Tobramycin nano transfer body as well as preparation method and application thereof
Tobramycin nanotransfers were prepared by oligoxylose modified liposomes, and tobramycin-in-situ gel was prepared by mixing them with the gel matrix, which solved the problems of poor permeability of tobramycin and insufficient stability of metastases, and achieved efficient targeted delivery and controlled release of ocular inflammation.
Patent Information
- Application Number
- CN202510236082.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-05-13
AI Technical Summary
As a hydrophilic drug, tobramycin has poor permeability and is difficult to efficiently pass through the cornea to reach the ocular lesions. The drug release ability and stability of existing metastases are insufficient.
Tobramycin nanotransfers were prepared by using xylooligosaccharides. Tobramycin-in-situ gel was prepared by mixing and swelling with the gel matrix, which increased the permeability and drug loading volume of the drug, and achieved long-term continuous release.
It improves the permeability and bioavailability of tobramycin, achieves targeted delivery and effective controlled release of ocular inflammation, extends the retention time of the drug at the drug site, and reduces the frequency of dosing and side effects.
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Figure CN119970626A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and specifically discloses a tobramycin nanotransfer body and a preparation method and application thereof. Background Art
[0002] Ophthalmic preparations made from tobramycin, a hydrophilic drug, need to penetrate the cornea to reach the lesion to exert their effects. The cornea is a lipid-water-lipid structure, and drug molecules penetrate the corneal epithelium through transcellular or paracellular pathways. However, due to the poor permeability of tobramycin, it is unable to efficiently penetrate the cornea to reach the lesion.
[0003] Transfersomes are modified liposomes composed of phospholipids and edge activators (such as surfactants). Compared with conventional liposomes, transfersomes are more flexible, less rigid, and more flexible, and can pass through smaller vesicle sizes or narrower pores than traditional liposomes. However, the drug release capacity and post-recondensation stability of conventional transfersomes still need to be improved, and the drug loading is unstable between batches. Summary of the invention
[0004] In view of the deficiencies in the prior art, the present invention provides a tobramycin nanotransfer body with sustained release, uniform and stable drug loading, and stability after re-coagulation, and then the tobramycin nanotransfer body is mixed with a gel matrix and swelled to prepare a tobramycin-in-situ gel. The in-situ gel has a uniform and stable drug loading, improved drug permeability and can be released continuously for a long time, and can be used to prepare an external preparation for treating ocular inflammation.
[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for preparing a tobramycin nanotransferase, the preparation method comprising the following steps: Step 1, mixing lipid, surfactant, xylo-oligosaccharide and alcohol solvent uniformly, and evaporating the alcohol solvent to obtain a film; Step 2, hydrating the film with tobramycin-PBS buffer to obtain a suspension; Step 3: subjecting the suspension to ultrasonic and graded filtration to obtain tobramycin nanotransfersomes.
[0006] According to the characteristics of tobramycin, an aminoglycoside drug, the present invention encapsulates it with lipid substances to prepare nano-transfer bodies, which effectively improves the permeability of hydrophilic drugs through biological barriers. The nano-transfer bodies have a small particle size, which can increase the contact area between the drug and the biomembrane, thereby quickly reaching the effective level of the drug. At the same time, the surface of the transfer body is modified by oligoxylose to improve the stability and overall performance of the nano-transfer body.
[0007] Xylo-oligosaccharides can penetrate into the transfer body through hydrogen bonds and physical methods. Due to the different hydrogen bond strengths between sugars and phospholipids, the present invention creatively finds that xylo-oligosaccharides can make the nano transfer body have a smaller particle size and PDI. Xylo-oligosaccharides are stable under pH 6-8 conditions and will not cause the transfer body to break and cause drug leakage. In addition, the introduction of oligofructose can not only give the transfer body the ability to fuse membranes or release drugs under specific pH conditions, but also improve its stability.
[0008] Preferably, the mass ratio of the lipid, surfactant and xylooligosaccharide is 4.5-5.5:0.8-1.2:0.4-0.6.
