Acute and Chronic Shear Stress Differently Regulate Endothelial Internalization of Nanocarriers Targeted to Platelet-Endothelial Cell Adhesion Molecule‑1

Intracellular delivery of nanocarriers (NC) is controlled by their design and target cell phenotype, microenvironment, and functional status. Endothelial cells (EC) lining the vascular lumen represent an important target for drug delivery. Endothelium <i>in vivo</i> is constantly or intermittently (as, for example, during ischemia-reperfusion) exposed to blood flow, which influences NC–EC interactions by changing NC transport properties, and by direct mechanical effects upon EC mechanisms involved in NC binding and uptake. EC do not internalize antibodies to marker glycoprotein PECAM(CD31), yet internalize multivalent NC coated with PECAM antibodies (anti-PECAM/NC) <i>via</i> a noncanonical endocytic pathway distantly related to macropinocytosis. Here we studied the effects of flow on EC uptake of anti-PECAM/NC spheres (∼180 nm diameter). EC adaptation to chronic flow, manifested by cellular alignment with flow direction and formation of actin stress fibers, inhibited anti-PECAM/NC endocytosis consistent with lower rates of anti-PECAM/NC endocytosis <i>in vivo</i> in arterial compared to capillary vessels. Acute induction of actin stress fibers by thrombin also inhibited anti-PECAM/NC endocytosis, demonstrating that formation of actin stress fibers impedes EC endocytic machinery. In contrast, acute flow without stress fiber formation, stimulated anti-PECAM/NC endocytosis. Anti-PECAM/NC endocytosis did not correlate with the number of cell-bound particles under flow or static conditions. PECAM cytosolic tail deletion and disruption of cholesterol-rich plasmalemma domains abrogated anti-PECAM/NC endocytosis stimulation by acute flow, suggesting complex regulation of a flow-sensitive endocytic pathway in EC. The studies demonstrate the importance of the local flow microenvironment for NC uptake by the endothelium and suggest that cell culture models of nanoparticle uptake should reflect the microenvironment and phenotype of the target cells

Delivering Nanoparticles to Lungs while Avoiding Liver and Spleen through Adsorption on Red Blood Cells

Nanoparticulate drug delivery systems are one of the most widely investigated approaches for developing novel therapies for a variety of diseases. However, rapid clearance and poor targeting limit their clinical utility. Here, we describe an approach to harness the flexibility, circulation, and vascular mobility of red blood cells (RBCs) to simultaneously overcome these limitations (cellular hitchhiking). A noncovalent attachment of nanoparticles to RBCs simultaneously increases their level in blood over a 24 h period and allows transient accumulation in the lungs, while reducing their uptake by liver and spleen. RBC-adsorbed nanoparticles exhibited ∼3-fold increase in blood persistence and ∼7-fold higher accumulation in lungs. RBC-adsorbed nanoparticles improved lung/liver and lung/spleen nanoparticle accumulation by over 15-fold and 10-fold, respectively. Accumulation in lungs is attributed to mechanical transfer of particles from the RBC surface to lung endothelium. Independent tracing of both nanoparticles and RBCs <i>in vivo</i> confirmed that RBCs themselves do not accumulate in lungs. Attachment of anti-ICAM-1 antibody to the exposed surface of NPs that were attached to RBCs led to further increase in lung targeting and retention over 24 h. Cellular hitchhiking onto RBCs provides a new platform for improving the blood pharmacokinetics and vascular delivery of nanoparticles while simultaneously avoiding uptake by liver and spleen, thus opening the door for new applications

Targeting to Endothelial Cells Augments the Protective Effect of Novel Dual Bioactive Antioxidant/Anti-Inflammatory Nanoparticles

Oxidative stress and inflammation are intertwined contributors to numerous acute vascular pathologies. A novel dual bioactive nanoparticle with antioxidant/anti-inflammatory properties was developed based on the interactions of tocopherol phosphate and the manganese porphyrin SOD mimetic, MnTMPyP. The size and drug incorporation efficiency were shown to be dependent on the amount of MnTMPyP added as well as the choice of surfactant. MnTMPyP was shown to retain its SOD-like activity while in intact particles and to release in a slow and controlled manner. Conjugation of anti-PECAM antibody to the nanoparticles provided endothelial targeting and potentiated nanoparticle-mediated suppression of inflammatory activation of these cells manifested by expression of VCAM, E-selectin, and IL-8. This nanoparticle technology may find applicability with drug combinations relevant for other pathologies

The Effect of Polymeric Nanoparticles on Biocompatibility of Carrier Red Blood Cells

