Publications

Biological barriers present a significant obstacle to treatment, especially when drugs are administered locally to increase their concentrations at the target site while minimizing unintended off-target effects. Among these barriers, mucus presents a challenge, as it serves as a protective layer in the respiratory, urogenital, and gastrointestinal tracts. Its role is to shield the underlying epithelial cells from pathogens and toxic compounds but also impedes the efficient delivery of drugs. Despite the exploration of mucolytic agents to improve drug delivery, overcoming this protective barrier remains a significant hurdle. In our study, we investigate an alternative approach involving the use of catalase-powered nanobots. We use an in vitro model that simulates intestinal mucus secretion to demonstrate the dual functionality of our nanobots. This includes their ability to disrupt mucus, which we confirmed through in vitro and ex vivo validation, as well as their self-propulsion to overcome the mucus barrier, resulting in a 60-fold increase compared with passive nanoparticles. Therefore, our findings highlight the potential utility of catalase-powered nanobots as carriers for therapeutic agents since they could enhance drug delivery efficiency by penetrating the mucus barrier.

JTD Keywords: Biological barrier, Biological barriers, Drug-delivery, Growth, Hydrogen-peroxide, Muci, Mucus, Nanobots, Nanomedicine, Nanomotors, Transport

Malaria, caused by different species of protists of the genus Plasmodium, remains among the most common causes of death due to parasitic diseases worldwide, mainly for children aged under 5. One of the main obstacles to malaria eradication is the speed with which the pathogen evolves resistance to the drug schemes developed against it. For this reason, it remains urgent to find innovative therapeutic strategies offering sufficient specificity against the parasite to minimize resistance evolution and drug side effects. In this context, nanotechnology-based approaches are now being explored for their use as antimalarial drug delivery platforms due to the wide range of advantages and tuneable properties that they offer. However, major challenges remain to be addressed to provide a cost-efficient and targeted therapeutic strategy contributing to malaria eradication. The present work contains a systematic review of nanotechnology-based antimalarial drug delivery systems generated during the last 10 years. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease

JTD Keywords: Adjuvant system, Antimalarial activities, Antimalarial agent, Antimalarial drug, Antimalarial drugs, Antimalarials, Artemisinin resistance, Causes of death, Child, Controlled drug delivery, Diseases, Drug delivery system, Drug delivery systems, Drug interactions, Drug side-effects, Drug-delivery, Experimental modelling, Heparan-sulfate, Human, Humans, In-vitro, Malaria, Malaria vaccine, Mannosylated liposomes, Medical nanotechnology, Models, theoretical, Nanocarriers, Nanomedicine, Nanotechnology, Parasite-, Parasitics, Plasmodium, Plasmodium-falciparum malaria, Red-blood-cells, Targeted delivery, Targeted drug delivery, Theoretical model, Therapeutic strategy

Microbial biofilms are complex three-dimensional structures where sessile microbes are embedded in a polymeric extracellular matrix. Their resistance toward the host immune system as well as to a diverse range of antimicrobial treatments poses a serious health and development threat, being in the top 10 global public health threats declared by the World Health Organization. In an effort to combat biofilm-related microbial infections, several strategies have been developed to independently eliminate biofilms or to complement conventional antibiotic therapies. However, their limitations leave room for other treatment alternatives, where the application of nanotechnology to biofilm eradication has gained significant relevance in recent years. Their small size, penetration efficiency, and the design flexibility that they present makes them a promising alternative for biofilm infection treatment, although they also present set-backs. This review aims to describe the main possibilities and limitations of nanomedicine against biofilms, while covering the main aspects of biofilm formation and study, and the current therapies for biofilm treatment. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

JTD Keywords: Anti-bacterial agents, Anti-infective agents, Antiinfective agent, Antimicrobial, Antimicrobials, Antimicrobials,bacteria,biofilm,infectious diseases,microorganism, Bacteria, Biofilm, Biofilm infections, Biofilms, Complex three dimensional structures, Diseases, Diverse range, Drug-delivery systems,in-vitro,cellular toxicity,nanoparticles,penetration,model,biocompatibility,perspectives,hyperthermia,diagnosi, Extracellular matrices, Global public health, Health risks, Infectious disease, Infectious diseases, Medical nanotechnology, Microbial biofilm, Microorganisms, Nanomedicine, Polymer, Polymers, Regulatory issues, Roadmap

The use of nanocarriers in medicine, so-called nanomedicine, is one of the most innovative strategies for targeting drugs at the action site and increasing their activity index and effectiveness. Phytomedicine is the oldest traditional method used to treat human diseases and solve health problems. The recent literature on the treatment of malaria infections using nanodelivery systems and phytodrugs or supplements has been analyzed. For the first time, in the present review, a careful look at the considerable potential of nanomedicine in promoting phytotherapeutic efficacy was done, and its key role in addressing a translation through a significant reduction of the current burden of malaria in many parts of the world has been underlined.

JTD Keywords: antiplasmodial activity, bioavailability, chloroquine, combination therapy, discovery, drug-delivery, drug-delivery systems, nanocapsules, nanomedicine, natural molecules, pharmacokinetics, phytomedicine, plasmodium-falciparum, Artemisinin-based combination therapy, Drug-delivery systems, Nanomedicine, Natural molecules, Phytomedicine, Solid lipid nanoparticles

Artemisinin, curcumin or quercetin, alone or in combination, were loaded in nutriosomes, special phospholipid vesicles enriched with Nutriose FM06®, a soluble dextrin with prebiotic activity, that makes these vesicles suitable for oral delivery. The resulting nutriosomes were sized between 93 and 146 nm, homogeneously dispersed, and had slightly negative zeta potential (around -8 mV). To improve their shelf life and storability over time, vesicle dispersions were freeze-dried and stored at 25 °C. Results confirmed that their main physico-chemical characteristics remained unchanged over a period of 12 months. Additionally, their size and polydispersity index did not undergo any significant variation after dilution with solutions at different pHs (1.2 and 7.0) and high ionic strength, mimicking the harsh conditions of the stomach and intestine. An in vitro study disclosed the delayed release of curcumin and quercetin from nutriosomes (∼53% at 48 h) while artemisinin was quickly released (∼100% at 48 h). Cytotoxicity assays using human colon adenocarcinoma cells (Caco-2) and human umbilical vein endothelial cells (HUVECs) proved the high biocompatibility of the prepared formulations. Finally, in vitro antimalarial activity tests, assessed against the 3D7 strain of Plasmodium falciparum, confirmed the effectiveness of nutriosomes in the delivery of curcumin and quercetin, which can be used as adjuvants in the antimalaria treatment. The efficacy of artemisinin was also confirmed but not improved. Overall results proved the possible use of these formulations as an accompanying treatment of malaria infections.Copyright © 2023. Published by Elsevier B.V.

