BBA - General Subjects (v.1810, #3)

Special issue on “Nanotechnologies: Emerging applications in biomedicine” by Agneta Richter-Dahlfors; Peter Kjäll (237-238).

Engineering systems for the generation of patterned co-cultures for controlling cell–cell interactions by Hirokazu Kaji; Gulden Camci-Unal; Robert Langer; Ali Khademhosseini (239-250).
Inside the body, cells lie in direct contact or in close proximity to other cell types in a tightly controlled architecture that often regulates the resulting tissue function. Therefore, tissue engineering constructs that aim to reproduce the architecture and the geometry of tissues will benefit from methods of controlling cell–cell interactions with microscale resolution.We discuss the use of microfabrication technologies for generating patterned co-cultures. In addition, we categorize patterned co-culture systems by cell type and discuss the implications of regulating cell–cell interactions in the resulting biological function of the tissues.Patterned co-cultures are a useful tool for fabricating tissue engineered constructs and for studying cell–cell interactions in vitro, because they can be used to control the degree of homotypic and heterotypic cell–cell contact. In addition, this approach can be manipulated to elucidate important factors involved in cell–matrix interactions.Patterned co-culture strategies hold significant potential to develop biomimetic structures for tissue engineering. It is expected that they would create opportunities to develop artificial tissues in the future.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Cell adhesion; Cell–cell interaction; Co-culture; Microfabrication; Micropatterning; Tissue engineering;

The design of biocompatible 2D surfaces and 3D nano/micro topographies based on self-organization has a variety of potential applications in medical devices and tissue engineering. We have reported that biocompatible 2D surface using poly(2-methoxyethyl acrylate) (PMEA) and honeycomb-patterned 3D films with regular interconnected pores that is formed by self-organization.We highlight that 1) the reasons for this compatibility by comparing the structure of water in hydrated PMEA to the water structure of other polymers and 2) the reasons that 3D films exerted a strong influence on normal, cancer and stem cell morphology, proliferation, differentiation, cytoskeleton, focal adhesion, and functions including matrix production profiles.1) We hypothesized that intermediate water, which prevents the biocomponents from directly contacting the polymer surface or non-freezing water on the polymer surface, plays an important role in the excellent biocompatibility. 2) The cellular response to 3D films originates from the regularly aligned adsorbed proteins determined by the pore structure of the film.It is expected that combining the biocompatible 2D surfaces and 3D nano/micro topographies will provide an effective strategy for medical devices and tissue engineering scaffolds.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Scaffold; Cell adhesion; Water structure; Freezing-bound water; Biocompatibility; Biointerface;

Cell-directed-assembly: Directing the formation of nano/bio interfaces and architectures with living cells by Helen K. Baca; Eric C. Carnes; Carlee E. Ashley; DeAnna M. Lopez; Cynthia Douthit; Shelly Karlin; C. Jeffrey Brinker (259-267).
The desire to immobilize, encapsulate, or entrap viable cells for use in a variety of applications has been explored for decades. Traditionally, the approach is to immobilize cells to utilize a specific functionality of the cell in the system.This review describes our recent discovery that living cells can organize extended nanostructures and nano-objects to create a highly biocompatible nano//bio interface [1].We find that short chain phospholipids direct the formation of thin film silica mesophases during evaporation-induced self-assembly (EISA) [2], and that the introduction of cells alter the self-assembly pathway. Cells organize an ordered lipid-membrane that forms a coherent interface with the silica mesophase that is unique in that it withstands drying—yet it maintains accessibility to molecules introduced into the 3D silica host. Cell viability is preserved in the absence of buffer, making these constructs useful as standalone cell-based sensors. In response to hyperosmotic stress, the cells release water, creating a pH gradient which is maintained within the nanostructured host and serves to localize lipids, proteins, plasmids, lipidized nanocrystals, and other components at the cellular surface. This active organization of the bio/nano interface can be accomplished during ink-jet printing or selective wetting–processes allowing patterning of cellular arrays–and even spatially-defined genetic modification.Recent advances in the understanding of nanotechnology and cell biology encourage the pursuit of more complex endeavors where the dynamic interactions of the cell and host material act symbiotically to obtain new, useful functions.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.► Cells serve as living colloids – creating localized chemical potential gradients that can direct self-assembly and self-catalyze fabrication of 3D microenvironments. ► Within cell built microenvironments, chemical and mechanical ‘signaling’ reinforced by cellular confinement induces genetic reprogramming and a spectrum of complex and potentially unknown cellular behaviors. ► Non-replicative persistence/permanent senescence developed in encapsulated cells may be an ideal state, allowing basic functionality at a very low metabolic cost – of potential interest for integrated cellular devices and platforms.
Keywords: Self-assembly; Yeast; E. coli; Silica; Encapsulation; Biosensors;

