CN114306241B - Microenvironment T cell function regulation and control nano delivery system and preparation method and application thereof - Google Patents
Microenvironment T cell function regulation and control nano delivery system and preparation method and application thereof Download PDFInfo
- Publication number
- CN114306241B CN114306241B CN202111635313.0A CN202111635313A CN114306241B CN 114306241 B CN114306241 B CN 114306241B CN 202111635313 A CN202111635313 A CN 202111635313A CN 114306241 B CN114306241 B CN 114306241B
- Authority
- CN
- China
- Prior art keywords
- placenta
- cell
- microenvironment
- nano
- delivery system
- 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
Links
Images
Abstract
The invention discloses a microenvironment T cell function regulation and control nano delivery system, 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 in contact with interstitial fluid high expression of placenta tissues is taken as a shell, a medicine carrier modified by an important immunoregulation cell helper T cell (Th) surface specificity high expression marker antibody in the placenta is taken as an inner core, and superparamagnetic ferroferric oxide SPIO nano particles, small molecular medicines for regulating the function of the helper T cell in the placenta, therapeutic genes or a combination thereof are loaded in the medicine carrier. The nanometer delivery system for microenvironment T cell function regulation and control can effectively avoid nonspecific drug absorption of other organs outside the maternal placenta and fetuses, and further realize specific drug delivery and function regulation and control of auxiliary T cells in the placenta.
Description
Technical Field
The invention relates to the field of chemical and biomedical engineering, in particular to a microenvironment T cell function regulation and control nano delivery system and a preparation method and application thereof.
Background
The threatened abortion is a common disease in pregnancy, and refers to that irregular bleeding appears in the vagina before 28 weeks of gestation of a lying-in woman, but abnormal pregnancy substances are not discharged, the cervix is not opened in obstetrics and gynecology department examination, and the rupture phenomenon of the fetal membrane is not found. If the condition is not controlled effectively, the fetal abortion can be caused finally, and the maternal health is even affected. The number of the diseases in the pregnancy is 15% counted. However, with the advent of the two-and-three-pregnancy policy, the proportion of the advanced pregnant women increases, the incidence and severity of the threatened abortion are on an obvious trend of rising year by year, the health of the pregnant women and the fetuses is seriously threatened, and even the birth rate of the population in China is affected [ Chinese women and children health care, 2019,34 (16): 3748-3751 ]. In those who develop threatened abortion, if effective treatment to improve placental function is available to avoid eventual abortion, the fetus still has a greater probability of survival. However, there is a lack of a definite and effective treatment means, and most patients can expect to self-heal to save the fetus only by means of rest and nutrition improvement. In order to achieve treatment of threatened abortion, the pathogenesis of threatened abortion needs to be clarified. The causes of threatened abortion found at present are very complex and include endocrine dyscrasia, chromosomal abnormality, maternal systemic infection, adverse environment, uterine defect, trauma, emotional injury, etc. However, the specific mechanism of how these causes affect the function of the placenta and ultimately cause placental damage has not been clarified, and therefore, no specific effective therapeutic means has been developed [ Chinese medicine report, 2019,16 (05): 30-33 ].
It is well known that pregnancy is accompanied by a complex maternal/fetal immune regulation and immune tolerance mechanism similar to that of inter-allogeneic transplantation, in addition to growth and development factors of previous interest. In successful pregnancies, maternal-fetal tolerance must be maintained at the maternal-fetal interface consisting of decidua and placental trophoblastic tissues until the fetus delivers [ Front immunol.2019 Oct 18; therefore, research means for reproductive immunology have been gradually emphasized in recent years, and experts in the field began to research immunological changes in placenta, decidua, and fetal tissues during abortion or threatened abortion. Threatened abortion or final abortion caused by a plurality of the above causes are found, and most of threatened abortion or final abortion are accompanied by impaired immune tolerance and activated immune attack of a placental [ Aust J Gen practice.2020 Jun;49 (6): 324-330. However, there is currently no definitive means to correct these newly discovered important immunopathological impairments and changes.
The most important cell population that regulates immune tolerance/immune attack in vivo (including the placental interna-fetal interface) is helper T cells (Th) among T lymphocytes [ Best practice Res Clin Obstet gynaecol.2017 Jul;42 ] of the formula. Helper T cells account for 65% of peripheral blood T cells, and their function is to broadly assist the response of immune cells including cytotoxic T cells, B cells, macrophages, DC cells, etc., and the most specific cell surface specific marker is CD4 (cluster of differentiation 4), and further CD3, TCR, CD28, CD2, CTLA-2, CD45, CD40L, LFA-1, etc. Th cells mainly comprise two subtypes of Th1 cells and Th2 cells [ Ann Allergy assay Immunol.2000 Jul;85 (1) 9-18; [ MEANS FOR solving PROBLEMS ] is provided.
Wherein, th1 cell participates in cellular immunity and delayed type hypersensitivity inflammatory reaction, is a proinflammatory subgroup, secretes cytokines such as IL-2, IFN-gamma, TNF-alpha and the like, can promote inflammation in placenta and resist bacterial infection. T-beta (T-Box transformation Factor 21) is the major Transcription Factor that promotes its differentiation [ cell.2000 Mar 17;100 (6): 655-69 ]. When Th1 is over activated in placenta and exceeds normal physiological level, it is very unfavorable to pregnancy and may cause pathological pregnancy such as abortion. Another subtype of Th, which is primarily associated with humoral immunity, is Th2 cells, which primarily function to suppress inflammation and have immunosuppressive properties. Such cells can be effected by expressing cytokines such as IL-4, IL-5, IL-6, IL-10, and the like. IL-4 and IL-2 are the major cytokines that contribute to their activation [ Annu Rev immunol.2006; 24. ] 607-56. It exerts its anti-inflammatory action in the placenta, inducing and maintaining immune tolerance to the fetus. Studies have demonstrated significant Th2 cell reduction and function suppression, th1 cell up-scaling and function activation in the placenta and blood of threatened abortion patients [ Int Arch Allergy immunol.2004 Jun;134 (2): 93-119 ].
