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Microspheres are commonly used as embolic materials in vascular interventional operations.However,due to the limitation of materials,almost all microspheres cannot be detected by imaging equipment in vivo.The visualization of microspheres refers to the addition of various materials to the microspheres that enable the microspheres to be displayed on the images of imaging equipment.In order to optimize the embolization process and facilitate postoperative follow-up,a lot of visualized microspheres have been reported so far,such as X-ray visualized microspheres,MRI visualized microspheres,ultrasonic visualized microspheres,etc.Clinical experience has shown that these microspheres can provide true spatial distribution and real-time intraoperative feedback,which contributes to the optimization,personalization,and improvement of vascular embolization technology.This paper aims to make a comprehensive review about the recent advances in researches about the above mentioned visualized microspheres.
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Objective To investigate the safety and efficacy of Embosphere microsphere in the treatment of massive hemoptysis during bronchial arterial embolization(BAE).To analyze the factors influencing the recurrence by Cox regression model.Methods Ninety patients with massive hemoptysis who underwent BAE using Embosphere microsphere were included.The immediate hemo-stasis rate within 24 hours,clinical success rate and prognostic factors were statistically analyzed.Results The immediate hemosta-sis rate within 24 hours was 94.4%(85/90).The clinical success rate was 56.7%(51/90).Cumulative hemoptysis-free recurrence con-trol rates at 6 months,1 year and 2 years postoperative were 81%,78%and 57%,respectively.Cox regression model analysis showed that the variables associated with recurrence were long-term hemoptysis,lung cancer,tuberculosis and lung destruction.Conclusion Embosphere microsphere are safe and effective embolic particles in the treatment of massive hemoptysis during BAE.Risk factors for hemoptysis recurrence include long-term hemoptysis,lung cancer,tuberculosis and lung destruction.
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BACKGROUND:Nucleus pulposus degeneration is an important pathological link of intervertebral disc degeneration.Melatonin has a protective effect on cells through anti-inflammatory and antioxidant pathways,but the effect of melatonin on the nucleus pulposus has been less studied.At present,the emergence of various biological scaffolders provides a new idea for the study of drug-material combinations.OBJECTIVE:To explore whether melatonin can improve the metabolic state of the nucleus pulposus by reducing oxidative stress damage as well as the effect of gelatin methacryloyl(GelMA)microspheres loaded with melatonin on intervertebral disc degeneration in vivo.METHODS:In vitro,melatonin was combined with GelMA solution,and GelMA hydrogel was prepared into microspheres by microfluidic technology to co-culture with nucleus pulposus cells.The cell proliferation activity was detected by cell counting kit-8 assay,the surface morphology of the microspheres was observed under scanning electron microscopy,and the rate of drug release was detected by ultraviolet spectrophotometer.Then,interleukin-1β was used to induce degeneration of the nucleus pulposus.After treatment,the expression levels of aggrecan,type Ⅱ collagen α1,matrix metalloproteinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs-5(ADAMTS5)in the nucleus pulposus were detected by qRT-PCR.In vivo,nucleus pulposus degeneration was induced by puncture.Subsequently,GelMA and GelMA@MT microspheres were injected.After 6 weeks,the specimens were taken for tissue staining,and the changes in tissue morphology were observed under the microscope for histological analysis and scoring.RESULTS AND CONCLUSION:(1)When the GelMA and GelMA@MT microspheres were observed under electron scanning microscope,melatonin binding did not change the morphology and external appearance of the microspheres.Drug release experiments showed that the drug release reached about 80%after 40 days.(2)Cell counting kit-8 assay results showed that both GelMA and GelMA@MT microspheres had no obvious cytotoxicity and promoted the proliferation of nucleus pulposus cells.(3)qRT-PCR results revealed that GelMA@MT microspheres increased the expression of aggrecan and type Ⅰ collagen α1 in the interleukin 1β environment by 42.1%and 27.1%,respectively,and decreased the expression of matrix metalloproteinase 13 and ADAMTS5 by 70.7%and 109.3%,respectively.(4)The level of reactive oxygen species was significantly lower in the interleukin 1β+GelMA@MT group than in the interleukin 1β and interleukin 1β+GelMA groups.(5)Histological staining of the sections showed that melatonin-loaded GelMA microspheres significantly delayed disc degeneration in vivo.(6)These findings indicate that GelMA@MT microspheres made by combining melatonin with GelMA hydrogel have good cytocompatibility in vitro and significantly delay nucleus pulposus degeneration in vitro and in vivo.
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BACKGROUND:The imbalance of matrix synthesis and degradation is the main cause of nucleus pulposus degeneration.Small molecule drug Kartogenin(KGN)can restore the balance of matrix synthesis and degradation.Sustained release of KGN using an appropriate drug delivery system is essential for the long-term and effective treatment of KGN.OBJECTIVE:To prepare the injectable hydrogel microspheres by encapsulating KGN with gelatin methacryloyl(GelMA)by microfluidic technology and to investigate the biocompatibility and biological function of nucleus pulposus cells.METHODS:β-Cyclodextrins(β-CD)and KGN were mixed firstly and then mixed with 10%GelMA at a volume of 1:9.Injectable hydrogel microspheres GelMA@β-CD@KGN were prepared by microfluidic technology.The micromorphology of the microspheres was characterized using a scanning electron microscope.The drug release of hydrogel microspheres immersed in PBS within one month was measured.Nucleus pulposus cells were isolated from SD rats and passage 1 cells were cultured in three groups.In the control group,nucleus pulposus cells were cultured separately.In the other two groups,GelMA@β-CD microspheres and GelMA@β-CD@KGN microspheres were co-cultured with nucleus pulposus cells.Cell proliferation was detected by CCK-8 assay and cell survival was detected by live/dead cell staining.Cells were cultured by two complete media with and without interleukin-1β with two kinds of microspheres.mRNA expressions of matrix synthesis and decomposing proteins in nucleus pulposus cells were detected by RT-PCR.RESULTS AND CONCLUSION:(1)Under the scanning electron microscope,the GelMA@β-CD@KGN microspheres after lyophilization were regularly spherical,highly dispersed,uniform in size and full in shape.GelMA@β-CD@KGN microspheres sustained drug release in vitro,reaching 62%of the total drug release at 30 days.(2)Live/dead cell staining showed that GelMA@β-CD@KGN could maintain the activity of nucleus pulposus cells.CCK-8 assay showed that GelMA@β-CD@KGN could promote the proliferation of nucleus pulposus cells.(3)In the complete media with and without interleukin-1β,mRNA expression of aggrecan and type Ⅱ collagen was higher in the GelMA@β-CD@KGN microsphere group than that in the GelMA@β-CD microsphere group(P<0.05,P<0.01);mRNA expression of matrix metalloproteinase 13 and platelet reactive protein disintegrin metallopeptidase 5 was lower than that in the GelMA@β-CD microsphere group(P<0.01).(4)These findings indicate that GelMA@β-CD@KGN microspheres have good biocompatibility and sustained drug release ability.As a drug delivery system,it is a kind of biomaterial with broad application prospects.
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BACKGROUND:A large number of studies have confirmed that tissue engineering scaffolds can almost completely repair osteochondral defects.However,when osteochondral defects are complicated with infection,even after thorough debridement in the early stage,the repair effect of simple osteochondral tissue engineering scaffolds is often unsatisfactory. OBJECTIVE:To prepare fibroin/chitosan/nano-hydroxyapatite scaffold loaded with vancomycin hydrochloride sustained release microspheres,and to investigate the repair effect on infected osteochondral defect in distal femur of rabbit. METHODS:(1)Vancomycin hydrochloride sustained release microspheres were prepared by emulsified solvent evaporation method.The sustained-release microspheres of different weights(7.5,10,and 12.5 mg)were mixed with fibroin protein-chitosan nanohydroxyapatite solution,and the scaffolds of fibroin protein/chitosan/nano-hydroxyapatite were prepared by chemical crosslinking method.The porosity,water absorption and expansion rate,hot water loss rate of the scaffolds,and drug sustained-release in vitro were characterized.(2)Forty-five New Zealand white rabbits were randomly divided into blank group,control group,and experimental group,with 15 rabbits in each group.The osteochondral defect and infection model of the distal femur of the right hind limb was established in both groups.The blank group was not treated,and the control group was implanted with fibroin protein-chitosan-nano-hydroxyapatite scaffold.Vancomycin hydrochloride sustained-release microspheres(10 mg)of fibroin/chitosan/nano-hydroxyapatite scaffold were implanted in the defect of the experimental group.The levels of C-reactive protein and leukocytes in blood samples were detected 1 week after operation.At 4,8 and 12 weeks after operation,the tissue of the operative area was taken for gross observation and pathological observation. RESULTS AND CONCLUSION:(1)With the increase of sustained-release microspheres content,the porosity of scaffolds decreased,and there was significant difference among groups(P<0.05).There were no significant differences in the pore size,water absorption expansion rate and hot water loss rate among the three groups(P>0.05).Vancomycin hydrochloride was released sustainably in vitro for more than 30 days in all three groups of scaffolds.(2)The levels of C-reactive protein and leukocytes in blood samples of the experimental group were lower than those of the blank group and control group(P<0.05).The repair of gross cartilage in the experimental group was significantly better than that in the blank group and the control group.Hematoxylin-eosin,Masson,Alcian blue and type Ⅱ collagen immunohistochemical stainings showed that the osteochondral repair effect of the experimental group was significantly better than that of the blank group and the control group at each time point.(3)The results showed that fibroin/chitosan/nano-hydroxyapatite scaffolds loaded with vancomycin hydrochloride sustained-release microspheres could effectively promote the repair of open osteochondral defects.
