CN105983100A - Protein delivery system of specific targeting macrophages - Google Patents

Abstract

The invention discloses a yeast hollow beta-glucan shell wrapped GMP-BSA protein targeting macrophage delivery system. The system allows BSA to be coated in a yeast shell through using a compact layer formed through the electrostatic adsorption effect of chitosan (CS), tripolyphosphate (TPP), sodium alginate and BSA. GMP-BSA particles prepared in the invention have very good protein release behavior in in-vitro experiments, and can avoid protein loss. The particles highly specifically target the macrophages, such as Raw 264.7 cells, primary BMDM cells (primary bone marrow macrophages) and peritoneal macrophages (PEMs), and are not devoured by NIH3T3, AD293, HeLa, Caco-2 and other non-macrophage tumor cells, and neutral granulocytes in blood. The macrophages play a great role in the pathogenesis of various diseases, and are very important potential target spots. The macrophages are key antigen transfer cells, and are very important in vaccine design. The system can highly selectively deliver various proteins to the macrophages in a targeting manner, and also has very wide application prospect in the field of targeting drug administration and the field of macrophage vaccine design.

Description

The protein transmission system of special target macrophage

Background technology:

Macrophage is the leukocyte of innate immune system, the most widely distributed.Macrophage plays respectively in innate immunity Plant immunologic function, activation and immunoreation.Therefore, macrophage is in various diseases such as Macrophage Activation Syndrome, rheumatic The Pathophysiology aspects such as arthritis, arteriosclerosis, enteritis and tumor generation play an important role.Based on macrophage in disease Regulating and controlling effect, they are likely to become important potential drug action target spot.This research is intended to research and develop the new drug of targeting macrophage Thus suppression and treatment relevant disease.

It addition, macrophage is important antigen presenting cell, it may induce specific CD8+ cytotoxic T cell effectively Play immunoreation, worked at the special defense mechanism initial stage of adaptive immunity (acquired immunity) by antigen processing and submission. In terms of the notable effective vaccine of design, targeted delivery to the macrophage of proteins and peptides class medicine is an effective strategy. Macrophage phagocytic pathogen, first passes through the identification receptor identification pathogen associated molecular pattern on Macrophage Surface as danger Danger signal causes activating and antigen presentation.

Special target macrophage transmission drug molecule treatment disease is a difficult problem, and further investigation builds novel vehicle systems and very must Want, carry out this research and first possess pattern recognition receptors based on Macrophage Surface, such as mannose receptor, Fc receptor and Complement receptors etc. remove receptor, and these receptors are used for removing the pathogen of phagocytosis by macrophage.Sometimes HDL receptor, Amyloid peptide receptor and hyaluronic acid receptor also can be used successfully to targeting target.Development targeting drug delivery system may make to reach The minimizing of the medicine (therapeutic agent) used by clinical effectiveness, it is also possible to effectively reduce drug-induced toxic and side effects and other side effect. Parcel or absorption drug molecule can protect medicine to carrier system in order to avoid degrading in the way arriving macrophage or inactivating.But, Relatively low cell selective and high heterogeneity are the subject matter that these systems face in actual applications, and carrier system is usual Need its carrier surface ligand modified of pattern recognition receptors.

Derive from the beta glucan granule bakeing yeast shell, be the microsphere of the porous about 2-4 μm of a kind of hollow, seek the mankind Support in learning and extensively apply as adjuvant as complementary goods and in vaccine design.This ghost is that the main pathogen of yeast is correlated with Molecular pattern, is identified by pattern recognition receptors Dectin-1, and this receptor mainly expresses agglutinin at Macrophage Surface.β-Portugal gathers Sugar granule can effectively be swallowed by macrophage in vitro.The hollow loose structure of beta glucan granule can realize high drug load, this Point is also this structure advantage as the carrier of targeting macrophage.In recent years, Aouadi etc. demonstrate beta glucan parcel SiRNA granule as oral transmission carrier efficient targeting macrophage, notable reticent Map4k4 gene, and protecting Animal from lipopolysaccharide cause lethal.But this method is owing to using Multiple components to cause preparation process loaded down with trivial details, and use The PEI that toxicity is bigger, therefore can reduce the repeatability of result and cause toxic and side effects.Huang et al. provides a kind of simple Parcel preparation method, directly absorb proteantigen enter glucan particles, this granule can cause as immunological adjuvant in vivo Strong immunoreation.But the shortcoming of this direct absorption preparation method is to discharge the albumen of absorption from carrier the most rapidly. Therefore we need to develop the New Policy that protein encapsulation enters glucan particles.

