ABSTRACT
Targeting of therapeutics or imaging agents to the endothelium has the potential to improve specificity and effectiveness of treatment for many diseases. One strategy to achieve this goal is the use of nanoparticles (NPs) targeted to the endothelium by ligands of protein determinants present on this tissue, including cell adhesion molecules, peptidases, and cell receptors. However, detachment of the radiolabel probes from NPs poses a significant problem. In this study, we devised polymeric NPs directly labeled with radioiodine isotopes including the positron emission tomography (PET) isotope (124)I, and characterized their targeting to specific endothelial determinants. This approach provided sizable, targetable probes for specific detection of endothelial surface determinants non-invasively in live animals. Direct conjugation of radiolabel to NPs allowed for stable longitudinal tracking of tissue distribution without label detachment even in an aggressive proteolytic environment. Further, this approach permits tracking of NP pharmacokinetics in real-time and non-invasive imaging of the lung in mice using micro-PET imaging. The use of this strategy will considerably improve investigation of NP interactions with target cells and PET imaging in small animals, which ultimately can aid in the optimization of targeted drug delivery.
Subject(s)
Drug Delivery Systems/methods , Endothelial Cells/diagnostic imaging , Nanoparticles , Polyvinyls , Positron-Emission Tomography/methods , Staining and Labeling , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Endothelial Cells/drug effects , Female , Iodine Radioisotopes , Lung/diagnostic imaging , Mice , Mice, Inbred C57BL , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Particle Size , Polyvinyls/chemical synthesis , Polyvinyls/chemistry , Time FactorsABSTRACT
Therapeutic proteins are prone to inactivation by aggregation, proteases and natural inhibitors, motivating development of protective delivery systems. Here we focus on protective encapsulation of the potent antioxidant enzyme, catalase, by filamentous polymer nanocarriers (f-PNC), with the specific goal of addressing whether polymer molecular weight (MW) controls formation and structural properties such as size and stiffness. While maintaining the same MW ratio of polyethylene glycol to polylactic acid, a series of PEG-b-PLA diblock copolymers were synthesized, with total MW ranging from about 10 kg/mol to 100 kg/mol. All diblocks formed f-PNC upon processing, which encapsulated active enzyme that proved resistant to protease degradation. Further, f-PNC stiffness, length, and thickness increased with increasing MW. Interestingly, heating above a polymer's glass transition temperature (<30 degrees C) increased f-PNC flexibility. Thus, we report here for the first time f-PNC that encapsulate an active enzyme with polymer MW-tunable flexibility, offering several potential therapeutic applications.
Subject(s)
Drug Carriers , Enzymes/chemistry , Lactic Acid/chemistry , Nanoparticles , Polyethylene Glycols/chemistry , Polymers/chemistry , Biocatalysis , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Polyesters , TemperatureABSTRACT
PURPOSE: Based on a unique phase alignment that occurs during formulation, we postulated that PEG-ylation of the cargo enzyme would enhance its encapsulation within diblock copolymer nanocarriers and thus resistance to proteases. METHODS: A freeze-thaw modified double emulsion technique was utilized to encapsulate either the catalytically active enzyme catalase (MW approximately 250 kDa) or PEG-catalase in PEG-PLA polymer nanocarriers (PNC). Spectrophotometer measurement of substrate depletion was utilized to monitor enzyme activity. Isotope labeling of the enzyme was used in conjunction with activity measurements to determine PNC loading efficiency and PNC-enzyme resistance to proteases. This labeling also enabled blood clearance measurements of PNC-loaded and non-loaded enzymes in mice. RESULTS: Non-loaded PEG-catalase exhibited longer circulation times than catalase, but was equally susceptible to proteolysis. Modulation of the ratio of relatively hydrophilic to hydrophobic domains in the diblock PEG-PLA copolymer provided either filamentous or spherical PNC loaded with PEG-catalase. For both PNC geometries, encapsulation and resistance to proteases of the resultant PNC-loaded enzyme were more effective for PEG-catalase than catalase. Isotope tracing showed similar blood levels of PNC-loaded and free PEG-catalase in mice. CONCLUSIONS: PEGylation enhances active catalase loading within PNC and resistance to protease degradation, relative to unloaded PEG-catalase.
Subject(s)
Catalase/chemistry , Catalase/pharmacokinetics , Nanoparticles/chemistry , Polyethylene Glycols/chemistry , Animals , Chemistry, Pharmaceutical , Diffusion , Drug Carriers/chemistry , Freezing , Hydrogen Peroxide/chemistry , Indicators and Reagents , Lactic Acid/chemistry , Mice , Mice, Inbred C57BL , Microspheres , Nanotubes , Particle Size , Peptide Hydrolases/chemistry , Polyesters , Polymers/chemistry , Tissue DistributionABSTRACT
The unique properties of synthetic nanostructures promise a diverse set of applications as carriers for drug delivery, which are advantageous in terms of biocompatibility, pharmacokinetics, targeting and controlled drug release. Historically, more traditional drug delivery systems have focused on spherical carriers. However, there is a growing interest in pursuing non-spherical carriers, such as elongated or filamentous morphologies, now available due to novel formulation strategies. Unique physiochemical properties of these supramolecular structures offer distinct advantages as drug delivery systems. In particular, results of recent studies in cell cultures and lab animals indicate that rational design of carriers of a given geometry (size and shape) offers an unprecedented control of their longevity in circulation and targeting to selected cellular and subcellular locations. This article reviews drug delivery aspects of non-spherical drug delivery systems, including material selection and formulation, drug loading and release, biocompatibility, circulation behavior, targeting and subcellular addressing.
Subject(s)
Drug Delivery Systems/methods , Drug Design , Polymers/chemistry , Chemistry, Pharmaceutical , Drug Compounding , Liposomes/chemistry , Micelles , Nanostructures/chemistryABSTRACT
Rapid clearance and proteolysis limit delivery and efficacy of protein therapeutics. Loading into biodegradable polymer nanocarriers (PNC) might protect proteins, extending therapeutic duration, but loading can be complicated by protein unfolding and inactivation. We encapsulated active enzymes into methoxy-poly(ethylene glycol- block-lactic acid) (mPEG-PLA) PNC with a freeze-thaw double emulsion ( J. Controlled Release 2005, 102 (2), 427-439). On the basis of concepts of amphiphile self-assembly, we hypothesized that the copolymer block ratio that controls spontaneous curvature would influence PNC morphology and loading. We examined PNC yield, shape, stability, loading, activity, and protease resistance of the antioxidant enzyme, catalase. PNC transitioned from spherical to filamentous shapes with increasing hydrophobic polymer fraction, consistent with trends for self-assembly of lower MW amphiphiles. Importantly, one diblock copolymer formed filamentous particles loaded with significant levels of protease-resistant enzyme, demonstrating for the first time encapsulation of an active therapeutic enzyme into filamentous carriers. PNC morphology also greatly influenced its degradation, offering a new means of controlled delivery.
