WO2019170061A1 - Method for encapsulating doxorubicin in hfn, and product thereof - Google Patents

Abstract

The invention relates to a method for encapsulating doxorubicin in HFn, and a product thereof. Specifically, the invention relates to exploring the optimal conditions for encapsulating doxorubicin in HFn by specifically using different concentrations of urea, different additives, different HFn/doxorubicin ratios, and/or different incubation conditions, such as the incubation temperature, the incubation time, the incubation buffer solution and the pH value. By using the encapsulation conditions obtained by the invention, the doxorubicin encapsulation in HFn can be fast, convenient, efficient, and sufficient.

Description

Method and product of HFn entrapped doxorubicin Technical field

The present invention relates to a method of encapsulating doxorubicin (DOX) of HFn (human H ferritin, a ferritin formed by self-assembly of the H subunit of human ferritin) and a product thereof. In particular, the present invention relates to methods and products for depolymerizing, refolding HFn and entraining doxorubicin using urea or guanidine hydrochloride.

Background technique

Ferritin is an important functional protein involved in and maintain iron metabolism balance. It is a kind of protein containing high iron content widely distributed in animals, plants and microbial cells. From bacteria to humans, although the ferritin amino acid sequences of different organisms are greatly different, their structures are similar. The typical ferritin structure is composed of a protein shell and an iron core. The protein shell is a cage structure formed by self-assembly of 24 subunits (outer diameter 12 nm, inner diameter 8 nm). The main component of the iron core is ferrihydrite. (5Fe ₂ O ₃ ·9H ₂ O). The ferritin shell is usually composed of two protein subunits (H and L). In different tissues and organs of the body, the proportion of H and L subunits in the ferritin molecule is different.

Human H ferritin (HFn) refers to ferritin formed by self-assembly of the H subunit of human iron eggs. The H subunit of human ferritin can self-assemble to form a cage protein, and the number of H subunits of human ferritin is usually 24. The full length amino acid sequence of human H ferritin is shown in SEQ ID NO: 1.

According to the literature, the drug loading of ferritin from different sources depends mainly on two ways: 1. The drug enters the inside of the protein shell through ion channels or hydrophobic channels on ferritin under specific treatment conditions; 2. Using low pH Or a high concentration of urea, the protein shell is depolymerized, and the drug to be entrapped is added during the subsequent recombination process, thereby realizing the loading of the drug inside the protein shell. Among the above-mentioned encapsulation methods, HFn entrapped doxorubicin is particularly efficient under high-concentration urea conditions, and 33 molecules of doxorubicin are loaded per molecule of HFn; the brief procedure of the method is: adding human HFn to a final concentration greater than 6M In the urea, react at room temperature for 30 minutes, then add appropriate amount of doxorubicin reagent, and protect from light for 10 minutes, then use dialysis method to gradually reduce the urea concentration in the reaction system to 0, thus achieving doxorubicin Envelope in HFn. Compared with other entrapment methods, although the high-concentration urea method effectively increases the loading of doxorubicin in HFn, there is still a large gap in the demand for clinical drugs. According to 1 molecule of ferritin, 33 molecules of doxorubicin are loaded. Calculated, the amount of protein required for a single clinical dose reached a level of grams, significantly exceeding the maximum dose of the existing protein drug (hundreds of milligrams). In order to meet the clinical drug demand, it is necessary in the art to develop a HFn drug entrapment method with a larger drug loading amount.

Doxorubicin is an anti-tumor antibiotic that inhibits the synthesis of RNA and DNA. It has the strongest inhibitory effect on RNA, has a broad anti-tumor spectrum, and has a role in various tumors. It is a non-specific drug for various growth. Periodic tumor cells have a killing effect. It is mainly used for acute leukemia and is effective for acute lymphoblastic leukemia and granulocyte leukemia. It is generally used as a second-line drug, that is, it can be considered when the drug is the drug of choice. For malignant lymphoma, it can be used as the drug of choice for alternate use. It has a certain effect on breast cancer, sarcoma, lung cancer, bladder cancer and other various cancers, and is often used in combination with other anticancer drugs.

