Peripheral arterial disease is atherosclerotic occlusive disease of the lower extremity arteries and afflicts hundreds of millions of individuals worldwide. Its most severe manifestation is chronic limb-threatening ischemia :1140–2, 2003)), which is associated with severe pain at rest in the limbs, which progresses to necrosis, limb amputation, and/or death of the patient. Consequently, the care of these patients is considered a financial burden for both patients and health systems. Multidisciplinary endeavors are required to address this refractory disease and to find definitive solutions that lead to improved living conditions. Revascularization is the cornerstone of therapy for preventing limb amputation, and both open vascular surgery and endovascular therapy play a key role in the treatment of patients with CLI. Around one-third of these patients are not candidates for conventional surgical treatment, however, leading to higher amputation rates (approaching 20–25% at one year) with high morbidity and lower quality of life. Advances in regenerative medicine have enabled the development of cell-based therapies that promote the formation of new blood vessels. Particularly, mesenchymal stem cells (MSCs) have emerged as an attractive therapeutic agent in various diseases, including CLI, due to their role in tissue regeneration and immunomodulation. This review discusses the characteristics of MSCs, as well as their regenerative properties and their action mechanisms on CLI. Critical limb ischemia  is the mThese patients also suffer a significant reduction in quality of life due to permanent local wound treatment and the chronic use of pain-relieving medications, plus other comorbidities, leading to a dependency on the support of caregivers. The poor clinical outcomes in these patients result in the increased use of medical resources, and high hospitalization rates of up to 375,000 admissions annually, leading to a considerable economic burden for national health care systems , 7. In tCurrently, standard therapeutic options include revascularization using a surgical or endovascular approach, depending on the patient's comorbidities, their vascular anatomy, and the location of the vascular lesions . MultidiStem cells have thus emerged as an attractive therapeutic agent in various diseases, including CLI, due to their angiogenic role, and their regenerative and immunomodulatory effects on tissue lesion. Autologous bone marrow stem cells (a-BM-SC) are considered the gold standard of cell therapy for CLI, but this therapy has several disadvantages that limit its use, such as the cardiovascular risk pattern common to CLI patients, and complications arising from invasive aspiration procedures. The angiogenic potential of transplanted cells also directly depends on the characteristics of the donor, which in this particular case may be impaired by the age and general health of CLI patients, and so a-BM-SC may not be the best therapeutic option for this condition . Other sMSCs are a heterogeneous subset of stromal cells distributed throughout the stroma of almost all tissues/organs in vivo , giving MSC isolation methods vary depending on their source: BM-MSCs are usually isolated using the density gradient procedure, or by direct cell plating on a solid surface due to their adhesion capacity , while AOn the other hand, fetal bovine serum (FBS) is the supplement to cell culture media more commonly used . Neverth, and (iii) MSC population must be positive (≥ 95%) for surface antigen markers such as CD29, CD73, CD90, CD44, and CD105, and MSCs must lack expression (≤ 2% positive) of endothelial markers (CD31), hematopoietic markers , human leukocyte antigen (HLA) class II, costimulatory molecules , and HLA-DR surface molecules [vs. BM-MSCs) [The International Society for Cell Therapy (ISCT) released a set of minimal criteria for laboratory-based scientific investigations . These golecules , althougarkers CD, hematopBM-MSCs) , 34, 35 Although it has been demonstrated that MSCs play a role in the angiogenic process on CLI, there is not currently a recommended approach for delivering MSCs as a treatment for this condition. Local administration is the most common route through which MSCs are applied, particularly intramuscular (into the gastrocnemius muscle) or intravascular  . Systemi2) after intramuscular injections of MSCs, results that were not obtained when cell therapy was performed through intra-arterial injections; however, no significant differences were reported between the routes of administration as regards significant pain relief and pain-free walking distance. It is also reported that although direct injection increases the localization of MSCs in their target tissue, it does not improve engraftment or the survival rate; this route can also cause further tissue damage from the bolus injection.There is still no clear consensus regarding the differential therapeutic effects of each route of administration . Indeed,Systemic administration (either IV or IA delivery) is a minimally invasive procedure that allows the wide distribution of cells throughout the body . HoweverAlthough IV delivery is the easiest and the most common systemic route in clinical practice, a frequently associated problem is the so-called pulmonary “first-pass” effect, which results in the significant entrapment of cells, leading to a higher absolute number of cells needed to ensure that a minimum number of cells reach the injury site distal to the lungs . The cauThe promotion of vascularization and angiogenesis is fundamental for efficient organ reconstitution and replacement ; therefoOther describe strategies that intensify angiogenesis potential include genetic manipulation and conjugation of pro-angiogenic factors . miRNA tDespite the remarkable intrinsic properties of MSCs for the treatment of CLI, there is still a lack of standardized routes and delivery methods to guarantee MSC optimal engraftment. Controlled studies may therefore be required to investigate appropriate approaches to delivering MSCs and ensure their survival at the ischemic sites.4 DBCO-Cy5-labeled, cells were monitored for two weeks using a 360º fluorescence tomographic imaging system. The authors found that the DBCO-Cy5-hAD-MSCs appeared to gradually converge at the inner thigh in the ischemic hindlimb, indicating cell migration toward the ischemic lesions; in contrast, a certain amount of the signal was initially observed but quickly disappeared in the normal hindlimb. These findings were confirmed by histological analysis two weeks post-transplantation, where DBCO-Cy5-hAD-MSCs were found in ischemic tissue, indicating the integration of the labeled cells into the host tissue [Some preclinical studies on the hindlimb ischemia model have shown MSC homing and engraftment by using local (intramuscular) or systemic routes. In particular, Lee et al.  labeled t tissue .6 cells) or rat bone marrow-derived mononuclear cells (rBM-MNC) (5 × 106 cells) to demonstrate the presence and viability of rBM-MSCs in the interstitial tissues three weeks after intramuscular injection, and the majority of rBM-MNC revealed severe organelle damage and disintegration. rBM-MSCs and rBM-MNC were also labeled with a fluorescent dye (PKH26 red fluorescent cell linker) and then transplanted into the ischemic thigh muscle in rats to examine cell differentiation. This subgroup of rats was euthanized three weeks after rBM-MSCs or rBM-MNC transplantation, and tissue sections were incubated with anti-von Willebrand factor (vWF) or anti-alpha-smooth muscle actin (α-SMA) antibodies, and endothelial and vascular smooth muscle cells markers, respectively. Histological studies revealed that PKH26-positive cells expressed vWF in both the rBM-MSCs and rBM-MNC groups, although quantitative analysis demonstrated that the number of PKH26/vWF-double-positive cells was significantly higher in the rBM-MSCs group than in the rBM-MNC group. In contrast, some of the transplanted rBM-MSCs were positive for α-SMA, but none of the rBM-MNC was stained for this antibody. rBM-MSCs thus survived well under an ischemic environment and differentiated not only into endothelial cells but also vascular smooth muscle cells.Similarly, Iwase et al.  used an 5 cells) into male C57BL/6 J mice. The mice had previously been intravenously injected with green fluorescence identified FITC-UEA-l to enhance the contrast of functional perfused vessels, and to test whether the vascular networks had connected to the mouse circulation. Ischemic hindlimbs treated with labeled hPMSCs were isolated and analyzed by fluorescent microscopy at Day 14. The merged images of both stainings (FITC-UEA-l and CM-DiI-labelled hPMSCs) showed the incorporation of hPMSCs into murine vascular networks or capillary networks, indicating their participation in angiogenesis in vivo. Immunostaining also showed that anti-human CD31 and anti-human α-SMA cells were detected in hPMSC-treated tissues after 21 days, indicating the endothelial and smooth muscle cell differentiation of hPMSCs in the ischemic limbs.Xie et al.  evaluatevs. normoxic conditions; the cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and the findings demonstrated the presence of CFSE-labelled cells in the ischemic tissue of mice receiving hypoxic rBM-MSCs, but not in the tissue of the mice that received normoxic rBM-MSCs at seven days post-transplantation, implying that hypoxia can further ameliorate blood flow by enhancing engraftment. A long-term tracking assay (four weeks post-transplantation) using double immunofluorescence for bromodeoxyuridine (BrdU) and CD31+  revealed that some of these BrdU+ cells were observed in the CD31+ blood vessels, indicating that some transplanted cells were incorporated into neo-vessels, and indeed functioned and contributed to blood perfusion. Some were also positive for α-SMA or desmin in the ischemic regions, also implying that some transplanted cells differentiated into muscle tissues [Huang et al. had similar results when comparing rBM-MSCs obtained from male C57BL/6 J (B6) and Balb/c mice cultured under hypoxic  tissues .It has also been reported that the homing process and engraftment depend on the MSC immunomodulatory capacity, which can be reduced by certain pathologic conditions such as diabetes, since hyperglycemia-mediated down-modulation of chemokine receptor expression in endothelial progenitor cells and other progenitor cells, resulting in defective angiogenesis and impaired reparative responses .Some preclinical studies have evidenced a shorter settlement time at the ischemic site after local administration , 49; howMSC angiogenic properties have been studied for a long time, but some of the underlying mechanisms of action remain unclear. MSCs belong to a special population of cells with homing ability, meaning they can selectively migrate to ischemic sites regardless of the delivery method in response to a variety of signals secreted by injured and immunological cells. Evidence suggests that MSCs can potentially move from their niche into the peripheral circulation and pass through vessel walls to reach target tissues. Once in the target site, they exert their effects either directly or through the secretion of paracrine factors .Usually, MSCs retain the ability to differentiate into a variety of mesenchymal lineages, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle, induced by specific medium conditions such as growth factors and cytokines . After dvs. fibroblasts, and observing that mBM-MSCs prevented the proliferation of CD4+ and CD8+ T cells, while fibroblasts did not produce significant suppression in either of the two immunological lineages. They subsequently evaluated the in vivo survival of luciferase-labeled mBM-MSCs (Luc+mBM-MSCs) in immunocompetent allogeneic recipients vs. immune-deficient recipients ). The analysis showed that Luc+mBM-MSC survival was significantly shorter in immunocompetent allogeneic recipients compared to that exhibited in immune-deficient recipients. These results demonstrate that under allogeneic conditions, mBM-MSCs cannot completely evade the immune system or induce immune memory and potential rejection [Although some studies have demonstrated the differentiation and/or transdifferentiation of MSCs in ischemic tissue –60, therejection .+) and CD31+). The outcomes revealed that the UA-hAD-MSC group induced significantly higher capillary density than EC-hAD-MSCs or a control group. Four weeks after transplantation, tissue was harvested and immunohistochemistry analysis revealed that the UA-hAD-MSCs group showed significantly higher levels of the representative pro-angiogenic genes, chemokines, and growth factors than the EC-hAD-MSCs group, as well as higher adhesion capacity, increased engraftment potential, and higher recovery of blood perfusion according to LDPI [On the other hand, Guo et al. explored whether EC differentiation from human AD-MSCs (EC-hAD-MSCs) was effective in improving therapeutic outcomes in the treatment of ischemic disease. In this study, hAD-MSCs were cultured under EC differentiation medium for 10 days. Flow cytometry analysis, western blot, and reverse transcription-polymerase chain reaction (RT-PCR) confirmed the EC-specific markers EC-hAD-MSCs relative to undifferentiated adipose MSCs (UA-hAD-MSCs). In vitro angiogenic studies showed the ability of UA-hAD-MSCs to express significantly higher levels of representative pro-angiogenic genes, chemokines, and growth factors than EC-hAD-MSCs. Analyses of engrafted cells in hindlimb sections after UA-hAD-MSC or EC-hAD-MSC injection marked with red fluorescent protein were carried out using NOD/severe combined immunodeficiency mice. Laser Doppler perfusion image (LDPI) analysis was performed, revealing a greater recovery of blood perfusion in the limbs injected with UA-hAD-MSC compared to those injected with EC-hAD-MSC. Vascular and capillary density in the ischemic hindlimb adductor muscle after cell injection was also measured using two endothelial markers , vWF, angiogenic factors CD31, stromal-derived factor-1 (SDF-1), angiopoietin-1 (ANG-1), erythropoietin, platelet-derived growth factor (PDGF), placental growth factor, interleukin-8 (IL-8), IL-6, hepatocyte growth factor (HGF), epidermal growth factor (EGF), MCP-1, macrophage colony-stimulating factor (M-CSF), interleukin-1 receptor antagonist (IL-1ra), and macrophage inflammatory protein-1alpha and beta, among others [The MSC factors that contribute to angiogenesis, tissue regeneration, and endothelial/progenitor cells stimulation on CLI are insulin growth factor-1 (IGF-1), VEGF, bFGF, transforming growth factor-beta  derived from MSCs has a great impact on the activation of different endothelial cell responses at injury sites, promoting angiogenesis and functional recovery .One mechanism that favors the increased paracrine effects that promote angiogenesis is the activation of the AKT signaling pathway. Chang et al. showed the AKT phosphorylation in an endothelial cell line (HAECs) by E69E7-MSCs conditioned medium (E6E7-CM) increasing the expression and release of IL-1β and VEGF-A in vitro. An ischemic model in Balb/c mice subsequently showed that E6E7-CM ameliorates limb loss and improves muscle fibrosis and endothelial density in ischemic limbs .A study by Lee involving hAD-MSCs treated with TNF-α showed that they secrete several proteins, growth factors, cytokines, proteases, and protease inhibitors in TNF-α-CM. The intramuscular injection of TNF-α-CM in the simulated chemotactic migration and in vivo homing of human endothelial progenitor cells (EPCs) promoted angiogenesis in the ischemia limb through IL-6 and IL-8 dependent mechanisms, which improved blood perfusion and inhibited tissue necrosis in the ischemia hindlimbs . These eRecent studies have shown that bone marrow-derived EPCs contribute to ischemic tissue repair by secreting paracrine factors. Liew et al. identified different angiogenesis-related factors in the CM of MSCs derived from B6 and C57BKS mice, such as matrix metalloproteinase (MMP)-3, C-X-C motif chemokine ligand (CXCL)-16, CXCL-4, CINC-10, insulin-like growth factor binding protein (IGFBP)-3, monocyte chemoattractant protein (MCP)-1, serpin e1, MMP-9, IGFBP-2, IGFBP-9, tissue inhibitor of metalloproteinases (TIMP)-1, pentraxin-3, and VEGF . These fOn the other hand, several studies have discussed the role of cell-to-cell interactions between MSCs and EC in angiogenesis and tissue regeneration. In animal models has been observed that once MSCs delivered, they are recruited toward ischemic tissue by chemostatic signaling and express a variety of specific cell surface molecules such as integrins, which regulate the rolling and adhesion of MSCs to EC. Later MSC transmigration into the vessel wall is mediated by platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31), junctional adhesion molecules such as VCAM-1, and cadherins, similar to leukocyte mechanisms. It has been described that soluble factors or lipid vesicles secreted by MSCs into the microenvironment play an important role in cross-talk, transfer of information, EC survival, transdifferentiation into EC, and mobilization of EPCs from the bone marrow . On the The immunomodulatory effect of MSCs has been reported in many studies and is mediated by paracrine mechanisms . MSCs alThe nature of the signals involved in the immunomodulatory effect of MSCs has been studied in several in vitro and animal models. Hypoxia-inducible factor-1 (HIF-1) is a key mediator of the hypoxic response complex. It regulates the transcription of several types of genes under hypoxic conditions related to chemokine secretion; the most important of these signals are SDF-1 and HGF, which are up-regulated during tissue damage . With spThe immunomodulatory effect of MSCs is communicated via MSC-secreted cytokines and has been proven to rely on the local microenvironment, as some effects depend on the pre-treatment of MSCs with inflammatory cytokines. These cytokine-mediated effects suggest a key role for regulatory T cells and monocytes in the overall pattern . MSCs caThe infiltration of neutrophils, macrophages, dendritic cells, and T cells not only contributes to chronic inflammation but also causes the release of elastase enzyme, which causes the inhibition of important healing factors such as PDGF and TGF-β . Liu et It remains unclear whether the phagocytosis of living MSCs occurs via the innate immune cells of the host, or whether the MSCs must undergo apoptosis to subsequently perform phagocytosis. Galleu et al. have shown that infused living MSCs are subject to perforin-induced apoptosis through recipient cytotoxic cells . Heat-inRecent studies have shown that intercellular communication using tunneling nanotubes can transfer mitochondria between neighboring cells. For example, MSCs have recently been shown to prevent apoptosis in EC by transferring mitochondria during hypoxic/ischemic stress . Recent Stem cell transplantation is expected to change the outcome of a damaged vascular system and the prognosis of patients in the early phase of acute ischemic vascular disease. Investigation of the protective effects of stem cell engraftment via TNT-mediated mitochondrial transfer could provide new insights into the therapeutics of ischemic vascular disease .Finally, the molecular mechanisms associated with the angiogenic potential of MSCs are through direct cell differentiation and/or transdifferentiation, cell contact interaction, paracrine signals (immunomodulation effect), and transfer of mitochondria Fig. .Fig. 5Th., who conducted a randomized controlled trial in 20 patients with established CLI, presenting Rutherford classification in Categories II-4, III-5, or III-6 with infra-inguinal arterial occlusive disease, and were not suitable or who had undergone failed revascularization treatment. Participants were randomized to receive 200 × 106 allogeneic BM-MSCs or placebo solution (each group n = 10), which were injected intramuscularly into the gastrocnemius muscle , and had a 6–24-month follow-up period. The study showed significant improvement in the rest ABPI and ankle pressure in participants treated with cell therapy relative to the patients treated with placebo. Wound healing, pain, and amputation rates were similar in both arms, and no related adverse events related to treatment were reported [8 ± 1.1 cells) and BM-MNC that BM-MSCs (9.6 × 108 ± 1.1 cells) were injected intramuscularly into the lower limb  that BM-MSCs were more potent than BM-MNC. Although BM-MSCs and BM-MNC implantation effectively increased blood flow in all 37 limbs, as assessed by the substantial improvement in rest pain, pain-free walking time, ABPI, TcO2, or the formation of new collateral vessels, BM-MSC transplantation was significantly more effective than BM-MNC for the treatment of type 2 diabetic patients with CLI and foot ulcers. There were no acute or chronic serious adverse events related to the BM-MSCs or BM-MNC injection during the 24-week follow-up period. The possible mechanism of therapeutic angiogenesis between the BM-MSCs and BM-MNC in this study was the delivery of angiogenic factors, which promote blood vessel growth and maturation and were detected from both cells in vitro. BM-MSCs from diabetic patients were also found to secrete more VEGF, FGF-2, and angiopoietin-1 than BM-MNC under normoxic and hypoxic conditions [A variety of clinical trials in CLI with MSC-based therapy have recently revealed their security profile and therapeutic potential Table . These ireported . In agrenditions .Table 1E., autologous AD-MSCs (1 × 108 cells) were intramuscularly administrated (15 sites for each muscle with the use of a standard grid) in seven diabetic and non-diabetic patients who were not suitable for vascular or endovascular surgery. No adverse event was associated with autologous AD-MSC transplantation during the follow-up. Six months after cell transplantation, a significant increase in TcPO2, reduction in rest pain, and wound healing were also observed. Nevertheless, no ABPI improvement or change in CLI grade was achieved [MSCs derived from other sources have also shown their angiogenic potential in CLI. In the study by Bura et alachieved . Similar2 were not statistically significant, the formation of new blood vessels was confirmed by angiography, suggesting that these may correspond to collateral small vessels which may improve perfusion outcomes but do not affect all clinical values [Other studies have proved the security and efficacy profile of combined cellular products; for example, Lasala et al. evaluated the intramuscular administration of a combination of autologous bone marrow-derived EPCs and BM-MSCs. No adverse events were reported during the clinical trial. At six-month follow-up, there was an increase in ABPI, walking time, pain relief, and physical functioning. Although changes in TcPOl values .n = 48) versus placebo (n = 28) in patients with CLI and no options for revascularization. This trial provides encouraging evidence that treatment with Ixmyelocel T is safe and beneficial in treating lower extremity CLI in a “no-option” population. Efficacy outcomes showed a statistically significant improvement in time to treatment failure (TTF) and in amputation-free survival (AFS) in Ixmyelocel T-treated patients relative to controls. The treatment effect for both TTF and AFS was even more pronounced in patients who entered the trial with baseline wounds, suggesting greater efficacy in more severe and advanced diseases. These results suggest that treatment with Ixmyelocel T is a promising treatment option for patients with CLI who are unable to undergo revascularization [On the other hand, Powell et al. evaluated Ixmyelocel T-treatment, which is a patient-specific, expanded, multicellular therapy containing autologous BM-MNC, BM-MSCs, and activated macrophages. This study was a Phase 2, double-blind, placebo-controlled, randomized trial conducted to assess both the safety and efficacy of intramuscular injections of Ixmyelocel T-treatment , or by transducing cytoprotective signals that preserve tissue structure.


Then, Lipofectamine/pDNA complexes were immobilized on the nanofibers through immersion in the solution containing the pDNA complexes, resulting in transfection and sustained delivery of pDNA. Cells cultured on the resultant nanofibers expressed genome-editing molecules including Cas9 protein and guide RNA (gRNA), resulting in targeted gene knock-in and knock-out. These results demonstrated the potential of Gelatin-Ph nanofibers obtained through electrospinning and subsequent HRP-mediated cross-linking for gene therapy and tissue regeneration by genome editing.The delivery of nucleic acids is indispensable for tissue engineering and gene therapy. However, the current approaches involving DNA/RNA delivery by systemic and local injections face issues such as clearance, off-target distribution, and tissue damage. In this study, we report plasmid DNA (pDNA) delivery using gelatin electrospun nanofibers obtained through horseradish peroxidase (HRP)-mediated insolubilization. The nanofibers were obtained through the electrospinning of an aqueous solution containing gelatin possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph) and HRP with subsequent HRP-mediated cross-linking of the Ph moieties by exposure to air containing 16 ppm H Delivery of nucleic acids such as DNA and RNA is an attractive technique for tissue engineering , wound hScaffolds, such as hydrogels ,10 and nElectrospinning is a technique for producing nanofibers by applying high voltage to a polymer solution. Electrospun nanofibers have several advantages, such as an ultrathin diameter and a high surface-to-volume ratio . In addi2O2 . T. T11]. T2O2 through HRP-mediated cross-linking. The cross-linked nanofibers showed excellent cytocompatibility. Immobilization of Lipofectamine/pDNA complexes on the nanofibers prevented the complexation of pDNA with serum proteins, which resulted in the sustained delivery of pDNA. Additionally, gene knock-in and knock-out could be achieved using the cross-linked nanofibers. Therefore, Gelatin-Ph electrospun nanofibers with Lipofectamine/pDNA complexes may be a potential candidate for gene delivery and genome editing. In summary, we developed Gelatin-Ph electrospun nanofibers with Lipofectamine/pDNA complexes for pDNA delivery. Gelatin-Ph electrospun nanofibers were insolubilized by exposure to air containing H


Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a rare form of vasculitis in children. SARS-CoV-2, the virus that causes COVID-19 infection, seems to trigger autoimmunity and new-onset autoimmune disease in pediatric and adult patients. We present a case of new-onset AAV following COVID-19 infection in an adolescent patient, and we review the literature of AAV following COVID-19 infection.An adolescent female with a history of asthma was diagnosed with mild COVID-19 infection and subsequently developed persistent cough, wheezing, hearing loss, arthralgias, and rash. Her imaging and laboratory workup showed pulmonary nodules and cavitary lesions, elevated inflammatory markers, negative infectious testing, and positive ANCA. She was treated with glucocorticoids, rituximab, and mycophenolate mofetil. At six-month follow-up, she had improvement in her symptoms, pulmonary function tests, imaging findings, and laboratory markers.We report the second case of new-onset anti-PR3, C-ANCA vasculitis and the fourth case of pediatric-onset AAV following COVID-19 infection. A systematic review of the literature found 6 cases of new-onset AAV in adults after COVID-19 infection. Pediatric and adult patients who develop AAV post COVID-19 infection have few, if any, comorbidities, and show marked radiographic and symptomatic improvement after treatment. There is increasing evidence for COVID-19-induced autoimmunity in children and our case highlights the importance of considering AAV in a child following a recent COVID-19 infection because timely treatment may improve clinical outcomes. Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a rare form of vasculitis in children with an estimated annual incidence of < 1 per one million children . GranuloCoronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused significant morbidity and mortality since it was first reported in late 2019 . SARS-CoA 16-year-old female with a past medical history significant for asthma was referred to the pediatric pulmonology clinic by her pediatrician for persistent cough and wheezing following COVID-19 infection. Her COVID-19 infection consisted of mild upper respiratory symptoms with anosmia and was diagnosed via PCR. Approximately 1 week after recovering from COVID-19, she developed wheezing and a prominent, non-productive cough. She was initially treated by her pediatrician with albuterol which helped her wheezing but not her cough. A chest x-ray at that time was normal. Over the next month, her symptoms progressed to include sinus pain, serosanguinous ear drainage, and a sensation of fullness in her ears. Her pediatrician treated her with courses of azithromycin, cefdinir, and doxycycline. The antibiotics did not resolve her symptoms and she was trialed on a 5-day course of prednisone (60 mg daily). She saw otolaryngology who diagnosed her with chronic bilateral serous otitis media and recommended tympanostomy tube placement. She underwent tympanostomy tube placement with some relief in her symptoms but reported ongoing bilateral hearing loss. She had a CT scan following tympanostomy tube placement that showed bilateral opacified mastoid air cells consistent with chronic inflammation.When she presented to pediatric pulmonology 6 weeks after the onset of her symptoms, she was still wheezing and had a daily paroxysmal cough with occasional post-tussive emesis. She also reported chest tightness and difficulty breathing. She had a chest x-ray which showed patchy airspace disease of the upper lungs, concerning for a multifocal infectious or inflammatory process. Her symptoms suggested an exacerbation of asthma for which she was prescribed another 5-day course of prednisone (40 mg twice daily), albuterol, and she was started on inhaled corticosteroids in combination with a long-acting beta agonist. Her symptoms improved initially but returned as soon as she stopped taking systemic corticosteroids. Over the next 5 weeks, the patient’s cough became productive of green sputum with worsening wheezing that was no longer responsive to bronchodilators. She was treated with a 28-day course of cefdinir for protracted bacterial bronchitis. When she returned for follow up three months later, she continued to complain of cough and wheezing with new onset myalgias. She underwent pulmonary function testing which showed moderate post-bronchodilation small airway obstruction.Chest x-ray revealed perihilar and bilateral upper lobe consolidations that represented a significant progression from her previous x-ray 3 months prior. High resolution chest CT demonstrated extensive multifocal pulmonary nodules and regions of consolidation with multiple areas of cavitation and central bronchiectasis with diffuse bronchial wall thickening as well as reactive mediastinal and hilar adenopathy Fig. A. CT finWhen she presented to pediatric rheumatology clinic, her review of systems was noteworthy for a 45-pound weight loss; intermittent conjunctivitis; sinus congestion, purulent nasal discharge, bilateral hearing loss, ear drainage, sinus headaches, recurrent nosebleeds; cough, wheezing and shortness of breath; progressively worsening bilateral arthralgias in her knees, feet and elbows that improved with activity; intermittent rashes, and sun sensitivity.She underwent lung tissue biopsy which showed non-specific findings with focal areas of parenchymal atelectasis as well as scattered intra-alveolar macrophages. There was no evidence of acute or granulomatous inflammation or vasculitis identified in the biopsied specimen. There were no yeast or fungal elements seen.Our patient’s clinical presentation and positive serology is most consistent with GPA. To induce remission, she was started on rituximab infusions which she tolerated well. She completed a glucocorticoid taper and was started on mycophenolate mofetil as maintenance therapy. At her most recent follow up, she reported a marked improvement in her symptoms although she continues to have bilateral conductive hearing loss necessitating hearing aids. Repeat CT imaging showed an overall improvement in the extent of previously seen multifocal consolidation, nodularity, and cavitation in her lungs Fig. B. She deHere we report the second case of new-onset anti-PR3, C-ANCA vasculitis and the fourth case of AAV following COVID-19 infection in the pediatric population. The gold standard for diagnosing childhood vasculitis is with either histopathology from tissue biopsy or by characteristic lesions detected by imaging studies. Multiple studies in adults with AAV have found that less than half of all biopsies show the characteristic inflammatory and vasculitic changes to confirm the diagnosis –17, and The authors conducted a systematic review of the literature from December 1, 2019 to January 1, 2022, in PubMed/MEDLINE, combining the MeSH search terms (COVID-19) AND (ANCA-associated vasculitis), filtered by age (birth-18) and date ; ((Adolescent) AND (COVID-19) AND (ANCA-associated vasculitis)); ((Pediatric) AND (COVID-19) AND (ANCA-associated vasculitis)). Two authors  independently screened titles, abstracts, and full texts of all relevant articles. All articles reporting ANCA-associated vasculitis after COVID-19 infection in the pediatric population were included. From each article, the authors collected data on publication year, age, gender, comorbidities, chronicity with COVID-19 infection, laboratory tests, serological profile, lung pathology, kidney pathology, AAV therapy, and outcomes.A review of the literature found three cases of PAAV following COVID-19 infection, and, with the present report, four cases were included in this review –29. Of tThere have been six reported cases of adults developing AAV following COVID-19 infection and a table summarizing these cases is well documented in an article by Izci Duran et al.  Similar The treatment strategy was similar in adults and children with all patients receiving systemic corticosteroids –33. The The clinical outcomes following treatment were generally favorable in both adult and pediatric patients: all patients had symptomatic and radiographic improvement; the patients with renal involvement had improvement or resolution of their hematuria, proteinuria and creatinine levels at follow-up; none required long-term dialysis; Two patients (one child and one adult) had bilateral hearing loss as part of their initial presentation . UnfortuOur case presentation and review of the pediatric and adult literature show that AAV may be another autoimmune-mediated sequela of COVID-19. We report the tenth case of AAV following COVID-19 infection. In general, patients who develop AAV after COVID-19 infection have few, if any, comorbidities, and show marked radiographic and symptomatic improvement after treatment.Autoimmunity may be generated by a combination of genetic, hormonal, and environmental factors in susceptible people , 40. One


