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Background: The manufacture of nanoparticles using manual methods is hampered by its challenging scale-up and poor reproducibility. To overcome this issue, the production of zein nanoparticles entrapping a lipophilic drug model, coumarin-6, by using a microfluidic system was assessed in this study. The influence of PEG density and chain length on zein nanoparticle characteristics, as well as their uptake efficacy in melanoma cancer cells, was also evaluated., Methods: Zein nanoparticles were prepared by both manual and microfluidic approaches to allow comparison between the two processes. PEGylated zein nanoparticles with various PEG densities and chain lengths were produced by nanoprecipitation and characterized. Their cellular uptake was evaluated on B16F10 melanoma cancer cells in vitro., Results: Zein nanoparticles have successfully been produced by both manual and microfluidic approaches. Parameters such as total flow rate and flow rate ratio of the aqueous and organic phases in microfluidic process, as well as the method preparation and aqueous to organic phase volume ratio during nanoprecipitation, have been shown to strongly influence the characteristics of the resulting nanoparticles. Continuous microfluidics led to the production of nanoparticles with low yield and drug entrapment, unlike nanoprecipitation, which resulted in zein nanoparticles with an appropriate size and an optimal drug entrapment efficiency of 64%. The surface modification of the nanoparticles produced by nanoprecipitation, with lower PEG density and shorter PEG chain length made mPEG5K-zein (0.5:1) the most favorable formulation in our study, resulting in enhanced stability and higher coumarin-6 uptake by melanoma cancer cells., Conclusion: mPEG5K-zein (0.5:1) nanoparticles prepared by nanoprecipitation were the most promising formulation in our study, exhibiting increased stability and enhancing coumarin-6 uptake by melanoma cancer cells., Competing Interests: The authors report no conflicts of interest in this work. Jamal Almowalad’s current affiliation is at the Department of Pharmaceutics, College of Pharmacy, Umm Al-Qura University, Al-Abidiyah, Makkah 21955, Kingdom of Saudi Arabia. Partha Laskar’s current affiliation is at the Department of Chemistry -Ångström, Uppsala University, 751 21 Uppsala, Sweden. The graphical abstract was created with BioRender.com., (© 2022 Meewan et al.)

Background: Plumbagin, a naphthoquinone extracted from the officinal leadwort presenting promising anti-cancer properties, has its therapeutic potential limited by its inability to reach tumors in a specific way at a therapeutic concentration following systemic injection. The purpose of this study is to assess whether a novel tumor-targeted, lipid-polymer hybrid nanoparticle formulation of plumbagin would suppress the growth of B16-F10 melanoma in vitro and in vivo., Methods: Novel lipid-polymer hybrid nanoparticles entrapping plumbagin and conjugated with transferrin, whose receptors are present in abundance on many cancer cells, have been developed. Their cellular uptake, anti-proliferative and apoptosis efficacy were assessed on various cancer cell lines in vitro. Their therapeutic efficacy was evaluated in vivo after tail vein injection to mice bearing B16-F10 melanoma tumors., Results: The transferrin-bearing lipid-polymer hybrid nanoparticles loaded with plumbagin resulted in the disappearance of 40% of B16-F10 tumors and regression of 10% of the tumors following intravenous administration. They were well tolerated by the mice., Conclusion: These therapeutic effects, therefore, make transferrin-bearing lipid-polymer hybrid nanoparticles entrapping plumbagin a highly promising anti-cancer nanomedicine., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Sakpakdeejaroen et al.)

Plumbagin, a natural naphthoquinone from the officinal leadwort, has recently been shown to exert promising anti-cancer effects. However, its therapeutic use is hampered by its failure to specifically reach tumors after intravenous administration, without secondary effects on normal tissues. Its poor solubility in water and rapid elimination following in vivo administration further limit its potential use. We hypothesize that the entrapment of plumbagin within PEGylated PLGA nanoparticles conjugated with transferrin, whose receptors are overexpressed on many types of cancer cells, could lead to a selective delivery of the drug to tumors following intravenous administration and enhance its chemotherapeutic effects. The objectives of this study were therefore to prepare and characterize transferrin-conjugated, PEGylated PLGA nanoparticles entrapping plumbagin, and to assess their anti-cancer efficacy in vitro as well as in tumor-bearing mice. The intravenous administration of transferrin-conjugated PEGylated PLGA nanoparticles resulted in the complete suppression of 10% of B16-F10 tumors and regression of 30% of the tumors, with improvement of the animal survival compared to controls. The treatment was well tolerated by the animals. Transferrin-bearing PEGylated PLGA nanoparticles entrapping plumbagin are therefore highly promising therapeutic systems, able to lead to tumor regression and even suppression after intravenous administration without visible toxicity.

