Phospholipids-based microbubbles sonoporation pore size and reseal of cell membrane cultured in vitro

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.     

声振脂质微泡作为反义寡核苷酸传递系统的研究

目的研究声振脂质微泡(PGM)作为反义寡核苷酸传递系统的可行性。方法应用PGM将结合荧光蛋白的反义核苷酸片段ZL转染到乳腺癌细胞SK-BR-3中，考察不同因素如：ZL浓度、PGM浓度、超声持续时间和机械指数(MI)等对ZL转染率及细胞存活率的影响。考察PGM的结果，并与其他脂质载体如lipofectamine和脂质体的转染效果比较。应用荧光显微镜观察转染率，碘化丙锭(PI)染色观察细胞存活率。结果考察因素中，PGM浓度，超声持续时间和MI对ZL转染率和细胞存活率影响较大。最佳超声条件为：PGM浓度为2%，超声持续时间为30 s和MI为1.0，在此条件下转染率为78%±10%，比未超声的PGM(4.0%)和lipofectamine(4.3%)的转染率增加了约18倍，而细胞存活率相近。结论在适宜条件下，声振脂质微泡可以有效促进体外反义核苷酸的转染。     

Phospholipids-based microbubbles sonoporation pore size and reseal of cell membrane cultured in vitro

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.     

脂质体和脂质微泡对细胞膜的声孔作用比较

目的研究两种脂质载体——脂质体和脂质微泡对细胞膜的声孔作用。方法应用体外培养的乳腺癌细胞SK-BR-3，考察脂质体和脂质微泡在低频超声条件下，对SK-BR-3细胞膜的声孔作用，应用原子力显微镜观察超声后即刻和24 h的SK-BR-3细胞表面变化。结果超声可使细胞丧失贴壁性能，形态变圆，脂质微泡的加入明显增强这种作用。原子力显微镜观察表明，对照组(正常细胞单纯超声照射)细胞膜表面孔道暂时增大，部分细胞膜表面孔道直径大于1 μm。2%和5%脂质体+超声照射组和对照组无明显差别。脂质微泡+超声照射组对细胞膜结构有明显影响，2%脂质微泡+超声照射组产生的细胞膜表面孔道直径在1～3 μm，24 h内可以恢复。5%脂质微泡+超声照射组产生的细胞膜孔道直径2～4 μm，24 h内不能完全恢复。结论2%脂质微泡可以产生较强的声孔作用，使细胞膜上出现可逆性孔道，有利于药物或基因递送到细胞内。     

Serum influence on in-vitro gene delivery using microbubble-assisted ultrasound

Abstract

Background: Plasmid DNA (pDNA) is attractive molecule for gene therapy. pDNA-targeted delivery by efficient and safe methods is required to enhance its intra-tissue bioavailability. Among non-viral methods, sonoporation has become a promising method for in-vitro and in-vivo pDNA delivery. The efficiency of non-viral delivery methods of pDNA is generally limited by the presence of serum.

Purpose: The aim of this study was to evaluate the influence of serum on in-vitro pDNA delivery using microbubble-assisted ultrasound.

Methods: The effects of a range of serum concentrations (0–50%) on efficiency of in-vitro pDNA delivery by sonoporation were determined on human glioblastoma cells. Furthermore, the influence of the serum on cell viability, membrane permeabilization, microbubble destruction, and pDNA topology were also assessed.

Results: In-vitro results showed that a low serum concentration (i.e. ≤1%) induced a significant increase in transfection level through an increase in cell viability. However, a high serum concentration (i.e. ≥5%) resulted in a significant decrease in cell transfection, which was not associated with a decrease in membrane permeabilization or loss in cell viability. This decrease in transfection level was in fact positively correlated to changes in pDNA topology.

Conclusion: Serum influences the efficiency of in-vitro pDNA delivery by sonoporation through change in pDNA topology.     

含气微泡介导反义寡核苷酸转染实验的参数优化

近年的研究表明，含气微泡在超声作用下的声孔效应（sonorporation）可明显增加基因的转染率，不仅安全性高，而且针对不同的基因具有定位和时空可控性。利用含气微泡作为基因与药物的超声靶向递送的载体，要求微泡的包膜材料在包裹气体的同时还必须具有足够的韧性和强度，使其在体内外都能保持一定的稳定性，特别是能克服体内动脉压力的影响。     

Comparison of the efficiency and toxicity of sonoporation with branched polyethylenimine-mediated gene transfection in various cultured cell lines

Objective. The objective of this study is to evaluate transfection efficiency and safety for gene delivery by sonoporation in comparison with cationic polymer gene carrier branched polyethylenimine (BPEI).

