WO2020258713A1 - 一种M细胞靶向和pH响应性的淀粉基载体材料及其制备方法与应用 - Google Patents

一种M细胞靶向和pH响应性的淀粉基载体材料及其制备方法与应用 Download PDF

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WO2020258713A1
WO2020258713A1 PCT/CN2019/121423 CN2019121423W WO2020258713A1 WO 2020258713 A1 WO2020258713 A1 WO 2020258713A1 CN 2019121423 W CN2019121423 W CN 2019121423W WO 2020258713 A1 WO2020258713 A1 WO 2020258713A1
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starch
carrier material
based carrier
cells
cell targeting
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PCT/CN2019/121423
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French (fr)
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李晓玺
张义平
陈玲
李琳
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华南理工大学
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    • 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
    • A61K47/61—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 the organic macromolecular compound being a polysaccharide or a derivative thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B31/00—Preparation of derivatives of starch
    • C08B31/08—Ethers
    • C08B31/12—Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch
    • C08B31/125—Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch having a substituent containing at least one nitrogen atom, e.g. cationic starch
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble

Definitions

  • the invention relates to a starch-based carrier material, in particular to a starch-based carrier material with M cell targeting and pH responsiveness, and a preparation method and application thereof.
  • the mucosal immune system is the largest component of the entire immune system and is the first line of defense of the human body. It contains about three-quarters of immune cells, of which M cells are the main entrance for mucosal uptake and capture of antigens. M cells are mainly distributed in the lymph node epithelium (FAE) of Peyer’s pathway. They are closely arranged with intestinal epithelial cells. The intestinal epithelium forms irregular microvilli, which helps it to take in antigenic substances from the intestinal lumen of the upper digestive tract. There are abundant swallowing vesicles and mitochondria in the cytoplasm of M cells, but less lysozyme. There is a membranous fold on the top of the M cell, and the base is deeply sunken into a pocket.
  • FAE lymph node epithelium
  • the intestinal epithelium forms irregular microvilli, which helps it to take in antigenic substances from the intestinal lumen of the upper digestive tract.
  • T cells There are T cells, B cells and macrophages in the pocket.
  • This structure shortens the distance between the antigen-containing vesicles across the M epithelium, and facilitates the rapid entry of the antigen into the subepithelial lymphatic tissue to induce mucosal immune response.
  • M cells transport a wide range of substances, including bacteria, viruses, antigens and particles. Since the distribution of M cells in the intestine is very small, specific targeting of M cells can enhance the absorption and transport of immune substance particles by M cells. Studies have shown that targeted delivery of antigens to M cells can increase the amount of antigen uptake in Peyer’s node and activate more T cells and B cells to participate in the immune response.
  • the intestinal mucosa can deliver antigens and other immune substances to the intestinal mucosa, and protect it from the gastrointestinal environment and local mucosal environment; Cells, improve the absorption and transport efficiency of immune substances by M cells, and enhance mucosal immune response.
  • M cells There are a variety of specific recognition receptors on the surface of M cells, such as protein peptides, glycoproteins, and phospholipids. Choosing appropriate ligands and using them for targeted modification of polymers can increase the transport of M cells to polymer particles. effectiveness.
  • Garinot et al. used photografting technology to modify RGD peptides with polyethylene glycol (PEG).
  • PEG polylactic acid
  • PCL-PEG polylactic acid
  • the amphiphilic copolymer is reprocessed by oil in water. After encapsulating the target functional protein in water, the transport efficiency of M cells to the target functional protein is enhanced.
  • the good water solubility and water absorption of PEG molecules themselves can improve the ability of tablets to release drugs in tablet formulations, and it is difficult to achieve controlled and sustained release of drugs.
  • PEG molecules can change the biofilm structure of various types of cells and have certain effects on cells. Damage.
  • the grafting rate of RGD is low, and the N element content after grafting is only 0.16%.
  • chitosan Targeted delivery of chitosan nanoparticles to Peyer's patch using M cell-homing peptide selected by phage display technology[J].Biomaterials, 2010, 31(30) : 7738-7747.)
  • M cell targeting peptide amino acid sequence is CKSTHPLSC
  • chitosan carrier material with the function of targeting M cells was prepared by ion gel method.
  • chitosan is easy to dissolve in an acidic environment, which is not conducive to protecting the functional activity of active substances in the gastrointestinal tract.
  • M cells were acylated by acylation.
  • the targeting peptide GRGDS was grafted onto the cationized ⁇ -glucan, and the nanoparticles formed by embedding the antigen PR8 had good targeting of M cells, but failed to control the release of antigen in the gastrointestinal environment during oral administration. the study.
  • M cell targeting carrier materials can protect immune active substances from being released and inactivated in the physiological environment of the gastrointestinal tract.
  • carrier materials is mostly synthetic polymer materials, which have potential hazards. Natural polymer carrier materials are mainly concentrated in chitosan. However, due to the solubility of the material itself, it is not conducive to the impact of immunologically active substances. Controlled release in the gastrointestinal tract.
  • M-cell carrier materials need to have the following characteristics: (1) It has a good embedding effect on immunologically active substances and can be combined with The active substance forms a stable transmission system; (2) It can resist the influence of strong acid, pH changes and enzymatic hydrolysis in the physiological environment of the gastrointestinal tract, so as to realize the controlled release of the active substance in the gastrointestinal environment; (3) With M Cell targeting function, through the recognition of specific receptors on the surface of M cells to achieve the function of targeting M cells, and improve the transport efficiency of M cells to active substances.
  • Starch is a kind of polysaccharide that exists widely in nature. It is non-toxic, biodegradable and has good biocompatibility. Studies have shown that starch-based carrier materials have good bioadhesion, but the drug delivery system stays longer in specific parts of the biofilm, which helps mucosal immune cells to absorb and transport it.
  • the purpose of the present invention is to overcome the above shortcomings and deficiencies of the prior art and develop a starch-based carrier material with M cell targeting and pH responsiveness.
  • the primary purpose of the present invention is to provide a starch-based carrier material for M cell targeting and pH responsiveness.
  • the starch-based carrier material not only has good M cell targeting properties, but also has good gastrointestinal pH responsiveness, certain resistance to enzymolysis, and high loading capacity.
  • Another object of the present invention is to provide a method for preparing the above-mentioned M cell targeting and pH-responsive starch-based carrier material.
  • Another object of the present invention is to provide the application of the starch-based carrier material for M cell targeting and pH responsiveness.
  • a starch-based carrier material with M cell targeting and pH responsiveness is as follows:
  • the starch-based carrier material for M cell targeting and pH responsiveness has a molecular weight of 7.04 ⁇ 10 4 to 2.11 ⁇ 10 6 g/mol, a degree of substitution of carboxymethyl groups of 0.04 to 0.28, and a targeting peptide GRGDS grafted The amount is 0.01% to 1.12% (calculated based on the N element content).
  • R being H or R 1 or R 2 does not mean that in the molecular structure, R is H at the same time or R is at the same time R 1 or R is at the same time R 2 at the same time, but it represents the starch of M cell targeting and pH responsiveness
  • a part of R is H, a part is R 1 , and a part is R 2 .
  • a method for preparing the above-mentioned M cell targeting and pH-responsive starch-based carrier material includes the following steps:
  • carboxymethyl starch is prepared by etherification reaction with monochloroacetic acid
  • the GRGDS short peptide is grafted onto the carboxymethyl starch molecular chain through an acylation reaction. After the reaction is completed, the unreacted catalyst and short peptide are removed by dialysis. Starch-based carrier material with M cell targeting function.
  • the molecular weight of the starch described in step (1) is 1.0 ⁇ 10 6 ⁇ 1.0 ⁇ 10 7 g/mol;
  • the etherification reaction in step (1) refers to the reaction at 40-50°C for 2 to 4 hours; the molar ratio of starch and monochloroacetic acid in step (1) is 1:0.1 to 0.4;
  • the degree of substitution of the carboxymethyl starch in step (2) is 0.04-0.27;
  • the acylation reaction in step (2) refers to the reaction at 25-35°C for 12-24 hours; the acylation reaction needs to be carried out in the presence of a catalyst, and the catalyst is 1-ethyl-(3-dimethyl A mixture of aminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), preferably 1-ethyl-(3-dimethylaminopropyl) with a molar ratio of 1:1 A mixture of carbodiimide (EDC) and N-hydroxysuccinimide (NHS).