[0009] The present invention prepares a solution according to the concentration of lipid in alcohol solvent of 9 mg / mL to 11 mg / mL, and then adds surfactant and xylo-oligosaccharide according to the mass ratio of the lipid, surfactant and xylo-oligosaccharide.
[0010] The volume ratio of the alcohol solvent to tobramycin-PBS buffer is 5:2-3.
[0011] Preferably, the lipid comprises at least one of dipalmitoylphosphatidylglycerol, soybean phosphatidylcholine, phospholipid 90G or phospholipid 90H; The surfactant includes at least one of Span 20, Span 80 or Tween 80; The alcohol solvent includes methanol.
[0012] Preferably, the tobramycin-PBS buffer comprises tobramycin, PBS buffer, a thickener and a humectant, and a preservative is optionally added; wherein the mass volume ratio of tobramycin to PBS buffer is 18 mg~22 mg:18 mL~20 mL.
[0013] Wherein, the thickener includes at least one of methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose E15, polyethylene glycol 400, and polycarbophil; the humectant includes at least one of glycerol, propylene glycol, or sorbitol; the pH of the PBS buffer is 7.35-7.45, and the pH of 7.4 is taken as an example for illustration in the embodiment of the present invention.
[0014] Preferably, the hydration temperature is 40°C to 50°C, and the time is 30min to 60min; The graded filtration includes filtering using filters with pore sizes of 20 μm to 22 μm, 400 nm to 700 nm, and 180 nm to 220 nm in sequence.
[0015] The present invention adopts extrusion to perform graded filtration to reduce the size of the transfer body and improve work efficiency and yield. The suspension can first be passed through a nylon filter of 20 μm to 22 μm for 4 to 6 cycles, and then the suspension can be passed through a filter with a pore size of 400 nm to 700 nm for 4 to 6 cycles and a filter with a pore size of 180 nm to 220 nm for 1 to 3 cycles under the protection of inert gas to obtain particles with uniform size distribution. The filtering conditions are controlled to ensure that the average particle size of the transfer body finally obtained is within the range of 145 nm to 160 nm.
[0016] In a second aspect, the present invention provides a tobramycin nanotransfer body prepared by the above-mentioned method for preparing the tobramycin nanotransfer body.
[0017] The tobramycin nanotransfer has not only good biocompatibility, drug loading capacity and controlled release capacity, but also good re-coagulation stability.
[0018] In a third aspect, the present invention provides a tobramycin-in-situ gel, comprising the following raw material components in weight fractions: 25% to 30% of a temperature-sensitive gel material, 0.5% to 1.5% of a thickener, 0.5% to 1.5% of a moisturizer, 0.5% to 1.5% of an isotonicity regulator, and 0.1% to 0.5% of the tobramycin nanotransfer body, with the remainder being PBS buffer.
[0019] The tobramycin-in-situ gel provided by the present invention uses phosphate buffer as a solvent and is prepared by using the above-mentioned tobramycin nanotransfer body and temperature-sensitive gel material as raw materials. Before application, the in-situ gel wraps tobramycin in a liquid state to form a semi-solid gel, which is uniform and stable; after application, due to changes in external conditions, a solid gel is formed at the administration site of the human body, thereby prolonging the retention time of the drug at the administration site, slowly releasing the drug, improving bioavailability, and achieving targeted delivery and effective controlled release of the target drug.
[0020] The present invention changes the traditional water carrier and uses a phosphate buffer, which can meet the pH requirement of the eye gel and increase the compliance of patients in clinical application.
[0021] Repeated gelation and degelation of the in-situ gel will cause the size of the transfer body to become larger. The present invention finds that the particle size increase of the tobramycin nano-transfer body modified with xylooligosaccharides is significantly smaller than that of the tobramycin nano-transfer body without xylooligosaccharides. Therefore, xylooligosaccharides can significantly improve the stability of tobramycin-in-situ gel.
