Scientists are focusing immense attention on polymeric nanocarriers as a prominent delivery vehicle for several biomedical applications including diagnosis of diseases, delivery of therapeutic agents, peptides, proteins, genes, siRNA, and vaccines due to their exciting physicochemical characteristics which circumvent degradation of unstable drugs, reduce toxic side effects through controlled release, and improve bioavailability. Polymers-based nanocarriers offer numerous benefits for in vivo drug delivery such as biocompatibility, biodegradability, non-immunogenicity, active drug targeting via surface modification, and controlled release due to their pH—and thermosensitive characteristics. Despite their potential for medicinal use, regulatory approval has been achieved for just a few. In this review, we discuss the historical development of polymers starting from their initial design to their evolution as nanocarriers for therapeutic delivery of drugs, peptides, and genes. The review article also expresses the applications of polymeric nanocarriers in the pharmaceutical and medical industry with a special emphasis on oral, ocular, parenteral, and topical application of drugs, peptides, and genes over the last two decades. The review further examines the practical, regulatory, and clinical considerations of the polymeric nanocarriers, their safety issues, and directinos for future research. A polymer represents several repeating units leading to the formation of a compound that has characteristics of high relative molecular mass and associated properties. In 1833, Jons Jakob Berzelius coined the term “polymer” which originated from the Greek word  and . Following major developments in polymer fabrication, the molecular properties of polymers were only recognized after the efforts of Hermann Staudinger in 1922, who first suggested that polymers consist of long chains of atoms bound together by covalent bonds. This took more than a decade to achieve acceptance among the scientific community. Initially, polymers were investigated according to the theory of associations or composite theory that emerged in 1861. It was suggested that cellulose and other polymers were colloids and molecular aggregates with small molecular weights bound by an unidentified intermolecular force. The use of polymers is not new in the medical field [®, a PEGylated adenosine deaminase enzyme for severe combined immunodeficiency disease. Doxil® is the first FDA-approved PEGylated nanoparticle (NP) based product that was commercialized in 1995. At present, eight other PEGylated protein therapeutics have been FDA approved for various diseases [Natural polymers are being used for decades as ingredients for herbal remedies. The case is very different when it comes to synthetic polymers. Water-soluble polymers can be considered a modern accomplishment as macromolecular drugs or as part of the inoculation-related drug delivery systems. The first polymer-drug conjugate in form of mescaline-N-vinylpyrrolidone conjugates was synthesized in 1955. A decade later, it was predicted that polyethylene glycol (PEG) can be attached to proteins using a method called PEGylation that comprises adding the covalent bond of polymer (ethylene glycol) polymer chains to some other entity, normally a therapeutically active drug or protein. In 1994, the first synthetic polymer-drug conjugate was clinically proven to treat cancer. This was made up of a doxorubicin copolymer conjugation of (N-(2-hydroxypropyl) methacrylamide . They rediseases ,7,8. Blodiseases . Since tdiseases . In 2013diseases . NK012 odiseases . All of −9 m) in diameter and have distinct physicochemical properties in comparison to macromaterials [Nanomaterials took the pharmaceutical, nutritional supplements, and food industries by storm. Such structures are 1–100 nm  under a valid concentration or temperature environment as versatile spherical structures . In nature, the core of micelles is hydrophobic and appropriate for incorporating hydrophobic moieties . They arVarshosaz et al., synthesized docetaxel-loaded polymeric micelles using folic acid grafted synperonic PE/F 127-cholesteryl hemisuccinate copolymer for precise delivery to cancerous cells. The in vitro cellular uptake observations in B16F10 melanoma cells revealed improved uptake of targeted polymeric micelles through folic acid receptor-mediated endocytosis . The micChida et al., fabricated epirubicin (EPI) loaded polymeric micelles armed with pH-sensitive drug release characteristics in contradiction to axillary lymph node metastasis (ALNM) of triple-negative breast cancer (TNBC) for improving the therapeutic effect. By accumulating selectively and penetrating both the vascularized ALNM and primary tumor, as well as effective drug trigger caused by intratumoral acidic atmosphere, the proposed formulation successfully prevented the growth of ALNM and the expansion of the primary tumor .Bae et al., designed environment-sensitive supramolecular structures for intracellular drug delivery of Adriamycin (ADR) via linking it to the polymer by a pH-sensitive coupler. The micelles treated carcinoma cells via their transportation into a cell by endocytosis, consequently avoiding cell-membrane transporters .Polymersomes are considered the next era of nanomedicine for clinical diagnosis and therapies due to their potential for controlling structure and features. These are the most favorable tools for a biomedical utility such as diagnosis and controlled drug delivery due to the compartmentalization of polymersomes. These are engineered amphiphilic block copolymers or vesicles, but in this circumstance they contain structures made up of one or more bilayers enclosing an aqueous center. In all conditions, the active load of molecules can be dissolved, dispersed, or chemically bound to nanocarriers. Their size is an important parameter for intracellular uptake. They offer better mechanical features and provide better structural and colloidal stability than liposomes in aqueous media . Their fp < 0.05) was revealed in MTT and flow cytometry results. Overall, the study demonstrated the theranostic application of targeted polymersomes with minimization of side effects [Polymermerosomes are presently being studied for the delivery of different probes for imaging target tissues/organs and cytotoxic medications to canceous cells and also for gene therapy . Zavvar  effects . Zhou et effects .Li et al., designed paclitaxel-loaded vesicles from matrix metalloproteinase (MMP)-cleavable peptide-linked triblock copolymer, polyethylene glycol-GPLGVRG-b-poly(ε-caprolactone)-b-poly(3-guanidinopropyl methacrylamide) that showed considerably improved cellular internalization effectiveness (~10 folds) in contrast to its pure form. The developed vesicles exhibited greater cytotoxicity against MMP-overexpressing HT1080 cells and multicellular spheroids. When MMP-2 was used for treatment it resulted in cleavage of stealthy PEG shell. The vesicles undergo morphological transformation into fused multicavity vesicles and small nanoparticles, accompanied by a redistribution of PGPMA segments with 76% exposed to the outside .To increase chemical reaction efficacy, Li et al., created polymeric nanoreactors (NRs). They created therapeutic vesicular NRs (theraNR) that were loaded with glucose oxidase (GOD). When injected into normal tissues, the theraNR was inactive. The tumor acidity activates them precisely at the target location. They effectively destroy cancer cells by inhibiting their antioxidant capacity and perfusing tumors through a synergistic action .Li et al., created a polymersome nanoreactor based on a block copolymer prodrug that may enable new cancer oxidation/chemotherapy via selective activation at tumor locations. GOD-loaded polymersome nanoreactors were created by optimizing block copolymers to self-accumulate into polymersomes in an aqueous solution for encapsulation of GOD. Through high tumor oxidative stress, oxidation/chemotherapy and released CPT medicines synergistically killed cancerous cells and inhibited tumor development . By simply combining a couple of differently charged block copolymers (poly(amino acid)s and PEG) in an aqueous medium, Koide et al., produced a PICsome  membrane) with a size range of up to 10 µm. Semipermeable characteristic of PICsomes membrane was discovered using spectral analysis and confocal laser scanning microscopic imaging. Even in the existence of serum proteins, the PICsomes maintained a high level of physiological stability, indicating that they may be used in medical applications such as therapeutic vehicles .By incorporating stimuli-sensitive linkers into a crosslinking membrane network, Li et al., created responsive nano-reactors built on polyion complex vesicles. GOD could be protected by the fabricated ROS-responsive nano-reactor with self-improving catalytic glucose oxidation, which is attributed to stimuli-reactive vesicle development short of breakage and size-selective payload release pattern, to attain cytocidal role by oxidative stress induction and glucose starvation. Pyroptosis, an immunostimulatory type of cell demise, was produced by the GOD-loaded therapeutic nanoreactor .Dendrimers  were incorporated as promising drug nanocarriers, although some authors seem to be more satisfactory in their description as “polymer therapeutics” . These aPillay et al., developed folic acid loaded dendrimer-functionalized selenium nanoparticles (FA-PAMAM-SeNPs) with size <150 nm and zeta potential of >25 mV. The developed formulation demonstrated greater cell viability (>85%) in contrast to placebo nanocomplexes (75%), confirming the