JTD Keywords: artemisinin, delivery, flavonol, formulations, liposomes, malaria infections, nanomedicine, nutriose (r) fm06, oral administration, plasmodium falciparum, In-vitro, Liposomes, Malaria infections, Nanomedicine, Nutriose® fm06, Oral administration, Plasmodium falciparum

Biodegradable periodic mesoporous organosilica nanoparticles (nanoPMOs) are widely used as responsive drug delivery platforms for targeted chemotherapy of cancer. However, the evaluation of their properties such as surface functionality and biodegradability is still challenging, which has a significant impact on the efficiency of chemotherapy. In this study, we have applied direct stochastic optical reconstruction microscopy (dSTORM), a single-molecule super-resolution microscopy technique, to quantify the degradation of nanoPMOs triggered by glutathione and the multivalency of antibody-conjugated nanoPMOs. Subsequently, the effect of these properties on cancer cell targeting, drug loading and release capability, and anticancer activity is also studied. Due to the higher spatial resolution at the nanoscale, dSTORM imaging is able to reveal the structural properties (i.e., size and shape) of fluorescent and biodegradable nanoPMOs. The quantification of nanoPMOs' biodegradation using dSTORM imaging demonstrates their excellent structure-dependent degradation behavior at a higher glutathione concentration. The surface functionality of anti-M6PR antibody-conjugated nanoPMOs as quantified by dSTORM imaging plays a key role in prostate cancer cell labeling: oriented antibody is more effective than random ones, while high multivalency is also effective. The higher biodegradability and cancer cell-targeting properties of nanorods conjugated with oriented antibody (EAB4H) effectively deliver the anticancer drug doxorubicin to cancer cells, exhibiting potent anticancer effects.

JTD Keywords: drug-delivery, nanocapsules, nanomaterials, nanomedicine, release, Silica nanoparticles

Drug and gene delivery systems based on polymeric nanoparticles offer a greater efficacy and a reduced toxicity compared to traditional formulations. Recent studies have evidenced that their internalization, biodistribution and efficacy can be affected, among other factors, by their mechanical properties. Here, we analyze by means of Atomic Force Microscopy force spectroscopy how composition, surface functionalization and loading affect the mechanics of nanoparticles. For this purpose, nanoparticles made of Poly(lactic-co-glycolic) (PLGA) and Ethyl cellulose (EC) with different functionalizations and loading were prepared by nano-emulsion templating using the Phase Inversion Composition method (PIC) to form the nano-emulsions. A multiparametric nanomechanical study involving the determination of the Young's modulus, maximum deformation and breakthrough force was carried out. The obtained results showed that composition, surface functionalization and loading affect the nanomechanical properties in a different way, thus requiring, in general, to consider the overall mechanical properties after the addition of a functionalization or loading. A graphical representation method has been proposed enabling to easily identify mechanically equivalent formulations, which is expected to be useful in the development of soft polymeric nanoparticles for pre-clinical and clinical use.Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.

JTD Keywords: afm, atomic-force microscopy, cell, delivery-systems, drug-delivery, emulsification approach, internalization, mechanics of nanoparticles, nanomedicine, nanoparticle functionalization, particles, protein corona, size, young?s modulus, Afm, Loaded plga nanoparticles, Mechanics of nanoparticles, Nanomedicine, Nanoparticle functionalization, Polymeric nanoparticles, Young’s modulus

Nanomedicine emerged some decades ago with the hope to be the solution for most unmet medical needs. However, tracking materials at nanoscale is challenging to their reduced size, below the resolution limit of most conventional techniques. In this context, we propose the use of direct stochastic optical reconstruction microscopy (dSTORM) to study time stability and cell trafficking after transfection of oligopeptide end-modified poly(?-aminoester) (OM-pBAE) nanoparticles. We selected different combinations of cationic end oligopeptides (arginine - R; histidine - H; and lysine - K) among polymer libraries, since the oligopeptide combination demonstrated to be useful for different applications, such as vaccination and gene silencing. We demonstrate that their time evolution as well as their cell uptake and trafficking are dependent on the oligopeptide. This study opens the pave to broad mechanistic studies at nanoscale that could enable a rational selection of specific pBAE nanoparticles composition after determining their stability and cell trafficking.© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.

JTD Keywords: cancer nanomedicine, cell trafficking, delivery, direct stochastic optical reconstruction microscopy (dstorm), nanoparticle stability, poly(beta-aminoester) nanoparticles, Direct stochastic optical reconstruction microscopy (dstorm), Poly(?-aminoester) nanoparticles, Poly(beta-amino ester)s, Poly(β-aminoester) nanoparticles

Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.