[0022] Preferably, the temperature-sensitive gel material comprises at least one of poloxamer 407, poloxamer 188, tyloxamer, chitosan, polylactic acid-polyethylene glycol block copolymer or poly-N-isopropylacrylamide; The thickener includes at least one of methylcellulose, hydroxyethylcellulose, hypromellose E15, polyethylene glycol 400 or polycarbophil; The humectant includes at least one of glycerin, propylene glycol or sorbitol; The isotonicity adjusting agent includes at least one of sodium chloride or mannitol; The pH of the PBS buffer is 7.35-7.45, and the pH of 7.4 is used as an example for illustration in the embodiments of the present invention.
[0023] In a fourth aspect, the present invention provides a method for preparing the above-mentioned tobramycin-in situ gel, the preparation method comprising the following steps: S1. mixing the temperature-sensitive gel material, a thickener, a humectant, an isotonicity adjusting agent and a PBS buffer to obtain a gel matrix; S2. Add the tobramycin nanotransfer body to the gel matrix under stirring, stir at 1°C~5°C for 25min~35min, and fully swell in a refrigerator at 0°C~4°C for 10h~15h to obtain the tobramycin-in-situ gel.
[0024] The preparation method of the tobramycin-in-situ gel provided by the invention has a simple process, does not require special equipment, and is convenient for large-scale promotion and application. The prepared tobramycin-in-situ gel has a small particle size, uniform drug loading, and high re-coagulation stability.
[0025] In the fifth aspect, in view of the good biocompatibility, stable drug loading capacity and controllable drug release performance of the tobramycin-in situ gel provided in the third aspect of the present invention, it can be used to prepare an external preparation for treating ocular inflammation.
[0026] Ocular drug delivery poses unique challenges to drug absorption. Drug absorption through the cornea is an effective route for topical ocular administration. Drugs reach the local vascular network through the cornea, aqueous humor, iris, and ciliary muscle, thereby exerting a local effect. Due to the special structure and environment, there are barriers to drug absorption in the eye, of which the cornea is the main barrier to drug absorption. The cornea has a lipid-water-lipid structure, and drug molecules penetrate the corneal epithelium through transcellular or paracellular pathways, while hydrophilic drugs have poor permeability and low bioavailability.
[0027] The tobramycin-in-situ gel prepared by tobramycin nanotransfer bodies and gel matrix of the present invention can penetrate the cornea to reach the lesion and exert a targeted effect, which can not only overcome eye obstacles, prolong retention time, increase eye permeability, have strong targeting and sustainable drug delivery, but also reduce the frequency of drug delivery, reduce side effects and improve bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0029] Figure 1 This is the liquid phase diagram of Tobramycin Nanotransferomer I in the effect example of the present invention. DETAILED DESCRIPTION
[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0031] In order to better illustrate the embodiments of the present invention, further examples are given below.
[0032] The xylooligosaccharide in the present invention is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and the specific parameters are: product name: xylobiose; product number: D131021; CAS: 6860-47-5.
[0033] Example 1 This embodiment provides a method for preparing a tobramycin nanotransferase, the preparation method comprising the following steps: Step 1: Add 0.5 g of phospholipid 90H to a round-bottom flask containing 50 mL of methanol to dissolve, prepare a methanol solution containing 10 mg / mL of lipid, add 0.1 g of Tween 80 and 0.05 g of oligoxylose, and mix well. In a rotary evaporator, evaporate the organic solvent at 45 ° C and 0.01 MP to form a thin film in the round-bottom flask; Step 2, the film in the round-bottom flask was placed under vacuum drying at 50°C and 0.05MP for 12 hours, 20 mL of tobramycin-PBS buffer was added, and hydrated at 45°C and 80 rpm for 60 minutes to obtain a suspension having a vesicle system; Step 3: ultrasonically treat the suspension in an ultrasonic instrument for 10 minutes, and perform graded filtration by extrusion to reduce the size of the transfer body, specifically: the sonicated suspension is first passed through a nylon filter with a pore size of 22 μm for 5 cycles, and then under the protection of inert gas, the suspension is passed through a filter with a pore size of 600 nm for 5 cycles and a filter with a pore size of 200 nm for 2 cycles to obtain 23.94 g of tobramycin nano-transfer body, recorded as tobramycin nano-transfer body I.
