Zhiliang Cheng

Multifunctional Nanoparticles: Cost Versus Benefit of Adding Targeting and Imaging Capabilities

Nanoparticle-based drug delivery systems have been developed to improve the efficacy and reduce the systemic toxicity of a wide range of drugs. Although clinically approved nanoparticles have consistently shown value in reducing drug toxicity, their use has not always translated into improved clinical outcomes. This has led to the development of “multifunctional” nanoparticles, where additional capabilities like targeting and image contrast enhancement are added to the nanoparticles. However, additional functionality means additional synthetic steps and costs, more convoluted behavior and effects in vivo, and also greater regulatory hurdles. The trade-off between additional functionality and complexity is the subject of ongoing debate and the focus of this Review.

Gadolinium‐Conjugated Dendrimer Nanoclusters as a Tumor‐Targeted T1 Magnetic Resonance Imaging Contrast Agent

Contrast agents are increasingly being used in diagnostic magnetic resonance (MR) imaging to help detect and characterize pathological abnormalities. In fact, it has been estimated that nearly 50% of all MR examinations already involve the use of MR contrast agents, with chelated gadolinium compounds being by far the most widely used.[1,2] Most clinically relevant Gd-based agents are small, non-targeted compounds that passively distribute into the intravascular and interstitial space.[3] However, there has recently been emerging interest in the development of paramagnetic contrast agents that are capable of probing the molecular profile of tissues via ligand targeting, enzymatic activity and multiplexing.[4,5] It is envisioned that these agents could be used to acquire a more specific clinical diagnosis and thus improve patient management.

Gold-Loaded Polymeric Micelles for Computed Tomography-Guided Radiation Therapy Treatment and Radiosensitization

Gold nanoparticles (AuNPs) have generated interest as both imaging and therapeutic agents. AuNPs are attractive for imaging applications since they are nontoxic and provide nearly three times greater X-ray attenuation per unit weight than iodine. As therapeutic agents, AuNPs can sensitize tumor cells to ionizing radiation. To create a nanoplatform that could simultaneously exhibit long circulation times, achieve appreciable tumor accumulation, generate computed tomography (CT) image contrast, and serve as a radiosensitizer, gold-loaded polymeric micelles (GPMs) were prepared. Specifically, 1.9 nm AuNPs were encapsulated within the hydrophobic core of micelles formed with the amphiphilic diblock copolymer poly(ethylene glycol)-b-poly(ε-capralactone). GPMs were produced with low polydispersity and mean hydrodynamic diameters ranging from 25 to 150 nm. Following intravenous injection, GPMs provided blood pool contrast for up to 24 h and improved the delineation of tumor margins via CT. Thus, GPM-enhanced CT imaging was used to guide radiation therapy delivered via a small animal radiation research platform. In combination with the radiosensitizing capabilities of gold, tumor-bearing mice exhibited a 1.7-fold improvement in the median survival time, compared with mice receiving radiation alone. It is envisioned that translation of these capabilities to human cancer patients could guide and enhance the efficacy of radiation therapy.

Paramagnetic Porous Polymersomes

The ability of chelated Gd to serve as an effective magnetic resonance (MR) contrast agent largely depends on fast exchange rates between the Gd-bound water molecules and the surrounding bulk water. Because water diffuses slowly across lipid bilayers, liposomes with encapsulated chelated Gd have not been widely adopted as MR contrast agents. To overcome this limitation, we have synthesized chemically stabilized, porous polymersomes with encapsulated gadolinium (Gd) chelates. The polymerosmes, 125 nm in diameter, were produced from the aqueous assembly of diblock copolymers, PEO(1300)-b-PBD(2500) (PBdEO), and phospholipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The PBdEO was cross-linked using a chemical initiator and the POPC was extracted with surfactant, generating a highly porous outer membrane. The encapsulated Gd chelates were attached to dendrimers to prevent their leakage through the pores. It was estimated that, on average, nearly 44 000 Gd were encapsulated within each polymersome. As a result of the slower rotational correlation time of Gd-labeled dendrimers and the porous outer membrane, the paramagnetic porous polymersomes exhibited an R1 relaxivity of 7.2 mM−1 s1− per Gd and 315 637 mM−1 s−1 per vesicle. This corresponds to a relaxivity that is amplified by a factor of ∼105 compared with Gd−DTPA.

Improved Tumor Targeting of Polymer-based Nanovesicles Using Polymer-Lipid Blends

Block copolymer-based vesicles have recently garnered a great deal of interest as nanoplatforms for drug delivery and molecular imaging applications due to their unique structural properties. These nanovesicles have been shown to direct their cargo to disease sites either through enhanced permeability and retention or even more efficiently via active targeting. Here, we show that the efficacy of nanovesicle targeting can be significantly improved when prepared from polymer-lipid blends compared with block copolymer alone. Polymer-lipid hybrid nanovesicles were produced from the aqueous coassembly of the diblock copolymer, poly(ethylene oxide)-block-polybutadiene (PEO-PBD), and the phospholipid, hydrogenated soy phosphatidylcholine (HSPC). The PEG-based vesicles, 117 nm in diameter, were functionalized with either folic acid or anti-HER2/neu affibodies as targeting ligands to confer specificity for cancer cells. Our results revealed that nanovesicles prepared from polymer-lipid blends led to significant improvement in cell binding compared to nanovesicles prepared from block copolymer alone in both in vitro cell studies and murine tumor models. Therefore, it is envisioned that nanovesicles composed of polymer-lipid blends may constitute a preferred embodiment for targeted drug delivery and molecular imaging applications.