Therefore, abortion (especially early threatened abortion) occurs due to activation of proinflammatory cells and release of proinflammatory factors in large quantities due to immune imbalance, and further, insufficient release of Th1 cells and Th2 proinflammatory factors, which inhibit inflammation, is induced. The normal secretion and release of these factors depends on the interaction between Th1 cell and Th2 cell to regulate the balance of maternal-fetal interface immune tolerance and thus maintain the normal pregnancy. Once the balance between them is broken, it leads to immunological damage to the placenta and fetus, ultimately resulting in poor pregnancy outcome such as miscarriage [ Am J Reprod immunol.1997 Jun;37 (6): 427-34 ].
Studies have shown that Th1/Th2 balance shifts to a Th2 predominance in pregnant women of normal pregnancy. Th1 dominance is the main cause of Th cells in the placenta after abortion including threatened abortion. In such cases, th2 cells and their associated cytokines favor pregnancy outcome, while Th1 cells and their associated cytokines favor pregnancy [ apmis.2011 Sep;119 (9): 597-604 ]. In CD4+ Th cells of patients with threatened abortion, key regulatory factors IL-4 and T-beta of Th2 and Th1 cells are respectively expressed and up-regulated, so that the cell proportion of Th2 is obviously reduced, the proportion of Th1 cells is obviously increased, and the proportion of Th2/Th1 is obviously down-regulated [ PLoS one.2014 Dec 19 ]; 9 (12): e115554.
In vivo and in vitro experimental studies of other immune-related diseases, many small molecules or gene therapy drugs that can regulate helper T cell (Th) function have been developed. As previously described, activation of Th1 cells is dependent on activation of the T-beta associated pathway. If the expression of transcription factor T-beta can be inhibited, the proportion of Th1 cells can be reduced [ J Immunol.2017 May 15;198 3939-3948. We can use siRNA technology for the exact suppression of CD4+ Th cell transcription factor T-beta expression, expected to achieve Th1 suppression in placental immunity. Armillarisin A is an immune function regulating drug, has anti-inflammatory effect in specific organs, and can exactly activate IL-4 pathway and up-regulate bioactivity of IL-4 [ Cell Biochem Biophys.2015 May;72 (1): 103-6 ]. IL-4 is a key inducing cytokine for Th2 cells. Armillarisin intervention is carried out on CD4+ Th cells, so that Th2 activation can be expected to be realized in placental immunity, immune tolerance is promoted, and immune imbalance in a threatened abortion state is reversed. More preferably, earlier studies have confirmed that the combined application of armillarisin and T-beta-siRNA to CD4+ Th cells can produce a synergistic effect, significantly increase the Th2/Th1 ratio, and produce immunomodulatory effects of inhibiting inflammation and promoting immune tolerance. Therefore, if armillarisin and T-beta-siRNA can be selectively delivered into CD4+ Th cells in the placenta under the premise of avoiding damaging other CD4+ cells outside the placenta, the inflammation of the placenta can be accurately and effectively inhibited, so that the placenta and fetus which are possibly aborted due to immune injury can be saved.
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. Helper T cells (Th) have some established surface markers (e.g., CD 4) that distinguish them from other placental stromal cells in placental tissue, and are distinguishable from other cells in the placenta. However, analysis of the expression quantity of the systemic multi-organ tissues shows that the surface marker is also obviously expressed in all CD4+ lymphocytes and partial somatic cells at other parts outside the placenta. If the antibody of CD4 is connected on the surface of the nano-drug carrier, the direct in vivo application will cause side effects on other CD4+ Th cells outside the placenta in vivo and other cells expressing the marker CD4. Therefore, only before entering the placenta in blood circulation, the auxiliary T cell (Th) recognition antibody of the nano-carrier is shielded, so that the distribution of the auxiliary T cell (Th) recognition antibody in the placenta can be avoided, and the distribution of the auxiliary T cell (Th) in the placenta is ensured.
The applicant has intensively developed the target identification of the circulating T cell marker in the early period, and further research on the delivery of the T cell nano-drug is carried out. It was recognized in the research that drug delivery to T cells in vivo including blood circulation and placenta is easier to achieve using antibody-modified nanocarriers [ ACS nano.2012 Dec 21;6 (12): 10646-57. However, it is known that CD4+ Th cells are distributed throughout the body along with blood, exert different immunoregulatory functions at different sites in the body, and cause systemic immune dysfunction when systemically killed. If the CD4+ Th cell function is directly and indiscriminately regulated in the body of the megastream pregnant woman, the placenta function of the pregnant woman can be regulated and controlled, and meanwhile, the whole body immunosuppression or the whole body autoimmune disease is generated. Therefore, how to selectively deliver the CD4+ Th cell function regulating and controlling medicine into the CD4+ Th cells in the placenta and the subgroup T helper cells (Th) thereof to carry out exact function regulation and control on the cells is a key scientific problem in the field, and ensures that the regulating and controlling means is only limited to the placenta and does not influence the CD4+ Th lymphocytes outside the placenta.
Development of T helper cell (Th) function modulating drugs presents pathophysiological disorders in both maternal and fetal aspects. 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 current drugs which may be effective on helper T cells (Th) in vitro experiments cannot regulate helper T cell (Th) functions under the condition of ensuring the safety of the mother and fetus, and may cause dysfunction of systemic immune cells and serious immune complications.
After small molecule drugs or gene therapy drugs which are possibly screened by in vitro experimental study and have the function regulation and control effect of helper T cells (Th) enter the circulation of a pregnant woman through injection, the drugs can take effect in CD4+ Th cells and other CD4+ cells of the whole body of the pregnant woman outside a placenta to generate side effects; meanwhile, after the medicine enters the placenta, the medicine rapidly penetrates 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 placenta-related diseases such as threatened abortion, placenta implantation and the like clinically. Doctors can only carry out passive symptomatic treatment aiming at the symptoms caused by the placenta dysfunction diseases. But can not realize the real placenta function recovery through the function regulation of immune cells such as helper T cells and the like. Therefore, how to avoid toxicity to the mother and fetus and realize effective delivery of the placenta-selective helper T cell function regulating and controlling medicine is the key to solve the threatened abortion caused by the functional disorder of the helper T cells in the placenta.
