Delivered online, this tool presented each challenge to participants. Participants had up to four response options at each turn of the conversation; the choice of response influenced the subsequent conversation, including coaching provided at the resolution of the situation. Participants were invited to complete pre-activity, immediate post-activity, and one-month follow-up questionnaires to assess satisfaction, self-efficacy, engagement, and knowledge change with the tool. Knowledge was assessed through situational judgment tests (SJTs). (3) Results: Thirty-two pharmacist preceptors participated. The frequency of participants reflecting on challenging learning situations increased significantly one-month post-simulation. Participants affirmatively responded that the tool was time-efficient, represented similar challenges they encountered in precepting, was easily navigable, and resulted in learning. Self-efficacy with skills in managing challenging learning situations increased significantly immediately post-simulation and at a one-month follow-up. Knowledge as measured through SJTs was not significantly changed. (4) Conclusions: Preceptors found an interactive narrative simulation a relevant, time-efficient approach for preceptor development for challenging non-academic learning situations. Post-simulation, preceptors more frequently reflected on challenging learning situations, implying behavior change. Self-efficacy and self-report of knowledge increased. Future research is needed regarding knowledge assessments. Health professions educators continually seek new and effective methods to engage preceptors. Preceptors, when surveyed, show greater preference for online versus live training due to flexibility, while self-study is valued due to accessibility ,2. ConsiSimulation through virtual role-playing represents an attractive research area for preceptor development due to its accessibility, interactivity, adaptability, and availability in self-study formats . SimulatVirtual or screen-based simulation has also been studied for other uses—such as for social interaction, change management, stress management, and job interview skills—due to its abilities to offer repetitive practice, individualization, immediate feedback, and an interpersonal safe environment ,20,21,22Screen-based simulation also allows for recreating situations that may be difficult or infeasible in person due to discomfort with participating in role play, inability to attend live training, or availability of facilitators. This capability could potentially be used to train preceptors for challenging non-academic learning situations such as professionalism concerns. Given preceptors’ level of involvement in pharmacy curricula, it is expected that preceptors may frequently encounter such situations ,25,26. AThis proof-of-concept study evaluated narrative-based simulation using a web-based tool designed to prepare preceptors for managing challenging non-academic learning situations. The simulation included three situations that preceptors may encounter in practice: professionalism, cross-cultural interactions, and student well-being. The research aims were to determine if simulation is an effective method for increasing preceptor knowledge, behavior, and self-efficacy, and to gauge level of preceptor engagement and satisfaction with simulation.Three challenging situations were created based on topics frequently offered in preceptor development programs, situations reported to the authors’ School, and topic requests from School preceptors . ChallenScripts were implemented using the ink editor from Inkle Studios . This narrative tool enables rapid writing and testing of branching dialog. In prior studies ,46, diffA convenience sample of participants was obtained by sending an email request for voluntary participation to 1275 preceptors at one school of pharmacy. Each participant had an individual session with the tool and research team member, either in person or via Zoom . Sessions were audio-recorded. Participants were asked to think aloud as they read descriptions and selected among options, providing a steady flow of detail of their observations and reasoning . The thrThree Kirkpatrick levels of training evaluation were considered in the outcome assessments: reaction, learning, and behavior . All part-tests when a sufficient sample size and normality of distribution was warranted, otherwise Wilcoxon signed rank tests were utilized, with significance set at 0.05, entries removed that had missing values, and Bonferroni correction for multiple tests. Qualitative data were analyzed using informal methods with patterns sought among participant responses to open-ended questions. The study was reviewed and deemed exempt by the School’s Institutional Review Board.The immediate post-activity questionnaire asked participants to rate satisfaction with the simulation, then through open-ended questions sought features liked most and least about the tool, suggestions for improvement, and desired additional resources. Participants were given the same self-efficacy, perceived behaviors, and knowledge questions. The one-month follow-up questionnaire included questions on perceived precepting behavior with challenging learning situations, whether coaching from the simulation had been used, and resources still needed. Participants also rated their self-efficacy, described behaviors, and completed the knowledge questions. The self-efficacy instrument was designed, using a five-point scale anchored by ‘strongly disagree’ and ‘strongly agree’, in accordance with recognized design principles and reviewed by education research and pharmacy practice experts before administration . DescripThirty-two preceptors were recruited to test the simulation tool . Of thesp < 0.04). Participants’ beliefs in their ability to successfully address challenging situations also changed, using the same test, from pre-activity to one-month post . Forty-two percent (N = 10) in the one-month post-activity survey indicated they had used strategies from the tool.Percentages of participants responding that they addressed tardiness, cross-cultural interactions, or mental health with students at least monthly prior to the simulation were 53% (N = 17), 50% (N = 16), and 38% (N = 12), respectively. Over their careers, 66% (N = 21), 53% (N = 17), and 53% (N = 17) indicated they had addressed these concerns. On a question asking about preceptor behavior related to regularly reflecting on challenging situations of all kinds with learners, 66% (N = 21) answered in the affirmative (agreeing or strongly agreeing) prior to the simulation experience while 80% (N = 20) answered so one-month after participation; a signed rank test comparing paired pre-activity and post-activity responses indicated that post-activity responses were significantly higher than pre-activity responses  of participants answered positively about time-efficiency and 92% (N = 22) felt navigation was easy. All agreed or strongly agreed that the experience was applicable to their practice, and 79% (N = 19) noted the challenges were similar to situations they experienced. Nearly all  claimed to have learned from using the tool. Open-ended positive comments included realism of challenges, ability to replay and explore dialog progression based on different response options, reflection on reactions, coaching provided at each challenge’s resolution, and engagement and ease of use. Constructive comments included need for a back button , desire to pinpoint which response selection(s) drove feedback , and some vagueness in response options. Forty-two percent (N = 10) of participants disagreed or strongly disagreed that there were right answers to the scenarios in the simulation, debating if just one answer fit, that response options were vague, or how certain answers were not considered “ideal”. Several participants requested additional challenging situations focused on good communication skills and social cues. One participant suggested using the tool to create virtual “rounds” for preceptors to share interactions within the situations and learn from each other how to manage the student. Additional resources requested included discussion with a School mentor, discussion with colleagues, continuing education programs on challenging situations, and handouts outlining resources.p > 0.12; see Moreover, participant self-efficacy significantly increased from pre-activity to immediate post-activity  and mainThis is one of the first studies to assess the effectiveness of web-based narrative simulation for preceptor development. Its strengths are in measuring preceptor satisfaction, self-reported behaviors, self-efficacy, and knowledge pre-post simulation. The simulation had a positive effect on preceptor self-efficacy and self-perception of knowledge but not on knowledge as measured by SJTs. Overall, most participants found the tool useful and the scenarios realistic; issues seemed to surround the sometimes-vague response options.Preceptors’ responses to their frequency of addressing challenging non-academic learning situations  indicated challenges selected for the simulation were relevant. Preceptors agreed these challenges were similar to situations they had experienced and applicable to their practice, indicating a higher prevalence of students requiring intervention than has been cited in the literature ,25,26. AOne-month after simulation, preceptors more frequently reflected on challenging learning situations, implying behavior change. Preceptors’ belief in their ability to successfully manage challenging learning situations increased after the simulation. Self-efficacy measures increased for challenging situations with learners having general professionalism, cultural awareness, and mental health concerns. Preceptors also believed the tool was time-efficient and easily navigable, an important consideration given time constraints that preceptors face ,53.Literature is presently lacking in assessment of preceptor self-efficacy with web-based simulation. Satisfaction data are also relatively lacking. A comparison of three simulation modalities  in Master of Pharmacy students found similar satisfaction with all modalities . Though Most preceptors reported learning using the web-based simulation tool. However, learning was not demonstrated in any systematic changes to their response prioritizations compared to expert response prioritizations. Participants and experts did not always agree on correct responses to the simulation scenarios nor the knowledge situations in the SJTs. Problem solution may be idiosyncratic; preceptors may learn strategies how to address a challenge, but each individual does so in their own way based on situational constraints. While the dynamic simulation allowed for variability in responses to complex situations, SJTs with a pre-selected best response may not have and therefore may not be optimal for assessing knowledge change for these types of situations. It is also possible that these preceptors were a self-selected group already knowledgeable in managing challenging situations, or that response options were too unclear in how they should be prioritized. Further investigation of the knowledge testing is warranted. There is evidence of learning using role play simulation for nurse educator development when comparing open-ended knowledge questions related to course objectives pre- and post-simulation . ParticiThis pilot study has several limitations. First, it was conducted with a small number of preceptors at one school of pharmacy to assess proof-of-concept; therefore, caution should be used when applying to other populations of preceptors who may have different experiences. Second, preceptors agreeing to participate may have opted in because they were open to using computer-based technology for preceptor development, perhaps causing bias in favor of simulation. Third, knowledge change assessed through SJTs did not show systematic effect. This form of assessment, in addition, was static, in contrast to the dynamic simulation. In addition, it is unclear if preceptors’ access to other training and resources after simulation could have impacted their self-efficacy. Further study is needed to confirm findings in larger populations of preceptors and determine best practices around use of simulation for training of preceptors in dealing with challenging situations and testing for knowledge change.Preceptors responded favorably to an interactive narrative simulation and demonstrated sustained increased self-efficacy with skills related to managing challenging non-academic learning situations from before the simulation to after. Preceptors reported an increase in reflecting on challenging situations. A test of change of knowledge from before to after the simulation did not show systematic effects when comparing participants’ prioritized response options to a SJT to a separate group of experts’ prioritizations. Future work with additional situations, preceptors, and formal knowledge assessments is warranted.


