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The stated objective of the present research investigation was to use a simultaneous nanodrug delivery approach to optimize the therapeutic effectiveness of anticancer drugs against breast cancer cells. For this purpose, Poly (lactic-co-glycolic acid) nanoparticles with two anticancer drugs; methotrexate (MTX) and doxorubicin (DOX) denoted as DOX/MTX@PLGA NPs was developed by nanoprecipitation method. The developed polymeric DOX/MTX@PLGA NPs exhibited hydrodynamic particle diameter of 170.6 ± 10.0 nm with a poly dispersity index (PDI) of 0.17 and zeta potential value of -9.2 ± 0.31 mV, and spherical geometry analyzed by TEM. Furthermore, the nanoparticles exhibited a pH-responsive drug release profile, outstanding encapsulation efficiency, excellent colloidal stability across various physiological media and pH responsive drug release profile. Additionally, polymeric nanoparticles demonstrated higher cell uptake, in-vitro cytotoxicity, and a high rate of apoptosis in comparison to free DOX and MTX through a synergistic effect, likely as a result of their small particle size. In conclusion, our work presents a novel and distinct approach for boosting the therapeutic efficacy of anticancer drugs by delivering drugs to breast cancer cells simultaneously. [ABSTRACT FROM AUTHOR]

Peptide coacervates self‐assembling via liquid‐liquid phase separation are appealing intracellular delivery vehicles of macromolecular therapeutics (proteins, DNA, mRNA) owing to their non‐cytotoxicity, high encapsulation capacity, and efficient cellular uptake. However, the mechanisms by which these viscoelastic droplets cross the cellular membranes remain unknown. Here, using multimodal imaging, data analytics, and biochemical inhibition assays, we identify the key steps by which droplets enter the cell. We find that the uptake follows a non‐canonical pathway and instead integrates essential features of macropinocytosis and phagocytosis, namely active remodeling of the actin cytoskeleton and appearance of filopodia‐like protrusions. Experiments using giant unilamellar vesicles show that the coacervates attach to the bounding membrane in a charge‐ and cholesterol‐dependent manner but do not breach the lipid bilayer barrier. Cell uptake in the presence of small molecule inhibitors – interfering with actin and tubulin polymerization – confirm the active role of cytoskeleton remodeling, most prominently evident in electron microscopy imaging. These findings suggest a peculiar internalization mechanism for viscoelastic, glassy coacervate droplets combining features of non‐specific uptake of fluids by macropinocytosis and particulate uptake of phagocytosis. The broad implications of this study will enable to enhance the efficacy and utility of coacervate‐based strategies for intracellular delivery of macromolecular therapeutics. [ABSTRACT FROM AUTHOR]

A hybrid lipid-natural source polysaccharides smart cubosome affords crystalline cubic. to inverse hexagonal phase transition and active membrane disturbances for prolonged. and triggered bioactive delivery. [Display omitted] Smart nanocarrier-based bioactive delivery systems are a current focus in nanomedicine for allowing and boosting diverse disease treatments. In this context, the design of hybrid lipid-polymer particles can provide structure-sensitive features for tailored, triggered, and stimuli-responsive devices. In this work, we introduce hybrid cubosomes that have been surface-modified with a complex of chitosan- N -arginine and alginate, making them pH-responsive. We achieved high-efficiency encapsulation of acemannan, a bioactive polysaccharide from Aloe vera , within the nanochannels of the bioparticle crystalline structure and demonstrated its controlled release under pH conditions mimicking the gastric and intestinal environments. Furthermore, an acemannan-induced phase transition from Im3m cubic symmetry to inverse hexagonal H II phase enhances the bioactive delivery by compressing the lattice spacing of the cubosome water nanochannels, facilitating the expulsion of the encapsulated solution. We also explored the bioparticle interaction with membranes of varying curvatures, revealing thermodynamically driven affinity towards high-curvature lipid membranes and inducing morphological transformations in giant unilamellar vesicles. These findings underscore the potential of these structure-responsive, membrane-active smart bioparticles for applications such as pH-triggered drug delivery platforms for the gastrointestinal tract, and as modulators and promoters of cellular internalization. [ABSTRACT FROM AUTHOR]

The focus of nanoparticles in vivo trafficking has been mostly on their tissue-level biodistribution and clearance. Recent progress in the nanomedicine field suggests that the targeting of nanoparticles to immune cells can be used to modulate the immune response and enhance therapeutic delivery to the diseased tissue. In the presence of tumor lesions, monocytic-myeloid-derived suppressor cells (M-MDSCs) expand significantly in the bone marrow, egress into peripheral blood, and traffic to the solid tumor, where they help maintain an immuno-suppressive tumor microenvironment. In this study, we investigated the interaction between PAMAM dendrimers and M-MDSCs in two murine models of glioblastoma, by examining the cell-level biodistribution kinetics of the systemically injected dendrimers. We found that M-MDSCs in the tumor and lymphoid organs can efficiently endocytose hydroxyl dendrimers. Interestingly, the trafficking of M-MDSCs from the bone marrow to the tumor contributed to the deposition of hydroxyl dendrimers in the tumor. M-MDSCs showed different capacities of endocytosing dendrimers of different functionalities in vivo. This differential uptake was mediated by the unique serum proteins associated with each dendrimer surface functionality. The results of this study set up the framework for developing dendrimer-based immunotherapy to target M-MDSCs for cancer treatment. [Display omitted] • Monocytic MDSC (M-MDSC) in the tumor show high capacity of taking up dendrimers. • The recruitment of M-MDSCs contribute to the dendrimer accumulation in the tumor. • Dendrimer surface chemistry affects their interactions with M-MDSCs. • Dendrimer protein corona mediates the interactions between dendrimers and M-MDSCs. [ABSTRACT FROM AUTHOR]

Alina Sigaeva,* Runrun Li,* Jan Jelle van Laar, Leon Wierenga, Romana Schirhagl Department of Biomaterials and Biotechnology, Groningen University, University Medical Center Groningen, Groningen, the Netherlands*These authors contributed equally to this workCorrespondence: Romana Schirhagl, Email romana.schirhagl@gmail.comIntroduction: As nanodiamonds become more and more widely used for intracellular labelling and measurements, the task of delivering these nanoparticles inside cells becomes more and more important. Certain cell types easily take up nanodiamonds, while others require special procedures.Methods: In previous research, we found that HT-29 cells (a colon cancer cell line), which are notoriously difficult in the context of nanodiamond internalization, show increased uptake rates, when pre-treated with trypsin- ethylenediaminetetraacetic acid (trypsin-EDTA). However, the uptake mechanism has not been studied before. This article focuses on a more detailed investigation of the reasons underlying this phenomenon. We start by identifying the timing of fluorescent nanodiamond (FND) uptake in trypsin-EDTA pre-treated cells. We then use a combination of chemical inhibitors and Immunocytochemistry to identify the main pathways employed by HT-29 cells in the internalization process.Results and Discussion: We investigate how these pathways are affected by the trypsin-EDTA pre-treatment and conclude by offering possible explanations for this phenomenon. We found that nanodiamonds are internalized via different pathways. Clathrin-mediated endocytosis proves to be the dominating mechanism. Trypsin-EDTA treatment increases particle uptake and affects the uptake mechanism.Keywords: nanodiamonds, cell uptake, cancer cells, HT29 cells, NV centers, imaging

Abstract Peptide coacervates self‐assembling via liquid‐liquid phase separation are appealing intracellular delivery vehicles of macromolecular therapeutics (proteins, DNA, mRNA) owing to their non‐cytotoxicity, high encapsulation capacity, and efficient cellular uptake. However, the mechanisms by which these viscoelastic droplets cross the cellular membranes remain unknown. Here, using multimodal imaging, data analytics, and biochemical inhibition assays, we identify the key steps by which droplets enter the cell. We find that the uptake follows a non‐canonical pathway and instead integrates essential features of macropinocytosis and phagocytosis, namely active remodeling of the actin cytoskeleton and appearance of filopodia‐like protrusions. Experiments using giant unilamellar vesicles show that the coacervates attach to the bounding membrane in a charge‐ and cholesterol‐dependent manner but do not breach the lipid bilayer barrier. Cell uptake in the presence of small molecule inhibitors – interfering with actin and tubulin polymerization – confirm the active role of cytoskeleton remodeling, most prominently evident in electron microscopy imaging. These findings suggest a peculiar internalization mechanism for viscoelastic, glassy coacervate droplets combining features of non‐specific uptake of fluids by macropinocytosis and particulate uptake of phagocytosis. The broad implications of this study will enable to enhance the efficacy and utility of coacervate‐based strategies for intracellular delivery of macromolecular therapeutics.

