Membrane Binding and Insertion of a pHLIP Peptide Studied by All-Atom Molecular Dynamics Simulations

Recent experiments in function mechanism study reported that a pH low-insertion peptide (pHLIP) can insert into a zwitterionic palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer at acidic pH while binding to the bilayer surface at basic pH. However, the atomic details of the pH-dependent interaction of pHLIP with a POPC bilayer are not well understood. In this study, we investigate the detailed interactions of pHLIP with a POPC bilayer at acidic and basic pH conditions as those used in function mechanism study, using all-atom molecular dynamics (MD) simulations. Simulations have been performed by employing the initial configurations, where pHLIP is placed in aqueous solution, parallel to bilayer surface (system S), partially-inserted (system P), or fully-inserted (system F) in POPC bilayers. On the basis of multiple 200-ns MD simulations, we found (1) pHLIP in system S can spontaneously insert into a POPC bilayer at acidic pH, while binding to the membrane surface at basic pH; (2) pHLIP in system P can insert deep into a POPC bilayer at acidic pH, while it has a tendency to exit, and stays at bilayer surface at basic pH; (3) pHLIP in system F keeps in an α-helical structure at acidic pH while partially unfolding at basic pH. This study provides at atomic-level the pH-induced insertion of pHLIP into POPC bilayer.     

Preparation and efficacy of tumor vasculature‐targeted doxorubicin cationic liposomes coated by N‐trimethyl chitosan

Cationic liposomes (CLs) can accumulate in tumor vascular endothelial cells (VECs) to show high selective targeting ability. Therefore, chemotherapeutic agent‐loaded CLs are considered as new therapeutic vehicles to enhance the treatment efficacy. This study investigated the effect of N‐trimethyl chitosan (TMC), one of derivatives of chitosan with positive charge determined by its degree of quaternization (DQ), on preparing doxorubicin (DOX)‐loaded CLs. TMCs with various DQ, i.e., 20% (TMC20), 40% (TMC40), and 60% (TMC60) were synthesized and characterized by ¹HNMR. DOX‐loaded liposomes (DOXL) were prepared by ammonium sulfate gradients followed by TMC‐coating to obtain TMC‐coated DOXL with various positive surface charges. The morphology, size, ζ‐potential and drug release in vitro of TMC‐coated DOXL were studied compared with those of DOXL. Human umbilical vein endothelial cells (HUVECs) as cell model, the vascular targeting ability of TMC‐coated DOXL was evaluated in vitro. A solid tumor, formed by implantationmurine hepatoma cells (H₂₂) into mice, as tumor model, the tumor inhibition rate and tumor histological sections stained by HE of TMC‐coated DOXL group were researched compared with those of free DOX and DOXL group. It was found that with the increase of TMC's DQ, the positive surface charge of TMC‐coated DOXL was enhanced accordingly, which had little effect on DOX release in vitro while led to the significant increase of DOX uptake by HUVECs in vitro and the treatment effect on solid tumor in vivo. Especially, TMC‐coated DOXL showed better targeting ability to the nuclei compared with free DOX and DOXL, which could further enhance the efficacy of DOX in vivo. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Acid-Sphingomyelinase Triggered Fluorescently Labeled Sphingomyelin Containing Liposomes in Tumor Diagnosis after Radiation-Induced Stress

In liposomal delivery, a big question is how to release the loaded material into the correct place. Here, we will test the targeting and release abilities of our sphingomyelin-consisting liposome. A change in release parameters can be observed when sphingomyelin-containing liposome is treated with sphingomyelinase enzyme. Sphingomyelinase is known to be endogenously released from the different cells in stress situations. We assume the effective enzyme treatment will weaken the liposome making it also leakier. To test the release abilities of the SM-liposome, we developed several fluorescence-based experiments. In in vitro studies, we used molecular quenching to study the sphingomyelinase enzyme-based release from the liposomes. We could show that the enzyme treatment releases loaded fluorescent markers from sphingomyelin-containing liposomes. Moreover, the release correlated with used enzymatic activities. We studied whether the stress-related enzyme expression is increased if the cells are treated with radiation as a stress inducer. It appeared that the radiation caused increased enzymatic activity. We studied our liposomes’ biodistribution in the animal tumor model when the tumor was under radiation stress. Increased targeting of the fluorescent marker loaded to our liposomes could be found on the site of cancer. The liposomal targeting in vivo could be improved by radiation. Based on our studies, we propose sphingomyelin-containing liposomes can be used as a controlled release system sensitive to cell stress.     

