Silica nanostructured platform for affinity capture of tumor-derived exosomes

Early diagnosis of prostate cancer and evaluation of appropriate treatment options requires development of effective and high-throughput selective capture technology for exosomes that are positive for the expression of enzyme-biomarker, prostate-specific membrane antigen (PSMA). Exosomes are small secreted vesicles that play a key role in intercellular communication and cancer progression. PSMA is highly enriched in exosomes excreted by PSMA+ prostate cancer cells. Using PSMA+ cells from the well-established prostate cancer cell line (LNCaP), the secreted exosomes were collected and isolated from the culture medium. The tumor-derived exosomes were selectively captured using a novel silica nanostructure support that had been functionalized with the small-molecule ligand TG97, a known inhibitor of PSMA enzymatic activity that binds irreversibly in the active site of PSMA. The concept was demonstrated using a single cancer type (i.e., prostate cancer), but based on the data obtained the approach may be applicable to a broad panel of biomarker ligands for selective capture of biomarker-positive exosomes from an array of cell types. The approach demonstrated herein overcomes many of the limitations of alternative methods that are often ineffective in isolating tumor-derived exosomes from those derived from normal tissue because of the low yield recovery and the time required for the process. A further advantage is the ability to isolate a specific subpopulation of exosomes relying on the expression of a specific surface marker as well as improved exosome recovery rate.     

Superporous Poly(ethylene glycol) Diacrylate Cryogel with a Defined Elastic Modulus for Prostate Cancer Cell Research

The physical and mechanical properties of the tumor microenvironment are crucial for the growth, differentiation and migration of cancer cells. However, such microenvironment is not found in the geometric constraints of 2D cell culture systems used in many cancer studies. Prostate cancer research, in particular, suffers from the lack of suitable in vitro models. Here a 3D superporous scaffold is described with thick pore walls in a mechanically stable and robust architecture to support prostate tumor growth. This scaffold is generated from the cryogelation of poly(ethylene glycol) diacrylate to produce a defined elastic modulus for prostate tumor growth. Lymph node carcinoma of the prostate (LNCaP) cells show a linear growth over 21 d as multicellular tumor spheroids in such a scaffold with points of attachments to the walls of the scaffold. These LNCaP cells respond to the growth promoting effects of androgens and demonstrate a characteristic cytoplasmic‐nuclear translocation of the androgen receptor and androgen‐dependent gene expression. Compared to 2D cell culture, the expression or androgen response of prostate cancer specific genes is greatly enhanced in the LNCaP cells in this system. This scaffold is therefore a powerful tool for prostate cancer studies with unique advantages over 2D cell culture systems.     

Selective cytotoxic effect of ZnO nanoparticles on glioma cells

In this study we examined the cytotoxic effect of ZnO nanoparticles on various human cancer and normal cells. We found that the ZnO nanoparticles exerted a cytotoxic effect on the human glioma cell lines A172, U87, LNZ308, LN18, and LN229, whereas no cytotoxic effect was observed on normal human astrocytes. Similarly, the ZnO nanoparticles induced cell death in breast and prostate cancer cell lines while no major effect was observed in the respective normal breast and prostate cell lines. Using the fluorescent dye 2,7-dichlorofluorescein diacetate, we found that treatment of the glioma cells with ZnO nanoparticles induced a large increase in the generation of reactive oxygen species (ROS) and treatment of the cells with N-acetyl cysteine decreased the cytotoxic effect of the ZnO nanoparticles. In contrast, a smaller effect on ROS generation was observed in the normal astrocytes. These results suggest that ZnO nanoparticles may be employed as a selective cytotoxic agent for the eradication of cancer cells.     

