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Abstract
The hydrodynamic method mimics the in vivo environment of the mechanical effect on cell stimulation, which not only modulates cell physiology but also shows excellent intracellular delivery ability. Herein, a hydrodynamic intracellular delivery system based on the gigahertz acoustic streaming (AS) effect is proposed, which presents powerful targeted delivery capabilities with high efficiency and universality. Results indicate that the range of cells with AuNR introduction is related to that of AS, enabling a tunable delivery range due to the adjustability of the AS radius. Moreover, with the assistance of AS, the organelle localization delivery of AuNRs with different modifications is enhanced. AuNRs@RGD is inclined to accumulate in the nucleus, while AuNRs@BSA tend to enter the mitochondria and AuNRs@PEGnK tend to accumulate in the lysosome. Finally, the photothermal effect is proved based on the large quantities of AuNRs introduced via AS. The abundant introduction of AuNRs under the action of AS can achieve rapid cell heating with the irradiation of a 785 nm laser, which has great potential in shortening the treatment cycle of photothermal therapy (PTT). Thereby, an efficient hydrodynamic technology in AuNR introduction based on AS has been demonstrated. The outstanding location delivery and organelle targeting of this method provides a new idea for precise medical treatment.
The generation and dispensing of microdroplets is a vital process in various fields such as biomedicine, medical diagnosis and chemistry. However, most methods still require the structures of nozzles or microchannels to assist droplet generation, which leads to limitations on system flexibility and restrictions on the size range of the generated droplets. In this paper, we propose a nozzle-free acoustic-based method for generating droplets using a gigahertz (GHz) bulk acoustic wave (BAW). Unlike most of the acoustofluidic approaches, the proposed method produces the droplet by pinching-off the liquid column generated by the acoustic body force at the oil-water interface. Benefitting from the focused acoustic energy and small footprint of the device, four orders of magnitude (ranging from 2 μm to 1800 μm) of droplet size could be produced by controlling the working time and power of the device. We also demonstrated cell encapsulation in the droplet and a high cell viability was achieved. The proposed acoustic-based droplet generation method exhibits capacity for generating droplets with a wide size range, versatility toward different viscosities, as well as biocompatibility for handling viable samples, which shows potential in miniaturization and scalability.
Near-infrared (NIR) detectors with high sensitivity and spectral selectivity are highly desired in various applications. In this work, a photothermal detector with high NIR sensitivity and spectral selectivity was developed by simply modifying a photothermal layer of reduced graphene oxide-Au nanorods (rGO-AuNRs) hybrid on a thermistor, which can convert the light energy into heat and reflect as resistance changes of a thermistor. Owing to the plasmon coupling of the two materials, the obtained rGO-AuNRs hybrid not only has remarkable photothermal conversion efficiency but also exhibits dependence on spectral response. Thus, benefiting from the excellent performance of the hybrid, the fabricated detector is sensitive to illumination in the wavelength range from 700 to 1000 nm with the highest photoresponsivity of 2.50 × 105 Ω·W−1. The photothermal detector presented in this work will provide a simple and inexpensive alternative for NIR detector development.
A surge of research in intracellular delivery technologies is underway with the increased innovations in cell‐based therapies and cell reprogramming. Particularly, physical cell membrane permeabilization techniques are highlighted as the leading technologies because of their unique features, including versatility, independence of cargo properties, and high‐throughput delivery that is critical for providing the desired cell quantity for cell‐based therapies. Amongst the physical permeabilization methods, sonoporation holds great promise and demonstrates to deliver a variety of functional cargos, such as biomolecular drugs, proteins, and plasmids, to various cells including cancer, immune, and stem cells. However, traditional bubble‐based sonoporation methods usually require special contrast agents. Bubble‐based sonoporation methods also have high chances of inducing irreversible damage to critical cell components, lowering the cell viability, and reducing the effectiveness of delivered cargos. To overcome these limitations, several novel non‐bubble‐based sonoporation mechanisms are under development. This review will cover both the bubble‐based and non‐bubble‐based sonoporation mechanisms being employed for intracellular delivery, the technologies being investigated to overcome the limitations of traditional platforms, as well as perspectives on the future sonoporation mechanisms, technologies, and applications.
Drug delivery systems (DDS) are extensively studied to improve the solubility, stability, pharmacokinetic, and biodistribution of chemotherapeutics. However, the drug delivery efficiency of traditional DDS is often limited by the complicated biological barriers in vivo. Herein, a multistage adaptive nanoparticle (MAN) that simultaneously overcomes multiple biological barriers to achieve tumor‐targeted drug delivery with high efficiency is presented. MAN has a core–shell structure, in which both the core and the shell are made of responsive polymers. This structure allows MAN to present different surface properties to adapt to its surrounding biological microenvironment, thereby achieving enhanced stability in blood circulation, improved tumor accumulation and cellular internalization in tumor tissues, and effective release of drug in cells. With these unique characteristics, the MAN loaded with docetaxel achieves effective tumor suppression with reduced systemic toxicity. Furthermore, MAN can load almost any hydrophobic drugs, providing a general strategy for the tumor‐targeted delivery of hydrophobic drugs to overcome the multiple biological barriers and improve the efficacy of chemotherapy.
Innate cell function can be artificially engineered and reprogrammed by introducing biomolecules, such as DNAs, RNAs, plasmid DNAs, proteins, or nanomaterials, into the cytosol or nucleus. This process of delivering exogenous cargos into living cells is referred to as intracellular delivery. For instance, clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 gene editing begins with internalizing Cas9 protein and guide RNA into cells, and chimeric antigen receptor‐T (CAR‐T) cells are prepared by delivering CAR genes into T lymphocytes for cancer immunotherapies. To deliver external biomolecules into cells, tools, including viral vectors, and electroporation have been traditionally used; however, they are suboptimal for achieving high levels of intracellular delivery while preserving cell viability, phenotype, and function. Notably, as emerging solutions, microfluidic and nanofluidic approaches have shown remarkable potential for addressing this open challenge. This review provides an overview of recent advances in microfluidic and nanofluidic intracellular delivery strategies and discusses new opportunities and challenges for clinical applications. Furthermore, key considerations for future efforts to develop microfluidics‐ and nanofluidics‐enabled next‐generation intracellular delivery platforms are outlined. This review provides an overview of recent advances in microfluidic and nanofluidic intracellular delivery strategies, enabling cellular engineering and cell therapy. New opportunities and challenges from micro(nano)fluidic intracellular delivery are highlighted, and key considerations on the establishment of microfluidics‐ and nanofluidics‐enabled next‐generation intracellular delivery platforms are also discussed.
