The Combination of icariin and constrained dynamic loading stimulation attenuates bone loss in ovariectomy-induced osteoporotic mice.

Osteoporosis is a disease characterized by low bone mass and progressive destruction of bone microstructure, resulting in increasing the risk of fracture. Icariin (ICA) as a phytoestrogen shows osteogenic effects, and the mechanical stimulation has been demonstrated the improving effect on osteoporosis. The objective of this study was to investigate the effect of ICA in combination with constrained dynamic loading (CDL) stimulation on osteoporosis in ovariectomized (OVX) mice. The serum hormone levels, bone turnover markers, trabecular architecture, ulnar biomechanical properties, and the expression of osteoblast-related gene (alkaline phosphatase, ALP; osteocalcin, OCN; bone morphogenetic protein-2, BMP-2; Collagen I (α1), COL1; osteoprotegerin, OPG) and osteoclast-related genes (receptor activators of NF-κB ligand, RANKL; tartrate-resistant acid phosphatase, TRAP) were analyzed. The results showed that ICA + CDL treatment could increase the osteocalcin (20.85%), estradiol levels (20.61%) and decrease the TRAP activity (26.27%) significantly than CDL treatment. The combined treatment attenuated bone loss and biomechanical decrease more than single use of CDL treatment. ICA + CDL treatment significantly up-regulated the level of osteoblast-related gene expression and down-regulated the osteoclast-related genes expression; moreover, the combined treatment increased the ratio of OPG/RANKL significantly compared to ICA (72.83%) or CDL (65.63%) treatment alone. The present study demonstrates that icariin in combination with constrained dynamic loading treatment may have a therapeutic advantage over constrained dynamic loading treatment alone for the treatment of osteoporosis, which would provide new evidence for the clinical treatment of osteoporosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1415-1424, 2018.

Effects of a novel photoactivated photosensitizer on MDR1 over-expressing human breast cancer cells.

Multidrug resistance (MDR) was the main reason of cancer chemotherapy failure. Photodynamic therapy (PDT) has been applied to the treatment of tumor and considered as a strategy for the overcoming of MDR phenomenon. Present study focused on a novel porphyrin-based photosensitizer DTP (meso-5-[p-diethylene triamine pentaacetic acid-aminophenyl]-10,15,20-triphenyl-porphyrin)-mediated photocytotoxicity on MDR1 highly expressing human breast cancer cell line MCF-7/ADR (adriamycin resistant) and the parental MCF-7 cell line. Experimental results indicated that DTP-PDT induced significant photocytotoxicity on MDR1 highly expressing MCF-7/ADR cell line, in spite of slightly weaker than on MCF-7 cell line, which was due to the relatively lower level of intracellular DTP in resistant MCF-7/ADR cells. Furthermore, intracellular DTP level in resistant MCF-7/ADR cells could not be altered with a Pgp inhibitor, verapamil and this indicated that DTP was not a possible substrate for the multidrug transporter Pgp. More importantly, photoactivated DTP could significantly reduce the expression of MDR1 gene at all the levels of mRNA, protein and function. The combined treatment with DTP-PDT and adriamycin was found to be more effective than adriamycin or DTP-PDT alone. In conclusion, our data demonstrated that DTP probably will be a potential photosensitizer in combating MDR phenomenon during the treatment of human breast cancer.

Photodynamic antimicrobial chemotherapy with the novel amino acid-porphyrin conjugate 4I: In vitro and in vivo studies.

Photodynamic antimicrobial chemotherapy (PACT), as a novel and effective therapeutic modality to eradicate drug resistant bacteria without provoking multidrug resistance, has attracted increasing attention. This study examined the antimicrobial efficacy of the novel cationic amino acid-porphyrin conjugate 4I with four lysine groups against two different clinical isolated strains (drug sensitive and multidrug resistant) of the Acinetobacter baumannii species and its toxicity on murine dermal fibroblasts in vitro, as well as the therapeutic effect of PACT on acute, potentially lethal multidrug resistant strain excisional wound infections in vivo. The PACT protocol exposed 4I to illumination, exhibiting high antimicrobial efficacy on two different strains due to a high yield of reactive oxygen species (ROS) and non-selectivity to microorganisms. The photoinactivation effects of 4I against two different strains were dose-dependent. At 3.9 µM and 7.8 µM, PACT induced 6 log units of inactivation of sensitive and multidrug resistant strains. In contrast, 4I alone and illumination alone treatments had no visibly antimicrobial effect. Moreover, cytotoxicity tests revealed the great safety of the photosensitizer 4I in mice. In the in vivo study, we found 4I-mediated PACT was not only able to kill bacteria but also accelerated wound recovery. Compared with non-treated mice, over 2.89 log reduction of multidrug resistant Acinetobacter baumannii strain was reached in PACT treat mice at 24 h post-treatment. These results imply that 4I-mediated PACT therapy is an effective and safe alternative to conventional antibiotic therapy and has clinical potential for superficial drug-resistant bacterial infections.