importance of selenium in the developed formulation. The developed formulation resulted in greater overall transgene expression that showed their improved receptor-mediated cellular uptake .Bartusik-Aebisher et al., developed a trastuzumab-dendrimer-fluorine DDS in which API was attached to dendrimers (fluorinated) via covalent bonding. They found that the potential of the developed formulation was more efficient than pure trastuzumab .Mota et al., encapsulated I-buthionine sulfoximine in folate-targeted polyurea dendrimers. Initially, the burst release was observed that was expected in these delivery systems. After 1 h, about 60% of drug release was found, reaching a plateau after 3 h. After 24 h, 90% drug release was found .Zhang et al., 2022 fabricated a third generation of poly(phosphorhydratzone) radical dendrimers and reported their action against the diagnosis of brain tumors . It was Knauer et al., 2022 designed a polycationic phosphorous dendrimer-based approach for the delivery of siRNA to target Lyn .Recently, the advancement of biological science managed novel developments in the area of in vitro cell culturing. Various novel innovations such as cells on chips/cell microarray need consistent supportive materials with decent biocompatibility, and cell adhesion and are simple to use . NanomatDespite their solute permeability and controllable release features, hydrogels are efficient carriers for medicines, proteins, and others. Hydrogels made of chitosan can create multilayered structures, which is why they demonstrate utility in distributing bioactive molecules including insulin and growth factors, and in cell and tissue organization. They can be administered to the body using very simple, minimal stress procedures for the administration such as injection and both ocular and nasal administration .Polymeric cubosomes are newly discovered inverse bicontinuous cubic mesophases. These are colloidal particles containing mesoporous block copolymers having well-defined reticulated pore networks. Kim et al., prepared polymer cubosomes using block copolymer which was synthesized by linking hydrophilic PEG and hydrophobic polyisoprene polymer blocks. The cubosomes were prepared via solution self-assembly of block copolymers . The bloZhang et al., developed the cubosomes loaded with a combination of anti-cancer drug cisplatin and paclitaxel for an effective targeting against cancer cells. In this work, a slow and sustained release was obtained by coating the cubosomes with poly-Ɛ-lysine. The coating prevented the initial burst of API resulting in a sustained drug release. TEM studies revealed very easily distinguishable spherical polyangular structures. Both initial reductions in burst discharge of drug and a slow, sustained discharge with time were revealed by in vitro studies. The cytotoxicity studies revealed the non-toxic behavior of cubosomes . The morBoge and co-workers developed the cubosomes for application in the delivery of peptide LL-37 for the ailment of skin infections. In the first strategy of development, the LL-37 was first added into a gel followed by dispersion into nanoparticles. The next strategy involved the adsorbing LL-37 onto preformed cubosomes. The last strategy consisted of the incorporation of LL-37 during their spontaneous formation in an ethanol/glycerol monooleate mixture. The pre-loaded cubosomes showed maximum efficiency in killing the bacteria .The merging of polymer technology with pharmaceutical research resulted in a quantum leap in terms of innovation in designing and developing novel DDSs such as nanotechnology-based delivery systems. The application of nanopolymers in pharmaceutical and biomedical fields are swiftly growing, such as developing scaffold in tissue engineering, designing a drug delivery system, ophthalmology, dentistry, bone repair, implantation of medical devices, prosthesis, and many other medical fields. It is possible to obtain desirable properties in a pharmaceutical polymer by modifying its physical and chemical characteristics such as molecular weight, type, composition, co-polymerization, biomimetic, and co-processed excipient .The discovery of the first synthetic drug delivery system sparked an interest in designing novel biodegradable polymers to replace the non-degradable polymer. The advent of bioadhesive polymer resulted in improved residence time and intimate contact between polymer and epithelial surface. Further advancements in polymer science led to the discovery of smart polymers that release the drug in presence of specific stimuli  . These nDrug molecules  are protected and stabilized by these polymers from environmental hazard degradation. Even though polymers have wide applications in the pharmaceutical industry, the criteria for proper selection and design of polymers is a challenging task and requires an in-depth understanding of its bulk properties  and surface properties  in addition to its physicochemical properties. The structure-activity relationship and polymeric matrices can be altered by changes in chemical composition and microstructural design . The polThe oral route has been explored as a non-invasive and preferred delivery route for numerous therapeutic agents. However, oral delivery suffers poor bioavailability and enzymatic instability ,86,87. MPolymeric materials being biocompatible, biodegradable, non-immunogenic, and functional that offer numerous advantages for drug delivery such as active drug targeting via surface functionalization and controlled release in the body . PolymerSeveral efforts have been made in ocular drug delivery to increase corneal assimilation by improving the drug residence time with the ocular surface through bioadhesive polymers . The pre® RS100 and Eudragit® RL100 which are official by USFDA as excipients for controlled drug delivery. The presence of a positive charge on nanoparticles resulted in ionic interaction with anionic mucin existing in the mucus layer of tear film leading to sustained release of drug and improved ocular penetration [Sharma et al., formulated polymeric nanoparticles using bioadhesive positively charged Eudragitetration . Mahor eetration . Mittal etration . Table 3etration ,147,148.IV infusion is one of the approaches for maintaining constant drug delivery which also avoids hepatic metabolism but the patient has to be hospitalized. Among methodologies to overcome traditional medical treatment, simulation of IV infusion by use of parenteral controlled release formulations has gained interest. This is the preferred route for delivery of active ingredients having a narrow therapeutic index and poor bioavailability. However, intramuscular and subcutaneous injection dosage form suffers rapid drug elimination. Continuous Active targeting can be achieved at different levels depending on the extent of penetration; it may occur at the organ, cell, or subcellular level. It is important to note that in solid tumors, even active targeting processes begin with the passive accumulation of the DDS in the tumor tissue, so any actively targeted carrier must satisfy the basic requirements outlined for passively targeted systems: the polymer/nanoparticle should be biocompatible, and the system must be stable in circulation, long-circulating, and of a size that permits efficient extravasation and accumulation in tumors. In addition, actively targeted systems are often more effective in general than passively targeted alternatives .Intravenous administration of biodegradable polymeric nanocarriers has been effectively employed for controlling and targeting drugs to a specific site of action. In particular, biodegradable nanocarriers formulated from PLGA have been extensively studied for sustained and targeted delivery of numerous agents . It is aPolymer-based nanocarriers have been extensively explored as topical formulations to improve cutaneous delivery owing to their physicochemical attributes which prevent degradation or denaturation of unstable drugs, reduce their toxic side effects through controlled release, and enhance their cutaneous penetration across the biological membrane more precisely skin barrier due to their ability to increase the concentration gradient . PolymerNatural polymers tend to develop hydrogels which render them suitable carriers for peptides, oligonucleotides, proteins, and water-soluble APIs for improving the absorption and targeted drug delivery of polymeric nanocarriers for topical administration. Chitosan-based nanocarriers have been widely used among the natural polymers for topical delivery. An N-deacetylated derivative of chitin, chitosan is a cationic polymer that is made up of glucosamine units. Its additional anti-oxidant, anti-inflammatory, and anti-microbial features make it an appropriate carrier for therapeutic delivery. Nanomedicine is a new area integrating nanotechnology with pharmaceutical and biomedical sciences, intending for the development of medicines with improved safety and toxicological profiles . In drugIn nanomedicine, polymeric nanoparticles serve two purposes  . Copaxoni/v administered formulations are shown in The composition and structure of polymeric micelles can be precisely modified to achieve specific release characteristics and drug loading. The only approved polymeric micelle is a traditional formulation of oestradiol (Estrasorb™). The use of modern co-block polymers led to the development of polymeric micelles at a lower critical micellar concentration with high stability . Few polThe various patented reported polymeric nanocarriers intended for oral, ocular, topical, and parenteral administration are enlisted in Most of the polymeric nanocarriers are generally regarded as safe owing to the interesting physicochemical characteristics of the polymer which render them biocompatible and biodegradable. Copolymers PLA and PLGA acid are the most extensively employed biomaterials for polymeric nanocarriers, that undergo hydrolytic degradation within the body leading to the generation of endogenous lactic acid and glycolic acid monomers which are metabolized via Kreb’s cycle leading to minimal systemic toxicity . HoweverGrabowski and Hillaireau studied the toxicity of surface-mediated polymeric nanoparticles on human-like THP-1 macrophages wherein cell mitochondrial activity, apoptosis detection, ROS, and pro-inflammatory cytokines production were chosen as toxicity criteria. Positively, negatively and neutral PLGA nanoparticles were fabricated with chitosan, poloxamer 188, and PVA stabilizers. The stabilizer-free PLGA nanoparticles did not exhibit any sign of toxicity at or above a therapeutic concentration of 0.1 mg/mL. However, PLGA nanoparticles prepared using stabilizers exhibited cytotoxicity above the therapeutic level of 0.1 mg/mL, thereby confirming the safety of the polymeric nanocarriers and the toxicological contribution of stabilizers used in the polymeric nanocarrier formulation .® S100 polymeric nanoparticles through genotoxicity studies [Rejinold and Muthunarayanan reported non-toxicity of the chitosan-g-poly-N-vinyl caprolactone nanoparticles in an array of cell lines at 100–1000 µg/mL . Similar studies . Singh a studies . PolymerPolymeric nanocarriers have become a prominent field of research in the arena of drug delivery since they can improve pharmacokinetic and bioavailability that results in effectiveness of various drugs, enhanced hydrophilicity, reduction of interaction with plasma and cellular proteins, and greater desposition of drug at target sites . Oral, iVarious factors govern the assimilation of the drug such as drug solubility, mucosal permeability, and stability in the GI tract milieu. Efforts are ongoing to tackle these issues have focused on physicochemical, biochemical, biological, and metabolic barriers that are responsible for limited assimilation. Different pharmaceutical technologies and DDSs such as nanocarriers, micelles, cyclodextrins, and lipid-based carriers have been discovered for improving the assimilation of a drug.GI tract leads to experience a varied range of pH, enzymatic degradation and low permeability across the membrane. Polymeric NPs may be an auspicious strategy that can improve stability to crossing the mucus barrier or interact with a mucus layer and increase residence and contact time .Oral, aerosol, and nasal vaccination is a novel immunization technique that employ nanocarriers to administer vaccine. Therapeutic agents and vaccines with lower penetration potential can be administer via transmucosal route while maintaining their biological activity. Nowdays, nanocarriers for transmucosal delivery are gaining more attention since delivery via oral mucosa helps evade first-pass metabolism, improves bioavailability and acts as a means of rapid drug transport to the systemic circulation. The oral transmucosal vaccine delivery by nanocarriers is the most upcoming innovation in efficient vaccine delivery ,200.Over the past few decades, polymer science has evolved to mediate safe and efficient drug delivery for the ailment of several health conditions. Over the years, the usage of polymers has been limited to oral delivery, but critical advances in polymer science such as the development of biodegradable nanosized carriers have accelerated their use in parenteral formulations. Further innovations in the application of polymers have led to a more sophisticated approach, thereby improving the pharmacokinetics, decreased toxicity, stealth effect, cell or organ-specific targeting, and controlled release of the drugs; these innovatioms have made it possible to administer two or more drugs or imaging/diagnostic agents within the same drug delivery system, leading to more effective therapy ,201,202.New technologies and advances in the synthesis and manufacturing process such as 3-D printing, supercritical fluid technology, and nanoengineering are revolutionizing polymer-based drug delivery systems . Scale-uNanomedicine has evolved over the last several decades from biologically inert substances to increasingly intelligent systems aiming at improving in vivo functioning. But, we must acknowledge that most systems depend on logical explanations-including some over-explanation rather than conclusive proof, which is a pivotal event in nanomedicine’s progress. The importance of investigating nano-bio interactions and desired functioning at the molecular, cellular, and tissue levels is often ignored .An overall change in global markets, technological innovations, and data on the pathophysiological and cell-based mechanisms should be considered that can outline the future progress for polymers in drug delivery.Polymeric nanocarriers have a great potential for clinical applications. They are expected to perform both diagnostic and therapeutic functions due to their interesting physiochemical characteristics which offer numerous advantages such as biocompatibility, biodegradation, non-immunogenicity, non-inflammatory, low toxicity, prolonged drug release, and active site targeting, rendering them safe for therapeutic applications at a certain concentration. At present, few polymeric systems can be employed for diagnostic and therapeutic applications. Among nanomaterials, polymeric nanocarriers have been the exceedingly explored delivery vehicles due to their wide range of solubility (hydrophilic and lipophilic surfaces) and self-assembling properties. They offer solubilization of hydrophobic or poorly soluble molecules, sustained drug release, and protection to encapsulated drugs from degradation and metabolism. The polymeric nanocarriers are considered as the best ones due to their good drug loading capacities, biodegradability, and stimuli-responsive control.Polymeric nanocarriers are safe for the parenteral route of administration than their surfactant counterparts. They are kinetically stable and hence dissociate very slowly in blood, resulting in extended circulation time. Recently biopolymeric nanocarriers have immerged as storms which are developed using polymers obtained from natural sources. These biopolymeric nanocarriers offer better biodegradability, biocompatibility and could be the next generation nanocarriers for drug delivery. In the literature, there are several studies for developing new generation polymeric nanocarriers to obtain future smart and multifunctional nanomedicine. Polymeric nanocarriers have many challenges for a carrier system such as biocompatibility, biodistribution, side effects, and biological barriers. Their biodegradability and biocompatibility profiles should be critically investigated before utilization in human clinical trials. One major concern that requires immediate attention is the regulatory concerns regarding the approval of polymer-based carriers since establishing new guidelines and policies seem to be in the initial stages of development. New criteria for manufacturing, testing, and storage of these nanosized polymeric materials should be defined by the regulatory agencies . So, the


We describe the challenges and lessons learned from two studies using linked administrative data from health, education and social care sectors to evaluate the Family Nurse Partnership (FNP), an intervention supporting adolescent mothers in England(E) and Scotland(S). We present recommendations for studies using linked administrative data to evaluate complex interventions.We constructed two cohorts of all mothers aged 13-19 giving birth in NHS hospitals in England and Scotland between 2010-2016/17 using linkage of mothers and babies in hospital admissions data , and identified FNP participation through linkage to FNP programme data. We additionally linked to health, educational and social care data for mothers and their babies . We used these data to identify key risk factors for enrolment in the FNP, assess the effect of the FNP on maternal and child outcomes, and determine programme characteristics modifying the effect of the FNP.Key challenges: characterising the intervention and usual care, understanding quality of multi-sector data linkage, data access delays, constructing appropriate comparator groups and interpreting outcomes captured in administrative data. Lessons learned: evaluations require detailed data on intervention activity (dates/geography), and assessment of usual care, which are rarely readily available and are time-consuming to gather; data linkage quality is variable/not available, making defining denominators challenging; data access delays impeded on data analysis time; unmeasured confounders not captured in administrative data may prevent generation of an appropriate comparator group. Recommendations: Characteristics informing targeting should be explicitly documented, and could be enhanced using linked primary care data and information on household members (e.g. fathers). Process evaluation and qualitative research could help to provide better understanding of mechanisms of effect.Linkage of administrative data presents exciting opportunities for efficient evaluation of large-scale, complex public health interventions. However, sufficient information is needed on programme meta-data, targeting and important confounders in order to generate meaningful results. Study findings should help stimulate exploration with practitioners about how programmes can be improved.


