Purpose: For decades, silver and its complexes have proven to be sufficient means of being antibacterial treatment methods. However, the rise of antibacterial resistant bacterial strains has become a leading threat to current treatments. Recently synthesized silver-furosemide (Ag-FSE) complex has shown promising antibacterial activity but has poor solubility. Therefore, the goal was to formulate chitosan nanoparticles (CSNPs) of Ag-FSE complex in order to enhance its solubility and antibacterial efficacy.
Methods: Synthesis of Ag-FSE complex was performed by dropwise addition of AgNO3 (0.362 g, 2.13 mmol) in water (2 mL) into a methanolic solution (15 mL) of furosemide (0.704 g, 2.13 mmol) and KOH (0.119 g, 2.13 mmol) under continuous stirring1. CSNPs were formulated via tripolyphosphate (TPP) ionotropic gelation and ultrasonication techniques2. Size, polydispersity index (PDI) and zeta potential (ZP) of Ag-FSE loaded CSNPs were determined using dynamic light scattering (DLS) technique. Encapsulation efficiency (EE%), morphological and structural characterizations were performed via scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and x-ray diffraction (XRD). Stability studies were performed to observe Ag-FSE CSNPs stability under different storage conditions. In vitro drug release studies were also performed to verify a release pattern of the drug-complex from the nanoparticles. Antibacterial activity of the nano-formulation was tested against E. coli, S. aureus, and P. aeruginosa bacteria.
Results: Primary goal was to obtain CSNPs with sizes < 300 nm along with a low PDI, and high ZP value to reduce risk of particulate aggregation. With 3 minutes sonication the results for size, PDI, and ZP were 344.6 ± 98.96 nm, 0.517 ± 0.107, and 44.1 ± 2.21 mV respectively. At 5 minutes sonication the results were 472.1 ± 42.81 nm, 0.507 ± 0.139, and 42.9 ± 3.86 mV respectively. At 10 minutes sonication the values were 261.3 ± 12.23 nm, 0.195 ± 0.023, and 42.8 ± 1.31 mV, respectively. Further, Ag-FSE loaded CSNPs were investigated for size, PDI and ZP. Size, PDI, and ZP were 257.5 ± 5.132 nm,0.185 ± 0.014, and 30.7 ± 5.48 mV respectively at 5 % loading whereas at 10 % loading the values were 216.6 ± 26.8 nm, 0.194 ± 0.021, and 25.8 ± 3.05 mV, respectively. SEM analysis exhibited that the Ag-FSE CSNPs were semi-spherical in shape and remained within the nano-size range (Figure 1). Furthermore, DSC and XRD results indicated solubility of the Ag-FSE complex had been improved as the nano-formulation underwent a shift from a crystalline nature to an amorphous nature. EE% for the Ag-FSE loaded CSNPs were calculated to be 88.24%. Stability studies for the Ag-FSE loaded CSNPs for both 5 % and 10 % loading indicated stable formulations in various conditions over the course of 120 days. In vitro drug release observed a slow and controlled release pattern of the encapsulated silver complex into CSNPs versus free drug in solution (Figure 2). In vitro antibacterial activity of the low concentration of Ag-FSE loaded into the CSNPs had observed effects on S. aureus and P. aeruginosa bacteria propagation that was formidable with comparison to that of free drug in solution.
Conclusion: We have successfully optimized Ag-FSE loaded CSNPs for size, PDI and ZP that were spherical with appropriate extended stability in various conditions. DSC and XRD results are indicative for improved solubility properties of the silver complex as shift from a crystalline nature to an amorphous nature was observed. The optimized formulation also exhibited controlled and sustained drug release characteristics that play crucial to the enhancement of antibacterial activity at low concentrations.
Rahul Kalhapure– Postdoc, University of Texas at El Paso, El Paso, Texas
Pradeep Kumar Bolla– Student, University of Texas at El Paso, El Paso, Texas
Julian Franco– Student, University of Texas, El Paso, El Paso, Texas
Jwala Renukuntla– Assistant Professor, University of Texas at El Paso, El Paso, Texas