Overcoming multidrug resistance with mesoporous silica nanorods as nanocarrier of doxorubicin

Multidrug resistance (MDR) is a major obstacle to the effective chemotherapy in many human malignancies. Nanoparticulate drug delivery systems (NDDSs) have been reported to be able to bypass MDR, but the cancer therapeutic efficacy is still limited. In this study, we firstly designed the nonspherical mesoporous silica nanorods (MSNRs) with aspect ratio (AR) of 1.5 and 5 as drug delivery systems of doxorubicin to overcome multidrug resistance. For drug loading, the long-rod MSNRs (NLR, AR = 5) showed higher drug loading capacity of doxorubicin (DOX) than the short-rod MSNRs (NSR, AR = 1.5). NLR encapsulated DOX had increased intracellular DOX accumulation in drug-resistant Chinese hamster ovary (CHO) cells compared with free DOX by observablly increased cellular uptake and significantly prolonged intracellular drug retention. It further exhibited increased cytotoxicity compared with free DOX under different drug concentrations. These findings may provide a new perspective for designing high-performance nanoparticulate drug delivery systems for bypassing multidrug resistance of cancer therapy.     

Efficiency against multidrug resistance by co-delivery of doxorubicin and curcumin with a legumain-sensitive nanocarrier

Multidrug resistance proteins (MDRPs), which are implicated in the mediation of multidrug resistance in tumors, represent the main obstacle to successful chemotherapy. As curcumin (Cur) exerts inhibitory effects on both the expression and function of MDRPs, a nanocarrier for the co-delivery of Cur and doxorubicin (DOX) was prepared to overcome MDR tumors through their synergistic effects. Owing to the overexpression of legumain in tumors, the release profile of DOX from this nanocarrier was designed to be legumain modulated, which was achieved by bridging DOX to a basic material (chitosan) with a legumain-sensitive peptide. Compared with nanoparticles that only contain DOX, the coadministration of DOX and Cur significantly inhibited multidrug resistance (P < 0.05) in a multidrug-resistant cancer cell model (MCF-7/ADR cell line), with cytotoxicity to normal cells (L929 cell line). Such inhibition could be ascribed to the increased DOX accumulation in the MCF-7/ADR nucleus. The co-delivery system exhibited good anticancer effects through prolonged circulation time, improved tumor-targeting efficiency, elevation of the tumor inhibition activity, and the suppression of MDRP expression. These data revealed the enormous potential of this co-delivery system for cancer therapy, especially in the later stages where multidrug resistance may develop.

Open image in new window

     

Regulation of particle morphology of pH-dependent poly(epsilon-caprolactone)-poly(gamma-glutamic acid) micellar nanoparticles to combat breast cancer cells

The advantage of polymeric drug carriers lies in the uptake of the polymer nanoparticles by cancer cells before they release the drug, thereby reducing its toxic effects on healthy cells. A poly(gamma-glutamic acid)-b-poly(epsilon-caprolactone)-b-poly(gamma-glutamic acid) block copolymer was synthesized to encapsulate the anti-cancer drug doxorubicin in the treatment of wild type human breast cancer cells (MCF-7/WT). This pH-controllable carrier is negatively-charged in the presence of healthy tissues leading to lower cellular uptake. On the other hand, it becomes more hydrophobic in the acidic environment of cancer tissues, increasing its cellular uptake through the lipid bilayer. The block copolymer was characterized using Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, differential scanning calorimetry and dynamic light scattering. The micelles formed at a critical concentration range of 62-130 microg/mL depending on the composition of poly(gamma-glutamic acid) and poly(epsilon-caprolactone) chains. The nano-sized micelles were found to have pH-dependent sizes in the range of 90-200 nm. The role of poly(gamma-glutamic acid) was to increase the hydrophilicity and decrease the particle size of the copolymer. The structures of micelles that were more compact and less anionic showed better stability in plasma. It was found that the drug loading content and drug loading efficiency were 12.14% and 97.22% respectively. The copolymer showed shrinking and aggregation at low pH which led to a slower drug release. These nano-sized micelles showed potential as effective drug delivery carriers for doxorubicin because of its accumulation and slow release inside the MCF-7/WT cells.     

