Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

<p>Abstract</p> <p>Background</p> <p>Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-<it>bl</it>-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA.</p> <p>Results</p> <p>Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R₂negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature.</p> <p>Conclusions</p> <p>This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques.</p

Polymeric synthetic nanoparticles for the induction of antigen-specific immunological tolerance

Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.Juvenile Diabetes Research Foundation Internationa

Superparamagnetic Nanoparticles as a Powerful Systems Biology. Characterization Tool in the Physiological Context

Recently, functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have been utilized for protein separation and therapeutic delivery of DNA and drugs. The development of new methods and tools for the targeting and identification of specific biomolecular interactions within living systems is of great interest in the fields of systems biology, target and drug identification, drug delivery, and diagnostics. Magnetic separation of organelles and proteins from complex whole-cell lysates allows enrichment and elucidation of intracellular interaction partners for a specific immobilized protein or peptide on the surface of SPIONs

Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage

The extracellular matrix of dense, avascular tissues presents a barrier to entry for polymer-based therapeutics, such as drugs encapsulated within polymeric particles. Here, we present an approach by which polymer nanoparticles, suffciently small to enter the matrix of the targeted tissue, here articular cartilage, are further modified with a biomolecular ligand for matrix binding. This combination of ultrasmall size and biomolecular binding converts the matrix from a barrier into a reservoir, resisting rapid release of the nanoparticles and clearance from the tissue site. Phage display of a peptide library was used to discover appropriate targeting ligands by biopanning on denuded cartilage. The ligand WYRGRL was selected in 94 of 96 clones sequenced after five rounds of biopanning and was demonstrated to bind to collagen II alpha 1. Peptide-functionalized nanoparticles targeted articular cartilage up to 72-fold more than nanoparticles displaying a scrambled peptide sequence following intra-articular injection in the mouse

A Novel Method for the Encapsulation of Biomolecules into Polymersomes via Direct Hydration

One of the major engineering challenges for the implementation of block copolymer vesicles, or polymersomes, as therapeutic drug carriers is obtaining high encapsulation efficiencies for biomolecules. Here we present it novel method for encapsulation of proteins with high encapsulation efficiency within polymersomes formed from block copolymers of poly(ethylene glycol)-bl-poly(propylene sulfide). By formulation of the neat block copolymer with a low molecular weight poly(ethylene glycol), direct hydration of the formulated mixture yielded polymersomes. We were able to achieve encapsulation efficiencies for ovalbumin at 37%. bovine serum albumin at 19%, and bovine gamma-globulin at 15% when the proteins were included in the hydration solution. The formulation process and the dispersion of polymersomes front the preparation in phosphate-buffered saline were characterized using confocal microscopy, cryogenic transmission electron microscopy, and fluorimetry. We were also successful in the encapsulation of proteinase K, a proteolytic enzyme, and demonstrated by SDS-PAGE that the enzyme was contained inside polymersomes when dispersed ill a solution of ovalbumin

Precision Intracellular Delivery Based on Optofluidic Polymersome Rupture

We present an optical approach for intracellular delivery of molecules contained within oxidation-sensitive polymersomes. The photosensitizer ethyl eosin is associated with the polymersome membrane to oxidatively increase the hydrophilicity of the hydrophobic block under optical excitation. This optofluidic interaction induces rapid polymersome rupture and payload release via the reorganization of the aggregate structure into smaller diameter vesicles and micelles. When the particles are endocytosed by phagocytes, such as RAW macrophages and dendritic cells, the polymersomes' payload escapes the endosome and is released In the cell cytosol within a few seconds of illumination. The released payload is rapidly distributed throughout the cytosol within milliseconds. The presented optofluidic method enables fast delivery and distribution throughout the cytosol of individual cells, comparable to photochemical internalization, but a factor of 100 faster than similar carrier mediated delivery methods (e.g., liposomes, polymersomes, or nanoparticles). Due to the ability to simultaneously induce payload delivery and endosomal escape, this approach can find applications in detailed characterizations of intra- and intercellular processes. As an example in quantitative cell biology, a peptide antigen was delivered in dendritic cells and MHC I presentation kinetics were measured at the single cell and single complex level

Synthesis of Pyridyl Disulfide-Functionalized Nanoparticles for Conjugating Thiol-Containing Small Molecules, Peptides, and Proteins

Previously we reported emulsion polymerization of propylene sulfide with Pluronic F127 as an emulsifier, yielding nanoparticles (NPs) in the 25 nm size range. Immunologically functional NPs were prepared by adding an antigen Pluronic conjugate to the polymerization mixture (Reddy, S. T., et al. (2007) Nat. Biotechnol. 25, 1159). We sought a more flexible scheme for conjugation of antigens and other biomolecules to the NP surfaces that would allow for milder reaction conditions than achievable during the polymerization step. Here, we present the synthesis of such functionalizable NPs in the form of NPs that carry thiol-reactive groups, to which thiol-containing antigens (peptide or protein) or other biomolecules can be conjugated under mild conditions to yield immunofunctional NPs. The Pluronic-stabilized poly(propylene sulfide) (PPS) NPs with thiol-reactive pyridyl disulfide groups are prepared in two steps by (1) emulsion polymerization of propylene sulfide in the presence of a carboxylate-Pluronic and (2) reaction of the carboxylic acid groups on the NP surface with cysteaminc pyridyl disulfide and a water-soluble carbodiimide reagent. We choose pyridyl disulfide groups to have a reduction-sensitive disulfide bond linking the antigen to the NP surface, allowing efficient release of antigen inside the cell in response to the reductive conditions within the endosome. The functionalizable NPs are characterized by proton NMR, dynamic light scattering (DLS), UV/vis spectroscopy, and transmission electron microscopy (TEM). Conjugation of small molecules and protein to the NP surface is presented

Aggregation Behavior of Poly(ethylene glycol-bl-propylene sulfide) Di- and Triblock Copolymers in Aqueous Solution

Block copolymers of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-PPS) have recently emerged as a new macromolecular amphiphile capable of forming a wide range of morphologies when dispersed in water. To understand better the relationship between stability and morphology in terms of the relative and absolute block compositions, we have synthesized a collection of PEG-PPS block copolymers and quantified their critical aggregation concentration and observed their morphology using cryogenic transmission electron microscopy after thin film hydration with extrusion and after solvent dispersion from tetrahydrofuran, a solvent for both blocks. By understanding the relationship between aggregate character and block copolymer architecture, we have observed that whereas the relative block lengths control morphology, the stability of the aggregates upon dilution is determined by the absolute block length of the hydrophobic PPS block. We have compared results obtained with PEG-BPS to those obtained with poly(ethylene glycol)-bl-poly(propylene oxide)-bl-poly(ethylene glycol) block copolymers (Pluronics). The results reveal that the PEG-PPS aggregates are substantially more stable than Pluronic aggregates, by more than an order of magnitude. PEG-PPS can form a wide variety of stable or metastable morphologies in dilute solution within normal time and temperature ranges, whereas Pluronics can generally form only spherical micelles under the same conditions. On the basis of these results, block copolymers of PEG with poly(propylene sulfide) may present distinct advantages over those with poly(propylene glycol) for a number of applications