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Single-molecule localization microscopy (SMLM) emerges as a powerful approach for super-resolution imaging that provides unprecedented resolution at the nanometer length scale. However, the development of appropriate probes with specific photophysical traits and characteristics is crucial for this approach. This study demonstrates two different fluorescent carbon dots (CDs) derived from the same molecular precursorâone emitting in red and the other in greenâas a SMLM-based super-resolution imaging probe for different applications with an average localization precision of 20 nm and a resolution of 60 nm. Both the CDs exhibit spontaneous blinking with high photon count and low duty cycle but with different blinking cycles. The red emissive CDs with a lower blinking cycle are ideal for quantitative analysis, whereas green emissive CDs with a higher blinking cycle are ideal for high-resolution imaging. We show that the difference in blinking features is linked to their chemical compositions, and the presence of much denser trap states in red emitting CDs is responsible for the reduction of its blinking cycle. This study shows that CDs can be designed as a potential probe for SMLM-based super-resolution imaging for diverse bioimaging applications.
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Nanoparticles usually enter cells through energy-dependent endocytosis that involves their cytosolic entry via biomembrane-coated endosomes. In contrast, direct translocation of nanoparticles with straight access to cytosol/subcellular components without any membrane coating is limited to very selective conditions/approaches. Here we show that nanoparticles can switch from energy-dependent endocytosis to energy-independent direct membrane penetration once an amphiphile is electrostatically bound to their surface. Compared to endocytotic uptake, this direct cell translocation is faster and nanoparticles are distributed inside the cytosol without any lysosomal trafficking. We found that this direct cell translocation option is sensitive to the charges of both the nanoparticles and the amphiphile. We propose that an electrostatically bound amphiphile induces temporary opening of the cell membrane, which allows direct cell translocation of nanoparticles. This approach can be adapted for efficient subcellular targeting of nanoparticles and nanoparticle-based drug delivery application, bypassing the endosomal trapping and lysosomal degradation.
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Nanopartículas , Citosol/metabolismo , Nanopartículas/metabolismo , Endocitosis , Endosomas/metabolismo , Sistemas de Liberación de MedicamentosRESUMEN
Ultrasound-based therapy is appealing as it can be used via a wireless approach at remote parts of the body including the brain. Microbubbles are commonly used in such therapy due to their highly sound-responsive property. However, the larger size of microbubbles limits selective targeting in vitro/in vivo. Here, we report the design of nanodroplets of 70-130 nm in size that can be easily converted to microbubbles via ultrasound exposure. The advantage of this approach is that smaller nanodroplets can be used for cell/subcellular targeting, and next, they can be used for therapy by converting to microbubbles. More specifically, folate/dopamine-terminated perfluorohexane nanodroplets are designed that are loaded with a molecular drug. These nanodroplets are used for selective cell targeting, followed by ultrasound-induced microbubble conversion that is associated with drug release and intracellular reactive oxygen species generation. This approach has been used for selective cell therapy applications. The designed nanodroplet and approach can be used for the enhanced therapeutic performance of existing drugs.
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Encéfalo , Microburbujas , Movimiento Celular , Tratamiento Basado en Trasplante de Células y Tejidos , DopaminaRESUMEN
Nonendocytic cell uptake of nanomaterials is challenging, which requires specific surface chemistry and smaller particle size. Earlier works have shown that an arginine-terminated nanoparticle of <10-20 nm size shows nonendocytic uptake via direct membrane penetration. However, the roles of surface arginine density and the arginine-arginine distance at the nanoparticle surface in controlling such nonendocytic uptake mechanism is not yet explored. Here we show that a higher arginine density at the nanoparticle surface with an arginine-arginine distance of <3 nm is the most critical aspect for such nonendocytic uptake. We have used quantum dot (QD)-based nanoparticles as a model for fluorescent tracking inside cells and for quantitative estimation of cellular uptake. We found that arginine-terminated nanoparticles of 10 nm size can opt for the energy-dependent endocytosis pathway if the arginine-arginine distance is >3 nm. In contrast, nanoparticles with <3 nm arginine-arginine distance rapidly enter into the cell via the nonendocytic approach, are freely available in the cytosol in large amounts to capture the cellular adenosine triphosphate (ATP), generate oxidative stress, and induce ATP-deficient cellular autophagy. This work shows that arginine-arginine distance at the nanoparticle surface is another fundamental parameter, along with the particle size, for the nonendocytic cell uptake of foreign materials and to control intracellular activity. This approach may be utilized in designing nanoprobes and nanocarriers with more efficient biomedical performances.
