Using design of experiments (DoE) to optimize performance and stability of biomimetic cell membrane-coated nanostructures for cancer therapy.

Introduction: Cell membrane-covered biomimetic nanosystems have allowed the development of homologous nanostructures to bestow nanoparticles with enhanced biointerfacing capabilities. The stability of these structures, however, still represents a challenge for the scientific community. This study is aimed at developing and optimizing cell derived membrane-coated nanostructures upon applying design of experiments (DoE) to improve the therapeutic index by homotypic targeting in cancer cells. Methods: Important physicochemical features of the extracted cell membrane from tumoral cells were assessed by mass spectrometry-based proteomics. PLGA-based nanoparticles encapsulating temozolomide (TMZ NPs) were successfully developed. The coating technology applying the isolated U251 cell membrane (MB) was optimized using a fractional two-level three-factor factorial design. All the formulation runs were systematically characterized regarding their diameter, polydispersity index (PDI), and zeta potential (ZP). Experimental conditions generated by DoE were also subjected to morphological studies using negative-staining transmission electron microscopy (TEM). Its short-time stability was also assessed. MicroRaman and Fourier-Transform Infrared (FTIR) spectroscopies and Confocal microscopy were used as characterization techniques for evaluating the NP-MB nanostructures. Internalization studies were carried out to evaluate the homotypic targeting ability. Results and Discussion: The results have shown that nearly 80% of plasma membrane proteins were retained in the cell membrane vesicles after the isolation process, including key proteins to the homotypic binding. DoE analysis considering acquired TEM images reveals that condition run five should be the best-optimized procedure to produce the biomimetic cell-derived membrane-coated nanostructure (NP-MB). Storage stability for at least two weeks of the biomimetic system is expected once the original characteristics of diameter, PDI, and ZP, were maintained. Raman, FTIR, and confocal characterization results have shown the successful encapsulation of TMZ drug and provided evidence of the effective coating applying the MB. Cell internalization studies corroborate the proteomic data indicating that the optimized NP-MB achieved specific targeting of homotypic tumor cells. The structure should retain the complex biological functions of U251 natural cell membranes while exhibiting physicochemical properties suitable for effective homotypic recognition. Conclusion: Together, these findings provide coverage and a deeper understanding regarding the dynamics around extracted cell membrane and polymeric nanostructures interactions and an in-depth insight into the cell membrane coating technology and the development of optimized biomimetic and bioinspired nanostructured systems.

The amount of dextran in PLGA nanocarriers modulates protein corona and promotes cell membrane damage.

Polymeric nanocarriers (NCs) are efficient vehicles to prevent drug unspecific biodistribution and increase the drug amounts delivered to tumor tissues. However, some toxicological aspects of NCs still lack a comprehensive assessment, such as their effects on cellular processes that lead to toxicity. We evaluate the interaction of poly(lactic-co-glycolic acid) (PLGA) NCs prepared using dextran (Dex) and Pluronic®-F127 as stabilizing agents with myocardial cells (H9C2), breast adenocarcinoma cells (MCF-7) and macrophages (RAW 264.7) to address the effect of Dex in PLGA NC formulations. By an emulsion diffusion method, doxorubicin-loaded NCs were prepared with no Dex (PLGA-DOX), 1% (w/v) Dex (Dex1/PLGA-DOX) and 5% (w/v) Dex (Dex5/PLGA-DOX). Uptake analyses revealed a significant reduction in Dex5/PLGA-DOX NC uptake by H9C2 and MCF-7, as in the case of Dex1/PLGA-DOX NCs in the absence of in vitro protein corona, revealing an effect of dextran concentration on the formation of protein corona. RAW 264.7 cells presented a greater uptake of Dex5/PLGA-DOX NCs than the other NCs likely because of receptor mediated endocytosis, since C-type lectins like SIGN-R1, mannose receptors and scavenger receptor type 1 that are expressed in RAW 264.7 can mediate Dex uptake. Despite the lower uptake, Dex5/PLGA-DOX NCs promote the generation of reactive oxygen species and oxidative membrane damage in MCF-7 and H9C2 even though cellular metabolic activity assessed by MTT was comparable among all the NCs. Our results highlight the importance of an in-depth investigation of the NC-cell interaction considering additional mechanisms of damage apart from metabolic variations, as nanoparticle-induced damage is not limited to imbalance in metabolic processes, but also associated with other mechanisms, e.g., membrane and DNA damage.

