(M1230-12-81) Ciprofloxacin-Loaded Albumin Nanoparticles as a New Strategy for the Treatment of Urinary Tract Infections and Catheter-Associated Urinary Tract Infections

Purpose: Urinary tract infections (UTIs) represent a significant global health concern (1). Uropathogenic Escherichia coli (UPEC) is the primary cause of these infections, which are becoming increasingly challenging to treat due to the emergence of antibiotic resistance and the formation of biofilms (2). UTIs are the most prevalent healthcare-associated infections; 80% of these infections are related to urinary catheters, leading to catheter-associated urinary tract infections (CAUTIs) (3). Nanotechnology presents a promising approach to the treatment of UTIs (4). Therefore, this research aimed at developing and characterizing ciprofloxacin-loaded albumin nanoparticles (CPF-BSA NPs) capable of effectively combating uropathogenic and their biofilms.

Methods: Albumin nanoparticles (BSA NPs) were developed by the desolvation method using ethanol as a desolvating agent and glutaraldehyde as crosslinker. To incorporate the drug, a required amount of ciprofloxacin (CPF) was dissolved in a solution previously prepared with polysorbate 80 and BSA at a final concentration of 1% and 4% (w/v) respectively. Then, the mixture was sonicated for 30 min at 40 °C and the other excipients were added. BSA NPs and CPF-BSA NPs were characterized by dynamic light scattering (DLS), laser-Doppler electrophoresis and transmission electron microscopy (TEM). The CPF entrapment efficiency (%EE) was determined indirectly by fluorescence. For this, the excitation wavelength was 295 nm, the emission wavelength registered was 456 nm, and the widths of both excitation and emission slit were set to 5 nm. The CPF release profile from CPF-BSA NPs was assessed by dialysis in two different media, PBS pH 7.4 and artificial urine pH 8. The release data were kinetically analyzed according to mathematical models using the program DD-Solver an add-in of Excel. The cytotoxicity of NPs was analyzed on HTB-4 cells using the resazurin assay. To evaluate the in vitro antibacterial activity of CPF-BSA NPS, we determined the minimum inhibitory concentration of CPF-BSA NPs against the clinically isolated E. coli 144 using the microdilution technique. Finally, we evaluated the ability of CPF-BSA NPs to prevent E. coli 144 biofilms using a stating biofilm assay and to eradicate E. coli 144 biofilms using the crystal violet technique.

Results: The physicochemical characteristics of BSA-NPs CPF-BSA NPs are detailed on table 1 suggesting a monodisperse size distribution. TEM micrographs revealed that BSA NPs and CPF-BSA NPs had a spherical and uniform shape indicating no significant changes after CPF incorporation. CPF-BSA NPs had an %EE of 87.460 ± 2.625 %, representing a payload of 838,289 ± 27.946 µg of CPF per synthesis. The release profile of CPF from CPF-BSA NPS showed a controlled release achieving complete release after 5 h in PBS and 3 h in artificial urine. Additionally, the best-fit model in both cases was the first-order model. The cell viability assay (Figure 1) demonstrated that CPF-BSA NPs exhibited significantly lower cytotoxicity than free CPF compared to the control (p≤0.05). The antibacterial analysis revealed that BSA NPs exhibited no antibacterial activity, while CPF and CPF-BSA NPs showed a minimum inhibitory concentration of 0.078 µg/mL. Finally, antibiofilm assays (Figure 2) indicated that 5 µg/mL of CPF, whether free or encapsulated in BSA-NPs, effectively prevented biofilm formation. Moreover, CPF-BSA NPs demonstrated a significantly higher (p≤0.01) reduction in biofilm biomass compared to free CPF.

Conclusion: BSA NPs and CPF-BSA NPs were successfully obtained by the desolvation method. The physicochemical properties of the obtained nanocarriers such as size (~ 120 nm), low PdI, and zeta potential (approximately -31 mV) suggest a highly stable colloidal system. Morphological analysis corroborated the formation of spherical NPs, and the %EE studies revealed a high incorporation of CPF into the BSA NPs. These characteristics together with the low cytotoxicity of CPF-BSA NPs, and the drug sustained release are desirable characteristics for drug delivery systems. Additionally, the synthetized CPF-BSA NPS demonstrated antibacterial and antibiofilm potential against the urinary clinical strain E. coli 144 suggesting that they could be employed for the treatment of UTI and CAUTI caused by uropathogens. Clearly, in vivo biological analysis is highly necessary to reach the correct conclusion about safety, doses, pharmacological activity, and fate of this nanosystem. Nevertheless, these preliminary results together suggest a suitable tool for future investigations in drug delivery, pharmaceutical, and veterinary industry.

References: 1. Foxman B. Urinary tract infection syndromes: occurrence, recurrence, bacteriology, risk factors, and disease burden. Infect Dis Clin North Am. marzo de 2014;28(1):1-13.
2. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol. mayo de 2015;13(5):269-84.
3. Clarke K, Hall CL, Wiley Z, Tejedor SC, Kim JS, Reif L, et al. Catheter-Associated Urinary Tract Infections in Adults: Diagnosis, Treatment, and Prevention. Journal of Hospital Medicine. 2020;15(9):552-6.
4. Sánchez SV, Navarro N, Catalán-Figueroa J, Morales JO. Nanoparticles as Potential Novel Therapies for Urinary Tract Infections. Frontiers in Cellular and Infection Microbiology [Internet]. 2021 [citado 31 de mayo de 2022];11. Disponible en: https://www.frontiersin.org/article/10.3389/fcimb.2021.656496

Acknowledgements: This work was funded by Regular FONDECYT Project 1231154, ANID/PIA/ACT192144, FONDAP Project 15130011, and National Doctoral Scholarship 21201831.

Table 1. Physicochemical characteristics of BSA NPs and CPF-BSA NPs

Figure 1. Cell viability of HTB-4 cells after exposure to CPF and CPF-BSA NPs for 24 hours. Data are expressed as a percentage of the viable control. *= statically significant differences between compared groups (p≤0.05).

Figure 2. Antibiofilm assays. A: Inhibition of biofilm formation of CPF and CPF-BSA NPs on Escherichia coli 144. B: Eradication of formed biofilm of CPF and CPF-BSA NPs on Escherichia coli 144.
**=statically significant differences between compared groups (p≤0.01). ****=statically significant differences between compared groups (p≤0.0001).