CN114377158B - Focus targeted distribution type nuclear magnetic resonance imaging functional probe and preparation method and application thereof - Google Patents

Abstract

The invention discloses a focus-targeted distributed nuclear magnetic resonance imaging functional probe and a preparation method and application thereof. The functional probe can effectively avoid the absorption of maternal placental external organ tissues and fetal nonspecific drugs, and further realize the delivery and function regulation of the specific drugs of the trophoblasts in the placenta.

Description

Focus targeted distribution type nuclear magnetic resonance imaging functional probe and preparation method and application thereof

Technical Field

The invention relates to the field of chemical and biomedical engineering, in particular to a focus targeted distribution nuclear magnetic resonance imaging functional probe and a preparation method and application thereof.

Background

Fetal Growth Restriction (FGR), which means that the weight of the fetus is below the tenth percentile of the average weight of its gestational age or below two standard deviations of its average weight, is an important cause of death in perinatal infants. In China, the death rate of FGR infants is 4-6 times of that of infants with normal body weight. Of the surviving patients, many exhibit physical and even intellectual development disorders during development. The specific expression is that the growth parameters, the neural development score, the intelligence quotient and the like of the FGR infant are obviously lower than those of a control group. Therefore, reducing the occurrence of FGR and its adverse consequences is a significant topic in the area of perinatal medicine. The development of FGR is closely related to placental dysfunction due to inhibition of placental trophoblast function [ Hum Reprod update.2020 Jun 18;26 (4): 501-513; placenta.2020 Jul; 96-18 ].

Current studies indicate the inflammatory state of placental trophoblasts [ Front immunol.2020 Nov 13;11 [ 531543 ] and apoptosis [ Life sci.2018 Aug 1; 206. ] is important in the development and development of placenta-associated diseases such as FGR. Previous studies found that Norgestrel has anti-inflammatory and anti-apoptotic effects, alleviates cellular and microenvironment inflammatory responses by reducing ROS production, and has antioxidant, anti-inflammatory, immunomodulatory, antiviral and other pharmacological activities beneficial to placental development. EXPH5 (Exophilin-5) is an effector of Rab27, involved in intracellular vesicle trafficking and secretion [ Nat Cell biol.2010 Jan;12 (1) 19-30; 1-13. Sup pp; biochim Biophys acta.2013 Dec;1833 3471-3480 ] previous studies found significantly high expression of EXPH5 in dysfunctional placental trophoblasts [ circulation.2017;136 ] 1824-1839. The research result proves that the function of the trophoblast in the FGR environment is expected to be recovered by knocking EXPH5 out.

Normal apoptotic, inflammatory and immune states of placental trophoblasts, closely related to the maintenance of normal vesicle trafficking function within cells [ J Reprod immunol.2007 Dec;76 (1-2): 61-7 ]. Therefore, the normal expression of the placenta is expected to be recovered through the EXPH5-siRNA, and the function recovery effect on the FGR placenta is generated by the synergistic effect of the ROS regulation function of Norgetrel in the trophoblasts.

However, these may realize TB function modulating drugs, which, when applied in vivo, present pathophysiological barriers both maternal and fetal. Drug use and new drug development in pregnant women requires consideration of both maternal and fetal distribution of the drug and toxicity issues. 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 may cause significant side effects to the pregnant woman. Therefore, the existing medicines which are possibly effective to TB in vitro experiments cannot realize the regulation and control of the TB function under the condition of ensuring the safety of a mother body and a fetus.

After micromolecule medicines or gene therapy medicines possibly having the TB function regulation and control function which are screened by in vitro experimental research enter the circulation of the pregnant woman through injection or oral administration, the medicines can take effect on the whole body cells of the pregnant woman outside the placenta to generate side effects; meanwhile, after the medicine enters the placenta, the medicine rapidly penetrates through a placenta barrier due to abundant blood supply to the side of the fetus, so that the fetus is damaged. Therefore, none of these drugs can be clinically applied. Therefore, at present, no exact pharmaceutical intervention means is provided for the diseases of restricted intrauterine growth of the fetus, placenta implantation and the like clinically. Doctors can only carry out passive symptomatic treatment 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 the pathological target cells in various diseases. Development of TB-specific delivery vectors presents significant difficulties as these vectors fail to address the potential for toxic side effects on maternal and fetal non-specific distribution. The approaches that researchers try to promote the delivery of TB-specific nano-drugs include 2, one is to increase the particle size of nano-drugs, so that the nano-drugs cannot pass through a fetal membrane barrier and are retained in placenta to generate drug delivery effect; the other is specific delivery of antibody modified nano-carriers aiming at TB cell membrane markers.