Comparing extracellular vesicles and cell membranes as biocompatible coatings for gold nanorods: Implications for targeted theranostics.

Extracellular vesicles (EVs) and cell membrane nanoghosts are excellent coatings for nanomaterials, providing enhanced delivery in the target sites and evasion of the immune system. These cell-derived coatings allow the exploration of the delivery properties of the nanoparticles without stimulation of the immune system. Despite the advances reported on the use of EVs and cell-membrane coatings for nanomedicine applications, there are no standards to compare the benefits and main differences between these technologies. Here we investigated macrophage-derived EVs and cell membranes-coated gold nanorods and compared both systems in terms of target delivery in cancer and stromal cells. Our results reveal a higher tendency of EV-coated nanorods to interact with macrophages yet both EV and cell membrane-coated nanorods were internalized in the metastatic breast cancer cells. The main differences between these nanoparticles are related to the presence or absence of CD47 in the coating material, not usually addressed in EVs characterization. Our findings highlight important delivery differences exhibited by EVs- or cell membranes- coated nanorods which understanding may be important to the design and development of theragnostic nanomaterials using these coatings for target delivery.

Modulation of beta-amyloid aggregation using ascorbic acid.

Studies have shown that the level of ascorbic acid (AA) is reduced in the brain of Alzheimer's disease (AD) patients. However, its effect on amyloid-ß 1-42 (Aß42) aggregation has not yet been elucidated. Here we investigated for the first time the effect of AA on Aß42 aggregation using fluorescence assay, circular dichroism, atomic force microscopy, isothermal titration calorimetry, ligand docking, and molecular dynamics. Our results showed that the fibril content decreases in the growth phase when the peptides are co-incubated with AA. AA molecules bind to Aß42 peptides with high binding affinity and a binding site for AA between the ß-strands of Aß42 oligomers prevents the stack of adjacent strands. We demonstrate the inhibitory effect of AA on the aggregation of Aß42 and its molecular interactions, which can contribute to the development of an accessible therapy for AD and also to the design of novel drugs for other amyloidogenic diseases.

Near-Infrared Photoactive Theragnostic Gold Nanoflowers for Photoacoustic Imaging and Hyperthermia.

Branched anisotropic gold nanostructures present distinguished performance acting both as contrast agents for photoacoustic imaging and as active agents for photothermal therapies. Despite advances in their fabrication methods, the synthesis of such gold nanomaterials in a simple and reproducible way is still a challenge. In this paper, we report the development of branched anisotropic gold nanoparticles, the so-called gold nanoflowers (AuNFs), as near-infrared active theragnostic materials for cancer therapy and diagnosis. In situ chemical synthesis of the AuNFs was optimized to obtain monodisperse nanoflowers with controllable size and optical properties. Upon varying the temperature and gold ion concentrations, it was possible to tune the optical activity of the nanoparticles from 590 to 960 nm. The AuNFs exhibited good stability in the cell culture medium, and under laser irradiation. Photoacoustic imaging revealed that the NFs could be imaged in phantom systems even at low concentrations. In vitro tests revealed that the nanoflowers were effective in the photothermal therapy of a rat hepatocarcinoma (HTC) cell lineage. In addition, no toxicity was observed to mouse fibroblast (FC3H) cells incubated with the AuNFs. Our results reveal a simple method to synthesize branched anisotropic gold nanostructures, which is a promising platform for photothermal and photoacoustic therapies.