RESUMO
Although nanotechnology often addresses biomedical needs, nanoscale tools can also facilitate broad biological discovery. Nanoscale delivery, imaging, biosensing, and bioreactor technologies may address unmet questions at the interface between chemistry and biology. Currently, many chemical biologists do not include nanomaterials in their toolbox, and few investigators develop nanomaterials in the context of chemical tools to answer biological questions. We reason that the two fields are ripe with opportunity for greater synergy. Nanotechnologies can expand the utility of chemical tools in the hands of chemical biologists, for example, through controlled delivery of reactive and/or toxic compounds or signal-binding events of small molecules in living systems. Conversely, chemical biologists can work with nanotechnologists to address challenging biological questions that are inaccessible to both communities. This Perspective aims to introduce the chemical biology community to nanotechnologies that may expand their methodologies while inspiring nanotechnologists to address questions relevant to chemical biology.
Assuntos
Biologia Molecular/tendências , Nanotecnologia/tendências , Animais , Materiais Biocompatíveis , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Enzimas/química , Humanos , Biologia Molecular/métodos , Imagem Molecular/métodos , NanopartículasRESUMO
Applications of single-walled carbon nanotubes (SWCNTs) in bioimaging and biosensing have been limited by difficulties with isolating single-chirality nanotube preparations with desired functionalities. Unique optical properties, such as multiple narrow near-infrared bands and several modes of signal transduction, including solvatochromism and FRET, are ideal for live cell/organism imaging and sensing applications. However, internanotube FRET has not been investigated in biological contexts. We developed single-chirality subcellular SWCNT imaging probes and investigated their internanotube FRET capabilities in live cells. To functionalize SWCNTs, we replaced the surfactant coating of aqueous two-phase extraction-sorted single-chirality nanotubes with helical polycarbodiimide polymers containing different functionalities. We achieved single-chirality SWCNT targeting of different subcellular structures, including the nucleus, to enable multiplexed imaging. We also targeted purified (6,5) and (7,6) chiralities to the same structures and observed internanotube FRET within these organelles. This work portends the use of single-chirality carbon nanotube optical probes for applications in biomedical research.
Assuntos
Nanotubos de Carbono , Diagnóstico por Imagem , Transferência Ressonante de Energia de Fluorescência , Humanos , Polímeros , TensoativosRESUMO
Fusion protein tags are widely used to capture and track proteins in research and industrial bioreactor processes. Quantifying fusion-tagged proteins normally requires several purification steps coupled with classical protein assays. Here, we developed a broadly applicable nanosensor platform that quantifies glutathione-S-transferase (GST) fusion proteins in real-time. We synthesized a glutathione-DNA-carbon nanotube system to investigate glutathione-GST interactions via semiconducting single-walled carbon nanotube (SWCNT) photoluminescence. We found that SWCNT fluorescence wavelength and intensity modulation occurred specifically in response to GST and GST-fusions. The sensor response was dependent on SWCNT structure, wherein mod(n - m, 3) = 1 nanotube wavelength and intensity responses correlated with nanotube diameter distinctly from mod(n - m, 3) = 2 SWCNT responses. We also found broad functionality of this sensor to diverse GST-tagged proteins. This work comprises the first label-free optical sensor for GST and has implications for the assessment of protein expression in situ, including in imaging and industrial bioreactor settings.
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Glutationa Transferase , Glutationa , Cromatografia de Afinidade , Glutationa Transferase/genética , ProteínasRESUMO
Development of targeted nanoparticle drug carriers often requires complex synthetic schemes involving both supramolecular self-assembly and chemical modification. These processes are generally difficult to predict, execute, and control. We describe herein a targeted drug delivery system that is accurately and quantitatively predicted to self-assemble into nanoparticles based on the molecular structures of precursor molecules, which are the drugs themselves. The drugs assemble with the aid of sulfated indocyanines into particles with ultrahigh drug loadings of up to 90%. We devised quantitative structure-nanoparticle assembly prediction (QSNAP) models to identify and validate electrotopological molecular descriptors as highly predictive indicators of nano-assembly and nanoparticle size. The resulting nanoparticles selectively targeted kinase inhibitors to caveolin-1-expressing human colon cancer and autochthonous liver cancer models to yield striking therapeutic effects while avoiding pERK inhibition in healthy skin. This finding enables the computational design of nanomedicines based on quantitative models for drug payload selection.
