Abstract
Background
Biofilms, such as those from Staphylococcus epidermidis, are generally insensitive to traditional antimicrobial agents, making it difficult to inhibit their formation. Although quercetin has excellent antibiofilm effects, its clinical applications are limited by the lack of sustained and targeted release at the site of S. epidermidis infection.
Objectives
Polyethylene glycol-quercetin nanoparticles (PQ-NPs)-loaded gelatin-N,O-carboxymethyl chitosan (N,O-CMCS) composite nanogels were prepared and assessed for the on-demand release potential for reducing S. epidermidis biofilm formation.
Methods
The formation mechanism, physicochemical characterization, and antibiofilm activity of PQ-nanogels against S. epidermidis were studied.
Results
Physicochemical characterization confirmed that PQ-nanogels had been prepared by the electrostatic interactions between gelatin and N,O-CMCS with sodium tripolyphosphate. The PQ-nanogels exhibited obvious pH and gelatinase-responsive to achieve on-demand release in the micro-environment (pH 5.5 and gelatinase) of S. epidermidis. In addition, PQ-nanogels had excellent antibiofilm activity, and the potential antibiofilm mechanism may enhance its antibiofilm activity by reducing its relative biofilm formation, surface hydrophobicity, exopolysaccharides production, and eDNA production.
Conclusions
This study will guide the development of the dual responsiveness (pH and gelatinase) of nanogels to achieve on-demand release for reducing S. epidermidis biofilm formation.
Keywords: Staphylococcus epidermidis, quercetin, biofilm, nanogels, on-demand release
INTRODUCTION
As a conditional pathogen, Staphylococcus epidermidis causes many infections in immunocompromised people, e.g., urinary system infection, bacterial endocarditis, infection after trauma and surgery, infection after the implantation of medical devices, and mastitis, in which the pathogenesis usually involves biofilm formation [1]. The formation of bacterial biofilms can prevent antimicrobial agents from inhibiting or killing S. epidermidis within the biofilm, resulting in recurrent infections and bacterial resistance that are difficult to cure. Therefore, biofilms help bacteria resist harsh environments and protect the bacteria inside the biofilm from the action of antimicrobial agents [2]. According to various reports, the bacteria inside biofilms have a 10 to 1,000-fold increased resistance to antibiotics compared to free bacteria, leading to recurrent clinical infections and increased difficulty in treatment [3]. Thus, new ways are needed to inhibit the formation of bacterial biofilms. S. epidermidis biofilms form in four stages: initial adhesion stage, aggregation stage, maturity stage, and shedding stage [4]. Generally, suppressing the maturity and shedding stages is challenging; thus, these two stages are not considered in the search for new ways to inhibit the formation of bacterial biofilms. The initial adhesion and aggregation stages have been studied widely to inhibit S. epidermidis biofilms. On the other hand, the formation of S. epidermidis biofilms is difficult to prevent using conventional antimicrobial agents because of their insensitivity to biofilms [3]. The resistance mechanism of biofilms mainly includes three aspects. (1) Biofilms hinder the penetration of antibiotics into the biofilms because they are three-dimensional aggregates of bacteria, and the extracellular matrix secreted by bacteria can adsorb antibiotics through hydrogen bonding and other interactions. At the same time, the hydrolase and catalase produced by bacteria adsorbed on the polysaccharide-protein complex can hydrolyze antibiotics, increasing the drug resistance of bacteria. (2) After bacteria form a biofilm, the bacteria inside the biofilm are limited by the nutrient supply and metabolic product excretion, and most are dormant. Thus, the bacteria inside the biofilm are insensitive to antimicrobial agents, leading to increased bacterial resistance. (3) Biofilms can resist the host immune responses and produce immune escape phenomena. Alginate secreted by bacteria can weaken the phagocytosis of neutrophils, avoiding the immune response of the body [5].
Quercetin, a natural flavonoid antioxidant, scavenges reactive species and hydroxyl radicals, making it a significant pharmaceutical compound owing to its anticarcinogenic, anti-inflammatory, and antimicrobial properties [6]. The antibiofilm effect of quercetin against S. epidermidis has been explored, exhibiting excellent antibiofilm effects [3]. On the other hand, its clinical application is limited by its lack of sustained and targeted release effects at the infection site of S. epidermidis. Nanogel drug-delivery systems with sustained and targeted release effects wrap drugs for antibacterial biofilms [7].
