CN114224870A - Placenta microenvironment targeted delivery probe and preparation method and application thereof - Google Patents
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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 morbidity and mortality in perinatal infants. 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-.
Most FGR pathogenesis is accompanied by a dysfunction of placental trophoblasts [ planta.2020 jul; 96:10-18 ]. Research currently shows that DNA methylation is critical in the maintenance of normal placenta function and the development of placenta-related diseases [ hypertension.2020 apr; 75(4) 1117-. 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 out CTSG. On the other hand, maintenance and promotion of placental function requires efficient activation of PKC pathway functions [ Cell Biol int.2020 dec; 2409 2415 ] Photol 12-myrisitate 13-acetate (PMA, 12-O-tetracyanophorbol-13-acetate) is a potent PKC activator, which can widely regulate the phosphorylation of various protein sites in trophoblast cells, and thus effectively regulate the function of trophoblast cells [ Biochem Biophys Res Commun.2012Jan 27; 417(4): 1127-32). Activation of PKC pathway and DNA methylation regulation are expected to produce synergy in cells [ J Cell biochem.2011jul; 112(7) 1761-72) to produce a functional regulation effect on the 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 side of the fetus, affecting the development of the fetus. Therefore, the medication of pregnant women including emergency drugs has many contraindications. The medicines for pregnant women are classified into 5 types according to teratogenic property, and except for a few medicines with the minimum toxicity which are classified into a type and a type b, most of the other medicines in the c type, the d type and the e type have obvious damage to fetuses. The pregnant women have heavy metabolism burden in vivo during pregnancy and complicated immune change. Therefore, even if the drug is not significantly toxic during non-pregnancy, it is likely to 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 aiming at the symptoms caused by the placenta dysfunction diseases. 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 the TB function regulating drug is the key to solving the 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 do not address the problem of potential 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, 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 means are needed to achieve retention of the nano-drug in the placenta and targeting of TB cells.
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 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 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 in targeted disintegration under the action of an enzyme which is in contact with 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 polypeptide applicable to matrix metalloproteinase 1, matrix metalloproteinase 8 and matrix metalloproteinase 14 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 invention can be synthesized by the prior art, for example, PEG is firstly reacted with polycation carrier to form the copolymer, and then the active group of polycation is reacted with the activated polyester segment to form the 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 for inhibiting the expression of CTSG (cathepsin G) gene.
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) 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 placenta microenvironment targeted delivery probe.
Preferably, in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15: 1.
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 side of the placenta matrix to release the medicine, so that the high-efficiency release and distribution of the medicine in the placenta matrix 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 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 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 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 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, 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 of the solution in step three), lyophilizing, and dissolving to 1mol L-1Standing in HCl solution for 24 hr to ionize Fe in SPIO sufficiently, detecting the absorbance of Fe atom at 248.3nm with atomic absorption spectrophotometer, calculating Fe concentration by substituting into standard curve made by Fe standard solution, and calculatingThe Fe content of the drug solution before lyophilization was determined.
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)2Continuing to dry for 0.5h, then adding 400mL of dried epsilon-caprolactone, and stirring and reacting for 24h at 105 ℃; cooling, adding 100mL ethanol to dissolve unreacted epsilon-caprolactone, filtering, dissolving the crude product in 250mL tetrahydrofuran, precipitating in a large amount of anhydrous ether, filtering, drying to obtain a white powdery product,the yield is 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 MFe2O4(M ═ Fe, Co, Mn) nanoparticies.J.am.chem.Soc.2004, 126,273-279 ] iron acetylacetonate Fe (acac)31.4126g (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 Phorbol 12-myritate 13-acetate (PMA), dissolving and mixing the PEG-PEI-PCL polymer and the Phorbol 12-myritate 13-acetate (PMA) uniformly 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 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-1Papain, 10 mmol. multidot.L-1Cysteine, 2 mmol. multidot.L-1The 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 the Fab fragment of Ecadrein (Mn. RTM.45 kDa) was weighed out 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 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, Synthesis of PEG-Polypeptides
0.05mmol of matrix metalloproteinase 1/8/14-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 aqueous acetonitrile (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 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 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 diagram of the prepared dual-targeting nano-drug delivery system is shown in figure 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 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 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).
