pH-responsive cationic polymer-functionalized poly-ε-caprolactone microspheres scavenge cell-free-DNA to alleviate intestinal ischemia/reperfusion harm by inhibiting M1 macrophage polarization | Journal of Nanobiotechnology

Affected person pattern assortment and knowledge recording

Ten sufferers who underwent laparoscopic gastrointestinal surgical procedure have been included on this examine. The affected person recruitment was permitted by the Ethics Committee of the Third Affiliated Hospital of Solar Yat-sen College (II2023-272-01). Samples of peritoneal drainage fluid and blood have been collected from sufferers instantly and 6 h after surgical procedure for evaluation. To attenuate the chance of blood contamination, we particularly chosen drainage fluid that was distant from the stomach incision drainage tube. Moreover, to make sure that the pattern remained unaffected by blood cells, we promptly subjected the collected drainage fluid to low-temperature centrifugation and subsequently saved it at -80℃. The operative period was recorded from the surgical anesthesia middle system, and the time of the primary postoperative flatus was obtained from the gastrointestinal surgical procedure nursing system.

Institution and remedy of the mouse intestinal I/R harm mannequin

This animal examine was permitted by the Animal Ethics Committee of the South China Agricultural College (2023F202). Male C57BL/6J mice (6–8 weeks outdated) have been bought from the Animal Middle of South China Agricultural College. The animals have been housed below particular pathogen-free circumstances with an ordinary 12/12 h mild/darkish cycle. The mice fasted the night time earlier than the experiment however have been permitted free entry to water. The experimental teams have been as follows: sham, I/R, and Micro DEA2k. As regards to the literature, after the mice have been anesthetized with 1% isoflurane (Shenzhen RWD Life Science), a midline incision was made, the layers have been opened, and the superior mesenteric artery (SMA) was remoted. A noninvasive microvascular arterial clamp was used to occlude the SMA for 45 min, after which the clamp was eliminated to revive SMA blood movement [17, 18]. The stomach incision was closed with steady sutures utilizing 3 − 0 silk thread. For the Micro DEA2k (0.4 mg/mL) group, 1 mL was injected intraperitoneally earlier than closing the incision. The I/R group was intraperitoneally injected with an equal quantity of 1× phosphate buffered saline (PBS) (ProCell). For the Sham group, the SMA was remoted with out clamping. Instantly after surgical procedure, every mouse was injected subcutaneously with preheated sterile saline (0.5 mL) for fluid resuscitation and was saved heat utilizing a heating pad. Mice have been monitored for twenty-four h to document their survival charges. Medical scoring was additionally carried out based mostly on the next standards: 0, no signs; 1, piloerection and huddling; 2, piloerection, huddling, and diarrhea; 3, lack of curiosity within the environment and extreme diarrhea; 4, decreased motion and lethargy; and 5, lack of self-righting reflexes [19]. The mice have been humanely euthanized at a rating of 5.

Molecular docking

The Drew-Dickerson dodecamer (1BNA) was used because the DNA mannequin for molecular docking. Its crystal construction file was obtained from the Protein Knowledge Financial institution database, which was saved as a pdbqt file after eradicating water molecules and including hydrogen utilizing AutoDock 4.2.6 and AutoDockTools 1.5.7 [20, 21]. Two consecutive repeating models of the cation section have been chosen because the mannequin ligands. After MM2 vitality minimization, they have been transformed into pdb format utilizing OpenBabel 3.1.1 (with an additional setting of hydrogen addition at pH = 1 for the protonated ligand mannequin) and imported into AutoDockTools 1.5.7, the place Gasteiger expenses have been added after the addition of hydrogen. After setting the torsional bonds and torsional facilities, the file was exported as a pdbqt file. The scale of the lattice level area was set to 60 nm × 80 nm × 120 nm, such that the docking lively website spanned the whole DNA molecular mannequin, with a spacing of 0.375 Å. After exporting the gpf file, AutoGrid was used to carry out lattice level calculations. The parameters associated to molecular docking have been set utilizing a genetic algorithm (GA) to generate 100 closing conformations. Every run had a most vitality analysis of two.5 million occasions and a most variety of steps of 27,000, with all different parameters retaining their default system settings. Within the docking run choices, parameters similar to random quantity technology, vitality, step measurement, and export format have been retained at their default settings. The dpf file was exported utilizing the Lamarckian GA. Lastly, the dpf file was used to carry out molecular docking, and the molecular docking binding energies have been calculated utilizing the dlg file. The binding pose with the bottom vitality was chosen and visualized utilizing UCSF Chimera 1.16. The unbound state is outlined because the state during which the ligand reaches equal free vitality at totally different positions when it’s far-off from the receptor, and it may be assumed that no interplay happens between the receptor and ligand.

