CN107129522B - Lipoic acid modified inherent disordered protein nano-carrier and preparation method and application thereof - Google Patents
Lipoic acid modified inherent disordered protein nano-carrier and preparation method and application thereof Download PDFInfo
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- CN107129522B CN107129522B CN201710202680.9A CN201710202680A CN107129522B CN 107129522 B CN107129522 B CN 107129522B CN 201710202680 A CN201710202680 A CN 201710202680A CN 107129522 B CN107129522 B CN 107129522B
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- lipoic acid
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- acid modified
- polypeptide
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Images
Classifications
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- A—HUMAN NECESSITIES
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Abstract
The invention relates to the technical field of medicines, in particular to a lipoic acid modified inherent disordered protein nano-carrier and a preparation method and application thereof. The nano-carrier provided by the invention is crosslinked by adopting a disulfide bond of lipoic acid, a formed polypeptide polymer can be rapidly degraded in cells and cannot be accumulated in the cells, amino acids forming the polypeptide carrier are all amino acids existing in the body, and the nano-carrier has no toxic or side effect on the cells and human bodies, and a CCK-8 method cell proliferation test shows that the prepared nano-carrier has very low cytotoxicity and simultaneously has better capacity of co-loading genes and chemotherapeutic drugs.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a lipoic acid modified inherent disordered protein nano-carrier and a preparation method and application thereof.
Background
Breast cancer is one of malignant tumors threatening female health, the current clinical treatment is mainly performed by operation, and is simultaneously combined with radiotherapy and chemotherapy drug therapy (chemotherapy for short) to perform comprehensive treatment, wherein chemotherapy plays a key role in breast cancer treatment, but conventional chemotherapy and radiotherapy drugs are not selective and cannot avoid damaging normal cells. Therefore, the development of targeting vectors for tumor cell therapy can greatly improve the effect on tumors. The introduction of the nano technology not only realizes the targeted therapy of the tumor, but also can reduce the toxic and side effects of the chemotherapeutic drugs. Therefore, the preparation of a nano drug delivery system capable of treating breast cancer in a targeted manner is of great significance.
The advent of nanotechnology has provided opportunities for the efficient delivery of drugs into cells. Nanotechnology products in the field of pharmacy include, but are not limited to, nanoparticles, micelles, liposomes, nanofibers, nanotubes, nano-chips, self-assembled polymer nanodevices, etc., which have various advantages: the particle size is small, the particle size distribution is narrow, targeted specific positioning can be carried out after surface modification, drug molecules are protected, the stability of the drug molecules is improved, physiological stimulation responsiveness and external energy (heat, light, sound and magnetism) responsiveness can be endowed, a single carrier can simultaneously encapsulate various therapeutic agents, and imaging and treatment can be combined to monitor the curative effect in real time.
Micelles (also known as micelles) are colloidal solutions formed by self-assembly of excess surfactant in water, and the lowest concentration at which surfactant molecules associate to form micelles is known as the Critical Micelle Concentration (CMC). The process of micelle formation is often referred to in the literature as "self-assembly". The nano-micelle is a nano-scale core-shell micelle formed by self-assembly of an amphiphilic block copolymer with a hydrophilic group and a hydrophobic group in water.
The polypeptide generally has no immunogenicity and can not pass through blood brain barrier, the most problem of natural polypeptide is that the half-life time is short, but the modified and modified long-acting polypeptide can be used as an effective drug carrier, Cell Penetrating Peptides (CPPs) are small molecule short peptides with strong cell membrane penetrating capability and can carry various macromolecules into cells, in 1988, Green L oewenstein et al report that TAT, an I-type immunodeficiency virus transcription activator, can enter cells alone, which is the first mile in the development history of CPPs, Fatowelling et al report that TAT protein can mediate proteins into cells, and the site responsible for exogenous transduction is amino acid 47-57 of TAT protein, which is called "TAT protein transduction structure" (peptide domain), is the first mile in 1994, Fatowelling et al report that TAT protein can mediate proteins into cells, and the site for exogenous transduction is the amino acid 47-57 of TAT protein, which is called "TAT protein transduction structure", so that the polypeptide is a small molecule, so that the polypeptide is easy to metabolize and clear away from the body, and has no obvious side effect, and the polypeptide is a hydrophilic protein, and the polypeptide, which can form a hydrophilic protein, a hydrophobic protein, a polypeptide-protein complex which can be used in the development of a biological cell-protein molecule, a biological molecule with the development and a biological membrane-protein molecule, a biological protein-protein molecule, a biological protein-protein molecule which can be used for the development, a biological protein-protein complex, a biological protein molecule with the development and a biological protein molecule, a biological protein-protein molecule, a biological protein-protein molecule with a biological protein-protein molecule, a biological protein-protein complex, a biological protein complex, a.
