Rejuvenation of an old organism was achieved in heterochronic parabiosis experiments, implicating different soluble factors in this effect. Extracellular vesicles (EVs) are the secretory effectors of many cells, including cardiosphere-derived cells (CDCs) with demonstrated anti-senescent effect. 1. To determine the role of EVs (versus other blood fractions) on the rejuvenating effect of the young blood. 2. To evaluate the anti-aging properties of therapeutically administered EVs secreted by young-CDCs in an old organism. Neonatal blood fractioned in 4 components  was used to treat old human cardiac stromal cells (CSPCs). CDCs were generated from neonatal rat hearts and the secreted CDC-EVs were purified. CDC-EVs were then tested in naturally-aged rats, using monthly injections over 4-months period. For validation in human samples, pediatric CDC-EVs were tested in aged human CSPCs and progeric fibroblasts. While the purified EVs reproduced the rejuvenating effects of the whole blood, CSPCs treated with EV-depleted serum exhibited the highest degree of senescence. Treatment with young CDC-EVs induce structural and functional improvements in the heart, lungs, skeletal muscle, and kidneys of old rats, while favorably modulating glucose metabolism and anti-senescence pathways. Lifespan was prolonged. EVs secreted by young CDCs exert broad-ranging anti-aging effects in aged rodents and in cellular models of human senescence. Our work not only identifies CDC-EVs as possible therapeutic candidates for a wide range of age-related pathologies, but also raises the question of whether EVs function as endogenous modulators of senescence. As a consequence, aging-related diseases, including cardiovascular disease, diabetes, and cancer, will increase dramatically in the coming decades. Today we treat these chronic conditions by addressing individual disease-specific pathways, rather than by targeting cellular senescence as a potentially treatable pathogenic mechanism. Cellular senescence is the phenomenon believed to underlie progressive aging-related tissue and organ dysfunction4.By 2050, nearly 1.5 billion people will be over age 659. Recently, we found that transplantation of young CDCs exerts anti-aging effects in old rodents, improving heart function10. Although we specifically targeted the heart in that study, multiple systemic benefits were evident, hinting that soluble factors might play a prominent role. In vitro experiments revealed that extracellular vesicles (EVs) from CDCs (CDC-EVs) mimicked the anti-senescent effects of CDCs, at least partially through activation of the telomerase-telomere axis. Together with the anti-tumorigenic effects of CDC-EVs in old rats with spontaneous leukemia11, it seems reasonable to hypothesize that anti-senescent properties of CDC-EVs may underlie the benefits. If so, EVs might be logical therapeutic candidates for a variety of aging-related diseases.Cardiosphere-derived cells (CDCs) are cardiac progenitor cells with broad-ranging bioactivity in preclinical and clinical studies12?Here, we address the following questions: Do CDC-EVs exert anti-aging properties in vivo? Are favorable changes durable, and do CDC-EVs affect longevity? Finally, do EVs (versus other blood fractions) mediate anti-senescent effects of young blood observed in heterochronic parabiosisFor detailed methods, please see the supplementary file.13. After initial evaluation with echocardiography and exercise testing, a total of 27 animals were divided randomly into two groups1: 13 rats treated with extracellular vesicles secreted by neonatal cardiosphere-derived cells (CDC-EVs)2, 14 rats receiving phosphate buffered saline . CDCs were grown from 30 freshly-explanted F344 neonatal rat hearts as described (suppl. ref.1). CDC-EVs were harvested from passage 2 CDCs, from serum-free medium conditioned for 15 days. An initial dose of 7.5 μg EV protein/g animal weight (based on our previous results11), equivalent to 1.3 × 108 particles/g animal weight followed by lower monthly doses  of CDC-EVs (resuspended in 300 µL PBS) or 300 µL PBS alone, injected percutaneously into the left ventricular (LV) cavity for systemic delivery. Cardiac echocardiography and exercise test were performed monthly and hemodynamic measurements were performed at endpoint. Blood samples were collected at baseline and endpoint. When appropriate (see supplementary file) animals received anesthesia with inhaled Isoflurane 4% for induction followed by 2%. After 16-weeks of follow-up animals were sacrificed, under general anesthesia hearts were arrested in diastole (intra-ventricular injection of KCl) and excised. Hearts and other organs were collected. In a separate group of 11 male and female 22-month-old Fisher 344 rats, glucose metabolism was evaluated with the glucose tolerance test (GTT).Twenty-two-month-old Fisher 344 rats  were obtained from the National Institute of Aging. All methods were carried out in accordance with relevant guidelines, approved by the Institutional Animal Care and Use Committee (IACUC) of the Cedars-Sinai Medical Center as per the National Institutes of Health , and are reported in accordance with ARRIVE guidelines. Old Fisher 344 rats were selected for this study since they mimic different human aging-related conditions such as cardiac fibrosis and diastolic dysfunctionFor human cells, the protocol was deemed exempt by the Institutional Review Board of the Cedars-Sinai Medical Center as it only involved the use of anonymized pathological specimens for tissue harvesting and CDC isolation. Old human cardiac stromal cells and human progeric fibroblasts were targeted with CDC-EVs isolated from human pediatric donors. Simulating heterochronic parabiosis in vitro, old human cardiac stromal cells were treated with different fractions of neonatal rat blood. Four different blood fractions were used in the consecutive experiments: total blood with cells, serum, EVs-depleted serum and purified EVs.p < 0.05 considered significant. IBM SPSS Statistics 20 was used for all analyses. For in vitro studies the lowest number of replicates per experiment was three.Pooled results are presented as mean ± SD (± SEM in figures) or percentages, for continuous and categorical variables, respectively. Significance of differences was assessed by Student t test or with 1-way ANOVA in case of multiple groups if the distribution of the variable was normal; otherwise, the Mann-Whitney or Kruskal-Wallis tests were used. Paired t-test was used to determine significance between baseline and endpoint in the same group of animals. All probability values reported are 2-sided, with CDC-EVs were isolated from neonatal rat CDCs as described in Methods and Fig. p < 0.001; Fig. p < 0.001; Fig. p = 0.03; Fig. 14, were all favorably modulated. Telomere length was ~2 times longer in CDC-EV treated versus control rats, as in our previous study using CDCs10. Ingenuity pathway analysis of the transcriptomes from CDC-EV and control rat hearts 16; immune B-cell receptor complex ; and extracellular signal-regulated protein kinases 1 and 2 . In the serum of CDC-EV treated rats, we observed an increase in alkaline phosphatase  and preservation of globulin levels compared with controls  in rats treated with CDC-EVs. Echocardiography showed progressive changes of E′ and E/E′ ratio, with improvements in the CDC-EV group but deterioration in controls  in a separate group of 22-month-old rats. The end-point test was done 48 h after CDC-EV or PBS infusion  and remained higher than in the PBS group (which deteriorated progressively) throughout follow-up  but decreased strikingly (to ~30% of control levels) in CDC-EV animals  on aged human cells. In vitro assays used cardiac stromal/progenitor cells (CSPC) obtained from > 55-year-old human donors and dermal fibroblasts from progeric patients. Aged human cells were treated with CDC-EVs resuspended in serum-free conditioned medium or serum-free medium alone (Methods).25 and an increase in anti-oxidative proteins27 . Since EVs are the soluble effectors of the CDCs32, we decided to compare their anti-senescent properties relative to those of other blood fractions in vitro, simulating heterochronic parabiosis model , two of which implicated the protein GDF11 as an anti-senescent mediator33.Repeated systemic administration of young CDC-EVs in aged rodents triggered broad-ranging functional improvements, with concordant structural changes in different organs and associated evidence of tissue rejuvenation Fig. . The ben4. Using a single cell-free therapeutic agent, young CDC-EVs, we demonstrated that all three pathologies can be favorably modulated. Moreover, tissue fibrosis contributing to