Size-controllable bacteriophage hydrogel microspheres and preparation method thereof

Abstract

This invention belongs to the field of biopharmaceutical technology and specifically discloses size-controlled bacteriophage hydrogel microspheres and their preparation method. The hydrogel microspheres are composed of bacteriophage, a polymer carrier, and a curing agent and are prepared using electrostatic spray technology. Compared with existing hydrogel microsphere preparation methods, these hydrogel microspheres can significantly prolong the sustained release time of bacteriophage in the intestinal environment and improve their stability in the gastric environment. Furthermore, by adjusting the preparation parameters, the size of the microspheres can be flexibly controlled to meet different application scenarios and needs.

Description

Technical Field

[0001] The present invention belongs to the field of biological preparations, and in particular relates to size-controllable bacteriophage hydrogel microspheres and a preparation method thereof. Background Art

[0002] The intestine is the body's primary site of digestion and absorption. It constantly interacts with the complex external environment and is highly susceptible to attack by pathogens, leading to intestinal infections. Currently, antibiotic therapy is the preferred treatment for intestinal bacterial infections. However, the number of antibiotic-resistant bacteria continues to soar worldwide, posing a serious threat to global public health. The development of new antimicrobial drugs is time-consuming and costly. Therefore, new alternative antimicrobial approaches are urgently needed.

[0003] As one of the oldest organisms on Earth, bacteriophages can specifically infect and lyse host bacteria. Bacteriophages have the characteristics of high host specificity, rapid proliferation, abundant resources, and easy modification. Therefore, bacteriophages can be used as an effective tool to address the crisis of bacterial resistance in the "post-antibiotic era." However, bacteriophages are extremely sensitive to low pH conditions and are easily destroyed by the gastric environment, resulting in complete inactivation of the phages. Microencapsulation technology, with the barrier and shielding effect of the wall material, can significantly improve the activity of sensitive substances in the stomach and achieve controlled release of encapsulated substances in the intestine through degradation of the wall material. Therefore, phage microencapsulation can be used as an effective strategy to enhance the survival of phages in the stomach, slowly release the encapsulated phages in the intestine, increase the coverage area of ​​phages, effectively lyse pathogens, and treat intestinal infections.

[0004] In the prior art, patent application number 202111069080.2 discloses a method for preparing cationic etherified starch / sodium alginate / xanthan gum / chitosan oligosaccharide / nano-TiO2 Salmonella phage microencapsulation microspheres. The prepared microspheres improve the acid stability of the phage in simulated gastric fluid and extend the release time of the phage in simulated intestinal fluid to 4 hours (h).

[0005] For example, patent application number 201110404475.3 discloses a sodium alginate / whey protein microsphere formulation for encapsulating Salmonella phages. This formulation protects phage activity in the gastric environment and extends phage release time to 3 hours in simulated intestinal fluid. Patent application number 202210109619.0 discloses phage microcapsules composed of sodium alginate / gelatin / carrageenan / maltodextrin / chitosan / hydroxymethylcellulose. These microcapsules exhibit excellent acid resistance and sustained release, releasing 79.33% of Salmonella phages within 2 hours in simulated intestinal fluid.

[0006] However, the above-mentioned existing technologies still have problems: 1. The release time of phage in phage microcapsules is short, resulting in a small treatment area of ​​phage microcapsules; 2. The size of currently available phage microcapsule preparations is relatively single and cannot be adjusted, which cannot flexibly meet different application scenarios and needs. Therefore, it is urgent to develop a simple, flexible, and effective method to prepare microencapsulated preparations for phage encapsulation, so as to regulate the size of phage microcapsules and achieve slow and sustained release of phage microcapsules, so as to better promote their elimination of drug-resistant bacteria in the intestine, reduce the colonization of pathogens, and promote intestinal and body health. Summary of the Invention

[0007] In response to the above-mentioned problems with the current preparation of phage microspheres, the present invention provides a size-controllable phage hydrogel microsphere and a preparation method thereof. The hydrogel microspheres are composed of phage, a polymer carrier, and a curing agent. By using electrostatic spraying technology, compared with the existing hydrogel microsphere preparation method, the sustained release time of phage in the intestinal environment can be significantly prolonged and its stability in the gastric environment can be improved. At the same time, by changing the solution concentration, the size of the microspheres can be flexibly controlled to meet different application scenarios and needs. The specific technical solution is as follows:

[0008] First, the present invention provides a size-controllable phage hydrogel microsphere, the preparation raw materials of which include phage, a polymer carrier and a curing agent; the polymer carrier includes sodium alginate, sodium hyaluronate and Eudragit S100.

