Quaternary Ammonium-Tethered Phenylboronic Acids Appended Supramolecular Nanomicelles as a Promising Bacteria Targeting Carrier for Nitric Oxide Delivery

The delivery of drugs to focal sites is a central goal and a key challenge in the development of nanomedicine carriers. This strategy can improve the selectivity and bioavailability of the drug while reducing its toxicity. To ensure the specific release of nitric oxide at the site of a bacterial infection without damaging the surrounding normal tissue, we designed a host-guest molecule containing a host molecule with a target moiety and a nitric oxide donor to release nitric oxide. The boronic acid group in the structure of this nanoparticle interacts strongly and specifically with the surface of E. coli. In addition, the quaternary amine salt can interact electrostatically with bacteria, indicating a large number of negatively charged cell membranes; altering the molecular structure of the cell membrane; increasing the permeability of the cell membrane; and causing cytoplasmic diffusion and cell lysis, resulting in lethal activity against most bacteria. The synthesised molecules were characterised by 1H NMR and mass spectrometry. The strong specific interaction of the boronic acid moiety with the surface of E. coli and the electrostatic interaction of the quaternary amine salt with the cell membrane were confirmed by antibacterial experiments on molecules with and without the targeting moiety. The targeting group-modified micelles enhanced the antibacterial effect of the micelles very effectively through specific interactions and electrostatic interactions. In addition, in vitro skin wound healing experiments also confirmed the targeting and antimicrobial effect of micelles. These results suggest that the specific release of nitric oxide at the site of bacterial infection is an important guide to further address the emergence of antibiotic-resistant strains of bacteria.