Apohemoprotein is focused on the field of theranostics, serving as a porphyrin carrier. Hemoglobin (Hb) consists of α2β2 tetramer with iron(II)‐protoporphyrin IX (heme) bound to each globin. However, heme‐removed Hb (apoHb) causes dissociation at αβ interfaces and aggregation under physiological conditions. We synthesized a stable apoHb derivative comprising intramolecular‐crosslinked apoHb (apoXHb) and human serum albumin (HSA), apoXHb‐HSA3. ApoXHb‐HSA3 engendered no aggregates in the physiological solutions. Moreover, apoXHb‐HSA3 was reconstituted with zinc(II)‐protoporphyrin IX (ZnP), generating ZnXHb‐HSA3, a potent photosensitizer for photodynamic therapy (PDT). The photophysical properties of ZnXHb‐HSA3 were identical to those of zinc‐substituted XHb (ZnXHb). Cellular uptake behavior was evaluated using various cancer cell lines. ZnXHb‐HSA3 released ZnP around the cells, and the free ZnP penetrated cell membranes. In contrast, protein units were not observed within the cells. ZnXHb‐HSA3 showed no cytotoxicity under dark conditions and demonstrated superior PDT activity in comparison to naked ZnXHb. ZnXHb‐HSA3 acts as an innovative porphyrin carrier for enhanced PDT. [ABSTRACT FROM AUTHOR]

Magnetic nanoparticles have gained attention as a potential structure for therapy and diagnosing oncological diseases. The key property of the magnetic nanoparticles is the ability to respond to an external magnetic field. It is known that magnetofection causes an increase in the cellular uptake of RNA and DNA in complexes with magnetic nanoparticles in the presence of a permanent magnetic field. However, the influence of a dynamic magnetic field on the internalization of MNPs is not clear. In this work, we propose the idea that applying external low-frequency dynamic magnetic fields may decrease the cellular uptake, such as macrophages and malignant neuroblastoma. Using fluorescence microscopy and atomic emission spectroscopy, we found that oscillating magnetic fields decreased the cellular uptake of magnetic nanoparticles compared to untreated cells by up to 46%. In SH-SY5Y tumor cells and macrophage RAW264.7 cells, the absolute values of Fe per cell differed by 0.10 pg/cell and 0.33 pg/cell between treated and untreated cells, respectively. These results can be applied in the control of the cellular uptake in different areas of biomedicine. [ABSTRACT FROM AUTHOR]

Phenylketonuria (PKU) is a rare inherited metabolic disease characterized by phenylalanine hydroxylase enzyme deficiency. In PKU patients, coenzyme Q10 (CoQ10) levels were found low. Therefore, we focused on the modification of CoQ10 to load the micelles and increase entry of micelles into the cell and mitochondria, and it is taking a part in ATP turnover. Micelles had produced by comparing two different production methods (thin-film layer and direct-dissolution), and characterization studies were performed (zeta potential, size, and encapsulation efficiency). Then, l-arginine (LARG) and poly-arginine (PARG) were incorporated with the micelles for subsequential release and PKU cell studies. The effects of these components on intracellular uptake and their use in the cellular cycle were analyzed by ELISA, Western blot, membrane potential measurement, and flow cytometry methods. In addition, both effects of LARG and PARG micelles on pharmacokinetics at the cellular level and their cell binding rate were determined. The thin-film method was found superior in micelle preparation. PARG/LARG-modified micelles showed sustained release. In the cellular and mitochondrial uptake of CoQ10, CoQ10-micelle + PARG > CoQ10-micelle + LARG > CoQ10-micelle > CoQ10 was found. This increased localization caused lowering of oxygen consumption rates, but maintaining mitochondrial membrane potential. The study results had showed that besides micelle formulation, PARG and LARG are effective in cellular and mitochondrial targeting. [ABSTRACT FROM AUTHOR]

[Display omitted] We apply a combination of polycaprolactone (PCL)-thiol ligand functionalization with flow-controlled microfluidic block copolymer self-assembly to produce biocompatible gold nanoparticle (GNP)-loaded micellar polymer nanoparticles (GNP-PNPs) in which GNPs are encapsulated within PCL cores surrounded by an external layer of poly(ethylene glycol) (PEG). By varying both the relative amount of block copolymer and the microfluidic flow rate, a series of GNP-PNPs are produced in which the mean number of GNPs per PNP in the < 50-nm fraction (Z ave, d < 50 nm) varies between 0.1 and 1.9 while the external PEG surface is constant. Z ave, d < 50 nm values are determined by statistical analysis of TEM images and compared with the results of cell uptake experiments on MDA-MB-231 cancer cells. For Z ave, d < 50 nm ≤ 1 (including a control sample of individual GNPs also with a PEG surface layer), cell uptake is relatively constant, but increases sharply for Z ave, d < 50 nm > 1, with a factor of 7 enhancement as Z ave, d < 50 nm increases from 1 to ∼2. Enabled by the shear processing control provided by the microfluidic chip, these results provide the first evidence that cellular uptake can be enhanced specifically by increasing the number of GNPs per vector, with other parameters, including polymeric material, internal structure, and external surface chemistry, held constant. They also demonstrate a versatile platform for packaging GNPs in biocompatible polymeric carriers with flow-controlled formulation optimization for various therapeutic and diagnostic applications. [ABSTRACT FROM AUTHOR]

The influence of a protein corona on the uptake of nanoparticles in cells has been demonstrated in various publications over the last years. Extracellular vesicles (EVs), can be seen as natural nanoparticles. However, EVs are produced under different cell culture conditions and little is known about the protein corona forming on EVs and its influence on their uptake by target cells. Here, we use a proteomic approach in order to analyze the protein composition of the EVs themselves and the protein composition of a human blood plasma protein corona around EVs. Moreover, we analyze the influence of the protein corona on EV uptake into human monocytes and compare it with the influence on the uptake of engineered liposomes. We show that the presence of a protein corona increases the uptake of EVs in human monocytes. While for liposomes this seems to be triggered by the presence of immunoglobulins in the protein corona, for EVs blocking the Fc receptors on monocytes did not show an influence of uptake. Therefore, other mechanisms of docking to the cell membrane and uptake are most like involved, demonstrating a clear difference between EVs and liposomes as technically produced nanocarriers. [ABSTRACT FROM AUTHOR]

