Martin D R Byrne M E Nanomedicine Nanotechnology Biology and Medicine Under Review

• Journal Listing
• Genes Dev
• five.27(22); 2013 Nov fifteen
• PMC3841729

Genes Dev. 2013 Nov 15; 27(22): 2397–2408.

Nanotechnology: emerging tools for biological science and medicine

Ian Y. Wong

¹Center for Biomedical Engineering, School of Engineering,

^twoInstitute for Molecular and Nanoscale Innovation, Brown Academy, Providence, Rhode Island 02912, USA;

Sangeeta N. Bhatia

³Sectionalisation of Wellness Sciences and Technology, Institute for Medical Engineering and Science,

⁴Electric Engineering and Information science,

⁵Koch Institute for Integrative Cancer Enquiry,

^viHoward Hughes Medical Establish, Massachusetts Institute of Applied science, Cambridge, Massachusetts 02139, USA;

Mehmet Toner

⁷BioMEMS Resource Center,

^viiiCenter for Engineering in Medicine,

^ixDepartment of Surgery, Massachusetts General Hospital, Harvard Medical School, Charlestown Massachusetts 02129, The states

Abstruse

Historically, biomedical enquiry has been based on two paradigms. First, measurements of biological behaviors have been based on bulk assays that average over large populations. Second, these behaviors have then been crudely perturbed by systemic administration of therapeutic treatments. Nanotechnology has the potential to transform these paradigms by enabling exquisite structures comparable in size with biomolecules besides as unprecedented chemic and physical functionality at modest length scales. Here, we review nanotechnology-based approaches for precisely measuring and perturbing living systems. Remarkably, nanotechnology can exist used to characterize single molecules or cells at extraordinarily high throughput and evangelize therapeutic payloads to specific locations as well equally showroom dynamic biomimetic behavior. These advances enable multimodal interfaces that may yield unexpected insights into systems biology as well as new therapeutic strategies for personalized medicine.

Keywords: Nanoparticle, microfluidics, single cell, biomarker, microneedle, targeted delivery

In the context of biology and medicine, nanotechnology encompasses the materials, devices, and systems whose structure and role are relevant for small length scales, from nanometers (10^−ix m) through microns (10^{−half dozen} m) (Whitesides 2003). This size regime is associated with intriguing phenomena in both living systems and artificial devices. In particular, the primal building blocks of life fall within this range, including biological macromolecules and cells. For example, dsDNA is a concatenation-like macromolecule with a diameter of 2 nm, prison cell membranes are sheet-like structures with a thickness of ∼10 nm, and (suspended) eukaryotic cells are approximately spherical with a diameter of ∼x um. Remarkably, artificial nanostructures can too exist constructed with comparable dimensions, including nanopores with ∼2-nm openings, inorganic nanowires of ∼10-nm diameter, and spherical nanoparticles of 10- to 100-nm diameter (Box 1). Moreover, this size regime is associated with unexpected chemistry and physics where molecular furnishings tin can play a significant role. This tin can be understood from a purely geometric statement—a width of ∼10 nm in a silicon nanowire corresponds to only ∼forty atoms. A event of this is that the surface area of these nanostructures is extremely large relative to the volume. If the aforementioned silicon nanowire has a square cross-department, then approximately 1 out of every 10 atoms is associated with the surface. In contrast, if we accept a macroscopic wire with a width of ten mm, only 1 in x,000 atoms is associated with the surface. Finally, every bit a event of these minor dimensions, electrons are spatially confined, resulting in exceptional electric, magnetic, optical, and thermal properties (Alivisatos 2004). An exciting prospect is to bridge across these biomolecular and physical realms, leveraging unique capabilities from one realm for application in the other.

Box i. Some assembly required: making nanostructures from molecular building blocks

The production of nanostructures occurs through two full general approaches (Madou 2012): (1) Top-down approaches impose a structure or design on a flat surface by successively adding or removing thin layers of materials. For instance, photolithography uses UV light to pattern light-sensitive polymer into nanoscale features. Increased resolution tin be accomplished using electron or focused ion beams to blueprint surfaces, particularly to mill or sculpt away material. (2) Lesser-up approaches are based on organizing atomic or molecular components into hierarchical assemblies. For case, cytoskeletal polymers grade through the sequential add-on of monomeric poly peptide units. Instead, carbon nanotubes or silicon nanowires are grown from chemical precursors in a gas or liquid phase. A key conceptual difference is that top-downwards fabrication is deterministic and well controlled, whereas bottom-up assembly is stochastic and may outcome in defects. Withal, top-down fabrication is optimized for highly specific processing conditions that are incompatible with biologics, necessitating the use of bottom-up assembly.

