CN114469891A - Micromolecule drug/therapeutic gene combined delivery system with enzyme sensitive shell and preparation method and application thereof - Google Patents
Micromolecule drug/therapeutic gene combined delivery system with enzyme sensitive shell and preparation method and application thereof Download PDFInfo
- Publication number
- CN114469891A CN114469891A CN202111645495.XA CN202111645495A CN114469891A CN 114469891 A CN114469891 A CN 114469891A CN 202111645495 A CN202111645495 A CN 202111645495A CN 114469891 A CN114469891 A CN 114469891A
- Authority
- CN
- China
- Prior art keywords
- placenta
- drug
- enzyme
- delivery system
- peg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
- A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
- A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
- A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
- A61K49/1875—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle coated or functionalised with an antibody
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention discloses a micromolecule drug/therapeutic gene combined delivery system with an enzyme sensitive shell, a preparation method and application thereof, wherein the delivery system is of a core-shell double-layer structure, an enzyme substrate polypeptide-PEG modified lipid bimolecular membrane which is subjected to targeted disintegration under the action of an enzyme which is contacted with interstitial fluid of a placenta and highly expressed is taken as a shell, a drug carrier modified by a placenta trophoblast surface specificity high expression marker antibody is taken as an inner core, and superparamagnetic ferroferric oxide SPIO nano particles, micromolecule drugs for regulating and controlling the function of the placenta trophoblast, therapeutic genes or a combination thereof are loaded in the drug carrier; the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester. The micromolecule drug/therapeutic gene combined delivery system of the enzyme sensitive shell can effectively avoid non-specific drug absorption of other organs outside the maternal placenta and the fetus, and further realizes delivery and function regulation of nourishing cell specific drugs in the placenta.
Description
Technical Field
The invention relates to the field of chemical and biomedical engineering, in particular to a micromolecule drug/therapeutic gene combined delivery system of an enzyme sensitive shell and a preparation method and application thereof.
Background
The placenta dysfunction is the root of serious diseases of pregnant women such as preeclampsia and the like. This disorder is mostly due to dysfunction of the most important cell in the placenta, the Trophoblast (TB). For example, TB has insufficient EMT and pro-angiogenic functions and can cause preeclampsia (causing malignant hypertension during pregnancy and the second leading cause of death in pregnant and lying-in women). TB is too aggressive and can lead to placental planting. TB, if malignant, results in hydatidiform mole or other trophoblastic tumors. In previous studies, many small molecules or gene therapy drugs have been developed that can modulate TB function. However, a drug delivery means which can exactly act on TB in placenta, perform function regulation on TB and further realize treatment of related diseases is lacked at present.
Applicants have also conducted extensive research in delivering drugs and generated valuable findings. In earlier studies, it was found that BML-284 plays a role of an effective Wnt/beta-Catenin signal activator by inducing TCF-dependent transcriptional activity, thereby promoting invasive transfer of cells [ Chem Biol interact.2020Jul 1; 325: 109110; am J Pathol.2020Nov; 190(11), 2237 and 2250. The applicant finds that the application of the polypeptide in preeclampsia placenta can play a remarkable function promoting role in TB with insufficient invasion function and angiogenesis induction function. Compared with normal placenta trophoblasts, the AHSP gene is obviously highly expressed in the preeclampsia placenta trophoblasts and is probably an important gene for the occurrence and development of preeclampsia in the placenta. Activation of AHSP in cells is closely related to changes in the expression level of the antioxidant regulatory protein STAT3 and the activity of the STAT3 signaling pathway activator interleukin 6(IL-6) [ Science China (Life Sciences). Previous studies by the applicant have demonstrated that the regulation of the Wnt/β -Catenin pathway and STAT3 pathway have close relationships and synergies [ Adv Sci (Weinh).2019Mar 7; 1801885 in (6) (9). The two pathways are applied in a combined way, and have close correlation in the regulation of cell invasion and transfer functions [ Cancer Res.2017Apr 15; 77(8), 1955-1967; biomed pharmacother.2018dec; 108: 618-.
There are pathophysiological disorders in both maternal and fetal aspects of developing TB function modulating drugs. Drug use and new drug development in pregnant women requires consideration of both the distribution of the drug in the mother and in the fetus itself, as well as creating toxicity problems in both. Most of the drugs can pass through the placenta and distribute into the side of the fetus, affecting the development of the fetus. Therefore, the medication of pregnant women including emergency drugs has many contraindications. The medicines for pregnant women are classified into 5 types according to teratogenic property, and except for a few medicines with the minimum toxicity which are classified into a type and a type b, most of the other medicines in the c type, the d type and the e type have obvious damage to fetuses. The pregnant women have heavy metabolism burden in vivo during pregnancy and complicated immune change. Therefore, even if the drug is not significantly toxic during non-pregnancy, it is likely to cause significant side effects to the pregnant woman. Therefore, the existing medicines which are possibly effective to TB in vitro experiments cannot realize the regulation and control of the TB function under the condition of ensuring the safety of a mother body and a fetus.
After micromolecule medicines or gene therapy medicines possibly having the TB function regulation and control function which are screened by in vitro experimental research enter the circulation of the pregnant woman through injection or oral administration, the medicines can take effect on the whole body cells of the pregnant woman outside the placenta to generate side effects; meanwhile, after the medicine enters the placenta, the medicine rapidly penetrates through a placenta barrier due to abundant blood supply to the side of the fetus, so that the fetus is damaged. Therefore, none of these drugs can be clinically applied. Therefore, at present, no exact pharmaceutical intervention means is provided for the diseases such as preeclampsia, placenta planting and the like clinically. Doctors can only carry out passive symptomatic treatment aiming at the symptoms caused by the placenta dysfunction diseases. But not through TB function regulation, realize the real placenta function recovery. Therefore, how to avoid toxicity to the mother and fetus and realize effective delivery of the TB function regulating drug is the key to solving the diseases caused by TB dysfunction.