Accurate Analysis of Tumor Margins Using a Fluorescent pH Low Insertion Peptide (pHLIP)

The recurrence of certain cancers remains quite high due to either incomplete surgical removal of the primary tumor or the presence of small metastases that are invisible to the surgeon. Near infrared (NIR) fluorescence imaging might improve surgical outcomes by providing sensitive, specific, and real-time visualization of normal and diseased tissues if agents can be found that discriminate between normal and diseased tissue and define tumor margins. We have developed a new approach for revealing tumor borders by using NIR fluorescently labeled pH Low Insertion Peptide (pHLIP) and have created a computational program for the quantitative assessment of tumor boundaries. The approach is tested in vivo by co-localization of GFP-tumors and NIR emission from the fluorescently labeled pHLIP, and it is found that boundaries are accurately reported and that sub-millimeter masses can be detected.     

Transferrin-Decorated Niosomes with Integrated InP/ZnS Quantum Dots and Magnetic Iron Oxide Nanoparticles: Dual Targeting and Imaging of Glioma

The development of multifunctional nanoscale systems that can mediate efficient tumor targeting, together with high cellular internalization, is crucial for the diagnosis of glioma. The combination of imaging agents into one platform provides dual imaging and allows further surface modification with targeting ligands for specific glioma detection. Herein, transferrin (Tf)-decorated niosomes with integrated magnetic iron oxide nanoparticles (MIONs) and quantum dots (QDs) were formulated (PEGNIO/QDs/MIONs/Tf) for efficient imaging of glioma, supported by magnetic and active targeting. Transmission electron microscopy confirmed the complete co-encapsulation of MIONs and QDs in the niosomes. Flow cytometry analysis demonstrated enhanced cellular uptake of the niosomal formulation by glioma cells. In vitro imaging studies showed that PEGNIO/QDs/MIONs/Tf produces an obvious negative-contrast enhancement effect on glioma cells by magnetic resonance imaging (MRI) and also improved fluorescence intensity under fluorescence microscopy. This novel platform represents the first niosome-based system which combines magnetic nanoparticles and QDs, and has application potential in dual-targeted imaging of glioma.     

pH‐sensitive Podophyllotoxin carrier for cancer cells specific delivery

A pH‐sensitive drug targeting system for solid tumors was established based on N‐isopropylacrylamide (NIPAAm) and chitosan conjugates. The mass ratio of NIPAAm and chitosan was adjusted to obtain super pH‐sensitive characteristic and the structure was studied by using Fourier transform infrared spectroscope to confirm the successful synthesis of the nanoparticles. The pH‐sensitive and drug release characteristics in vitro were studied as well. Human lung cancer cells A‐549 and human fibroblast were used to test the biocompatibility of blank and Podophyllotoxin (POD) loaded nanoparticles further to certificate the reliability of targeting acidic tumor extracellular pH. Results revealed that when charge ratio between NIPAAm and CS achieve 4:1(w/w), the drug‐loaded nanoparticles, which diameters ranged from 50 to 150 nm, exhibited super pH‐sensitive responses to tumor pH. Encapsulation and loading efficiencies were 63.7% and 2.4%, respectively. The cumulative release rate of POD, which significantly enhanced at pH 6.8 while decreased rapidly either below pH 6.5 or above pH 6.9 at 37°C. At pH 6.8, POD‐loaded nanoparticles showed cytotoxicity in MTT test and fluorescence microscopic study, comparable to that of free POD at the same POD concentrations, whereas at pH 7.4 there was little cytotoxicity at the tested concentration range. Thereby, the atoxic PNIPAAm‐g‐chitosan nanoparticle has the potentiality as a novel anticancer drugs carrier. POLYM. COMPOS., 2010. © 2009 Society of Plastics Engineers     

Cytotoxic Activity of LLO Y406A Is Targeted to the Plasma Membrane of Cancer Urothelial Cells

Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.     

