CN114605502A - Hepatitis B virus sample particle nano-carrier and drug delivery system - Google Patents

Hepatitis B virus sample particle nano-carrier and drug delivery system Download PDF

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CN114605502A
CN114605502A CN202210067904.0A CN202210067904A CN114605502A CN 114605502 A CN114605502 A CN 114605502A CN 202210067904 A CN202210067904 A CN 202210067904A CN 114605502 A CN114605502 A CN 114605502A
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column
delivery system
hepatitis
eluent
drug delivery
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CN114605502B (en
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王云龙
李玉林
王继创
张怡青
王敏
王运从
程蕾
韩笑
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Henan Longxing Biotechnology Co ltd
HENAN BIOENGINEERING RESEARCH CENTER
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HENAN BIOENGINEERING RESEARCH CENTER
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Abstract

The invention belongs to the technical field of drug nano-carriers, and particularly relates to a hepatitis B virus-like particle nano-carrier and a drug delivery system. The vector has an amino acid sequence as described below: the method is characterized in that 79-81 th amino acid of hepatitis B core protein HBc-170 is replaced by RGD sequence, and two ends of RGD are respectively connected with 78 th and 82 th amino acid of HBc-170 by adopting connecting peptide to construct the hepatitis B core protein HBc-170. After the hepatitis B virus-like particle nano-carrier encapsulates the drug, the encapsulation stability is obviously improved.

Description

Hepatitis B virus sample particle nano-carrier and drug delivery system
Technical Field
The invention belongs to the technical field of drug nano-carriers, and particularly relates to a hepatitis B virus-like particle nano-carrier and a drug delivery system.
Background
In clinical medicine at present, nanotechnology has made considerable progress, and is increasingly used in many fields such as medicine, material science, biology, and the like, and plays a very important role in each field. The application of nanotechnology reflects the popularity of a new mode of disease treatment and has achieved great success in many academic research and clinical practices. The method for conveying the tumor treatment drug by taking the nano-particles as the carrier overcomes the defects of the traditional chemotherapy drug, and provides possibility for improving the curative effect and reducing the side effect of the chemotherapy drug.
Among the various types of VLPs currently in existence, the hepatitis b virus core protein (HBc) -based virus-like particle is the most flexible vector model. Hepatitis B virus-like particles are simple in source and self-assembled, thus avoiding complex chemical synthesis steps. They are uniform in form and proper in size; has a monodisperse structure and can stably exist under a wide range of pH and temperature conditions; the surface and the interior of the structure have a plurality of reactive sites, and the structure can be self-assembled into a regular icosahedron structure with the size of about 35 nm. In addition, the hepatitis B virus-like particle is used as a protein carrier, has good universality, can be correctly assembled into a complete spherical particle structure in expression systems of pronuclei, eukaryon, mammals and the like, and can be used as a vaccine, an antigen carrier or an adjuvant.
Research proves that the traditional anti-tumor drug is loaded into the HBc-VLP, the DOX is transported to the tumor cells in a targeted manner, the circulation time of the drug in vivo can be prolonged, the toxic and side effects of the drug on normal tissues are reduced, the application in the aspect of anti-tumor drug carriers is better, and the potential in the aspect of patent drug future is greater. However, some problems still remain to be solved in the HBc-VLP, such as low drug loading, poor stability and the like, when forming an encapsulation complex.
It has been proved that the full length of the core protein of hepatitis B virus consists of 183-185 amino acids, while the first 144 amino acids are necessary for correct assembly into the capsid dimer structure. : an N-terminal self-assembly domain (SA, aa 1-150) and a C-terminal poly-arginine domain (CTD, aa 151-183), wherein the SA mainly performs a self-assembly function, and a secondary structure of the SA mainly comprises an alpha helix, the CTD is a random sequence, and the 145 th to 183 th amino acid sequences of the HBc-VLP which have no influence on the assembly of the HBc are rich in arginine.
