Biguanide-functionalized peptide mimics effectively combat drug-resistant ESKAPE pathogens and meningitis

Domain: physics
Published: 2025-12-09
Editorial outcome: minor_revision

Abstract
ESKAPE pathogens-induced meningitis seriously threatens human health due to antimicrobial resistance and low permeability of blood-brain barrier (BBB). It’s a promising strategy to combat drug-resistant pathogens using synthetic mimics of host defense peptide (HDP) bearing positive charges that are mainly provided by amine or guanidine. Here, we report that biguanide serves as a type of positively charged moiety to design HDP mimics. Biguanide exerts a stronger interaction with bacterial membrane phospholipids via bidentate hydrogen bonds than do amine and guanidine. The biguanide-functionalized HDP mimic, PBGProOx20, targets bacterial membrane phospholipids to show potent activity against all ESKAPE pathogens and does not induce antimicrobial resistance. PBGProOx20 exhibits promising BBB-penetrating property and displays potent therapeutic effects in female mice full-thickness infection model, sub-cutaneous infection model, kidney infection model, peritonitis model, and meningitis model. This study provides a promising strategy for designing HDP mimics to combat ESKAPE pathogens and meningitis.

Introduction
Bacterial meningitis poses a serious threat to human health with a mortality rate reaching up to 54%1, particularly when caused by multidrug-resistant ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species2,3. The blood-brain barrier (BBB) further limits the clinical selection and efficacy of antibiotics4. Thus, it is urgent to develop potent antimicrobials with broad-spectrum activity and BBB penetrating property for the treatment of ESKAPE pathogens-induced infections, especially meningitis. Host defense peptides (HDPs) have been widely studied as potential anti-infective alternatives of antibiotics owing to their broad-spectrum antimicrobial activity and low tendency to induce antimicrobial resistance5–9. However, inherent limitations of HDPs such as low protease stability, high cost, and cumbersome synthesis have restricted their applications10. Consequently, HDPs mimics are actively studied, aiming to address drug-resistant bacterial infections11–22. However, currently there are few reports displaying therapeutic efficacy in bacterial meningitis23.

Cationic HDPs and their mimics exert antimicrobial activity through initial interactions with the negatively charged phospholipids of bacteria, primarily using amine or guanidine moieties as cationic groups (Fig. 1a)24–37. However, achieving dual-functional HDP mimics with potent anti-ESKAPE activity and BBB-penetrating ability using these two moieties remains challenging38–44. We turn attention to an alternative positively charged moiety, biguanide, which possesses higher positive charge density than amine and guanidine, as revealed by density functional theory calculations (Fig. 1b). The planar, conjugated cationic structures of biguanide enables simultaneous donation of two hydrogen bonds to anionic phosphate head groups of bacterial membrane phospholipids (Fig. 1c). This dual-point attachment generates cooperative electrostatic interaction and bidentate hydrogen bonds, significantly enhancing membrane binding affinity and disruption efficiency45. The higher localized positive charge density also endows biguanide-based compounds more efficient initial electrostatic interaction with negatively charged luminal surface of BBB, thereby potentially enhancing its BBB penetration efficiency via adsorptive-mediated transcytosis, which is a crucial pathway for positively charged molecules to enter the brain46,47. Thus, we hypothesize that biguanide can serve as a type of positively charged moiety for HDP mimics to exert broad-spectrum activity and BBB-penetrating property to combat drug-resistant infections caused by ESKAPE pathogens, especially meningitis (Fig. 1a).

In this study, we explore biguanide-functionalized HDP mimics using poly(2-oxazoline)s as the backbone structure that was recently reported to be a peptide-mimicking scaffold by our group48–51. The optimal compound, PBGProOx20, demonstrates broad-spectrum activities against multi-drug resistant bacteria and mature biofilms, including all ESKAPE pathogens, via a membrane-disrupting mechanism (Fig. 1d). Notably, the repeated use of PBGProOx20 doesn’t induce bacteria to develop resistance against PBGProOx20 and cross-resistance against commonly used antibiotics. PBGProOx20 displays potent therapeutic efficacy against ESKAPE pathogens in multiple models of drug-resistant bacterial infections in mice, including full-thickness wound infections, subcutaneous infections, kidney infections, and peritonitis (Fig. 1d). Moreover, PBGProOx20 effectively penetrates BBB through transcytosis across brain endothelial cells and exerts promising therapeutic effects in treating bacterial meningitis. Our findings suggest that biguanide-functionalized HDP mimics represent a promising strategy to develop antimicrobials with potent activities against all ESKAPE pathogens and BBB penetration to treat bacterial meningitis.

Results
Synthesis of biguanide-functionalized poly(2-oxazoline)s with variable spacer groups
A series of HDP-mimicking poly(2-oxazoline)s were synthesized with positively charged biguanide groups and variable sidechain spacer groups. Briefly, amine-functionalized poly(2-oxazoline)s were synthesized through methyl trifluoromethanesulfonate (MeOTf)-initiated polymerization of 2-oxazolines featuring diverse side chains, followed by trifluoroacetic acid (TFA) treatment to remove the N-Boc protecting groups (Fig. 2a, and Supplementary Figs. 1~15). The obtained amine-functionalized poly(2-oxazoline)s were treated with N-Amidinopyrazole-1-carboxamidine to afford the biguanide-functionalized poly(2-oxazoline)s in a single step (Supplementary Figs. 16~19)52. The resulting poly(2-oxazoline)s, featuring biguanide groups and variable sidechain spacer groups (methan-, ethan-, propan-, butan-, pentan-), were obtained at around 20 mer (DP=20) at a narrow dispersity (1.07−1.26), according to the characterization using gel permeation chromatography (GPC) (Fig. 2b, Supplementary Fig. 20, Table S1). The successful functionalization of poly(2-oxazoline)s with biguanide groups was confirmed by 1H NMR spectra (Supplementary Figs. 21–30). The strong electron-withdrawing effect and high charge density of the biguanide moiety induced a notable downfield shift of the proton peak from ‘H N’ (δ~3.0 ppm for −CH2−amine) to ‘H B’ (δ~3.2 ppm for −CH2−biguanide), with no residual upfield peaks attributable to unconverted amine groups observed, indicating virtually complete functionalization (Fig. 2c).

As described above, the synthetic process of biguanide-functionalized poly(2-oxazoline)s can be completed in as fast as 12 h. In contrast, solid-phase-peptide synthesis (SPPS) is the most common method for HDPs synthesis53. The sequential grafting of amino acids and repeated deprotection steps, along with complex purification schemes, significantly increase synthetic difficulty, period and limit quantities of the final product. Solid-phase synthesis of HDPs often continuously consumes excess expensive coupling reagents and protected amino acids. In contrast, the fast synthesis and cheap reactants make poly(2-oxazoline)s significantly more cost-effective and accessible, overcoming key limitations of HDPs such as high cost and difficult synthesis.

In vitro antimicrobial activity and toxicity of biguanide-functionalized poly(2-oxazoline)s
The antimicrobial efficacy of the biguanide-functionalized poly(2-oxazoline)s with various side chain spacers against six ESKAPE pathogens was quantified using the minimum inhibitory concentrations (MIC). These polymers exhibited potent activity against gram-positive bacteria (S. aureus and E. faecium), with MICs ranging between 6.25 and 12.5 μg/mL (Fig. 2d). Notably, the biguanide-functionalized poly(2-oxazoline) bearing a propane side-chain spacer, PBGProOx20, outperformed other polymers against gram-negative bacteria, including K. pneumoniae, A. baumannii, P. aeruginosa, and E. coli, with MICs ranging from 6.25 to 25 μg/mL. Moreover, these polymers displayed negligible hemolysis against red blood cells and cytotoxicity toward COS7 kidney cells, with HC10 and IC50 values exceeding 2000 μg/mL and 400 μg/mL, respectively (Fig. 2e). The antimicrobial selectivity index (SI) of these polymers was calculated from HC10/MIC and IC50/MIC, respectively. Among these polymers, PBGProOx20 demonstrated superior antimicrobial selectivity against all drug-resistant ESKAPE pathogens, with SI surpassing 80 (HC10/MIC) and 32 (IC50/MIC) (Tables S2 and S3).

Poly(hexamethylene biguanide) (PHMB) is a broad-spectrum antimicrobial agent and is approved as a disinfectant used for topical wound care. However, the antimicrobial activity of PHMB is primarily attributed to the dense linear arrangement of biguanide groups, resulting in potential toxicity that limits its in vivo applications. The HDP-mimicking poly(2-oxazoline)s deliberately incorporate biguanide moieties sparsely and controllably onto the highly tunable, flexible, and biocompatible poly(2-oxazoline) backbone. The poly(2-oxazoline) was recently reported to be a peptide-mimicking scaffold by our group50,51, which provides a modular platform to fine-tune the hydrophobic/hydrophilic balance, chain length, and biguanide spacing, enabling the optimization of antimicrobial activity and toxicity. We also compared the antimicrobial activity, hemolysis, cytotoxicity and antimicrobial selectivity of PHMB (Table S4). While PHMB showed potent antimicrobial efficacy (MIC=0.39-6.25 μg/mL), substantial hemolysis and cytotoxicity (HC10=3.13 μg/mL, IC50=25 μg/mL) greatly weakened its antimicrobial selectivity. PBGProOx20 demonstrates significantly better antimicrobial selectivity against ESKAPE pathogens and has greater potential for treating drug-resistant bacterial infections.

To further elucidate the impact of polymer chain length on antimicrobial performance, we synthesized biguanide-functionalized poly(2-oxazoline)s (PBGProOxn) bearing a propane side chain spacer and variable chain lengths (5, 10, 20, and 40 mer) at a narrow dispersity (1.10−1.23) (Supplementary Fig. 31, and Table S5). Among these polymers, PBGProOx20 and PBGProOx40 demonstrated potent antimicrobial efficacy against all six ESKAPE pathogens with MICs in the range of 6.25 to 25 μg/mL, superior to 5 and 10 mer polymers (Fig. 2f). Moreover, PBGProOx20 exhibited lower cytotoxicity (IC50 >400 μg/mL) than PBGProOx40 (Fig. 2g). We also evaluated the cytotoxicity of PBGProOx20 on primary cells and found that bone marrow mesenchymal stromal cells (BMSCs) retained ~100% viability even after 24-h treatment with 400 μg/mL of PBGProOx20 (Supplementary Fig. 32). Collectively, these results suggest PBGProOx20 as the optimal antimicrobial polymer for further study.

The broad-spectrum antimicrobial activities of PBGProOx20 were further assessed against multiple clinically isolated bacterial strains, comprising 13 gram-positive strains and 17 gram-negative strains. Most of these pathogens were drug-resistant or even multidrug-resistant to tested antibiotics, including ampicillin, vancomycin, streptomycin, imipenem, and norfloxacin (Fig. 2h, i, and Tables S6 and S7). In sharp contrast, PBGProOx20 was active against all these drug-resistant bacteria, with MIC values ranging from 1.56 to 25 μg/mL against gram-positive bacteria and from 12.5 to 25 μg/mL against gram-negative bacteria (Fig. 2h, i).

To evaluate PBGProOx20’s proteolytic stability, trypsin was used as a representative enzyme for enzymatic degradation studies. Following trypsin treatment, PBGProOx20 retained its broad-spectrum antimicrobial activity against S. aureus and E. coli (Supplementary Fig. 33). This contrasts sharply with the classical HDP melittin, which completely lost its antimicrobial activity (MIC increased from 6.25 μg/mL to >200 μg/mL). Furthermore, NMR analysis showed no structural changes in PBGProOx20 after 7 days exposure to trypsin (Supplementary Fig. 34). These results demonstrate the poly(2-oxazoline)’s significantly greater resistance to enzymatic degradation compared to HDPs.

In vitro broad-spectrum antimicrobial performance of PBGProOx20
We further studied the time-killing kinetics of PBGProOx20 against S. aureus and E. coli in the exponential growth phase. It was found that PBGProOx20 at a concentration of 2×MIC and 4×MIC completely eradicated S. aureus and E. coli within three or two hours, respectively (Fig. 2j). PBGProOx20 exhibited rapid killing efficacy comparable to that of colistin against E. coli; whereas, vancomycin at a concentration of 4×MIC was unable to eradicate S. aureus after 5-h treatment. Compared with exponential-phase bacteria, persister cells pose a significant challenge due to their dormant state and increased resistance to antibiotics, frequently leading to recurrent infections post-therapy54. Notably, PBGProOx20 eliminated persister cells within 24 h at a concentration of 4×MIC, achieving a reduction exceeding 6 logs in colony-forming units (CFU) (Fig. 2k). In sharp contrast, persister cells of S. aureus and E. coli still maintained sustained viability even after treatment with high-dose ciprofloxacin or ampicillin.

The established biofilms, predominantly composed of dormant bacterial persister cells, render conventional antibiotics largely ineffective55. The efficacy of PBGProOx20 in eradicating mature biofilms was evaluated and compared with vancomycin and colistin, two critical last-resort antibiotics. Vancomycin demonstrated poor anti-biofilm activity against S. aureus biofilm even at a high concentration of 512×MIC (Fig. 2l). Similarly, colistin exhibited effectiveness against E. coli biofilm only at a high concentration of 256×MIC, underscoring the robust tolerance of biofilms to antibiotic treatments (Fig. 2l). In contrast, PBGProOx20 effectively eradicated mature biofilms of S. aureus and E. coli at remarkably lower concentrations of 16×MIC and 8×MIC, respectively. Confocal microscopy images of live/dead-stained biofilms unequivocally demonstrated the capability of PBGProOx20 to effectively eliminate bacterial persister cells within biofilms (Fig. 2m). Consequently, PBGProOx20 exhibited broad-spectrum and potent antimicrobial activities superior to conventional antibiotics, particularly in targeting persister cells and mature biofilms.

Antimicrobial mechanism of PBGProOx20
The superior antimicrobial performance of PBGProOx20 against drug-resistant bacteria motivated us to investigate the antimicrobial mechanism. The fluorescent dye (morpholino-naphthalimide) labeled polymer (dye-PBGProOx20) with green fluorescence was synthesized via bromide-initiated polymerization on 2-oxazoline ProNH-Boc, followed by N-Boc deprotection and biguanidylation steps (Supplementary Figs. 35a and 36a). Time-lapse fluorescence confocal imaging was employed to visualize the intracellular localization of dye-PBGProOx20 within S. aureus and E. coli, using propidium iodide (PI, red fluorescence) as the indicator probe for cell death (Fig. 3a, b). The fluorescent images and intensity analysis revealed rapid accumulation of dye-PBGProOx20 on the membrane of S. aureus and E. coli within 1 min (Fig. 3c, d). The fluorescence signals from both dye-PBGProOx20 and PI were observed in the cytoplasm of S. aureus after around 11 min of incubation, indicating membrane damage and subsequent entry of dye-PBGProOx20 and PI into the bacterial cells (Fig. 3c, e). In the case of E. coli, the fluorescence of dye-PBGProOx20 was mainly observed on the cell membrane throughout our observation, despite PI had penetrated into the cytoplasm (Fig. 3d, e).

The PBGProOx20-treated bacterial cells also showed membrane-associated discontinuous red fluorescence puncta after staining with the membrane dye FM4-64, indicating perturbations to membrane lipid structure (Fig. 3f). Additionally, SYTOX was used as a probe for membrane integrity to show that PBGProOx20 treatment damaged bacterial membranes in a concentration-independent manner (Fig. 3g). Scan electron microscopy (SEM) was employed to characterize the morphology changes of bacterial membrane. It was found that PBGProOx20 treatment caused obvious wrinkling and collapse of the membrane in both S. aureus and E. coli (Fig. 3h). Transmission electron microscope (TEM) further validated that PBGProOx20 caused significant membrane breakage, leakage of intracellular contents, and formation of extensive internal voids (Fig. 3i).