[0034] The preparation method of the tobramycin-PBS buffer is as follows: take 20 mg of tobramycin, 0.1 g of polyethylene glycol 4000, 0.1 g of propylene glycol and 20 mg of potassium sorbate, and add PBS buffer with a pH of 7.4 to make the volume to 20 mL.
[0035] Example 2 This embodiment provides a method for preparing a tobramycin nanotransferase, the preparation method comprising the following steps: Step 1: Dissolve 0.55 g of phospholipid PC90 in a round-bottom flask containing 50 mL of methanol to prepare a methanol solution containing 11 mg / mL of lipid, add 0.08 g of Span 80 and 0.06 g of oligoxylose, and mix well. Evaporate the organic solvent in a rotary evaporator at 45 ° C and 0.01 MP to form a thin film in the round-bottom flask; Step 2: After the film in the round-bottom flask is placed in a vacuum dryer at 50° C. and 0.05 MP for 12 h, 20 mL of tobramycin-PBS buffer is added, and hydrated at 50° C. and 100 rpm for 30 min to obtain a suspension having a vesicle system; Step 3: ultrasonically treat the suspension in an ultrasonic instrument for 10 minutes, and perform graded filtration by extrusion to reduce the size of the transfer body, specifically: the sonicated suspension is first passed through a nylon filter with a pore size of 20 μm for 4 cycles, and then under the protection of inert gas, the suspension is passed through a filter with a pore size of 700 nm for 6 cycles and a filter with a pore size of 180 nm for 2 cycles to obtain 24.35 g of tobramycin nano-transfer body, recorded as tobramycin nano-transfer body II.
[0036] The preparation method of the tobramycin-PBS buffer is as follows: take 22 mg of tobramycin, 0.12 g of hydroxypropyl methylcellulose E15, 0.085 g of sorbitol and 20 mg of potassium sorbate, and add PBS buffer with a pH of 7.4 to make the volume to 20 mL.
[0037] Example 3 This embodiment provides a method for preparing a tobramycin nanotransferase, the preparation method comprising the following steps: Step 1: Dissolve 0.45 g of dipalmitoylphosphatidylglycerol in a round-bottom flask containing 50 mL of methanol to prepare a methanol solution containing 9 mg / mL of lipid, add 0.12 g of Span 20 and 0.04 g of oligoxylose, and mix well. Evaporate the organic solvent in a rotary evaporator at 45 ° C and 0.01 MP to form a thin film in the round-bottom flask; Step 2, the film in the round-bottom flask was placed in a vacuum dryer at 50°C and 0.05MP for 12 hours, 20 mL of tobramycin-PBS buffer was added, and hydrated at 40°C and 100 rpm for 60 minutes to obtain a suspension having a vesicle system; Step 3: ultrasonically treat the suspension in an ultrasonic instrument for 10 minutes, and perform graded filtration using extrusion to reduce the size of the transfer body, specifically: the sonicated suspension is first passed through a nylon filter with a pore size of 22 μm for 6 cycles, and then under the protection of inert gas, the suspension is passed through a filter with a pore size of 400 nm for 4 cycles and a filter with a pore size of 220 nm for 3 cycles to obtain 23.55 g of tobramycin nano-transfer body, recorded as tobramycin nano-transfer body III.
[0038] The preparation method of the tobramycin-PBS buffer is as follows: take 28 mg of tobramycin, 0.08 g of polycarbophil, 0.12 g of propylene glycol and 20 mg of potassium sorbate, and add PBS buffer with a pH of 7.4 to make the volume to 20 mL.
[0039] Example 4 This embodiment provides a tobramycin-in situ gel, which comprises the following components in weight fractions: 27% of poloxamer 407, 2% of poloxamer 188, 0.5% of polyethylene glycol, 0.5% of propylene glycol, 0.5% of sodium chloride, 0.1% of tobramycin nanotransfer body I and 0.1% of potassium sorbate, and the balance is PBS buffer.
