Objective To combine the active poly lactic co-glycolic acid (PLGA) nanosperes with adriamycin in side (ADM-NP) on the surface of ultrasound micrbubbles in covalent bonding and to prepare a novel drug-loading ultrasound micrbubble,and observe the physicochemical property.To quantify drug encapsulation efficiency and drug-loading amounts and drug-release properties in vitro and the effect of tumor imaging.Methods ADM-NP were prepared with double emulsification method,The surface carboxyl of ADM-NP was activated with carbodiimide method.Amino ultrasound microbubbles (MB-NH2) were prepared with mechanical shaking.The carboxyl of ADM-NP and the MB-NH2 could take place condensation reaction under a certain condition.Light microscope was used to observe the shape and distribution of the novel micrbubble.High performance liquid chromatography (HPLC) was used to quantify drug encapsulation efficiency and drug loading amounts.This novel microbubbles were put in 2 dialysis bag to observe the releasing properties of ADM in vitro,and one of the bag was using low frequency ultrasound irradiation for 120 s.The effect of tumor imaging using this novel microbubble was observed.Results After 48 hours,a number of ADM-NP attacted to the MB-NH2 like a gar land.Determination of entrapment efficiency and drug loading of it by HPLC weare (86.11 ± 6.76)％ and (8.71 ± 0.46)％.The sustained release in vitro can last for more than 48 hours.More than 90％ of ADM encapsulated in the 2 groups was sustained released for 48 hours.And the release characteristics of the 2 groups in vitro was in accord with Higuchi equation,and no difference was observed in the 2 groups (P ＞0.05).The tumor showed typical enhancement pattems of quick wash-in and quick wash-out.Conclusions To combine the nanospheres to the surface of microbubbles with covalent bonding,it could prepare a novel efficient drug-loading microbubbles for a original technique of ultrasound-targeted microbubble destruction(UTMD).

Objective To screen cardiac-specific short-acting peptides on live myocardial slices using phage display technology,so as to improve the targeted delivery efficiency of drugs in myocardium and provide effective candidates for the targeted therapy of Duchenne muscular dystrophy (DMD) and other cardiomyopathies.Methods Myocardial tissue slices were prepared and cultured in vitro.The protein activities of the tissues were examined by immunohistochemistry.The in vitro cultured myocardial tissue slices were co-incubated with phage library (1×1012 pfu),and the phages that bound to the myocardium were recovered and amplified.The cardiac-specific targeting phages were identified by five rounds of in vitro phage biopanning.The candidate phage-related insertion sequence was sequenced,and the in vivo tissue distribution of the highly enriched phages was verified.Results A platform for in vitro culturing of live myocardial slices was established.Myocardial slices with good biological activity were obtained.After 48 hours of culturing,the normal expression and localization of Dystrophin protein were detected.Using phage library,candidate phages were screened after five rounds of phage biopanning.The results of the sequencing analyses and in vivo tissue distribution verification indicated that the selected candidate phages showed significant enrichment in myocardium and skeletal muscle,and showed low levels in liver and kidney tissues.Conclusions The candidate phages showed higher binding efficiency in both myocardium and skeletal muscle,indicating that the candidate peptides had myocardial targeting property,and that can provide a new method for myocardial targeting therapy of DMD.

OBJECTIVE: To establish a HPLC method for simultaneous determination of metronidazole and chloram. phcnicol in comedo cream. METHODS: The method included a Diamonsil C18 column (250mm ? 4.6mm, 5?m) and methanol water(50 : 50,v/v)as the mobile phase and an ultraviolet detecting wavelength of 280nm .RESULTS:The calibralion curve was linear in a concentration range of ( 10 - 250)?g/ml of both metronidazole and chloramphenicol (r = 0.9 998) .The average recoveries of metronidazole and chloramphenicol were 100.53% , 100.53% and RSD were 0.79% , 0.56% respectively .CONCLUSION:The method for the determination of metronidazole and chloramphenicol was rapid and convenient. It can be used for the quality control of metronidazole and chloramphenicol in cream.