  • the catalyst is 1-ethyl-(3-dimethyl
  • EDC aminopropyl)carbodiimide
  • NHS N-hydroxysuccinimide
  • step (2) The amount of carboxymethyl starch, catalyst, and GRGDS peptide in step (2) meets the following requirements: in molar terms, the ratio of carboxymethyl group: catalyst: GRGDS peptide is 1:1:1 to 4:1 :1 (wherein the molar ratio of EDC and NHS in the catalyst is 1:1)
  • the carboxymethyl starch after the reaction in step (1) can be subjected to enzymolysis, enzyme inactivation, and lyophilization, and then used as the raw material carboxymethyl starch in step (2) for acylation reaction.
  • the enzymatic hydrolysis refers to the enzymatic hydrolysis of pullulanase followed by high-temperature ⁇ -amylase enzymatic hydrolysis.
  • the conditions of pullulanase enzymatic hydrolysis are: unit enzyme activity 15U/g (carboxymethyl starch dry basis), Enzymatic hydrolysis at 50°C for 16-24 hours; high-temperature resistant ⁇ -amylase enzymatic hydrolysis conditions: unit enzyme activity 100U/g (Carboxymethyl starch dry basis), enzymatic hydrolysis at 80°C for 10-30 minutes.
  • the active substance in the oral preparation is preferably a positively charged immunologically active substance.
  • the present invention has the following advantages and beneficial effects:
  • the method for preparing a starch-based carrier material with M cell targeting and pH response provided by the present invention has simple operation, mild reaction conditions and strong controllability.
  • the present invention provides a starch-based carrier material with M cell targeting and pH response.
  • the grafted M cell targeting peptide GRGDS can specifically recognize and bind to M cells, which helps to improve M cells.
  • the bioavailability of active substances enhances the immune response.
  • the present invention provides a starch-based carrier material with M cell targeting and pH response.
  • the stomach of the carrier material can be adjusted by adjusting the degree of substitution of carboxymethyl groups and the grafting amount of the M cell targeting peptide GRGDS.
  • the intestinal controlled release effect and M cell targeting ability are suitable for the oral immune function of different active substances.
  • Figure 1 is an infrared spectrogram of the starch-based carrier materials prepared in Examples 1 to 3.
  • Figure 2 is a hydrogen nuclear magnetic resonance spectrum of the starch-based carrier materials prepared in Examples 1 to 3.
  • Figure 3 shows the zeta potential of starch-based carrier materials prepared in Examples 1-3 under different pH conditions.
  • Figure 4 shows the cytotoxicity of starch-based carrier materials prepared in Examples 1 to 3.
  • Fig. 5 is a laser confocal image of the co-culture of the starch-based carrier material prepared in Examples 1 to 3 and the cell monolayer composed of M cells and Caco-2 cells.
  • Figure 6 shows the M cell transport efficiency of starch-based carrier materials prepared in Examples 1 to 3.
  • Figure 7 shows the encapsulation efficiency of the starch-based carrier materials prepared in Examples 1 to 3 on the target protein.
  • Fig. 8 is a laser confocal image of co-culture of assembled microcapsules obtained by embedding OVA with starch-based carrier materials prepared in Examples 1 to 3 and cell monolayers composed of M cells and Caco-2 cells.
  • Figure 9 shows the M cell transport efficiency of the assembled microcapsules obtained by embedding OVA with starch-based carrier materials prepared in Examples 1 to 3.
  • the reagents used in the examples can be conventionally purchased from the market unless otherwise specified.
  • GRGDS short peptide was purchased from Gil Biochemical Co., Ltd.
  • OVA egg protein
  • the cells used in the experiment were human colon cancer cell Caco-2 cells (ATCC: HTB37) and human lymphoma cell Raji B cells (ATCC: CCL-86), both of which were purchased from the China Type Culture Collection (CCTCC).
  • test conditions of the zeta potential in the examples refer to the literature "Zhang Y, Chi C, Huang X, et al. Starch-based nanocapsules fabricated through layer-by-layer assembly for oral delivery of protein to lower gastrointestinal tract[J].Carbohydrate polymers,2017, 171:242-251.”, the specific test conditions are: the sample concentration is 5mg/mL, the solvent is a phosphate buffer (0.01M) with different pH, and the test instrument is a nano laser particle size analyzer (Malvern Instruments Co., Ltd., UK Company), the test temperature is 25°C, the number of tests is 3 times, the equilibrium time is 2min, and the equilibrium temperature is 25°C.
  • the Caco-2 cell suspension was placed on the filter membrane (Corning, USA, 3 ⁇ m, filter membrane surface area 1.12cm 2 ) at the bottom of the 12-well polycarbonate Transwell cell in an amount of 0.5 mL per well, at 37°C, 5% CO 2. Incubate for 14 days under saturated steam conditions. During the culture process, 0.5 mL of high-glycemic DMEM medium was added to the inner chamber (above the filter membrane), and 1 mL of high-glycemic DMEM medium was added to the bottom side of the chamber (under the filter membrane), and the medium was replaced every two days.
  • the cell resistance of the monolayer is tested by a transmembrane cell resistance meter, and the cells with cell resistance greater than 300 ⁇ /cm are selected for further co-cultivation.
  • DMEM/RPMI 1640 mixed medium add 0.5 mL of DMEM/RPMI 1640 mixed medium to the chamber, and 1.5 mL of mixed medium to the bottom of the chamber. The medium needs to be replaced every day.
  • a layer is formed on the top of the filter membrane at the bottom of the Transwell chamber.
  • the starch-based carrier material was fluorescently labeled with cationized fluorescent nanoparticles (Thermo Fisher Scientific, INC), and the fluorescently labeled samples were diluted with DMEM (2% FBS) culture solution to a sample concentration of 1.5 ⁇ 10 5 Cells/mL, after equilibrating at 37°C for a period of time, add 0.5mL to the inner chamber of the Transwell chamber with cell monolayers of M cells and Caco-2 cells, and add 1.5mL DMEM (2% FBS) to the outer chamber. ) Culture medium.
  • DMEM 2% FBS
  • the cells were washed with PBS, and then the corresponding secondary antibody (Anti rat IgG-488) (Jackson ImmunoRasearch Laboratories, INC.) was added, and the cells were treated at 37°C for 1 h. After the end, wash with PBS, and finally add Hoechst 33342 staining agent to stain all nuclei for 2 minutes. After cleaning with PBS, remove the filter membrane of the chamber and place it on a glass slide, cover it with a cover glass, and test with a laser confocal microscope to observe the targeting of starch-based carrier materials to M cells. If the different fluorescently labeled starch-based carrier materials and the fluorescent positions of M cells overlap, it indicates that the starch-based carrier material has M cell targeting.
  • M cell targeted transport experiment reference "Garinot M, Fiévez V, Pourcelle V, et al. PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination[J].Journal of Controlled Release,2007,120(3):195- 204.” method with slight modifications, the specific steps are as follows: the M cell targeted transport experiment was performed on the bottom filter membrane of the Transwell cell with the cell monolayer of M cells and Caco-2 cells.
  • the starch-based carrier material is fluorescently labeled with cationized fluorescent nanoparticles (Thermo Fisher Scientific, INC), and the fluorescently labeled sample is diluted with DMEM (2% FBS) broth to a sample concentration of 1.5 ⁇ 10 5 pcs/mL
  • DMEM 2% FBS
  • the culture plate was placed in a 37°C, 5% CO 2 incubator and incubated for 4 hours, and the fluorescent-labeled starch-based carrier material after targeted transport by M cells entered the culture medium of the outer chamber.
  • the transport efficiency of M cells to the starch-based carrier material can be calculated by the following formula. The calculation method of transport efficiency is as follows:
  • dQ/dt is the number of fluorescently labeled starch-based carrier materials transported in the outer chamber per unit time (pieces/s)
  • A is the area of the cell monolayer in the Transwell plate (cm 2 )
  • C 0 is the addition to the inner chamber
  • the method of in vitro simulated release of target protein (OVA) by starch-based carrier materials refers to the literature "Zhang Y, Chi C, Huang X, et al. Starch-based nanocapsules fabricated through layer-by-layer assembly for oral delivery of protein to lower gastrointestinal tract[J].Carbohydrate polymers,2017,171:242-251.”, the specific steps are: the assembled microcapsules obtained after the starch-based carrier material is embedded in OVA are placed in 150mL simulated gastric juice for 2h, and the sample solution 2mL is immediately centrifuged (10000rpm/min, 10, min), take the supernatant and measure the OVA content to calculate the release rate; then centrifuge the assembled microcapsules in the simulated gastric juice (10000rpm/min, 10, min), and place them in 150mL of simulated small intestinal fluid 4h, immediately centrifuge 2mL of the sample solution (10000rpm/min, 10min), take the supernatant and measure the OVA content to calculate the release
  • Simulated gastric juice Dissolve 7mL concentrated HCl in a small amount of water, shake well in a beaker and transfer to a 1L volumetric flask to make the volume constant with double distilled water.