COVID-19 affects children to a lesser extent than adults but they can still get infected and transmit SARS-CoV-2 to their contacts. Field deployable non-invasive sensitive diagnostic techniques are needed to evaluate the infectivity dynamics of SARS-CoV-2 in pediatric populations and guide public health interventions, particularly if this population is not fully vaccinated. We evaluated the utility of high-throughput Luminex assays to quantify saliva IgM, IgA and IgG antibodies against five SARS-CoV-2 spike (S) and nucleocapsid (N) antigens in a contacts and infectivity longitudinal study in 122 individuals (52 children and 70 adults). We compared saliva versus serum/plasma samples in infected children and adults diagnosed by weekly RT-PCR over 35 days (n=62), and those who consistently tested negative over the same follow up period (n=60), in the Summer of 2020 in Barcelona, Spain. Saliva antibody levels in SARS-CoV-2 RT-PCR positive individuals were significantly higher than in negative individuals and correlated with those measured in sera/plasmas. Asymptomatic infected individuals had higher levels of anti-S IgG than symptomatic individuals, suggesting a protective anti-disease role for antibodies. Higher anti-S IgG and IgM levels in serum/plasma and saliva, respectively, in infected children compared to infected adults could also be related to stronger clinical immunity in them. Among infected children, males had higher levels of saliva IgG to N and RBD than females. Despite overall correlation, individual clustering analysis suggested that responses that may not be detected in blood could be patent in saliva, and vice versa.In conclusion, measurement of SARS-CoV-2-specific saliva antibodies should be considered as a complementary non-invasive assay to serum/plasma to determine COVID-19 prevalence and transmission in pediatric populations before and after vaccination campaigns. Since the start of the COVID-19 pandemic, it has become apparent that pediatric populations are less affected than adult or older populations , althougIn this context, a question of high interest has been whether children who become infected might be less efficient transmitters to their immediate contacts , as thisThe presence of the virus in nasal or nasopharyngeal samples can be detected with real time polymerase chain reaction (RT-PCR) and antigen-based sensitive methods, while prior exposure has to be assessed by detecting antibodies to SARS-CoV-2 antigens. The latter is usually done by measuring specific immunoglobulins in serum or plasma samples, which requires obtaining blood samples by venous or capillary punctures, less amenable to large field studies. Several commercial rapid diagnostic tests (RDT) exist that are useful as point of care kits, but they may be limited by their sensitivity and specificity in the case of asymptomatic infections, which usually induce lower levels of antibodies , 12. A nBeing a respiratory pathogen, the mucosal immunity has a key importance, and thus the role of IgA in controlling the virus is becoming increasingly important . TherefoIn this study we adapted and evaluated three Luminex-based antibody assays to quantify the levels of IgM, IgA and IgG against several SARS-CoV-2 antigens in saliva samples, and compared them to the levels of antibodies obtained using serum/plasma samples from the same individuals who had a positive or negative RT-PCR diagnosis over a five week follow up period. We tested the applicability of the Luminex saliva assays in children and adult volunteers, having different demographic characteristics and clinical presentations, participating in a contacts and infectivity study after the first peak of the COVID-19 pandemic in the Summer of 2020 in Barcelona.via three recruitment pathways: (i) active surveillance in 22 Summer schools, as previously reported  independently of the presence of COVID-19 compatible symptoms and subjects with negative nasopharyngeal RT-PCR  and seroreported , (ii) pareported . Blood s® instrument. Crude median fluorescent intensities (MFI) and background fluorescence from blank wells were exported using the xPONENT software.Quantitative suspension array technology (qSAT) assays to measure IgM, IgA and IgG against SARS-CoV-2 were adapted from our previous standardized serum/plasma protocols  to a sal10MFI) of each antibody/antigen pair between study groups. Radar charts were used to compare median log10MFI of all antibodies together between study groups by Mann-Whitney U test. Heatmaps with hierarchical clustering (Euclidean method) were used to evaluate patterns of responses at the individual level depending on clinical and demographic variables. Due to the unavailability of pre-pandemic saliva samples, we explored calculating seropositivity cutoffs by the mean plus 3 standard deviations (SD) of pandemic negative samples for serum/plasma and for saliva samples  and non-infected (n=60) individuals in whom saliva and/or serum/plasma samples were analyzed, are shown in 10MFI) of IgM, IgA and IgG antibodies to five SARS-CoV-2 antigens in saliva and serum/plasma samples from RT-PCR positive and negative individuals. IgG levels to all antigens in saliva were statistically significantly higher (p<0.05 for N CT and p<0.001 for the rest) in RT-PCR positive than negative individuals  , and significantly higher saliva levels of IgM to RBD and S than adults (p<0.05) Figure 2Among RT-PCR positive individuals, IgG levels to RBD and S were significantly higher in serum/plasma from asymptomatic than symptomatic subjects (p<0.01)  and depended on the Ig isotype and antigen pair. These variable correlations would indicate different dynamics of antibody responses in blood versus mucosal tissues , 27, 28.We examined potential reasons for less discrimination between infected and non-infected subjects in saliva than in serum/plasma samples. On one hand, low antibody levels in some infected individuals could be due to non-responsiveness  or becauWe investigated other factors that could be associated with the antibody responses in saliva. We observed different patterns to what is reported in cross-sectional population studies, where adults and symptomatic individuals tend to have higher antibody levels than children and asymptomatic ones, respectively. This could be related to the population under study, who are mostly children infected cases and their contacts. Higher levels of anti-S IgG among asymptomatic individuals could indicate protection against disease in infected individuals, and higher IgG and IgM levels in children could also be related to a stronger immunity. High antibody titers in saliva in vaccinated individuals have been related to lower transmission potential . The levThe significant role of mucosal immunity and, particularly, of secretory and circulating IgA antibodies in COVID-19, is becoming more apparent, and could be exploited for beneficial diagnostic, therapeutic, or prophylactic purposes (vaccines) . This suA study limitation was that seropositivity thresholds could not be estimated with pre-pandemic saliva samples due to lack of access to them, and thus sensitivity and specificity could not be established for the saliva assays by standard methods. Using RT-PCR negative pandemic samples was somewhat useful for serum/plasma samples, but with saliva there was substantial overlap between antibody levels in infected and non-infected individuals. In addition, being pandemic samples, we cannot ascertain that they were not previously exposed at low levels and therefore this approach is not optimal. This constraint could be overcome in follow up studies by assessing seroconversion in consecutive samples calculating the fold change increase in levels (e.g. ≥4) over a given study period , 28 and,In conclusion, antibody levels in saliva measured with our high-throughput qSAT assays largely correlated with those in serum/plasma from individuals with confirmed RT-PCR diagnosis of SARS-CoV-2 infection, but the degree of correlation depended on the isotype and antigen. Higher antibody levels found in asymptomatic individuals and in children, particularly among males, could indicate protection against disease and stronger immunity. This non-invasive field deployable antibody screening could be useful to establish the percentage of people who have been exposed to SARS-CoV-2 in epidemiological surveys, and to assess the longitudinal maintenance of antibodies , particuThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Institutional Review Board and the Hospital Sant Joan de Déu Ethics Committee (Ref. PIC 153-20) and the Hospital Clínic ethics committee (Ref. CEIC-7455). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.CD and SAl wrote the first draft of the manuscript. RA, GM, and IJ contributed to the manuscript write up. NR, CC, and LI produced proteins for immunoassays. DB, LP, and AC managed samples. MV, AJ, RR, and SAl performed antibody assays. RA, GM, and CD coordinated immunology and data analysis. RS and SAl performed data analysis. CMA performed microbiology analyses. GP, MM, MH, MG-A, AG-M, EB-C, JC, MC, CF, VF, QB, BB, PM-M, SAj, NB, SAr, MF, EG, JG-G, and IJ performed the clinical and epidemiological studies. All read approved the final version of the manuscript.The project has been funded by Stavros Niarchos Foundation (SNF), Banco Santander and other private donors of KidsCorona, and Fundació Privada Daniel Bravo Andreu. RR had the support of the Health Department, Catalan Government (PERIS SLT017/20/000224). Development of SARS-CoV-2 reagents was partially supported by the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C. ISGlobal receives support from the Spanish Ministry of Science and Innovation through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. CISM is supported by the Government of Mozambique and the Spanish Agency for International Development (AECID).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Self-assembling peptides could be considered a novel class of agents able to harvest an array of micro/nanostructures that are highly attractive in the biomedical field. By modifying their amino acid composition, it is possible to mime several biological functions; when assembled in micro/nanostructures, they can be used for a variety of purposes such as tissue regeneration and engineering or drug delivery to improve drug release and/or stability and to reduce side effects. Other significant advantages of self-assembled peptides involve their biocompatibility and their ability to efficiently target molecular recognition sites. Due to their intrinsic characteristics, self-assembled peptide micro/nanostructures are capable to load both hydrophobic and hydrophilic drugs, and they are suitable to achieve a triggered drug delivery at disease sites by inserting in their structure’s stimuli-responsive moieties. The focus of this review was to summarize the most recent and significant studies on self-assembled peptides with an emphasis on their application in the biomedical field. Molecular self-assembly is a natural process driven by various non-covalent interactions, such as electrostatic and/or hydrophobic, aromatic stacking, hydrogen bonding, or metal coordination interactions . The eleIn nature, the self-assembly is a common process essential to the life activities of cells; for example, the formation of cytoskeletons requires the self-assembly of actins and tubulins or the formation of double-stranded DNA molecules ,5. ErrorConsidering these physiological roles, in recent years, the self-assembling process has been largely studied; in particular, self-assembling peptides (SAPs) are arousing great attention as novel biomaterials in different fields such as nanomedicine, nanomaterials, and nanobiotechnology ,9,10,11.It is well known that peptide-based structures, with respect to synthetic small molecules, possess several advantages like high chemical and biological diversity, higher potency and selectivity for their targets, low toxicity and good membrane penetration, and they are used in different fields ,13. UnfoThanks to their characteristics, SAPs show unique biochemical and physicochemical activities that can overcome the disadvantages of common peptides. Indeed, the modification of the SAPs’ chemical structure allows to regulate their size and morphology  ,16. By mFurthermore, when assembled, SAPs can be used as scaffolds for cell and tissue regeneration  and carrCurrently, SAPs are also used as a three-dimensional cell culture and as biIn this review, we describe how to design SAPs using different building blocks