Chinese patent document CN109529044A discloses a tumor targeting drug and a preparation method thereof, the tumor targeting drug is prepared by encapsulating doxorubicin with a targeting nanocarrier, wherein the targeting nanocarrier has an amino acid sequence as described below: the 72 th-82 th amino acid of HBc-144 is replaced by RGD sequence, and two ends of the RGD sequence are respectively connected with the 77 th and 83 th amino acids of HBc-144 through connecting peptides; the C-terminal tail of HBc-144 can also be connected with a polyhistidine polypeptide. Paragraph [0075] of the patent literature specification and the attached figure 3 of the specification describe that the tumor targeting drug prepared by encapsulating adriamycin by using the targeting nano-carrier is incubated for 4-48h in phosphate buffer solution with pH of 5-8, and sampling is carried out at different time points to calculate the release efficiency. The experimental result shows that under the condition of pH7-8 (close to physiological pH), the drug release efficiency is gradually increased within 4-48 h; the drug release efficiency reaches about 10% in 4 hours; the release efficiency of the drug reaches about 30% when the cells are incubated with phosphate buffer solution with pH7 and pH8 for 24 h. The encapsulation stability of the targeting nanocarrier disclosed in the patent document CN109529044A to the drug is poor under the condition of approaching physiological pH, and needs to be further improved.
Disclosure of Invention
The invention provides a hepatitis B virus-like particle nano-carrier, which is constructed by taking HBc-170 as a main body, and can remarkably improve the encapsulation stability of an encapsulated compound prepared by encapsulating a medicament in physiological conditions and reduce the release rate of the encapsulated medicament in physiological conditions.
It is a second object of the present invention to provide a drug delivery system.
It is a third object of the present invention to provide a method for the preparation of a delivery system as described above.
It is a fourth object of the present invention to provide a method of purifying a drug delivery system as described above.
A fifth object of the present invention is to provide a method for purity detection of a drug delivery system as described above.
It is also an object of the present invention to provide a method of concentration detection for a drug delivery system as described above.
The hepatitis B virus-like particle nano-carrier adopts the following technical scheme: a hepatitis b virus-like particle nanocarrier, the nanocarrier having an amino acid sequence as set forth in seq id no: the method is characterized in that 79-81 th amino acid of hepatitis B core protein HBc-170 is replaced by RGD sequence, and two ends of RGD are respectively connected with 78 th and 82 th amino acid of HBc-170 by adopting connecting peptide to construct the hepatitis B core protein HBc-170.
In a further preferred embodiment, the carrier further comprises a polyhistidine polypeptide linked to the end of the C-terminus.
As a further preferred embodiment, the amino acid sequence of the linker peptide is GTSGTSGSSGSGGT and/or GGSGSSGSTG.
The drug delivery system adopts the following technical scheme: a delivery system comprising a carrier according to any preceding claim and a drug encapsulated within the carrier.
As a further preferred technical scheme, the medicament includes but is not limited to antineoplastic drugs; the anti-tumor drug is an anthracycline drug and is selected from any one or a combination of a plurality of adriamycin, daunorubicin, pirarubicin, epirubicin, aclarubicin and medicinal derivatives thereof.
The preparation method of the drug delivery system adopts the following technical scheme: the method comprises the following steps: (1) incubating the hepatitis B pseudovirus particle nano-carrier with a dissociation solution at 25 ℃ for 2 h; (2) adding a medicine into the mixed solution obtained in the step (1), slightly shaking, and incubating for 30min at 4 ℃; (3) transferring the mixed solution obtained in the step (2) into a dialysis bag with the molecular weight of 3500Da, and firstly putting the dialysis bag into 10% polymerization solution for overnight dialysis at 4 ℃; (4) after 12h, putting the dialysis bag into new 10% polymerization solution and continuing dialysis for 2 h; (5) putting the dialysis bag into 0% of the polymer solution, dialyzing at 4 ℃, replacing 0% of the polymer solution as required in the dialysis process until the dialysate is clear and transparent and does not turn red any more, and stopping dialysis to obtain the drug delivery system; the dissociation liquid is prepared according to the following method: 27g of NaCl, 30g of glycine, 480g of urea, 150ml of 1M Tris-HCl (pH8.0), deionized water and the mixture were added, stirred and dissolved to a constant volume of 1L; the 10% polymerization solution was prepared as follows: 9g of NaCL, 10g of glycine, 100ml of glycerol and 50ml of 1M Tris-HCL (pH8.0), adding deionized water, stirring and dissolving to a constant volume of 1L; the 0% polymerization solution is prepared according to the following method: weighing NaCl 9g and glycine 10g, adding 1M Tris-HCl (pH8.0) 50ml, adding deionized water, stirring and dissolving to volume of 1L.
