Synthesis and chemical modification of degradable polymers to enhance gene delivery

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2007.Includes bibliographical references.Poly([beta]-amino ester)s are a class of cationic, degradable polymers that have shown significant promise as gene delivery agents, more effective than the state-of-the-art, commercially available non-viral systems. The main objective of this thesis is to synthesize new poly([beta]-amino ester)s and modify existing ones to further improve their gene delivery properties for clinical applications. This has been accomplished by developing both side- and end-chain chemistries for poly([beta]-amino ester)s. A series of novel poly([beta]-amino ester)s were prepared using a new amine monomer 2-(2-pyridyldithio)-ethylamine. The polymer side chains display fast and selective reactivity towards thiol ligands, as demonstrated using mercaptoethylamine (MEA) and RGDC, a ligand that binds with high affinity to certain integrin receptors on angiogenic endothelial cells. The MEA derivatives in particular, were able to self-assemble with plasmid DNA to form nano-complexes that can partially disassemble in response to intracellular glutathione concentrations. These polymers also displayed low cellular toxicity and were able to mediate transfection at high levels in human hepatocellular carcinoma cells. It is envisioned that the PDA poly([beta]-amino ester)s can serve as cationic, degradable platforms to attach targeting ligands, viral peptides and other molecules to a single chain to improve gene delivery.(cont.) A two-step end-modification strategy is also presented to optimize the functionality at the polymer end points. Conditions were developed so that many structurally diverse end groups could be explored, without the need for polymer purification. Using a highly efficient poly(3-amino ester), C32, optimization of the terminal amine group improved in vitro gene transfection by 30% and reduced the polymer:DNA ratio 5-fold. Differences of single carbons and functional groups at the polymer ends were shown to affect many polymer-DNA properties, including the binding affinity, complex size and surface charge, levels of endocytosis, cytotoxicity and transfection. Intraperitoneal gene delivery in mice using several end-modified C32 polymers proved an order-of-magnitude more effective than unmodified C32, as measured in whole body scans and harvested organs. The end- and side-chain modification strategies presented here have led to the discovery of improved poly([beta]-amino ester)s for gene delivery and may aid in their future development into clinically useful delivery systems.by Gregory Thomas Zugates.Ph.D

Nanoparticle-Delivered Multimeric Soluble CD40L DNA Combined with Toll-Like Receptor Agonists as a Treatment for Melanoma

Stimulation of CD40 or Toll-Like Receptors (TLR) has potential for tumor immunotherapy. Combinations of CD40 and TLR stimulation can be synergistic, resulting in even stronger dendritic cell (DC) and CD8+ T cell responses. To evaluate such combinations, established B16F10 melanoma tumors were injected every other day X 5 with plasmid DNA encoding a multimeric, soluble form of CD40L (pSP-D-CD40L) either alone or combined with an agonist for TLR1/2 (Pam3CSK4 ), TLR2/6 (FSL-1 and MALP2), TLR3 (polyinosinic-polycytidylic acid, poly(I:C)), TLR4 ( monophosphoryl lipid A, MPL), TLR7 (imiquimod), or TLR9 (Class B CpG phosphorothioate oligodeoxynucleotide, CpG). When used by itself, pSP-D-CD40L slowed tumor growth and prolonged survival, but did not lead to cure. Of the TLR agonists, CpG and poly(I:C) also slowed tumor growth, and the combination of these two TLR agonists was more effective than either agent alone. The triple combination of intratumoral pSP-D-CD40L + CpG + poly(I:C) markedly slowed tumor growth and prolonged survival. This treatment was associated with a reduction in intratumoral CD11c+ dendritic cells and an influx of CD8+ T cells. Since intratumoral injection of plasmid DNA does not lead to efficient transgene expression, pSP-D-CD40L was also tested with cationic polymers that form DNA-containing nanoparticles which lead to enhanced intratumoral gene expression. Intratumoral injections of pSP-D-CD40L-containing nanoparticles formed from polyethylenimine (PEI) or C32 (a novel biodegradable poly(B-amino esters) polymer) in combination with CpG + poly(I:C) had dramatic antitumor effects and frequently cured mice of B16F10 tumors. These data confirm and extend previous reports that CD40 and TLR agonists are synergistic and demonstrate that this combination of immunostimulants can significantly suppress tumor growth in mice. In addition, the enhanced effectiveness of nanoparticle formulations of DNA encoding immunostimulatory molecules such as multimeric, soluble CD40L supports the further study of this technology for tumor immunotherapy

Epidermal Growth Factor–PEG Functionalized PAMAM-Pentaethylenehexamine Dendron for Targeted Gene Delivery Produced by Click Chemistry

Aim of this study was the site-specific conjugation of an epidermal growth factor (EGF)-polyethylene glycol (PEG) chain by click chemistry onto a poly(amido amine) (PAMAM) dendron, as a key step toward defined multifunctional carriers for targeted gene delivery. For this purpose, at first propargyl amine cored PAMAM dendrons with ester ends were synthesized. The chain terminal ester groups were then modified by oligoamines with different secondary amino densities. The oligoamine-modified PAMAM dendrons were well biocompatible, as demonstrated in cytotoxicity assays. Among the different oligoamine-modified dendrons, PAMAM-pentaethylenehexamine (PEHA) dendron polyplexes displayed the best gene transfer ability. Conjugation of PAMAM-PEHA dendron with PEG spacer was conducted via click reaction, which was performed before amidation with PEHA. The resultant PEG-PAMAM-PEHA copolymer was then coupled with EGF ligand. pDNA transfections in HuH-7 hepatocellular carcinoma cells showed a 10-fold higher efficiency with the polyplexes containing conjugated EGF as compared to the ligand-free ones, demonstrating the concept of ligand targeting. Overall gene transfer efficiencies, however, were moderate, suggesting that additional measures for overcoming subsequent intracellular bottlenecks in delivery have to be taken

