Hypersound-Enhanced Intracellular Delivery of Drug-Loaded

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Biological and Medical Applications of Materials and Interfaces

Hypersound-Enhanced Intracellular Delivery of Drug-Loaded Mesoporous Silica Nanoparticles in a Non-Endosomal Pathway Yao Lu, Loganathan Palanikumar, Eun Seong Choi, Jurriaan Huskens, Ja-Hyoung Ryu, Yanyan Wang, Wei Pang, and Xuexin Duan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02447 • Publication Date (Web): 15 May 2019 Downloaded from http://pubs.acs.org on May 16, 2019

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ACS Applied Materials & Interfaces

Hypersound-Enhanced Intracellular Delivery of Drug-Loaded Mesoporous Silica Nanoparticles in a Non-Endosomal Pathway

Yao Lu,†,‡ Loganathan Palanikumar,# Eun Seong Choi,# Jurriaan Huskens, ‡ Ja-Hyoung Ryu,#,* Yanyan Wang, † Wei Pang, †,* and Xuexin Duan †,*

†State

Key Laboratory of Precision Measuring Technology & Instruments, Tianjin

University, Tianjin 300072, China. ‡Molecular

Nanofabrication group, MESA+ Institute for Nanotechnology, University of

Twente, 7500 AE, Enschede, The Netherlands. #Department

of Chemistry, School of Natural Science, Ulsan National Institutes of

Science and Technology (UNIST), Ulsan, 44919, Korea.

ACS Paragon Plus Environment

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KEYWORDS: Gigahertz Acoustic Streaming, NEMS Resonator, Mesoporous Silica Nanoparticle, Penetration, Drug Delivery

ABSTRACT: 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


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without the limitation of carrier materials, which provides a versatile way of enhanced drug delivery for biomedical applications.

1. INTRODUCTION

Effective chemotherapy depends on efficient drug delivery aiming to realize rapid apoptosis of cancer cells as well as minimum side effects on normal cells.1-3 The treatment with free drugs may cause homogeneous accumulation in non-tumor tissues, thus decrease disease targeting.4-6 Drug carriers were developed to surmount the nonspecific toxicity and to increase the lifetime of the loaded drug molecules in a blood circulation.7,8 Among these carrier systems, mesoporous silica nanoparticles (MSNs) have triggered a lot of research interest since their well-defined pores can hold numerous drug molecules to provide an efficient drug concentration.9,10 In particular, polymerwrapped MSNs (PMSNs) have been developed by capping the pores with disulfide crosslinked polymers to achieve stable and highly efficient loading of drugs.11,12 It has been reported that the drug molecules loaded in the PMSNs can be released via degradation of the polymer shell in the intracellular reducing microenvironment.13,14 The carrier-


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mediated methods can reduce the non-specific toxicity effects from free drugs, but at the same time limit the therapeutic efficacy due to the inefficient release of drugs hindered by biological barriers.15,16 In a carrier-mediated system, efficient drug delivery relies on the ability of overcoming the first fence to transport through the cell membrane and subsequently release drugs across the second or third barrier of the endosomal system and the nuclear envelope.17,18 However, in most cases, nanoparticle-based carriers are internalized through an endocytotic pathway, which makes them trapped in endo-lysosomal vesicles and prevents their further transport to the cytoplasm and/or nucleus, the active site of drugs.19,20 Thus, the efficacy of MSN-based intracellular chemotherapy still remains challenging for medical practice. Existing chemical methods are mainly focused on suppressing the second barrier of endosomes through a process of endosomal escape, like utilizing of pH-sensitive materials

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or incorporating of the “proton sponge”

effect.23,24 On the other hand, physical methods based on the membrane-disruption principle have been applied to overcome the first barrier of the cell membrane by changing the membrane permeability.25,26 In our recent work, hypersound of gigahertz (GHz)


