CN112442454A - Nano fluorescence-labeled artificial bacteria and preparation method thereof - Google Patents
Nano fluorescence-labeled artificial bacteria and preparation method thereof Download PDFInfo
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Abstract
The invention provides an artificial bacterium and a preparation method thereof, which marks a nano material on an artificial bacterium biological carrier, and particularly relates to a salmonella nano fluorescence marking method with strong specificity, rapidness, convenience and the like. According to the invention, salmonella YB1 with high safety and strong anaerobic targeting is selected as a bacterial biological carrier, a reducing agent tri (2-carboxyethyl) phosphine (TCEP) is adopted to mildly reduce disulfide bonds (S-S) on the outer membrane protein surface of salmonella YB1 into Sulfydryl (SH), and the salmonella YB1 rich in free-SH and phospholipid-polymer nano probes (INPs) loaded with ICG and having maleimide groups on the surface are subjected to chemical crosslinking to construct YB1-INPs with fluorescence imaging capability. The method stably marks the nano fluorescent probes INPs on the surface of the salmonella YB1, can avoid the reduction of the reaction efficiency of the nano probes and the surface of bacteria due to cross-linking, and the obtained YB1-INPs can meet the practical requirements of basic research and clinical application on tracking and monitoring bacteria and real-time evaluation of tumor treatment effect.
Description
Technical Field
The invention relates to the field of nano medicine, in particular to a nano fluorescence labeled artificial bacterium and a preparation method and application thereof.
Background
The tumor is one of stubborn diseases which people need to overcome urgently, people use the modified engineering bacteria to treat the tumor in recent years, a good curative effect is obtained, the tumor targeting targeted release of therapeutic genes by using attenuated bacteria becomes a new bright point in the field of tumor gene therapy, wherein the salmonella typhimurium is a typical representative of the bacteria. The salmonella typhimurium is used as a facultative anaerobic gram-negative bacterium, has potential tumor targeting and tumor killing capacity, and also has the advantages of simple genetic operation and the like. Rough flesh ("research on tumor metastasis inhibition mechanism of Salmonella typhimurium YB 1", 2018, university of mansion Master's academic paper) reports Salmonella typhimurium YB1, which is an attenuated strain with strict anaerobic property after being genetically modified, and has excellent tumor targeting property and tumor metastasis inhibition capability.
Although the bacterial therapy is widely concerned by researchers due to the advantages of targeting tumors, invading tumor cells, having strong plasticity and the like, the problems of unclear treatment mechanism, weak controllability and the like of the bacterial therapy limit the development and application of the bacterial therapy as tumor treatment medicines. Therefore, there is a need to find a safe and efficient method to track, monitor, and assess the effectiveness of treatment of bacteria. The method of adding nano-materials on the surface of bacteria to provide a protective layer for the bacteria, or labeling the bacteria with nanoprobes has become a hot research point for bacterial therapy.
In the prior art, CN200910188814 discloses a nanoparticle and a preparation method thereof, wherein a core is formed by polylactic-co-glycolic acid (PLGA), an intermediate layer formed by phospholipid surrounds the surface of the core, and a shell part formed by distearoyl phosphatidyl ethanolamine-polyethylene glycol containing amino or carboxyl is inserted in the intermediate layer. This document does not relate to the labeling of fluorescent probes and has a limited range of use. CN201010607708 discloses a fluorescent nanoprobe, which also has an inner core formed by polylactic acid-glycolic acid copolymer, a middle layer formed by phospholipid surrounding the surface of the inner core, and an outer shell portion formed by distearoyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG) containing amino or carboxyl interspersed in the middle layer, wherein indocyanine green (ICG) is dispersed in the inner core. The two methods for preparing the nano particles in the prior art both adopt a stirring and mixing mode, so that the preparation efficiency is low, and the time consumption is long. CN201210424026 and CN201210574535 disclose two kinds of nano-drug particles and a preparation method thereof, which combine the advantages of a nano-encapsulation technology, a chemotherapeutic drug and a near-infrared photothermal conversion reagent for thermotherapy, and the two methods solve the technical problems of combined action of thermotherapy and chemotherapy, loading of the near-infrared photothermal conversion reagent and the chemotherapeutic drug at the same time, and slow release. None of the above prior art uses attenuated bacteria for targeted therapy of tumors, and even less related to salmonella typhimurium.
