nanoscale intracellular delivery

NPE-00045; No of Pages 14 Nanotechnology and Precision Engineering xxx (xxxx) xxx

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Recent advances in micro/nanoscale intracellular delivery Mengjie Sun, Xuexin Duan ⁎ State Key Laboratory of Precision Measuring Technology & Instruments, Tianjin University, Tianjin, 300072, China

a r t i c l e

i n f o

Available online xxxx

Keywords: Drug delivery Physical approaches Cell membrane disruption Low dimension

a b s t r a c t 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. Copyright © 2019 Tianjin University. Publishing Service by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction The safe and efficient intracellular delivery of biologically active macromolecules into living cells is a critical and challenging process in biotechnology.1,2 For many different applications, such as cell analysis,3 drug therapy, and gene transfection,4 a range of materials, including small molecules, nucleic acids, proteins, and nanomaterials, must be delivered to different kinds of cells. Given the strict regulation of the plasma membrane, direct translocation of external materials is largely prevented.5 For example, the cell membrane is an impassable barrier for small hydrophilic molecules. Macromolecules (such as DNA, RNA, and proteins) are hardly uptaken by cells without external help.6 Table 1 summarizes the typical molecules and reagents that are currently important in cell biology and their challenges for intracellular delivery. Current transfection methods still feature many limitations. For example, the delivery of large molecules into immune cells, stem cells, and neurons remains difficult.7,8 The delivery normally requires vectors, such as viruses or peptides, specific to target molecules.9 In addition, batch processing of cells often results in cell damage or drug residue heterogeneity. Thus, the precise dose control and drug therapy must be critically provided at the single-cell level. The development of delivery methods that can improve safety, speed, cost, and efficiency

⁎ Corresponding author. E-mail address: [email protected] (X. Duan).

to achieve efficient delivery of various molecules to various cells remains a long-term challenge.10 Current intracellular delivery can be generally divided into two approaches: carrier-based methods and membrane disruption techniques (Fig. 1). In the delivery of materials with a carrier, the carrier can completely pack the cargo and prevent its degradation. A carrier can also utilize its own properties to enter the desired intracellular compartment and release the payload at appropriate time and conditions. As a promising delivery method with a long history of research, viral vectors are delivered into cells by means of viral infection, which requires no external assistance.12,13 Although the viral vectors possess the advantages of high efficiency and specificity, the nature of viruses causes inevitable problems, such as in vivo immune response, vector safety, and manufacturing complexity. Given these challenges, biomimic lipid nanocarriers have become the most advanced nonviral vectors in nucleic acid delivery, because they avoid the effects of counterpart limitations. Nonetheless, the exact escaping mechanism remains unclear. Most carriers require cellular uptake through endocytic pathways.14–16 Thus, only limited combinations of cargo materials and cell types are available. Certain carriers can fuse with the target membrane through membrane fusion process assisted by membrane proteins.17 Therefore, the direct delivery of cargo by the carrier with fusion capability can avoid endocytosis.18 Vesicles or microvesicles may fuse with target cells and deliver macromolecules, such as DNA or proteins, to avoid the delivery process and cytotoxicity of synthetic vectors.19,20 The exact fusion mechanism still needs further study. Target cells and potential cargo materials may be inapplicable or escape pathways.

https://doi.org/10.1016/j.npe.2019.12.003 2589-5540/Copyright © 2019 Tianjin University. Publishing Service by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Table 1 Common target materials for intracellular delivery. 11 Cargo

Category

Small molecules

All the small molecular structures that have biological significance.

Challenge

Structural diversity. Chemical diversity. Dependent transfer technique. Nanomaterials Quantum dots, nanoparticles (NPs), Structural diversity. and carbon nanotubes. Chemical diversity. Dependent transfer technique. Cytotoxicity. The immune response. Nucleic acids All oligonucleotides including DNA, DNA needs to be delivered RNA, siRNA, mRNA, and miRNA. to the nucleus. RNA is unstable. Integration of DNA and genome causes security accidents. Proteins All amino acid combinations, Difficult to produce and to including antibodies, short peptides, purify. structural proteins, and May cause overexpression. transcription factors. Features structural diversity and very sensitive tertiary structure.

By contrast, membrane disruption uses physical methods, including mechanical, electrical, thermal, optical, chemical stimulation, etc. These methods could generate discontinuous and transient nanopores in the plasma membrane, resulting in the increased pore size of the membrane to allow the diffusion of exogenous molecules or direct penetration to cell membranes with solid conduits or carriers to release cargo in cells. This approach is based on the rapid perturbation and healing of the cell membrane, by which almost any type of submicron material can be delivered regardless of carrier properties. Membrane disruption not only facilitates nucleic acid delivery but also protein delivery, including the delivery of antibodies,21 transcription factors, and genome-editing nucleases.22,23 However, traditional membrane disruption methods often pose challenges, such as (1) irreversible damages to cells due to excessively strong physical disruption and reduced delivery efficiency due to inadequate stress; (2) limited throughput and scalability of several methods; (3) physical disruption to whole cell population, resulting in passive operation of non-target cells. Continuous efforts have been made to improve intracellular delivery efficiency to solve these issues. In recent years, with the rapid development of micro- and nanotechnologies, including microfluidic system and lab-on-chip techniques, membrane disruption technologies at

Fig. 1. Two approaches for intracellular delivery and their basic mechanisms.

