Antisense Peptide Research Articles

Cell-penetrating anti-sense peptide nucleic acids targeting sulfatase 2 inhibit adipogenesis in human mesenchymal stem cells

Targeting the genes regulate the lineage commitment of human mesenchymal stem cells (hMSCs) to adipocytes provides a promising strategy for addressing obesity. In this study, we investigated the therapeutic potential of cell-penetrating anti-sense peptide nucleic acids (PNAs) designed to enhance solubility and hybridization properties, specifically targeting sulfatase 2 (SULF2), a potential reciprocal regulator of adipocyte and osteoblast differentiation in hMSCs. Cell-penetrating modified PNA oligomers effectively inhibit SULF2 gene transcription, leading to significant reductions in adiponectin protein synthesis and intracellular lipid droplet accumulation during adipogenesis in human bone marrow-derived MSCs (hBM-MSCs). Notably, PNA oligomer compound 5 exhibited the most potent anti-adipogenic activity, with an IC50 value of 0.28 μM. These findings show the potential of SULF2-targeting cell-penetrating PNA oligomers as novel therapeutic agents for obesity-related metabolic diseases.

Engineering an oncolytic adenoviral platform for precise delivery of antisense peptide nucleic acid to modulate PD-L1 overexpression in cancer cells

Cancer immunotherapy is focused on stimulating the immune system against cancer cells by exploiting immune checkpoint mechanisms. PD-1/PD-L1 is one of the most known immune checkpoints due to the widespread upregulation of the Programmed Death Ligand 1 (PD-L1) transmembrane protein in cancer tissues. Accordingly, taking advantage of the ability of oncolytic adenoviruses (OAd) to specifically infect and kill tumor cells over healthy ones, here, we developed a targeted delivery platform based on OAd to selectively deliver in cancer cells an antisense peptide nucleic acid (PNA) targeting the PD-L1 mRNA. The antisense PNA was modified with a six-lysine tail to improve water solubility and binding affinity to the polyanionic surface of the OAd carrier. Dynamic light scattering measurements confirmed the effective binding of the PNA cargo to OAd. Flow cytometry analysis evaluated the impact on PD-L1 protein expression in A549 and SK-OV3 cancer cell lines post-incubation with the OAd/PNA system. Statistically significant PD-L1 downregulation was observed in SK-OV3 cells treated with OAd-delivered PNA, surpassing the effect of free PNA. Confocal microscopy showed the cytoplasmic localization of OAd-delivered PNA, supporting the proposed antisense mechanism for PD-L1 downregulation. This targeted delivery system holds potential for enhancing the effectiveness of cancer immunotherapy.

Efficient and selective kidney targeting by chemically modified carbohydrate conjugates

We investigated a renal tubule-targeting carbohydrate (RENTAC) that can selectively deliver small-molecule and nucleic acid analogs to the proximal convoluted tubules of the kidney following systemic delivery in mice. We comprehensively evaluated anti-miR-21-peptide nucleic acid-RENTAC, and fluorophore-RENTAC conjugates in cell culture and invivo. We established that RENTAC conjugates showed megalin- and cubilin-dependent endocytic uptake in the immortalized kidney cell line. Invivo biodistribution studies confirmed the retention of RENTAC conjugates in the kidneys for several days compared with other organs. Immunofluorescence staining confirmed the selective distribution of the RENTAC conjugates in proximal convoluted tubules. We further demonstrated proximal convoluted tubule targeting features of RENTAC conjugates in a folic acid-induced kidney fibrosis mouse model. As a biological readout, we targeted miR-33 using antisense peptide nucleic acid (PNA) 33-RENTAC conjugates in the fibrotic kidney disease model. The targeted delivery of PNA 33-RENTAC resulted in slower fibrosis progression and decreased collagen deposition. We also confirmed that the RENTAC ligand did not exert any adverse reactions. Thus, we established that the RENTAC ligand can be used for broad clinical applications targeting the kidneys selectively.

