Although sub-100 nm nanoclusters of metal nanoparticles are of interest in many fields including biomedical imaging, sensors and catalysis, it has been challenging to control their morphologies and chemical properties. Herein, a new concept is presented to assemble equilibrium Au nanoclusters of controlled size by tuning the colloidal interactions with a polymeric stabilizer, PLA(1k)-b-PEG(10k)-b-PLA(1k). The nanoclusters form upon mixing a dispersion of ~5 nm Au nanospheres with a polymer solution followed by partial solvent evaporation. A weakly adsorbed polymer quenches the equilibrium nanocluster size and provides steric stabilization. Nanocluster size is tuned from ~20 nm to ~40 nm by experimentally varying the final Au nanoparticle concentration and the polymer/Au ratio, along with the charge on the initial Au nanoparticle surface. Upon biodegradation of the quencher, the nanoclusters reversibly and fully dissociate to individual ~5 nm primary particles. Equilibrium cluster size is predicted semi-quantitatively with a free energy model that balances short-ranged depletion and van der Waals attractions with longer-ranged electrostatic repulsion, as a function of the Au and polymer concentrations. The close spacings of the Au nanoparticles in the clusters produce strong NIR extinction over a broad range of wavelengths from 650 to 900 nm, which is of practical interest in biomedical imaging.
nanoclusters; plasmonic nanoparticles; colloidal forces; depletion attraction; biodegradable nanoparticles; equilibrium assembly
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.
(170.5120) Photoacoustic imaging; (170.3880) Medical and biological imaging
Clusters of metal nanoparticles with an overall size less than 100 nm and high metal loadings for strong optical functionality, are of interest in various fields including microelectronics, sensors, optoelectronics and biomedical imaging and therapeutics. Herein we assemble ~5 nm gold particles into clusters with controlled size, as small as 30 nm and up to 100 nm, which contain only small amounts of polymeric stabilizers. The assembly is kinetically controlled with weakly adsorbing polymers, PLA(2K)-b-PEG(10K)-b-PLA(2K) or PEG (MW = 3350), by manipulating electrostatic, van der Waals (VDW), steric, and depletion forces. The cluster size and optical properties are tuned as a function of particle volume fractions and polymer/gold ratios to modulate the interparticle interactions. The close spacing between the constituent gold nanoparticles and high gold loadings (80–85% w/w gold) produce a strong absorbance cross section of ~9×10−15 m2 in the NIR at 700 nm. This morphology results from VDW and depletion attractive interactions that exclude the weakly adsorbed polymeric stabilizer from the cluster interior. The generality of this kinetic assembly platform is demonstrated for gold nanoparticles with a range of surface charges from highly negative to neutral, with the two different polymers.
Monoclonal antibodies continue to command a large market for treatment of a variety of diseases. In many cases, the doses required for therapeutic efficacy are large, limiting options for antibody delivery and administration. We report a novel formulation strategy based on dispersions of antibody nanoclusters that allows for subcutaneous injection of highly concentrated antibody (~190 mg/ml). A solution of monoclonal antibody 1B7 was rapidly frozen and lyophilized using a novel spiral-wound in situ freezing technology (SWIFT) to generate amorphous particles. Upon gentle stirring, a translucent dispersion of ~430 nm protein clusters low apparent viscosity (~24 cp) formed rapidly in buffer containing the pharmaceutically acceptable crowding agents, trehalose, polyethylene glycol and n-methyl-2-pyrrolidone. Upon in vitro dilution of the dispersion, the nanoclusters rapidly reverted to monomeric protein with full activity, as monitored by dynamic light scattering and antigen binding. When administered to mice as an intravenous solution, subcutaneous solution or subcutaneous dispersion at similar (4.6-7.3 mg/kg) or ultra-high dosages (51.6 mg/kg), the distribution and elimination kinetics were within error and the protein retained full activity. Overall, this method of generating high-concentration, low-viscosity dispersions of antibody nanoclusters could lead to improved administration and patient compliance, providing new opportunities for the biotechnology industry.
Background and Objectives
The macrophage is an important early cellular marker related to risk of future rupture of atherosclerotic plaques. Two-channel two-photon luminescence (TPL) microscopy combined with optical coherence tomography (OCT) was used to detect, and further characterize the distribution of aorta-based macrophages using plasmonic gold nanorose as an imaging contrast agent.
Study Design/Materials and Methods
Nanorose uptake by macrophages was identified by TPL microscopy in macrophage cell culture. Ex vivo aorta segments (8 × 8 × 2 mm3) rich in macrophages from a rabbit model of aorta inflammation were imaged by TPL microscopy in combination with OCT. Aorta histological sections (5 µm in thickness) were also imaged by TPL microscopy.
Merged two-channel TPL images showed the lateral and depth distribution of nanorose-loaded macrophages (confirmed by RAM-11 stain) and other aorta components (e.g., elastin fiber and lipid droplet), suggesting that nanorose-loaded macrophages are diffusively distributed and mostly detected superficially within 20 µm from the luminal surface of the aorta. Moreover, OCT images depicted detailed surface structure of the diseased aorta.
Results suggest that TPL microscopy combined with OCT can simultaneously reveal macrophage distribution with respect to aorta surface structure, which has the potential to detect vulnerable plaques and monitor plaque-based macrophages overtime during cardiovascular interventions.
atherosclerosis; macrophage; nanorose; two-photon luminescence microscopy; optical coherence tomography; photothermal wave imaging
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 trafficking 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 trafficking of endocytosed magnetic nanoparticles. In pulsed magneto-motive ultrasound 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 signal from individual nanoparticles. Here we further demonstrate that pMMUS imaging can identify interaction between magnetic nanoparticles and living cells, i.e. intracellular aggregation 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 trafficking non-invasively and in real-time.
Pulsed magneto-motive ultrasound imaging; superparamagnetic iron-oxide nanoparticles; macrophage; endocytosis; intracellular trafficking
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 ultra-thin 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 sub-monolayer to multilayer aggregates of Abs on the nanorose surface was observed for 54 Abs and an overall particle diameter of ~60 to 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.
Metal nanoparticles with surface plasmon resonance (SPR) in the near-infrared region (NIR) are of great interest for imaging and therapy. Presently, gold nanoparticles with NIR absorbance are typically larger than 50nm, above the threshold size of ~5 nm required for efficient renal clearance. As these nanoparticles are not biodegradable, concerns about long-term toxicity have restricted their translation into the clinic. Here, we address this problem by developing a flexible platform for the kinetically-controlled assembly of sub-5 nm ligand-coated gold particles to produce metal/polymer biodegradable nanoclusters smaller than 100 nm with strong NIR absorbance for multimodal application. A key novel feature of the proposed synthesis is the use of weakly adsorbing biodegradable polymers that allows tight control of nanocluster size and, in addition, results in nanoclusters with unprecedented metal loadings, and thus optical functionality. Over time, the biodegradable polymer stabilizer degrades under physiological conditions that leads to disassembly of the nanoclusters into sub-5nm primary gold particles which are favorable for efficient body clearance. This synthesis of polymer/inorganic nanoclusters combines the imaging contrast and therapeutic capabilities afforded by the NIR-active nanoparticle assembly with the biodegradability of a polymer stabilizer.
plasmonic nanoparticles; biodegradation of nanoparticles; near-infrared nanoparticles; clearance of nanoparticles; nanoclusters
Plasmonic metal nanoparticles are used in photoacoustic imaging as contrast agents because of their resonant optical absorption properties in the visible and near-IR regions. However, the nanoparticles could accumulate and result in long-term toxicity in vivo, because they are generally not biodegradable. Recently, biodegradable plasmonic gold nanoclusters, consisting of sub-5 nm primary gold nanoparticles and biodegradable polymer stabilizer, were introduced. In this Letter, we demonstrate the feasibility of biodegradable nanoclusters as a photoacoustic contrast agent. We performed photoacoustic and ultrasound imaging of a tissue-mimicking phantom with inclusions containing nanoclusters at various concentrations. The results indicate that the biodegradable gold nanoclusters can be used as effective contrast agents in photoacoustic imaging.