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Objective To develop a time-resolved fluorescent immunoassay kit for the rapid,accurate and quantitative detection of S100B protein in serum and to evaluate its performance.Methods The test strip was prepared using time-resolved fluorescent microsphere-labeled anti-S100B polyclonal antibody and rabbit IgG antibody,labeling pads,sample pads,S100B nitrocellulose films and absorbent paper,and an S100B time-resolved fluorescence immunoassay kit was obtained by assembling the cartridge.The performance of the kit developed was evaluated by standard curve,accuracy,minimum detection limit,linear interval,specificity,reproducibility and stability.The reference intervals of 199 pieces of healthy human serum and plasma samples from a certain region were detected with the kit,and the clinical performance of the kit and Roche Elecsys S100 kit was tested by synchronous blind method to assess the consistency of the results of the two kits for 142 samples.Results The S100B time-resolved fluorescence immunoassay kit had the standard curve beingy=(1.133 02+1.752 24)/[1+(x/1.082 20)×(-0.603 52)]-1.752 24,R2=0.999 08 and the linear range being[0.05,30]ng/mL,which met the requirements of the relative deviation of the accuracy within±15%,the minimum detection limit not hgier than 0.05 ng/mL,the relative deviation of specificity within±15%and the coefficient of variation of intra-and inter-batch difference less than 15%.The stability test results indicated that the kit was valid for 12 months at 2-30 ℃ conditions.The reference intervals of serum and plasma samples measured by the kit were both lower than 0.3 ng/mL.Clinical trials showed that the results by the kit and Roche Elecsys S100 Assay Kit were in high agreement(Kappa=0.906 1>0.80)and met the requirements.Conclusion The kit developed detects the concentration of S100B protein in serum quickly,accurately and quantitatively,and provides references for the diagnosis and treatment of neurological diseases,autoimmune diseases,cerebrovascular diseases and etc.[Chinese Medical Equipment Journal,2024,45(1):47-55]
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Objective:To explore the radiological protection measures for yttrium-90 ( 90Y)-loaded resin microsphere therapy in clinical application. Methods:The surgical operation process for 90Y-loaded resin microsphere therapy was simulated, involving measurement of ambient dose equivalent rates at various stages: preoperative preparation (dominated by drug package), drug transfer, intraoperative procedures (drug operation and injection), and postoperative care and observation within the hospital. Based on the simulation, the protection measures in clinical application were analyzed. Results:The dose equivalent rate ranged from 0.12 to 0.42 μSv/h around the active chamber and from 1.04 to 3.32 μSv/h in the fume hood. Around the digital subtraction angiography (DSA) room, the maximum dose equivalent rate was 0.78 μSv/h when 90Y and DSA were applied simultaneously and 0.36 μSv/h when 99Tc m and DSA were applied. For the first operating position in the fluoroscopy protection area, the maximum dose equivalent rate was 13.19 μSv/h at 155 cm height when only 90Y was applied, and 315.01 μSv/h at 80 cm height when 90Y and DSA were applied. For the second operating position, the maximum dose equivalent rate was 6.28 μSv/h at 155 cm height when only 90Y was applied and 291.03 μSv/h at the same height when 90Y and DSA were applied. The dose-equivalent rates ranged from 0.11 to 0.58 μSv/h around the dedicated ward for postoperative patients. Conclusions:The existing shielding measures, such as those in the nuclear medicine department and interventional room, meet the radiation protection requirements for 90Y-loaded resin microsphere therapy. However, it is still necessary to conduct a scientific assessment based on the actual situation. Additionally, radiation protection measures and surface contamination treatment should be enhanced during drug operation.
