Arginine functionalized hydroxyapatite nanoparticles and its bioactivity for gene delivery

In order to further improve the transfection efficiency of hydroxyapatite nanoparticle （HAp）, arginine functionalized hydroxyapatite （HAp/Arg） was synthesized by hydrothermal synthesis. The morphology, crystallite size and zeta potential of the HAp/Arg were characterized by transmission electron microscopy （TEM）, atomic force microscopy （AFM） and zeta potential analyzer. The loading and protecting properties of HAp/Arg to DNA were tested by electrophoresis. Its cytotoxicity was also measured in Hela cells and HAEC cells by MTT and LDH, and its transfection efficiency was examined by fluorescence microscope and flow cytometry. The results reveal that HAp/Arg is short rod-like and nano single crystal, the mean diameter is 50-90 nm and zeta potential is 35.8 mV at pH 7.4. HAp/Arg to DNA can be condensed by electrostatic effect and protect DNA against degradation in DNase I, and shows high transfection efficiency without cytotoxicity. These results suggest that HAp/Arg can be a promising alternative as a novel gene delivery system.

Effect of hydroxyapatite nanoparticles on the growth and p53/c-Myc protein expression of implanted hepatic VX_2 tumor in rabbits by intravenous injection

AIM： To evaluate the effect of hydroxyapatite nano- particles （Nano HAP） by intravenous injection on the inhibition of implanted hepatic VX2 tumor growth in rabbits and cell p53/c-Myc protein expression. METHODS： 60 hepatic VX2 tumor-bearing rabbits was randomly divided into five groups. Nano HAP collosol 20 mglkg, 40 mg/kg, 5-FU solutions 20 mg/mL, mixed liquor of 5-FU solution 20 mg/mL and Nano HAP collosol 20 mg/kg were infused by vein, normal saline conducted as the control. The general state, weight, liver function and gross tumor volume were detected dynamically. The expression of p53 and c-Myc gene protein in tumor tissue was detected by immunohistochemistry methods. RESULTS： The growth of implanted hepatic VX2 tumors was significantly inhibited in all therapy groups, 3 wk after the injection, the tumor control rates in Nano HAP collosol groups were 25.5% and 32.5% respectively, and the gross tumor volumes were obviously less than that of control group. （24.81 ± 5.17 and 22.73 ± 4.23 vs 33.32 ± 5.26, P 〈 0.05）. The tumor control rate of 5-FU group was 43.7% （18.74 4± 4.40 vs 33.32 ± 5.26, P 〈 0.05）, but the general state of the animals after injection aggravated; and the adverse reaction in the drug combination group obviously decreased. Due to the effect of Nano HAP, the positive expression of tumor associated the mutated p53 and c-Myc in tumor tissue was decreased obviously compared with the control group. CONCLUSION： Nano HAP has evident inhibitory action on rabbit implanted hepatic VX2 tumor in vivo, which may be the result of decreasing the expression of the mutated p53 and c-myc, and drug combination can obviously decrease the adverse reaction of 5-FU.

Morphologies of hydroxyapatite nanoparticles adjusted by organic additives in hydrothermal synthesis

Properties of hydroxyapatite （HA, Ca10（PO4）6（OH）2）, including bioactivity, biocompatibility, solubility and adsorption could be tailored over wide ranges by the control of particle composition, particle size and morphology. In order to satisfy various applications, well-crystallized pure HA nanoparticles were synthesized at moderate temperatures by hydrotherrnal synthesis, and HA nanoparticles with different lengths were obtained by adding organic additives. X-ray diffractometry （XRD） and Fourier transform infrared （FTIR） spectrometry were used to characterize these nanoparticles, and the morphologies of the HA particles were observed by transmission electron microscopy （TEM）. The results demonstrate that shorter rod-like HA particles can be prepared by adding cetyltrimethylammonium bromide （CTAB）, as the additive of CTAB can block the HA crystal growth along with c-axis. And whisker HA particles are obtained by adding ethylenediamine tetraacetic acid （EDTA）, since EDTA may have effect on the dissolution-repreeipitation process of HA.

