Optimization of intracellular macromolecule delivery by nanoparticle-mediated photoporation.

Nanoparticle-mediated photoporation is a novel delivery platform for intracellular molecule delivery. We studied the dependence of macromolecular delivery on molecular weight and sought to enhance delivery efficiency. DU145 prostate cancer cells were exposed to pulsed laser beam in the presence of carbon-black nanoparticles. Intracellular uptake of molecules decreased with increasing molecular weight. Attributing this dependence to molecular diffusivity, we hypothesized that macromolecular delivery efficiency could be enhanced by increasing either laser fluence or laser exposure duration at low fluence. We observed increased percentages of macromolecule uptake by cells in both cases. However, trade-off between cell uptake and viability loss was most favorable at low laser fluence (25-29 mJ/cm2) and longer exposure durations (4-5 min). We conclude that long exposure at low laser fluence optimizes intracellular macromolecule delivery by nanoparticle-mediated photoporation, which may be explained by longer time for macromolecules to diffuse into cells, during and between laser pulses.

Serum Protects Cells and Increases Intracellular Delivery of Molecules by Nanoparticle-Mediated Photoporation.

INTRODUCTION: Intracellular delivery of molecules is central to applications in biotechnology, medicine, and basic research. Nanoparticle-mediated photoporation using carbon black nanoparticles exposed to pulsed, near-infrared laser irradiation offers a physical route to create transient cell membrane pores, enabling intracellular delivery. However, nanoparticle-mediated photoporation, like other physical intracellular delivery technologies, necessitates a trade-off between achieving efficient uptake of exogenous molecules and maintaining high cell viability. METHODS: In this study, we sought to shift this balance by adding serum to cells during nanoparticle-mediated photoporation as a viability protectant. DU-145 prostate cancer cells and human dermal fibroblasts were exposed to laser irradiation in the presence of carbon black (CB) nanoparticles and other formulation additives, including fetal bovine serum (FBS) and polymers. RESULTS: Our studies showed that FBS can protect cells from viability loss, even at high-fluence laser irradiation conditions that lead to high levels of intracellular delivery in two different mammalian cell types. Further studies revealed that full FBS was not needed: viability protection was achieved with denatured FBS, with just the high molecular weight fraction of FBS (>30 kDa), or even with individual proteins like albumin or hemoglobin. Finally, we found that viability protection was also obtained using certain neutral water-soluble polymers, including Pluronic F127, polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline), and polyethylene glycol, which were more effective at increased concentration, molecular weight, or hydrophobicity. CONCLUSION: Altogether, these findings suggest an interaction between amphiphilic domains of polymers with the cell membrane to help cells maintain viability, possibly by facilitating transmembrane pore closure. In this way, serum components or synthetic polymers can be used to increase intracellular delivery by nanoparticle-mediated photoporation while maintaining high cell viability.

Tailoring Optical Properties of Luminescent Semiconducting Nanocrystals through Hydrostatic, Anisotropic Static, and Dynamic Pressures.

Luminescent semiconductor nanocrystals are a fascinating class of materials because of their size-dependent emissions. Numerous past studies have demonstrated that semiconductor nanoparticles with radii smaller than their Bohr radius experience quantum confinement and thus size-dependent emissions. Exerting pressure on these nanoparticles represents an additional, more dynamic, strategy to alter their size and shift their emission. The application of pressure results in the lattices becoming strained and the electronic structure altered. In this Minireview, colloidal semiconductor nanocrystals are first introduced. The effects of uniform hydrostatic pressure on the optical properties of metal halide perovskite (ABX3 ), II-VI, III-V, and IV-VI semiconductor nanocrystals are then examined. The optical properties of semiconductor nanocrystals under static and dynamic anisotropic pressure are then summarized. Finally, future research directions and applications utilizing the pressure-dependent optical properties of semiconductor nanocrystals are discussed.

Unraveling the Role of Interfaces on the Spall Failure of Cu/Ta Multilayered Systems.