A especially exciting example of bottom-up self-associates has been the utilize of one-dimensional (1D) nucleic acid strands that organize into designed ii-dimensional (2nd) and iii-dimensional (3D) architectures based on molecular recognition (Aldaye et al. 2008). Initial work based on the formation of branches and junctions inspired the design of 2D tiles that organized into hierarchical lattices based on logical rules (Seeman 2003). Subsequently, in "DNA origami," a unmarried long DNA strand could exist guided with shorter "staple" strands to fold into more complex 2D shapes (Rothemund 2006). Most recently, these approaches have been extended to 3D structures. For instance, cuboidal "Lego-similar" bricks have been produced, where the free ends are recognition sites that can demark uniquely to detail bricks in solution with a defined orientation (Fig. 2A; Ke et al. 2012) (). Nearly arbitrary "pixelated" objects can be designed by removing brick-like elements from a solid cube of ∼10 pixels per side (∼25 nm), with 100 dissimilar geometries demonstrated. Alternatively, at Dna strands can be woven into canvas-like structures that tin can be rolled, curved, and twisted (Fig. 2B; Dietz et al. 2009). One promising application for these techniques is to construct scaffolds where DNA-linked proteins, peptides, and inorganic nanostructures tin exist precisely positioned and dynamically actuated. Building on these concepts, a drug delivery "robot" has been demonstrated where logical aptamer recognition of cancer-associated antigens led to unveiling of antibody fragments that could molecularly interact with cell surface receptors (Douglas et al. 2012). Overall, Deoxyribonucleic acid nanotechnology is extremely powerful for rationally designing 2D and 3D nanostructures with precise command that cannot be achieved using any other techniques. All the same, a continuing challenge is to reduce the formation of defective and misfolded structures every bit well as establish the biocompatibility of these structures in vivo (Shih and Lin 2010).

(A) DNA "Lego-like" bricks for three-dimensional cuboid structures. (B) DNA sheets assembled into curved and anisotropic geometries. (C) Nanoparticles of different materials, shapes, and sizes fabricated using PRINT. (D) Multicomponent barcoded particles made using menstruum lithography. From Pregibon et al. (2007) and Ke et al. (2012). Reprinted with permission from AAAS. Reprinted from Shih and Lin (2010), with permission from Elsevier. Reprinted from Wang et al. (2011a); © 2011 Wiley-VCH Verlag GmbH & Co., KGaA, Weinheim.

A number of hybrid schemes have been developed that combine top-down and bottom-up approaches. For instance, in soft lithography, pinnacle-down features are replicated into plastic "stamps," which tin can be coated with proteins or inorganic nanomaterials to be transferred to specific locations on a surface (Whitesides et al. 2001). A variation of this approach (PRINT) has been used to make polymeric particles, which are cast and removed using gyre-to-roll manufacturing (Wang et al. 2011a). This arroyo can independently control particle size, shape, stiffness, material, and surface chemical science (Fig. 2C). This was used to systematically explore design rules for ruby blood cell-shaped particles, showing that eightfold softer particles displayed xxx-fold enhanced circulation times (Merkel et al. 2011). Another case is the employ of photolithography to cross-link hydrogels in microfluidic channels, allowing for layered composite architectures based on interfacial flows (Helgeson et al. 2011). This approach is especially powerful for constructing complex geometries with different functionalities (Fig. 2D); for instance, multicompartment particles with barcode identifiers and regions of distinct nucleic acid probes for multiplexed analyte detection (Pregibon et al. 2007). This functionality could be further practical to generate heterogeneous textures, chemical patchiness, and continuous molecular gradients. Overall, these hybrid schemes take advantage of both bottom-upward and top-down approaches for preparing nanostructures with greater consistency under more than biocompatible conditions. Nevertheless, a standing challenge for widespread adoption volition exist to calibration product for high-volume manufacturing.

A conceptual framework for nano–bio interfaces tin be structured based on two overarching themes (Fig. 1). First, nanotechnology enables new ways to measure and detect biology both in vitro and in vivo. For instance, nanoscale devices can sense minute differences at the level of single molecules and single cells. This exquisite sensitivity can be used, for case, to characterize single-cell heterogeneity at extremely high throughput, revealing distinct hierarchies and subpopulations. This level of detail is often lost in traditional bulk assays, which measure "average" information about populations pooled from large numbers. 2d, nanotechnology enables new ways to perturb cells and treat patients. For example, nanomaterials can be designed to precisely deliver therapeutics to targeted locations while overcoming or evading biological barriers, thus altering the inherent pharmacokinetics and biodistribution of the cargo (typically a drug). This avoids the complications associated with conventional delivery using soluble drugs, which are associated with low and inconsistent uptake. An exciting prospect is the synergistic integration of these 2 themes: nanotechnologies that can simultaneously detect and perturb biology. The design and construction of such biomimetic nanosystems, driven past feedback between nanomaterials and their environment, will requite ascent to emergent behaviors such as adaptation, self-organization, and amplification. These capabilities are potentially transformative for confronting the spatial and temporal complexity of living systems.

Key themes underlying nanotechnology in biological science and medicine, including new capabilities to measure or adjy cells in vitro as well as diagnose or treat patients. An heady prospect is the future integration of these different capabilities for biomimetic behaviors such as adaption, self-organization, and amplification.

In this review, nosotros review some illustrative examples of nanotechnology applied to biology and medicine. This is intended to make the new capabilities of nanotechnology accessible for nonexperts (Box1, Fig. two), with particular emphasis on our respective areas of expertise in nanotechnology and microfluidics for mammalian prison cell biology and medicine. In item, we accost recent advances for characterizing biological systems: precision measurement of biomolecules and cells in vitro, manipulation of picoliter droplets, and the clinical evaluation of complex samples. We also examine innovative approaches for perturbing biological systems, including minimally invasive nanoneedles too as rationally designed multifunctional nanoparticles. Due to infinite constraints, we simply included a express sampling of recent developments; we refer readers to more comprehensive reviews elsewhere for each topic. Finally, we consider the future prospects and challenges for these technologies.