Hydrogels with sustained and targeted release effects effectively wrap antimicrobial agents to inhibit bacterial biofilm formation. For example, bioactive hydrogels with high adhesion ability and excellent biocompatibility were developed by loading the active components berberine and Spirulina platensis into the non-toxic carboxymethyl chitosan (CS)/sodium alginate substrates and further cross-linking them with genipin to produce oxygen under laser irradiation to relieve biofilm hypoxia and reduce biofilm resistance, improving the sensitivity of biofilms to antimicrobial agents [8]. Multifunctional hydrogels prepared by guar gum downregulated the related pathways that promoted biofilm formation and activated the expression of the agr family genes to accelerate biofilm damage [9]. In addition, enrofloxacin composite nanogels formulated by gelatin and sodium alginate reduced the minimum membrane inhibition concentration of the bacterium [10]. Therefore, polysaccharides, such as CS and its derivatives, gelatin, are used widely to inhibit the formation of bacterial biofilms. Although CS has excellent antibacterial properties, its poor solubility limits its widespread application [11]. N,O-carboxymethyl chitosan (N,O-CMCS) was obtained from the carboxylation of monochloroacetic acid (MCAA) to improve the characteristics of CS with poor solubility [12]. Therefore, it was assumed that quercetin-loaded N,O-CMCS-gelatin nanogels (Q-nanogels) would be prepared by electrostatic interaction between gelatin (positive charge) and N,O-CMCS (negative charge) using ionic crosslinkers. On the other hand, the infected site of S. epidermidis will show a weak acid environment (pH 5.5) and can release gelatinase [12,13]. Q-nanogels are hydrolyzed immediately by gelatinase in a weak acid environment (pH 5.5), rapidly releasing quercetin. Therefore, although Q-nanogels have targeted properties, they lack a sustained release effect.
Polyethylene glycol (PEG) is used widely to encapsulate drugs for sustained release. For example, after enrofloxacin was encapsulated by PEG, the PEG-enrofloxacin nanoparticles formed exhibited significant sustained-release effects at the Staphylococcus aureus infection site [10]. Accordingly, quercetin may show an excellent slow-release effect after being encapsulated by PEG. Furthermore, PEG-quercetin nanoparticles (PQ-NPs) were encapsulated by electrostatic interactions between gelatin (positive charge) and N,O-CMCS (negative charge) using ionic crosslinkers to form PQ-NP-loaded gelatin-N,O-CMCS composite nanogels (PQ-nanogels) for on-demand release, improving the antibiofilm activity of PQ-nanogels against S. epidermidis. At the infected site of S. epidermidis, the PQ-nanogels may be hydrolyzed, and PQ-NPs are released continuously because of the action of a weak acid environment (pH 5.5) and gelatinase. Quercetin is released slowly because of the sustained-release effects of PQ-NPs. The slowly released quercetin may help reduce the relative biofilm formation, surface hydrophobicity, exopolysaccharides (EPS) production, and eDNA production, inhibiting the formation of S. epidermidis biofilm. Therefore, the formation mechanism, physicochemical characterization, and antibiofilm activity of PQ-nanogels against S. epidermidis were studied to achieve on-demand release.
MATERIALS AND METHODS
Materials
CS (molecular weight (MW): 161.16 KDa, viscosity: 50–800 mPa.s, degree of acetylation 80%–95%), sodium tripolyphosphate (TPP, MW: 367.86), gelatin (≥ 99%; MW: 5,000), isopropyl alcohol (≥ 99.5%; MW: 60.01), MCAA (98%; MW: 94.50), Tryptone Soya Broth, and quercetin (95%–99%; MW: 302.20) were purchased from ChuangXin Pharmaceutical Co., Ltd. (China). PEG (MW: 697.61) was obtained from Dingyuan Biotechnology Co., Ltd (China). The antibodies used for live/dead bacterial staining analysis are supplied by Pumoke Biotechnology Inc. (China). A strong biofilm-positive strain S. epidermidis ATCC 35984 was obtained from the Engineering Laboratory for Tarim Animal Diseases Diagnosis and Control of Tarim University (China).
Synthesis of N,O-CMCS
N,O-CMCS was synthesized using a slight modification of the procedure described elsewhere [12]. Briefly, 10 g of CS was dispersed into 100 mL of isopropyl alcohol, and the suspension was stirred at room temperature. Subsequently, 25 mL of a 10 M sodium hydroxide aqueous solution was divided into five equal parts and added to the mixed suspension over 25 min. The alkaline suspension was stirred continuously at 800 rpm for an additional 30 min. Subsequently, 20 g of MCAA was added dropwise to each of the five equal parts at 5-min intervals. The reaction temperature was maintained at 60°C with stirring for 3 h. The solid product of N,O-CMCS was obtained by filtering the reaction mixture and rinsed three times with 80% v/v ethanol solution and then several times with 100% ethanol. The dry product of N,O-CMCS was obtained using a lyophilizer (FDU-1200; Shanghai Lingyi Biotechnology Co., Ltd, Japan). Finally, the lyophilized N,O-CMCS was determined by Fourier transform infrared (FTIR, Nicolet iS50; Thermo Scientific Inc., USA) spectroscopy.
Formulation of PQ-nanogels
PQ-nanogels were formulated by hot melt homogenization with the ultrasonic method and electrostatic interaction [14]. Briefly, 1.0 g PEG was added to 10 mL of ultrapure water with magnetic stirring and heat using a magnetic stirrer (GL-3250A; Kylin-Bell Lab Instruments, China) for complete dissolution to obtain the PEG solution. Subsequently, 0.1 g quercetin was slowly added to the PEG solution to obtain a primary PEG-quercetin solution. The primary PEG-quercetin solution was sonicated using 6 mm microprobes with 90% amplitude for 3 min to form PQ-NPs. N,O-CMCS (0.1, 0.2, and 0.3 g), gelatin (0.5, 1.0, and 1.5 g), and TPP (0.1, 0.2, and 0.3 g) were then added to 5, 5, and 2 mL ultrapure water with magnetic stirring and heat for complete dissolution to obtain N,O-CMCS solution, gelatin solution, and TPP solution, respectively. Subsequently, PQ-NPs were added dropwise to the gelatin solution to obtain a gelatin mixture solution. Finally, a N,O-CMCS solution and a TPP solution were added dropwise into a gelatin mixture solution with magnetic stirring at 1,500 RPM to form the PQ-nanogels. Similarly, blank nanogels (quercetin-free) were formulated by PEG, N,O-CMCS, gelatin, and TPP. Q-nanogels (containing quercetin) were formulated from N,O-CMCS, gelatin, and TPP. The optimal amount of N,O-CMCS, gelatin, and TPP was evaluated by an orthogonal experiment using the loading capacity (LC) as indices. Subsequently, the encapsulation efficiency (EE) of the optimal formula of PQ-nanogels was determined. Each sample was formulated three times, and the data are expressed as the mean ± SD.