Introduction
Microbial contamination caused by pathogens poses a serious threat to human health and has posed an unprecedented challenge to antimicrobial research in recent decades [1]. Usually, the use of antibiotics is the contemporary treatment of choice for bacterial infections. However, the misuse of antibiotics is inducing the emergence of more and more drugresistant bacteria [2]. The antibiotic resistance crisis is one of the greatest challenges to global health, and if not actively addressed, it will have permanent and enormous humanitarian and economic consequences [3,4]. Notably, antibiotic resistance is associated not only with structural and genetic mutations in mayfly bacteria, but also with bacterial biofilms [5]. Therefore, the development of more effective antimicrobial agents, as well as the proposal of new antimicrobial strategies, are urgent needs for academic research and clinical treatment.
To date, many approaches with significant antimicrobial effects have emerged in various medical fields. Among these, nanomaterials technology offers powerful broadspectrum antibacterial strategies. In contrast with the specific structures of antibiotics, potential in the biological field. However, due to the presence of the non-specific targeted delivery of NO, the rapid systemic clearance of low molecular weight donors and concerns about non-essential toxicity have hindered the further clinical application of low molecular weight NO donors. In order to precisely deliver effective therapeutic concentrations to the lesion and reduce adverse effects, there is a need to develop NO drug release platforms that can be externally regulated and targeted.
Recently, there has been increasing interest in developing targeted antimicrobial materials and methods [16], which can effectively improve antimicrobial efficiency by applying targeted antimicrobial materials and strategies. For example, Nesha et al. designed a versatile and stimulating (NIR laser-activated) antimicrobial platform by combining the inherent photothermal capability and the excellent biocompatibility of polydopamine nanoparticles with the membrane targeting and cleavage activity of antimicrobial peptides (AMP) [17]. Cheng et al. proposed a new approach for targeting bacteria by using formic acid (a cellular metabolite found in certain bacterial species) to activate the antimicrobial drug and selectively inhibit its growth [18]. The synthesis of biocompatible enzyme-responsive silver nanoparticle assemblies and their application in the efficient targeting of methicillinresistant Staphylococcus aureus for antimicrobial therapy has been demonstrated in a report by Yang et al. [19]. Herein, we designed functional nanomicelles particles with the dual-targeting delivery of nitric oxide antimicrobial agents to bacterial cell membranes, which can precisely target the cell membranes of Gram-negative bacteria. Quaternary ammonium salt (QAS) compounds have been shown to be powerful antimicrobial agents against spectroscopic bacteria. Bacterial surfaces are negatively and positively charged quaternary ammonium salts (QAS) can bind to negatively charged bacterial or biofilm surfaces through electrostatic interactions, altering the cell membrane molecular structure, increasing cell membrane permeability, which can even lead to cytoplasmic diffusion and cell lysis, and thus can be lethal to most bacteria [20,21]. Compared to commonly used antibioticbased antimicrobial agents, QAS compounds are rarely resistant to bacteria [22]. However, Gram-negative bacteria, which consist of complex multilayered outer membranes, are difficult to be inserted and penetrated by QAS compound-based antimicrobial agents compared to Gram-positive bacteria that contain simple cell wall structures [23]. Consequently, achieving antimicrobial activity comparable to Gram-positive bacteria requires greater concentrations of QAS compound-based antimicrobial agents, increasing the toxic side effects arising from in vivo use [24]. Therefore, this challenge highlights the need for alternative platforms for effective control, effective targeting, or combination therapy. Boronic acid (BA), a compound with a boron centre connected to three hydroxyl groups through an oxy-boron bond, has been widely used in the development of biochemical sensors [25,26] and targeted antitumours [27,28] because of its high sensitivity for covalent binding to cisdiols in sugars. The rich lipopolysaccharide (LPS) on the cell wall surface of Gram-negative bacteria contains a cis-diol molecular structure, while the boronic acid group covalently couples to the cis-diol structure, and the boronic acid-based functional materials have been shown to have excellent recognition and targeting of bacterial properties [29,30]. Although phenylboronic acids can specifically recognize and target bacterial cell membranes, the antibacterial activity of nanoparticles modified by the phenylboronic acid groups with only a single antimicrobial mechanism is still insufficient for in vivo antimicrobial repair applications [31][32][33]. Therefore, the construction of a multimodal targeting therapeutic platform is a promising strategy to combat bacterial-associated infections.
Based on the construction of a multimodal targeted therapeutic platform to achieve a selective fight against bacterial-associated infections, we reported a targeted pro-drug nanoparticle with efficient antibacterial activity. In this study, a natural compound β-CD and its derivatives (maltoheptose) with good biocompatibility and abundant functional groups were selected as backbone molecules. Based on the host-guest interaction between cyclodextrin and adamantane molecules, an antibacterial nanomaterial that not only targets bacterial cell membranes, but also can release NO, was designed and synthesized. The antimicrobial process of the designed pro-drug nanoparticles is as follows(Scheme 1): pro-drug nanoparticles bind to the negatively charged outer membrane of bacteria with lipopolysaccharide (LPS) + NO production from the NO donor → bacterial apoptosis. a selective fight against bacterial-associated infections, we reported a targeted pro-drug nanoparticle with efficient antibacterial activity. In this study, a natural compound β-CD and its derivatives (maltoheptose) with good biocompatibility and abundant functional groups were selected as backbone molecules. Based on the host-guest interaction between cyclodextrin and adamantane molecules, an antibacterial nanomaterial that not only targets bacterial cell membranes, but also can release NO, was designed and synthesized. The antimicrobial process of the designed pro-drug nanoparticles is as follows(Scheme 1): pro-drug nanoparticles bind to the negatively charged outer membrane of bacteria with lipopolysaccharide (LPS) + NO production from the NO donor → bacterial apoptosis. Scheme 1. Schematic diagram of supramolecular assembly and targeted antibacterial mechanism.

Measurements
The 1 H NMR spectra were recorded on a JNM-ECS 400M NMR (JEOL, Akishima-shi, Japan) instrument using dimethyl sulfoxide-d6 (DMSO-d 6 ) or deuterium oxide (D 2 O) as the solvent and TMS as the internal reference. The transmission electrons (TEM) were measured on a Talos F200C (FEI, Hillsboro, OR, USA). A mass spectrometric analysis was also performed on a Bruker Daltonics autoflex III smartbeam. Scanning electron microscopy was recorded on a Hitachi S-4800.
All animal experiments were conducted in compliance with the Animal Ethical Procedures and Guidelines of the People's Republic of China and were approved by the Animal Ethics Committee of Lanzhou University (China) (No. 2022-G04).

