| Chemotherapy is the most commonly used and effective way for cancer treatment.At present, intravenous administration of anticancer drug remains to be the mostprevalent approach for the treatment of tumors. It is effective for cancer therapy, butmany disadvantages such as long time consuming, side effects and bad therapeuticeffect for continuous treatment can be encountered. Oral administration of anticancerdrug is a viable alternative to intravenous injections, since it can simplify treatmentand save time, in addition, patients are more receptive to it. Nevertheless, deliveringanticancer drug orally often leads to a low bioavailability owing to first-pass effectand barriers to physical absorption in the epithelium. Chitosan (CS), a natural cationicpolysaccharide, attracts increasing concern in oral drug delivery field because of itsfavorable biocompatibility, histocompatibility as well as its ability to open tightjunctions (TJs) between intestinal epithelial cells and facilitate paracellular transportof drugs.Carboxymethyl chitosan (CMCS) was synthesized from the carboxymethylationreaction of chitosan under soft conditions and could be soluble in neutral pH. Therelusts of fourier transform infrared spectrum showed that carboxymethyl groups werecovalently connected onto chitosan molecular skeleton. Molecular weight of CMCSwas12kDa, which was measured by ubbelohde viscosity method. Elemental analysisresults showed that the degree of deacetylation and the degree ofcarboxymethyl substitution were81%and92%, respectively. Cy3NHS Ester andfluorescein isothiocyanate used as were respectively labeled onto the main chain ofCS (Cy3-CS) and CMCS (FITC-CMCS), for further fluorescence localization.CS-nanogels (CS-NGs) were prepared by ionic gelation method and their particlesize and zeta potential were187.8nm and+33.4mV, respectively.CS/CMCS-nanogels,(CS/CMCS-NGs) were prepared by polyelectrolyte complexation method and their average particle size and zeta were202.4nm and-40.7mV, respectively. The transmission electron microscope images showed thatCS-NGs and CS/CMCS-NGs were all spherical. CS-NGs and CS/CMCS-NGsexhibited low hemolysis rate (﹤5%) and BSA adsorption rate. MTT assay showedthat CS-NGs and CS/CMCS-NGs were nontoxic to MEFs, HUVEC, Caco-2and toxicto MCF-7, when their concentration reached1000μg/mL.Doxorubicin hydrochloride (DOX), a model anticancer drug, was loaded inCS-NGs (DOX:CS-NGs) and CS/CMCS-NGs (DOX:CS/CMCS-NGs). The particlesize, zeta potential, loading efficiency and loading content of DOX:CS-NGs were209.6nm,32.8mv,35.43%and14.9%, respectively. The particle size, zeta potential,loading efficiency and loading content of DOX:CS/CMCS-NGs were279.3nm and-33.8mv,72.87%and21.4%, respectively. Drug release of DOX:CS-NGs andDOX:CS/CMCS-NGs was studied in various pH media with different enzyme,simulating the complete GI environment by the dynamic dialysis method, the resultsindicated that the DOX:CS/CMCS-NGs exhibited favorable gastric acid tolerance andsustained-release behavior in simulating small intestinal fluid. Ex vivo intestinaladhesion and permeation results showed that DOX:CS/CMCS-NGs exhibit highefficiency in enhancing absorption of DOX throughout the entire small intestine. Theapparent permeability coefficient (Papp) and mucoadhesive capacity were1-3.2folds and1.1-2.4folds than that of DOX:CS-NGs.Different formulations were administered to the rats: DOX aqueous solution,DOX:CS/CMCS-NGs and DOX:CS-NGs were given orally. DOX were administeredby intravenous injection and used as control. Pharmacokinetics and tissue distributionresults showed that DOX:CS/CMCS-NGs exhibited the highest absolutebioavailability (42%), nearly6-folds and1.75folds higher than those obtained byDOX and DOX:CS-NGs. Organs were excised at24h after administration ofDOX:CS/CMCS-NGs to rats. Liver, spleen and lung in DOX:CS/CMCS-NGs showedsignificantly higher DOX level compare