Effect of Tb/Mg doping on composition and physical properties of hydroxyapatite nanoparticles for gene vector application

Transfection efficiency of hydroxyapatite nanoparticles(HAnps)is relative to the particle size,morphology,surface charge,surface modifier and so on.This study prepared HAnps with doped Tb/Mg by hydrothermal synthesis method(HTSM)and investigated the effects of different Tb/Mg contents on the morphology,particle size,surface charge,composition and cellular endocytosis of HAnps.The results showed that Mg-HAnps possessed better dispersion ability than Tb-HAnps.With increasing doping content of Tb/Mg-HAnps,the granularity of Tb-HAnps increased,while that of Mg-HAnps declined.Both particle size and zeta potential of Mg-HAnps were lower than those of Tb-HAnps.7.5%Mg-doping HAnps presented relatively uniform slender rod morphology with average size of30nm,while10%Mg-doping HAnps were prone to agglomeration.Moreover,Mg-HAnps-GFP(green fluorescent protein)endocytosed by MG63cells was dotted in the perinuclear region,while Tb-HAnps were more likely to aggregate.In conclusion,as gene vectors,Mg-HAnps showed enhanced properties compared to Tb-HAnps.

Effects of Hydroxyapatite Nanoparticles on Apoptosis and Invasion of Human Renal Cell Carcinoma 786-0 Cells

Renal cell carcinoma is the most common cancer of the kidney, and resistant to traditional therapies. The aim of this study is to investigate the effects of hydroxyapatite nanoparticles on human renal cell carcinoma 786-0 cells. Cell proliferation was assessed with an 3-（4,5-dimethylthiazol-2-yl） 2,5-diphenyltetrazolium bromide（MTT） staining kit. The apoptosis assay was assessed with an FITC Annexin V Apoptosis Detection Kit. Caspase-3 and caspase-12 were detected by immunocytochemical staining and semi-quantitative RT-PCR. Cell wound healing assay was used to ensure cell motility. Matrigel invasion assay was analysed via transwell chambers. Our results showed that hydroxyapatite nanoparticles significantly reduced cell proliferation, invasion and induced apoptosis of 786-0 cells. The inhibiting action may have relation with up-regulated caspase-12, leading the cells to apoptosis. This study suggests that hydroxyapatite nanoparticles may be an effective and delivery system for renal cell carcinoma therapy.

Effects of the Interaction between Hydroxyapatite Nanoparticles and Hepatoma Cells

To gain a better understanding of the anticancer effects of hydroxyapatite （HAP） nanoparticles in vivo and in vitro, the effects of the interaction of HAP nanoparticles with hepatoma cells were explored. HAP nanoparticles were prepared by homogeneous precipitation and characterized by laser particle analysis and transmission electron microscopy （TEM）. HAP nanoparticles were observed to be uniformly distributed, with rod-like shapes and diameters in the range of 42.1-87.1 nm. Overnight attached, suspended, and proliferating Bel-7402 cells were incubated with HAP nanoparticles. Inverted microscopy observation revealed that HAP nanoparticles with a cell membrane showed good adsorption. TEM demonstrated that HAP nanoparticles were present on the surface of cells, continuously taken up by cells through endocytosis, and transported in vesicles close to the nucleus. Fluorescence microscopy showed that the concentrations of intracellular Ca2＋ labeled with Fluo-3 calcium fluorescent probe were significantly enhanced. In addition, inverted microscopy observation revealed that suspended cells treated with HAP nanoparticles did not adhere to the culture bottle, resulting in cell death. After the overnight attached cells were treated with HAP nanoparticles for 96 h with increasing doses of HAP nanoparticles, inverted microscopy observation revealed that cell proliferation was slowed and ceU-ceU adhesion was weakened. Feulgen staining and image analysis indicated that the nuclear DNA content of the cells was markedly reduced, and argyrophilic nucleolar organizer region （AgNOR） staining and image analysis indicated that the number of AgNORs was significantly decreased. Therefore, hepatoma cells brought about the adsorption, uptake, transport and degradation of HAP nanoparticles. In addition, HAP nanoparticles affected hepatoma cells with regard to cell-cell adhesion, cell and extracellular matrix adhesion, and DNA and protein synthesis; thus inhibiting cell proliferation. This understanding of the effects of interaction between HAP nanoparticles and hepatoma cells is useful for further study of the anticancer mechanisms of HAP nanoparticles.