However, when used directly in the injection, doxorubicin has a wide range of biochemical effects on the body due to its diffuse distribution throughout the blood, and has a strong cytotoxic effect. The main toxic reactions are: white blood cells and thrombocytopenia, about 60% to 80% of patients can occur; 100% of patients have varying degrees of hair loss, can resume growth after stopping the drug; cardiotoxicity, manifested as arrhythmia, ST- T changes, more often 1 to 6 months after withdrawal; nausea, loss of appetite; drug spillage outside the blood vessels can cause tissue ulcers and necrosis. In addition, urine can appear red after administration.

Therefore, there is a need in the art for a method for efficiently loading doxorubicin with HFn and corresponding products.

Summary of the invention

A first aspect of the invention relates to a method of HFn-loaded doxorubicin comprising the steps of:

1) Depolymerization of HFn in a solution containing 5-9 M urea, an additive for promoting doxorubicin dissolution and doxorubicin for 8-30 h at an incubation temperature of 30 to 60 ° C, preferably, the incubation temperature is 35 to 50 ° C More preferably, 38 to 48 ° C, more preferably 40 to 45 ° C; and / or, preferably, the incubation time is 8 to 20 h, more preferably, 8 to 16 h, more preferably, 10 to 14 h;

2) re-polymerizing HFn and entrapping doxorubicin by removing the denaturant in the solution;

3) optionally, removing residual urea, additives and doxorubicin in the solution;

4) Optionally, drying the above solution to obtain an HFn solid entrapped with doxorubicin;

The additive in which the dissolution of doxorubicin is promoted is selected from the group consisting of glycerin, Tween-20, Triton-100, sucrose, glucose, arginine, betaine or a mixture thereof.

In some embodiments, the amino acid sequence of the H subunit comprising HFn is set forth in SEQ ID NO.

In some embodiments, the additive that promotes doxorubicin dissolution is selected from the group consisting of glycerin or sucrose.

In some embodiments, the mass ratio of HFn to doxorubicin in the solution is from 1:2 to 5:1, preferably from 1:1 to 4:1, more preferably from 8:2.5 to 8:3.5.

In some embodiments, the removal of urea in solution, the additive to promote doxorubicin dissolution, and residual doxorubicin are carried out by desalting, preferably using a desalting column.

In some embodiments, the mass ratio of HFn-loaded doxorubicin is 5% to 60%, preferably, at least 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or higher, more preferably at least 20%, 25%, 30% or 35%.

In some embodiments, the HFn content in the solution ranges from 0.1 to 100 mg/ml, preferably from 0.2 to 10 mg/ml, more preferably from 0.3 to 5 mg/ml, more preferably from 0.5 to 2 mg/ml, more preferably , 0.7 to 1.4 mg/ml; and/or, the content of doxorubicin ranges from 0.1 to 50 mg/ml, preferably from 0.2 to 10 mg/ml, more preferably from 0.3 to 5 mg/ml, more preferably from 0.5 to 2 mg/ml, more preferably 0.7 to 1.4 mg/ml.

In some embodiments, the concentration of urea is 8 M, and/or depolymerization is 9-20 h, more preferably 10-14 h, and/or the incubation temperature is 35 °C to 48 °C, preferably 39 °C to 45 °C.

In some embodiments, the solution is Tris-HCl buffer, citric acid-sodium citrate buffer or acetic acid-sodium acetate buffer, preferably, the pH of the solution is 4-10, more preferably 7-9, More preferably, 7.5 to 8.5; and/or, preferably, the concentration of the solution is 20 to 200 mM, more preferably 25 to 100 mM, more preferably 25 to 50 mM.

A second aspect of the invention relates to an adriamycin-encapsulated HFn obtained by the method of HFn-loaded doxorubicin described in the above first aspect.

A third aspect of the invention relates to a pharmaceutical composition comprising doxorubicin-containing HFn obtained by the method of HFn-loaded doxorubicin described in the above first aspect.

The method for enrolling doxorubicin in the HFn of the present invention can greatly increase the entrapment amount of doxorubicin, and the clinical administration dose will be greatly reduced under the premise of achieving the same therapeutic effect.

