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Basic fibroblast growth factor (bFGF) has shown great therapeutic effects for diabetic nephropathy (DN). However, its clinical applications are limited due to its short half-life, low stability and poor penetration. Herein, a bFGF-loaded liposome (bFGF-lip) was constructed and combined with ultrasound-targeted microbubble destruction (UTMD) to overcome these drawbacks. bFGF-lip exhibited spherical morphology with a diameter of 171.1 ± 14.2 nm and a negative zeta potential of -5.15 ± 2.08 mV, exhibiting a sustained-release profile of bFGF. DN rat models were successfully induced by streptozotocin. After treatment with bFGF-lip + UTMD, the concentration of bFGF in kidney of DN rats was significantly enhanced in comparison with free bFGF treatment. Additionally, the morphology and the function of the kidneys were obviously recovered after bFGF-lip + UTMD treatment as shown by ultrasonography and histological analyse. The molecular mechanism was associated with the inhibition of renal inflammation. After treatment with bFGF-lip + UTMD, the activation of NF-κB was obviously reduced in the renal tissues, and downstream inflammatory mediators including TGF-β1, MCP-1, IL-6 and IL-1β were also down regulated. In addition, inflammation-induced cellular apoptosis of renal tubular cells was also significantly inhibited by detecting Bax, caspase-3 and Bcl-2. Therefore, bFGF-lip in combination with UTMD might be a potential strategy to reverse the progression of early DN.

The present study seeks to observe the preventive effects of doxorubicin-induced cardiomyopathy (DOX-CM) in rats using targeted non-mitogenic acidic fibroblast growth factor (MaFGF) mediated by nanoparticles (NP) combined with ultrasound-targeted MB destruction (UTMD). DOX-CM rats were induced by intraperitoneally injected doxorubicin. Six weeks after intervention, the indices from the transthoracic echocardiography and velocity vector imaging showed that the left ventricular function in the MaFGF-loaded NP (MaFGF-NP) + UTMD group was significantly improved compared with the DOX-CM group. The increased malondialdehyde and decreased superoxide dismutase were observed in the DOX-CM group, while a significant increase in superoxide dismutase and a decrease in malondialdehyde were detected in the groups treated with MaFGF-NP + UTMD. From the Masson staining, the MaFGF-NP + UTMD group showed a significant difference from the DOX-CM group. The cardiac collagen volume fraction and the ratio of the perivascular collagen area to the luminal area number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling positive cells in the MaFGF-NP + UTMD group decreased to 8.9%, 0.55-fold, compared with the DOX-CM group (26.5%, 1.7-fold). From terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling staining, the results showed the strongest inhibition of apoptosis progress in MaFGF-NP + UTMD group. The immunohistochemical staining of the TGF-β1 in MaFGF-NP + UTMD group reached 3.6%, which was much lower than that of the DOX-CM group (12.6%). These results confirmed that the abnormalities, including left ventricular dysfunction, myocardial fibrosis, cardiomyocytes apoptosis and oxidative stress, could be suppressed by twice weekly MaFGF treatments for 6 consecutive weeks (free MaFGF or MaFGF-NP+/UTMD), with the strongest improvements observed in the MaFGF-NP + UTMD group. Western blot analyses of the heart tissue further revealed the highest pAkt levels, highest anti-apoptosis protein (Bcl-2) levels and strongest reduction in proapoptosis protein (Bax) levels in the MaFGF-NP + UTMD group. This study confirmed the preventive effects of DOX-CM in the rats with MaFGF-NP and UTMD by retarding myocardial fibrosis, inhibiting oxidative stress, and decreasing cardiomyocyte apoptosis., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Diabetic nephropathy (DN) is one of the most common and lethal microvascular complications of diabetes. This study aimed to explore whether coenzymeQ10 (CoQ10) as an antioxidant combined with ultrasound-targeted microbubble destruction (UTMD) could reverse the progress of early diabetic nephropathy (DN). CoQ10 has great potential to treat early DN. However, the clinical application of CoQ10 has been limited because of its low aqueous solubility and non-specific distribution. Therefore, CoQ10-loaded liposomes (CoQ10-lip) were prepared and combined with ultrasound microbubbles for the early theranostics of DN. CoQ10-lip exhibited a good round morphology with a diameter of 183±1.7nm and a negative zeta potential of -25.3mV, which was capable of prolonging the release of the encapsulated CoQ10. The early DN rat models were induced by streptozotocin (STZ) and confirmed by contrast-enhanced ultrasound (CEUS) and 24-h urinary albumin. After the administration of CoQ10-lip combined with the UTMD technique to rats with early DN, the morphology and function of the kidney were evaluated by ultrasonography, histological and molecular analyses. The renal hemodynamics were significantly improved, moreover, 24-h urinary protein, and oxidative stress indexes were modulated after treatment with CoQ10-lip+UTMD indicating recovery of renal function. An elevated level of Nphs2 protein and reduced caspase 3 level indicated the preservation of podocytes and inhibition of cell apoptosis after CoQ10-lip+UTMD treatment. The molecular mechanism was associated with the upregulation of Bcl-2 and the downregulation of Bax. Moreover, the combination of CoQ10-lip and ultrasound microbubbles demonstrated a better protective effect on the damaged kidney than the other groups (free CoQ10 or CoQ10-lip+/- UTMD). Conclusively, CoQ10-lip in combination with ultrasound microbubbles might be a potential strategy to reverse the progress of early DN., (Copyright © 2017. Published by Elsevier B.V.)

