"Eat me" imaging and therapy.

Clearance of apoptotic debris is a vital role of the innate immune system. Drawing upon principles of apoptotic clearance, convenient delivery vehicles including intrinsic anti-inflammatory characteristics and specificity to immune cells can be engineered to aid in drug delivery. In this article, we examine the use of phosphatidylserine (PtdSer), the well-known "eat-me" signal, in nanoparticle-based therapeutics making them highly desirable "meals" for phagocytic immune cells. Use of PtdSer facilitates engulfment of nanoparticles allowing for imaging and therapy in various pathologies and may result in immunomodulation. Furthermore, we discuss the targeting of the macrophages and other cells at sites of inflammation in disease. A thorough understanding of the immunobiology of "eat-me" signals is requisite for the successful application of "eat-me"-bearing materials in biomedical applications.

Hybrid nanoparticles improve targeting to inflammatory macrophages through phagocytic signals.

Macrophages are innate immune cells with great phenotypic plasticity, which allows them to regulate an array of physiological processes such as host defense, tissue repair, and lipid/lipoprotein metabolism. In this proof-of-principle study, we report that macrophages of the M1 inflammatory phenotype can be selectively targeted by model hybrid lipid-latex (LiLa) nanoparticles bearing phagocytic signals. We demonstrate a simple and robust route to fabricate nanoparticles and then show their efficacy through imaging and drug delivery in inflammatory disease models of atherosclerosis and obesity. Self-assembled LiLa nanoparticles can be modified with a variety of hydrophobic entities such as drug cargos, signaling lipids, and imaging reporters resulting in sub-100nm nanoparticles with low polydispersities. The optimized theranostic LiLa formulation with gadolinium, fluorescein and "eat-me" phagocytic signals (Gd-FITC-LiLa) a) demonstrates high relaxivity that improves magnetic resonance imaging (MRI) sensitivity, b) encapsulates hydrophobic drugs at up to 60% by weight, and c) selectively targets inflammatory M1 macrophages concomitant with controlled release of the payload of anti-inflammatory drug. The mechanism and kinetics of the payload discharge appeared to be phospholipase A2 activity-dependent, as determined by means of intracellular Förster resonance energy transfer (FRET). In vivo, LiLa targets M1 macrophages in a mouse model of atherosclerosis, allowing noninvasive imaging of atherosclerotic plaque by MRI. In the context of obesity, LiLa particles were selectively deposited to M1 macrophages within inflamed adipose tissue, as demonstrated by single-photon intravital imaging in mice. Collectively, our results suggest that phagocytic signals can preferentially target inflammatory macrophages in experimental models of atherosclerosis and obesity, thus opening the possibility of future clinical applications that diagnose/treat these conditions. Tunable LiLa nanoparticles reported here can serve as a model theranostic platform with application in various types of imaging of the diseases such as cardiovascular disorders, obesity, and cancer where macrophages play a pathogenic role.

Noninvasive detection of macrophages in atheroma using a radiocontrast-loaded phosphatidylserine-containing liposomal contrast agent for computed tomography.

PURPOSE: Macrophage plays an important role in plaque destabilization in atherosclerosis. By harnessing the affinity of macrophages to certain phospholipid species, a liposomal contrast agent containing phosphatidylserine (PS) and X-ray computed tomographic (CT) contrast agent was prepared and evaluated for CT imaging of plaque-associated macrophages in rabbit models of atherosclerosis. PROCEDURES: Liposomes containing PS and iodixanol were evaluated for their physicochemical characteristics, in vitro macrophage uptake, in vivo blood pool clearance, and organ distribution. Plaque enhancement in the aorta was imaged with CT in two atherosclerotic rabbit models. RESULTS: In vitro macrophage uptake of PS liposomes increased with increasing amount of PS in the liposomes. Overall clearance of PS liposomes was more rapid than control liposomes. Smaller PS liposomes (d = 112 ± 4 nm) were more effective than control liposomes of similar size or larger control and PS liposomes (d = 172 ± 17 nm) in enhancing aortic plaques in both rabbit models. CONCLUSIONS: Proper liposomal surface modification and appropriate sizing are important determinant for CT-based molecular imaging of macrophages in atheroma.

In vitro uptake of apoptotic body mimicking phosphatidylserine-quantum dot micelles by monocytic cell line.

A new quantum dot (QD) PEGylated micelle laced with phosphatidylserine (PS) for specific scavenger receptor-mediated uptake by macrophages is reported. The size and surface chemistry of PS-QD micelles were characterized by standard methods and the effects of their physicochemical properties on specific targeting and uptake were comprehensively studied in a monocytic cell line (J774A.1).

Quantum dots as a platform for nanoparticle drug delivery vehicle design.