(1) Background: In neuroendocrine tumors (NETs), somatostatin receptor subtype 2 is highly expressed, which can be targeted by a radioactive ligand such as [177Lu]Lu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid-[Tyr3,Thr8]-octreotide (177Lu-DOTA-TOC) and, more recently, by a lead specific chelator (PSC) containing 203/212Pb-PSC-PEG2-TOC (PSC-TOC). The molar activity (AM) can play a crucial role in tumor uptake, especially in receptor-mediated uptake, such as in NETs. Therefore, an investigation of the influence of different molar activities of 203/212Pb-PSC-TOC on cell uptake was investigated. (2) Methods: Optimized radiolabeling of 203/212Pb-PSC-TOC was performed with 50 µg of precursor in a NaAc/AcOH buffer at pH 5.3–5.5 within 15–45 min at 95° C. Cell uptake was studied in AR42 J, HEK293 sst2, and ZR75-1 cells. (3) Results: 203/212Pb-PSC-TOC was radiolabeled with high radiochemical purity >95% and high radiochemical yield >95%, with AM ranging from 0.2 to 61.6 MBq/nmol. The cell uptake of 203Pb-PSC-TOC (AM = 38 MBq/nmol) was highest in AR42 J (17.9%), moderate in HEK293 sstr (9.1%) and lowest in ZR75-1 (0.6%). Cell uptake increased with the level of AM. (4) Conclusions: A moderate AM of 15–40 MBq/nmol showed the highest cell uptake. No uptake limitation was found in the first 24–48 h. Further escalation experiments with even higher AM should be performed in the future. It was shown that AM plays an important role because of its direct dependence on the cellular uptake levels, possibly due to less receptor saturation with non-radioactive ligands at higher AM. [ABSTRACT FROM AUTHOR]

Abstract The influence of a protein corona on the uptake of nanoparticles in cells has been demonstrated in various publications over the last years. Extracellular vesicles (EVs), can be seen as natural nanoparticles. However, EVs are produced under different cell culture conditions and little is known about the protein corona forming on EVs and its influence on their uptake by target cells. Here, we use a proteomic approach in order to analyze the protein composition of the EVs themselves and the protein composition of a human blood plasma protein corona around EVs. Moreover, we analyze the influence of the protein corona on EV uptake into human monocytes and compare it with the influence on the uptake of engineered liposomes. We show that the presence of a protein corona increases the uptake of EVs in human monocytes. While for liposomes this seems to be triggered by the presence of immunoglobulins in the protein corona, for EVs blocking the Fc receptors on monocytes did not show an influence of uptake. Therefore, other mechanisms of docking to the cell membrane and uptake are most like involved, demonstrating a clear difference between EVs and liposomes as technically produced nanocarriers.

Here, we report a novel dispersion polymerization for the preparation of cross‐linked poly[N‐(2‐hydroxypropyl) methacrylamide] (PHPMA)‐based nanogels in water/2‐methoxyethanol mixture (H2O/MetCel), initiated with potassium persulfate (KPS), and stabilized with poly(vinyl alcohol) 25/140 (PVA) and sodium dodecyl sulfate (SDS). Obtained nanogels were characterized using transmission (TEM) and cryogenic transmission electron microscopy (cryo‐TEM), dynamic light scattering (DLS), asymmetric flow field‐flow fractionation (A4F), nuclear magnetic resonance spectroscopy (NMR), and Raman spectroscopy methods in terms of size, particle size distribution, morphology, and structure. N‐(2‐hydroxypropyl) methacrylamide (HPMA) was copolymerized with 20 wt% ethylene dimethacrylate (EDMA) resulting in 138 nm poly[N‐(2‐hydroxypropyl) methacrylamide‐co‐ethylene dimethacrylate] (PHPMA‐EDMA) nanogel dispersion with irregular shape and core‐shell type structure. Next, we copolymerized HPMA with 20 wt% EDMA and 10 wt% propargyl methacrylate (PMA) to incorporate reactive functionality into the final core‐shell type 120 nm poly[N‐(2‐hydroxypropyl) methacrylamide‐co‐ethylene dimethacrylate‐co‐propargyl methacrylate] (PHPMA‐EDMA‐PMA) nanogel dispersion. Then, the biocompatibility of PHPMA‐EDMA‐PMA nanogel was proved using rat mesenchymal stem cells (rMSC), and human foreskin fibroblasts (BJ). PHPMA‐EDMA‐PMA nanogel was fluorescently labeled with sulfo‐cyanine3 azide resulting in 131 nm nanogel. We performed in vitro uptake studies with fluorescently labeled PHPMA‐EDMA‐PMA nanogel using rMSC showing that the fluorescently labeled PHPMA‐EDMA‐PMA nanogel was well‐distributed in the cytosol and taken up into lysosomes. [ABSTRACT FROM AUTHOR]

Multidrug resistance (MDR) is the main challenge in cancer treatment. In this sense, we designed transferrin (Tf)-conjugated PLGA nanoparticles (NPs) containing an organoselenium compound as an alternative to enhance the efficacy of cancer therapy and sensitize MDR tumor cells. Cytotoxicity studies were performed on different sensitive tumor cell lines and on an MDR tumor cell line, and the Tf-conjugated NPs presented significantly higher antiproliferative activity than the nontargeted counterparts in all tested cell lines. Due to the promising antitumor activity of the Tf-decorated NPs, further studies were performed using the MDR cells (NCI/ADR-RES cell line) comparatively to one sensitive cell line (HeLa). The cytotoxicity of NPs was evaluated in 3D tumor spheroids and, similarly to the results achieved in the 2D assays, the Tf-conjugated NPs were more effective at reducing the spheroid's growth. The targeted Tf-NPs were also able to inhibit tumor cell migration, presented a higher cell internalization and induced a greater number of apoptotic events in both cell lines. Therefore, these findings evidenced the advantages of Tf-decorated NPs over the nontargeted counterparts, with the Tf-conjugated NPs containing an organoselenium compound representing a promising drug delivery system to overcome MDR and enhance the efficacy of cancer therapy. [ABSTRACT FROM AUTHOR]

Improving the stability of drugs in the gastrointestinal tract and their penetration ability in the mucosal layer by implementing a nanoparticle delivery strategy is currently a research focus in the pharmaceutical field. However, for most drugs, nanoparticles failed in enhancing their oral absorption on a large scale (4 folds or above), which hinders their clinical application. Recently, several researchers have proved that the intestinal epithelial cell membrane crossing behaviors of nanoparticles deeply influenced their oral absorption, and relevant reviews were rare. In this paper, we systematically review the behaviors of nanoparticles in the intestinal epithelial cell membrane and mainly focus on their intracellular mechanism. The three key complex intracellular processes of nanoparticles are described: uptake by intestinal epithelial cells on the apical side, intracellular transport and basal side exocytosis. We believe that this review will help scientists understand the in vivo performance of nanoparticles in the intestinal epithelial cell membrane and assist in the design of novel strategies for further improving the bioavailability of nanoparticles. [ABSTRACT FROM AUTHOR]

The interaction between collagen/collagen-like peptides and the commonly expressed immune cell receptor LAIR-1 (leukocyte-associated immunoglobulin-like receptor-1) regulates and directs immune responses throughout the body. Understanding and designing these interactions within the context of biomaterials could advance the development of materials used in medical applications. In this study, we investigate the immunomodulatory effects of biomaterials engineered to display a human collagen III-derived ligand peptide (LAIR1-LP) that targets LAIR-1. Specifically, we examine the effects of LAIR1-LP functionalized surfaces on uptake of polymeric particles and cell debris by macrophages polarized toward inflammatory or healing phenotypes. We observed that culture of macrophages on LAIR1-LP functionalized surfaces increased their uptake of PLGA micro- and nano-particles, as well as apoptotic fibroblasts, while reducing their secretion of TNFα in response to LPS/IFNγ pro-inflammatory stimulation, when compared to cells seeded on control surfaces. To investigate the role of LAIR-1 in the observed LAIR1-LP-induced effects, we used siRNA to knock down LAIR-1 expression and found that cells lacking LAIR-1 exhibited enhanced particle uptake on LAIR1-LP and control surfaces. Furthermore, analysis of gene expression showed that LAIR-1 knockdown led to increase expression of other receptors involved in cell uptake, including CD-36, SRA-1, and beta-2 integrin. Together, our study suggests that LAIR1-LP enhances macrophage uptake potentially through interactions with collagen-domain binding surface receptors, and inhibits inflammation through interaction with LAIR-1.

