Micro- and nanobubbles: A versatile non-viral platform for gene delivery

Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1–8 μm, characterised by their core–shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or ‘loaded’ in purposed-built bubble systems – indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described.     

Preparation and characterization of dextran nanobubbles for oxygen delivery

Dextran nanobubbles were prepared with a dextran shell and a perfluoropentan core in which oxygen was stored. To increase the stability polyvinylpirrolidone was also added to the formulation as stabilizing agent. Rhodamine B was used as fluorescent marker to obtain fluorescent nanobubbles. The nanobubble formulations showed sizes of about 500 nm, a negative surface charge and a good capacity of loading oxygen, no hemolytic activity or toxic effect on cell lines. The fluorescent labelled nanobubbles could be internalized in Vero cells. Oxygen-filled nanobubbles were able to release oxygen in different hypoxic solutions at different time after their preparation in in vitro experiments. The oxygen release kinetics could be enhanced after nanobubble insonation with ultrasound at 2.5 MHz. The oxygen-filled nanobubble formulations might be proposed for therapeutic applications in various diseases.     

超声微泡造影剂介导基因靶向治疗肝细胞癌的研究进展

随着超声造影及微泡制备技术的不断发展,超声微泡造影剂携带基因和药物的靶向治疗成为当前医学领域中的研究热点.利用超声波与微泡造影剂的相互作用及所产生的生物学效应,可实现微泡携带基因向目标组织转移释放,使肿瘤细胞局部目的基因的浓度大大增高,达到靶向治疗目的.     

Microbubbles in ultrasound-triggered drug and gene delivery

Ultrasound contrast agents, in the form of gas-filled microbubbles, are becoming popular in perfusion monitoring; they are employed as molecular imaging agents. Microbubbles are manufactured from biocompatible materials, they can be injected intravenously, and some are approved for clinical use. Microbubbles can be destroyed by ultrasound irradiation. This destruction phenomenon can be applied to targeted drug delivery and enhancement of drug action. The ultrasonic field can be focused at the target tissues and organs; thus, selectivity of the treatment can be improved, reducing undesirable side effects. Microbubbles enhance ultrasound energy deposition in the tissues and serve as cavitation nuclei, increasing intracellular drug delivery. DNA delivery and successful tissue transfection are observed in the areas of the body where ultrasound is applied after intravascular administration of microbubbles and plasmid DNA. Accelerated blood clot dissolution in the areas of insonation by cooperative action of thrombolytic agents and microbubbles is demonstrated in several clinical trials.     

Toxicological effects of cationic nanobubbles on the liver and kidneys: Biomarkers for predicting the risk

Nanobubbles with acoustical activity are used as both diagnostic and therapeutic carriers for detecting and treating diseases. We aimed to prepare nanobubbles and assess toxic responses to them in the liver and kidneys. The cytotoxicity of nanobubbles was determined by examining the viability of liver (HepG2) and kidney (293T) cell lines after a 24-h treatment at various concentrations (0.01–2%). Toxic effects of different formulations were compared by determining functional markers such as γ-glutamyl transferase (γ-GT) and blood urea nitrogen (BUN) after intravenous administration of nanobubbles. Cationic nanobubbles caused concentration-dependent cytotoxicity against cultured cells with a more significant effect in the liver than in the kidneys. A significant reduction of viability was revealed at a concentration as low as 0.1%. Cational systems with soyaethyl morpholinium ethosulfate (SME) exhibited the greatest γ-GT level at 6-fold higher than the control. Immunohistochemistry detected liver fibrosis and inflammation with nanobubbles treatment, especially SME-containing ones at higher doses. According to plasma proteomic profiles, gelsolin and fetuin-B were significantly downregulated 3-fold in the high-dose SME-treated group. Transthyretin decreased by 6-fold in this group. The fibrinogen gamma chain expression was highly elevated. The results suggest that these protein biomarkers are sensitive for assessing the risk of nanobubble exposure. This study is the first to systematically evaluate the possible toxicity of nanobubbles in the liver and kidneys.     

Response of contrast agents to ultrasound

Microbubbles are used as ultrasonic contrast agents that enhance the ultrasound signals of the vascular bed. The recent development of site-targeted microbubbles opened up the possibility for molecular imaging as well as localised drug and gene delivery. Initially the microbubbles' physical properties and their response to the ultrasound beam were not fully understood. However, the introduction of fast acquisition microscopy has allowed the observation of the microbubble behaviour in the presence of ultrasound. In addition, acoustical techniques can determine the scatter of single microbubbles. Sonoporation experiments promise high-specificity drug and gene delivery, but the responsible physical mechanisms, particularly for in vivo applications, are not fully understood. An improvement of microbubble technology may address variability related problems in both imaging and drug/gene delivery.     

