Advanced three-dimensional in vitro liver models to study the activity of anticancer drugs.

The liver is one of the most important organs in the human body. It performs many important functions, including being responsible for the metabolism of most drugs, which is often associated with its drug-induced damage. Currently, there are no ideal pharmacological models that would allow the evaluation of the effect of newly tested drugs on the liver in preclinical studies. Moreover, the influence of hepatic metabolism on the effectiveness of the tested drugs is rarely evaluated. Therefore, in this work we present an advanced model of the liver, which reflects most of the morphologically and metabolically important features of the liver in vivo, namely: three-dimensionality, cellular composition, presence of extracellular matrix, distribution of individual cell types in the structure of the liver model, high urea and albumin synthesis efficiency, high cytochrome p450 activity. In addition, the work, based on the example of commonly used anticancer drugs, shows how important it is to take into account hepatic metabolism in the effective assessment of their impact on the target organ, in this case cancer. In our research, we have shown that the most similar to liver in vivo are 3D cellular aggregates composed of three important liver cells, namely hepatocytes (HepG2), hepatic stellate cells (HSCs), and hepatic sinusoidal endothelial cells (HSECs). Moreover, we showed that the cells in 3D aggregate structure need time (cell-cell interactions) to improve proper liver characteristic. The triculture model additionally showed the greatest ability to metabolize selected anticancer drugs.

Review: 3D cell models for organ-on-a-chip applications.

Two-dimensional (2D) cultures do not fully reflect the human organs' physiology and the real effectiveness of the used therapy. Therefore, three-dimensional (3D) models are increasingly used in bioanalytical science. Organ-on-a-chip systems are used to obtain cellular in vitro models, better reflecting the human body's in vivo characteristics and allowing us to obtain more reliable results than standard preclinical models. Such 3D models can be used to understand the behavior of tissues/organs in response to selected biophysical and biochemical factors, pathological conditions (the mechanisms of their formation), drug screening, or inter-organ interactions. This review characterizes 3D models obtained in microfluidic systems. These include spheroids/aggregates, hydrogel cultures, multilayers, organoids, or cultures on biomaterials. Next, the methods of formation of different 3D cultures in Organ-on-a-chip systems are presented, and examples of such Organ-on-a-chip systems are discussed. Finally, current applications of 3D cell-on-a-chip systems and future perspectives are covered.

Vascularized tumor-on-chip microplatforms for the studies of neovasculature as hope for more effective cancer treatments.

Angiogenesis is the development of new blood vessels from pre-existing vasculature. Multiple factors control its course. Disorders of the distribution of angiogenic agents are responsible for development of solid tumors and its metastases. Understanding of the molecular interactions regulating pathological angiogenesis will allow for development of more effective, even personalized treatment. A simulation of angiogenesis under microflow conditions is a promising alternative to previous studies conducted on animals and on 2D cell cultures. In this review, we summarize what has been discovered so far in the field of vascularized tumor-on-a-chip platforms. For this purpose, we describe different vascularization techniques used in microfluidics, present various attempts to induce angiogenesis-on-a-chip and report some approaches to recapitulate vascularized tumor microenvironment under microflow conditions.

Oxidation-derived anticancer potential of sumanene-ferrocene conjugates.

An effective synthetic protocol towards the oxidation of sumanene-ferrocene conjugates bearing one to four ferrocene moieties has been established. The oxidation protocol was based on the transformation of FeII from ferrocene to FeIII-containing ferrocenium cations by means of the treatment of the title organometallic buckybowls with a mild oxidant. Successful isolation of these ferrocenium-tethered sumanene derivatives 5-7 gave rise to the biological evaluation of the first, buckybowl-based anticancer agents, as elucidated by in vitro assays with human breast adenocarcinoma cells (MDA-MB-231) and embryotoxicity trials in zebrafish embryos supported with in silico toxicology studies. The designed ferrocenium-tethered sumanene derivatives featured attractive properties in terms of their use in cancer treatments in humans. The tetra-ferrocenium sumanene derivative 7 featured especially beneficial biological features, elucidated by low (<40% for 10 µM) viabilities of MDA-MB-231 cancer cells together with a 1.4-1.7-fold higher viability of normal cells (human mammary fibroblasts, HMF) for respective concentrations. Compound 7 featured significant cytotoxicity against cancer cells thanks to the presence of sumanene and ferrocenium moieties; the latter motif also provided the selectivity of anticancer action. The biological properties of 7 were also improved in comparison with those of native building blocks, which suggested the effects of the presence of the sumanene skeleton towards the anticancer action of this molecule. Ferrocenium-tethered sumanene derivatives exhibited potential towards the generation of reactive oxygen species (ROS), responsible for biological damage to the cancer cells, with the most efficient generation of the tetra-ferrocenium sumanene derivative 7. Derivative 7 also did not show any embryotoxicity in zebrafish embryos at the tested concentrations, which supports its potential as an effective and cancer-specific anticancer agent. In silico computational analysis also showed no chromosomal aberrations and no mutation with AMES tests for the compound 7 tested with and without microsomal rat liver fractions, which supports its further use as a potent drug candidate in detailed anticancer studies.

