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1.
Expert Opin Drug Deliv ; 14(8): 913-925, 2017 08.
Article in English | MEDLINE | ID: mdl-28643528

ABSTRACT

BACKGROUND: This paper describes the synthesis of thiolated chitosan-based hydrogels with varying degrees of crosslinking that has been utilized to modulate release kinetics of two clinically relevant FDA-approved anti-VEGF protein drugs, ranibizumab and aflibercept. These hydrogels have been fabricated into disc shaped structures for potential use as patches on ocular surface. METHODS: Protein conformational changes and aggregation after loading and release was evaluated by circular dichroism (CD), steady-state tryptophan fluorescence spectroscopy, electrophoresis and size-exclusion chromatography (SEC). Finally, the capacity of both released proteins to bind to VEGF was tested by ELISA and surface plasmon resonance (SPR) technology. RESULTS: The study demonstrates the versatility of thiolated chitosan-based hydrogels for delivering proteins. The effect of various parameters of the hydrogel on protein release kinetics and mechanism of protein release was studied using the Korsmeyer-Peppas release model. Furthermore, we have studied the stability of released proteins in detail while comparing it with non-entrapped proteins under physiological conditions to understand the effect of formulation conditions on protein stability. CONCLUSIONS: The disc-shaped thiolated chitosan-based hydrogels provide a potentially useful platform to deliver ranibizumab and aflibercept for the treatments of ocular diseases such as wet AMD, DME and corneal neovascularization.


Subject(s)
Chitosan/chemistry , Hydrogels/chemistry , Ranibizumab/chemistry , Receptors, Vascular Endothelial Growth Factor/chemistry , Recombinant Fusion Proteins/chemistry , Chitosan/administration & dosage , Chitosan/pharmacology , Drug Liberation , Eye/pathology , Hydrogels/administration & dosage , Hydrogels/pharmacology , Neovascularization, Pathologic/drug therapy , Ranibizumab/administration & dosage , Ranibizumab/pharmacology , Receptors, Vascular Endothelial Growth Factor/administration & dosage , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/pharmacology , Vascular Endothelial Growth Factor A/metabolism
2.
Sci Rep ; 5: 12116, 2015 Jul 10.
Article in English | MEDLINE | ID: mdl-26159518

ABSTRACT

Previous publications on stapled peptide inhibitors against Mdm2/Mdm4-p53 interactions have established that this new class of drugs have the potential to be easily optimised to attain high binding affinity and specificity, but the mechanisms controlling their cellular uptake and target engagement remain elusive and controversial. To aid in understanding the rules of peptide and staple design, and to enable rapid optimisation, we employed the newly-developed cellular thermal shift assay (CETSA). CETSA was able to validate stapled peptide binding to Mdm2 and Mdm4, and the method was also used to determine the extent of cellular uptake, cellular availability, and intracellular binding of the endogenous target proteins in its native environment. Our data suggest that while the stapled peptides engage their targets intracellularly, more work is needed to improve their cellular entry and target engagement efficiency in vivo. CETSA now provides a valuable tool to optimize such in vivo properties of stapled peptides.


Subject(s)
Nuclear Proteins/metabolism , Peptides/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Proto-Oncogene Proteins/metabolism , Biological Assay/methods , Cell Cycle Proteins , Cell Line, Tumor , HCT116 Cells , Humans , Protein Binding/physiology , Tumor Suppressor Protein p53/metabolism
3.
Mol Ther Nucleic Acids ; 3: e217, 2014 Dec 16.
Article in English | MEDLINE | ID: mdl-25514650

ABSTRACT

Development of DNA aptamer screens that are both simple and informative can increase the success rate of DNA aptamer selection and induce greater adoption. High eIF4e levels contribute to malignancies, thus eIF4e presents itself as a valuable target for DNA aptamer-based inhibition screen. Here, we demonstrate a method for the rapid selection of looped DNA aptamers against eIF4e by combining negative selection and purification in a single step, followed by characterization with high throughput sequencing. The resulting aptamers show functional binding to eIF4e and inhibit translation initiation in biochemical assays. When transfected into cells, eIF4e aptamers cause a dramatic loss of cell proliferation in tumor cells as seen with eIF4e knockdown with antisense oligonucleotides, shRNAs, and siRNAs, hinting at therapeutic possibilities. With the large data set provided by high throughput sequencing, we demonstrate that selection happens in waves and that sequencing data can be used to infer aptamer structure. Lastly, we show that ligation of looped aptamers can enhance their functional effects. These results demonstrate a rapid protocol to screen and optimize aptamers against macromolecules of interest.

4.
J Am Chem Soc ; 136(17): 6159-62, 2014 Apr 30.
Article in English | MEDLINE | ID: mdl-24494589

ABSTRACT

We demonstrate the use of fluorescent molecular rotors as probes for detecting biomolecular interactions, specifically peptide-protein interactions. Molecular rotors undergo twisted intramolecular charge transfer upon irradiation, relax via the nonradiative torsional relaxation pathway, and have been typically used as viscosity probes. Their utility as a tool for detecting specific biomolecular interactions has not been explored. Using the well characterized p53-Mdm2 interaction as a model system, we designed a 9-(2-carboxy-2-cyanovinyl) julolidine-based p53 peptide reporter, JP1-R, which fluoresces conditionally only upon Mdm2 binding. The reporter was used in a rapid, homogeneous assay to screen a fragment library for antagonists of the p53-Mdm2 interaction, and several inhibitors were identified. Subsequent validation of these hits using established secondary assays suggests increased sensitivity afforded by JP1-R. The fluorescence of molecular rotors contingent upon target binding makes them a versatile tool for detecting specific biomolecular interactions.


Subject(s)
Fluorescent Dyes/metabolism , Nitriles/metabolism , Peptides/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Quinolizines/metabolism , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Drug Evaluation, Preclinical/methods , Fluorescent Dyes/chemistry , Humans , Models, Molecular , Molecular Sequence Data , Nitriles/chemistry , Peptides/chemistry , Protein Interaction Mapping/methods , Protein Interaction Maps/drug effects , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Quinolizines/chemistry , Spectrometry, Fluorescence/methods , Tumor Suppressor Protein p53/antagonists & inhibitors , Viscosity
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