Angiogenesis and airway reactivity in asthmatic Brown Norway rats.

Expanded and aberrant bronchial vascularity, a prominent feature of the chronic asthmatic airway, might explain persistent airway wall edema and sustained leukocyte recruitment. Since it is well established that there are causal relationships between exposure to house dust mite (HDM) and the development of asthma, determining the effects of HDM in rats, mammals with a bronchial vasculature similar to humans, provides an opportunity to study the effects of bronchial angiogenesis on airway function directly. We studied rats exposed bi-weekly to HDM (Der p 1; 50 µg/challenge by intranasal aspiration, 1, 2, 3 weeks) and measured the time course of appearance of increased blood vessels within the airway wall. Results demonstrated that within 3 weeks of HDM exposure, the number of vessels counted within airway walls of bronchial airways (0.5-3 mm perimeter) increased significantly. These vascular changes were accompanied by increased airway responsiveness to methacholine. A shorter exposure regimen (2 weeks of bi-weekly exposure) was insufficient to cause a significant increase in functional vessels or reactivity. Yet, 19F/1H MR imaging at 3T following αvß3-targeted perfluorocarbon nanoparticle infusion revealed a significant increase in 19F signal in rat airways after 2 weeks of bi-weekly HDM, suggesting earlier activation of the process of neovascularization. Although many antigen-induced mouse models exist, mice lack a bronchial vasculature and consequently lack the requisite human parallels to study bronchial edema. Overall, our results provide an important new model to study the impact of bronchial angiogenesis on chronic inflammation and airways hyperreactivity.

Molecular photoacoustic imaging of angiogenesis with integrin-targeted gold nanobeacons.

Photoacoustic tomography (PAT) combines optical and acoustic imaging to generate high-resolution images of microvasculature. Inherent sensitivity to hemoglobin permits PAT to image blood vessels but precludes discriminating neovascular from maturing microvasculature. α(v)ß(3)-Gold nanobeacons (α(v)ß(3)-GNBs) for neovascular molecular PAT were developed, characterized, and demonstrated in vivo using a mouse Matrigel-plug model of angiogenesis. PAT results were microscopically corroborated with fluorescent α(v)ß(3)-GNB localization and supporting immunohistology in Rag1(tm1Mom) Tg(Tie-2-lacZ)182-Sato mice. α(v)ß(3)-GNBs (154 nm) had 10-fold greater contrast than blood on an equivolume basis when imaged at 740 nm to 810 nm in blood. The lowest detectable concentration in buffer was 290 nM at 780 nm. Noninvasive PAT of angiogenesis using a 10-MHz ultrasound receiver with α(v)ß(3)-GNBs produced a 600% increase in signal in a Matrigel-plug mouse model relative to the inherent hemoglobin contrast pretreatment. In addition to increasing the contrast of neovessels detected at baseline, α(v)ß(3)-GNBs allowed visualization of numerous angiogenic sprouts and bridges that were undetectable before contrast injection. Competitive inhibition of α(v)ß(3)-GNBs with α(v)ß(3)-NBs (no gold particles) almost completely blocked contrast enhancement to pretreatment levels, similar to the signal from animals receiving saline only. Consistent with other studies, nontargeted GNBs passively accumulated in the tortuous neovascular but provided less than half of the contrast enhancement of the targeted agent. Microscopic studies revealed that the vascular constrained, rhodamine-labeled α(v)ß(3)-GNBs homed specifically to immature neovasculature (PECAM(+), Tie-2(-)) along the immediate tumor periphery, but not to nearby mature microvasculature (PECAM(+), Tie-2(+)). The combination of PAT and α(v)ß(3)-GNBs offered sensitive and specific discrimination and quantification of angiogenesis in vivo, which may be clinically applicable to a variety of highly prevalent diseases, including cancer and cardiovascular disease.

Lipid membrane editing with peptide cargo linkers in cells and synthetic nanostructures.

Current strategies for deploying synthetic nanocarriers involve the creation of agents that incorporate targeting ligands, imaging agents, and/or therapeutic drugs into particles as an integral part of the formulation process. Here we report the development of an amphipathic peptide linker that enables postformulation editing of payloads without the need for reformulation to achieve multiplexing capability for lipidic nanocarriers. To exemplify the flexibility of this peptide linker strategy, 3 applications were demonstrated: converting nontargeted nanoparticles into targeting vehicles; adding cargo to preformulated targeted nanoparticles for in vivo site-specific delivery; and labeling living cells for in vivo tracking. This strategy is expected to enhance the clinical application of molecular imaging and/or targeted therapeutic agents by offering extended flexibility for multiplexing targeting ligands and/or drug payloads that can be selected after base nanocarrier formulation.

