Abstract Ligand targeted therapy (LTT) is a precision medicine strategy that can selectively target diseased cells while minimizing off‐target effects on healthy cells. Integrin‐targeted LTT has been developed recently for angiogenesis‐related diseases. However, the clinical success is based on the optimal design of the nanoparticles for inducing receptor clustering within the cell membrane. The current study focused on determining the surface density of Ser‐Asp‐Val containing anti‐integrin heptapeptide on poly (ethylene glycol)‐b‐poly(propylene sulfide) micelles (MC) required for anti‐angiogenic effects on HUVECs. Varying peptide density on PEG‐b‐PPS/Pep‐PA MCs (Pep‐PA‐Peptide‐palmitoleic acid) was used in comparison to a random peptide (SGV) and cRGD (cyclic‐Arginine‐Glycine‐Aspartic acid) construct at 5%‐density on MCs. Immunocytochemistry using CD51/CD31 antibody was performed to study the integrin blocking by MCs. In addition, the expression of VWF and PECAM‐1, cell migration and tube formation was evaluated in the presence of PEG‐b‐PPS/Pep‐PA MCs. The results show PEG‐b‐PPS/SDV‐PA MCs with 5%‐peptide density to achieve significantly higher αvβ3 blocking compared to random peptide as well as cRGD. In addition, αvβ3 blocking via MCs further reduced the expression of vWF and PECAM‐1 angiogenesis protein expression in HUVECs. Although a significant level of integrin blocking was observed for 1%‐peptide density on MCs, the cell migration and tube formation were not significantly affected. In conclusion, the results of this study demonstrate that the peptide surface density on PEG‐b‐PPS/Pep‐PA MCs has a significant impact in integrin blocking as well as inhibiting angiogenesis during LTT. The outcomes of this study provides insight into the design of ligand targeted nanocarriers for various disease conditions.

Controlling nanocarrier interactions with the immune system requires a thorough understanding of the surface properties that modulate protein adsorption in biological fluids, since the resulting protein corona redefines cellular interactions with nanocarrier surfaces. Albumin is initially one of the dominant proteins to adsorb to nanocarrier surfaces, a process that is considered benign or beneficial by minimizing opsonization or inflammation. Here, we demonstrate the surface chemistry of a model nanocarrier can be engineered to stabilize or denature the three-dimensional conformation of adsorbed albumin, which respectively promotes evasion or non-specific clearance in vivo. Interestingly, certain common chemistries that have long been considered to convey stealth properties denature albumin to promote nanocarrier recognition by macrophage class A1 scavenger receptors, providing a means for their eventual removal from systemic circulation. We establish that the surface chemistry of nanocarriers can be specified to modulate adsorbed albumin structure and thereby tune clearance by macrophage scavenger receptors.

Primary open-angle glaucoma is associated with elevated intraocular pressure (IOP) that damages the optic nerve and leads to gradual vision loss. Several agents that reduce the stiffness of pressure-regulating Schlemm's canal (SC) endothelial cells, in the conventional outflow pathway of the eye, lower IOP in glaucoma patients and are approved for clinical use. However, poor drug penetration and uncontrolled biodistribution limit their efficacy and produce local adverse effects. Compared to other ocular endothelia, FLT4/VEGFR3 is expressed at elevated levels by SC endothelial cells and can be exploited for targeted drug delivery. Here, we validate FLT4 receptors as clinically relevant targets on SC cells from glaucomatous human donors and engineer polymeric self-assembled nanocarriers displaying lipid-anchored targeting ligands that optimally engage this receptor. Targeting constructs were synthesized as lipid-PEGx-peptide, differing in the number of PEG spacer units (x), and were embedded in micelles. We present a novel proteolysis assay for quantifying ligand accessibility that we employ to design and optimize our FLT4-targeting strategy for glaucoma nanotherapy. Peptide accessibility to proteases correlated with receptor-mediated targeting enhancements. Increasing the accessibility of FLT4-binding peptides enhanced nanocarrier uptake by SC cells while simultaneously decreasing the uptake by off-target vascular endothelial cells. Using a paired longitudinal IOP study in vivo, we show that this enhanced targeting of SC cells translates to IOP reductions that are sustained for a significantly longer time as compared to controls. Confocal microscopy of murine anterior segment tissue confirmed nanocarrier localization to SC within 1 h after intracameral administration. This work demonstrates that steric effects between surface-displayed ligands and PEG coronas significantly impact the targeting performance of synthetic nanocarriers across multiple biological scales. Minimizing the obstruction of modular targeting ligands by PEG measurably improved the efficacy of glaucoma nanotherapy and is an important consideration for engineering PEGylated nanocarriers for targeted drug delivery.

