CN116617408A - Microorganism and functional nucleic acid co-delivery system and preparation method and application thereof - Google Patents

Microorganism and functional nucleic acid co-delivery system and preparation method and application thereof Download PDF

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CN116617408A
CN116617408A CN202310335101.3A CN202310335101A CN116617408A CN 116617408 A CN116617408 A CN 116617408A CN 202310335101 A CN202310335101 A CN 202310335101A CN 116617408 A CN116617408 A CN 116617408A
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nucleic acid
functional nucleic
bacteria
tumor
acid
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CN116617408B (en
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任怡琳
薛育政
盛颖玥
戴圆圆
吴铁龙
许心怡
郭丽娜
周文虎
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Affiliated Hospital of Jiangnan University
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Abstract

The invention provides a microorganism and functional nucleic acid co-delivery system, a preparation method and application thereof, comprising bacteria, functional nucleic acid and an acid-sensitive coating, wherein the acid-sensitive coating is formed by oxidizing and self-polymerizing poly-5-hydroxytryptamine on the surface of the bacteria, and the acid-sensitive coating adsorbs the functional nucleic acid through multiple acting forces; the bacteria are anaerobic or facultative anaerobes; the functional nucleic acid is formed by loading nucleic acid medicine with maleimide modified functional manganese hyaluronate nanoparticle, and the nucleic acid medicine takes PD-L1 as a target point. The co-delivery system disclosed by the invention is simple and controllable in preparation process, can protect bacteria from being rapidly cleared in the systemic circulation, realizes the synergistic targeting delivery of bacteria and functional nucleic acid under the biological chemotactic effect of the bacteria, and enables an acid-sensitive coating to respond to degradation and shedding of tumor microenvironment so as to recover the antitumor active tumor of the bacteria; meanwhile, the released functional nucleic acid is taken up by tumor cells at the periphery of the tumor, the expression of intracellular immune checkpoint PD-L1 is regulated down, the immune escape of the tumor is blocked, and the two are combined to realize the accurate and efficient treatment of the tumor.

Description

Microorganism and functional nucleic acid co-delivery system and preparation method and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a microorganism and functional nucleic acid co-delivery system, and a preparation method and application thereof.
Background
The incidence of malignant tumors is high, which constitutes a great threat to human health. Drug therapy is the most common therapeutic modality, but is mostly accompanied by serious toxic side effects. Targeted drug delivery is a precondition for good efficacy and avoidance of toxicity. A large number of researches show that anaerobic or facultative anaerobe has natural targeting to tumor area, the accumulation amount in the tumor area can reach over thousand times of that of normal tissue, and the anaerobe can play an anti-tumor role in competing nutrition, directly attacking tumor cells, activating immune system and the like. In addition, in view of its excellent drug carrying capacity, bacteria can be a good tumor targeted delivery vehicle. Bacteria are therefore uniquely advantageous in tumor therapy.
The anti-tumor effect of bacteria is proved on an animal model and then rapidly goes to clinic, but the clinic treatment effect is far from that of animal, and patients do not benefit. The mechanism research shows that bacteria entering the organism can be identified by an anti-infection immune system represented by neutrophils and can be rapidly cleared, and the targeting and the implantation of the bacteria at a tumor position are limited; bacteria that reach the tumor site will be bound by neutrophils to the central tumor area, limiting their killing effect on the peripheral active tumor area. Thus, immune clearance and immune barrier are two important factors limiting the clinical efficacy of bacteria.
Surface modification is an important strategy to solve the above-mentioned problem of immune clearance. Researchers wrap bacteria through erythrocyte membranes, so that immune clearance of the bacteria is effectively reduced, and circulation time of the bacteria in blood is prolonged, but the wrapping is unstable, and the bacteria are easy to fall off uncontrollably along with proliferation of the bacteria; also, researchers form polydopamine coatings on bacterial surfaces through dopamine oxidation autopolymerization, which also reduces bacterial immune clearance, but is not easily degraded, and is difficult to effectively restore the anti-tumor activity of bacteria. Therefore, the development of novel bacterial surface modification strategies is of great practical significance.
Poly (5-hydroxytryptamine) (PST) is a structural analog of polydopamine, and our previous studies have found that PST can be coated on the surface of almost any material as well, including glass, metals, nano-inorganic/organic materials, etc. Notably, unlike polydopamine, PST has acid sensitive degradable properties, and under physiological pH conditions, PST can be stably coated on the surface of the material, while in a slightly acidic environment of tumor, the PST shell gradually falls off. In addition, PST has bioadhesion property, and can adsorb biological macromolecule and micromolecular medicine through hydrogen bond, pi-pi stacking, electrostatic acting force, metal coordination acting force and the like. These properties provide the possibility to solve the above mentioned problems of bacteria in antitumor applications.
