Anti-cancer Activity of Biogenic Nat-ZnO Nanoparticles Synthesized Using Nyctanthes arbor-tristis (Nat) Flower Extract.

Inorganic nanoparticles (NPs) have played an important role as nano-drug delivery systems during cancer therapy in recent years. These NPs can carry cancer therapeutic agents. Due to this, they are considered a promising ancillary to traditional cancer therapies. Among inorganic NPs, Zinc Oxide (ZnO) NPs have been extensively utilized in cellular imaging, gene/drug delivery, anti-microbial, and anti-cancerous applications. In this study, a rapid and cost-effective method was used to synthesize Nat-ZnO NPs using the floral extract of the Nyctanthes arbor-tristis (Nat) plant. Nat-ZnO NPs were physicochemically characterized and tested further on in vitro cancer models. The average hydrodynamic diameter (Zaverage) and the net surface charge of Nat-ZnO NPs were 372.5 ± 70.38 d.nm and -7.03 ± 0.55 mV, respectively. Nat-ZnO NPs exhibited a crystalline nature. HR-TEM analysis showed the triangular shape of NPs. Furthermore, Nat-ZnO NPs were also found to be biocompatible and hemocompatible when tested on mouse fibroblast cells and RBCs. Later, the anti-cancer activity of Nat-ZnO NPs was tested on lung and cervical cancer cells. These NPs displayed potent anti-cancer activity and induced programmed cell death in cancer cells.

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.

Current aspects of organoid technology for biomaterial toxicity analysis.

Organoids provide us an opportunity to understand how diseases affect cellular physiology, human tissues or organs. They are indespensible tools for biomaterial toxicity analysis, drug discovery and regenerative medicine.

TiO2 nanostructures - a double edged sword: current progress on their role in stem cells' differentiation, cancer therapy, and their toxicity issues.

Titanium dioxide (TiO2)-based nanostructures have wide applications in cosmetics, toothpastes, pharmaceuticals, coatings, papers, inks, plastics, food products, textiles, and many others. Recently, they have also been found to have huge potential as stem cells' differentiation agents as well as stimuli-responsive drug delivery systems in cancer therapy. In this review, we present some of the recent progress in the role of TiO2-based nanostructures toward the above-mentioned applications. We also present recent studies on the toxicity issues of these nanomaterials and the mechanisms behind the toxicity issues. We have reviewed the recent progress of TiO2-based nanostructures on their stem cells' differentiation potentials, their photo- and sono-dynamic capabilities, as stimuli-responsive drug delivery systems, and finally their toxicity issues with mechanistic understanding on the same. We believe that this review will help researchers be aware of the latest progress in the applications as well as few toxicity issues associated with TiO2-based nanostructures, which will help them design better nanomedicine for future applications.

Isolongifolene-loaded chitosan nanoparticles synthesis and characterization for cancer treatment.

Recent breakthroughs in the field of nanoparticle-based therapeutic delivery methods have changed the standpoint of cancer therapy by effectively delaying the process of disease development. Nanoparticles have a unique capacity of good penetrating ability than other therapeutic leads used in traditional therapeutics, and also, they have the highest impact on disease management. In the current study isolongifolene-loaded Chitosan nanoparticles have been formulated, synthesized and then characterized by the use of Fourier Transform Infrared Spectroscopy, X-ray Diffraction, Scanning Electron Microscopy and Transmission Electron Microscopy. Further, the characterized chitosan nano formulation was evaluated for hemocompatibility, plasma stability, and in-vitro release. Isolongifolene-loaded chitosan nanoparticles were found to be compatible with plasma and also, they exhibited a constant release pattern. Hence, chitosan-loaded nanoparticles could be employed as an excellent adjuvant in cancer therapeutic, to combat the multi-drug resistance in solid tumors.

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.

Recent Advances in Cardiac Tissue Engineering for the Management of Myocardium Infarction.

Myocardium Infarction (MI) is one of the foremost cardiovascular diseases (CVDs) causing death worldwide, and its case numbers are expected to continuously increase in the coming years. Pharmacological interventions have not been at the forefront in ameliorating MI-related morbidity and mortality. Stem cell-based tissue engineering approaches have been extensively explored for their regenerative potential in the infarcted myocardium. Recent studies on microfluidic devices employing stem cells under laboratory set-up have revealed meticulous events pertaining to the pathophysiology of MI occurring at the infarcted site. This discovery also underpins the appropriate conditions in the niche for differentiating stem cells into mature cardiomyocyte-like cells and leads to engineering of the scaffold via mimicking of native cardiac physiological conditions. However, the mode of stem cell-loaded engineered scaffolds delivered to the site of infarction is still a challenging mission, and yet to be translated to the clinical setting. In this review, we have elucidated the various strategies developed using a hydrogel-based system both as encapsulated stem cells and as biocompatible patches loaded with cells and applied at the site of infarction.

Recent trends in biodegradable polyester nanomaterials for cancer therapy.

Biodegradable polyester nanomaterials-based drug delivery vehicles (DDVs) have been largely used in most of the cancer treatments due to its high biological performance and wider applications. In several previous studies, various biodegradable and biocompatible polyester backbones were used which are poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL), poly(propylene fumarate) (PPF), poly(lactic-co-glycolic acid) (PLGA), poly(propylene carbonate) (PPC), polyhydroxyalkanoates (PHA), and poly(butylene succinate) (PBS). These polyesters were fabricated into therapeutic nanoparticles that carry drug molecules to the target site during the cancer disease treatment. In this review, we elaborately discussed the chemical synthesis of different synthetic polyesters and their use as nanodrug carriers (NCs) in cancer treatment. Further, we highlighted in brief the recent developments of metal-free semi-aromatic polyester nanomaterials along with its role as cancer drug delivery vehicles.

Cardiac Differentiation of Mesenchymal Stem Cells: Impact of Biological and Chemical Inducers.

Cardiovascular disorders (CVDs) are the leading cause of global death, widely occurs due to irreparable loss of the functional cardiomyocytes. Stem cell-based therapeutic approaches, particularly the use of Mesenchymal Stem Cells (MSCs) is an emerging strategy to regenerate myocardium and thereby improving the cardiac function after myocardial infarction (MI). Most of the current approaches often employ the use of various biological and chemical factors as cues to trigger and modulate the differentiation of MSCs into the cardiac lineage. However, the recent advanced methods of using specific epigenetic modifiers and exosomes to manipulate the epigenome and molecular pathways of MSCs to modify the cardiac gene expression yield better profiled cardiomyocyte like cells in vitro. Hitherto, the role of cardiac specific inducers triggering cardiac differentiation at the cellular and molecular level is not well understood. Therefore, the current review highlights the impact and recent trends in employing biological and chemical inducers on cardiac differentiation of MSCs. Thereby, deciphering the interactions between the cellular microenvironment and the cardiac inducers will help us to understand cardiomyogenesis of MSCs. Additionally, the review also provides an insight on skeptical roles of the cell free biological factors and extracellular scaffold assisted mode for manipulation of native and transplanted stem cells towards translational cardiac research.

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.

Glycogen synthase kinase 3ß inhibitor- CHIR 99021 augments the differentiation potential of mesenchymal stem cells.

AIM: Mesenchymal stem cells (MSCs) are immunomodulatory, non-teratogenic and multipotent alternatives to embryonic or induced pluripotent stem cells (ESCs or iPSCs). However, the potency of MSCs is not equivalent to the pluripotency of ESCs or iPSCs. We used CHIR 99021 to improve current protocols and methods of differentiation for the enhanced transdifferentiation potency of MSCs. MAIN METHODS: We used Flurescence activated cell sorter (FACS) for MSC immunophenotyping and biochemical assay for demonstrating the trilineage potential of MSCs. We used real-time polymerase chain reaction, immunocytochemistry and Western blotting assay for analyzing the expression of lineage-specific markers. KEY FINDINGS: CHIR 99021 treatment of MSCs resulted in enhanced transdifferentiation into neurological, hepatogenic and cardiomyocyte lineages with standardized protocols of differentiation. CHIR 99021-treated MSCs showed increased nuclear localization of ß-catenin. These MSCs showed a significantly increased deposition of active histone marks (H3K4Me3, H3K36Me3), whereas no change was observed in repressive marks (H3K9Me3, H3K27Me3). Differential methylation profiling showed demethylation of the transcription factor OCT4 promoter region with subsequent analysis revealing increased gene expression and protein content. The HLA-DR antigen was absent in CHIR 99021-treated MSCs and their differentiated cell types, indicating their immune-privileged status. Karyotyping analysis showed that CHIR 99021-treated MSCs were genomically stable. Teratoma analysis of nude mice injected with CHIR 99021-treated MSCs showed the increased presence of cell types of mesodermal origin at the site of injection. SIGNIFICANCE: MSCs pretreated with CHIR 99021 can be potent, abundant alternative sources of stem cells with enhanced differentiation capabilities that are well suited to cell-based regenerative therapy.