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micro/nanoscale are gradually emerging as alternative approaches for intracellular delivery. Owing to their high-precision mechanical operation provided by modern micromachining technology, micro/nanoscale membrane disruptions can now accurately manipulate membrane perturbations at the single-cell and subcellular levels. Micro-nano systems can also provide accurate drug release rate and time control, avoiding the unnecessary toxic reactions caused by excessive drug concentration at the macro scale. Furthermore, given the size compatibility, these technologies can be easily integrated with other microfluidics or labon-chips, largely facilitating the downstream cell analysis. In the following sections, we will introduce the current methods of membrane disruption of interest in the field of micro/nanotechnology and their advantages and challenges. 2. Recent advances in micro/nanoscale intracellular delivery The earliest membrane disruption-based intracellular delivery can be traced back to the microinjection technique in 191128. Electroporation transfection DNA technique was proven to be viable in 1982,24 which led to the development of other membrane disruption techniques, including optoporation25 and sonoporation.26 Many membrane destruction techniques were later abandoned due to drawbacks, such as low throughput, high cost, and technical operation, except electroporation, which was widely adapted. During the last decade, membrane disruption technology has been extensively combined with micro/ nanotechnology, microfluidics, and lab-on-chip approaches and is being developed to create more new opportunities. 2.1. Micro/nanoneedles Intracellular delivery dates back to the work of Barber et al. 25 who used a very thin glass needle filled with injection solutions to inoculate living cells with substances such as bacteria. Traditional microinjection techniques typically require cells to be immobilized on a substrate or to be held in place by additional precision devices. The injection process also requires the technician to possess precise and skilled operation, which is slow and can only inject one cell at a time, thus limiting the throughput. In recent years, the introduction of technologies, such as automation equipment28 and robotic systems,29 has considerably improved the therapeutic efficiency of microinjection technology, but considerable throughput and scalability still need to be realized. Based on microinjection research, studies have shown that one-dimensional microscale structures (microneedles) with sharp tips can penetrate skin, cells, and other tissues, which enabled drug delivery and biological therapy. Microneedles with a wide range of lengths (50–500 μm) have been applied in intradermal delivery applications.30,31 Until the mid-1990s, with the development of microelectronic industry, microneedles were considered as an important research topic for drug delivery.32 Microneedles can easily penetrate the human skin without irritate the nerves, allowing drug penetration in a gentle and painless manner.33 Micromachining technology can fabricate microneedles with different types of materials, including semiconductors, metals, and polymers.34 In general, polymer microneedles are widely used owing to advantages of low toxicity, production cost, and risk of waste and good biocompatibility.35 Integrating microneedles onto traditional patches has shown that a variety of drugs36–39, including proteins, antibodies, and vaccines can be successfully achieved. Polymeric microneedle patches are used in four different ways using to deliver drugs (Fig. 2a). (1) Coated microneedles. The drug molecules are coated on the microneedle indication; then, the microneedle could penetrate the skin cuticle to release the drugs. (2) Dissolvable microneedles. Microneedles are made by mixtures of soluble polymers and therapeutic drugs. Microneedles require hours or days to fully dissolve and release the drug after being inserted into the skin. (3) Degradable microneedles. Microneedles and patches are made from biodegradable polymers. The drug will be released into the skin as the polymer

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hydrolyzes. Thus, the sustained release of the therapeutic agent in a constant dose can be achieved. (4) Bioresponsive microneedles. Microneedle patches can respond to specific biosignals or environmental changes. Microneedles can deliver insulin and growth hormone through animal skin. In recent years, studies have also shown that microneedles can carry degradable NPs and release browning agents for obesity treatment.40,41 In addition, glycemic control of mouse models is achieved using bioresponsive microneedle patches.42 Most microneedles contain tips, which measure tens of microns, that cannot be precisely located to a single cell, thus resulting in the nonuniform delivery of plasmids or other macromolecules. Nanoneedles with shorter lengths (b1 μm) and sharp tips (b100 nm) were then developed to solve the issue. They provide better precision at the singlecell level. In 2007, Kim et al.43 first demonstrated the penetration of nanowire (NW) arrays into cells, which allowed the gene delivery to mammalian stem cells. On this basis, different nanostructures, including nanoneedles, nanopores, nanostraws, and other nanoscale structures, have been developed. These works use needle-like (wire) structures with high aspect ratio to directly penetrate the cells under the action of gravity, the cells are inserted to form pores by nanoscale wires. Kwak et al. described in detail the key role of NW arrays in cell function-related applications. Penetrating NWs could efficiently deliver biomacromolecules and photoelectric stimulation, and high-density non-penetrating NWs could be used to explore the interactions between nanostructured substrate and cell surface.44 When the NW penetrates the cell, the molecules could dissociate into the cytoplasm. Therefore, the NW structure can be modified or doped with the target molecules by direct fabrication through micro/nanotechnology.45 In this case, biological macromolecules can enter cells without chemical modification or virus packaging. Shalek et al.46 prepared vertical NWs by chemical vapor deposition or standard semiconductor technology. Previous studies have shown that cells could be spontaneously penetrated while being placed on the NW.43,47–50 In Shalek's work, 1,1Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine(DID)-labeled membrane HeLa cells were inoculated on green fluorescently labeled NW and observed this penetration process (Fig. 2b). This platform could introduce siRNA, peptides, DNA, proteins, and impermeable inhibitors into challenging cell types, such as neurons and immune cells. However, the NWs failed to penetrate cell immediately after contact. They required the principal stress caused by cell diffusion or the tension created by the adhesion of the cell membrane to the NWs.51 The nanostraw works similarly to microinjection. The high aspect ratio of nanostraws allows their direct insertion into cells. Then, biomolecules can diffuse directly into the cell from the external environment through the hollow structure, and the molecular delivery efficiency can be accelerated with electrophoresis.52 Xu et al.53 created the rapid transmission of second messenger Ca2+ by using a nanostraw device, without chemical carrier and genetic modification. They cultured the cells for 24 h on the nanostraw array (Fig. 2c) and then combined the nanostraw array with the flow channel; thus, the flowing solution could flow through the bottom of the array, enabling Ca2+ from the solution to codiffuse into hundreds of cells over several seconds. This method can achieve the effective control of both intracellular delivery time and drug dosage. However, nanoscale straws are relatively difficult to use in terms of manufacturing process and cost, and their fixed size is limited for specific applications. Given these conditions, breakthroughs and simplification are required in processing technology. In view of these problems, He et al.54 proposed a simpler preparation method by using O2 plasma etching to prepare nanostraws with controllable parameters of different structures; they proved that DNA transfection can be achieved by combining a nanopipette with a microfluidic device or external technology. This work advanced the use of nanostraws for a wide range of biomedical devices. Nanoneedles can directly penetrate the cell membrane to form nanoscale holes. After the needle is withdrawn, exogenous molecules could diffuse into the cytoplasm before the holes are healed. Park et al.

