Controlled intracellular delivery of biomolecular cargo is critical for developing targeted therapeutics and cell reprogramming.Conventional delivery approaches(e.g.,endocytosis of nano-vectors,microinjection,and elec...Controlled intracellular delivery of biomolecular cargo is critical for developing targeted therapeutics and cell reprogramming.Conventional delivery approaches(e.g.,endocytosis of nano-vectors,microinjection,and electroporation)usually require time-consuming uptake processes,labor-intensive operations,and/or costly specialized equipment.Here,we present an acoustofluidics-based intracellular delivery approach capable of effectively delivering various functional nanomaterials to multiple cell types(e.g.,adherent and suspension cancer cells).By tuning the standing acoustic waves in a glass capillary,our approach can push cells in flow to the capillary wall and enhance membrane permeability by increasing membrane stress to deform cells via acoustic radiation forces.Moreover,by coating the capillary with cargo-encapsulated nanoparticles,our approach can achieve controllable cell-nanoparticle contact and facilitate nanomaterial delivery beyond Brownian movement.Based on these mechanisms,we have successfully delivered nanoparticles loaded with small molecules or protein-based cargo to U937 and HeLa cells.Our results demonstrate enhanced delivery efficiency compared to attempts made without the use of acoustofluidics.Moreover,compared to conventional sonoporation methods,our approach does not require special contrast agents with microbubbles.This acoustofluidics-based approach creates exciting opportunities to achieve controllable intracellular delivery of various biomolecular cargoes to diverse cell types for potential therapeutic applications and biophysical studies.展开更多
Ultrasonic imaging is becoming the most popular medical imaging modality,owing to the low price per examination and its safety.However,blood is a poor scatterer of ultrasound waves at clinical diagnostic transmit freq...Ultrasonic imaging is becoming the most popular medical imaging modality,owing to the low price per examination and its safety.However,blood is a poor scatterer of ultrasound waves at clinical diagnostic transmit frequencies.For perfusion imaging,markers have been designed to enhance the contrast in B-mode imaging.These so-called ultrasound contrast agents consist of microscopically small gas bubbles encapsulated in biodegradable shells.In this review,the physical principles of ultrasound contrast agent microbubble behavior and their adjustment for drug delivery including sonoporation are described.Furthermore,an outline of clinical imaging applications of contrast-enhanced ultrasound is given.It is a challenging task to quantify and predict which bubble phenomenon occurs under which acoustic condition,and how these phenomena may be utilized in ultrasonic imaging.Aided by high-speed photography,our improved understanding of encapsulated microbubble behavior will lead to more sophisticated detection and delivery techniques.More sophisticated methods use quantitative approaches to measure the amount and the time course of bolus or reperfusion curves,and have shown great promise in revealing effective tumor responses to anti-angiogenic drugs in humans before tumor shrinkage occurs.These are beginning to be accepted into clinical practice.In the long term,targeted microbubbles for molecular imaging and eventually for directed anti-tumor therapy are expected to be tested.展开更多
Objective： To explore the safety of ultrasound and microbubbles for enhancing the chemotherapeutic sensitivity of malignant tumors in the digestive system in a clinical trial, as well as its efficacy.Methods： From O...Objective： To explore the safety of ultrasound and microbubbles for enhancing the chemotherapeutic sensitivity of malignant tumors in the digestive system in a clinical trial, as well as its efficacy.Methods： From October 2014 to June 2016, twelve patients volunteered to participate in this study. Eleven patients had hepatic metastases from tumors of the digestive system, and one patient had pancreatic carcinoma. According to the mechanical index （MI） in the ultrasound field, patients were classified into four groups with MIs of 0.4, 0.6, 0.8 and 1.0. Within half an hour after chemotherapy, patients underwent ultrasound scanning with ultrasound microbubbles （SonoVue） to enhance the efficacy of chemotherapy. All adverse reactions were recorded and were classified in 4 grades according to the Common Terminology Criteria for Adverse Events version 4.03 （CTCAE V4.03）. Tumor responses were evaluated by the Response Evaluation Criteria in Solid Tumors version 1.1 criteria. All the patients were followed up until progression.Results： All the adverse reactions recorded were level 1 or level 2. No local pain occurred in any of the patients. Among all the adverse reactions, fever might be related to the treatment with ultrasound combined with microbubbles. Six patients had stable disease （SD）, and one patient had a partial response （PR） after the first cycle of treatment. At the end of follow-up, tumor progression was restricted to the original sites, and no new lesions had appeared.Conclusions： Our preliminary data showed the potential role of a combined treatment with ultrasound and microbubbles in enhancing the chemotherapeutic sensitivity of malignant tumors of the digestive system. This technique is safe when the MI is no greater than 1.0.展开更多