<div><p>Red blood cells (RBCs) can be used for vascular delivery of encapsulated or surface-bound drugs and carriers. Coupling to RBC prolongs circulation of nanoparticles (NP, 200 nm spheres, a conventional model of polymeric drug delivery carrier) enabling their transfer to the pulmonary vasculature without provoking overt RBC elimination. However, little is known about more subtle and potentially harmful effects of drugs and drug carriers on RBCs. Here we devised high-throughput <i>in vitro</i> assays to determine the sensitivity of loaded RBCs to osmotic stress and other damaging insults that they may encounter <i>in vivo</i> (<i>e</i>.<i>g</i>. mechanical, oxidative and complement insults). Sensitivity of these tests is inversely proportional to RBC concentration in suspension and our results suggest that mouse RBCs are more sensitive to damaging factors than human RBCs. Loading RBCs by NP at 1:50 ratio did not affect RBCs, while 10–50 fold higher NP load accentuated RBC damage by mechanical, osmotic and oxidative stress. This extensive loading of RBC by NP also leads to RBCs agglutination in buffer; however, addition of albumin diminished this effect. These results provide a template for analyses of the effects of diverse cargoes loaded on carrier RBCs and indicate that: i) RBCs can tolerate carriage of NP at doses providing loading of millions of nanoparticles per microliter of blood; ii) tests using protein-free buffers and mouse RBCs may overestimate adversity that may be encountered in humans.</p></div

Mechanical fragility of red blood cells with 200nm nanoparticles adsorbed onto their surface at 1.0% hematocrit.

<p>Percent hemolysis of mouse NP:RBCs (A) and human NP:RBCs (C) (50:1; 200:1; and 2000:1) were obtained after constant rotation with glass beads at 24 rpm at 37°C for up to 8h and 24h, respectively. Percent hemolysis of different NP:RBC ratios for mouse RBC at 0.5h (B) and human RBC at 2h (D). Values are means (n = 4–5) ± SD. Please note that some deviation bars are too small to be evident.</p

Oxidative fragility of red blood cells with 200nm nanoparticles adsorbed onto their surface at 1.0% hematocrit.

<p>Percent hemolysis of mouse (A,B,C) and human (D,E) RBC/NP:RBCs (50:1; 200:1; and 2000:1) were obtained after rotating at 24 rpm at 37°C for up to 24h and 46h, respectively, after being challenged with 3mM H₂O₂. Percent hemolysis of different NP:RBC ratios for mouse RBC at 4h (B), 24h (C) and human RBC at 32h (E). Values are means (n = 4–5) ± SD. (***,P<0.001 vs naïve RBC). Please note in (D) lysis is caused by adsorption of NPs, not by H₂O₂. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152074#pone.0152074.g002" target="_blank">Fig 2B</a> for controls without H₂O₂.</p

Osmotic fragility of red blood cells with 200nm nanoparticles adsorbed onto their surface at 1.0% hematocrit.

<p>Hemolytic curves for mouse (A) and human (B) NP:RBCs were obtained after immediate exposure to different [NaCl]. Curves were determined for NP:RBC ratio (50:1, 200:1, and 2000:1). Percent hemolysis of different RBC/NP:RBC ratios at 73mM NaCl (C). Values are means (n = 4–6) ± SD. (***, P<0.001 vs naïve RBC). Please note that some deviation bars are too small to be evident.</p

Complement lysis of human RBCs with 200nm nanoparticles adsorbed onto their surface at 1.0% hematocrit.

<p>Hemolytic curves for human RBC/NP:RBCs were obtained after the addition of complement obtain from serum rotating at 37°C for 4h. The addition of streptavidin-biotin (SA-B) was used as a control. Curves were determined for NP:RBC ratio (200:1; 400:1; 2000:1). Values are means (n = 4) ± SD. (**, P<0.01 ***, P<0.001 vs. naïve RBC). Please note that some deviation bars are too small to be evident.</p

Effect of low stress over time on red blood cells with 200nm nanoparticles adsorbed onto their surface at 1.0% hematocrit.

<p>Hemolytic curves for mouse (A) and (B) human RBC/NP:RBCs were obtained after constant rotation at 24 rpm at 37°C for up to 46h. Curves were determined for NP:RBC ratio (50:1; 200:1; 2000:1). Percent hemolysis of different NP:RBC ratios at 24h (C). Values are means (n = 5) ± SD. (***, P<0.001 vs naïve mouse RBC) (###,P<0.001 vs naïve human RBC).</p