Methods. The cDNA expressing VEGF¹⁶⁵ was cloned under chicken β-actin promoter. The plasmid DNA was transfected into the CHO, HEK293, and NIH3T3 cells using microbubble-based sonoporation and BPEI (25 kDa) under various conditions. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expressed protein level. Cytotoxicities of transfection methods were compared by Cell Counting Kit-8.

Results. At 1 MHz intensity, transfection efficiency of sonoporation was enhanced by microbubble concentration with no detrimental effects. By contrast, BPEI exacerbated cell viability, despite its high transgene expression efficiency.

Conclusion. Sonoporation gene therapy might be the safest technique to be used in actual clinical practice.     

星点设计结合效应面法优化声学脂质微泡的制备

声学微泡是近年发展起来的药物靶向递送系统，利用超声波作用促进药物或基因定位释放到细胞或组织中。本文利用星点设计(CCD)结合效应面法(RSM)优化声学脂质微泡制备条件。蛋黄磷脂、Tween 80和聚乙二醇1500是影响2～8 μm微泡浓度的主要因素。本文应用星点设计综合考察这些因素，利用效应面优化法得到最佳处方。实验评价指标为2～8 μm粒径的微泡浓度。采用多元二次方程对实验结果进行拟合，从而产生三维效应曲面图，最佳处方条件可从三维效应曲面的顶点得到。优化实验得到的最佳处方进行加速试验，考察稳定性。通过体内造影效果实验，研究本品的声学效应，并与国外上市产品SonoVue进行对照。结果表明，3个考察因素对2～8 μm微泡浓度均有影响， 最佳处方配比为： 蛋黄磷脂8.35 mg， Tween 8021.68 mg和聚乙二醇1500201 mg。所制备的2～8 μm微泡浓度平均值达到8.60×10⁹·mL^-1。加速试验结果显示脂质微泡物理稳定性良好。本品最佳处方体内造影强度(相对强度)和持续时间分别为4.47±0.15和(302±7)s，与国外上市产品SonoVue［4.28±0.13和(309±8)s］无明显差异。星点设计结合效应面法筛选出的声学脂质微泡浓度高，物理稳定性和声学造影效果好。     

Ultrasound-mediated gene delivery

Importance of the field: The use of ultrasound with microbubbles raises the possibility of an efficient and safe gene delivery.

Areas covered in this review: This review summarizes the current state of the art of gene delivery by sonoporation under the following topics. First, the basic ultrasound parameters and the characteristics of microbubble in biological systems are discussed. Second, the extensions of sonoporation to other fields of gene delivery such as viral and non-viral vector are briefly reviewed. Finally, recent applications in an animal model for various diseases are introduced.

What the reader will gain: Information and comments on gene delivery by sonoporation or enhanced cell membrane permeability by means of ultrasound.

Take home message: Ultrasound-mediated gene delivery combined with microbubble agents provides significant safety advantages over other methods of local gene delivery.     

Bleomycin delivery into cancer cells in vitro with ultrasound and SonoVue® or BR14® microbubbles

Abstract

Background: Cell exposure to ultrasound (US) in the presence of contrast agent microbubbles (MBs) can result in cell sonoporation that can be exploited for drug or gene delivery. Anticancer drug bleomycin (BLM), used in sonoporation, can effectively eliminate tumor cells in vitro and in vivo. Nevertheless, sonoporation mechanism is not known, thus different US parameters and MB types are used. Recently, we proposed that efficiency of cell sonoporation can be related to the efficiency of MB sonodestruction.

Purpose: We analyzed human tumor cells viability in response to BLM, US and MB treatment.

Methods: Human glioblastoma astrocytoma (U-87 MG) or colon cancer (HCT-116) cells were exposed to US in the presence of BLM and either SonoVue® or BR14® MBs. MB sonodestruction was evaluated according to US signal attenuation.

Results: Both HCT-116 and U-87 MG cell viability following US exposure decreased up to 30%. Decrease in cell viability followed similar tendency as MB sonodestruction, which suggests direct relationship between MB sonodestruction and BLM intracellular delivery.

Conclusion: Sonoporation is a feasible method to deliver BLM in to several types of human cancer cell lines. Efficiency of cell sonoporation correlated well with MB sonodestruction, providing a possibility to optimize US parameters by measuring MB sonodestruction.     