Macropinocytosis is a consequence of oncogenic alterations of cancer cells while most healthy cells are non-macropinocytic. It is currently unclear whether macropinocytic cancer cells can be targeted rather than healthy cells, by adjusting the shape and size of nanoparticles. Herein, the endocytosis of two differently shaped nanoparticles; nanorods and nanospheres are compared in cancer and healthy cells. The cells are breast epithelial cancer cells (MCF7) and breast epithelial healthy cells (MCF10A) and pancreas cancer cells (PANC-1 cells) and non-tumourogenic patient-derived cancer-associated fibroblasts (CAFs). The endocytosis pathway is quantified by a combination of pair correlation microscopy and endocytosis inhibitors. MCF7 cells use clathrin-mediated endocytosis and macropinocytosis to take up the nanorods while MCF10A cells use predominantly clathrin-mediated endocytosis. Based on the comparison of endocytic behavior of cancer and healthy cells, MCF7 cells can be induced to take up more nanorods and suppress the metabolism and endocytosis of nanorods in MCF10A cells. The nanorods allow targeting to breast cancer MCF7 cells and pancreas cancer cells over the healthy cells. This study opens exciting possibilities for shape to target the cancer cells over healthy cells, by adjusting nanoparticle shape.
Supramolecular approaches have opened up vast possibilities to construct versatile materials, especially those with stimuli‐responsiveness and integrated functionalities of multi‐modal diagnosis and synergistic therapeutics. In this study, a hybrid theranostic nanosystem named TTPY‐Py⊂CP5@AuNR is constructed via facile host–guest interactions, where TTPY‐Py is a photosensitizer with aggregation‐induced emission and CP5@AuNR represents the carboxylatopillar[5]arene (CP5)‐modified Au nanorods. TTPY‐Py⊂CP5@AuNR integrates the respective advantages of TTPY‐Py and CP5@AuNR such as the high performance of reactive oxygen species generation and photothermal conversion, and meanwhile shows fluorescence responses to both temperature and pH stimuli. The successful modification of CP5 macrocycles on AuNRs surfaces can eliminate the cytotoxicity of AuNRs and enable them to serve as the nanocarrier of TTPY‐Py for further theranostic applications. Significantly, in vitro and in vivo evaluations demonstrate that this supramolecular nanotheranostic system possesses multiple modalities including intensive fluorescence imaging (FLI), photoacoustic imaging (PAI), efficient photodynamic therapy (PDT), and photothermal therapy (PTT), indicating its great potential for FLI‐PAI imaging‐guided synergistic PDT‐PTT therapy. Moreover, TTPY‐Py can be released upon activation by the acidic environment of lysosomes and then specifically light up mitochondria. This study demonstrates a new strategy for the design of versatile nanotheranostics for accurate tumor imaging and cancer therapies.
Near infrared (NIR)‐resonant gold nanoparticles (AuNPs) hold great promise in cancer diagnostics and treatment. However, translating the theranostic potential of AuNPs into clinical applications still remains a challenge due to the difficulty to improve the efficiency and specificity of tumor delivery in vivo as well as the clearance from liver and spleen to avoid off target toxicity. In this study, endothelial colony forming cells (ECFCs) are exploited as vehicles to deliver AuNPs to tumors. It is first demonstrated that ECFCs display a great capability to intake AuNPs without losing viability, and exert antitumor activity per se. Using a human melanoma xenograft mouse model, it is next demonstrated that AuNP‐loaded ECFCs retain their capacity to migrate to tumor sites in vivo 1 day after injection and stay in the tumor mass for more than 1 week. In addition, it is demonstrated that ECFC‐loaded AuNPs are efficiently cleared by the liver over time and do not elicit any sign of damage to healthy tissue.
Exploiting a comprehensive strategy that processes diagnosis and therapeutic functions is desired for eradicating tumors. In this study, two versatile nanoparticles are introduced: one is polyethylene glycol‐ and polyethyleneimine‐modified gold nanorods (mPEG–PEI–AuNRs), and the other is formed by electrostatic interactions between mPEG–PEI and calcium carbonate nanoparticles (mPEG–PEI/CaNPs). These two nanoparticles possess following favorable properties: 1) mPEG–PEI–AuNRs and mPEG–PEI/CaNPs show not only high cell uptake in acidic tumoral pH, but also efficient accumulation in tumors with prolonged circulation. 2) mPEG–PEI/CaNPs can generate carbon dioxide (CO2) bubbles in acidic tumoral environment and the photoacoustic (PA) signals from mPEG–PEI–AuNRs can be enhanced with the generation of CO2 bubbles. 3) The tumors can be eradicated by combining photothermal therapy (PTT) with ultrasonic therapy (UST) under the near‐infrared (NIR) laser and ultrasonic irradiation with the presence of mPEG–PEI–AuNRs and CO2 bubbles from mPEG–PEI/CaNPs. The detailed evaluation of cellular uptake, photothermal property of mPEG–PEI–AuNRs, CO2 bubbles’ generation from mPEG–PEI/CaNPs, imaging, and combined PTT and UST are carried out in vitro or in vivo. This work has great potential usage for diagnosis and treatment in the future.
Hydrodynamic force loading platforms for controllable cell mechanical deformation play an essential role in modern cell technologies. Current systems require assistance from specific microstructures thus limiting the controllability and flexibility in cell shape modulation, and studies on real‐time 3D cell morphology analysis are still absent. This article presents a novel platform based on acoustic streaming generated from a gigahertz device for cell shape control and real‐time cell deformation analysis. Details in cell deformation and the restoration process are thoroughly studied on the platform, and cell behavior control at the microscale is successfully achieved by tuning the treating time, intensity, and wave form of the streaming. The application of this platform in cell membrane permeability modulation and analysis is also exploited. Based on the membrane reorganization during cell deformation, the effects of deformation extent and deformation patterns on membrane permeability to micro‐ and macromolecules are revealed. This technology has shown its unique superiorities in cell mechanical manipulation such as high flexibility, high accuracy, and pure fluid force operation, indicating its promising prospect as a reliable tool for cell property study and drug therapy development.