Molecular dynamics (MD) simulations are carried out to investigate the effects of the type and spacing of FCC/BCC interfaces on the deformation and spall behavior. The simulations are carried out using model Cu/Ta multilayers with six different types of interfaces. The results suggest that interface type can significantly affect the structure and intensity of the incoming shock wave, change the activated slip systems, alter dislocation slip and twinning behavior, affect where and how voids are nucleated during spallation and the resulting spall strength. Moreover, the above aspects are significantly affected by the interface spacing. A transition from homogeneous to heterogeneous dislocation nucleation occurs as the interface spacing is decreased to 6 nm. Depending on interface type and spacing, damage (voids) nucleation and spall failure is observed to occur not only at the Cu/Ta interfaces, but also in the weaker Cu layer interior, or even in the stronger Ta layer interior, although different mechanisms underlie each of these three distinct failure modes. These findings point to the fact that, depending on the combination of interface type and spacing, interfaces can lead to both strengthening and weakening of the Cu/Ta multilayered microstructures.

Unconventional route to dual-shelled organolead halide perovskite nanocrystals with controlled dimensions, surface chemistry, and stabilities.

The past few years have witnessed rapid advances in the synthesis of high-quality perovskite nanocrystals (PNCs). However, despite the impressive developments, the stability of PNCs remains a substantial challenge. The ability to reliably improve stability of PNCs while retaining their individual nanometer size represents a critical step that underpins future advances in optoelectronic applications. Here, we report an unconventional strategy for crafting dual-shelled PNCs (i.e., polymer-ligated perovskite/SiO2 core/shell NCs) with exquisite control over dimensions, surface chemistry, and stabilities. In stark contrast to conventional methods, our strategy relies on capitalizing on judiciously designed star-like copolymers as nanoreactors to render the growth of core/shell NCs with controlled yet tunable perovskite core diameter, SiO2 shell thickness, and surface chemistry. Consequently, the resulting polymer-tethered perovskite/SiO2 core/shell NCs display concurrently a stellar set of substantially improved stabilities (i.e., colloidal stability, chemical composition stability, photostability, water stability), while having appealing solution processability, which are unattainable by conventional methods.

Enabling Tailorable Optical Properties and Markedly Enhanced Stability of Perovskite Quantum Dots by Permanently Ligating with Polymer Hairs.

Instability of perovskite quantum dots (QDs) toward humidity remains one of the major obstacles for their long-term use in optoelectronic devices. Herein, a general amphiphilic star-like block copolymer nanoreactor strategy for in situ crafting a set of hairy perovskite QDs with precisely tunable size and exceptionally high water and colloidal stabilities is presented. The selective partition of precursors within the compartment occupied by inner hydrophilic blocks of star-like diblock copolymers imparts in situ formation of robust hairy perovskite QDs permanently ligated by outer hydrophobic blocks via coprecipitation in nonpolar solvent. These size- and composition-tunable perovskite QDs reveal impressive water and colloidal stabilities as the surface of QDs is intimately and permanently ligated by a layer of outer hydrophobic polymer hairs. More intriguingly, the readily alterable length of outer hydrophobic polymers renders the remarkable control over the stability enhancement of hairy perovskite QDs.

All-Inorganic Perovskite Nanocrystals with a Stellar Set of Stabilities and Their Use in White Light-Emitting Diodes.