As a further preferable technical scheme, the dosage of the medicament is 75% of the mass of the hepatitis B virus-like particle nano-carrier.
The purification method of the drug delivery system adopts the following technical scheme: purifying the prepared drug delivery system by DEAE ion exchange chromatography, comprising the following steps:
(1) column equilibration with buffer solution: washing the column with 10 column volumes of a base solution containing 20mM Tris-HCl pH8.0 until the effluent pH is consistent with the base solution pH;
(2) loading: when the numerical value of the ultraviolet detector is zero, the prepared drug delivery system is loaded at the flow rate of 4 mL/min;
(3) and (3) eluting by eluent I: washing the column by using eluent I with 5 times of column volume, and collecting an elution peak to obtain the purified drug delivery system; the eluent I contains 20mM Tris-Cl pH8.0.
As a further preferred technical solution, the purification method further comprises the following steps after elution of eluent I:
(4) and (3) eluting with an eluent II: washing the column with 5 times of the column volume of eluent II, and collecting an elution peak; the eluent II contains 20mM Tris-Cl pH8.0 and 400mM NaCl;
(5) and (3) eluting with eluent III: washing the column with 5 column volumes of eluent III; the eluent III is 0.5M NaOH;
(6) and (3) eluting by eluent I: the pH was equilibrated to pH8.0 with 5 column volumes of the eluent I.
The purity detection method of the drug delivery system adopts the following technical scheme: detecting by adopting HPLC, wherein a chromatographic column is an Ultrahydrogel 2000 chromatographic column; the mobile phase is as follows: 20mmol/L PBS and 150mmol/L NaCl; the length of the chromatographic column is 300nm, the inner diameter of the chromatographic column is 7.8mm, the temperature of the chromatographic column is 25 ℃, and the flow rate of a mobile phase is 0.6 mL/min; the detection wavelengths were 280nm and 480 nm.
The purity detection method of the drug delivery system adopts the following technical scheme: HPLC is adopted for detection, and the chromatographic column is a C18 column.
In a further preferable technical scheme, the chromatographic column is a waters Tnatural C18 column, the length of the column is 250mm, the inner diameter of the column is 4.6mm, the particle size of the filler is 5mm, the temperature of the column is 25 ℃, and the detection wavelength of the detector is 480 nm.
The beneficial effects of the invention are: the hepatitis B virus-like particle nano-carrier constructed by selecting HBc-170 as a main body has obviously better encapsulation stability to drugs than the targeting nano-carrier constructed by taking HBc-144 as a main body. The hepatitis B virus-like particle nano-carrier constructed by increasing the HBc-VLP amino acid sequence has better encapsulation stability, and can be used for encapsulating a plurality of chemotherapeutic drugs such as adriamycin and the like.
The hepatitis B virus-like particle nano-carrier can be prepared in large batch and has higher purity, and because the carrier particles have more arginine sequences, the targeted drug has higher encapsulation amount and better stability, and the drug is not easy to leak in vivo along with blood circulation. The hepatitis B virus-like particle nano-carrier has high encapsulation efficiency, good stability and simple production process, can be prepared in large batch and has great potential in the aspect of future patent medicine.
The release efficiency of the encapsulated compound prepared after the hepatitis B virus-like particle nano-carrier encapsulates the adriamycin in phosphate buffer (physiological pH) with pH7.4 within 24h is less than 10%, and the encapsulation stability is high (the encapsulation stability of the targeted nano-carrier disclosed by Chinese patent document CN109529044A to the medicine is poor under the condition of being close to the physiological pH, and the release efficiency in the phosphate buffer with pH7-8 for 24h is about 30%).
The encapsulation amount of the hepatitis B virus-like particle nano-carrier to adriamycin can reach up to 250 mug/mL (the concentration of the hepatitis B virus-like particle nano is 0.5 mg/mL).
The drug delivery system can realize the targeted transportation of the drug in vivo and has good stability.
Compared with free adriamycin, the adriamycin-encapsulated drug delivery system has better stability, higher bioavailability and small side effect, and can be prepared in a large scale.
The purification method of the drug delivery system has good purification effect and high yield, and is suitable for purifying large-batch samples.