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Fig. 2. (a) Schematic of the mechanism by which a polymer microneedle patches deliver drugs; (b) Si NWs as nanoneedles for intracellular delivery at the single-cell level46; (c) microfluidic device integrated with nanostraw structures.53

fabricated nanoneedle arrays55,56 with 12 μm height and tapered to b30 nm in diameter by etching of silicon.57 The density of the nanoneedle arrays ensures that each cell could be punctured by at least one nanoneedle. The single-layer adherent cells were loaded with a puncture (pressed into the cells by controlling the loading force and puncture speed) or centrifugation (fixing the nanoneedle array on the centrifuge device, adjusting the different centrifugation speed and time, and rotating the suspension cells in the solution) to compare the efficiencies of intracellular delivery of the two loading methods.58 Zhu et al. selected a diamond with superior mechanical properties and chemical inertness to prepare the nanoneedle arrays.59 They rinsed the suspension cells onto a dense array of nanoneedles for a one-time, high-throughput, cellular mechanical poration of the cells. Given that the nanoneedles are short and condensed adequately, the cell membranes were slightly disrupted to enhance molecular diffusion without

puncturing cells. This work had led to effective drug delivery to resistant cancer cells and is expected to be used to treat multiple resistant cancer cells.59 All three forms of one-dimensional nanostructures significantly improve the accuracy of drug delivery beyond traditional microneedle transdermal delivery and risky microinjection. The NW/needle can be fixed on the support base, effectively avoiding the accumulation of toxicity of suspended nanomaterials (nanotubes, NPs, and suspended NWs) in cells.60–62 However, the area of the nanoneedle substrate limits the number of cells cultured on the nanotip structure, and cell culture and membrane permeation also require a long time. Collecting the released cells from nanoneedle substrates is a difficulty that still requires resolution.63–65 When delivering naked plasmid DNA that is not complexed with lipofectamine, the transfection rate is extremely low, probably because the nanoneedle array could not simultaneously process the cell and nuclear membranes. The diffusion of delivered DNA in the

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cytoplasm is restricted by structural proteins,66 which might degrade and prevent their entrance to the nucleus. Notably, the direct penetration of the cell membrane through the mechanical structure often causes irreversible cell damages. This condition requires the continuous innovation and optimization of preparation processes and materials of micro/nanoneedles to provide high delivery efficiency while reducing cell damage. Therefore, noncontact stimulation of the external field to induce cell membrane permeability can bring more interesting benefits and more application prospects. 2.2. Electroporation Electroporation is a relatively trivial physical method. This process exposes the cells to an electric field, applying a voltage on the cell membrane to form a transmembrane potential difference of about 0.5 V67 to induce transient pores in cell membrane, whereas exogenous substances in the surrounding environment are effectively delivered into the cell by electrophoresis or promotion of the diffusion effect (Fig. 3a). The earliest electroporation report was released in 1982. Neumann et al. used a pair of parallel plates to apply a certain voltage to disperse suspended cells and proved that electroporation could be used for mammalian DNA transfection. The advantages and disadvantages of three electroporation scales (the earliest developed bulk electroporation method and micro−/nano-electroporation technology) were summarized by Yang et al.68 In contrast to macroscale methods, electroporation combined with micro/nanotechnology can locate