To figure out the mode of action of PBGProOx20 on bacterial cell membranes and how biguanide outperforms amine or guanidine, multiply studies were conducted to compare polymer-bacterial membrane interaction using poly(2-oxazolie)s with positively charged moieties, PNProOx20, PGProOx20 and PBGProOx20 (Fig. 4a). In the octanol-water partition study, all three polymers, dye-PNProOx20, dye-PGProOx20, and dye-PBGProOx20, partitioned almost exclusively into the water layer due to their charged nature in the absence of any additives (Supplementary Fig. 37). However, dye-PBGProOx20 exhibited significant partitioning into the octanol phase at lower equivalents of sodium laurate surrogate (a membrane-bound fatty acid salt) than dye-PNProOx20 and dye-PGProOx20. As sodium laurate concentration increased, dye-PBGProOx20 was almost entirely partitioned into the octanol layer. This finding demonstrated that PBGProOx20 exhibited greater affinity for the membrane and more effectively translocated into the membrane-mimic lipophilic layer.

We then assessed the impact of these polymers on cytoplasmic membrane potential using DiSC (5)3, and found that PBGProOx20 induced more significant membrane depolarization in both S. aureus and E. coli at different concentrations compared to PNProOx20 and PGProOx20 (Fig. 4b). Moreover, the membrane zeta potential of both S. aureus and E. coli shifted from below −15 mV to above +5 mV after treatment with PBGProOx20, even at a low concentration of 0.5×MIC (Fig. 4c). PBGProOx20 induced larger membrane zeta potential shifts than PNProOx20 and PGProOx20, indicating its greater accumulation on bacterial membranes. We then detected membrane fluidity using Laurdan as a fluorescent probe (Fig. 4d). Compared to PNProOx20 and PGProOx20, PBGProOx20 treatment induced a greater increase in Laurdan fluorescence, revealing a more pronounced reduction in membrane fluidity in both S. aureus and E. coli. The ΔpH of bacterial cells, a key component of the proton motive force, was also evaluated using the fluorescent probe BCECF-AM (Fig. 4e). A more marked dissipation of ΔpH was observed in both S. aureus and E. coli after treatment with PBGProOx20. These findings demonstrate that biguanide-functionalized PBGProOx20 accumulates more effectively in bacterial membranes, inducing membrane potential derangement and decreasing membrane rigidity. This disturbs bacterial homeostasis and leads to critical metabolic dysfunctions, notably the dissipation of the proton motive force56,57.

The above results highlighted the interaction between PBGProOx20 and the negatively charged bacterial membrane. Considering that the negative charges of bacterial membranes are primarily provided by phospholipids58, we explored the effect of three main negatively charged bacterial phospholipids on the antibacterial potential of PBGProOx20. Among these, phosphatidylglycerol (PG), a major anionic phospholipid in the bacterial membrane, significantly reduced the antimicrobial activity of PBGProOx20 against both S. aureus and E. coli (Fig. 4f). Conversely, negatively charged cardiolipin (CL) and phosphatidylethanolamine (PE), along with the neutral phosphatidylcholine (PC), had little effect on the antimicrobial efficacy of PBGProOx20 even at high concentrations (Fig. 4f, Supplementary Fig. 38). We then performed UV-vis binding study to determine the interaction between PBGProOx20 and PG (Fig. 4g). The addition of PG induced a notable red shift in the UV-vis spectrum of PBGProOx20, suggesting that PBGProOx20 binds with PG to form complexes. We then quantified the binding affinity between polymers and phospholipids using isothermal titration calorimetry (ITC). The equilibrium dissociation constant (KD) between PBGProOx20 and PG was determined to be 4.5 μmol L−1, indicating a high affinity between PBGProOx20 and the negatively charged PG (Fig. 4h); whereas, PBGProOx20 showed poor binding affinity with neutral PC (Supplementary Fig. 39). Moreover, the KD values between PG and PNProOx20 and between PG and PGProOx20 were 69 μmol L−1 and 11 μmol L−1, respectively, demonstrating that the biguanide group exhibited a substantially higher affinity for negatively charged phospholipids than do amine and guanidine groups (Fig. 4h).

Large unilamellar vesicles (LUVs) composed of PG/PC (1:1) were employed to simulate bacterial membranes and figure out the interactions of PBGProOx20 with phospholipid bilayer. PBGProOx20 induced potassium ion leakage from these LUVs, with the extent of leakage proportional to the polymer concentration (Fig. 4i). In contrast, PBGProOx20 exhibited virtually no effect on PC-based LUVs, which simulate mammalian cell membranes. We also found that PBGProOx20 markedly enhanced the permeability of the outer membrane in E. coli, using N-Phenyl-1-naphthylamine (NPN) as the fluorescent probe (Supplementary Fig. 40). Notably, PBGProOx20 induced the accumulation of endogenous reactive oxygen species (ROS) in treated S. aureus and E. coli (Supplementary Fig. 41). ROS may correspondingly exacerbate membrane damage, further destabilizing bacterial homeostasis. These results suggest that the biguanide moiety plays an essential role in binding phosphatidylglycerol, enabling the PBGProOx20 to disrupt the phospholipid bilayer and induce leakage of intracellular contents.

Prevention of drug resistance evolution
We subsequently evaluated whether bacteria develop antimicrobial resistance following prolonged exposure to PBGProOx20. Neither S. aureus nor E. coli developed resistance to PBGProOx20 after 32 passages of continuous treatment at 0.5×MIC (Fig. 5a). In contrast, under the same conditions, ciprofloxacin and moxifloxacin exhibited significant increases in MIC values against S. aureus (512-fold) and E. coli (32-fold), respectively. Furthermore, ciprofloxacin-treated S. aureus (S. aureus-ciprofloxacin-P32) and moxifloxacin-treated E. coli (E. coli-moxifloxacin-P32) all acquired resistance to four quinolone antibiotics and ampicillin (β-lactam antibiotic) in our study (Fig. 5b). Notably, PBGProOx20-treated bacteria, both S. aureus-PBGProOx20-P32 and E. coli-PBGProOx20-P32, remained susceptible to all antibiotics tested (Fig. 5b). To elucidate the bacterial responses to PBGProOx20 and antibiotic treatments, RNA-seq analysis was performed on S. aureus and E. coli following above drug resistance assays. In comparison to the S. aureus control group without antimicrobial treatment, the volcano plots revealed that PBGProOx20 treatment resulted in 22 differentially expressed genes (DEGs), including 14 downregulated genes and 8 upregulated genes; whereas, ciprofloxacin treatment affected the expression of 82 DEGs, including 41 downregulated genes and 41 upregulated genes [log2-fold change ≥ 1; 5% false discovery rate (FDR)] (Fig. 5c). For E. coli, PBGProOx20 treatment altered 446 DEGs, including 302 downregulated genes and 144 upregulated genes; whereas compared to E. coli control group without antimicrobial treatment, moxifloxacin treatment affected the expression of 795 DEGs, including 494 downregulated genes and 301 upregulated genes (Fig. 5d). The fewer DEGs observed in the PBGProOx20 treatment group compared to antibiotic treatments suggests a lower likelihood of mutations contributing to the development of drug resistance.

Further Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to categorize the gene products into standardized terms. The term Two-Component System (TCS) was identified among the top 15 enriched KEGG pathways in the ciprofloxacin-treated S. aureus group and moxifloxacin-treated E. coli group (Fig. 5e, f). TCS represents a major mechanism by which microorganisms sense and respond to external and/or intracellular stimuli by regulating the expression of genes involved in pathogenic pathways and antibiotic resistance59. Given the strong correlation between TCS and multidrug efflux pumps, we conducted a scan of gene expression related to various efflux pump families (Fig. 5g). Specifically, the efflux pumps genes were upregulated to varying extents in ciprofloxacin-treated S. aureus group, including NorA and NorB from the major facilitator super family (MFS) family, hrtA and hrtB from the ATP-binding cassette (ABC) family, mepA and mepR from the multidrug and toxic compound extrusion (MATE) family59,60. In the moxifloxacin-treated E. coli group, a large population of efflux pump genes were upregulated, including acrA and acrB from the resistance-nodulation-cell division (RND) families, cysW and cysU from the ABC family, mdtD, mdtG and mdtL from the MFS family61. In sharp contrast, almost none of the genes related to efflux pump families were upregulated in PBGProOx20-treated groups. Conversely, PBGProOx20 significantly downregulated several efflux pumps genes, such as hrtA and hrtB in S. aureus, and mdtG in E. coli. Overall, these results demonstrated that PBGProOx20 and antibiotics exert distinct effects on the expression of drug-resistant genes, which can be attributed to their different antimicrobial mechanisms. This is also consistent with above antimicrobial resistance studies, which showed that bacteria rapidly acquired severe resistance to antibiotics, but not to PBGProOx20.

KEGG and Gene Ontology (GO) analysis of PBGProOx20-treated groups also revealed that the regulated pathways were mainly related to substance and energy metabolism, including amino acid biosynthesis and metabolism, fatty acid degradation and metabolism, ATPase activity, and ion transport (Fig. 5e, f, and Supplementary Figs. 42–44). These pathways are essential for bacterial survival and complex physiological functions, such as cell growth, biological production, resource uptake, and stress resistance. Besides, the expression of chemotaxis- and flagellum-related genes were significantly altered in PBGProOx20-treated E. coli group, which indicated that PBGProOx20 could regulate locomotion and adhesion of E. coli during pathogen-host interaction. It was also worth mentioning that the GO term related to “hemolysis in other organism” was enriched in the PBGProOx20-treated S. aureus group (Fig. 5h). Bacteria often produces different virulence factors to facilitate the infections, such as leukocidins and hemolysins62. A further analysis showed that genes related to hemolysin including hlgA, hlgB, and hlgC, were significantly downregulated in PBGProOx20-treated cells. These genes encode the gamma-hemolysin subunits HlgA, HlgB, and HlgC, respectively (Fig. 5i)63. Additionally, Efb and Ecb possess the ability to inhibit complement activation as well as neutrophil adhesion to fibrinogen, playing a crucial role in evading the host immune system for bacteria during colonization, spread, and distribution60. In present study, Efb and Ecb encoding gene efb and ecb were also significantly downregulated. Therefore, PBGProOx20 could inhibit virulence factor secretion to reduce the inflammatory damage caused by drug-resistant bacteria, which may contribute to the in vivo therapeutic efficacy.

Broad-spectrum therapeutic efficacy of PBGProOx20 in mice models of local and systemic bacterial infections
In light of the potent in vitro antimicrobial performance of PBGProOx20, we further investigated the in vivo therapeutic efficacy using multiple mice models. In the mice models of full-thickness wound infections (Fig. 6a), saline-treated wounds showed bacterial burdens with 7.6 log CFU/g for S. aureus and 8.9 log CFU/g for E. coli (Fig. 6b). The topical application of PBGProOx20 achieved 2.2 and 2.6 logs reduction of S. aureus and E. coli, respectively, in the infected wounds, which outperformed vancomycin (1.2 logs reduction) and imipenem (2.1 logs reduction). In the mice models of subcutaneous infections, bacterial loads in the infected skin exceeded 10 log CFU/g. PBGProOx20 treatment led to a reduction of more than 1 log CFU/g for both S. aureus and E. coli, showing efficacy comparable to colistin but surpassing that of vancomycin (less than 0.5 log reduction) (Fig. 6c, d). We further evaluated the anti-infective efficacy of PBGProOx20 in a mice models of renal infections induced by S. aureus and E. coli (Fig. 6e). Systemic administration with PBGProOx20 significantly decreased the bacterial loads in the infected kidneys from 7 log CFU/g to 4.6 log CFU/g for S. aureus, and from 8.5 log CFU/g to 5.5 log CFU/g for E. coli (Fig. 6f). Besides, PBGProOx20 substantially alleviated necrosis in the glomeruli and renal tubules, and improved the condition of renal lesions (Fig. 6g). In addition, the therapeutic efficacy of PBGProOx20 was also evaluated in a mice models of peritonitis (Fig. 7a). Intraperitoneal injection with four different bacterial suspensions resulted in 100% death of mice within 3 days (Fig. 7b). A single dose of PBGProOx20 significantly improved survival rates, rescuing 83% of S. aureus infected mice, as well as 100% of the mice infected with S. epidermidis, E. coli, and A. baumannii. Furthermore, PBGProOx20 treatments significantly reduced bacterial loads within the blood, peritoneal fluid and major organs, including heart, liver, spleen, lung, and kidney. The demonstrated therapeutic efficacy was comparable to or even superior to that of vancomycin and imipenem (Fig. 7c–f). Histological examination of major organs through hematoxylin and eosin (H&E) staining revealed that PBGProOx20 treatment effectively mitigated organ lesions, including hepatocyte necrosis, loss of splenic white pulp, alveolar wall thickening, and glomerular necrosis (Fig. 7g).

Cell-penetrating and BBB-penetrating properties of PBGProOx20
We further used rhodamine-labelled polymer (RB-PBGProOx20, red fluorescence) to explore the cell-penetrating behavior of PBGProOx20 in brain microvascular endothelial cells (bEnd.3) (Supplementary Figs. 35b and 36b). The obvious increase in intracellular fluorescence over time suggested efficient internalization of PBGProOx20 toward bEnd.3 cells (Fig. 8a). Live-cell fluorescence imaging showed that PBGProOx20 predominantly localized within the endolysosomal compartments, rather than in mitochondria (Fig. 8b), which prevents interaction between PBGProOx20 and mitochondria, contributing to the low cytotoxicity. Considering that the mitochondrial membrane also contains negatively charged phospholipids, the mitochondrial membrane potential was also characterized using rhodamine 123 (Supplementary Fig. 45). It was found that the mitochondrial membrane potential in bEnd.3 cells remained normal after PBGProOx20 treatment, which further ruled out any adverse effects of PBGProOx20 on mitochondria.

To figure out the cellular uptake mechanism of PBGProOx20, we compared the internalization of PBGProOx20 in bEnd.3 cells at both 4°C and 37°C. The absence of the RB-PBGProOx20 fluorescent signal at 4°C suggested that the internalization of PBGProOx20 relies on energy-dependent endocytosis, rather than energy-independent direct penetration (Fig. 8c). Endocytosis can occur through various pathways, including macropinocytosis, clathrin-dependent endocytosis, and caveolin-dependent endocytosis42. We then blocked these pathways individually with different inhibitors to further explore the specific endocytosis pathway of PBGProOx20. Ethyl isopropyl amiloride (EIPA) and chlorpromazine (CPZ), but not genistein (GEN), significantly inhibited the cellular uptake of PBGProOx20 in bEnd.3 cells, indicating that macropinocytosis and clathrin-dependent endocytosis together dominate the internalization of PBGProOx20 (Fig. 8d). Furthermore, we observed an obvious exocytosis process of PBGProOx20 in bEnd.3 cells over time (Fig. 8e). This continuous transcytosis behavior further underscores the potential of PBGProOx20 to penetrate BBB.

The in vitro transwell model was further used to quantify the BBB-penetrating efficiency of PBGProOx20 (Fig. 8f). The initial transendothelial electrical resistance (TEER) values in transwell model exceeded 150 Ω·cm2, indicating tight junctions of bEnd.3 cells (Fig. 8g)64. Notably, PBGProOx20 did not disrupt the tight junctions of bEnd.3 cells, as TEER values remained stable in the presence of PBGProOx20. Importantly, PBGProOx20 exhibited an increased transport ratio over incubation time, reaching ~26% after 24 h (Fig. 8h). Moreover, the transport ratio of PBGProOx20 in the transwell model was inhibited by EIPA and CPZ in a manner consistent with the results of cellular uptake study (Fig. 8i). These findings demonstrated that PBGProOx20 could effectively penetrate the BBB through a transcytosis process, which involved macropinocytosis and clathrin-dependent endocytosis, and subsequent exocytosis.