A significant barrier to the application of nanoparticles for precision medicine is the mononuclear phagocyte system (MPS), a diverse population of phagocytic cells primarily located within the liver, spleen and lymph nodes. The majority of nanoparticles are indiscriminately cleared by the MPS via macropinocytosis before reaching their intended targets, resulting in side effects and decreased efficacy. Here, we demonstrate that the biodistribution and desired tissue accumulation of targeted nanoparticles can be significantly enhanced by co-injection with polymeric micelles containing the actin depolymerizing agent latrunculin A. These macropinocytosis inhibitory nanoparticles (MiNP) were found to selectively inhibit non-specific uptake of a second "effector" nanoparticle in vitro without impeding receptor-mediated endocytosis. In tumor bearing mice, co-injection with MiNP in a single multi-nanoparticle formulation significantly increased the accumulation of folate-receptor targeted nanoparticles within tumors. Furthermore, subcutaneous co-administration with MiNP allowed effector nanoparticles to achieve serum levels that rivaled a standard intravenous injection. This effect was only observed if the effector nanoparticles were injected within 24 h following MiNP administration, indicating a temporary avoidance of MPS cells. Co-injection with MiNP therefore allows reversible evasion of the MPS for targeted nanoparticles and presents a previously unexplored method of modulating and improving nanoparticle biodistribution following subcutaneous administration.

Diabetic retinopathy (DR), Retinopathy of Pre-maturity (ROP), and Age-related Macular Degeneration (AMD) are multifactorial manifestations associated with abnormal growth of blood vessels in the retina. These three diseases account for 5% of the total blindness and vision impairment in the US alone. The current treatment options involve heavily invasive techniques such as frequent intravitreal administration of anti-VEGF (vascular endothelial growth factor) antibodies, which pose serious risks of endophthalmitis, retinal detachment and a multitude of adverse effects stemming from the diverse physiological processes that involve VEGF. To overcome these limitations, this current study utilizes a micellar delivery vehicle (MC) decorated with an anti-angiogenic peptide (aANGP) that inhibits αvβ3 mediated neovascularization using primary endothelial cells (HUVEC). Stable incorporation of the peptide into the micelles (aANGP-MCs) for high valency surface display was achieved with a lipidated peptide construct. After 24 h of treatment, aANGP-MCs showed significantly higher inhibition of proliferation and migration compared to free from aANGP peptide. A tube formation assay clearly demonstrated a dose-dependent angiogenic inhibitory effect of aANGP-MCs with a maximum inhibition at 4 μg/mL, a 1000-fold lower concentration than that required for free from aANGP to display a biological effect. These results demonstrate valency-dependent enhancement in the therapeutic efficacy of a bioactive peptide following conjugation to nanoparticle surfaces and present a possible treatment alternative to anti-VEGF antibody therapy with decreased side effects and more versatile options for controlled delivery.

Increased stiffness of the Schlemm's canal (SC) endothelium in the aqueous humor outflow pathways has been associated with elevated intraocular pressure (IOP) in glaucoma. Novel treatments that relax this endothelium, such as actin depolymerizers and rho kinase inhibitors, are in development. Unfortunately, these treatments have undesirable off-target effects and a lower than desired potency. To address these issues, a targeted PEG-b-PPS micelle loaded with actin depolymerizer latrunculin A (tLatA-MC) is developed. Targeting of SC cells is achieved by modifying the micelle surface with a high affinity peptide that binds the VEGFR3/FLT4 receptor, a lymphatic lineage marker found to be highly expressed by SC cells relative to other ocular cells. During in vitro optimization, increasing the peptide surface density increased micellar uptake in SC cells while unexpectedly decreasing uptake by human umbilical vein endothelial cells (HUVEC). The functional efficacy of tLatA-MC, as measured by decreased SC cell stiffness compared to non-targeted micelles (ntLatA-MC) or targeted blank micelles (tBL-MC), is verified using atomic force microscopy. tLatA-MC reduced IOP in an in vivo mouse model by 30-50%. The results validate the use of a cell-softening nanotherapy to selectively modulate stiffness of SC cells for therapeutic reduction of IOP and treatment of glaucoma.

Increased stiffness of Schlemm's canal endothelial cells (SC cells) is a major contributing factor to the increased pressure characteristic of primary open-angle glaucoma. New treatments for glaucoma are being developed using actin depolymerizers and rho kinase inhibitors to address this increased stiffness. However, these agents have off-target effects and are not as potent as had been hoped. We have developed a micellar nanocarrier assembled from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers capable of encapsulating latrunculin A (Lat A) with the goal of modulating SC cell stiffness. Lat A-loaded nanocarriers were similar in size and morphology to unloaded poly (ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) micelles, loaded Lat A at 62% encapsulation efficiency, and retained loaded Lat A for at least 22 days. The continued functional activity of Lat A following encapsulation within micelles was verified in murine macrophages, which are known to display decreased endocytosis in response to Lat A-dependent cytoskeletal disruption. Endocytic inhibition remained unchanged when comparing equal concentrations of micelle-loaded versus free form Lat A. Uptake of Lat A-loaded micelles by human SC cells was verified in vitro with no sign of cytotoxicity, and modulation of SC cell stiffness was measured by atomic force microscopy. Lat A-loaded micelles significantly decreased SC cell stiffness, which resulted in visible changes in cell morphology as observed by confocal microscopy. Our results demonstrate that PEG-bl-PPS micelles represent a tunable platform for the controlled intracellular delivery of latrunculin. These self-assembled polymeric nanobiomaterials may support the rational design and engineering of delivery systems for the treatment of glaucoma. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1771-1779, 2018.