SnoRNAs are frequently processed into snoRNA-derived RNAs (sdRNAs) that function much like traditional microRNAs (miRNAs). That said, our analyses suggest a global switch from DICER-dependent (predominately miRNA) to DICER-independent (predominately sdRNA) biogenesis/gene regulation in colon cancer. Whereas the expressions of 259 of 288 appreciably expressed miRNAs are significantly decreased (avg. 6.4% of WT) in human colon cancer DICER-KOs, 95 of 103 sdRNAs are conversely, significantly increased (avg. 679.3%) in DICER-KOs as compared to WT. As many diseases are characterized by DICER deficiency, this putative global switch to DICER-independent sdRNA regulations may contribute to an array of human diseases. Figure 1A).Mature microRNAs (miRNAs) are single-stranded, ~20 nucleotide (nt) noncoding RNAs (ncRNAs). MiRNAs are primarily excised from longer precursors via DICER to produce mature single-stranded miRNAs that are loaded into the RNA-induced silencing complex (RISC) to direct binding to complementary protein-coding mRNA transcripts for post-translational gene silencing . Notably, several studies have shown that although some individual miRNAs can be upregulated and have an oncogenic function, miRNA expression is frequently globally suppressed in tumor cells compared with normal tissue . Furthermore, loss of DICER itself is known to contribute to several severe human diseases including multiple malignancies, as well as some autoimmune, neurological, reproductive, cardiovascular, and neoplastic disorders  . Relatedly, global downregulation of DICER-dependent miRNA processing and concurrent reciprocal upregulation of AGO2-dependent, DICER-independent miRNA processing is required for efficient erythropoiesis . Specifically, miR-451 is one of the only a few miRNAs whose maturation is currently known to be wholly independent of DICER , and expression of the well-conserved mature miR-451 directly mediates erythroid progenitor differentiation into red blood cells. In short, DICER-independent, AGO2/SLICER processing of the pre-miR-451 hairpin separates it from the biogenesis of standard DICER-dependent miRNAs. The independent regulation of these two separate miRNA-excision pathways allows miR-451 to escape the global downregulation of miRNAs observed during erythropoiesis   and other ncRNAs in the nucleolus, which ensures accurate translation of proteins by ribosomes. For nearly five decades, it was generally accepted that these housekeeping activities were the primary, if not the sole, function of these molecules (Tollervey and Kiss 1997). Recent discoveries by our lab  and other groups , however, indicate that snoRNAs are frequently further processed into shorter ncRNA species ~20 nt in length. These snoRNA-derived RNAs (sdRNAs) likely number in the thousands  and have distinct functions from their snoRNA precursors. Rather than guiding riboside modification of ribosomal and transfer RNAs, sdRNAs appear to guide post-transcriptional silencing of mRNA expression in a homology-driven process that is reminiscent of canonical miRNA-mediated RISC gene silencing .In stark contrast to miRNAs, however, recent studies indicate that sdRNAs are often over-expressed in human diseases, including common cancers . As examples, snoRNA sdRNA-93 over-expression contributes to specific breast cancer subtype progressions , and similar sdRNA -A24 and -D19b over-expressions have been reported to be associated with enhanced prostate cancer metastasis . That said, perhaps the most striking distinction between conventional miRNAs and sdRNAs is that sdRNAs are apparently excised from specific snoRNAs through a DICER-independent process .Figure 1B-D).The data presented here strongly agree with previous reports  suggesting that sdRNAs are excised from snoRNAs by a DICER-independent, AGO2/SLICER-driven pathway clearly distinct from the typical DICER-dependent biogenesis of miRNAs. Independent analysis of the small RNA transcriptomes of human colon cancer HCT116 WT and DICER-KO cells identified 288 miRNAs and 103 sdRNAs to be appreciably expressed (≥ 30 RPM) in WT and/or KOs. Notably, whereas we find the expressions of 259 of 288 miRNAs to be significantly decreased (avg. 6.4% of WT) in HCT116 DICER-KOs, we find 95 of 103 sdRNA expressions to be conversely, significantly increased (avg. 679.3%) in DICER-KO HCT116 cells as compared to WT , we have recently reassessed how (and if) sdRNAs differ from miRNAs electing to take a much broader look at sdRNAs and miRNAs in health and disease. That said, we were stunned to find  that the near total loss of miRNA expression observed during DICER impairment is accompanied by a reciprocal, global increase in sdRNA expressions. While our and others’ previously published sdRNA analyses strongly implicate a RNAi-based mechanism for sdRNA that is reminiscent of miRNAs , the data presented here indicate that unlike miRNAs, many sdRNAs are excised from snoRNAs by a DICER-independent mechanism. Importantly, DICER deficiency and/or global reductions in miRNAs occur across many human diseases and our current findings strongly suggest a fundamental switch from DICER-dependent miRNA regulation of gene expression to AGO2-dependent sdRNA regulation when DICER activity is diminished. Our data now raise the possibility of a global switch from DICER-dependent miRNA regulations associated with normal cellular metabolism to DICER-independent sdRNA regulations triggered during malignant transformation when DICER activity is diminished ).A single FASTA file comprising all annotated human miRNAs contained within the miRNA registry (Kozomara and Griffiths-Jones 2011) and all human snoRNAs currently annotated in Ensembl  was assembled. Alignments between snoRNAs and miRNAs and individual small RNA-seq reads were performed on the Alabama Supercomputer Center SGI UV 2000 and DMC cluster and obtained via Basic Local Alignment Search Tool (BLAST+) using the following parameters: 100% identity, word_size = 6, ungapped, and evalue = 0.001 . All accepted BLAST+ alignments were restricted to perfect matches (100% identity) between 16 and 32 nts. The frequency of alignments to putative sdRNA loci across each full-length snoRNA was calculated by counting reads defined as ≥16 nts and perfect matches (100% identity) and sdRNAs called as previously defined . Publicly available next-generation small RNA deep-sequencing libraries were obtained from the NCBI Sequence Read Archive (SRA) (www.ncbi.nlm.nih.gov/sra/). These included HCT116 WT (SRR3174964) and DICER-KO (SRR3174968) small RNA transcriptomes. Individual miRNAs and sdRNAs not expressed at ≥30 RPM in either library were excluded. BLAST-based alignments were confirmed by independently employing SURFR . The Short Uncharacterized RNA Fragment Recognition (SURFR) tool comprehensively profiles ncRNA-derived RNAs from input RNA-seq data. SURFR analysis of HCT116 WT (SRR3174964) and DICER-KO (SRR3174968) small RNA transcriptomes returned expression in reads per million (RPM) for each sdRNA and miRNA detected with 100% of miRNA and sdRNA expressions agreeing within 3.1% of BLAST alignment-based values. RStudio was used to calculate differential expression of BLAST alignment-based values. Returned results strictly required sdRNAs and miRNAs to be expressed at ≥ 30 RPM in at least one library and all sdRNAs and miRNAs were classified as (1) upregulated; ≥ 150% expression in DICER-KO as compared to WT, (2) downregulated; ≤ 50% expression in DICER-KO as compared to WT, or (3) unchanged; expression in DICER-KO within 50% of WT.


Furthermore, such imaging traceable nanosystems could provide real-timely information of drug accumulation and therapeutic effects in a non-invasive and safe manner. Herein, the article highlights the recent prominent applications based on the metal ions self-assembly in cancer treatment. This strategy may open up new research directions to develop novel drug delivery systems for cancer theranostics.Metal-ion-based self-assembly supramolecular theranostics exhibit excellent performance in biomedical applications owing to their potential superiorities for simultaneous precise diagnosis, targeted drug delivery, and monitoring the response to therapy in real-time. Specially, the rational designed systems could achieve specific  The usefulness of small-molecule therapeutic dyes has been widely studied in various therapeutic paradigms ; however, their efficacy is limited by instability, rapid clearance, and low tissue selectivity . Supramo3+), zinc (Zn2+), and manganese (Mn2+) ions have been confirmed to interact with sulfonic acid and Lewis base groups at the terminal of Indocyanine green (ICG), a US FDA-approved theranostic dye for clinical use, to self-assemble into multi-level supramolecular systems (II-dipicolylamine-Arg-Gly-Asp (Bis(DPA-Zn)-RGD) complex showed more efficient ability to carry surviving small interfering RNA and to target pathological corneal tissues. Moreover, this ICG-linked complex, can self-assemble into a metal-organic nanostructures (MONs) to realize multimodal imaging-guided phototherapy and gene therapy, thus improving the corneal neovascularization synergistic therapy effect  play important roles in numerous physiological functions such as cellular homeostasis and enzymatic activities, and can also coordinate with functional molecules to spontaneously form an organized self-assembled supramolecular nanostructure with excellent biostability, biocompatibility, and safety . Iron, a systems . Such me systems . For exay effect . Further systems .in situ self-assembly ICG-based supramolecular system for simultaneous detection and treatment of hepatocellular carcinoma or other iron-overload disorders. Fe3+ was confirmed to coordinate with the sulfonic acid from ICG and form supramolecular nanoassemblies, which significantly decreased the single-MRI intensity and enabled rapid clearance from the body (approximately 50-fold lower than that of free Fe3+), thereby, addressing the clinical requirement for high-performance iron quantification while facilitating excess iron drainage   of Fe3+, producing a marked correlation between the T1SIR and the inner Fe3+ concentration, which could be repurposed as an MRI contrast and quantitative liver iron concentration measure  of ICG/Leci-assembly of Fe3+ and MRI contrast changes  . Meanwhi measure . The metoperties . After i changes , which cisorders . Furthernslation . Collectin vivo self-assembly. Chu et al. proposed a tumor stimuli-responsive co-assembly strategy to improve cancer therapy by in situ oxygen and generation of the novel sinoporphyrin sodium (DVDMS) nanotheranostic photosensitizer (nanoDVD). DVDMS is widely applied in phototherapy due to its ability to transform near-infrared (NIR) laser into heat and generate copious amounts of reactive oxygen species (ROS)  production, thus improving the phototherapy effect. Forthermore, as shown in 2/DVDMS can self-assemble into nanoDVD in vivo, which can be monitored by activated photoacoustic/fluorescence/magnetic resonance imaging  . However imaging . The tumin situ assembled nanoplatform to achieve precise orthotopic multimodal imaging and imaging-guided thermal ablation through coordination-driven self-assembly supramolecular systems  and the blood–brain tumor barrier . Gao et  systems . The aut systems . Specifi systems . Simulta systems . MoreoveIn summary, rationally designed nanotheranostics that harness the metal-coordination-assisted molecular assembly of therapeutic agents can simultaneously diagnose, deliver drugs, and monitor the response to therapy in real-time. The high specificity and sensitivity of theranostic dye-bearing supramolecular nanoplatforms integrate multimodal imaging functions as well as synergetic therapy, which demonstrates significant potential for clinical early-stage diagnosis and effective treatment . Compare