Physicochemical characterization
Surface morphology
The appearance, lyophilized powder, reconstituted solution, and optical microscopy images of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels were evaluated. Freshly prepared PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels were placed in an inclined bottle to observe their gel state. These freshly prepared samples were then placed in a lyophilizer for freeze-drying to obtain a lyophilized powder, which was observed by optical microscopy (Nikon SMz745T; Nikon, Japan). The lyophilized powder samples were redissolved in ultrapure water to obtain a reconstituted solution for evaluating its resolubility. Furthermore, scanning electron microscopy (SEM, APREO; Thermo Scientific Inc.) and transmission electron microscopy (TEM, JEM-2100Plus; JEOL, Japan) were used to observe the surface morphology of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels. Briefly, PQ-NPs were diluted 100 fold and placed into the ultrasonic cleaner (BILON3-120A; Shanghai Xinnuo Instrument Group Co., Ltd, China) to ultrasonic for 1 h. The blank nanogels, Q-nanogels, and PQ-nanogels were then freeze-dried using a lyophilizer. Subsequently, the PQ-NPs suspension, freeze-dried blank nanogels, Q-nanogels, and PQ-nanogels were placed on a silicon wafer and coated with gold by ion sputtering and observed by SEM at an accelerating voltage of 20 kV after oven-drying. Freshly prepared PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels were placed on copper grids with thin slices and dried at 25°C by negative staining using sodium phosphotungstate (2%). The morphology of these freshly prepared samples was observed by TEM.
Size, zeta potential (ZP), and polydispersity index (PDI)
In this study, the mean size, ZP, and PDI of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels were determined using a Zetasizer ZX3600 (Malvern Instruments, UK) at 25°C. Briefly, all freshly prepared samples were diluted 100-fold with ultrapure water, and the mean size, ZP, and PDI were measured three times using a Zetasizer ZX3600.
Powder X-ray diffraction (PXRD) and FTIR spectroscopy
After the PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels were freeze-dried, the PXRD (Ultima IV; Beijing Lihua Saisi Technology Co., Ltd, Japan) patterns and FTIR spectra were obtained.
In vitro responsive release
The PQ-nanogels were diluted with ultrapure water and placed into a dialysis bag (MW: 3,500). The dialysis bags containing the PQ-nanogels (100 mg/mL) were placed into 500 mL phosphate buffered saline (PBS, pH 5.5/7.4) without and with gelatinase at 37°C ± 0.5°C. At different times (0.5, 1, 2, 4, 8, 12, and 24 h), 1 mL of the dialysate was removed, and blank PBS with an equal temperature (37°C ± 0.5°C) and volume (1 mL) was added. The quercetin concentrations were then determined using an ultraviolet spectrophotometer. Finally, the in vitro release curves of PQ-nanogels in different micro-environments (pH 5.5 and 7.4; without and with gelatinase) were drawn according to the cumulative release percentage.
Antibiofilm activity
Relative biofilm formation
The relative biofilm formation of blank nanogels, quercetin, and PQ-nanogels on biofilm formation of S. epidermidis ATCC 35984 was evaluated by crystal violet staining. Briefly, S. epidermidis ATCC 35984 (1 × 106 CFU/mL) were cultured with different concentrations (0, 32, 64, 128, 256, 512, and 1,024 μg/mL) of blank nanogels, quercetin and PQ-nanogels for 24 h at 37°C. The S. epidermidis ATCC 35984 biofilms treated with different concentrations of the quercetin formulations were stained with crystal violet and dissolved in 95% ethanol. The optical density (OD) of the samples was determined using an enzyme-linked immunosorbent analyzer (Thermo Fisher Scientific Inc.) at 570 nm. The relative biofilm formation was calculated using the following formula:
| Relative Biofilm Formation = [Treated OD570/Untreated OD570] × 100% |
Cell surface hydrophobicity
The cell surface hydrophobicity of S. epidermidis ATCC 35984 treated with different concentrations (0, 32, 64, 128, 256, 512, and 1,024 μg/mL) of the blank nanogels, quercetin, and PQ-nanogels were evaluated, as described elsewhere [3]. Briefly, the S. epidermidis ATCC 35984 (1 × 106 CFU/mL) treated with different quercetin formulations were collected and washed three times with phosphate-urea-magnesium (PUM) buffer. Using the PUM buffer as blank control, the absorbance value of S. epidermidis ATCC 35984 was controlled in the range of 0.4–0.6 and recorded as ODcontrol. The ODsamples absorbance values of the S. epidermidis ATCC 35984 treated with different quercetin formulations at 400 nm were obtained. The cell surface hydrophobicity was calculated using the formula:
| Hydrophobicity = [(ODcontrol − ODsamples)/ODcontrol] × 100% |
EPS production
The EPS production of S. epidermidis ATCC 35984 treated with blank nanogels, quercetin, and PQ-nanogels was determined as reported elsewhere [15]. The EPS of S. epidermidis ATCC 35984 (1 × 106 CFU/mL) treated with different concentrations (64 and 128 μg/mL) of quercetin and PQ-nanogels were extracted and precipitated using 95% ethanol. Subsequently, proteinase K and n-butyl alcohol were used to remove the proteins. Subsequently, the aqueous layer was collected, followed by dialysis with distilled water overnight. Finally, the liquid was lyophilized to obtain the EPS production of S. epidermidis ATCC 35984 treated with quercetin and PQ-nanogels.
