Phage nano-particles for targeted ablation of fat cells as well as preparation method and application of phage nano-particles

Abstract

The invention relates to the technical field of biology, and particularly discloses a bacteriophage nano-particle for targeted ablation of adipocytes and a preparation method and application thereof, the bacteriophage nano-particle comprises an M13 bacteriophage modified by genetic engineering, and an adipocyte targeted peptide ATS is displayed on the surface of the bacteriophage nano-particle; the functional molecule curcumin is entrapped in the phage nanoparticles; according to the invention, functional molecule curcumin is carried by a bacteriophage nano-material, so that efficient killing is realized at the local part of fat cells, and redundant fat cells in the body can be accurately, efficiently, safely and minimally invasively eliminated, so that various pain points of the existing obesity treatment means are solved; the treatment scheme is relatively simple and convenient to operate, the dependence on the technology of doctors is lower than that of complex liposuction surgery, and clinical popularization and application are easier.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a bacteriophage nanoparticle for targeted ablation of adipocytes and a preparation method and application thereof. BACKGROUND

[0002] Obesity has become a major global public health challenge, and is a key risk factor for inducing cardiovascular diseases, type II diabetes, metabolic syndrome, and various cancers. At present, the treatment methods for obesity or local fat accumulation mainly include lifestyle intervention, drug treatment, physical therapy, and surgery. However, these existing means have their own limitations, and it is difficult to achieve an ideal balance between effectiveness, specificity, safety, and minimally invasive.

[0003] Specifically, at present, the main methods for reducing fat mainly include:

[0004] 1. Physical method (such as freezing, radio frequency, ultrasonic wave): destroying fat through physical energy such as cold and heat. However, the targeting is poor, and the surrounding normal tissues are easily damaged, leading to pain, uneven effect, and the need for multiple treatments.

[0005] 2. Surgical method (such as liposuction, injection of fat-dissolving): directly removing or chemically dissolving fat. However, it is traumatic, slow to recover, and has risks of infection, bleeding, and uneven contour, and the effect is highly dependent on the skill of the doctor.

[0006] The common defect of these methods is that they cannot accurately, efficiently, and safely target adipocytes, and it is difficult to balance between effect and safety. In summary, the common bottleneck of existing fat ablation technologies is that they cannot achieve efficient, accurate, and safe targeted removal of adipocytes.

[0007] To solve the above problems, the present application aims to provide a bacteriophage nanomaterial-based targeted removal of adipocytes. SUMMARY

[0008] The present application aims to provide a bacteriophage nanoparticle for targeted ablation of adipocytes and a preparation method and application thereof, to solve the problem that the common bottleneck of existing fat ablation technologies is that they cannot achieve efficient, accurate, and safe targeted removal of adipocytes, as proposed in the background.

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0010] The bacteriophage nanoparticle for targeted ablation of adipocytes comprises a genetically engineered M13 bacteriophage, which displays a fat cell targeting peptide ATS on the surface; a functional molecule curcumin loaded inside the bacteriophage nanoparticle; and a functional polymer P4VP shell for stabilizing the loading.

[0011] Preferably, the M13-ATS phage is prepared by extracting M13-KO7 plasmid and performing PCR amplification; inserting ATS sequence into M13-KO7 plasmid, performing site-specific mutation; performing DpnI digestion and gel recovery on the amplification product; using Exnase II for recombination and circularization, transforming into competent cells; screening positive clones and expanding culture, and purifying to obtain M13-ATS phage.

[0012] Preferably, the enrichment stages are 2-10 stages, each stage has an enrichment time of 1.5-3 hours, and the operation temperature is 20-30°C; the solution remaining after enrichment of the intermediate chamber is used for salt production or recycled to the pretreatment step.

[0013] The preparation method of the phage nanoparticle targeting and ablating adipocytes, the M13-ATS phage is self-assembled with P4VP and curcumin in a DMF / water mixed solution; the organic solvent is removed by dialysis, and the M13-ATS / P4VP / Cur nanoparticle is purified by centrifugation.