  • Simulated small intestinal juice Weigh 6.8g KH 2 PO 4 and add a certain amount of water to dissolve it, drop its pH to 6.8 with NaOH lye, and add a small amount of pancreatin (Singma-Aldrich Technology Co., Ltd.) (10g) aqueous solution to it. Transfer to a 1L volumetric flask to make the volume constant with double distilled water.
  • RGD-CMS1 The infrared spectrum and the hydrogen nuclear magnetic resonance spectrum of the starch-based carrier material (RGD-CMS1) prepared in this example are shown in Figure 1 and Figure 2 respectively.
  • RGD-CMS1 Figure 1
  • Figure 2 The infrared spectrum of RGD-CMS1 ( Figure 1), RGD-CMS1 has two new absorption peaks at 1730 and 1250 cm -1 , which are the absorption peaks of the amide I band and the amide III band, which proves that RGD-CMS1 In the presence of amide bonds.
  • RGD-CMS1 is negatively charged, while protonation at pH (1.2) in the stomach is almost uncharged, and protonation at pH (6.8) in the small intestine, with a zeta potential of -21.85 mV (the results are shown in Figure 3).
  • the carboxymethyl starch and starch-based carrier material RGD-CMS1 without branched polypeptide were co-cultured with Caco-2 cells and M cells in the cell monolayer on the bottom filter membrane of the Transwell chamber.
  • the results are shown in Figure 5, where A1-A4 are laser confocal micrographs of unbranched carboxymethyl starch co-cultured with Caco-2 cells and M cells, and B1-B4 are starch-based carrier materials RGD-CMS1 and Caco-2 cells and M Confocal laser micrograph of cells co-cultured.
  • A1 and B1 are the nuclei of Caco-2 cells and M cells in the cell monolayer on the filter membrane at the bottom of the Transwell chamber (the bright spots in the figure);
  • A2 and B2 are the M cells in the cell monolayer (specific for M cells). Observation of sex antibody labeling, bright spot in the figure);
  • A3 and B3 are fluorescently labeled carboxymethyl starch without grafted polypeptide and starch-based carrier material RGD-CMS1 (bright spot in the figure);
  • A4 and B4 are the top 3 respectively Overlay of each figure. It can be seen from A4 that the carboxymethyl starch of ungrafted polypeptide does not overlap with the fluorescent position of M cells (the positions of different arrows in the figure), and they are scattered.
  • the carboxymethyl starch of ungrafted polypeptide does not have M Cell targeting. From B4, it can be concluded that the starch-based carrier material RGD-CMS1 overlaps with the fluorescent position of M cells in the cell monolayer on the bottom of the Transwell cell on a large area (arrow 1 in the figure points to M cells, and arrow 2 points to fluorescently labeled Starch-based carrier material, the overlapping part of M cells and starch-based carrier material is marked with a circle), indicating that the starch-based carrier material RGD-CMS1 exhibits good M targeting ability.
  • the carboxymethyl starch with a carboxymethyl substitution degree of 0.04 prepared in step (1) is first enzymatically hydrolyzed by pullulanase (unit enzyme activity 15U/g (carboxymethyl starch dry basis)) at 50°C for 24 hours Then, it was enzymatically hydrolyzed with high temperature resistant ⁇ -amylase (unit enzyme activity 100U/g (carboxymethyl starch dry basis)) at 80°C for 30 min to obtain carboxymethyl starch with a molecular weight of 6.99 ⁇ 10 4 g/mol ( CMS2);
  • RGD-CMS2 The infrared spectrum and hydrogen nuclear magnetic resonance spectrum of RGD-CMS2 obtained in this example are shown in Figure 1 and Figure 2, respectively.
  • RGD-CMS2 In the infrared spectrum of RGD-CMS2 ( Figure 1), RGD-CMS2 is at 1730 and 1250 cm -1 Two new absorption peaks appeared, namely the absorption peaks of amide I band and amide III band, which proved the existence of amide bond in RGD-CMS2.
  • RGD-CMS2 is negatively charged, but protonated at pH (1.2) in the stomach without charge, and deprotonated at pH (6.8) in the small intestine, with a zeta potential of -1.98 mV (the result is shown in Figure 3).
  • the starch-based carrier material RGD-CMS2 was co-cultured with Caco-2 cells and M cells in the cell monolayer on the bottom filter membrane of the Transwell cell.
  • the results are shown in Figure 5, where C1 is the cell monomolecular on the bottom filter membrane of the Transwell cell
  • C2 is the M cell in the cell monolayer (observed by the M cell specific antibody labeling, the bright spot in the figure);
  • C3 is the fluorescently labeled starch-based
  • the carrier material RGD-CMS2 (the bright spot in the picture);
  • C4 is the superimposed picture of the first 3 pictures respectively.
  • the starch-based carrier material RGD-CMS2 partially overlaps with the fluorescent position of M cells in the cell monolayer on the bottom filter membrane of the Transwell chamber (the arrow 1 in the figure points to the M cell, and the arrow 2 points to the fluorescently labeled starch-based
  • the carrier material, the overlapping part of the M cell and the starch-based carrier material is marked with a circle), indicating that the starch-based carrier material RGD-CMS2 exhibits a certain M targeting ability.
  • the transport efficiency of M cells to starch-based carrier materials before and after grafting the targeting peptide is shown in Figure 6. It can be seen from the figure that compared to the carboxymethyl starch obtained in step (2), the M cells have a positive effect on RGD-CMS2. (Referred to as R-CMS2 in the figure), the transport efficiency is increased by 1.05 times.
  • step (1) The carboxymethyl starch with a carboxymethyl substitution degree of 0.24 obtained in step (1) is first enzymatically hydrolyzed by pullulanase (unit enzyme activity 15U/g (carboxymethyl starch dry basis)) at 50°C for 16 hours Then, use high-temperature-resistant ⁇ -amylase (unit enzyme activity 15U/g (carboxymethyl starch dry basis)) to enzymolyze at 80°C for 10 min to obtain carboxymethyl starch with a molecular weight of 4.56 ⁇ 10 5 g/mol ( CMS3);
  • a starch-based carrier material with a molecular weight of 4.58 ⁇ 10 5 g/mol, a degree of carboxymethyl substitution of 0.25, and a grafting amount of the targeting peptide GRGDS of 0.71% (calculated based on the N element content) was obtained ( RGD-CMS3).
  • RGD-CMS3 is negatively charged, but protonated at pH (1.2) in the stomach without charge, and deprotonated at pH (6.8) in the small intestine, with a zeta potential of -27.3 mV (the result is shown in Figure 3).
  • the starch-based carrier material RGD-CMS3 was co-cultured with Caco-2 cells and M cells in the cell monolayer on the bottom filter membrane of the Transwell cell, as shown in Figure 5, where D1 is the cell monolayer on the bottom filter membrane of the Transwell cell The nuclei of Caco-2 cells and M cells in the middle (the bright spot in the figure); D2 is the M cell in the cell monolayer (observed by M cell specific antibody labeling, the bright spot in the figure); D3 is a fluorescently labeled starch-based carrier Material RGD-CMS3 (the bright spot in the picture); D4 is the superimposed picture of the first 3 pictures respectively.
  • the starch-based carrier material RGD-CMS3 partially overlaps with the fluorescent position of M cells in the cell monolayer on the bottom filter membrane of the Transwell chamber (the arrow 1 in the figure points to the M cell, and the arrow 2 points to the fluorescently labeled starch-based
  • the carrier material, the overlapping part of the M cell and the starch-based carrier material is marked with a circle), indicating that the starch-based carrier material RGD-CMS3 exhibits a certain M targeting ability.
  • the transport efficiency of M cells to starch-based carrier materials before and after grafting the targeting peptide is shown in Figure 6. It can be seen from the figure that compared to the carboxymethyl starch obtained in step 2, the M cells have a positive effect on RGD-CMS3 ( Figure 6). (Referred to as R-CMS3 in Chinese), the transport efficiency is increased by 2.1 times.
  • concentrations 5mg/mL and 2mg/mL, respectively .
  • Take a certain amount of starch-based carrier material solution in a beaker, according to the mass ratio of OVA: starch-based carrier material 1:2, use a dropper to slowly drop a certain amount of OVA solution into the carrier solution, and the dripping process will not stop.