organ dysfunction34 was broadly ameliorated  in CDC-EV treated rats. Based on these findings, CDC-EVs emerge as a strategy capable of targeting pathophysiologic mechanism(s) underlying many age-related chronic conditions. Both, MiR-146 and miR-92a highly enriched in CDC-EVs32 known to be implicated in aging-related pathways36 may have played a role in rejuvenating effects observed in our study.Cardiovascular diseases, diabetes, and cancer are aging-related conditions which underlie much morbidity and mortality in the elderly population25. Senescent cells are characterized by extensive transcriptional changes, telomere attrition, chronically stimulated DNA damage response, mitochondrial dysfunction, the senescence-associated secretory phenotype (SASP), and cell-cycle arrest39. Previously, we described an anti-senescent effect of young CDC-EVs in vitro10. Here, we confirm that cellular rejuvenation, conceived as partial or total reversal of senescence, can be also achieved in vivo in old animals injected with young CDC-EVs. Benefits include telomere elongation in heart cells, less-active DNA damage response , lower IL-6 levels, and changes in protein levels suggestive of enhanced mitochondrial biogenesis in skeletal muscle. Extensive transcriptomic differences in treated versus control groups were consistent with the observed upregulation of the transcription factor NANOG and extracellular signal-regulated kinase ERK 1/2. Both are recognized regulators and stabilizers of the pluripotency gene regulatory network15. Accordingly, we speculate that the mechanism of action of young CDC-EVs is related in part to the control of the dynamic state of pluripotency and reprogramming41, a strategy that has been touted in pursuit of rejuvenation42. This idea is also supported by the increased self-assembly potential of young CDC-EV-primed senescent human heart cells in vitro.Cellular senescence is thought to contribute to progressive age-related organ dysfunction45, the use of young blood or its components has gained particular traction, including trials in humans . Much effort has been put into identifying the blood component(s) responsible for the rejuvenating effects48, without conclusive results to date. While acknowledging the limitations of our in vitro “parabiosis” experiment, we observed that EVs are required for the rejuvenating effects of neonatal serum. In fact, treatment with neonatal CDC-EVs in old animals, and in vitro on senescent human cells, induced multiple broad anti-aging effects.Among the various approaches being tested to promote rejuvenationSeveral limitations of our study need to be considered when interpreting the results. Although we selected a naturally-aged rat model, rodents may do not fully recapitulate the aging process in larger mammals. We did, however, confirm the rejuvenating effects of CDC-EVs in models of human cellular senescence  in vitro. Additionally, the functional studies were performed sequentially, allowing us to evaluate CDC-EV-induced changes in the same animal, but histopathological evaluation could only be performed at study end-point, so only the differences between groups at that specific time point were assessed. In the in vitro experiment simulating parabiosis, although the EV fraction was purified and separated from free proteins by the density gradient method, we cannot rule out that some individual proteins may be different quantitatively in the EV-depleted serum fraction. Another limitation of the simulated in vitro parabiosis was the lack of true cross-circulation; however, it would have been impossible experimentally, using currently available methods, to deplete EVs selectively in vivo in one paired animal and not the other.Aging-related diastolic dysfunction and the associated heart failure represent unmet clinical needs. Since classical therapeutic targets pursued in systolic heart failure have failed, novel biological interventions are being explored. Here, we find that EVs secreted by young donors’ cells can reverse various aging-related pathologies in senescent rats. Infusion of young CDC-EVs trigger systemic rejuvenation as manifested by improved glucose metabolism, exercise capacity, and survival. In the heart, signs of cellular rejuvenation were associated with structural (less fibrosis and hypertrophy) and functional (decreased stiffness) improvements. Rejuvenating effects of young CDC-EVs were validated in human models of cellular senescence. Given that allogeneic CDCs are already in advanced testing and have proven safe to date, such cells can be used as manufacturing platforms for EVs, enabling rapid progress to clinical testing in a variety of aging-related disorders.49. EVs can influence multiple pathways synergistically; indeed, our findings indicate or hint that CDC-EVs modulate not only the telomere-telomerase axis10 and the p53-p38-MAPK pathway, but also the DNA damage response, oxidative stress, mitochondrial function, and pluripotency dynamics. Even more processes, yet to be explored, may underlie the observed benefits on lifespan, metabolic health, tissue composition and cardiac function. In conclusion, EVs are nanoparticles containing thousands of known and potential signaling molecules Supplementary Information.Supplementary Figures.


Quantificationwas done using liquid chromatography tandem mass spectrometry on cellularand naked genomic DNA of human bronchial epithelial cells (BEAS-2B)exposed to BPDE (N2-BPDE-dG adduct was identifiedin both samples, with naked DNA showing a higher abundance. Overalladduct levels increased with higher concentrations of BPDE. The behavioraldiscrepancy between naked and cellular DNA presumably involves cellularmachinery such as histone, transcription, and repair proteins.The authors devisedseparate workflows to quantify the prevalence of  to BPDE 1b. The NN2-BPDE-dG, theauthors developed BPDE-dG-Damage-seq (N2-BPDE-dG. These fragments are then barcoded, sequenced,and aligned to the human genome to determine the location of damage. The high frequency of G at the called damage site across various BPDE concentrations, along with the lack of nucleotide enrichment at adjacent sites, supports the fidelity of BPDE-dG-Damage-seq.To reveal locations of mage-seq 1c. BriefN2-BPDE-dG in cellular and naked DNA (8In line with the mass spectrometrydata, the level of damage in cellular DNA was almost always lowerthan that of naked DNA. The impact of cellular processes on damageaccumulation was inferred from the relative abundance of aked DNA 1d. OverlN2-BPDE-dG-bearing trinucleotide sequences observedby the authors were remarkably similar to those of mutational signaturesfound in cancer patients.2N2-BPDE-dG was detected consistently in lung cancer hot-spotgenes, including the chromatin modifying gene SMARCA4 and the tumorsuppressor genes CDKN2A, KEAP1, and TP53. In particular, BPDE damagefound in CDKN2A matches the most frequently mutated site of this genein lung adenocarcinoma, which is also one of the top ranked genome-widemutations observed in lung carcinomas. Almost all lung-carcinoma patients with mutations at this site are smokers, indicating a strong correlation between BPDE-mediated formation of DNA lesions and the progression of lung cancer.With this informationin hand, the authors compared their genomic map with mutations identifiedin smoking-related lung cancers. The relative frequencies of N2-BPDE-dG that links carcinogen-DNA adducts to mutational signaturesseen in medical cases. The study also provides a general method togenetically map DNA alkylation events and predict cancer mutations.A key insight of the study is that genomic features shape the formationand retention of alkylation products. DNase data from BEAS-2B cells,instead of a generic DNase map of the human genome as used by thepresent study, would enable a more specific analysis.Collectively, Sturla andco-workers construct a genome-wide map for The factthat open DNA carries fewer adducts raises an intriguing paradox:unlike nucleosome-packed DNA, open DNA is less protected from damage,but is also more actively repaired. Results of the current study suggestthat it is the repair process, rather than reactivity, that dominatesthe accumulation of alkylation products in the genome. It would beinteresting to determine if such a trend persists with shorter treatmenttime or in different cell types. Many of the observed phenomena canbe mechanistically dissected through time-resolved profiling and bycomparing damage profiles in cells of active and disabled repair machinery.Ultimately, these studies will provide important insights into therelationship between DNA alkylation, damage accumulation, and cancerprogression.