Ultrasound microbubble-carried PNA targeting to c-myc mRNA inhibits the proliferation of rabbit iliac arterious smooth muscle cells and intimal hyperplasia

Objective: To elucidate the transfected effect of albumin ultrasound microbubbles carrying peptide nucleic acids (PNAs) against c-myc gene to the vascular walls and their effect on the intimal proliferation induced by vascular denudation.

Methods: A rabbit iliac artery intimal proliferation model was constructed and PNA against c-myc mRNA was designed and synthesized and was added to albumin solution before ultrasound microbubbles were prepared and encapsulated in matrix of albumin. The ultrasound microbubbles carrying PNA were transfected to intima under ultrasound exposure. The transfected effect was identified by a histochemical method and the expression of c-myc was detected by in situ hybridization. The proliferation of intimal smooth muscle cells was estimated by the expression of proliferative cell nuclear antigen (PCNA) of them. The intimal area and thickness were judged morphologically for intimal hyperplasia.

Results: The ultrasound microbubbles with PNA were successfully prepared and c-myc PNA was transfected to vascular intimal cells. The expression of c-myc and PCNA by intimal vascular smooth muscle cells (vSMCs) was inhibited significantly and the intimal thickness and area were reduced remarkably.

Conclusion: Transfection of c-myc PNA could inhibit proliferartion of vSMCs and intima in the rabbit iliac artery intimal proliferation model and the targeted transfection of albumin ultrasound microbubbles carrying PNA offers a feasible way to facilitate its access to specific cells in vivo and produce bioavailability.     

Anti-tumor effect of ultrasound-induced Nordy-loaded microbubbles destruction

Background: Synthesized dl-Nordihydroguaiaretic acid (dl-NGDA or “Nordy”) can inhibit the growth of malignant human tumors, especially the tumor angiogenesis. However, its liposoluble nature limits its in vivo efficacy in the hydrosoluble circulation of human.

Purpose: We tried to use the ultrasonic microbubble as the carrier and the ultrasound-induced destruction for the targeted release of Nordy and evaluate its in vitro and in vivo anti-tumor effect.

Methods: Nordy-loaded lipid microbubbles were prepared by mechanical vibration. Effects of ultrasound-induced Nordy-loaded microbubbles destruction on proliferation of human umbilical vein endothelial cells (HUVECs), tumor derived endothelial cells (Td-ECs), and rabbit transplanted VX2 tumor models were evaluated.

Results: The ultrasound-induced Nordy-loaded microbubbles destruction inhibited the proliferations of HUVECs and Td-ECs in vitro, and inhibited the tumor growth and the microvasculature in vivo. Its efficacy was higher than those of Nordy used only and Nordy with ultrasound exposure.

Conclusion: Ultrasonic microbubbles can be used as the carrier of Nordy and achieve its targeted release with improved anti-tumor efficacy in the condition of ultrasound-induced microbubbles destruction.     

Vascular gene transfer and drug delivery in vitro using low-frequency ultrasound and microbubbles

Aim:

To determine the effects of ultrasound exposure in combination with a microbubble contrast agent (SonoVue) on the cellular uptake and delivery of drugs/genes into human umbilical vein endothelial cells (HUVECs) as well as their biological effects on migration.

Methods:

HUVECs in suspension were exposed to pulsed ultrasound with a 10% duty cycle in combination with various concentrations of a microbubble contrast agent (SonoVue) using a digital sonifier at a frequency of 20 kHz and an intensity of 3.77 W/cm² on the surface of a horn tip. Cell culture inserts were used to determine the cell migration ability.

Results:

Exposure to pulsed ultrasound resulted in enhanced green fluorescent protein (EGFP) gene transfection efficiencies ranging from 0.2% to 2%. The transfection efficiency of HUVECs was approximately 3-fold higher in the presence of SonoVue than in its absence at the effective exposure time of 6 s. For drug delivery to HUVECs using ultrasound, the delivery efficiencies of a low-molecular-weight model drug (TO-PRO^®−1, M_W 645.38) were significantly higher when compared to drug delivery without ultrasound, with a maximum efficiency of approximately 34%. However, the delivery efficiencies of a high-molecular-weight model drug (Dextran-Rhodamine B, M_W 70 000) were low, with a maximum delivery efficiency of nearly 0.5%, and gene transfection results were similarly poor. The migration ability of HUVECs exposed to ultrasound was also lower than that of the control (no exposure).