eDNA production
The eDNA production of S. epidermidis ATCC 35984 treated with blank nanogels, quercetin, and PQ-nanogels was determined, as reported previously [15]. Briefly, after S. epidermidis ATCC 35984 (1 × 106 CFU/mL) was grown in contact with quercetin and PQ-nanogels (64 and 128 μg/mL) at 37°C for 24 h, the samples were washed with PBS, and mixed with a Tris-ethylenediaminetetraacetic acid (EDTA) buffer solution. The samples were centrifuged at 10,000 RPM for 10 min, and the pellet was suspended by a Tris-EDTA buffer solution. The absorbance of the S. epidermidis ATCC 35984 treated with quercetin and PQ-nanogels supernatants (260 nm) was measured after centrifugation at 10,000 RPM for 15 min. The inhibition percentage of eDNA relative to the untreated control (Tris-EDTA buffer solution was used as the control) was measured.
Morphological analysis
The formation of S. epidermidis ATCC 35984 biofilm treated with blank nanogels, quercetin, and PQ-nanogels was observed by SEM. Briefly, S. epidermidis ATCC 35984 (1 × 106 CFU/mL) with quercetin and PQ-nanogels (64 and 128 μg/mL) were grown on a cover glass at 37°C for 24 h, respectively. The adherent bacteria were fixed and dehydrated. The samples were fixed with 2.5% glutaraldehyde for 2 h at 4°C. The surfaces were washed three times with PBS for 15 min, and the sample was dehydrated using a graded series of ethanol (30%, 50%, 70%, 90%, 95%, and 100%) for 20 min each. After critical-point drying and coating by gold sputtering, all samples were observed by SEM.
Statistical analysis
The experiment data are expressed as the mean ± SD. and analyzed by one-way analysis of variance using the SPSS 19.0 software (IBM Corp., USA). A p value < 0.05 was considered significant.
RESULTS
Synthesis of N,O-CMCS
N,O-CMCS was carboxylated through carboxylation of MCAA and determined by FTIR spectroscopy (Fig. 1). The distinctive peaks for CS in the FTIR spectrum of N,O-CMCS at 2,920 cm−1 vanished, and new characteristic peaks (2,945 and 2,873 cm−1) were observed. N,O-CMCS exhibited a characteristic absorption peak of carboxymethyl sodium salt at 1,450 cm−1, which was assigned to the symmetric stretching vibration absorption peak of COO−, suggesting that CS underwent a carboxymethylation reaction. The C–O stretching vibration absorption peak of the primary hydroxyl group at 1,157 cm−1 was weakened significantly, indicating that CS introduced carboxymethyl groups at the N and O positions to varying degrees, confirming the product N,O-CMCS.
Fig. 1. Synthesis of N,O-CMCS. (A) Schematic diagram of N,O-CMCS chemical structure; (B) Fourier transform infrared spectrum of N,O-CMCS.
CS, chitosan; N,O-CMCS, N,O-carboxymethyl chitosan.
Optimization of PQ-nanogels
The optimized formulation of PQ-nanogels comprised 0.3 g N,O-CMCS, 1.5 g gelatin, and 0.1 g TPP. The amount of N,O-CMCS (R = 4.2) and gelatin (R = 3.6) had a more significant effect on the LC of optimized PQ-nanogels compared to TPP (R = 0.2). In this study, the LC and EE of optimal PQ-nanogels were 18.9% ± 0.4% and 77.3% ± 0.5%, respectively (Table 1).
Table 1. Optimization of PQ-nanogels by orthogonal experiment (L934) (n = 3).
| Sample | N,O-CMCS (g) | Gelatin (g) | TPP (g) | LC (%) |
|---|---|---|---|---|
| 1 | 0.1 | 0.5 | 0.1 | 10.7 ± 0.4 |
| 2 | 0.1 | 1.0 | 0.2 | 12.7 ± 0.3 |
| 3 | 0.1 | 1.5 | 0.3 | 15.0 ± 0.2 |
| 4 | 0.2 | 1.5 | 0.1 | 17.2 ± 0.4 |
| 5 | 0.2 | 1.0 | 0.3 | 14.9 ± 0.1 |
| 6 | 0.2 | 0.5 | 0.2 | 13.7 ± 0.5 |
| 7 | 0.3 | 1.5 | 0.2 | 18.3 ± 0.5 |
| 8 | 0.3 | 1.0 | 0.1 | 17.5 ± 0.6 |
| 9 | 0.3 | 0.5 | 0.3 | 15.2 ± 0.7 |
| K1 | 12.8 | 13.2 | 15.1 | |
| K2 | 15.3 | 15.0 | 14.9 | |
| K3 | 17.0 | 16.8 | 15.0 | |
| R | 4.2 | 3.6 | 0.2 | |
| Optimum | K3 | K3 | K1 |
Values are presented as mean ± SD.
