The calibration curve was constructed in a linear operating range from 0.67 μM to 121 μM UA. The proposed sensor displayed good reproducibility with an RSD of 3.65% calculated for five subsequent measurements, and a low detection limit of 64.28 nM, determined using the 3 S/m method. Interference studies and the real sample analysis of allergen-treated cell lines proved that the proposed sensing platform possesses excellent sensitivity, reproducibility, and stability. Therefore, it can potentially be used to evaluate stress factors in medical research and clinical practice.This paper aims to develop an amperometric, non-enzymatic sensor for detecting and quantifying UA as an alert signal induced by allergens with protease activity in human cell lines (HEK293 and HeLa). Uric acid (UA) has been classified as a damage-associated molecular pattern (DAMP) molecule that serves a physiological purpose inside the cell, while outside the cell it can be an indicator of cell damage. Cell damage or stress can be caused by different health problems or by environmental irritants, such as allergens. We can act and prevent the events that generate stress by determining the extent to which cells are under stress. Amperometric calibration measurements were performed with a carbon paste electrode modified with La(OH) Various events can strain the human body, putting stress on the cells and tissues. Oxidative stress, heat shock, hypersensitivity, autoimmune diseases and other disorders can cause cell dysfunction and lead to apoptosis . BesidesUric acid, being a final product of purine metabolism, has a high impact on human health. It is produced in the liver, intestines, kidneys, vascular endothelium, and muscles by the metabolism of purine .Both high and low human plasma UA concentrations can indicate a pathological state. On the one hand, UA acts as a scavenger of peroxyl radicals, hydroxyl radicals, and singlet oxygen, thus exhibiting antioxidative features. It is a specific inhibitor of radicals generated by the decomposition of peroxynitrite, preventing cell injuries and nitration of tyrosine residues in proteins. Furthermore, it can protect against oxidative damage by chelating metal ions such as iron and copper, lowering their catalytic activity in free-radical reactions . UA alsoUA also acts as an initiator and amplifier of allergic inflammation and can cause hypertension and cardiovascular diseases via the induction of growth factors, hormones, cytokines, and autacoids. UA penetrates vascular smooth muscle fibers and activates signal transduction, increasing the expression of inflammatory mediators. Furthermore, urate crystals can deposit in the connective tissues of the joints, tendons, kidneys, and rarely in heart valves and the pericardium. Consequently, UA is a risk factor for renal disorders (kidney injuries and kidney stones), acute and chronic inflammatory arthritis, gout, myocardial infarction, and stroke ,5. ElevaThe important UA feature is that it is produced in higher concentrations when the cells suffer from stress. Both live and dying cells degrade their nucleic acids in deamination and dephosphorylation processes. Purine nucleoside phosphorylase converts the relevant degradation products, inosine and guanosine, to the purine bases, hypoxanthine and guanine, which further metabolize to xanthine. Oxidation of xanthine via xanthine oxidase leads to the formation of UA. Therefore, although uric acid is regularly present in cells, it increases in concentration when the cells are damaged . The UA’Considering its importance and abundance in the human body, it is no wonder that numerous methods have been developed for UA detection and determination. The separation and detection methods commonly involve chromatography or electrophoresis coupled with UV/VIS ,7,8,9,10Enzyme-based UA biosensors use uricase as an enzyme for the oxidation of uric acid ,28. The 3 for the simultaneous determination of dopamine, uric acid and ascorbic acid, thus proving the materials’ compatibility with these analytes. La doping can affect lattice structures and phase transformations of compounds due to its larger atomic radius and unique electron structure, thus enhancing the catalytic and electrochemical properties of La-doped materials /K4[Fe(CN)6] (1:1) in 0.1 M KCl solution. EIS measurements were conducted in the frequency range from 10 kHz to 10 mHz, at 0.3 V. The used experimental parameters for CV measurements were: potential range from −0.5 V to 1 V and the scan rate of 50 mV/s. The CV measurements were also performed in the 0.1 M PB (pH 6) solution containing 10 µM UA Chronoamperometry (CA) was the detection and quantification method of choice. Measurements were taken in 0.1 M PB at pH 6, with the potential set on 0.3 V while adding an increasing quantity of UA during the period of 600 s. High-performance liquid chromatography (HPLC) for uric acid analysis was conducted  with photodiode array detection on a Hypersil Gold aQ C18 analytical column .All electrochemical measurements were carried out using a CH Instruments analyzer  driven by voltammetric software CHI (Version 4.03). A three-electrode system was employed, with a modified carbon paste (CP) working electrode (WE), a Calomel reference electrode (RE), and a Pt wire as the counter electrode (CE). The measurements were conducted in 10 mL of 0.1 phosphate buffer (PB) at pH 6. Electrochemical characterization of working electrodes was conducted using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) in 5 mM K3@MWCNT powder was prepared by means of the method reported in the literature /K4[Fe(CN)6] and 0.1 M KCl support electrolyte, at the scan rate of 50 mV/s 3@MWCNTs/CP, respectively. Consequently, La(OH)3@MWCNTs/CP was determined to have the best electrocatalytic properties and was chosen for further examination. The next step in the electrode’s surface optimization was varying the modifier percentage in CP. The resulting voltammograms  = 0.69 V, 0.47 V and 0.36 V and oxidation/reduction current ratios (Ia/Ic) = 1.02, 1.01 and 1.04 for 2%, 5% and 10% La(OH)3@MWCNTs/CP, respectively. Higher values for the peak-to-peak separation in this system, in comparison with the theoretical value of 59 mV, is a common phenomenon and it is the product of the heterogeneity of the hand-made carbon paste electrodes. The reported decrease in the ΔE value, as a result of the increase in the amount of the modifier, indicate excellent properties of the selected composite regarding the diffusion properties of the electrode surface. Since oxidation occurs on lower potentials and the other parameters point out that the electrode reaction is reversible when using 10% La(OH)3@MWCNTs/CP, this electrode material is assumed to have the best electrocatalytic properties.Characterization of the electrodes’ surfaces during the optimization of the final electrode modification was performed using CV and EIS. CV was employed to determine the interfacial properties of electrode materials and examine electron transfer kinetics between the electrode surface and the electrolyte. Firstly, the unmodified carbon paste electrode was compared to La(OH) 50 mV/s . The oxiectrodes . The oximmograms  show a s2, 1.50 mm2, 2.04 mm2, 2.20 mm2, 2.83 mm2 and 3.72 mm2 for CP, La(OH)3/CP, MWCNT/CP, 2% La(OH)3@MWCNT/CP, 5% La(OH)3@MWCNT/CP and 10% La(OH)3@MWCNT/CP, respectively. This proves, once again, that composite material formation is a crucial part of the modification process. Not only does it enhance the material’s electrocatalytic properties, but also leads to an increase in the effective surface area.To further support the abovementioned assumptions, effective surface areas were estimated for each electrode. Calculations were performed using the Randles–Sevcik Equation and the specific surface areas of the electrodes were 2.02 mm2+/3+ redox pair from cyano complexes and 0.1 M KCl support electrolyte. Rct values of CP, La(OH)3/CP, MWCNTs/CP and La(OH)3@MWCNTs/CP  and a linear (low frequency) region. The semicircle radius is electron transport resistance-dependent and is defined by its Rct value, while the linear part is diffusion-dependent. The measurements were conducted in PB pH 6, in the 5 mM redox probe containing FeWCNTs/CP  were 40,3 encourages the electron shuttle and thus has the lowest Rct value, while the composite material exhibits improved properties when compared to the unmodified paste and MWCNTs alone. Moreover, as previously discussed, this material shows the best current response in the cyclic voltammetry measurements, which confirms that La(OH)3@MWCNTs exhibits the optimal electrocatalytic activity. Furthermore, the increase in the La(OH)3@MWCNTs amount in the paste leads to the reduction in Rct and thus the improvement of the overall electrochemical performance, which is evident from the experimental data tal data . The obt3/CP, MWCNTs/CP, and La(OH)3@MWCNTs/CP electrodes show the oxidation peak current values 0.31 µA, 0.28 µA, 0.27 µA, and 0.27 µA, respectively ectively . Conside3/CP, and La(OH)3@MWCNT/CP) provide a significantly improved voltammetric peak and higher currents (I) toward UA detection. The CV signals of UA using these modified electrodes were similar, so for the selection of the optimal electrode for further experiments, we used the data obtained from the electrochemical characterization of all electrodes, as previously described. The La(OH)3/CP electrode had the poorest CV response based on these measurements, most likely due to a decrease in electrode active surface area. MWCNTs promoted electron transfer and decreased the interfacial resistance, and better CV response was achieved when MWCNTs/CP were used. The La(OH)3 likely makes the entire complex of MWCNTs and La(OH)3 more porous, which can help improve the response of the modified electrode. All of this led to the conclusion that the synergistic effect of MWCNTs and La(OH)3 improved the modified electrode’s electrochemical properties, which will contribute to the sensor’s analytical performance for uric acid detection. The electrochemical behavior of La(OH)3@MWCNTs/CP electrodes, with different weight percent of the modifier (from 2% to 10%) was compared using CV using CV . It is eusing CV .3@MWCNTs/CP electrode containing 10% of the modifier mixed in the carbon paste. Measurements were taken in 0.1 M PB, in the pH range from 2 to 9, at the scan rate of 50 mV/s, and they confirm that the reaction is pH-sensitive. The more alkaline the solution becomes, the oxidation peak potential shifts to lower values, from 0.56 V at pH 2 to 0.24 V at pH 9  at pH 9 A. On the at pH 9  55]..3@MWCNTs+ deprotonation and the oxidation of uric acid, the hydration of the intermediate diimine and its subsequent decomposition into allantoin and CO2. While this mechanism infers that the reaction is pH-dependent and that alkaline conditions allow the initial deprotonation to proceed, it is less obvious as to why the pH values higher than pH 6 are less favorable. Since pKa values for uric acid are 5.4 and 9.8, we can assume that the elevation of pH above pH 6 leads to further deprotonation of urate or diimine, thus introducing new intermediate species and broadening the oxidation peak, lowering its resolution and decreasing the maximal peak current.The reported mechanism can be divided into three main steps—2e-/2HThe best performance of the electrochemical system at pH 6, and its similarity with the physiological conditions are the reasons why this pH is used for all further measurements.2 F2)/4RT vAΓ *, we can calculate the surface coverage of the adsorbed species (Γ*) [2, 44.08 nmol/cm2, 22.04 nmol/cm2, 11.02 nmol/cm2, 7.35 nmol/cm2, 5.51 nmol/cm2, 4.41 nmol/cm2, 2.94 nmol/cm2 and 2.20 nmol/cm2. These values indicate that, although the adsorption of the UA on the electrode