Systemic sclerosis (SSc) is an autoimmune disease characterised by severe vasculopathy and fibrosis of various organs including the lung. Targeted treatment options for SSc-associated interstitial lung disease (SSc-ILD) are scarce. We assessed the effects of pirfenidone in a mouse model of SSc-ILD.In vitro, pirfenidone effects on human eosinophil and endothelial cell function were analysed using flow cytometry-based assays and electric cell-substrate impedance measurements, respectively.Pulmonary function, inflammation and collagen deposition in response to pirfenidone were assessed in Fra-2-overexpressing transgenic (Fra-2 TG) and bleomycin-treated mice. In Fra-2 TG mice, lung transcriptome was analysed after pirfenidone treatment. in vitro. A pronounced immune response with high levels of cytokines/chemokines and disturbed endothelial integrity with low vascular endothelial (VE)-cadherin levels was observed in pirfenidone-treated Fra-2 TG mice. In contrast, eosinophil and VE-cadherin levels were unchanged in bleomycin-treated mice and not influenced by pirfenidone. In vitro, pirfenidone exacerbated the IL-4 induced reduction of endothelial barrier resistance, leading to higher leukocyte transmigration.Pirfenidone treatment attenuated pulmonary remodelling in the bleomycin model, but aggravated pulmonary inflammation, fibrosis and vascular remodelling in Fra-2 TG mice. Pirfenidone increased interleukin (IL)-4 levels and eosinophil numbers in lung tissue of Fra-2 TG mice without directly affecting eosinophil activation and migration This study shows that antifibrotic properties of pirfenidone may be overruled by unwanted interactions with pre-injured endothelium in a setting of high T-helper type 2 inflammation in a model of SSc-ILD. Careful ILD patient phenotyping may be required to exploit benefits of pirfenidone while avoiding therapy failure and additional lung damage in some patients. Pirfenidone worsens vascular and pulmonary dysfunction in a SSc-ILD mouse model due to its negative effects on primed endothelial cells and may lead to unfavourable effects in patients with underlying type 2 inflammation as seen in SSc-ILDhttps://bit.ly/3Jk821j Systemic sclerosis (SSc) is an autoimmune disease leading to severe vasculopathy and fibrosis of various organs. It is characterised by vascular and fibrotic abnormalities, due to dysregulation of innate and adaptive immunity leading to high levels of inflammatory and pro-fibrotic mediators such as interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-α or transforming growth factor (TGF)-β, as well as the T-helper type 2 (Th2) cytokines IL-4 and IL-13 , 6. The A second antifibrotic drug, pirfenidone, has long been discussed for SSc-ILD therapy. Pirfenidone reduces pro-inflammatory and fibrotic responses by decreasing levels of inflammatory mediators and growth factors such as IL-1β or TGF-β, as shown in multiple cell culture studies, animal models and in IPF patients –14. AlthThe RELIEF study investigated the effects of pirfenidone in patients with non-IPF progressive fibrotic ILD, but was terminated prematurely due to slow enrolment . In thisWe tested the hypothesis that pirfenidone might be beneficial in SSc-ILD due to its anti-inflammatory and antifibrotic effects, by applying a mouse model overexpressing the AP-1 transcription factor Fos-related antigen-2 (Fra-2 transgene) . This moIn contrast to our expectations, pirfenidone aggravated the vascular and pulmonary phenotype in the Fra-2 mouse model of SSc-ILD. We provide evidence that in certain specific inflammatory settings, pirfenidone may have detrimental effects on endothelial cell function and lung permeability.supplementary material.A detailed description of all methods is provided in the −1 . Pirfenidone was provided by Hoffmann–La Roche. Food was weighed regularly and mean pirfenidone uptake was calculated. Mean pirfenidone uptake was ∼400 mg·kg−1 bodyweight per day, a frequently used dosage in mouse studies, leading to pirfenidone exposure ratios and plasma concentrations similar to those in patients treated with pirfenidone [Female Fra-2 overexpressing/transgenic (Fra-2 TG) mice and wild-type (WT) littermates were maintained under specific pathogen-free conditions in isolated ventilated cages with 12-h light/dark cycles. All animal experiments met European Union guidelines 2010/63/EU) and were approved by the local authorities . Bleomycin was given intratracheally at a dose of 0.8 units per kg bodyweight, as described previously , 25. Pir/63/EU anfenidone , 28. Pirsupplementary figure S1). Collagen quantification by Western blot analysis and hydroxyproline measurements revealed significantly elevated collagen levels in the lungs of pirfenidone-treated Fra-2 TG mice as compared to WT control mice . This highlights the role of inflammation in the development of pulmonary fibrosis in this mouse model.Of note, intranasal application of the glucocorticoid budesonide in Fra-2 TG mice ameliorated pulmonary inflammation, improved lung function  and decrsupplementary figure S3a) or reactive oxygen species production (supplementary figure S3b). Eotaxin-induced shape change and chemotaxis remained unaltered by pirfenidone pre-treatment (supplementary figure S3c and d). Survival analysis showed that pirfenidone, alone or in combination with pro-survival factor IL-5, had no influence on eosinophil longevity in vitro (supplementary figure S3e). In summary, pirfenidone did not alter eosinophil properties or function by its own, nor did it modulate eotaxin or IL-5 mediated effects on shape change, chemotaxis or survival. Therefore, increased eosinophil abundance in the lungs of pirfenidone-treated Fra-2 TG mice cannot be explained by direct pirfenidone effects on eosinophils.To investigate whether increased influx of eosinophils into the lung tissue might be due to direct effects of pirfenidone on eosinophils, we analysed stimulation-induced shape change, chemotaxis, reactive oxygen species production and survival of human eosinophils isolated from blood in response to pirfenidone. Pirfenidone alone did not affect eosinophil activation as measured by shape change  in response to pirfenidone. Pirfenidone or its vehicle control (dimethyl sulfoxide (DMSO)) alone did not affect HMVEC resistance . Knockdown of CEACAM1 is known to modulate endothelial barrier leakiness, to increase leukocyte–endothelial interaction [Our transcriptomic analysis revealed that, in Fra-2 TG mice, pirfenidone activated several pathways involved in chemotaxis of granulocytes and lymphocytes. This was on the one hand due to the increased expression of chemoattractants, such as members of the C-C and C-X-C family of chemokines as well as classical cytokines such as IL-4 or IL-6, and on the other hand due to lower expression of genes involved in endothelial barrier formation (eraction , and to eraction . These oIndeed, Fra-2 TG mice exhibit prominent, partially Th2-driven inflammation with high levels of IL-4 , 20. OurIn line with this, we showed that Fra-2 TG mice exhibited lower expression of VE-cadherin and increased lung permeability compared to WT mice . This deIn addition, VE-cadherin is downregulated in the lung in response to inflammatory stimuli causing decreased permeability that correlates with poor prognosis , 46, 47.Since vascular and endothelial alterations are a major disease component of SSc, it is vital to understand the mode of action of pirfenidone, in particular its effect on the endothelium. In the carefully balanced interactions between endothelium and immune system, pirfenidone might trigger a vicious cycle of endothelial injury and immune cell recruitment, leading to exacerbated damage of the endothelium. Based on our experimental data, we speculate that Th2-predominant eosinophilic inflammation and endothelial barrier disturbance may be triggers for unfavourable pirfenidone effects in patients. The clinical relevance of our findings is highlighted by case reports describing increased wheezing and cough, eosinophilic pneumonia and increased numbers of lymphocytes and eosinophils after the administration of pirfenidone in IPF patients –52. Therin vitro data confirmed the endothelial barrier loss upon pirfenidone treatment in inflammatory settings seen in vivo in Fra-2 TG mice. In conclusion, this study shows that the well-established antifibrotic properties of pirfenidone may be overruled by unwanted interactions with pre-injured endothelium in a setting of high Th2 inflammation. Therefore, assessment of Th2 cytokine and eosinophil levels, together with markers of an activated/injured endothelium could be useful to discriminate patients at risk of adverse effects and potential disease exacerbations from patients who may benefit from pirfenidone treatment.A limitation of our study is that it is purely based on animal models and lacks clinical data. Nonetheless, it has valuable translational potential, as the Fra-2 TG mouse model closely resembles human SSc-ILD and has been successfully used to assess the efficacy of nintedanib, recently approved for the treatment of SSc-ILD, or IL-13 blocking antibodies , 21, 23.10.1183/13993003.02347-2021.Supp1Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.ERJ-02347-2021.SupplementSupplementary material 10.1183/13993003.02347-2021.Shareable1This one-page PDF can be shared freely online.ERJ-02347-2021.ShareableShareable PDF


Nanoparticles offer numerous advantages in various fields of science, particularly in medicine. Over recent years, the use of nanoparticles in disease diagnosis and treatments has increased dramatically by the development of stimuli-responsive nano-systems, which can respond to internal or external stimuli. In the last 10 years, many preclinical studies were performed on physically triggered nano-systems to develop and optimize stable, precise, and selective therapeutic or diagnostic agents. In this regard, the systems must meet the requirements of efficacy, toxicity, pharmacokinetics, and safety before clinical investigation. Several undesired aspects need to be addressed to successfully translate these physical stimuli-responsive nano-systems, as biomaterials, into clinical practice. These have to be commonly taken into account when developing physically triggered systems; thus, also applicable for nano-systems based on nanomaterials. This review focuses on physically triggered nano-systems (PTNSs), with diagnostic or therapeutic and theranostic applications. Several types of physically triggered nano-systems based on polymeric micelles and hydrogels, mesoporous silica, and magnets are reviewed and discussed in various aspects. In the last few years, the increased incidence of chronic diseases such as cancer and metabolic disorders has triggered the need for more efficient, specific, and localized treatments that can deliver drugs at the target site in a highly controlled manner and also allow a precise and early diagnosis . Nanotecin vivo distribution, and toxicity  mostly undergo a phase transition when they experience temperature above their LCST. In contrast, other polymeric systems that become soluble over heating have an upper critical solution temperature (UCST) . Thermo-1O2) or reactive oxygen species (ROS), which will be able to destroy cells , is usually applied as an excitation source. In the case of converting light to heat, known as photothermal therapy (PTT) or photodynamic therapy (PDT), the light source  is applied to kill cancer cells . PDT indoy cells .Alternating magnetic field (AMF) is another source for designing magneto-responsive DDS or gene delivery systems. Magneto-responsive nano-systems can generate heat upon sensing AMF and can be utilized for magnetic hyperthermia treatment. On the other hand, such particles can be used as contrasting agents to provide the signal-to-noise ratio in magnetic resonance imaging (MRI). Magnetic nanoparticles (MNPs) are a suitable carrier for designing therapeutic systems and have been applied as a diagnostic tool for MRI. MNPs have been applied widely in designing theranostic agents, participating in therapy and diagnosis through MRI .Sonography is a well-known imaging method based on ultrasound waves created by mechanical oscillations of a piezoelectric material when an alternating current exerts. In the last decades, 3D ultrasound imaging has developed to have three-dimensional images and a better concept of organ volume/area, resulting in an advanced diagnosis of abnormalities in the early stages .X-ray computed tomography (CT) is a non-invasive clinical imaging modality that combines X-ray images from different body angles with computer processing techniques and provides valuable anatomical information with high spatial resolution.In continuous of our previous review articles in the field of pharmaceutics , in thisIn the last 10 years, many preclinical studies were performed on PTNS to develop and optimize stable, precise, and selective therapeutic or diagnostic agents. The systems must meet the requirements of efficacy, toxicity, pharmacokinetics, and safety before clinical investigation.Interestingly, several PTNS have entered clinical studies, and