The principle of increasing the particle size of the nano-drugs and promoting the distribution of the drugs in the placenta is that experimental research finds that the nano-drugs less than 300nm cannot be retained in the placenta and easily enter the fetus through the placenta. Researchers have therefore attempted to synthesize nanomedicines with particle sizes > 300nm, which are retained in the placenta, resulting in functional regulation of various cells including placental TB. However, an excessively large particle size (> 100 nm) of the drug is disadvantageous for the in vivo distribution of the drug. Most of the nano-drugs with the particle size of more than 300nm are captured by a reticuloendothelial system in maternal circulation, generate side effects everywhere in the whole body, can reach the placenta and realize low proportion of specific distribution of TB. Therefore, other 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., vimentin) that distinguish it from other placental stromal cells in placental tissue, and from other cells in the placenta. However, analysis of the expression level of multi-organ tissues throughout the body revealed that some cells of the surface marker were expressed in other parts of the placenta. The expression abundance on the surface of a small number of high-expression cells is not significantly different from TB. If the antibody of Vimentin is connected to the surface of the nano-drug carrier, the direct in vivo application can cause side effects on other cells expressing the marker Vimentin 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 absent at present.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a focus-targeted nuclear magnetic resonance imaging functional 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 nourishing cell membrane marker to reduce the distribution of the medicament in a fetal organ tissue after passing through the placenta in a targeted manner, so that the absorption of maternal placenta organ tissue and fetal nonspecific medicaments 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 targeted distribution nuclear magnetic resonance imaging functional probe.

The invention is realized by the following technical scheme:

a focus-targeted nuclear magnetic resonance imaging functional probe comprises a shell, an inner core, superparamagnetic ferroferric oxide SPIO nano particles loaded in the inner core, micromolecular drugs for regulating and controlling the function of placenta trophoblasts, therapeutic genes or a combination thereof;

the outer shell is an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is subjected to targeted disintegration under the action of an enzyme which is in contact with high expression of placenta interstitial fluid; the enzyme highly expressed in placenta interstitial fluid is one or more of histone deacetylase, lysozyme, kininase, histaminase or oxytocin;

the inner core is a drug carrier modified by a marker antibody with high surface specificity expression of placenta trophoblast, and the drug carrier is a copolymer formed by a polycation carrier modified by polyethylene glycol and hydrophobic degradable polyester; the marker antibody with high surface specificity expression of the placenta trophoblast is an Fab segment of a Vimentin antibody.

The placenta of pregnant women is rich in a plurality of enzymes for promoting placenta development and fetal nutrition, the enzymes highly expressed in the placenta interstitial fluid are one or more of histone deacetylase, lysozyme, kininase, histaminase, oxytocin or histone deacetylase, wherein Histone Deacetylase (HDAC) is extremely high in expression level in the placenta interstitial fluid and hardly expressed in normal human blood and interstitial fluid, and therefore, the histone deacetylase is preferred.

The histone deacetylase substrate polypeptide can be selected from Ac-Lys-AMC, molecular weight: 345.39Da.

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 Norgetrel, and the therapeutic gene is siRNA inhibiting expression of EXPH5 (Exophilin 5) gene.

The average particle size of the focus-targeted nuclear magnetic resonance imaging functional 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 difficulty is increased, and the loading of drugs and genes is not beneficial.

The invention also provides a preparation method of the focus targeted distribution nuclear magnetic resonance imaging functional 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-targeted nuclear magnetic resonance imaging functional 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 HDAC substrate polypeptide-PEG modified lipid bilayer membrane is used as the shell, so that the nano-delivery system is ensured to be stably distributed in enzyme-free blood before entering a placental enzyme environment, drug leakage is reduced, and other cells outside the placenta are reduced or avoided from phagocytosis. Thereby ensuring the safety of other tissues and organs outside the maternal placenta; the HDAC enzyme sensitive shell is disintegrated in a microenvironment containing enzymes at the placenta matrix side to release the medicine, so that the high-efficiency release and distribution of the medicine in the matrix placenta can be ensured; due to the introduction of the enzyme sensitive shell, the distribution efficiency of the placenta can be ensured without adopting a large-particle-size nano-carrier structure, the particle size of the nano-carrier is effectively reduced, the stable circulation distribution of the medicine before entering the placenta is ensured, and the reticuloendothelial system is ensured not to phagocytize a large amount of carriers to cause the reduction of curative effect and the increase of side effect.