[0009] Furthermore, the phage titer is 10 5 ~10 12 PFU / g.

[0010] Furthermore, the curing agent is a calcium chloride solution, and the concentration of the calcium chloride solution is 0.01 to 1 mol / L.

[0011] Furthermore, the particle size of the phage hydrogel microspheres is 100 to 900 μm; and the release time of the phage hydrogel microspheres is 3 to 8 hours.

[0012] The present invention also provides a method for preparing size-controllable phage hydrogel microspheres, comprising the following steps:

[0013] S1: preparing phage fluid: mixing phage with its host bacterial fluid, culturing to obtain phage proliferation fluid, then centrifuging and filtering the phage proliferation fluid and collecting the supernatant to obtain phage fluid;

[0014] S2: Prepare carrier solution: weigh sodium alginate, sodium hyaluronate, and Eudragit S100, stir and dissolve to obtain carrier solution, and store for later use;

[0015] S3: preparing a precursor solution: mixing the phage solution and the carrier solution, stirring them evenly to obtain a precursor solution;

[0016] S4 electrostatic atomization: The precursor solution is atomized using an electrostatic spray platform, and the solidifying agent is used to absorb, solidify and cross-link to obtain phage-release hydrogel microspheres.

[0017] Furthermore, in step S1, the shaking culture temperature is 20-37° C., the culture time is 12-96 h, the centrifugal speed is 8000-15000 rpm, and the centrifugation time is 5-30 min.

[0018] Furthermore, in step S2, the mass ratio of sodium alginate, sodium hyaluronate and Eudragit S100 is 1-7:1-7:1-7; and the stirring time is 12-24 hours.

[0019] Furthermore, the total mass concentration of sodium alginate, sodium hyaluronate and Eudragit S100 in the carrier solution of step S2 is 1% to 5%, and the storage temperature is 0 to 4°C.

[0020] Furthermore, in the step S3, the volume ratio of the phage solution to the carrier solution is 1:9 to 7:3; the stirring speed is 100 to 200 rpm; and the stirring time is 5 to 30 min.

[0021] Furthermore, the electrostatic spray platform in step S4 includes a syringe pump, an electrostatic sprayer and a receiver; the syringe pump is connected to the inlet of the electrostatic sprayer, and the receiver is located below the outlet of the electrostatic sprayer; the injection speed of the syringe pump in step S4 is 1 to 2 mL / h.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The phage sustained-release hydrogel microspheres prepared in the present invention use safe, non-toxic, biocompatible and biodegradable sodium alginate, sodium hyaluronate and Eudragit S100 as carrier materials. The prepared hydrogel microspheres can be safely applied to organisms and can be used for oral delivery.

[0024] 2) Compared to existing methods that utilize electrostatic spraying during the molding process, the phage-derived hydrogel microspheres prepared by this invention exhibit highly uniform particle size and can produce microspheres with diameters as low as 100 μm, enabling the application of phage microcapsules in a wider range of biotherapeutic applications. Furthermore, by adjusting the concentration of the carrier solution during preparation, microspheres of varying sizes can be prepared, with flexibility ranging from 100 μm to 900 μm. This addresses the current limitations of controllable microsphere preparation technology. This method has the potential for scalable production and can address diverse application scenarios.

[0025] 3) This invention utilizes electrostatic spray technology, combined with a defined curing agent concentration and injection spray speed, to ensure an encapsulation efficiency of approximately 90% in the prepared microspheres, thereby improving the utilization efficiency and therapeutic efficacy of the phage raw material. Furthermore, the preparation process does not involve the use of organic reagents or heat treatment, fully preserving the biological activity of the phage.