[0014] The composite hydrogel of the phage nanoparticle, the hydrogel is formed by crosslinking sodium alginate and calcium gluconate, and is used for loading the phage nanoparticle to achieve a slow-release effect.

[0015] Preferably, the concentration of sodium alginate is 2-4 wt%, the concentration of calcium gluconate is 2-4 wt%, and the volume mixing ratio of the two is 3:1.

[0016] The preparation method of the composite hydrogel of the phage nanoparticle, comprising the following steps:

[0017] S1, weigh 231.95 mg of sodium alginate and dissolve it in 7.5 mL of ultrapure water to obtain mixture A, and the concentration of Alg is 3 wt%;

[0018] S2, weigh 77.32 mg of calcium gluconate and dissolve it in 2.5 mL of ultrapure water to obtain mixture B, and the concentration of Ca is 3 wt%;

[0019] S3, under stirring, drop mixture B into mixture A at a volume ratio of 1:3; continue stirring for 10 min to prepare 10 mL of Gel;

[0020] S4, add 1 ml of purified M13-ATS / P4VP / Cur nanoparticle to 3 ml of Gel to prepare M13-ATS / P4VP / Cur phage nanoparticle hydrogel.

[0021] Preferably, the phage nanoparticle targeting and ablating adipocytes is used in the preparation of a drug for ablating adipocytes. The adipocytes are subcutaneous adipocytes or visceral adipocytes.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] Highly targeted and specific: This invention utilizes genetically engineered or chemically modified phage nanoparticles. Specific peptides displayed on their surface act like "biological missiles," precisely identifying and binding with high affinity to specific markers on the surface of adipocytes. This active targeting capability fundamentally overcomes the shortcomings of existing physical therapies (cold, heat, sound waves) in terms of poor targeting and easy damage to surrounding tissues, achieving a "precision strike" on target adipocytes and greatly reducing the risk of accidental damage to normal tissues such as skin, nerves, and blood vessels.

[0024] The treatment is highly effective and thorough: high concentrations of effector molecules (curcumin) are delivered directly to adipocytes via phage vectors, achieving efficient enrichment and release at the target site. This localized mode of action avoids the dilution and metabolism problems associated with traditional systemic drug administration, achieving a stronger killing effect with a smaller drug dose, thus more thoroughly clearing adipocytes and potentially reducing the number of treatments required and increasing the efficacy of a single treatment.

[0025] Superior safety and minimal invasiveness: Due to its high targeting specificity, the method of this invention can minimize off-target effects. Treatment can be achieved through simple local injection, resulting in far less trauma than traditional liposuction surgery, while also avoiding inflammation and induration of surrounding tissues caused by the diffusion of chemical lipolysis agents. This minimally invasive characteristic gives it a unique advantage in fat treatment of delicate areas such as the face and neck, leading to faster postoperative recovery and fewer complications.

[0026] The technology platform boasts strong scalability: The phage nanoplatform in this invention is highly modular and programmable. By replacing the target peptides displayed on its surface or the functional molecules it carries, it can be easily adapted to different depths and types of adipose tissue (such as subcutaneous fat and visceral fat), and even provides a universal technology platform for treating other cell-specific diseases (such as tumors), with broad application prospects.

[0027] Cost and operational advantages: Bacteriophages can be produced on a large scale and at low cost through bio-fermentation, exhibiting good stability and ease of storage and transportation. The treatment regimen is relatively simple to administer, requiring less skill from the physician than complex liposuction surgery, making it easier to promote and apply in clinical practice. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the assembly of M13-ATS / P4VP / Cur and a schematic diagram of the M13-ATS / P4VP / Cur hydrogel of the present invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1:

[0031] Preparation of M13-ATS phage targeting adipocytes

[0032] Plasmid extraction:

[0033] ① Take the bacterial culture that has been cultured overnight and centrifuge at 8000 rpm for 10 min. Discard the supernatant and collect the bacterial cells;

[0034] ② Add 250 μL of buffer RS, and resuspend the bacterial pellet by pipetting or vortexing;

[0035] ③ Add 250 μL of lysis buffer LB, gently invert 6-8 times to fully lyse the cells, and incubate at room temperature for 4 minutes. Cell lysis should be performed gently to avoid genomic DNA shearing breaks.