TM-augmented anterior cruciate ligament repair (ACL-IB) and between patients after ACL-IB and ACL reconstruction (ACL-R), and controls.Little is known about knee mechanics and muscle control after augmented ACL repair. Our aim was to compare knee biomechanics and leg muscle activity during walking between the legs of patients 2 years after InternalBraceTwenty-nine ACL-IB, 27 sex- and age-matched ACL-R (hamstring tendon autograft) and 29 matched controls completed an instrumented gait analysis. Knee joint angles, moments, power, and leg muscle activity were compared between the involved and uninvolved leg in ACL-IB (paired t-tests), and between the involved legs in ACL patients and the non-dominant leg in controls  using statistical parametric mapping . Means and 95% confidence intervals (CI) of differences in discrete parameters  were calculated.Significant differences were observed in ACL-IB only in minimum knee flexion angle  and maximum knee flexion moment during stance , and differences between ACL-IB and ACL-R only in maximum knee flexion during swing . Compared to controls, ACL-IB  and ACL-R  had lower (maximum) anterior tibia position around heel strike. ACL-R also had lower maximum knee extension moment  and internal knee rotation moment  during stance, and greater maximum semitendinosus activity before heel strike  than controls.Our results suggest comparable ambulatory knee function 2 years after ACL-IB and ACL-R, with ACL-IB showing only small differences between legs. However, the differences between both ACL groups and controls suggest that function in the involved leg is not fully recovered and that ACL tear is not only a mechanical disruption but also affects the sensorimotor integrity, which may not be restored after surgery. The trend toward fewer abnormalities in knee moments and semitendinosus muscle function during walking after ACL-IB warrants further investigation and may underscore the importance of preserving the hamstring muscles as ACL agonists.Level III, case-control study.clinicaltrials.gov, NCT04429165 (12/06/2020).The online version contains supplementary material available at 10.1186/s12891-023-06916-7. TM-augmentation consists of a polyethylene tape that is placed on the femorally reattached native ACL and fixed to the femur and the tibia [Anterior cruciate ligament (ACL) tears can lead to knee instability  and – evhe tibia .The advantages of ACL repair procedures (augmented or non-augmented) over traditional ACL-R (where the torn ligament is replaced with an autologous muscle tendon ) are a lThe aim of this study was to compare knee biomechanics and leg muscle activity during walking.between the legs of patients 2 years after ACL-IB (side-to-side difference (SSD) in ACL-IB), andbetween the involved leg of patients 2 years after ACL-IB, the involved leg of patients 2 years after ACL-R, and the non-dominant leg of healthy controls (leg difference between groups).Considering the less invasive procedure and greater preservation of natural knee joint structures in ACL-IB, we expect no SSD in patients after ACL-IB (first hypothesis) and more natural (closer to control) biomechanics after ACL-IB than after ACL-R (second hypothesis).This study is a substudy of a larger non-randomised comparative umbrella study with retrospective data collection .TM-augmented ACL repair after proximal rupture  performI and II , 24. Thel groups .Twenty-nine patients after unilateral primary ACL-IB, 27 sex- and age-matched patients after ACL-R with hamstring autograft , and 29 sex- and age-matched healthy controls were included Table . To compAll participants were examined in a single visit to the Laboratory of Functional Biomechanics at the University Hospital Basel (Switzerland). Anthropometric parameters were measured including all parameters required for the underlying gait models , and participants were prepared for the subsequent gait analysis. Participants were then asked to walk in their own shoes at self-selected speeds along a 10-m walkway with two embedded force plates until at least four valid trials per leg were obtained . Anthropometric data were managed using REDCap© , 29. GaiKnee biomechanics (kinematics and kinetics) during walking were recorded using a motion capture system  and two force plates . Reflective markers Ø 16 mm) were placed on the skin at defined bony landmarks , with additional nine and six cluster markers on the femur and the tibia, respectively  in NexuMuscle activity of the gluteus medius, vastus lateralis, vastus medialis, semitendinosus, tibialis anterior and gastrocnemius medialis muscles was recorded using surface electromyography (EMG) electrodes . EMG electrodes were attached bilaterally to shaved (if necessary) and alcohol-cleaned skin according to the standards of the SENIAM project  Surface.Raw EMG data were filtered , full-wave rectified, and smoothed  . EMG ampTrajectories of knee kinetics, kinematics and muscle activity data were time-normalised to the gait cycle ) and averaged across trials for each participant and leg for further statistical analysis.Group characteristics, including anthropometric and clinical data and spatio-temporal parameters, were compared between groups using analysis of variance (ANOVA), and Kruskal-Wallis or Mann-Whitney U tests (patient data only) when quantile-quantile-plots did not yield a normal distribution. Based on our sample size estimation , 28 subjBiomechanical and muscle activity patterns  are commonly described and analysed using discrete parameters computed from the respective time series data  , 3 or thTrajectories. Trajectories of knee kinematics and kinetics, and muscle activity data were compared using SPM  [M.0.4.7) . Side-toM.0.4.7) . Only siM.0.4.7) , 41. ForSSD in ACL-IB: mean and SD of the maximum difference between the trajectory of the involved and uninvolved leg for each patient.Between ACL-IB and ACL-R: mean and SD of the maximum difference between the trajectory of each ACL-IB and the mean trajectory of ACL-R.Between patient groups  and controls: mean and SD of the maximum difference between the trajectory of each patient and the mean trajectory of controls.Discrete parameters. Frequently studied discrete parameters  of knee angles, moments and power after ACL-R were calculated  was significantly higher and the maximum knee flexion moment (-0.07 ± 0.17Nm/kg) was significantly lower in the involved leg than in the uninvolved leg in ACL-IB  and knee internal rotation moment  differed significantly between groups , and to compare the same parameters between the involved legs of patients 2 years after ACL-IB, the involved legs of patients 2 years after ACL-R, and the non-dominant leg of healthy controls. Our results confirmed our first hypothesis that there would be no SSDs in patients 2 years after ACL-IB in almost all parameters. Our second hypothesis, that knee biomechanics and muscle activation patterns would be more natural (closer to control) after ACL-IB than after ACL-R was partially confirmed, particularly for knee moments and semitendinosus muscle activity.During loading response, patients after ACL-IB had a lower maximum knee flexion moment in the involved leg compared to the uninvolved leg, but not compared to the non-dominant leg in our control group. During push-off, in ACL-IB, the involved knee was 2.4° more flexed, resulting in a lower range of motion during the stance phase of walking. However, regardless of statistical significance, this difference was less than the minimal clinically important difference of 3° reported in the literature for differences between limbs in a healthy population , and lesDuring loading response, patients after ACL-IB had a lower maximum knee flexion moment in the involved leg compared to the uninvolved leg, but not compared to the non-dominant leg in our control group. The external knee flexion moment is internally controlled by the quadriceps muscle, whose contraction is associated with anterior tibial shear force and tibia translation . The ACLBased on these results and the thresholds reported in the literature, we consider the observed interlimb differences in minimum knee extension angle during push-off and in maximum knee flexion moment during loading response in ACL-IB to be not meaningful, suggesting that there is no relevant asymmetry in knee kinematics, kinetics, and muscle activity 2 years after ACL-IB. Nevertheless, these differences should be considered in the context of possible subsequent injury or the onset of osteoarthritis and may continue to decrease, stagnate, or even increase over time.The greater knee flexion angle during swing in ACL-IB compared to ACL-R was the only significant difference observed between patient groups. As mentioned previously, the observed difference of < 4° in knee flexion angle was rather small and within the error for knee flexion angles recorded using skin-based motion capture , suggestThe comparison between ACL patients and the matched healthy control group with comparable knee-related activity levels shown in the TAS indicated similar gait adaptation in knee biomechanics after ACL-IB and ACL-R, especially in anterior tibia position and knee moments. Although only patients after ACL-IB showed a lower anterior tibia position around foot strike compared with controls when comparing trajectories (SPM), a significant difference was also observed between ACL-R and controls in maximum anterior tibia position (discrete parameter). Therefore, the translational behaviour of the tibia does not seem to be fully recovered even 2 years after ACL-IB or ACL-R. As stated previously, differences in the results between the SPM analysis and the discrete parameter analysis may occur due to variations in motion velocity affected by time normalisation. Therefore, peak values may be shifted (rather than coinciding in time), which may have resulted in non-significant (masked) differences in the SPM analysis between ACL-R and controls when comparing each time point between groups.Inconsistent results have been reported for dynamic anterior-posterior translation in the knee during walking after ACL-R, mostly comparing between legs in patient and not to