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Portadores de Fármacos/química , Nanomedicina/métodos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Portadores de Fármacos/metabolismo , Portadores de Fármacos/farmacocinética , Endocitose , Indóis/química , Camundongos , Nanopartículas/química , Tamanho da Partícula , Distribuição TecidualRESUMO
The use of single-walled carbon nanotubes (SWCNTs) as near-infrared optical probes and sensors require the ability to simultaneously modulate nanotube fluorescence and functionally derivatize the nanotube surface using noncovalent methods. We synthesized a small library of polycarbodiimides to noncovalently encapsulate SWCNTs with a diverse set of functional coatings, enabling their suspension in aqueous solution. These polymers, known to adopt helical conformations, exhibited ordered surface coverage on the nanotubes and allowed systematic modulation of nanotube optical properties, producing up to 12-fold differences in photoluminescence efficiency. Polymer cloaking of the fluorescent nanotubes facilitated the first instance of controllable and reversible internanotube exciton energy transfer, allowing kinetic measurements of dynamic self-assembly and disassembly.
Assuntos
Carbodi-Imidas/química , Transferência de Energia , Nanotubos de CarbonoRESUMO
In this study, we report on the preparation, characterization, and cytocompatibility of hydrogels for biomedical applications made from two different molecular weights of chitosan (CS) blended with poly(vinyl alcohol) (PVA) and chemically cross-linked with tetraethyl orthosilicate (TEOS) followed by freeze-drying. A series of CS-PVA hydrogels were synthesized with different amounts of chitosan (1%, 2%, and 3% by weight). The structure of these CS-PVA hydrogels was characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The hydrogel samples were also characterized for tensile strength, contact angle, swelling behavior, and degradation at physiological body temperature. Their physicochemical properties, biocompatibility, and cell viability when cultured with human dermal fibroblasts were assessed using alamarBlue and live/dead assays and compared to optimize their functionality. SEM analysis showed that the concentration and molecular weight of the chitosan component affected the pore size. Furthermore, the contact angle decreased with increasing chitosan content, indicating that chitosan increased its hydrophilic properties. The in vitro degradation study revealed a nonlinear time-dependent relationship between chitosan concentration or molecular weight, and the rate of degradation was affected by the pore size of the hydrogel. All of the CS-PVA hydrogels exhibited good cell proliferation, particularly with the high molecular weight chitosan samples.
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Imbalance of potassium-ion levels in the body can lead to physiological dysfunctions, which can adversely impact cardiovascular, neurological, and ocular health. Thus, quantitative measurement of potassium ions in a biological system is crucial for personal health monitoring. Nanomaterials can be used to aid in disease diagnosis and monitoring therapies. Optical detection technologies along with molecular probes emitting within the near-infrared (NIR) spectral range are advantageous for biological measurements due to minimal interference from light scattering and autofluorescence within this spectral window. Herein, we report the development of NIR fluorescent nanosensors, which can quantitatively detect potassium ions under biologically relevant conditions. The optical nanosensors were developed by using photoluminescent single-walled carbon nanotubes (SWCNTs) encapsulated in polymers that contain potassium chelating moieties. The nanosensors, polystyrene sulfonate [PSS-SWCNTs, nanosensor 1 (NS1)] or polystyrene-co-polystyrene sulfonate [PS-co-PSS-SWCNTs, nanosensor 2 (NS2)], exhibited dose-dependent optical responses to potassium ion level. The nanosensors demonstrated their biocompatibility via the evaluation of cellular viability, proliferation assays, and expression of cytokeratin 12 in corneal epithelial cells (CEpiCs). Interestingly, the nanosensors' optical characteristics and their responses toward CEpiCs were influenced by encapsulating polymers. NS2 exhibited a 10 times higher fluorescence intensity along with a higher signal-to-noise ratio as compared to NS1. NS2 showed an optical response to potassium ion level in solution within 5 min of addition and a limit of detection of 0.39 mM. Thus, NS2 was used for detailed investigations including potassium ion level detection in serum. NS2 showed a consistent response to potassium ions at the lower millimolar range in serum. These results on optical sensing along with biocompatibility show a great potential for nanotube sensors in biomedical research.
