Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases. Mitochondria in cells play a crucial role in programmed cell death and redox processes. Nicotinamide adenine dinucleotide (NAD(P)H) is the primary producer of energy in mitochondria, changing NAD(P)H can directly reflect the physiological state of mitochondria. Therefore, NAD(P)H can be used to evaluate metabolic response. In this paper, we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy (TP-FLIM) to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H. The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH, serum content, and oxygen concentration in the cell culture environment, and by the treatment with reagents such as H2O2 and paclitaxel. Taxol (PTX). This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.
Biomaterials that promote angiogenesis are required for repair and regeneration of bone. In-situ formed injectable hydrogels functionalised with bioactive agents, facilitating angiogenesis have high demand for bone regeneration. In this study, pH and thermosensitive hydrogels based on chitosan (CS) and hydroxyapatite (HA) composite materials loaded with heparin (Hep) were investigated for their pro-angiogenic potential. Hydrogel formulations with varying Hep concentrations were prepared by sol–gel technique for these homogeneous solutions were neutralised with sodium bicarbonate (NaHCO3) at 4 °C. Solutions (CS/HA/Hep) constituted hydrogels setting at 37 °C which was initiated from surface in 5–10 minutes. Hydrogels were characterised by performing injectability, gelation, rheology, morphology, chemical and biological analyses. Hydrogel solutions facilitated manual dropwise injection from 21 Gauge which is highly used for orthopaedic and dental administrations, and the maximum injection force measured through 19 G needle (17.191 ± 2.296N) was convenient for manual injections. Angiogenesis tests were performed by an ex-ovo chick chorioallantoic membrane (CAM) assay by applying injectable solutions on CAM, which produced in situ hydrogels. Hydrogels induced microvascularity in CAM assay this was confirmed by histology analyses. Hydrogels with lower concentration of Hep showed more efficiency in pro-angiogenic response. Thereof, novel injectable hydrogels inducing angiogenesis (CS/HA/Hep) are potential candidates for bone regeneration and drug delivery applications.
An intelligent narrow pH-triggered multilayer film was prepared on magnesium alloys, aiming to solve the implant infections during the implantation period and improve the corrosion resistance of magnesium alloys. The encapsulation of ibuprofen by chitosan (IBU@CS) makes the release of IBU sensitive to narrow pH (pH 6.8-7.4). Positive charged IBU@CS was assembled with heparin (Hep) to fabricate (Hep/IBU@CS)10 film on AZ31 alloys using layer-by-layer method. The microstructure, composition and anticorrosion properties of the film were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and electrochemical experiments. Cellular activity was studied by MTT cell viability assay. The results showed that the Hep/IBU@CS multilayer films improved the corrosion resistance of magnesium alloys. The in vitro test demonstrated that the release of IBU in the film presented narrow pH sensitivity. The films showed no obvious signs of cytotoxicity conformed by the MTT assay and presented antibacterial properties. These preliminary results demonstrate the potential use of the Hep/IBU@CS multilayer films on magnesium-based implants.
J. Emami, Moloud Kazemi, Farshid Hasanzadeh
et al.
Abstract In this study, pH-triggered polymeric micelle comprising α-tocopherol (TOC) and heparin (HEP) was developed and loaded with docetaxel (DTX). The amphiphilic copolymer was synthesized by grafting TOC onto HEP backbone by a pH-cleavable bond. DTX-loaded micelles were characterized in terms of critical micelle concentration (CMC), particle size, zeta potential, entrapment efficiency (EE), pH-responsive behavior, and drug release. In vitro cytotoxicity of the micelles against breast cancer cells was investigated by MTT assay. The cellular uptake of coumarin-loaded micelles was also evaluated. Furthermore, the pharmacokinetics of DTX-loaded micelles was evaluated and compared with that of Taxotere®. HEP–CA–TOC copolymers showed low CMC values and high EE. At pH 7.4, the micelles remained stable in size and shape, whereas considerable changes in particle size and morphology were observed at pH 5.5. DTX-loaded micelles showed pH-dependent drug release profiles. Coumarin-loaded micelles showed higher cellular uptake than free coumarin. Therefore, the DTX-loaded micelles showed more toxicity against breast cancer cells than free DTX. A significant increase in T1/2 β, AUC0-∞ and MRT was observed in DTX-loaded micelle treated group as compared to the group treated with Taxotere®. The results suggest that the pH-sensitive HEP-modified micelles could be promising for enhanced intracellular drug delivery of DTX for cancer treatment.
