Clot-Targeted Micellar Formulation Improves Anticoagulation Efficacy of Bivalirudin

Application of anticoagulants remains the primary strategy for prevention and treatment of thrombosis. However, high rate of bleeding complications limits their use. The peptide anticoagulant bivalirudin has been reported to exhibit a lower rate of bleeding complications than heparin, and it also has the advantage of not causing thrombocytopenia, which is a problem with heparin. Nonetheless, hemorrhage is the most common complication of bivalirudin therapy, and there is no effective antidote. Here we use a thrombus-binding peptide, CR(<i>N</i>Me)EKA, to accomplish selective delivery of the bivalirudin-carrying micellar nanocarrier to sites of thrombosis. Bivalirudin and CR(<i>N</i>Me)EKA, each with a PEG-lipid tail, spontaneously assembled into 30 nm micelles, which almost completely retained the anticoagulant activity of bivalirudin. The micellar formulations exhibited high stability both <i>in vitro</i> and <i>in vivo</i>. In a thromboplastin-induced mouse thrombosis model, the targeted micelles accumulated in lung thrombi 10-fold more than nontargeted micelles. Moreover, the micellar formulation significantly prolonged the half-life and thereby increased the bioavailability of bivalirudin. The micellar bivalirudin had significantly higher anticoagulant activity than free bivalirudin in both the lung thrombosis model and a ferric chloride-induced carotid artery thrombosis model. The specific targeting of thrombi demonstrated here makes it possible to increase the efficacy of bivalirudin as an anticoagulant. Alternatively, the dose could be reduced without loss of efficacy to lower the systemic exposure and improve safety

Tumor-Penetrating Nanosystem Strongly Suppresses Breast Tumor Growth

Antiangiogenic and vascular disrupting compounds have shown promise in cancer therapy, but tend to be only partially effective. We previously reported a potent theranostic nanosystem that was highly effective in glioblastoma and breast cancer mouse models, retarding tumor growth and producing some cures [Agemy, L. et al. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 17450−17455. Agemy, L. et al. Mol. Ther. 2013, 21, 2195–2204.]. The nanosystem consists of iron oxide NPs (“nanoworms”) coated with a composite peptide with tumor-homing and pro-apoptotic domains. The homing component targets tumor vessels by binding to p32/gC1qR at the surface or tumor endothelial cells. We sought to further improve the efficacy nanosystem by searching for an optimally effective homing peptide that would also incorporate a tumor-penetrating function. To this effect, we tested a panel of candidate p32 binding peptides with a sequence motif that conveys tumor-penetrating activity (CendR motif). We identified a peptide designated as Linear TT1 (Lin TT1) (sequence: AKR­GAR­STA) as most effective in causing tumor homing and penetration of the nanosystem. This peptide had the lowest affinity for p32 among the peptides tested. The low affinity may have moderated the avidity effect from the multivalent presentation on nanoparticles (NPs), such that the NPs avoid getting trapped by the so-called “binding-site barrier”, which can hinder tissue penetration of compounds with a high affinity for their receptors. Treatment of breast cancer mice with the LinTT1 nanosystem showed greatly improved efficacy compared to the original system. These results identify a promising treatment modality and underscore the value of tumor penetration effect in improving the efficacy tumor treatment

Composite Porous Silicon–Silver Nanoparticles as Theranostic Antibacterial Agents

A theranostic nanoparticle with biochemically triggered antibacterial activity is demonstrated. Metallic silver is deposited onto porous silicon nanoparticles (pSiNPs) by galvanic displacement. When aqueous diaminesilver ([Ag­(NH₃)₂]⁺) is used as a silver source, the pSiNPs template the crystalline silver as small (mean diameter 13 nm) and well-dispersed nanoparticles embedded within and on the larger (100 nm) pSiNPs. The silver nanoparticles (AgNPs) quench intrinsic photoluminescence (PL) from the porous silicon (pSi) matrix. When exposed to an aqueous oxidant, the AgNPs are preferentially etched, Ag⁺ is released into solution, and PL from the pSi carrier is recovered. The released Ag⁺ results in 90% killing of (Gram-negative) <i>Pseudomonas aeruginosa</i> and (Gram-positive) <i>Staphylococcus aureus</i> within 3 h. When conjugated with the TAT peptide (sequence RKKRRQRRR), the silver-deposited porous silicon (pSi-Ag) nanocomposite shows distinct targeting toward <i>S. aureus</i> bacteria in vitro. Intravenously injected TAT-conjugated pSi-Ag nanoparticles accumulate in the liver, spleen, and lungs of mice, and the in vivo release of Ag⁺ and recovery of PL from pSi are demonstrated by the subsequent intraperitoneal administration of a hexacyanoferrate solution. The released Ag⁺ leads to a significant bacterial count reduction in liver tissue relative to the control. The data demonstrate the feasibility of the targeted and triggered delivery of antibacterial Ag⁺ ion in vivo, using a self-reporting and nontoxic nanocarrier