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electric fields at the single-cell level, considerably reduce the voltage and heat effect, and avoid cell damages caused by high voltage.69 Chang et al.70 designed a microfluidic system which contained nanopore array and a series of interlaced U-shaped microcap structures (Fig. 3b and c). After immersing and lifting the chip vertically into the cell suspension, the U-shaped structure kept pointing upward. The cells could be efficiently captured in the U-shaped structure by gravity and hydrodynamics and enabled good contact with the 400 nm nanopores. The cells contacting with the nanopores can be electroporated directly by the pulsed electric field, and the surfacecharged macromolecule can quickly and directly enter the cells through the nanopores by electrophoresis. This platform can effectively capture and transfect primary mouse cardiomyocytes, a process that is almost impossible to achieve by traditional electroporation because of the large number of ion-transport proteins on sarcolemma. This condition often leads to ion channel activation or abnormalities, resulting in high cell death rate and low transfection efficiency. Given that the pore size of electroporation is extremely small to be observed with an optical microscope, Guo et al.71 adopted an electrical measurement method to detect physiological behaviors inside and outside of the cell and comprehensively studied the cell electroporation process. The novel microarray chip comprised four electrode units, each of which had a pair of central electrodes at its center. The chip was assembled on the printed circuit board, and the polydimethylsiloxane (PDMS) cavity with inlet and outlet was attached to the chip to form a cell sample chamber (Fig. 3d). After the cell suspension was inserted

Fig. 3. (a) Schematics of microfluidic electroporation68; (b) rapid capture of cells onto nanostructures by dipping-trap method; (c) nanochannel electroporation platform70; (d) schematic of the microarray chip combining (i) cell positioning, (ii) electroporation, and (iii) impedance measurement.71

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into the chip and stabilized, it was captured by the negative dielectrophoresis force and positioned to the center electrode. Then, the cells were electroporated in situ by the two center electrodes. After electroporation, impedance analysis can be performed to detect the cellular dynamic processes.71 This setup is considerably less complicated compared with a patch clamp72,73 that could characterize singlecell electroporation. Moreover, impedance analysis integrates multiple functions, such as selective in situ electroporation, cell array localization, and real-time electrical, measurement, and might have potential applications in tumor therapy and pathological analysis. Recently, microfluidic electroporation device has been developed for better transfection efficiency and cell viability.74,75 However, given the limited size of microfluidic channels, the cell processing speed must be sacrificed to achieve the balance between high flux and low voltage, which is particularly important in the research of micro-nanoscale drug delivery. 2.3. Optoporation Using light in the form of a focused laser for cellular drug delivery, optical transfection has also received research attention. As a noncontact method, optics-based technology is important for the manipulation of biological materials at the micron and submicron scale.76 Small transient pores can be created by a variety of light forms to allow the delivery of plasmid DNA and other macromolecules. Light energy leads to electron plasma, which causes the photochemically induced degradation of the membrane. Meanwhile, light energy can also generate cavitation bubbles with an ultrashort lifetime on the membrane. Several laser systems, including continuous-wave, pulsed picosecond, and nanosecond lasers, have been used for optically mediated membrane.77 These techniques afford a noncontact, fast, and sterile method to introduce membrane impermeable molecules or uses a vector to deliver the

drug of interest. The efficiency of delivery strongly depends on the cell and/or drug types. Studies have used different forms of light to directly introduce macromolecules into cultured cells. Using ultrafast pulsed light for optical transfection offers selective targeting and high efficiency and viability.78 The ultrafast laser beam operates over a very small area of action on cells, which limits its capability for full transfection. Dhakal et al.79 used ultrafast near-infrared ray (NIR) laser microbeam to deliver both single opsins and large multi-opsin constructs to target cells. The optical delivery of multiple opsin-encoding genes leads to targeted expression and white-light activation.80 The delivery of large fusion constructs of multiple spectrally separated opsins was achieved by ultrafast NIR laser-mediated optoporation to obtain a high cell sensitivity to ambient broadband light, leading to visual restoration. This technology provides a novel idea for the functionalization of optical transfection. Although various optical technologies are constantly evolving, many problems remain regarding the intracellular delivery of genes/transcripts, including the low quality and reproducibility. Combining optical transfection with microfluidic technology has been popularly used to increase the transfection efficiency and throughput. Uchugonova et al.81 built an ultrashort femtosecond (fs) lasermicrofluidic cell transfection platform to achieve optical reprogramming of large cell populations. In a microfluidic tube with multiple genes, ultrashort laser pulses induce transient membrane permeabilization, which enables the production of high-quality and contamination-free induced pluripotent stem cells (Fig. 4a).81 Schomaker et al. 82 proposed another optical transfection method combined photosensitive materials with laser treatment (Fig. 4b). They incubated gold NPs (AuNP) with cells and exposed them to fslaser pulses. Photosensitive chemicals localized to the membranes of endocytic vesicles could be activated by laser stimulation to induce localized membrane permeabilization of the cell.82 Cell processing for

Fig. 4. (a) Illustration of fs-laser-microfluidic transfection platform81; (b) schematic of AuNP-mediated optoporation principle.82