In the prior art, for the nanoprobe labeling technology of bacteria, the nanoprobe and a functional group in the outer membrane protein of the bacteria are generally crosslinked through a chemical reaction, for example, an amide bond is formed through a carbodiimide reaction, but the efficiency and the specificity of the reaction between the nanoprobe and the surface of the bacteria may be reduced by the cross-linking between a carboxyl group and an amino group. When artificial functional groups (such as ketone and azide) are introduced on the surface of the bacterial outer membrane by a crosslinking method like glycoengineering, the time for expressing the specific functional groups is long, even days are needed.
Based on various defects and deficiencies of the prior art, the inventor creatively combines a nano fluorescence labeling technology with the targeted treatment of tumors by engineering bacteria, provides a fluorescence labeling method which is strong in specificity, rapid, simple and convenient, and efficiently and stably labels nano fluorescence probes INPs on the surface of salmonella YB1, thereby realizing the tracking and monitoring of bacteria in the treatment of bacterial tumors, evaluating the treatment effect in real time, providing great convenience for the development and clinical treatment of tumors and the research of related antitumor drugs, and having wide application prospects.
Disclosure of Invention
The invention provides an artificial bacterium, wherein the surface of the artificial bacterium is marked with nano fluorescent probes (INPs), and the INPs are chemically cross-linked through a maleimide group on the surface and a thiol group on the surface of salmonella treated by a reducing agent; the INPs comprise amphoteric compounds, monolayer lipid molecules, near-infrared photothermal conversion reagents and hydrophobic polymers; the amphoteric compound contains a maleimide group; the bacteria are salmonella.
The invention also provides a method for labeling salmonella by using the nano fluorescent probe, which is strong in specificity, rapid, simple and convenient, and comprises the steps of chemically crosslinking a maleimide group on the surface of the nano fluorescent probe (INPs) and a thiol group on the surface of the salmonella treated by a reducing agent, wherein the INPs comprise a hydrophilic outer shell, an intermediate layer and a hydrophobic inner shell, the hydrophilic outer shell contains an amphoteric compound, the intermediate layer contains a single-layer lipid molecule, and the hydrophobic inner shell contains a near-infrared photothermal conversion reagent and a hydrophobic polymer; the amphoteric compound is inserted into the middle layer to form a hydrophobic shell; the near-infrared photothermal conversion reagent is adsorbed on the hydrophobic polymer to form a hydrophobic inner shell.
Preferably, the amphoteric compound is distearoyl phosphatidyl ethanolamine-carboxy polyethylene glycol (DSPE-PEG) or distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleic amide (DSPE-PEG-Mal), preferably DSPE-PEG-Mal; preferably, the monolayer lipid molecules comprises lecithin or cephalin, preferably vegetable lecithin, and more preferably soy lecithin.
Preferably, the outer shell comprises soybean lecithin and DSPE-PEG-Mal in a mass ratio of 2: 3.
preferably, the near-infrared photothermal conversion reagent comprises one or more of indocyanine green (ICG), gold nanorods and carbon nanotubes, preferably ICG; preferably, the hydrophobic polymer is selected from polylactic-co-glycolic acid (PLGA), polylactic acid, polycaprolactone, preferably PLGA.
Preferably, the INPs further comprise chemotherapeutic agents adsorbed to the lipid ends and monolayer lipid molecules of the amphipathic compounds; preferably, the chemotherapeutic drug comprises one or more of adriamycin, epirubicin, paclitaxel, catharanthine and platinum drugs.
Preferably, the salmonella is salmonella YB1 strain; preferably, the reducing agent is tris (2-carboxyethyl) phosphine (TCEP).