The promising in vitro cell-penetrating capabilities of PBGProOx20 prompted us to explore the in vivo BBB-penetrating ability. We investigated the plasma pharmacokinetics of RB-PBGProOx20 using fluorescent quantification method. Following a 20-mg/kg bolus dose, the plasma concentrations of PBGProOx20 achieved were above those required for antimicrobial activity in vitro and a half-life of ~4 h was defined (Fig. 8j, and Table S8). After systemic administration in mice, PBGProOx20 was found with significant amount within the brain, showing distribution levels comparable to that in the heart, spleen, and lung (Fig. 8k). The in vivo BBB-penetrating ability of PBGProOx20 was further confirmed using the well-established zebrafish model by observing obvious fluorescence signal in the zebrafish brain region after administration with RB-PBGProOx20 (Fig. 8l)65. These results altogether demonstrated the ability of PBGProOx20 to penetrate the BBB, showing potential in treating bacterial meningitis caused by ESKAPE pathogens.

Broad-spectrum and potent antimicrobial efficacy in bacterial meningitis and low in vivo toxicity of PBGProOx20
Bacterial suspensions of S. aureus and E. coli were directly injected into mice brains to induce bacterial meningitis (Fig. 9a). The saline-treated mice all showed a poor survival rate of 17%. Systemic administrations with PBGProOx20 significantly improved survival rates, rescuing 83% of both S. aureus-infected and E. coli-infected mice (Fig. 9b), superior to that of vancomycin (50%) on S. aureus-infected mice but slightly inferior to that of meropenem (100%) on E. coli-infected mice. Quantitative analysis of the bacterial load in the mice brain indicated that saline-treated mice showed high bacterial burdens, with 8.0 log CFU/brain for S. aureus and 8.9 log CFU/brain for E. coli (Fig. 9c). PBGProOx20 treatment led to a 2 log and 4.1 log decrease in bacterial loads for S. aureus and E. coli, respectively, which was superior to that of vancomycin and comparable to that of meropenem. Histological examination through H&E staining revealed that PBGProOx20 treatment effectively mitigated inflammation of the infected brain, especially infiltration of neutrophils (Fig. 9d).

We also conducted a single-dose tolerability study to evaluate the in vivo biosafety of PBGProOx20. After intraperitoneal injection with high dose (200 and 300 mg/kg) of PBGProOx20, the mice demonstrated 100% survival without any sign of malaise within 14 days. PBGProOx20 at 400 mg/kg caused 33% death and the maximum tolerated dose (MTD) of PBGProOx20 was determined to be ~300 mg/kg (Fig. 9e). PHMB exhibited stronger systemic toxicity, with a MTD of ~20 mg/kg, making it difficult to meet the biosafety requirements for in vivo infection treatment. Moreover, the normal increase in mice body weight was also observed over a 14-day period post-administration with PBGProOx20 (Fig. 9f). The immunohistology analysis of the liver and kidney at 2 days and 14 days post injection showed that PBGProOx20 caused little toxicity to organs critical for metabolism (Fig. 9g). Further investigations into the complete blood count revealed that hematological parameters in PBGProOx20-treated mice were consistent with those observed in saline-treated mice, encompassing red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) counts (Fig. 9h). Besides, the blood biochemical parameters also remained within normal ranges, including liver function indicators (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)), renal function indicators (blood urea nitrogen (BUN) and creatinine (CREA)), and electrolyte balance (potassium (K+), sodium (Na+), and chloride (Cl−)) (Fig. 9h). These findings collectively demonstrate the biosafety of PBGProOx20 for systemic administration, particularly in treating bacterial meningitis.

Methods
All animal research was performed in accordance with the Guidelines for Care and Use of Laboratory Animals of East China University of Science and Technology. All procedures in this study were approved by the Animal Research Bioethics Committee, East China University of Science and Technology (ECUST-2023-012). Mice were raised in an individually ventilated cage (IVC) system at 20–26°C and 40–70% humidity, with a dark/light cycle of 12 h.

Mice wound infection model
This model was conducted according to the previously reported method with slight modification66. Female ICR mice (6–8 weeks) were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 75 mg/kg and the hair on the mice back was shaved. S. aureus USA300 LAC or E. coli ATCC 25922 were cultured in LB medium at 37°C for 9 h, then collected and suspended in saline at a cell density of 3×106 CFU/mL or 1×108 CFU/mL, respectively. The full-thickness wounds (6-mm diameter) were obtained using biopsy punch, and bacterial suspensions were dropped to wound sites. After 24-h post infection, 15 μL saline containing PBGProOx20 (4 mg/mL), vancomycin (4 mg/mL for treatment of S. aureus infection) or imipenem (4 mg/mL for treatment of E. coli infection) was applied to the infected wounds. Saline treatment was used as the control. The treatment was conducted every 4 h for a total of 3 times. The treated wound sites were excised for CFU quantification of bacterial load at 4 h after the last treatment (n=10 in each group).

Mice subcutaneous infection model
This model was conducted according to the previously reported method with slight modification67. Female ICR mice (6–8 weeks) were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 75 mg/kg and the hair on the mice back was shaved. S. aureus USA300 LAC or E. coli ATCC 25922 were cultured in LB medium at 37°C for 9 h, then collected and suspended in saline at a cell density of 1.5×109 CFU/mL or 1.5×108 CFU/mL, respectively. 100 μL of bacterial suspensions were injected into the subcutaneous area of the mice back. After 24-h post infection, 50 μL saline containing PBGProOx20 (4 mg/mL), vancomycin (4 mg/mL for treatment of S. aureus infection) or colistin (4 mg/mL for treatment of E. coli infection) was applied to the infected sites through subcutaneous injection. Saline treatment was used as the control. The treatment was conducted every 12 h for a total of 3 times. The treated wound sites were excised for CFU quantification of bacterial load at 12 h after the last treatment (n=10 in each group).

Mice kidney infection model
This model was conducted according to the previously reported method with slight modification68. Female ICR mice (6–8 weeks) were intravenously injected with 0.2 mL carrageenan solution (2 mg/mL) at 7 days prior to infection. S. aureus USA300 LAC or E. coli ATCC 25922 were cultured in LB medium at 37°C for 9 h, then collected and suspended in saline at a cell density of 1.5×107 CFU/mL or 6×107 CFU/mL, respectively. 200 μL of bacterial suspensions were intravenously injected into above mice. After 4-h post infection, saline containing PBGProOx20 (40 mg/kg), vancomycin (40 mg/kg for treatment of S. aureus infection) or imipenem (40 mg/kg for treatment of E. coli infection) was intraperitoneally injected into the infected mice. Saline treatment was used as the control. The treatment was conducted every 24 h for a total of 3 times. The mice kidneys were excised for CFU quantification of bacterial load and histological analysis at 24 h after the last treatment (n=6 in each group).

Mice peritonitis models
This model was conducted according to the previously reported method with slight modification27,69. S. aureus USA300 LAC, S. epidermidis 9397, E. coli 2904 and A. baumannii 0403 were cultured in LB medium at 37°C for 9 h, then collected and suspended in 5% mucin solution at a cell density of 7.5×108 CFU/mL, 5.1×109 CFU/mL, 2×105 CFU/mL or 5×106 CFU/mL, respectively. For S. epidermidis-induced peritonitis model, female ICR mice (6–8 weeks) were immunosuppressed by intraperitoneal injection of cyclophosphamide (150 mg/kg) at 4 days and cyclophosphamide (100 mg/kg) at 1 day prior to infection. The mice used in other bacteria-induced peritonitis models were normal. 200 μL of bacterial suspensions were intraperitoneally injected into female ICR mice (6–8 weeks). After 30-min post infection, saline containing PBGProOx20 (40 mg/kg), vancomycin (40 mg/kg for treatment of S. aureus and S. epidermidis infections) or imipenem (40 mg/kg for treatment of E. coli and A. baumannii infections) was intraperitoneally injected into the infected mice. Saline treatment was used as the control. The mice organs were excised for quantification of bacterial load and histological analysis at 24 h after the infection and the survival of remaining mice was recorded for 7 days (n=6 in each group).

Mice meningitis models
This model was conducted according to the previously reported method with slight modification70. Female ICR mice (6–8 weeks) were anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 75 mg/kg and the hair on the mice head was shaved. S. aureus USA300 LAC and E. coli ATCC 25922 were cultured in LB medium at 37°C for 9 h, then collected and suspended in saline at a cell density of 2.5×107 CFU/mL or 1.6×106 CFU/mL, respectively. Female mice were infected intraventricularly (20 μL of bacterial suspensions) via a burr hole (coordinates 0.6 mm dorsal to bregma, 1.2 mm lateral to middle line and 2 mm ventral). After 12-h post infection, saline containing PBGProOx20 (40 mg/kg), vancomycin (40 mg/kg for treatment of S. aureus infections) or meropenem (40 mg/kg for treatment of E. coli infections) was intraperitoneally injected into the infected mice. Saline treatment was used as the control. The treatment was conducted every 24 h for a total of 6 times. The mice brains were excised for quantification of bacterial load and histological analysis at 24 h after the last treatment, and the survival of remaining mice was recorded for 14.5 days (n=6 in each group).

In vivo toxicity evaluation on PGMeOx10
In mice dose tolerance study, female ICR mice (6–8 weeks) were intraperitoneally administered with PBGProOx20 (200 mg/kg, 300 mg/kg, and 400 mg/kg) and PHMB (20 mg/kg, 40 mg/kg, and 100 mg/kg). The survival of treated mice was recorded for 14 days after the first treatment (n=6 in each group). The body weight of mice treated with PBGProOx20 (40 mg/kg) were recorded until 14 days after the first treatment, and the mice blood were collected at 2 days or 14 days after the first treatment for complete blood count and blood biochemical parameters analysis, including red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT), aspartate alanine aminotransferase (ALT), aminotransferase (AST), blood urea nitrogen (BUN), creatinine (CREA), and three ions (K+, Na+, Cl−). The mice liver and kidney were also excised for histological analysis. Saline treatment was used as the control.