Acidic fibroblast growth factor (aFGF) has shown the great potential to prevent the structural and functional injuries caused by diabetic cardiomyopathy (DCM). The present study sought to investigate the preclinical performance and mechanism of the combination therapy of aFGF-nanoparticles (aFGF-NP) and ultrasound-targeted microbubble destruction (UTMD) technique for DCM prevention. From Mason staining and TUNEL staining, aFGF-NP+UTMD group showed significant differences from the diabetes group and other groups treated with aFGF or aFGF-NP. The cardiac collagen volume fraction (CVF) and cardiac myocyte apoptosis index in aFGF-NP+UTMD group reduced to 4.15% and 2.31% respectively, compared with those in the diabetes group (20.5% and 11.3% respectively). Myocardial microvascular density (MCD) in aFGF-NP+UTMD group was up to 35n/hpf, much higher than that in the diabetes group (14n/hpf). The diabetes group showed similar results (MCD, CVF and cardiac myocyte apoptosis index) to other aFGF treatment groups (free aFGF±UTMD or aFGF-NP). Indexes from transthoracic echocardiography and hemodynamic evaluation also proved the same conclusion. These results confirmed that the abnormalities including diastolic dysfunctions, myocardial fibrosis and metabolic could be suppressed by the different extents of twice weekly aFGF treatments for 12 consecutive weeks (free aFGF or aFGF-NP±UTMD), with the strongest improvements observed in the aFGF-NP+UTMD group. Western blot and immunohistochemical analyses of heart tissue samples further revealed the high efficiency of heart-targeted delivery and effective cardioprotection with this combination approach. Overall, this study has generated supportive data that are critical for the translation of a promising DCM prevention strategy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ultrasonic microbubbles were used to open blood-brain barriers (BBB) with a reversed and limited behavior feature in the study, which could improve the brain-targeted delivery of anti-tumor drugs. The glioma rat model was prepared. Low-frequency ultrasound was combined with microbubbles to affect the permeability of BBB compared with the permeability of independently administered Evans blue (EB) crossing BBB. Time point and length of ultrasound were investigated whether they affect the permeability of BBB and the damage of brain tissue. The effect of the growth time of glioma on BBB permeability was explored. Only glioma had a very little impact on BBB permeability. However, ultrasonic microbubbles opened the BBB with the features of temporary, limited and reversed behavior and improved EB and magnetic resonance imaging contrast agent penetrating BBB. A length of 30 s ultrasound is appropriate for opening BBB and no damage of brain tissue. Drugs should be injected before ultrasound so that they enter into brain as BBB opening. Ultrasonic microbubbles can open BBB effectively and safely, which improve drugs penetrating BBB under proper time point and length.