and their potential application as drug delivery systems. Furthermore, we summarize the most recent and significant studies on SAPs with an emphasis on their application as drugs in the clinical and biomedical field.Normally, to design functional peptide assemblies, the first step involves the morphological control; indeed, it is important to understand the nature of peptide folding that drives the self-assembly process and creates a range of diverse structures. Depending on the nature of building blocks, it is possible to synthesize SAPs with distinctive properties that are largely used in the biomedical area. The building blocks can be categorized according to their distinctive constituent amino acids and/or to the various bound chains  units).Herein, the most-used building blocks, intended as peptide monomers able to auto-assemble to obtain SAPs with different morphologies, are schematically reported in Single amino acids can provide similar advantages of peptides but with the additional advantage of lower costs of production . A first2-FF-COOH , derives from the central hydrophobic cluster of the amyloidogenic polypeptide Aβ-42, and it is considered as the smallest homo-aromatic peptide able to self-assemble thanks to intramolecular aromatic interactions. It is able to form highly ordered nanostructures, such as nanotubes, nanowires, nanocrystals, and nanoparticles   generates small unnatural transmembrane nanochannels (<1 nm) on the cell membrane for the delivery of anti-cancer drugs such as 5-fluorouracil (5-FU) .Normally, self-assembling short peptides can form ordered assemblies rich in β-sheet structures. However, α-helical structures, the most common secondary structures of natural proteins, are less exploited for the creation of assembled structures. Short peptides containing unmodified amino acids present weak intrinsic helical stability, which hinders their use to generate self-assembled helical structures . One of 12-VVAAG, Nap-C12-VVAAD, and Nap-C12-VVAADD) with varying tail groups and C-terminal hydrophilic amino acids have been tested for their capability to self-assemble and form hydrogels to release the antitumoral drug doxorubicin (DOX) ..75].According to the purpose, several strategies could be used to design co-assembled structures. One approach involves the association of two peptides with the same sequence but one of them bearing typical motifs, such as the EAK16-II peptide that mixed with its histidinylated form, which is used to induce a strong aggregation of epithelial cells and wound closure .It is also possible to mix one peptide with a longer one formed by two sequences of the same peptide and connected by a spacer: for example, the peptide FEFEFKFK linked to its double-length peptide, FEFEFKFK-GG-FKFKFEFE, generates fibers with increased stability and elasticity .Another strategy involves the use of different types of peptides combined with different non-peptidic structural elements . RecentlInterestingly, Ajovalasit A. et al. very recently described an innovative co-assembled structure constituted by a peptide amphiphile and a natural product, the tamarind seed xyloglucan (XG). This natural compound has anti-inflammatory properties and positive effects on skin re-epithelization. Generally, the combination of cationic PAs with polysaccharides can create structures similar to membranes, which are useful for skin applications. In this work, a cationic PA  and a carboxylated XG (CXG) were co-assembled through the injection of PA solution drops inside CXG solution. The resulting CXG-PA hydrogel revealed to be able to significantly close wounds in in vivo studies, acting as a potent therapeutic for skin lesions .As demonstrated by these examples, the co-assembly process is a novel and interesting approach to obtain innovative and multifunctional biomaterials.Magnetic resonance imaging (MRI) is a growing research discipline that allows to image intact and opaque tissues in three dimensions at cellular scale in a non-invasive manner . It is bCao et al. designed a gadolinium (Gd)-based MR contrast agent that is susceptive to furin, a protease that is overexpressed in cancer cells. After intracellular disulfide reduction, the structure condenses into amphiphilic dimers, which subsequently self-assemble into Gd-nanoparticles (NPs). Gd-NPs assembly causes the increase in the local intracellular concentration of Gd with a higher molecular weight with consequent higher relaxivity. In vivo studies on tumor-xenografted mice reported the preferential presence of assembled Gd-NPS in the tumors, which enhanced the contrast of MRI images as compared to non-cleavable Gd-containing peptides . In anotFluorescence bioimaging plays an essential role in biomedical research and treatment, being a widely used technique, for example, in cellular studies to visualize individual functional sites, in applied therapeutics to identify tumors, and to track conformation processes in real time . The maiInterestingly, for applications of light diagnostics and therapy in precision medicine, Apter et al. developed biocompatible visible fluorescence light (FL)-delivering probes. Indeed, PEGylated phenylalanine peptide derivatives (PEG-F6) self-assembled into hybrid nanostructures forming β-sheets that acquire the visible FL property found in β-sheet amyloid fibrils associated with neurodegenerative diseases .Self-assembly peptides have even been used for light-activated mechanical actuation in living systems. These light-responsive materials are composed by peptide amphiphile supramolecular polymers with a crosslinked spiropyran network, which expel water in response to visible light. Shaped films of these materials were able to generate a bending and crawling motion under photo-actuation .Three-dimensional (3D) bioprinting enables to design complex architectures including different cell types and microenvironments in a rapid, precise, and reproducible manner. One major challenge in bioprinting technologies is the development of suitable bioinks that maintain the shape of the printed construct and that provide a microenvironment allowing cell survival and physiological functions . DiffereUltrashort peptides composed of three to seven amino acids are attractive candidates as bioinks since they can retain high water content preventing cellular dehydration during the printing process ,92. Such2 (IVZK) and Ac-Ile-Val-Phe-Lys-NH2 (IVFK)) forming hydrogels during 3D bioprinting utilizing physiological buffers at body temperature   .Negatively charged-amphiphilic SAPs based on three glutamic acid (E) units represent ideal templates for the deposition of hydroxyapatite crystals creating a bioactive nanofibrous scaffold, which induced calcium phosphate mineralization and consequently enhanced osteogenic differentiation of osteoblast-like cells .Functional peptide sequences were used to create 3D graphene foam (GF)-based hydroxyapatite minerals. GF foam was prepared by coating polyurethane foam (PUF) template in graphene oxide (GO) solution, followed by PUF removal by pyrolysis. The peptide molecule LLVFGAKMLPHHGA was first self-assembled forming 2D peptide nanosheets (PNSs) that were then noncovalently conjugated onto the graphene foam . The obtTo provide sufficient bioactivity meeting the requirements of clinical applications, Quan et al. developed 3D self-assembled hydrogel scaffolds based on bone morphogenetic protein-2 biomimetic peptide (BMPBP), which is a powerful osteoinductive cytokine. These supports significantly accelerated the in vivo bone regeneration in rat cranial bone defects .In the field of neural tissue engineering, Motamed et al. developed self-assembly peptides composed of β-amino acid building blocks, which, when N-acetylated, generated a network of nanofibers in aqueous solution, forming mechanically stable hydrogel at physiological conditions. Such hydrogels had similar stiffness to brain tissue and promoted neural cell adhesion and proliferation .A self-assembling peptide hydrogel was reported also as a cell carrier for nucleus pulposus of intervertebral discs engineering. The peptide was based on FEFEFKFK  chains, which possess hydrophilic and hydrophobic faces. Therefore, when solution ionic strength and pH increased, the chains self-assembled into anti-parallel β-sheet nanofibers. After 3D culture of nucleus pulposus cells, upregulation of nucleus pulposus-specific genes pointed out successful tissue regeneration with high cell viability .Interestingly, for suturing small vessels, Smith et al. developed a multiphase transitioning peptide hydrogel that could directly be injected into the lumen. The SAP contained a photocaged glutamic acid and formed a solid hydrogel in the syringe based on β-hairpin interactions. Upon application of a shear, peptides unfolded, and hydrogel dissolved and could be delivered to the lumen of the collapsed vessel. Upon removal of the sheer, the hydrogel network quickly recovered. After suturing was complete, intravascular gel was dissolved initiating the gel–sol phase transition by irradiation with light .As demonstrated by these examples, the self-assembly of various types of peptides shows promising applications in tissue engineering.The translation of SAPs into commercial drugs requires: (1) an improvement of activity over current drugs on the market; (2) lower or similar costs than the competing products; (3) no toxicity for animal and human tissues; and (4) approval by regulatory agencies  for widespread uses .Currently, SAPs are increasingly used in biomedical fields, and many are already under clinical studies ,141. HerRADA16 was designed with the intention of mimicking the self-assembly properties of a natural SAP named EAK16. This SAP was unexpectedly discovered in a natural protein as an unusual repetitive fragment (AEAEAKAKAEAEAKAK) ,145. To 2; carboxylic acid functional group, –COOH) allow the formation of exceptionally stable and highly ordered β-sheet structure through the formation of different types of bonding  -3-(2-naphthyl)-D-alanyl-L-cysteinyl-L-tyrosyl-Dtryptophyl-L-lysyl-L-valyl-L-cysteinyl-L-threoninamide, acetate salt], and its structure is reported in This cyclic octapeptide in water can form noncovalently bonded dimers. The dimers thanks to noncovalent forces  can, in turn, form β-sheet-rich filaments leading to the formation of nanotubes with a defined diameter (24 nm) and thickness and can subsequently lead to the formation of hydrogels. The structure of the semi-solid gel results from a very dense packing of the nanotubes ,155. TheLanreotide was licensed by the FDA in 2007, and it is currently used for the relief of neuroendocrine symptoms and in particular for the treatment of radiotherapy-resistant acromegaly or in cases where surgery has failed to restore normal growth hormone secretion ,157.® Depot Injection. Somatuline Depot is a prescription medicine available as sterile and ready-to-use prefilled syringes containing lanreotide acetate supersaturated bulk solution of 24.6% w/w lanreotide base. It is safe and effective in adults, but, currently, it is not indicated in children [The trade name of the product is Somatulinechildren ,143,158.Peptide self-assembly mechanisms represent a powerful tool to produce structures of varying size, scales, and functions and with exclusive physical properties. Protein structures are present in several biological systems as cargo transport, microbial defense, or structural support, ranging from oligomers and nanospheres to tubes or hierarchical assemblies. Usually, these structures are formed by non-covalent forces that give them dynamism and flexibility. Great attempts have been committed for exploring self-assembly proteins, which allow the formation of functional materials; however, the results are expensive and difficult to produce. In this context, short peptide-based structures have been shown to possess great potential as biomaterials representing a great promise for future research. The recent examples of successful clinical trials of the self-assembling peptide scaffolds for accelerating wound healing and regenerative medicine and surgical uses provide a glimpse of what is coming for their worldwide widespread use. In the future, SAP nanomaterials are required to be translated into commercial products providing, thanks to properties as specific as targetability, therapy, and eventual imaging ability, a functional improvement over existing products on the market. We believe that exploring new modifications in SAP sequences could be the key to creating novel structures able to be used in diverse fields.


