CN115120737A - Cationic polymer modified phage, preparation method, application and biological medicine - Google Patents
Cationic polymer modified phage, preparation method, application and biological medicine Download PDFInfo
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Abstract
Bacteriophages are widely used as antibacterial agents for the treatment of bacterial infectious diseases due to their specificity and high efficacy in infecting and inhibiting host bacteria. However, the use of bacteriophages to kill intracellular pathogens is largely limited by their inability to enter eukaryotic cells. According to the cationic polymer modified phage, the preparation method, the application and the biological medicine, through electrostatic interaction, the cationic polymer can be selectively coated on the negatively charged head part of the phage instead of the positively charged tail part, so that the charge reversal phage with unaffected vitality can be prepared. Cationic polymers are used to reverse the surface charge of bacteriophages, thereby facilitating their entry into bacteria-infected cells and enabling the killing of bacteria within the cells.
Description
Technical Field
The invention belongs to the technical field of biology and nano-medicine, and particularly relates to a cationic polymer modified phage, a preparation method, application and a biological medicine.
Background
The rapid emergence of bacterial resistance to various antibiotics is the current greatest threat to human health, and research on replacing traditional antibacterial drugs has attracted extensive attention of researchers. Bacteriophages are bacteria-specific viruses that can infect and inhibit host bacteria, and represent an important alternative to current multidrug-resistant pathogen times against antibiotic resistance. Due to its high specificity, acceptable safety, and the ability to overcome antibiotic resistance, the use of bacteriophages as antibacterial therapies has been successfully tested in various phase I and phase II clinical trials. However, the efficacy of a bacteriophage in killing intracellular pathogens is often low due to insufficient entry into eukaryotic cells, and although a bacteriophage can be internalized by macropinocytosis, clathrin-mediated endocytosis, or pit-mediated endocytosis, it is inactive during internalization and fails to perform bactericidal function intracellularly.
Researchers have now developed many sophisticated methods such as surface chemical conjugation and physical encapsulation to enhance the bioavailability of bacteriophages by introducing exogenous functions. For example, it has been reported that attachment of stealth polymers via covalent bonds reduces phage elimination and reduces T-helper type 1 immune responses. Loading onto nanoparticles or liposomes has been used to enhance the resistance of phage to external damage. Unfortunately, physical encapsulation is always affected by release-dependent phage viability, while covalent modification readily interferes with the structure of the phage tail, which plays a key role in binding to host bacterial receptors. More importantly, previous methods are insufficient to promote phage entry into eukaryotic cells and to inhibit host pathogens harbored within their cells.
Disclosure of Invention
In order to solve the technical problems, the invention provides a cationic polymer modified bacteriophage, a preparation method, application and a biological medicine, wherein the cationic polymer is selectively adsorbed on the head of the bacteriophage through electrostatic interaction, and is beneficial to the bacteriophage to enter cells and generate an intracellular antibacterial effect.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a cationic polymer-modified bacteriophage comprising a bacteriophage and a cationic polymer, wherein the head of the bacteriophage has a positive charge and the tail has a negative charge, and the cationic polymer is adsorbed to the head of the bacteriophage.
The surface potential of the modified phage has negative charges reversed to positive charges.
The cationic polymer is linear polyethyleneimine with the molecular weight of 10kDa, branched polyethyleneimine with the molecular weight of 25kDa, chitosan or hyperbranched poly (beta-amino ester) and the like.
The invention also provides a preparation method of the cationic polymer modified phage, which comprises the steps of mixing the aqueous solution of the cationic polymer with the phage suspension, vibrating at the temperature of 20-35 ℃, and then performing ultrafiltration purification to obtain the modified phage.
The concentration of the cationic polymer aqueous solution was 2 mg/mL.
The aqueous solution of the cationic polymer was mixed with the phage suspension in a volume ratio of 10: 1.
Use of the above cationic polymer-modified bacteriophage or the cationic polymer-modified bacteriophage produced by the above production method for the preparation of a biopharmaceutical for the treatment of bacterially infected cells.
A biological medicine comprises the cationic polymer modified phage or the cationic polymer modified phage prepared by the preparation method.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages and positive effects:
the invention utilizes the difference of the surface charges of the head and the tail of the bacteriophage to selectively form a cationic coating on the surface of the head with negative charges through electrostatic interaction. With the help of the coating, the whole surface potential can be reversed to be positively charged, the bacteriophage is promoted to enter the eukaryotic cell, and the remarkable antibacterial effect is shown in the cell.
