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Title: TPGS-based and S-thanatin functionalized nanorods for overcoming drug resistance in Klebsiella pneumonia

Journal：《Nature Communications》

Link：https://www.nature.com/articles/s41467-022-31500-3

Repoter：Kangyi, master of 2020

Tigecycline is regarded as the last line of defense to combat multidrug-resistant Klebsiella pneumoniae. However, increasing utilization has led to rising drug resistance and treatment failure. Here, the reporter design a D-alpha tocopheryl polyethylene glycol succinate-modified and S-thanatin peptide-functionalized nanorods based on calcium phosphate nanoparticles for tigecycline delivery and pneumonia therapy caused by tigecycline-resistant Klebsiella pneumoniae. After incubation with bacteria, the fabricated nanorods can enhance tigecycline accumulation in bacteria via the inhibitory effect on efflux pumps exerted by D-alpha tocopheryl polyethylene glycol succinate and the targeting capacity of S-thanatin to bacteria. The synergistic antibacterial capacity between S-thanatin and tigecycline further enhances the antibacterial activity of nanorods, thus overcoming the tigecycline resistance of Klebsiella pneumoniae. After intravenous injection, nanorods significantly reduces the counts of white blood cells and neutrophils, decreases bacterial colonies, and ameliorates neutrophil infiltration events, thereby largely increasing the survival rate of mice with pneumonia. These findings may provide a therapeutic strategy for infections caused by drug-resistant bacteria.

Klebsiella pneumonia (KPN) is regarded as one of the most serious nosocomial pathogens threatening public health. As a gram-negative bacterium, KPN can induce multiple infections, such as pneumonia, liver abscesses, urinary tract infections, meningitis, and bacteremia in hospitalized patients with insufficient immune systems. In recent years, KPN strains with high virulence and mucus phenotypes have gradually become an important pathogen clinically that can cause serious infections in healthy people and significantly threaten their health. Recently, nanodrug delivery systems (DDSs) have emerged as novel therapeutic means for deadly infections. Combination therapy of nanomaterials and antibiotics might contribute to a better therapeutic index. Numerous studies have constructed DDSs based on Ag, Au, Cu, Fe, Ti, and mesoporous silica nanoparticles to treat infectious diseases17, and their clinical use in vivo is hindered by safety. More efficient nanomaterial-based delivery strategies, such as pH-triggered, enzyme-sensitive, and bacterial toxin-triggered DDSs, could potentially allow the release of antibiotics in a spatiotemporally controlled fashion and have gain attention worldwide. However, this process is complex and sophisticated, and the released antibiotics lack specificity for bacteria. Therefore, a safe DDS with targeting efficacy to bacteria might be an effective strategy for the treatment of infection caused by TRKP.

Fig. 1: Preparation and characterization of TTCT.

The crystal structure and the chemical composition of the nanorods were confirmed by comparing the X-ray diffraction (XRD) spectrum of nanorods with the typical crystal diffraction peaks of hydroxylapatite crystals (Fig. 1A). Afterwards, TIG was encapsulated into the Ts-TPGS/Cap and TPGS/Cap nanorods by the dialysis method. The particle size of the formulations was maintained at ∼25 nm and showed a narrow size distribution (Fig. 1B). All the formulations exhibited monomodal zeta potential distributions with a negative charge (Fig. 1C). However, the zeta potential of the nanorods decreased from −18.56 ± 1.66 mV to −14.99 ± 1.66 mV after Ts modification, possibly because some hydroxyl groups of TPGS were replaced by Ts. Moreover, TIG loading further decreased the zeta potential, which was possibly due to the electrostatic interaction between the drug and nanorods. Transmission electron microscopy (TEM) images showed that all of the formulations had uniform and rod-like morphologies (Fig. 1D). The stability of the formulations was explored as a function of time by dynamic light scattering (DLS). It was observed that the prepared nanorods were stable with small changes in particle size and PDI after storage for 20 days (Fig. 1E, F). Furthermore, the particle size and PDI of TCT and TTCT remained almost constant in 10% fetal bovine serum (FBS)-containing PBS within 24 h (as shown in Fig. 1G), indicating that TCT and TTCT would not disassemble in circulation.

Fig. 2: Evaluation of the antibacterial activity of TTCT towards KPN and TRKP in vitro.

As shown in Fig. 2A, the minimal inhibitory concentrations (MICs) of TIG towards KPN and TRKP were 1 µg/mL and 4 µg/mL, respectively, indicating that TRKP was not susceptible to TIG. Antibacterial capacity of TTCT was the best as evidenced by its lowest MIC value. It was possibly related to the combinational effect between TIG and Ts peptide. For TRKP, the MIC values of TIG and TCT were 4 µg/mL and 2 µg/mL, respectively, indicating that the TPGS-based nanodrug delivery system could ameliorate the TIG resistance of TRKP. It is worth mentioning that the MIC value of TTCT was 1 µg/mL, which was equal to that of free TIG against KPN, suggesting that TTCT was effective in overcoming drug resistance. TIG, TCT, and Ts-TPS/Cap could not prevent the growth of bacteria (Fig. 2B, C). As a whole, TTCT demonstrated better antibacterial activity than TIG and TCT in both KPN and TRKP.