[0040] This embodiment also provides a method for preparing the above-mentioned tobramycin-in-situ gel, and the preparation method comprises the following steps: S1. Take 13.5 g of poloxamer 188, 1 g of poloxamer 188, 0.25 g of polyethylene glycol, 0.25 g of propylene glycol, 0.25 g of sodium chloride and 50 mg of potassium sorbate, add PBS buffer with a pH of 7.4 to make up to 50 mL, stir evenly, and obtain a gel matrix; S2. Add 50 mg of tobramycin nanotransfer body I to the gel matrix under magnetic stirring at 600 rpm in an environment of 4°C ± 0.5°C, mix well and continue stirring for 30 minutes under this condition until the solution is clear, transparent and uniform. Swell in a refrigerator at 4°C ± 0.5°C for 12 hours to obtain tobramycin-in situ gel, which is recorded as tobramycin-in situ gel I.
[0041] Example 5 This embodiment provides a tobramycin-in-situ gel, which comprises the following components in weight fractions: 25% tyloxamer, 1.5% hydroxypropyl methylcellulose E15, 1.5% sorbitol, 1% mannitol, 0.5% tobramycin nanotransfer body II and 0.5% potassium sorbate, with the remainder being PBS buffer.
[0042] This embodiment also provides a method for preparing the above-mentioned tobramycin-in-situ gel, and the preparation method comprises the following steps: S1. Take 12.5 g of tyloxamer, 0.75 g of hypromellose E15, 0.75 g of sorbitol, 0.5 g of mannitol and 250 mg of potassium sorbate, add PBS buffer with a pH of 7.4 to make up to 50 mL, stir evenly, and obtain a gel matrix; S2. In the process of magnetically stirring the gel matrix at 800 rpm in an environment of 0℃±0.5℃, add 250 mg of tobramycin nanotransfer body II, mix well and continue stirring for 30 minutes under this condition until the solution is clear, transparent and uniform. Swell in a refrigerator at 0℃±0.5℃ for 10 hours to obtain tobramycin-in situ gel, which is recorded as tobramycin-in situ gel II.
[0043] Example 6 This embodiment provides a tobramycin-in situ gel, which comprises the following components by weight: 27% poloxamer 407, 3% poly (N-isopropylacrylamide), 1% polycarbophil, 1% propylene glycol, 0.75% sodium chloride, 0.3% tobramycin nanotransfer body III and 0.3% benzalkonium bromide, and the balance is PBS buffer.
[0044] This embodiment also provides a method for preparing the above-mentioned tobramycin-in-situ gel, and the preparation method comprises the following steps: S1. Take 13.5 g of poloxamer 407, 1.5 g of poly (N-isopropylacrylamide), 0.5 g of polycarbophil, 0.5 g of propylene glycol, 0.375 g of sodium chloride and 150 mg of benzalkonium bromide, add PBS buffer with a pH of 7.4 to make up to 50 mL, stir evenly, and obtain a gel matrix; S2. In the process of magnetically stirring the gel matrix at 700 rpm in an environment of 2°C ± 0.5°C, add 150 mg of tobramycin nanotransfer body III, mix well and continue stirring for 30 minutes under this condition until the solution is clear, transparent and uniform. Swell in a refrigerator at 2°C ± 0.5°C for 15 hours to obtain tobramycin-in situ gel, which is recorded as tobramycin-in situ gel III.
[0045] Example 7 This embodiment provides a tobramycin-in situ gel, which comprises the following components by weight: 25% poloxamer 407, 3% poloxamer 188, 1.5% hydroxyethyl cellulose, 1.5% propylene glycol, 1% sodium chloride, 0.5% tobramycin nanotransfer body I and 0.5% potassium sorbate, and the balance is PBS buffer.
[0046] This embodiment also provides a method for preparing the above-mentioned tobramycin-in-situ gel, and the preparation method comprises the following steps: S1. Take 12.5 g of poloxamer 407, 1.5 g of poloxamer 188, 0.75 g of hydroxyethyl cellulose, 0.75 g of propylene glycol, 0.5 g of sodium chloride and 250 mg of potassium sorbate, add PBS buffer with a pH of 7.4 to make up to 50 mL, stir evenly, and obtain a gel matrix; S2. In the process of magnetically stirring the gel matrix at 700 rpm in an environment of 4°C ± 0.5°C, add 250 mg of tobramycin nanotransfer body I, mix well and continue stirring for 30 minutes under this condition until the solution is clear, transparent and uniform. Swell in a refrigerator at 4°C ± 0.5°C for 12 hours to obtain tobramycin-in situ gel, which is recorded as tobramycin-in situ gel IV.
