Gene editing tool-loaded biomimetic cationic vesicles with highly efficient bacterial internalization for in vivo eradication of pathogens

doi:10.21203/rs.3.rs-5222230/v1

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Gene editing tool-loaded biomimetic cationic vesicles with highly efficient bacterial internalization for in vivo eradication of pathogens

https://doi.org/10.21203/rs.3.rs-5222230/v1

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In the post-COVID-19 era, drug-resistant bacterial infections emerge as one of major death causes, where multidrug-resistant Acinetobacter baumannii (MRAB) and drug-resistant Pseudomonas aeruginosa (DRPA) represent primary pathogens. However, the classical antibiotic strategy currently faces the bottleneck of drug resistance. We develop an antimicrobial strategy that applies the selective delivery of CRISPR/Cas9 plasmids to pathogens by biomimetic cationic hybrid vesicles (BCVs), irrelevant to bacterial drug resistance. The CRISPR/Cas9 plasmids were constructed, replicating in MRAB or DRPA and expressing ribonucleic proteins, leading to irreparable chromosomal lesions; however, delivering the negatively charged plasmids with extremely large molecular weight to the pathogens at the infection site became a huge challenge. We found that the BCVs integrating the bacterial out membrane vesicles and cationic lipids efficiently delivered the plasmids in vitro/in vivo to the pathogens followed by internalization. The BCVs were used by intratracheal or topical application in hydrogels against MRAB pulmonary infection or DRPA wound infection, and both of the two pathogens were eradicated from the lung or the wound. CRISPR/Cas9 plasmid-loaded BCVs become a promising medication for drug-resistant bacteria infections.

CRISPR/Cas9 plasmid

Outer membrane vesicle

Cationic lipid

Acinetobacter baumannii

Pseudomonas aeruginosa

Pulmonary infection

Wound infection

Antimicrobial resistance (AR) in bacterial infections has emerged as one of the primary public health threats in the 21st century[1]. A report indicates that AR bacteria could kill 10 million individuals annually by 2050, and six pathogens were responsible for almost 80% of the 1.27 million deaths attributed directly to AR in 2019, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, in order of number of deaths[1–3]. Among of them, A. baumannii and P. aeruginosa are respectively notorious for their infection of the lung and wounds, and both are challenging to eradicate in vivo, due to the rapid dissemination of AR genes and the robustness of biofilms[4, 5]. Although antibiotics remain the primary treatment for bacterial infections, their abuse has resulted in AR mutations[6, 7]. Alternatives to antibiotics, such as metal ions[8], antimicrobial peptides[9], bacteriophages[10], and Bdellovibrio bacteriovorus[11], have shown promise; however, they come with limitations, such as toxicity, specificity, hemolysis risk, and preservation challenges. Therefore, it is urgent to develop new alternative therapies to eradicate AR bacteria.

CRISPR/Cas9 is an efficient and specific gene editing tool and has been studied as a potential treatment of human diseases[12–14]. It functions as a ribonucleic protein (RNP) to perform DNA recognition and cleavage[15–17]. Recent studies indicate that CRISPR/Cas9 systems can effectively eliminate AR bacteria by causing irreparable chromosomal lesions, reprogramming toxin-antitoxin systems, and inactivating drug-resistant genes[18–20]. Compared to the traditional antimicrobial mechanisms in which compounds competitively inhibit the activity of bacterial macromolecules, CRISPR/Cas9 directly targets bacterial DNA to exert its effects, thereby being unrelated to bacterial AR[21]. More importantly, a single RNP causing a genomic double stranded break (DSB) is sufficient to induce bacterial death due to the introduction of irreparable chromosomal lesions[22, 23]. CRISPR/Cas9 can be encoded by plasmids or phages, allowing it to replicate and even spread within bacteria for eradication of bacteria[24]. Consequently, the non-concentration-dependent bactericidal effect of CRISPR/Cas9 exhibits unparalleled efficacy compared to other antimicrobials. However, the in vivo antimicrobial efficacy of CRISPR/Cas9 has been rarely reported due to the absence of efficient delivery vectors. CRISPR/Cas9 plasmids have extremely large molecular weight and electronegativity, leading to the in vivo bacterial internalization efficiency too low to eradicate bacteria.

Up to now, two carriers, genetically engineered bacteriophage and conjugative bacteria, are reported to deliver CRISPR/Cas9 systems to pathogens for antibacterial[18, 21, 25]. Nevertheless, the high specificity of bacteriophages to host bacteria makes it difficult to deal with the infections caused by different strains of the same species, while finding suitable bacteriophages is time-consuming and laborious work[26]. The conjugative bacteria are low delivery efficiency and have possibility of additional infections, leading to even more severe cases[18]. A novel strategy for safe and efficient in vivo delivering CRISPR/Cas9 plasmids to bacteria is necessary to improve the awkward situation of antibacterial CRISPR/Cas9 agents.

Outer membrane vesicles (OMVs) are small spherical lipid vesicles (20–300 nm) that derive from the outer membranes of Gram-negative bacteria[6, 27, 28]. This unique structure makes it easy to fuse with the homologous bacteria[29, 30]. OMVs can carry specific cargos, including DNA, proteins, and compounds, to complete communication between bacteria[27]. Thus, OMVs are used to deliver antibiotics to AR bacteria[27, 31, 32], while their ability to deliver CRISPR/Cas9 plasmids to bacteria has not been explored. Unfortunately, the low yield of OMVs may limit their future commercial application as drug delivery carriers[29]. The more readily accessible bacterial membranes are also a promising drug delivery carrier, although its equivalence to OMVs merits further investigation[33]. Cationic lipids have been widely used to enhance the delivery efficiency of nucleic acid drugs in mammalian cells[34, 35]. They can ionize to produce cations, thereby changing the zeta potential of vesicles or nanoparticles to facilitate the transport of nucleic acids into cells[36]. Lipid nanoparticles (LNPs) prepared from cationic lipids have played a prominent role in mRNA vaccines and have been extensively utilized during the COVID-19 pandemic, but concerns arise from their toxicity and lack of targeting capabilities[37–39]. A biomimetic vesicle, derived from a hybrid of bacterial membrane derivatives and cationic lipids, serves as an effective and low-toxic plasmid delivery vector for in vivo targeting bacteria, which is indeed promising.