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frequency generated by a nanoelectromechanical (NEMS) resonator has been successfully applied to induce transient nanopores in lipid membranes,27 vesicles28 and cell membranes.29 Different from the conventional ultrasound of megahertz (MHz) frequency, the main working mechanism of hypersound is based on the acoustic streaming effect that can directly couple with the cell membrane at a sub-micron length scale.27 The high speed acoustic streaming induced by the GHz acoustics provides a dedicate tool to mechanically disrupt the cell membrane which can reversibly control the membrane permeability on demand.30 In this work, we applied hypersound of GHz frequency to assist the transmembrane delivery of Dox-loaded PMSNs. It is assumed that the acoustic streaming effects induced by hypersound can reversibly change the permeability of the cell membrane, thereby facilitating the direct penetration of drug carriers in avoid of the endosomal trapping issue. The cellular uptake of Dox-loaded PMSNs and the corresponding cell viability were tested with hypersound of different powers and durations to evaluate the therapeutic efficacy of this acoustic method. The mechanism of the carrier-based direct translocation was analyzed with inhibition experiments by suppressing their traditional endocytotic


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pathways. This study improves our understanding of hypersonic poration effects on enhanced cellular uptake and confirms the potential of hypersound as a non-invasive modality to change the pathways of extracellular substances.

2. RESULTS AND DISCUSSION 2.1. Hypersound-Controlled Distribution of Dox-load PMSNs. The schematic representation of hypersound-enhanced delivery of nanoparticles in a cellular system is illustrated in Figure 1. To enter into cells, polymer-wrapped mesoporous silica nanoparticles (PMSNs) are conventionally transported across the cell membrane via endocytosis (Barrier 1) and will be trapped into endosomes, which behave as another barrier (Barrier 2). Here, hypersound of gigahertz (GHz) frequency is applied to overcome these two biological barriers by reversibly disrupting the cell membrane. It is expected that the doxorubicin (Dox)-loaded PMSNs reach the cytoplasm though hypersound-induced transient openings by avoiding the formation of endosomes. In this way, the drug carriers will eject the loaded Dox molecules directly into the cytoplasm


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within a short time, and the small Dox molecules can subsequently be transported to the nucleus by diffusion.

Figure 1. Schematic representation of hypersound-triggered intracellular delivery of Dox-loaded PMSNs into cells. Conventionally, Dox-loaded PMSNs get through the cell membrane (Barrier 1) in an endocytotic pathway, and are subsequently limited by the endosome (Barrier 2) and envelope (Barrier 3) from reaching the nucleus. Once hypersound is turned on, the permeability of the cell membrane is enhanced, during which Dox-loaded PMSN can directly penetrate into the cytoplasm and gradually release the loaded Dox to the nucleus. The zoom-in images illustrate the chemical structure of the PDS-PEG polymer shell of the Dox-loaded PMSNs. As a model drug carrier for evaluation of the delivery efficiency, the structure of Doxloaded PMSNs is described as the cartoon nanoparticle in Figure 1. Dox molecules are


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encapsulated into the pores of MSNs that are non-covalently capped with copolymers containing 2-pyridyl disulfide hydrochloride (PDS) and poly(ethylene glycol) (PEG). Characterizations of the PDS-PEG polymer by nuclear magnetic resonance (NMR) spectra and gel permeation chromatography (GPC) were shown in Figure S1 (Supporting Information). Here, PEG is used to provide good water solubility and to prevent the nonspecific interactions with bio-macromolecules upon further clinical use. The PDS groups are used to facilitate wrapping of MSNs through multiple weak electrostatic interactions. Subsequently, the copolymers are crosslinked by the disulfide exchange to obtain a stable polymer shell for drug encapsulations (as depicted in the zoom-in image). After entering into the cytoplasm, the Dox molecules previously loaded within the PMSNs are released by the degradation of the polymer shell in the intracellular reducing microenvironment. However, the release rate of this polymer-wrapped MSN was quite low. The release profile in our previous published results showed that even at a higher concentration of reducing agent (10 mM GSH), it would take around 10 hours to reach a total release of the encapsulated Dox molecules. Dynamic light scattering (DLS) and zeta-potential measurements were used to check their size (177.2 nm, Figure S2) and surface charge