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Sodium alginate (SA) is a kind of natural polymer material extracted from kelp, which has excellent biocompatibility, non-toxicity, biodegradability and abundant storage capacity. The formation condition of sodium alginate gel is mild, effectively avoiding the inactivation of active substances. After a variety of preparation methods, sodium alginate microspheres are widely used in the fields of biomaterials and tissue engineering. This paper reviewed the common methods of preparing alginate microspheres, including extrusion, emulsification, electrostatic spraying, spray drying and coaxial airflow, and discussed their applications in biomedical fields such as bone repair, hemostasis and drug delivery.
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Alginates , Biocompatible Materials , Drug Delivery Systems , Microspheres , Plastic Surgery ProceduresABSTRACT
OBJECTIVE@#To prepare a novel hyaluronic acid methacrylate (HAMA) hydrogel microspheres loaded polyhedral oligomeric silsesquioxane-diclofenac sodium (POSS-DS) patricles, then investigate its physicochemical characteristics and in vitro and in vivo biological properties.@*METHODS@#Using sulfhydryl POSS (POSS-SH) as a nano-construction platform, polyethylene glycol and DS were chemically linked through the "click chemistry" method to construct functional nanoparticle POSS-DS. The composition was analyzed by nuclear magnetic resonance spectroscopy and the morphology was characterized by transmission electron microscopy. In order to achieve drug sustained release, POSS-DS was encapsulated in HAMA, and hybrid hydrogel microspheres were prepared by microfluidic technology, namely HAMA@POSS-DS. The morphology of the hybrid hydrogel microspheres was characterized by optical microscope and scanning electron microscope. The in vitro degradation and drug release efficiency were observed. Cell counting kit 8 (CCK-8) and live/dead staining were used to detect the effect on chondrocyte proliferation. Moreover, a chondrocyte inflammation model was constructed and cultured with HAMA@POSS-DS. The relevant inflammatory indicators, including collagen type Ⅱ, aggrecan (AGG), matrix metalloproteinase 13 (MMP-13), recombinant A disintegrin and metalloproteinase with thrombospondin 5 (Adamts5), and recombinant tachykinin precursor 1 (TAC1) were detected by immunofluorescence staining and real-time fluorescence quantitative PCR, with normal cultured chondrocytes and the chondrocyte inflammation model without treatment as control group and blank group respectively to further evaluate their anti-inflammatory activity. Finally, by constructing a rat model of knee osteoarthritis, the effectiveness of HAMA@POSS-DS on osteoarthritis was evaluated by X-ray film and Micro-CT examination.@*RESULTS@#The overall particle size of POSS-DS nanoparticles was uniform with a diameter of about 100 nm. HAMA@POSS-DS hydrogel microspheres were opaque spheres with a diameter of about 100 μm and a spherical porous structure. The degradation period was 9 weeks, during which the loaded POSS-DS nanoparticles were slowly released. CCK-8 and live/dead staining showed no obvious cytotoxicity at HAMA@POSS-DS, and POSS-DS released by HAMA@POSS-DS significantly promoted cell proliferation (P<0.05). In the chondrocyte anti-inflammatory experiment, the relative expression of collagen type Ⅱ mRNA in HAMA@POSS-DS group was significantly higher than that in control group and blank group (P<0.05). The relative expression level of AGG mRNA was significantly higher than that of blank group (P<0.05). The relative expressions of MMP-13, Adamts5, and TAC1 mRNA in HAMA@POSS-DS group were significantly lower than those in blank group (P<0.05). In vivo experiments showed that the joint space width decreased after operation in rats with osteoarthritis, but HAMA@POSS-DS delayed the process of joint space narrowing and significantly improved the periarticular osteophytosis (P<0.05).@*CONCLUSION@#HAMA@POSS-DS can effectively regulate the local inflammatory microenvironment and significantly promote chondrocyte proliferation, which is conducive to promoting cartilage regeneration and repair in osteoarthritis.
