One-step synthesis of orange-red emissive carbon dots: photophysical insight into their excitation wavelength-independent and dependent luminescence.

A low-temperature method was developed to synthesize orange-red luminescence phosphor-doped carbon dots (CDs) without complicated purification procedures. These CDs showed excitation wavelength-independent narrow emission (photo-luminescence quantum yield, Φf ∼ 12 to 22%) with single exponential time-resolved decay in weakly polar/non-polar solvents, indicating the presence of one kind of chromophore. In contrast, the same CDs showed excitation wavelength-dependent broad emission (Φf ∼ 1 to 8%) with multi-exponential fluorescence decay in polar solvents. These CDs exhibited poor solubility in polar solvents, resulting in CD aggregates contributed by excitation wavelength-dependent weak luminescence. The CDs embedded in polymethyl methacrylate (PMMA) polymer film displayed bright orange-red fluorescence under UV 365 nm illumination, indicating their potential application in solid-state luminescence. Further, an analytical method was developed for the naked-eye detection of trifluoracetic acid (red emission) and triethylamine (green emission) under UV 365 nm illumination with reversible two switch-mode luminescence. Additionally, this efficient orange-red luminescence of CDs was utilized for possible bioimaging applications with negligible cytotoxicity in 3T3 mouse fibroblast cells.

Differential immunomodulation of human mesenchymal stromal cells from various sources in an inflammation mimetic milieu.

Mesenchymal stromal cells (MSCs) are very advantageous in the field of regenerative medicine because of their immunomodulatory properties. However, reports show that these properties vary from source to source. Hence, understanding the source-dependent specificity of MSCs and their immunomodulatory abilities will enable optimal use of MSCs in cell-based therapies. Here, we studied human MSCs from three different sources, adipose tissue (AT), bone marrow (BM) and Wharton's jelly (WJ), with respect to phenotypic responses of human peripheral blood mononuclear immune cells (hPBMCs/MNCs) and the concurrent changes in cytokine expression in MSCs, under mitogen-stimulated co-culture conditions. We used cytometric analysis to study the immunoregulatory properties of MSCs on MNCs and cytokine profiling of MSCs using a customized PCR array and solid-phase sandwich enzyme-linked immunosorbent assay. Our results reveal differential modulation of immune cells as well as MSCs upon activation by the mitogen phytohemagglutinin, independently and in co-culture. Notably, we observed source-specific MSC-cytokine signatures under stimulated conditions. Our results show that AT-MSCs up-regulate VEGF, BM-MSCs up-regulate PTGS-2 and WJ-MSCs increase expression of IDO considerably compared with controls. This remarkable modulation in source-specific cytokine expression was also validated at a functional level by quantitative protein expression studies. In our hands, even though MSCs from AT, BM and WJ sources exhibit characteristic immunomodulatory properties, our results highlight that MSCs sourced from different tissues may exhibit unique cytokine signatures and thus may be suitable for specific regenerative applications.

Generation and transplantation of hepatocytes-like cells using human origin hepatogenic serum for acute liver injury treatment.

Liver failure is a critical disease for which regenerative therapies are still being explored. The major limitation in the use of a clinical grade, viable cell-based therapy approach is the scarce availability of sufficient number of in-vitro differentiated hepatocyte-like cells (HLC) that can induce regeneration and ameliorate liver injury. Here, we report for the first time an approach to engineer HLCs using sera of hyperbilirubin patients that act as a reservoir of differentiation factor. Utilizing our humanized approach, mesenchymal stem cells (hMSC) derived from umbilical cord tissue were transdifferentiated into HLC using patient-derived serum along with dimethyl sulfoxide (DMSO). We studied the effects of serum on the proliferation, cell cycle analysis, and apoptosis of hMSC by various differentiation combinations. We optimized the hepatic transdifferentiation ability of hMSC with hyperbilirubin serum treatment for a period of 7 days. Assessment of HLC functionalities was shown by quantifying the HLC spent medium for albumin and urea secretions. Transplantation of HLC in an acute liver injury (ALI) rat model showed an effective improvement in the liver function and histological changes in the liver. The results of this study suggest that hMSC-derived HLC using humanized hepatogenic serum holds a promising potential for cell transplantation, as an efficient therapy modality for liver failure in humans.

Defective cell proliferation is an attribute of overexpressed Notch1 receptor and impaired autophagy in Fanconi Anemia.