Novel polymer biomaterials and interfaces inspired from cell membrane functions by Kazuhiko Ishihara; Yusuke Goto; Madoka Takai; Ryosuke Matsuno; Yuuki Inoue; Tomohiro Konno (268-275).
Materials with excellent biocompatibility on interfaces between artificial system and biological system are needed to develop any equipments and devices in bioscience, bioengineering and medicinal science. Suppression of unfavorable biological response on the interface is most important for understanding real functions of biomolecules on the surface. So, we should design and prepare such biomaterials.One of the best ways to design the biomaterials is generated from mimicking a cell membrane structure. It is composed of a phospholipid bilayered membrane and embedded proteins and polysaccharides. The surface of the cell membrane-like structure is constructed artificially by molecular integration of phospholipid polymer as platform and conjugated biomolecules. Here, it is introduced as the effectiveness of biointerface with highly biological functions observed on artificial cell membrane structure.Reduction of nonspecific protein adsorption is essential for suppression of unfavorable bioresponse and achievement of versatile biomedical applications. Simultaneously, bioconjugation of biomolecules on the phospholipid polymer platform is crucial for a high-performance interface.The biointerfaces with both biocompatibility and biofunctionality based on biomolecules must be installed on advanced devices, which are applied in the fields of nanobioscience and nanomedicine.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Cell membrane; Phospholipid polymer; Biomaterial; Biointerface; Biocompatibility;

Organic bioelectronics in nanomedicine by Karl Svennersten; Karin C. Larsson; Magnus Berggren; Agneta Richter-Dahlfors (276-285).
Nanomedicine is a research area with potential to shape, direct, and change future medical treatments in a revolutionary manner over the next decades. While the common goal with other fields of biomedicine is to solve medical problems, this area embraces an increasing number of technology platforms as they become miniaturized. Organic electronics has over the past two decades developed into an exciting and thriving area of research.Today, the organic electronics field stands at the interface with biology. As the area of organic bioelectronics advances, it holds promise to make major contributions to nanomedicine. The progress made in this direction is the topic of this review.We describe the inherent features of conducting polymers, and explain the usefulness of these materials as active scaffolds in cell biology and tissue engineering. We also explain how the combined ionic and electronic conductive nature of the polymers is used to precisely control the delivery of signal substances. This unique feature is key in novel devices for chemical communication with cells and tissues.This review highlights the results from the creative melting pot of interdisciplinary research in organic bioelectronics.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Organic bioelectronics; Tissue engineering; Drug delivery; Ca2+ signalling; Spatial–temporal gradients;

Conjugated polymers for enhanced bioimaging by Therése Klingstedt; K. Peter R. Nilsson (286-296).
Conjugated polymers (CPs) have been used for creating bioimaging tools or biosensors that provide a direct link between spectral signal and different biological processes. The detection schemes of these sensors are mainly employing the efficient light harvesting properties or the conformation sensitive optical properties of the CPs. Hence, the presence of biomolecules or biological events can be detected through fluorescence resonance energy transfer (FRET) between the CP and an acceptor molecule, or through their impact on the conformation of the conjugated backbone, which is seen as an alteration of the optical properties of the CP.In this review, the utilization of CPs for sensitive detection of DNA and protein conformational changes will be presented. The main part will be focused on the specific binding of CPs to protein deposits associated with protein misfolding diseases, such as Alzheimer's disease (AD), and the discovery that tailor-made CPs can be used for in vivo optical imaging of protein aggregates will be discussed.The unique optical properties of CPs can be used as molecular tools for sensitive detection of genetic material and for characterization of the pathological hallmarks associated with protein misfolding disorders, such as AD.CPs are novel molecular tools that can be used for sensitive bioimaging of biological processes and these tools offer the possibility to study biological events in a complementary fashion to conventional techniques.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Conjugated Polymers; Bioimaging; Fluorescence; DNA; Protein Aggregates;

A common strategy of microbial pathogens is to invade host cells during infection. The invading microbes explore different intracellular compartments to find their preferred niche.Imaging has been instrumental to unravel paradigms of pathogen entry, to identify their exact intracellular location, and to understand the underlying mechanisms for the formation of pathogen-containing niches. Here, we provide an overview of imaging techniques that have been applied to monitor the intracellular lifestyle of pathogens, focusing mainly on bacteria that either remain in vacuolar-bound compartments or rupture the endocytic vacuole to escape into the host's cellular cytoplasm.We will depict common molecular and cellular paradigms that are preferentially exploited by pathogens. A combination of electron microscopy, fluorescence microscopy, and time-lapse microscopy has been the driving force to reveal underlying cell biological processes. Furthermore, the development of highly sensitive and specific fluorescent sensor molecules has allowed for the identification of functional aspects of niche formation by intracellular pathogens.Currently, we are beginning to understand the sophistication of the invasion strategies used by bacterial pathogens during the infection process- innovative imaging has been a key ingredient for this.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Host-pathogen interactions; Membrane trafficking; Bacterial entry; Imaging techniques; Intracellular localization;