Herein, we propose a strategy for the in vivo eradication of drug-resistant bacteria by delivering CRISPR/Cas9 plasmids to pathogens, using biomimetic cationic hybrid vesicles (BCVs) prepared from cationic lipids and homologous bacterial membrane derivatives. The lung and epidermal wounds are susceptible sites for pathogens invasion, representing the typical models of superficial and deep infections[40, 41], respectively. The models of multidrug-resistant A. baumannii (MRAB) infection in the lung and drug-resistant P. aeruginosa (DRPA) infection in wounds were utilized to evaluate the effectiveness and universality of the antimicrobial strategy. The CRISPR/Cas9 plasmid-loaded BCVs, including pABK-loaded cationic lipid hybrid OMVs (PCOMVs) and pPAK-loaded cationic hybrid membrane vesicles (PCMVs), demonstrated high bactericidal efficiency and good safety in the aforementioned models, respectively, providing a novel platform for in vivo targeting gene delivery to bacteria and offering a new strategy for the treatment of drug-resistant bacteria infections.

A. baumannii CRISPR/Cas9 plasmid design and OMV-mediated plasmid transfer

We initially validated the antimicrobial strategy of CRISPR/Cas9 plasmids combining with BCVs against MRAB. Based on the previously reported shuttle plasmid pBECAb-apr between A. baumannii and E. coli[42], we constructed pABCas9 (Fig. S1), which enables the continuous production of Cas9 proteins in A. baumannii. Different sgRNA fragments can be conveniently introduced into pABCas9 and transcribed constitutively, after the plasmid digestion using BsaI (Fig 1A). To investigate the impact of different targeting sites on the bactericidal efficacy, 6 plasmids bearing sgRNA were constructed; pABK1-3 targeted distinct sites within the coding regions in the genome, while pABK4-6 targeted different sites at non-coding regions in the genome (Fig. 1A); all the plasmids were verified for accuracy through sequencing and transformed into MRAB via electroporation (Fig. 1B). With the exception of pABCas9, the transformation of pABKs resulted in no colony growth (Fig. 1B), indicating that CRISPR/Cas9 targeting different loci within MRAB genome led to the equivalent bactericidal effect. pABK-2 was randomly selected for subsequent experiments and written as pABK (Fig. S2). The pABK was checked by the agarose gel electrophoresis (Fig. 1C).

To further elucidate the cause of bacterial death, we investigated the function of pABK. The MRAB harboring pABCas9 produced Cas9 but free MRAB did not, indicating that pABK also led to Cas9 production (Fig. 1D). The observed protein bands were smaller than the expected size of Cas9 (theoretically 150 kDa), which might be attributed to the degradation of Cas9 in sample preparation. Other scientists also report this phenomenon[43]. Then, the sgRNA was chemically synthesized and incubated with the Cas9 protein to form an RNP complex; the DNA fragment containing the sgRNA cleavage site was subsequently amplified and incubated with the RNP (Fig. 1E), resulting in the cleavage of the original DNA (~2600 bp) into 2 fragments (~1800 bp and ~800 bp), also called DSB, that leaded to the introduction of irreparable chromosomal lesions[25]. Therefore, the mechanism of the MRAB death induced by pABK has been confirmed.

Next, we explored the efficiency of plasmid transfer mediated by OMVs of MRAB. The pABCas9 bearing the apramycin resistance gene (bla) was electroporated into MRAB, and the mutant harboring pABCas9 was obtained after apramycin screening; the OMVs of this mutant, namely SOMVs, were subsequently isolated. The wild type MRAB acquired apramycin resistance through co-incubation with SOMVs (Fig. 1F), and the PCR results indicated that both SOMVs and the MRAB post-co-incubation harbored the plasmid (Fig. 1G). To investigate the transformation capabilities of SOMVs towards different bacterial strains, SOMVs were separately incubated with MRAB, E. coli DH5α, and probiotic E. coli Nissle 1917 (EcN). Both MRAB and EcN were capable of growing on the resistance plates (Fig. 1H), yet the transformation efficiency of MARB, i.e., 2.526%, was significantly higher than that of EcN, i.e., 0.007% (Fig. 1I). We conducted a proteomic analysis of the OMVs of MRAB, and discovered that kinases, outer membrane proteins, and chaperone proteins were abundantly present (Fig. S3 & S4, Table S2), suggesting a potential significant role in the plasmid transfer mediated by OMVs. Then, we evaluated the antibacterial effects of pABK-loaded OMVs (POMVs). MRAB was incubated with POMVs, the natural OMVs, and the naked pABK, respectively, and then plated on agar plates. However, there was no significant difference in the growth of these groups of colonies (Fig. 1J), indicating that POMVs were not competent in exhibiting significant antimicrobial activity.

Characteristics and bacterial internalization of cationic hybrid OMVs

The transformation efficiency of natural OMVs was low although they shared a similar composition to the outer membrane of homologous bacteria. The negative surface charge of OMVs should contribute to the low efficiency due to electrostatic repulsion with negatively charged bacteria. Given the great success of cationic lipids in the gene delivery field, we aimed to improve the plasmid delivery efficiency of OMVs by cationic lipid hybridization. A common cationic lipid, N-decyl-N,N-dimethyldecan-1-aminium bromide (DDAB), was used for the positive charge modification of OMVs. DDAB was used to prepare cationic liposomes (CLs), which were then co-incubated with OMVs for fusion (Fig. 2A). Ultrasound was used for improving the fusion of CLs and OMVs, which was frequently applied in preparation of hybrid vesicles[44]. The successful fusion of CLs and OMVs was confirmed by several detections. Firstly, in the FRET spectra, the fluorescence at 534 nm (NBD-PE) increased with the increase of OMV ratio, and was compared with the fluorescence of pure CLs and fully disaggregated CLs (Triton X-100 dissociated CLs) (Fig. 2B), indicating the FRET effect of the dye labeled on the CL lipid was blocked by the hybridization of the OMV membrane molecules. Then, the DiD-labeled CLs and the DiO-labeled OMVs were fused under ultrasound. We found the DiD red fluorescent area and the DiO green fluorescent area overlaying in the CLSM images (Fig. 2C). The ImageJ graphs showed the co-localization of red and green area (Fig. 2D), demonstrating the fully hybridization of CLs and OMVs. The COMVs with the CL/OMV ratio of 1:1 (v/v) were prepared due to their strongest antibacterial activity compared to the COMVs with other CL/OMV ratios (Fig. S5). SDS-PAGE analysis showed that COMVs inherited almost all of the protein of OMVs (Fig. 2E), and the Western blotting showed that the MRAB outer membrane characteristic protein Omp38 was detected in both OMVs and COMVs (Fig. 2F). In summary, the above results demonstrated the successful fusion of CLs and OMVs to generate COMVs.

