This review is conceived as a guide for material science researchers and conservators aiming to face the problem of deterioration of contemporary artworks entirely or partially made of plastics. It initially illustrates the analytical approaches for identifying polymeric material components in 3D art objects, such as sculptures and installations, and provides a perspective of their limits and advantages. Subsequently, the methodologies used for studying the deterioration of contemporary art plastics are reviewed, emphasising the main effects of the different types of degradation  and suggesting the appropriate techniques for their detection. Finally, the application of artificial ageing tests is critically assessed. All the concepts are elaborated through case studies and examples. Artists and designers have used materials produced for industrial purposes since the last part of the nineteenth century . EspeciaConcerning a more general presence of polymers in cultural heritage materials, polymeric components have been added to modern paints as binders since the 1930s and several studies have addressed their long-term stability ,5,6,7,8.In addition, plastics’ durability is an especially relevant requisite for collectability. To date, the majority of plastics collected, and hence potentially researched by material scientists, are design objects; however, knowledge advancement about objects with a greater artistic and economic value, such as sculptures, could have a higher impact on current societal concerns . First, Private and public collectors keep investing a considerable amount of money in contemporary art objects, and undoubtedly synthetic materials have a prominent role in the fabrication of such works. Artprice.com’s most recent report shows that in the last 20 years, the international market of contemporary art had an average growth of 1370% .Contemporary art is among the healthiest sectors of the modern economy, and even in times of global financial crisis, it has shown exponential growth. Therefore, heritage scientists are urged to fully understand synthetic polymers’ long-term behaviours in artworks to respond to inevitable upcoming losses, and upgrade their knowledge on emerging materials  . The repIn this review, the first research question relates to the analytical techniques used to identify and assess the durability of polymer components in artworks. The second research question aims at evaluating whether artificial ageing tests are the most efficient investigation method to predict artwork durability. Its overall aim is to critically present and discuss all the scientific literature about polymer analysis and degradation from a conservation science perspective but also be of general interest for a polymer scientist readership. The objectives are: (i) to summarise previous research about synthetic polymers used for the fabrication of contemporary art objects; (ii) to compare and contrast data-based research about polymer degradation behaviours; (iii) select the best methodologies of analysis and characterisation, and suggest further improvements.Trademarks, registration marks, and patent numbers are the preliminary data to collect on plastic objects. However, they are seldom available for artworks, apart from the cases in which artists used plastic objects instead of raw materials. On the other hand, visual features as transparency/opacity might be of aid in sorting plastics. Odour is also considered a preliminary tool for polymer identification if materials are already showing extensive deterioration ,19,24,25Instrumental analysis allows identifying and characterising the different plastic components in artefacts, comprising polymeric blends, and additives (including dyes and pigments). Further, additives may migrate from the polymeric material and degrade at different rates compared to bulk polymers, and being able to identify and quantify them has proven to be extremely informative . MoleculVibrational spectroscopies, especially infrared spectroscopy, are the techniques of choice for polymer structure determination . DiffereMore specifically, attenuated total reflectance (ATR) FTIR spectroscopy has successfully identified plastics and additives in collections since it provides reliable results with limited sampling. The use of polymers with common and well-defined structures, mostly vinyl polymers, has been unequivocally revealed, e.g., in the case of PMMA-made vacuum plastics by C. Kauffman , in the −1 were ascribed to talc, whereas the presence of C=O stretching bands (1710–1740 cm−1) could indicate the presence of plasticisers or the occurrence of products of polymer degradation, as discussed in the next review section.The recent availability of relatively inexpensive portable spectroscopy systems has also paved the way to fast and reliable identification of plastic components in museum collections, with particular attention on the systems that differ from those based on ATR-FTIR which do not require contact of the crystal with the object, exposing the artwork to the risk of visible indentation. Portable ATR coupled with NIR spectroscopy has been used for the identification of reference historical polymers , but it FTIR spectroscopy also offers invaluable information for identifying many condensation polymers through the recognition of the main functional groups. PUR, ER, UP and PA are sometimes challenging to unequivocally identify even by using comprehensive databases and applying a complementary analytical technique, such as Raman spectroscopy, py-GC/MS or nuclear magnetic resonance (NMR) spectroscopy may become necessary. That is, without forgetting that any of these techniques may be, and have indeed been, used alone for direct compositional analysis.−1 (C–O–CO stretching of esters) [−1 (C–O–C stretching of polyether polyol) [Due to their lightness and softness, PUR foams have attracted artists and designers since the 1960s. The fact that the two families of foams, i.e., poly(ester urethane) and poly(ether urethane), show different degradative pathways and a wide variety of possible compositions within each family makes their analysis a critical conservation issue. Although FTIR analysis easily detects the family of the PUR used in many contemporary artworks through the presence of characteristic main ester or ether peaks, at around 1120–1150 cm esters)  or 1050– polyol) ,43, resp polyol) . In the 1H MAS) NMR [1H-13C correlation experiments or total correlation spectroscopy (TOCSY) and heteronuclear multiple quantum coherence (HMQC) maps from solution, indicated the presence of diisocyanatotoluene constituting the hard phase of the PUR, and clarified the detailed microstructure of the poly(propylene oxide)-poly(ethylene oxide) copolymer.The formulation components of a reference poly(ester urethane) foam used for artefacts could also be revealed by a combination of FTIR and (py-)GC/MS, and confirmed by proton magic angle spinning (MAS) NMR . The py-MAS) NMR . Solid sOther materials with a wide compositional diversity are UP resins, used in contemporary art and design, because of their lightness and low cost. UP resins are prepared from a saturated dicarboxylic acid or its anhydride  and an unsaturated dicarboxylic acid or anhydride , which are reacted with one or more dialcohols, such as ethylene glycol or propylene glycol. Since the 1960s, it has been popular to use such resins to produce fibreglass-reinforced polymer composite artworks with a three-dimensional shape which often suffer problems of degradability, strictly related to chemical composition and manufacturing process of the polymer composite. An investigation preliminary to conservation treatment of a floating sculpture by M. Pan  very efficiently showed that the cured resin was pyrolysed into styrene, phthalic acid, short glycols and cinnamic acid. Most compounds relate to the original building blocks of the polymer, with styrene being the vinyl monomer used for the copolymerisation, and the cinnamic acid the linkage between the reactive double bonds (fumaric acid) and styrene . In otheConcerning PA, different artists have occasionally used these polymers since the 1960s, with a special mention for the early linear constructions by N. Gabo, and may be easily recognised by FTIR spectroscopy. Nevertheless, identifying their exact nature, which strongly influences their degradability and long-term behaviour, may require further characterisation through non-spectroscopic techniques. As examples, the linen used in Gabo’s sculpture showed DSC curves with the typical features of polyamide 6,6 , and alsIt is also worth citing a preliminary investigation by Necemer et al. aiming to access the capabilities of energy dispersive X-ray fluorescence spectrometry (EDXRF) with monochromatic excitation for the characterisation of the plastic materials used in artefacts from museum collections, e.g., PE, PP, PMMA, PET and PVC . A nondeFinally, The study of degradation mechanisms and stabilisation processes has been fundamental, since the Second World War with the introduction of new classes of synthetic polymers, and later in the 1950s and 1960s, when polymers became omnipresent in our daily life, in defining polymer uses and industrial applications. Researchers interested in fundamental properties of polymeric materials such as ageing performance or mechanical properties, including dimensional stability and shrinkage, may refer to any polymer encyclopedia or reference book on polymer degradation and stability . In addiOn the other hand, the interest in issues relating to the durability of modern and contemporary artworks, entirely or partially realised in synthetic polymers, is relatively recent. Only in recent decades have museums, conservators and heritage scientists focused their attention on the extent of the degradation of plastics in collections and the factors causing their breakdown ,30. It iPhysical factors of polymer deterioration that are especially important in heritage collections include migration of additives  and interaction with the surroundings, particularly with light and oxygen, in some cases quickened by mechanical stress due to specific uses or handling. Before moving on to reviewing the analytical methodology applied for studying any of these different factors, it is essential to stress the following, often misunderstood, points: (i) the problem of additive migration is particularly important only for objects/artworks made of/containing some specific polymer types, e.g., plasticised PVC or plasticised elastomers, where the plasticiser content may be relevant; (ii) ageing by oxidation, accelerated by the effect of light, heat, mechanical stress or secondary components is ubiquitous and occurs in all the organic materials, as well as under the mild and controlled conditions found in museums and art collections; (iii) the long-term process of elimination of small molecules from polymeric structures such as those of PVC and PVAc, i.e., by dehydrochlorination and deacetylation, respectively, forming chromophores, occurs spontaneously even at room temperature but is very structurally specific.A slow release over time of plasticisers from polymer bulk, such as the typical phthalates extensively used in PVC and many other polymers, causes the exudation of a sticky and oily film onto the artefact surface before complete evaporation takes place. Moisture and dust also accumulate on the films and may initiate further degradative processes. Other examples of migration are those related to the presence of lubricants, such as stearic acid. It is used up to 3% by weight in PVC and other polymers, and yellows and separates from the polymer, causing embrittlement of the plastic surface with time. Further, blooming is caused by salt residues derived from the exudation of flame retardants and those plasticisers that turn solid at room temperature .To extend the lifespan of plasticised polymers used in a variety of industrial products, extensive experimental research has been carried out to understand their thermal or photochemical degradation ,53, ofte−1 peak is characteristic of