The purity detection method and the concentration detection method of the drug delivery system can provide reliable guarantee for the detection of the concentration and the purity of the drug delivery system, and are convenient for quantifying the preparation effect of the drug delivery system.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a HPLC purity detection profile of a drug delivery system of the present invention;
FIG. 2 is a HPLC concentration profile of the drug delivery system of the present invention;
FIG. 3 is a regression equation of the DOX standard curve for encapsulation in a drug delivery system of the present invention;
FIG. 4 is a graph of the results of a dynamic dialysis assay to determine the stability of the drug delivery system of the present invention in a physiological environment;
FIG. 5 is a HPLC purity detection profile of the delivery system of comparative example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 preparation of hepatitis B Virus-like particle nanocarriers of the present invention
The method comprises the following steps:
(1) the nucleotide sequence of the hepatitis B virus-like particle nano-carrier is obtained by a gene synthesis method according to the amino acid sequence of the hepatitis B virus-like particle nano-carrier, the nucleotide sequence is constructed into an expression vector plasmid pEt43.1(a) through an Xho I/Nde I enzyme cutting site, an RGD sequence is inserted between 78 th amino acid and 82 th amino acid of HBc-170 protein (79 th-81 th amino acid is removed), a connecting peptide GTSGTSGSSGSGGT is inserted between the 78 th amino acid and the RGD sequence, and a connecting peptide GGSGSSGSTG is inserted between the RGD and the 82 th amino acid, so that the amino acid sequence of the RGD-HBc-170 protein (shown as SEQ ID No. 1) is obtained; adding a polyhistidine polypeptide (HHHHHHHH) to the C-terminal tail of the RGD-HBc-170 protein; synthesizing a nucleotide sequence encoding the hepatitis B virus-like particle nanocarrier of the present invention according to the above description; the synthesized nucleotide sequence was ligated to a plasmid (pET21 a).
(2) After obtaining the plasmid containing the target gene, the following steps are carried out: adding the obtained plasmid containing the target gene (1 ng plasmid is added into 100. mu.L of competent cells) into the Escherichia coli competent cell suspension, placing in 42 deg.C water bath for 60-90s, transferring to ice, cooling for 2-3min, and adding LB culture medium for culturing at 37 deg.C after conversion.
(3) And (3) activation: and (3) selecting a monoclonal colony from a recombinant bacterium plate, adding the monoclonal colony into 5ml of a culture medium, carrying out constant-temperature shaking culture at 37 ℃ for 4 hours, and adding a new liquid for bottle expansion culture.
(4) Induction: the temperature of the shaking table is reduced to 26 ℃, the temperature is reduced to the set temperature, 300 mu l of IPTG (isopropyl-beta-thiogalactoside) is added into each bottle of bacterial liquid for induction, shaking table cultivation is carried out at constant temperature, the rotation speed of the shaking table is 200rpm/min, the temperature is 26 ℃, and the cultivation time is 12 hours.
(5) Collecting bacteria, crushing and crude extracting: pouring the induced bacterial liquid into a centrifuge tube, centrifuging at 4 ℃ at the rotation speed of 4000rpm/min for 4min, and discarding the supernatant. Adding high-salt lysis solution into the centrifuged thallus precipitate, blowing the precipitate by a suction tube, and then carrying out ultrasonic crushing.
(6) Ammonium sulfate precipitation: centrifuging the suspension at 12000rpm/min for 10min, pouring the supernatant into a test tube, adding equal volume of precooled pure water into the test tube, stirring in ice bath, adding 1.2 times volume of saturated ammonium sulfate slowly, stirring for 5min, centrifuging, re-suspending the 10min centrifuged precipitate with 20ml of 20mm Tris, ultrasonically crushing, continuing to centrifuge, discarding the precipitate, and retaining the supernatant
(7) Purifying a molecular sieve: the molecular sieves were loaded after previously balancing the pH to 8.0 with an equilibration solution (20mmpH8.0Tris-HCl 300 mmNaCl). Collecting: and after the sample is loaded for about 1h, the reading of the ultraviolet detector begins to rise, the reading rises to more than 20, the effluent protein begins to be collected, 50ml sterile test tubes are used for collection, 50ml of each test tube is used, the number and the OD value are written on the tube body, and the collected protein is placed on ice. And (4) the second peak is collected, and the peristaltic pump is closed after the third peak is completely reduced.
