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Marina Simón,1,* Jesper Tranekjær Jørgensen,1,* Kamilla Norregaard,1 Jonas Rosager Henriksen,2 Gael Clergeaud,2 Thomas L Andresen,2 Anders Elias Hansen,2 Andreas Kjaer1 1Department of Clinical Physiology and Nuclear Medicine & Cluster for Molecular Imaging, Copenhagen University Hospital – Rigshospitalet & Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; 2Department of Health Technology, Section for Biotherapeutic Engineering and Drug Targeting, Technical University of Denmark, Kongens Lyngby, Denmark*These authors contributed equally to this workCorrespondence: Andreas Kjaer, Department of Clinical Physiology and Nuclear Medicine & Cluster for Molecular Imaging, Copenhagen University Hospital – Rigshospitalet & Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark, Email akjaer@sund.ku.dkIntroduction: Traditional cancer treatments, such as chemotherapy, are often incapable of achieving complete responses as standalone therapies. Hence, current treatment strategies typically rely on a combination of several approaches. Nanoparticle-based photothermal therapy (PTT) is a technique used to kill cancer cells through localized, severe hyperthermia that has shown promise as an add-on treatment to multiple cancer therapies. Here, we evaluated whether the combination of gold nanoshell (NS)-based PTT and liposomal doxorubicin could improve outcome in a mouse model of colorectal cancer.Methods: First, NS-based PTT was performed on tumor-bearing mice. Radiolabeled liposomes were then injected at different timepoints to follow their accumulation in the tumor and determine the ideal injection time after PTT. In addition, fluorescent liposomes were used to observe the liposomal distribution in the tumor after PTT. Finally, we combined PTT and doxorubicin-loaded liposomes and studied the effect of the treatment strategy on the mice by following tumor growth and survival.Results: PTT significantly improved liposomal accumulation in the tumor, but only when the liposomes were injected immediately after the therapy. The liposomes accumulated mostly in regions adjacent to the ablated areas. When PTT was combined with liposomal doxorubicin, the mice experienced a slowdown in tumor growth and an improvement in survival.Conclusion: According to our preclinical study, NS-based PTT seems promising as an add-on treatment for liposomal chemotherapy and potentially other systemic therapies, and could be relevant for future application in a clinical setting.Graphical Abstract: Keywords: liposomes, PET imaging, chemotherapy, hyperthermia, gold nanoparticles

Diseases caused by S. pneumoniae are the leading cause of child mortality. As antibiotic resistance of S. pneumoniae is rising, vaccination remains the most recommended solution. However, the existing pneumococcal polysaccharides vaccine (Pneumovax ® 23) proved only to induce T-independent immunity, and strict cold chain dependence of the protein conjugate vaccine impedes its promotion in developing countries, where infections are most problematic. Affordable and efficient vaccines against pneumococcus are therefore in high demand. Here, we present an intranasal vaccine Lipo + CPS12F&αGC, containing the capsular polysaccharides of S. pneumoniae 12F and the iNKT agonist α-galactosylceramide in cationic liposomes. In BALB/cJRj mice, the vaccine effectively activates iNKT cells and promotes B cells maturation, stimulates affinity-matured IgA and IgG production in both the respiratory tract and systemic blood, and displays sufficient protection both in vivo and in vitro. The designed vaccine is a promising, cost-effective solution against pneumococcus, which can be expanded to cover more serotypes and pathogens., (© 2024. The Author(s).)