Detection, characterization, staging, and response assessment are key steps in the imaging pathway of ovarian cancer. The most common type, high grade serous ovarian cancer, often presents late, so that accurate disease staging and response assessment are required through imaging in order to improve patient management. Currently, computerized tomography (CT) is the most common method for these tasks, but due to its poor soft-tissue contrast, it is unable to quantify early response within lesions before shrinkage is observed by size criteria. Therefore, quantifiable techniques, such as diffusion-weighted magnetic resonance imaging (DW-MRI), which generates high contrast between tumor and healthy tissue, are increasingly being explored. This article discusses the basis of diffusion-weighted contrast and the technical issues that must be addressed in order to achieve optimal implementation and robust quantifiable diffusion-weighted metrics in the abdomen and pelvis. The role of DW-MRI in characterizing adnexal masses in order to distinguish benign from malignant disease, and to differentiate borderline from frankly invasive malignancy is discussed, emphasizing the importance of morphological imaging over diffusion-weighted metrics in this regard. Its key role in disease staging and predicting resectability in comparison to CT is addressed, including its valuable use as a biomarker for following response within individual lesions, where early changes in the apparent diffusion coefficient in peritoneal metastases may be detected. Finally, the task of implementing DW-MRI into clinical trials in order to validate this biomarker for clinical use are discussed, along with the trials that include it within their protocols. High grade serous ovarian cancer (HGSOC) is the leading cause of death from gynecologic malignancy , borderline or other malignant (immature teratomas and granulosa cell tumors) ovarian masses is essential to establish the correct management pathway. Standard management involves primary debulking (cytoreductive) surgery (PDS) followed by adjuvant taxane–platinum combination chemotherapy . SuboptiMaking a surgical decision on resectability relies heavily on imaging assessment of disease extent and location. To date, contrast enhanced computerized tomography (CT) scans of the abdomen and pelvis have formed the mainstay of this imaging assessment and are considered standard-of-care as CT is easily available and the imaging modality is robust and relatively cheap. However, even when contrast enhanced, CT lacks the soft-tissue contrast to detect disease at potentially “difficult-to-resect” sites. The superior soft tissue contrast of T2-W magnetic resonance imaging (MRI) together with techniques such as diffusion-weighted (DW-) MRI provide coverage of the abdomen and pelvis with high contrast between tumor and non-tumor tissue ,15 and, −3 mm2/s and evolution time of ~50 ms has a root-mean-square displacement of ~30 µm, which means that in cells (dimension ~1–15 µm), water molecules will encounter many cellular and subcellular impediments.In DW-MRI, the MR signal is sensitized to motion by the inclusion of two additional magnetic field gradients within the pulse sequence . The firb-value . These parameters can be adjusted to alter the sensitivity of the sequence to diffusion. The higher the b-value, the more sensitive an image is to the effects of diffusion. An optimal high b-value to image peritoneal metastases from ovarian cancer is around 1000 s/mm2  ,48 47,48CT of the abdomen and pelvis extended to the chest is the first line imaging modality for staging ovarian cancer as recommended by the European Society of Uro-Genital Radiology (ESUR) . Its exc18FDG-PET/CT, WB-DWI/MRI allowed more accurate tumor characterization and detection of peritoneal, mesenteric, and serosal metastases in a small cohort of 32 patients [18FDG-PET/CT was similar [18FDG-PET/CT or CT [A whole-body (WB) DW-MRI technique has recently been advocated for staging and assessing operability . Comparepatients . For the similar . For two similar . The sup similar . MoreoveCT or CT . InterclCT or CT . NeverthDW-MRI may be used qualitatively for evaluating clinical response to NACT . QualitaUse of the quantitative biomarker, the ADC, derived from DW-MRI for response assessment requires more rigorous technical attention during image acquisition and analysis. In order to be reliable enough for clinical decision-making, the derived ADC needs to be both accurate (close to the reference standard or ground-truth it represents) and precise (both repeatable and reproducible). Repeatability assesses the “closeness of the agreement between the results of successive measurements of the same measurand carried out under the same conditions of measurement”, while reproducibility assess the “closeness of the agreement between the results of measurements of the same measurand carried out under changed conditions of measurement”, such as a different MRI scanner . Thus, tQuantifiable treatment-induced changes measured by ADC may occur earlier than conventional morphologic alterations ,67. The p = 0.03) [An analysis of the distribution of ADC values within a lesion provides another approach to assessing disease response. An early study using histogram analysis of ADC values showed that in responders, all centile ADC values increased significantly after the first and third cycle, while skew and kurtosis decreased significantly after the third cycle, but that in non-responders, no parameter changed significantly . A follo = 0.03) . A histo = 0.03) ,74.Quantitative diffusion-weighted imaging also may be useful for characterizing postsurgical findings, with high ADC values being more likely to represent areas of edema or inflammation and low ADC values being suggestive of the presence of active tumor cells , though As with imaging in all clinical trials, when introducing DW-MRI into clinical trials of HGSOC, standardization of all stages of image acquisition, manipulation, analysis, and quantitative assessment is mandatory. Pre-specified imaging protocols, and variations in these must be recorded: poor protocol compliance or inaccurate recording has the potential to alter the images and, therefore, the measured outputs. This is essential because clinical trials are key in informing changes in clinical practice. Rigorous trial conduct and an audit trail with absolute transparency are, therefore, mandatory elements for a successful and reliable trial.b-values, and sequence parameters across scanner platforms need to be based on assessments of scanner capability in order to achieve the best possible signal-to-noise ratio in all cases. Some parameters can be fixed across all platforms, for example, b-values. Three b-values, in addition to b = 0 s/mm2, are ideal for ADC quantification and allow non-linear models to be used; advanced diffusion sequences  acquired at a single station through the largest lesion may be used to enable more sophisticated diffusion modelling [Standard protocols for DW-MRI in ovarian cancer have been developed based on initial measurements using test-objects and volunteers to produce images of an acceptable quality for use in multicenter clinical trials . Standarodelling .Following protocol development, protocol optimization requires site visits to assess the capabilities of all scanners in the study and refinement of the protocol based on the results from all sites. Test-objects must be used to assess fat suppression, geometric distortion, ghosting, contrast-to-noise, presence of artefacts, and accuracy of ADC estimates between scanners at different sites . Volunteclinicaltrials.gov website currently lists only eight trials in ovarian cancer that utilize DW-MRI (The e DW-MRI . Only onb-values allows modelling of the signal decay to derive a quantitative biomarker-the ADC. A low b-value of 100 s/mm2 ensures that perfusional elements are excluded, while a high b-value of 1000 s/mm2 is ideal to avoid signal decay that lies within the image noise floor. Characterization of adnexal masses, however, based on quantitative ADC assessments alone, is unreliable; here morphological imaging and assessment of cystic and solid components is the diagnostic key. However, for malignant adnexal masses, ADC is inversely correlated with cellularity and is indicative of poor prognostic outcome.DW-MRI provides qualitative and quantitative evaluation of disease in ovarian cancer and must be implemented with an understanding of its strengths and limitations . A technQualitative assessment of abdomino-pelvic DW-MRI is increasingly recognized as valuable in ovarian cancer staging. Its role in comparison to currently performed CT staging will emerge from direct comparison of these modalities in on-going multi-center trials. The ADC may be incorporated into clinical trials of ovarian cancer as a response biomarker but requires careful quality assurance and quality control procedures during the conduct of the trial to minimize measurement variability and ensure reproducibility. The ADC has potential to indicate response in individual peritoneal lesions after one cycle of NACT and prior to size changes being evident. In future, it’s potential for guiding tailored management of chemotherapy and targeted agents not only within clinical trial protocols but also in clinical routine will become established.