JTD Keywords: active targeting, active targeting dstorm, antibodies, dstorm, heterogeneity, multivalency, nanomedicine, nanoparticle functionality, size, super-resolution microscopy, surface, Active targeting, Antibodies, Cell membranes, Cell receptors, Cytology, Direct stochastic optical reconstruction microscopy, Dstorm, Heterogeneity, Ligands, Medical nanotechnology, Molecules, Nanomedicine, Nanoparticle functionality, Nanoparticle targeting, Nanoparticles, Optical reconstruction, Single molecule, Stochastic systems, Stochastics, Super-resolution microscopy, Superresolution microscopy

Nanomedicine involves the use of nanotechnology for clinical applications and holds promise to improve treatments. Recent developments offer new hope for cancer detection, prevention and treatment; however, being a heterogenous disorder, cancer calls for a more targeted treatment approach. Personalized Medicine (PM) aims to revolutionize cancer therapy by matching the most effective treatment to individual patients. Nanotheranostics comprise a combination of therapy and diagnostic imaging incorporated in a nanosystem and are developed to fulfill the promise of PM by helping in the selection of treatments, the objective monitoring of response and the planning of follow-up therapy. Although well-established imaging techniques, such as Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT), are primarily used in the development of theranostics, Optical Imaging (OI) offers some advantages, such as high sensitivity, spatial and temporal resolution and less invasiveness. Additionally, it allows for multiplexing, using multi-color imaging and DNA barcoding, which further aids in the development of personalized treatments. Recent advances have also given rise to techniques permitting better penetration, opening new doors for OI-guided nanotheranostics. In this review, we describe in detail these recent advances that may be used to design and develop efficient and specific nanotheranostics for personalized cancer drug delivery. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: 5-aminolevulinic acid, cancer, contrast agents, in-vivo, malignant gliomas, multifunctional nanoparticles, nanomedicine, optical imaging, ovarian-cancer, personalized medicine, quantum dots, silica nanoparticles, targeted probes, theranostics, Cancer, Nanomedicine, Optical imaging, Personalized medicine, Superparamagnetic iron-oxide, Theranostics

Malaria eradication has for decades been on the global health agenda, but the causative agents of the disease, several species of the protist parasite Plasmodium, have evolved mechanisms to evade vaccine-induced immunity and to rapidly acquire resistance against all drugs entering clinical use. Because classical antimalarial approaches have consistently failed, new strategies must be explored. One of these is nanomedicine, the application of manipulation and fabrication technology in the range of molecular dimensions between 1 and 100 nm, to the development of new medical solutions. Here we review the current state of the art in malaria diagnosis, prevention, and therapy and how nanotechnology is already having an incipient impact in improving them. In the second half of this review, the next generation of antimalarial drugs currently in the clinical pipeline is presented, with a definition of these drugs’ target product profiles and an assessment of the potential role of nanotechnology in their development. Opinions extracted from interviews with experts in the fields of nanomedicine, clinical malaria, and the economic landscape of the disease are included to offer a wider scope of the current requirements to win the fight against malaria and of how nanoscience can contribute to achieve them. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: antibody-bearing liposomes, antimalarial drugs, combination therapies, drug-delivery strategies, malaria diagnosis, malaria prophylaxis, malaria therapy, nanocarriers, nanomedicine, nanoparticles, nanotechnology, plasmodium, plasmodium-falciparum, red-blood-cells, targeted delivery, targeted drug delivery, vitro antimalarial activity, Antimalarial drugs, Isothermal amplification lamp, Malaria diagnosis, Malaria prophylaxis, Malaria therapy, Nanocarriers, Nanomedicine, Nanotechnology, Plasmodium, Targeted drug delivery

The functionalization of nanoparticles with functional moieties is a key strategy to achieve cell targeting in nanomedicine. The interplay between size and ligand number is crucial for the formulation performance and needs to be properly characterized to understand nanoparticle structure-activity relations. However, there is a lack of methods able to measure both size and ligand number at the same time and at the single particle level. Here, we address this issue by introducing a correlative light and electron microscopy (CLEM) method combining super-resolution microscopy (SRM) and transmission electron microscopy (TEM) imaging. We apply our super-resCLEM method to characterize the relationship between size and ligand number and density in PLGA-PEG nanoparticles. We highlight how heterogeneity found in size can impact ligand distribution and how a significant part of the nanoparticle population goes completely undetected in the single-technique analysis. Super-resCLEM holds great promise for the multiparametric analysis of other parameters and nanomaterials.

JTD Keywords: cellular uptake, correlative light and electron microscopy (clem), density, electron microscopy (em), functionalization, heterogeneity, nanomedicine, nanoparticles, pegylation, plga, progress, quantification, size, Correlative light and electron microscopy (clem), Electron microscopy (em), Heterogeneity, Nanomedicine, Nanoparticles, Physicochemical characterization, Super-resolution microscopy (srm)

Combination therapies, where two drugs acting through different mechanisms are administered simultaneously, are one of the most efficient approaches currently used to treat malaria infections. However, the different pharmacokinetic profiles often exhibited by the combined drugs tend to decrease treatment efficacy as the compounds are usually eliminated from the circulation at different rates. To circumvent this obstacle, we have engineered an immunoliposomal nanovector encapsulating hydrophilic and lipophilic compounds in its lumen and lipid bilayer, respectively. The antimalarial domiphen bromide has been encapsulated in the liposome membrane with good efficiency, although its high IC50 of ca. 1 μM for living parasites complicates its use as immunoliposomal therapy due to erythrocyte agglutination. The conjugation of antibodies against glycophorin A targeted the nanocarriers to Plasmodium-infected red blood cells and to gametocytes, the sole malaria parasite stage responsible for the transmission from the human to the mosquito vector. The antimalarials pyronaridine and atovaquone, which block the development of gametocytes, have been co-encapsulated in glycophorin A-targeted immunoliposomes. The co-immunoliposomized drugs have activities significantly higher than their free forms when tested in in vitro Plasmodium falciparum cultures: Pyronaridine and atovaquone concentrations that, when encapsulated in immunoliposomes, resulted in a 50% inhibition of parasite growth had no effect on the viability of the pathogen when used as free drugs.