  • the calculation method of the encapsulation rate is as follows:
  • m 0 the total mass of OVA in the input system
  • m 1 the mass of OVA in the supernatant
  • the encapsulation efficiency of the starch-based carrier material prepared in Examples 1 to 3 on the target protein is shown in Figure 7. It can be seen from Figure 7 that when the degree of carboxymethyl substitution is high, the starch-based carrier material has a higher degree of encapsulation of the target protein. The encapsulation rate is higher.
  • the assembled microcapsules obtained by embedding the starch-based carrier materials prepared in Examples 1 to 3 with OVA were co-cultured with Caco-2 cells and M cells in the cell monolayer on the bottom filter membrane of the Transwell chamber.
  • the laser confocal diagram is shown in the figure As shown in 8, A1 ⁇ C1 are the nuclei of Caco-2 cells and M cells in the cell monolayer on the filter membrane at the bottom of the Transwell chamber (the bright spots in the figure); A2 ⁇ C2 are the M cells in the cell monolayer (by M Observed by cell-specific antibody labeling, bright spots in the figure); A3 ⁇ C3 are the assembled microcapsules (highlights) constructed by embedding OVA with starch-based carrier materials prepared in Examples 1 to 3 with fluorescent labels; A4 ⁇ C4 are respectively An overlay of the first 3 images.
  • the assembled microcapsules constructed by the starch-based carrier material embedded OVA partially overlap with the fluorescent position of M cells in the cell monolayer on the bottom filter membrane of the Transwell chamber (arrow 1 in the figure points to M cells, arrow 2
  • the overlapping part of M cells and starch-based carrier material is marked with a circle), and as the amount of GRGDS peptide grafting is larger, the overlapping part is more, indicating that the starch-based carrier material is embedding OVA Afterwards, it showed a certain M targeting ability, and the grafting amount of GRGDS affected the M cell targeting ability of the assembled microcapsules constructed by the starch-based carrier material embedded OVA.

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Abstract

本发明公开了一种M细胞靶向和pH响应性的淀粉基载体材料及其制备方法与应用。该淀粉基载体材料的分子结构如下所示，该淀粉基载体材料的分子量为7.04×104～2.11×106g/mol，羧甲基基团取代度为0.04～0.28，靶向肽GRGDS接枝量为0.01％～1.12％(以N元素含量计算)。其在胃部pH (pH＝1.2)条件下质子化不溶于水，在小肠pH(pH＝6.8)条件下去质子化缓慢溶于水，因此具有良好的胃肠道pH响应性，可通过静电相互作用对带正电荷的活性物质进行高效包埋，能够保护活性物质在胃肠道中不被破坏而失活；同时可靶向到M细胞，提高M细胞对活性物质的转运效率，进而提高其生物利用度。 (I)

Description

一种M细胞靶向和pH响应性的淀粉基载体材料及其制备方法与应用 技术领域
本发明涉及一种淀粉基载体材料，特别涉及一种M细胞靶向和pH响应性的淀粉基载体材料及其制备方法与应用。
背景技术
粘膜免疫系统是整个免疫系统最大的组成部分，是人体免疫的第一道防线，包含了约四分之三的免疫细胞，其中M细胞是粘膜摄取和捕获抗原的主要入口。M细胞主要分布于Peyer’s通路的淋巴结上皮(FAE)部位，其与肠上皮细胞紧密排列在一起，肠上皮形成不规则的微绒毛，有助于其从上消化道的肠腔中摄取抗原物质。M细胞胞浆内有丰富的吞饮小泡和线粒体，但溶菌酶较少。M细胞顶部有膜性褶皱，基底部深深凹陷成一个口袋，袋中有T细胞、B细胞及巨噬细胞等。这种结构缩短了含有抗原的吞饮小泡跨越M上皮的距离，有利于抗原快速进入上皮下的淋巴组织诱导粘膜免疫应答。除此之外，M细胞转运物质的范围很广，包括细菌、病毒、抗原以及微粒。由于M细胞在肠道中的分布极少，因此，特异性靶向M细胞能够增强M细胞对免疫物质颗粒的吸收和转运。研究表明，将抗原靶向传递至M细胞，可以提高Peyer’s结的抗原摄取量，激活更多的T细胞和B细胞参与免疫反应。
随着人们对营养和健康的关注度与日俱增，除了通过食物获得基本的营养成分之外，人们还会摄入大量的营养功能活性物质，如功能性蛋白质和肽类、多糖和寡糖、脂质和脂肪酸、益生菌、多分类物质等，来提高自身的健康水平。然而，由于口服传输过程中，游离的功能活性物质易受胃肠道中物理、化学和生理屏障的影响，不能有效启动粘膜免疫反应。除此之外，粘膜局部纤毛的频繁摆动、粘膜局部水解酶的大量存在，也使得外来的免疫物质迅速降解失活，使得粘膜部位接种抗原很难诱导粘膜免疫应答。同时，由于M细胞在肠道中 的分布极少，大多数传输系统主要经粘膜上皮细胞随机吞噬穿越黏膜层，不能有效启动粘膜免疫应答。因此，需要一种能够主动靶向M细胞的载体，一方面能够将抗原等免疫物质传输至肠道粘膜部位，免受胃肠道环境及粘膜局部环境的破坏；另一方面能够靶向到M细胞，提高M细胞对免疫物质的吸收和转运效率，增强粘膜免疫应答。