in this review, we are reporting some examples of PTNS currently under investigation.In the following sections, we will expand on four types of PTNS. Starting from polymeric micelles and nanogels and we will move into mesoporous silica nanoparticles (MSNs) and MNPs. Conclusions and future outlook present an overall view that PTNS has the potential to be used in medical applications.co-polymers) that are self-assembled in aqueous media. At the same time, their concentration is more than critical micelle concentration (CMC). Some procedures have been applied for micelle production like oil-in-water emulsion, solubilization of the copolymer, and subsequent solvent evaporation, dialysis, and film casting. Variously reported morphologies for PMs are spherical, star, worm, crew-cut, flower-like, unimolecular, toroids, helices, cylindrical, lamellae, and vesicles configurations (Polymeric micelles (PMs) are composed of hydrophilic–hydrophobic segments , poly, pluronics, tetronics, polysaccharide-, phosphazene, and chitosan-derivatives  graft co-polymer that is successfully crosslinked with genipin as a natural crosslinking agent (PNIPAAm-co-PLLys)-X genipin  were conjugated with FA and loaded with DOX to provide DOX-FA-PMs. The cellular uptake and MMT assay study demonstrated effective internalization of these PMs into C6 glioma cancer cells with perfect biocompatibility. The in vivo assay set expressed high prevention of tumor growth in C6 glioma TBM significantly. The location analysis of DOX in the C6 glioma-induced rat model was accomplished through fluorescent imaging  & PLA  & PLA  for the -PNIPAAm  and star-PNIPAAm  as tempeco- 1,4,8-trioxa{4.6}spiro-9-undecanone)-b-PEG-b-poly (PECT) triblock co-polymer for loading of two therapeutic and diagnostic agents; DOX and 131I isotope and their subsequent co-delivery was developed. Temperature-induced self-aggregation of PECT triblock PMs shows synergetic treatment due to the combined chemoradiotherapy. The results confirmed that PECT could be recognized as a potent agent in the co-delivery of DOX and 131I, in which the system is diagnosable through fluorescent imaging (Synthesis of the thermo-responsive micellar-hydrogel system based on poly(CL- imaging .N-(2-benzoyloxypropylmethacrylamide (HPMAm-Bz) or naphthoyl analog (HPMAm-Nt), with N-(2-hydroxypropyl) methacrylamide monolactate (HPMAm-Lacy) via a PEG-based macro-initiator were synthesized [mPEG-p(HPMAm-Bz/Nt-co-HPMAm-Lacy)]. These PMs were loaded with anticancer drugs, PTX and DTX, and their temperature-sensitive release profile was probed. The authors designed these thermosensitive PMs to show that the π–π stacking consequence developed by aromatic functionality improves the loading capacity and stability of their DDSs (L-glutamate) (PLG) grafted on 2-(2-methoxyethoxy) ethyl methacrylate (MEO2MA) or 2-(2-(2-methoxyethoxy)ethoxy) ethyl methacrylate (MEO3MA) via click reaction was reported. These “hairy-rod” polypeptides were self-assembled into PMs for loading and releasing of DOX (N-vinylcaprolactam (NVCL), N-vinylpyrrolidone (py), and vinyl acetate (VAc) monomers through RAFT polymerization was developed. Star polymers of PNVCL with variation in homopolymeric arms were created. The aggregation behavior of PMs and their solubility were related to the block order in the arms of star polymers. Interestingly, these libraries of thermo-responsive six arms star polymers displayed dissimilar operations in MTX encapsulation and delivery  (PLGA/HA) was synthesized following the loading of PpIX as an effective photosensitizer. PLGA/HA-PpIX micelles have considerable capability for in vitro CD44-targeted PDT treatment toward A549 cells  with urethane, urea, and thiourea functional groups in their structures to induce hydrogen bonding was disclosed. Subsequent encapsulation of anticancer drug, DOX and photothermal agent, and ICG in PMs provided mPEG-b-PLys@DOX-ICG. Constructive effects of strong H-bonding among the inner hydrophobic segment of PMs and at the same time between the hydrophobic parts and drugs include reducing CMCs, increasing micelle stability, improving the drug loading capacity, declining the size of the micelle, and decelerated drug release pattern, respectively. The ICG photothermal effect triggered destabilization of H-bonding and DOX release and considerable enhancement of cytotoxicity under NIR laser irradiation (in vitro without loading any drugs. (7-diethylaminocoumarin-4-yl)methyl (DEACM) carbonate was chosen as the photo-sensitive group and conjugated with PEG to PEGylated DEACM. Photolysis of DEACM upon UV irradiation can produce carbon dioxide bubbles during micelles degradation. Indeed, in vitro, multiple tumor ablation was induced without any anticancer drug through 8-s UV exposure  s modified cholesterol and CXCR4 molecule inhibitor (PAMDCh). NIR dye/IR780 was loaded in cationic PMADCh, which were applied to form IR780@micelle/siRNA polyplexes. Upon laser irradiation, the photothermal effects of IR780 resulted in the disruption of endosomal membranes and facilitated endosomal escape while promoting siRNA transfection . Synthesadiation . A novelexposure .b-PCL-b-poly(2-(piperidin-1-yl)ethyl methacrylate) (PEG-b-PCL-b-PPEMA) triblock co-polymer and subsequent co-encapsulation with DPAE (diphenylanthracene endoperoxide) and cypate as a fluorescent dye. By NIR irradiation at tumor cells, the PMs induced efficient hyperthermia by cypate through PTT. They triggered large intracellular singlet O2 production by DPAE through PDT via local photothermal consequence in the interior cores of PMs (co-assembly of PCL-b-poly(methoxytri(ethylene glycol) methacrylate-co-N-(2-methacrylamido)ethyl folate amide) (PCL-b-P(TEGMA-co-NMFA) and PCL-b-poly(diethyleneglycolmonomethylether methacrylate) (PCL-b-PDEGMA), which is targeted with folate receptor and encapsulated ICG. Upon NIR irradiation, the toxicity induction by the micelle@ICG has effectively inhibited the growth of HeLa cells  waves as an external physical stimulus for controlling the drug release from ultrasound-sensitive polymer micelles has achieved growing interest due to ultrasound’s safety, inexpensiveness, and non-invasive nature . In addiFive amphiphilic diblock and triblock co-polymers were produced using hydrophilic and hydrophobic oxazoline-based segments. The synthesized PMs were encapsulated with dexamethasone, and their spontaneous and US-mediated release profile was investigated. The results demonstrated that the US intensifies the amount of dexamethasone release by 6%–105% with due attention to the copolymer type, dexamethasone loading on the carrier, and the time, location, and intensity of stimulation .in vitro and in vivo. Furthermore, NB diagnosable material in this system has successfully resulted in extravascular ultrasonic imaging (An engaging system of siRNA micelle-nanobubble (NB) complexes was designed based on gene delivery for tumor therapy. NB was selected as a promising nanocarrier due to the prosperous production of NBs accompanied by its US sensitivity and the potential for passive accumulation in tumor cells. SiRNA micelle-NB was prepared by the interaction of siRNA micelles with a positive charge based on mPEG-b-PLys diblock co-polymer and gas-cored liposomes with a negative charge. The US-mediated siRNA transfection, which gives rise to surpassed therapeutic operation and cancer cell apoptosis, was investigated  imaging . Later,  imaging .2-responsive  that was grafted onto glycol chitosan (GC) and then subsequent crosslinking with glutaraldehyde (GA) lead to the formation of the dual light/pH-responsive-crosslinked PMs (CPMs). Fast release of CPT was observed during light irradiation at low pH (b-poly (tertbutylmethylacrylate-co-hydroxylmethacrylate), photosensitizer Ce6, Gd3+ as MR T1-weighted MR agent, and prodrug DOX. Upon NIR laser irradiation, the micelle induced ROS creation and local heat production for PDT and PTT and diagnosed via PAI. In addition, the micelle could construct a magnetic resonance signal at an acidic medium to implement MRI (Combining pH-responsive co-polymer with a photosensitizer was established to overcome the drug resistance in targeted tumor therapy through chemo-PTT. Light-sensitive t low pH . A multiment MRI .® F127 copolymers and various amine-containing methacrylate monomers for combined delivery of microRNA (miR-345) and GEM  based on a pentablock copolymer system composed of amphiphilic pentablock copolymers based on Pluronic  and GEM .g-PLC-b-PNIPAAm block co-polymer. The first block (PCL) is prepared via ROP, and the chain extension with the second block (PNIPAAm) is performed via RAFT polymerization. DTX was loaded on these biodegradable PMs with high encapsulation efficiency, and the release rate was controlled via pH and temperature . The delivery of DOX from supramolecular PMs was accelerated at low pH at 37°C . Anotherperature .co-PNBM core and hydrophilic PEG coronas that are multi-responsive to temperature, light (UV), pH, and reduction . The in vitro release profile was investigated by the use of hydrophobic NR drug. Each stimulus has a specific effect on these multi-responsive PMs; as the temperature changed the size of the micelle, the micelle swelled in low pH, a few amounts of DTT disarranged the micelle configuration, and irradiation of UV light caused the dissolution of the micelle construction (A creative quadruple responsive copolymer based on PEG-ss-poly (DMAEMA)-co-poly (2-nitrobenzyl methacrylate) [PEG-ss-(PDMAEMA-co-PNBM)] comprising disulfides unit was developed. Self-assembling this copolymer generates PMs with hydrophobic PDMAEMA-truction .The term “nanogels” (NanoGel™) is used to define nanosized particles (10–100 nm) formed by crosslinked polymer networks, which are able to swell in a suitable solvent and absorb large quantities of water .Crosslinks are essential for nanohydrogel structural stability because they prevent polymer chain dissolution in the aqueous environment. NGs, also called hydrogel nanoparticles, are found in a wide variety of applications in biomedical fields, such as DDS and bioimaging .via reaction among hydrophilic monomers and difunctional cross-linkers or physical cross-linking agents  through the paracellular or transcellular passages .• Both types of drugs (hydrophilic and hydrophobic) could be entrapped in NG networks.NGs are good carriers for DDS due to their specific properties :• The smHowever, using expensive techniques for preparing some NGs and removing solvents and surfactants from final products are disadvantages of such systems .NGs can be classified as non-responsive and stimuli-responsive. Non-responsive NGs swell consequently to the water absorption. In contrast, responsive NGs can swell/deswell after exposure to environmental changes  and radioisotope (131I) labeled albumin simultaneously. The NGs showed a proper responsivity to temperature and pH so as it was in a solution form at room temperature while transformed into the gel near body physiological temperature. Moreover, the drug release accelerated in tumor cells due to pH effect. Surprisingly, the nanocarrier exhibited a biodegradable manner at 37°C and pH of 5.8. Furthermore, MTT assay on 4T1 cells revealed that it could be biocompatible and cause significant growth inhibition by sustained drug release. Administration of PDMAEMA gel @njection .Recently, HIV drug lopinavir was loaded in a polymeric NG (PNIPAAm) prepared in different sizes . The injMoreover, they observed cell cycle arrest triggering apoptosis death in MCF-7 cells by the nano-carriers . Poly N.Thermo- and pH-responsive NG delivery system based on lysine modified-polyvinylcaprlactam (PVCL) conjugated with DOX was established and showed high uptake in the MCF-7 cell line. The synthetic strategy depicted in co-PHPMA-co-PADMA-PAMAM-CD NG was fabricated to load with ICG and DOX, exhibiting strong NIR-light sensitivity. Using PTT and chemotherapy simultaneously leads to effective results in in vitro and in vivo assays (b-PAA NG was studied. The NG has a significant destructive effect on the