CN114404367B - Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof - Google Patents

Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof Download PDF

Info

Publication number
CN114404367B
CN114404367B CN202111644114.6A CN202111644114A CN114404367B CN 114404367 B CN114404367 B CN 114404367B CN 202111644114 A CN202111644114 A CN 202111644114A CN 114404367 B CN114404367 B CN 114404367B
Authority
CN
China
Prior art keywords
placenta
carrier
nano
drug
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.)
Active
Application number
CN202111644114.6A
Other languages
Chinese (zh)
Other versions
CN114404367A (en
Inventor
郭宇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
First Affiliated Hospital of Sun Yat Sen University
Original Assignee
First Affiliated Hospital of Sun Yat Sen University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by First Affiliated Hospital of Sun Yat Sen University filed Critical First Affiliated Hospital of Sun Yat Sen University
Priority to CN202111644114.6A priority Critical patent/CN114404367B/en
Publication of CN114404367A publication Critical patent/CN114404367A/en
Application granted granted Critical
Publication of CN114404367B publication Critical patent/CN114404367B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • 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
    • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
    • 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
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • 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
    • A61K47/6845—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 cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • 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
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nanotechnology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Endocrinology (AREA)
  • Medical Informatics (AREA)
  • Biophysics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dispersion Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Reproductive Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention discloses a nano-carrier distributed to lesion cells in a targeted manner, a preparation method and application thereof, wherein the nano-carrier comprises a shell and a core, and the shell is an enzyme substrate polypeptide-PEG modified lipid bilayer membrane disintegrated in a targeted manner under the action of an enzyme highly expressed by contacting interstitial fluid of placenta tissues; the inner core is a drug carrier modified by a marker antibody with high placenta trophoblast surface specificity expression; the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester; the drug carrier is loaded with superparamagnetic ferroferric oxide SPIO nano particles, micromolecule drugs for regulating and controlling the function of placenta trophoblasts, therapeutic genes or a combination thereof. The nano-carrier can effectively avoid the nonspecific drug absorption of other organs outside the maternal placenta and fetuses, thereby realizing the delivery and function regulation of the specific drug of the trophoblasts in the placenta.

