CN114469890B - Focus microenvironment sensitive delivery nuclear magnetic probe and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a focus microenvironment sensitive delivery nuclear magnetic probe and a preparation method and application thereof, wherein the preparation method comprises the following steps: the enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is in targeted disintegration under the action of enzyme which is in contact with placenta interstitial fluid and highly expressed is used as a shell, a medicine carrier modified by a placenta trophoblast surface specificity high expression marker antibody is used as an inner core, and superparamagnetic ferroferric oxide SPIO nano particles, micromolecular medicines for regulating and controlling the function of the placenta trophoblast, therapeutic genes or a combination thereof are loaded in the medicine carrier. The delivery nuclear magnetic probe can effectively avoid the nonspecific drug absorption of other organs outside the placenta of the mother body and the fetus, thereby realizing the delivery and function regulation of the specific drug of the nourishing cells in the placenta.
Description
Technical Field
The invention relates to the field of chemical and biomedical engineering, in particular to a focus microenvironment sensitive delivery nuclear magnetic probe and a preparation method and application thereof.
Background
Gestational Diabetes Mellitus (GDM) refers to an abnormality in sugar metabolism that occurs or is discovered first during pregnancy. With the improvement of living standard, the change of dietary structure and the increase of the age of the lying-in women, the incidence rate of GDM tends to increase year by year, and about 20% of pregnant women are diagnosed as GDM at present. GDM has major damage to both parturient and fetus Nat Rev Dis primers.2019jul 11;5 (1): 47 ]. Is easy to cause infection, hydramnion, baby's birth difficulty, birth canal injury, postpartum hemorrhage, cesarean section yield increase, fetal deformity, etc. GDM newborns are prone to neonatal respiratory distress syndrome, hypoglycemia, hyperbilirubinemia, and the like. Moreover, the incidence of diabetes in GMD fetuses after adulthood is much higher than normal, and the incidence of hyperglycemia in GDM parturients after re-gestation is multiplied. More seriously, GDM parturients are 7 times more likely to develop type 2 diabetes in the future than normoglycemic pregnant women. Existing clinical studies prove that if GDM is effectively controlled, the risk of postpartum diabetes is significantly reduced [ Nat Rev endocrinol.2012nov;8 (11): 639-49 ]. The earliest studies demonstrated that the onset of GDM is associated with systemic insulin resistance in patients. Recent studies have found that abnormalities in trophoblast function, including Wnt/β -catenin pathway inactivation [ Mol Med rep.2017aug;16 1007-1013. ] secretion of tumor necrosis factor (tumor necrosis factor- α, TNF- α) -associated insulin resistance [ Int J Mol sci.2020jan 12;21 479 ], and the placenta and in vivo wide inflammatory response triggered by the secretion of interleukin6 (interleukin 6, IL-6) [ Acta Obstet Gynecol Scand.2011May;90 And (5) 524-30 ] are related at the same time. Together, these changes lead to placental dysfunction that may be the initiating factor in the onset of GDM. The simultaneous treatment of Wnt/beta-catenin pathway activators such as WAY-262611 and siRNA inhibiting IL-6 expression is expected to produce better therapeutic effect on GDM when the changes are treated simultaneously [ Diabetol Metab Syndr.2020Sep17; 12; j Diabetes Res.2020Mar 23; 2020.
However, the existing treatment modes of common blood sugar control medicines such as oral metformin and the like cannot solve the fundamental pathogenic factors of GDM from the placenta and even trophoblasts, and the treatment effect on GDM is generally poor. Therefore, the existing treatment dilemma of GDM is more prominent under the policy of the advanced age of the parturient and the encouragement of multiple births in China. Therefore, there is an urgent need to develop drugs that can directly restore the function of TB cells by directly targeting the function change of Trophoblasts (TB) when GDM is attacked, so as to improve the prognosis of the vast GDM pregnant women and fetuses. Therefore, the small molecule or gene medicine can be used in combination, the expression or the function of TNF-alpha and IL-6 can be inhibited, and the recovery of the trophoblast function in GDM can be realized, and the placenta function can be saved. In vitro experiments aiming at the trophoblast cells prove that the combined use of the TNF-alpha inhibitor and the IL-6siRNA has the regulation and control effects on the corresponding molecular pathway and the cell function of the trophoblast cells. However, the use of these therapies in pregnant women faces significant obstacles.