MicroRNAs (miRNAs) are small noncoding RNAs that are critical for the regulation of multiple physiological and pathological processes, thus holding great clinical potential. However, the therapeutic applications of miRNAs are severely limited by their biological instability and poor intracellular delivery. Herein, we describe a dual-layers surface engineering strategy to design an efficient miRNA delivery nanosystem based on metal-organic frameworks (MOFs) incorporating lipid coating. The resulting nanoparticle system was demonstrated to protect miRNA from ribonuclease degradation, enhance cellular uptake and facilitate lysosomal escape. These ensured effective miRNA mediated gene therapy, which synergized with MOF-specific photodynamic therapy and pre-encapsulated doxorubicin (Dox) chemotherapy to provide a multifunctional with therapeutic effectiveness against cencer cells The mechanisms of miRNA binding and Dox loading were revealed, demonstrating the potential of the present MOFs surface-engineered strategy to overcome their inherent pore-size restriction for macromolecular miRNA carrying, enableefficient co-delivery. In vitro studies revealed the potential of our multifunctional system for miRNA delivery and the demonstrated the therapeutic effectiveness against cancer cells, thereby providing a versatile all-in-one MOFs strategy for delivery of nucleic acids and diverse therapeutic molecules in synergistic therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

iNKT cells - often referred as the "Swiss Army knife" of the immune system - have emerged as central players in cancer vaccine therapies. Glycolipids activating iNKT cells, such as α-galactosylceramide (αGalCer), can enhance the immune response against co-delivered cancer antigens and have been applied in the design of self-adjuvanting anti-tumor vaccines. In this context, this work focuses on the chemical synthesis of ganglioside tumor-associated carbohydrate antigens (TACAs), namely GM3 and (Neu5Gc)GM3 antigens, their conjugation to αGalCer, and their formulation into liposomes as an efficient platform for their in vivo delivery. Liposomes containing GM3-αGalCer, (Neu5Gc)GM3-αGalCer, and equimolar amounts of the two conjugates have been fully characterized and their ability to activate iNKT cell has been confirmed ex vivo in mouse and human cell assays. The candidates were tested in in vivo immunization studies, demonstrating an ability to induce both T H 1 and T H 2 cytokines leading to the production of all subclasses of IgG antibodies. Notably, the study also demonstrated that serum antibodies raised against the two TACAs, alone and in combination, were cross-reactive. This finding has consequences for future vaccine designs - even if a highly tumor-selective antigen is chosen, the resulting antibody response may be broader than anticipated., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Nucleic acid-based therapeutics encapsulated into lipid nanoparticles (LNPs) can potentially target the root cause of genetic skin diseases. Although nanoparticles are considered impermeable to skin, research and clinical studies have shown that nanoparticles can penetrate into skin with reduced skin barrier function when administered topically. Studies have shown that epidermal keratinocytes express the low-density lipoprotein receptor (LDLR) that mediates endocytosis of apolipoprotein E (apoE)-associated nanoparticles and that dermal fibroblasts express mannose receptors. Here we prepared LNPs designed to exploit these different endocytic pathways for intracellular mRNA delivery to the two most abundant skin cell types, containing: (i) labile PEG-lipids (DMG-PEG2000) prone to dissociate and facilitate apoE-binding to LNPs, enabling apoE-LDLR mediated uptake in keratinocytes, (ii) non-labile PEG-lipids (DSPE-PEG2000) to impose stealth-like properties to LNPs to enable targeting of distant cells, and (iii) mannose-conjugated PEG-lipids (DSPE-PEG2000-Mannose) to target fibroblasts or potentially immune cells containing mannose receptors. All types of LNPs were prepared by vortex mixing and formed monodisperse (PDI ∼ 0.1) LNP samples with sizes of 130 nm (±25%) and high mRNA encapsulation efficiencies (≥90%). The LNP-mediated transfection potency in keratinocytes and fibroblasts was highest for LNPs containing labile PEG-lipids, with the addition of apoE greatly enhancing transfection via LDLR. Coating LNPs with mannose did not improve transfection, and stealth-like LNPs show limited to no transfection. Taken together, our studies suggest using labile PEG-lipids and co-administration of apoE when exploring LNPs for skin delivery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Particle-based systems have become a state-of-the-art method for in vitro expanding cytotoxic T cells by tailoring