One-Pot, One-Step Synthesis of Drug-Loaded Magnetic Multimicelle Aggregates.

Lipid-based formulations provide a nanotechnology platform that is widely used in a variety of biomedical applications because it has several advantageous properties including biocompatibility, reduced toxicity, relative ease of surface modifications, and the possibility for efficient loading of drugs, biologics, and nanoparticles. A combination of lipid-based formulations with magnetic nanoparticles such as iron oxide was shown to be highly advantageous in a growing number of applications including magnet-mediated drug delivery and image-guided therapy. Currently, lipid-based formulations are prepared by multistep protocols. Simplification of the current multistep procedures can lead to a number of important technological advantages including significantly decreased processing time, higher reaction yield, better product reproducibility, and improved quality. Here, we introduce a one-pot, single-step synthesis of drug-loaded magnetic multimicelle aggregates (MaMAs), which is based on controlled flow infusion of an iron oxide nanoparticle/lipid mixture into an aqueous drug solution under ultrasonication. Furthermore, we prepared molecular-targeted MaMAs by directional antibody conjugation through an Fc moiety using Cu-free click chemistry. Fluorescence imaging and quantification confirmed that antibody-conjugated MaMAs showed high cell-specific targeting that was enhanced by magnetic delivery.

Role of alkylated residues in the tetrapeptide self-assembly-A molecular dynamics study.

Designing peptide sequences that self-assemble into well-defined nanostructures can open a new venue for the development of novel drug carriers and molecular contrast agents. Current approaches are often based on a linear block-design of amphiphilic peptides where a hydrophilic peptide chain is terminated by a hydrophobic tail. Here, a new template for a self-assembling tetrapeptide (YXKX, Y = tyrosine, X = alkylated tyrosine, K = lysine) is proposed with two distinct sides relative to the peptide's backbone: alkylated hydrophobic residues on one side and hydrophilic residues on the other side. Using all-atom molecular dynamics simulations, the self-assembly pathway of the tetrapeptide is analyzed for two different concentrations. At both concentrations, tetrapeptides self-assembled into a nanosphere structure. The alkylated tyrosines initialize the self-assembly process via a strong hydrophobic effect and to reduce exposure to the aqueous solvent, they formed a hydrophobic core. The hydrophilic residues occupied the surface of the self-assembled nanosphere. Ordered arrangement of tetrapeptides within the nanosphere with the backbone hydrogen bonding led to a beta sheet formation. Alkyl chain length constrained the size and shape of the nanosphere. This study provides foundation for further exploration of self-assembling structures that are based on peptides with hydrophobic and hydrophilic moieties located on the opposite sides of a peptide backbone.

Longitudinal elastic wave imaging using nanobomb optical coherence elastography: erratum.

We present an erratum to correct an inadvertent error made during the calculations of the in-focus fluence of pulsed laser used to excite nanoparticles [Opt. Lett.44, 3162 (2019)OPLEDP0146-959210.1364/OL.44.003162] and to update the conclusion regarding laser safety limits achieved with this type of excitation.

Longitudinal elastic wave imaging using nanobomb optical coherence elastography.

Wave-based optical coherence elastography (OCE) is a rapidly emerging technique for elasticity assessment of tissues having high displacement sensitivity and simple implementation. However, most current noncontact wave excitation techniques are unable to target a specific tissue site in 3D and rely on transversal scanning of the imaging beam. Here, we demonstrate that dye-loaded perfluorocarbon nanoparticles (nanobombs) excited by a pulsed laser can produce localized axially propagating longitudinal shear waves while adhering to the laser safety limit. A phase-correction method was developed and implemented to perform sensitive nanobomb elastography using a ∼1.5 MHz Fourier domain mode-locking laser. The nanobomb activation was also monitored by detecting photoacoustic signals. The highly localized elastic waves detected by the nanobomb OCE suggest the possibility of high-resolution 3D elastographic imaging.

Nanobomb optical coherence elastography.