JTD Keywords: Combination therapy, Immunoliposomes, Malaria, Nanomedicine, Nanotechnology, Plasmodium, Targeted drug delivery

Understanding how polyprotic compounds distribute within liposome (LP) suspensions is of major importance to design effective drug delivery strategies. Advances in this research field led to the definition of LP-based active drug encapsulation methods driven by transmembrane pH gradients with evidenced efficacy in the management of cancer and infectious diseases. An accurate modeling of membrane-solution drug partitioning is also fundamental when designing drug delivery systems for poorly endocytic cells, such as red blood cells (RBCs), in which the delivered payloads rely mostly on the passive diffusion of drug molecules across the cell membrane. Several experimental models have been proposed so far to predict the partitioning of polyprotic basic/acid drugs in artificial membranes. Nevertheless, the definition of a model in which the membrane-solution partitioning of each individual drug microspecies is studied relative to each other is still a topic of ongoing research. We present here a novel experimental approach based on mathematical modeling of drug encapsulation efficiency (EE) data in liposomal systems by which microspecies-specific partition coefficients are reported as a function of pH and phospholipid compositions replicating the RBC membrane in a simple and highly translatable manner. This approach has been applied to the study of several diprotic basic antimalarials of major clinical importance (quinine, primaquine, tafenoquine, quinacrine, and chloroquine) describing their respective microspecies distribution in phosphatidylcholine-LP suspensions. Estimated EE data according to the model described here closely fitted experimental values with no significant differences obtained in 75% of all pH/lipid composition-dependent conditions assayed. Additional applications studied include modeling drug EE in LPs in response to transmembrane pH gradients and lipid bilayer asymmetric charge, conditions of potential interest reflected in our previously reported RBC-targeted antimalarial nanotherapeutics.

JTD Keywords: Distribution coefficient, Liposomal systems, Malaria therapy, Nanomedicine, Partition coefficient, PH-controlled drug encapsulation, Polyprotic drug, Targeted drug delivery

AFMBioMed is the founding name under which international conferences and summer schools are organized around the application of atomic force microscopy in life sciences and nanomedicine. From its inception at the Atomic Energy Commission in Marcoule near 2004 to its creation in 2007 and to its 10th anniversary conference in Krakow, a brief narrative history of its birth and rise will demonstrate how and what such an organization brings to laboratories and the AFM community. With the current planning of the next AFMBioMed conference in Münster in 2019, it will be 15 years of commitment to these events.

JTD Keywords: Atomic Force Microscopy, Single molecules, Biomechanics, Force spectroscopy, High-speed AFM, Imaging, Nanoindentation, Nanomedicine, Nanotoxicology

Endocytosis and vesicular trafficking are cellular processes that regulate numerous functions required to sustain life. From a translational perspective, they offer avenues to improve the access of therapeutic drugs across cellular barriers that separate body compartments and into diseased cells. However, the fact that many factors have the potential to alter these routes, impacting our ability to effectively exploit them, is often overlooked. Altered vesicular transport may arise from the molecular defects underlying the pathological syndrome which we aim to treat, the activity of the drugs being used, or side effects derived from the drug carriers employed. In addition, most cellular models currently available do not properly reflect key physiological parameters of the biological environment in the body, hindering translational progress. This article offers a critical overview of these topics, discussing current achievements, limitations and future perspectives on the use of vesicular transport for drug delivery applications.

JTD Keywords: Cellular vesicles, Vesicle fusion, Fission and intracellular trafficking, Drug delivery systems and nanomedicines, Transcytosis and endocytosis of drugs carriers, Disease effects on vesicular trafficking, Drug effects on vesicular trafficking, Role of the biological environment

The efficacy and tolerability of systemically administered anticancer agents are limited by their off-target effects. Precise spatiotemporal control over their cytotoxic activity would allow improving chemotherapy treatments, and light-regulated drugs are well suited to this purpose. We have developed phototrexate, the first photoswitchable inhibitor of the human dihydrofolate reductase (DHFR), as a photochromic analog of methotrexate, a widely prescribed chemotherapeutic drug to treat cancer and psoriasis. Quantification of the light-regulated DHFR enzymatic activity, cell proliferation, and in vivo effects in zebrafish show that phototrexate behaves as a potent antifolate in its photoactivated cis configuration, and that it is nearly inactive in its dark-relaxed trans form. Thus, phototrexate constitutes a proof-of-concept to design light-regulated cytotoxic small molecules, and a step forward to develop targeted anticancer photochemotherapies with localized efficacy and reduced adverse effects.

JTD Keywords: Photopharmacology, Photodynamic therapy, Antiproliferative, Arthritis, Psoriasis, Nanomedicine

Current strategies for the mass administration of antimalarial drugs demand oral formulations to target the asexual Plasmodium stages in the peripheral bloodstream, whereas recommendations for future interventions stress the importance of also targeting the transmission stages of the parasite as it passes between humans and mosquitoes. Orally administered polyamidoamine (PAA) nanoparticles conjugated to chloroquine reached the blood circulation and cured Plasmodium yoelii-infected mice, slightly improving the activity of the free drug and inducing in the animals immunity against malaria. Liquid chromatography with tandem mass spectrometry analysis of affinity chromatography-purified PAA ligands suggested a high adhesiveness of PAAs to Plasmodium falciparum proteins, which might be the mechanism responsible for the preferential binding of PAAs to Plasmodium-infected erythrocytes vs. non-infected red blood cells. The weak antimalarial activity of some PAAs was found to operate through inhibition of parasite invasion, whereas the observed polymer intake by macrophages indicated a potential of PAAs for the treatment of certain coinfections such as Plasmodium and Leishmania. When fluorescein-labeled PAAs were fed to females of the malaria mosquito vectors Anopheles atroparvus and Anopheles gambiae, persistent fluorescence was observed in the midgut and in other insect’s tissues. These results present PAAs as a versatile platform for the encapsulation of orally administered antimalarial drugs and for direct administration of antimalarials to mosquitoes, targeting mosquito stages of Plasmodium.