Diabetic cardiomyopathy (DCM) is the leading cause of morbidity and mortality among the diabetic patients and currently there is no effective means to reverse its pathological progress. Basic fibroblast growth factor (bFGF) has shown promise as a molecular therapy for DCM, but its delivery is inefficient and non-specific. In the present study, a therapy combining nanoparticle (NP) carrier and ultrasound-targeted microbubble destruction (UTMD) was reported the first time for bFGF delivery to the heart of diabetic rats. bFGF-loaded NP (bFGF-NP) were prepared with Poloxamer 188-grafted heparin copolymer using water-in-water technique, and the morphology, encapsulation efficiency, and bioactivity of bFGF-NP were studied. The cellular uptake and cytotoxicity of bFGF-NP were evaluated with primary cultures of the left ventricular (LV) cardiomyocytes in vitro. Therapeutic effects of bFGF-NP/UTMD on the heart of DCM rats were studied by measuring LV systolic and diastolic functions, hemodynamic characteristics and indicators of cardiac remodeling including myocardial collagen volume fraction and capillary density. Results demonstrated that bFGF-NP showed good round morphology, efficient bFGF encapsulation and stable bioactivity of bFGF in vitro. bFGF-NP/UTMD combined treatment significantly enhanced the efficiency of bFGF cellular uptake (P<0.05) without obvious cytotoxicity. Significant improvements (P<0.05) in both cardiac functions and tissue morphology in the DCM rats were observed in bFGF-NP/UTMD group. These were not achievable using free bFGF, bFGF-NP or UTMD treatment alone. Our results show that combining a non-viral vector with UTMD technique is an effective strategy to deliver bFGF to the heart, and the resulting growth factor therapy has demonstrated potential to reverse the progress of DCM by restoring the cardiac functions and even the structure of damaged cardiac tissues., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Characterization and antitumor activity of basic fibroblast growth factor-mediated active targeting doxorubicin microbubbles (bFGF-DOX-MB) were investigated. Pluronic F68 with chemical conjugation of doxorubicin (DOX-P) and peptide KRTGQYKLC-conjugated DSPE-PEG2000 were prepared. bFGF-DOX-MB had a normal distribution of particle size, with average particle size of 2.7 μm. Using A549 mouse model, bFGF-DOX-MB combined ultrasound showed the best inhibition effect on tumor volume growth among all the test groups. Similar conclusion was obtained from experimental measurements of tumor weight change and blood cell count. From the results, chemotherapeutic drug inhibition on tumor growth could be enhanced by local ultrasound combined with active targeting bFGF-DOX-MB, which might provide a potential application for ultrasound-mediated chemotherapy.

Ultrasound (US)-mediated cavitation of microbubbles has evolved into a new tool for organ-specific gene and drug delivery. This paper was to investigate the feasibility of acidic fibroblast growth factor (aFGF) intravenous delivery to the ischemic myocardium of rats by ultrasonic microbubbles modified with heparin. Heparin modified microbubbles (HMB) were prepared by the freeze-dried method. Acute myocardial infarction (AMI) model was established and the cardio protective effect of the aFGF combing with HMB (aFGF-HMB) under US-mediated cavitation technique was investigated. aFGF-HMB combined with US-mediated cavitation technique was examined by ECG. Ejection fraction (EF), fractional shortening (FS) and left ventricular diastolic diameter (LVDd) were measured to monitor the improvement of global myocardial contractile function. Myocardial tissue was stained with hematoxylin and eosine (HE) to evaluate the elaborate general morphology of the ischemic myocardium. From morphologic observation and echocardiography in rat heart, aFGF-HMB had suitable size distribution, physical stability and good acoustic resonance function. From AMI rat experiments, aFGF-HMB under US-mediated cavitation technique exerted aFGF cardio protective effect in ischemic myocardium. From histological evaluation, US-mediated cavitation of aFGF-HMB showed improvement of myocardial ischemia. With the visual imaging and US-triggered drug release advantages, US-mediated cavitation of aFGF-HMB might be developed as a novel technique for targeting delivery of aFGF into ischemic myocardium.