[0036] ④ Add 350 μL of binding buffer BD. Immediately and gently invert 6–8 times to mix thoroughly. Incubate at room temperature for 2 min. Centrifuge at 13,000 rpm for 10 min and carefully collect the supernatant.

[0037] ⑤ Add 100 μL of buffer AC to DNA adsorption column P3, centrifuge at 13,000 rpm for 1 min, and discard the waste liquid.

[0038] ⑥ Insert the DNA adsorption column P3 into the 2mL collection tube for later use.

[0039] ⑦ Add the supernatant to DNA adsorption column P3, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0040] ⑧ Place the DNA adsorption column P3 back into the collection tube, add 600 μL of wash buffer W, centrifuge at 12,000 rpm at room temperature for 30 s, and discard the waste liquid.

[0041] 9. Repeat step 5.

[0042] ⑩ Place the DNA adsorption column P3 back into the collection tube, and centrifuge the empty column at 13,000 rpm at room temperature for 2 min to remove residual wash buffer W.

[0043] ⑪ Place the DNA adsorption column P3 into a new 1.5mL centrifuge tube, add 50-100uL of elution buffer to the center of the DNA adsorption column P3, and incubate for 2 minutes.

[0044] ⑫ Then centrifuge at 12,000 rpm for 1 min. Collect the filtrate, which is the plasmid DNA solution.

[0045] PCR amplification

[0046] Prepare the PCR reaction mixture in the following proportions, with a total volume of 20 μL: 10 μL 2X Mix, 0.5 μL P3F primer, 0.5 μL P3R primer, 8 μL H2O, and 1 μL of the extracted plasmid DNA template. Gently pipette and mix well, then briefly centrifuge to collect the reaction mixture at the bottom of the tube.

[0047] Place the PCR reaction tubes into the PCR instrument and set the following reaction program: 95℃ pre-denaturation for 3 min; then perform 38 cycles, each cycle including 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s; finally, 72℃ complete extension for 5 min. After the reaction is complete, remove the PCR products for later use.

[0048] This includes identification methods using nucleic acid electrophoresis:

[0049] Nucleic acid electrophoresis: Preliminary identification of the M13-KO7 plasmid

[0050] To prepare a 1% agarose gel: add 0.35g agarose to 35ml TAE buffer in an Erlenmeyer flask, and microwave to fully dissolve it each time.

[0051] After heating is complete, let the temperature drop to about 50°C, then add the nucleic acid dye and mix thoroughly.

[0052] Pour the agarose gel into the pre-installed gel casting plate (about 15 minutes) and wait for the gel to solidify.

[0053] Install the electrophoresis tank, pour in 1X TAE buffer, place the solidified gel into the electrophoresis tank, and remove the comb;

[0054] Sample;

[0055] Electrophoresis, 120V, 20min;

[0056] M13-KO7 plasmid site mutation (ATS linker)

[0057] ATS F / R primers were designed and synthesized, with sequences based on the sequences of the M13-KO7 plasmid and ATS to ensure specific binding.

[0058] The purity of the extracted M13-KO7 plasmid was determined using a nucleic acid concentration analyzer to ensure that the plasmid purity was ≤1 ng / μL, which meets the requirements for subsequent experiments.