Disclosure of Invention
In order to solve the technical problems, the invention provides a microorganism and functional nucleic acid co-delivery system, a preparation method and application thereof, wherein an acid-sensitive coating is formed on the surface of bacteria by poly-5-hydroxytryptamine so as to ensure that the bacteria cannot be easily removed in the process of systemic circulation; the acid-sensitive coating adsorbs functional nucleic acid, and after bacteria target tumor positions, the bacteria and the functional nucleic acid can be released rapidly in response to tumor microenvironment, and the tumor immune barrier is broken through by the cooperation of the bacteria and the functional nucleic acid, so that the efficient treatment of tumors is realized.
In order to achieve the above object, the present invention provides a co-delivery system of a microorganism and a functional nucleic acid, comprising a bacterium, the functional nucleic acid, and an acid-sensitive coating formed by oxidative autopolymerization of poly-5-hydroxytryptamine on the surface of the bacterium, the acid-sensitive coating adsorbing the functional nucleic acid by multiple forces;
the bacteria are anaerobic or facultative anaerobes;
the functional nucleic acid is formed by loading nucleic acid medicines with maleimide modified functionalized manganese hyaluronate nanoparticles, and the nucleic acid medicines take PD-L1 as targets.
Preferably, the bacterium is one of attenuated salmonella, escherichia coli, listeria, clostridium, lactobacillus and vibrio desulphurisation.
Preferably, the nucleic acid drug is one of antisense nucleic Acid (ASO), small interfering RNA (siRNA) and DNAzyme.
Based on a general inventive concept, the present invention also provides a method for preparing a microorganism and functional nucleic acid co-delivery system, comprising the steps of:
s1, preparing bacterial suspension, adding 5-hydroxytryptamine into the bacterial suspension, adjusting the pH of the solution to be alkaline, and stirring in a dark place to obtain acid-sensitive coating modified bacteria;
s2, preparing maleimide modified functionalized manganese hyaluronate nanoparticles by a template method, adding sulfhydryl modified nucleic acid drugs, and incubating to obtain functional nucleic acid;
and S3, incubating the bacteria modified by the acid-sensitive coating prepared in the step S1 and the functional nucleic acid prepared in the step S2 together, and centrifuging to obtain the bacteria and functional nucleic acid co-delivery system.
Preferably, the concentration of the bacterial suspension in step S1 is 1X 10 8 ～2×10 9 CFU/mL, preferably 1.6X10 9 CFU/mL。
Preferably, the final concentration of 5-hydroxytryptamine in step S1 is 1-10 mg/mL, preferably 2mg/mL.
Preferably, the final concentration of the functional nucleic acid in step S3 is 1 to 5. Mu.M, preferably 5. Mu.M.
Preferably, the specific preparation steps of the maleimide modified functionalized manganese hyaluronate nanoparticle in the step S2 include:
s201, dripping N- (2-aminoethyl) maleimide solution into mixed solution of hyaluronic acid, EDC and NHS for reaction, and dialyzing and freeze-drying to obtain maleimide modified functional hyaluronic acid;
s202, redissolving the maleimide modified functional hyaluronic acid obtained in the step S201, adding sodium hydroxide and manganese chloride solution, and centrifuging and collecting precipitate after ultrasonic treatment to obtain the maleimide modified functional hyaluronic acid manganese nanoparticle.
Based on a general inventive concept, the invention also provides an application of the microorganism and functional nucleic acid co-delivery system in preparing tumor immunotherapy medicaments.
Preferably, the tumor immunotherapy medicine is an injection.
Preferably, the injection comprises intravenous injection of the microorganism and functional nucleic acid co-delivery system and pharmaceutically acceptable auxiliary materials.
Compared with the prior art, the invention has the following beneficial effects:
1. the microorganism and functional nucleic acid co-delivery system provided by the invention forms poly-5-hydroxytryptamine (PST) on the surface of bacteria by oxidizing 5-hydroxytryptamine, and adsorbs functional nucleic acid by hydrogen bond, pi-pi stacking, electrostatic acting force, metal coordination acting force and the like.
2. After targeting to the tumor acidic microenvironment, the acid-sensitive coating PST of the microorganism and functional nucleic acid co-delivery system is degraded and shed, and bacteria are released and recovered to the central area of the tumor to play the role of resisting tumor of the Trojan horse, wherein the method comprises the steps of directly inducing apoptosis of tumor cells, promoting gap connection between the tumor cells and DC cells to perform antigen presentation so as to activate the DC cells, and the activated DC cells can activate the T cells to perform immune killing on the tumor cells; meanwhile, released functional nucleic acid is taken up by tumor cells at the periphery of the tumor, the expression of intracellular immune checkpoint PD-L1 is regulated down, the immune escape of the tumor is blocked, and the bacteria and the functional nucleic acid are combined to realize the accurate and efficient treatment of the tumor; the delivery system is simple and convenient to prepare, high in biological safety, and provides a new solution for clinical application bottlenecks of bacteria and a new idea for tumor immunotherapy.