Purifying by a molecular sieve to obtain the purified hepatitis B virus-like particle nano-carrier.
Example 2 construction of a drug delivery System of the invention
2.1 drug Loading
100mL of the target protein (0.5mg/mL) was incubated with 50mL of the prepared dissociation solution at 25 ℃ for 2 hours, and the total volume of the solution was 150 mL. At this point 37.5mg of doxorubicin was added to the above mixed solution and the resulting mixed solution was incubated at 4 ℃ for an additional 30min with shaking. After that, it was transferred to a dialysis bag with a molecular weight of 3500Da, and first placed in 10% of the polymer solution (1L) and dialyzed overnight at 4 ℃, after 12 hours the polymer solution was changed to 10% and dialyzed at 4 ℃, after 2 hours the polymer solution was changed to 0% polymer solution and dialyzed further at 4 ℃, during which the polymer solution was changed 5 times until the dialysate became clear and transparent and did not turn red any more, and the dialysis was stopped, i.e., an encapsulated complex was preliminarily formed (the drug delivery system of the present invention).
Wherein the dissociation solution is prepared according to the following method: 27g of NaCl, 30g of glycine, 480g of urea, 150ml of 1M Tris-HCl (pH8.0), deionized water and the mixture were added, stirred and dissolved to a constant volume of 1L;
the 10% polymerization solution was prepared as follows: 9g of NaCL, 10g of glycine, 100ml of glycerol and 50ml of 1M Tris-HCL (pH8.0), adding deionized water, stirring and dissolving to a constant volume of 1L;
the 0% polymerization solution was prepared as follows: weighing NaCl 9g and glycine 10g, adding 1M Tris-HCl (pH8.0) 50ml, adding deionized water, stirring and dissolving to volume of 1L.
2.2 Complex purification-DEAE ion exchange chromatography
(1) Column equilibration with buffer solution: the column was washed with 10 column volumes of a base solution containing 20mM Tris-HCl (pH8.0) until the effluent pH was consistent with the base solution pH.
(2) Loading: when the UV detector value is set to zero, the encapsulation complex is loaded at a flow rate of 4 mL/min.
(3) And (3) eluting by eluent I: the column was washed with 5 column volumes of eluent I (containing 20mM Tris-Cl (pH8.0)) and the elution peak (i.e., the purified encapsulated complex) was collected.
(5) And (3) eluting with an eluent II: the column was washed with 5 column volumes of eluent II (containing 20mM Tris-Cl (pH8.0), 400mM NaCl) and the eluted peak was collected.
(6) And (3) eluting with eluent III: the column was washed with 5 column volumes of eluent III (0.5M NaOH).
(7) And (3) eluting by eluent I: the ph was equilibrated to pH8.0 with 5 column volumes of eluent I containing 20mM Tris-Cl (pH 8.0).
2.3 Complex concentration
(1) Putting the sample eluted by the eluent I into a 3500Da dialysis bag, attaching dry polyethylene glycol (PEG20000) powder outside, and concentrating in a refrigerator at 4 ℃;
(2) during the concentration process, adding a small amount of dried polyethylene glycol powder for multiple times to increase the concentration speed, and replacing the saturated polyethylene glycol with new polyethylene glycol until the required concentration volume is reached;
(3) after the concentration is finished, the dialysis bag is washed clean by distilled water, and the concentrated protein solution is taken out, so that the purified compound (the drug delivery system of the invention) is obtained. The protein concentration can be quantitatively determined by HPLC as required, and the morphological structure of the resulting complex (the drug delivery system of the present invention) is observed by electron microscopy.
Example 3: the purity of the complex (delivery system of the invention) was verified by means of HPLC
(1) The detection wavelength of the detector of the high performance liquid chromatograph is 280nm (the absorption peak of the hepatitis B virus sample particle nano-carrier of the invention) and 480nm (DOX absorption peak);
(2) the chromatographic column is an Ultrahydrogel 2000 chromatographic column;
(3) the length of the chromatographic column is 300mm
(4) The inner diameter of the chromatographic column is 7.8mm
(5) The column temperature of the chromatographic column is 25 DEG C
(6) The chromatographic column mobile phase is as follows: 20mmol/L PBS, 150mmol/L NaCl;
(7) the flow rate of the chromatographic column: 0.6 mL/min;
(8) by detecting the ultraviolet absorption values of the compound at 280nm and 480nm, a single absorption peak appears at about 14min, no other miscellaneous peak exists, and the peak types of 280nm and 480nm almost completely coincide, which indicates that the compound (prepared in example 2) is successfully prepared and has high purity (shown in figure 1).