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Animals , Rats , Matrix Metalloproteinase 13 , Microspheres , Hydrogels , Collagen Type II , Diclofenac , Inflammation , Osteoarthritis, Knee/drug therapy , Hyaluronic Acid , AggrecansABSTRACT
OBJECTIVE@#To construct polyhydroxyalkanoate (PHA) microspheres loaded with bone morphogenetic protein 2 (BMP-2) and human β-defensin 3 (HBD3), and evaluate the antibacterial activity of microspheres and the effect of promoting osteogenic differentiation, aiming to provide a new option of material for bone tissue engineering.@*METHODS@#The soybean lecithin (SL)-BMP-2 and SL-HBD3 were prepared by SL-mediated introduction of growth factors into polyesters technology, and the functional microsphere (f-PMS) containing BMP-2 and HBD3 were prepared by microfluidic technology, while pure microsphere (p-PMS) was prepared by the same method as the control. The morphology of microspheres was observed by scanning electron microscopy and the water absorption was detected; the release curves of BMP-2 and HBD3 in f-PMS were detected by ELISA kit. The antibacterial effect of microspheres in Staphylococcus aureus and Escherichia coli was tested with the LIVE/DEADTM BacLightTM bacterial staining kit; the biocompatibility of microspheres was tested using Transwell and cell counting kit 8 (CCK-8). The effect of microspheres on osteogenic differentiation was determined by collagen type Ⅰ (COL-1) immunofluorescence staining and alkaline phosphatase (ALP) concentration.@*RESULTS@#In this experiment, the f-PMS and p-PMS were successfully constructed. Morphological characteristics showed that p-PMS surface was rough and distributed with micropores of 1-3 μm, while f-PMS surface was smooth and existed white granular material. There was no significant difference in water absorption between the two groups (P>0.05). The release curves of BMP-2 and HBD3 in the f-PMS and p-PMS were basically the same, showing both early sudden release and late slow release. The antibacterial activity of f-PMS was significantly higher than that of p-PMS in the test that against Staphylococcus aureus and Escherichia coli (P<0.05), but there was no significant difference in biocompatibility between the two groups (P>0.05). The results of osteogenic differentiation of human BMSCs showed that the fluorescence intensity of osteogenic specific protein COL-1 of f-PMS was significantly higher than that in p-PMS, and the activity of ALP in f-PMS was also significantly higher than that in p-PMS (P<0.05).@*CONCLUSION@#The p-PHA have good antibacterial activity and biocompatibility, and can effectively promote the osteogenic differentiation of human BMSCs, which is expected to be applied to bone tissue engineering in the future.
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Humans , Osteogenesis , Polyhydroxyalkanoates , Microspheres , Alkaline Phosphatase , Anti-Bacterial Agents/pharmacology , Coloring Agents , Escherichia coliABSTRACT
Electrospray technology is a method to prepare microspheres by electrostatic force. Electrospray technology has the advantages of simple operation and mild reaction conditions. The polymer microspheres prepared by electrospray technology have uniform morphology and good monodispersity, which is a new and promising method. In this paper, the devices and principles of electrospray technology, the factors affecting the morphology and particle size of the prepared polymer microspheres by the electrospray process, and the types of polymer solutions commonly used in electrospray technology were described, and the applications of electrospray technology for drug delivery, loaded nanoparticles, cell therapy, and bioactive substance delivery were reviewed. It can be concluded that electrospray technology has a broad application prospect and potential application value.
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@#Chitosan-based microspheres use chitosan as the main material to obtain particles with special structures through microsphere processing technology. They have the ability of slow and controlled release of drugs and the role of scaffolding, which have great application prospect in stomatology, but the application of chitosan-based microspheres is still in the research stage and has not yet been applied in clinical practice. This article reviews progress of domestic and foreign research on chitosan-based microspheres, in aspects of treatment of oral and jawbone tissue defects, periodontal diseases, dental pulp diseases and nerve tissue injury, in order to provide reference for follow-up research.
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Abstract To investigate osteoclast formation in vivo and if leukotriene B4 (LTB4) loaded in microspheres (MS) could be used as a therapeutical strategy to promote a sustained delivery of the mediator and prevent osteoclast differentiation. Methods: In vivo, apical periodontitis was induced in mice to investigate osteoclast differentiation and signaling in absence of 5-lipoxygenase (5-LO). In vitro, LTB4-MS were prepared using an oil-in-water emulsion solvent extraction-evaporation process. Characterization and efficiency of LTB4 encapsulation were investigated. J774A.1 macrophages were cultured in the presence of monocyte colony-stimulating factor (M-CSF) and ligand for receptor activator of nuclear factor kappa B (RANKL) and then stimulated with LTB4-MS. Cytotoxicity, in vitro MS-LTB4 uptake, osteoclast formation and gene expression were measured. Results: We found that 5-LO negatively regulates osteoclastic formation in vivo during apical periodontitis development. In vitro, LTB4-MS were up-taken by macrophages and were not cytotoxic to the cells. LTB4-MS inhibited osteoclast formation and the synthesis of osteoclastogenic genes Acp5, Mmp9, Calcr and Ctsk. LTB4-MS inhibited differentiation of macrophages into an osteoclastic phenotype and cell activation under M-CSF and RANKL stimulus.