3. The functional nucleic acid is formed by loading nucleic acid medicines on maleimide modified functional manganese hyaluronate nanoparticles, the carrier can obviously increase the uptake of the nucleic acid medicines by tumor cells through an endocytic mechanism, then the nucleic acid medicines and divalent manganese ions are released simultaneously under the action of intracellular GSH, and the divalent manganese ions can be used as cofactors to activate the mRNA cleavage activity of the nucleic acid medicines, so that the expression level of PD-L1 in the tumor cells is obviously reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of Sal@PST/DzMN construction process and anti-tumor effect in example 1 of the present invention;
FIG. 2 is a graph showing the particle size distribution and potential of Sal@PST in example 1 of the present invention, wherein FIGS. 2A and 2B are respectively the particle size distribution and potential of Sal@PST, FIGS. 2C and 2D are respectively the transmission electron microscope image and the optical microscope image thereof, and FIGS. 2E and 2F are respectively the ultraviolet and infrared spectrograms thereof;
fig. 3 is a representation of DzMN in embodiment 1 of the present invention, wherein fig. 3A is a transmission electron microscope image of HMN and DzMN, fig. 3B and 3C are particle size distribution and potential diagrams thereof, fig. 3D is an ultraviolet absorption spectrum thereof, and fig. 3E is an element scan thereof;
FIG. 4 is a representation of Sal@PST/DzMN in example 1 of the present invention, wherein FIG. 4A is a transmission electron microscope plot of Sal@PST/DzMN, FIG. 4B is a potential diagram thereof, FIG. 4C is a scanned elemental plot of Sal@PST and Sal@PST/DzMN, and FIG. 4D is a desorption rate plot of Sal@PST/DzMN in different media;
FIG. 5 is a graph showing phagocytosis results of Sal or Sal@PST by macrophages and neutrophils in experimental example 1 of the present invention, wherein FIG. 5A is a graph showing phagocytosis results of macrophages and FIG. 5B is a graph showing phagocytosis results of neutrophils;
FIG. 6 is a result of investigating pH response degradation characteristics of PST in experimental example 2 of the present invention, wherein FIG. 6A is an external appearance and an ultraviolet absorption spectrum of a supernatant after incubation, FIG. 6B is a transmission electron microscope image after incubation, FIGS. 6C to 6E are growth graphs of Sal, sal@PDA, sal@PST at different pH values, respectively, and FIG. 6F is a release graph of DzMN in Sal@PST/DzMN;
FIG. 7 is an in vitro antitumor effect study of the bacteria Sal@PST/DzMN in experimental example 3 of the invention, wherein FIG. 7A is a graph showing apoptosis results of B16F10 cells after different treatments, FIG. 7B is a schematic diagram showing a gap junction formation experiment, FIG. 7C is a fluorescence intensity distribution diagram of Calcein AM in DC2.4 cells in the gap junction formation experiment, FIG. 7D is a graph showing expression conditions of CD86 and MHC II of DC2.4 cells after different treatments, and FIG. 7E is a graph showing quantitative results of double positive DC2.4 cells of CD86 and MHC II;
FIG. 8 is a graph showing the results of examining the in vitro antitumor effect of DzMN in Sal@PST/DzMN in experimental example 4 of the present invention, wherein FIGS. 8A and 8B are a fluorescence imaging graph and a flow chart showing the uptake of free Dz or DzMN by B16F10 cells, and FIG. 8C is the expression of intracellular PD-L1, respectively;
FIG. 9 is a graph showing the survival rate and tumor growth of mice after various treatments in experimental example 5 of the present invention, wherein FIG. 9A is the survival rate of mice in each group during the treatment period, FIG. 9B is the tumor volume-time curve of mice in each group during the treatment period, and FIG. 9C is the tumor appearance of mice in each group after the treatment is completed;
FIG. 10 is a graph of H & E staining and TUNEL staining of tumor sections of mice of each group in Experimental example 5 of the present invention;
FIG. 11 is a graph showing the change in body weight during administration of each group of mice in Experimental example 6 of the present invention;
FIG. 12 shows the results of analysis of serum biochemical indicators of mice of each group in Experimental example 6 of the present invention, wherein FIG. 12A shows the results of ALT and AST analysis, and FIG. 12B shows the results of BUN and Cre analysis;
FIG. 13 shows the results of detection of serum inflammatory factors in mice of each group in Experimental example 6 of the present invention, wherein FIGS. 13A to 13D show the results of detection of TNF- α, IFN- γ, IL-6 and IL-10, respectively;
FIG. 14 is an analysis of heart, liver, spleen, lung, kidney pathological section of each group of mice in experimental example 6 of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated; the reagents used in the examples were all commercially available unless otherwise specified.
