Microfluidics single-cell encapsulation reveals that poly-l-lysine-mediated stem cell adhesion to alginate microgels is crucial for cell-cell crosstalk and its self-renewal.

Microfluidic cell encapsulation has provided a platform for studying the behavior of individual cells and has become a turning point in single-cell analysis during the last decade. The engineered microenvironment, along with protecting the immune response, has led to increasingly presenting the results of practical and pre-clinical studies with the goals of disease treatment, tissue engineering, intelligent control of stem cell differentiation, and regenerative medicine. However, the significance of cell-substrate interaction versus cell-cell communications in the microgel is still unclear. In this study, monodisperse alginate microgels were generated using a flow-focusing microfluidic device to determine how the cell microenvironment can control human bone marrow-derived mesenchymal stem cells (hBMSCs) viability, proliferation, and biomechanical features in single-cell droplets versus multi-cell droplets. Collected results show insufficient cell proliferation (234 % and 329 %) in both single- and multi-cell alginate microgels. Alginate hydrogels supplemented with poly-l-lysine (PLL) showed a better proliferation rate (514 % and 780 %) in a comparison of free alginate hydrogels. Cell stiffness data illustrate that hBMSCs cultured in alginate hydrogels have higher membrane flexibility and migration potency (Young's modulus equal to 1.06 kPa), whereas PLL introduces more binding sites for cell attachment and causes lower flexibility and migration potency (Young's modulus equal to 1.83 kPa). Considering that cell adhesion is the most important parameter in tissue engineering, in which cells do not run away from a 3D substrate, PLL enhances cell stiffness and guarantees cell attachments. In conclusion, cell attachment to PLL-mediated alginate hydrogels is crucial for cell viability and proliferation. It suggests that cell-cell signaling is good enough for stem cell viability, but cell-PLL attachment alongside cell-cell signaling is crucial for stem cell proliferation and self-renewal.

Safety and immunogenicity of COReNAPCIN, a SARS-CoV-2 mRNA vaccine, as a fourth heterologous booster in healthy Iranian adults: A double-blind, randomized, placebo-controlled, phase 1 clinical trial with a six-month follow-up.

The recurrent COVID-19 infection, despite global vaccination, highlights the need for booster doses. A heterologous booster has been suggested to enhance immunity and protection against emerging variants of concern of the SARS-CoV-2 virus. In this report, we aimed to assess the safety, and immunogenicity of COReNAPCIN, as a fourth booster dose after three doses of inactivated vaccines. METHODS: The study was conducted as a double-blind, randomized, placebo-controlled phase 1 clinical trial of the mRNA-based vaccine candidate, COReNAPCIN. The vaccine was injected as a heterologous booster in healthy Iranian adults aged 18-50 who had previously received three doses of inactivated SARS-CoV-2 vaccines. In the study, 30 participants were randomly assigned to receive either COReNAPCIN in two different doses (25 µg and 50 µg) or placebo. The vaccine candidate contained mRNA encoding the complete sequence of the pre-fusion stabilized Spike protein of SARS-CoV-2, formulated within lipid nanoparticles. The primary endpoint was safety and the secondary objective was humoral immunogenicity until 6 months post-vaccination. The cellular immunogenicity was pursued as an exploratory outcome. RESULTS: COReNAPCIN was well tolerated in vaccinated individuals in both doses with no life-threatening or other serious adverse events. The most noticeable solicited adverse events were pain at the site of injection, fatigue and myalgia. Regarding the immunogenicity, despite the seroprevalence of SARS-CoV-2 antibodies due to the vaccination history for all and previous SARS-CoV-2 infection for some participants, the recipients of 25 and 50 µg COReNAPCIN, two weeks post-vaccination, showed 6·6 and 8·1 fold increase in the level of anti-RBD, and 11·5 and 21·7 fold increase in the level of anti-spike antibody, respectively. The geometric mean virus neutralizing titers reached 10.2 fold in the 25 µg group and 8.4 fold in 50 µg group of pre-boost levels. After 6 months, the measured anti-spike antibody concentration still maintains a geometric mean fold rise of 2.8 and 6.3, comparing the baseline levels in 25 and 50 µg groups, respectively. Additionally, the significant increase in the spike-specific IFN-ϒ T-cell response upon vaccination underscores the activation of cellular immunity. CONCLUSION: COReNAPCIN booster showed favorable safety, tolerability, and immunogenicity profile, supporting its further clinical development (Trial registration: IRCT20230131057293N1).

