Intracellular delivery can be achieved by bombarding cells or tissues with accelerated molecules or bacteria without the need for carrier particles

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Intracellular delivery can be achieved by bombarding cells or tissues with accelerated molecules or bacteria without the need for carrier particles Wei-Nan Lian a , Cheng-Hung Chang b,c , Yin-Jhen Chen d , Ro-Lan Dao a , Yun-Cin Luo d , Jun-Yi Chien e , Shie-Liang Hsieh a , Chi-Hung Lin a,d,f,g,⁎ a

Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan Institute of Physics, National Chiao-Tung University, Hsinchu 300, Taiwan c National Center for Theoretical Sciences, Hsinchu 300, Taiwan d Department of Medical Education and Research, Taipei City Hospital, Taipei, Taiwan e Center for Neural Regeneration, Department of Neurosurgery, Neurological Institute, Taipei Veteran General Hospital, Taipei, Taiwan f Department of Surgery, Taipei Veteran General Hospital, Taipei, Taiwan g National Nano Device Laboratory, Hsinchu, Taiwan b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

To deliver non-permeable molecules into cells, one can utilize protocols such as

Received 2 August 2006

microinjection, electroporation, liposome-mediated transfection or virus-mediated

Revised version received

transfection. However, each method has its own limitations. Here we have developed a

15 September 2006

new molecular delivery technique where live cells or tissues are bombarded with highly

Accepted 18 September 2006

accelerated molecules directly and without the need to conjugate the molecules onto carrier

Available online 4 October 2006

particles, which is essential in conventional “gene gun” experiments. Gene bombardments can be applied to well-differentiated cells, primary cultured cells/neurons or tissue explants,

Keywords:

all of which are notoriously difficult to transfect. Exogenously made proteins and even

Transfection

bacteria can be effectively introduced into cells where they can execute their function or

Gene gun

replicate. Our experimental results and physical model support the notion that accelerated

Living cells

chemicals, proteins, or microorganisms carry enough momentum to penetrate the plasma

Permeability

membrane. The bombardment process is associated with a transient (∼10 min) increase in cell permeability, but such membrane leakage has a minimal adverse effect on cell survival.

Abbreviations:

© 2006 Elsevier Inc. All rights reserved.

F-actin, filamentous actin IF, immunofluorescence D3K, dextran (MW:3K) D40K, dextran (MW:40K) D70K, dextran (MW:70K) D500K, dextran (MW:500K) CHO cell, Chinese Hamster Ovary cell

⁎ Corresponding author. Institute of Microbiology and Immunology, National Yang-Ming University, 155, Li-Non St. Sec. 2, Taipei 112, Taiwan. Fax: +886 2 28212880. E-mail address: [email protected] (C.-H. Lin). 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.09.028