However, the existing drugs which may have the function of regulating TB function are inevitably distributed in all organs of the mother and are distributed to the fetus through the placenta. These drugs, while modulating TB function, can produce maternal and fetal toxicity. The drug administration for pregnant women including emergency drugs at present has a lot of contraindications, and the problems of drug distribution and toxicity of the drugs in the mother and fetus need to be considered in the drug use and new drug development of the pregnant women are the first problems. Most of the drugs can pass through the placenta and distribute into the side of the fetus, affecting the development of the fetus. Therefore, the existing medicines for realizing the function regulation of TB such as EMT, angiogenesis promotion and the like in-vitro experiments cannot really realize the function regulation of the TB in the placenta on the premise of ensuring the safety of medication. Therefore, how to avoid toxicity to the mother and fetus and realize the accurate delivery of the TB function regulating drug to TB in the placenta is the key to solve the disease symptoms of gestational diabetes and the like caused by TB dysfunction.
The approaches that researchers try to promote the delivery of TB-specific nano-drugs include 2, one is to increase the particle size of nano-drugs, so that the nano-drugs cannot pass through a fetal membrane barrier and are retained in placenta to generate drug delivery effect; the other is specific delivery of antibody modified nano-carriers aiming at TB cell membrane markers.
The principle of increasing the particle size of the nano-drug and promoting the distribution of the drug in the placenta is that experimental research finds that the nano-drug less than 300nm cannot be retained in the placenta and easily enters the fetus through the placenta. Therefore, researchers try to synthesize nano-drugs with the particle size of more than 300nm, so that the nano-drugs are retained in the placenta to generate the functional regulation and control on various cells including the placental TB. However, too large a particle size (> 100 nm) 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 everywhere in 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., keratin 7, cytokine-7, CK 7) that distinguish it from other placental stromal cells in the 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 CK7 is connected to the surface of the nano-drug carrier, the direct in vivo application of the antibody will cause side effects on other cells expressing the marker CK7 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 and shortcomings of the prior art, the primary object of the invention is to provide a focus microenvironment sensitive delivery nuclear magnetic probe, which utilizes a placenta microenvironment to reduce the distribution of a medicament in a maternal organ tissue before entering a placenta in a targeted manner, and utilizes a trophoblast marker to reduce the distribution of the medicament in a fetal organ tissue after passing the placenta in a targeted manner, so that the non-specific medicament absorption of other organs outside the maternal placenta and the fetus can be effectively avoided, and the delivery and the function regulation of a nourishing cell specific medicament in the placenta can be further realized.
The invention also aims to provide a preparation method of the focus microenvironment sensitive delivery nuclear magnetic probe.
The invention is realized by the following technical scheme:
a focal microenvironment sensitive delivery nuclear magnetic probe comprising:
the enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is in targeted disintegration under the action of enzyme which is in contact with placenta interstitial fluid and highly expressed is used as a shell, a drug carrier modified by a marker antibody with placenta trophoblast surface specificity and high expression is used as an inner core, and superparamagnetic ferroferric oxide SPIO nano particles, micromolecular drugs for regulating and controlling the function of placenta trophoblasts, therapeutic genes or a combination thereof are loaded in the drug carrier;
wherein the enzyme highly expressed in placenta interstitial fluid is one or more of AMP activated protein kinase, lysozyme, kininase, histaminase, oxytocin or matrix metalloproteinase;
the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester;
the marker antibody specifically and highly expressed on the surface of the placenta trophoblast is an Fab segment of a keratin 7 (CK 7) antibody.