Activated human neutrophil response to perfluorocarbon nanobubbles: oxygen-dependent and -independent cytotoxic responses

Nanobubbles, a type of nanoparticles with acoustically active properties, are being utilized as diagnostic and therapeutic nanoparticles to better understand, detect, and treat human diseases. The objective of this work was to prepare different nanobubble formulations and investigate their physicochemical characteristics and toxic responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-activated human neutrophils. The nanobubbles were prepared using perfluoropentane and coconut oil as the respective core and shell, with soybean phosphatidylcholine (SPC) and/or cationic surfactants as the interfacial layers. The cytotoxic effect of the nanobubbles on neutrophils was determined by extracellular O₂⁻ release, intracellular reactive oxygen species (ROS), lactate dehydrogenase (LDH), and elastase release. Particle sizes of the nanobubbles with different percentages of perfluorocarbon, oil, and surfactants in ranged 186-432 nm. The nanobubbles were demonstrated to inhibit the generation of superoxide and intracellular ROS. The cytotoxicity of nanobubbles may be mainly associated with membrane damage, as indicated by the high LDH leakage. Systems with Forestall (FE), a cationic surfactant, or higher SPC contents exhibited the greatest LDH release by 3-fold compared to the control. The further addition of an oil component reduced the cytotoxicity induced by the nanobubbles. Exposure to most of the nanobubble formulations upregulated elastase release by activated neutrophils. Contrary to this result, stearylamine (SA)-containing systems slightly but significantly suppressed elastase release. FE and SA in a free form caused stronger responses by neutrophils than when they were incorporated into nanobubbles. In summary, exposure to nanobubbles resulted in a formulation-dependent toxicity toward human neutrophils that was associated with both oxygen-dependent and -independent pathways. Clinicians should therefore exercise caution when using nanobubbles in patients for diagnostic and drug targeting aims.     

Gold-capped mesoporous silica nanoparticles as an excellent enzyme-responsive nanocarrier for controlled doxorubicin delivery

Abstract

Mesoporous silica nanoparticles (MSNs) have ideal characteristics as next generation of controlled drug delivery systems. In this study, a MSN-based nanocarrier was fabricated and gold nanoparticle (GNP)-biotin conjugates were successfully grafted onto the pore outlets of the prepared MSN. This bioconjugate served as a capping agent with a peptide-cleavable linker sensitive to matrix metalloproteinases (MMPs), which are overexpressed extracellular proteolytic enzymes in cancerous tissue. The prepared nanocarriers were fully characterised by scanning electron microscopy (SEM), transmission electron microscopy (TEM), nitrogen adsorption/desorption, Fourier transform infra-red spectroscopy (FTIR), dynamic light scattering (DLS) and thermo gravimetric analysis (TGA). In vitro release studies showed efficient capping of MSNs with gold gate and controlled release of Doxorubicin (DOX) in the presence of matrix metalloproteinase-2 (MMP-2) and acidic pH values. High DOX-loading capacity (21%) and encapsulation efficiency (95.5%) were achieved using fluorescence technique. DOX-loaded nanocarriers showed high cytocompatibility and could efficiently induce cell death and apoptosis in the MMP-2 overexpressed cell lines. Moreover, Haemolysis, platelet activation and inflammatory responses assessment approved excellent hemocompatibility and minimal side effects by encapsulation of DOX in MSNs carrier.     

Chitosan-based hydrogels for controlled, localized drug delivery

Hydrogels are high-water content materials prepared from cross-linked polymers that are able to provide sustained, local delivery of a variety of therapeutic agents. Use of the natural polymer, chitosan, as the scaffold material in hydrogels has been highly pursued thanks to the polymer's biocompatibility, low toxicity, and biodegradability. The advanced development of chitosan hydrogels has led to new drug delivery systems that release their payloads under varying environmental stimuli. In addition, thermosensitive hydrogel variants have been developed to form a chitosan hydrogel in situ, precluding the need for surgical implantation. The development of these intelligent drug delivery devices requires a foundation in the chemical and physical characteristics of chitosan-based hydrogels, as well as the therapeutics to be delivered. In this review, we investigate the newest developments in chitosan hydrogel preparation and define the design parameters in the development of physically and chemically cross-linked hydrogels.     