Synthesis of DEA2k and Micro DEA2k

Synthesis of α-bromo-ℇ-caprolactone (CL-Br). Initially, 57.12 g of N-bromosuccinimide (NBS, 99.00%, Aladdin) and 5.81 g of p-toluenesulfonic acid (98.5%, Aladdin) have been dissolved in 200 mL of dichloromethane (DCM, 99.5%, Guanghua Sci-Tech) and stirred in an ice-water tub. Then, 30.00 g of cyclohexanone (99.0%, Macklin) was added dropwise. The combination was subjected to chilly vacuum filtration to take away the salts, adopted by extraction and washing. The extraction was carried out utilizing a saturated sodium chloride answer, adopted by washing and drying over anhydrous sodium sulfate to acquire 38.52 g of 2-bromocyclohexanone. Subsequent, the 2-bromocyclohexanone was dissolved in 250 mL of DCM, and m-chloroperoxybenzoic acid (mCPBA, 55.07 g, 239.35 mmol) was added. The combination was refluxed in a single day at 50 °C after which cooled. The answer was extracted with saturated sodium thiosulfate answer, saturated sodium bicarbonate answer, and deionized water. It was then extracted with saturated sodium chloride answer to take away water and dried over anhydrous sodium sulfate in a single day. The ensuing crystals have been recrystallized from a DCM/n-hexane combination at -20 °C to acquire 23.48 g of white crystals, recognized as CL-Br. The product was positioned in a microwave reactor (Initiator+, Biotage) with 3.41 g of the macroinitiator PEG (common Mn 2000, Aladdin) and reacted at 110 °C for 4 h for ring-opening polymerization (ROP), which produced 8.84 g of the triblock copolymer P(CL-Br)-b-PEG-b-P(CL-Br). Following this course of, the triblock copolymer initiated ROP in 2.57 g of ε-caprolactone (97%, Bide Pharmatech), which yielded the pentablock copolymer PCL-b-P(CL-Br)-b-PEG-b-P(CL-Br)-b-PCL. Subsequent, 2 g of the pentablock copolymer was weighed and modified by including 1.51 g of N, N-diethyl-N’-methyl ethylenediamine (98%, Macklin); the ensuing product was named DEA2k. After 1 mg/mL of DEA2k was blended evenly with 1 mg/mL of PCL microspheres, centrifugation was carried out utilizing a tabletop centrifuge (Centrifuge 5810R, Eppendorf) at 15,000 rpm for 10 s to precipitate the microspheres. The supernatant containing free DEA2k was eliminated. The mass m_m of the microspheres was obtained by lyophilization utilizing the load discount technique, and the whole mass m_t was obtained by lyophilization after direct mixing. The floor polymer content material per unit mass of microspheres was calculated to be 85.7% utilizing the next formulation:

The overwhelming majority of the polymers have been efficiently hooked up to the floor of the microspheres, and the product was named Micro DEA2k.

Characterization of DEA2k and Micro DEA2k

Proton nuclear magnetic resonance (NMR) spectra have been recorded at room temperature utilizing an NMR spectrometer (AVANCE III 400 MHz, Bruker). The relative molecular weight and dispersity have been measured by gel permeation chromatography (GPC) (1260 Infinity, Agilent). The morphology was characterised utilizing transmission electron microscopy (TEM, JEOL1400+). The hydrated measurement (Measurement), polydispersity index (PDI), and zeta potential have been measured by dynamic mild scattering (DLS, Malvern Zetasizer Nano ZS) at 25 ºC.

