Studies On Wheat Germ Agglutinin-Mediated Bioadhesive Nanoparticles For Oral Delivery Of Thymopentin

The purpose of this study was to design and evaluate specific bioadhesive poly （lactic-co-glycolic） acid （PLGA） nanoparticles for oral delivery of thymopentin （TP5）. Wheat germ agglutinin （WGA）, with the ubiquitous presence of binding sites along the gastrointestinal tract, was selected here as a model lectin to take cytoadhesive properties. The scope of this thesis research includes: （1） the study of pharmaceutical preformulation; （2） the preparation and characterization of TP5-loaded PLGA nanoparticles （TP5-NPs）; （3） the preparation and characterization of TP5-loaded specific bioadhesive nanoparticles （WGA-TP5-NPs）; （4） the evaluation of in vitro bioadhesive activity; （5） the investigation of bioadhesion and biodistribution following oral administration; and （6） the pharmacodynamical study in vivo.Firstly, a quantitative RP-HPLC method was developed to determine the concentration of TP5 in vitro. The linear calibration curve was obtained in a concentration range of 5～400μg·mL^-1. The precision, recovery, limit of detection and limit of quantitation accorded with the requirements of methodology. It was observed that TP5 had the best stability in solution under frozen conditions, and light could not destroyed it at room temperature for 24 h. TP5 was found to be stable in solution at pH 5.0～9.2, while the selections of buffers did not affect its stability.Using a double emulsion-solvent evaporation method, TP5-NPs were prepared and the preparation techniques were optimized by means of an orthogonal test design with entrapment efficiency as index of investigation. The average diameter and zeta potential of performed TP5-NPs were （150.3±9.6） nm and （-13.6±1.3） mV. The drug loading and entrapment efficiency were determined to be （0.481±0.011） % and （28.12±0.60） %, respectively. TP5-NPs were found to be stable in distilled water, artificial gastric and intestinal solution within 24 h. After 6 months of storage at 4℃, no significant changes in physiochemical characteristics of nanoparticles occurred. After a burst effect, the TP5 in vitro release from TP5-NPs was consistent with a Higuchi or Ritger-Peppas diffusion mechanism. Lowry’s method and RBC agglutination test were established to determine the content and bioactivity of WGA, respectively. The extraction conditions of WGA were optimized. WGA was purified from the extracts consecutively by use of selective heating treatment, salting-out with （NH₄）₂SO₄, and affinity chromatography with partially hydrolyzed chitin. The resulting WGA preparations showed a single band on SDS-PAGE, demonstrated same mobility as that of Sigma standard.Novel WGA-PLGA conjugates were synthesized by coupling the amino groups of WGA to the carbodiimide-activated carboxylic groups of PLGA, and the identification of the conjugates was performed using MALDI-TOF-MS. The content of WGA in WGA-PLGA was determined to be （75.56±1.64） %. The conjugates were found to be soluble in DMSO and partially soluble in acetone, but could not dissolve in water and other organic solvents. The complete retention of bioactivity of WGA after covalent coupling was confirmed.WGA-TP5-NPs with different WGA density were prepared by using an emulsion-solvent evaporation technique. Compared to conventional TP5-NPs, the average diameter, zeta potential and drug entrapment efficiency of WGA-TP5-NPs were found to be slightly increased. WGA-TP5-NPs were stable in artificial gastric and intestinal solution, being capable to protect the loaded drugs from acidic pH and enzymatic degradation. The release profiles of TP5 from WGA-TP5-NPs in vitro were noted to be typically biphasic. At the first 2 h, amounting to （26.72±2.01）～（32.90±1.97） % of TP5 were released in artificial gastric solution, and （39.08±2.32）～（44.65±2.40） % of TP5 were released in artificial intestinal solution after 24 h. When nanoparticles were incubated in pH 7.4 PBS and rat serum solution, （43.60±2.30）～（47.91±2.12） % and （67.84±2.42）～（73.02±2.60） % of entrapped TP5 were released from WGA-TP5-NPs after 4 d. The release data were evaluated by model-dependent methods, and the results indicated that after a burst effect, the TP5 release from WGA-TP5-NPs was consistent with a Higuchi or Ritger-Peppas diffusion mechanism.The in vitro bioadhesive activities of nanoparticles were evaluated by pig gastric mucin （PM） binding experiments. The conjugation of WGA enhanced the interaction about 1.8～4.2 fold compared with that of the non-conjugated one. A mathematical model was used based on the Langmuir equation, and the rate constants of interaction （k） between WGA-TP5-NPs with different WGA density and PM were calculated to be 1.563×10^-3, 2.601×10^-3 and 3.184×10^-3 (μg·min·mL^-1)^-1, respectively. These interactions could be competitively inhibited by NAcGlc, a specific sugar of WGA.For preparation of fluorescent nanoparticles, FITC was covalently coupled to PLGA by a carbodiimide method. TP5-F-NPs and WGA-TP5-F-NPs were prepared using an emulsion-solvent evaporation technique. The main physicochemical characteristics of fluorescent nanoparticles were found to be similar with those unlabeled. The FITC was stable in artificial gastric and intestinal solution, as well as rat tissue homogenates, and the binding of fluorescent marker to nanoparticles was also found to be stable.The specific bioadhesion of nanoparticles on rat intestinal mucosa was studied ex vivo. An important increase of interaction between WGA-TP5-F-NPs and the intestinal segments （jejunum and ileum） was observed compared with that of TP5-F-NPs （P<0.05）. No specific binding of WGA-TP5-F-NPs to Peyer’s patches region was found, and NAcGIc inhibited their binding effectively. Fluorescence photomicrographs confirmed the bioadhesion of WGA-TP5-F-NPs on intestinal villous epithelium and Peyer’s patches, as well as the internalization of nanoparticles into mucus layer.Quantitative study of the bioadhesion and distribution of nanoparticles in rat tissues after oral administration was conducted by means of fluorescence spectrophotometer. Noticeable amounts of WGA-TP5-F-NPs bioadhered to small intestine with a significant increase of resistance time. Following oral administration of a single dose or daily for 7 days, higher amounts of WGA-TP5-F-NPs bioadhered in small intestine and distributed in all tissues. The percents of distribution in per gram of tissue and the percents of distribution in each tissue were noticed to be increased. Compared to TP5-F-NPs, of the total dose administered, 1.4～2.8 and 1.5～3.1 times as much WGA-TP5-F-NPs were transported from rat gastrointestinal tract into the circulation.The pharmacodynamical studies on oral administration of nanoparticles were performed in multicolor flow cytometry. The changes of CD4⁺/CD8⁺ ratios in the blood of cyclophosphamide immunosuppressant rats were determined. There was a little enhancement on the oral uptake of TP5 when TP5 solution or TP5-NPs administered, while the oral gavages of WGA-TP5-NPs had a remarkable immunomodulating effect as the same as what the TP5 injection acted. It was also suggested that the enhanced oral uptake was related to the increasing of WGA density on nanoparticles.These results confirmed that WGA-TP5-NPs prepared in this study could effectively encapsulate TP5 as oral drug delivery system. The conjugation of WGA led a specific bioadhesion in small intestine and prolonged the resistance time of nanoparticles in the intestinal tract, therefore improved the oral absorption of TP5 due to the receptor-mediated internalization.