CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form

Oueslati, Nadia; Leblanc, Pierrick; Harscoat–Schiavo, Christelle; Rondags, Emmanuel; Meunier, Stéphane; Kapel, Romain; Marc, Ivan

doi:10.1016/j.carbpol.2014.05.039

Cited by 66 publications

(44 citation statements)

References 22 publications

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“…Furthermore, a hit was stringently identified for having at least a 1.5‐fold improvement in titre over that of the WT. Previous studies have used the CTAB assay in a 96‐well MTP format; this reiterates its suitability as a screening system platform for the pmHAS KnowVolution campaign.…”

Section: Discussionsupporting

confidence: 92%

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Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High‐Molecular‐Weight Hyaluronic Acid

et al. 2018

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Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain-length specificity and a twofold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

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Section: Discussionsupporting

confidence: 92%

“…The 96‐well MTP screening of HA by CTAB turbidimetric assay : The CTAB turbidimetric assay based on a 96‐well format was slightly modified . This method depended on the formation of an insoluble complex between HA and CTAB, for which the turbidity was linearly proportional to the quantity of HA in the system .…”

Section: Methodsmentioning

confidence: 99%

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High‐Molecular‐Weight Hyaluronic Acid

et al. 2018

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“…To determine the content of GAGs in pdECM and native tissue, 1,9-dimethyl methylene blue (DMMB) (Sigma-Aldrich) assay was used as previously described 31 . The HA content in the pdECM and native tissue was quantified by carbazole assay as previously described 32 . The collagen content in the pdECM and native tissue was quantified by hydroxyproline assay as previously described 33 .…”

Section: Methodsmentioning

confidence: 99%

The mode and dynamics of glioblastoma cell invasion into a decellularized tissue-derived extracellular matrix-based three-dimensional tumor model

Koh

Cha

Park

et al. 2018

Sci Rep

136

100

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Glioblastoma multiforme (GBM) is the most common brain tumor with very aggressive and infiltrative. Extracellular matrix (ECM) plays pivotal roles in the infiltrative characteristics of GBM.To understand the invasive characteristic of GBM, it is necessary to study cell-ECM interaction in the physiologically relevant biomimetic model that recapitulates the GBM-specific ECM microenvironment. Here, we propose biomimetic GBM-specific ECM microenvironment for studying mode and dynamics of glioblastoma cell invasion. Using tissue decellularization process, we constructed a patient tissuederived ECM (pdECM)-based three-dimensional in vitro model. In our model, GBM cells exhibited heterogeneous morphology and altered the invasion routes in a microenvironment-adaptive manner. We further elucidate the effects of inhibition of ECM remodeling-related enzymatic activity (Matrix metalloproteinase (MMP) 2/9, hyaluronan synthase (HAS)) on GBM cell invasion. Interestingly, after blocking both enzyme activity, GBM cells underwent morphological transition and switch the invasion mode. Such adaptability could render cell invasion resistant to anti-cancer target therapy. There results provide insight of how organ-specific matrix differentially regulates cancer cell phenotype, and have significant implications for the design of matrix with appropriate physiologically relevant properties for in vitro tumor model.Invasion and dissemination of cancer cells cause migration of neoplastic cells into surrounding tissues, resulting in mortality in tumor patients 1-3 . In particular, cancer cell invasion is of enormous clinical importance, since it involves both distant metastasis and local spreading, whereby cancer cells degrade and migrate through the tissue. The significance of cancer cell invasion is evident in the glioblastoma multiforme (GBM), which shows infiltrative and rapid growth into the surrounding brain tissue. The invasive and migratory characteristics of GBM cells provide a wealth of information about tumors within a patient 3 .Three-dimensional (3D) in vitro culture systems are increasingly employed to assess cell-ECM interactions and invasion of tumor cells. Indeed, cell behavior and responsiveness are dramatically different in a 3D physiological environment versus two-dimensional (2D) Petri dish conditions 4,5 . Importantly, in 3D cultures, ECM is an essential determinant of the cellular response to invasion and migration processes 6 . Brain ECM has a distinct composition from ECM in other tissues and organs, with a low stiffness and loosely connected cellular network. The ECM component of brain tissues contains high amounts of hyaluronic acid (HA), glycosaminoglycans (GAGs), and proteoglycans, but lacks fibrous materials such as collagen, fibronectin, etc 7,8 .In fact, the interaction between GBM cells and unique extracellular environment of the brain could impact on the invasive characteristics of GBM cells. Accumulated experimental and clinical data demonstrate that invasion

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“…The hexadecyltrimethylammonium bromide turbidimetric method (CTM) has been demonstrated as an accurate, sensitive and specific method for HA quantification. 37,38 Thus, it was applied to quantify HA amount on the surface of HA-KPV-NPs in the present study. We found that the HA amount on HA-KPV-NPs was 63.1 ± 1.4 mg/ mg of NPs.…”

Section: Physicochemical Characterization Of Npsmentioning

confidence: 99%

Orally Targeted Delivery of Tripeptide KPV via Hyaluronic Acid-Functionalized Nanoparticles Efficiently Alleviates Ulcerative Colitis

et al. 2017

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Overcoming adverse effects and selectively delivering drug to target cells are two major challenges in the treatment of ulcerative colitis (UC). Lysine-proline-valine (KPV), a naturally occurring tripeptide, has been shown to attenuate the inflammatory responses of colonic cells. Here, we loaded KPV into hyaluronic acid (HA)-functionalized polymeric nanoparticles (NPs). The resultant HA-KPV-NPs had a desirable particle size (∼272.3 nm) and a slightly negative zeta potential (∼-5.3 mV). These NPs successfully mediated the targeted delivery of KPV to key UC therapy-related cells (colonic epithelial cells and macrophages). In addition, these KPV-loaded NPs appear to be nontoxic and biocompatible with intestinal cells. Intriguingly, we found that HA-KPV-NPs exert combined effects against UC by both accelerating mucosal healing and alleviating inflammation. Oral administration of HA-KPV-NPs encapsulated in a hydrogel (chitosan/alginate) exhibited a much stronger capacity to prevent mucosa damage and downregulate TNF-α, thus they showed a much better therapeutic efficacy against UC in a mouse model, compared with a KPV-NP/hydrogel system. These results collectively demonstrate that our HA-KPV-NP/hydrogel system has the capacity to release HA-KPV-NPs in the colonic lumen and that these NPs subsequently penetrate into colitis tissues and enable KPV to be internalized into target cells, thereby alleviating UC.

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CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form

Cited by 66 publications

References 22 publications

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High‐Molecular‐Weight Hyaluronic Acid

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High‐Molecular‐Weight Hyaluronic Acid

The mode and dynamics of glioblastoma cell invasion into a decellularized tissue-derived extracellular matrix-based three-dimensional tumor model

Orally Targeted Delivery of Tripeptide KPV via Hyaluronic Acid-Functionalized Nanoparticles Efficiently Alleviates Ulcerative Colitis

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