Photoinduced Endosomal Escape Mechanism: A View from Photochemical Internalization Mediated by CPP-Photosensitizer Conjugates

Endosomal escape in cell-penetrating peptide (CPP)-based drug/macromolecule delivery systems is frequently insufficient. The CPP-fused molecules tend to remain trapped inside endosomes and end up being degraded rather than delivered into the cytosol. One of the methods for endosomal escape of CPP-fused molecules is photochemical internalization (PCI), which is based on the use of light and a photosensitizer and relies on photoinduced endosomal membrane destabilization to release the cargo molecule. Currently, it remains unclear how this delivery strategy behaves after photostimulation. Recent findings, including our studies using CPP-cargo-photosensitizer conjugates, have shed light on the photoinduced endosomal escape mechanism. In this review, we discuss the structural design of CPP-photosensitizer and CPP-cargo-photosensitizer conjugates, and the PCI mechanism underlying their application.     

An Intrinsically Disordered Peptide Facilitates Non‐Endosomal Cell Entry

Many cell‐penetrating peptides (CPPs) fold at cell surfaces, adopting α‐ or β‐structure that enable their intracellular transport. However, the same structural folds that facilitate cellular entry can also elicit potent membrane‐lytic activity, limiting their use in delivery applications. Further, a distinct CPP can enter cells through many mechanisms, often leading to endosomal entrapment. Herein, we describe an intrinsically disordered peptide (CLIP6) that exclusively employs non‐endosomal mechanisms to cross cellular membranes, while being remarkably biocompatible and serum‐stable. We show that a single anionic glutamate residue is responsible for maintaining the disordered bioactive state of the peptide, defines its mechanism of cellular entry, and is central to its biocompatibility. CLIP6 can deliver membrane‐impermeable cargo directly to the cytoplasm of cells, suggesting its broad utility for delivery of drug candidates limited by poor cell permeability and endosomal degradation.     

Modular Redesign of a Cationic Lytic Peptide To Promote the Endosomal Escape of Biomacromolecules

Endocytosis is an important route for the intracellular delivery of biomacromolecules, wherein their inefficient endosomal escape into the cytosol remains a major barrier. Based on the understanding that endosomal membranes are negatively charged, we focused on the potential of cationic lytic peptides for developing endosomolysis agents to release such entrapped molecules. As such, a venom peptide, Mastoparan X, was employed and redesigned to serve as a delivery tool. Appending a tri‐glutamate unit to the N‐terminus attenuates the cytotoxicity of Mastoparan X by about 40 fold, while introduction of a Ni^II‐dipicolylamine complex enhances cellular uptake of the peptide by about 17 fold. Using the optimized peptide, various fluorescently labeled macromolecules were successfully delivered to the cytosol, enabling live‐cell imaging of acetylated histones.     

A Glycosylated Covalent Organic Framework Equipped with BODIPY and CaCO3 for Synergistic Tumor Therapy

Ca²⁺, a ubiquitous but nuanced modulator of cellular physiology, is meticulously controlled intracellularly. However, intracellular Ca²⁺ regulation, such as mitochondrial Ca²⁺ buffering capacity, can be disrupted by ¹O₂. Thus, the intracellular Ca²⁺ overload, which is recognized as one of the important cell pro‐death factors, can be logically achieved by the synergism of ¹O₂ with exogenous Ca²⁺ delivery. Reported herein is a nanoscale covalent organic framework (NCOF)‐based nanoagent, namely CaCO₃@COF‐BODIPY‐2I@GAG ( 4 ), which is embedded with CaCO₃ nanoparticle (NP) and surface‐decorated with BODIPY‐2I as photosensitizer (PS) and glycosaminoglycan (GAG) targeting agent for CD44 receptors on digestive tract tumor cells. Under illumination, the light‐triggered ¹O₂ not only kills the tumor cells directly, but also leads to their mitochondrial dysfunction and Ca²⁺ overload. An enhanced antitumor efficiency is achieved via photodynamic therapy (PDT) and Ca²⁺ overload synergistic therapy.     

Lanthanum Chloride Promoted Proliferation with Enhanced S‐phase Entry and Inhibited Potassium Currents of NIH 3T3 Cells

The effects of La³⁺ on proliferation, cell cycles, apoptosis and ion channels were investigated in mouse embryo fibroblast NIH 3T3 cells and its possible mechanisms were explored. Our data showed that La³⁺ promoted cell proliferation with increased S‐phase entry and inhibited the outward potassium currents in a concentration‐dependent manner in NIH 3T3 cells. La³⁺ and Ca²⁺ had synergistic effect on cell proliferation and cell cycles. It showed that Ca²⁺ was needed for La³⁺‐promoted cell cycle progression. Using the whole‐cell voltage‐clamp technique, we found that La³⁺ blocked the outward potassium current in a concentration‐dependent manner in NIH 3T3 cells. Lanthanum ions can increase intracellular Ca²⁺ concentration through inhibition of potassium currents, which induce a series of physiological changes and improve proliferation of cells. This may be one of the molecular mechanisms of lanthanum ions induced cell proliferation. The present work provides a new perspective for understanding the biological and toxicological effects of lanthanum.     

Probing Endosomal Escape Using pHlexi Nanoparticles

The effective escape of nanocarriers from endosomal compartments of the cell remains a major hurdle in nanomedicine. The endosomal escape of pH‐responsive, self‐assembled, dual component particles based on poly[2‐(diethylamino)ethyl methacrylate)(PDEAEMA) and poly(ethylene glycol)‐b‐poly[2‐(diethylamino)ethyl methacrylate) (PEG‐b‐PDEAEMA) has been recently reported. Herein, we report that polymer molecular weight (M _n) can be used to tune endosomal escape of nanoparticle delivery systems. PDEAEMA of M _n 7 kDa, 27 kDa, 56 kDa and 106 kDa was synthesized via reversible addition‐fragmentation chain transfer (RAFT) polymerization and co‐assembled with PEG‐b‐PDEAEMA (16 kDa) via nanoprecipitation. All particles had similar size, displayed pH‐responsive behaviour, and low toxicity regardless of molecular weight. Ovalbumin was loaded in the particles to demonstrate loading and release capabilities and as a marker to study internalization and endosomal escape. Association and endosomal escape was found to depend on molecular weight, with enhanced escape observed for high M _n PDEAEMA: 42% of cells with particle induced endosomal escape for 106 kDa nanoparticles, compared to minimal escape for 7 kDa particles. The results show that a simple variation in molecular weight can enhance the endosomal escape of polymeric carriers, and thus improve their effectiveness for intracellular delivery of therapeutics.

     

Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca2+‐ATPase

Calcium ATPase is a member of the P‐type ATPase, and it pumps calcium ions from the cytoplasm into the reticulum against a concentration gradient. Several X‐ray structures of different conformations have been solved in recent years, providing basis for elucidating the active transport mechanism of Ca²⁺ ions. In this work, molecular dynamics (MD) simulations were performed at atomic level to investigate the dynamical process of calcium ions moving from the outer mouth of the protein to their binding sites. Five initial locations of Ca²⁺ ions were considered, and the simulations lasted for 2 or 6 ns, respectively. Specific pathways leading to the binding sites and large structural rearrangements around binding sites caused by uptake of calcium ions were identified. A cooperative binding mechanism was observed from our simulation. Firstly, the first Ca²⁺ ion binds to site I , and then, the second Ca²⁺ ion approaches. The interactions between the second Ca²⁺ and the residues around site I disturb the binding state of site I and weaken its binding ability for the first bound Ca²⁺. Because of the electrostatic repulsion of the second Ca²⁺ and the electrostatic attraction of site II , the first bound Ca²⁺ shifts from site I to site II . Concertedly, the second Ca²⁺ binds to site I , forming a binding state with two Ca²⁺ ions, one at site I and the other at site II . Both of Glu908 and Asp800 coordinate with the two Ca²⁺ ions simultaneously during the concerted binding process, which is believed to be the hinge to achieve the concerted binding. In our simulations, four amino acid residues that serve as the channel to link the outer mouth and the binding sites during the binding process were recognized, namely Tyr837, Tyr763, Asn911, and Ser767. The analyses regarding the activity of the proteins via mutations of some key residues also supported our cooperative mechanism. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009     