Tissue-Specific Gene Delivery via Nanoparticle Coating

Author Manuscript: 2010 August 1.The use of biomaterials for gene delivery can potentially avoid many of the safety concerns with viral gene delivery. However, the efficacy of polymeric gene delivery methods is low, particularly in vivo. One significant concern is that the interior and exterior composition of polymeric gene delivery nanoparticles are often coupled, with a single polymer backbone governing all functions from biophysical properties of the polymer/DNA particle to DNA condensation and release. In this work we develop electrostatically adsorbed poly(glutamic acid)-based peptide coatings to alter the exterior composition of a core gene delivery particle and thereby affect tissue-specificity of gene delivery function in vivo. We find that with all coating formulations tested, the coatings reduce potential toxicity associated with uncoated cationic gene delivery nanoparticles following systemic injection. Particles coated with a low 2.5:1 peptide:DNA weight ratio (w/w) form large 2 μ sized particles in the presence of serum that can facilitate specific gene delivery to the liver. The same particles coated at a higher 20:1 w/w form small 200 nm particles in the presence of serum that can facilitate specific gene delivery to the spleen and bone marrow. Thus, variations in nanoparticle peptide coating density can alter the tissue-specificity of gene delivery in vivo.National Institutes of Health (U.S.) (BRP: 1R01CA124427-01)National Institutes of Health (U.S.) (EB 000244)National Institutes of Health (U.S.) (U54 CA119349-01)David & Lucile Packard Foundation (Fellowship 1999-1453A

Polymer-lipid nanoparticles for systemic delivery of mRNA to the lungs

Therapeutic nucleic acids hold great promise for the treatment of disease but require vectors for safe and effective delivery. Synthetic nanoparticle vectors composed of poly(β‐amino esters) (PBAEs) and nucleic acids have previously demonstrated potential utility for local delivery applications. To expand this potential utility to include systemic delivery of mRNA, hybrid polymer–lipid nanoformulations for systemic delivery to the lungs were developed. Through coformulation of PBAEs with lipid–polyethylene glycol (PEG), mRNA formulations were developed with increased serum stability and increased in vitro potency. The formulations were capable of functional delivery of mRNA to the lungs after intravenous administration in mice. To our knowledge, this is the first report of the systemic administration of mRNA for delivery to the lungs using degradable polymer–lipid nanoparticles

Double-degradable responsive self-assembled multivalent arrays-temporary nanoscale recognition between dendrons and DNA

This article reports self-assembling dendrons which bind DNA in a multivalent manner. The molecular design directly impacts on self-assembly which subsequently controls the way these multivalent nanostructures bind DNA-this can be simulated by multiscale modelling. Incorporation of an S-S linkage between the multivalent hydrophilic dendron and the hydrophobic units responsible for self-assembly allows these structures to undergo triggered reductive cleavage, with dithiothreitol (DTT) inducing controlled breakdown, enabling the release of bound DNA. As such, the high-affinity self-assembled multivalent binding is temporary. Furthermore, because the multivalent dendrons are constructed from esters, a second slow degradation step causes further breakdown of these structures. This two-step double-degradation mechanism converts a large self-assembling unit with high affinity for DNA into small units with no measurable binding affinity-demonstrating the advantage of self-assembled multivalency (SAMul) in achieving highly responsive nanoscale binding of biological targets

New Methods for Characterizing the Complex Neural Circuitry of the Adult Drosophila Brain

141 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007.Recently improved genetic mosaic methods permit unprecedented resolution for study of neural circuitry formation in Drosophila melanogaster. This study utilizes new mosaic methods to investigate conserved mechanisms that underlie the development of adult-specific neurons. In addition to providing several novel insights about the genetics of neural circuitry development, this study also provides a framework for increased resolution in examination of lineage and morphology in the Drosophila brain. First, mosaic analysis with a repressible cell marker (MARCM) reveals stereotyped lineage and morphogenesis in the ellipsoid body (EB), a center of locomotor regulation, resembling development of the mushroom bodies (MB's), sites involved heavily in olfactory learning. In addition, EB neuron birth and axon development are widely separated in time making them ideal model neurons for further studies. Second, comparative genetic mosaic analysis of EB and dorsal cluster (DC) neurons suggests that TGF-beta signaling is widely required for maturation and morphogenesis of post-mitotic adult-specific neurons. Third, combined application of MARCM with a newly developed technique, called "Flip-out" MARCM, reveals that the Drosophila Down syndrome cell adhesion molecule (Dscam) is required for suppressing ectopic bifurcation MB axons and for promoting divergent guidance of growth cones. Knockout of Dscam in EB neurons reveals a general role in formation and guidance of axonal branches. Finally, combining mosaic methods to study both cellular genetic requirements and phenotypes suggest a non-autonomous requirement for the atypical receptor tyrosine kinase (RTK), Linotte/Derailed (Lio/Drl), in patterning MB axon lobe architecture. In the development of MB circuitry, Lio/Drl may act to counter Wnt5 signaling, which appears to be generally required for axon extension across the brain midline.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD