The process of freezing protein solutions can perturb the conformation of the protein and potentially lead to aggregate formation during long-term storage in the frozen state. Radial macroscopic freeze concentration and temperature profiles for bovine serum albumin (BSA) solutions in small cylindrical stainless steel vessels were determined for various freezing rates. The measured concentrations of both BSA and immunoglobulin G2, as well as trehalose in sampled ice sections, increased by up to twofold to threefold toward the bottom and radial center for slow freezing rates produced in stagnant air freezers. The concentration and temperature profiles result in density gradients that transport solutes by convective flow. For faster external cooling by either forced convection of air or a liquid coolant, the increased freezing rate raised the ice front velocity resulting in enhanced dendritic ice growth. The ice trapped the solutes more effectively before they were removed from the ice front by diffusion and convection, resulting in more uniform solute concentration profiles. The dynamic temperature profiles from multiple radial thermocouples were consistent with the independently measured freeze concentration profiles. The ability to control the protein concentration profile in the frozen state offers the potential to improve stability of protein in long-term frozen storage. (c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:1316-1329, 2011
Macrophages are one of the most important cell types involved in the progression of atherosclerosis which can lead to myocardial infarction. To detect macrophages in atherosclerotic plaques, plasmonic gold nanorose is introduced as a nontoxic contrast agent for fluorescence imaging. We report macrophage cell culture and ex vivo tissue studies to visualize macrophages targeted by nanorose using scanning confocal microscopy. Atherosclerotic lesions were created in the aorta of a New Zealand white rabbit model subjected to a high cholesterol diet and double balloon injury. The rabbit was injected with nanoroses coated with dextran. A HeNe laser at 633 nm was used as an excitation light source and a acousto-optical beam splitter was utilized to collect fluorescence emission in 650-760 nm spectral range. Results of scanning confocal microscopy of macrophage cell culture and ex vivo tissue showed that nanoroses produce a strong fluorescence signal. The presence of nanorose in ex vivo tissue was further confirmed by photothermal wave imaging. These results suggest that scanning confocal microscopy can identify the presence and location of nanorose-loaded macrophages in atherosclerotic plaques.
Recently, pulsed magneto-motive ultrasound (pMMUS) imaging augmented with ultra-small magnetic nanoparticles has been introduced as a tool capable of imaging events at molecular and cellular levels. The sensitivity of a pMMUS system depends on several parameters, including the size, geometry and magnetic properties of the nanoparticles. Under the same magnetic field, larger magnetic nanostructures experience a stronger magnetic force and produce larger displacement, thus improving the sensitivity and signal-to-noise ratio (SNR) of pMMUS imaging. Unfortunately, large magnetic iron-oxide nanoparticles are typically ferromagnetic and thus are very difficult to stabilize against colloidal aggregation. In the current study we demonstrate improvement of pMMUS image quality by using large size superparamagnetic nanoclusters characterized by strong magnetization per particle. Water-soluble magnetic nanoclusters of two sizes (15 and 55 nm average size) were synthesized from 3 nm iron precursors in the presence of citrate capping ligand. The size distribution of synthesized nanoclusters and individual nanoparticles was characterized using dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM). Tissue mimicking phantoms containing single nanoparticles and two sizes of nanoclusters were imaged using a custom-built pMMUS imaging system. While the magnetic properties of citrate-coated nanoclusters are identical to those of superparamagnetic nanoparticles, the magneto-motive signal detected from nanoclusters is larger, i.e. the same magnetic field produced larger magnetically induced displacement. Therefore, our study demonstrates that clusters of superparamagnetic nanoparticles result in pMMUS images with higher contrast and SNR.
The importance of noncovalent interactions in the realm of biological materials continues to inspire efforts to create artificial supramolecular polymeric architectures. These types of self-assembled materials hold great promise as environmentally stimuli-responsive materials because they are capable of adjusting their various structural parameters, such as chain length, architecture, conformation, and dynamics, to new surrounding environments upon exposure to appropriate external stimuli. Nevertheless, in spite of considerable advances in the area of responsive materials, it has proved challenging to create synthetic self-assembled materials that respond to highly disparate analytes and whose environmentally induced changes in structure can be followed directly through both various spectroscopic and X-ray diffraction analyses. Herein, we report a new set of artificial self-assembled materials obtained by simply mixing two appropriately chosen, heterocomplementary macrocyclic receptors, namely a tetrathiafulvalene-functionalized calixpyrrole and a bis(dinitrophenyl)-meso-substituted calix pyrrole. The resulting polymeric materials, stabilized by combination of donor-acceptor and hydrogen bonding interactions, undergo dynamic, reversible dual guest-dependent structural transformations upon exposure to two very different types of external chemical inputs, namely chloride anion and trinitrobenzene. The structure and dynamics of the copolymers and their analyte-dependent responsive behavior was established via single crystal X-ray crystallography, SEM, heterocomplementary isodesmic analysis, 1- and 2D NMR, and dynamic light scattering spectroscopies. Our results demonstrate the benefit of using designed heterocomplementary interactions of two functional macrocyclic receptors to create synthetic, self-assembled materials for the development of "smart" sensory materials that mimic the key biological attributes of multianalyte recognition and substrate-dependent multisignaling.
Iron oxide nanoparticles, in the form of sub-100-nm clusters, were synthesized in the presence of poly(acrylic acid) (PAA) or poly(styrene sulfonate-alt-maleic acid) (PSS-alt-MA) to provide electrosteric stabilization. The superparamagnetic nanoclusters were characterized using a superconducting quantum interference device (SQUID), transmission electron microscopy (TEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA), and zeta potential measurements. To anchor the polymer shell on the nanoparticle surface, the polymer was cross-linked for a range of cross-linking densities. For nanoclusters with only 12% (w/w) PSS-alt-MA, electrosteric stabilization was sufficient even in 8 wt % NaCl. For PAA, the cross-linked polymer shell was essentially permanent and did not desorb even upon dilution of the nanoparticles for iron oxide concentrations down to 0.014 wt %. Without cross-linking, over half of the polymer desorbed from the particle surfaces. This general approach of the adsorption of polymer stabilizers onto nanoparticles followed by cross-linking may be utilized for a wide variety of cross-linkable polymers without the need to form covalent bonds between the nanoparticles and polymer stabilizer. Thus, this cross-linking approach is an efficient and inexpensive method of stabilizing nanoparticles for large-scale applications, including the electromagnetic imaging of subsurface reservoirs, even at high salinity.
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.
Melanoma accounts for 75% of all skin cancer deaths. Pulsed photothermal radiometry (PPTR), optical coherence tomography (OCT) and ultrasound (US) are non-invasive imaging techniques that may be used to measure melanoma thickness, thus, determining surgical margins. We constructed a series of PDMS tissue phantoms simulating melanomas of different thicknesses. PPTR, OCT and US measurements were recorded from PDMS tissue phantoms and results were compared in terms of axial imaging range, axial resolution and imaging time. A Monte Carlo simulation and three-dimensional heat transfer model was constructed to simulate PPTR measurement. Experimental results show that PPTR and US can provide a wide axial imaging range (75 mu m-1.7 mm and 120-910 mu m respectively) but poor axial resolution (75 and 120 mu m respectively) in PDMS tissue phantoms, while OCT has the most superficial axial imaging range (14-450 mu m) but highest axial resolution (14 mu m). The Monte Carlo simulation and three-dimensional heat transfer model give good agreement with PPTR measurement. PPTR and US are suited to measure thicker melanoma lesions (> 400 mu m), while OCT is better to measure thin melanoma lesions (< 400 mu m).
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 similar to 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.