Photodynamic therapy with a novel porphyrin-based photosensitizer against human gastric cancer.

The objective of the present study was to evaluate the effects of novel porphyrin-based photosensitizer meso-5-[ρ-diethylene triamine pentaacetic acid- aminophenyl]-10,15,20-triphenyl-porphyrin (DTP)-mediated photodynamic therapy (PDT) on the HGC27 and SNU-1 human gastric cancer cell lines. The absorption spectrum of DTP was analyzed using a microplate spectrophotometer. The HGC27 or SNU-1 cells were incubated with DTP and exposed to illumination by a 650-nm laser. The experiments were divided into four groups: A blank control, cells treated with DTP without light, cells exposed to laser light without DTP and cells treated with a combination of DTP and light together. The phototoxicity of DTP was analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Cell apoptosis was detected by flow cytometry and Hoechst 33342 staining. In addition, the intracellular distribution of DTP was investigated by laser scanning confocal microscopy. DTP-PDT demonstrated marked phototoxicity towards HGC27- and SNU-1 cells. The rate of cell death increased significantly in a DTP concentration-dependent and light dose-dependent manner, with maximum mortality rates of 74.14 and 67.76%, respectively. There were significant differences between the therapeutic and control groups (P<0.01). In addition, the growth of cells treated with DTP or laser light alone was not inhibited. Further evaluation revealed that, following DTP-PDT, HGC27 and SNU-1 cells demonstrated notable apoptotic changes, including condensed chromatin, fragmented nuclei and apoptotic bodies, and the percentage of apoptotic cells was significantly higher than that of the control groups (P<0.01). Furthermore, confocal laser scanning microscopy revealed that DTP localized to the lysosomes but not mitochondria in the two types of tumor cell. In conclusion, significant phototoxicity and reduced cytotoxicity in dark conditions make the novel photosensitizer DTP a promising potential PDT drug for future use in the treatment of human gastric cancer.

In vitro and in vivo antitumor activity of a novel porphyrin-based photosensitizer for photodynamic therapy.

PURPOSE: Photodynamic therapy (PDT) is a promising treatment in cancer therapy, based on the use of a photosensitizer activated by visible light in the presence of oxygen. Nowadays significant research efforts have been focused on finding a new photosensitizer. In the present paper, the antitumor effects of a novel porphyrin-based photosensitizer, {Carboxymethyl-[2-(carboxymethyl-{[4-(10,15,20-triphenylporphyrin-5-yl)-phenylcarbamoyl]-methyl}-amino)-ethyl]-amino}-acetic acid (ATPP-EDTA) on two types of human malignant tumor cells in vitro and a gastric cancer model in nude mice, were evaluated. METHODS: The PDT efficacy with ATPP-EDTA in vitro was assessed by MTT assay. The intracellular accumulation was detected with fluorescence spectrometer, and the intracellular distribution was determined by laser scanning confocal microscopy. The mode of cell death was investigated by Hoechst 33342 staining and flow cytometer. BGC823-derived xenograft tumor model was established to explore the in vivo antitumor effects of ATPP-EDTA. RESULTS: ATPP-EDTA exhibited intense phototoxicity on both cell lines in vitro in concentration- and light dose-dependent manners meanwhile imposing minimal dark cytotoxicity. The accumulation of ATPP-EDTA in two malignant cell lines was time-dependent and prior compared to normal cells. It was mainly localized at lysosomes, but induced cell death by apoptotic pathway. ATPP-EDTA significantly inhibited the growth of BGC823 tumors in nude mice (160 mW/cm(2), 100 J/cm(2)). CONCLUSIONS: Present studies suggest that ATPP-EDTA is an effective photosensitizer for PDT to tumors. It distributed in lysosomes and caused cell apoptosis. ATPP-EDTA, as a novel photosensitizer, has a great potential for human gastric cancer treatment in PDT and deserves further investigations.