surface decreases with the increase in the scan rate, it is not negligible and can influence the accuracy of the measurements. To prevent introducing a systematic error in the measurements, the surface of the electrode was renewed before each measurement . The simple and reproducible restoration of the clean electrode surface was one of the major advantages of using the carbon paste electrode in this study.The CV measurements in PB pH 6 were conducted with optimized electrode surface parameters at various scan rates (from 2 mV/s to 100 mV/s) to determine the reaction kinetics. The oxidation peak potential and ΔE are constant for all scan rates in the measured range B, while ies (Γ*) . The surEven though the anodic peak does not show potential shift and its current linearly increases with the scan rate, the reduction peak is almost absent in all voltammograms, while the cathodic capacitive current increases with the increase in the scan rate. This indicates that the electrode reaction is characterized by a reversible electron transfer followed by an irreversible chemical reaction . In this3@MWCNTs/CP electrode, containing 10% of nanomaterial mixed in the CP, toward different standard solutions of the UA, was recorded in 0.1 M PB pH 6, at the fixed potential at 0.3 V at 0.3 V A. Succesat 0.3 V B. The liFive successive measurements of 2 μM UA standard solution were performed, to test the repeatability of the proposed sensing platform for UA determination and monitoring, and the obtained current values were 6.01 nA, 5.71 nA, 5.70 nA, 5.71 nA and 5.42 nA, giving an RSD of 3.65% . The rep3 nanoparticles to modify the CP electrode, a very good sensitivity of the UA detection method was achieved. This is probably due to the joint action of the modifier—the alkaline properties of La(OH)3, which facilitate the binding of UA, as well as the increase in the active surface area and electron transfer efficiency by MWCNTs.Furthermore, we conducted an extensive literature overview and compared the linear working range, LOD and RSD of our method to the best methods documented in the literature . The resOne of the essential features of the sensor is its selectivity. CV measurements of a 10 µM UA solution in 0.1 M PB at pH 6 were conducted in the presence of common interferents found in biological matrices. Both organic and inorganic interfering species which exist in the lysed cell culture were taken into account. The measurements are performed in the potential range from −0.5 V to 1 V, at the scan rate of 50 mV/s, with the ratio of UA and potential organic interferents 1:1. The chemical behavior of UA in the presence of ascorbic acid (AA), citric acid (CA), dopamine (DOPA), gallic acid (GA) and glucose (GLU) is shown in the 2, MgOAc and NaOAc in 0.1 M PB at pH 6 on detection of 10 mM UA was studied. The results show that none of the mentioned salts in a 1000:1 ratio to UA influence UA detection. The interference study proves that the proposed sensing platform shows satisfying selectivity towards UA in the presence of common organic and inorganic interferents.Considering the fact that high concentrations of salts can be present in the sample, the influence of 0.1 M KCl, NaCl, CaClThe practicability estimation of the developed method after the cell stress.To analyse the applicability of the proposed sensing platform in biological samples, measurements were conducted in a real sample matrix. CV measurements were performed in the potential range from −0.5 V to 1 V at the scan rate of 50 mV/s in the presence and absence of UA to scan for potential interferents . The basAfter the calibration, real sample analysis was conducted. The samples were obtained by treating the human HEK293 and HeLa cell cultures with the papain, and the actinidin allergen, respectively, during a time frame of 12 h. Ten microliters of the sample was added to 5 mL of 0.1 PB at pH 6, and the change in the amperometric current at 0.3 V was measured. However, the UA concentration in the sample was found to be lower than the limit of quantification. Therefore, the sample was spiked with UA standard solution, so the final concentration of UA (spiked) was 100 µM. The measurements were repeated five times with the spiked sample, and the mean value of the rise in the amperometric signal corresponded to the 0.2010 mM UA concentration in the 5 mL of PB, which agrees with the total UA concentration in the sample of 100.5 µM. That proves that the proposed method is applicable in real sample analysis. However, it requires either more concentrated samples, larger quantities of samples, or spiking the samples with a known amount of UA.To further support this claim, a series of HeLa and Hek293 cell cultures were treated with papain and EG4 inhibited papain for prolonged periods. The cell cultures were prepared in duplicate with each of the abovementioned allergens independently and the amperometric response at 0.3 V was recorded immediately after the addition of allergens, and after incubation periods of 3 h, 6 h and 12 h. The obtained results are given as mean values of the measurements in The acquired values indicate that the stress on the cells after the adequately long (6 h and 12 h) exposure to papain is sufficient to release considerable amounts of UA. Furthermore, the quantity of UA released is directly proportional to the incubation period, proving that the developed method is suitable for real-time detection or monitoring of cell damage or stress.For method validation, the same real samples were analyzed by using HPLC as a standard method. The obtained results display good agreement with those achieved in the developed electrochemical method. The recoveries of the determination are 94.3–103.0%, indicating good accuracy of the developed method in real sample analysis.https://mostwiedzy.pl/AGREE (accessed on 7 June 2022) [While developing an analytical procedure, attention is being shifted more and more to its ecological acceptability. Several methods can assess the green aspects of an analytical procedure in which the green analytical procedure index (GAPI) A and Anane 2022) . For GAPne 2022) .In This paper developed and tested a sensing platform for UA determination in the biological sample. Knowing that the electrode surface modifications are the key enabler for next-generation chemistries based on the interface reactions, we offered a sensing platform based on the novel composite material, optimized to have the best electrochemical performance. Their compatibility with the UA as the analyte was tested and the calibration was performed in the optimal conditions, showing remarkable sensitivity and a wide dynamic working range. Reproducibility and repeatability tests have shown excellent accuracy and precision of the method as a limiting and key factor for practical application. The disadvantage of this method is its limited selectivity in the presence of the common interfering species in the biological matrix. However, this drawback can be bypassed by good experiment planning and/or the use of chemometrics. In the end, the proposed sensing platform was tested in the human cell cultures exposed to the allergens. In the first series of samples , concentrations of UA were too low to perform the direct measurement, so the samples had to be spiked. In spite of that, we were able to determine the UA concentration and to repeat the measurements with the unspiked samples stressed with papain. In this way, we not only proved that the stress induced in the cells can be measured by this method, but clearly distinguished the levels of stress induced in HeLa and Hek 293 cell cultures by the use of three different allergens . Since UA is released when cells suffer from stress, sensing devices that can measure a change in the UA concentration can provide us with plenty of information on both the environmental factors and pathological states that cause stress to the human organism. Furthermore, the simplicity of this method and the device itself opens up opportunities for its commercial use. Hence, we think that this sensing device, as it is, can potentially find applications in medical research and clinical practice.


Central apnea (CA) events always can be seen in the polysomnographic (PSG) reports of children with obstructive sleep apnea (OSA), and sometimes the central apnea index (CAI) is higher than the obstructive apnea and hypopnea index (OAHI). Commonly, the clinicians only attribute it to the age. This study aims to elucidate the distribution characteristics and major factors associated with CA in pediatric OSA.A retrospective chart review of PSG data of children with OSA from January 2017 to March 2018 was performed.p < 0.001). The CAI increased with an increasing OAHI (p = 0.003). The possibility of a CAI ≤ 1 increased with age (p < 0.001), and boys were more likely to have a CAI ≤ 1 (p = 0.001).856 children  were involved. 50.1% (429/856) had a CAI > 1, and 2.9% (25/856) had a CAI >5. Children with a CAI >1 had a higher OAHI, arousal index (AI), oxygen desaturation index (ODI), and a longer REM period, but a younger age and a shorter slow-wave sleep (SWS) phase. Multivariate binary logistic regression showed that with a 1% increased REM period, the risk of the CAI being >1 increased by 5.3% (In addition to obstructive apnea (OA), almost all children with OSA also had CA, and a CAI > 1 was most likely to occur. The OAHI and REM period were risk factors for an increased CAI, and age and male sex were protective factors. The sleep related breathing disorders are characterized by respiratory disorders during sleep and some daytime symptoms, such as snoring, mouth breathing, behavioral and learning issues, which can lead to serious consequences if unidentified and untreated , 2. The The diagnosis is usually based on the dominant respiratory disorders of polysomnography (PSG) . The maiThe pathogenesis and clinical manifestations of central apnea and obstructive apnea are different. The current research evidences have not clarified whether the pathophysiological characteristics of children with OSA affect the stability of respiratory central ventilation, which leads to the occurrence of central events. We hypothesized that there may be a certain correlation between central apnea and obstructive apnea, and some factors may drive the occurrence of central apnea in pediatric OSA. The purpose of this study is to summarize the distribution characteristics and influencing factors of CA events in children with OSA to guide clinical practice.A retrospective chart review of polysomnographic data of children diagnosed with OSA from January 2017 to March 2018 was performed. All patients had at least one sleep complaint, such as snoring, apnea, open-mouth breathing, or daytime sleepiness, and underwent PSG in the Sleep Respiration Monitoring Center in our hospital. OSA was diagnosed based on the criteria of the ICSD-3. Patients were classified into mild (1 ≤ OAHI < 5), moderate (5 ≤ OAHI < 10) and severe groups (OAHI ≥ 10) , including electroencephalography (EEG), left and right electrooculography (EOG), submental electromyography (EMG), snoring recording, body position recording, finger pulse oximetry (SpO2), electrocardiography (ECG), heart rate recording, and thoracoabdominal movement measurement by thoracic and abdominal effort belts. Nasal air flow was recorded utilizing a thermistor and a pressure nasal cannula. Respiratory effort was measured by respiratory inductance plethysmography. According to the guidelines of the AASM , apnea wThe main indicators and definitions are as follows. The obstructive apnea index (OAI) and central apnea index (CAI) were computed as the number of obstructive apnea events and the number of central apnea events per hour of sleep, respectively. The OAHI was defined as