Glioblastoma remains the most malignant and intrinsically resistant brain tumour in adults. Despite intensive research over the past few decades, through which numerous potentially druggable targets have been identified, virtually all clinical trials of the past 20 years have failed to improve the outcome for the vast majority of GBM patients. The observation that small subgroups of patients displayed a therapeutic response across several unsuccessful clinical trials suggests that the GBM patient population probably consists of multiple subgroups that probably all require a distinct therapeutic approach. Due to extensive inter- and intratumoral heterogeneity, assigning the right therapy to each patient remains a major challenge. Classically, bulk genetic profiling would be used to identify suitable therapies, although the success of this approach remains limited due to tumor heterogeneity and the absence of direct relationships between mutations and therapy responses in GBM. An attractive novel strategy aims at implementing methods for functional precision oncology, which refers to the evaluation of treatment efficacies and vulnerabilities of (ex vivo) living tumor cells in a highly personalized way. Such approaches are currently being implemented for other cancer types by providing rapid, translatable information to guide patient-tailored therapeutic selections. In this review, we discuss the current state of the art of transforming technologies, tools and challenges for functional precision oncology and how these could improve therapy selection for GBM patients. Targeted therapies hold the promise to eradicate cancer cells through the inhibition of specific oncogenic proteins . The effGlioblastoma (GBM) , still tTo identify appropriate drug targets for GBM, large-scale sequencing programs were initiated to uncover disease causing genetic aberrations ,12,13. OThe failure of these trials could in part be explained by a lack of sufficiently precise selection procedures to enroll the appropriate patients that could actually benefit from the given therapy . So far,Finally, GBM tumor cells can also acquire de novo resistance upon therapy. Indeed, in an initially TMZ-responsive tumor cell population, resistance can easily be acquired by upregulating DNA-repairing enzymes such as MGMT or by inactivating the DNA mismatch repair (MMR) system, eventually leading to tumor recurrence . At this®) [In spite of the overall inability to treat GBM with durable clinical outcomes, clinical trials sometimes describe small groups of patients that did show a clinical response. For instance, in the multicentric INTELLANCE trial (NCT01800695), a small group of patients did experience clinical benefits from the treatment. Recurrent GBM patients that harboured an EGFR amplification were treated with a combination of TMZ and anti-EGFR monoclonal antibody coupled to a toxin  ,26, Thes®) . The ove®) , but bei®) . This isfunctional interpretation [dynamic, faster, more detailed and potentially longitudinal insights into the ability of cells to respond to therapy in a genotype agnostic way, although methods to do so remain difficult. In this light, approaches for assessing differential drug responses are gaining traction by which live tumor cells are ex vivo exposed to various therapeutic insults, while a chosen cellular response is carefully monitored—an approach coined functional diagnostics, functional oncology or functional precision medicine [Major efforts are currently being put in matching specific (genetic) cancer features to drug responses . Howeverretation   and baseline pathological (tissue) analyses. Indeed, coming to a proper patient-tailored interpretation will require that the different levels of information  are integrated into an overarching framework to steer clinical decision making. Overall, the goal of functional testing is to bring forward personalized medicine to patients diagnosed with complex disease entities, where treatment options are rather limited. In other words, functional tests ought to facilitate the matching of each patient to the most beneficial treatment. This being said, ex vivo drug exposure of freshly isolated tumor biopsies can directly inform on cell death, alterations in signaling networks, cellular phenotype and morphology or even tumor cell–tumor microenvironment (TME) crosstalk and adverse events in normal tissue. Certainly, the type of functional readout informing on tumor and non-malignant cellular vulnerabilities would largely depend on the mechanism of action of the given treatment. Typically, investigators rely on commonly available, FDA-approved therapies or drugs in clinical trials where dose-escalating studies where safety and tolerability of the therapy of interest has been already assessed and approved.In the particular case of GBM, the functional screening method should not only be able to map each tumor in great detail—given GBM’s high degree of intra-tumoral heterogeneity —it shoulThe development of functional diagnostic assays strongly depends on the availability of representative cancer models that maximally capture the genetic and phenotypic features of patients’ tumor. So far, in vitro cancer research has been relying on so-called conventional cancer cell lines , which, https://clue.io/about, accessed on 2 July 2022), comprising extensive and continuously expanding connectivity maps of protein, RNA expression and/or morphological changes in cancer cells, as a response to drug perturbation in addition to drug repurposing library of FDA-approved drugs, clinical trial drugs and pre-clinical compounds. All these large drug-screening resources are invaluable for exploring and understanding the mechanisms of various classes of compounds, drug repurposing and matching (combinations of) genomic mutations with functional responses over time [For the initial identification and evaluation of drug targets, compound design and efficacy testing, patient-derived cell cultures and organoids provide an excellent platform to preclinically explore and evaluate pharmacological responses across individual tumors. Given the fact that such models more faithfully recapitulate features of the tumor-of-origin, drug screening across cohorts of such models, offers the identification of therapeutic options that can be immediately linked to particular features present in the identified models . As suchver time . Howeverver time . Similarver time , reducinver time  and co-cver time ,43,44.Drug screening in PDX clinical trials were executed for various cancer types and solid tumors . With thTo circumvent these issues and in line with the rapidly advancing organoid technology, organoid cultures were successfully established from patient specimens or through pluripotent stem cell reprogramming. In either case, organoids represent self-organizing, 3D systems which highly resemble the tissue from which they were derived. PDOs conserve tumor heterogeneity and TME components, tissue architecture, molecular and functional features. PDOs can be efficiently expanded over time while conserving patient-specific genomic features and intra-tumor heterogeneity, which could be reliably correlated with functional responses to therapeutics . Non-malIn general, tumors including GBM, release cells and cellular content into the bloodstream or cerebrospinal fluid (CSF). These biomarkers are shed from the tumor residing site in form of circulating tumor cells (CTCs), proteins, cell-free nucleic acids and extracellular vesicles (EVs), accordingly systemized as liquid biopsies . As suchCurrent research is focused on refining methods for CTC isolation, ex vivo expansion and the establishment of CTC cell lines ,79. Addi9 blood cells); therefore, efficient CTC isolation which recapitulates intra-tumoral heterogeneity and enables functional assessment is still far beyond the reach of GBM patients [Owing to their location, brain tumors are challenging for surgical resection. Even when the tumor is accessible, the invasive surgery and biopsy collection present a risk of swelling and neurological damage. As patients receive an MRI scan within 12 weeks of treatment, contrast-enhancing lesions that are revealed on the images can indicate tumor progression, but might also be caused by post-radiotherapy edema, termed as pseudoprogression, which can spontaneously resolve ,81. At tpatients ,80,81.Hence, the ideal model for rapid functional assessment of drug sensitivity in GBM would be a system which maximally preservers the native cellular integrity  and inteAn auspicious high-throughput drug screening methodology has emerged with microfluidic devices . The chiCurrently, numerous clinical trials are testing the efficacy of functional diagnostic methods in the prediction of patients’ outcomes . For insFor GBM, several clinical centers are engaging into functional measurements and ex vivo tumor profiling. At the Oslo University Hospital, GBM patients recurring or progressing after first-line treatment are recruited in the ISM-GBM study, in which individual cancer stem cells (CSCs) are first propagated in vitro as PDCL models, and subsequently subjected to high-throughput screening (HTS) towards FDA/EMA approved drugs (NCT05043701). An individualized drug combination would be prescribed to each patient, based on the outcomes from functional tests ,92. SimiA handful of preclinical studies have been initiated for GBM, where drug vulnerabilities of organoids or PDCLs are going to be measured in in vitro assays . In the case of NCT04180046, primary GBM cell lines are going to be established from patient samples, in order to pathologically characterize the presence of GBM-related hallmarks  and measure dose-response effects of natural and synthetic drugs . Similar3D-PREDICT (NCT03561207) is a multicenter prospective study which among other cancer types  is also enrolling patients diagnosed with adult high-grade glioma (anaplastic astrocytoma and GBM). Here, extensive molecular profiling and direct ex vivo drug testing of patient tumor materials are carried out, with the ultimate goal being to make personalized medicine recommendations. Thus, the primary outcome measure of this trial is correlation between patient outcomes and functional results. Initially, four patients have been included in this study generating PDCLs and organoids for these patients. This enables the characterization and comparison of patients’ biopsies and individual tumor-derived models at genomic and transcriptomic level and further performing functional tests, such as clonogenic assays and 3D-PREDICT assays. The latter is practically a viability test of 3D spheroids treated with a mono-drug library at different concentrations for an adapted period of time, which extrapolates IC50 values and stratifies the response predictions as unresponsive, moderate and responsive. Based on these results, tumor spheroids derived from one of the four included patients  diagnosed with GBM featuring ependymoma regions was found sensitive to JAK/STAT and mTOR inhibition. In this manner, clinicians opted to treat this patient with a combination therapy consisting of ruxolitinib and everolimus. After his seventh progression, the patient was classified as having stable disease for more than 4 months post-treatment . A seconAn Ex Vivo DEtermiNed Cancer Therapy (EVIDENT) trial has been recently initiated (NCT05231655), which aims at determining the efficacy and feasibility of ex vivo screening in prediction of standard-of-care therapy outcome and novel therapy identification, including patients diagnosed with solid tumors . This trial seems most prominent because solid tumor biopsies will be directly screened and the response would be quantified and correlated to patient clinical outcome.Conventional genetic-based matching of patients and treatments may be beneficial for only a fraction of cancers, where the oncogene driver mutation is uniform and maintained at a stable level among the cancer cell population. Such examples are seen in HER2-positive breast cancers , BRAF muA growing appreciation of biobanking, the generation of living biobanks of patient-derived models and ex-vivo treatments have the potential to enhance the development of rationally selected combined therapies and guide prospective clinical trials. As the number of clinical trials and assessments of functional diagnostic platforms increase, we anticipate the implementation of this strategy in standard clinical oncology practice. Importantly, the integration of molecular characterization data, functional profiles, clinical parameters and patient follow-up from a multitude of individuals into a single database might even enable informing clinical decisions for patients from whom sufficient tumor material may not be available. Finally, while multiple endeavors are ongoing to implement functional diagnostics to select appropriate therapeutic options for GBM, it still remains to be seen which approach will prove to be the most predictive and clinically relevant.


