Protein Corona Formed from Different Blood Plasma Proteins Affects the Colloidal Stability of Nanoparticles Differently.

Significant progress in the characterization of protein corona has been made. However, insights on how the corona affects the aggregation of nanoparticles (NPs) and consequent biological identity are still lacking. Here, we examined how the corona formed from four major serum proteins, immunoglobulin G (IgG), fibrinogen (FBG), apolipoprotein A1 (ApoA1), and human serum albumin (HSA), over a range of concentrations affects the aggregation of gold NPs (AuNPs). We found that at physiological pH of 7.4, all four proteins aggregated the AuNPs at low concentrations but conferred colloidal stability at high concentrations due to the complete "corona coat" around individual AuNPs. Due to their immune-related functions, IgG and FBG aggregated the AuNPs to a greater extent compared to HSA and ApoA1 which were mostly involved in transport of small molecules. We then introduced the AuNP-corona formed from each protein into an acidic solution at pH 6.2 with high ionic concentration for up to 24 h as a model of the tumor microenvironment to examine for changes in their aggregation. We observed that protein corona formation sterically stabilized the AuNP-corona for all four proteins, resulting in a smaller increase in aggregation and size compared to citrate-capped AuNPs. This was especially true for corona formed at high protein:AuNP ratios. Our study therefore showed that the formation of a complete "corona coat" around NPs at sufficiently high protein:NP ratio was required for colloidal stability of designed NP systems in both physiological and cancer microenvironment to maintain efficiency and efficacy in cancer drug delivery.

Influence of protein corona and caveolae-mediated endocytosis on nanoparticle uptake and transcytosis.

Transcytosis of nanoparticles (NPs) is emerging as an attractive alternative to the paracellular route in cancer drug delivery with studies suggesting targeting caveolae-mediated endocytosis to maximize NP transcytosis. However, there are limited studies on transcytosis of NPs, especially for corona-coated NPs. Most studies focused on cellular uptake as an indirect measure of the NP's transcellular permeability (Pd). Here, we probed the effect of protein corona on the uptake and transcytosis of 20, 40, 100, and 200 nm polystyrene nanoparticles (pNP-PC) across HUVECs in a microfluidic channel that modelled the microvasculature. We observed increased cell uptake with size of pNP-PC although it was the smallest 20 nm pNP-PC that exhibited the highest transcellular Pd. In the absence of corona however, cell uptake decreased with size, and the largest 200 nm pNP-PEG exhibited the lowest transcellular Pd. By inhibiting caveolae-mediated endocytosis in HUVECs, smaller pNPs had a larger drop in cell uptake than larger pNPs, regardless of surface coating. However, only the smallest (20 nm) and largest (200 nm) pNP-PC had a decrease in Pd following inhibition with MßCD. Our findings showed that the protein corona affected the transcytosis of NPs, and their uptake by caveolae-mediated endocytosis did not necessarily lead to transcytosis.

Quantifying Vascular Distribution and Adhesion of Nanoparticles with Protein Corona in Microflow.

The protein corona has emerged as an important determinant of biological response in nanoparticle (NP) drug delivery. However, there is presently no reported study on how the protein corona affects the behavior of NPs in microflow and its subsequent interactions with the vascular endothelium, which could affect their delivery to the target tumor site regardless of its targeting mechanism. Furthermore, a consensus on the role of physical and surface characteristics of NPs in affecting the margination of NPs is lacking due to different methods of quantifying margination. In this study, we examine how the particle adhesion (PA) method and particle distribution (PD) method quantify the margination of 20, 40, 100, and 200 nm polystyrene NPs (pNPs) differently in fibronectin or pluronic F-127-coated microfluidic straight channels. We found that PA reduced with increasing pNP size, whereas the PD was similar across all pNP sizes regardless of channel coating. We then formed a protein corona on all pNPs (pNPs-PC) and found that the protein corona increased the adhesion of 40-200 nm pNPs in fibronectin-coated channels, with no size dependence between them except for 40 nm, which had significantly higher particle adhesion. The PA method was also dependent on channel coating, whereas the PD method was independent of channel coating. These results suggested that the PA method was more amenable to surface interactions between the pNPs and the channel wall while providing a measure of the amount of NPs that interacted with the channel walls, whereas the PD method provided a representation of their distribution across the channel due to margination. The two methods complement each other to elucidate a more holistic understanding of how different factors might affect a NP's margination in future studies.

Preparation and characterization of an amylase-triggered dextrin-linked graphene oxide anticancer drug nanocarrier and its vascular permeability.

