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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2016)

High molecular weight and highly-charged biomolecules are emerging as drugs with high selectivity and efficacy; however, new strategies are needed to improve their efficiency and targeted delivery in biological systems. One potential solution is the conjugation or association of the biologic with a molecular transporter.

Inspired by proteins, such as HIV-Tat, with cellular translocation abilities, numerous guanidinium-rich molecular transporters have been synthesized from a diverse range of nonpeptidic scaffolds. While their repertoire has expanded tremendously in the past two decades, new transporters can provide unique intracellular distributions, targeting effects, or pharmacological properties.

Polymyxin B is a cyclic polypeptide antibiotic containing five primary amines and a hydrophobic tail that has not been exploited as a delivery module. Here, we synthesized functionalized derivatives of polymyxin and its per-guanidinylated derivative and evaluated their cellular uptake in mammalian cells. Both polymyxin and its guanidinylated form effectively enter mammalian cells at nanomolar concentrations and can facilitate the cellular delivery of large biomolecules and liposomal assemblies.

Guanidinoglycosides are a non-oligomeric class of molecular transporters developed in our lab that permeate the cell membrane through heparan sulfate-dependent pathways at low nanomolar concentrations. To further advance guanidinoglycosides as transporters, guanidinylated neomycin (GNeo) derivatives containing different fatty acids were synthesized and incorporated into liposomes. A small molecule dye or a lysosomal enzyme were encapsulated in the liposomes and delivered to Chinese hamster ovary cells or human fibroblasts, respectively. Incorporation of stearyl- or di-oleyl-GNeo lipids into liposomes resulted in the greatest enhancement of uptake. The delivery of α-L-iduronidase, a lysosomal enzyme, was able to restore enzyme function in fibroblasts lacking endogenous enzyme. As an alternative approach to enhance GNeo as a molecular transporter, oligomers of GNeo were synthesized using ring-opening metathesis polymerization. The synthesis of the reactive monomer, formation of GNeo oligomers, and preliminary cellular uptake studies are presented. In addition to using GNeo for new delivery systems, we also sought to explore the effect the linker connecting the cargo to the carrier has on conjugation and uptake. Varying the length and hydrophobic properties of the linker joining GNeo to biotin resulted in diverse conjugation efficiencies to streptavidin and differences in cellular uptake, highlighting the importance of the linker when designing and studying new molecular transporters.

    Cover page: Overcoming Biological Barriers: Synthesis and Evaluation of Molecular Transporters
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2016)
    Two methods for assembling guanidinoneomycin-decorated liposomes are presented and their ability to deliver an active enzyme to the lysosomes and restore enzyme function in diseased cells is compared.
      Cover page: Delivery of an active lysosomal enzyme using GNeosomes
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2016)
      Polymyxin B is an antibiotic used against multi-resistant gram negative infections, despite observed nephrotoxicity. Here we report the synthesis of functionalized derivatives of polymyxin B and its per-guanidinylated derivative in order to further explore the structural requirements necessary to facilitate uptake of the antibiotic into mammalian cells. We also investigate the possibility of using these novel scaffolds as molecular transporters. At nanomolar concentrations, both are capable of delivering large cargo (>300 kDa) into living cells. Their uptake depends exclusively on cell surface heparan sulfate. Mechanistic studies indicate these novel transporters are internalized through caveolae-mediated pathways and confocal microscopy show colocalization with lysosomes. The polymyxin-based transporters demonstrate cytosolic delivery through the delivery of a ribosome-inactivating toxin. Furthermore, the natural polymyxin scaffold can be incorporated into liposomes and enhance their intracellular uptake. In addition to demonstrating the ability of the polymyxin scaffold to facilitate internalization into mammalian cells, these observations suggest the potential use of polymyxin and guanidinopolymyxin for intracellular delivery.
        Cover page: Polymyxins facilitate entry into mammalian cells
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2016)
        Polymyxin B is an antibiotic used against multi-resistant gram negative infections, despite observed nephrotoxicity. Here we report the synthesis of functionalized derivatives of polymyxin B and its per-guanidinylated derivative in order to further explore the structural requirements necessary to facilitate uptake of the antibiotic into mammalian cells. We also investigate the possibility of using these novel scaffolds as molecular transporters. At nanomolar concentrations, both are capable of delivering large cargo (>300 kDa) into living cells. Their uptake depends exclusively on cell surface heparan sulfate. Mechanistic studies indicate these novel transporters are internalized through caveolae-mediated pathways and confocal microscopy show colocalization with lysosomes. The polymyxin-based transporters demonstrate cytosolic delivery through the delivery of a ribosome-inactivating toxin. Furthermore, the natural polymyxin scaffold can be incorporated into liposomes and enhance their intracellular uptake. In addition to demonstrating the ability of the polymyxin scaffold to facilitate internalization into mammalian cells, these observations suggest the potential use of polymyxin and guanidinopolymyxin for intracellular delivery.
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2017)
          Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain.
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2017)
            Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain.
              Cover page: Guanidinylated Neomycin Conjugation Enhances Intranasal Enzyme Replacement in the Brain
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2021)
              Heparan sulfate (HS) proteoglycans bind extracellular proteins that participate in cell signaling, attachment and endocytosis. These interactions depend on the arrangement of sulfated sugars in the HS chains generated by well-characterized biosynthetic enzymes; however, the regulation of these enzymes is largely unknown. We conducted genome-wide CRISPR-Cas9 screens with a small-molecule ligand that binds to HS. Screening of A375 melanoma cells uncovered additional genes and pathways impacting HS formation. The top hit was the epigenetic factor KDM2B, a histone demethylase. KDM2B inactivation suppressed multiple HS sulfotransferases and upregulated the sulfatase SULF1. These changes differentially affected the interaction of HS-binding proteins. KDM2B-deficient cells displayed decreased growth rates, which was rescued by SULF1 inactivation. In addition, KDM2B deficiency altered the expression of many extracellular matrix genes. Thus, KDM2B controls proliferation of A375 cells through the regulation of HS structure and serves as a master regulator of the extracellular matrix.
                Cover page: Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate
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