Calcium Carbonate Mineralized Nanoparticles as an Intracellular Transporter of Cytochrome c for Cancer Therapy

A new intracellular delivery system based on an apoptotic protein‐loaded calcium carbonate (CaCO₃) mineralized nanoparticle (MNP) is described. Apoptosis‐inducing cytochrome c (Cyt c) loaded CaCO₃ MNPs (Cyt c MNPs) were prepared by block copolymer mediated in situ CaCO₃ mineralization in the presence of Cyt c. The resulting Cyt c MNPs had a vaterite polymorph of CaCO₃ with a mean hydrodynamic diameter of 360.5 nm and exhibited 60 % efficiency for Cyt c loading. The Cyt c MNPs were stable at physiological pH (pH 7.4) and effectively prohibited the release of Cyt c, whereas, at intracellular endosomal pH (pH 5.0), Cyt c release was facilitated. The MNPs enable the endosomal escape of Cyt c for effective localization of Cyt c in the cytosols of MCF‐7 cells. Flow cytometry showed that the Cyt c MNPs effectively induced apoptosis of MCF‐7 cells. These findings indicate that the CaCO₃ MNPs can meet the prerequisites for delivery of cell‐impermeable therapeutic proteins for cancer therapy.     

Fura-2探针对希土Y3+跨PC12细胞膜行为研究

使用AR-MIC-CM阳离子测定系统,发展Fura-2荧光测定技术,将其应用于测定细胞内游离希土离子Y³⁺,并以此研究了Y³⁺跨PC12细胞(大鼠嗜铬细胞瘤细胞)膜的行为。结果表明：在模拟细胞内各离子组分,pH=7.05的溶液中,测得表观解离常数为4.5p mol·L^-1。对于PC12细胞,静息条件下Y³⁺不能跨越细胞膜进入胞内。与钙离子通道相关的KCl和去甲肾上腺素均不能刺激希土Y³⁺过膜。用Ouabain(哇巴因)使胞内Na⁺超载后,Y³⁺可过膜进入细胞内,且过膜量与胞外Y³⁺浓度和胞内Na⁺超载程度有一定的浓度依赖关系,提示Y³⁺可以经由Na⁺/Y³⁺交换机制过膜而进入细胞内。     

A Protein‐Based Encapsulation System with Calcium‐Controlled Cargo Loading and Detachment

Protein‐based encapsulation systems have a wide spectrum of applications in targeted delivery of cargo molecules and for chemical transformations in confined spaces. By engineering affinity between cargo and container proteins it has been possible to enable the efficient and specific encapsulation of target molecules. Missing in current approaches is the ability to turn off the interaction after encapsulation to enable the cargo to freely diffuse in the lumen of the container. Separation between cargo and container is desirable in drug delivery applications and in the use of capsids as catalytic nanoparticles. We describe an encapsulation system based on the hepatitis B virus capsid in which an engineered high‐affinity interaction between cargo and capsid proteins can be modulated by Ca²⁺. Cargo proteins are loaded into capsids in the presence of Ca²⁺, while ligand removal triggers unbinding inside the container. We observe that confinement leads to hindered rotation of cargo inside the capsid. Application of the designed container for catalysis was also demonstrated by encapsulation of an enzyme with β‐glucosidase activity.     

Effects of Cations on HPTS Fluorescence and Quantification of Free Gadolinium Ions in Solution; Assessment of Intracellular Release of Gd3+ from Gd-Based MRI Contrast Agents