Description

Nano-carrier for targeted distribution of diseased cells and preparation method and application thereof
Technical Field
The invention relates to the field of chemical and biomedical engineering, in particular to a nano-carrier for targeted distribution of diseased cells and a preparation method and application thereof.
Background
Placental implantation is a group of diseases in which placental villi invade the myometrium to varying degrees, and is the leading cause of perinatal massive hemorrhage, urgent hysterectomy, and maternal mortality. The average blood loss of the implanted placenta operation is 3630 +/-2216 milliliters, the complications such as uterine rupture, neonatal death and infection, and ureter and bladder injury caused by difficult operation treatment are easy to combine, so that fistula and other complications are formed, the life health of pregnant and lying-in women and neonates is seriously threatened, and the death rate of the lying-in women can reach 7 to 10 percent. The implantation of the placenta is related to the common factors of multiple artificial abortion and uterine curettage history, scar uterine pregnancy after cesarean section, advanced age, preposed placenta, last placenta residue and the like, and the number of patients is gradually increased under the condition that the number of current two-fetus three-fetus and old-age puerperae is increased.
Although some degree of prediction can be performed on placenta implantation by imaging means such as MRI, there is no reliable means for realizing placenta implantation therapy for the pathogenesis in clinical practice. Even if diagnosed in advance, treatment cannot be achieved. In terms of pathological mechanism, placenta implantation is a pathological state which shows that part or all of decidua basalis layer is lost, and part or even the whole placenta villus tissue abnormally invades into the myometrium. This abnormally invasive state causes the placenta to become attached to the uterine wall. Therefore, the disease is initiated by invasion of placental trophoblasts and over-activation of EMT function, which in turn results in a disregulation of placental function. The regulation of the placenta function must be carried out according to the mechanism, and the clinical significant problem is expected to be improved.
Placental dysfunction is the source of the onset of a number of major diseases in pregnant women, including placental implantation. This disorder is mostly due to dysfunction of the most important cell in the placenta, the placental Trophoblast (TB). For example, excessive activation of the EMT and pro-angiogenic functions of TB can trigger placental implantation. Sulfasalazine has been shown in prior studies to be a compound that can achieve a definitive inhibition of the NF- κ B pathway in trophoblasts [ gastroenterology.2000 Nov;119 1209-18, further realizing the inhibition of the function of the trophoblast EMT by inhibiting the NF-kB pathway, further reducing the invasive ability of the trophoblast and improving the placenta function [ Placenta.2010 Nov;31 And (11): 997-1002 ]. CORO6 (Coronin 6) is a potent activator of the wnt pathway, closely related to cellular ubiquitination [ Front Cell Dev biol.2021 Jun 22;9 ] whose high expression can promote wnt pathway activity that is closely related to invasive metastasis [ Front Cell Dev biol.2021 May 7; 9. If the siRNA technology can be utilized to realize the definite inhibition of the CORO6 expression of the trophoblasts, the inhibition of the EMT and the invasion of the trophoblasts can be realized through the inhibition of the wnt pathway. Research shows that if the inhibition of NF-kB and wnt pathway can be realized simultaneously in EMT regulation, the EMT function of cells can be reliably reduced [ Food Chem toxicol.2019 Feb;124, 219-230 ]. Therefore, it is desired to achieve the above trophoblast function controlling drug, which is effectively delivered to trophoblasts in vivo.
However, there is currently no drug carrier means capable of exactly delivering drugs into TB in placenta, performing functional regulation on TB, and further realizing treatment of related diseases. 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 the fetus itself, as well as creating toxicity problems in both respects. Most of the drugs can pass through the placenta and distribute into the fetus, affecting the development of the fetus. Therefore, pregnant women including emergency drugs have a lot of contraindications. The drugs for pregnant women are classified into 5 types according to teratogenicity, and except for a few drugs with the minimum toxicity which are classified into a and b types, most of the other drugs with c, d and e types have obvious damage to fetuses. The 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 available for the diseases such as the placenta implantation. 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 TB function control drugs is the key to solving diseases caused by TB dysfunction.
The current macromolecule nano-carrier drug can realize the specific drug delivery to pathological target cells in various diseases. However, these vectors do not address the potential for toxic side effects on maternal and fetal non-specific distribution, and development of TB-specific delivery vectors is therefore a major challenge. 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. Therefore, researchers try to synthesize nano-drugs with the particle size of more than 300nm, so that the nano-drugs are retained in the placenta to generate the functional regulation and control on various cells including the placental TB. However, too large a particle size (> 100 nm) of the drug is detrimental to the in vivo distribution of the drug. Most of the nano-drugs with the particle size of more than 300nm are captured by a reticuloendothelial system in maternal circulation, generate side effects everywhere in the whole body, can reach the placenta and realize low proportion of specific distribution of TB. Therefore, other means are needed to achieve retention of the nano-drug in the placenta and targeting of TB cells.
The nano-drug can adopt a nano-drug linked antibody to target and identify the cell membrane marker of the target cell, thereby realizing the specific delivery of the target cell. TB has some established surface markers (e.g., placental Growth Factor, PLGF) 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 PLGF 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 PLGF 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 nano-carrier for targeted distribution of diseased cells, wherein the nano-carrier utilizes the placenta microenvironment to reduce the distribution of a drug in maternal organ tissues before entering the placenta and utilizes a trophoblast marker to reduce the distribution of the drug in fetal organ tissues after passing through the placenta, so that the absorption of non-specific drugs in other organs outside the maternal placenta and in the fetus can be effectively avoided, and the delivery and the function regulation of the specific drug of the nourishing cells in the placenta can be further realized.
