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Attritional evaluation of lipophilic and hydrophilic metallated phthalocyanines for oncological photodynamic therapy.
Dias, LM, Sharifi, F, de Keijzer, MJ, Mesquita, B, Desclos, E, Kochan, JA, de Klerk, DJ, Ernst, D, de Haan, LR, Franchi, LP, et al
Journal of photochemistry and photobiology. B, Biology. 2021;:112146
Abstract
BACKGROUND AND AIM Oncological photodynamic therapy (PDT) relies on photosensitizers (PSs) to photo-oxidatively destroy tumor cells. Currently approved PSs yield satisfactory results in superficial and easy-to-access tumors but are less suited for solid cancers in internal organs such as the biliary system and the pancreas. For these malignancies, second-generation PSs such as metallated phthalocyanines are more appropriate. Presently it is not known which of the commonly employed metallated phtahlocyanines, namely aluminum phthalocyanine (AlPC) and zinc phthalocyanine (ZnPC) as well as their tetrasulfonated derivatives AlPCS4 and ZnPCS4, is most cytotoxic to tumor cells. This study therefore employed an attritional approach to ascertain the best metallated phthalocyanine for oncological PDT in a head-to-head comparative analysis and standardized experimental design. METHODS ZnPC and AlPC were encapsulated in PEGylated liposomes. Analyses were performed in cultured A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and EGFR overexpression. First, dark toxicity was assessed as a function of PS concentration using the WST-1 and sulforhodamine B assay. Second, time-dependent uptake and intracellular distribution were determined by flow cytometry and confocal microscopy, respectively, using the intrinsic fluorescence of the PSs. Third, the LC50 values were established for each PS at 671 nm and a radiant exposure of 15 J/cm2 following 1-h PS exposure. Finally, the mode of cell death as a function of post-PDT time and cell cycle arrest at 24 h after PDT were analyzed. RESULTS In the absence of illumination, AlPC and ZnPC were not toxic to cells up to a 1.5-μM PS concentration and exposure for up to 72 h. Dark toxicity was noted for AlPCS4 at 5 μM and ZnPCS4 at 2.5 μM. Uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. After 60 min, the entire non-nuclear space of the cell was photosensitized, with PS accumulation in multiple subcellular structures, especially in case of AlPC and AlPCS4. PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 μM, 0.04 μM, and 0.81 μM, respectively, 24 h post-PDT (based on sulforhodamine B assay). ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. At 4 h post-PDT, the mode of cell death comprised mainly apoptosis for ZnPC and AlPC, the extent of which was gradually exacerbated in AlPC-photosensitized cells during 8 h. ZnPC-treated cells seemed to recover at 8 h post-PDT compared to 4 h post-PDT, which had been observed before in another cell line. AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested at 2 h after PDT. During the course of 8 h, necrotic cell death transitioned into mainly late apoptotic cell death. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). Cell cycle arrest was most profound in cells that had been photosensitized with AlPC and subjected to PDT. CONCLUSIONS Liposomal AlPC is the most potent PS for oncological PDT, whereas ZnPCS4 was photodynamically inert in A431 cells. AlPC did not induce dark toxicity at PS concentrations of up to 1.5 μM, i.e., > 37 times the LC50 value, which is favorable in terms of clinical phototoxicity issues. AlPC photosensitized multiple intracellular loci, which was associated with extensive, irreversible cell death signaling that is expected to benefit treatment efficacy and possibly immunological long-term tumor control, granted that sufficient AlPC will reach the tumor in vivo. Given the differential pharmacokinetics, intracellular distribution, and cell death dynamics, liposomal AlPC may be combined with AlPCS4 in a PS cocktail to further improve PDT efficacy.
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Fat-Soluble Vitamin Supplementation Using Liposomes, Cyclodextrins, or Medium-Chain Triglycerides in Cystic Fibrosis: A Randomized Controlled Trial.