8-Hydroxypyrene-1,3,6-trisulfonate (HPTS) is a small, hydrophilic fluorescent molecule. Since the pKa of the hydroxyl group is close to neutrality and quickly responds to pH changes, it is widely used as a pH-reporter in cell biology for measurements of intracellular pH. HPTS fluorescence (both excitation and emission spectra) at variable pH was measured in pure water in the presence of NaCl solution or in the presence of different buffers (PBS or hepes in the presence or not of NaCl) and in a solution containing BSA. pKa values have been obtained from the sigmoidal curves. Herein, we investigated the effect of mono-, di-, and trivalent cations (Na⁺, Ca²⁺, La³⁺, Gd³⁺) on fluorescence changes and proposed its use for the quantification of trivalent cations (e.g., gadolinium ions) present in solution as acqua-ions. Starting from the linear regression, the LoD value of 6.32 µM for the Gd³⁺ detection was calculated. The effects on the emission were also analyzed in the presence of a combination of Gd³⁺ at two different concentrations and the previously indicated mono and di-valent ions. The study demonstrated the feasibility of a qualitative method to investigate the intracellular Gd³⁺ release upon the administration of Gd-based contrast agents in murine macrophages.     

Towards Understanding the Involvement of H+-ATPase in Programmed Cell Death of Psammosilene tunicoides after Oxalic Acid Application

Psammosilene tunicoides is a unique perennial medicinal plant species native to the Southwestern regions of China. Its wild population is rare and endangered due to over-excessive collection and extended growth (4–5 years). This research shows that H⁺-ATPase activity was a key factor for oxalate-inducing programmed cell death (PCD) of P. tunicoides suspension cells. Oxalic acid (OA) is an effective abiotic elicitor that enhances a plant cell’s resistance to environmental stress. However, the role of OA in this process remains to be mechanistically unveiled. The present study evaluated the role of OA-induced cell death using an inverted fluorescence microscope after staining with Evans blue, FDA, PI, and Rd123. OA-stimulated changes in K⁺ and Ca²⁺ trans-membrane flows using a patch-clamp method, together with OA modulation of H⁺-ATPase activity, were further examined. OA treatment increased cell death rate in a dosage-and duration-dependent manner. OA significantly decreased the mitochondria activity and damaged its electron transport chain. The OA treatment also decreased intracellular pH, while the FC increased the pH value. Simultaneously, NH₄Cl caused intracellular acidification. The OA treatment independently resulted in 90% and the FC led to 25% cell death rates. Consistently, the combined treatments caused a 31% cell death rate. Furthermore, treatment with EGTA caused a similar change in intracellular pH value to the La³⁺ and OA application. Combined results suggest that OA-caused cell death could be attributed to intracellular acidification and the involvement of OA in the influx of extracellular Ca²⁺, thereby leading to membrane depolarization. Here we explore the resistance mechanism of P. tunicoides cells against various stresses endowed by OA treatment.     

Optimizing Charge Switching in Membrane Lytic Peptides for Endosomal Release of Biomacromolecules

Endocytic pathways are practical routes for the intracellular delivery of biomacromolecules. Along with this, effective strategies for endosomal cargo release into the cytosol are desired to achieve successful delivery. Focusing on compositional differences between the cell and endosomal membranes and the pH decrease within endosomes, we designed the lipid-sensitive and pH-responsive endosome-lytic peptide HAad. This peptide contains aminoadipic acid (Aad) residues, which serve as a safety catch for preferential permeabilization of endosomal membranes over cell membranes, and His-to-Ala substitutions enhance the endosomolytic activity. The ability of HAad to destabilize endosomal membranes was supported by model studies using large unilamellar vesicles (LUVs) and by increased intracellular delivery of biomacromolecules (including antibodies) into live cells. Cerebral ventricle injection of Cre recombinase with HAad led to Cre/loxP recombination in a mouse model, thus demonstrating potential applicability of HAad in vivo.     

DNAzyme‐Loaded Metal–Organic Frameworks (MOFs) for Self‐Sufficient Gene Therapy

DNAzymes have been recognized as potent therapeutic agents for gene therapy, while their inefficient intracellular delivery and insufficient cofactor supply precludes their practical biological applications. Metal–organic frameworks (MOFs) have emerged as promising drug carriers without in‐depth consideration of their disassembled ingredients. Herein, we report a self‐sufficient MOF‐based chlorin e6‐modified DNAzyme (Ce6‐DNAzyme) therapeutic nanosystem for combined gene therapy and photodynamic therapy (PDT). The ZIF‐8 nanoparticles (NPs) could efficiently deliver the therapeutic DNAzyme without degradation into cancer cells. The pH‐responsive ZIF‐8 NPs disassemble with the concomitant release of the guest DNAzyme payloads and the host Zn²⁺ ions that serve, respectively, as messenger RNA‐targeting agent and required DNAzyme cofactors for activating gene therapy. The auxiliary photosensitizer Ce6 could produce reactive oxygen species (ROS) and provide a fluorescence signal for the imaging‐guided gene therapy/PDT.     