Background: Gas-filled phospholipid-based ultrasonic microbubbles (PUMs) are widely used in diagnostic imaging. The micro- or nanoparticle size and the physiochemical nature of shell provide the potential for a new way to improve pulmonary absorption for peptides and proteins., Methods: Male Sprague-Dawley rats were fasted for 12 h. Then insulin solution and insulin-PUM mixture solution were administered by intratracheal instillation. The hypoglycemic effect was observed to evaluate insulin absorption after lung administration. Fluorescein isothiocyanate-dextran (molecular mass, 4 kDa) was used as the index of evaluating drug alveolar deposition and absorption by visualization techniques., Results: Administration of insulin solution containing PUMs significantly reduced the blood glucose levels of Sprague-Dawley rats, compared with administration of insulin-only solution. The minimum reductions of the blood glucose concentration produced by insulin solution containing PUMs and by an insulin-only solution reached 60.81% and 34.60% of the initial glucose levels, respectively, and their bioavailabilities relative to subcutaneous injection were 48.58% and 29.09%, respectively. Histopathological study of the lung showed no changes in the morphology of the pulmonary alveoli after administration to these drugs. Only a slight inflammatory cell infiltration in the alveoli could be found in some rats., Conclusion: These results suggested that PUMs might be used as an effective way to improve pulmonary absorption for peptides and proteins.

Objective: An in vivo study on enhancing antitumor effect of intravenous epirubicin hydrochloride (EPI) by acoustic cavitation in situ combined with phospholipid-based microbubbles (PMB) was reported., Methods: Five-week-old male nude mice were used, and HCT-116 cells were s.c. (subcutaneous injection) inoculated in axilla of these mice. Five groups were designed and five consecutive treatments were applied to investigate the tumor growth, body weight growth, and normal organ growth., Results: Inhibition effects on tumor growth were observed in all groups treated by EPI. However, the potency of tumor growth retardation caused by EPI depended on the application of PMB and US in situ. An effective retardation on tumor growth and improved mice survival status were observed when intravenous EPI combined with sonicated PMB in situ. Short-term lingering sensitive period for chemotherapeutic drugs after acoustic cavitation was also observed in experiment under asynchronous administration of EPI and sonicated PMB., Conclusion: Intravenous EPI combined with PMB-mediated cavitation in situ might provide a potential application for US-mediated chemotherapy.

Background: The present study investigated the enhancement efficiency between liposomes and microbubbles for insulin pulmonary absorption., Methods: Two types of phospholipid-based vesicle-liposomes and microbubbles-were prepared, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cytotoxicity test was used to evaluate their in vitro toxicity in A549 cells. Cellular uptake of insulin combined with liposomes or microbubbles was determined using A549 cells. With intratracheal insufflation of Sprague-Dawley rats, an insulin mixture with liposomes or microbubbles was administered to assess its potential for promoting drug pulmonary absorption., Results: Both liposomes and microbubbles had a narrow and monodispersed size distribution with average diameter of 3.1 μm and 1.0 μm, respectively. From the MTT cytotoxicity test, a phospholipid-based vesicle concentration of <25% (vol/vol) in the final volume was the safe dosage range that could avoid severe cytotoxic effects. The intracellular uptake amount of insulin in the insulin-microbubble mixture was significantly higher than that in the insulin-liposome mixture. The minimum reductions of the blood glucose concentration produced by insulin-microbubble and insulin-liposome mixtures were 60.8% and 35.0% of the initial glucose levels, respectively, and their bioavailabilities relative to subcutaneous injection were 48.6% and 30.8%, respectively., Conclusions: Microbubbles have much better efficiency than liposomes in the rate and extent of insulin pulmonary absorption. Microbubbles might be recommended as a potential agent for enhancing protein intrapulmonary absorption.