Intrinsic dual emissive insulin capped Au/Ag nanoclusters as single ratiometric nanoprobe for reversible detection of pH and temperature and cell imaging.

pH and temperature are two important characteristics in cells and the environment. These, not only in the well-done regulation of cell functions but also in diagnosis and treatment, have a key role. Protein-protected bimetallic nanoclusters are abundantly used in the building of biosensors. However, insulin-stabilized Au-Ag nanoclusters with dual intrinsic emission have not been investigated yet. In this work, Dual emissive insulin templated Au-Ag nanocluster (Ins(Au/Ag)NCs) were first synthesized in a simple and green one-put manner. The two emission wavelengths of, as-prepared NCs centered at 410 and 630 nm, excited in one excitation wavelength (330 nm). These two emission peaks were assigned to the di-Tyrosine cross-linked formation and bimetallic nanoclusters respectively. Further analysis displayed that each emission band of Ins(Au/Ag)NCs responded to one variable whilst another peak remained constant; For blue and red emission wavelengths, pH dependency and thermo-responsibility were observed respectively. As-prepared nanoprobe with the intrinsic dual emissive feature was used for ratiometric determination of these parameters, each with a discrete response from another. The linear range of 6.0-9.0 for pH and 1 to 71 °C for temperature was obtained, which comprises the physiological range of pH and temperature and afforded intracellular sensing and imaging capability. As-prepared NCs probe show excellent biocompatibility and cell membrane permeability, and so were successfully applied as direct ratiometric pH and temperature probes in HeLa and HFF cells. More interestingly, this dual emissive nanoprobe is capable of distinguishing cancer cells from normal ones.

Hemoglobin bio-adhesive nanoparticles as a colon-specific delivery system for sustained release of 5-aminosalicylic acid in the effective treatment of inflammatory bowel disease.

A colonic drug delivery system was developed to specifically deliver 5-aminosalicylic acid (5-ASA) to the inflamed site by conjugating with hemoglobin nanoparticles (HbNPs). The 5-ASA-HbNPs (eight 5-ASA molecules per Hb molecule) with the size of 220 nm and zeta potential of -14.6 mV is a tailored nanoparticle able to pass through the mucus layer. The 5-ASA-HbNPs do not undergo chemical and enzymatic hydrolysis in the simulated gastrointestinal fluids over 6 h. Significantly higher cellular uptakes and prolonged release was seen for the 5-ASA-HbNPs in Caco-2 cells, compared to free 5-ASA over 72 h. In addition, 5-ASA-HbNPs revealed similar therapeutic effectiveness with free 5-ASA against tumor necrosis factor and showed less inhibitory concentration (IC50) for myeloperoxidase enzyme activity. In vivo imaging of mouse demonstrated the localization of drug in the descending colon after oral administration and about 15% of the administered dose was recovered as 5-ASA from urine in 6 h. The use of these nanoparticles with the mucus adhesion properties and permeability to intestinal epithelial cells can be a good candidate with potential application in the colonic drug delivery field.

Microfluidic investigation of the effect of graphene oxide on mechanical properties of cell and actin cytoskeleton networks: experimental and theoretical approaches.

