A layered cancer-on-a-chip system for anticancer drug screening and disease modeling.

Recent advances in the development of microfluidic systems for the culture of complex and three-dimensional cell, tissue, and organ models allow their use in toxicity studies and mimicking many diseases. These types of in vitro models are important because of the huge advantages over standard two-dimensional cell cultures: better mimicking of in vivo conditions and more reliable response to the tested drugs. This report presents a new approach to modeling skin cancer (melanoma-on-a-chip) and breast cancer (breast cancer-on-a-chip) using the microfluidic systems. We designed a microfluidic device to co-culture cancer cells with non-malignant cells, which are the main component of the cancer microenvironment. In the construction of the microsystem, we used a scaffold in the form of a porous membrane made of poly(ethylene terephthalate), which enables the regular and reproducible arrangement of cells in the culture and maintains intercellular communication. To demonstrate the functionality of the microsystem, we used it to analyze the effectiveness of photodynamic therapy in the treatment of melanoma and chemotherapy in the treatment of breast cancer. The developed microsystem can be successfully used to model cancer diseases, especially with a layered arrangement of cells in the cancerous tissue, such as melanoma, ductal breast cancer, or breast cancer metastases to the skin.

A Novel Approach for the Creation of Electrically Controlled LC:PDMS Microstructures.

This work presents research on unique optofluidic systems in the form of air channels fabricated in PDMS and infiltrated with liquid crystalline material. The proposed LC:PDMS structures represent an innovative solution due to the use of microchannel electrodes filled with a liquid metal alloy. The latter allows for the easy and dynamic reconfiguration of the system and eliminates technological issues experienced by other research groups. The paper discusses the design, fabrication, and testing methods for tunable LC:PDMS structures. Particular emphasis was placed on determining their properties after applying an external electric field, depending on the geometrical parameters of the system. The conclusions of the performed investigations may contribute to the definition of guidelines for both LC:PDMS devices and a new class of potential sensing elements utilizing polymers and liquid crystals in their structures.

The 10th anniversary of MXenes: Challenges and prospects for their surface modification toward future biotechnological applications.

A broad family of two-dimensional (2D) materials - carbides, nitrides, and carbonitrides of early transition metals, called MXenes, became a newcomer in the flatland at the turn of 2010 and 2011 (over ten years ago). Their unique physicochemical properties made them attractive for many applications, highly boosting the development of various fields, including biotechnological. However, MXenes' functional features that impact their bioactivity and toxicity are still not fully well understood. This study discusses the essentials for MXenes's surface modifications toward their application in modern biotechnology and nanomedicine. We survey modification strategies in context of cytotoxicity, biocompatibility, and most prospective applications ready to implement in medical practice. We put the discussion on the material-structure-chemistry-property relationship into perspective and concentrate on overarching challenges regarding incorporating MXenes into nanostructured organic/inorganic bioactive architectures. It is another emerging group of materials that are interesting from the biomedical point of view as well. Finally, we present an influential outlook on the growing demand for future research in this field.

Degradable nanohydrogel with high doxorubicin loadings exhibiting controlled drug release and decreased toxicity against healthy cells.

A new nanogel/drug carrier of 100-150 nm size, based on poly(N-isopropylacrylamide-co-sodium acrylate) and degradable crosslinker (cystine derivative), was synthesized. Using the electrostatic interactions between the carboxylic groups in the polymer network and the protonated amine groups of doxorubicin it was possible to load the drug into the carrier to a very high level of 28-30% relative to the dry mass of the polymer. The presence of the -S-S- groups made the polymer network susceptible to degradation by glutathione. The size of the nanoparticles was small enough to enable them to easily penetrate the cells. The MTT assay indicated that compared to free doxorubicin the nanogel particles loaded with doxorubicin were more cytotoxic against the MCF-7 and A2780 cancer cells, while they were 150 times less toxic against the MCF-10A healthy cells. The new carrier nanoparticles appeared also to be useful for prolonged drug delivery.