The invention also provides a preparation method of INPs, which comprises the following steps:
(1) mixing soybean lecithin ethanol solution and DSPE-PEG-Mal ethanol solution at a ratio of 2: 3, the total weight of the lecithin and the DSPE-PEG-Mal is 15 percent of the total weight of the PLGA;
(2) adding ICG 4% ethanol solution into 2mL 4% ethanol solution;
(3) mixing the solutions in the steps (1) and (2) to obtain a mixed solution, and ultrasonically dispersing the mixed solution for 5 minutes by using an ultrasonic crusher;
(4) in the ultrasonic process of the step (3), dropwise adding a PLGA acetone solution into the mixed solution to form a nano probe dispersion liquid;
(5) and (5) transferring the nano probe dispersion liquid obtained in the step (4) into a 3500KDa dialysis bag for dialysis and concentration to obtain purified INPs.
Preferably, the method for labeling salmonella by using the nano fluorescent probe comprises the following steps:
(1) preparing INPs;
(2) will have a density of 107-108Collecting CFU/mL salmonella in a centrifuge tube, and centrifuging at 3500rpm gravity;
(3) resuspending the Salmonella in a 20-30mM TCEP in PBS and incubating at 37 ℃ for 20-120 min;
(4) the bacteria were washed twice with PBS;
(5) adding 1mL of INPs prepared in the step (1) into the Salmonella obtained in the step (4), and incubating at 37 ℃ for 20-120 minutes;
(6) and (4) centrifuging and washing to obtain the salmonella marked by the nano fluorescent probe.
The invention also provides salmonella marked by the nano fluorescent probe prepared by any one of the methods.
The invention also provides application of the salmonella in preparation or screening of drugs for treating and/or preventing tumors.
The technical principle and the beneficial effects of the invention are as follows:
the invention discloses a salmonella nano fluorescence labeling method with strong specificity, rapidness, convenience and the like, and aims to provide a method for obtaining artificial salmonella with simple preparation process and stable property, and realize effective fluorescence tracing on an artificial salmonella carrier. According to the invention, salmonella YB1 with high safety and strong anaerobic targeting property is selected as a bacterial biological carrier, and a reducing agent tris (2-carboxyethyl) phosphine (TCEP) is adopted to mildly reduce disulfide bonds (S-S) on the outer membrane protein surface of salmonella YB1 into Sulfydryl (SH); then, carrying out chemical crosslinking on salmonella YB1 rich in free-SH and phospholipid-polymer nanoprobes (INPs) loaded with near-infrared fluorescent dye ICG and provided with maleimide groups (Mal) on the surface to construct YB1-INPs with fluorescence imaging capability. The method is rapid, simple, safe and efficient, the nano fluorescent probes INPs are stably marked on the surface of the salmonella YB1, the reduction of the reaction efficiency of the nano probes and the surface of bacteria due to cross-linking can be avoided, and the obtained YB1-INPs can meet the practical requirements of basic research and clinical application on tracking and monitoring bacteria and real-time evaluation of tumor treatment effect.
Drawings
FIG. 1 shows the surface mercapto number (A) of YB1, YB1 and YB1-INPs treated by TCEP, and SEM images (B) of YB1 and YB 1-INPs.
FIG. 2 is a fluorescence spectrum diagram of nano fluorescent probes INPs and nano fluorescence labeled Salmonella YB 1-INPs.
Detailed Description
The present invention will be described in further detail with reference to specific examples below so that those skilled in the art can better understand the present invention and practice the present invention, but the examples are not intended to limit the present invention.
The experimental procedures used in the following examples are conventional unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.
