Tumor-On-A-Chip Models for Predicting In Vivo Nanoparticle Behavior.

Nanosized drug formulations are broadly explored for the improvement of cancer therapy. Prediction of in vivo nanoparticle (NP) behavior, however, is challenging, given the complexity of the tumor and its microenvironment. Microfluidic tumor-on-a-chip models are gaining popularity for the in vitro testing of nanoparticle targeting under conditions that simulate the 3D tumor (microenvironment). In this review, following a description of the tumor microenvironment (TME), the state of the art regarding tumor-on-a-chip models for investigating nanoparticle delivery to solid tumors is summarized. The models are classified based on the degree of compartmentalization (single/multi-compartment) and cell composition (tumor only/tumor microenvironment). The physiological relevance of the models is critically evaluated. Overall, microfluidic tumor-on-a-chip models greatly improve the simulation of the TME in comparison to 2D tissue cultures and static 3D spheroid models and contribute to the understanding of nanoparticle behavior. Interestingly, two interrelated aspects have received little attention so far which are the presence and potential impact of a protein corona as well as nanoparticle uptake through phagocytosing cells. A better understanding of their relevance for the predictive capacity of tumor-on-a-chip systems and development of best practices will be a next step for the further refinement of advanced in vitro tumor models.

Mandibular Canal Branching Assessed With Cone-Beam Computed Tomography.

This study aimed to evaluate the prevalence and type of mandibular canal branching in a sample of the Iranian population through cone-beam computed tomography (CBCT) images. This cross-sectional study was performed on CBCT records of 300 patients (112 males and 188 females; mean age 30.14 ± 10.96 y). The presence of mandibular canal branch (MCB) was evaluated by reconstructing multiple panoramic views and buccolingual cross-sections, according to Naitoh's classification. The prevalence of MCB, the type of MCB, and the demographic information of all patients were recorded. The maximum number of branching and the maximum type of branching were also registered for each patient. Mandibular canal branching was observed in 131(43.7%) of 300 CBCT images without side or sex differences ( P > 0.05). The most frequent type was dental, followed by the retromolar, forward, and buccolingual types, respectively. There was a statistically higher frequency of retromolar canal in females than in males (odds ratio: 1.14 ± 4.25; P = 0.013). About 46% of patients with MCB had more than one accessory canal and 29% displayed more than one type of MCB. The coincidence of dental and retromolar canals in a patient was the most prevalent combination. Considering the relatively high prevalence of MCB (43.7%), it is recommended to keep in mind the possibility of anatomical variations of the inferior alveolar nerve during dental procedures to avoid postsurgical complications. The high possibility of observing multiple numbers and types of branching in the patients should also be considered during preoperative assessments.

Reverse relation between cytotoxicity and Polyethylenimine/DNA ratio, the effect of using HEPES-buffered saline (HBS) medium in gene delivery.

Polyethyleneimine (PEI) is considered a promising cationic polymer in non-viral gene delivery. DNA binding properties and other biochemical characteristics of PEI such as the proton sponge phenomenon, offered the branched 25 kDa PEI to be widely used for therapeutic DNA delivery, although the possible cytotoxic effects and the best conditions of PEI preparation are not still well recognized. While higher PEI/Plasmid ratios have increased transfection efficiencies, it induces more cell stress and toxicity. Considering that the PEI particle size and resulting cytotoxicity are affected by media ions, we used Neuro2A cells to assess the cell stress properties of PEI/Plasmid complexes prepared in a HEPES-buffered saline medium. Delivery of a plasmid containing EGFP happened in all increasing ratios of PEI/plasmid from 0.5, 2, 4, and 6, while higher ratios induced less unfolded protein response as evidenced by lower transcription of ER stress markers Grp78, Atf4, Chop, Xbp1, and induced Xbp1 splicing. These data were also supported by MTT cytotoxicity assay results. These findings indicate that preparing higher PEI/plasmid ratio complexes (using the equivalent of 200 ng DNA) in the HBS medium leads to less cytotoxicity.

Optimizing the synthesis and purification of MS2 virus like particles.

Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated. In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100 mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30 nm and suitable homogeneity with minimum 12-month stability at 4 °C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 h in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents' delivery.

Evaluating polyethyleneimine/DNA nanoparticles-mediated damage to cellular organelles using endoplasmic reticulum stress profile.

Gene therapy has emerged as an influential tool for treating the genetic and specific acquired disorders. Among all kinds of gene delivery systems, the cationic polymer polyethyleneimine (PEI) is considered as a promising non-viral gene delivery vector, although there are still concerns about its magnitude of cytotoxicity. While any cell insult leads to unfolded/misfolded protein accumulation and its consequent unfold protein response, evaluating the expression profile of ER-stress genes would be a sensitive indicator of cell stress. Beside cytotoxicity assays, real-time RT-PCR was used to investigate the effects of PEI nanoparticles on the endoplasmic reticulum. Treating Neuro2A cells revealed that PEI can induce cell toxicity in a concentration-dependent manner. Also, It increased the transcript levels of Grp78 (Bip), Atf4 and Chop, and splicing of Xbp1. To further optimize the transfection properties in Neuro2A cells, PEI was used to deliver a plasmid DNA containing GFP reporter. While different PEI/plasmid ratios revealed similar transfection efficiency, increasing the PEI/plasmid ratio led to induction of ER-stress markers. These results underscored that beside the effectiveness of PEI, using the lowest possible ratio of PEI/plasmid would minimize the detrimental effects of PEI on cells and confer it a beneficial therapeutic importance in nucleic acid delivery.