The invention adopts the medicine carrier modified by the placenta trophoblast surface marker antibody as the inner core, the medicine is modified by the TB cell surface marker antibody, and the TB cell membrane in the placenta can be anchored exactly after being released, thereby ensuring the specific administration of the TB cell in a complex placenta environment, and simultaneously avoiding unnecessary placenta function damage caused by the administration of other cells in the placenta; most of the medicines entering the placenta are targeted by the antibody and are exactly anchored in TB cells, so that the medicines are ensured to leak through a placenta barrier and enter the side of the fetus, and the safety of the fetus is ensured; after the medicine is anchored on the TB cell membrane, the therapeutic medicine and the therapeutic gene are promoted to be swallowed into the TB cell, so that the function regulation is realized, and the exact TB function regulation is ensured.

The invention also provides application of the focus-targeted nuclear magnetic resonance imaging functional probe in preparing a medicine for regulating and controlling the placenta trophoblast dysfunction disease, wherein the placenta trophoblast dysfunction disease is fetal growth restriction.

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 focus-targeted distributed nuclear magnetic resonance imaging functional probe with a double-layer structure. The double-layer structure can ensure that the liposome shell has stable structure and keeps stable circulation in the blood circulation of the pregnant woman, so that the liposome is not easily captured by other tissues and cells including a reticuloendothelial system, the distribution and release of other tissues except a placenta, which are influenced by the body of the pregnant woman, and the toxic and side effects are reduced;

(2) After the transmission system enters the placenta along with blood circulation, an enzyme substrate in the outer shell of the transmission system is decomposed by corresponding enzyme highly expressed in placenta tissues, and the protective lipid bilayer outer shell is quickly disintegrated in the placenta to release the antibody modified nano-medicament 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 a TB cell membrane in a placenta and is further specifically endocytosed by the TB cell to generate a function regulation and control effect, so that exact treatment on TB cell diseases is ensured;

(3) Through exact 'antigen-antibody reaction', the medicament is retained in the placenta rich in TB cells after the lipid bimolecular shells are disintegrated, so that the medicament leakage is reduced, the medicament passes through a placenta barrier, and the toxic and side effects on a fetus are reduced; and also avoids affecting vascular endothelial cells, immune cells and other stromal cells in the placenta.

Drawings

Fig. 1 is a schematic structural diagram of a lesion targeted nuclear magnetic resonance imaging functional 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 ^-1 The solution was left to stand for 24 hours to ionize Fe sufficiently in SPIO, and the absorbance of Fe atoms at 248.3nm was measured with an atomic absorption spectrophotometer and substituted into a standard curve prepared with a Fe standard solution to calculate the Fe concentrationAnd then calculating the Fe content in the medicine solution before freeze-drying.

The particle size test method comprises the following steps:

the particle size of the samples was measured with a Zeta-Plus potential particle sizer (Brooken Haven) with an incident laser wavelength λ =532nm, an incident angle θ =90 ° and a temperature of 25 ℃; the average of the three measurements was taken.

Example 1:

s1, synthesis of polyethyleneimine grafted polyethylene glycol (PEG-PEI)

The method adopts a two-step method to synthesize polyethyleneimine grafted polyethylene glycol (PEG-PEI), firstly uses carbonyldiimidazole to activate the terminal hydroxyl of monomethyl ether polyglycol, and then reacts with the amino of polyethyleneimine to generate PEG-PEI. The specific operation is as follows: monomethyl ether glycol (8.0 g, mn = 2kDa) was weighed into a reaction flask, dried under vacuum at 80 ℃ for 6 hours, and dissolved by adding THF (60 mL) under an argon atmosphere. Carbonyldiimidazole (CDI, 6.4 g) was weighed into another reaction flask, and THF with mPEG-OH dissolved therein was slowly dropped into the CDI flask using an isopiestic dropping funnel, and the reaction was stirred at room temperature overnight. Distilled water (0.648 mL) was added to inactivate the excess CDI and stirring was continued for 30min. Precipitating the solution into a large amount of cold ether, filtering, and drying in vacuum to obtain white powdery solid mPEG-CDI;

weighing PEI (4.4 g, MW = 1.8kDa) and adding the PEI into a two-mouth bottle (50 mL), adding trichloromethane (20 mL) to dissolve the PEI and adding PEG-CDI (3.2 g), stirring at room temperature for reaction for 24h, filling the solution into a dialysis bag (MWCO =3.5 kDa), dialyzing the solution in the trichloromethane for 24h, concentrating the solution in the dialysis bag under reduced pressure, precipitating the solution in a large amount of cold ether, filtering and drying to obtain white powder packaged product mPEG-PEI;

s2, synthesis of poly (acetimide) grafted polyethylene glycol grafted polycaprolactone (PEG-PEI-PCL)