Biophysical studies and intracellular destabilization of pH-sensitive liposomes

We examined changes in membrane properties upon acidification of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes and evaluated their potential to deliver entrapped tracers in cultured macrophages. Membrane permeability was determined by the release of entrapped calcein or hydroxypyrene-1,3,6-trisulfonic acid (HPTS)-p-xylene-bis-pyridinium bromide (DPX); membrane fusion, by measuring the change in size of the liposomes and the dequenching of octadecylrhodamine-B fluorescence; and change in lipid organization, by³¹P nuclear magnetic resonance spectroscopy. Measurement of cell-associated fluorescence and confocal microscopy examination were made on cells incubated with liposomes loaded with HPTS or HPTS-DPX. The biophysical studies showed (i) a lipid reorganization from bilayer to hexagonal phase progressing from pH 8.0 to 5.0, (ii) a membrane permeabilization for pH<6.5, (iii) an increase in the mean diameter of liposomes for pH<6.0, and (iv) a mixing of liposome membranes for pH<5.7. The cellular studies showed (i) an uptake of the liposomes that were brought from pH 7.5–7.0 to 6.5–6.0 and (ii) a release of ∼15% of the endocytosed marker associated with its partial release from the vesicles (diffuse localization). We conclude that the permeabilization and fusion of pH-sensitive liposomes occur as a consequence of a progressive lipid reorganization upon acidification. These changes may develop intracellular after phagocytosis and allow for the release of the liposome content in endosomes associated with a redistribution in the cytosol.     

Poly(l‐histidine)‐containing polymer bioconjugate hybrid materials as stimuli‐responsive theranostic systems

For decades, researchers have aspired to develop materials for noninvasive treatment and monitoring of pathological conditions. Various organs, tissues, subcellular compartments, and their pathophysiological states can be characterized by their pH values. pH‐dependent intracellular tumor targeting has received particular attention due to the unique acidic environment of the solid tumors created by physiological and metabolical abnormalities. Responsive nanocarriers, when exposed to these pH stimuli, respond quickly to the physicochemical changes by undergoing structural deformations, such as swelling and phase transition, which favors the drug release specifically at the diseased site. Recently, researchers have developed several new poly(L ‐histidine) (p(His))‐based pH responsive systems for sustained drug release and molecular targeting. This review focuses on the p(His)‐based pH responsive nanocarriers, which are utilized in biomedical applications such as anti‐cancer drug delivery and nucleic acid delivery. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40796.     

Selective Display of a Chemoattractant Agonist on Cancer Cells Activates the Formyl Peptide Receptor 1 on Immune Cells**

Current immunotherapeutics often work by directing components of the immune system to recognize biomarkers on the surface of cancer cells to generate an immune response. However, variable changes in biomarker distribution and expression can result in inconsistent patient response. The development of a more universal tumor-homing strategy has the potential to improve selectivity and extend therapy to cancers with decreased expression or absence of specific biomarkers. Here, we designed a bifunctional agent that exploits the inherent acidic microenvironment of most solid tumors to selectively graft the surface of cancer cells with a formyl peptide receptor ligand (FPRL). Our approach is based on the pH(Low) insertion peptide (pHLIP), a unique peptide that selectively targets tumors in vivo by anchoring to cancer cells in a pH-dependent manner. We establish that selectively remodeling cancer cells with a pHLIP-based FPRL activates formyl peptide receptors on recruited immune cells, potentially initiating an immune response towards tumors.     

Bifunctional bacterial magnetic nanoparticles for tumor targeting

Bifunctional bacterial magnetic nanoparticles (BBMPs), which present both magnetic drug targeting and tumor bio-targeting properties, have been developed by chemically coupling both doxorubicin and a galactosyl ligand on to the membrane surface of the bacterial magnetic nanoparticles (BMPs). The BBMP product has a high drug load ratio and magnetic respondence, and exhibits a narrow size distribution and is sensitive to pH to enable drug release. In comparison to doxorubicin-coupled BMPs, without modification with a galactosyl ligand, BBMPs present a higher uptake by the target asialoglycoprotein receptor (ASGP-R) expressed by HepG2 cells and display stronger cytotoxicity.     

Folate Functionalized Boron Nitride Nanotubes and their Selective Uptake by Glioblastoma Multiforme Cells: Implications for their Use as Boron Carriers in Clinical Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is increasingly being used in the treatment of several aggressive cancers, including cerebral glioblastoma multiforme. The main requirement for this therapy is selective targeting of tumor cells by sufficient quantities of ¹⁰B atoms required for their capture/irradiation with low-energy thermal neutrons. The low content of boron targeting species in glioblastoma multiforme accounts for the difficulty in selective targeting of this very malignant cerebral tumor by this radiation modality. In the present study, we have used for the first time boron nitride nanotubes as carriers of boron atoms to overcome this problem and enhance the selective targeting and ablative efficacy of BNCT for these tumors. Following their dispersion in aqueous solution by noncovalent coating with biocompatible poly-l-lysine solutions, boron nitride nanotubes were functionalized with a fluorescent probe (quantum dots) to enable their tracking and with folic acid as selective tumor targeting ligand. Initial in vitro studies have confirmed substantive and selective uptake of these nanovectors by glioblastoma multiforme cells, an observation which confirms their potential clinical application for BNCT therapy for these malignant cerebral tumors.     