Drawings
FIG. 1 is a schematic diagram of the ability of the cationic polymer of the present invention to enter cells and kill intracellular bacteria after adsorption on the head of a bacteriophage
FIG. 2a is a TEM image of bare N2 of an embodiment of the present invention;
FIG. 2b is a TEM image of PEI @ P of an embodiment of the present invention;
FIG. 3 is a graph of the particle size of bare N2 and PEI @ P in accordance with an embodiment of the present invention;
FIG. 4 is a Zeta potential of bare N2 and PEI @ P according to an embodiment of the present invention;
FIG. 5 is a typical confocal image of PEI @ P of an embodiment of the present invention with green and red showing FITC labeled PEI and rhodamine B stained phage;
FIG. 6 shows MODE-K cells (5X 10) in example of the present invention 4 ) Respectively exposed to 1X 10 9 Representative confocal images after 2 and 4 hours for naked phage and PEI @ P1; blue and green represent Hoechst stained nuclei and FITC labeled phage, scale bar: 10 mu m;
FIG. 7 shows MODE-K cells (5X 10) in example of the present invention 4 ) At 1X 10 10 Typical confocal images after incubation of (a)1 and (b)2 hours, respectively, with PEI @ P, nuclei and lysosomes were stained with Hoechst and red lysosomal tracer, respectively, scale bar: 10 mu m;
FIG. 8 shows MODE-K cells (5X 10) in example of the present invention 4 ) At 1X 10 9 Flow cytometry histograms of naked N2, PEI @ P after incubation, respectively; blank cells were used as negative controls;
FIG. 9 shows MODE-K cells (5X 10) in example of the present invention 4 ) At 1X 10 9 MFI after 1, 2 and 4 hours incubation of uncoated N2 or PEI @ P, respectively; phage were labeled with FITC and blank cells were used as negative controls;
FIG. 10a shows GFP-SL1344 infected MODE-K cells (5X 10) 4 ) At 1X 10 9 MFI after 2 hours incubation with naked N2 or PEI @ P, respectively;
FIG. 10b is an illustration of GFP-SL13 in an embodiment of the invention44 infected MODE-K cells (5X 10) 4 ) At 1X 10 9 MFI after incubation for 4 hours with naked N2 or PEI @ P, respectively;
FIG. 11 shows GFP-SL 1344-infected MODE-K cells (5X 10) in example of the present invention 4 ) At 1X 10 9 Counting mCherry-SL1344 in MODE-K cells after incubation of naked N2 or PEI @ P for 4 hours respectively;
FIG. 12 shows GFP-SL1344 infected MODE-K cells (5X 10) 4 ) At 1X 10 9 Respectively incubating naked N2 or PEI @ P for 4 hours, and counting naked phages in MODE-K cells;
FIG. 13 is a graph of in vivo inhibition of intestinal intracellular infection by PEI @ P of GFP-SL1344 in mice of the present invention, a is a design of experiments to evaluate in vivo efficacy of PEI @ P in a mouse intestinal infection model, representative IVIS images of b liver, c spleen and d intestinal tissues taken from mice treated with PBS, naked N2 and PEI @ P, respectively;
FIG. 14a is a quantitative statistical data of GFP-SL1344 localization in liver IVIS images of GFP-SL1344 infected mice;
FIG. 14b is a statistical quantification of GFP-SL1344 localization in spleen IVIS images of GFP-SL1344 infected mice;
FIG. 14c is a graph showing the quantitative statistics of GFP fluorescence localization of GFP-SL1344 in small intestine IVIS images in GFP-SL1344 infected mice;
FIG. 15a is a statistical data of plate counts for GFP-SL1344 localization in liver in GFP-SL1344 infected mice;
FIG. 15b is a statistical data of plate counts for GFP-SL1344 localization to the spleen in GFP-SL1344 infected mice;
FIG. 15c is a statistical data of plate counts for GFP-SL1344 localization in the small intestine of GFP-SL1344 infected mice;
FIG. 16 is a representative immunofluorescence image of a liver, b spleen and c small intestine sections co-stained with phalloidin (red), anti-silenced protein UshA (0)/AF594 (green) and DAPI (blue), tissue harvested from SL1344 infected mice after treatment with PBS, N2 and PEI @ P, respectively; scale bar: 60 μm (PBS, N2 and PEI @ P, three sets of which are shown in two columns, the left column being the original photograph and the right column being the enlarged view on the original, and two rows of the three sets a, b and c, viewed transversely, the first row being a merged image of phalloidin (red), anti-silenced protein UshA (0)/AF594 (green) and DAPI (blue), the second row being a green probe image of SL1344, highlighting the distribution of bacteria in the tissue);
FIG. 17 is a plaque count chart of PEI @ P and naked phage N2 cultured with mCherry-SL1344, respectively, in an embodiment of the invention.