The invention also aims to provide a preparation method of the nano-carrier for targeted distribution of diseased cells.
The invention is realized by the following technical scheme:
a nano-carrier distributed by targeting pathological cells, which comprises an outer shell and an inner core, wherein the outer shell is an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is disintegrated in a targeted manner under the action of an enzyme highly expressed by contacting placenta interstitial fluid; the enzyme highly expressed in placenta interstitial fluid is one or more of matrix metalloproteinase 9, lysozyme, kininase, histaminase or oxytocin;
the inner core is a drug carrier modified by a marker antibody with high placenta trophoblast surface specificity expression; 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 an Fab segment of a placenta growth factor (PLGF) antibody;
the drug carrier is loaded with superparamagnetic ferroferric oxide SPIO nano particles, micromolecule drugs for regulating and controlling the function of placenta trophoblasts, therapeutic genes or a combination thereof.
The placenta of the pregnant woman is rich in a plurality of enzymes for promoting the development of the placenta and nutrition of a fetus, the enzymes highly expressed in the placenta interstitial fluid are one or more of Matrix metalloproteinase 9 (Matrix metalloproteinase 9, MMP-9), lysozyme, kininase, histaminase, oxytocin or a family of Matrix metalloproteinase, wherein the expression level of the Matrix metalloproteinase 9 in the placenta interstitial fluid is very high, and the Matrix metalloproteinase 9 is hardly expressed in the blood and interstitial fluid of a normal human body, so that the Matrix metalloproteinase 9 is preferred.
The matrix metalloproteinase 9 substrate polypeptide can be selected from DNP-Pro-Cha-Gly-Cys (Me) -His-Ala-Lys (N-Me-Abz) -NH2, molecular weight: 1077.22Da.
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, micromolecular drugs for regulating and controlling the function of placenta trophoblasts, therapeutic genes or a combination thereof. The small molecular drug is sulfasalazine, and the treatment gene is siRNA for inhibiting CORO6 (Coronin 6) gene expression.
The average particle size of the nano-carrier 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 increase the preparation difficulty and to be beneficial to loading drugs and genes.
The invention also provides a preparation method of the nano-carrier for targeted distribution of diseased cells, 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 nano carrier which is distributed to the diseased cells in a targeted manner.
Preferably, in the step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15.
The MMP-9 substrate polypeptide-PEG modified lipid bilayer membrane is used as a shell, so that the distribution of a nano transmission system in enzyme-free blood is stable before entering a placental enzyme environment, 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 MMP-9 enzyme sensitive shell is disintegrated in a microenvironment containing enzymes at the side of the placenta matrix to release the medicine, so that the high-efficiency release and distribution of the medicine in the placenta matrix can be ensured; due to the introduction of the enzyme sensitive shell, the distribution efficiency of the placenta can be ensured without adopting a large-particle-size nano carrier structure, the particle size of the nano carrier is effectively reduced, the stable circulation distribution of the medicine before entering the placenta is ensured, and the reticuloendothelial system is ensured not to 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 medicine carrier modified by the placenta trophoblast surface marker antibody as the inner core, the medicine is modified by the TB cell surface marker antibody, and the TB cell membrane in the placenta can be anchored exactly after being released, thereby ensuring the specific administration of the TB cell in a complex placenta environment, and simultaneously avoiding unnecessary placenta function damage caused by the administration of other cells in the placenta; most of the medicines entering the placenta are targeted by the antibody and are exactly anchored in TB cells, so that the medicines are ensured to leak through a placenta barrier and enter the side of the fetus, and the safety of the fetus is ensured; after the medicine is anchored on the TB cell membrane, the therapeutic medicine and the therapeutic gene are promoted to be swallowed into the TB cell, so that the function regulation is realized, and the exact TB function regulation is ensured.
The invention also provides application of the double-targeting nano-drug delivery system in preparation of a drug for regulating and controlling the placenta trophoblastic dysfunction disease, wherein the disease for regulating and controlling the placenta trophoblastic dysfunction is placenta implantation.
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 the nano-carrier with a double-layer structure. The double-layer structure can ensure that the liposome shell has stable structure and keeps stable circulation in the blood circulation of the pregnant woman, so that the liposome is not easily captured by other tissues and cells including a reticuloendothelial system, the distribution and release of other tissues except a placenta, which are influenced by the body of the pregnant woman, and the toxic and side effects are reduced;
(2) After the transmission system enters the placenta along with blood circulation, an enzyme substrate in the outer shell of the transmission system is decomposed by corresponding enzyme highly expressed in placenta tissues, and the protective lipid bimolecular outer shell is rapidly disintegrated in the placenta to release the antibody modified nano-drug capable of anchoring TB cell membrane surface marks. The nano-drug is prevented from being absorbed by other tissue cells of a parent body, is specifically anchored on a TB cell membrane in a placenta and is further specifically endocytosed by the TB cell to generate a function regulation and control effect, so that exact treatment on TB cell diseases is ensured;
(3) Through an exact antigen-antibody reaction, the medicament is retained in the placenta rich in TB cells after the lipid bimolecular shell is disintegrated, so that the medicament leakage is reduced and the toxic and side effects on fetuses are reduced; and 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 nano-carrier prepared in example 1 of the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the scope of the invention.
The raw materials of the invention are as follows:
Figure BDA0003443619640000051