The OMVs, CLs, COMVs, and PCOMVs, were spherical (Fig. 2G), with the comparable particle sizes of 160.9 ± 2.5 nm, 146.9 ± 1.0 nm, 181.4 ± 4.5 nm and 192.6 ± 0.9 nm (n = 3), respectively (Fig. 2H). OMVs had the negative zeta potential of -10.5 ± 0.8 mV (n = 3), while CLs, COMVs, and PCOMVs had the positive zeta potentials of 22.0 ± 1.3 mV, 17.6 ± 1.2 mV, and 16.4 ± 1.4 mV, respectively, due to the hybridization of cationic lipids (Fig. 2I). Electroporation is a commonly used method to load genes in vesicular carriers[45]. We co-incubated plasmids with vesicles and then used electroporation to load plasmids into vesicles. To verify the plasmid being loaded in COMVs, we performed different treatments on PCOMVs and detected them with the agarose gel electrophoresis. After comparing with the electrophoresis results of COMVs and naked pABK, we found that PCOMVs did not migrate in the gel and exhibited nucleic acid bands in the loading hole (Fig. 2J); after DNase treatment, PCOMVs still did not migrate and the band at the loading hole disappeared (Fig. 2J), indicating that the surface of PCOMVs has adsorbed the plasmid, and due to their positive charge, they were difficult to migrate in the gel; after DNase digesting and Triton X-100 processing, the same band was shown as the pABK band (Fig. 2J). We concluded that pABK was simultaneously present on the surface and in the inner phase of PCOMVs.

The bacterial internalization of vesicles was studied. DiD and DAPI were used to label vesicles (CLs, OMVs, COMVs) and bacterial nucleic, respectively. DiD-labeled OMVs did not appear around the bacterial cells (Fig. 2K-L), while both DiD-labeled CLs and COMVs appeared around the bacterial cells, indicating both CLs and COMVs were easily adsorbed by electronegative bacteria to fulfill the first step of internalization. CLs significantly led to bacterial aggregation and abnormal biofilm formation, while bacteria co-incubated with COMVs exhibited normal morphology (Fig. 2K-L), indicating that the strong cationic property of CLs may damage the bacterial surface, making them difficult to internalize plasmid. Overall, the hybrid product of cationic lipids and OMVs, COMVs, demonstrated superior ability to promote the internalization of plasmids by bacteria.

pABK-loaded COMVs lead to MRAB death

To demonstrate the effective bacterial internalization of the plasmid and bactericidal action of PCOMVs, MRAB was incubated with the naked pABK, POMVs, PCLs and PCOMVs for 24 h, respectively, and then recovered in LB broth for 24 h; subsequently, the OD₆₀₀ of the cultures were recorded and the colonies were counted after plating the cultures. Only PCOMVs exhibited bactericidal activity and showed the best antibacterial effect at a plasmid concentration of 0.4 ng·μL^-1 (p < 0.0001) (Fig. 3A), while the other formulations did not demonstrate detectable antibacterial effects with varying plasmid concentrations (Fig. 3A-B), indicating that COMVs effectively mediated the bacterial internalization and bactericidal activity of the plasmid, while the naked plasmid, OMVs, and CLs were useless.

To further confirm that the bactericidal effect of PCOMVs was mediated by the internalization of pABK plasmids by COMVs, MRAB was treated with PBS, SM, COMVs, pABK and PCOMVs, respectively, following the aforementioned process. Among them, SM was an MRAB resistant aminoglycoside antibiotic. The colony count results showed that COMVs and pABK alone did not cause significant antibacterial activity, consistent with the PBS and SM groups, while after loading pABK, PCOMVs did exhibit antibacterial activity (Fig. 3C). The results of bacterial quantification based on the qPCR (Fig. 3D) also supported this conclusion. The live/dead bacterial assay exhibited the bacterial state in different formulations. SYTO-9 was used to label all bacteria, while PI only labeled dead bacteria with damaged cell membranes. Only PCOMVs caused PI staining of bacteria, indicating that they caused bacterial death and the death process involved membrane damage (Fig. 3E). SEM images recorded the morphology of MRAB after different treatments; the treatment of PCOMVs led to the rumpled bacterial surface (Fig. 3F), indicating the programmed death of bacteria[46, 47]. The phenomenon is consistent with DNA replication disruption caused by DSB induced by pABK.

Biocompatibility and biodistribution of PCOMVs

Given the potential toxicity of excessive cationic lipids[48], we first evaluated the effect of PCOMVs on the viability of Beas-2B cells, a type of human normal lung epithelial cells. For ease of description, we used the concentration of the plasmid in PCOMVs to represent the formulation concentration. PCOMVs at a concentration of 0.25 ng·μL^-1 and below had no effect on cell growth (Fig. 4A), while a concentration of 0.5 ng·μL^-1 reduced cell viability to 60% (data not shown). Although the safe concentration of cells was relatively low, the dose was appropriately increased when used in vivo, and PCOMVs had been proven in the subsequent experiments to eliminate MRAB with only a single low-dose administration. The elimination of pABK in mammalian cells was validated. After co-incubation with PCOMVs, the cells were passaged multiple times, and the cell morphology remained normal during the process (data not shown). All the cell cultures were collected for qPCR to detect the content of the plasmid. As the cells passed through multiple passages, the C_T value of the sample gradually increased until the third passage reaching 35.5 ± 0.4 (Fig. 4B); subsequently, the C_T remained above 35 or undetectable, indicating that the plasmid had been cleared by the cells.

The hemolysis of PCOMVs was assayed, showing a very little hemolysis (6.6%) (Fig. 4C) when the concentration of pABK in PCOMVs reached 7 ng·μL^-1, i.e., 2 ng·μL^-1 DDAB was contained. However, when PCOMVs were intratracheally (i.t.) administered to mice, PCOMVs of the same concentration did not induce the significant changes in WBC, RBC, and PLT (Fig. 4D-F). Moreover, the CRE levels in the serum did not change compared with that in the healthy mice when a series of concentrations of PCOMVs were used (Fig. 4G). The ALT level increased compared with the healthy mice when 7 ng·μL^-1 pABK in PCOMVs were used (Fig. 4H). This observation raised the possibility that cationic lipids might be entering the liver when the dosage was high. Nevertheless, the vital organs, including the heart, liver, spleen, lung, and kidney, showed no significant structural damages or lesions even with the highest dosing (Fig. 4I).