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(-9.87 mV, Table S1). Transmission electron microscopy (TEM, Figure S3) was applied to study the morphology of the nanoparticles. These results show that the synthesized Dox-loaded PMSNs are uniform and stable in aqueous suspension. The cellular uptake of Dox-loaded PMSNs under the stimulation of hypersound (1.6 GHz) was evaluated by confocal laser scanning microscopy (CLSM, Figure 2a). For a clear comparison, a group of HeLa cells that were incubated under the same conditions (i.e. cell density, culture medium, drug amount, and reaction time) but without hypersonic treatment is defined as the control group. As images of the control group (top row in Figure 2a) show, the intensity of the red fluorescence emitted by Dox was quite low in the cellular region. However, after exposure to hypersound of 250 mW for 10 min (images in the middle row of Figure 2a), the cells presented considerably increased Dox fluorescence inside their interior, indicating that a detectable quantity of Dox-loaded PMSNs had been internalized into the cells within a very short time. In a previous study, Dox-loaded PMSNs were found to reach the interior of cells after 30-min incubation without external stimulation,14 which in turn confirms the improved delivery efficiency of Dox-loaded PMSNs with the assistance of hypersound. Subsequently, the intensity of Dox


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fluorescence increased upon increasing the input power of hypersound to 500 mW. This power-dependent property implies that the delivered amount of drug carriers can be controlled on demand by tuning the input power of hypersound, which can potentially be applied to realize different release profiles, such as a burst release at higher power31 or a sustained release at lower power.32 Light field

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hypersound-treated groups, HeLa cells were exposed to hypersound of 250 or 500 mW for 10 min in the presence with the same amount of Dox-loaded PMSNs. For the control group, HeLa cells were incubated with Dox-loaded PMSNs without any hypersonic treatment for 10 min. The concentration of Dox-loaded PMSNs (Dox loading: 24 wt.%) in 0.1 M phosphate-buffered saline (PBS, pH 7.4) was 10 μg mL-1. Dox fluorescence (red) and nuclear DAPI fluorescence (blue) were captured by confocal microscopy. (b) The profiles represent the fluorescence distributions at indicated sections (white arrows) in the merged channel with 63x magnification, which shows the degree of colocalization between Dox and the nucleus. Furthermore, the cell nuclei were selectively stained with 4’,6-diamidino-2phenylindole (DAPI) to show the distribution of Dox-loaded PMSNs within the cells. Interestingly, the distribution of Dox fluorescence across cells stimulated by hypersound of different input powers presents different trends. As the merged images in the right column show, for cells exposed to hypersound of 250 mW for 10 min, the Dox fluorescence concentrated around the DAPI-stained areas without any overlap, indicating that the Dox-loaded PMSNs remained in the cytoplasm during the acoustic treatment. However, in the case of a power of 500 mW for 10 min, the localized Dox fluorescence was enhanced in the border of the nucleus and even a small fraction of red fluorescence overlapped with the DAPI signal, which suggests that Dox has entered into the nucleus


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with more intensive hypersound. These results are in accordance with the corresponding profiles shown in Figure 2b. 2.2. Hypersound-Enhanced Therapeutic Efficacy. Since the polymer shell of Doxloaded PMSNs can be dissolved due to the reducing conditions in the cell interior environment,14 the red fluorescence within the nucleus can be either from the Dox-loaded carriers or from released Dox. We further tested the therapeutic efficacy to figure out the issue. The cell viability of HeLa cells treated with Dox-loaded PMSNs was assessed using the Presto Blue assay.33 As shown in Figure 3a, Dox-loaded PMSNs show more toxicity under hypersound of 250 mW for 10 min in comparison with the non-hypersound treated group. An even higher cell apoptosis was observed by increasing the input power to 500 mW for 10 min. This can be related to the rapid internalization of Dox-loaded PMSNs triggered by hypersound, which is in accordance with the results shown in Figure 2a. Here, the cells exposed to hypersound (500 mW, 10 min) but in absence of Dox-loaded PMSNs were set as a control to verify that the hypersonic treatment itself does not cause cytotoxicity. The nontoxic nature of the MSN carriers was verified in a previous study14 and the cell viability assay of PMSN carriers in absence of Dox molecules was tested as


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well to show its biocompatible properties (Figure S5, Supporting Information). All-in-all, these results show the synergistic and specific effects of the combined use of Dox-loaded PMSNs and the acoustic treatment. It is likely that the insensitive acoustic streaming induced by high-frequency GHz hypersound provides strong shear stresses and disrupt the cell membrane,29 which enables the Dox-loaded PMSNs to reach the cytoplasm by escaping from the endosomal system and can thus release loaded drugs to the nucleus. The acoustically assisted release events can therefore be triggered as a fast ejection into the cytoplasm, providing a high local concentration of released Dox and inducing high efficient cell death, which is in contrast to the slow diffusion through the endosomal vesicle in the absence of hypersound.


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Figure 3. Comparison of the cell viability between Dox-loaded PMSNs and free Dox with hypersound treatment of (a) 10 and (b) 20 min. Blank: cells were incubated with cell culture medium without any hypersonic treatment. Control: cells were exposed to hypersound of 500 mW for 10 min. Dox-loaded PMSN group: cells were incubated with Dox-loaded PMSNs without or with the treatment of hypersound. Free Dox molecules group: cells were incubated with free Dox molecules without or with the treatment of hypersound. The input power of hypersound was 250 and 500 mW. The concentration of Dox-loaded PMSNs (Dox loading: 24 wt.%) was 10 μg mL-1 (i.e. the concentration of Dox encapsulated within the MSN carriers was 2.4 μg mL-1). The concentration of free Dox molecules was 2 μg mL-1. Data present mean ± SD (n = 6). *P < 0.05; **P < 0.01, analyzed by student t-test. The * indications above the light blue columns refer to the comparison between groups treated with free Dox molecules and the blank group. (c)


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Fluorescence microscopy images for the distributions of Dox-loaded PMSNs and the free Dox molecules after treatment with hypersound of 600 mW for 20 min. Drug carriers are widely used in chemotherapeutics since drugs that freely diffuse and distribute throughout the blood circulation will result in undesirable side effects and limit achievement of proper doses required for efficacious responses.34 However, the conventional therapeutic efficacy of carrier-formulated drugs is limited due to the carrier effect.15 Here, the cell viability of the group either treated with Dox-loaded PMSNs or free Dox was compared with hypersound of different intensities. In Figure 3a, free Dox shows more cytotoxicity than Dox-loaded PMSNs with or without hypersound treatment. This can be attributed to the carrier effect of PMSNs.16 After entering into cells, Dox-loaded PMSNs need to gradually open the polymer shells within the reducing intracellular microenvironment, then release the encapsulated Dox to the cytoplasm. In contrast, free Dox can directly interact with cells by diffusing all the way to the nucleus thus correlating with much greater toxicity. Note that when the input power was increased to 600 mW and extended for 20 min (Figure 3b), there is no marked statistical difference in cell viability between Dox-loaded PMSNs and free Dox, suggesting that the therapeutic efficacy of


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drug carriers and free drugs has achieved a nearly equal level. This is related to their similar subcellular distributions patterns as shown in Figure 3c. The red fluorescence emitted either from Dox-loaded PMSNs or free Dox was homogeneously distributed within the cells and totally overlapped with the DAPI-stained nucleus. It indicates that the limitation of carrier effects from MSN was overcome as the acoustic streaming was strong enough to trigger more cellular accumulation of Dox-loaded PMSNs and therefore achieve a compensation for the degradation process of the polymer shell. The delivery site of Dox-loaded PMSNs was verified later to be the cytoplasm instead of the endosomes, which could also be a possible reason for the enhanced therapeutic effect. The fluorescence intensities of Dox-loaded PMSNs before and after the stimulation of hypersound (600 mW, 20 min) were also compared (Figure S6, Supporting Information) to show the stability of PMSNs under the acoustic stimulation. The sight decreased fluorescence intensity indicated that most Dox were still preserved in PMSNs with only few leakages under hypersound. This equivalent efficiency achieved by hypersound is significant for the intracellular delivery of carrier-formulated drugs. Actually, localizing the drug-loaded nanoparticles


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does not provide information on the site of drug release, since only the transport of released drugs to the nucleus is the major approach for most anticancer drugs to achieve their neucleotropic ability.35 However, for nanoparticles with a diameter larger than ~9-12 nm, the bidirectional passive diffusion through the nucleus is restricted by the nuclear pore complex (NPX).36,37 With enhanced membrane permeability induced by the GHz acoustic streaming, the drug carriers can directly penetrate the cell membrane and release the loaded drugs into the cytoplasm without trapping by endosomes. From this site, the released drug molecules will efficiently diffuse into the nucleus to improve the final therapeutic efficacy. 2.3. Uptake Mechanism of Hypersound-Enhanced Delivery. To further evaluate how hypersound directly enhanced the cellular uptake of Dox-loaded PMSNs, a type of more stable mesoporous silica nanoparticles coated with crosslinked, layer-by-layer (LbL) assembled polymers (CLM-MSNs)38,39 were prepared and tested for this hypersoundresponsive process. As shown in Figure 4a, raspberry-type MSNs were wrapped with two oppositely

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negatively charged poly(methacrylate-co-2-mercaptoethyl methacrylate) (PMA-coPMEM) were used to form the LbL films. The crosslinking in the polyelectrolyte thin films was achieved by thiol exchange between the pyridyl disulfide in PDMAEM-co-PPDEM and the thiol in PMA-co-PMEM.39 Since the polymer shell of the MSNs was covalently crosslinked, the polymer layer is rather stable and cannot be removed by the physical acoustic treatment.40 The covalent crosslinked LBL polyelectrolyte film can stably hold the lipophilic dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) and 3,3'dioctadecyloxacarbocyanine (DiO) which are physically encapsulated in the pores of MSNs. Nor the hypersound-mediated pathway MSNs would be affected due to this different surface polymer layer.


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Figure 4. (a) Schematic and chemical structure of the cross-linked LbL-system of PDMAEM-co-PPDEM and PMA-co-PMEM (x: 0.33; y: 0.67). (b) Fluorescence microscopy images of intracellular uptake and distributions of dye-labeled CLM-MSNs in HeLa cells. For the hypersound-treated groups, HeLa cells were exposed to hypersound of 500 mW for 10 min in presence of dye-labeled CLM-MSNs. For the control group, HeLa cells were incubated with CLM-MSNs without any hypersonic treatment for 10 min. The concentration of CLM-MSNs (Dye loading capability: DiI 0.9 wt.%, DiO 0.8 wt.%) is 10 μg mL-1. As shown in Figure 4b, after exposure to hypersound of 500 mW for 10 min, the fluorescence emitted from both DiI and DiO can be clearly observed within the cells. Since the dyes cannot be released from the CLM-MSNs due to the stable encapsulation by covalently crosslinked polymer shell, the florescence emitted inside the cell can only be


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induced by the cellular uptake of the CLM-MSNs and it proves that hypersound can trigger the direct cellular uptake of MSNs as large as to 200 nm. In the control experiment, whereas the fluorescence intensity of the cells incubated with the same dye-labeled CLMMSNs in the absence of acoustic treatment was much lower. This again confirms the enhancement of the cellular uptake effect by hypersound and indicates the change of the uptake pathway of the MSNs. To further understand the mechanism of the cellular uptake enhancement by the hypersound, the pathways of Dox-loaded PMSNs were analyzed with inhibition tests. Two inhibitors (permethylated-β-cyclodextrin (m-βCD) and sucrose)41 were used to identify the particular uptake pathway of Dox-loaded PMSNs into cells in the absence of hypersound. The internalization of Dox-loaded PMSNs was monitored after the pretreatment with inhibitors of different concentrations for 30 min. In Figure 5a, the intensity of Dox fluorescence randomly improved with m-βCD of increased concentrations, suggesting that m-βCD, an inhibitor of caveolin-dependent endocytosis,42 did not reduce the cellular uptake of Dox-loaded PMSNs. In contrast, the inhibitor sucrose largely prevented the


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uptake of Dox-loaded PMSNs at higher concentrations, indicating that the primary uptake pathway of Dox-loaded PMSNs was clathrin-mediated endocytosis.43

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Figure 5. (a) Fluorescence intensity of HeLa cells pretreated with inhibitors (m-βCD or sucrose) for 30 min and then incubated with Dox-loaded PMSNs for 20 min. The applied concentrations of m-βCD were: 10, 20, 40, 60 and 80 mM (saturated). The concentrations of sucrose were: 225, 450, 900, 1350 and 1800 mM (saturated). Data present mean ± SD (n = 10). *P < 0.05 and **P < 0.01 compared with the control group (the first bar). (b) Fluorescence microscopy images of intracellular uptake and distributions of Dox-loaded PMSNs in HeLa cells with or without the pretreatment with


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inhibitors. The control group: cells with or without pretreatment with inhibitor were incubated with Dox-loaded MSNs (10 μg mL-1). The hypersound group: cells with or without pretreatment with inhibitor were exposed to hypersound of 500 mW for 10 min. To further determine the change of the uptake pathway in response to hypersound, cells pretreated with inhibitors were incubated with Dox-loaded PMSNs with or without the acoustic stimulation. As shown in Figure 5b, for the groups without exposure to hypersound, cells pretreated with m-βCD (80 mM) emitted nearly equal Dox fluorescence compared to the cells incubated without any inhibitors, while the cells pretreated with sucrose (1800 mM) presented nearly no Dox fluorescence. This is in accordance with the results shown in Figure 5a by confirming the inhibition effect of sucrose on the cellular uptake of Dox-loaded PMSNs. However, for cells that were stimulated by hypersound of 500 mW for 10 min, all the groups emitted significantly stronger Dox fluorescence compared with the non-acoustic treatment groups, regardless whether they were pretreated with inhibitors or not. This indicates that the uptake pathway of Dox-loaded PMSNs triggered by hypersound was no longer affected by the inhibitors. Particularly for the sucrose-treated group, since the formation of endosome has been disabled due to the blocking of the endocytosis functionality, it is likely that the Dox-loaded PMSNs inside


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the cell interior accumulated strongly into the cytoplasm instead of the endosomal system, which in turn shows that the uptake pathway of Dox-load PMSNs affected by the hypersound-triggered GHz acoustic streaming is no longer the endocytotic pathway. This can also be attributed to the acoustic poration effects29 It has been found that the cell membrane will be reversibly disrupted by the acoustic streaming resulted from the highfrequency GHz hypersound, during which some transient nanopores of 100-200 nm can be generated in the cell membrane28 to allow for the direct translocation of nanoparticles of limited sizes into the cytoplasm. It is therefore presumed that the conventional trapping of drug carriers by endosomes can be avoided by changing the uptake pathway with hypersound-enhanced membrane permeability, which will improve the therapeutic efficacy of drug carriers by directly releasing the loaded drug molecules in site of the cytoplasm. Different from the chemistry methods that normally incorporates a certain type of chemical agents to realize endosomal escape after an endocytosis process, hypersound just changes the endocytotic pathway to direct penetration via a physical membrane-disruption process, which has no limitations to the type of drug carriers or targeted cells.


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3. CONCLUSIONS In this work, hypersound of gigahertz (GHz) frequency was used to enhance the intracellular delivery of drug carriers by overcoming the conventional biological barriers of endosomal trapping inside the cell interior. Polymer-wrapped mesoporous silica nanoparticles (PMSNs) encapsulated with doxorubicin (Dox) were employed as the model carrier to demonstrate the acoustically improved delivery efficiency under hypersound of different intensities with the fluorescence and the cell viability tests. The mechanism studies illustrated that the GHz acoustic streaming induced by hypersound facilitated the internalization of PMSNs into cells following a hypersonic poration process, which changes the cellular uptake of drug carriers with a size of 100-200 nm from an endocytotic pathway to direct penetration. Since hypersonic poration is a transient, reversible and power-dependent process, the delivery of Dox-loaded PMSNs becomes a non-toxic and tunable process that can be controlled on demand through the power or duration of hypersound. Compared with the traditional endocytosis, this hypersoundtriggered translocation avoids the formation of endosomes, which logically improves the delivery efficiency by stepping over the biological barriers inside cells and directly deliver


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the drugs to nucleus. In principle, this membrane-disruption method is not limited to one single kind of drug carrier or drug, which can be potentially applied to different nanocontainers encasing multiple drug combinations for various therapeutic applications. The further applications of this acoustic method on different cell lines or tumor tissues in vivo would be worth trying and can be evaluated in the future work.

4. EXPERIMENTAL SENCTION Materials: Dulbecco's modified eagle medium (DMEM), 1,1’-dioctadecyl-3,3,3’,3’tetramethylindocarbocyanine (DiI) and 3,3’-dioctadecyloxacarbocyanine (DiO) were purchased from Thermo Fisher Scientific Inc. Doxorubicin hydrochloride (Dox) was purchased from Ontario Chemicals Inc. Phosphate-buffered saline (PBS) solution was composed of 0.1 M sodium dihydrogen phosphate and 0.15 M sodium chloride. The pH was adjusted to 7.4 with sodium hydroxide. Preparation of Dox-loaded MSNs: Mesoporous silica nanoparticles (MSNs, MCM-41, 160 nm) and PDS-PEG polymer were synthesized according to a previously published result, and the same batch of MSNs and polymers were used here.14 Then, MSNs (10


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mg) were added to ultrapure water (1 mL) and sonicated to disperse the MSNs until a uniform colloidal solution was observed. An amount of 5 mg Dox was dissolved in this solution and allowed to stir for 3 h. Then, the dispersion was centrifuged to collect the Dox-loaded MSNs and the supernatant was used to calculate the drug loading content. In order to remove the Dox from the exterior surface of the MSNs, the drug-loaded MSNs were washed twice with ultrapure water and collected by centrifugation. After that, the polymer solution of PDS-PEG was added to Dox-loaded MSNs and stirred for 3 h at room temperature. Then dithiothreitol (DTT) (60 mol % against PDS) was added to the mixture and stirred for 3h to allow for in-situ crosslinking. The polymer cross-linked PMSNs were collected by centrifugation. The unabsorbed and unreacted DTT were removed by washing with DI water by centrifugation and re-dispersion cycles. Preparation of CLM-MSNs: Raspberry-type MSNs (rMSNs),14 PDMAEM-co-PPDEM and PMA-co-PMEM polymers36 were synthesized according to a previously published result and same batch of MSNs and polymers were used here. Then, rMSNs were alternately immersed in the phosphate buffer (0.1 M, pH 7.4) of PDMAEM-co-PPDEM (1 mg mL-1) and PMA-co-PMEM (1 mg mL-1) for 5 min for each step. rMSNs were washed


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with the 0.15 M NaCl solution after each step. Finally, the layer-by-layer (LbL) film was crosslinked by the thiol-exchange reaction and functionalized with the fluorescent dyes by placing them respectively in the solution of DiI (1 mg mL-1) and DiO (1 mg mL-1). Characterizations of CLM-MSNs were illustrated in Figure S4 and Table S2 in Supporting Information. Transmission electron microscopy: Transmission electron microscopy (TEM) analysis was performed using JEOL-2100 microscope at an accelerating voltage of 200 kV. To prepare the sample for TEM, a droplet of Dox-loaded PMSNs/CLM-MSNs was casted on a 200 mesh copper grid for 2 min, and then gently wiped with a sterile paper. Dynamic light scattering and zeta-potential measurements: Dynamic light scattering (DLS) and zeta-potential measurements were carried out simultaneously using Malvern ZS 90 Zetasizer instrument. 0.1% suspensions of Dox-loaded PMSNs/CLM-MSNs were prepared in MilliQ water and these suspensions were sonicated for 5 min before the analysis. Setup of intracellular delivery system: The setup for an intracellular delivery system triggered by hypersound is shown in Figure S7. A polydimethylsiloxane (PDMS) chamber