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Introduction Being able to introduce bioactive molecules such as DNA, proteins or chemical reagents into living cells and then examine the consequent responses is critical to the advancements of modern biology. Certain compounds are readily “membrane permeable”, they can enter the cells by simple diffusion. On the other hand, delivery of membrane impermeable molecules to living cells or tissues can be achieved by a variety of chemical, physical, or biological methods. Each technique has its own advantages and drawbacks. In general, molecular delivery can be better done in relatively undifferentiated rather than differentiated cells. Cell lines are more effective targets for transfection than primary cultured cells or tissue explants and proteins are more difficult to be delivered into cells than DNA. Here, we contrive a new molecular bombardment method to tackle some of these challenges. Soluble compounds in the aqueous environment can be “internalized” by various endocytosis pathways, but the internalized molecules are typically trapped or even undesirably metabolized within certain intracellular compartments. In general, almost any given substance can be “microinjected” into cells, but the technical hurdle is high and throughput is low. Membrane impermeable DNA and most proteins can enter cells by diffusion if membrane permeability is changed by mild detergent or ultrasound treatments [1,2]. However, a large (sometimes unaffordable) amount of materials is needed in these experiments to create a high enough concentration gradient to facilitate the diffusion process. Conventional “gene transfection” protocols using either calcium phosphate precipitation or liposomes and are very useful for DNA transfer [3,4]; however, the procedures have not been successfully applied to the delivery of proteins. Moreover, transfection in certain types of cells is very difficult. For these cells, one may consider using viruses for gene delivery [5–7], but again this biological means is incapable of delivering exogenously made or purified proteins. Intracellular delivery by physical methods, such as the momentum associated with highly accelerated objects, can be more widely applied and is less restricted by the target. In socalled particle-mediated delivery technology (the gene gun), biomolecules (such as DNA) are linked to the surface of solid substrate carriers (such as micron-scale gold particles). These microparticle carriers can then be accelerated to a very high speed and are able to traverse a solution and penetrate the plasma membrane. By doing so, they help to deliver the molecules into the cells [8]. Although this method has been successfully applied to the introduction of genes in plants and animal tissues, the presence of carrier particles within the targeted cells may cause some adverse effects. In addition, the conjugation protocols for linking biomolecules to carriers were developed mainly for DNA. The surface coupling procedures can, in some cases, interfere with the functions of proteins delivered. To our knowledge, no report has been published on using accelerated molecules alone to penetrate and “transfect” cells. In this report we demonstrate that highly accelerated molecules alone carry enough momentum to penetrate the

plasma membrane. Using such “molecular bombardment”, small chemicals, DNA, proteins or even bacteria can be readily introduced into cultured cell lines, primary cultured neurons and tissue explants. Furthermore, upon the delivery of the compounds, the target cells remain functionally alive. A physical scheme has been developed to describe the penetration event by these accelerated molecules.

Materials and methods Cell and tissue culture CHO (Chinese hamster ovary), Hep G2, and HeLa cell lines were used. Standard cell culture protocols were applied [9]. Primary cultured fish keratocytes were isolated from the scales [10], and kept in HEPES–DMEM (GIBCO Life Technology) supplemented with 10% fetal bovine serum, and 0.1% gentamicin. The cells that migrated out of the fish scales were harvested by trypsin–EDTA treatment and further cultured on sterilized coverslips. The retinal explants were prepared as previously described [11]. Optic nerve crush was carried out on anesthetized goldfish. Seven days later, the fish were dark-adapted for 30 min then anesthetized to remove the eye. The retina was isolated in Hanks' buffer (Sigma), chopped into 0.4-mm square pieces and plated on poly-L-lysine-coated coverslips for microscopic studies.

The molecular bombardment assay The molecular bombardment assay was performed using a gene gun machine (PDS-1000/He system, Bio-Rad) following standard operation protocols, but the coupling procedure for the molecules to the carrier particles was omitted. Solutions (6 μl) containing the molecules to be delivered were first deposited onto the holding membrane. The membrane was ruptured by high-pressured helium gas (450 psi) whereby the molecules on the holding membrane were accelerated and used to directly bombard the target cells or tissues in a closed chamber that was at 253.17 Torr vacuum. After the bombardment, the cells were immediately washed with sterilized PBS and fresh medium to remove excessive molecules in the extracellular environment. The presence of bombarding (fluorescent) molecules inside the cells was detected and quantified under a fluorescence microscope equipped with a digital camera (Orca ER, Hamammatsu) using the Metamorph program (Universal Imaging Corporation, West Chester, PA), or by the confocal microscopy (Leica SP2, Heidelberg, Germany). To detect molecular entry by diffusion, equal amount of non-accelerated fluorescent molecules were added to the culture medium after the bombardment. Hoechst dye, Lucifer yellow (LY), fluorescently labelled dextrans of different molecular weights were all obtained from Molecular Probes (Eugene, Oregon). The organelle targeted sequences (Clontech, Takara Bio company) used in Fig. 2 were the nuclear localization signal of the simian virus 40 large T-antigen (pEYFP-nu), the plasma membrane anchor of

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neuromodulin (pEYFP-pm), a mitochondrial targeting sequence derived from human cytochrome C oxidase (pEYFP-mito). Rhodamine-conjugated actin and tubulin proteins were from Cytoskeleton (Denver, USA).

