Biofunctionalized nanoneedles for the direct and site-selective delivery of probes into living cells

Background

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.

Scope of review

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.

Major conclusions

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.

General significance

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.     

The application of atomic force microscopy to the study of living vertebrate cells in culture

Atomic force microscopy (AFM), a relatively new variant of scanning probe microscopy developed for the material sciences, is becoming an increasingly important tool in other disciplines. In this review I describe in nontechnical terms some of the basic aspects of using AFM to study living vertebrate cells. Although AFM has some unusual attributes such as an ability to be used with living cells, AFM also has attributes that make its use in cell biology a real challenge. This review was written to encourage researchers in the biological and biomedical sciences to consider AFM as a potential (and potent) tool for their cell biological research.     

Fluorescence microscopy today

Fluorescence microscopy has undergone a renaissance in the last decade. The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues. The impact of these and other new imaging methods in biophysics, neuroscience, and developmental and cell biology has been remarkable. Further advances in fluorophore design, molecular biological tools and nonlinear and hyper-resolution microscopies are poised to profoundly transform many fields of biological research.     

In vivo bioluminescence imaging for integrated studies of infection

Understanding biological processes in the context of intact organ systems with fine temporal resolution has required the development of imaging strategies that reveal cellular and molecular changes in the living body. Reporter genes that confer optical signatures on a given biological process have been used widely in cell biology and have been used more recently to interrogate biological processes in living animal models of human biology and disease. The use of internal biological sources of light, luciferases, to tag cells, pathogens, and genes has proved to be a versatile tool to provide in vivo indicators that can be detected externally. The application of this technology to the study of animal models of infectious disease has not only provided insights into disease processes, but has also revealed new mechanisms by which pathogens may avoid host defences during infection.     

Bicarbonate stimulated adenylyl cyclases

Bicarbonate ion is fundamental to the biology of all living organisms. HCO(3)(-) is vital to such diverse physiological processes as carbon fixation, cellular homeostasis, sperm maturation, and nucleotide synthesis. A defined subset of adenylyl cyclases identified in eukaryotes and prokaryotes are directly activated by HCO(3)(-). As such, cAMP represents the first identified biological effector for fluctuations in intracellular inorganic carbon levels. The identification of a signal transduction pathway activated by HCO(3)(-) has far reaching implications for understanding how the cell responds to fluctuations in this essential anion.     

Detection of living cervical cancer cells by transient terahertz spectroscopy

The photonic energy of terahertz wave is in the same order of magnitude as the rotational and vibrational energy levels of organic and biological macromolecules, so it has unique advantages in detecting cells and biological macromolecules. However, in the life environment, the dynamic time scale of cell-environment interaction and structural conformation change of biological macromolecules are within picosecond to millisecond, and water has strong absorption to terahertz wave, which has become the bottleneck problem for the detection of cells and biological macromolecules by terahertz technology. In this article, we developed a set of terahertz single measurement system based on the tilt wave front of grating pulse technique. The system was employed for the terahertz detection of trace living cervical cancer cells. We achieved transient detection of the terahertz pulse time-domain waveform of the living HeLa cells. The characteristic absorption peaks were identified by Lambert-Beer law, respectively, at 0.49, 0.71, 1.04, 1.07, 1.26 and 1.37 THz. The absorbance is proportional to the cell concentration.     

Noncontact measurement of the local mechanical properties of living cells using pressure applied via a pipette

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.     

Single-molecule visualization in cell biology

Recent progress in single-molecule detection techniques has allowed us to visualize the dynamic behaviour and reaction kinetics of individual biological molecules inside living cells. Single-molecule visualization provides a direct way to quantify, with a high spatial and temporal resolution, biological events inside cells at the single-molecule level. In this article, we discuss how single-molecule visualization can be used in cell biology.     

The cell theory--history, present state and prospects (on the 150th anniversary of the development of the cell theory)

The main stages of history of this most important biological conception are presented and the state of the modern cell theory and its future prospects are considered. Since 1839, when T. Schwann expounded his conception of the cell, a long pathway in cognition of the cell function and organization has been covered. From the original picture of the complex organism as a "cellular state", made up of relatively independent "elementary organisms", i.e. cells the modern biology has come to the idea of the cell as an integral system either being a part of a complex organism, or living free in the nature (protists). The cell represents certain qualitatively peculiar level in a complex evolutionary established hierarchy of biological systems. Some particular tight relations, existing between cytology, as a fundamental biological science and molecular biology, genetics, ecology and other biological disciplines are considered. The importance of the cell conception is ascertained for practical aims, especially in medicine.     