In recent nanobiotechnology developments, a wide variety of functional nanomaterials and engineered biomolecules have been created, and these have numerous applications in cell biology. For these nanomaterials to fulfill their promises completely, they must be able to reach their biological targets at the subcellular level and with a high level of specificity. Traditionally, either nanocarrier- or membrane disruption-based method has been used to deliver nanomaterials inside cells; however, these methods are suboptimal due to their toxicity, inconsistent delivery, and low throughput, and they are also labor intensive and time-consuming, highlighting the need for development of a next-generation, intracellular delivery system. This study reports on the development of an intracellular nanomaterial delivery platform, based on unexpected cell-deformation phenomena via spiral vortex and vortex breakdown exerted in the cross- and T-junctions at moderate Reynolds numbers. These vortex-induced cell deformation and sequential restoration processes open cell membranes transiently, allowing effective and robust intracellular delivery of nanomaterials in a single step without the aid of carriers or external apparatus. By using the platform described here (termed spiral hydroporator), we demonstrate the delivery of different nanomaterials, including gold nanoparticles (200 nm diameter), functional mesoporous silica nanoparticles (150 nm diameter), dextran (hydrodynamic diameters between 2-55 nm), and mRNA, into different cell types. We demonstrate here that the system is highly efficient (up to 96.5%) with high throughput (up to 1 × 106 cells/min) and rapid delivery (∼1 min) while maintaining high levels of cell viability (up to 94%).
Intracellular delivery enables the efficient drug delivery into various types of cells and has been a long-term studied topics in modern biotechnology. Targeted delivery with improved delivery efficacy requires considerable requirements. This process is a critical step in many cellular-level studies, such as cellular drug therapy, gene editing delivery, and a series of biomedical research applications. The emergence of micro- and nanotechnology has enabled the more accurate and dedicated intracellular delivery, and it is expected to be the next generation of controlled delivery with unprecedented flexibility. This review focuses on several represented micro- and nanoscale physical approaches for cell membrane disruption-based intracellular delivery and discusses the mechanisms, advantages, and challenges of each approach. We believe that the deeper understanding of intracellular delivery at such low dimension would help the research community to develop more powerful delivery technologies for biomedical applications. Keywords: Drug delivery, Physical approaches, Cell membrane disruption, Low dimension
Sonoporation is a targeted drug delivery technique that employs cavitation microbubbles to generate transient pores in the cell membrane, allowing foreign substances to enter cells by passing through the pores. Due to the broad size distribution of microbubbles, cavitation events appear to be a random process, making it difficult to achieve controllable and efficient sonoporation. In this work a technique is reported using a microfluidic device that enables in parallel modulation of membrane permeability by an oscillating microbubble array. Multirectangular channels of uniform size are created at the sidewall to generate an array of monodispersed microbubbles, which oscillate with almost the same amplitude and resonant frequency, ensuring homogeneous sonoporation with high efficacy. Stable harmonic and high harmonic signals emitted by individual oscillating microbubbles are detected by a laser Doppler vibrometer, which indicates stable cavitation occurred. Under the influence of the acoustic radiation forces induced by the oscillating microbubble, single cells can be trapped at an oscillating microbubble surface. The sonoporation of single cells is directly influenced by the individual oscillating microbubble. The parallel sonoporation of multiple cells is achieved with an efficiency of 96.6 ± 1.74% at an acoustic pressure as low as 41.7 kPa. This work demonstrates a newly developed microfluidic device that enables parallel modulation of membrane permeability using an oscillating microbubble array. This parallel sonoporation device can generate an array of air microbubbles with the same diameter and the oscillation of the microbubble array is controllable.
Acoustofluidics: From lab to clinic Research into using sound waves to separate biological particles from fluid moving through tiny channels is rapidly advancing, but more is needed to develop real-life clinical applications. Tony Jun Huang of Duke University in the US and colleagues reviewed the latest studies in the rapidly growing field of ‘acoustofluidic separation’. While progress has been made in using the technology to separate micro-sized objects like cells and bacteria from fluids, few breakthroughs have been achieved for separating nanoparticles, like lipids and viruses. Also, advances are needed to speed up the separation process. Still, the field shows much promise for the development of label-free, point-of-care devices for cancer diagnosis, placental health monitoring, and isolating bacteria from water or food. Focusing on product development and technological improvements will enable this technology to find real-world applications, the researchers conclude.
a r t i c l e i n f o Even as gigahertz (GHz) acoustic streaming has developed into a multi-functional platform technology for biochemical applications, including ultrafast microfluidic mixing, microparticle operations, and cellar or vesicle surgery , its theoretical principles have yet to be established. This is because few studies have been conducted on the use of such high frequency acoustics in microscale fluids. Another difficulty is the lack of velocimetry methods for microscale and nanoscale fluidic streaming. In this work, we focus on the basic aspects of GHz acoustic streaming, including its micro-vortex generation principles, theoretical model, and experimental characterization technologies. We present details of a weak-coupled finite simulation that represents our current understanding of the GHz-acoustic-streaming phenomenon. Both our simulation and experimental results show that the GHz-acoustic-induced interfacial body force plays a determinative role in vortex generation. We carefully studied changes in the formation of GHz acoustic streaming at different acoustic powers and flow rates. In particular, we developed a microfluidic-particle-image velocimetry method that enables the quantification of streaming at the microscale and even nanoscale. This work provides a full map of GHz acoustofluidics and highlights the way to further theoretical study of this topic.
In 1961, Segre and Silberberg first reported the tubular pinch effect and numerous theoretical studies were subsequently published to explain the inertial particle migration phenomenon. Presently, as fluid mechanics meets micro- and nanotechnology, theoretical studies on intrinsic particle migration and flow phenomena associated with inertia are being experimentally tested and validated. This collective study on the fluid-particle-structure phenomena in microchannels involving fluid inertia is called, “inertial microfluidics”. Beyond theoretical studies, now inertial microfluidics has been gaining much attention from various research fields ranging from biomedicine to industry. Despite the positive contributions, there is still a lack of clear understanding of intrinsic inertial effects in microchannels. Therefore, this minireview introduces the mechanisms and underlying physics in inertial microfluidic systems with specific focuses on inertial particle migration and secondary flow, and outlines the opportunities provided by inertial microfluidics, along with an outlook on the field.
Hierarchical assemblies of nanomaterial superstructures with controlled orientation affords a multitude of novel properties of plasmonics and broad applications. Yet constructing multi-functional superstructures with nanoparticles positioned in desired locations remains challenging. Herein, gold nanorods (GNRs) assembled in stripe patterns with controlled orientation and structures in millimeter scale for versatile application have been achieved. Applications of patterned GNRs in sensing enhancement and engineering mammalian cells alignment are investigated experimentally. The performance of patterned GNRs in surface enhanced Raman scattering (SERS) and electrical sensing are found in orientational dependence. The SERS signals of vertically arranged GNR arrays exhibit double the folder intensity than those horizontally arranged. In contrast, the horizontally arranged GNRs exhibit twice as much electrical conductivity. The system is further explored to pattern mammalian cells. For the first time, we reveal the nanostructured topography of GNR confined cells to a specific region, and direct the adhesion and extension of living cells, which opens up broad applications in tissue engineering and biosensing.