Development of 5-fluorouracil-dichloroacetate mutual prodrugs as anticancer agents.

5-Fluorouracil (5-FU) is one of the most widely applied chemotherapeutic agents with a broad spectrum of activity. However, despite this versatile activity, its use poses many limitations. Herein, novel derivatives of 5-FU and dichloroacetic acid have been designed and synthesized as a new type of codrugs, also known as mutual prodrugs, to overcome the drawbacks of 5-FU and enhance its therapeutic efficiency. The stability of the obtained compounds has been tested at various pH values using different analytical techniques, namely HPLC and potentiometry. The antiproliferative activity of the new 5-FU derivatives was assessed in vitro on SK-MEL-28 and WM793 human melanoma cell lines in 2D culture as well as on A549 human lung carcinoma, MDA-MB-231 breast adenocarcinoma, LL24 normal lung tissue, and HMF normal breast tissue as a multicellular 3D spheroid model cultured in standard (static) conditions and with the use of microfluidic systems, which to a great extent resembles the in vivo environment. In all cases, new mutual prodrugs showed a higher cytotoxic activity toward cancer models and lower to normal cell models than the parent 5-FU itself.

Cellular uptake of biotransformed graphene oxide into lung cells.

Due to its high surface area and convenient functionalization, graphene oxide has many potential applications in biomedicine, especially as a drug carrier. However, knowledge about its internalization inside mammalian cells is still limited. Graphene oxide cellular uptake is a complex phenomenon affected by factors such as the size of the particle and modifications of its surface. Moreover, nanomaterials introduced into living organisms interact with biological fluids' components. It may further alter its biological properties. All these factors must be considered when the cellular uptake of potential drug carriers is considered. In this study, the effect of graphene oxide particle sizes on internalization efficiency into normal (LL-24) and cancerous (A549) human lung cells was investigated. Moreover, one set of samples was incubated with human serum to determine how the interaction of graphene oxide with serum components affects its structure, surface, and interaction with cells. Our findings indicate that samples incubated with serum enhance cell proliferation but enter the cells with lesser efficiency than their counterparts not incubated with human serum. What is more affinity towards the cells was higher for larger particles.

Graphene oxide internalization into mammalian cells - a review.

Due to the high surface area and convenient functionalization, graphene oxide has a significant potential application in biomedicine. It can serve as multi-purpose platform for bioimaging, gene and drug delivery, photothermal and photodynamic therapy. To implement graphene oxide in diagnostics and therapeutics successfully, it is essential to investigate its mechanisms of uptake into mammalian cells thoroughly. Herein, up to date knowledge about graphene oxide internalization pathways is presented. Nanomaterial lateral dimensions, surface modifications and biotransformation phenomenon as well as a type of the cell may play a pivotal role in a graphene oxide cellular uptake. Hence, the impact of these factors is comprehensively discussed based on so far published studies. Although great progress has been made in elucidating graphene oxide internalization pathway, there are still challenges to overcome. These are discussed along with the prospects concerning further studies in this field of science.

Exploring Endothelial Expansion on a Chip.

Angiogenesis is the development of new blood vessels from the existing vasculature. Its malfunction leads to the development of cancers and cardiovascular diseases qualified by the WHO as a leading cause of death worldwide. A better understanding of mechanisms regulating physiological and pathological angiogenesis will potentially contribute to developing more effective treatments for those urgent issues. Therefore, the main goal of the following study was to design and manufacture an angiogenesis-on-a-chip microplatform, including cylindrical microvessels created by Viscous Finger Patterning (VFP) technique and seeded with HUVECs. While optimizing the VFP procedure, we have observed that lumen's diameter decreases with a diminution of the droplet's volume. The influence of Vascular Endothelial Growth Factor (VEGF) with a concentration of 5, 25, 50, and 100 ng/mL on the migration of HUVECs was assessed. VEGF's solution with concentrations varying from 5 to 50 ng/mL reveals high angiogenic potential. The spatial arrangement of cells and their morphology were visualized by fluorescence and confocal microscopy. Migration of HUVECs toward loaded angiogenic stimuli has been initiated after overnight incubation. This research is the basis for developing more complex vascularized multi-organ-on-a-chip microsystems that could potentially be used for drug screening.

Graphene 2D platform is safe and cytocompatibile for HaCaT cells growing under static and dynamic conditions.