Summary of the invention:

It is huge that the present invention constructs a kind of yeast shell coating protein matter " GMP-BSA " structure targeting based on beta glucan granule Phagocyte transferrin matter system.Utilize and develop this carrier system from bakery yeast isolated beta glucan granule.This system profit Lean on the compacted zone of electrostatic adsorption formation by BSA with chitosan (CS), tripolyphosphate (TPP), sodium alginate with BSA It is coated in yeast shell.This system can greatly reduce protein and discharge and lose in conveying way.In vitro and in vivo cellular uptake Experiment shows the recognition mechanism of BSA-loaded glucan particles (GMP-BSA).Coating protein matter has no effect on beta glucan The recognition mechanism of grain itself.GMP-BSA granule is absorbed by macrophage selectivity, such as Raw 264.7 cell, primary BMDM Cell (Primary bone marrow macrophage) and peritoneal macrophage (PECs), and this granule not by such as NIH3T3, AD293, The non-huge neutrophil phagocytosis bitten in tumor cell and blood such as HeLa and Caco-2.It is considered that this system can high selectivity The various albumen of targeted, to macrophage, is administered at targeted drug and is also widely used in terms of macrophage vaccine design Prospect.

Detailed description of the invention:

1. prepare beta glucan granule

Yeast S.cerevisiae obtains beta glucan granule through a series of alkalescence acidic treatment steps.3.75g yeast is dissolved in 50 In mL pure water, with 1.0M NaOH regulation pH value to about 12.0-12.5, then 60 DEG C heat while stirring, 1 hour Rear 2000g is centrifuged 10min and collects yeast shell precipitation, and this precipitation is dissolved in 50mL pure water, regulates pH value with 1.0M HCl To about 4-5, after heating while stirring lasting 1 hour at 55 DEG C, centrifugal yeast shell precipitation of collecting, washing yeast ghost 3 Time, isopropanol washes yeast ghost 4 times, and acetone is washed 2 times, obtains 1.72g yeast shell powder after vacuum drying.

2. fluorescein and rhodamine marks beta-glucan particles and BSA

500mg GMP is dissolved in 50mL 0.1M Na₂CO₃(pH 9.2), 5mg FITC or rhodamine B are dissolved in 2mL DMSO In, then dripping in GMP, under room temperature, lucifuge is stirred overnight.10mL 1.0M Tris solution (pH 8.3) joins State the stirring unreacted fluorescent dye of 15-30min cancellation in solution, wash and be centrifuged and collect labeled GMP, until supernatant Without color.Dehydrated alcohol and acetone are respectively washed one time and are anhydrated, and lucifuge is vacuum dried.

10mg BSA is dissolved in 10mL 5mM EDTA and 1.2mL 0.1M Na₂CO₃(pH 9.2), 1mg FITC or Luo Dan Bright B is dissolved in 0.4mLDMSO, then drips in BSA solution, and under room temperature, lucifuge is stirred overnight.2mL 1.0M Tris Solution (pH 8.3) joins and stirs the unreacted fluorescent dye of 15-30min cancellation in above-mentioned solution under room temperature, and dialysis is removed not The fluorescent dye of reaction, lucifuge lyophilizing obtains the BSA of labelling.

3. prepare the GMP granule (GMP-BSA) of capsule contracting BSA

20 μ L 50mg/mL BSA join 10mg dry yeast shell powder, incubated at room temperature 10-30min, add 20 μ L 0.4% 2 hours capsule contracting BSA of pH5.0 low-molecular-weight CS incubated at room temperature enter yeast shell.1.0mL excess TPP/Alg (1.0mg/mL TPP, 0.4mg/mL sodium alginate) join in yeast shell, ultrasonic 10s discrete particles, stirs 1 hour.2000g from Heart 5min collects GMP-BSA, PBS and washes three times, 70% ethanol wash one time degerming, then wash three times with PBS, vacuum freeze-drying obtains To GMP-BSA powder.

The Morphological characterization of 4.GMP-BSA

Laser Scanning Confocal Microscope and JSM-7500F scanning electron microscope (Tokyo, Japan) is used to detect GMP-BSA granule Profile and surface character.Rhodamine B in yeast shell surface markers, on BSA labelling, FITC or FITC is marked at yeast shell table Face, rhodamine B is marked on BSA, utilizes Laser Scanning Confocal Microscope detection particle shape and protein distribution in granule.? Grain size Malvern laser particle analyzer Nano ZS90.Result proves that BSA capsule well contracts inside GMP, and GMP-BSA Granule all oval states in 2-4 μm.