DRAWINGS

Figure 1. AKTA map of a separate HFn sample.

Figure 2. AKTA pattern of a HFn sample entrained with DOX. The concentrations of HFn and DOX are consistent. HFn has no special absorption peak at 485nm wavelength, and DOX has a special absorption peak at 485nm wavelength. It can be clearly seen from this figure that there is an absorption peak at 485nm in the peak position of HFn, indicating that HFn is encapsulating DOX. .

Detailed ways

definition

The HFn of the present invention refers to a fully human heavy chain (H subunit) ferritin having an amino acid sequence as shown in SEQ ID NO.

The depolymerization of the present invention means that the tight closed spherical structure of the ferritin natural 24-mer is opened, and the visual representation is that the peak time on the gel exclusion column is significantly delayed, so that the depolymerized protein peak and the unagglomerated protein peak occur. Separation. In the present invention, the depolymerization ratio is at least 50%, such as at least 55%, 60%, 65%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, up to achievable 100%.

Repolymerization means that after the substance causing depolymerization is removed or reduced to a sufficiently low concentration, the depolymerized ferritin returns to the tightly closed globular structure of the natural 24-mer, which is visually represented as a gel-exhaust column. The peak time is reduced to a peak time similar to that of natural ferritin. In the present invention, the polycondensation ratio is at least 50%, such as 55%, 60%, 65%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, up to 100%.

The HFn-loaded doxorubicin in the present invention refers to a process in which doxorubicin is carried in a cage structure of HFn, and is in the same solution system in the process of HFn depolymerization and recombination in a solution system. The doxorubicin loaded will be encased in a cage structure that is re-formed after recombination.

The upper limit of the doxorubicin encapsulation in the HFn of the present invention is the maximum amount of drug that can be contained in the internal volume of the HFn cage structure, i.e., the amount of drug that can be contained in a volume of about 268 cubic nanometers. In some embodiments, the drug loading is 5 to 60%, preferably 10 to 50%, 20 to 40%, 25 to 35%, such as at least 8%, 10%, 15%, in terms of mass ratio. 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or higher. In some embodiments, 50-500 doxorubicin molecules are entrained per molecule of HFn, such as 60, 80, 90, 100, 110, 120, 130, 140, 150, 170, 190, 200, 220, 240, 260 , 280, 240, 260, 280, 300, 350, 400, 450, 500 or more.

The method for enrolling doxorubicin in the HFn of the present invention can greatly increase the entrapment amount of doxorubicin, and the clinical administration dose will be greatly reduced under the premise of achieving the same therapeutic effect.

The buffer system of the doxorubicin-containing solution of the present invention may include, but is not limited to, Tris-HCl, citric acid-sodium citrate, acetic acid-sodium acetate buffer. The buffer concentration is in the range of 20 mM to 200 mM, preferably, 30 mM to 150 mM, more preferably 40 mM to 80 mM, and the pH is in the range of 6.9.0 to 9.0, preferably 7.2 to 8.5, more preferably 7.5 to 8.3. .

The doxorubicin-containing buffer system of the present invention also contains an additive which promotes the dissolution of doxorubicin, and any additive known in the art for promoting the dissolution of doxorubicin can be used in the method of the present invention. In some embodiments, the additive is selected from the group consisting of glycerin, Tween-20, Triton-100, sucrose, glucose, arginine, betaine, or mixtures thereof, and in some embodiments, the additive is selected from the group consisting of glycerin or sucrose. Or a mixture of glycerin and sucrose, in some embodiments, the additive has a volume ratio or weight ratio of from 8 to 40%, preferably from 10 to 25%, more preferably from 12 to 20%.

The doxorubicin-encapsulated HFn of the present invention may be provided in the form of a pharmaceutical composition, i.e., in addition to the HFn encapsulating doxorubicin, which also contains a pharmaceutically acceptable carrier. These pharmaceutical compositions may be formulated according to conventional techniques using pharmaceutically acceptable carriers or diluents, as well as any other known excipients, such as those disclosed in Remington: The Science and Practice of Pharmacy, 22nd edition, edited by Gennaro, Mack Publishing Co., 2013.