Future Applications of MXenes in Biotechnology, Nanomedicine, and Sensors.

The past few years have seen significant developments in the chemistry and potential biological applications of 2D materials. This review focuses on recent advances in the biotechnological and biomedical applications of MXenes, which are 2D carbides, nitrides, and carbonitrides of transition metals. Nanomaterials based on MXenes can be used as therapeutics for anticancer treatment, in photothermal therapy as drug delivery platforms, or as nanodrugs without any additional modification. Furthermore, we discuss the potential use of these materials in biosensing and bioimaging, including magnetic resonance and photoacoustic imaging techniques. Finally, we present the most significant examples of the use of MXenes as efficient agents for environmental and antimicrobial treatments, as well as a brief discussion of their future prospects and challenges.

Magnetic field-assisted selective delivery of doxorubicin to cancer cells using magnetoliposomes as drug nanocarriers.

Magnetoliposomes are promising candidates for the development of selective drug delivery systems in the treatment of cancer. Those nanosystems were tested as carriers of a strong chemotherapeutic agent, doxorubicin, which is used against breast cancer. Herein, the magnetic properties of hydrophobic iron oxide nanoparticles located exclusively in the lipid bilayer were used to release this drug from the magnetoliposomes. The cytotoxic activity of the nanostructures against the normal and cancer cell lines was determined on the basis of cells viability measurement after incubation with different concentrations of these nanomaterials. In the same way, the effectiveness of killing cancer cells in combination with exposure to magnetic field was also evaluated. These experiments confirmed that the nanostructures composed of liposomes and magnetic nanoparticles are not cytotoxic. However, magnetoliposomes loaded with doxorubicin were effective and selective in reducing the viability of human breast tumor cell lines. In this paper, we demonstrated the promising application of the studied magnetoliposomes as carriers of doxorubicin released under the influence of magnetic field in tumor cells.

2D Ti2C (MXene) as a novel highly efficient and selective agent for photothermal therapy.

Photothermal therapy (PTT) has shown significant potential for anti-cancer modality. In this report, according to our best knowledge, we explore for the first time Ti2C-based MXene as a novel, highly efficient and selective agent for photothermal therapy (PTT). Ti2C superficially modified with PEG was obtained from the layered, commercially available Ti2AlC MAX phase in the process of etching aluminum layers using concentrated HF, and characterized by scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HREM) as well as X-ray photoelectron spectroscopy for chemical analysis (ESCA-XPS). The PEG-coated Ti2C flakes showed a satisfactory photothermal conversion efficacy (PTCE) and good biocompatibility in wide range of the tested concentrations. Through in vitro studies, the PEG-modified Ti2C demonstrated notable NIR-induced ability to cancerous cells' ablation with minimal impact on non-malignant cells up to the concentration of 37.5 µg mL-1. The applied doses of Ti2C_PEG in our work were even 24 times lower comparing other MXene-based photothermal agents. This work is expected to expand the utility of 2D MXenes to biomedical applications through the development of entirely novel agents for photothermal therapy. This work is expected to expand the utility of 2D MXenes to biomedical applications through the development of entirely novel agents for photothermal therapy.

The influence of selected ω-mercaptocarboxylate ligands on physicochemical properties and biological activity of Cd-free, zinc­copper­indium sulfide colloidal nanocrystals.

In this work core/shell cadmium-free zinc­copper­indium sulfide ZnCuInS/ZnS quantum dots (QDs) originally stabilized with hydrophilic alkanethiol were modified with 3-mercaptopropionic acid (MPA) or 6-mercaptohexanoic acid (MHA) via two-step ligand exchange method. The obtained QDs were further characterized by TEM, UV Vis, and fluorescence spectroscopy. Both types of QDs were non-toxic in a wide range of concentrations. To our knowledge, our studies are the first attempt to determine the type of cell death and reactive oxygen species production level as a result of incubation of cell cultures with ZnCuInS/ZnS QDs. Furthermore, the accumulation of QDs in vitro was examined on three human cell lines by fluorescence intensity measurements and visualized by confocal microscopy. The modification of QDs with a ligand characterized by slightly longer aliphatic chain (MHA), instead of typically used MPA turns out to be beneficial both from the point of colloidal stability, preservation of optical properties during ligand exchange as well as reflects in a higher cellular uptake. This contribution can be beneficial from the point of view of the selection of the optimal ligands and concentrations in the case of ZnCuInS/ZnS core-shell QDs for biological applications.