Lectin-based biosensor strategy for electrochemical assay of glycan expression on living cancer cells

In this article, we report a novel lectin-based biosensor for electrochemical assay of cancer-associated glycosylation by comparative study of mannose and sialic acid expression on normal and cancer cells derived from human lung, liver, and prostate. Using a sandwich format, high sensitivity and selectivity were achieved by combining the lectin-based biosensor with the {lectin-Au-Th} bioconjugates featuring lectin and thionine (Th) labels linked to gold nanoparticles (AuNPs) for signal amplification. The proposed strategy demonstrated that mannose exhibited high expression levels in both normal and cancer cells, while sialic acid was more abundant in cancer cells as compared to normal ones. The results were in good agreement with those from fluorescent microscopy studies. The differences in the two glycan expression indicated that sialic acid could serve as a potential biomarker for early cancer detection. The lectin-based biosensor was also successfully used to quantify cancer cells and evaluate the average amount of sialic acid on single cell surface, which could supply significant information on glycan functions in cancer progression. Overall, the lectin-based electrochemical biosensor provides an effective pathway to analyze glycan expression on living cells and may greatly facilitate the medical diagnosis and treatment in early process of cancer.     

Cancer‐Cell‐Phenotype‐Dependent Differential Intracellular Trafficking of Unconjugated Quantum Dots

A diverse array of nanoparticles, including quantum dots (QDs), metals, polymers, liposomes, and dendrimers, are being investigated as therapeutics and imaging agents in cancer diseases. However, the role of the cancer‐cell phenotype on the uptake and intracellular fate of nanoparticles in cancer cells remains poorly understood. Reported here is that differences in cancer‐cell phenotypes can lead to significant differences in intracellular sorting, trafficking, and localization of nanoparticles. Unconjugated anionic QDs demonstrate dramatically different intracellular profiles in three closely related human‐prostate‐cancer cells used in the investigation: PC3, PC3‐flu, and PC3‐PSMA. QDs demonstrate punctated intracellular localization throughout the cytoplasm in PC3 cells. In contrast, the nanoparticles localize mainly at a single juxtanuclear location (“dot‐of‐dots”) inside the perinuclear recycling compartment in PC3‐PSMA cells, where they co‐localize with transferrin and the prostate‐specific membrane antigen. The results indicate that nanoparticle sorting and transport is influenced by changes in cancer‐cell phenotype and can have significant implications in the design and engineering of nanoscale drug delivery and imaging systems for advanced tumors.     

Versatile, fully automated, microfluidic cell culture system

There is increasing demand for automated and quantitative cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambers and maintains cell viability for weeks. Individual culture conditions are customized in terms of cell seeding density, composition of culture medium, and feeding schedule, and each chamber is imaged with time-lapse microscopy. Using this device, we perform the first quantitative measurements of the influence of transient stimulation schedules on the proliferation, osteogenic differentiation, and motility of human primary mesenchymal stem cells.     

Interaction of fibroblast with poly(p-dioxanone) and its degradation products

In vitro techniques were used to evaluate the interactions between Fibroblastic cells and Poly(p-Dioxanone) PPDX and compared its performance with that of other polymeric substrates. In vitro biocompatibility was assessed by studying cell adhesion and cell growth of cells on the polymer films themselves as well as in media enriched with the degradation products of PPDX and Poly(glycolic)/Poly(L-lactide 90:10 copolymer (PGLA-910). Our results show that althought all polymers tested were suitable for initial attachment, PPDX proved to be the most favorable surface for cell growth; as cell density after 48 h of culture was similar to that obtained on tissue culture Polystyrene TCPS (control). No signs of cell damage were detected using scanning electron microscopy (SEM) where after 48 h. of cell seeding on PPDX, fibroblast exhibited a confluent cell multilayer similar to TCPS. In addition, the products of the hydrolytic degradation of PPDX had no citotoxic effect on the adherence and proliferation of fibroblastic cell on TCPS. The hydrolytic degradation starts in the amorphous regions, as the tie-chain segments in these regions degrade into fragments causing the pH decrease in buffer solution and weight loss of degradable polymers. The in vitro evaluation suggests that PPDX may be candidate biomaterial for the construction a cell-polymer matrix.     