Due to the difficulty and complexity of tumor therapy, the cooperative delivery system of chemotherapeutic drugs and gene drugs has become a hot spot for tumor therapy research in recent years. The co-delivery system can improve the transfection efficiency and the synergistic effect of the curative effect of the medicament, thereby improving the curative effect of tumor treatment. One of the challenges in the transport of chemotherapeutic and genetic drugs is the design and development of these co-delivery systems. An effective delivery system can span a variety of obstacles and deliver drugs into cells in the body to produce an anti-tumor effect. Thus, these delivery systems must be multifunctional, and must be stable, specific for long periods of time, and capable of enhanced endosomal escape.
Polypeptide vectors mainly refer to various cell penetrating peptides, which have been researched in the past 20 years due to the high gene therapy ability and low toxicity of the polypeptide vectors, arginine in the polypeptide vectors is found to be capable of effectively adsorbing negatively charged gene substances due to surface-enriched positive charges to form small-particle-size and structurally stable vector/gene complexes ([1] Ma, Y.; Gong, C.; Ma, Y.; Fan, F.; al. direct cytological delivery of cargos, vitamin b. a vector conjugation of D-and L-arginine residues.J. controlled delivery 2012,162,286, 294.[2] belonor 2012, J.; Choi, C.S.; Nam, H.Y.; Park, M.K., Kim.H., Jasson.S. A.201294.; 2. belonor.2012, J. Choi, C.S. S. Pat. No. 9, K.; Nanom, H.Y.; Park. S.S. K., J. 10. Pat. 10. A.S.7, J. 10. endophagostimulsion, W.A.A.A.A.A.22, K., coding, K, and N.A. 9. A.7. A.A. 5. endophagostimulatory, and S.A.A.A.A.A.A. 9. A.A. 9. A. 7, a.A. 7. a.9, a.A. 7. a gene of a gene, a.9, a.A.9. a gene, a.A.9, a. 7. a gene, a.9, a gene encoding, a.9. a.A.A.9, a.9, a.A.A.9, a.9, a.A.A.A.A.A.A.A.A.9, a, a.9, a.A.A.A.A. 9, a gene, a.A.A.A.A.A.A.A.A.A.A.9, a gene, a.A.A.A.A. 9, a.9, a, a.A. 9, a.
In the field of protein science research, the paradigm of "sequence-structure-function" has been the dominance, however, in recent decades the discovery of intrinsically disordered proteins has broken this Kiscoyu law. As early as the early 90 s, several proteins were found to have biological functions, although they did not have secondary and tertiary structures. At The end of The 20 th century, scientists have proposed The concept of Protein triads (The Protein Trinity), considering that in The native state, intracellular proteins and functional Protein domains exist in one of 3 thermodynamic states, namely, The ordered molten globular state and The random coil state, in which The Protein functions directly or by a triplet transition. In 1999, Wright et al proposed the concept of intrinsically disordered proteins.
Intrinsically disordered normal proteins (IDPs) are a class of proteins that have an indeterminate three-dimensional structure in their native state, but are still capable of performing normal biological functions. The regions of such proteins that do not have a stable structure are called disordered regions, and the regions that have a stable structure are called ordered regions. The amino acid residues in the disordered region of IDPs have low hydrophobicity and large electrostatic repulsion, which results in the disordered region assuming a loose state. This form of presentation has many advantages, such as a large contact surface area for the intrinsically disordered protein, conformational flexibility, the possibility of interaction with several ligands, etc. When the disordered regions of IDPs bind to ligands, the disordered regions of IDPs tend to form folded states due to the reduction in static charge. Recent studies have indicated that disordered regions of IDPs play an important role in the interaction with biomolecules. The disordered regions of IDPs may participate in one-to-many or many-to-one interactions, which is an important reason for the functional diversity of IDPs. IDPs play a key role in a variety of physiological activities such as molecular recognition, cell cycle regulation, cell signal transduction, intracellular regulation, and the like.
Although the one-time transmembrane of the CPPs relates to a plurality of mechanisms, the CPPs applied so far have the main transmembrane endocytosis effect, and have the problems of endocytosis capture and lysosome degradation when the endocytosis is carried out.