5.BSA drug loading and the detection of encapsulation efficiency

The BSA drug loading of GMP-BSA and the method for encapsulation efficiency trace bicinchoninic acid directly detect BCA in supernatant and contain Amount.Collect the centrifugal supernatant trace BCA quantification kit analysis obtained in GMP-BSA preparation process.At 1.5mL In test tube, the trace BCA of every 60 μ L of supernatant mixing equal volume detects liquid, hatches 1 hour for 60 DEG C, takes 100 μ L mixed liquors Sample uses microplate reader to weigh the light absorption value of sample at 570nm.In supernatant, the concentration of BSA is bent according to the BSA of concentration known Line computation, empty GMP adsorbs chitosan/TPP/ sodium alginate, is not added with BSA same method and prepares the supernatant dissolving that microsphere obtains BSA prepares standard curve.The BSA drug loading of GMP-BSA is 5.00% ± 0.25%, encapsulation efficiency is 49.67% ± 2.12%.

6.GMP-BSA release in vitro BSA

10mg GMP-BSA and 1mL is hatched containing 0.02% Hydrazoic acid,sodium salt PBS (pH 7.4) at room temperature shaking table 150rpm. At suitable time point, 8000rpm is centrifuged 5min and collects supernatant, adds the fresh release liquid of equivalent, in release supernatant BSA content use trace BCA quantification kit quantitative, use the change of Origin 8.0 software analysis BSA content.? In initial 12h, BSA burst size is less than 20%, and due to the slow reduction of chitosan sodium alginate, this granule can delay continuously On The Drug Release BSA2 time-of-week.

7.SDS-PAGE electrophoresis and CD spectrum prove the integrity of albumen

Taking different time points 15 μ L and make sample from the BSA supernatant that GMP-BSA discharges, naked BSA is positive control.Gel It is all under conditions of trishydroxymethylaminomethane/disposable buffer of glycine SDS, uses energy digital display type voltage stabilizing in Tanon days steady The constant voltage mode of flow electrophoresis instrument (EPS-300) 180V runs the integrity of the protein that glue, gel and coomassie brilliant blue staining detect, Result proves the BSA not degraded of release.The secondary structure circular dichroism spectra of the BSA of release goes detection, and naked BSA is sun Property comparison, cuvette is 2mm quartz cylinder pond, and Sample Scan wave-length coverage is 200-260nm.This result and gel result Unanimously.

The separation of 8.BMDM and cultivation

10 weeks big female Mus of C57BL/6J are used for preparing Primary bone marrow origin macrophage, this cell separation in mice both sides Thigh bone and Tibia, crosses film and removes impurity, and the macrophage of every mice is inoculated in 10cm culture dish, with containing 20%FBS, 1% dual anti- Cultivate the DMEM culture medium culturing of the supernatant of 3d with 30% l cell strain L929, within every 2 days, change liquid to cell, 5 Subsequent experimental is gone to after it.

9. the separation of peritoneal macrophage and cultivation

10 weeks big female Mus of C57BL/6J shift to an earlier date 4 days first lumbar injection 2mL4% peptone broth enrichment peritoneal macrophages, then Peritoneal macrophage is obtained by PBS order peritoneal lavage peritoneal cavity.This cell RPMI1640 culture medium is resuspended with 6 × 10⁵ The every hole of density be inoculated in 24 orifice plates, wash non-adherent cell off with PBS after 3 hours, after staying adherent macrophage to carry out Continuous experiment.

10. the separation of M1 and the M2 type macrophage activated and cultivation

The bone marrow origin macrophage deriving from 10 weeks big female Mus of C57BL/6J is inoculated in 24 orifice plates cultivation 5 days, with IFN- γ (30ng/mL) and LPS (10ng/mL) Co stituation cultivate 36h, induce M1 type macrophage, with IL-4 (20 Ng/mL) stimulate cultivation 36h, induce M2 type macrophage, prepare subsequent experimental.

11. Laser Scanning Confocal Microscope detection cell in vitro phagocytosiss

GMP-BSA is carried out in the phagocytosis of cellular level in macrophage and non-macrophage simultaneously.For macrophage, RAW 264.7, primary BMDMs, PECs are with 1 × 10⁶Individual cell/mL is inoculated in 24 orifice plates, the GMP-BSA of variable concentrations after 6h Granule joins hatches 12h in cell.PBS defines macrophage with IF488-F4/80 monoclonal antibody after washing 2 times, and DAPI contaminates core. M1 and the M2 type macrophage of activation add 12 μ g/mL hatch 6 respectively, 12,24h.Non-adherent cell NIH3T3, AD293, HeLa, and Caco-2 cell line carries out identical operation as negative control.PBS washes 3 times, and fixing cell DAPI contaminates core, Laser Scanning Confocal Microscope detects.Result shows this granule height specially targeting macrophage, such as Raw 264.7 cell, primary BMDM Cell (Primary bone marrow macrophage) and peritoneal macrophage (PEMs), and this granule not by such as NIH3T3, AD293, HeLa and Caco-2 etc. are non-, and huge tumor cell of biting swallows.