The abnormal concentrations of both biothiols and pH in lysosomes are seriously related to many major diseases, such as Parkinson's and Alzheimer's diseases. Up to now, there are few reports that clearly illustrate the relationship between lysosomal pH and biothiols via fluorescence assay. Herein, novel carbon dots (Scy-CDs) are prepared with good water dispersibility and excellent photostability, and a large Stokes shift of 106 nm is exhibited under an excitation wavelength of 450 nm. The remarkable pH-dependent behavior of Scy-CDs is presented with the fluorescence quenching based on the donor-excited photoinduced electron transfer (d-PET) process. The pKa value is 5.30, which is in good agreement with the range of the normal and abnormal lysosomal pH. Upon the addition of cysteine (Cys) or homocysteine (Hcy), the d-PET process is effectively inhibited with fluorescence recovery totally. The significant co-localization of Scy-CDs with Lyso-Tracker Deep Red in HEp-2 cells and the Pearson correlation coefficient 0.88 strongly suggest that the Scy-CDs can target lysosomal pH and Cys/Hcy in living cells.
Moloud Kazemi, J. Emami, Farshid Hasanzadeh
et al.
Abstract DTX-loaded folate targeted micelles (DTX/FA-PEG-HEP-CA-TOC) were prepared using a dialysis method and the impact of various processing variables were evaluated on micelle properties. In vitro cytotoxicity and in vivo pharmacokinetics of the optimized formulation were evaluated. DTX/FA-PEG-HEP-CA-TOC exhibited average particle size of 90–185 nm with high entrapment efficiency (53–93%) and pH-dependent drug release behavior. DTX/FA-PEG-HEP-CA-TOC micelles exhibited higher toxicity against SKOV3 cells than DTX/HEP-CA-TOC micelles and free DTX. Significant increase in the T 1/2 β, AUC and MRT and decrease in Vd as well as CL values was observed in DTX/FA-PEG-HEP-CA-TOC micelle treated group compared to free DTX treated group. Graphical Abstract
Artepillin C is the main compound present in propolis from Baccharis dracunculifolia, whose antitumor activity has been the focus of many studies. Herein, we shall investigate the Artepillin C mechanisms of action against cells derived from the oropharyngeal carcinoma (HEp-2). Cytotoxicity tests revealed that the concentrations of Artepillin C required to reduce cell viability by 50% (CC50) are dependent on the incubation time, decreasing from 40.7 × 10-5 mol/L to 15.7 × 10-5 mol/L and 9.05 × 10-5 mol/L considering 12, 24 and 48 h, respectively. Hydrophobic interactions on neutral species of Artepillin C induce aggregation over the HEp-2 plasma membrane, given the acid conditions of the cellular culture. Indeed, Langmuir monolayers mimicking cellular membranes of tumor cells revealed Artepillin C affinity to interact with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) containing 20 mol% of 1,2-dipalmitoyl-sn-glychero-3-phosphoserine (DPPS), leading aggregation on giant unilamellar vesicles (GUVs) at pH 3.2. Moreover, leakage experiments on GUVs have shown that the presence of DPPS enhances the efflux of the fluorescent probe signaling the membrane permeabilization, which is the origin of the necrotic pathway triggered in HEp-2 cells, as observed by flow cytometry assays.
Abstract A new poly (3-arginylamino propylene succinate-b-polypropylene glycol) (PAPS-PPG) diblock copolymer was synthesized by one-pot ring opening polymerization (ROP) technique. 1H NMR spectral technique was used to determine arginine conjugation efficiency and molecular weight of final polycations by using 4,4-dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA) as an internal standard. The PAAPS-PPG polycation was electrostatically complexed with heparin (HEP) at an isoelectric point to obtain PAAPS-PPG/HEP coacervate microparticles (Coa MPs). The PAAPS-PPG/HEP Coa MPs exhibit acid induced dissociation at pH 5, but PAAPS/HEP Coa MPs without PPG block do not show acid induced dissociation. The PPG block in PAAPS-PPG/HEP Coa MPs increased 20 folds of cell attachment efficiency on human mesenchymal stem cells (hMSCs). The PAAPS-PPG/HEP Coa MPs could effectively encapsulate siRNA and exhibit more than 95% cell viability up to 200 μg/mL. Hence, the pH responsive PAAPS-PPG/HEP Coa MPs platform with enhanced stem cell attachment ability would find potential application in gene delivery.