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Trehalose Glycopolymers for Protein ...

Lau, Uland Y.

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Trehalose Glycopolymers for Protein and Cell Stabilization.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Trehalose Glycopolymers for Protein and Cell Stabilization./
作者:	Lau, Uland Y.
面頁冊數:	161 p.
附註:	Source: Dissertation Abstracts International, Volume: 77-07(E), Section: B.
Contained By:	Dissertation Abstracts International77-07B(E).
標題:	Biomedical engineering. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10027115
ISBN:	9781339526263

Trehalose Glycopolymers for Protein and Cell Stabilization.
Lau, Uland Y.

Trehalose Glycopolymers for Protein and Cell Stabilization. - 161 p.

Source: Dissertation Abstracts International, Volume: 77-07(E), Section: B.

Thesis (Ph.D.)--University of California, Los Angeles, 2016.

Proteins, biomolecules, and cells play an important role for their use as therapeutics and reagents for research applications. However, one main limitation is their inherent instability. Proteins can easily denature and lose activity under a variety of stresses, and cells can easily lose viability due to cytotoxic compounds and conditions. This has led to the investigation of excipients that can aid in preserving activity, including polymers, osmolytes, proteins, and sugars. Among the sugars, trehalose has been shown to have remarkable properties for general protein and cell stabilization. This dissertation presents the utilization of trehalose glycopolymers for the stabilization of cells and proteins in three different applications. The polymers were used for protein stabilization during protein micro- and nano-patterning, stabilization of a therapeutic-relevant protein, and for cell preservation.

ISBN: 9781339526263Subjects--Topical Terms:

535387
Biomedical engineering.

Trehalose Glycopolymers for Protein and Cell Stabilization.
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245 1 0 $a Trehalose Glycopolymers for Protein and Cell Stabilization.
300 $a 161 p.
500 $a Source: Dissertation Abstracts International, Volume: 77-07(E), Section: B.
500 $a Adviser: Heather D. Maynard.
502 $a Thesis (Ph.D.)--University of California, Los Angeles, 2016.
520 $a Proteins, biomolecules, and cells play an important role for their use as therapeutics and reagents for research applications. However, one main limitation is their inherent instability. Proteins can easily denature and lose activity under a variety of stresses, and cells can easily lose viability due to cytotoxic compounds and conditions. This has led to the investigation of excipients that can aid in preserving activity, including polymers, osmolytes, proteins, and sugars. Among the sugars, trehalose has been shown to have remarkable properties for general protein and cell stabilization. This dissertation presents the utilization of trehalose glycopolymers for the stabilization of cells and proteins in three different applications. The polymers were used for protein stabilization during protein micro- and nano-patterning, stabilization of a therapeutic-relevant protein, and for cell preservation.
520 $a Direct write patterning of multiple biological molecules on surfaces has tremendous potential in applications of tissue engineering, diagnostics, proteomics and biosensors. Precise control over the position and arrangement of proteins, especially on the micro- and nano- scale level is a particular challenge towards the improvement of miniaturized devices. A trehalose glycopolymer was utilized as a resist material that protects proteins during electron beam lithography, and thus enables direct write multiplexed protein patterns with micro- and nano- scale features and alignment capabilities. The trehalose glycopolymers behave as negative resists, crosslinking to the substrate upon electron beam irradiation, and provide stabilization to proteins against the harsh processing conditions. Patterns with user-defined shapes were generated with a variety of protein types, such as antibodies, enzymes, and growth factors, and were shown to retain their activity. To further demonstrate the technique, fabrication of antibody patterns for multiplexed cytokine detection from live cells was achieved. A sandwich immunoassay was developed for the detection of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) secreted directly from stimulated macrophage cells. Multiplexing with both IL-6 and TNFalpha on a single chip was demonstrated successfully with high specificity and in relevant cell culture conditions. The ability to monitor cytokine release over time following cell stimulation was also demonstrated. Simultaneous detection of these extracellular signaling markers demonstrates the potential application towards disease profiling and diagnostics.
520 $a Next, the trehalose glycopolymers were used for the stabilization and enhancement of the pharmacokinetic properties of a therapeutic protein, granulocyte colony-stimulating factor (G-CSF). G-CSF was designed with a polyhistidine-tag, maltose binding protein for solubility, and an enzyme cleavage site, and was expressed in E. coli and purified for the studies. Trehalose polymers with polystyrene or polymethacrylate backbones (P1, P2, and P3), as well as a degradable trehalose polymer (p(BMDO-co-trehalose)) were synthesized to investigate their potential for therapeutic use. The trehalose polymers were shown to be non-cytotoxic in mouse and human cell lines up to a concentration of 8 mg/mL. However, the degraded products of p(BMDO- co-trehalose) exhibited cytotoxicity at a lower concentration of 1 mg/mL. Stability screening with the trehalose glycopolymers on G-CSF showed that styrenyl acetal linked trehalose polymer, P1, best stabilized G-CSF compared to the other polymers and was selected for G-CSF conjugation. Therefore, P1 with a benzaldehyde end group was synthesized and conjugated to G-CSF via reductive amination. The resulting G-CSF-P1 conjugate retained higher bioactivity compared to native G-CSF when subjected to heat and lyophilization stressors.
520 $a Finally, the trehalose glycopolymers were investigated for the stabilization and preservation of cells. Three different approaches were evaluated. First, extracellular protection by the covalent attachment of trehalose polymers to cells using modified sugars was tested. To evaluate the effectiveness of extracellular trehalose on cell stabilization, the trehalose polymers were added as excipients. Jurkat cells were heated at 50 °C and the added trehalose polymer did not significantly stabilize the cells at longer stress times. Cryopreservation with a combination of the polymer and dimethylsulfoxide (DMSO) showed slight increase in cell viabilities (65.7% +/- 3.1 with polymer and DMSO compared to 55.6% +/- 2.1 with DMSO only), but the results suggest that trehalose polymers are not likely to fully replace DMSO as a cryopreservative agent. The final approach utilized a bacterial pore-forming toxin, streptolysin O (SLO), to permeabilize human dermal fibroblast (HDF) cells for loading trehalose polymers. Permeabilization and loading was characterized by fluorescence with a fluorophore-labeled trehalose polymer and by the anthrone assay. No significant improvements in cell preservation were observed through trehalose polymer loading by this approach. Other techniques for cytosolic uptake of trehalose polymers should be considered for future work.
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