Insects constitute the largest group in the animal kingdom, and include many economically important species as well as pests. This abundance provides large-scale genetic resources, and research on insects is beneficial from the perspectives of human health, agricultural production and basic science Fig. .Drosophila melanogaster a crucially important model organism for basic biology research. However, many interesting biological hallmarks lie in non-Drosophila species, in which gene manipulation and related in vivo systems are still difficult. The recent establishment of genome editing techniques provides the capacity for gene knock-out (KO), knock-in (KI), and/or knock-down (KD) in non-model systems. Two types of genome editing tools, including transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, are available and are frequently applied. The CRISPR/Cas9 system involves the invariable Cas9 protein and easily designed single-guide RNAs (sgRNAs), while TALEN needs to be redesigned to target different loci each time. Thus, the CRISPR/Cas9 is technically simple and more widely used.The availability of many genetic tools has made Drosophila as a model insect, genome editing brings great convenience and reliability to gene function research in Drosophila. For example, CRISPR-based screening has lower off-target effects than RNA interference (RNAi) [Although scientists have exploited a series of substantial genetic tools using e (RNAi) . The pree (RNAi) . KI can e (RNAi) .et al. used transgenic CRISPR/Cas9-mediated KO of nuclear polyhedrosis virus (NPV) genes to develop a new silkworm strain with a high degree of resistance to Bombyx mori nuclear polyhedrovirus infection [et al. replaced the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) from the spider Nephila clavipes [Gene editing tools can be used to generate novel economic insect strains more rapidly and with greater efficiency than traditional breeding methods . Chen etnfection . Using Tclavipes . This edclavipes .doublesex, causing complete population suppression in malaria vector Anopheles gambiae mosquito populations and all-male population collapse of the agricultural pest Ceratitis capitata [Gene drive targets with functional or structural constraints that might prevent the development of resistant variants could offer a route to successful vector or pest population control. Bier and colleagues have established a new CRISPR/Cas9-based method, called ‘active genetics’ that can greatly bias the transmission of genetic traits, thereby bypassing the traditional constraints of Mendelian inheritance . This necapitata ,9. TheseGenome editing provides an easy and effective approach to generate loss-of-function mutants for functional genetic studies. CRISPR/Cas9 has provided answers regarding wing color mimicry of butterflies and social behavior in ants ,11.Despite a large amount of research effort, genome editing tools have still only been applied to a limited number of insect species, including the silkworm, butterflies, a few moths, mosquitoes and locusts. This is due to the difficulty of applying microinjection techniques in insect eggs. Many parameters need to be explored and optimized in each species, including (but not limited to) appropriate penetration techniques, egg collection time, injection timing, injection placement, the method for sealing eggs after injection and incubation conditions. The embryos of many insects have a hard shell, which makes microinjection difficult. For example, a double-needle system, in which a tungsten needle pierces the egg shell and a capillary glass needle injects plasmids, is used for microinjection in the silkworm. One group has developed a technology termed Receptor-Mediated Ovary Transduction of Cargo to deliver Cas9 ribonucleoprotein (RNP) to the arthropod germline by injection into adult female mosquitoes without injecting eggs .The major obstacle is that it is not easy to select an edited insect from the brood population. Such screening problems can be resolved using transgenic-based genome editing with select marker genes, such as fluorescent protein genes or body color genes. One method involves the use of the ribozyme-gRNA-ribozyme (RGR) structure with multiple sgRNAs—some for the target genes and others for genes involved in body color—in a single vector, thus allowing selection of mutants based on readily detectable visual characteristics (i.e. fluorescence or body color).Cpf1 has been reported to produce cohesive ends after DSBs that are suitable for HDR repair [CRISPR-based KI has failed many times in the silkworm and other lepidopteran insects. Compared with successful TALEN-based KI, HDR has a higher probability of occurrence than non-homologous end joining (NHEJ), which induces DNA double-strand breaks (DSBs) in CRISPR/Cas9. Inhibition of the NHEJ pathway factors Ku70 and DNA ligase 4 can improve KI efficiency . The endR repair .https://crispr.dbcls.jp/) can reduce the likelihood of off-target effects [Although the probability of off-target effects with CRISPR/Cas9 is much lower than it is with RNAi, the potential occurrence of such effects also leads to experimental inaccuracies. Computer-aided sgRNA design (CRISPRdirect:  effects . In someDrosophila insects, future work should focus on the development of precise genome editing methodologies, with high efficiency and low probabilities of off-target effects for large-fragment KI and single-base pair editing. It will also be necessary for RNA function to be studied and novel techniques to be developed to edit non-coding RNA, piwi-interacting RNA, microRNAs and so on. Additionally, due to their ecological importance, the effects of transferring gene-edited insects from the laboratory to the wild will require further study, monitoring and the development of specific regulations.In entomological research, especially in non-


Some clinical studies have shown promising effects of transcranial direct current stimulation (tDCS) over the primary motor cortex (M1) on pain relief. Nevertheless, a few studies reported no significant analgesic effects of tDCS, likely due to the complexity of clinical pain conditions. Human experimental pain models that utilize indices of pain in response to well-controlled noxious stimuli can avoid many confounds that are present in the clinical data. This study aimed to investigate the effects of high-definition tDCS (HD-tDCS) stimulation over M1 on sensitivity to experimental pain and assess whether these effects could be influenced by the pain-related cognitions and emotions. A randomized, double-blinded, crossover, and sham-controlled design was adopted. A total of 28 healthy participants received anodal, cathodal, or sham HD-tDCS over M1 (1 mA for 20 min) in different sessions, in which montage has the advantage of producing more focal stimulation. Using a cold pressor test, several indices reflecting the sensitivity to cold pain were measured immediately after HD-tDCS stimulation, such as cold pain threshold and tolerance and cold pain intensity and unpleasantness ratings. Results showed that only anodal HD-tDCS significantly increased cold pain threshold when compared with sham stimulation. Neither anodal nor cathodal HD-tDCS showed significant analgesic effects on cold pain tolerance, pain intensity, and unpleasantness ratings. Correlation analysis revealed that individuals that a had lower level of attentional bias to negative information benefited more from attenuating pain intensity rating induced by anodal HD-tDCS. Therefore, single-session anodal HD-tDCS modulates the sensory-discriminative aspect of pain perception as indexed by the increased pain threshold. In addition, the modulating effects of HD-tDCS on attenuating pain intensity to suprathreshold pain could be influenced by the participant’s negative attentional bias, which deserves to be taken into consideration in the clinical applications. Non-invasive brain stimulation techniques, such as transcranial magnetic stimulation and transcranial electrical stimulation, are neuromodulation approaches that can regulate the cortical activity . TranscrPrevious studies have shown tDCS effects on attenuating pain perception in experimental pain and clinical pain conditions, such as neuropathic pain, fibromyalgia, and migraine . ApplyinMost studies that investigated the effects of tDCS over M1 upon pain perception have adopted the conventional montage with the target electrode placed at the M1 and the reference electrode placed at the contralateral supraorbital area. Nevertheless, the spatial distribution of the electrical field for conventional tDCS configurations has been critically discussed. Conventional tDCS stimulation modulates cortical activation in a large cortical area beyond the cortical region underlying the target electrode . In addiIn light of the advantages of HD-tDCS, some studies attempted to assess whether HD-tDCS targeted on M1 can effectively alleviate clinical pain . For insPain perception is greatly dependent upon psychological factors, such as pain-related cognitions and emotions . These pCold pressor pain, induced by submerging a non-dominant hand into cold water, is a well-validated test to mimic clinical pain, because of the more sustained and higher level of pain intensity and unpleasantness that it evokes . It showA priori power analysis using G*Power software was conducted to determine the appropriate sample size for a within-participant design with two factors (2 × 3 = 6 conditions). It yielded a sample size of n = 28 to detect a medium effect size of f = 0.25 at a standard error probability of α = 0.05 with a power of 0.95. Therefore, we recruited 28 participants . It suggests successful blinding of HD-tDCS. Ratings of adverse events were compared among the three HD-tDCS sessions. As shown in p > 0.05 for all comparisons), except for ratings of burning sensation . Nevertheless, ratings to sham stimulation were not different from anodal or cathodal stimulation . It suggests that cathodal HD-tDCS causes more adverse effects on eliciting burning sensation.Immediately after tDCS intervention, the blinding of stimulation type was evaluated using the questionnaires. The effectiveness of blinding HD-tDCS  was analyzed using a Cochran’s F2,48 = 3.83, p = 0.035, Post hoc paired-sample t-tests showed that cold pain threshold was greater after anodal HD-tDCS than after sham stimulation . The main effect of the Stimulation Side was also significant .In contrast, repeated measure ANOVA did not show any significant main effects or interaction on pain tolerance, pain intensity, and unpleasantness ratings evoked by cold pressor stimulus . A negative value for the pain rating or a positive value for pain threshold and tolerance indicates analgesia induced by active HD-tDCS. Finally, the relationships between the analgesic effects of HD-tDCS and pain-related cognitions/emotions  were estimated using Pearson correlation analysis. As shown in r = 0.59, p = 0.002). This result suggested that individuals with a lower level of attentional bias to negative information would benefit more from attenuating pain intensity rating induced