proliferation of C6 glioma cancer cells and an inhibition effect on tumor growth of C6 tumor-bearing Sprague–Dawley rats (co-NHMAAm-co-SCC) NG was loaded with 5-FU and sodium copper chlorophyllin (SCC), which produces heat exposure to green light  on PEI-PNIPAAM-PEI NG resulted in an ultrasound-responsive structure that releases DOX immediately after cavitation induced by ultrasonic waves. The DOX-loaded NG showed comparable cytotoxicity on HEK293 and Huh7 cell lines compared to free DOX. It also demonstrated more cell growth inhibition in tumor cells than in normal ones .Improving wound healing and increased therapeutic gain for frostbite were reported after treatment of rats’ skin with GLT NG containing triterpenoids drug in combination with ultrasound waves . Pressurvia the disordering of lipid shells of the liposomes.Among the other nanostructures to deliver the cargo to the desired site, liposomes are one of the first and most investigated nanocarriers due to their biocompatibility, low side effects or cytotoxicity, ease in biodegradation under physiological conditions, and desired properties in loading and delivery of the entrapped cargo. The spherical shape of liposomes formed by the bilayer of lipids allows them to mimic the cell membrane and load both hydrophobic and hydrophilic drugs . Due to Using particular physical sources to increase the temperature above the physiological condition but not more than 42°C converted mild hyperthermia into a powerful tool for targeting drug delivery purposes. As the sources can act selectively in ON/OFF modes during a predefined interval and irradiate in a user-adjusted location, they have shown promising results in combination with chemotherapy agents . Hyperthc) while experiencing heating  were designed and investigated at 37°C or 42°C (using an RF generator) on H22 cancer cells. The in-vitro study exhibited that both Free-Vin and Thermo-Vin groups significantly decreased the H22 cell viability after 24 h. Moreover, the accumulation of the drug into the tumor was 15 times greater for the Thermo-Vin+RF group than the Free-Vin one. The Kaplan–Meier analysis showed that the Thermo-Vin+RF group owned the longest mice survival among all groups  in a burst manner, allowing the liposomes to be more clinically applicable .c while triggering a rapid release of the drug due to their accumulation at the boundary and making stable pores -loaded liposomes showed the most efficient therapeutic gain of photocoagulation  transforms into solid silica .This section reports some examples of MSNs responsive to exogenous physical triggers and their application in the biomedical fields.Within the unique properties of MSNs’ pores to be capped by gatekeepers, the possibility of designing smart engineered delivery agents becomes possible. MSNs have found diverse applications in medicine, catalysts, adsorbents, and sensors .etc.), have made them a good platform for DDSs. Today, researchers are exploring oral and injection formulas of MSNs for therapeutic purposes. MSNs utilization for cargo loading like small molecules  and macromolecules such as proteins and genetic materials , the MSNs with devoted properties of metal cores have been prepared . Such nob-PNIPAAm was utilized both as structure-directing and thermo-responsive agents while loaded with Ibu, and the system proved to have a thermosensitive release profile  was developed for co-delivery of paclitaxel, 5-Fu, cis-platin, and siRNA (targeting ABCG2) while also targeted with FA and the system was abbreviated as MSN@pNIPAAm-pMA/FA. The results demonstrated that both anticancer drug and siRNA were successfully delivered to CD133+ cancer cells by designed MSNs. aryngeal . Anotherin vitro DOX release pattern was assessed in differentiated human neurons and epithelial HeLa cells (A remotely targeted thermo/pH on-demand delivery and diagnosis system based on gold and magnetic NPs embedded silica nano-shuttles (MGNSs) coated with pNIPAM-co-pMA (MGNS@ PNIPAM-co-PAA) loaded with DOX was created, and the assembled structure was named as nano golf balls. Distinguished applications of the aforementioned structure were investigated through magnetic-field transport in; 1) glass capillary tubes as simulator of delivery through blood vessels, 2) porous hydrogels as simulator of human tissue . The La cells .The presence of diagnosable materials in the core of thermo-responsive MSNs has resulted in the formation of theranostic systems. MNP core-modified thermo-responsive agent coated with MSN (MNP@PNIPAAm-co-DMAEMA@MSN) was successfully created for MTX delivery and MR imaging.MSN capped with paraffin showed to have a thermo-sensitive cargo release profile since paraffin could be melted at a certain temperature . By thisLight is one of the most powerful sources of exogenous stimuli and could be considered a safe treatment protocol . On the Cyclodextrins (CD) with a self-assembled structure capable of host and guest interplay could potentially be applied as a gatekeeper of MSNs’ pores . Azo-ben2S, and Mo2S), and also organic-based fluorophores NPs like graphene QDs and carbon-dots with photo and thermal sensitivity have been applied as MSNs gatekeepers in designing DDSs  that could be de-hybridized in encountering localized heat . Anothervia PTT .via carbamate photoresponsive linkage)  . Carbon linkage) .2S-embedded MSN has exhibited to be an excellent PT agent and while fabricated with fluorogen PhENH2 and targeted with folic acid (FA) showed to be a potent agent in targeted PTT and fluorescence imaging (2S3) NPs have also been recognized as a NIR light absorbing and contrast agent of CT imaging. Recently, two novel types of theranostic light-responsive MSNs systems with Bi2S3 have been reported (2S3 targeted with trastuzumab (Bi2S3-MSN-trastuzumab) loaded with DOX was created for simultaneous PTT and CT imaging (2S3-MSN-RGD) to the system for DOX delivery and malignant tumor PTT (Mo imaging . A novel imaging . Other e imaging . Bismuthreported . MSN-coa imaging . Anotherumor PTT .Other developments in the establishment of light-responsive nano-carriers lay in the formation of core/shell structure of common responsive elements even with MSN as a core (MSN@Shell) or in the opposite, MSN as a shell (core@MSN). Metallic-based NPs with emitting properties have played a distinct role in designing theranostic agents. In this regard, gold-coated MMSNs (MNP-coated MSN) were designed for PTT and dual MR and CT imaging . Polydopin vivo injection, and PTT and dual US/PA imaging are presented in A dual imaging nanotheranostic system based on biodegradable gold nanorattle (GNR)-coated MSN filled (GNRs@MSN) with perfluoropentane (PFP) was developed for melanoma PTT and ultrasound (US) and photoacoustic (PA) imaging. A schematic illustrating synthesis, Newly established up-converted NPs (UCNPs), known as an unusual combination of lanthanide ions, showed to be potent photo-responsive agents due to their capacity in adsorbing NIR and changing to UV or visible emission . UCNPs c4:Yb3+/Er3+ with MSN coating and thermoresponsive capped were developed for DOX delivery  @ 0.6 capped with azobenzene and loaded with RB and DOX was developed for DDs via PDT. The UCNPs capable of emitting UV: blue and green: red multi-band light was acted as a stimulus of light-responsive Azo and photo-sensitizer of RB. The mechanism of action of this multifunctional nano platform in blood vessels is depicted in Multifunctional luminescent-based UCNPs of NaYFdelivery . Multi-cIn vitro MRI analysis showed the high relaxivity of the NPs in the magnetic field, potent theranostic agents . Some examples of these potent systems are discussed here. Multifunctional superparamagnetic iron oxide-based MSN with dibenzo-crown ethers (MMSN-crown ethers) periphery with trimodal responsiveness to pH/ultrasound and magnetic field was developed for DOX delivery. Ultrasonic waves could stimulate sodium or cesium ions binding with crown ethers with pH-responsive binding that act as gatekeepers of the system. c agents .via external magnetic field was created based on magnetic/Fe3O4 core and zinc oxide/ZnO interlayer coated by mesoporous silica (MNP@ZnO2@MS). The ZnO interlayer could act as an absorber of microwave irradiation with an excellent thermal response. These magneto-responsive particles showed sustained control release upon microwave irradiation  while loaded with DOX and zinc phthalocyanine (ZnPc) (MMSN-lipid-PEG-MTX/DOX&ZnPc) as a photosensitizer. The presence of lipid-PEG-MTX in periphery of MMSN was effective in increasing dispersion stability and preventing drug leakage while decreasing hemolytic activity. The magnetic core provided magneto targeting to the tumor site, and the MTX conjugate would selectively kill cancer cells by overexpressing folate receptors. ZnPc was loaded in the lipid layer and served as a fluorescence tracking agent in FI and a source of PDT ROS.A nanoassembly of MMSN coated with chitosan (charge converted polymer-g-FA and citraconic anhydride) targeted by TAT peptide (MMSN@CS/TAT) and loaded by CMP as DNA-toxin antitumor drug was established. MRI observed a high accumulation of NPs in the tumor site. Upon this phenomenon, the FA acts as a target for receptor-mediated endocytosis in the cancer cells. Through endocytosis into the lysosomes, the polymeric layer charges’ reversed from negative to positive and separated from MMSN, and TAT peptide assisted the carrier entrance to nucleus, where CMP could induce apoptosis . A noveladiation . A maghe at 40°C . A multiThe original idea of using magnetic particles to deliver medicinal agents to a specific body area was formed in the late 1970s. This method injects the whole drug/magnetic carrier into the body through venous or arterial vessels . Then, bTargeted drug delivery is more relevant to cancer treatment because the main challenge in cancer treatment is to target and kill cancer cells so that they have as little effect on healthy cells as possible . One of in vivo, and the presence of some nanoparticles with nanocarriers enhances their effect on cancer cells , after 6 hours of the synthesized particles reaching the nucleus of MCF 7 cancer cells. Then, after 12 h, it was observed that the cell death rate was reduced by 50%. In another study by 3O4 in the form of liquid-to-solid transition state was used. This system will be able to suppress the MB-231 breast cancer xenograft model in mice from the injection route in the liquid state. It was seen that after injecting this system, the cancer site was considered with high accuracy, without showing any leakage in other locations -Feocations . Other cocations .In the end, it is necessary to mention some of the nano-systems that have been able to enter the field of treatment. Among the types of nanoparticles mentioned so far, nanocrystals, liposome and lipid-based nano-systems, polymer-based nanoparticles, protein-based nano-systems, and metal-based nanopharmaceuticals have been successful in the field of therapy . The folThe key properties of physical stimuli-responsive therapeutic/diagnostic systems compared to the direct delivery of therapeutic agents are their ability to release the drug in a stimuli-responsive manner, high drug loading capability, lower toxicity, synergistic therapeutic efficacy, and biocompatibility. Another promising approach is to focus on nanoparticles with the potential to respond to multiple triggers. For instance, the light source has a high point focusing accuracy but is very poor at penetrating tissue. On the other hand, ultrasound waves have the best permeability to the tissue, but they are weak to have the accuracy of targeting like light. It is possible to generate a system with the positive points of both origin systems and synergistically plays significant strengthening effects in the treatment process by combining these two physical systems. Although physical stimuli-responsive nanoparticles are primarily evaluated for preclinical applications, they will shortly be an essential part of the clinician’s armory in addressing drug delivery and diagnosis.