Counting one by one: precision measurements of biomolecules and cells

Historically, measurements of biomolecules and cells have occurred using bulk assays, such as lysates pooled from populations of thousands or millions of cells at the completion of the experiment. Instead, the modest sizes associated with nanostructures enable them to probe and manipulate the dynamics of single cells and molecules with unprecedented resolution. 1 example of this is the use of nanoscale pores that can discriminate between molecules on the footing of size and biochemical characteristics (Branton et al. 2008). In particular, biological channels and pores are capable of regulating ion transport with a selectivity on the order of angstroms (0.1 nm or 10⁻¹⁰ yard) (Bayley and Cremer 2001). Inspired by this biological functionality, it has been hypothesized that nanopores could unwind and unzip DNA then that individual nucleotides translocate sequentially in single file. Still, edifice precisely controlled pores with ∼1-nm diameter has been challenging (Venkatesan and Bashir 2011). Biological poly peptide pores such every bit α-haemolysin (αHL) present well-defined apertures only generally crave incorporation into mechanically delicate lipid bilayers to maintain stability. In dissimilarity, artificial nanopores lack the chemic complexity of proteins and exhibit reduced selectivity. A hybrid scheme was recently implemented by directly inserting an αHL protein into a slightly larger inorganic nanopore (Fig. 3A; Hall et al. 2010). This scheme displays the benefits of a biological pore with increased selectivity and sensitivity but also the mechanical stability of an inorganic scaffold. This device could potentially be scaled so that large numbers of nanopores could operate in parallel. In principle, this approach could enable long reads of single molecules at high translocation velocities. An ongoing claiming for this technology is to accomplish sensitivity with single-base-pair resolution due to the stochastic motion of DNA too as the measurement sensitivity at fast translocation speeds.

(A) Sequencing DNA with hybrid biological–bogus nanopores. (B) Measuring cell-secreted ROS using carbon nanotubes. (C) Electrophysiology in three-dimensonal (3D) scaffolds using nanowire arrays. (D) Mechanical deformation of 3D scaffolds using traction force microscopy. (Eastward) Growth of adherent cells measured using microresonators. (F) Growth of cells in solution measured using suspended mass resonators. Reprinted past permission from Macmillan Publishers Ltd. from Hall et al. (2010), Jin et al. (2010), Legant et al. (2010), Son et al. (2012), and Tian et al. (2012). Reproduced with permission from Park et al. (2010).

The small sizes of nanostructure-based biosensors tin exist harnessed nearly finer when they are patterned at high densities around single cells, enabling highly localized measurements at submicron and subcellular length scales. For example, existing technologies cannot measure the cellular secretion of growth factors, cytokines, and other signaling molecules into extracellular infinite (Love 2010). Nanostructures are advantageous not only considering they are comparable in size with biomolecules but also because their size enables enhanced optical characteristics (Brolo 2012). One recent written report used carbon nanotube sensors to detect reactive oxygen species (ROS) with single-molecule resolution based on optical fluorescence quenching (Jin et al. 2010). In response to epidermal growth factor (EGF) stimulation of A431 carcinoma cells, transient "hot spots" of high ROS concentration were observed in the proximity of the prison cell membrane (Fig. 3B) even when cells were fixed. An analogous approach was used to measure intracellular cytochrome C dynamics using plasmonic spectroscopy of golden nanoparticles (Choi et al. 2009). Remarkably, in response to proapoptotic stimuli, they were able to mensurate the release of cytochrome C from mitochrondria into the cytoplasm. Finally, an array of nanoplasmonic resonators patterned with submicron spacing was used to narrate the autocrine signaling of IL-2 in T lymphocytes, revealing a highly localized secretion contour (Wang et al. 2011c). Overall, these optical nanostructures enable exquisite molecular sensitivity with loftier spatial and temporal resolution, capable of distinguishing betwixt molecules secreted by a particular jail cell and those in the groundwork. All the same, producing these nanostructures requires specialized fabrication techniques that are challenging to calibration upward, which will need to be addressed before they tin can be widely used.

Another readout of interest for single cells is electric and ion aqueduct action. Such elecrophysiological measurements are highly relevant for neuroscience (Alivisatos et al. 2013) likewise as pharmacological testing (Dunlop et al. 2008). Yet, existing patch clamps and electrodes are relatively beefy and cumbersome so that they integrate poorly with iii-dimensional (3D) tissues and do not achieve single-prison cell resolution. Nanostructures add new capabilities hither, since their high surface area to volume ratios make them extremely sensitive to electrical changes. There has been all-encompassing piece of work to develop integrated circuits that are highly flexible, stretchable, and biocompatible (Kim et al. 2012a). One recent study incorporated a flexible mesh-like array of electrical sensors that formed an interpenetrating network with a 3D scaffold (Fig. 3C). Subsequent culturing of neurons showed good viability over several weeks, and simultaneous recordings of electrical activity from different cells was possible. This degree of seamless integration may have occurred because the synthetic architectural elements were comparable in size with the neurons. Future work with increased densities of electrical sensors could potentially be used to map out neural circuits (Alivisatos et al. 2013). I difficulty with this arroyo is the positioning of each electrode to interface with specific cells of involvement.

