Chemically Modified Bifidobacterium bifidum for Cancer Immunotheranostics Using Photothermal Therapy

Conventional cancer therapies face a myriad of shortcomings which has hampered their widespread application. Bacteria-driven cancer therapy has emerged as a very promising therapeutic strategy, offering numerous advantages such as high selectivity, specificity, and high-targeting efficiency. They possess numerous unique mechanisms for treating cancer that are unachievable with standard methods.^[1]<br/>In this project, we report the modification of an anaerobic bacteria,<i> Bifidobacterium bifidum</i>, which exhibits an innate anticancer efficiency.^[2] The bacteria were modified by incorporating a photothermal agent, indocyanine green with cremophor EL, by incubation process.^[3] Cremophor EL, an FDA-approved polyoxyethylene castor oil derivative, commonly used in poorly water-soluble drugs was added to indocyanine green molecules for encapsulation thereby forming indocyanine green with cremophor EL nanoparticles. After overnight incubation with <i>Bifidobacterium bifidum</i>, indocyanine green with cremophor EL molecules penetrated the bacterial membrane forming the modified bacteria, thereby changing their color from white to green. These modified bacteria were capable of absorbing light in the near-infrared (NIR) region thereby making them provide an excellent photothermal conversion efficiency. Characterization of these chemically modified bacteria was carried out using UV spectroscopy, transmission electron microscopy, fluorescence spectroscopy, and confocal microscopy to determine their absorption range, structural and morphological changes, and to elucidate their mechanism resulting in their modification. Furthermore, they exhibited low toxicity and high temperature elevation in <i>in-vitro</i> studies with and without laser irradiation. <i>In vivo</i> studies were conducted in syngenic tumor mice models to study the tumor targeting efficiency using colon26 cancers. The modified anaerobic bacteria were intratumorally injected and their accumulation was observed solely in the hypoxic environment inside the tumor leaving other organs, and their distribution was determined using an NIR fluorescent imaging system. Additionally, the treatment of these colon cancers was carried out by photothermal therapy, by irradiating the tumors at 0.7W using an 808nm NIR laser after 24 hours of intratumoral injection. The application of photothermal therapy was observed by the temperature elevation caused by the NIR laser on the tumor surface resulted in excellent anticancer efficiency causing the disappearance of the tumor after a few days. The blood test results in tumor mice models also revealed that the modified bacteria were completely non-toxic and biocompatible in mice. Various tissue staining techniques were also carried out to explain the mechanism of tumor suppression.<br/>In summary, this study demonstrates a very simple mechanism of nanoengineering the bacteria without any complicated genetic manipulation, rather a much simpler process to modify bacteria to increase their efficiency at the same time maintaining their morphology, function, and structure. This study demonstrates the potential for bacteria therapy in cancer theranostics as they can provide a paradigm-shifting knowledge for the development of next-generation anticancer agents by nanoengineering the bacteria.<br/><br/><b>References</b><br/>1. Forbes, N. S. Nat. Rev. Cancer, 2010,10, 785−794.<br/>2. Abdolalipour, E. et. al., Microbial Pathogenesis, 2020, 145, 104207.<br/>3. Reghu, S. and Miyako E. Nano Letters, 2022, 22, 1880-1888.