Statistical analysis
Statistical analysis was performed using Origin software. Significance analysis between two groups were performed using two-tailed student’s t-test. Significance analysis for multiple comparison of more than two groups were performed using one-way analysis of variance of (ANOVA) with Turkey post-test. All mean±standard deviation was indicated by the error bars. The differences were regarded as statistically not significant (ns) with probability P > 0.05 and significant with probability P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript describes an effective strategy in designing host defense peptide (HDP) mimics to combat ESKAPE-induced infections, especially meningitis. The authors use biguanide as a new type of positively charged moiety to design HDP mimics. The optimal compound PBGProOx20 demonstrates both BBB-penetrating properties and potent broad-spectrum antimicrobial performance against all ESKAPE pathogens. PBGProOx20 targets bacterial membrane phospholipids to disrupt the bacterial membrane and does not induce antimicrobial resistance. PBGProOx20 also displays effective anti-infective activity in multiple ESKAPE-induced infection models, including meningitis. Overall, the manuscript reveals an exciting novel antimicrobial drug candidate and should advance the field significantly. I strongly recommend this work for publication in Nature Communications after the following minor revisions.\n1. It is suggested to mark the specific MIC values in Figures 2d and 2f.\n2. In Figure 2j, the time-kill studies lack positive controls.\n3. Details of treatment procedures in the animal experiments need to be provided in the figure captions, including concentration, frequency, and treatment method.\n4. Provide statistical analysis (e.g., log-rank test) to confirm survival differences in the systemic infection and meningitis models.\n5. The dose-tolerance study was performed for 7 days in Figure 9e. This may not be indicative of adverse effects. Survival ratio at longer time points (such as 14 days) need to be included.\n6. Relevant references need to be provided to support the methods used for the wound and subcutaneous infection models.", "reviewer_2": "This manuscript presents an interesting study on the development of biguanide-functionalized host defense peptide (HDP) mimics with broad-spectrum antimicrobial activity and the ability to penetrate the blood-brain barrier (BBB), which is highly relevant for treating infections caused by drug-resistant ESKAPE pathogens, especially bacterial meningitis. The design of HDP mimics incorporating biguanide as a positively charged moiety is novel and presents a promising strategy to address the urgent need for new therapeutics against multidrug-resistant pathogens. However, there are several points that need to be clarified and further elaborated to enhance the manuscript's overall quality and impact.\nMajor Comments:\n- The introduction outlines the context and the relevance of the study, highlighting the challenges in treating ESKAPE pathogens and bacterial meningitis. However, the manuscript could benefit from further clarification and elaboration on some key points.\nThe relationship between the properties of biguanide and its potential for BBB penetration should be better defined. It is mentioned that biguanide has a \"higher positive charge density,\" but the impact of this on BBB penetration is not fully explained. A more in-depth explanation of how charge density correlates with BBB penetration would be beneficial. The discussion on previous research about amine and guanidine-modified HDP mimics should be more concise, focusing on how biguanide differs or improves upon these moieties.\n- The results and discussion sections present the key findings of the study clearly, but some areas need additional detail or clarification to strengthen the overall argument.\nThe manuscript describes the membrane-disrupting mechanism of PBGProOx20 but does not provide sufficient details on the exact molecular interactions between PBGProOx20 and bacterial membranes. Including additional data or references supporting the mechanism of action would strengthen this part of the manuscript.\nThe manuscript claims that PBGProOx20 penetrates the BBB via transcytosis, but further clarification on how this was measured would be useful. Was this observed through in vivo imaging, or is it inferred from the therapeutic efficacy in meningitis models? A more detailed description of the experimental setup for BBB penetration would be valuable.\nThe statement that PBGProOx20 does not induce antimicrobial resistance should be supported by data showing a lack of upregulation in resistance-related genes, as well as results from long-term treatment studies. The manuscript mentions that PBGProOx20 does not induce cross-resistance, but additional data comparing resistance development between PBGProOx20 and traditional antibiotics would further substantiate this claim.\nWhile the therapeutic efficacy of PBGProOx20 in various infection models is promising, the manuscript lacks a clear description of how these models were conducted. Were bacterial loads measured? What was the dosing regimen? Including such details would improve the rigor of the study.\n- The conclusion is concise but could benefit from a more forward-looking statement regarding the potential clinical applications and future directions for research. It would be helpful to briefly mention potential challenges or areas of improvement in the development of PBGProOx20 or similar compounds and how the findings of this study could influence future strategies for treating other types of resistant infections or CNS diseases.\nMinor Comments:\n- The abstract provides a good summary of the study, but it could benefit from a clearer structure and more focus on key findings.\n- The abstract mentions \"bidentate hydrogen bonds\" without clearly explaining their significance or how they contribute to the enhanced interaction with bacterial membranes. A brief clarification of this point would help readers understand the novelty of biguanide's role. It would be useful to briefly mention the specific infection models used in vivo (i.e., types of mouse models) to provide more context on the therapeutic potential of PBGProOx20.\n- The manuscript would benefit from clearer figures and legends that better illustrate the molecular interactions, experimental models, and results. Some figures, particularly Figure 1, could be more descriptive in showing how biguanide interacts with bacterial membranes.\n- There is some repetition in the introduction, particularly regarding the challenges posed by ESKAPE pathogens and BBB penetration. Streamlining this section could improve readability.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "This manuscript by Jiang et al. presents the design and evaluation of biguanide-functionalized host defense peptide (HDP) mimics as a strategy to combat drug-resistant ESKAPE pathogens and treat meningitis. The Authors introduce biguanide as a novel positively charged moiety on peptidomimetics, which have the potential of enhancing the interactions between polymers and bacterial membranes, and thus facilitating blood–brain barrier (BBB) penetration. The Authors conducted a comprehensive set of experiments, including computational modeling, in vitro antibacterial activity studies, mechanistic analysis, toxicity assessments (both in vitro and in vivo), and efficacy studies in multiple in vivo models, such as a zebrafish brain penetration model, murine wound infection model, meningitis model, and several other mice models using different infection routes. The study is impressively thorough, and the multidisciplinary approach significantly enhances the impact of this study. In particular, breaking the blood–brain barrier is a challenging but crucial task, given the limited treatment options for related infections.\nOverall, the manuscript provides sounding support for the lead compound (PBGProOx20) as a non-resistance-inducing and membrane-disruptive antimicrobial. The Authors have also demonstrated that PBGProOx20’s impressive antibiotic properties are achieved through an endocytosis-based mechanism. The Authors’ findings suggest that PBGProOx20 is a potential next-generation antibiotic. The manuscript should be considered for publication following revisions to address the following minor points:\n1. While the use of biguanide as a positively charged moiety is new in the context of HDP mimics, the broader strategy of incorporating cationic functional groups to enhance antimicrobial activity is well-established. The manuscript would benefit from a clearer discussion and/or experimental evidence of how biguanide outperforms amine or guanidinium modifications (in addition to the computational comparisons that the Authors already mentioned). This is particularly important given the synthetic complexity of biguanide-functionalized mimics. Are there any unique advantages in terms of stability, selectivity, or protease resistance of biguanide-polymers? As mentioned by the Authors (line 45), HDPs often suffer from low protease stability, high cost, and difficult synthesis. It is unclear whether biguanide-functionalized mimics can address these limitations.\n2. Please specify the detection limit (in CFU) of the time-kill assays shown in Figure 2. Additionally, please clarify how the kill ratio was calculated.", "reviewer_5": "The authors introduce biguanide-functionalized poly(2-oxazoline) (PBGProOx20) as a host-defense-peptide (HDP) mimic that displays broad bactericidal activity against all ESKAPE pathogens, no detectable resistance development, and the ability to cross the blood brain barrier (BBB) and cure murine bacterial meningitis. The conceptual advance lies in replacing the classic amine/guanidinium cation of HDP mimics with a biguanide moiety that offers a higher charge density and bidentate H-bonding to anionic phospholipids, thereby enhancing membrane binding and BBB transport. Authors showed activity against >30 multidrug-resistant clinical isolates, eradication of persisters and mature biofilms, and absence of cross-resistance after 16 passages. They also demonstrated that PBGProOx20 outperforms meropenem at equi-doses and is well tolerated in in full-thickness wounds, skin abscess, kidney infection, peritonitis, and crucially meningitis. Comprehensive investigations\nConcerns:\n1- Novelty positioning vs PHMB and other biguanide polymers is not adequately discussed. PHMB is known to permeabilize membranes and is approved for wound care. How does PBGProOx20 differ in structure–activity, toxicity, and BBB permeability? Maybe add a paragraph comparing physicochemical parameters, MIC values and systemic safety relative to PHMB.\n2- Therapeutic doses up to 40 mg kg⁻¹ i.p. were used in in vivo models. Plasma half-life, brain exposure, and maximum tolerated dose are not reported. For dose justification and pharmacokinetics, please provide basic PK (plasma and brain) and MTD data if available, or discuss ongoing studies.\n3- Selective toxicity on mammalian membranes was tested only on COS-7 cells for 24 h. Mechanism focused on PG but mammalian mitochondria are also PG-rich. Please include data on mitochondrial membrane potential or cytotoxicity in primary human cells if possible or at least discuss it.\n4- Resistance study was for 16 passages (~24 days), which may be too short to detect low-frequency resistance. I suggest to either extend evolution experiment to ≥30 passages or add genomic analysis to confirm low mutational burden.\nMinor comments\n1. Typographical errors: “amime-functionalized” (Fig. 2c caption).\n2. Figure 1d legend abbreviations (BBB, HDP) should be defined in the caption.\n3. Provide chemical structures of comparator amine- and guanidine-POx controls in the main text, not only in Supplementary Fig. S4.\n4. Supplementary Methods page numbers are missing; add running headers for easy cross-reference.\n5. Clarify whether biguanidylation is quantitative (NMR integration appears sub-unity)."}, "author_response": "23 July 2025\nDear all reviewers,\nWe are submitting the revision of our manuscript “Biguanide-Functionalized Peptide Mimics\nEffectively Combat Drug-Resistant ESKAPE Pathogens and Meningitis”. We greatly thank all reviewers for\nyour suggestive comments and suggestions, following which we have fully addressed all questions and\nsubstantially improved our manuscript.\nOur responses to your comments are detailed below; for reference, the comments/questions are\npresented in italics, with our response following in blue text. New data and new text in our revision was\nhighlighted in yellow.\nReviewer 1:\nThis manuscript describes an effective strategy in designing host defense peptide (HDP) mimics to combat\nESKAPE-induced infections, especially meningitis. The authors use biguanide as a new type of positively\ncharged moiety to design HDP mimics. The optimal compound PBGProOx20 demonstrates both\nBBB-penetrating properties and potent broad-spectrum antimicrobial performance against all ESKAPE\npathogens. PBGProOx20 targets bacterial membrane phospholipids to disrupt the bacterial membrane and\ndoes not induce antimicrobial resistance. PBGProOx20 also displays effective anti-infective activity in\nmultiple ESKAPE-induced infection models, including meningitis. Overall, the manuscript reveals an\nexciting novel antimicrobial drug candidate and should advance the field significantly. I strongly recommend\nthis work for publication in Nature Communications after the following minor revisions.\nResponse: We thank you for the favorable comments and high recognition of our manuscript.\n1. It is suggested to mark the specific MIC values in Figures 2d and 2f.\nResponse: We thank you for the suggestion and have marked the corresponding MIC values in figure 2d\nand 2f. The new figures were updated in our revised manuscript, as shown below (revised Figure 2d, 2f).\n“Figure 2. (d) Antimicrobial activity of biguanide-functionalized poly(2-oxazoline)s against ESKAPE\npathogens, including E. faecium (E), S. aureus (S), K. pneumoniae (K), A. baumannii (A), P. aeruginosa (P),\nE. coli (E). All these bacteria are drug resistant or multi-drug resistant strains.”\n“Figure 2. (f) Antimicrobial activity of biguanide-functionalized poly(2-oxazoline)s PBGProOx with\nn\ndifferent chain lengths against ESKAPE pathogens.”\n2. In Figure 2j, the time-kill studies lack positive controls.\nResponse: We thank you for this suggestion and have supplemented positive controls using two\nantibiotics, vancomycin and colistin. The time killing kinetics of vancomycin and colistin at a concentration\nof 4×MIC were examined against S. aureus and E. coli, respectively. The new figures and related description\nwere updated in our revised manuscript, as shown below (revised Figure 2j).\n“It was found that PBGProOx at a concentration of 2×MIC and 4×MIC completely eradicated S.\n20\naureus and E. coli within three or two hours, respectively (Figure 2j). PBGProOx exhibited rapid killing\n20\nefficacy comparable to that of colistin against E. coli; whereas, vancomycin at a concentration of 4×MIC was\nunable to eradicate S. aureus after 5-hour treatment.”\n“Figure 2. (j) Time killing kinetics of PBGProOx (2×MIC, 4×MIC) against S. aureus and E. coli at\n20\nexponential growth phase, using vancomycin (4×MIC) and colistin (4×MIC) as the antibiotic controls,\nrespectively. The detection limit for viable bacterial concentration is 10 CFU/mL, as indicated by the dashed\nline. Data points below this threshold are designated as 0 CFU/mL.”\n3. Details of treatment procedures in the animal experiments need to be provided in the figure captions,\nincluding concentration, frequency, and treatment method.\nResponse: We thank you for reminding us to supplement the treatment procedures for animal\nexperiments. We have added detailed dosing regimen in the schematic diagrams of all animal experiments\nand corresponding figure legends in our revised manuscript, as shown below (revised Figure 6a, 6c, 6e, 7a,\n9a).\n“Figure 6 (a) Schematic representation of the full-thickness infection model. The infected wounds were\ntreated topically with 15 μL of saline, PBGProOx (4 mg/mL), vancomycin (4 mg/mL) or imipenem (4\n20\nmg/mL) every 4 hours for triple applications.”\n“Figure 6. (c) Schematic representation of the sub-cutaneous infection model. The infected tissues were\ntreated subcutaneously with 100 μL of saline, PBGProOx (4 mg/mL), vancomycin (4 mg/mL) or colistin (4\n20\nmg/mL) every 12 hours for triple applications.”\n“Figure 6. (e) Schematic representation of the kidney infection model. The infected mice were treated\nintraperitoneally with saline, PBGProOx (40 mg/kg), vancomycin (40 mg/kg) or imipenem (40 mg/kg)\n20\ndaily for triple applications.”\n“Figure 7. (a) Schematic representation of the peritonitis model. The infected mice were treated\nintraperitoneally with saline, PBGProOx (40 mg/kg), vancomycin (40 mg/kg) or imipenem (40 mg/kg) for\n20\nonce application.”\n“Figure 9. (a) Schematic representation of the meningitis model. The infected mice were treated\nintraperitoneally with saline, PBGProOx (40 mg/kg), vancomycin (40 mg/kg) or meropenem (40 mg/kg)\n20\ndaily for six applications.”\n4. Provide statistical analysis (e.g., log-rank test) to confirm survival differences in the systemic infection\nand meningitis models.\nResponse: We thank you for the suggestion. We have conducted statistical analysis in figure 7b and\nfigure 9b in the revised manuscript using log-rank test, and confirmed that PBGProOx treatment\n20\nstatistically improved the survival rate of the infected mice in the systemic infection and meningitis models\n(revised Figure 7b, 9b).