The doxorubicin-encapsulated HFn obtained by the method of the present invention can be used to treat and/or prevent a disease or condition in a subject. The disease or condition in which the doxorubicin-coated HFn of the present invention can treat and/or prevent depends on the doxorubicin contained, i.e., it is known in the art to be treated and/or prevented by doxorubicin. The disease or condition can be treated and/or prevented by the doxorubicin-containing HFn of the present invention. Without being bound by any theory, the doxorubicin-encapsulated HFn of the present invention can be targeted to a tumor to release the entrained doxorubicin at the tumor site after administration to achieve prevention and/or treatment of the tumor. In some embodiments, the disease or condition is selected from the group consisting of acute leukemia (lymphocytic and granulocyte), malignant lymphoma, breast cancer, bronchial lung cancer (undifferentiated small cell and non-small cell), ovarian cancer, Soft tissue sarcoma, osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, stomach cancer, liver cancer.

The pharmaceutical compositions of this invention may be administered by any suitable route and mode, including, without limitation, intravenous or subcutaneous injection or infusion.

The pharmaceutical compositions of the invention may also be used in combination with other drugs, such as ABVD, CAF, CAOP, FAM, AC, AOP, ACP, CY-VA-DIC, MACC, or in combination with radiation therapy.

The invention is further illustrated by the following specific examples, which are intended to illustrate the invention and are not intended to limit the scope of the invention. Any changes, modifications, or implementations of the equivalents of the embodiments, which are within the scope of the present invention, should be understood to be within the scope of the present invention.

Example

Example 1 Study on the process of HFn-loaded doxorubicin

1 main equipment

1.1 AKTA avant (with G75 desalting column) (GE Healthcare)

1.2 constant temperature water bath

1.3 Agilent 1260 HPLC (with Agilent SEC-5 300A Gel Exclusion Column) (Agilent Technologies)

2 process conditions research

2.1 Effect of pH on HFn-loaded doxorubicin

2.1.1 Sample preparation

First, HFn (the subunit sequence is as shown in SEQ ID NO. 1, a cage structure composed of 24 subunits, Nature Nanotechnology, 2012, 7(7), 459-464) (50 mM Tris-HCl pH 7.2). Concentrated to 25-30 mg/mL to make it a HFn mother liquor. Prepare 20 mg/mL DOX mother liquor for use in ultrapure water.

Then, using a 50 mM acetic acid-sodium acetate buffer solution having a pH of 4, 5, and 6 and a 50 mM Tris-HCl buffer solution having a pH of 7, 8, 9, 10, respectively, 1000 times of the replacement HFn mother liquid was exchanged. .

Next, weighed 7 parts of 0.960 g of urea, and added 12 mg of HFn with different pH values (4, 5, 6, 7, 8, 9, 10) after each exchange, and then added 0.2 mL of DOX mother liquor, respectively. Add a buffer solution with the same HFn pH value to each sample and dilute to 2 mL.

Each sample was mixed for 30 s using an oscillator, then placed in a 37 ° C water bath for 30 s, and the operation was repeated continuously until the sample was completely mixed and the solid urea was completely dissolved.

Finally, the sample was placed in an incubator at 25 ° C and incubated for 18 h.

2.1.2 Sample Processing

Each sample was directly eluted with urea and free doxorubicin on a 13.5 mL G75 column using the same buffer as the sample pH.

2.1.3 Sample determination

The sample was subjected to SDS-PAGE (determination of the presence of protein), NANODROP (measured by the value of doxorubicin, manufacturer Gene Company Limited Gene Co., Ltd., model ND2000), BCA protein assay (the amount of HFn can be obtained, kit manufacturer) : Thermo Fisher Scientific (China) Co., Ltd., Item No. Prod #23227), HPLC (received monomer after HFn-loaded doxorubicin, manufacturer: Agilent Technologies (China) Co., Ltd., Model High Performance Liquid Chromatography 1260Infinity ) Determination. The results are shown in Table 1.