their surface with activating molecules. However, commonly used methods utilize facile carbodiimide chemistry leading to uncontrolled orientation of the immobilized antibodies on the particle surface that can lead to poor binding to target cells. To address this, selective coupling strategies utilizing regioselective chemical groups such as disulfide bridges offer a simple approach. In this work we present a set of methods to investigate the effect of polymeric nanoparticles, conjugated with either regioselective- or randomly-immobilized antiCD3 and antiCD28 antibodies, on the activation potential, expansion and expression of activation markers in T cells. We show that nanoparticles with well-oriented monovalent antibodies conjugated via maleimide require fewer ligands on the surface to efficiently expand T cells compared to bivalent antibodies randomly-immobilized via carbodiimide conjugation. Analysis of the T cell expression markers reveal that the T cell phenotype can be fine-tuned by adjusting the surface density of well-oriented antibodies, while randomly immobilized antibodies showed no differences despite their ligand density. Both conjugation techniques induced cytotoxic T cells, evidenced by analyzing their Granzyme B secretion. Furthermore, antibody orientation affects the immunological synapse and T cell activation by changing the calcium influx profile upon activation. Nanoparticles with well-oriented antibodies showed lower calcium influx compared to their bivalent randomly-immobilized counterparts. These results highlight the importance of controlling the antibody density and orientation on the nanoparticle surface via controlled coupling chemistries, helping to develop improved particle-based expansion protocols to enhance T cell therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Systemically delivered hedgehog inhibitors including vismodegib and sonidegib are widely used to treat basal cell carcinomas (BCCs). Ablative fractional laser (AFL)-assisted topical delivery of vismodegib has been demonstrated in preclinical studies. The aim of this explorative clinical study was to evaluate intratumoral vismodegib concentrations and effect on hedgehog pathway gene expression following AFL-assisted topical vismodegib delivery to BCCs., Methods: In an open-label clinical trial, 16 nodular BCCs (in n = 9 patients) received one application of CO 2 -AFL (40 mJ/microbeam, 10% density) followed by topical vismodegib emulsion. After 3-4 days, vismodegib concentrations in tumor biopsies (n = 15) and plasma were analyzed and compared with samples from patients receiving oral treatment (n = 3). GLI1, GLI2, PTCH1, and PTCH2 expression was determined by quantitative polymerase chain reaction (n = 7) and GLI1 additionally by in situ hybridization (n = 3)., Results: Following AFL-assisted topical administration, vismodegib was detected in 14/15 BCCs and reached a median concentration of 6.2 µmol/L, which compared to concentrations in BCC tissue from patients receiving oral vismodegib (9.5 µmol/L, n = 3, p = 0.8588). Topical vismodegib reduced intratumoral GLI1 expression by 51%, GLI2 by 55%, PTCH1 and PTCH2 each by 73% (p ≤ 0.0304) regardless of vismodegib concentrations (p ≥ 0.3164). In situ hybridization demonstrated that GLI1 expression was restricted to tumor tissue and downregulated in response to vismodegib exposure., Conclusion: A single AFL-assisted topical application of vismodegib resulted in clinically relevant intratumoral drug concentrations and significant reductions in hedgehog pathway gene expressions., (© 2024 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.)

Cell-mediated drug delivery by conjugating nanomedicine to the surface of living cells is a promising strategy for enhancing the efficacy of both drug delivery and cell therapy. It exploits the tissue homing properties of the specific cell types to overcome in vivo barriers and forms a drug depot by directly putting the therapeutic payload in target cells. An important concern of developing this system is the method to conjugate nanoparticles on cells. Herein, we developed a bioorthogonal T cell conjugation strategy using SPAAC click chemistry, which allows controllable and highly efficient conjugation without affecting the viability and functions of the cytotoxic T lymphocytes. Azide groups were incorporated on the surface of T cells through metabolic glycoengineering, followed by reacting with dibenzylcyclooctyne (DBCO) modified lipid nanoparticles (LNPs). LNPs can be conjugated to T cells, allowing for the loading of different drug molecules on the cells. The metabolic engineering and click reaction approach provides a simple and versatile strategy to conjugate NPs to living cells and enable the development of sophisticated therapeutic cell products., (© 2024 Wiley-VCH GmbH.)