Subcutaneous injection of concentrated protein and peptide solutions, in the range of 100–400 mg/mL, is often not possible with a 25- to 27-gauge needle, as the viscosity can be well above 50 cP. Apparent viscosities below this limit are reported for suspensions of milled lysozyme microparticles up to nearly 400 mg/mL in benzyl benzoate or benzyl benzoate mixtures with safflower oils through a syringe with a 25- to 27-gauge needle at room temperature. These apparent viscosities were confirmed using a cone-and-plate rheometer. The intrinsic viscosity regressed from the Kreiger–Dougherty model was only slightly above the Einstein value of 2.5, indicating the increase in viscosity relative to that of the solvent was caused primarily by the excluded volume. Thus, the increases in viscosity from electrical double layer interactions (electroviscous effects), solvation of the particles, or deviations of the particle shape from a spherical geometry were minimal, and much smaller than typically observed for proteins dissolved in aqueous solutions. The small electroviscous effects are expected given the negligible zeta potential and thin double layers in the low dielectric constant organic solvent. The suspensions were resuspendable after a year, with essentially constant particle size after two months as measured by static light scattering. The lower apparent viscosities for highly concentrated protein suspensions relative to protein solutions, coupled with these favorable characteristics upon resuspension, may offer novel opportunities for subcutaneous injection of therapeutic proteins.
The ability of 20–50 nm nanoparticles to target and modulate the biology of specific types of cells will enable major advancements in cellular imaging and therapy in cancer and atherosclerosis. A key challenge is to load an extremely high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. Herein we report ~30 nm stable uniformly sized near-infrared (NIR) active, superparamagnetic nanoclusters formed by kinetically controlled self-assembly of gold-coated iron oxide nanoparticles. The controlled assembly of nanocomposite particles into clusters with small primary particle spacings produces collective responses of the electrons that shift the absorbance into the NIR region. The nanoclusters of ~70 iron oxide primary particles with thin gold coatings display intense NIR (700–850 nm) absorbance with a cross section of ~10−14 m2. Because of the thin gold shells with an average thickness of only 2 nm, the r2 spin–spin magnetic relaxivity is 219 mM−1 s−1, an order of magnitude larger than observed for typical iron oxide particles with thicker gold shells. Despite only 12% by weight polymeric stabilizer, the particle size and NIR absorbance change very little in deionized water over 8 months. High uptake of the nanoclusters by macrophages is facilitated by the dextran coating, producing intense NIR contrast in dark field and hyperspectral microscopy, both in cell culture and an in vivo rabbit model of atherosclerosis. Small nanoclusters with optical, magnetic, and therapeutic functionality, designed by assembly of nanoparticle building blocks, offer broad opportunities for targeted cellular imaging, therapy, and combined imaging and therapy.
gold; iron oxide; nanocluster; near-infrared; macrophage targeted imaging; MRI; atherosclerosis; cancer
Application of photothermal Optical Coherence Tomography (OCT) to detect macrophages in ex vivo rabbit arteries which have engulfed nanoclusters of gold coated iron oxide (nanorose) is reported. Nanorose engulfed by macrophages associated with atherosclerotic lesions in rabbit arteries absorb incident laser (800nm) energy and cause optical pathlength (OP) variation which is measured using photothermal OCT. OP variation in polydimethyl siloxane tissue phantoms containing varying concentrations of nanorose match values predicted from nanoparticle and material properties. Measurement of OP variation in rabbit arteries in response to laser excitation provides an estimate of nanorose concentration in atherosclerotic lesions of 2.5x109 particles/ml. OP variation in atherosclerotic lesions containing macrophages taking up nanorose has a different magnitude and profile from that observed in control thoracic aorta without macrophages and is consistent with macrophage presence as identified with RAM-11 histology staining. Our results suggest that tissue regions with macrophages taking up nanorose can be detected using photothermal OCT.
(170.4500) Optical Coherence Tomography; (170.3880) Medical and Biological Imaging
The objective of the study was to produce rapidly dissolving formulations of the poorly water-soluble drug repaglinide using an innovative new technology, ultra-rapid freezing (URF), and to investigate the influence of excipient type on repaglinide stability. Repaglinide compositions containing different types and levels of excipients and different drug potencies (50%–86%) were produced by the URF technology. Repaglinide/excipient solutions were frozen on a cryogenic substrate, collected, and lyophilized to form a dry powder. Surfactants, including sodium dodecyl sulfate, and alkalizing agents such as diethanolamine (DEA) and tromethamine (TRIS) were incorporated into the compositions. Forced degradation of repaglinide was conducted under stressed conditions (eg, elevated temperature, exposure to peroxide) to determine the stability of the drug in such environments. The solubility of repaglinide increased as a function of increasing pH; therefore, incorporation of an alkalizing agent into the URF formulations increased the drug's solubility. Drug instability resulted when the drug was exposed to pH values above 9.0. URF formulations containing alkalizing agents showed no degradation or spontaneous recrystallization in the formulation, indicating that increased stability was afforded by processing. URF processing created nanostructured drug/excipient particles with higher dissolution rates than were achieved for unprocessed drug. Alkalizing agents such as TRIS and DEA, present at levels of 25% to 33% wt/wt in the formulations, did not cause degradation of the drug when processed using URF. URF processing, therefore, yielded fast-dissolving formulations that were physically and chemically stable, resistant to alkali degradation or spontaneous recrystallization in the formulation.