Poly(Vinyl Alcohol)/Collagen/Hydroxyapatite Nanoparticles Hybrid System Containing Yttrium-90 as a Potential Agent to Treat Osteosarcoma

Osteosarcoma is the most common primary malignant bone tumors, affecting mostly children, adolescents and young adults. This is an aggressive tumor that results in a high mortality rate and poor prognosis. Due to the low sensitivity of osteosarcoma to ionizing radiation, such treatment is not used very often and it can be recommended only to postsurgical therapy. As an alternative therapy, functionalized nanomaterials allow their accumulation in tumor tissues due to their unique properties, making them good agents to act as stable carriers for radionuclides. In this work, mesoporous hydroxyapatite nanoparticles were synthesized and the functionalization process with poly(vinyl alcohol) and collagen was investigated. The samples were characterized by X-ray diffraction (XRD), N2 adsorption, elemental analysis (CHN), Fourier Transform Infrared Spectroscopy (FTIR), Nitrogen Adsorption, Transmission Electron Microscopy (TEM), and Thermal Analysis. Also, the yttrium incorporation potential and its release kinetics in the hydroxyapatite matrix were evaluated to study the capacity of this system to treat osteosarcomas. The results indicate that this material has a promisor potential to treat this kind of tumor.

A systematic assessment of hydroxyapatite nanoparticles used in the treatment of melanoma

Nano Research volume 13,pages2106–2117(2020)Cite this article 106 Accesses 1 Altmetric Metrics details Abstract Melanoma is a highly malignant skin tumor which is prone to recurrence and metastasis.Hydroxyapatite nanoparticles(nHAPs)were reported to possess a suppressive effect on proliferation of various tumor cells in vitro.This study aimed to assess in vitro and in vivo anti-tumor ability and biosafety of the nHAPs used in the treatment of melanoma.Three types of nHAPs with different morphology and crystallinity were synthesized.In vitro cell viability and proliferation studies demonstrated that all three types of nHAPs can inhibit viability and proliferation of A375 and SK-MEL-28 melanoma cells in a concentration-dependent manner.In addition,the rod-shape nHAPs with a crystallinity of 45.60%had the most prominent suppressive effect on the two melanoma cells tested.An important positive regulator of G1/S phase transition in cell cycle,Cyclin D1 protein,was reduced by nHAPs treatment in vivo.We further discovered that the migration ability of the nHAPs treated melanoma cells was greatly decreased.RNA sequencing result revealed that melanoma metastasis related genes were down-regulated by nHAPs,including MMP2,MMP14,ITGA9,ITGB3,ITGB4 and S100B.High concentration of nHAPs treatment in melanoma-bearing nude mice showed a strong inhibitory effect on tumor size and weight.Most importantly,hemolysis,electrolyte disturbance or inflammation response was not discovered in the experimental animals from nHAPs treated groups.We proved that the nHAPs synthesized in the current study has a selective effect to suppress melanoma tumor proliferation and was safe with regard to normal cells and tissue.

Controlled Preparation and Formation Mechanism of Hydroxyapatite Nanoparticles under Different Hydrothermal Conditions

Hydroxyapatite （HAP） nanoparticles with uniform morphologies and controllable size were synthesized successfully by molecular template hydrothermal approach. The organic alcohols including ethanol, glycol, glycerol and butanol were used as templates to regulate the nucleation and crystal growth. The synthesized powders were characterized by X-ray diffraction, Fourier infrared spectrum and trans- mission electron microscopy. The results showed that the obtained HAP particles were uniform rod-like crystals, and the template molecular structures had significant effect on the morphology and size of HAP particles. The template molecules with longer hydrophobic groups resulted in longer particle length and larger aspect ratio. Compared with the concentration of template molecules, the template molecular structure showed larger influence on controlling the HAP morphology and size. Furthermore, the for- mation mechanism of these rod-like HAP particles prepared by alkyl alcohol templates was discussed. Moreover, hydrothermal treatment temperature and time could be also used for controlled preparation of HAP nanoparticles.