The biodistribution of i.t. PCOMVs was verified by DiR fluorescent imaging of the major organs within 24 h. At 6 h after administration, the fluorescent intensities of the PCOMV group maintained a high level in the lung and no fluorescence was detected in the other organs, which was similar to those of the free DiR group (Fig. 4J). At 12 h after administration, the fluorescence intensity in the PCOMV group was higher than that in the free DiR group, and the difference became even more pronounced at 24 h after administration (Fig. 4J), indicating the long-term retention of PCOMVs in the lung, which benefited to enhance the local bactericidal effect.

Eradication of MRAB in the lung of infected mice by PCOMVs

On the basis of the above biocompatibility of PCOMVs, the proper concentrations of PCOMVs were further screened by comparing the body weight of mice with pneumonia after administration of different concentrations of PCOMVs. All the PCOMVs containing 1, 3, and 5 ng·μL^-1 pABK showed anti-MRAB pneumonia effects (Fig. S6), so that the lowest concentration of PCOMVs containing 1 ng/μL pABK was selected in the pharmacodynamic study.

The MRAB pneumonic mice experienced gradual body weight loss during the period of immune suppression (day -4 to day 0) (Fig. 5A). From day 1 to day 4, the mouse body weight in the PCOMV group had gradual increase with the similar profile to that in the healthy group (Fig. 5A). However, the other groups, including the model, SM, and pABK groups, had gradual body weight decrease though the extent in the pABK group was little. On day 4, the body weight in the PCOMV group had statistical differences with that in the model group (p < 0.01) and that in the SM group (p < 0.05).

SpO₂ indicates the level of oxygen binding to hemoglobin in the blood. Respiratory dysfunction can lead to a decrease in SpO₂. Infection with MRAB caused a significant drop in the SpO₂ level of mice, down to about 70% (Fig. 5B). SM and pABK did not improve SpO₂ levels, but PCOMVs made SpO₂recovering to the healthy level (Fig. 5B). Respiratory function affects the exercise capacity. An open-field experiment revealed significant reductions in the exercise capacity for the model, SM, and pABK groups compared to the healthy mice, as indicated by decreased total distance (Fig. 5C). PCOMV treatment restored the total distance to the level of the healthy group (Fig. 5C).

PCOMVs exhibited superior antibacterial efficacy, greatly reducing bacterial burden to 2.7×10⁵ CFU·mg^-1 with a seven-order magnitude decrease compared to that in the model group (1×10¹² CFU·mg^-1) (Fig. 5D), highlighting them in vivo efficacy against MRAB. SM and pABK also showed anti-MRAB effects even though their efficiencies were far weaker than PCOMVs. Notably, although the C_T value (~32) of the healthy group exceeded the linear range, the C_T value (~31) of the PCOMV group was only about 1 higher than that of the healthy group, indicating that PCOMVs had essentially eradicated the lung pathogens. TNF-α is an important pro-inflammatory factor. The TNF-α levels in the model and SM groups were high. PCOMVs made the TNF-α level greatly decreasing, indicating that the inflammatory symptoms of MRAB pneumonia were highly ameliorated (Fig. 5E). The lung appearance in the PCOMV-treated mice closely resembled the healthy state, indicating the strong therapeutic effect (Fig. 5F). The H&E staining of lung tissue sections further confirmed the high therapeutic efficiency of PCOMVs (Fig. 5F). Obvious alveolar collapse, a lot of exudates, and infiltration of inflammatory cells were observed in the model, SM, and pABK groups, demonstrating the typical histological alteration due to lung infection. In contrast, all the above symptoms were alleviated and the lung status almost recovered to the healthy status after PCOMV treatment (Fig. 5F). TUNEL staining revealed extensive apoptosis in the model, SM, and pABK groups, while the PCOMV group had significantly reduced positive areas (Fig. 5G). Therefore, i.t. PCOMVs greatly reduced lung infections and relieved inflammation due to the eradication of MRAB from the lung.

Characteristics of bacteria membrane and cationic lipid hybrid vesicles

To demonstrate the versatility of CRISPR/Cas9 plasmid-loaded BCVs, the eradication strategy for DRPA was explored. Given the low yield of OMVs[6], the cationic lipid hybrid bacterial membrane vesicles (CMVs) were further used for plasmid delivery.

The previously reported pCasPA was engineered following the construction process of pABK to generate pPAK[49], whose cleavage site located in the coding region of DRPA_3247. Compared with the plasmid without sgRNA sequence, the electroporation of pPAK resulted in no colony growth (Fig. 6A), indicating that pPAK mediated the death of DRPA. Then, the bacterial membranes (MVs) were isolated and prepared into cationic lipid hybrid vesicles (CMVs). SDS-PAGE results showed that CMVs retained the same protein composition as MVs (Fig. 6B). FRET spectra indicated thorough hybridization and fusion of cationic lipids with bacterial membranes due to the reduced FRET effect (Fig. 6C).

Before fusion, the sizes of CLs and MVs were 155.8 ± 1.8 nm and 134.2 ± 1.3 nm (n = 3), respectively; while the size of CMVs increased to 183.9 ± 6.3 nm; the size of PCMVs was 198.2 ± 11.4 nm, slightly larger than that before loading pPAK (Fig. 6D). The zeta potential of MVs was -10.4 mV, while CLs, CMVs, and PCMVs exhibited positive electrical charges with gradually decreasing zeta potentials from 20.8 mV to 17.2 mV (Fig. 6E). As the aforementioned description, we screened the optimal ratio of CLs to MVs and found that the CMVs prepared at a ratio of 10:3 (v/v) had the highest plasmid delivery efficiency. The DRPA viability was examined after co-incubation with kanamycine (Kan), the naked pPAK, CMVs, and PCMVs. PCMVs resulted in the lowest bacterial vitality, while both the naked pPAK and CMVs did not decrease bacterial vitality (Fig. 6F). Considering the difficulty of PCMVs staying in the wound, a biocompatible PVA hydrogel was used as a carrier of PCMVs. We compared the inhibitory effects of the naked pPAK, blank PVA hydrogels, Kan, and PCMVs on DRPA. The naked pPAK and blank PVA hydrogels did not show significant antibacterial effects, indicating that plasmids cannot enter DRPA, and proving that PVA hydrogels can be used as an inactive drug carrier of PCMVs; conversely, the co-incubation of PCMVs and DRPA resulted in minimal colony growth, indicating the high antibacterial effect of PCMVs (Fig. 6G). PVA hydrogels are an appropriate carrier of PCMVs for wound infection treatment.