We report a simple, robust, and inexpensive strategy to enable all-inorganic CsPbX3 perovskite nanocrystals (NCs) with a set of markedly improved stabilities, that is, water stability, compositional stability, phase stability, and phase segregation stability via impregnating them in solid organic salt matrices (i.e., metal stearate; MSt). In addition to acting as matrices, MSt also functions as the ligand bound to the surface of CsPbX3 NCs, thereby eliminating the potential damage of NCs commonly encountered during purification as in copious past work. Quite intriguingly, the resulting CsPbX3-MSt nanocomposites display an outstanding suite of stabilities. First, they retain high emission in the presence of water because of the insolubility of MSt in water, signifying their excellent water stability. Second, anion exchange between CsPbBr3-MSt and CsPbI3-MSt nanocomposites is greatly suppressed. This can be ascribed to the efficient coating of MSt, thus effectively isolating the contact between CsPbBr3 and CsPbI3 NCs, reflecting notable compositional stability. Third, remarkably, after being impregnated by MSt, the resulting CsPbI3-MSt nanocomposites sustain the cubic phase of CsPbI3 and high emission, manifesting the strikingly improved phase stability. Finally, phase segregation of CsPbBr1.5I1.5 NCs is arrested via the MSt encapsulation (i.e., no formation of the respective CsPbBr3 and CsPbI3), thus rendering pure and stable photoluminescence (i.e., demonstration of phase segregation stability). Notably, when assembled into typical white light-emitting diode architecture, CsPbBr1.5I1.5-MSt nanocomposites exhibit appealing performance, including a high color rendering index ( Ra) and a low color temperature ( Tc). As such, the judicious encapsulation of perovskite NCs into organic salts represents a facile and robust strategy for creating high-quality solid-state luminophores for use in optoelectronic devices.

Effect of laser fluence, nanoparticle concentration and total energy input per cell on photoporation of cells.

Intracellular delivery of molecules can be increased by laser-exposure of carbon black nanoparticles to cause photoporation of the cells. Here we sought to determine effects of multiple laser exposure parameters on intracellular uptake and cell viability with the goal of determining a single unifying parameter that predicts cellular bioeffects. DU145 human prostate cancer cells in suspension with nanoparticles were exposed to near-infrared nanosecond laser pulses over a range of experimental conditions. Increased bioeffects (i.e., uptake and viability loss determined by flow cytometry) were seen when increasing laser fluence, number of pulses and nanoparticle concentration, and decreasing cell concentration. Bioeffects caused by different combinations of these four parameters were generally predicted by their cumulative energy input per cell, which served as a unifying parameter. This indicates that photoporation depends on what appears to be the cumulative effect of multiple cell-nanoparticle interactions from neighboring nanoparticles during a series of laser pulses.

Photoporation Using Carbon Nanotubes for Intracellular Delivery of Molecules and Its Relationship to Photoacoustic Pressure.

Exposure of carbon-black (CB) nanoparticles to near-infrared nanosecond-pulsed laser energy can cause efficient intracellular delivery of molecules by photoporation. Here, cellular bioeffects of multi-walled carbon nanotubes (MWCNTs) and single-walled carbon nanotubes (SWCNTs) are compared to those of CB nanoparticles. In DU145 prostate-cancer cells, photoporation using CB nanoparticles transitions from (i) cells with molecular uptake to (ii) nonviable cells to (iii) fragmented cells with increasing laser fluence, as seen previously. In contrast, photoporation with MWCNTs causes uptake and, at higher fluence, fragmentation, but does not generate nonviable cells, and SWCNTs show little evidence of bioeffects, except at extreme laser conditions, which generate nonviable cells and fragmentation, but no significant uptake. These different behaviors cannot be explained by photoacoustic pressure output from the particles. All particle types emit a single, ≈100 ns, mostly positive-pressure pulse that increases in amplitude with laser fluence. Different particle types emit different peak pressures, which are highest for SWCNTs, followed by CB nanoparticles and then MWCNTs, which does not correlate with cellular bioeffects between different particle types. This study concludes that cellular bioeffects depend strongly on the type of carbon nanoparticle used during photoporation and that photoacoustic pressure is unlikely to play a direct mechanistic role in the observed bioeffects.

Design and fabrication of distributed Bragg reflector multilayers for dynamic pressure sensing.