The current macromolecule nano-carrier drug can realize the specific drug delivery to pathological target cells in various diseases. The development of placenta-specific helper T cell-specific delivery vectors presents significant difficulties, as these vectors do not address the problem of potentially toxic side effects on maternal and fetal nonspecific distribution. The approaches that researchers try to promote the delivery of helper T cell specific nano-drugs at present include 2, one is to enlarge the particle size of the nano-drugs, so that the nano-drugs cannot pass through a fetal membrane barrier and are retained in placenta to generate the drug delivery effect; the other is specific delivery of antibody modified nano-carriers aiming at helper T 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 have tried to synthesize nano-drugs with a particle size of > 300nm, which are retained in the placenta, and generate functional regulation on various cells including helper T cells in the placenta. 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 throughout the whole body, can reach the placenta and realize the specific distribution ratio of only auxiliary T cells in the placenta. Therefore, there is a need to achieve retention of the nanomedicine in the placenta and selective targeting of helper T cells within the placenta in other ways.
In summary, there is a lack of nanocarrier systems that can effectively avoid the nonspecific drug absorption of the mother and fetus, and further achieve the specific drug delivery and function control of the helper T cells in the placenta.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a microenvironment T cell function regulation and control nano delivery system, which utilizes the placenta microenvironment targeting to reduce the distribution of systemic immune cells and organ tissues of a parent before a drug enters the placenta, utilizes the targeting of an auxiliary T cell membrane marker in the placenta to reduce the distribution of the drug in a fetal immune system and organ tissues after the drug passes through the placenta, can effectively avoid the absorption of nonspecific drugs of other organs outside the maternal placenta and the fetus, and further realizes the delivery and function regulation and control of the specific drug of the auxiliary T cell in the placenta.
The invention also aims to provide a preparation method of the microenvironment T cell function regulating nano delivery system.
The invention is realized by the following technical scheme:
a microenvironment T cell function regulation and control nano delivery system is of a core-shell double-layer structure, takes an enzyme substrate polypeptide-PEG modified lipid bimolecular membrane which is targeted to disintegrate under the action of an enzyme which is contacted with high expression of placental interstitial fluid as an outer shell, takes a marker antibody modified drug carrier which is specifically high expressed on the surface of an auxiliary T cell in a placenta as an inner core, and loads superparamagnetic ferroferric oxide SPIO nano particles, micromolecular drugs which regulate and control the function of the auxiliary T cell in the placenta, therapeutic genes or a combination thereof; the enzyme highly expressed in placenta interstitial fluid is one or more of matrix metalloproteinase 3, matrix metalloproteinase 9, lysozyme, kininase, histaminase, oxytocin or protein kinase C; 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 auxiliary T cells in the placenta is an Fab section of a CD4 antibody.
The placenta of the pregnant woman is rich in a plurality of placenta and enzymes highly expressed by tissue microenvironment thereof, the enzymes highly expressed by placenta interstitial fluid are one or more of matrix metalloproteinase 3, matrix metalloproteinase 9, lysozyme, kininase, histaminase, oxytocin, protein kinase C or other matrix metalloproteinase, wherein the matrix metalloproteinase 3 has extremely high expression level in the placenta interstitial fluid, and is hardly expressed in the blood and the interstitial fluid of a normal human body, so the matrix metalloproteinase 3 is preferably selected.
The substrate polypeptide of matrix metalloproteinase 3 can be selected from Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys (Dnp) -NH2, molecular weight: 1675.84Da.
The placental helper T cell (Th) cell membrane marker is CD4, CD3, TCR, CD28, CD2, CTLA-2, CD45, CD40L, LFA-1, etc., preferably CD4. The invention adopts Fab fragment of CD4 antibody to realize the target of Th cell.
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 auxiliary T cells in placenta, therapeutic genes or the combination thereof. The small molecular drug is armillarisin, and the therapeutic gene is siRNA for inhibiting the expression of T-beta (T-Cell-Specific T-Box transformation Factor) gene.
In the research, the synergistic effect on the immunoregulation function can be generated by activating IL-4 and the channel activity thereof and inhibiting the expression of T-beta in the helper T cells through the regulation and control effect of key transcription factors, so that the proportional change and the immunoregulation function of the helper T cells are more effectively promoted, the recovery of the placental immune state of the threatened abortion lesion is promoted, the placental inflammation is reduced, and the prognosis is improved.
The average particle size of the microenvironment T cell function-regulated nano delivery system is 80nm-300nm, preferably 100nm-210nm, the particle size is too large to be beneficial to in vivo circulation, and the particle size is too small to be difficult to prepare and to load drugs and genes.
The invention also provides a preparation method of the microenvironment T cell function regulating and controlling nano delivery system, which comprises the following steps:
s1, loading superparamagnetic ferroferric oxide (SPIO) nanoparticles, micromolecular drugs for regulating and controlling functions of auxiliary T cells in placenta and/or genes to a copolymer to obtain composite nanoparticles;
s2, linking the auxiliary T cell surface marker antibody in the placenta to the composite nano particle;
s3, linking the substrate polypeptide of the enzyme highly expressed by the microenvironment of the placenta and tissues thereof 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 microenvironment T cell function-controlled nano delivery system.
Preferably, in the step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15.
The matrix metalloproteinase 3 enzyme substrate polypeptide-PEG modified lipid bilayer membrane is used as the shell, so that the distribution of a nano transmission system in enzyme-free blood is stable before the nano transmission system enters a placental enzyme environment, the drug leakage is reduced, and other cells outside the placenta are reduced or avoided from phagocytosis. Thereby ensuring the safety of the maternal extraplacental immune system and other tissues and organs; the enzyme sensitive shell is disintegrated in a microenvironment containing enzyme at the placenta matrix side to release the medicine, so that the high-efficiency release and distribution of the medicine in the matrix placenta can be ensured; due to the introduction of the enzyme sensitive shell, the distribution efficiency of the placenta can be ensured without adopting a large-particle-size nano-carrier structure, the particle size of the nano-carrier is effectively reduced, the stable circulation distribution of the medicine before entering the placenta is ensured, and the reticuloendothelial system is ensured not to phagocytize a large amount of carriers to cause the reduction of curative effect and the increase of side effect.