In vitro cytotoxicity assay

RAW264.7 cells, bought from the ATCC cell financial institution, have been cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Applied sciences), which contained 10% fetal bovine serum (FBS; Gibco Life Applied sciences) and 1% bispecific antibody (Pen Strep, Penicillin Streptomycin, Gibco) in an incubator (Steri-Cycle 371, Thermo Scientific) at 37 °C and 5% carbon dioxide. Cells in good situation have been chosen and seeded right into a 96-well plate at roughly 10⁴ cells per effectively. After culturing in a single day, the tradition medium was changed with recent medium containing totally different concentrations of cationic polymers. After 24 h of incubation, 10 µL of 5 mg/mL methylthiazolyldiphenyl-tetrazolium bromide answer (MTT, 98%, Sigma-Aldrich) was added and incubated for one more 4 h. After eradicating the supernatant, 10 µL of dimethyl sulfoxide (DMSO, 99.7%, Guangzhou Chemical Reagent Manufacturing unit) was added to every effectively to dissolve the pellet. Lastly, the absorbance was measured at 570 nm utilizing a microplate reader (BioTek Synergy2 Gen 5). The cell survival price was calculated by (Q–Q₀)/(Q₁–Q₀)×100%, the place Q is the optical density (OD) of cells with the polymer, Q₀ is the OD of wells with out cells, and Q₁ is the OD of cells with out the polymer. Every experiment was repeated 3 times. The IC50 was calculated by becoming a logistic equation.

Protein adsorption capability assay

Resistance to protein adsorption was assessed utilizing the BCA Protein Assay Package (Pierce™ BCA Protein Assay Package, Thermo Scientific). Firstly, 50 µL of two mg/mL bovine serum albumin (BSA, Aladdin) answer was blended evenly with 50 µL of 1 mg/mL cationic materials. The management group was established by mixing 50 µL of deionized water with 50 µL of BSA answer. After incubation at 37 °C in a shaker for 1 h, the samples have been centrifuged at 8000 rpm for five min, and the supernatant was collected. Calibration curves have been obtained for BSA options with totally different concentrations, and the residual BSA focus within the pattern supernatant was decided based mostly on ultraviolet absorption (280 nm). The quantity of BSA adsorbed onto the cationic polymer was calculated utilizing (C_C–C_T)V/m, the place C_C is the BSA focus of the management group (1 mg/mL), C_T is the BSA focus of the supernatant after the addition of the cationic polymer, V is the quantity of the answer (100 µL), and m is the load of the cationic polymer (50 µg). Every experiment was repeated 3 times.

Potentiometric acid-base titration

DEA2k was dissolved in 10 mL of hydrochloric acid customary answer to yield a focus of 500 µM (amine group content material: 10 mM), and 0.1 M sodium hydroxide customary answer was step by step added dropwise to yield a pH of two.00; this served as the start line for the titration. Subsequent, a 0.1 M sodium hydroxide customary answer was added dropwise with fixed stirring, and the cumulative quantity of sodium hydroxide answer added and the corresponding pH worth have been measured, and the titration curve was plotted. The height of the first-order spinoff of the curve is the purpose of sudden pH change, outlined because the beginning and ending factors of amine protonation. The connection between the diploma of protonation α and pH through the course of was calculated by utilizing the next equation:

the place Φ is the pH worth of the answer, V is the cumulative quantity of sodium hydroxide answer added at that pH, and subscripts i and t denote the beginning and ending factors of deprotonation, respectively. The protonation curve of the cationic polymer was fitted by a mixture of two logistic equations, calculated as follows:

the place B₁ and C₁ correspond to easier-to-protonate amine teams, B₂ and C₂ correspond to harder-to-protonate amine teams, and E represents the proportion of easier-to-protonate amine teams. The buffer capability (β) at totally different pH values will be calculated from the fitted diploma of protonation of the cationic polymer, and the formulation is as follows:

Measurement of DNA binding capability

The binding capability of the cationic polymer to CpG1826 was measured by agarose gel electrophoresis. Cationic polymer options with totally different concentrations have been blended with 0.4 µL of 1 mg/mL CpG1826 answer (ODN 1826, 5’-tccatgacgttcctgacgtt-3,’ GenScript) to acquire complicated options with totally different nitrogen-to-phosphorus ratios, and the CpG1826 aqueous answer was used as a clean management. After permitting the options to face at room temperature for 0.5 h, they have been added to 2 µL of 1% agarose gel containing a nucleic acid dye (Goldview nucleic acid dye (10,000 × DMSO), Biosharp, China). Electrophoresis was carried out in 1% tris-acetate-EDTA (TAE) buffer answer (voltage 60 V, 45 min, DYY-6 C, Beijing Liuyi). Lastly, the gels have been photographed and analyzed utilizing a gel imaging system (Gel Doc XR+; Bio-Rad).