\n“Figure 7. (b) Survival rates of infected mice after treatment with saline, PBGProOx20, vancomycin or\nimipenem in the peritonitis model. The mice were infected by S. aureus, S. epidermidis, E. coli and A.\nbaumannii, respectively. The survival benefit was determined using a log-rank test. **P < 0.01, ***P <\n0.001.”\n“Figure 9. (b) Survival rates of infected mice after treatment with saline, PBGProOx , vancomycin or\n20\nimipenem in the meningitis model. The mice were infected by S. aureus and E. coli, respectively. The\nsurvival benefit was determined using a log-rank test. *P < 0.05, ***P < 0.01, ***P < 0.001.”\n5. The dose-tolerance study was performed for 7 days in Figure 9e. This may not be indicative of adverse\neffects. Survival ratio at longer time points (such as 14 days) need to be included.\nResponse: We thank you for this question and suggestion. We have followed the suggestion to\nconducted survival rates studies of the uninfected mice after 14 days treatment with PBGProOx at different\n20\nconcentrations. The new figures and related description were updated in our revised manuscript, as shown\nbelow (revised Figure 9e).\n“After intraperitoneal injection with high dose (200 and 300 mg/kg) of PBGProOx , the mice\n20\ndemonstrated 100% survival without any sign of malaise within 14 days. PBGProOx at 400 mg/kg caused\n20\n33% death and the maximum tolerated dose (MTD) of PBGProOx was determined to be ~300 mg/kg\n20\n(Figure 9e). PHMB exhibited stronger systemic toxicity, with a MTD of ~20 mg/kg, making it difficult to\nmeet the biosafety requirements for in vivo infection treatment.”\n“Figure 9. (e) Survival rates of the uninfected mice at day 14 after the single-dose intraperitoneal injection\nof PBGProOx and poly(hexamethylene biguanide) (PHMB) at different concentrations.”\n20\n6. Relevant references need to be provided to support the methods used for the wound and subcutaneous\ninfection models.\nResponse: We thank you for reminding us on this and we have added supporting references for the\nwound and subcutaneous infection models in our revised manuscript, as shown below (ref. 66, 67).\n“Mice wound infection model. …This model was conducted according to the previously reported\nmethod with slight modification.66 …”\n“Mice subcutaneous infection model. This model was conducted according to the previously reported\nmethod with slight modification.67 …”\n“66. Xu, Z., et al. Converting organosulfur compounds to inorganic polysulfides against resistant\nbacterial infections. Nat. Commun. 9, 3713 (2018).”\n“67. Lu, M., et al. Bacteria-specific phototoxic reactions triggered by blue light and phytochemical\ncarvacrol. Sci. Transl. Med. 13, eaba3571 (2021).”\nReviewer #2 (Remarks to the Author)\nThis manuscript presents an interesting study on the development of biguanide-functionalized host defense\npeptide (HDP) mimics with broad-spectrum antimicrobial activity and the ability to penetrate the\nblood-brain barrier (BBB), which is highly relevant for treating infections caused by drug-resistant ESKAPE\npathogens, especially bacterial meningitis. The design of HDP mimics incorporating biguanide as a\npositively charged moiety is novel and presents a promising strategy to address the urgent need for new\ntherapeutics against multidrug-resistant pathogens. However, there are several points that need to be\nclarified and further elaborated to enhance the manuscript's overall quality and impact.\nResponse: We thank you for the constructive comments and suggestions on our manuscript.\nMajor Comments:\n- The introduction outlines the context and the relevance of the study, highlighting the challenges in treating\nESKAPE pathogens and bacterial meningitis. However, the manuscript could benefit from further\nclarification and elaboration on some key points.\nThe relationship between the properties of biguanide and its potential for BBB penetration should be better\ndefined. It is mentioned that biguanide has a \"higher positive charge density,\" but the impact of this on BBB\npenetration is not fully explained. A more in-depth explanation of how charge density correlates with BBB\npenetration would be beneficial. The discussion on previous research about amine and guanidine-modified\nHDP mimics should be more concise, focusing on how biguanide differs or improves upon these moieties.\nResponse: Thank you for your valuable suggestions to remind us provide a sufficiently explanation on\nhow the high positive charge density of biguanide affects BBB penetration efficiency. As reported in the\nliterature, BBB penetration involves multiple pathways, among which adsorptive-mediated transcytosis\nserves as a crucial pathway for positively charged molecules to enter the brain. This pathway relies on strong\nelectrostatic interactions between the positive charges on the molecular surface and negatively charged\nmembrane components (such as heparan sulfate) on the luminal surface of BBB. Therefore, we hypothesis\nthat the higher localized positive charge density of biguanide endows biguanide-based compounds more\nefficient initial electrostatic interaction with negatively charged luminal surface of BBB, thereby potentially\nenhancing its BBB penetration efficiency via adsorptive-mediated transcytosis.\nWhile the proposed mechanism offers a plausible theoretical explanation, we acknowledge that\ninvestigating how molecular structures affect blood-brain barrier penetration efficiency represents a\nsignificant independent research direction requiring dedicated experimental design and resources. Our\ncurrent focus in this manuscript lies in the discovery of dual-functional peptide mimics with broad-spectrum\nantimicrobial activity and demonstrable BBB-penetrating capability. More details of how charge structures\nmediate BBB penetration efficiency will be explored in future studies. Therefore, we have added a dedicated\nparagraph in the revised Introduction explaining the correlations between charge density and BBB\npenetration, and outlined this mechanistic investigation as a key future research direction in the Discussion\nsection. The revised content is shown below:\n“The higher localized positive charge density also endows biguanide-based compounds more efficient\ninitial electrostatic interaction with negatively charged luminal surface of BBB, thereby potentially\nenhancing its BBB penetration efficiency via adsorptive-mediated transcytosis, which is a crucial pathway\nfor positively charged molecules to enter the brain.46,47 ... Future research will focus on investigating how\ncationic and hydrophobic groups affect the BBB-penetrating efficiency, and explore diverse molecular\nstructures to enhance antimicrobial potency and BBB penetration efficiency.”\nBy following your suggestion, we have also condensed the background on amine/guanidine-modified\nHDP mimics. The updated Introduction now focuses directly on the potential advantages of the biguanide\ngroup. The revised content was shown below:\n“Cationic HDPs and their mimics exert antimicrobial activity through initial interactions with the\nnegatively charged phospholipids of bacteria, primarily using amine or guanidine moieties as cationic groups\n(Figure 1a).24-37 However, achieving dual-functional HDP mimics with potent anti-ESKAPE activity and\nBBB-penetrating ability using these two moieties remains challenging.38-44 ... The planar, conjugated cationic\nstructures of biguanide enable simultaneous donation of two hydrogen bonds to anionic phosphate\nheadgroups of bacterial membrane phospholipids (Figure 1c). This dual-point attachment generates\ncooperative electrostatic interaction and bidentate hydrogen bonds, significantly enhancing membrane\nbinding affinity and disruption efficiency.The planar, conjugated cationic structures of biguanide enable\nsimultaneous donation of two hydrogen bonds to anionic phosphate headgroups of bacterial membrane\nphospholipids (Figure 1c). This dual-point attachment generates cooperative electrostatic interaction and\nbidentate hydrogen bonds, significantly enhancing membrane binding affinity and disruption efficiency.45”\n- The results and discussion sections present the key findings of the study clearly, but some areas need\nadditional detail or clarification to strengthen the overall argument.\nThe manuscript describes the membrane-disrupting mechanism of PBGProOx20 but does not provide\nsufficient details on the exact molecular interactions between PBGProOx20 and bacterial membranes.\nIncluding additional data or references supporting the mechanism of action would strengthen this part of the\nmanuscript.\nResponse: We thank you for your suggestion on strengthening the molecular interactions underlying\nPBGProOx 's membrane-disrupting mechanism. In our manuscript, we demonstrated the\n20\nmembrane-disrupting behavior of PBGProOx by time-lapse fluorescence imaging, FM4-64 membrane\n20\nlabeling, and electron microscopy characterization (Figure 3). We also observed that PBGProOx caused\n20\nsignificant membrane depolarization, a substantial reversal of membrane zeta potential, leading to reduced\nmembrane stiffness and disruption of intracellular pH balance (Figure 4b, 4c, 4d, 4e). To elucidate the\nmolecular interactions of PBGProOx and bacterial membranes, we focused on negatively charged\n20\nphospholipids in bacterial membranes and found that adding exogenous phosphatidylglycerol (PG)\nsignificantly reduced PBGProOx 's broad-spectrum antimicrobial activity (Figure 4f). Isothermal titration\n20\ncalorimetry (ITC) experiments further confirmed binding affinity between PBGProOx and\n20\nphosphatidylglycerol (Figure 4h).\nTo figure out the direct evidence that PBGProOx binds with phosphatidylglycerol and how this\n20\nsubsequently induces membrane disruption, we performed additional experiments. First, an octanol-water\npartition assay confirmed PBGProOx 's high membrane affinity (revised Figure S21). Then a UV-Vis\n20\nbinding assay with spectral shift analysis verified PBGProOx 's specific binding to phosphatidylglycerol on\n20\nbacterial membranes (revised Figure 4g). Finally, using potassium-loaded large unilamellar vesicles (LUV)\ncomposed of phosphatidylglycerol and phophatidylcholine as bacteria mimicking membranes, we found that\nPBGProOx triggers significant potassium ion leakage (revised Figure 4i). These results suggest that\n20\nof the phospholipid bilayer, potentially forming transmembrane pores to ultimately leading to leakage of\nessential cellular contents. We have incorporated these new experimental data and mechanistic analysis as\nwell as supporting references into the revised manuscript, as shown below.\n“... In the octanol-water partition study, all three polymers, dye-PNProOx , dye-PGProOx , and\n20 20\ndye-PBGProOx , partitioned almost exclusively into the water layer due to their charged nature in the\n20\nabsence of any additives (Figure S21). However, dye-PBGProOx exhibited significant partitioning into the\n20\noctanol phase at lower equivalents of sodium laurate surrogate (a membrane-bound fatty acid salt) than\ndye-PNProOx and dye-PGProOx . As sodium laurate concentration increased, dye-PBGProOx was\n20 20 20\nalmost entirely partitioned into the octanol layer. This finding demonstrated that PBGProOx exhibited\n20\ngreater affinity for the membrane and more effectively translocated into the membrane-mimic lipophilic\nlayer. ...”\n“... We then performed UV-vis binding study to determine the interaction between PBGProOx and PG\n20\n(Figure 4g). The addition of PG induced a notable red shift in the UV-vis spectrum of PBGProOx ,\n20\nsuggesting that PBGProOx binds with PG to form complexes. ...”\n20\n“... Large unilamellar vesicles (LUVs) composed of PG/PC (1:1) were employed to simulate bacterial\nmembranes and figure out the interactions of PBGProOx with phospholipid bilayer. PBGProOx induced\n20 20\npotassium ion leakage from these LUVs, with the extent of leakage proportional to the polymer\nconcentration (Figure 4i). In contrast, PBGProOx exhibited virtually no effect on PC-based LUVs, which\n20\nsimulate mammalian cell membranes. ... These results suggest that the biguanide moiety plays an essential\nrole in binding phosphatidylglycerol, enabling PBGProOx to disrupt the phospholipid bilayer and induce\n20\nleakage of intracellular contents.”\n“Figure S21. The octanol-water partition study of amine-functionalized polymer dye-PNProOx ,\n20\nguanidine-functionalized polymer dye-PGProOx , biguanide-functionalized polymer dye-PBGProOx . The\n20 20\nresults were shown as fluorescent images and the percentage of dye-polymer in octanol.”\n“Figure 4. PBGProOx binds more strongly with phosphatidylglycerol to damage bacterial\n20\nmembranes. (a) The structure of amine-functionalized, guanidine-functionalized and\nbiguanide-functionalized poly(2-oxazoline)s with propyl spacer arm. (b) Cytoplasmic membrane\ndepolarization of S. aureus and E. coli after 30-minute treatment with PNProOx , PGProOx and\n20 20\nPBGProOx at different concentrations. (c) Zeta potential of S. aureus and E. coli before (the controls) and\n20\nafter 30-minute treatment with PNProOx , PGProOx and PBGProOx at different concentrations. (d)\n20 20 20\nMembrane fluidity of S. aureus and E. coli before (the controls) and after 2-hour treatment with PNProOx ,\n20 20\nbefore (the controls) and after 2-hour treatment with PNProOx , PGProOx and PBGProOx at different\n20 20 20\nconcentrations. (f) MIC changes of PBGProOx against S. aureus and E. coli in the presence of different\n20\nmembrane phospholipids. (g) The UV–vis spectra of PBGProOx titrated with phosphatidylglycerol (PG).\n20\n(h) ITC analysis of the interaction between poly(2-oxazoline)s with different positively charged moieties and\nphosphatidylglycerol (PG). (i) Potassium ion leakage from large unilamellar vesicles (LUVs) with varying\nphospholipid compositions after 30min-treatment with PBGProOx at different concentrations. Data are\n20\nmean ± s.d. in b, c, d, e and i. Data are presented as mean ± s.d., statistical analysis was conducted using\ntwo-tailed t-test in b, c, d, e and i. *P < 0.05, **P < 0.01, ***P < 0.001.”\nThe manuscript claims that PBGProOx20 penetrates the BBB via transcytosis, but further clarification on\nhow this was measured would be useful. Was this observed through in vivo imaging, or is it inferred from the\ntherapeutic efficacy in meningitis models? A more detailed description of the experimental setup for BBB\npenetration would be valuable.\nResponse: We thank you for the inspiring question on how PBGProOx penetrates the BBB via\n20\ntranscytosis was measured. In this manuscript, we found that PBGProOx could penetrate the BBB based on\n20\nextensive in vivo and in vitro studies, including intracellular uptake and efflux study, BBB-mimicking\ntranswell model, mice organs distribution, and in vivo imaging in zebrafish.\nFirst, we observed that PBGProOx (red fluorescence) penetrated the cell membrane and trapped\n20\nwithin lysosomes of brain microvascular endothelial cells (bEnd.3), key constituents of the blood-brain\nbarrier (BBB) (Figures 8a, 8b). Furthermore, we observed an obvious exocytosis process of PBGProOx in\n20\nbEnd.3 cells over time (Figures 8e). This continuous transcytosis behavior underscores the potential of\nPBGProOx to penetrate the BBB. We further employed a well-established transwell model to simulate the\n20\nin vivo structure of the BBB and quantify the BBB-penetrating efficiency of PBGProOx (Figure 8f, 8g).\n20\n24-hour incubation (Figure 8h). These findings demonstrate that PBGProOx effectively penetrates the BBB\n20\nvia a transcytosis process.\nWe also investigated the in vivo BBB-penetrating ability of PBGProOx . Following systemic\n20\nadministration in mice, PBGProOx exhibited significant accumulation within the brain, reaching\n20\ndistribution levels comparable to those observed in the heart, spleen, and lungs (Figure 8k). Furthermore, in a\nwell-established zebrafish model, distinct fluorescence signals from dye-labeled PBGProOx were clearly\n20\nobserved within the zebrafish brain region (Figure 8l). Collectively, these in vitro and in vivo results\ndemonstrate the ability of PBGProOx to penetrate the BBB.\nof revised Discussion as shown below. The related evidence that PBGProOx could penetrate the BBB is\n20\nalso provided to ensure accessibility.\n“... PBGProOx also exhibited continuous transcytosis behavior within brain microvascular endothelial\n20\ncells, along with substantial penetrating efficiency in tanswell model. Consequently, PBGProOx effectively\n20\npenetrated the BBB to distribute in the brain tissues of mice and zebrafish, achieving potent therapeutic\nefficacy in treating drug-resistant bacterial meningitis.”\n“Figure 8. (a) Fluorescent images (left) and flow cytometry analyses (right) on mean fluorescence intensity\n(MFI) of bEnd.3 cells before and after treatment with RB-PBGProOx (12.5 μg/mL). (b) Mitochondrial and\n20\nlysosomal colocalization in bEnd.3 cells after treatment with RB-PBGProOx (12.5 μg/mL) for 4h.”\n20\n“Figure 8. (e) Flow cytometry analyses of RB-PBGProOx -treated bEnd.3 cells after different exocytosis\n20\ntime.”\n“Figure 8. (f) Schematic representation of the in vitro transwell model. (g) TEER values of bEnd.3 cells\nmonolayer in the transwell model after treatment with RB-PBGProOx (12.5 μg/mL) at different time points.\n20\n(h) The transport ratio of RB-PBGProOx in the transwell model after different incubation time. (i) Percent\n20\ntransport ratio of RB-PBGProOx in the transwell model at the presence of different endocytosis inhibitors.”\n20\n“Figure 8. (k) In vivo distribution of RB-PBGProOx in mice organs after administration of 20 mg/kg via\n20\nintraperitoneal injection. (l) Confocal images of the zebrafish larvae before (Control) and after systemic\nadministration of RB-PBGProOx (3.13 μg/mL) for 1 hour.”