Example 4: the concentration of the complex (prepared in example 2) was verified by means of HPLC
(1) The detection wavelength of a detector of the high performance liquid chromatograph is 480 nm;
(2) the chromatographic column is a C18 column;
(3) the column length of the C18 column is 250 mm;
(4) the column inner diameter of the C18 column is 4.6 mm;
(5) the grain diameter of the C18 column packing is 5 mm;
(6) the chromatography column is a waters Tnature C18 column;
(7) the column temperature of the chromatographic column is 25 ℃;
(8) by detecting the ultraviolet absorption value of the compound at 480nm, a single absorption peak appears at about 6min (as shown in figure 2), and the peak area is calculated and substituted into a regression equation (as shown in figure 3, y (peak area) is 8874.4x-19973, R20.9981) the concentration (x) of DOX was calculated.
Through calculation, the encapsulating amount of the hepatitis B virus-like particle nano-carrier to the adriamycin can reach:
(2196708+19973)/8874.4 ═ 250 μ g/mL (concentration of hepatitis B virus-like particle nanocarrier of the present invention was 0.5 mg/mL).
Example 5: verification of the stability of the complexes in physiological environments by dynamic dialysis
The encapsulated drug was tested by HPLC for leakage of DOX in PBS (7.4) over time. DOX-loaded virus-like particles (3mL, DOX concentration 250. mu.g/mL) were transferred into a dialysis bag (molecular weight cut-off: 3500Datons) and placed in phosphate buffer pH7.4 (30mL) under gentle and constant stirring at 37 ℃. 1mL of release medium was collected at 3h intervals and replenished with the same volume of fresh release medium. The compound has better stability under physiological environment and basically no leakage of the drug can be seen by detecting the content of free DOX in different time periods through HPLC (as shown in figure 4).
As can be seen from FIG. 4, in the phosphate buffer solution with pH7.4, the encapsulated drug is basically not leaked within 9-24h, the maximum release efficiency of DOX is below 10%, and the hepatitis B virus-like particle nano-complex of the present invention has good stability.
Comparative example 1
The only difference from example 2 is: the step of "2.2 Complex purification-DEAE ion exchange chromatography" was omitted (the remaining steps were in accordance with example 2), and the prepared complex (drug delivery system) was tested for purity according to the purity test method of example 3. The detection results are shown in the HPLC profile shown in FIG. 5.
As can be seen from fig. 1 and 5, the purification method of the drug delivery system of the present invention has a good purification effect on the prepared complex (drug delivery system).
Comparative example 2 examination of the Effect on yield of the purification method of the drug delivery system of the present invention
1. Sample pretreatment: the encapsulated complex prepared in section 2.1 above was concentrated to a protein concentration of 1mg/mL and DOX concentration of 440. mu.g/mL before purification and was used.
2. And purifying the standby sample obtained by pretreating the sample by an ultracentrifugation method, a molecular sieve and a DEAE ion exchange chromatography method.
(1) The specific steps of ultracentrifugation purification are as follows: different sucrose concentration gradients (10%, 20%, 40%, 60%) are set, centrifugation is carried out at 29000/r for 3h, the obtained target encapsulation complex is subjected to a 40% sucrose gradient, the concentration and the volume of the purified target encapsulate are measured, and the target encapsulation complex (the yield of the drug delivery system of the invention) is calculated.
Determination of sample concentration after purification: firstly, adjusting the protein concentration of the purified target encapsulation compound to 0.5mg/ml, then detecting DOX in the target encapsulation compound by using a liquid phase, and substituting the peak area at 480nm into a regression equation: the concentration of DOX was calculated as y (peak area) 8874.4 x-19973.
The experimental results are as follows: the loading volume was 35ml, and when the protein concentration after ultracentrifugation was 0.5mg/ml, the volume of the complex was 12ml and the DOX concentration was 130 ug/ml.
(2) Agarose gel chromatography column (molecular sieve) purification: the flow rate was 3.5ml/min, the sample volume was 20ml, the UV value was raised to 20 to start the sample receiving, and the first elution peak (eluent 20mM Tris-HCl pH8.0) was received, where the target encapsulation complex was present.