Ultrasound mediated drug-loaded nanoparticles crossing cell membranes as a new strategy to reverse cancer multidrug resistance

The emergence of the multidrug resistance (MDR) phenomenon in tumor has rendered many currently available chemotherapeutic drugs ineffective. Although many strategies have been explored to overcome MDR, the results have been disappointing to the obstacle. The aim of this study was to investigate whether the new strategy of combining drug-loaded nanoparticles (Nps) and ultrasound (US) would show useful effects on the reversal of MDR in tumor. The MDR leukemia K562/A02 cells were treated with the daunorubicin (DNR)-loaded TiO2 Nps drug carrier and US exposure. We observed good biocompatibility of the therapeutic approach, and the fresh evidence from the electrochemical studies, MTT assays, and caspase-3 immunocytochemistry demonstrated that the strategy could significantly increase the uptake of DNR by drug-resistant leukemia cells, and enhance the sensitivity of the MDR cells to the chemotherapeutic agents after released in the cells. The resisting fold became obviously lower and the apoptosis was induced in the cells as well. It was therefore concluded that the strategy could have good reversal ability of MDR in tumor. These findings reveal that the reversal of MDR in tumor by US mediated drug-loaded Nps crossing cell membranes could represent promising approach in cancer therapy.     

A TEM study of functionalized magnetic nanoparticles targeting breast cancer cells

In this paper, a transmission electron microscopy (TEM) study was carried out to investigate the ability of magnetic nanoparticles (MNPs) to target breast cancer cells in mice. MNPs were functionalized using Luteinizing Hormone Releasing Hormone (LHRH), whose receptors are expressed in most types of breast cancer cells. LHRH conjugated MNPs (LHRH-MNPs) were injected intravenously into female nude mice bearing MDA-MB-435S.luc tumors for thirty days. These mice were sacrificed 20 h after MNP injection. Tumors and periphery organs including livers, lungs and kidneys were collected for analysis. A dedicated transmission electron microscopy (TEM) study was then carried out to investigate the distribution of nanoparticles in cells. We found that dispersive LHRH-MNPs were distributed in tumor cells and cells in lungs and livers. No LHRH-MNPs were observed in kidney cells. Furthermore, LHRH-MNPs tend to aggregate and form clusters in tumor cells and cells in lungs where metastases were developed. These suggest that MNPs functionalized using LHRH can be used to target both primary cancer cells and the metastatic cells. The study also indicates that TEM is a useful tool to study the sub-cellular distribution of functionalized magnetic nanoparticles in mice bearing breast cancers.     

Efficacy of lytic peptide-bound magnetite nanoparticles in destroying breast cancer cells

A 23-amino-acid synthetic lytic peptide (Hecate) was covalently linked to magnetite nanoparticles and the lytic peptide-bound nanoparticles were characterized by X-ray absorption near-edge structure spectroscopy, transmission electron microscopy, and electron diffraction. Investigation of magnetic properties with a superconducting quantum interference device (SQUID) magnetometer has shown a reduction in the saturation magnetization (Ms) of magnetite nanoparticles after binding with lytic peptide. An in vitro cell culture assay with breast cancer cell lines MDA-MB-435S revealed that the lytic peptide-bound magnetite nanoparticles were therapeutically active.     

MUC1 aptamer-conjugated mesoporous silica nanoparticles effectively target breast cancer cells

In the present study, we developed aptamer (Apt) conjugated mesoporous silica nanoparticles (MSNs) for specific delivery of epirubicin (EPI) to breast cancer cells. MSNs were synthesized and functionalized with 3-mercaptopropyltrimethoxysilane (3-MPTMS), followed by MUC1 aptamer conjugation through disulfide bonds. The nanoparticles were analyzed by transmission electron microscopy (TEM), particle size analyzer, zeta potential, elemental analysis (CHNS), aptamer conjugation efficiency, drug loading efficiency, and drug release profile. Cell uptake and in vitro cytotoxicity of different formulations were performed. The results of MSNs characterization confirmed spherical nanoparticles with thiol functional groups. Particle size of obtained nanoparticles was 163?nm in deionized water. After conjugation of MUC1 aptamer and EPI loading (MSN-MUC1-EPI), particle size increased to 258?nm. The aptamer conjugation to MSNs with disulfide bonds were confirmed using gel retardation assay. Cellular uptake studies revealed better cell uptake of MSN-MUC1-EPI compared to MSN-EPI. Moreover, cytotoxicity study results in MCF7 cell lines showed improved cytotoxicity of MSN-MUC1-EPI in comparison with MSN-EPI or EPI at the same concentration of drug. These results exhibited that MSN-MUC1-EPI has the potential for targeted drug delivery into MUC1 positive breast cancer cells to improve drug efficacy and alleviate side effects.     