Glycosyl-modified diporphyrins for in vitro and in vivo fluorescence imaging.

The application of probes for optical imaging is becoming popular as they have high safety and good biocompatibility. We prepared two kinds of glycosyl-modified diporphyrins, and their potentials as fluorescent probes were tested for the first time. After preparation of the glycosyl-modified porphyrin monomers, Ag-promoted coupling of the monomers was used to obtain glucose-modified porphyrin dimer (GPD) and lactose-modified porphyrin dimer (LPD). The strong interaction between the two porphyrin rings achieves red-shifted emission, and thus circumvents autofluorescence and light-scattering in biological samples. Although the glycosylation improves solubility, it also yielded selective attachment to cell membranes, and to chorions of early developmental-stage zebrafish. Patch-clamp experiments revealed the biocompatibility and low toxicity of GPD and LPD. Moreover, an in vivo imaging experiment provided direct evidence that zebrafish chorion contains sugar-binding proteins. The modification and derivatization make porphyrins potential bioimaging probes for specific optical imaging.

[A new photodynamic therapy drug toward gastric cancer MGC803 cell].

OBJECTIVE: To investigate the treatment efficiency of a new photodynamic therapeutic(PDT) drug synthesized by our laboratory toward MGC803 cells and related mechanisms. METHODS: Bleaching method was used to evaluate the photostability of drug upon repetitive illumination. MTT assay was used to determine the ability of new drug killing MGC803 cells after PDT. Laser scanning confocal microscopy (LSCM) was applied to investigate the subcellular localization of drug in MGC803 cells (mitochondria and/or lysosomes). Hoechst staining and flow cytometry(Annexin V/PI double-staining) were performed to detect the death mode of MGC803 cells after PDT. RESULTS: This new PDT drug had good stability to light irradiation after repetitive illumination. MTT assay showed no cytotoxicity towards MGC803 cells only by drug or only by irradiation(P>0.05), but intense lethal effect was observed with drug and light combination(P<0.05). The phototoxicity of medicine increased with the elevation of concentration, the LD50 was 1.74 µmol/L, and reaching plateau at the concentration of 3.12 µmol/L, even increasing the concentration. LSCM found that drug localized in lysosomes of MGC803 cells. Hoechst staining showed that the death mode of cells was mainly necrosis and Annexin V/PI double-staining proved this result further. CONCLUSION: This new PDT drug is an effective PDT sensitizer for MGC803 cells and the death mode of cells is mainly necrosis.

Transmyocardial drilling revascularization combined with heparinized bFGF-incorporating stent activates resident cardiac stem cells via SDF-1/CXCR4 axis.

OBJECTIVE: To investigate whether transmyocardial drilling revascularization combined with heparinized basic fibroblast growth factor (bFGF)-incorporating degradable stent implantation (TMDRSI) can promote myocardial regeneration after acute myocardial infarction (AMI). METHODS: A model of AMI was generated by ligating the mid-third of left anterior descending artery (LAD) of miniswine. After 6 h, the animals were divided into none-treatment (control) group (n=6) and TMDRSI group (n=6). For TMDRSI group, two channels with 3.5 mm in diameter were established by a self-made drill in the AMI region, into which a stent was implanted. Expression of stromal cell-derived factor-1(α) (SDF-1(α)) and CXC chemokine receptor 4 (CXCR4), cardiac stem cell (CSC)-mediated myocardial regeneration, myocardial apoptosis, myocardial viability, and cardiac function were assessed at various time-points. RESULTS: Six weeks after the operation, CSCs were found to have differentiated into cardiomyocytes to repair the infarcted myocardium, and all above indices showed much improvement in the TMDRSI group compared with the control group (P<0.001). CONCLUSIONS: The new method has shown to be capable of promoting CSCs proliferation and differentiation into cardiomyocytes through activating the SDF-1/CXCR4 axis, while inhibiting myocardial apoptosis, thereby enhancing myocardial regeneration following AMI and improving cardiac function. This may provide a new strategy for myocardial regeneration following AMI.