the sum of obstructive apnea events, mixed apnea events and obstructive hypopnea events per hour of sleep. The oxygen desaturation index (ODI) was defined as the number of hourly decreases in oxygen saturation exceeding 3%. Min Sat/Mean Sat was defined as the minimum/average percutaneous oxygen saturation (SpO2) during night sleep.p < 0.05 were screened by univariate binary logistic regression analysis. We eliminated the variables with collinearity problems and ultimately included age, sex (reference = girl), rapid eye movement (REM) period, slow-wave sleep (SWS) period, and OAHI in the multivariate logistic regression analysis. Considering CAI as the dependent variable, age, sex (1 = boy), REM period, SWS period, and the OAHI were considered covariables. Using the forward-stepwise method, in which the probability for conditional entry of predictors was 0.05 and that for the removal of predictors was 0.10, The final model contains all the factors significantly related to the results. All tests were two-tailed, and differences were considered statistically significant at p < 0.05. IBM SPSS Statistics 24.0 was used for the statistical analyses.Data of continuous quantitative variables are represented as the means ± standard deviations. The Kolmogorov-Smirnov test was used to determine whether the distribution of all variables was normal. All the baseline characteristics of the subject population were not normally distributed; therefore, non-parametric statistical analysis was used. The chi-square test and Kruskal-Wallis test were used to analyze the differences in variables among the mild, moderate and severe groups, and the Mann-Whitney test was used to analyze the differences in continuous variables between the CAI ≤ 1 group and the CAI >1 group. The population distribution among the different degrees of CAI is displayed graphically. Associations between the CAI and potential possible influencing factors were evaluated by Spearman rank-order correlation analysis, and a significant correlation was considered at 0.40 or higher. The independent variables with In total, 856 children were eligible for inclusion; there were 317 girls and 539 boys, with a mean age of 4.9 ± 2.4 years. There were 374 mild cases, 261 moderate cases, and 221 severe cases. The baseline characteristics are shown in p < 0.001), AI (p < 0.001), OAI (p < 0.001), and ODI (p < 0.001); and a longer REM period (p < 0.001) but a younger age (p < 0.001); lower Min Sat (p = 0.019); and shorter SWS period (p = 0.016) than the CAI ≤ 1 group.Among all the included OSA patients, 50.1% (429/856) had a CAI > 1, and 2.9% (25/856) had a CAI >5. CAI distribution proportions are presented in By calculating the simple correlation coefficient (Spearman test), we evaluated the correlation strength between the CAI and variables with significant differences. There were positive correlations between CAI and sex, OAHI, AI, OAI, ODI, and REM period. In particular, there were significant negative correlations between the CAI and SWS period .First, we used a univariate binary logistic regression model  to examine the factors related to CA . Among tp < 0.001). The CAI increased with the increase in the OAHI. Older age was associated with an increased probability of a CAI ≤ 1, and boys were more likely to be diagnosed with a CAI ≤ 1 than girls.Second, the independent significant variables without collinear problems in the univariate binary logistic regression analysis were entered into the multivariate binary logistic regression analysis . For eacThe pathogenesis and clinical manifestations of CA and OA are different. Children with OSA are characterized by OA during nocturnal sleep, which affects their sleep structure and blood oxygen , 5, but In our study, we included 856 children with OSA and found that almost all children with OSA had CA at night, with a CAI > 1 in 50.1% and a CAI > 5 in 2.9%. These indicate that OA and CA coexist in children with OSA, and abnormal CAI is not uncommon in healthy children with OSA.Central apnea is generally considered a normal physiological phenomenon in healthy infants and children and is considered benign, but there is a lack of research on abnormal CAI thresholds in children with OSA. In this study, the demographic characteristics and sleep breathing parameters of the CAI > 1 group and the CAI ≤ 1 group were further compared, and it was found that the indicators in the two groups is different. This suggests that 1 can roughly be used as a CAI threshold to reflect abnormal central apnea in children with OSA. In clinical practice, when a child with OSA has a CAI > 5, it is recommended to carefully review the underlying disease conditions of the child, paying attention to whether there is a concurrent CSA, and it is recommended to evaluate neuroanatomical abnormalities. For other children with specific underlying medical conditions, further monitoring should be carried out.In both our clinic and the already research, we found that central apnea events often occur during sleep in children with OSA. But it is still unknown that the potential influencing factors of central apnea events in children with OSA. Until now, few studies have focused on this topic.In our study, we observed that the severe OSA group had the highest CAI, and the CAI increased as the OAHI and OAI increased. Regression analysis results further confirmed that increases in OAHI and OAI were risk factors for a CAI > 1, and CAI increased with the severity of OSA; thus, we speculate that the CAI may reflect the severity of OSA in children to some extent. Therefore, in children with OSA, while there may be central apneas which don't merit a diagnosis of central sleep apnea syndrome, central apnea events can certainly be a by product of OSA, higher depending on the severity of OSA. For OSA children with higher CAI, clinicians need to pay attention to the fact that their conditions may be more serious. Especially for children with CAI > 5, we not only need to pay attention to the possibility of comorbidity of OSA and CSAS, but also need to consider the severity of OSA. Genoveva's latest large-sample study also found similar results. They analyzed PSG data of 1,279 children between 1 and 14 years of age over a period of 10 years. They found that patients with OSA had a higher CAI (2.16) than those without OSA (1.17), and this correlation increased with increasing degrees of OA severity . The ODITraeger et al.  also fouIn our study, no sex ratio difference was observed among the children with different severities of OSA. Rosemary's study also showed that there was no sex difference in children with OSA; he noted that females with moderate-severe OSA exhibited more internalized problems  . Howevern = 98, P < 0.01). The mechanisms underlying CA in pediatric OSA are not clear. McLaren explained the different mechanisms contributing to CA, Controller defect, ventilator defect and PaCO2 changes are three primary mechanisms , and the univariate regression analysis showed that a shorter SWS period  resulted in a lower CAI value. There was no significant difference between the CAI > 1 group and the CAI ≤ 1 group in the LS period. Therefore, we can conclude that sleep structure and sleep phase play roles in the occurrence of CA in pediatric OSA patients.In our study, the correlation analysis showed that there was a significant negative correlation between the CAI and SWS period  of the children was not taken into account. The association between obesity and CA is unclear. We did not obtain data on PaCO2, as this information was unavailable for many of the cases. The medical history length was not considered in this study. The severity of OSA will increase with the prolongation of the medical history; will CA become increasingly frequent as the medical history length increases in children with OSA? This will be investigated in further research.As described above, we found that almost all children with OSA experienced CA at night, and a CAI > 1 was more likely to occur in children with OSA, sometimes a CAI >5 may occur in OSA children. The OAHI and REM period were risk factors for CA. Age, male sex were protective factors. CAI could be a supplementary index to reflect the severity of OSA in children. So, in clinical practices, for OSA children with higher CAI, especially CAI > 5, clinicians not only consider the age of children and the possibility of comorbidity of OSA and CSAS, but also to pay attention to the severity of OSA and the sleep construction.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.This study was approved by the Ethics Committee of the Capital Institute of Pediatrics (SHERLL2019051). In the course of our research, we abided by the strict ethical principles stipulated in the requirements of the committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.JL collected and analyzed data and drafted the first manuscript. LCh and LCa designed the project and revised the manuscript. GH analyzed data. All authors have read and approved the final version of the manuscript.This work was supported by the CAMS Initiative for Innovative Medicine (2016-I2M-1-008).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The Hedgehog (HH) signaling network is one of the main regulators of invertebrate and vertebrate embryonic development. Along with other networks, such as NOTCH and WNT, HH signaling specifies both the early patterning and the polarity events as well as the subsequent organ formation via the temporal and spatial regulation of cell proliferation and differentiation. However, aberrant activation of HH signaling has been identified in a broad range of malignant disorders, where it positively influences proliferation, survival, and therapeutic resistance of neoplastic cells. Inhibitors targeting the HH pathway have been tested in preclinical cancer models. The HH pathway is also overactive in other blood malignancies, including T-cell acute lymphoblastic leukemia (T-ALL). This review is intended to summarize our knowledge of the biological roles and pathophysiology of the HH pathway during normal T-cell lymphopoiesis and in T-ALL. In addition, we will discuss potential therapeutic strategies that might expand the clinical usefulness of drugs targeting the HH pathway in T-ALL. Drosophila melanogaster, where HH mutations disrupt the polarity of the larval segments [The Hedgehog (HH) signaling pathway was discovered more than 40 years ago in the fruit fly segments . The HH segments ,3. Howevsegments . An HH psegments . T-ALL isegments . Neverthsegments . HH signsegments ,10, whersegments . TherefoThe HH signaling pathway is a highly regulated, concerted network consisting of extracellular ligands, membrane receptors, cytoplasmic signaling molecules, co-regulators, and transcription factors. The interactions between components of this signaling cascade are spatially and temporally regulated to provide pathway activation only in the proper cell and tissue context. Typically, this signaling network is not active in mature cells, whereas its activation is necessary for tissue development and homeostasis . HH signThe HH signaling network is extremely intricate due to the existence of canonical and non-canonical mechanisms of activation. In mammals, the activation of the canonical HH signaling pathway starts with the binding of one of three HH ligands, Sonic HH (SHH), Desert HH (DHH), or Indian HH (IHH), to a 12-pass transmembrane receptor, referred to as Patched (PTCH), which comprises two homologs, PTCH1 and PTCH2 . InteresIn the absence of HH ligands, PTCH receptors localize to the primary cilium, where they prevent activation of the seven-pass transmembrane signaling protein Smoothened (SMO). SMO inhibition is achieved through the export of sterols from the inner leaflet of the plasma