\nshowing a lack of upregulation in resistance-related genes, as well as results from long-term treatment\nstudies. The manuscript mentions that PBGProOx20 does not induce cross-resistance, but additional data\ncomparing resistance development between PBGProOx20 and traditional antibiotics would further\nsubstantiate this claim.\nResponse: We thank you for this insightful suggestion regarding resistance induction evidence. We have\nextended the duration of drug resistance development assays for PBGProOx and traditional antibiotics.\nPBGProOx (revised Figure 5a). In addition, PBGProOx -treated bacteria did not develop cross-resistance\n20 20\nto multiple conventional antibiotics (revised Figure 5b). Ciprofloxacin-treated S. aureus and\nmoxifloxacin-treated E. coli not only acquired high resistance to their respective antibiotics, but also\ndeveloped resistance to other quinolone antibiotics (levofloxacin and norfloxacin) as well as β-lactam\nantibiotics (ampicillin). Furthermore, as presented in our manuscript (Figure 5c-5i), transcriptomic analysis\n(RNA-seq) of bacteria after long-term exposure revealed variation of gene expression: PBGProOx induced\n20\nfewer gene expression changes with no significant upregulation in any key resistance gene families. In\ncontrast, quinolone antibiotics triggered massive upregulation of resistance determinants, such as NorA and\nNorB from the major facilitator superfamily (MFS) family, hrtA and hrtB from the ATP-binding cassette\n(ABC) family, mepA and mepR from the multidrug and toxic compound extrusion (MATE) family, and acrA\nand acrB from the resistance-nodulation-cell division (RND) family. Collectively, these resistance assays\nrobustly demonstrate PBGProOx ’s exceptionally low propensity for inducing antimicrobial resistance\n20\ncompared to conventional antibiotics.\nThe updated data and results on long-term resistance development and cross-resistance comparisons\nhave been incorporated into our revised manuscript below (revised Figure 5a, 5b). The RNA-seq data are\nalso provided to ensure accessibility (Figure 5c-5i).\n“We subsequently evaluated whether bacteria develop antimicrobial resistance following prolonged\nexposure to PBGProOx . Neither S. aureus nor E. coli developed resistance to PBGProOx after 32\n20 20\npassages of continuous treatment at 0.5×MIC (Figure 5a). In contrast, under the same conditions,\nciprofloxacin and moxifloxacin exhibited significant increases in MIC values against S. aureus (512-fold)\nand E. coli (32-fold), respectively. Furthermore, ciprofloxacin-treated S. aureus (S. aureus-ciprofloxacin-P )\n32\nand moxifloxacin-treated E. coli (E. coli-moxifloxacin-P ) all acquired resistance to four quinolone\n32\nantibiotics and ampicillin (β-lactam antibiotic) in our study (Figure 5b). Notably, PBGProOx -treated\n20\nbacteria, both S. aureus-PBGProOx -P and E. coli-PBGProOx -P , remained susceptible to all antibiotics\n20 32 20 32\ntested (Figure 5b).”\n“To elucidate the bacterial responses to PBGProOx and antibiotic treatments, RNA-seq analysis was\n20\nperformed on S. aureus and E. coli following above drug resistance assays. In comparison to the S. aureus\ncontrol group without antimicrobial treatment, the volcano plots revealed that PBGProOx treatment\n20\nresulted in 22 differentially expressed genes (DEGs), including 14 down-regulated genes and 8 up-regulated\ngenes; whereas, ciprofloxacin treatment affected the expression of 82 DEGs, including 41 down-regulated\ngenes and 41 up-regulated genes [log2-fold change ≥ 1; 5% fasle discovery rate (FDR)] (Figure 5c). For E.\ncoli, PBGProOx treatment altered 446 DEGs, including 302 down-regulated genes and 144 up-regulated\n20\ngenes; whereas compared to E. coli control group without antimicrobial treatment, moxifloxacin treatment\naffected the expression of 795 DEGs, including 494 down-regulated genes and 301 up-regulated genes\n(Figure 5d). The fewer DEGs observed in the PBGProOx treatment group compared to antibiotic\n20\ntreatments suggests a lower likelihood of mutations contributing to the development of drug resistance.”\n“Further Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to\ncategorize the gene products into standardized terms. The term Two-Component System (TCS) was\nidentified among the top 15 enriched KEGG pathways in the ciprofloxacin-treated S. aureus group and\nmoxifloxacin-treated E. coli group (Figure 5e, 5f). TCS represents a major mechanism by which\nmicroorganisms sense and respond to external and/or intracellular stimuli by regulating the expression of\ngenes involved in pathogenic pathways and antibiotic resistance. Given the strong correlation between TCS\nand multidrug efflux pumps, we conducted a scan of gene expression related to various efflux pump families\n(Figure 5g). Specifically, the efflux pumps genes were up-regulated to varying extents in\nciprofloxacin-treated S. aureus group, including NorA and NorB from the major facilitator super family\n(MFS) family, hrtA and hrtB from the ATP-binding cassette (ABC) family, mepA and mepR from the\nmultidrug and toxic compound extrusion (MATE) family. In the moxifloxacin-treated E. coli group, a large\npopulation of efflux pump genes were up-regulated, including acrA and acrB from the\nresistance-nodulation-cell division (RND) families, cysW and cysU from the ABC family, mdtD, mdtG and\nmdtL from the MFS family. In sharp contrast, almost none of the genes related to efflux pump families were\nup-regulated in PBGProOx -treated groups. Conversely, PBGProOx significantly down-regulated several\n20 20\nefflux pumps genes, such as hrtA and hrtB in S. aureus, and mdtG in E. coli. Overall, these results\ndemonstrated that PBGProOx and antibiotics exert distinct effects on the expression of drug-resistant genes,\n20\nwhich can be attributed to their different antimicrobial mechanisms. This is also consistent with above\nantimicrobial resistance studies, which showed that bacteria rapidly acquired severe resistance to antibiotics,\nbut not to PBGProOx .”\n20\n“Figure 5. Susceptibility of PBGProOx to antimicrobial resistance and gene transcription analysis.\n20\n(a) Drug resistance development of S. aureus and E. coli after continuous treatment with PBGProOx and\n20\nantibiotics at a concentration of 0.5×MIC for 32 passages. (b) MIC changes of PBGProOx and different\n20\nantibiotics against S. aureus and E. coli, which were treated with PBGProOx and antibiotics at a\n20\nconcentration of 0.5×MIC for 32 passages. (c) Volcano plot of DEGs in ciprofloxacin-treated and\nPBGProOx -treated S. aureus compared with untreated S. aureus. (d) Volcano plot of DEGs in\n20\nmoxifloxacin-treated and PBGProOx -treated E. coli compared with untreated E. coli. (e) KEGG\n20\nenrichment analysis of DEGs in ciprofloxacin-treated and PBGProOx -treated S. aureus compared with\n20\nuntreated S. aureus. The top 15 most enriched KEGG categories were shown in a scatter diagram in the\ncomparison groups. (f) KEGG enrichment analysis of DEGs in moxifloxacin-treated and\nPBGProOx -treated E. coli compared with untreated E. coli. (g) Heat map of efflux pump-associated DEGs\n20\nbetween PBGProOx -treated, antibiotic-treated and untreated S. aureus and E. coli. Difference clustering\n20\nheatmap of regulated efflux pump genes with the treatment of PBGProOx and different antibiotics. Blue,\n20\nred, and white bars represent the down-regulated genes, up-regulated genes, and the genes without\nsignificant changes, respectively. (h) GO enrichment analysis of DEGs in PBGProOx -treated S. aureus\n20\ncompared with untreated S. aureus. (i) Heat map of virulence-associated DEGs between PBGProOx -treated\n20\nand untreated S. aureus.”\nWhile the therapeutic efficacy of PBGProOx20 in various infection models is promising, the manuscript\nlacks a clear description of how these models were conducted. Were bacterial loads measured? What was the\ndosing regimen? Including such details would improve the rigor of the study.\nResponse: Thank you for the valuable suggestion on the animal study. Following your suggestion,\nwe have added detailed dosing regimen in the schematic diagrams of all animal experiments and corresponding\nfigure legends in our revised manuscript, as shown below (revised Figure 6a, 6c, 6e, 7a, 9a).\n“Figure 6. (a) Schematic representation of the full-thickness infection model. The infected wounds were\ntreated topically with 15 μL of saline, PBGProOx (4 mg/mL), vancomycin (4 mg/mL) or imipenem (4\n20\nmg/mL) every 4 hours for triple applications.”\n“Figure 6. (c) Schematic representation of the sub-cutaneous infection model. The infected tissues were\ntreated subcutaneously with 100 μL of saline, PBGProOx (4 mg/mL), vancomycin (4 mg/mL) or colistin (4\n20\nmg/mL) every 12 hours for triple applications.”\n“Figure 6. (e) Schematic representation of the kidney infection model. The infected mice were treated\nintraperitoneally with saline, PBGProOx (40 mg/kg), vancomycin (40 mg/kg) or imipenem (40 mg/kg)\n20\ndaily for triple applications.”\n“Figure 7. (a) Schematic representation of the peritonitis model. The infected mice were treated\nintraperitoneally with saline, PBGProOx (40 mg/kg), vancomycin (40 mg/kg) or imipenem (40 mg/kg) for\n20\nonce application.”\n“Figure 9. (a) Schematic representation of the meningitis model. The infected mice were treated\nintraperitoneally with saline, PBGProOx (40 mg/kg), vancomycin (40 mg/kg) or meropenem (40 mg/kg)\n20\ndaily for six applications.”\n- The conclusion is concise but could benefit from a more forward-looking statement regarding the potential\nclinical applications and future directions for research. It would be helpful to briefly mention potential\nchallenges or areas of improvement in the development of PBGProOx20 or similar compounds and how the\nfindings of this study could influence future strategies for treating other types of resistant infections or CNS\ndiseases.\nResponse: We thank you for your valuable suggestion on enhancing the forward-looking perspective of\nthis study. We have incorporated the following points as recommended in the revised conclusion section.\n“Our study provides an alternative designing option of the positively charged moiety to develop potent\nantimicrobial and BBB-penetrating HDP mimics. PBGProOx holds promise as a potential therapeutic\n20\nagainst life-threatening drug-resistant systemic infections and CNS infections like meningitis, particularly\nwhere conventional antibiotics fail. However, realizing this clinical potential will require further exploration,\nincluding scalable synthesis, comprehensive long-term toxicity profiling, and detailed\npharmacokinetic/pharmacodynamic optimization. Furthermore, developing stable formulations suitable for\ncorresponding administration routes will be critical for clinical translation. Beyond PBGProOx , the\n20\nbiguanide design paradigm provides a robust platform for developing next-generation antimicrobials against\na wider spectrum of multi-drug resistant pathogens, while its BBB-penetrating efficacy opens new avenues\nfor treating other CNS infections or repurposing as a neurotherapeutic delivery platform. Future research will\nfocus on investigating how cationic and hydrophobic groups affect the BBB-penetrating efficiency, and\nexplore diverse molecular structures to enhance antimicrobial potency and BBB penetration efficiency.”\nMinor Comments:\n- The abstract provides a good summary of the study, but it could benefit from a clearer structure and more\nfocus on key findings.\nResponse: We thank you for the valuable comment and we have streamlined some cumbersome content\nand elaborated more on the key points in the revised abstract, as shown below.\n“ESKAPE pathogens-induced meningitis seriously threaten human health due to antimicrobial\nresistance and low permeability of blood-brain barrier (BBB). It’s a promising strategy to combat\ndrug-resistant pathogens using synthetic mimics of host defense peptide (HDP) bearing positive charges that\nare mainly provided by amine or guanidine. Here, we report that biguanide serves as a new type of positively\ncharged moiety to design HDP mimics. Biguanide exerts a stronger interaction with bacterial membrane\nphospholipids via bidentate hydrogen bonds than do amine and guanidine. The biguanide-functionalized\nHDP mimic, PBGProOx , targets bacterial membrane phospholipids to show potent activity against all\nsub-cutaneous infection model, kidney infection model, peritonitis model, and meningitis model. This study\nprovides a promising strategy for designing HDP mimics to combat ESKAPE pathogens and meningitis.”\n- The abstract mentions \"bidentate hydrogen bonds\" without clearly explaining their significance or how\nthey contribute to the enhanced interaction with bacterial membranes. A brief clarification of this point\nwould help readers understand the novelty of biguanide's role. It would be useful to briefly mention the\nspecific infection models used in vivo (i.e., types of mouse models) to provide more context on the therapeutic\npotential of PBGProOx20.\nResponse: We thank you for your inspiring suggestions. Considering the word restriction of abstract,\nwe have added a detail clarification into the revised introduction section to specify the contribution of\nbidentate hydrogen bonds on enhanced interaction with bacterial membranes. We also followed your suggestion\nto supply the specific infection models in the abstract. These revisions provide clearer mechanistic insight and\nexperimental context in our revised manuscript, as shown below.\n“... The planar, conjugated cationic structures of biguanide enable simultaneous donation of two\nhydrogen bonds to anionic phosphate headgroups of bacterial membrane phospholipids (Figure 1c). This\ndual-point attachment generates cooperative electrostatic interaction and bidentate hydrogen bonds,\nsignificantly enhancing membrane binding affinity and disruption efficiency. ...”\n“...PBGProOx exhibits promising BBB-penetrating property and displays potent therapeutic effects in\n20\nfull-thickness infection model, sub-cutaneous infection model, kidney infection model, peritonitis model, and\nmeningitis model.”\n- The manuscript would benefit from clearer figures and legends that better illustrate the molecular\ninteractions, experimental models, and results. Some figures, particularly Figure 1, could be more\ndescriptive in showing how biguanide interacts with bacterial membranes.\nResponse: We sincerely appreciate your valuable suggestion. We have comprehensively revised all\nfigures and legends to provide more detailed illustrations, including molecular interactions, experimental\nmodels, and key results. Specifically, we have redesigned Figure 1d to include a detailed schematic model\nillustrating the proposed interaction between biguanide-functionalized poly(2-oxazoline)s and bacterial\nmembrane phospholipids, as well as the process leading to bacterial death. We have also added appropriate\ndescriptions of experimental models to the figure legends to facilitate interpretation of the results,\nparticularly supplementary details (dose, number and route of administrations) for animal model illustrations.\nRepresentative revised figures and corresponding legends are presented below.\n“Figure 1. (d) The biguanide-functionalized poly(2-oxazoline) possesses broad-spectrum antimicrobial\nactivity and blood-brain barrier (BBB) penetrating property, thus showing potent therapeutic efficacy in\nmultiple infection models, including meningitis.”\n“Figure 6 (a) Schematic representation of the full-thickness infection model. The infected wounds were\ntreated topically with 15 μL of saline, PBGProOx (4 mg/mL), vancomycin (4 mg/mL) or imipenem (4\n20\nmg/mL) every 4 hours for triple applications.”\n“Figure 6. (c) Schematic representation of the sub-cutaneous infection model. The infected tissues were\ntreated subcutaneously with 100 μL of saline, PBGProOx (4 mg/mL), vancomycin (4 mg/mL) or colistin (4\n20\nmg/mL) every 12 hours for triple applications.”\n“Figure 6. (e) Schematic representation of the kidney infection model. The infected mice were treated\nintraperitoneally with saline, PBGProOx (40 mg/kg), vancomycin (40 mg/kg) or imipenem (40 mg/kg)\n20\ndaily for triple applications.”\n“Figure 7. (a) Schematic representation of the peritonitis model. The infected mice were treated\nintraperitoneally with saline, PBGProOx (40 mg/kg), vancomycin (40 mg/kg) or imipenem (40 mg/kg) for\n20\nonce application.”\n“Figure 9. (a) Schematic representation of the meningitis model. The infected mice were treated\nintraperitoneally with saline, PBGProOx (40 mg/kg), vancomycin (40 mg/kg) or meropenem (40 mg/kg)\n20\ndaily for six applications.”\n- There is some repetition in the introduction, particularly regarding the challenges posed by ESKAPE\npathogens and BBB penetration. Streamlining this section could improve readability.\nResponse: We thank you for pointing out this. We have streamlined the introduction accordingly and\nremoved repetition about ESKAPE pathogen and BBB penetration challenges to improve readability of the\nmanuscript, as shown below.\n“Bacterial meningitis poses a serious threat to human health with a mortality rate reaching up to 54%,\nparticularly when caused by multidrug-resistant ESKAPE pathogens, including Enterococcus faecium,\nStaphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and\nEnterobacter species. The blood-brain barrier (BBB) further limited the clinical selection and efficacy of\nantibiotics. ...”\nReviewer #3 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nResponse: We thank you for the valuable comments and suggestions to help us substantially improve\nour manuscript.