M细胞表面有多种特异性识别受体，如蛋白质多肽类、糖蛋白以及磷脂类，选择合适的配体，利用其对聚合物进行靶向性修饰，能增加M细胞对聚合物颗粒的转运效率。Garinot等人在PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination(Journal of Controlled Release,2007,120(3):195-204)利用光接枝技术将RGD肽与聚乙二醇(PEG)修饰的聚乳酸(PCL-PEG)中的PEG共价连接，合成了能够靶向M细胞的表面接枝有RGD短肽的PCL-PEG两亲性共聚物，该两亲性共聚物通过水包油再包水的方法包埋目标功能蛋白后，增强了M细胞对目标功能蛋白的转运效率。但是PEG分子本身良好的水溶解性和吸水性，在药片制剂中可提高片剂释放药物的能力，难以实现药物的控缓释，PEG分子可改变各类细胞的生物膜结构，对细胞有一定的损伤。除此之外，RGD的接枝率较低，接枝后N元素含量仅为0.16％。Yoo等人(Yoo M K,Kang S K,Choi J H,et al.Targeted delivery of chitosan nanoparticles to Peyer’s patch using M cell-homing peptide selected by phage display technique[J].Biomaterials,2010,31(30):7738-7747.)采用M细胞靶向肽(氨基酸序列为CKSTHPLSC)与壳聚糖通过离子凝胶的方法制备了具有靶向M细胞的功能的壳聚糖载体材料。然而，根据文献研究，壳聚糖在酸性环境下易于溶解，不利于在胃肠道中保护活性物质的功能活性。Lee等人在Oral Gavage Delivery of PR8 Antigen with β-Glucan-Conjugated GRGDS Carrier to Enhance M-Cell Targeting Ability and Induce Immunity(Biomacromolecules,2017,18(4):1172-1179)中通过酰化反应将M细胞靶向肽GRGDS接枝到阳离子化的β-葡聚糖上，包埋抗原PR8形成的纳米颗粒具有良好的靶向M细胞，但未能对口服过程中胃肠道环境下抗原的控制释放的研究。
目前关于靶向M细胞的载体系统的研究主要集中于靶向M细胞的功能的 研究，而对于M细胞靶向载体材料能否保护免疫活性物质在胃肠道生理环境中不被释放而失活少有研究。除此，载体材料的选择多为人工合成高分子材料，存在潜在的危害，天然高分子载体材料主要集中于壳聚糖，但又由于材料本身的溶解性等问题，不利于对免疫活性物质在胃肠道中的控制释放。因此，为实现免疫活性物质的口服M细胞靶向，进而提高口服免疫活性物质生物利用率，M细胞载体材料需具备以下特征：(1)对免疫活性物质有较好的包埋效果，能够与活性物质形成稳定的传输体系；(2)在胃肠道生理环境中能够抵抗强酸、pH变化以及酶水解的影响，以便在胃肠道环境中实现对活性物质的控制释放；(3)具有M细胞靶向功能，通过与M细胞表面特异性受体识别达到靶向M细胞的功能，提高M细胞对活性物质的转运效率。
淀粉是一种广泛存在于自然界的多糖，无毒、生物可降解、生物相容性好。研究表明，淀粉基载体材料具有良好的生物粘附性，可是给药系统在生物膜的特定部位滞留时间延长，有助于粘膜免疫细胞对其吸收和转运。本发明的目的是为了克服上述现有技术的缺点与不足，开发一种M细胞靶向和pH响应性的淀粉基载体材料。
发明内容
为了克服上述现有技术的缺点与不足，本发明的首要目的在于提供一种M细胞靶向和pH响应性的淀粉基载体材料。该淀粉基载体材料除了具有良好的M细胞靶向性以外，还具有良好的胃肠道pH响应性、一定的抗酶解性以及装载能力高等特点。
本发明另一目的在于提供上述M细胞靶向和pH响应性的淀粉基载体材料的制备方法。
本发明再一目的在于提供上述M细胞靶向和pH响应性的淀粉基载体材料的应用。
本发明的目的通过下述方案实现：
一种M细胞靶向和pH响应性的淀粉基载体材料，其分子结构如下所示：
Figure PCTCN2019121423-appb-000001
所述的M细胞靶向和pH响应性的淀粉基载体材料的分子量为7.04×10 4～2.11×10 6g/mol，羧甲基基团取代度为0.04～0.28，靶向肽GRGDS接枝量为0.01％～1.12％(以N元素含量计算)。
所述的R为H或R 1或R 2并不代表分子结构中R同时为H或者R同时为R 1或者R同时为R 2的情况，而是代表M细胞靶向和pH响应性的淀粉基载体材料的分子结构中多个R一部分为H，一部分为R 1、一部分为R 2。
一种上述的M细胞靶向和pH响应性的淀粉基载体材料的制备方法，包括以下步骤：
(1)以淀粉为原料，利用一氯乙酸通过醚化反应制备羧甲基淀粉；
(2)以羧甲基淀粉为原料，通过酰化反应将GRGDS短肽接枝到羧甲基淀粉分子链上，反应结束后通过透析除去未反应的催化剂和短肽，经冻干后获得具有M细胞靶向功能的淀粉基载体材料。
步骤(1)中所述的淀粉的分子量为1.0×10 6～1.0×10 7g/mol；
步骤(1)中所述的醚化反应是指在40～50℃反应2～4h；步骤(1)中所述的淀粉和一氯乙酸的摩尔比为1：0.1～0.4；
步骤(2)中所述的羧甲基淀粉的取代度为0.04～0.27；
步骤(2)中所述的酰化反应是指在25～35℃反应12～24h；酰化反应需要在催化剂存在条件下进行，所述的催化剂为1-乙基-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基丁二酰亚胺(NHS)的混合物，优选为摩尔比为1:1的1-乙基-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基丁二酰亚胺(NHS)的 混合物。
步骤(2)中所述的羧甲基淀粉、催化剂、GRGDS短肽的用量满足：以摩尔量计，羧甲基基团：催化剂：GRGDS短肽的比值为1:1:1～4:1:1(其中催化剂中EDC和NHS的摩尔比为1:1)
步骤(2)中酰化反应体系中羧甲基淀粉的浓度为0.5～2％(w/v)(g/mL)溶剂为磷酸盐缓冲液(0.1M，pH＝7.5)。
步骤(1)反应结束后的羧甲基淀粉还可以经过酶解、灭酶活、冻干后再作为步骤(2)的原料羧甲基淀粉进行酰化反应。
所述的酶解是指先经过普鲁兰酶酶解后再进行耐高温α-淀粉酶酶解，普鲁兰酶酶解条件为：单位酶活15U/g(羧甲基淀粉干基)，在50℃下酶解16～24h；耐高温α-淀粉酶酶解的条件为：单位酶活100U/g(羧甲基淀粉干基)，在80℃下酶解10～30min。
上述的M细胞靶向和pH响应性的淀粉基载体材料在制备口服制剂中的应用。
所述的口服制剂中的活性物质优选为带正电荷免疫活性物质。
本发明相对于现有技术，具有如下的优点及有益效果：
(1)本发明提供的一种具有M细胞靶向和pH响应的淀粉基载体材料的制备方法，操作简单，反应条件温和，可控性强。
(2)本发明一种具有M细胞靶向和pH响应的淀粉基载体材料，其在胃部pH(pH＝1.2)条件下质子化不溶于水，在小肠pH(pH＝6.8)条件下去质子化缓慢溶于水，因此具有良好的胃肠道pH响应性，可通过静电相互作用对带正电荷的活性物质进行高效包埋，能够较好地保护活性物质在胃肠道中不被破坏而失活；同时可靶向到M细胞，提高M细胞对活性物质的转运效率，进而提高其生物利用度。
(3)本发明提供一种具有M细胞靶向和pH响应的淀粉基载体材料，通过所接枝的M细胞靶向肽GRGDS，能够与M细胞特异性识别并结合，有助于提高M细胞对活性物质的生物利用率，增强免疫应答。
(4)本发明提供一种具有M细胞靶向和pH响应的淀粉基载体材料，可 通过调节羧甲基基团取代度以及M细胞靶向肽GRGDS接枝量，来调节该载体材料的胃肠道控释效果和M细胞靶向能力，以适用于不同活性物质的口服免疫功能。
附图说明
图1为实施例1～3制备的淀粉基载体材料的红外光谱图。
图2为实施例1～3制备的淀粉基载体材料的核磁共振氢谱图。
图3为实施例1-3制备的淀粉基载体材料在不同pH条件下的ζ-电位。
图4为实施例1～3制备的淀粉基载体材料的细胞毒性。
图5为实施例1～3制备的淀粉基载体材料与M细胞和Caco-2细胞组成的细胞单分子层共培养的激光共聚焦图。
图6为实施例1～3制备的淀粉基载体材料的M细胞转运效率。
图7为实施例1～3制备的淀粉基载体材料对目标蛋白的包封率。
图8位实施例1～3制备的淀粉基载体材料包埋OVA获得的组装微囊与M细胞和Caco-2细胞组成的细胞单分子层共培养的激光共聚焦图。
图9为实施例1～3制备的淀粉基载体材料包埋OVA获得的组装微囊的M细胞转运效率。
具体实施方式
下面结合实施例和附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例中所用试剂如无特殊说明均可从市场常规购得。其中GRGDS短肽购自吉尔生化有限公司，鸡卵蛋白(OVA)购自Sigma试剂有限公司。实验所用细胞为人体结肠癌细胞Caco-2细胞(ATCC：HTB37)和人淋巴瘤细胞Raji B细胞(ATCC：CCL-86)，均购自中国典型培养物保藏中心(CCTCC)。
实施例中ζ-电位的测试条件参考文献“Zhang Y,Chi C,Huang X,et al.Starch-based nanocapsules fabricated through layer-by-layer assembly for oral delivery of protein to lower gastrointestinal tract[J].Carbohydrate polymers,2017, 171:242-251.”，具体测试条件为：样品浓度5mg/mL，溶剂为不同pH的磷酸盐缓冲液(0.01M)，测试仪器为纳米激光粒度仪(英国马尔文仪器有限公司)，测试温度为25℃，测试次数为3次，平衡时间为2min，平衡温度为25℃。