N-H stretching modes of a secondary amine while the bands at 1565 cm−1 could correspond to the N-H deformation mode and the 1636 cm−1 band to the carbonyl group of an amide. The advanced contact angle increases from 84 °C for the as-collected PVC to around 107 °C and simply emphasises the additive migration.Plasticised PVC films aged for about 30 years in museum conditions, and already showing surface exudates, were submitted to an artificial ageing test consisting in a temperature cycle (2 days at 80 °C then 1 day at 25 °C) under a relative humidity of 65%, and the physico-chemical changes, examined by weighing, FTIR and UV visible spectroscopies, GC/MS, dynamic mechanical analysis, contact angle measurements and other techniques. The effect of a preliminary mechanical surface cleaning was also studied. As expected, samples either freely hung or placed in a closed glass vessel displayed both a progressive loss of plasticiser , and a cAlthough further studies are necessary to extend the conclusions to other plasticised polymers, the study by Royaux et al. concludes that, at least for PVC, an efficient preservation strategy may pursue a longer conservation of properties, including visual appearance, while maintaining the artefacts in closed containers. The results also provide crucial information on applying treatments, removing additive surface accumulation to improve their visual aspect without any subsequent degradation.Another example of migration of plasticisers in contemporary artworks has been revealed in an installation by L. Cecchini, composed of many rubber objects. GC/MS identified the yellowish sticky liquid exuded from their surface as a mixture of phthalates contained in one of the components used to prepare the poly(ether urethane) elastomer, identified by FTIR spectroscopy . An artiAgeing of most artworks entirely or partially made of plastics is essentially due to oxidation through an auto-accelerating mechanism, where after a certain induction period with no significant changes, the degradation rate increases rapidly. Based on the well-known cycle of oxidation of hydrocarbons and, by extension, of polymers , a series of oxygen-containing species such as ketones, aldehydes, acids, esters and alcohols are formed. Such degradation products have functional groups that were not present in the pristine material; they may be chromophores and eventually have molecular weights far different from the initial ones due to either secondary cross-linking or scission reactions. Fragments may have molecular weight down to some thousands of Da  or even be volatile .If oxidation is especially important for vinyl polymers, the specific structural features of condensation polymers also make them suitable for hydrolysis, usually as a first degradation step. The hydrolytic degradation consists in the reaction of polymers with water, resulting in the cleavage of susceptible functional groups, commonly into chemical species that resemble the monomers used in the polycondensation synthesis. Polymers inherently susceptible to hydrolysis are those forming water during polymerisation, such as UP, PA and PUR, and also semi-synthetic polymers such as cellulose esters. To a lesser extent, ER and other thermosetting resins are also exposed to hydrolytic degradation.The formation of new functional groups as an effect of oxidation or hydrolysis is limited by oxygen or water diffusion, respectively, to the surface of the artworks and may be easily detected by direct methods such as different vibrational spectroscopy techniques, or indirectly through, e.g., colour detection, UV-vis or fluorescence spectroscopy , in some−1 and carbonyl absorption centered around 1720 cm−1 due to ketones, with shoulders at lower and higher wavenumbers. Additional information on the conservation state of the artwork was also revealed by py-GC/MS analysis, also shown in A representative example of applying basic analytical techniques to oxidation studies concerns the monitoring of artworks made of easily oxidisable polydiene elastomers, with natural PI being the most commonly used. The faster than expected  deterior13C solid state NMR added structural details. Both 13C distortionless enhancement by polarisation transfer (DEPTH) and especially cross-polarisation experiments, where the signals of the rigid components are amplified from the protons, show the presence of the aliphatic polyisoprene signals and many new peaks, indicating that there are several processes of chemical alteration responsible for the apparent degradation. New signals were identified as follows: (i) a minor signal peaking at 208 ppm corresponding to ketones and aldehydes, usually considered the product of the main product of the dominating process; (ii) a signal at 178 ppm corresponding to carboxylic acids; (iii) a signal at 105 ppm due to the formation of new double-bonded carbons; (iv) the dominating signal at 83 ppm with a side peak at 73 ppm, that constitutes 15% of all carbon in the quantitative DEPTH spectrum, corresponding to carbons with a single bond to oxygen, possibly generated through the formation of oxygen cross-links that would explain the hardening of the material.The effects of the oxidation on the same polymer but in another work of art were also investigated by two types of NMR , performAnother family of vinyl polymers, the so-called polyolefins, essentially consisting of PE and PP, are the leading industrial polymers for a wide range of commercial applications but were only rarely used by artists. Recently, the limited stability of a 50-year-old artwork partially made of low-density PE has been revealed by micro-FTIR analysis on diamond cells . SamplesAmong polycondensation polymers, it is well known that PUR, especially in the form of foams, may deteriorate in a few years as an effect of moisture and light, showing discolouration, loss of flexibility and crumbling. Studies on materials for industrial purposes demonstrate that poly(ester urethane) is more sensitive to hydrolysis while poly(ether urethane) is more prone to oxidation ,63. ConcMore detailed studies on the complex mechanism of degradation of PUR foams, consisting of parallel processes of photolysis, oxidation and hydrolysis, together with cross-linking of the hydrolysed fractions, required the use of techniques that enable the analysis of microstructures. Py-GC/MS in combination with EGA-MS of the material used in “Contenitoreumano” and “Moon Surface” by Turcato enabled not only the identification of the components discussed in the previous section but also suggested a change in the structure of the polymer as a result of 50 years of ageing ,44. SimiA different methodological approach that is worth discussing in detail is that applied to follow the deterioration due to hydrolysis in cellulose plastics, specifically tested in CN and CA sculptures realised by N. Gabo and A. Pevsner, and L. Moholy-Nagy and M. Duchamp, respectively , to addrIn all the cases in which in situ surface analysis techniques or invasive sampling may not be performed, the identification of chain scission products considered as markers of degradation provided a new powerful diagnostic tool. Curran and colleagues introduced VOC analysis to classify polymeric museum artefacts according to their degradation . Solid p−10 g, whereas the use of novel 3D SERS substrates [More recently, the combination of a nano-destructive sampling procedure with the surface enhanced Raman spectroscopy (SERS) sensitivity allowed for achieving the detection of non-volatile low molecular weight degradation products formed at the surface of reference polymers and was applied to follow the deterioration of PI-made artworks . Siliconbstrates  providesIn addition to assessing the current state of the conservation of artworks, possibly through the detection of the effects of degradation on the constituent materials, the prediction of their long-term behaviour plays an important role to ultimately address conservation and management strategy. It is usually assumed that the polymeric parts of museum objects are the more degradable interface with the environment and that their overall durability is directly related to the ageing of such critical components. A common material science practice to investigate polymers’ weathering, in times shorter than those necessary under natural conditions, consists in the exposure of reference samples to simulated oxidative conditions . Ageing sampled from the artwork;supplied directly by the artist as material remainders available in their studio;prepared from the commercial materials stated by the artist;selected among commercial materials similar to the one used in the artwork.Specimens for the assays may be:The effect of the accelerated ageing on polymer structure and molecular weight may be followed over the treatment by any spectroscopic, chromatographic or thermal analysis techniques, including those that are not usually applied to artworks due to limitations in terms of sample amount or surface area. In addition, the evolution of volatiles, weight and surface changes may also be monitored to follow the progress of degradation reactions.Illustrative examples of the procedure are represented by two studies of the degradative behaviour of different types of ER, namely, an aliphatic ER and an epoxy vinyl ester, used for surface finishing of sculptures realised in 2003 and 1998, respectively ,30,71. RIn the specific case of the ER, it has been supposed that the degradation takes place through the typical auto-oxidation process. The initial step consists of the abstraction of some labile hydrogen, e.g., the one adjacent to the amine group in the aliphatic ER , and thePhenomenological and mechanistic aspects concerning the behavior of the critical polymeric component could be corroborated through a comparison with the results from the evaluation of the actual state of the conservation of sculptures. ATR-FTIR spectroscopy alone provided a clear correspondence between artificial and natural ageing of both ERs, and also the formation of chromophores has been progressing quickly in a museum environment, as predicted. For example, the yellowing due to artificial photo-ageing of the epoxy vinyl ester is compared in In addition, other predictions on the durability of the following reference polymers could be compared with the natural ageing behaviour of other artworks: PI  , poly offers an alternative, often unique, option to enable identification. The further development and broader application of instruments actually in use for other cultural heritage objects and barely experimented on plastic artworks, such as handheld Raman spectroscopy , in situg  and weight loss are both excellent indicators to track the loss of plasticisers, while contact angle measurements are the best choice to follow the blooming of higher molecular weight additives. Visual inspection, UV-vis spectroscopy or colour measurements  should also always be carried out to follow surface deterioration, as colour changes due to the formation of very small amounts of chromophores must be considered as early markers of polymer degradation, and enable the investigation of oxidation/hydrolysis kinetics.When IR analysis does not unequivocally identify polymer structures  or functional groups resulting from degradation processes, other techniques must be applied, alone or in combination with vibrational spectroscopies. Py-GC/MS not only revealed the composition of different types of PUR foams, polyesters and many polyaddition polymers used by diverse artists over recent decades but also allowed the evaluation of the ageing of, e.g., PUR and PI artworks. NMR spectroscopy is a potentially excellent technique to detect structural changes in polymers and may be used as a diagnostic alternative to follow any type of degradation but has the limitation of requiring larger amounts of sample than py-GC/MS. Notwithstanding, several different NMR experiments already showed their potential for polymer oxidation studies, with a special mention for the first application of a noninvasive tool, i.e., unilateral NMR, to follow chemical processes in situ, through direct analysis of artworks. Changes of TAll these methods disclose the structural and molecular changes taking place due to ageing and permit the proposal of detailed degradation mechanisms. This knowledge, resulting from either the evaluation of the current state of conservation or through simulation tests of model polymers, is essential to predict further ageing and durability of artworks.With respect to future trends, further efforts are expected in the fine-tuning of the analytical procedures aiming to detect the low molecular weight products of degradation of artwork plastics, either in the form of VOCs or released as oligomeric compounds onto the surface. The development of easy-to-use museum kits to sample on site and instrumentally detect such products may be considered as the final objective of an innovative strategy to diagnose surface degradation in polymeric museum artefacts. In addition, the application of a reflectance hyperspectral imaging technique initially introduced to perform, at once, noninvasive diagnostics, analysis and documentation of artworks such as paintings and polychrome surfaces ,77 is al