Imaging breast cancer cells and tissues using peptide-labeled fluorescent silica nanoparticles

The imaging of tumor cells and tumor tissue samples is very important for cancer detection and therapy. We have taken advantages of fluorescent silica nanoparticles (FSiNPs) coupled with a molecular recognition element that allows for effective in vitro and ex vivo imaging of tumor cells and tissues. In this study, we report on the targeting and imaging of MDA-MB-231 human breast cancer cells using arginine-glycine-aspartic acid (RGD) peptide-labeled FSiNPs. When linked with RGD peptide using the cyanogen bromide (CNBr) method, the FSiNPs exhibited high target binding to alphavbeta3 integrin receptor (ABIR)-positive MDA-MB-231 breast cancer cells in vitro. Further study regarding the ex vivo imaging of tumor tissue samples was also carried out by intravenously injecting RGD peptide-labeled FSiNPs into athymic nude mice bearing the MDA-MB-231 tumors. Tissue images demonstrated that the high integrin alphavbeta3 expression level of the MDA-MB-231 tumors was clearly visible due to the special targeting effects of the RGD peptide-labeled FSiNPs, and the tumor fluorescence reached maximum intensity at 1 h postinjection. Our results break new ground for using FSiNPs to optically image tumors, and may also broaden the applications of silica nanoparticles in biomedicine.     

Atorvastatin-loaded lipid nanoparticles: antitumor activity studies on MCF-7 breast cancer cells

Atorvastatin is a synthetic statin commonly used in the treatment of hypercholesterolemia. Apart from this, statins appear to have pleiotropic effects, including modulation of cell growth, apoptosis. Through modulation of these pathways, statins have the potential to influence a wide range of disease processes, including cancer. However, poor aqueous solubility (0.1?mg/mL) and poor oral bioavailability has limited therapeutic application of atorvastatin. Present work is an attempt to improve tumor targeting of atorvastatin by incorporating in nanostructured lipid carriers (NLCs) and studying its anticancer activity on MCF-7 cell lines. NLCs of atorvastatin were formulated by high-speed homogenization followed by probe sonication method. The optimized batch of NLCs had a mean size of 130.02?±?3.1?nm and entrapment efficiency of 90.42?±?3.7%. The in vitro drug release study by dialysis method indicated that drug entrapped in the NLCs remains entrapped at acidic pH as well as in phosphate buffer of pH 7.4 for a prolonged period of time as compared to plain drug. In vitro cytotoxicity studies on MCF-7 (mammary adenocarcinoma human cell lines) cell lines showed that concentration of drug required for total growth inhibition (TGI) and 50% growth inhibition (GI50) of MCF-7 cells was found to be 27.4?µg/mL and <10?µg/mL respectively, in case of atorvastatin- NLCs which is less than that required in case of plain atorvastatin and almost similar to that of adriamycin. All these findings reinforce the fact that atorvastatin loaded NLCs are promising novel delivery system for treating breast cancer.     

Measuring the doxorubicin content of single nuclei by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

Individual nuclei isolated from the human leukemia CCRF-CEM and CEM-C2 cells treated with doxorubicin (DOX) were in-column lysed with a sodium dodecyl sulfate (SDS) containing buffer, their contents were then separated by micellar electrokinetic capillary chromatography using the same lysing buffer, and the DOX content was detected by laser-induced fluorescence. Use of a microscope for the selection of one nucleus from the nuclear preparation decreases the possibility of introduction of other subcellular components that are commonly found as impurities in subcellular fractions. The presence of SDS in the running buffer made negligible the DNA's quenching effect on DOX fluorescence, which often compromises quantification of DOX by direct imaging, making it possible to carry out the first direct measurement of the doxorubicin content of isolated nuclei. On average, nuclei from CCRF-CEM and CEM/C2 cell lines contained 85 +/-64 (n = 6) and 91 +/- 51 (n =7) amol of DOX, respectively. These values correspond to 74 and 65% of the average total cellular content as determined by single-cell analysis of the corresponding cell types. It is envisioned that this approach could become an important bioanalytical tool to investigate the effect of treatments with fluorescent drugs targeting the nucleus.     