Figure BDA0003443619640000061
c57BL/6J-Marveld1 knockout mice (strain No.: KOCMP-277010-Marveld 1-B6J-VA) Seiko (Suzhou) Biotechnology Co., ltd.
The Fe content determination method comprises the following steps:
the Fe content in the nano-drug system is measured by an atomic absorption spectrophotometer method and is used for measuring the dosage of the nano-drug. Weighing a certain amount of prepared drug solution (such as 1mL in step III), lyophilizing, and dissolving to 1mol L -1 The HCl solution is placed for 24 hours to ensure that Fe in the SPIO is fully ionized, an atomic absorption spectrophotometer is used for detecting the absorbance of Fe atoms at 248.3nm, the absorbance is substituted into a standard curve made by using a Fe standard solution to calculate the concentration of Fe, and then the content of Fe in the medicine solution before freeze-drying is calculated in a reverse mode.
The particle size test method comprises the following steps:
the particle size of the samples was measured with a Zeta-Plus potential particle sizer (Brooken Haven) with an incident laser wavelength λ =532nm, an incident angle θ =90 ° and a temperature of 25 ℃; the average of the three measurements was taken.
Example 1:
s1, synthesis of polyethyleneimine grafted polyethylene glycol (PEG-PEI)
The method adopts a two-step method to synthesize polyethyleneimine grafted polyethylene glycol (PEG-PEI), firstly uses carbonyldiimidazole to activate the terminal hydroxyl of monomethyl ether polyglycol, and then reacts with the amino of polyethyleneimine to generate PEG-PEI. The specific operation is as follows: monomethyl ether glycol (8.0 g, mn = 2kDa) was weighed into a reaction flask, dried under vacuum at 80 ℃ for 6 hours, and dissolved by adding THF (60 mL) under an argon atmosphere. Carbonyldiimidazole (CDI, 6.4 g) was weighed into another reaction flask, and THF with mPEG-OH dissolved therein was slowly dropped into the CDI flask using an isopiestic dropping funnel, and the reaction was stirred at room temperature overnight. Distilled water (0.648 mL) was added to inactivate the excess CDI and stirring was continued for 30min. Precipitating the solution into a large amount of cold diethyl ether, filtering, and vacuum drying to obtain white powdery solid mPEG-CDI;
weighing PEI (4.4 g, MW = 1.8kDa) and adding the PEI into a two-mouth bottle (50 mL), adding trichloromethane (20 mL) to dissolve the PEI and adding PEG-CDI (3.2 g), stirring at room temperature for reaction for 24h, filling the solution into a dialysis bag (MWCO =3.5 kDa), dialyzing the solution in the trichloromethane for 24h, concentrating the solution in the dialysis bag under reduced pressure, precipitating the solution in a large amount of cold ether, filtering and drying to obtain white powder packaged product mPEG-PEI;
s2, synthesis of poly (acetimide) grafted polyethylene glycol grafted polycaprolactone (PEG-PEI-PCL)
Firstly, synthesizing PCL-OH, adding 15g of dried dodecanol into a two-mouth bottle, drying at 70 ℃ for 8 hours in vacuum, adding 2ml of Sn (Oct) 2 Continuously drying for 0.5h, then adding 400mL of dried epsilon-caprolactone, and stirring and reacting for 24h at 105 ℃; after cooling, adding 100mL of ethanol to dissolve unreacted epsilon-caprolactone, filtering, dissolving the crude product in 250mL of tetrahydrofuran, precipitating in a large amount of anhydrous ether, filtering, and drying to obtain a white powdery product with the yield of 96%;
then PCL-CDI is synthesized, 10g of PCL-OH (Mn = 5000) is added into a two-mouth bottle, vacuum drying is carried out for 8h at 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 room temperature are carried out, and a white powdery product is obtained, wherein the yield is 90%;
finally, reacting the PCL-CDI with PEG-PEI to prepare PEG-PEI-PCL, adding 1.6g of PEG-PEI into a 50mL two-mouth bottle, adding 30mL of trichloromethane to dissolve the PEG-PCL, then slowly dripping 10mL of trichloromethane solution containing 200mg of PCL-CDI, stirring and reacting for 24h at room temperature, dialyzing for 24h in 1000mL of trichloromethane by using a dialysis bag (MWCO =5 kDa), decompressing to remove part of trichloromethane, then precipitating in anhydrous ether, filtering and drying to obtain a white powder product with the yield of 86%;
s3, preparation of polyethylene glycol-polyethyleneimine-polycaprolactone loaded SPIO nano particles and drugs (PEG-PEI-PCL-SPIO/drug)
SPIO (superparamagnetic ferroferric oxide) according to the literature [ S.H.Sun, H.Zeng, D.B.Robinson, S.Raoux, P.M.Rice, S.X.Wang, G.X Li.Monodisperse MFe 2 O 4 (M = Fe, co, mn) nanoparticles.J.am.chem.Soc.2004,126, 273-279.) iron acetylacetonate Fe (acac) 3 1.4126g (4 mmol), 5.16g (20 mmol) of 1, 2-hexadecanediol, 3.8ml (12 mmol) of oleic acid and 3.8ml (12 mmol) of oleylamine are added into a 200ml three-necked bottle, then 40ml of dibenzyl ether is added under the protection of nitrogen and stirred for dissolution, the mixture is heated to 200 ℃ in a sand bath and stirred under reflux for 2h, 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 the normal hexane solution of SPIO, collecting 5mg of SPIO nano particles in a serum bottle (10 mL), weighing 50mg of PEG-PEI-PCL polymer and 5mg of sulfasalazine, dissolving and mixing the PEG-PEI-PCL polymer and the sulfasalazine uniformly by using dimethyl sulfoxide (3 mL), dropwise adding the solution into 20mL of distilled water under ultrasonic dispersion, placing the reaction solution in a dialysis bag (MWCO =3.5 kDa) for dialysis for 24h to remove the dimethyl sulfoxide, centrifuging at the rotating speed of 12000r/mim, collecting precipitates, and discarding 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 PLGF antibody is first cleaved by the methods in the prior literature to obtain the Fab segment of PLGF, which is then purified. Then, linking the PLGF-Fab to mal-PEG-COOH, and reacting the PEG connected with the antibody and amino on the PEG-PEI-PCL-SPIO nano particles by amidation reaction to prepare Fab-PEG-PEI-PCL-SPIO;
the specific operation is as follows: 10mg of PLGF antibody was weighed out at 0.5 mg. Multidot.ml -1 Papain, 10 mmol. L -1 Cysteine, 2 mmol. 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 Fab fragment of PLGF;
1mg of the Fab fragment of PLGF (Mn =45 kDa) was weighed out and pretreated with EDTA solution (500. Mu.L 0.5M) for 15min at 4 ℃.5ml of PBS solution was added to dissolve the solution, and 1mg of dithiothreitol was added thereto to react at 25 ℃ for 30min. After removing dithiothreitol by centrifugation with a centrifugal ultrafiltration tube with a molecular weight cutoff of 1k, 5ml of PBS solution was added for dissolution, mal-PEG-COOH (2mg, mn = 4k) was added and mixed uniformly, and the mixture was left overnight at 4 ℃. And then centrifuging by using a centrifugal ultrafiltration tube with the molecular weight cutoff of 5k to remove excessive mal-PEG-COOH. Activating carboxyl in Fab-PEG-COOH by 500 micrograms of EDC and NHS respectively for 15min, then adding the PEG-PEI-PCL-SPIO/drug 1695l prepared in the step 3, reacting overnight at 4 ℃, finally removing excessive small molecular impurities of EDC and NHS by ultrafiltration and centrifugation, removing unconnected antibodies by centrifugation at 12000r/min, collecting solid solution, ultrasonically dispersing the solid solution into distilled water, and regulating the concentration of Fab-PEG-PEI-PCL-SPIO/drug nanoparticles to a constant volume until the Fe content is 0.145mg/mL for later use;