Red blood cells (RBC) have great potential as drug delivery systems, capable of producing unprecedented changes in pharmacokinetics, pharmacodynamics, and immunogenicity. Despite this great potential and nearly 50 years of research, it is only recently that RBC-mediated drug delivery has begun to move out of the academic lab and into industrial drug development. RBC loading with drugs can be performed in several ways—either via encapsulation within the RBC or surface coupling, and either ex vivo or in vivo—depending on the intended application. In this review, we briefly summarize currently used technologies for RBC loading/coupling with an eye on how pharmacokinetics is impacted. Additionally, we provide a detailed description of key ADME  changes that would be expected for RBC-associated drugs and address unique features of RBC pharmacokinetics. As thorough understanding of pharmacokinetics is critical in successful translation to the clinic, we expect that this review will provide a jumping off point for further investigations into this area. The idea of using red blood cells (RBC) as carriers for drug delivery initially emerged about half a century ago as an approach to improve enzyme replacement therapy . HoweverNevertheless, approaches to use RBCs as carriers for pharmacological agents have recently gained significant and rapidly growing attention. Progress in this field is rapidly diversifying and accelerating towards potentially clinically useful products. Many groups are now investigating the use of RBCs in drug delivery and are making significant contributions, leading to breakthrough findings and upbeat investments. Several RBC-based drug delivery approaches have entered clinical trials, including RBC-encapsulated asparaginase  and dexamethasone . Novel advanced strategies are emerging, including genetic molecular modifications of RBC ,3, modulBoth encapsulation into and coupling to the surface of RBC fundamentally transform the key parameters of absorption, distribution, metabolism, and elimination (ADME) of drugs and drug delivery systems (DDS), including diverse nanocarriers. To our knowledge, studies of the pharmacokinetics (PK) and pharmacodynamics (PD) of RBC-based DDS are lacking, despite great relevance for industrial development and clinical utility.In order to help to close this gap of knowledge, in this paper we undertook the first attempt to define specific, salient parameters controlling behavior of RBC/DDS in the body. Our goal is to provide the modular framework for experimental and theoretical pre-clinical and clinical investigations of ADME-PK-PD features of RBC-based drug delivery.Loading drugs into the carrier RBC at the present time can be achieved only in isolated RBCs. The most advanced approach involves osmotic swelling, causing transient pores in the RBC membrane (see below). Novel experimental approaches include attempts to use cell-penetrating peptides to import therapeutic proteins in the carrier RBC  and fusiDrug encapsulation into RBCs for use in humans is currently achieved either in vitro or ex vivo using either autologous blood or matching donor blood as a source for RBCs. Washed RBCs are loaded with drugs via transient pores formed in the membrane of RBC during osmotic swelling in hypotonic buffer containing a high concentration of drugs, with subsequent washing with an excess amount of drug ,11. NotaThere are several clinical trials utilizing RBC-based drug delivery systems , pursuinEncapsulation into the inner volume of RBC (A) limits interaction of the drug retained in the RBC ghost with compounds that do not diffuse through the plasma membrane; (B) helps to protect drug cargo from the body and vice versa; and (C) involves the minor fraction of RBC in blood, since this procedure takes place ex vivo, followed by the infusion of loaded RBC ghosts.In the original prototypes, surface loading was achieved by chemical conjugation of cargoes to isolated RBCs ex vivo ,14. NeweIn several advanced iterations, drugs and carriers can be conjugated to or fused with antibodies, antibody fragments, peptides, or other ligands that bind to the RBC surface ,16,17,18Drug delivery systems based on surface loading can find utility for the prolongation and redistribution of agents that are supposed to work in the bloodstream and other sites accessible for RBCs , includiIn addition, RBC drug delivery systems can be surface-modified to confer specific affinity to therapeutic sites of interest, including those of vascular injury ,14,23,24Surface RBC loading is pursued for many drugs including anti-thrombotic and anti-inflammatory agents . For exaBoth encapsulation into and surface-loading onto RBCs may modulate the response of immune and innate host defense system to biotherapeutic agents (see below). Both methods dramatically alter the PK of the drug cargoes.Drug loading into RBCs physically separates cargoes from the body. Surface coupling to a large RBC carrier markedly limits the ability of drugs to interact with cognate receptors and other counterparts . This miTherefore, drug loading to carrier RBCs profoundly alters their pharmacological profile, in some cases enabling effects simply unavailable to free drugs. It is tempting to postulate that RBCs may also significantly and favorably modulate the delivery and therapeutic effects of nanocarriers, among other artificial drug delivery systems, and these interactions with nanocarriers are detailed below.RBCs have also been membrane-engineered to reduce RBC antigen immunogenicity. The ability to camouflage allogenic erythrocytes and create a universal RBC, in theory, would minimize the risk of potentially life-threatening hemolytic transfusion reactions as well as shortages of matched blood groups. Various polymer types, such as polyethylene glycol (PEG) and hyperbranched polyglycerol, have been covalently conjugated to the RBC surface ,48. For Given the utility of polymers in RBC membrane engineering, polymers have also been employed for therapeutic surface loading. In a recent work, PEG was used to engineer immunoglobin-based therapeutics to the RBC surface . The sysApart from the different advantages of RBCs mentioned above, RBCs offer unique opportunities from an engineering standpoint. First, mature RBCs are enucleated, allowing the attachment of immunomodulatory drugs, which are known to play a role in switching phenotypes, without altering physiological functions of the RBC or worsening disease pathology. This feature provides an edge over other malleable cell-based therapies such as macrophages, which can lead to disease progression due to switched phenotypes. Second, RBCs (and RBC-associated drugs) have access to every tissue in the body. Tissue-specific targeting can be achieved by optimizing a known set of parameters and the site of injection. Moreover, the versatile biomechanical properties of an RBC allow for squeezing through blood vessels of a diameter smaller than itself, creating the opportunity to target vascular endothelia of high-density capillary beds. Lastly, the ability of RBCs to negotiate immune cell clearance in the liver and spleen, until they are senescent, offers a different pathway for presenting antigens to the immune cells in these organs ,54. ThisOne such approach is to physically adsorb nanocarriers onto the surface of RBCs (RBC hitchhiking) ,6,58. BiRBC hitchhiking has been shown to significantly alter the PK profile of attached nanocarriers ,58. In p+ HPCs collected from a healthy O negative donor are genetically engineered to express one or more biotherapeutic proteins inside cells or on cell surface. These engineered cells are expanded, differentiated, and enucleated in a bioreactor to obtain the final RBCs carrying therapeutics. RBCs generated by this approach seem to have many unique properties. For example, RBCs with enzymes expressed inside cells show promise in treating metabolic diseases such as phenylketonuria . In addition, RBCs with proteins expressed on cell surfaces can have specific interactions with diverse arms of the immune system, depending on the properties of the surface-expressed proteins. For example, RBCs carrying a specific set of surface proteins can activate T cells or NK cells, which are being investigated for therapy of solid tumors and leukemia [Another emerging approach towards creating RBC DDS is to manufacture RBCs carrying therapeutics via genetic engineering of hematopoietic precursor cells (HPCs) . In thisleukemia ,62,63. TPreviously, genetic engineering of RBCs has been employed to coat RBC membranes with viral snares. RBCs lack the key machinery necessary for viral gene expression, making them impervious to infection. In a study on coxsackievirus B, investigators engineered murine RBCs to express the virus’s target receptor protein, enabling entrapment of the virus inside the cell . Mice exCmax), time of Cmax (tmax), and bioavailability (F)  . As extrVss)  tissue perfusion, (2) diffusion, (3) protein/cell binding, (4) active transport, and (5) vascular permeability. Tissue perfusion is largely governed by blood flow to the tissue. In Following the entry of the drug into the systemic circulation, the drug is able to move between blood and tissues, in a process termed distribution. The primary metric that is used to describe the extent of drug distribution is the volume of distribution at steady state (Vss) . It shouThe removal of an active drug substance from the circulation can occur either as direct elimination of the unchanged drug or via metabolism of the drug molecule and subsequent elimination of the metabolite. The primary metric used to describe the efficiency of elimination is clearance (CL), which describes the volume from which drug is removed per unit time. While there is no theoretical lower bound on clearance, the upper limit to this parameter is the sum of blood flows to the eliminating organs. A summary of key routes of elimination for a variety of drug classes is provided in Proper characterization and understanding of PK properties of drug candidates is critical for effective drug development. In the early 1990s, poor understanding of pharmacokinetics early in development resulted in PK being the most common reason for attrition (40%) during clinical trials; however, by the end of the decade, this had fallen to less than 10% of failures, as early, thorough characterization of PK became more common . AdditioRegardless of the method of attachment of drugs to carrier erythrocytes, proper analysis of PK is complicated by the number of potential analytes. Complete characterization of PK would include measurements of RBC-associated drug, released drug (plasma or serum), and survival of the infused RBCs. As summarized in As highlighted above , PK expeImmediately after injection of RBC-associated drugs, the drug will be exclusively localized within the cellular component of blood A. This iMost small molecule and protein therapeutics are eliminated from