Eva Harth

Targeted Nanoparticles That Deliver a Sustained, Specific Release of Paclitaxel to Irradiated Tumors

To capitalize on the response of tumor cells to XRT, we developed a controlled-release nanoparticle drug delivery system using a targeting peptide that recognizes a radiation-induced cell surface receptor. Phage display biopanning identified Gly-Ile-Arg-Leu-Arg-Gly (GIRLRG) as a peptide that selectively recognizes tumors responding to XRT. Membrane protein extracts of irradiated glioma cells identified glucose-regulated protein GRP78 as the receptor target for GIRLRG. Antibodies to GRP78 blocked the binding of GIRLRG in vitro and in vivo. Conjugation of GIRLRG to a sustained-release nanoparticle drug delivery system yielded increased paclitaxel concentration and apoptosis in irradiated breast carcinomas for up to 3 weeks. Compared with controls, a single administration of the GIRLRG-targeted nanoparticle drug delivery system to irradiated tumors delayed the in vivo tumor tripling time by 55 days (P = 0.0001) in MDA-MB-231 and 12 days in GL261 (P < 0.005). This targeting agent combines a novel recombinant peptide with a paclitaxel-encapsulating nanoparticle that specifically targets irradiated tumors, increasing apoptosis and tumor growth delay in a manner superior to known chemotherapy approaches.

Molecular Dendritic Transporter Nanoparticle Vectors Provide Efficient Intracellular Delivery of Peptides

We present the synthesis of a modular delivery system that is composed of two main macromolecular building blocks, dendritic molecular transporter molecules and a polymeric scaffold in a size dimension of 5-10 nm. The conjugated dendritic molecular transporter units proved to be critical for the delivery of the polymer nanoparticle into 3T3 cells and illustrates the dendritic molecular transporter promoted intracellular uptake of polymer particles derived from intramolecular chain collapse processes. In a sequence of modification steps, pyridinyldithio linker was introduced to undergo thiol-disulfide exchange reactions with peptide sequences containing cysteine amino acid units to furnish peptide-nanoparticle conjugates with cleavable disulfide linkers. The intracellular uptake of the nanoparticle conjugates and the delivery of the peptidic cargo were studied via dual labeling of the nanoparticle with Alexa Fluor 568 dye and fluorescein (FITC) markers on the peptide in mammalian cell lines such as NIH 3T3 cells via confocal microscopy. In this work, we have demonstrated the assembly of a novel nanoscopic delivery system in which the conjugated dendritic molecular transporter molecules facilitated the rapid cellular uptake of a nanoparticle-peptide conjugate with up to 25 copies of peptidic cargo to establish new venues for the implementation of protein and oligonucleotide drugs.

High relaxivity MRI imaging reagents from bimodal star polymers

Star polymer architectures were developed as multimodal imaging systems as fluorescence and magnetic resonance imaging (MRI) agents. Acrylate star polymers in hydrodynamic diameters of 10 ± 2 nm comprising fluorene derivatives as core units were modified with dopamine derivatives in adjustable quantities to chelate lanthanides such as Gd3+ and Eu3+. Ionic relaxivity values of 84 mM−1s−1 in an applied magnetic field of 0.5T at 37 °C confirmed the rapid water exchange of the highly hydrated star polymer. The utility is extended by the consecutive modification with allyl groups to perform thiol-ene reactions to conjugate thiol modified targeting units such as (cRGD) and dendritic molecular transporter as cell penetrating units. Cell uptake experiments in NIH 3T3 cells showed a rapid uptake of the star polymer independently of the presence of molecular transporter unit as it was detected through both the blue fluorescence of the fluorine core and the red fluorescence of the complexed Eu3+ to the linear polymer arms. In vivo mouse models confirmed the star polymer imaging reagent altered the image contrast significantly. In this work, we have adapted macromolecules as imaging agents for fluorescence microscopy and magnetic resonance imaging in conjunction with intracellular delivery and targeting.

Tailored polyester nanoparticles: post-modification with dendritic transporter and targeting units via reductive amination and thiol-ene chemistry

We present synthetic strategies for the utilization of functionalized polyester nanoparticles that enable efficient chemistries to conjugate targeting units and dendritic molecular transporter entities to form potent carrier systems for targeted drug delivery and transport across biological barriers. Polyester nanoparticles that feature functionalities such as keto, amine and allyl groups to react with moieties present in bioactive compounds, could be prepared in a one-pot procedure that improves the prior-developed drop-in cross-linking method. Integrated keto functionalities were utilized with amines of the N-terminus of peptide targeting units in high yielding reductive amination reactions. Thiol-ene reaction conditions were developed to perform mild addition reactions with targeting units, such as a novel c-RGD, but also cell penetrating dendritic transporter structures. The synthesis of targeting peptide–nanoparticle conjugates (NP–P–dye), dendritic molecular transporter nanoparticle conjugates (NP–MT–dye) as well as conjugates that contained both bioactive entities (NP–P–MT–dye), could be achieved. To study these systems in vitro, imaging units such as Alexa Fluor® 594 were introduced as another additional component to label primarily the nanoparticle backbone. This work describes several efficient post-modification strategies to accommodate the demands of biomaterials for mild conjugation chemistries utilizing amine and thiol units to form polyester bioconjugates with specific functionalities as a platform for an array of therapeutic applications.