Studies on effectiveness of PTT on 3D tumor model under microfluidic conditions using aptamer-modified nanoshells.

Herein, we present the research focused on the synthesis and application of aptamer-modified gold nanoshells for photothermal therapy (PTT). NIR-absorbing hollow gold nanoshells were synthetized and conjugated with anti-MUC1 aptamer (HGNs@anti-MUC1). MUC1 (Mucin 1) is a transmembrane glycoprotein, which is overexpressed in a variety of epithelial cancers (eg. breast, lung, pancreatic). In order to evaluate the efficiency of PTT with HGNs@anti-MUC1 we used 3D cell culture model - multicellular spheroids. The selected cell culture model is considered as the best in vitro model for cancer research (similar morphology, metabolite and oxygen gradients, cellular interactions and cell growth kinetics in the spheroids are similar to the early stage of a nonvascular tumor). We conducted our research on human normal (MRC-5, MCF-10A) and tumor (A549, MCF-7) cell lines using a microfluidic system. Aptamer-modified nanoparticles were accumulated selectively in tumor cells (A549, MCF-7) and this fact contributed to the reduction of tumor spheroids viability and size. It should be underlined, that it is the first example of photothermal therapy carried out in a microsystem on multicellular spheroids.

Biological characterization of the modified poly(dimethylsiloxane) surfaces based on cell attachment and toxicity assays.

Poly(dimethylsiloxane) (PDMS) is a material applicable for tissue and biomedical engineering, especially based on microfluidic devices. PDMS is a material used in studies aimed at understanding cell behavior and analyzing the cell adhesion mechanism. In this work, biological characterization of the modified PDMS surfaces based on cell attachment and toxicity assays was performed. We studied Balb 3T3/c, HMEC-1, and HT-29 cell adhesion on poly(dimethylsiloxane) surfaces modified by different proteins, with and without pre-activation with plasma oxygen and UV irradiation. Additionally, we studied how changing of a base and a curing agent ratios influence cell proliferation. We observed that cell type has a high impact on cell adhesion, proliferation, as well as viability after drug exposure. It was tested that the carcinoma cells do not require a highly specific microenvironment for their proliferation. Cytotoxicity assays with celecoxib and oxaliplatin on the modified PDMS surfaces showed that normal cells, cultured on the modified PDMS, are more sensitive to drugs than cancer cells. Cell adhesion was also tested in the microfluidic systems made of the modified PDMS layers. Thanks to that, we studied how the surface area to volume ratio influences cell behavior. The results presented in this manuscript could be helpful for creation of proper culture conditions during in vitro tests as well as to understand cell response in different states of disease depending on drug exposure.

3D lung spheroid cultures for evaluation of photodynamic therapy (PDT) procedures in microfluidic Lab-on-a-Chip system.

The purpose of this paper is to present a fully integrated microchip for the evaluation of PDT procedures efficiency on 3D lung spheroid cultures. Human lung carcinoma A549 and non-malignant MRC-5 spheroids were utilized as culture models. Spheroid viability was evaluated 24 h after PDT treatment, in which 5-aminolevulinic acid (ALA) had been used as a precursor of a photosensitizer (protoporphyrin IX - PpIX). Moreover, spheroid viability over a long-term (10-day) culture was also examined. We showed that the proposed PDT treatment was toxic only for cancer spheroids. This could be because of a much-favoured enzymatic conversion of ALA to PpIX in cancer as opposed normal cells. Moreover, we showed that to obtain high effectiveness of ALA-PDT on lung cancer spheroids additional time of spheroid after light exposure was required. It was found that PDT had been effective 5 days after PDT treatment with 3 mM ALA. To the best of our knowledge this has been the first presentation of such research performed on a 3D lung spheroids culture in a microfluidic system.