A novel 2D-surface shock pressure sensor is designed and tested based on 1D-Photonic Crystal, i.e., Distributed Bragg Reflector Multilayer (DBR/ML) structures. The fast opto-mechanical response of these structures to changes in layer thicknesses and refractive indices are ideally suited for dynamic pressure sensing. They offer the potential to minimize acoustic impedance mismatch between the material layers, and most importantly, the potential to monitor both temporal and spatial (lateral) variations during shock compression. In this feasibility study, different materials and device designs are investigated to identify material/device design combinations with optimum response to dynamic loading. Structural and material effects are studied in terms of spectral and mechanical properties, structure stability, and the ease of fabrication process. Structures comprising of different numbers of SiO1.5/SiO1.7 bilayer stacks are modeled, and fabricated. A 10-bilayer structure placed under a dynamic compressive load of ~7.2 GPa, exhibits a blueshift of 29 nm with a response time of ~5 ns which is well within the shock pressure rise time measured with PDV velocimetry. This promising result successfully demonstrates the feasibility of the specifically designed DBR/ML structure as a dynamic pressure sensor.

Unconventional Route to Uniform Hollow Semiconducting Nanoparticles with Tailorable Dimensions, Compositions, Surface Chemistry, and Near-Infrared Absorption.

Despite impressive recent advances in the synthesis of lead chalcogenide solid nanoparticles, there are no examples of lead chalcogenide hollow nanoparticles (HNPs) with controlled diameter and shell thickness as current synthetic approaches for HNPs have inherent limitations associated with their complexity, inability to precisely control the dimensions, and limited possibilities with regard to applicable materials. Herein, we report on an unconventional strategy for crafting uniform lead chalcogenide (PbS and PbTe) HNPs with tailorable size, surface chemistry, and near-IR absorption. Amphiphilic star-like triblock copolymers [polystyrene-block-poly(acrylic acid)-block-polystyrene and polystyrene-block-poly(acrylic acid)-block-poly(3,4-ethylenedioxythiophene)] were rationally synthesized and exploited as nanoreactors for the formation of uniform PbS and PbTe HNPs. Compared to their solid counterparts, the near-IR absorption of the HNPs is blue-shifted owing to the hollow interior. This strategy can be readily extended to other types of intriguing low-band-gap HNPs for diverse applications.

Role of cytoskeletal mechanics and cell membrane fluidity in the intracellular delivery of molecules mediated by laser-activated carbon nanoparticles.

Exposure of cells and nanoparticles to near-infrared nanosecond pulsed laser light can lead to efficient intracellular delivery of molecules while maintaining high cell viability by a photoacoustic phenomenon known as transient nanoparticle energy transduction (TNET). Here, we examined the influence of cytoskeletal mechanics and plasma membrane fluidity on intracellular uptake of molecules and loss of cell viability due to TNET. We found that destabilization of actin filaments using latrunculin A led to greater uptake of molecules and less viability loss caused by TNET. Stabilization of actin filaments using jasplakinolide had no significant effect on uptake or viability loss caused by TNET. To study the role of plasma membrane fluidity, we increased fluidity by depletion of membrane cholesterol using methyl-ß-cyclodextrin and decreased fluidity by enrichment of the membrane with cholesterol using water-soluble cholesterol. Neither of these membrane fluidity changes significantly altered cellular uptake or viability loss caused by TNET. We conclude that weakening mechanical integrity of the cytoskeleton can increase intracellular uptake and decrease loss of cell viability, while plasma membrane fluidity does not appear to play a significant role in uptake or viability loss caused by TNET. The positive effects of cytoskeletal weakening may be due to an enhanced ability of the cell to recover from the effects of TNET and maintain viability. Biotechnol. Bioeng. 2017;114: 2390-2399. © 2017 Wiley Periodicals, Inc.

Energy Transfer Mechanisms during Molecular Delivery to Cells by Laser-Activated Carbon Nanoparticles.