Nowak, JK, Sobkowiak, P, Drzymała-Czyż, S, Krzyżanowska-Jankowska, P, Sapiejka, E, Skorupa, W, Pogorzelski, A, Nowicka, A, Wojsyk-Banaszak, I, Kurek, S, et al
Nutrients. 2021;(12)
Abstract
Fat-soluble vitamin deficiency remains a challenge in cystic fibrosis (CF), chronic pancreatitis, and biliary atresia. Liposomes and cyclodextrins can enhance their bioavailability, thus this multi-center randomized placebo-controlled trial compared three-month supplementation of fat-soluble vitamins in the form of liposomes or cyclodextrins to medium-chain triglycerides (MCT) in pancreatic-insufficient CF patients. The daily doses were as follows: 2000 IU of retinyl palmitate, 4000 IU of vitamin D3, 200 IU of RRR-α-tocopherol, and 200 µg of vitamin K2 as menaquinone-7, with vitamin E given in soybean oil instead of liposomes. All participants received 4 mg of β-carotene and 1.07 mg of vitamin K1 to ensure compliance with the guidelines. The primary outcome was the change from the baseline of all-trans-retinol and 25-hydroxyvitamin D3 concentrations and the percentage of undercarboxylated osteocalcin. Out of 75 randomized patients (n = 28 liposomes, n = 22 cyclodextrins, and n = 25 MCT), 67 completed the trial (89%; n = 26 liposomes, n = 18 cyclodextrins, and n = 23 MCT) and had a median age of 22 years (IQR 19-28), body mass index of 20.6 kg/m2 [18.4-22.0], and forced expiratory volume in 1 s of 65% (44-84%). The liposomal formulation of vitamin A was associated with the improved evolution of serum all-trans-retinol compared to the control (median +1.7 ng/mL (IQR -44.3-86.1) vs. -38.8 ng/mL (-71.2-6.8), p = 0.028). Cyclodextrins enhanced the bioavailability of vitamin D3 (+9.0 ng/mL (1.0-17.0) vs. +3.0 ng/mL (-4.0-7.0), p = 0.012) and vitamin E (+4.34 µg/mL (0.33-6.52) vs. -0.34 µg/mL (-1.71-2.15), p = 0.010). Liposomes may augment the bioavailability of vitamin A and cyclodextrins may strengthen the supplementation of vitamins D3 and E relative to MCT in pancreatic-insufficient CF but further studies are required to assess liposomal vitamin E (German Clinical Trial Register number DRKS00014295, funded from EU and Norsa Pharma).
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Development of Lipid Nanoparticles for the Delivery of Macromolecules Based on the Molecular Design of pH-Sensitive Cationic Lipids.
Sato, Y
Chemical & pharmaceutical bulletin. 2021;(12):1141-1159
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Abstract
Considerable efforts have been made on the development of lipid nanoparticles (LNPs) for delivering of nucleic acids in LNP-based medicines, including a first-ever short interfering RNA (siRNA) medicine, Onpattro, and the mRNA vaccines against the coronavirus disease 2019 (COVID-19), which have been approved and are currently in use worldwide. The successful rational design of ionizable cationic lipids was a major breakthrough that dramatically increased delivery efficiency in this field. The LNPs would be expected to be useful as a platform technology for the delivery of various therapeutic modalities for genome editing and even for undiscovered therapeutic mechanisms. In this review, the current progress of my research, including the molecular design of pH-sensitive cationic lipids, their applications for various tissues and cell types, and for delivering various macromolecules, including siRNA, antisense oligonucleotide, mRNA, and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system will be described. Mechanistic studies regarding relationships between the physicochemical properties of LNPs, drug delivery, and biosafety are also summarized. Furthermore, current issues that need to be addressed for next generation drug delivery systems are discussed.
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Liposomes for malaria management: the evolution from 1980 to 2020.