\nReviewer #4 (Remarks to the Author)\nThis manuscript by Jiang et al. presents the design and evaluation of biguanide-functionalized host defense\npeptide (HDP) mimics as a strategy to combat drug-resistant ESKAPE pathogens and treat meningitis. The\nAuthors introduce biguanide as a novel positively charged moiety on peptidomimetics, which have the\npotential of enhancing the interactions between polymers and bacterial membranes, and thus facilitating\nblood–brain barrier (BBB) penetration. The Authors conducted a comprehensive set of experiments,\nincluding computational modeling, in vitro antibacterial activity studies, mechanistic analysis, toxicity\nassessments (both in vitro and in vivo), and efficacy studies in multiple in vivo models, such as a zebrafish\nbrain penetration model, murine wound infection model, meningitis model, and several other mice models\nusing different infection routes. The study is impressively thorough, and the multidisciplinary approach\nsignificantly enhances the impact of this study. In particular, breaking the blood–brain barrier is a\nchallenging but crucial task, given the limited treatment options for related infections.\nOverall, the manuscript provides sounding support for the lead compound (PBGProOx20) as a\nnon-resistance-inducing and membrane-disruptive antimicrobial. The Authors have also demonstrated that\nPBGProOx20’s impressive antibiotic properties are achieved through an endocytosis-based mechanism. The\nAuthors’ findings suggest that PBGProOx20 is a potential next-generation antibiotic. The manuscript should\nbe considered for publication following revisions to address the following minor points:\nResponse: We thank you for the favorable comments and recognition on our manuscript.\n1. While the use of biguanide as a positively charged moiety is new in the context of HDP mimics, the\nbroader strategy of incorporating cationic functional groups to enhance antimicrobial activity is\nwell-established. The manuscript would benefit from a clearer discussion and/or experimental evidence of\nhow biguanide outperforms amine or guanidinium modifications (in addition to the computational\ncomparisons that the Authors already mentioned).\nResponse:We thank you for your valuable comments on how biguanide outperforms amine or\nguanidinium, which is an important support for the hypothesis of our manuscript. Following your suggestion,\nwe conducted additional mechanism studies confirming that biguanide exhibits superior\nmembrane-disrupting activity compared to amine or guanidinium, as described below and updated in the\nrevised manuscript.\n“To figure out the mode of action of PBGProOx on bacterial cell membranes and how biguanide\n20\noutperforms amine or guanidine, multiply studies were conducted to compare polymer-bacterial membrane\ninteraction using poly(2-oxazolie)s with positively charged moieties, PNProOx , PGProOx and\n20 20\nPBGProOx (Figure 4a). In the octanol-water partition study, all three polymers, dye-PNProOx ,\n20 20\ndye-PGProOx , and dye-PBGProOx , partitioned almost exclusively into the water layer due to their\n20 20\ncharged nature in the absence of any additives (Figure S21). However, dye-PBGProOx exhibited\n20\nsignificant partitioning into the octanol phase at lower equivalents of sodium laurate surrogate (a\nmembrane-bound fatty acid salt) than dye-PNProOx and dye-PGProOx . As sodium laurate concentration\n20 20\nincreased, dye-PBGProOx was almost entirely partitioned into the octanol layer. This finding demonstrated\n20\nthat PBGProOx exhibited greater affinity for the membrane and more effectively translocated into the\n20\nmembrane-mimic lipophilic layer.”\n“We then assessed the impact of these polymers on cytoplasmic membrane potential using DiSC (5),\n3\nand found that PBGProOx induced more significant membrane depolarization in both S. aureus and E. coli\n20\nat different concentrations compared to PNProOx and PGProOx (Figure 4b). Moreover, the membrane\n20 20\nzeta potential of both S. aureus and E. coli shifted from below −15 mV to above +5 mV after treatment with\nPBGProOx , even at a low concentration of 0.5×MIC (Figure 4c). PBGProOx induced larger membrane\n20 20\nzeta potential shifts than PNProOx and PGProOx , indicating its greater accumulation on bacterial\n20 20\nmembranes. We then detected membrane fluidity using Laurdan as a fluorescent probe (Figure 4d).\nCompared to PNProOx and PGProOx , PBGProOx treatment induced a greater increase in Laurdan\n20 20 20\nfluorescence, revealing a more pronounced reduction in membrane fluidity in both S. aureus and E. coli. The\nΔpH of bacterial cells, a key component of the proton motive force, was also evaluated using the fluorescent\nprobe BCECF-AM (Figure 4e). A more marked dissipation of ΔpH was observed in both S. aureus and E.\ncoli after treatment with PBGProOx . These findings demonstrate that biguanide-functionalized\n20\nderangement and decreasing membrane rigidity. This disturbs bacterial homeostasis and leads to critical\nmetabolic dysfunctions, notably the dissipation of the proton motive force.”\n“... We further performed UV-vis binding study to determine the interaction between PBGProOx and\n20\nsuggesting that PBGProOx binds with PG to form complexes. We then quantified the binding affinity\n20\nbetween polymers and phospholipids using isothermal titration calorimetry (ITC). The equilibrium\ndissociation constant (KD) between PBGProOx and PG was determined to be 4.5 µmol L-1, indicating a\n20\nhigh affinity between PBGProOx and the negatively charged PG (Figure 4h); whereas, PBGProOx\n20 20\nshowed poor binding affinity with neutral PC (Figure S23). Moreover, the KD values between PG and\nPNProOx and between PG and PGProOx were 69 µmol L-1 and 11 µmol L-1, respectively, demonstrating\n20 20\nthat the biguanide group exhibited a substantially higher affinity for negatively charged phospholipids than\ndo amine and guanidine groups (Figure 4h).”\n... These results suggest that the biguanide moiety plays an essential role in binding\nphosphatidylglycerol, enabling PBGProOx to disrupt the phospholipid bilayer and induce leakage of\n20\nintracellular contents.”\n“Figure S21. The octanol-water partition study of amine-functionalized polymer dye-PNProOx ,\n20\nguanidine-functionalized polymer dye-PGProOx , biguanide-functionalized polymer dye-PBGProOx . The\n20 20\nresults were shown as fluorescent images and the percentage of dye-polymer in octanol.”\n“Figure 4. PBGProOx binds more strongly with phosphatidylglycerol to damage bacterial\n20\nmembranes. (a) The structure of amine-functionalized, guanidine-functionalized and\nbiguanide-functionalized poly(2-oxazoline)s with propyl spacer arm. (b) Cytoplasmic membrane\ndepolarization of S. aureus and E. coli after 30-minute treatment with PNProOx , PGProOx and\n20 20\nPBGProOx at different concentrations. (c) Zeta potential of S. aureus and E. coli before (the controls) and\n20\nafter 30-minute treatment with PNProOx , PGProOx and PBGProOx at different concentrations. (d)\n20 20 20\nMembrane fluidity of S. aureus and E. coli before (the controls) and after 2-hour treatment with PNProOx ,\n20 20\nbefore (the controls) and after 2-hour treatment with PNProOx , PGProOx and PBGProOx at different\n20 20 20\nconcentrations. (f) MIC changes of PBGProOx against S. aureus and E. coli in the presence of different\n20\nmembrane phospholipids. (g) The UV–vis spectra of PBGProOx titrated with phosphatidylglycerol (PG).\n20\n(h) ITC analysis of the interaction between poly(2-oxazoline)s with different positively charged moieties and\nphosphatidylglycerol (PG). (i) Potassium ion leakage from large unilamellar vesicles (LUVs) with varying\nphospholipid compositions after 30min-treatment with PBGProOx at different concentrations. Data are\n20\nmean ± s.d. in b, c, d, e and i. Data are presented as mean ± s.d., statistical analysis was conducted using\ntwo-tailed t-test in b, c, d, e and i. *P < 0.05, **P < 0.01, ***P < 0.001.”\nThis is particularly important given the synthetic complexity of biguanide-functionalized mimics. Are there\nany unique advantages in terms of stability, selectivity, or protease resistance of biguanide-polymers?\nAs mentioned by the Authors (line 45), HDPs often suffer from low protease stability, high cost, and difficult\nsynthesis. It is unclear whether biguanide-functionalized mimics can address these limitations.\nResponse: We thank you for your questions regarding the unique advantages of\nbiguanide-functionalized mimics. As suggested, we conducted the protease stability studies on PBGProOx\n20\ncompared to HDPs and provided a detailed explanation that poly(2-oxazoline)s outperform HDPs, which\noften suffer from low protease stability, high cost, and difficult synthesis. The new figures and related\ndescription were updated in our revised manuscript, as shown below (revised Figure S16, S17).\n“As described above, the synthetic process of biguanide-functionalized poly(2-oxazoline)s can be\ncompleted in as fast as 12 hours. In contrast, solid-phase-peptide synthesis (SPPS) is the most common\nmethod for HDPs synthesis.53 The sequential grafting of amino acids and repeated deprotection steps, along\nwith complex purification schemes, significantly increase synthetic difficulty, period and limit quantities of\nthe final product. Solid-phase synthesis of HDPs often continuously consumes excess expensive coupling\nreagents and protected amino acids. In contrast, the fast synthesis and cheap reactants make\npoly(2-oxazoline)s significantly more cost-effective and accessible, overcoming key limitations of HDPs\nsuch as high cost and difficult synthesis. ... 53. Deo, S., Turton, K.L., Kainth, T., Kumar, A. & Wieden, H.-J.\nStrategies for improving antimicrobial peptide production. Biotechnol. Adv. 59, 107968 (2022).”\n“... To evaluate PBGProOx ’s proteolytic stability, trypsin was used as a representative enzyme for\n20\nenzymatic degradation studie. Following trypsin treatment, PBGProOx retained its broad-spectrum\n20\nantimicrobial activity against S. aureus and E. colis (Figure S17). This contrasts sharply with the classical\nHDP melittin, which completely lost its antimicrobial activity (MIC increased from 6.25 μg/mL to >200\nμg/mL). Furthermore, NMR analysis showed no structural changes in PBGProOx after 7 days exposure to\n20\ntrypsin (Figure S18). These results demonstrate the poly(2-oxazoline)’s significantly greater resistance to\nenzymatic degradation compared to HDPs.”\n“Figure S17. Antimicrobial activities changes of PBGPenOx and melittin before and after treatment with\n20\ntrypsin, shown as MIC values against S. aureus ans E. coli.”\n“Figure S18. Structural changes of PBGPenOx before and after treatment with trypsin, characterized by 1H\n2\n2. Please specify the detection limit (in CFU) of the time-kill assays shown in Figure 2. Additionally, please\nclarify how the kill ratio was calculated.\nResponse: We thank you for this suggestion. In the original manuscript, the detection limit for viable\nbacterial concentration was 10 CFU/mL, and the bacterial killing ratio of PBGPenOx was calculated as the\n20\nratio of bacterial concentration after polymer treatment to the initial concentration before treatment.\nFollowing the suggestion from reviewer 1, we have re-conducted the time-kill kinetics study using the\nantibiotics vancomycin and colistin as positive controls in the revised manuscript. To show more details,\nthe updated results are presented as curves showing residual viable bacterial concentration variation with\ntreatment time. In revised Figure 2j, the detection limit is indicated by a dashed line, with corresponding\ndescription added to the figure legend as shown below.\n“It was found that PBGProOx at a concentration of 2×MIC and 4×MIC completely eradicated S.\n20\naureus and E. coli within three or two hours, respectively (Figure 2j). PBGProOx exhibited rapid killing\n20\nefficacy comparable to that of colistin against E. coli; whereas, vancomycin at a concentration of 4×MIC was\nunable to eradicate S. aureus after 5-hour treatment.”\n“Figure 2. (j) Time killing kinetics of PBGProOx (2×MIC, 4×MIC) against S. aureus and E. coli at\n20\nexponential growth phase, using vancomycin (4×MIC) and colistin (4×MIC) as the antibiotic controls,\nrespectively. The detection limit for viable bacterial concentration is 10 CFU/mL, as indicated by the dashed\nline. Data points below this threshold are designated as 0 CFU/mL.”\nReviewer #5 (Remarks to the Author)\nThe authors introduce biguanide-functionalized poly(2-oxazoline) (PBGProOx20) as a host-defense-peptide\n(HDP) mimic that displays broad bactericidal activity against all ESKAPE pathogens, no detectable\nresistance development, and the ability to cross the blood brain barrier (BBB) and cure murine bacterial\nmeningitis. The conceptual advance lies in replacing the classic amine/guanidinium cation of HDP mimics\nwith a biguanide moiety that offers a higher charge density and bidentate H-bonding to anionic\nphospholipids, thereby enhancing membrane binding and BBB transport. Authors showed activity\nagainst >30 multidrug-resistant clinical isolates, eradication of persisters and mature biofilms, and absence\nof cross-resistance after 16 passages. They also demonstrated that PBGProOx20 outperforms meropenem at\nequi-doses and is well tolerated in in full-thickness wounds, skin abscess, kidney infection, peritonitis, and\ncrucially meningitis. Comprehensive investigations\nResponse: We thank you for the favorable comments and suggestions below.\nConcerns:\n1- Novelty positioning vs PHMB and other biguanide polymers is not adequately discussed. PHMB is known\nto permeabilize membranes and is approved for wound care. How does PBGProOx20 differ in structure–\nactivity, toxicity, and BBB permeability? Maybe add a paragraph comparing physicochemical parameters,\nMIC values and systemic safety relative to PHMB.\nResponse: We appreciate your insightful comment regarding the novelty positioning of PBGProOx\n20\nrelative to established biguanide polymers like PHMB. Following your suggestion, we have supplemented\nstudies on activity, toxicity and in vivo biosafety of PHMB, confirming that PBGProOx significantly\n20\noutperforms PHMB in antimicrobial selectivity. Besides, we have also made a description to show the\nnovelty of PBGProOx . The new data and related description were updated in our revised manuscript, as\n20\nshown below (revised Table S4, Figure 9e).\n“Poly(hexamethylene biguanide) (PHMB) is a broad-spectrum antimicrobial agent and approved as a\ndisinfectant used for topical wound care. However, the antimicrobial activity of PHMB is primarily\nattributed to the dense linear arrangement of biguanide groups, resulting in potential toxicity that limits its in\nvivo applications. The HDP-mimicking poly(2-oxazoline)s deliberately incorporate biguanide moieties\nsparsely and controllably onto the highly tunable, flexible, and biocompatible poly(2-oxazoline) backbone.\nThe poly(2-oxazoline) was recently reported to be a novel peptide-mimicking scaffold by our group, which\nprovides a modular platform to fine-tune the hydrophobic/hydrophilic balance, chain length, and biguanide\nspacing, enabling the optimization of antimicrobial activity and toxicity. We also compared the antimicrobial\nactivity, hemolysis, cytotoxicity and antimicrobial selectivity of PHMB (Table S4). While PHMB showed\npotent antimicrobial efficacy (MIC = 0.39~6.25 μg/mL), substantial hemolysis and cytotoxicity (HC = 3.13\n10\nμg/mL, IC = 25 μg/mL) greatly weakened its antimicrobial selectivity. PBGProOx demonstrates\n50 20\nsignificantly better antimicrobial selectivity against ESKAPE pathogens and has greater potential for treating\ndrug-resistant bacterial infections.”\n“... After intraperitoneal injection with high dose (200 or 300 mg/kg) of PBGProOx , the mice\n20\ndemonstrated 100% survival without any sign of malaise within 14 days. PBGProOx at 400 mg/kg caused\n20\n33% death and the maximum tolerated dose (MTD) of PBGProOx was determined to be ~300 mg/kg\n20\n(Figure 9e). PHMB exhibited stronger systemic toxicity, with a MTD of ~20 mg/kg, making it difficult to\nmeet the biosafety requirements for in vivo infection treatment.”\n“Table S4. Antimicrobial activities, toxicity, and selectivity index of Poly(hexamethylene biguanide)\n(PHMB).”\nMIC HC IC SI SI\n10 50\nStrains\n(μg/mL) (μg/mL) (μg/mL) (HC /MIC) (IC /MIC)\n10 50\nE. faecium 0.39 \\ \\ 8 64\nS. aureus 0.39 \\ \\ 8 64\nK. pneumoniae 1.56 \\ \\ 2 16\nA. baumannii 1.56 \\ \\ 2 16\nP. aeruginosa 6.25 \\ \\ 0.5 4\nE. coli 0.78 \\ \\ 4 32\nRed blood cells \\ 3.13 \\ \\ \\\nCOS7 cells \\ \\ 25 \\ \\\n“Figure 9. (e) Survival rates of the uninfected mice at day 14 after the single-dose intraperitoneal injection of\nPBGProOx and poly(hexamethylene biguanide) (PHMB) at different concentrations.”\n20\n2- Therapeutic doses up to 40 mg kg⁻¹ i.p. were used in in vivo models. Plasma half-life, brain exposure, and\nmaximum tolerated dose are not reported. For dose justification and pharmacokinetics, please provide basic\nPK (plasma and brain) and MTD data if available, or discuss ongoing studies.\nResponse: We thank you for emphasizing the study of pharmacokinetics and safety. In our manuscript,\nwe tested the plasma concentrations of PBGProOx at different time points following a single administration\n20\n(i.p., 20 mg/kg) (Figure 8j). To achieve detailed pharmacokinetics of PBGProOx , we have supplemented a\n20\nsummary of some important pharmacokinetic parameters (such as plasma half-life) according to plasma\nconcentration-time profile, using the non-compartmental model analysis (revised Table S8). We also\nconducted a mice organ distribution study and found that PBGProOx showed remarkable brain exposure at\n20\nboth 2 and 6 hours post a single administration (i.p., 20 mg/kg) (Figure 8k). Additionally, we followed your\nsuggestions to evaluate the maximum tolerated dose (MTD) of PBGProOx (revised Figure 9e), confirming\n20\nthe safety of the concentrations administered in the animal models. These results collectively support the\npotential of PBGProOx to address the challenges in treating meningitis. Relevant figures, table and\n20\ndescriptions have been updated in the revised manuscript, as detailed below.