Synthesis and biological assay of erlotinib analogues and BSA-conjugated erlotinib analogue.

A series of erlotinib analogues that have structural modification at 6,7-alkoxyl positions is efficiently synthesized. The in vitro anti-tumor activity of synthesized compounds is studied in two non-small cell lung cancer (NSCLC) cell lines (A549 and H1975). Among the synthesized compounds, the iodo compound 6 (ETN-6) exhibits higher anti-cancer activity compared to erlotinib. An efficient method is developed for the conjugation of erlotinib analogue-4, alcohol compound, with protein, bovine serum albumin (BSA), via succinic acid linker. The in vitro anti-tumor activity of the protein attached erlotinib analogue, 8 (ETN-4-Suc-BSA), showed stronger inhibitory activity in both A549 and H1975 NSCLC cell lines.

Microwave-assisted efficient conjugation of nanodiamond and paclitaxel.

Nanodiamond has recently received considerable attention due to the various possible applications in medical field such as drug delivery and bio-labeling. For this purpose suitable and effective surface functionalization of the diamond material are required. A versatile and reproducible surface modification method of nanoscale diamond is essential for functionalization. We introduce the input of microwave energy to assist the functionalization of nanodiamond surface. The feasibility of such a process is illustrated by comparing the biological assay of ND-paclitaxel synthesized by conventional and microwave irradiating. Using a microwave we manage to have approximately doubled grafted molecules per nanoparticle of nanodiamond.

Osimertinib Induces the Opposite Effect of Proliferation and Migration in the Drug Resistance of EGFR-T790M Non-small Cell Lung Cancer Cells.

BACKGROUND: Lung cancer has become one of the leading causes of cancer incidence and mortality worldwide. Non-small cell lung carcinoma (NSCLC) is the most common type among all lung cancer cases. NSCLC patients contained high levels of activating epidermal growth factor receptor (EGFR) mutations, such as exon 19 deletion, L858R and T790M. Osimertinib, a third-generation of EGFR tyrosine kinase inhibitor (EGFR-TKI), has therapeutic efficacy on the EGFR-T790M mutation of NSCLC patients; however, treatment of osimertinib still can induce drug resistance in lung cancer patients. Therefore, investigation of the drug resistance mechanisms of osimertinib will provide novel strategies for lung cancer therapy. METHODS: The H1975OR osimertinib-resistant cell line was established by prolonged exposure with osimertinib derived from the H1975 cells. The cell proliferation ability was evaluated by the cell viability and cell growth assays. The cell migration ability was determined by the Boyden chamber assays. The differential gene expression profile was analyzed by genome-wide RNA sequencing. The protein expression and location were analyzed by western blot and confocal microscopy. RESULTS: In this study, we established the osimertinib-resistant H1975 (T790M/L858R) cancer cells, named the H1975OR cell line. The cell growth ability was decreased in the H1975OR cells by comparison with the H1975 parental cells. Conversely, the cell migration ability was elevated in the H1975OR cells. We found the differential gene expression profile of cell proliferation and migration pathways between the H1975OR and H1975 parental cells. Interestingly, the protein levels of phospho-EGFR, PD-L1, E-cadherin and ß-catenin were decreased, but the survivin and N-cadherin proteins were increased in the H1975OR drug-resistant cells. CONCLUSION: Osimertinib induces the opposite effect of proliferation and migration in the drug resistance of EGFRT790M lung cancer cells. We suggest that differential gene and protein expressions in the cell proliferation and migration pathways may mediate the drug resistance of osimertinib in lung cancer cells. Understanding the molecular drugresistant mechanisms of proliferation and migration pathways of osimertinib may provide novel targets and strategies for the clinical treatment of EGFR-TKIs in lung cancer patients.

Targeting EGFR and Monitoring Tumorigenesis of Human Lung Cancer Cells In Vitro and In Vivo Using Nanodiamond-Conjugated Specific EGFR Antibody.