K1, K2, and K3 are the average grade for three levels in each factor; R is the different value between the maximum and minimum of K1, K2, and K3 in each level; Optimal formulation of PQ-nanogels: 0.3 g N,O-CMCS, 1.5 g gelatin and 0.1 g TPP.
PQ-nanogels, polyethylene glycol-quercetin nanoparticle-loaded gelatin-N,O-carboxymethyl chitosan composite nanogels; N,O-CMCS, N,O-carboxymethyl chitosan; TPP, tripolyphosphate; LC, loading capacity.
Morphological evaluation
The appearance, lyophilized powder, reconstituted solution, optical microscopy images, SEM, and TEM of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels were evaluated (Fig. 2). The appearance of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels was clear and transparent, faint yellow, dark yellow, and light yellow in inclined bottle, respectively. The PQ-NPs presented a solution form, whereas blank nanogels, Q-nanogels, and PQ-nanogels all showed a gel form in an inclined bottle. Hence, the PQ-nanogels were prepared. After these freshly prepared samples were freeze-dried, the lyophilized powder of the PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels in a bottle were white, white, light yellow, and white, respectively. The lyophilized samples were observed by optical microscopy. The PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels all exhibited a network structure to varying degrees. In particular, PQ-nanogels showed an obvious dense network structure. Subsequently, the lyophilized samples were redissolved in ultrapure water to obtain a reconstituted solution. The reconstituted solutions of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels were clear and transparent, clear and transparent, light yellow, and clear and transparent in a bottle, respectively. Hence, the prepared PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels were water-soluble. SEM images of freshly prepared PQ-NPs revealed spherical particles clustered together in an orderly manner. On the other hand, the SEM of freeze-dried blank nanogels, Q-nanogels, and PQ-nanogels revealed a three-dimensional network structure, particularly for PQ-nanogels. TEM images of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels revealed spherical particles.
Fig. 2. Appearance, lyophilized powder, reconstituted solution, optical microscopy images (scale bars: 400 nm), SEM (scale bars: 500 nm), and TEM (scale bars: 100 nm) of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels.
SEM, scanning electron microscopy; TEM, transmission electron microscopy; PQ-NPs, polyethylene glycol-quercetin nanoparticles; Q-nanogels, quercetin-loaded N,O-carboxymethyl chitosan-gelatin nanogels; PQ-nanogels, polyethylene glycol-quercetin nanoparticle-loaded gelatin-N,O-carboxymethyl chitosan composite nanogels.
Physicochemical characterization
The particle size, ZP, and PDI of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels were 28.7 ± 2.1 nm (Fig. 3A), −0.6 ± 0.2 mV (Fig. 3E), and 0.45 ± 0.02; 52.5 ± 2.7 nm (Fig. 3B), 13.7 ± 0.4 mV (Fig. 3F), and 0.35 ± 0.11; 89.4 ± 1.8 nm (Fig. 3C), 14.2 ± 0.6 mV (Fig. 3G), and 0.32 ± 0.04; 121.5 ± 2.3 nm (Fig. 3D), 14.8 ± 0.4 mV (Fig. 3H), and 0.28 ± 0.02, respectively (Table 2).
Fig. 3. Characteristics of the PQ-nanogels. Size distribution of PQ-NPs (A), blank nanogels (B), Q-nanogels (C), and PQ-nanogels (D); Zeta potential of PQ-NPs (E), blank nanogels (F), Q-nanogels (G) and PQ-nanogels (H); FTIR (I); PXRD (J); In vitro responsive release (K).
PQ-nanogels, polyethylene glycol-quercetin nanoparticle-loaded gelatin-N,O-carboxymethyl chitosan composite nanogels; Q-nanogels, quercetin-loaded N,O-carboxymethyl chitosan-gelatin nanogels; PQ-NPs, polyethylene glycol-quercetin nanoparticles; TPP, tripolyphosphate; PEG, polyethylene glycol ; N,O-CMCS, N,O-carboxymethyl chitosan.
Table 2. Size, ZP and PDI values of PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels (n = 3).
| Sample | Size (nm) | ZP (mV) | PDI |
|---|---|---|---|
| PQ-NPs | 28.7 ± 2.1 | −0.6 ± 0.2 | 0.45 ± 0.02 |
| Blank nanogels | 52.5 ± 2.7 | 13.7 ± 0.4 | 0.35 ± 0.11 |
| Q-nanogels | 89.4 ± 1.8 | 14.2 ± 0.6 | 0.32 ± 0.04 |
| PQ-nanogels | 121.5 ± 2.3 | 14.8 ± 0.4 | 0.28 ± 0.02 |
Values are presented as mean ± SD.