Antibacterial effect of PCMVs on DRPA in the burn wound

The burn wound infection model was successfully established. The healing rate of infected wounds was significantly lower than that of uninfected wounds (Fig. 6H). Compared with Kan, PCMVs significantly accelerated wound healing, and there was no difference in wound healing rate between the PCMV group and the uninfected group on days 14 and 21 after modeling (Fig. 6H-I), indicating that PCMVs effective inhibited the growth of DRPA. Histopathological analyses of wound tissues showed that PCMVs significantly reduced inflammatory infiltration (H&E stained sections), increased collagen deposition (Masson stained sections), and suppressed pathogen density (Giemsa stained sections), compared to the infected wounds (Fig. 6J). Although the Kan group also exhibited reduced inflammatory infiltration and increased collagen deposition, the pathogen density within the wounds remained relatively high (Fig. 6J). Furthermore, the levels of inflammatory factors, TNF-α and IL-6, in the wound tissues significantly decreased after PCMV treatment (Fig. 6K-L). The results demonstrated that PCMVs eradicated DRPA in wounds, thereby controlling wound inflammation and accelerating wound healing.

The previous reports had shown the lethal effect of RNP on bacteria[19, 23]. Over the past decade, despite the RNP antimicrobial strategies have further developed[20, 21, 25, 50], few delivery tools have been able to selectively in vivo deliver the CRISPR/Cas9 systems with extremely large molecular weight to pathogens. So far, bacteriophages are the only proven carrier that can deliver the CRISPR/Cas9 system to targeted bacteria in vivo[25], which limits the clinical translation of CRISPR/Cas9 antimicrobial agents. In this study, we have established a CRISPR/Cas9 plasmid delivery platform based on biomimetic cationic lipid hybrid vesicles (BCVs) for the in vivo eradication of drug-resistant bacteria.

Plasmids are employed as vectors for the CRISPR/Cas9 system due to their high stability and ease of programming[51]. The stability of plasmids enhances the druggability of the nanomedicine, while the easy reprogramming facilitates the replacement of targeted sgRNA sequences for different pathogens. Furthermore, we have demonstrated that the CRISPR/Cas9 plasmid, i.e., pABK, can express RNP in MRAB to cleave the target DNA, thereby inducing replication abnormalities and abnormal bacterial membranes, leading to bacterial death. However, the more detailed death process of the bacteria remains to be revealed. Moreover, the horizontal transfer of plasmids mediated by OMVs was observed in this study, indicating that the replicable pABCas9 could spread between bacteria and produce a cascade bactericidal effect, although the efficiency was not verified. When discussing CRISPR/Cas9 therapy, it is imperative to address its off-target risks, such as spontaneous mutations in bacterial target sequences. Plasmids can be easily reprogramed to simultaneously target multiple sequences in the bacterial genome. Another concern is the possibility of the CRISPR/Cas9 plasmids entering eukaryotic cells. The plasmids operate exclusively in target pathogens, and our data showed they did not replicate within eukaryotic cells and were consequently eliminated (Fig. 3B). Therefore, the CRISPR/Cas9 therapy is safe.

We tailored a CRISPR/Cas9 plasmid delivery platform, BCVs, for bacteria. The biomimetic carrier results from the hybridization of cationic lipids with bacterial membrane derivatives, which can be derived from OMVs or MVs, benefiting potential large-scale production. Previous studies have suggested that OMVs carry various functional proteins on their surface, indicating their homologous bacterial homing function[33]. Some researchers have used OMVs to deliver traditional antibiotics into bacteria, effectively addressing antibiotic resistance[27, 31]. However, the efficiency of OMV-mediated plasmid internalization by bacteria is very limited, leading to insufficient antimicrobial effects (Fig. 1J, 3A, 3B). Given the success of cationic lipids in nucleic acid delivery, cationic lipid hybridization is employed to enhance the internalization efficiency of OMVs. Finally, under specific proportions, COMVs showed high plasmid delivery efficiency. Inhalation of PCOMVs eradicated the infected MRAB from the lung, thereby alleviating pulmonary inflammation and facilitating the recovery of respiratory and motor functions of the mice (Fig. 4).

The low yield of OMVs limits the clinical translation of COMVs, and the applicability of this strategy to other pathogen infections remains to be verified. Homologous bacterial membranes are utilized in the formulation (CMVs) to load CRISPR/Cas9 plasmids for combating wound infections caused by DRPA. The PCMVs significantly inhibited wound infection and promoted wound healing. Therefore, we develop a kind of novel hybrid vesicles composed of bacterial membrane derivatives and cationic lipids, capable of mediating efficient plasmid internalization at the sites of infection, offering a novel antimicrobial strategy based on CRISPR/Cas9. The bacterial membrane derivatives, including OMVs and MVs, significantly expand the sources of hybrid vesicles. The BCVs, as targeted bacterial delivery platform, are worth further exploration for various cargo delivery.

Although there have been concerns about the safety of CLs and OMVs,[52–54] this study does not show any noticeable side effects of PBCVs in the mice (Fig. 3). We attribute the high safety profile of PBCVs to the efficient delivery of CRISPR/Cas9 plasmids and the natural metabolism of mammal cells. Moreover, we validate the efficacy of PBCVs in two common pathogens-induced infection models, including lung infection and wounds infection. Provided that the contact between PBCVs and pathogens is ensured, PBCVs can work whether they are inhaled or loaded in a hydrogel, demonstrating the versatile potential of PBCVs in the treatment of various drug-resistant bacterial infections.

In summary, our study has demonstrated that the CRISPR/Cas9 plasmid-loaded BCVs are an effective strategy for the eradication of in vivo pathogens. To our knowledge, PBCVs represent the first CRISPR/Cas9 plasmid delivery platform targeting bacteria, and are validated in infection models caused by two common drug-resistant pathogens at different sites. The plasmids and carriers within this platform can be easily reprogrammed to accommodate the infections by unknown pathogens and genetic mutations. Their efficiency and versatility are impressive, and the accessibility and controllability of materials make clinical translation feasible.

Animals

Male BALB/c mice (19–21 g) were obtained from the SPF Biotechnology Co., Ltd. (Beijing, China). Mice were housed under the constant condition of humidity (50 ± 5%) and temperature (25 ± 1°C) with 12‒12 h light‒dark cycles. Food and water were available ad libitum.

