Wolf Wrasidlo

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 MARCOs 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.

Magnetic resonance and fluorescence imaging of doxorubicin-loaded nanoparticles using a novel in vivo model

We report here the in vivo combined-modality imaging of multifunctional drug delivery nanoparticles. These dextran core-based stealth liposomal nanoparticles (nanosomes) contained doxorubicin, iron oxide for magnetic resonance imaging (MRI) contrast, and BODIPY for fluorescence. The particles were long-lived in vivo because of surface decoration with polyethylene glycol and the incorporation of acetylated lipids that were ultraviolet cross-linked for physical stability. We developed a rodent dorsal skinfold window chamber that facilitated both MRI and non-invasive optical imaging of nanoparticle accumulation in the same tumors. Chamber tumors were genetically labeled with DsRed-2, which enabled co-localization of the MR images, the red fluorescence of the tumor, and the blue fluorescence of the nanoparticles. The nanoparticle design and MR imaging developed with the window chamber were then extended to orthotopic pancreatic tumors expressing DsRed-2. The tumors were MR-imaged using iron oxide-dextran liposomes and by fluorescence to demonstrate the deep imaging capability of these nanoparticles.

Assembly and targeting of liposomal nanoparticles encapsulating quantum dots.

Quantum dots (QDs) are attracting intense interest as fluorescence labeling agents for biomedical imaging because biocompatible coatings and relatively nontoxic rare earth metal QDs have emerged as possible options. QD photoemissions are bright, of narrow wavelength range, and very stable. We sought to encapsulate QDs within targeted PEGylated liposomes to reduce their propensity for liver uptake and to amplify the already strong QD emission signal. A novel lipid-QD conjugate initialized a process by which lipids in solution coalesced around the QDs. The liposomal structure was confirmed with size measurements, SEM, and IR spectroscopy. PEGylated QD liposomes injected into a xenograft tumor model largely cleared from the body within 24 h. Residual liver labeling was low. Targeted QD liposomes exhibited robust tumor labeling compared with controls. This study highlights the potential of these near IR emitting QD liposomes for preclinical/clinical applications.

Original ArticleMagnetic resonance and fluorescence imaging of doxorubicin-loaded nanoparticles using a novel in vivo model

We report here the in vivo combined-modality imaging of multifunctional drug delivery nanoparticles. These dextran core-based stealth liposomal nanoparticles (nanosomes) contained doxorubicin, iron oxide for magnetic resonance imaging (MRI) contrast, and BODIPY for fluorescence. The particles were long-lived in vivo because of surface decoration with polyethylene glycol and the incorporation of acetylated lipids that were ultraviolet cross-linked for physical stability. We developed a rodent dorsal skinfold window chamber that facilitated both MRI and non-invasive optical imaging of nanoparticle accumulation in the same tumors. Chamber tumors were genetically labeled with DsRed-2, which enabled co-localization of the MR images, the red fluorescence of the tumor, and the blue fluorescence of the nanoparticles. The nanoparticle design and MR imaging developed with the window chamber were then extended to orthotopic pancreatic tumors expressing DsRed-2. The tumors were MR-imaged using iron oxide-dextran liposomes and by fluorescence to demonstrate the deep imaging capability of these nanoparticles.

High-efficiency liposomal encapsulation of a tyrosine kinase inhibitor leads to improved in vivo toxicity and tumor response profile

Staurosporine (STS) is a potent pan-kinase inhibitor with marked activity against several chemotherapy-resistant tumor types in vitro. The translational progress of this compound has been hindered by poor pharmacokinetics and toxicity. We sought to determine whether liposomal encapsulation of STS would enhance antitumor efficacy and reduce toxicity, thereby supporting the feasibility of further preclinical development. We developed a novel reverse pH gradient liposomal loading method for STS, with an optimal buffer type and drug-to-lipid ratio. Our approach produced 70% loading efficiency with good retention, and we provide, for the first time, an assessment of the in vivo antitumor activity of STS. A low intravenous dose (0.8 mg/kg) inhibited U87 tumors in a murine flank model. Biodistribution showed preferential tumor accumulation, and body weight data, a sensitive index of STS toxicity, was unaffected by liposomal STS, but did decline with the free compound. In vitro experiments revealed that liposomal STS blocked Akt phosphorylation, induced poly(ADP-ribose) polymerase cleavage, and produced cell death via apoptosis. This study provides a basis to explore further the feasibility of liposomally encapsulated STS, and potentially related compounds for the management of resistant solid tumors.

