ConspectusThe use of nanoparticles (NPs) in biology and medicine requires a molecular-level understanding of how NPs interact with cells in a physiological environment. A critical difference between well-controlled in vitro experiments and in vivo applications is the presence of a complex mixture of extracellular proteins. It has been established that extracellular serum proteins present in blood will adsorb onto the surface of NPs, forming a “protein corona”. Our goal was to understand how this protein layer affected cellular-level events, including NP binding, internalization, and transport. A combination of microscopy, which provides spatial resolution, and spectroscopy, which provides molecular information, is necessary to probe protein–NP–cell interactions. Initial experiments used a model system composed of polystyrene NPs functionalized with either amine or carboxylate groups to provide a cationic or anionic surface, respectively. Serum proteins adsorb onto the surface of both cationic and anionic NPs, forming a net anionic protein–NP complex. Although these protein–NP complexes have similar diameters and effective surface charges, they show the exact opposite behavior in terms of cellular binding. In the presence of bovine serum albumin (BSA), the cellular binding of BSA–NP complexes formed from cationic NPs is enhanced, whereas the cellular binding of BSA–NP complexes formed from anionic NPs is inhibited. These trends are independent of NP diameter or cell type. Similar results were obtained for anionic quantum dots and colloidal gold nanospheres. Using competition assays, we determined that BSA–NP complexes formed from anionic NPs bind to albumin receptors on the cell surface. BSA–NP complexes formed from cationic NPs are redirected to scavenger receptors. The observation that similar NPs with identical protein corona compositions bind to different cellular receptors suggested that a difference in the structure of the adsorbed protein may be responsible for the differences in cellular binding of the protein–NP complexes. Circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy show that the structure of BSA is altered following incubation with cationic NPs, but not anionic NPs. Single-particle-tracking fluorescence microscopy was used to follow the cellular internalization and transport of protein–NP complexes. The single particle-tracking experiments show that the protein corona remains bound to the NP throughout endocytic uptake and transport. The interaction of protein–NP complexes with cells is a challenging question, as the adsorbed protein corona controls the interaction of the NP with the cell; however, the NP itself alters the structure of the adsorbed protein. A combination of microscopy and spectroscopy is necessary to understand this complex interaction, enabling the rational design of NPs for biological and medical applications.
Nanoparticles used for biological and biomedical applications encounter a host of extracellular proteins. These proteins rapidly adsorb onto the nanoparticle surface, creating a protein corona. Poly(ethylene glycol) can reduce, but not eliminate, the nonspecific adsorption of proteins. As a result, the adsorbed proteins, rather than the nanoparticle itself, determine the cellular receptors used for binding, the internalization mechanism, the intracellular transport pathway, and the subsequent immune response. Using fluorescence microscopy and flow cytometry, we first characterize a set of polystyrene nanoparticles in which the same adsorbed protein, bovine serum albumin, leads to binding to two different cell surface receptors: native albumin receptors and scavenger receptors. Using a combination of circular dichroism spectroscopy, isothermal titration calorimetry, and fluorescence spectroscopy, we demonstrate that the secondary structure of the adsorbed bovine serum albumin protein controls the cellular receptors used by the protein–nanoparticle complexes. These results show that protein secondary structure is a key parameter in determining the cell surface receptor used by a protein–nanoparticle complex. We expect this link between protein structure and cellular outcomes will provide a molecular basis for the design of nanoparticles for use in biological and biomedical applications.
Nanoparticles are increasingly important for biological applications ranging from drug delivery to cellular imaging. In the course of these applications, nanoparticles are exposed to a complex environment of extracellular proteins that can be adsorbed onto the surface of the nanoparticle, altering nanoparticle-cell interactions. We have investigated how proteins found in blood serum affect the binding of nanoparticles to the surface of cells. Using fluorescence microscopy, we find that the cellular binding of cationic nanoparticles is enhanced by the presence of serum proteins while the binding of anionic nanoparticles is inhibited. We have determined that this difference in cellular binding is due to the use of distinct cellular receptors. Competition assays, quantified with flow cytometry, show that the protein-nanoparticle complex formed from the cationic nanoparticles binds to scavenger receptors on the cell surface. Interestingly, the protein-nanoparticle complex formed from anionic nanoparticles binds to native protein receptors. As nanoparticles become increasingly important for in vivo applications, we expect these results will inform the design of nanoparticles with improved cellular binding.
Inflammation and altered glutamate metabolism are two pathways implicated in the pathophysiology of depression. Interestingly, these pathways may be linked given that administration of inflammatory cytokines such as interferon-α to otherwise non-depressed controls increased glutamate in the basal ganglia and dorsal anterior cingulate cortex (dACC) as measured by magnetic resonance spectroscopy (MRS). Whether increased inflammation is associated with increased glutamate among patients with major depression is unknown. Accordingly, we conducted a cross-sectional study of 50 medication-free, depressed outpatients using single-voxel MRS, to measure absolute glutamate concentrations in basal ganglia and dACC. Multivoxel chemical shift imaging (CSI) was used to explore creatine-normalized measures of other metabolites in basal ganglia. Plasma and cerebrospinal fluid (CSF) inflammatory markers were assessed along with anhedonia and psychomotor speed. Increased log plasma C-reactive protein (CRP) was significantly associated with increased log left basal ganglia glutamate controlling for age, sex, race, body mass index, smoking status and depression severity. In turn, log left basal ganglia glutamate was associated with anhedonia and psychomotor slowing measured by the finger-tapping test, simple reaction time task and the Digit Symbol Substitution Task. Plasma CRP was not associated with dACC glutamate. Plasma and CSF CRP were also associated with CSI measures of basal ganglia glutamate and the glial marker myoinositol. These data indicate that increased inflammation in major depression may lead to increased glutamate in the basal ganglia in association with glial dysfunction and suggest that therapeutic strategies targeting glutamate may be preferentially effective in depressed patients with increased inflammation as measured by CRP.