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gene and cell therapies generally use several separate procedures for gene transfer and cell separation or elimination. Lukianova-Hleb et al.83 developed a new approach of using plasmonic nanobubble (PNB) for simultaneous transfection of target cells and elimination of unwanted subsets of other cells. Transient PNBs were generated around a AuNP after a short-term laser irradiation. Laser energy was converted into heat, causing a nanoscale explosion for transmembrane injection of molecular cargo. Depending on cell specificity, PNBs of different sizes were generated around cell-targeted gold nanoshells and nanospheres. Simultaneously, high efficacy, selectivity, and no-damage delivery of both molecular cargo and cell destruction were achieved using laser pulse bulk treatment. Compared with other existing methods, this technology provides simultaneous and cell-specific multifunctionality. More importantly, laser pulse bulk treatment has also been used in hard-totransfected cells, such as primary neurons and stem cells, and in difficult scenarios such as in a living embryo.84 The instrumentations required, however, are generally expensive, complicated, and bulky. 2.4. Sonoporation Although light/electroporation can solve several intracellular delivery challenges, their application in delivering some specific proteins and nanomaterials is still limited. The damage caused by the energy field and cytotoxicity are also unsolved. By contrast, ultrasound as a mild technology can promote the drug/gene delivery to cells (nuclei).85–89 Ultrasonic stimulation can alter the cell membrane permeability, thereby allowing the extracellular molecular absorption in a phenomenon called sonoporation. In 1986, Fechheimer M et al. first used ultrasound to load exogenous macromolecules into suspension cells90 and then transfect DNA of mammalian cells. Sonoporation can interfere with cell membrane permeability in two ways: ultrasound microbubbles (cavitation) and acoustic stimulation. In past studies, ultrasound was often used in combination with microbubbles, because the addition of microbubbles causes cavitation and enhances the effect of sonoporation. Cavitation uses the pressure phase of ultrasonic waves to cause the microbubbles to alternately contract and expand.91 Cavitation bubbles generally refer to sealed bubbles (10 μm in diameter) which were originally developed for use in ultrasonic imaging.92 The microbubbles vibrate steadily or collapse violently when exposed to ultrasound, causing the liquid flowing around to oscillate to generate sufficient shear force to open up nearby cell membranes.93 An ultrasound field can focus on local tissues and organs in the microbubbles, thus improving targeted drug delivery.94,95 This technology has been extensively studied and is expected to be an effective tool for gene delivery.96 Wang et al.97 incorporated a green fluorescence protein–αtubulin fusion protein to label the alpha-tubulin cytoskeleton of HeLa cells and then stimulated these cells with a single 1 MHz pulsed ultrasound and microbubbles (Fig. 5a and b). When the acoustic pressure increased, or when the distance between the microbubbles and the cells decreased, significant cell deformation could be observed, hence enhancing the membrane permeability and disintegration of the cytoskeleton. Therefore, the proper control of acoustic energy and microbubblecell distance can effectively improve the efficiency and safety of ultrasound therapy.97 Horsley et al.98 used ultrasound to activate microbubbles to deliver high concentrations of drugs into urothelial cells. As a common disease, urinary tract infection still lacks efficient treatment. Oral antibiotics cannot penetrate the bladder wall well enough to accumulate to an effective concentration.99 Therefore, in their work, the drugs and cultured cells were exposed to the ultrasound chamber100 with effective parameters, proving that the amount of ultrasound-activated microbubbles released in the cells was 16 times higher than that without microbubbles. This finding could affect traditional oral antibiotic treatments. Although evidence supports the therapeutic efficacy of ultrasound-driven microbubbles, problems with the transformation from basic research to clinical research still need to be addressed. Roovers et al.101 focused on acoustic settings and

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microbubble-related parameters and envisioned new technologies that provide additional control over treatment to provide better microbubble-assisted ultrasonic treatment program. However, sonoporation often causes irreversible cell damage,102 and many studies have discovered that the regeneration and colony formation ability of cells after ultrasonic radiation are also relatively reduced.103 The precise control of microbubbles and the exogenous chemicals used to generate microbubbles remains urgent. Without microbubbles, the acoustic formed by high-frequency sound pressure (N10 MHz) only stimulates cell membrane permeability, which has attracted people's attention. For example, Ding et al.104 specifically discussed the basic principle and classification of surface acoustic waves (SAWs). Compared with cell exposure to low-frequency (b1 MHz) bulk ultrasonic wave, SAWs with highfrequency (N10 MHz) electromechanical Rayleigh waves can effectively eliminate cavitation and excessive shear damage to cells. Xie et al.105 (2019) specifically described the theory of SAW and its interaction with particles and contact fluids and divided the SAW fluids into two types: the traveling saw (TSAW) and the standing saw (SSAW). The SAW device is composed of a lithium niobate singlecrystal piezoelectric substrates and alternating finger pairs of straight interdigitated transducer (IDT) placed on a piezoelectric base. For SAW-promoted drug delivery, AC electrical signal is applied to the IDT, and acoustic waves are generated and transmitted to the well plate where cells are cultured. The cells are then exposed to the transient stimulation of high-frequency acoustic pressure, which would effectively promote membrane lipid reorganization. AuNPs and macromolecules have been delivered into cells assisted with SAW. 106 The experimental results showed that this method increased the delivery efficiency of 20 kDa dextran by N2-fold and that of 250 kDa dextran by 1.5-fold (Fig. 5c). Given that cell membranes reseal almost instantly after acoustic stimulation stops, this technique can efficiently ensure cell viability. Yoon et al.107 developed an acoustic-transfection technique using a high-frequency ultrasonic transducer with a center frequency of N150 MHz in combination with a fluorescence microscope. The transducer relied on a programmed displacement platform to accurately adjust the position, and a fluorescence microscope could detect changes in the fluorescence intensity of the treated cells. In general, the focused area of low-frequency ultrasound at 1–5 MHz usually reaches the millimeter level, which often affects a large number of cells. However, this acoustic-transfection technique can restrain the area with a 10 μm diameter, and the transfection technology can be realized at the single-cell level through this focusing capability (Fig. 5d). This technology enables the intracellular delivery of a variety of DNA plasmids, mRNA. and recombinant proteins without microbubbles. Acoustic transfection can also provide a CRISPR/Cas9 system to modify and reprogram the genome of a single living cell.107 Hypersound, defined as ultrasound with frequency N 1 GHz and generated by bulk acoustic wave resonator, has been recently reported for drug delivery applications.108 In this type of device, thin-film piezoelectric material is sandwiched between two metal electrodes to achieve a high resonate frequency. Such structure also guarantees the device stability at high power input (up to a few watts). The generated acoustic waves propagate along the axial plane, further actuating the fluid motion.109 When the GHz resonator contacts with the liquid, the acoustic wave energy could be attenuated rapidly, and a strong body force is generated at the device–liquid interface.110 Lu et al.112 fabricated the GHz resonator with a field effect transistor (FET) on the same chip. Such composite device was applied to investigate the hypersound poration effect on supported lipid bilayers (SLBs) in real time. Cyclic voltammetry, atomic force microscopy, and laser scanning microscopy were used together to characterize the nanopores.114 The relationship between membrane deformation and poration induced by hypersound was carefully studied. As shown in Fig. 5e, SLB was covered on the resonator, and the gold electrode connected with FET. The results showed