Example 1
Preparation of microorganism and functional nucleic acid Co-delivery System (Sal@PST/DzMN)
The method comprises the steps of carrying out surface acid-sensitive coating modification on bacteria, then incubating the bacteria with functional nucleic acid, and adsorbing the functional nucleic acid on the surfaces of the bacteria through multiple acting forces, wherein the specific construction process of the system and the anti-tumor mode of the system are shown in figure 1.
The bacterial and functional nucleic acid co-delivery system (Sal@PST/DzMN) of this example was prepared using the following procedure:
(1) Acid-sensitive coating modification of bacteria:
the attenuated salmonella was shake-cultured in a thermostated shaker at 37℃and 180 rpm. Collecting bacteria by centrifugation, adding physiological saline, and re-suspending according to OD 600 The concentration was adjusted to 1.6X10 9 CFU/mL. 0.05mL of the bacterial suspension was taken, 0.2mL of ethanol, 0.045mL of ammonia water, 0.21mL of a 100mM Tween-80 aqueous solution, 0.02mL of a 225mg/mL 5-hydroxytryptamine aqueous solution, and 1.67mL of water were added, and after mixing uniformly, stirring was performed at 37℃in the dark for 4 hours. And (3) centrifuging at 3000rpm for 5min to obtain bacteria modified by the poly-5-hydroxytryptamine coating (Sal@PST). Particle size and potential before and after bacterial modification were measured by Marlven Nano ZS instrument, morphology was observed by transmission electron microscope and optical microscope, and uv and ir absorption spectra were scanned by uv-vis spectrophotometer and ir spectrometer, respectively, and the results are shown in fig. 2.
FIG. 2 is a graph showing the characteristics of Sal@PST, wherein FIGS. 2A and 2B are the particle size distribution and potential diagram of Sal and Sal@PST, respectively, and the particle size of bacteria after being modified by a coating is increased from 1040nm to 1241nm, and the absolute value of the negative potential is reduced; FIGS. 2C and 2D are a transmission electron microscope image and an optical microscope imaging image, respectively, wherein the Sal@PST is visible as an obvious shell under the transmission electron microscope, and the Sal@PST is enhanced in contrast under the optical microscope; FIGS. 2E and 2F are ultraviolet and infrared spectra, respectively, each showing a characteristic peak of PST. The above results also indicate the successful construction of Sal@PST.
(2) Preparation of functional nucleic acid:
200.5mg of HA, 115.0mg of EDC and 69.1mg of NHS were dissolved in 22mL of 0.1M MES buffer pH 4.5 and stirred at room temperature for 30min. Then, 176.6mg of Mal HCl was weighed, dissolved in 1mL of ultrapure water, and added dropwise to the above system, followed by a reaction for 24 hours. After the completion of the reaction, the reaction solution was collected and dialyzed (mwco=3500 Da) in ultrapure water for 3 days, and lyophilized for 24 hours to obtain HA-Mal. Taking 4mL of HA-Mal solution with the concentration of 5mg/mL, adding 80 mu L of NaOH solution with the concentration of 1M and 40 mu L of MnCl with the concentration of 20mg/mL under stirring 2 The solution was then sonicated for 10min and centrifuged at 20000rpm for 10min, and the precipitate (maleimide modified functionalized manganese hyaluronate nanoparticles, denoted HMN) was collected. 10mM HEPES buffer, pH 7.4 was added for resuspension,thiol-modified PD-L1 DNAzyme (Dz) was added thereto to give a final concentration of 20. Mu.M, and incubated overnight at room temperature. Centrifuging at 20000rpm for 10min to obtain functional nucleic acid (DzMN). The forms of HMN and DzMN are observed through a transmission electron microscope, element scanning is carried out, the particle size and the potential of the HMN and DzMN are measured through a Marlven Nano ZS instrument, and the ultraviolet absorption spectrum of the HMN and DzMN is scanned through an ultraviolet-visible spectrophotometer.
Fig. 3 is a representation of DzMN, wherein fig. 3A is a transmission electron microscope image of HMN and DzMN, fig. 3B and 3C are particle size distribution and potential diagrams thereof, respectively, fig. 3D is an ultraviolet absorption spectrum thereof, and fig. 3E is an elemental scan thereof. The results in FIG. 3 show that the particle size of DzMN is increased, the potential is not significantly changed, the UV absorption at 200-400nm is enhanced, and the characteristic P element is present, compared with HMN, indicating successful synthesis of functional nucleic acid.