Monitoring of dopamine release in single cell using ultrasensitive ITO microsensors modified with carbon nanotubes

The study of single cell dynamics has been greatly adapted in biological and medical research and applications. In this work a novel microfluidic electrochemical sensor with carbon nanotubes (CNTs) modified indium tin oxide (ITO) microelectrode was developed for single cells release monitoring. The sensitivity of the electrochemical sensor after CNTs surface modification was improved by 2.5-3 orders of magnitude. The developed CNTs modified ITO sensor was successfully employed to monitor the dopamine release from single living rat pheochromocytoma (PC 12) cells. Its ultrahigh sensitivity, transparency and need for fewer agents enable this smart electrochemical sensor to become a powerful tool in recording dynamic release from various living tissues and organs optically and electrically.     

Kinetics of the light flash of the living firefly: a supramolecular process.

The light intensity vs. time curve of the light flash of the living firefly has been measured. Unlike the purified firefly enzyme system in aqueous solution, the living system does not show light decay conforming to a double exponential time curve, to simple first or second order decay, or to solid-state Elovich kinetics. Light decay of the living flash does show linearity in a probit vs. square root of time plot, which may indicate a reaction rate-limited by cooperative interactions of a biological phase transition. The observation that the kinetics of the firefly light system differ in the living cell from those in the purified system suggests that in the living system supramolecular factors control the rate of the reaction.     

A new xanthene‐based two‐photon fluorescent probe for the imaging of 1,4‐dithiothreitol (DTT) in living cells

1,4‐Dithiothreitol (DTT) has wide applications in cell biology and biochemistry. Development of effective methods for monitoring DTT in biological systems is important for the safe handling and study of toxicity to humans. Herein, we describe a two‐photon fluorescence probe (Rh‐DTT) to detect DTT in living systems for the first time. Rh‐DTT showed high selectivity and sensitivity to DTT. Rh‐DTT can be successfully used for the two‐photon imaging of DTT in living cells, and also can detect DTT in living tissues and mice.     

Biological atomism and cell theory

Biological atomism postulates that all life is composed of elementary and indivisible vital units. The activity of a living organism is thus conceived as the result of the activities and interactions of its elementary constituents, each of which individually already exhibits all the attributes proper to life. This paper surveys some of the key episodes in the history of biological atomism, and situates cell theory within this tradition. The atomistic foundations of cell theory are subsequently dissected and discussed, together with the theory's conceptual development and eventual consolidation. This paper then examines the major criticisms that have been waged against cell theory, and argues that these too can be interpreted through the prism of biological atomism as attempts to relocate the true biological atom away from the cell to a level of organization above or below it. Overall, biological atomism provides a useful perspective through which to examine the history and philosophy of cell theory, and it also opens up a new way of thinking about the epistemic decomposition of living organisms that significantly departs from the physicochemical reductionism of mechanistic biology.     

Effective intracellular inhibition of the cAMP-dependent protein kinase by microinjection of a modified form of the specific inhibitor peptide PKi in living fibroblasts

In order to obtain a peptide retaining its biological activity following microinjection into living cells, we have modified a synthetic peptide [PKi(m)(6-24)], derived from the specific inhibitor protein of the cAMP-dependent protein kinase (A-kinase) in two ways: (1) substitution of the arginine at position 18 for a D-arginine; (2) blockade of the side chain on the C-terminal aspartic acid by a cyclohexyl ester group. In an in vitro assay, PKi(m) has retained a specific inhibitory activity against A-kinase as assessed against six other kinases, with similar efficiency to that of the unmodified PKi(5-24) peptide. Microinjection of PKi(m) into living fibroblasts reveals its capacity to prevent the changes in cell morphology and cytoskeleton induced by drugs which activate endogenous A-kinase, whereas the original PKi peptide failed to do so. This inhibition of A-kinase in vivo by PKi(m) lasts between 4 and 6 h after injection. In light of its effective half-life, this modified peptide opens a route for the use of biologically active peptides in vivo, an approach which has been hampered until now by the exceedingly short half-life of peptides inside living cells. By providing a direct means of inhibiting A-kinase activity for sufficiently long periods to observe effects on cellular functions in living cells, PKi(m) represents a powerful tool in studying the potential role of cAMP-dependent phosphorylation in vivo.     

Dead cells don't dance: insights from live-cell imaging in plants

Live-cell imaging has yielded surprising pictures of subcellular structures and dynamics in living plant cells. Recent studies illustrate the power of live-cell observation for revealing new biological phenomena and for generating new questions about plant cell structure and function.     