The placenta of the pregnant woman is rich in a plurality of enzymes for promoting placenta development and fetal nutrition, the enzymes highly expressed by the placenta interstitial fluid are one or more of AMP activated protein kinase (AMPK), lysozyme, kininase, histaminase, oxytocin or matrix metalloproteinase, wherein the AMPK has extremely high expression level in the placenta interstitial fluid and is hardly expressed in normal human blood and interstitial fluid, so the AMPK is preferred.
AMPK substrate polypeptide can be selected from HIS-MET-ARG-SER-ALA-MET-SER-GLY-LEU-HIS-LEU-VAL-LYS-ARG-ARG-NH2, molecular weight: 1779.15Da, which are commercially available.
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 molecular drug is WAY-262611, and the therapeutic gene is siRNA for inhibiting IL-6 (Interleukin-6) gene expression.
The average particle size of the delivery nuclear magnetic probe is 80nm-300nm, preferably 100nm-210nm, the particle size is too large to be beneficial to in vivo circulation, the particle size is too small to be prepared, the preparation difficulty is increased, and the loading of drugs and genes is not beneficial.
The invention also provides a preparation method of the focus microenvironment sensitive delivery nuclear magnetic probe, which comprises the following steps:
s1, loading superparamagnetic ferroferric oxide (SPIO) nano particles, micromolecular medicines for regulating and controlling placenta trophoblast functions 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 focus microenvironment sensitive delivery nuclear magnetic probe.
Preferably, in the step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15.
According to the invention, the AMPK substrate polypeptide-PEG modified lipid bilayer membrane is used as a shell, so that the distribution of a nano transmission system in the blood without AMPK enzyme is stable before the nano transmission system enters the placental enzyme environment, the drug leakage is reduced, and other cells outside the placenta are reduced or avoided from phagocytosis. Thereby ensuring the safety of other tissues and organs outside the maternal placenta; the AMPK enzyme sensitive shell is disintegrated in a microenvironment containing enzyme at the placenta matrix side to release the medicine, so that the high-efficiency release and distribution of the medicine in the matrix placenta can be ensured; due to the introduction of the enzyme sensitive shell, the distribution efficiency of the placenta can be ensured without adopting a large-particle-size nano-carrier structure, the particle size of the nano-carrier is effectively reduced, the stable circulation distribution of the medicine before entering the placenta is ensured, and the reticuloendothelial system is ensured not to phagocytize a large amount of carriers to cause the reduction of curative effect and the increase of side effect.
The invention adopts the medicine carrier modified by the placenta trophoblast surface marker antibody as the inner core, the medicine is modified by the TB cell surface marker antibody, and the TB cell membrane in the placenta can be anchored exactly after being released, thereby ensuring the specific administration of the TB cell in a complex placenta environment, and simultaneously avoiding unnecessary placenta function damage caused by the administration of other cells in the placenta; most of the medicines entering the placenta are exactly anchored in TB cells through antibody targeting, so that the medicines are ensured to rarely leak through a placenta barrier and rarely enter the side of a fetus, and the safety of the fetus is ensured; after the medicine is anchored on TB cell membranes, the therapeutic medicine and the therapeutic gene are promoted to be swallowed into the TB cells, 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 placenta trophoblast dysfunction disease is gestational diabetes (gestational hyperglycemia).
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; delivery of a nuclear magnetic probe with a synthetic bilayer 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 nano-drug delivery system enters the placenta along with blood circulation, an enzyme substrate in the outer shell of the nano-drug delivery system is decomposed by corresponding enzyme highly expressed in placenta tissue, and the protective lipid bilayer outer shell is quickly disintegrated in the placenta to release the antibody modified nano-drug capable of anchoring the TB cell membrane surface marker. The nano-drug is prevented from being absorbed by other tissue cells of a parent body, is specifically anchored on TB cell membranes in a placenta, is prevented from being absorbed by other cells in the placenta, avoids leakage through a maternal-fetal barrier, is further specifically endocytosed by the TB cells, generates a function regulation and control effect, and ensures that TB cell diseases are treated exactly;
(3) Through an exact antigen-antibody reaction, the medicament is retained in the placenta rich in TB cells after the lipid bimolecular shell is disintegrated, so that the medicament leakage is reduced and the toxic and side effects on fetuses are reduced; and avoids affecting vascular endothelial cells, immune cells and other stromal cells in the placenta.