(3) Construction of acid sensitive coatings and functional nucleic acid modified bacteria:
and (3) re-suspending the collected Sal@PST in 10mM HEPES buffer with pH of 7.4, adding DzMN, incubating for 2 hours at 37 ℃ to obtain the acid-sensitive coating and the functional nucleic acid modified bacteria (Sal@PST/DzMN) after incubation for 5 minutes at 3000 rpm. Observing the form of Sal@PST/DzMN by a transmission electron microscope, scanning elements, and measuring the potential by a Marlven Nano ZS instrument; sal@PST/DzMN was placed in different media (HEPES, PBS, FBS, BSA, SDS, naCl and urea), incubated at 37℃for 2h, centrifuged at 3000rpm for 5min, and the desorption behavior of DzMN was examined by measuring the absorbance of the supernatant at 340 nm.
FIG. 4 is a representation of Sal@PST/DzMN, wherein FIG. 4A is a transmission electron microscope image of Sal@PST/DzMN, FIG. 4B is a potential diagram thereof, FIG. 4C is a scanned view of elements of Sal@PST and Sal@PST/DzMN, and FIG. 4D is a desorption rate diagram of Sal@PST/DzMN in different media. The results in FIG. 4 show that the apparent particle attachment is seen in the transmission electron microscopy, the absolute value of the negative potential of Sal@PST/DzMN is increased compared with that of Sal@PST, and special P and Mn elements appear in the element scan, indicating the successful construction of Sal@PST/DzMN. In various mediums examined, the desorption rate of DzMN is low, which indicates that the DzMN has good adsorption stability.
Experimental example 1
The anti-recognition/phagocytosis effect of the bacterial and functional nucleic acid co-delivery system of example 1 (Sal@PST/DzMN) was examined.
(1) Log-grown RAW264.7 cells (mouse peritoneal macrophages, purchased from the xiangya medical laboratory center at the university of south China) were taken, counted for digestion, and diluted 1.5x10 with an appropriate amount of RPMI-1640 complete medium containing 10% FBS 5 Cell suspensions at each mL, 0.5mL per well, were seeded in 24-well plates and incubated overnight for adherence. Then Sal or Sal@PST (Sal expressing green fluorescent protein) was added to the cell culture broth, and after incubation for 1.5h, the supernatant was discarded and washed 2 times with PBS. Cells were collected, resuspended in PBS and flow tested.
(2) Taking peripheral blood of a mouse, extracting neutrophils in the peripheral blood by using a neutrophil extraction kit of the peripheral blood of the mouse, re-suspending the neutrophils by using PBS, adding Cell Tracker Deep Red, incubating at 37 ℃ for 30min to dye the cells, centrifuging at 2000rpm for 5min, collecting the cells, and re-suspending the cells by using a complete culture medium. Sal or Sal@PST (Sal expressing green fluorescent protein) was added to the cell culture broth, and after incubation for 1.5h, each sample was collected for flow-through detection.
FIG. 5 is a graph showing phagocytosis results of Sal or Sal@PST by macrophages and neutrophils, wherein FIG. 5A is a macrophage and FIG. 5B is a neutrophil. The results in FIG. 5 show that macrophages and neutrophils engulf less Sal@PST than Sal, indicating that the acid sensitive coating modification of the present invention reduces bacterial recognition by immune cells and reduces immune clearance.
Experimental example 2
The pH responsive degradation characteristics of PST in the bacterial and functional nucleic acid co-delivery system of example 1 (Sal@PST/DzMN) were examined.
(1) Sal@PST was placed in buffers at pH 7.4, 6.0, 5.0, respectively, and after overnight incubation, centrifuged at 3000rpm for 5min, and the supernatant was collected. The color of the supernatant was observed and the absorption spectrum of the supernatant at 380-800nm was scanned by an ultraviolet-visible spectrophotometer. The results are shown in FIG. 6A: as the pH of the buffer solution is reduced, the color of the supernatant gradually deepens, and the ultraviolet absorption is enhanced.
(2) Sal@PST was placed in a buffer pH 5.0 and after overnight incubation, the morphology was observed by transmission electron microscopy. The results are shown in FIG. 6B: apparent dissolution of the shell is visible in the transmission electron microscopy.
(3) Bacterial growth curve: sal, sal@PDA (polymeric material control, no pH responsive degradation property), sal@PST were placed in LB broth pH 7.0, 6.0, 5.0, respectively, and cultured with shaking at 37℃and sampled every 2 h. Determination of OD of sample by means of an enzyme-labelling instrument 600 Values, bacterial growth curves were plotted. The results are shown in FIGS. 6C to 6E: PDA or PST modifications delayed bacterial growth under neutral conditions and the PST modified bacteria gradually recovered to conform to unmodified bacteria under acidic conditions, whereas PDA modified bacteria did not recover.