Biomechanical and morphological analysis of the cells is a novel approach for monitoring the environmental features, drugs, and toxic compounds' effects on cells. Graphene oxide (GO) has a broad range of medical applications such as tissue engineering and drug delivery. However, the effects of GO nanosheets on biological systems have not been completely understood. In this study, we focused on the biophysical characteristics of cells and their changes resulting from the effect of GO nanosheets. The biophysical properties of the cell population were characterized as follows: cell stiffness was calculated by atomic force microscopy, cell motility and invasive properties were characterized in the microfluidic chip in which the cells are able to visualize cell migration at a single-cell level. Intracellular actin was stained to establish a quantitative picture of the intracellular cytoskeleton. In addition, to understand the molecular interaction of GO nanosheets and actin filaments, coarse-grained (CG) molecular dynamics (MD) simulations were carried out. Our results showed that GO nanosheets can reduce cell stiffness in MCF7 cells and MDA-MB-231 cell lines and highly inhibited cell migration (39.2%) in MCF-7 and (38.6%) in MDA-MB-231 cell lines through the GO nanosheets-mediated disruption of the intracellular cytoskeleton. In the presence of GO nanosheets, the cell migration of both cell lines, as well as the cell stiffness, significantly decreased. Moreover, after GO nanosheets treatment, the cell actin network dramatically changed. The experimental and theoretical approaches established a quantitative picture of changes in these networks. Our results showed the reduction of the order parameter in actin filaments was 23% in the MCF7 cell line and 20.4% in the MDA-MB-231 cell line. The theoretical studies also showed that the GO nanosheet-actin filaments have stable interaction during MD simulation. Moreover, the 2D free energy plot indicated the GO nanosheet can induce conformational changes in actin filaments. Our findings showed that the GO nanosheets can increase the distance of actin-actin subunits from 3.22 to 3.5 nm and in addition disrupt native contacts between two subunits which lead to separate actin subunits from each other in actin filaments. In this study, the biomechanical characteristics were used to explain the effect of GO nanosheets on cells which presents a novel view of how GO nanosheets can affect the biological properties of cells without cell death. These findings have the potential to be applied in different biomedical applications.

Imidazolium-based ionic liquid functionalized mesoporous silica nanoparticles as a promising nano-carrier: response surface strategy to investigate and optimize loading and release process for Lapatinib delivery.

Imidazolium-based ionic liquid functionalized PEGylated mesoporous silica nanoparticles MCM-41 (denoted as [ImIL-PEGylated@MCM-41] NPs) is synthesized and evaluated as an efficient and reliable pH-sensitive nano-carrier for controlled release of cationic Lapatinib (Lap) drug. This nano-DDS was fully characterized by dynamic light scattering, scanning electron microscopy, X-ray powder diffraction, Fourier transform infrared spectroscopy, N2 adsorption-desorption measurement, and differential scanning calorimeter. Furthermore, the drug loading content and in-vitro drug release profile were studied. The entrapment and loading efficiency of the optimized formulation for Lap were 91 ± 2.0% and 32.21 ± 2.70%, respectively. The results of cytotoxicity assay demonstrated that ImIL-PEG@MCM-41 has no significant toxicity on both cancerous and normal cell lines and the anticancer activity of Lap@ImIL-PEG@MCM-41 was comparable to free drug in case of human breast cells (SKBR3) and human embryonic kidney 293 cells (HEK-293). Meanwhile, three-dimensional (3D) cell culture was performed by multicellular tumor spheroids for understanding of cell response to drugs in physiologically 3D microenvironments. The results of Lap@ImIL-PEG@MCM-41 uptake during 48 hours showed a gradual release of the Lap through the multicellular tumor spheroids. This showed that the pH-responsive controlled release of Lapatinib leads to the satisfactory results in the in vitro breast cancer therapy.

Seed-mediated Electrochemically Developed Au Nanostructures with Boosted Sensing Properties: An Implication for Non-enzymatic Glucose Detection.

A new approach has been developed to improve sensing performances of electrochemically grown Au nanostructures (AuNSs) based on the pre-seeding of the electrode. The pre-seeding modification is simply carried out by vacuum thermal deposition of 5 nm thin film of Au on the substrate followed by thermal annealing at 500 °C. The electrochemical growth of AuNSs on the pre-seeded substrates leads to impressive electrochemical responses of the electrode owing to the seeding modification. The dependence of the morphology and the electrochemical properties of the AuNSs on various deposition potentials and times have been investigated. For the positive potentials, the pre-seeding leads to the growth of porous and hole-possess networks of AuNSs on the surface. For the negative potentials, AuNSs with carved stone ball shapes are produced. The superior electrode was achieved from AuNSs developed at 0.1 V for 900 s with pre-seeding modification. The sensing properties of the superior electrode toward glucose detection show a high sensitivity of 184.9 µA mM-1 cm-2, with a remarkable detection limit of 0.32 µM and a wide range of linearity. The excellent selectivity and reproducibility of the sensors propose the current approach as a large-scale production route for non-enzymatic glucose detection.

PDMS Nano-Modified Scaffolds for Improvement of Stem Cells Proliferation and Differentiation in Microfluidic Platform.