[0047] Comparative Example 1 This comparative example provides a method for preparing a tobramycin nanotransfer, which is basically the same as that in Example 1, except that in step 1, only 0.5 g of xylo-oligosaccharide is not added, and the amounts of the remaining raw materials and the preparation parameters are the same as those in Example 1. The finally prepared tobramycin nanotransfer is recorded as tobramycin nanotransfer pair I.
[0048] Comparative Example 2 This comparative example provides a tobramycin-in situ gel and a preparation method thereof. The component composition and preparation method of the tobramycin-in situ gel are basically the same as those of Example 7, with the only difference being that the "tobramycin nanotransfer body I" in Example 7 is replaced by an equal amount of "tobramycin nanotransfer body pair I" prepared in comparative example 1, and the amounts of the remaining raw materials and the preparation parameters are the same as those in Example 7. The finally prepared tobramycin-in situ gel is recorded as tobramycin-in situ gel pair I.
[0049] Comparative Example 3 This comparative example provides a tobramycin-in situ gel and a preparation method thereof. The component composition and preparation method of the tobramycin-in situ gel are basically the same as those in Example 7, except that the "Poloxamer 407 27%" in Example 7 is replaced by "Poloxamer 407 17%", and the amounts of the remaining raw materials and the preparation parameters are the same as those in Example 7. The finally prepared tobramycin-in situ gel is recorded as tobramycin-in situ gel pair II.
[0050] Effect Example 1 This effect example measures the particle size, Zeta potential, polydispersity index (PDI), encapsulation efficiency, drug loading and stability of the tobramycin nanotransfer bodies prepared in Examples 1-3 and Comparative Example 1. The specific measurement method is as follows: (1) Determination of average particle size, polydispersity index and zeta potential The tobramycin transfer body was diluted 10 times in distilled water, and its average particle size, polydispersity index (PDI) and Zeta potential were measured using a Malvern laser nanoparticle size analyzer. The measurement results of the particle size, Zeta potential and PDI of tobramycin nanotransfer bodies Ⅰ~Ⅲ and tobramycin nanotransfer body pair Ⅰ are shown in Table 1.
[0051] Table 1
[0052] As shown in Table 1, the average particle size of tobramycin nanotransfers Ⅰ~Ⅲ is 148.2nm~156.2nm, the PDI is 0.210~0.221, and the Zeta potential is within -32.46mV~-31.26mV. The particle size and PDI of tobramycin nanotransfers Ⅰ are significantly higher than those of tobramycin nanotransfers Ⅰ, and the absolute value of Zeta potential is significantly lower than that of tobramycin nanotransfers Ⅰ. Therefore, the stability of tobramycin nanotransfers Ⅰ is better. The reason may be that xylo-oligosaccharides can penetrate into the nanotransfers through hydrogen bonds and physical methods, which can stabilize the particle size and PDI of the transfers.
[0053] (2) Determination of encapsulation efficiency and drug loading The encapsulation efficiency of tobramycin nanotransfersomes was determined by ultrafiltration centrifugation. The specific method is as follows: 0.5 mL of the nanotransfersome solution was placed in the top sleeve of an ultrafiltration centrifuge tube, ultrafiltration centrifuged at 6000 r / min for 15 minutes, the ultrafiltration liquid in the tube was aspirated, and the mass of the free tobramycin nanotransfersomes was calculated. The experiment was repeated 3 times to obtain the average value. The encapsulation efficiency calculation formula is shown in Formula 1.
[0054] Encapsulation efficiency = ×100%Formula 1 In formula 1, m is the total mass of tobramycin added when preparing tobramycin nanotransfersomes, mg; m1 is the mass of free tobramycin, mg.