Nanoprobes provide advantages for real-time monitoring of tumor markers and tumorigenesis during cancer progression and development. Epidermal growth factor receptor (EGFR) is a key protein that plays crucial roles for tumorigenesis and cancer therapy of lung cancers. Here, we show a carbon-based nanoprobe, nanodiamond (ND), which can be applied for targeting EGFR and monitoring tumorigenesis of human lung cancer cells in vitro and in vivo. The optimal fluorescent intensities of ND particles were observed in the human lung cancer cells and nude mice under in vivo imaging system. The fluorescence signal of ND particles can be real-time detected in the xenografted human lung tumor formation of nude mice. Moreover, the ND-conjugated specific EGFR antibody cetuximab (Cet) can track the location and distribution of EGFR proteins of lung cancer cells in vitro and in vivo. ND-Cet treatment increased cellular uptake ability of nanocomposites in the EGFR-expressed cells but not in the EGFR-negative lung cancer cells. Interestingly, single ND-Cet complex can be directly observed on the protein G bead by immunoprecipitation and confocal microscopy. Besides, the EGFR proteins were transported to lysosomes for degradation. Together, this study demonstrates that ND-conjugated Cet can apply for targeting EGFR and monitoring tumorigenesis during lung cancer progression and therapy.

A novel EGFR inhibitor suppresses survivin expression and tumor growth in human gefitinib-resistant EGFR-wild type and -T790M non-small cell lung cancer.

Tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR-TKIs) are currently used therapy for non-small cell lung cancer (NSCLC) patients; however, drug resistance during cancer treatment is a critical problem. Survivin is an anti-apoptosis protein, which promotes cell proliferation and tumor growth that highly expressed in various human cancers. Here, we show a novel synthetic compound derived from gefitinib, do-decyl-4-(4-(3-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)propyl) piper-azin-1-yl)-4-oxobutanoate, which is named as SP101 that inhibits survivin expression and tumor growth in both the EGFR-wild type and -T790M of NSCLC. SP101 blocked EGFR kinase activity and induced apoptosis in the A549 (EGFR-wild type) and H1975 (EGFR-T790M) lung cancer cells. SP101 reduced survivin proteins and increased active caspase 3 for inducing apoptosis. Ectopic expression of survivin by a survivin-expressed vector attenuated the SP101-induced cell death in lung cancer cells. Moreover, SP101 inhibited the gefitinib-resistant tumor growth in the xenograft human H1975 lung tumors of nude mice. SP101 substantially reduced survivin proteins but conversely elicited active caspase 3 proteins in tumor tissues. Besides, SP101 exerted anticancer abilities in the gefitinib resistant cancer cells separated from pleural effusion of a clinical lung cancer patient. Consistently, SP101 decreased the survivin proteins and the patient-derived xenografted lung tumor growth in nude mice. Anti-tumor ability of SP101 was also confirmed in the murine lung cancer model harboring EGFR T790M-L858R. Together, SP101 is a new EGFR inhibitor with inhibiting survivin that can be developed for treating EGFR wild-type and EGFR-mutational gefitinib-resistance in human lung cancers.

Covalent linkage of nanodiamond-paclitaxel for drug delivery and cancer therapy.

A nanoparticle-conjugated cancer drug provides a novel strategy for cancer therapy. In this study, we manipulated nanodiamond (ND), a carbon nanomaterial, to covalently link paclitaxel for cancer drug delivery and therapy. Paclitaxel was bound to the surface of 3-5 nm sized ND through a succession of chemical modifications. The ND-paclitaxel conjugation was measured by atomic force microscope and nuclear magnetic resonance spectroscopy, and confirmed with infrared spectroscopy by the detection of deuterated paclitaxel. Treatment with 0.1-50 microg ml(-1) ND-paclitaxel for 48 h significantly reduced the cell viability in the A549 human lung carcinoma cells. ND-paclitaxel induced both mitotic arrest and apoptosis in A549 cells. However, ND alone or denatured ND-paclitaxel (after treatment with strong alkaline solution, 1 M NaOH) did not induce the damage effects on A549 cells. ND-paclitaxel was taken into lung cancer cells in a concentration-dependent manner using flow cytometer analysis. The ND-paclitaxel particles were located in the microtubules and cytoplasm of A549 cells observed by confocal microscopy. Furthermore, ND-paclitaxel markedly blocked the tumor growth and formation of lung cancer cells in xenograft SCID mice. Together, we provide a functional covalent conjugation of ND-paclitaxel, which can be delivered into lung carcinoma cells and preserves the anticancer activities on the induction of mitotic blockage, apoptosis and anti-tumorigenesis.