s5, preparation of therapeutic gene composite nano particle
The PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle with positive charge and CORO6-siRNA with negative charge can be compounded to prepare the nano compound through electrostatic interaction. The specific operation is as follows: mu.g of CORO6-siRNA was diluted with PBS to a final volume of 1.5mL and shaken well. Taking 1.5mL of the PEG-PEI-SPIO prepared in the step (3) (or 1.6mL of the Fab-PEG-PEI-SPIO prepared in the step (4)) nanoparticles, ultrasonically dispersing the nanoparticles uniformly, mixing a CORO6-siRNA diluted solution and a PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle solution uniformly, fixing the volume of the composite system to 0.061mg/mL, blowing, uniformly mixing and standing for 30 minutes to prepare a uniform composite;
s6, PEG-polypeptide synthesis
0.05mmol of matrix metalloproteinase 9 sensitive polypeptide (DNP-Pro-Cha-Gly-Cys (Me) -His-Ala-Lys (N-Me-Abz) -NH2, molecular weight: 1077.22 Da), 5mmol of EDC and 5mmol of DMAP were dissolved in 10mL of acetonitrile in water (acetonitrile: water = 1), and the mixture was magnetically stirred at 500rpm for 2h on an ice-water bath under N2 protection to activate the Peptide. After 2h 0.5mmol PEG-NHS (molecular weight 3000 Da) was added and the reaction was continued for 72h. After the reaction is finished, putting the reaction solution into a dialysis bag (MWCO =3.5 kDa), dialyzing for 72h, and freeze-drying to obtain a product PEG-polypeptide;
preparation of S7 and PEG-polypeptide modified liposome shell @ therapeutic gene composite nanoparticle
PEG-polypeptide and cholesterol (20 mg each) were dissolved in 5mL of methylene chloride and the methylene chloride was spun dry using a vacuum rotary vacuum spinner to form a thin film of liposomes on the wall of the round bottom flask. 2mL of the therapeutic gene composite nanoparticle prepared in 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% NaCl) solution was added to dissolve the PEG-polypeptide modified liposome shell @ therapeutic gene composite nanoparticle, the filtration rate of a syringe filter with a pore size of 220nm was adjusted to a constant volume of 0.061mg/mL, and the mixture was stored at 4 ℃ for further use.
The specific structural schematic diagram of the prepared nano-carrier is shown in fig. 1.
Examples 2-4, comparative examples 1-6:
compared with example 1, examples 2-4 or comparative examples 1-6 can be prepared by changing the 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
Figure BDA0003443619640000091
Function evaluation test
1. Magnetic Resonance Imaging (MRI) assay to evaluate the placental-specific delivery function of drugs
Marveld1 (Marvel Domain containment 1) is a gene having an EMT inhibitory function. Earlier studies have demonstrated that Marveld1 knockout mice develop uterine bleeding and dystocia. It is found by dissection that this is because the placental trophoblasts of this type of animal invade into the uterine wall, causing the placenta to adhere tightly to the uterine wall, which in turn causes dystocia. Even if a part of the fetus is delivered, the placenta can not be automatically stripped, and the placenta draws the uterus, thereby causing the symptoms of uterus deformation, uterus hemorrhage and birth canal blockage. The Marveld1 knockout mouse can be used for pregnancy, simulating the pathophysiological process of placenta implantation, and detecting the distribution and function regulation and control effects of the carrier.
Establishing a model:
8-week-old C57BL/6J-Marveld1 gene knockout mice and control group SPF-grade C57BL/6 mice (purchased from Guangdong provincial center for medical laboratory animals), female mice 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). The pregnant C57BL/6J-Marveld1 gene knockout mice are placenta implantation models, and the pregnant C57BL/6 mice are normal control groups.
MRI imaging to detect placental distribution of drugs:
the placenta implantation model animal was subjected to chloral hydrate anesthesia on day 11, and then scanned with the MRIT2 sequence 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);
c57BL/6j mouse uterus MRI imaging was performed using a Philips Intera 1.5T MRI scanner, and its animal specific coils. The evolution of signal intensity in the uterus and embryonic region in mice was observed on MRIbTFE sequence, and T2map imaging technique was used to measure the change in T2 relaxation time with SPIO in drug distributed in the uterus, placenta, embryo and other organs in vivo, and the respective relaxation rates R2 at 0h and 2h were calculated. The Relative increase ratio of R2 at 2h after drug injection (RSI (Relative Signal Intensity)% = R2) was calculated 2h /R2 0h ) The results are shown in Table 2.
Table 2 evaluation results of placenta-specific delivery function
Figure BDA0003443619640000101
Figure BDA0003443619640000111
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 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 PLGF antibody targets other cells with PLGF expressed on various cell membranes in vivo including TB, and the cell membrane targeting is not strong; therefore, lower placental RSI and lower liver RSI were detected; the drug without lipid membrane had a smaller particle size and entered the placenta, and passed the placental barrier in a larger proportion, and a higher RSI of the embryo was detected.
The delivery systems of comparative examples 3 and 4, which did not contain PLGF antibody, failed to target and anchor the drug into the placenta to TB cells, failed to obtain placental retention, leaked a significant amount of the placental 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 MMP-9 substrate polypeptide-PEG was used as a shell, and a drug carrier modified by placental trophoblast surface marker antibody was used as a core to synthesize a bilayer structure nanocarrier, with a particle size ranging from 80 to 210nm. The particle size of the liposome is about 100nm, and the outer negative electricity lipid bilayer membrane is convenient to avoid being phagocytized by a reticuloendothelial system in a large amount, so that the in vivo circulation time is prolonged, and the in vivo effective circulation is realized. The substrate polypeptide-PEG modified lipid bilayer membrane shell is stable in circulation of other tissues and organs in vivo, reaches a placenta microenvironment with high specificity expression MMP-9, disintegrates along with degradation of the polypeptide, and realizes drug specificity distribution in the placenta tissue. The outer shell of the drug is disintegrated in the placenta microenvironment, and the inner core of the drug containing the PLGF antibody fragments is revealed. The PLGF antibody fragment can be anchored in a TB cell with cell membrane specificity and high expression PLGF in a placenta, promote the medicine to realize TB function regulation after being specifically endocytosed by the TB cell, reduce the distribution in other cells of the placenta and reduce the influence on the function of the placenta. The PLGF antibody enables the drug in the placenta to be anchored in TB cells, thereby effectively reducing the drug from leaking through the maternal-fetal barrier and reducing the drug from reaching the embryo.