Targeting synthesis of the Chromosome Replication Initiator Protein DnaA by antisense PNA-peptide conjugates in Escherichia coli

Initiation of chromosome replication is an essential stage of the bacterial cell cycle that is controlled by the DnaA protein. With the aim of developing novel antimicrobials, we have targeted the initiation of DNA replication, using antisense peptide nucleic acids (PNAs), directed against DnaA translation. A series of anti-DnaA PNA conjugated to lysine-rich bacterial penetrating peptides (PNA-BPPs) were designed to block DnaA translation. These anti-DnaA PNA-BPPs inhibited growth of wild-type Escherichia coli cells at low micromolar concentrations, and cells exposed to anti-DnaA PNA-BPPs exhibited characteristic hallmarks of chromosome replication inhibition. These results present one of very few compounds successfully targeting initiation of chromosome replication, an essential step in the bacterial cell cycle.

Overcoming MTDH and MTDH-SND1 complex: driver and potential therapeutic target of cancer

<p class="MsoNormal" style="margin-top: 12pt; text-align: justify;"><span style="font-family: arial, helvetica, sans-serif;"><span lang="EN-US">Metadherin (MTDH), also known as LYRIC or AEG-1, is an oncogene that enhances tumor progression, metastasis, drug resistance, and immune escape in various cancers by modulating multiple oncogenic pathways, including </span><span lang="EN-US">NF-κB, PI3K/AKT, Wnt/β-catenin, MAPK, and AMPK</span><span lang="EN-US">. Due to the unknown of the complete structure of MTDH, the deep mechanisms of MTDH and selective inhibitors targeting MTDH remain to be explored. The Protein-Protein interaction (PPI) with the Staphylococcal nuclease domain containing 1 (SND1) is a crucial mechanism underlying the function of MTDH. Current studies have demonstrated that inhibitors, including antisense oligonucleotides, peptides, and small molecules targeting MTDH or MTDH-SND1 interactions, provide novel strategies to inhibit the oncogenetic effects of MTDH. This review summarizes and discusses the structure, function, and regulation of MTDH in cancers, providing the potential therapeutic perspectives of MTDH or MTDH-SND1 PPI for drug discovery.</span></span></p>

Subtractive modification of bacterial consortium using antisense peptide nucleic acids.

Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of Escherichia coli and Pseudomonas putida in an artificial bacterial consortium comprising E. coli, P. putida, Pseudomonas fluorescens, and Lactiplantibacillus plantarum. Moreover, the growth inhibition of P. putida promoted the growth of P. fluorescens and inhibited the growth of L. plantarum. These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome.

Investigating the Effectiveness of mqsR-Peptide Nucleic Acid as a Novel Solution for the Eradication of Persister Cells in Clinical Isolates of Escherichia coli.

Bacterial persisters are non- or slow-growing phenotypic variants that may be responsible for recalcitrance and relapse of persistent infections and antibiotic failure. In Escherichia coli, mqsRA is a well-known type II toxin-antitoxin system associated with persister cell formation. This study aimed to investigate the efficiency of an antisense peptide nucleic acid (PNA) targeting mqsRA in eliminating E. coli persisters. The study included 600 non-duplicated urine samples from adult patients with suspected urinary tract infections. The isolates were subjected to antimicrobial susceptibility testing and bacterial persister cells assay. The presence of mqsRA in the isolates was evaluated by polymerase chain reaction. Finally, expression of the mqsR and mqsA genes was assessed after exposure to normal conditions, stress, and different concentrations of mqsR-PNA (1 - 35 μM). The mqsR gene was significantly overexpressed under stress conditions, which was compensated by the PNA treatment. Complete inhibition of E. coli persister cells was achieved after overnight treatment with the anti-mqsR-PNA at concentrations ≥ 15 μM. The growth of E. coli persister cells can be inhibited by the anti-mqsR-PNA. Further studies are required to evaluate the effectiveness of this antisense PNA in both preclinical and clinical settings.