controls. Beard et al.  observedWhile lower maximum knee extension (higher knee flexion) moment and lower (maximum) internal rotation moments were observed in ACL-R compared to controls in SPM and/or discrete parameters, ACL-IB did not differ significantly from controls in these parameters. Our results after ACL-R are comparable to two meta-analyses , 3 that Interestingly, most of the differences in the involved leg of ACL patients were observed around foot strike and in terminal stance around ipsilateral push-off, compared to the healthy contralateral leg or with controls. During a complete gait cycle, the maximum length of the ACL or its posteromedial/anterolateral bundle was measured immediately before, at, or shortly after foot strike –55, duriDuring walking, patients after ACL-R used up to 11% more of their maximal voluntary semitendinosus activation than controls . The tendency for higher semitendinosus activation in ACL-R compared to controls was also observed in SPM, although not significantly. This could be due to the discrepancy between SPM and discrete parameters (discussed in 4.2.1) or the high variability in semitendinosus activity in ACL-R compared to controls and ACL-IB. A previous meta-analysis –59 and pThe loading of the knee joint or ACL depends not only on the magnitude and direction of the external forces, i.e., ground reaction force, but also on the magnitude and direction of the internal forces, such as the forces exerted by the muscles spanning the knee , and theIn conclusion, the similarity in muscle activity after ACL-IB compared to controls but the presence of differences after ACL-R, suggests a reduced or even absent muscular compensatory mechanism after ACL-IB. Consequently, our results highlight that preservation of native ACL fibres and the semitendinosus muscle including its tendon may be beneficial.TM-augmented ACL repair as an alternative treatment modality for a specific subgroup of ACL patients . However, it is not known whether these results hold true when compared to patients with other tendon grafts. Regardless of ACL-IB or ACL-R, neither group achieved normal walking biomechanics (translation and kinetics). Therefore, 2 years postoperatively, gait still appears to be affected by the original ACL rupture and may not fully normalize thereafter. This is consistent with the notion that ACL rupture not only represents a mechanical disruption in terms of loss of stability, but also affects sensorimotor integrity, including neuroplastic adaptations [These results indicate that neither ACL group was functionally worse or better, as demonstrated by comparable ambulatory knee mechanics, and muscle activity patterns in a direct comparison between ACL-IB and ACL-R. Regarding the surgical procedure, ACL-IB achieved a similar walking outcome while being less invasive and preserving the surrounding knee musculature, including its function. Thus, the absence of harvesting and associated complications  with ACLptations . TherefoThe major strength of this study is that, for the first time, we provide information on knee biomechanics and leg muscle activity patterns after ACL-IB and compare them with age- and sex-matched patients after ACL-R with hamstring tendon grafts and a healthy matched control group. This study also has some limitations. Based on surgical recommendations, we only included patients with a proximal rupture for augmented ACL repair , whereasTwo years after ACL-IB, patients have no relevant leg asymmetry in knee biomechanics or muscle activity. The similar walking outcome after ACL-IB and ACL-R, but the less invasive surgery of ACL-IB , underlines the value of augmented ACL repair as a possible alternative to ACL-R for patients with a proximal rupture and a moderate activity level . Nevertheless, both groups of patients still showed gait adaptations compared to controls at 2 years postoperatively. Fewer differences in knee kinetics and semitendinosus muscle activation after ACL-IB than after ACL-R indicates that preservation of native ACL fibres and the muscle-tendon complex may be beneficial. Biomechanical and neurological sensorimotor studies are desirable to better understand the differences in functional outcome between ACL-IB and ACL-R and the presumed benefits of ACL-IB.Below is the link to the electronic supplementary material.Supplementary Material 1


Fluorescence and photoacoustic imaging techniques offer valuable insights into cell- and tissue-level processes. However, these optical imaging modalities are limited by scattering and absorption in tissue, resulting in the low-depth penetration of imaging. Contrast-enhanced imaging in the near-infrared window improves imaging penetration by taking advantage of reduced autofluorescence and scattering effects. Current contrast agents for fluorescence and photoacoustic imaging face several limitations from photostability and targeting specificity, highlighting the need for a novel imaging probe development. This review covers a broad range of near-infrared fluorescent and photoacoustic contrast agents, including organic dyes, polymers, and metallic nanostructures, focusing on their optical properties and applications in cellular and animal imaging. Similarly, we explore encapsulation and functionalization technologies toward building targeted, nanoscale imaging probes. Bioimaging applications such as angiography, tumor imaging, and the tracking of specific cell types are discussed. This review sheds light on recent advancements in fluorescent and photoacoustic nanoprobes in the near-infrared window. It serves as a valuable resource for researchers working in fields of biomedical imaging and nanotechnology, facilitating the development of innovative nanoprobes for improved diagnostic approaches in preclinical healthcare. Fluorescence and photoacoustic (PA) imaging are two distinct but related optical methods for visualizing cells and tissue. Fluorescence imaging is based on the use of targeted tracers that absorb light energy and re-emit light of a different wavelength . FluoresThe advantages of fluorescence and PA imaging are further enhanced using near-infrared II  light. NIR-II, also known as short-wave infrared (SWIR), has been demonstrated to achieve tissue imaging at a higher depth and lower auto-fluorescence and light scattering effects than lower-wavelength imaging windows. NIR-I (700–1000 nm), NIR-II (1000–2000 nm), and NIR-III 2000–2400 nm) are different near-infrared (NIR) imaging windows, each characterized by specific wavelength ranges and distinct applications in biomedical imaging [00 nm areThis review article provides an up-to-date overview of the current state of research on NIR-II nanoprobes for fluorescence and photoacoustic imaging, highlighting recent advances, as well as the use of targeted agents for cell-specific imaging. NIR-II nanoprobes are a class of imaging agents designed to function in the near-infrared-II (NIR II) window (1000–1700 nm) and exhibit unique optical properties that enable them to overcome limitations observed in traditional NIR-I and visible-light imaging probes. Their distinct features and imaging capabilities make them valuable tools in fluorescence and photoacoustic imaging. This review covers a range of nanoprobes that demonstrate either NIR-II fluorescence, photoacoustic properties under NIR-II excitation, or possess both capabilities. The main components of NIR-II imaging probes are described: fluorophores, delivery technology, and targeting conjugates. Two main families of contrast agents, organic and inorganic, will be discussed. Transgenic PA and fluorescence contrast agents will not be discussed in this paper. Applications outside of PA and fluorescence imaging will not be discussed. Important considerations for probe construction are pharmacokinetic properties and the method of clearance . UnderstFörster Resonance Energy Transfer (FRET)-based processes are used extensively in medical imaging . FRET isThis process of intramolecular electron donation and acceptance has led to the rational design of novel fluorescence probes with red-shifted emission spectra into the NIR-II. The donor–π–acceptor (D–π–A) class of fluorophores has attracted recent attention for fluorescence in the NIR region . The π-cFluorescence imaging relies on the emission of fluorescent light by fluorophores when excited by a specific wavelength of light. The process involves the absorption of photons at a higher energy level, followed by the subsequent emission of photons at a lower energy level. This emission occurs within the NIR-II spectral range, making it suitable for in vivo imaging. Fluorescence imaging offers high sensitivity and excellent spatial resolution, enabling the detection of specific targets labeled with fluorescent probes. Photoacoustic imaging (PAI) combines the benefits of optical and ultrasound imaging. It relies on the photoacoustic effect, where short laser pulses are used to illuminate tissue or samples. The absorbed light energy induces a transient thermoelastic expansion, generating ultrasound waves that are detected by an ultrasound transducer. These ultrasound waves are then converted into images, providing information on the optical absorption properties of the tissue. PAI offers a deeper penetration than pure optical techniques. Combining the two modalities provides complementary information; fluorescence imaging excels at detecting specific molecular targets, while PAI enhances the visualization of tissue structures at depth. Additionally, both modalities can allow for simultaneous functional and molecular imaging to provide information about biological processes in real time. fl, QY) indicates a greater efficiency in converting absorbed photons into fluorescent photons [Optimal fluorochromes are characterized by several key photophysical properties. A high quantum yield (Φ photons . The mol photons . Underst photons .The absorbance cross-sections (ACS) for one-photon e sample . The proe sample ,13,14. Ae sample ,16. The Like fluorochromes for fluorescence imaging, the ideal PA contrast agent will exhibit a large molar extinction coefficient, a sharp-peaked absorption spectrum, peak