Mesenchymal stem cells are multipotent adult stem cells capable of generating bone, cartilage and fat, and are thus currently being exploited for regenerative medicine. When considering osteogenesis, developments have been made with regards to chemical induction (e.g. differentiation media) and physical induction (e.g. material stiffness, nanotopography), targeting established early transcription factors or regulators such as runx2 or bone morphogenic proteins and promoting increased numbers of cells committing to osteo-specific differentiation. Recent research highlighted the involvement of microRNAs in lineage commitment and terminal differentiation. Herein, gold nanoparticles that confer stability to short single stranded RNAs were used to deliver MiR-31 antagomiRs to both pre-osteoblastic cells and primary human MSCs in vitro. Results showed that blocking miR-31 led to an increase in osterix protein in both cell types at day 7, with an increase in osteocalcin at day 21, suggesting MSC osteogenesis. In addition, it was noted that antagomiR sequence direction was important, with the 5 prime reading direction proving more effective than the 3 prime. This study highlights the potential that miRNA antagomiR-tagged nanoparticles offer as novel therapeutics in regenerative medicine. [ABSTRACT FROM AUTHOR]

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies. [ABSTRACT FROM AUTHOR]

Aims: RNAi is a powerful tool for gene silencing that can be used to reduce undesirable overexpression of oncogenes as a novel form of cancer treatment. However, when using RNAi as a therapeutic tool there is potential for associated gene effects. This study aimed to utilize gold nanoparticles to deliver siRNA into HeLa cells. Results: Knockdown of the c-myc oncogene by RNAi, at the RNA, protein and cell proliferation level was achieved, while also identifying associated gene responses. Discussion: The gold nanoparticles used in this study present an excellent delivery platform for siRNA, but do note associated gene changes. Conclusion: The study highlights the need to more widely assess the cell physiological response to RNAi treatment, rather than focus on the immediate RNA levels. [ABSTRACT FROM AUTHOR]

Nanotechnology plays an increasingly important role in the biomedical arena. In particular, magnetic nanoparticles (mNPs) have become important tools in molecular diagnostics, in vivo imaging and improved treatment of disease, with the ultimate aim of producing a more theranostic approach. Due to their small sizes, the nanoparticles can cross most of the biological barriers such as the blood vessels and the blood brain barrier, thus providing ubiquitous access to most tissues. In all biomedical applications maximum nanoparticle uptake into cells is required. Two promising methods employed to this end include functionalization of mNPs with cell-penetrating peptides to promote efficient translocation of cargo into the cell and the use of external magnetic fields for enhanced delivery. This study aimed to compare the effect of both penetratin and a static magnetic field with regards to the cellular uptake of 200 nm magnetic NPs and determine the route of uptake by both methods. Results demonstrated that both techniques increased particle uptake, with penetratin proving more cell specific. Clathrinmedicated endocytosis appeared to be responsible for uptake as shown via PCR and western blot, with Pitstop 2 (known to selectively block clathrin formation) blocking particle uptake.Interestingly, it was further shown that a magnetic field was able to reverse or overcome the blocking, suggesting an alternative route of uptake. [ABSTRACT FROM AUTHOR]

Nanoparticle entry into the cell depends on the surface charge and also on their size. Here, we report the entry of large magnetic nanoparticles (500 nm mean diameter) into the cell, being mediated by a mechanical stimulus supplied to the culture flasks. Investigations were carried out at 2-10 Hz frequency range with the vertical excursions ranging from 5 to 20 nm. Mechanical stimulation was found to aid the entry of both positive and negatively charged nanoparticles over a frequency range of 2-10 Hz. Transmission electron microscopy analysis indicated that, the stimulated samples could possibly mediate particle uptake through membrane invaginations, while the control samples indicated particles at the cell periphery, just outside the cell membrane. Mechanical stimulation had no significant effect on the cell morphology. Bromodeoxyuridine incorporation resulted in an increase in the proportion of S-phase in the stimulated samples compared with the controls, suggesting a reduction in the cell cycle duration. Mechanical stimulation could very well extend its effects to nanoscale cellular movements, and also facilitate the entry of large magnetic nanoparticle. This could be an interesting prospect for nanoparticle mediated drug delivery. [ABSTRACT FROM AUTHOR]

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies. [ABSTRACT FROM AUTHOR]

Abstract: Magnetic nanoparticles are widely used in bioapplications such as imaging (MRI), targeted delivery (drugs/genes) and cell transfection (magnetofection). Historically, the impermeable nature of both the plasma and nuclear membranes hinder potential. Researchers combat this by developing techniques to enhance cellular and nuclear uptake. Two current popular methods are using external magnetic fields to remotely control particle direction or functionalising the nanoparticles with a cell penetrating peptide (e.g. tat); both of which facilitate cell entry. This paper compares the success of both methods in terms of nanoparticle uptake, analysing the type of magnetic forces the particles experience, and determines gross cell response in terms of morphology and structure and changes at the gene level via microarray analysis. Results indicated that both methods enhanced uptake via a caveolin dependent manner, with tat peptide being the more efficient and achieving nuclear uptake. On comparison to control cells, many groups of gene changes were observed in response to the particles. Importantly, the magnetic field also caused many change in gene expression, regardless of the nanoparticles, and appeared to cause F-actin alignment in the cells. Results suggest that static fields should be modelled and analysed prior to application in culture as cells clearly respond appropriately. Furthermore, the use of cell penetrating peptides may prove more beneficial in terms of enhancing uptake and maintaining cell homeostasis than a magnetic field. [Copyright &y& Elsevier]