Co-delivery of antibiotic and baicalein by using different polymeric nanoparticle cargos with enhanced synergistic antibacterial activity

Abstract

To evaluate the effect of polymer structures on their unique characteristics and antibacterial activity, this study focused on developing amphiphilic copolymers by using three different molecules through RAFT polymerization. Three amphiphilic copolymers, namely, PBMA-b-(PDMAEMA-r-PPEGMA) (BbDrE), (PBMA-r-PDMAEMA)-b-PPEGMA (BrDbE), and PBMA-r-PDMAEMA-r-PPEGMA (BrDrE), are successfully self-assembled into spherical or oval shaped nanoparticles in aqueous solution and remain stable in PBS, LB, and 10% FBS solutions for at least 3 days. The critical micelle concentrations are 0.012, 0.025, and 0.041 mg/mL for BbDrE, BrDbE, and BrDrE, respectively. The zeta potential values under pH 5.5 and pH 7.4 conditions are 3.18/0.19, 8.57/0.046, and 2.54/-0.69 mV for BbDrE, BrDbE, and BrDrE nanoparticles, respectively. The three copolymers with similar monomer compositions show similar molecular weight and thermostability. Baicalein (BA) and ciprofloxacin (CPX) are encapsulated into the three nanoparticles to obtain BbDrE@BA/CPX, BrDbE@BA/CPX, and BrDrE@BA/CPX nanocomposites, with LC values of 63.9/78.3, 63.9/74.7, and 55.3/64.8, respectively. The two drugs are released from the three drug-loaded nanocomposites with 60%–95% release in pH 5.5 over 24 h and 15%–30% release in pH 7.4. The drug-loaded nanocomposites show synergistic antibacterial activity than the naked drug (2–8 fold reduction for CPX) or single drug-loaded nanocomposites (4–8 fold reduction for CPX) against Pseudomonas aeruginosa and Staphylococcus aureus. The drug-loaded nanocomposites inhibit the formation of bacterial biofilms above their MIC values and eliminate bacterial biofilms observed by fluorescent microscope. Finally, the nanocomposites improve the healing of infection induced by P. aeruginosa and S. aureus on rat dermal wounds. These results indicate that antimicrobial agents with different structures could be an alternative treatment strategy for bacteria-induced infection.

Introduction

The emergence and dramatic development of antimicrobial resistance to pathogenic bacteria, especially the formation of bacterial biofilm, remain one of the dominant challenges and threats to human health. Antibiotics are considered the most efficient drugs to treat bacterial infections, and have saved millions of patients’ lives in the last decades (Davies and Davies, 2010). However, the overuse and abuse of antibiotics lead to a growing number of multidrug-resistant species and severe bacterial infections, which will cause more than 10 million annual deaths by 2050 based on recent projections (Willyard, 2017). The long and slow development of new antibiotics expands the problem, and the world is on the verge of “post-antibiotic era” (Gupta et al., 2019). Hence, innovative treatment strategies should be discovered and developed to combat resistant bacteria.