JTD Keywords: Anopheles, Antimalarial drugs, Malaria, Mosquitoes, Nanomedicine, Nanotechnology, Plasmodium, Polyamidoamines, Polymers, Targeted drug delivery

Curcumin is an antimalarial compound easy to obtain and inexpensive, having shown little toxicity across a diverse population. However, the clinical use of this interesting polyphenol has been hampered by its poor oral absorption, extremely low aqueous solubility and rapid metabolism. In this study, we have used the anionic copolymer Eudragit® S100 to assemble liposomes incorporating curcumin and containing either hyaluronan (Eudragit-hyaluronan liposomes) or the water-soluble dextrin Nutriose® FM06 (Eudragit-nutriosomes). Upon oral administration of the rehydrated freeze-dried nanosystems administered at 25/75 mg curcumin·kg−1·day−1, only Eudragit-nutriosomes improved the in vivo antimalarial activity of curcumin in a dose-dependent manner, by enhancing the survival of all Plasmodium yoelii-infected mice up to 11/11 days, as compared to 6/7 days upon administration of an equal dose of the free compound. On the other hand, animals treated with curcumin incorporated in Eudragit-hyaluronan liposomes did not live longer than the controls, a result consistent with the lower stability of this formulation after reconstitution. Polymer-lipid nanovesicles hold promise for their development into systems for the oral delivery of curcumin-based antimalarial therapies.

JTD Keywords: Malaria, Curcumin, Nanomedicine, Oral administration, Lipid nanovesicles, Eudragit, Nutriose, Hyaluronan, Plasmodium yoelii

JTD Keywords: Extracellular vesicles, Hemozoin, Malaria, Nanomedicine, Plasmodium, Red blood cell, Targeted drug delivery

Most drugs currently entering the clinical pipeline for severe malaria therapeutics are of lipophilic nature, with a relatively poor solubility in plasma and large biodistribution volumes. Low amounts of these compounds do consequently accumulate in circulating Plasmodium-infected red blood cells, exhibiting limited antiparasitic activity. These drawbacks can in principle be satisfactorily dealt with by stably encapsulating drugs in targeted nanocarriers. Here this approach has been adapted for its use in immunocompetent mice infected by the Plasmodium yoelii 17XL lethal strain, selected as a model for human blood infections by Plasmodium falciparum. Using immunoliposomes targeted against a surface protein characteristic of the murine erythroid lineage, the protocol has been applied to two novel antimalarial lipophilic drug candidates, an aminoquinoline and an aminoalcohol. Large encapsulation yields of >90% were obtained using a citrate-buffered pH gradient method and the resulting immunoliposomes reached in vivo erythrocyte targeting and retention efficacies of >80%. In P. yoelii-infected mice, the immunoliposomized aminoquinoline succeeded in decreasing blood parasitemia from severe to uncomplicated malaria parasite densities (i.e. from ≥25% to ca. 5%), whereas the same amount of drug encapsulated in non-targeted liposomes had no significant effect on parasite growth. Pharmacokinetic analysis indicated that this good performance was obtained with a rapid clearance of immunoliposomes from the circulation (blood half-life of ca. 2 h), suggesting a potential for improvement of the proposed model.

JTD Keywords: Immunoliposomes, Malaria, Nanomedicine, Plasmodium falciparum, Plasmodium yoelii 17XL, Targeted drug delivery

The adaptation of existing antimalarial nanocarriers to new Plasmodium stages, drugs, targeting molecules, or encapsulating structures is a strategy that can provide new nanotechnology-based, cost-efficient therapies against malaria. We have explored the modification of different liposome prototypes that had been developed in our group for the targeted delivery of antimalarial drugs to Plasmodium-infected red blood cells (pRBCs). These new models include: (i) immunoliposome-mediated release of new lipid-based antimalarials; (ii) liposomes targeted to pRBCs with covalently linked heparin to reduce anticoagulation risks; (iii) adaptation of heparin to pRBC targeting of chitosan nanoparticles; (iv) use of heparin for the targeting of Plasmodium stages in the mosquito vector; and (v) use of the non-anticoagulant glycosaminoglycan chondroitin 4-sulfate as a heparin surrogate for pRBC targeting. The results presented indicate that the tuning of existing nanovessels to new malaria-related targets is a valid low-cost alternative to the de novo development of targeted nanosystems.

JTD Keywords: Glycosaminoglycans, Malaria, Nanomedicine, Plasmodium, Targeted drug delivery

Heparin is one of the oldest drugs, which nevertheless remains in widespread clinical use as an inhibitor of blood coagulation. The history of its identification a century ago unfolded amid one of the most fascinating scientific controversies turning around the distribution of credit for its discovery. The composition, purification and structure-function relationship of this naturally occurring glycosaminoglycan regarding its classical role as anticoagulant will be dealt with before proceeding to discuss its therapeutic potential in, among other, inflammatory and infectious disease, cancer treatment, cystic fibrosis and Alzheimer's disease. The first bibliographic reference hit using the words 'nanomedicine' and 'heparin' is as recent as 2008. Since then, nanomedical applications of heparin have experienced an exponential growth that will be discussed in detail, with particular emphasis on its antimalarial activity. Some of the most intriguing potential applications of heparin nanomedicines will be exposed, such as those contemplating the delivery of drugs to the mosquito stages of malaria parasites.

JTD Keywords: Anopheles, Antimalarial drugs, Heparin, Malaria, Mosquitoes, Nanomedicine, Nanotechnology, Plasmodium, Targeted drug delivery

Parasite proteins exported to the surface of Plasmodium falciparum-parasitized red blood cells (pRBCs) have a major role in severe malaria clinical manifestation, where pRBC cytoadhesion and rosetting processes have been strongly linked with microvascular sequestration while avoiding both spleen filtration and immune surveillance. The parasite-derived and pRBC surface-exposed PfEMP1 protein has been identified as one of the responsible elements for rosetting and, therefore, considered as a promising vaccine candidate for the generation of rosette-disrupting antibodies against severe malaria. However, the potential role of anti-rosetting antibodies as targeting molecules for the functionalization of antimalarial drug-loaded nanovectors has never been studied. Our manuscript presents a proof-of-concept study where the activity of an immunoliposomal vehicle with a dual performance capable of specifically recognizing and disrupting rosettes while simultaneously eliminating those pRBCs forming them has been assayed in vitro. A polyclonal antibody against the NTS-DBL1α N-terminal domain of a rosetting PfEMP1 variant has been selected as targeting molecule and lumefantrine as the antimalarial payload. After 30 min incubation with 2 μM encapsulated drug, a 70% growth inhibition for all parasitic forms in culture (IC50: 414 nM) and a reduction in ca. 60% of those pRBCs with a rosetting phenotype (IC50: 747 nM) were achieved. This immunoliposomal approach represents an innovative combination therapy for the improvement of severe malaria therapeutics having a broader spectrum of activity than either anti-rosetting antibodies or free drugs on their own.