Despite the fact of availability of several treatments for breast cancer, most of them fail to attain the desired therapeutic response due to their poor bioavailability, high doses, non-selectivity and as a result systemic toxicity. Here in an attempt made to study the transdermal effect of leflunomide (LEF) against breast cancer. In order to improve the poor physicochemical properties of LEF, it was loaded into cubosomes. Cubosomes were prepared by the emulsification method. Colloidal characteristics of cubosomes including particle size, ζ-potential, entrapment efficiency, in-vitro release profile and ex-vivo permeation were studied. In addition, morphology, stability, cytotoxicity and cell uptake in MDA-MB-231 cell line were carried out for the selected cubosomal formulation. The selected LEF loaded cubosomal formulation showed a small particle size (168 ± 1.08) with narrow size distribution (PI 0.186 ± 0.125) and negative ζ potential (–25.5 ± 0.98). Its Entrapment efficiency (EE%) was 93.2% and showed sustained release profile that extended for 24 h. The selected formulation showed stability when stored at 25 °C for three months in terms of size and EE%. TEM images illustrated the cubic structure of the cubosome. Cell culture results revealed the superiority of LEF cubosomes compared to LEF suspension in their cytotoxic effects with an IC50 close to that of doxorubicin. Furthermore, LEF cell uptake was significantly higher for LEF cubosomes. This may be attributed to the effect of nano-encapsulation on enhancing drug pharmacological effects and uptake indicating the potential usefulness of LEF cubosomes for breast cancer management.

Polystyrene (PS) and di-(2-ethylhexyl) phthalate (DEHP) exist widely in the environment. However, their distribution in organisms remains unclear. We used three sizes (50 nm, 500 nm, and 5 μm) of PS and DEHP to study the distribution and accumulation of PS, DEHP, and mono(2-ethylhexyl) phthalate (MEHP) in mice and nerve cell models (HT22 and BV2 cells) and their potential toxicity. Results showed that PS entered the blood of mice, and the distribution of different particle sizes in different tissues was different. After the combined exposure to PS and DEHP, PS carried DEHP, which significantly increased the DEHP content and MEHP content and the highest content of MEHP was in the brain. With the decrease in PS particle size, the contents of PS, DEHP, and MEHP in the body increased. The levels of inflammatory factors were increased in the serum of the PS or/and DEHP group. In addition, 50 nm polystyrene can carry MEHP into nerve cells. These results suggest for the first time that PS and DEHP combined exposure can induce systemic inflammation, and the brain is an important target organ of PS and DEHP combined exposure. This study may serve as a reference for further evaluation of the neurotoxicity induced by combined exposure to PS and DEHP. [ABSTRACT FROM AUTHOR]

Magnetic nanoparticles have gained attention as a potential structure for therapy and diagnosing oncological diseases. The key property of the magnetic nanoparticles is the ability to respond to an external magnetic field. It is known that magnetofection causes an increase in the cellular uptake of RNA and DNA in complexes with magnetic nanoparticles in the presence of a permanent magnetic field. However, the influence of a dynamic magnetic field on the internalization of MNPs is not clear. In this work, we propose the idea that applying external low-frequency dynamic magnetic fields may decrease the cellular uptake, such as macrophages and malignant neuroblastoma. Using fluorescence microscopy and atomic emission spectroscopy, we found that oscillating magnetic fields decreased the cellular uptake of magnetic nanoparticles compared to untreated cells by up to 46%. In SH-SY5Y tumor cells and macrophage RAW264.7 cells, the absolute values of Fe per cell differed by 0.10 pg/cell and 0.33 pg/cell between treated and untreated cells, respectively. These results can be applied in the control of the cellular uptake in different areas of biomedicine.

Plant virus-based nanoparticles (VNPs) offer a bioinspired approach to the delivery of drugs and imaging agents. The chemical addressability, biocompatibility, and scalable manufacturability of VNPs make them a promising alternative to synthetic delivery platforms. However, VNPs, just like other proteinaceous or synthetic nanoparticles (NPs), are readily recognized and cleared by the immune system and mechanisms such as opsonization and phagocytosis. Shielding strategies, such as PEGylation, are commonly used to mitigate premature NP clearance. Here, we investigated polyethylene glycol (PEG) coatings on the tobacco mosaic virus (TMV), which was used as a model nanocarrier system. Specifically, we evaluated the effects of linear and multivalent PEG coatings at varying chain lengths on serum protein adsorption, antibody recognition, and macrophage uptake. Linear and multivalent PEGs of molecular weights 2,000 and 5,000 Da were successfully grafted onto the TMV at ≈ 20%–60% conjugation efficiencies, and the degree of cross-linking as a function of PEG valency and length was determined. PEGylation resulted in the modulation of TMV–macrophage interactions and reduced corona formation as well as antibody recognition. Linear and multivalent PEG 5,000 formulations (but not PEG 2,000 formulations) reduced α-TMV antibody recognition, whereas shorter, multivalent PEG coatings significantly reduced α-PEG recognition—this highlights an interesting interplay between the NP and the PEG itself in potential antigenicity and should be an important consideration in PEGylation strategies. This work provides insight into the PEGylation of VNPs, which may improve the possibility of their implementation in clinical applications.

How many nanoparticles are taken up by human cells is a key question for many applications, both within medicine and safety. While many methods have been developed and applied to this question, microscopy-based methods present some unique advantages. However, the laborious nature of microscopy, in particular the consequent image analysis, remains a bottleneck. Automated image analysis has been pursued to remedy this situation, but offers its own challenges. Here we tested the recently developed deep-learning based cell identification algorithm Cellpose on fluorescence microscopy images of HeLa cells. We found that the algorithm performed very well, and hence developed a workflow that allowed us to acquire, and analyse, thousands of cells in a relatively modest amount of time, without sacrificing cell identification accuracy. We subsequently tested the workflow on images of cells exposed to fluorescently-labelled polystyrene nanoparticles. This dataset was then used to study the relationship between cell size and nanoparticle uptake, a subject where high-throughput microscopy is of particular utility.

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The lack of knowledge about the uptake of NPs by biological cells poses a significant problem for drug delivery. For this reason, designing an appropriate model is the main challenge for modelers. To address this problem, molecular modeling studies that can describe the mechanism of cellular uptake of drug-loaded nanoparticles have been conducted in recent decades. In this context, we developed three different models for the amphipathic nature of drug-loaded nanoparticles (MTX-SS-γ-PGA), whose cellular uptake mechanism was predicted by molecular dynamics studies. Many factors affect nanoparticle uptake, including nanoparticle physicochemical properties, protein–particle interactions, and subsequent agglomeration, diffusion, and sedimentation. Therefore, the scientific community needs to understand how these factors can be controlled and the NP uptake of nanoparticles. Based on these considerations, in this study, we investigated for the first time the effects of the selected physicochemical properties of the anticancer drug methotrexate (MTX) grafted with hydrophilic-γ-polyglutamic acid (MTX-SS-γ-PGA) on its cellular uptake at different pH values. To answer this question, we developed three theoretical models describing drug-loaded nanoparticles (MTX-SS-γ-PGA) at three different pH values, such as (1) pH 7.0 (the so-called neutral pH model), (2) pH 6.4 (the so-called tumor pH model), and (3) pH 2.0 (the so-called stomach pH model). Exceptionally, the electron density profile shows that the tumor model interacts more strongly with the head groups of the lipid bilayer than the other models due to charge fluctuations. Hydrogen bonding and RDF analyses provide information about the solution of the NPs with water and their interaction with the lipid bilayer. Finally, dipole moment and HOMO-LUMO analysis showed the free energy of the solution in the water phase and chemical reactivity, which are particularly useful for determining the cellular uptake of the NPs. The proposed study provides fundamental insights into molecular dynamics (MD) that will allow researchers to determine the influence of pH, structure, charge, and energetics of NPs on the cellular uptake of anticancer drugs. We believe that our current study will be useful in developing a new model for drug delivery to cancer cells with a much more efficient and less time-consuming model. [ABSTRACT FROM AUTHOR]