The SARS-CoV-2 vaccine roll-out has been successful around the world. However, there are increasing concerns about adverse events. We report two pediatric cases of Multisystem-Inflammatory-Syndrome (MIS-C) with neurological involvement that occurred after SARS-CoV-2 vaccination and unknown recent SARS-CoV-2 infection. Brain magnetic resonance revealed mild-encephalopathy with reversible-splenial-lesion in both cases and complete resolution within 4 weeks. In conclusion, this report aims to describe rare emerging clinical entities that can help pediatricians to make an early diagnosis and to provide appropriate treatment. Multisystem-Inflammatory-Syndromes following COVID-19 vaccination remain rare events. When a history of a recent contact with SARS-CoV-2 is present, a careful evaluation by the clinicians in charge of immunization activities is suggested prior to proceeding with the vaccination. Multisystem Inflammatory Syndrome (MIS) in children and adolescent (MIS-C) is a condition that usually occurs 2–6 weeks after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. MIS-C is a febrile syndrome with generalized inflammation and multi-organ involvement including abdominal symptoms like pain, vomiting and diarrhea, skin rash and often hypotension and cardiac dysfunction .Besides late inflammatory condition, neurological disorders related to SARS-CoV-2 infection were also described. These include the mild encephalitis/encephalopathy with reversible splenial lesion (MERS) recently reported in children with MIS-C .The introduction of vaccines to prevent SARS-CoV-2 infection has been one of the key strategies in the global control of the COVID-19 pandemic.Adverse effects from COVID-19 vaccination are usually minor, including local pain at injection site, fever, asthenia or general musculoskeletal pain .In particular, neurological adverse events following vaccination are generally mild and transient, although rare major neurological complications, such as cerebral venous sinus thrombosis, Bell’s palsy, acute transverse myelitis, acute disseminated encephalomyelitis, and acute demyelinating polyneuropathy, have been described ,6Recently, a multisystem inflammatory syndrome following SARS-CoV-2 vaccination (MIS-V) both in adults  and chilMERS is a clinic-radiological syndrome characterized by a transient mild encephalopathy and a reversible lesion in the splenium of the corpus callosum on MRI. Patients with MERS presented with mild central nervous system symptoms such as consciousness disturbance, seizures and headache, and recovered completely within a month . The patHereby we described two children who presented MERS and MIS-C after SARS-CoV-2 vaccine and unknown SARS-CoV-2 infection.A 14-year-old previously healthy girl presented with history of an episode of catatonia revealed after 2 days of fever and vomit, which arose within 24 h after the first shot of Pfizer/Biontech COVID-19 vaccine. The neurological manifestation resolved in about 7–8 min and was followed by agitation and mental confusion. A month earlier, a familiar cluster of SARS-CoV-2 infection was documented, but the patient’s nasopharyngeal swab for SARS-CoV-2 with real-time polymerase chain reaction (npRT-PCR) resulted negative. Family history resulted positive for autoimmunity: mother affected by Systemic Lupus Erythematosus (SLE). In the past medical history, weight loss associated with skin hyperpigmentation and asthenia had been reported. Physical examination showed skin hyperpigmentation, a mild cutaneous rash and signs of hypotensive shock associated with severe metabolic acidosis and dyselecrolytemia . The npRT-PCR was negative, but SARS- COV-2 nucleocapsid IgG turned positive. Clinical, laboratory, and imaging findings are shown in Brain magnetic resonance imaging (MRI) showed hyperintensity on T2-weighted images in the splenium of the corpus callosum with restricted diffusion, suggestive of MERS . ElectroDiagnostic lumbar puncture was performed, and chemical–physical analysis of cerebrospinal fluid resulted negative as well as microbiological assays.According with the “Centers for Disease Control and Prevention” (CDC) case definition criteria, the diagnosis of MIS-C was made and intravenous immunoglobulin (IVIG) (2 gr/Kg) was administered. In the suspicion of primary adrenal insufficiency (increased ACTH) intravenous hydrocortisone (100 mg/mq/day) therapy and fluids were promptly started. After 24 h of stress dose of steroid, the patient had significant improvement in mental status. On day 3 of hospitalization, the girl presented respiratory difficulties. Chest X-ray showed right pleural effusion with extension up to the middle of the hemithorax. She started nasal cannula oxygen, human albumin infusion, diuretic and anticoagulant therapy with enoxaparin with gradual improvement of symptoms and complete resolution of pleural effusion after 5 days. The search for anti-adrenal antibodies was subsequently performed with a positive result confirming primary adrenal insufficiency.She started substitutive therapy with three daily doses of hydrocortisone (15 mg/mq/day) and one daily dose of fludrocortisone (0.1 mg/day). During hospitalization, the symptoms progressively improved, and the patient was discharged from hospital on day 16 after complete recovery. Four weeks after the first image, a brain MRI was repeated, showing complete resolution of the lesion in the splenium of the corpus callosum .A previously healthy 9-year-old boy presented with vomiting and diarrhea following 4 days of fever and 10 days after 2nd mRNA-SARS-CoV2 vaccine. In the previous month, he had COVID-19 exposure from a family cluster, but npRT-PCR SARS-CoV-2 resulted negative. Family history resulted positive for autoimmunity, with his mother being affected by lupus nephritis.At arrival to the emergency department, he was conscious but agitated and hypotensive (85/45 mmHg). He had fever, conjunctivitis, palmar rash, abdominal pain and headache. On admission, npRT-PCR was negative for SARS-CoV-2, but nucleocapsid and spike protein IgG resulted positive. Clinical, laboratory, and imaging findings are shown in Due to the clinical status and the need to undertake cardiologic supportive therapy, he was admitted to the pediatric intensive care unit (PICU) where IVIG treatment (2 gr/kg) was started in combination with methylprednisolone and enoxaparin. On day 4, considering the persistence of hypotension and the mild improvement of inflammatory markers, treatment with Anakinra was administered at the dosage of 2 mg/kg twice daily.Subsequently, the patient’s clinical features and vital signs progressively improved and therapy was slowly reduced.Agitation regressed and echocardiography revealed normalization of ventricular systolic function.The following brain MRI, performed four weeks after the first imagine, showed no abnormalities , and theTo our knowledge, this is the first case series describing MIS-C after SARS-CoV-2 vaccination (MIS-V) and previous unknown SARS-CoV-2 infection, presenting with neurological findings of MERS. Such condition has been recently named as MIS-V due to its clinical presentation, which results quite similar to MIS-C. The affected patients suffer from febrile illness and present multinflammatory symptoms affecting the gastrointestinal, cardiovascular and neurological system associated with elevated markers of inflammation and altered coagulation ,14.The pathophysiology and case definition of MIS-V are still under evaluation . The cytOverall, these hypotheses must be evaluated with caution. Indeed, with the widespread COVID-19 vaccination program, some cases of MIS-C caused by SARS-CoV-2 infections acquired before full vaccine are expected to occur post-vaccination, and will appear to be temporally associated with the vaccine. It is worth highlighting that a detailed medical history focused on individualization of recent familial cluster of COVID-19 and predisposition to autoimmunity could drive the timing of immunization. Indeed, a recent study reported 15 patients including pediatric cases and young adults with earlier diagnoses of MIS-V, but with past or recent SARS-CoV-2 infection revealed by anti-nucleocapsid antibodies .The overlapping neurological findings of MERS observed in both cases is of particular interest. As more patients gain access to COVID-19 vaccines, a wide spectrum of neurological complications is continuously being reported . SeveralMERS has been recently described in two children with MIS-C without history of COVID-19 vaccination . These cIn conclusion, this report and the few previously reported cases emphasize the extreme rarity of MIS after COVID-19 vaccination. Strict surveillance of adverse events related to vaccination is needed because a misattribution of MIS or MERS to vaccination can lead to increased vaccine hesitancy. Therefore, a careful evaluation is required to consider alternative diagnoses and to determine if previous SARS-CoV-2 infection, especially when familial history of autoimmunity is present. In this scenario, serological tests aiming to detect the presence of anti-nucleocapsid antibodies might be informative.This report has some limitations. It involved a small number of patients, and further study of larger cohorts is necessary to confirm the observations assumed in this report. In particular, additional studies will be needed to investigate SARS-CoV-2 vaccination as a trigger of hyperinflammation and the pathogenic mechanisms underlying transient central nervous system involvement in MIS. Finally, it is of pivotal importance to unravel if many autoimmune conditions are associated with gene variants that could predispose susceptible individuals to mount an abnormal immunological response to SARS-CoV-2 infection or vaccination leading to MIS.