absorption in the SWIR window, and high photostability. However, unlike fluorochromes, ideal PAI signaling compounds must exhibit a low quantum yield to efficiently convert heat energy to acoustic waves . This efThis section discusses organic and inorganic fluorophores for fluorescence and photoacoustic bioimaging in the NIR-II window. It begins by highlighting the features of organic fluorophores and their limitations, such as low photostability and the lack of biocompatible dyes emitting wavelengths exceeding 1200 nm. It then delves into various classes of organic fluorophores, such as cyanine dyes, organic electron donors and acceptors, commercial donor–acceptor IR dyes, aggregation-induced emitters, and semiconducting polymer nanoparticles. For each class, specific examples, applications, and challenges are discussed. The section then transitions to inorganic fluorophores, focusing on semiconductor quantum dots, graphene-based materials, single-walled carbon nanotubes, rare-earth nanoparticles, nanogolds and gold nanomaterials, and carbon dots. Throughout the discussion, attention is paid to their unique properties, applications, and prospects in NIR-II fluorescence and photoacoustic imaging. Organic dyes are the first class of contrast agents developed for fluorescence bioimaging. Organic dyes generally have low photostability but have low cost and well-established functionalization chemistry and safety assessments. There is also a lack of organic, biocompatible dyes that emit wavelengths exceeding 1200 nm . Major c−1cm−1 [Cyanide dyes represent a sub-type of organic small-molecule fluorescence probes. NIR fluorophore design is commonly based on the modification of polymethine dyes. The most well-known cyanine dye, indocyanine green (ICG), is an FDA-approved imaging contrast used for angiography in ophthalmology. ICG has a half-life of 2–4 min in aqueous solution and binds rapidly to serum proteins, resulting in fast clearance . ICG als−1cm−1 ,70.Although ICG exhibits peak fluorescence emission at 820 nm, it is known to exhibit long-tail fluorescence in the SWIR wavelengths of up to 1500 nm . This prICG has also been studied as a contrast agent in PA cancer imaging . Lee et Benzobisthiadiazole  is an organic electron acceptor applied to NIR-II functional imaging with a 3 mm image depth ,72. BBTDCoupled with a strong electron donor, the strong donor–strong acceptor strategy has yielded NIR-II-sensitive polymers based on the ICT effect. Typical electron donors coupled with BBT or thiophene-based acceptors include cyclopentadithiophene (CDT), dithienosilole (DTS), and dithienopyrrole (DTP) .Several polymethine molecules such as IR-26, IR-783, IR-1048, IR-1051, and IR-1061 exhibit fluorescence in the NIR-II region . Many, lKilian et al. obtained and screened 13 commercially available dyes with NIR-II absorbance for in vivo fluorescence and PA imaging . Of thesKilian et al. continued to investigate BIBDAH as a NIR-II PA contrast agent for longitudinal pre-clinical imaging . Here, BSemiconducting polymer nanoparticles (SPNs) are a subclass of organic semiconducting materials composed of highly π-conjugated, electron-delocalized polymer backbones, which may be modified for bioimaging . Like quRelatively few SPNs have been engineered toward NIR-II fluorescence imaging as small-molecule fluorophores in NIR-II fluorescence imaging are limited by low fluorescence quantum yields. However, Hong et al. synthesized a polymer fluorophore to emit in the NIR-II window—the first to do so . The polThe generally high photothermal conversion efficiencies of SPNs are advantageous for PA imaging. Zhen et al. outlined a screening process to elucidate the relationship between SP molecular structure and PA properties . They deMany organic fluorophores exhibit aggregation-induced quenching, which may preclude their use as solid-state nanoparticles. On the other hand, some fluorophores become brighter in the solid state, a phenomenon called aggregation-induced emission (AIE) . FluoropWu et al. designed a novel AIEgen with emission in the NIR-II region based on donor–acceptor–donor (D–A–D)-structured small molecules . The absSingh et al. reported J-aggregates (JA) made of indocyanine green dye as a candidate for a biocompatible NIR contrast agent class suitable for photoacoustic imaging . They inPorphyrins are large, heterocyclic compounds consisting of four subunits of pyrrole linked by methane bridges, which may be substituted by functional groups at the β-position . PorphyrBoron-dipyrromethene (BODIPY)-based compounds are a class of NIR-II-emitting pyrroles . Zou et Quantum dots (QDs) are inorganic semiconductor nanocrystals with narrow, tunable fluorescence emission bands and broad absorption bands. QDs are bright, highly photostable, and easily conjugated, making them desirable for fluorescence bioimaging. QDs have potential for heavy metal toxicity and for relatively short circulation time .Lead sulfide (PbS) QDs have the highest fluorescence quantum yield (60%) and can be tuned to emit in the NIR-II wavelengths. However, they have a relatively low chemical stability and have been criticized for potential toxicity from the leaking of heavy metal ions resulting from sulfide oxidation. Zebibula et al. addressed the issue of toxicity by encapsulating PbS QDs in dual layers of Pluronic F-127 polymer . Zhang ePbS/CdS nanoparticles have a limited water solubility due to a coating of oleylamine during synthesis, which poses a challenge for deployment in living systems. Jin et al. developed several PbS QDs to overcome water solubility issues, including coating the QDs with glutathione (GSH) and recombinant proteins . Zhao etSilver sulfide (AgS) QDs are among the most successful for live cell tracking due to high fluorescence quantum yield in the NIR-II window . Althoug2 hybrid bound carbon atoms in a hexagonal configuration [2-hybridized structure, generating wrinkles in the sheet. Reduced graphene oxide (rGO) features further functional defects across the basal plane to enable the loading of various other materials [Graphene is one of the most well-studied nanomaterials, consisting of a two-dimensional sheet of spguration . Graphenaterials . However2 domains more efficiently absorb NIR light [Reduced GO shows strong fluorescence quenching when absorbing NIR light; however, NIR-II emission has not been studied. Of the graphene-based contrasts, rGOs are especially suited toward PAI because their larger spIR light . This prIR light . The rGOIR light .2-hybridized carbon atoms, constructed from graphene sheets rolled into tubes at a chiral angle [Single-walled carbon nanotubes (SWCNTs) are a family of highly elongated tubular structures composed of covalently bonded carbon atoms arranged in ordered forms. Carbon nanotubes are a high-aspect ratio nanostructure of spal angle . The resThe optical properties of SWCNTs offer unique advantages, including high photostability under a prolonged NIR fluorescence. This non-photobleaching property of SWCNTs lends advantage for long-term measurements in real-time. SWCNTs have a relatively low fluorescence quantum yield due in part to quenching by structural defects . FluoresHong et al. developed a dual-color SWCNT to characterize differences between NIR-I and NIR-II fluorescence imaging . Their NPhotoacoustic signal has been recorded from SWCNTs of lengths 50–300 nm and diameters of 1–2 nm . Photoac− ions, resulting in aqueous quenching. Up-conversion luminescence has been well studied for decades in the context of imaging. Down-converting RENPs are less studied yet hold greater potential for NIR-II imaging.Rare-earth (RE) nanoparticles are composed of biocompatible lanthanide metals and feature high photostability and narrow-band fluorescence emission . In term3+ ion with 980 nm light produces the resonance energy required for a down-conversion fluorescence emission at 1550 nm. Erbium lattices may be structured in a compact, hexagonal configuration (β-phase) or cubic configuration (α-phase). These structural phases have been shown to influence NIR-II fluorescence of ErNPs. Zhong et al. reported that zinc-doped alpha-phase ErNPs produced an eleven-fold enhancement of down-conversion fluorescence [Erbium-based nanoparticles (ErNPs) have been studied as promising NIR-IIb fluorescence contrast agents. The excitation of the Errescence . The RE ion may be excited directly via absorption, or receive energy from a neighboring ion, a process known as sensitization. Ytterbium ions have been shown to be sensitizing agents in nanoparticle formulations for down-conversion luminescence, first demonstrated by Naczynski et al. . The res3+ ions and featuring a core-shell structure (NaYF4:5%Nd@NaGdF4). This nanoprobe exhibited a long-tailed emission spectrum in the NIR-II range. Surface modification of the nanocrystals using DSPE-PEG2k extended blood circulation time and enhanced biocompatibility and biosafety when tested in vivo. The nanoprobe exhibited a relatively high quantum yield of 0.52%, surpassing that of the fluorescence dye IR-26. Utilizing NaYF4:5%Nd@NaGdF4 as the basis for NIR-II imaging, the successful visualization and monitoring of tumor blood vessels were achieved in breast tumor models [Few studies have investigated the NIR-II excitation of PA RENPs. However, He et al. increased the PA efficiency of up-converting RENPs by functionalization with acrylic acid . This plr models .Nanogolds represent a family of nanostructures  studied for potential in cancer theranostics and NIR-II imaging. Gold nanomaterials are biocompatible, chemically inert, and typically have high molar extinction coefficients. Nanogolds in the size range of 5–200 nm are captured by the reticuloendothelial system, which may place internal organs at risk. However, atomic gold nanoclusters (AuNCs) have sizes below the kidney filtration threshold, which allow for rapid renal clearance.Kong et al. developed AuNCs tailored for NIR-II fluorescence imaging in a xenograft-bearing mouse model . Such AuGold nanomaterials have been studied as an NIR-II-absorbing PA contrast. Of these, miniature gold nanorods (solid or hollow) that are 5–10 times smaller than