membrane, thereby depriving SMO of the sterols required for its activation . PrimaryUpon binding of an HH ligand to a two-molecule PTCH complex, the N-terminal palmitate of the ligand is inserted into the sterol tunnel of one of the PTCH molecules, thereby blocking sterol export. Then, binding of a ligand to the second PTCH molecule induces endocytosis of the complex away from the primary cilium .Once PTCH proteins leave the cilium, they lose their repressing activity on SMO. When SMO is inactive, Suppressor of Fused (SUFU) binds to the three GLI (from glioma-associated) zinc finger transcription factors  and prevents their nuclear translocation and gene regulatory activities . SUFU isUpon HH ligand binding to PTCH, SMO accumulates in the primary cilium, thereby initiating the canonical HH signaling cascade ,35,36,37In addition to the canonical pathway of activation, GLI transcription factors could be activated via noncanonical mechanisms (which bypass the HH ligand–PTCH/SMO axis), mostly in cancer cells , as we sInitially, the HH pathway was linked to development anomalies ,47,48. HGLI1, PTCH1, PTCH2, SNAI1, HHIP1, MYCN, CCND1, CCND2, BCL2, CFLAR, FOXF1, FOXL1, FOXC2, TWIST2, PRDM1, JAG2, GREM1, ZEB1, and ZEB2 [Several lines of evidence suggest that aberrant activation of the HH signaling network drives cancer progression by regulating neoplastic cell proliferation and survival, metastasis, drug-resistance, and the expansion of cancer stem cells (CSCs) which are thought to be behind late-stage tumor recurrence ,53. In fand ZEB2 .PTCH1, SMO, and SUFU genes, as in the cases of BCC [Four distinct mechanisms have been proposed to explain dysregulation of canonical HH signaling in neoplastic cells. However, these mechanisms are not necessarily mutually exclusive, which further complicates our understanding of the HH pathway in cancer . 1. Types of BCC , rhabdoms of BCC , and meds of BCC . 2. Types of BCC ,60,61,62s of BCC ,64. 4. Ts of BCC , multipls of BCC , and soms of BCC . A schemNoncanonical activation of the HH network, where PTCH/SMO are bypassed, is a typical driver in several malignant disorders . The exiGLI1 and GLI2 promoters, thereby increasing gene transcription [Moreover, epigenetic regulation has been recently implicated in noncanonical HH signaling. For instance, while acetylation negatively modulates GLI1 and GLI2 transcriptional activity , deacetycription . Examples of cancers displaying noncanonical HH signaling include, but are not limited to, esophageal carcinoma , glioblaThe aberrant activation of HH signaling in cancer cells has prompted academic groups and pharmaceutical companies to develop several antagonists targeting the key components of this signaling pathway . We willThe development of HH signaling pathway antagonists has primarily focused on targeting SMO. The first SMO inhibitor was cyclopamine, a natural occurring steroid alkaloid from the corn lily Veratrum californicum . SeveralWhen Glasdegib was combined with intensive induction (intravenous ARA-C and daunorubicin) in untreated AML patients, CR rates were similar to historical controls, while there was a benefit in OS, with a median OS of 14.7 months in patients ≥ 55 years old vs. 8.7 months in historical controls. About 40% of patients were alive at 36 months . In TablThis could be the most promising strategy for inhibiting HH signals, as therapeutics targeting GLI and inhibiting its DNA-binding activity may also blunt the noncanonical HH signaling route . GLI-antArsenic trioxide (ATO) is capable of antagonizing the HH signaling pathway . The mecIt is becoming increasingly appreciated that nanoparticle-based drug delivery platforms have the potential for ameliorating the current anti-neoplastic treatments. For instance, nanoparticles offer several advantages including enhanced solubility, stability, and bioavailability of the transported drugs . NanocarLymphopoiesis refers to the production of B-cells, T-cells, and natural killer (NK) cells. It comprises a series of maturational steps from hematopoietic stem cells (HSCs) to common lymphoid progenitors (CLPs), to fully differentiated B-, T-, and NK cells ,121,122.The relevance of HH signaling in adult HSCs is still controversial and ill-defined . Some stAs to lymphopoiesis, the development of T- and B-cells is blocked in the BM at the level of CLPs if PTCH expression is abrogated in mice, whereas cells of myeloid lineage develop normally . AnotherRegarding T-cell lymphopoiesis, multiple lines of evidence indicate that HH signals are fundamental for T-cell development in both the embryonic and the adult thymus . In-CD8-), double positive , and, finally, single positive  mature T-cell stages [+ immature single positive (ISP) subpopulation [-CD44+, DN2 thymocytes are CD25+CD44+, DN3 thymocytes are CD25+CD44-, and DN4 thymocytes are CD25-CD44- [+ or CD8+ SP cells. Finally, SP CD4+ and CD8+ cells leave the thymus and populate the peripheral lymphoid tissue/organs [The T-cell differentiation process comprises several phases, consisting of double negative  and neg25-CD44- ) selectie/organs ,140,141.+ ISP stage to the CD4+CD8+ double positive stage [+ ISP maturation to CD4+CD8+ DP cells [The maturation of T-cells is strictly dependent on the interactions between the developing lymphocytes and the thymic stroma, which consists of thymic epithelial cells (TECs) and mesenchyme-derived cells . Two popve stage ,152. In DP cells . Components of HH signaling have been identified in both human  and mousRegarding T-cell progenitors, IHH is expressed by thymocytes at the DP stage . PTCH1 acip, is increased [-/- or Gli3-/- thymi [There is a general agreement that HH signaling, through SHH (secreted by cTECs), SMO, GLI2, and GLI3, is an essential positive regulator of the early stages of thymocyte maturation in the fetal thymus, where it controls proliferation and survival of DN1 cells as well as their differentiation to DN2 thymocytes ,159,162./- thymi ,161, whe/- thymi   treated with recombinant SHH, demonstrated that thymocyte development was blocked at the DN3 stage . Moreovel thymus ,161. ThiAlthough earlier studies have focused on the importance of SHH produced by TECs during early stages of T-cell development ,162, a s-/-, Gli2-/-, Gli2A-transgenic, Gli2R-transgenic, Gli2ΔC-tramsgenic, Gli3-/-) [+ CD8+ DP cells to CD4+ CD8− SP lymphocytes [Several studies have demonstrated that mTEC-secreted SHH also negatively regulates the transition from the DP to SP mature T-cells, most likely via a lowering of the TCR signaling strength. These investigations took advantage of several murine embryonic models (ShhGli3-/-) ,165,166.phocytes . The finphocytes ,169. + thymocyte precursors while inhibiting their proliferation and maturation to DP cells [+ progenitor cells induced by IL-7 signaling. In a subsequent study, FTOC experiments indicated that HH signaling attenuated the percentage of both CD4+ SP cells and CD8+SP cells, as well as increased the proportion of DP cells in thymi of children [Most of the studies on HH signaling in the thymus have been performed using animal models. However, a couple of studies have been accomplished in vitro using human cells. An early investigation reached the conclusion that SHH increases the survival of CD34DP cells . These echildren . − CD8− DN phenotype and the expression of both stem cell and myeloid-associated markers [GATA3, ETV6, RUNX1), to epigenetic regulators , and signaling pathways, including FLT3 and NRAS [+CD8+ DP phenotype), are typically associated with genetic alterations in TLX1, TLX3, NKX.2.1, and NKX.2.2 homeobox genes, as well as in CDKN2A, TAL1 and LMO1,2 [TAL1 and LMO1,2 expression are also associated with mature T-ALLs displaying surface CD3 [NOTCH1 oncogenic anomalies are frequent in cortical T-ALLs and can be detected in up to 40% of mature T-ALLs [NOTCH1 mutations [T-ALL comprises 10–15% of pediatric and 20–25% of adult cases of ALL and is considered to be a high-risk subtype of ALL . In rece markers , commonland NRAS . Corticad LMO1,2 ,185. TALface CD3 ,181. NOTutations . Althougutations . In factutations . Moreoveutations , is stilutations , therebyGLI3 transcription via hypermethylation [CBFA2T3-GLIS2 fusion gene which leads to upregulation of HH-related genes  [GLI1 [Regarding hematological cancers, aberrant HH signaling has been mainly investigated in AML , where ihylation , express, NCAM1) , and ove1) [GLI1 . Importa1) [GLI1 . In Nove1) [GLI1 . Deregul1) [GLI1 , multipl1) [GLI1 , chronic1) [GLI1 , and B-c1) [GLI1 . The first reports suggesting a possible activation of the HH network in T-ALL cells were published in 2009 and 2011. They showed that the SMO inhibitor cyclopamine synergized with either quercetin  or a NOTThe causative factors contributing to HH signaling upregulation in T-ALL cells are far from being clear. The available evidence suggests that activation could depend on genetic aberrations, autocrine signaling, and ligand-independent noncanonical mechanisms.Mutations in critical components of the HH network have been identified in T-ALL patients. Dagklis and coworkers  discoverIn a subsequent study, 20 monoallelic anomalies of genes encoding components of the HH pathway were identified in 17 of 109 (16%) childhood T-ALL samples . Two patAnother mechanism of HH network activation in T-ALL could be the existence of an autocrine loop, as suggested by a study, based on RNA sequencing, in which 139 T-ALL samples were analyzed and compared with healthy T-cell progenitors . An ectoA few therapeutically relevant molecular pathways which crosstalk with HH signaling have been identified in T-ALL cells. Some of these crosstalks are indicative of the existence of noncanonical mechanisms of HH signaling upregulation. Two signaling pathways which are frequently overactive in T-ALL are the PI3K/Akt/mTOR and MEK/ERK networks ,209. EitThe HH pathway also crosstalks with Forkhead box C1 (FOXC1), a transcription factor which plays a pivotal role in mesenchymal lineage specification, normal organ development, and longevity . HoweverNOTCH signaling, which is aberrantly upregulated in >65% of T-ALL patients by activating mutations in NOTCH1, is a major regulator of leukemic cell growth, survival, and rewired metabolism. The oncogenic activity of NOTCH1 is critically dependent on the enhanced expression of c-MYC . TherapeThe interactions between the glucocorticoid receptor nuclear receptor subfamily 3 group C member 1 (NR3C1) and the HH pathway  are of pFurthermore, DEX treatment reduces GLI1 stability in T-ALL cells via enhanced ubiquitination and proteasomal degradation  Figure . OverallAnother pathway known for its interactions with HH signaling is the AMP-activated protein kinase (AMPK) network. For instance, AMPK is capable of directly phosphorylating and destabilizing GLI1 protein, thereby blocking GLI1 transcriptional activity in medulloblastoma . AMPK-deIn T-ALL cells, AMPK signaling was the most consistently downregulated pathway under serum-depleted conditions, and this coincided with increased GLI1 expression and enhanced sensitivity to HH inhibitors, especially GANT 61. In contrast, mTORC1/p70SK1 activity was upregulated. Serum starvation in vitro mimics, at least in part, the growth of metabolically stressed cancer cells trying to adapt in vivo to nutrient-depleted environments, such as BM and the thymus . The incFor the sake of completeness, it should be recalled that in T-lymphoblastic lymphoma (T-LBL), HH activation is dependent on the downregulation of some miRNAs  which positively impact on the transcription of SMO . The dis+ leukemic T-cells [T-ALL is characterized by the transformation of immature T-cells, which accumulate a wide array of genetic and epigenetic anomalies. These alterations lead to the sustained proliferation and survival of abnormal