Endowment of pH Responsivity to Anticancer Peptides by Introducing 2,3-Diaminopropionic Acid Residues

Endowment of pH responsivity to anticancer peptides is a promising approach to achieve better selectivity to cancer tissues. In this research, a template peptide was designed based on magainin 2, an antimicrobial peptide with anticancer activity, and a series of peptides were designed by replacing different numbers of lysine with the unnatural amino acid, 2,3diaminopropionic acid (Dap), which has a positive charge at weakly acidic pH in cancer tissues, but is neutral at physiological pH 7.4. These Dap-containing peptides are expected to interact more strongly with tumor cells than with normal cells because 1) weakly acidic conditions form in tumors, and 2) the membrane of tumor cells is more anionic than that of normal cells. Although all examined peptides showed potent cytotoxicities to multidrug-resistant cancer cells at a weakly acidic pH (ED₅₀≈5 μm ), the toxicity decreased with an increase in the number of Dap at pH 7.4 (8 Dap residues resulted in ED₅₀≈60 μm ). Furthermore, the introduction of Dap reduced cytotoxicity against normal cells. Thus, Dap led to significantly improved cancer targeting due to a pH-dependent charge shift. Fluorescence imaging and model membrane experiments supported this charge-shift model.     

Development of pH‐sensitive nanogels for cancer treatment using crosslinked poly(aspartic acid‐graft‐imidazole)‐block‐poly(ethylene glycol)

pH‐sensitive nanogels (NGs) based on poly(aspartic acid‐graft‐imidazole)‐poly(ethylene glycol) were developed using linear PEG with different molecular weights (2000 and 4000 Da) as crosslinkers. The pH‐sensitive NGs showed reversible size changes during continuously alternating pH changes. The anticancer treatment potential of pH‐sensitive NGs was studied using a model drug, irinotecan (IRI). IRI‐loaded NGs (ILNs) showed different drug release kinetics in acidic versus neutral pH, in addition to pH‐dependent cytotoxicity. Due to its longer crosslinker, ILN 4 (crosslinked with PEG 4000) showed faster IRI release and a greater magnitude of IRI release than ILN 2 (crosslinked with PEG 2000), resulting in greater cytotoxicity against HCT 116 colorectal cancer cells. These pH‐sensitive NGs could potentially be used in cancer treatment by mediating the accumulation and release of IRI from ILNs in the acidic tumor environment and by reducing systemic toxicity due to reversible swelling–shrinkage. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46268.     

Egg phosphatidylcholine and dioleoylphosphatidylethanolamine liposomes containing acid proteinoid: Comparison of pH‐sensitivity

pH‐Sensitive liposomes were prepared by modifying the surfaces of egg phosphatidylcholine (EPC) liposomes and dioleoylphosphatidylethanolamine (DOPE) liposomes with an acidic proteinoid. The acidic proteinoid (Prot AL) was prepared by a melt‐condensation of aspartic acid and leucine (98.5:1.5 mol/mol). The maximum amount of Prot AL accommodated by EPC liposomes without loss of the fluorescence quenching of calcein occurred when the ratio of Prot AL to EPC was 1:2. The EPC liposomes exhibited pH‐dependent release but the degree of release in 5 min was less than 10% in the range of pH 6.0–8.0. A marked increase in release was observed at pH 5.5 and the degree of release was about 38%. Acidification‐induced contraction of Prot AL may impose a mechanical stress on the liposomal membrane, deforming and demaging the membrane. On the other hand, a high fluorescence quenching, more than 60%, was obtained when the ratio of Prot AL to DOPE was 5.5:10. The pH‐sensitivity of DOPE liposomes bearing the proteinoid was much higher than that of egg PC liposomes bearing the same proteinoid. Following the changes in the size with varying pH, DOPE liposomes seemed to be disintegrated.     

Monomethyl Auristatin E Grafted-Liposomes to Target Prostate Tumor Cell Lines

Novel nanomedicines have been engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or short half-life. Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. A Monomethyl Auristatin E (MMAE) warhead was grafted on a lipid derivative and integrated in fusogenic liposomes, following the model of antibody drug conjugates. By modulating the liposome composition, we designed a set of particles characterized by different membrane fluidities as a key parameter to obtain selective uptake from fibroblast or prostate tumor cells. Only the fluid liposomes made of palmitoyl-oleoyl-phosphatidylcholine and dioleoyl-phosphatidylethanolamine, integrating the MMAE-lipid derivative, showed an effect on prostate tumor PC-3 and LNCaP cell viability. On the other hand, they exhibited negligible effects on the fibroblast NIH-3T3 cells, which only interacted with rigid liposomes. Therefore, fluid liposomes grafted with MMAE represent an interesting example of drug carriers, as they can be easily engineered to promote liposome fusion with the target membrane and ensure drug selectivity.     