Resumo O objetivo deste trabalho foi Investigar a formação de osteoclastos in vivo e se o leucotrieno B4 (LTB4) incorporado em microesferas (MS) poderia ser usado como estratégia terapêutica para promover uma entrega sustentada do mediador e prevenir a diferenciação dos osteoclastos. Métodos: In vivo, a periodontite apical foi induzida em camundongos para investigar a diferenciação e sinalização de osteoclastos na ausência de 5-lipoxigenase (5-LO). In vitro, LTB4-MS foi preparado usando um processo de evaporação e extração de solvente de emulsão de óleo em água. A caracterização e a eficiência do encapsulamento do LTB4 foram investigadas. Macrófagos J774A.1 foram cultivados na presença de fator estimulador de colônia de monócitos (M-CSF) e ligante para o receptor ativador do fator nuclear kappa B (RANKL) e, então, estimulados com LTB4-MS. Citotoxicidade, captação in vitro de MS-LTB4, formação de osteoclastos e expressão gênica foram avaliadas. Resultados: A via 5-LO regula negativamente a formação de osteoclastos in vivo durante o desenvolvimento da periodontite apical. In vitro, LTB4-MS foram fagocitadas pelos macrófagos e não foram citotóxicos para as células. LTB4-MS inibiu a formação de osteoclastos e a síntese dos genes pró-osteoclastogênicos Acp5, Mmp9, Calcr e Ctsk. Conclusões: LTB4-MS inibiu a diferenciação de macrófagos em um fenótipo osteoclástico e a ativação celular sob estímulo de M-CSF e RANKL.
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The electrochemical performance of zinc cobaltite–based nano/micromaterial depends on its shape and morphology. Here we report on the electrochemical performance of zinc cobaltite (ZnCo O ) material synthesized via a facile 2 4 hydrothermal method. The synthesized material was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) analysis. It was found to be a single-phase zinc cobaltite material with a cubic spinel crystal structure. The electrochemical performance of the synthesized zinc cobaltite microstructure material was evaluated by cyclic voltammetry, cyclic chronopotentiometry and electrochemical impedance -1 -1 spectroscopy. The zinc cobaltite microspheres material displayed a high specific capacitance of 600.37 F g at a current density of 1 A g . Such electrochemical performance may qualify the zinc cobaltite microspheres material as a potential electroactive material in supercapacitors.
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@#Recently, in situ gel has been widely used as a local delivery system for periodontitis treatment because of its lesion injectability, local drug depot function, and drug sustained-release effect.Different therapeutic purposes can be achieved by loading different types of drugs such as antibiotics, bioactive factors, etc.In this review, different types of in situ gel with temperature-sensitive, ion-sensitive, pH-sensitive and solvent-exchanged characteristics were introduced for their applications and limitations in the delivery of drug for periodontitis;and the elimination of periodontal inflammation, periodontal tissue repair and the long-term role after loading microsphere achieved by the in situ gel system were also reviewed.
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A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine the plasma concentration of progesterone in Beagle dogs, and apply it to the study of the pharmacokinetics of progesterone sustained-release formulation in Beagle dogs. The plasma samples were processed by protein precipitation method and megestrol acetate was used as an internal standard (IS). The quantitation analysis was performed using multiple-reaction monitoring (MRM) mode at the specific ion transitions of m/z 315.2→97.0 for progesterone and m/z 385.2→267.1 for megestrol acetate (IS) under the positive ion condition. Male Beagle dogs were injected intramuscularly with progesterone sustained-release microspheres and the plasma samples were collected at different time points after administration. The relevant pharmacokinetic parameters were calculated by WinNonlin 8.1 software. A good linearity over the range of 0.1-500.0 ng·mL-1 was yielded by this method. The intra- and inter-day precision (RSD) were all less than 13.25% and the accuracy (RE) was within 8.92%. Stability test showed that progesterone in dog plasma was stable at room temperature for 12 h, up to 60 days at -20 ℃ and after three cycles of freeze-thaw. The recovery of it ranged from 71.43%-77.97%. After intramuscular injection of progesterone sustained-release microspheres in Beagle dogs, tmax was 19.00 ± 25.36 h, Cmax was 137.72 ± 11.59 ng·mL-1, t1/2 was 83.83 ± 26.43 h. The drug was released continuously in vivo and in a continuous absorption process for many times with good sustained-release effect. The method developed in this study is sensitive, rapid and stable. It is suitable for the determination of progesterone plasma concentration in Beagle dogs, and can be applied to the preclinical pharmacokinetic study of progesterone-related formulations. The animal experiment scheme of this study was approved by the Animal Ethics Committee of the Academy of Military Medical Sciences.