Wave-based optical elastography is rapidly emerging as a powerful technique for quantifying tissue biomechanical properties due to its noninvasive nature and high displacement sensitivity. However, current approaches are limited in their ability to produce high-frequency waves and highly localized mechanical stress. In this Letter, we demonstrate that the rapid liquid-to-gas phase transition of dye-loaded perfluorocarbon nanodroplets ("nanobombs") initiated by a pulsed laser can produce highly localized, high-frequency, and broadband elastic waves. The waves were detected by an ultra-fast line-field low-coherence holography system. For comparison, we also excited waves using a focused micro-air-pulse. Results from tissue-mimicking phantoms showed that the nanobombs produced elastic waves with frequencies up to ∼9 kHz, which was much greater than the ∼2 kHz waves excited by the air-pulse. Consequently, the nanobombs enabled more accurate quantification of sample viscoelasticity. Combined with their potential for functionalization, the nanobombs show promise for accurate and highly specific noncontact all-optical elastography.

Synthesis of Stable Citrate-Capped Silver Nanoprisms.

Citrate-stabilized silver nanoprisms (AgNPrs) can be easily functionalized using well-developed thiol based surface chemistry that is an important requirement for biosensor applications utilizing localized surface plasmon resonance (LSPR) and surface-enhanced Raman Scattering (SERS). Unfortunately, currently available protocols for synthesis of citrate-coated AgNPrs do not produce stable nanoparticles thus limiting their usefulness in biosensing applications. Here we address this problem by carrying out a systematic study of citrate-stabilized, peroxide-based synthesis of AgNPrs to optimize reaction conditions for production of stable and reproducible nanoprisms. Our analysis showed that concentration of secondary reducing agent, l-ascorbic acid, is critical to AgNPr stability. Furthermore, we demonstrated that optimization of other synthesis conditions such as stabilizer concentration, rate of silver nitrate addition, and seed dilution result in highly stable nanoprisms with narrow absorbance peaks ranging from 450 nm into near-IR. In addition, the optimized reaction conditions can be used to produce AgNPrs in a one-pot synthesis instead of a previously described two-step reaction. The resulting nanoprisms can readily interact with thiols for easy surface functionalization. These studies provide an optimized set of parameters for precise control of citrate stabilized AgNPr synthesis for biomedical applications.

Control of Primary Particle Spacing in Gold Nanoparticle Clusters for Both High NIR Extinction and Full Reversibility.

Reversible NIR-active nanoparticle clusters with controlled size from 20 to 100 nm were assembled from 5 nm gold nanoparticles (Au NP), with either citrate (CIT) or various binary ligands on the surface, by tuning the electrostatic repulsion and the hydrogen bonding via pH. The nanoclusters were bound together by vdW forces between the cores and the hydrogen bonds between the surface ligands and dissociated to primary nanoparticles over a period of 20 days at pH 5 and at pH 7. When high levels of citrate ligands were used on the primary particle surfaces, the large particle spacings in the nanoclusters led to only modest NIR extinction. However, a NIR extinction (E1000/525) ratio of up to ∼0.4 was obtained for nanoclusters with binary ligand mixtures composed of citrate and either cysteine (CYS), glutathione (GSH), or thioctic acid zwitterion (TAZ) while maintaining full reversibility to primary particles. The optimum ligand ratio for both an E1000/525 of ∼0.4 and full reversibility decreased with increasing length of the secondary ligand (1.5/1 for CYS/CIT, 0.75/1 for GSH/CIT, and 0.5/1 for TAZ/CIT) because a longer secondary ligand maintains a sufficient interparticle spacing required for dissociation more effectively. Interestingly, the zeta potential and the first-order rate constant for nanocluster dissociation were similar for all three systems at the optimum ligand ratios. After incubation in 10 mM GSH solution (intracellular concentration), only the TAZ/CIT primary nanoparticles were resistant to protein opsonization in 100% fetal bovine serum, as the bidentate binding and zwitterion tips of TAZ resisted GSH exchange and protein opsonization, respectively.

Intratumoral Biosynthesis of Gold Nanoclusters by Pancreatic Cancer to Overcome Delivery Barriers to Radiosensitization.