DNA methylation microarray uncovers a permissive methylome for cardiomyocyte differentiation in human mesenchymal stem cells.

Differentiation of Wharton's Jelly-Mesenchymal Stem cells (WJ-MSCs) into cardiomyocytes (CMs) in vitro has been reported widely although contradictions remain regarding the maturation of differentiated MSCs into fully functioning CMs. Studies suggest that use of epigenetic modifiers like 5'Azacytidine (5-AC) in MSCs de-methylates DNA and results in expression of cardiac-specific genes (CSGs). However, only partial expression of the CSG set leads to incomplete differentiation of WJ-MSCs to CMs. We used the Agilent 180 K human DNA methylation microarray on WJ-MSCs, 5-AC treated WJ-MSCs and human cardiac tissue (hCT) to analyze differential DNA methylation profiles which were then validated by bisulfite sequencing PCR (BSP). BSP confirmed that only a limited number of CSGs were de-methylated by 5-AC in WJ-MSCs. It also revealed that hCT displays a methylation profile similar to promoter regions of CSG in untreated WJ-MSCs. Thus, the presence of hypo-methylated CSGs indicates that WJ-MSCs are ideal cell types for cardiomyogenic differentiation.

Human Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells Labeled with Mn2+ and Gd3+ Co-Doped CuInS2-ZnS Nanocrystals for Multimodality Imaging in a Tumor Mice Model.

Mesenchymal stem cell (MSCs) therapy has recently received profound interest as a targeting platform in cancer theranostics because of inherent tumor-homing abilities. However, the terminal tracking of MSCs engraftment by fluorescent in situ hybridization, immuno-histochemistry, and flow-cytometry techniques to translate into clinics is still challenging because of a dearth of inherent MSCs-specific markers and FDA approval for genetic modifications of MSCs. To address this challenge, a cost-effective noninvasive imaging technology based on multifunctional nanocrystals (NCs) with enhanced detection sensitivity, spatial-temporal resolution, and deep-tissue diagnosis is needed to be developed to track the transplanted stem cells. A hassle-free labeling of human umbilical cord Wharton's Jelly (WJ)-derived MSCs with Mn2+ and Gd3+ co-doped CuInS2-ZnS (CIS-ZMGS) NCs has been demonstrated in 2 h without requiring an electroporation process or transfection agents. It has been found that WJ-MSCs labeling did not affect their multilineage differentiation (adipocyte, osteocyte, chondrocyte), immuno-phenotypes (CD44+, CD105+, CD90+), protein (ß-actin, vimentin, CD73, α-SMCA), and gene expressions. Interestingly, CIS-ZMGS-NCs-labeled WJ-MSCs exhibit near-infrared (NIR) fluorescence with a quantum yield of 84%, radiant intensity of ∼3.999 × 1011 (p/s/cm2/sr)/(µW/cm2), magnetic relaxivity (longitudinal r1 = 2.26 mM-1 s-1, transverse r2 = 16.47 mM-1 s-1), and X-ray attenuation (78 HU) potential for early noninvasive multimodality imaging of a subcutaneous melanoma in B16F10-tumor-bearing C57BL/6 mice in 6 h. The ex vivo imaging and inductively coupled plasma mass-spectroscopy analyses of excised organs along with confocal microscopy and immunofluorescence of tumor results also significantly confirmed the positive tropism of CIS-ZMGS-NCs-labeled WJ-MSCs in the tumor environment. Hence, we propose the magnetofluorescent CIS-ZMGS-NCs-labeled WJ-MSCs as a next-generation nanobioprobe of three commonly used imaging modalities for stem cell-assisted anticancer therapy and tracking tissue/organ regenerations.

Metal-free semi-aromatic polyester as nanodrug carrier: A novel tumor targeting drug delivery vehicle for potential clinical application.