Table 1. Effect of pH on HFn-encapsulated DOX

Summary: Since DOX degrades under alkaline conditions above pH 8.0, the optimum pH is chosen to be 8.0.

Figures 1 and 2 show the AKTA spectra of individual HFn and DOX-enhanced HFn, respectively, which clearly demonstrate the case of confirming whether HFn encloses DOX.

2.2 The effect of HFn and DOX ratio on HFn entrapped DOX

2.2.1 Sample preparation

The HFn solution (50 mM Tris-HCl pH 8.0) was first concentrated to 25-30 mg/mL to make it a HFn mother liquor. Prepare 20 mg/mL DOX mother liquor for use in ultrapure water. Since many samples need to be configured, the configuration of the samples is recorded in the form of Table 2.

Table 2. HFn: DOX sample ratio table

Note: 2 mL of each sample was prepared and finally made up to 2 mL with a buffer solution of 50 mM Tris-HCl pH 8.0.

Each sample was mixed for 30 s using an oscillator, then placed in a 37 ° C water bath for 30 s, and the operation was repeated continuously until the sample was completely mixed and the solid urea was completely dissolved.

Finally, the sample was placed in an incubator at 25 ° C and incubated for 18 h.

2.2.2 Sample Processing

The sample was directly eluted with urea and free doxorubicin on a 13.5 mL G75 column using 50 mM Tris-HCl pH 8.0 buffer solution.

2.2.3 Sample determination

The sample was subjected to SDS-PAGE (determination of the presence of protein), NANODROP (measured by the value of doxorubicin), BCA protein assay (the amount of HFn can be obtained), and HPLC (the monomer after HFn-loaded doxorubicin) Case) Determination. The results are shown in Table 3.

Table 3. HFn: DOX Data Sheet

Summary: Comprehensive HFn encapsulation of DOX results, from the perspective of the use of DOX, the final choice of HFn is 8mg / mL, DOX is 3mg / mL.

2.3 Effect of time and temperature on HFn entrapped DOX

2.3.1 Sample preparation

The HFn solution (50 mM Tris-HCl pH 8.0) was first concentrated to 25-30 mg/mL to make it a HFn mother liquor. Prepare 20 mg/mL DOX mother liquor for use in ultrapure water.

Each sample was 0.960 g of urea, 16 mg of HFn was added to each urea, and 0.3 mL of DOX mother liquor was added separately. Finally, each sample was separately added to a buffer solution to a volume of 2 mL.

Each sample was mixed for 30 s using an oscillator, then placed in a 37 ° C water bath for 30 s, and the operation was repeated continuously until the sample was completely mixed and the solid urea was completely dissolved. Since the items examined this time are temperature and time, there are 20 samples, and Table 4 provides this information.

Table 4. Temperature and time sample tables

2.3.2 Sample Processing

The sample was directly eluted with urea and free doxorubicin on a 13.5 mL G75 column using 50 mM Tris-HCl pH 8.0 buffer solution.

2.3.3 Sample determination

The sample was subjected to SDS-PAGE (determination of the presence of protein), NANODROP (measured by the value of doxorubicin), BCA protein assay (the amount of HFn can be obtained), and HPLC (the monomer after HFn-loaded doxorubicin) Case) Determination. The results are shown in Tables 5 and 6.

Table 5. Time and temperature data sheets

Summary: The incubation temperature was determined to be 42 ° C and the incubation time was determined to be 12 h.

Table 6. Time and temperature refinement experimental data sheet

Summary: The results showed that the incubation temperature was 42 ° C, and the incubation time was 12 h.

2.4 Effect of additives on HFn entrapped DOX

The present invention also investigates the effect of additives on HFn entrapped DOX. The present inventors have discovered that the additive may primarily address the problem of aggregation of HFn upon entrapment, reducing the amount of HFn entrapped DOX when the additive inhibits aggregation, and increases the amount of HFn entrapped DOX when the additive promotes aggregate generation.

The present invention tested various additives including, but not limited to, glycerin, Tween-20, Triton-100, sucrose, glucose, arginine, betaine or mixtures thereof, and the additives which were found to be more effective were glycerin and sucrose.

