Internalization Kinetics Research Articles

A multidisciplinary design method and application for complex systems

A reasonable electrochemical mechanism model is the basis for accurately describing the actual operation of hydrogen fuel cell (HFC). In this paper, a multidisciplinary modeling and simulation method is proposed. Based on the internal electrochemical reaction kinetics and electrochemical thermodynamics mechanism of the HFC, a voltage output model and a back pressure regulation model are constructed. By analyzing the relationship between the back pressure of the anode and cathode of the HFC and the effective partial pressure of the reactant gas, a voltage output model and back pressure adjustment model are integrated to form an HFC electrochemical mechanism model, which realizes the dynamic and accurate description of the HFC output performance. The final experimental results show that the electrochemical mechanism model of the HFC can achieve good results.

THE STUDY OF HEAVY METAL ION SORPTION KINETICS

Phosphorus-containing polymeric sorbent based on butadiene-styrene rubber was used to remove zinc, iron and chromium ions from water. The research was carried out to study the sorption characteristics by determining the effects of various parameters, such as the pH of the solution, the initial concentration of metal ions, the sorbent mass, the phase contact time, and temperature. The regularities of the kinetics of the sorption of zinc, iron and chromium ions on the phosphorus-containing sorbent synthesized on the basis of butadiene-styrene rubber were studied. On the basis of the results it was defined that the process of sorption of zinc, iron and chromium ions on the cation exchanger proceeds by an ion-exchange mechanism. The kinetics of this process is a combination of external and internal diffusion kinetics (with some predominance of external diffusion kinetics) and is better described by the pseudo-second order reaction model. In addition, a certain contribution to the overall speed of the process is made by the interaction of the sorbed ions with the functional groups of the cation exchanger. Keywords: phosphorus-containing cation exchanger, zinc, iron, chromium, sorption, kinetics.

Sandwich-like high-load MXene/polyaniline film electrodes with ultrahigh volumetric capacitance for flexible supercapacitors

Two-dimensional transition metal carbides/nitrides (MXene) have excellent physicochemical properties, but the restacking of MXene films restricts their development towards flexible supercapacitors with high energy density. Introducing Polyaniline (PANI) into the MXene layer and expanding the interlayer distance of the MXene can reduce the effect of restacking on the MXene after compounding. However, despite the excellent electronic conductivity of the composited MXene/PANI (MP), its internal slow ionic kinetics becomes a fundamental limitation of the electrochemical performance after the MP loading increases. To compensate for this weakness, MP films are often scaled down to a few micrometers in size (<2 mg cm−2), which limits their development. Here, we introduce α-Fe2O3/MnO2 (FM) into MP for the first time by designing a sandwich structure, which significantly improves the bulk capacitance. Due to a large number of active sites and good hydrophilic properties on the MXene surface, FM can interact with the MP. By complexing with MP, the accumulation and loss of FM can be reduced. At the same time, the effect of increasing loading on the electrochemical performance of MP can be compensated. The MXene-PANI/α-Fe2O3-MnO2/MXene-PANI (MP/FM/MP) electrode still exhibits high capacitive performance (661 F g−1, 3138 mF cm−3) when the MP loading reaches 5 mg cm−2, with excellent mechanical properties and increased flexibility. In addition, the corresponding symmetric supercapacitor also shows a remarkable energy density of 53.32 Wh·L−1 (17.45 Wh kg−1). This study provides a way to fabricate MXene-based electrodes with high loadings by designing sandwich-structured electrodes.

Effect of Hydrogen on the Internal Oxidation of a Pd–Cr Alloy in Dual-Atmosphere Conditions

The effect of hydrogen on oxygen permeability has been studied in a diluted Pd–Cr alloy in dual- and single- atmosphere conditions between 600 and 950 °C. The 0.3 mm thick Pd–1.5Cr foil was exposed in dry and humid air as well as in dual-atmosphere conditions, with one sample surface being exposed to air and one to hydrogen, as encountered in solid oxide fuel cells. At all temperatures, Cr oxidized internally forming internal oxidation zones which were measured in metallographic cross sections. Below 800 °C, an external layer of PdO formed on the surface decreasing the internal oxidation kinetics. No measurable effect of hydrogen on the internal oxidation of Cr in Pd has been detected.