Today, cells are commonly analyzed in ensembles, i.e. thousands of cells per sample, yielding results on the average response of the cells. However, cellular heterogeneity implies the importance of studying how individual cells respond, one by one, in order to learn more about drug targeting and cellular behavior.This review discusses general aspects on miniaturization of biological assays and in particular summarizes single-cell assays in microwell formats. A range of microwell-based chips are discussed with regard to their well characteristics, cell handling, choice of material etc. along with available detection systems for single-cell studies. History and trends in microsystem technology, various commonly used materials for device fabrication, and conventional methods for single-cell analysis are also discussed, before a closing section with a detailed example from our research in the field.A range of miniaturized and microwell devices have shown useful for studying individual cells. In vitro assays offering low volume sampling and rapid analysis in a high-throughput manner are of great interest in a wide range of single-cell applications. Size compatibility between a cell and micron-sized tools has encouraged the field of micro- and nanotechnologies to move into areas such as life sciences and molecular biology. To test as many compounds as possible against a given amount of patient sample requires miniaturized tools where low volume sampling is sufficient for accurate results and on which a high number of experiments per cm2 can be performed.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Miniaturization; Microwell; Single cell; Well design; Well-based assay; In vitro cell assay;

Multi-stage delivery nano-particle systems for therapeutic applications by Rita E. Serda; Biana Godin; Elvin Blanco; Ciro Chiappini; Mauro Ferrari (317-329).
The daunting task for drug molecules to reach pathological lesions has fueled rapid advances in Nanomedicine. The progressive evolution of nanovectors has led to the development of multi-stage delivery systems aimed at overcoming the numerous obstacles encountered by nanovectors on their journey to the target site.This review summarizes major findings with respect to silicon-based drug delivery vectors for cancer therapeutics and imaging. Based on rational design, well-established silicon technologies have been adapted for the fabrication of nanovectors with specific shapes, sizes, and porosities. These vectors are part of a multi-stage delivery system that contains multiple nano-components, each designed to achieve a specific task with the common goal of site-directed delivery of therapeutics.Quasi-hemispherical and discoidal silicon microparticles are superior to spherical particles with respect to margination in the blood, with particles of different shapes and sizes having unique distributions in vivo. Cellular adhesion and internalization of silicon microparticles is influenced by microparticle shape and surface charge, with the latter dictating binding of serum opsonins. Based on in vitro cell studies, the internalization of porous silicon microparticles by endothelial cells and macrophages is compatible with cellular morphology, intracellular trafficking, mitosis, cell cycle progression, cytokine release, and cell viability. In vivo studies support superior therapeutic efficacy of liposomal encapsulated siRNA when delivered in multi-stage systems compared to free nanoparticles.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Multi-stage vectors; Drug delivery; Microparticle; Porous silicon; Nanoparticle;

Biofunctionalized nanoneedles for the direct and site-selective delivery of probes into living cells by Kyungsuk Yum; Min-Feng Yu; Ning Wang; Yang K. Xiang (330-338).
Accessing the interior of live cells with minimal intrusiveness for visualizing, probing, and interrogating biological processes has been the ultimate goal of much of the biological experimental development.The recent development and use of the biofunctionalized nanoneedles for local and spatially controlled intracellular delivery brings in exciting new opportunities in accessing the interior of living cells. Here we review the technical aspect of this relatively new intracellular delivery method and the related demonstrations and studies and provide our perspectives on the potential wide applications of this new nanotechnology-based tool in the biological field, especially on its use for high-resolution studies of biological processes in living cells.Different from the traditional micropipette-based needles for intracellular injection, a nanoneedle deploys a sub-100-nm-diameter solid nanowire as a needle to penetrate a cell membrane and to transfer and deliver the biological cargo conjugated onto its surface to the target regions inside a cell. Although the traditional micropipette-based needles can be more efficient in delivery biological cargoes, a nanoneedle-based delivery system offers an efficient introduction of biomolecules into living cells with high spatiotemporal resolution but minimal intrusion and damage. It offers a potential solution to quantitatively address biological processes at the nanoscale.The nanoneedle-based cell delivery system provides new possibilities for efficient, specific, and precise introduction of biomolecules into living cells for high-resolution studies of biological processes, and it has potential application in addressing broad biological questions.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Nanoneedle; Cargo delivery into living cell; Imaging; Single-molecule study; Subcellular; Compartment; Nucleus; Cytoplasm;