The current macromolecule nano-carrier drug can realize the specific drug delivery to pathological target cells in various diseases. Development of TB-specific delivery vectors presents significant difficulties as these vectors do not address the problem of potential toxic side effects on maternal and fetal non-specific distribution. The approaches that researchers try to promote the delivery of TB-specific nano-drugs include 2, one is to increase the particle size of nano-drugs, so that the nano-drugs cannot pass through a fetal membrane barrier and are retained in placenta to generate drug delivery effect; the other is specific delivery of antibody modified nano-carriers aiming at TB cell membrane markers.
The principle of increasing the particle size of the nano-drugs and promoting the distribution of the drugs in the placenta is that experimental research finds that the nano-drugs less than 300nm cannot be retained in the placenta and easily enter the fetus through the placenta. Researchers have therefore attempted to synthesize nanomedicines with particle sizes > 300nm, which are retained in the placenta, resulting in functional regulation of various cells including placental TB. However, too large a particle size (> 100nm) of the drug is detrimental to the in vivo distribution of the drug. Most of the nano-drugs with the particle size of more than 300nm are captured by a reticuloendothelial system in maternal circulation, generate side effects at all parts of the whole body, can reach the placenta and realize low proportion of specific distribution of TB. Therefore, other means are needed to achieve retention of the nano-drug in the placenta and targeting of TB cells.
The nano-drug can adopt a nano-drug linked antibody to target and identify the cell membrane marker of the target cell, thereby realizing the specific delivery of the target cell. TB has some established surface markers (e.g., epidermal growth factor receptor, EGFR for short) that distinguish it from other placental stromal cells in placental tissue, and from other cells in the placenta. However, analysis of the expression level of multiple organ tissues throughout the body revealed that some cells of the surface marker were expressed in other parts of the placenta. The expression abundance on the surface of a small number of high-expression cells is not significantly different from TB. If the antibody of the EGFR is connected to the surface of the nano-drug carrier, the direct in vivo application will cause side effects on other cells expressing the marker EGFR in vivo. Therefore, only before entering the placenta in blood circulation, the TB cell recognition antibody of the nano-carrier is shielded, so that the TB cell recognition antibody can be prevented from being distributed in cells outside the placenta, and the TB cell recognition antibody can be ensured to be distributed in the placenta.
In summary, a nano-carrier system capable of effectively avoiding the nonspecific drug absorption of the mother and the fetus and further realizing the specific drug delivery and function regulation of the TB cells in the placenta is lacking at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a micromolecular drug/therapeutic gene combined delivery system with an enzyme sensitive shell, which utilizes the placenta microenvironment to reduce the distribution of the drug in maternal organ tissues before entering the placenta and utilizes the trophoblast markers to reduce the distribution of the drug in fetal organ tissues after passing through the placenta in a targeted manner, thereby effectively avoiding the absorption of nonspecific drugs of other organs outside the maternal placenta and the fetus and further realizing the delivery and function regulation of specific drugs of nourishing cells in the placenta.
The invention also aims to provide a preparation method of the small molecule drug/therapeutic gene combined delivery system of the enzyme sensitive shell.
The invention is realized by the following technical scheme:
a micromolecule drug/therapeutic gene combined delivery system of an enzyme sensitive shell is of a core-shell double-layer structure, an enzyme substrate polypeptide-PEG modified lipid bimolecular membrane which is subjected to targeted disintegration under the action of an enzyme which is contacted with high expression of placenta interstitial fluid is used as a shell, a drug carrier modified by a marker antibody of high expression of the surface specificity of placenta trophoblasts is used as an inner core, and superparamagnetic ferroferric oxide (SPIO) nano particles, micromolecule drugs for regulating and controlling the function of the placenta trophoblasts, therapeutic genes or a combination of the micromolecule drugs and the therapeutic genes are loaded in the drug carrier; the enzyme highly expressed in placenta interstitial fluid is one or more of protein kinase C, 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 with high surface specificity expression of the placenta trophoblast is a Fab segment of an Epidermal Growth Factor Receptor (EGFR) antibody.
The placenta of pregnant women is rich in a plurality of enzymes for promoting the development of the placenta and nutrition of fetuses, the enzymes highly expressed in the placenta interstitial fluid are one or more of protein kinase C, lysozyme, kininase, histaminase, oxytocin or matrix metalloproteinase, wherein Protein Kinase C (PKC) is extremely high in expression level in the placenta interstitial fluid and hardly expressed in normal human blood and interstitial fluid, and therefore, the protein kinase C is preferred.
The protein kinase C substrate polypeptide may be Glu-Arg-Met-Arg-Pro-Arg-Lys-Arg-Gln-Gly-Ser-Val-Arg-Arg-Arg-Val, molecular weight: 2067.43 Da.
The drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester, the copolymer is one or more of polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL, polyethylene glycol-polyethyleneimine-polylactic acid PEG-PEI-PLA or polyethylene glycol-polyethyleneimine-polylactic acid-glycolic acid PEG-PEI-PLGA, and preferably polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL.
The copolymer of the invention can be synthesized by the prior art, for example, PEG is firstly reacted with polycation carrier to form the copolymer, and then the active group of polycation is reacted with the activated polyester segment to form the copolymer.
The copolymers of the present invention are also commercially available.
The drug carrier of the invention is loaded with superparamagnetic ferroferric oxide SPIO nano particles, micromolecule drugs for regulating and controlling the function of placenta trophoblasts, therapeutic genes or the combination thereof. The small molecular drug is BML-284, and the therapeutic gene is siRNA for inhibiting AHSP (Alpha-hemoglobin-stabilizing protein) gene expression. Therefore, in the research, the expression of AHSP in the trophoblast is inhibited, and the STAT3 pathway is regulated to generate a synergistic effect on the BML-284 function, so that the invasion and angiogenesis of the trophoblast are more effectively promoted, the restoration of the pathological placenta function in preeclampsia is promoted, the symptoms are relieved, and the prognosis is improved.