[0026] 4) The present invention utilizes electrostatic spraying technology combined with a limited carrier concentration to increase the surface thickness and internal crosslinking density of the prepared phage-release hydrogel microspheres, ensuring their biostability in the gastric environment and protecting them from damage and degradation by gastric acid and pepsin. This allows for controlled, slow, and sustained release of phage in the intestinal tract, with a release time of up to eight hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of a size-controllable bacteriophage hydrogel microsphere and its preparation method of the present invention;

[0028] Figure 2 This is a schematic diagram of a size-controllable bacteriophage hydrogel microsphere and a preparation method thereof of the present invention;

[0029] Figure 3 This is an optical microscope photograph of the phage sustained-release hydrogel microspheres in Example 1 of the present invention;

[0030] Figure 4 This is an optical microscope photograph of the phage sustained-release hydrogel microspheres in Example 2 of the present invention;

[0031] Figure 5 This is an optical microscope photograph of the phage sustained-release hydrogel microspheres in Example 3 of the present invention;

[0032] Figure 6 This is an optical microscope photograph of the phage sustained-release hydrogel microspheres in Comparative Example 5 of the present invention.

[0033] Description of the drawings: syringe pump 1, electrostatic sprayer 2, receiver 3. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with embodiments.

[0035] Example 1

[0036] Basically Figure 1-Figure 3 As shown, this embodiment provides a size-controllable phage hydrogel microsphere and a preparation method thereof, comprising the following steps:

[0037] S1 Preparation of phage liquid: This example uses Salmonella phage LPST94 and LPST153 as representatives for the preparation of phage liquid. The method for preparing phage hydrogel microspheres provided by the present invention is a universal technology that can be used for the preparation of various types of phage hydrogel microspheres, and is not limited to Salmonella phage. The specific steps are as follows: pick single colonies from the LA plates of Salmonella Typhimurium UK-1 and Salmonella Typhimurium ATCC13311, inoculate them into LB liquid culture medium, and culture them with shaking at 37°C until the logarithmic growth phase (OD is about 0.7). Take 100 μL of the above bacterial solution and mix it with 100 μL of phage storage solution of phage LPST94 and LPST153 (titer is 10 8 PFU / mL) were mixed, and then 20 mL of LB liquid medium was added and cultured at 37°C with shaking for 12 h to obtain a phage lysate. Subsequently, the supernatant was collected by centrifugation at 4°C (10,000 rpm, 10 min) and filtration through a 0.22 μm membrane to obtain a phage solution.

[0038] S2. Prepare carrier solution: Weigh a certain amount of sodium alginate, sodium hyaluronate, and Eudragit S100, respectively, with a mass ratio of 1-7:1-7:1-7. Dissolve sodium alginate, sodium hyaluronate, and Eudragit S100 in sterile water to a total concentration of 3% (w / v). Stir magnetically for 12 hours to completely dissolve. Store at 4°C until ready to use.

[0039] S3: Preparing a precursor solution: The phage solution obtained in step S1 and the carrier solution obtained in step S2 are mixed at a ratio of 1:9 to obtain a phage / hydrogel microsphere precursor solution.

[0040] S4 electrostatic atomization: The syringe pump (1), electrostatic sprayer (2) and receiver (3) are assembled, the outlet of the syringe pump (1) is connected to the inlet of the electrostatic sprayer (2) through a hose, and the receiver (3) is placed below the outlet of the electrostatic sprayer (2). The precursor solution is added to the syringe pump (1), and then 0.01-1 mol / L calcium chloride solution is added to the receiver (3) as a solidifying agent. The syringe pump (1) injects the precursor solution into the electrostatic sprayer (2) at a rate of 1-2 mL / h. The electrostatic sprayer (2) atomizes the precursor solution into tiny droplets. The calcium chloride solution receives and solidifies the atomized droplets to obtain sodium alginate / sodium hyaluronate / Eudra S100 sustained-release hydrogel microspheres loaded with bacteriophage.

[0041] Example 2

[0042] The difference between this embodiment and embodiment 1 is that the total concentration of sodium alginate, sodium hyaluronate and Eudragit S100 in the carrier solution is 5%, and the other raw materials, preparation methods and parameters are the same as those in embodiment 1. Figure 4 shown.

