Investigation of Optimized Treatment Conditions for Acoustic-Transfection Technique for Intracellular Delivery of Macromolecules.
ABSTRACT: Manipulation of cellular functions and structures by introduction of genetic materials inside cells has been one of the most prominent research areas in biomedicine. High-frequency ultrasound acoustic-transfection has recently been developed and confirmed by intracellular delivery of small molecules into HeLa cells at the single-cell level with high cell viability. After we proved the concept underlying the acoustic-transfection technique, treatment conditions for different human cancer cell lines have been intensively investigated to further develop acoustic-transfection as a versatile and adaptable transfection method by satisfying the requirements of high-delivery efficiency and cell membrane permeability with minimal membrane disruption. To determine optimal treatment conditions for different cell lines, we developed a quantitative intracellular delivery score based on delivery efficiency, cell membrane permeability and cell viability after 4 and 20?h of treatment. The intracellular delivery of macromolecules and the simultaneous intracellular delivery of two molecules under optimal treatment conditions were successfully achieved. We found that DNA plasmid was delivered by acoustic-transfection technique into epiblast stem cells, which expressed transient mCherry fluorescence.
Project description:Controlling cell functions for research and therapeutic purposes may open new strategies for the treatment of many diseases. An efficient and safe introduction of membrane impermeable molecules into target cells will provide versatile means to modulate cell fate. We introduce a new transfection technique that utilizes high frequency ultrasound without any contrast agents such as microbubbles, bringing a single-cell level targeting and size-dependent intracellular delivery of macromolecules. The transfection apparatus consists of an ultrasonic transducer with the center frequency of over 150?MHz and an epi-fluorescence microscope, entitled acoustic-transfection system. Acoustic pulses, emitted from an ultrasonic transducer, perturb the lipid bilayer of the cell membrane of a targeted single-cell to induce intracellular delivery of exogenous molecules. Simultaneous live cell imaging using HeLa cells to investigate the intracellular concentration of Ca(2+) and propidium iodide (PI) and the delivery of 3?kDa dextran labeled with Alexa 488 were demonstrated. Cytosolic delivery of 3?kDa dextran induced via acoustic-transfection was manifested by diffused fluorescence throughout whole cells. Short-term (6?hr) cell viability test and long-term (40?hr) cell tracking confirmed that the proposed approach has low cell cytotoxicity.
Project description:A high-throughput non-viral intracellular delivery platform is introduced for the transfection of large cargos with dosage-control. This platform, termed Acoustic-Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min<sup>-1</sup> per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage-controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.
Project description:Efficient intracellular delivery of biologically active macromolecules has been a challenging but important process for manipulating live cells for research and therapeutic purposes. There have been limited transfection techniques that can deliver multiple types of active molecules simultaneously into single-cells as well as different types of molecules into physically connected individual neighboring cells separately with high precision and low cytotoxicity. Here, a high frequency ultrasound-based remote intracellular delivery technique capable of delivery of multiple DNA plasmids, messenger RNAs, and recombinant proteins is developed to allow high spatiotemporal visualization and analysis of gene and protein expressions as well as single-cell gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9), a method called acoustic-transfection. Acoustic-transfection has advantages over typical sonoporation because acoustic-transfection utilizing ultra-high frequency ultrasound over 150?MHz can directly deliver gene and proteins into cytoplasm without microbubbles, which enables controlled and local intracellular delivery to acoustic-transfection technique. Acoustic-transfection was further demonstrated to deliver CRISPR-Cas9 systems to successfully modify and reprogram the genome of single live cells, providing the evidence of the acoustic-transfection technique for precise genome editing using CRISPR-Cas9.
Project description:Active transport of gas molecules is critical to preserve the physiological functions of organisms. Oxygen, as the most essential gas molecule, plays significant roles in maintaining the metabolism and viability of cells. Herein, we report a nanomotor-based delivery system that combines the fast propulsion of acoustically propelled gold nanowire nanomotors (AuNW) with the high oxygen carrying capacity of red blood cell membrane-cloaked perfluorocarbon nanoemulsions (RBC-PFC) for active intracellular delivery of oxygen. The oxygen delivery capacity and kinetics of the AuNW nanomotors carrying RBC-PFC (denoted as "Motor-PFC") are examined under ultrasound field. Specifically, the fast movement of the Motor-PFC under an acoustic field accelerates intracellular delivery of oxygen to J774 macrophage cells. Upon entering the cells, the oxygen loaded in the Motor-PFC is sustainably released, which maintains the cell viability when cultured under hypoxic conditions. The acoustically propelled Motor-PFC leads to significantly higher cell viability (84.4%) over a 72 h period, compared to control samples with free RBC-PFC (44.4%) or to passive Motor-PFC (32.7%). These results indicate that the Motor-PFC can act as an effective delivery vehicle for active intracellular oxygen transport. While oxygen is used here as a model gas molecule, the Motor-PFC platform can be readily expanded to the active delivery of other gas molecules to various target cells.