The average particle size of the micromolecule drug/therapeutic gene combined delivery system of the enzyme sensitive shell is 70nm-300nm, preferably 90nm-200nm, the particle size is too large to be beneficial to in vivo circulation, the particle size is too small to be beneficial to increase preparation difficulty and loading drugs and genes.
The invention also provides a preparation method of the small molecule drug/therapeutic gene combined delivery system of the enzyme sensitive shell, which comprises the following steps:
s1, loading superparamagnetic ferroferric oxide (SPIO) nanoparticles, micromolecular drugs for regulating and controlling the function of placenta trophoblasts and/or genes to the copolymer to obtain composite nanoparticles;
s2, linking the placenta trophoblast surface marker antibody to the composite nanoparticle;
s3, linking enzyme substrate polypeptide for promoting placenta development and fetal nutrition with PEG to obtain polypeptide-PEG;
s4, mixing the polypeptide-PEG and the liposome to form a polypeptide-PEG modified lipid bilayer membrane;
s5, assembling the polypeptide-PEG modified lipid bilayer membrane and the composite nanoparticles into a small molecule drug/therapeutic gene combined delivery system with an enzyme sensitive shell.
Preferably, in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15: 1.
According to the invention, the PKC substrate polypeptide-PEG modified lipid bilayer membrane is used as the shell, so that the nano transmission system is ensured to be stably distributed in enzyme-free blood before entering a placental enzyme environment, the drug leakage is reduced, and the phagocytosis of other cells outside the placenta is reduced or avoided. Thereby ensuring the safety of other tissues and organs outside the maternal placenta; the PKC 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 phagocytose a large amount of carriers to cause the reduction of the curative effect and the increase of the side effect.
The invention adopts the drug carrier modified by the placenta trophoblast surface marker antibody as an inner core, the drug is modified by the TB cell surface marker antibody or antibody fragment, and the drug can be released in placenta interstitial fluid to exactly anchor the TB cell membrane in the placenta, thereby ensuring the specific drug delivery to the TB cell in a complex placenta environment, and simultaneously avoiding unnecessary placenta function damage caused by drug 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 small molecule drug/therapeutic gene combined delivery system of the enzyme sensitive shell in preparing a drug for regulating and controlling the placenta trophoblast dysfunction disease, wherein the disease for regulating and controlling the placenta trophoblast dysfunction disease is preeclampsia (gestational hypertension).
Compared with the prior art, the invention has the following beneficial effects:
(1) in the research, expression of AHSP in trophoblasts is inhibited, and a synergistic effect on the activity regulation function of Wnt/beta-Catenin pathway of BML-284 can be generated by regulating and controlling STAT3 pathway, so that invasion and angiogenesis of trophoblasts are promoted more effectively, restoration of pathological placenta function in preeclampsia is promoted, symptoms are relieved, and prognosis is improved;
(2) 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; a small molecule drug/therapeutic gene combined delivery system of an enzyme sensitive shell with a synthetic double-layer structure. The double-layer structure can ensure that the liposome shell structure is stable and keeps stable circulation in the blood circulation of the pregnant woman, so that the nano-drug is not easily captured by other tissues and cells including a reticuloendothelial system, the distribution and release of other tissues except a placenta which are influenced in the body of the pregnant woman are reduced, and the toxic and side effects are reduced;
(3) after the transmission system enters the placenta along with blood circulation, an enzyme substrate in the outer shell of the transmission system is decomposed by corresponding enzyme highly expressed in placenta tissues, and the protective lipid bimolecular outer shell is rapidly disintegrated in the placenta to release the antibody modified nano-drug capable of anchoring TB cell membrane surface marks. The nano-drug is prevented from being absorbed by other tissue cells of a parent body, is specifically anchored on a TB cell membrane in a placenta, is further specifically endocytosed by the TB cell, generates a function regulation and control effect, and ensures that TB cell diseases are treated exactly;
(4) 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 small molecule drug/therapeutic gene combined delivery system with an enzyme-sensitive shell 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-1The 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 potential particle size meter (Brooken Haven) at 25 ℃ at an incident laser wavelength λ of 532nm, an incident angle θ of 90 ° and a temperature of 532 ℃; the average of the three measurements was taken.