Overcoming pancreatic cancer immune resistance by codelivery of CCR2 antagonist using a STING-activating gemcitabine-based nanocarrier

STING agonist has recently gained much attention for cancer treatment, but the therapeutic potential of STING agonist is hampered by STING-associated tumor immune resistance. In this work, guided by both bioinformatics and computer modeling, we rationally designed a “one stone hits two birds” nanoparticle-based strategy to simultaneously activate STING innate immune response while eliminating STING-associated immune resistance for the treatment of pancreatic ductal adenocarcinoma (PDAC). We discovered that the ultra-small sized micellar system based on gemcitabine-conjugated polymer (PGEM), which showed superior capacity of penetration in pancreatic tumor spheroid model and orthotopic tumor model, could serve as a novel “STING agonist”. The activation of STING signaling in dendritic cells (DCs) by PGEM increased both innate nature killer (NK) and adaptive anti-tumor T cell response. However, activation of STING signaling by PGEM in tumor cells also drove the induction of chemokines CCL2 and CCL7, resulting in immune resistance by recruiting tumor associated macrophage (TAM) and myeloid-derived suppressor cells (MDSCs). Through the combination of computer modeling and experimental screening, we developed a dual delivery modality by incorporating a CCR2 (the receptor shared by both CCL2 and CCL7) antagonist PF-6309 (PF) into PGEM micellar system. Our studies demonstrated that PGEM/PF formulation significantly reduced pancreatic tumor burden and induced potent anti-tumor immunity through reversing the CCL2/CCL7-mediated immunosuppression. Moreover, PGEM/PF sensitized PDAC tumors to anti-PD-1 therapy, leading to complete suppression/eradication of the tumors. Our work has shed light to the multi-faceted role of STING activation and provided a novel immunotherapy regimen to maximize the benefit of STING activation for PDAC treatment. In addition, this work paved a new way for bioinformatics and computer modeling-guided rational design of nanomedicine.     

Luteinizing hormone‐releasing hormone targeted poly(methyl vinyl ether maleic acid) nanoparticles for doxorubicin delivery to MCF‐7 breast cancer cells

The purpose of this study was to design a targeted anti‐cancer drug delivery system for breast cancer. Therefore, doxorubicin (DOX) loaded poly(methyl vinyl ether maleic acid) nanoparticles (NPs) were prepared by ionic cross‐linking method using Zn²⁺ ions. To optimise the effect of DOX/polymer ratio, Zn/polymer ratio, and stirrer rate a full factorial design was used and their effects on particle size, zeta potential, loading efficiency (LE, %), and release efficiency in 72 h (RE₇₂, %) were studied. Targeted NPs were prepared by chemical coating of tiptorelin/polyallylamin conjugate on the surface of NPs by using 1‐ethyl‐3‐(3‐dimethylaminopropyl) carboiimid HCl as cross‐linking agent. Conjugation efficiency was measured by Bradford assay. Conjugated triptorelin and targeted NPs were studied by Fourier‐transform infrared spectroscopy (FTIR). The cytotoxicity of DOX loaded in targeted NPs and non‐targeted ones were studied on MCF‐7 cells which overexpress luteinizing hormone‐releasing hormone (LHRH) receptors and SKOV3 cells as negative LHRH receptors using Thiazolyl blue tetrazolium bromide assay. The best results obtained from NPs prepared by DOX/polymer ratio of 5%, Zn/polymer ratio of 50%, and stirrer rate of 960 rpm. FTIR spectrum confirmed successful conjugation of triptorelin to NPs. The conjugation efficiency was about 70%. The targeted NPs showed significantly less IC₅₀ for MCF‐7 cells compared to free DOX and non‐targeted NPs.Inspec keywords: nanoparticles, polymer blends, cancer, cellular biophysics, drug delivery systems, drugs, biomedical materials, zinc, positive ions, Fourier transform infrared spectra, nanomedicine, proteinsOther keywords: luteinizing hormone‐releasing hormone, poly(methyl vinyl ether maleic acid), doxorubicin delivery, MCF‐7 breast cancer cell, anticancer drug delivery system, doxorubicin‐loaded PVM‐MA nanoparticle, ionic cross‐linking method, zinc ion, doxorubicin‐polymer ratio effect, zinc‐polymer ratio effect, particle size, zeta potential, loading efficiency, release efficiency, chemical coating, tiptorelin‐polyallylamin conjugation, PVM‐MA nanoparticle surface, 1‐ethyl‐3‐(3‐dimethylaminopropyl) carboiimid HCl, cross‐linking agent, Bradford assay, Fourier transform infrared spectroscopy, cytotoxicity, LHRH receptor, SKOV3 cell, Thiazolyl blue tetrazolium bromide assay, conjugation efficiency, time 72 h, Zn²⁺      