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Nanopartículas , Puntos Cuánticos , Arginina , Nanopartículas/metabolismo , Autofagia , Adenosina Trifosfato , Tamaño de la Partícula , EndocitosisRESUMEN
Although photodynamic therapy is a promising approach for cancer treatment, it has limited clinical application due to the poor performance of conventional photosensitizers. In this study, we present a carbon nanoparticle-based photosensitizer for efficient photodynamic cell therapy. The nanoparticles have been synthesized from a steel industry-based waste material, exhibiting strong fluorescence in the visible region, rapidly entering the cell via non-endocytic uptake, and localizing within the mitochondria. Light exposure of nanoparticle-labeled cells offers efficient photodynamic therapy and induces cytotoxicity. Overall, this study highlights the utility of carbon nanoparticles in efficient photodynamic therapy via rapid cellular uptake and subcellular targeting.
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Nanopartículas , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Nanopartículas/uso terapéutico , FluorescenciaRESUMEN
Cellular membranes are constantly bombarded with biomolecules and nanoscale particles, and cell functionality depends on the fraction of the bound/internalized entities. Understanding the biophysical parameters underlying this complex process is very difficult in live cells. Model membranes provide an ideal platform to obtain insight into the minimal and essential parameters involved in determining cell membrane-nanoparticle (NP) interaction. Here we report spontaneous binding and unbinding of semiconductor NPs, carrying different net charges and interacting with model biomembranes, using in situ neutron reflectivity (NR) and fluorescence microscopy studies. We observe a critical concentration of NPs above which they spontaneously unbind along with lipids from lipid monolayer membranes, leaving behind fewer bound NPs. This critical concentration varies depending on whether the NPs carry a net charge or are neutral, and is also governed by the extent of NP crowding for a fixed NP charge. The observations suggest a subtle interplay between electrostatics, membrane fluidity, and NP crowding effects, which eventually determines the adsorbed concentration for unbinding transition. Our study provides valuable microscopic insight into the parameters that could determine the biophysical process underlying NP uptake and ejection by cells which, in turn, can be utilized for their potential applications in bioimaging and drug delivery.
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Nanopartículas , Electricidad Estática , Nanopartículas/química , Membrana Celular/metabolismo , Sistemas de Liberación de Medicamentos , Fluidez de la MembranaRESUMEN
Biomolecular piezoelectric materials are envisioned for advanced biomedical applications for their robust piezoelectricity, biocompatibility, and flexibility. Here, we report the piezoelectric property of amyloid fibrils derived from three distinct proteins: lysozyme, insulin, and amyloid-ß. We found that piezoelectric properties are dependent on the extent of the ß-sheet structure and the extent of fibril anisotropy. We have observed the piezoelectric constant value in the range of 24-42 pm/V for fibrils made of lysozyme/insulin/amyloid-ß, and for the sheet/bundle-like structure of lysozyme aggregates, the value becomes 62 pm/V. These piezoelectric constant values are 4-10 times higher than the native lysozyme/insulin/amyloid proteins. Computational studies show that extension of the ß-sheet structure produces an asymmetric arrangement of charges (in creating dipole moment) and mechanical stress induces an aligned orientation of these dipoles that results in a piezoelectric effect. It is shown that these piezoelectric fibrils can harvest mechanical as well as ultrasound-based energy to produce a voltage of up to 1 V and a current of up to 13 nA. These fibrils are employed for reactive oxygen species (ROS) generation under ultrasound exposure and utilized for ultrasonic degradation of organic pollutants or killing of cancer cells via intracellular ROS generation under ultrasound exposure. Our findings demonstrate that the piezoelectric property of protein fibrils has potential for wireless therapeutic applications and may have physiological roles that are yet to be explored.