Novel Drug to Target HK2 Overcomes Therapeutic Resistance in Preclinical B-Cell Lymphoma Models

Background: The Warburg's effect is a hallmark in cancer. Hexokinase II (HK2) is a glycolic kinase to phosphorylate glucose to glucose-6-phosphate, which can be utilized as the energy source for cells. HK2 is predominantly overexpressed in cancer cells and has been associated with poor clinical outcomes in cancer patients. Previously, we demonstrated that HK2 mRNA and protein levels were increased in diffuse large B-cell lymphoma patients, and were significantly associated with inferior progression free survival (PFS) and overall survival (OS). In our rituximab and chemotherapy-resistant cell lines (TRCL), we demonstrated an upregulation of HK2 expression level is association with voltage-dependent anion channel (VDAC) resulting in a) repress mitochondrial potential and b) global therapy resistance. Knocking out HK2 overcame the chemo-resistance phenotype observed in TRCL. Here, we designed a novel specific inhibitor of HK2 and tested its activity in lymphoma pre-clinical models. Material and Methods: A HK2 specific inhibitor (HK2-001) was designed and generated according to antisense peptide technology, CABS-docking system and ROSWTTA analyzing. A panel of B-cell lymphoma cell lines including rituximab/chemo-sensitive and resistant cell lines (Raji, Raji 4RH, RL and RL 4RH), diffuse large B-cell lymphoma (DLBCL) (DHL4, DOHH2, TMD8 and U2932) and mantle cell lymphoma (MCL) (HBL2, Granta, Mino and Z138) were used. In addition, primary tumor cells isolated from chronic lymphocytic lymphoma (CLL) or other B-cell lymphoma patients were used. Briefly, cell lines or primary tumor cells were exposed to escalating doses of HK2-001 alone or in combination with different chemotherapy agents (doxorubicin, dexamethasone), targeted agents (i.e. burton tyrosine kinase inhibitors, BH3 mimetics) or monoclonal anti-CD20 antibodies. Cell viability was evaluated using the Presto Blue assay in experiments testing pharmacological interactions between HK2-001 and chemotherapy or targeted agents. All experiments were performed in triplicates in three separate experiments. The coefficient of synergy was calculated using CalcuSyn. Results: In vitro exposure of lymphoma cell lines to HK2-001 induced cell death in a panel of B-cell lymphoma cell lines representing rituximab/chemo-sensitive and resistant lymphoma. In addition, synergistic activity was observed between HK2-001 and doxorubicin, ibrutinib, and Venetoclax. These effects were also observed in primary tumor cells isolated from B-cell lymphoma patients. In addition, HK2-001 potentiated the anti-tumor activity of Rituximab and Obinutuzumab ex vivo. Conclusion: We developed a novel peptide targeting HK2. Pre-clinical studies suggested anti-tumor activity as a single agent. More importantly, HK2-001 enhanced the anti-tumor activity of chemotherapy agents, BTK inhibitors, BH3 mimetics and monoclonal antibodies targeting CD20. HK2-001 is a promising novel therapeutic strategy for against B-cell lymphoma, especially in refractory or relapsed patients. Future studies aimed to evaluate the effects of HKII-001 drug combinations in lymphoma animal models are planned and to evaluate its effects on Chimeric antigen receptor T-cells (CART) or bi-specific antibodies.