Determination of sample concentration after purification: firstly, regulating the protein concentration of the target compound to 0.5mg/ml, then detecting DOX in the target compound by using a liquid phase, and substituting the peak area at 480nm into a regression equation: the concentration of DOX was calculated as y (peak area) 8874.4 x-19973.
And (3) purification result: the sample loading volume is 20 ml; when the protein concentration after molecular sieve purification is 0.5mg/ml, the volume of the complex is 7ml, and the concentration of DOX is 114 ug/ml.
(3) DEAE purification: the flow rate is 4ml/min, the sample loading volume is 100ml, the ultraviolet instrument value rises to 20, the sample is inoculated, the first elution peak is inoculated, the target encapsulation compound is in the first elution peak (DEAE purification is carried out according to the method of the 2.2 ' compound purification-DEAE ion exchange chromatography ' part, the step (3) eluent elution ' step collects the obtained sample which is the purified sample)
Determination of sample concentration after purification: firstly, regulating the protein concentration of the target compound to 0.5mg/ml, then detecting DOX in the target compound by using a liquid phase, and substituting the peak area at 480nm into a regression equation: the concentration of DOX was calculated as y (peak area) 8874.4 x-19973.
And (3) purification result: the loading volume was 100ml, and when the protein concentration after DEAE purification was 0.5mg/ml, the volume of the complex was 105ml, and the DOX concentration was 250 ug/ml.
(4) The yields of the encapsulation complexes of the present invention after purification by the purification methods of the above (1) to (3) were calculated according to the following formulas, respectively:
yield ═ yield (concentration of DOX after purification × volume of sample) ÷ (concentration of DOX before purification × volume of sample) × 100%
The calculation shows that the yield of the ultracentrifugation purification method is 10 percent; the yield of the molecular sieve purification method is 9 percent; the yield of the DEAE purification method of the present invention is 60%.
(5) And (4) experimental conclusion: the purification method of the drug delivery system has good purification effect, and the yield is obviously superior to other purification methods in the field; in addition, the DEAE column ion exchange chromatography purification method can be suitable for purifying large-batch samples, can effectively improve the purification efficiency, and is used for mass production.
Remarking: the 3 purification methods (1) to (3) can all lead the purified encapsulation compound (drug delivery system) to reach the purity requirement; the ultracentrifugation purification method described in (1) above has a highest yield in many experiments, and even when the ultracentrifugation method is used for purification, a better yield cannot be obtained by adjusting experimental parameters; the molecular sieve purification method described in (2) above is the case where the yield is the highest among a plurality of experiments, and even when purification is performed using a molecular sieve, a better yield cannot be obtained by improving experimental parameters.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> research center of bioengineering technology in Henan province
Longxing Biotech. Co., Ltd, Henan province
<120> hepatitis B virus-like particle nano-carrier and drug delivery system
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 194
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Asp Ile Asp His Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu
1 5 10 15
Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp
20 25 30
Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys
35 40 45
Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu
50 55 60
Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Gly Thr
65 70 75 80
Ser Gly Thr Ser Gly Ser Ser Gly Ser Gly Gly Thr Arg Gly Asp Gly
85 90 95
Gly Ser Gly Ser Ser Gly Ser Thr Gly Lys Ser Arg Glu Leu Val Val
100 105 110
Gly Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Ile Leu Trp
115 120 125
Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr
130 135 140
Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro
145 150 155 160
Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg
165 170 175
Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg
180 185 190
Arg Ser

Claims (10)

1. A hepatitis b virus-like particle nanocarrier, wherein the nanocarrier has an amino acid sequence as follows: the method is characterized in that 79-81 th amino acid of hepatitis B core protein HBc-170 is replaced by RGD sequence, and two ends of RGD are respectively connected with 78 th and 82 th amino acid of HBc-170 by adopting connecting peptide to construct the hepatitis B core protein HBc-170.
2. The hepatitis B virus-like particle nanocarrier of claim 1, wherein the C-terminus of the nanocarrier is further linked to a polyhistidine polypeptide.
3. The hepatitis B virus-like particle nanocarrier of claim 1 or 2, wherein the amino acid sequence of the linker peptide is GTSGTSGSSGSGGT and/or GGSGSSGSTG.