The Cellular Behavior, Intracellular Signaling Profile and Nuclear-Targeted Potential Functions of Porcine Growth Hormone (pGH) in Swine Testicular Cells.

Porcine growth hormone (pGH) has many important biological functions and roles, and the biological activity of pGH is closely related with its cell behavior and characteristics. However, so far, the behavior of pGH in swine testicular cell remains unclear. For this, in the current work, the swine testicular cell line (ST) was used as an in vitro model, and CLSM (Confocal laser scanning microscope), IFA (Indirect immunofluorescence assay), FCM (Flow cytometry) and WB (Western-blotting) were used to explore the pGH's cell behivior and function, and the results showed that pGH and GHR could internalize into ST cell and transported to the nucleus. Furthermore, we studied the internalization kinetics of pGH and GHR on ST cell, and found that pGH and GHR internalizes into ST cell in a time-dependent manner. More importantly, we also investigated the potential molecular functions of pGH-GHR after it entered into the cell nuclei. The results indicated that nuclear-localized GHR could participate in cell proliferation by regulating the signal intensity of STAT5. In summary, our current research shows that the nuclear-localized pGH-GHR participates in the cell proliferation of ST cell, which lays a solid foundation for further research on the regulatory effect of pGH on testicular tissue.

Methylamine gas healing of perovskite films: a short review and perspective

We summarize the internal chemical mechanism, crystallization kinetics and future prospects of the MA0 healing process.

Real-Time Quantification of Cell Internalization Kinetics by Bioluminescent Probes.

Alongside the intracellular transport of nutrients needed for cellular homeostasis, great efforts exist to effectively deliver substances such as proteins and genes into the cell for therapy, gene editing, disease diagnosis, and more. To evaluate the intracellular delivery of such substances, conventional methods impose semi-quantifications and discrete measures of the dynamic process of cellular internalization. Herein, we detail the methods to quantify cell internalization kinetics in real-time using individually nano-encapsulated bioluminescent Firefly Luciferase (FLuc) enzymes as probes. We include a comprehensive protocol to synthesize and characterize the encapsulated FLuc, assay the real-time bioluminescence (BL) in cells, and analyze the real-time BL profile to extract key parameters of cell internalization kinetics. Quantifying the kinetics of intracellular delivery offers the opportunity to resolve the underlying mechanisms governing membrane translocation and provide measures reflecting cellular state and metabolism while playing a critical role in the clinical development of effective vectors.

Modulating internal transition kinetics of responsive macromolecules by collective crowding.

Packing and crowding are used in biology as mechanisms to (self-)regulate internal molecular or cellular processes based on collective signaling. Here, we study how the transition kinetics of an internal "switch" of responsive macromolecules is modified collectively by their spatial packing. We employ Brownian dynamics simulations of a model of Responsive Colloids, in which an explicit internal degree of freedom-here, the particle size-moving in a bimodal energy landscape self-consistently responds to the density fluctuations of the crowded environment. We demonstrate that populations and transition times for the two-state switching kinetics can be tuned over one order of magnitude by "self-crowding." An exponential scaling law derived from a combination of Kramers' and liquid state perturbation theory is in very good agreement with the simulations.

Hyaluronan-coated nanoparticles for active tumor targeting: Influence of polysaccharide molecular weight on cell uptake

Here we aimed to correlate different molecular weights of hyaluronic acid (HA), 200, 800 and 1437 kDa, used to decorate poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs), to their cell uptakes. NP internalization kinetics in CD44-overexpressing breast carcinoma cells were quantified, using healthy fibroblast cells as reference. Actually, NP uptake and selectivity by tumor cells were maximized for NPs HA 800 kDa, while being minimum for NPs HA1400 kDa. This unexpected result could be explained considering that the interaction between NPs and tumor cells is dictated by rearrangement and conformation of that segment of HA chain that actually protrudes from the NPs. Overall, results obtained in this work point at how HA molecular weight, is pivotal project parameter in NP formulation to promote active targeting in the CD44 overexpressing cancer cells.