The objective of this study was to investigate the factors for enhancing the susceptibility of cancer cells to chemotherapeutic drug by ultrasound microbubbles. Ultrasound (US) combined with phospholipid-based microbubbles (MB) was used to enhance the susceptibility of colon cancer cell line SWD-620 to anticancer drugs Topotecan hydrochloride (TOP). Experiments were designed to investigate the influence of main factors on cell viability and cell inhibition, such as US intensity, MB concentration, drug combination with MB, asynchronous action between US triggered cavitation and drug entering cell, MB particle size. US exposure for 10 sec with US probe power at 0.6 W/cm(2) had satisfied cell viability. Treated with US combined with 15% MB, cell viability maintained more than 85% and cell inhibition 86.16%. Under optimal US combined with MB, TOP showed much higher cell inhibition than that of only TOP group. Cell inhibition under short delayed time (<2 h) for TOP addition did not show obvious difference. In terms of MB particle size, the order of cell inhibition was: Mixture > Micron bubble part > Nanometer bubble part. US combined with MB can enhance the susceptibility of cancer cells to chemotherapeutic drug, which may provide a potential method for US-mediated tumor chemotherapy.

This work was to compare the encapsulation efficiency and ultrasound-triggered release for protein between microbubbles and liposomes. Bovine serum albumin (BSA) was used as a model. Final ratios between BSA and HPC in microbubbles and liposomes were 1:5, 1:7 and 1:10, respectively. Morphologic characteristics and contrast enhancement of loaded microbubbles and liposomes were measured. Encapsulation efficiency and ultrasound-stimulated release profile were detected. The mean size of loaded microbubbles and liposomes was 3.4 microm and 1.7 microm, respectively. Encapsulation efficiency of microbubbles had an inverse relationship with the ratio between BSA and HPC, while loaded liposomes remained nearly unchanged in the designed range of the ratio between BSA and HPC. Microbubbles resulted in significant enhancement of CnTi images. After ultrasound, more than 90% of the entrapped BSA was released from microbubbles, but less than 5% of BSA released from liposomes. Microbubbles are a promising delivery system for proteins.

Sonogenic microbubble agent is a newly developed drug targeting delivery system, which uses ultrasonic beam to enhance the delivery of drug and gene to targeted cells and tissues. In this paper, the preparation of sonogenic phospholipids-based microbubbles was optimized by using central composite experimental design (CCD) and response surface methodology (RSM). Hydrogenated egg phosphatidylcholine (EPC), Tween 80 and polyethylene glycol 1500 (PEG 1500) were important components affecting the concentration of 2 - 8 microm microbubbles in the preparation. The combined effects of these three factors were analyzed by CCD and optimized by RSM. Evaluation variable was the concentration of 2 - 8 microm microbubbles. Overall desirability was fitted to a second-order polynomial equation, through which three dimensional response surface graphs were produced. Optimal experimental conditions were selected from the stationary point of the response surfaces. The stability of the sonogenic phospholipids-based microbubbles by the optimal formulation was investigated by accelerated experiment. The contrast effect in vivo of the optimal formulation was investigated. Foreign market product SonoVue was used as the control. From the results, all the three factors had positive effects on the concentration of 2 - 8 microm microbubbles. The optimal condition in the preparation of phospholipids-based microbubbles was obtained as following: EPC 8.35 mg, Tween 80 21.68 mg and PEG 1500 201 mg. The mean value of the concentration of 2 - 8 microm microbubbles in rechecking experiment reached 8.60 x 10(9) x mL(-1). From the accelerated experiment, phospholipids-based microbubbles showed good physical stability. The intensity (relative unit) and duration of the contrast effect by the optimal formulation were 4.47 +/- 0.15 and (302 +/- 7) s respectively, which showed little difference with foreign market product SonoVue [4.28 +/- 0.13, (309 +/- 8) s]. The optimal formulation selected by CCD and RSM showed high microbubble concentration, good physical stability and effective sonogenic contrast effect.

The aim of this study was to investigate the application of ultrasonic gas-filled liposomes in enhancing transfer for breast cancer-related antisense oligonucleotides in vitro. An antisense oligodeoxynucleotide (AS-ODN) sequence, HA2741, modified with luciferase reporter plasmid, was used in evaluating the enhancing effect of gas-filled liposomes for gene transfer in breast cancer cells. Some important factors on HA 2741 transfection efficiency, such as wave intensity, ultrasound duration, gas-filled liposome concentration, and HA2741 concentration, were tested, respectively. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by propidium iodide assay. Reverse-transcriptase polymerase chain reaction and immunocytochemistry were used to detect the inhibitory effect of HA2741 on HER-2 expression. All the four factors (wave intensity, ultrasound duration, gas-filled liposome concentration, and HA2741 concentration) showed a positive effect on AS-ODN transfection efficiency. However, these factors had a negative effect on cell viability. Considering all the factors investigated, the maximum transfection efficiency with minimum cell viability achieved under 2% gas-filled liposome mixed with 80 nmol/L HA2741 for 30-second ultrasound exposure at -3.0 dB wave intensity, which gave an overall transfection efficiency exceeding 90% and a cell viability near 90%. Under controlled conditions, ultrasound-mediated AS-ODN transfer, enhanced by gas-filled liposomes, may represent an effective, safe avenue for cancer-related gene delivery.