PCSK9 Targeting in the Management of Hypercholesterolaemia

It has long been established that elevated plasma concentrations of low-density lipoprotein cholesterol (LDL-C) are among the prominent contributors leading to the development of atherosclerotic plaques and, ultimately, cardiovascular disease. In the current era of optimal risk factor modification, proprotein convertase subtilisin/kexin type 9 (PCSK9) targeting has emerged as a potent therapeutic approach in the management of hypercholesterolaemia, addressing several substantial, unmet clinical needs. PCSK9 monoclonal antibodies, evolocumab and alirocumab, as well as inclisiran, which is a small interfering RNA that halts the transcription of PCSK9 mRNA, are being increasingly used in current clinical practice, as they induce intensive LDL-C reductions without any significant safety and tolerability concerns. Based on the success of these agents, the concept of PCSK9 targeting with novel agents with enhanced biological properties, or via different administration routes, has received considerable attention. In this regard, numerous antisense oligonucleotides, peptides, and proteins are currently under evaluation in randomised controlled trials, yielding propitious results up to date; they may enter clinical use in the coming years. Meanwhile, a PCSK9 vaccine, as well as genome editing via clustered regularly interspaced palindromic repeats/Cas9, hold great promise to eradicate LDL-C altogether as a cardiovascular risk factor. This review aims to present and discuss the current clinical and scientific evidence pertaining to the field of medications that exert their biological effect by targeting PCSK9, which are either in use in clinical practice, or are currently being evaluated in pre-clinical or clinical studies, and may prove beneficial in the near future.

Pyrenebutyrate Enhances the Antibacterial Effect of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pyogenes.

Antisense peptide nucleic acids (PNAs) inhibit bacterial growth in several infection models. Since PNAs are not spontaneously taken up by bacteria, they are often conjugated to carriers such as cell-penetrating peptides (CPPs) in order to improve translocation. Hydrophobic counterions such as pyrenebutyrate (PyB) have been shown to facilitate translocation of peptides over natural and artificial membranes. In this study, the capability of PyB to support translocation of CPP-coupled antisense PNAs into bacteria was investigated in Streptococcus pyogenes and Streptococcus pneumoniae. PyB enhanced the antimicrobial activity of CPP-conjugated antisense PNAs in S. pyogenes. The most significant effect of PyB was observed in combination with K8-conjugated anti-gyrA PNAs. In contrast, no significant effect of PyB on the antimicrobial activity of CPP-conjugated PNAs in S. pneumoniae was detected. Uptake of K8-FITC into S. pyogenes, Escherichia coli, and Klebsiella pneumoniae could be improved by pre-incubation with PyB, indicating that PyB supports the antimicrobial effect of CPP-antisense PNAs in S. pyogenes by facilitating the translocation of peptides across the bacterial membrane.

Combined mutations of the penA with ftsX genes contribute to ceftriaxone resistance in Neisseria gonorrhoeae and peptide nucleic acids targeting these genes reverse ceftriaxone resistance

To investigate the gene mutations associated with ceftriaxone (CRO) resistance among gonococcal isolates, and to determine the effects of the mutated genes on CRO minimum inhibitory concentrations (MICs) with transformation assays and antisense peptide nucleic acids (asPNAs). Ceftriaxone-resistant (CROR) and ceftriaxone-susceptible (CROS) isolates were identified using EUCAST and paired according to similarity in their MICs to other antimicrobials. The two groups of gonococci were sequenced and analysed. Mutated genes that showed a statistical difference between the two groups were transformed into gonococcal reference strains to determine their functions. AsPNAs were designed and transformed into the former transformant to further confirm the effects of the mutated genes. Twenty-two paired CROR and CROS isolates were obtained. The incidence of the penA-A501T and penA-G542S mutations individually, as well as combined mutations (penA-A501T and ftsX-R251H, penA-G542S and ftsX R251H), was statistically different between the two groups. The MIC of ATCC43069 (A43) increased 2 times following transformation with penA-A501T, and the MICs of A43 and ATCC49226 (A49) increased 32 times and 2 times following transformation with penA-A501T and ftsX-R251H, respectively. Antisense PNA-P3 reduced the MIC of the A43 transformant most significantly when transformed individually. PNA-P3 and PNA-F1 (asPNAs of the penA and ftsX) restored CRO susceptibility. PenA-A501T and penA-G542S mutations are important in CRO resistance among gonococci isolates. The ftsX-R251H mutation is also related to CRO resistance, and combined mutations of ftsX-R251H and penA-A501T comediate a significant reduction in CRO susceptibility. The combined application of PNA-P3 and PNA-F1 could effectively reverse the resistance to CRO in N. gonorrhoeae.

Antisense Oligonucleotides Selectively Enter Human-Derived Antibiotic-Resistant Bacteria through Bacterial-Specific ATP-Binding Cassette Sugar Transporter.

