Bacteria-Driven Hypoxia Targeting for Combined Biotherapy and

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Bacteria-Driven Hypoxia Targeting for Combined Biotherapy and Photothermal Therapy Wenfei Chen, Ying Wang, Ming Qin, Xudong Zhang, Zhirong Zhang, Xun Sun, and Zhen Gu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02235 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018

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Bacteria-Driven Hypoxia Targeting for Combined Biotherapy and Photothermal Therapy Wenfei Chen1, Ying Wang1, Ming Qin1, Xudong Zhang2, Zhirong Zhang1, Xun Sun*1 and Zhen Gu2 1

Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West

China School of Pharmacy, Sichuan University, Chengdu 610041, P. R. China 2

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and

North Carolina State University, Raleigh, North Carolina 27695, USA Correspondence to Prof. Xun Sun, E-mail: [email protected]

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ABSTRACT: The facultative anaerobe Salmonella strain VNP20009 selectively colonizes into tumors following systemic injection due to its preference for the hypoxia in the tumor cores. However, the Phase 1 Clinical Trial of VNP20009 has been terminated mainly due to its weak anti-tumor effects and exhibition of dose-dependent toxicity. Here we leveraged the advantages of VNP20009 biotherapy together with polydopamine-mediated photothermal therapy in order to enhance the antitumor efficacy toward malignant melanoma. VNP20009 was coated with polydopamine via oxidation and self-polymerization, which was then injected into tumor-bearing mice via the tail vein. Polydopamine coated VNP20009 targeted hypoxic areas of the solid tumors, and near-infrared laser irradiation of the tumors induced heating due to polydopamine. This combined approach eliminated the tumors without relapse or metastasis with only one injection and laser irradiation. More importantly, we found both VNP and pDA potentiate the therapeutic ability of each other, resulting in a superior anticancer effect.

KEYWORDS: salmonella, polydopamine, hypoxia targeting, photothermal therapy, malignant melanoma


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Over a century ago, scientists proposed that bacteria, as the "enemy of our enemy", could serve as friendly anti-tumor agents.1 In the late 19th century, the German physicians W. Busch and F. Fehleisen2 as well as the American physician W. Coley3,4 systematically investigated cancer regression due to bacterial infection. Since then, various genetically modified bacterial strains, which offer reduced toxicity and immunogenicity5,6 and can deliver therapeutic genes, proteins, and small molecules to the disease sites, including tumors,7 have been developed in several cases with enough efficiency to merit clinical trials.1 The facultatively anaerobic Salmonella typhimurium VNP20009 is a particularly attractive genetically modified strain because it can target tumors by virtue of its preference for the hypoxia in the tumor cores, in contrast to the more oxygenated outer tumor regions.8 VNP20009 also prefers to colonize necrotic tumor tissues because of the abundance of nutrients in these sites.9 In VNP20009, part of the purI gene has been deleted, allowing genetically stable attenuation of virulence, and part of the msbB gene has been deleted, reducing the risk of septic shock; these deletions endow the bacterial strain with an excellent safety profile.10,11 Its safety and anti-cancer activity in pre-clinical animal studies led researchers to examine VNP20009 in a phase 1 clinical trial,12,13 which, unfortunately, had to be terminated because of few tumor regression and undesired dose-dependent side effects.14,15 We hypothesized that combination of the tumor-targeting ability of VNP20009 and photothermal therapy could achieve enhanced specificity and anti-tumor effectiveness. In photothermal therapy, photothermal agents in the target tissue under irradiation can cause local temperature elevation because they can convert the incident light into heat, which then kills the surrounding tumor cells.16-18 Photothermal therapy has attracted substantial interest because it is highly selective (limited primarily to the irradiated tissue)19,20 and minimally invasive.21,22


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Hence, when attached to VNP20009, the appropriate photothermal agents would target selectively to hypoxic areas of tumors, causing effective photothermal ablation of tumors by tumor-restricted irradiation with limited side effects. Compared with widely used photothermal agents, such as metal (e.g., Au, Ag, and Pd23-25) nanoparticles, Cu-based semiconductor nanoparticles,26 and carbon-based nanomaterials,27 melanin-like polydopamine (pDA) exhibit better biocompatibility due to its excellent biodegradability.28 pDA nanoparticles have been shown to efficiently convert near-infrared light into heat and kill tumor cells both in vitro and in vivo,29,30 suggesting their potential for photothermal therapy. Therefore we sought to harness the tumor-targeting ability of VNP20009 to deliver the photothermal agent pDA to hypoxic and necrotic tumor areas. We aimed to coat VNP20009 partially with the pDA molecules,31 which would not compromise the bacteria's tumor-targeting ability. Hypoxia-targeting VNP20009 is particularly attractive for cancer treatment because the hypoxic microenvironment in tumors renders them less vulnerable to anticancer drugs and free radicals.32 We also hypothesized that the biotherapeutic efficacy of VNP20009 would be promoted as tumor cells lyse in response to pDA-mediated photothermal therapy, acting as nutrients that attract the bacteria.33,34 RESULTS AND DISCUSSION The pDA-coated VNP20009 (hereafter pDA-VNP; Figure 1) was prepared in a single step within 2 h via dopamine oxidation and self-polymerization.35 VNP20009 itself had an average size of 942.0 nm and zeta potential of -0.25 mV. In contrast, as the dopamine concentration increased from 250 to 2,000 µg/mL, pDA-VNP showed the average size of 974.9 to 1251.5 nm


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and the zeta potential of -7.19 mV to -16.45 mV (Figure S1A), owing to there were more hydroxyl groups on the surface with a thicker coating of polydopamine. The color of the pDAVNP suspension went from clear to black as the dopamine concentration increased from 62.5 to 2,000 µg/mL (Figure S1B). Transmission electron microscopy (TEM) showed VNP20009 on its own had a typical rod shape (Figure 2A), and pDA clearly sticked on the Salmonella surface after 2-h self-polymerization (Figure 2B). TEM micrographs of the microtome-sliced VNP20009 and pDA-VNP also revealed the pDA-coating on the bacteria (Figure 2C-D). Thicker polydopamine layers were obtained with longer polymerization time or higher dopamine concentrations. FTIR spectroscopy of pDA-VNP showed several peaks between 1,000 and 1,700 cm-1 (Figure S1C), most of which were also present in the pDA reference spectrum. Specifically, the spectrum of pDA-VNP contained more obvious peaks at 1,535 cm-1 (N–H shearing vibrations) and 1,235 cm-1 (C–OH stretching vibrations). The peak at 3,297 cm-1 was assigned to aromatic hydrogen stretching vibrations. These peaks were consistent with successful preparation of pDA. Then we analyzed the weight percent and atomic percent (%) of three chemical elements (C, N, O) in VNP20009 and pDA-VNP (prepared at 1,000 µg/mL dopamine) by Energy dispersive X-ray spectroscopy (EDX) ， roughly measuring the content of pDA (Figure S1D). The pDA-VNP microparticles absorbed broadly over the range of 200-900 nm, and their absorption was much stronger with increased dopamine concentration (i.e., 125 to 2,000 µg/mL; Figure 2E). In particular, the absorption at the photothermal therapy wavelength of 808 nm was stronger at higher dopamine concentrations. Next, we assessed the efficiency of photothermal conversion by pDA-VNP. Temperature change (∆T) in a pDA-VNP suspension increased with irradiation time and with dopamine concentration (Figure 2F). For example, the temperature of a


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suspension of pDA-VNP prepared at 1,000 µg/mL dopamine increased by 23.0 °C after irradiation for 300 s. In contrast, the temperature of deionized water increased only 8.2 °C under the same conditions. Heating at 42 °C for at least 10 min or at temperatures >50 °C for only 5 min is believed to kill cancer cells effectively.36 We assumed that pDA-VNP prepared at 1,000 µg/mL dopamine could easily induce heating to temperatures above 50 °C within 5 min in vivo, starting from a basal temperature of 36-37 ºC. Next, we evaluated whether irradiating VNP20009 or pDA-VNP at 808 nm for 5 min would affect the viability of VNP20009, since this might affect the efficiency of pDA delivery to the tumor sites. We found that laser irradiation (808 nm, 1.18 W cm-2, 5 min) showed minimal effects on VNP20009 viability (Figure 2G). We also checked whether the pDA coating would compromise the ability of VNP20009 to replicate or to invade tumor cells. We found that pDAVNP prepared at 1,000 µg/mL dopamine had the same growth profile as compared with VNP20009 alone (Figure S2A-B), indicating that the coating of pDA did not hinder the bacterial duplication. Moreover, incubating B16F10 cells for 2 h with pure VNP20009 or pDA-VNP prepared at 500 to 2,000 µg/mL dopamine led to similar levels of bacterial invasion, which increased with longer incubation such as 4 h (Figure 3A). Then we examined the growth of VNP20009 with or without 808-nm irradiation for 1 or 5 min on B16F10 cells. As shown in Figure S3, there were more bacteria in the cells after irradiation for 5 min than other treatments. We can assume that tumor cell lysis generated by pDA-VNP-based photothermal therapy could release nutrients for bacteria growing, resulting the significantly enhanced amount of VNP20009 in B16F10 cells. Furthermore, after 2-h incubation with pDA-VNP prepared from 0.5 to 2.0 mg/mL dopamine, mouse fibroblast cells L929 showed more than 88% viability, indicating the low toxicity of pDA-VNP towards normal cells (Figure S4).


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Given the promising photothermal conversion efficiency and bacterial invasion capacity of pDA-VNP, we tested its ability to kill B16F10 tumor cells in vitro. First, we quantitated the cytotoxicity of pure VNP20009 and pDA-VNP prepared at various dopamine concentrations with or without 808-nm irradiation for 5 min (Figure 3B). Cell viability was 75.4%-84.2% after 4-h incubation with VNP20009 or pDA-VNP but without irradiation, consistent with the proapoptotic effects of VNP20009. B16F10 viability after irradiation was significantly lower in the presence of pDA-VNP than in the presence of pure VNP20009, and the cell-killing effect of pDA-VNP increased with dopamine concentration till 2,000 µg/mL. At a dopamine concentration of 2,000 µg/mL, B16F10 viability was only 34.5%, which showed no significant differences with that at 1,000 µg/mL. Cells treated with the same groups in the cell viability study were then stained with FITC-Annexin V and propidium iodide and analyzed by flow cytometry. The results indicated that most cells were in end-stage apoptosis or already dead. Irradiating B16F10 cells incubated with pDA-VNP prepared at 1.0 mg/mL dopamine led 80.7% of B16F10 cells to progress to late-stage apoptosis or to die (Figure 3C). The corresponding percentage was much lower with pure VNP20009 or pDA-VNP without irradiation. Similar results were obtained when we examined the treated B16F10 cells using confocal laser scanning microscopy. Cell death (red stain) was much greater when cells were treated with pDA-VNP prepared at 1.0 mg/mL dopamine under irradiation than that of other groups (Figure 3D). Interestingly, incubating B16F10 cells for 4 h with pDA-VNP led to less cell death than incubating them with pure VNP20009 at the same bacterial concentration. This may reflect shielding effect by the pDA coating. Collectively, these in vitro results suggested that photothermal therapy mediated by pDA-VNP could efficiently kill cancer cells under laser irradiation.


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Thereafter, we tested the safety and efficacy of the pDA-VNP approach in vivo using C57BL/6 mice bearing B16F10 allografts. First, we examined the in vivo biodistribution of VNP20009 before and after pDA-coating and laser treatment. Mice were intravenously injected with 0.1 mL of normal saline containing 105 bacterial colony forming units (CFUs) of pure VNP20009 or pDA-VNP prepared at 1.0 mg/mL dopamine on day 0, followed by 808-nm irradiation (or not) for 5 min on day 3. On days 1, 4, 8, and 20 after injection, hearts, livers, spleens, lungs, kidneys, and tumors were collected. Bacteria in these tissues were quantitated by serial dilution plating and then counted on solid LB agar plates. In the absence of irradiation, mice injected with pure VNP20009 showed a higher amount of bacterial colony (normalized by weight; CFUs/g) in tumors than those in other organs from day 1, with the differences becoming significant (p

Bacteria-Driven Hypoxia Targeting for Combined Biotherapy and

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