Sonoporation mediated by microbubbles is being extensively studied as a promising technology to facilitate gene/drug delivery to cells. However, the theoretical study regarding the mechanisms involved in sonoporation ...Sonoporation mediated by microbubbles is being extensively studied as a promising technology to facilitate gene/drug delivery to cells. However, the theoretical study regarding the mechanisms involved in sonoporation is still in its infancy.Microstreaming generated by pulsating microbubble near the cell membrane is regarded as one of the most important mechanisms in the sonoporation process. Here, based on an encapsulated microbubble dynamic model with considering nonlinear rheological effects of both shell elasticity and viscosity, the microstreaming velocity field and shear stress generated by an oscillating microbubble near the cell membrane are theoretically simulated. Some factors that might affect the behaviors of microstreaming are thoroughly investigated, including the distance between the bubble center and cell membrane（d）, shell elasticity（χ）, and shell viscosity（κ）. The results show that（i） the presence of cell membrane can result in asymmetric microstreaming velocity field, while the constrained effect of the membrane wall decays with increasing the bubble-cell distance;（ii） the bubble resonance frequency increases with the increase in d and χ, and the decrease in κ,although it is more dominated by the variation of shell elasticity; and（iii） the maximal microstreaming shear stress on the cell membrane increases rapidly with reducing the d, χ, and κ. The results suggest that microbubbles with softer and less viscous shell materials might be preferred to achieve more efficient sonoporation outcomes, and it is better to have bubbles located in the immediate vicinity of the cell membrane.展开更多
A model of an ultrasound-driven encapsulated microbubble（EMB） oscillation near biomaterial wall is presented and used for describing the microflow-induced shear stress on the wall by means of a numerical method. The...A model of an ultrasound-driven encapsulated microbubble（EMB） oscillation near biomaterial wall is presented and used for describing the microflow-induced shear stress on the wall by means of a numerical method. The characteristic of the model lies in the explicit treatment of different types of wall for the EMB responses. The simulation results show that the radius-time change trends obtained by our model are consistent with the existing models and experimental results. In addition, the effect of the elastic wall on the acoustic EMB response is stronger than that of the rigid wall, and the shear stress on the elastic wall is larger than that of the rigid wall. The closer the EMB to the wall, the greater the shear stress on the wall. The substantial shear stress on the wall surface occurs inside a circular zone with a radius about two-thirds of the bubble radius. This paper may be of interest in the study of potential damage mechanisms to the microvessel for drug and gene delivery due to sonoporation.展开更多
Objective： To investigate the feasibility of ultrasound （US） mediated enhanced green fluorescent protein （EGFP） gene delivery in subcutaneous transplanted tumors of human cervical carcinoma （He/a） and the contr...Objective： To investigate the feasibility of ultrasound （US） mediated enhanced green fluorescent protein （EGFP） gene delivery in subcutaneous transplanted tumors of human cervical carcinoma （He/a） and the contribution of lipid shell microbubble （LSMB） on gene transfection. Methods： LSMB and plasmid were injected into nude mice by tail vein followed local US irradiation （P ＋ LSMB ＋ US group）. US exposure parameter was set at 2.0 W/cm2, 2 rain, duty cycle 20%. EGFP expression was evaluated by imaging for 7 days. Nude mice undergoing plasmid injection alone （P group）, plasmid injection and US exposure （P ＋ US group）, plasmid and LSMB injection （P ＋ LSMB group） were used as controls. Frozen section and histological examinations were conducted. Expression of EGFP was scored. Kinetics of protein expression post transfection and localization in vivo were evaluated. Results： Plasmid injection with LSMB plus US exposure strongly increased gene transfer efficiency. Strong EGFP expression was mainly seen in LSMB ＋ P ＋ US group. It was significantly higher than any of the following groups, P group, US ＋ P group, or LSMB ＋ P group （P 〈 0.01）./n vivo expression level of post-US 3 days was significantly higher than any other time points （P 〈 0.01）. There was not significant expression level of EGFP in other organs or tissues regardless of US exposure. No tissue damage was seen histologically. Conclusion： The combination of LSMB and US exposure could effectively transfer plasmid DNA to transplanted tumors without causing any apparently adverse effect. LSMB could be effective as a non-viral vector system in in vivo gene delivery. It would be a safe gene delivery method and provide an alternative to current clinical gene therapy.展开更多
Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthes...Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix(ECM) and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications.However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fi brosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process.Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble–mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.展开更多
Lipid-coated microbubbles are widely used as an ultrasound contrast agent,as well as drug delivery carriers.However,the two main limitations in ultrasound diagnosis and drug delivery using microbubbles are the short h...Lipid-coated microbubbles are widely used as an ultrasound contrast agent,as well as drug delivery carriers.However,the two main limitations in ultrasound diagnosis and drug delivery using microbubbles are the short half-life in the blood system,and the difficulty of surface modification of microbubbles for active targeting.The exosome,a type of extracellular vesicle,has a preferentially targeting ability for its original cell.In this study,exosome-fused microbubbles(Exo-MBs)were developed by embedding the exosome membrane proteins into microbubbles.As a result,the stability of Exo-MBs is improved over the conventional microbubbles.On the same principle that under the exposure of ultrasound,microbubbles are cavitated and self-assembled into nano-sized particles,and Exo-MBs are self-assembled into exosome membrane proteins-embedded nanoparticles(Exo-NPs).The Exo-NPs showed favorable targeting properties to their original cells.A photosensitizer,chlorin e6,was loaded into Exo-MBs to evaluate therapeutic efficacy as a drug carrier.Much higher therapeutic efficacy of photodynamic therapy was confirmed,followed by cancer immunotherapy from immunogenic cell death.We have therefore developed a novel ultrasound image-guided drug delivery platform that overcomes the shortcomings of the conventional ultrasound contrast agent and is capable of simultaneous photodynamic therapy and cancer immunotherapy.展开更多
基金the support from the National Institutes of Health(R01GM141055)the National Science Foundation(CMMI2104295)+7 种基金the China Scholarship Councilthe NIH/NCATS UCLA CTSI(UL1TR001881)through the UC Center for Accelerated Innovationsupported by the National Science Foundation Graduate Research Fellowship(1644868)support from the UCLA Innovation Fund MedTech Innovator Awardthe Challenge Initiative at UCLAseed funding provided through a UCLA David Geffen School of Medicine Regenerative Medicine Theme Awardthe support provided by the NIH Common Fund through an NIH Director's Early Independence Award co-funded by the National Institute of Dental and Craniofacial Research and Office of the Director,NIH under award number(DP50D028181)the NIH for a predoctoral fellowship supported by the National Heart,Lung,and Blood Institute of the National Institutes of Health under Award Number(F31HL149356)。
文摘Controlled intracellular delivery of biomolecular cargo is critical for developing targeted therapeutics and cell reprogramming.Conventional delivery approaches(e.g.,endocytosis of nano-vectors,microinjection,and electroporation)usually require time-consuming uptake processes,labor-intensive operations,and/or costly specialized equipment.Here,we present an acoustofluidics-based intracellular delivery approach capable of effectively delivering various functional nanomaterials to multiple cell types(e.g.,adherent and suspension cancer cells).By tuning the standing acoustic waves in a glass capillary,our approach can push cells in flow to the capillary wall and enhance membrane permeability by increasing membrane stress to deform cells via acoustic radiation forces.Moreover,by coating the capillary with cargo-encapsulated nanoparticles,our approach can achieve controllable cell-nanoparticle contact and facilitate nanomaterial delivery beyond Brownian movement.Based on these mechanisms,we have successfully delivered nanoparticles loaded with small molecules or protein-based cargo to U937 and HeLa cells.Our results demonstrate enhanced delivery efficiency compared to attempts made without the use of acoustofluidics.Moreover,compared to conventional sonoporation methods,our approach does not require special contrast agents with microbubbles.This acoustofluidics-based approach creates exciting opportunities to achieve controllable intracellular delivery of various biomolecular cargoes to diverse cell types for potential therapeutic applications and biophysical studies.