Subcellular organelles, e.g., nucleus, mitochondria and lysosome, are the vital organelles with responsibilities that maintain cell operation and metabolism. Owing to their roles in energy production and programmed cell death, these organelles have become prime therapeutic targets in different diseases and states. In this study, biocompatible, organelle-targeting nanoprobes were developed by modifying gold nanorods (AuNRs) with specific targeting peptides. These nanoprobes were employed to directly profile subcellular biomolecules and vital organelles in situ by surface-enhanced Raman scattering (SERS) spectroscopy. Macromolecular spectral profiles of subcellular organelles were achieved and compared. Further, these organelle-targeting AuNRs were used for the photothermal treatment of cancer cells (HepG2, Hela and MCF-7 cell lines). The ell viability assays show that nucleus- and mitochondria-targeting AuNRs provide higher photothermal efficiencies under an 808 nm laser relative to the lysosome-targeting ones. This study makes critical insight into the spectral profiles of subcellular organelles, and also inspires people in the development of high-efficacy cancer therapeutic strategies by subcellular organelle-targeting drugs.
Hydrodynamic phenomena are ubiquitous in living organisms and can be used to manipulate cells or emulate physiological microenvironments experienced in vivo. Hydrodynamic effects influence multiple cellular properties and processes, including cell morphology, intracellular processes, cell–cell signaling cascades and reaction kinetics, and play an important role at the single-cell, multicellular, and organ level. Selected hydrodynamic effects can also be leveraged to control mechanical stresses, analyte transport, as well as local temperature within cellular microenvironments. With a better understanding of fluid mechanics at the micrometer-length scale and the advent of microfluidic technologies, a new generation of experimental tools that provide control over cellular microenvironments and emulate physiological conditions with exquisite accuracy is now emerging. Accordingly, we believe that it is timely to assess the concepts underlying hydrodynamic control of cellular microenvironments and their applications and provide some perspective on the future of such tools in in vitro cell-culture models. Generally, we describe the interplay between living cells, hydrodynamic stressors, and fluid flow-induced effects imposed on the cells. This interplay results in a broad range of chemical, biological, and physical phenomena in and around cells. More specifically, we describe and formulate the underlying physics of hydrodynamic phenomena affecting both adhered and suspended cells. Moreover, we provide an overview of representative studies that leverage hydrodynamic effects in the context of single-cell studies within microfluidic systems.
Mechanistic understanding of the endocytosis and intracellular trafficking of nanoparticles is essential for designing smart theranostic carriers. Physico-chemical properties, including size, clustering and surface chemistry of nanoparticles regulate their cellular uptake and transport. Significantly, even single nanoparticles could cluster intracellularly, yet their clustering state and subsequent trafficking are not well understood. Here, we used DNA-decorated gold (fPlas-gold) nanoparticles as a dually emissive fluorescent and plasmonic probe to examine their clustering states and intracellular transport. Evidence from correlative fluorescence and plasmonic imaging shows that endocytosis of fPlas-gold follows multiple pathways. In the early stages of endocytosis, fPlas-gold nanoparticles appear mostly as single particles and they cluster during the vesicular transport and maturation. The speed of encapsulated fPlas-gold transport was critically dependent on the size of clusters but not on the types of organelle such as endosomes and lysosomes. Our results provide key strategies for engineering theranostic nanocarriers for efficient health management.
Targeting nanoparticles to malignant tissues for improved diagnosis and therapy is a popular concept. However, after surveying the literature from the past 10 years, only 0.7% (median) of the administered nanoparticle dose is found to be delivered to a solid tumour. This has negative consequences on the translation of nanotechnology for human use with respect to manufacturing, cost, toxicity, and imaging and therapeutic efficacy. In this article, we conduct a multivariate analysis on the compiled data to reveal the contributions of nanoparticle physicochemical parameters, tumour models and cancer types on the low delivery efficiency. We explore the potential causes of the poor delivery efficiency from the perspectives of tumour biology (intercellular versus transcellular transport, enhanced permeability and retention effect, and physicochemical-dependent nanoparticle transport through the tumour stroma) as well as competing organs (mononuclear phagocytic and renal systems) and present a 30-year research strategy to overcome this fundamental limitation. Solving the nanoparticle delivery problem will accelerate the clinical translation of nanomedicine.
Cancer metastasis accounts for the high mortality of many types of cancer. Owing to the unique advantages of high specificity and minimal invasiveness, photothermal therapy (PTT) has been evidenced with great potential in treating cancer metastasis. In this review, we outline the current approaches of PTT with respect to its application in treating metastatic cancer. PTT can be used alone, guided with multimodal imaging, or combined with the current available therapies for effective treatment of cancer metastasis. Numerous types of photothermal nanotherapeutics (PTN) have been developed with encouraging therapeutic efficacy on metastatic cancer in many preclinical animal experiments. We summarize the design and performance of various PTN in PTT alone and their combinational therapy. We also point out the lacking area and the most promising approaches in this challenging field. In conclusion, PTT or their combinational therapy can provide an essential promising therapeutic modality against cancer metastasis.
In this manuscript we describe the preparation of an oxygen-loaded microbubble (O2MB) platform for the targeted treatment of pancreatic cancer using both sonodynamic therapy (SDT) and antimetabolite therapy. O2MB were prepared with either the sensitiser Rose Bengal (O2MB-RB) or the antimetabolite 5-fluorouracil (O2MB-5FU) attached to the microbubble (MB) surface. The MB were characterised with respect to size, physical stability and oxygen retention. A statistically significant reduction in cell viability was observed when three different pancreatic cancer cell lines (BxPc-3, MIA PaCa-2 and PANC-1), cultured in an anaerobic cabinet, were treated with both SDT and antimetabolite therapy compared to either therapy alone. In addition, a statistically significant reduction in tumour growth was also observed when ectopic human xenograft BxPC-3 tumours in SCID mice were treated with the combined therapy compared to treatment with either therapy alone. These results illustrate not only the potential of combined SDT/antimetabolite therapy as a stand alone treatment option in pancreatic cancer, but also the capability of O2-loaded MBs to deliver O2 to the tumour microenvironment in order to enhance the efficacy of therapies that depend on O2 to mediate their therapeutic effect. Furthermore, the use of MBs to facilitate delivery of O2 as well as the sensitiser/antimetabolite, combined with the possibility to activate the sensitiser using externally applied ultrasound, provides a more targeted approach with improved efficacy and reduced side effects when compared with conventional systemic administration of antimetabolite drugs alone.