Design and Applications of Small Molecular Probes for Calcium Detection

The physiological significance of calcium ions such as the role in cellular signalling, cell growth, etc. have driven the development of methods to detect and monitor the level of Ca²⁺ ions, both in vivo and in vitro. Although various approaches for the detection of calcium ions have been reported, methods based on small molecular fluorescent probes have unique advantages including small probe size, easy monitoring of detection processes and applicability in biological systems. In this review article, we will discuss the progress in the development of Ca²⁺‐binding fluorescent probes by taking into account the types of chelating groups that have been employed for Ca²⁺ binding.     

A Highly Efficient Chemiluminescence Probe for the Detection of Singlet Oxygen in Living Cells

Singlet oxygen is among the reactive oxygen species (ROS) with the shortest life‐times in aqueous media because of its extremely high reactivity. Therefore, designing sensors for detection of ¹O₂ is perhaps one of the most challenging tasks in the field of molecular probes. Herein, we report a highly selective and sensitive chemiluminescence probe ( SOCL‐CPP ) for the detection of ¹O₂ in living cells. The probe reacts with ¹O₂ to form a dioxetane that spontaneously decomposes under physiological conditions through a chemiexcitation pathway to emit green light with extraordinary intensity. SOCL‐CPP demonstrated promising ability to detect and image intracellular ¹O₂ produced by a photosensitizer in HeLa cells during photodynamic therapy (PDT) mode of action. Our findings make SOCL‐CPP the most effective known chemiluminescence probe for the detection of ¹O₂. We anticipate that our chemiluminescence probe for ¹O₂ imaging would be useful in PDT‐related applications and for monitoring ¹O₂ endogenously generated by cells in response to different stimuli.     

Effect of Toxic Metal Ions on Photosensitized Singlet Oxygen Generation for Photodegradation of Polyaromatic Hydrocarbon Derivatives and Inactivation of Escherichia coli

Here, we report an experimental study of the effect of toxic metal ions on photosensitized singlet oxygen generation for photodegradation of PAH derivatives, Anthracene‐9,10‐dipropionic acid disodium salt (ADPA) and 1,5‐dihydroxynapthalene (DHN) and photoinactivation of Escherichia coli bacteria by using cationic meso‐tetra(N‐methyl‐4‐pyridyl)porphine tetrachloride (TMPyP) as a singlet oxygen photosensitizer. Three s‐block metals ions, such as Na⁺, K⁺ and Ca²⁺ and five toxic metals such as Cd²⁺, Cu²⁺, Hg²⁺, Zn²⁺ and Pb²⁺ were studied. The s‐block metal ions showed no change in the rate of photodegradation of ADPA or DHN by TMPyP, whereas a dramatic change in the photodegradation of ADPA and DHN was observed in the presence of toxic metals. The maximum photodegradation rate constants of ADPA and DHN were observed for Cd²⁺ ions [(3.91 ± 0.20) × 10^?3 s^?1 and (7.18 ± 0.35) × 10^?4 s^?1, respectively]. Strikingly, the photodegradation of ADPA and DHN was almost completely inhibited in the presence of Hg²⁺ ions and Cu²⁺ ions. A complete inhibition of growth of E. coli was observed upon visible light irradiation of E. coli solutions with TMPyP and toxic metal ions particularly, Cd²⁺, Hg²⁺, Zn²⁺ and Pb²⁺ ions, except for Cu²⁺ ions where a significantly slow inhibition of E. coli's growth was observed.     