文摘Ultrasonic imaging is becoming the most popular medical imaging modality,owing to the low price per examination and its safety.However,blood is a poor scatterer of ultrasound waves at clinical diagnostic transmit frequencies.For perfusion imaging,markers have been designed to enhance the contrast in B-mode imaging.These so-called ultrasound contrast agents consist of microscopically small gas bubbles encapsulated in biodegradable shells.In this review,the physical principles of ultrasound contrast agent microbubble behavior and their adjustment for drug delivery including sonoporation are described.Furthermore,an outline of clinical imaging applications of contrast-enhanced ultrasound is given.It is a challenging task to quantify and predict which bubble phenomenon occurs under which acoustic condition,and how these phenomena may be utilized in ultrasonic imaging.Aided by high-speed photography,our improved understanding of encapsulated microbubble behavior will lead to more sophisticated detection and delivery techniques.More sophisticated methods use quantitative approaches to measure the amount and the time course of bolus or reperfusion curves,and have shown great promise in revealing effective tumor responses to anti-angiogenic drugs in humans before tumor shrinkage occurs.These are beginning to be accepted into clinical practice.In the long term,targeted microbubbles for molecular imaging and eventually for directed anti-tumor therapy are expected to be tested.
基金sponsored by National Key Research and Development Plan(No.2017YFC0107300 and No.2017YFC0107303)
文摘Objective： To explore the safety of ultrasound and microbubbles for enhancing the chemotherapeutic sensitivity of malignant tumors in the digestive system in a clinical trial, as well as its efficacy.Methods： From October 2014 to June 2016, twelve patients volunteered to participate in this study. Eleven patients had hepatic metastases from tumors of the digestive system, and one patient had pancreatic carcinoma. According to the mechanical index （MI） in the ultrasound field, patients were classified into four groups with MIs of 0.4, 0.6, 0.8 and 1.0. Within half an hour after chemotherapy, patients underwent ultrasound scanning with ultrasound microbubbles （SonoVue） to enhance the efficacy of chemotherapy. All adverse reactions were recorded and were classified in 4 grades according to the Common Terminology Criteria for Adverse Events version 4.03 （CTCAE V4.03）. Tumor responses were evaluated by the Response Evaluation Criteria in Solid Tumors version 1.1 criteria. All the patients were followed up until progression.Results： All the adverse reactions recorded were level 1 or level 2. No local pain occurred in any of the patients. Among all the adverse reactions, fever might be related to the treatment with ultrasound combined with microbubbles. Six patients had stable disease （SD）, and one patient had a partial response （PR） after the first cycle of treatment. At the end of follow-up, tumor progression was restricted to the original sites, and no new lesions had appeared.Conclusions： Our preliminary data showed the potential role of a combined treatment with ultrasound and microbubbles in enhancing the chemotherapeutic sensitivity of malignant tumors of the digestive system. This technique is safe when the MI is no greater than 1.0.
基金Projects supported by the National Basic Research Program,China(Grant No.2011CB707900)the National Natural Science Foundation of China(Grant Nos.81127901,81227004,81271589,11374155,11161120324,11074123,11174141,11274170,11104140,11474001,and 11474161)+1 种基金the National High Tech Research and Development Program,China(Grant No.2012AA022702)the Program for New Century Excellent Talents in University of Ministry of Education of China(Grant No.NCET-11-0236)
文摘Sonoporation mediated by microbubbles is being extensively studied as a promising technology to facilitate gene/drug delivery to cells. However, the theoretical study regarding the mechanisms involved in sonoporation is still in its infancy.Microstreaming generated by pulsating microbubble near the cell membrane is regarded as one of the most important mechanisms in the sonoporation process. Here, based on an encapsulated microbubble dynamic model with considering nonlinear rheological effects of both shell elasticity and viscosity, the microstreaming velocity field and shear stress generated by an oscillating microbubble near the cell membrane are theoretically simulated. Some factors that might affect the behaviors of microstreaming are thoroughly investigated, including the distance between the bubble center and cell membrane（d）, shell elasticity（χ）, and shell viscosity（κ）. The results show that（i） the presence of cell membrane can result in asymmetric microstreaming velocity field, while the constrained effect of the membrane wall decays with increasing the bubble-cell distance;（ii） the bubble resonance frequency increases with the increase in d and χ, and the decrease in κ,although it is more dominated by the variation of shell elasticity; and（iii） the maximal microstreaming shear stress on the cell membrane increases rapidly with reducing the d, χ, and κ. The results suggest that microbubbles with softer and less viscous shell materials might be preferred to achieve more efficient sonoporation outcomes, and it is better to have bubbles located in the immediate vicinity of the cell membrane.