regular nanorods have emerged as an attractive choice due to high structural aspect ratios, as plasmonic absorption is closely related to particle size and structure. Cai et al. developed such a miniature hollow gold nanorod (~46 nm) for cancer-targeted PAI in the mouse model . FunctioCarbon dots (CDs) are small carbon nanoparticles typically ranging from 2 to 10 nanometers in size. They are derived from carbon-rich precursors through a simple and low-cost synthesis process, often involving hydrothermal or microwave-assisted methods . The exaCarbon dots are primarily characterized by their intrinsic fluorescence, with the latest property under investigation being NIR-II fluorescence. Carbon dots doped with iron (Fe-CDs) developed by Ci et al. exhibited a linear, pH-dependent fluorescence intensity peaking at 1000 nm . These wThe main contents discussed in this section focus on different types of delivery systems for imaging contrast agents. The first part discusses lipid-based delivery systems, which offer formulation versatility, biocompatibility, high payload content, and stability for NIR-II contrast agents. The second part introduces microbubble-based delivery, which involves microspheres of encapsulated gas that can be used as ultrasound contrast agents and drug delivery platforms. NIR-II fluorescence imaging using microbubbles has been explored, showing potential for multi-modal imaging applications. Next, nanomicelle-based delivery systems are discussed, focusing on polymerized micelles. Lastly, protein assemblies for imaging are presented, involving supramolecular complexes of proteins loaded with fluorophores. These various delivery systems, illustrated in Lipid-based carrier systems are an emerging technology designed to address issues with biocompatibility and water solubility in delivered biomolecules . Lipid-bLipid encapsulation techniques are being applied to NIR-II contrast agents. In a study, IR-1061 dye was encapsulated within a lipid nanoparticle functionalized with PEG for the PA imaging of hepatocellular carcinoma in mice . This Po3F8 gas core loaded with Sudan Black (BNB) and DiD fluorescent dye. This was applied as a photoacoustic contrast agent for breast cancer imaging and showed the potential of BNBs as multi-modal contrast agents capable of specialized tumor imaging in vivo [Microbubbles are microspheres of encapsulated gas commonly used as ultrasound contrast agents but have gained recent attention as a platform technology for drug delivery and multi-modal imaging ,115. The in vivo .Polymerized micelles, also known as nanocarriers, have potential for delivering imaging contrast agents. These nanomicelles are self-assembled, colloidal dispersions with a hydrophobic core and hydrophilic shell, typically 10–100 nm in diameter . MicelleSupramolecular assemblies of protein complexes may be loaded with fluorophores for labeled imaging. These bioengineered platforms can be tuned to enhance the brightness of the fluorophore in addition to modifying the pharmacokinetic and clearance properties of the probe. Antaris et al. functionalized the NIR-II dye CH-4T to form bright supramolecular assemblies with plasma proteins . Such prThis section focuses on the functionalization and surface treatment of imaging contrast agents. PEGylation is explored as a biochemical modification process involving the attachment of polyethylene glycol (PEG), which allows for the retention of imaging contrast agents without the loss of function. The second part discusses the use of monoclonal antibodies and antibody mimetics for contrast-enhanced imaging. The third part covers targeting peptides, which are small peptide sequences capable of binding to cells and tissue targets with high specificity and affinity. Aptamers, which are synthetic RNA or single-stranded DNA molecules, represent an emerging class of ligands for contrast agent delivery. Lastly, cell membrane encapsulation is discussed. Each of these are illustrated in PEGylation is a biochemical modification process of covalent or non-covalent attachment of polyethylene glycol (PEG) to biomolecules . ComposePEG is synthesized from ethylene oxide monomers by means of ring-opening polymerization. The reaction produces a mixture of bi-functional and mono-functional PEG chains of varying molecular weights. The size and position of PEG molecules strongly affect the properties of the conjugated biomolecule. First-generation PEGylation relied on the non-specific binding of PEG chains of varied sizes, but additional purification steps are employed in recent methods to improve the specificity of function.Monocolonal antibodies (mAbs) are highly site-specific proteins developed for biotherapeutics . The affCell-targeting peptides (CTPs) are small peptide sequences capable of binding to cells and tissue targets with high affinity and specificity . CTPs reSeveral peptide-conjugated NIR-II fluorophores have been reported. Wen et al. applied an amphiphilic peptide to AgS quantum dots conjugated with the tumor targeting RGD peptide . This chAptamers are synthetic sequences of RNA or single-stranded DNA molecules that represent an emerging class of ligands for contrast agent delivery. The SELEX  method is used to generate a nucleic acid sequence from a combinatorial library . CompareAn analysis by Kelly et al. compared the binding properties of fifteen aptamers to eleven different cancer lines as well as to that of antibody controls . This stHowever, aptamers have been successfully conjugated on fluorescence and PAI probes. An in vivo study by Shi et al. demonstrated a high specificity of tumor cell binding using a cyanide-based dye labeled with the TD05 aptamer . PA probBiomimetic functionalization is an emerging approach of NIR-II nanoprobe surface treatment involving the encapsulation of fluorophores within cell membrane components. Often referred to as cell membrane-coated nanoparticles (CCNPs) or camouflaged nanoparticles, biomimetic materials are encapsulated within sections of an extracted cell membrane, granting them native cell functionalities. This membrane can be further treated with additional surface ligands and modifications. Biomimetic encapsulation may be considered both a delivery system and a surface treatment, as the cell membrane imparts properties of both functionalities to the nanoprobe. Biomimetic encapsulation is particularly advantageous for the delivery of nanoprobes across the blood–brain barrier (BBB). Men et al. recently demonstrated specific NIR-II fluorescence imaging of glioma in a mouse model using membrane-coated polymer dots . SimilarNot only can membrane encapsulation be applied toward the targeting of specific tissue types, but it can also be used for photoacoustic imaging. NIR-II-absorbing polypyrroles enveloped in cancer cell membranes demonstrated a guided PA imaging of tumors in the mouse model . The NIRThe development of next-generation NIR-II-sensitive imaging probes will require a detailed understanding of the latest nanobiotechnology. The brightness of a fluorescent molecule is determined by the product of its fluorescence quantum yield and its absorption cross-section at the excitation wavelength. Similarly, for ideal photoacoustic imaging (PAI) contrast agents, a large molar extinction coefficient, a sharp-peaked absorption spectrum, peak absorption in the SWIR window, and high photostability are desired. Emerging NIR-II-sensitive materials will be made to be targeted, stable, and safe for in vivo imaging. Functionalization technology opens possibilities to tailor imaging contrast agents for improved specificity, sensitivity, and performance in fluorescence and PA imaging modalities. The combinations of transport and functionalization technologies will be specific to the use case and tissue target. To date, NIR-II nanoprobes have been used in fluorescence and photoacoustic angiography, tumor imaging, the tracking of cellular migration, examining biological distribution and clearance pathways, tracking the location and differentiation of transplanted stem cells, the monitoring of collagen degradation, the imaging of inflammation in the brain and lymph nodes, and the monitoring of bacterial infections. NIR-II contrast agents will continue to be a platform for studying tissue structures and disease pathologies.It is essential to acknowledge that NIR-II optical imaging technology is still in its early stages and faces some key challenges. One major concern is the limited stability of many organic NIR-II nanoprobes in water, restricting their clinical utility. Additionally, a comprehensive understanding of the biocompatibility, metabolic pathways, and long-term toxicity of NIR-II nanoprobes within the body is crucial, with many inorganic probes falling short in this regard. Furthermore, conventional NIR-II optical imaging systems suffer from drawbacks such as a narrow field of view and low resolution. These limitations must be overcome for the widespread use of NIR-II optical imaging in clinical settings.As technology continues to evolve, the development of novel nanoprobes with improved photophysical properties, enhanced brightness, and better stability is expected. Researchers will likely focus on fine-tuning the design of NIR-II fluorophores and exploring various functionalization techniques to enable the targeted and specific imaging of disease biomarkers. Additionally, efforts to improve cytotoxicity evaluation and biocompatibility of imaging probes will be crucial for their successful translation into clinical settings. The integration of multimodal imaging approaches, by combining fluorescence and photoacoustic modalities, is anticipated to provide complementary information, enabling more accurate and comprehensive assessments of biological processes. Moreover, advancements in instrumentation and imaging systems will likely facilitate a higher spatial and temporal resolution, enabling real-time imaging and the dynamic monitoring of biological events at the molecular level. The future of NIR-II imaging probes in fluorescence and photoacoustic imaging is bright, with the potential to revolutionize non-invasive imaging in preclinical research.