Role of Calcium in Photodynamically Induced Cell Damage of Human Fibroblasts

Photodynamically induced changes in the cytoplasmic free calcium concentration ([Ca²⁺]_i) and its role in cell damage were investigated in human skin fibroblasts using confocal laser microscopy. Fluorescence and absorbance spectrophotometry measurements indicate that the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS₄) binds to the plasma membrane and only after irradiation is able to enter the cells, causing massive morphologic alterations. Upon irradiation of sensitizer-treated cells, the increase in [Ca²⁺]_i is related to the amount of light and extracellular [Ca²⁺]_e. The increase in [Ca²⁺]_i was substantially reduced in the absence of [Ca²⁺]_e. Cell damage or death after photodynamic treatment was prevented and shifted toward higher fluence by increasing [Ca²⁺]_i at high [Ca²⁺]_e and was greater at low [Ca²⁺]_e. Application of Ca²⁺ channel blockers, such as Co²⁺, Cd²⁺ or verapamil, could not prevent the increase of [Ca²⁺]_i. Our results indicate that activation of the photosensitizer, AlPcS₄, causes an influx of Ca²⁺, which protects cells from photodamage. At low [Ca²⁺]_e and high fluence values, release of Ca²⁺ from internal stores probably as a protective measure occurs in order to increase the [Ca²⁺]_i.     

Effect of pH on complexation in mixed dilute aqueous solutions of poly(acrylic acid), poly(ethylene glycol), and Cu2+ ions

The phase states of mixed dilute solutions of PAA, PEG, and Cu²⁺ ions largely determines the mechanism governing the growth of metal nanoparticles during the subsequent reduction of copper ions. Mixtures with PAA: PEG > 1 base-mol/base-mol and PAA: Cu²⁺ ≥ 5 base-mol/mol are studied. It is shown that the simultaneous complexation of PAA with PEG and Cu²⁺ ions in these mixtures at pH values below the intrinsic pH of a solution is accompanied by phase separation related to insolubility of PAA-PEG interpolymer complexes. A decrease in the pH of the ternary mixture is caused by the release of a strong low-molecular-mass acid due to complexation with Cu²⁺ ions. The minimum pH value, above which the PAA-PEG-Cu²⁺ system becomes single-phase (a transparent solution), depends on the concentration ratio between PAA and PEG chains (the mean degree of polymerization). This value is either 6.8–7.0 (if all macromolecules are incorporated in the insoluble interpolymer complex with PEG) or 4.0 (if chains occur in excess). Methods of preparing single-phase systems in the pH range 4.0–7.0 via exchange reactions of the PAA-Cu²⁺ complex with PEG or the nonstoichiometric soluble interpolymer complex PAA-PEG are developed. Viscometry, electron microscopy, and dynamic light scattering are used to investigate the compositions and structures of soluble complexes, in which either each chain (if the chain is long) may be linked with both PEG and Cu²⁺ ions or PAA chains are redistributed between two complexes (at comparable lengths of PAA and PEG chains).     

Cadmium(II) specifically interacts with cellular signaling to induce proto-oncogenes c-fos and c-jun in rat PC12 cells

Cadmium(II) compounds are carcinogens but only weakly mutagens. Because Cd(II) at low micromolar concentrations stimulates cell growth and induces some proto-oncogenes with several mammalian cell lines, the stimulation of cell proliferation is discussed as a major mechanism of the carcinogenicity of this metal. In the present work, the induction of the cellular proto-oncogenes c-fos and c-jun by Cd(II) was studied in rat PC12 cells. Several cellular signal transduction elements were tested as mediators of the proto-oncogene induction by cadmium. Cd(II) does not evoke mobilization of free intracellular Ca²⁺ in PC12 cells, and there was no impairment of the effect of Cd(II) by inhibitors of protein kinase A or of MAPK kinase. However, bisindolylmaleimide I, a specific inhibitor of protein kinase C abolished the proto-oncogene induction by Cd(II). Hence, a critical role for protein kinase C in the mitogenic effect of Cd(II) is inferred, and a substitution of structural Zn²⁺ ions by Cd²⁺ ions in this enzyme is discussed as the putative mechanism. Received: 30 July 1997 / Revised: 9 October 1997 / Accepted: 11 October 1997