An intein-mediated site-specific click conjugation strategy for improved tumor targeting of nanoparticle systems.

The ability to modify and directly target nanoparticulate carriers has greatly increased their applicability in diagnostic and therapeutic studies. Generally essential to the targeting of nanoparticles is the bioconjugation of targeting ligands to the agents surface. While bioconjugation techniques have steadily improved in recent years, the field is still plagued with inefficient conjugations reactions and/or the lack of site-specific coupling. To overcome these limitations, click chemistry and expressed protein ligation (EPL) are combined to produce a highly efficient, site-specific reaction. This new EPL-click conjugation strategy is applied to create superparamagnetic iron oxide nanoparticles (SPIO) labeled with HER2/neu affibodies. These HER2-SPIO nanoparticles prove to be highly potent and receptor-specific in both in vitro cell studies and murine tumor models. Moreover, when EPL-click-derived HER2-SPIO are compared with SPIO that had been labeled with HER2 affibodies using other popular bioconjugation methods, they produce a statistically significant improvement in contrast enhancement upon cell binding. The EPL-click system is also successfully extended to other nanoparticle platforms (i.e., liposomes and dendrimers) highlighting the versatility of the approach.

A pH‐Responsive Drug‐Delivery Platform Based on Glycol Chitosan–Coated Liposomes

Currently, a substantial amount of effort is focused on developing actively targeted, receptor-specific nanoparticles for the delivery of anticancer drugs and diagnostic imaging contrast agents.[1–7] Receptor-targeted nanoparticles hold much promise and are often shown to provide nanoparticle delivery beyond that seen with the enhanced permeability and retention (EPR) effect alone. However, these nanoparticles still face considerable challenges as a result of the significant heterogeneity in receptor expression, not only between patients and tumor types, but also within individual tumors.[8] In many cases, the overexpressed receptor may also be present on normal tissues leading to detrimental off-target effects. Perhaps even more troubling is that several recent studies have shown that cancer stem cells may not even possess any known up-regulated receptor.[9] Therefore, targeting strategies that are more generalizable across a broad range of tumors than receptor-specific targeting are highly desirable.

Assessing the sensitivity of commercially available fluorophores to the intracellular environment.

The use of fluorescence has become commonplace in the biological sciences, with many studies utilizing probes based on commercially available fluorophores to provide insight into cell function and behavior. As these imaging applications become more advanced, it becomes increasingly important to acquire accurate quantitative measurements of the fluorescence signal. Absolute quantification of fluorescence, however, requires the fluorophores themselves to be insensitive to environmental factors such as nonspecific protein interactions and pH. Here, we present a method for characterizing the sensitivity of fluorophores to the cytosolic environment by comparing their fluorescent intensity to an environment-insensitive reference signal before and after intracellular delivery. Results indicated that although the fluorescent intensity of a few fluorophores, e.g., fluorescein, were highly susceptible to the intracellular environment, other fluorophores, e.g., Dylight 649, Alexa647, and Alexa750, were insensitive to the intracellular environment. It was also observed that the sensitivity of the fluorophore could be dependent on the biomolecule to which it was attached. In addition to assessing the environmental sensitivity of fluorophores, a method for quantifying the amount of fluorophores within living cells is also introduced. Overall, the present study provides a means to select fluorophores for studies that require an absolute quantification of fluorescence in the intracellular environment.

Nanometre-sized molecular oxygen sensors prepared from polymer stabilized phospholipid vesicles

Nanometre-sized, chemically-stabilized phospholipid vesicle sensors have been developed for detection of dissolved molecular oxygen. Sensors were prepared by forming 150 nm phospholipid vesicles from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or DOPC doped with small (<1%) mole percentages of 1,2-dioleoyl-sn-glycero-3-phosphoethanol amine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE). Sensors were stabilized via cross-linking polymerization of hydrophobic methacrylate monomers partitioned into the hydrophobic interior of the DOPC bilayer. The resultant unilamellar, nanometre-sized, polymer-lipid vesicles are spherical, biocompatible and protect sensing components that are loaded into the aqueous interior of the vesicle from interfering species in the exterior environment. For O(2) detection, the oxygen-sensitive fluorescent dye, tris(1,10-phenanthroline)ruthenium(II) chloride (Ru(phen)(3)) was encapsulated into the aqueous interior of the polymerized phospholipid vesicle. NBD-PE was introduced into the phospholipid bilayer of the sensor as a reference dye, allowing ratiometric sensors to be constructed. The resultant sensors show high sensitivity, excellent reversibility and excellent linearity over a physiological range of dissolved oxygen concentrations. These results suggest that polymerized phospholipid vesicle sensors can be used for monitoring intracellular O(2) dynamics.

Facile Method for the Site‐Specific, Covalent Attachment of Full‐Length IgG onto Nanoparticles

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled the crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzocyclooctyne-modified nanoparticles, via copper-free click chemistry.