Construction of Hyaluronic Tetrasaccharide Clusters Modified Polyamidoamine siRNA Delivery System
<p>Synthetic scheme of polyamidoamine (PAMAM)-gallic acid triethylene glycol (GATG)- hyaluronan tetrasaccharide clusters (HA4).</p> "> Figure 2
<p>(<b>a</b>) Agarose gel electrophoresis of PAMAM-3GATG-HA4/siRNA and PAMAM-6GATG-HA4/siRNA nanocomplexes at different N/P ratios. (<b>b</b>) Particle size and zeta potential of PAMAM-3GATG-HA4/siRNA and PAMAM-6GATG-HA4/siRNA nanocomplexes at different N/P ratios. siRNA concentration in nanocomplexes was 100 nM. (<b>c</b>) Transmission electron microscopy (TEM) images of PAMAM-3GATG-HA4/siRNA and PAMAM-6GATG-HA4/siRNA nanocomplexes. The scale was 2 μm in the original and 100 nm in the magnified image. (<b>d</b>) Serum stability of nanocomposites at 37 °C for 24 h. Results are expressed as mean ± SD (<span class="html-italic">n</span> = 3).</p> "> Figure 3
<p>(<b>a</b>) Effects of vectors PAMAM, PAMAM-3GATG-HA4 and PAMAM-6GATG-HA4 on cell viability within 48 h of MDA-MB-231 cells and MCF-7 cells. Results are expressed as mean ± SD (<span class="html-italic">n</span> = 4). (<b>b</b>) Uptake of nanocomplexes in MDA-MB-231 cells and MCF-7 cells by flow cytometry. FAM-siRNA concentration was 200 nM. In the competition experiments, pre-incubated with free HA was added for incubation. Results are expressed as mean ± SD (<span class="html-italic">n</span> = 3). (<b>c</b>) Laser confocal images used to observe uptake of nanocomplexes in MDA-MB-231 cells. FAM-siRNA concentration was 200 nM. Blue denotes the nucleus and †green denotes FAM-siRNA (scale 25 μm). (<b>d</b>) The lysosomal escape of siRNA after 0.5 and 2 h uptake of PAMAM-6GATG-HA4/siRNA nanocomplexes in MDA-MB-231 cells. FAM-siRNA concentration was 200 nM. Green denotes siRNA and red denotes lysosome (scale 25 μm). Statistical analysis was performed with one-way ANOVA and Bonferroni post-hoc testing with * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.005.</p> "> Figure 4
<p>The gene silencing efficacy of PAMAM-GATG-HA4 nanocomplexes analyzed by flow-cytometry and quantitative real-time polymerase chain reaction (qRT-PCR) in MDA-MB-231-GFP cells. The gene silencing efficacy without adding hyaluronan tetrasaccharide was analyzed (<b>a</b>) by flow-cytometry and (<b>b</b>) with adding hyaluronan tetrasaccharide analyzed by flow-cytometry. (<b>c</b>) The gene silencing efficacy without adding hyaluronan tetrasaccharide analyzed by qRT-PCR and (<b>d</b>) with adding hyaluronan tetrasaccharide analyzed by qRT-PCR. Statistical analysis was performed with one-way ANOVA and Bonferroni post-hoc testing with * <span class="html-italic">p</span> < 0.05 and *** <span class="html-italic">p</span> < 0.01. The “negative” represents the blank control group using MDA-MB-231 cells without GFP; the “control” means the MDA-MB-231-GFP cells were not treated with siGFP or nanocomplexes; and the “free” and “lipo” mean the MDA-MB-231-GFP cells were treated with free siGFP or lipofectamine 2000 containing siGFP respectively.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Synthesis of PAMAM-Gallic Acid-Triethylene Glycol (PAMAM-GATG)
2.3. Synthesis of PAMAM-GATG-Hyaluronan Tetrasaccharide Clusters (PAMAM-GATG-HA4)
2.4. Cell Culture
2.5. Preparation and Characterization of Nanocomplexes
2.6. Serum Stability Assay
2.7. Cell Viability Assay
2.8. Cellular Uptake in Different Cell Lines
2.9. Intracellular Trafficking and Endosomal Escape
2.10. Gene Silencing
2.11. Statistical Analysis
3. Results and Discussion
3.1. Synthesis and Characterization of PAMAM-GATG and PAMAM-GATG-HA4
3.2. Characterization of the Nanocomplexes
3.2.1. Gel Retardation Assay
3.2.2. Size and Surface Property of PAMAM-GATG-HA/siRNA Nanocomplexes
3.2.3. Serum Stability of PAMAM-GATG-HA/siRNA Nanocomplexes
3.3. Cytotoxicity Assay
3.4. Cellular Uptake
3.5. Intracellular Colocalization and Endosomal Escape
3.6. Gene Silencing
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Ma, Y.; Sha, M.; Cheng, S.; Yao, W.; Li, Z.; Qi, X.-R. Construction of Hyaluronic Tetrasaccharide Clusters Modified Polyamidoamine siRNA Delivery System. Nanomaterials 2018, 8, 433. https://doi.org/10.3390/nano8060433
Ma Y, Sha M, Cheng S, Yao W, Li Z, Qi X-R. Construction of Hyaluronic Tetrasaccharide Clusters Modified Polyamidoamine siRNA Delivery System. Nanomaterials. 2018; 8(6):433. https://doi.org/10.3390/nano8060433
Chicago/Turabian StyleMa, Yingcong, Meng Sha, Shixuan Cheng, Wang Yao, Zhongjun Li, and Xian-Rong Qi. 2018. "Construction of Hyaluronic Tetrasaccharide Clusters Modified Polyamidoamine siRNA Delivery System" Nanomaterials 8, no. 6: 433. https://doi.org/10.3390/nano8060433