We developed a new instrumental method by which human melanoma cells (LU1205) are sonoporated via radiation pressures exerted by highly-confined ultrasonic waves produced by high lateral-resolution ultrasonic micro-transducer arrays (UMTAs). The method enables cellular-level site-specific sonoporation within the cell monolayer due to UMTAs and can be applicable in the delivery of drugs and gene products in cellular assays. In this method, cells are seeded on the biochip that employs UMTAs for high spatial resolution and specificity. UMTAs are driven by 30-MHz sinusoidal signals and the resulting radiation pressures induce sonoporation in the targeted cells. The sonoporation degree and the effective lateral resolution of UMTAs are determined by performing fluorescent microscopy and analysis of carboxylic-acid-derivatized CdSe/ZnS quantum dots passively transported into the cells. Models representing the transducer-generated ultrasound radiation pressure, the ultrasound-inflicted cell membrane wound, and the transmembrane transport through the wound are developed to determine the ultrasound-pressure-dependent wound size and enhanced cellular uptake of nanoparticles. Model-based calculations show that the effective wound size and cellular uptake of nanoparticles increase linearly with increasing ultrasound pressure (i.e., at applied radiation pressures of 0.21, 0.29, and 0.40 MPa, the ultrasound-induced initial effective wound radii are 150, 460, and 650 nm, respectively, and the post-sonoporation intracellular quantum-dot concentrations are 7.8, 22.8, and 29.9 nM, respectively) and the threshold pressure required to induce sonoporation in LU1205 cells is ∼0.12 MPa.
Nanostructures of different sizes, shapes and material properties have many applications in biomedical imaging, clinical diagnostics and therapeutics. In spite of what has been achieved so far, a complete understanding of how cells interact with nanostructures of well-defined sizes, at the molecular level, remains poorly understood. Here we show that gold and silver nanoparticles coated with antibodies can regulate the process of membrane receptor internalization. The binding and activation of membrane receptors and subsequent protein expression strongly depend on nanoparticle size. Although all nanoparticles within the 2-100 nm size range were found to alter signalling processes essential for basic cell functions (including cell death), 40- and 50-nm nanoparticles demonstrated the greatest effect. These results show that nanoparticles should no longer be viewed as simple carriers for biomedical applications, but can also play an active role in mediating biological effects. The findings presented here may assist in the design of nanoscale delivery and therapeutic systems and provide insights into nanotoxicity.
The ability of collapsing (cavitating) bubbles to focus and concentrate energy, forces and stresses is at the root of phenomena such as cavitation damage, sonochemistry or sonoluminescence. In a biomedical context, ultrasound-driven microbubbles have been used to enhance contrast in ultrasonic images. The observation of bubble-enhanced sonoporation--acoustically induced rupture of membranes--has also opened up intriguing possibilities for the therapeutic application of sonoporation as an alternative to cell-wall permeation techniques such as electroporation and particle guns. However, these pioneering experiments have not been able to pinpoint the mechanism by which the violently collapsing bubble opens pores or larger holes in membranes. Here we present an experiment in which gentle (linear) bubble oscillations are sufficient to achieve rupture of lipid membranes. In this regime, the bubble dynamics and the ensuing sonoporation can be accurately controlled. The use of microbubbles as focusing agents makes acoustics on the micrometre scale (microacoustics) a viable tool, with possible applications in cell manipulation and cell-wall permeation as well as in microfluidic devices.
Cell mechanical motion is a key physiological process that relies on the dynamics of actin filaments. Herein, a localized shear-force system based on gigahertz acoustic streaming (AS) is proposed, which can simultaneously realize intracellular delivery and cellular mechanical regulation. The results demonstrate that gold nanorods (AuNRs) can be delivered into the cytoplasm and even the nuclei of cancer and normal cells within a few minutes by AS stimulation. The delivery efficiency of AS stimulation is four times higher than that of endocytosis. Moreover, AS can effectively promote cytoskeleton assembly, regulate cell stiffness and change cell morphology. Since the inhibitory effect of AuNRs on cytoskeleton assembly, this AuNRs-AS system is able to inhibit or promote cell mechanical motion in a controlled manner by regulating the mechanical properties of cells. The bidirectional regulation of cell motion is further verified via scratch experiments, in which AuNRs-treated cells recover their motion ability through AS stimulation. In particular, the results of AuNRs-AS mechanical regulation on cell are related to the intrinsic properties of cell lines, revealing to more obvious effects on the cells with higher motor capacities. In summary, this acoustic technology has shown superiorities in controllable cell-motion manipulation, indicating its potential in building a multifunctional, integrated cytomechanics regulation platform.
The development of rapid and efficient tools to modulate neurons is vital for the treatment of nervous system diseases. Here, a novel non-invasive neurite outgrowth modulation method based on a controllable acoustic streaming effect induced by an electromechanical gigahertz resonator microchip is reported. The results demonstrate that the gigahertz acoustic streaming can induce cell structure changes within a 10 min period of stimulation, which promotes a high proportion of neurite bearing cells and encourages longer neurite outgrowth. Specifically, the resonator stimulation not only promotes outgrowth of neurites, but also can be combined with chemical mediated methods to accelerate the direct entry of nerve growth factor (NGF) into cells, resulting in higher modulation efficacy. Owing to shear stress caused by the acoustic streaming effect, the resonator microchip mediates stress fiber formation and induces the neuron-like phenotype of PC12 cells. We suggest that this method may potentially be applied to precise single-cell modulation, as well as in the development of non-invasive and rapid disease treatment strategies.
The internalization of therapeutic molecules into cells-a critical step in enabling a suite of autologous ex vivo gene and cell therapies-is highly regulated by the lipid barrier imposed by the cell membrane. Strategies to increase the efficiency of delivering these exogenous payloads into the cell, while maintaining the integrity of both the therapeutic molecules to be delivered as well as the host cells they are delivered to, are therefore required. This is especially the case for suspension cells that are particularly difficult to transfect. In this work, we show that it is possible to enhance the uptake of short interfering RNA (siRNA) into nonadherent Jurkat and HuT 78 cells with a rapid poration-free method involving high-frequency (MHz order) acoustic excitation. The 2-fold enhancement in gene knockdown is almost comparable with that obtained with conventional nucleofection, which is among the most widely used intracellular delivery methods, but with considerably higher cell viabilities (>91% compared to approximately 76%) owing to the absence of pore formation. The rapid and effective delivery afforded by the platform, together with its low cost and scalability, therefore renders it a potent tool in the cell engineering pipeline.