Project description:Intracellular delivery is essential to therapeutic applications such as genome engineering and disease diagnosis. Current methods lack simple, noninvasive strategies and are often hindered by long incubation time or high toxicity. Hydrodynamic approaches offer rapid and controllable delivery of small molecules, but thus far have not been demonstrated for delivering functional proteins. In this work, we developed a robust hydrodynamic approach based on gigahertz (GHz) acoustics to achieve rapid and noninvasive cytosolic delivery of biologically active proteins. With this method, GHz-based acoustic devices trigger oscillations through a liquid medium (acoustic streaming), generating shear stress on the cell membrane and inducing transient nanoporation. This mechanical effect enhances membrane permeability and enables cytosolic access to cationic proteins without disturbing their bioactivity. We evaluated the versatility of this approach through the delivery of cationic fluorescent proteins to a range of cell lines, all of which displayed equally efficient delivery speed (≤20 min). Delivery of multiple enzymatically active proteins with functionality related to apoptosis or genetic recombination further demonstrated the relevance of this method.
Project description:Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure-function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F(0)F(1)-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F(0)F(1)-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.
Project description:Suspended cells are difficult to be transfected by common biochemical methods which require cell attachment to a substrate. Mechanical oscillations of suspended cells at certain frequencies are found to result in significant increase in membrane permeability and potency for delivery of nano-particles and genetic materials into the cells. Nanomaterials including siRNAs are found to penetrate into suspended cells after subjecting to short-time mechanical oscillations, which would otherwise not affect the viability of the cells. Theoretical analysis indicates significant deformation of the actin-filament network in the cytoskeleton cortex during mechanical oscillations at the experimental frequency, which is likely to rupture the soft phospholipid bilayer leading to increased membrane permeability. The results here indicate a new method for enhancing cell transfection.
Project description:Delivery of large and structurally complex target molecules into cells is vital to the emerging areas of cellular modification and molecular therapy. Inadequacy of prevailing in vivo (viral) and in vitro (liposomal) gene transfer methods for delivery of proteins and a growing diversity of synthetic nanomaterials has encouraged development of alternative physical approaches. Efficacy of injury/diffusion-based delivery via shear mechanoporation is largely insensitive to cell type and target molecule; however, enhanced flexibility is typically accompanied by reduced gene transfer effectiveness. We detail a method to improve transfection efficiency through coordinated mechanical disruption of the cell membrane and electrophoretic insertion of DNA to the cell interior. An array of micromachined nozzles focuses ultrasonic pressure waves, creating a high-shear environment that promotes transient pore formation in membranes of transmitted cells. Acoustic Shear Poration (ASP) allows passive cytoplasmic delivery of small to large nongene macromolecules into established and primary cells at greater than 75% efficiency. Addition of an electrophoretic action enables active transport of target DNA molecules to substantially augment transfection efficiency of passive mechanoporation/diffusive delivery without affecting viability. This two-stage poration/insertion method preserves the compelling flexibility of shear-based delivery, yet substantially enhances capabilities for active transport and transfection of plasmid DNA.
Project description:Advances in gene editing are leading to new medical interventions where patients' own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.
Project description:Ultrasound-driven microbubble activities have been exploited to transiently disrupt the cell membrane (sonoporation) for non-viral intracellular drug delivery and gene transfection both in vivo and in vitro. In this study, we investigated the dynamic behaviors of a population of microbubbles exposed to pulsed ultrasound and their impact on adherent cells in terms of intracellular delivery and cell viability. By systematically analyzing the bubble activities at time scales relevant to pulsed ultrasound exposure, we identified two quantification parameters that categorize the diverse bubble activities subjected to various ultrasound conditions into three characteristic behaviors: stable cavitation/aggregation (type I), growth/coalescence and translation (type II) and localized inertial cavitation/collapse (type III). Correlation of the bubble activities with sonoporation outcome suggested that type III behavior resulted in intracellular delivery, whereas type II behavior caused the death of a large number of cells. These results provide useful insights for rational selection of ultrasound parameters to optimize outcomes of sonoporation and other applications that exploit the use of ultrasound-driven bubble activities.