Tiffany P. Gustafson

Improving paclitaxel delivery: in vitro and in vivo characterization of PEGylated polyphosphoester-based nanocarriers.

Nanomaterials have great potential to offer effective treatment against devastating diseases by providing sustained release of high concentrations of therapeutic agents locally, especially when the route of administration allows for direct access to the diseased tissues. Biodegradable polyphosphoester-based polymeric micelles and shell cross-linked knedel-like nanoparticles (SCKs) have been designed from amphiphilic block-graft terpolymers, PEBP-b-PBYP-g-PEG, which effectively incorporate high concentrations of paclitaxel (PTX). Well-dispersed nanoparticles physically loaded with PTX were prepared, exhibiting desirable physiochemical characteristics. Encapsulation of 10 wt% PTX, into either micelles or SCKs, allowed for aqueous suspension of PTX at concentrations up to 4.8 mg/mL, as compared to <2.0 μg/mL for the aqueous solubility of the drug alone. Drug release studies indicated that PTX released from these nanostructures was defined through a structure-function relationship, whereby the half-life of sustained PTX release was doubled through cross-linking of the micellar structure to form SCKs. In vitro, physically loaded micellar and SCK nanotherapeutics demonstrated IC50 values against osteosarcoma cell lines, known to metastasize to the lungs (CCH-OS-O and SJSA), similar to the pharmaceutical Taxol formulation. Evaluation of these materials in vivo has provided an understanding of the effects of nanoparticle structure-function relationships on intratracheal delivery and related biodistribution and pharmacokinetics. Overall, we have demonstrated the potential of these novel nanotherapeutics toward future sustained release treatments via administration directly to the sites of lung metastases of osteosarcoma.

Polycarbonates derived from glucose via an organocatalytic approach.

An organocatalyzed ring-opening polymerization methodology was developed for the preparation of polycarbonates derived from glucose as a natural product starting material. The cyclic 4,6-carbonate monomer of glucose having the 1, 2, and 3 positions methyl-protected was prepared in three steps from a commercially available glucose derivative, and the structure was confirmed by means of NMR and IR spectroscopies, electrospray ionization mass spectrometry (MS), and single-crystal X-ray analysis. Polymerization of the monomer, initiated by 4-methylbenzyl alcohol in the presence of 1,5,7-triazabicyclo[4.4.0]dec-5-ene as the organocatalyst, proceeded effectively in a controlled fashion to afford the polycarbonate with a tunable degree of polymerization, narrow molecular weight distribution, and well-defined end groups, as confirmed by a combination of NMR spectroscopy, gel-permeation chromatography, and MALDI-TOF MS. A distribution of head-to-head, head-to-tail, and tail-to-tail regiochemistries was determined by NMR spectroscopy and tandem MS analysis by electron transfer dissociation. These polycarbonates are of interest as engineering materials because of their origination from renewable resources combined with their amorphous character and relatively high glass transition temperatures as determined by X-ray diffraction and differential scanning calorimetry studies.

Poly(D-glucose carbonate) block copolymers: a platform for natural product-based nanomaterials with Solvothermatic characteristics.

A natural product-based polymer platform, having the characteristics of being derived from renewable materials and capable of breaking down, ultimately, into natural byproducts, has been prepared through the ring-opening polymerization (ROP) of a glucose-based bicyclic carbonate monomer. ROP was carried out via chain extension of a polyphosphoester (PPE) macroinitiator in the presence of 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) organocatalyst to afford the PPE-b-poly(D-glucose carbonate) (PDGC) block copolymer. This new copolymer represents a functional architecture that can be rapidly transformed through thiol-yne reactions along the PPE segment into a diverse variety of amphiphilic polymers, which interestingly display stimuli-sensitive phase behavior in the form of a lower critical solution temperature (LCST). Below the LCST, they undergo self-assembly to form spherical core-shell nanostructures that display a poorly defined core-shell morphology. It is expected that hydrophobic patches are exposed within the micellar corona, reminiscent of the surface complexity of proteins, making these materials of interest for triggered and reversible assembly disassembly processes.