Intracerebroventricular transplantation of human amniotic epithelial cells ameliorates spatial memory deficit in the doubly transgenic mice coexpressing APPswe and PS1ΔE9-deleted genes.

BACKGROUND: Human amniotic epithelial cells (HAECs), which have characteristics of both embryonic and pluripotent stem cells, are therefore a candidate in cell therapy without creating legal or ethical problems. In the present study, we aimed to investigate the effects of intracerebroventricular transplantation of HAECs on doubly transgenic mice of Alzheimer's disease (AD) coexpressing presenilin-1 (PS1) and mutant Sweden amyloid precursor protein (APPswe) genes. METHODS: The offspring mice genotypes were detected using PCR identification of APPswe and PS1 gene. The doubly transgenic (TG) mice (n = 20) and wild-type (WT) mice (n = 20) were randomly divided into two groups respectively: the transplantation group treated with HAECs and the control group with phosphate buffered saline. Six radial arm water maze test was used to assess the spatial memory in the TG and WT mice. Amyloid plaques and neurofibrillary tangles were analyzed using congo red and acid-silver methenamine staining respectively. Immunofluorescence cytochemistry was used to track the survival of HAECs. Immunohistochemistry was used to determine the expression of octamer-binding protein 4 (Oct-4) and Nanog in the HAECs. High performance liquid chromatography was used to measure acetylcholine in hippocampus. The density of cholinergic neurons in basal forebrain and nerve fibers in hippocampus was measured using acetylcholinesterase staining. RESULTS: Amyloid deposition occurred in hippocampus and frontal cortex in the double TG mice aged 8 months, but not in WT mice. The results also showed that transplanted HAECs can survive for at least 8 weeks and migrate to the third ventricle without immune rejection. The graft HAECs can also express the specific marker Oct-4 and Nanog of stem cell. Compared with the control group, transplantation of HAECs can not only significantly improve the spatial memory of the TG mice, but also increase acetylcholine concentration and the number of hippocampal cholinergic neurites. CONCLUSIONS: These results demonstrate that intracerebroventricular transplantation of HAECs can improve the spatial memory of the double TG mice. The higher content of acetylcholine in hippocampus released by more survived cholinergic neurites is one of the causes of this improvement.

Mechanisms of the protective effects of BMSCs promoted by TMDR with heparinized bFGF-incorporated stent in pig model of acute myocardial ischemia.

This study investigates the mechanism by which transmyocardial drilling revascularization combined with heparinized basic fibroblast growth factor incorporated degradable stent implantation (TMDRSI) enhanced effects of bone marrow mesenchymal stem cells (BMSCs) transplantation against acute ischemic myocardial injury. After the mid-third of left anterior descending artery was ligated, miniswine were divided into none-treatment group (control, n = 6), BMSCs implantation group (C, n = 6), TMDRSI group (TS, n = 6) and TMDRSI and BMSCs implantation group (TSC, n = 6). Two channels of 3.5 mm diameter were established by a self-made drill in the ischemic region, into which a stent was implanted for the TS and TSC groups. Autologous BMSCs were transplanted into the ischemic region in C group or around the channels in TSC group. Expression of von Willebrand factor, vascular endothelial growth factor, interleukin-1ß, transforming growth factor-ß(3) , cell proliferation and apoptosis, histological and morphological analyses, myocardial remodelling and cardiac function were evaluated at different time-points. Six weeks after the operation, the above indices were significantly improved in TSC group compared with others (P < 0.05), though C and TS groups also showed better results than the control group (P < 0.05). The new method was shown to have activated paracrine pathway of transplanted BMSCs, increased survival and differentiation of such cells, and enhanced effects of BMSCs transplantation on myocardial remodelling, which may provide a new strategy for cell therapies against acute ischemic myocardial injury.

Mid-term effect of stem cells combined with transmyocardial degradable stent on swine model of acute myocardial infarction.