The population of childhood cancer survivors (CCS) has grown rapidly in recent decades. Although cured of their original malignancy, these individuals are at increased risk of serious late effects, including age-associated complications. An impaired immune system has been linked to the emergence of these conditions in the elderly and CCS, likely due to senescent immune cell phenotypes accompanied by low-grade inflammation, which in the elderly is known as “inflammaging.” Whether these observations in the elderly and CCS are underpinned by similar mechanisms is unclear. If so, existing knowledge on immunosenescent phenotypes and inflammaging might potentially serve to benefit CCS. We summarize recent findings on the immune changes in CCS and the elderly, and highlight the similarities and identify areas for future research. Improving our understanding of the underlying mechanisms and immunosenescent markers of accelerated immune aging might help us to identify individuals at increased risk of serious health complications. Each year, ∼300,000 children are diagnosed with a form of cancer globally. . Cancer Many conditions affecting CCS in early/mid-adulthood are age-associated; cardiovascular diseases, diabetes mellitus, hypertension, frailty and cancer increase during aging . Aging iThe immune systems of young adult CCS exhibit common features with those of the elderly . High prIn this review, we discuss the possible adverse effects of childhood cancer treatment on the immune system and the potential links to enduring ill health in CCS. We compare these effects to the senescent immune system in the “healthy” elderly, assessing the evidence for parallel versus distinct mechanisms in these two populations. We conclude by speculating on how we might exploit our knowledge of cellular, molecular and epigenetic mechanisms of immunosenescence to improve health and wellbeing in CCS and we highlight possible directions for future research in the field.Many of the initial studies defining immunosenescence were performed in unique cohorts of centenarians, allowing the identification of immunosenescent cellular phenotypes/markers that enable successful aging . These dImmune changes in both the elderly and CCS have now been identified ; below w+ and CD8+ T cells are well established , central memory , effector memory  and terminal effector T cells  (Age-associated changes in the subpopulation frequencies of CD4ablished . Multi-pd CCR7−) . In gened CCR7−) . Interesd CCR7−) , T-cell d CCR7−) .INK4a expression, and elevated secretion of inflammatory cytokines , IgM memory (IgD+/CD27+), switched memory (IgD−/CD27+), and late/exhausted memory . While total B-cell counts remain relatively unchanged in the peripheral blood in healthy adults,  was shown to accumulate in aged mice . These cThe profound age-associated changes in the immune system underlying immunosenescence are mainly linked to adaptive immunity. However, research conducted over the past two decades has demonstrated many functional age-related alterations in innate immune cells . We outlMonocyte/macrophage activation status and function are essential for appropriate immune responses against pathogens and transformed cells and for mounting the adaptive immune response. Data from human and animal studies show age-related dysfunction of the monocyte/macrophage system . A detai+ populations, have been observed in healthy males and females (20–84 yo)  . The CD1d in CCS   upon in vitro stimulation with retinoic acid in healthy elderly (Dendritic cells (DCs) are professional antigen-presenting cells that can prime naive T-cell activation and effector differentiation. In general, the phenotypes and numbers of DCs are largely unaffected by aging,  although elderly . Since p elderly . The samConcerning DC function, the following alterations have been reported in elderly individuals: decreased capacity to phagocytose antigens,  migrate,bright NK cells as a possible consequence of limited production of new NK cells,  cells are a subset of cytotoxic non-T lymphocytes that promote an early innate immune response by recognizing and killing virus-infected and tumor cells. Thus, quantitative and functional NK cell defect may lower resistance to infections and protecting effect against tumors in elderly/CCS. While the distribution of NK cell subsets seems to be altered in elderly individuals, the data are inconsistent . A decreK cells,  with a c elderly . Similar elderly . The agi elderly . If alteThus far, we have discussed how age-related alterations of individual immune cell types and their function underlie aging. However, there are also data to suggest that the age-related dysfunction of effector immune cells is in fact inherited from their progenitors. Throughout our lifespan, encounters with various stressors  progressively impair various cellular repair mechanisms. A consequence of inadequate repair is hematopoietic stem and progenitor cell (HSPCs) aging . Key marAged HSPCs in humans are associated with perturbed lymphopoiesis  and incrAnother age-related phenomenon of clonal hematopoiesis is associated with a risk of developing hematologic malignancies and an increase in specific somatic mutations in peripheral blood cells with age . These m+CD38− and CD34+CD38+,  inhibitors, and caloric restriction . AMPK acreceptor . Regardiutophagy . Sirtuinutophagy . For exautophagy  and decrutophagy . Althougutophagy . Thus, butophagy .de novo T cells and increasing thymic output. Another thymic regeneration strategy involves reprogramming fibroblasts with forkhead box N1 (FOXN1) to form thymic epithelial cells and create a functional thymic stroma, ) and 4) presence of health complications   and newlications . DetectiOne notable effect of cancer therapies, including chemotherapy,  radiotheHere, we hypothesize that the crosstalk of telomere control mechanisms and chronic inflammatory disorders is another crucial factor in the development of accelerated senescence in CCS. The fact that telomere shortening is induced by anticancer therapy, and at the same time clearly correlates as an additional risk for further development of cancer makes telomere length assessment a strong candidate for stratification of CCS at different risks of further adverse effects.INK4a and epigenetic changes. Moreover, p16INK4a expression in T cells correlates with intensity of chemotherapy and frailty in the CCS, and thus cellular senescence has been proposed to be associated with premature aging in CCS  of healthy elderly,  and low risk (reduced monitoring) subpopulation groups. The appropriate patient stratification would enable increased screening and pro-active early clinical management of these patients.The parallels between immune senescence in the elderly and immune alterations seen in CCS are, in some cases, quite notable; however, the definition of altered immune states in CCS is incomplete. The specific effects of different cancer treatments on the young immune system are yet to be fully understood, and long-term follow-up studies are overall lacking. Moreover, another hurdle is that the data available for CCS immune cell phenotype and CLGI consist almost exclusively of leukemia survivors, while the comorbidities cover multiple cancer types—leukemias, lymphomas, brain and nervous system tumors etc. The question is, whether the studies were done on leukemia survivors on purpose due to the highest incidence reaching up to 25% of all pediatric neoplasms,  and thusAnother possible explanation for the differences in the immune features between CCS and healthy peers is that immune alterations may pre-dated treatment, or even the cancer itself. Therefore, future research should be directed to investigate mechanisms behind these alterations resulting in identification of immune parameters allowing prognosis prediction. One such predictive pre-treatment immune cell characteristic has been already attributed to the neutrophil-to-lymphocyte ratio in pediatric patients with solid brain tumors who have elevated neutrophils and decreased lymphocyte counts compared to children with unrelated diagnoses prior to surgery . These dIn summary, we consider that the late adverse effects in CCS are similar to the immunosenescence-related age-associated morbidities observed in the elderly. Our belief is that immunosenescent phenotyping could provide crucial information for patients struggling with the therapy-induced morbidities that occur in CCS. This link is especially relevant considering the growing research emphasis on ways to ameliorate immunosenescence in the elderly. If relevant parallels exist between these two patient groups, then similar strategies could markedly benefit CCS. The increasing numbers of CCS are an undisputable testament to one of the biggest successes of oncological research in previous decades. The challenge moving forward is to ensure that cancer cures are not necessarily accompanied by a future of chronic conditions with the potential to limit both quantity and quality of life.