T-cells . For exa T-cells . Interes T-cells , which s T-cells . Yet ano T-cells . These oAnother clear indication that the activation of the HH pathway contributes to the survival and maintenance of T-ALL cells comes from a study performed in a zebrafish model, where notch1a promotes T-cell transformation . Burns a50 of cyclopamine is around 1 μM [Preclinical studies performed in vitro or in vivo have demonstrated the efficacy of HH signaling inhibitors in T-ALL cells. Hou and coworkers  reportedund 1 μM . Howeverund 1 μM . These fIn a subsequent study, two SMO inhibitors (cyclopamine and GDC-0449) caused a concentration-dependent inhibition of proliferation in three of nine T-ALL cell lines, while GANT61 inhibited proliferation in six of nine cell lines. Importantly, cell lines in which the drugs led to the strongest decrease in GLI1 expression also displayed the strongest inhibition of proliferation . MoreoveOverall, the studies discussed in this Section and in In the past several years, the outcomes of T-ALL patients  have dramatically improved, despite the fact that the mainstays of therapy have not changed. Nevertheless, therapy treatment-related deaths and long-term detrimental adverse effects for leukemia survivors remain serious issues which still await a solution. Moreover, the prognosis for most refractory and relapsed patients remains dismal due to the few available treatment options. Therefore, there is an urgent need for novel and less toxic targeted therapeutics for T-ALL patients. The evidence we have reviewed in this article indicates that targeting the HH signaling pathway may present a new therapeutic opportunity in patients who are resistant to conventional drugs such as ARA-C or GCs. Adapting T-ALL therapy to patients’ individual needs requires a growing arsenal of small molecule inhibitors and chemotherapeutic agents for the simultaneous targeting of multiple tumor-promoting processes and oncogenic signaling pathways. As we have summarized in this review, we now have at our disposal several drugs targeting HH signals. While acknowledging the relevance of these therapeutics, much is left to be uncovered for translating them in the clinic for treatment of T-ALL patients. Moreover, although some of these drugs have been approved for cancer treatment, there is a need for new therapeutics to overcome drug resistance and adverse effects of the inhibitors currently used. In particular, given the modest activity of the SMO inhibitors in human T-ALL cells and the well-known issue of emergence of resistance to these therapeutics in other tumor clinical trials, it will be crucial to determine which signaling pathways alter leukemic cell sensitivity to SMO inhibitors. These findings will be of the utmost importance to design rational combination treatments incorporating HH pathway inhibitors in T-ALL patients.We need to achieve a much better understanding of the complex molecular interactions involving HH signaling and other pathways in both healthy and neoplastic cells as well as the best ways to employ drugs targeting HH in T-ALL. As we learn more about the roles of HH in T-ALL, it may be possible to develop drugs which mainly target the functions of HH in leukemic cells and which could be more amenable to a therapeutic intervention than HH signaling itself. For example, a recent study performed in melanoma cells has revealed that a novel SOX2-BRD4 transcriptional complex drives the expression of GLI1 via non-canonical HH signaling . InteresFurthermore, biomarkers indicating which patients may benefit the most from drugs targeting HH are still awaiting definitive identification, although some progress has been made, as we have summarized above. In conclusion, despite some significant issues which remain to be solved, the findings we have summarized here provide a compelling rationale for future clinical trials aimed at testing the potential of HH inhibitors in T-ALL patients, especially in combination with either chemotherapeutics or other targeted drugs .


Bone-related diseases are major contributors to morbidity and mortality in elderly people and the current treatments result in insufficient healing and several complications. One of the promising areas of research for healing bone fractures and skeletal defects is regenerative medicine using stem cells. Differentiating stem cells using agents that shift cell development towards the preferred lineage requires activation of certain intracellular signaling pathways, many of which are known to induce osteogenesis during embryological stages. Imitating embryological bone formation through activation of these signaling pathways has been the focus of many osteogenic studies. Activation of osteogenic signaling can be done by using small molecules. Several of these agents, e.g., statins, metformin, adenosine, and dexamethasone have other clinical uses but have also shown osteogenic capacities. On the other hand, some other molecules such as T63 and tetrahydroquinolines are not as well recognized in the clinic. Osteogenic small molecules exert their effects through the activation of signaling pathways known to be related to osteogenesis. These pathways include more well-known pathways including BMP/Smad, Wnt, and Hedgehog as well as ancillary pathways including estrogen signaling and neuropeptide signaling. In this paper, we review the recent data on small molecule-mediated osteogenic differentiation, possible adjunctive agents with these molecules, and the signaling pathways through which each small molecule exerts its effects. Bone diseases are a principal medical problem for the elderly population which lead to lower quality of life. Osteoporosis is one of these disorders which is becoming increasingly more common, resulting in higher numbers of bone fractures and mortality rates. Bone fractures are more common in women compared to men and cause a variety of complications including decreased mobility and increased long-term care needs. About 1.9%-4.9% of bone fractures in the US have insufficient healing which in turn can be categorized as delayed union or nonunion. Currently, there are no known pharmaceutical agents that single-handedly accelerate nonunion fracture healing. The gold standard of treatment for nonunion fractures is autologous bone grafting. . AlthougBone tissue engineering proposes a potential means toward acceleration of healing of bone defects . Stem ceIn the developmental stages, osteogenesis happens through migration of mesenchymal stem cells to the site in which bone is to be formed. Moreover, the process of bone formation takes place through either of the following methods: direct osteoblast differentiation of cells also known as intramembranous ossification, or indirect bone formation through chondrocyte differentiation followed by later bone formation, also known as endochondral ossification . The majThe other building block in tissue regeneration is growth factors which are proteins that direct cellular fate into specific paths including differentiation and gene expression. In spite of being major players, growth factors are expensive and usually, high doses are required in order for efficient bone regeneration. For example, bone morphogenic protein 2 (BMP-2) is considered a potent osteoinductive factor (BMPs are among the most widely used growth factors for induction of bone formation). However, treatment with high dose BMP-2 has been reported to have various side effects such as vertebral osteolysis with cyst formation, ectopic bone, cancer and life-threatening inflammatory cervical swelling. One method to overcome the disadvantages of growth factors is utilization of small molecular compounds instead of them. Small molecules have been reported to show great potential in regenerative studies due to low cost, easy membrane passage, and cellular activation. Furthermore, small molecules are generally easier to synthesize and store compared with natural growth factors –14. AnotSmall molecules and growth factors drive stem cells into various lineages through activation of intracellular signaling pathways , 18. MulSmall molecules exert their osteogenic effects by activating certain signaling cascades in cells. Activation of these cascades, in turn, results in regulations and expression of their specific target genes . Therefo2+ dependent Wnt pathway contributes to osteogenesis of stem cells. This pathway is triggered by binding of Wnt ligands to the FZD receptors. Then, activation of G protein results in Ca2+ ions being released from the endoplasmic reticulum, which results in initiation of protein kinase C (PKC) signaling pathway. Wnt-5a is a noncanonical Wnt ligand which has an important action in osteogenesis of BMP-2. It is known that Ca2+ dependent Wnt signaling is involved in osteogenesis of Wnt-5a [Many signaling pathways are known to induce osteogenesis in stem cells . One of f Wnt-5a –21. Therf Wnt-5a .Fig. 21.BMP/Smad signaling is considered another important osteogenic signaling pathway. BMP/Smad signaling is known as a pro-osteogenic, pro-adipogenic signaling pathway . This paUnlike the BMP signaling pathway, Adenosine-signaling is a pro-osteogenic, anti-adipogenic signaling pathway . The relDrosophila and consists of three members: Desert Hedgehog, Indian hedgehog and Sonic hedgehog. Hedgehog signaling is initiated by binding of the hedgehog ligand to the transmembrane protein receptor known as Patch (PTCH)  signaling is known as a pro-osteogenic, anti-adipogenic signaling pathway . This siCH) Fig. . This biProtein kinases are a group of enzymes that are responsible for phosphorylation of proteins. They are generally known as on/off switches of various cellular functions including differentiation . MitogenThe PKC family includes 15 molecules which are divided into 3 different groups based on the substrate required for their activation. Certain members of this family are known to exert different effects on hard tissue regeneration; e.g., PKC δ and β are considered pro-osteogenic kinases, whereas PKC α and μ downregulate osteogenesis . ProteinAdenosine monophosphate-activated kinase (AMPK) signaling is known to be responsible for maintaining cellular energy homeostasis. This signaling pathway has been found to be an important factor in bone formation and inhibition of adipogenic differentiation. Chava et al. have recently demonstrated that activation of AMPK results in phosphorylation of RUNX2 at serine 118. The phosphorylation of RUNX2 is crucial for osteogenic differentiation and dephosphorylation of AMPK results in adipogenic differentiation. These results indicate that AMPK phosphorylation modulates homeostasis between osteogenic and adipogenic differentiation and shifts stem cells towards osteogenic lineages  Fig. 3)3).Several other signaling pathways have been found to be responsible in small molecule mediated osteogenesis. These pathways include Estrogen receptor (ER) signaling, RhoA/actin, RANKL/RANK/OPG, mTOR signaling and neuropeptide signaling –50.One of the pathways responsible for fracture healing is neuropeptide Y (NPY) signaling Fig. . PreviouOsteoprotegerin (OPG), Receptor activator of NF-kB ligand (RANKL) and Receptor activator of NF-kB (RANK) are the main components of the osteoclast maturation RANKL/RANK/OPG signaling pathway. Osteoclast differentiation and bone resorption are mainly modulated by secretion of OPG and RANKL. Binding of OPG to RANK can block binding of RANKL to RANK; this means that an increase in OPG/RANKL ratio can block osteoclast differentiation and bone resorption . It is aRhoA/actin signaling is another pathway found to be responsible in osteogenesis of stem cells. Rho GTPase family is the most important modulator of actin cytoskeleton rearrangement. RhoA is one of the twenty Rho proteins which plays