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  • br The hydrodynamic diameters of the

    2019-10-29

    
    The hydrodynamic diameters of the Ap-Dox complex was 5.8 nm. The nuclear delivery efficiency is greatly dependent on the particle size. It was reported that NPs with size smaller than 39 nm could cross nu-clear pore complex (NPC) to enter the cell nucleus [33]. Therefore, it is probable that Ap-Dox complex enter the cell nuclear with the suitable size.
    3.2. Physiochemical properties of Lip(Ap-Dox)
    From the Fig. 3A, it was observed that the free aptamer AS1411 clearly migrated through the gel. However, the Lip(Ap) (mliposome/m Aptamer 1411 = 125) displayed a clear and bright band at the origin site and no band at the free Ap site, indicating that aptamer AS1411 was completely encapsulated in the liposome at this mass ratio. Therefore, at the optimized ratio Ap-Dox complex was successfully encapsulated in liposome with an efficient loading of Dox (LC = 81.2% (μg/mg)). Lip (Ap(mut)-Dox) encapsulated Ap(mut)-Dox with a similar LC (LC = 80.5% (μg/mg)).
    The mean particle sizes of Lip(Ap(mut)-Dox) and Lip(Ap-Dox) were 135.4 nm and 128.6 nm, respectively, which was suitable for the ac-cumulation at tumor site and at the same time less recognizable from the RES with a consequent longer blood circulation [34]. In addition, the zeta potentials were detected as -5.6 mV and -6.1 mV for Lip(Apt (mut)-Dox) and Lip(Apt-Dox). The TEM images revealed that the mor-phology of liposomes were spherical and had a smooth surface (Fig. 3B).
    Drug release can be considered as a surrogate of the stability of the bilayer against the loss of the entrapped drug. The in vitro drug release study of Lip(Ap(mut)-Dox) and Lip(Ap-Dox) performed at various pH values (pH 7.4, 6.5, 5.0) displayed the enhanced drug release rate at acidic condition (Fig.3C).
    3.3. The nucleolin immunofluorescence and immunochemistry
    It is reported that nucleolin is found in the cytoplasm of various types of cancer cells, whereas it is generally not found outside the nu-cleus of normal Belinostat (PXD101) [35]. The immunofluorescence and im-munochemistry were carried out to investigate the location of nucleolin in MCF-7/Adr and MCF-10A cells. In Fig. 4A, it was observed that the fluorescent of anti-nucleolin antibody mainly distributed in nucleus in normal breast (MCF-10A) cells, whereas in MCF-7/Adr cells, the fluorescent distributed both in nucleus and cytoplasm. The distribution of nucleolin in immunochemistry was essentially in agreement with the immunofluorescence, which demonstrated that the nucleolin expressed not only in nucleus, but also in cytoplasm in MCF-7/Adr cells. In principle, nucleolin can be used to deliver a vehicle into cancer cells nucleus.
    Fig. 4. Subcellular localization of nucleolin in MCF-10A and MCF-7/Adr cells. (A) The dis-tribution of nucleolin was determined by im-munofluorescence. Shown are immuno-fluorescence staining using monoclonal antibody to nucleolin (green), nuclear stain with DAPI (blue), and merged images (Scale bar =10 μm). (B) The distribution of nucleolin was determined by immunochemistry (Scale bar =20 μm). (For interpretation of the refer-ences to colour in this figure legend, the reader is referred to the web version of this article).
    Fig. 5. Intracellular trafficking of liposomes to the nucleus was monitored by confocal fluor-escence microscope. (A) MCF-7/Adr cells treatment with Lip(Ap(mut)-FAM) and Lip(Ap-FAM) for 2 h. (B) Nucleus uptake of Lip(Ap (mut)-FAM) and Lip(Ap-FAM) was determined by flow cytometry (Statistical significance: *P < 0.05, All the data are represented as mean ± SD).
    3.4. The distribution and nuclear uptake of liposomes
    On the basis of the fluorescence quenching of Dox by intercalating into the GC duplex, an additional marker FAM linked on aptamer AS1411 and aptamer(mut) was used as fluorescent probe to investigate intracellular distribution of liposome. Then confocal laser scanning microscopy (CLSM) measurements were showed in Fig. 5A. It was found that the fluorescence of Lip(Ap-FAM) was mainly localized in nucleus for 2 h, indicating that Ap-FAM can enter the nucleus. In con-trast, the fluorescence of Lip(Ap(mut)-FAM) was mainly localized in the cytoplasm, as indicated by the FAM fluorescence signal outside the nucleus. By taking advantage of the shuttling protein nucleolin, ap-tamer AS1411 was transferred to the nucleus, suggesting the Ap-Dox complex could be delivered to the nucleus bypassing the efflux-medi-ated MDR effect. In addition, the fluorescent intensity of Lip(Ap(mut)-FAM) and Lip(Ap-FAM) in the nuclear was exploited using flow cyto-metry. The flow cytometry study results showed that the nuclear as-similation of Lip(Ap-FAM) was 2.02-fold greater than that of Lip(Ap (mut)-FAM) in MCF-7/Adr cells after 2 h of exposure (P < 0.05) (Fig. 5B).