The preparation and characteristics of sterically stabilized liposomes containing paclitaxel and super-paramagnetic iron oxide nanoparticles

Theranostics, combining therapy and diagnosis, is an appealing approach for chemotherapy. In the present study, we selected paclitaxel (PTX) as a therapeutic agent, super-paramagnetic iron oxide nanoparticles (SPIO) as a diagnostic agent and sterically stabilized liposomes as a carrier to prepare theranostic liposomes. The SPIO were prepared and characterized. Moreover, the sterically stabilized liposomes containing PTX and SPIO (PTX/SPIO-SSL) were prepared. The characteristics of PTX/SPIO-SSL were investigated. The results indicated that prepared SPIO exhibited super-paramagnetic and could be used for MRI. The average particle size of PTX/SPIO-SSL was about 170 nm, with a polydispersity index (PDI) less than 0.3. The zeta potential of PTX/SPIO-SSL was negative. The PTX entrapment efficiency of PTX/SPIO-SSL was more than 98%. The TEM results indicated the spherical structure and dense SPIO content in PTX/SPIO-SSL. The in vitro release of PTX from PTX/SPIO-SSL and PTX-SSL was almost identical at both pH 6.8 and 7.4. In conclusion, the PTX/SPIO-SSL were prepared and characterized in vitro. The anti-tumor and diagnostic activity of PTX/SPIO-SSL should be investigated deeply in future study.     

Stimuli-responsive LbL capsules and nanoshells for drug delivery

Review of basic principles and recent developments in the area of stimuli responsive polymeric capsules and nanoshells formed via layer-by-layer (LbL) is presented. The most essential attributes of the LbL approach are multifunctionality and responsiveness to a multitude of stimuli. The stimuli can be logically divided into three categories: physical (light, electric, magnetic, ultrasound, mechanical, and temperature), chemical (pH, ionic strength, solvent, and electrochemical) and biological (enzymes and receptors). Using these stimuli, numerous functionalities of nanoshells have been demonstrated: encapsulation, release including that inside living cells or in tissue, sensors, enzymatic reactions, enhancement of mechanical properties, and fusion. This review describes mechanisms and basic principles of stimuli effects, describes progress in the area, and gives an outlook on emerging trends such as theranostics and nanomedicine.     

Magnetic nanomaterials for hyperthermia-based therapy and controlled drug delivery

Previous attempts to review the literature on magnetic nanomaterials for hyperthermia-based therapy focused primarily on magnetic fluid hyperthermia (MFH) using mono metallic/metal oxide nanoparticles. The term “hyperthermia” in the literature was also confined only to include use of heat for therapeutic applications. Recently, there have been a number of publications demonstrating magnetic nanoparticle-based hyperthermia to generate local heat resulting in the release of drugs either bound to the magnetic nanoparticle or encapsulated within polymeric matrices. In this review article, we present a case for broadening the meaning of the term “hyperthermia” by including thermotherapy as well as magnetically modulated controlled drug delivery. We provide a classification for controlled drug delivery using hyperthermia: Hyperthermia-based controlled drug delivery through bond breaking (DBB) and hyperthermia-based controlled drug delivery through enhanced permeability (DEP). The review also covers, for the first time, core-shell type magnetic nanomaterials, especially nanoshells prepared using layer-by-layer self-assembly, for the application of hyperthermia-based therapy and controlled drug delivery. The highlight of the review article is to portray potential opportunities for the combination of hyperthermia-based therapy and controlled drug release paradigms -towards successful application in personalized medicine.     

Cell microenvironment stimuli-responsive controlled-release delivery systems based on mesoporous silica nanoparticles

To develop novel tumor cell microenvironment stimuli-responsive smart controlled-release delivery systems is one of the current common interests of materials science and clinical medicine. Meanwhile, mesoporous silica nanoparticles as a promising drug carrier have become the new area of interest in the field of biomedical application in recent years because of their unique characteristics and abilities to efficiently and specifically entrap cargo molecules. This review describes the more recent developments and achievements of mesoporous silica nanoparticles in drug delivery. In particular, we focus on the stimuli-responsive controlled-release systems that are able to respond to tumor cell environmental changes, such as pH, glucose, adenosine-5′-triphosphate (ATP), glutathione (GSH), and H₂O₂.     

Experiment on the factors for enhancing the susceptibility of cancer cells to chemotherapeutic drug by ultrasound microbubbles

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/cm² 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.