The mechanical forces exerted by cells to sense and remodel their environs are extremely important for understanding how they regulate cell and tissue function (Nelson and Bissell 2006). This has been previously explored in the context of cells adhering to apartment two-dimensional (2D) substrates. Recently, there has been increasing interest in measuring how cells interact with 3D microenvironments, which may be more than physiologically relevant (Bakery and Chen 2012). The forces applied by cells to deform 3D scaffolds was quantified by tracking the motion of tens of thousands of embedded fluorescent nanoparticles (Fig. 3D; Legant et al. 2010). Remarkably, they establish that fibroblasts in 3D exerted strong traction forces most the tips of extensions, analogous to forces measured in 2D well-nigh lamellipodia. An important adjacent stride will exist to conform this technique to systems more reminiscent of the extracellular matrix (ECM), which exhibit highly heterogeneous and dynamic architectures (Even-Ram and Yamada 2005).

Finally, nanostructures can be used to straight measure single-cell phenotypes. In detail, i source of cell-to-jail cell variability is the cell bike, driving growth dynamics and partition events (Snijder and Pelkmans 2011). Resonating sensors were used to measure the mass of adherent cells over l h (Fig. 3E; Park et al. 2010). Alternatively, a resonating sensor with suspended geometry was used to mensurate the mass of suspended cells over 35 h (Fig. 3F; Son et al. 2012). Remarkably, both groups observed that growth rate is not constant but increases with increasing jail cell size. These growth rates varied at sure checkpoints in the jail cell cycle and besides varied at the single-cell level across the population. These techniques may help to reveal mechanically driven feedback mechanisms used to constrain variability and maintain homeostasis at the jail cell and tissue level (Kafri et al. 2013).

Overall, nanostructures enable sensitive measurements of single molecular and cellular behaviors, enabled by sizes comparable with these biological building blocks and enhanced optical, electronic, and mechanical characteristics. Nevertheless, the general schemes described here crave shut proximity to cells, which oft raises questions of biocompatibility for extended long-term measurements. Moreover, these techniques require highly specialized fabrication techniques as well as measurement infrastructure. Every bit such, they are currently much more effective for in vitro measurements compared with in vivo.

Divide and conquer: droplets for high-throughput biology

Another approach for accessing single-molecule dynamics and single-cell heterogeneity is based on using microfluidics to partition majority solutions into smaller volumes that comprise detached numbers of molecules or cells. For example, droplets of aqueous solution can be formed with volumes of ∼one pL (10⁻¹² L) or ∼x-μm diameter. At these small-scale sizes, the effective concentration of reagents is increased, and the improvidence distance is considerably decreased (Leamon et al. 2006; Vincent et al. 2010). This may be transformative for loftier-throughput assays (∼x^nine aerosol per milliliter) where extremely big numbers of reactions must be performed in parallel while efficiently using expensive reagents (Guo et al. 2012; Schneider et al. 2013).

1 device is based on the electrical manipulation of aerosol on flat surfaces (digital microfluidics) (Wheeler 2008). A modular set of operations could be performed on droplets, including moving, merging, mixing, splitting, and dispensing from different reservoirs (Fig. 4A). This scheme is particularly advantageous for solid-phase or liquid–liquid extraction using nonaqueous solvents, which are more often than not incompatible with conventional plastic microfluidic devices. For instance, estrogen could exist straight extracted from one μL of tissue, blood, or serum in ∼ten min without user intervention, corresponding to a 1000-fold reduction in sample volume and a xx-fold increment in speed (Mousa et al. 2009). These analytes were and so sensitively quantified using mass spectrometry. Dried blood spots could too be analyzed using a similar technique, avoiding the issues of fouling and clogging that challenge channel-based microfluidic devices (Jebrail et al. 2011).

(A) One-picoliter aerosol tin exist dispensed, moved, merged, mixed, and split up in an open geometry using electric fields. (B) Droplet libraries can be challenged confronting a reactant so sorted at ultrahigh throughput in channels. From Wheeler (2008). Reprinted with permission from AAAS. Reproduced from Guo et al. (2012) with permission of The Royal Society of Chemistry.

Alternatively, a channel geometry was implemented where droplets of aqueous solution are compartmentalized by an oil phase and lined up unmarried file (Fig. 4B). Libraries of droplets encoding unlike analytes such as drug compounds, viruses, antibodies, or enzymes could be screened against cells or other reactants (Guo et al. 2012). These droplets can exist interrogated past fluorescence and sorted at high speeds on the order of 10⁸ droplets per twenty-four hours. This engineering was practical to the directed evolution of the enzyme horseradish peroxidase, which generated mutants with catalytic rates >10 times faster than the parent, with a one thousand-fold increase in speed and a ane million-fold reduction in cost (Agresti et al. 2010). Another awarding is digital PCR, where DNA can exist compartmentalized in the single-molecule limit and amplified without bias (Bakery 2012). 1 challenge for droplet microfluidics is to interface with the macroscopic world, whether to isolate individual droplets of interest or for highly combinatorial assays (Guo et al. 2012). 1 possibility to address these difficulties could be the use of on-chip logic and ciphering (Prakash and Gershenfeld 2007) for big-calibration integration (Melin and Quake 2007).