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Amiloide , Muramidasa , Amiloide/química , Especies Reactivas de Oxígeno , Muramidasa/química , Pliegue de Proteína , Insulina/químicaRESUMEN
The delivery of cell impermeable exogenous material into live cells by external stimuli is critical for both biological research and therapeutic applications. Although electroporation-based delivery of foreign materials inside the cell is a powerful approach, cell viability is often compromised due to the requirement of high voltage. Here, we report a piezoelectric hydroxyapatite nanowire-embedded poly(vinylidene fluoride) (PVDF) film for ultrasonic electroporation-based delivery of foreign materials to adherent cells. We found that 9 wt % loading of hydroxyapatite nanowires into PVDF can enhance the piezoelectric property by 2-3 times (with a piezoelectric constant value of 58 pm/V) than pure PVDF/nanowire, which is comparable to commonly known piezoelectric ceramics. These films can harvest mechanical as well as ultrasound-based energy to produce electrical potential up to 2 V. This biocompatible film can be used to grow cells on top of it and for subsequent application of ultrasound to exert electric voltage on cell membrane. We found that ultrasonic exposure to adhered cells leads to reversible pore formation on cell membrane that offers intracellular delivery of FITC-dextran with 75% efficiency. The developed piezoelectric film-based ultrasonic electroporation can be used for wireless electroporation in remote areas.
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Nanocables , Ultrasonido , Electroporación , DurapatitaRESUMEN
Although efficient cell nucleus delivery of exogenous materials can greatly improve their biochemical activity, this is strictly restricted by cellular uptake and intracellular trafficking processes. In the current approach, synthetic carriers are designed for cell delivery of exogenous materials via endocytosis, and nucleus delivery can be achieved via endosomal escape. Here, we demonstrate that a nonendocytic cell uptake approach can be adapted for protein delivery to the cell nucleus. We have designed a phenylboronic acid-terminated micellar carrier that can bind with protein in the presence of green tea polyphenol and deliver protein into the cytosol via the nonendocytic approach. Using this approach, four different proteins are delivered to the cytosol within 15 min, and low-molecular weight proteins are delivered to the nucleus. The designed approach can be extended for delivering macromolecular drugs to subcellular targets for a more efficient therapy.
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Although subcellular targeting can enhance the therapeutic performance of most drugs, such targeting requires appropriate carrier-based delivery that can bypass endosomal/lysosomal trafficking. Recent works show that nanocarriers can be designed for direct cell membrane translocation and nonendocytic uptake, bypassing the usual endocytosis processes. Here we show that this approach can be adapted for the rapid cell nucleus delivery of molecular drugs. In particular, a guanidinium-terminated nanocarrier is used to create a weak interaction-based carrier-drug nanoassembly for direct membrane translocation into the cytosol. The rapid and extensive entry of a drug-loaded nanocarrier into the cell without any vesicular coating and affinity of the drug to the nucleus allows their nucleus labeling. Compared to endocytotic uptake that requires more than hours for cell uptake followed by predominant lysosomal entrapment, this nonendocytic uptake labels the nucleus within a few minutes without any lysosomal trafficking. This approach may be utilized for nanocarrier-based subcellular targeting of drugs for more effective therapy.
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Núcleo Celular , Nanopartículas , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Citosol/metabolismo , Lisosomas/metabolismo , Endocitosis , Portadores de Fármacos/farmacología , Sistemas de Liberación de MedicamentosRESUMEN
Although mitochondria have been identified as a potential therapeutic target for the treatment of various diseases, inefficient drug targeting to mitochondria is a major limitation for related therapeutic applications. In the current approach, drug loaded nanoscale carriers are used for mitochondria targeting via endocytic uptake. However, these approaches show poor therapeutic performance due to inefficient drug delivery to mitochondria. Here, we report a designed nanoprobe that can enter the cell via a nonendocytic approach and label mitochondria within 1 h. The designed nanoprobe is <10 nm in size and terminated with arginine/guanidinium that offers direct membrane penetration followed by mitochondria targeting. We found five specific criteria that need to be adjusted in a nanoscale material for mitochondria targeting via the nonendocytic approach. They include <10 nm size, functionalization with arginine/guanidinium, cationic surface charge, colloidal stability, and low cytotoxicity. The proposed design can be adapted for mitochondria delivery of drugs for efficient therapeutic performance.