Study of rotational dynamics of receptor-targeted contrast agents in cancerous and normal prostate tissues using time-resolved picosecond emission spectroscopy

We studied the time-resolved polarization-dependent fluorescence spectroscopy of receptor-targeted contrast agents (Cybesin and Cytate) bound with prostate cancer cells in prostate tissue. An analytical model dealing with highly viscous tissue media was developed and used to investigate the rotation times and fluorescence anisotropies of the receptor-targeted contrast agents in prostate tissue. The differences of rotation times and fluorescence anisotropies were observed for Cybesin (Cytate) in cancerous and normal prostate tissues, which reflect changes of the microstructures of cancerous and normal tissues and their different bound affinity with contrast agents. The preferential uptake of Cytate (Cybesin) in cancerous tissue was used to image and distinguish cancerous tissue areas from normal tissue areas. The fluorescence polarization difference imaging technique was used to enhance the image contrast between the cancerous and normal tissue areas. This research may help to introduce a new optical approach and criteria for prostate cancer detection.     

Intact endothelial cell sheet harvesting from thermoresponsive surfaces coated with cell adhesion promoters.

Recently, with the development of smart polymers, research has looked to using thermoresponsive polymers as cell culture substrates. These novel surfaces allow the cultivation of cells without enzymes using the thermoresponsive phase transition property of poly(N-isopropylacrylamide) (PNIPAAm). However, this requires expensive techniques to generate a sufficiently thin film that allows cell adhesion. In this study, we looked at simple solvent cast films which normally show poor cell adhesion, but here the films are coated with cell adhesion promoters (CAPs) to improve cell growth without altering the copolymer thermoresponsive behaviour.A copolymer of PNIPAAm and N-tert-butylacrylamide (NtBAm) with a ratio of 85:15, respectively, was synthesized and solvent cast. The copolymer films were coated with CAPs, such as collagen, fibronectin and laminin, to increase their cell adhesion and growth properties. Cell activity measured by the alamarBlue assay showed similar results for coated copolymer films and standard tissue culture plastic controls. Deposition of CAPs on to the copolymer films was characterized by scanning electron microscopy and atomic force microscopy. Cell detachment from the copolymer films is not affected by the surface coatings of CAPs, and endothelial cells are recovered as an intact sheet, which has great potential for uses in tissue engineering applications. The results demonstrate a versatile method for the cultivation of cells while eliminating the need for the use of digestive enzymes such as trypsin. This study shows that cultivation on physically bonded PNIPAAm copolymers is viable and achievable by relatively simple methods.     

Image and fractal analysis of osteoblastic cells in viscous media

The aim of the present study was to determine whether osteoblastic morphology was affected by the viscosity of culture media and whether any morphological differences could be readily quantified. A cytochemical stain was used for alkaline phosphatase and a combination of image and fractal analysis, utilising seven morphological parameters to assess the morphology of osteoblasts. Using regression analysis it was determined that as the viscosity of the culture media increased the area of the cells significantly decreased and the fractal dimension of the cell profiles significantly increased. A discriminant function analysis was used to examine whether cell populations could be classified according to culturing time or the viscosity of culturing media based on the seven morphological parameters. It was determined that the cells could be classified up to 93% correct according to the viscosity of the media they were cultured in and up to 93.5% correct according to the culturing time. This study demonstrated that viscous media affects the morphology of osteoblastic cells and that discriminant function analysis can be used to classify these cells based on their morphological parameters.     

Highly-efficient single-cell capture in microfluidic array chips using differential hydrodynamic guiding structures

This paper presents a highly efficient single cell capture scheme using hydrodynamic guiding structures in a microwell array. The implemented structure has a capturing efficiency of >80%, and has a capacity to place individual cells into separated microwells, allowing for the time-lapse monitoring on single cell behavior. Feasibility was tested by injecting microbeads (15 μm in diameter) and prostate cancer PC3 cells in an 8×8 microwell array chip and >80% of the microwells were occupied by single ones. Using the chips, the number of cells required for cell assays can be dramatically reduced and this will facilitate overcoming a huddle of assays with scarce supply of cells.     