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In order to meet the clinical needs of long-acting sustained-release thienorphine, injectable thienorphine loaded microspheres were developed, and the accelerated stability study was carried out to explore the suitable storage and transportation conditions of the microspheres. Using poly(lactic-co-glycolic acid) (PLGA) as carrier material, 3 batches of microspheres were prepared in pilot scale with emulsion solvent evaporation method. By investigating the in vitro release of thienorphine loaded microspheres at 37, 45, 52, and 60 ℃, and applying the Arrhenius equation, the linear relationship between the release rate constant (lgk) and the temperature (1/T) was established to obtain the equation: lgk = -8.073/T + 24.35 (R2 = 0.985 3), which showed that the release of microspheres at high temperature can be used to predict the release in vitro at 37 ℃, and 52.0 ± 0.5 ℃ was selected as the accelerated release condition in vitro. The quality research methods were established to investigate the changes of critical quality attributes such as microsphere morphology, drug loading, particle size and distribution, polymer molecular weight, and the related substances under accelerated conditions. The difference factor f1 and similarity factor f2 were used to assess the similarity of release behavior under accelerated conditions. The results showed that under the accelerated experimental conditions of 25 ± 2 ℃ and relative humidity (RH) 60% ± 5%, the critical quality attributes of injectable thienorphine loaded microspheres had no significant change in 6 months, suggesting that the long-term storage condition could be 5 ± 3 ℃.
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Objective:To develop a novel sustained-release local anesthetic microspheres and evaluate the effects on sciatic nerve block in rabbits.Methods:The magnetic lidocaine microspheres were prepared by W 1/O/W 2 compound emulsion method, investigating their external morphology, measuring the magnetic response characteristics by the VSM and draw the hysteresis loop.The encapsulation efficiency and drug-loading rate were calculated, and the cumulative release curves in vitro were drawn.Fifteen healthy rabbits (half male and half female), aged 5-6 months, weighing 3.0-3.5 kg, were selected for sciatic nerve block and divided into 3 groups ( n=5 each) using a random number table method: magnetic response lidocaine microspheres group (PL group), normal saline control group (C group) and lidocaine group (L group). Magnetic response lidocaine microsphere buffer 2 ml, normal saline 2 ml and 2% lidocaine 2 ml were injected around the rabbit sciatic nerve through a catheter in PL, C and L groups, respectively.The applied magnetic field was withdrawn at 60 h after injection.Before injection (T 0) and at 30 min and 2 , 8, 16, 24, 48, 60, 62 and 64 h after injection (T 1-9), the compound action potentials and conduction velocities of bilateral sciatic nerve trunks were measured, and block was assessed using toe reflex score and modified Tarlov score. Results:The magnetic lidocaine microspheres were brown in color and observed as monodisperse, regular spheres with a diameter of (9±3) μm, an encapsulation rate of 46.18%, a drug loading of 6.02%, and a superparamagnetic release rate of 97% in vitro at 60 h. The hysteresis loop passed through the origin and no hysteresis occurred with the absence of an external magnetic field.Compared with C group, the action potentials and conduction velocities of the sciatic nerve, toe reflex score and modified Tarlov score were significantly decreased at T 1-T 8 in PL group ( P<0.05). Compared with L group, the action potentials and conduction velocities of the sciatic nerve were significantly increased at T 1, the action potential was decreased at T 2-T 8, the conduction velocity was decreased at T 3-T 8, the toe reflex score was increased at T 1 and decreased at T 3-T 8, and the modified Tarlov score was increased at T 1 and T 2 and decreased at T 3-T 8 in PL group ( P<0.05). Conclusions:Magnetic response lidocaine microsphere is successfully developed with good magnetic responsiveness and release and can prolong the sciatic nerve block time in rabbits.