Calcium ion is vital for the regulation of many cellular functions and serves as a second messenger in the signal transduction pathways. Once the intracellular Ca2+ level exceeds the tolerance of cells (called Ca2+ overload), oxidative stress, mitochondrial damage, and cell/mitochondria apoptosis happen. Therefore, Ca2+ overload has started to be deeply exploited as a new strategy for cancer therapy due to its high efficiency and satisfactory safety. This review aims to highlight the recent development of Ca2+‐based nanomaterials (such as Ca3(PO4)2, CaCO3, CaO2, CaH2, CaS, and others) able to trigger intracellular Ca2+ overload and apoptosis in cancer therapy. The intracellular mechanisms of varied Ca2+‐based nanomaterials and the different types of strategies to enhance Ca2+ overload are discussed in detail. Moreover, the design of more efficient Ca2+ overload‐mediated cancer therapies is prospected mainly based on 1) the enhanced cellular uptake by surface modification and morphology optimization of nanomaterials, 2) the accelerated Ca2+ release from nanomaterials by increasing the intracellular H+ level and by photothermal effect, and 3) the overload maintenance by Ca2+ efflux inhibition, Ca2+ influx promotion, or promoting Ca2+ release from the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Gelatin is a biocompatible, biodegradable, cheap, and nontoxic material, which is already used for pharmaceutical applications. Nanoparticles from gelatin (GNPs) are considered a promising delivery system for hydrophilic and macromolecular drugs. Mechanical properties of particles are recognized as an important parameter affecting drug carrier interaction with biological systems. GNPs offer the preparation of particles with different stiffness. GNPs were loaded with Fluorescein isothiocyanate-labeled 150 kDa dextran (FITC-dextran) yielding also different elastic properties. GNPs were visualized using atomic force microscopy (AFM), and force–distance curves from the center of the particles were evaluated for Young's modulus calculation. The prepared GNPs have Young's moduli from 4.12 MPa for soft to 9.8 MPa for stiff particles. Furthermore, cytokine release (IL-6 and TNF-α), cell viability, and cell uptake were determined on macrophage cell lines from mouse (RAW 264.7) and human (dTHP-1 cells, differentiated human monocytic THP-1 cells) origin for soft and stiff GNPs. Both particle types showed good cell compatibility and did not induce IL-6 and TNF-α release from RAW 264.7 and dTHP-1 cells. Stiffer GNPs were internalized into cells faster and to a larger extent. [ABSTRACT FROM AUTHOR]

[Display omitted] Uniform sodium-dysprosium double molybdate (NaDy(MoO 4) 2) nanoparticles having different morphologies (spheres and ellipsoids) and tunable size have been synthesized for the first time in literature. The procedure is based on a homogeneous precipitation process at moderated temperatures (≤220 °C) from solutions containing appropriated precursors dissolved in ethylene glycol-water mixtures, in the absence (spheres) or the presence (ellipsoids) of tartrate anions. The effects of the morphological characteristics (size and shape) of the nanoparticles on the magnetic relaxivity at high field (9.4 T) have been evaluated finding that the latter magnitude was higher for the spheres than for the ellipsoids, indicating their better suitability as contrast agents for high-field magnetic resonance imaging. Such nanoparticles have been successfully coated with polymers bearing carboxylate functional groups through a layer-by-layer process, which improves the colloidal stability of the nanoparticles in physiological media. It has been also found that the coating layer had no significant effects on the nanoparticles relaxivity and that such coated nanoparticles exhibited a high biocompatibility and a high chemical stability. In summary, we have developed NaDy(MoO 4) 2 based bioprobes which meet the required criteria for their use as contrast agents for high-field magnetic resonance imaging. [ABSTRACT FROM AUTHOR]

Computational microscopy is a crucial tool for studying internalization of Au nanohexapods (AuNHs) and can be applied in designing nanostructures for biomedical applications. To further explore the molecular insights into interactions between AuNHs and a plasma membrane (PM), we conducted a coarse-grained molecular dynamic (CGMD) simulation. This simulation aimed to investigate the cellular internalization pathways of AuNHs, considering factors such as sizes (6, 8, 10, and 12 nm diameters) varying rod lengths (LR_S0.6) and spheres at end edges (LR_S0.9), shapes (varying sphere diameters (DS) end edges, and varying pyramid cores (DC) for fixed size at 8 nm AuNH), and surface-charge modifications (neutral and negatively charged). The simulation results indicated that size, shape, and surface modification significantly affect cellular uptake. Each AuNH size facilitated interaction and translocation through the PM by direct permeation and an endocytosis process, utilizing sharp ends or edges to disrupt/penetrate the PM and subsequently enter the inner cell. Direct translocations of shapes (DS and DC AuNHs) were not observed. The surface-charged modification (8 nm sized AuNHs) showed the direct translocation process. When comparing the cellular uptake of shaped AuNHs, the DC AuNH exhibited a higher free-energy barrier. Notably, anionic AuNHs demonstrated the lowest free-energy barrier among the various surface-charged AuNHs compared with neutrally charged AuNHs and bare AuNHs, resulting in anionic charges that were more easily translocated into the inner cell than other shapes and charges. Additionally, the permeability of the surface covering was lower than the unmodified surface due to charges and hydrocarbons facilitating translocation. These findings offer a comprehensive understanding of interactions between AuNHs with various characteristics and a realistic PM, thereby providing insights for the design of nanostructures with biological applications. [Display omitted] [ABSTRACT FROM AUTHOR]

Morin, a bioflavonoid, is known for its wide range of pharmacological benefits. However, poor aqueous solubility and oral bioavailability are significant disadvantages of morin. In this study, the morin/hydroxypropyl-β-cyclodextrin inclusion complexes (MCD IC-1 and MCD IC-2) were prepared, and their bioavailability, cell uptake, toxicity were studied. MCD IC-1 and MCD IC-2 showed enhanced aqueous solubility and oral bioavailability than free morin. Free morin showed limited aqueous solubility (0.104 ± 0.37 ng/μl), while MCD IC-1 and MCD IC-2 significantly enhanced solubility to 136.36 ± 0.18 ng/μl and 188.35 ± 0.32 ng/μl respectively. The oral bioavailability of MCD IC-2 was 4.55 times higher than free morin, with a Cmax of 860.95 ng/ml compared to 250.70 ng/ml of free morin, and area under the concentration-time curve from 0 to 24 h (AUC 0-24) was 4779.88 ng/ml.h for MCD IC-2 compared to 1050.32 ng/ml.h for free morin. In vitro cell uptake study in LX-2 cells revealed enhanced uptake of MCD IC-2. Also, MCD IC-2 treatment to culture activated LX-2 cells showed dose-dependent cytotoxicity. All these results highlighted that the MCD IC-2 is likely to improve in vivo oral bioavailability and inhibition of LX-2 cell growth. Further cell culture and pre-clinical studies are essential to determine the anti-fibrotic effect of MCD IC-2 against experimental liver fibrosis cells and animal models. [ABSTRACT FROM AUTHOR]

