Nanoscale Cargo Research Articles

Magnetically Driven Nanotransporter-Assisted Intracellular Delivery and Autonomous Release of Proteins.

Biofunctional proteins such as active enzymes and therapeutic proteins show tremendous promise in disease treatment. However, intracellular delivery of proteins is facing substantial challenges owing to their vulnerability to degradation and denaturation and the presence of various biological barriers such as their low membrane transport efficiency. Herein, we report a magnetically driven and redox-responsive nanotransporter (MRNT) for highly efficient intracellular delivery of biofunctional proteins. The MRNT has remarkably high cargo capacity, compared with that without nanoscale cargo compartments. We have demonstrated the directional and dynamic motion of the MRNT using both nanoparticle tracking analysis and magnetic driving evaluation. Moreover, the active MRNT can translocate into the cytosol and sense the reducing cytosolic environment to discharge protein cargoes autonomously. The internalization mechanism of the MRNT has been studied using endocytosis inhibitors. Under the magnetic drive, the MRNT can promote a protein transduction efficiency of over 95%, and the intracellular protein delivery by the active MRNT shows significantly higher (∼4 times) enzymatic activity and therapeutic efficiency than those achieved by the static ones. Our proof-of-concept study provides a valuable tool for intracellular protein transduction and contributes to biotechnology and protein therapeutics.

Design considerations for effective thermal management in mobile nanotweezers

Controlled manipulation of nanoscale objects in fluids is relevant to both fundamental studies and technological advances in nanotechnology. While standard techniques of nanomanipulation, such as optical and plasmonic tweezers have limitations in simultaneous trapping and transport of nanoscale cargo, magnetically driven plasmonic nanorobots under optical illumination provide a promising solution. These so called mobile nanotweezers (MNT) use strongly localized electromagnetic field near plasmonic nanostructures to trap objects with high efficiency and can simultaneously be driven by magnetic fields to selectively trap, transport and release colloidal cargo. Upon illumination, apart from strong optical gradient forces due to local electric field enhancement, additional fluidic forces arise due to the heat generated by absorption of light. Here, we present a method to understand and engineer thermally induced fluidic forces in mobile nanotweezers. The temperature enhancement and associated thermofluidic forces are studied as a function of MNT geometry. We also discuss illumination at wavelengths slightly detuned from plasmon resonance frequency, which produces sufficient field enhancement with negligible generation of heat, and therefore much reduced thermophoretic and convective forces. This allowed us to engineer thermoplasmonic forces in MNTs for enhanced trapping performance and diverse applications.

Transport and programmed release of nanoscale cargo from cells by using NETosis.

Cells can take up nanoscale materials, which has important implications for understanding cellular functions, biocompatibility as well as biomedical applications. Controlled uptake, transport and triggered release of nanoscale cargo is one of the great challenges in biomedical applications of nanomaterials. Here, we study how human immune cells (neutrophilic granulocytes, neutrophils) take up nanomaterials and program them to release this cargo after a certain time period. For this purpose, we let neutrophils phagocytose DNA-functionalized single-walled carbon nanotubes (SWCNTs) in vitro that fluoresce in the near infrared (980 nm) and serve as sensors for small molecules. Cells still migrate, follow chemical gradients and respond to inflammatory signals after uptake of the cargo. To program release, we make use of neutrophil extracellular trap formation (NETosis), a novel cell death mechanism that leads to chromatin swelling, subsequent rupture of the cellular membrane and release of the cell's whole content. By using the process of NETosis, we can program the time point of cargo release via the initial concentration of stimuli such as phorbol 12-myristate-13-acetate (PMA) or lipopolysaccharide (LPS). At intermediate stimulation, cells continue to migrate, follow gradients and surface cues for around 30 minutes and up to several hundred micrometers until they stop and release the SWCNTs. The transported and released SWCNT sensors are still functional as shown by subsequent detection of the neurotransmitter dopamine and reactive oxygen species (H2O2). In summary, we hijack a biological process (NETosis) and demonstrate how neutrophils transport and release functional nanomaterials.