Nanoparticle-based drug delivery (NDD) has emerged as a promising approach to improving upon the efficacy of existing drugs and enabling the development of new therapies. Proof-of-concept studies have demonstrated the potential for NDD systems to simultaneously achieve reduced drug toxicity, improved bio-availability, increased circulation times, controlled drug release, and targeting. However, clinical translation of NDD vehicles with the goal of treating particularly challenging diseases, such as cancer, will require a thorough understanding of how nanoparticle properties influence their fate in biological systems, especially in vivo. Consequently, a model system for systematic evaluation of all stages of NDD with high sensitivity, high resolution, and low cost is highly desirable. In theory, this system should maintain the properties and behavior of the original NDD vehicle, while providing mechanisms for monitoring intracellular and systemic nanocarrier distribution, degradation, drug release, and clearance. For such a model system, quantum dots (QDots) offer great potential. QDots feature small size and versatile surface chemistry, allowing their incorporation within virtually any NDD vehicle with minimal effect on overall characteristics, and offer superb optical properties for real-time monitoring of NDD vehicle transport and drug release at both cellular and systemic levels. Though the direct use of QDots for drug delivery remains questionable due to their potential long-term toxicity, the QDot core can be easily replaced with other organic drug carriers or more biocompatible inorganic contrast agents (such as gold and magnetic nanoparticles) by their similar size and surface properties, facilitating translation of well characterized NDD vehicles to the clinic, maintaining NDD imaging capabilities, and potentially providing additional therapeutic functionalities such as photothermal therapy and magneto-transfection. In this review we outline unique features that make QDots an ideal platform for nanocarrier design and discuss how this model has been applied to study NDD vehicle behavior for diverse drug delivery applications.

siRNA-aptamer chimeras on nanoparticles: preserving targeting functionality for effective gene silencing.

SiRNA-aptamer chimeras are emerging as a highly promising approach for cell-type specific delivery of siRNA due to the outstanding targeting capability of aptamers and the compatibility of chimeras with native ribonuclease (Dicer) processing. For efficient RNA interference (RNAi), however, additional challenges must be addressed, in particular how to get siRNA out of the endosome after cell entry and how to preserve aptamer targeting specificity when chimeras are combined with delivery carriers. Here, we report a rationally designed nanoparticle vector that simultaneously displays large surface area for high siRNA payload, exposed aptamer for specific targeting, proton sponge effect for endosome escape, and fluorescence for imaging and quantification. A key concept of this work is to graft chimeras onto nanoparticle surface via a two-step process: first immobilizing siRNA onto nanoparticle via noncovalent interactions to facilitate intracellular unpackaging and reduce nanoparticle surface charge (avoiding nonspecific electrostatic interactions between aptamers and nanoparticles) and then coupling siRNA and aptamer with retained conformation and high accessibility. Compared with conventional one-step adsorption of siRNA-aptamer chimeras onto nanoparticles with random orientations and conformations, which does not elicit much improved RNAi effect than nontargeted nanoparticle-siRNA complexes (∼6-8% improvement of the total cell population), under the same RNA concentration our approach shows selective gene silencing and enables 34% more silenced cells of the total cell population over nontargeted nanoparticle-siRNA complexes. This remarkable difference in RNAi efficiency using nanoparticle-chimera complexes is directly related to cell uptake discrepancy resulting from aptamer conformation on the nanoparticle surface (intact vs random).

A combined chemoimmunotherapy approach using a plasmid-doxorubicin complex.

We report a combined chemoimmunotherapy vehicle consisting of plasmid loaded with doxorubicin and evaluate its efficacy in two different tumor models. A stable complex was formed with a 1300:1 ratio of doxorubicin bound to native plasmid via intercalation. Pharmacokinetics of the complex showed much slower clearance from plasma up to 3 h compared to 10 min for free doxorubicin. In mice bearing NCI-H358 xenografts, lower doses of complex (doxorubicin 0.5 mg/kg, plasmid 4 mg/kg) effectively reduced tumor growth compared to high doses (5 mg/kg) of free doxorubicin (68% versus 77%). Similar results were observed in mice bearing 4T1 murine allografts; the complex (doxorubicin 2 mg/kg, plasmid 8 mg/kg) was effective and caused similar reduction of tumor compared to free doxorubicin (4 mg/kg) (47% versus 46%). The complex showed no signs of severe systemic toxicity or cardiotoxicity compared to the free doxorubicin in mice as indicated by body weights and heart tissue histology. Elevated levels of cytokines (IL-12, IL-6, and IFN-gamma) were observed in serum as well as in tumor tissue after intravenous injection of complex when compared to plasmid or doxorubicin alone. This approach simultaneously delivers both chemotherapeutic and immunotherapeutic agents without time delay, improves pharmacokinetics of the free drug, lowers drug toxicity, upregulates a variety of cytokines, and is effective against different tumors.

Quantum dot-aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on bi-fluorescence resonance energy transfer.

We report a novel quantum dot (QD)-aptamer(Apt)-doxorubicin (Dox) conjugate [QD-Apt(Dox)] as a targeted cancer imaging, therapy, and sensing system. By functionalizing the surface of fluorescent QD with the A10 RNA aptamer, which recognizes the extracellular domain of the prostate specific membrane antigen (PSMA), we developed a targeted QD imaging system (QD-Apt) that is capable of differential uptake and imaging of prostate cancer cells that express the PSMA protein. The intercalation of Dox, a widely used antineoplastic anthracycline drug with fluorescent properties, in the double-stranded stem of the A10 aptamer results in a targeted QD-Apt(Dox) conjugate with reversible self-quenching properties based on a Bi-FRET mechanism. A donor-acceptor model fluorescence resonance energy transfer (FRET) between QD and Dox and a donor-quencher model FRET between Dox and aptamer result when Dox intercalated within the A10 aptamer. This simple multifunctional nanoparticle system can deliver Dox to the targeted prostate cancer cells and sense the delivery of Dox by activating the fluorescence of QD, which concurrently images the cancer cells. We demonstrate the specificity and sensitivity of this nanoparticle conjugate as a cancer imaging, therapy and sensing system in vitro.