实施例中淀粉基载体材料的细胞毒性的测试方法参考文献“He B,Lin P,Jia Z,et al.The transport mechanisms of polymer nanoparticles in Caco-2 epithelial cells[J].Biomaterials,2013,34(25):6082-6098.”，具体为：将Caco-2细胞用含10％胎小牛血清的DMEM培养液配成单个细胞悬浮液，以1×10 3个/孔的密度接种于96孔板，37℃培养24h，弃去旧的培养液。每孔加10微升淀粉基载体材料溶液(溶剂为含10％胎小牛血清的DMEM培养液，0.1M,pH＝7.4)，37℃培养24h。每孔加10微升CCK-8试剂(碧云天生物有限公司)，37℃培养4h后，选择450nm波长，在酶免疫检测仪上测定各孔吸光值。对照孔不加淀粉基载体材料溶液，空白孔不加细胞和淀粉基载体材料溶液。细胞毒性计算公式如下：
Figure PCTCN2019121423-appb-000002
其中，As为实验孔的吸光值，Ab为空白孔吸光值，Ac为对照孔吸光值。
Raji B细胞与Caco-2细胞共培养构建体外模型M细胞参照文献“des Rieux A,Fievez V,Théate I,et al.An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells[J].European journal of pharmaceutical sciences,2007,30(5):380-391.”)，具体步骤如下：选取对数生长期的Caco-2细胞，利用0.25％胰蛋白酶对细胞进行消化，经无菌离心(800rpm/min，3min)，用高糖DMEM培养基(其中含有10％牛血清，1％非必需氨基酸)将细胞重悬成1×10 6个/mL Caco-2细胞悬浮液。以每孔0.5mL的量将Caco-2细胞悬浮液置于12孔聚碳酸酯Transwell小室底部的滤膜上(Corning，USA，3μm，滤膜表面积1.12cm 2)，在37℃、5％CO 2以及饱和水蒸气条件下培养14天。培养过程中在内室(滤膜上方)加入0.5mL高糖DMEM培养基，在小室底侧(滤膜下侧)加入1mL高糖DMEM培养基，并每两天更换一次培养基。单层培养结束后，利用跨膜细胞电阻仪对单层细胞 电阻进行测试，选取细胞电阻大于300Ω/cm的小室细胞进行进一步的共培养。共培养时，选取对数生长期的Raji B细胞，以DMEM/RPMI 1640＝2：1混合培养基将细胞分散成5×10 5个/mL的细胞悬浮液，以每孔1mL的量加入到小室底侧进行共培养。共培养时小室内加入0.5mL DMEM/RPMI 1640混合培养基，小室底侧加入1.5mL混合培养基，培养基需每天更换，共培养持续4～5天后在Transwell小室底部滤膜上端形成一层分布有M细胞和Caco-2细胞的细胞单分子层。
激光共聚焦显微镜观察淀粉基载体材料的M细胞靶向性方法参考文献“des Rieux A,Fievez V,Théate I,et al.An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells[J].European journal of pharmaceutical sciences,2007,30(5):380-391.”，具体步骤如下：在上述具有M细胞和Caco-2细胞的细胞单分子层的Transwell小室底部滤膜上加入淀粉基载体材料溶液，采用激光共聚焦显微镜来观察淀粉基载体材料的M细胞靶向性。实验前，首先用阳离子化的荧光纳米颗粒(Thermo Fisher Scientific，INC)对淀粉基载体材料进行荧光标记，将荧光标记的样品用DMEM(2％FBS)培养液稀释至样品浓度为1.5×10 5个/mL，37℃下平衡一段时间后，取0.5mL加入已构建好的具有M细胞和Caco-2细胞的细胞单分子层的Transwell小室的内室，外室加1.5mL DMEM(2％FBS)培养液。将培养板放入37℃、5％CO 2孵箱中孵育4h，吸除培养基，用PBS清洗Transwell板孔内室底部的滤膜，加入4％多聚甲醛溶液对Caco-2细胞和M细胞进行固定处理15min，固定后吸除多聚甲醛，使用PBS于低温下清洗细胞，随后加入M细胞单抗(NKM 16-2-4)(Medical&Biological Laboratories Co.,LTD)溶液，4℃下处理24h。处理结束后用PBS清洗细胞，然后加入相应的二抗(Anti rat IgG-488)(Jackson ImmunoRasearch Laboratories,INC.)，于37℃下处理1h。结束后用PBS清洗干净，最后加入Hoechst 33342染色剂对全部细胞核进行染色处理2min。用PBS清洗干净后，将小室的滤膜揭下置于载玻片上，盖上盖玻片，用激光共聚焦显微镜进行测试，观察淀粉基载体材料对M细胞的靶向性。若不同荧光标记的淀粉基载体材料和M细胞的荧光位置发生重合即可表 示淀粉基载体材料具有M细胞靶向性。
M细胞靶向转运实验参考文献“Garinot M,Fiévez V,Pourcelle V,et al.PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination[J].Journal of Controlled Release,2007,120(3):195-204.”的方法并稍作修改，具体步骤如下：在上述具有M细胞和Caco-2细胞的细胞单分子层的Transwell小室底部滤膜上进行M细胞靶向转运实验。首先用阳离子化的荧光纳米颗粒(Thermo Fisher Scientific，INC)对淀粉基载体材料进行荧光标记，将荧光标记的样品用DMEM(2％FBS)培养液稀释至样品浓度为1.5×10 5个/mL，37℃下平衡一段时间后，取0.5mL加入已构建好的具有M细胞和Caco-2细胞的细胞单分子层的Transwell小室的内室，外室加1.5mLDMEM(2％FBS)培养液。将培养板放入37℃、5％CO 2孵箱中孵育4h，经M细胞靶向转运后的荧光标记的淀粉基载体材料进入外室培养液中。测定外室培养液中的荧光标记的淀粉基载体材料的荧光粒子数量，可通过下式计算M细胞对淀粉基载体材料的转运效率。转运效率计算方法如下所示：
Figure PCTCN2019121423-appb-000003
其中，dQ/dt为外室中单位时间内转运的荧光标记的淀粉基载体材料数量(个/s)，A为Transwell板中细胞单分子层的面积(cm 2)，C 0为加入内室的荧光标记的淀粉基载体材料稀释液的初始浓度(个/mL)。
淀粉基载体材料对目标蛋白(OVA)的体外模拟释放的方法参考文献“Zhang Y,Chi C,Huang X,et al.Starch-based nanocapsules fabricated through layer-by-layer assembly for oral delivery of protein to lower gastrointestinal tract[J].Carbohydrate polymers,2017,171:242-251.”，具体步骤为：将淀粉基载体材料包埋OVA后获得的组装微囊置于150mL模拟胃液中2h后取样液2mL立即离心(10000rpm/min，10，min)，取上清液测量OVA的含量计算释放率；然后将模拟胃液中组装微囊离心出来(10000rpm/min，10，min)，置于150mL的模拟小肠液中4h，取样液2mL立即离心(10000rpm/min，10，min)，取上清液测量OVA的含量计算释放率；溶出仪参数设置：桨叶底部距溶出杯的 内底部25±2mm；温度37℃；转速75rpm。
模拟胃液：取7mL浓HCl溶于少量水中，在烧杯中摇匀后移至1L容量瓶中用双蒸水定容。
模拟小肠液：称取6.8g KH 2PO 4加一定量水溶解后，用NaOH碱液将其pH值滴至6.8后，取少量胰酶(Singma-Aldrich科技公司)(10g)水溶液加入其中，转至1L容量瓶中用双蒸水定容。
实施例1
(1)按照一氯乙酸：原淀粉(分子量为2.0×10 6g/mol)＝0.4的摩尔比进行醚化反应，反应温度50℃，反应时间4h，制得羧甲基取代度为0.27的羧甲基淀粉(CMS1)；
(2)按照摩尔比，羧甲基基团：催化剂：GRGDS短肽＝1:1:1的比例进行酰化反应，反应温度为35℃，反应时间为24h，其中催化剂为EDC和NHS(摩尔比为1:1)，反应体系中羧甲基淀粉的浓度为1％(w/v，g/mL)，溶剂为磷酸盐缓冲液(0.1M，pH＝7.5)。反应结束经透析、冷冻干燥，得到分子量为2.11×10 6g/mol，羧甲基取代度为0.27，靶向肽GRGDS接枝量为1.12％(以N元素含量计算)的淀粉基载体材料(RGD-CMS1)。
