Synthesis and Characterization of Pyrazole-Enriched Cationic Nanoparticles as New Promising Antibacterial Agent by Mutual Cooperation

Abstract

A pyrazole derivative (CB1) was previously evaluated in vivo for various pharmacological activities (with the exception of antimicrobial effects), using DMSO as the administrative medium, mainly due to its water insolubility. Considering the global necessity for new antimicrobial agents, CB1 attracted our attention as a candidate to meet this need, mainly because the secondary amine group in its structure would make it possible to obtain its hydrochloride salt (CB1H), thus effortlessly solving its water-solubility drawbacks. In preliminary microbiologic investigations on Gram-negative and Gram-positive bacteria, CB1H displayed weak antibacterial effects on MDR isolates of Gram-positive species, nonetheless better than those displayed by the commonly-used available antibiotics. Therefore, aiming at improving such activity and extending the antibacterial spectrum of CB1H to Gram-negative pathogens, in this first work CB1 was strategically formulated in nanoparticles using a cationic copolymer (P7) previously developed by us, possessing potent broad-spectrum bactericidal activity. Using the nanoprecipitation method, CB1H-loaded polymer nanoparticles (CB1H-P7 NPs) were obtained, which were analyzed by attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to confirm the successful loading. Additionally, CB1H-P7 NPs were fully characterized in terms of morphology, size, polydispersity indices, surface charge, DL%, and EE%, as well as release and potentiometric profiles.

Enhanced Antibacterial Activity of a Cationic Macromolecule by Its Complexation with a Weakly Active Pyrazole Derivative

“…Interestingly, not considering the nano-formulation in its totality but the amount in weight of P7 and CB1H present in the amount of CB1H-P7 NPs which were necessary to inhibit bacteria, we noted that by complexing CB1H with P7, not only had we greatly improved the weak antibacterial effects of pristine CB1H as desired, but unexpectedly, we had also improved those of P7. Indeed, since, as previously reported [23] (Table S1), CB1H-P7 NPs contain CB1H at 48.6% (w/w) and P7 at 51.4% (w/w), the actual MIC values (µg/mL) of CB1H and P7, when in the complex, are those observable in columns 5 and 6 of Tables 3 and 4. In this regard, the MICs of complexed P7 were lower than those of P7 when administered alone by two times, against 7 out of 12 Gram-positive isolates and on 12 out of 24 Gram-negative, four times against five out of 24 Gram-negative isolates, and 8 times on one of 24 Gram-negative strains (bold red data in columns 2 and 5 of Tables 3 and 4).…”

“…Although the dendrimers used as solubilizing agents were free of any antibacterial effect, following the encapsulation, the antibacterial properties of pristine pyrazoles were significantly improved and their cytotoxicity reduced, achieving pyrazole-based NPs endowed with potent antibacterial/bactericidal activity against several species of MDR bacteria [6,22]. Now, we first synthetized a new highly substituted pyrazole derivative, in the form of ammonium hydrochloride salt (CB1H), to resolve the poor water solubility of the free amine in advance [23]. Then, after obtaining considerably high MIC values (MIC = 128 µg/mL and >256 µg/mL on Gram-positive and Gram-negative isolates, respectively), CB1H was formulated into nanoparticles (CB1H-P7 NPs) using a cationic copolymer (P7) with strong broad-spectrum bactericidal effects to enhance its weak antibacterial activity [23].…”

“…Table 3. MIC values of P7 and CB1H when administered alone, MIC values of CB1H-P7 NPs, and those of P7 and CB1H in the complex according to the (DL%) of CB1H-P7 NPs [23] against bacteria of Gram-positive species, obtained from experiments carried out at least in triplicate, expressed as µM and µg/mL. Original P7 (13,719) 1 Original CB1H (371.9) 1 average molecular mass (Mn); 2 MW; 3 refers to CB1H-P7 NPs; # denotes KPC-producing isolates; § denotes New Delhi metallo carbapenemases (NDMs)-producing isolate; P. aeruginosa, S. maltophilia and A. baumannii were all MDR bacteria; 1V, 5V, 6V, 7G = MDR strains isolated from patients with cystic fibrosis; CR = MDR (P. aeruginosa) or KPC-producing (K. pneumoniae) strains resistant also to colistin; py pyomelanin producers; * resistant to the combination avibactam-ceftazidime; ## resistant to carbapenems; COR co-trimoxazole resistant; COI co-trimoxazole intermediate; red bold data evidence the MICs of original P7 and complexed P7 according to which the latter was more potent than the first; light blue bold data evidence the MICs of pristine CB1H and complexed CB1H, according to which, while the first one was inactive against all isolates considered (MICs > 128 µg/mL), the latter, except for two isolates, was active against all strains considered; underlined data refer to the MICs of CB1H-P7 NPs active against CR strains of P. aeruginosa and K. pneumoniae.…”

“…and against NDMs, thus being more efficacious than ceftolozane [22]. More recently, aiming at improving the weak antibacterial activity (MICs = 128 µg/mL and > 256 µg/mL on selected Gram-positive and Gram-negative isolates respectively) of a new pyrazole hydrochloride salt (CB1H) (Figure 2a), CB1H-loaded nanoparticles (CB1H-P7 NPs) were developed by using a cationic copolymer (P7) (Figure 2b) [23]. In this case, P7 prepared by us [24], had proved to possess very potent broadspectrum bactericidal activity (MICs = 0.6-4.6 µM and 2.3-9.3 µM against Gram-positive and Gram-negative bacteria, respectively) [25], even if also considerable cytotoxicity on human tumor cells (LD50 = 4.1-5.1 µM) [24].…”

“…MIC values of P7 and CB1H when administered alone, MIC values of CB1H-P7 NPs, and those of P7 and CB1H in the complex according to the DL% of CB1H-P7 NPs[23], against bacteria of Gram-negative species, obtained from experiments carried out at least in triplicate, expressed as µM and µg/mL.…”