12. cell toxicity tests

RAW 264.7 is with 5 × 10³Individual cell/mL is inoculated in 96 orifice plates, and GMP-BSA granule is with different concentration (3-400 μ g/mL) hatch 24h, every hole adds 10 μ L MTT (5.0mg/mL) 37 DEG C and hatches 4h, abandons supernatant, adds 100 μ L DMSO Dissolve first a ceremonial jade-ladle, used in libation, at 490nm, read light absorption value by microplate reader.Result display GMP-BSA granule is up to 400 μ g/mL's Under concentration, cell do not had toxic and side effects.

The enforcement of GMP-BSA phagocytosis in 13. bodies

10 weeks big female Mus of C57BL/6J shift to an earlier date 4 days first lumbar injection 2mL 4% peptone broth enrichment peritoneal macrophages, then Lumbar injection or the GMP-BSA granule of intravenous injection 150 μ L 30mg/kg parcel rhodamine B labelling BSA, after 3h, carry Taking spleen, liver, lung and peritoneal macrophage, the neutrophilic granulocyte separated in blood does negative control, and peritoneal macrophage is from abdominal cavity After proposition, adherent 2h washes away non-adherent cell, and IF488-F4/80 monoclonal antibody dyeing 30min defines macrophage, mounting copolymerization Focusing microscope testing result.Spleen, liver, lung are cut into small pieces, with 100U/mL deoxyribonuclease I and collagenase 4 at 37 DEG C Hatch 30min.The tissue digested removes impurity by 100mm filter membrane, and centrifugal collecting cell is inoculated in 24 porose discs, adherent After 2-3h, washing away non-attached cell, IF488-F4/80 monoclonal antibody dyeing 30min defines macrophage, and mounting Laser Scanning Confocal Microscope is examined Survey result.Neutrophilic granulocyte extracts from the blood of intravenous injection into mice, DAPI by gradient centrifugation neutrophilic granulocyte separating kit Mounting Laser Scanning Confocal Microscope testing result after dye core.Result shows this granule height specially targeting macrophage, the macrophage of organ With peritoneal macrophage (PECs), and this granule do not have by the neutrophil phagocytosis in blood.

Claims (7)

1. the transmission of a novel protein targeting macrophage based on yeast hollow beta glucan shell parcel GMP-BSA System.This system utilizes chitosan (CS), tripolyphosphate (TPP), sodium alginate and bovine serum albumin (BSA) by quiet BSA is coated in yeast shell by the compacted zone that electro-adsorption effect is formed.Preparation method for first BSA is joined in sky yeast shell, Incubated at room temperature 10-30min, adds 2 hours capsule contracting BSA of low-molecular-weight CS incubated at room temperature and enters yeast shell.It is eventually adding Excess TPP/ sodium alginate in yeast shell, centrifugal collects GMP-BSA, and vacuum freeze-drying obtains GMP-BSA powder.This Grain high selectivity specially targeted various albumen macrophage, such as Raw264.7 cell, primary BMDM cell (primary bone Marrow macrophage) and peritoneal macrophage (PEMs), and this granule not by such as NIH3T3, AD293, HeLa and The non-huge neutrophil phagocytosis bitten in tumor cell and blood such as Caco-2.

The preparation method of protein targeted delivery systems the most according to claim 1, it is characterised in that: with outside yeast hollow Shell capsule pix protein matter is as targeted delivery protein system.

The preparation method of protein targeted delivery systems the most according to claim 1, it is characterised in that: protein TPP, Sodium alginate and chitosan are wrapped in sky yeast shell.

The characteristic of protein targeted delivery systems the most according to claim 1, this system is in vitro by macrophage system Raw 264.7 cells, mice separate peritoneal macrophage and bone marrow origin macrophage selected by property phagocytosis.

The characteristic of protein targeted delivery systems the most according to claim 1, this system mice live body by abdominal cavity huge bite thin Property phagocytosis selected by born of the same parents and intraorganic macrophage.

The characteristic of protein targeted delivery systems the most according to claim 1, this system is not by non-macrophage phagocytic.

The characteristic of protein targeted delivery systems the most according to claim 1, this system is not thin by the neutral grain in blood Born of the same parents are swallowed.