ZP, zeta potential; PDI, polydispersity index; PQ-NPs, polyethylene glycol-quercetin nanoparticles; Q-nanogels, quercetin-loaded N,O-carboxymethyl chitosan-gelatin nanogels; PQ-nanogels, polyethylene glycol-quercetin nanoparticle-loaded gelatin-N,O-carboxymethyl chitosan composite nanogels.
Fig. 3I presents the FTIR spectra of gelatin, TPP, quercetin, PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels. The characteristic FTIR peaks of quercetin were consistent with another study [16]. In the spectrum of PQ-NPs, the characteristic peaks for quercetin at 3,336 and 1,663 cm−1 disappeared, and the characteristic peaks for PEG were observed at 2,885, 1,116, and 972 cm−1. This also suggested that quercetin had been completely wrapped with PEG, and the PQ-NPs were prepared. In the spectrum of blank nanogels, new characteristic peaks (3,490 cm−1) were observed, which were attributed to the interaction between PEG, N,O-CMCS, gelatin, and TPP. In the spectrum of Q-nanogels, new characteristic peaks (1,530 cm−1) were observed, and the distinctive peaks for quercetin at 2,885, 1,116, and 972 cm−1 disappeared, which may be due to the electrostatic interaction between gelatin and N,O-CMCS using TPP. The distinctive peaks for quercetin at 2,885 and 972 cm−1 were not observed from the spectrum of PQ-nanogels, and a new characteristic peak (1,530 cm−1) was visible, which may be attributed to the electrostatic interaction between gelatin and N,O-CMCS using TPP.
PXRD was performed to determine the crystallinity and physical state of CS, N,O-CMCS, gelatin, TPP, quercetin, PQ-NPs, blank nanogels, Q-nanogels, and PQ-nanogels (Fig. 3J). The PXRD patterns of PQ-NPs showed characteristic XRD peaks of quercetin (17.1°, 20.5°, 28.6°, and 35.4° 2θ) disappeared, and the characteristic XRD peaks of PEG (22.0° and 27.5° 2θ) were showed. This suggests that quercetin was completely wrapped into PEG. The characteristic XRD peaks of quercetin were almost absent in blank nanogels, Q-nanogels, and PQ-nanogels. Thus, blank nanogels, Q-nanogels, and PQ-nanogels were amorphous because of the electrostatic interaction between gelatin and N,O-CMCS using TPP. The disappeared characteristic XRD peaks of quercetin in the PQ-nanogels can be attributed to the formation of PQ-nanogels.
In this study, 58.9% ± 2.8% of the PQ-nanogels were released at pH 7.4 after 24 h, compared to 86.2% ± 3.0% at pH 5.5 without gelatinase; 72.3% ± 3.1% of the PQ-nanogels were released at pH 7.4 with gelatinase after four hours, compared to 98.0% ± 1.2% at pH 5.5 with gelatinase (Fig. 3K).
Suppressed biofilm formation by PQ-nanogels
The relative biofilm formation, cell surface hydrophobicity, EPS production, eDNA production, and morphological analysis of S. epidermidis ATCC 35984 treated with blank nanogels, quercetin, and PQ-nanogels were systematically studied to assess the antibiofilm activity of PQ-nanogels against the biofilm formation of S. epidermidis (Fig. 4A). The relative biofilm formation of S. epidermidis ATCC 35984 treated with blank nanogels, were decreased by ≥ 93.3% ± 4.6% at 64 µg/mL and by ≥ 92.3% ± 2.6% at 128 µg/mL. The relative biofilm formation of S. epidermidis ATCC 35984 treated with quercetin decreased by ≥ 15.7% ± 4.0% at 64 µg/mL and by ≥ 53.0% ± 5.1% at 128 µg/mL. The relative biofilm formation of S. epidermidis ATCC 35984 treated with PQ-nanogels decreased by ≥ 58.3% ± 1.7% (p < 0.0001) at 64 µg/mL and by ≥ 66.7% ± 2.9% (p < 0.05) at 128 µg/mL (Fig. 4B). The concentrations of the blank nanogels were 64 and 128 μg/mL. The cell surface hydrophobicities of S. epidermidis ATCC 35984 were 91.3% ± 1.9% and 92.3% ± 1.7%, respectively. The quercetin concentrations were 64 and 128 μg/mL, and the cell surface hydrophobicities of S. epidermidis ATCC 35984 were 72.3% ± 4.2% and 60.3% ± 5.2%, respectively. On the other hand, the concentration of the PQ-nanogels were 64 and 128 μg/mL, and the cell surface hydrophobicities of S. epidermidis ATCC 35984 were 58.3% ± 5.4% (p < 0.05) and 41.0% ± 4.9% (p < 0.0001), respectively (Fig. 4C). The concentrations of the blank nanogels were 64 and 128 μg/mL, and the levels of EPS production of S. epidermidis ATCC 35984 were 20.0% ± 0.8% and 20.3% ± 2.0%, respectively. The quercetin concentrations were 64 and 128 μg/mL, and the levels of EPS production of S. epidermidis ATCC 35984 were 10.3% ± 1.2% and 5.3% ± 1.1%, respectively. In contrast, the levels of EPS production of S. epidermidis ATCC 35984 were 5.3% ± 0.5% (p < 0.01) and 1.3% ± 0.5% (p < 0.05) when the concentrations of the PQ-nanogels were 64 and 128 μg/mL, respectively (Fig. 4D). The concentration of blank nanogels were 64 and 128 μg/mL. The eDNA levels of S. epidermidis ATCC 35984 were 0.93% ± 0.03% and 0.94% ± 0.01%, respectively. The concentrations of quercetin were 64 and 128 μg/mL, and the eDNA levels of S. epidermidis ATCC 35984 were 0.63% ± 0.08% and 0.41% ± 0.04%, respectively. On the other hand, the