Project description:Diabetic wounds require continuous and coordinated modulation of the microenvironment concurrent with tissue regeneration, yet this remains a significant challenge. As a proof of concept, we herein propose to use dimeric copper peptide for diabetic wound treatment. The dimeric copper peptide (D-CuP) was first synthesized and then incorporated into a reactive oxygen species (ROS)-responsive hydrogel matrix to improve therapeutic compliance, culminating in the formulation of G/D-CuP. Compared to monomer copper peptide (CuP), a wound healing agent, D-CuP exhibits multivalency, enhanced biologic stability against proteases and the broad biological activities, such as anti-inflammatory and antioxidative properties, while promoting angiogenesis and fibroblast proliferation and migration. Meanwhile, the hydrogel matrix, exhibiting excellent ROS-scavenging capabilities, has been engineered to an intelligent drug reservoir for wound-responsive release of D-CuP at the wound site while simultaneously attenuating inflammatory responses. Ultimately, the G/D-CuP group demonstrated superior therapeutic efficacy, achieving in 97.2% closure of infected wounds.In this study, we present a proof-of-concept study to optimize the treatment of diabetic wounds. This method integrates a dimeric copper peptide hydrogel (G/D-CuP) to address several critical factors: i) The design and synthesis of the dimeric copper peptide (D-CuP) feature a broad range of targets and enhanced biological activities, such as anti-inflammatory and antioxidative properties, while promoting angiogenesis and fibroblast proliferation and migration; ii) Utilizing the steric hindrance conferred by dimerization reduces protease access to the active site and significantly improving stability against proteases; iii) A ROS-responsive hydrogel matrix has been synthesized, exhibiting superior ROS-scavenging capabilities, functioning as an intelligent drug reservoir that enables the controlled release of D-CuP at the wound interface while simultaneously attenuating inflammatory responses in the microenvironment; iv) G/D-CuP possesses outstanding deformability and spreadability, allowing it to adapt to any wound shape; v) The synthesis and preparation process of G/D-CuP are straightforward and cost-effective, making it highly promising for clinical translation. This multifunctional treatment platform is anticipated to accommodate the complex and dynamic biological processes involved in diabetic wound healing, thereby significantly improving the overall efficacy of wound healing treatments.

Project description:Aim The experiment aimed to analyse genome-wide DNA methylation re-patterning in regenerating ear pinnae in mice in response to zebularine and retinoic acid treatment. Animals The experiments were conducted on 8 to 10-week-old females of the BALB/c mouse strain in the Tri-City Academic Laboratory Animal Centre. After the mice were anaesthetised with isoflurane, through-and-through holes of 2 mm diameter were made in the ear pinna centre using a scissor-style ear punch (Hammacher Solingen; LOT FTC-15/8670/1). Next, the animals were randomised into groups of six. Immediately after wounding, the mice were administered subcutaneous injections of alginated hydrogel formulations. Mice in one group received zebularine (48 mg in 200 µl of 2% alginate hydrogel) and retinoic acid (0.8 mg in 200 µl of 2% alginate hydrogel), mice in the control group received 400 alginate hydrogel carriers each. The protocol for animal experiments was approved by the Local Ethics Committee for Animal Experimentation in Bydgoszcz (approval no. 51/2020). Tissue collection and genomic DNA extraction The tissues were collected 7 days after the injury, placed immediately in liquid nitrogen and stored at -80°C. Genomic DNA was extracted from 3 mm rings surrounding the initial punch wounds using DNeasy Blood and Tissue kit (Qiagen.) Each DNA sample was extracted from a pair of ear pinna rings from the same animal. BSseq DNA samples from a pair of ear pinnae from each animal were pooled. Extracted DNA was quantified spectrophotometrically on the Varioskan Lux (ThermoFisher Scientific), and DIN (DNA integrity number) was determined with Genomic DNA Screen Tape Analysis on 4510 TapeStation System (Agilent). All samples with DIN >7.0 were further processed. Reduced-representation bisulfite sequencing (RRBS) was performed by the Genomic Core Facility at the University of Alabama at Birmingham using the Ovation RRBS Methyl-Seq kit (Tecan Genomics), followed by DNA libraries sequencing on NextSeq 500 platform (Illumina). Data processing Following Tecan's Analysis Guide (https://github.com/nugentechnologies/NuMetRRBS), Trim Galore (version 0.6.7) was used to remove residual adapter sequences from the reads. Diversity trimming and filtering with their trimming script were then applied to the trimmed FASTQ files.

Project description:Aim The experiment aimed to determine transcriptome changes in regenerating ear pinnae in mice in response to zebularine and retinoic acid treatment. Animals The experiments were conducted on 8 to 10-week-old females of the BALB/c mouse strain in the Tri-City Academic Laboratory Animal Centre. After the mice were anaesthetised with isoflurane, through-and-through holes of 2 mm diameter were made in the ear pinna centre using a scissor-style ear punch (Hammacher Solingen; LOT FTC-15/8670/1). Next, the animals were randomised into groups of six. Immediately after wounding, the mice were administered subcutaneous injections of alginated hydrogel formulations. Mice in one group received zebularine (48 mg in 200 µl of 2% alginate hydrogel) and retinoic acid (0.8 mg in 200 µl of 2% alginate hydrogel), mice in the control group received 400 alginate hydrogel carriers each. The protocol for animal experiments was approved by the Local Ethics Committee for Animal Experimentation in Bydgoszcz (approval no. 51/2020). Tissue collection and RNA extraction The tissues were collected 7 days after the injury, placed immediately in liquid nitrogen and stored at -80°C. Total RNA was extracted from 3 mm rings surrounding the initial punch wounds using RNeasy Mini kit (Qiagen). Each RNA sample was extracted from a pair of ear pinna rings from the same animal. RNAseq RNA samples from a pair of ear pinnae from each animal were pooled. The quantity of the extracted RNA was determined fluorometrically on Qubit RNA assay (ThermoFisher Scientific), while the RIN (RNA integrity number) was determined using RNA Screen Tape Analysis on 4150 TapeStation System (Agilent). TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat kit (Illumina) was used for library construction, followed by 5' and 3' adapter ligation. The cDNA libraries were sequenced in a paired-end mode on the Illumina platform as a service provided by Macrogen. Data processing Sequencing data was converted into FASTQ format with Illumina's bcl2fastq converter. All samples were trimmed using Trim Galore (version 0.6.7), and these trimmed reads were successfully aligned to Gencode's GRCm39 Release M28 mouse reference genome using STAR (version 2.7.10a). Transcript assembly and estimation of the relative abundances were performed with HTSeq-count (version 2.0.2). Raw counts were then normalised using DESeq2 (version 1.36.0).

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Wound healing and anti-oxidant activities induced by Dersani Hydrogel with Alginate and Dersani Hydrogel ointments

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