As displayed in Fig. 2D, The bacterial morphology was obviously changed after treatment with various preparations. Ts-TPGS/Cap nanorods led to noticeable holes in their cell walls in both KPN and TRKP, which was possibly attributed to the antibacterial mechanisms of the Ts peptide. The bacterial structures were massively destroyed by TTCT in both the KPN and TRKP groups, further confirming the superior antibacterial effect of TTCT. In conclusion, all of the results shown in Fig. 2 revealed that TTCT was a superior preparation with excellent antibacterial function against both KPN and TRKP when compared with TIG, TCT, and Ts-TPGS/Cap nanorods.

Fig. 3: Investigation of the mechanisms by which TTCT overcomes drug resistance.

As shown in Fig. 3A, both NT-TPGS/Cap and Ts-TPGS/Cap nanorods displayed a gradual increase in the intra-bacteria fluorescent signal with a prolonged incubation time. Ts-TPGS/Cap nanorods displayed stronger bacterial internalization capacity than NT-TPGS/Cap nanorods in both KPN and TRKP at 2 and 6 h. The results were consistent with those obtained by flow cytometry (Fig. 3B, C). The fluorescent signals in KPN after incubation with Ts-TPGS/Cap nanorods were ~2-fold greater than those of NT-TPGS/Cap nanorods at both 2 h and 6 h (Fig. 3D). For TRKP, similar results were observed, suggesting that Ts-TPGS/Cap nanorods exhibited better bacterial internalization activity, which was possibly attributed to the targeting moiety Ts peptide.

Fig. 4: Evaluation of the biodistribution and targeting efficacy of Ts-TPGS/Cap in vivo.

Using tracheal injection of KPN or TRKP bacteria into the lungs of healthy mice, the author created acute pneumonia mouse models and investigated the in vivo targeting efficiency of Ts-TPGS/Cap nanorods. As shown in Fig. 4A, B, Ts-TPGS/Cap nanorods displayed similar bio-distribution characteristics between pneumonia mice infected by KPN and TRKP. In pneumonia mice caused by both KPN and TRKP, the quantitative signals of fluorescence (expressed by fluorescence intensity per gram of tissue, ID/g) of Ts-TPGS/Cap nanorods in the lung were ~1.5-fold and ~2.5-fold higher than those of TPGS/Cap at 5 h and 24 h, respectively (Fig. 4C, D). Notably, the Ts-TPGS/Cap nanorods were primarily distributed to the liver, lung, and kidney of the pneumonia mice at 24 h. The ID/g of the lungs were higher than those of the liver and kidneys.

Fig. 5: Investigation of the anti-infective efficacy of TTCT in vivo.

As shown in Fig. 5A, B, the pneumonia mice infected by TRKP survived 33.3% at 5 days, compared with the KPN-infected mice at 16.7%. KPN-infected mice receiving TIG and TCT treatment (TIG dose: 15 mg/kg) survived 66.7% and 83.3%, respectively, indicating that encapsulating TIG in nanorods boosted its antibacterial activity. Moreover, Ts-TPGS/Cap nanorod treatment also enhanced the survival rate of KPN-infected mice.The results showed that bacterial infection caused an obvious increase of WBCs and neutrophils (Fig. 5C–D). As shown in Fig. 5E, the total cells in BALF excessively increased in KPN- and TRKP-infected mice. TTCT displayed the highest reduction among these groups. The percentages of neutrophils in total cells were determined by flow cytometry. It was observed that neutrophils accounted for ~85% and ~75% of the total cells in KPN-infected and TRKP-infected mice, respectively (Fig. 5F). The percentages of neutrophils were apparently decreased after administration of TIG, TCT, TTCT, and Ts-TPGS/Cap nanorods, clearly indicating that pulmonary neutrophil infiltration was ameliorated. It is worth mentioning that TTCT treatment still exhibited the best effects among the treatments. Overall, TTCT therapy consistently displayed the best therapeutic effect, with lower counts of WBCs and neutrophils in blood, lower pulmonary neutrophil infiltration, and higher reductions in CRP levels and bacterial colonies.

In summary, a TPGS-based and Ts-modified nanodrug delivery system with LPS targeting and synergistic antibacterial activity was designed for TIG delivery and therapy of acute pneumonia caused by MDR bacteria. The prepared Ts-TPGS/Cap nanorods could effectively encapsulate TIG and achieve sustained drug release. Through the binding between Ts and LPS, Ts-TPGS/Cap exhibited targeting and enhanced accumulation in both KPN and TRKP. TPGS could exert its inhibitory capacity on the activity of efflux pumps and the expression of acrA, acrB and ramA in TRKP. In this way, the TIG concentration inside bacteria was significantly higher in the TTCT group than in the TIG and TTCT groups. The synergistic antibacterial capacity between Ts and TIG further enhanced the antibacterial activity TTCT, thus overcoming the drug resistance of TRKP.

In mice with pneumonia, TTCT administration could significantly reduce the WBC and neutrophil counts in blood samples and decrease the total cell and CRP levels in BALF. Moreover, TTCT was capable of ameliorating neutrophil infiltration in the lungs and reducing bacterial colonies from BALF, thus apparently increasing the survival rates of mice with pneumonia caused by both KPN and TRKP. In addition, TTCT led to little toxicity to the liver. These results indicated that TTCT could overcome the drug resistance of TRKP.