Protein-engineered biomaterials: Nanoscale mimics of the extracellular matrix by Nicole H. Romano; Debanti Sengupta; Cindy Chung; Sarah C. Heilshorn (339-349).
Traditional materials used as in vitro cell culture substrates are rigid and flat surfaces that lack the exquisite nano- and micro-scale features of the in vivo extracellular environment. While these surfaces can be coated with harvested extracellular matrix (ECM) proteins to partially recapitulate the bio-instructive nature of the ECM, these harvested proteins often exhibit large batch-to-batch variability and can be difficult to customize for specific biological studies. In contrast, recombinant protein technology can be utilized to synthesize families of 3 dimensional protein-engineered biomaterials that are cyto-compatible, reproducible, and fully customizable.Here we describe a modular design strategy to synthesize protein-engineered biomaterials that fuse together multiple repeats of nanoscale peptide design motifs into full-length engineered ECM mimics.Due to the molecular-level precision of recombinant protein synthesis, these biomaterials can be tailored to include a variety of bio-instructional ligands at specified densities, to exhibit mechanical properties that match those of native tissue, and to include proteolytic target sites that enable cell-triggered scaffold remodeling. Furthermore, these biomaterials can be processed into forms that are injectable for minimally-invasive delivery or spatially patterned to enable the release of multiple drugs with distinct release kinetics.Given the reproducibility and flexibility of these protein-engineered biomaterials, they are ideal substrates for reductionist biological studies of cell–matrix interactions, for in vitro models of physiological processes, and for bio-instructive scaffolds in regenerative medicine therapies.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Biomaterial; Protein engineering; Stem cell niche; Extracellular matrix; Tissue engineering; Regenerative medicine;

The goal of tissue engineering is to restore tissue function using biomimetic scaffolds which direct desired cell fates such as attachment, proliferation and differentiation. Cell behavior in vivo is determined by a complex interaction of cells with extracellular biosignals, many of which exist on a nanoscale. Therefore, recent efforts in tissue engineering biomaterial development have focused on incorporating extracellular matrix- (ECM) derived peptides or proteins into biomaterials in order to mimic natural ECM. Concurrent advances in nanotechnology have also made it possible to manipulate protein and peptide presentation on surfaces on a nanoscale level.This review discusses protein and peptide nanopatterning techniques and examples of how nanoscale engineering of bioadhesive materials may enhance outcomes for regenerative medicine.Synergy between ECM-mimetic tissue engineering and nanotechnology fields can be found in three major strategies: (1) Mimicking nanoscale orientation of ECM peptide domains to maintain native bioactivity, (2) Presenting adhesive peptides at unnaturally high densities, and (3) Engineering multivalent ECM-derived peptide constructs.Combining bioadhesion and nanopatterning technologies to allow nanoscale control of adhesive motifs on the cell–material interface may result in exciting advances in tissue engineering.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Biomaterials; Nanotechnology; Cell adhesion; Regenerative medicine; Integrins;

Toxicology of engineered nanomaterials: Focus on biocompatibility, biodistribution and biodegradation by Andrea Kunzmann; Britta Andersson; Tina Thurnherr; Harald Krug; Annika Scheynius; Bengt Fadeel (361-373).
It is widely believed that engineered nanomaterials will be increasingly used in biomedical applications. However, before these novel materials can be safely applied in a clinical setting, their biocompatibility, biodistribution and biodegradation needs to be carefully assessed.There are a number of different classes of nanoparticles that hold promise for biomedical purposes. Here, we will focus on some of the most commonly studied nanomaterials: iron oxide nanoparticles, dendrimers, mesoporous silica particles, gold nanoparticles, and carbon nanotubes.The mechanism of cellular uptake of nanoparticles and the biodistribution depend on the physico-chemical properties of the particles and in particular on their surface characteristics. Moreover, as particles are mainly recognized and engulfed by immune cells special attention should be paid to nano–immuno interactions. It is also important to use primary cells for testing of the biocompatibility of nanoparticles, as they are closer to the in vivo situation when compared to transformed cell lines.Understanding the unique characteristics of engineered nanomaterials and their interactions with biological systems is key to the safe implementation of these materials in novel biomedical diagnostics and therapeutics. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.
Keywords: Engineered nanomaterials; Biomedical applications; Nanotoxicology; Nano–immuno interactions;