Enhanced localization of anticancer drug in tumor tissue using polyethylenimine-conjugated cationic liposomes

Liposome-based drug delivery systems hold great potential for cancer therapy. However, to enhance the localization of payloads, an efficient method of systemic delivery of liposomes to tumor tissues is required. In this study, we developed cationic liposomes composed of polyethylenimine (PEI)-conjugated distearoylglycerophosphoethanolamine (DSPE) as an enhanced local drug delivery system. The particle size of DSPE-PEI liposomes was 130 ± 10 nm and the zeta potential of liposomes was increased from -25 to 30 mV by the incorporation of cationic PEI onto the liposomal membrane. Intracellular uptake of DSPE-PEI liposomes by tumor cells was 14-fold higher than that of DSPE liposomes. After intratumoral injection of liposomes into tumor-bearing mice, DSPE-PEI liposomes showed higher and sustained localization in tumor tissue compared to DSPE liposomes. Taken together, our findings suggest that DSPE-PEI liposomes have the potential to be used as effective drug carriers for enhanced intracellular uptake and localization of anticancer drugs in tumor tissue through intratumoral injection.     

Prodrug strategies in anticancer chemotherapy

The majority of clinically approved anticancer drugs are characterized by a narrow therapeutic window that results mainly from a high systemic toxicity of the drugs in combination with an evident lack of tumor selectivity. Besides the development of suitable galenic formulations such as liposomes or micelles, several promising prodrug approaches have been followed in the last decades with the aim of improving chemotherapy. In this review we elucidate the two main concepts that underlie the design of most anticancer prodrugs: drug targeting and controlled release of the drug at the tumor site. Consequently, active and passive targeting using tumor-specific ligands or macromolecular carriers are discussed as well as release strategies that are based on tumor-specific characteristics such as low pH or the expression of tumor-associated enzymes. Furthermore, other strategies such as ADEPT (antibody-directed enzyme prodrug therapy) and the design of self-eliminating structures are introduced. Chemical realization of prodrug approaches is illustrated by drug candidates that have or may have clinical importance.     

The selective delivery of anticancer agents using target-sensitive liposomes

Target-sensitive (TG-S) liposomes having modified antibodies on their surface were employed to study the release of calcein and the selective delivery of the anticancer agents, doxorubicin (DOX) and methotrexate (MTX). The release of calcein from TG-S liposome occurred when the various target cells were contacted with liposomes and it was proportionally increased with the increase of antibody affinity to the target cells. Increasing the concentration of antigen molecules (major histocompatibility, MHC) on the surface of RMA-S, the release of calcein and drugs from TG-S liposomes contacting with RMA-S also rised. The destabilization of TG-S liposomes was only induced above a threshold density of surface antigen on the target cell membrane. The growth inhibition of specific target cells by the liposomal drugs was always stronger than that of the non-specific ones. For specific target cells, the IC₅₀ of liposomal DOX was about 2 times greater than that of free DOX, on the while, for non-specific target cells, more than 5 times. This indicates that the liposomal drugs were transferred preferentially to the specific target cells than the non-specific ones. Based on this phenomenon, the TG-S liposomal MTX were also applied for the selective elimination of the specific target cells in the mixed culture of specific and non-specific target cells.     

Accessible Synthetic Probes for Staining Actin inside Platelets and Megakaryocytes by Employing Lifeact Peptide

Lifeact is a 17‐residue peptide that can be employed in cell microscopy as a probe for F‐actin when fused to fluorescent proteins, but therefore is not suitable for all cell types. We have conjugated fluorescently labelled Lifeact to three different cell‐penetrating systems (a myristoylated carrier (myr), the pH low insertion peptide (pHLIP) and the cationic peptide TAT) as a strategy to deliver Lifeact into cells and developed new tools for actin staining with improved synthetic accessibility and low toxicity, focusing on their suitability in platelets and megakaryocytes. Using confocal microscopy, we characterised the cell distribution of the new hybrids in fixed cells, and found that both myr– and pHLIP–Lifeact conjugates provide efficient actin staining upon cleavage of Lifeact from the carriers, without affecting cell spreading. This new approach could facilitate the design of new tools for actin visualisation.