Microfluidics cell-based assays require strong cell-substrate adhesion for cell viability, proliferation, and differentiation. The intrinsic properties of PDMS, a commonly used polymer in microfluidics systems, regarding cell-substrate interactions have limited its application for microfluidics cell-based assays. Various attempts by previous researchers, such as chemical modification, plasma-treatment, and protein-coating of PDMS revealed some improvements. These strategies are often reversible, time-consuming, short-lived with either cell aggregates formation, not cost-effective as well as not user- and eco-friendly too. To address these challenges, cell-surface interaction has been tuned by the modification of PDMS doped with different biocompatible nanomaterials. Gold nanowires (AuNWs), superparamagnetic iron oxide nanoparticles (SPIONs), graphene oxide sheets (GO), and graphene quantum dot (GQD) have already been coupled to PDMS as an alternative biomaterial enabling easy and straightforward integration during microfluidic fabrication. The synthesized nanoparticles were characterized by corresponding methods. Physical cues of the nanostructured substrates such as Young's modulus, surface roughness, and nanotopology have been carried out using atomic force microscopy (AFM). Initial biocompatibility assessment of the nanocomposites using human amniotic mesenchymal stem cells (hAMSCs) showed comparable cell viabilities among all nanostructured PDMS composites. Finally, osteogenic stem cell differentiation demonstrated an improved differentiation rate inside microfluidic devices. The results revealed that the presence of nanomaterials affected a 5- to 10-fold increase in surface roughness. In addition, the results showed enhancement of cell proliferation from 30% (pristine PDMS) to 85% (nano-modified scaffolds containing AuNWs and SPIONs), calcification from 60% (pristine PDMS) to 95% (PDMS/AuNWs), and cell surface marker expression from 40% in PDMS to 77% in SPION- and AuNWs-PDMS scaffolds at 14 day. Our results suggest that nanostructured composites have a very high potential for stem cell studies and future therapies.

Encapsulation of an endostatin peptide in liposomes: Stability, release, and cytotoxicity study.

The endostatin protein is a potent inhibitor of angiogenesis and tumor growth. The anti-angiogenic and antitumor properties of full-length endostatin can be mimicked by its N-terminal segment, including residues 1-27. Therefore, our previous studies have shown that a mutant N-terminal peptide which the Zn-binding loop was replaced by a disulfide loop (referred to as the ES-SS peptide) has preserved antiangiogenic and antitumor properties compared to the native peptide. To increase stability and plasma half-life of the ES-SS peptide, the nano-sized liposomal formulations of the peptide with different ratio of phosphocholine (PC) were synthesized. The liposomal peptide formulations possessed an average size of around 100 nm with (-4 to -36 mv) in zeta potential. The encapsulation efficiency of the ES-SS peptide was in the range of 24-54% with different lipid: peptide molar ratios. In vitro release of the peptide from liposomes indicated a complete peptide release after 7 days. Cytotoxicity assay was evaluated using the human umbilical vein endothelial cells (HUVECs) for various concentrations of the liposomal peptide. The results depicted the gradual release of the peptide through liposomes. By comparing with the free peptide, the liposomal peptide formulations have indicated higher cell viability with IC50 value about 0.1 µM. The peptide-liposome interactions, as well as the peptide effect on the liposome structure, were also investigated through coarse-grained molecular dynamics (CG-MD) simulation. The results revealed that the peptides were assembled in the hydrophilic core of the liposome. The peptide behavior in liposome can stabilize the liposome structure and be a response to the observed low peptide release rate. The investigation is promising for designing a liposome-based anti-angiogenesis peptide delivery system.

A novel aspect of functionalized graphene quantum dots in cytotoxicity studies.

Graphene quantum dots (GQDs) represent a new generation of graphene-based nanomaterials with enormous potential for use and development of a variety of biomedical applications. However, up to now little studies have investigated the impact of GQDs on human health in case of exposure. GQDs were synthesized from citric acid as carbon precursor by hydrothermal treatment at 160 °C for 4 h. The synthesized GQDs showed strong blue emission under UV-Irradiation with fluorescence quantum yield of 9.8%. The obtained GQDs were further carbonized, activated and functionalized by nitric acid vapor method. Nitrogen adsorption/desorption isotherms were used to analyze the surface area and porous structures of GQDs. The results revealed that compared to GQDs, the specific surface area of functionalized graphene quantum dots (fGQDs) has been increased from 0.0667 to 2.5747 m2/g and pore structures have been enhanced significantly. The potential cytotoxic effect of GQDs, fGQDs and GO suspensions was evaluated on HFF cell line using MTT assays and flow cytometry method after 24 h incubation. We have for the first time demonstrated that by carbonization, activation and functionalization of GQDs they still showed cytocompatible properties. We observed excellent biocompatibility of GQDs and fGQDs at low concentrations. Moreover, the results suggested that modification of GQDs yields product suspensions with high surface area, enhanced pore volume and loading capacities. Thus, fGQDs represent an attractive candidate for further use in drug delivery systems and bio-imaging application.