Methotrexate-conjugated L-lysine coated iron oxide magnetic nanoparticles for inhibition of MCF-7 breast cancer cells

Methotrexate (MTX), a stoichiometric inhibitor of dihydrofolate reductase enzyme, is a chemotherapeutic agent for treating a diversity of neoplasms. In this study, we design and developed a new formulation of MTX that serves as drug carrier and examined its cytotoxic effect in vitro. This target drug delivery system is dependent on the release of the MTX within the lysosomal compartment. The iron oxide magnetic nanoparticles (IONPs) were first surface-coated with L-lysine and subsequently conjugated with MTX through amidation between the carboxylic acid end groups on MTX and the amine groups on the IONPs surface. MTX-conjugated L-lysine coated IONPs (F-Lys-MTX NPs) was characterized by X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, Fourier transform infrared spectroscopy, vibrating sample magnetometer, and transmission electron microscopy techniques. The cytotoxicity of the void of MTX and F-Lys-MTX NPs were compared to each other by MTT assay of the treated MCF-7 cell lines. The results showed that the ζ-potential of F-Lys-MTX NPs was about ?5.49?mV and the average size was 43.72?±?4.73?nm. Model studies exhibited the release of MTX via peptide bond cleavage in the presence of proteinase K and at low pH. These studies specify that F-Lys-MTX NPs have a very remarkable anticancer effect, for breast cancer cell lines.     

Doxorubicin-loaded polymeric micelle overcomes multidrug resistance of cancer by double-targeting folate receptor and early endosomal pH

An optimized, pH-sensitive mixed-micelle system conjugated with folic acid is prepared in order to challenge multidrug resistance (MDR) in cancers. The micelles are composed of poly(histidine (His)-co-phenylalanine (Phe))-b-poly(ethylene glycol) (PEG) and poly(L-lactic acid) (PLLA)-b-PEG-folate. Core-forming, pH-sensitive hydrophobic blocks of poly(His-co-Phe) of varying composition are synthesized. The pH sensitivity of the micelles is controlled by the copolymer composition and is fine tuned to early endosomal pH by blending PLLA(3K)-b-PEG(2K)-folate in the presence of a basic anticancer drug, doxorubicin (DOX). In vitro tests are conducted against both wild-type (A2780) and DOX-resistant ovarian carcinoma cell lines. A mixed-micelle system composed of poly(His-co-Phe (16 mole%))-b-PEG (80 wt%) and PLLA-b-PEG-folate (20 wt%) is selected to target early endosomal pH. DOX-loaded micelles effectively kill both wild-type sensitive (A2780) and DOX-resistant ovarian MDR cancer-cell lines (A2780/DOX(R)) through an instantaneous high dose of DOX in the cytosol, which results from active internalization, accelerated DOX release triggered by endosomal pH, and an endosomal membrance disruption.     

PAMAM-modified citric acid-coated magnetic nanoparticles as pH sensitive biocompatible carrier against human breast cancer cells

Denderimer-modified magnetic nanoparticles are a promising drug delivery nanosystem which can improve the therapeutic efficacy of chemotherapy drugs and can also be beneficial as magnetic resonance (MR) images contrast agent. The present study introduces the preparation and characterization of the potential therapeutic efficiency of curcumin (CUR)-loaded denderimer-modified citric acid coated Fe₃O₄ NPs. Polyamidoamine (PAMAM, generation G₅) was used to encapsulate citric acid coated Fe₃O₄ nanoparticles. The successful preparation of CUR-loaded nanocarriers were confirmed by X-ray diffraction (XRD), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM), and transmission electron microscopy (TEM) techniques. The loading capacity and encapsulation efficiency of CUR molecules were 12?±?0.03% and 45.58?±?0.41%, respectively. The anticancer effect of void CUR and CUR-loaded nanocarriers were compared to each other by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on treated MCF-7 cell line. It can be concluded that application of nanoparticles can be more effective strategy for controlled and slow release of CUR in human breast cancer treatment.     

Simple monitoring of cancer cells using nanoparticles

Here we present a new strategy for a simple and fast detection of cancer circulating cells (CTCs) using nanoparticles. The human colon adenocarcinoma cell line (Caco2) was chosen as a model CTC. Similarly to other adenocarcinomas, colon adenocarcinoma cells have a strong expression of EpCAM, and for this reason this glycoprotein was used as the capture target. We combine the capturing capability of anti-EpCAM functionalized magnetic beads (MBs) and the specific labeling through antibody-modified gold nanoparticles (AuNPs), with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction (HER) detection technique. The fully optimized process was used for the electrochemical detection of Caco2 cells in the presence of monocytes (THP-1), other circulating cells that could interfere in real blood samples. Therefore we obtained a novel and simple in situ-like sensing format that we applied for the rapid quantification of AuNPs-labeled CTCs in the presence of other human cells.     