by anodal HD-tDCS stimulation.Correlation analysis was conducted to determine whether the analgesic effects of HD-tDCS were influenced by pain-related cognitions and emotions. Firstly, cold pain sensitivity measured at hands contralateral and ipsilateral to the HD-tDCS side was grand averaged for each tDCS type , thus yielding three values for each pain sensitivity measure and for each participant. Next, the analgesic effects of HD-tDCS were evaluated by calculating the contrast between active and sham stimulation (i.e., anodal The current study evaluated the effects of single-session HD-tDCS over M1 on cold pain sensitivity among the healthy population. Anodal HD-tDCS showed to be effective in increasing cold pain threshold when compared with sham stimulation. Neither anodal nor cathodal HD-tDCS significantly modulated cold pain tolerance and pain intensity and unpleasantness ratings. Analgesic effects of anodal HD-tDCS on cold pain intensity rating could be influenced by the level of attentional bias to negative information. These results suggested that anodal HD-tDCS over M1 can modulate the sensory-discriminative aspect of experimental pain perception and that analgesic effects of anodal HD-tDCS on the perception of suprathreshold pain may be influenced by psychological factors, such as negative attentional bias.Single-session 20-min anodal HD-tDCS targeted on the M1, relative to sham stimulation, increased cold pain threshold, regardless of whether a painful stimulus was delivered to the hand contralateral or ipsilateral to the tDCS target site. This finding is in line with via HD-tDCS stimulating M1. Nevertheless, we did not measure neurophysiological data that could allow us to assess the mechanisms underlying the analgesic effects. Future neuroimaging studies are recommended to further explore whether and how HD-tDCS over M1 modulates the endogenous pain inhibitory pathway and subjective pain perception.Although previous studies have reported analgesic effects of M1-tDCS in both experimental and clinical pain settings , the undHigh-definition tDCS over M1 did not modulate cold pain tolerance and pain intensity and unpleasantness ratings. Human pain is a subjective and multidimensional experience involving sensory-discriminative, affective-motivational, and cognitive-evaluation aspects . AlthougCorrelation analysis showed that analgesic effects of anodal HD-tDCS on pain intensity ratings were strongly associated with the participant’s negative attentional bias. It is manifested that those healthy participants with a lower level of attentional bias to negative information tend to have greater effects of HD-tDCS on relieving pain intensity. With the knowledge that patients with chronic pain have more negative attitudes and beliefs, such as pain catastrophizing and fear of pain, as well as negative bias toward pain , the affIn conclusion, relative to sham stimulation, single-session anodal HD-tDCS over M1 increased cold pain threshold in the healthy population. It indicates anodal HD-tDCS targeted on the M1 can effectively modulate the sensory-discriminative aspect of pain perception. In addition, the effectiveness of anodal HD-tDCS in attenuating pain intensity ratings to suprathreshold pain could be influenced by the level of attentional bias to negative information. Our findings support the potential application of HD-tDCS interventions in pain relief among the clinical pain patients and highlight that individual attentional bias to negative information should be well taken into consideration. Given that multiple-session tDCS may be more effective than single-session stimulation , future The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Medical Ethics Committee, Health Science Center, Shenzhen University. The patients/participants provided their written informed consent to participate in this study.XYL and RJ: conception and design of the study and drafting the manuscript. XYL and JY: data acquisition. XYL and XXL: data analysis. XYL, SC, YH, JL, and RJ: writing—reviewing and editing. All authors read and approved the final manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Liver cancer is one of the most common causes of cancer mortality worldwide. Chemotherapy and radiotherapy are the conventional therapies generally employed in patients with liver tumors. The major issue associated with the administration of chemotherapeutics is their high toxicity and lack of selectivity, leading to systemic toxicity that can be detrimental to the patient’s quality of life. An important approach to the development of original liver-targeted therapeutic products takes advantage of the employment of biologically active ligands able to bind specific receptors on the cytoplasmatic membranes of liver cells. In this perspective, glycyrrhetinic acid (GA), a pentacyclic triterpenoid present in roots and rhizomes of licorice, has been used as a ligand for targeting the liver due to the expression of GA receptors on the sinusoidal surface of mammalian hepatocytes, so it may be employed to modify drug delivery systems (DDSs) and obtain better liver or hepatocyte drug uptake and efficacy. In the current review, we focus on the most recent and interesting research advances in the development of GA-based hybrid compounds and DDSs developed for potential employment as efficacious therapeutic options for the treatment of hepatic cancer. Liver cancer is one of the most common cancers and among the most common causes of cancer mortality worldwide . The higAn enormously important approach to the development of original therapeutic products actively targeting liver cancer takes advantage of the employment of biologically active ligands able to bind specific receptors on cytoplasmatic membranes of liver cells. In fact, these ligands may be used to synthesize hybrid compounds based on traditional chemotherapy drugs or to functionalize the surface of micro- and nano-drug delivery systems (DDSs) since they can allow a specific and efficient drug internalization into tumoral cells .In particular, regarding DDSs, more extensive angiogenesis is typical of tumors since more oxygen and nutrients are needed for their rapid growth . HoweverGlycyrrhiza genus (Fabaceae family), also known as licorice, is extensively spread in the Mediterranean basin of Africa, Europe, and Asia, and Glycyrrhiza glabra L. is commercially the most important species belonging to this genus, largely employed as food and for medicinal purposes. Glycyrrhetinic acid (GA), also known as enoxolone, is a triterpenoid derivative of beta-amyrin and is the aglycone derived from intestinal hydrolysis of glycyrrhizin, a pentacyclic triterpenoid present in roots and rhizomes of licorice [The licorice . GA occulicorice . It is rlicorice . GA has been used as a ligand for liver targeting due to the expression of GA receptors on the sinusoidal surface of mammalian hepatocytes, so it may be employed to modify DDSs and obtain better liver or hepatocyte drug uptake and efficacy ,16. PrevIn the current review, we focus on the most recent and interesting research advances in the development of GA-based hybrid compounds and DDSs developed for potential employment as efficacious therapeutic options for the treatment of hepatic cancer. Since in some delivery systems GA is employed both as a ligand to target HCC cells and as a cytotoxic drug, in this paragraph, we have briefly reviewed the molecular mechanisms underlying GA’s anticancer effects. GA exerts a remarkable antitumoral effect against various cancers, including lung cancer, pituitary adenoma cells, glioblastoma cells, and prostate cancer, as well as HCC. By analyzing the anticancer activity of a large number of GA derivatives as related to the structural features, Xu et al.  have sugAt the cellular level, GA acts, through multiple mechanisms, by inhibiting cell proliferation, invasion, and metastasis and inducing autophagy, apoptosis, and cell cycle arrest ,26,27,28More in particular, as to HCC, GA exerts an active anticancer effect by inhibiting cell proliferation, inducing apoptosis, and arresting cell cycle in the G1-phase. The apoptotic effect of GA seems to be related to the activation of caspase-8 and reduction of the antiapoptotic proteins B-cell lymphoma 2 (Bcl-2) and Bcl-xL, with consequent activation of downstream mitochondrial pathways and caspase-3. GA can reverse immunosuppression due to hepatic stellate cells (HSCs) in the tumor microenvironment and abrogate their invasiveness, thus potentiating an immune microenvironment in tumors. The antiangiogenic and antimetastatic effect of GA in HCC could be related to downregulation of the expression of vascular endothelial growth factor (VEGF), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) (a marker for lymphatic metastasis), and matrix metalloproteinase-2 (MMP-2) protein . However, there is evidence that the autophagic response induced by GA via ERK  activation could weaken its anti-HCC activity. This effect of GA has to be taken into consideration, especially in the case of combinatorial therapy ,29. FurtA particularly interesting issue is represented by the research about triterpenoids, as well as other natural compounds, as mitochondria-targeted anticancer drugs. Mitochondria represent the main cellular source of adenosine triphosphate (ATP) produced through oxidative phosphorylation. Under aerobic conditions, to produce ATP, normal cells use the glycolytic pathway to transform glucose into pyruvate in the cytosol; then, pyruvate is converted into acetyl-CoA within the mitochondria. Conversely, under anaerobic conditions, a lower amount of pyruvate is employed to produce ATP, and cytoplasmatic glycolysis is preferred. However, cancer cells show a particular biochemical behavior (“aerobic glycolysis”), since under aerobic conditions, they produce energy mainly through the glycolytic pathway that should be employed to produce the macromolecules needed for biosynthesis; furthermore, in this way, cells produce more lactic acid which has an important role in cancer progression . BesidesPharmacophore hybridization is a new strategy for designing and developing new bioactive molecules based on the combination of pharmacophoric moieties of different bioactive compounds by covalent bonds to generate an original hybrid entity with better pharmacokinetic and pharmacodynamic properties in comparison with the parent drugs. Furthermore, this approach can result in compounds exhibiting modified selectivity profiles, different and/or dual modes of action, and reduced side effects . Notably, 5-fluorouracil (5-FU) is a cell cycle-specific anticancer drug that interferes with DNA synthesis and inhibits RNA formation. Despite its useful activity, 5-FU exhibits evident adverse effects which limit its clinical usefulness. For this reason, structural modification of 5-FU was performed to improve its selectivity and reduce the toxic side effects. Alkyl chains are usually used as the linkers of two pharmacophores in order to change the physical and chemical properties and increase liposolubility  Table 1Table 1. Similarly, furoxans, an important class of