Example 1:
s1 synthesis of polyethylenimine grafted polyethylene glycol (PEG-PEI)
The method adopts a two-step method to synthesize polyethyleneimine grafted polyethylene glycol (PEG-PEI), firstly uses carbonyldiimidazole to activate the terminal hydroxyl of monomethyl ether polyglycol, and then reacts with the amino of polyethyleneimine to generate PEG-PEI. The specific operation is as follows: monomethyl ether glycol (8.0g, Mn ═ 2kDa) was weighed into a reaction flask, dried under vacuum at 80 ℃ for 6h, and dissolved by adding THF (60mL) under an argon atmosphere. Carbonyldiimidazole (CDI, 6.4g) was weighed into another reaction flask, and THF with mPEG-OH dissolved therein was slowly dropped into the CDI flask using an isopiestic dropping funnel, and the reaction was stirred at room temperature overnight. Distilled water (0.648mL) was added to inactivate excess CDI and stirring was continued for 30 min. Precipitating the solution into a large amount of cold ether, filtering, and drying in vacuum to obtain white powdery solid mPEG-CDI;
weighing PEI (4.4g, MW 1.8kDa) and adding into a two-mouth bottle (50mL), adding chloroform (20mL) to dissolve and add PEG-CDI (3.2g), stirring at room temperature for 24h, filling the solution into a dialysis bag (MWCO 3.5kDa), dialyzing with chloroform for 24h, concentrating the solution in the dialysis bag under reduced pressure, then precipitating in a large amount of cold ether, filtering and drying to obtain white powder packaged product mPEG-PEI;
s2 synthesis of poly (acetimide) grafted polyethylene glycol grafted polycaprolactone (PEG-PEI-PCL)
Firstly, synthesizing PCL-OH, adding 15g of dried dodecanol into a two-mouth bottle, vacuum-drying at 70 ℃ for 8h, adding 2ml of Sn (Oct)2Continuing to dry for 0.5h, then adding 400mL of dried epsilon-caprolactone, and stirring and reacting for 24h at 105 ℃; cooling, adding 100mL of ethanol to dissolve unreacted epsilon-caprolactone, filtering, dissolving the crude product in 250mL of tetrahydrofuran, precipitating in a large amount of anhydrous ether, filtering, and drying to obtain a white powdery product with the yield of 96%;
then PCL-CDI is synthesized, 10g of PCL-OH (Mn is 5000) is added into a two-mouth bottle, vacuum drying is carried out for 8h at the temperature of 50 ℃, 7.2g (10eq.) of Carbonyl Diimidazole (CDI) is added after the PCL-CDI is dissolved in 50mL of tetrahydrofuran, argon protection is carried out, room temperature reaction is carried out for 24h, precipitation is carried out in a large amount of anhydrous ether, filtration and vacuum drying at the room temperature are carried out, and a white powdery product is obtained, wherein the yield is 90%;
finally, reacting the PCL-CDI with PEG-PEI to prepare PEG-PEI-PCL, adding 1.6g of PEG-PEI into a 50mL two-mouth bottle, adding 30mL of trichloromethane to dissolve the PEG-PCL, slowly dropping 10mL of trichloromethane solution containing 200mg of PCL-CDI, stirring at room temperature to react for 24h, dialyzing in 1000mL of trichloromethane by using a dialysis bag (MWCO ═ 5kDa) for 24h, removing part of trichloromethane under reduced pressure, then precipitating in anhydrous ether, filtering and drying to obtain a white powder product, wherein the yield is 86%;
s3, preparation of polyethylene glycol-polyethyleneimine-polycaprolactone loaded SPIO nano-particles and drugs (PEG-PEI-PCL-SPIO/drug)
SPIO (superparamagnetic ferroferric oxide) according to the literature [ S.H.Sun, H.Zeng, D.B.Robinson, S.Raoux, P.M.Rice, S.X.Wang, G.X Li.Monodisperse MFe2O4(M ═ Fe, Co, Mn) nanoparticies.J.am.chem.Soc.2004, 126,273-279 ] iron acetylacetonate Fe (acac)31.4126g (4mmol), 5.16g (20mmol) of 1, 2-hexadecanediol, 3.8ml (12mmol) of oleic acid and 3.8ml (12mmol) of oleylamine are added into a 200ml three-necked bottle, then 40ml of dibenzyl ether is added under the protection of nitrogen gas to be stirred and dissolved, the mixture is heated to 200 ℃ in a sand bath and stirred under reflux for 2h, then heated to 300 ℃ and refluxed for 1h, and the reaction system slowly turns from dark red to black; naturally cooling in air, precipitating in 150ml ethanol, centrifuging at 10000rpm for 5min, discarding the supernatant, dissolving the lower precipitate in 70ml n-hexane containing 4 drops of oleic acid and oleylamine, centrifuging at 10000rpm for 10min to remove insoluble part, precipitating the solution in 200ml ethanol, centrifuging at 10000rpm for 10min, dissolving the lower precipitate in 60ml n-hexane, introducing argon gas for protection, and storing at 4 deg.C;
drying and weighing the normal hexane solution of the SPIO, collecting 5mg of SPIO nano particles in a serum bottle (10mL), weighing 50mg of PEG-PEI-PCL polymer and BML-2845 mg (CAS number: 853220-52-7), dissolving and uniformly mixing the PEG-PEI-PCL polymer and the BML-2845 mg (CAS number: 853220-52-7) by using dimethyl sulfoxide (3mL), dropwise adding the solution into 20mL of distilled water under ultrasonic dispersion, placing the reaction solution into a dialysis bag (MWCO is 3.5kDa), dialyzing for 24h to remove the dimethyl sulfoxide, centrifuging at the rotating speed of 12000r/mim, collecting precipitates, and discarding the supernatant. Dissolving the precipitate with water, ultrasonically dispersing, repeating centrifugal operation, ultrasonically dispersing the prepared PEG-PEI-PCL-SPIO/drug nanoparticles into water, filtering with a needle filter with the aperture of 220nm, adding purified water, adjusting the concentration of the PEG-PEI-PCL-SPIO/drug nanoparticles to 0.145mg/mL with constant volume, and storing the product at 4 ℃ for later use;
s4, preparation of antibody-targeted polyethylene glycol-polyethyleneimine-polycaprolactone-loaded SPIO nano-particle/drug (Fab-PEG-PEI-PCL-SPIO/drug)
The EGFR antibody is first cleaved by methods known in the literature to obtain the Fab fragment of EGFR, which is then purified. Then linking EGFR-Fab to mal-PEG-COOH, and reacting PEG connected with 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 EGFR antibody was weighed out at 0.5 mg. multidot.ml-1Papain, 10 mmol. multidot.L-1Cysteine, 2 mmol. multidot.L-1The enzyme is hydrolyzed for 4 hours under the condition of pH7.6. Separating the enzymolysis product by ProteinA affinity chromatography, further purifying the penetration peak by DEAE anion exchange chromatography, dialyzing, desalting and freeze-drying to obtain a Fab fragment of the EGFR with higher purity;