the circulation with half-lives of minutes to hours, necessitating frequent dosing. While this may not be a significant concern for drugs with good bioavailability and/or wide therapeutic indices, this is often a limiting factor in efficacy. Approaches to extend the circulation time and to improve targeted delivery of therapeutics, such as encapsulation in liposomes (hours–days) and conjugation to mAbs (days–weeks), have been applied clinically with some success ,109. HowAs summarized in 51Cr labeling).Thorough analysis of PK of RBC-associated drugs requires information describing the blood and tissue PK of the RBC-associated drug, released drug, and carrier RBCs. While this degree of characterization may seem daunting, in reality it only requires two distinct bioassays, typically liquid chromatography-mass spectroscopy for small molecule drugs and a method to trace infused RBCs , or antibody binding to RBC membrane proteins ,111,112.ted RBCs .2 fragment, the mAb caused more rapid elimination of coated RBCs, likely due to the presence of the Fc fragment [However, it should be noted that the relative potency of a given coupling strategy on RBC circulation will likely be highly dependent on the method of coupling. For example, at equivalent loading doses of Ter119 mAb and its (Fab)fragment . This sefragment . This sut1/2 ~ minutes) to RBCs via affinity binding [Increasing the affinity for target epitopes is generally expected to increase residence time at the target in the absence of confounding factors , largely due to reductions in the rate of dissociation from the target. Due to their lack of endocytic capacity and extremely long circulation time relative to most  drugs, RBCs present an attractive system to study this phenomenon. It is clear that providing a molecule that previously had negligible affinity for RBCs with nanomolar or better affinity for an RBC membrane protein is capable of generating remarkable improvements in PK. Our group has previously demonstrated that coupling plasminogen activators  onto the surface of RBCs ex vivo are taken up rapidly and in large numbers in the first vascular bed encountered following injection  ,6,7,115.A key advantage to using affinity ligands to attach a therapeutic to the surface of RBCs is the potential for in vivo loading, as compared to covalent conjugation and internal loading, which require ex vivo manipulation of RBCs and reinfusion. The loading of drugs in vivo could greatly reduce the possibility of damaging RBCs, leading to rapid elimination or severe toxicities related to hemolysis. It is important to consider how the location of loading could affect PK of carrier RBCs and associated drugs.Ex vivo manipulation and loading of RBCs, followed by reinfusion into patients, will result in the entire injected dose being present on a small fraction of total RBCs in the bloodstream. On the other hand, in vivo loading allows tuning the degree of RBC loading by adjusting the infusion rate. For example, if the dose is administered very quickly, as an IV bolus, loading will be similar as what would be expected for ex vivo loading. However, a very slow IV infusion, over minutes to hours, would provide a relatively lower load on each individual RBC, but a more homogeneous distribution across the entire RBC population, potentially leading to fewer adverse effects on the RBCs, and longer circulation times .For almost 50 years, academic researchers have been intrigued by the potential of using autologous RBC as a drug delivery system for a myriad of therapeutics. The initial concept of loading enzymes into the RBC has been extended and is now applied to a diverse range of loading strategies  and therapeutic modalities . Attachment of drugs to the RBC can lead to unprecedented changes in circulation time, biodistribution, elimination pathways, pharmacodynamics, and immunogenicity. Therapeutics that are attached to the surface of the RBC are able to directly interact with and transfer to the endothelium, particularly within capillaries where the RBC is deformed and squeezes through the vessel. Recent advances have allowed this field to move from an academic curiosity into a potentially clinically used strategy. These include approaches for rapid ex vivo loading of RBC, development of new affinity ligands for coupling to membrane proteins, and genetic engineering of the RBC. Despite these significant technological advances, there is a clear gap of knowledge in many areas related to the PK/BD of RBC-associated drugs that we hope to begin to close through careful investigation of the unique aspects of RBC PK that we have described.


Hypercoagulability and thrombosis remain a challenge to diagnose and treat in severe COVID-19 infection. The ability of conventional global coagulation tests to accurately reflect in vivo hypo- or hypercoagulability is questioned. The currently available evidence suggests that markedly increased D-dimers can be used in identifying COVID-19 patients who may need intensive care unit (ICU) admission and close monitoring or not. Viscoelastic methods (VMs), like thromboelastography (TEG) and rotational thromboelastometry (ROTEM), estimate the dynamics of blood coagulation. The evaluation of coagulopathy by VMs in severe COVID-19 infection seems an increasingly attractive option. Available evidence supports that COVID-19 patients with acute respiratory failure suffer from severe hypercoagulability rather than consumptive coagulopathy often associated with fibrinolysis shutdown. However, the variability in definitions of both the procoagulant profile and the clinical outcome assessment, in parallel with the small sample sizes in most of these studies, do not allow the establishment of a clear association between the hypercoagulable state and thrombotic events. VMs can effectively provide insight into the pathophysiology of coagulopathy, detecting the presence of hypercoagulability in critically ill COVID-19 patients. However, it remains unknown whether the degree of coagulopathy can be used in order to predict the outcome, establish a diagnosis or guide anticoagulant therapy. Critically ill coronavirus disease 2019 (COVID-19) patients are at risk of developing not only hypoxia and excessive inflammation but, also, frequent thrombotic manifestations [In sepsis, the disturbance between components of the coagulation and fibrinolytic system leads to a variable clinical picture, tilting from an initial hypercoagulability towards a subsequent hypocoagulable disease state, depending on the phase of septic coagulopathy . BleedinCOVID-19-related proinflammatory cytokines induce an endothelial injury, leading to primary hemostasis activation and the overexpression of the tissue factor . The resThe recommended management of COVID-19 coagulopathy is based on the currently available evidence that markedly increased D-dimer levels can be used as a predictor of the development of ARDS, the need for admission in intensive care units (ICU) and death. Increased D-dimer levels in COVID-19 patients have been attributed to excess thrombin generation secondary to endothelial activation induced by the infectious stimuli, severe hypoxemia and local pulmonary microthrombosis .In this context, the International Society of Thrombosis and Haemostasis (ISTH) recommends measuring the D-dimers, prothrombin time and platelet count (decreasing order of importance) in all patients who present with COVID-19 infection [The ability of conventional global coagulation tests to reflect in vivo hypo- or hypercoagulability accurately is questioned , as thesAn electronic search on the PubMed and Scopus databases was carried out for studies published in 2019 and 2020. Our search strategy combined terms or keywords that are related to COVID-19 infection  with those related to the viscoelastic methods  for the identification of relevant articles. The reference lists of the extracted articles were also pursued for relevant studies that could be further included in the review. An inclusion and exclusion methodology was formed. Articles concerning the application of viscoelastic methods in the management of patients with COVID-19 infection were included. Exclusion criteria included studies published in a language other than English, studies with no full text available and case reports. A limitation of this study is that, given that this review is a narrative review and not a systematic review or meta-analysis, it lacks a quantitative data synthesis of the results of the included studies. However, even though this is not a systematic review, to the best of our knowledge, almost all of the prominent relevant studies have been included in this narrative review. Additionally, given the great heterogeneity of the included studies, the results of such a meta-analysis would not be of great value for the readers. In a future time, a meta-analysis of published studies with more consistent methodology would be more valuable.Both TEG and TEM assess clot formation/lysis kinetics and clot strength by measuring the amount of a continuously applied rotational force, transmitted by the developing clot to an electromechanical transduction system. TEG assesses the viscoelastic properties of clotting native whole blood upon the activation of hemostasis by exogenously added triggers (kaolin). The following variables are measured: R (minutes), which is a measure of the clotting time from the point of coagulation ignition to the appearance of the clot, K (minutes) and K angle, which define the velocity of the clot formation, MA (mm), which defines the maximal amplitude of the clot and Lys-30, which defines the percentage decrease of the clot amplitude at 30 min post-MA.2+, phospholipids and ellagic acid to activate and assess coagulation through the intrinsic pathway. The tissue factor (TF) used in EXTEM assay activates and assesses the extrinsic pathway, while FIBTEM assay is applied to differentiate between thrombocytopenia and fibrin polymerization. The following parameters are assessed: the clotting time , determined as the time elapsed from the start of the measurement until the formation of a clot 2 mm in amplitude; clot formation time  as the time elapsed from the end of the CT (amplitude of 2 mm) until a clot firmness of 20 mm is achieved; the amplitude recorded at 10, 20 and 30 min ; α-angle, which is the angle between the central line (x-axis) and the tangent of the TEM tracing at the amplitude point of 2 mm, describing the kinetics of clot formation; maximum clot firmness , which reflects the final strength of the clot; lysis index at 60 minutes (LI60), defined as the percentage of remaining clot stability in relation to the MCF following the 60-min observation period after CT and indicating the speed of fibrinolysis, and maximum lysis (ML), which reflects the percent decrease of the maximal amplitude over time.A ROTEM analysis includes intrinsically-activated test using ellagic acid (INTEM), extrinsically-activated test with tissue factor (EXTEM) and fibrin-based extrinsically activated test with tissue factor and the platelet inhibitor cytochalasin D (FIBTEM) assays. The INTEM assay uses CaCurrently, coagulation point-of-care viscoelastic tests are increasingly used to characterize the coagulation profile of these patients. All of them have reported a procoagulant profile on ICU admission in the COVID-19 patients with ARDS, as indicated by the detection of increased clot strength. The findings and design characteristics of all relevant studies are presented in Panigada et al. were the first who noted decreased or increased TEG variables in 24 COVID-19 ICU patients by comparing them with the mean of the respective reference ranges previously established in 40 healthy adult subjects . The R aDespite the ongoing debate regarding the degree that COVID-19 behaves differently than other well-studied critical illnesses, it is probably reasonable to assume that COVID-19 affects the pathways of inflammation and coagulation perhaps in a different scale and time frame. Therefore, established laboratory markers of deregulated inflammation and coagulation are