本实施例中所制备的淀粉基载体材料(RGD-CMS1)的红外光谱图和核磁共振氢谱图分别如图1和图2所示。在RGD-CMS1的红外谱图(图1)中，RGD-CMS1在1730和1250cm -1处出现了两个新的吸收峰，分别为酰胺I带和酰胺III带的吸收峰，证明RGD-CMS1中有酰胺键的存在。在RGD-CMS1的核磁氢谱图中(图2)，在1.63ppm、2.84ppm以及3.56ppm处出现了新的化学位移，分别为精氨酸分子上的亚甲基(—CH 2—)、天冬氨酸分子上的次甲基(—CH 2—CH(COOH)—CH 2—)以及甘氨酸分子上的亚甲基(—CH 2—)的质子化学位移，证明RGD短肽成功接枝到CMS分子上。
RGD-CMS1带负电荷，而在胃部pH(1.2)下质子化几乎不带电荷，在小肠pH(6.8)条件下去质子化，ζ-电位为-21.85mV(结果如图3所示)。
RGD-CMS1与Caco-2细胞共培养后，在所有实验浓度下(10-100μg/mL) Caco-2细胞的存活率均在95％以上(结果如图4所示)，证明该淀粉基载体材料安全无毒。
将未接支多肽的羧甲基淀粉和淀粉基载体材料RGD-CMS1分别与Transwell小室底部滤膜上细胞单分子层中Caco-2细胞和M细胞进行共培养，结果如图5所示，其中A1-A4为未接支多肽的羧甲基淀粉与Caco-2细胞和M细胞进行共培养的激光共聚焦显微图，B1-B4为淀粉基载体材料RGD-CMS1与Caco-2细胞和M细胞进行共培养的激光共聚焦显微图。图5中A1、B1为Transwell小室底部滤膜上细胞单分子层中Caco-2细胞和M细胞的细胞核(图中亮点)；A2、B2为细胞单分子层中的M细胞(由M细胞特异性抗体标记观察得到，图中亮点)；A3、B3分别为荧光标记的未接枝多肽的羧甲基淀粉和淀粉基载体材料RGD-CMS1(图中亮点)；A4、B4分别为各自前3个图的叠加图。从A4可以看出，未接枝多肽的羧甲基淀粉与M细胞的荧光位置并未重合(图中不同箭头的位置)，分散开来，因此未接枝多肽的羧甲基淀粉不具有M细胞靶向性。而从B4可以得出，淀粉基载体材料RGD-CMS1与Transwell小室底部滤膜上细胞单分子层中M细胞的荧光位置发生大面积重合(图中箭头1指向M细胞，箭头2指向荧光标记的淀粉基载体材料，M细胞与淀粉基载体材料重合部分用圆圈标出)，说明淀粉基载体材料RGD-CMS1呈现较好的M靶向靶向性。
接枝靶向肽前后M细胞对淀粉基载体材料的转运效率如图6所示，从图中可以看出，相比于步骤(1)中得到的羧甲基淀粉，M细胞对RGD-CMS1(图中简称R-CMS1)的转运效率提高了2.75倍。
实施例2
(1)按照一氯乙酸：原淀粉(分子量为1.0×10 6g/mol)＝0.1的摩尔比进行醚化反应，反应温度40℃，反应时间2h，制得羧甲基取代度为0.04的羧甲基淀粉；
(2)将步骤(1)制备的羧甲基取代度为0.04的羧甲基淀粉先经普鲁兰酶(单位酶活15U/g(羧甲基淀粉干基))在50℃酶解24h后，再用耐高温α -淀粉酶(单位酶活100U/g(羧甲基淀粉干基))在80℃下酶解30min，得到分子量为6.99×10 4g/mol的羧甲基淀粉(CMS2)；
(3)按照摩尔比羧甲基基团：催化剂：GRGDS短肽＝4:1:1的比例进行酰化反应，反应温度为25℃，反应时间为12h，其中催化剂为EDC和NHS(摩尔比为1:1)，反应体系中羧甲基淀粉的浓度为0.5％(w/v，g/mL)，溶剂为磷酸盐缓冲液(0.1M，pH＝7.5)。反应结束经透析、冷冻干燥，得到分子量为7.04×10 4g/mol，羧甲基取代度为0.04，靶向肽GRGDS接枝量为0.01％(以N元素含量计算)的淀粉基载体材料(RGD－CMS2)。
本实施例所得RGD-CMS2的红外光谱图和核磁共振氢谱图分别如图1和图2所示，RGD-CMS2的红外谱图(图1)中，RGD-CMS2在1730和1250cm -1处出现了两个新的吸收峰，分别为酰胺I带和酰胺III带的吸收峰，证明RGD-CMS2中有酰胺键的存在。在RGD-CMS2的核磁氢谱图(图2)中，在1.63ppm、2.84ppm以及3.56ppm处出现了新的化学位移，分别为精氨酸分子上的亚甲基(—CH 2—)、天冬氨酸分子上的次甲基(—CH 2—CH(COOH)—CH 2—)以及甘氨酸分子上的亚甲基(—CH 2—)的质子化学位移，证明RGD短肽成功接枝到CMS分子上。
RGD-CMS2带负电荷，而在胃部pH(1.2)下质子化不带电荷，在小肠pH(6.8)条件下去质子化，ζ-电位为-1.98mV(结果如图3所示)。
RGD-CMS2与Caco-2细胞共培养后，在所有实验浓度下(10-100μg/mL)Caco-2细胞的存活率均在95％以上(结果如图4所示)，证明该淀粉基载体材料安全无毒。
将淀粉基载体材料RGD-CMS2与Transwell小室底部滤膜上细胞单分子层中Caco-2细胞和M细胞进行共培养，结果如图5所示，其中C1为Transwell小室底部滤膜上细胞单分子层中Caco-2细胞和M细胞的细胞核(图中亮点)；C2为细胞单分子层中的M细胞(由M细胞特异性抗体标记观察得到，图中亮点)；C3为荧光标记的淀粉基载体材料RGD-CMS2(图中亮点)；C4分别为各自前3个图的叠加图。从C4可以看出，淀粉基载体材料RGD-CMS2与Transwell小室底部滤膜上细胞单分子层中M细胞的荧光位置发生部分重合 (图中箭头1指向M细胞，箭头2指向荧光标记的淀粉基载体材料，M细胞与淀粉基载体材料重合部分用圆圈标出)，说明淀粉基载体材料RGD-CMS2呈现一定的M靶向靶向性。
接枝靶向肽前后M细胞对淀粉基载体材料的转运效率如图6所示，从图中可以看出，相比于步骤(2)中得到的羧甲基淀粉，M细胞对RGD-CMS2(图中简称R-CMS2)的转运效率提高了1.05倍。
实施例3
(1)按照一氯乙酸：原淀粉(分子量为1.0×10 7g/mol)＝0.3的摩尔比进行醚化反应，反应温度45℃，反应时间3h，制得羧甲基取代度为0.24的羧甲基淀粉；
(2)将步骤(1)中所得羧甲基取代度为0.24的羧甲基淀粉先经普鲁兰酶(单位酶活15U/g(羧甲基淀粉干基))在50℃酶解16h后，再用耐高温α-淀粉酶(单位酶活15U/g(羧甲基淀粉干基))在80℃下酶解10min，得到分子量为4.56×10 5g/mol的羧甲基淀粉(CMS3)；
(3)按照摩尔比羧甲基基团：催化剂：GRGDS短肽＝2:1:1的比例进行酰化反应，反应温度为30℃，反应时间为18h。其中催化剂为EDC和NHS(摩尔比为1:1)，反应体系中羧甲基淀粉的浓度为2％(w/v，g/mL)，溶剂为磷酸盐缓冲液(0.1M，pH＝7.5)。反应结束经透析、冷冻干燥，得到分子量为4.58×10 5g/mol，羧甲基取代度为0.25，靶向肽GRGDS接枝量为0.71％(以N元素含量计算)的淀粉基载体材料(RGD-CMS3)。
本实施例中所制备的淀粉基载体材料(RGD-CMS3)的红外光谱图和核磁共振氢谱图分别如图1和图2所示。在RGD-CMS3的红外谱图(图1)中，在1730和1250cm -1处出现了两个新的吸收峰，分别为酰胺I带和酰胺III带的吸收峰，证明RGD-CMS3中有酰胺键的存在。在RGD-CMS3的核磁氢谱图中(图2)，在1.63ppm、2.84ppm以及3.56ppm处出现了新的化学位移，分别为精氨酸分子上的亚甲基(—CH 2—)、天冬氨酸分子上的次甲基(—CH 2—CH(COOH)—CH 2—)以及甘氨酸分子上的亚甲基(—CH 2—)的质子化学 位移，证明RGD短肽成功接枝到CMS分子上。
RGD-CMS3带负电荷，而在胃部pH(1.2)下质子化不带电荷，在小肠pH(6.8)条件下去质子化，ζ-电位为-27.3mV(结果如图3所示)。
RGD-CMS3与Caco-2细胞共培养后，在所有实验浓度下(10-100μg/mL)Caco-2细胞的存活率均在95％以上(结果如图4所示)，证明该淀粉基载体材料安全无毒。
将淀粉基载体材料RGD-CMS3与Transwell小室底部滤膜上细胞单分子层中Caco-2细胞和M细胞进行共培养，如图5所示，其中D1为Transwell小室底部滤膜上细胞单分子层中Caco-2细胞和M细胞的细胞核(图中亮点)；D2为细胞单分子层中的M细胞(由M细胞特异性抗体标记观察得到，图中亮点)；D3为荧光标记的淀粉基载体材料RGD-CMS3(图中亮点)；D4分别为各自前3个图的叠加图。从D4可以看出，淀粉基载体材料RGD-CMS3与Transwell小室底部滤膜上细胞单分子层中M细胞的荧光位置发生部分重合(图中箭头1指向M细胞，箭头2指向荧光标记的淀粉基载体材料，M细胞与淀粉基载体材料重合部分用圆圈标出)，说明淀粉基载体材料RGD-CMS3呈现一定的M靶向靶向性。
接枝靶向肽前后M细胞对淀粉基载体材料的转运效率如图6所示，从图中可以看出，相比于步骤2中得到的羧甲基淀粉，M细胞对RGD-CMS3(图中简称R-CMS3)的转运效率提高了2.1倍。
实施例4
将实施例1～3中得到的淀粉基载体材料和目标蛋白(OVA)分别溶于磷酸盐缓冲液(0.01M，pH＝3.0)中，分别配置成浓度为5mg/mL和2mg/mL的溶液。取一定量的淀粉基载体材料溶液于烧杯中，按照质量比为OVA:淀粉基载体材料＝1:2，用滴管向载体溶液中缓慢滴入一定量的OVA溶液，滴加过程中不停轻轻摇晃烧杯，滴加结束后使混合液充分混合后，于25℃下静置2h。将组装液离心(10000r/min，10min)，弃去上清液，沉淀物即为淀粉基载体材料包埋后的目标蛋白。