Nanoparticle delivery to solid tumors is a prime challenge in nanomedicine. Here, we approach this challenge through the lens of biogeochemistry, the field that studies the flow of chemical elements within ecosystems as manipulated by living cellular organisms and their environments. We leverage biogeochemistry concepts related to gold cycling against pancreatic cancer, considering mammalian organisms as drivers for gold nanoparticle biosynthesis. Sequestration of gold nanoparticles within tumors has been demonstrated as an effective strategy to enhance radiotherapy; however, the desmoplasia of pancreatic cancer impedes nanoparticle delivery. Our strategy overcomes this barrier by applying an atomic-scale agent, ionic gold, for intratumoral gold nanoparticle biosynthesis. Our comprehensive studies showed the cancer-specific synthesis of gold nanoparticles from externally delivered gold ions in vitro and in a murine pancreatic cancer model in vivo; a substantial colocalization of gold nanoparticles (GNPs) with cancer cell nuclei in vitro and in vivo; a strong radiosensitization effect by the intracellularly synthesized GNPs; a uniform distribution of in situ synthesized GNPs throughout the tumor volume; a nearly 40-day total suppression of tumor growth in animal models of pancreatic cancer treated with a combination of gold ions and radiation that was also associated with a significantly higher median survival versus radiation alone (235 vs 102 days, respectively).

Antibody-Conjugated Polymersomes with Encapsulated Indocyanine Green J-Aggregates and High Near-Infrared Absorption for Molecular Photoacoustic Cancer Imaging.

Imaging plays a critical role in all stages of cancer care from early detection to diagnosis, prognosis, and therapy monitoring. Recently, photoacoustic imaging (PAI) has started to emerge into the clinical realm due to its high sensitivity and ability to penetrate tissues up to several centimeters deep. Herein, we encapsulated indocyanine green J (ICGJ) aggregate, one of the only FDA-approved organic exogenous contrast agents that absorbs in the near-infrared range, at high loadings up to ∼40% w/w within biodegradable polymersomes (ICGJ-Ps) composed of poly(lactide-co-glycolide-b-polyethylene glycol) (PLGA-b-PEG). The small Ps hydrodynamic diameter of 80 nm is advantageous for in vivo applications, while directional conjugation with epidermal growth factor receptor (EGFR) targeting cetuximab antibodies renders molecular specificity. Even when exposed to serum, the ∼11 nm-thick membrane of the Ps prevents dissociation of the encapsulated ICGJ for at least 48 h with a high ratio of ICGJ to monomeric ICG absorbances (i.e., I895/I780 ratio) of approximately 5.0 that enables generation of a strong NIR photoacoustic (PA) signal. The PA signal of polymersome-labeled breast cancer cells is proportional to the level of cellular EGFR expression, indicating the feasibility of molecular PAI with antibody-conjugated ICGJ-Ps. Furthermore, the labeled cells were successfully detected with PAI in highly turbid tissue-mimicking phantoms up to a depth of 5 mm with the PA signal proportional to the amount of cells. These data show the potential of molecular PAI with ICGJ-Ps for clinical applications such as tumor margin detection, evaluation of lymph nodes for the presence of micrometastasis, and laparoscopic imaging procedures.

Charged gold nanoparticles with essentially zero serum protein adsorption in undiluted fetal bovine serum.

The adsorption of even a single serum protein molecule on a gold nanosphere used in biomedical imaging may increase the size too much for renal clearance. In this work, we designed charged ~5 nm Au nanospheres coated with binary mixed-charge ligand monolayers that do not change in size upon incubation in pure fetal bovine serum (FBS). This lack of protein adsorption was unexpected in view of the fact that the Au surface was moderately charged. The mixed-charge monolayers were composed of anionic citrate ligands modified by place exchange with naturally occurring amino acids: either cationic lysine or zwitterionic cysteine ligands. The zwitterionic tips of either the lysine or cysteine ligands interact weakly with the proteins and furthermore increase the distance between the "buried" charges closer to the Au surface and the interacting sites on the protein surface. The ~5 nm nanospheres were assembled into ~20 nm diameter nanoclusters with strong near-IR absorbance (of interest in biomedical imaging and therapy) with a biodegradable polymer, PLA(1k)-b-PEG(10k)-b-PLA(1k). Upon biodegradation of the polymer in acidic solution, the nanoclusters dissociated into primary ~5 nm Au nanospheres, which also did not adsorb any detectable serum protein in undiluted FBS.

