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An investigation of alkaline phosphatase enzymatic activity after electrospinning and electrospraying.

ABSTRACT: The high target specificity and multifunctionality of proteins has led to great interest in their clinical use. To this end, the development of delivery systems capable of preserving their bioactivity and improving bioavailability is pivotal to achieve high effectiveness and satisfactory therapeutic outcomes. Electrohydrodynamic (EHD) techniques, namely electrospinning and electrospraying, have been widely explored for protein encapsulation and delivery. In this work, monoaxial and coaxial electrospinning and electrospraying were used to encapsulate alkaline phosphatase (ALP) into poly(ethylene oxide) fibres and particles, respectively, and the effects of the processing techniques on the integrity and bioactivity of the enzyme were assessed. A full morphological and physicochemical characterisation of the blend and core-shell products was performed. ALP was successfully encapsulated within monolithic and core-shell electrospun fibres and electrosprayed particles, with drug loadings and encapsulation efficiencies of up to 21% and 99%, respectively. Monoaxial and coaxial electrospinning were equally effective in preserving ALP function, leading to no activity loss compared to fresh aqueous solutions of the enzyme. While the same result was observed for monoaxial electrospraying, coaxial electrospraying of ALP caused a 40% reduction in its bioactivity, which was attributed to the high voltage (22.5 kV) used during processing. This demonstrates that choosing between blend and coaxial EHD processing for protein encapsulation is not always straightforward, being highly dependent on the chosen therapeutic agent and the effects of the processing conditions on its bioactivity.

SUBMITTER: Onyekuru LC

PROVIDER: S-EPMC8312041 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundComprehensive characterization of the phosphoproteome in living cells is critical in signal transduction research. But the low abundance of phosphopeptides among the total proteome in cells remains an obstacle in mass spectrometry-based proteomic analysis. To provide a solution, an alternative analytic strategy to confidently identify phosphorylated peptides by using the alkaline phosphatase (AP) treatment combined with high-resolution mass spectrometry was provided. While the process is applicable, the key integration along the pipeline was mostly done by tedious manual work.ResultsWe developed a software toolkit, iPhos, to facilitate and streamline the work-flow of AP-assisted phosphoproteome characterization. The iPhos tookit includes one assister and three modules. The iPhos Peak Extraction Assister automates the batch mode peak extraction for multiple liquid chromatography mass spectrometry (LC-MS) runs. iPhos Module-1 can process the peak lists extracted from the LC-MS analyses derived from the original and dephosphorylated samples to mine out potential phosphorylated peptide signals based on mass shift caused by the loss of some multiples of phosphate groups. And iPhos Module-2 provides customized inclusion lists with peak retention time windows for subsequent targeted LC-MS/MS experiments. Finally, iPhos Module-3 facilitates to link the peptide identifications from protein search engines to the quantification results from pattern-based label-free quantification tools. We further demonstrated the utility of the iPhos toolkit on the data of human metastatic lung cancer cells (CL1-5).ConclusionsIn the comparison study of the control group of CL1-5 cell lysates and the treatment group of dasatinib-treated CL1-5 cell lysates, we demonstrated the applicability of the iPhos toolkit and reported the experimental results based on the iPhos-facilitated phosphoproteome investigation. And further, we also compared the strategy with pure DDA-based LC-MS/MS phosphoproteome investigation. The results of iPhos-facilitated targeted LC-MS/MS analysis convey more thorough and confident phosphopeptide identification than the results of pure DDA-based analysis.

Project description:BACKGROUND:Necrotizing enterocolitis (NEC) is a devastating disease of intestinal inflammation that primarily affects premature infants. A potential risk factor for necrotizing enterocolitis is exposure of the premature neonatal intestine to environmental bacteria and their proinflammatory products such as lipopolysaccharide. The metalloenzyme alkaline phosphatase (ALP) has been shown to reduce lipopolysaccharide-mediated inflammation. Additionally, premature rat pups have reduced alkaline phosphatase activity and expression as compared to full term pups. To explore the possibility that the human premature neonatal intestine has a paucity of alkaline phosphatase activity, we measured endogenously produced intestinal alkaline phosphatase activity in meconium as a function of gestational age. To test whether breast milk could serve as a source of exogenous alkaline phosphatase to the neonatal intestine through ingestion, we measured alkaline phosphatase activity in breast milk across a range of time points post-birth. METHODS:Alkaline phosphatase activity was quantified in 122 meconium samples from infants of gestational ages ranging from 24 to 40?weeks and in 289 breast milk samples collected from 78 individual mothers between days 2-49 post-birth. RESULTS:We observed a strong positive correlation between the meconium alkaline phosphatase activity and gestational age, with preterm infants having lower meconium alkaline phosphatase activities than early term or term infants. Breast milk alkaline phosphatase activity was highest in the first week post-birth, with peak alkaline phosphatase activity at day 2 post-birth, followed by relatively low alkaline phosphatase activity in weeks 2-7. CONCLUSIONS:Our results are consistent with the two major risk factors for necrotizing enterocolitis development, preterm birth and lack of breast milk feeding, both contributing to a paucity of alkaline phosphatase activity and impaired capacity to detoxify proinflammatory bacterial products such as lipopolysaccharide.

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An investigation of alkaline phosphatase enzymatic activity after electrospinning and electrospraying.

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