Holistic assessment of covalently labeled core-shell polymeric nanoparticles with fluorescent contrast agents for theranostic applications.

The successful development of degradable polymeric nanostructures as optical probes for use in nanotheranostic applications requires the intelligent design of materials such that their surface response, degradation, drug delivery, and imaging properties are all optimized. In the case of imaging, optimization must result in materials that allow differentiation between unbound optical contrast agents and labeled polymeric materials as they undergo degradation. In this study, we have shown that use of traditional electrophoretic gel-plate assays for the determination of the purity of dye-conjugated degradable nanoparticles is limited by polymer degradation characteristics. To overcome these limitations, we have outlined a holistic approach to evaluating dye and peptide-polymer nanoparticle conjugation by utilizing steady-state fluorescence, anisotropy, and emission and anisotropy lifetime decay profiles, through which nanoparticle-dye binding can be assessed independently of perturbations, such as those presented during the execution of electrolyte gel-based assays. This approach has been demonstrated to provide an overall understanding of the spectral signature-structure-function relationship, ascertaining key information on interactions between the fluorophore, polymer, and solvent components that have a direct and measurable impact on the emissive properties of the optical probe. The use of these powerful techniques provides feedback that can be utilized to improve nanotheranostics by evaluating dye emissivity in degradable nanotheranostic systems, which has become increasingly important as modern platforms transition to architectures intentionally reliant on degradation and built-in environmental responses.

Defining a Polymethine Dye for Fluorescence Anisotropy Applications in the Near-Infrared Spectral Range

Fluorescence anisotropy in the near-infrared (NIR) spectral range is challenging because of the lack of appropriate NIR fluorescent labels. We have evaluated polymethine fluorescent dyes to identify a leading candidate for NIR anisotropy applications. The NIR dye LS601 demonstrated low fluorescence anisotropy values (r) as a result of its relatively long fluorescent lifetime 1.3 ns. The r value of LS601 unbound and coupled to biological macromolecules was found to have a sufficient dynamic range from 0.24 to 0.37, demonstrating the feasibility of fluorescence anisotropy in the NIR. The viability of fluorescence anisotropy using a NIR label was demonstrated by characterization of dye-protein conjugates. These results open the door to a number of applications in drug discovery, fluorescence anisotropy imaging and contrast agent development.

A NIR dye for development of peripheral nerve targeted probes

Current imaging modalities lack the ability to quickly assess and classify nerve injury for predicting favourable versus unfavourable healing outcomes, which could minimize episodes of chronic pain and loss of function by allowing for early intervention. Thus, the development of a technique to noninvasively assess peripheral nerve damage is of critical importance. While the development of nerve specific near infrared (NIR) molecular probes capable of such diagnostics constitutes our long term goal, initial studies to identify a NIR dye for constructing such a probe are required. We have evaluated the properties of a novel highly hydrophilic and functionalizable polymethine dye, and its more hydrophobic analogue indocyanine green, within the sciatic nerve of rats following intra-nerve injection. The reporting ability of both dyes at critical depths for nerve imaging, the importance of hydrophilicity on dye transport through nervous tissue, and their toxicity - or lack thereof - to the neural environment have been evaluated. The results suggest that the novel NIR dye is an appropriate fluorescent reporter for use in designing nerve-specific optical molecular probes for non-invasive diagnosis and classification of nerve injury.