Microfluidic, label-free enrichment of prostate cancer cells in blood based on acoustophoresis

Circulating tumor cells (CTC) are shed in peripheral blood at advanced metastatic stages of solid cancers. Surface-marker-based detection of CTC predicts recurrence and survival in colorectal, breast, and prostate cancer. However, scarcity and variation in size, morphology, expression profile, and antigen exposure impairs reliable detection and characterization of CTC. We have developed a noncontact, label-free microfluidic acoustophoresis method to separate prostate cancer cells from white blood cells (WBC) through forces generated by ultrasonic resonances in microfluidic channels. Implementation of cell prealignment in a temperature-stabilized (±0.5 °C) acoustophoresis microchannel dramatically enhanced the discriminatory capacity and enabled the separation of 5 μm microspheres from 7 μm microspheres with 99% purity. Next, we determined the feasibility of employing label-free microfluidic acoustophoresis to discriminate and divert tumor cells from WBCs using erythrocyte-lysed blood from healthy volunteers spiked with tumor cells from three prostate cancer cell-lines (DU145, PC3, LNCaP). For cells fixed with paraformaldehyde, cancer cell recovery ranged from 93.6% to 97.9% with purity ranging from 97.4% to 98.4%. There was no detectable loss of cell viability or cell proliferation subsequent to the exposure of viable tumor cells to acoustophoresis. For nonfixed, viable cells, tumor cell recovery ranged from 72.5% to 93.9% with purity ranging from 79.6% to 99.7%. These data contribute proof-in-principle that label-free microfluidic acoustophoresis can be used to enrich both viable and fixed cancer cells from WBCs with very high recovery and purity.     

Osteogenic Response of C2C12 Cells on Thermoreversible Polymers

Bone Morphogenetic Proteins (BMPs) in combination with biomaterials are being explored for clinical bone regeneration. The current biomaterials used for BMPs delivery are not specifically designed to support BMP‐induced osteogenesis. Towards this goal, we designed synthetic N‐isopropylacrylamide (NiPAM)‐based thermosensitive polymers and investigated their ability to support osteogenic transformation of pluripotent C2C12 cells. Cell attachment to the polymers was limited as compared to attachment to the plastic surfaces optimized for cell culture. Short‐term (<7 days) studies indicated relatively little cell growth on the polymer surfaces. However, C2C12 cells retained their ability to respond to BMP‐2, as determined by alkaline phosphatase (ALP) induction, when cultured on thermoreversible polymers. Some polymers supported ALP induction that was far superior (～10‐fold) to cells grown on tissue culture surfaces. We conclude that thermosensitive polymers, although limited in their ability to support cell attachment and growth, did support the pluripotent cells' ability to be transformed under the influence of BMP‐2. The ALP induction was dependent on the compositional details of the polymers, suggesting that in vivo osteoinduction was likely to be influenced by the physicochemical properties of the polymers.     

Quantum dot-aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on bi-fluorescence resonance energy transfer

We report a novel quantum dot (QD)-aptamer(Apt)-doxorubicin (Dox) conjugate [QD-Apt(Dox)] as a targeted cancer imaging, therapy, and sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed a targeted QD imaging system (QD-Apt) that is capable of differential uptake and imaging of prostate cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results in a targeted QD-Apt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET mechanism. A donor-acceptor model fluorescence resonance energy transfer (FRET) between QD and Dox and a donor-quencher model FRET between Dox and aptamer result when Dox intercalated within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD, which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.     

Ibrutinib conjugated surface-functionalized multiwalled carbon nanotubes and its biopolymer composites for targeting prostate carcinoma

In this report, nanocomposites are composed of surface-functionalized multi-walled carbon nanotubes (f-MWCNTs) and polymers (polyethylene glycol, chitosan) or silver nanoparticles (AgNPs) were successfully synthesized and used as nanocarriers for drug delivery. The drug (Ibrutinib, Ibr) encapsulated with different nanocomposites was used for effective prostate cancer treatment. The as-prepared bioconjugates were confirmed by Fourier transform infrared spectroscopy, X-ray diffraction, and Raman spectroscopy. The drug loading efficiency of 95.5% was achieved for f-MWCNTs/AgNPs composite. The drug release profile showed that the f-MWCNTs/AgNPs composite released 79% in 84 h at pH 5.5 indicating the sustainable drug release. Further, these Ibr-loaded nanocomposites were conjugated with T₃₀ oligonucleotides (T₃₀ ODN) for targeting over-expressed prostate-specific membrane antigen in the prostate gland. The prostate anti-cancer activity was evaluated using PC-3 and MDA-MB-231 cancer cells and the results indicated that the Ibr- loaded nanocomposites conjugated with T₃₀ ODN exhibited higher cell killing efficiency compared to the free Ibr. Therefore, these conjugated nanocomposites are effective drug delivery systems for prostate cancer disease targeted therapy.