BACKGROUND: We aimed to confirm the mid-term results of the new method combined with bone marrow-derived mesenchymal stem cells (MSCs) transplantation and transmyocardial drilling revascularization (TMDR) with degradable stent incorporated with basic fibroblast growth factor and heparin. METHODS: The miniswine underwent acute myocardial infarction by ligation of the left anterior descending coronary artery. Transmyocardial channels with 3.5 mm diameter (TMDR) were made by mechanical drilling in the infarction territory and basic fibroblast growth factor stents were implanted into the channels. Animals were randomly divided into the following four groups (n=6 in each): control; II: MSCs implantation; III: TMDR+stent implantation; IV: TMDR+stent implantation+MSCs implantation. Three months postoperatively, ECG-gated single photon emission computed tomography, histopathological examination, and reverse transcription-polymerase chain reaction were carried out. RESULTS: Left ventricular ejection fraction and myocardial perfusion were significantly improved in group IV than that in other groups (P<0.05). Compared with other groups, vessel density was augmented and cell apoptosis was reduced in group IV (P<0.01). Reverse transcription-polymerase chain reaction results showed that the expression levels of von Willebrand factor, transforming growth factor-beta3, vascular endothelial growth factor, and interleukin-1beta were much higher in group IV than that in other groups (P<0.05). CONCLUSION: Three months after operation, MSCs transplantation combined with TMDR and degradable stent significantly improved cardiac function, enhanced neovascular density, reduced infarcted size, improved ventricular remodeling, and reduced cardiac myocyte apoptosis, and therefore provides strong information for clinical trial.

Heparin- and basic fibroblast growth factor-incorporated stent: a new promising method for myocardial revascularization.

BACKGROUND: The problem for transmyocardial revascularization is the occlusion of transmural channels. We have developed a novel heparinized basic fibroblast growth factor (bFGF)-incorporating tubular stent by polymer materials of poly D, L-lactic/glycolic acid (PLGA) and polycaprolactone (PCL) with preferable biocompatibility and mechanical elasticity. This study investigated the effect of this new stent on transmyocardial perfusion. METHODS: Miniswine were grouped into bare stent group (BS), heparinized stent group (HS), and bFGF- and heparin-incorporated stent group (HBS). Two stents were implanted into the ischemic region. At 6 wk postoperatively, von Willebrand Factor (vWF) and Ki-67 antigen were immunohistologically stained. The expression of transforming growth factor-beta3 (TGF-ß3), vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1ß), and vWF mRNA from the cardiac tissue around the stent were performed by reverse transcription polymerase chain reaction (RT-PCR). Left ventricular function and myocardial perfusion by echocardiography and nuclear scanning were also documented. RESULTS: Ki-67 positively-staining proliferating cells, expressions of proangiogenic factors of TGF-ß3, VEGF, vWF, IL-1ß, and vascular density in HBS group were significantly different from those in HS and BS groups (P < 0.05). Neovascularization with endothelization, improvement in perfusion, and fractional shortening in BHS and HS groups were significantly better than those in BS group (P < 0.001). CONCLUSIONS: Heparinized bFGF-incorporating stent developed in the present study may keep the myocardial channel open with full luminal endothelization. By inducing cellular migration and expression of angiogenic factors, this novel stent may further enhance neovascular formation, increase myocardial perfusion, and improve cardiac function.

A new transmyocardial degradable stent combined with growth factor, heparin, and stem cells in acute myocardial infarction.

AIMS: We developed a new method-transmyocardial drilling revascularization (TMDR) with absorbable stent incorporated with basic fibroblast growth factor (bFGF) and heparin. The present study tested the effect of this method with transplantation of bone marrow-derived stem cells (BMSCs) in acute myocardial infarction. METHODS AND RESULTS: Infarction was produced in mini-swine by ligating the left anterior descending (LAD) coronary artery. TMDR of 3.0 mm in diameter was made by mechanical drilling in the infarcted area. The animals that had LAD ligation were divided into six groups according to the procedures followed (n = 6 in each): control; T (TMDR); C (cell implantation); TS (TMDR+stent implantation); TC (TMDR+cell implantation); TSC (TMDR+stent implantation+cell implantation). Left ventricular (LV) function, myocardial perfusion, vascular density, and histological and morphological analyses were evaluated pre-operatively and at 30 min and 6 weeks post-operatively. Six weeks after operation, the above indices were significantly better in the TSC group than in other groups (P < 0.001 compared with the control group, and P < 0.05 or 0.01 compared with the TS and TC groups), although TS and TC also showed better results than the control group (P < 0.05). CONCLUSION: We have demonstrated in a pig model that an intramyocardial stent implanted with slow release of bFGF, heparin, and BMSC transplantation may significantly increase LV function, cardiac blood flow, and vascular density. Therefore, the present study may provide a new method for the surgical treatment of myocardial infarction.