All optical dynamic nanomanipulation with active colloidal tweezers

Manipulation of colloidal objects with light is important in diverse fields. While performance of traditional optical tweezers is restricted by the diffraction-limit, recent approaches based on plasmonic tweezers allow higher trapping efficiency at lower optical powers but suffer from the disadvantage that plasmonic nanostructures are fixed in space, which limits the speed and versatility of the trapping process. As we show here, plasmonic nanodisks fabricated over dielectric microrods provide a promising approach toward optical nanomanipulation: these hybrid structures can be maneuvered by conventional optical tweezers and simultaneously generate strongly confined optical near-fields in their vicinity, functioning as near-field traps themselves for colloids as small as 40 nm. The colloidal tweezers can be used to transport nanoscale cargo even in ionic solutions at optical intensities lower than the damage threshold of living micro-organisms, and in addition, allow parallel and independently controlled manipulation of different types of colloids, including fluorescent nanodiamonds and magnetic nanoparticles.

Autochemophoretic DNA Motors Generate 100+ Piconewton Forces

DNA walkers, which “walk” along linear or planar tracks via burnt-bridge hybridization interactions, are promising synthetic analogs of motor proteins such as kinesin, myosin, and dynein. DNA walkers can precisely transport nanoscale cargo but cannot generate piconewton-scale force. This primary function of motor proteins is necessary for countless processes including muscle contraction, clotting, immunosensing, embryogenesis, and mechanosensation. We present progress towards the design of force-generating nanomachines by showing that highly polyvalent DNA motors (HPDMs) generate 100+ piconewtons of force via a novel mechanism that we term autochemophoresis. HPDMs are DNA-coated microparticles that connect to planar RNA-functionalized surfaces via DNA-RNA hybridization (Yehl & Salaita et. al., Nature Nanotechnology 2016). Ribonuclease H enables translocation by cleaving the RNA-DNA duplexes, resulting in an RNA depletion track in the HPDM's wake. Translocating for hours at micron/minute speeds, HPDMs are the fastest, most processive DNA-based motors reported to date. To test HPDMs’ force generation capability we designed a single molecule fluorescence microscopy experiment which enables direct visualization of mechanically-ruptured molecular bonds. Surprisingly, we found that HPDMs generate forces that mechanically rupture 25 basepair DNA duplexes and biotin-streptavidin bonds (the strongest noncovalent bonds found in nature with 100+ pN force thresholds). HPDMs lack directed tracks and conformational switching such as ATP-fueled powerstrokes, thus underscoring the novelty of this result. To study this fundamental mechanism of force-generation, we developed a simulation method that accurately reproduces most properties of HPDM motion via direct modeling of the distance-dependant biophysics of DNA-RNA interactions. These simulations highlight the mechanism of HPDM force generation and demonstrate that motion is driven by autochemophoresis, which has been observed in biological systems (Sugawara & Kaneko, Biophysics 2011). Our work suggests that autochemophoresis may be a third fundamental method of force generation in molecular motors and living systems.

Tailoring Surface Opening of Hollow Nanocubes and Their Application as Nanocargo Carriers.

Hollow nanoparticles (NPs) are of broad interest for biomedical, optical, and catalytic applications due to their unique geometry-related physicochemical properties. The ability to engineer hollow structures with surface openings is particularly attractive since emergent properties are promised by the design of shell porosity and encapsulation of guest materials. However, it still remains challenging to precisely control the opening of the hollow structure, in terms of shape, size, and location. Here, we report a facile one-step strategy to synthesize a hollow nanostructure with well-defined cubic-shape openings at the corners, by regulating nanoscale galvanic replacement processes with specific surface-capping agents. The final product is a single-crystalline AuAg alloy which morphologically features three “belts” orthogonally wrapping around a virtual cube, denoted by nanowrapper. We demonstrate a structural tunability of our synthetic method for tailoring nanowrapper and the corresponding tuning of its plasmonic band from the visible to near-infrared (NIR) range. Advanced electron tomography techniques provide unambiguous three-dimensional (3D) visualizations to reveal an unconventional transformation pathway of sharp-cornered Ag nanocube to nanowrapper and correlate its structure with measured and computed spectroscopic properties. Importantly, we find that the surfactant, i.e., cetylpyridinium chloride (CPC), is crucial for the openings to be localized at the corners of the hollow cube and be tailored to a cubic shape in our one-step process. Furthermore, such a well-defined hollow architecture also allows a guest nano-object to be contained within, while the large openings at corners enable controlled loading/release of nanoscale cargo, a DNA-coated particle, using change of ionic conditions. This work expands our understanding of surface engineering in nanoscale galvanic replacement reactions and opens new ways toward the shape control of hollow NPs.