Results and Discussion
In order to effectively improve the targeting performance, as well as synergistic and precise bactericidal anti-infective therapy, we report a nanoparticle with efficient antimicrobial targeting precursors. In this study, a natural compound β-CD and its derivatives (maltoheptose) with good biocompatibility and abundant functional groups were selected as backbone molecules. Based on the host-guest interaction between cyclodextrin and adamantane molecules, an antibacterial nanomaterial that not only targets bacterial cell membranes but also can release NO was designed and synthesized. NO can penetrate the membranes of bacteria and induce oxidative stress damage in bacteria without resistance to bacteria, and it is a widely used gaseous antibacterial agent. However, the high activity and the short half-life of NO have forced the development of various delivery systems to increase the local NO concentration and to achieve effective antibacterial activity. In the study, a quaternary ammonium-tethered phenylboronic acids appended supramolecular nanomicelle was constructed as a promising bacteria-targeting carrier for NO delivery. First, two targeting host molecules CD-QAS-B 7 and CD-QAS-B 1 with different numbers of dual-targeting functional groups were synthesized. Next, the guest molecules containing multiple NO donors (Ad-MH-NO) were prepared via nitrosation modification of maltoheptose. Finally, the amphiphilic coupled supramolecules containing hydrophilic and hydrophobic chain segments were assembled into supramolecular nanomicelles in water via host-guest interaction coupling.

Design, Synthesis, and Characterisation of CD-QAS-BA and Ad-MH-NO
The host molecules CD-QAS-B 7 and CD-QAS-B 1 with targeting groups were synthesized by a multistep synthesis, as shown in Figure 1. CD-QAS-B 1 was synthesized by the substitution of the 6-hydroxyl group of β-CD with the p-toluenesulfonyl chloride and subsequent amination with the dimethylamine solution to give CD-(dma) 1 . CD-(dma) 1 was reacted with 4-(bromomethyl) benzeneboronic acid to construct CD-QAS-B 1 containing one target group. CD-QAS-B 7 was synthesized by substituting the 6-hydroxyl group of β-CD with iodine, and subsequent amination with dimethylamine solution to obtain CD-(dma) 7 , which was reacted with 4-(bromomethyl) benzeneboronic acid to construct CD-QAS-B 7 containing seven target groups. The molecular structures of the intermediates and the target molecules were verified by 1 H NMR and MS spectra (Figures 2 and S1). The experimental results showed that we successfully synthesized the dual-targeting host molecules CD-QAS-B 7 and CD-QAS-B 1 . The preparation process of Ad-MH-NO is shown in Figure 3. First, maltoheptose (MH) was prepared by the ring-opening reaction of β-cyclodextrin, which provided the key sugar segment for the next step of the synthesis. Then, Ad-MH was obtained by modifying the adamantane amine to the reduced terminal hydroxyl group of MH, and the remaining modifiable hydroxyl groups were then reacted with acetyl bromide to obtain Ad-MH-Br with multiple alkyl bromides. Finally, the alkyl bromide was reacted with AgNO 3 to obtain the Ad-MH-NO guest molecule containing multiple NO donors. The details of the synthesis of various molecules are in the Experiment Section (Supporting Information), and the molecular structures of the intermediates and the target molecules were verified by 1 H NMR and FTIR (Figure 4). bromide was reacted with AgNO3 to obtain the Ad-MH-NO guest molecule containing multiple NO donors. The details of the synthesis of various molecules are in the Experiment Section (Supporting Information), and the molecular structures of the intermediates and the target molecules were verified by 1 H NMR and FTIR ( Figure 4).

Transmission Electron Microscopy (TEM) of Assembled Nanoparticles
To obtain antibacterial materials with integrated targeting and NO donors, we assembled amphiphilic coupled supramolecules containing hydrophilic and hydrophobic chain segments into nanomicelles particles in water. As shown in Figure 5, the TEM images showed that the prepared nanomicelles had a uniform size and a well-dispersed circular structure, indicating that both of the main molecules of one and seven modified dualtargeting functional groups on cyclodextrins could couple with the guest molecules of NO donors to self-assemble and form regular nanostructures. Furthermore, CMC values were studied by the widely reported pyrene-probe based fluorescence technique. The ratio of the fluorescence intensity ratio of micelles I 403 /I 373 to the logarithm of concentration is shown in Figure S2, the CMC value for CD-QAS-B1 was 1.175 × 10 −7 mg/mL, the CMC value for CD-QAS-B 7 was 5.754 × 10 −7 mg/mL.