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: 11mg) 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 mice were placed in a closed container of 5L volume daily, and pregnant mice without passive smoking treatment served as normal control groups.
MRI imaging to detect placental distribution of drugs:
on day 11, after chloral hydrate anesthesia, the MRIT2 sequence was scanned at time points before (0h) and 2h (2h) after drug injection to observe the in vivo distribution of the nano-drug containing SPIO. 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 calculated2h/R20h) 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 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 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, lower placental RSI and lower 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 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 MMP 1/8/14 was used as a shell, a drug carrier modified by a placental trophoblast surface marker antibody was used as a core, and a placental microenvironment targeted delivery probe with a bilayer structure was synthesized, wherein the particle size range was 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 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 Ecadrein antibody fragment can be anchored in a placenta to a TB cell with a cell membrane specificity and high expression of Ecadrein, promotes the drug to be specifically endocytosed by the TB cell to realize the regulation of the TB function, reduces the distribution in other cells of the placenta, and reduces the influence on the function of the placenta. 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 (treatment dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline) are injected into D3, D6, D9, D12 and D15, and a series of tests are performed in D17, and 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 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. Cutting placenta tissue, and storing at-80 deg.C in liquid nitrogen.
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 each treated group of fetal mice was calculated as (FGR count/total number of animals in this group) × 100%
TABLE 3 evaluation of the therapeutic Effect of animal models with restricted intrauterine growth of fetus
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 in TB cells to obtain placenta retention, and a large amount of drug leaks through the placenta barrier, so that the therapeutic effect is poor, the number of dead fetus + absorbed fetus is high, the occurrence rate of FGR 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, failed to target and anchor the drug entering the placenta to TB cells, failed to obtain placenta retention, resulted in a large number of leaks across the placenta barrier, and detected poor therapeutic effect, higher number of dead and absorbed fetuses, higher FGR incidence, lower weight of the litter, and lower 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 in placenta is reduced, 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. 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 was used as a shell, a drug carrier modified by a placental trophoblast surface marker antibody was used as a core, and a placental microenvironment targeted delivery probe with a bilayer structure was synthesized, wherein the particle size range was 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 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 Ecadrein antibody fragment can be anchored in a TB cell with cell membrane specificity and high expression of Ecadrein in a placenta, promotes the drug to be specifically endocytosed by the TB cell to realize TB function regulation and control, reduces the distribution in other cells of the placenta, reduces the influence on the function of the placenta, and realizes 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 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 placenta microenvironment targeted delivery probe prepared by the invention has no obvious toxic or side effect on the mother and the fetus.
Claims (9)
1. A placental microenvironment targeted delivery probe, comprising:
A) the 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 high expression of placenta interstitial fluid, and the enzyme in high expression of the 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.
2. The placental microenvironment targeted delivery probe of claim 1, wherein the enzyme highly expressed in placental interstitial fluid is one or a mixture of matrix metalloproteinase 1, matrix metalloproteinase 8, or matrix metalloproteinase 14.
3. The placental microenvironment targeted delivery probe of claim 1, wherein the copolymer is one or more of polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL, polyethylene glycol-polyethyleneimine-polylactic acid PEG-PEI-PLA or polyethylene glycol-polyethyleneimine-polylactic acid-glycolic acid PEG-PEI-PLGA, preferably polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL.
4. The placental microenvironment-targeted delivery probe of claim 1, wherein the placental microenvironment-targeted delivery probe has an average particle size of 80nm to 300nm, preferably 100nm to 210 nm.
5. The placental microenvironment-targeted delivery probe of claim 1, wherein the small molecule drug is Phorbol 12-myristate 13-acetate and the therapeutic gene is siRNA that inhibits expression of CTSG gene.
6. The method of making a placental microenvironment targeted delivery probe of any one of claims 1-5, comprising the steps of:
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 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;
s5, assembling the polypeptide-PEG modified lipid bilayer membrane and the antibody composite nanoparticles into a placenta microenvironment targeted delivery probe.
7. The preparation method of the placental microenvironment targeted delivery probe of claim 6, wherein in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15: 1.
8. The use of the placental microenvironment targeted delivery probe of any one of claims 1-5 in the manufacture of a medicament for modulating a disease of placental trophoblast dysfunction.
9. The use of claim 8, wherein the disease that modulates placental trophoblast dysfunction is fetal growth restriction.
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