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(thickness: 600 μm) with a diameter of 6 mm was sealed on the NEMS resonator (1.6 GHz) and filled with cell culture medium containing Dox-loaded PMSNs (typical 50 µL). A glass slide that seeded with approximately 1 × 104 HeLa cells was covered on top of the PDMS chamber, aligning above the center of the BAW resonator. Sinusoidal signal of 1.6 GHz was generated by a signal generator (Agilent, N5181A) and amplified by a power amplifier (Mini-Circuits, ZHL-5W-422), which then transduced the electrical signal to mechanical vibrations of resonator and generated hypersound. The input power of hypersound was conveniently controlled by adjusting the power of the signal generator. Cell viability analysis: In vitro cytotoxicity was evaluated by the Presto Blue assay (Thermo Fisher Scientific Inc.). HeLa cells were first seeded into a 96-well plate at a density of 1.0 × 104 cells per well in 100 µL DMEM and incubated at 37 °C in 5% CO2 atmosphere for 24 h. Afterwards, the culture medium was replaced with 10 mg mL−1 Doxloaded PMSNs (Dox loading density 24 wt.%, the equivalent concentration of Dox was 2.4 μg mL-1) or 2 μg mL-1 Dox molecules that dissolved in DMEM and treated with hypersound of different input powers. Here, the equivalent dosage of free Dox molecules was introduced as a comparison with Dox-loaded MSNs to show the hypersound effects


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on delivery efficiency of different types of drugs, in which the slight difference would not affect the evaluation of the delivery efficacy. HeLa cells that incubated with Dox-loaded PMSNs/Dox but without any treatment of hypersound were used as a negative control. HeLa cells that that incubated with DMEM and treated with hypersound were used as a positive control. After incubation, the Presto Blue stock solution (5 mg mL−1, 10 µL) was added to each well and incubated for another 20 min. The absorbance was monitored using a microplate reader (Thermal Scientific, Multiskan GO) at the excitation wavelength of 540 nm. The efficiency was assayed in terms of the percentage of cell viability compared to untreated blank control cells. Fluorescence measurements: Confocal laser scanning microscope (CLSM, Nikon A1) and fluorescence microscope (Olympus, BX53) were used to evaluate the intracellular uptake and distribution of Dox-loaded PMSNs or free Dox molecules. After each hypersonic treatment, the HeLa cells were washed with PBS buffer three times to remove the redundant nanoparticles or drug molecules. The HeLa cells were then fixed with 4% paraformaldehyde aqueous solution for 10 min at room temperature and rinsed with PBS buffer again. Afterwards, the HeLa cells were stained with DAPI at room


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temperature for 5 min and rinsed with PBS buffer twice. The prepared slides of fixed samples were examined by CLSM and fluorescence microscope at excitation wavelength (Ex) of 485 and 403 nm, emission wavelength of (Em) 550 and 450 nm respectively for Dox and DAPI. To quantify the cellular uptake of Dox-loaded PMSNs with inhibitors, the fluorescence intensity of HeLa cells was measured by a fluorescence spectrophotometer (Thermal Scientific, Varioksan LUX).

ASSOCIATED CONTENT

Supporting Information. Characterizations of the Dox-loaded MSN and the CLM-MSN, setup of the drug delivery system. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

* Tel. /Fax: +86 2227401002. E-mail: [email protected], [email protected], [email protected].


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ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (NSFC No. 61674114, 91743110, 21861132001), National Key R&D Program of China (2017YFF0204600), Tianjin Applied Basic Research and Advanced Technology (17JCJQJC43600), the Foundation for Talent Scientists of Nanchang Institute for Microtechnology of Tianjin University, the 111 Project (B07014), and the Netherlands Organization for Scientific Research (NWO-NSFC grant to J. H. and X. D.). J.H.R. was supported by the National Research Foundation of Korea (NRF) (2016R1A5A1009405) and we thank Prof. S. Joo for kind provide of mesoporous silica nanoparticles.

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TOC


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Hypersound-Enhanced Intracellular Delivery of Drug-Loaded

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