The morphology of hydroxyapatite nanoparticles regulates clathrinmediated endocytosis in melanoma cells and resultant anti-tumor efficiency

Clathrin-mediated endocytosis plays a critical role for hydroxyapatite nanoparticles(HANPs)to enter tumor cells,induce mitochondrial apoptosis,and inhibit tumor growth.This study was aimed to investigate how the morphology of HANPs impacts the endocytosis of the particles in melanoma cells,and their anti-tumor effect by using in vitro cell experiments and in vivo tumor animal model.Three shapes of HANPs,including granular HANPs(G-HANPs),rod-like HANPs(R-HANPs),and needle-like HANPs(N-HANPs),were successfully prepared by wet chemical method.All the three HANPs could be internalized into A375 melanoma cells as indicated by cellular transmission electron microscopy images.Among these HANPs,only G-HANPs induced morphological change of mitochondria and loss of mitochondrial membrane potential(Δψm),and exhibited the greatest intracellular internalization efficiency in the tumor cells.Furthermore,the results of immunofluorescence staining and western blotting indicated that the level of adaptin-2(AP2)protein was up-regulated by all the HANPs,and highest in G-HANPs treated A375 cells.Moreover,in the tumor-bearing mouse model,we found that tumor growth was delayed by all the three HANPs,of which,G-HANPs delayed tumor growth most efficiently and presented a highest expression level of AP2 protein in tumor tissues.Therefore,this study suggested that the morphology of HANPs regulated their endocytosis efficiency and their effect to inhibit tumor growth.This work facilitates to direct the rational design of nano-materials for tumor therapy.

Hydroxyapatite nanoparticles drive the potency of Toll-like receptor 9 agonist for amplified innate and adaptive immune response

The potency of Toll-like receptor 9(TLR9)agonist to drive innate immune response was limited due to immune suppression or tolerance during TLR9 signaling activation in immune cells.Herein we addressed this problem by introducing hydroxyapatite nanoparticles(HANPs)to CpG ODN(CpG),a TLR9 agonist.The study revealed that HANPs concentration and durationdependently reprogramed the immune response by enhancing the secretion of immunostimulatory cytokines(tumor necrosis factorα(TNFα)or IL-6)while reducing the production of immunosuppressive cytokine(IL-10)in macrophages in response to CpG.Next,the enhanced immune response benefited from increased intracellular Ca2+in macrophage by the addition of HANPs.Further,we found exposure to HANPs impacted the mitochondrial function of macrophages in support of the synthesis of adenosine triphosphate(ATP),the production of nicotinamide adenine dinucleotide(NAD),and reactive oxygen species(ROS)in the presence or absence of CpG.In vaccinated mice model,only one vaccination with a mixture of CpG,HANPs,and OVA,a model antigen,allowed the development of a long-lasting balanced humoral immunity in mice without any histopathological change in the local injection site.Therefore,this study revealed that HANPs could modulate the intracellular calcium level,mitochondrial function,and immune response in immune cells,and suggested a potential combination adjuvant of HANPs and TLR9 agonist for vaccine development.

Anti-melanoma effect and action mechanism of a novel chitosan-based composite hydrogel containing hydroxyapatite nanoparticles

Hydroxyapatite nanoparticles(HANPs)have been increasingly regarded and reported due to their potential anti-tumor ability.Previously,we found that the rod-like HANPs had good application potential for cutaneous melanoma(CMM).To satisfy the actual requirements in repairing post-operative skin defects and inhibiting CMM recurrence after tumorectomy,we constructed a novel chitosan/alginate(CS/Alg)hydrogel containing the aforementioned HANPs.The in vitro cell experiments confirmed that activated mitochondrial-dependent apoptosis was tightly related to the anti-tumor ability of HANPs.Specifically,we further discovered several target proteins might be involved in abnormal activating Wnt,proteoglycans in cancer,oxidative phosphorylation and p53 signaling pathways.The in vivo animal experiments demonstrated that the HANPsloaded CS/Alg hydrogel(CS/Alg/HANPs)had a similar effect on inhibiting tumor growth as HANPs,and CS/Alg hydrogel as well as phosphate buffered saline(PBS)group(control)not showed any effect,proving the key role of HANPs.The immunohistochemical staining demonstrated a tumor inhibition via the mitochondria-mediated apoptosis pathway,consistent with the in vitro evaluation.Moreover,CS/Alg/HANPs exhibited no additional biosafety risk to the functions of major organs.Overall,this CS/Alg/HANPs hydrogel has substantial application potential for treating CMM.