The study concerns the influence of graphene monolayer, as a 2 D platform, on cell viability, cytoskeleton, adhesions sites andmorphology of mitochondria of keratinocytes (HaCaT) under static conditions. Based on quantitative and immunofluorescent analysis, it could be stated that graphene substrate does not cause any damage to membrane or disruption of other monitored parameters. Spindle poles and cytokinesis bridges indicating proliferation of cells on this graphene substrate were detected. Moreover, the keratinocyte migration rate on the graphene substrate was comparable to control glass substrate when the created wound was completely closed after 38 hours. HaCaT morphology and viability were also assessed under dynamic conditions (lab on a chip - micro scale). For this purpose, microfluidic graphene system was designed and constructed. No differences as well as no anomalies were observed during cultivation of these cells on the graphene or glass substrates in relation to cultivation conditions: static (macro scale) and dynamic (micro scale). Only natural percentage of dead cells was determined using different methods, which proved that the graphene as the 2 D platform is cytocompatible with keratinocytes. The obtained results encourage the use of the designed lab on a chip system in toxicity testing of graphene also on other cells and further research on the use of graphene monolayers to produce bio-bandages for skin wounds in animal tests.

Multiorgan-on-a-Chip: A Systemic Approach To Model and Decipher Inter-Organ Communication.

Multiorgan-on-a-chip (multi-OoC) platforms have great potential to redefine the way in which human health research is conducted. After briefly reviewing the need for comprehensive multiorgan models with a systemic dimension, we highlight scenarios in which multiorgan models are advantageous. We next overview existing multi-OoC platforms, including integrated body-on-a-chip devices and modular approaches involving interconnected organ-specific modules. We highlight how multi-OoC models can provide unique information that is not accessible using single-OoC models. Finally, we discuss remaining challenges for the realization of multi-OoC platforms and their worldwide adoption. We anticipate that multi-OoC technology will metamorphose research in biology and medicine by providing holistic and personalized models for understanding and treating multisystem diseases.

A biocompatible poly(amidoamine) (PAMAM) dendrimer octa-substituted with α-cyclodextrin towards the controlled release of doxorubicin hydrochloride from its ferrocenyl prodrug.

Facile and efficient methods for the synthesis of the first poly(aminodamine) PAMAM G1.0 dendrimer octa-substituted with α-cyclodextrin and a novel ferrocenyl prodrug of doxorubicin hydrochloride are developed. This vector is non-toxic and can bind the designed ferrocenyl prodrug. It also shows a controlled drug release profile and high cytotoxicity against breast cancer cells (MCF-7), as elucidated by the in vitro biological studies performed with an innovative cell-on-a-chip microfluidic system.

Well-defined Graphene Oxide as a Potential Component in Lung Cancer Therapy.

BACKGROUND: Graphene oxide (GO) has unique physical and chemical properties that can be used in anticancer therapy - especially as a drug carrier. Graphene oxide, due to the presence of several hybrid layers of carbon atoms (sp2), has a large surface for highly efficient drug loading. In addition, GO with a large number of carboxyl, hydroxyl and epoxy groups on its surface, can charge various drug molecules through covalent bonds, hydrophobic interactions, hydrogen bonds and electrostatic interactions. OBJECTIVE: The aim of our work was to evaluate the possibility of future use of graphene oxide as an anticancer drug carrier. METHODS: In this paper, we present GO synthesis and characterization, as well as a study of its biological properties. The cytotoxic effect of well-defined graphene oxide was tested on both carcinoma and non-malignant cells isolated from the same organ, which is not often presented in the literature. RESULTS: The performed research confirmed that GO in high concentrations (> 300 µgmL-1) selectively decreased the viability of cancer cell line. Additionally, we showed that the GO flakes have a high affinity to cancer cell nucleus which influences their metabolism (inhibition of cancer cell proliferation). Moreover, we have proved that GO in high concentrations can cause cell membrane damage and generate reactive oxygen species on a low level mainly in cancer cells. CONCLUSION: The proposed GO could be useful in anticancer therapy. A high concentration of GO selectively causes the death of tumor cells, whereas GO with low concentration could be a potential material for anticancer drug loading.

Functionalization of graphene: does the organic chemistry matter?

Reactions applying amidation- or esterification-type processes and diazonium salts chemistry constitute the most commonly applied synthetic approaches for the modification of graphene-family materials. This work presents a critical assessment of the amidation and esterification methodologies reported in the recent literature, as well as a discussion of the reactions that apply diazonium salts. Common misunderstandings from the reported covalent functionalization methods are discussed, and a direct link between the reaction mechanisms and the basic principles of organic chemistry is taken into special consideration.

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.

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.

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).