Whereas adoptive T cell therapy has been extensively studied for cancer treatment, the response is still limited primarily due to immune dysfunction related to poor cell engraftment, tumor infiltration and engagement, and lack of a target. In addition, the modification of therapeutic T cells often suffers from being complex and expensive. Here, we present a strategy to load T cells with SHP099, an allosteric SHP2 inhibitor, to enhance the therapeutic efficacy of the T cells. Remote-loading of SHP099 into lipid nanoparticles decorated with triarginine motifs resulted in nanocrystal formation of SHP099 inside the lipid vesicles and allowed high loading efficiency and prolonged retention of SHP099 nanocrystals within T cells. Cell-loaded SHP099 enabled sustained inhibition of the PD-1/PD-L1 signaling and increased cytolytic activity of the T cells. We show in a mouse model that tumor-homing T cells can circulate with the cargos, improving their tumor accumulation compared to systemically administered lipid nanoparticles. On an established solid tumor model, adoptively transferred SHP099 loaded T cells induced complete tumor eradication and durable immune memory against tumor rechallenging on all treated mice by effectively inhibiting the PD-1/PD-L1 checkpoint signal. We demonstrate that the combination of T cell therapy with SHP2 inhibition is a promising therapeutic strategy, and the lipid nanocrystal platform could be generalized as a promising approach for T cell loading of immunomodulatory drugs.

Adoptive T-cell transfer (ACT) offers a curative therapeutic option for subsets of melanoma and hematological cancer patients. To increase response rates and broaden the applicability of ACT, it is necessary to improve the post-infusion performance of the transferred T cells. The design of improved treatment strategies includes transfer of cells with a less differentiated phenotype. Such T cell subsets have high proliferative potential but require stimulatory signals in vivo to differentiate into tumor-reactive effector T cells. Thus, combination strategies are needed to support the therapeutic implementation of less differentiated T cells. Here we show that systemic delivery of tumor-associated antigens (TAAs) facilitates in vivo priming and expansion of previously non-activated T cells and enhance the cytotoxicity of activated T cells. To achieve this in vivo priming, we use flexible delivery vehicles of TAAs and a TLR7/8 agonist. Contrasting subcutaneous delivery systems, these vehicles accumulate TAAs in the spleen, thereby achieving close proximity to both cross-presenting dendritic cells and transferred T cells, resulting in robust T-cell expansion and anti-tumor reactivity. This TAA delivery platform offers a strategy to safely potentiate the post-infusion performance of T cells using low doses of antigen and TLR7/8 agonist, and thereby enhance the effect of ACT., (© 2021. The Author(s).)

While transfusion of donor blood is a reasonably safe and well-established procedure, artificial oxygen carriers offer several advantages over blood transfusions. These benefits include compatibility with all blood types, thus avoiding the need for cross matching, availability, lack of infection, and long-term storage. Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being explored as an "oxygen bridge" to replace or complement standard blood transfusions in extreme, life-threatening situations such as trauma in remote locations or austere battlefield or when blood is not an option due to compatibility issues or patient refusal due to religious objections. Herein, a novel HBOC was prepared using the layer-by-layer technique. A poly(lactide- co -glycolide) core was fabricated and subsequently decorated with Hb and nanozymes. The Hb was coated with poly(dopamine), and preservation of the protein structure and functionality was demonstrated. Next, cerium oxide nanoparticles were incorporated as nanozymes, and their ability to deplete reactive oxygen species (ROS) was shown. Finally, decorating the nanocarrier surface with poly(ethylene glycol) decreased protein adsorption and cell association/uptake. The as-prepared Hb-based oxygen nanocarriers were shown to be hemo- and bio-compatible. Their catalytic potential was furthermore demonstrated in terms of superoxide radical- and peroxide-scavenging abilities, which were retained over multiple cycles. Overall, these results demonstrate that the reported nanocarriers show potential as novel oxygen delivery systems with prolonged catalytic activity against ROS.