Implantable medical devices have been developed to provide multifunctional ability to numerous bioapplications. In the scope of orthopaedics, four methodologies were already proposed to design implant technologies: non-instrumented passive implants, non-instrumented active implants, instrumented passive implants and instrumented active implants. Even though bone replacements are among the most performed surgeries worldwide, implant failure rates can still exceed 10%. Controversial positions multiply in the scientific community about the potential of each methodology to minimize the burden related to implant failures. In this perspective paper, we argue that the next technological revolution in the field of implantable bone devices will most likely emerge with instrumented active implants as multifunctional smart devices extracorporeally controlled by clinicians/surgeons. Moreover, we provide a new perspective about implant technology: the essence of instrumented implants is to enclose a hybrid architecture in which optimal implant performances require both smart instrumentation and smart coatings, although the implant controllability must be ensured by extracorporeal systems. Although only non-instrumented passive and active technologies are available in the market, no agreement was achieved so far concerning the best methodology to minimize implant failures. At stake are millions of total hip replacement (THR) and total knee replacement (TKR) surgeries performed per year worldwide  highlights their worldwide societal impact. In the scope of orthopaedics, technology innovation has triggered the development of four methodologies to design intracorporeal biomedical devices: 1) non-instrumented passive implants, only designed to restore mobility and reduce pain, and performance optimization based on geometry and materials; 2) non-instrumented active implants, focus on (bio)chemical modifications of surfaces; 3) instrumented passive implants, designed to monitor biomechanical quantities orldwide , as a reorldwide . Althougorldwide . This isorldwide . The socorldwide . Besidesorldwide ; (b) surorldwide ). Ultimaorldwide .Osseointegration is an essential process to establish an asymptomatic and stable long-term fixation . An accuThis paper then provides a deep discussion concerning the next technological revolution in the field of implantable bone devices. All analyses will be focused on uncemented implants, as: (i) long-term cemented fixations are harder to achieve; (ii) an increasing number of young and active patients is expected for the forthcoming decades; (iii) periprosthetic interfaces with bone-implant biocontact are easier to control. Our conclusions can be generalized for a wide range of implantable medical devices, including for orthopedics, neurology, ophthalmology and psychiatry.Current methodologies used to improve the performance of these bone implants have been based on the optimization of their geometry and materials . ImplantChemical and biochemical modifications of the implants’ surfaces have been considered the most effective methodology to design non-instrumented active implants . Two genin vivo  optimization the implant designs and materials; 3) performing preclinical testing of new implant technologies; 4) monitoring the rehabilitation process after implant insertion.The concept of Instrumented Implant is another approach that aims to optimize the performance of implants ; 2015). in vivo . The devi) Their design can be very complex, increasing as their multifunctional ability increases.ii) Controllability of the bone-implant interface behaviour is rather reduced: (a) the delivery dynamics does not consider the bone-implant  states; (ii) bioactivity, osteoconductivity and osteoinductivity cannot be changed after implant insertion (pre-established actuation mode); (c) long-term release of bioactive substances  most likely will be quite hard to implement and according to non-personalized patterns.iii) The ability to deliver different stimulations to target tissue peri-implant regions will most likely be quite difficult to attain.iv) Time-dependent dosing release can be significantly hard to monitor.Although all these technological breakthroughs hold potential for future implementation of high sophisticated biodevices, the most effective methodology to ensure revision-free outcomes is not obvious to identify. Indeed, clinical outcomes have not been able to suggest the ineffectiveness of passive implants or the effectiveness of active implants. Besides, no strong evidences explain why research must be focused on geometry and biomaterials rather than in the design of instrumented implants. In order to emphasize what research lines must be primarily pursued to implement failure-free bone implants, the potential of each methodology to design implants capable of optimal performances was recently inferred. By conducting an optimality analysis to passive and active implants using the Pontryagin Maximum Principle, it was demonstrated that optimal implant performances require some kind of sensing, actuation, communication and self-powering systems : to chanCurrent non-instrumented active implants are designed without implant-clinician communication. Monitoring of the bone-implant fixation cannot be performed throughout the daily life of patients as only imaging methods  are used . Even thAlthough instrumented passive implants strongly contributed to the development of smart implants, they did not bring about a technological revolution in the field of implantable bone devices. Their monitoring systems were not designed to support therapeutic actuations in peri-implant regions : their fInstrumented active technologies has emerged as a leading research topic that aims to design implants comprising biophysical therapeutic actuation, bone-implant interface sensing, implant-clinician communication and self-powering ability  Soares ; 2015). Staphylococcus aureus , such as the three-electrode cathodic voltage-controlled electrical stimulation  remain an unsolved problem. Besides, small-scale electromagnetic harvesters are not currently able to harvest voltages exceeding 10 V for low-frequency-excited operation. Piezoelectric energy harvesting are being investigated to overcome this limitation by incorporating stacked piezoelectric elements with multiple layers , even thUp to today, no miniaturized processing system was already developed to manage sensing, stimulation and self-powering operations, most likely because fundamental research is still being conducted to explore the effectiveness, feasibility and controllability of sensing and stimulation systems. It should be noted that all instrumentation and electronics incorporated within smart implants can be disabled, such that they only operate as non-instrumented passive or non-instrumented active implants. In fact, instrumented implants will only perform optimal trajectories from failure states to non-failure states if implant geometries, materials and textures are optimized. Moreover, multifunctional instrumented active implants are not technologies apart from non-instrumented active implants: the former includes the latter . The truMultifunctional smart implants establish a wide range of new means of therapeutic actuations. The expertise of clinicians/surgeons can be used to administrate appropriated stimulatory therapies, taking advantage of decision-making based on previous research studies and on outcomes obtained from other patients. Further sophistication may be attained if active implants are designed with the ability to autonomously define the stimulatory therapies. Nowadays, available technology allows transferring the human knowledge to data storage systems located inside instrumented implants, and artificial intelligence algorithms can learn from the inherent variability of bone-implant interface states of each patient. Smart therapeutic actuation can be explored as a time-dependent complementarity: biophysical stimuli delivery can be exploited as the main method to avoid implant failures, although it can be programmed as an adjuvant strategy to enhance the therapeutic ability of smart biocoatings. The capability for osteogenic, osteoconductive and osteoinductive stimulations are thus inherent to instrumented active implants.This new concept model of Instrumented Active Implant as a hybrid technology also presents some constraints. The decision-making of clinicians/surgeons is not error-free, and the probability to be administrated non-optimized therapies increases as the number of monitored bone-implant states decreases. Other risks cannot be disregarded, such as the cytotoxic and genotoxic risks related to implant fractures, which are significantly higher when implant systems are biointegrated in young/active patients. Biocompatible cosurface stimulators can be already constructed, but manufacturing of biocompatible sensing, processing, energy storage and communication systems is still a hot topic in chemistry and material sciences. Risks can be minimized if instrumentation and electronic systems were fully encapsulated using biocompatible materials, as well as by miniaturizing these inner systems to minimize the hollowed structures inside implants. The higher the number of sensing/acting structures, the more complex electronic systems will be. Therefore, the most reliable scheme seems to be the incorporation of sensors and stimulators in the peri-implant regions where more pronounced bone loss usually occurs . Another underestimate issue is the inherent osteogenic effects that can be promoted by extracorporeally-induced magnetic fields stimulating the bone-implant interface when communication and/or powering operations are performed. Although it seems reasonable to assume much lower exposure times to these stimuli when comparing with those delivered for therapeutic actuation, these extracorporeally-originated stimuli can interfere with administrated control trajectories imposed to bony proliferation, differentiation and mineralization. Finally, although smart implants will be more expensive than passive implants, the societal burden will be significantly reduced, as the number of years lived with disability will most likely be minimized.Several challenges will arise in the development of instrumented active implants. Promising results were achieved concerning the performance of cosurface capacitive stimulation to control bone remodeling, but the osteogenic effects of a wide variety of stimuli are still unknown. Idiosyncrasies of patients may difficult the identification process towards optimality of therapeutic actuations. The therapeutic complementarity approach between biophysical stimulation and active biocoating stimulation will require deep multidisciplinary research, in particular in patients suffering from severe bone remodeling disorders (e.g. osteoporosis). To ensure everlasting operation of instrumented implants, smart adaptive self-powering systems must be designed, since the performance of electromagnetic harvesters is function of time-varying body motion dynamics driving the harvesters. Moreover, such harvesters must be designed considering geometric optimization prior to fabrication, taken into account the tridimensional body motions during the routine activities of patients and according to their ageing processes. Indeed, the amount of energy harvested may impose limits to the magnitude of biophysical stimuli that can be delivered to the peri-implant bone volume and, consequently, the amount of therapeutic actuation trajectories may be reduced. Storage systems will most likely be necessary, mainly to power implants during the perioperative period, in old ages and when demanding therapies are required . Finally, as these implants always have to be manufactured with inner hollowed structures, it is imperative to research new materials with bulk properties that can minimize fracture risks. An overview of the main strengths and limitations of multifunctional smart implants is presented in Despite constraints and challenges to be addressed in future research, there are strong indicators highlighting a coming technological revolution in the field of implantable bone devices: implant technologies emerging as hybrid instrumented multifunctional devices with ability to promote both smart biophysical stimulation and smart active biocoating stimulation.