Finding a needle in a haystack: extreme clinical diagnostics

Blood and other biofluids encode a tremendous amount of information about the good for you or diseased country of patients (Toner and Irimia 2005). Dissimilar in vitro measurements, deciphering clinically relevant information oft requires isolating sure biomarkers from an extraordinarily complex and heterogeneous mixture of proteins (Anderson and Anderson 2002) or cells (Mach et al. 2013). These biomarkers of interest may exist extremely rare and obscured past the circuitous background of the overall population. Moreover, constructive point-of-care diagnostics must be rapid, accurate, and low cost to serve patients and inform clinical diagnostics (Mentum et al. 2012). Nanostructures are advantageous, since their high surface areas can be used to capture clinically relevant biomarkers through molecular recognition processes. The enhanced chemic and physical properties can be then be used to detect or isolate these biomarkers.

Rapid, multiplexed proteomic analysis has been demonstrated from microliter quantities of claret using integrated microfluidic chips (Fan et al. 2008). A robust and modular patterning scheme was adult where nonoverlapping sets of DNA linkers immobilize antibodies at discrete locations (Fig. 5A). The highly reproducible patterning of these biomolecules at high density was critical to achieve this multiplexed readout. These antibodies are and so used in a sandwich assay with a secondary antibody, allowing measurement of twelve separate biomarkers with loftier dynamic range. The correlations in cytokine, growth factor, and antigen expression could be used to stratify patient disease land. This approach was also practical to measure out secreted signals from allowed cells, revealing high functional heterogeneity at the single-cell level (Ma et al. 2011).

(A) Integrated barcode bit for proteomic biomarkers from blood. (B) Protein typing of circulating microvesicles using miniaturized NMR. (C) Antigen-dependent and -independent enrichment of rare tumor cells using magnetic nanoparticles. (D) Multiplexed synthetic biomarkers are amplified by enzymatic cleavage near tumors followed past renal concentration for noninvasive monitoring of illness progression. Reprinted by permission from Macmillan Publishers Ltd. from Fan et al. (2008) and Shao et al. (2012).

Microvesicles secreted from tumors have also been characterized equally biomarkers (Shao et al. 2012). Microvesicles were labeled using clusters of target-specific magnetic nanoparticles, which could be sensitively detected using micro-NMR (nuclear magnetic resonance) technology based on changes in spin–spin relaxation time (T₂) (Fig. 5B; Lee et al. 2008). This approach revealed hit heterogeneity in single microvesicles compared with the bulk analyses that have historically been performed. Remarkably, the proteomic signature of these microvesicles was consequent with the cells of origin. Furthermore, microvesicle expression profiles tracked efficacy of drug treatment and were predictive of differences in treatment mechanisms.

Circulating tumor cells (CTCs) shed by metastatic tumors into the claret have generated much contempo interest. However, CTCs are extremely rare, intermixed with blood cells at concentrations of approximately 1 per billion in clinical samples (Yu et al. 2011). An established biomarker for CTCs is EpCAM, which is expressed by cells of epithelial origin but defective in blood cells. One arroyo for "CTC-Chips" was based on the enhanced surface surface area associated with micro/nanostructures for adhesion-based capture using arrays of EpCAM-functionalized micropillars (Nagrath et al. 2007). This engineering science was clinically validated for tracking illness progression and informing therapeutic treatments (Maheswaran et al. 2008). Nevertheless, one difficulty posed by laminar fluid period at these length scales is relatively slow mixing (Stone et al. 2004), which limited the transport of CTCs to the capture surface. The 2nd generation "HB-Scrap" addressed this result by using topographically patterned "herringbones" to promote chaotic mixing (Stott et al. 2010). Remarkably, this engineering science also captured multicellular CTC "clusters," raising the possibility that collective or cooperative behaviors may be advantageous for metastatic dissemination. Additional texturing of the capture surface using silicon nanowires has also demonstrated splendid capture efficiencies (Wang et al. 2011b).

The third-generation CTC-iChip was based on the separation of cells bound with ferromagnetic nanoparticles (Ozkumur et al. 2013). This approach was highly controlled using deterministic particle ordering through inertial focusing (Di Carlo et al. 2007), allowing precise deflection of selected cells into drove channels (Fig. 5C) at extremely loftier throughput (8 mL/h ∼ ten⁷ cells/sec). The small size of these nanoparticles is advantageous for binding efficiently to surface receptors on CTCs—even those with relatively depression expression that may not be captured using existing immunoaffinity techniques. Furthermore, a negative depletion approach enriches for cancer cells that do not express established epithelial cell surface markers, including triple-negative breast cancers, pancreatic cancers, and melanoma. These isolated CTCs displayed morphological similarities to tumors of origin but considerable variability in size. This single-cell heterogeneity was further interrogated past multigene microfluidic quantitative RT–PCR of 15 different prostate cancer cells, revealing distinct subsets with variable expression of epithelial, mesenchymal, and stalk cell biomarkers as well as androgen receptors. This approach is highly promising for monitoring and guiding personalized therapies.