To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide (AS-ODNs) by insonated gas-filled lipid microbubbles, SF6-filled microbubbles were prepared by sonication-lyophilization method. An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles. Three levels of mixing speed, different durations of mixing and various delay time before ultrasound were examined, separately. Transfection efficiency was detected by fluorescence microscopy. Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared. From the results, there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells. AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration, but there is a negative relationship with delay time before ultrasound. The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r x min(-1) for 30-60 s with less than 60 s delay before ultrasound. For a successful transfection, long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids, because these vectors depend on endocytosis and membrane fusion to realize transfection. Unlike liposomes and cationic lipid-polymer hybrids, gas-filled lipid microbubbles depend on sonorporation effect to realize transfection. Therefore, the incubation of gene and microbubbles before mixing cells may not be necessary. Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.

Ultrasonic beam can be focused on a particular tissue. As ultrasound contrast agents for medical ultrasound imaging, gas-filled microbubbles can also be used as drugs or gene carriers. Therefore, ultrasonic microbubbles become a topic of intense interest in drug delivery because they can be used as gene targeting delivery systems. Based on the relevant materials, the applications of ultrasonic microbubbles as gene delivery systems in various tissues in vivo were reviewed, such as cardiac, vascular, skeletal muscle and tumor. Their potential problems in future use were also discussed.

Aim: To compare sonoporation effect of two phospholipids-based vectors-liposomes and microbubbles on cultured cell membrane., Methods: A breast cancer cell line SK-BR-3 was exposed to ultrasound alone, 2% or 5% liposome + ultrasound and 2% or 5% microbubble + ultrasound, separately. Immediately after the experiment and 24 h after ultrasound exposure, atomic-force microscopy (AFM) scanning was used to observe the membrane change of SK-BR-3 cells., Results: After ultrasound exposure, normal SK-BR-3 cells more or less lost their natural shape, showing elliptic outline with obtuse curved boundary. In groups added with phospholipids-based microbubbles, more obtuse curved boundary of cells was observed. The membrane pores of SK-BR-3 cells had apparent changes after ultrasound exposure. With AFM technique, membrane pores under ultrasound alone or ultrasound with liposomes conditions were enlarged, the diameter of some pores exceeding 1 microm. But all the membrane pores in these conditions returned to normal appearance after 24 hours. In ultrasound with 2% microbubble condition, most membrane pores were about 1 - 3 microm in size and returned to normal appearance after 24 h. In ultrasound with 5% microbubble condition, however, pores of most cell membrane porosity was about 2 - 4 pm and did not totally return to normal appearance after 24 h., Conclusion: At 2% concentration, phospholipids-based microbubble could enhance ultrasonic sonoporation effect and produce reparable membrane pores on SK-BR-3 cells, which appeared to be a promising vehicle for drug and gene delivery.

Aim: To investigate the feasibility of transfer antisense oligodeoxynucleotides (AS-ODNs) by the phospholipids-based gas-filled microbubbles (PGM) under ultrasound activation., Methods: An antisense oligodeoxynucleotides sequence ZL combined with luciferase reporter plasmid was used. A breast cancer cell line SK-BR-3 was exposed to different conditions to investigate the effects of such factors as ZL concentration, PGM concentration, mechanical index (MI) and ultrasound exposure duration on transfection efficiency and cell viability. The transfection efficiency and cell viability by other lipid vectors such as lipofectamine and liposome were also tested, whose results were comparied with that of PGM. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by PI (propidium iodide) assay., Results: Among the factors tested, ultrasound exposure duration, MI and PGM concentration had obvious impacts on transfection efficiency and cell viability. The results showed that the optimal ultrasound condition was the exposure to ultrasound at MI 1.0 for 30 s with 2% PGM concentration, which gave an overall transfection efficiency of 78% +/- 10%, increased nearly 18 folds over the transfection by PGM (4.0%) or lipofectamine (4.3%) without ultrasound. Under same ultrasound conditions, different vectors showed significant difference in transfection efficiency while there are similar results in cell viability., Conclusion: Under proper ultrasound conditions, PGM can markedly enhance AS-ODNs transfection efficiency.