The acronym MINOCA  refers to myocardial infarction with normal or near-normal coronary arteries on invasive angiography. The broad spectrum of pathological mechanisms responsible for myocardial injury in MINOCA makes defining the exact underlying etiology challenging. We report the uncommon case of an acute myocardial infarction with normal coronary arteries suggestive of MINOCA caused by paradoxical coronary embolism due to a wide right-to-left shunting through a patent fossa ovalis. Integrated multimodality imaging diagnostic work-up, including cardiac magnetic resonance, transesophageal contrast echocardiography, and transcranial contrast Doppler, has been crucial for identifying the most likely mechanism underlying MINOCA. She had active smoking and hypertension as cardiovascular risk factors. On admission, the patient was asymptomatic, physical examination was unremarkable, and vital parameters were within normal limits; laboratory tests showed normal values for both blood cell counts and C-reactive protein; a nasopharyngeal swab was obtained resulting negative for severe acute respiratory syndrome-coronavirus-2 infection. Transthoracic echocardiography showed normal morphology and function of the cardiac chambers and excluded valvular and pericardial diseases The recent medical history revealed a hospitalization for the same clinical picture three months earlier with a final diagnosis of non-ST segment elevation myocardial infarction (NSTEMI) with fully normal coronary arteries on a comprehensive diagnostic invasive angiography, including testing for epicardial coronary spasm and microvascular dysfunction. On that occasion, the patient was discharged on dual antiplatelet therapy, which she was still taking at the time of the second hospital admission for chest pain. Of relevance, the patient reported a recent episode of transient edema of the right lower limb and recurrent episodes of dizziness in the last few months.A 68-year-old female was admitted to the Coronary Care Unit after being evaluated at the Emergency Room for sudden-onset oppressive chest pain lasting 15 min and radiating to the back combined with subtle nonspecific ST-segment changes at the lateral site on a 12-lead electrocardiogram (ECG) and elevated high-sensitive troponin I .Given the characteristics of the chest pain, we performed an urgent computed tomographic angiography which ruled out both acute aortic syndrome and pulmonary embolism. Therefore, coronary angiography was repeated, confirming normal coronary arteries , and raiSupplementary Video 1.Click here for additional data file.Then, owing to the anatomic predisposing condition to paradoxical embolism and the recent history of transient oedema of the right calf and episodes, the patient was screened for deep vein thrombosis, which resulted in negative. Moreover, given the large MES detected by TCD and the reported relatively recent history of dizziness, the patient underwent brain magnetic resonance imaging which showed minute multiple areas of impaired signaling of the cerebral white matter, from lacunar ischemic outcomes. We excluded silent paroxysmal atrial fibrillation, as a potential cause of systemic embolism, with ECG telemetry throughout the entire 13 day hospitalization. Finally, although recommended in younger patients, given the history of spontaneous abortions, she was screened for acquired, inherited, and malignancy-associated thrombophilia, which yielded negative results.e.g., myocarditis), MINOCA caused by paradoxical coronary embolism due to a large right-to-left shunting through a PFO was considered the most plausible diagnostic hypothesis.Based on the result of the overall clinical-instrumental evaluation combined with the lack of clinically apparent alternative diagnosis  is the current term referring to acute myocardial injury caused by an ischemic mechanism without obstructive coronary atherosclerotic disease (if any stenosis<50%) on invasive angiography.e.g., fibroelastoma), thrombophilia, silent atrial fibrillation, and right-to-left shunting . Paradoxical embolism is a rare case of coronary embolism which may occur in case of right-to-left shunting . In patient diagnosed with paradoxical coronary embolism, PFO and deep venous thrombosis are the most common causes of intracardiac shunt and embolic source, respectively. However, although the prevalence of PFO in the general population has been estimated as high as 25%, the true incidence of PFO-associated paradoxical coronary embolism is an unknown and likely underestimated phenomenon. Based on scarce pathologic and clinical series and case reports in the literature, a paradoxical embolism through a PFO as the cause of acute myocardial infarction should be suspected and investigated in younger or low cardiovascular risk profile patients, especially with concomitant pulmonary embolism.A broad spectrum of coronary-related pathological mechanisms responsible for MINOCA has been reported. These include plaque disruption/erosion, spontaneous dissection, epicardial spasm, microvascular dysfunction, and coronary embolism. In general, coronary embolism as the mechanism responsible for MINOCA is considered uncommon. However, this may be due to the lack of systematic screening for predisposing conditions such as aortic valve diseases  meaning coronary embolism as probable cause of acute myocardial infarction.A diagnosis of paradoxical coronary embolism is applicable in the case of direct visualization of embolic occlusion on angiography, without embolic source in the left-sided cardiac chambers but, conversely, with evidence of deep vein thrombosis potentially flowing into the systemic circulation through a right-to-left intracardiac shunt. Nevertheless, these criteria are rarely satisfied fully, especially in the case of embolism affecting distal and small coronary vessels, which may be missed on angiography. Since our patient was clinically stable without specific ECG patterns for an acute coronary syndrome, invasive coronary angiography was performed 48–72 h after admission. Consequently, spontaneous coronary recanalization might have occurred during such a time frame, preventing the direct visualization of the embolism. However, Shibata et al have recently proposed a scoring system for diagnosis of definite or probable coronary embolism. In the recent prospective multicenter Stockholm Myocardial Infarction with Normal Coronaries (SMINC-2) study, 77% of patients with MINOCA received a definitive diagnosis when investigated early with comprehensive CMR imaging. The updated 2020 European guidelines for management of patients with NSTEMI recommend CMR (Class 1B) in all MINOCA cases with undefined or uncertain cause.CMR imaging proved highly effective in identifying the underlying pathological mechanism in most MINOCA patients and, therefore, in guiding to choose the most appropriate therapy. In our clinical case, CMR imaging could also detect an ASA, appearing as anomalous mobility of the membrane of the fossa ovalis with excursions greater than 10 mm into both atria during the cardiac cycle. ASA is present in approximately 4% of PFOs and is closely associated with the causal role of a PFO in patients with cryptogenic ischemia, as well as with a worse long-term prognosis in patients treated with antiplatelet against whom treated with the closure of the septal defect with device. Interestingly, Wöhrle et al showed a not negligible prevalence of subclinical myocardial infarction, as high as 11%, in PFO patients with a first cryptogenic cerebral ischemia who underwent CMR imaging.Although TDC or TOE with microbubble test are the gold standard to diagnose PFO , a growing body of data in literature underlines the increasing diagnostic value of CMR. CMR imaging provides an accurate detailing of the IAS anatomical variants, presenting as a complete fusion of septum primum and secundum, or vice versa an incomplete fusion determining a “channel-like” tubular morphology, the latter present in 15–38% of the general population and rarely associated with PFO. Moreover, channel length and free limb of the septum primum, are predictive of PFO-associated shunt. The specificity for PFO diagnosis is 100% if three criteria are applied: recognizability of the septum primum flap, continuous interatrial contrast column in correspondence of the tunnel, contrast jet from the column towards the right atrium. Conversely, the diagnostic sensitivity is poor, because of the impossibility of performing the Valsalva maneuver correctly to provoke right-to-left shunting.The acronym MINOCA  refers to myocardial infarction with normal or near-normal coronary arteries on invasive angiography. Among patients diagnosed with acute myocardial infarction, 6–8% is labeled as MINOCA.The broad spectrum of the pathological mechanisms responsible for myocardial injury in MINOCA makes defining the exact underlying etiology challenging. On the other hand, correctly identifying the culprit mechanism fosters tailored therapies, thus improving the clinical outcome.Cardiac magnetic resonance (CMR) imaging proved highly effective in identifying the underlying pathological mechanism in most MINOCAs with uncertain etiology.


Because of the increased burden of liver disease worldwide, there is a growing demand for 3D in vitro liver models—Liver Organoids. Based on the type of initiation cells, the liver organoid can be classified as PSC-derived or ASC-derived. Liver organoids originated from ASC or primary sclerosing cholangitis, which are co-cultured in matrix gel with components such as stromal cells or immune cells, and eventually form three-dimensional structures in the presence of cytokines. Liver organoids have already made progress in drug screening, individual medicine and disease modeling with hereditary liver diseases, alcoholic or non-alcoholic liver diseases and primary liver cancer. In this review, we summarize the generation process of liver organoids and the current clinical applications, including disease modeling, drug screening and individual medical treatment, which provide new perspectives for liver physiology and disease research.As the largest internal organ, the liver is the key hub for many physiological processes. Previous research on the liver has been mainly conducted on animal models and cell lines, in which not only there are deficiencies in species variability and retention of heritable material, but it is also difficult for primary hepatocytes to maintain their metabolic functions after  As the largest internal organ, the liver is the key hub for many physiological processes. It not only participates in the metabolism of major nutrients, but also has diverse roles in the regulation of the immune system and the decomposition of heterogeneous biological compounds , includiin vitro model, bile duct cells and hepatocytes complete cyclic renewal every 60 and 150 days, respectively  cultured cell lines. Cell lines from benign or malignant tumours of the liver and primary human hepatocytes (PHHs) are the main sources for these in vitro studies because cancer-derived hepatocyte lines lack their normal liver cell counterparts, despite their ability to proliferate indefinitely. Most importantly, they lack most of the cell types that differentiate into primary tissues  are a type of cell that can self-renew and differentiate into specialized cell types that comprise one of the three germ layers in vivo . Thus, Pin vivo . PSCs arin vivo .IPSC generated by reprogramming of skin fibroblasts  can diffTo address the lack of homologous substrate components, Adult