Drawings
Fig. 1 is a schematic structural diagram of a lesion microenvironment-sensitive delivery nuclear magnetic 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:
the Fe content in the nano-drug system is measured by an atomic absorption spectrophotometer method and is used for measuring the dosage of the nano-drug. Weighing a certain amount of prepared drug solution (such as 1mL in step III), lyophilizing, and dissolving to 1mol L -1 The HCl solution is placed for 24 hours to ensure that Fe in 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 sample was measured with a Zeta-Plus potentiometric particle sizer (Brooken Haven) with an incident laser wavelength λ =532nm, an incident angle θ =90 °, temperature 25 ℃; the average of the three measurements was taken.
Example 1:
s1, synthesis of polyethyleneimine grafted polyethylene glycol (PEG-PEI)
The method comprises the steps of synthesizing polyethyleneimine grafted polyethylene glycol (PEG-PEI) by a two-step method, activating the terminal hydroxyl of monomethyl ether polyglycol by carbonyl diimidazole, and reacting 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 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-necked bottle (50 mL), adding trichloromethane (20 mL) to dissolve the PEI, adding PEG-CDI (3.2 g), stirring at room temperature for 24h, filling the solution into a dialysis bag (MWCO =3.5 kDa), dialyzing the solution with 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 packed product mPEG-PEI;
s2, synthesis of poly (acetimide) grafted polyethylene glycol grafted polycaprolactone (PEG-PEI-PCL)
Firstly, synthesizing PCL-OH, adding 15g of dried dodecanol into a two-mouth bottle, vacuum-drying at 70 ℃ for 8h, adding 2ml of Sn (Oct) 2 Continuing to dry for 0.5h, then adding 400mL of dried epsilon-caprolactone, and stirring and reacting for 24h at 105 ℃; cooling, adding 100mL of ethanol to dissolve unreacted epsilon-caprolactone, filtering, dissolving a 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) 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;
blowing and weighing a normal hexane solution of SPIO, collecting 5mg of SPIO nano particles in a serum bottle (10 mL), weighing 50mg of PEG-PEI-PCL polymer and WAY-262611 mg, dissolving and mixing the PEG-PEI-PCL polymer and WAY-262611 mg uniformly by using dimethyl sulfoxide (3 mL), dropwise adding the solution into 20mL of distilled water under ultrasonic dispersion, putting the reaction solution into a dialysis bag (MWCO =3.5 kDa) for dialysis for 24h to remove the dimethyl sulfoxide, 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)
CK7 antibody is firstly cracked by the method in the prior literature, and Fab segment of CK7 is obtained and purified. Then, linking the CK7-Fab to mal-PEG-COOH, and reacting the PEG connected with the antibody with amino on PEG-PEI-PCL-SPIO nano particles by amidation reaction to prepare Fab-PEG-PEI-PCL-SPIO;
the specific operation is as follows: 10mg of CK7 antibody was weighed out at 0.5 mg. Multidot.ml -1 Papain, 10 mmol. 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 high-purity CK7 Fab fragment;
1mg of Fab fragment of CK7 (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 using 500 mu g of EDC and NHS respectively for 15min, then adding the PEG-PEI-PCL-SPIO/drug 1698 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 IL-6-siRNA with negative electricity can be compounded to prepare the nano compound through electrostatic interaction. The specific operation is as follows: mu.g of IL-6-siRNA was diluted with PBS to a final volume of 1.5mL and shaken well. Taking 1.5mL of the PEG-PEI-SPIO prepared in the step (3) (or 1.6mL of the Fab-PEG-PEI-SPIO prepared in the step (4)) nanoparticles, ultrasonically dispersing the nanoparticles uniformly, mixing the IL-6-siRNA diluted solution and the PEG-PEI-SPIO (or the Fab-PEG-PEI-SPIO) nanoparticle solution uniformly, fixing the volume of the composite system to 0.061mg/mL, blowing, mixing uniformly and standing for 30 minutes to prepare a uniform composite;