[0043] Example 3

[0044] The difference between this embodiment and embodiment 1 is that the total concentration of sodium alginate, sodium hyaluronate and Eudragit S100 in the carrier solution is 1%, and the other raw materials, preparation methods and parameters are the same as those in embodiment 1. Figure 5 shown.

[0045] Example 4

[0046] The difference between this embodiment and embodiment 1 is that the volume ratio of the phage solution to the carrier solution in the precursor solution in step S3 is 5:5, and other raw materials, preparation methods, and parameters are consistent with those in embodiment 1.

[0047] Example 5

[0048] The difference between this embodiment and embodiment 1 is that the volume ratio of the phage solution to the carrier solution in the precursor solution in step S3 is 7:3, and other raw materials, preparation methods, and parameters are the same as those in embodiment 1.

[0049] Comparative Example 1

[0050] The difference between this comparative example and Example 1 is that the total concentration of sodium alginate, sodium hyaluronate and Eudragit S100 in the carrier solution is 6%, and other raw materials, preparation methods and parameters are the same as those in Example 1.

[0051] Comparative Example 2

[0052] The difference between this comparative example and Example 1 is that the total concentration of sodium alginate, sodium hyaluronate and Eudragit S100 in the carrier solution is 0.5%, and other raw materials, preparation methods and parameters are the same as those in Example 1.

[0053] Comparative Example 3

[0054] The difference between this comparative example and Example 1 is that the volume ratio of the phage solution to the carrier solution in the precursor solution in step S3 is 8:2, and other raw materials, preparation methods, and parameters are the same as those in Example 1.

[0055] Comparative Example 4

[0056] The difference between this comparative example and Example 1 is that the volume ratio of the phage solution to the carrier solution in the precursor solution is 9:1, and other raw materials, preparation methods, and parameters are the same as those in Example 1.

[0057] Comparative Example 5

[0058] The difference between this comparative example and Example 1 is that the preparation method is different. In this comparative example, injection preparation is adopted in step S4, and the specific preparation steps are as follows: the precursor solution is added to the injection pump (1), and 0.01-1 mol / L calcium chloride solution is added to the receiver (3) as a curing agent. The injection pump (1) drips the precursor solution into the calcium chloride solution at a rate of 1-2 mL / h for curing and cross-linking, and finally obtains sodium alginate / sodium hyaluronate / Eutrac S100 sustained-release hydrogel microspheres loaded with bacteriophages. The image of the bacteriophage sustained-release hydrogel microspheres is shown in FIG. Figure 6 shown.

[0059] Experimental Example 1: Determination of embedding efficiency

[0060] This experimental example is to test the encapsulation efficiency of the phage sustained-release hydrogel microspheres prepared in the above examples and comparative examples.

[0061] Experimental methods:

[0062] 100 mg of hydrogel microspheres prepared in the above examples and comparative examples were weighed and added to 900 μL of microsphere disruption solution. The solution was incubated at 37° C. for 4 h to dissolve the microspheres. The phage content in the microsphere disruption solution was then determined, and the phage entrapment rate in the hydrogel microspheres was calculated.

[0063] The phage embedding efficiency was calculated according to the following formula:

[0064]

[0065] The experimental results are shown in Table 1 below:

[0066] Table 1: Encapsulation efficiency of phage sustained-release hydrogel microspheres in Examples and Comparative Examples

[0067]

[0068] Experimental results analysis:

[0069] As shown in Table 1, the results of the encapsulation efficiency show that the encapsulation efficiency of the hydrogel microspheres in different examples and comparative examples varies. The phage encapsulation efficiencies of Example 1 (3% polymer concentration, phage solution and carrier solution volume ratio of 1:9), Example 2 (5% polymer concentration, phage solution and carrier solution volume ratio of 1:9), Example 3 (1% polymer concentration, phage solution and carrier solution volume ratio of 1:9), Example 4 (3% polymer concentration, phage solution and carrier solution volume ratio of 5:5), and Example 5 (3% polymer concentration, phage solution and carrier solution volume ratio of 7:3) were 91.61%, 92.31%, 86.97%, 90.22%, and 88.57%, respectively. Overall, the encapsulation efficiency was close to 90%, indicating that electrostatic spraying is an efficient phage encapsulation method.