The invention adopts the medicine carrier modified by the auxiliary T cell surface marker antibody in the placenta as the inner core, the medicine is modified by the auxiliary T cell surface marker antibody or the antibody fragment, and the auxiliary T cell membrane in the placenta can be anchored exactly after being released, thereby ensuring the specific administration of the auxiliary T cell in the complicated placenta environment, and simultaneously avoiding unnecessary placenta function damage caused by the administration of other cells in the placenta;
most of the drugs entering the placenta are targeted by the antibody and are exactly anchored in the auxiliary T cells, so that the drugs are ensured to leak through the placenta barrier and enter the side of the fetus, and the safety of the fetus is ensured; after the drug is anchored on the helper T cell membrane, the therapeutic drug and the therapeutic gene are promoted to swallow helper T cells, the regulation and control of the key pathway activity and the transcription factor activity are realized, and the definite helper T cell proportion and the immune function regulation are ensured.
The invention also provides application of the microenvironment T cell function regulation and control nano delivery system in preparation of a medicament for regulating and controlling the placental internal helper T cell dysfunction disease, wherein the placental internal helper T cell dysfunction disease is threatened abortion.
Compared with the prior art, the invention has the following beneficial effects:
(1) In the research, the synergistic effect of the regulation and control function of IL-4 related immunoregulation pathway activity of armillarisin is generated by inhibiting the expression of T-beta in the helper T cell and regulating and controlling the key transcription factor, so that the proportion change and the immunocompetence of the helper T cell are more effectively promoted, the recovery of the placenta immune state of lesion caused by threatened abortion is promoted, the symptom is relieved, and the 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; taking a drug carrier modified by a marker antibody with high specificity and expression on the surface of a helper T cell in the placenta as an inner core; synthesizing a microenvironment T cell function-regulated nano delivery system with a double-layer structure. The double-layer structure can ensure that the liposome shell structure of the nano-drug 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 CD4+ Th cells outside the placenta) including a reticuloendothelial system, the function of the Th cells outside the placenta is not easily influenced, the distribution and release of other tissues outside the placenta, which are influenced by the body of the pregnant woman, are reduced, and 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 auxiliary T cell membrane surface marks. The nano-drug is prevented from being absorbed by other tissue cells of a parent body, is specifically anchored on an auxiliary T cell membrane in a placenta and is further specifically endocytosed by the auxiliary T cell to generate a function regulation and control effect, so that the definite treatment of the auxiliary T cell dysfunction disease is ensured;
(4) Through exact 'antigen-antibody reaction', the medicament is retained in the placenta rich in helper T cells after the lipid bimolecular shell is disintegrated, so that the medicament leakage is reduced and the toxic and side effects on the fetus are reduced; and also avoids affecting trophoblasts, vascular endothelial cells, other immune cells and other stromal cells in the placenta.
Drawings
Fig. 1 is a schematic structural diagram of a microenvironment T cell function-controlled nano delivery system 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:
monomethyl ether polyethylene glycol (mPEG, mn =2000 Da) Sigma;
branched polyethyleneimine (hy-PEI Mw =25000 Da) BASF;
n, N-Carbonyldiimidazole (CDI) AR Sigma;
epsilon-caprolactone Sigma-Aldrich;
armillarisin A (CAS No.: 53696-74-5) MedChemexpress;
mal-PEG-COOH Beijing Kazheng medicine;
recombinant Anti-CD4 antibody (ab 288725) abcam;
matrix metalloproteinase 3 substrate polypeptide (cat 1956-2-26) from Shanghai Qiaozhizao Biotech, inc.;
siRNA of T-bet (ID 108405) Sammerley Feishell technology (China) Co., ltd.;
hydrocortisone (CAS No.: 50-23-7) MedChemexpress;
mifepristone (CAS No.: 84371-65-3) MedChemexpress.
The Fe content determination method comprises the following steps:
and measuring the Fe content in the nano-drug system by using an atomic absorption spectrophotometer method, wherein the Fe content is used for measuring the dosage of the nano-drug. Weighing a certain amount of prepared drug solution (such as 1mL of the solution in step three), lyophilizing, and dissolving to 1mol L -1 The HCl solution is placed for 24 hours to ensure that Fe in the SPIO is fully ionized, an atomic absorption spectrophotometer is used for detecting the absorbance of Fe atoms at 248.3nm, the absorbance is substituted into a standard curve made by using a Fe standard solution to calculate the concentration of Fe, and then the content of Fe in the medicine solution before freeze-drying is calculated in a reverse mode.
The particle size test method comprises the following steps:
the particle size of the sample was measured with a Zeta-Plus potentiometric particle sizer (Brooken Haven) with an incident laser wavelength λ =532nm, an incident angle θ =90 °, temperature 25 ℃; the average of the three measurements was taken.