1mg of Fab fragment of EGFR (Mn. RTM.45 kDa) was weighed and pretreated with EDTA solution (500. mu.L 0.5M) for 15min at 4 ℃.5ml of PBS solution was added to dissolve the solution, 1mg of dithiothreitol was added thereto, and the reaction was carried out at 25 ℃ for 30 min. After removing dithiothreitol by centrifugation in a centrifugal ultrafiltration tube having a molecular weight cut-off of 1k, 5ml of a PBS solution was added to dissolve the dithiothreitol, and mal-PEG-COOH (2mg, Mn 4k) was added thereto and mixed well, followed by standing at 4 ℃ overnight. And then centrifuging by using a centrifugal ultrafiltration tube with the molecular weight cutoff of 5k to remove excessive mal-PEG-COOH. Activating carboxyl in Fab-PEG-COOH by using 500 mu g of EDC and NHS respectively for 15min, then adding 16mL of PEG-PEI-PCL-SPIO/drug prepared in the step 3, reacting overnight at 4 ℃, finally performing ultrafiltration and centrifugation to remove excessive small molecular impurities of EDC and NHS, performing centrifugation at 12000r/min to remove unconnected antibodies, collecting a solid solution, performing ultrasonic dispersion on the solid solution into distilled water, and performing constant volume adjustment on the concentration of Fab-PEG-PEI-PCL-SPIO/drug nanoparticles until the Fe content is 0.145mg/mL for later use;
s5 preparation of therapeutic gene composite nano particle
The PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle with positive electricity and the AHSP-siRNA with negative electricity can be compounded into a nano compound through electrostatic interaction. The specific operation is as follows: 400 μ g of AHSP-siRNA was diluted to a final volume of 1.5mL with PBS and shaken well. Taking 1.5mL of the PEG-PEI-SPIO prepared in the step (3) (or 1.6mL of the Fab-PEG-PEI-SPIO prepared in the step (4)) nanoparticles, ultrasonically dispersing uniformly, uniformly mixing an AHSP-siRNA diluted solution and a PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle solution, fixing the volume of the composite system to 0.061mg/mL, blowing, uniformly mixing and standing for 30 minutes to prepare a uniform composite;
s6, Synthesis of PEG-Polypeptides
0.05mmol of protein kinase C-sensitive polypeptide (Glu-Arg-Met-Arg-Pro-Arg-Lys-Arg-Gln-Gly-Ser-Val-Arg-Arg-Arg-Val, molecular weight: 2067.43Da), 5mmol of EDC and 5mmol of DMAP were dissolved in 10mL of an aqueous acetonitrile solution (acetonitrile: water ═ 1:1), and the mixture was magnetically stirred at 500rpm for 2h in an ice-water bath under protection of N2 to activate Peptide. After 2h 0.5mmol PEG-NHS (molecular weight 3000Da) was added and the reaction was continued for 72 h. After the reaction is finished, putting the reaction solution into a dialysis bag (MWCO is 3.5kDa), dialyzing for 72h, and freeze-drying to obtain a product PEG-polypeptide;
s7 preparation of PEG-polypeptide modified liposome shell @ therapeutic gene composite nanoparticle
PEG-polypeptide and cholesterol (20 mg each) were dissolved in 5mL of methylene chloride and the methylene chloride was spun dry using a vacuum rotary vacuum spinner to form a thin film of liposomes on the wall of the round bottom flask. 2mL of the therapeutic gene composite nanoparticle prepared in the step 5 is added dropwise into the liposome film formed by the PEG-polypeptide and cholesterol at the speed of 0.5mL/min under slow stirring. And (3) continuing stirring for 30min after the dropwise addition is finished, fully assembling the liposome and the therapeutic gene composite nanoparticles, and finally separating the liposome loaded with the therapeutic gene composite nanoparticles from the empty liposome by using strong magnets. And finally, adding 2mL of physiological saline (0.9% NaCl) solution to dissolve the PEG-polypeptide modified liposome shell @ therapeutic gene composite nano particles, wherein the aperture is 220nm, the filtration rate of a syringe filter is constant volume until the Fe content is 0.061mg/mL, and the solution is stored at 4 ℃ for later use.
The specific structural schematic diagram of the prepared small molecule drug/therapeutic gene combined delivery system of the enzyme sensitive shell is shown in figure 1.
Examples 2-4, comparative examples 1-6:
compared with example 1, examples 2-4 or comparative examples 1-6 can be prepared by changing the dosage of the polymer, the drug and the SPIO in step S3 or omitting one of steps S3, S4, S5, S6 and S7, and the following table 1 specifically shows:
table 1: examples and comparative examples
Function evaluation test
1. Magnetic Resonance Imaging (MRI) assay to evaluate the placental-specific delivery function of drugs
Establishing a model:
SPF grade C57BL/6 mice (purchased from guangdong provincial medical laboratory animal center) 8 weeks old, female and male mice 2: 1 mating in estrus coops, carrying out Papanicolaou staining on vaginal secretion smears of female mice on the next day, and marking the vaginal sperm-positive person of the sample as pregnancy when the diagnosis is observed under an optical microscope as pregnancy (D0). Sterile water-soluble 2mg/mL nitroso-L-arginine methyl ester is fed to pregnant mice to establish preeclampsia/pregnancy hypertension models, and the pregnant mice fed with equivalent double distilled water are normal control groups.
MRI imaging to detect placental distribution of drugs:
preeclampsia model animals were scanned on day 11, after chloral hydrate anesthesia, for the MRIT2 sequence at time points before (0h) and 2h (2h) after drug injection to observe the in vivo distribution of the nano-drugs containing SPIO. The dosage of the tail vein injection nano-drug is as follows: (therapeutic dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline);
c57BL/6j mouse uterus MRI imaging was performed using a Philips Intera 1.5T MRI scanner, with its animal specific coils. The evolution of signal intensity in the uterine and embryonic regions of C57BL/6j mice was observed on MRIbTFE sequences and the relaxation time changes of T2 with SPIO in the drug distributed in the uterus, placenta, embryo and other organs in vivo were measured using T2map imaging techniques, calculating the relaxation rates R2 at 0h and 2h, respectively. The relative increase rate of R2 (rsi (relative Signal intensity)%) at 2h after drug injection was calculated as R22h/R20h) The results are shown in Table 2.