\n“We investigated the plasma pharmacokinetics of RB-PBGProOx using fluorescent quantification\n20\nmethod. Following a 20-mg/kg bolus dose, the plasma concentrations of PBGProOx achieved were above\n20\nthose required for antimicrobial activity in vitro and a half-life of ~4 hours was defined (Figure 8j, Table S8).\nAfter systemic administration in mice, PBGProOx was found with significant amount within the brain,\n20\nshowing distribution levels comparable to that in the heart, spleen, and lung (Figure 8k). ... These results\naltogether demonstrated the ability of PBGProOx to penetrate the BBB, showing potential in treating\n20\nbacterial meningitis caused by ESKAPE pathogens.”\n“After intraperitoneal injection with high dose (200 or 300 mg/kg) of PBGProOx , the mice\n20\ndemonstrated 100% survival without any sign of malaise within 14 days. PBGProOx at 400 mg/kg caused\n20\n33% death and the maximum tolerated dose (MTD) of PBGProOx was determined to be ~300 mg/kg\n20\n(Figure 9e). PHMB exhibited stronger systemic toxicity, with a MTD of ~20 mg/kg, making it difficult to\nmeet the biosafety requirements for in vivo infection treatment.”\n“Figure 8. (j) Curves of plasma concentrations of RB-PBGProOx over time after administration of\n20\n20 mg/kg via intraperitoneal injection.”\n“Table S8. Pharmacokinetic parameters of RB-PBGProOx after intraperitoneal administration (20 mg/kg)\n20\nin mice by noncompartmental analysis.”\nParameter Unit Value\nt min 254.1052365\n1/2\nAUC 0-t μg/ml*min 4272.652195\nAUC 0-∞ μg/ml*min 6222.944854\nAUMC 0-∞ μg/ml*min^2 1913014.656\nMRT 0-∞ min 307.413082\nVz_obs ml 11.78208624\nCl_obs ml/min 0.032139125\nVss_obs ml 9.879987342\n“Figure 8. (k) In vivo distribution of RB-PBGProOx in mice organs after administration of 20 mg/kg via\n20\nintraperitoneal injection.”\n“Figure 9. (e) Survival rates of the uninfected mice at day 14 after the single-dose intraperitoneal injection of\nPBGProOx and poly(hexamethylene biguanide) (PHMB) at different concentrations.”\n20\n3- Selective toxicity on mammalian membranes was tested only on COS-7 cells for 24 h. Mechanism focused\non PG but mammalian mitochondria are also PG-rich. Please include data on mitochondrial membrane\npotential or cytotoxicity in primary human cells if possible or at least discuss it.\nResponse: We thank you for this valuable suggestion. The primary human cells and the required ethical\napprovals are currently unavailable in our research setting. As an alternative, we conducted cytotoxicity\nassessments using primary rat cells (revised Figure S16). Besides, we also evaluated mitochondrial\nmembrane potential changes induced by PBGProOx (revised Figure S29). The results align with our\n20\nprevious findings and support the low cytotoxicity of PBGProOx . Furthermore, as shown in our manuscript,\n20\nintracellular co-localization studies revealed that PBGProOx penetrated the mammalian cell membrane to\n20\nlocalize primarily in lysosomes, with minimal co-localization observed with mitochondria (Figure 8b). This\ndistinct localization pattern provides further evidence that the PBGProOx does not interact with or affect\n20\nmitochondria, reinforcing its favorable safety profile. Relevant figures and descriptions have been updated in\nthe revised manuscript, as detailed below.\n“We also evaluated the cytotoxicity of PBGProOx on primary cells and found that bone marrow\n20\nmesenchymal stromal cells (BMSCs) retained ~100% viability even after 24-hour treatment with 400 μg/mL\nof PBGProOx (Figure S16).”\n20\n“Live-cell fluorescence imaging showed that PBGProOx predominantly localized within the\n20\nendolysosomal compartments, rather than in mitochondria (Figure 8b), which prevents interaction between\nPBGProOx and mitochondria, contributing to the low cytotoxicity. Considering that the mitochondrial\n20\nmembrane also contains negatively charged phospholipids, the mitochondrial membrane potential was also\ncharacterized using rhodamine 123 (Figure S29). It was found that the mitochondrial membrane potential in\nbEnd.3 cells remained normal after PBGProOx treatment, which further ruled out any adverse effects of\n20\n“Figure S16. The cytotoxicity of PBGPenOx at different concentrations toward bone marrow mesenchymal\n20\nstromal cells (BMSCs) assessed the methyl thiazolyl tetrazolium (MTT) assay.”\n“Figure 8. (b) Mitochondrial and lysosomal colocalization in bEnd.3 cells after treatment with\nRB-PBGProOx (12.5 μg/mL) for 4 hours.”\n20\n“Figure S29. Fluorescence images of bEnd.3 cells before and after PBGProOx (12.5 μg/mL) treatment for\n20\n4 hours, using rhodamine 123 (1 μM) as a mitochondrial membrane potential indicator. Carbonyl\nCyanide3-ChloroPhenylhydrazone (CCCP) served as positive control. Normal mitochondria exhibit bright\ngreen fluorescence, while loss of mitochondrial membrane potential leads to fluorescence attenuation or\ndisappearance.”\n4- Resistance study was for 16 passages (~24 days), which may be too short to detect low-frequency\nresistance. I suggest to either extend evolution experiment to ≥30 passages or add genomic analysis to\nconfirm low mutational burden.\nResponse: We thank you for this suggestion. We have extended the duration of drug resistance\ndevelopment assays for PBGProOx and traditional antibiotics from 16 passages to 32 passages, and S.\n20\naureus and E. coli did not acquire any resistance to PBGProOx (revised Figure 5a). Besides,\nresistance to their respective antibiotics, but also developed resistance to other quinolone antibiotics\n(levofloxacin and norfloxacin) as well as β-lactam antibiotics (ampicillin). Furthermore, as presented in our\nmanuscript (Figure 5c-5i), transcriptomic analysis (RNA-seq) of bacteria after long-term exposure revealed\nvariation of gene expression: PBGProOx induced fewer gene expression changes with no significant\n20\nupregulation in any key resistance gene families. In contrast, quinolone antibiotics triggered massive\nupregulation of resistance determinants, such as NorA and NorB from the major facilitator superfamily (MFS)\nfamily, hrtA and hrtB from the ATP-binding cassette (ABC) family, mepA and mepR from the multidrug and\ntoxic compound extrusion (MATE) family, and acrA and acrB from the resistance-nodulation-cell division\n(RND) family. Collectively, these resistance assays robustly demonstrate PBGProOx ’s exceptionally low\n20\npropensity for inducing antimicrobial resistance compared to conventional antibiotics.\nUpdated data and results on long-term resistance or cross-resistance development have been\nincorporated into our revised manuscript below (revised Figure 5a, 5b). The RNA-seq data are also provided\nto ensure accessibility (Figure 5c-5i).\n“We subsequently evaluated whether bacteria develop antimicrobial resistance following prolonged\nexposure to PBGProOx . Neither S. aureus nor E. coli developed resistance to PBGProOx after 32\n20 20\npassages of continuous treatment at 0.5×MIC (Figure 5a). In contrast, under the same conditions,\nciprofloxacin and moxifloxacin exhibited significant increases in MIC values against S. aureus (512-fold)\nand E. coli (32-fold), respectively. Furthermore, ciprofloxacin-treated S. aureus (S. aureus-ciprofloxacin-P )\n32\nand moxifloxacin-treated E. coli (E. coli-moxifloxacin-P ) all acquired resistance to four quinolone\n32\nantibiotics and ampicillin (β-lactam antibiotic) in our study (Figure 5b). Notably, PBGProOx -treated\n20\nbacteria, both S. aureus-PBGProOx -P and E. coli-PBGProOx -P , remained susceptible to all antibiotics\n20 32 20 32\ntested (Figure 5b).”\n“To elucidate the bacterial responses to PBGProOx and antibiotic treatments, RNA-seq analysis was\n20\nperformed on S. aureus and E. coli following above drug resistance assays. In comparison to the S. aureus\ncontrol group without antimicrobial treatment, the volcano plots revealed that PBGProOx treatment\n20\nresulted in 22 differentially expressed genes (DEGs), including 14 down-regulated genes and 8 up-regulated\ngenes; whereas, ciprofloxacin treatment affected the expression of 82 DEGs, including 41 down-regulated\ngenes and 41 up-regulated genes [log2-fold change ≥ 1; 5% fasle discovery rate (FDR)] (Figure 5c). For E.\ncoli, PBGProOx treatment altered 446 DEGs, including 302 down-regulated genes and 144 up-regulated\n20\ngenes; whereas compared to E. coli control group without antimicrobial treatment, moxifloxacin treatment\naffected the expression of 795 DEGs, including 494 down-regulated genes and 301 up-regulated genes\n(Figure 5d). The fewer DEGs observed in the PBGProOx treatment group compared to antibiotic\n20\ntreatments suggests a lower likelihood of mutations contributing to the development of drug resistance.”\n“Further Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to\ncategorize the gene products into standardized terms. The term Two-Component System (TCS) was\nidentified among the top 15 enriched KEGG pathways in the ciprofloxacin-treated S. aureus group and\nmoxifloxacin-treated E. coli group (Figure 5e, 5f). TCS represents a major mechanism by which\nmicroorganisms sense and respond to external and/or intracellular stimuli by regulating the expression of\ngenes involved in pathogenic pathways and antibiotic resistance. Given the strong correlation between TCS\nand multidrug efflux pumps, we conducted a scan of gene expression related to various efflux pump families\n(Figure 5g). Specifically, the efflux pumps genes were up-regulated to varying extents in\nciprofloxacin-treated S. aureus group, including NorA and NorB from the major facilitator super family\n(MFS) family, hrtA and hrtB from the ATP-binding cassette (ABC) family, mepA and mepR from the\nmultidrug and toxic compound extrusion (MATE) family. In the moxifloxacin-treated E. coli group, a large\npopulation of efflux pump genes were up-regulated, including acrA and acrB from the\nresistance-nodulation-cell division (RND) families, cysW and cysU from the ABC family, mdtD, mdtG and\nmdtL from the MFS family. In sharp contrast, almost none of the genes related to efflux pump families were\nup-regulated in PBGProOx -treated groups. Conversely, PBGProOx significantly down-regulated several\n20 20\nefflux pumps genes, such as hrtA and hrtB in S. aureus, and mdtG in E. coli. Overall, these results\ndemonstrated that PBGProOx and antibiotics exert distinct effects on the expression of drug-resistant genes,\n20\nwhich can be attributed to their different antimicrobial mechanisms. This is also consistent with above\nantimicrobial resistance studies, which showed that bacteria rapidly acquired severe resistance to antibiotics,\nbut not to PBGProOx .”\n20\n“Figure 5. Susceptibility of PBGProOx to antimicrobial resistance and gene transcription analysis.\n20\n(a) Drug resistance development of S. aureus and E. coli after continuous treatment with PBGProOx and\n20\nantibiotics at a concentration of 0.5×MIC for 32 passages. (b) MIC changes of PBGProOx and different\n20\nantibiotics against S. aureus and E. coli, which were treated with PBGProOx and antibiotics at a\n20\nconcentration of 0.5×MIC for 32 passages. (c) Volcano plot of DEGs in ciprofloxacin-treated and\nPBGProOx -treated S. aureus compared with untreated S. aureus. (d) Volcano plot of DEGs in\n20\nmoxifloxacin-treated and PBGProOx -treated E. coli compared with untreated E. coli. (e) KEGG\n20\nenrichment analysis of DEGs in ciprofloxacin-treated and PBGProOx -treated S. aureus compared with\n20\nuntreated S. aureus. The top 15 most enriched KEGG categories were shown in a scatter diagram in the\ncomparison groups. (f) KEGG enrichment analysis of DEGs in moxifloxacin-treated and\nPBGProOx -treated E. coli compared with untreated E. coli. (g) Heat map of efflux pump-associated DEGs\n20\nbetween PBGProOx -treated, antibiotic-treated and untreated S. aureus and E. coli. Difference clustering\n20\nheatmap of regulated efflux pump genes with the treatment of PBGProOx and different antibiotics. Blue,\n20\nred, and white bars represent the down-regulated genes, up-regulated genes, and the genes without\nsignificant changes, respectively. (h) GO enrichment analysis of DEGs in PBGProOx -treated S. aureus\n20\ncompared with untreated S. aureus. (i) Heat map of virulence-associated DEGs between PBGProOx -treated\n20\nand untreated S. aureus.”\nMinor comments\n1. Typographical errors: “amime-functionalized” (Fig. 2c caption).\nResponse: We thank you for reminding us on this and we have corrected \"amime\" to \"amine\" in Figure\n2c legend.\n“Figure 2. (c) 1H NMR characterization of biguanide-functionalized poly(2-oxazoline) PBGProOx and\n20\namine-functionalized poly(2-oxazoline) PNProOx .”\n20\n2. Figure 1d legend abbreviations (BBB, HDP) should be defined in the caption.\nResponse: We thank you for this reminder. We have added full name of “BBB” and “HDP” in Figure 1a\nand 1d legends as shown below.\n“Figure 1. (a) The proposed antimicrobial design strategy that biguanide serves as a new type of\npositively charged moiety to design host defense peptides (HDPs) mimics. ... (d) The\nbiguanide-functionalized poly(2-oxazoline) possesses broad-spectrum antimicrobial activity and blood-brain\nbarrier (BBB) penetrating property, thus showing potent therapeutic efficacy in multiple infection models,\nincluding meningitis.”\n3. Provide chemical structures of comparator amine- and guanidine-POx controls in the main text, not only\nin Supplementary Fig. S4.\nResponse: We thank you for this suggestion. We have added the structure of amine-functionalized POx\nand guanidine-functionalized POx with same propyl spacer arm as PBGProOx in revised Figure 4a as\n20\nshown below.\n“Figure 4. (a) The structure of amine-functionalized, guanidine-functionalized and biguanide-functionalized\npoly(2-oxazoline)s with propyl spacer arm.”\n4. Supplementary Methods page numbers are missing; add running headers for easy cross-reference.\nResponse: We thank you for reminding the absence of page numbers in the Supplementary Methods.\nWe have added corresponding page numbers and running headers in the revised supplementary file. Additionally,\nthe table of contents has been included in the supporting information section to facilitate navigation. These\nmodifications ensure efficient cross-reference between the main manuscript and supplementary materials.\n5. Clarify whether biguanidylation is quantitative (NMR integration appears sub-unity).\nResponse: We thank you for this questions regarding NMR quantification. In our manuscript,\ncomparative NMR analysis of the amine-functionalized poly(2-oxazoline)s precursor and\nbiguanide-functionalized poly(2-oxazoline)s confirmed near-quantitative conversion, as evidenced by the\ncomplete downfield shift of protons adjacent to the cationic groups. Specifically, protons near amine groups\nresonate at upfield positions (δ ~3.0 ppm for −CH −amine). The strong electron-withdrawing effect and high\n2\ncharge density of the biguanide moiety induced a significant downfield shift (δ ~3.2 ppm for\n−CH −biguanide), with no residual upfield peaks attributable to unconverted amine groups observed,\n2\nindicating virtually complete functionalization. To enhance clarity, we have annotated all NMR peak\nassignments in the Supporting Information (revised Figure S5-S14) and expanded the spectral interpretation\nin the main manuscript to explicitly correlate this displacement of proton signals with conversion efficiency\n(revised Figure 2c), as shown below.\n“The successful functionalization of poly(2-oxazoline)s with biguanide groups was confirmed by 1H\nNMR spectra (Figure S5-S14). The strong electron-withdrawing effect and high charge density of the\nbiguanide moiety induced a notable downfield shift of the proton peak from ‘H ’ (δ~3.0 ppm for\nN\n−CH −amine) to ‘H ’ (δ~3.2 ppm for −CH −biguanide), with no residual upfield peaks attributable to\nunconverted amine groups observed, indicating virtually complete functionalization (Figure 2c).”\n“Figure 2. (c) 1H NMR characterization of biguanide-functionalized poly(2-oxazoline) PBGProOx and\n20\namine-functionalized poly(2-oxazoline) PNProOx .”\n20\n“Figure S7. 1H NMR spectrum of polymer PNProOx in D O, 600 MHz.”\n20 2\n“Figure S12. 1H NMR spectrum of polymer PBGProOx in D O, 600 MHz.”\n20 2\nWe greatly thank all the reviewers for those valuable comments, which help us substantially improved\nour manuscript. We hope that the revised manuscript will prove to be acceptable for publication in Nature\nCommunications.\nSincerely,\nRunhui Liu\nDistinguihsed Professor of Chemistry & Biomaterials, ECUST"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed my concerns properly, and the revised manuscript is suggested for publication.", "reviewer_2": "Thank you for your revised manuscript and your detailed responses.\nAuthors have addressed all the comments, clarification requests, suggestions for textual improvements, and the additional\nexplanations related to the experimental/results section in a thorough and comprehensive manner.\nFrom my point of view, the manuscript is now suitable for acceptance.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_4": "The authors have nicely addressed my comments. I recommend it for publication.", "reviewer_5": "Authors responded satisfactorily to raised comments; revised manuscript is greatly improved"}, "author_response": ""}]

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