Example 1:
s1, synthesis of polyethyleneimine grafted polyethylene glycol (PEG-PEI)
The method comprises the steps of synthesizing polyethyleneimine grafted polyethylene glycol (PEG-PEI) by a two-step method, activating the terminal hydroxyl of monomethyl ether polyglycol by carbonyl diimidazole, and reacting with the amino of polyethyleneimine to generate PEG-PEI. The specific operation is as follows: monomethyl ether ethylene glycol (8.0 g, mn = 2kda) was weighed into a reaction flask, dried under vacuum at 80 ℃ for 6h, and dissolved by adding THF (60 mL) under argon atmosphere. Carbonyldiimidazole (CDI, 6.4 g) was weighed into another reaction flask, and THF with mPEG-OH dissolved therein was slowly dropped into the CDI flask using an isopiestic dropping funnel, and the reaction was stirred at room temperature overnight. Distilled water (0.648 mL) was added to inactivate excess CDI and stirring was continued for 30min. Precipitating the solution into a large amount of cold ether, filtering, and drying in vacuum to obtain white powdery solid mPEG-CDI;
weighing PEI (4.4 g, MW = 1.8kDa) and adding the PEI into a two-necked bottle (50 mL), adding trichloromethane (20 mL) to dissolve the PEI, adding PEG-CDI (3.2 g), stirring at room temperature for 24h, filling the solution into a dialysis bag (MWCO =3.5 kDa), dialyzing the solution with the trichloromethane for 24h, concentrating the solution in the dialysis bag under reduced pressure, precipitating the solution in a large amount of cold ether, filtering and drying to obtain white powder packed product mPEG-PEI;
s2, synthesis of poly (acetimide) grafted polyethylene glycol grafted polycaprolactone (PEG-PEI-PCL)
Firstly, synthesizing PCL-OH, adding 15g of dried dodecanol into a two-mouth bottle, vacuum-drying at 70 ℃ for 8h, adding 2ml of Sn (Oct) 2 Continuing to dry for 0.5h, then adding 400mL of dried epsilon-caprolactone, and stirring and reacting for 24h at 105 ℃; cooling, adding 100mL of ethanol to dissolve unreacted epsilon-caprolactone, filtering, dissolving a crude product in 250mL of tetrahydrofuran, precipitating in a large amount of anhydrous ether, filtering, and drying to obtain a white powdery product with the yield of 96%;
then PCL-CDI is synthesized, 10g of PCL-OH (Mn = 5000) is added into a two-mouth bottle, vacuum drying is carried out for 8h at 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, slowly dropping 10mL of trichloromethane solution containing 200mg of PCL-CDI, stirring at room temperature to react for 24h, dialyzing in 1000mL of trichloromethane by using a dialysis bag (MWCO =5 kDa) for 24h, removing part of trichloromethane by reducing pressure, precipitating in anhydrous ether, filtering and drying to obtain a white powder product, wherein the yield is 86%;
s3, preparation of polyethylene glycol-polyethyleneimine-polycaprolactone loaded SPIO nano particles and medicine (PEG-PEI-PCL-SPIO/drug)
SPIO (superparamagnetic ferroferric oxide) according to the literature [ S.H.Sun, H.Zeng, D.B.Robinson, S.Raoux, P.M.Rice, S.X.Wang, G.X Li.Monodisperse MFe 2 O 4 (M = Fe, co, mn) nanoparticles.J.am.chem.Soc.2004,126,273-279 ] iron acetylacetonate Fe (acac) 3 1.4126g (4 mmol), 5.16g (20 mmol) of 1, 2-hexadecanediol, 3.8ml (12 mmol) of oleic acid and 3.8ml (12 mmol) of oleylamine are added into a 200ml three-necked bottle, then 40ml of dibenzyl ether is added under the protection of nitrogen and stirred for dissolution, the mixture is heated to 200 ℃ in a sand bath and stirred under reflux for 2h, then heated to 300 ℃ and refluxed for 1h, and the reaction system slowly changes from dark red to black; naturally cooling in air, precipitating in 150ml ethanol, centrifuging at 10000rpm for 5min, discarding the supernatant, dissolving the lower precipitate in 70ml n-hexane respectively added with 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 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 (10 mL), weighing 50mg of PEG-PEI-PCL polymer and 5mg of armillarisin A (CAS number: 53696-74-5), dissolving and mixing the PEG-PEI-PCL polymer and the armillarisin A uniformly with dimethyl sulfoxide (3 mL), dropwise adding the solution into 20mL of distilled water under ultrasonic dispersion, placing the reaction solution into a dialysis bag (MWCO =3.5 kDa) for dialysis for 24h to remove the dimethyl sulfoxide, centrifuging at the rotating speed of 12000r/mim, collecting precipitates, and discarding 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 CD4 antibody is first cleaved by methods known in the literature to obtain the Fab fragment of CD4, which is then purified. Then linking the CD4-Fab to mal-PEG-COOH, and reacting the PEG connected with the antibody with the 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 CD4 antibody was weighed and subjected to enzymatic hydrolysis for 4 hours under conditions of 0.5 mg. Ml-1 of papain, 10 mmol. L-1 of cysteine, 2 mmol. L-1 of EDTA, pH 7.6. 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 CD4 with high purity;
1mg of Fab fragment of CD4 (Mn =45 kDa) was weighed and pretreated with EDTA solution (500. Mu.L 0.5M) for 15min at 4 ℃.5ml of PBS solution was added to dissolve the solution, and 1mg of dithiothreitol was added thereto to react at 25 ℃ for 30min. After removing dithiothreitol by centrifugation with a centrifugal ultrafiltration tube with a molecular weight cutoff of 1k, 5ml of PBS solution was added for dissolution, mal-PEG-COOH (2mg, mn = 4k) was added and mixed uniformly, and the mixture was left overnight at 4 ℃. And then centrifuging by using a centrifugal ultrafiltration tube with the molecular weight cutoff of 5k to remove excessive mal-PEG-COOH. Activating carboxyl in Fab-PEG-COOH by using 500 mu g of EDC and NHS respectively for 15min, then adding the PEG-PEI-PCL-SPIO/drug 1698 prepared in the step 3, reacting overnight at 4 ℃, finally performing ultrafiltration and centrifugation to remove excessive small molecular impurities of EDC and NHS, performing centrifugation at 12000r/min to remove unconnected antibodies, collecting a solid solution, performing ultrasonic dispersion on the solid solution into distilled water, and performing constant volume adjustment on the concentration of Fab-PEG-PEI-PCL-SPIO/drug nanoparticles until the Fe content is 0.145mg/mL for later use;
s5, preparation of therapeutic gene composite nano particle
The PEG-PEI-SPIO (or Fab-PEG-PEI-SPIO) nanoparticle with positive electricity and the T-beta-siRNA with negative electricity can be compounded to prepare the nano compound through electrostatic interaction. The specific operation is as follows: mu.g of T-beta-siRNA was diluted with PBS to a final volume of 1.5mL and shaken well. Taking 1.5mL of the PEG-PEI-SPIO (or 1.6mL of the net weight of the Fab-PEG-PEI-SPIO prepared in the step (4) nano particles prepared in the step (3)) to be dispersed uniformly by ultrasonic, uniformly mixing the T-bet-siRNA diluted solution and the PEG-PEI-SPIO (or the Fab-PEG-PEI-SPIO) nano particle 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;
synthesis of S6, PEG-polypeptide
0.05mmol of matrix metalloproteinase 3-sensitive polypeptide (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys (Dnp) -NH2, molecular weight: 1675.84 Da), 5mmol of EDC and 5mmol of DMAP are dissolved in 10mL of acetonitrile in water (acetonitrile: water = 1), protected by N2 on an ice-water bath, and magnetically stirred at 500rpm for 2h to activate Peptide. After 2h 0.5mmol PEG-NHS (molecular weight 3000 Da) was added and the reaction was continued for 72h. After the reaction is finished, putting the reaction solution into a dialysis bag (MWCO =3.5 kDa), dialyzing for 72h, and freeze-drying to obtain a product PEG-polypeptide;
preparation of S7 and PEG-polypeptide modified liposome shell @ therapeutic gene composite nanoparticle
PEG-polypeptide and cholesterol (20 mg each) were dissolved in 5mL of methylene chloride and the methylene chloride was spun dry using a vacuum rotary vacuum spinner to form a thin film of liposomes on the wall of the round bottom flask. 2mL of the therapeutic gene composite nanoparticle prepared in step 5 was added dropwise to the liposome film formed of the above PEG-polypeptide and cholesterol at a rate of 0.5mL/min with slow stirring. And (3) continuing stirring for 30min after the dropwise addition is finished, fully assembling the liposome and the therapeutic gene composite nanoparticles, and finally separating the liposome loaded with the therapeutic gene composite nanoparticles from the empty liposome by using strong magnets. Finally, 2mL of physiological saline (0.9% 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 microenvironment T cell function-regulated nano delivery system is shown in figure 1.
