Functionalization of Halloysite Nanotubes via Grafting of Dendrimer for Efficient Intracellular Delivery of siRNA

Here, polyamidoamine grafted halloysite nanotubes (PAMAM-<i>g</i>-HNTs) were synthesized for loading of siRNA in order to intracellular delivery of siRNA and treat of breast cancer via gene therapy. The successful grafting of PAMAM on HNTs was confirmed by various analytical methods. The size, zeta potential, and grafting ratio of PAMAM-<i>g</i>-HNTs is ∼206.2 nm, +19.8 mV, and 3.04%, respectively. PAMAM-<i>g</i>-HNTs showed good cytocompatibility toward HUVECs (84.7%) and MCF-7 cells (82.3%) even at high concentration of 100 μg/mL. PAMAM-<i>g</i>-HNTs/siRNA exhibited enhanced cellular uptake efficiency of 94.3% compared with Lipofectamine 2000 (Lipo2000)/siRNA (83.6%). PAMAM-<i>g</i>-HNTs/small interfering RNA-vascular endothelial growth factor (siVEGF) led to 78.0% knockdown of cellular VEGF mRNA and induced 33.6% apoptosis in the MCF-7 cells, which is also much higher than that of Lipo2000/siVEGF. In vivo anti-cancer results demonstrated that PAMAM-<i>g</i>-HNTs/siVEGF treated 4T1-bearing mice showed enhanced anti-cancer efficacy than Lipo2000/siVEGF group. Also, the nanocarrier system showed negligible toxic effects toward the major organs of mice. In vivo fluorescence imaging studies showed that there is a slight decrease in the fluorescence signal of PAMAM-<i>g</i>-HNTs/cy5-siVEGF after 72 h post-injection. Therefore, PAMAM-<i>g</i>-HNTs show promising application as novel nanovectors for siRNA delivery and gene therapy of cancer

Effect of carnosine on mitochondria membrane potentials of spleen lymphocytes in restraint-stressed mice.

<p>Spleen lymphocyte samples containing 5×10⁵ cells were stained with rhodamine-123. After incubation and wash, cells were resuspended in PBS. <i>ΔΨm</i> was determined using a flow cytometer. Data represented mean ± S.E.M. obtained from 10 animals in each group. The significance of differences from normal group is at ^##p<0.01, and from model group at ^*p<0.05, ^**p<0.01.</p

<i>In vitro</i> effect of carnosine on the number of spleen lymphocytes (including NK cells) treated with corticosterone.

<p>Carnosine had no tendency to inhibit the reduction of total lymphocytes (A) and NK cells (B) induced by corticosterone <i>in vitro</i>. Data represented mean ± S.E.M. of triplicate assays. The significance of differences from untreated cells is at ^##p<0.01.</p

<i>In vivo</i> and <i>in vitro</i> effects of carnosine on the proliferative response (stimulation index) of spleen lymphocytes stimulated by ConA.

<p>(A) Carnosine increased ConA-stimulated lymphocyte proliferation <i>in vivo</i>. Data represented mean ± S.E.M. obtained from 10 animals in each group. The significance of differences from normal is at ^##p<0.01, and from model at ^*p<0.05, ^**p<0.01. (B) Carnosine increased ConA-stimulated lymphocyte proliferation by treatment <i>in vitro</i>. Data represented mean ± S.E.M. of triplicate assays. The significance of differences from normal is at ^##p<0.01, and from model at ^*p<0.05, ^**p<0.01.</p

Effect of carnosine on gene expressions of GR, Bax and Bcl-2 mRNAs in spleen lymphocytes of restraint-stressed mice.

<p>RT-PCR was employed to determine the effect of carnosine on the expression of GR, Bax and Bcl-2 mRNAs. Relative expression values for each target gene were expressed as a ratio of target gene expression level to 18S expression level. Data represented mean ± S.E.M. obtained from 10 animals in each group. The significance of differences from normal is at ^##p<0.01, and from model at ^*p<0.05, ^**p<0.01.</p

Effect of carnosine on lymphocyte apoptosis confirmed by TUNEL assay.

<p>(A) The TUNEL assay was carried out by One Step TUNEL Apoptosis Assay Kit. The images of TUNEL positive cells were captured by a fluorescence microscope (200×). (B) Quantitative result of TUNEL assay was analyzed. Data represented mean ± S.E.M. obtained from 10 animals in each group. The significance of differences from normal is at ^##p<0.01, and from model at ^**p<0.01.</p

Effect of carnosine on spleen index and spleen lymphocyte number in restraint-stressed mice.

<p>On the 7^th day of the experiment, all mice except normal control were physically restrained in a 50 ml polypropylene centrifuge tube with holes for 18 h. Spleens were collected from mice and weighted. Spleen index was expressed as spleen weight (g) over body weight (g). Spleens were disrupted with a grinder. After debris separation and erythrocytes lysis, spleen lymphocytes were obtained and counted. The results represent mean ± S.E.M. obtained from 10 animals in each group. The significance of differences from normal group is at</p>##<p>p<0.01, and from model group at</p>*<p>p<0.05,</p>**<p>p<0.01, as determined by ANOVA analysis.</p

Effects of carnosine on brain histamine concentration (pmol/g tissue) in restraint-stressed mice.

<p>Brain regions including cerebral cortex, hippocampus and hypothalamus were rapidly dissected and homogenized in PBS containing 3% perchloric acid. Histamine level in brain regions was determined by HPLC-ECD. The results represent mean ± S.E.M. obtained from 10 animals in each group. The significance of differences from normal group was at</p>##<p>p<0.01, and model group at</p>**<p>p<0.01, as determined by ANOVA analysis.</p

Effect of carnosine on cytochrome c (Cyt c) contents in mitochondria and cytoplasm in restraint-stressed mice.

<p>Spleen lymphocytes samples containing (2×10⁶) were gently lysed with lysis buffer. Lysates were centrifuged to obtain supernatants (cytosolic extracts free of mitochondria) and the pellets (fraction that contains mitochondria). These two fractions were adjusted to 2 mg protein/ml in the buffer. Contents of cytochrome c were determined using difference spectra at the wavelength pairs of 550∼535, 554∼540 and 563∼577 respectively. The results represent mean ± S.E.M. obtained from 10 animals in each group. The significance of differences from normal group is at</p>##<p>p<0.01, and from model group at</p>*<p>p<0.05,</p>**<p>p<0.01, as determined by ANOVA analysis.</p

Effect of carnosine on plasma corticosterone level of restraint-stressed mice.

<p>Blood containing heparin was centrifuged to obtain plasma. Corticosterone was extracted from plasma by acetic ether, and its content was determined by HPLC with a UV detector at 254 nm. Data represented mean ± S.E.M. obtained from 10 animals in each group. The significance of differences from normal group is at ^##p<0.01, and from model group at ^**p<0.01.</p