2. Establishment of animal model with placenta implantation for evaluating therapeutic effect
Drugs (therapeutic dose 0.31mg/Kg iron equivalent drug or physiological saline with equal volume) are injected into D3, D6, D9, D12 and D15, and serial detection is carried out during production, and the detection results are shown in Table 3:
placenta and litter examination: after production, the number of normal born baby is recorded, the abdominal cavity of the pregnant mouse is opened, the uterus is dissected, the residual baby and the placenta are sequentially taken out, and the number of the baby difficult to be born is 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.
Judgment of dystocia caused by placenta implantation: when the mice are delivered, the placenta is tightly adhered to the uterine wall and cannot be peeled from the implantation site in the process of delivery, and the adhered placenta pulls the uterus towards the cervix, so that the cervix is blocked, the uterine bleeding is increased finally, and the mice with dead mothers and fetuses are marked as dystocia. The difficult-to-produce rate = (number of difficult-to-produce cases/total number of cases) 100% for each group of pregnant mice.
TABLE 3 evaluation of treatment efficacy in animal model of placental implantation
Group of Hard yield (%) Number born per fetus
Normal control group 2.52 8.59
Example 1 11.27 8.32
Example 2 11.86 5.94
Example 3 11.53 8.17
Example 4 12.28 8.11
Comparative example 1 62.75 3.98
Comparative example 2 64.67 3.83
Comparative example 3 64.61 3.52
Comparative example 4 65.17 3.21
Comparative example 5 65.94 3.21
Comparative example 6 66.35 3.06
From the above results, it can be seen that in comparative example 1, the placenta trophoblast surface marker antibody is not linked, and after the polypeptide-PEG modified lipid bilayer is disintegrated, the drug in the content cannot be anchored in TB cells to obtain placenta retention, and a large amount of drug leaks through the placenta barrier, so that the treatment effect is poor, the dystocia rate is high, and the number born is low; at the same time, the drug accumulates in the embryo, resulting in embryotoxicity and also in 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 of the placenta microenvironment cannot be achieved; the PLGF antibody targets in vivo expression PLGF cells including TB, and has weak cell targeting, poor detection treatment effect, high dystocia rate and low litter size. Meanwhile, the medicine without lipid membrane has smaller particle size, enters the placenta and passes through the placenta barrier in a larger proportion, so that the embryo toxicity is caused, and the litter size is also lower.
The delivery systems of comparative examples 3 and 4, which did not contain PLGF antibody, failed to target and anchor the drug into the placenta to TB cells, failed to obtain placental retention, resulted in a large number of leaks across the placental barrier, and detected poor therapeutic effect, high dystocia rate, and low litter size. Meanwhile, the lipid bilayer membrane outer shell of the comparative example 3 is not modified by enzyme-sensitive polypeptide, so that the distribution of the lipid bilayer membrane outer shell in placenta is reduced, and the detection shows that the treatment effect is poor, the dystocia rate is high, and the litter size is low. Comparative example 4, without the lipid bilayer membrane shell, was less effective than comparative example 3.
The comparative examples 5 and 6 have excessively large particle sizes, resulting in extremely poor in vivo circulation distribution effect, and the drugs are mainly phagocytosed by the reticuloendothelial system of the liver in large amounts, resulting in insufficient distribution of the placenta drugs, poor treatment effect, high dystocia rate, and low litter size. Comparative example 6, which has a particle size much larger than comparative example 5, has a poorer circulation distribution, and thus has a poorer therapeutic effect than comparative example 5.
In examples 1 to 4, a lipid bilayer membrane modified by MMP-9 substrate polypeptide-PEG was used as a shell, and a drug carrier modified by placental trophoblast surface marker antibody was used as a core to synthesize a bilayer structure nanocarrier, with a particle size ranging from 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 substrate polypeptide-PEG modified lipid bilayer membrane shell is stable in circulation of other tissues and organs in vivo, reaches a placenta microenvironment with high specificity expression MMP-9, disintegrates along with degradation of the polypeptide, and realizes drug specificity distribution in placenta tissues. The drug shell is disintegrated in the placenta microenvironment, and the drug inner core containing the PLGF antibody fragment is exposed. The PLGF antibody fragment can be anchored in a TB cell with cell membrane specificity and high expression of PLGF in a placenta, promotes the medicine 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 function of the placenta, and realizes better treatment effect through effective regulation of the TB function. The PLGF antibody makes the medicine in the placenta anchored to TB cell, and this can reduce the leakage of medicine to the mother and fetus barrier and reduce the medicine reaching the fetus with less toxicity to fetus.
3. Drug for toxicity evaluation of animal models
At 72 hours after the injection of the drug, the mice in the normal control group were bled from the tail vein, and liver function index glutamic pyruvic transaminase (ALT), total bilirubin (TBil) and kidney function index Blood Urea Nitrogen (BUN) and serum creatinine (sCr) were measured. The detection instrument is a Hitachi 7600 type full-automatic biochemical analyzer, and the detection result is shown in Table 4.
TABLE 4 toxicity evaluation results
Figure BDA0003443619640000141
According to the results, the nano-carrier prepared by the invention has no obvious toxic or side effect on the mother and the fetus.

