Liposome preparation for anticoagulant thrombolytic difunctional fusion protein and preparation method thereof

A technology of fusion proteins and liposomes, which is applied in the directions of liposome delivery, peptide/protein components, and medical preparations of inactive ingredients, etc. Useful for intravenous injections

Inactive Publication Date: 2011-08-10
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This greatly consumes the amount of drug, reduces the utilization rate of the drug, and increases the cost, and t...

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  • Liposome preparation for anticoagulant thrombolytic difunctional fusion protein and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The factors affecting the encapsulation efficiency of liposomes were screened, and 4 factors with greater influence were selected: the ratio of soybean lecithin to cholesterol (A), the drug concentration (B), the volume of PBS (C), and the ultrasonic time in water bath ( D). Taking these four factors as the investigation factors, each factor takes three levels, and adopts the orthogonal design method L 9 (34) to carry out the experiment. The experimental design is shown in Table 1.

[0016] Table 1 Four-factor three-level orthogonal design table

[0017]

[0018] The experimental results are shown in Table 2:

[0019] Table 2 Orthogonal experiment results

[0020] No.

[0021] Through the analysis in Table 2, it can be seen from the range R that the influence of the four factors on the encapsulation efficiency is in the following order: C>A>D>B. Based on the above results, it is determined that the best recipe process combination for the prepara...

Embodiment 2

[0023] The liposome emulsion obtained by film dispersion is further reduced and uniform in particle size by probe ultrasound. The ultrasonic power of the probe was 50W, the interval was 2S, and the cumulative time was 5min, 10min, and 15min.

[0024] Table 3 Effect of probe ultrasound time on encapsulation efficiency, particle size and thrombolytic activity of HV12p-rPA liposomes (n=3)

[0025] The ultrasonic probe time(min)

[0026] It can be seen from Table 3 that with the extension of ultrasonic time, the encapsulation rate of liposomes decreased, and the probe ultrasonication for 10 min had little effect on the encapsulation rate compared with ultrasonic 5 min. As the ultrasonic time prolongs, the particle size gradually decreases between 170-140nm. When the ultrasonic time increased from 5 min to 10 min, the thrombolytic activity did not change, but when the ultrasonic time was extended to 15 min, the thrombolytic activity decreased significantly. Considering th...

Embodiment 3

[0028] Determination of encapsulation efficiency: BCA reagent method The principle is similar to that of the Lowery method for protein quantification, that is, under alkaline conditions, the protein and Cu 2+ Complexation and reduction of Cu 2+ into Cu 1+ . BCA and Cu 1+ Combined to form a stable purple-blue complex, there is a high light absorption value at 570 nm and is proportional to the protein concentration, according to which the protein concentration can be determined.

[0029] Encapsulation efficiency = (total protein * dilution factor - free protein) / total protein * dilution factor * 100%

[0030] Take two liposome suspensions, one is to adjust the Zeta potential to flocculate the liposomes, after refrigerated centrifugation, extract the upper supernatant, and measure the free protein content outside the liposome; the other is added to the demulsifier TritonX-100 to destroy The phospholipid bilayer of the plastid releases the protein drug encapsulated in the li...

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Abstract

The invention relates to the field of biopharmaceutics, in particular to a liposome preparation for anticoagulant thrombolytic difunctional fusion protein, namely anticoagulant thrombolytic difunctional fusion protein of 12 peptides of hirudin and reteplase (HV12p-rPA) and a preparation method thereof. The anticoagulant thrombolytic difunctional fusion protein (HV12p-rPA) constructed in the laboratory has the dual effects of anticoagulation and thrombolysis by structural identification, expression, chromatography renaturation, purification and extracorporal and intracorporal pharmacodynamic experiments. In the preparation method, the liposome preparation is prepared from the anticoagulant thrombolytic difunctional fusion protein serving as a raw material, and a liposome is prepared by an optimized membrane dispersion-probe ultrasonic method; and the optimized membrane dispersion-probe ultrasonic method is characterized in that a prescription which is most suitable for improving the envelop rate of the HV12p-rPA liposome is selected by taking a ratio of phospholipid to cholesterol, the concentration of protein medicaments, the volume of buffer solution and water-bath ultrasonic time as influence factors, and the grain diameter of the HV12p-rPA liposome is reduced and homogenized further by probe ultrasonic, so that the HV12p-rPA liposome of which the grain diameter is between 140 and 145 nanometers and which is used for intravenous injection is prepared. By the preparation method, the envelop rate of the prepared HV12p-rPA liposome is over 90 percent, and extracorporal thrombolytic activity and extracorporal anticoagulant activity are 23,810 international unit (IU) .mg<1> and 414 antithrombin unit (ATU) .mg<1> respectively.

Description

technical field [0001] The present invention relates to the field of biopharmaceuticals, in particular to liposome preparations of novel anticoagulant and thrombolytic bifunctional fusion protein—hirudin 12 peptide and reteplase anticoagulant and thrombolytic bifunctional fusion protein (HV12p-rPA) and preparation method. Background technique [0002] Reteplase (r-PA), an isomer of human tissue plasminogen activator (t-PA), is a third-generation thrombolytic drug that has been used clinically. The 12 peptides (53~64 aa) at the C-terminus of hirudin are the minimum peptides necessary for hirudin to have maximum anticoagulant activity, and its smaller molecular weight is more conducive to reducing bleeding compared with the full-length hirudin protein side effect. In the previous work, our laboratory constructed and expressed the recombinant HV12p-rPA fusion gene (patent number: ZL 2006 1 0022457.8 CN1970574 B). Through gene sequencing, protein structure analysis, chromatog...

Claims

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Application Information

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IPC IPC(8): A61K9/127A61K38/49A61K38/10A61K47/28A61P7/02
Inventor 余蓉邓璇梁兰
Owner SICHUAN UNIV
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