CN114796123B - Self-assembled peptide nano-preparation with anti-hepatic fibrosis function and preparation method and application thereof - Google Patents
Self-assembled peptide nano-preparation with anti-hepatic fibrosis function and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a self-assembled peptide nano preparation with anti-liver fibrosis function, which is formed by self-assembling hydrophilic anti-liver fibrosis polypeptide with 2-20 hydrophobic amino acids in aqueous solution; the hydrophobic amino acid is amino acid containing benzene ring. The invention also provides a preparation method of the following steps: preparing the designed peptide into a solution in a good solvent; adding the obtained solution into a poor solvent; and (3) regulating the pH value to 6.5-7.5 by using an alkaline solution, and then aging to obtain the self-assembled peptide nano preparation. According to the preparation method, the anti-liver fibrosis nano particles are prepared from the anti-liver fibrosis hydrophilic polypeptide through modification of the hydrophobic amino acid, the self-assembled nano particles are formed, the hydrophobic amino acid can form hydrogen bonds with the hydrophilic polypeptide on one hand, and on the other hand, stable dispersion is formed in a poor solvent through accumulation acting force, and the nano particles in the preparation method have the advantages of being strong in targeting, capable of remarkably prolonging the enrichment time of a drug in the liver, stable in property, remarkably improving the drug effect, high in biosafety and the like.
Description
Technical Field
The invention relates to the field of nano biological medicine, in particular to a self-assembled peptide nano preparation with an anti-hepatic fibrosis function, and a preparation method and application thereof.
Background
Polypeptide self-assembly is a technique by which polypeptide molecules can be assembled to form nanoparticles having a uniform distribution of specific dimensions. The polypeptide nano material has wide application prospect, such as wide application in tissue engineering, drug delivery and nanoelectronics.
The polypeptide has very wide application as a therapeutic drug, but the amino acid sequence of the polypeptide determines whether the application is favorable for clinical transformation, for example, the peptide sequence with strong hydrophilicity is easy to degrade in vivo, has short half-life period, poor targeting and short acting time, and also greatly limits the clinical application.
Peptides are important structural units naturally existing in nature, and the amino acid sequence of peptide molecules determines that the peptide molecules can self-assemble to form nano particles with various morphologies under certain conditions. The reasonably designed peptide sequence can self-assemble to form nano particles with a certain size under a certain condition, thereby improving the defects of rapid degradation, lack of targeting and the like of hydrophilic peptide.
Liver fibrosis is a process of excessive accumulation and scarring of extracellular matrix induced by chronic liver injury, an early stage of liver cirrhosis, and no effective therapeutic drug has been clinically used at present. The self-assembled nano particles of the polypeptide molecules have the advantages of no immunogenicity, good biocompatibility, small toxic and side effects and the like when being used in biological medicine. However, the application of reasonably designed polypeptide molecules and preparing nano-preparations based on self-assembly in the aspect of liver fibrosis treatment is not reported yet.
Based on the self-assembled peptide nano preparation with the anti-hepatic fibrosis function, and a preparation method and application thereof are provided.
Disclosure of Invention
The invention aims at designing a nanoparticle formed by introducing hydrophobic amino acid based on hydrophilic polypeptide and self-assembling, and an application and a preparation method thereof in the aspect of liver fibrosis treatment. The nano particles formed by the invention are stable in aqueous solution, have uniform particle size distribution, can prolong the enrichment time of hydrophilic polypeptide in liver, and have the capacity of passively targeting the liver.
To achieve the purpose, the invention adopts the following technical scheme:
a self-assembled peptide nano-preparation with anti-liver fibrosis function, which is formed by self-assembling hydrophilic anti-liver fibrosis polypeptide added with 2-20 hydrophobic amino acids in aqueous solution; the hydrophobic amino acid is amino acid containing benzene ring.
The nanometer preparation is formed by self-assembly in water after hydrophilic polypeptide is introduced into hydrophobic amino acid. It is emphasized that the designed peptide sequences are novel polypeptide molecules formed by covalently introducing amino acids into hydrophilic polypeptides, unlike previous co-assembled forms of hydrophilic and hydrophobic polypeptides.
