Extracellular vesicles (EVs) are under evaluation as therapeutics or as vehicles for drug delivery. Preclinical studies of EVs often use mice or other animal models to assess efficacy and disposition. However, as most EVs under evaluation are derived from human cells, they may elicit immune responses which may contribute to toxicities or enhanced EV clearance. Furthermore, EVs from different cell sources or EVs comprising various cargo may differ with respect to immunogenicity or toxicity. To assess EV‐induced immune response and toxicity, we dosed C57BL/6 mice with EVs intravenously and intraperitoneally for 3 weeks. EVs were harvested from wild type or engineered HEK293T cells which were modified to produce EVs loaded with miR‐199a‐3p and chimeric proteins. Blood was collected to assess hematology, blood chemistry, and immune markers. Spleen cells were immunophenotyped, and tissues were harvested for gross necropsy and histopathological examination. No signs of toxicity were observed, and minimal evidence of changes in immune markers were noted in mice dosed with engineered, but not with wild type EVs. This study provides a framework for assessment of immunogenicity and toxicity that will be required as EVs from varying cell sources are tested within numerous animal models and eventually in humans.

Extracellular vesicles (EVs) hold great potential as novel systems for nucleic acid delivery due to their natural composition. Our goal was to load EVs with microRNA that are synthesized by the cells that produce the EVs. HEK293T cells were engineered to produce EVs expressing a lysosomal associated membrane, Lamp2a fusion protein. The gene encoding pre‐miR‐199a was inserted into an artificial intron of the Lamp2a fusion protein. The TAT peptide/HIV‐1 transactivation response (TAR) RNA interacting peptide was exploited to enhance the EV loading of the pre‐miR‐199a containing a modified TAR RNA loop. Computational modeling demonstrated a stable interaction between the modified pre‐miR‐199a loop and TAT peptide. EMSA gel shift, recombinant Dicer processing and luciferase binding assays confirmed the binding, processing and functionality of the modified pre‐miR‐199a. The TAT‐TAR interaction enhanced the loading of the miR‐199a into EVs by 65‐fold. Endogenously loaded EVs were ineffective at delivering active miR‐199a‐3p therapeutic to recipient SK‐Hep1 cells. While the low degree of miRNA loading into EVs through this approach resulted in inefficient distribution of RNA cargo into recipient cells, the TAT TAR strategy to load miRNA into EVs may be valuable in other drug delivery approaches involving miRNA mimics or other hairpin containing RNAs.

Penicillin-binding proteins (PBPs) are the high-affinity target sites of all β-lactam antibiotics in bacteria. It is well known that each β-lactam covalently binds to and thereby inactivates different PBPs with various affinities. Despite β-lactams serving as the cornerstone of our therapeutic armamentarium against Klebsiella pneumoniae , PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae . PBP binding was determined using isolated membrane fractions from K. pneumoniae strains ATCC 43816 and ATCC 13883. Binding reactions were conducted using β-lactam concentrations from 0.0075 to 256 mg/liter (or 128 mg/liter). After β-lactam exposure, unbound PBPs were labeled by Bocillin FL. Binding affinities (50% inhibitory concentrations [IC ₅₀ ]) were reported as the β-lactam concentrations that half-maximally inhibited Bocillin FL binding. PBP occupancy patterns by β-lactams were consistent across both strains. Carbapenems bound to all PBPs, with PBP2 and PBP4 as the highest-affinity targets (IC ₅₀ , <0.0075 mg/liter). Preferential PBP2 binding was observed by mecillinam (amdinocillin; IC ₅₀ , <0.0075 mg/liter) and avibactam (IC ₅₀ , 2 mg/liter). Aztreonam showed high affinity for PBP3 (IC ₅₀ , 0.06 to 0.12 mg/liter). Ceftazidime bound PBP3 at low concentrations (IC ₅₀ , 0.06 to 0.25 mg/liter) and PBP1a/b at higher concentrations (4 mg/liter), whereas cefepime bound PBPs 1 to 4 at more even concentrations (IC ₅₀ , 0.015 to 2 mg/liter). These PBP binding data on a comprehensive set of 13 clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae enable, for the first time, the rational design and optimization of double β-lactam and β-lactam–β-lactamase inhibitor combinations.

Pancreatic cancer is the fourth largest cause of cancer deaths in the Unites States and the prognosis is grim with <5% survival chances upon diagnosis. The objective of this study was to assess the combined chemopreventive effect of solid lipid nanoparticle (SLN) encapsulated drugs aspirin (ASP), curcumin (CUR) and free sulforaphane (SFN) for the chemoprevention of pancreatic cancer. Experiments were carried out (1) to evaluate the feasibility of encapsulation of these chemopreventive agents within solid lipid systems and (2) to measure the synergistic effects of a combination of ASP with CUR in SLNs mixed with free SFN against cell proliferation and apoptosis in pancreatic cancer cells, MIA PaCa-2 and Panc-1. The SLNs were prepared using a modified solvent evaporation technique and were characterized for particle sizing, encapsulation efficiency and drug release. ASP and CUR SLNs were formulated within the particle size range of 150‑250 nm and were found to have an encapsulation efficiency of 85 and 69%, respectively. Sustained release of drugs over a 96 h period from SLNs was observed. The SLNs were stable over a 3-month storage period at room temperature. Cell viability studies demonstrated that combinations of low doses of ASP SLN (25 µM), CUR SLN (2.5 µM) and free SFN (5 µM) significantly reduced cell viability by 43.6 and 48.49% in MIAPaca-2 and Panc-1 cell lines, respectively. Furthermore, increased apoptosis of 61.3 and 60.37% was found in MIA Paca-2 and Panc-1 cell lines, respectively, in comparison to the individual doses administered. Synergistic effects were demonstrated using MTS and apoptosis assays. Thus, this study successfully demonstrated the feasibility of using a solid lipid nanoparticulate system for the first time to deliver this novel combination chemoprevention regimen, providing valuable evidence for the usability of nanotechnology-based drug regimens towards pancreatic cancer chemoprevention.

Globally increased ultraconserved noncoding RNA expression in pancreatic adenocarcinoma