Designing of drug nanocarriers to aid delivery of therapeutics is an expanding field that can improve medical treatments. Nanocarriers are often functionalized with elements that recognize cell-surface molecules involved in subcellular transport to improve targeting and endocytosis of therapeutics. Combination-targeting using several affinity elements further modulates this outcome. The most studied example is endothelial targeting via multiple cell adhesion molecules (CAMs), which mimics the strategy of leukocytes to adhere and traverse the vascular endothelium. Yet, the implications of this strategy on intracellular transport and in vivo biodistribution remain uncharacterized. We examined this using nanocarriers functionalized for dual- or triple- targeting to intercellular, platelet-endothelial, and/or vascular CAMs (ICAM-1, PECAM-1, VCAM-1). These molecules differ in expression level, location, pathological stimulation, and/or endocytic pathway. In endothelial cells, binding of PECAM-1/VCAM-1-targeted nanocarriers was intermediate to single-targeted counterparts and enhanced in disease-like conditions. ICAM-1/PECAM-1-targeted nanocarriers surpassed PECAM-1/VCAM-1 in control, but showed lower selectivity toward disease-like conditions. Triple-targeting resulted in binding similar to ICAM-1/PECAM-1 combination and displayed the highest selectivity in disease-like conditions. All combinations were effectively internalized by cells, with slightly better performance when targeting receptors of different endocytic pathways. In vivo, ICAM-1/PECAM-1-targeted nanocarriers outperformed PECAM-1/VCAM-1 in control and disease-like conditions, and triple-targeted counterparts slightly enhanced this outcome in some organs. As a result, delivery of a model therapeutic cargo (acid sphingomyelinase, deficient in Niemann-Pick disease A-B) was enhanced to all affected organs by triple-targeted nanocarriers, particularly in disease-like conditions. Therefore, multi-CAM targeting may aid optimization of some therapeutic nanocarriers, where the combination and multiplicity of the affinity moieties utilized allow modulation of targeting performance.
To improve properties such as thermal conductivity, low temperature thermal strain, and creep resistance of ultra-high molar mass polyethylene (UHMMPE) fibers, several researchers have previously undertaken efforts to crosslink these fibers using radiation. Ionizing radiation is commonly used to crosslink bulk UHMMPE in other applications, such as artificial joints. However, UHMMPE fibers differ from bulk UHMMPE in that they have a higher crystallinity (approximately 85% to 90%) and are very highly oriented during manufacturing in which the fibers are stretched 50 to 100 times their original length. Thus, the amorphous fraction of the UHMMPE fibers is also highly ordered. Several experiments were conducted to crosslink the UHMMPE fibers using both low dose rate (gamma) and high dose rate (electron beam) irradiation, all in the absence of oxygen. In all cases, the tensile strength of the fiber was greatly reduced by the irradiation. The oxidation index was also measured for the irradiated samples, and oxidation was not found to play a major role in the reduction of tensile strength in the fibers after irradiation. While this work did not achieve the desired result of improving the mechanical properties of the UHMMPE fiber, a significant result was found. The electron paramagnetic resonance (EPR) spectrum of the UHMMPE fibers was measured shortly after irradiation, and a mixture of allyl and alkyl radicals were detected. The irradiated samples were stored in dark ambient conditions for at least six years, then reexamined using EPR for free radical characterization. Surprisingly, the gamma-irradiated samples showed clear evidence of long-lived polyenyl radicals present in the material. Free radicals are very reactive species that will typically migrate to the surface of the crystalline domain and decay in a relatively short time through various reactions in the amorphous regions. It is hypothesized herein that due to the high crystallinity and large anisotropy of the highly drawn UHMMPE fiber, the polyenyl radicals were trapped in the crystal phase and were unable to migrate and decay. An experiment was performed to test this hypothesis, by which samples of the irradiated fibers were heated to temperatures above first the alpha relaxation and then melting point of polyethylene, and EPR measurements were taken. Results showed that the polyenyl radical signal persisted below the Tm, but was rapidly eliminated upon melting of the crystals. These experiments support the hypothesis that the long-lived polyenyl radicals are trapped in the crystalline region of the polyethylene fibers.
Nanocarriers (NCs) help improve the performance of therapeutics, but their removal by phagocytes in the liver, spleen, tissues, etc. diminishes this potential. Although NC functionalization with polyethylene glycol (PEG) lowers interaction with phagocytes, it also reduces interactions with tissue cells. Coating NCs with CD47, a protein expressed by body cells to avoid phagocytic removal, offers an alternative. Previous studies showed that coating CD47 on non-targeted NCs reduces phagocytosis, but whether this alters binding and endocytosis of actively-targeted NCs remains unknown. To evaluate this, we used polymer NCs targeted to ICAM-1, a receptor overexpressed in many diseases. Co-coating of CD47 on anti-ICAM NCs reduced macrophage phagocytosis by ~50% up to 24 h, while increasing endothelial-cell targeting by ~87% over control anti-ICAM/IgG NCs. Anti-ICAM/CD47 NCs were endocytosed via the CAM-mediated pathway with efficiency similar to (0.99-fold) anti-ICAM/IgG NCs. Comparable outcomes were observed for NCs targeted to PECAM-1 or transferrin receptor, suggesting broad applicability. When injected in mice, anti-ICAM/CD47 NCs reduced liver and spleen uptake by ~30–50% and increased lung targeting by ~2-fold (~10-fold over IgG NCs). Therefore, co-coating NCs with CD47 and targeting moieties reduces macrophage phagocytosis and improves targeted uptake. This strategy may significantly improve the efficacy of targeted drug NCs.
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