Polyester nanomaterials have been widely used in drug delivey application from a longer period of time. This study reports the synthesis of metal-free semi-aromatic polyester (SAP) nanomaterial for drug delivery and evaluate its in vivo acute and systemic toxicity for potential clinical application. The ring opening coplymerization of commercially available cyclohexene oxide (CHO) and phthalic anhydride (PA) monomers was carried out to synthesize fully alternating poly(CHO-co-PA) copolymer using metal-free activators. The obtained low Mn SAP was found to be biocompatible, hemocompataible and biodegradable nature. This copolymer was first-time used to fabricate curcumin (CUR) loaded nanoparticles (NPs). These NPs were physicochemically characterized by thermogravimetric analyzer (TGA), X-ray diffraction (XRD), and UV/visible spectrophotometer analysis. Further, these negatively charged core-shell spherical NPs exhibited slow sustained release behavior of CUR with anomalous transport and further displayed its higher intracellular uptake in SiHa cells at different time-periods compared to free CUR. In vitro anti-cancer therapeutic effects of free CUR and poly(CHO-alt-PA)-CUR NPs were evaluated on different cancer cells. We observed the increased cytotoxicity of CUR NPs with low IC50 values compared to free CUR. These results were further substantiated with ex vivo data where, a significant reduction was observed in CUR NPs treated tumor spheroid's size as compared to free CUR. Furthermore, the different doses of metal-free poly(CHO-alt-PA) nanomaterial were tested for its acute and systemic toxicity in BALB/c mice. We did not observe any significant toxicity of tested nanomaterial on vital organs, blood cells and the body weight of mice. Our study suggest that this metal-free SAP nanomaterial can be used for potential clinical application.

Noninvasive Tracking and Regenerative Capabilities of Transplanted Human Umbilical Cord-Derived Mesenchymal Stem Cells Labeled with I-III-IV Semiconducting Nanocrystals in Liver-Injured Living Mice.

Acute liver injury is a critical syndrome ascribed to prevalent death of hepatocytes and imperatively requires liver transplantation. Such a methodology is certainly hampered due to the deficit of healthy donors. In this regard, stem cell-based regenerative therapies are attractive in repairing injured tissues and organs for medical applications. However, it is crucial to understand the migration, engraftment, and regeneration capabilities of transplanted stem cells in the living animal models. For the first time, we demonstrate rapid labeling of umbilical cord-derived mesenchymal stem cells (MSCs) with near-infrared (NIR)-fluorescent CuInS2-ZnS nanocrystals (CIZS-NCs) to develop innovative nanobioconjugates (MSCs-CIZS-NBCs) that exhibit 98% labeling efficiency. Before nanobioconjugate synthesis, the pristine CIZS-NCs were prepared via a two-step, hot-injection, rapid and low-cost domestic-microwave-refluxing (MW-R) method within 6 min. The as-synthesized CIZS-NCs display high photoluminescence quantum yield (∼88%) and long-lived lifetime (23.4 µs). In contrast to unlabeled MSCs, the MSCs-CIZS nanobioconjugates show excellent biocompatibility without affecting the stemness, as confirmed by cell viability, immunophenotyping (CD44+, CD105+, CD90+), multi-lineage-specific gene expressions, and differentiation into adipocytes, osteocytes, and chondrocytes. The in vivo fluorescence tracking analyses revealed that the MSCs-CIZS-NBCs after tail-vein injection were initially trapped in the lungs and gradually engrafted in the injured liver within 2 h. The regeneration potential of MSCs-CIZS-NBCs was confirmed via renewal of the portal tract composed of portal veins, bile ducts, and hepatic arteries around the hepatocytes. Consequently, no apparent inflammations, necrosis, or apoptosis was observed in the acetaminophen (APAP)-induced liver-injured BALB/c mice model over 3 days after transplantation, as corroborated using laser-scanning confocal microscopy and histopathological and hematological analyses. Hence, our innovative NIR-fluorescent MSCs-CIZS-NBCs offer an off-the-self technology for noninvasive tracking of transplanted MSCs in an acute-liver-injured animal model for future image-guided cell-therapies.

Microwave-Assisted Synthesis of Quasi-Pyramidal CuInS2 -ZnS Nanocrystals for Enhanced Near-Infrared Targeted Fluorescent Imaging of Subcutaneous Melanoma.