Linear release nanoparticle devices for advanced targeted cancer therapies with increased efficacy

The cross-linked supramolecular structure of prepared polyester based nanoparticles enable increased and efficient small molecule drug loading after nanoparticle formation and post-modification with targeting peptides via thiol-ene ‘click’ chemistry. It could be demonstrated that the drug loading does not influence the structural integrity of the particle and its diameter was not significantly changed. A prolonged linear release profile of the drug without the typical ‘burst effect’ was observed in emulsified particles and mirrored the linear degradation profile of the investigated particles, which are critical properties for controlled and predictable pharmacokinetics in cancer therapies. Furthermore, the final peptide-targeted and drug-loaded particles were found to be readily dispersed in buffer or water, and with the confirmed biocompatibility, these novel and adjustable drug delivery systems are promising vectors for the treatment of various cancers.

Synthesis of low-temperature benzocyclobutene cross-linker and utilization

We present the synthesis of a benzocyclobutene based cross-linking reagent that allows a 100 °C lower processing temperature in comparison with typical benzocyclobutenes as demonstrated with the preparation of polyacrylate nanoparticles through the intramolecular chain collapse reaction.

Stretchable Gas Barrier Achieved with Partially Hydrogen-Bonded Multilayer Nanocoating

Super gas barrier nanocoatings are recently demonstrated by combining polyelectrolytes and clay nanoplatelets with layer-by-layer deposition. These nanobrick wall thin films match or exceed the gas barrier of SiOx and metallized films, but they are relatively stiff and lose barrier with significant stretching (≥ 10% strain). In an effort to impart stretchability, hydrogen-bonding polyglycidol (PGD) layers are added to an electrostatically bonded thin film assembly of polyethylenimine (PEI) and montmorillonite (MMT) clay. The oxygen transmission rate of a 125-nm thick PEI-MMT film increases more than 40x after being stretched 10%, while PGD-PEI-MMT trilayers of the same thickness maintain its gas barrier. This stretchable trilayer system has an OTR three times lower than the PEI-MMT bilayer system after stretching. This report marks the first stretchable high gas barrier thin film, which is potentially useful for applications that require pressurized elastomers.

Combined MEK Inhibition and BMP2 Treatment Promotes Osteoblast Differentiation and Bone Healing in Nf1Osx−/− Mice

Neurofibromatosis type I (NF1) is an autosomal dominant disease with an incidence of 1/3000, caused by mutations in the NF1 gene, which encodes the RAS/GTPase‐activating protein neurofibromin. Non‐bone union after fracture (pseudarthrosis) in children with NF1 remains a challenging orthopedic condition to treat. Recent progress in understanding the biology of neurofibromin suggested that NF1 pseudarthrosis stems primarily from defects in the bone mesenchymal lineage and hypersensitivity of hematopoietic cells to TGFβ. However, clinically relevant pharmacological approaches to augment bone union in these patients remain limited. In this study, we report the generation of a novel conditional mutant mouse line used to model NF1 pseudoarthrosis, in which Nf1 can be ablated in an inducible fashion in osteoprogenitors of postnatal mice, thus circumventing the dwarfism associated with previous mouse models where Nf1 is ablated in embryonic mesenchymal cell lineages. An ex vivo–based cell culture approach based on the use of Nf1flox/flox bone marrow stromal cells showed that loss of Nf1 impairs osteoprogenitor cell differentiation in a cell‐autonomous manner, independent of developmental growth plate–derived or paracrine/hormonal influences. In addition, in vitro gene expression and differentiation assays indicated that chronic ERK activation in Nf1‐deficient osteoprogenitors blunts the pro‐osteogenic property of BMP2, based on the observation that only combination treatment with BMP2 and MEK inhibition promoted the differentiation of Nf1‐deficient osteoprogenitors. The in vivo preclinical relevance of these findings was confirmed by the improved bone healing and callus strength observed in Nf1osx−/− mice receiving Trametinib (a MEK inhibitor) and BMP2 released locally at the fracture site via a novel nanoparticle and polyglycidol‐based delivery method. Collectively, these results provide novel evidence for a cell‐autonomous role of neurofibromin in osteoprogenitor cells and insights about a novel targeted approach for the treatment of NF1 pseudoarthrosis.

Synthesis of amino acid-based polymers via atom transfer radical polymerization in aqueous media at ambient temperature.

Well-defined acryloyl beta-alanine (ABA) polymers were synthesized directly via atom transfer radical polymerization (ATRP) under near physiological conditions using various water soluble initiators with high yield and narrow molecular weight distributions.

Matrices for combined delivery of proteins and synthetic molecules

With the increasing advancement of synergistic, multimodal approaches to influence the treatment of infectious and non-infectious diseases, we witness the development of enabling techniques merging necessary complexity with leaner designs and effectiveness. Systems- and polypharmacology ask for multi-potent drug combinations with many targets to engage with the biological system. These demand drug delivery designs for one single drug, dual drug release systems and multiple release matrices in which the macromolecular structure allows for higher solubilization, protection and sequential or combined release profiles. As a result, nano- and micromaterials have been evolved from mono- to dual drug carriers but are also an essential part to establish multimodality in polymeric matrices. Surface dynamics of particles creating interfaces between polymer chains and hydrogels inspired the development not only of biomedical adhesives but also of injectable hydrogels in which the nanoscale material is both, adhesive and delivery tool. These complex delivery systems are segmented into two delivery subunits, a polymer matrix and nanocarrier, to allow for an even higher tolerance of the incorporated drugs without adding further synthetic demands to the nanocarrier alone. The opportunities in these quite novel approaches for the delivery of small and biological therapeutics are remarkable and selected examples for applications in cancer and bone treatments are discussed.