[0070] The phage encapsulation efficiency of Comparative Example 1 (6% polymer concentration, phage solution to carrier solution volume ratio of 1:9) was 85.91%. Due to the low polymer concentration in Comparative Example 2 (0.5% polymer concentration), hydrogel microspheres could not be formed. Furthermore, the proportion of carrier solution in Comparative Example 3 (3% polymer concentration, phage solution to carrier solution volume ratio of 8:2) and Comparative Example 4 (3% polymer concentration, phage solution to carrier solution volume ratio of 9:1) was too low, and hydrogel microspheres could not be formed. The phage encapsulation efficiency of Comparative Example 5 (3% polymer concentration, injection extrusion preparation) was 88.54%, indicating a decrease in encapsulation efficiency.

[0071] Experimental Example 2: Release rate determination

[0072] This experimental example is to determine the release rate and action time of the phage sustained-release hydrogel microspheres prepared in the above examples and comparative examples.

[0073] Experimental methods:

[0074] 100 mg of the phage sustained-release hydrogel microspheres prepared in the above examples and comparative examples were weighed and added to 900 μL of simulated gastric fluid. After incubation for 2 hours, the simulated gastric fluid was removed. Next, 900 μL of simulated intestinal fluid was added. After incubation with shaking for a specified period of time (1, 2, 3, 4, 5, 6, 8, 10, or 12 hours), the phage titer was determined.

[0075] The experimental results are shown in Table 2 below:

[0076]

[0077] Experimental results analysis:

[0078] As shown in Table 2, the release rate results indicate that the phage release profiles of the hydrogel microspheres in different examples and comparative examples vary. The phage release times for Example 1 (3% polymer concentration, phage solution to carrier solution volume ratio of 1:9), Example 2 (5% polymer concentration, phage solution to carrier solution volume ratio of 1:9), Example 3 (1% polymer concentration, phage solution to carrier solution volume ratio of 1:9), Example 4 (3% polymer concentration, phage solution to carrier solution volume ratio of 5:5), and Example 5 (3% polymer concentration, phage solution to carrier solution volume ratio of 7:3) were 5 hours, 8 hours, 3 hours, 4 hours, and 3 hours, respectively. The phage release time for Comparative Example 1 (6% polymer concentration, phage solution to carrier solution volume ratio of 1:9) was 8 hours. Due to the low polymer concentration in Comparative Example 2 (0.5% polymer concentration), hydrogel microspheres could not be formed. However, the carrier solution ratio in Comparative Example 3 (3% polymer concentration, 8:2 volume ratio of phage solution to carrier solution) and Comparative Example 4 (3% polymer concentration, 9:1 volume ratio of phage solution to carrier solution) was too low, and hydrogel microspheres could not be formed. The phage release time in Comparative Example 5 (3% polymer concentration, injection extrusion preparation) was 5 hours.

[0079] Experimental Example 3: Microsphere Particle Size Measurement

[0080] This experimental example is to determine the average particle size of the phage sustained-release hydrogel microspheres prepared in the above examples and comparative examples.

[0081] Experimental methods:

[0082] The morphology of the hydrogel microspheres was observed and photographed using an optical microscope, and a certain number of microspheres were randomly selected from the microsphere images to measure their sizes and calculate the average particle size using ImageJ software.

[0083]

[0084] The experimental results are shown in Table 3 below:

[0085]

[0086]

[0087] Analysis of experimental results:

[0088] As shown in Table 3, the microsphere particle size results show that the microsphere particle sizes of Example 1 (3% polymer concentration, phage solution and carrier solution volume ratio of 1:9), Example 2 (5% polymer concentration, phage solution and carrier solution volume ratio of 1:9), Example 3 (1% polymer concentration, phage solution and carrier solution volume ratio of 1:9), Example 4 (3% polymer concentration, phage solution and carrier solution volume ratio of 5:5), and Example 5 (3% polymer concentration, phage solution and carrier solution volume ratio of 7:3) are 687.98 μm, 883.77 μm, 133.04 μm, 224.90 μm, and 143.46 μm, respectively. The size of the phage microspheres prepared in the present invention can be controlled within the range of 133.04 μm to 883.77 μm by adjusting the range conditions, and the minimum size can reach 133.04 μm, which can meet the needs of more biological treatments and solve the current technical problem of uncontrollable phage microsphere size.