Blood triggered rapid release porous nanocapsules

Rapid-release drug delivery systems present a new paradigm in emergency care treatments. Such systems combine a long shelf life with the ability to provide a significant dose of the drug to the bloodstream in the shortest period of time. Until now, development of delivery formulations has concentrated on slow release systems to ensure a steady concentration of the drug. To address the need for quick release system, we created hollow polyacrylate nanocapsules with nanometer-thin porous walls. Burst release occurs upon interaction with blood components that leads to escape of the cargo. The likely mechanism of release involves a conformational change of the polymer shell caused by binding albumin. To demonstrate this concept, a near-infrared fluorescent dye indocyanine green (ICG) was incorporated inside the nanocapsules. ICG-loaded nanocapsules demonstrated remarkable shelf life in aqueous buffers with no release of ICG for twelve months. Rapid release of the dye was demonstrated first in vitro using albumin solution and serum. SEM and light scattering analysis demonstrated the retention of the nanocapsule architecture after the release of the dye upon contact with albumin. In vivo studies using fluorescence lifetime imaging confirmed quick discharge of ICG from the nanocapsules following intravenous injection.

Functional sugar-based polymers and nanostructures comprised of degradable poly(D-glucose carbonate)s

Fundamental synthetic methodology was advanced to allow for the preparation of a reactive glucose-based block copolycarbonate, which was conveniently transformed into a series of amphiphilic block copolymers that underwent aqueous assembly into functional nanoparticle morphologies having practical utility in biomedical and other applications. Two degradable D-glucose carbonate monomers, with one carrying alkyne functionality, were designed and synthesized to access well-defined block polycarbonates (Đ < 1.1) via sequential organocatalytic ring opening polymerizations (ROPs). Kinetic studies of the organocatalyzed sequential ROPs showed a linear relationship between the monomer conversion and the polymer molecular weight, which indicated the controlled fashion during each polymerization. The pendant alkyne groups underwent two classic click reactions, copper-catalyzed azide–alkyne dipolar cycloaddition (CuAAC) and thiol–yne addition reactions, which were employed to render hydrophilicity for the alkyne-containing block and to provide a variety of amphiphilic diblock poly(D-glucose carbonate)s (PGCs). The resulting amphiphilic PGCs were further assembled into a family of nanostructures with different sizes, morphologies, surface charges and functionalities. These non-ionic and anionic nanoparticles showed low cytotoxicity in RAW 264.7 mouse macrophage cells and MC3T3 healthy mouse osteoblast precursor cells, while the cationic nanoparticles exhibited significantly higher IC50 (162 μg mL−1 in RAW 264.7; 199 μg mL−1 in MC3T3) compared to the commercially available cationic lipid-based formulation, Lipofectamine (IC50 = 31 μg mL−1), making these nanomaterials of interest for biomedical applications.

Tungsten-loaded SMP foam nanocomposites with inherent radiopacity and tunable thermo-mechanical properties

Shape memory polymer (SMP) foams have been developed for use in neurovascular occlusion applications. These materials are predominantly polyurethanes that are known for their biocompatibility and tunable properties. However, these polymers inherently lack X-ray visibility, which is a significant challenge for their use as implantable materials. Herein, low density, highly porous shape memory polyurethane foams were developed with tungsten nanoparticles dispersed into the foam matrix, at increasing concentrations, to serve as a radiopaque agent. Utilizing X-ray fluoroscopy sufficient visibility of the foams at small geometries was observed. Thermal characterization of the foams indicated altered thermal response and delayed foam actuation with increasing nanoparticle loading (because of restricted network mobility). Mechanical testing indicated decreased toughness and strength for higher loading because of disruption of the SMP matrix. Overall, filler addition imparted x-ray visibility to the SMP foams and allowed for tuned control of the transition temperature and actuation kinetics for the material.

Externally sensitized deprotection of PPG-masked carbonyls as a spatial proximity probe in photoamplified detection of binding events.

Externally sensitized electron-transfer fragmentation in dithiane PPG (photoremovable protecting group)-protected carbonyls is adopted for detection and amplification of molecular recognition events. The new methodology allows for detection of as low as 50 attomoles of avidin utilizing an imager based on a low sensitivity mass-produced consumer CCD camera. Numeric modelling is carried out to demonstrate the intrinsic limitations of 2D amplification on surfaces and the advantages of unconstrained amplification in a compartmentalized volume of spatially addressable 3D solutions.