JTD Keywords: Combination therapy, Immunoliposomes, Malaria, Nanomedicine, Rosetting, Targeted drug delivery

Lysosomal storage disorders are currently treated by enzyme replacement therapy (ERT) through the direct administration of the unprotected recombinant protein to the patients. Herein we present an ionically cross-linked polyelectrolyte complex (PEC) composed of trimethyl chitosan (TMC) and α-galactosidase A (GLA), the defective enzyme in Fabry disease, with the capability of directly targeting endothelial cells by incorporating peptide ligands containing the RGD sequence. We assessed the physicochemical properties, cytotoxicity, and hemocompatibility of RGD-targeted and untargeted PECs, the uptake by endothelial cells and the intracellular activity of PECs in cell culture models of Fabry disease. Moreover, we also explored the effect of different freeze-drying procedures in the overall activity of the PECs. Our results indicate that the use of integrin-binding RGD moiety within the PEC increases their uptake and the efficacy of the GLA enzyme, while the freeze-drying allows the activity of the therapeutic protein to remain intact. Overall, these results highlight the potential of TMC-based PECs as a highly versatile and feasible drug delivery system for improving the ERT of lysosomal storage disorders.

JTD Keywords: Enzyme replacement therapy, Fabry disease, Lysosomal delivery, Nanomedicine, Polyelectrolyte complexes, Trimethyl chitosan, α-galactosidase A

JTD Keywords: Anopheles, Antimalarial drugs, Combination therapies, Heparin, Malaria, Mosquitoes, Nanomedicine, Nanotechnology, Plasmodium, Poly(amidoamine)s, Polymers, Targeted drug delivery

One of the most important factors behind resistance evolution in malaria is the failure to deliver sufficiently high amounts of drugs to early stages of Plasmodium-infected red blood cells (pRBCs). Despite having been considered for decades as a promising approach, the delivery of antimalarials encapsulated in immunoliposomes targeted to pRBCs has not progressed towards clinical applications, whereas in vitro assays rarely reach drug efficacy improvements above 10-fold. Here we show that encapsulation efficiencies reaching >96% are achieved for the weak basic drugs chloroquine (CQ) and primaquine using the pH gradient loading method in liposomes containing neutral saturated phospholipids. Targeting antibodies are best conjugated through their primary amino groups, adjusting chemical crosslinker concentration to retain significant antigen recognition. Antigens from non-parasitized RBCs have also been considered as targets for the delivery to the cell of drugs not affecting the erythrocytic metabolism. Using this strategy, we have achieved unprecedented complete nanocarrier targeting to early intraerythrocytic stages of the malaria parasite for which there is a lack of specific extracellular molecular tags. Immunoliposomes studded with monoclonal antibodies raised against the erythrocyte surface protein glycophorin A were capable of targeting 100% RBCs and pRBCs at the low concentration of 0.5 μM total lipid in the culture, with >95% of added liposomes retained on cell surfaces. When exposed for only 15 min to Plasmodium falciparum in vitro cultures of early stages, free CQ had no significant effect on the viability of the parasite up to 200 nM, whereas immunoliposomal 50 nM CQ completely arrested its growth. In vivo assays in mice showed that immunoliposomes cleared the pathogen below detectable levels at a CQ dose of 0.5 mg/kg, whereas free CQ administered at 1.75 mg/kg was, at most, 40-fold less efficient. Our data suggest that this significant improvement is in part due to a prophylactic effect of CQ found by the pathogen in its host cell right at the very moment of invasion.

JTD Keywords: Immunoliposomes, Malaria, Nanomedicine, Plasmodium, Targeted drug delivery

The malaria parasite, Plasmodium spp., is a delicate unicellular organism unable to survive in free form for more than a couple of minutes in the bloodstream. Upon injection in a human by its Anopheles mosquito vector, Plasmodium sporozoites pass through the liver with the aim of invading hepatocytes. Those which succeed spend inside their host cell a recovery time before replicating and entering the blood circulation as fragile merozoites, although their exposure to host defenses is extraordinarily short. Quick invasion of red blood cells (RBCs) in a process lasting just a few minutes allows the parasite to escape immune system surveillance. For most of its erythrocytic cycle the pathogen feeds mainly on hemoglobin as it progresses from the early blood stages, termed rings, to the late forms trophozoites and schizonts. Early stages are ideal targets for antimalarial therapies because drugs delivered to them would have a longer time to kill the parasite before it completes its development. However, only 6 h after invasion does the permeability of the infected erythrocyte to anions and small nonelectrolytes, including some drugs, start to increase as the parasite matures [1]. During this maturation process the parasite hydrolyzes hemoglobin in a digestive vacuole, which is the target of many amphiphilic drugs that freely cross the RBC membrane and accumulate intracellularly. As a result, most antimalarials start affecting the infected cell relatively late in the intraerythrocytic parasite life cycle, when their effect is probably often too short to be lethal to Plasmodium.