The nanoparticle formed by self-assembly through hydrogen bond and pi-pi interaction has the advantages of stable property, controllable particle size and capability of prolonging the enrichment time of water-soluble peptide in liver and liver targeting. Thus being applicable to the field of liver fibrosis treatment, and the characteristic greatly promotes the clinical transformation of water-soluble peptides.
Further, the hydrophilic polypeptide chain comprises one or more of Leu-Ser-Lys-Leu (LSKL), his-Ser-Asp-Val-His-Lys (HSDVHK), gln-Ala-Gly-Asp-Val (QAGDV), arg-Leu-Thr-Arg-Lys-Arg-Gly-Leu-Lys (RLTRKRGLK), cys-Pro-Ile-Glu-Asp-Arg-Pro-Met-Cys (CPIEDRPMC), cys-Cys-Gln-Asp-Ser-Glu-Thr-Arg-Thr-Phe-Tyr (CCQDSETRTFY), leu-Arg-Glu-Leu-His-Leu-Asn-Asn-Asn (LRELHLNNN), asp-His-Asn-Asn-Pro-Gln-Ile-Arg (DHNNPQIR).
Further, the hydrophobic amino acid is one or more of phenylalanine (F), tryptophan (W), proline (P) and tyrosine (Y).
The preparation method of the self-assembled peptide nano-preparation with the anti-hepatic fibrosis function comprises the following steps:
(1) Preparing the designed peptide into a solution in a good solvent; wherein the designed peptide is formed by assembling hydrophilic anti-liver fibrosis polypeptide and hydrophobic amino acid;
(2) Adding the solution obtained in the step (1) into a poor solvent;
(3) And (3) regulating the pH value to 6.5-7.5 by using an alkaline solution, and then aging to obtain the self-assembled peptide nano preparation.
Further, the designed peptide has a concentration of 0.1-20mg/mL in the good solvent.
Further, the designed peptide has a concentration of 1-10mg/mL in the good solvent.
Further, the designed peptide has a concentration of 0.01-5mg/mL in the poor solution.
Further, the designed peptide has a concentration of 0.1-3mg/mL in the poor solution.
Further, the good solvent is one or more of tetrahydrofuran, N-dimethylformamide and dimethyl sulfoxide.
Further, the poor solvent is one or more of water, phosphate buffer solution, normal saline and glucose solution for injection.
The self-assembled peptide nano-preparation with the anti-hepatic fibrosis function can be applied to the anti-hepatic fibrosis aspect.
Further, the anti-hepatic fibrosis comprises anti-oxidative stress, anti-hepatic stellate cell activation, induction of hepatic stellate cell apoptosis, inhibition of extracellular matrix synthesis, anti-inflammatory, inhibition of synthesis and activation of undesirable cytokines.
Further, the undesirable cytokines include: transforming growth factor beta, platelet derived growth factor, leptin, interferon-gamma, adiponectin, integrin.
Compared with the existing anti-hepatic fibrosis polypeptide, the invention has the following beneficial effects: first, the nanoparticles provided by the invention enhance the enrichment time of hydrophilic anti-hepatic fibrosis polypeptides in the liver; secondly, the nano particles provided by the invention endow the hydrophilic anti-hepatic fibrosis polypeptide with targeting ability in liver; thirdly, the nano particles provided by the invention have the characteristics of controllable particle size, stable dispersion in a water system, high biological safety, no immunogenicity and the like. The use of other carriers is avoided by the nanoparticles formed by carrier-free self-assembly.
Drawings
FIG. 1 is a graph showing the particle size distribution of nanoparticles in example 1;
FIG. 2 is a graph showing the size distribution of nanoparticles in different tetrahydrofuran systems according to example 1;
FIG. 3 is a transmission electron microscope image of the nanoparticle of example 1;
FIG. 4 is a graph of in vivo mouse targeting versus time fluorescence for nanoparticles of example 1;
FIG. 5 is a statistical plot of the tissue distribution of nanoparticles in example 1;
FIG. 6 is a statistical graph showing the in vivo anti-liver fibrosis serological characterization of liver function for the nanoparticles of example 1;
FIG. 7 is a statistical graph of hydroxyproline effects of in vivo anti-hepatic fibrosis liver histological characterization of nanoparticles of example 1;
FIG. 8 is an H & E staining chart of anti-hepatic fibrosis liver tissue sections in vivo of nanoparticles of example 1;
FIG. 9 is a map of Masson staining of liver tissue sections with anti-liver fibrosis in vivo with nanoparticles of example 1;
FIG. 10 is a graph showing the particle size distribution of nanoparticles in example 2;
FIG. 11 is a transmission electron microscope image of the nanoparticle of example 2;
FIG. 12 is a graph showing the particle size distribution of nanoparticles in example 3;
FIG. 13 is a transmission electron microscope image of the nanoparticle of example 3.