a role in myosin 2 activation and production of actomyosin force and stress fiber. It is known that formation of stress fibers and stabilization and polymerization of actin shifts stem cell fate toward osteogenic ends  Fig. 4)4).Mechanistic target of rapamycin (mTOR) signaling is a nutrient-sensing pathway which plays roles in cell cycle progression and survival. This pathway consists of two separate complexes known as mTORC1 and mTORC2, each of which is responsible for certain cellular functions. It has been recently found that mTOR signaling plays an important role in osteogenic differentiation of stem cells Fig. . HoweverAnother pathway which has recently been found to play an important role in osteogenesis is estrogen signaling Fig. . EstrogeStatins are a group of cholesterol lowering drugs which primarily target HMG-CoA reductase. They are also known as osteo-inductive agents . SimvastSeveral signaling pathways are thought to be related with statin-mediated osteogenesis. Zhang et al. found that addition of Dickkopf-1(DKK-1), a Wnt inhibitor, to the medium one hour prior to SM, can significantly downregulate osteogenic differentiation of rat BMMSCs. This drug was also found to increase expression of β-catenin protein. Furthermore, shRNA mediated inhibition of β-catenin protein expression attenuated SM-induced osteogenesis. These findings suggest that statins activate Wnt/β-catenin signaling pathway to achieve osteogenic differentiation . There iEstrogen signaling is another pathway which plays role in stem cell differentiation by statins. Chuang et al. studied the interconnection of statin-related osteogenic differentiation and estrogen signaling in mouse bone marrow MSCs. They utilized fulvestrant as an estrogen receptor antagonist to block estrogen signaling. It was found that blockade of ER signaling downregulated SM-mediated osteogenesis; however, mRNA expression of ER-alpha was not altered. This suggests that SM administration does not affect gene expression of ER-alpha, but increased ER function. To further elucidate whether ER-alpha and/or GPER-1 is responsible for SM mediated osteogenesis, ER-alpha and GPER-1 were inhibited by their specific antagonists. While it was found that GPER-1 antagonism did not affect osteogenesis, ER-alpha antagonism significantly reduced osteogenic markers and BMP-2 expression induced by SM. Furthermore, it was found that SM can act as a weak receptor agonist for ER-alpha .BMP/Smad signaling is found to be another pathway responsible for statin mediated osteogenic differentiation. SM can increase Smad1/5/8 protein phosphorylation level and reverse TNF-α related inhibition of Smad1/5/8 phosphorylation in MC3T3-E1 cells . Also, aSeveral studies have focused on combination of treatment with statins and other osteogenic agents or materials and whether this combination can enhance osteogenic differentiation , 63, 68.The small molecule adenosine is a purine nucleoside, widely used as a vasodilator and neuromodulator. Recently, a growing body of attention has been focused on adenosine and adenosine receptors as factors implicating in bone regeneration . ExogenoAlthough not quite as much as A2b receptor, A1 adenosine receptors have also been found to contribute to increase in bone tissue formation. Activation of A1 receptor has been found to be connected with activation of MAPK and PI3K pathways . MoreoveBisphosphonates (BPs) are a drug class commonly used as treatment of osteoporosis, Paget’s disease and malignancy-related hypercalcemia due to being able to decrease the process of bone resorption. Although BP administration has resulted in improvement of osteointegration and implant stability, its application has been limited due to osteonecrosis of jaw. One of the safer techniques which is used in order to limit such side effects is pretreatment of cells with BPs. Wen et al. studied pretreatment of BMMSCs with zoledronic acid (ZA) in order to develop a method through which this complication could be avoided. Pretreatment of stem cells with BPs before implantation has been suggested as a possible means through which osteonecrosis can be avoided. To evaluate the outcomes of ZA pretreatment, BMMSCs were pretreated with ZA and then cultured in an osteogenic medium. It was observed that 0.5 and 1 Micromolar of ZA lead to enhancement of osteogenic differentiation. Furthermore, it was found that unlike high dose, low dose ZA administration resulted in an increase in proliferation of BMMSCs in the first 72 h of treatment. Moreover, they investigated the mechanism through which ZA leads cells towards osteogenesis. They found that osteogenic induction of stem cells with ZA significantly increased OPG without altering Rankl level. This leads to an increase in OPG/Rankl ratio . In anotDexamethasone (DEX) is a synthetic corticosteroid agent which is known as an inducer of osteogenic differentiation. DEX-mediated bone formation is thought to be linked with Wnt and MAPK signaling pathways in stem cells derived from human exfoliated deciduous teeth (SHEDs) . TreatmeDEX has been reported to enhance response of stem cells to BMP-2 mediated osteogenic differentiation. Recently, BMP-2 and DEX have demonstrated augmented osteogenic differentiation in muscle-derived stromal cells. Animal experiment has shown similar results. In vivo implantation of BMP-2 and DEX showed synergistically enhanced ectopic bone formation compared to implants containing BMP-2 only . AnotherMelatonin (MLT) is a hormone secreted from the hypothalamus which is involved in regulation of the circadian rhythms . ApplicaAlthough MLT is considered as a potential osteogenic agent, its water insolubility has restrained its intracellular uptake. Therefore, improving MLT water solubility may contribute to a wider application in the clinic. Cyclodextrin is an oligosaccharide which has previously shown improved water solubility of simvastatin. Terauchi et al. used 2-hydroxypropyl beta-Cyclodextrin to form an inclusion complex with MLT. 2-hydroxypropyl beta-Cyclodextrin increased intracellular concentration of MLT and its metabolites and enhanced osteogenesis in MC3T3-E1 cells. Therefore, utilization of 2-hydroxypropyl beta-Cyclodextrin with MLT may be a promising method for bone regeneration .Application of other molecules alongside MLT has also been shown to contribute to enhancement of osteogenesis. Maioli et al. used a combination of hyaluronic, butyric and retinoic acid with MLT to induce differentiation. The combination was found to upregulate expression of osteogenic specific genes in a synergistic manner. This synergistic effect is possibly through upregulation of vascular endothelial growth factor. In addition, MLT + Retinoic acid (RA) + Butyric acid (BU) + Hyaluronic acid (HA) medium exhibited higher levels of osteogenic gene expression compared to a traditional dexamethasone-based osteogenic medium. The results from this study suggest that adding the combination of RA, BU and HA to MLT can increase MLT induced osteogenic differentiation . AdditioCompelling evidence suggests that metformin, a biguanide widely known as a type 2 diabetic agent, can effectively lower risk of bone fractures. Several studies have demonstrated that application of metformin can contribute to proliferation and osteogenic differentiation of stem cells . Gu et aSince metformin is a hydrophilic agent, its cellular uptake depends on cell membrane organic cation transformers (OCTs). Jofi et al. hypothesized that presence of functional OCTs would be essential in order to induce metformin-mediated osteogenesis of cells. To this end, they harvested MSCs from human umbilical cord Wharton’s jelly (UC-MSCs) and studied whether or not OCTs are found in the membrane of these cells and whether they can affect osteogenic differentiation induced by metformin. It was observed that different OCT isoforms were present in UC-MSCs. After subjecting these cells to different doses of metformin, the osteogenic involved LKB1/AMPK signaling pathway was activated. Furthermore, cell lines treated with metformin revealed an increased osteogenic commitment with upregulation of bone specific genes and mineralized nodules. However, these results were abolished when the pan-OCT inhibitor quinidine was used. This suggests that functional presence of OCT is required for achieving effective osteogenesis by metformin .Phenamil is a derivative of the diuretic agent, amiloride, which targets sodium channels in the cell membrane and the Na/H exchanger for their function. Other than its diuretic effects, Phenamil is known to induce osteogenic differentiation through BMP/Smad signaling pathway , 103. UsPhenamil is known to induce osteogenic differentiation through BMP/Smad signaling pathway. Phenamil-induced osteoblastogenesis is related with upregulated Trib3 expression. Tribble’s homologs family members (Tribs) are involved in different cellular actions including cell differentiation. Although the mechanism by which phenamil exerts Trib3 gene expression during osteogenesis is not fully understood, which is thought to be through ion channel function inhibition. In another study regarding vascular remodeling, phenamil induced Trib3 gene expression by acid-sensing ion channel inhibition. Increase in Trib3 expression downregulates the BMP antagonist, Smurf1, which is consistent with enhanced Smad protein levels and osteogenic genes expression. In addition, phenamil-mediated expression of Trib3 abrogates BMP-2-induced adipogenesis by suppression of PPARγ expression. Moreover, phenamil upregulates BMP-2 expression in hBMMSCs. Therefore, phenamil induces osteogenesis by engagement of BMP-Smad pathway and therefore attenuates adipogenesis .2+) is another agent which has been used together with phenamil to induce osteogenesis. Co-delivery of Sr2+ and phenamil resulted in enhanced osteo/odontogenesis of stem cells. It was further found that co-incubation with Sr and phenamil resulted in higher Smad1/5/8 phosphorylation which possibly contributed to this additive effect. It has been shown that co-treatment of animals with mal-calcification conditions resulted in higher level of new hard tissue. These results suggest that co-delivery of phenamil and Sr2+ may be a possible strategy for adjuvant treatment for bone tissue engineering [Since phenamil has been proven to increase BMP level in stem cells, Fan et al. investigated whether incubation of cells with Phenamil and BMP-2 can enhance osteogenic differentiation. It was found concomitant treatment of AD-MSCs with Phenamil and BMP-2 can accelerate osteogenic differentiation . Additioineering .Parathyroid hormone (PTH) is a bone anabolic agent which has been approved for treatment of osteoporosis. Application of PTH results in increased bone strength and mass. Moreover, utilization of PTH is known to enhance bone regeneration through increased osteocyte activity and osteogenic differentiation of MSCs . AlthougSignaling of PTH occurs through a G αs/αq-protein-coupled receptor and sequentially cAMP and PKA signaling. Although most studies have shown cAMP as an osteoblastogenic factor, some have shown contradicting results. These results are likely due to differences in the mechanisms through which cAMP is modulated, concentration level of cAMP and the duration of elevated cAMP level . It seemResveratrol is a natural polyphenol which is found in various plants and red wine and has bone-protective characteristics , 111. RePreviously, it was known that resveratrol can induce osteogenic differentiation possibly through estrogen receptor-mediated activation of ERK1/2 MAPK signaling pathway. Also, resveratrol can abolish osteoclast differentiation by inhibition of RANKL signaling in DPSCs , 114. HoSupplementation of Vitamin D3 (Vit D3) is recommended in all treatment and prevention