JTD Keywords: Malaria, Nanomedicine, Plasmodium, Red blood cell, Targeted drug delivery

Nanotechnological devices for therapeutic applications are massively addressed to diseases prevalent in the developed world, particularly cancer, because of the wrong assumption (for both ethical and technical reasons) that nanomedicines are too expensive and thus they can not be applied to diseases of poverty. Here we have applied quantum dots to study at the cellular level the delivery of the contents of liposomes to erythrocytes infected by the malaria parasite Plasmodium falciparum, and to macrophages infected by the leishmaniasis causative agent Leishmania infantum. A number of works have reported on the encapsulation in liposomes of drugs against both diseases as a strategy to increase therapeutic efficacy and decrease unspecific toxicity. Liposome-carried drugs end up inside Plasmodium-infected red blood cells (pRBCs) and in the phagolysosome system of Leishmania-infected macrophages but some knowledge gaps still obscure subcellular events related to these processes. As a proof of concept, we have used confocal fluorescence microscopy to follow the fate in pRBCs and infected macrophages of quantum dots encapsulated in liposomes, and of lysosomes, leishmaniasis and malaria parasites, nuclei, and phagosomes. Our data indicate that liposomes merge their lipid bilayers with pRBC plasma membranes but are engulfed by macrophages, where they fuse with lysosomes. Lysosomes have not been observed to join with phagosomes harboring single Leishmania parasites, whereas in phagosomes where the parasite has divided there is lysosome-specific fluorescence with a concomitant disappearance of lysosomes from the cytosol. In later stages, all the lysosome-specific label is found inside phagosomes whereas the phagosomal marker cadaverine strongly stains the macrophage nucleus, suggesting that Leishmania infection induces in its later stages nuclear degeneration and, possibly, apoptosis of the host cell. These results indicate that induction of macrophage apoptosis should be explored as a possible strategy used by Leishmania to prepare its egress.

JTD Keywords: Leishmania infantum, Leishmaniasis Liposomes, Malaria, Nanomedicine, Nanotechnology, Plasmodium falciparum, Quantum dots

JTD Keywords: antimalarial, heparin, magic bullet, malaria, nanomedicine, nanotechnology, nanovector, Plasmodium, polymers, targeted drug delivery, chloroquine, immunoliposome, liposome, nanoparticle, solid lipid nanoparticle, Anopheles, antimalarial activity, drug delivery system, drug efficacy, erythrocyte, human, IC50, malaria, malaria control, nanoencapsulation, nonhuman, pathophysiology, Plasmodium, Review

It can be foreseen that in a future scenario of malaria eradication, a varied armamentarium will be required, including strategies for the targeted administration of antimalarial compounds. The development of nanovectors capable of encapsulating drugs and of delivering them to Plasmodium-infected cells with high specificity and efficacy and at an affordable cost is of particular interest. With this objective, dendritic derivatives based on 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) and Pluronic® polymers have been herein explored. Four different dendritic derivatives have been tested for their capacity to encapsulate the antimalarial drugs chloroquine (CQ) and primaquine (PQ), their specific targeting to Plasmodium-infected red blood cells (pRBCs), and their antimalarial activity in vitro against the human pathogen Plasmodium falciparum and in vivo against the rodent malaria species Plasmodium yoelii. The results obtained have allowed the identification of two dendritic derivatives exhibiting specific targeting to pRBCs vs. non-infected RBCs, which reduce the in vitro IC50 of CQ and PQ by ca. 3- and 4-fold down to 4.0 nm and 1.1 μm, respectively. This work on the application of dendritic derivatives to antimalarial targeted drug delivery opens the way for the use of this new type of chemicals in future malaria eradication programs.

JTD Keywords: Antimalarial targeted drug delivery, Dendrimers, Malaria, Nanomedicine, Plasmodium, Polymeric nanoparticles

Current malaria therapeutics demands strategies able to selectively deliver drugs to Plasmodium-infected red blood cells (pRBCs) in order to limit the appearance of parasite resistance. Here, the poly(amidoamines) AGMA1 and ISA23 have been explored for the delivery of antimalarial drugs to pRBCs. AGMA1 has antimalarial activity per se as shown by its inhibition of the in vitrogrowth of Plasmodium falciparum, with an IC50 of 13.7 μM. Fluorescence-assisted cell sorting data and confocal fluorescence microscopy and transmission electron microscopy images indicate that both polymers exhibit preferential binding to and internalization into pRBCs versus RBCs, and subcellular targeting to the parasite itself in widely diverging species such as P. falciparum and Plasmodium yoelii, infecting humans and mice, respectively. AGMA1 and ISA23 polymers with hydrodynamic radii around 7 nm show a high loading capacity for the antimalarial drugs primaquine and chloroquine, with the final conjugate containing from 14.2% to 32.9% (w/w) active principle. Intraperitoneal administration of 0.8 mg/kg chloroquine as either AGMA1 or ISA23 salts cured P. yoelii–infected mice, whereas control animals treated with twice as much free drug did not survive. These polymers combining into a single chemical structure drug carrying capacity, low unspecific toxicity, high biodegradability and selective internalization into pRBCs, but not in healthy erythrocytes for human and rodent malarias, may be regarded as promising candidates deserving to enter the antimalarial therapeutic arena.

JTD Keywords: Malaria, Nanomedicine, Plasmodium, Polyamidoamines, Polymer-drug carriers, Targeted drug delivery

The first part of this paper reviews the current development and key issues on implantable multi-sensor devices for in vivo theranostics. Afterwards, the authors propose an innovative biomedical multisensory system for in vivo biomarker monitoring that could be suitable for customized theranostics applications. At this point, findings suggest that cross-cutting Key Enabling Technologies (KETs) could improve the overall performance of the system given that the convergence of technologies in nanotechnology, biotechnology, micro&nanoelectronics and advanced materials permit the development of new medical devices of small dimensions, using biocompatible materials, and embedding reliable and targeted biosensors, high speed data communication, and even energy autonomy. Therefore, this article deals with new research and market challenges of implantable sensor devices, from the point of view of the pervasive system, and time-to-market. The remote clinical monitoring approach introduced in this paper could be based on an array of biosensors to extract information from the patient. A key contribution of the authors is that the general architecture introduced in this paper would require minor modifications for the final customized bio-implantable medical device.