Ultra-rapid freezing; dissolution enhancement; stability enhancement; repaglinide; alkalizing agents
A new spinning oil film (SOF) solid-in-oil-in-oil emulsion process was developed to produce uniform-sized proteinloaded biodegradable microspheres. A thin SOF on a cylindrical rotor was used to shear droplets from a nozzle tip to control droplet size. The resulting microspheres with low polydispersity (6%) produced a low burst (6%–11%) release even at high loadings (13%–18% encapsulated solids, 8%–12% protein). The SOF process had a high yield and did not require the presence of water, which can cause protein denaturation, or surfactants, which may be unwanted in the final product. Amorphous protein and crystalline excipient solids were encapsulated into 3 different polymers, giving a homogenous drug distribution throughout the microspheres, and an essentially complete protein encapsulation efficiency (average=99%). In contrast, large burst release was observed for polydisperse microspheres produced by a conventional emulsification technique, particularly for microspheres smaller than 25 μm in diameter, which gave 93% burst at 15% loading. The uniform encapsulation of high loadings of proteins into microspheres with low polydispersity in an anhydrous process is of practical interest in the development of controlled-release protein therapeutics.
microsphere size control; monodisperse emulsions; spray freezing into liquid process; bovine serum albumin; solid-in-oil-in-oil processing; PLGA; initial burst
Aerosolized evaporative precipitation into aqueous solution and spray freezing into liquid nanostructured formulations of itraconazole as prophylaxis significantly improved survival relative to commercial itraconazole oral solution and the control in a murine model of invasive pulmonary aspergillosis. Aerosolized administration of nanostructured formulations also achieved high lung tissue concentrations while limiting systemic exposure.
The purpose of this research was to form stable suspensions of submicron particles of cyclosporine A, a water-insoluble drug, by rapid expansion from supercritical to aqueous solution (RESAS). A solution of cyclosporine A in CO2 was expanded into an aqueous solution containing phospholipid vesicles mixed with nonionic surfactants to provide stabilization against particle growth resulting from collisions in the expanding jet. The products were evaluated by measuring drug loading with high performance liquid chromatography (HPLC), particle sizing by dynamic light scattering (DLS), and particle morphology by transmission electron microscopy (TEM) and x-ray diffraction. The ability of the surfactant molecules to orient at the surface of the particles and provide steric stabilization could be manipulated by changing process variables including temperature and suspension concentration. Suspensions with high payloads (up to 54 mg/mL) could be achieved with a mean diameter of 500 nm and particle size distribution ranging from 40 to 920 nm. This size range is several hundred nanometers smaller than that produced by RESAS for particles stabilized by Tween 80 alone. The high drug payloads (≈10 times greater than the equilibrium solubility), the small particle sizes, and the long-term stability make this process attractive for development.
supercritical fluid; carbon dioxide; rapid expansion; water-insoluble
The objective of this study was to investigate the influence of processing parameters on the morphology, porosity, and crystallinity of polymeric polyethylene glycol (PEG) microparticles by spray freezing into liquid (SFL), a new particle engineering technology. Processing parameters investigated were the viscosity and flow rate of the polymer solution, nozzle diameter, spray time, pressure, temperature, and flow rate of the cryogenic liquid. By varying the processing parameters and feed composition, atomization and heat transfer mechanisms were modified resulting in particles of different size distribution, shape, morphology, density, porosity, and crystallinity. Median particle diameter (M50) varied from 25 μm to 600 μm. Particle shape was spherical or elongated with highly irregular surfaces. Granule density was between 0.5 and 1.5 g/mL. In addition to producing particles of pure polymer, drug particles were encapsulted in polymeric microparticles. The encapsulation efficiency of albuterol sulfate was 96.0% with a drug loading of 2.4%, indicating that SFL is useful for producing polymeric microparticles for drug delivery applications. It was determined that the physicochemical characteristics of model polymeric microparticles composed of PEG could be modified for use as a drug delivery carrier.
particle engineering; spray-freezing into liquid; polymeric carrier