Memvanga, PB, Nkanga, CI
Malaria journal. 2021;(1):327
Abstract
Malaria is one of the most prevalent parasitic diseases and the foremost cause of morbidity in the tropical regions of the world. Strategies for the efficient management of this parasitic infection include adequate treatment with anti-malarial therapeutics and vaccination. However, the emergence and spread of resistant strains of malaria parasites to the majority of presently used anti-malarial medications, on the other hand, complicates malaria treatment. Other shortcomings of anti-malarial drugs include poor aqueous solubility, low permeability, poor bioavailability, and non-specific targeting of intracellular parasites, resulting in high dose requirements and toxic side effects. To address these limitations, liposome-based nanotechnology has been extensively explored as a new solution in malaria management. Liposome technology improves anti-malarial drug encapsulation, bioavailability, target delivery, and controlled release, resulting in increased effectiveness, reduced resistance progression, and fewer adverse effects. Furthermore, liposomes are exploited as immunological adjuvants and antigen carriers to boost the preventive effectiveness of malaria vaccine candidates. The present review discusses the findings from studies conducted over the last 40 years (1980-2020) using in vitro and in vivo settings to assess the prophylactic and curative anti-malarial potential of liposomes containing anti-malarial agents or antigens. This paper and the discussion herein provide a useful resource for further complementary investigations and may pave the way for the research and development of several available and affordable anti-malarial-based liposomes and liposomal malaria vaccines by allowing a thorough evaluation of liposomes developed to date for the management of malaria.
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Antagonist Effects of l-Phenylalanine and the Enantiomeric Mixture Containing d-Phenylalanine on Phospholipid Vesicle Membrane.
Nandi, S, Pyne, A, Ghosh, M, Banerjee, P, Ghosh, B, Sarkar, N
Langmuir : the ACS journal of surfaces and colloids. 2020;(9):2459-2473
Abstract
One of the congenital flaws of metabolism, phenylketonuria (PKU), is known to be related to the self-assembly of toxic fibrillar aggregates of phenylalanine (Phe) in blood at elevated concentrations. Our experimental findings using l-phenylalanine (l-Phe) at millimolar concentration suggest the formation of fibrillar morphologies in the dry phase, which in the solution phase interact strongly with the model membrane composed of 1,2-diacyl-sn-glycero-phosphocholine (LAPC) lipid, thereby decreasing the rigidity (or increasing the fluidity) of the membrane. The hydrophobic interaction, in addition to the electrostatic attraction of Phe with the model membrane, is found to be responsible for such phenomena. On the contrary, various microscopic observations reveal that such fibrillar morphologies of l-Phe are severely ruptured in the presence of its enantiomer d-phenylalanine (d-Phe), thereby converting the fibrillar morphologies into crushed flakes. Various biophysical studies, including the solvation dynamics experiment, suggest that this l-Phe in the presence of d-Phe, when interacting with the same model membrane, now reverts the rigidity of the membrane, i.e., increases the rigidity of the membrane, which was lost due to interaction with l-Phe exclusively. Fluorescence anisotropy measurements also support this reverse rigid character of the membrane in the presence of an enantiomeric mixture of amino acids. A comprehensive understanding of the interaction of Phe with the model membrane is further pursued at the single-molecular fluorescence detection level using fluorescence correlation spectroscopy (FCS) experiments. Therefore, our experimental conclusion interprets a linear correlation between increased permeability and enhanced fluidity of the membrane in the presence of l-Phe and certifies d-Phe as a therapeutic modulator of l-Phe fibrillar morphologies. Further, the study proposes that the rigidity of the membrane lost due to interaction with l-Phe was reinstated-in fact, increased-in the presence of the enantiomeric mixture containing both d- and l-Phe.
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Effects of liposomal nasal spray with vitamins A and E on allergic rhinitis.
Lauriello, M, di Marco, GP, Necozione, S, Tucci, C, Pasqua, M, Rizzo, G, Eibenstein, A
Acta otorhinolaryngologica Italica : organo ufficiale della Societa italiana di otorinolaringologia e chirurgia cervico-facciale. 2020;(3):217-223
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Preparation of nanoliposomes by microfluidic mixing in herring-bone channel and the role of membrane fluidity in liposomes formation.