Detection of Atomic Scale Changes in the Free Volume Void Size of Three-Dimensional Colorectal Cancer Cell Culture Using Positron Annihilation Lifetime Spectroscopy

Positron annihilation lifetime spectroscopy (PALS) provides a direct measurement of the free volume void sizes in polymers and biological systems. This free volume is critical in explaining and understanding physical and mechanical properties of polymers. Moreover, PALS has been recently proposed as a potential tool in detecting cancer at early stages, probing the differences in the subnanometer scale free volume voids between cancerous/healthy skin samples of the same patient. Despite several investigations on free volume in complex cancerous tissues, no positron annihilation studies of living cancer cell cultures have been reported. We demonstrate that PALS can be applied to the study in human living 3D cell cultures. The technique is also capable to detect atomic scale changes in the size of the free volume voids due to the biological responses to TGF-β. PALS may be developed to characterize the effect of different culture conditions in the free volume voids of cells grown in vitro.     

Cell electrospinning: a unique biotechnique for encapsulating living organisms for generating active biological microthreads/scaffolds

Jet-based technologies are increasingly being explored as potential high-throughput and high-resolution methods for the manipulation of biological materials. Previously shown to be of use in generating scaffolds from biocompatible materials, we were interested to explore the possibility of using electrospinning technology for the generation of scaffolds comprised of living cells. For this, it was necessary to identify appropriate parameters under which viable threads containing living cells could be produced. Here, we describe a method of electrospinning that can be used to deposit active biological threads and scaffolds. This has been achieved by use of a coaxial needle arrangement where a concentrated living biosuspension flows through the inner needle and a medical-grade poly(dimethylsiloxane) (PDMS) medium with high viscosity (12,500 mPa s) and low electrical conductivity (10-15 S m-1) flows through the outer needle. Using this technique, we have identified the operational conditions under which the finest cell-bearing composite microthreads are formed. Collected cells that have been cultured, postelectrospinning, have been viable and show no evidence of having incurred any cellular damage during the bionanofabrication process. This study demonstrates the feasibility of using coaxial electrospinning technology for biological and biomedical applications requiring the deposition of living cells as composite microthreads for forming active biological scaffolds.     

Scale-free flow of life: on the biology, economics, and physics of the cell

The present work is intended to demonstrate that most of the paradoxes, controversies, and contradictions accumulated in molecular and cell biology over many years of research can be readily resolved if the cell and living systems in general are re-interpreted within an alternative paradigm of biological organization that is based on the concepts and empirical laws of nonequilibrium thermodynamics. In addition to resolving paradoxes and controversies, the proposed re-conceptualization of the cell and biological organization reveals hitherto unappreciated connections among many seemingly disparate phenomena and observations, and provides new and powerful insights into the universal principles governing the emergence and organizational dynamics of living systems on each and every scale of biological organizational hierarchy, from proteins and cells to economies and ecologies.     

On the impact on biochemical research of the discovery of stable isotopes: the outcome of the serendipic meeting of a refugee with the discoverer of heavy isotopes at Columbia University

As late as the 1930s, approaches to biochemical research not only were rather primitive, but a certain amount of mysticism still surrounded the biochemical events that occur in the living cell. To a great extent, this was due to the lack of techniques needed to uncover the subtle reactions in the living cell. In the early 1930s, an accidental meeting of two scientists revolutionized approaches in biochemical studies and led to the scientific explosion in molecular biology that has occurred during the last few decades. The dark political storm in Germany deposited Dr. Rudolf Schoenheimer on the New York shore, where he met Professor Urey, who recently had discovered "heavy" hydrogen. Schoenheimer suggested that biological compounds tagged with heavy atoms of hydrogen would enable investigators to follow their metabolic pathways. This intellectual leap revolutionized the thinking and design of experiments and made it possible to uncover the myriad reactions that occur in the living cell.     

Synthesis of a near-infrared fluorescent probe and its application in imaging of MCF-7 cells

IR-789, a novel near-infrared fluorescent probe, was designed, synthesized, and applied to living cells. The probe exhibited better response fluorescence characteristics than the only FDA-approved agent, indocyanine green. Cell experiments showed that the probe had high affinity and without apparent cytotoxicity. Fluorescent image experiments in living MCF-7 cells (human breast adenocarcinoma cell line) further demonstrated the potential applications of the probe in biological systems. The probe effectively prevented the influence of autofluorescence and native cellular species in biological systems. It also exhibited high sensitivity, good photostability, and excellent cell membrane permeability.