Objective:To investigate the correlation between white matter hyperintensities (WMHs) and stroke etiology classification in patients with acute isolated penetrating artery territory infarction.Methods:Patients with first-ever acute isolated penetrating artery territory infarction admitted to the Department of Neurology, the Affiliated Hospital of Xuzhou Medical University from January 2017 to May 2020 were enrolled retrospectively. According to the Chinese Ischemic Stroke Subclassification (CISS) system, they were divided into large artery atherosclerosis (LAA) and perforating artery disease (PAD). According to the distribution of infarcts, they were divided into lenticulostriate artery (LSA) territory infarction and paramedian pontine artery (PPA) territory infarction. The demographics, vascular risk factors, baseline clinical data, WMHs location, and Fazekas Scale scores were documented. Multivariate logistic regression analysis was used to identify the independent influencing factors of stroke etiology classification. Results:A total of 440 patients with acute isolated penetrating artery territory infarction were enrolled, including 120 (27.3%) in the LAA group, and 320 (72.7%) in the PAD group; 213 (48.4%) with LSA territory infarction, and 227 (51.6%) with PPA territory infarction. The proportion of patients with total Fazekas score 3-6 and periventricular WMHs (PWMHs) score 2-3 in the PAD group was significantly higher than those in the LAA group (all P<0.05). In patients with LSA territory infarction, the proportion of the patients with hypertension, WMHs total Fazekas score 3-6 and PWMHs score 2-3 in PAD subgroup was significantly higher than those in the LAA subgroup, while the proportion of the patients with hyperlipidemia was significantly lower than that in LAA subgroup (all P<0.05). In patients with PPA territory infarction, the levels of low-density lipoprotein cholesterol and homocysteine in the PAD subgroup were significantly lower than those in the LAA subgroup. Multivariate logistic regression analysis showed that PWMHs score 2-3 was an independent correlation factor of PAD (odds ratio [ OR] 2.220, 95% confidence interval [ CI] 1.085-4.541; P=0.029). In patients with LSA territory infarction, hyperlipidemia was independently correlated with LAA ( OR 0.432, 95% CI 0.192-0.972; P=0.042), and PWMHs score 2-3 was independently correlated with PAD ( OR 3.846, 95% CI 1.193-12.397; P=0.024). In patients with PPA territory infarction, higher low-density lipoprotein cholesterol ( OR 0.660, 95% CI 0.494-0.883; P=0.005), homocysteine ( OR 0.958, 95% CI 0.930-0.987; P=0.005) and C-reactive protein ( OR 0.987, 95% CI 0.977-0.997; P=0.008) were independently correlated with LAA. Conclusions:WMHs are common in patients with acute isolated perforating territory infarction caused by LAA and PAD, and more severe PWMHs suggest that PAD is more likely to be the cause of the acute isolated perforating territory infarction, especially in patients with LSA territory infarction.

OBJECTIVE:To optimize the decoction process of agkistrodon. METHODS:RP-HPLC with pre-column derivatiza-tion was adopted. With the contents of 4 main amino acids in agkistrodon as index,the decoction process(decocting times,heating time,water quantity and medicinal material crushing granularity) was optimized by orthogonal tests and verified. RESULTS:The optimal technology of the decoction process of agkistrodon was as 3 times of decoction,60 min of heating time,50 ml of water consumption for 0.90 g medicinal material and No.6 sieve. The results of verification test showed the total extraction of 4 kinds of amino acids was 72.68 mg/g(RSD=3.77%,n=3). CONCLUSIONS:The decoction technology can be used for the decoction pro-cess of agkistrodon,and it is stable and feasible.

Objective To investigate the antitumor effect and its mechanism of microbubble contrast combined with Mitoxantrone exposed to low-frequency ultrasound on human breast cancer cell MCF-7. Methods MTT method was applied to examine the growth inhibition of MCF-7 treated with Mitoxantrone. MCF-7 cells were randomly divided into 4 groups:Mitoxantrone group (group D), ultrasound+Mitoxantrone group (group U+D), ultrasound+microbuble +Mitoxantrone group (group U+M+D) and control group (group C). The cytoactive of each group was examined with MTT. The intracellular drug content in each group was measured with high performance liquid chromatography. The morphology of MCF-7 cells apoptosis was observed with transmission electron microscopy (TEM). Results The IC_(50) of Mitoxantrone was 2.87 μg/ml. The differences of cytoactive among all groups were significant (P<0.05). The intracellular drug content of group U+M+D was higher than that of group U+D, and the latter was higher than that of group D. The morphological changes of apoptosis were observed with TEM. Conclusion Low-frequency ultrasound can promote intracellular drug content as to enhance the sensitivity of chemotherapy drugs on tumor cells, and this effect can be enhanced by microbubble contrast exposure to low-frequency ultrasound.

Recently, the droplet microfluidic system attracts interests due to its high throughput and low cost to detect and screen. The picoliter micro-droplets from droplet microfluidics are uniform with respect to the size and shape, and could be used as monodispensed micro-reactors for encapsulation and detection of single cell or its metabolites. Therefore, it is indispensable to characterize micro-droplet and its application from droplet microfluidic system. We first constructed the custom-designed droplet microfluidic system for generating micro-droplets, and then used the micro-droplets to encapsulate important amino acids such as glutamic acid, phenylalanine, tryptophan or tyrosine to test the droplets' properties, including the stability, diffusivity and bio-compatibility for investigating its application for amino acid detection and sorting. The custom-designed droplet microfluidic system could generate the uniformed micro-droplets with a controllable size between 20 to 50 microm. The micro-droplets could be stable for more than 20 h without cross-contamination or fusion each other. The throughput of detection and sorting of the system is about 600 micro-droplets per minute. This study provides a high-throughput platform for the analysis and screening of amino acid-producing microorganisms.
Amino Acids ; isolation & purification ; Microfluidic Analytical Techniques ; Microfluidics ; instrumentation

The most effective sequence of small interfering RNA (siRNA) silencing STAT3 of psoriatic keratinocytes (KCs) was screened out,and the effects of the most effective siRNA combined with ultrasonic irradiation and SonoVue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic siRNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective siRNA was selected for the subsequent experiments.The negative controls of siRNA (siRNA-NC) labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective siRNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and SonoVue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective siRNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group) was compared with that only carried by Lipofectamine 3000 (L group).The results showed that siRNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue mierobubbles could effectively knock down the STAT3 expression at mRNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on mRNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of siRNA-3 could serve for further research on gene therapy of psoriasis.