Detailed Description
The following description will further describe the cationic polymer modified bacteriophage, its preparation method, application and biological medicine in detail by combining the attached drawings and specific examples. The advantages and features of the present invention will become more apparent from the following description.
According to the invention, the head part of the bacteriophage is provided with positive charges, the tail part of the bacteriophage is provided with negative charges, and the head part and the tail part of the bacteriophage have surface potential difference, so that the cationic polymer is selectively adsorbed on the head part of the bacteriophage through electrostatic interaction, the bacteriophage can keep a complete tail structure, the surface potential of the bacteriophage is reversed from the negative charges to the positive charges, the bacteriophage is facilitated to enter eukaryotic cells and generate intracellular antibacterial action, and as shown in figure 1, the cationic polymer is adsorbed on the head part of the bacteriophage, and then the cells can enter and kill intracellular bacteria. The phage can be modified by the method or the structure of the invention as long as the phage can have a head-tail structure or has a positive charge at the head and a negative charge at the tail, and no matter the phage can be cracked or not cracked, and the adopted cationic polymer can be linear polyethyleneimine with the molecular weight of 10kDa, branched polyethyleneimine with the molecular weight of 25kDa, chitosan, hyperbranched poly (beta-amino ester) or the like.
The following detailed description of the aspects of the invention is given by way of specific examples:
1. preparation of cationic Polymer-modified phage
A specific lytic bacteriophage (named N2) against Salmonella typhimurium (SL1344) was selected from a sewage sample collected from Shanghai local hospitals, and a cationic polymer of Polyethyleneimine (PEI) was selected.
An aqueous polyethyleneimine solution having a concentration of 2mg/mL was mixed with the phage suspension at a volume ratio of 10: 1. After shaking for 30 minutes at room temperature, the mixture was purified by ultrafiltration to remove excess PEI to give the polyethyleneimine modified phage PEI @ P, hereafter PEI @ P is used to represent the polyethyleneimine modified phage.
2. Verification of cationic Polymer adsorption on phage
The microstructure of the naked phage (FIG. 2a), and the microstructure of PEI @ N2 (FIG. 2b) were observed using a transmission electron microscope and the average size of the naked N2 was about 165nm as measured by Dynamic Light Scattering (DLS) and increased to about 210nm after PEI coating (FIG. 3). Accordingly, the zeta potential of bare N2 reversed from-26.5 mV to 24.4mV after PEI coating due to the presence of the positively charged PEI coating (FIG. 4). The resulting PEI @ P was further characterized by Confocal Laser Scanning Microscopy (CLSM). For ease of observation, PEI and N2 were labeled with Fluorescein Isothiocyanate (FITC) and rhodamine B, respectively. Representative confocal images showed complete overlap between the fluorescence signals of FITC and rhodamine B (the left panel in fig. 5 represents the FITC labeled PEI confocal image, the middle panel represents the rhodamine B stained phage confocal image, and the right panel is a typical confocal image of PEI @ P), indicating that PEI was successfully coated onto N2 via electrostatic interaction.
3. Verification of PEI Selective adsorption on N2 header
Stock solutions of naked phage and PEI @ P were dropped onto a copper mesh, respectively, and observed by Transmission Electron Microscopy (TEM) after negative staining with uranyl acetate. As shown in fig. 2a and 2b, typical TEM images show that bare N2 consists of an icosahedral head of about 60nm in diameter and a tail of about 90nm in length. PEI @ P showed a bright, rounded outer ring located only around the N2 head, compared to the sharp edges and corners of the phage head, indicating that the phage had a selective coating. To further demonstrate that PEI was not coated onto the tail of N2, PEI @ P was cultured with mCherry-SL1344(SL1344 is a salmonella number, mCherry is a red fluorescent protein, a plasmid expressing mCherry was introduced into salmonella SL1344, and the bacteria were allowed to glow) and the viability of the coated phage was assessed by plaque counting. The results are shown in figure 17, where PEI @ P exhibited comparable activity to naked N2. Unaffected viability after coating in turn reflects the selective coating of PEI onto the head of N2, since binding requires a complete phage tail structure, a critical step for invading bacteria.
4. Research on the fact that PEI can promote N2 to enter eukaryotic cells
Will be 1 × 10 9 FITC labeled PEI @ P incorporation containing 5X 10 4 MODE-K cells in culture and the mixture incubated at 37 ℃ for a predetermined time point. FITC-labeled N2 was used as a control under the same experimental conditions. As shown in FIG. 6, cellular internalization of uncoated phage could not be detected even with an extended incubation time of 4 hours, indicating inefficient entry of N2 into MODE-K cells. In contrast, PEI @ P only observed strong fluorescent signals around each cell after 1 hour incubation. We speculate that the charge-reversing phage activated by the PEI coating enhanced the interaction of N2 with the negatively charged surface of MODE-K cells. As the culture time increased to 2 hours, a visible fluorescent signal was found within the cultured cells. After 4 hours of culture, the green fluorescence was well distributed in these cells, which means that PEI @ P was sufficient for entering MODE-K cells. By lysosomal tracking dyes we also found that PEI @ P is able to escape from lysosomes, especially in case the incubation time is extended to 2 hours (fig. 7), which can be explained by the proton sponge mechanism of PEI. These results demonstrate that PEI coating not only enables N2 to enter cells, but also confers its ability to escape from lysosomes.
The incubated MODE-K cells were analyzed by flow cytometry. In addition to cells incubated with naked N2, blank cells were also used as controls. In close agreement with the results of confocal imaging, MODE-K cells showed a similar flow cytometry histogram to the blank cells after incubation with naked N2, indicating limited cellular uptake (fig. 8). The inability of N2 to enter the cells was further confirmed by the lack of statistical difference between the Mean Fluorescence Intensity (MFI) of the blank and N2-treated cells (fig. 9). The shift of the flow cytometry histograms to higher fluorescence intensity is expected to increase with the incubation time of PEI @ P. The MFI values of PEI @ P treated cells were significantly higher than those of N2 treated cells, respectively. MFI values after 1(p <0.05), 2(p <0.0001) and 4 (p <0.01) hours of incubation are shown in fig. 9. In sharp contrast to uncoated N2, the apparent fluorescence in PEI @ P treated MODE-K cells indicates that PEI coating may facilitate cellular entry of N2.
5. Verification of the Sterilization Effect of PEI @ P
To verify the bactericidal effect of PEI @ P, the amount of mCherry-SL1344 remaining in MODE-K cells after treatment was determined by bacterial count. MODE-K cells not infected with SL1344 (Salmonella typhimurium) and infected MODE-K cells treated with PBS were used as negative and positive controls, respectively. GFP-SL1344(GFP Green fluorescent protein) -infected MODE-K cells (5X 10) 4 ) At 1X 10 9 The MFI after 2 hours (fig. 10a) and 4 hours (fig. 10b) incubation with naked N2 or PEI @ P, respectively, it can be seen that the MFI after incubation with PEI @ P is lower than that of naked N2, indicating that PEI @ P can kill pathogens within infected cells. FIG. 11 is a count of intracellular mCherry-SL1344 after 4 hours of treatment and shows that PEI @ P kills approximately 85% of the pathogens in MODE-K cells compared to naked N2 which has no inhibitory effect on intracellular mCherry-SL 1344. FIG. 12 is a count of intracellular N2 after 4 hours of treatment, and inhibition of intracellular mCherry-SL1344 by PEI @ P is further supported by plaque count results, with a colony inhibition effect 44-fold higher than that of naked N2. Indicating that PEI @ P has excellent antibacterial efficacy.
6. Verification of bactericidal ability of PEI @ P to enter infected cells in organisms
Firstly, 3X 10 is filled by an oral intragastric administration method 7 GFP-SL1344 from CFUs was given to mice to establish mouse animal models of Salmonella typhimurium intracellular infection to verify the inhibitory effect of PEI @ P on Salmonella intracellularis in the gut.
Then using PBS, 1X 10 11 PFUs N2 and equal amounts of PEI @ P were orally administered to mice and at designated time points, bacterial distributions in organs of gut, liver and spleen were collected and the fluorescence of GFP-SL1344 was quantified using an In Vivo Imaging System (IVIS), the number of GFP-1344 plates counted and immunofluorescence imaged in the organsBacterial distribution and relative fluorescence intensity to assess the invasion and persistence of GFP-SL1344 in each organ. As shown in FIG. 13, IVIS images and quantitative analysis showed that GFP-SL1344 had the weakest fluorescence signal in these organs sampled from mice treated with PEI @ P, indicating a significant increase in PEI @ P inhibitory efficacy (FIGS. 13b-d and 14). The results show that oral administration of naked N2 attenuates the GFP signal negligibly compared to PBS, reflecting the ineffectiveness of using naked phage. Bacterial counts further confirmed the minimal retention of GFP-SL1344 in PEI @ P treated mice (figure 15). The inhibition of GFP-SL1344 by PEI @ P was increased by 3.6, 9.0 and 19.7 fold in the small intestine, liver and spleen, respectively, compared to nude N2 treated mice.