Conjugation of antibodies to gold nanorods through Fc portion: synthesis and molecular specific imaging.

Anisotropic gold nanorods provide a convenient combination of properties, such as tunability of plasmon resonances and strong extinction cross sections in the near-infrared to red spectral region. These properties have created significant interest in the development of antibody conjugation methods for synthesis of targeted nanorods for a number of biomedical applications, including molecular specific imaging and therapy. Previously published conjugation approaches have achieved molecular specificity. However, the current conjugation methods have several downsides including low stability and potential cytotoxicity of bioconjugates that are produced by electrostatic interactions, as well as lack of control over antibody orientation during covalent conjugation. Here we addressed these shortcomings by introducing directional antibody conjugation to the gold nanorod surface. The directional conjugation is achieved through the carbohydrate moiety, which is located on one of the heavy chains of the Fc portion of most antibodies. The carbohydrate is oxidized under mild conditions to a hydrazide reactive aldehyde group. Then, a heterofunctional linker with hydrazide and dithiol groups is used to attach antibodies to gold nanorods. The directional conjugation approach was characterized using electron microscopy, zeta potential, and extinction spectra. We also determined spectral changes associated with nanorod aggregation; these spectral changes can be used as a convenient quality control of nanorod bioconjugates. Molecular specificity of the synthesized antibody targeted nanorods was demonstrated using hyperspectral, optical and photoacoustic imaging of cancer cell culture models. Additionally, we observed characteristic changes in optical spectra of molecular specific nanorods after their interactions with cancer cells; the observed spectral signatures can be explored for sensitive cancer detection.

Immunomagnetic nanoscreening of circulating tumor cells with a motion controlled microfluidic system.

Combining the power of immunomagnetic assay and microfluidic microchip operations, we successfully detected rare CTCs from clinical blood samples. The microfluidic system is operated in a flip-flop mode, where a computer-controlled rotational holder with an array of microfluidic chips inverts the microchannels. We have demonstrated both theoretically and experimentally that the direction of red blood cell (RBC) sedimentation with regards to the magnetic force required for cell separation is important for capture efficiency, throughput, and purity. The flip-flop operation reduces the stagnation of RBCs and non-specific binding on the capture surface by alternating the direction of the magnetic field with respect to gravity. The developed immunomagnetic microchip-based screening system exhibits high capture rates (more than 90%) for SkBr3, PC3, and Colo205 cell lines in spiked screening experiments and successfully isolates CTCs from patient blood samples. The proposed motion controlled microchip-based immunomagnetic system shows great promise as a clinical tool for cancer diagnosis and prognosis.

Excretion and toxicity of gold-iron nanoparticles.

Though gold nanoparticles have been considered bio-inert, recent studies have questioned their safety. To reduce the potential for toxicity, we developed a nanoclustering of gold and iron oxide as a nanoparticle (nanorose) which biodegrades into subunits to facilitate rapid excretion. In this present study, we demonstrate acid and macrophage lysosomal degradation of nanorose via loss of the near-infrared optical shift, and clearance of the nanorose in vivo following i.v. administration in C57BL/6 mice by showing gold concentration is significantly reduced in 11 murine tissues in as little as 31 days (P < 0.01). Hematology and chemistry show no toxicity of nanorose injected mice up to 14 days after administration. We conclude that the clustering design of nanorose does enhance the excretion of these nanoparticles, and that this could be a viable strategy to limit the potential toxicity of gold nanoparticles for clinical applications. FROM THE CLINICAL EDITOR: The potential toxicity of nanomaterials is a critically important limiting factor in their more widespread clinical application. Gold nanoparticles have been classically considered bio-inert, but recent studies have questioned their safety. The authors of this study have developed a clustering gold and iron oxide nanoparticle (nanorose), which biodegrades into subunits to facilitate rapid excretion, resulting in reduced toxicity.

Nanobomb optical coherence elastography in multilayered phantoms.