Biological research and many cell-based therapies rely on the successful delivery of cargo materials into cells. Intracellular delivery in an in vitro setting refers to a variety of physical and biochemical techniques developed for conducting rapid and efficient transport of materials across the plasma membrane. Generally, the techniques that are time-efficient (e.g., electroporation) suffer from heterogeneity and low cellular viability, and those that are precise (e.g., microinjection) suffer from low-throughput and are labor-intensive. Here, we present a novel in vitro microfluidic strategy for intracellular delivery, which is based on the acoustic excitation of adherent cells. Strong mechanical oscillations, mediated by Lamb waves, inside a microfluidic channel facilitate the cellular uptake of different size (e.g., 3-500 kDa, plasmid encoding EGFP) cargo materials through endocytic pathways. We demonstrate successful delivery of 500 kDa dextran to various adherent cell lines with unprecedented efficiency in the range of 65-85% above control. We also show that actuation voltage and treatment duration can be tuned to control the dosage of delivered substances. High viability (≥91%), versatility across different cargo materials and various adherent cell lines, scalability to hundreds of thousands of cells per treatment, portability, and ease-of-operation are among the unique features of this acoustofluidic strategy. Potential applications include targeting through endocytosis-dependant pathways in cellular disorders, such as lysosomal storage diseases, which other physical methods are unable to address. This novel acoustofluidic method achieves rapid, uniform, and scalable delivery of material into cells, and may find utility in lab-on-a-chip applications.
Cell therapy and cellular engineering begin with internalizing synthetic biomolecules and functional nanomaterials into primary cells. Conventionally, electroporation, lipofection, or viral transduction has been used; however, these are limited by their cytotoxicity, low scalability, cost, and/or preparation complexity, especially in primary cells. Thus, a universal intracellular delivery method that outperforms the existing methods must be established. Here, we present a versatile intracellular delivery platform that leverages intrinsic inertial flow developed in a T-junction microchannel with a cavity. The elongational recirculating flows exerted in the channel substantially stretch the cells, creating discontinuities on cell membranes, thereby enabling highly effective internalization of nanomaterials, such as plasmid DNA (7.9 kbp), mRNA, siRNA, quantum dots, and large nanoparticles (300 nm), into different cell types, including hard-to-transfect primary stem and immune cells. We identified that the internalization mechanism of external cargos during the cell elongation-restoration process is achieved by both passive diffusion and convection-based rapid solution exchange across the cell membrane. Using fluidic cell mechanoporation, we demonstrated a transfection yield superior to that of other state-of-the-art microfluidic platforms as well as current benchtop techniques, including lipofectamine and electroporation. In summary, the intracellular delivery platform developed in the present study enables a high delivery efficiency (up to 98%), easy operation (single-step), low material cost (<$1), high scalability (1 × 106 cells/min), minimal cell perturbation (up to 90%), and cell type/cargo insensitive delivery, providing a practical and robust approach anticipated to critically impact cell-based research.
Here, we present a high performance uncooled near-infrared (NIR) detector comprising of a giga hertz (GHz) solidly mounted resonator (SMR) and gold nanorods (GNRs) arrays. By coupling the localized surface plasmon resonances of GNRs, the resonator system exhibits optimized optical response to vis-NIR region. Both simulation and experiments demonstrate the hybrid GNRs-SMR exhibit significantly enhanced optical responsive sensitivity of NIR, the tunable aspect ratios (AR) of GNRs enable resonator respond sensitively to selected light. Specially, taking advantage of the acoustofluidic effect of SMR, the GNRs can be controllably and precisely modified on the microchip surface in an ultra-short time, which addresses one of the most fundamental challenges in the localized functionalization of micro/nano scale surface. The presented work opens new directions in development of novel miniaturized, tunable NIR detector.
Intracellular delivery is essential to therapeutic applications such as genome engineering and disease diagnosis. Current methods lack simple, non-invasive 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 non-invasive 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 delivery of cationic fluorescent proteins to a range of cell lines, all of which displayed equally efficient delivery speed (≤ 20 minutes). Delivery of multiple enzymatically active proteins with functionality related to apoptosis or genetic recombination further demonstrated the relevance of this method.
In 1995, the year the first cancer nanomedicine, Doxil, was approved by the Food and Drug Administration (FDA), only 23 manuscripts appeared in a PubMed search for "nanoparticles for cancer" keywords. Now, over 25 000 manuscripts can be found using those same keywords, yet only 15 nanoparticle-based cancer nanomedicines are approved globally. Based on the clinicaltrials.gov database, a total of 75 cancer nanomedicines are under clinical investigation involving 190 clinical trials summarized here. In this Account, we focus on cancer nanomedicines that have been approved or reached clinical trials to understand this high attrition rate. We classify the various nanomedicines, summarize their clinical outcomes, and discuss possible reasons for product failures and discontinuation of product development efforts. Among ongoing and completed clinical trials, 91 (48 completed) are phase 1, 78 (59 completed) phase 2, and 21 (11 completed) phase 3. The success rate of phase 1 trials has been high-roughly 94%. Of those phase 1 trials with identified outcomes, 45 showed positive safety and efficacy results, with only one negative result (low efficacy) and two terminated due to adverse reactions. In some cases, findings from these trials have not only shown improved pharmacokinetics, but also avid drug accumulation within tumor tissues among active-targeting nanoparticles, including BIND-014, CALAA-01, and SGT-94. However, the success rate drops to ∼48% among completed phase 2 trials with identified outcomes (31 positive, 15 negative, and 4 terminated for toxicity or poor efficacy). A majority of failures in phase 2 trials were due to poor efficacy (15 of 19), rather than toxicity (4 of 19). Unfortunately, the success rate for phase 3 trials slumps to a mere ∼14%, with failures stemming from lack of efficacy. Although the chance of success for cancer nanomedicines starting from the proof-of-concept idea in the laboratory to valuable marketed product may seem daunting, we should not be discouraged. Despite low success rates, funding from the government, foundations, and research organizations are still strong-an estimated > $130 M spent by the National Institutes of Health (NIH) on R01s focused on nanomedicine in 2018 alone. In addition, the NIH created several special initiatives/programs, such as the National Cancer Institute (NCI) Alliance, to facilitate clinical translation of nanomedicines. Companies developing cancer nanomedicines raised diverse ranges of funds from venture capital, capital markets, and industry partnerships. In some cases, the development efforts resulted in regulatory approvals of cancer nanomedicines. In other cases, clinical failures and market pressure from improving standard of care products resulted in product terminations and business liquidation. Yet, recent approvals of nanomedicine products for orphan cancers and continuing development of nanoparticle based drugs for immune-oncology applications fuel continuing industrial and academic interest in cancer nanomedicines.