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This study presents the development of stable-isotope labeled hydrophobic, hydrazide reagents for the relative quantification of N-linked glycans. The P2GPN "light" ((12)C) and "heavy" ((13)C(6)) pair are used to differentially label two N-linked glycan samples. The samples are combined 1:1, separated using HILIC, and then mass differentiated and quantified using mass spectrometry. These reagents have several benefits: (1) impart hydrophobic character to the glycans affording an increase in electrospray ionization efficiency and MS detection; (2) indistinguishable chromatographic, MS, and MS/MS performance of the "light" and "heavy" reagents affording relative quantification; and (3) analytical variability is significantly reduced due to the two samples being mixed together after sample preparation. Obtaining these analytical benefits only requires ~4 h of sample preparation time. It is shown that these reagents are capable of quantifying changes in glycosylation in simple mixtures, and the analytical variability of the reagents in pooled plasma samples is shown to be less than ±30%. Additionally, the incorporation of an internal standard allows one to account for the difference in systematic error between the two samples due to the samples being processed in parallel and not mixed until after derivatization.
Assuntos
Hidrazonas/química , Polissacarídeos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Isótopos de Carbono/química , Glicopeptídeos/análise , Glicosilação , Interações Hidrofóbicas e Hidrofílicas , Marcação por IsótopoRESUMO
Carbon nanotube-based donor-acceptor devices are used in applications ranging from photovoltaics and sensors to environmental remediation. Non-covalent contacts between donor dyes and nanotubes are often used to optimize sensitization and scalability. However, inconsistency is often observed despite donor dye studies reporting strong donor-acceptor interactions. Here, we demonstrate that the dye binding location is an important factor in this process: we used coated-acceptor chromatic responses and find that dye binding is affected by the coating layer. The emission response to free- and protein-sequestered porphyrin was tested to compare direct and indirect dye contact. An acceptor complex that preferentially red-shifts in response to sequestered porphyrin was identified. We observe inconsistent optical signals that suggest porphyrin-dye interactions are best described as coating-centric; therefore, the coating interface must be considered in application and assay design.
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Microalbuminuria is an important clinical marker of several cardiovascular, metabolic, and other diseases such as diabetes, hypertension, atherosclerosis, and cancer. The accurate detection of microalbuminuria relies on albumin quantification in the urine, usually via an immunoturbidity assay; however, like many antibody-based assessments, this method may not be robust enough to function in global health applications, point-of-care assays, or wearable devices. Here, we develop an antibody-free approach using synthetic molecular recognition by constructing a polymer to mimic fatty acid binding to the albumin, informed by the albumin crystal structure. A single-walled carbon nanotube, encapsulated by the polymer, as the transduction element produces a hypsochromic (blue) shift in photoluminescence upon the binding of albumin in clinical urine samples. This complex, incorporated into an acrylic material, results in a nanosensor paint that enables the detection of microalbuminuria in patient samples and comprises a rapid point-of-care sensor robust enough to be deployed in resource-limited settings.
