Journal Pre-proofs Bacteria-propelled microrockets to promote the tumor accumulation and intracellular drug uptake Songzhi Xie, Tian Xia, Shang Li, Chuanfei Mo, Maohua Chen, Xiaohong Li PII: DOI: Reference:
S1385-8947(19)33201-2 https://doi.org/10.1016/j.cej.2019.123786 CEJ 123786
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
15 October 2019 5 December 2019 9 December 2019
Please cite this article as: S. Xie, T. Xia, S. Li, C. Mo, M. Chen, X. Li, Bacteria-propelled microrockets to promote the tumor accumulation and intracellular drug uptake, Chemical Engineering Journal (2019), doi: https://doi.org/ 10.1016/j.cej.2019.123786
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Bacteria-propelled microrockets to promote the tumor accumulation and intracellular drug uptake
Songzhi Xie,a,1 Tian Xia,b,1 Shang Li,a Chuanfei Mo,a Maohua Chen,a Xiaohong Li a,*
a Key
Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials
Science and Engineering, Southwest Jiaotong University, Chengdu 610031, P.R. China b Department
of Pathology, Western Theater Command Air Force Hospital, Chengdu 610021, P. R.
China
1 Songzhi
Xie and Tian Xia contributed equally to this work.
* Corresponding Author. E-mail:
[email protected]. Tel: +8628-87634068, Fax: +8628-87634649.
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ABSTRACT: To address the biological or pathological barriers, the self-propulsion abilities of drug carriers may bring distinct improvements when compared to passive diffusion. In the current study bacteriapropelled microtubular rockets are proposed to enhance the extravasation from blood circulation, penetration in the tumor matrix and intracellular drug uptake. Microtubes with loaded doxorubicin (MTDOX) are constructed via layer-by-layer deposition of oxidized alginate (O-Alg) and chitosan in sacrificial porous membranes, followed by crosslinking via schiff’s base bonds to regulate drug release. Under a concentration gradient of L-aspartate as the chemoattractant, E. coli Nissle1917 (EcN) are embeddded into microtubes as bioengines to obtain MTDOX@EcN microrockets. The assembled EcN provide aligned propulsion forces and directional trajectories like a microrocket, exhibiting high motion velocities in both water and viscous media. The uptake efficiency of MTDOX@EcN by tumor cells shows over 2.1 folds higher than that of MTDOX, indicating that the EcN propulsion could promote the cell internalization of MTDOX. After intravenous injection of MTDOX@EcN, the DOX levels in tumors reach 6.1 folds higher than those of MTDOX after 7 days, suggesting that the EcN-propelled rocket-like motion could promote the extravasation from blood circulation and the retention in tumors. Compared with other treatment, MTDOX@EcN could significantly inhibit the tumor growth, increase the survival rate of animals, and avoid side effects like hematologic, hepatic and renal toxicities. Herein, the bacteria-propelled microrockets demonstrate a feasible strategy to boost therapeutic efficacy of anticancer agents by combating multiple biological barriers in the drug delivery pathway.
Keywords: microtubular rocket; bacterial propulsion; layer-by-layer deposition; tumor accumulation enhancement; intracellular uptake promotion
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1. Introduction: Chemotherapy is an important and potent treatment method to destroy or slow down the growth of cancer cells. Unfortunately, cancer chemotherapy has shown limitations such as poor biocompatibility, inefficiently delivery and rapid blood clearance. The development of nanoparticles, polymeric micelles and liposomes has made significant progress in prolonging the blood circulatin, improving the tumor accumulation, and increasing the bioavailability of anticancer drugs [1]. However, an effective drug delivery system has to overcome a set of transport barriers from the administration site to the intracellular target. It is known that 85% of human cancers are derived from solid tumors, and the nanocarriers are confronted with biological or pathological barriers in blood circulation, blood-to-tumor extravasation, tumor interstitial penetration, cellular uptake and intracellular drug release [2]. The tumor targeting of nanocarriers is mainly taking advantages of the enhanced permeation and retention (EPR) effect, resulted from the leaky blood vessels and poor functional lymphatics in tumor tissues. Despite well control over the size, geometry and surface characteristics, the tumor accumulation of nanocarriers is usually lower than 5% and rarely higher than 10% of the administered dose [3]. After extravasation into tumors, nanocarriers have to penetrate the tumor extracellular matrix (ECM) to reach tumor cells. There are many substances such as collagen, hyaluronic acid, and glycoproteins filled in solid tumors, resisting the delivery diffusion of chemotherapeutic drugs [4]. Experimental approaches have been investigated to improve the accumulation and distribution of nanocarriers in solid tumors, including enzymatic degradation of ECM and normalization of tumor vasculatures. Zhou et al. coated nanoparticles with collagenases, elastases and hyaluronidase, promoting the accumulation in the core of multicellular spheroids and the deep penetration into tumor tissues [5]. However, the enzymatic degradation approach meets challenges in utilization of optimal types and doses due to the variations of tumor ECM. In addition, the inappropriate destruction of tumor microenvironment is usually associated with tumor invasion and cancer metastasis [6].
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To address the transportation barriers, the self-propulsion abilities of drug carriers may bring distinct improvements when compared to passive diffusion [7]. Namely, self-propelled drug carriers may provide continuous driving force to cross diffusion barriers, distribute in the whole tumors and interact with tumor cells [8]. The motion is mainly drived via conversion of external or chemical energy into mechanical force. External energy sources such as high magnetic field, strong electric field and ultrasound have been utilized to control the movement. Shao et al. constructed Janus particles with a magnetic head and a mesoporous SiO2 body. After treatment under magnetic field, the tumor growth was notably suppressed with a significant reduction of systematic toxicity [9]. But these techniques often require trained professsionals, special equipment and hospital environment [10]. Alternatively, microscale particles and rods are decorated with catalytic metals and enzymes for converting of chemical sources into propelling forces. Hu et al. indicated that platinum nanoparticles on polyelectrolyte multilayer microtubes could produce oxygen bubbles as the self-propelling force via catalytic decomposition of hydrogen peroxide [11]. It should be noted that the hydrogen peroxide fuel is highly toxic to cells and tissues, thus restricting the application in biomedical fields [12]. The enzymatic reaction with glucose and urea as fuels could produce chemotaxic motions, but they are still lack of self-guided force and targeted motion toward tumors [13]. A dynamical alternative to overcome these barriers is the development of self-guided micromotors based on the motility of natural organisms (cells and bacteria). It is known that the hypoxic zone in the tumor core reduces the susceptibility of tumor cells to anticancer drugs, while the lower oxygen microenvironment provides an appreciable habitat for bacteria targeting [14]. Several flagellar bacteria have been applied as an actuator part, and drugs or drug-loading cargoes are integrated on bacteria via different ways such as electrostatic adhesion, chemical bonding, and antibody linking [15]. Bacteria demonstrate the ability to carry particles of 500 nm to 10 μm, and the biohybrid could swim at a speed of several body lengths per second [16]. Stanton et al. prepared Janus polystyrene microspheres (2 μm) by selective capping of metal layers, and bacteria were attached via preferential interaction with the 4
metal caps. The bacterial attachment on one side of microparticles produced directional forces to propel the motion of microparticles [17], while the excessive bacteria attachment around the entire particle disturbed the directional motion [18]. In addition, bacterial micromotors meet several challenges in the construction and biomedical applications. Many bacteria used need attenuated process to avoid virulence. Despite lack of pathogenicity, the potential immunogenicity of attenuated bacteria should be addressed referring to clinical applications [19]. Second, the cargoes in the micromotor investigation are composed of nonbiodegradable polystyrene and polydimethylsiloxane, indicating limitations in in vivo application. Nontoxic bacteria power and biodegradable and biocompatible matrix materials should be exported for in vivo application of bacteria-based micromotors [20]. Third, up to now most studies have focused on in vitro motion profiles and in vivo distribution properties of bacteria with loaded metal or polymer nanoparticles, whereas few report focused on in vivo evaluation of therapeutic efficacy [21]. Recently Park et al. coated polyelectrolyte multilayer on polystyrene microparticles (1 μm) for bacterial attachment and drug loading. The bacteria-propelled motion increased the targeting efficiency of microparticles to breast cancer cells in vitro [22]. In the current study, E. coli Nissle1917 (EcN) was integrated into microtubes to enhance the drug accumulation and distribution in tumor tissues. EcN is a normal probiotics existed in human and animals and absence of protein toxin expression, exhibiting little mannose-resistant hemagglutinating adhesions and causing no immune attack [23]. In the previous studies, free doxorubicin (DOX) or DOX-polymer conjugates were chemically linked onto the surfaces of EcN. EcN could accumulate and proliferate in the solid tumor, and the preferential drug accumulation achieved a better chemotherapeutic effect of tumors [24, 25]. However, the drug loading levels were relatively low, at around 4.3% and 5.8% after chemically linking free DOX and DOX-polymer conjugates, respectively. In the current study, DOX was loaded in microtubes via layer-by-layer deposition, and the drug loadings and wall thickness can be selectively tuned by varying the deposition layers. Scheme 1a illustrates the preparation process of EcN-powered DOX-loaded microtublular rocket (MTDOX@EcN), 5
using polycarbonate (PC) membranes as the master template. MTDOX were obtained by layer-by-layer assembly of oxidized alginate (O-Alg) and chitosan, and crosslinked via schiff’s base bonds to achieve acid-sensititive drug release. EcN were used as bioengines to push the motion after embedment into microtubes under a concentration gradient of L-aspartate (Asp) as chemoattractant in the inner walls. As shown in Scheme 1b, the self-propelled rocket-like motion was expected to enhance the extravasation of MTDOX@EcN from blood circulation, penetration in the tumor matrix and uptake into tumor cells. The EcN viability, motion behavior, drug release, intracellular uptake and cytoxicity of MTDOX@EcN were investigated. The biodistribution of EcN and DOX and the antitumor efficacy of MTDOX@EcN were evaluated on tumor-bearing mice.
2. Materials and methods 2.1. Materials EcN bacteria were donated from Southwest University (Chongqing, China), and DOX was from Melone Pharmaceutical Co., Ltd. (Dalian, China). PC membranes (pore size: 2 μm), dialysis bags (MW cutoff: 3.5 kDa), Asp, poly(ethylene imine), sodium alginate, chitosan, hyaluronic acid (HA), deuterated dimethyl sulfoxide (DMSO-d6), carboxylic acid functionalized Fe3O4 nanoparticles (20 nm), 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI), Triton X-100 and trypsin were procured from Sigma-Aldrich Inc (St. Louis, MO). Rabbit antimouse antibodies of Ki-67 and caspase-3, goat antirabbit IgG–horseradish peroxidase (HRP) and 3,3-diaminobenzidine (DAB) developer were purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Rabbit anti-E. coli polyclonal antibody and donkey anti-rabbit IgG-fluorescein isothiocyanate (FITC) were obtained from Sangon Biotech Co., Ltd (Shanghai, China). All other chemicals were analytical grade and received from Changzheng Regents Company (Chengdu, China), unless otherwise indicated.
2.2. Preparation of MTDOX 6
Scheme 1a illustrates the preparation process of MTDOX via layer-by-layer assembly of alginate and chitosan in micropores of PC membrane [26], and alginate was oxidized to increase the biodegradability as previously described with slight modifications [27]. Briefly, 15 mL of sodium metaperiodate solution (10%, w/w) in water was added into 15 mL of sodium alginate solution (20%, w/w) in ethanol. After reaction for 4 h at room temperature, the mixtures were dialyzed against water for 2 days to completely remove unreacted sodium metaperiodate. The dialysis solution was freezedried to obtain O-Alg. PC membrane was immersed into poly(ethylene imine) (1 mg/mL) and NaCl (0.5 M) solution in water for 30 min, followed by gentle sonication for 2 min. After water rinsing for 10 min, the PC membrane was placed into a film-changeable filter, follows by successively squeezing chitosan solution (1.5 mg/mL in water containing 0.1 M NaCl and 0.02 M acetic acid) and O-Alg solution (1.5 mg/mL in water containing 0.1 M NaCl). Total 16 layers of O-Alg/chitosan were deposited to obtain microtubes, and CaCl2 (0.5 mg/mL) was used to crosslink O-Alg. In each deposition intermittence, 0.1 M NaCl solution was used to wash and wet cotton swabs were used to wipe the surface of PC membranes to abandon the non-assembled residues. DOX was added in chitosan solution and used to assemble with O-Alg solution, and successive 12 layers (from layer 3 to 14) were deposited to obtain MTDOX. In addition, O-Alg solution containing Fe3O4 nanoparticles were deposited in the middle of microtubes (layer 9), and chitosan solution containing Asp (0.1 mg/mL) were deposited in the layer 11. The resulting PC membranes were polished by plasma etching at a pressure of 100 mTorr (ZJL400-I, Chengdu CVAC Vacuum Technology, China) for 4 min to remove the complexes formed on the surface. The PC template was dissolved in methylene dichloride, and microtubes were collected via centrifugation and magnetic field, followed by dispersion in ethanol/water mixtures (1/7, v/v).
2.3. Preparation of MTDOX@EcN
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EcN was loaded into the hollow pores of MTDOX using Asp as the chemoattractant (Scheme 1a). Briefly, EcN was incubated overnight in Luria-Bertani (LB) broth containing 1% of tryptone, 0.5% of yeast extract and 1% of NaCl, followed by collection via centrifugation. EcN were resuspended in phosphate buffered saline (PBS) at an optical density (OD) of 0.8 and mixed with microtube suspensions (1 mL) in a centrifuge tube for 30 min. A magnet was placed under the centrifuge tube for 5 min to collect MTDOX and MTDOX@EcN and the unbound EcN was removed from the supernatant. MTDOX were separated by centrifugation under 4000 rpm for 1 min and then 3000 rpm for 3 min to obtain MTDOX@EcN. Different Asp amounts were loaded in microtubes as the chemoattractant to determine the effect on the EcN loading efficiency.
2.4. Characterization of MTDOX and MTDOX@EcN The morphologies of MTDOX and MTDOX@EcN were detected by using a scanning electron microscope (SEM, FEI Quanta 200, The Netherlands), and the length, inner diameter and wall thickness were evaluated from SEM images. The formation of schiff’s base bonds between O-Alg and chitosan was characterized by using a Fourier transform infrared (FTIR) spectroscope (Thermo Nicolet 6700). The oxidation degree of O-Alg was examined from the aldehyde densities via the hydroxylamine hydrochloride method [28]. The drug loading of DOX and Asp in microtubes was determined from the residual in the washing solutions and the initial addition amount. The DOX levels in the media were measured by using a fluorescence spectrophotometer (Hitachi F-7000, Japan) with the excitation/emission wavelengths of 485/590 nm. The Asp concentration was determined via reaction with ninhydrin (1.0 M) and measured by using an ultraviolet-visible (UV-vis) spectrophotometer (UV-2550, Shimadzu, Japan) at 560 nm. The amount of EcN loaded in microtubes was calculated according to the concentration difference before and after bacterial assembly. In order to directly observe EcN, MTDOX@EcN were mixed with rabbit anti-E. coli polyclonal antibody for 1 h at 37 C, followed by PBS washing and then incubation 8
with FITC-conjugated donkey anti-rabbit IgG [24]. The immunostained EcN and inoculation of DOX in MTDOX@EcN were examined by using a confocal laser scanning microscope (CLSM, Leica TCS SP2, Germany). The viability of bacterial cells in MTDOX@EcN was estimated by the Live/Dead BacLight Bacterial Viability Kit (Invitrogen, Grand Island, NY) according to the manufacture’s instructions. The live and dead bacteria were observed by CLSM at the excitation/emission wavelengths of 485/498 (SYTO 9) and 535/617 nm (propidium iodide).
2.5. Motion behaviors of MTDOX@EcN The motion of MTDOX@EcN was examined in LB media using Asp as the chemoattractant [29]. Briefly, polydimethylsiloxane (PDMS) was put along four sides of a glass slide to construct a cuboid chamber and Asp was included in one side. The cuboid chamber with filled with LB media, and the motion of MTDOX@EcN was recorded by using an inverted video microscope (Olympus IX51, Japan) under a 40× objective. The bacterial positions were tracked from series trajectory frames, and the motion velocity was determined via ImageJ analysis. To estimate the penetration ability of MTDOX@EcN in viscous media, the culture media were added with HA to simulate the viscous networks of ECM [30]. The chamber was filled with media containing 3 mg/mL of HA and a capillary tube was connected with the chamber wall so that media could fill up the capillary tube from the chamber. At the end of the capillary tube, far away from chamber, was filled with agar containing Asp. MTDOX or MTDOX@EcN with an equal amount of microtubes were added into the chambers. After incubation for 60 min, the capillary tubes were detected at different sites under CLSM to estimate the motion and permeation behaviors.
2.6. Drug release and degradation of MTDOX@EcN The pH-sensitive drug release and degradation of MTDOX@EcN were investigated after incubation in pH 7.4, 6.5 and 5.5 buffers, using MTDOX as control. Briefly, MTDOX@EcN were inoculated into a 9
dialysis bag and immersed in release buffers (20 mL), which were kept at 37 C in a thermostated shaking water bath. At regular time intervals, 1.0 mL of the release media were retrieved, and an equal volume of fresh buffers were added back for continuing incubation. The DOX levels in the release media were examined by using a fluorescence spectrophotometer as above. In addition, MTDOX@EcN were collected after incubation for 1 and 7 days in pH 6.5 buffers and observed by SEM to estimate the microtube degradation.
2.7. Cellular uptake of MTDOX@EcN The cellular uptake of MTDOX@EcN was detected on 4T1 and RAW267.4 cells as described previously [31], using DOX and MTDOX as control. Briefly, 4T1 and RAW267.4 cells from American Type Culture Collection (Rockville, MD) were incubated in a 24-well tissue culture plate (TCP) at a density of 1 × 105 cells per well for 24 h. Cells were treated with DOX, MTDOX and MTDOX@EcN for 8 h at an equivalent DOX dose of 1 μg/mL. In order to evaluate the cellular uptake of MTDOX@EcN by 4T1 cells in the tumor ECM, the individual group was incubated in media of pH 6.5 [32]. After trypsin digestion and Triton X-100 lysis, DOX levels in the cell lysate were detected by using a fluorescence spectrophotometer and compared with those initially added to obtain the uptake efficiency. In addition, 4T1 cells were treated with MTDOX and MTDOX@EcN at an equivalent DOX dose of 1 μg/mL for 12 h. Cells were fixed in 4% paraformaldehyde for 30 min, followed by DAPI staining and PBS rinsing. After incubation with trypan blue to quench the fluorescence of outer membranes, cells were observed under CLSM at the excitation/emission wavelengths of 405/462 nm. To estimate the potential endocytosis pathways for the cellular uptake of DOX, MTDOX and MTDOX@EcN, 4T1 cells were treated individual inhibitors as described previously [33]. Briefly, 4T1 cells were pretreated with chlorpromazine (25 µM), nystatin (25 µM), or amiloride (100 µM) for 2 h at 37 °C. DOX, MTDOX or MTDOX@EcN were added into media at an equivalent DOX dose of 1 μg /mL,
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and cells were incubated for 8 h at 37 °C or 4 °C, using cells without inhibitor pretreatment as control. Then cells were digested and the cellular uptake efficiency was detected as above.
2.8. Cytotoxicity and haemolysis testing of MTDOX@EcN The cellular cytotoxicity and apoptosis induction of DOX, MTDOX and MTDOX@EcN were evaluated on 4T1 and RAW267.4 cells as described previously [31]. Briefly, cells were seeded in 96-well TCPs at a density of 1 × 104 cells per well. After culture for 24 h in pH 6.5 or pH 7.4 media, cells were incubated with series concentrations of DOX, MTDOX and MTDOX@EcN for 72 h, using cells without treatment as control. Cells were cultured in MTT-containing media for 4 h before measuring the absorbance in dimethyl sulfoxide by using a Quant microplate spectrophotometer (Elx-800, Bio-Tek Instrument Inc., Winooski, VT). To mmeasure the cell apoptosis, 4T1 cells were grown in 12-well TCPs at a density of 2 × 105 cells per well before treatment with DOX, MTDOX and MTDOX@EcN for 48 h. Cells were collected via trypsin digestion and stained by using an Annexin V-FITC apoptosis detection kit (Beijing 4A Biotech Ltd., Beijing, China) according to the manufacture’s instructions, followed by flow cytometry analysis (BD Accuri C6, BD Biosciences, CA). The blood compatibility of MTDOX@EcN was measured from haemolysis as described previously [34]. Briefly, red blood cells (RBCs) were harvested from rat blood via centrifugation, followed by PBS washing to remove any hemoglobin. RBC suspensions were incubated with EcN, MTDOX and MTDOX@EcN with an equivalent dose of DOX or EcN, using deionized water and PBS treatment as positive and negative controls, respectively. After incubating at 37 C for 1 h, supernatants were collected from via centrifugation, and the hemoglobin levels were estimated by using an UV-vis spectrophotometer at 540 nm. The haemolysis rate was obtained from the absorbance difference between samples with negative control, in comparison with that between positive and negative controls.
2.9. Tissue distribution of MTDOX@EcN 11
The biodistribution of MTDOX@EcN was determined from the EcN and DOX levels in major tissues of 4T1 tumor-bearing mice as described previously [25], compared with those of free DOX, EcN, and MTDOX. All animal procedures were performed in accordance with the National Institutes Health Guide for the Care and Use of Laboratory Animals of China and approved by the Animal Care and Use Committee of Southwest Jiaotong University. Briefly, female Balb/c mice (20–22 g) were received from Sichuan Dashuo Biotech Inc. (Chengdu, China), and 4T1 cells were injected into the mammary fat pad at 5 × 106 cells per mouse to establish tumor models. After tumor growth to around 100 mm3, mice were intravenously injected with free DOX, MTDOX and MTDOX@EcN at an equivalent DOX dose of 1.0 mg/kg. Another group of tumor-bearing mice were treated with free EcN at the same number of MTDOX@EcN. After 3 h and 1, 3 and 7 days of treatment, animals were sacrificed to retrieve tumors, lungs, livers, spleens, kidneys and hearts. To determine the bacterial levels, tissues were ground and spread onto LB agar plates for bacteria counting. Tissues were also processed into sections and immunostained with anti-E. coli polyclonal antibody as above, and the DOX and FITC fluorescence was observed via CLSM to examine the drug and EcN distributions. To examine the DOX levels, tissues were homogenized in PBS before extraction with dimethyl formamide. The drug contents in the extract were detected as above, and calibrated after adding known amount of DOX in the tissue homogenates from untreated mice. The percentage of injected dose (ID%) was defined as the ratio of the drug content detected in a tissue to the administration dose.
2.10. In vivo toxicity and antitumor efficiency of MTDOX@EcN The tumor-bearing mice were divided randomly into 5 groups with 7 mice per group. DOX, EcN, MTDOX and MTDOX@EcN were intravenously adminstered through tail veins at an equivalent DOX dose of 1.0 mg/kg, using saline injection as control. The tumor volumes, body weights and survival rates of mice were monitored every 3 days after treatment as described previously [24]. The number of survival mice was plotted against treatment time, the 50% mean survival time was calculated from the 12
survival curves. Mice were sacrificed after treatment for 24 days to retrieve tumors and hearts, followed by processing into sections for histological analysis. Hematoxylin and eosin (H&E) staining was performed on tissue sections to determine tumor necroses and cardiotoxicities and observed under a light microscope (Nikon Eclipse E400, Japan). Immunohistochemical (IHC) staining of Ki-67 and caspase-3 was perfromed to examine the proliferation and apoptosis of tumor cells. The proliferation or apoptosis ratios were obtained from the number of positively stained cells in the staining images and compared with the total number of cells in the same areas [25]. To monitor the cytotoxicity of MTDOX@EcN treatment, whole bloods were collected after 1 and 3 days for routine blood testing. The numbers of RBCs, white blood cells (WBCs) and platelets (PLTs) were detected by using a Sysmex XT-2000i automated hematology analyzer (Sysmex Corp., Hyogo, Japan). The serum levels of alaninetransaminase (ALT), aspartate transarninase (AST), creatinine (CRE) and blood urea nitrogen (BUN) were measured by using an automatic biochemistry analyzer (AU5800, Beckman Coulter, Brea, CA).
2.11. Statistical analysis Data are expressed as mean ± standard deviation (SD). Whenever appropriate, comparisons among multiple groups were performed by analysis of variance (ANOVA), while a two-tailed Student’s t-test was used to discern the statistical difference between two groups. A probability value (p) of less than 0.05 was considered to be statistically significant.
3. Results and discussion 3.1. Characterization of MTDOX and MTDOX@EcN MTDOX were prepared by layer-by-layer assembly of O-Alg and chitosan in porous templates, followed by removal of the PC membrane (Scheme 1a). The inoculation of DOX in chitosan solutions was used to load drugs in microtubes after assembly. Fe3O4 nanoparticles were included at around 13
0.7% for the separation and purification of MTDOX. Fig. 1a shows SEM images of MTDOX, displaying average length of 3.0 μm, inner tube size of 1.5 μm and wall thickness of 0.1 μm (inset of Fig. 1a). As shown in Fig. 1b, CLSM observations indicated a hollow structure and DOX loadings in MTDOX. Asp was loaded into microtubes as chemoattractant to capture EcN for construction into MTDOX@EcN. Different Asp amounts of were loaded in microtubes, and Fig. 1c shows the loading efficiency of EcN as a function of Asp concentrations used in the assembly process. The assembly efficiency between EcN and microtubes was increased with increasing the Asp contents and reached a plateau at Asp loading levels of over 0.5%. Fig. 1d shows an SEM image of MTDOX@EcN, indicating that a bacterium was drilled into MTDOX and about half of the body was out of the microtube. It was suggested that EcN had the optimal status compromised under the guidance of chemotactic agents and the electrostatic interaction with microtubes. EcN were IHC stained with FITC-labeled antibody (Fig. 1e), showing the efficient assembly of EcN (green) and MTDOX (red). MTDOX@EcN were stained with SYTO 9 green and propidium iodide red to indicate the integrity of bacteria wall. SYTO 9 penetrated intact membranes and generally stained all alive and dead bacteria, while propidium iodide was generally excluded from viable cells with intact membranes [35]. As shown in Fig. 1f, rare red signals in CLSM images confirmed that most EcN kept alive after MTDOX@EcN assembly. Thus, integral EcN morphologies were kept in MTDOX@EcN due to the gentle condition in the assembly process, and should have the ability to drive MTDOX.
3.2. DOX release profiles from MTDOX@EcN An efficient drug loading was one of the challenges in bacteria-derived drug delivery system. In the previous study, DOX was grafted onto EcN and the drug loading contents was 4.3% [24]. To achieve a higher therapeutic efficiency, DOX was conjugated with promicelle polymers and then immobilized on EcN, acheving a DOX loading levels of around 5.8% [25]. In the current study, DOX was mixed in the multiple layers of microtubes, reaching a loading content of around 20.6%, which was remarkably 14
higher than those of DOX-encapsulated microspheres [36]. The DOX loading content in MTDOX@EcN was around 8.1%. Fig. 2a shows the DOX release profiles from MTDOX and MTDOX@EcN after incubation in pH 7.4, 6.5 and 5.5 buffers. MTDOX@EcN indicated only around 6.2% of DOX release at pH 7.4 during 24 h, while around 67.9% and 42.7% of accumulated releases were detected after incubation in pH 5.5 and 6.5 buffers, respectively. MTDOX showed similar release profiles of DOX in response to media pH. It was indicated that MTDOX and MTDOX@EcN were kept stable during circulation and gradually released DOX in tumor tissues. Fig. 2b shows SEM images of MTDOX@EcN after incubation in pH 6.5 for 24 h and 6 days. MTDOX@EcN kept the rough shape with little floccular thing attach on the tube after 24 h. After incubation for 6 days, with peel or patch falling off, the thickness of tube walls was decreased with significant degradations of microtubes. In the current study, alginate was oxidized into O-Alg to increase the biodegradability [27]. As shown in Fig. 2c, except the absorption peaks of carboxylate groups at 1412 cm−1, FTIR analysis showed a new peak at 1726 cm−1 ascribed to the carbonyl groups from O-Alg. The oxidation degree of O-Alg was about 39.3%, defined as the percentage of oxidized uronic acid units. After being mixed with chitosan by layer-by-layer assembly, aldehyde groups from O-Alg were crosslinked via the schiff’s base reaction with amino groups from chitosan (Fig. 2d), indicating an absorption peak at 1638 cm−1 of schiff’s base bonds [37]. The schiff’s base structure was stable in neutral environment but broken under acid environment. Zhang et al. constructed pH-sensitive DOX-carried nanoparticles using schiff’s base linkages, acquiring expected drug release under the mild acidic microenvironment in tumor cells [38].
3.3. Motility and penetration of MTDOX@EcN As one of flagellated E. coli bacteria, EcN-loaded cargoes had fine motion ability, leading to the target accumulation and efficient penetration in tumors [24]. In this study, the motility of MTDOX@EcN was estimated by tracking and analyzing the trajectories in culture media and the penetration in viscous 15
media. Fig. 3a shows the motion trajectories of MTDOX@EcN. The average velocity was around 6.8 μm/s, indicating that EcN kept the motion capability after confinement into microtubes. Compared with that of free EcN (9.8 μm/s) [24], the lower velocity of MTDOX@EcN was due to the slight restriction of the flagella movement in microtubes. It was indicated that the attachment of multiple bacteria led to twisting and rotational motion of micromotors, thus abating the net translational motion [39]. In the current study, only one bacterium was captured inside a microtube along with the length of the microtube, and the aligned propulsion forces induced directional trajectories like a microrocket [40]. The penetration of MTDOX@EcN was the vital feature for cargo delivery to enhance the drug distribution in target tumor tissues and the interaction with tumor cells. Fischer et al. constructed magnetic micropropellers and estimated the propulsion velocity in water and viscoelastic media under a magnetic field. The result showed micropropellers lost over 90% of motion velocity when changing the buffer from water to HA solutions, which was one type of specific thickening agents to simulate tumor ECM [30]. In the current study, the average velocity of MTDOX@EcN was around 2.9 μm/s in HA solutions, indicating that EcN kept the motion capability in viscous media. In addition, we designed a chamber connected with a capillary tube filled with HA to observe the penetration of MTDOX@EcN (Fig. 3b). Fig. 3c shows CLSM images after 60 min of incubation, indicating red fluorescence signals of DOX close to the chamber. There was no significant difference in the fluorescence signals between MTDOX@EcN and MTDOX in the proximal range. Few fluorescence signals were detected for MTDOX in the capillary tube away from the chamber, while strong fluorescence signals were observed for MTDOX@EcN. The results proved that EcN had the ability to assist the penetration of MTDOX in viscous media.
3.4. In vitro cytotoxicity of MTDOX@EcN The cell viability of MTDOX@EcN and MTDOX was tested on 4T1 and RAW264.7 cells after incubation in pH 6.5 and 7.4 buffers, and compared with those of equivalent free DOX and microtubes 16
without drug inoculation. As shown in Fig. 4a, over 90% of cell viabilities were kept after incubation with microtubes for 72 h, indicating no significant cytotoxicity for microtubes assembled by layer-bylayer deposition of alginate and chitosan. The incubation with MTDOX and MTDOX@EcN at pH 7.4 led to no apparent cytotoxicity on 4T1 cells, due to few DOX release during the time period (Fig. 2a). The IC50 value of free DOX against 4T1 cells was around 1.7 μg/mL, which was lower than that of MTDOX@EcN (4.1 μg/mL) after incubation in pH 6.5 buffer. This difference was ascribed to the gradual release of DOX from MTDOX@EcN, and the initial drug concentration was lower than that of free DOX. It should be noted that MTDOX@EcN had significantly higher cytotoxicities than MTDOX at each drug concentration at pH 6.5. In addition, microtubes, MTDOX, and MTDOX@EcN showed no apparent cytotoxicities on RAW264.7 cells compared with free DOX (Fig. 4b). The cell apoptosis was estimated via Annexin V-FITC and propidium iodide staining. Fig. 4c shows the flow cytometry images of apoptosis analysis, indicating that microtubes did not cause any cell apoptosis according to the apoptosis rate of 3.5%. Free DOX treatment led to the most significant apoptosis induction of 4T1 cells at a ratio of 56.3%, attributed to the high initial concentration of DOX. It was worthy noticing that MTDOX@EcN caused a higher apoptosis rate (49.3%) than MTDOX (31.9%) after incubation at pH 6.5. The interaction of MTDOX@EcN with blood should be a significant aspect of the biocompatibility after intravenous injection for tumor treatment. The haemolysis effect was measured from the absorption value of haemoglobin after treatment with EcN, MTDOX, and MTDOX@EcN. As shown in Fig. 4d, the haemolysis ratios of EcN, MTDOX and MTDOX@EcN were about 4.6%, 1.0% and 2.2%, respectively. The haemolysis ratio of below 5% proved the good haemocompatibility and safety for in vivo application [34].
3.5. Cellular uptake of MTDOX@EcN
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Fig. 4e summarizes the cellular uptake efficiency of MTDOX and MTDOX@EcN after incubation in pH 6.5 and pH 7.4 buffers for 72 h. Free DOX showed the maximal internalization by both 4T1 and Raw264.7 at uptake efficiency of 46%50%. The uptake efficiency of MTDOX@EcN by 4T1 cells showed over 2.1 folds higher than those of MTDOX at both pH 6.5 and 7.4 (p < 0.05), indicating EcN could drive and promote internalization of microtubes into cells. Macrophages indicated significantly lower uptake of MTDOX@EcN and significantly higher uptake of MTDOX than 4T1 (p < 0.05). Thus there was less significant difference in the macrophage uptake of MTDOX@EcN and MTDOX (p > 0.05). The cellular uptake of MTDOX and MTDOX@EcN was observed by CLSM after 12 h of incubation with 4T1 cells. As shown in Fig. 4f, scattered red fluorescence even columnar signals were observed in the cytoplasm of 4T1 cells, indicating that both MTDOX and MTDOX@EcN were taken up into cells more or less. The fluorescence signals for MTDOX@EcN were much stronger than those of MTDOX, suggesting that EcN as the microrocket engines had more capability to promote the cellular uptake of microtubes. The endocytosis pathway of MTDOX@EcN was determined by pretreatment of cells with different inhibitors. Chlorpromazine, nystatin and amiloride were used as the inhibitors of clathrin- and caveolin-mediated endocytosis and macropinocytosis, respectively [33]. As shown in Fig. 4g, the cellular uptake of MTDOX@EcN and MTDOX were significantly inhibited by amiloride pretreamtent, indicating an uptake mechanism of macropinocytosis. In the previous report, Teo et al. reported that the strongest macropinocytosis was observed in the cellular uptake of 2 μm-sized poly(methly methacrylate) pillars compared with other internalization avenues, suggesting that the larger size was the chief factor when referring to the cell uptake way of macropinocytosis [41]. Faille et.al used amiloride to pretreat the uptake process of microscopical platelet-derived microparticles, indicating that over 70% of decrease in the uptake levels by endothelial cells [42]. In addition, due to the energy dependent process, the upake efficiency of MTDOX@EcN and MTDOX was significantly decreased at 4 °C (p < 0.05). As the control, free DOX molecules entered the cell interior by free diffusion under less restriction of the inhibitors. 18
3.6. Tissue distribution of drugs and EcN after injection of MTDOX@EcN The drug distributions in 4T1 tumor-bearing mice were investigated after intravenous injection of MTDOX@EcN, MTDOX and free DOX. Fig. 5a shows DOX levels in tumors and other major tissues (liver, spleen, lung, heart and kidney) after injection for 3 h, 1, 3 and 7 days. After 3 h of injection, almost all tissues had DOX signals presenting a wide distribution. However, DOX levels decreased rapidly after 1 and 3 days and were only detectable in liver and tumors after 7 days. After administration of MTDOX@EcN and MTDOX for 3 h, the DOX levels in livers were around 20.2% and 24.4% of the injected doses, respectively. They were remarkably higher than that of free drug (11.5%, p < 0.05) due to the preferential accumulation in the reticuloendothelial system. The free drug injection showed higher DOX levels in kidney than other formulation, suggesting the quick elimination of free drugs. The DOX level in tumors after 1 days of MTDOX@EcN administration reached around 6.6%, almost 10 times higher than that of free DOX. After 3 days of MTDOX@EcN administration, around 5.9 % and 5.1 % of injected doses were detected in liver and tumor, showing around 14.3 % and 4.6 % of reductions, respectively. After MTDOX@EcN treatment for 7 days, tumors displayed a significantly higher DOX level (2.5 %) than those of other tissues (p < 0.5). Moreover, the DOX levels in tumors after 3 h, 1, 3 and 7 days of treatment with MTDOX@EcN were 3.7, 4.6, 5.0 and 6.1 folds higher than those of MTDOX, respectively (p < 0.05). It was demonstrated that the EcN-propelled rocket-like motion could promote the extravasation of MTDOX@EcN from blood circulation and the retention in tumors. The tissue distribution of bacteria was evaluated after intravenous injection of MTDOX@EcN and free EcN. As shown in Fig. 5b, bacteria were rapidly distributed in multiple organs via circulation, and significantly higher bacteria levels were detected in livers than other tissues after 3 h and 1 and 3 days of treatment (p < 0.05). The EcN levels indicated a rapid decline over time in tissues except an increase in tumors. After treatment for 7 days, no bacteria signal was detected in tissues except tumors (1.2 × 109 cfu) and livers (5.1 × 103 cfu). EcN could accumulate and expand in tumors rather than other 19
tissues, demonstrating the capability as tumor-targeted vehicles. The EcN numbers in tumors after treatment with free EcN for 3 h, 1, 3 and 7 days were 1.4, 5.1, 4.4 and 1.3 folds higher than those of MTDOX@EcN, respectively. This result should be caused by the higher growth restriction of EcN in microtubes in the early stage than the later one. Fig. 5c shows the immunostained images of EcN in tumor sections after MTDOX@EcN treatment for 3 h, 1, 3 and 7 days. After treatment for 3 h and 1 day, the yellow green fluorescence signals could be found in tumor sections but very rare, showing the EcN accumulation in tumors. The fluorescence signals indicated significant increases after 3 and 7 days, suggesting further multiplication of EcN in tumors.
3.7. In vivo antitumor efficiency of MTDOX@EcN treatment The antitumor efficiency of MTDOX@EcN was evaluated with respect to the tumor growth and survival rate of 4T1 tumor-bearing mice. Fig. 6a shows the tumor volumes after different treatment. The tumor volume showed an unconfined grow and reached about 2380 mm3 after saline treatment for 30 days. The tumor growth was suppressed during the initial 2 weeks after injection of free EcN, DOX and MTDOX, while remarkable tumor growths were observed during the followed period. Free EcN and DOX had more or less antitumor abilities, showing around 20.6% and 36.1% of tumor growth inhibition after 1 month, respectively. The sustained release of DOX from MTDOX led to a higher tumor growth inhibition (60.9%), while the treatment with MTDOX@EcN further promoted the inhibition of tumor growth (75.6%), due to the EcN-drived tumor accumulation and intracellular uptake of DOX. In addition, the self-propelled motion indicated more significant effect on the tumor growth inhibtion during the following time peroid (Fig. 6a). Fig. 6b summarizes the Kaplan–Meier plotting for animal survivals. Compared with that of saline treatment (21 days), the EcN treatment indicated more or less antitumor effect with the median survival time of 24 days. After MTDOX treatment, 50% of the mice died within 33 days, which was higher than that of free DOX (27 days). After treatment with
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MTDOX@EcN, the median survival time was 42 days, and 17% of mice survived after 54 days, showing significantly stronger antitumor capability than other treatment. The antitumor capability was further examined via H&E and IHC staining after 24 days of treatment with MTDOX@EcN, MTDOX, EcN and free DOX, using saline as control. In the H&E staining images of tumors, necrotic cells showed unclear cellular morphologies, darker chromatin and pyknotic even obliterate nuclei, and the increase of necrotic regions suggested the underlying tumor shrinkage [43]. As shown in Fig. 6c, a plenty of living cells were observed in tumor sections after saline treatment, indicating few antitumor efficacy. Mild necrosis was observed after free EcN and DOX treatment, while remarkable necrosis was recognized after treatment with MTDOX@EcN. IHC staining of Ki-67 and caspase-3 was implemented to track the cell proliferation and apoptosis in tumors [44]. Fig. 6d shows typical IHC staining images of Ki-67, indicating the lowest proliferative cells in tumors after MTDOX@EcN treatment. Compared with that of 13.2 1.4% for MTDOX@EcN, the percentage of positively stained cells were significantly higher after MTDOX (31.3 2.5%) and free DOX treatment (57.1 4.2%) (p < 0.05). After treatment with saline and free EcN the cell proliferation index reached 79.5 3.4% and 90.1 3.8%, respectively. The apoptotic cells were counted in the IHCstained images of tumors on the expression of caspase-3 (Fig. 6e). The percentage of caspase-3-positive cells in the MTDOX@EcN group was 84.3 ± 3.8%, which was significantly higher than those after MTDOX (67.8 ± 4.3%) and free DOX treatment (48.6 ± 2.9%) (p < 0.05). The treatment with EcN and saline showed lower apoptotic indices of 32.5 ± 4.3% and 11.7 ± 3.9%, respectively. From these histological evaluations, the MTDOX@EcN treatment could arrest the proliferation and promote the apoptosis of tumor cells, indicating excellent superiority in the tumor treatment.
3.8. In vivo safety of MTDOX@EcN treatment
21
The clinical use of DOX has been long noted that patients may suffer cardiotoxicity [45]. The treatment safety of MTDOX@EcN was evaluated with respect to the body weight variation, HE staining of heart tissues and haematological analysis. The body weight of all groups showed roughly a tendency to increase. Due to virulence of free DOX, the treatment with free DOX showed a lower body weight than other groups. The MTDOX@EcN treatment showed body weight features similar to the saline group, indicating the absence of side effects. Fig. 6f shows H&E staining images of heart sections after different treatment. Histopathological abnormalities, such as hyperemia and myocardial fiber breakage were identified after treatment with free DOX, while no obvious degenerations or lesions were observed in other groups. It was indicated that MTDOX@EcN could help relieving the cardiotoxicity of DOX treatment. The treatment toxicity of MTDOX@EcN was evaluated from the hematological and biochemical analysis of blood retrieved. After MTDOX@EcN treatment for 1 day, the blood count results showed the numbers of RBCs, WBCs and PLTs at 6.7 1012/L, 6.9 109/L, and 1.0 1012/L, respectively. There was no significant difference compared with RBC (6.8 1012/L), WBC (6.7 109/L) and PLT counts (1.1 1012/L) in blood from normal mice (p > 0.05), indicating that the intravenous administration of MTDOX@EcN led to no apparent haematologic toxicity. In addition, the physiological status of liver and kidney could be reflected from ALT, AST, CRE, and BUN levels. Among them hepatocellular damage caused high levels of ALT and AST, while BUN and CRE levels were associated with renal injury [46]. The biochemical analysis indicated ALT (17.0 ± 2.1 U/L), AST (30.5 ± 2.8 U/L), BUN (6.3 ± 1.0 mmol/L) and CRE levels (26.5 ± 2.2 mol/L) after 1 day of MTDOX@EcN treatment. Compared with ALT (16.3 ± 1.2 U/L), AST (29.6 ± 2.4 U/L), BUN (5.9 ± 0.3 mmol/L) and CRE levels (25.3 ± 3.5 mol/L) of normal mice, mice after treatment with MTDOX@EcN indicated no hepatic and renal toxicity (p > 0.05). All the hematologic and biochemical analysis results showed no significant difference after treatment for 3 days compared with those of 1 day (p > 0.05). Thus, EcN as one of 22
probiotics introduced no toxicity and were effective engines for construction of biohybrid microtors. Zhang et al. decorated live macrophage cells with magnesium microparticles to prepare another form of biohybrid motors. The magnesium microparticles drove the cell motion and macrophages maintained the endotoxin neutralization functionality for biological tasks [47]. In the current study, MTDOX@EcN biohybrid microrockets used live EcN bacteria as the engines, the DOX-loaded microtubes as the cargoes. The reverse design was relied on the functional element of EcN such as tumor targeting and self-propeled motion. The layer-by-layer-assembled microtubes were kept stable in physiological environment and could release the entrapped drug from the cargoes in response to the acidic signals in tumor cells (Fig. 2). The bacteria-propelled rocket-like motion could enhance the accumulation in tumors (Fig. 5), the penetration in the tumor matrix (Fig. 3) and the intracellular uptake of MTDOX@EcN (Fig. 4), promoting the antitumor efficacy (Fig. 6).
4. Conclusion Bacterial microrockets are prepared by assembly of EcN into microtubes. Microtubes are constructed by layer-by-layer assembly of O-Alg and chitosan in the micropores of PC membrane templates. Microtubes are crosslinked via Schiff’s base and have the ability of acid-sensitive release in the tumor areas. The inherent motility and tumor targeting of EcN inspire the self-propelled rocket-like motion of MTDOX@EcN to enhance the penetration in the tumor matrix and intracellular uptake into tumor cells. The in vivo antitumor efficacy indicates that MTDOX@EcN have the ability to inhibit the tumor development, prolong the animal survival and avoid side effects like hematologic, hepatic and renal toxicities. This study demonstrates the capabilities of bacterial microrockets in promoting the tumor accumulation, intracellular uptake and stimuli-responsive release of therapeutic drugs for cancer treatment.
Acknowledgements 23
This work is supported by National Natural Science Foundation of China (31771034), and the Analytical and Testing Center of Southwest Jiaotong University for SEM and CLSM analysis..
References [1]
M. Karimi, A. Ghasemi, Z.P. Sahandi, R. Rahighi, B.S. Moosavi, H. Mirshekari, M. Amiri, P.Z. Shafaei, A. Aslani, M. Bozorgomid, D. Ghosh, A. Beyzavi, A. Vaseghi, A. Aref, L. Haghani, S. Bahrami, M. Hamblin, Smart micro/nanoparticles in stimulus-responsive drug/gene delivery systems, Chem. Soc. Rev. 45 (2016) 1457-1501.
[2]
S. Qin, A. Zhang, S. Cheng, L. Rong, X. Zhang, Drug self-delivery systems for cancer therapy, Biomaterials 112 (2017) 234-247.
[3]
H. Kobayashi, R. Watanabe, P.L. Choyke, Improving conventional enhanced permeability and retention (EPR) effects; what is the appropriate target? Theranostics 4 (2014) 81-89.
[4]
J. Liu, M. Li, Z. Luo, L. Dai, X. Guo, K. Cai, Design of nanocarriers based on complex biological barriers in vivo for tumor therapy, Nano Today 15 (2017) 56-90.
[5]
H. Zhou, Z. Fan, J. Deng, P.K. Lemons, D.C. Arhontoulis, W.B. Bowne, H. Cheng, Hyaluronidase embedded in nanocarrier PEG shell for enhanced tumor penetration and highly efficient antitumor efficacy, Nano Lett. 16 (2016) 3268-3277.
[6]
J. Wang, Z. Lu, Y. Gao, M.G. Wientjes, J.L. Au, Improving delivery and efficacy of nanomedicines in solid tumors: role of tumor priming, Nanomedicine 6 (2011) 1605-1620.
[7]
Z. Wu, J. Troll, H. Jeong, Q. Wei, M. Stang, F. Ziemssen, Z. Wang, M. Dong, S. Schnichels, T. Qiu, P. Fischer, A swarm of slippery micropropellers penetrates the vitreous body of the eye, Sci. Adv. 4 (2018) eaat4388.
[8]
Z. Wu, X. Lin, X. Zou, J. Sun, Q. He, Biodegradable protein-based rockets for drug transportation and light-triggered release, ACS Appl. Mater. Interfaces 7 (2015) 250−255.
24
[9]
D. Shao, J. Li, X. Zheng, Y. Pan, Z. Wang, M. Zhang, Q. Chen, W. Dong, L. Chen, Janus “nanobullets” for magnetic targeting liver cancer chemotherapy, Biomaterials 100 (2016) 118-133.
[10] B. Esteban-Fernández, C. Angell, F. Soto, M.A. Lopez-Ramirez, D.F. Báez, S. Xie, J. Wang, Y. Chen, Acoustically propelled nanomotors for intracellular siRNA delivery, ACS Nano 10 (2016) 4997-5005. [11] N. Hu, M. Sun, X. Lin, C. Gao, B. Zhang, C. Zheng, H. Xie, Q. He, Self-propelled rolled-up polyelectrolyte multilayer microrockets, Adv. Funct. Mater. 28 (2018) 1705684. [12] M. Luo, Y. Feng, T. Wang, J. Guan, Micro-/nanorobots at work in active drug delivery, Adv. Funct. Mater. 28 (2018) 1706100. [13] F. Peng, Y. Tu, D.A. Wilson, Micro/nanomotors towards in vivo application: cell, tissue and biofluid, Chem. Soc. Rev. 46 (2017) 5289-5310. [14] Z. Hosseinidoust, B. Mostaghaci, O. Yasa, B.W. Park, A.V. Singh, M. Sitti, Bioengineered and biohybrid bacteria-based systems for drug delivery, Adv. Drug Delivery Rev. 106 (2016) 27-44. [15] R.W. Carlsen, M. Sitti, Bio-hybrid cell-based actuators for microsystems, Small 10 (2015) 38313851. [16] T. Samira, M. Mahmood, D. Jamal, M. Sylvain, T. Maryam, Covalent binding of nanoliposomes to the surface of magnetotactic bacteria for the synthesis of self-propelled therapeutic agents, ACS Nano 8 (2014) 5049-5060. [17] M.M. Stanton, J. Simmchen, X. Ma, A. Miguellopez, S. Sanchez, Biohybrid Janus motors driven by Escherichia coli, Adv. Mater. Interfaces 3 (2016) 1500505. [18] S.J. Park, Y.K. Lee, S. Cho, S. Uthaman, I. Park, J. Min, S.Y. Ko, J. Park, S. Park, Effect of chitosan coating on a bacteria-based alginate microrobot, Biotechnol. Bioeng. 112 (2015) 769776. [19] J.W. Yoo, D.J. Irvine, D.E. Discher, M. Samir, Bio-inspired, bioengineered and biomimetic drug delivery carriers, Nat. Rev. Drug Discov. 10 (2011) 521-535. 25
[20] M.M. Stanton, B.W. Park, D. Vilela, K. Bente, D. Faivre, M. Sitti, S. Sanchez, Magnetotactic bacteria powered biohybrids target E. coli biofilms, ACS Nano 11 (2017) 9968-9978. [21] W. Park, S. Cho, X. Huang, A.C. Larson, D. Kim, Branched gold nanoparticle coating of Clostridium novyi-NT spores for CT-guided intratumoral injection, Small 13 (2017) 1602722. [22] B. Park, J. Zhuang, O. Yasa, M. Sitti, Multifunctional bacteria-driven microswimmers for targeted active drug delivery, ACS Nano 11 (2017) 8910-8923. [23] J. Henker, M.W. Laass, B.M. Blokhin, Y.K. Bolbot, V.G. Maydannik, M. Elze, C. Wolff, J. Schulze, The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhoea in infants and toddlers, Eur. J. Pediatr. 166 (2007) 311-318. [24] S.Z. Xie, L. Zhao, X.J. Song, M.S. Tang, C.F. Mo, X.H. Li, Doxorubicin-conjugated Escherichia coli Nissle 1917 swimmers to achieve tumor targeting and responsive drug release, J. Control. Release 268 (2017) 390-399. [25] S.Z. Xie, M.H. Chen, X.J. Song, Z. Zhang, Z.L. Zhang, Z.J. Chen, X.H. Li, Bacterial microbots for acid-labile release of hybrid micelles to promote the synergistic antitumor efficacy, Acta Biomater. 78 (2018) 198-210. [26] Z. Wu, Y. Wu, W. He, X. Lin, J. Sun, Q. He, Self-propelled polymer-based multilayer nanorockets for transportation and drug release, Angew. Chem. Int. Edit. 52 (2013) 7000-7003. [27] C. Gao, M. Liu, J. Chen, X. Zhang, Preparation and controlled degradation of oxidized sodium alginate hydrogel, Polym. Degrad. Stabil. 94 (2009) 1405-1410. [28] J.M. Chen, Z.G. Liu, M.H. Chen, H. Zhang, X.H. Li, Electrospun gelatin fibers with a multiple release of antibiotics accelerate dermal regeneration in infected deep burns, Macromol. Biosci. 16 (2016) 1368-1380. [29] A. Be'Er, R. Harshey, Collective motion of surfactant-producing bacteria imparts superdiffusivity to their upper surface, Biophys. J. 101 (2011) 1017-1024.
26
[30] S. Debora, A.G. Mark, J.G. Gibbs, M. Cornelia, K.I. Morozov, A.M. Leshansky, F. Peer, Nanopropellers and their actuation in complex viscoelastic media, ACS Nano 8 (2014) 87948801. [31] H. Zhang, Y. Liu, M.H. Chen, X.M. Luo, X.H. Li, Shape effects of electrospun fiber rods on the tissue distribution and antitumor efficacy, J. Control. Release 244 (2016) 52-62. [32] B. Tang, J.L. Zaro, S. Yan, C. Qian, Y. Yu, P. Sun, Y. Wang, W.C. Shen, J. Tu, C. Sun, Acidsensitive hybrid polymeric micelles containing a reversibly activatable cell-penetrating peptide for tumor-specific cytoplasm targeting, J. Control. Release 279 (2018) 147-156. [33] Z. Sulin, G. Huajian, B. Gang, Physical principles of nanoparticle cellular endocytosis, ACS Nano 9 (2015) 8655-8671. [34] R. He, C. Yin, Trimethyl chitosan based conjugates for oral and intravenous delivery of paclitaxel, Acta Biomater. 53 (2017) 355-366. [35] P. Sepulvedamedina, Y. Katsenovich, V. Musaramthota, M. Lee, B. Lee, R. Dua, L. Lagos, The effect of uranium on bacterial viability and cell surface morphology using atomic force microscopy in the presence of bicarbonate ions, Res. Microbiol. 166 (2015) 419-427. [36] J. Li, X. Zhang, M. Zhao, L. Wu, K. Luo, Y. Pu, B. He, Tumor-pH-sensitive PLLA-based microsphere with acid cleavable acetal bonds on the backbone for efficient localized chemotherapy, Biomacromolecules 19 (2018) 3140-3148. [37] C. Chen, M. Liu, S. Li, C. Gao, J. Chen, In vitro degradation and drug-release properties of water-soluble chitosan cross-linked oxidized sodium alginate core-shell microgels, J. Biomat. Sci-Polym. E. 23 (2012) 2007-2024. [38] Y. Zhang, F. Huang, C. Ren, L. Yang, J. Liu, Z. Cheng, L. Chu, J. Liu, Targeted chemophotodynamic combination platform based on the DOX prodrug nanoparticles for enhanced cancer therapy, ACS Appl Mater. Inter. 9 (2017) 13016-13028.
27
[39] A. Sahari, D.M. Headen, B. Behkam, Effect of body shape on the motile behavior of bacteriapowered swimming microrobots (BacteriaBots), Biomed. Microdevices 14 (2012) 999-1007. [40] M.M. Stanton, B. Park, A. Miguellopez, X. Ma, M. Sitti, S. Sanchez, Biohybrid microtube swimmers driven by single captured bacteria, Small 13 (2017) 1603679. [41] B.K.K. Teo, G. Seok-Hong, T.S. Kustandi, L.W. Wei, L.H. Yee, E.K.F. Yim, The effect of micro and nanotopography on endocytosis in drug and gene delivery systems, Biomaterials 32 (2011) 9866-9875. [42] D. Faille, F. Elassaad, A.J. Mitchell, M.K. Alessi, G. Chimini, T. Fusai, G.E. Grau, V. Combes, Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells, J. Cell. Mol. Med. 16 (2012) 1731-1738. [43] J. Ding, C. Li, Y. Zhang, W. Xu, J. Wang, X. Chen, Chirality-mediated polypeptide micelles for regulated drug delivery, Acta Biomater. 11 (2015) 346-355. [44] R. Anjum, A.M. Ali, Z. Begum, J. Vanaja, A. Khar, Selective involvement of caspase-3 in ceramide induced apoptosis in AK-5 tumor cells-FEBS Letters, Febs Lett. 439 (1998) 81-84. [45] F.S. Carvalho, B. Ana, G. Rita, A.J. Moreno, A. Rui, Carvalho, P.J. Oliveira, Doxorubicininduced cardiotoxicity: from bioenergetic failure and cell death to cardiomyopathy, Med. Res. Rev. 34 (2013) 106-135. [46] Q. Chen, L. Yang, M. Han, E. Cai, Y. Zhao, Synthesis and pharmacological activity evaluation of arctigenin monoester derivatives, Biomed. Pharmacother. 84 (2016) 1792-1801. [47] F. Zhang, R. Mundaca-Uribe, H. Gong, B. Esteban-Fernández de Ávila, M. Beltrán-Gastélum, E. Karshalev, A. Nourhani, Y. Tong, B. Nguyen, M. Gallot, Y. Zhang, L. Zhang, J. Wang, A macrophage-magnesium hybrid biomotor: fabrication and characterization, Adv. Mater. 31 (2019) 1901828.
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Figure captions: Scheme 1. (a) Schematic illustration of the preparation process of MTDOX@EcN microrockets. O-Alg and chitosan (Chi) are deposited layer-by-layer in the micropores of PC membrane templates, and DOX, Fe3O4 nanoparticles and Asp are loaded in the multilayers. After removal of the PC template via dissolution in methylene dichloride, microtubes are collected via centrifugation and magneti field, followed by assembly of EcN into microtubes using Asp as the chemoattractant. (b) The bacteriapropelled motion enhances the extravasation of MTDOX@EcN from blood circulation, penetration in the tumor matrix and intracellular uptake into tumor cells, followed by DOX release from microtubes in response to acidic signals. Fig. 1. Characterization of MTDOX and MTDOX@EcN. (a) Typical SEM and (b) CLSM images of MTDOX. (c) The assembly efficiency of EcN with MTDOX with different Asp contents. (d) Typical SEM and (e) CLSM images of MTDOX@EcN. (f) CLSM images of EcN in MTDOX@EcN after Live/Dead staining. Fig. 2. Acid-labile drug release and matrix degradation of MTDOX@EcN. (a) In vitro DOX release from MTDOX and MTDOX@EcN after incubation in pH 7.4, 6.5 and 5.5 buffers (n = 3). (c) Typical SEM images of MTDOX@EcN after incubation in pH 6.5 for 24 h and 6 days. (c) FTIR analysis of alginate, O-Alg and the complex with chitosan (O-Alg/Chi). (d) Schematic drawing of Schiff’s base formation between O-Alg and chitosan in the microtube matrix. Fig. 3. Motility and penetration of MTDOX@EcN. (a) Motion trajectories of MTDOX@EcN in culture media recorded by using an inverted microscope. Each line shows the motility trajectory of a single MTDOX@EcN. Numbers on the trajectory line represent the position of MTDOX@EcN at 0, 1, 2 and 3 s. (b) Schematic illustration of the penetration testing of MTDOX@EcN in viscose HA media. (c) Typical CLSM images of MTDOX@EcN and (d) MTDOX located in the far (L1) and close sites (L2) of the capillary tube after 60 min of incubation.
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Fig. 4. Cellular toxicity and uptake of MTDOX@EcN. (a) Cytotoxicities of MTDOX@EcN, MTDOX, MT and DOX to 4T1 and (b) RAW264.7cells after incubation in pH 7.4 and pH 6.5 buffers for 72 h (n = 5). (c) Flow cytometry analysis of 4T1 cells after incubation for 48 h with MTDOX@EcN, MTDOX, MT and DOX. Lower left of each image, living cells; Lower right, early apoptotic cells; Upper right, late apoptotic cells; Upper left, necrotic cells. Inserted numbers indicate the percentage of cells present in each area. (d) Haemolysis of EcN, MTDOX and MTDOX@EcN, and insert illustrates the visual images of haemolysis analysis. (e) Cellular uptake of MTDOX@EcN, MTDOX and DOX after incubation with 4T1 and RAW267.4 cells in pH 6.5 and pH 7.4 buffers for 24 h (n = 5). (f) Typical CLSM images of 4T1 cells, counterstained by DAPI, after incubation with MTDOX and MTDOX@EcN. (g) Cellular uptake of DOX, MTDOX and MTDOX@EcN after pretreatment with chlorpromazine (CPZ), nystatin (NS), or amiloride (ALR) as endocytosis inhibitors and after incubation at 4 C (n = 5). Fig. 5. Tissue distribution of drugs and EcN after injection of MTDOX@EcN. (a) Percent injected dose (ID%) of DOX in hearts (He), livers (Li), spleens (Sp), lungs (Lu), kidneys (ki) and tumors (Tu) from 4T1 tumor-bearing mice after intravenous injection of MTDOX@EcN, MTDOX and DOX for 3 h, 1, 3 and 7 days (n = 3). (b) EcN counts in above tissues after intravenous injection of MTDOX@EcN and EcN for 3 h, 1, 3 and 7 days (n = 3). (c) Typical IHC staining images of EcN in tumors after MTDOX@EcN treatment for 3 h, 1, 3 and 7 days. Fig. 6. Antitumor efficiency of MTDOX@EcN treatment. (a) Tumor volumes and (b) survival rates of tumor-bearing mice after intravenous administration of MTDOX@EcN, MTDOX and free DOX at a dose of 1 mg DOX/kg, using saline and free EcN as control (n = 6). (c) Typical H&E staining images (‘N’ represents necrotic area, ‘T’ represents tumor mass) of tumors after treatment for 24 days. (d) IHC staining images of Ki-67 and (e) caspase-3 of tumors retrieved after treatment for 24 days. (f) Typical H&E staining images of heart tissues after treatment for 24 days. Circle area indicates the presence of hyperemia.
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Highlights
Microrocket is constructed by assembly of one bacterium as the bioengine into a microtube.
Microtubes are fabricated by layer-by-layer assembly of alginate and chitosan in porous template.
Bacteria drive the rocket-like directional motion of microtubes in both water and viscous media.
Microrockets enhance the blood-tumor tissue extravasation and internalization into tumor cells.
Demonstrate a novel strategy to combat multiple biological barriers in the drug delivery pathway.
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