Optimization of Dendritic Polypeptide Delivery System for Antisense Antibacterial Agents Targeting ftsZ.

There is an urgent requirement for a novel treatment strategy for drug-resistant Staphylococcus aureus (S. aureus) infection. Antisense antimicrobials are promising antimicrobials, and efficient drug delivery systems are necessary for the further development of antisense antimicrobials. To develop new antisense drugs and further improve delivery efficiency and safety, we designed and screened new antisense sequences and optimized dendritic polypeptide nanoparticles (DP-AD) discovered in previous studies. The N/P ratio is optimized from 8:1 to 6:1, and the positive charge number of the optimized DP-AD is studied comprehensively. The results show that the N/P ratio and positive charge number have no significant effect on the particle size distribution and transport efficiency of DP-AD. Reducing the N/P ratio can significantly reduce the cytotoxicity of DP-AD, but it does not affect its delivery efficiency and antibacterial activity. However, in drug-resistant strains, the antibacterial activity of DP-AD76:1 with 10 positive charges is higher than that of DP-AD86:1 with 8 positive charges. Our research discovered a novel ASOs targeting ftsZ and concluded that DP-AD76:1 with 10 positive charges was the optimal choice at the current stage, which provided a promising strategy for the treatment of drug-resistant S. aureus.

Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs).

Introduction: Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. Methods: HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. Results: In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. Discussion: iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

Pharmacological Characters and Toxicity Evaluation of Coumarin Derivative LP4C as Lead Compound against Biofilm Formation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa-induced biofilm infection is difficult to treat and poses a significant threat to public health. Our previous study found a new coumarin derivative LP4C which exerted potent in vitro and in vivo anti-biofilm activity against Pseudomonas aeruginosa; however, the underlying molecular mechanism and drug-likeness of LP4C is unclear. In this study, we confirmed that LP4C could inhibit the biofilm in dose-dependent manner without bactericidal activity. The transcriptomic profiling and RT-PCR result revealed that bacterial pyrimidine mediated the inhibitory activity of LP4C. The cell viability was not affected in LP4C treatment groups with the concentration under 200 µg/mL, and no death or toxicity sign was observed in mice treated by 20, 40 and 80 mg/kg LP4C during the three-week test period. Ames test presented that LP4C had no effect on the bacterial reverse mutation. In additional, pharmacokinetic results showed that LP4C was likely to have the orally bioavailable properties. Our data indicate that LP4C is a possible lead compound for the development of new anti-biofilm infection agents against Pseudomonas aeruginosa.

Evaluation of Antibacterial Activity of Thiourea Derivative TD4 against Methicillin-Resistant Staphylococcus aureus via Destroying the NAD+/NADH Homeostasis.

To develop effective agents to combat bacterial infections, a series of thiourea derivatives (TDs) were prepared and their antibacterial activities were evaluated. Our results showed that TD4 exerted the most potent antibacterial activity against a number of Staphylococcus aureus (S. aureus), including the methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis and Enterococcus faecalis strains, with the minimum inhibitory concentration (MIC) at 2-16 µg/mL. It inhibited the MRSA growth curve in a dose-dependent manner and reduced the colony formation unit in 4× MIC within 4 h. Under the transmission electron microscope, TD4 disrupted the integrity of MRSA cell wall. Additionally, it reduced the infective lesion size and the bacterial number in the MRSA-induced infection tissue of mice and possessed a good drug likeness according to the Lipinski rules. Our results indicate that TD4 is a potential lead compound for the development of novel antibacterial agent against the MRSA infection.

Antibacterial Activity of Squaric Amide Derivative SA2 against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA)-caused infection is difficult to treat because of its resistance to commonly used antibiotic, and poses a significant threat to public health. To develop new anti-bacterial agents to combat MRSA-induced infections, we synthesized novel squaric amide derivatives and evaluated their anti-bacterial activity by determining the minimum inhibitory concentration (MIC). Additionally, inhibitory activity of squaric amide 2 (SA2) was measured using the growth curve assay, time-kill assay, and an MRSA-induced skin infection animal model. A scanning electron microscope and transmission electron microscope were utilized to observe the effect of SA2 on the morphologies of MRSA. Transcriptome analysis and real-time PCR were used to test the possible anti-bacterial mechanism of SA2. The results showed that SA2 exerted bactericidal activity against a number of MRSA strains with an MIC at 4-8 µg/mL. It also inhibited the bacterial growth curve of MRSA strains in a dose-dependent manner, and reduced the colony formation unit in 4× MIC within 4-8 h. The infective lesion size and the bacterial number in the MRSA-induced infection tissue of mice were reduced significantly within 7 days after SA2 treatment. Moreover, SA2 disrupted the bacterial membrane and alanine dehydrogenase-dependent NAD+/NADH homeostasis. Our data indicates that SA2 is a possible lead compound for the development of new anti-bacterial agents against MRSA infection.