Oral administration of vismodegib for basal cell carcinoma treatment is limited by significant class-specific systemic side effects. We investigated the approach of combining ablative fractional laser-assisted drug delivery with an extended-release microemulsion formulation of vismodegib to provide efficient cutaneous delivery in vivo. The developed formulation consisted of an oil-in-water microemulsion stabilized by Tween-80. Pig skin was exposed to ablative fractional laser followed by topical application of vismodegib microemulsion for 4 hours. At 4 hours, 2 days, 5 days, and 9 days, we evaluated vismodegib biodistribution in superficial, mid, and deep dermis and plasma (n = 189 measurements) and assessed local skin reactions. Sustained topical delivery of vismodegib was detected in all depths of ablative fractional laser-exposed skin over the course of the study, with peak concentrations found at 5 days and 9 days. The highest vismodegib concentrations reached 1,409.7 μmol/liter in superficial dermis and 62.3 μmol/liter in deep dermis, exceeding steady-state plasma concentrations previously reported for oral administration of vismodegib (5.5-56.0 μmol/liter). Ablative fractional laser increased vismodegib uptake up to 16.6-fold compared with intact skin. Only mild local skin responses to vismodegib were observed, and no vismodegib was detected in plasma. We report sustained topical delivery of vismodegib in vivo at high concentrations with favorable skin tolerability, suggesting a future safer vismodegib treatment., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Autonomously propelled/externally guided micromotors overcome current drug delivery challenges by providing (a) higher drug loading capacity, (b) localized delivery (less toxicity), (c) enhanced tissue penetration and (d) active maneuvering in vivo. These microscale drug delivery systems can exploit biological fluids, as well as exogenous stimuli, like light-NIR, ultrasound and magnetic fields (or a combination of these), towards propulsion/drug release. Ability of these wireless drug carriers towards localized targeting and controlled drug release, makes them a lucrative candidate for drug administration in complex microenvironments (like solid tumors or gastrointestinal tract). In this report, we discuss these microscale drug delivery systems for their therapeutic benefits under in vivo setting and provide a design-application rationale towards greater clinical significance. Also, a proof-of-concept depicting 'microbots-in-a-capsule' towards oral drug delivery has been discussed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Objective: Hedgehog inhibitors such as vismodegib are targeted drugs widely used for the treatment of basal cell carcinomas; however, their use is significantly limited by frequent systemic side effects due to oral administration route. We aim to use ablative fractional laser (AFL) to enable the topical delivery of vismodegib to relevant dermal depths., Materials and Methods: Pig skin was treated in vitro with a fractional 10,600 nm CO 2 laser at 0 or 80 mJ/microbeam and exposed to vismodegib (6.4 mmol/L) in Franz-diffusion cells for 0.5, 4, and 24 hours (n = 54 samples), either formulated in a micro-emulsion composed of soybean oil and Tween 80 or dissolved in ethanol as vehicle control. Vismodegib biodistribution was studied at specific skin depths from 0 to 1,800 µm (incremental steps of 300 µm) by mass spectrometry., Results: Combination of AFL and vismodegib emulsion substantially enhanced the delivery of drug into the skin. Emulsion formulation alone yielded higher vismodegib skin concentrations compared to vehicle control in superficial and mid-dermis (0-900 µm, P = 0.002-0.015). The over-all highest concentration found (554.5 µmol/L) was reached at 24 hours in superficial (0-300 µm) AFL exposed skin, 7.6-fold higher than vehicle control (P = 0.002) and 9.7-101.6 fold higher than previously reported steady-state plasma concentrations in patients treated with oral vismodegib (5.5-56.9 µmol/L). Compared to intact skin, AFL exposure significantly increased skin concentrations of vismodegib even in deep skin layers (24 h, 900-1,800 µm, emulsion: 8.7-74.3 µmol/L vs. 0.0-0.0 µmol/L, P = 0.004-0.048; vehicle control: 23.7-50.6 µmol/L vs. 0.0-1.6 