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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.

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.

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.

The objective of this study was to investigate the possibility of increasing the entrapment of protein-loaded liposomes with a modified freeze-thaw technique. Blank liposomes were prepared by ethanol injection method. Then recombinant human growth hormone (rhGH) was added to blank liposome suspension. rhGH encapsulation efficiency was enhanced by modified freeze-thaw technique. We separated each step and studied the effect of each parameter on encapsulation: incubation temperature (water bath from 0 to 15 degrees C), incubation duration (from 5 to 90 min), number of freeze-thaw (from 0 to 4 cycle), and the ratio between rhGH and phospholipids (from 1:5 to 1:10). The effect of cryoprotectants on the encapsulation and particle size distribution was finally examined. rhGH encapsulation efficiency was determined by Bradford's dye-binding assay. Morphology and size distribution of rhGH liposomes were also observed. The optimum parameters for rhGH encapsulation were incubation temperature of 5 degrees C, incubation duration of 40 min, and three to four cycles of freeze-thaw. The ratio between rhGH and phospholipids did not affect the encapsulation percentage. Trehalose exhibited the highest integrated value among the cryoprotectants investigated. From the results, this study demonstrates the suitability of the modified freeze-thaw technique for obtaining rhGH liposomes with high entrapment.

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.

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 microm. 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 (C(3)F(8) or SF(6))-filled microbubble groups exceeded 1 microm, which were significantly larger than that of air-filled microbubble group. Membrane pores were about 2-3 microm 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.

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]

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]

Abstract: This work investigated the influence of some factors on the property in vitro of a self-made lipid ultrasound (US) contrast agent (LCA) and evaluated the relationship of acoustic pressure and enhancement effect in normal rabbit kidney parenchyma. In the in vitro studies, filling gas, solvent and concentration of LCA solution were investigated. Morphologic characteristics, concentration and mean diameter of microbubbles were considered as indices. In the in vivo studies, contrast-tuned imaging (CnTi) technique was used to investigate the enhancement effects in kidney parenchyma under nine acoustic pressure levels. Among the samples saturated with different filling gases, perfluoropropane (C3F8) resulted in the highest concentration of microbubbles and air, the lowest. Microbubbles filled with C3F8 or sulfur hexafluoride (SF6) were quite stable and remained at a high level of concentration (above 2 × 109 microbubbles per mL) much longer than did air-filled microbubbles. Among the four solutions tested, 5% glucose solution and 0.9% saline solution showed higher initial concentrations and greater longevity than dextran 40 glucose solution (6%) or distilled water. The concentration of LCA solution had a positive correlation with the microbubble concentration. All microbubble samples under different test conditions remained shape-complete and no aggregation or fusion was observed. The mean diameter of microbubble samples was about 3.4 μm. Contrast intensity and longevity of CnTi enhancement in vivo showed an acoustic-pressure-dependent decrease. At 1 kPa, contrast intensity increased 224-fold (4.47/0.02) and the longevity of CnTi enhancement in the kidney parenchyma remained longer. (E-mail: lctuua@yahoo.com.cn) [Copyright &y& Elsevier]