基金Projects supported by the National Natural Science Foundation of China(Grant Nos.11174077 and 11474090)the Natural Science Foundation of Hunan Province,China(Grant No.13JJ3076)+1 种基金the Science Research Program of Education Department of Hunan Province,China(Grant No.14A127)the Doctoral Fund of University of South China(Grant No.2011XQD46)
文摘A model of an ultrasound-driven encapsulated microbubble（EMB） oscillation near biomaterial wall is presented and used for describing the microflow-induced shear stress on the wall by means of a numerical method. The characteristic of the model lies in the explicit treatment of different types of wall for the EMB responses. The simulation results show that the radius-time change trends obtained by our model are consistent with the existing models and experimental results. In addition, the effect of the elastic wall on the acoustic EMB response is stronger than that of the rigid wall, and the shear stress on the elastic wall is larger than that of the rigid wall. The closer the EMB to the wall, the greater the shear stress on the wall. The substantial shear stress on the wall surface occurs inside a circular zone with a radius about two-thirds of the bubble radius. This paper may be of interest in the study of potential damage mechanisms to the microvessel for drug and gene delivery due to sonoporation.
基金a grant from the National Natural Sciences Foundation of China (No. 30670548).
文摘Objective： To investigate the feasibility of ultrasound （US） mediated enhanced green fluorescent protein （EGFP） gene delivery in subcutaneous transplanted tumors of human cervical carcinoma （He/a） and the contribution of lipid shell microbubble （LSMB） on gene transfection. Methods： LSMB and plasmid were injected into nude mice by tail vein followed local US irradiation （P ＋ LSMB ＋ US group）. US exposure parameter was set at 2.0 W/cm2, 2 rain, duty cycle 20%. EGFP expression was evaluated by imaging for 7 days. Nude mice undergoing plasmid injection alone （P group）, plasmid injection and US exposure （P ＋ US group）, plasmid and LSMB injection （P ＋ LSMB group） were used as controls. Frozen section and histological examinations were conducted. Expression of EGFP was scored. Kinetics of protein expression post transfection and localization in vivo were evaluated. Results： Plasmid injection with LSMB plus US exposure strongly increased gene transfer efficiency. Strong EGFP expression was mainly seen in LSMB ＋ P ＋ US group. It was significantly higher than any of the following groups, P group, US ＋ P group, or LSMB ＋ P group （P 〈 0.01）./n vivo expression level of post-US 3 days was significantly higher than any other time points （P 〈 0.01）. There was not significant expression level of EGFP in other organs or tissues regardless of US exposure. No tissue damage was seen histologically. Conclusion： The combination of LSMB and US exposure could effectively transfer plasmid DNA to transplanted tumors without causing any apparently adverse effect. LSMB could be effective as a non-viral vector system in in vivo gene delivery. It would be a safe gene delivery method and provide an alternative to current clinical gene therapy.
文摘Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix(ECM) and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications.However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fi brosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process.Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble–mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.
基金supported by the Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education,Science,and Technology(NRF-2016R1A6A1A03012845,NRF-2022M3E5F1017553),Republic of Koreathe Ministry of Food and Drug Safety grant(22213MFDS421),Republic of Korea.
文摘Lipid-coated microbubbles are widely used as an ultrasound contrast agent,as well as drug delivery carriers.However,the two main limitations in ultrasound diagnosis and drug delivery using microbubbles are the short half-life in the blood system,and the difficulty of surface modification of microbubbles for active targeting.The exosome,a type of extracellular vesicle,has a preferentially targeting ability for its original cell.In this study,exosome-fused microbubbles(Exo-MBs)were developed by embedding the exosome membrane proteins into microbubbles.As a result,the stability of Exo-MBs is improved over the conventional microbubbles.On the same principle that under the exposure of ultrasound,microbubbles are cavitated and self-assembled into nano-sized particles,and Exo-MBs are self-assembled into exosome membrane proteins-embedded nanoparticles(Exo-NPs).The Exo-NPs showed favorable targeting properties to their original cells.A photosensitizer,chlorin e6,was loaded into Exo-MBs to evaluate therapeutic efficacy as a drug carrier.Much higher therapeutic efficacy of photodynamic therapy was confirmed,followed by cancer immunotherapy from immunogenic cell death.We have therefore developed a novel ultrasound image-guided drug delivery platform that overcomes the shortcomings of the conventional ultrasound contrast agent and is capable of simultaneous photodynamic therapy and cancer immunotherapy.