strategies of osteoporosis. A wide range of bone catabolic and anabolic mechanisms are known to be mediated through the active form of vit D3 which is 1,25-dihydroxyvitamin D3 . It acts2O2-induced oxidative stress. While H2O2 administration reduced calcium deposition and mRNA level of osteogenic genes, Vit D3 restored this injury. Application of Vit D3 was consistent with upregulation of Sirt1/FOXO1 axis. When the Sirt1 inhibitor, sirtinol, was utilized, Vit D3-induced enhancement of osteogenesis in oxidative condition was attenuated. Therefore, Sirt1/FOXO1 enhancement appears to be responsible for improvement of osteogenic differentiation by Vit D3 under oxidative stress [Oxidative stress is one of the notable obstacles in the process of osteogenesis. Xiong et al. demonstrated that utilization of Vit D3 can promote osteogenesis of mouse BMMSCs under oxidative stress induced by high glucose in diabetic patients. They found that Vit D3 can inactivate FOXO1 through PI3K/Akt signaling pathway and binding of FOXO1 with β-catenin is crucial for FOXO1-mediated transcription. This suggests that Vit D3 can enhance osteogenesis in oxidative stress of high-glucose condition through Wnt-β-catenin signaling pathway . Zhou ete stress .T63 is a small molecular compound that has recently been identified as a promoter of osteogenesis and suppressor of adipogenesis in MC3T3-E1 cells. T63-mediated osteogenic differentiation seems to take place through activation of two intracellular signaling pathways: 1- Canonical Wnt signaling is activated when T63 is administered. Incubation of cells with T63 results in upregulation of β-catenin and phosphorylation of GSK-3β. In addition, when cells were treated with the Wnt inhibitor, DKK-1, osteogenic differentiation was attenuated. 2- Utilization of T63 resulted in increased level of pSmad1/5/8 and BMPs 2, 4 and 7. Thus, T63 regulates osteogenesis by activating BMP signaling. Furthermore, application of BMP-specific antagonist Noggin abrogated osteogenesis of T63. This suggests that BMP signaling possibly acts as the upstream pathway in activation of Wnt signaling by T63 .Tetrahydroquinolines (THQs) and pyrazolopyrimidines (PPs) are two classes of molecules which have previously been found to enhance Wnt signaling pathway .Chen et al. investigated possible roles of THQs and PPs in promoting osteogenic differentiation. They synthesized 24 derivatives (12 THQs and 12 PPs) and studied their osteogenic potentials on C2C12 myoblast cells. After culturing cells with these agents, it was found that one of the THQ derivatives, named THQ-1a and one of the PPs, named PP-9 were the most potent osteogenic inducers. Incubation of C2C12 myoblast cells with both of the above-mentioned substances was consistent with increase in β-catenin protein levels after 3 days. Thus, application of PP-9 and THQ-1a induced osteogenic differentiation possibly through activation of Wnt signaling .Smoothened is a transmembrane signaling transducer of Hedgehog signaling pathway and its deletion is related with diminished bone formation in mice. Kashiwagi et al. studied the effects of a Smoothened agonist  (SAG) on bone formation in mice. They found that local single-shot SAG injection increased callus formation during bone regeneration. To examine the effect of Hh agonism in fracture healing, a single dose of SAG was delivered to the site of defect in mice with tibial fractures. It was observed that SAG application caused an increase in mineralization of calluses. Although the cellular morphology was the same between treatment and control group, an increase in the number of cartilaginous cells was found in the former. Immunostaining of the cells showed an increase in proliferation as the mechanism of soft callus growth. Furthermore, exogenous Hh agonism resulted in an advance in formation of hard callus . MoreovePurmorphamine is another agonist of the smoothened receptor and thus activator of Hh signaling pathway . ActivatRecently, the mTOR inhibitor rapamycin has been found to induce osteogenic differentiation. Through affecting the PI3K/AKT/mTOR signaling pathway, rapamycin can alter RunX2 and BMP-2 levels and thus contribute to osteogenic differentiation. Carpentieri et al. investigated rapamycin’s potential in inducing osteoblast differentiation of blood-derived stem cells (BDSCs). After ten days, it was found that BDSCs’ morphology had altered into osteoblast like cells. Furthermore, there were Calcium-containing granules and Calcium phosphate containing vesicles in the mitochondria which was a sign of bone mineralization .Small molecules, as key players in tissue engineering, are a major factor in the field of osteogenic differentiation and an important component in improving osteogenesis. Activation of intracellular osteogenic signaling cascades is the method through which these agents induce osteoblast differentiation. For majority of these agents, more than one signaling pathway plays a role in osteogenic differentiation. For example, BMP/Smad and canonical Wnt signaling are both crucial in simvastatin-mediated osteogenesis. Additionally, both BMP/Smad signaling and OPG/Rankl signaling influence differentiation outcomes induced by bisphosphonates. However, some pathways such as ERK ½ and p38 have shown contradictory results during different molecules-mediated osteoblast differentiation. For instance, while ERK ½ positively correlates with DEX-induced osteogenesis, this pathway has been found to have a negative correlation with statin-mediated osteogenesis. These results point out the need for more extensive research on the mechanisms by which small molecules induce osteogenic differentiation. Of the currently studied small molecules, Dex, Vit-D3, and Simvastatin have been evaluated more extensively and yield the greatest potential for application in the clinic. Moreover, it seems that combination of small molecules together or with growth factors is needed to achieve a more specific osteogenic differentiation of stem cells. Several cell lines including BMMSCs, AD-MSCs, DPSCs, and Amniotic epithelial cells have been previously differentiated to the osteogenic cells by using small molecules . EmployiAnother necessary factor for successful tissue regeneration is scaffolds that mimic the natural cellular niche and regulate cell proliferation and differentiation. The incorporation of several small molecules in natural and synthesized scaffolds such as Polyethylene glycol diacrylate (PEGDA), Poly (lactic-co-glycolic acid) (PLGA), calcium phosphate, and gelatin has been previously studied , 134–136Although natural growth factors such as BMP have been previously studied in fracture healing studies, small molecules have rarely been utilized to do so . The res


Chemotherapeutic drugs are traditionally used for the treatment of cancer. However, chemodrugs generally induce side effects and decrease anticancer effects due to indiscriminate diffusion and poor drug delivery. To overcome these limitations of chemotherapy, in this study, ultrasound-responsive liposomes were fabricated and used as drug carriers for delivering the anticancer drug doxorubicin, which was able to induce cancer cell death. The ultrasound-sensitive liposome demonstrated a size distribution of 81.94 nm, and the entrapment efficiency of doxorubicin was 97.1 ± 1.44%. The release of doxorubicin under the ultrasound irradiation was 60% on continuous wave and 50% by optimizing the focused ultrasound conditions. In vivo fluorescence live imaging was used to visualize the doxorubicin release in the MDA-MB-231 xenografted mouse, and it was demonstrated that liposomal drugs were released in response to ultrasound irradiation of the tissue. The combination of ultrasound and liposomes suppressed tumor growth over 56% more than liposomes without ultrasound exposure and 98% more than the control group. In conclusion, this study provides a potential alternative for overcoming the previous limitations of chemotherapeutics. The current strategies for the treatment of cancer involve surgical dissection, radiotherapy, and chemotherapy. Among the means of cancer therapy, chemotherapy is one of the most used methods compared to the other strategies. Conventional chemotherapy induces a toxic effect in tumor cells and a suppression of tumor growth. However, chemotherapeutics also cause side effects in healthy tissues and biological systems due to the random or wide distribution of drugs. To overcome this disadvantage, drug carriers have received great attention for the treatment of tumors ,2. Drug-There are several types of drug carriers for anticancer drugs. Among the nanosized drug carriers, liposomes lead the market because of their unique properties and broad range of biomedical applications ,9,10. AmUltrasound has been actively applied for the clinical field, especially high-intensity focused ultrasound, which has been approved for the ablation of uterine fibroids in the human clinic ,16. In aRegarding the advantage of the ultrasound-triggered release of drugs, an ultrasound-sensitive liposome encapsulating doxorubicin (IMP301) was developed in this study. IMP301 was designed for doxorubicin release under the specific condition of focused ultrasound pressure by the composition and ratio of phospholipids. This characteristic is able to not only reduce the side effects of doxorubicin but also enhances the anticancer effect. In this study, the physicochemical characteristics and sono-sensitivity of IMP301 were verified by in vitro experiments with a comparison to conventional liposomal doxorubicin (DOXIL). In addition, the anticancer effect of IMP301 was investigated by its pharmacokinetics, biodistribution, and in vivo efficacy.N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and sodium salt) (DSPE-mPEG2000), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Lipoid AG . 1-Stearoyl-2-lyso-sn-glycero-3-phosphocholine  was purchased from NOF America Corporation . Doxorubicin hydrochloride (DOX) was purchased from Gemini Pharmaceuticals Inc., . Cholesterol, ammonium sulfate, hydrochloric acid, sodium hydroxide, 1--3,5-diphenylformazan, thiazolyl blue formazan (MTT), and L-histidine were purchased from Sigma-Aldrich . Sucrose was purchased from CheilJedang . An Oasis HLB 3 cc Vac Cartridge  was purchased from Waters .1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), . The temperature of the vesicles was maintained at 50 °C during the extrusion. The dispersion of extruded liposomes was exchanged with pH 6.5 10% sucrose and a 10 mM histidine buffer using a 12–14 kDa dialysis membrane. The ammonium gradient across the liposomal membrane was generated by exchanging ammonium sulfate with the buffer. DOX was encapsulated into the intraliposomal aqueous phase using the remote loading method. DOX was added to the liposome dispersion at a ratio of 1:8 to liposomes and stirred at 37 °C for 2 h. DOX-loaded liposomes were diluted with a buffer solution, so that the DOX concentration was 2 mg/mL, and stored at 2–8 °C.The lipid composition of IMP301, which was obtained from a pilot study, was DSPC/DSPE-PEG/cholesterol/DOPE/MSPC. IMP301 was fabricated by ethanol injection followed by extrusion. Briefly, 1.50 g of DSPC, 2.66 g of DSPE-PEG, 2.20 g of cholesterol, 9.16 g of DOPE, and 0.50 g of MSPC were dissolved in 62.5 mL of ethanol. The organic phase was gently heated to 60 °C to dissolve the lipid components. Then, the lipid-containing ethanol was injected into 437.5 mL of 250 mM ammonium sulfate solution at 250 rpm. Multilamellar vesicles were assembled and dispersed during ethanol injection and downsized by serial extrusion cycles with polycarbonate filter pore sizes ranging from 200 to 80 nm using a LIPEX® XT, Advanced Instruments, Norwood, MA, USA). The temperature was maintained at 25 °C. DOX and lipids were quantified by a high-performance liquid chromatography  system with a diode array detector and an evaporative light scattering detector. The extraction of free DOX for the measurement of the encapsulation efficacy was carried out using the same method as that used for the quantification of released DOX. The morphology of liposomes was examined using cryo-transmission electron microscopy (cryo-TEM). Specimens for cryo-TEM were prepared using an FEI Vitrobot-type FP5350/60. A liposome solution (5 µL) was applied to a carbon TEM grid  blotted with filter paper and plunged into liquid ethane. In addition, the encapsulation efficiency of doxorubicin was analyzed with a UV-vis system after purifying the unloaded DOX using a PD-10 column. The quantification of doxorubicin was measured by the absorbance at 475 nm.The size distribution of IMP301 was measured using dynamic light scattering . The osmolarity of IMP301 was measured using an osmometer . An additional 4 mL of distilled water was added to the desalting column and collected in a cuvette to measure the absorbance of liposomal DOX at 475 nm. A reduced absorbance compared with non-irradiated liposomes indicated the amount of DOX released. The percentage of released drug was calculated using the following equation:rA and oA represent the absorbance intensities of the sonicated liposome suspension and of the original one, respectively.To conduct the DOX release test of IMP301, a plane wave ultrasound was irradiated by a pin-type ultrasonicator for 1 min at a frequency of 24 kHz and a continuous ultrasound wave intensity of 92 kW/cmTo analyze the effect of FUS on DOX release, focused ultrasound (FUS) machinery  was used with a water bath equipped with a temperature controller and degasser. The center frequency of the FUS transducer was 1 MHz, and the beam resolution of the focal zone at 6 dB demonstrated a circular shape with a 1 mm diameter circle and a length of 1 cm. To investigate FUS-triggered DOX release, IMP301 was exposed to FUS with varying FUS parameters of intensity, duty cycle, pulse repetition frequency (PRF), and exposure time/spot. Prior to FUS irradiation, IMP301 was contained in the dialysis membrane and was kept at 37 °C by degassing water for 1 h. The quantification of DOX release was analyzed by measuring the optical absorbance at 475 nm. After FUS irradiation, the released DOX was purified using a desalting column, and its amount was calculated as described above. Commercial liposomal doxorubicin, DOXIL, was used as an ultrasound-insensitive liposome.Stability tests were carried out in 10% sucrose and 10 mM histidine buffer solutions at a pH of 6.5. IMP301 was filtered through a 0.2 µm bottle top filter to prevent germ growth caused by the dispersant. Then, 10 mL of IMP301 (2 mg/mL) was packed in a sterilized 20 mL vial with aluminum seals. Aliquots of IMP301 were stored at 2–8 °C in the dark. To assess stability, each vial was used for measuring the DOX content, entrapment, lipid content, size, and polydispersity index (PDI) of IMP301 at 0, 1, 3, 6, and 9 months. The amount of DOX released was analyzed with an HPLC system after extracting the released DOX using a serum-coated SPE column. The extraction of DOX from the SPE column was performed using a 1:1 mixture of distilled water and acetonitrile. The concentration of DOX in the collected solution was obtained by measuring the absorbance at 254 nm in an HPLC system equipped with a C-18 reverse phase column. The phospholipids were quantified using an evaporative laser scattering detector.4 cells/well 24 h prior to treatment on 8-well glass chamber slides with culture medium. IMP301, DOXIL, and free DOX were added to each well for 30 min. To compare the efficiency of DOX uptake, exposure to each agent was carried out for 1 min at low-frequency ultrasounds (24 kHz). MDA-MB-231 cells were washed with phosphate-buffered saline  and fixed with 4% paraformaldehyde for 3 min. The cellular uptake was observed using a confocal laser scanning microscope. A cellular uptake study was performed using MDA-MB-231 human breast cancer cells. Three groups treated with IMP301, DOXIL (Batch No. LBZSC00), and free DOX were compared. MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic. MDA-MB-231 cells were seeded at a density of 2.5 × 104) were seeded in a 96-well plate. After overnight incubation, 2, 4, 6, 8, and 10 µg/mL IMP301 and DOXIL were applied to each well. Ultrasound-treated particles were prepared using the same method used for the cellular uptake investigation. Cells were incubated with a medium containing the drugs for 3 h, which was then replaced with fresh culture medium after washing three times with PBS. After 72 h of incubation, the MTT assay was conducted to evaluate cell viability.The MDA-MB-231 cell line was used for the in vitro cytotoxicity test. Cells  of Seoul National University Bundang Hospital . ICR mice (6 weeks old) were prepared for the pharmacokinetic studies. Mice were grouped into the free-DOX and IMP301 groups and injected intravenously with the respective drug preparation. Blood samples were collected at different time points . DOX was quantitatively measured in blood plasma. Briefly, mouse plasma mixed with DOX solution was prepared as a standard solution, which was mixed with daunorubicin as an internal standard, followed by centrifugation at 13,000 rpm. The supernatant was diluted with a 30% acetonitrile solution containing 0.1% formic acid. Quantitative measurements of DOX were performed using LC-MS/MS. The DOX calibration curve was plotted using the DOX and daunorubicin area ratio. The DOX concentration in mouse plasma was measured after dilution with PBS containing 20% methanol and 5% BSA.2 intensity, 2.0% duty cycle, 250 Hz PRF, and 10s/spot was irradiated after each agent administration. To quantify the release of DOX in tumors, an in vivo fluorescence imaging system was used to visualize the fluorescence images of the mice. The fluorescence intensity was mainly detected at λEx/λEm = 470/560 nm. To eliminate the auto-fluorescence biomolecules from the mouse body, spectral unmixing was conducted, and only the fluorescence intensity of DOX was observed.A subcutaneous MDA-MB-231 human breast cancer xenograft model  was used to evaluate DOX release to the tumor region under FUS irradiation using in-house-developed FUS machinery, IMD-10R. IMP301 and DOXIL (20 mg/kg) were administered intravenously. FUS with a 2.0 kW/cmn = 3) were prepared for in vivo anticancer efficacy experiments. MDA-ME-231 cells were inoculated at 5 × 106 per mouse. To assess the synergistic effect of IMP301 and ultrasounds, nine mice were randomly divided into three groups: (i) control, (ii) IMP301 , and (iii) IMP301 under FUS irradiation using VIFU 2000 . Saline was injected in the control group (i). FUS was irradiated with the condition of 2.8 kW/cm2, 30 s/spot, PRF 250 Hz, and duty cycle 5%. All groups were treated with the drugs on days 1, 4, and 8. The dose of doxorubicin was administrated at 2 mg/kg. In addition, the FUS exposure group was irradiated with the ultrasound at the tumor region after 1 h of administration. To investigate the tumor suppression efficiency, tumor volume and body weight were measured every 3 days for 1 month. The tumor volume was calculated using the equation 0.5 × short length × long length × height, and the distributions of DOX, DOXIL, and IMP301 to the heart were quantitively analyzed by LC-MS/MS.MDA-MB-231 xenografted mice  and seems to induce destabilization by converting lamellar to the inverted hexagonal phase under acoustic wave pressure [To compare the sonosensitivity of IMP301 and DOXIL, both liposomes was exposed to continuous ultrasound waves, and the release amount of doxorubicin was quantitatively measured. Under ultrasound irradiation with a 92 W/cmpressure . Convers2 is required . The DOX release tended to increase as a function of ultrasound strength. To effectively release DOX from the inner liposome, a specific intensity threshold is required; in the case of IMP301, at least 2.8 kW/cmrequired a. In parrequired b. PRF isrequired c. Furtheuired . The starting masses of DOX and all phospholipids for liposome production were standard. The standard ranges of DOX content, lipid content, and entrapment efficiency were set as 90–110% and verified by the doxorubicin release ratio under the ultrasound exposure (data not shown). The results showed a range of 90–110% in the initial month, and the IMP301 composition remained consistently in this standard range of 90–110% for 1, 3, 6, and 9 months . The appTo investigate the effect of the combination of IMP301 and ultrasound, a cellular uptake study was performed using confocal microscopy and MDA-MB-231 cells. Nuclei were stained with DAPI (blue fluorescence), and red fluorescence showed DOX uptake and localization in cell nuclei. In the ultrasound-exposure IMP301 group, the fluorescence intensity by DOX was co-localized to the DAPI-stained area . This reTo verify ultrasound-triggered cell cytotoxicity, an in vitro MTT assay was carried out using the MDA-MB-231 cell line. Ultrasound-insensitive liposomal DOX, DOXIL, was used for comparison with IMP301. Each drug was divided into groups irradiated with ultrasound and a non-irradiated group. All groups were treated with cells at concentrations ranging from 2 to 10 µg/mL . In the max values of the fDOX- and IMP301-treated groups were 7978.6 and 80,824.7 ng/mL, respectively. As DOX showed low levels of cytotoxicity in the in vitro test, it is expected that there will be little concern, even if the blood concentration is high.To study the pharmacokinetics, IMP301 and free DOX (fDOX) were administered via the intravenous route. The concentration of DOX in the plasma was assessed using LC-MS/MS. A rapid reduction in the DOX concentration in the plasma was observed in the free DOX (fDOX) group. The results in this group confirmed that DOX showed rapid clearance, and small amounts of DOX were measured at all time points. However, the IMP301 treatment group showed a relatively higher concentration of DOX in the plasma than the fDOX treatment group . It was IMP301 and DOXIL ultrasound-triggered release were compared using live fluorescence imaging analysis. The DOX fluorescence emission of IMP301 and DOXIL was quenched in the core of both liposomes, whereas the fluorescence intensity of DOX was considerably enhanced by its release . TherefoNone of the groups showed significant body weight loss, indicating low systemic toxicity b. The FUIn this study, ultrasound-sensitive liposomal DOX was developed to enhance cancer treatment. IMP301 was uniformly produced with liposomes with diameters of less than 100 nm and a production yield of over 95%. To determine the characteristics of the ultrasound response, in vitro DOX release was analyzed using different FUS parameters . The release of DOX was proportional to the total ultrasound energy with respect to the intensity and exposure time. Interestingly, the release of DOX from IMP301 was critically related to the intensity and PRF. For effective DOX release, the intensity is more important than the duty cycle, and PRF for cavitation is essential to generate strong acoustic wave pressure. The release ratio of DOX under FUS irradiation was two times higher than that of DOXIL. The in vivo fluorescence imaging showed that in IMP301 the drug was released from the liposomes in response to FUS. The combination of IMP301 and FUS showed great therapeutic efficacy in MDA-MB-231 tumor-bearing mice. DOX released from liposomes accumulated in tumor tissues and killed cancer cells, leading to tumor suppression.Ultrasound combination therapy could facilitate the delivery of drugs into specific areas in the body with ultrasound imaging ,26,27,28