eDNA of S. epidermidis ATCC 35984 were 0.50% ± 0.04% (p > 0.05) and 0.23% ± 0.02% (p < 0.01) when the concentrations of the PQ-nanogels were 64 and 128 μg/mL, respectively (Fig. 4E). The formation of S. epidermidis ATCC 35984 biofilm treated with quercetin and PQ-nanogels was observed by SEM to explore antibiofilm activity further. Morphological analysis revealed many extracellular substances on the surface of untreated S. epidermidis ATCC 35984. Hence, extracellular polysaccharides were generated, indicating that the biofilm of S. epidermidis was formed. The extracellular substances on the surface of S. epidermidis ATCC 35984 treated with quercetin began to decrease, and the aggregation phenomenon between bacteria decreased. Some bacteria underwent slight collapse. The extracellular substances on the surface of S. epidermidis ATCC 35984 treated with blank nanogels showed almost no change, and aggregation still occurred among bacteria, resulting in a large number of bacteria. Few extracellular substances were found on the surface of S. epidermidis ATCC 35984 treated with PQ-nanogels, and less aggregation occurred among bacteria, resulting in a decrease in the number of bacteria. Bacteria collapse and cell membrane shrinkage or even rupture (Fig. 4F).
Fig. 4. Antibiofilm activity of quercetin and PQ-nanogels against S. epidermidis (n = 3; The dots within the bars represent each measure).
(A) Pattern diagram of reduced cell surface hydrophobicity, EPS production, and eDNA production. Reduced relative biofilm formation (B), cell surface hydrophobicity (C), EPS production (D), and eDNA production (E) by blank nanogels, quercetin, and PQ-nanogels. (F) Scanning electron microscopy images of S. epidermidis treated with different concentrations (0, 64 and 128 μg/mL) of blank nanogels, quercetin and PQ-nanogels (scale bars: 4 μm).
EPS, exopolysaccharides; NS, not significant; PQ-nanogels, polyethylene glycol-quercetin nanoparticle-loaded gelatin-N,O-carboxymethyl chitosan composite nanogels.
*p < 0.05; **p < 0.01; ***p < 0.001.
DISCUSSION
CS was successfully carboxylated by introducing carboxymethyl groups to the N-terminal and O-terminal of CS to improve its water-solubility. Hence, N,O-CMCS was prepared successfully through the carboxylation of MCAA. The concentration of excipients is crucial for drug preparations because it can determine the LC [10]. In this study, the formula of PQ-nanogels was optimized through orthogonal experiments (L934) using LC as indices to obtain the optimal formula. The amount of N,O-CMCS (0.1, 0.2, and 0.3 g), gelatin (0.5, 1.0, and 1.5 g), and TPP (0.1, 0.2, and 0.3 g) were used as variables to screen the formula of the PQ-nanogels. The optimized formulation of PQ-nanogels comprised 0.3 g N,O-CMCS, 1.5 g gelatin, and 0.1 g TTP. The amount of N,O-CMCS (R = 4.2) and gelatin (R = 3.6) had a more significant effect on the LC of optimized PQ-nanogels compared to TTP (R = 0.2). This was attributed to the role of N,O-CMCS and gelatin as the anion and cation carriers to construct the structure of PQ-nanogels, respectively. On the other hand, the LC and EE are higher, meaning that more drugs are in the composite nanogels system.
The optical microscopy images, SEM, and TEM of PQ-nanogels showed that these interconnected three-dimensional network structures could provide sufficient space for the migration and proliferation of cells, and the transport of oxygen and nutrients. This result may be attributed to the electrostatic interaction between gelatin (positive charge) and N,O-CMCS (negative charge) using TPP. The size of the PQ-nanogels was the largest, possibly due to the electrostatic interaction between gelatin and N,O-CMCS using TPP. With a nanoscale particle size, the pharmaceutical preparations had a larger surface area, facilitating drug interactions with bacteria or biofilms. The ZP and PDI are the indices used to evaluate the stability of the preparation. The preparation is generally stable When the absolute value of the ZP is larger, and the PDI is smaller [17]. Therefore, the prepared PQ-nanogels were stable and could be in broader contact with bacteria, play a stronger antibacterial activity, and inhibit the effects on bacterial biofilm [2]. FTIR spectroscopy and PXRD showed that PQ-nanogels were prepared by electrostatic interactions between gelatin and N,O-CMCS with sodium TPP. Considering the micro-environment (pH 5.5 and gelatinase) of S. epidermidis, the release of the PQ-nanogels in PBS (pH 5.5/7.4) with and without gelatinase at 37°C ± 0.5°C was estimated to determine the environmental pH and gelatinase-responsiveness. The PQ-nanogels displayed a dual responding effect (pH and gelatinase) to achieve the on-demand release performance because of the pH-responsiveness of N,O-CMCS, and the gelatinase-responsiveness of gelatin. Therefore, the prepared PQ-nanogels could inhibit bacterial growth and bacterial biofilm formation.