The objective of this work was to prepare echogenic phospholipid-based gas-filled microbubbles (PGM) and investigate their physical characteristics, echogenicity and loading ability of hirudin under various NaCl concentrations. PGM were prepared by a sonication-lyophilization method. Hirudin was used as a model drug to evaluate the drug encapsulation efficiency of the PGM. PGM loaded with hirudin were prepared by dissolving lyophilized powder with hirudin solution. The morphology, particle size and microbubble concentration of PGM were measured. The hirudin encapsulation efficiency as a function of NaCl concentration was determined. The mean particle size and microbubble concentration of PGM were unchanged by the presence of hirudin for at least 60 min after preparation. Hirudin encapsulation quantity was proportional to the hirudin concentration until saturation occurred at high concentration, and the encapsulation efficiency had an inverse relationship. Hirudin encapsulation efficiency was affected by NaCl concentration. When NaCl concentration was increased from 10 mg mL(-1) to 20 mg mL(-1) in PGM solution, hirudin encapsulation efficiency decreased from 35.8 to 26.7%, and microbubble concentration decreased from 2.7 x 10(8) to 1.7 x 10(8) microbubbles per mL. The PGM were shown easily to be visible in in vivo rabbit liver. There was no difference in echogenicity between the loaded and unloaded bubbles. PGM prepared by the sonication-lyophilization method exhibited satisfactory physical characteristics and loading ability and are suitable for use in imaging and ultrasound-triggered delivery.

Experiments in vitro and in vivo were designed to investigate tumor growth inhibition of chemotherapeutics-loaded liposomes enhanced by acoustic cavitation. Doxorubicin-loaded liposomes (DOX liposomes) were used in experiments to investigate acoustic cavitation mediated effects on cell viability and chemotherapeutic function. The influence of lingering sensitive period after acoustic cavitation on tumor inhibition was also investigated. Animal experiment was carried out to verify the practicability of this technique in vivo. From experiment results, blank phospholipid-based microbubbles (PBM) combined with ultrasound (US) at intensity below 0.3 W/cm2 could produce acoustic cavitation which maintained cell viability at high level. Compared with DOX solution, DOX liposomes combined with acoustic cavitation exerted effective tumor inhibition in vitro and in vivo. The lingering sensitive period after acoustic cavitation could also enhance the susceptibility of tumor to chemotherapeutic drugs. DOX liposomes could also exert certain tumor inhibition under preliminary acoustic cavitation. Acoustic cavitation could enhance the absorption efficiency of DOX liposomes, which could be used to reduce DOX adverse effect on normal organs in clinical chemotherapy. [ABSTRACT FROM AUTHOR]

Background: Ultrasonic microbubbles are used as ultrasound-triggered delivery carriers for protein drugs. Aim: This work was to prepare stabilized protein-loaded phospholipid-based ultrasonic microbubbles (PUM) and to determine its value as a protein delivery system. Method: Bovine serum albumin (BSA) was used as a model protein drug. BSA-containing PUM were prepared by dissolving lyophilized PUM powder in BSA solution. The particle size and microbubble concentration of BSA-containing PUM were measured. The BSA encapsulation efficiency as a function of BSA concentration was determined. Contrast enhancement of BSA-containing PUM in vivo was detected. The release profile of BSA from PUM was also investigated. Results: The mean particle size and microbubble concentration of PUM were unchanged by the presence of BSA for at least 30 minutes after preparation. The net amount of BSA entrapped in PUM was maintained unchanged with increasing BSA concentration. BSA-containing PUM were shown easily to be visible in in vivo rabbit kidney. There was no difference in echogenicity between the loaded and unloaded PUM. Ultrasound duration had a positive relationship with BSA release. Ultrasound of 30 seconds stimulated 94.1% and 93.3% of BSA release from PUM solutions containing 0.3% and 1.5% BSA, respectively. Conclusions: Protein-loaded PUM exhibited satisfactory physical characteristics and were potent for using in ultrasound-triggered delivery. [ABSTRACT FROM AUTHOR]