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staining. The stained samples were mounted using antiphotobleaching medium containing 20 mM n-propyl-gallate (Sigma) in 80% glycerol/20% PBS.

Bacterial bombardment Immunostaining Immunofluorescence staining was carried out as previously described [12]. Briefly, cells were fixed with 4% paraformaldehyde/2 mM EGTA/400 mM sucrose/PBS at room temperature for 15 min, then permeabilized with 0.5% Triton X-100 in fix solution for 5 min. The samples were then incubated with 5 mg/ml BSA/PBS and then with primary antibody at room temperature for 1hr. After extensive PBS washes, fluorophoreconjugated secondary antibodies (Jackson Immuno Research, West Grove, PA) were added at the concentrations recommended by the manufacturer at room temperature for 1 h. FITC-conjugated phalloidin at 1 U/ml was used for F-actin

Ampicillin-resistant E. coli bacteria (strain BL21-DE) transformed with the EGFP gene (a gift from Dr. Wan-Jr Shu of NYMU) was adjusted to an OD600 of 0.2 and concentrated 100-fold before bombardments. Six μl of the concentrated bacteria was used either for each bacterial bombardment, or add to the cell culture medium as a control for phagocytosis or other non-specific cellular entry. The number and viability of the intracellular E. coli were quantified by a colony formation assay. Target cells were washed twice by PBS after the bacteria bombardment and treated with 100 μg/ml gentamicin to eliminate the bacteria in the extracellular medium. The cells were then lysed with NET

Fig. 1 – Chemicals of different sizes and properties can be delivered by the molecular bombardment assays. CHO cells were subjected to molecular bombardment by accelerated DNA dye Hoechst 33258 (A), Lucifer yellow (B), TRITC-D70K (C), or FITC-D500K (D). Successful delivery of these fluorescent molecules was verified by fluorescence microscopy. Scale bar = 20 μm.

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Table 1 – Efficiency of intracellular delivery by the direct molecular bombardment assay Target cells CHO

Molecules to be delivered

Concentrations (μg/μl) a

Weight used per shot (μg)

Delivery effciency b (targeted cells/500 cells)

Hoechst 33258 (MW: 623.96)

1 0.5 0.1 2.5 1.25 0.5 10 5 2.5 10 5 2.5 1 1 3 3

6 3 1 15 7.5 3 60 30 15 60 30 15 6 6 18 18

65.48 ± 18.81 57.7 ± 25.56 27.25 ± 7.22 35.47 ± 8 19.9 ± 2.64 11.95 ± 3.36 38.16 ± 8.19 25.44 ± 9.46 19.04 ± 4.61 39.1 ± 8.79 8.28 ± 2.19 4.35 ± 2.66 34.82 ± 5.07 9.06 ± 1.85 67.15 ± 13.04 62.36 ± 5.89

Lucifer yellow (MW:491.57)

Dextran (MW:70K)

Dextran (MW:500K)

CHO Hep G2 CHO CHO a b

DNA (GFP-tubulin gene) DNA (GFP-tubulin gene) Actin proteins Tubulin proteins

Concentrations of molecules to be delivered are shown; volume per shot = 6 μl. Mean ± STDEV are shown.

buffer (150 mM NaCl, 0.5% NP-40, 50 mM Tris, 1 mM EDTA and 1% Triton X-100, supplemented with protease inhibitors) at 4°C to release the intracellular bacteria. After 12,000×g centrifugation at 4°C for 30 min, the pellets containing the intracellular bacteria were collected and spread on the LB plates containing 40 μg/ml ampicillin; they were then incubated overnight at 37°C. The resulting bacterial colonies were inspected and counted.

bombardment than those present for the same weight of D70K, yet both the D500K and D70K dextrans exhibited a similar degrees of intracellular delivery. This finding that larger molecules, namely D500K, were more readily introduced by bombardment than smaller molecules, namely D70K, suggests that the momentum associated with the highly accelerated molecules might account for the penetration of the molecules through the cell membrane.