(1) Background: In neuroendocrine tumors (NETs), somatostatin receptor subtype 2 is highly expressed, which can be targeted by a radioactive ligand such as [177Lu]Lu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴,-tetraacetic acid-[Tyr3,Thr8]-octreotide (177Lu-DOTA-TOC) and, more recently, by a lead specific chelator (PSC) containing 203/212Pb-PSC-PEG2-TOC (PSC-TOC). The molar activity (AM) can play a crucial role in tumor uptake, especially in receptor-mediated uptake, such as in NETs. Therefore, an investigation of the influence of different molar activities of 203/212Pb-PSC-TOC on cell uptake was investigated. (2) Methods: Optimized radiolabeling of 203/212Pb-PSC-TOC was performed with 50 µg of precursor in a NaAc/AcOH buffer at pH 5.3–5.5 within 15–45 min at 95° C. Cell uptake was studied in AR42 J, HEK293 sst2, and ZR75-1 cells. (3) Results: 203/212Pb-PSC-TOC was radiolabeled with high radiochemical purity >95% and high radiochemical yield >95%, with AM ranging from 0.2 to 61.6 MBq/nmol. The cell uptake of 203Pb-PSC-TOC (AM = 38 MBq/nmol) was highest in AR42 J (17.9%), moderate in HEK293 sstr (9.1%) and lowest in ZR75-1 (0.6%). Cell uptake increased with the level of AM. (4) Conclusions: A moderate AM of 15–40 MBq/nmol showed the highest cell uptake. No uptake limitation was found in the first 24–48 h. Further escalation experiments with even higher AM should be performed in the future. It was shown that AM plays an important role because of its direct dependence on the cellular uptake levels, possibly due to less receptor saturation with non-radioactive ligands at higher AM.

Despite the fact of availability of several treatments for breast cancer, most of them fail to attain the desired therapeutic response due to their poor bioavailability, high doses, non-selectivity and as a result systemic toxicity. Here in an attempt made to study the transdermal effect of leflunomide (LEF) against breast cancer. In order to improve the poor physicochemical properties of LEF, it was loaded into cubosomes. Cubosomes were prepared by the emulsification method. Colloidal characteristics of cubosomes including particle size, f-potential, entrapment efficiency, in-vitro release profile and exvivo permeation were studied. In addition, morphology, stability, cytotoxicity and cell uptake in MDAMB-231 cell line were carried out for the selected cubosomal formulation. The selected LEF loaded cubosomal formulation showed a small particle size (168 ± 1.08) with narrow size distribution (PI 0.186 ± 0.125) and negative f potential (-25.5 ± 0.98). Its Entrapment efficiency (EE%) was 93.2% and showed sustained release profile that extended for 24 h. The selected formulation showed stability when stored at 25 °C for three months in terms of size and EE%. TEM images illustrated the cubic structure of the cubosome. Cell culture results revealed the superiority of LEF cubosomes compared to LEF suspension in their cytotoxic effects with an IC50 close to that of doxorubicin. Furthermore, LEF cell uptake was significantly higher for LEF cubosomes. This may be attributed to the effect of nanoencapsulation on enhancing drug pharmacological effects and uptake indicating the potential usefulness of LEF cubosomes for breast cancer management. [ABSTRACT FROM AUTHOR]

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno‐)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross‐reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid‐based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell–single vesicle level, thereby enabling high content EV cell uptake assays and plate‐based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open‐source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities. [ABSTRACT FROM AUTHOR]

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Chagas disease, caused by the protozoan Trypanosoma cruzi, is an important public health problem in Latin America. Nanoencapsulation of anti-T. cruzi drugs has significantly improved their efficacy and reduced cardiotoxicity. Thus, we investigated the in vitro interaction of polyethylene glycol-block-poly(D,L-lactide) nanocapsules (PEG-PLA) with trypomastigotes and with intracellular amastigotes of the Y strain in cardiomyoblasts, which are the infective forms of T. cruzi, using fluorescence and confocal microscopy. Fluorescently labeled nanocapsules (NCs) were internalized by non-infected H9c2 cells toward the perinuclear region. The NCs did not induce significant cytotoxicity in the H9c2 cells, even at the highest concentrations and interacted equally with infected and non-infected cells. In infected cardiomyocytes, NCs were distributed in the cytoplasm and located near intracellular amastigote forms. PEG-PLA NCs and trypomastigote form interactions also occurred. Altogether, this study contributes to the development of engineered polymeric nanocarriers as a platform to encapsulate drugs and to improve their uptake by different intra- and extracellular forms of T. cruzi, paving the way to find new therapeutic strategies to fight the causative agent of Chagas disease. [ABSTRACT FROM AUTHOR]

Macrophages are the main cells present in the synovial membrane. They play an important role in the development and progression of osteoarthritis (OA). After the establishment of the disease macrophages mostly adopt a pro-inflammatory secretory phenotype (OA phenotype) further inducing cartilage degradation. Indomethacin (IND) is a non-steroidal anti-inflammatory drug (NSAID) able to inhibit the synthesis of prostaglandins mediated by both cyclooxygenase isoforms depicting a potent anti-inflammatory capacity. However, the lack of specificity and short half-like of free drugs within the joint cavity limits its utility in controlling inflammation after intra-articular administration. This study aims at developing IND loaded glycosylated nanostructured lipid carriers (NLCs) to selectively target macrophages and promote their reprogramming to an anti-inflammatory phenotype. This approach focused on the local administration of the NLCs, offers a promising therapeutic strategy for treating OA by modulating the inflammatory environment within the joint. NLCs will be designed by combining experimental and in silico docking analyses, and thoroughly characterized to obtain drug delivery systems with high stability and suitable physicochemical properties. The proposed mannose-functionalized systems exhibited adequate particle sizes (≈ 70 nm) and positive surface charges (> 20 mV) to be efficiently retained in the joint cavity. Moreover, the developed NLCs demonstrated effective and specific uptake by OA-like macrophages leading to a significant decrease in the secretion of the pro-inflammatory cytokines IL-6, IL-8 and TNF-α similarly to the free drug. Therefore, these systems effectively reprogrammed OA-associated macrophages to adopt a more regenerative phenotype, offering a promising strategy for managing inflammation in OA., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Patricia Diaz-Rodriguez reports financial support was provided by Spanish Ministry of Science and Innovation. Mariana Landin reports financial support was provided by Xunta de Galicia. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper]., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The potential health implications of environmental micro/nanoplastics (MNPLs) are increasingly concerning. Beyond environmental exposure, other sources such as food packaging, including herbal/teabags, may also be significant. This study investigates the release of MNPLs from three commercially available teabags. By simulating tea preparation, MNPL samples were extracted and characterized using a range of analytical techniques: scanning electron microscopy (SEM), transmission electron microscopy (TEM), attenuated total reflectance/Fourier transform infrared spectroscopy (ATR-FTIR), dynamic light scattering (DLS), laser Doppler velocimetry (LDV), and nanoparticle tracking analysis (NTA). The results confirmed that the teabags were made of nylon-6 (NY6), polypropylene (PP), and cellulose (CL) and that microfibers and nano-range particles (NPLs) were present in the leachates. NTA data revealed that the number of released NPLs was 1.20 × 10 9 /mL (PP; 136.7 nm), 1.35 × 10 8 /mL (CL; 244 nm), and 8.18 × 10 6 /mL (NY6; 138.4). The leachate particles were then stained with iDye Poly-Pink and used to expose three human intestine-derived cell types (Caco-2, HT29, and HT29-MTX) to assess their biointeractions and the role of the mucosubstances in vitro. The results demonstrated that after 24 h of exposure to 100 μg/mL NPLs, there was significant uptake of PP-NPLs in HT29-MTX cells, as a model of cells segregating high amount of mucus. A similar uptake was observed for CL-NPLs in HT29 and HT29-MTX cells, while NY6-NPLs were internalized preferentially in Caco-2 cells. These findings underscore the importance of identifying new environmentally relevant MNPL exposure sources, for developing realistic MNPLs samples, and further investigating their potential human health effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Polymeric micelles have widely been used as drug delivery carriers, and recently, single-chain nanoparticles (SCNPs) emerged as potential, smaller-sized, alternatives. In this work, we are comparing both NPs side by side and evaluate their ability to be internalized by breast cancer cells (MCF-7) and macrophages (RAW 264.7). To be able to generate these NPs on demand, the polymers were assembled by flow, followed by the stabilization of the structures by photocross-linking using blue light. The central aim of this work is to evaluate how the type of solvent affects self-assembly and ultimately the structure of the final NP. Therefore, a library of copolymers with different sequences, including block copolymers (AB, ABA, BAB), and statistical copolymers (rAB and rAC) was synthesized using PET-RAFT with A denoting poly(ethylene glycol) methyl ether acrylate (PEGMEA), B as 2-hydroxyethyl acrylate (HEA), and C as 4-hydroxybutyl acrylate (HBA). The polymers were conjugated with a quinoline derivative to enable the formation of cross-linked structures by photocross-linking during flow assembly. Using water as the dispersant for photocross-linking led to the preassembly of these amphiphilic polymers into compact SCNPs and cross-linked micelles, resulting in a quick photoreaction. In contrast, acetonitrile led to fully dissolved polymers but a low rate of the photoreaction. These intramolecularly cross-linked polymers were then placed in water to result in more dynamic micelles and looser SCNPs. Small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and size exclusion chromatography (SEC) coupled with a viscosity detector show that cross-linking in acetonitrile results in better-defined NPs with a shell rich in PEGMEA. Cross-linking in acetonitrile led to NPs with significantly higher cellular uptake. Interestingly, passive transport was identified as the main pathway for the delivery of our NPs on MCF-7 cells, confirmed by the uptake of NPs on cells treated with inhibitors and by red blood cells. This work underscored the importance of the polymer precursor's structure and the choice of solvent during intramolecular cross-linking in determining the drug delivery efficiency and biological behavior of SCNPs.

