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

Here, polyamidoamine grafted halloysite nanotubes (PAMAM- g-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- g-HNTs is ∼206.2 nm, +19.8 mV, and 3.04%, respectively. PAMAM- g-HNTs showed good cytocompatibility toward HUVECs (84.7%) and MCF-7 cells (82.3%) even at high concentration of 100 µg/mL. PAMAM- g-HNTs/siRNA exhibited enhanced cellular uptake efficiency of 94.3% compared with Lipofectamine 2000 (Lipo2000)/siRNA (83.6%). PAMAM- g-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- g-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- g-HNTs/cy5-siVEGF after 72 h post-injection. Therefore, PAMAM- g-HNTs show promising application as novel nanovectors for siRNA delivery and gene therapy of cancer.

In vitro and in vivo toxicity evaluation of halloysite nanotubes.

Because of their outstanding properties, increasing numbers of research studies and emerging applications for manufacturing products are currently in progress for halloysite nanotubes (HNTs). Therefore, the impact of HNTs on the environment and human health should be taken into consideration. In order to clearly show the cell uptake of HNTs and the biodistribution of HNTs in zebrafish, HNTs are labeled with fluorescein isothiocyanate (FITC-HNTs). The cytotoxicity assays showed that the cell viabilities of human umbilical vein endothelial cells (HUVECs) and human breast adenocarcinoma (MCF-7) cells were above 60% after being treated with different concentrations of HNTs (2.5-200 µg mL-1) for 72 h. Confocal laser scanning microscopy (CLSM) results showed the uptake of HNTs by HUVECs and MCF-7 cells. The in vivo toxicity of HNTs was then investigated in the early development of zebrafish embryos. The percent survival of zebrafish embryos and larvae showed no significant changes at different developmental stages (24, 48, 72, 96, and 120 hpf) when treated with various concentrations of HNTs (0.25-10 mg mL-1). Besides, HNTs could promote the hatchability of zebrafish embryos and did not affect the morphological development of zebrafish at a concentration of ≤25 mg mL-1. HNTs could also be ingested by zebrafish larvae and accumulated predominantly in the gastrointestinal tract. The fluorescence intensity of FITC-HNTs decreased gradually with time, which suggested that HNTs could be excreted by zebrafish larvae through the gastrointestinal metabolism. Therefore, it can be concluded that HNTs are relatively biocompatible nanomaterials, which can be utilized in many fields.

Large-area assembly of halloysite nanotubes for enhancing the capture of tumor cells.

Here, polystyrene sulfonate sodium (PSS) modified Halloysite nanotubes (HNTs) were self-assembled into a patterned coating on a glass substrate with ordered nanotube arrays in a slit-like confined space. The microstructure of the formed patterned HNTs coating was investigated. The formed strips are more regular and almost parallel to each other with an increase in HNTs concentration. The HNTs coating formed from the 2% PSS-HNTs dispersion has the maximum nanotube alignment degree. The patterned HNTs coating was employed to capture tumor cells. The tumor cells can be captured by the HNTs coating effectively compared with a smooth glass surface due to the enhanced topographic interactions between the HNTs coating and cancer cells. The HNTs coating prepared from the 2% PSS-HNTs dispersion has the highest capture yield which is due to the ordered nanotube arrangement and the appropriate surface roughness. The HNTs coating was further conjugated with anti-EpCAM, which leads to the capture yield of MCF-7 cells reaching 92% within 3 h. The HNTs coating can capture 8 MCF-7 cells from 1 mL artificial blood samples spiked with 10 MCF-7 cells, showing the promising applications of HNTs in clinical circulating tumor cell capture for early diagnosis and monitoring of cancer patients.

Effects of halloysite nanotubes on physical properties and cytocompatibility of alginate composite hydrogels.

Sodium alginate (SA)/halloysite nanotubes (HNTs) composite hydrogels were successfully prepared by solution blending and cross-linking with calcium ions. HNTs can improve the physical properties and cytocompatibility of composite hydrogels. The static and shear viscosity of SA/HNTs solution increase by the addition of HNTs. FTIR suggests the presence of hydrogen bond interactions between HNTs and SA. The crystal structure of HNTs is retained in the composites as showed by the X-ray diffraction result. A porous structure with pore size of 100-250µm is found in the hydrogels, which can provide a space for cell growth and migration. The compressive mechanical properties of composite hydrogels significantly increase compared to the pure SA hydrogel. The SA/HNTs composite hydrogels with 80% HNTs loading exhibit the compressive stress at 80% strain of 2.99MPa, while the stress at 80% strain of pure SA hydrogel is only 0.8MPa. The dynamic storage modulus of composite hydrogels also markedly increases with HNTs concentration. The differential scanning calorimetry endothermic peak area and swelling ratios in NaCl solution of the composite hydrogels decrease by the addition of HNTs. Preosteoblast (MC3T3-E1) culture results reveal that the SA/HNTs composites especially at relatively low HNTs loading show a significant increase in cells adhesion and proliferation compared to the pure SA hydrogel. All the results demonstrate that the SA/HNTs composite hydrogels show a promising application in bone tissue engineering.

Polyethyleneimine grafted short halloysite nanotubes for gene delivery.

Inorganic nanoparticles have attracted much attentions in gene delivery because of their desirable characteristics including low toxicity, well-controlled characteristics, high gene delivery efficiency, and multi-functionalities. Here, natural occurred halloysite nanotubes (HNTs) were developed as a novel non-viral gene vector. To increase the efficiency of endocytosis, HNTs were firstly shortened into an appropriate size (~200nm). Then polyethyleneimine (PEI) was grafted onto HNTs to bind green fluorescence protein (GFP) labeled pDNA. The structure and physical-chemical properties of PEI grafted HNTs (PEI-g-HNTs) were characterized by various methods. PEI-g-HNTs show lower cytotoxicity than PEI. PEI-g-HNTs are positively charged and can bind DNA tightly at designed N/P ratio from 5:1 to 40:1. PEI-g-HNTs/pDNA complexes show much higher transfection efficiency towards both 293T and HeLa cells compared with PEI/pDNA complexes at the equivalent N/P ratio. The transfection efficiencies of PEI-g-HNTs/pDNA complex towards HeLa cell can reach to 44.4% at N/P ratio of 20. PEI-g-HNTs/pDNA complexes possess a higher GFP protein expression than PEI/pDNA from simple western immunoblots. So, PEI-g-HNTs are potential gene vectors with good biocompatibility and high transfection efficiency, which have promising applications in cancer gene therapy.