One unresolved question for biomedical diagnostics is how early on biomarkers tin can exist detected during affliction progression. Recently, a quantitative framework was proposed for estimating measurable levels of biomarker based on their secretion from tumors (relative to healthy tissues), transport into vasculature, and subsequent dilution, degradation, and elimination (Hori and Gambhir 2011). A troubling determination was a sizable mismatch between tumor size and detection limits, translating to a period of years before a tumor could exist diagnosed.

A new approach was recently demonstrated for amplifying biomarkers so that they tin be more easily detected (Kwong et al. 2013). A library of synthetic, mass-encoded peptides was linked to nanoparticles using protease-cleavable linkers (Fig. 5D). During liver fibrosis or colorectal cancer, increased expression of proteases such every bit matrix metalloproteins (MMP) led to repeated catalytic cleavage of synthetic linkers, leading to liberation and concentration of peptides in the urine. Samples were then tested using mass spectroscopy for highly sensitive measurements of the mass-encoded "synthetic biomarkers." This scheme overcomes the limitations of conventional biomarker detection by amplifying the initial point, increasing sensitivity using stable constructed peptides that practice non occur biologically (low background), and multiplexing. This initial demonstration in minor animals shows swell promise for further clinical applications with humans.

The integration of microfluidics and nanotechnology has enabled new modalities for point-of-intendance diagnostics too as implantable devices for monitoring patients, specially for early detection and to mensurate drug efficacy. In item, microfluidic technologies with integrated sensors for processing bodily fluids such as claret or saliva tin can be manufactured very reliably in a high-throughput format and also are very robust. Nanostructures, by virtue of their small dimensions, tin interact precisely with molecular and cellular components besides as exhibit exceptional electronic, magnetic, and optical properties. As a consequence, nanostructures can considerably raise the sensitivity of these assays. However, these nanostructures may require highly specialized product techniques that are hard to scale up (Box 1). A crucial challenge for the hereafter volition exist to translate these technologies to the bedside or point of intendance with sufficiently high accuracy and low toll.

Lying on a bed of nails: minimally invasive delivery using nanoneedles

A continuing challenge for therapeutic applications is targeted commitment of agents to defined locations in cells and tissues. Biological barriers such as the cell membrane, mucosal layers, or skin have evolved to exist highly efficient at excluding unrecognized materials. For instance, eukaryotic jail cell membranes sectionalisation the interior of a jail cell from the surrounding extracellular environment and are comprised of amphiphilic lipids with a hydrophilic head and a hydrophobic tail (De Weer 2000). These lipids are bundled to grade a hydrophobic cadre region that restricts the transport of hydrophilic and polar molecules across the bilayer. As a consequence, using such molecules to perturb cellular role often requires special techniques to bypass the prison cell membrane, such as electroporation or delivery agents, which may have a detrimental effect on cell viability. Equally a consequence, molecular agents such as plasmid Dna, siRNA, peptides, proteins, and small molecule drugs may be ineffective when delivered systemically. Nanostructures with small diameters but a high aspect ratio are a highly promising alternative for bypassing these barriers in order to evangelize agents to divers locations.

A nanopatterned surface consisting of an array of silicon nanowires with an ∼50-nm diameter and ∼1-μm height was functionalized with a variety of molecular agents in a microarray format (Shalek et al. 2010, 2012). Remarkably, these nanowires spontaneously inserted into cells without inducing apoptosis (Fig. 6A). This led to robust delivery of molecular agents (>95%) into the cellular interior to guide neuronal growth, siRNA knockdown, and inhibition of apoptosis as well as target proteins to organelles. These nanopatterned surfaces are fully compatible with existing printing technologies, enabling "living-cell microarrays" for high-throughput screens of cell stimuli with greatly increased efficiency. All the same, 1 limitation of this technique is that the concentration and release kinetics of drugs are non well controlled. An alternate arroyo was based on an array of hollow alumina "nanostraws" that served as fluid conduits from an adjacent reservoir (VanDersarl et al. 2012). The limerick of molecular species in the reservoir could be defined using microfluidics, assuasive spatial gradients or transient pulses of molecular species. Farther control over molecular delivery and intracellular transport was achieved by applying spatially localized electric pulses, which enabled >seventy% transfection efficiency (Xie et al. 2013). Future piece of work will be necessary to explore the role of physical nanotopography on cell behavior (Kim et al. 2012b) as well as confirm long-term cell viability.

(A) Arrays of silicon nanowires can deliver siRNA into the interior of dendritic cells. Dissolving polymer microneedles (B) tin can evangelize influenza vaccine to divers locations beneath the peel (C). Reprinted by permission from Macmillan Publishers Ltd. from Sullivan et al. (2010). Reprinted with permission from Shalek et al. (2012). © 2012 American Chemic Lodge.