Cell-Penetrating Peptides.

In this introductory chapter, we first define cell-penetrating peptides (CPPs), give short overview of CPP history and discuss several aspects of CPP classification. Next section is devoted to the mechanism of CPP penetration into the cells, where direct and endocytic internalization of CPP is explained. Kinetics of internalization is discussed more extensively, since this topic is not discussed in other chapters of this book. At the end of this section some features of the thermodynamics of CPP interaction with the membrane is also presented. Finally, we present different cargoes that can be transferred into the cells by CPPs and briefly discuss the effect of cargo on the rate and efficiency of penetration into the cells.

Quantificational 4D Visualization of Industrial Electrodeposition.

Electrodeposition is a fundamental technology in modern society and has been widely used in metal plating and extraction, etc. However, extreme reaction conditions, including wide operation temperature ranges and corrosive media (molten salt/oxide systems as a particular example), inhibit direct in situ observation of the electrodeposition process. To visualize the electrode kinetics in such “black box,” X‐ray tomography is employed to monitor the electrochemical processes and three‐dimensional (3D) evolution of morphology. Benefiting from the excellent penetration of X‐ray, a non‐destructive and non‐contact in situ four‐dimensional (4D) visualization of Ti deposition is realized. Real‐time 3D reconstructed images reveal that the counterintuitive nucleation and growth process of a mesoscale Ti dendrite at both solid and liquid cathodes. According to 3D morphology evolution, unusual mechanism based on synergetic effect of the diffusion of metallic Ti and local field enhancement is achieved utilizing a simulation method based on a finite element method. This approach allows for timely and accurately regulating the electrodeposition process upon in situ monitored parameters. More importantly, the 4D technique upon operando X‐ray tomography and numerical simulation can be easily applied to other electrodeposition systems, which will help deeply understand the internal kinetics and the precise optimization of the electrodeposition conditions.

Strategies for improving electrochemical reaction kinetics of cathode materials for subzero-temperature Li-ion batteries: A review

Lithium-ion batteries (LIBs) have been widely used as the main power sources for consumer electronics and electric vehicles. However, LIBs are usually suffered from serious reduction in energy and cruising ability when operated below 0 °C, owing to the sluggish Li+ion transport kinetics in the bulk electrodes, electrolyte, and their interfaces. In this review, the recent important advances in improving the electrochemical reaction kinetics for the subzero operations of LIBs based on the cathode perspective are summarized. Firstly, we mainly discussed the influences of subzero temperatures on the electrochemical reaction kinetics for several typical cathode materials, including olivine LiFePO4 (LFP) and Li3V2(PO4)3 (LVP) phosphates, layered LiCoO2 (LCO), Ni-rich LiNi1-x-yCoxMnyO2 (NCM) and LiNi1-x-yCoxAlyO2 (NCA) oxides, as well as Li- and Mn-rich Li1-xNi1-x-y-zCoyMnzO2 (LMR) oxides. Then, various strategies developed for enhancing the interfacial and internal electrochemical reaction kinetics for these typical cathode materials under subzero temperatures are detailly discussed in detail. Subsequently, a short comparison and overview of studies and related properties of the LFP, LVP, NCM and LMR cathode materials are provided. Finally, the specific suggestions and ideas for dealing with the challenges of the slow kinetics for cathode materials under subzero temperatures are put forward, inspiring the further developments of LIBs and other devices for subzero-temperature applications.