Fanconi Anemia (FA) is an inherited bone marrow failure syndrome caused by mutation in FA pathway proteins, involved in Interstrand Cross Link (ICL) repair. FA cells exhibit in vitro proliferation arrest due to accumulated DNA damage, hence understanding the rescue mechanism that renders proliferation advantage is required. Gene expression profiling performed in FA patients Peripheral Blood Mononuclear Cells (PBMCs) revealed a wide array of dysregulated biological processes. Functional enrichment and gene clustering analysis showed crippled autophagy process and escalated Notch signalling pathway in FA clinical samples and cell lines. Notch pathway mediators overexpression were reverted in FANCA mutant cells when treated with Rapamycin, an autophagy inducer. Additionally, Rapamycin stabilized cell viability after treatment with the DNA damaging agent, MitomycinC (MMC) and enhanced cell proliferation genes expression in FANCA mutant cells. Inherently FANCA mutant cells express impaired autophagy; thus activation of autophagy channelizes Notch signalling cascade and sustains cell viability.

A Multiparametric Fluorescence Probe to Understand the Physicochemical Properties of Small Unilamellar Lipid Vesicles in Poly(ethylene glycol)-Water Medium.

FDAPT (2-formyl-5-(4'-N,N-dimethylaminophenyl)thiophene) efficiently senses the minimum alteration of lipid bilayer microenvironment with all six different fluorescence parameters namely emission wavelength, fluorescence intensity, steady-state anisotropy, and their corresponding time-dependent parameters (Sahu et al., J. Phys. Chem. B 2018, 122, 7308-7318). In the present work, the effect of poly(ethylene glycol) on the small unilamellar vesicle is demonstrated with the emission behavior of the FDAPT probe. A medium and a high molecular weight PEG were chosen to perturb the lipid vesicles. The alteration of the bilayer polarity, water content inside bilayer, lipid packing density in the perturbed vesicles reflect significant changes in different fluorescence parameters of FDAPT probe. The effect of PEG on the unilamellar vesicle was rationalized with the alteration of the emission behavior, fluorescence lifetime, steady-state anisotropy and anisotropy decay of the probe. The simple and convenient fluorescence measurements provide new insights into the effect of PEG on the packing density, water volume, micro polarity, and microviscosity of the small unilamellar vesicle. The physiological understanding was extended to rationalize the cryoprotecting behavior of PEG.

Anti-cancer Application of Nat-ZnFe2O4 Nanoparticles on 2D Tumor Models.

Metal/Metal Oxide nanoparticles (M/MO NPs) exhibit potential biomedical applications due to their tunable physicochemical properties. Recently, the biogenic synthesis of M/MO NPs has gained massive attention due to their economical and eco-friendly nature. In the present study, Nyctanthes arbor-tristis (Nat) flower extract-derived Zinc Ferrite NPs (Nat-ZnFe2O4 NPs) were synthesized and physicochemically characterized by FTIR, XRD, FE-SEM, DLS, and other instruments to study their crystallinity, size, shape, net charge, presence of phytocompounds on NP's surface and several other features. The average particle size of Nat-ZnFe2O4 NPs was approx. 25.87 ± 5.67 nm. XRD results showed the crystalline nature of Nat-ZnFe2O4 NPs. The net surface charge on NPs was -13.28 ± 7.18 mV. When tested on mouse fibroblasts and human RBCs, these NPs were biocompatible and hemocompatible. Later, these Nat-ZnFe2O4 NPs exhibited potent anti-neoplastic activity against pancreatic, lung, and cervical cancer cells. In addition, NPs induced apoptosis in tested cancer cells through ROS generation. These in vitro studies confirmed that Nat-ZnFe2O4 NPs could be used for cancer therapy. Moreover, further studies are recommended on ex vivo platforms for future clinical applications.

Understanding the aggregation of excitation wavelength independent emission of amphiphilic carbon dots for bioimaging and organic acid sensing.

Herein, excitation wavelength-independent, tunable emissive and amphiphilic CDs with high quantum yield were synthesized by a low-temperature oxidation method employing banana peel waste as a carbon source. These CDs showed longer wavelength emissions (green to yellow) independent of the excitation wavelength when dispersed in different polar to non-polar solvents. The quantum yields of the same CDs were 9-32% in different solvent polarities for different emissions. On the other hand, a large stokes-shifted emission (∼9606 cm-1) was observed for CDs in the non-polar and weak polar solvents. The particle size of CDs increases from a hydrophobic to a hydrophilic environment with the change in emission colour from yellow to green. A polar and a non-polar host matrix were used to overcome the limitation of aggregation-caused quenching of CDs in the solid state to obtain bright emissions. These CDs were potentially used for naked-eye detection of trifluoroacetic acid (TFA) by changing the emission colour from yellow to orange under UV 365 nm. Sensing of TFA was also shown reversibly switch emission colour and average lifetime for multiple cycles. Additionally, the highly emissive CDs show negligible cytotoxicity in 3T3 fibroblast cells, indicating possible bioimaging applications in 3T3 cells.