The binding capability of the cationic polymer was quantitatively assessed by aggressive binding to ethidium bromide (EtBr). Firstly, 2.17 µL of 1 mg/mL EtBr (94%, Acros Organics) was added to three mL of PBS or sodium acetate buffered answer (SABS) to simulate totally different pH values, and the fluorescence emission spectrum of the answer was measured. Then, 10 µL of 1 mg/mL ctDNA (Sigma-Aldrich) answer was added and blended evenly to extend the fluorescence depth of the answer. Subsequently, totally different volumes of cationic polymer answer (1 mg/mL) have been added sequentially and blended. The fluorescence emission curve was measured utilizing a fluorescence spectrometer (excitation wavelength: 280 nm, detection emission wavelength: 500–800 nm).

In vitro anti-inflammatory assay

RAW264.7 macrophages have been added to 96-well plates at a density of 1 × 10⁴ cells/effectively. Cells have been cultured in DMEM containing 10% FBS for 12 h. The tradition medium was changed with recent medium containing CpG1826 (0.5 µM), and the cells have been co-cultured for 4 h earlier than discarding the medium. After washing 3 times with PBS, recent medium containing cationic polymer (25 µg/mL) was added, and the cells have been incubated for twenty-four h. Then, the supernatant was collected for TNF-α and IL-6 assays. The group with out CpG1826 and cationic polymer within the medium served because the management group.

Western blot

RAW264.7 macrophages have been added to 6-well plates at a density of two × 10⁵ cells/effectively. Cells have been cultured in DMEM containing 10% FBS for 12 h. The tradition medium was changed with recent medium containing CpG1826 (0.5 µM), and the cells have been co-cultured for 4 h earlier than discarding the medium. After washing 3 times with PBS, recent medium containing cationic polymer (25 µg/mL) was added, and the cells have been incubated for twenty-four h. Centrifugation was carried out to gather the cells, and RIPA lysis buffer (Beijing Solarbio Expertise Co., Ltd.) was added, boiled, and centrifuged. The supernatant was collected to acquire the protein samples. The totally different protein samples have been separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins on the PAGE gel have been transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membrane bands of the corresponding protein molecular weights and applicable sizes have been excised and blocked in an answer of skim milk powder for 1 h. Then, they have been incubated in a single day on a shaker at 4 °C with the next main antibodies: anti-p65 (1:1000) (Cell Signaling Expertise, 8242 S, USA), anti-pp65 (1:1000) (Cell Signaling Expertise, 3033 S, USA), anti-TLR9 (1:1000) (ABclonal, A14642, China), anti-MyD88 (1:1000) (ABclonal, A0980, China), and anti-β-actin (1:50000) (ABclonal, AC026, China). After the first antibodies have been recovered, the samples have been washed thrice with tris-buffered saline with Tween 20 (TBST) answer (Biosharp, BL315B, China) for six min every and incubated with an anti-rabbit IgG secondary antibody (1:20000) (Cell Signaling Expertise, 7074 S, USA) at room temperature for 1 h. After discarding the secondary antibody, the samples have been washed 3 times with TBST for six min every, and enhanced chemiluminescence (ECL) publicity answer (Shanghai Beyotime Biotechnology Co., Ltd.) was added dropwise for imaging.