[0055] 1 g of each of tobramycin nanotransfer bodies I to III and tobramycin nanotransfer body pair I were taken and dissolved in a phosphate buffer with a pH of 7.4 to prepare a solution with a concentration of 0.5 g / mL. The tobramycin content was determined using a liquid chromatography equipped with an evaporative light scattering detector to calculate the drug loading.
[0056] After measurement and calculation, the encapsulation efficiency and drug loading of different tobramycin nanotransfer bodies are shown in Table 2 below.
[0057] Table 2
[0058] As shown in Table 2, the encapsulation efficiency of tobramycin nanotransfersomes Ⅰ to Ⅲ is in the range of 49.78% to 50.47%, and the drug loading is in the range of 9.25% to 9.43%. The encapsulation efficiency and drug loading of tobramycin nanotransfersomes Ⅰ are significantly lower than those of tobramycin nanotransfersomes Ⅰ. The reason may be that xylo-oligosaccharides are stable under pH 6 to 8 and will not break the transfersomes and cause drug leakage.
[0059] (3) Stability test This effect example investigates the average particle size, encapsulation efficiency and drug loading content of tobramycin nanotransfersome Ⅰ and tobramycin nanotransfersome pair Ⅰ after storage under different conditions (-4°C refrigerator and 25°C constant temperature box) for 7 days, 14 days and 21 days, as shown in Table 3.
[0060] Table 3
[0061] As shown in Table 3, compared with tobramycin nanotransfer pair I, tobramycin nanotransfer I is more stable. The reason may be that the introduction of oligoxylose increases the hydrogen bond strength and gives the transfer body a small size and stable PDI. At the same time, the increase of oligoxylose has a significant effect on the gelation and degelation of in-situ gel and stability investigation. It effectively reduces the leakage of drugs.
[0062] Effect Example 2 This effect example measures the gelation temperature and re-gelation stability of the tobramycin-in-situ gel prepared in Examples 4-7 and Comparative Examples 2-3. The specific measurement method is as follows: (1) Determination of the gelation temperature of tobramycin-in situ gel by the test tube inversion method First, pour an appropriate amount of tobramycin-in situ gel into a vial, place it in a water bath, and slowly heat it up; then tilt the vial to observe and record the condition of the drug solution; when the drug solution flows as a semi-solid colloid and there is slight resistance when stirring with a glass rod, record the temperature at this time.
[0063] The gelation temperatures of tobramycin-in-situ gels Ⅰ to Ⅳ and tobramycin-in-situ gel pair Ⅰ were all 30℃~35℃. Among them, the bottle wall of tobramycin-in-situ gel Ⅰ, tobramycin-in-situ gel Ⅱ and tobramycin-in-situ gel pair Ⅰ began to gel 30s~40s after reaching 30℃, and did not flow at all after inverting for 100s; the bottle wall of tobramycin-in-situ gel Ⅲ began to gel 30s~40s after reaching 31℃, and did not flow at all after inverting for 120s; the bottle wall of tobramycin-in-situ gel Ⅳ began to gel 30~40s after reaching 32℃, and did not flow at all after inverting for 120s. The combination of poloxamer 407 and poloxamer 188 can effectively regulate the temperature.
[0064] Tobramycin-in-situ gel pair II did not gel in a hot water bath at 40℃, 50℃, and 60℃ for 5 minutes. The reason may be that the preparation of in-situ gel has certain requirements on the concentration of the gel matrix, and the thermosensitive in-situ gel has a critical phase transition temperature. Only when the concentration reaches the requirement can its structure and other properties change with environmental changes. The degree of cross-linking of the thermosensitive gel material contained in the tobramycin-in-situ gel pair II did not constitute the gel concentration, so it did not gel at 60℃.
[0065] (2) Recondensation stability Place the tobramycin-in-situ gel in a water bath at 40°C to make it a semi-solid gel preparation, keep it for ten minutes, take it out and place it in a cold water bath to restore the fluidity of the in-situ gel, keep it for ten minutes. Continue to heat and re-coagulate at 40°C, and then place it in a cold water bath to restore its shape. Repeat the experiment. One time the gel turns into a liquid is recorded as one re-coagulation. When the re-coagulation effect reaches more than 50 times, the regulations meet the requirements. The specific re-coagulation times of tobramycin-in-situ gels I~IV and tobramycin-in-situ gel pair Ⅰ are shown in Table 4.