unsurprisingly affected by this new infectious disease.The main interactions between coagulation and inflammation involve the procoagulant, anticoagulant and fibrinolytic pathways. Regarding adult sepsis, in most relevant studies, VMs showed sepsis-induced coagulopathy, but changes in coagulation parameters were heterogeneous, with patients showing both hyper- and hypocoagulability . VariabiHowever, this sequence of events is not always the case. For instance, using VMs, a clear hypocoagulable profile was noted at the early phase of neonatal sepsis ,33 assocAccordingly, the pathophysiology for COVID-19-related systemic microthrombosis seems to be specific and, in particular, different from DIC ,7.In-line with fibrinolysis shutdown, often reported in COVID-19 critically ill patients, hypofibrinolysis has also been demonstrated in several studies in adult sepsis ,27,35,36Based on these data, it can be assumed that the type of coagulopathy and the relevant clinical symptoms appearing, due to the interaction between inflammation and coagulation, are closely dependent on the intensity and duration of inflammatory stimuli, the previous hemostatic balance and the timing of the measurements. VMs have the capacity of assessing this delicate equilibrium between procoagulant, anticoagulant and fibrinolytic factors, revealing the coagulation disturbances that might lead either to bleeding or thrombotic disorders. However, they are considered inappropriate to assess each hemostatic component individually and independently . Based oThe following principle has to be considered when performing VMs: Thromboelastometric tests are not designed to predict thrombotic or bleeding events but only to help determine the cause of bleeding . Thus, bAll these limitations are also in place regarding VM measurements in the severe COVID-19 infection. Characteristically, in almost all relevant studies, different criteria were used to define the hypercoagulability. Furthermore, the corresponding parameters of each assay probably were not equally affected by the administration of several anticoagulant treatments. Thus, these results are difficult to be standardized and used in order to “quantify” the hypercoagulability and define the prognosis or optimal treatment strategy. Given the background, and keeping in mind that VMs provide overall information on coagulation and fibrinolysis equilibrium, it would rather not be a prudent approach to apply an antiplatelet or fibrinolytic or enhanced anticoagulant therapy to treat hypercoagulability, based exclusively on the VM findings and without the use of specific assays to evaluate certain hemostatic components. Considering these characteristics and limitations of VMs, the results of these assays should be evaluated in a particular context. They can effectively provide insight into the pathophysiology of coagulopathy, detecting the presence of hypercoagulability in critically ill COVID-19 patients. However, the type and degree of coagulopathy might be inadequate in order to predict the outcome, establish a diagnosis or guide an antigoagulant therapy. To achieve these goals, much more needs to be done. Studies with adequate sample sizes to establish reference ranges for each viscoelastic test or multicenter study properly designed to define the common cut-off values of hypo- and hypercoagulability or threshold values to predict prognosis are necessary. Finally, regarding the utility of VMs in order to guide anticoagulant, antiplatelet or fibrinolytic therapy, the parallel use of other specific assays evaluating the hemostatic components affected by the corresponding treatment, in association with the clinical outcome, should be considered a prerequisite before their application in clinical practice.


Arthritis, including osteoarthritis (OA) and rheumatoid arthritis (RA), is the leading cause of years lived with disability (YLD) worldwide. Although pain is the cardinal symptom of arthritis, which is directly related to function and quality of life, the elucidation of the mechanism underlying the pathogenesis of pain in arthritis has lagged behind other areas, such as inflammation control and regulation of autoimmunity. The lack of therapeutics for optimal pain management is partially responsible for the current epidemic of opioid and narcotic abuse. Recent advances in animal experimentation and molecular biology have led to significant progress in our understanding of arthritis pain. Despite the inherent problems in the extrapolation of data gained from animal pain studies to arthritis in human patients, the critical assessment of molecular mediators and translational studies would help to define the relevance of novel therapeutic targets for the treatment of arthritis pain. This review discusses biological and molecular mechanisms underlying the pathogenesis of arthritis pain determined in animal models of OA and RA, along with the methodologies used. Musculoskeletal diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA), are the leading causes of years lived with disability (YLD) worldwide. Furthermore, YLD due to OA increased by 31.5% from 2006 to 2016, in association with the aging of the population . SimilarRadiographic changes in OA are poorly correlated with pain and physical function, and the risk factors for radiographic OA are not the same as those for OA pain ,10,11. AIn addition to nociceptive mechanisms arising from structural changes, recent evidence indicates that neurophysiological pathways contribute to OA pain, such that persistent tissue damage and inflammation in the joint induce mechanisms of peripheral and central sensitization . In a stAlthough a variety of OA animal models have been developed, there is no single model that can replicate all aspects of human OA. The most commonly used animal models employ surgical or chemical induction . SurgicaThere are many approaches for assessing the behaviors associated with OA pain in animal models . The mosIt is notable that, until recently, the most commonly used induction methods to study OA pain were distinct from those most often used to investigate the pathophysiology and regulation of structural joint damage . DMM andIn animal models, as in humans, the extent of joint damage and pain are not always correlated . For exaOA animal models differ in terms of the inflammatory response evoked in the synovium. In general, chemical-induced models tend to exhibit more inflammation in the synovium compared to surgical models. As synovitis is observed before the thinning of the articular cartilage or subchondral bone changes in the MIA model, the abovementioned augmentation of pain behavior in the MIA model may reflect the influence of inflammation on pain . The colSubchondral changes in bone are common in most OA animal models. A recent study showed that osteoclast-initiated subchondral bone remodeling plays an important role in the generation of pain in ACLT-induced animal OA . Netrin-Obesity has been identified as a key risk factor for the development and progression of OA in numerous epidemiological studies in different ethnic populations. Three studies evaluated the relationship between obesity and OA development via the inhibition of transient receptor potential cation channel, subfamily V, member 4 (TRPV-4), lecithin-cholesterol acyltransferase (Lcat), apolipoprotein A1 (Apoa1), and low-density lipoprotein receptor (Ldlr) in mice fed with a high-fat diet , Western-type diet  or cholesterol-rich diet ,110,111.Age is another strong risk factor for OA in human patients. Although it varies by strain and sex, some inbred strains of mice, such as STR/ort and C57BL/6, spontaneously develop OA with age, and spontaneous joint damage does develop in the majority of mouse strains >12 months of age . AlthougAlthough female sex is a risk factor for both OA and OA-associated pain, female mice have been infrequently used for the study of OA , with onCompared to OA, which is considered a type of noninflammatory arthritis, research endeavors in RA have focused on the regulation of abnormal immune responses and inflammation, with pain considered a byproduct of inflammation that would be controlled by reducing disease activity. Although biological disease-modifying anti-rheumatic drugs (DMARDs) have revolutionized disease control and long-term outcomes of RA, substantial numbers of patients still suffer from pain despite low disease activity. The use of biological DMARDs has provided new insights into the unique qualities of pain manifestations independent of inflammation in RA . The bloThere has been some speculation on the mechanism underlying persistent pain despite the absence of inflammation. It may result from irreversible joint damage and from changes in the CNS processing signals from the joint . CentralConditioned pain modulation, a measure of inhibitory pathways leading to a diffuse decrease in pain in response to acutely painful stimuli, is impaired in RA patients, and this association is mediated by sleep problems . PatientAnimal models of RA are divided into immunization and transfer models . The forExperimental methods for assessing pain behaviors in RA models are largely similar to those used for OA models, although there are discrepancies in response patterns. In addition, because RA models generally exhibit polyarthritis, modalities useful for measuring asymmetric joint pathology sometimes cannot be used for RA models. Compared to OA, RA models exhibit a greater degree of joint inflammation, which is reflected in their pain behaviors. Mechanical allodynia measured with von Frey fiber and thermal hypersensitivity measured with hot plate response time tend to appear earlier in RA models compared to OA models. While static weight bearing is useful for pain assessment in OA models, because of its polyarticular nature, it is not appropriate for assessment of many RA models. It is notable that in human TNF-Tg (hTNF-Tg) mice, gait parameters measured using catwalk analyses have revealed the association between gait abnormalities and the extent of cartilage damage and bone erosions, but not with the extent of synovitis . These oBoth the CIA and CAIA models clearly exhibit mechanical allodynia and thermal hypersensitivity in the early phase of arthritis . While mCompared to research on the pathogenesis of aberrant immunity and inflammation, the mechanism of pain in RA animal models has been explored only recently .Although pain behavior largely parallels the development of inflammation, as in human patients, it is not always concordant with the degree of inflammation. As in humans, pain outlasts inflammation in monophasic RA models induced with antibodies. In the K/BxN serum transfer arthritis model, mechanical allodynia develops congruent with joint swelling and persists after the resolution of inflammation . A transInflammation and suppression of pain-related spontaneous behaviors, such as locomotor activity, operant responses, and place avoidance, are strongly correlated in rapid-onset models of arthritis . HoweverRecent advances in animal experimentation and molecular biology have led to significant progress in our understanding of the mechanisms underlying the pathogenesis of arthritis pain. The concept that pain is a mere byproduct of joint damage and nociceptive response has become outdated. Aside from DAMPs produced from joint destruction, various nociceptive players, such as NGF- TrkA axis, CGRP, TRPV-1, and SASP, have been proposed as molecular mediators of OA pain. For RA, pain independent of inflammation has been correlated with ATF-3, TLR-4, and FcγR1. With the advances in cutting edge molecular techniques such as single cell polymerase chain reaction (PCR) and CRISPR/Cas9 mediated gene regulation, the knowledge base for pain pathogenesis would be significantly and rapidly expanded. Despite the inherent difficulty in extrapolating data gained in animal pain behavior studies to human arthritis, the critical assessment of various molecular mediators and translational studies would help to define the biological relevance of novel therapeutic targets for the treatment of arthritis pain.