(4) pH-responsive release of DzMN: sal@PST/DzMN was placed in a buffer solution at pH 7.4 and 6.0, respectively, and the mixture was oscillated at 37℃and 100rpm in a constant temperature water bath oscillation tank. Sampling at 0, 0.5, 1, 2, 4, 8 hr, centrifuging at 3000rpm for 5min, collecting supernatant, centrifuging at 20000rpm for 10min, re-dispersing the precipitate with equal volume of water, and measuring its absorbance at 340nm by ultraviolet-visible spectrophotometer. From this the cumulative release rate of DzMN is calculated. The results are shown in FIG. 6F: dzMN releases significantly faster at pH 6.0 than at pH 7.4, exhibiting pH responsive release characteristics
Together, the above results in fig. 6 demonstrate that PST has good pH response degradation characteristics, can stably entrap bacteria under physiological conditions, and can release bacteria and DzMN in response to degradation and shedding of tumor acidic microenvironment after reaching tumor sites.
Experimental example 3
Examination of the anti-tumor Effect of the bacteria in the Co-delivery System of bacteria and functional nucleic acid (Sal@PST/DzMN) of example 1
(1) Apoptosis study: logarithmic growth of B16F10 cells (mouse melanoma cells, purchased from Xiangya medical laboratory center, university of south China) was taken, counted, and diluted 1.5X10 with an appropriate amount of DMEM complete medium containing 10% FBS 5 Cell suspensions at each mL, 2mL per well, were seeded in 6-well plates and incubated overnight to allow adherence. At the same time Sal@PST was incubated overnight in buffer at pH 7.4 or pH 6.0, respectively. Sal or pretreated Sal@PST was then added to the cell culture broth, respectively, and after 24h incubation, the supernatant was collected in a 5mL Ep tube, followed by digestion of the collected cells, and mixing with the supernatant.It was centrifuged at 1000rpm for 5min, the supernatant was discarded, and washed 2 times with PBS. Then, 100 mu L of 1×binding buffer is used for resuspension, 5 mu LAnnexin-FITC is added, and the mixture is incubated for 10min at room temperature and in a dark place; then 5. Mu.L PI was added and incubated at room temperature for 5min in the dark. Finally, the sample volume was made up to 300 μl with PBS for flow-through detection.
(2) Formation of gap connection: B16F10 cells and DC2.4 cells (mouse bone marrow derived dendritic cells, purchased from xiangya medical laboratory center, university of south China) were inoculated into 6-well plates, respectively, and incubated overnight for adherence, according to the methods described above. At the same time Sal@PST was incubated overnight in buffer at pH 7.4 or pH 6.0, respectively. Sal or pretreated Sal@PST was then added to the cell culture broth, respectively, and after incubation for 4h, the broth was discarded and washed 3 times with PBS. Fresh complete medium containing 50. Mu.g/mL gentamicin was added and the culture continued for 24h. The cells of each group were then individually collected, counted after being resuspended in PBS, and their densities were adjusted to be uniform. The dye Calcein AM (2. Mu.M) was added to B16F10 cells, DDAO (10. Mu.M) was added to DC2.4 cells, stained at 37℃for 30min, and centrifuged at 1000rpm for 5min to remove the remaining dye. Two cell pellets were resuspended in RPMI-1640 complete medium, incubated at a ratio of b16f10:dc2.4=2:1, cells were collected by centrifugation after 1h, and flow-detection was performed after PBS resuspension, as shown in fig. 7C:
(3) DC cell activation study: cells were treated as in (2) and then each group of cells was collected separately and inoculated into 12-well plates for co-incubation. After 24h, the cells from each well were collected and resuspended in 100. Mu.L PBS. Then 0.5. Mu.Lanti-MHC II-FITC (1 mg/mL) and 2.5. Mu.L anti-CD86-PE (0.2 mg/mL) were added to each well, after incubation at 4℃for 30min, cells were collected by centrifugation at 1500rpm for 5min, and after PBS was resuspended for flow detection.
FIG. 7 is an in vitro antitumor effect study of bacteria in Sal@PST/DzMN, wherein FIG. 7A is a graph showing apoptosis results of B16F10 cells after various treatments: sal can induce apoptosis of tumor cells, and Sal@PST has reduced anti-tumor effect after pretreatment by a pH 7.4 buffer solution, and has recovered to the effect that Sal can induce apoptosis of tumor cells after pretreatment by a pH 6.0 buffer solution, which also shows that Sal@PST stably exists in a neutral environment, effectively degrades in an acidic environment and recovers bacterial activity. FIG. 7B is a schematic diagram of a gap junction formation experiment, FIG. 7C is a graph showing a fluorescence intensity distribution of Calcein AM in DC2.4 cells in the gap junction formation experiment, FIG. 7D is a graph showing the expression of CD86 and MHC II in DC2.4 cells after different treatments, and FIG. 7E is a graph showing the quantitative results of DC2.4 cells positive for both CD86 and MHC II. As can be seen from the results of fig. 7, sal induces apoptosis of tumor cells, promotes formation of gap junctions between tumor cells and DC cells (dendritic cells), and further promotes activation of DC cells; PST modification can attenuate the above activities of bacteria, which are restored after pretreatment under acidic conditions. Therefore, the bacteria in the embodiment 1 of the invention have multiple anti-tumor activities such as direct induction of tumor cell apoptosis, induction of immune response and the like, and can be activated as required after PST modification, and the activity is recovered in response to tumor microenvironment.