In the last decade, immune checkpoint blockade (ICB) has revolutionized the standard of treatment for solid tumors. Despite success in several immunogenic tumor types evidenced by improved survival, ICB remains largely unresponsive, especially in “cold tumors” with poor lymphocyte infiltration. In addition, side effects such as immune-related adverse events (irAEs) are also obstacles for the clinical translation of ICB. Recent studies have shown that focused ultrasound (FUS), a non-invasive technology proven to be effective and safe for tumor treatment in clinical settings, could boost the therapeutic effect of ICB while alleviating the potential side effects. Most importantly, the application of FUS to ultrasound-sensitive small particles, such as microbubbles (MBs) or nanoparticles (NPs), allows for precise delivery and release of genetic materials, catalysts and chemotherapeutic agents to tumor sites, thus enhancing the anti-tumor effects of ICB while minimizing toxicity. In this review, we provide an updated overview of the progress made in recent years concerning ICB therapy assisted by FUS-controlled small-molecule delivery systems. We highlight the value of different FUS-augmented small-molecules delivery systems to ICB and describe the synergetic effects and underlying mechanisms of these combination strategies. Furthermore, we discuss the limitations of the current strategies and the possible ways that FUS-mediated small-molecule delivery systems could boost novel personalized ICB treatments for solid tumors. Cancers are devastating life-threatening diseases. Globally, an estimated 19.3 million new cancer cases, and nearly 10 million cancer deaths, occurred in 2020. By 2040, the global cancer burden is expected to reach 28.4 million cases . SurgeryAmong these combination strategies, the recently much-discussed technology, focused ultrasound (FUS), has been found to enhance the anti-tumor immune response in humans . SonodynDue to the great potential of FUS-mediated therapy in boosting immunotherapy especially ICB, in this review we summarize the recent research progress of FUS-controlled small-molecule delivery systems in ICB therapy. In addition, we discuss the possible mechanisms by which FUS-mediated small-molecule delivery systems could facilitate personalized ICB therapy for solid tumors.Physiologically, immune checkpoints are points in cellular pathways at which key molecules maintain the immune homeostasis and protect healthy tissues from immune attack . To dateThe clinical impact of ICB has grown considerably over recent decades yet several challenges remained to be overcome. Notably, the response rate to ICB varies for different types of cancers . ResultsAnother difficult problem is that systemic administration of ICB can lead to severe irAEs in numerous organs . Early t+ DCs, which promote T Cell infiltration through cross-presentation , medium intensity, or low intensity (<3 W/cm2) (The exploration of the application of ultrasound in medicine began in the early 20th century in an attempt to detect the presence of tumors in brain tissue based on tissue density . In 19503 W/cm2) . High-in3 W/cm2) . It was 3 W/cm2) . Since t3 W/cm2) . Basical3 W/cm2) , increas3 W/cm2) .FUS can induce biological effects within deep tissue in a minimally or non-invasive manner . The speDue to the thermal effects of HIFU, the most commonly explored application is thermal ablation . MonitorFUS techniques can also be applied in neuromodulation therapies. Low-intensity focused ultrasound (LIFU) plays a useful role in neuromodulation compared with the ablative effect caused by HIFU . Indeed,in situ by FUS are taken up by dendritic cells  changes, as well as concurrent downregulation of protumor regulators such as regulatory T Cells, IL10, TGFb, and VEGF-A  elicited stronger systemic cellular antitumor immunity and suppression of tumor growth than thermal-HIFU (T-HIFU) in murine breast cancer models because M-HIFU induced an inflammatory, M1-biased, macrophage population which contributed to enhanced antitumor immunity in M-HIFU-treated tumors . Howeverd VEGF-A . In thisraftment . Anotherraftment . It was raftment .Overall, the combination of FUS and ICB has been shown to exert a superior anti-tumor effect by inducing a local or systemic inflammatory immune activation, and enhancing the intratumoral infiltration and maturity of innate and adaptive immune cells, as well as concurrently downregulating protumor regulators in different types of solid tumors.Accumulative evidence suggests that FUS has potential in small-molecule drug delivery  because + T Cells across evaluated organs significantly, but upregulated checkpoint molecules (PD-1 and TIM-3) on these cells were detected at 1-week post-FUS, suggesting the increased activation of the T Cells. Similarly, the study of Lee et al. indicated that a closed-loop controlled MB-FUS system could lead to a better penetration and distribution of the anti-PD1 antibody in the GL261 glioma mouse model  loaded nanocomplex-conjugated microbubbles (ncMBs) that target antigen-presenting cells  APCs, F. cGAMP cin vivo  effect to sustainably deliver small-molecule drugs to solid tumors after intravenous administration . However+ and CD8+ T Cells and tumor-suppressing M1 phenotype macrophages, and increased the expression of PD-1 and PD-L1 in T Cells. Moreover, CFUS markedly inhibited primary B16 melanoma growth and prevented tumor growth in distal untreated sites. These results suggested that the potential combination of CFUS and PD-1/PD-L1 blockade may provide a better anti-tumor effect.It has been extensively reported that the release of DAMPs such as calreticulin (CRT) in solid tumors can lead to local activation of ICD . CRT enh2O2 economizer was developed for on-demand O2 supply and sonosensitizer-mediated ROS production during ultrasound activation was developed. Both in vitro and in vivo experiments demonstrated that MFC could be effectively accumulated in tumor cells and tumor tissue via homologous targeting mechanisms, and ultrasound irradiation promoted MFC dissociation and the exposure of Fe-PDAP for a more robust O2 supply  the genome of which was optimized to express anti-CTLA-4 and anti-PD-L1  may increase the risk of inducing a strong immune response , and modinsitu αCD40 combinatorial therapy on ICB refractory murine melanoma , a chemotherapy, in murine metastatic TNBC models . The treThese studies provide timely and provocative insights into the immunogenicity of FUS and the role of FUS-ICB-based combinatorial strategies in solid tumors. To this end, future studies are expected to optimize treatment frequency, and drug dosage in larger animal models and clinical trials.Although the field of drug delivery systems as a whole is advancing at a rapid pace, the design of FUS-mediated delivery technologies for this field is still in its infancy, especially when in combination with ICB for human solid tumors. The major challenges still remain in the TME, with high interstitial fluid pressure, compressed vasculature and dense fibrotic tissue surrounding solid tumors that hinders T Cell infiltration and dampens the efficacy of ICB. Small molecule agents that change the physical conditions of TME could be the future targets of the FUS-mediated delivery systems. Losartan (an angiotensin inhibitor), which is under investigation in patients with pancreatic cancer in a phase II clinical trial (NCT01821729), was shown to potentiate the therapeutic delivery of chemotherapeutic agents to several solid tumor types . TreatmeDespite its potential, several limitations of FUS-assisted drug delivery remain to be solved, including the short duration of the effect and erratic drug uptake . In clinICB has been extensively explored as one of the most promising cancer immunotherapies, yet it has failed to acquire satisfactory clinical outcomes for solid tumors. Small-molecule drugs could assist ICB through different mechanisms, but need precise and safe delivery. In this review, we summarized the current frontline studies of FUS-assisted small-molecule drug delivery in combination with ICB to treat solid tumors. This research provides a proof-of-concept for FUS-mediated delivery serving as a pioneer and effective combinatorial regimen for ICB. By further understanding the underlying mechanisms of the TME and developing novel technologies, FUS-mediated small-molecule delivery could definitely boost the efficacy and the safety of ICB in solid tumors.


