Materials and reagents.
The biodegradable Poly(DL-lactic-co-glycolic acid) (50:50) (PLGA, MW: kDa), heterobifunctional NH2-PEG-COOH (MW: 534.6 Da) and OH-PEG-maleimide (MW: 617 Da) polymers were purchased from Nanosoft Polymers. Ac2-26 peptide (AMVSEFLKQAWFIENEEQEYVQTVK) was purchased from Tocris Biosciences. The Collagen IV targeting peptide (KLWVLPKGGGC) and the scrambled Ac2-26 peptide (WLKQKFQESVEQIAYVMENATEFEV) were purchased from Mimotopes. All NPs included a 5 molar % of PLGA.Cy5.5 fluorescent polymer for tracking studies. All methods were performed in accordance with the relevant guidelines and regulations of both Imperial College London and the Technical University of Munich. All animal procedures were approved by the local animal welfare committee of the regional administration of Upper Bavaria, Germany (Protocol No. 55.2–2532.Vet_02-17-203).
Synthesis of NPs.
PLGA-PEG-COOH, PLGA-PEG-Col IV and PLGA-PEG-Cy5.5 were synthesised as previously reported.30 Ac2-26 or scrambled peptide loaded PLGA NPs were prepared by a single-step nanoprecipitation self-assembly method. Either Ac2-26 or scrambled peptide (120 µg) were added to the polymer mixture containing PLGA-PEG-COOH, PLGA-PEG-Col IV, PLGA-Cy5.5 (3 mg total polymer mass) in 1 mL acetonitrile, and the solution was gently vortexed overnight at RT. This polymer and peptide mixture was then added in a dropwise manner to 10mL of distilled water under gentle stirring and the solution stirred at RT for 4 hours and filtered through sterile 0.45 µm syringe filters (regenerated cellulose, 17 mm, Cole Palmer Instruments). The NPs were concentrated by centrifugation at 3000 x g for 20 min using Amicon Ultra-15 centrifugal filter units (MWCO 100KDa, Millipore Ltd), washed with deionized water, and resuspended in 1mL of H2O, and then further diluted with PBS prior to injection at a concentration of 1 µg Ac2-26. This solution was then dropwise added to a 1% wt ratio chitosan solution. The solution was stirred overnight and filtered through sterile 0.45 µm syringe filters (regenerated cellulose, 17 mm, Cole Palmer Instruments). The chitosan coated NPs were concentrated by centrifugation at 3000 x g for 20 min using Amicon Ultra-15 centrifugal filter units (MWCO 100KDa, Sigma-Aldrich), washed with deionized water, and resuspended in 1 mL of nuclease free H2O (total 3.15 mg/mL chitosan coated NP). For pectin coating the chitosan coated NPs were added dropwise to a 1 wt% solution of pectin and the NPs stirred overnight and filter the solution through sterile 0.45 µm syringe filters (regenerated cellulose, 17 mm, Cole Palmer Instruments). The pectin, chitosan coated NPs were concentrated by centrifugation at 3000 x g for 20 min using Amicon Ultra-15 centrifugal filter units (MWCO 100KDa, Sigma-Aldrich), washed with deionized water, and resuspended in 1 mL of nuclease free H2O (total 3.18 mg/mL pectin, chitosan coated NP).
NP characterization
Ac2-26 and scrambled peptide quantification measurements were carried out on a Spark Multimode Microplate Reader. The NP mean sizes, size distribution, and z-potentials were determined by Malvern Nano ZS Zetasizer at 25 oC. All measurements were carried out in triplicates. The average particle size was expressed in intensity mean diameter and the reported value was represented as mean ± average (n = 3). For TEM, a 10 µl solution of 1mg/ml freshly prepared NPs in H2O was deposited on carbon coated copper grids, the excess solution was blotted, and the grids were immersed in a solution of 0.75% uranyl formate stain. The stain was blotted, and the sample imaged within 1 hour of preparation on a Tecnai™ G2 Spirit BioTwin electron microscope equipped with an AMT 2k CCD camera and low-dose software (80kV, direct mag. 98000x).
Ac2-26 and Scrm-Ac2-26 release kinetics from NPs
To quantify the Ac2-26 release profile, 3.12 mg/mL NP samples (either P-C-Col IV-Ac2-26-NPs or P-C-Col IV-Scrm-NPs) were made in PBS, and the NPs were incubated at 37°C. At defined time intervals, the NPs were removed, transferred to Amicon Ultra-15 centrifugal filter units (MWCO 10 kDa; Sigma-Aldrich), and centrifuged at 3,000 x g for 20 min. The NPs were then resuspended in PBS, and incubation at 37 oC was continued until the designated time point. The filtrate (10 µL) was analyzed with a nanodrop UV-Vis spectrometer, and absorbance was measured at 220 nm to determine the amount of released peptide at each time point and a cumulative % release curve generated.