2.4.1 Effect of glycerol on HFn entrapped DOX

2.4.1.1 Experimental conditions

Incubation of HFn: DOX mass concentration ratio of 8:3.

Incubation buffer: 50 mM Tris-HCl pH: 8.0 (containing 1%, 5%, 10%, 15%, 20% glycerol).

Urea concentration during incubation: 8M, incubation temperature: 42 ° C, incubation time: 12h, 24h.

2.4.1.2 Sample preparation:

2.4.1.2.1 The purified HFn protein was concentrated and then exchanged using a buffer of 50 mM Tris-HCl pH: 8.0, the volume was changed 1000-fold, and finally HFn was concentrated to about 20 mg/mL for use.

2.4.1.2.2 Weigh 5 parts of 0.960g of urea, then add 6.0mg of DOX per part of urea, mix well, add 16mg of HFn to each mixture of urea and DOX, dissolve in 40°C water bath, then go to 20 μL, 100 μL, 200 μL, 300 μL, and 400 μL of 100% glycerol were added to each sample, and finally each sample was dissolved to 2 mL.

2.4.1.2.3 The sample and the blank control were separately placed at 42 ° C for 12 h, 24 h and the sample was analyzed.

2.4.1.3 Sample Analysis:

2.4.1.3.1 Samples Urea and excess DOX were eluted directly on a G75 column using 50 mM Tris-HCl pH: 8.0 buffer containing the same concentration of glycerol as the sample.

2.4.1.3.2 Perform SDS-PAGE on the sample (determine the presence of protein), NANODROP (measure the value of doxorubicin), BCA protein determination (the amount of HFn can be obtained), HPLC (obtained HFn-loaded mold) The monomer case after the prime) was measured. The results are shown in Table 7.

2.4.2 Effect of sucrose on HFn-encapsulated DOX

2.4.2.1 Experimental conditions

Incubation of HFn: DOX mass concentration ratio of 8:3.

Incubation buffer: 50 mM Tris-HCl pH: 8.0 (containing 1%, 3%, 5%, 10% sucrose)

Urea concentration during incubation: 8M, incubation temperature: 42 ° C, incubation time: 12h, 24h.

2.4.2.2 Sample preparation:

2.4.2.2.1 The purified HFn protein was concentrated and then exchanged using a buffer of 50 mM Tris-HCl pH: 8.0, the volume was changed 1000-fold, and finally HFn was concentrated to about 20 mg/mL for use.

2.4.2.2.2 Weigh 4 parts of 0.960g of urea, then add 6.0mg of DOX per part of urea, mix well, add 16mg of HFn to each mixture of urea and DOX, dissolve in 40°C water bath, then go to 20 mg, 60 mg, 100 mg, and 200 mg of sucrose were added to each sample, and finally each sample was dissolved to 2 mL.

2.4.2.2.3 The sample and the blank control were separately placed at 42 ° C for 12 h, 24 h after the sample was analyzed.

2.4.2.3 Sample Analysis:

2.4.2.3.1 Samples Urea and excess DOX were eluted directly on a G75 column using 50 mM Tris-HCl pH: 8.0 buffer containing the same concentration of sucrose as the sample.

2.4.2.3.2 SDS-PAGE (determination of the presence of protein), NANODROP (measuring the value of doxorubicin), BCA protein determination (the amount of HFn can be obtained), HPLC (obtaining HFn-encapsulated mold) The monomer case after the prime) was measured. The results are shown in Table 7.

2.4.3 Effect of Tween-20 on HFn-packaged DOX

2.4.3.1 Experimental conditions

Incubation of HFn: DOX mass concentration ratio of 8:3.

Incubation buffer: 50 mM Tris-HCl pH: 8.0 (containing 0.01%, 0.05%, 0.10%, 0.50% Tween)

Urea concentration during incubation: 8M, incubation temperature: 42 ° C, incubation time: 12h, 24h.

2.4.3.2 Sample preparation:

2.4.3.2.1 The purified HFn protein was concentrated and then exchanged using a buffer of 50 mM Tris-HCl pH: 8.0, the volume was changed 1000-fold, and finally HFn was concentrated to about 20 mg/mL for use.