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Albuminas/química , Albuminúria/diagnóstico , Técnicas Biossensoriais/métodos , Albuminas/isolamento & purificação , Albuminúria/urina , Biomarcadores/sangue , Biomarcadores/urina , Proteínas Sanguíneas/análise , Ácidos Graxos , Humanos , Imobilização , Nanoestruturas/química , Pintura , Espectrometria de Fluorescência , Urina/químicaRESUMO
Single-walled carbon nanotubes are of interest in biomedicine for imaging and molecular sensing applications and as shuttles for various cargos such as chemotherapeutic drugs, peptides, proteins, and oligonucleotides. Carbon nanotube surface chemistry can be modulated for subcellular targeting while preserving photoluminescence for label-free visualization in complex biological environments, making them attractive materials for such studies. The cell nucleus is a potential target for many pathologies including cancer and infectious diseases. Understanding mechanisms of nanomaterial delivery to the nucleus may facilitate diagnostics, drug development, and gene-editing tools. Currently, there are no systematic studies to understand how these nanomaterials gain access to the nucleus. Herein, we developed a carbon nanotube based hybrid material that elucidate a distinct mechanism of nuclear translocation of a nanomaterial in cultured cells. We developed a nuclear-targeted probe via cloaking photoluminescent single-walled carbon nanotubes in a guanidinium-functionalized helical polycarbodiimide. We found that the nuclear entry of the nanotubes was mediated by the import receptor importin ß without the aid of importin α and not by the more common importin α/ß pathway. Additionally, the nanotube photoluminescence exhibited distinct red-shifting upon entry to the nucleus, potentially functioning as a reporter of the importin ß-mediated nuclear transport process. This work delineates a noncanonical mechanism for nanomaterial delivery to the nucleus and provides a reporter for the study of nucleus-related pathologies.
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Núcleo Celular/metabolismo , Substâncias Luminescentes/metabolismo , Nanotubos de Carbono/análise , Imagem Óptica , Transporte Ativo do Núcleo Celular , Células HeLa , Humanos , Substâncias Luminescentes/química , Microscopia de Fluorescência , Estrutura Molecular , Nanotubos de Carbono/química , Polímeros/química , Células Tumorais CultivadasRESUMO
Alkylating agents such as cisplatin play an essential role in chemotherapy regimens, but initial and acquired resistance in many cancer types often dampen therapeutic response. The poor understanding of the mechanisms of resistance highlight the need for quantitative measurements of alkylating agent distribution at both the tissue and subcellular levels. Sensors for use in live animals and cells would allow for more effective study of drug action and resistance. Toward this end, single-walled carbon nanotubes suspended with single-stranded DNA have suitable optical properties for in vivo sensors, such as near-infrared emission and sensitivity to the local environment via solvatochromic responses. Currently, solvatochromic changes of such sensors have been limited by the chemical nature of the analyte, making it impossible to control the direction of energy emission changes. Here, we describe a new approach to control the direction and magnitude of solvatochromic responses of carbon nanotubes. We found that the alkylation of DNA on the nanotube surface can result in small changes in DNA conformation that allow the adsorption of amphiphiles to produce large differences (>14 nm) in response to different drugs. The technique surprisingly revealed differences among drugs upon alkylation. The ability to control carbon nanotube solvatochromism as desired may potentially expand the application of nanotube-based optical sensors for new classes of analytes.
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Nanotubos de Carbono , Adsorção , Antineoplásicos , DNA de Cadeia SimplesRESUMO
Carbon nanotube-based molecular probes, imaging agents, and biosensors in cells and in vivo continue to garner interest as investigational tools and clinical devices due to their unique photophysical properties. Surface chemistry modulation of nanotubes plays a critical role in determining stability and interaction with biological systems both in vitro and in vivo. Among the many parameters that influence the biological fate of nanomaterials, surface charge is particularly influential due to direct electrostatic interactions with components of the cell membrane as well as proteins in the serum, which coat the nanoparticle surface in a protein corona and alter nanoparticle-cell interactions. Here, we modulated functional moieties on a helical polycarbodiimide polymer backbone that non-covalently suspended the nanotubes in aqueous media. By derivatizing the polymer with either primary amine or carboxylic acid side chains, we obtained nanotube complexes that present net surface charges of opposite polarity at physiological pH. Using these materials, we found that the uptake of carbon nanotubes in these cells is highly dependent on charge, with cationic nanotubes efficiently internalized into cells compared to the anionic nanotubes. Furthermore, we found that serum proteins drastically influenced cell uptake of the anionic nanotubes, while the effect was not prominent for the cationic nanotubes. Our findings have implications for improved engineering of drug delivery devices, molecular probes, and biosensors.