Detailed Description
The invention will be further illustrated with reference to the following specific embodiments, which are intended to illustrate the invention and not to limit it further.
The invention provides a self-assembled peptide nano preparation with anti-liver fibrosis function, which is formed by adding 2-20 hydrophobic amino acids into hydrophilic anti-liver fibrosis polypeptide and self-assembling in aqueous solution; the hydrophobic amino acid is amino acid containing benzene ring.
Wherein the hydrophilic polypeptide chain comprises one or more of Leu-Ser-Lys-Leu (LSKL), his-Ser-Asp-Val-His-Lys (HSDVHK), gln-Ala-Gly-Asp-Val (QAGDV), arg-Leu-Thr-Arg-Lys-Arg-Gly-Leu-Lys (RLTRKRGLK), cys-Pro-Ile-Glu-Asp-Arg-Pro-Met-Cys (CPIEDRPMC), cys-Cys-Gln-Asp-Ser-Glu-Thr-Arg-Thr-Phe-Tyr (CCQDSETRTFY), leu-Arg-Glu-Leu-His-Leu-Asn-Asn-Asn (LRELHLNNN), asp-His-Asn-Pro-Gln-Ile-Arg (DHNNPQIR).
The hydrophobic amino acid is one or more of phenylalanine (F), tryptophan (W), proline (P) and tyrosine (Y).
The preparation method of the self-assembled peptide nano-preparation with the anti-hepatic fibrosis function comprises the following steps:
(1) Preparing the designed peptide into a solution in a good solvent; wherein the designed peptide is formed by assembling hydrophilic anti-liver fibrosis polypeptide and hydrophobic amino acid;
(2) Adding the solution obtained in the step (1) into a poor solvent;
(3) And (3) regulating the pH value to 6.5-7.5 by using an alkaline solution, and then aging to obtain the self-assembled peptide nano preparation.
Wherein the concentration of the designed peptide in the good solvent is 0.1-20mg/mL; for example, 0.1mg/mL,1mg/mL,2mg/mL,4mg/mL,6mg/mL,8mg/mL,10mg/mL,12mg/mL,14mg/mL,16mg/mL,18mg/mL,20mg/mL. The concentration of the designed peptide in the poor solution is 0.01-5mg/mL; for example, 0.01mg/mL,0.1mg/mL,0.5mg/mL,1mg/mL,2mg/mL,3mg/mL,4mg/mL,5mg/mL.
The good solvent comprises but is not limited to one of tetrahydrofuran, N-dimethylformamide or dimethyl sulfoxide; the poor solvent includes, but is not limited to, any one of water, phosphate buffer, physiological saline, and glucose solution for injection.
Illustratively, the polypeptides contemplated in the present invention have the structural formula:
the self-assembled peptide nanometer preparation prepared by the invention has the shape of a sphere, the diameter of the sphere is 50-300 nm, and the nanometer particles with the particle diameter are not directly released into the whole body circulation after entering the circulation system, but are absorbed by the reticuloendothelial system, so that most of medicines are accumulated in the liver to realize targeting. Namely, the nano particles in the range can be effectively enriched in the liver for 48 hours or more, and in addition, the nano particles can also realize liver targeting function, effectively realize aggregation and enhance the drug effect.