Different Effect of Hydrogelation on Antifouling and Circulation Properties of Dextran–Iron Oxide Nanoparticles

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this “stealth” effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by cross-linking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages <i>in vivo</i>. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles’ long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles

⁶⁴Cu-Labeled LyP‑1-Dendrimer for PET-CT Imaging of Atherosclerotic Plaque

The ability to detect and quantify macrophage accumulation can provide important diagnostic and prognostic information for atherosclerotic plaque. We have previously shown that LyP-1, a cyclic 9-amino acid peptide, binds to p32 proteins on activated macrophages, facilitating the visualization of atherosclerotic plaque with PET. Yet, the in vivo plaque accumulation of monomeric [¹⁸F]­FBA-LyP-1 was low (0.31 ± 0.05%ID/g). To increase the avidity of LyP-1 constructs to p32, we synthesized a dendritic form of LyP-1 on solid phase using lysine as the core structural element. Imaging probes (FAM or 6-BAT) were conjugated to a lysine or cysteine on the dendrimer for optical and PET studies. The N-terminus of the dendrimer was further modified with an aminooxy group in order to conjugate LyP-1 and ARAL peptides bearing a ketone. Oxime ligation of peptides to both dendrimers resulted in (LyP-1)₄- and (ARAL)₄-dendrimers with optical (FAM) and PET probes (6-BAT). For PET-CT studies, (LyP-1)₄- and (ARAL)₄-dendrimer-6-BAT were labeled with ⁶⁴Cu (<i>t</i>_1/2 = 12.7 h) and intravenously injected into the atherosclerotic (ApoE^–/–) mice. After two hours of circulation, PET-CT coregistered images demonstrated greater uptake of the (LyP-1)₄-dendrimer-⁶⁴Cu than the (ARAL)₄-dendrimer-⁶⁴Cu in the aortic root and descending aorta. Ex vivo images and the biodistribution acquired at three hours after injection also demonstrated a significantly higher uptake of the (LyP-1)₄-dendrimer-⁶⁴Cu (1.1 ± 0.26%ID/g) than the (ARAL)₄-dendrimer-⁶⁴Cu (0.22 ± 0.05%ID/g) in the aorta. Similarly, subcutaneous injection of the LyP-1-dendrimeric carriers resulted in preferential accumulation in plaque-containing regions over 24 h. In the same model system, ex vivo fluorescence images within aortic plaque depict an increased accumulation and penetration of the (LyP-1)₄-dendrimer-FAM as compared to the (ARAL)₄-dendrimer-FAM. Taken together, the results suggest that the (LyP-1)₄-dendrimer can be applied for in vivo PET imaging of plaque and that LyP-1 could be further exploited for the delivery of therapeutics with multivalent carriers or nanoparticles

Different Effect of Hydrogelation on Antifouling and Circulation Properties of Dextran–Iron Oxide Nanoparticles

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this “stealth” effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by cross-linking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages <i>in vivo</i>. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles’ long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles

Different Effect of Hydrogelation on Antifouling and Circulation Properties of Dextran–Iron Oxide Nanoparticles

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this “stealth” effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by cross-linking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages <i>in vivo</i>. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles’ long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles

Different Effect of Hydrogelation on Antifouling and Circulation Properties of Dextran–Iron Oxide Nanoparticles

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this “stealth” effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by cross-linking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages <i>in vivo</i>. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles’ long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles

Recognition of Dextran–Superparamagnetic Iron Oxide Nanoparticle Conjugates (Feridex) via Macrophage Scavenger Receptor Charged Domains

Dextran-coated superparamagnetic iron oxide nanoparticles (dextran–SPIO conjugates) offer the attractive possibility of enhancing MRI imaging sensitivity so that small or diffuse lesions can be detected. However, systemically injected SPIOs are rapidly removed by macrophages. We engineered embryonic cells (HEK293T) to express major macrophage scavenger receptor (SR) subtypes including SR-AI, MARCO, and endothelial receptor collectin-12. These SRs possess a positively charged collagen-like (CL) domain and they promoted SPIO uptake, while the charge neutral lipoprotein receptor SR-BI did not. In silico modeling indicated a positive net charge on the CL domain and a net negative charge on the cysteine-rich (CR) domain of MARCO and SR-AI. In vitro experiments revealed that CR domain deletion in SR-AI boosted uptake of SPIO 3-fold, while deletion of MARCO’s CR domain abolished this uptake. These data suggest that future studies might productively focus on the validation and further exploration of SR charge fields in SPIO recognition