expression;
the average particle size of the placenta microenvironment targeted delivery probe is 80nm-300nm.
2. The placental microenvironment-targeted delivery probe of claim 1, wherein the placental microenvironment-targeted delivery probe has an average particle size of 100nm to 210nm.
3. The method of making a placental microenvironment targeted delivery probe of any one of claims 1-2, comprising the steps of:
s1, loading superparamagnetic ferroferric oxide (SPIO) nano particles, micromolecular drugs for regulating and controlling functions of placenta trophoblasts and/or genes to a copolymer to obtain composite nano particles;
s2, linking the placenta trophoblast surface marker antibody to the composite nanoparticles to obtain antibody composite nanoparticles;
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;
and S5, assembling the polypeptide-PEG modified lipid bilayer membrane and the antibody composite nanoparticles into a placenta microenvironment targeted delivery probe.
4. The preparation method of the placental microenvironment targeted delivery probe according to claim 3, wherein in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15.
5. The use of the placental microenvironment targeted delivery probe of any one of claims 1-2 in the manufacture of a medicament for modulating a disease of placental trophoblast dysfunction that is fetal growth restriction.
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