s6, PEG-polypeptide synthesis
0.05mmol of AMP-activated protein kinase (AMPK) -sensitive polypeptide (HIS-MET-ARG-SER-ALA-MET-SER-GLY-LEU-HIS-LEU-VAL-LYS-ARG-ARG-NH 2, MW: 1779.15 Da), 5mmol of EDC and 5mmol of DMAP were dissolved in 10mL of acetonitrile in water (acetonitrile: water = 1), protected with N2 on an ice-water bath and magnetically stirred at 500rpm for 2h to activate 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 diagram of the prepared delivery nuclear magnetic probe 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, as shown in table 1 below:
table 1: examples and comparative examples
Functional evaluation experiment
1. Magnetic Resonance Imaging (MRI) assay for evaluating placenta-specific delivery function of drugs in gestational diabetes model
Model establishment and weight detection:
8 week-old SPF grade C57BL/6 mice (purchased from Guangdong provincial animal center for medical laboratory) were housed in an SPF light-controlled rearing environment, subjected to a light/dark cycle at a constant temperature of 22 ℃. + -. 2 ℃ and a humidity of 60% for 12 hours, and freely received food and drinking water and reared in metabolic cages. Female and male mice 2:1 mating in estrus in coop, carrying out Papanicolaou staining on vaginal secretion smears of female mice on the next day, and observing a vaginal sperm-positive person of a specimen under an optical microscope to diagnose that the person is pregnant, wherein the diagnosis is marked as the 0 th day of pregnancy (D0). 4 days of pregnancy 8:00 after fasting for 12h to 5 days of pregnancy, 50mg/kg STZ (0.1 mol/L solution freshly prepared with citrate-sodium citrate buffer pH = 4.2-4.5) was injected rapidly into the tail vein of the model group. An equal volume of STZ-free buffer was injected as a normal control.
Determination of fasting blood glucose: after fasting for 12h in advance, in 8 days earlier than 10 days of pregnancy, cutting the tail to collect blood and detect the fasting blood sugar of the pregnant rat, regarding the pregnant rat with the fasting blood sugar of more than or equal to 11.1mmol/L as successful model building, and building a gestational diabetes model.
As before, after 12h of prior fasting, 17 days of pregnancy 8:00 the fasting blood sugar of the fasting pregnant mouse is measured by cutting the tail and collecting blood.
MRI imaging to detect placental distribution of drugs:
on day 11 of gestational diabetes model animals, after chloral hydrate anesthesia, MRIT2 sequences are scanned at time points before (0 h) and 2h (2 h) after drug injection, and distribution of the nano-drug containing SPIO in vivo is observed. 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);
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 region in mice was observed on MRIbTFE sequence, and T2map imaging technique was used to measure the change in T2 relaxation time with SPIO in drug distributed in the uterus, placenta, embryo and other organs in vivo, and the respective relaxation rates R2 at 0h and 2h were calculated. The Relative increase ratio of R2 at 2h after drug injection (RSI (Relative Signal Intensity)% = R2) was calculated 2h /R2 0h ) The results are shown in Table 2.