Many tissues are composed of layered structures, and a better understanding of the changes in the layered tissue biomechanics can enable advanced guidance and monitoring of therapy. The advent of elastography using longitudinally propagating shear waves (LSWs) has created the prospect of a high-resolution assessment of depth-dependent tissue elasticity. Laser activation of liquid-to-gas phase transition of dye-loaded perfluorocarbon (PFC) nanodroplets (a.k.a., nanobombs) can produce highly localized LSWs. This study aims to leverage the potential of photoactivation of nanobombs to incudce LSWs with very high-frequency content in wave-based optical coherence elastography (OCE) to estimate the elasticity gradient with high resolution. In this work, we used multilayered tissue-mimicking phantoms to demonstrate that highly localized nanobomb (NB)-induced LSWs can discriminate depth-wise tissue elasticity gradients. The results show that the NB-induced LSWs rapidly change speed when transitioning between layers with different mechanical properties, resulting in an elasticity resolution of ∼65 µm. These results show promise for characterizing the elasticity of multilayer tissue with a fine resolution.

Thermal stability of biodegradable plasmonic nanoclusters in photoacoustic imaging.

The photothermal stability of plasmonic nanoparticles is critically important to perform reliable photoacoustic imaging and photothermal therapy. Recently, biodegradable nanoclusters composed of sub-5 nm primary gold particles and a biodegradable polymer have been reported as clinically-translatable contrast agents for photoacoustic imaging. After cellular internalization, the nanoclusters degrade into 5 nm primary particles for efficient excretion from the body. In this paper, three different sizes of biodegradable nanoclusters were synthesized and the optical properties and photothermal stability of the nanoclusters were investigated and compared to that of gold nanorods. The results of our study indicate that 40 nm and 80 nm biodegradable nanoclusters demonstrate higher photothermal stability compared to gold nanorods. Furthermore, 40 nm nanoclusters produce higher photoacoustic signal than gold nanorods at a given concentration of gold. Therefore, the biodegradable plasmonic nanoclusters can be effectively used for photoacoustic imaging and photothermal therapy.

Prospecting Cellular Gold Nanoparticle Biomineralization as a Viable Alternative to Prefabricated Gold Nanoparticles.

Gold nanoparticles (GNPs) have shown considerable potential in a vast number of biomedical applications. However, currently there are no clinically approved injectable GNP formulations. Conversely, gold salts have been used in the clinic for nearly a century. Further, there is evidence of GNP formation in patients treated with gold salts (i.e., chrysiasis). Recent reports evaluating this phenomenon in human cells and in murine models indicate that the use of gold ions for in situ formation of theranostic GNPs could greatly improve the delivery within dense biological tissues, increase efficiency of intracellular gold uptake, and specificity of GNP formation within cancer cells. These attributes in combination with safe clinical application of gold salts make this process a viable strategy for clinical translation. Here, the first summary of the current knowledge related to GNP biomineralization in mammalian cells is provided along with critical assessment of potential biomedical applications of this newly emergent field.

Effect of perfluorocarbon composition on activation of phase-changing ultrasound contrast agents.

BACKGROUND: While microbubble contrast agents (MCAs) are commonly used in ultrasound (US), they are inherently limited to vascular targets due to their size. Alternatively, phase-changing nanodroplet contrast agents (PNCAs) can be delivered as nanoscale agents (i.e., small enough to extravasate), but when exposed to a US field of sufficient mechanical index (MI), they convert to MCAs, which can be visualized with high contrast using nonlinear US. PURPOSE: To investigate the effect of perfluorocarbon (PFC) core composition and presence of cholesterol in particle coatings on stability and image contrast generated from acoustic activation of PNCAs using high-frequency US suitable for clinical imaging. METHODS: PNCAs with varied core compositions (i.e., mixtures of perfluoropentane [C5] and/or perfluorohexane [C6]) and two coating formulations (i.e., with and without cholesterol) were characterized and investigated for thermal/temporal stability and postactivation, nonlinear US contrast in phantom and in vivo environments. Through hydrophone measurements and nonlinear numerical modeling, MI was estimated for pulse sequences used for PNCA activation. RESULTS: All PNCA compositions were characterized to have similar diameters (249-267 nm) and polydispersity (0.151-0.185) following fabrication. While PNCAs with majority C5 core composition showed higher levels of spontaneous signal (i.e., not due to US activation) in phantoms than C6-majority PNCAs, all compositions were stable during imaging experiments. When activating PNCAs with a 12.3-MHz US pulse (MI = 1.1), C6-core particles with cholesterol-free coatings (i.e., CF-C6-100 particles) generated a median contrast of 3.1, which was significantly higher (p < 0.001) than other formulations. Further, CF-C6-100 particles were activated in a murine model, generating US contrast ≥ $ \ge $ 3.4. CONCLUSION: C6-core PNCAs can provide high-contrast US imaging with minimal nonspecific activation in phantom and in vivo environments.

