Anti-EGFR bioengineered bacterial outer membrane vesicles as targeted immunotherapy candidate in triple-negative breast tumor murine model.

Cancer immunotherapy employing checkpoint inhibitors holds great promise across diverse cancers; nonetheless, a substantial proportion of patients (ranging from 55 to 87%) remain unresponsive to this treatment. To amplify therapeutic efficiency, we propose a synergistic therapeutic strategy that entails the deployment of targeted nano-sized particles carrying Toll-like receptor (TLR) agonists to the tumor site. This innovative approach seeks to activate intratumoral antigen-presenting cells using bioengineered outer membrane vesicles (OMVs) derived from gram-negative bacteria. These OMVs possess inherent attributes of surface-exposed immune stimulators and TLR-activating components, rendering them intriguing candidates for investigation. These OMVs were meticulously designed to selectively target cancer cells exhibiting an overexpression of epidermal growth factor receptor (EGFR). To gauge the precision of this targeting, the conducted affinity-based assays aimed at determining the equilibrium dissociation constant of the single-chain variable fragment employed for this purpose. In vitro experiments confirmed the OMVs' proficiency in adhering to EGFR-overexpressed cancer cells. Moreover, the evaluation extended to an in vivo context, where the therapeutic effect of nanovesicles was appraised within the tumor microenvironment of the triple-negative breast cancer mouse model. Notably, both intraperitoneal and intratumoral administrations of nanovesicles exhibited the ability to activate natural killer cells and skew M2 macrophage towards an M1 phenotype. The combined scrutiny of in vitro and in vivo findings underscores the potential efficiency of OMVs as a promising strategy for future anti-tumor endeavors.

Aptamer-based diagnosis of various SARS-CoV2 strains isolated from clinical specimens.

The emergence of the SARS-CoV-2 virus, an unknown strain of coronavirus, has resulted in severe acute respiratory syndrome with high mortality rates worldwide. Due to the possibility of asymptomatic carriers, late diagnosis of infected individuals can lead to uncontrollable transmission of the disease, making early and accurate detection crucial in controlling the spread of the virus. In this study we identified high-binding-affinity aptamers targeting various strains of the SARS-CoV2 (COVID-19) virus, using the GO-Cell-SELEX (Graphene Oxide- Systematic Evolution of Ligands by Exponential Enrichment) strategy. A total of 96 aptamers were developed through 11 rounds of GO-Cell-SELEX from a random 40 nucleotide single-strand DNA (ssDNA) aptamer library. Using the surface plasmon resonance (SPR) method, the dissociation constant (Kd) values of all aptamers were calculated and two aptamers 52 and 91 with Kd 50 and 61 were selected for enzyme-linked apta-sorbent assay (ELASA). Aptamer 91 could detect various strains of the virus in above 97% of clinical samples obtained from nasopharyngeal swaps (NPS) specimens kept in viral transport media (VTM), confirmed by real-time PCR assay at COVID-19 Reference Diagnostic Laboratory of Iran, Pasture Institute. Aptamer 52 could detect the SARS-CoV2 virus in a competitive lateral flow assay (LFA) to be considered for a future designed kit. These two simple, specific, and sensitive tests can be used in combination for rapid and early diagnosis of various strains of the COVID-19 virus. Our results suggest that these two discovered aptamers present an opportunity for developing a new rapid aptamer-based coronavirus diagnostic kit.

Aptamer-based enzyme-linked oligonucleotide assay for specific detection of clinical bacterial strains isolated from cerebrospinal fluid samples.

Meningitis, acute infection of the meninges, is the 10th leading cause of mortality among infectious diseases. Although many different causes for meningitis (viruses and bacteria) have been diagnosed, the most common ones are Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. The effort to find a new method for detection of bacterial meningitis is an urgent need for clinical treatment. DNA aptamers generated by cell-systematic evolution of ligands by exponential enrichment (SELEX) against bacterial cells provide a novel cell labeling and biosensing technique. Here, we isolated single-stranded DNA aptamers during the SELEX method with a high affinity for different bacterial genera. This approach was demonstrated on H. influenzae type B, N. meningitidis serogroups A, B, C, and Y, and Streptococcus pneumoniae serotypes 18, 14, 19A, 6A, and 6B which served as targets in 20 rounds of cell-SELEX. After 20 rounds of SELEX, a total of 93 aptamers were identified. Among these, aptamers C65 and C50 showed the highest affinity toward targets with a dissociation constant of 6.98 and 15.79, respectively. Selected aptamers were able to successfully detect clinical bacterial strains isolated from cerebrospinal fluid samples of meningitis patients by double-aptamer sandwich enzyme-linked oligonucleotide assay (ELONA). Our findings demonstrated that aptamers with broad affinity to bacterial taxa in different genera can be isolated for the development of diagnostic tools for multiple targets. We further showed that sandwich ELONA based on single-stranded DNA aptamer is sensitive and specific enough for detection of the superior cause of bacterial meningitis.