Detecting vascular biosignatures with a colloidal, radio-opaque polymeric nanoparticle.

A synthetic methodology for developing a polymeric nanoparticle for targeted computed tomographic (CT) imaging is revealed in this manuscript. The work describes a new class of soft type, vascularly constrained, stable colloidal radio-opaque metal-entrapped polymeric nanoparticle using organically soluble radio-opaque elements encapsulated by synthetic amphiphile. This agent offers several-fold CT signal enhancement in vitro and in vivo demonstrating detection sensitivity reaching to the low nanomolar particulate concentration range.

Exploiting lipid raft transport with membrane targeted nanoparticles: a strategy for cytosolic drug delivery.

The ability to specifically deliver therapeutic agents to selected cell types while minimizing systemic toxicity is a principal goal of nanoparticle-based drug delivery approaches. Numerous cellular portals exist for cargo uptake and transport, but after targeting, intact nanoparticles typically are internalized via endocytosis prior to drug release. However, in this work, we show that certain classes of nanoparticles, namely lipid-coated liquid perfluorocarbon emulsions, undergo unique interactions with cells to deliver lipophilic substances to target cells without the need for entire nanoparticle internalization. To define the delivery mechanisms, fluorescently-labeled nanoparticles complexed with alphav beta 3-integrin targeting ligands were incubated with alphav beta 3-integrin expressing cells (C32 melanoma) under selected inhibitory conditions that revealed specific nanoparticle-to-cell interactions. We observed that the predominant mechanism of lipophilic delivery entailed direct delivery of lipophilic substances to the target cell plasma membrane via lipid mixing and subsequent intracellular trafficking through lipid raft-dependent processes. We suggest that local drug delivery to selected cell types could be facilitated by employing targeted nanoparticles designed specifically to utilize alternative membrane transport mechanisms.

Contact-facilitated drug delivery with Sn2 lipase labile prodrugs optimize targeted lipid nanoparticle drug delivery.

Sn2 lipase labile phospholipid prodrugs in conjunction with contact-facilitated drug delivery offer an important advancement in Nanomedicine. Many drugs incorporated into nanosystems, targeted or not, are substantially lost during circulation to the target. However, favorably altering the pharmacokinetics and volume of distribution of systemic drug delivery can offer greater efficacy with lower toxicity, leading to new prolonged-release nanoexcipients. However, the concept of achieving Paul Erhlich's inspired vision of a 'magic bullet' to treat disease has been largely unrealized due to unstable nanomedicines, nanosystems achieving low drug delivery to target cells, poor intracellular bioavailability of endocytosed nanoparticle payloads, and the substantial biological barriers of extravascular particle penetration into pathological sites. As shown here, Sn2 phospholipid prodrugs in conjunction with contact-facilitated drug delivery prevent premature drug diffusional loss during circulation and increase target cell bioavailability. The Sn2 phospholipid prodrug approach applies equally well for vascular constrained lipid-encapsulated particles and micelles the size of proteins that penetrate through naturally fenestrated endothelium in the bone marrow or thin-walled venules of an inflamed microcirculation. At one time Nanomedicine was considered a 'Grail Quest' by its loyal opposition and even many in the field adsorbing the pains of a long-learning curve about human biology and particles. However, Nanomedicine with innovations like Sn2 phospholipid prodrugs has finally made 'made the turn' toward meaningful translational success.

Novel paramagnetic contrast agents for molecular imaging and targeted drug delivery.

Molecular biology and genomic sciences are revealing the early biological signatures for many diseases. In response, the Molecular Imaging community is rapidly developing contrast agents to visualize the nascent pathological changes and to concomitantly deliver treatment directly to the site of disease. The evaluation, development and use of these new agents require a complementary understanding of contrast chemistry and imaging techniques. The fundamental issues surrounding magnetic contrast agent development, rational drug delivery, MR molecular imaging, and their interdependence are elucidated.

Improvements in the ultrasonic contrast of targeted perfluorocarbon nanoparticles using an acoustic transmission line model.