Kotouček, J, Hubatka, F, Mašek, J, Kulich, P, Velínská, K, Bezděková, J, Fojtíková, M, Bartheldyová, E, Tomečková, A, Stráská, J, et al
Scientific reports. 2020;(1):5595
Abstract
Introduction of microfluidic mixing technique opens a new door for preparation of the liposomes and lipid-based nanoparticles by on-chip technologies that are applicable in a laboratory and industrial scale. This study demonstrates the role of phospholipid bilayer fragment as the key intermediate in the mechanism of liposome formation by microfluidic mixing in the channel with "herring-bone" geometry used with the instrument NanoAssemblr. The fluidity of the lipid bilayer expressed as fluorescence anisotropy of the probe N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) was found to be the basic parameter affecting the final size of formed liposomes prepared by microfluidic mixing of an ethanol solution of lipids and water phase. Both saturated and unsaturated lipids together with various content of cholesterol were used for liposome preparation and it was demonstrated, that an increase in fluidity results in a decrease of liposome size as analyzed by DLS. Gadolinium chelating lipids were used to visualize the fine structure of liposomes and bilayer fragments by CryoTEM. Experimental data and theoretical calculations are in good accordance with the theory of lipid disc micelle vesiculation.
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Nanoemulsion and Nanoliposome Based Strategies for Improving Anthocyanin Stability and Bioavailability.
Chen, BH, Stephen Inbaraj, B
Nutrients. 2019;(5)
Abstract
BACKGROUND Anthocyanins, a flavonoid class of water-soluble pigments, are reported to possess several biological activities, including antioxidant, anti-inflammatory, and anti-cancer. However, anthocyanins are highly susceptible to degradation in high pH, light, heat, and oxygen during processing and storage. Conventional microencapsulation techniques fail to provide stability to anthocyanins under physiological environments mainly because of their large particle size as well as low zeta potential and encapsulation efficiency. METHODS Nanotechnology provides novel strategies for preparing nanoformulations to enhance the physicochemical stability of anthocyanins. Nanoemulsion and nanoliposome are the two most commonly used nanosystems in pharmaceutical and food-related fields. In this review, an overview of various nanoemulsion and nanoliposome systems reported recently for enhancing stability, bioavailability, and bioactivity of anthocyanins is presented. RESULTS Anthocyanin nanoemulsions with different oil, water, surfactant, and cosurfactant ratios were prepared from extracts of mangosteen peel, purple sweet potato, cranberry, red cabbage, blueberry, jaboticaba peel, and acai berry and evaluated for their antioxidant activity, enhancement of physicochemical stability, topical skin application, and urinary tract infection. Likewise, unilamellar and multilamellar nanoliposomes were prepared using different types and levels of lecithin without or with cholesterol from anthocyanin standards and extracts of Hibiscus sabdariffa, mulberry, elderberry, black carrot, and pistachio green hull for the evaluation of physicochemical and oxidative stability, in vitro bioaccessibility, and melanogenic activity, as well as protective effects against diabetes mellitus and cataract. CONCLUSION This review provides an insight into the current nanotechnology updates on enhancement of anthocyanin stability and biological activity.
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Interpreting non-linear drug diffusion data: Utilizing Korsmeyer-Peppas model to study drug release from liposomes.