Experimental example 4
Examination of the anti-tumor Effect of the functional nucleic acid in the Co-delivery System of bacteria and functional nucleic acid of example 1 (Sal@PST/DzMN)
(1) Cell uptake study: the B16F10 cells grown logarithmically were digested with pancreatin and diluted to a density of 1.5X10 with DMEM medium containing 10% FBS 5 Cell suspensions at each mL were seeded at 0.5mL per well in 24 well plates and incubated overnight for adherence. FAM-labeled free Dz or DzMN (Dz concentration 400 nM) was then added, respectively, and after incubation for 4h, the culture broth was aspirated and washed 2 times with PBS. Each well was fixed with 0.5mL of 4% paraformaldehyde for 15min and discarded, and washed 2 times with PBS. Then adding Hoechst 33342 dye solution (diluted 100 times) to dye the nucleus for 5min, and imaging under a fluorescence microscope. In addition, cells were collected after the above treatment, and were subjected to flow assay after being resuspended in PBS.
(2) Expression study of PD-L1: B16F10 cells were seeded in 6-well plates at 2mL per well and incubated overnight for adherence as described above. Then HMN or DzMN (Dz concentration 400 nM) was added respectively, and after 24h incubation, total cell proteins were extracted and the intracellular PD-L1 protein expression was detected by Western Blot.
FIG. 8 is an in vitro antitumor effect study of DzMN in Sal@PST/DzMN, wherein FIGS. 8A and 8B are respectively a fluorescence imaging diagram and a flow chart of free Dz or DzMN uptake by B16F10 cells, and FIG. 8C is an expression condition of intracellular PD-L1. The result shows that the free nucleic acid medicine is difficult to freely cross cell membranes to enter tumor cells due to the strong hydrophilicity and electronegativity, and DzMN can obviously increase the uptake of Dz by the tumor cells through an endocytic mechanism and obviously lower the expression level of intracellular PD-L1.
Experimental example 5
Examine the in vivo antitumor activity of the bacterial and functional nucleic acid co-delivery system of example 1 (Sal@PST/DzMN):
(1) Construction of mouse subcutaneous tumor model: B16F10 cells with good growth state are digested and collected into a 15mL sterile centrifuge tube, and are centrifuged at 1000rpm for 5min to collect cell sediment, and PBS is added for resuspension after 2 times of PBS washing to make the density of the cell sediment be 1 multiplied by 10 7 The samples were placed on ice at a volume of one mL. The C57BL/6 mice were shaved and the cell suspension was aspirated with a 1mL syringe, and 100. Mu.L of each mouse was subcutaneously injected into the armpit. The growth of the tumor in the mice was observed periodically, and the length and width of the tumor were measured with a vernier caliper, and the tumor volume= (length x width) 2 ) Tumor volume was calculated.
(2) The tumor volume of the mice is 100mm 3 At this time (noted as day 0), tumor-bearing mice were randomly divided into 7 groups, and 100. Mu.L of PBS, sal, sal@PST or Sal@PST/DzMN was injected intravenously at the tail on day 0 and 3, respectively, at a dose of 6X 10 in Sal 6 CFU/or 2X 10 only 7 CFU/CFU.
(3) The survival number of each group of mice is recorded every day, and the survival rate of the mice is calculated; the length and width of each group of mice tumor are measured by a vernier caliper, the tumor volume is calculated according to a formula, and a mouse tumor volume-time curve is drawn.
(4) Mice were sacrificed on day 7, tumors were removed, and the appearance of the tumors was recorded by photographing.
(5) The removed mouse tumors were fixed overnight in 4% paraformaldehyde, and after paraffin-embedded sections, H & E staining and TUNEL staining were performed, respectively.
Fig. 9 shows the survival rate and tumor growth of mice after various treatments, wherein fig. 9A shows the survival rate of mice in each group during the treatment period, fig. 9B shows the tumor volume-time curve of mice in each group during the treatment period, and fig. 9C shows the tumor appearance of mice in each group after the treatment. The results show that the Sal group mice are all dead within 3 days, the Sal@PST and Sal@PST/DzMN can improve the survival rate of the mice and inhibit the tumor growth of the mice to a certain extent, and the Sal@PST/DzMN is more effective, so that the bacterial and functional nucleic acid can jointly promote the anti-tumor treatment effect.
FIG. 10 is a graph of H & E staining and TUNEL staining of tumor sections of mice in each group. The results showed that various degrees of nuclear atrophy, necrosis and apoptosis occurred in each group, with the Sal@PST/DzMN group being most pronounced at high doses, indicating good anti-tumor effects.