Firstly, synthesizing PCL-OH, adding 15g of dried dodecanol into a two-mouth bottle, drying at 70 ℃ for 8 hours in vacuum, adding 2ml of Sn (Oct) ₂ Continuously drying 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 anhydrous ether, filtering, and drying to obtain white powderProduct as such, yield 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, 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 =5 kDa) for 24h, removing part of trichloromethane by reducing pressure, 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) is described in documents [ 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, and then heated to 300 ℃ and refluxed for 1h, and the reaction system slowly changes from dark red to black; naturally cooling in air, precipitating in 150ml ethanol, centrifuging at 10000rpm for 5min, discarding the supernatant, dissolving the lower precipitate in 70ml n-hexane containing 4 drops of oleic acid and oleylamine, centrifuging at 10000rpm for 10min to remove insoluble part, precipitating the solution in 200ml ethanol, centrifuging at 10000rpm for 10min, dissolving the lower precipitate in 60ml n-hexane, introducing argon gas for protection, and storing at 4 deg.C;

drying and weighing an n-hexane solution of SPIO, collecting 5mg of SPIO nano particles in a serum bottle (10 mL), weighing 50mg of PEG-PEI-PCL polymer and 5mg of Norgetrel, dissolving and uniformly mixing the PEG-PEI-PCL polymer and the Norgetrel with chloroform (3 mL), dropwise adding the solution into 20mL of distilled water under ultrasonic dispersion, volatilizing to remove the chloroform, centrifuging at the rotating speed of 12000r/mim, collecting precipitates, and removing a supernatant. Dissolving the precipitate with water, ultrasonically dispersing, repeating the centrifugal operation, ultrasonically dispersing the prepared PEG-PEI-PCL-SPIO/drug nano particles into water, filtering by using a needle filter with the aperture of 220nm, adding purified water, adjusting the concentration of the PEG-PEI-PCL-SPIO/drug nano particles to a constant volume until the content of Fe is 0.145mg/mL, 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)

Firstly, the Vimentin antibody is cracked by adopting the method in the prior literature, the Fab segment of the Vimentin is obtained, and the Fab segment is purified. Then linking Vimentin-Fab to mal-PEG-COOH, and reacting PEG connected with the antibody and 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 Vimentin 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 enzymolysis product by ProteinA affinity chromatography, further purifying penetration peak by DEAE anion exchange chromatography, dialyzing, desalting, and lyophilizing to obtain high-purity Vimentin Fab fragment;

1mg of Vimentin Fab fragment (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 charge and the EXPH5-siRNA with negative charge can be compounded into a nano compound through electrostatic interaction. The specific operation is as follows: mu.g of EXPH5-siRNA was diluted with PBS to a final volume of 1.5mL and shaken well. Taking 1.5mL of the PEG-PEI-SPIO (or 1.6mL of the net weight of the Fab-PEG-PEI-SPIO) prepared in the step (3) to be dispersed uniformly by ultrasonic, uniformly mixing an EXPH5-siRNA diluted solution and a PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle solution, fixing the volume of the composite system to 0.061mg/mL, blowing, uniformly mixing and standing for 30 minutes to prepare a uniform composite;

s6, PEG-polypeptide synthesis

0.05mmol of histone deacetylase sensitive polypeptide (Ac-Lys-AMC, molecular weight: 345.39 Da), 5mmol of EDC and 5mmol of DMAP were dissolved in 10mL of acetonitrile in water (acetonitrile: water = 1), protected by 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 =1.0 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 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. Finally, 2mL of physiological saline (0.9 percent NaCl) solution is added to dissolve the PEG-polypeptide modified liposome shell @ therapeutic gene composite nano particles, the filtration rate of a syringe filter with the aperture of 220nm is determined, the volume is fixed until the Fe content is 0.061mg/mL, and the solution is stored at 4 ℃ for later use.