Near-infrared (NIR) fluorescent CuInS2 -ZnS nanocrystals (CIZS NCs) are synthesized via an ultra-fast, non-injection microwave (MW)-assisted nanoalloying process at 230 ºC within 5 min using 1-dodecanethiol (DDT) as both the sulfur source and solvent under solvothermal (ST) condition. The structural and surface analyses reveal that DDT-functionalized CIZS NCs exhibit quasi-pyramids of tetragonal-phase with well-defined facets. The DDT-functionalized CIZS NCs present a photoluminescence quantum yield (PLQY) of 76% and a long-lived fluorescence lifetime of ≈0.6 µs in organic-phase. Subsequently, DDT-functionalized CIZS NCs are phase-transferred via ligand-exchange using 11-mercaptoundecanoic acid (MUA) into water-soluble MUA-CIZS NCs that exhibit a substantial PLQY of 55%. In addition, the NIR-fluorescent MUA-functionalized CIZS NCs in conjugation with folic acid (FA), as a tumor-targeting ligand, demonstrates enhanced tumor-targeted imaging ability. The FA-MUA-CIZS NC conjugates exhibit a cell viability of ≈75% even at the highest concentration of 1 mg mL-1 and a labeling efficiency of 95.4%. The in vivo imaging results corroborate that FA-MUA-CIZS NCs conjugates are actively targeted to folate receptor-positive B16F10 tumor-bearing C57BL/6 mice in 2 h. The histopathological and hematological studies confirm no significant changes in tissue architecture and blood biochemical parameters. The confocal microscopy studies reveal deep penetration and uniform distribution of FA-MUA-CIZS NCs conjugates in subcutaneous melanoma.

Expression Profiling of Differentially Regulated Genes in Fanconi Anemia.

Gene expression analysis mainly helps to study gene quantification methods by using various downstream detection approaches like imaging, amplification, probe hybridization, or sequencing. With respect to DNA, which is less static, mRNA levels vary over time, between cell types under divergent conditions. Gene expression analysis is principally focused on determination of mRNA levels transcribed from DNA. DNA microarrays are one of the robust and powerful tools to detect changes in multiple transcripts in larger cohorts in parallel. The basic principle of DNA microarray hybridization is complementary base pairing of single-stranded nucleic-acid sequences. On a microarray platform (also called a chip), known sequences called targets are attached at fixed locations (spots) to a solid surface such as glass using robotic spotting. Since a large number of samples (variables) are used in a typical hybridization experiment, which often leads to impreciseness for example, target mRNA transcribed from the same source should be identical every time. In such cases, developing an optimized protocol for microarray platform to study the expression profiling of differentially regulated genes is a challenging task. Thus genome-wide expression array analysis yields data about candidate genes that may be involved in disease acquisition progression, and helps in better understanding the pathophysiology of the disease. In this chapter we describe in detail the microarray technique, a well-accepted method for understanding the development and progression of Fanconi anemia (FA), a genetic disorder which is characterized by progressive bone marrow failure and a predisposition to cancer.

An efficient protocol for deriving liver stem cells from neonatal mice: validating its differentiation potential.

The success of liver regeneration depends on the availability of suitable cell types and their potential to differentiate into functional hepatocytes. To identify the stem cells which have the ability to differentiate into hepatocytes, we used neonatal liver as source. However, the current protocol for isolating stem cells from liver involves enzymes like collagenase, hyaluronidase exposed for longer duration which limits the success. This results in the keen interest to develop an easy single step enzyme digestion protocol for isolating stem cells from liver for tissue engineering approaches. Thus, the unlimited availability of cell type favors setting up the functional recovery of the damaged liver, ensuring ahead success towards treating liver diseases. We attempted to isolate liver stem derived cells (LDSCs) from mouse neonatal liver using single step minimal exposure to enzyme followed by in vitro culturing. The cells isolated were characterized for stem cell markers and subjected to lineage differentiation. Further, LDSCs were induced to hepatocyte differentiation and validated with hepatocyte markers. Finally, we developed a reproducible, efficient protocol for isolation of LDSCs with functional hepatocytes differentiation potential, which further can be used as in vitro model system for assessing drug toxicity assays in various preclinical trials.