Primers, bacteria and plasmids

Bacteria, plasmids, and primers used in this study are described in Table S1 and Table S2. MRAB and DRPA were used for modeling in this study. MRAB (ATCC17978) was purchased from American Type Culture Collection (ATCC, Rockefeller, USA), and DRPA was isolated from clinical samples and kindly given by Prof. Xie Fei. All bacteria were grown at 37°C in LB broth or on LB plates solidified with 2% agar. If necessary, an appropriate antibiotic (100 µg·mL^− 1 apramycin, or 10 µg·mL^− 1 tetracycline) was added to the solid or liquid media to screen engineered bacteria.

CRISPR/Cas9 plasmid construction and electrotransformation

The CRISPR/Cas9 plasmids, pABK against MRAB and pPAK against DRPA, were constructed by Gibson Assembly (NEB, Ipswich, USA), and the construction details can be found in the Supporting Information. Both pABK and pPAK can be digested by BsaI enzyme and then introduced into different sgRNA fragments through T4 ligase. Both pABKs and pPAK were verified by Sanger sequencing (GENEWIZ, Suzhou, China). Electrocompetent cells of MRAB and DRPA were prepared according to the previous reports[42, 49]. An aliquot (5 µL) of CRISPR/Cas9 plasmids was added to the electrocompetent cells for electrotransformation with 2.5 kV, 200 Ω, and 25 µF. The bacteria were recovered in the antibiotic-free LB media (1 mL) and shaken at 37°C for 1 h before spreading on the LB plates supplemented with appropriate antibiotics. The plates were incubated at 37°C overnight for colony imaging.

Bacterial membrane derivative isolation and formulation preparation

OMVs isolation was conducted as follows. A single colony of bacteria was cultured in LB media at 37°C and 220 rpm overnight until OD₆₀₀ reached 1.0. The bacterial suspension was centrifuged at 5000 ×g for 15 min and the supernatant was filtrated sequentially through 0.45-µm and 0.22-µm filters. The filtrate was concentrated with Millipor® centrifugal filters (MW cutoff, 100 kDa, Burlington, USA) and the concentrates underwent ultracentrifugation (Optima XPN-80, Beckman, Brea, USA) at 150000 ×g and 4°C for 70 min. The OMVs in the bottom were collected and washed twice with phosphate buffered solutions (PBS). The final precipitate was suspended with PBS, wherein the protein was determined with the BCA Protein Assay Kit and diluted to 0.25 mg·mL^− 1 proteins in the OMVs with PBS. The OMVs were stored at -80°C. The OMVs of MRAB harboring pABCas9 were isolated as the process, but they were named SOMVs and resuspended in LB media.

Membrane vesicles (MVs) isolation was conducted as follows. A single colony of DRPA was cultured in LB media at 37°C and 220 rpm until OD₆₀₀ reached 0.6. The bacterial suspension was centrifuged at 5000 ×g for 15 min and washed by PBS for three times, and then resuspended in PBS. The bacteria resuspension solution was placed on ice and underwent intermittent ultrasonication at 300 W for 100 times, followed by 10000 ×g centrifugation for 20 min and the supernatant was collected and filtered through 0.22-µm filters for three times. Finally, the MVs of DRPA were obtained and stored at -80°C.

Cationic liposomes (CLs) were prepared using the film method. Briefly, DDAB (30 mg), EPC (20 mg), and cholesterol (10 mg) were dissolved in absolute ethanol under ultrasound and transferred to a 250-mL round flask. The ethanol was removed by rotary evaporation under vacuum, resulting in a film at the bottom. The film was hydrated with PBS (12 mL) to obtain a suspension that was ultrasound-processed using a probe ultrasound instrument (JY92-11, Ningbo Xinzhi Biotechnology Co. Ltd.; Ningbo, China) with the following conditions: 300 W, ultrasound for 10 s, stop for 10 s, and repeating 45 times in an ice-cold bath. CLs were obtained after the final suspension was filtered through 0.22-µm filters.

Cationic lipid hybrid OMVs (COMVs) and cationic lipid hybrid MVs (CMVs) were prepared with the membrane fusion method. The CLs and the OMVs were mixed with the volume ratios of 1:0.25, 1:0.5, 1:0.75, 1:1, 1:1.25, and 1:1.5, respectively. The mixtures were ultrasound-processed as above and filtered through 0.22-µm filters to obtain COMVs. The CLs and the MVs were mixed with the volume ratios of 10:3 following ultrasound-processed as above and filtered through 0.22-µm filters to obtain CMVs. COMVs or CMVs (100 µL) were mixed with the CRISPR/Cas9 plasmid (5 µg) and electroporated with two pulses at 500 V, 200 Ω, and 25 µF. The mixture was 4-fold diluted with PBS. The pABK-loaded CLs, OMVs, and pPAK-loaded MVs were also prepared by the same method to generate pABK- loaded CLs (PCLs), POMVs, and PCMVs.

PCMV-loaded hydrogels were prepared for in vivo efficacy test. PVA-1788 was dissolved in water and stirred until completely dissolved, and then underwent repeated freeze-thawing to form a cross-linked structure for freeze-drying. The PCMV suspension with an adjusted concentration was added to the freeze-dried PVA gel to obtain the PCMV-loaded hydrogel for wound topical application.

Agarose gel electrophoresis

Several nucleic acid samples were prepared and the details were descripted in the Supporting Information. All the nucleic acid samples were detected by agarose gel electrophoresis at 110 V for 30 min, and the nucleic acid was stained with Goldview^® dye (Yeasen, Shanghai, China). The agarose gels were observed by an imager (OI600MF, BIO-OI, Guangzhou, China).

Western Blotting and SDS-PAGE

MRAB harboring pABCas9 was cultured until OD₆₀₀ reached 1, and then the culture was collected and centrifuged at 4°C and 5000 ×g for 15 min. The cell precipitates were lysed after 100°C heating for 10 min and proteins were determined using the BCA method. The samples were analyzed with the Western blotting (WB) method using the Cas9 antibodies (Bioss, Beijing, China). The MRAB bearing no pABCas9 was used as the control. CLs, OMVs, and COMVs were analyzed with the same WB method using Omp38 antibodies (AmyJet Scientific, Wuhan, China).

All samples were normalized to an equal protein concentration with the BCA method. An aliquot (20 µL) sample (including CLs, OMVs, COMVs, MVs, CMVs) was mixed with loading buffer and heated to 100°C for 10 min, and then the samples were loaded onto a 10% acrylamide gel and subjected to electrophoresis at 80 V for 1 h followed by 120 V for 2 h. The gels were stained with Coomassie blue (Epizyme, Shanghai, China) for photographing.