[0059] Prepare the PCR reaction mixture in the following proportions, with a total volume of 50 μL: ddH2O to 50 μL, 2×MaxBuffer 25 μL, dNTP Mix (10 mM each) 1 μL, M13-KO7 plasmid template DNA 1 μL, ATS F primer 2 μL, ATS R primer 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL. Gently mix and briefly centrifuge.

[0060] Place the PCR reaction tubes into the PCR instrument and set the reaction program: 95℃ pre-denaturation for 3 min; then perform 35 cycles, each cycle including 95℃ denaturation for 15 s, 68℃ annealing for 15 s, 72℃ extension for 5 min; and finally 72℃ complete extension for 7 min.

[0061] Nucleic acid electrophoresis was performed according to the method for identification of nucleic acid electrophoresis, and the connection of ATS was identified to observe whether the target band of the expected size appeared.

[0062] Digestion of amplified product DpnI

[0063] Add 45 μL of the PCR amplification product to a 1.5 mL centrifuge tube, then add 1 μL of DpnI enzyme, mix gently, and centrifuge briefly to collect the reaction solution at the bottom of the tube. Then place the centrifuge tube in a 37°C water bath and incubate for 90 min to remove methylated template plasmids.

[0064] Gel recovery (removal of DpnI enzyme)

[0065] The product after DpnI digestion was subjected to nucleic acid electrophoresis. After electrophoresis, the gel containing the target DNA fragment was quickly cut off under UV light and placed into a clean 1.5mL centrifuge tube, removing as much of the gel without the target fragment as possible.

[0066] Add an equal volume of Buffer GDP to the centrifuge tube, place the centrifuge tube in a 50-55℃ constant temperature water bath for 7-10 minutes, and invert the centrifuge tube every 2-3 minutes during the process to accelerate the complete dissolution of the gel block.

[0067] Briefly centrifuge to collect the droplets on the tube wall to the bottom. Place the FastPure DNA Mini Columns-G adsorption column in a 2mL collection tube, transfer the dissolved sol solution to the adsorption column, place it in a centrifuge, set the speed to 12000rpm, centrifuge for 30-60s, and discard the filtrate in the collection tube.

[0068] After discarding the filtrate, place the adsorption column back into the collection tube, add 300 μL of Buffer GDP, let it stand at room temperature for 1 min, then centrifuge at 12000 rpm for 30-60 s and discard the filtrate.

[0069] Add 700 μL of Buffer GW to the adsorption column, set the rotation speed to 12000 rpm, centrifuge for 30-60 seconds, discard the filtrate, and repeat this step once.

[0070] Place the adsorption column back into the collection tube, set the rotation speed to 12000 rpm, and centrifuge for 2 minutes to completely remove any residual buffer solution from the adsorption column.

[0071] Place the adsorption column into a new 1.5 mL sterile centrifuge tube, add 25 μL of Elution Buffer to the center of the column, incubate at room temperature for 2 min, then centrifuge at 12000 rpm for 1 min. Discard the adsorption column and store the collected DNA solution at -20°C for later use.

[0072] Recombination reaction:

[0073] Take 0.8 μL of the above gel recovery product (the concentration was detected to be 340.7 ng / μL) and add it to a 1.5 mL centrifuge tube. Then add 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and 13.2 μL of ddH2O in sequence. Gently pipette and mix well. After a short centrifugation, collect the reaction solution to the bottom of the tube.

[0074] Place the centrifuge tubes in a 37°C water bath and incubate for 30 minutes to allow the amplification products to undergo in vitro circularization of linear DNA under the catalysis of Exnase II. After the reaction, immediately place the centrifuge tubes on ice to cool or store them at 4°C to prevent the enzyme activity from continuing to affect subsequent experiments.

[0075] Transformation of recombinant products:

[0076] Remove the XL1-Blue clone competent cells from the -80℃ freezer and quickly place them on ice to thaw. After the cells are completely thawed, add 100 μL of competent cells to a 1.5 mL centrifuge tube.