Programmable Peptide-Cross-Linked Nucleic Acid Nanocapsules as a Modular Platform for Enzyme Specific Cargo Release.

Herein we describe a modular assembly strategy for photo-cross-linking peptides into nucleic acid functionalized nanocapsules. The peptides embedded within the nanocapsules form discrete nanoscale populations capable of gating the release of molecular and nanoscale cargo using enzyme-substrate recognition as a triggered release mechanism. Using photocatalyzed thiol-yne chemistry, different peptide cross-linkers were effectively incorporated into the nanocapsules and screened against different proteases to test for degradation specificity both in vitro and in cell culture. By using a combination of fluorescence assays, confocal and TEM microscopy, the particles were shown to be highly specific for their enzyme targets, even between enzymes of similar protease classes. The rapid and modular nature of the assembly strategy has the potential to be applied to both intracellular and extracellular biosensing and drug delivery applications.

Depicting Binding-Mediated Translocation of HIV-1 Tat Peptides in Living Cells with Nanoscale Pens of Tat-Conjugated Quantum Dots.

Cell-penetrating peptides (CPPs) can translocate across cell membranes, and thus have great potential for the cellular delivery of macromolecular cargoes. However, the mechanism of this cellular uptake process is not yet fully understood. In this study, a time-lapse single-particle light-sheet microscopy technique was implemented to obtain a parallel visualization of the translocating process of individual human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) peptide conjugated quantum dots (TatP-QDs) in complex cellular terrains. Here, TatP-QDs served as nanoscale dynamic pens, which depict remarkable trajectory aggregates of TatP-QDs on the cell surface. Spectral-embedding analysis of the trajectory aggregates revealed a manifold formed by isotropic diffusion and a fraction of directed movement, possibly caused by interaction between the Tat peptides and heparan sulfate groups on the plasma membrane. Further analysis indicated that the membrane deformation induced by Tat-peptide attachment increased with the disruption of the actin framework in cytochalasin D (cyto D)-treated cells, yielding higher interactions on the TatP-QDs. In native cells, the Tat peptides can remodel the actin framework to reduce their interaction with the local membrane environment. Characteristic hot spots for interaction were detected on the membrane, suggesting that a funnel passage may have formed for the Tat-coated particles. This finding offers valuable insight into the cellular delivery of nanoscale cargo, suggesting an avenue for direct therapeutic delivery.

Adhesion of an Ultrasmall Nanoparticle on a Bilayer Membrane is Still Size and Shape Dependent

It has been found recently that an ultrasmall nanoparticle whose size is smaller than the thickness of a cell membrane has unique roles in biomedical applications including the development of next generation of drugs or advanced nanoscale cargo carriers. However, the effect of physical properties of an ultrasmall nanoparticle on its adhesion to a bilayer membrane, which is a key step for Nano–Bio interaction as well as the biomedical applications, is still largely unknown. By using molecular dynamics, we find that both size and shape of an ultrasmall nanoparticle strongly affect its adhesion states on a bilayer membrane (e.g., adhesion, separation or entwined by polymer chains). Interestingly, our simulations show that with decreasing particle size, the effect of particle shape becomes even more evident for the adhesion behavior. It is indicated that the competition between nanoparticle-polymer binding and polymer chain deformation, both of which are influenced by particle size and shape, determines the final adhesion states of an ultrasmall nanoparticle. Our results are helpful for the full understanding of interaction mechanism between nanoparticles and cell membranes and the practical applications of such ultrasmall nanoparticles.

Development of Protein‐Cage‐Based Delivery Nanoplatforms by Polyvalently Displaying β‐Cyclodextrins on the Surface of Ferritins Through Copper(I)‐Catalyzed Azide/Alkyne Cycloaddition

Protein cages are spherical hollow macromolecules that are attractive platforms for the construction of nanoscale cargo delivery vehicles. Human heavy-chain ferritin (HHFn) is modified genetically to control the number and position of functional groups per cage. 24 β-CDs are conjugated precisely to the modified HHFn in specific locations through thiol-maleimide Michael-type addition followed by copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC). The resulting human ferritins displaying β-CDs (β-CD-C90 HHFn) can form inclusion complexes with FITC-AD, which can slowly release the guest molecule reversibly in a buffer solution via non-covalent β-CD/AD interactions. β-CD-C90 HHFn can potentially be used as delivery vehicles for insoluble drugs.