Claims (5)

1. A nano-carrier for targeted distribution of diseased cells, wherein the nano-carrier comprises an outer shell and an inner core,
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 placenta interstitial fluid is matrix metalloproteinase 9; the enzyme substrate polypeptide is DNP-Pro-Cha-Gly-Cys (Me) -His-Ala-Lys (N-Me-Abz) -NH2;
the inner core is a drug carrier modified by a marker antibody with high placenta trophoblast surface specificity expression;
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 placenta growth factor (PLGF) antibody;
superparamagnetic ferroferric oxide SPIO nano particles, micromolecule medicines for regulating and controlling the function of placenta trophoblasts and therapeutic genes are loaded in the medicine carrier; the small molecular drug is sulfasalazine, and the therapeutic gene is siRNA for inhibiting CORO6 gene expression;
the average grain diameter of the nano-carrier is 80nm-300nm.
2. The nanocarrier for targeted distribution of diseased cells according to claim 1, wherein the average particle size of the nanocarrier is in the range of 100nm to 210nm.
3. The method for preparing nanocarriers to diseased cells via targeted distribution to diseased cells according to any of claims 1-2, comprising the steps of:
s1, loading superparamagnetic ferroferric oxide (SPIO) nano particles, micromolecular medicines for regulating and controlling placenta trophoblast functions and therapeutic genes to a copolymer to obtain composite nano particles;
s2, linking the placenta trophoblast surface marker antibody to the composite nanoparticles to obtain antibody composite nanoparticles;
s3, linking enzyme substrate polypeptide for promoting placenta development and fetal nutrition with PEG to obtain polypeptide-PEG;
s4, mixing the polypeptide-PEG and the liposome to form a polypeptide-PEG modified lipid bilayer membrane;
and S5, assembling the polypeptide-PEG modified lipid bilayer membrane and the antibody composite nanoparticles into a nano carrier which is distributed to the diseased cells in a targeted manner.
4. The preparation method of the nanocarrier for targeted distribution of diseased cells according to claim 3, wherein in step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15.
5. Use of the nanocarrier of any of claims 1-2 for modulating a dysfunction of placental trophoblasts by targeting distribution of diseased cells in the preparation of a medicament for modulating a dysfunction of placental trophoblasts as a placental implant.
CN202111644114.6A 2021-12-29 2021-12-29 Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof Active CN114404367B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111644114.6A CN114404367B (en) 2021-12-29 2021-12-29 Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111644114.6A CN114404367B (en) 2021-12-29 2021-12-29 Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN114404367A CN114404367A (en) 2022-04-29
CN114404367B true CN114404367B (en) 2023-02-21