Fluid shear stress in a logarithmic microfluidic device enhances cancer cell stemness marker expression.

Fluid shear stress (FSS) is crucial in cancer cell survival and tumor development. Noteworthily, cancer cells are exposed to several degrees of FSS in the tumor microenvironment and during metastasis. Consequently, the stemness marker expression in cancer cells changes with the FSS signal, although it is unclear how it varies with different magnitudes and during metastasis. The current work explores the stemness and drug resistance characteristics of the cervical cancer cell line HeLa in a microfluidic device with a wide range of physiological FSS. Hence, the microfluidic device was designed to achieve a logarithmic flow distribution in four culture chambers, realizing four orders of biological shear stress on a single chip. The cell cycle analysis demonstrated altered cell proliferation and mitotic arrest after FSS treatment. In addition, EdU staining revealed increased cell proliferation with medium to low FSS, whereas high shear had a suppressing effect. FSS increased competence to withstand higher intracellular ROS and mitochondrial membrane potential in HeLa. Furthermore, stemness-related gene (Sox2, N-cadherin) and cell surface marker (CD44, CD33, CD117) expressions were enhanced by FSS mechanotransduction in a magnitude-dependent manner. In summary, these stemness-like properties were concurrent with the drug resistance capability of HeLa towards doxorubicin. Overall, our microfluidic device elucidates cancer cell survival and drug resistance mechanisms during metastasis and in cancer relapse patients.

Genome-wide methylome pattern predictive network analysis reveal mesenchymal stem cell's propensity to undergo cardiovascular lineage.

Mesenchymal stem cells (MSCs) differentiation toward cardiovascular lineage prediction using the global methylome profile will highlight its prospective utility in regenerative medicine. We examined the propensity prediction to cardiovascular lineage using 5-Aza, a well-known cardiac lineage inducer. The customized 180 K microarray was performed and further analysis of global differentially methylated regions by Ingenuity pathway analysis (IPA) in both MSCs and 5-AC-treated MSCs. The cluster enrichment tools sorted differentially enriched genes and further annotated to construct the interactive networks. Prediction analysis revealed pathways pertaining to the cardiovascular lineage found active in the native MSCs, suggesting its higher propensity to undergo cardiac, smooth muscle cell, and endothelial lineages in vitro. Interestingly, gene interaction network also proposed majorly stemness gene network NANOG and KLF6, cardiac-specific transcription factors GATA4, NKX2.5, and TBX5 were upregulated in the native MSCs. Furthermore, the expression of cardiovascular lineage specific markers such as Brachury, CD105, CD90, CD31, KDR and various forms of ACTIN (cardiac, sarcomeric, smooth muscle) were validated in native MSCs using real time PCR and immunostaining and blotting analysis. In 5-AC-treated MSCs, mosaic interactive networks were observed to persuade towards osteogenesis and cardiac lineage, indicating that 5-AC treatment resulted in nonspecific lineage induction in MSCs, while MSCs by default have a higher propensity to undergo cardiovascular lineage. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03058-2.

Establishing the promising role of novel combination of triple therapeutics delivery using polymeric nanoparticles for Triple negative breast cancer therapy.

Introduction: Triple-negative breast cancer (TNBC) is a lethal tumor with an advanced degree of metastasis and poor survivability as compared to other subtypes of breast cancer. TNBC which consists of 15 % of all types of breast cancer is categorized by the absence of expression of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor-2 (HER2). This is the main reason for the failure of current hormonal receptor-based therapies against TNBCs, thus leading to poor patient outcomes. Therefore, there is a necessity to develop novel therapies targeting this devastating disease. Methods: In this study, we have targeted TNBC by simultaneous activation of apoptosis through DNA damage via cytotoxic agent such as paclitaxel (PAC), inhibition of PARP activity via PARP inhibitor, olaparib (OLA) and inhibiting the activity of FOXM1 proto-oncogenic transcription factor by using RNA interference technology (FOXM1-siRNA) in nanoformulations. Experiments conducted in this investigation include cellular uptake, cytotoxicity and apoptosis study using MDA-MB-231 cells. Results: The present study validates that co-delivery of two drugs (PAC and OLA) along with FOXM1-siRNA by cationic NPs, enhances the therapeutic outcome leading to greater cytotoxicity in TNBC cells. Conclusion: The current investigation focuses on designing a multifunctional drug delivery platform for concurrent delivery of either PAC or PARP inhibitor (olaparib) and FOXM1 siRNA in chitosan-coated poly(D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) with the ability to emerge as a front runner therapeutic for TNBC therapy.