Heart-on-a-Chip: An Investigation of the Influence of Static and Perfusion Conditions on Cardiac (H9C2) Cell Proliferation, Morphology, and Alignment.

Lab-on-a-chip systems are increasingly used as tools for cultures and investigation of cardiac cells. In this article, we present how the geometry of microsystems and microenvironmental conditions (static and perfusion) influence the proliferation, morphology, and alignment of cardiac cells (rat cardiomyoblasts-H9C2). Additionally, studies of cell growth after incubation with verapamil hydrochloride were performed. For this purpose, poly(dimethylsiloxane) (PDMS)/glass microfluidic systems with three different geometries of microchambers (a circular chamber, a longitudinal channel, and three parallel microchannels separated by two rows of micropillars) were prepared. It was found that static conditions did not enhance the growth of H9C2 cells in the microsystems. On the contrary, perfusion conditions had an influence on division, morphology, and the arrangement of the cells. The highest number of cells, their parallel orientation, and their elongated morphology were obtained in the longitudinal microchannel. It showed that this kind of microsystem can be used to understand processes in heart tissue in detail and to test newly developed compounds applied in the treatment of cardiac diseases.

Studies of anticancer drug cytotoxicity based on long-term HepG2 spheroid culture in a microfluidic system.

Cell-on-a-chip systems have become promising devices to study the effectiveness of new anticancer drugs recently. Several microdevices for liver cancer culture and evaluation of the drug cytotoxicity have been reported. However, there are still no proven reports about high-throughput and simple methods for the evaluation of drug cytotoxicity on liver cancer cells. The paper presents the results of the effects of the anticancer drug (5-fluorouracil, 5-FU) on the HepG2 spheroids as a model of liver cancer. The experiments were based on the long-term 3D spheroid culture in the microfluidic system and monitoring of the effect of 5-FU at two selected concentrations (0.5 mM and 1.0 mM). Our investigations have shown that the initial size of the spheroids has influence on the drug effect. With the increase of the spheroids diameter, the drug resistance (for the two tested 5-FU concentrations) decreases. This phenomenon was observed both through cells metabolism analysis, as well as changes in spheroids sizes. In our research, we have shown that the lower 5-FU (0.5 mM) concentration causes higher decrease in HepG2 spheroids viability. Moreover, due to the microsystem construction, we observe the drug resistance effect (10th day of culture) regardless of the initial size of the created spheroids and the drug concentration.

Evaluation of nanoencapsulated verteporfin's cytotoxicity using a microfluidic system.

A new-generation of nanoencapsulated photosensitizers could be a good solution to perform effective photodynamic therapy (PDT). In this paper, we present physicochemical characterization and cellular investigation of newly prepared long-sustained release oil-core polyelectrolyte nanocarriers loaded with verteporfin (nano VP) in relation to free VP. For this purpose, a macroscale multiwell plates and multifunctional microfluidic system (for three types of cell cultures: monoculture, coculture and mixed culture) were used. A physical analysis of nano VP showed its high stability, monodispersity with unimodal shape and highly positive charge, what made them good candidates for cancer treatment. Biological properties (cellular internalization and uptake as well as cytotoxicity) of nano and free VP were evaluated using both carcinoma (A549) and normal (MRC-5) human lung cells. It was investigated that verteporfin was accumulated in cancer cells preferentially. Low cytotoxicity of the tested photosensitizer was observed in both macro, and microscale. However, in experiments performed in the microsystem, nano VP allowed the reduction of cytotoxic effect, especially in relation to the normal cells. It could result from the specific environment of cell growth in the microsystem which can quite closely mimic the in vivo conditions. Our results suggest that the presented microsystem could be a very useful microtool for testing of new generation of photosensitizers in various configurations of cell cultures, which are difficult to perform in the macroscale. Moreover, the prepared nano VP could be successfully used for further research i.e. evaluation of PDT procedures.