Objective To explore the mechanism and inhibition of botulinum toxin type A (BTXA) on hypertrohic scar fibroblasts.Methods The cells were treated by 0 (control),0.2,0.4,0.8 U/ml BTXA for 48 h.Cell viability was detected by MTT assay.Cell apoptosis was detected by Hoechst staining.Cell cycle was detected by flow cytometry.The level of cell cycle related protein D1 (Cyclin D1),proliferation nuclear antigen (PCNA) and activation of phosphatidylinositol 3 kinase (PI3K) / protein kinase B (AKT) signaling pathway were assayed by western blot.Results Compared with control group(0.75±0.07),0.2,0.4,0.8 U/mL BTXA(0.59 ± 0.06,0.43 ± 0.04,0.34± 0.03) inhibited hypertrohic scar fibroblasts cell viability,increased cell apoptotic rate[control group(2.38±0.24)％;BTXA(15.79±1.54)％,(27.32±2.69)％,(38.46±3.90)％],down-regulated the expression of Cyclin D1(control group 1.57±0.18;BTXA 0.93±0.07,0.42±0.04,0.35±0.03) and PCNA(control group 1.46±0.16;BTXA 0.50±0.05,0.59±0.05,0.37±0.03),inhibited the expression of PI3K(control group 0.98±0.06;BTXA 0.49±0.04,0.50±0.04,0.39±0.03) and the phosphorylation of AKT(control group 1.38±0.08;BTXA 0.97±0.06,0.60±0.04,0.29± 0.02),made cell cycle arrested in G1 phase,The difference was statistically significant (P＜0.05).Conclusion These results suggested BTXA inhibit proliferation via blocking the activation of PI3K/AKT signal pathway and down-stream related cell cycle related protein.

BackgroundDry eye is a multi-factorial-induced tear film and ocular surface disorder.Immunoinflammation plays a key role in the pathogenesis of dry eye.As inhibitor of the cyclo-oxygenase pathway,nonsteroidal anti-inflammatory drugs play an anti-inflammatory and anti-hypersensitivity role,and it can be a potential treatment for dry eyes.ObjectiveThis study was to investigate the effectiveness of nonsteroidal anti-inflammatory drugs (0.1％topical pranoprofen) on moderate to severe dry eyes and its mechanism.MethodsThis was a small sample of randomized controlled clinical trial.Thirty right eyes of 30 patients with moderate to severe dry eyes were included in the study according to the diagnosis criteria and randomized into two groups.The patients of the trial group received topical administration of 0.1％ pranoprofen plus 0.1％ sodium hyaluronate,and those of the control group received the topical 0.1％ sodium hyaluronate only.Ocular surface inflammation index scores (OSDI) and ocular surface fluorescine staining (OSS) scores were measured under the slit lamp,and tear film break-up time (BUT),Schirmer Ⅰ test values were evaluated.The expression of human leucocyte antigen-DR (HLA-DR) and CD11b in conjunctiva epithelial cells were detected by impression cytology and flow cytometry (FCM).All the indexes were compared between the two groups before and after treatment.Informed consent was obtained from all patients.ResultsThere were no significant differences in terms of age and gender and their baseline values between the trial group and control group (t=0.412,P=0.684;x2=0.240,P=0.624),and so were all the indexes (P＞0.05).Compared with the control group,the OSDI,OSS scores and cells positive for HLA-DR were lowered but the BUT was delayed in the trial group on day 15 ( t=2.43,P=0.03;t=2.83,P=0.01;t=3.29,P=0.00;t=3.23,P=0.00 ).No significant differences were found in the Schirmer Ⅰ test value and CD11b expression between these two groups (t=0.17,P=0.87;t=0.28,P=0.79).The OSDI,OSS scores and BUT were significantly improved,and the number of cells positive for HLA-DR were reduced 15 days after administration of drugs in comparison with before treatment in the trial group ( t =12.30,10.70,6.10,7.92,P =0.00 ).However,there were no comparable alteration seen in these indexes before and after the usage of drugs in the control group ( P＞0.05).Positive correlations were found in HLADR expression with OSDI and OSS ( r =0.601,P =0.018 ; r =0.586,P =0.022 ) and a negative correlation in HLADR expression with BUT (r=-0.697,P=0.004) on day 15 in the trial group.ConclusionsTopical usage of 0.1％ pranoprofen is beneficial for remitting the ocular signs and symptoms in moderate to severe dry eyes.This study illustrates that topical usage of 0.1％ pranoprofen can down-regulate the expression of inflammatory markers in conjunctival epithelial cells.