Table 2 evaluation results of placenta-specific delivery function
| Group of | Placenta RSI (relative signal multiple) | Embryo RSI (relative signal multiple) | Liver RSI (relative multiple of signal) |
| Physiological saline group | 1.00 | 1.00 | 1.00 |
| Example 1 | 25.11 | 1.06 | 3.36 |
| Example 2 | 24.42 | 2.23 | 2.07 |
| Example 3 | 21.00 | 1.37 | 3.08 |
| Example 4 | 27.16 | 0.99 | 3.78 |
| Comparative example 1 | 10.12 | 16.74 | 5.29 |
| Comparative example 2 | 7.74 | 10.16 | 8.46 |
| Comparative example 3 | 3.46 | 2.65 | 8.31 |
| Comparative example 4 | 3.10 | 2.40 | 10.54 |
| Comparative example 5 | 12.53 | 1.06 | 16.67 |
| Comparative example 6 | 9.20 | 1.01 | 27.39 |
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 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 CK7 antibody targets other cells with CK7 expressed by various cell membranes in vivo including TB, and the cell membrane targeting property is not strong; therefore, lower placental RSI and lower liver RSI were detected; the drug without lipid membrane has smaller particle size, enters the placenta, passes the placenta barrier in larger proportion, and the embryo RSI is detected to be higher.
The delivery systems of comparative examples 3 and 4, which contained no CK7 antibody, were unable to target and anchor the drug entering the placenta to TB cells, unable 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 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 to 4, a bilayer structure delivery nuclear magnetic probe was synthesized using a lipid bilayer membrane modified with the substrate polypeptide of AMPK, PEG, as the outer shell and a drug carrier modified with an antibody against a marker on the surface of placental trophoblasts as the inner core, and the particle size ranged from 80 to 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 lipid bilayer membrane shell modified by the substrate polypeptide-PEG is stable in the circulation of other tissues and organs in vivo, reaches a placenta microenvironment with high specificity expression AMPK, is disintegrated along with the degradation of the polypeptide, and realizes the drug specificity distribution in the placenta tissues. After the drug shell is disintegrated in the placenta microenvironment, the drug core containing the CK7 antibody fragment is revealed. The CK7 antibody fragment can be anchored in a cell membrane specific high-expression CK7 TB cell in a placenta, promote the specific endocytosis of a drug by the TB cell to realize the regulation of the TB function, reduce the distribution in other cells of the placenta and reduce the influence on the function of the placenta. The CK7 antibody enables the medicine in the placenta to be anchored to the TB cell, so that the medicine leakage through a maternal-fetal barrier is effectively reduced, and the medicine reaching the embryo is reduced.
2. Establishment of gestational diabetes animal model for evaluating treatment effect
The injection of D3, D6, D9, D12 and D15 for drug therapy (therapeutic dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline) and the serial detection at D17, 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. Cutting placenta tissue, and storing at-80 deg.C in liquid nitrogen.
TABLE 3 evaluation of the treatment Effect in the animal model of gestational diabetes
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 treatment effect is poor, the blood sugar is high, the weight of the fetus is low, and the number born is low; meanwhile, the drug is accumulated in the embryo, which causes embryo toxicity, lower weight of the fetus and lower litter size.
The delivery system of comparative example 2 does not contain a polypeptide-PEG modified lipid bilayer membrane as a shell, and targeted release aiming at the placenta microenvironment cannot be realized; the CK7 antibody targets CK7 cells expressed in vivo including TB, the cell targeting is weak, the detection result is poor, the blood sugar is high, the litter weight is low, and the litter size is low. Meanwhile, the medicine without lipid membrane has smaller particle size, and the medicine enters the placenta and passes through the placenta barrier in a larger proportion, so that the weight of the fetus is lower, and the litter size is lower.
The delivery systems of comparative examples 3 and 4, which did not contain the CK7 antibody, failed to target and anchor the drug into the placenta to TB cells, failed to obtain placental retention, resulted in a large number of leaks across the placental barrier, and detected poor therapeutic efficacy, higher blood glucose, lower litter weight, and lower litter size. 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, and the detection shows that the treatment effect is poor, the blood sugar is high, the litter weight is low, and the litter size is low. Comparative example 4, which has no lipid bilayer membrane shell, is less effective than comparative example 3.