Theoretical Modeling of the Weaving of Clathrin into Nanoscale Baskets

Many biological systems are capable of spontaneously assembling a diverse set of molecular architectures from a single subunit, without the need to pre-pattern the assembly. Cellular uptake of external substances is accomplished by a highly adaptive endocytosis process that accommodates a wide range of cargo shapes and sizes. Clathrin-mediated endocytosis involves the formation of a pit that is surrounded by a honeycomb coating whose pinwheel-shaped subunit is a clathrin-protein complex. We develop a theoretical model for the thermodynamics and kinetics of clathrin assembly, addressing the behavior in 2 and 3 dimensions, relevant to membrane and bulk assembly, respectively. The clathrin triskelions are modeled as effective flexible pinwheels that form leg-leg associations and resist elastic deformation. Thus, the pinwheels are capable of forming a range of ring structures, including 5-, 6-, and 7-member rings that are observed experimentally. Our theoretical model employs Monte Carlo simulations to address thermodynamic behavior and Brownian dynamics simulations to track the motion of clathrin pinwheels at sufficiently long time scales to achieve complete assembly. With this theoretical model, we predict the phase diagram for clathrin assembly incorporating binding interactions, elastic deformation, and defect-pair coupling, utilizing Kosterlitz-Thouless theory of defect-induced melting in 2 dimensions. Using analytical theory and computational simulations, we explore the role of binding strength and clathrin elasticity in the ability for clathrin lattices to dynamically reorganize due to local changes in membrane elasticity and tension. We then proceed to discuss the dynamics of lattice reorganization during the process of a clathrin-coated membrane wrapping around a nanoscale cargo.

Computer simulation of the translocation of nanoparticles with different shapes across a lipid bilayer

Understanding how nanoparticles with different shapes interact with cell membranes is important in drug and gene delivery, but this interaction remains poorly studied. Using computer simulations, we investigate the physical translocation processes of nanoparticles with different shapes (for example, spheres, ellipsoids, rods, discs and pushpin-like particles) and volumes across a lipid bilayer. We find that the shape anisotropy and initial orientation of the particle are crucial to the nature of the interaction between the particle and lipid bilayer. The penetrating capability of a nanoparticle across a lipid bilayer is determined by the contact area between the particle and lipid bilayer, and the local curvature of the particle at the contact point. Particle volume affects translocation indirectly, and particle rotation can complicate the penetration process. Our results provide a practical guide to geometry considerations when designing nanoscale cargo carriers.

The influence of mediators of intracellular trafficking on transgene expression efficacy of polymer–plasmid DNA complexes

Polymer-mediated gene delivery is an attractive alternative to viral vectors, but is limited by low efficacies of transgene expression. We report that polymers possess differential efficacies for transfecting closely related human prostate cancer cells, which correlates with dramatically different intracellular fate of nanoscale cargo in these cells. Sequestration of nanoscale cargo (27 nm quantum dots and 150–250 nm polyplexes) at a single location near the microtubule organizing compartment (MTOC) in PC3–PSMA human prostate cancer cells correlated with lower polymer-mediated transgene expression compared to PC3 cells, which showed distributed localization throughout the cytoplasm. We show, for the first time, that treatment with the histone deacetylase 6 (HDAC6) inhibitor tubacin, which acetylates tubulin of microtubules in the cytoplasm, abolished quantum dot and polyplex sequestration at the perinuclear recycling compartment/microtubule organizing center (PNRC/MTOC) and increased polymer-mediated transgene expression by up to forty-fold compared to cells not treated with the HDAC6 inhibitor drug. Treatment with the class I and II HDAC inhibitor trichostatin A (TSA) demonstrated similar levels of transgene expression enhancement. These results indicate that mediators of intracellular trafficking can be employed to modulate nanoparticle fate and enhance the efficacy of nanoscale therapeutics in cells. Simultaneous use of high-efficacy polymers along with mediators of intracellular trafficking is an attractive synergistic strategy for enhancing polymer-mediated transgene expression.