[0066] Table 4
[0067] As shown in Table 4, the recoagulation effect of tobramycin-in-situ gel I~IV reached more than 50 times. However, the recoagulation effect of tobramycin-in-situ gel on I was worse, and the particle size increased significantly after 28 recoagulations. The reason may be that oligoxylose can improve the stability of tobramycin nanotransfer bodies, thereby improving the recoagulation stability of tobramycin-in-situ gel.
[0068] In view of the advantages of good biocompatibility, stable drug loading capacity, controllable drug release performance and high re-coagulation stability provided by the present invention, the tobramycin-in-situ gel can be used to prepare an external preparation for treating ocular inflammation.
[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing a tobramycin nanotransferase, characterized in that: The preparation method comprises the following steps: Step 1, mixing lipid, surfactant, xylo-oligosaccharide and alcohol solvent uniformly, and evaporating the alcohol solvent to obtain a film; Step 2, hydrating the film with tobramycin-PBS buffer to obtain a suspension; Step 3: subjecting the suspension to ultrasonic and graded filtration to obtain tobramycin nanotransfersomes.
2. The method for preparing the tobramycin nanotransferase according to claim 1, characterized in that: The mass ratio of the lipid, surfactant and xylooligosaccharide is 4.5-5.5:0.8-1.2:0.4-0.
6.
3. The method for preparing the tobramycin nanotransferase according to claim 1, characterized in that: The lipid comprises at least one of dipalmitoylphosphatidylglycerol, soybean phosphatidylcholine, phospholipid 90G or phospholipid 90H; and / or The surfactant comprises at least one of Span 20, Span 80 or Tween 80; and / or The alcohol solvent includes methanol.
4. The method for preparing the tobramycin nanotransferase according to claim 1, characterized in that: The tobramycin-PBS buffer comprises tobramycin, PBS buffer, a thickener and a moisturizer.
5. The method for preparing the tobramycin nanotransferase according to claim 1, characterized in that: The hydration temperature is 40°C to 50°C and the hydration time is 30min to 60min; and / or The graded filtration includes filtering using filters with pore sizes of 20 μm to 22 μm, 400 nm to 700 nm, and 180 nm to 220 nm in sequence.
6. A tobramycin nanotransfer, characterized in that: The tobramycin nanotransferase is prepared by the preparation method of any one of claims 1 to 5.
7. A tobramycin-in situ gel, characterized in that: The invention comprises the following raw material components in weight fractions: 25% to 30% of a temperature-sensitive gel material, 0.5% to 1.5% of a thickener, 0.5% to 1.5% of a moisturizer, 0.5% to 1.5% of an isotonicity regulator and 0.1% to 0.5% of the tobramycin nanotransfer body, and the balance is PBS buffer.
8. The tobramycin-in situ gel according to claim 7, characterized in that The temperature-sensitive gel material comprises at least one of poloxamer 407, poloxamer 188, tyloxamer, chitosan, polylactic acid-polyethylene glycol block copolymer or poly-N-isopropylacrylamide; and / or The thickener comprises at least one of methylcellulose, hydroxyethylcellulose, hypromellose E15, polyethylene glycol 400 or polycarbophil; and / or The humectant comprises at least one of glycerin, propylene glycol or sorbitol; and / or The isotonicity adjusting agent includes at least one of sodium chloride or mannitol.
9. The method for preparing tobramycin-in situ gel according to claim 7 or 8, characterized in that: The preparation method comprises the following steps: S1. mixing the temperature-sensitive gel material, a thickener, a humectant, an isotonicity adjusting agent and a PBS buffer to obtain a gel matrix; S2. The tobramycin nanotransfer body is mixed evenly with the gel matrix, and swelled at 0°C to 4°C for 10h to 15h to obtain the tobramycin-in-situ gel.
10. Use of the tobramycin-in situ gel according to claim 7 or 8 in the preparation of an external preparation for treating ocular inflammation.