Label free phosphate functionalized semiconducting polymer dots for detection of iron(III) and cytochrome c with application to apoptosis imaging.

We report on facile synthesis and characterization of phosphate-functionalized polymer dots (PDs) by doping tributyl phosphate (TBP) in a semiconducting polymer poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,10-3}-thiadiazole)] (PFBT). Then, the prepared TBP@PFBT PDs were used to develop a very high sensitive probe for detection Fe3+, Cu2+ ions and Cytochrome c based on aggregation induced fluorescence off mechanism. The PDs exhibited a linear dynamic range for Fe3+ from 0.1 to 2 nM with a detection limit of 30 pM and for Cu2+ from 2.0 to 50.0 nM with a detection limit of 0.35 nM. Meanwhile, this probe showed a linear dynamic range for Cyt c from 175 to 1750 pM with a detection limit of 32.7 pM. The TBP@PFBT PDs is a simple, one-step, fast, non-invasive, label-free, and inexpensive probe that is capable of online apoptosis monitoring response to drugs with an ever-present opportunity to contribute in a variety of in-vitro and in-vivo biological applications. We also obtained sharp, specific 2D and 3D imaging results for early stage apoptosis in breast cancer cells. Moreover, this technique possesses the advantage of rapid determination of Fe3+ ion in biological or environmental samples. Importantly, this label-free assay provides short determination time of only a few min, easy operation and very low LOD allowing 100-4000 times increased in sensitivity over previously reported probes, together with high selectivity without need to using biorecognition elements like enzymes, antibodys and/or aptamers. Such excellent features make the TBP@PFBT PDs an excellent probe for successful apoptosis imaging in live cells.

Hemoglobin-incorporated iron quantum clusters as a novel fluorometric and colorimetric probe for sensing and cellular imaging of Zn(II) and cysteine.

The authors describe a novel water-soluble, stable, biocompatible, and highly fluorescent probe consisting of iron quantum clusters incorporated into human adult hemoglobin (Hb-FeQCs). The Hb-FeQCs were characterized by various spectroscopic techniques. The probe displays strong absorption and yellow fluorescence with a peak centered at 567 nm (photo-excited at 460 nm). The Hb-FeQCs show excellent photostability over a wide range of pH values (5-12), even in the presence of high electrolyte concentrations. A colorimetric and a fluorometric method were worked out for the quantitation Zn(II) and cysteine in aqueous solution. Zinc ions induce a visible color change from brown to yellow. The sensitivity of Hb-FeQCs towards other metal ions was negligible, with the exception of Co2+ and Cu2+, which caused a modest interference. The Hb-FeQCs were exploited in a sensitive and selective turn-on fluorescence assay for Zn2+. It is also found that cysteine quenches the fluorescence of the Hb-FeQCs/Zn(II) complex. Under the optimized conditions, the probe has a linear response in the 0.04 to 2.2 µM Zn(II) concentration range, with a 48 nM detection limit. Response to cysteine is linear in the 1-60 µM concentration range, with a 0.25 µM limit of detection. This fluorescent probe undergoes fluorescent emission intensity enhancement upon binding to zinc ions in living normal human fibroblast cells under visible lamp. The cellular imaging capability and very low cytotoxicity of this soluble iron quantum clusters can be potentially extended as an exciting sub-nanoplatform with promising biomaterial applications. Graphical abstract Schematic of yellow-emitting iron quantum clusters in hemoglobin matrix (Hb-FeQCs) were characterized and successfully applied for sensing zinc(II) and cysteine. The act as an on-off fluorescent probe and can be applied to image zinc ions in human fibroblast cells under visible light.