Transmission Electron Microscopy (TEM) of Assembled Nanoparticles
To obtain antibacterial materials with integrated targeting and NO donors, w sembled amphiphilic coupled supramolecules containing hydrophilic and hydroph chain segments into nanomicelles particles in water. As shown in Figure 5, the TEM ages showed that the prepared nanomicelles had a uniform size and a well-dispersed cular structure, indicating that both of the main molecules of one and seven mod

Transmission Electron Microscopy (TEM) of Assembled Nanoparticles
To obtain antibacterial materials with integrated targeting and NO donors, we assembled amphiphilic coupled supramolecules containing hydrophilic and hydrophobic chain segments into nanomicelles particles in water. As shown in Figure 5, the TEM images showed that the prepared nanomicelles had a uniform size and a well-dispersed circular structure, indicating that both of the main molecules of one and seven modified dual-targeting functional groups on cyclodextrins could couple with the guest molecules of NO donors to self-assemble and form regular nanostructures. Furthermore, CMC values were studied by the widely reported pyrene-probe based fluorescence technique. The ratio of the fluorescence intensity ratio of micelles I403/I373 to the logarithm of concentration is shown in Figure S2, the CMC value for CD-QAS-B1 was 1.175 × 10 −7 mg/mL, the CMC value for CD-QAS-B7 was 5.754 × 10 −7 mg/mL.

Detection of NO In Vitro
Real-time NO release was measured using a Griess reagent colour reaction. The kinetics of NO release from CD-QAS-B was determined under physiological conditions (pH 7.40, 37 °C). The initial concentration of NO can be obtained by quantifying the resulting colour by UV/VIS spectroscopy, and the resulting NO release is shown in Figure 6A. Overall, MH-NO has NO payload capability. NO release was most pronounced during the first few hours in PBS (pH 7.4) at 37 °C. The initial high concentration of bactericidal NO ensures an effective initial attack on microorganisms. As shown in Figure S3, the kinetic analysis of NO formation for all materials was found to be in accordance with the kinetic quasi first order equation.

Detection of NO In Vitro
Real-time NO release was measured using a Griess reagent colour reaction. The kinetics of NO release from CD-QAS-B was determined under physiological conditions (pH 7.40, 37 • C). The initial concentration of NO can be obtained by quantifying the resulting colour by UV/VIS spectroscopy, and the resulting NO release is shown in Figure 6A. Overall, MH-NO has NO payload capability. NO release was most pronounced during the first few hours in PBS (pH 7.4) at 37 • C. The initial high concentration of bactericidal NO ensures an effective initial attack on microorganisms. As shown in Figure S3, the kinetic analysis of NO formation for all materials was found to be in accordance with the kinetic quasi first order equation.

Antibacterial Properties
Due to the specificity of the cell membrane structure of the Gram-negative bacteria, E. coli was chosen as the test subject of this work in order to investigate the antibacterial activity of our designed dual-targeting NO delivery system containing quaternary positive ions and boronic acid moieties. As shown in Figure 6C, the number of viable colonies of E. coli on solid LB agar plates varied after interacting with different materials for a period of time, respectively. Compared with the PBS solution control, although there was no functional group in Ad-MH-NO to target the bacterial cell membrane, the continuous release of NO also induced rapid apoptosis of bacteria, and thus the number of surviving bacterial colonies was much lower than that in the PBS control. When bacteria interacted with B 1 -QAS@MH-NO or B 7 -QAS@MH-NO dual-targeting nanoparticles, the results showed that the number of surviving bacterial colonies on the plates was much lower than that of the system without targeted property, and the number of colonies in the B 7 -QAS@MH-NO group was reduced by about half compared to the B 1 -QAS@MH-NO group. However, a large number of bacteria survived after interaction with cyclodextrins with a single target group. The results also showed that the dual-targeting functional groups could significantly enhance the antibacterial activity of the NO-delivered nanoplatform with the same NO content, and they were proportional to the number of targeting groups. As shown in Figure 6B, it can also be seen that the bacterial cell membrane integrity was barely visible after the interaction with Ad-MH-NO compared to the control group, while the bacterial cell membrane integrity was severely disrupted after the interaction with the NO delivery nanoparticles with targeting groups. This phenomenon is in line with our original idea of using the targeting strategy to increase the extent of NO damage to bacterial cell membranes in order to improve the antibacterial activity of NO.