The lipoic acid is an amphiphilic substance with an intramolecular five-membered ring disulfide bond structure and a terminal carboxyl group, has better affinity with a lipid bilayer of a cell membrane, has better effect of encapsulating chemotherapeutic drugs, and can crack disulfide bonds under the reductive condition of cells so as to provide conditions for the effective release of the drugs at tumor parts.
Based on the theory, the invention provides a lipoic acid modified degradable polypeptide gene vector of IDPs (C L IP6) which enter cells through a non-endocytosis mechanism, and overcomes the defects of weak endocytosis body capture, lysosome degradation, weak capability of introducing gene fragments into cells, low gene transfection efficiency and nondegradable property of the current polypeptide vector.
Chinese patent document CN105727307A discloses a lipoic acid modified nano-polypeptide carrier, which consists of arginine, histidine, lipoic acid and cysteine, disulfide bonds carried by the lipoic acid are adopted for crosslinking, and the formed polypeptide polymer can be rapidly degraded in cells, has very low cytotoxicity, simultaneously has better gene and chemotherapeutic drug co-loading capability, and can specifically enhance the sensitivity of drug-resistant cells to chemotherapeutic drugs and promote the apoptosis of breast cancer cells in the drug-resistant treatment of breast cancer.
At present, no lipoic acid modified inherent disordered protein nano-carrier exists, and a nano delivery carrier capable of treating breast cancer in a targeted manner is available.
Disclosure of Invention
The invention aims to provide a biodegradable polypeptide nano-carrier with high gene transfection efficiency, wherein a chemotherapy drug is wrapped in an inner cavity, and a gene is wrapped outside the nano-carrier. Another object of the present invention is to provide a method for preparing the nanocarrier; the third purpose of the invention is to provide the application of the nano-carrier in co-carrying chemotherapeutic drugs and gene therapy drugs.
The invention aims to solve the main technical problems that: how to improve the capability of the polypeptide nano-carrier for guiding the gene segment to enter the cell and how to improve the capability of the polypeptide nano-carrier for carrying the chemotherapeutic drug and the gene together, and simultaneously ensure that the material has the biodegradable characteristic.
The invention designs a lipoic acid modified polypeptide nano carrier of inherent disordered protein, which is a polypeptide consisting of lysine, valine, arginine, proline, threonine and glutamic acid, wherein arginine with positive charge can be combined with a gene fragment with negative charge and has a membrane penetrating effect, and an anionic glutamic acid residue in the inherent disordered polypeptide is a key amino acid for maintaining the disordered biological activity state of the inherent disordered polypeptide and is also related to the unique non-endocytosis mechanism and better biocompatibility of the inherent disordered protein. The lipoic acid part can increase the affinity of the carrier and cell membranes and can entrap chemotherapeutic drugs to achieve co-loading, and the cross-linking of disulfide bonds of the lipoic acid part increases the drug loading and transfection capacities, and can be cracked under the bioreductive condition to realize the purpose of drug release.
Compared with polypeptide L AHR modified by lipoic acid in CN105727307A, the invention only uses the lipoic acid of the prior invention continuously, realizes tumor microenvironment sensitivity between disulfide bonds of the lipoic acid, and the cell-penetrating peptide C L IP6 used in the invention is a degradable polypeptide gene vector of IDPs (C L IP6) entering cells by a non-endocytosis mechanism, wherein glutamic acid plays a role, the cell-penetrating peptide enters the cells through a non-endocytosis approach, wherein the membrane-penetrating effect and the DNA or RNA carrying capacity can be endowed by rich arginine, D-proline enables the protease hydrolysis stability of the polypeptide gene vector to be higher, and the drug molecules with weak cell permeability and lysosome degradation can be expected to enter the cells, so that the defects that the existing polypeptide vector has endocytosis capture, lysosome degradation, the introduction of gene fragments into the cells is not strong, the gene transfection efficiency is not high, the non-degradable performance is overcome, the lipoic acid modified C L IP6 is provided by the invention, the result of laser confocal inspection of the in vivo distribution of the lipoic acid also proves that the C L IP can effectively enter the cells through a micelle-labeled fluorescent micelle-labeled micelle concentration measurement method, and the critical fluorescence of the fluorescent micelle concentration of the siRNA 0.00327 is very small.
In a first aspect of the present invention, a lipoic acid modified polypeptide is provided, where the polypeptide is an internalization peptide 6(C L IP6) with IDPs cytoplasmically located, and the amino acid sequence of the polypeptide is as follows:
CLIP6：KVRVRVRVDPTRVRERVK(Dp is D-proline) (SEQ ID NO: 1), the amino acids are linked by peptide bonds, and the polypeptide can be abbreviated as C L IP6 and C L.