Examples 2-4, comparative examples 1-6:
compared with example 1, examples 2-4 or comparative examples 1-6 can be prepared by changing the amounts of polymer, drug and SPIO fed in step S3 or omitting one of steps S3, S4, S5, S6, S7, as shown in table 1 below:
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 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). Feeding pregnant mice with sterile water-soluble hydrocortisone 25mg/Kg and mifepristone 4mg/Kg by intragastric feeding every day to establish a threatened abortion model; pregnant mice receiving equal amount of sterile water and gavage were used as normal control group.
MRI imaging to detect placental distribution of drugs:
on day 11, after chloral hydrate anesthesia, the animal model for threatened abortion was scanned by the MRIT2 sequence at time points before (0 h) and 2h (2 h) after drug injection, and the in vivo distribution of the nano-drug containing SPIO was observed. The dosage of the tail vein injection nano-drug is as follows: (therapeutic dose 0.31mg/Kg iron equivalent drug, or equal volume of physiological saline);
MRI imaging of the uterus of C57BL/6j mice was performed using a Philips Intera 1.5T MRI scanner, with its animal specific coils. The evolution of signal intensity of the uterine placenta and embryonic region in C57BL/6j mice was observed on MRIbTFE sequence, and the T2 relaxation time change caused by the distribution of SPIO in the drug in the uterus, placenta, embryo and other organs in vivo was measured using T2map imaging technique, and the respective relaxation rates R2 at 0h and 2h were calculated. The Relative increase rate of R2 (RSI (Relative Signal Intensity)% = R2) at 2h after drug injection was calculated 2h /R2 0h ) The results are shown in Table 2。
Table 2 evaluation results of placenta-specific delivery function
From the above results, it can be seen that, in comparative example 1, the helper T cell (Th) surface marker antibody is not linked, and after the polypeptide-PEG modified lipid bilayer is disintegrated in the placenta, the contained drug cannot be anchored to the helper T cell to obtain placenta retention, a large amount of drug leaks through the placenta barrier, and low placenta RSI is detected; the drug is gathered in the embryo, which results in high embryo RSI; the drug can not be anchored to the auxiliary T cell to obtain placenta retention, and part of the drug is separated from the placenta and distributed systemically, so that the liver RSI is high.
The delivery system of comparative example 2 does not contain a polypeptide-PEG modified lipid bilayer membrane as a shell, and cannot achieve targeted release for the placenta microenvironment; in addition, the CD4 antibody targets other cells with various cell membranes expressing CD4 in vivo including helper T cells (Th), and the placenta selection 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 CD4 antibody, failed to target anchoring of the drug into the placenta to helper T cells (Th), 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, without the lipid bilayer envelope, had lower RSI for placenta and 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 has poorer circulation, so its liver RSI is higher than that of comparative example 5; the particle size was larger and less prone to leakage through the maternal-fetal barrier, so placenta RSI was lower than comparative example 5.
In examples 1 to 4, a lipid bilayer membrane modified by enzyme substrate polypeptide-PEG was used as a shell, a drug carrier modified by a T helper cell (Th) surface marker antibody in placenta was used as a core, and a bilayer-structured microenvironment T cell function-controlled nano delivery system was synthesized, having a particle size range of 80 to 210nm. The particle size of about 100nm and the outer negative electricity lipid bilayer membrane are convenient to avoid being phagocytized by a reticuloendothelial system in quantity, so that the in vivo circulation time is prolonged, and the in vivo effective circulation is realized. The substrate polypeptide-PEG modified lipid bilayer membrane shell is stable in the circulation of other tissues and organs in vivo, reaches the placenta microenvironment of specific high expression matrix metalloproteinase, disintegrates along with the degradation of polypeptide, and realizes the drug specific distribution in the placenta tissues. The drug shell disintegrates in the placental microenvironment, revealing the inner drug core containing the CD4 antibody fragment. The CD4 antibody fragment can be anchored in a cell membrane specificity high expression CD4 helper T cell (Th) in a placenta, promote the drug to be specifically endocytosed by the helper T cell (Th) to realize the function regulation of the helper T cell (Th), reduce the distribution in other cells of the placenta and reduce the influence on the function of the placenta. The CD4 antibody enables the drug in the placenta to be anchored to a helper T cell (Th), and also effectively reduces the drug from leaking through the maternal-fetal barrier and reduces the drug from reaching the embryo.
2. Establishment of threatened abortion animal model for evaluating treatment effect
Injecting medicine (therapeutic dose is 0.31mg/Kg iron equivalent medicine or physiological saline with equal volume) into D3, D6, D9, D12 and D15, and detecting embryo loss rate at D17:
the uterus of the mice is observed after the abdomen is opened, and the survival of the embryo and the loss of the placenta are judged. The embryo is judged to be lost if the volume of the embryo is obviously reduced to less than 50 percent of the average volume or the fetal placenta unit has obvious hemorrhage or necrosis. Further, embryo Loss Rate (ELR) = number of lost embryos/(number of lost embryos + number of surviving embryos) × 100% was calculated.