Example 1
INPs were prepared by one-step ultrasound. First, an ethanol solution of soybean lecithin (10mg/mL, 12. mu.L) and an ethanol solution of DSPE-PEG-Mal (10mg/mL, 18. mu.L) were mixed at a ratio of 2: 3 (total weight of lecithin and DSPE-PEG-Mal is 15% of total weight of PLGA), ICG 4% ethanol solution (1mg/mL, 1mL) was added to 2mL 4% ethanol solution. Then, the above mixed solution was ultrasonically dispersed for 5 minutes at a frequency of 20kHz and a power of 130W using a VCX130 ultrasonicator, and a PLGA acetone solution (4mg/mL, 0.5mL) was dropwise added to the above mixed solution during the ultrasonic process to form a nanoprobe dispersion. And finally, transferring the nano probe dispersion liquid into a 3500KDa dialysis bag for dialysis and concentration to obtain purified INPs.
Will have a density of 108CFU/mL of Salmonella YB1 was collected in a centrifuge tube, centrifuged at 3500rpm by gravity, and resuspended in 30mM TCEP in PBS. The bacteria were then incubated at 37 ℃ for 20 minutes and washed twice with PBS. 1mL of INPs was then added to 108CFU/mL bacteria were incubated at 37 ℃ for 2 hours and washed by centrifugation to obtain YB 1-INPs.
Example 2
INPs were prepared by one-step ultrasound. First, an ethanol solution of soybean lecithin (10mg/mL, 12. mu.L) and an ethanol solution of DSPE-PEG-Mal (10mg/mL, 18. mu.L) were mixed at a ratio of 2: 3 (total weight of lecithin and DSPE-PEG-Mal is 15% of total weight of PLGA), ICG 4% ethanol solution (0.8mg/mL, 1mL) was added to 2mL 4% ethanol solution. Then, the above mixed solution was ultrasonically dispersed for 5 minutes at a frequency of 20kHz and a power of 130W using a VCX130 ultrasonicator, and a PLGA acetone solution (4mg/mL, 0.5mL) was dropwise added to the above mixed solution during the ultrasonic process to form a nanoprobe dispersion. And finally, transferring the nano probe dispersion liquid into a 3500KDa dialysis bag for dialysis and concentration to obtain purified INPs.
Will have a density of 107CFU/mL of Salmonella YB1 was collected in a centrifuge tube, centrifuged at 3500rpm by gravity, and resuspended in 20mM TCEP in PBS. The bacteria were then incubated at 37 ℃ for 20 minutes and washed twice with PBS. 1mL of INPs was then added to 107CFU/mL bacteria were incubated at 37 ℃ for 2 hours and washed by centrifugation to obtain YB 1-INPs.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. An artificial bacterium, the surface of which is labeled with nano fluorescent probes (INPs), wherein the INPs are chemically cross-linked through maleimide groups on the surface and thiol groups on the surface of salmonella treated by a reducing agent; the INPs comprise amphoteric compounds, monolayer lipid molecules, near-infrared photothermal conversion reagents and hydrophobic polymers; the amphoteric compound contains a maleimide group; the bacteria are salmonella.
2. A method for labeling salmonella with nano fluorescent probes comprises the steps of chemically crosslinking maleimide groups on the surfaces of nano fluorescent probes (INPs) with thiol groups on the surfaces of salmonella treated by a reducing agent, wherein the INPs comprise a hydrophilic outer shell, an intermediate layer and a hydrophobic inner shell, the hydrophilic outer shell contains an amphoteric compound, the amphoteric compound contains the maleimide groups, the intermediate layer contains a monolayer of lipid molecules, and the hydrophobic inner shell contains a near-infrared photothermal conversion reagent and a hydrophobic polymer; the amphoteric compound is inserted into the middle layer to form a hydrophobic shell; the near-infrared photothermal conversion reagent is adsorbed on the hydrophobic polymer to form a hydrophobic inner shell.
3. The artificial bacterium according to claim 1 or the method according to claim 2, said amphoteric compound being distearoylphosphatidylethanolamine-carboxypolyethylene glycol (DSPE-PEG) or distearoylphosphatidylethanolamine-polyethylene glycol-maleamide (DSPE-PEG-Mal), preferably DSPE-PEG-Mal; the monolayer lipid molecules comprise lecithin or cephalin, preferably vegetable lecithin, and more preferably soybean lecithin.