stem cells (ASCs) are undifferentiated lineage-committed cells. Terminally differentiated cholangiocytes or hepatocytes can also become activated by appropriate liver injury, which causes them to restart the cell cycle and induce liver regenerative repair by forming ASCs . HoweverASCs are located in a very specific microenvironment, namely, the stem cell niche, which ensures that the cells are able to renew themselves. Stem cell niches are functional domains within stem cell populations capable of controlling the dynamics of tissue homeostasis under different conditions , and thein vitro for long periods of time  in the Hering duct that emanates from the biliary tree, trigger regeneration by restoring both hepatocytes and cholangiocytes, suggesting that they are an alternative source for new cell therapies. Following acute liver injury, such as partial hepatectomy (PHx), total liver chemical-induced damage or infection , mature  of time .in vitro expansion  and hepatic encephalopathy (HE) due to cirrhosis, have been reported to associate with microbial characteristics. For example, microbes play an essential role in liver inflammation, with the liver impacting and communicating with microbes through mediators such as bile acids or inflammatory signals. This connection is known as the liver-microbiome axis . Therefoin vitro infection model with hepatitis B virus (HBV) after co-culture of PSCs with stromal cells on 3D microtiter plates, which developed into vascularised and functional tissue after transplantation. The significant release of viruses from infected IPSC-derived organoids in this assay and the high expression of the infection-promoting factors GPC5, PPARA and CEBPA in IPSC-derived liver organoids demonstrated the stability of this differentiated infection model.The co-culture of organoids with viruses, which can mimic virus infection, has potential for infectious disease models. For example, in vitro model, provide an effective way to investigate the human tissue response to SARS-CoV-2 infection.During the COVID-19 epidemic, organoids were similarly applied to related research areas. Researchers reported that liver organoids were highly sensitive to SARS-CoV-2 infection and showed high expression of chemokines following infection, which is consistent with findings in lung specimens from human COVID-19 autopsies . In addiThe co-culture of organoids and fibroblasts has been widely used in tumour patient-derived organoids. Cancer associated fibroblasts (CAF), which are derived from tissue-residual fibroblasts, endothelial cells and vascular smooth muscle cells, play an essential role in the establishment of the tumour microenvironment . CAFs caIn addition to the growth promoting effects on CSCs and liver tumour-derived organoids, CAFs can induce tumour drug resistance . CAFs cain vitro. However, due to the heterogeneity of IPSCs, common culture protocols do not guarantee that the cells used for organoid generation have the same characteristics, even if they come from the same cohort. To demonstrate the difference between the TGFβ1-induced artificial liver fibrosis model and the liver fibrosis caused by the natural development of NAFLD, Ali et al. established HepaRG-based bioengineered multicellular liver microtissues (BE-MLMs). HepaRG (cell line), HUVECs (human primary), KCs (human primary), and HSCs (human primary) were co-cultured in spheroid-laden hydrogels at different ratios while simulating liver tissue native configuration, such as nutrients/O2 gradients and direct cell-cell contacts between different cell types inside spheroids  culture of hepatocytes primarily consists of monolayers on collagen gels and cultures covered with matrix or collagen gels, which are defined as “sandwich cultures” , but hepMatrigel is an extract from Engelbreth-Holm Swarm mouse sarcoma with components similar to those of a true basement membrane that can form a hydrogel at temperatures of 30°C and above , and theThe matrix gels prepared by decellularization, as described above, are of animal origin, and their residual animal protein content may induce an immune response in the host . TherefoWith the development of organoid chips in recent years, liver chips have received much attention and investigation. The types of cells and culture approaches used for liver microarrays are constantly improving, and the overall model can be classified as simple microwell microarrays , complexThe liver organoids generated of the rat, dog and human involved using micro-engineered organoids containing primary hepatocytes and stromal cells. Primary hepatocytes of the three sources were located in the porous membrane with two parallel microchannels within the ECM sandwich, while stromal cells were located on the other side of the sandwich. In vitro cells self-organizing into organoids are limited by the lack of circulatory system as well as oxygen and nutrients, which leads to the appearance of central organoid necrosis. To address this problem, vascularization methods have been applied to the construction of organoids, and interspersing fibers of endothelial cells between fibers containing parenchymal cells can facilitate vascularization and anastomosis with the host tissue in vivo. In addition to this, 3D printing techniques have been applied to construct vascularized frameworks for tissue development and differentiation of organoids in response to endothelial-parenchymal interactions  is an autosomal dominant disorder that results in multiple organ abnormalities due to mutations in le ducts . In a stASS1 was not detected in patient-derived organoids, but it could be detected in healthy donors, indicating the importance of ASS1 defects in CTLN1. The ability to reverse disease-associated ammonia accumulation by genetic manipulation techniques to achieve the expression of ASS1 suggests that the model is suitable for genetic manipulation. This study also provides early evidence for the role of organoids in genetically-corrected treatments for genetic disorders.Citrullinemia type 1 (CTLN1) is a relatively rare inherited metabolic disorder that causes severe and fatal neonatal hyperammonemia. It is mainly due to a mutation in arginosuccinate synthetase (ASS1) that affects the detoxification function of the liver, thereby blocking the urea cycle and causing a significant accumulation of ammonia in the body that cannot be converted to urea, ultimately leading to hyperammonemia . ResearcDGUOK. The liver, as the main storage site for iron in the body, is more sensitive to the oxidative stress caused by iron overload, which ultimately leads to severe liver damage. However, the process from iron overload to liver failure is not known. Recently, DGUOK mutation to generate liver organoids in vitro, a model that not only has the characteristics of hepatocytes, but also includes the patient’s genetic context. In addition, corrections by CRISPR/Cas9 gene editing enable more accurate controls. The ability of n-acetylcysteine (NAC) to reduce the sensitivity of NCOA4-dependent ferritin degradation-mediated iron pendant disease in lysosomes to iron overload has led to the further study of MDS.Mitochondrial DNA deficiency syndrome (MDS) is a serious genetic disorder caused by iron overload in the body, which is mainly due to mutations in Alcoholic liver disease (ALD) is a liver disease that is closely related to alcohol use. Chronic alcohol abuse (>60 g/day) plays an important role in the development of the disease, and genetic factors, such as patatin-like phospholipase domain containing 3 (PNPLA3) and transmembrane six superfamily member 2 (TM6SF2) expression, viral infections, obesity or malnutrition, are also associated with ALD . ESC-derUnlike ALD, the causative factors for non-alcoholic fatty liver diseases (NAFLD) may include high-energy diet, sedentary lifestyle, obesity, diabetes and hyperlipidemia . When liViral hepatitis, mainly caused by hepatophilic viruses, causes acute hepatocyte necrosis, degeneration and inflammation. Hepatitis B virus (HBV), which causes chronic infection in more than 200 million individuals worldwide, is the predominant infectious agent of the liver . HBV exhDue to the ACE2+/TMPRSS2+ expressing cell population in human bile ducts, human liver ductal organoids can be used for SARS-CoV-2 disease modelling. By co-culturing with SARS-CoV-2, human liver ductal organoids exhibited susceptibility and supported robust viral replication . In addiHepatic fibrosis is a dynamic pathological process that essentially results from the excessive accumulation of heterologous hepatic myofibroblastic components, leading to abnormal production of connective tissue in the liver . InitialARPKD demonstrated that the increased TGFβ expression generated by this mutation in bile duct cells could stimulate myofibroblasts to form collagen fibers  are common sources of 3D odelling . Patientantation .P53, RB, MYC2 and other oncogenes, Current sources of organoid models for Cholangiocarcinoma (CCA) include biopsy tissue from CCA patients and genetically engineered human cholangiocyte, and have been useful in disease metabolism studies, drug screening and individualized medicine . BRCA1-aPolycystic liver disease is a type of cystic lesion caused by the presence of fetal cholestatic cells in the liver , which lin vitro studies of hepatic lipid deposition.Hepatic lipid deposition is a unique and common condition in cats in which fats are broken down into free fatty acids and then transported through the bloodstream to the liver, where they accumulate as triacylglycerols in hepatocytes. Using a feline liver organoid system to examine the effects of drugs on treating hepatic lipid deposition in cats, In addition, traditional Chinese medicines play important roles in the treatment of liver diseases. After applying cholesterol + MIX , As the toxicity of certain drugs, such as the proprietary compound JNJ-2, can associate with significant species variability, liver organoids provide a considerably more accurate tool for drug toxicity testing than animal models . For druLiver organoids are also used to assess drug toxicity in cells of different origins. For example, The properties of organoids derived from normal tissues have a unique role in the study of liver physiology. The normal state of the hepatopancreatic islet axis is closely related to the normal functions of the liver and other organs of the mammalian body. Regenerative medicine is a field of medical research based on the natural healing ability of tissues that each organism possesses within itself. The liver, due to its powerful regenerative capacity, has opened up a wide range of prospects for the clinical application of hepatocyte transplantation and artificial liver generation using hepatocytes. Monogenic inherited liver diseases are caused by genetic defects in hepatocyte function in the absence of stem cell damage, and thus, liver transplantation can play an important role in the treatment of monogenic inherited diseases .in vitro models for clinical studies of the liver due to their ability to better mimic the in vivo environment. Organoids have demonstrated their potential in modeling infectious, genetic, and alcoholic liver diseases, as well as in drug screening and personalized medicine.In summary, PSC- or ASC-derived liver organoids are gradually replacing cell lines and animal models as promising IPSC-derived organoids have the advantage of not relying on primary tissue resection because patient-derived iPSC lines can generate multiple cell types indefinitely and repeatedly . Unfortu