Previous studies have shown that exposure of carbon black nanoparticles to nanosecond pulsed near-infrared laser causes intracellular delivery of molecules through hypothesized transient breaks in the cell membrane. The goal of this study is to determine the underlying mechanisms of sequential energy transfer from laser light to nanoparticle to fluid medium to cell. We found that laser pulses on a timescale of 10 ns rapidly heat carbon nanoparticles to temperatures on the order of 1200 K. Heat is transferred from the nanoparticles to the surrounding aqueous medium on a similar timescale, causing vaporization of the surrounding water and generation of acoustic emissions. Nearby cells can be impacted thermally by the hot bubbles and mechanically by fluid mechanical forces to transiently increase cell membrane permeability. The experimental and theoretical results indicate that transfer of momentum and/or heat from the bubbles to the cells are the dominant mechanisms of energy transfer that results in intracellular uptake of molecules. We further conclude that neither thermal expansion of the nanoparticles nor a carbon-steam chemical reaction play a significant role in the observed effects on cells, and that acoustic pressure appears to be concurrent with, but not essential to, the observed bioeffects.

Asymmetrical optical microcavity structures for dynamic pressure sensing: design, fabrication, validation.

Optical microcavity (OMC) structures have spectral properties that are directly related to their physical dimensions and material refractive indices. Their intrinsically fast optical response to mechanically-induced changes in these parameters makes OMCs uniquely suited for dynamic sensing when paired with a suitably fast streak camera and spectrograph. Various designs and processes of fabrication for asymmetrical OMC (AOMC) structures were investigated to optimize and assess their feasibility for dynamic sensing. Structural and material effects were studied in terms of spectral properties, structure stabilities and fabrication process. From this study, it was shown that an AOMC structure with a SiO2 cavity layer and Ag mirror layers, fabricated with thin adhesion Al2O3 layers exhibited the best structural stability and spectral properties. Under dynamic compressive loading of ~4 GPa, the structure exhibited a blueshift of 22 nm and a temporal response time of < 3.3 ns, thus demonstrating the potential of AOMC based dynamic pressure sensing.

Synthesis and characterization of a BaGdF5:Tb glass ceramic as a nanocomposite scintillator for x-ray imaging.

Transparent glass ceramics with embedded light-emitting nanocrystals show great potential as low-cost nanocomposite scintillators in comparison to single crystal and transparent ceramic scintillators. In this study, cubic structure BaGdF5:Tb nanocrystals embedded in an aluminosilicate glass matrix are reported for potential high performance MeV imaging applications. Scintillator samples with systematically varied compositions were prepared by a simple conventional melt-quenching method followed by annealing. Optical, structural and scintillation properties were characterized to guide the design and optimization of selected material systems, aiming at the development of a system with higher crystal volume and larger crystal size for improved luminosity. It is observed that enhanced scintillation performance was achieved by tuning the glass matrix composition and using GdF3 in the raw materials, which served as a nucleation agent. A 26% improvement in light output was observed from a BaGdF5:Tb glass ceramic with addition of GdF3.

Poloxamer surfactant preserves cell viability during photoacoustic delivery of molecules into cells.

Efficient intracellular delivery of molecules is needed to modulate cellular behavior for laboratory and medical applications, but is often limited by trade-offs between achieving high intracellular delivery and maintaining high cell viability. Here, we studied photoacoustic delivery of molecules into cells by exposing DU145 human prostate carcinoma cells to nanosecond laser pulses in the presence of carbon black nanoparticles. Under strong laser exposure conditions, less than 30% of cells were viable and exhibited uptake. Addition of poloxamer surfactant at those laser exposure conditions increased cell viability to almost 90%, with intracellular uptake in >80% of cells. This remarkable increase in efficiency of intracellular delivery and cell viability may be attributed to enhanced cell membrane resealing by poloxamer surfactant after photoacoustic delivery. While F-68 poloxamer was effective, the larger, more-hydrophobic F-127 poloxamer provided the best results. There was no significant protective effect from addition of Ca(2+) , BAPTA-AM, ATP, fetal bovine serum or glycine betaine, which were expected to promote active cell membrane repair mechanisms and other active intracellular protective processes. We conclude that poloxamer surfactant preserves cell viability during photoacoustic delivery of molecules into cells, thereby enabling highly efficient intracellular delivery.

Efficient intracellular delivery of molecules with high cell viability using nanosecond-pulsed laser-activated carbon nanoparticles.

Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5-9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability.