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Precise regulation of near-infrared light, defeating tumors with "bacteria"

In recent years, the rapid development of synthetic biology has brought new opportunities for the in-depth optimization of tumor bacterial therapy. Based on synthetic biology methods, scientists can use genetically engineered microorganisms or cells instead of traditional chemical small molecules or biological agents as the basis for the development of new disease treatments. Artificially designed microorganisms or cells carrying synthetic gene circuits can respond to disease markers or external signals, and realize the control of drug release location, release time, and release dose, which has gradually developed into a powerful weapon for humans to fight against diseases.


On the basis of traditional bacterial transformation, the rationally designed gene pathway endows chassis bacteria with more diverse therapeutic capabilities and makes up for the defects of natural strains in tumor treatment. Tumor bacterial therapy has therefore become a new type of tumor with great application prospects therapy. The optimization strategy of existing tumor bacterial therapy mainly focuses on the application of new drugs and the introduction of new controllable release methods, etc., but ignores the influence of bacteria’s own behavior on tumor treatment effect, and lacks the continuous control of bacterial behavior in the process of tumor treatment . Tumor treatment is a long-term process. Controlled and sustained drug release is the key to improving the efficacy of tumor treatment. Although the sustained release of drugs has been achieved in the field of materials through polymer hydrogels and lipids, but in tumor bacterial therapy Still a challenge.


Through the design of synthetic biology, the researchers successfully transformed the Pseudomonas aeruginosa strain into an engineered bacterium with therapeutic efficacy for solid tumors. During the treatment process, the global phenotype of the engineered bacteria can be precisely controlled by the irradiation program of near-infrared light, that is, the three phenotypes of weak colonization, colonization and cleavage can be switched, so as to ablate the tumor more effectively Body to achieve therapeutic effect, has great potential application value.


In nature, Pseudomonas aeruginosa is in a planktonic state in a favorable environment, and its ability to colonize the surface is weak; while in an unfavorable environment, it will enter a biofilm state, and its surface colonization ability is greatly enhanced. Inspired by the way bacteria live in nature, the research team designed the planktonic state and biofilm state for the engineered bacteria to control their colonization ability. Among them, the colonization ability of planktonic bacteria is weak and can reduce the damage to normal tissues ; while bacteria in the biofilm state have a strong colonization ability, which can increase their colonization in tumor tissues. The research team constructed an attenuated Pseudomonas aeruginosa strain as a new chassis strain in tumor bacterial therapy by knocking out the virulence factor regulatory protein coding gene vfr and the type III secretion system-related genes exoS and exoT. The switching of Pseudomonas aeruginosa between the planktonic state and the biofilm state is closely related to the concentration of the second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). To this end, the research team used two The gene module controls the c-di-GMP concentration in the bacterial cell, respectively 1) expressing the phosphodiesterase PA2133 through a constitutive promoter to decompose c-di-GMP, so that the bacteria maintain a low intracellular c-di -GMP level, enter the planktonic state; 2) Introduce the photosensitive protein BphS that can synthesize c-di-GMP under near-infrared light irradiation, near-infrared light irradiation can increase the level of c-di-GMP in the bacterial cell, and the bacteria enter the biological film state. In addition, in order to realize the controlled release of therapeutic drugs, the research team designed a third way of life for the engineered bacteria, that is, the lytic state, expressing the lytic gene through the c-di-GMP responsive promoter, which can make the bacteria in the c-di-GMP After rising, it enters the lysis state (Figure 1). In this way, the three lifestyles of bacteria are all related to the concentration of c-di-GMP. By changing the irradiation intensity of near-infrared light, the amount of activated BphS protein can be controlled, and then the concentration of c-di-GMP in the engineering bacteria can be adjusted, thereby realizing the control of the bacterial lifestyle. In addition to the effect of light intensity, the results of theoretical simulations indicated that the intensity of the ribosome binding site (RBS1) upstream of PA2133 and the ribosome binding site (RBS2) upstream of anti-terminator protein Q would be important for the bacteria to enter different lifestyles. The intensity of the near-infrared light required has a huge impact.


Figure 1 Design of the gene circuit for programming the bacterial lifestyle


In order to obtain engineering bacteria that can display three lifestyles under different intensities of near-infrared light, the research team replaced RBS1 and RBS2 in batches, and screened candidate strains in batches through a 96-well lighting device, and then verified them with a microscope. The resulting engineered strain was named H017 (Fig. 2). The lifestyle of H017 can be programmed by adjusting the irradiation intensity of near-infrared light hierarchically. When H017 is subjected to a medium-intensity-high-intensity cycle of near-infrared light irradiation program, the bacteria will enter the biofilm state-lysis state lifestyle cycle.


Fig. 2 Programming bacterial lifestyle by adjusting the intensity of near-infrared light hierarchically


In order to verify the role of the three lifestyles of engineered bacteria in tumor therapy in vitro, the research team conducted bacteria-cell co-culture experiments in microfluidic channels (Figure 3). The research team first confirmed that when cells in a designated area are illuminated with different intensities of near-infrared light, the engineered bacteria can precisely enter the corresponding lifestyle in the area. When a medium-intensity-high-intensity light program is applied to cells in a designated area, H017 first enters a biofilm state and colonizes a large number of cells on the surface, and then enters a lysed state to release therapeutic drugs and cause cell necrosis. The above results show that the lifestyle of engineered bacteria can be controlled with high spatio-temporal resolution by near-infrared light. In addition, based on the results achieved by the control group, the research team found that the drug accumulation process in the biofilm state and the drug release process in the cleavage state are the keys for H017 to kill tumor cells.


Figure 3 Programmed bacterial "biofilm-lysis" lifestyle transition to achieve controlled release of drugs


Subsequently, the research team used the mouse subcutaneous tumor model to explore whether the life style of engineered bacteria in the tumor could be controlled by near-infrared light and achieve the expected function (Figure 4). The results showed that after intratumoral injection of bacteria, there was no significant difference in the number of bacteria in the tumor tissue of the mice cultured in the dark for three days (D3) compared with the PBS injection group, indicating that the bacteria that entered the planktonic state of life were difficult to colonize in the tumor; Compared with the D3 group, the number of bacteria in the tumor tissue of mice irradiated with medium light intensity for three days (M3) increased significantly, indicating that the bacteria entering the biofilm state had a greatly enhanced colonization ability in tumor tissue; medium light intensity Compared with the M3 group, the number of bacteria in the mice irradiated with high light intensity for two days and one day group (M2-H1) was significantly reduced, and obvious necrotic areas could be seen in the tumor tissue sections of the M2-H1 group, indicating that the bacteria were lysed and the drug was released And killed the tumor cells; medium light intensity for two days, high light intensity for one day and medium light intensity for two days group (M2-H1-M2) compared with M2-H1 group, the number of bacteria increased again, which indicated that the bacteria in After being irradiated with medium light intensity, it enters the colonization growth state again. In addition, after seven days of exposure to medium-intensity near-infrared light, the engineered bacteria in the tumor still had the ability to lyse, indicating that the gene circuit had better stability. Based on the above results, it can be seen that different intensities of near-infrared light irradiation can make the intratumoral bacteria enter the corresponding lifestyle and achieve the expected function, and the continuous control of the intratumoral bacterial lifestyle can be achieved by continuously changing the light program.


Fig. 4 Manipulation of bacterial lifestyle in solid tumors by near-infrared light


Finally, the research team explored whether it can enhance its tumor treatment effect by programming the lifestyle of engineered bacteria (Figure 5). When H017 is only used as a drug delivery carrier, high-intensity near-infrared light irradiation can cause H017 to crack and release drugs to complete tumor treatment. During the 20-day experimental period, the team effectively inhibited tumor growth through 8 injections of bacteria. Next, the research team controlled the bacteria's lifestyle through light programs to achieve sustained control over drug accumulation and drug release. The results showed that after two cycles of moderate-intensity 2-day-high-intensity 1-day near-infrared light program (M2-H1×2) were applied to the mice, the tumor growth of the mice was inhibited, while that of the control group could not be inhibited. Tumor growth in mice. In a longer-term experiment (M2-H1×6), tumor treatment by programming bacterial lifestyle with light time inhibited tumor growth in all mice, and tumors completely disappeared in 30% of the mice, while those in the control group were small. Tumors in mice continued to grow. The above results show that the programmed bacterial lifestyle has significant advantages in the long-term treatment of tumors, and can achieve better tumor suppression effects with fewer bacterial injections.



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