Current vehicles used to deliver antisense oligonucleotides (ASOs) cannot distinguish between bacterial and mammalian cells, greatly hindering the preclinical or clinical treatment of bacterial infections, especially those caused by antibiotic-resistant bacteria. Herein, bacteria-specific ATP-binding cassette (ABC) sugar transporters are leveraged to selectively internalize ASOs by hitchhiking them on α (1-4)-glucosidically linked glucose polymers. Compared with their cell-penetrating peptide counterparts, which are non-specifically engulfed by mammalian and bacterial cells, the presented therapeutics consisting of glucose polymer and antisense peptide nucleic-acid-modified nanoparticles are selectively internalized into the human-derived multidrug-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus, and they display a much higher uptake rate (i.e., 51.6%). The developed strategy allows specific and efficient killing of nearly 100% of the antibiotic-resistant bacteria. Its significant curative efficacy against bacterial keratitis and endophthalmitis is also shown. This strategy will expand the focus of antisense technology to include bacterial cells other than mammalian cells.

High-order framework nucleic acid for targeted-delivery of antisense peptide nucleic acids to overcome drug resistance.

A sophisticated high-order framework nucleic acid (FNA) was engineered for the targeted delivery and responsive release of environment tolerant antisense peptide nucleic acids (asPNAs). The dendritic FNA-asPNAs system was constructed via simple one-pot modular assembly and demonstrated a good synergistic effect with chemotherapy on drug resistant cancer cells.

DNA Nanoribbon for Efficient Anti-miRNA Peptide Nucleic Acid Delivery and Synergistic Enhancement of Cancer Cell Apoptosis.

Antisense peptide nucleic acid (asPNA), an effective antisense drug, has been employed as a gene therapy agent and a useful tool in molecular biology. Gaining control over the delivery of asPNA to target tissues has been a major hindrance to its wide application in clinical practice. A simple and efficient DNA nanoribbon (DNR)-based drug delivery process has been designed in this study that releases the asPNA agent to inhibit oncogenic microRNAs (miRNAs). Furthermore, we demonstrated how the AS1411 aptamer that binds nucleolin on the cell membranes works as a control mechanism capable of identifying target cancer cells and enhancing the enrichment capacity of DNR. With the biodegradability of DNR, we can efficiently initiate the release of asPNA into the cytoplasm, particularly targeting the intended miR-21 and synergistically increasing programmed cell death 4 (PDCD4) expression to enhance cell apoptosis. We assume that this well-defined delivery mechanism will aid in designing antisense site-specific treatments for various diseases, including cancer.

Establishment of anti-DKK3 peptide for the cancer control in head and neck squamous cell carcinoma (HNSCC)

BackgroundHead and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck. We identified cancer-specific genes in HNSCC and focused on DKK3 expression. DKK3 gene codes two isoforms of proteins (secreted and non-secreted) with two distinct cysteine rich domains (CRDs). It is reported that DKK3 functions as a negative regulator of oncogenic Wnt signaling and, is therefore, considered to be a tumor suppressor gene. However, our series of studies have demonstrated that DKK3 expression is specifically high in HNSCC tissues and cells, and that DKK3 might determine the malignant potentials of HNSCC cells via the activation of Akt. Further analyses strongly suggested that both secreted DKK3 and non-secreted DKK3 could activate Akt signaling in discrete ways, and consequently exert tumor promoting effects. We hypothesized that DKK3 might be a specific druggable target, and it is necessary to establish a DKK3 inhibitor that can inhibit both secreted and non-secreted isoforms of DKK3.MethodsUsing inverse polymerase chain reaction, we generated mutant expression plasmids that express DKK3 without CRD1, CRD2, or both CRD1 and CRD2 (DKK3ΔC1, DKK3ΔC2, and DKK3ΔC1ΔC2, respectively). These plasmids were then transfected into HNSCC-derived cells to determine the domain responsible for DKK3-mediated Akt activation. We designed antisense peptides using the MIMETEC program, targeting DKK3-specific amino acid sequences within CRD1 and CRD2. The structural models for peptides and DKK3 were generated using Raptor X, and then a docking simulation was performed using CluPro2. Afterward, the best set of the peptides was applied into HNSCC-derived cells, and the effects on Akt phosphorylation, cellular proliferation, invasion, and migration were assessed. We also investigated the therapeutic effects of the peptides in the xenograft models.ResultsTransfection of mutant expression plasmids and subsequent functional analyses revealed that it is necessary to delete both CRD1 and CRD2 to inhibit Akt activation and inhibition of proliferation, migration, and invasion. The inhibitory peptides for CRD1 and CRD2 of DKK3 significantly reduced the phosphorylation of Akt, and consequently suppressed cellular proliferation, migration, invasion and in vivo tumor growth at very low doses.ConclusionsThis inhibitory peptide represents a promising new therapeutic strategy for HNSCC treatment.

