Preparation Pectin Fragment RG-I Based On Its Binding With Gal-3 And The Cancer Celltargeted Drug Delivery Of RG-I Nano Selfassembly

In recent years,pectin fragments originated from various degradation methods have attracted extensive attention from both academic and medical fields,owing to the wide biological functions as a result of their specific binding to galectin-3（Gal-3）.More and more evidences have indicated that rhamnogalactan-I（RG-I）is the main active fragment of pectin binding to Gal-3.As proven by clinical,Gal-3 is highly expressed in a subset of metastatic cancer cells and is a new target for cancers therapy.For these reasons,RG-I is proposed to be used as a carrier material to construct a novel anticancer drug nano-delivery system based on its binding with the Gal-3 on the surface of cancer cells.The main content of this thesis is as follows:（1）Set up of a regio-selective degradation method for citrus pectin and the optimization.Citrus pectin was degraded by enzymatic and ultraviolet light catalytic hydrogen peroxide oxidation（UHP）method,respectively.The optimal conditions by enzymatic degradation were p H 5.8,30℃and endo-polygalacturonase dosage 20μL/g citrus pectin,resulting in hydrolysates with low molecular weight（Mw 7-185 k Da）and high degree of methylation（DM58-73%）.For UHP degradation,degraded pectins with low Mw（55-191 k Da）and low DM（24-54%）were produced under 30 m M H₂O₂ for 1-7 h UV irradiation.Upon combining enzymatic degradation with UHP,pectin was further degraded with final Mw（5-50 k Da）and DM（6%-47%）.Using Gal-3-mediated hemagglutination（G3H）assay,two degraded pectin products with the strongest binding ability to Gal-3 were selected,i.e.,En2 produced by enzymatic hydrolysis under 2 h and UHP4-5 by UHP under 5 h,with minimum Gal-3 inhibition concentration（MIC）of 0.125 mg/m L.However,the Gal-3 binding ability of the degraded pectin by a combined degradation method was decreased.（2）Isolation,purification and structural characterization of pectin fragments and the bio-effect on cell level for their binding to Gal-3.En2 and UHP4-5 were purified by DEAE-cellulose anion exchange and dextran gel column chromatography,respectively.Two active components with Gal-3 binding ability,En2-0.3P and UHP4-5-0.3P eluted by 0.3 M Na Cl,were screened by G3H method.The MIC values of two samples were both 16μg/m L.Based on the structural characterization techniques,such as GPC,HPLC,HPAEC-PAD,FT-IR,¹H NMR and HSQC,En2-0.3P was composed of methoxy-substituted（DM 44.3%）homogalacturonan（HG～55.1%）backbone and RG-I region（37.6%）consisting of low substituted（18 chains）and length（6 sugar residues）branches with Mw 101 k Da.UHP4-5-0.3P was composed of 55.4%HG backbone（DM 48.5%）and RG-I region（33.8%）,consisting of high substituted（46 chains）and length（8 sugar residues）branches with Mw 327.0 k Da.CCK8 method was used to determine the cell proliferation activity.By comparing 293A human normal kidney cell,which has low Gal-3 expression,and the Gal-3 over-expressed cells Hep G2,MCF-7 and Hela cancer cells,it was concluded that the obtained RG-I fragments produced a bio-effect at cellular level by inhibiting the proliferation of cancer cells through binding to Gal-3.（3）Construction of cancer cells targeted drug delivery system with RG-I based on its binding to Gal-3 and the application.First,En2-0.3P and UHP4-5-0.3P were hydrophobically modified by a conjugating reaction with curcumin（Cur）,resulting in the conjugated Cur of2.15%and 1.60%,respectively,under 15 mg/m L Cur at 60℃for 12 h.As confirmed by FT-IR and ¹H NMR spectra,carboxyl group in pectin fragment was reacted with hydroxyl group of Cur through an esterification reaction.The obtained amphiphilic RG-I unit assembled in aqueous solution to form RG-I assembly with diameter around 300-400 nm.Second,Cur,an anticancer drug model,was loaded under RG-I 20 mg/m L,Cur 4 mg/m L and 240 W ultrasound to obtain nanoparticles UHPNP and En NP with Cur loading of 2.05%and 1.02%,respectively.SEM observation indicated that they appeared cubic and ellipsoid,respectively,with particle size in the range of 200 nm to 250 nm.In addition,the stability trial showed that the particle size of Cur nanoparticles remained unchanged（p<0.01）under room temperature for 3 weeks.In vitro drug release simulation indicated that the release of Cur at p H 5.0,6.4 and 7.4 followed zero-order,first-order and Higuchi kinetics,respectively,and the maximum drug release rate was 76.1%（UHPNP）.（4）Cancer cell-targeted delivery and bio-function of the Cur-loaded RG-I nano assembly.Laser confocal microscope showed that UHPNP was easier to be captured by Hep G2 than En NP,with the average fluorescence intensity being 2.58 times that of En NP at the same concentration as disclosed by flow cytometric analysis.The competitive Gal-3 binding experiment with lactose indicated that the UHPNP nano-delivery system was capable of delivering Cur to liver cancer cell based on the specific recognition between UHPNP and Gal-3,which is high expressed on the of surface Hep G2.The CCK8 assay showed that UHPNP at low concentration（10-100μg/m L）inhibited the survival of the three tumor cells with the inhibition rate above 80%.