Confocal laser scanning microscopy

RAW264.7 macrophages have been seeded at a focus of 4 × 10⁵/mL in glass-bottom confocal dishes and incubated in a single day. After washing 3 times with PBS, serum-free DMEM was added, which contained 0.5 µM FAM fluorescently labeled CpG1826 (FAM-CpG1826, GenScript Biotech). After co-culturing for 4 h, the cells have been washed 3 times with PBS, and serum-free DMEM was added, which contained 25 µg/mL Micro DEA2k. After culturing for one more 4 h, the medium was discarded and the cells have been washed 3 times with PBS. Subsequent, the cells have been incubated with 0.2/mL of lysosomal inexperienced fluorescent probe (LysoTracker™ Inexperienced DND-99, Invitrogen) in serum-free DMEM for 30 min at midnight, adopted by 1 µg/mL of bisbenzimide H 33,342 trihydrochloride (Hoechst 33342, Sigma) in PBS for 10 min at midnight for staining. The colocalization of lysosomes, CpG1826, and Micro DEA2k was analyzed utilizing a laser confocal microscope (TCS SP8, Leica Microsystems, GmbH).

cfDNA purification and quantification

cfDNA was purified from serum, peritoneal drainage, and lavage fluid utilizing a DNA purification equipment (PCR Purification Package/DNA Purification Package, D0033, Shanghai Beyotime Biotechnology Co., Ltd.), and the focus of cfDNA was measured utilizing an ultra-micro spectrophotometer (Thermo NanoDrop 2000, Thermo Fisher Scientific Inc.).

HE staining

After fixation for twenty-four h, the tissue samples have been embedded in paraffin and sectioned utilizing a paraffin microtome (Leica) to acquire tissue sections with a thickness of two μm. Sections have been deparaffinized, stained with hematoxylin and eosin (HE), washed, dehydrated, and mounted. Routine HE staining was used to evaluate the histomorphological modifications in main organs, and an clever pathology imaging system microscope (Vectra, PerkinElmer) was used for picture acquisition and evaluation. The diploma of small intestinal harm after reperfusion was assessed utilizing the modified Chiu’s technique, which is predicated on modifications within the intestinal mucosal villi and glands [22]. The histological harm to the lungs, kidneys, and liver was assessed utilizing their respective scoring methods [23,24,25]. Automated picture evaluation was carried out on 5 randomly chosen 200× magnification photographs from every pattern.

Immunohistochemistry

After making ready paraffin sections in accordance with the steps above, the expression and site of F4/80 have been detected utilizing a main anti-F4/80 antibody (1:500) (GB113373, Servicebio) and a labeled goat anti-rabbit secondary antibody (1:200) (GB23303, Servicebio). The expression and localization of TLR9 have been detected utilizing a main anti-TLR9 antibody (1:300) and a labeled goat anti-rabbit secondary antibody (1:200) (GB23303, Servicebio). The expression and site of MyD88 have been detected utilizing a main anti-MyD88 antibody (1:1000) (GB111554, Servicebio) and a labeled goat anti-rabbit secondary antibody (1:200) (GB23303, Servicebio). The expression and localization of p65 have been detected utilizing a main anti-p65 antibody (1:500) (#8242, Abcam) and labeled goat anti-rabbit secondary antibody (1:200) (GB23303, Servicebio). Lastly, utilizing an clever pathology imaging system microscope (Vectra, PerkinElmer) for picture acquisition and evaluation, automated picture evaluation was carried out on 5 randomly chosen 200× photographs from every pattern to quantify the relative intensities of protein staining for F4/80, TLR9, MyD88, and p65.

Enzyme-linked immunosorbent assay

The concentrations of TNF-α and IL-6 within the tradition supernatant of RAW 264.7 cells have been measured utilizing the Mouse TNF-α Enzyme-Linked Immunosorbent Assay (ELISA) Package (JL10484) and the Mouse IL-6 ELISA Package (JL20268), respectively. The concentrations of TNF-α, IL-6 and MCP-1 in mouse serum and peritoneal lavage fluid have been measured utilizing the Mouse TNF-α ELISA Package (JL10484), Mouse IL-6 ELISA Package (JL20268), and Mouse MCP-1 ELISA Package (JL20304), respectively. The concentrations of LPS and FABP2 within the mouse serum have been decided utilizing mouse LPS ELISA kits (JL20691) and mouse FABP2 ELISA kits (JL20694). All of the reagents have been sourced from Shanghai Jianglai Biotechnology Co., Ltd.