Objective: The objective of this work was to examine the application of catechins-containing phospholipids-based ultrasonic microbubbles (PUM) in ultrasound-induced delivery. Methods: Catechins-containing PUM were prepared by dissolving lyophilized PUM powder in catechins solution. Morphologic characteristics of catechins-loaded microbubbles were measured compared with blank microbubbles. The contrast enhancement in vivo of loaded microbubbles was measured by contrast-tuned imaging (CnTi) technique. Encapsulation efficiency and ultrasound-stimulated release profile in vitro were detected. Results: Incorporation of catechins in PUM had little effect on the mean particle size and microbubble concentration. Catechins encapsulation efficiency maintained relatively stable with increasing catechins concentration. However, the net amount of catechins encapsulated per milliliter of PUM solution showed a positive relationship with increasing catechins concentration. Effective enhancement in ultrasonic images was observed under CnTi technique combined with catechins-containing PUM. There was no significant difference in the contrast intensity and the longevity of CnTi enhancement between the images of the loaded and unloaded PUM. From ultrasound-stimulated release profile in vitro, 30 s ultrasound stimulated more than 90% of catechins released from loaded PUM. Conclusions: PUM exhibited satisfactory morphologic characteristics, loading ability, and acoustical activity, which could be recommended as a potential carrier for ultrasound-induced delivery. [ABSTRACT FROM AUTHOR]

Objective: To investigate phospholipids-based microbubbles induced sonoporation and cell membrane reseal in vitro under various conditions. Methods: A breast cancer cell line SK-BR-3 was used to investigate ultrasonic sonoporation under various conditions. Atomic force microscopy (AFM) scanning techniques were employed to observe the change of membrane pores. Results: Normal SK-BR-3 cells membrane pores were evenly distributed and less than 1 μm. After ultrasound exposure, membrane pores were enlarged at different degree depending on ultrasound exposure durations, filling gas species and microbubble suspension concentration. With microbubble suspension concentration being increased to 5% or ultrasound exposure reached 30 s, membrane pores in fluorocarbon (C3F8 or SF6)-filled microbubble groups exceeded 1 μm, which were significantly larger than that of air-filled microbubble group. Membrane pores were about 2-3 μm under ultrasound 60 s with 5% fluorocarbon-filled microbubble suspension. After 24 h of incubation, most of the enlarged membrane pores could reseal to normal size, which corresponded to cell viability. Conclusions: Membrane pores can be obviously enlarged by ultrasonic sonoporation of fluorocarbon-filled microbubbles, whose reseal time depended on ultrasound exposure duration and microbubble suspension concentration. [ABSTRACT FROM AUTHOR]

Objective: To compare transfection efficiency and safety for antisense oligodeoxynucleotides (AS-ODNs) between two type of phospholipids-based vectors.Methods: An AS-ODNs sequence HA824 combined with luciferase reporter plasmid was used. Under low intensity ultrasound (US), a breast cancer cell line SK-BR-3 was exposed to different concentration of microbubbles and liposomes. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by propidium iodide assay. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the inhibitory effect of HA824 on HER-2 expression at mRNA level. Atomic force microscopy (AFM) scanning techniques was employed to observe the change of membrane pore size.Results: AS-ODNs transfection efficiency showed an increasing tend with microbubble concentration, but not with liposome concentration. Maximum transfection efficiency with minimum cell viability was achieved under 2% microbubble concentration. Too strong sonoporation activity would enlarge membrane pores significantly and cause low cell viability.Conclusion: US-mediated AS-ODNs transfection enhanced by phospholipids-based microbubbles represents an effective and safe avenue. [ABSTRACT FROM AUTHOR]