Synthesis of 111In-p-SCN-Bn-DTPA-nimotuzumab and its preclinical evaluation in EGFR positive NSCLC animal model

Abstract The aim of this study was to investigate labeling of nimotuzumab (h-R3) with 111In using p-SCN-Bn-DTPA as bifunctional chelate, evaluate its targeting potential against SK-LU-1, H226, H650, H661, and HCC4006 non-small cell lung carcinoma (NSCLC) cell lines and correlate epidermal growth factor receptor (EGFR) expression level with internalization kinetics, biodistribution and imaging accuracy using Balb/c mice and New Zealand White rabbit (NZWR) animal model. The amount of p-SCN-Bn-DTPA attached to h-R3 was assessed by measuring relative absorbance at 652 nm with ultraviolet (UV) spectrophotometer. High-performance liquid chromatography (HPLC) was used to determine percent radiochemical purity (%RCP) and in vitro stability using excess amount of diethylenetriamine pentaacetate (DTPA). The in vitro stability in rat serum was estimated using iTLC-SG. EGFR expression level in each tumor was assessed by chemiluminescence. In vivo uptake in different organs of Balb/c mice and non-invasive imaging potential using NZWR bearing HCC4006 tumor, was evaluated with gamma camera. UV spectroscopy has confirmed the attachment of five p-SCN-Bn-DTPA (chelate) with one antibody. The HPLC indicated 98.85 ± 0.14% (n = 3) %RCP with high yield (&gt;96%), specific activity 3.5 ± 0.0.25 mCi per mg and 94.25 ± 0.34% in vitro stability at 37 °C in mice serum. In excess DTPA no considerable transchelation was experiential from the 111In labeled p-SCN-Bn-DTPA-h-R3 to the challenger. The EGFR expression in HCC4006 was higher amongst all with band density of 23.53 relative to 1.00 of H226. Initially internalization was lower which went up 1.05 × 104 molecules per HCC4006 cell in 48 h. The optimal concentration of h-R3 for maximum uptake was 15 μg per animal. Higher uptake in target organ was observed in animal infected with HCC4006 cells. However, in excess pure h-R3 the uptake was significantly reduced indicating tumor specificity. HCC4006 target site was undistinguishable relative to background activity in the initial phase of imaging due to poor uptake. However, within 60 h the HCC4006 tumor was quite apparent. This experiment suggests that at optimal dosage of 111In labeled h-R3 can be used for localization and identification of EGFR positive NSCLC using gamma camera.

An original methodology to study polymeric nanoparticle-macrophage interactions: Nanoparticle tracking analysis in cell culture media and quantification of the internalized objects

Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) are among the most employed (co)polymers for the preparation of drug nanocarriers for the treatment of cancer and infectious diseases. Before considering any clinical use, it is necessary to understand the interactions between polymeric nanoparticles (NPs) and their physiological environment, especially immune cells. Here, we propose a simple, yet precise method to assess NPs internalization kinetics in macrophages, based on the direct analysis of the cell culture media after different incubation times. The proof of concept is given here by using fluorescent PLGA NPs. Nanoparticle tracking analysis (NTA) was a method of choice, enabling detecting each individual NP and analyzing its trajectory while in Brownian motion. As compared to dynamic light scattering (DLS), NTA enabled a more precise determination of NP size distribution. The uptake process was rapid: in one hour, around a third of the NPs were internalized. In addition, the internalized NPs were visualized by confocal microscopy. The fluorescent cellular stacks were analyzed using a freely available macro for ImageJ software, Particle_In_Cell-3D. The internalized objects were localized and counted. This methodology could serve for further studies while analyzing the effects of NPs size, shape and surface properties on their interaction with various cell lines.

Death Receptors DR4 and DR5 Undergo Spontaneous and Ligand-Mediated Endocytosis and Recycling Regardless of the Sensitivity of Cancer Cells to TRAIL.