Results

Gene transfection was achieved by DNA bombardment of cultured cells or tissue explants

Molecules of different sizes and properties can be delivered into cultured live cells by bombardment The molecular bombardment experiments described here were very easy to operate. Typically, molecules in solution were accelerated by the standard gene gun operation protocol. Compounds of different sizes and physicochemical properties can be successfully delivered by this procedure. As shown in Fig. 1A, the cell impermeable bisbenzimide dye Hoechst 33258 (MW = 623.96) could be delivered by bombardment staining the nucleus in a dose-dependent manner (Table 1). Nonaccelerated Hoechst 33258 added to the culture medium gave no detectable nuclear staining (data not shown). Similarly, the polar trace molecule Lucifer yellow (LY, MW = 491.57) and the biologically inert dextrans of various different sizes were also found to penetrate the cells by the molecular bombardment (Figs. 1B–D, and Table 1). Note in Table 1 that D500K had only about 1/7 of molecules present per

The conventional gene gun method utilizes particles to carry DNA into the cells [13,14]. Here, purified plasmid DNA (6 μg per shot) in a solution of distilled water was used for direct (carrierfree) DNA bombardment. As shown in Figs. 2A–B, EYFP fusion genes such as pEYFP-actin and pEYFP-tub could be effectively bombarded into and subsequently transfect the well-differentiated and polarized hepatic Hep G2 cells [9]. The resulting production of fluorescent actin proteins was able to label the entire intracellular actin cytoskeleton, including the highly concentrated actin filaments in the microvilli of the apical domain (arrowheads). Among the different types of cell lines tested, the efficiency of the DNA bombardments ranged from about 10% (well-differentiated Hep G2 cells) to 35% (CHO cells; see Table 1). Similarly, DNA bombardment could successfully insert an EYFP gene fused with specific targeting sequences for the nucleus/nucleolus (Fig. 2C), plasma membrane (Fig. 2D), or mitochondria (Fig. 2E), into cells, as shown by the correct subcellular targeting of the introduced proteins.

Fig. 2 – DNA bombardment can be used for gene transfection in cultured cells and tissue explants. DNA bombardments were performed on well-differentiated and polarized hepatic Hep G2 cells (A–E) and the retina explants of the goldfish (F) using plasmid DNA containing the β-actin gene fused with EYFP (A), the β-tubulin gene fused with EYFP (B and F) and the EYFP gene conjugated with specific targeting sequences for the nucleus (C), the plasma membrane (D), and the mitochondria (E). Images were taken by confocal microscopy. Scale bar = 10 μm (A–E). Scale bar = 200 μm (F).

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Tissue explants are notoriously difficult to transfect with DNA using conventional chemical methods. We demonstrate in Fig. 2F that the accelerated EYFP–tubulin fusion gene could be successfully delivered to the neurons of goldfish retina explants by direct DNA bombardment. The transfected neurons expressed fluorescently labeled tubulin proteins that were incorporated to the bundled microtubules inside the outgrowing neurites (arrows).

Cytoskeletal proteins were introduced into live cells by direct protein bombardments Compared to DNA, there are even fewer reliable protocols to deliver proteins into cells. Here we demonstrate that acceler-

ated proteins or polypeptides carry enough momentum to enter live cells without severely damaging the hosts. Purified monomeric actin proteins (43 kDa) labeled with rhodamine (Rh-actin) were accelerated and used to bombard CHO cells (Fig. 3A) or primary cultured fish keratocytes (Fig. 3B). After entering the soluble actin protein pools inside the cell, the exogenously introduced Rh-actin participated in the assembly–disassembly kinetics of the actin cytoskeleton, and was found to have incorporated into the intracellular F-actin structures such as stress fibers (arrows) or lamellapodia (arrowheads). Fish keratocytes containing the bombarding Rh-actin proteins remain intact with respect to their migration activities. The introduced Rh-actin proteins actively participated in the dynamic redistribution and reorganization of the