4. The artificial bacterium of claim 1 or the method of claim 2, wherein the near-infrared photothermal conversion agent comprises any one or more of indocyanine green (ICG), gold nanorods, carbon nanotubes, preferably ICG; the hydrophobic polymer is selected from polylactic-co-glycolic acid (PLGA), polylactic acid, polycaprolactone, preferably PLGA.
5. The artificial bacterium of claim 1 or the method of claim 2, wherein said INPs further comprise chemotherapeutic agents adsorbed to the lipid-terminal and unilamellar lipid molecules of said amphipathic compound, said chemotherapeutic agents comprising one or more of doxorubicin, epirubicin, paclitaxel, catharanthine, and platinoids.
6. The artificial bacterium of claim 1 or the method of claim 2, the salmonella being the salmonella YB1 strain; the reducing agent is tris (2-carboxyethyl) phosphine (TCEP).
7. A method for preparing nanometer fluorescent probes (INPs) comprises the following steps:
(1) mixing soybean lecithin ethanol solution and DSPE-PEG-Mal ethanol solution at a ratio of 2: 3, the total weight of the lecithin and the DSPE-PEG-Mal is 15 percent of the total weight of the PLGA;
(2) adding ICG 4% ethanol solution into 2mL 4% ethanol solution;
(3) mixing the solutions in the steps (1) and (2) to obtain a mixed solution, and ultrasonically dispersing the mixed solution for 5 minutes by using an ultrasonic crusher;
(4) in the ultrasonic process of the step (3), dropwise adding a PLGA acetone solution into the mixed solution to form a nano probe dispersion liquid;
(5) and (5) transferring the nano probe dispersion liquid obtained in the step (4) into a 3500KDa dialysis bag for dialysis and concentration to obtain purified INPs.
8. The method of claim 2, comprising the steps of:
(1) preparing INPs according to the method of claim 8;
(2) will have a density of 107-108Collecting CFU/mL salmonella in a centrifuge tube, and centrifuging at 3500rpm gravity;
(3) resuspending the Salmonella in a 20-30mM TCEP in PBS and incubating at 37 ℃ for 20-120 min;
(4) the bacteria were washed twice with PBS;
(5) adding 1mL of INPs prepared in the step (1) into the Salmonella obtained in the step (4), and incubating at 37 ℃ for 20-120 minutes;
(6) and (4) centrifuging and washing to obtain the salmonella marked by the nano fluorescent probe.
9. The nano fluorescent probe-labeled salmonella prepared according to any one of the methods of claims 2 to 9, which is salmonella YB1 strain.
10. Use of the salmonella labeled with the nano fluorescent probe prepared according to any one of the methods of claims 2 to 9 or the salmonella of claim 9 or the artificial bacterium of claim 1 in preparing or screening a drug for treating and/or preventing tumors.
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| CN103040757A (en) * | 2012-12-26 | 2013-04-17 | 深圳先进技术研究院 | Core-shell drug nano-particles, as well as preparation method and application thereof |
| CN103099784A (en) * | 2012-10-29 | 2013-05-15 | 深圳先进技术研究院 | Nanometer medicine particle, preparation method and application thereof |
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| CN103099784A (en) * | 2012-10-29 | 2013-05-15 | 深圳先进技术研究院 | Nanometer medicine particle, preparation method and application thereof |
| CN103040757A (en) * | 2012-12-26 | 2013-04-17 | 深圳先进技术研究院 | Core-shell drug nano-particles, as well as preparation method and application thereof |
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| Title |
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| FUMING CHEN 等: "Nanophotosensitizer-engineered Salmonella bacteria with hypoxia targeting and photothermal-assisted mutual bioaccumulation for solid tumor therapy", 《BIOMATERIALS》 * |
| 邢婕华 等: "基于迈克尔加成反应的沙门氏菌纳米荧光标记研究", 《化学通报》 * |
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