TABLE 3 animal model for threatened abortion evaluation of therapeutic effect
| Group of | Number of lost embryos | Number of surviving embryos | Embryo loss Rate (%) |
| Normal control group | 0.26 | 8.75 | 2.89％ |
| Example 1 | 1.39 | 5.85 | 19.17％ |
| Example 2 | 3.00 | 5.48 | 35.38％ |
| Example 3 | 2.47 | 5.80 | 29.84％ |
| Example 4 | 2.67 | 5.68 | 31.95％ |
| Comparative example 1 | 4.67 | 5.04 | 48.08％ |
| Comparative example 2 | 4.79 | 4.97 | 49.07％ |
| Comparative example 3 | 5.01 | 4.83 | 50.92％ |
| Comparative example 4 | 5.25 | 4.69 | 52.84％ |
| Comparative example 5 | 5.12 | 4.77 | 51.78％ |
| Comparative example 6 | 5.37 | 4.62 | 53.79％ |
From the above results, it can be seen that in comparative example 1, the T helper cell (Th) surface marker antibody in the placenta is not linked, and after the polypeptide-PEG-modified lipid bilayer is disintegrated, the drug in the content cannot be anchored to the T helper cell to obtain placenta retention, and a large amount of drug leaks through the placenta barrier, and it is detected that the treatment effect is poor, the number of lost embryos is high, the embryo loss rate is high, the number of surviving embryos is low, and the estimated number of born is low; meanwhile, the drugs aggregate in the embryos, causing embryotoxicity and also causing a lower number of surviving embryos, which is expected to be lower in litter size.
The delivery system of comparative example 2 does not contain a polypeptide-PEG modified lipid bilayer membrane as a shell, and selective targeted release for the placenta microenvironment cannot be achieved; the CD4 antibody is widely targeted to in vivo expression CD4 cells including helper T cells, the placenta selection targeting is weak, the detected treatment effect is poor, the number of lost embryos is high, the embryo loss rate is high, the number of surviving embryos is low, and the estimated litter size is low. 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 number of surviving embryos is lower, and the expected litter size is lower.
The delivery systems of comparative examples 3 and 4, which do not contain CD4 antibody, do not target anchoring of the drug entering the placenta to helper T cells, and placenta retention is not obtained, resulting in a large number of leaks across the placenta barrier, and detection of insufficient treatment accuracy, poor efficacy, higher number of lost embryos, higher embryo loss rate, lower number of surviving embryos, and lower expected litter size. Meanwhile, the lipid bilayer membrane outer shell of the comparative example 3 is not modified by enzyme-sensitive polypeptide, the distribution in the placenta is reduced, the detected treatment effect is poor, the number of lost embryos is high, the embryo loss rate is high, the number of surviving embryos is low, and the estimated 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 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 number of lost embryos, high embryo loss rate, low number of surviving embryos, and low estimated litter size. The particle size of comparative example 6 is much larger than that of comparative example 5, and the circulation distribution is worse, so that the therapeutic effect is worse than that of comparative example 5.
In examples 1 to 4, a lipid bilayer membrane modified by enzyme substrate polypeptide-PEG was used as a shell, and a drug carrier modified by a T-helper cell surface marker antibody in placenta was used as a core to synthesize a bilayer-structured microenvironment T cell function-regulated nano delivery system with a particle size range of 80 to 210nm. The particle size of the liposome is about 100nm, and the outer negative electricity lipid bilayer membrane is convenient to avoid being phagocytized by a reticuloendothelial system in a large amount, so that the in vivo circulation time is prolonged, and the in vivo effective circulation is realized. The lipid bilayer membrane shell modified by the substrate polypeptide-PEG is stable in the circulation of other tissues and organs in vivo, reaches a placenta microenvironment of specific high-expression matrix metalloproteinase, is disintegrated along with the degradation of the polypeptide, realizes the selective and specific distribution of the drug in the placenta tissue, and accurately and effectively regulates and controls the Th1/Th2 cell ratio, promotes the immune tolerance maintenance and recovers the threatened abortion. The drug shell disintegrates in the placental microenvironment, revealing the inner drug core containing the CD4 antibody fragment. The CD4 antibody fragment can be anchored in a cell membrane specificity high expression CD4 helper T cell in a placenta, and after the drug is promoted to be specifically endocytosed by the helper T cell, the regulation and control of helper T cell proportion and immune function are realized, and the normal immune state of the placenta is recovered; meanwhile, the distribution in other cells of the placenta is reduced, the influence on the function of the placenta is reduced, a better treatment effect is realized through effective regulation and control of the proportion of auxiliary T cells and the immune function, and the recovery of the threatened abortion is promoted. The CD4 antibody enables the medicine in the placenta to be anchored in the helper T cell, so that the medicine leakage through a maternal-fetal barrier is effectively reduced, the medicine 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 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
According to the results, the nano delivery system for microenvironment T cell function regulation and control prepared by the invention has no obvious toxic or side effect on the mother and the fetus.
Claims (5)
1. A microenvironment T cell function regulation and control nano delivery system is characterized in that the nano delivery system is of a core-shell double-layer structure, an enzyme substrate polypeptide-PEG modified lipid bilayer membrane which is targeted to disintegrate under the action of an enzyme which is contacted with high expression of placental interstitial fluid is used as a shell, a marker antibody modified drug carrier which is specifically high expressed on the surface of an auxiliary T cell in a placenta is used as an inner core, and superparamagnetic ferroferric oxide SPIO nano particles, small molecular drugs which regulate and control the function of the auxiliary T cell in the placenta, therapeutic genes or a combination of the small molecular drugs and the therapeutic genes are loaded in the drug carrier;
the enzyme highly expressed in placenta interstitial fluid is matrix metalloproteinase 3;
the enzyme substrate polypeptide is Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys (Dnp) -NH2;
the drug carrier is a copolymer formed by a polyethylene glycol modified polycation carrier and hydrophobic degradable polyester; the copolymer is polyethylene glycol-polyethyleneimine-polycaprolactone PEG-PEI-PCL;
the marker antibody with high surface specificity expression of the helper T cells in the placenta is a Fab section of a CD4 antibody;
the small molecular drug is armillarisin A; the therapeutic gene is siRNA inhibiting the expression of T-bet gene;
the average particle size of the microenvironment T cell function-regulated nano delivery system is 80nm-300nm.