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Fig. 5. (a) Schematic of cavitation100; (b) schematic of the acoustic exposure apparatus used to investigate the intracellular delivery of fluorescent marker and cytoskeleton dynamics induced by sonoporation97; (c) side (top) and perspective (bottom) view schematics of the experimental setup105; (d) acoustic transfection of adherent cells106; (e) schematic of the integrated sensing system111; (f) hypersonic wave generated by a nanoelectromechanical resonator.107

that the hypersound propagation deformed SLB and produced nanopores. Thus, ions in the buffer solution could diffuse across the membrane and induce potential changes across the membrane.

Zhang et al.108 used the GHz resonator to develop a novel cell poration method. Hypersound was used to stimulate cells and induce the transient nanopores to achieve efficient delivery of exogenous

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molecules. In this platform, cells were cultured at the bottom of the chamber, and the target solution was added into the chamber. The hypersound device was placed on the top of the chamber, and the position of the device was adjusted to optimize poration. DOX is a typical drug usually delivered into different cell lines, such as HeLa and 3T3 cells, at appropriate input power and treatment time (Fig. 5f). The application of hypersound not only promotes the entry of DOX into cells but also facilitates the uniform distribution of DOX in the nucleus. The resulting strong acoustic streaming could exert large normal and shear stress on cells and induce the transient nanopores in cell membrane to improve the membrane permeability. Hyper-sonication requires no micro bubble assistance. Thus, this process features advantages compared with the conventional sonoporation. This work is a breakthrough in the field of cell uptake, especially in solving the nuclear membrane barrier and cytoplasmic transport. Following this work, polymerwrapped mesoporous silica NPs encapsulated with DOX were successfully delivered.113 This research showed that hypersound could promote drug carriers of 100–200 nm to penetrate the cell membrane directly, avoiding the slow release of endocytosis and the formation of endosomes.114,115 This acoustic method is fast and efficient for cell or tissue treatment, showing great potential for further delivery of larger molecules or cargo. 2.5. Microfluidic system Electroporation and sonoporation cause low cell viability or may limit the delivery of materials due to electrical charges. Thus, recent microfluidic delivery methods based on rapid cellular mechanical deformation have become a prominent alternative in certain applications.116–118 In general, microfluidic constriction results in mechanical deformation of cells by passing them through a narrow area 30%–80% smaller than the cell diameter. The shear force and pressure generated by this process can induce transient holes and promote passive diffusion of macromolecules into cytoplasm. Membrane pores can be rapidly sealed after intracellular delivery.119 This method is simple, controllable, fast, high-throughput, and suitable for delivery of almost any macromolecule into almost any cell type. This system shows potential in previously challenging cell types (primary immune cells and stem cells) and materials. Szeto et al.120 devised a simple approach that relied on microscale cell squeezing and passive diffusion (Fig. 6a) to deliver the entire protein antigen directly into B cells with low non-specific antigen uptake capacity. This approach showed a limited success for electroporation, because it relies on engineering and precisely controlled electric fields. Sharei et al.121 designed a cytoplasmic delivery method based on rapid mechanical deformation of cells. They believed that the size and frequency of pores created by membrane rupture are related to the shear and compression forces that the cells are subjected to. Therefore, 45 parallel microfluidics channels with different shrinkage sizes and quantities were made by etching silicon and then sealed by a Pyrex layer (Fig. 6b). The width and length of the constricted area differed, resulting in varied degrees and duration of shear and compression forces on the cell. This device was based on a parallel channel design that could achieve a cell throughput as high as 20,000/s. This approach successfully delivered a range of cargos, including carbon nanotubes, proteins and siRNA, into 11 types of cells, including primary fibroblasts, embryonic stem cells, and a range of immune cells. Lam et al.122 developed a less expensive but simple and rapid method using PDMS as a replacement device to complete the construction of the cell squeezing platform. They designed a cytoplasmic PDMS-based delivery and modification system called cyto-PDMS. The design principle was similar to that of Sharei et al's.121. PDMS was molded into microchannels and bonded to a glass sheet. The cells and macromolecular materials entered from the air inlet through the contraction area and exited from the air outlet. The transparency of PDMS allowed direct and real-time characterization of cells as they were passing through the constriction area. This platform exhibited minimal buckling in the

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constriction area but can withstand high shear forces, thus overcoming the previous challenges of limiting flow rates due to the high pressure sensitivity of PDMS.123 The results indicated that this platform could deliver cargos of different sizes into the cytoplasm of human fibroblasts with minimal effect on cell viability. In addition, the results demonstrated that the recombinant enzyme-active Cre-protein could be transferred into nucleus of the recipient fibroblast through appropriate genome recombination. Squeezing as a platform eases the use of a wide range of cargos without concerning complications, such as the size and charge action. However, given the cell size differences and transmission inconsistency, cell clogging of microchannels and cargo delivery residual heterogeneity are still unsolved.118,124 Meacham et al.125 reported a method for coordinating mechanical disruption of cell membranes and electrophoresis of DNA into cells to increase transfection efficiency. In this platform, the acoustic shear force (ASP) technique was used to form a pressure gradient at the tip of the nozzle by focusing the acoustic wave forcing the cell to pass through the cell-scale hole and withstanding high shear force in a short time, thereby forming a continuous transient poration126 (Fig. 6c). In this process, only the high shear environment was used to cause cell poration. The sound field was insufficient to destroy the cell membrane. Next, the deformed cells were exposed to a low-intensity electric field, which forced DNA into the cells by electrophoresis.127,128 In this work, ASP technology achieved transfection of peripheral blood mononuclear cells, and the ASP-ep coupling method significantly improved transfection efficiency. It demonstrated the potential of ASP platform in large-scale integration applications. This approach further optimized the delivery efficiency of microfluidic squeezing and shearing of cell membrane poration by both the diffusion of material molecules into cells through poration and electrophoretic movement, which can be actively driven to accelerate the delivery efficiency. Different from the constriction function that disrupts the cell membrane to increase permeability, Kizer et al.129 designed a clog-free and sheathless inertial microfluidic platform, named hydroporator, based on a rapid mechanical cell deformation relying solely on hydrodynamic cell shearing. This device utilized the inertia effect to first mix the cell suspensions with target materials and inserted them into the microchannel. As shown in Fig. 6d, given their interactions with the secondary flow, cells could be aligned with the central channel and uniformly migrated to the stagnation point at the cross-junction where they were stretched by fluid dynamics. This process resulted in transient discontinuous porations in the cell membrane. Thus, targets in the solution could enter the cytoplasm before the cell membrane was resealed.130,131 This work has successfully delivered 2000 kDa dextran and for the first time showed the delivery capacity of different large DNA nanostructures and their biostability in living cells. Given that delivery efficiency is related to flow rate, a high flow rate is required to achieve efficient delivery of larger molecules. However excessive flow rates may exacerbate cell death. Li et al. designed a droplet microfluidic132 platform for single-cell transfection to achieve efficient and consistent plasmid delivery of lipoplex-mediated suspension cells.133 Lipoplex-mediated intracellular delivery is extremely inefficient (usually b5%) in transfection of suspended cells (such as lymphocytes and hematopoietic cells for immunotherapy).134,135 The efficiency of lipoplex-mediated transfection depends on two factors: endocytic capability of target cells136,137 and lipoplex size.138,139 Through their devices, a single cell was wrapped in a dispersed droplet with negatively and positively charged lipids and passed through a winding structure channel to experience chaotic advection. Plasmids and liposomes are self-assembled into lipoplexes in this process. Chaotic advection could aggravate the collision of lipoplexes with cells. Thus, the shear force exerted by fluid compression on cells increased when cells passed through the droplet pinch-off orifice, leading to increased membrane permeability and enhanced lipoplex delivery into cells through endocytosis (Fig. 6e). The results showed that the delivery efficiency of three suspended cells increased

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from 5% to 50%, solving the problem of intercellular variation. This work is expected to reprocess the transfected single cells to minimize immune rejection. In addition, the combination of microfluidics with electroporation has overcome a series of deficiencies, such as heat dissipation of traditional electroporation. Extremely short electrode spacing also could reduce the voltage requirements. Xu et al.140 fabricated a microchannel device containing both inlet and outlet electrodes to generate an electric field. High-speed fluid shear stress and electroporation were used to induce cell membrane pores and promote DNA delivery into cardiomyocytes. Hollow gold electrodes with high aspect ratios could be combined with electroporation or optoporation to induce transient nanopores on the cell membrane with controlled timing and cellular localization.142,143 Monitoring cell electrical signals during intracellular delivery is critical to the overall characterization of delivery effects.144 However, the manufacturing complexity of such type of devices often limits this idea. Drug delivery with microfluidic system is often used at several or single-cell level, allowing the accurate recording of electrical signals during delivery to reveal the specific behaviors between the such and the unaffected cells. In the work of Cerea et al.144 , realtime electrical recording was achieved in combination with singlecell drug delivery. They designed a structure that could be used to record large cell populations. Hollow nanostructures and bottom microchannels could deliver specific reagents at specific locations in cell culture. This work filled the gap in the microfluidic process of individual cells in the microelectrode array.145,146

Table 2 Advantages and challenges for membrane disruption methods.