2.4.3.2.2 Weigh 4 parts of 0.960g of urea, then add 6.0mg of DOX per part of urea, mix well, add 16mg of HFn to each mixture of urea and DOX, dissolve in 40°C water bath, then go to 0.2 μL, 1 μL, 2 μL, and 10 μL of 100% Tween-20 were added to each sample, and finally each sample was dissolved to 2 mL.

2.4.3.2.3 The sample and the blank control were separately placed at 42 ° C for 12 h, 24 h and the sample was analyzed.

2.4.3.3 Sample Analysis:

2.4.3.3.1 Samples Urea and excess DOX were eluted directly on a G75 column using 50 mM Tris-HCl pH: 8.0 buffer containing the same concentration of Tween-20 as the sample.

2.4.3.3.2 SDS-PAGE (determination of the presence of protein), NANODROP (measuring the value of doxorubicin), BCA protein determination (the amount of HFn can be obtained), HPLC (obtaining HFn-loaded mold) The monomer case after the prime) was measured. The results are shown in Table 7.

2.4.4 Effect of arginine on HFn-encapsulated DOX

2.4.4.1 Experimental conditions

Incubation of HFn: DOX mass concentration ratio of 8:3.

Incubation buffer: 50 mM Tris–HCl pH: 8.0 (containing 1%, 2%, 4% arginine)

Urea concentration during incubation: 8 M, incubation temperature: 42 ° C, incubation time: 12 h.

2.4.4.2 Sample preparation:

2.4.4.2.1 The purified HFn protein was concentrated and then exchanged using a buffer of 50 mM Tris-HCl pH: 8.0, the volume was changed 1000-fold, and finally the HFn was concentrated to about 25-30 mg/mL for use.

2.4.4.2.2 Weigh 3 parts of 0.480g of urea, then add 0.1mL of DOX mother liquor with a concentration of 30mg/mL to each urea, and then add 84μL, 166.5μL, 333μL of 12% fine to each sample. The mother liquor with a pH of 8.0 was mixed well, and 8 mg of HFn was added to each mixture of urea and DOX, and finally each sample was dissolved to 1 mL.

2.4.4.2.3 The sample and the blank control were separately placed at 42 ° C for 12 h and the samples were analyzed.

2.4.4.3 Sample Analysis:

2.4.4.3.1 Samples Urea and excess DOX were eluted directly on a G75 column using 50 mM Tris-HCl pH: 8.0 buffer containing the same concentration of arginine as the sample.

2.4.4.3.2 Perform SDS-PAGE on the sample (determine the presence of protein), NANODROP (measure the value of doxorubicin), BCA protein determination (the amount of HFn can be obtained), HPLC (obtained HFn-encapsulated mold) The monomer case after the prime) was measured. The results are shown in Table 7.

Table 7. Effect of glycerol, sucrose, Tween-20, and arginine on HFn-encapsulated DOX

Effect of 2.5 ionic strength on HFn entrapped DOX

2.5.1 Sample preparation

The HFn solution (50 mM Tris-HCl pH 8.0) was first concentrated to 25-30 mg/mL to make it a HFn mother liquor.

Then weighed 4 parts of 4.800 g of urea, added 60.0 mg of DOX per part, and thoroughly mixed the two, then added 80 mg of HFn per part, and finally added an appropriate amount of 1000 mM Tris-HCl pH 8.0 mother liquor and an appropriate amount of ultrapure water. The volume of the sample was made up to 10 mL, and the Tris-HCl concentration of each sample was 20, 30, 40, 50 mM, respectively.

Each sample was mixed for 30 s using an oscillator, then placed in a 37 ° C water bath for 30 s, and the operation was repeated continuously until the sample was completely mixed and the solid urea was completely dissolved.

Finally, the sample was placed in an incubator at 42 ° C and incubated for 12 h.

2.5.2 Sample Processing

Each sample was directly eluted with urea and free doxorubicin on a 13.5 mL G75 column using 50 mM Tris-HCl pH 8.0 buffer.