The specific structural schematic diagram of the prepared focus target distribution nuclear magnetic resonance imaging functional 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

Function evaluation test

1. Magnetic Resonance Imaging (MRI) experiment to evaluate placenta-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 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). Upon induction of FGR formation, pregnant mice were exposed to 300ppm CdCl via drinking water during full pregnancy, starting 7 days pre-mating ₂ And replacing drinking water of the pregnant mouse in each cage every two days to establish a fetal intrauterine growth restricted model. Control mice were given double distilled water.

MRI imaging to detect placental distribution of drugs:

on day 11, after chloral hydrate anesthesia, the MRIT2 sequence was scanned at time points before (0 h) and 2h (2 h) after drug injection to observe the in vivo distribution of the nano-drug containing SPIO. The dosage of the tail vein injection nano-medicament is as follows: (therapeutic dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline);

MRI imaging of the uterus of C57BL/6j mice was performed using a Philips Intera 1.5T MRI scanner, with its animal specific coils. The evolution of signal intensity in the uterus and embryonic regions in mice was observed on the MRIbTFE sequence and the T2 relaxation time changes due to SPIO distribution in drugs in the uterus, placenta, embryo and other organs in vivo were measured using T2map imaging technique, calculating the relaxation rates R2 at 0h and 2h, respectively. The Relative 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	25.09	1.06	3.03
				Example 2	24.40	2.22	1.87
Example 3	20.98	1.37	2.78
				Example 4	27.14	0.99	3.41
Comparative example 1	9.13	16.73	4.77
				Comparative example 2	6.98	10.16	7.64
Comparative example 3	3.12	2.35	7.50
				Comparative example 4	2.80	2.16	9.50
Comparative example 5	11.30	1.05	15.04
				Comparative example 6	8.30	1.02	24.71

From the above results, in comparative example 1, the placenta trophoblast surface marker antibody is not linked, and after the polypeptide-PEG modified lipid bilayer is disintegrated, the drug in the content cannot be anchored in TB cells to obtain placenta retention, a large amount of drug leaks through the placenta barrier, and low placenta RSI is detected; the drugs are accumulated in the embryo, which results in high embryo RSI; the drug can not be anchored in TB cells to obtain placenta retention, and part of the drug is separated from the placenta and distributed systemically, so that the liver RSI is high.

The delivery system of comparative example 2 does not contain a polypeptide-PEG modified lipid bilayer membrane as a shell, and targeted release to the placenta microenvironment cannot be achieved; in addition, the Vimentin antibody targets other cells with various cell membranes expressing Vimentin 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, without the Vimentin 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 to 4, a lipid bilayer membrane modified by HDAC substrate polypeptide-PEG was used as a shell, and a drug carrier modified by placental trophoblast surface marker antibody was used as an inner core to synthesize a bilayer-structured focal-targeted nuclear magnetic resonance imaging functional probe with a particle size range of 80 to 210nm. The particle size of about 100nm and the outer negative electricity lipid bilayer membrane are convenient to avoid being phagocytized by a reticuloendothelial system in quantity, 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 HDAC, is disintegrated along with the degradation of the polypeptide, and realizes the drug specificity distribution in the placenta tissues. The drug shell disintegrates in the placental microenvironment, revealing the inner drug core containing the Vimentin antibody fragments. The Vimentin antibody fragment can be anchored in a placenta to a TB cell with a cell membrane specificity and high expression of Vimentin, so that the medicine is promoted to be specifically endocytosed by the TB cell to realize the regulation of the TB function, the distribution of the Vimentin antibody fragment in other cells of the placenta is reduced, and the influence on the function of the placenta is reduced. The Vimentin antibody enables the medicine in the placenta to be anchored in TB cells, so that the medicine leakage through a maternal-fetal barrier is effectively reduced, and the medicine reaching the embryo is reduced.

2. Establishing animal model with restricted intrauterine growth of fetus for evaluating treatment effect

Drugs (therapeutic dose 0.31mg/Kg iron equivalent drug, or physiological saline of the same volume) 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 sequence, and the number of surviving litters was recorded. Removing a fetal membrane and an umbilical cord on the placenta, shearing the umbilical cord at the end of the fetus along the root of the umbilical cord, respectively placing the placenta and the fetus on sterile gauze, sucking out amniotic fluid on the surface, and weighing the placenta and the fetus by an analytical balance. Cutting placenta tissue, and storing at-80 deg.C in liquid nitrogen.