[0089] Compared with Example 1, Comparative Example 1 changed the total concentration of the total polymer in the carrier solution (6% polymer concentration), and the average particle size of the prepared microspheres increased to 890.71 μm, but there was no significant difference from Example 2. Since the polymer concentration of Comparative Example 2 (0.5% polymer concentration) was too low, hydrogel microspheres could not be formed. The proportion of carrier solution in Comparative Example 3 (3% polymer concentration, 8:2 volume ratio of phage solution and carrier solution) and Comparative Example 4 (3% polymer concentration, 9:1 volume ratio of phage solution and carrier solution) was too low, and hydrogel microspheres could not be formed. Compared with Example 1, Comparative Example 5 was prepared by extrusion, and its microspheres had an average particle size of 2371.22 μm, which was significantly increased in particle size. However, the excessive size limited its clinical and preclinical applications to a certain extent.

[0090] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be viewed as exemplary and non-restrictive in all respects. Furthermore, it should be understood that although this specification is described in terms of implementation methods, it does not encompass only one technical solution. This narrative is provided for clarity only, and those skilled in the art should consider the specification as a whole. The technical solutions in the embodiments may also be appropriately combined to form other implementation methods that are understandable to those skilled in the art.

Claims

1. A size-controllable bacteriophage hydrogel microsphere, characterized in that: The preparation raw materials include bacteriophage, polymer carrier and curing agent; the polymer carrier includes sodium alginate, sodium hyaluronate and Eudragit S100; the particle size of the bacteriophage hydrogel microspheres is 100-900 μm; and the release time of the bacteriophage hydrogel microspheres is 3-8 hours.

2. The size-controllable bacteriophage hydrogel microspheres according to claim 1, characterized in that: The phage titer is 10 5 ~10 12 PFU / g.

3. The size-controllable bacteriophage hydrogel microspheres according to claim 1, characterized in that: The curing agent is a calcium chloride solution, and the concentration of the calcium chloride solution is 0.01-1 mol / L.

4. The method for preparing size-controllable phage hydrogel microspheres according to any one of claims 1 to 3, wherein: The following steps are involved: S1: preparing phage fluid: mixing phage with its host bacterial fluid, culturing to obtain phage proliferation fluid, then centrifuging and filtering the phage proliferation fluid and collecting the supernatant to obtain phage fluid; S2 Preparation of carrier solution: Weigh sodium alginate, sodium hyaluronate and Eudragit S100, stir and dissolve to obtain carrier solution. For storage and standby use, the mass ratio of sodium alginate, sodium hyaluronate and Eudragit S100 is 1-7:1-7:1-7; the stirring time is 12-24 hours; the total mass concentration of sodium alginate, sodium hyaluronate and Eudragit S100 in the carrier solution is 1%-5%, and the storage temperature is 0-4°C; S3: Preparing a precursor solution: Mixing the phage solution and the carrier solution in proportion, stirring evenly to obtain a precursor solution, wherein the volume ratio of the phage solution to the carrier solution in the precursor solution is 1:9 to 7:3; stirring at a speed of 100 to 200 rpm; and stirring for 5 to 30 minutes; S4 electrostatic atomization: The precursor solution is atomized using an electrostatic spray platform, and the curing agent is used to receive, solidify and cross-link to obtain phage hydrogel microspheres.

5. The method for preparing size-controllable phage hydrogel microspheres according to claim 4, characterized in that: In step S1, the culture temperature is 20-37°C and the culture time is 12-96 hours; The centrifugal speed is 8000-15000 rpm, and the centrifugal time is 5-30 minutes.

6. The method for preparing size-controllable phage hydrogel microspheres according to claim 4, characterized in that: The electrostatic spray platform in step S4 includes a syringe pump, an electrostatic sprayer and a receiver; the outlet of the syringe pump is connected to the inlet of the electrostatic sprayer, and the receiver is located below the outlet of the electrostatic sprayer; the injection speed of the syringe pump is 1 to 2 mL / h.

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