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BACKGROUND: Some recent studies have shown that the three-dimensional (3D) model of HepaRG cells can better mimic the in vivo microenvironment and show better liver differentiation and function compared with two-dimensional culture. OBJECTIVE: HepaRG was selected to prepare 3D collagen microspheres, and the adaptive culture and functional expression of cells in the collagen microspheres were evaluated. METHODS: Collagen hydrogel was used as the scaffold for 3D HepaRG and HepG2 microspheres. Stable cell spheres were formed. HepaRG microspheres, HepG2 microspheres, HepaRG two-dimensional culture and HepG2 two-dimensional culture were used as controls. At 1, 6, and 12 days of culture, cell survival was detected by the Live/Dead assay staining. After 1, 6, 12, and 16 days of culture, the urea synthesis and CYP3A4 secretion of the supernatant were detected in each group. After 12 days of culture, relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA was detected by qPCR. The expression levels of hepatocyte marker albumin and CYP3A4 protein were determined using western blot assay. RESULTS AND CONCLUSION: (1) In 12 days of culture, Live/Dead assay staining showed that the cell viability in the 3D collagen microsphere was well-maintained and the amount of central necrotic cells was small, with high cell viability. In the 3D collagen microsphere, especially HepaRG cells, multiple cellular clusters formed and adjacent clusters were connected closely, which created a cross-linked structure. (2) After 1, 6, and 12 days of culture, the urea content of HepaRG 3D collagen microspheres was higher than that of HepaRG two-dimensional culture (P < 0.05). After 1, 6, 12, and 16 days of culture, the urea content of HepG2 3D collagen microspheres was higher than that of HepG2 two-dimensional culture (P < 0.05). After 1, 6, and 12 days of culture, the secretion of CYP3A4 in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional culture (P < 0.05). After 6 and 12 days of culture, the secretion of CYP3A4 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (3) The relative expression of CYP3A4, CYP1A2, UGT1A1, and CPS1 mRNA in HepaRG 3D collagen microspheres was higher than that in HepaRG two-dimensional cells (P < 0.05), and the relative expression of CYP1A2 in HepG2 3D collagen microspheres was higher than that in HepG2 two-dimensional culture (P < 0.05). (4) The expression levels of albumin and CYP3A4 protein in HepaRG 3D collagen microspheres were higher than those of HepG2 3D collagen microspheres, ordinary microspheres, and two-dimensional culture (P < 0.05). (5) These results indicated the high-level expression of hepatocyte functions in 3D collagen HepaRG microsphere, which could be taken as a reference in drug metabolism evaluation in vitro and tissue engineering application.
ABSTRACT
BACKGROUND: The free enzyme has the problems of poor stability and inability to be reused during application. Using magnetic polymer microspheres as the carrier of the binding enzyme to prepare immobilized enzyme can maintain the natural activity of the enzyme, and can be reused, but also provide convenient conditions for automatic production management. OBJECTIVE: Magnetic chitosan microspheres as carrier of binding enzyme were used to prepare the immobilized lactase, which is easy to recycle, can be reused, and has high enzyme activity and stability. METHODS: By inputting a certain amount of magnetic chitosan microspheres into the phosphoric acid buffer for swelling for 2 hours, the swelling magnetic chitosan microspheres were collected with a magnet and added to a certain concentration of lactase phosphate buffer. They were shocked in a constant temperature shaker for 1 hour and preserved in refrigerator at 4 °C. The microspheres were precipitated with a magnet to pour out the supernatant. After full washing with buffer solution, the immobilized lactase was obtained. The properties of the magnetic microspheres (the amount of glutaraldehyde used in the preparation of microspheres was 2, 4, 6, 8, 10, 12, 14 mL), the amount of enzymes (0.5, 1, 1.5, 2 g/L) added, the pH (6.4, 6.8, 7.0, 7.2) of the buffer, and the immobilization time (1, 2, 5, 10, 15, 20 hours) were tested to determine the optimal immobilization conditions. RESULTS AND CONCLUSION: (1) The optimum conditions for immobilized lactose with magnetic chitosan microspheres were as follows: magnetic chitosan microspheres prepared with 10 mL of glutaraldehyde were selected as the immobilized carrier of lactase. The amount of enzyme added was 0.3 g/L, pH 7.0 and the immobilization time was 5 hours. (2) Compared with the free enzyme, immobilized lactase showed a wider range of reaction temperature and pH value. (3) The ability of immobilized enzyme binding substrate was enhanced. (4) After repeated use of the immobilized enzyme five times, the enzyme activity remained 65%. (5) The storage stability of lactase was also improved after immobilization.