And (4) counting the weight of all normal fetal mice, and setting the fetal mice with the weight lower than the tenth percentile of all the fetal mice as FGR. The FGR incidence rate of each treated group of fetal mice = (FGR amount/total number of animals in this group) × 100% was calculated

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 Vimentin antibody targets Vimentin 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 vismentin antibodies, failed to target and anchor the drug entering the placenta to TB cells, failed to obtain placental retention, and resulted in a large number of leaks across the placental 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 viable fetuses. Meanwhile, the lipid bilayer membrane outer shell of the comparative example 3 is not modified by enzyme-sensitive polypeptide, the distribution of the lipid bilayer membrane outer shell in placenta is reduced, the detected treatment effect is poor, the number of dead fetus and absorbed fetus is high, the FGR incidence rate is high, the weight of fetus is low, and the number of live fetus is low. Comparative example 4, which has no lipid bilayer membrane shell, is less effective than comparative example 3.

The particle sizes of the comparative examples 5 and 6 are too large, so that the in vivo circulation distribution effect is extremely poor, the medicines are mainly phagocytosed by the reticuloendothelial system of the liver in a large amount, the placenta medicine is not distributed enough, the treatment effect is poor, the number of dead fetuses and absorbed fetuses is high, the FGR incidence rate is high, the weight of the fetuses is low, and the number of live fetuses is low. 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 lipid bilayer membrane modified by HDAC substrate polypeptide-PEG was used as a shell, and a drug carrier modified by placental trophoblast surface marker antibody was used as an inner core to synthesize a bilayer-structured focal-targeted nuclear magnetic resonance imaging functional probe with a particle size range of 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 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 HDAC, disintegrates along with degradation of the polypeptide, and realizes specific distribution of the drug in the placenta tissue. The drug shell disintegrates in the placental microenvironment, revealing the drug core containing the Vimentin antibody fragment. The Vimentin antibody fragment can be anchored in a cell membrane specific high expression Vimentin TB cell in a placenta, 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, reduces the influence on the placenta function, and realizes better treatment effect through effective regulation of the TB function. The Vimentin antibody enables the drug in the placenta to be anchored in TB cells, so that the drug leakage through a maternal-fetal barrier is effectively reduced, the drug reaching the embryo is reduced, and the toxicity to the fetus is low.

3. Drug for toxicity evaluation of animal models

At 72 hours after the injection of the drug, the tail vein was bled and liver function index glutamic pyruvic transaminase (ALT), total bilirubin (TBil) and kidney function index Blood Urea Nitrogen (BUN) and serum creatinine (srr) 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 focus-targeted nuclear magnetic resonance imaging functional probe prepared by the invention has no obvious toxic or side effect on the mother and the fetus.

Claims (5)

1. A focus targeted distributed nuclear magnetic resonance imaging functional probe is characterized in that the functional probe comprises a shell, an inner core, superparamagnetic ferroferric oxide SPIO nano particles loaded in the inner core, micromolecular drugs for regulating and controlling the function of placenta trophoblasts, therapeutic genes or a combination thereof; the small molecule drug isNorgestrelThe therapeutic gene is inhibitionEXPH5siRNA for gene expression;

the shell is an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is subjected to targeted disintegration under the action of an enzyme which is highly expressed by contacting placenta interstitial fluid; the enzyme highly expressed in the placenta interstitial fluid is histone deacetylase; the enzyme substrate polypeptide is Ac-Lys-AMC;

the inner core is a drug carrier modified by a marker antibody with high surface specificity and expression of placenta trophoblasts, and 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 segment of a Vimentin antibody;

the average particle size of the functional probe is 80nm-300nm.

2. The functional nuclear magnetic resonance imaging probe with lesion-targeted distribution according to claim 1, wherein the average particle size of the functional probe is 100nm to 210nm.

3. The method for preparing the focus-targeted nuclear magnetic resonance imaging functional probe according to any one of claims 1 to 2, which is characterized by 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-targeted nuclear magnetic resonance imaging functional probe.

4. The preparation method of the focus-targeted nuclear magnetic resonance imaging functional probe according to claim 3, characterized in that in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15.

5. Use of the foci-targeted nuclear magnetic resonance imaging functional probe of any one of claims 1-2 in the preparation of a medicament for modulating placental trophoblast dysfunction disease, which is fetal growth restriction.

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