Abstract Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed ‘EVAnalyzer’, an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno‐)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross‐reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid‐based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell–single vesicle level, thereby enabling high content EV cell uptake assays and plate‐based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open‐source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

Multidrug resistance (MDR) is the main challenge in cancer treatment. In this sense, we designed transferrin (Tf)-conjugated PLGA nanoparticles (NPs) containing an organoselenium compound as an alternative to enhance the efficacy of cancer therapy and sensitize MDR tumor cells. Cytotoxicity studies were performed on different sensitive tumor cell lines and on an MDR tumor cell line, and the Tf-conjugated NPs presented significantly higher antiproliferative activity than the nontargeted counterparts in all tested cell lines. Due to the promising antitumor activity of the Tf-decorated NPs, further studies were performed using the MDR cells (NCI/ADR-RES cell line) comparatively to one sensitive cell line (HeLa). The cytotoxicity of NPs was evaluated in 3D tumor spheroids and, similarly to the results achieved in the 2D assays, the Tf-conjugated NPs were more effective at reducing the spheroid’s growth. The targeted Tf-NPs were also able to inhibit tumor cell migration, presented a higher cell internalization and induced a greater number of apoptotic events in both cell lines. Therefore, these findings evidenced the advantages of Tf-decorated NPs over the nontargeted counterparts, with the Tf-conjugated NPs containing an organoselenium compound representing a promising drug delivery system to overcome MDR and enhance the efficacy of cancer therapy.

Despite the undisputable role of the protein corona in the biointeractions of liposome drug carriers, the field suffers from a lack of knowledge regarding the patterns of protein deposition on lipid surfaces with different compositions. Here, we investigated the protein coronas formed on liposomes of basic compositions containing combinations of egg phosphatidylcholine (PC), palmitoyloleoyl phosphatidylglycerol (POPG), and cholesterol. Liposome−protein complexes isolated by size-exclusion chromatography were delipidated and analyzed using label-free LC-MS/MS. The addition of the anionic lipid and cholesterol both affected the relative protein abundances (and not the total bound proteins) in the coronas. Highly anionic liposomes, namely those containing 40% POPG, carried corona enriched with cationic proteins (apolipoprotein C1, beta-2-glycoprotein 1, and cathelicidins) and were the least stable in the calcein release assay. Cholesterol improved the liposome stability in the plasma. However, the differences in the corona compositions had little effect on the liposome uptake by endothelial (EA.hy926) and phagocytic cells in the culture (U937) or ex vivo (blood-derived monocytes and neutrophils). The findings emphasize that the effect of protein corona on the performance of the liposomes as drug carriers occurs through compromising particle stability rather than interfering with cellular uptake.

Air liquid interface (ALI) exposure systems are gaining interest, and studies suggest enhanced response of lung cells exposed to particles at ALI as compared to submerged exposure, although the results have been somewhat inconsistent. Previous studies have used monocultures and measured particle deposition using assumptions including consistent particle deposition, particle density, and shape. This study exposed co-cultures of A549 and differentiated THP-1 cells to flame-generated particles using three exposure methods: ALI, pseudo-ALI, and submerged. The dose at ALI was measured directly, reducing the need for assumptions about particle properties and deposition. For all exposure methods an enhanced pro-inflammatory response (TNFα) and Cytochrome P450 (CYP1A1) gene expression, compared to their corresponding negative controls, was observed. ALI exposure induced a significantly greater TNFα response compared to submerged exposure. The submerged exposures exhibited greater induction of CYP1A1 than other exposure methods, although not statistically significant. Some of the factors behind the observed difference in responses for the three exposure methods include differences in physicochemical properties of particles in suspending media, delivered dose, and potential contribution of gas-phase species to cellular response in ALI exposure. However, given the difficulty and expense of ALI exposures, submerged exposure may still provide relevant information for particulate exposures. [ABSTRACT FROM AUTHOR]

Nearly four million yearly deaths can be attributed to respiratory diseases, prompting a huge worldwide health emergency. Additionally, the COVID-19 pandemic's death toll has surpassed six million, significantly increasing respiratory disease morbidity and mortality rates. Despite recent advances, it is still challenging for many drugs to be homogeneously distributed throughout the lungs, and specifically to reach the lower respiratory tract with an accurate sustained dose and minimal systemic side effects. Engineered nanocarriers can provide increased therapeutic efficacy while lessening potential biochemical adverse reactions. Poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer, has attracted significant interest as an inhalable drug delivery system. However, the influence of the nanocarrier surface charge and its intratracheal instillation has not been addressed so far. In this study, we fabricated red fluorescent PLGA nanocapsules (NCs)—Cy5/PLGA—with either positive (Cy5/PLGA+) or negative surface charge (Cy5/PLGA-). We report here on their excellent colloidal stability in culture and biological media, and after cryo-storage. Their lack of cytotoxicity in two relevant lung cell types, even for concentrations as high as 10 mg/mL, is also reported. More importantly, differences in the NCs' cell uptake rates and internalization capacity were identified. The uptake of the anionic system was faster and in much higher amounts—10-fold and 2.5-fold in macrophages and epithelial alveolar cells, respectively. The in vivo study demonstrated that anionic PLGA NCs were retained in all lung lobules after 1 h of being intratracheally instilled, and were found to accumulate in lung macrophages after 24 h, making those nanocarriers especially suitable as a pulmonary immunomodulatory delivery system with a marked translational character. [ABSTRACT FROM AUTHOR]