µmol/L, P = 0.002). The total delivery of vismodegib-emulsion into mid-deep dermal skin layers from 600 to 1,800 µm was for AFL exposed skin 8.2 fold higher than intact skin. Also, delivery of emulsion vismodegib by AFL was time-dependent as seen by the continuous increase in concentrations found over time, with highest uptake detected after 24 hours (4-24 hours, 0-900 µm, P = 0.002-0.004)., Conclusion: AFL enhances topical delivery of micro-emulsion formulated vismodegib, reaching concentrations similar to or above plasma concentrations previously reported in patients receiving oral vismodegib. Lasers Surg. Med. 51:79-87, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

The enhanced permeability and retention (EPR) effect increases tumor accumulation of liposomal chemotherapy and should, in theory, increase anticancer effects and lower toxicity. Unfortunately, liposomal chemotherapy has generally not met the expected potential, perhaps because the EPR effect is not ubiquitous. PET imaging using radiolabeled liposomes can identify cancers positive for the EPR effect. In the current study, we show in clinical canine cancer patients that repeated imaging with radiolabeled liposomes ( 64 Cu-liposome) induces the accelerated blood clearance (ABC) phenomenon. This was observed even with very long intervals between PEGylated liposome injections, which contradict previous reporting in experimental animal models. The induction of ABC may be devastating for the theranostic use of liposomal imaging, as this could vaccinate patients against therapeutic efficacy. To investigate and solve this important problem, an additional study part was designed in which rats were subjected to repeated liposomal administrations, including stealth 64 Cu-liposome PET imaging and Caelyx chemotherapy. Most importantly, it was found that, by increasing the lipid dose at the first injection or by supplying a small predose before the second 64 Cu-liposome injection, ABC could be prevented. Importantly, signs of liposome tracer breakdown with subsequent renal excretion were observed. These findings highlight the importance of the ABC phenomenon for liposomal predictive imaging in a clinically relevant setting and show that carefully planned application is central to avoid potential detrimental effects on patient benefit.

The clinical use of liposomal drug delivery vehicles is often hindered by insufficient drug release. Here we present the rational design of liposomes optimized for secretory phospholipase A 2 (sPLA 2 ) triggered drug release, and test their utility in vitro and in vivo. We hypothesized that by adjusting the level of cholesterol in anionic, unsaturated liposomes we could tune the enzyme specificity based on membrane fluidity, thus obtaining liposomes with an improved therapeutic outcome and reduced side effects. Cholesterol is generally important as a component in the membranes of liposome drug delivery systems due to its stabilizing effects in vivo. The incorporation of cholesterol in sPLA 2 sensitive liposomes has not previously been possible due to reduced sPLA 2 activity. However, in the present work we solved this challenge by optimizing membrane fluidity. In vitro release studies revealed enzyme specific drug release. Treatment of two different cancer cell lines with liposomal oxaliplatin revealed efficient growth inhibition compared to that of clinically used stealth liposomes. The in vivo therapeutic effect was evaluated in nude NMRI mice using the sPLA 2 secreting mammary carcinoma cell line MT-3. Three days after first treatment all mice having received the novel sPLA 2 sensitive liposome formulation were euthanized due to severe systemic toxicity. Thus the present study demonstrates that great caution should be implemented when utilizing sPLA 2 sensitive liposomes and that the real utility can only be disclosed in vivo. The present studies have clinical implications, as sPLA 2 sensitive formulations are currently undergoing clinical trials (LiPlaCis®)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The first developed secretory phospholipase A 2 (sPLA 2 ) sensitive liposomal cisplatin formulation (LiPlaCis®) is currently undergoing clinical evaluation. In the present study we revisit and evaluate critical preclinical parameters important