Apoptosis of Cancer Cells Induced by HAP Nanoparticles

To confirm apoptosis is one of the hepatoma cells death pathways after HAP nanoparticles absorption,hepatoma cells were collected for ultrathin sections preparation and examined under a transmission electron microscope（TEM）after 1 h incubation with HAP nanoparticle.Apoptosis was detected by TUNEL technique.After absorption.some vacuoles with membrane containing HAP nanoparticles were found in cytoplasma.The nuclear enrelope shrinked.and some area pullulated from nucleus.The karyotin became pycnosis and assembled at the edge.An apoptosis body was found.and the data of IOD and numbers of the positive apoptosic signals in nuclear area of slides could illustrate much more apoptosis in the HAP group than those in the control group（P〈0.001）.The experimental results indicate that the HAP nanoparticles can induce cancer cells apoptosis.

Effects of Antihepatocarcinoma with Apatite Nanoparticles in vivo

The inhibition effect of hydroxyapatite （ HAP ） nanoparticles on hepatocarcinoma was investigated in vivo. The human hepatocarcinoma cell line Bel- 7402 was transplanted subcutaneously into nude mice. Hydroxyapatite nanoparticles suspension at a dose of 0. 2 mL was injected into the transplanted tumors every day for 2 weeks, and saline was used us control. The efficacy of hydroxyapatite nanoparticles on this carcinoma was surveyed and morphological changes of tissue and cells were observed by light microscopy and transmission electron microscopy （TEM）. Experimental results show that hydroxyapatite nanoparticles have a visible destructive effect on the structures of hepatocarcinoma cells and tissue. The inhibition rates of tumor growth were 77.21% and 51. 32% after intra-tumor injection of hydroxyapatite nanoparticles for 1 week and 2 weeks, respectively. Compared with the control group, hydroxyapatite nanoparticles can also prolong the survival time of the nude mice bearing this cancer significantly. This indicates that hydroxyapatite nanoparticles have the therapeutic potential on hepatoma in vivo.

Effect of Hydroxyapatitc Nanoparticles on K562 Cells in vitro

Stable and single-dispersed hydroxyapatite （HAP） nanoparticles were synthesized with ultrasonic-assisted method. HAP nanoparticles were characterized by dynamic light scattering, XRD （X-ray diffraction） and TEM （Transmission Electron Microscopy）. The effect of HAP nanoparticles on the K562 human myelogenous leukemia cell line was investigated by MTT assay and cell count test, and the mechanism was studied through the changes of cell cycle and ultrastructure. The results showed that HAP nanoparticles inhibited the proliferation of K562 cells dramatically in vitro. HAP nanoparticles entered the cytoplasm of K562 cells and the cells were arrested at G/M phase, thus, the cells died directly.

Study on the Variation of Calcium and Phosphorus in Nano Hydroxyapatie Treated Cancer Cells through Synchrotron Radiation XRF

The HAP nanoparticles were synthesized with a chemical co-precipitation method. It was monodispersed in needle shape, Its average diameter was 30 nm and length was 70 nm. Human liver cancer cells and normal hepatic cells were treated by hydroxyapatite nanopartides. SR-XRF analysis was used to detect the content variation of calcium and phosphor elements in cancer cells when the cells were treated by hydroxyapatite nanoparticles. It was introduced to reveal the anti-cancer mechanism of HAP nanoparticles. Experiments verified that the contents of calcium and phosphor elements in hepatocelltdar carcinoma had been obviously increased after the cells were treated by hydroxyapatite nanoparticles. And the contents increased with hydroxyapatite concentration and treatment time increasing. The Ca/P molar ratio in treated cell was different from both that in hydroxyapatite and that in untreated cancer cell. Apatite nanoparticles evidently altered the calcium and phosphor environmeats in treated hepatocellalar carcinoma cells. That finally halted the proliferation of carcinoma cells.