包封率计算方法如下：
Figure PCTCN2019121423-appb-000004
其中，m 0—投入体系内OVA的总质量，m 1—上清液中OVA的质量
实施例1～3制备的淀粉基载体材料对目标蛋白的包封率如图7所示，从图7中可以看出，当羧甲基取代度较高时，淀粉基载体材料对目标蛋白的包封率较高。
将实施例1～3制备的淀粉基载体材料包埋OVA获得的组装微囊分别与Transwell小室底部滤膜上细胞单分子层中Caco-2细胞和M细胞进行共培养，激光共聚焦图如图8所示，其中A1～C1为Transwell小室底部滤膜上细胞单分子层中Caco-2细胞和M细胞的细胞核(图中亮点)；A2～C2为细胞单分子层中的M细胞(由M细胞特异性抗体标记观察得到，图中亮点)；A3～C3依次为荧光标记的实施例1～3制备的淀粉基载体材料包埋OVA构建的组装微囊(亮点)；A4～C4分别为各自前3个图的叠加图。从A4～C4可以看出，淀粉基载体材料包埋OVA构建的组装微囊与Transwell小室底部滤膜上细胞单分子层中M细胞的荧光位置发生部分重合(图中箭头1指向M细胞，箭头2指向荧光标记的淀粉基载体材料，M细胞与淀粉基载体材料重合部分用圆圈标出)，且随着GRGDS肽接枝量较多时，重合部分越多，说明淀粉基载体材料在包埋OVA后呈现一定的M靶向靶向性，且GRGDS的接枝量影响淀粉基载体材料包埋OVA构建的组装微囊的M细胞靶向性。
M细胞对实施例1～3中制备的淀粉基载体材料包埋OVA获得的组装微囊的转运效率如图9所示，结果表明M细胞对其具有较高的转运效率。
经检测，实施例1～3中淀粉基载体材料包埋的目标蛋白(OVA)，在胃和小肠中仅有35.35％、40.47％、43.80％的目标蛋白(OVA)在到达M细胞之前被释放出来。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种M细胞靶向和pH响应性的淀粉基载体材料，其特征在于分子结构如下所示：
    Figure PCTCN2019121423-appb-100001
    所述的M细胞靶向和pH响应性的淀粉基载体材料的分子量为7.04×10 4～2.11×10 6g/mol，羧甲基基团取代度为0.04～0.28，靶向肽GRGDS接枝量以N元素含量计算为0.01％～1.12％；
    所述的R为H或R 1或R 2代表分子结构中一部分R为H，一部分R为R 1，一部分为R 2。
  2. 一种根据权利要求1所述的M细胞靶向和pH响应性的淀粉基载体材料的制备方法，其特征在于包括以下步骤：
    (1)以淀粉为原料，利用一氯乙酸通过醚化反应制备羧甲基淀粉；
    (2)以羧甲基淀粉为原料，通过酰化反应将GRGDS短肽接枝到羧甲基淀粉分子链上，反应结束后通过透析除去未反应的催化剂和短肽，经冻干后获得具有M细胞靶向功能的淀粉基载体材料。
  3. 根据权利要求2所述的M细胞靶向和pH响应性的淀粉基载体材料的制备方法，其特征在于：
    步骤(1)反应结束后的羧甲基淀粉经过酶解、灭酶活、冻干后再作为步骤(2)的原料羧甲基淀粉进行酰化反应。
  4. 根据权利要求3所述的M细胞靶向和pH响应性的淀粉基载体材料的制备方法，其特征在于：
    所述的酶解是指先经过普鲁兰酶酶解后再进行耐高温α-淀粉酶酶解，普鲁兰酶酶解条件为：单位酶活15U/g，在50℃下酶解16～24h；耐高温α-淀粉酶酶解的条件为：单位酶活100U/g，在80℃下酶解10～30min。
  5. 根据权利要求2或3所述的M细胞靶向和pH响应性的淀粉基载体材料的制备方法，其特征在于：
    步骤(1)中所述的淀粉的分子量为1.0×10 6～1.0×10 7g/mol；
    步骤(1)中所述的醚化反应是指在40～50℃反应2～4h；
    步骤(1)中所述的淀粉和一氯乙酸得摩尔比为1：0.1～0.4。
  6. 根据权利要求2或3所述的M细胞靶向和pH响应性的淀粉基载体材料的制备方法，其特征在于：
    步骤(2)中所述的羧甲基淀粉的取代度为0.04～0.27。
  7. 根据权利要求2或3所述的M细胞靶向和pH响应性的淀粉基载体材料的制备方法，其特征在于：
    步骤(2)中所述的酰化反应是指在25～35℃反应12～24h；酰化反应在催化剂存在条件下进行，所述的催化剂为1-乙基-(3-二甲基氨基丙基)碳二亚胺和N-羟基丁二酰亚胺的混合物。
  8. 根据权利要求7所述的M细胞靶向和pH响应性的淀粉基载体材料的制备方法，其特征在于：
    步骤(2)中所述的羧甲基淀粉、催化剂、GRGDS短肽的用量满足：以摩尔量计，羧甲基基团：催化剂：GRGDS短肽的比值为1:1:1～4:1:1，其中催化剂中1-乙基-(3-二甲基氨基丙基)碳二亚胺和N-羟基丁二酰亚胺的摩尔比为1:1；
    步骤(2)中酰化反应体系中羧甲基淀粉的浓度满足每100mL的酰化体系中含有0.5～2g的羧甲基纤维素钠淀粉。
  9. 根据权利要求1所述的M细胞靶向和pH响应性的淀粉基载体材料在制备口服制剂中的应用。
  10. 根据权利要求9所述的M细胞靶向和pH响应性的淀粉基载体材料在制备口服制剂中的应用，其特征在于口服制剂中的活性物质为带正电荷免疫 活性物质。
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Publication number Priority date Publication date Assignee Title
CN103341179A (zh) * 2013-07-08 2013-10-09 苏州大学 一种粘膜m细胞靶向性病毒性心肌炎基因疫苗及其制备
CN104159573A (zh) * 2011-10-13 2014-11-19 卡斯西部储备大学 用于控制出血和药物递送的纳米粒子
CN110256585A (zh) * 2019-06-28 2019-09-20 华南理工大学 一种M细胞靶向和pH响应性的淀粉基载体材料及其制备方法与应用

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CN102688498B (zh) * 2012-06-02 2014-04-16 山东大学 紫杉烷类药物-羧甲基壳聚糖两亲性大分子前药、胶束制剂和冻干胶束制剂，及其制备方法
CN106581686A (zh) * 2016-12-15 2017-04-26 中国药科大学 一种透明质酸修饰的肿瘤微环境特异性释药的两亲性壳聚糖衍生物载体的制备和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104159573A (zh) * 2011-10-13 2014-11-19 卡斯西部储备大学 用于控制出血和药物递送的纳米粒子
CN103341179A (zh) * 2013-07-08 2013-10-09 苏州大学 一种粘膜m细胞靶向性病毒性心肌炎基因疫苗及其制备
CN110256585A (zh) * 2019-06-28 2019-09-20 华南理工大学 一种M细胞靶向和pH响应性的淀粉基载体材料及其制备方法与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DONG-YI LEE ET AL.: "Oral Gavage Delivery of PR8 Antigen with β-Glucan-Conjugated GRGDS Carrier to Enhance M-Cell Targeting Ability and Induce Immunity", BIOMACROMOLECULES, vol. 18, no. 4, 9 March 2017 (2017-03-09), XP055773404, DOI: 20200325103709 *
ELIAS ASSAAD ET AL.: "The influence of protonation ratio on properties of carboxymethyl starch excipient at various substitution degrees: Structural insights and drug release kinetics", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 394, no. 1-2,, 8 May 2010 (2010-05-08), XP027083828, DOI: 20200325103909 *

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