This study examined the antibiofilm activity of PQ-nanogels against the biofilm formation of S. epidermidis, relative biofilm formation, cell surface hydrophobicity, EPS production, eDNA production, and morphological analysis of S. epidermidis ATCC 35984 treated with blank nanogels, quercetin, and PQ-nanogels. PQ-nanogels can enhance the relative biofilm formation of S. epidermidis, enhancing the antibiofilm activity of quercetin. Furthermore, the PQ-nanogels had concentration-dependent antibiofilm effects. The cell surface hydrophobicity contributes to S. epidermidis adhering to hydrophobic tissue surfaces, such as breasts and skin. Therefore, cell surface hydrophobicity is crucial to forming S. epidermidis biofilms. In general, a higher cell surface hydrophobicity indicates a stronger ability of S. epidermidis to form biofilms. Therefore, biofilm formation can be weakened by reducing the cell surface hydrophobicity. In addition, PQ-nanogels can reduce the cell surface hydrophobicity of S. epidermidis significantly, enhancing the antibiofilm activity of quercetin. The potential antibiofilm mechanism of PQ-nanogels against S. epidermidis may be enhanced antibiofilm activity owing to its cell surface hydrophobicity [18]. The adhered bacteria will thicken the biofilm through bacterial proliferation, during which various EPS, such as polysaccharide intercellular adhesin (the main component of EPS), were synthesized. Therefore, reducing the formation of biofilm by decreasing EPS production is significant.
Furthermore, the PQ-nanogels can reduce EPS production of S. epidermidis significantly, enhancing the antibiofilm activity of quercetin. Similarly, the potential antibiofilm mechanism of PQ-nanogels against S. epidermidis may enhance its antibiofilm activity by reducing its EPS production [19]. Biofilm formation will increase bacterial resistance by enabling the horizontal transfer of antibiotic resistance genes through eDNA. Thus, it is essential to decrease biofilm formation by reducing the eDNA of S. epidermidis. More importantly, PQ-nanogels can significantly reduce the eDNA of S. epidermidis, thereby enhancing the antibiofilm activity of quercetin. Therefore, the potential antibiofilm mechanism of PQ-nanogels against S. epidermidis may enhance its antibiofilm activity by reducing its eDNA [7]. On the other hand, the relative biofilm formation, cell surface hydrophobicity, EPS production, eDNA production, and morphological analysis of S. epidermidis ATCC 35984 treated with blank nanogels had not undergone any significant changes. Morphological analysis indicated that PQ-nanogels had a stronger antibiofilm activity against S. epidermidis. The potential antibiofilm mechanism of PQ-nanogels against S. epidermidis may be to enhance its antibiofilm activity by reducing its relative biofilm formation, cell surface hydrophobicity, EPS production, and eDNA production (Fig. 5). Therefore, PQ-nanogels play the role of quercetin by inhibiting biofilms through the nanogels drug delivery systems.
Fig. 5. Schematic diagram of the dual responsiveness (pH and gelatinase) of PEG-quercetin nanoparticles-loaded gelatin-N,O-CMCS composite nanogels to achieve on-demand release for reducing the formation of S. epidermidis biofilm.
EPS, exopolysaccharides; PEG, polyethylene glycol; N,O-CMCS, N,O-carboxymethyl chitosan; TPP, tripolyphosphate.
ACKNOWLEDGMENTS
The authors acknowledge the assistance of Instrumental Analysis Center of Tarim University (Ma Guocai, Wang Lijun and Du Ning). The authors would like to thank Wang Wenqian from Shiyanjia Lab (http://www.shiyanjia.com) for the FTIR analysis.
Footnotes
Funding: The article was supported financially by Opening Foundation of Xinjiang Key Laboratory of Animal Infectious Diseases (2023KLA008), Opening Foundation of Engineering Laboratory for Tarim Animal Diseases Diagnosis and Control of Xinjiang Production & Construction Corps (ELDC202303), Innovation and Entrepreneurship Training Program for College Students (202310757001), the Program Nanjing Agricultural University-Tarim University Joint Fund (NNLH202301) and The second group of Tianshan Talent Training Program: Youth Support Talent Project (2023TSYCQNTJ0033).
Conflict of Interest: The authors declare no conflicts of interest.
- Conceptualization: Luo W, Guo D, Gao X, Wei J, Wei Y.
- Data curation: Luo W.
- Formal analysis: Luo W.
- Funding acquisition: Luo W.
- Investigation: Luo W.
- Methodology: Luo W, Jiang Y, Liu J, Sun B.
- Project administration: Luo W, Jiang Y, Wei J, Wei Y.
- Resources: Luo W.
- Software: Luo W.
- Supervision: Luo W.
- Validation: Luo W.
- Visualization: Luo W.
- Writing - original draft: Luo W, Guo D, Algharib SA.
- Writing - review & editing: Luo W, Algharib SA.
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