| Objective:To investigate the mechanisms of targeted delivery of FA-CS-LZ-NPs in vitro,folate acid modified chitosan nonoparticles loaded with ligustrazine（FA-CS-LZ-NPs）was prepared and investigated for in vitro study.Methods:1. Chitosan was degradated by hydrogen peroxide oxidation at60℃. The relativeviscosities were measured with Ostwald viscometer and the relative viscosities valueswere used to calculate average molecular weights of Chitosan from differentdepolymerized samples with Mark–Houwink’s equation. Infrared absorption spectrawas used to analyze the chemical composition of chitosan and its degradation.2. Folate modified chitosan was prepared by the conjugation of the activated folateester with the amine group on the chitosan. The conjugation had been analyzed byinfrared spectroscopy and nuclear magnetic resonance spectroscopy. The number offolates in each chitosan molecule was measured by UV spectrophotometry.3. FA-CS-LZ-NPs were prepared using ionic gelation method to conjugatedifferent coupled number of folate modified chitosan of different molecular weight. Theencapsulation efficiency （EE）, loading capacity （LC） and the cumulative release amountof LZ-CS-NPs were detected by HPLC. The FA-CS-LZ-NPs were characterized bytransmission electron microscopy and photon correlation spectroscopy.4. Measure the cytotoxic activity of FA-CS-LZ-NPs by MTT assay. Two cell lines,a human breast cancer cell line MCF-7overexpressed with folate receptor and A549, ahuman lung adenocarcinoma epithelial cell line which lacks folate receptor were used inthis study. The two cell lines were incubated with FA-CS-LZ-NPs and the nonoparticles without ligustrazine with different concentrations respectively. Non-toxic dosage ofFA-CS-LZ-NPs was measured with this measurement （Dosage for95%cells viability）.5. HPLC assay was used to determine intracellular concentration of ligustrazine incell exposed to FA-CS-LZ-NPs for different hours. FA-CS-LZ-NPs uptake by two celllines were visualized by laser confocal microscopy. MCF-7cells were incubated withnon-toxic dosage of FA-CS-LZ-NPs, chitosan nonoparticles loaded with ligustrazine（CS-LZ-NPs）, ligustrazine solution （LZ） were defined as control groups,FA-CS-LZ-NPs with A549cells was defined as negative control group.6. The mechanism of FA-CS-LZ-NPs targeted delivery in vitro was detected withsaturated receptor assay. MCF-7cells were treated with folate solution ofFA-CS-LZ-NPs and FA-CS-LZ-NPs respectively. Negative control group was A549cells treated with folate solution of FA-CS-LZ-NPs and FA-CS-LZ-NPs. HPLC assaywas used to determine intracellular concentration of ligustrazine in cell exposued toFA-CS-LZ-NPs for4hours.Results:1. The molecular weight of chitosan depend on the degradation time. When thereaction time lasted to2hours, the molecular weight of chitosan was50kDa, and thechemical structure didn’t change, but the β-glucosidic bond was collapsed.2. The IR spectrum of folate acid modified chitosan was compared with that offolate acid and chitosan, there is a new IR band at1560cm-1, which represent amidelinkage. The1H NMR spectrum of folate acid modified chitosan and chitosan showedthat the peaks were enhanced to about3.26ppm and3.72ppm. The appearance of thesepeaks confirmed a successful conjugation of folate acid with chitosan. When theconjugation rate of folate acid and chitosan was170:1（mol/mol）, the number of folatesin each chitosan （molecular weight5×104Da） was34.3. FA-CS-LZ-NPs were prepared with molecular weight at5kDa and coupledwith22modified folate in each chitosan molecule. The average particle size ofFA-CS-LZ-NPs was182.7±0.56nm, the EE and LC were59.6±0.23%and15.3±0.16%.The delivery system reached release equilibrium after released in vitro for3days.Thecumulative release proportion was about95%. The released degree was faster in themetaacid medium（pH6.5） than the neutral medium（pH7.4）.4. FA-CS-LZ-NPs’ non-toxic dosage for MCF-7and A549cell lines were0.25mg·mL-1(the concentration of ligustrazine is50μg·mL-1). The nonoparticleswithout drugs were non-toxic for the two cell lines in the concentrations’ range. 5. Intracellular ligustrazine was45.47±0.32μg·mL-1for4hours’ incubation andthe concentration was saturate for MCF-7cells, which was significant higher than theCS-LZ-NPs group (22.10±1.02μg·mL-1),LZ group(4.57±0.76μg·mL-1),and the negativecontrol group of FA-CS-LZ-NPs for A549cells(20.10±0.92·mL-1). There was a highfluorescence intensity of ligustrazine in MCF-7cells treated with FA-CS-LZ-NPs bylaser confocal microscopy.6. The intracellular concentration of ligustrazine for MCF-7cells incubated withfolate solution and FA-CS-LZ-NPs group were8.00±0.40μg·mL-1,45.47±0.32μg·mL-1,63.00±3.44μg·mL-1, the MCF-7cells exposed to single FA-CS-LZ-NPs group were4.30±0.40μg·mL-1,26.47±0.86μg·mL-1,43.2±2.82μg·mL-1. MCF-7cells could noteffectively uptake ligustrazine with folate solution by a significant difference of twogroups （p<0.01）. The negative control group of folate solution with FA-CS-LZ-NPs andwith FA-CS-LZ-NPs for A549cells had no significant difference. Folate solution didnot impact A549cells on uptake nonoparticles.Conclusions:In this study, a simple process has been developed to synthesize folate modifiedchitosan, which could be selectively absorbed by folate receptor. The FA-CS-LZ-NPshave suitable diameter, high encapsulation efficiency and loading capacity, uniformspherical morphology,slow release in vitro. The FA-CS-LZ-NPs could successfullytarget MCF-7cells overexpressing the folate receptor, which is mediated by folatereceptor. |
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