Animal experiments and study design
Female BALB/c mice of 11–16 weeks with body weight between 19 and 24 g were used for the experiments (Charles River, Sulzfeld, Germany). The local animal facility environment had a temperature of 22°C and a humidity of 45–60% followed a 12-hour light/dark cycle. Animals were kept at specific-pathogen free (SPF) conditions throughout the procedure. An initial period of 1 week prior to initiation of the experiments was allowed for adaption to housing conditions.
DSS was administered for 7 days in drinking water for colitis induction which causes an inflammatory reaction by permeabilization of the mucosa for bacterial influx to the lamina propria36. DSS was offered ad libitum in drinking water at 2 percent (w/v) of DSS (molecular weight 36,000–50,000, MP Biomedicals, Heidelberg, Germany). The DAI is an established score to semi quantitatively score severity of experimental colitis by scoring weight loss, blood in stolls and stool consistency was measured prior to surgery and before sacrification.
After 7 days of DSS, the surgical anastomosis was performed and postoperatively drinking water was switched to normal water. Mice were treated throughout the whole procedure by verum ( P-C-Col IV-Ac2-26 NPs, either 1µg or 10µg ) and placebo ( P-C-Col IV-Scrm-NP 10 µg) every 3.5 days throughout the whole procedure. Medication was administered by oral gavage by a 20G plastic cannula. Tissue was harvested at either POD 3 or 7 after endocopy and sacrification by isoflurane general anaesthesia and cervical dislocation.
Surgical procedure
Anaesthesia was initiated by a mixture of 5% isoflurane (CP-Pharma, Burgdorf, Germany) and remainder of oxygen, and was maintained under reduced isoflurane concentrations of 1–3% to allow spontaneous breathing. Body temperature was maintained at 37°C by a heating pad. Subcutaneous injections of meloxicam (Boehringer Ingelheim Pharma, Ingelheim, Germany) and buprenorphine (Indivior, Berkshire, UK) were used for analgesia. After laparotomy in supine position, the colon was transected at the height of the lower renal pole, preserving the vasculature. Using an operating microscope (Carl Zeiss, Oberkochen, Germany), a standardized end-to-end anastomosis was performed in microsurgical technique by 12 single stitches of monofilamentous Ethilon 9 − 0 (Ethicon, Norderstedt, Germany). Postoperatively, mice had free access to regular drinking water and food ad. libitum. Postoperative daily assessment included body weight measurements, observation of wound healing and administration of analgesics .
Murine colonoscopy and dehiscence scoring
Before sacrification at POD 3 or 7, colonoscopy was performedusing the Coloview system Mainz (Karl Storz, Tuttingen, Germany) with a 7 Charriere examination shaft prior to evaluation in isoflurane sedation. Instillagel (FARCO-Pharma, Cologne, Germany) was used as lubricant.The mucosa and anastomosis were evaluated under retraction of the colonoscope. Wound healing was scored according to the following score: 0 = no dehiscence, 1 = suture protruding into the lumen, 2 = dehiscence less than 25% of the circumference, 3 = dehiscence more than 25% of the circumference, 4 = complete dehiscence, as described before34.
Tissue harvesting and analysis
Mice were sacrificed by cervical dislocation under isoflurane sedation. The abdomen was opened and scoring of the adhesions was performed (Range 0–7): Adhesion of mesenterial fat, the uterus, intestine or other organs was scored with 1 point each. No adhesions was scored with 0, bluntly completely removable tissue with 1, partially removable tissue with 2 and non-removable adhesions with 3. For tissue harvesting, the descending colon was resected at least 1 cm proximally and distally from the anastomosis. Prior to histological assessment, the bowel was cut in half lengthwise. Consecutive sections (4 µm thickness) of the colon bearing the anastomosis were prepared and subjected to H&E staining. Anastomotic healing was in histological sections of the anastomotic area by a pathology specialist by a healing score35: Scores for inflammatory cells (0–4 points, this score was inversed as higher inflammatory cell numbers reflect impaired healing), angiogenesis (0–4 points), collagen synthesis (0–4 points), fibroblast ingrowth (0–4 points) were summed up. A functional score evaluating the closure of the bowel wall was performed by adding the following items34: first closed layer counted from extraluminal side (0 = no healed layer, 1 = serosa layer, 2 = muscular layer, 3 = submucosal layer, 4 = mucosal layer), number of healed layers (counting mucosa, submucosa, muscularis, serosa) (0–4), epithelial layer closed (0 = no, 1 = yes) and intact crypt architecture (0 = no, 1 = yes) were added for the functional healing score.
Statistics
The results are presented as mean ± standard error of the mean (SEM) for parametric or median for non-parametric results. Unpaired two-tailed t-test or one-way analysis of variance (ANOVA) combined with Bonferroni post-test were applied to determine statistical significance for parametric data. For non-parametric data Mann-Whitney-U test were used. Data plotting and statistical analyses were performed in Prism (GraphPad, San Diego, USA).