Monoclonal antibodies have evolved from research tools to powerful therapeutics in the past 30 years. Clinical success rates of antibodies have exceeded expectations, resulting in heavy investment in biologics discovery and development in addition to traditional small molecules across the industry. However, protein therapeutics cannot drug targets intracellularly and are limited to soluble and cell-surface antigens. Tremendous strides have been made in antibody discovery, protein engineering, formulation, and delivery devices. These advances continue to push the boundaries of biologics to enable antibody conjugates to take advantage of the target specificity and long half-life from an antibody, while delivering highly potent small molecule drugs. While the “magic bullet” concept produced the first wave of antibody conjugates, these entities were met with limited clinical success. This review summarizes the advances and challenges in the field to date with emphasis on antibody conjugation, linker-payload chemistry, novel payload classes, absorption, distribution, metabolism, and excretion (ADME), and product developability. We discuss lessons learned in the development of oncology antibody conjugates and look towards future innovations enabling other therapeutic indications. With  in 1986,® . In. In51]. Linker-payload design is also critical to the successful implementation of high-DAR ADCs, via reduced aggregation and improved overall pharmacokinetics profiles. Even though increasing DAR instinctively increases in vitro potency of ADCs, it may not translate to an improvement of in vivo potency since the plasma clearance of ADCs rises along with DAR . The agg® (gemtuzumab ozogamicin) which is composed of a CD33 mAb linked to the cytotoxic drug calicheamicin via an acid-liable hydrazine linker. While initially approved for the treatment of acute myeloid leukemia (AML) in 2000, it was pulled from the market at the request of regulatory agencies in 2010 due to safety concerns and the failure to reproduce the clinical benefit connected to linker stability in AML patients. Following additional interrogation of exposure-response relationships, examining alternative lower dosing and scheduling, Mylotarg found a path back to the market and received a new FDA approval for newly diagnosed CD-33 positive acute AML patients in 2017.As discussed in the sections above, considerable advancements in next generation ADCs are anticipated to further explore both the chemical and biological design elements for ADCs. This includes, but is not limited to, antibody engineering to facilitate direct site-specific conjugation, modification of conjugation chemistry and introduction of novel linker compositions to confer enhanced stability, as well as, the exploration of additional existing and novel chemical entities/modalities as conjugates to increase the pharmacological applications of ADCs. Many of these advancements to design better molecules are intricately interdependent with optimizing or improving the ADME drug-ability properties of ADCs, such as linker-payload stability, distribution, and pharmacokinetics (PK). This is because defining the exposure-response relationship for both safety and efficacy has been intimately tied with ADC peripheral PK, target tissue or site of action concentration and the disposition of the payload at the intended site. The criticality of understanding the exposure-response relationship and therapeutic index (TI) for ADCs is exemplified by MylotargDissecting the ADME properties of ADCs is a complex endeavor given the unique properties of each component within the molecules. ADC ADME involves delineating the intertwined properties of linker-payload stability, pharmacokinetics, clearance, metabolism, and disposition of mAb, conjugate , as well as, the ADC entity itself. The ADME properties of ADCs are influenced by the mAb, target antigen, linker, site of conjugation, DAR number, and the conjugated species . The antibody component of ADCs is the primary driver of the slow clearance, long systemic half-life and restricted tissue distribution of these modalities compared to their payload counterparts. Similar to mAbs, the properties related to target , as well as, antibody structure  that affect antibody PK and disposition also impact ADCs. An anti-drug antibody (ADA) or neutralizing antibody (Nab) response against the therapeutic antibody component can affect the PK profile and shorten the half-life of the ADCs in the body ,19. NeveIn addition to the antibody, the linker and payload components of ADCs are also subject to their own clearance mechanisms. The stability of the linker to premature release of the payload in the systemic circulation has been demonstrated to be a critical ADC ADME component for determining the exposure-response and exposure-toxicity relationships . From a Like mAbs, ADCs are likely trafficked via the vascular and lymphatic systems. The biodistribution of ADCs follows that of the antibody component. ADC are removed from the systemic circulation by target-mediated drug disposition (TMDD); thus, highly vascularized organs or tissues that express the target antigen are involved in the clearance of ADCs from the periphery. The TMDD is believed to be followed by intracellular trafficking of the target: ADC complex to lysosomes where degradation of the ADC occurs, and the payload is released from the mAb to elicit its activity. In addition, nonspecific uptake of ADCs by pinocytosis also facilitates their systemic depletion. This form of uptake could lead to degradation and/or recycling of the ADC by the neonatal Fc receptor (FcRn). Indeed, in terms of tissue distribution the preponderance of ADCs are observed in four organs including the liver, kidneys, lungs, and skin ,146; howThe elimination of ADCs involves two processes. First, intracellular catabolism through proteolysis in the tissues . Second, complete deconjugation of the payload which can result in both mAb or mAb with a partial linker along with free drug . While tAnother area of intense research is dissecting the noted phenomenon of resistance against ADCs. A few mechanisms of resistance have been noted including to the antibody portion of the ADCs by mutation and/or down-regulation of the target antigen, as well as, to the payload via drug efflux transporters that remove the payload from cells . ChangesAn antibody conjugate combines an inherently complex antibody with a small synthetic molecule drug to create an even more complex large molecule. Despite the relatively small addition in molecular weight, the small molecule drug has a profound impact on the characteristics and properties of the conjugate.Over the last decade, significant advancements in analytical methods have been made to characterize ADCs and have been extensively reviewed ,153,154.It is well-documented that the addition of a small molecule drug to an otherwise soluble and stable antibody can cause aggregation and other physicochemical instability in the ADC ,163. ThiIn addition to all the issues encountered during antibody formulation development, the formulation development of ADC drugs must find suitable pH and excipient conditions to simultaneously maintain the stability of the antibody, the linker, and the small molecule drug . EvAll the current ADC drugs on the market are lyophilized products suitable for intravenous administration. Such a freeze-dried state protects the ADCs from chemical degradation and aggregation which occur under long-term solution storage conditions. Although interest has been growing for liquid formulation based on the increased experience with ADCs and the improved solubility and stability of the new generation of ADCs, few such feasibility studies have been reported in the literature to date. It can be particularly challenging to prevent payloads from falling off the antibody over a long period of time in solution. In addition, the currently approved ADC drugs are reconstituted to 0.25–20 mg/mL in solution, significantly below the concentrations for most therapeutic antibody products. While the above is a viable approach for intravenous administration commonly used for oncology therapies, the emerging non-oncology application of the ADCs will likely demand stable liquid formulation and subcutaneous administration as commonly expected for many antibody drugs to increase convenience for patients. This growing trend exerts pressure on linker-payload design and conjugation methods in addition to the properties of the parental antibodies, as well as on formulation and device development. The current pre-clinical data for non-oncology ADCs suggest that the ADC doses might not be significantly lower than those for antibodies ,45,47,52Antibody conjugates in oncology have thus far delivered several successfully approved therapeutics. Extensive research into novel payloads, more developable linkers and conjugation chemistries further enable the field of oncology conjugates to cross the finish line. These learnings and advances also help propel the next generation of conjugates for non-oncology indications. Additional challenges such as in vivo stability, formulation and delivery will drive the field to seek solutions to broaden the therapeutic horizon to include payloads like nucleic acids. Overall, the rise of non-oncology ADC therapeutics offers a huge opportunity for innovation at multiple fronts of drug discovery and development for years to come.