Experimental example 6
Examine the in vivo safety of the bacterial and functional nucleic acid co-delivery system of example 1 (Sal@PST/DzMN):
mice were treated as in experimental example 5, and changes in body weight of the mice were recorded during the administration period; mouse serum was collected at day 7 for biochemical index analysis and inflammatory factor detection; the heart, liver, spleen, lung and kidney of the mice were taken, washed with physiological saline, the filter paper was blotted to remove water, and after fixation with 4% paraformaldehyde for 24 hours, paraffin-embedded, sectioned, H & E stained, and observed for pathological changes using an optical microscope.
Figure 11 shows the body weight change during dosing for each group of mice. As can be seen, the body weight of the Sal group mice decreased significantly, while the body weight of the remaining groups did not change significantly during the dosing period, indicating that the PST surface modification improved bacterial biosafety.
FIG. 12 shows the results of serum biochemical index analysis of mice in each group. As can be seen from the graph, there was no obvious difference in each index value for each group of mice.
FIG. 13 shows the results of serum inflammatory factor detection in each group of mice. As can be seen from the figures, there is no significant difference in the levels of serum TNF- α, IFN- γ, IL-6 and IL-10 in each group of mice, indicating that the acid sensitive coatings and functional nucleic acid modified bacteria of the present invention do not cause systemic inflammation.
FIG. 14 is an analysis of heart, liver, spleen, lung, kidney pathology sections of each group of mice. No obvious pathological changes are seen in each organ of each group of mice, which indicates that the bacterial and functional nucleic acid co-delivery system (Sal@PST/DzMN) has good biological safety in vivo.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A co-delivery system of a microorganism and a functional nucleic acid, comprising a bacterium, the functional nucleic acid, and an acid-sensitive coating formed by oxidative autopolymerization of poly-5-hydroxytryptamine on the surface of the bacterium, the acid-sensitive coating adsorbing the functional nucleic acid by multiple forces;
the bacteria are anaerobic or facultative anaerobes;
the functional nucleic acid is formed by loading nucleic acid medicines with maleimide modified functionalized manganese hyaluronate nanoparticles, and the nucleic acid medicines take PD-L1 as targets.
2. The co-delivery system of a microorganism and a functional nucleic acid of claim 1, wherein the bacterium is one of attenuated salmonella, escherichia coli, listeria, clostridium, lactobacillus, and vibrio desulphurisation.
3. The co-delivery system of a microorganism and a functional nucleic acid of claim 1, wherein the nucleic acid drug is one of an antisense nucleic acid, a small interfering RNA, and a deoxyribozyme.
4. A method for preparing a co-delivery system of a microorganism and a functional nucleic acid according to any one of claims 1 to 3, comprising the steps of:
s1, preparing bacterial suspension, adding 5-hydroxytryptamine into the bacterial suspension, adjusting the pH of the solution to be alkaline, and stirring in a dark place to obtain acid-sensitive coating modified bacteria;
s2, preparing maleimide modified functionalized manganese hyaluronate nanoparticles by a template method, adding sulfhydryl modified nucleic acid drugs, and incubating to obtain functional nucleic acid;
and S3, incubating the bacteria modified by the acid-sensitive coating prepared in the step S1 and the functional nucleic acid prepared in the step S2 together, and centrifuging to obtain the bacteria and functional nucleic acid co-delivery system.
5. The method according to claim 4, wherein the concentration of the microorganism suspension in the step S1 is 1X 10 8 ～2×10 9 CFU/mL。
6. The method according to claim 4, wherein the final concentration of 5-hydroxytryptamine in the step S1 is 1-10 mg/mL.
7. The method according to claim 4, wherein the final concentration of the functional nucleic acid in the step S3 is 1 to 5. Mu.M.
8. The preparation method according to claim 4, wherein the specific preparation step of the maleimide modified functionalized manganese hyaluronate nanoparticle in the step S2 comprises the following steps:
s201, dripping N- (2-aminoethyl) maleimide solution into mixed solution of hyaluronic acid, EDC and NHS for reaction, and dialyzing and freeze-drying to obtain maleimide modified functional hyaluronic acid;
s202, redissolving the maleimide modified functional hyaluronic acid obtained in the step S201, adding sodium hydroxide and manganese chloride solution, and centrifuging and collecting precipitate after ultrasonic treatment to obtain the maleimide modified functional hyaluronic acid manganese nanoparticle.
9. Use of a co-delivery system of a microorganism according to any one of claims 1 to 3 with a functional nucleic acid or a co-delivery system of a microorganism according to any one of claims 4 to 9 with a functional nucleic acid in the manufacture of a medicament for tumour immunotherapy.
10. The use according to claim 9, wherein the tumor immunotherapeutic agent is an injection.
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