Antimicrobial Activity of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pneumoniae.

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for multiple other infectious diseases, such as meningitis and otitis media, in children. Resistance to penicillins, macrolides, and fluoroquinolones is increasing and, since the introduction of pneumococcal conjugate vaccines (PCVs), vaccine serotypes have been replaced by non-vaccine serotypes. Antisense peptide nucleic acids (PNAs) have been shown to reduce the growth of several pathogenic bacteria in various infection models. PNAs are frequently coupled to cell-penetrating peptides (CPPs) to improve spontaneous cellular PNA uptake. In this study, different CPPs were investigated for their capability to support translocation of antisense PNAs into S. pneumoniae. HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs efficiently reduced the viability of S. pneumoniae strains TIGR4 and D39 in vitro. Two essential genes, gyrA and rpoB, were used as targets for antisense PNAs. Overall, the antimicrobial activity of anti-gyrA PNAs was higher than that of anti-rpoB PNAs. Target gene transcription levels in S. pneumoniae were reduced following antisense PNA treatment. The effect of HIV-1 TAT- and (RXR)4XB-anti-gyrA PNAs on pneumococcal survival was also studied in vivo using an insect infection model. Treatment increased the survival of infected Galleria mellonella larvae. Our results represent a proof of principle and may provide a basis for the development of efficient antisense molecules for treatment of S. pneumoniae infections. IMPORTANCE Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for the deaths of up to 2 million children each year. Antibiotic resistance and strain replacement by non-vaccine serotypes are growing problems. For this reason, S. pneumoniae has been added to the WHO "global priority list" of antibiotic-resistant bacteria for which novel antimicrobials are most urgently needed. In this study, we investigated whether CPP-coupled antisense PNAs show antibacterial activity in S. pneumoniae. We demonstrated that HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs were able to kill S. pneumoniae in vitro. The specificity of the antimicrobial effect was verified by reduced target gene transcription levels in S. pneumoniae. Moreover, CPP-antisense PNA treatment increased the survival rate of infected Galleria mellonella larvae in vivo. Based on these results, we believe that efficient antisense PNAs can be developed for the treatment of S. pneumoniae infections.

INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors.

Ribosome profiling (Ribo-seq) is a powerful method for the transcriptome-wide assessment of protein synthesis rates and the study of translational control mechanisms. Yet, Ribo-seq also has limitations. These include difficulties with the analysis of translation-modulating molecules such as antibiotics, which are often toxic or challenging to deliver into living cells. Here, we have developed in vitro Ribo-seq (INRI-seq), a cell-free method to analyze the translational landscape of a fully customizable synthetic transcriptome. Using Escherichia coli as an example, we show how INRI-seq can be used to analyze the translation initiation sites of a transcriptome of interest. We also study the global impact of direct translation inhibition by antisense peptide nucleic acid (PNA) to analyze PNA off-target effects. Overall, INRI-seq presents a scalable, sensitive method to study translation initiation in a transcriptome-wide manner without the potentially confounding effects of extracting ribosomes from living cells.