Table 2 evaluation results of placenta-specific delivery function
| Group of | Placenta RSI (relative signal multiple) | Embryo RSI (relative signal multiple) | Liver RSI (relative multiple of signal) |
| Normal group of physiological saline | 1.00 | 1.00 | 1.00 |
| Example 1 | 23.8 | 1.21 | 3.35 |
| Example 2 | 22.5 | 3.25 | 2.05 |
| Example 3 | 20.5 | 1.43 | 3.02 |
| Example 4 | 24.8 | 1.02 | 3.82 |
| Comparative example 1 | 10.2 | 15.53 | 5.22 |
| Comparative example 2 | 7.21 | 10.35 | 7.36 |
| Comparative example 3 | 3.52 | 3.78 | 7.38 |
| Comparative example 4 | 3.23 | 3.08 | 9.52 |
| Comparative example 5 | 13.5 | 1.03 | 15.85 |
| Comparative example 6 | 9.23 | 0.99 | 24.02 |
From the above results, in comparative example 1, the placental trophoblast cell surface marker antibody is not linked, and after the polypeptide-PEG modified lipid bilayer is disintegrated, the drug in the content cannot be anchored in TB cells to obtain placental retention, a large amount of drug leaks through the placental barrier, and low placental RSI is detected; the drug is gathered in the embryo, which results in high embryo RSI; the failure of the drug to anchor to TB cells to achieve placental retention also results in partial drug detachment from the placenta and systemic distribution, resulting in higher liver RSI.
The delivery system of comparative example 2 does not contain a polypeptide-PEG modified lipid bilayer membrane as a shell, and cannot achieve targeted release for the placenta microenvironment; in addition, the EGFR antibody targets various other cells with cell membranes expressing EGFR in vivo including TB, and the cell membrane targeting is not strong; therefore, low placenta RSI and low liver RSI were detected; the drug without lipid membrane had a smaller particle size and entered the placenta, and passed the placental barrier in a larger proportion, and a higher RSI of the embryo was detected.
The delivery systems of comparative examples 3 and 4, which did not contain EGFR 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 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 a much poorer circulation, so its liver RSI is higher than that of comparative example 5; the placenta has a larger particle size and is less likely to leak through the maternal-fetal barrier, so placenta RSI is lower than comparative example 5.
In examples 1-4, a PKC substrate polypeptide-PEG modified lipid bilayer membrane was used as a shell, a drug carrier modified by a placental trophoblast surface marker antibody was used as a core, and a bilayer structure enzyme-sensitive shell small molecule drug/therapeutic gene combined delivery system was synthesized with a particle size range of 70-200 nm. The particle size of the liposome is about 100nm, and the outer negative electricity lipid bilayer membrane is convenient to avoid being phagocytized by a reticuloendothelial system in a large amount, so that the in vivo circulation time is prolonged, and the in vivo effective circulation is realized. The substrate polypeptide-PEG modified lipid bilayer membrane shell is stable in circulation of other tissues and organs in vivo, reaches a placenta microenvironment with specificity and high expression of PKC, disintegrates along with degradation of the polypeptide, and realizes drug specificity distribution in the placenta tissue. The drug shell disintegrates in the placental microenvironment, revealing the drug core containing the EGFR antibody fragment. The EGFR antibody fragment can be anchored in a TB cell of a cell membrane specificity high expression EGFR in a placenta, promote the drug to be specifically endocytosed 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 EGFR 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 preeclampsia animal model to evaluate treatment effect
Drugs (treatment dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline) are injected into D3, D6, D9, D12 and D15, and a series of tests are performed in D17, and the test results are shown in Table 3:
and (3) blood pressure detection: a BP-2000 blood pressure analysis system is applied, a tail sleeve method is adopted to measure the pregnant mouse Systolic pressure (SBP) noninvasively, the room temperature is kept at 26 ℃, a channel 1 is set to be 1V (1V is equivalent to 300mmHg), and a channel 2 is set to be SmV. The mouse is fixed in a mouse cage, the mouse tail is placed in a 17mm tail sleeve, and the center of the bottom of the mouse tail is contacted with the sensor. Continuously pressurizing and measuring the pregnant mouse for 10 times in a quiet state, and taking an average value and recording the average value at an interval of 1s every time;
and (3) urine protein determination: the pregnant mouse is placed in a sterile metabolism cage for 24h, urine is collected for 24h by using a sterile glass bottle, and the supernatant is obtained by centrifuging at 2500rpm for 15 min. Protein detection was performed using a fully automatic biochemical analyzer, and the absorbance value (a value) was measured. The calculation formula of the urine protein concentration is as follows:
urine protein concentration (mg/L) ═ sample assay tube a value/standard tube a value × standard concentration (mg/L);
remarks, standard substance concentration (1300 mg/L);
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 efficacy of treatment in preeclampsia animal models
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 blood pressure is high, the urine protein is high, the weight of the fetus is low, and the litter size is low; meanwhile, the drug is accumulated in the embryo, which causes embryo toxicity, lower weight of the fetus and lower litter size.
The delivery system of comparative example 2 does not contain a polypeptide-PEG modified lipid bilayer membrane as a shell, and targeted release aiming at the placenta microenvironment cannot be realized; the EGFR antibody targets in vivo expression EGFR cells including TB, the cell targeting is weak, the detected treatment effect is poor, the blood pressure is high, the urine protein 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 EGFR antibody, failed to target and anchor the drug into the placenta to TB cells, failed to acquire placental retention, resulted in a large number of leaks across the placental barrier, and detected poor therapeutic efficacy, higher blood pressure, higher urine protein, 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 pressure is high, the urine protein 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.