The invention is illustrated below by means of more detailed examples:
example 1
In this embodiment, LSKL is used as a basic peptide of a hydrophilic anti-liver fibrosis polypeptide, and a designed polypeptide sequence FFLSKL is obtained by introducing an FF sequence, and the structural formula is as follows:
the sequence is synthesized by a polypeptide solid-phase synthesis method, and specifically comprises the following steps:
(1) Using chloromethyl polystyrene resin as insoluble solid phase carrier, firstly, covalent connecting leucine (L) amino acid with amino group protected by blocking group on the solid phase carrier;
(2) Removing the amino protecting group under the action of trifluoroacetic acid, so that the first amino acid L is connected to the solid phase carrier;
(3) Then the carboxyl of the second amino acid lysine (K) with blocked amino is activated by N, N' -Dicyclohexylcarbodiimide (DCC), the second amino acid K with activated carboxyl reacts with the amino L of the first amino acid connected to the solid phase carrier to form peptide bond, thus generating a dipeptide with protecting group on the solid phase carrier, repeating the steps until the synthesis of the sequence FFLSKL is completed.
5mg of the designed polypeptide FFLSKL is taken and dissolved in 1mL of tetrahydrofuran, ultrasonic treatment is carried out for 10min under the condition of 70HZ, 4mL of pre-prepared sodium hydroxide aqueous solution (100 microlitres of 0.1mol/L sodium hydroxide is added into 4mL of water) is added, and after uniform mixing, the pH is continuously adjusted to 6.80-7.20 by using 0.1mol/L sodium hydroxide solution. In the shade, age for 48h, use a 1kDa dialysis bag in solution: water = 1: dialysis was performed in water for 24h at 100 f to give a nanoparticle dispersion of 1 mg/mL.
The sample was taken and subjected to dynamic light scattering detection, and the result is shown in FIG. 1, which shows that the diameter of the nanoparticle was about 150 nm.
Among these, pH is critical in the system, as amphiphilic polypeptide self-assembly is primarily dependent on the forces of hydrogen bonding. The pH can change the charge distribution in the polypeptide sequence, thereby forming positively or negatively charged polypeptide chains in solution, affecting the formation of hydrogen bonds, and thus affecting the stability and morphology of the self-assembled polypeptide.
In addition, the amount of tetrahydrofuran can influence the size of the nano particles, and when the content of tetrahydrofuran in the system (tetrahydrofuran/water) is adjusted to be 5% -40%, the size of the nano particles is sequentially increased as shown in fig. 2.
Taking 20 microliters of the nanoparticle solution in the example 1, dripping the solution on a copper mesh, adsorbing for 5 minutes, taking out the copper mesh, airing, and carrying out negative dyeing by using 2% phosphotungstic acid, wherein the appearance is observed by a transmission electron microscope as shown in figure 3. The results show that: FFLSKL can self-assemble into uniform nano particles, and the shape is spherical, and the diameter is about 150 nm.
To further examine the performance of the self-assembled peptide nano-preparation with anti-liver fibrosis function prepared in this example, the following tests were performed, respectively:
nanoparticle targeting ability test:
4mg of the designed polypeptide FFLSKL is taken and dissolved in 1mL of tetrahydrofuran, ultrasonic treatment is carried out for 10min under the condition of 70HZ, 4mL of pre-prepared sodium hydroxide aqueous solution (100 microlitres of 0.1mol/L sodium hydroxide is added into 4mL of water) is added, and after uniform mixing, the pH is continuously adjusted to 7.0 by using 0.1mol/L sodium hydroxide solution. In the shade, after aging for 24 hours, 3% of DiR fluorescent dye relative to the mass of the polypeptide was added and aging was continued for 24 hours. 250mg of glucose was added to the solution to prepare an isotonic solution.
The same mass of DiR was dissolved in 10% (dimethyl sulfoxide/water) solution, and glucose was adjusted to isotonicity as a control, and mice were intravenously injected with 0.2mL, and in vivo images of the mice were performed at each time point of 0.5, 1, 2, 4, 6, 8, 12, 24, 48 hours, respectively, as shown in fig. 4. After the last shooting, mice were sacrificed under anesthesia, and the heart, liver, spleen, lung, kidney were collected and fluorescence imaging statistics were performed as shown in fig. 5.
The results show that: nanoparticles are targeted to the liver in a time-dependent manner by passive targeting. The average fluorescence intensity of the region of interest reached a maximum at 48 hours post injection, indicating a longer nanoparticle aggregation time in the liver. The fluorescence intensity detection is carried out on the liver, heart, lung, spleen and kidney of the tissue organs of the mice, and the result shows that the fluorescence intensity in the liver is stronger than that of other tissue organs, which indicates that the nano particles are mainly gathered in the liver.