[0077] Add 10 μL of the recombinant product to a centrifuge tube, gently tap the tube wall to mix, avoiding vigorous shaking, and then let it stand on ice for 30 min.

[0078] Place the centrifuge tubes in a 42°C constant temperature water bath and heat shock for 45 seconds. Keep the centrifuge tubes stationary during the heat shock process, and then immediately place them on ice to cool for 2-3 minutes to prevent cells from dying due to excessive heat.

[0079] Add 900 μL of antibiotic-free LB liquid medium to a centrifuge tube, place the centrifuge tube in a 37°C constant temperature shaker, set the speed to 200-250 rpm, and shake for 1 hour to allow the cells to regain activity and express the resistance gene.

[0080] Place the LB solid medium plates containing the corresponding resistance (matching the resistance of the recombinant plasmid) into a 37°C constant temperature incubator and preheat for 30 minutes.

[0081] Place the culture solution after shaking into a centrifuge, set the speed to 5000 rpm, and centrifuge for 5 minutes. After centrifugation, carefully discard 900 μL of supernatant and gently resuspend the bacterial pellet in the remaining 100 μL of culture medium.

[0082] In a clean bench, use a sterile spreader to evenly spread the resuspended bacterial cells onto a preheated LB solid medium plate. Then place the plate in a 37°C incubator and incubate upside down for 12-16 hours.

[0083] Identification of recombinant products: After overnight culture, several single clones were selected and inoculated into LB liquid medium containing appropriate antibiotics and cultured overnight. Plasmids were then extracted for first-generation sequencing.

[0084] Add the successfully sequenced bacterial culture to a 10ml shake tube for expansion culture, and simultaneously shake 200ml of culture medium with 2ml of ER2738 until the logarithmic phase OD value is about 0.6.

[0085] Add 10 ml of the modified supernatant (M13-ATS) to the above culture medium and shake for 16-18 h;

[0086] Collect the supernatant and purify it.

[0087] Transfer the phage to a centrifuge bottle, filling the volume to no more than 1 / 3, centrifuge at 7500 rpm for 15 min, and collect the supernatant into a 2L bottle;

[0088] Add NaCl / PEG-6000 (w / v) to a wide-mouth bottle at a volume ratio of 4:1;

[0089] Incubate at room temperature for 1 hour, then incubate overnight at 4°C.

[0090] Centrifuge at 12000 rpm for 15-20 min, then discard the supernatant;

[0091] Resuspend the particles in PBS, centrifuge at 8000 rpm for 5 min, and collect the supernatant.

[0092] Add 200 μL of NaCl / PEG-6000 and incubate on ice for 1 hour;

[0093] Centrifuge at 12000-14000 rpm for 15 min, then discard the supernatant;

[0094] The purified phage was obtained by resuspending it in PBS.

[0095] Example 2:

[0096] Preparation of phage nanoparticles: 1 mg P4VP and 1 mg curcumin were added to 1 mL DMF to prepare the feeding solution;

[0097] While stirring, add 149.1 uL of M13-ATS phage (titer: 2.2 x 10¹³) to 4.5 mL of pure water;

[0098] Slowly add the P4VP solution prepared in step 1, and stir at room temperature for 30 min;

[0099] The dialysis bag (3500 Da) was used for dialysis in 2 L of pure water. The dialysis fluid was changed every 8 hours, and the dialysis was performed for 48 hours.

[0100] Centrifugation purification yielded M13-ATS / P4VP / Cur phage nanoparticles.

[0101] Example 3:

[0102] Preparation of M13-ATS / P4VP / Cur phage nanoparticle hydrogel

[0103] Accurately weigh 231.95 mg of sodium alginate and add it to 7.5 mL of ultrapure water. Stir with a magnetic stirrer (300 rpm) for 2 hours until the sodium alginate is completely dissolved to obtain a 3 wt% Alg solution. Take care to avoid generating bubbles during the process.

