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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Core/shell nanoparticles for pH-sensitive delivery of doxorubicin

Core/shell nanoparticles with lipid core were prepared and characterized as pH-sensitive delivery system of anticancer drug. The lipid core is composed of drug-loaded lecithin and the polymeric shell is composed of Pluronics (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) tri-block copolymer, F-127). Based on the preparation method in the previous report by us, the freeze-drying of drug-loaded lecithin was performed in the F-127 aqueous solution containing trehalose used as a cryoprotectant to form stabilized core/shell nanoparticles. For the application of core/shell nanoparticles as a pH-sensitive drug delivery system for anticancer drug, doxorubicin was loaded into the core/shell nanoparticles and the drug loading amount and drug release behavior in response to pH change were observed.     

Aptamer-conjugated nanoparticles for the collection and detection of multiple cancer cells

We have extended the use the aptamer-conjugated nanoparticles for the collection and detection of multiple cancer cells. The aptamers were selected using a cell-based SELEX strategy in our laboratory for cancer cells that, when utilized in this method, allow for the selective recognition of the cells from complex mixtures including fetal bovine serum samples. Aptamer-conjugated magnetic nanoparticles were used for the selective targeting cell extraction, and aptamer-conjugated fluorescent nanoparticles were employed for sensitive cellular detection. Employing both types of nanoparticles allows for selective and sensitive detection not possible by using the particles separately. Fluorescent nanoparticles amplify the signal intensity versus a single fluorophore label resulting in improved sensitivity. In addition, aptamer-conjugated magnetic nanoparticles allow for extraction and enrichment of target cells not possible with other separation methods. Fluorescent imaging and a microplate reader were used for cellular detection to demonstrate the wide applicability of this methodology for medical diagnostics and cell enrichment and separation.     

Development of CaP nanocomposites as photothermal actuators for doxorubicin delivery to enhance breast cancer treatment

Breast cancer is the most common one in women worldwide and doxorubicin(Dox)is one of the most commonly used and effective drugs for breast cancer treatment.Unfortunately,Dox-based chemotherapy faces irreversible cardiotoxicity and unsatisfactory therapy efficiency.It is desirable to devise Dox nanoformulations with less adverse effects and greater therapeutic efficacy for this cancer treatment.In this work,a multifunctional calcium phosphate nanoformulation(ICG-Dox/DNA@CaP)was developed by co-loading Dox/DNA complexes and indocyanine green(ICG)molecules for photothermal therapy(PTT)-enhanced chemotherapy.In this nanocomposite,using DNA as Dox carrier facilitated Dox loading into the CaP matrix,and significantly reduced Dox leakage as well as cytotoxicity in comparison with that of free Dox in physiological medium(pH 7.4).In specific,ICG-Dox/DNA@CaP only released Dox in a weakly acidic nuclease-containing environment,such as tumor microenvironment and endosome/lysosome.Moreover,Dox/DNA complexes exhibited synergistic interactions with ICG-based photothermal effect on tumor cell apoptosis in this ICG-Dox/DNA@CaP nanocomposite.This work has demonstrated a new strategy to combine FDA-approved therapeutics(Dox and ICG)in CaP-based nanomaterials for reduced cytotoxicity and enhanced therapeutic effect,and provided a new way to engineer CaP carriers as multifunctional delivery systems for clinical anti-cancer therapy.     

Graphene oxide used as a carrier for adriamycin can reverse drug resistance in breast cancer cells

This study evaluates the reversal effects of graphene oxide (GO) used as a carrier for adriamycin (ADR) in cancer drug resistance, and provides a preliminary investigation into the reversal mechanism. ADR was loaded onto the GO surface (ADR-GO) by physical mixing and drug loading content was found to be high, up to 93.6%. In vitro releases of ADR from ADR-GO were studied using a dialysis method, and they exhibited a significant pH-sensitive property. Cell experiments showed that GO significantly enhanced the accumulation of ADR in MCF-7/ADR cells (an ADR resistant breast cancer cell line) and exhibited much higher cytotoxicity than free ADR, suggesting that ADR-GO could effectively reverse ADR resistance of MCF-7/ADR, with the reversal index reaching 8.35. Microscopy studies found that GO could effectively carry drug molecules into cells in both endocytosis-dependent and independent manners. In conclusion, use of GO as a carrier for chemotherapeutic agents is favorable for the treatment of drug resistant cancers.     

Diagnosis of breast cancer by analysis of sialic acid concentrations in human saliva by surface-enhanced Raman spectroscopy of silver nanoparticles

Breast cancer is the most common type of malignant tumor among women and their second leading cause of cancer-related deaths.The most common method for screening and diagnosis is mammography.Nonetheless,two main problems have been identified.First,the dose of radiation received during the test prevents the method from the use on women who are ＜ 40 years old.Second,there can be mammogram failure owing to the lack of tumor contrast with the fibrous tissue.Therefore,there is a need for screening methods that will help to identify high-risk cases.We developed a biological marker test that can help to identify them.Increased levels of sialic acid (SA) in saliva are known to correlated with breast cancer.In this study,we evaluated the feasibility of Raman spectroscopy as a method for quantification of SA in saliva,using citrate-reduced silver nanoparticles (cit-Ag-NPs) as a surface-enhanced Raman spectroscopy (SERS) substrate.Quantification of SA was accomplished by measuring its intensity in saliva and comparing it with a calibration curve of SA standards.The mean SA concentration in saliva was found to be significantly higher among 100 breast cancer patients (18.3 ± 9.4 mg·dL-1;mean ± SD) than among 106 healthy controls (3.5 ± 1.0 mg·dL-1).The SERS test showed sensitivity of 94％ and specificity 98％ for detection of patients with breast cancer,assuming that SA concentration ＞ 7 mg·dL-1 is a cutoff for positive test results.Our findings prove the usefulness of this SERS technique as a simple,convenient,and highly sensitive method of quantitative analysis of SA in saliva.The simplicity of this nanotechnological test may help to substantially reduce the mortality among patients with breast cancer by providing women with a simple,noninvasive screening test that can be applied regardless of age or density of breast tissue.