Acoustically driven bubbles produce a range of mechanical, thermal and chemical effects that can be exploited in drug delivery applications. Significant improvements in the targeting, distribution and efficacy of both current and emerging therapeutics can be achieved, from small molecules to biologics and nucleic-acid-based drugs. This Review describes how specially designed cavitation nuclei in the form of solid, liquid or gas particles can enable the triggered release of drugs, promote the permeabiliziation of challenging biological barriers and enhance drug delivery through tissue regions where diffusion alone is inadequate. Scalable strategies for mapping and controlling cavitation activity to harness its therapeutic potential at depth within the body are discussed, alongside current and emerging applications for the treatment of diseases, including cancer and stroke.
The intracellular delivery efficiency of drug-loaded nanocarriers is often limited by biological barriers arising from the plasma membrane and the cell interior. In this work, the entering of doxorubicin (Dox)-loaded mesoporous silica nanoparticles (MSNs) into cytoplasm was acoustically enhanced through direct penetration with the assistance of hypersound of gigahertz (GHz) frequency. Both fluorescence and cell viability measurements revealed that the therapeutic efficacy of Dox-loaded MSNs were significantly improved by tuning the power and duration of hypersound on demand with a nanoelectromechanical (NEMS) resonator. Mechanism studies with inhibitors illustrated that the membrane defects induced by the hypersound-triggered GHz acoustic streaming facilitated the Dox-loaded MSNs of 100-200 nm to directly penetrate through the cell membrane instead of via the traditional endocytosis, which highly increased the delivery efficiency by avoiding the formation of endosomes. This acoustic method enables the drug carriers to overcome biological barriers of the cell membrane and the endosomes without the limitation of carrier materials, which provides a versatile way of enhanced drug delivery for biomedical applications.
The successful intracellular delivery of exogenous macromolecules is crucial for a variety of applications ranging from basic biology to the clinic. However, traditional intracellular delivery methods such as those relying on viral/non-viral nanocarriers or physical membrane disruptions suffer from low throughput, toxicity, and inconsistent delivery performance and are time-consuming and/or labor-intensive. In this study, we developed a single-step hydrodynamic cell deformation-induced intracellular delivery platform named "hydroporator" without the aid of vectors or a complicated/costly external apparatus. By utilizing only fluid inertia, the platform focuses, guides, and stretches cells robustly without clogging. This rapid hydrodynamic cell deformation leads to both convective and diffusive delivery of external (macro)molecules into the cell through transient plasma membrane discontinuities. Using this hydroporation approach, highly efficient (∼90%), high-throughput (>1 600 000 cells per min), and rapid delivery (∼1 min) of different (macro)molecules into a wide range of cell types was achieved while maintaining high cell viability. Taking advantage of the ability of this platform to rapidly deliver large molecules, we also systematically investigated the temporal biostability of vanilla DNA origami nanostructures in living cells for the first time. Experiments using two DNA origami (tube- and donut-shaped) nanostructures revealed that these nanostructures can maintain their structural integrity in living cells for approximately 1 h after delivery, providing new opportunities for the rapid characterization of intracellular DNA biostability.
Nanomaterials can offer a chance to integrate many of excellent physical and chemical performances into a single carrier for smart responsive drug delivery. Herein, gold nanorods/mesoporous manganese dioxide (Au/MnO2) hybrid nanoparticles were prepared to combine the photothermal effect of gold nanorods (AuNRs) with glutathione (GSH)-responsive and pH-responsive performances of MnO2. The near-infrared (NIR) responsive constituent of Au/MnO2 nanoparticle was AuNRs. Doxorubicin hydrochloride (DOX), a widely used anticancer drug, was loaded into the Au/MnO2 hybrid nanoparticle via electrostatic force, hydrogen bond and physical absorption with a drug loading up to 99.1%. The results revealed that the mesoporous MnO2 was degraded in the media with high concentrations of GSH and acid microenvironment. The Au/MnO2 nanoparticles displayed satisfying drug release kinetics (ca. 47% of loaded drug released in 12 h) and showed excellent GSH-responsive, pH-responsive and NIR-responsive performances. This multi-responsive nanoplatform is expected to have wide biomedical application for cancer therapy such as photothermal therapy, drug delivery, and tumor microenvironment improvement.
Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo types—small molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery.
To develop and realize sonoporation-based macromolecule delivery, it is important to understand the underlying cellular bioeffects involved. It is known that an appropriate level of reactive oxygen species (ROS) is necessary to maintain normal physiologic function, but excessive ROS triggers adverse downstream bioeffects. However, it is still unclear whether a relationship exists between intracellular ROS levels and sonoporation. Using a customized platform for 1.5-MHz ultrasound exposure (13.33 µs duration and 0.70 MPa peak negative pressure) and imaging the dynamics of sonoporation and intracellular ROS at the single-cell level, we quantified the exogenous molecular uptake and the concentration of intracellular ROS indicator to evaluate the extent of sonoporation and ROS change, respectively. Our results revealed that the intracellular ROS level was correlated with the degree of the sonoporation. (i) Within ~120 s of the onset of ultrasound, during which membrane perforation and complete membrane resealing occurred, intracellular ROS rapidly decreased because of extracellular diffusion of dichlorofluorescein through the perforated membrane and positively correlated with the degree of the sonoporation. (ii) In the following 270 s (120-390 s post-exposure), ROS generation in reversibly sonoporated cells gradually increased and was positively correlated with the degree of the sonoporation. (iii) The ROS level in irreversibly sonoporated cells reduced to depletion during this time interval. It is possible that ROS generation in reversibly sonoporated cells can impact their long-term fate. These results thus provide new insight into the biological response to sonoporation.