Nanoparticles used in biological applications encounter a complex mixture of extracellular proteins. Adsorption of these proteins on the nanoparticle surface results in the formation of a “protein corona,” which can dominate the interaction of the nanoparticle with the cellular environment. The goal of this research was to determine how nanoparticle composition and surface modification affect the cellular binding of protein-nanoparticle complexes. We examined the cellular binding of a collection of commonly used anionic nanoparticles: quantum dots, colloidal gold nanoparticles, and low-density lipoprotein particles, in the presence and absence of extracellular proteins. These experiments have the advantage of comparing different nanoparticles under identical conditions. Using a combination of fluorescence and dark field microscopy, flow cytometry, and spectroscopy, we find that cellular binding of these anionic nanoparticles is inhibited by serum proteins independent of nanoparticle composition or surface modification. We expect these results will aid in the design of nanoparticles for in vivo applications.
Background and Purpose Cerebral thermoregulation remains poorly understood. Temperature dysregulation is deeply implicated in potentiation of cerebrovascular ischemia. We present a multi-phasic, MR thermographic study in a non-human primate (NHP) model of MCA infarction, hypothesizing detectable brain temperature disturbances and brain-systemic temperature decoupling. Materials and Methods Three rhesus macaque NHP were sourced for three-phase MRI: 1) baseline MRI (t-7); 2) seven-hour continuous MRI following minimally-invasive, endovascular MCA stroke induction (t0); 3) post-stroke day MRI (t1) follow-up. MR thermometry was achieved by multi-voxel spectroscopy (semi-LASER MRSI) using the proton resonance frequency chemical shift. Relationship of brain and systemic temperatures with time and infarction volumes was characterized using a mixed-effects model. Results Following MCA infarction progressive cerebral hyperthermia was observed in all three subjects, significantly outpacing systemic temperature fluctuations. Highly significant associations were observed for systemic, hemispheric, and global brain temperatures (F-stat p.0005 for all regressions) relative to time from stroke induction. Significant differences in the relationship between temperature and time following stroke onset were detected when comparing systemic temperatures with ipsilateral (p=.007), contralateral (p=.004), and infarction core (p=.003) temperatures following multiple comparisons correction. Significant associations were observed between infarction volumes and both systemic (p≤.01) and ipsilateral (p=.04) brain temperatures, but not contralateral brain temperature (p=.08). Conclusion Successful physiologic and continuous post-ischemic cerebral MR thermography was conducted, and prescribed in an NHP infarction model to facilitate translatability. The results confirm hypothesized temperature disturbance and decoupling of physiologic brain-systemic temperature gradients. These findings inform a developing paradigm of brain thermoregulation, and the applicability of brain temperature as a neuroimaging biomarker in CNS injury.
Understanding and subsequently controlling non-specific interactions between engineered nanomaterials and biological environment have become increasingly important for further developing and advancing nanotechnology for biomedical applications. Such non-specific interactions, also known as the biofouling effect, mainly associate with the adsorption of biomolecules (such as proteins, DNAs, RNAs, and peptides) onto the surface of nanomaterials and the adhesion or uptake of nanomaterials by various cells. By altering the surface properties of nanomaterials the biofouling effect can lead to changes of physicochemical properties, pharmacokinetics, functions, and toxicity of nanomaterials. This review provides discussions on the current understanding of the biofouling effect, the factors that affect the non-specific interactions associated with biofouling, and the impact of the biofouling effect on the performances and functions of nanomaterials. An overview of the development and applications of various anti-biofouling coating materials to preserve and improve the properties and functions of engineered nanomaterials for intended biomedical applications is also provided.
BACKGROUND AND PURPOSE Brain temperature is critical for homeostasis, relating intimately to cerebral perfusion and metabolism. Cerebral thermometry is historically challenged by the cost and invasiveness of clinical and laboratory methodologies. We propose the use of non-invasive MR thermometry in patients with cerebrovascular disease, hypothesizing the presence of a measurable brain thermal response reflecting the tissue hemodynamic state. MATERIALS AND METHODS Contemporaneous imaging and MR thermometry was performed in 10 patients (ages 32–68 years) undergoing acetazolamide challenge for chronic, anterior circulation steno-occlusive disease. Cerebrovascular reactivity was calculated using blood oxygen level-dependent imaging and arterial spin labeling MR methods. Brain temperature was calculated pre- and post-acetazolamide using previously established chemical shift thermometry. Mixed-effects models of the voxel-wise relationships between the brain thermal response and cerebrovascular reactivity were computed, and significance of model coefficients were determined with an F-test (p<.05). RESULTS We observed significant, voxel-wise quadratic relationships between cerebrovascular reactivity (from blood oxygen-level dependent imaging) and the brain thermal response (x coefficient = 0.052, p<.001; x2 coefficient = 0.0068, p<.001) and baseline brain temperatures (x coefficient = 0.59, p=.008; x2 coefficient = −0.13, p<.001). A significant linear relationship was observed for the brain thermal response with cerebrovascular reactivity (from arterial spin labeling) (p=.001). CONCLUSIONS The findings support the presence of a brain thermal response exhibiting complex but significant interactions with tissue hemodynamics, which we posit to reflect a relative balance of heat producing versus heat dissipating tissue states. The brain thermal response is a potential noninvasive biomarker for cerebrovascular impairment.
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