Human skin too displays barrier backdrop that limit molecular ship (Prausnitz et al. 2004). The outermost layer of the epidermis, known as the stratum corneum, displays a high degree of structural heterogeneity. Every bit a consequence, molecular ship across this layer must occur through highly tortuous pathways and can be strongly impeded. Ane approach to deliver molecular agents is to use hypodermic needles that inject past the stratum corneum. However, this is a painful, highly invasive procedure that cannot be self-administered. Instead, an array of polymeric microneedles with a smaller diameter can painlessly insert into skin, delivering drugs to a controlled depth (Fig. 6B,C; Sullivan et al. 2010). This is particularly useful for vaccination, since immunogenic Langerhans cells are frequently localized ∼100 μm from the surface. These microneedles degrade inside minutes, allowing the skin to reseal and recover barrier part to prevent infection. Moreover, these microneedles were found to amend on antibody and cellular immune response in mice compared with conventional needles. Variations on this concept include multilayer-coated microneedles, which tin can be used for programmed and sustained delivery profiles (DeMuth et al. 2013).

Deliver to zip lawmaking: multifunctional nanoparticles for therapy and diagnostics

Drug-loaded nanoparticles can also be advantageous for delivering concentrated doses to target sites through prolonged apportionment times and increased uptake combined with reduced toxicity elsewhere (Shi et al. 2010). Size appears to play an important part in spherical particle send, since micrometer-scale particles are often cleared by macrophage phagocytosis or filtration in the liver or spleen (Mitragotri and Lahann 2009). Nevertheless, these behaviors are also affected by physical characteristics such as shape, mechanics, and surface chemistry (Nel et al. 2009). For instance, pathogens such equally leaner and viruses display distinctive asymmetric and anisotropic shapes, which enable specific interactions with target cells and evasion of the immune response (Yoo et al. 2011). In contrast, scarlet blood cells exhibit a characteristic flexible, discoidal shape that avoids filtration in the spleen, despite their relatively large size (Skalak and Branemark 1969). Inspired past this physical miracle, long, flexible polymeric micelles were designed that persist in apportionment for extended periods with minimal phagocytosis (Geng et al. 2007). Recent advances in synthesis and fabrication take enabled the design of biomimetic nanosystems that can cooperate to synergistically amend their functionality (Box 1).

Nanoparticles can too incorporate advanced functionality such as targeting to specific organs and cells, responsiveness to external stimuli, imaging capabilities, and drug delivery (Bao et al. 2013). For example, passive targeting of tumors based on enhanced permeability and retentivity (EPR) in "leaky vasculature" (Chauhan et al. 2011) tin can be complimented by "active" targeting of cancer cells. For instance, tumor cells and vasculature often brandish distinct molecular "zip codes" that can be targeted past short peptide sequences (Ruoslahti 2012). Remarkably, certain peptides tin actually penetrate deep into tissues, thereby overcoming elevated interstitial pressures (Sugahara et al. 2009). These findings, which originated in phage display and farther developed through the imaging of targeted nanomaterials, take led to new key insights on the trafficking of particulate materials in vivo, termed the CendR pathway (Teesalu et al. 2009).

This approach was clinically translated as a nanoparticle that uses a targeting ligand for serum membrane antigen (PSMA) (Fig. 7A; Hrkach et al. 2012). This nanoparticle incorporates a "stealth" polymer castor crush that limits immunosurveilance as well as a degradable hydrophobic cadre that could exist loaded with drugs. These optimized characteristics promoted circulation times while reducing liver accumulation. Moreover, these nanoparticles exhibited increased tumor accumulation relative to solvent-based drugs and prolonged tumor suppression. In preliminary patient trials, tumor shrinkage was observed fifty-fifty at diminished dosages of nanoparticles relative to solvent-based drugs.

(A) Nanoparticle consisting of a degradable polymeric cadre loaded with drugs protected past a "stealth" polymer brush crush with targeting ligands. (B) Amplified targeting of nanoparticles by locally induced coagulation. From Hrkach et al. (2012). Reprinted with permission from AAAS. Reprinted by permission from Macmillan Publishers Ltd. from von Maltzahn et al. (2011).

A specially challenging but attractive therapeutic cargo is siRNA. In principle, it can silence so-called "undruggable" targets just must be delivered repeatedly to the cytosol of the target jail cell to engage the silencing mechanism in the RNA-induced silencing complex (RISC) (Whitehead et al. 2009). Moreover, siRNA is vulnerable to degradation by serum nucleases and renal filtration. Nanomaterials take the potential to accost these problems by enabling targeted delivery of enhanced payloads while protecting the siRNA until needed (Davis et al. 2008). Exciting early on clinical results take been demonstrated in cancer patients using a synthetic delivery based on a transferrin-targeted cyclodextran particle with a "stealth" polymer castor shell (Davis et al. 2010). Recently, tumor-penetrating nanocomplexes exploiting the CendR pathway have been developed to target essential oncogenes such as ID4 (Ren et al. 2012). An important next pace volition be to enhance the potency of siRNA, which has been demonstrated through the addition of lipid-like nanomaterials, enabling the silencing of multiple genes simultaneously (Love et al. 2010). It should be noted that nanomaterials have been used extensively to silence targets related to liver office, including for liver cancer, hypercholesterolemia, diabetes Ii, HBV, etc. (Whitehead et al. 2009) Notwithstanding, boosted work will exist needed to extend delivery platforms beyond the liver and tumors to other normal and diseased tissues.