Development of Surface Chemical Strategies for Synthesizing Redox-Responsive Diatomite Nanoparticles as a Green Platform for On-Demand Intracellular Release of an Antisense Peptide Nucleic Acid Anticancer Agent.

Redox-responsive silica drug delivery systems are synthesized by aeco-friendly diatomite source to achieve on-demand release of peptide nucleic acid (PNA) in tumor reducing microenvironment, aiming to inhibit the immune checkpoint programmed cell death 1 receptor/programmed cell death receptor ligand 1 (PD-1/PD-L1) in cancer cells. The nanoparticles (NPs) are coated with polyethylene glycol chains as gatekeepers to improve their physicochemical properties and control drug release through the cleavable disulfide bonds (S-S) in a reductive environment. This study describes different chemical conditions to achieve the highest NPs' surface functionalization yield, exploring both multistep and one-pot chemical functionalization strategies. The best formulation is used for covalent PNA conjugation via the S-S bond reaching a loading degree of 306 ± 25µg PNA mg-1 DNPs . These systems are used for in vitro studies to evaluate the kinetic release, biocompatibility, cellular uptake, and activity on different cancer cells expressing high levels of PD-L1. The obtained results prove the safety of the NPs up to 200µg mL-1 and their advantage for controlling and enhancing the PNA intracellular release as well as antitumor activity. Moreover, the downregulation of PD-L1 observed only with MDA-MB-231 cancer cells paves the way for targeted immunotherapy.

The novel putative target to the eradication of Acinetobacter baumannii persister cells

BackgroundBacterial persister cells are dormant, slow-growing or growth-arrested phenotypic variants of normal cells in bacterial populations and are suggested that occurrence of the persisters leads to the recalcitrance and relapse of different chronic infections. Acinetobacter baumannii associated-infections are often correlated with high rates of drug-tolerant persisters, triggering a leading human health concern. MethodsIn our study, mqsR (as the toxin) and mqsA (as antitoxin) were considered as novel antimicrobial targets and silencing with antisense peptide nucleic acid (PNA) assay to eradicate the A. baumannii persister cells. Firstly, mqsR/A genes were molecularly evaluated. The functionality was assessed by normal and stress conditions in A. baumannii persister cells. ResultsExpression of mqsR significantly increased the under stress. A high expression of mqsR TA type II system was detected in A. baumannii persister cells. The mqsR gene fully silenced by PNA and A. baumannii persisters is eradicated. ConclusionsAntisense mqsR-PNA in 15 and 20 μM concentrations could eradicate A. baumannii persister cells. Moreover, it is proposed that other TA loci in A. baumannii are surveyed by antisense PNA to detect their functionality. However, conferring to importance of persisters in human infections, ex vivo, in vivo, preclinical and clinical settings could be highlighted.

Antisense Peptide Nucleic Acid-Diaminobutanoic Acid Dendron Conjugates with SbmA-Independent Antimicrobial Activity against Gram-Negative Bacteria.

Precision antisense antibacterial agents may be developed into novel antibiotics in the fight against multidrug-resistant Gram-negative bacteria. In this study, a series of diaminobutanoic acid (DAB) dendrons are presented as novel carriers for the delivery of antisense antibacterial peptide nucleic acids (PNAs). The dendron–PNA conjugates targeting the essential acpP gene exhibit specific antisense antimicrobial bactericidal activity against Escherichia coli and Klebsiella pneumoniae at one-digit micromolar concentrations, while showing low toxicity to human cells. One compound selected from a structure–activity relationship series showed high stability in mouse and human serum (t1/2 ≫ 24 h) as well as in vivo activity against a multidrug-resistant, extended spectrum beta-lactamase-producing E. coli in a murine peritonitis model. The compound was also well tolerated in mice upon i.v. administration up to a dose of 20 mg/kg, and in vivo fluorescence imaging indicated clearance via renal excretion with slight accumulation in the kidneys and liver. Thus, DAB-based dendrons constitute a promising new chemistry platform for development of effective delivery agents for antibacterial drugs with possible in vivo use.