nitric oxide (NO) donors, were derivatized with GA to produce novel furoxan/GA hybrids to selectively target the liver by producing high concentrations of NO in HCC cells and to overcome furoxan’s severe adverse effects  Figure . NO, a pIn order to improve GA solubility and bioavailability, a novel conjugate combining GA, tetramethylpyrazine, and a small-molecule amino acid (TOGA) was studied . TOGA reStandard chemotherapy is centered on low-molecular-weight drugs, such as DOX, cisplatin, or gemcitabine, with a short half-life and off-target accumulation in healthy organs . Due to d,l-lactide-co-glycolide) (PLGA)) or of natural origin  and chitosan (CS)). Homopolymers are chains of one type of monomer that are chemically linked, while copolymers are characterized by different units. Due to their high molecular weight, polymers have been classified as macromolecules since they present more than ten repeating units.Polymers are very large molecules containing many repeating subunits called monomers, and may be either synthetic  or poly . In the Similarly, GA-decorated polymeric NPs loaded with norcantharidin (NCTD), a chemically synthesized and FDA-approved drug for cancer treatment , were teArtemisia annua with anticancer activity [Similarly, Pan and coworkers tested GA-targeted PEG-PLGA NPs to improve the therapeutic efficiency of artesunate (ART) in the treatment of HCC. ART is an antimalarial drug extracted from the traditional herb activity . Data coN-acetyl-d-glucosamine and d-glucuronic disaccharides, displays exceptional properties such as biocompatibility, biodegradability, and low toxicity and represents an ideal carrier polymer to produce NPs for the targeted delivery of drugs. In addition, HA can actively target the surfaces of cancer cells, such as HCC cells, by binding the CD44 receptor. Xue and coworkers tested in vitro GA-modified HA NPs as carriers for the model drug docetaxel (DTX) [HA, a naturally occurring hydrophilic acid mucopolysaccharide containing repeating el (DTX) . DTX is el (DTX)  but is aUsing the same DDS, Wu and coworkers  tried toAmphiphilic copolymers may be used to produce micellar DDSs. In particular, one has to note that the micelle surface can be modified with GA to target cancer cells. Furthermore, micellar DDSs can be taken into consideration as a promising strategy for multi-drug delivery. In fact, the development of combination therapies is essential to achieve synergic therapeutic effects by simultaneously affecting multiple pathways involved in cancer promotion and progression and by reducing toxicity and side effects. Wu and coworkers  used triMore recently, Yang and coworker  tested atrans form 18α- and the cis form 18β-GA) as well as of other derivatives obtained by the removal of 11-carbonyl in the ring structure of GA (11-deoxy-18β-GA) or the removal of the hydroxyl at C3 through C3-acetylation (3-Ace-GA). In particular, 11-deoxy-18β-GA does not induce a pseudoaldosterone effect and possesses better anti-inflammatory, antiulcer, and antiallergic activities [An innovative kind of DDS is represented by a combination of lipids and amphiphilic polymers. Liposomes (Lip), spherical bilayer vesicles formed spontaneously with phospholipids dispersed in water, are well known for their application as DDSs, and their surface may be modified by GA to target liver cancer cells. However, liposomes have critical problems such as drug leakage during storage, burst drug release, normal tissue toxicity, and immune response, so their performance may be improved by using polymers, which are more stable than lipid molecules and less prone to undergo oxidative degradation phenomena ,89. A vetivities . In compSome papers reported the targeting properties of 3-succinyl-30-stearyl GA using conventional liposomes based on phospholipids and cholesterol in order to deliver wogonin (WG)  or oxaliSimilarly, Chang and coworkers ,90 testeRecently, liposomes provided a promising chance to overcome the problem of an effective codelivery of multiple drugs, since, as said before, chronic administration of a single drug is often associated with undesirable side effects or drug resistance, which may cause failure in cancer therapy. This approach was examined by Jiang and coworkers , who evaDue to the limited quantity of receptors on the target cell membrane, the cellular uptake of single-ligand modified NPs is restricted. Even if the dose increases, at a certain point, the response becomes saturated, the cellular uptake will not increase, and all biological responses reach a maximum . To overEndogenous lectins are present on many normal and cancer cells and participate in several biological functions, working as receptors and mediating endocytosis of specific glycoconjugates. Carbohydrate-specific receptors present in the cell membrane, able to recognize carbohydrate epitopes, play an important role in cellular adhesion and recognition processes, distinguishing specific complex oligosaccharides with an elevated affinity and avidity. Recent research studies have shown that ASGP-R is a special lectin expressed on the cell membrane of liver cells and overexpressed in several human tumoral hepatocytes ; it can Li and coworkers  confirmeAn innovative dual ligand-modified TDDS was recently proposed by Qi et al., who developed GA and Gal-functionalized DSPE-PEG liposomes for the co-delivery of CUR and capsaicin (CAPS), known for its anticarcinogenic, antiproliferative, and antioxidative effects , for livN-nitrosodiethylamine receiving a chloroform dose. The drug-loaded NPs were able to significantly improve the levels of various serum biomarkers by reducing circulating α-fetoprotein and transaminases. In addition, compared to the free drug, dual-ligands CS NPs showed higher therapeutic efficiency as indicated by their ability to induce apoptosis rather than necrosis. These NPs also had the advantage of allowing regeneration of the tissues and restoring the normal liver structure, as well as also possessing a better safety profile compared to the conventional DOX. Interestingly, an effect was also observed for dual-ligand CS NPs without DOX, probably due to the hepatoprotective and anticancer effects of the two attached ligands as well as CS.Lactobionic acid (LA), containing gluconic acid and a Gal moiety, has rapidly emerged as an alternative targeting molecule thanks to the selective cellular uptake capacity of hepatoma-targeting chemotherapy due to its ability to bind ASGP-R . HefnawyHowever, the introduction of a second ligand molecule in the DDS, besides GA, can lead to effects opposite to those desired. Du and coworkers tested GA-LA dual-ligand properties of DOX-loaded NPs, prepared from a low-molecular-weight heparin (LMWH)-derived polymer . In thisAngelica sinensis polysaccharides (APS), the main active components of this plant [Since there is an increased interest in the research of materials for targeted delivery, a new DDS has been developed by producing nanomicelles from is plant . APSs red-galactosyl-(1–3)-N-acetyl-d-galactosamine  GalNA), were used to prepare dual-ligand-modified DOX-loaded liposomes (DOX/GA-PNA-Lip) to improve liver targeting and anticancer efficacy [Using a similar approach, GA and peanut agglutinin (PNA), a plant lectin protein able to bind to β-efficacy . Gal-β can be used to improve cellular internalization of materials with thiol-reactive groups . Thanks +-ATPases, such that the lysosomal pH value in cancer cells was reported to be in the range low as 3.8–5.0 for cancer cells and in the range 4.5–6.0 for normal cells [Of particular interest in the field of TDDSs is the development of materials that respond to the acidic pH of tumor extracellular tissues and intracellular organelles such as endosomes and lysosomes. In fact, the dysregulation of cellular pH, closely connected to hypoxic conditions, is common in solid tumors, and this tumoral acidic microenvironment can promote migration, invasion, and metastasis through various mechanisms . The pH al cells ,104,105.CS and HA show pH-responsive properties via swelling or shrinking in the external media, producing valuable DDSs able to induce a burst release by a modification in the pH.DOX-loaded HA-GA succinate (HSG) conjugate-based NPs (HSG/DOX NPs) were tested for their liver-targeting and pH-sensitive properties . Both HATian and coworkers  preparedRecently, pH-sensitive polymeric micelles modified with a hydrazone bond have been considered a promising tool for effective cancer therapy. Hydrazone bonds, formed by the carbonyl group with hydrazine, have an acid-sensitive chemical structure; they are stable under physiological pH conditions but are rapidly hydrolyzed in the slightly acidic microenvironment of hepatoma cells. Zheng and coworkers tested the pharmacokinetics of GA liver-targeted delivery using a pH-sensitive polymeric DDS based on PEG-PLA hydrazone bond-modified micelles (GA-PEG-HZ-PLA)  loaded wAn intelligent pH-sensitive DDS of CAPS/GA-sulfated HA (sHA) DOX-loaded NPs was developed for liver-targeting co-delivery of DOX and CAPS . The potAdditionally, Li et al. used sHA to develop a DDS based on sHA-DOX/HA-GA micelles, which showed HCC-targeted and pH-responsive properties both in vitro against HepG2 cells and in vivo in H22 cell-bearing BALB/c mice .l-histidine) (PHIS) due to the protonation of its imidazole groups in acidic cytoplasm (pH < 6.5), other than its ability to escape from the endosome. Using GA and PHIS conjugated with PEG-PLGA , the liver-targeting properties of andrographolide (AGP)-loaded micelles were tested [Another excellent candidate polymer for pH-sensitive drug release is poly-based nano-vehicle for the dual delivery of DOX and camptothecin-PEG (CPT-PEG) has been prepared and decorated with GA (CPT-PEG@MSN-DOX-GA) to target HepG2 cells . The comMetal-organic frameworks (MOFs) are a new generation of materials consisting of metal ion clusters linked by multitopic organic ligands, forming extended network structures. Due to their capability to be used as DDSs, there is an increased interest in the development of nano MOFs (NMOFs). MOF materials are considered good candidates for small-molecule drug delivery thanks to their good biological safety and porous nature, which is responsible for their high drug loading and long drug release time. In particular, zirconium MOFs exhibit high stability and low toxicity . In the A pH-sensitive DDS based on GA-functionalized graphene oxide (GA-GO) was prepared by Zhang and coworkers and used as an effective nanocarrier for the targeted delivery of DOX into liver cancer cells . In addi50 value for GA-PPI-DOX, which was found to be dragging more cells in the apoptotic phase as compared to GA-MWCNT-DOX. This effect is probably due to more conjugation of GA on the PPI periphery than on MWCNT formulations. Furthermore, the in vitro release of GA-MWCNT-DOX was slower and sustained as compared to GA-PPI-DOX.Chopdey and coworkers investigated the drug-targeting potential of GA-conjugated polypropylene imine (PPI) dendrimers (GA-PPI) and multi-walled carbon nanotubes (GA-MWCNTs) for the liver targeting of DOX . Higher Today, there is a