We synthesized a dextrin (DEX)-conjugated graphene oxide (GO) nanocarrier (GO100-DEX) as a potential drug delivery system to respond to a tumor-associated stimulus, α-amylase, that has high permeability through the fenestrated endothelial barrier to the tumor site. At acidic pH and in the presence of α-amylase to simulate tumor conditions, GO100-DEX released a 1.5-fold higher amount of doxorubicin (DOX) than of GO100. Under the same conditions, the cytotoxic effects of GO100-DEX/DOX were 2-fold greater than those of free DOX and 2.9-fold greater than those of GO100/DOX. Employing an in vitro biomimetic microfluidic blood vessel model lined with human umbilical vein endothelial cells, we evaluated the tumor vasculature endothelial permeation of GO100-DEX and GO100 using dextrans of 10 and 70kDa for comparison and as standards to validate the microfluidic blood vessel model. The results showed that the permeabilities of GO100-DEX and GO100 were 4.3- and 4.9-fold greater than that of 70kDa dextran and 2.7- and 3.1-fold higher than that of 10kDa dextran, thus demonstrating the good permeability of the GO-based nanocarrier through the fenestrated endothelial barrier.

A Facile Method to Probe the Vascular Permeability of Nanoparticles in Nanomedicine Applications.

The effectiveness of nanoparticles (NP) in nanomedicine depends on their ability to extravasate from vasculature towards the target tissue. This is determined by their permeability across the endothelial barrier. Unfortunately, a quantitative study of the diffusion permeability coefficients (Pd) of NPs is difficult with in vivo models. Here, we utilize a relevant model of vascular-tissue interface with tunable endothelial permeability in vitro based on microfluidics. Human umbilical vein endothelial cells (HUVECs) grown in microfluidic devices were treated with Angiopoietin 1 and cyclic adenosine monophosphate (cAMP) to vary the Pd of the HUVECs monolayer towards fluorescent polystyrene NPs (pNPs) of different sizes, which was determined from image analysis of their fluorescence intensity when diffusing across the monolayer. Using 70 kDa dextran as a probe, untreated HUVECs yielded a Pd that approximated tumor vasculature while HUVECs treated with 25 µg/mL cAMP had Pd that approximated healthy vasculature in vivo. As the size of pNPs increased, its Pd decreased in tumor vasculature, but remained largely unchanged in healthy vasculature, demonstrating a trend similar to tumor selectivity for smaller NPs. This microfluidic model of vascular-tissue interface can be used in any laboratory to perform quantitative assessment of the tumor selectivity of nanomedicine-based systems.

Nanoparticle drug delivery systems and their use in cardiac tissue therapy.

Cardiovascular diseases make up one of the main causes of death today, with myocardial infarction and ischemic heart disease contributing a large share of the deaths reported. With mainstream clinical therapy focusing on palliative medicine following myocardial infarction, the structural changes that occur in the diseased heart will eventually lead to end-stage heart failure. Heart transplantation remains the only gold standard of cure but a shortage in donor organs pose a major problem that led to clinicians and researchers looking into alternative strategies for cardiac repair. This review will examine some alternative methods of treatment using chemokines and drugs carried by nanoparticles as drug delivering agents for the purposes of treating myocardial infarction through the promotion of revascularization. We will also provide an overview of existing studies involving such nanoparticulate drug delivery systems, their reported efficacy and the challenges facing their translation into ubiquitous clinical use.

An instantaneous colorimetric protein assay based on spontaneous formation of a protein corona on gold nanoparticles.

Commercial protein assays used ubiquitously in laboratories typically require long incubation times due to the inherently slow protein-reagent reactions. In this study, we report a novel facile technique for the instantaneous measurement of total protein concentration by exploiting the rapid aggregation dynamics of gold nanoparticles (NPs). By adsorbing different amounts of proteins on their surface to form a protein corona, these NPs can be sterically stabilized to different degrees by aggregation, thus exhibiting a spectrum of color change which can be quantitatively characterized by UV-Vis absorption spectroscopy. We evaluated this technique on four model proteins with different structures: bovine serum albumin (BSA), normal mouse immunoglobulin G (IgG), fibrinogen (FBG) and apolipoprotein A-I (Apo-A1) using two approaches, sequential and simultaneous. We obtained an approach-dependent linear concentration range up to 80 µg mL(-1) and 400 µg mL(-1) for sequential and simultaneous approaches, respectively. This linear working range was wider than that of the commercial Bradford assay and comparable to the Micro BCA assay. The simultaneous approach was also able to produce a linear working range of 200 to 1000 µg mL(-1) (R(2) = 0.995) in human urine, while the sequential approach was non-functional in urine. Similar to Micro BCA, the NP-based protein assay was able to elicit a linear response (R(2) > 0.87) for all four proteins with different structures. However, unlike Micro BCA which requires up to 120 min of incubation, we were able to obtain the read-out almost instantaneously without the need for incubation. The NP-based technique using the simultaneous approach can thus be exploited as a novel assay for instantaneous protein quantification to increase the productivity of laboratory processes.