Conclusion:

The use of low-frequency and low-energy ultrasound in combination with microbubbles could be a potent physical method of increasing drug/gene delivery efficiency. This technique is a promising nonviral approach that can be used in cardiovascular disease therapy.     

Improving transfection efficiency of ultrapure oligochitosan/DNA polyplexes by medium acidification

Abstract

Context: Ultrapure oligochitosans (UOCs) have recently been reported as efficient nonviral vectors for corneal and retinal gene delivery. However, the influence of some physicochemical factors on the transfection efficiency, such as the pH, remains unclear. Deeper in vitro research of these factors could provide valuable information for future clinical applications.

Objective: The aim of this study is to determine the influence of the pH decrease on the transfection efficiency of UOC/pDNA polyplexes in HEK293 and ARPE19 cells.

Materials and methods: We elaborated self-assembled UOC/pCMS-EGFP polyplexes. The influence of the most important factors on the particle size and the zeta potential was studied by an orthogonal experimental design. We evaluated, in vitro, the cellular uptake and the transfection efficiency by flow cytometry, and the cytotoxicity of the vectors by CCK-8 assay.

Results and discussion: The pH of the medium strongly influences the physicochemical properties of the polyplexes, and by its modulation we are able to control their superficial charge. A significant increase on the cellular uptake and transfection efficiency of UOCs was obtained when the pH was acidified. Neither of our UOC/pCMS-EGFP polyplexes caused cytotoxicity; however, cells treated with Lipofectamine 2000? showed decreased cell viability.

Conclusion: This kind of UOC vectors could be useful to transfect cells that are in an acidic environment, such as tumor cells. However, additional in vivo studies may be required in order to obtain an effective and safe medicine for nonviral gene therapy purpose.     

Pharmaceutical and medical aspects of hyaluronic acid–ketorolac combination therapy in osteoarthritis treatment: radiographic imaging and bone mineral density

Abstract

The objective of this study was to formulate novel painless combined hyaluronic acid (HA)–ketorolac (KT) membrane for the management of osteoarthritis with rapid analgesic onset, thus avoiding HA frequent invasive intra-articular injections and KT gastrointestinal complaints associated with all non-steroidal anti-inflammatory drugs. HA was chemically crosslinked with carbodiimide/glutaraldehyde to yield membrane of low water content. Different in vitro aspects (mechanical properties, water content and in vitro release) were studied leading to an optimized soft, flexible K8 HA membrane containing 30?mg KT that achieved the desired balance of excellent elasticity and low water content. Moreover, a successful retardation of KT release rate was achieved (82%) after 48?h with favored initial fast drug release in the first hour (32.7%) to attain rapid analgesic effect. The clinical assessments in arthritic rats revealed apparent improvement in joint space narrowing, highest increase in bone mineral density at the proximal tibia and distal femur joints with the absence of osteophytosis only in animal group treated with combined HA–KT membrane. Application of K8 membrane was able to preserve KT plasma concentration above its minimum effective concentration for 48?h therefore, would able to replace six commercial tablets each of 10?mg KT.     

Gene silencing of Rac1 with RNA interference mediated by ultrasound and microbubbles in human LoVo cells: evaluation of cell invasion inhibition and metastatic

Objective: The objective of this study was to assess the change of cytoskeleton and cell cycle in LoVo (human colorectal cancer) cell via gene silencing of Rac1 with RNA interference mediated by microbubble (SonoVue) and ultrasound (US).

Methods: The compound of plasmid Rac1-shRNA, LoVo cells, and SonoVue was exposed to US (1?MHz, 2?W/cm², 5?min). The expression of Rac1 mRNA and Rac1 protein was detected by RT-PCR and Western blot. Cytoskeleton was taken by confocal microscope in a random fashion. Cell invasion was assayed using modified Boyden chambers, and cell cycle and apoptosis were analyzed by flow cytometry in LoVo cells.

Results: Rac1 gene is overexpressed in human colorectal cancer cells, gene silencing of Rac1 with RNA interference mediated by microbubble and US strongly inhibited lamellipodia formation, cell invasion, and delayed cell cycle, as well as enhanced cell apoptosis of LoVo cells in vitro.

Conclusion: Silencing Rac1 gene mediated by microbubble and US may become a new treatment option for the inhibition of the invasion and metastasis of colorectal cancer cells.     