for the therapeutic potential and safety of platinum drugs, here oxaliplatin (L-OHP), formulated in sPLA 2 sensitive liposomes. We show the mole percentage of negatively charged phospholipid needed to obtain enzyme-sensitivity for saturated systems is ≥25% for 16-carbon chain lipid membranes, and >40% for 18-chain lipid membranes, which was surprising as 25% is used clinically in LiPlaCis®. Efficient sPLA 2 -dependent growth inhibition of colorectal cancer cells was demonstrated in vitro, where cell membrane degradation and cytolysis depends on the sensitivity of the formulation towards the enzyme and is governed by the amount of lysolipids generated and the presence of serum proteins. We found that serum proteins did not affect the lipase activity of the enzyme towards the membranes but instead sequester the lysolipid byproducts consequently inhibiting their detergent-like cytotoxic properties. In vivo therapeutic potential and safety of the liposomes was investigated in nude mice bearing sPLA 2 -deficient FaDu squamous carcinoma and sPLA 2 -expressing Colo205 colorectal adenocarcinoma. After intravenous injections, the tumor growth was suppressed for liposomal L-OHP relative to free drug, but only a weak response was observed for both slow- and fast-releasing sPLA 2 -sensitive formulations compared to non-sensitive liposomes. Also, the mice did not show longer survival. In turn, for the highly sPLA 2 -sensitive liposomes, multiple high doses caused petechial cutaneous hemorrhages, along with multifocal hepatonecrotic lesions, suggestive of premature activation in skin and liver irrespective of sPLA 2 -status of the tumor engraft. These results indicate that although liposomal carriers can improve the antitumor efficacy of platinum drugs, sPLA 2 -triggered release suffers from a narrow therapeutic index and has safety concerns., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An efficient liposomal system for screening the zinc ionophore activity of a selected library consisting of the most relevant dietary polyphenols is presented. The zinc ionophore activity was demonstrated by exploring the use of zinc-specific fluorophore FluoZin-3 loaded liposomes as simple membrane tools that mimic the cell membrane. The zinc ionophore activity was demonstrated as the capacity of polyphenols to transport zinc cations across the liposome membrane and increase the zinc-specific fluorescence of the encapsulated fluorophore FluoZin-3. In addition, the zinc chelation strength of the polyphenols was also tested in a competition assay based on the fluorescence quenching of zinc-dependent fluorescence emitted by zinc-FluoZin-3 complex. Finally, the correlation between the chelation capacity and ionophore activity is demonstrated, thus underlining the sequestering or ionophoric activity that the phenolic compounds can display, thus, providing better knowledge of the importance of the structural conformation versus their biological activity. Furthermore, the assays developed can be used as tools for rapid, high-throughput screening of families of polyphenols towards different biometals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

As one of the building blocks of the cell membrane, lipids and their interaction with neighboring lipids and other molecules, as well as their ability to form different kinds of structures, have garnered immense interest. By exploiting the effective shape and thermal-phase behavior of lipids, we have prepared lipid superstructures such as twisted ribbons and rectangular and hexagonal shaped lipidic nanostructures using the curvature tuned preparation method. These lipidic superstructures were then used as nanoreactor templates for the inorganic synthesis of diversely shaped and sized gold nanostructures exploring different administration routes of reducing agents, citrate, and tetrachloroauric acid, which as a result formed different organizations of gold nanoparticles aligned and guided by the template structure. Tailor-designed metallic nanostructures can be obtained through a careful selection of lipids and conditions for lipid superstructure preparation and their consequent use as template nanoreactors. The diversely sized and shaped gold nanostructures obtained have great potential for catalysis and plasmonics.