GFP-SL1344 is reported to be harbored in intestinal epithelial cells and eventually spread to major organs such as the liver and spleen through mesenteric lymph nodes. Thus, the greatly enhanced efficacy of PEI @ P may be explained by their ability to enter intestinal epithelial cells and subsequently inhibit contained GFP-SL1344, which reduces GFP-SL1344 translocation to major organs. Furthermore, PEI @ P may be able to penetrate the intestinal mucus layer and intestinal vascular barrier, eliminating SL1344 transferred into cells of these organs after translocation to the liver and spleen.
To further elucidate the inhibitory efficacy of PEI @ P enhancement on salmonellae intracellulare, tissue sections were paraffin-embedded from small intestine, liver and spleen for immunofluorescence staining. Sections were stained for cytoskeleton, nuclei and GFP-SL1344 with rhodamine-labeled phalloidin, 4', 6-diamidino-2-phenylindole (DAPI) and the anti-silenced protein UshA (0)/AF594, a primary antibody specific for salmonella O antigen, and the corresponding FITC-labeled secondary antibody. As shown in FIG. 16, the immunofluorescence image directly shows the minimal fluorescence signal of intracellular GFP-SL1344 in each organ section of PEI @ P treated mice, in sharp contrast to PBS and naked N2 treated mice that exhibited severe infection signals in the corresponding tissues.
In conclusion, the cationic polymer is adsorbed on the head of the phage, so that the surface charge can be reversed, the phage can be promoted to enter eukaryotic cells, and the activity of the phage invading host bacteria can be maintained. Therefore, the bacteriophage modified by the cationic polymer can be prepared into a biological medicine which can inhibit the activity of bacteria.
The embodiments of the present invention have been described in detail with reference to the drawings, but the present invention is not limited to the embodiments. Even if various changes are made to the present invention, it is still within the scope of the present invention if they fall within the scope of the claims of the present invention and their equivalents.
Claims (8)
1. A cationic polymer-modified bacteriophage comprising a bacteriophage and a cationic polymer, wherein the head of said bacteriophage has a positive charge and the tail of said bacteriophage has a negative charge, and said cationic polymer is adsorbed to the head of said bacteriophage.
2. The cationic polymer-modified bacteriophage of claim 1, wherein the surface potential of said modified bacteriophage reverses a negative charge to a positive charge.
3. The cationic polymer-modified bacteriophage of claim 1, wherein said cationic polymer is a linear polyethyleneimine, a branched polyethyleneimine, a chitosan, or a hyperbranched poly (β -amino ester) having a molecular weight.
4. A method for preparing a cationic polymer-modified phage according to any of claims 1-3, wherein an aqueous solution of a cationic polymer is mixed with a phage suspension, purified by ultrafiltration after shaking at a temperature of 20-35 ℃ to obtain the modified phage.
5. The method for producing a cationic polymer-modified phage according to claim 4, wherein the concentration of the aqueous cationic polymer solution is 2 mg/mL.
6. The method for producing a cationic polymer-modified phage according to claim 5, wherein the aqueous solution of the cationic polymer is mixed with the phage suspension at a volume ratio of 10: 1.
7. Use of a cationic polymer-modified bacteriophage according to any one of claims 1 to 3 or prepared by the preparation method according to any one of claims 4 to 6 for the preparation of a biopharmaceutical for the treatment of a bacterially infected cell.
8. A biopharmaceutical comprising the cationic polymer-modified bacteriophage of any one of claims 1 to 3 or produced by the production method of any one of claims 4 to 6.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003077953A2 (en) * | 2002-03-12 | 2003-09-25 | Phage Genomics Inc. | Bacteriophage composition |
| CN105793428A (en) * | 2013-05-15 | 2016-07-20 | 皇家创新有限公司 | Bacteriophage |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003077953A2 (en) * | 2002-03-12 | 2003-09-25 | Phage Genomics Inc. | Bacteriophage composition |
| CN105793428A (en) * | 2013-05-15 | 2016-07-20 | 皇家创新有限公司 | Bacteriophage |
Non-Patent Citations (1)
| Title |
|---|
| 王吉 等: "基于噬菌体的电化学生物传感器在检测食源性病原菌中的研究进展", 食品科学, vol. 43, no. 7, pages 254 - 262 * |
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