A vast amount of mobile apps have been developed during the past few months in an attempt to “flatten the curve” of the increasing number of COVID-19 cases.This systematic review aims to shed light into studies found in the scientific literature that have used and evaluated mobile apps for the prevention, management, treatment, or follow-up of COVID-19.We searched the bibliographic databases Global Literature on Coronavirus Disease, PubMed, and Scopus to identify papers focusing on mobile apps for COVID-19 that show evidence of their real-life use and have been developed involving clinical professionals in their design or validation.Mobile apps have been implemented for training, information sharing, risk assessment, self-management of symptoms, contact tracing, home monitoring, and decision making, rapidly offering effective and usable tools for managing the COVID-19 pandemic.Mobile apps are considered to be a valuable tool for citizens, health professionals, and decision makers in facing critical challenges imposed by the pandemic, such as reducing the burden on hospitals, providing access to credible information, tracking the symptoms and mental health of individuals, and discovering new predictors. The COVID-19 outbreak, which first emerged in China, has spread worldwide. On March 11, 2020, the World Health Organization (WHO) declared COVID-19 as a pandemic [Although mobile apps are successfully used for managing chronic diseases ,4, the oDespite increasing reliance on mobile health  solutions as part of COVID-19–related response plans, major knowledge gaps exist about their utility and efficacy during the current pandemic for both health professionals as well as for the general population. To this direction, this systematic review aims to shed light into studies found in the scientific literature on the use and evaluation of mobile apps for the prevention, management, treatment, or follow-up of COVID-19.Other recent reviews have focused merely on the exploration of COVID-19 mobile apps in app stores in general  or were The bibliographic databases of PubMed and Scopus, along with the global research database on COVID-19 developed by the WHO , were seIn this context, the inclusion criteria for study selection were the following: features of the COVID-19 mobile app should be described, the study should show evidence of the implementation of the COVID-19 mobile app in real life and provide quantitative outcomes, the study should show that clinical professionals were involved in the design or validation of the mobile app, the paper describing the study must have been written in English. Case reports, letters to editors, preprint papers, qualitative studies, surveys or reviews, simulation studies, and studies describing protocols were excluded from the review.The string “ OR  OR (smartphone) OR (mobile phone) OR (mobile application) OR (mobile app) OR (app) AND (COVID-19),” was used for a search within the title, abstract, and keywords of the manuscripts. Authors HK, AT, AK, FL, DGK, and IK independently screened the identified papers to minimize possible errors and bias in the selection process. Any disagreements were resolved by discussion between the authors to reach consensus. The authors first screened the abstracts of the candidate papers for inclusion and assessed their eligibility according to the defined inclusion and exclusion criteria. Moreover, the authors selected the final papers for inclusion after reading the full manuscripts of the eligible papers, as well as their references.The Effective Public Health Practice Project (EPHPP) tool was adopted to assess the methodological quality of the included studies. The EPHPP tool is suitable for evaluating quantitative studies in a wide range of health-related topics, and it has demonstrated reliability . The incOur last search in June 2020 returned 165 manuscripts from the PubMed database, 188 manuscripts from the Scopus database, and another 375 manuscripts from the WHO research database. All the retrieved records were imported in the Zotero bibliography management software (Center for History and New Media at George Mason University), which identified 252 duplicates. We screened the abstracts of the remaining 476 papers according to our inclusion and exclusion criteria, and 22 papers were found to be eligible. After reading the full text of the papers, the authors agreed to include 12 papers. The screening procedure along with reasons for excluding papers are shown in the PRISMA flow diagram in On the basis of the EPHPP criteria for selection bias, design, confounders, blinding, data collection, and dropouts, we found the methodological quality to be moderate for 2 of the 12 (17%) studies ,24 and wIn the following sections, we provide a comparison of the included studies, in terms of intervention target and main features, study design, and outcome and clinical implications.The reviewed papers varied in intervention target and main features . One papHome monitoring was handled based on telephone outreach , smart vstudy design, almost all selected studies were proof of concept, observational studies, or both, aiming to evaluate the practical use, usability, or user satisfaction with the corresponding apps that were developed to support individuals, health care providers, and policy makers during the COVID-19 pandemic , 3 out of the 12 selected studies [Concerning the  studies ,25,27, d studies  reported studies  reported studies  reported studies  reported studies  reported studies  reported studies  reported studies  mentioneOutcome assessments were varied across studies. An overview is shown in In most of the cases, app users were satisfied with the educational and risk assessment information provided in the mobile apps , despite the short time period covered (December 2019 to June 2020), thereby showing the high interest of the scientific community in the research of mobile apps for COVID-19.Our main finding is that, despite that the current research evidence is fragmented and requires greater methodological rigor, mobile apps have been found to benefit citizens, health professionals, and decision makers in facing the COVID-19 pandemic. In particular, mobile apps can help in solving several COVID-19–related challenges by increasing the reach of reliable information to both citizens and health professionals, decreasing misinformation and confusion, tracking symptoms and mental health of citizens, home monitoring and isolation, discovering new predictors, optimizing health care resource allocation, and reducing the burden of hospitals.The participants in the studies were mainly young and middle-aged adults. Further studies are needed that will involve older participants, who are in greater risk of developing serious complications due to COVID-19. Understanding the needs of older individuals in the COVID-19 pandemic period would be the first necessary step toward designing and developing mobile apps to encourage their physical and mental well-being .Our review, in contrast to other reviews that have not examined the evaluation of COVID-19 mobile apps in pragmatic studies , identifThe implications for clinical practice for each one of the discussed works is shown in detail in Timmers et al eHealth mobile apps for COVID-19 that support several functionalities such as tracking of symptoms and provision of accurate and timely information as well as self-assessment can be implemented in a short time to be used by individuals.Those apps provide valuable information to both governments and health care providers, since they support monitoring of patient health status and provide summary statistics regarding the progress of health.Such apps could be used in future outbreaks of other viruses to support all involved stakeholders.Yamamoto et al Such apps could be used in future outbreaks of other viruses to support all involved stakeholders.Zamberg et al Mobile health  apps could help solve some of the COVID-19 challenges by providing more accurate and timely information to health care professionals. This benefit is sourced by the centralized management and storage of main, up-to-date, validated, and easily accessed information in one platform.mHealth apps used in health care organizations as communication tools of validated information should be assessed in the context of clinical studies, with regards to their impact on clinical outcomes.Kodali et al According to users, the app should be enhanced with additional functionalities including tracking of location, provision of up-to-date information on COVID-19 as well as information on areas with high/medium/low epidemiological burden, and deployment in nonmobile platforms.Huckins et al According to the findings, since the beginning of the COVID-19 pandemic, there has been an extended negative impact both on the physical health of individuals, resulting in an increased number of deaths, and in their mental health, resulting in changes in their behavior.Drew et al The research of a broader range of potential risk factors for COVID-19 results will be largely supported by the use of the app within several large epidemiology cohorts for which there are a large amount of data on lifestyle, diet and health factors, and genetic information.It will be useful to deploy the tool in several clinical studies, in centralized actions related to biobanking, and in health care worker monitoring programs.Medina et al The intervention impacts several organizational matters and provides answers regarding the following questions related to COVID-19:Which situations should we focus on to provide effective care to prevent patient admission to intensive care unit and mechanical support?How can those situations be better supported by increased patient self-reporting?Can we predict, based on time data, future inpatient demand?How can we manage not having conflicts between patient choice and availability of treatment?Menni et al Routine screening for COVID-19 could also include loss of sense of taste and smell. These two symptoms should also be included in the related symptom list provided by the World Health Organization.Ros and Neuwirth The implementation and deployment of the described digital tutorials, as an effective and swift global public health educational tool, help alleviate the burden that hospitals, health care professionals/responders, and patients face due to the COVID-19 pandemic.Bae et al Episode triage, timely diagnosis, isolation of patients, and their treatment can be largely supported by telemedicine solutions.Ben Hassen et al Mobile apps can be used by patients, their caregivers, and health care professionals to better monitor and manage patient health status in the context of patient hospitalization. Moreover, such apps are cost-effective, reliable, and safe, providing important economic benefits to hospitals. Overall, they are accepted to a great extent by patients and individuals.Bourdon et al The use of telemedicine tools for consultation purposes improved the access to health care services for patients with ophthalmological problems. At the same time, such tools preserved social distancing and sanitary measures.Such tools can be used in emergency situations by ophthalmological patients who have limited access to specialized care.As reported in the textbox, eHealth solutions can be implemented rapidly and can offer essential tools in supporting the COVID-19 pandemic for all stakeholders including citizens, health care providers, policy makers, and governments. The impact of the COVID-19 pandemic goes beyond the illness and deaths that are directly associated with the SARS-CoV-2 pathogen and toward an expanded scope encompassing mental health and several behavioral changes.However, there are still several areas to explore, such as better geolocation tracking; timely COVID-19 updates; deployment on nonsmartphone platforms; better incidence visualization and prediction; investigation of a much broader range of putative risk factors for COVID-19 outcomes ; and the integration with clinical systems, digital health literacy, and engagement with mobile apps.Apps such as the ones presented in these studies can give the research community the opportunity to monitor long term effects of COVID-19; answer questions such as the number of people truly affected, why some people get sicker than others, and how long people can stay immune to the disease; and perform better resource allocation of medical equipment.In case of future disease outbreaks, the mobile apps already developed for COVID-19 can be valuable tools ready to support people, health care providers, and policy makers. This study can provide a guide for future developers and researchers regarding the current methodological gaps and challenges that need to be addressed to develop well-designed and evaluated apps for similar future circumstances.We used terms related to mobile apps, mHealth, and mobile phones for our literature search according to the review objectives and did not use related terms such as “telehealth,” which might have resulted in an inadvertent omission of studies. Our literature search was conducted in a limited number of bibliographic databases , which nevertheless have been largely used worldwide. The gray literature was not explored. The interrater reliability between the authors was not assessed. A meta-analysis was not possible due to the heterogeneity of the included studies. For many cases, the quality of the study might not reflect the quality or the effectiveness of the developed mobile app. As such, useful and effective apps might have not been included in this review study due to the limited quality of the related studies.