Microfluidic platform for photodynamic therapy cytotoxicity analysis of nanoencapsulated indocyanine-type photosensitizers.

The application of nanotechnology is important to improve research and development of alternative anticancer therapies. In order to accelerate research related to cancer diagnosis and to improve the effectiveness of cancer treatment, various nanomaterials are being tested. The main objective of this work was basic research focused on examination of the mechanism and effectiveness of the introduction of nanoencapsulated photosensitizers to human carcinoma (A549) and normal cells (MRC-5). Newly encapsulated hydrophobic indocyanine-type photosensitizer (i.e., IR-780) was subjected to in vitro studies to determine its release characteristics on a molecular level. The photosensitizers were delivered to carcinoma and normal cells cultured under model conditions using multiwell plates and with the use of the specially designed hybrid (poly(dimethylsiloxane) (PDMS)/glass) microfluidic system. The specific geometry of our microsystem allows for the examination of intercellular interactions between cells cultured in the microchambers connected with microchannels of precisely defined length. Our microsystem allows investigating various therapeutic procedures (e.g., photodynamic therapy) on monoculture, coculture, and mixed culture, simultaneously, which is very difficult to perform using standard multiwell plates. In addition, we tested the cellular internalization of nanoparticles (differing in size, surface properties) in carcinoma and normal lung cells. We proved that cellular uptake of nanocapsules loaded with cyanine IR-780 in carcinoma cells was more significant than in normal cells. We demonstrated non cytotoxic effect of newly synthesized nanocapsules built with polyelectrolytes (PEs) of opposite surface charges: polyanion-polysodium-4-styrenesulphonate and polycation-poly(diallyldimethyl-ammonium) chloride loaded with cyanine IR-780 on human lung carcinoma and normal cell lines. However, the differences observed in the photocytotoxic effect between two types of tested nanocapsules can result from the type of last PE layer and their different surface charge.

Adhesion of MRC-5 and A549 cells on poly(dimethylsiloxane) surface modified by proteins.

PDMS is a very popular material used for fabrication of Lab-on-a-Chip systems for biological applications. Although PDMS has numerous advantages, it is a highly hydrophobic material, which inhibits adhesion and proliferation of the cells. PDMS surface modifications are used to enrich growth of the cells. However, due to the fact that each cell type has specific adhesion, it is necessary to optimize the parameters of these modifications. In this paper, we present an investigation of normal (MRC-5) and carcinoma (A549) human lung cell adhesion and proliferation on modified PDMS surfaces. We have chosen these cell types because often they are used as models for basic cancer research. To the best of our knowledge, this is the first presentation of this type of investigation. The combination of a gas-phase processing (oxygen plasma or ultraviolet irradiation) and wet chemical methods based on proteins' adsorption was used in our experiments. Different proteins such as poly-l-lysine, fibronectin, laminin, gelatin, and collagen were incubated with the activated PDMS samples. To compare with other works, here, we also examined how ratio of prepolymer to curing agent (5:1, 10:1, and 20:1) influences PDMS hydrophilicity during further modifications. The highest adhesion of the tested cells was observed for the usage of collagen, regardless of PDMS ratio. However, the MRC-5 cell line demonstrated better adhesion than A549 cells. This is probably due to the difference in their morphology and type (normal/cancer).

Effect of downscaling on the linearity range of a calibration curve in spectrofluorimetry.