Efficient delivery of genes and therapeutic agents to the interior of the cell is critical for
modern biotechnology. Herein, a new type of chemical-free cell poration method—
hypersonic poration—is developed to improve the cellular uptake, especially the
nucleus uptake. The hypersound (≈GHz) is generated by a designed piezoelectric
nano-electromechanical resonator, which directly induces normal/shear stress and
“molecular bombardment” effects on the bilayer membranes, and creates reversible
temporal nanopores improving the membrane permeability. Both theory analysis
and cellular uptake experiments of exogenous compounds prove the high delivery
efficiency of hypersonic poration. Since target molecules in cells are accumulated with
the treatment, the delivered amount can be controlled by tuning the treatment time.
Furthermore, owing to the intrinsic miniature of the resonator, localized drug delivery
at a confined spatial location and tunable arrays of the resonators that are compatible
with multiwell plate can be achieved. The hypersonic poration method shows great
delivery efficacy combined with advantage of scalability, tunable throughput, and
simplification in operation and provides a potentially powerful strategy in the field of
molecule delivery, cell transfection, and gene therapy.
As a minimally invasive therapeutic strategy, gold nanorod (AuNRs) -based plasmonic photothermal therapy (PPT) has shown significant promise for the selective ablation of cancer cells. However, the heat stress experienced by cells during the PPT treatment produces significant amounts of reactive oxygen species (ROS), which could harm healthy, untreated tissue near the point of care by inducing irreversible damage to DNA, lipids and proteins, potentially causing cellular dysfunction or mutation. In this study, we utilized biocompatible Pt-coated AuNRs (PtAuNRs) with different platinum shell thicknesses as an alternative to AuNRs often used for the treatment. We show that the PtAuNRs maintain the efficacy of traditional AuNRs for inducing cell death while scavenging the ROS formed as a byproduct during PPT treatment, thereby protecting healthy, untreated cells from indirect death resulting from ROS formation. The synergistic effect of PtAuNRs in effectively killing cancer cells through hyperthermia with the simultaneous removal of heat stress induced ROS during PPT was validated in vitro using cell viability and fluorescence assays. Our results suggest that the high photothermal efficiency and novel ROS scavenging activity of PtAuNRs makes them ideal candidates to improve the therapeutic efficacy of PPT treatment while reducing the risk of undesired side effects due to the heat-stress induced ROS formation.
For mitochondria-targeting delivery, a coupling reaction between poly(ε-caprolactone) diol (PCL diol) and 4-carboxybutyltriphenylphosphonium (4-carboxybutyl TPP) results in the synthesis of amphiphilic TPP-PCL-TPP (TPCL) polymers with a bola-like structure. In aqueous environments, the TPCL polymer self-assembled via cosolvent dispersion and film hydration, resulting in the formation of cationic nanoparticles (NPs) less than 50 nm in size with zeta-potentials of approximately 40 mV. Interestingly, different preparation methods for TPCL NPs result in various morphologies such as nanovesicles, nanofibers, and nanosheets. In vitro cytotoxicity results with TPCL NPs indicate IC50 values of approximately 10–60 μg mL−1, suggesting their potential as anticancer nanodrugs. TPCL NPs can be loaded both with hydrophobic doxorubicin (Dox) and its hydrophilic salt form (Dox·HCl), and their drug loading contents are approximately 2–10 wt% depending on the loading method and the hydrophilicity/hydrophobicity of the drugs. Although Dox·HCl exhibits more cellular and nuclear uptake, resulting in greater antitumor effects than Dox, most drug-loaded TPCL NPs exhibit higher mitochondrial uptake and approximately 2–7-fold higher mitochondria-to-nucleus preference than free drugs, resulting in superior (approximately 7.5–18-fold) tumor-killing activity for most drug-loaded TPCL NPs compared with free drugs. In conclusion, TPCL-based nanoparticles have potential both as antitumor nanodrugs themselves and as nanocarriers for chemical therapeutics.
Nanoparticles have been proposed as carriers for drugs, genes and therapies to treat various diseases. Many strategies have been developed to target nanomaterials to specific or over-expressed receptors in diseased cells, and these typically involve functionalizing the surface of nanoparticles with proteins, antibodies or other biomolecules. Here, we show that the targeting ability of such functionalized nanoparticles may disappear when they are placed in a biological environment. Using transferrin-conjugated nanoparticles, we found that proteins in the media can shield transferrin from binding to both its targeted receptors on cells and soluble transferrin receptors. Although nanoparticles continue to enter cells, the targeting specificity of transferrin is lost. Our results suggest that when nanoparticles are placed in a complex biological environment, interaction with other proteins in the medium and the formation of a protein corona can 'screen' the targeting molecules on the surface of nanoparticles and cause loss of specificity in targeting.
Noble metal nanoparticles are capable of confining resonant photons in such a manner as to induce coherent surface plasmon oscillation of their con- duction band electrons, a phenomenon leading to two important properties. Firstly, the confinement of the photon to the nanoparticle's dimensions leads to a large increase in its electromagnetic field and consequently great enhancement ofall the nanoparticle's radiative properties, such as absorption and scattering. Moreover, by confining the photon's wavelength to the nanoparticle's small dimensions, there exists enhanced imaging resolving powers, which extend well below the diffraction limit, a property of con- siderable importance in potential device applications. Secondly, the strongly absorbed light by the nanoparticles is followed by a rapid dephasing of the coherent electron motion in tandem with an equally rapid energy transfer to the lattice, a process integral to the technologically relevant photothermal properties of plasmonic nanoparticles. Of all the possible nanoparticle shapes, gold nanorods are especially intriguing as they offer strong plasmonic fields while exhibiting excellent tunability and biocompatibility. We begin this review of gold nanorods by summarizing their radiative and nonradiative properties. Their various synthetic methods are then outlined with an emphasis on the seed-mediated chemical growth. In particular, we describe nanorod spontaneous self-assembly, chemically driven assembly, and poly- mer-based alignment. The final section details current studies aimed at applications in the biological and biomedical fields.
A novel two-step protocol for intracellular drug delivery has been evaluated in vitro. As a first step TO-PRO-3 (a cell-impermeable dye that displays a strong fluorescence enhancement upon binding to nucleic acids) encapsulated in thermosensitive liposomes was released after heating to 42°C. A second step consisted of ultrasound-mediated local permeabilization of cell membrane allowing TO-PRO-3 internalization observable as nuclear staining. Only the combination of two consecutive steps - heating and sonication in the presence of SonoVue microbubbles led to the model drug TO-PRO-3 release from the thermosensitive liposomes and its intracellular uptake. This protocol is potentially beneficial for the intracellular delivery of cell impermeable drugs that suffer from rapid clearance and/or degradation in blood and are not intrinsically taken up by cells.