Evaluation of biochemical parameters

Serum creatinine (CRE) content material was calculated by measuring the absorbance at 546 nm utilizing a Mouse Creatinine Assay Package (Sarcosine Oxidase Technique; JL-T0928, Shanghai Jianglai Biotechnology Co., Ltd.). Serum blood urea nitrogen (BUN) content material was calculated by measuring the absorbance at OD = 625 nm utilizing a Mouse Urea Nitrogen Assay Package (Enzyme Technique; JL-T1014, Shanghai Jianglai Biotechnology Co, Ltd). Serum alanine aminotransferase and aspartate aminotransferase (ALT/AST) ranges have been calculated by measuring the absorbance at 340 nm utilizing a Mouse Serum ALT/AST Assay Package (JL-T2637, Shanghai Jianglai Biotechnology Co, Ltd.).

Stream cytometry evaluation

RAW264.7 macrophages have been added to 6-well plates at a density of two × 10⁵ cells/effectively. Cells have been cultured in DMEM containing 10% FBS for 12 h. The tradition medium was changed with recent medium containing CpG1826 (0.5 µM), and the cells have been co-cultured for 4 h earlier than discarding the medium. After washing 3 times with PBS, recent medium containing cationic polymer (25 µg/mL) was added, and the cells have been incubated for twenty-four h, then centrifuged and resuspended. The cells have been then handled with a viability dye (1:500) (423101, Biolegend), blocked with CD16/32 (1:50) (101302, Biolegend), and stained with a CD86 antibody (1:80) (105012, Biolegend).

Peritoneal lavage fluid was collected 6 h after reperfusion utilizing the tactic described above and centrifuged to acquire a single-cell suspension. The supernatant was discarded, leaving the cell pellet, which was handled with a viability dye (1:500) (423101, BioLegend) and blocked with CD16/32 (1:50) (101302, BioLegend). This was adopted by staining with mouse CD45 (1:80) (103154, BioLegend, USA), F4/80 (1:80) (123137, BioLegend, USA), CD11b (1:200) (101206, BioLegend, USA), and CD86 (1:80) (105012. Biolegend, USA).

After sacrificing the mice, the spleen was bluntly dissected and the floor capsule and adipose tissue have been discarded. After grinding the tissue pattern, pink blood cell lysis buffer was added, adopted by resuspension and centrifugation. The cell pellet was then handled with a viability dye (1:500) (423101, BioLegend) and blocked with CD16/32 (1:50) (101302, BioLegend). This was adopted by staining with mouse CD45 (1:80) (103154, BioLegend, USA), F4/80 (1:80) (123137, BioLegend, USA), CD11b (1:200) (101206, BioLegend, USA), and CD86 (1:80) (105012. Biolegend, USA).

After staining, the cells have been washed and resuspended in 1% BSA buffer. Lastly, movement cytometry knowledge have been collected utilizing a movement cytometer (Attune NxT, Thermo Fisher Scientific) and analyzed utilizing FlowJo software program (10.8.1).

In vivo imaging

To elucidate the in vivo distribution of Micro DEA2k in regular and mannequin mice, 1 mL of Cy7-labeled Micro DEA2k (0.4 mg/mL) was injected intraperitoneally into mannequin mice previous to stomach closure. Regular mice have been injected intraperitoneally with 1 mL of Cy7-labeled Micro DEA2k (0.4 mg/mL) because the management group (n = 3). After injection, they have been monitored with an in vivo imaging system (IVIS Spectrum, PerkinElmer, Inc.), and in vivo imaging was carried out at 0, 6, 24, and 48 h. Three extra mice have been taken from every group and have been sacrificed and dissected at two time factors (6 and 24 h). The mind, coronary heart, lungs, liver, kidneys, testes, spleen, and small intestines have been excised for ex vivo imaging. To look at the in vivo distribution of Micro DEA2k and DEA2k within the mannequin mice, three extra mannequin mice have been injected intraperitoneally with 1 mL of Cy7-labeled DEA2k (0.2 mg/mL). The mice have been euthanized and dissected at two time factors (6 and 24 h), and the organs have been eliminated for ex vivo imaging.

Statistical analyses

Statistical evaluation was carried out utilizing GraphPad Prism 9.4.1, and the outcomes have been expressed as imply ± SEM. Between-group variations have been assessed utilizing the Scholar’s t-test (for easy two-sample or paired pattern comparisons) or one-way evaluation of variance and Tukey’s submit hoc check (for a number of comparisons). The Kaplan-Meier technique was used to check the variations in survival charges. The Spearman correlation coefficient (R²) was used to judge correlations within the scientific knowledge.