Fluorescence detection of formulations

The fusion of CLs and OMVs was verified with the Förster resonance energy transfer (FRET) method. CLs were labeled with NBD-PE (excitation/emission = 470/534 nm) and Rhod-PE (excitation/emission = 560/583 nm). The dye-labeled CLs were mixed with OMVs and processed as above. The fluorescent spectra (500─700 nm) were recorded at the excitation wavelength of 470 nm with a microplate reader (SPARK, Tecan, Männedorf, Switzerland). The fusion of CLs and the MVs derived from DRPA was verified by the same method. For visualization, DiD and DiO dyes were used to label CLs and OMVs, respectively. The two membranes were mixed and processed as above. Fluorescent images were captured using a confocal laser scanning microscope (CLSM 880, Carl Zeiss AG, Oberkochen, Germany).

Physical property determination of CLs, OMVs, COMVs, and PCOMVs

CLs, OMVs, COMVs, and PCOMVs were stained with uranyl acetate (1%) and observed under a transmission electron microscope (TEM, H-7650, Hitachi, Tokyo, Japan), respectively. The size distribution and zeta potentials of them were measured by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, Malvern, UK) at 25°C.

Co-localization of formulations and bacteria

CLs, OMVs, and COMVs were labeled with DiD (10^− 5 M) for 20 min, respectively, and washed with PBS thrice after centrifugation to remove excess DiD. The labeled CLs, OMVs, and COMVs were incubated with MRAB (10⁸ CFU·mL^− 1) at 37°C for 6 h. The MRAB was stained with 4,6’-diamidino-2-phenylindol (DAPI) (2 µg·mL^− 1) and images were captured using Zeiss CLSM 880.

Bacterial viability staining and scanning electron microscopy

PBS, SM, COMVs, pABK, and PCOMVs were co-incubated with MRAB as described above, respectively, and then the bacteria were collected and washed thrice with PBS; the bacteria were stained with SYTO-9 (5 µM) and propidium iodide (PI, 1 µg·mL^− 1) at 37°C for 15 min, following observation under a microscope (CKX53, Olympus Corporation, Tokyo, Japan). The bacteria in the precipitates were fixed with glutaraldehyde, dehydrated with alcohol, coated with gold, and observed under a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan) at 5 kV.

CCK-8 assay

Beas-2B cells were seeded in 96-well plates at a density of 2 × 10⁴ cells/well and incubated at 37°C for 24 h. A series of PCOMVs in BEpiCM media with pABK concentrations ranging from 0.063 ng·µL^− 1 to 0.250 ng·µL^− 1 were added into the wells, respectively, and the cells were sequentially incubated at 37°C for 24 h. The culture media containing 10% cell counting kit-8 (CCK-8) were added into each well followed by incubation for 1 h. The absorbance of wells was measured at 450 nm with a microplate reader and the cell viability was calculated as Eq. (1).

Cell viability (%) = (OD_sample−OD_blank)/(OD_control−OD_blank) × 100% Eq. (1)

Toxicity tests of pcomvs

The hemolysis assays of formulations were conducted with rat red blood cells (RBCs).[55] Briefly, a 2% RBC suspension was mixed with different concentrations of PCOMVs (3, 5, 7 ng·µL^− 1 pABK in PCOMVs), PBS (negative control), and 1% Triton X-100 solutions (positive control) at 37 °C for 2 h, respectively. After the suspensions were centrifuged at 2000 rpm and 4 °C for 10 min in a centrifuge (Sorvall™ Legend™ Micro 21R, Thermo Scientific, Waltham, USA), the OD of supernatants was measured using a microplate reader (Spark, TECAN, Männedorf, Switzerland) at 540 nm. The hemolysis rates were calculated according to Eq. (2).

Hemolysis rate (%) = (OD_sample–OD_negative)/(OD_positive–OD_negative) × 100% Eq. (2)

The in vivo toxicity of inhaled PCOMVs was assessed on mice after i.t. administration of PCOMVs referred our previous study.[56] Briefly, mice were anesthetized and fixed on a mouse operating table; under a laryngoscope, PCOMVs were sprayed into the lung of mice through the trachea via a thin tube. Mice were randomly divided into 4 groups, including the healthy group without any process, the other three groups with i.t. administration of 20 µL PCOMVs containing 3, 5, and 7 ng·µL^− 1 pABK for every mouse, respectively. After 24 h, whole blood was collected. Primary blood cells, including white blood cells (WBC), RBC, and platelets (PLT) were measured using a blood cell analyzer (DF52-Vet, Shenzhen Mindray Bio-Medical Electronics Co. Ltd.; Shenzhen, China). Glutamic-pyruvic transaminase (ALT) and creatinine (CRE) in the serum were also measured.

IVIS imaging

PCOMVs (1 mL) were mixed with a DiR solution (5 µM, 1 µL) in dimethyl sulphoxide (DMSO) at 37°C for 0.5 h to obtain DiR-labeled PCOMVs that were i.t. administered to mice. The mice were sacrificed after 6, 12, and 24 h, respectively, and their major organs, including the heart, liver, spleen, lung, and kidney, were excised and imaged (748/780 nm) using an in vivo imaging system (IVIS) (ABL-X5 Plus, Tanon, Shanghai, China).

Pneumonia model and treatment

Mouse bacterial pneumonia model was established as referred to our previous research.[57] Briefly, a dexamethasone acetate injection (5 mg·mL^− 1, 0.1 mL) was intraperitoneally (i.p.) injected into mice for 5 consecutive days (from day − 4 to day 0), and then anesthetized with isoflurane. MRAB (25 µL, 10⁶ CFU·mL^− 1) was i.t. administered to the mice. Six hour later, the mice were treated with formulations (20 µL) via i.t. dosing.

The mice were randomly divided in 5 groups with 5 mice in every group. The formulations included PBS, SM (32 µg·mL^− 1), COMVs, pABK (1 ng·µL^− 1), and PCOMVs (1 ng·µL^− 1 pABK). The healthy mice in the healthy group were only treated with i.t. PBS as above. The body weight of mice during the experiment was recorded. On day 4, the blood oxygen saturation (SpO₂) was examined with pulse oximeter (HS20A, Heal Force Bio-Meditech Holdings Limited, Shanghai, China). Their behavioral open-field test was performed to assess the exercise capacity of mice with each trial lasting 5 min. The total distance covered by each mouse was recorded. A meticulous cleaning of the apparatus was carried out between animals using a 20% (v/v) ethanol solution. The upper lobes of the right lung were processed as above. Tissues were deparaffinized and rehydrated followed by standard terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining and microscopic examination. Their pulmonary histopathology was conducted as above descripted.