Tumor necrosis factor-associated ligand inducing apoptosis (TRAIL) induces apoptosis through the death receptors (DRs) 4 and 5 expressed on the cell surface. Upon ligand stimulation, death receptors are rapidly internalized through clathrin-dependent and -independent mechanisms. However, there have been conflicting data on the role of death receptor endocytosis in apoptotic TRAIL signaling and possible cell type-specific differences in TRAIL signaling have been proposed. Here we have compared the kinetics of TRAIL-mediated internalization and subsequent recycling of DR4 and DR5 in resistant (HT-29 and A549) and sensitive (HCT116 and Jurkat) tumor cell lines of various origin. TRAIL stimulated the internalization of both receptors in a concentration-dependent manner with similar kinetics in sensitive and resistant cell lines without affecting the steady-state expression of DR4 and DR5 in cell lysates. Using the receptor-selective TRAIL variant DR5-B, we have shown that DR5 is internalized independently of DR4 receptor. After internalization and elimination of TRAIL from culture medium, the receptors slowly return to the plasma membrane. Within 4 h in resistant or 6 h in sensitive cells, the surface expression of receptors was completely restored. Recovery of receptors occurred both from newly synthesized molecules or from trans-Golgi network, as cycloheximide and brefeldin A inhibited this process. These agents also suppressed the expression of cell surface receptors in a time- and concentration-dependent manner, indicating that DRs undergo constitutive endocytosis. Inhibition of receptor endocytosis by sucrose led to sensitization of resistant cells to TRAIL and to an increase in its cytotoxic activity against sensitive cells. Our results confirm the universal nature of TRAIL-induced death receptor endocytosis, thus cell sensitivity to TRAIL can be associated with post-endocytic events.

Exploring the photocatalytic conversion mechanism of gaseous formaldehyde degradation on TiO2–x-OV surface

To understand the conversion mechanism of photocatalytic gaseous formaldehyde (HCHO) degradation, strontium (Sr)-doped TiO2–x–OV catalysts was designed and synthesized in this study, with comparable HCHO removal performance. Our results proved that foreign-element doping reduced Ti4+ to the lower oxidation state Ti(4– x)+, and that the internal charge kinetics was largely facilitated by the unbalanced electron distribution. Oxygen vacancies (OVs) were developed spontaneously to realize an electron-localized phenomenon in TiO2–x–OV, thereby boosting O2 adsorption and activation for the enhanced generation of reactive oxygen species (ROS). At the chemisorption stage, in-situ DRIFTS spectra and density functional theory calculation results revealed that surface adsorbed O2 (Oads) and lattice O (Olat) engaged in the isomerisation of HCHO to dioxymethylene (DOM) on TiO2–x–OV and TiO2, respectively. Time-resolved DRIFTS spectra under light irradiation revealed that the DOM was then converted to formate and thoroughly oxidized to CO2 and H2O in TiO2–x–OV. While bicarbonate byproducts were detected from DOM hydroxylation or possible side conversion of CO2 in TiO2, owing to insufficient consumption of surface hydroxyl. Our study enhances the understanding on the photocatalytic oxidation of HCHO, thereby promoting the practical application in indoor air purification.

Influence of iron binding in the structural stability and cellular internalization of bovine lactoferrin

Lactoferrin (Lf) is an iron-binding glycoprotein and a component of many external secretions with a wide diversity of functions. Structural studies are important to understand the mechanisms employed by Lf to exert so varied functions. Here, we used guanidine hydrochloride and high hydrostatic pressure to cause perturbations in the structure of bovine Lf (bLf) in apo and holo (unsaturated and iron-saturated, respectively) forms, and analyzed conformational changes by intrinsic and extrinsic fluorescence spectroscopy. Our results showed that the iron binding promotes changes on tertiary structure of bLf and increases its structural stability. In addition, we evaluated the effects of bLf structural change on the kinetics of bLf internalization in Vero cells by confocal fluorescence microscopy, and observed that the holo form was faster than the apo form. This finding may indicate that structural changes promoted by iron binding may play a key role in the intracellular traffic of bLf. Altogether, our data improve the comprehension of bLf stability and uptake, adding knowledge to its potential use as a biopharmaceutical.

Antibody-Drug Conjugate Efficacy in Neuroblastoma: Role of Payload, Resistance Mechanisms, Target Density, and Antibody Internalization.