The comparative examples 5 and 6 have an excessively large particle size, resulting in poor in vivo circulation distribution effect, and the drug is mainly phagocytosed by the reticuloendothelial system of the liver in large amounts, resulting in insufficient distribution of the placenta drug, poor therapeutic effect, high blood pressure, high urine protein, low litter weight, and low litter size. Comparative example 6, which has a particle size much larger than comparative example 5, has a poorer circulation distribution, and thus has a poorer therapeutic effect than comparative example 5.
In examples 1-4, a PKC substrate polypeptide-PEG modified lipid bilayer membrane was used as a shell, a drug carrier modified by a placental trophoblast surface marker antibody was used as a core, and a bilayer structure enzyme-sensitive shell small molecule drug/therapeutic gene combined delivery system was synthesized with a particle size range of 70-200 nm. The particle size of the liposome is about 100nm, and the outer negative electricity lipid bilayer membrane is convenient to avoid being phagocytized by a reticuloendothelial system in a large amount, so that the in vivo circulation time is prolonged, and the in vivo effective circulation is realized. The substrate polypeptide-PEG modified lipid bilayer membrane shell is stable in circulation of other tissues and organs in vivo, reaches a placenta microenvironment with specificity and high expression of PKC, disintegrates along with degradation of the polypeptide, and realizes drug specificity distribution in the placenta tissue. The drug shell disintegrates in the placental microenvironment, revealing the drug core containing the EGFR antibody fragment. The EGFR antibody fragment can be anchored in a TB cell of a cell membrane specificity high expression EGFR in a placenta, the medicine is promoted to be specifically endocytosed by the TB cell to realize the regulation of the TB function, the distribution in other cells of the placenta is reduced, the influence on the function of the placenta is reduced, and a better treatment effect is realized through the effective regulation of the TB function. The EGFR 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 mice in the normal control group were bled from the tail vein, and liver function indices glutamic-pyruvic transaminase (ALT), total bilirubin (TBil), and kidney function indices Blood Urea Nitrogen (BUN) and serum creatinine (sCr) were measured. The detection instrument is a Hitachi 7600 type full-automatic biochemical analyzer, and the detection result is shown in Table 4.
TABLE 4 toxicity evaluation results
According to the results, the small molecule drug/therapeutic gene combined delivery system of the enzyme sensitive shell prepared by the invention has no obvious toxic or side effect on the mother and the fetus.
Claims (9)
1. An enzyme sensitive shell small molecule drug/therapeutic gene combined delivery system, which is characterized in that the delivery system is core-shell
The placenta trophoblast cell is of a shell double-layer structure, 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 interstitial fluid of placenta and highly expressed is used as a shell, a drug carrier modified by a marker antibody with high surface specificity expression of placenta trophoblasts is used as an inner core, and superparamagnetic ferroferric oxide SPIO nano particles, micromolecular drugs for regulating and controlling the function of the placenta trophoblasts, therapeutic genes or a combination of the micromolecular drugs and the therapeutic genes are loaded in the drug carrier;
the enzyme highly expressed in placenta interstitial fluid is one or more of protein kinase C, 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 with high surface specificity expression of the placenta trophoblast is a Fab segment of an Epidermal Growth Factor Receptor (EGFR) antibody.
2. The enzyme-sensitive shelled small molecule drug/therapeutic gene combination delivery system of claim 1, wherein the enzyme highly expressed in placental interstitial fluid is protein kinase C.
3. The enzyme-sensitive shell small molecule drug/therapeutic gene combined delivery system according to claim 1, wherein the copolymer is one or more of polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL, polyethylene glycol-polyethyleneimine-polylactic acid PEG-PEI-PLA or polyethylene glycol-polyethyleneimine-polylactic acid-glycolic acid PEG-PEI-PLGA, preferably polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL.
4. The enzyme-sensitive shell small molecule drug/therapeutic gene combination delivery system according to claim 1, wherein the average particle size of the enzyme-sensitive shell small molecule drug/therapeutic gene combination delivery system is 70nm to 300nm, preferably 90nm to 200 nm.
5. The enzyme-sensitive shelled small molecule drug/therapeutic gene combination delivery system of claim 1, wherein the small molecule drug is BML-284; the therapeutic gene is siRNA for inhibiting AHSP gene expression.
6. The method of preparing a small molecule drug/therapeutic gene combination delivery system of an enzyme sensitive sheath of any one of claims 1-5, comprising the steps of:
s1, loading superparamagnetic ferroferric oxide (SPIO) nanoparticles, micromolecular drugs for regulating and controlling the function of placenta trophoblasts and/or genes to the copolymer to obtain composite nanoparticles;
s2, linking the placenta trophoblast surface marker antibody to the composite nanoparticles to obtain antibody composite nanoparticles;
s3, linking enzyme substrate polypeptide for promoting placenta development and fetal nutrition with PEG to obtain polypeptide-PEG;
s4, mixing the polypeptide-PEG and the liposome to form a polypeptide-PEG modified lipid bilayer membrane;
s5, assembling the polypeptide-PEG modified lipid bilayer membrane and the antibody composite nanoparticle into a small molecule drug/therapeutic gene combined delivery system with an enzyme sensitive shell.
7. The preparation method of the small molecule drug/therapeutic gene combined delivery system of the enzyme-sensitive shell according to claim 6, wherein in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15: 1.
8. Use of the small molecule drug/therapeutic gene combination delivery system of an enzyme sensitive envelope according to any of claims 1-5 for the manufacture of a medicament for the modulation of placental trophoblast dysfunction.