     

AFM-based analysis of human metastatic cancer cells

Recently biomechanics of cancer cells, in particular stiffness or elasticity, has been identified as an important factor relating to cancer cell function, adherence, motility, transformation and invasion. We report on the nanomechanical responses of metastatic cancer cells and benign mesothelial cells taken from human body cavity fluids using atomic force microscopy. Following our initial study (Cross et al 2007 Nat. Nanotechnol. 2 780-3), we report on the biophysical properties of patient-derived effusion cells and address the influence of cell morphology on measured cell stiffness. Using a cytocentrifugation method, which yields morphologically indistinguishable cells that can be prepared in 1?min and avoids any possible artifacts due to 12?h ex?vivo culture, we find that metastatic tumor cells are more than?80% softer than benign cells with a distribution over six times narrower than that of normal cells. Consistent with our previous study, which yielded distinguishable cell populations based on ex?vivo growth and morphological characteristics, our results show it is unlikely that morphology alone is sufficient to explain the difference in elastic moduli for these two cell types. Moreover, analysis of non-specific cell adhesion inherent to tumor and normal cells collected from patients show surface adhesion of tumor cells is ～33% less adhesive compared to that of normal cells. Our findings indicate that biomechanical-based functional analysis may provide an additional platform for cytological evaluation and diagnosis of cancer in the future.     

Dielectric cell response in highly conductive buffers

We present a novel method for the identification of live and dead T-cells, dynamically flowing within highly conductive buffers. This technique discriminates between live and dead (heat treated) cells on the basis of dielectric properties variations. The key advantage of this technique lies in its operational simplicity, since cells do not have to be resuspended in isotonic low conductivity media. Herein, we demonstrate that at 40 MHz, we are able to statistically distinguish between live and dead cell populations.     

Seamless signal transduction from live cells to an NO sensor via a cell-adhesive sensing matrix

A smart live-cell assay was developed as a cellular biosensing system. This system is based on novel tactics: the direct assembly of human cultured cells onto a cell-adhesive sensing matrix. This novel design provides considerable advantages, among them the possibility of capturing molecular signals immediately after they are secreted from living cells. The design also helps preserve all cellular characteristics intact. In this study, a cell-adhesive NO sensing matrix, acting as both an NO-permeable membrane and a cell-adhesive scaffold, was designed using functional polymers and a short peptide sequence derived from extracellular matrix (ECM) proteins. Using the cell-adhesive NO sensing matrix, we constructed a cellular biosensing system based on in situ monitoring of NO released from a human umbilical vein endothelial cell (HUVEC) layer. HUVECs were employed as an organ-functional model of a blood vessel in view of screening vasodilatory substances for clinical purposes. In our novel system, the electrochemical NO sensor is adjacent to the NO-producing cells, which allows the sensing device to achieve superior sensitivity and precise response to a very low number of NO molecules. Our design enables the fixing of the exact distance between the organ-functional model and the chemical sensor without cumbersome manipulations. Consequently, this cellular biosensing system may be readily applicable to high-throughput analysis in the field of drug screening.     

Compatibility of different polymers for cord blood-derived hematopoietic progenitor cells

The low yield of hematopoietic progenitor cells (HPC) present in cord blood grafts limits their application in clinics. A reliable strategy for ex vivo expansion of functional HPC is a present goal in regenerative medicine. Here we evaluate the capacity of several two-dimensional polymers to support HPC proliferation. Basic compatibility was tested by measuring cell viability, cytotoxicity and apoptosis of CD34⁺ progenitors that were short and long-term exposed to sixteen bio and synthetic polymers. Resomer^? RG503, PCL and Fibrin might be good alternatives to tissue culture plastic for culture of CB-derived CD34⁺ progenitors. Further, these polymers will be produced in three-dimensional structures and tested for their cytocompatibility.