Targeted acoustic contrast agents offer the potential for sensitive ultrasonic detection of pathologic tissues. We have previously reported the development of a ligand-targeted, lipid-encapsulated, liquid perfluorodichlorooctane ultrasonic contrast system with a small nominal particle size (approximately 250-nm diameter). Perfluorocarbon nanoparticles substantially increase reflectivity when bound to targeted surfaces, and we propose that this system can be approximated physically as a simple, thin layer, acoustic transmission line. In this study, we evaluate this model and compare the ultrasonic reflectivity of different perfluorocarbon formulations with widely varying acoustic impedances targeted to either nitrocellulose membranes or plasma thrombi in vitro. Five perfluorocarbons were investigated: perfluorohexane (PFH), perfluorooctane (PFO), perfluorooctyl bromide (PFOB), perfluorodichlorooctane (PFDCO), and perfluorodecalin (PFD). Ultrasonic reflection was measured by acoustic microscopy (17 to 35 MHz). Acoustic reflectivity was increased (P < 0.05) by all targeted perfluorocarbon formulations, and the magnitude of the contrast effect was inversely correlated with the perfluorocarbon acoustic impedance. PFH nanoparticles exhibited the greatest enhancement, and PFD nanoparticles showed the least. The acoustic transmission line model predicted well the relative differences in acoustic reflectivity and frequency dependence among the perfluorocarbon formulations. For future clinical applications, PFO nanoparticles may provide the best combination of acoustic enhancement, in vivo physical stability, and safety.

Surface passivation of carbon nanoparticles with branched macromolecules influences near infrared bioimaging.

A superior and commercially exploitable 'green synthesis' of optically active carbon nanoparticle (OCN) is revealed in this work. The naked carbon particles (<20 nm) were derived from commercial food grade honey. The fluorescence properties of these particles were significantly enhanced by utilizing hyberbranched polymer for surface passivation. A dramatic increase in near infrared emission was achieved compared to a linear polymer (PEG) coated carbon nanoparticles. Interestingly, as passivating agent becomes more extensively branched (pseudo generation 2 to 4), the average radiant efficiency amplifies considerably as a direct result of the increasing surface area available for light passivation. The particles showed negligible loss of cell viability in presence of endothelial cells in vitro. Preliminary in vivo experiment showed high contrast enhancement in auxiliary lymphnode in a mouse model. The exceptionally rapid lymphatic transport of these particles suggests that such an approach may offer greater convenience and reduced procedural expense, as well as improved surgical advantage as the patient is positioned on the table for easier resection.

Nanotechnology in interventional cardiology.

High-grade atherosclerotic stenoses are reduced to zero or minimal residual stenosis grades by a single or a series of balloon angioplasties. Currently, stents are implanted to prevent immediate vascular recoil and elution of an antimitotic drug from the stent struts minimizes restenosis. An unwanted side-effect of this drug elution is delayed re-endothelialization which requires treatment with two anti-platelet drugs, in many cases for a minimum of 1 year to prevent acute in-stent thrombosis. Advances in stent design and drug elution technology, now in its fourth generation, have not abated this issue. Nanotechnology-based local drug delivery has the potential to achieve restenosis prevention while not impeding endothelial healing. Molecularly targeted drugs can be aimed to specifically bind to epitopes in the injured media and adventitia. Thus, endothelial healing may progress unhindered. To prevent restenosis, this technology may be used with bare metal or biodegradable stents. In this article novel nanoparticulate agents will be compared regarding their potential to deliver drugs to molecular targets within the vascular wall. Potential molecular targets, targeting mechanisms, drug-delivery propensities, and biocompatibility will be reviewed.

Molecularly targeted nanocarriers deliver the cytolytic peptide melittin specifically to tumor cells in mice, reducing tumor growth.

The in vivo application of cytolytic peptides for cancer therapeutics is hampered by toxicity, nonspecificity, and degradation. We previously developed a specific strategy to synthesize a nanoscale delivery vehicle for cytolytic peptides by incorporating the nonspecific amphipathic cytolytic peptide melittin into the outer lipid monolayer of a perfluorocarbon nanoparticle. Here, we have demonstrated that the favorable pharmacokinetics of this nanocarrier allows accumulation of melittin in murine tumors in vivo and a dramatic reduction in tumor growth without any apparent signs of toxicity. Furthermore, direct assays demonstrated that molecularly targeted nanocarriers selectively delivered melittin to multiple tumor targets, including endothelial and cancer cells, through a hemifusion mechanism. In cells, this hemifusion and transfer process did not disrupt the surface membrane but did trigger apoptosis and in animals caused regression of precancerous dysplastic lesions. Collectively, these data suggest that the ability to restrain the wide-spectrum lytic potential of a potent cytolytic peptide in a nanovehicle, combined with the flexibility of passive or active molecular targeting, represents an innovative molecular design for chemotherapy with broad-spectrum cytolytic peptides for the treatment of cancer at multiple stages.