Targeting EGFR of triple-negative breast cancer enhances the therapeutic efficacy of paclitaxel- and cetuximab-conjugated nanodiamond nanocomposite.

Breast cancer is the most common malignancy and a leading cause of cancer-related mortality among women worldwide. Triple-negative breast cancer (TNBC) is characterized by the lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). However, epidermal growth factor receptor (EGFR) is highly expressed in most of the TNBCs, which may provide a potential target for EGFR targeting therapy. Nanodiamond (ND) is a carbon-based nanomaterial with several advantages, including fluorescence emission, biocompatibility, and drug delivery applications. In this study, we designed a nanocomposite by using ND conjugated with paclitaxel (PTX) and cetuximab (Cet) for targeting therapy on the EGFR-positive TNBC cells. ND-PTX inhibited cell viability and induced mitotic catastrophe in various human breast cancer cell lines (MDA-MB-231, MCF-7, and BT474); in contrast, ND alone did not induce cell death. ND-PTX inhibited the xenografted human breast tumors in nude mice. We further investigated ND-PTX-Cet drug efficacy on the TNBC of MDA-MB-231 breast cancer cells. ND-PTX-Cet could specifically bind to EGFR and enhanced the anticancer effects including drug uptake levels, mitotic catastrophe, and apoptosis in the EGFR-expressed MDA-MB-231 cells but not in the EGFR-negative MCF-7 cells. In addition, ND-PTX-Cet increased the protein levels of active caspase-3 and phospho-histone H3 (Ser10). Furthermore, ND-PTX-Cet showed more effective on the reduction of TNBC tumor volume by comparison with ND-PTX. Taken together, these results demonstrated that ND-PTX-Cet nanocomposite enhanced mitotic catastrophe and apoptosis by targeting EGFR of TNBC cells, which can provide a feasible strategy for TNBC therapy. STATEMENT OF SIGNIFICANCE: Current TNBC treatment is ineffective against the survival rate of TNBC patients. Therefore, the development of new treatment strategies for TNBC patients is urgently needed. Here, we have designed a nanocomposite by targeting on the EGFR of TNBC to enhance therapeutic efficacy by ND-conjugated PTX and Cet (ND-PTX-Cet). Interestingly, we found that the co-delivery of Cet and PTX by ND enhanced the apoptosis, mitotic catastrophe and tumor inhibition in the EGFR-expressed TNBC in vitro and in vivo. Consequently, this nanocomposite ND-PTX-Cet can be applied for targeting EGFR of human TNBC therapy.

Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway.

Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.

Co-delivery of paclitaxel and cetuximab by nanodiamond enhances mitotic catastrophe and tumor inhibition.

The poor intracellular uptake and non-specific binding of anticancer drugs into cancer cells are the bottlenecks in cancer therapy. Nanocarrier platforms provide the opportunities to improve the drug efficacy. Here we show a carbon-based nanomaterial nanodiamond (ND) that carried paclitaxel (PTX), a microtubule inhibitor, and cetuximab (Cet), a specific monoclonal antibody against epidermal growth factor receptor (EGFR), inducing mitotic catastrophe and tumor inhibition in human colorectal cancer (CRC). ND-PTX blocked the mitotic progression, chromosomal separation, and induced apoptosis in the CRC cells; however, NDs did not induce these effects. Conjugation of ND-PTX with Cet (ND-PTX-Cet) was specifically binding to the EGFR-positive CRC cells and enhanced the mitotic catastrophe and apoptosis induction. Besides, ND-PTX-Cet markedly decreased tumor size in the xenograft EGFR-expressed human CRC tumors of nude mice. Moreover, ND-PTX-Cet induced the mitotic marker protein phospho-histone 3 (Ser10) and apoptotic protein active-caspase 3 for mitotic catastrophe and apoptosis. Taken together, this study demonstrated that the co-delivery of PTX and Cet by ND enhanced the effects of mitotic catastrophe and apoptosis in vitro and in vivo, which may be applied in the human CRC therapy.

Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds.

Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker ß-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

The effect of fluorescent nanodiamonds on neuronal survival and morphogenesis.

Nanodiamond (ND) has emerged as a promising carbon nanomaterial for therapeutic applications. In previous studies, ND has been reported to have outstanding biocompatibility and high uptake rate in various cell types. ND containing nitrogen-vacancy centers exhibit fluorescence property is called fluorescent nanodiamond (FND), and has been applied for bio-labeling agent. However, the influence and application of FND on the nervous system remain elusive. In order to study the compatibility of FND on the nervous system, neurons treated with FNDs in vitro and in vivo were examined. FND did not induce cytotoxicity in primary neurons from either central (CNS) or peripheral nervous system (PNS); neither did intracranial injection of FND affect animal behavior. The neuronal uptake of FNDs was confirmed using flow cytometry and confocal microscopy. However, FND caused a concentration-dependent decrease in neurite length in both CNS and PNS neurons. Time-lapse live cell imaging showed that the reduction of neurite length was due to the spatial hindrance of FND on advancing axonal growth cone. These findings demonstrate that FNDs exhibit low neuronal toxicity but interfere with neuronal morphogenesis, and should be taken into consideration when applications involve actively growing neurites (e.g. nerve regeneration).

The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent.

Nanocarbon is a promising type of biomaterial for diagnostic and therapeutic applications. Fluorescent nanodiamond (FND) containing nitrogen-vacancy centers as built-in fluorophores is a new addition to the nanocarbon family. Here, we study the long-term stability and biocompatibility of 100-nm FNDs in rats through intraperitoneal injection over 5 months and develop the potential application of this biomaterial for sentinel lymph node mapping in a mouse model. From both in vivo and ex vivo fluorescence imaging as well as transmission electron microscopy, we found that the intradermally administered FND particles can be drained from the injection sites by macrophages and selectively accumulated in the axillary lymph nodes of the treated mice. Our measurements of water consumption, fodder consumption, body weight, and organ index showed no significant difference between control and FND-treated groups of the rats. Histopathological analysis of various tissues and organs indicated that FNDs are non-toxic even when a large quantity, up to 75 mg/kg body weight, of the particles was administered intraperitoneally to the living animals. With the properties of wide-ranging biocompatibility and perfect chemical and photophysical stability, FND is well suited for use as a contrast agent for long-term in vivo imaging.

Cancer cell labeling and tracking using fluorescent and magnetic nanodiamond.

Nanodiamond, a promising carbon nanomaterial, develops for biomedical applications such as cancer cell labeling and detection. Here, we establish the nanodiamond-bearing cancer cell lines using the fluorescent and magnetic nanodiamond (FMND). Treatment with FMND particles did not significantly induce cytotoxicity and growth inhibition in HFL-1 normal lung fibroblasts and A549 lung cancer cells. The fluorescence intensities and particle complexities were increased in a time- and concentration-dependent manner by treatment with FMND particles in lung cancer cells; however, the existence of FMND particles inside the cells did not alter cellular size distribution. The FMND-bearing lung cancer cells could be separated by the fluorescent and magnetic properties of FMNDs using the flow cytometer and magnetic device, respectively. The FMND-bearing cancer cells were identified by the existence of FMNDs using flow cytometer and confocal microscope analysis. More importantly, the cell morphology, viability, growth ability and total protein expression profiles in the FMND-bearing cells were similar to those of the parental cells. The separated FMND-bearing cells with various generations were cryopreservation for further applications. After re-thawing the FMND-bearing cancer cell lines, the cells still retained the cell survival and growth ability. Additionally, a variety of human cancer types including colon (RKO), breast (MCF-7), cervical (HeLa), and bladder (BFTC905) cancer cells could be used the same strategy to prepare the FMND-bearing cancer cells. These results show that the FMND-bearing cancer cell lines, which reserve the parental cell functions, can be applied for specific cancer cell labeling and tracking.