Wu, IY, Bala, S, Škalko-Basnet, N, di Cagno, MP
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 2019;:105026
Abstract
The aim of this work was to clarify the dynamics behind the influence of ionic strength on the changes in drug release from large unilamellar vesicles (LUVs). For this purpose, we have investigated the transport of two different model drugs (caffeine and hydrocortisone) formulated into liposomes through different types of barriers with different retention properties (regenerated cellulose and the newly introduced biomimetic barrier, Permeapad®). Drug release from liposomes was studied utilizing the standard Franz diffusion cells. LUV dispersions were exposed to the isotonic, hypotonic and hypertonic environment (difference of 300 mOsm/kg between the initial LUVs and the environment) and experimental data treated with both linear and non-linear (Korsmeyer-Peppas) regression models. To alter the rigidity of the liposomal membranes, cholesterol was introduced in the liposomal barriers (up to 25% w/w). Korsmeyer-Peppas model was proven to be suited to analyse experimental data throughout the experimental time frame, providing important additive information in comparison to standard linear approximation. The obtained results are highly relevant as they improve the interpretation of drug release kinetics from LUVs under osmotic stress. Moreover, the findings can be utilized in the development of liposomal formulations intended for nose-to-brain targeted drug delivery.
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Safety and immunogenicity of the chlamydia vaccine candidate CTH522 adjuvanted with CAF01 liposomes or aluminium hydroxide: a first-in-human, randomised, double-blind, placebo-controlled, phase 1 trial.
Abraham, S, Juel, HB, Bang, P, Cheeseman, HM, Dohn, RB, Cole, T, Kristiansen, MP, Korsholm, KS, Lewis, D, Olsen, AW, et al
The Lancet. Infectious diseases. 2019;(10):1091-1100
Abstract
BACKGROUND Chlamydia is the most common sexually transmitted bacterial infection worldwide. National screening programmes and antibiotic treatment have failed to decrease incidence, and to date no vaccines against genital chlamydia have been tested in clinical trials. We aimed to assess the safety and immunogenicity, in humans, of a novel chlamydia vaccine based on a recombinant protein subunit (CTH522) in a prime-boost immunisation schedule. METHODS This phase 1, first-in-human, double-blind, parallel, randomised, placebo-controlled trial was done at Hammersmith Hospital in London, UK, in healthy women aged 19-45 years. Participants were randomly assigned (3:3:1) to three groups: CTH522 adjuvanted with CAF01 liposomes (CTH522:CAF01), CTH522 adjuvanted with aluminium hydroxide (CTH522:AH), or placebo (saline). Participants received three intramuscular injections of 85 μg vaccine (with adjuvant) or placebo to the deltoid region of the arm at 0, 1, and 4 months, followed by two intranasal administrations of 30 μg unadjuvanted vaccine or placebo (one in each nostril) at months 4·5 and 5·0. The primary outcome was safety and the secondary outcome was humoral immunogenicity (anti-CTH522 IgG seroconversion). This study is registered with Clinicaltrials.gov, number NCT02787109. FINDINGS Between Aug 15, 2016, and Feb 13, 2017, 35 women were randomly assigned (15 to CTH522:CAF01, 15 to CTH522:AH, and five to placebo). 32 (91%) received all five vaccinations and all participants were included in the intention-to-treat analyses. No related serious adverse reactions were reported, and the most frequent adverse events were mild local injection-site reactions, which were reported in all (15 [100%] of 15) participants in the two vaccine groups and in three (60%) of five participants in the placebo group (p=0·0526 for both comparisons). Intranasal vaccination was not associated with a higher frequency of related local reactions (reported in seven [47%] of 15 participants in the active treatment groups vs three [60%] of five in the placebo group; p=1·000). Both CTH522:CAF01 and CTH522:AH induced anti-CTH522 IgG seroconversion in 15 (100%) of 15 participants after five immunisations, whereas no participants in the placebo group seroconverted. CTH522:CAF01 showed accelerated seroconversion, increased IgG titres, an enhanced mucosal antibody profile, and a more consistent cell-mediated immune response profile compared with CTH522:AH. INTERPRETATION CTH522 adjuvanted with either CAF01 or aluminium hydroxide appears to be safe and well tolerated. Both vaccines were immunogenic, although CTH522:CAF01 had a better immunogenicity profile, holding promise for further clinical development. FUNDING European Commission and The Innovation Fund Denmark.