[0104] Accurately weigh 77.32 mg of calcium gluconate and add it to 2.5 mL of ultrapure water. Stir with a magnetic stirrer (200 rpm) for 30 min until the calcium gluconate is completely dissolved to obtain a Ca solution with a concentration of 3 wt%.

[0105] Place the Alg solution on a magnetic stirrer and turn on the stirrer (100-300 rpm). While stirring, slowly add the Ca solution dropwise to the Alg solution at a volume ratio of 1:3. After the addition is complete, continue stirring for 10 minutes to allow the solution to react fully and obtain 10 mL of Gel (hydrogel).

[0106] Take 1 mL of the purified M13-ATS / P4VP / Cur phage nanoparticles obtained in step 3, and slowly add them to 3 mL of the prepared gel. Gently mix the gel with a pipette to avoid generating air bubbles, and prepare the M13-ATS / P4VP / Cur phage nanoparticle-hydrogel. Store it in a refrigerator at 4°C for later use.

[0107] In summary, this invention provides a phage nanoparticle system that can specifically target and efficiently ablate adipocytes. This system can be prepared into a therapeutic agent suitable for local minimally invasive injection using a hydrogel carrier, and has great application potential in the treatment of obesity and related diseases.

[0108] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0109] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Phage nanoparticles for targeted ablation of adipocytes, characterized in that, The phage nanoparticles include genetically engineered M13 phages with adipocyte-targeting peptide ATS on their surface; and curcumin, a functional molecule encapsulated within the phage nanoparticles. And a functional polymer P4VP housing for stable loads.

2. The phage nanoparticles for targeted ablation of adipocytes according to claim 1, characterized in that: The M13-ATS phage was obtained by extracting the M13-KO7 plasmid and performing PCR amplification; inserting the ATS sequence into the M13-KO7 plasmid and performing site-specific mutations; digesting the amplification product with DpnI and recovering it with gel; performing recombinant circularization using Exnase II and transforming it into competent cells; screening positive clones and expanding the culture to obtain the M13-ATS phage.

3. The method for preparing phage nanoparticles targeting and ablating adipocytes according to any one of claims 1-2, characterized in that: Includes the following steps: The M13-ATS phage was self-assembled with P4VP and curcumin in a DMF / water mixture; The organic solvent was removed by dialysis, and the M13-ATS / P4VP / Cur nanoparticles were obtained by centrifugation purification.

4. The composite hydrogel of phage nanoparticles according to any one of claims 1-2, characterized in that: The hydrogel is formed by cross-linking sodium alginate and calcium gluconate, and is used to encapsulate the phage nanoparticles to achieve a sustained release effect.

5. The composite hydrogel of phage nanoparticles according to claim 4, characterized in that: The sodium alginate concentration is 2-4 wt%, the calcium gluconate concentration is 2-4 wt%, and the volume mixing ratio of the two is 3:

1.

6. The method for preparing the phage nanoparticle composite hydrogel according to claim 4, characterized in that: Includes the following steps: S1. Weigh 231.95 mg of sodium alginate and dissolve it in 7.5 mL of ultrapure water to obtain mixture A, with an Alg concentration of 3 wt%. S2. Weigh 77.32 mg of calcium gluconate and dissolve it in 2.5 mL of ultrapure water to obtain mixture B, with a Ca concentration of 3 wt%. S3. While stirring, add mixture B dropwise to mixture A at a volume ratio of 1:3; continue stirring for 10 minutes to obtain 10 mL of gel. S4. Add 1 ml of purified M13-ATS / P4VP / Cur nanoparticles to 3 ml of GEL to prepare M13-ATS / P4VP / Cur phage nanoparticle hydrogel.

7. The use of the phage nanoparticles for targeted ablation of adipocytes according to claim 1 in the preparation of drugs for ablation of adipocytes.

8. The application according to claim 6, characterized in that: The fat cells are subcutaneous fat cells or visceral fat cells.

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