JTD Keywords: Biocompatible, Biosensor, Biotelemetry, Implantable multi-sensor, Innovation, KET, Nanomedicine, Personalized medicine, Biotelemetry, Innovation, Medical nanotechnology, Biocompatible, Implantable system, In-vivo, KET, Multi sensor, Personalized medicines, Theranostics, Biosensors

JTD Keywords: Antimalarial, Erythrocyte, Glycosaminoglycan, Heparin, Magic bullet, Malaria, Nanomedicine, Nanovector, Plasmodium, Targeted drug delivery

JTD Keywords: Antimalarial, Erythrocyte, Glycosaminoglycan, Heparin, Magic bullet, Malaria, Nanomedicine, Nanovector, Plasmodium, Targeted drug delivery

Nanotechnological devices for therapeutic applications are massively addressed to diseases prevalent in the developed world, particularly cancer, because of the wrong assumption (for both ethical and technical reasons) that nanomedicines are too expensive and thus they can not be applied to diseases of poverty. Here we have applied quantum dots to study at the cellular level the delivery of the contents of immunoliposomes to erythrocytes infected by the malaria parasite Plasmodium falciparum, and to macrophages infected by the leishmaniasis causative agent Leishmania infantum. A number of works have reported on the encapsulation in liposomes of drugs against both diseases as a strategy to increase therapeutic efficacy and decrease unspecific toxicity. Liposome-carried drugs end up inside Plasmodium-infected red blood cells (pRBCs) and in the phagolysosome system of Leishmania-infected macrophages but some knowledge gaps still obscure subcellular events related to these processes. As a proof of concept, we have used confocal fluorescence microscopy to follow the fate in pRBCs and L. infantum-infected macrophages of quantum dots encapsulated in liposomes, and of lysosomes, Leishmania and Plasmodium parasites, nuclei, and phagosomes. Our data indicate that liposomes merge their lipid bilayers with pRBC plasma membranes but are engulfed by macrophages, where they fuse with lysosomes. Lysosomes have not been observed to join with phagosomes harboring single L. infantum parasites, whereas in phagosomes where the parasite has divided there is lysosome-specific fluorescence with a concomitant disappearance of lysosomes from the cytosol. In later stages, all the lysosome-specific label is found inside phagosomes whereas the phagosomal marker cadaverine strongly stains the macrophage nucleus, suggesting that L. infantum infection induces in its later stages nuclear degeneration and possibly, apoptosis of the host cell. These results indicate that induction of macrophage apoptosis should be explored as a possible strategy used by L. infantum to prepare its egress.

JTD Keywords: Leishmania infantum, Leishmaniasis, Liposomes, Malaria, Nanomedicine, Nanotechnology, Plasmodium falciparum, Quantum dots

Antimicrobial peptide drugs are increasingly attractive therapeutic agents as their roles in physiopathological processes are being unraveled and because the development of recombinant DNA technology has made them economically affordable in large amounts and high purity. However, due to lack of specificity regarding the target cells, difficulty in attaining them, or reduced half-lives, most current administration methods require high doses. On the other hand, reduced specificity of toxic drugs demands low concentrations to minimize undesirable side-effects, thus incurring the risk of having sublethal amounts which favour the appearance of resistant microbial strains. In this scenario, targeted delivery can fulfill the objective of achieving the intake of total quantities sufficiently low to be innocuous for the patient but that locally are high enough to be lethal for the infectious agent. One of the major advances in recent years has been the size reduction of drug carriers that have dimensions in the nanometer scale and thus are much smaller than -and capable of being internalized by- many types of cells. Among the different types of potential antimicrobial peptide-encapsulating structures reviewed here are liposomes, dendritic polymers, solid core nanoparticles, carbon nanotubes, and DNA cages. These nanoparticulate systems can be functionalized with a plethora of biomolecules providing specificity of binding to particular cell types or locations; as examples of these targeting elements we will present antibodies, DNA aptamers, cell-penetrating peptides, and carbohydrates. Multifunctional Trojan horse-like nanovessels can be engineered by choosing the adequate peptide content, encapsulating structure, and targeting moiety for each particular application.

JTD Keywords: Antibodies, Aptamers, Dendrimers, Liposomes, Nanomedicine, Nanoparticles, Nanovectors, Targeting

Current administration methods of antimalarial drugs deliver the free compound in the blood stream, where it can be unspecifically taken up by all cells, and not only by Plasmodium-infected red blood cells (pRBCs). Nanosized carriers have been receiving special attention with the aim of minimizing the side effects of malaria therapy by increasing drug bioavailability and selectivity. Liposome encapsulation has been assayed for the delivery of compounds against murine malaria, but there is a lack of cellular studies on the performance of targeted liposomes in specific cell recognition and on the efficacy of cargo delivery, and very little data on liposome-driven antimalarial drug targeting to human-infecting parasites. We have used fluorescence microscopy to assess in vitro the efficiency of liposomal nanocarriers for the targeted delivery of their contents to pRBCs. 200-nm liposomes loaded with quantum dots were covalently functionalized with oriented, specific half-antibodies against P. falciparum late form-infected pRBCs. In less than 90 min, liposomes dock to pRBC plasma membranes and release their cargo to the cell. 100.0% of late form-containing pRBCs and 0.0% of non-infected RBCs in P. falciparum cultures are recognized and permeated by the content of targeted immunoliposomes. Liposomes not functionalized with antibodies are also specifically directed to pRBCs, although with less affinity than immunoliposomes. In preliminary assays, the antimalarial drug chloroquine at a concentration of 2 nM, >= 10 times below its IC50 in solution, cleared 26.7 ± 1.8% of pRBCs when delivered inside targeted immunoliposomes.

JTD Keywords: Antimalarial chemotherapy, Chloroquine, Half-antibodies, Immunoliposomes, Malaria, Nanomedicine

Ligand-activated proteins can be controlled with light by means of synthetic photoisomerizable tethered ligands (PTLs). The application of PTLs to ligand-gated ion channels, including the nicotinic acetylcholine receptor and ionotropic glutamate receptors, is reviewed with emphasis on rational photoswitch design and the mechanisms of optical switching. Recently reported molecular dynamic methods allow simulation with high reliability of novel PTLs for any ligand-activated protein whose structure is known.

JTD Keywords: Nicotinic acetylcholine receptor, Kainate receptor, Glutamate receptor, Photoisomerizable tether ligand (PTL), Optical switch, Nanotoggle, Azobenzene, Neurobiology,, Nanoengineering, Nanomedicine