Nanoparticle in vivo potency (serological level) test:
the nanoparticles prepared in example 1 were taken and prepared into a 1mg/mL solution in a glucose injection having a concentration of 5%. Mice were taken 20, and divided into 4 groups: normal group, liver fibrosis model group, FFLSKL nanoparticle treated group, unmodified peptide LSKL treated group.
The treatment groups were tail-injected with 200 μl of nanoparticles and unmodified peptide LSKL, respectively. The treatment was continued for 14 days. After completion, serum was collected and liver was collected. Serum is used for detecting liver function glutamic pyruvic transaminase (ALT), and the detection method comprises the following steps: the results of 50. Mu.l of mouse serum were measured by a biochemical analyzer using a glutamic acid Aminotransferase (ALT) assay kit (IFCC method) (Michael medical treatment) according to the methods described in the corresponding specifications, and are shown in FIG. 6.
The results show that: the ALT content of the model group is obviously increased, the ALT content of the FFLSKL nanoparticle treatment group is obviously reduced and is obviously lower than that of an unmodified peptide LSKL treatment group, and the difference P is obviously less than 0.05. The modified nanoparticle has a significantly stronger therapeutic effect on liver fibrosis than the unmodified nanoparticle.
Nanoparticle in vivo potency (tissue protein level) test:
hydroxyproline (HYP) is unique to collagen fibers, so the tissue HYP content directly reflects the degree of fibrosis. The liver tissue is taken for HYP content measurement, and the measurement method comprises the following steps: 50mg of fresh liver tissue was taken, 1mL of physiological sodium chloride was added, the tissue was crushed by using a tissue refiner, and after centrifugation at 3000 x for 20min, 50. Mu.L of the supernatant was taken and examined according to the method described in the hydroxyproline kit (alkaline hydrolysis method) (Nanjing built Bio Inc.) specification, and the examination results are shown in FIG. 7.
The results show that: the HYP content of the model group is obviously increased, and the average value reaches 0.19 micrograms/mg liver weight. The FFLSKL nanoparticle treatment group is only 0.12 micrograms/mg liver weight, and the average value of the LSKL treatment group reaches 0.17 micrograms/mg liver weight, which shows that the FFLSKL nanoparticle group has good treatment effect on liver fibrosis and is obviously stronger than the unmodified peptide LSKL treatment group.
Nanoparticle in vivo efficacy (histopathology) test:
the liver tissue is taken and soaked in 4% paraformaldehyde for 48 hours for tissue fixation, paraffin is used for embedding the tissue, and then frozen sections of the liver tissue are carried out for H & E staining.
The specific steps of H & E dyeing include:
1. paraffin sections dewaxed to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II 5min-75% ethanol 5min, and washing with tap water;
2. hematoxylin staining: slicing, dyeing with hematoxylin dye solution for 3-5min, washing with tap water, differentiating with differentiation solution, washing with tap water, and washing with running water;
3. eosin staining: sequentially dehydrating the slices in 85% and 95% gradient alcohol for 5min, and dyeing in eosin dye solution for 5min;
4. and (3) removing the water sealing piece: sequentially slicing, adding absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-absolute ethyl alcohol III 5 min-dimethyl I5 min-dimethyl II 5min, and sealing with neutral resin;
5. microscopic examination, image acquisition and analysis, and the final result is shown in fig. 8.
The results show that: the liver cell cable structure disorder degree, the number of false leaves, the enlargement of cell nuclei, interstitial inflammatory infiltration and the like of the model group are all obviously higher than those of the normal group, which shows that the model is successful and the FFLSKL nanoparticle treatment group shows obviously reduced. While the LSKL treatment group was similar to the model group. The FFLSKL nano particles can well reduce liver injury and fibrosis, and the effect is stronger than that of unmodified peptide LSKL.
Nanoparticle in vivo efficacy (histopathology) test:
the liver tissue is taken and soaked in 4% paraformaldehyde for 48 hours for tissue fixation, paraffin is used for embedding the tissue, and then frozen sections of the liver tissue are carried out for Masson staining.