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Arginina , Mitocondrias , Arginina/metabolismo , Guanidina/metabolismo , Mitocondrias/metabolismo , Sistemas de Liberación de Medicamentos , Portadores de Fármacos , Membrana Celular/metabolismoRESUMEN
Trehalose is a disaccharide that is capable of inhibiting protein aggregation and activating cellular autophagy. It has been shown that a polymer or nanoparticle form, terminated with multiple trehalose units, can significantly enhance the anti-amyloidogenic performance and is suitable for the treatment of neurodegenerative diseases. Here, we report a trehalose-conjugated polycarbonate-co-lactide polymer and formulation of its nanoparticles having multiple numbers of trehalose exposed on the surface. The resultant poly(trehalose) nanoparticle inhibits the aggregation of amyloid beta peptides and disintegrates matured amyloid fibrils into smaller fragments. Moreover, the poly(trehalose) nanoparticle lowers extracellular amyloid ß oligomer-driven cellular stress and enhances cell viability. The presence of biodegradable polycarbonate components in the poly(trehalose) nanoparticle would enhance their application potential as an anti-amyloidogenic material.
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Nanopartículas , Enfermedades Neurodegenerativas , Humanos , Péptidos beta-Amiloides/metabolismo , Trehalosa/farmacología , Nanopartículas/uso terapéutico , PolímerosRESUMEN
Lipid nanotubules (LNTs) are conduits that form on the membranes of cells and organelles, and they are ubiquitous in all forms of life from archaea and bacteria to plants and mammals. The formation, shape and dynamics of these LNTs are critical for cellular functions, supporting the transport of myriad cellular cargoes as well as communication within and between cells, and they are also widely believed to be responsible for exploitation of host cells by pathogens for the spread of infection and diseases. In vitro kinetic control of LNT formation can considerably enhance the scope of utilization of these structures for disease control and therapy. Here we report a new paradigm for spontaneous lipid nanotubulation, capturing the dynamical regimes of growth, stabilization and retraction of the tubes through the binding of synthetic nanoparticles on supported lipid bilayers (SLBs). The tubulation is determined by the spontaneous binding-unbinding of nanoparticles on the LNTs. The presented methodology could be used to rectify malfunctioning cellular tubules or to prevent the pathogenic spread of diseases through inhibition of cell-to-cell nanotubule formation.
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Membrana Dobles de Lípidos , Nanopartículas , Cinética , Membrana Dobles de Lípidos/química , Nanopartículas/químicaRESUMEN
Piezocatalytic materials have considerable application potential in wireless therapy. Most of these applications require biocompatible nanomaterials for in vivo targeting and control of intracellular processes. However, the piezocatalytic performance of a material decreases at a nanometer size regime, and most of the biocompatible materials have poor piezocatalytic efficiency. In particular, hydroxyapatite or calcium phosphate-based nanomaterials have weak piezocatalytic properties that limit the biomedical application potential. Here, we show that anisotropic shape and Au nanoparticle conjugation can enhance the piezocatalytic property of a calcium phosphate nanomaterial by 10 times and the performance approaches that of the bulk/nanoparticle form of well-known BaTiO3. The colloidal form of calcium phosphate nanowires/nanorods/nanospheres (2-5 nm diameter and 30-1000 nm length) and their Au nanoparticle (5-8 nm) composites are prepared, and their piezoelectric properties have been investigated with piezoresponse force microscopy. It has been observed that the anisotropic nanowire structure of calcium phosphate can enhance the piezoelectric property by 2 times and Au nanoparticle conjugation can enhance it up to 10 times with a piezoelectric constant value of 72 pm/V, which is close to the value of the bulk/nanoparticle form of BaTiO3. This enhanced piezoelectric property is shown to enhance the piezocatalytic reactions by 10 times. The approach has been used to design colloidal nano-bioconjugate for selective labeling of cancer cells, followed by wireless cell therapy via medical-grade ultrasound-based intracellular reactive oxygen species generation. The developed approach and material can be extended for wireless therapeutic applications and for controlling intracellular processes.
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The antimicrobial and cytotoxic effects of zinc oxide nanomaterials are popularly thought to be occurring due to zinc ion leaching, but the exact mechanism of cytotoxicity is controversial and not fully understood. Recent studies have shown that oxygen vacancy defects in the nanoscale zinc oxide can generate reactive oxygen species (ROS) under dark conditions and may induce cytotoxicity. In this work, we show that the cytotoxicity of zinc oxide nanoparticles is directly correlated with oxygen vacancy defects that generate ROS under dark conditions. More specifically, we designed zinc oxide nanoparticles with controlled oxygen vacancy defects by controlled gallium doping and showed that the ROS generation property of zinc oxide nanoparticles under dark conditions is directly correlated with oxygen vacancy defects. Further studies show that superoxide radicals and hydrogen peroxide are the primary ROS that are produced under dark conditions. These colloidal nanoparticles are used for cell labeling and therapy via intracellular ROS generation without any light exposure. The designed nanoparticle can be used for the formulation of advanced antibacterial and antimicrobial materials and other cell therapy applications.