Ultrasound targeted microbubble destruction for drug and gene delivery

Background: Gas-filled microbubbles have been used as ultrasound contrast agents for some decades. More recently, such microbubbles have evolved as experimental tools for organ- and tissue-specific drug and gene delivery. When sonified with ultrasound near their resonance frequency, microbubbles oscillate. With higher ultrasound energies, oscillation amplitudes increase, leading to microbubble destruction. This phenomenon can be used to deliver a substance into a target organ, if microbubbles are co-administered loaded with drugs or gene therapy vectors before i.v. injection. Objective: This review focuses on different experimental applications of microbubbles as tools for drug and gene delivery. Different organ systems and different classes of bioactive substances that have been used in previous studies will be discussed. Methods: All the available literature was reviewed to highlight the potential of this non-invasive, organ-specific delivery system. Conclusion: Ultrasound targeted microbubble destruction has been used in various organ systems and in tumours to successfully deliver drugs, proteins, gene therapy vectors and gene silencing constructs. Many proof of principle studies have demonstrated its potential as a non-invasive delivery tool. However, too few large animal studies and studies with therapeutic aims have been performed to see a clinical application of this technique in the near future. Nevertheless, there is great hope that preclinical large animal studies will confirm the successful results already obtained in small animals.     

Ovarian cancer targeted hyaluronic acid-based nanoparticle system for paclitaxel delivery to overcome drug resistance

Abstract

Objective: Most primary human ovarian tumors and peritoneal implants, as well as tumor vascular endothelial cells, express the CD44 family of cell surface proteoglycans, the natural ligand for which is hyaluronic acid (HA). Paclitaxel (PTX) is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including ovarian cancer. This study aimed to develop a HA-based PTX-loaded nanoparticle system to improve the ovarian cancer therapeutic effects.

Methods: PTX-loaded cationic nanostructured lipid nanoparticles (PTX-NLCs) were prepared. HA-PE was then coated onto the PTX-NLCs by electrostatic adsorption to form HA-PTX-NLCs. In vitro tumor cell inhibition efficiency was analyzed on SKOV3 human ovarian cancer cells (SKOV3 cells) and PTX-resistant SKOV3 cells (SKOV3/PTX cells). In vivo anticancer ability was evaluated with mice bearing SKOV3 ovarian cancer cells xenografts.

Results: HA-PTX-NLCs had an average diameter of 163?nm, and PTX was incorporated with an efficiency of over 80%. The in vitro viability of SKOV3 cells and SKOV3/PTX cells was obviously inhibited by HA-PTX-NLCs. In the ovarian cancer cells model, significant reduction in tumor growth was observed, whereas the conventional PTX injection group did not achieve significance.

Conclusion: This study demonstrated that significantly improved results were obtained by the newly constructed HA-PTX-NLCs, in terms of in vitro and in vivo therapeutic efficacy. These findings strongly support the superiority of HA based nano-system for the PTX delivery, thus enhance the efficacy of ovarian cancer chemotherapy.     

Efficacy of combined therapy with paclitaxel and low-level ultrasound in human chronic myelogenous leukemia cell line K562

Abstract

Background: Sonochemotherapy, which applies ultrasound in cancer chemotherapy, has been proven to be a promising therapeutic modality by some previous researches.

Purpose: To investigate the interaction between an antineoplastic agent – paclitaxel (PTX) and low-level ultrasound in human chronic myelogenous leukemia cell line K562, their combined efficacy, and the potential mechanisms in sonochemotherapy.

Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis and Guava Viacount assay were adopted to examine cell viability. Apoptosis was analyzed using Annexin V-PE/7-amino-actinomycin D staining. Changes in plasma membrane permeability were monitored by FD500-uptake and lactate dehydrogenase (LDH) release assays. Additionally, the ultrastructure changes were evaluated under scanning electron microscope (SEM).

Results: When 5?ng/ml PTX was combined with sonication at Load Power (LP)?=?2?W, the expected synergistic effects on cell viability loss (p?<?0.05) and apoptosis enhancement could be significantly detected, the plasma membrane permeability showed the best response, and relatively serious cell damages were observed under SEM.

Conclusion: The interaction between these two therapies depended on specific parameter settings, such as PTX dose and ultrasound intensity. Under synergistic conditions, ultrasound significantly potentiated the efficacies of PTX, including cytotoxicity, apoptosis and cell damage induction, which may be due to the enhanced membrane permeability and the increased intracellular PTX level.