The specific steps of the Masson dyeing include:
1. paraffin sections dewaxed to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II 5min-75% ethanol 5min, and washing with tap water;
2. immersing the slices in MassonA solution overnight, and washing with tap water;
3. slicing, immersing in dye solution mixed by Masson B solution and Masson C solution in equal ratio for 1min, washing with tap water, differentiating with 1% hydrochloric acid alcohol, and washing with tap water;
4. immersing the slices in Masson D solution for 6min, and rinsing with tap water;
5. dipping and dyeing with Masson E solution for 1min;
6. washing is not performed, and the Masson F liquid is directly inoculated for dyeing for 2-30s after being slightly drained;
7. rinsing and differentiating the slices with 1% glacial acetic acid, and dehydrating with two cylinders of absolute ethyl alcohol;
8. transparent sealing piece: slicing, placing into a third jar of anhydrous ethanol for 5min, allowing xylene to be transparent for 5min, and sealing with neutral resin;
9. microscopic examination, image acquisition and analysis, and the final result is shown in fig. 9.
The results show that: the model group (blue part) had significant collagen deposition, but was almost absent in the normal group. The FFLSKL nanoparticle treated group showed a significant decrease in the amount of collagen compared to the model group. In contrast, a large amount of collagen deposition was observed in the LSKL-treated group. These results demonstrate that FFLSKL nanoparticles can reduce CCl 4 The development of induced liver injury and liver fibrosis has significant therapeutic advantages over LSKL.
Example 2
In this example, QAGDV is used as a base peptide for hydrophilic anti-hepatic fibrosis polypeptide, and by introducing FFY sequence, the designed polypeptide sequence FFYQAGDV is obtained with the following structural formula:
the sequence is synthesized by a polypeptide solid-phase synthesis method, and specifically comprises the following steps:
(1) The chloromethyl polystyrene resin is used as an insoluble solid phase carrier, and firstly, amino acid valine (V) with an amino group protected by a blocking group is covalently connected to the solid phase carrier;
(2) Removing the amino protecting group under the action of trifluoroacetic acid, so that the first amino acid V is connected to the solid phase carrier;
(3) The carboxyl group of the second amino acid aspartic acid (D) with blocked amino group is activated by N, N' -Dicyclohexylcarbodiimide (DCC), the second amino acid D with activated carboxyl group is reacted with the amino group of the first amino acid V connected to the solid phase carrier to form peptide bond, thus forming a dipeptide DV with protecting group on the solid phase carrier, and the steps are repeated until the synthesis of the sequence FFYQAGDV is completed.
5mg of the designed peptide FFYQAGDV was dissolved in 0.5mL of dimethyl sulfoxide, 2mL of water was added, the pH was adjusted to 6.5, and the mixture was placed in a refrigerator at 4℃and aged for 48 hours, and the mixture was dissolved in a nano-solution in neutral water using a dialysis bag: water = 1:100 by volume for 48 hours to obtain 2mg/mL nanoparticle dispersion.
The sample was taken and subjected to dynamic light scattering detection, and the result is shown in FIG. 10, which shows that the diameter of the nanoparticle was about 60 nm.
And (3) taking 20 mu L of the nanoparticle solution, dripping the nanoparticle solution on a copper mesh, adsorbing for 5min, taking out the copper mesh, airing, and carrying out negative dyeing by using 2% phosphotungstic acid, wherein the appearance is observed by a transmission electron microscope as shown in figure 11.
The results show that: FFYQAGDV can self-assemble into uniform nano particles, and has spherical shape and diameter of about 60 nm.
Example 3
In this embodiment, LRELHLNNN is used as a basic peptide of a hydrophilic anti-liver fibrosis polypeptide, and a designed polypeptide sequence FFFFWPPLRELHLNNN is obtained by introducing an FFFFWPP sequence, and the structural formula is as follows:
the sequence is synthesized by a polypeptide solid-phase synthesis method, and comprises the following specific steps:
(1) The chloromethyl polystyrene resin is used as insoluble solid phase carrier, and one amino acid asparagine (N) with amino group protected by blocking group is first connected to the solid phase carrier.
(2) Under the action of trifluoroacetic acid, the amino protecting group is removed, so that the first amino acid N is attached to the solid support.