2.5.3 Sample determination

The sample was subjected to SDS-PAGE (determination of the presence of protein), NANODROP (measured by the value of doxorubicin), BCA protein assay (the amount of HFn can be obtained), and HPLC (the monomer after HFn-loaded doxorubicin) Case) Determination. The results are shown in Table 8.

Table 8. Effect of ionic strength on HFn-encapsulated DOX

Summary: In order to ensure the buffering capacity of the liquid, the ionic strength of the selected system was 30 mM Tris-HCl.

Claims (11)

A method for HFn-loaded doxorubicin comprising the steps of: 1) Depolymerization of HFn in a solution containing 5-9 M urea, an additive for promoting doxorubicin dissolution and doxorubicin for 8-30 h at an incubation temperature of 30 to 60 ° C, preferably, the incubation temperature is 35 to 50 ° C More preferably, 38 to 48 ° C, more preferably 40 to 45 ° C; and / or, preferably, the incubation time is 8 to 20 h, more preferably, 8 to 16 h, more preferably, 10 to 14 h; 2) re-polymerizing HFn and entrapping doxorubicin by removing the denaturant in the solution; 3) optionally, removing residual urea, additives and doxorubicin in the solution; 4) Optionally, drying the above solution to obtain an HFn solid entrapped with doxorubicin; The additive in which the dissolution of doxorubicin is promoted is selected from the group consisting of glycerin, Tween-20, Triton-100, sucrose, glucose, arginine, betaine or a mixture thereof. The method of HFn-loaded doxorubicin according to claim 1, wherein the amino acid sequence of the H subunit constituting HFn is as shown in SEQ ID NO. The method of HFn-loading doxorubicin according to claim 1 or 2, wherein the additive for promoting doxorubicin dissolution is selected from the group consisting of glycerin or sucrose. The method for HFn-loaded doxorubicin according to any one of claims 1 to 3, wherein the mass ratio of HFn to doxorubicin in the solution is from 1:2 to 5:1, preferably from 1:1 to 4: 1, more preferably, 8:2.5 to 8:3.5. The method of HFn-loaded doxorubicin according to any one of claims 1 to 4, wherein the urea in the solution, the additive which promotes the dissolution of doxorubicin and the residual doxorubicin are carried out by desalting, preferably, desalting The column is carried out. The method for HFn-loaded doxorubicin according to any one of claims 1 to 5, wherein the mass ratio of HFn-loaded doxorubicin is 5% to 60%, preferably at least 8%, 10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or higher, more preferably at least 20%, 25%, 30% or 35%. The method for HFn-loaded doxorubicin according to any one of claims 1 to 6, wherein the HFn content in the solution ranges from 0.1 to 100 mg/ml, preferably from 0.2 to 10 mg/ml, more preferably from 0.3 to 5 mg. /ml, more preferably, 0.5 to 2 mg/ml, more preferably 0.7 to 1.4 mg/ml; and/or the doxorubicin content is in the range of 0.1 to 50 mg/ml, preferably 0.2 to 10 mg/ml, More preferably, it is 0.3 to 5 mg/ml, more preferably 0.5 to 2 mg/ml, and more preferably 0.7 to 1.4 mg/ml. The method of HFn-loaded doxorubicin according to any one of claims 1 to 7, wherein the concentration of urea is 8M, and/or depolymerization is 9-20h, more preferably 10-14h, and/or incubation temperature It is 35 ° C to 48 ° C, preferably 39 ° C to 45 ° C. The method for HFn-loaded doxorubicin according to any one of claims 1 to 8, wherein the solution is Tris-HCl buffer, citric acid-sodium citrate buffer or acetic acid-sodium acetate buffer, preferably, a solution The pH is from 4 to 10, more preferably from 7 to 9, more preferably from 7.5 to 8.5; and/or, preferably, the concentration of the solution is from 20 to 200 mM, more preferably from 25 to 100 mM, more preferably 25 to 50 mM. An adriamycin-loaded HFn obtained by the method of HFn-loaded doxorubicin according to any one of claims 1 to 9. A pharmaceutical composition comprising doxorubicin-containing HFn obtained by the method of HFn-loaded doxorubicin according to any one of claims 1-9.

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