Self-assembled dual-drug loaded core-shell nanoparticles based on metal-free fully alternating polyester for cancer theranostics.

Recent research has been directed to the use of biocompatible and biodegradable metal-free fully alternating polyester nanomaterial in drug delivery application. The practice of triethyl borane (Et3B)/Bis(triphenylphosphoranylidene)ammonium chloride (PPNCl) Lewis pair as non-metallic catalyst was carried out to synthesize alternating copolymer of commercially available tert-butyl glycidyl ether (tBGE) and phthalic anhydride (PA) (poly(tBGE-alt-PA) copolymer) of low Mnvia nearly controlled ring-opening copolymerization (ROCOP) reaction. This biocompatible, hemocompatible, and biodegradable copolymer was used in the fabrication of different nanodrug formulations (NDFs) loaded with doxorubicin (DOX), curcumin (CUR) and their combination. Transmission electron microscope (TEM) imaging showed the spherical shape and core-shell internal structure for all NDFs with an average particle diameter ranging between 200 and 250 nm. X-ray diffraction (XRD) analysis displayed the amorphous nature of both DOX and CUR after their entrapment into the copolymer matrix. Differential scanning colorimetry (DSC) analysis presented no potential chemical interactions between the drug and copolymer. The cellular drug uptake study showed the increased uptake for all NDFs compared to free drug and exhibited higher DOX and CUR accumulation in dual-drug loaded nanoparticles treated pancreatic cancer (MIA PaCa-2) cells. The in vitro drug release kinetic study displayed the slow sustained drug release behavior with anomalous transport for both DOX and CUR in a defined physiological environment. Further, the anti-tumor efficacy of all NDFs was examined on several different cancer cell lines and maximum cytotoxicity was observed in MIA PaCa-2 cells with low inhibitory concentration (IC50) values. These NDFs inhibited the proliferation of MIA PaCa-2 cells due to cell cycle arrest in G2/M phase. In result, MIA PaCa-2 cells underwent apoptosis with significant changes in mitochondrial membrane potential and increased reactive oxygen species (ROS) level. In future, this study will open several novel insights related to the use of such biocompatible and biodegradable metal-free polyesters in targeted drug delivery, tissue engineering and other biomedical applications.

Simulated microgravity increases polyploid giant cancer cells and nuclear localization of YAP.

Physical cues are vital in determining cellular fate in cancer. In vitro 3D culture do not replicate forces present in vivo. These forces including tumor interstitial fluid pressure and matrix stiffness behave as switches in differentiation and metastasis, which are intricate features of cancer stem cells (CSCs). Gravity determines the effect of these physical factors on cell fate and functions as evident from microgravity experiments on space and ground simulations. Here, we described the role of simulation of microgravity (SMG) using rotary cell culture system (RCCS) in increasing stemness in human colorectal cancer cell HCT116. We observed distinct features of cancer stem cells including CD133/CD44 dual positive cells and migration in SMG which was not altered by autophagy induction or inhibition. 3D and SMG increased autophagy, but the flux was staggered under SMG. Increased unique giant cancer cells housing complete nuclear localization of YAP were observed in SMG. This study highlights the role of microgravity in regulating stemness in CSC and importance of physical factors in determining the same.

Evaluation of platelet lysate as a substitute for FBS in explant and enzymatic isolation methods of human umbilical cord MSCs.

Mesenchymal stem cells (MSCs) have immense potential for cell-based therapy of acute and chronic pathological conditions. MSC transplantation for cell-based therapy requires a substantial number of cells in the range of 0.5-2.5 × 106 cells/kg body weight of an individual. A prolific source of MSCs followed by in vitro propagation is therefore an absolute prerequisite for clinical applications. Umbilical cord tissue (UCT) is an abundantly available prolific source of MSC that are fetal in nature and have higher potential for ex-vivo expansion. However, the ex-vivo expansion of MSCs using a xenogeneic supplement such as fetal bovine serum (FBS) carries the risk of transmission of zoonotic infections and immunological reactions. We used platelet lysate (PL) as a xeno-free, allogeneic replacement for FBS and compared the biological and functional characteristics of MSC processed and expanded with PL and FBS by explant and enzymatic method. UCT-MSCs expanded using PL displayed typical immunophenotype, plasticity, immunomodulatory property and chromosomal stability. PL supplementation also showed 2-fold increase in MSC yield from explant culture with improved immunomodulatory activity as compared to enzymatically dissociated cultures. In conclusion, PL from expired platelets is a viable alternative to FBS for generating clinically relevant numbers of MSC from explant cultures over enzymatic method.