4. A delivery system comprising a carrier according to any of claims 1 to 3 and a drug encapsulated within the carrier.
5. Delivery system according to claim 4, wherein the drug includes, but is not limited to, antineoplastic drugs; the anti-tumor drug is an anthracycline drug and is selected from any one or a combination of a plurality of adriamycin, daunorubicin, pirarubicin, epirubicin, aclarubicin and medicinal derivatives thereof.
6. A method of preparing a delivery system according to claim 4 or 5, comprising the steps of: (1) incubating the hepatitis B virus-like particle nano-carrier with a dissociation solution at 25 ℃ for 2 h; (2) adding a medicine into the mixed solution obtained in the step (1), slightly shaking, and incubating for 30min at 4 ℃; (3) transferring the mixed solution obtained in the step (2) into a dialysis bag with the molecular weight of 3500Da, and firstly putting the dialysis bag into 10% polymerization solution for overnight dialysis at 4 ℃; (4) after 12h, putting the dialysis bag into new 10% polymerization solution and continuing dialysis for 2 h; (5) putting the dialysis bag into 0% of the polymer solution, dialyzing at 4 ℃, replacing 0% of the polymer solution as required in the dialysis process until the dialysate is clear and transparent and does not turn red any more, and stopping dialysis to obtain the drug delivery system; the dissociation solution is prepared according to the following method: 27g of NaCl, 30g of glycine, 480g of urea, 150ml of 1M Tris-HCl (pH8.0), deionized water and the mixture were added, stirred and dissolved to a constant volume of 1L; the 10% polymerization solution was prepared as follows: 9g of NaCL, 10g of glycine, 100ml of glycerol and 50ml of 1M Tris-HCL (pH8.0), adding deionized water, stirring and dissolving to a constant volume of 1L; the 0% polymerization solution is prepared according to the following method: weighing NaCl 9g and glycine 10g, adding 1M Tris-HCl (pH8.0) 50ml, adding deionized water, stirring and dissolving to volume of 1L.
7. The method for preparing the drug delivery system according to claim 6, wherein the drug is doxorubicin, and the amount of doxorubicin is 75% by mass of the hepatitis B virus-like particle nanocarrier.
8. A method of purifying a drug delivery system according to claim 4 or 5, wherein the prepared drug delivery system is purified by DEAE ion exchange chromatography, comprising the steps of:
(1) column equilibration with buffer: washing the column with 10 column volumes of a base solution containing 20mM Tris-HCl pH8.0 until the effluent pH is consistent with the base solution pH;
(2) loading: when the numerical value of the ultraviolet detector is zero, the prepared drug delivery system is loaded at the flow rate of 4 mL/min;
(3) and (3) eluting by eluent I: washing the column with an eluent I with 5 times of column volume, and collecting an elution peak to obtain the purified drug delivery system; the eluent I contains 20mM Tris-Cl pH8.0;
preferably, the purification method further comprises the following steps after elution of eluent I: (4) and (3) eluting with an eluent II: washing the column with 5 times of the column volume of eluent II, and collecting an elution peak; the eluent II contains 20mM Tris-Cl pH8.0 and 400mM NaCl; (5) and (3) eluting with eluent III: washing the column with 5 column volumes of eluent III; the eluent III is 0.5M NaOH; (6) and (3) eluting by eluent I: the pH was equilibrated to pH8.0 with 5 column volumes of the eluent I.
9. The method for detecting the purity of a drug delivery system according to claim 4 or 5, wherein HPLC is adopted for detection, and the chromatographic column is an Ultrahydrogel 2000 chromatographic column; the mobile phase is as follows: 20mmol/L PBS and 150mmol/L NaCl; the length of the chromatographic column is 300nm, the inner diameter of the chromatographic column is 7.8mm, the temperature of the chromatographic column is 25 ℃, and the flow rate of a mobile phase is 0.6 mL/min; the detection wavelengths were 280nm and 480 nm.
10. The method of detecting the concentration of a drug delivery system according to claim 4 or 5, wherein the detection is performed by HPLC, and the chromatographic column is a C18 column; the chromatographic column is a waters Tnature C18 column, the length of the column is 250mm, the inner diameter of the column is 4.6mm, the particle size of the filler is 5mm, the temperature of the column is 25 ℃, and the detection wavelength of the detector is 480 nm.
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