To begin, prepare zinc, calcium, copper, and ferric alginate hydrogel micro-spheres. For the antimicrobial performance test, add 10 milliliters of each of the two bacterial solutions into a 15 milliliter sterile centrifuge tube. Then incubate the cultures with 0.5 milliliters of hydrogel microspheres in a constant temperature shaker.
Dilute each bacterial solution, 10 to the power of five times, with sterile water. Using sterile glass beads, evenly spread 200 microliters of bacterial solution on an LB solid medium. Place the plates in an incubator at 37 degrees Celsius for 12 hours to assess colony formation.
Add one milligram per milliliter of bovine serum albumin or BSA suspension, to 500 microliters of each microsphere and incubate for 24 hours. Add each saturated sample to one milliliter PBS, and agitate for 15 minutes at 37 degrees Celsius with continuous shaking at 80 revolutions per minute. After aliquoting, replace the medium with a fresh one at one, three, six, 12, 24, 36, and 48 hours.
Quantify the supernatant protein concentrations at specific times. Add the prepared ALG microspheres to PBS, and incubate them at 37 degrees Celsius for 24 hours. Obtain whole blood from healthy mice in anticoagulation tubes containing sodium citrate.
After vortexing centrifuge the tube for 15 minutes at 1, 500 revolutions per minute to obtain red blood cells. After flushing the cells, prepare a 10%red blood cell solution with PBS, and add 20 microliters of blood cell solution to the microsphere experimental and control groups. Incubate the samples at 37 degrees Celsius for four hours, and centrifuge them at 1, 500 RPM for 15 minutes.
After keeping the supernatant aside, photograph the red blood cells in each group following hemolysis. Finally, measure the absorbance values of the supernatants in each group and compute the hemolysis rate using the formula. Different microspheres exhibited antibacterial activity against staphylococcus aureus and escherichia coli with copper and zinc, ALG microspheres, showing the strongest antibacterial properties.
The drug release assay revealed that the release rate of the iron microsphere was relatively faster than that of the other three ions. Biocompatibility assessment showed that the red blood cells in the suspension remained intact upon contact with different microspheres, indicating minimal hemolysis by the microspheres.