Degradable PLGA scaffolds with basic fibroblast growth factor: experimental studies in myocardial revascularization.

Our goal was to investigate the efficacy of degradable poly(D,L-lactic-coglycolic acid) (PLGA) scaffolds loaded with basic fibroblast growth factor (bFGF) in inducing cardiac neovascularization, increasing perfusion, and improving cardiac function.For ease of scaffold implantation into the ventricular wall, we developed a channel-producing device. Mini-swine, established as the animal model, were grouped as follows: channels-alone (control) group, channels and blank scaffolds (CBS) group, and channels and bFGF-incorporating scaffolds (CFS) group. Two scaffolds were implanted in each animal in the CBS and CFS groups. Six weeks postoperatively, endothelial cells were immunohistologically stained for von Willebrand factor, and proliferating cells for Ki-67 antigen. The density of new vessels was counted by image-analysis software. Left ventricular function and myocardial perfusion were documented by echocardiography and nuclear scanning, respectively, before implantation and 6 weeks postoperatively.The combined application of PLGA and bFGF ensured sustained release of growth factor in the target region. In the CFS group, Ki-67-positively stained cells, vascular density, and perfusion-defect percentage all showed significant improvement (P < 0.001), compared with the control and CBS groups, which did not. Moreover, the left ventricular fractional shortening percentage in the CFS group (28.98% +/- 1.24%) showed a significant increase, compared with the control group (26.57% +/- 1.92%, P = 0.009) and the CBS group (27.11% +/- 0.71%, P = 0.033), neither of which showed a difference (P = 0.508).The bFGF-incorporating PLGA scaffold can promote neovascular formation, enhance blood-flow perfusion, and improve myocardial function, although the original scaffold lumina were eventually occluded by inflammatory cells and scar tissue.

[Preparation of a biodegradable drug-eluting stent in myocardium channel].

OBJECTIVE: To prepare a biodegradable drug-eluting stent in myocardium channel and evaluate its effect on myocardium channel after transmyocardial revascularization (TMR). METHODS: A biodegradable drug-eluting stent was prepared using poly (epsilon-caprolactone) (PCL), bovine serum albumin (BSA), and poly (D, L-lactide-co-glycolide) (PLGA) as material of stent, model protein drug, and drug carrier respectively. The amount of BSA in stent and in vitro released BSA of stent were determined by the Coomassie brilliant blue assay. The mechanical strength of stent was tested by universal material testing machines. The material and structure of stent was characterized by nuclear magnetic resonance spectroscopy. The effect of stent on myocardium channel after TMR was evaluated in vivo by a standard animal model of chronic myocardial ischemia in miniswine. RESULTS: The stent could carry 13.1 microg BSA per mg of stent and the stent could release about 95% of BSA after 30 days. The stent diminished 80% of initial scale under the stress of 1.7 Mpa. It also kept the myocardium channel patency after TMR. CONCLUSIONS: A biodegradable drug-eluting stent in myocardium channel was successfully prepared. It can sustain the pressure from the heart and achieve the controlled release of drug. The stent can ensure the myocardium channel patency after TMR.

[Preparation and evaluation of microbubble ultrasound contrast agent with N-carboxymethyl chitosan].

OBJECTIVE: To prepare microbubble, made of N-carboxymethyl chitosan, as ultrasound contrast agent and evaluate its characteristics and acoustic effects in vivo. METHODS: Oil-Water-Oil multiple emulsion/solvent evaporation method was used to prepare the microbubble contrast agent. Both optical micrography and scanning electron micrography were performed to determine the bubble size and morphology. The acoustic effect of the N-carboxymethyl chitosan echo contrast agent was evaluated in vivo in rabbit. Liver echo images were recorded with ultrasound machine before and after intravenous bolus injecting 0.5 ml of the agent. RESULTS: The novel N-carboxymethyl chitosan echo contrast agent was formulated as lyophilized product, with a mean diameter of 2-3 microm and a shell thickness of 250-300 nm. Its size is relatively uniform. The imaging effect was remarkably enhanced with the ultrasonic contrast agent when applied in rabbit livers. CONCLUSION: It is feasible to prepare excellent microbubble ultrasound contrast agent with N-carboxymethyl chitosan as membrane components.