(3) The carboxyl group of the second amino acid asparagine (N) with blocked amino group is then activated by N, N' -Dicyclohexylcarbodiimide (DCC), the second amino acid N with activated carboxyl group is reacted with the amino group of the first amino acid N attached to the solid support to form a peptide bond, thus forming a dipeptide NN with a protecting group on the solid support, and the above steps are repeated until the synthesis of the sequence FFFFWPPLRELHLNNN is completed.
Taking FFFFWPPLRELHLNNN mg of modified peptide, dissolving in 0.5mL of tetrahydrofuran, adding 3mL of water, adjusting the pH to 7.5, placing in a refrigerator at 4 ℃, aging for 24 hours to obtain a nano solution, and using a dialysis bag to prepare the nano solution in neutral water: water = 1:100 by volume for 48 hours to give 1mg/mL nanoparticle dispersion.
The sample was taken and subjected to dynamic light scattering detection, and the result is shown in FIG. 12, which shows that the diameter of the nanoparticle was about 170 nm.
And (3) taking 20 mu L of the nanoparticle solution, dripping the nanoparticle solution on a copper mesh, adsorbing for 5min, taking out the copper mesh, airing, and carrying out negative dyeing by using 2% phosphotungstic acid, wherein the appearance is observed by a transmission electron microscope and is shown in figure 13.
The results show that: FFFFWPPLRELHLNNN can self-assemble into uniform nano particles, and has spherical shape and diameter of about 170 nm.
According to the anti-liver fibrosis nanoparticle, the anti-liver fibrosis hydrophilic polypeptide is modified by hydrophobic amino acid to form the self-assembled nanoparticle, the hydrophobic amino acid can form a hydrogen bond with the hydrophilic polypeptide on one hand, and on the other hand, stable dispersion is formed in a poor solvent through pi-pi stacking acting force. Compared with the polypeptide before design, the nano-particle has the advantages of strong targeting, capability of remarkably prolonging the enrichment time of the drug in the liver, stable property, remarkably improved drug effect, high biological safety and the like.
The applicant states that the present invention is illustrated by the above examples as a product and a detailed preparation method of the present invention, but the present invention is not limited to the above product and detailed preparation method, i.e., it does not mean that the present invention must be practiced depending on the above product and detailed preparation method. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary materials, selection of specific modes, etc., fall within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present invention within the scope of the technical concept of the present invention. These simple variants are all within the scope of protection of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (7)
1. The self-assembled peptide nano preparation with the anti-hepatic fibrosis function is characterized in that the preparation is formed by self-assembling a polypeptide sequence FFLSKL in an aqueous solution; the nano particles in the nano preparation are spherical, and the diameter of the nano particles is in the range of 50nm-300 nm.
2. A method for preparing a self-assembled peptide nano-preparation with anti-hepatic fibrosis function according to claim 1, comprising the following steps:
(1) Preparing a solution of the polypeptide sequence FFLSKL in a good solvent;
(2) Adding the solution obtained in the step (1) into a poor solvent;
(3) And (3) regulating the pH value to 6.5-7.5 by using an alkaline solution, and then aging to obtain the self-assembled peptide nano preparation.
3. The method for preparing the self-assembled peptide nano-preparation with the anti-hepatic fibrosis function according to claim 2, wherein the concentration of the polypeptide sequence FFLSKL in a good solvent is 0.1-20mg/mL.
4. The method for preparing the self-assembled peptide nano-preparation with the anti-hepatic fibrosis function according to claim 2, wherein the concentration of the polypeptide sequence FFLSKL in a poor solution is 0.01-5mg/mL.
5. The method for preparing a self-assembled peptide nano-preparation with anti-hepatic fibrosis function according to claim 2, wherein the good solvent is one or more of tetrahydrofuran, N-dimethylformamide and dimethyl sulfoxide.
6. The method for preparing a self-assembled peptide nano-preparation with anti-hepatic fibrosis function according to claim 4, wherein the poor solvent is one or more of water, phosphate buffer solution, physiological saline and glucose solution for injection.
7. The use of a self-assembled peptide nano-preparation with anti-liver fibrosis function according to claim 1 for preparing anti-liver fibrosis drugs.
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