Antibacterial Properties
Due to the specificity of the cell membrane structure of the Gram-negative bacteria, E. coli was chosen as the test subject of this work in order to investigate the antibacterial activity of our designed dual-targeting NO delivery system containing quaternary positive ions and boronic acid moieties. As shown in Figure 6C, the number of viable colonies of E. coli on solid LB agar plates varied after interacting with different materials for a period of time, respectively. Compared with the PBS solution control, although there was no functional group in Ad-MH-NO to target the bacterial cell membrane, the continuous release of NO also induced rapid apoptosis of bacteria, and thus the number of surviving bacterial colonies was much lower than that in the PBS control. When bacteria interacted with B1-QAS@MH-NO or B7-QAS@MH-NO dual-targeting nanoparticles, the results showed that the number of surviving bacterial colonies on the plates was much lower than that of the system without targeted property, and the number of colonies in the B7-QAS@MH-NO group was reduced by about half compared to the B1-QAS@MH-NO group. However, a large number of bacteria survived after interaction with cyclodextrins with a single target group. The results also showed that the dual-targeting functional groups could significantly enhance the antibacterial activity of the NO-delivered nanoplatform with the same NO content, and they were proportional to the number of targeting groups. As shown in Figure 6B, it can also be seen that the bacterial cell membrane integrity was barely visible after the interaction with Ad-MH-NO compared to the control * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ***** p < 0.0001.

Bacterial Staining Test
DAF-FM can cross the membrane of living cells. After entering the cells, it can be catalysed by the cytoplasmic esterase to form DAF-FM, which cannot cross the cell membrane [34]. DAF-FM has very weak fluorescence, but after reacting with NO, it will produce the fluorescein benzotriazole, with an excitation wavelength of 495 nm and an emission wavelength of 515 nm. As shown in Figure 7, there was almost no fluorescence in the bacterial cell membrane after the interaction with Ad-MH-NO. Additionally, the fluorescence in the bacterial cell membrane after the interaction with B-QAS@MH-NO was significantly enhanced, and the fluorescence intensity was positively correlated with the number of the targeting groups. The stronger fluorescence in the bacterial cell membrane indicated that the additional NO-related content in the membrane could lead to serious damage to the bacterial cell membrane and could cause oxidative damage to DNA and liposomes, thus accelerating the bacterial death process. This result is consistent with the results of the in vitro antibacterial experiments described above, indicating that the dualtargeting NO delivery nanoplatform can effectively target bacterial cell membranes; deliver NO donors to the vicinity of bacteria for a cumulative release in a targeted manner; enhance local NO concentration; and thus improve the antibacterial activity of the nanoplatform.
damage to the bacterial cell membrane and could cause oxidative damage to DNA and liposomes, thus accelerating the bacterial death process. This result is consistent with the results of the in vitro antibacterial experiments described above, indicating that the dualtargeting NO delivery nanoplatform can effectively target bacterial cell membranes; deliver NO donors to the vicinity of bacteria for a cumulative release in a targeted manner; enhance local NO concentration; and thus improve the antibacterial activity of the nanoplatform.

Live/Dead Staining of Biofilms with 3D CLSM
The live/dead staining of the biofilm was analysed using a three-dimensional confocal scanning microscope, as shown in Figure 8. While propidium iodide can reach the nucleus and embed DNA to produce red fluorescence that marks dead bacteria with damaged cell membranes, SYTO9 can enter all bacteria. In general, the fluorescence intensity of SYTO9 (green) decreases when both dyes penetrate the bacteria. Therefore, bacteria with intact membranes show green fluorescence, while bacteria with damaged membrane structure mainly show red fluorescence. Compared with PBS and Ad-MH-NO, a large number of red fluorescence spots appeared in B1-QAS@MH-NO and B7-QAS@MH-NO groups, indicating that the enhanced antibiofilm activity was based on the dual-targeting reaction.

Live/Dead Staining of Biofilms with 3D CLSM
The live/dead staining of the biofilm was analysed using a three-dimensional confocal scanning microscope, as shown in Figure 8. While propidium iodide can reach the nucleus and embed DNA to produce red fluorescence that marks dead bacteria with damaged cell membranes, SYTO9 can enter all bacteria. In general, the fluorescence intensity of SYTO9 (green) decreases when both dyes penetrate the bacteria. Therefore, bacteria with intact membranes show green fluorescence, while bacteria with damaged membrane structure mainly show red fluorescence. Compared with PBS and Ad-MH-NO, a large number of red fluorescence spots appeared in B 1 -QAS@MH-NO and B 7 -QAS@MH-NO groups, indicating that the enhanced antibiofilm activity was based on the dual-targeting reaction.