2. The microenvironment T cell function-regulated nano-delivery system of claim 1, wherein the microenvironment T cell function-regulated nano-delivery system has an average particle size of 100nm to 210nm.
3. The method of preparing a microenvironment T cell function-modulating nano delivery system of any one of claims 1-2, comprising the steps of:
s1, loading superparamagnetic ferroferric oxide (SPIO) nanoparticles, micromolecular drugs for regulating and controlling functions of auxiliary T cells in placenta and/or genes to a copolymer to obtain composite nanoparticles;
s2, linking the marker antibody on the surface of the auxiliary T cell in the placenta to the composite nano particle to obtain an antibody composite nano particle;
s3, linking the substrate polypeptide of the enzyme highly expressed by the microenvironment of the placenta and tissues thereof 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 microenvironment T cell function-controlled nano delivery system.
4. The method for preparing the microenvironment T cell function-regulated nano delivery system of claim 3, wherein in the step S1, the mass ratio of the copolymer to the superparamagnetic ferroferric oxide SPIO nanoparticles is 5-15.
5. Use of the nano delivery system for microenvironment T cell dysfunction regulation of any one of claims 1-2 in the manufacture of a medicament for regulating a placental disorder of helper T cells, wherein the placental disorder of helper T cell dysfunction is threatened abortion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111635313.0A CN114306241B (en) | 2021-12-29 | 2021-12-29 | Microenvironment T cell function regulation and control nano delivery system and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111635313.0A CN114306241B (en) | 2021-12-29 | 2021-12-29 | Microenvironment T cell function regulation and control nano delivery system and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114306241A CN114306241A (en) | 2022-04-12 |
| CN114306241B true CN114306241B (en) | 2022-10-04 |
Family
ID=81017282
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111635313.0A Active CN114306241B (en) | 2021-12-29 | 2021-12-29 | Microenvironment T cell function regulation and control nano delivery system and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114306241B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1004578A2 (en) * | 1998-11-05 | 2000-05-31 | Pfizer Products Inc. | 5-oxo-pyrrolidine-2-carboxylic acid hydroxamide derivatives |
| CN1895225A (en) * | 2006-06-26 | 2007-01-17 | 裴泽军 | Brilliant mycomycin elaioplast preparation and its making method |
-
2021
- 2021-12-29 CN CN202111635313.0A patent/CN114306241B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1004578A2 (en) * | 1998-11-05 | 2000-05-31 | Pfizer Products Inc. | 5-oxo-pyrrolidine-2-carboxylic acid hydroxamide derivatives |
| CN1895225A (en) * | 2006-06-26 | 2007-01-17 | 裴泽军 | Brilliant mycomycin elaioplast preparation and its making method |
Non-Patent Citations (3)
| Title |
|---|
| CD4~+T细胞在原因不明复发性流产中的研究进展;王文娟等;《国际妇产科学杂志》;20131215(第06期);525-529 * |
| 胎盘细胞嗜酸性粒细胞趋化因子和T淋巴细胞激活上调性表达分泌因子S表达与自然流产的相关性;肖世金等;《上海医学》;20091025(第10期);906-909 * |
| 胎盘辅助性T细胞因子IL-4、IL-6和IFN-γ与子痫前期关系的研究;韩莉等;《中国妇幼保健》;20100501(第13期);1849-1851 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114306241A (en) | 2022-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020267191A1 (en) | Polymeric bile acid nanocompositions targeting the pancreas and colon | |
| 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 | |
| CN114306241B (en) | Microenvironment T cell function regulation and control nano delivery system and preparation method and application thereof | |
| CN113876964B (en) | Tumor cell membrane drug-carrying system and construction method and application thereof | |
| CN114425044B (en) | Disease focus microenvironment immune cell function regulation and delivery system and preparation method and application thereof | |
| CN114377158B (en) | Focus targeted distribution type nuclear magnetic resonance imaging functional probe and preparation method and application thereof | |
| CN114404367B (en) | Nano-carrier for targeting distribution of diseased cells and preparation method and application thereof | |
| CN114366819B (en) | Placenta focus specific drug distribution delivery system and preparation method and application thereof | |
| CN114376974B (en) | Microenvironment enzyme targeting combined antibody Fab cell targeting vector 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 | |
| CN114366724B (en) | Microenvironment macrophage regulation and delivery system and preparation method and application thereof | |
| CN114469890B (en) | Focus microenvironment sensitive delivery nuclear magnetic probe and preparation method and application thereof | |
| CN114272390B (en) | Microenvironment targeted combined cell targeted tumor inhibition carrier 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 | |
| CN114377150B (en) | MMP enzyme-sensitive placenta microenvironment and tumor microenvironment targeting vector, and preparation method and application thereof | |
| CN114748433A (en) | Anti-inflammatory and antioxidant pharmaceutical composition as well as preparation method and application thereof | |
| CN112546236A (en) | PH-sensitive double-drug-framework polymer prodrug and preparation method and application thereof | |
| Fang et al. | Metronidazole-loaded nanoparticulate thermoreversible gel for gynecologic infection of Trichomonas vaginalis | |
| CN108743956B (en) | Albumin-bound anthracycline antitumor antibiotic preparation and preparation method thereof | |
| Mu et al. | Neutrophil Targeting Platform Reduces Neutrophil Extracellular Traps for Improved Traumatic Brain Injury and Stroke Theranostics | |
| CN110721315B (en) | Preparation method of FK506 sustained-release nano-micelle and application of FK506 sustained-release nano-micelle in preparation of dry eye medicine | |
| CN117919430A (en) | Cell membrane bionic nano material and preparation and application thereof | |
| US20220168230A1 (en) | Nanoparticle mediated therapy |
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 |