Numerous approaches and antibiotic replacements have been established to reduce the formation of antibiotic resistance (Guo et al., 2019, Li et al., 2014, Wang et al., 2021, Zhao et al., 2019). The use of polymeric nanoparticles as drug delivery vehicles is a powerful tool to circumvent bacterial infections (Abri et al., 2019, Hao et al., 2021, Kyzioł et al., 2020, Preem et al., 2017, Weldrick et al., 2019) Polymeric drug delivery systems can deliver drugs to the demanding sites at a controlled rate and appropriate drug concentration for an extended period of time (Boateng et al., 2008, Rambhia and Ma, 2015). Polymeric nanoparticles can respond to the unique microenvironment of relevant diseases, resulting in on-demand drug release at desired sites (Xiong et al., 2012). One of the most obvious characteristics in bacterial infections is the low pH in the microenvironment (Radovic-Moreno et al., 2012). Some antibiotics will lose their activity under acidic conditions (Gonzalez Gomez et al., 2019, Mercier et al., 2002), whereas bacteria may quickly adapt to acidic environment (Radovic-Moreno et al., 2012); this phenomenon is a barrier that hinders antibiotics to combat bacteria in the bacterial infection environment. Cationic polymers show excellent antibacterial activity against bacterial species by destructing bacterial cells. However, the toxicity of polycationic antibacterial agents in vivo remain a concern. Therefore, a pH-dependent surface charge-switching polymeric nanoparticle is essential to treat bacterial infections with fewer side effects. These polymeric nanoparticles are neutral or negatively charged under physiological conditions and switch to positive charge under acidic condition. This behavior cannot only decrease the toxicity to host cells but also improve the treatment effect by enhancing the interaction between the nanoparticles and bacterial cells. Meanwhile, the pH-sensitive charge-switching behavior provides convenience for drug delivery and on-demand drug release. For example, Radovic-Mereno et al. designed a polymeric nanoparticle drug delivery system that exhibits surface charge-switching behavior under acidic condition. The nanoparticle was designed to shield nontarget interactions at pH 7.4 but bind avidly to bacteria in acidity. The system could target bacterial cell wall and potentially treat bacterial infections in an acidic environment (Radovic-Moreno et al., 2012).

Combining antibiotics with other drugs is another approach used to improve treatment outcomes. The combination shows synergistic effects on treatment of bacterial infections (2020, Kim et al., 2017, Kumar et al., 2012). For example, Deepika et al. used PEG-PLGA nanoparticle system to simultaneously encapsulate benzamide and rutin as drug candidates. The obtained system displayed the synergistic effect of rutin and benzamide on anti-biofilm activity (2020). Chan et al. found that baicalein (BA) can reverse the resistance of methicillin-resistant Staphylococcus aureus by inhibiting the overexpression of the drug-resistant pump, and restore the antibacterial properties of CPX against the resistant bacteria (Chan et al., 2011). BA, a flavonoid, is mainly derived from the roots of the traditional Chinese medicinal plant Scutellaria. BA shows broad-spectrum antibacterial effect, and can enhance or restore the sensitivity of drug-resistant bacteria to antibiotics (Luo et al., 2016, Qian et al., 2015, Richards et al., 2018). Luo et al. found that BA could inhibit the formation of biofilms by interfering with the signaling pathways of quorum sensing of Pseudomonas aeruginosa. Meanwhile, BA could reduce the inflammatory response factors in bacterial infections (Luo et al., 2016). However, BA has poor water solubility and is easily oxidized leading to low bioavailability, which limiting its application in clinic. Therefore, a method for effective delivery of BA should be developed to increase its concentration in bacterial infection.

Although the combination between antibiotics and BA can improve treatment outcomes, few researches related drug delivery systems are performed to study their synergistic effects on treating bacterial infection. Therefore, we hope to obtain a system that can not only simultaneously carried antibiotic and BA, but also release two drugs respond to bacteria- infected microenvironment. Importantly, it is hopeful to decrease the antibacterial dose of antibiotics in the presence of BA, thereby reducing or delaying the occurrence and development of bacterial resistance. It has been demonstrated that the structure and composition of copolymer plays an important role in the property of nano formulations (Qianqian et al., 2015, Rahman et al., 2020). Herein we designed and synthesized three different copolymers with different structures and distributions based on butyl methacrylate (BMA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), and poly(ethylene glycol) methyl ether methacrylate (PEGMA). BMA offers the hydrophobic segment, PEGMA provides the hydrophilic and biocompatible segment, and DMAEMA acts as the pH-dependent charge-switching and target bacterial cell monomer. The three copolymers self-assemble into nanoparticles in water solution, and their size, morphology, and change in charges are explored. CPX and BA are loaded into the three copolymeric nanoparticles, and the obtained drug-loaded nanocomposites are used to evaluate antibacterial activity against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus). The synergistic effects of CPX and BA in the nanocomposites on antibacterial activity are determined. The potential treatment effect of the drug-loaded nanocomposites is also explored in vivo.