Thymus immunosuppressive pentapeptide (TIPP) is a novel anti-inflammatory peptide with high efficacy and low toxicity. This study aims to establish a selective LC-MS/MS method for analyzing the analyte TIPP in biological samples, laying the foundation for further PK and PD studies of TIPP. Protein precipitation was conducted in acetonitrile supplemented with 2% formic acid and 25 mg/mL dithiothreitol as a stabilizer, which was followed by backwashing the organic phase using dichloromethane. The chromatographic separation of TIPP was achieved on a C18 column with a gradient elution method. During positive electrospray ionization, TIPP was analyzed via multiple-reaction monitoring. The linear relationships between the concentration of TIPP and peak area in murine plasma cell lysates, supernatants, and the final cell rinse PBS were established within the ranges of 20–5000 ng/mL, 1–200 ng/mL, 10–200 μg/mL, and 0.1–20 ng/mL, respectively (r2 > 0.99). Validated according to U.S. FDA guidelines, the proposed method was proved to be acceptable. Such a method had been successfully applied to investigate the pharmacokinetics of TIPP in mice via subcutaneous injection. The plasma half-life in mice was 5.987 ± 1.824 min, suggesting that TIPP is swiftly eliminated in vivo. The amount of TIPP uptake by RBL-2H3 cells was determined using this method, which was also visually verified by confocal. Furthermore, the effective intracellular concentration of TIPP was deduced by comparing the intracellular concentration of TIPP and degrees of inflammation, enlightening further investigation on the intracellular target and mechanism of TIPP. [ABSTRACT FROM AUTHOR]

In three novel peptidoids based on the tryptophan—histidine—tryptophan (WHW) peptide, the central histidine was replaced by Ala-(triazole), and two derivatives also had one tryptophan replaced with pyrene-alkyls of different lengths and flexibility. Pyrene analogues show strong fluorescence at 480–500 nm, attributed to intramolecular exciplex formation with tryptophan. All three peptidoids bind Cu2+ cation in water with strong affinity, with Trp- Ala-(triazole)-Trp binding comparably to the parent WHW, and the pyrene analogues even stronger, demonstrating that replacement of histidine with triazole in peptides does not hamper Cu2+ coordination. The studied peptidoids strongly bind to ds-DNA and ds-RNA, whereby their complexes with Cu2+ exhibit distinctively different interactions in comparison to metal-free analogues, particularly in the stabilization of ds-DNA against thermal denaturation. The pyrene peptidoids efficiently enter living cells with no apparent cytotoxic effect, whereby their red-shifted emission compared to the parent pyrene allows intracellular confocal microscopy imaging, showing accumulation in cytoplasmic organelles. However, irradiation with 350 nm light resulted in evident antiproliferative effect on cells treated with micromolar concentrations of the pyrene analogues, presumably attributed to pyrene-induced production of singlet oxygen and consecutive cellular damage. [ABSTRACT FROM AUTHOR]

Investigations on the effects of newly constructed glucose-PEG2000-DSPE modified carbamazepine nano system on oxidative stress damage and cell apoptosis in epilepsy. The nano system was constructed by egg yolk lecithin, cholesterol, GLUPEG2000-DSPE, and carbamazepine as per the molar ratio of 95:20:5:6.35. The particle size, zeta potential, and release rate of carbamazepine was determined using a microscope and a microplate reader. The cells toxicity was detected for determine the optimal concentration of carbamazepine nano system. Cell uptake, cell apoptosis ratio and ROS level was determined by flow cytometry analysis. The in vivo studies were performed using male Wistar rats. H&E staining was employed for histological evaluation. Immunofluorescence was utilized for measure the expression level of GLUT1. ELISA assay was obtained for detecting the levels of SOD, GSH-Px and MDA. The results shown the average particle size was 108.57 ± 3.42 nm, and the mean zeta potential was −52.75 ± 1.48 mV. The modified carbamazepine liposomes exhibited higher release rate. Cell uptake indicated that carbamazepine nano system could be successfully internalized into cells. Flow cytometry analysis revealed the carbamazepine nano system dramatically decreased cell apoptosis rate and downregulated ROS level. Moreover, carbamazepine nano system improved histological status, increased GLUT1 expression and decreased oxidative stress in vivo. In conclusion, glucose-PEG2000-DSPE modified carbamazepine nano system ameliorated cell apoptosis and oxidative stress damage in epilepsy. [ABSTRACT FROM AUTHOR]

Black phosphorus nanosheets (BPs) show great potential for various applications including biomedicine, thus their potential side effects and corresponding improvement strategy deserve investigation. Here, in vitro and in vivo biological effects of BPs with and without titanium sulfonate ligand (TiL4 ) modification are investigated. Compared to bare BPs, BPs with TiL4 modification (TiL4 @BPs) can efficiently escape from macrophages uptake, and reduce cytotoxicity and proinflammation. The corresponding mechanisms are also discussed. These findings may not only guide the applications of BPs, but also propose an efficient strategy to further improve the biocompatibility of BPs.

1,5-Disubstituted indole-2-carboxaldehyde derivatives 1a–h and glycine alkyl esters 2a–c are shown to undergo a novel cascade imination-heterocylization in the presence of the organic base DIPEA to provide 1-indolyl-3,5,8-substituted γ-carbolines 3aa–ea in good yields. The γ-carbolines are fluorescent and exhibit anticancer activities against cervical, lung, breast, skin, and kidney cancer cells.

When nanoparticles were introduced into the biological media, the protein corona would be formed, which endowed the nanoparticles with new bio-identities. Thus, controlling protein corona formation is critical to in vivo therapeutic effect. Controlling the particle size is the most feasible method during design, and the influence of media pH which varies with disease condition is quite important. The impact of particle size and pH on bovine serum albumin (BSA) corona formation of solid lipid nanoparticles (SLNs) was studied here. The BSA corona formation of SLNs with increasing particle size (120–480 nm) in pH 6.0 and 7.4 was investigated. Multiple techniques were employed for visualization study, conformational structure study and mechanism study, etc. “BSA corona-caused aggregation” of SLN2‒3 was revealed in pH 6.0 while the dispersed state of SLNs was maintained in pH 7.4, which significantly affected the secondary structure of BSA and cell uptake of SLNs. The main interaction was driven by van der Waals force plus hydrogen bonding in pH 7.4, while by electrostatic attraction in pH 6.0, and size-dependent adsorption was confirmed. This study provides a systematic insight to the understanding of protein corona formation of SLNs.

Improving the stability of drugs in the gastrointestinal tract and their penetration ability in the mucosal layer by implementing a nanoparticle delivery strategy is currently a research focus in the pharmaceutical field. However, for most drugs, nanoparticles failed in enhancing their oral absorption on a large scale (4 folds or above), which hinders their clinical application. Recently, several researchers have proved that the intestinal epithelial cell membrane crossing behaviors of nanoparticles deeply influenced their oral absorption, and relevant reviews were rare. In this paper, we systematically review the behaviors of nanoparticles in the intestinal epithelial cell membrane and mainly focus on their intracellular mechanism. The three key complex intracellular processes of nanoparticles are described: uptake by intestinal epithelial cells on the apical side, intracellular transport and basal side exocytosis. We believe that this review will help scientists understand the in vivo performance of nanoparticles in the intestinal epithelial cell membrane and assist in the design of novel strategies for further improving the bioavailability of nanoparticles.