Evaluation of Wound Healing Effect of Bacterial Infection
In order to further verify that the dual-targeting nanoparticles can promote w healing in mice infected with E. coli. As shown in Figure 9, mice were divided int gaderm 3M , Tegaderm 3M + sponge, B7-QAS@MH-NO, B1-QAS@MH-NO, and Ad-MH groups, and wound healing was recorded at different times. Compared with the co group, the infected wounds of mice treated with B7-QAS@MH-NO and B1-QAS@MH gradually formed scars and healed faster than the other three groups.

Evaluation of Wound Healing Effect of Bacterial Infection
In order to further verify that the dual-targeting nanoparticles can promote wound healing in mice infected with E. coli. As shown in Figure 9, mice were divided into Tegaderm 3M , Tegaderm 3M + sponge, B 7 -QAS@MH-NO, B 1 -QAS@MH-NO, and Ad-MH-NO groups, and wound healing was recorded at different times. Compared with the control group, the infected wounds of mice treated with B 7 -QAS@MH-NO and B 1 -QAS@MH-NO gradually formed scars and healed faster than the other three groups.
In order to further verify that the dual-targeting nanoparticles can promote wound healing in mice infected with E. coli. As shown in Figure 9, mice were divided into Tegaderm 3M , Tegaderm 3M + sponge, B7-QAS@MH-NO, B1-QAS@MH-NO, and Ad-MH-NO groups, and wound healing was recorded at different times. Compared with the control group, the infected wounds of mice treated with B7-QAS@MH-NO and B1-QAS@MH-NO gradually formed scars and healed faster than the other three groups.

Histological Analysis
There are four overlapping steps in wound healing: inflammation, tissue formation, stroma formation, and remodelling. H&E, Masson, and CD31 were used for the histological analysis of infected wounds to study the subtle changes of the infected wounds. As shown in Figure 10, compared with the control group, inflammatory cells in the B7-QAS@MH-NO and B1-QAS@MH-NO groups were significantly reduced, indicating that the material could promote skin regeneration. Collagen synthesis by dermal fibroblasts is also important for wound healing. The results of the Masson trichromatic staining showed that the amount of collagen deposition on the wound surface in B7-QAS@MH-NO and B1-QAS@MH-NO groups gradually increased, and the intensity of collagen staining was significantly enhanced.

Histological Analysis
There are four overlapping steps in wound healing: inflammation, tissue formation, stroma formation, and remodelling. H&E, Masson, and CD31 were used for the histological analysis of infected wounds to study the subtle changes of the infected wounds. As shown in Figure 10, compared with the control group, inflammatory cells in the B 7 -QAS@MH-NO and B 1 -QAS@MH-NO groups were significantly reduced, indicating that the material could promote skin regeneration. Collagen synthesis by dermal fibroblasts is also important for wound healing. The results of the Masson trichromatic staining showed that the amount of collagen deposition on the wound surface in B 7 -QAS@MH-NO and B 1 -QAS@MH-NO groups gradually increased, and the intensity of collagen staining was significantly enhanced. Immunohistochemical staining of endothelial cell marker CD31 showed that the expression of CD31 increased gradually and concentrated in the dermis, especially in the dermal papilla layer. The CD31 staining was more intense in the B7-QAS@MH-NO and B1-QAS@MH-NO groups, indicating that B-QAS@MH-NO stimulated angiogenesis more, promoted blood vessel formation, and aided nutrient transport in the process of wound healing. Figure 10. Histological micrograph of exposed abscess treated with Tegaderm 3M (control), Tegaderm 3M + sponge, B7-QAS@MH-NO, B1-QAS@MH-NO, Ad-MH-NO, Masson's trichromatic staining, and immunohistochemical CD31 staining.

Conclusions
In conclusion, we designed dual-targeting micelles loaded with NO and modified with phenylboronic acid and quaternary ammonium salts against cyclodextrins. We combined the specific interaction of the boronic acid moiety with Escherichia coli and the electrostatic interaction of quaternary ammonium salts with bacterial cell membranes, resulting in the specific release of nitric oxide at the site of bacterial infection. The results showed that micelles modified by the phenylboronic acid moiety with the quaternary am- Immunohistochemical staining of endothelial cell marker CD31 showed that the expression of CD31 increased gradually and concentrated in the dermis, especially in