Fig. 3 – Accelerated actin or tubulin proteins penetrate cell membranes and are incorporated into the intracellular cytoskeletal structures. (A–B) Protein bombardment using rhodamine-labeled β-actin protein (Rh-actin) was carried out in CHO cells (A) or primary cultured fish keratocytes (B). After the protein bombardment, the target cells were fixed and stained with fluorescein-labeled phalloidin (FITC-Ph). The exogenous Rh-actin was found to be incorporated into the intracellular actin architecture, including stress fibers (arrows) and lamellapodia (arrowheads). (C) Time-lapsed recording of the Rh-actin dynamics in fish keratocyte by fluorescence microscopy. (D) Rhodamine-labeled β-tubulin proteins (Rh-tub) were accelerated and used to bombard fish keratocytes. The introduced Rh-tub could participate in assembly and thus label the intracellular microtubule arrays. The time-lapsed tiled images demonstrate a single labeled microtubule exhibiting the dynamic instability process: fast shrinkage followed by slow growth (arrowhead). Scale bar = 10 μm.

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actin cytoskeleton during cell motility (Fig. 3C, and supplementary movie S1), suggesting that the acceleration procedure did not perturb the cytoskeletal protein's functions. Similarly, rhodamine-labeled tubulin proteins (Rh-tub, 65 kDa) could be introduced into fish keratocytes by protein bombardment (Fig. 3C). The resulting fluorescently labeled microtubule arrays exhibited a full-range of activities, including dynamic instability (Supplementary movies S2). Typically, more than 60% of cells residing within the area of protein bombardment were successfully loaded with the protein of interest (Table 1).

Accelerated bacteria penetrated cells and replicate in the eukaryotic cytoplasm We then tested if the bacteria bombardment would result in cells taking in bacteria without any involvement of the phagocytosis pathway. Pathogenic E. coli bacteria were first transformed with the EGFP gene. The EGFP–E. coli bacteria were accelerated and targeted at cultured Hep G2 cells. The presence of EGFP–E. coli inside the cell were detected by fluorescence microscopy (arrow, Fig. 4A). The viability of the bacteria delivered by the bombardment was determined by a bacterial colony formation assay. The target cells were subjected to the bacteria bombardment, then extensively washed and finally treated with antibiotic to eliminate any

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extracellular bacteria. The cells were then lysed and the intracellular bacteria were quantified by counting colony forming units (CFU). We typically obtain about 50 CFU from about 500 cells after each bombardment with bacteria. Background entry mainly through phagocytosis within the 10-min observation period was negligible. The proliferation of the intracellular bacteria was also determined. After the bacteria bombardment and the removal of extracellular bacteria (by addition of antibiotic to the medium), the cells were further incubated for 1, 2, 3, or 4 h during which the CFUs of the intracellular bacteria were quantified. As shown in Fig. 4B, the rate of bacteria proliferation inside the cells (black lines, 3 independent experiments) was about the same as for E. coli growing in the DMEM cell culture medium (dashed lines, 4 independent experiments). Non-accelerated bacteria added to the antibiotic-containing culture medium did not grow (gray lines, 4 independent experiments). These results indicated that acceleration and bombardment did not damage the bacteria based on their survival and continuous proliferation inside the eukaryotic cytoplasm.

Molecular bombardment caused some of the bombarded cells to experience a transient increase in cell permeability The high-pressure helium when hitting the water surface might cause a sound wave that could lead to an increase in

Fig. 4 – Bacterial bombardment allows E. coli to enter and replicate inside a eukaryotic cell. (A) EGFP–E. coli were accelerated and used to bombard cultured Hep G2 cells. The presence of the intracellular bacteria was detected using phase contrast and fluorescence microscopy. Scale bar = 10 μm. (B) To quantify the intracellular bacteria that have entered cells by the bombardment, the host Hep G2 cells were subjected to a bacterial colony formation assay after elimination of the extracellular bacteria by antibiotic treatment (dashed lines, 4 independent experiments). The growth of bacteria in the cell culture medium DMEM in the presence (gray lines, 4 independent experiments) or absence of gentamicin (black lines, 3 independent experiments) was also measured.

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cell permeability, similar to the shock wave generated by a lithotripter or laser [15–17]. In a series of experiments, one of which is shown in Fig. 5, we tested the changes in cell permeability following molecular bombardments. Accelerated FITC-D70K was used to bombard the cells and then washed out. An equal amount of non-accelerated TRITCD70K was then added to the medium and the mixture incubated for 14 min. Cellular entry by TRITC-D70K was mediated by diffusion rather than endocytosis, since the latter effect is negligible within the 14-min incubation period employed and the homogenous distribution of TRITC-D70K rather than punctate spots inside the cell argues against their entry via endocytosis. If we compare the presence of intracellular FITC-D70K and TRITC-D70K (Fig. 5A), we noticed that TRITC-D70K existed only in the cells that also contained FITC-D70K. Some cells had FITC-D70K only (crosses), but almost no cell contained TRITC-D70K alone. The ratio among cells having molecules delivered by bombardment, by diffusion, or by both mechanisms remained about the same across the 19 experiments (Table 2), even if the fluorescent labels of D70K were swapped. From these results, we concluded that about half of the cells hit by D70K experienced an increase in cell permeability that could facilitate the entry of non-accelerated D70K molecules by diffusion. This diffusion-based entry was dependent on the size and physicochemical properties of the molecule. In Figs. 5B–D, the ratio of cells containing molecules that entered by bombardment (orange bars), or by diffusion for 1 min (pink bars) or 10 min (blue bars) were calculated. The diffusion entry was induced by permeating cells with the water droplet bombardment, then allowing molecule to enter the cell from the surrounding medium. From these results, we noticed a significant diffusions entry exerted by Hoechst dyes, LY, D3K or D40K in the majority of the water dropletbombarded cells after incubation for 10 min, while diffusion entry by D70K occurred only in about half of the waterbombarded cells and diffusion entry by D500K was almost negligible. No calcium influx occurred after the bombardment or during the subsequent incubation period (data not shown). Time-lapsed recordings were performed to monitor the dynamics of molecular entry by diffusion and an example is shown in Fig. 5C. In the image taken immediately after the bombardment (1 min), cells containing molecules delivered

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by the bombardment are highlighted (cells framed by green dashed lines). Changes in fluorescence intensity indicate the accumulation of fluorescent dextrans that have entered by diffusion; these were measured in some of the bombarded cells (asterisks, Fig. 5C), and plotted as a function of time (black traces, Fig. 5D). Diffusion entry in cells that had not been bombarded with molecules was also measured (crosses, Fig. 5C and red traces, Fig. 5D). It should be noted that most of the diffusion entry took place in the first 10 min after the bombardment then reached a plateau. If the tracing molecules for diffusion were added to the surrounding medium after the bombarded cells were allowed to recover (for 2–14 min), the ratio of the cells that allowed diffusion entry gradually decreased (Fig. 5E). As shown, the majority of bombarded cells had already recovered within 10 min of the bombardment and exhibited very little membrane leakage.

Discussion We demonstrate here that cell impermeable bioactive chemicals or macromolecules (including DNA and proteins) can be conveniently introduced into live cells by direct bombardment of cultured cells or tissues by these molecules, while the cells are maintained under standard culture conditions. Notably, the molecules to be delivered by this novel bombardment method are kept in the solution phase throughout the entire experiment; there is no need to conjugate the molecules to the surface of microparticle carriers as required by the conventional “gene gun” method [8,14,18]. The surface chemistry associated with the conjugation procedures is technical challenging and, in some cases, can damage the molecule's bioactivity [16,19]. The molecular bombardment experiments are easy to carry out. The simple physical principles behind the bombardment method make it applicable to almost all kind of biological samples, including cultured cells that are notoriously difficult to transfect (such as well-differentiated and polarized epithelia cells), primary cultured cells/neurons, or tissue explants. In our hands, more than 80% of the cells survive the molecule bombardments. The overall delivery efficiency for DNA bombardments is comparable to conventional transfection methods using chemicals or liposomes.

Fig. 5 – Molecular bombardment causes a transient increase of cell permeability. (A) Accelerated FITC-D70K was used to bombard Hep G2 cells then removed; the cells containing the bombarding molecules are highlighted (green dashed lines). After the bombardment, TRITC-D70K was added to the culture medium and incubated for 14 min; the cells containing TRITC-D70K that had entered by diffusion are indicated (red dashed lines). In the color-merged panel, note every TRITC-D70K containing cells also contained FITC-D70K (yellow dashed lines), while some FITC-D70K cells contained no TRITC-D70K (crosses). Scale bar = 20 μm. (B) The ratio of cells containing molecules delivered by bombardment (orange bars), by diffusion for 1 min (pink bars), or 10 min (blue bars) was calculated. (C–D) Time-lapsed recording of TRITC-D70K entering cells by diffusion (C) and the associated changes in fluorescence intensity over time (D). Cells containing the bombarding FITC-D70K are highlighted (green dashed lines). The entry of TRITC-D70K by diffusion progressively increased in some of the FITC-D70K-positive cells (asterisks in panel C, black traces in panel D); no TRITC-70K entry is visible in the cells that were FITC-70K-free (crosses in panel C, red traces in panel D). Scale bar = 20 μm. (E) Cells were bombarded with accelerated FITC-D70K. After a recovery period (2 to 14 min), TRITC-D70K was added to the medium for 10 min and the cells exhibiting TRITC-D70K entry by diffusion were quantified as percent of the FITC-70K-positive cells. Three independent experiments were carried out for each of the three cell types. Mean ± SD is shown.

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0.65 0.36 0.65 0.44 0.41 0.69 0.5 0.49 0.66 0.48 0.53 0.12 31 10 30 16 14 33 25 21 33 26 23.9 8.31 1 0 0 0 0 0 3 2 2 2 1 1.15 The number indicates the positive cells found in the total of 100 cells observed in each experiment.

0.78 1.3 17.11 7.46

20.22 6.32

0.54 0.1

16 18 16 20 20 15 22 20 15 25 18.7 3.3 0.46 0.48 0.42 0.48 0.61 0.65 0.45 0.61 0.71 24 26 19 21 20 22 10 11 29 1 3 0 3 0 0 0 0 0 27 25 26 20 13 12 12 7 12

1 2 3 4 5 6 7 8 9 10 Mean SD

Diffusion Bombardment + diffusion only (FITC) (FITC + TRITC) D70K D70K Bombardment only (TRITC) D70K Ratio of permeable cells post bombardment Number of Bombardment Diffusion only Bombardment + diffusion experiment only (FITC) (TRITC) D70K (FITC + TRITC) D70K D70K

Table 2 – A transient increase of cell permeability was found in 50% of cells subjected to molecular bombardments

Ratio of permeable cells post bombardment

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Yet most intriguingly, purified or recombinant proteins can be readily bombarded into live cells to study their function. The material needed for proteins bombardment (typically 6 μg per shot) is much less than that needed for methods such as electroporation [20], shock wave [15], or ultrasound-mediated delivery [1]. The bombardment assay can also be applied to deliver bacteria into eukaryotic cells. Many bacteria can be engulfed by macrophages via phagocytosis. In this process, the internalized bacteria are kept in vesicular compartments such as phagosomes [21,22]. The bacteria bombardment assay described here provides a unique avenue to deliver bacteria into the eukaryotic cytoplasm, totally bypassing the phagocytotic pathway. In the eukaryotic cytoplasm, the bacteria survive and proliferate (Fig. 4). We attribute the penetration of the accelerated molecules through the cell membrane to two major factors: (i) the final velocity (or the momentum) of the molecule before penetrating the cells, and (ii) the permeability of the cell membrane. How essential are these two factors depends on the intensity of the pressure pulse that is created at the beginning of the experiment. As shown in the cartoon of Fig. 6, this pulse triggers a wave front together with fast advancing molecule droplets that contain molecules to be delivered (blue dots) towards the culture medium surface. When this wave front touches the water surface, it creates a sound wave in the water (1482 m/s at 20°C) propagating through the water and reaching cells sitting on the bottom of the substrate. Depending on the energy of the hit, the membranes could become detached and therefore unstable, thus entering a more permeable configuration. Following the wave front, the molecular droplets (blue dots) collide with the water surface. Since they can easily fuse with water, these droplet-coated molecules encounter less surface tension and can penetrate into the water medium with less velocity reduction than dry material. Suppose the molecule is a prolate ellipsoid with the semimajor axis a and the semiminor axis b. This molecule will encounter a frictional force F when moving in the water. This force at least takes the value F = 6πηrv, as described in Stokes law, when the molecule velocity v is small [23], where r = (ab2)1/3 is the effective radius of the molecule and η is the viscosity of the water (8.9 × 10− 4 kg/m s at 25°C). If the molecule moves along in still water, its original velocity will soon reduce to the diffusion velocity due to the force F. However, in our bombardment assays the molecules are companied by droplets. Right after penetrating into the water medium these droplets still carry a considerable momentum and company the molecules to move a distance. These moving droplets largely screen out the frictional force of the still water on the molecules. Therefore the droplet-coated molecules can rush much faster than the uncoated ones through the thin water medium. When they hit the membrane, the residual velocity of the molecules together with their heavy weight (in the order of kDa) gives a momentum large enough to penetrate the cell membranes. Thus, the accelerated molecule in factor (i) indeed could play an essential role in the molecular penetration under the right intensity of the initial pressure pulse and a proper thickness of the water medium.

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Fig. 6 – The hypothetic model for the molecular bombardment assay. Molecules in small water droplets (blue dots) are accelerated by the high-pressure gas and used to bombard the cells. The combination of wave front and plasma membrane penetration by high speed molecules causes some of the affected cells (cells #2 and #3, but not cell #1) to become transiently permeable to non-accelerated molecules (red dots) added to the medium, allowing their entry by diffusion. The membranes reseal within 10 min after the bombardment.

Now the question is whether a wounded membrane in factor (ii) is also important. Our data reveal that nonaccelerated molecules added to the water (red dots) can only enter by diffusion into those cells already subjected to direct penetration by accelerated molecules (Fig. 6, cell #2 and cell #3), although some bombarded cells do not support such diffusion entry (Fig. 6, cell #1). Our results show that wave front is not strong enough to render cells permeable to allow entry of non-accelerated molecules by diffusion (Fig. 6, cell #4). That is, the pressure pulse used in the bombardment assay is not high enough to cause any significant damage to the cell membranes under water. This scenario is consistent with the high rate of cell survival after the molecule bombardment.

Acknowledgments We thank Dr. Arthur Chiou and Dr. Yin Yeh for fruitful discussion. We thank Dr. Wan-Jer Shyr for providing the GFP– E. coli, and Weber Chen for technical support. Special thanks also to Dr. Chi Keung Chan for an inspiring discussion about hydrodynamics. This work is supported by grants from National Science Council, UST-CNST, National Research Program for Genomic Medicine and National Nano Science and Technology Program, Taiwan, awarded to C.H.L.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2006.09.028.

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