9. The use of claim 8, wherein said disease that modulates placental trophoblast dysfunction is gestational hypertension.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111645495.XA CN114469891B (en) | 2021-12-29 | 2021-12-29 | Micromolecule drug/therapeutic gene combined delivery system with enzyme sensitive shell and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111645495.XA CN114469891B (en) | 2021-12-29 | 2021-12-29 | Micromolecule drug/therapeutic gene combined delivery system with enzyme sensitive shell and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114469891A true CN114469891A (en) | 2022-05-13 |
| CN114469891B CN114469891B (en) | 2023-01-24 |
Family
ID=81508825
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111645495.XA Active CN114469891B (en) | 2021-12-29 | 2021-12-29 | Micromolecule drug/therapeutic gene combined delivery system with enzyme sensitive shell and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114469891B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003022991A2 (en) * | 2001-09-07 | 2003-03-20 | Board Of Regents, The University Of Texas System | Compositions and methods of use of targeting peptides against placenta and adipose tissues |
| WO2011133617A1 (en) * | 2010-04-23 | 2011-10-27 | The Board Of Trustees Of The University Of Illinois | Nano-hybrid delivery system for sequential utilization of passive and active targeting |
| CN106177986A (en) * | 2016-08-16 | 2016-12-07 | 国家纳米科学中心 | A kind of lipopolymer medicine-carried nano particles and its preparation method and application |
| CN107715121A (en) * | 2017-09-19 | 2018-02-23 | 暨南大学 | A kind of magnetic resonance imaging nano-medicament carrier, nano medicament carrying system and preparation method thereof |
| CN109453187A (en) * | 2018-09-27 | 2019-03-12 | 天津医科大学 | Antibody nucleic acids drug conjugates and its preparation method and application with double enzyme sensitivity characteristic |
| CN110898066A (en) * | 2018-09-17 | 2020-03-24 | 中国科学院上海药物研究所 | Pharmaceutical composition, liposome containing pharmaceutical composition and application |
-
2021
- 2021-12-29 CN CN202111645495.XA patent/CN114469891B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003022991A2 (en) * | 2001-09-07 | 2003-03-20 | Board Of Regents, The University Of Texas System | Compositions and methods of use of targeting peptides against placenta and adipose tissues |
| WO2011133617A1 (en) * | 2010-04-23 | 2011-10-27 | The Board Of Trustees Of The University Of Illinois | Nano-hybrid delivery system for sequential utilization of passive and active targeting |
| CN106177986A (en) * | 2016-08-16 | 2016-12-07 | 国家纳米科学中心 | A kind of lipopolymer medicine-carried nano particles and its preparation method and application |
| CN107715121A (en) * | 2017-09-19 | 2018-02-23 | 暨南大学 | A kind of magnetic resonance imaging nano-medicament carrier, nano medicament carrying system and preparation method thereof |
| CN110898066A (en) * | 2018-09-17 | 2020-03-24 | 中国科学院上海药物研究所 | Pharmaceutical composition, liposome containing pharmaceutical composition and application |
| CN109453187A (en) * | 2018-09-27 | 2019-03-12 | 天津医科大学 | Antibody nucleic acids drug conjugates and its preparation method and application with double enzyme sensitivity characteristic |
Non-Patent Citations (1)
| Title |
|---|
| QUANYIN HUA 等: "Enzyme-Responsive Nanomaterials for Controlled Drug Delivery", 《NANOSCALE》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114469891B (en) | 2023-01-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103446588B (en) | Targeting type diagnosis and treatment coupling medicine and its preparation method and application | |
| CN101249266B (en) | Nano liver target direction amphipathic nature block copolymers drug administration system and preparation | |
| CN114224870B (en) | Placenta microenvironment targeted delivery probe and preparation method and application thereof | |
| CN114288278A (en) | Drug-loaded microalgae, preparation method and application thereof | |
| CN113876964B (en) | Tumor cell membrane drug-carrying system and construction method and application thereof | |
| CN106310297B (en) | Multifunctional macromolecule prodrug nanoscale medicine delivery system and preparation method and purposes | |
| CN114288249A (en) | Nano immune regulation medicine for regulating proportion of Treg/Th17 cells in focus microenvironment and preparation method and application thereof | |
| CN114469891B (en) | Micromolecule drug/therapeutic gene combined delivery system with enzyme sensitive shell and preparation method and application thereof | |
| CN114209652B (en) | Microenvironment NK cell immune regulation delivery system and preparation method and application thereof | |
| CN114366819B (en) | Placenta focus specific drug distribution delivery system and preparation method and application thereof | |
| CN114425044B (en) | Disease focus microenvironment immune cell function regulation and delivery system and preparation method and application thereof | |
| CN114404367B (en) | Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof | |
| CN114469890B (en) | Focus microenvironment sensitive delivery nuclear magnetic probe and preparation method and application thereof | |
| CN114366724B (en) | Microenvironment macrophage regulation and delivery system and preparation method and application thereof | |
| CN114272390B (en) | Microenvironment targeted combined cell targeted tumor inhibition carrier and preparation method and application thereof | |
| CN114377150B (en) | MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, and preparation method and application thereof | |
| CN114376974B (en) | Microenvironment enzyme targeting combined antibody Fab cell targeting vector and preparation method and application thereof | |
| CN114377158B (en) | Focus targeted distribution type nuclear magnetic resonance imaging functional probe and preparation method and application thereof | |
| CN114344477B (en) | Small molecule drug/therapeutic gene delivery system modified by cell surface marker antibody Fab segment, preparation method and application | |
| CN111249254B (en) | Preparation method and application of baicalin-entrapped folic acid coupled albumin nanoparticles | |
| CN101632834A (en) | Magnetic nano-carrier with targeted hydrophobic drug delivery to tumor and preparation method thereof | |
| CN114306241B (en) | Microenvironment T cell function regulation and control nano delivery system and preparation method and application thereof | |
| CN111821469A (en) | Homing targeting RSGRVSN peptide modified polyethylene glycol-polydopamine-Prussian blue composite nanoparticle and preparation method thereof | |
| CN112546236A (en) | PH-sensitive double-drug-framework polymer prodrug and preparation method and application thereof | |
| CN111840324B (en) | Au DENPs-macrophage complex applied to osteosarcoma cell imaging or treatment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |