Photosensitizer-Bacteria Bio-Hybrids Promote Photodynamic Cancer

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Photosensitizer-Bacteria Bio-Hybrids Promote Photodynamic Cancer Cell Ablation and Intracellular Protein Delivery Min Wu, Wenbo Wu, Yukun Duan, Xueqi Li, Guobin Qi, and Bin Liu Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.9b01518 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 12, 2019

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Chemistry of Materials

Photosensitizer-Bacteria

Bio-Hybrids

Promote

Photodynamic Cancer Cell Ablation and Intracellular Protein Delivery Min Wu †#, Wenbo Wu†#, Yukun Duan†, Xueqi Li†, Guobin Qi†, and Bin Liu†* †Department

of Chemical and Biomolecular Engineering, National University of

Singapore, 4 Engineering Drive 4, 117585, Singapore ABSTRACT Live bacteria have drawn widespread interests as carriers to deliver genes and proteins into eukaryotic cells for the treatment of various cancer types owing to their good biocompatibility and active targeting ability. However, how to realize effective gene and protein release remains an issue and whether the bacteria could efficiently deliver therapeutic agents has not been successfully realized. Herein, we report a new bio-hybrid system composed of aggregation-induced emission photosensitizer (PS) nanoparticles TDNPP coated Escherichia coli (E. coli), which serves as a PSs delivery vector for effective imaging and ablation of tumor cells. TDNPP coating layer on the surface of E. coli could facilitate bacteria to invade cancer cell and efficiently release protein through the production of reactive oxygen species (ROS) upon light irradiation. Furthermore, multifunctional TDNPPs delivered by bacteria have also achieved enhanced cancer cell imaging and effective light-mediated cancer killing in vitro as compared to the same PS NPs without the bacteria carrier. Our study thus presents an alternative strategy to optimize bacteria-mediated cancer therapy and intracellular protein delivery. 1

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INTRODUCTION Selective delivery of specific therapeutic agents into cancer cells with low toxic side effects poses a challenge in cancer therapy. 1 Over the past decades, researchers have made an array of attempts in the development of several effective carriers such as polymers, liposomes, inorganic porous materials and bacterial vectors to achieve active or passive targeted delivery of therapeutics.2 Among these, bacteria such as Escherichia coli (E. coli),3 Salmonella,4 and Listeria monocytogenes have been explored in cancer treatment for over a century. Bacteria as therapeutic agent carriers present multiple advantages over artificially synthetic carriers, which include (i) their unique ability to preferentially colonize tumors in an active motility by aerotaxis or chemotaxis pathway; 5 (ii) due to the intrinsic genetic system, live bacteria could be genetically engineered to delivery tumoricidal agents such as gene or protein;6, 7 (iii) as a natural protein-making factory, bacteria vectors are cost-effective compared with most artificial synthetic carriers.8 Despite the outstanding performance made by bacteria-mediated cancer therapy, several challenges remain to be further explored,

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such as the balance between efficacy and safety.10 The widespread application of attenuated bacteria such as attenuated Salmonella and Listeria monocytogenes reveals that potential immunogenicity might cause host to develop autoimmune-associated toxic response after administration which remains a matter of concern.11 To address this safety concern, non-pathogen bacteria such as E. coli is considered as a category of relatively safe bio-vectors to deliver plasmid DNA, protein and drug into cancer cells owing to their good biocompatibility and biodegradability.12, 2


13

However,

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non-pathogen bacteria as therapeutic agent vectors often suffer from low invasion efficiency into cancer cells due to energetically unfavorable mutual interaction between bacteria and cancer cell.14

Besides, the effective release of protein from

bacteria inside cancer cells is very challenging. Very often, membrane-disrupting lipopeptide antibiotic polymyxin B is used to facilitate the protein release from E. coli.3, 15 However, the choice of such a method has raised concerns about the potential toxicity and tissue reaction with this agent.16 Therefore, it is of great importance to develop a highly effective and safe strategy, which can collectively overcome the current hurdles of bacteria-mediated cancer therapy. Surface modification of bacteria with functional nanomaterial is considered as an effective strategy to improve bacteria-mediated tumor therapy.17,

18

Synthetic

polymer-coated on live bacteria is able to facilitate bacteria invasion into cancer to damage cancer cells with multimodal features.19, 20 However, there has been no report to date that the existing system could significantly improve the protein release of bacteria into target cells. Recent reports unveiled that the light-induced generation of ROS from membrane-bound photosensitizers (PSs) could damage bacterial membrane with special and temporal control,

21-23

therefore, we envision that photosensitizer

nanoparticles modified on bacterial surface might help to achieve controllable release of proteins from bacteria. Photosensitizer (PS) nanoparticles have been extensively studied and applied for photodynamic therapy (PDT).24-27 They rely on PSs to convert oxygen into cytotoxic reactive oxygen species (ROS) under light irradiation to kill cancer cells or bacteria 3



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nearby.28-30 Recently, one category of prominent PSs with aggregation-induced emission (AIE) is attracting growing attention in biomedical field due to their high brightness and strong photosensitization in the aggregate state.31,

32

By taking

advantages of both AIE PS nanoparticles and bacteria, we herein demonstrate a hybrid bio-system comprised of AIE PSs nanoparticles and non-invasive E. coli acting as an efficient vector to deliver PSs, which can fulfill the two requirements for bacteria-mediated cancer therapy, i.e. efficient cancer cell invasion and enhanced protein release (Scheme 1). To achieve this objective, we firstly designed and synthesized an AIE PS (TD) with emission in the NIR range for imaging-guided PDT. Encapsulation of TD with biocompatible block lipid-PEG co-polymer (DSPE-PEG2000) as the polymer matrix formed TD nanoparticles (TDNPs). TDNPs showed a far-red/NIR emission maximum located at 670 nm, and effective 1O2 production capability which is better as compared with that of commercial PSs (Ce6 and Rose Bengal). Furthermore, a cationic polymer polyethyleneimine (PEI) was employed to assist the coating of TDNPs on the surface of E. coli (denoted as TDNPP-E. coli) by electrostatic interaction. In vitro results showed that TDNPP-E. coli could invade and image cancer cell more effectively than free TDNPs and TDNPPs. The bacteria mediated photosensitizer delivery not only increases the protein release from E. coli but also kills cancer cells by the efficient production of ROS upon light irradiation.

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Scheme 1 Schematic illustration of TDNPP coated live bacteria (E. coli) for intracellular killing of cancer cell. (A) The structure of TD and illustration of nanoparticle formation. (B) Process of TDNPP coated live E. coli. (C) Intracellular trafficking of nanoparticle-coated live E. coli and photosensitizer delivery.

RESULTS AND DISCUSSION Synthesis of AIE PS and the NP Formation. In our design of TD, triphenylamine and benzothiadiazole moiety were used as the electron donor and the auxiliary acceptor, respectively, while dicyanovinyl group was used as the real electron acceptor. This D-A’-π-A structure can simultaneously improve the light-harvesting ability and photosensitization as compared to the traditional PSs with D-A or D-π-A 5



structure.33 In addition, the thiophene ring next to dicyanovinyl group can help localize the LUMO distribution in the acceptor, leading to good HOMO-LUMO separation to yield a small ΔEst value (the energy gap between the lowest singlet state and the lowest triplet state) of 0.197 eV (Figure 1A), which is also beneficial for 1O2 generation.34 As a typical AIE active unit, tetraphenylethene was further linked to the donor of TD, to endow TD with AIE features. The synthesis of TD is presented in Scheme S1. By two continuous steps of Suzuki coupling reaction, the intermediate of S14 was synthesized from compounds S6 and S11, which were prepared according to the literature report.34,

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Subsequently, through the reaction between S14 and

malononitrile in the presence of titanium tetrachloride as the catalyst, the target compound of TD was obtained with a yield of 81.8%. The chemical structure of TD has been confirmed by NMR spectra (Figures S1 and S2) and high-resolution mass spectrometry (Figure S3). The AIE property of TD was studied by measuring its PL spectra in tetrahydrofuran (THF)/water mixtures. As showed in Figure S4, TD showed negligible emission in pure THF solution. Upon increasing water content of the THF/water mixture from 0 to 60%, the far-red/NIR emission around 680 nm is slightly enhanced. Further addition of water to 90% led to a significant PL enhancement, which confirmed the AIE characteristic of TD. TDNPs were prepared via a modified nanoprecipitation method36 using a biocompatible block lipid-PEG co-polymer, DSPE-PEG2000, to form NPs with TD as the hydrophobic core. The size and morphology of TDNPs were detected by transmission electron microscope (TEM) and dynamic light scattering (DLS). The 6


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results show that TDNPs exhibit a spherical shape and a uniform size of ~45 nm (Figure 1B). As shown in Figure 1C, a visible absorption peak of TDNP centered at 485 nm and a maximum far-red/NIR emission at 670 nm were observed. To detect the ROS generation efficiency of TDNPs, the decomposition rates of ABDA (an indicator of singlet oxygen, 50 × 10−6 M) by TDNPs and two commercial PSs (Ce6 and Rose Bengal) were tested in aqueous media under light irradiation (60 mW/cm2, 400-700 nm), respectively (Figure 1D). Under the same condition, TDNPs decomposed 14.9 nmol of ABDA per minute, which was much higher than that of Ce6 and Rose Bengal (6.7 and 11.6 nmol of ABDA per minute, respectively), suggesting that TDNPs are more effective PSs for ROS generation.

Figure 1 (A) HOMO-LUMO distribution of TD. The optimized molecular structures of HOMO and LUMO were computed by TD-DFT (Gaussian 09/B3LYP/6-31G (d)). (B) Particle size and TEM image (inset) of TDNP. (C) UV-vis absorption and emission (λex = 488 nm) spectra of TDNP. (D) The decomposition rates of ABDA by different PSs under light irradiation (60

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mW/cm2, 400-700 nm) in water (A0, A refer to the absorbance at 378 nm of ABDA), [PS] = 0.01 mg/mL, [ABDA] = 5×10-5 M.

Characterization of TDNPP-E .coli Hybrids. To coat TDNPs onto the surface of E. coli, a cationic polymer polyethylenimine (PEI) with a molecular weight of 600 Da which has little toxicity to cells and bacteria within a reasonable concentration (lower than 50 μg/mL, as showed in Figures S5 and S6) was added to reverse the surface charge of TDNPs. The surface charge of TDNPs is gradually turned to be positive with the increasing amount of PEI, and the potential is changed from -20 mV to +20 mV when weight ratio of PEI to TDNP reached 1:1 (Figure S7A). Furthermore, no obvious change was observed for the size and morphology of PEI modified TDNPs (TDNPPs) when compared with TDNPs (Figures S7B, S8). As shown in Figures S7C and S9, E. coli can be coated well by TDNPPs at PEI/TDNPs 1:1, which was therefore chosen for further study. The formation of TDNPP-E. coli complex and co-localization of TDNPP with E. coli were observed by confocal laser scanning microscopy (CLSM) at excitation/emission wavelengths of ∼480/670 nm (Figure 2A). The red fluorescence around bacteria indicated TDNPPs attachment on the surface of E. coli. To confirm confocal analysis results, field emission scanning electron microscopy (FE-SEM) was performed. As shown in Figure 2B, bare E. coli exhibited typical rod shape with smooth surface, whereas in the case of TDNPP-coated E. coli, TDNPPs could be clearly observed on the E. coli surface membrane after multiple washing steps. The difference in SEM images between TDNPP-E. coli and uncoated naked E. coli therefore confirmed the stable attachment of TDNPP onto the E. coli 8


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surface. To further examine the coating efficiency of TDNPP on E. coli, fluorescence-activated cell sorting (FACS) was carried out. The percentage of the TDNPP-E. coli complexes was tested by extracting its fluorescence intensity from bare E. coli. The fluorescence intensity in distribution histograms as a percentage of the total of 50,000 events was presented by FlowJo software. The narrower and much higher intensity distributions of TDNPP-E. coli as compared to those of bare bacteria (Figure 2C) indicate that relatively uniform amount of nanoparticles were bound to bacteria and more than 95% of E. coli were associated with TDNPPs.

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Figure 2 (A) Confocal microscopy images of TDNNP coated E. coli. Scale bar show 2 μm. (B) Field emission scanning electron microscopy images of bare E. coli and TDNPP-E. coli. The scale bar represents 1μm. (C) FACS histograms obtained from TDNPP-E. coli.

Biological Characterization of TDNPP-E .coli Bio-Hybrids. Since the surfaces potential of bacteria and cancer cell are both negative, proximity to each other is an energy disadvantage. The coating of TDNPP on the surface of E. coli brought a positive shift of zeta potential from -16.0 mV to 1.84 mV (Figure S10) which indicated TDNPP coating layer increased the surface potential of bacteria and overcome the energetically unfavorable barrier of E. coli to approach cancer cells and facilitates the subsequent invasion process. To assess the cellular uptake efficiency of TDNPP-E. coli, fluorescence microscopy and flow cytometry experiments were carried out. Fluorescence microscopy images (Figure 3A) of TDNPs, TDNPPs and TDNPP-E. coli treated HeLa cells at the same concentration of TD showed that all three groups entered HeLa cells, while TDNPP-E. coli were internalized considerably more than TDNPs and TDNPPs (blue, cell nucleus; red, TD groups). Subsequently, we analyzed the fluorescence intensity of TDNPs, TDNPPs and TDNPP-E. coli in the HeLa cells by flow cytometry. The results shown in Figures 3B and 3C are in accordance with those of the fluorescence microscopy. These results indicate that TDNPP-E. coli could be transported to HeLa cells in vitro and can promote the delivery of photosensitizer TD to tumor cells.

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Figure 3 (A) Confocal microscopy images of TDNPs, TDNPPs, TDNPP-E. coli uptake by HeLa cells at infection ratio (bacteria to cell) of 50:1 (Scale bar represented 40 μm). The red fluorescence of TDNPPs was detected in the range of 600 nm to 800 nm at 480 nm excitation, while the blue signal of Hoechst was detected in the range of 420 nm to 480 nm at 405 nm excitation. (B) Quantitative analysis and (C) relative uptake of the TD for HeLa cell after cultured with TDNPs, TDNPPs and TDNPP-E. coli by flow cytometry, [TD] = 0.1 μM. All samples were collected from 10,000 cells for analysis.

To investigate intracellular localization of TDNPP-E. coli, the time dependent endocytosis in HeLa cells was imaged by confocal microscope (Figure S11). The red fluorescent signal of TD was first observed after 4h incubation and the signal 11



gradually increased until 12 h incubation. Interestingly, TDNPP-E. coli were internalized and transported to the nucleus of cells following a time-dependent manner. At 24 h, red signal was observed in the cell nuclei area, implying the accumulation of TDNPP-E. coli in the nucleus. A growing number of studies showed that various bacteria can secrete and deliver factors to help them attack or even enter the nucleus of eukaryocyte cells.37,

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To further demonstrate that the entry of

TDNPP-E. coli into the nucleus was caused by the active bacteria, we evaluated the cellular uptake of TDNPP coated active E. coli and TDNPP coated inactive E. coli (inactivated by high temperature) in HeLa cells. Confocal images and the intensity profiles in Figure 4A and Figure S12 clearly showed that most of the fluorescent signals were observed from TDNPP coated active E. coli inside the nuclei. However, for that of TDNPP coated inactive E. coli, majority of the fluorescence signal was distributed surrounding the cytoplasm with fewer fractions in nuclei. The z-stacking confocal image of TDNPP-E. coli internalized HeLa cells in Figure S13 confirmed that TDNPP-E. coli was indeed internalized by the cells and accumulated inside the nuclei. Nucleus, as the center of a cell, has been regarded as the most suitable and sensitive subcellular device for efficient treatment outcome using PDT.39 In addition, ROS possesses the intrinsic drawbacks including a limited diffusion region (10–20 nm) and a very short half-life (0.03–0.18 ms) in biological systems.40 Therefore, the delivery and accumulation of PSs in nuclei should promote the photodynamic efficiency for cancer therapy. Subsequently, the effect of PDT was quantitative evaluated using standard MTT (methylthiazolyldiphenyltetrazolium bromide) cell 12


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viability test. After incubating the HeLa cells after 6 h, 12 h or 24 h using different concentrations of TDNPP-E. coli, white light with a dose of 60 mWcm-2 was used to induce the light-activated cell ablation. Figure 4B showed that the killing efficiency was enhanced with the increase of incubation time and the highest killing efficiency of tumor cell could reach over 80%. The TDNPP-E. coli showed more obvious cytotoxicity to HeLa cancer cells compared with TDNPP under irradiation (Figure 4C).

However, the dark groups have little effect on the cell viability, indicating

TDNPPs coated bacteria were biocompatible.

Figure 4 (A) Confocal microscopy imaging results (left) and line-scan profiles (right) of ﬂuorescence-strength of HeLa cells after TDNPP coated active and inactive E. coli treatments for 13



24 h. The red fluorescence of TDNPP was detected in the range of 600 nm to 800 nm at excitation of 480 nm, and the blue fluorescence of Hoechst was detected in the range of 430 and 470 nm at excitation of 405 nm. The scar bar is 20 μm. (B) Inhibitory effects of TDNPP coated E. coli formulation on viability of tested HeLa cell under light irradiation at 60 mW/cm2 for 5 min at different time 6 h, 12 h and 24 h. (C) inhibitory effects of TDNPP and TDNPP-E. coli under dark or irradiation at 24 h.

Promotion of Protein Delivery of Bacteria by TDNPPs. Owing to their good biocompatibility and biodegradability, non-invasive E. coli has been employed as a category of relatively safe bio-vectors to deliver plasmid DNA, protein and drug into cancer cells.41 However, poor cell invasion is considered as a critical challenge for bacteria vector application in tumor therapy. To better observe bacterial ingestion, GFP protein expressing E. coli (E. coli-GFP) was applied as a template to be coated with TDNPP to demonstrate our design. The successful formation of TDNPP-E. coli-GFP hybrids was confirmed by FACS. As shown in Figure S14, TDNPP-E. coli-GFP plots combined the TDNPP fluorescence together with GFP fluorescence and more than 90% of E. coli-GFP bacteria were coated with TDNPPs. Confocal microscopy study (Figure S15) showed that intracellular trafficking of some TDNPP-E. coli-GFP colocalize with lysosome and a portion of GFP fluorescence was monitored to spread out from red fluorescence of LysoTracker which have indicated that TDNPP-E. coli-GFP have escaped from lysosomes and delivered GFP protein into cytoplasm. To study the invasion efficiency of TDNPP coated E. coli-GFP in HeLa cells, confocal laser scanning microscopy (CLSM) was performed. After 14


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incubating TDNPP coated E. coli-GFP with HeLa cells for 4 h, the green fluorescence of GFP and red fluorescence of TDNPP merged well inside HeLa cells with an overlay color of yellow. On the contrary, the naked E. coli-GFP was rarely ingested by HeLa cells (Figure 5A). Such results revealed that coating TDNPP on the E. coli-GFP significantly enhanced the cancer cell invasion and GFP protein delivery. During intracellular protein delivery process, inefficient protein release is a barrier inhibiting protein delivery of E. coli. The outer membrane is a fundamental permeability barrier of E. coli and destruction of it can facilitate the protein release out of bacteria.42,

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Therefore, coating TDNPP on the bacteria surface with light

irradiation to damage bacteria membrane should be an effective method to promote protein release. E. coli-GFP was again used as the model to facilitate monitoring of protein release. After irradiation of TDNPP-E. coli-GFP with white light for 5 min at a power density of 60 mWcm-2, the bacteria were collected for the following SEM imaging and the supernatant was collected for fluorescence measurements. Results demonstrated that TDNPP-E. coli-GFP without irradiation possessed clear edges and coarse surfaces as a result of the coating of TDNPPs. After irradiation, the membranes of most TDNPP-E. coli-GFP became collapsed, split and merged (Figure 5B). As showed in Figure S16, TDNPP-E. coli-GFP group had the highest fluorescence intensity under irradiation, which indicated that the GFP release from bacteria in PBS was promoted by irradiation. For the detection of GFP release into eukaryotic cells from bacteria, the naked or coated E. coli-GFP were co-cultured with HeLa cells for 12 h, exposed to light irradiation, and then cultivated for another 24 h. The cells were 15



lysed with 1 % Triton X-100 and centrifuged to collect the supernatant for further fluorescence measurements. As a mild lysis agent, 1 % Triton X-100 is good for eukaryocyte cells protein analysis by cell lysis, but it was difficult to degrade bacteria (Figure S17). Results in Figure 5C demonstrated that the fluorescence intensity of TDNPP-E. coli-GFP group significantly increased under irradiation, which indicated that the GFP release from bacteria to HeLa cells was promoted by irradiation. Besides, confocal microscopy study was performed to further monitor the GFP release inside HeLa cells, as showed in Figure S18, green fluorescence of GFP was colocalized with red fluorescence of TDNPP-E. coli before irradiation which indicated that GFP protein is still inside the bacteria. However, the green fluorescence of GFP protein dispersed throughout the whole HeLa cells after irradiation indicating that the protein is released from bacteria inside the cells. The measurements of GFP release matched well with the SEM results, which confirmed the intensive protein release effect induced by light-triggered ROS generation of TDNPPs.

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Figure 5 (A) Confocal microscopy images of E. coli-GFP and TDNPP- E. coli-GFP incubated with HeLa cells after 4 h at multiplicity of infection (MOI) ratio of 50:1. The scar bar is 25 μm. (B) Field emission scanning electron microscopy images of TDNPP coated E. coli-GFP with or without light irradiation for 5 min at a power density of 60 mWcm-2. The scar bar is 1μm. (C) The GFP release from E. coli-GFP within HeLa cells after irradiating the samples for 5 min using 60 mWcm-2 power density.

CONCLUSION In conclusion, an effective AIE PS TD and its delivery system based on nanoparticle-coated bacteria have been developed. The TDNPPs were prepared to display far-red/NIR emission around 670 nm with effective ROS generation. TDNPP coating on the surface of E. coli yielded a bio-hybrid material, which facilitated bacteria to invade cancer cell and release protein efficiently. Furthermore, 17



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multifunctional TDNPP delivered by bacteria have also achieved better cancer cell imaging and effectively light-mediated cancer killing in vitro as compared to the same PS NPs without the bacteria carrier. This work demonstrates that coating live bacteria with PSs containing nanoparticles not only presents an alternative strategy to optimize bacteria-mediated cancer therapy, but also offers a promising opportunity for intracellular protein delivery. The same strategy should be broadly applied to other bacteria-mediated and light controlled precise therapy. EXPERIMENTAL SECTION Materials and Instruments. Dichloromethane (DCM) and Tetrahydrofuran (THF) were dried by distillation using sodium as drying agent and benzophenone as indicator. Compound S635 and S1133 presented in Scheme S1 were prepared using the same synthetic

procedures

which

were

reported

in

previous

1,2-Distearoyl-sn-glycero-3-phosphoe-thanolamine-N-[Methoxy

literatures. (polyethylene

glycol)-2000] (DSPE-mPEG2000) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were acquired from Invitrogen (Waltham, MA, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2,

5-diphenyl

tetrazolium

bromide

(MTT),

penicillin-streptomycin (PS) solution and dimethyl sulfoxide (DMSO) and other chemicals were obtained from Sigma-Aldrich. Ultrapure Milli-Q water (Millipore, 18.2 MΩ) was provided by Milli-Q System (Millipore Corporation, Bedford, USA). 10× phosphate buffered saline (PBS) stock buffer with pH 7.4 was acquired from 1st Base in Singapore.

Escherichia coli (E. coli, BL 21), GFP expressing E. coli (E. 18


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coli-GFP) and HeLa human cervical carcinoma cell line were purchased from American Type Culture Collection (ATCC, Manassas, VA). NMR spectra were measured on a Bruker ARX 400 spectrometer and tetramethylsilane (TMS) as the internal standard. UV-vis absorption spectra were recorded on Shimadzu UV-1700 spectrometer and photoluminescence spectra were taken on a Perkin-Elmer LS 55 spectrometer, respectively. Particle size measurements were recorded on dynamic light scattering (DLS, Brookhaven Instruments Co., USA). After DLS measurements, the morphologies of all samples were observed on a transmission electron microscopy (TEM, JEM-2010F, JEOL, Japan). Synthesis of compound TD. Compound S14 (164 mg, 0.20 mmol) and malononitrile (39.6 mg, 0.60 mmol) were dissolved in DCM (20 mL), and TiCl4 (80 μ L, 0.70 mmol) was added slowly at 0 oC. After stirred for 0.5 h, pyridine (0.06 mL, 0.70 mmol) was injected and stirred for another 0.5 h. After that, the reaction solution was heated at 40 oC and stirred for another 4 h. after Cooled down to ambient temperature, the reaction was terminated with pure water (50 mL) and the solution was extracted by dichloromethane. The collected DCM layers were washed by brine, dried with Na2SO4, and then concentrated at vacuum distillation. The target residues were purified by chromatography with n-hexane/DCM (1/1 ~1/4, v/v) to obtain the target product TD as red solid (142 mg, 81.8 %).

1H

NMR (400 MHz, CDCl3) δ

(TMS, ppm): 8.17 (d, J = 7.6 Hz, 4H, ArH), 7.90-7.77 (m, 6H, ArH), 7.70 (d, J = 7.6 Hz, 4H, ArH), 7.30-7.25 (m, 3H, ArH), 7.19-7.02 (m, 20H, ArH), 9.65-9.88 (m, 4H, ArH).

13C

NMR (100 MHz, CDCl3) δ (ppm): 164.8, 154.1, 153.8, 141.2, 138.5, 19



137.0, 136.3, 135.5, 134.0, 132.4, 131.4, 130.5, 130.2, 130.0, 129.4, 129.1, 129.0, 127.7, 127.6, 127.2, 126.5, 125.1, 123.8, 122.8, 114.6, 114.1, 77.9. HRMS (ESI), calcd for (C58H37N5S2): m/z [M+Na]+: 867.2485; found: m/z 867.2507. Preparation of TDNPP. The TDNP were made by the modified nano-precipitation method. 1 mL THF mixture including 1 mg of TD and 2 mg of DSPE-PEG2000 was dropwise added to 10 mL pure water with the sonication using a microtip probe sonicator (XL2000, Misonix Incorporated, NY) at 18 W power output for 2 min. After THF evaporation by stirring with 600 rpm at room temperature overnight, TDNP solution (9 mL, 0.1 mg/mL based on TD) was collected and filtered via 0.22 µm filter membrane. Subsequently, The TDNP solution was centrifuged through an ultracentrifugal filter with a molecular weight cut-off (MWCO) of 100 kDa, and TDNP (0.9 mL, 1 mg/mL based on TD) was acquired. 10 μg, 50 μg, 100 μg, 200 μg of polyethyleneimine (PEI) were added to 100 μL of the resulting TDNP solution, respectively, to obtain the surface positive charge of TDNPP for the further study. Preparation of TDNPP coated bacteria. The E. coli and GFP-E. coli cells were routinely grown at 37 °C and 220 rpm in LB medium. After overnight culture, the bacteria were collected and the optical density at 600 nm (OD600) was adjusted to 1.0 in PBS. The bacterial suspension was added into 20 µM TDNPP solution with the same volume, mixed and cocultured at 37 °C for 30 min. The mixtures were centrifuged at 6000 rpm for 4 min and the supernatants were discarded, washed twice and the TDNPP coated bacteria were obtained.

