ABSTRACT
INTRODUCTION: Diabetic foot infection (DFI) is one of the complications of diabetes mellitus. Clindamycin (CLY) is one of the antibiotics recommended to treat DFI, but CLY given orally and intravenously still causes many side effects. METHODS: In this study, we encapsulated CLY in a bacteria sensitive microparticle system (MP-CLY) using polycaprolactone (PCL) polymer. MP-CLY was then delivered in a separable effervescent microarray patch (MP-CLY-SEMAP), which has the ability to separate between the needle layer and separable layer due to the formation of air bubbles when interacting with interstitial fluid in the skin. RESULT: The characterization results of MP-CLY proved that CLY was encapsulated in large amounts as the amount of PCL polymer used increased, and there was no change in the chemical structure of CLY. In vitro release test results showed increased CLY release in media cultured with Staphylococcus aureus bacteria and showed controlled release. The characterization results of MPCLY-SEMAP showed that the developed formula has optimal mechanical and penetration capabilities and can separate in 56 ± 5.099 s. An ex vivo dermatokinetic test on a bacterially infected skin model showed an improvement of CLY dermatokinetic profile from MP-CLY SEMAP and a decrease in bacterial viability by 99.99%. CONCLUSION: This research offers proof of concept demonstrating the improved dermatokinetic profile of CLY encapsulated in a bacteria sensitive MP form and delivered via MP-CLY-SEMAP. The results of this research can be developed for future research by testing MP-CLY-SEMAP in vivo in appropriate animal models.
Subject(s)
Anti-Bacterial Agents , Clindamycin , Diabetic Foot , Skin , Staphylococcus aureus , Clindamycin/administration & dosage , Diabetic Foot/drug therapy , Diabetic Foot/microbiology , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/pharmacology , Staphylococcus aureus/drug effects , Animals , Skin/microbiology , Skin/metabolism , Polyesters/chemistry , Drug Delivery Systems/methods , Drug Liberation , Administration, Cutaneous , Transdermal Patch , Humans , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Drug Carriers/chemistryABSTRACT
The Hippo tumor suppressor pathway is an important regulator of cell proliferation and apoptosis, and signal transduction occurs through phosphorylation of the effector protein TAZ by the serine/threonine kinase LATS1/2. Here, we report the biophysical and computational studies to characterize the interaction between TAZ and LATS1/2 through WW domain-PPxY motif binding. We show that the TAZ WW domain exhibits a binding preference for the second of the two PPxY motifs of LATS1 in vitro. We modelled the structure of the domain in complex with LATS1 PPxY2 peptide and, through molecular dynamics simulations, show that WW domain-PPxY2 complex is stable with some flexibility in the peptide region. Next, we predict and verify that L143 and T150 of the WW domain are important for TAZ binding with the PPxY2 peptide using mutational and isothermal titration calorimetric studies. Furthermore, we suggest that the electrostatic potential of charged residues within the binding pocket may influence the ligand affinity among otherwise highly similar WW domains.
Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Amino Acid Substitution , Biophysical Phenomena , Humans , Intracellular Signaling Peptides and Proteins/genetics , Models, Molecular , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/genetics , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Structural Homology, Protein , Trans-Activators , Transcription Factors , Transcriptional Coactivator with PDZ-Binding Motif Proteins , WW Domains/geneticsABSTRACT
Diabetes mellitus can cause diabetic foot infection (DFI) complications. DFI is generally caused by infection from bacteria and Methicillin-Resistant Staphylococcus aureus (MRSA) which is resistant to several antibiotics. Application therapy of clindamycin (CLY) administration with the oral route has low bioavailability and non-selective distribution of antibiotics towards bacteria intravenously. In this research, CLY was developed into bacterially sensitive microparticles (MPs) which were further incorporated into a separable effervescent microarray patch (SEMAP) system to increase the selective and responsive to DFI-causing bacteria of CLY. To support this formulation, we explore the potential of silver nanoparticles (AgNPs) towards the UV-Vis spectrophotometry method. The analytical method was validated in phosphate-buffered saline (PBS), tryptic soy broth (TSB), and skin tissue to quantify CLY, CLY loaded in microparticle, and SEMAP system. The developed analytical method was suitable for the acceptance criteria of ICH guidelines. The results showed that the correlation coefficients were linear ≥ 0.999. The values of LLOQ towards PBS, TSB, and skin tissue were 2.02 µg/mL, 4.29 µg/mL, and 2.31 µg/mL, respectively. These approaching methods were also found to be accurate and precise without being affected by dilution integrity. The presence of Staphylococcus aureus bacteria culture can produce lipase enzymes that can lysing the microparticle matrix. Drug release studies showed that bacterial infection in the high drug release microparticle sensitive bacteria and high drug retention in ex vivo dermatokinetic in rat skin tissue media. In addition, in vivo studies were required to quantify the CLY inside in further analytical validation methods.