great interest in next-generation therapies that use biological macromolecules, such as plasmid DNA, short interfering RNA (siRNA) or antisense nucleotides, so that new DDS techniques able to improve therapeutic efficacy by taking into account the molecular mechanisms of these new therapeutic agents are required. However, since these products are biological macromolecules, they cannot simply pass cell membranes, so it is essential to develop new technologies to protect them from nuclease degradation and allow them to be easily introduced into cells. CS has been shown to protect siRNA from serum degradation and deliver it to tumor cells. Zheng and coworkers  tested aFurthermore, the co-delivery of chemotherapeutic drugs and siRNA can improve antitumor efficacy compared to a single administration. Some siRNAs, such as Bcl-2 siRNA, can target the Bcl-2 gene, inhibit Bcl-2 protein synthesis, and induce apoptosis of tumor cells. CS-PEI hybrid systems have been prepared to enhance the transfection efficiency of genomic medicines. Yan and coworkers  tested t50 value) of siRNA/DOX/DPP and siRNA/DOX/GH-DPP NPs against HepG2 cell viability was 1.02 and 0.76 DOX µg/mL, respectively, which were lower than that of free DOX (1.86 DOX µg/mL), supporting an improved cellular uptake of DOX and siRNA via GA receptor-mediated endocytosis and the better sensitivity of HepG2 cells to DOX owing to down-regulation of Bcl-2 by RNA interference. In vivo data confirmed liver-targeting delivery and decreased uptake by normal cells of siRNA/DOX/GH-DPP in H22 tumor-bearing mice, resulting in higher anti-hepatoma efficacy than siRNA/DOX/DPP NPs and less systemic toxicity compared to free DOX.The same strategy has been tested using liver-targeted NPs composed of DSPE-PEG-PEI (DPP) with GA-modified HA (GH) for the co-delivery of DOX and Bcl-2 siRNA (siRNA/DOX/GH-DPP) . The hal50 of free DOX, PEI-GA/DOX, and PEI−GA/DOX/shAkt1 NPs in HepG2 was estimated to be approximately 4.56, 2.07, and 0.99 μg/mL, respectively, confirming the higher drug delivery and the synergic activity of combination therapy. In addition, after treatment with PEI-GA/DOX/shAkt1 NPs, the reduction of Akt1 protein level in HepG2 cells induced autophagy, as an alternative pathway to cell death (type II cell death), via LC3B−II protein upregulation. Furthermore, in vivo treatment with PEI-GA/DOX/shAkt1 NPs indicated improved tumor growth inhibition in Hepa-1.6 cell grafted tumor-bearing C57BL-6J mice.Since HCC is associated with the activation of the PI3K/Akt/mTOR signaling pathway, facilitating the development of tumor cell proliferation, angiogenesis, metastasis, and invasion, Wang and coworkers  tested aHowever, low-molecular-weight PEIs  are known for their low toxicity but poor transfection. Cao and coworkers designed GA-modified LMWH PEIs to demonstrate that modification of LMWH PEIs with GA could give high transfection efficiency and allow liver targeting .GO represents a potentially useful material in gene therapy due to its 2D planar structure with the high presence of surface oxygenated functional groups and due to its ability to easily cross cell membranes. To ameliorate these properties, the GO surface may be modified by positively charged cationic polymers. Polyamidoamine (PAMAM) dendrimers are biodegradable cationic and highly branched spherical polymeric macromolecules with a peptide bond backbone, and their conjugation with GO has been explored as an innovative approach for gene delivery. Liu and coworkers  demonstrPhotodynamic therapy (PDT) is a promising alternative for cancer treatment, especially due to its painless and non-invasive modalities. A photosensitizer (PS), systemically or topically administered, accumulates in the target site during a predetermined duration time , after which the target site is irradiated by the light of appropriate wavelength and energy, producing PS photo-excitation. This excited PS has to transfer its energy to surrounding intracellular oxygen, forming cytotoxic reactive oxygen species (ROS). However, the light energy absorbed by a PS can also be released through fluorescence or heat generation. The main targets of irradiated PS-induced damage are represented by mitochondria, lysosomes, plasma membranes, nuclei, and blood vessels around cancer cells . To be c−2 for 1 h), it showed light phototoxicity in HepG2 cells when compared to cells maintained in the dark, suggesting that TPP-based nanomaterials could be applied for the PDT of cancer cells [For example, since GA can improve the uptake of PS to cancer cells, Wang and coworkers studied the properties of the amphiphilic GA-porphyrin (TPP) conjugate self-assembled into NPs (TPP-GA NPs). TPP, one of the most widely used photosensitizers, is hydrophobic, while GA is water soluble, so the amphiphilic conjugate TPP-GA can self-assemble into NPs. In vitro experiments demonstrated that TPP-GA NPs are uptaken by endocytosis into tumoral cells, and under opportune irradiation  for 80 s. After intravenous administration in HepG2 tumor-bearing mice, GA-PEG-SiPC accompanied with PDT revealed liver cancer-targeted accumulation and anticancer properties via apoptosis and necrosis without side effects and resistance to treatment.Phthalocyanine has a high absorbance at 600−700 nm, high ROS generation efficiency, and is also stable from chemical and photochemical degradation. Silicon phthalocyanine (SiPC) was linked to GA using PEG and 3-(ethoxydimethylsilyl)propylamine (APDES) . GA-PEG-DDSs projected for the combinate use of chemotherapeutics and PS are an innovative strategy for cancer treatment, also because ROS produced by PS irradiation can disrupt the lysosome membrane and thus induce lysosome escape of the drugs. Then, DOX and the photosensitizer pheophorbide A (PHA) were loaded into micelles made of the poly-ε-caprolactone-cystamine-carboxymethyl CS-GA (PCL-SS-CMC-GA) polymer, where the switchable disulfide bonds were predisposed to be degraded in the high redox potential of cancer cells and trigger the release of therapeutic agents . The redChemotherapy is the main treatment for hepatic tumors, including HCC, but it is still challenging due to several problems, including its nonselective biodistribution and effects, as well as multidrug chemoresistance. Active TDDSs, based on the interaction between a ligand present on the surface of the DDS and specific receptors present on the target cells, hold enormous potential in cancer therapy for their capability to improve DS internalization and drug uptake into specific cells with higher drug bioavailability and lower systemic toxicity.Taking this into account, our review systematically describes the more recent advances in the development of biomaterials based on GA for active drug delivery targeted to liver cancer. Besides the capability to target hepatic cells, some of these systems can offer, due to their unique chemical characteristics, several additional advantages.First, innovative systems aimed at the simultaneous delivery of more therapeutic agents acting on different targets or mechanisms involved in cancer growth and progression have been developed. In particular, one has to remember that GA itself has well-documented anti-cancer properties. Another important aspect involves delivering nucleic acids such as siRNA/pDNA, since these therapeutic agents can enter target cells, usually via endocytosis, and their functionality strongly depends on the efficiency of endosomal escape. Particularly interesting is the development of GA-based biomaterials that incorporate PS drugs to be used in liver cancer PDT.Special attention is focused on dual functionalization and on the design of stimuli-responsive systems. In fact, a recent trend in the surface functionalization of DDSs involves their decoration with two ligands , enabling them to deliver more drugs to specific cells and further reduce normal tissue toxicity. There has been increased development of GA-based systems responsive to intrinsic characteristics of tumor microenvironments, in particular the lower pH.Thus, these results highlight the potential therapeutic efficacy of rationally designed TDDSs based on GA and lead us to hypothesize that the research about these products will continue to increase in the future. However, at this phase of research, the translation of the existing data from the laboratory to the clinical field appears to still be very problematic. A particular problem is represented by the limited scientific knowledge about the hepatic GA receptors. Although several papers have demonstrated the existence of these proteins called GA receptors on the membranes of normal and tumoral liver cells, we know very little about the physiological and biochemical aspects related to their functionality.On the other hand, the efficacy of all the products reported in this review has been demonstrated in vitro on opportune cancer cell lines, and in a few cases, in vivo using experimental animals. Thus, the difficulty of extrapolating these experimental findings from isolated cell lines to complex biological systems and then to humans is evident, and only further studies can allow us to demonstrate their actual clinical efficacy, as well as their safety and biocompatibility.One has also to point out that the increased complexity of methodologies employed to realize the described TDDSs can also introduce significant obstacles in reproducibility, scale-up/out, and quality control, and consequently significantly higher production costs, all aspects that make their applicability to the clinic more complex.Finally, several obstacles have limited the clinical application of licorice-derived therapeutics. The metabolism of a large part of marketed drugs is regulated by cytochrome P450 (CYP) isoforms , which are most frequently involved in drug phase I biotransformation, and by uridine 5′-diphospho-glucuronosyltransferases (UGTs), which play a main role in phase II metabolism. Furthermore, the activity of the transmembrane ATP-binding cassette transporter P-glycoprotein, predominantly expressed in the intestinal tract, brain, liver, and kidney, is also crucial for drug metabolism and bioavailability. It has been reported that GA can significantly affect the activity of some metabolic enzymes, including several CYP450 isoforms and UGTs, as well as of P-gp, so it could mediate potential drug-drug interactions. In addition, a further complication due to licorice uptake seems to be pseudohyperaldosteronism, a clinical condition characterized by hypertension, hypokalemia, and suppression of plasma renin and aldosterone levels. This effect is due to its component, GA, which acts mainly through two different mechanisms: by blocking the enzyme 11-β-hydroxysteroid dehydrogenase type 2 (11-β-HSD2), which inactivates cortisol to cortisone, and by directly binding the mineralocorticoid receptor as an agonist .In conclusion, active targeted biomaterials based on GA represent a great promise for the development of an innovative therapeutic strategy in the treatment of liver cancer, although there is an imperative need for further studies to demonstrate their efficacy and safety.