Nakalanga syndrome is a clinical manifestation of onchocerciasis-associated epilepsy characterized by stunting, delayed or absent secondary sexual development and skeletal deformities, and is often accompanied by epileptic seizures. The pathophysiology of Nakalanga syndrome is unknown. Here, we describe the post-mortem findings of a 17-year-old female who died with Nakalanga syndrome in northern Uganda. Macroscopic and histopathological examination of all major organs , including the brain and the pituitary gland, was performed. The suspected cause of death was malaria, and all major organs and pituitary gland appeared normal, except the lungs, which were edematous consistent with the malaria. Neuropathological changes include signs of neuro-inflammation (gliosis and activated microglia), which co-localized with tau-reactive neurofibrillary tangles and threads. The pathology was most abundant in the frontal cortex, thalamic and hypothalamic regions, and mesencephalon. The choroid plexus showed psammoma bodies. These findings indicate accelerated aging, probably due to repeated seizures. The neuropathological findings were similar to other persons who died with onchocerciasis-associated epilepsy. Examination of the pituitary gland did not reveal new information concerning the underlying pathophysiological mechanism of Nakalanga syndrome. Therefore, more post-mortem studies should be performed. The Nakalanga syndrome has been reported in many onchocerciasis-endemic regions, including South America , Uganda In this case report, we describe the post-mortem findings of a 17-year-old female with Nakalanga syndrome who died during an acute febrile illness in northern Uganda. A complete post-mortem exam was performed as before . SamplesThe deceased belonged to the Acholi tribe and lived in the Pader district of northern Uganda. She had an older sister who suffered from nodding syndrome. The deceased had head nodding seizures induced by the sight of food or by cold weather which started in 2005, at the age of 4 years. Several years later, the disease progressed to generalized tonic–clonic seizures and episodes of blank staring (absences) and wandering away from home were reported. There was no history of any severe disease, such as severe malaria or febrile illness, head trauma or abnormal development before the onset of the seizures, which could explain the development of seizures and the disease. Despite treatment with sodium valproate, carbamazepine and folic acid, three epileptic episodes weekly  were reported, as well as an episode of status epilepticus. She did not present onchocerciasis skin lesions or nodules, but she took ivermectin treatment during the bi-annual mass distributions in the region. Prior to death, the deceased was unable to perform normal household tasks, had a poor appetite and sometimes needed assistance with eating. She was very weak but able to walk without assistance, dress, wash and groom herself and was not bedridden. Around the time of death, she developed fever, headache and cough and was started on antimalarial treatment after a positive rapid diagnostic test. However, her clinical condition deteriorated and she was admitted to the hospital where she died three days later. External examination showed general wasting, with reduced subcutaneous fat and muscle mass, poor secondary sexual development, crippled legs, kyphosis and stunting (short stature). There was severe pallor, dehydration, mild jaundice and anemia but no palpable peripheral lymph nodes. Internal organs were normal, but smaller than expected for her age. The lungs were heavy and edematous with thick mucus secretions in the parenchyma and petechiae on the serous surfaces, which may have been related to the malaria infection prior to death . Examination of the visceral organs  showed no abnormalities. Histological examination of the brain showed neuronal loss, especially the Purkinje cells and granular cells in the tops of the folia in the cerebellum. Spongiosis and an increased amount of glial cells were observed in the sub-pial layer at the top of the gyri of the left and right frontal cortex. Large neuronal bodies with inclusions, pseudo-inclusions and vacuolization were observed in the paraventricular nuclei on the right side A. The naThe pathophysiology of Nakalanga syndrome remains unknown. Two main characteristics of the syndrome include stunted growth and delayed or absence of secondary sexual development, pointing towards an endocrinological cause. Endocrinological investigations were performed earlier in eight persons with nodding syndrome in Uganda . Here, sIn this case report, macroscopic investigation of the brain of the 17-year-old woman did not show any abnormalities; microscopic investigation of the brain revealed overlapping foci of tau-reactive neurofibrillary tangles, gliosis and activated microglia, as was previously observed in persons with OAE , furtherO. volvulus microfilariae [One major drawback of post-mortem studies is that the disease is in its end stage, and factors underlying the pathological mechanism might no longer be present. However, the normal appearance of the pituitary gland in this case report and an earlier post-mortem study suggest that Nakalanga syndrome may not be caused by a structural dysfunction or lesion of the pituitary gland. Our post-mortem examination of the brain and pituitary gland did not provide further indication for the underlying pathophysiological mechanism. In agreement with Marshal and Cherry (1962), there was no evidence supporting an earlier suggestion that pituitary dysfunction in Nakalanga syndrome is the result of accumulating filariae . Additiofilariae  informat


Backgrounds: Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique for the treatment of several psychiatric disorders, e.g., mood disorders and schizophrenia. Therapeutic effects of tDCS are suggested to be produced by bi-directional changes in cortical activities, i.e., increased/decreased cortical excitability via anodal/cathodal stimulation. Although tDCS provides a promising approach for the treatment of psychiatric disorders, its neurobiological mechanisms remain to be explored.Objectives: To review recent findings from neurophysiological, chemical, and brain-network studies, and consider how tDCS ameliorates psychiatric conditions.Findings: Enhancement of excitatory synaptic transmissions through anodal tDCS stimulation is likely to facilitate glutamate transmission and suppress gamma-aminobutyric acid transmission in the cortex. On the other hand, it positively or negatively modulates the activities of dopamine, serotonin, and acetylcholine transmissions in the central nervous system. These neural events by tDCS may change the balance between excitatory and inhibitory inputs. Specifically, multi-session tDCS is thought to promote/regulate information processing efficiency in the cerebral cortical circuit, which induces long-term potentiation (LTP) by synthesizing various proteins.Conclusions: This review will help understand putative mechanisms underlying the clinical benefits of tDCS from the perspective of neurotransmitters, network dynamics, intracellular events, and related modalities of the brain function. Transcranial direct current stimulation (tDCS) is a non-invasive method that modulates neural activities in the brain by delivering low-amplitude  over a short period (no more than 30 min) between electrodes (anode and cathode). At least, one of the electrodes is placed on the scalp, through which electronic currents penetrate the skull to enter the brain and facilitate or inhibit spontaneous neural activities in the vicinity of electrodes  has been reported  by itself is not strong enough to depolarize the membrane potential of neurons to the firing threshold, and only increases the rate of spontaneous combustion and their excitability   and NMDA (N-methyl-D-aspartate) receptors, both of which are coupled with ion-channels. The AMPA receptor is involved in the intracellular influx of sodium ion during neuronal depolarization, causes transient action potentials, and accounts for most of the excitatory synaptic transmissions. On the other hand, the NMDA receptor is involved in the intracellular influx of calcium ion during depolarization, produces prolonged action potentials, and mediates neural circuits governing memory and learning. Therefore, actions on NMDA receptors, inducing plasticity of neurons, play a dominant role in improving symptoms of psychiatric disorders. Accordingly, dextromethorphan (NMDA receptor inhibitor) suppresses the effect of anodal stimulation , continuous enhancement of signal transduction between neurons, is thought to mediate the effect of tDCS . First, without significant change in BDNF concentrations in the blood of patients with major depressive disorder  may also mediate the development of LTP  binds to BDNF promoter I, and recruits of CREB-binding proteins. These findings suggest that anodal tDCS increases hippocampal LTP and memory via mechanisms related to BDNF genes (Podda et al., BDNF binds to TrkB receptors that regulate the growth and synaptic activity of neurons, and are thought to be involved in the formation of LTP Stephen, . For exaIn this review, we discussed the electrophysiological understanding of tDCS on the basis of the stimulation-dependent model. Biochemically, enhancement of excitatory synaptic transmissions through anodal stimulation is likely to facilitate glutamate transmission and suppress gamma-aminobutyric acid transmission in the cortex. Accordingly, tDCS may positively or negatively regulate dopamine, serotonin, and acetylcholine transmissions. These neural events may change the balance between excitatory and inhibitory inputs. In this way, anodal stimulation may modulate activity levels of multiple network systems.LTP may also provide putative mechanisms underlying the ability of tDCS to treat psychiatric disorders. Future studies should consider other domains of symptoms of psychiatric conditions of schizophrenia and mood disorders, e.g., social cognition and meta-cognition (Nishida et al., The current review is narrative and the articles were not systematically searched. Moreover, many of the papers presented in this review targeted healthy individuals rather than those with psychiatric disorders. It should be noted that patients with mental illnesses might respond differently to tDCS from healthy people. Furthermore, some articles included in this review were on the effect of tDCS over motor cortex. Further study is warranted to examine evidence for stimulation on prefrontal cortex that has been the main target of psychiatric disorders, such as depression and schizophrenia (Mezger et al., YY and TS planned and designed the review, made substantial contributions, and approved the final manuscript. YY collected the data and drafted the first manuscript. TS critically reviewed the draft and revised it. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.