Labile zinc, a tiny fraction of total intracellular zinc that is loosely bound to proteins and easily interchangeable, modulates the activity of numerous signaling and metabolic pathways. Dietary plant polyphenols such as the flavonoids quercetin (QCT) and epigallocatechin-gallate act as antioxidants and as signaling molecules. Remarkably, the activities of numerous enzymes that are targeted by polyphenols are dependent on zinc. We have previously shown that these polyphenols chelate zinc cations and hypothesized that these flavonoids might be also acting as zinc ionophores, transporting zinc cations through the plasma membrane. To prove this hypothesis, herein, we have demonstrated the capacity of QCT and epigallocatechin-gallate to rapidly increase labile zinc in mouse hepatocarcinoma Hepa 1-6 cells as well as, for the first time, in liposomes. In order to confirm that the polyphenols transport zinc cations across the plasma membrane independently of plasma membrane zinc transporters, QCT, epigallocatechin-gallate, or clioquinol (CQ), alone and combined with zinc, were added to unilamellar dipalmitoylphosphocholine/cholesterol liposomes loaded with membrane-impermeant FluoZin-3. Only the combinations of the chelators with zinc triggered a rapid increase of FluoZin-3 fluorescence within the liposomes, thus demonstrating the ionophore action of QCT, epigallocatechin-gallate, and CQ on lipid membrane systems. The ionophore activity of dietary polyphenols may underlay the raising of labile zinc levels triggered in cells by polyphenols and thus many of their biological actions.

The synthesis of highly stable ultrasmall monodisperse populations of palladium nanoparticles in the range of 1-3 nm in size was achieved via polyol reduction within 1,2-dioleoyl-sn-glycero-3-phosphor-rac-(1-glycerol) liposomal nanoreactors exploiting glycerol as both reducing and stabilizing agent. The liposome-based green method was compared with synthesis in solution, and the reducing agent concentration and the lipidic composition of the liposomal nanoreactors were demonstrated to have a strong effect on the final size and homogeneity of the palladium nanoparticles. Glycerol molecules acting as capping agent demonstrated the ability to stabilize the palladium nanoparticles over a long period of time, maintaining their homogeneity in size and shape. The obtained palladium nanoparticles were characterized using transmission electron microscopy, selected area electron diffraction, Fourier transform infrared and Raman spectroscopies, X-ray diffraction, and dynamic light scattering to determine their morphology, size, charge, surface chemistry, and crystal structure. The catalytic activity of the palladium nanoparticles was also tested for a reduction reaction.

There has been enormous interest in the last decade in development methods for the inorganic synthesis of metallic nanoparticles of desired sizes and shapes because of their unique properties and extensive applications in catalysis, electronics, plasmonics, and sensing. Here we report on an environmentally friendly, one-pot synthesis of metallic nanoparticles, which avoids the use of organic solvents and requires mild experimental conditions. The developed method uses liposomes as nanoreactors, where the liposomes were prepared by encapsulating chloroauric acid and exploited the use of glycerol, incorporated within the lipid bilayer as well as in its hydrophilic core, as a reducing agent for the controlled preparation of highly homogeneous populations of gold nanoparticles. The effects of temperature, the presence of a capping agent, and the concentration of glycerol on the size and homogeneity of the nanoparticles formed were investigated and compared with solution-based glycerol-mediated nanoparticle synthesis. Well-distributed gold nanoparticle populations in the range of 2-8 nm were prepared in the designed liposomal nanoreactor with a clear dependence of the size on the concentration of glycerol, the temperature, and the presence of a capping agent whereas large, heterogeneous populations of nanoparticles with amorphous shapes were obtained in the absence of liposomes. The particle morphology and sizes were analyzed using transmission electron microscopy imaging, and the liposome size was measured using photon correlation spectroscopy., (© 2011 American Chemical Society)