Myelin oligodendrocyte glycoprotein (MOG)-associated disease (MOGAD) is a rare, antibody-mediated inflammatory demyelinating disorder of the central nervous system (CNS) that has varying phenotypes. FLAIR (fluid-attenuated inversion recovery)-hyperintense Lesions in Anti-MOG-associated Encephalitis with Seizures (FLAMES) is a much rarer manifestation of cortical encephalitis encountered in MOGAD. We report a rare case of a nine-year-old girl who presented with a drop in her academic performance and right-sided Epilepsia partialis continua. Magnetic resonance imaging (MRI) of the brain detected evidence for unilateral (left) cortical encephalitis with peri-ictal juxtacortical edema. An electroencephalogram revealed a hemi-generalized poly spike and wave discharges in the left hemisphere, several of which correlated with myoclonic jerks. The cerebrospinal fluid (CSF) analysis was normal. Autoimmune workup resulted in a positive serum MOG-immunoglobulin G (IgG), which confirmed the diagnosis of FLAMES. The child showed an excellent clinical response to intravenous methylprednisolone and intravenous immunoglobulins therapy. Myelin oligodendrocyte glycoprotein (MOG)-associated disease (MOGAD) is a rare antibody-mediated inflammatory demyelinating disorder of the central nervous system (CNS). Despite earlier studies describing MOGAD as similar to neuromyelitis optica spectrum disorders (NMOSD), recent evidence describes MOGAD as a distinct disease with unique immunological characteristics -3. It maEncephalitis is a relatively rare manifestation of MOGAD compared to optic neuritis and myelitis. Till recently, encephalitis in MOGAD was thought to involve subcortical structures similar to acute disseminated encephalomyelitis (ADEM). Recently, a distinct and much rarer manifestation of MOGAD, cortical encephalitis, was described which is FLAIR (Fluid attenuated inversion recovery)-hyperintense Lesions in Anti-MOG-associated Encephalitis with Seizures (FLAMES). It was first described by Ogawa et al. in 2017 as a specific clinico-radiological syndrome, separate from other anti-MOG antibody-associated inflammatory demyelinating disorders. It often has an indolent clinical course, particularly in children -5.Seizures, headaches, fevers, and cortical symptoms referable to the FLAMES location are the most common clinical manifestations. A vast majority have two or more of the four above-mentioned findings simultaneously ,5.We report a rare case of a nine-year-old girl who presented with a drop in her academic performance and right-sided weakness of her upper and lower limbs and face. Early recognition and prompt treatment led to an exceptional outcome.A nine-year-old girl was brought to our neurology department by her parents with a 2.5-month history of a drop in scholastic performance. Initially, her scholastic decline and inattention were perceived to result from a behavioral issue by her teacher, and the parents were intimated. She was taken to a child psychiatrist for evaluation. The child’s psychiatric evaluation was normal and was advised close monitoring to seek any evolution of symptoms. Two weeks later, she was noted to have brief episodes of right upper and lower limb jerks with preserved consciousness and awareness. She was initially started on oral carbamazepine by a pediatrician, which resulted in a transient cessation of the jerks.In the last month, despite being on medication, the frequency increased to such a degree that the jerking was nearly continuous. The jerks also started affecting the right side of her face. This was associated with a drop in verbal output with intact comprehension and weakness of right-sided limbs with consequent walking difficulty. At this stage, the child was referred to our care. At the initial examination, the child was conscious, alert, and comprehending. She was having left hemispheric epilepsia partialis continua (EPC). Magnetic resonance imaging (MRI) of the brain detected evidence of unilateral (left) cortical encephalitis with peri-ictal juxtacortical edema Figures -3.An electroencephalogram revealed a hemi-generalized poly spike and wave discharges in the left hemisphere, several of which correlated with myoclonic jerks. Cerebrospinal fluid (CSF) demonstrated normal cells, proteins, and glucose. Initial possibilities of early Rasmussen encephalitis (prior to the development of atrophy) Vs. MOG-related unilateral encephalitis (FLAMES) were considered. Autoimmune workup for CSF N-methyl-D-aspartate receptor (NMDAR) antibody, serum NMDAR, voltage-gated potassium channel (VGKC), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, and gamma-aminobutyric acid (GABA) antibodies were negative.Serum MOG cell-based indirect immunofluorescence assay was positive. Serum metabotropic glutamate receptor 3 (mGLUR3) was negative. The child was started on a five-day 30 mg/kg/day pulsed intravenous methylprednisolone (IVMP) along with 2 g/kg intravenous immunoglobulins (IVIg). She had a significant improvement in EPC and started walking independently. She was maintained on a monthly 1g/kg IVIg pulse. At her one-month follow-up, there were no neurological deficits. The patient was followed up for four months during which there was no recurrence of symptoms. She lost follow-up later on.We report a case of a young girl with FLAMES, a novel clinco-radiographic entity. FLAMES is a subtype of the MOGAD spectrum.In 2017, Ogawa et al. performed a retrospective cross-sectional study among a cohort of 24 consecutive adult patients with steroid-responsive encephalitis of unknown etiology after encountering an index case of MOG antibody-positive unilateral cortical encephalitis with epileptic seizure. Four MOG-IgG-positive patients were identified, all of whom had similar distinct unilateral cortical T2-FLAIR hyperintensities, which resolved with high-dose IVMP. In contrast, none of the MOG-IgG-negative patients showed neuroimaging findings suggesting anti-MOG-associated encephalitis [The discovery of a disease-specific serum IgG antibody that selectively binds MOG has increased the understanding of the diverse spectrum of MOGAD. MOG is a member of the immunoglobulin superfamily and a lesser component of myelin. MOG is expressed on the surface of oligodendrocytes, on the outermost part of the myelin sheath. It is highly immunogenic, making it a potential target for MOG antibodies ,7. The fThe mean presenting age is 29 years range 11-46 years), with a male predominance (60%) [-46 yearsIn a few of the earlier reports, subcortical FLAIR hyperintensities on MRI were considered as an exclusion criterion. Later reports, however, placed little emphasis on these findings. In our case, MRI was done while the child was having EPCs and the juxtacortical FLAIR hyperintensities are likely because of peri-ictal edema, as there is no corresponding diffusion-weighted imaging (DWI) restriction suggesting that it is a vasogenic pathology. While the cortical gyral FLAIR hyperintensities showed corresponding DWI restriction suggesting a cytotoxic pathology secondary to encephalitis, there is also associated sulcal effacement and corresponding focal thin leptomeningeal enhancement, as is noted in a few of the earlier reports .Cell-based assays with full-length human MOG as the target antigen are considered the gold standard method for detecting anti-MOG antibodies. Serum is a better sampling source than CSF, which has a lower concentration of antibodies . The timThe concept of MOGAD and FLAMES are relatively novel entries into the domain of the antibody-mediated inflammatory disorder spectrum. The low prevalence, varied clinical features, and age-related differences in clinical presentation all contribute to the sparsity of medical literature on this topic. To date, no large-scale, multicentric treatment trial has been conducted. As a result, there are no evidence-based guidelines. Since MOGAD/FLAMES have only recently been delineated from NMOSD, there is an overlap in the treatment approaches. The treatment guidelines are derived from expert opinions.High-dose IVMP (30 mg/kg/day for 5-10 days) is the treatment modality of choice, particularly for acute exacerbations. For patients with steroid unresponsiveness or an aggressive clinical course, early initiation of IVIg or plasma exchange may be beneficial ,14,15. TGlatiramer acetate, natalizumab, and interferon beta, which are classically used as disease-modifying drugs in MS, have been ineffective or worsened the disease ,17.Immunosuppressants such as azathioprine, methotrexate, mycophenolate mofetil, rituximab, and IVIg (repeated cycles) have been studied to combat the potentially hazardous consequences of long-term steroid therapy. A recent retrospective multicentric study reported that IVIg infusions demonstrated the most promising results (relapse rate of ≤ 20%) compared to other agents (relapse rate 59-74%). Hence, we opted for monthly pulse IVIg therapy ,18.This case report adds to the literature evidence for the recently described FLAMES clinico-radiographic entity. Though previously considered a variant of NMOSD, MOGAD is now recognized as a separate, antibody-mediated inflammatory demyelinating pathology. The discovery of novel antibodies has redefined the thought process about inflammatory demyelinating CNS disorders. The initial clinical presentation may be varied, which can add to the diagnostic difficulty. Hence, physicians should remain cognizant of this. Physicians should notice FLAMES as a unique sub-entity of the MOGAD spectrum with distinctive clinical and radiological findings and could be a differential diagnosis for several focal encephalitis-like presentations. The method and timing of testing for MOG antibodies are crucial. Cell-based assays have the highest sensitivity and are considered the gold standard. Repeat testing might be beneficial even after initial negative results if the clinical suspicion is strong. Early recognition and prompt treatment are crucial for achieving the best possible outcomes.