Interest in the microfluidic environment, owing to its unique physical properties, is increasing in much innovative chemical, biological, or medicinal research. The possibility of exploiting and using new phenomena makes the microscale a powerful tool to improve currently used macroscopic methods and approaches. Previously, we reported that an increase in the surface area to volume ratio of a measuring cell could provide a wider linear range for fluorescein (Kwapiszewski et al., Anal. Bioanal. Chem. 403:151-155, 2012). Here, we present a broader study in this field to confirm the assumptions we presented before. We studied fluorophores with a large and a small Stokes shift using a standard cuvette and fabricated microfluidic detection cells having different surface area to volume ratios. We analyzed the effect of different configurations of the detection cell on the measured fluorescence signal. We also took into consideration the effect of concentration on the emission spectrum, and the effect of the surface area to volume ratio on the limit of linearity of the response of the selected fluorophores. We observed that downscaling, leading to an increase in the probability of collisions between molecules and cell walls with no energy transfer, results in an increase in the limit of linearity of the calibration curve of fluorophores. The results obtained suggest that microfluidic systems can be an alternative to the currently used approaches for widening the linearity of a calibration curve. Therefore, microsystems can be useful for studies of optically dense samples and samples that should not be diluted.

A microfluidic system to study the cytotoxic effect of drugs: the combined effect of celecoxib and 5-fluorouracil on normal and cancer cells.

We have investigated the response of normal and cancer cells to exposure a combination of celecoxib (Celbx) and 5-fluorouracil (5-FU) using a lab-on-a-chip microfluidic device. Specifically, we have tested the cytotoxic effect of Celbx on normal mouse embryo cells (Balb/c 3T3) and human lung carcinoma cells (A549). The single drugs or their combinations were adjusted to five different concentrations using a concentration gradient generator (CGG) in a single step. The results suggest that Celbx can enhanced the anticancer activity of 5-FU by stronger inhibition of cancer cell growth. We also show that the A549 cancer cells are more sensitive to Celbx than the Balb/c 3T3 normal cells. The results obtained with the microfluidic system were compared to those obtained with a macroscale in vitro cell culture method. In our opinion, the microfluidic system represents a unique approach for an evaluation of cellular response to multidrug exposure that also is more simple than respective microwell plate assays. Figureᅟ

Long-term three-dimensional cell culture and anticancer drug activity evaluation in a microfluidic chip.

In this work, we present a microfluidic array of microwells for long-term tumor spheroid cultivation and anticancer drug activity evaluation. The three-dimensional microfluidic system was obtained by double casting of poly(dimethylsiloxane). Spheroids of HT-29 human carcinoma cells were cultured in the microsystem for four weeks. After two weeks of the culture growth slowdown and stop were observed and high cell viability was determined within next two weeks. The characteristics of a homeostasis-like state were achieved. A cytostatic drug (5-fluorouracil) was introduced into the microsystem with different frequency (every day or every second day) and different concentrations. The geometry and construction of the microsystem enables flushing away of unaggregated (including dead) cells while viable spheroids remain inside microwells and decreasing spheroid diameter can be observed and measured as an indicator of decreasing cell viability. The results have shown differences in response of spheroids to different concentrations of 5-fluorouracil. It was also observed, that higher frequency of drug dosing resulted in more rapid spheroid diameter decrease. The presented microfluidic system is a solution for cell-based studies in an in vivo-like microfluidic environment. Moreover, observation of decreasing spheroid dimensions is a low-cost, label-free and easy-to-conduct mean of a quantitative determination of a 3D cellular model response to a applied drug. It is suitable for long-term observation of spheroid response, in a contrary to other viability assays requiring termination of a culture.

Effect of a high surface-to-volume ratio on fluorescence-based assays.

In the work discussed in this paper, the effect of a high surface-to-volume ratio of a microfluidic detection cell on fluorescence quenching was studied. It was found that modification of the geometry of a microchannel can provide a wider linear range. This is a phenomenon which should be taken into consideration when microfluidic systems with fluorescence detection are developed. The dependence of the linear range for fluorescein on the surface-to-volume ratio was determined. Both fluorescence inner-filter effects and concentration self-quenching were taken into consideration. It was found that inner-filter effects have little effect on the extent of the linear range on the microscale.