| Background:Helicobacter Pylori is a key etiological factor in gastric and duodenal ulcers and gastric adenocarcinoma, one of the most common forms of cancer in humans. A widely recommended regimen is the triple therapy. The short residence time and rapid destruction of antimicrobial drugs in the stomach acid(p H 1.2) caused an insufficient antimicrobial concentration, thus limiting the application of the ordinary preparations. The oral nanoparticle drug delivery system could effectively inhibit the destruction of stomach acid and prolong the residence time of the drugs to strength the therapeutic efficacy of the antimicrobial drugs. Nevertheless, higher specific and controllable delivery system for the treatment of Helicobacter Pylori infection remains a challenge.Objective:Considering the excellent mucoadhesive properties, biocompatibility and easily modification of chitosan, we designed and synthized two series of chitosan derivativesUACs-1/2/3 and UECs-1/2/3 by conjugating C2 amino group and C6 hydroxyl group of chitosan with ureido group through various fatty chains, hoping that we can get a potential targeting materials for the treatment of Helicobacter Pylori in vitro and in vivo.Method:(1) The intermediate products U1/2/3 were prepared by reacting urea with hydantoic acid,6-aminocaproic acid or 12-aminododecanoic acid, respectively. Then, chitosan derivatives UACs-1/2/3 and UECs-1/2/3 was obtained through modifying the amino group and hydroxyl group of chitosan with intermediate products U1/2/3. The structure of all synthesized chitosan derivatives was confirmed by MS, 1H NMR and FTIR. The substitution degree of ureido group was calculated by 1H NMR.(2) The chitosan derivatives/TPP nanoparticles were prepared using the ionic gelationmethod with chitosan derivatives and cross-linking agent TPP. The process of Chitosan derivatives/TPP weight ratio and chitosan derivatives concentration was optimized. The particles size, zeta potential and morphology of the chitosan derivatives/TPP nanoparticles under different p H values were obversed. And the surface element distribution characteries of chitosan derivatives/TPP nanoparticles were analyzed by X-ray photoelectron spectroscopy(XPS). The cytotoxicity of the chitosan derivatives/TPP nanoparticles was evaluated by MTT. Using amoxicillin as model drug, the drug loaded nanoparticles were prepared and the drug loading efficiency was characterized. The release profiles of amoxicillin from drug-loaded nanoparticles were evaluated at different p H values.(3) The in vitro antibacterial activity against Helicobacter Pylori of amoxicillin-loadednanoparticles was quantified by turbidimetric method and the effect of urea addition on antibactericidal capability was evaluated. The specific Helicobacter Pylori uptake of FITC-labeled UECs-3/TPP nanoparticles under different p H values was tested by flow cytometry. The effect of urea on the uptake of FITC-labeled UECs-3/TPP nanoparticles was also evaluated by flow cytometry and confocal laser scanning microscopy. The in vivo antimicrobial activity was evaluated through Helicobacter Pylori-infected Balb/C mice model and H&E staining were performed. Thedistribution of gastrointestinal tract of the IR783-labeled UECs-3/TPP nanoparticles were also observed in the nude mice.Results:(1) Two series of chitosan derivatives UACs-1/2/3 and UECs-1/2/3 were synthetized. Thestructure of 6 chitosan derivatives were consistent with that of targeting onesaccording to 1H NMR, MS and FTIR. The degree of substitution of the ureido groupwas calculated and listed as following: UACs-1(6.56% ± 0.60%), UACs-2(3.92% ±0.47%), UACs-3(2.69% ± 0.31%), UECs-1(6.81% ± 0.62%), UECs-2(5.53% ±0.56%), UECs-3(2.92% ± 0.47%).(2) The results of particle size and zeta potential showed that the optimized conditions topreapare chitosan derivatives/TPP nanoparticles were chitosan derivatives/TPP at3:1 and chitosan derivatives concentration at 1.8 mg/m L. All of the optimized chitosanderivatives/TPP nanoparticles had a particle size of 123.2 nm- 268.6 nm and positivezeta potential of 18.07 m V- 30.53 m V. The XPS results showed that the ureido groupwas on the surface of the nanoparticles. The MTT results indicated that the chitosanderivatives/TPP nanoparticles were sucure enough and the drug loading efficiency ofthe nanopartilces were among 21.7%- 28.7%. The drug release profiles of theamoxicillin-loaded nanoparticles exhibited favorable p H-sensitive characteristics,which could hinder the release of amoxicillin at p H 1.2 and enable a more effectiverelease at p H 6.0 and p H 7.0.(3) The in vitro antibacterial experiment demonstrated that, compared withAMX-Cs-3/TPP nanoparticles, a more specific and effective Helicobacter Pylorigrowth inhibition of AMX-UACs-3/TPP nanoparticles and AMX-UECs-3/TPPnanoparticles was observed. Moreover, the growth inhibition of Helicobacter Pyloritreated with AMX-UACs-3/TPP nanoparticles and AMX-UECs-3/TPP nanoparticlesexhibited a decline with the addition of urea. The results of Helicobacter Pylori uptakeshowed that, with the p H values decreased from 7.4 to 4.0, the Ure I channels wouldgradually open and the uptake of FITC-UACs-3/TPP nanoparticles increased(MFIincreased from 51.6 to 114.3). The uptake of FITC-UACs-3/TPP nanoparticlesdecreased after the addition of urea(MFI decreased from 114.3 to 91.8(p H 4.0) and 91.7 to76.1(p H5.5)). The low temperature also reduced the uptake of the FITC-UACs-3/TPP nanoparticles with the ATP enzyme activity fading. The results of in vivo antimicrobial evaluation showed that the antibacterial activity of AMX-UECs-3/TPP nanoparticles exhibited a superior activity than AMX-Cs/TPP nanoparticles and AMX-UACs-3 nanoparticles(p < 0.05). H&E staining verified that AMX-UECs-3/TPP nanoparticles could successfully repair gastric mucosal damage caused by Helicobacter Pylori. With the in vivo imaging system, the prepared IR783-UECs-3/TPP nanoparticles were able to retain in stomach more than 6 h, which facilitates the nanoparticles to penetrate the gastric mucosa layer to arrive the area of gastric epithelial cells and finally target to clear the Helicobacter Pylori.Conclusion:In this thesis, two series of chitosan derivatives including UACs-1/2/3 and UECs-1/2/3 were constructed through introducing ureido group to C2 amino group and C6 hydroxyl group of chitosan to preapare the chitosan derivatives/TPP nanoparticiels. The in vitro and in vivo antibacterial activity against Helicobacter Pylori was evaluated. The results showed that the prepared chitosan derivatives/TPP nanoparticles had the characteristic of p H sensitivity, low cytotoxicity and biocompatibility. Among them, AMX-UECs-3/TPP nanoparticles exhibited more specific and effective Helicobacter Pylori growth inhibition, which can be used as a new promising drug delivery system targeted to Helicobacter Pylori. |
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