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GFP protein release induced by TDNPP under radiation. E. coli-GFP culture was centrifuged, washed and resuspended in PBS (1 mL) and cultured with 20 μM TDNPP at 37 °C for 30 min. As a comparison, E. coli-GFP solution without TDNPP was also prepared. Next, both bacterial solutions were divided into four copies (0.25 mL) and added to 0.25 mL PBS, then radiation of 60 mW cm−2 white light for 10 min. The bacterial culture in dark was used as the control. After radiation or not, the bacterial solutions were shaken at 37 °C for another 6 h in dark and then centrifuged at 14000g for 2 min. All supernatants were applied for fluorescence measurements and the bacteria were collected for TEM detection. For the detection of GFP release into eukaryotic cells from bacteria, The E. coli and E. coli-GFP coated with TDNPP was cultured with HeLa cells for 12 h. After that, the bacteria were exposed under white light at 60 mW cm−2 for 5 min and then continue to cultivate for 24 h.

After the co-culture, the HeLa cells were washed thrice and

treated with 200 μL of 1 % Triton X-100 (for eukaryotic cell lysing without destroying bacteria) for 15 min, then centrifuged the lysate at 14000g for 10 min at 4 °C. The supernatant was removed carefully to a fresh tube for further fluorescence measurements. Intracellular trafficking assay. The cellular uptake of TDNPP coated E. coli in HeLa cell lines was evaluated. HeLa cells were plated on 8-well plates at a density of 2×104/well and grown for 18-22 h at 37 °C at a humidified 5% CO2 incubator. The medium was replaced with 300 µL of fresh serum-free medium and 100 µL of the TDNPP coated E. coli at MOI ratio of 20 (bacteria/cell) were added to each well. 21



After 1 h, 4 h, 6 h, 12 h, and 24 h incubation, the medium was discarded. The cells were then washed twice and then treated with 10 μg/mL of Hoechst 33342 in PBS for 15 min. The images were taken on a confocal scanning laser microscope. The delivery efficiency of TDNP, TDNPP, and TDNPP- E. coli to HeLa cells was tested by flow cytometry was used to compare. In vitro cell viability Evaluation. HeLa cells were plated in 96-well plates at a density of 1.0 × 104 cells/well and incubated 18-22 h for cells attachment. After that, cells were cultured with different amounts of TDNPP coated bacteria for 6 h, 12 h, and 24 h, respectively, and then radiation of 60 mW cm−2 white light for 10 min (compared with the group under dark). After irradiation, all cells were cultivated for another 24 h. After incubation, the medium was replaced with fresh MTT containing medium at a final concentration of 0.5 mg/mL for 4 h. After the removal of MTT medium, 100 μL of DMSO was added to dissolve formazam crystals. The microplates were gently shaken for 5 min and measured at a wavelength of 590 nm on a microplate reader. ASSOCIATED CONTENT Supporting Information Synthesis and characterization data of TD, some additional optical properties, TEM images, zeta potential, particle size and CLSM images of TDNP and TDNPP was measured, as well as the supplementary experimental section. AUTHOR INFORMATION Corresponding Author 22


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* E-mail: [email protected]. ORCID Min Wu: 0000-0002-3956-0107 Wenbo Wu: 0000-0002-6794-217X Yukun Duan: 0000-0003-3299-0822 Bin Liu: 0000-0002-0956-2777 Author Contributions # M.

W. and W. W. contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank the financial support from the Singapore NRF Competitive Research Program

(grant

no.

R279-000-483-281),

NRF

Investigatorship

(grant

no.

R279-000-444-281), and the National University of Singapore (grant no. R279-000-482-133). REFERENCES (1)

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Photosensitizer-Bacteria Bio-Hybrids Promote Photodynamic Cancer

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