7-Chloro-6-piperidin-1-yl-quinoline-5,8-dione (PT-262), a novel ROCK inhibitor blocks cytoskeleton function and cell migration.

The 5,8-quinolinediones are precursors for producing multiple types of bioactive products. In this study, we investigated a new compound derived from 5,8-quinolinediones, 7-chloro-6-piperidin-1-yl-quinoline-5,8-dione (designated as PT-262), which markedly induced cytoskeleton remodeling and migration inhibition in lung carcinoma cells. Comparison with various cytoskeleton inhibitors, including paclitaxel, colchicine and phallacidin, the cell morphology following treatment with PT-262 was similar to phallacidin on the cell elongation and abnormal actin polymerization. However, PT-262 did not directly bind to actin filaments. ROCK (Rho-associated coiled-coil forming protein kinase) is a downstream effector of RhoA to mediate the phosphorylation of myosin light chain (MLC) and cytoskeleton reorganization. The RhoA-ROCK-MLC pathway has been shown to promote cancer cell migration and metastasis. Interestingly, PT-262 was more effective on inhibiting ROCK kinase activities than specific ROCK inhibitors Y-27632 and H-1152. PT-262 induced cytoskeleton remodeling and migration inhibition in A549 lung carcinoma cells. The total MLC and phosphorylated MLC proteins and stress fibers were blocked after treatment with PT-262. Nonetheless, the RhoA protein and GTPase activity were not altered by PT-262. A computational model suggests that PT-262 interacts with the ATP-binding site of ROCK protein. Together, these findings demonstrate that PT-262 is a new ROCK inhibitor.

Endocytic carboxylated nanodiamond for the labeling and tracking of cell division and differentiation in cancer and stem cells.

Nanodiamond (ND) is carbon nanomaterial developing for biological applications in recent years. In this study, we investigated the location and distribution of 100 nm carboxylated ND particles in cell division and differentiation. ND particles were taken into cells by macropinocytosis and clathrin-mediated endocytosis pathways. However, the cell growth ability was not altered by endocytic ND particles after long-term cell culture for 10 days in both A549 lung cancer cells and 3T3-L1 embryonic fibroblasts. ND particles were equal separating into two daughter cells of cell division approximately. Finally, the cell retained a single ND's cluster in cytoplasm after sub-cultured for several generations. Interestingly, ND's clusters were carried inside of cell but without inducing damages after long-term cell culture. Moreover, ND particles did not interfere with the gene or protein expressions on the regulation of cell cycle progression and adipogenic differentiation. Together, these findings provide that endocytic ND particles are non-cytotoxic in cell division and differentiation, which can be applied for the labeling and tracking of cancer and stem cells.

Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond.

Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin (α-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (α7-nAChR). The electrostatic binding of cND-α-BTX was mediated by the negative charge of the cND and the positive charge of the α-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled α-BTX presented a yellow color at the same location, which indicated that α-BTX proteins were conjugated with cND particles. Xenopus laevis's oocytes expressed the human α7-nAChR proteins by microinjection with α7-nAChR mRNA. The cND-α-BTX complexes were bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked α7-nAChR-mediated inward currents of the oocytes were blocked by cND-α-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-α-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated α-BTX still preserves its biological activity in blocking the function of α7-nAChR, and provide a visual system showing the binding of α-BTX to α7-nAChR.

Nanometer-sized diamond particle as a probe for biolabeling.

A novel method is proposed using nanometer-sized diamond particles as detection probes for biolabeling. The advantages of nanodiamond's unique properties were demonstrated in its biocompatibility, nontoxicity, easily detected Raman signal, and intrinsic fluorescence from its natural defects without complicated pretreatments. Carboxylated nanodiamond's (cND's) penetration ability, noncytotoxicity, and visualization of cND-cell interactions are demonstrated on A549 human lung epithelial cells. Protein-targeted cell interaction visualization was demonstrated with cND-lysozyme complex interaction with bacteria Escherichia coli. It is shown that the developed biomolecule-cND complex preserves the original functions of the test protein. The easily detected natural fluorescent and Raman intrinsic signals, penetration ability, and low cytotoxicity of cNDs render them promising agents in multiple medical applications.