Cesium carbonate supported on hydroxyapatite coated Ni_(0.5)Zn_(0.5)Fe_2O_4 magnetic nanoparticles as an efficient and green catalyst for the synthesis of pyrano[2,3-c]pyrazoles

Cesium carbonate supported on hydroxyapatite coated Nio.sZno.sFe2O4 magnetic nanoparticles （Nio.sZno.sFe2O4@Hap-Cs2CO3） was found to be magnetically separable, highly efficient, green and recyclable heterogeneous catalyst. The synthesized nanocatalyst has been characterized with several methods （FT-IR, SEM, TEM, XRD and XRF） and these analyzes confirmed which the cesium carbonate is well supported to catalyst surface. After full characterization, its catalytic activity was investigated in the synthesis of pyranopyrazole derivatives and the reactions were carried out at room temperature in 50：50 water/ethanol with excellent yields （88-95%）. More importantly, the Nio.5Zno.sFe204@Hap-Cs2CO3 was easily separated from the reaction mixture by external magnetic field and efficiently reused at least six runs without any loss of its catalytic activity. Thus, the developed nanomagnetic base catalyst is potentially useful for the green and economic production of organic compounds.

Enamel matrix proteins in promoting saliva lubrication

Anti-wear performance of human enamel in the mouth is closely related to the lubrication of salivary pellicle.It is well known that the inorganic hydroxyapatite(HA)of the enamel plays an important role in the adsorption and pellicle-forming of salivary proteins on the enamel,but the role of enamel matrix proteins remains unclear.In this study,the adsorption and lubrication behavior of salivary proteins on original,heated,and deproteinated enamel surfaces was comparatively investigated using an atomic force microscopy and nano-indentation/scratch techniques.Compared with that on the original enamel surface,the adsorption and lubrication behavior of salivary proteins remains almost unchanged on the heated enamel surface(where the enamel matrix proteins are denatured but the size of HA crystalline nanoparticles keeps constant)but exhibits an obvious compromise on the deproteinated enamel surface(where the enamel matrix proteins are removed and agglomeration of HA crystallites occurs).The HA agglomeration weakens the electrostatic interaction of enamel surfaces with salivary proteins to cause a distinct negative influence on the adsorption and pellicle-forming of salivary proteins.Further,the negative effect is confirmed with a quartz crystal microbalance with dissipation.In summary,by regulating enamel nanostructure for appropriate electrostatic interactions between salivary proteins and enamel surfaces,the enamel matrix proteins play an essential role in the adsorption and pellicle-forming of salivary proteins on human enamel,and then contribute to saliva lubrication,which provides the enamel with an anti-wear mechanism.The findings will promote and assist the design of enamel-inspired anti-wear materials.

A CS-based composite scaffold with excellent photothermal effect and its application in full-thickness skin wound healing

The development of natural polymer-based scaffolds with excellent biocompatibility,antibacterial activity,and blood compatibility,able to facilitate full-thickness skin wound healing,remains challenging.In this study,we have developed three chitosan(CS)-based porous scaffolds,including CS,CS/CNT(carbon nanotubes)and CS/CNT/HA(nano-hydroxyapatite,n-HA)using a freeze-drying method.All three scaffolds have a high swelling ratio,excellent antibacterial activity,outstanding cytocompatibility and blood compatibility in vitro.The introduction of CNTs exhibited an obvious increase in mechanical properties and exerts excellent photothermal response,which displays excellent healing performance as a wound dressing in mouse full-thickness skin wound model when compared to CS scaffolds.CS/CNT/HA composite scaffolds present the strongest ability to promote full-thickness cutaneous wound closure and skin regeneration,which might be ascribed to the synergistic effect of photothermal response from CNT and excellent bioactivity from n-HA.Overall,the present study indicated that CNT and n-HA can be engineered as effective constituents in wound dressings to facilitate full-thickness skin regeneration.