with other groups, demonstrating thatDOX:CS/CMCS-NGs could significantly increase circulation time of DOX in body. DOX (i.v.), DOX:CS/CMCS-NPs (oral), or CS/CMCS-NPs (oral) were administeredto relevant groups on day1and on day15. At the end of25th day, animals wereweighted, euthanatized and the hearts and kidneys were dissected and weighed.Biochemical parameters of hearts and kidneys were tested. The results showed thatoral DOX:CS/CMCS-NGs can significantly reduce the heart and renal toxicity ofDOX.The results of uptake of Cy3-CS/FITC-CMCS-NGs by Caco-2cells showed that theCS/CMCS-NGs could be likely internalized via active endocytosis particularlythrough clathrin-mediated pathway, which enhanced transcellular transport of DOX insmall intestine. The Ca2+binding capacity of CS, CMCS and CMCS/CS-NGs wastested at different pH values simulating the GI tract environment. CMCS exhibitedexcellent Ca2+binding capacity compared to CS. And, the3D structure of nanogelfurther strengthened this binding capacity which was at least4-fold and1.5-foldhigher than that of CS and CMCS at either pH value, respectively. These resultsdemonstrated that CS/CMCS-NGs had a strong binding ability of Ca2+. Paracellulartransport mechanism of DOX:CS/CMCS-NGs was studied by Caco-2cellsmonolayers model. The results showed that CS and CMCS displayed a transient andreversible decrease in transepithelial electrical resistance (TEER), which couldfacilitate paracellular transport of drugs into systemic circulation. The extents inTEER reduction produced by CS were decreased with pH value increasing. But theextents in TEER reduction produced by CMCS were increased with pH valueincreasing. The introduction of Ca2+into DOX:CS/CMCS-NGs obviously inhibitedthe extents of TEER reduction at either pH value. Above results demonstrated that thesynergistic effect of CS and CMCS in NGs promoted paracellular permeation of DOX.In the duodenum (pH <7), protonated CS in NGs enhanced paracelluar transport ofDOX by TJs opening. Meanwhile, the CMCS in NGs deprived Ca2+from AJs byfavor of carboxylic groups and further promoted the TJs disruption. In the jejunumand ileum (pH>7), CS in NGs was deprotonated while the CMCS in NGs still couldguarantee continuous and enhancing drug absorption. Nanogels immobilized multilayer alginate beads (NGs-M-ALG-Beads) wereprepared by immobilization of DOX:CS/CMCS-NGs in the core and layers of themultilayer sodium alginate beads. Optimization of CMs-MALG-Beads was completedusing response surface methodology, which is a statistical and mathematical methodused to fit the experimental data to the model for optimization processes. The resultsshowed that the DOX loading efficiency of NGs-M-ALG-Beads had a correlationwith the concentration of ALG, CaCl2and DOX:CS/CMCS-NGs and was not relatedto the number of layers. the highest DOX loading efficiency were97.21%, which wasobtained at ALG3.44(%), CaCl23.23(%) and DOX:CS/CMCS-NGs0.3(%),respectively. The swelling characteristic and drug release results indicated that4-layerNGs-M-ALG-Beads possessed favorable gastric acid tolerance (the swelling rate=5%, the cumulative drug release rate=3.8%). In small intestine, the intactDOX:CS/CMCS-CMs were able to rapidly release from NGs-M-ALG-Beads with thedissolution of ALG matrix. Ex vivo intestinal mucoadhesive and permeation showedthat CMs-M-ALG-Beads exhibited continued growth for Pappvalues of DOX, whichwas1.071.15folds and1.281.38folds higher than DOX:CS:CMCS-CMs in ratjejunum and ileum, respectively, demonstrating that NGs-M-ALG-Beads were able toenhance the absorption of DOX by controlled releasing DOX:CS/CMCS-CMs andprolonging the contact time between the DOX:CS/CMCS-CMs and small intestinalmucosa.Aboce results obtained in the present work implied that CS/CMCS-NGs had greatpotential to be applied as safe and effective oral drug delivery. |
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