Wound infection model and treatment

Mouse bacterial wound infection models were established as referred to our previous reports.[11, 58] The mice were anesthetized with i.p. injection of 1% pentobarbital sodium (50 mg·kg^− 1), and their dorsum hair was removed. The mice were scalded at 100 °C for 6 s using an instrument (YLS-5Q, Bio-will Co., Ltd., Shanghai, China) to form a circular, deep, partial-thickness burns wound with the diameter of ~ 1 cm on the mucous layer. A DRPA suspension (6 × 10⁸ CFU·mL^− 1, 20 µL) in PBS was evenly dripped onto the wound, and Tegaderm™ Film (1624W, 3M, Saint Paul, USA) was applied to the surface of the skin until yellow-green pus was produced.

The mice were randomly divided into 4 groups, including the uninfected control, infected control, kanamycin-loaded PVA hydrogel (Kan, 0.5 mg·kg^− 1), and PCMV-loaded PVA hydrogel (PCMV, pPAK 0.4 ng·µL^− 1) groups (n = 6). The wounds in the 2 treatment groups were covered with the hydrogels of 0.2 mL, respectively. Both Kan and PCMVs were refreshed on day 3 and 7. The wounds were photographed on day 0, 3, 7, 14, and 21, and the wound area was analyzed using ImageJ software (the National Institute of Health, USA). Wound recovery rates were calculated as Eq. (3). A₀ represents the wound area on day 0, and A_t indicates the wound area at measuring time points. All the mice were sacrificed on day 21, and the wound tissues were excised. The skin samples of 4 mice each group were used to determine inflammatory cytokines, and the other samples were used in histopathological study as previous described.

Wound recovery rate (%) = (A₀ − A_t)/A₀ × 100% Eq. (3)

Inflammatory cytokine assays

The upper lobes of the left lung or the skin tissues were homogenized with sterile saline to get 10% (w/w) homogenates. The homogenates were centrifuged at 5000 ×g for 10 min at 4°C, and the supernatant was collected for TNF-α and IL-6 detection following the ELISA kit operating manuals (Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). OD₄₅₀ was determined using a microplate reader (Spark, TECAN, Männedorf, Switzerland).

Data analysis

All data were presented as mean ± standard deviation (SD). The statistical analysis was performed with GraphPad Prism (Version 8.0, GraphPad Software, San Diego, USA). One-way analysis of variance (ANOVA) was employed to identify significant differences between different groups. Significance levels were denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

AR, antibiotic-resistant; AST, aminotransferase; BCVs, biomimetic cationic hybrid vesicles; CLs, cationic liposomes; CLSM, confocal laser scanning microscopy; CRE, creatinine; DiD, 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate; DiO, 3,3’-dioctadecyloxacarbocyanine; DSB, double-strand break; EcN, E. coli Nissle 1917; ELISA, Enzyme-linked immunosorbent assay; FRET, Förster resonance energy transfer; GHT, gene horizontal transfer; H&E, hematoxylin and eosin staining; HTH, helix-turn-helix; LB, Luria-Bertani; LNPs, lipid nanoparticles ; MRAB, multidrug-resistant Acinetobacter baumannii; OMVs, outer membrane vesicles; pABCas9, CRISPR/Cas9-based genome-editing plasmids; PCOMVs, pABK-loaded cationic lipid hybrid OMVs; PCMVs, pPAK-loaded cationic hybrid membrane vesicles; PDI, small polydispersity index; RHS, rearrangement hotspot; RNP, ribonucleic protein complex; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; sgRNA, single guide RNA; SM, streptomycin; SpO2, blood oxygen saturation; TA, toxin-antitoxin; TEM, transmission electron microscope; TNF-α, tumor necrosis factor-α; TUNEL, terminal-deoxynucleotidyl transferase mediated nick end labeling; WB, Western blotting; WBC, white blood cells.

Acknowledgment

This work was supported by the Beijing Natural Science Foundation of China (7232261).

Author Contributions

X. Jia, B. Yuan, and Y. Jin designed the research, conducted experiments, and performed data analysis. K. Wang, D. Ling, and M. Wei participated in parts of the formulations preparation and in vivo antibacterial experiments. Y. Hu and W. Guo participated in the in vitro antibacterial experiments and in vivo biocompatibility experiments. Z. Chen participated in part of CRISPR/Cas9 plasmid construction experiments. L. Du directed parts of the experiments. X. Jia and B. Yuan wrote the manuscript. B. Yuan and Y. Jin revised the manuscript. All of the authors have read and approved the final manuscript. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Data availability

All data generated and analyzed during this research are included in this published article.

Ethics approval and consent to participate

The animal experimental operation was performed at the Beijing institute of radiation medicine. All animal experiments had been reviewed and approved by the Laboratory Animal Ethics Committee of Beijing institute of radiation medicine (Beijing, China) (Approval No. IACUC-DWZX-2022-752)

Competing interests

The authors declare no competing financial interest.

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Additional file Materials and Reagents; Construction of CRISPR/Cas9 Plasmids; Sample Preparation for Agarose Gel Electrophoresis; Colony Photograph; Proteome Detection of OMVs; Real-time Fluorescence Quantitative PCR; Histopathologic Examination; Figs. S1−S6; Tables S1−S3 (PDF)

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Gene editing tool-loaded biomimetic cationic vesicles with highly efficient bacterial internalization for in vivo eradication of pathogens

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Version 1

Abstract

Figures

Introduction

Results

Discussion

Methods

Animals

Primers, bacteria and plasmids

CRISPR/Cas9 plasmid construction and electrotransformation

Bacterial membrane derivative isolation and formulation preparation

Agarose gel electrophoresis

Western Blotting and SDS-PAGE

Fluorescence detection of formulations

Physical property determination of CLs, OMVs, COMVs, and PCOMVs

Co-localization of formulations and bacteria

Bacterial viability staining and scanning electron microscopy

CCK-8 assay

Toxicity tests of pcomvs

IVIS imaging

Pneumonia model and treatment

Wound infection model and treatment

Inflammatory cytokine assays

Data analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1