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Lipid accumulation within the lumen of endolysosomal vesicles is observed in various pathologies including atherosclerosis, liver disease, neurological disorders, lysosomal storage disorders, and cancer. Current methods cannot measure lipid flux specifically within the lysosomal lumen of live cells. We developed an optical reporter, composed of a photoluminescent carbon nanotube of a single chirality, that responds to lipid accumulation via modulation of the nanotube's optical band gap. The engineered nanomaterial, composed of short, single-stranded DNA and a single nanotube chirality, localizes exclusively to the lumen of endolysosomal organelles without adversely affecting cell viability or proliferation or organelle morphology, integrity, or function. The emission wavelength of the reporter can be spatially resolved from within the endolysosomal lumen to generate quantitative maps of lipid content in live cells. Endolysosomal lipid accumulation in cell lines, an example of drug-induced phospholipidosis, was observed for multiple drugs in macrophages, and measurements of patient-derived Niemann-Pick type C fibroblasts identified lipid accumulation and phenotypic reversal of this lysosomal storage disease. Single-cell measurements using the reporter discerned subcellular differences in equilibrium lipid content, illuminating significant intracellular heterogeneity among endolysosomal organelles of differentiating bone-marrow-derived monocytes. Single-cell kinetics of lipoprotein-derived cholesterol accumulation within macrophages revealed rates that differed among cells by an order of magnitude. This carbon nanotube optical reporter of endolysosomal lipid content in live cells confers additional capabilities for drug development processes and the investigation of lipid-linked diseases.
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Aterosclerose/sangue , DNA de Cadeia Simples/química , Lipídeos/química , Nanotubos de Carbono/química , Aterosclerose/patologia , DNA de Cadeia Simples/sangue , Endossomos/química , Humanos , Medições Luminescentes , Lisossomos/química , Lisossomos/metabolismo , Macrófagos/química , Macrófagos/metabolismo , Monócitos/química , Monócitos/metabolismo , Doença de Niemann-Pick Tipo C , Óptica e Fotônica/instrumentação , Análise de Célula Única/métodos , Vesículas Transportadoras/química , Vesículas Transportadoras/metabolismoRESUMO
A family of guanidinium-side-chain functionalized polycarbodiimides has been synthesized by allowing an azido guanidinium salt to react with alkyne polycarbodiimides via the copper catalyzed [3 + 2] cycloaddition (Click) reaction. Poly-2(a-d) are cationic/amphiphilic polymers in which the global hydrophilic/hydrophobic balance has been tailored by local alteration of the length of alkyl side chain in the repeat unit of polymers prior to polymerization. The shorter alkyl chains yield water-soluble polymers, Poly-2c, -2d, and -2e. Antibacterial activities of these cationic polycarbodiimides have been investigated for Gram-positive and Gram-negative bacteria that include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Acinetobacter baumannii. It was observed that the influence of hydrophobic-hydrophilic balance per repeat unit of these polymers have profound effects for both antimicrobial and hemolytic activities. In addition, these polycarbodiimide-guanidinium-triazole conjugates offered moderate to significant antibacterial activity and rapid interaction with red blood cells causing blood precipitation without significant hemolysis in case of Poly-2(b-e). This latter property has the potential to be exploited in the polymer coatings or wound protection.