Comparative examples 5 and 6 have an excessively large particle size, resulting in extremely poor distribution of circulation in vivo, and the drug is mainly phagocytosed by the reticuloendothelial system of the liver in a large amount, resulting in insufficient distribution of the placental drug, poor therapeutic effect, high blood sugar, low litter weight, and low litter size. Comparative example 6, which has a particle size much larger than comparative example 5, has a poorer circulation distribution, and thus has a poorer therapeutic effect than comparative example 5.
In examples 1 to 4, a bilayer structure delivery nuclear magnetic probe was synthesized using a lipid bilayer membrane modified with the substrate polypeptide of AMPK, PEG, as the outer shell and a drug carrier modified with an antibody against a marker on the surface of placental trophoblasts as the inner core, and the particle size ranged from 80 to 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 lipid bilayer membrane shell modified by the substrate polypeptide-PEG is stable in circulation of other tissues and organs in vivo, reaches a placenta microenvironment with specific high expression of AMPK, disintegrates along with degradation of the polypeptide, and realizes drug specific distribution in placenta tissues. The drug shell disintegrates in the placental microenvironment, revealing the drug core containing the CK7 antibody fragment. The CK7 antibody fragment can be anchored in a TB cell with cell membrane specificity and high expression of CK7 in a placenta, promotes the medicine 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 CK7 antibody enables the medicine in the placenta to be anchored in TB cells, so that medicine leakage through a maternal-fetal barrier is effectively reduced, medicine reaching the embryo is reduced, and toxicity to the fetus is low.
3. Drug for toxicity evaluation of animal models
At 72 hours after the injection of the drug, the mice were bled from the tail vein, and liver function indices, alanine transaminase (ALT), total bilirubin (TBil), and kidney function indices, blood Urea Nitrogen (BUN) and serum creatinine (sCr), were examined. 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 delivery nuclear magnetic probe prepared by the invention has no obvious toxic or side effect on the mother and the fetus.
Claims (5)
1. A focal microenvironment-sensitive delivery nuclear magnetic probe, comprising:
the enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is in targeted disintegration under the action of the enzyme highly expressed by contacting placenta interstitial fluid is taken 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, and superparamagnetic ferroferric oxide SPIO nano particles, micromolecule drugs for regulating and controlling the function of the placenta trophoblast, therapeutic genes or the combination of the small molecule drugs and the therapeutic genes are loaded in the drug carrier;
wherein the enzyme highly expressed in the placenta interstitial fluid is protein kinase activated by AMP;
the enzyme substrate polypeptide is HIS-MET-ARG-SER-ALA-MET-SER-GLY-LEU-HIS-LEU-VAL-LYS-ARG-ARG-NH2;
the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester; the copolymer is polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL;
the marker antibody with high surface specificity expression of the placenta trophoblast is an Fab section of a keratin 7 antibody;
the small molecular drug is WAY-262611, and the therapeutic gene is siRNA for inhibiting the expression of IL-6 gene;
the average particle size of the delivery nuclear magnetic probe is 80nm-300nm.
2. The focal microenvironment-sensitive delivery nuclear magnetic probe according to claim 1, wherein the delivery nuclear magnetic probe has an average particle size of 100nm to 210nm.
3. The method for preparing a focal microenvironment sensitive delivery nuclear magnetic probe according to any one of claims 1-2, comprising the following steps:
s1, loading superparamagnetic ferroferric oxide (SPIO) nano particles, micromolecular drugs for regulating and controlling functions of placenta trophoblasts and/or genes to a copolymer to obtain composite nano particles;
s2, linking the placenta trophoblast surface marker antibody to the composite nano particle to obtain an antibody composite nano particle;
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 focus microenvironment sensitive delivery nuclear magnetic probe.
4. The preparation method of the focus microenvironment sensitive delivery nuclear magnetic probe according to claim 3, wherein in the step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15.
5. Use of the focal microenvironment sensitive delivery nuclear magnetic probe of any one of claims 1-2 in the preparation of a medicament for modulating placental trophoblast dysfunction disease, wherein the modulated placental trophoblast dysfunction disease is gestational diabetes.
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