Folic acid-functionalized niosomal nanoparticles for selective dual-drug delivery into breast cancer cells: An in-vitro investigation

In this study, a folic acid-functionalized niosome was formulated and loaded with letrozole and curcumin as a promising drug carrier system for chemotherapy of the breast cancer cells. The formulation process was optimized by varying the type of Span 80 and total lipid to drug ratio, where Span 80 and lipid to drug molar ratio of 10 resulted in the niosomes with maximum encapsulation of both drugs but minimum size. The developed niosomal formulation showed a great storage stability up to one month with the small changes in drug encapsulation efficiency and size during the storage. In addition, they showed a pH-dependent release behaviour with slow drug release at physiological pH (7.4) while considerable drug release in acidic conditions (pH = 3), making it a promising candidate for breast cancer treatment. The cytotoxicity study shows the niosomal formulation has high biocompatibility with HEK-293 healthy cells, while having remarkable inhibitory effects on MCF-7 and MDA-MB-231 breast cancer cells due to the presence of folic acid in formulation, and in turn, selective internalization of the as-developed nanocarrier through folate receptor-mediated endocytosis. The double drug-loaded niosomes affect the gene expression by studied breast cancer cell lines; down-regulates the expression of Bcl2, cyclin D, and cyclin E genes while they up-regulate the expression of p53, Bax, caspase-3, and caspase-9 genes. The flow cytometry results showed that letrozole/curcumin-loaded niosomes enhanced the apoptosis rate in both MCF-7 and MDA-MB-231 cells compared to the mixture of letrozole and curcumin, which was due to the synergic effect between the two drugs as well as higher cell uptake by niosomal formulation. The findings of our study show the importance of developing highly biocompatible niosomal formulations in the future of nanomedicine that enables the co-delivery of two hydrophobic drugs into the cancer cells improves the efficiency of chemotherapy due to the synergic effect between the drugs.     

Synthesis of silica-coated rhodium nanoparticles in reversed micellar solution

Silica (SiO₂)-coated rhodium (Rh) nanoparticles were prepared using a water-in-oil microemulsion of polyoxyethylene (15) cetyl ether, cyclohexane and water. SiO₂-coated Rh nanoparticles were obtained by hydrolyzing metal alkoxide (tetraethylorthosilicate, TEOS) in the solution containing Rh complex nanoparticles followed by thermal and reduction treatments. In the SiO₂-coated Rh nanoparticle, a Rh particle with an average diameter of 4.1 nm was located nearly at the center of each spherical SiO₂ particle. The SiO₂ layer was approximately 15 nm thick. Since the Rh particle was wholly surrounded by SiO₂, the Rh particle of the SiO₂-coated Rh nanoparticle exhibited an extremely high thermal stability. Furthermore, the porous structure of the SiO₂ layer could be controlled by the hydrolysis conditions of TEOS.     

Internalization of hydroxyapatite nanoparticles in liver cancer cells

Hydroxyapatite (HAP) is the main inorganic component of hard tissues and shows excellent biocompatibility and osteoconductivity properties. Nanoparticles of HAP can be synthesised by the precipitation method in distilled water. The needle shaped particles are below 100 nm in size with low-crystallinity and high-surfacial activation. Recent studies showed toxic effects of HAP nanoparticles on cancer cells. Other studies focus on the application of HAP nanoparticles as drug and gene delivery system or cell marker. However, to date, the exact internalization pathway of HAP nanoparticles into cells has not been determined. When HAP nanoparticles were added to cell culture medium, the particles immediately became instable and formed agglomerates with a size of about 500–700 nm. Hence, cells seldom encounter single HAP nanoparticles in the environment of cell culture or body fluid. The TEM showed internalized HAP captured by vacuoles in the cytoplasm of the hepatocellular carcinoma cells. The invaginations in the cell membrane before nanoparticle uptake suggested endocytic pathways as internalization mechanism. This study revealed that agglomerated HAP nanoparticles were internalized by cells through the energy-dependent process of clathrin-mediated endocytosis. Depletion of intracellular potassium arrested the formation of coated pit, which inhibited the uptake of HAP.