Selective targeting of antibody conjugated multifunctional nanoclusters (nanoroses) to epidermal growth factor receptors in cancer cells.

The ability of smaller than 100 nm antibody (Ab) nanoparticle conjugates to target and modulate the biology of specific cell types may enable major advancements in cellular imaging and therapy in cancer. A key challenge is to load a high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. A versatile method called thin autocatalytic growth on substrate (TAGs) has been developed in our previous study to form ultrathin and asymmetric gold coatings on iron oxide nanocluster cores producing exceptional near-infrared (NIR) absorbance. AlexaFluor 488 labeled Abs were used to correlate the number of Abs conjugated to iron oxide/gold nanoclusters (nanoroses) with the hydrodynamic size. A transition from submonolayer to multilayer aggregates of Abs on the nanorose surface was observed for 54 Abs and an overall particle diameter of ∼60-65 nm. The hydrodynamic diameter indicated coverage of a monolayer of 54 Abs, in agreement with the prediction of a geometric model, by assuming a circular footprint of 16.9 nm diameter per Ab molecule. The targeting efficacy of nanoclusters conjugated with monoclonal Abs specific for epidermal growth factor receptor (EGFR) was evaluated in A431 cancer cells using dark field microscopy and atomic absorbance spectrometry (AAS) analysis. Intense NIR scattering was achieved from both high uptake of nanoclusters in cells and high intrinsic NIR absorbance of individual nanoclusters. Dual mode imaging with dark field reflectance microscopy and fluorescence microscopy indicates the Abs remained attached to the Au surfaces upon the uptake by the cancer cells. The ability to load intense multifunctionality, specifically strong NIR absorbance, conjugation of an Ab monolayer in addition to a strong r2 MRI contrast that was previously demonstrated in a total particle size of only 63 nm, is an important step forward in development of theranostic agents for combined molecular specific imaging and therapy.

Pulsed magneto-motive ultrasound imaging to detect intracellular trafficking of magnetic nanoparticles.

As applications of nanoparticles in medical imaging and biomedicine rapidly expand, the interactions of nanoparticles with living cells have become an area of active interest. For example, intracellular accumulation of nanoparticles-an important part of cell-nanoparticle interaction-has been well studied using plasmonic nanoparticles and optical or optics-based techniques due to the change in optical properties of the nanoparticle aggregates. However, magnetic nanoparticles, despite their wide range of clinical applications, do not exhibit plasmonic-resonant properties and therefore their intracellular aggregation cannot be detected by optics-based imaging techniques. In this study, we investigated the feasibility of a novel imaging technique-pulsed magneto-motive ultrasound (pMMUS)-to identify intracellular accumulation of endocytosed magnetic nanoparticles. In pMMUS imaging a focused, high intensity, pulsed magnetic field is used to excite the cells labeled with magnetic nanoparticles, and ultrasound imaging is then used to monitor the mechanical response of the tissue. We demonstrated previously that clusters of magnetic nanoparticles amplify the pMMUS signal in comparison to the signal from individual nanoparticles. Here we further demonstrate that pMMUS imaging can identify interaction between magnetic nanoparticles and living cells, i.e. intracellular accumulation of nanoparticles within the cells. The results of our study suggest that pMMUS imaging can not only detect the presence of magnetic nanoparticles but also provides information about their intracellular accumulation non-invasively and in real-time.