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Nanopartículas , Óxido de Zinc , Antibacterianos/farmacología , Supervivencia Celular , Nanopartículas/toxicidad , Oxígeno , Especies Reactivas de Oxígeno , SuperóxidosRESUMEN
Direct cytosolic delivery of large biomolecules that bypass the endocytic pathways is a promising strategy for therapeutic applications. Recent works have shown that small-molecule, nanoparticle, and polymer-based carriers can be designed for direct cytosolic delivery. It has been shown that the specific surface chemistry of the carrier, nanoscale assembly between the carrier and cargo molecule, good colloidal stability, and low surface charge of the nano-assembly are critical for non-endocytic uptake processes. Here we report a guanidinium-terminated polyaspartic acid micelle for direct cytosolic delivery of protein and DNA. The polymer delivers the protein/DNA directly to the cytosol by forming a nano-assembly, and it is observed that <200 nm size of colloidal assembly with near-zero surface charge is critical for efficient cytosolic delivery. This work shows the importance of size and colloidal property of the nano-assembly for carrier-based cytosolic delivery of large biomolecules.
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Materiales Biocompatibles/química , Citosol/química , ADN/genética , Nanopartículas del Metal/química , Péptidos/química , Albúmina Sérica Bovina/química , Animales , Bovinos , Coloides/química , ADN/química , Guanidina/química , Humanos , Células KB , Ensayo de Materiales , Micelas , Estructura Molecular , Tamaño de la PartículaRESUMEN
Although arginine-rich polymers and peptides are extensively used as delivery carriers for drugs/proteins/nanoparticles, their cell delivery mechanism is not clearly understood. Recent studies show that arginine-terminated nanoparticles can enter into a cell via a nonendocytic approach that involves direct membrane penetration. However, poor colloidal stability of arginine-terminated nanoparticles under physiological conditions restricts their application potential. Here, we show that the nonendocytic cell delivery of arginine-terminated nanoparticles is controlled by their colloidal stability in the presence of phosphates. We have designed arginine-terminated quantum dots (QDs) of 10-15 nm hydrodynamic size, which enter into the cell via a nonendocytic approach, provided that they are colloidal and dispersed during cellular uptake. We have demonstrated that arginine-terminated QDs rapidly precipitate in the presence of monophosphates or polyphosphates, and polyphosphates have a stronger effect than monophosphates. Introducing polyethylene glycol at the QD surface can improve the colloidal stability against phosphates. Control experiments show that amine/ammonium-terminated cationic QDs of similar sizes do not have such a type of phosphate-dependent precipitation issue. We propose that arginine-terminated colloidal nanoparticles have a unique advantage over amine/ammonium-terminated nanoparticles as they can bind with the cell membrane phosphate via guanidinium-phosphate salt bridging. Bulk phosphate provides reversibility in this binding interaction so that nonendocytic cell uptake occurs via charge compensation of cationic nanoparticles without membrane damage. The developed surface chemistry approach and the proposed mechanisms can be adapted to other nanoparticles for efficient cell delivery and for designing delivery carriers.
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Nanopartículas , Puntos Cuánticos , Arginina , Coloides , FosfatosRESUMEN
Nanoparticles are widely used in various biomedical applications as drug delivery carriers, imaging probes, single-molecule tracking/detection probes, artificial chaperones for inhibiting protein aggregation, and photodynamic therapy materials. One key parameter of these applications is the ability of the nanoparticles to enter into the cell cytoplasm, target different subcellular compartments, and control intracellular processes. This is particularly the case because nanoparticles are designed to interact with subcellular components for the required biomedical performance. However, cells are protected from their surroundings by the cell membrane, which exerts strict control over entry of foreign materials. Thus, nanoparticles need to be designed appropriately so that they can readily cross the cell membrane, target subcellular compartments, and control intracellular processes.In the past few decades there have been great advancements in understanding the principles of cellular uptake of foreign materials. In particular, it has been shown that internalization of foreign materials (small molecules, macromolecules, nanoparticles) is size-dependent: endocytotic uptake of materials requires sizes greater than 10 nm, and materials with sizes of 10-100 nm usually enter into cells by energy-dependent endocytosis via biomembrane-coated vesicles. Direct access to the cytosol is limited to very specific conditions, and endosomal escape of material appears to be the most practical approach for intracellular processing.In this Account, we describe how cellular uptake and intracellular processing of nanoscale materials can be controlled by appropriate design of size and surface chemistry. We first describe the cell membrane structure and principles of cellular uptake of foreign materials followed by their subcellular trafficking. Next, we discuss the designed surface chemistry of a 5-50 nm particle that offers preferential lipid-raft/caveolae-mediated endocytosis over clathrin-mediated endocytosis with minimum endosomal/lysosomal trafficking or energy-independent direct cell membrane translocation (without endocytosis) followed by cytosolic delivery without endosomal/lysosomal trafficking. In particular, we emphasize that the zwitterionic-lipophilic surface property of a nanoparticle offers preferential interaction with the lipid raft region of the cell membrane followed by lipid raft uptake, whereas a lower number of affinity biomolecules (<25) on the nanoparticle surface offers caveolae/lipid-raft uptake, while an arginine/guanidinium-terminated surface along with a size of <10 nm offers direct cell membrane translocation. Finally, we discuss how nanoprobes can be designed by adapting these surface chemistry and size preference principles so that they can readily enter into the cell, label different subcellular compartments, and control intracellular processes such as trafficking kinetics, exocytosis, autophagy, amyloid aggregation, and clearance of toxic amyloid aggregates. The Account ends with a Conclusions and Outlook where we discuss a vision for the development of subcellular targeting nanodrugs and imaging nanoprobes by adapting to these surface chemistry principles.
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Membrana Celular/metabolismo , Nanopartículas/metabolismo , Transporte Biológico/fisiología , Caveolas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Microdominios de Membrana/metabolismo , Nanopartículas/química , Tamaño de la Partícula , Pinocitosis/fisiología , Propiedades de SuperficieRESUMEN
Cells respond to external stress by altering their membrane lipid composition to maintain fluidity, integrity and net charge. However, in interactions with charged nanoparticles (NPs), altering membrane charge could adversely affect its ability to transport ions across the cell membrane. Hence, it is important to understand possible pathways by which cells could alter zwitterionic lipid composition to respond to NPs without compromising membrane integrity and charge. Here, we report in situ synchrotron X-ray reflectivity (XR) measurements to monitor the interaction of cationic NPs in the form of quantum dots, with phase-separated supported lipid bilayers of different compositions containing an anionic lipid and zwitterionic lipids having variable degrees of stiffness. We observe that the extent of NP penetration into the respective membranes, as estimated from XR data analysis, is inversely related to membrane compression moduli, which was tuned by altering the stiffness of the zwitterionic lipid component. For a particular membrane composition with a discernible height difference between ordered and disordered phases, we were able to observe subtle correlations between the extent of charge on the NPs and the specificity to bind to the charged and ordered phase, contrary to that observed earlier for phase-separated model biomembranes containing no charged lipids. Our results provide microscopic insight into the role of membrane rigidity and electrostatics in determining membrane permeation. This can lead to great potential benefits in rational designing of NPs for bioimaging and drug delivery applications as well as in assessing and alleviating cytotoxicity of NPs.
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Designing of nanoparticles (NPs) for biomedical applications or mitigating their cytotoxic effects requires microscopic understanding of their interactions with cell membranes. Such insight is best obtained by studying model biomembranes which, however, need to replicate actual cell membranes, especially their compositional heterogeneity and charge. In this work we have investigated the role of lipid charge density and packing of phase separated Langmuir monolayers in the penetration and phase specificity of charged quantum dot (QD) binding. Using an ordered and anionic charged lipid in combination with uncharged but variable stiffness lipids we demonstrate how the subtle interplay of zwitterionic lipid packing and anionic lipid charge density can affect cationic nanoparticle penetration and phase specific binding. Under identical subphase pH, the membrane with higher anionic charge density displays higher NP penetration. We also observe coalescence of charged lipid rafts floating amidst a more fluidic zwitterionic lipid matrix due to the phase specificity of QD binding. Our results suggest effective strategies which can be used to design NPs for diverse biomedical applications as well as to devise remedial actions against their harmful cytotoxic effects especially against respiratory diseases.