Family

ID=81269780

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111644114.6A Active CN114404367B (en) 2021-12-29 2021-12-29 Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN114404367B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070087001A1 (en) * 2005-10-19 2007-04-19 Center For Molecular Medicine And Immunology Inhibition of placenta growth factor (PLGF) mediated metastasis and/or angiogenesis
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
CN106633084A (en) * 2016-10-14 2017-05-10 中国药科大学 All-solid-phase synthesis method for high-polymer materials with tumor site enzyme sensitive characteristics for constructing vesicae

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070087001A1 (en) * 2005-10-19 2007-04-19 Center For Molecular Medicine And Immunology Inhibition of placenta growth factor (PLGF) mediated metastasis and/or angiogenesis
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
CN106633084A (en) * 2016-10-14 2017-05-10 中国药科大学 All-solid-phase synthesis method for high-polymer materials with tumor site enzyme sensitive characteristics for constructing vesicae

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
基质金属蛋白酶及其组织抑制剂与滋养细胞侵袭性生长关系的研究;武卫平等;《诊断病理学杂志》;20060630;第13卷(第3期);第205-208页 *

Also Published As

Publication number Publication date
CN114404367A (en) 2022-04-29

Similar Documents

Publication Publication Date Title
CN114585396B (en) Injectable hydrogel with anti-inflammatory and repair promoting functions, preparation method thereof and application thereof in heart repair
CN114224870B (en) Placenta microenvironment targeted delivery probe and preparation method and application thereof
CN114288249A (en) Nano immune regulation medicine for regulating proportion of Treg/Th17 cells in focus microenvironment and preparation method and application thereof
CN113876964B (en) Tumor cell membrane drug-carrying system and construction method and application thereof
CN114288278A (en) Drug-loaded microalgae, preparation method and application thereof
CN114404367B (en) Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof
CN108524951A (en) A kind of Sorafenib nanometer formulation with liver tumour targeting
CN106310297B (en) Multifunctional macromolecule prodrug nanoscale medicine delivery system and preparation method and purposes
CN114469890B (en) Focus microenvironment sensitive delivery nuclear magnetic probe and preparation method and application thereof
CN114376974B (en) Microenvironment enzyme targeting combined antibody Fab cell targeting vector and preparation method and application thereof
CN114425044B (en) Disease focus microenvironment immune cell function 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
CN114377158B (en) Focus targeted distribution type nuclear magnetic resonance imaging functional probe 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
CN114377150B (en) MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, and preparation method and application thereof
CN114366724B (en) Microenvironment macrophage regulation and delivery system and preparation method and application thereof
CN114306241B (en) Microenvironment T cell function regulation and control nano delivery system 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
CN109954144A (en) Twin-stage pH response nano grain and preparation method thereof based on the poly- beta-amino ester material of modification
Sahni et al. Nanotechnologies for the detection and treatment of endometriosis
CN111249254A (en) Preparation method and application of baicalin-entrapped folic acid coupled albumin nanoparticles
CN111840324B (en) Au DENPs-macrophage complex applied to osteosarcoma cell imaging or treatment
CN112402382B (en) Preparation method and application of co-assembled nano-drug for cooperatively regulating and controlling renin angiotensin system by double ligand targeting

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