Antibody-drug conjugates (ADC) are a targeted cancer therapy that utilize the specificity of antibodies to deliver potent drugs selectively to tumors. Here we define the complex interaction among factors that dictate ADC efficacy in neuroblastoma by testing both a comprehensive panel of ADC payloads in a diverse set of neuroblastoma cell lines and utilizing the glypican 2 (GPC2)-targeting D3-GPC2-PBD ADC to study the role of target antigen density and antibody internalization in ADC efficacy in neuroblastoma. We first find that DNA binding drugs are significantly more cytotoxic to neuroblastomas than payloads that bind tubulin or inhibit DNA topoisomerase 1. We additionally show that neuroblastomas with high expression of the ABCB1 drug transporter or that harbor a TP53 mutation are significantly more resistant to tubulin and DNA/DNA topoisomerase 1 binding payloads, respectively. Next, we utilized the GPC2-specific D3-GPC2-IgG1 antibody to show that neuroblastomas internalize this antibody/GPC2 complex at significantly different rates and that these antibody internalization kinetics correlate significantly with GPC2 cell surface density. However, sensitivity to pyrrolobenzodiazepine (PBD) dimers primarily dictated sensitivity to the corresponding D3-GPC2-PBD ADC, overall having a larger influence on ADC efficacy than GPC2 cell surface density or antibody internalization. Finally, we utilized GPC2 isogenic Kelly neuroblastoma cells with different levels of cell surface GPC2 expression to define the threshold of target density required for ADC efficacy. Taken together, DNA binding ADC payloads should be prioritized for development for neuroblastoma given their superior efficacy and considering that ADC payload sensitivity is a major determinant of ADC efficacy.

In vivo high-contrast visualization of upconversion nanoparticle labeled virus using time-resolved approach

Virus labeling is an ideal strategy to explore the interaction between virus and host cells. However, existed probes are limited in in vivo studies due to the poor imaging effects caused by tissue-depth and autofluorescence. Lanthanide-doped upconversion nanoparticles (UCNP) can be effectively excited in deeper bio-tissues, and its long fluorescence life-time is suitable for in vivo imaging using time-resolved luminescence imaging technology. In this study, lanthanide-doped UCNP was used to label viral envelope and nucleic acid, which could be used for in vitro and in vivo virus tracking. The in vitro results demonstrated that the UCNP-labeled Influenza A virus (UCNP-IAV) could be used for the study of virus internalization kinetics, and proved that the movement of UCNP labeled IAV was lysosome- and microtube-dependent. For in vivo study, pre-labeling and in situ labeling strategies were implemented to monitor the IAV infection. The results showed that the UCNP probe could provide high-quality images using time-gated strategy to effectively monitor the viral infection in vivo. Further, simultaneous tracking of IAV and adenovirus type-5 (Ad5) labeled by UCNP with different life-time was realized using time-resolved luminescence imaging technology. Hence, this paper provided a novel labeling method for virus tracking using UCNP, which was optimal to study the mechanism of virus infection in vitro and in vivo.

Targeted Mass Spectrometry-Based Approach for the Determination of Intrinsic Internalization Kinetics of Cell-Surface Membrane Protein Targets.

Successful development of targeted therapeutics aimed at the elimination of diseased cells relies on the target properties and the therapeutics that target them. Currently, target properties have been evaluated through antibody-dependent semiquantitative approaches such as flow cytometry, Western blotting, or microscopy. Since antibodies can alter target properties following binding, antibody-dependent approaches provide at best skewed measurements for target intrinsic properties. To circumvent, here we attempted to develop an antibody-free targeted mass spectrometry-based (ATM) strategy to measure the surface densities and the intrinsic rates (Kint) of CD38 internalization in multiple myeloma cell lines. Using cell-surface biotinylation in conjunction with differential mass tagging to separate inward CD38 molecules from the outbound and nascent ones, the ATM approach revealed diversities in measured CD38 Kint values of 0.239 min-1 S.E. ± 0.076, 0.109 min-1 S.E. ± 0.032, and 0.058 min-1 S.E. ± 0.001 for LP1, NCIH929, and MOLP8 cell lines, respectively. Together with CD38 surface densities, intrinsic Kint values aligned well with the tumor penetration model and supported the outcomes for tumor regression in mouse xenografts upon drug treatment. Additionally, the ATM approach can evaluate molecules with fast Kint as we determined for CTLA4 protein. We believe that the ATM approach has the potential to evaluate diverse cell-surface targets as part of the pharmacological assessment in drug discovery.