I unresolved issue is whether the additional functionality of these nanoparticles has merchandise-offs by increasing manufacturing complexity and the likelihood of unforeseen effects in vivo (Cheng et al. 2012). For instance, loading multiple drugs may result in cross-reactivity and difficulties in coordinating release profiles. Alternatively, given the finite volume available in a nanoparticle, loading both imaging and therapeutic agents may mean diminished effectiveness due to insufficient quantities of both. Rather that increment the complexity of individual nanoparticles, researchers have designed cooperative nanosystems where the action of uncomplicated, specialized particles are spatially and temporally coordinated. One approach relies on nanoparticles that can communicate through the environment to amplify the recruitment of therapeutics to a tumor site (von Maltzahn et al. 2011). In this system, gilded nanorods passively accumulate in tumors, where they activate a coagulation response upon heating (Fig. 7B). This coagulation serves as a "bespeak" for "receiver" nanoparticles that target the transglutaminase or polymerized fibrin present in the coagulated surround. This communication enhances accumulation of receiving nanoparticles by 40-fold relative to noncommunicating controls, corresponding to an amplification of 35,000 molecules per signaling entity. In a similar fashion, a ii-particle system was designed with ane nanoparticle increasing tumor permeability upon heating and inducing stress-related upward-regulation of p32 surface receptors (Park et al. 2010). These receptors were so targeted by a second doxorubicin-loaded liposome, thereby amplifying therapeutic commitment to the permeated tumor. Such cooperative, synergistic behaviors offer an alternative route for the design of new therapeutic strategies.

Discussion

Nanotechnology offers heady new approaches for both measuring living systems, including characterizing distinctive phenotypes and enriching clinically relevant biomarkers, and perturbing living systems, such equally targeted drug delivery modalities that tin can overcome biological barriers. These diverse examples tin can exist generalized as structural or functional interfaces for communicating data between biotic and abiotic systems. In this context, several fundamental themes emerge that motivate the ongoing development of nanotechnology for biological science and medicine. Starting time, nanotechnology enables interfaces between the macroscale of humans down to the nanoscale of molecules and cells. These tools can be used to precisely examine and perturb individuals within a population, rather than the relatively crude majority methods that have been used historically. Second, nanotechnology is based on translation and conversion between different types of signals. For case, cells and tissues often signal using biochemical messengers in ionic solution, which is very unlike from the conventional technologies used by humans. Nevertheless, the infrequent chemical and concrete properties of nanomaterials, including enhanced electronic, optical, and thermal functionality, tin be used to locally detect or perturb biological phenomena. Finally, many biomedical applications crave the compatibility of artificial nanomaterials with biological systems. Artificial nanomaterials are often synthesized and fabricated under conditions that are highly toxic to cells and tissues. Thus, these artificial nanomaterials may non role as designed under complex physiological weather condition. Maintaining the structure and function of both bogus and biological elements over extended time periods remains an ongoing area of inquiry.

Moving forward, translating these proof-of-concept technologies from a controlled laboratory environs to widespread usage will require extensive testing and validation. In item, biomedical diagnostics have to meet relatively loftier standards for performance and accuracy, especially compared with existing technologies. Another claiming is manufacturing, since nanotechnological systems are ofttimes synthesized or made using highly specialized techniques that are non amenable for commercial calibration-upward. I arroyo, reminiscent of constructed biology (Cheng and Lu 2012), is to begin standardization of nanotechnological "parts," which would enable the ease of comparing between different studies equally well as integration of these different modular components.

Finally, every bit artificial nanotechnologies go increasingly capable of interacting with each other and their biological counterparts, the resulting behaviors may be highly unpredictable and counterintuitive. For example, signaling and feedback mechanisms between the bogus and biological systems may generate emergent behaviors that would not be observed otherwise. Nonetheless, harnessing this complexity tin can enable cooperative systems capable of performing tasks beyond the private functionalities of the elective parts. For instance, under the proper conditions, biomimetic behaviors could occur, such as distension, optimization, mapping, self-assembly, commonage motion, synchronization, and decision-making. Ultimately, a systems approach (Csete and Doyle 2002) to model the interactions between bogus and biological systems may aid in understanding modularity, robustness, and fragility, which could inform biological investigations and guide new therapeutic strategies.

Acknowledgments

We repent for those chief works that are non cited due to the scope of this review and infinite constraints. We thank Due south. Hauert and Due east. Seker for helpful comments, and M. Karabacak, G. Kwong, and A. Rotem for assistance with figures. I.Y.Westward. was a Merck Boyfriend of the Damon Runyon Cancer Enquiry Foundation (DRG-2065-x). S.Due north.B. is a Howard Hughes Medical Investigator. Additional support was provided by the Lustgarten Foundation (to S.N.B.) and the National Institutes of Health (U54 CA151884, Massachusetts Institute of Technology-Harvard Eye of Cancer Nanotechnology Excellence, to S.North.B., and P41-EB002503, BIoMEMS Resource Center, to M.T.).

Footnotes

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