Novel therapeutic strategy for neurodegeneration by blocking Aβ seeding mediated aggregation in models of Alzheimer"s disease

Aβ accumulation plays a central role in the pathogenesis of Alzheimers disease (AD). Recent studies suggest that the process of Aβ nucleated polymerization is essential for Aβ fibril formation, pathology spreading and toxicity. Therefore, targeting this process represents an effective therapeutic strategy to slow or block disease progression. To discover compounds that might interfere with the Aβ seeding capacity, toxicity and pathology spreading, we screened a focused library of FDA-approved drugs in vitro using a seeding polymerization assay and identified small molecule inhibitors that specifically interfered with Aβ seeding-mediated fibril growth and toxicity. Mitoxantrone, bithionol and hexachlorophene were found to be the strongest inhibitors of fibril growth and protected primary cortical neuronal cultures against Aβ-induced toxicity. Next, we assessed the effects of these three inhibitors in vivo in the mThy1-APPtg mouse model of AD (8-month-old mice). We found that mitoxantrone and bithionol, but not hexachlorophene, stabilized diffuse amyloid plaques, reduced the levels of Aβ42 oligomers and ameliorated synapse loss, neuronal damage and astrogliosis. Together, our findings suggest that targeting fibril growth and Aβ seeding capacity constitutes a viable and effective strategy for protecting against neurodegeneration and disease progression in AD.

Molecular Determinants of α-Synuclein Mutants’ Oligomerization and Membrane Interactions

Parkinsons disease (PD) is associated with the formation of toxic α-synuclein oligomers that can penetrate the cell membrane. Familial forms of PD are caused by the point mutations A53T, A30P, E46K, and H50Q. Artificial point mutations E35K and E57K also increase oligomerization and pore formation. We generated structural conformations of α-synuclein and the above-mentioned mutants using molecular dynamics. We elucidated four main regions in these conformers contacting the membrane and found that the region including residues 39-45 (Zone2) may have maximum membrane penetration. E57K mutant had the highest rate of interaction with the membrane, followed by A53T, E46K, and E35K mutants and wild type (wt) α-synuclein. The mutant A30P had the smallest percentage of conformers that contact the membrane by Zone 2 than all other mutants and wt α-synuclein. These results were confirmed experimentally in vitro. We identified the key amino acids that can interact with the membrane (Y38, E62, and N65 (first hydrophilic layer); E104, E105, and D115 (second hydrophilic layer), and V15 and V26 (central hydrophobic layer)) and the residues that are involved in the interprotein contacts (L38, V48, V49, Q62, and T64). Understanding the molecular interactions of α-synuclein mutants is important for the design of compounds blocking the formation of toxic oligomers.

Localized In Vivo Activation of a Photoactivatable Doxorubicin Prodrug in Deep Tumor Tissue

Sparing sensitive healthy tissue from chemotherapy exposure is a critical challenge in the treatment of cancer. The work described here demonstrates the localized in vivo photoactivation of a new chemotherapy prodrug of doxorubicin (DOX). The DOX prodrug (DOX‐PCB) was 200 times less toxic than DOX and was designed to release pure DOX when exposed to 365 nm light. This wavelength was chosen because it had good tissue penetration through a 1 cm diameter tumor, but had very low skin penetration, due to melanin absorption, preventing uncontrolled activation from outside sources. The light was delivered specifically to the tumor tissue using a specialized fiber‐optic LED system. Pharmacokinetic studies showed that DOX‐PCB had an α circulation half‐life of 10 min which was comparable to that of DOX at 20 min. DOX‐PCB demonstrated resistance to metabolic cleavage ensuring that exposure to 365 nm light was the main mode of in vivo activation. Tissue extractions from tumors exposed to 365 nm light in vivo showed the presence of DOX‐PCB as well as activated DOX. The exposed tumors had six times more DOX concentration than nearby unexposed control tumors. This in vivo proof of concept demonstrates the first preferential activation of a photocleavable prodrug in deep tumor tissue.

Neuroprotective effects of the anti-cancer drug sunitinib in models of HIV neurotoxicity suggests potential for the treatment of neurodegenerative disorders

Anti‐retrovirals have improved and extended the life expectancy of patients with HIV. However, as this population ages, the prevalence of cognitive changes is increasing. Aberrant activation of kinases, such as receptor tyrosine kinases (RTKs) and cyclin‐dependent kinase 5 (CDK5), play a role in the mechanisms of HIV neurotoxicity. Inhibitors of CDK5, such as roscovitine, have neuroprotective effects; however, CNS penetration is low. Interestingly, tyrosine kinase inhibitors (TKIs) display some CDK inhibitory activity and ability to cross the blood–brain barrier.

Extraction Protocol and Mass Spectrometry Method for Quantification of Doxorubicin Released Locally from Prodrugs in Tumor Tissue

The localized conversion of inactive doxorubicin prodrug chemotherapeutics to pharmacalogically active forms is difficult to quantify in mouse tumor models because it occurs only in small regions of tissue. The tumor tissue extraction protocol and LC-MS/MS analysis method described here were optimized to obtain a detection limit of 7.8 pg for the activated doxorubicin and 0.36 ng for the doxorubicin prodrug. This method can be useful for determining the biodistribution and activation efficiency for many different doxorubicin prodrugs. It can also be used for quantification of doxorubicin from tumor models that have poor vascularization resulting in low tissue accumulation.