3. Features of an ideal intracellular delivery system

• Biocompatibility and safety

Over the years, we have developed several techniques to overcome the barriers to intracellular delivery. Different methods feature their own advantages (Table 2), and comparing their performances is difficult. We aim to develop new methods that can meet the requirements of high precision, large scale, and high flexibility. Here, we present guidelines that can be used by researchers to develop new technologies for intracellular delivery.

Compatibility is required for all drug delivery systems to avoid any problem that might be caused by immune response. Safety is important for clinical application. NPs, for example, can deliver drugs efficiently, but the toxicity has been a barrier to clinical use.

Methods

Advantages

Nanoneedle

Provides cellular nanoscale features. Directly penetrates the nucleus. Can be used for many cell types. Electroporation High transfection efficiency. High-throughput. Optoporation Local operation. High accuracy at single-cell level. Sonoporation Transient exposure to membrane disruption and low cell damages. Locate microbubbles and target cavitation. Suitable for a wide range of cell types. Microfluidic Simple equipment and easy manufacture. Independent of physical field or the carrier. Hydrodynamics does not block the flow passage.

Challenges High-precision manufacturing requirement. Difficult to implement on a large scale. Easily causes cell lysis. Not suitable for high-throughput. High cell death rate. Slow microflow processing. Complex and costly equipment. Damage to some molecules (e.g., protein denaturation). Loss of cytoplasmic content. Cavitation could produce reactive oxygen species that damage DNA, requires additional chemicals in conventional sonoporation.

The efficiency of the squeeze cell method correlates with cell size. Works on suspended cells only and considers the molecular residual heterogeneity of materials. Incompatible with cell characterization methods. The size of membrane poration depends on precise shear forces.

• Control mechanism

• Minimal cell perturbation

Exogenous materials and physical forces can cause off-target effects and toxicity to cells. The environmental factors, such as temperature, that cause Brownian kicks may exert instability onto delivery systems. Minimizing the exogenous decoration and manipulation is critical to successful drug delivery systems. • Scalability

A delivery system should be scalable given that the number of cells requiring treatment could vary considerably. Not only the target but also the production of delivery system should be scalable. • Suitability to cell types

An ideal delivery system should be compatible to any types of cells of interest, including hard-to-transfect cells. This feature relies on a delivery mechanism independent of cell type or at least one appropriate mechanism for each cell type.

A reliable control mechanism is essentially critical for nextgeneration drug delivery system. Controlling drug delivery via signaling is difficult to achieve but is important for sophisticated delivery systems. The specificity and dosage rely largely on control mechanism. Furthermore, an ideal drug delivery system should control its behavior by the environmental factors, such as the temperature, pH, or both. • Cost

Delivery systems should be economically reasonable and inexpensive. 4. Outlook Compared with chemical methods, physics-based membrane disruption technology overcomes many challenges and avoids the side effects caused by viral vectors. This process holds potential to solve the problems of treating difficult-to-transfect cells. In particular, the development of micronanotechnology shows promise among emerging technologies in the fields of biomedicine and clinical therapy. This discipline also exhibits new scalability and controllability to treat a few cells and at

Fig. 6. (a) Schematic representation of microfluidic squeezing for macromolecule delivery to cells120; (b) illustration of delivery mechanism showing the rapid deformation of a cell generating transient membrane holes, as it passes through microfluidic constriction. The illustration is an electron micrograph of current parallel channel design with blue cells121; (c) schematic of combined mechanoporation/electrophoresis gene transfer method125; (d) schematic of the design and operating principles of the vector-free intracellular delivery system129; (e) chip design and working mechanism. Scale bar: 100 μm.133

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the single-cell level. Researchers may select different methods for various applications. For example, electrical methods are more accessible to single-channel independent control; acoustic cavitation and optical methods are difficult to perform in microarrays, microneedles and microchannels have certain limitations in terms of the flexibility of cell type, etc. The physical mechanisms of various methods exhibit potential advantages and limitations. The combination two or more of these techniques may provide more sustainable and innovative options for intracellular delivery systems. A more thorough understanding of the intracellular delivery mechanism will help the biomedical research community to further develop more powerful technologies in medical and industrial applications. Micro-nanoscale manufacturing technology has become the key to promote micro-systems to be more miniaturized and diversified and integrated in large scale. If we could reasonably use their unique advantages to cooperate, improvements in the intracellular delivery system can be possibly achieved.

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Further Reading 1. Messina GC, Dipalo M, La Rocca R, et al. Spatially, temporally, and quantitatively controlled delivery of broad range of molecules into selected cells through plasmonic nanotubes. Adv Mater 2015;27(44):7145-9. https://doi.org/10.1002/adma. 201503252. Mengjie Sun received the B.S. degree from Tianjin University in 2017, majoring in measurement and control technology and instruments. She is currently pursuing a M.S. degree in Tianjin University. Her research interests focus on intracellular delivery at the micro/nanoscale based on hypersound.

Xuexin Duan received his PhD degree at University of Twente, Netherland (2010). After Postdoc studies at Yale University, he moved to Tianjin University. Currently, he is a full professor at State Key Laboratory of Precision Measuring Technology & Instruments, Department of Precision Instrument Engineering of Tianjin University. His research is about MEMS/NEMS devices, microsystem, microfluidics and their interfaces with chemistry, biology, medicine, and environmental science.

Please cite this article as: M. Sun and X. Duan, Recent advances in micro/nanoscale intracellular delivery, , https://doi.org/10.1016/j. npe.2019.12.003