The lipoic acid modification means that the carboxyl of the lipoic acid is connected with the amino of the first lysine through an amide group.
The lipoic acid modified polypeptide can be abbreviated as L A-C L IP6, which is abbreviated as L A-C L, wherein L A is lipoic acid, and C L is C L IP6 (cytosol-localization interaction peptide 6).
In a second aspect of the present invention, a lipoic acid modified inherently disordered protein nanocarrier is provided, wherein the nanocarrier is a polymer of the lipoic acid modified polypeptide, and the polymer is formed by crosslinking lipoic acid disulfide bonds through cysteine.
The chemical structural formula of the monomer of the polymer is shown as a formula (I), the chemical structural formula of the monomer of the polymer is reduced as a formula (II), and the chemical structure of the lipoic acid modified inherent disordered protein nano carrier is shown as a formula (III):
in formula (II), KVRVRVRVRVRVRVDPTRVRERVK, K is lysine; v valine; r is arginine;Dp is D-proline; t is threonine; and E, glutamic acid.
The polypeptide C L IP6 KVRVRVRVRVRV of the inventionDPTRVRERVK, K is lysine; v valine; r is arginine;Dthe polypeptide comprises 17 peptides consisting of P, D-proline, T, threonine and E, wherein amino acids are connected through peptide bonds and are abbreviated as C L in English, lipoic acid and amino groups are connected through amide bonds at the N end of the 17 peptides, the polypeptide modified by the lipoic acid is abbreviated as L A-C L in English, and the sulfhydryl group of the lipoic acid is oxidized and crosslinked through cysteine to form a polymer which is abbreviated as L A-C L ss in English.
Preferably, the molecular weight of the polymer is 2000-20000Da, and the polymer with the molecular weight is not suitable, so that the transfection efficiency of the gene vector is reduced; most preferably 15000-.
Preferably, the molar amount of cysteine in the polymer is 10% of the lipoic acid modified polypeptide.
In a third aspect of the present invention, there is provided a preparation method of the lipoic acid modified inherently disordered protein nano-carrier, wherein the preparation method comprises the following steps:
(A) synthesizing the lipoic acid modified polypeptide L A-C L;
(B) and (2) preparing the lipoic acid modified inherent disordered protein nano carrier, namely dissolving the lipoic acid modified polypeptide L A-C L synthesized in the step (A) in methanol, adding cysteine hydrochloride to ensure that the molar weight of cysteine is 5-20% of that of the lipoic acid modified polypeptide, and stirring and reacting for 12 hours in the absence of light.
In a preferred embodiment of the invention, the step (B) is specifically that the lipoic acid modified polypeptide L A-C L synthesized in the step (A) and cysteine hydrochloride are dissolved in methanol, so that the molar amount of cysteine is 10% of that of the lipoic acid modified polypeptide, and the lipoic acid modified polypeptide is stirred away from light and reacted for 12 hours.
Preferably, the solution after the reaction in step (B) is treated with N2And (5) drying. With N2The dried samples were stored at-20 ℃.
Transferring the solution reacted in the step (B) into a dialysis bag with the molecular weight cutoff of 1000, wherein the dialysate is distilled water, and dialyzing for 12 hours.
In order to maintain the higher activity of the nano carrier material, the dialyzed solution is freeze-dried and stored at-20 ℃, and the nano material can be stored for a long time at 4 ℃ after being redissolved.
In a fourth aspect of the present invention, the lipoic acid modified intrinsically disordered protein nano-carrier is provided for use in the preparation of a combined chemotherapeutic drug or a genetic drug.
Furthermore, the invention provides an application of the lipoic acid modified intrinsically disordered protein nano-carrier in preparing a breast cancer treatment drug.
Furthermore, the invention also provides the application of the lipoic acid modified inherent disordered protein nano-carrier co-carried gene and chemotherapeutic drugs in the preparation of breast cancer treatment drugs.
The application refers to that arginine in the nano carrier is positively charged and can be combined with negatively charged genes.
The application refers to that the fat solubility of the lipoic acid in the nano carrier can carry fat-soluble chemotherapeutic drugs.
The gene is DNA or siRNA.
The chemotherapy medicine is liposoluble adriamycin, and can also be docetaxel, cyclophosphamide, etc.
The application is as follows:
preparing nano micelles by using ultrasonic emulsification of the nano carrier and the chemotherapeutic drugs, and mixing the nano micelles with DNA to prepare a co-loading system;
the encapsulation rate of the chemotherapeutic drug is 40% -50%, and the nitrogen-phosphorus ratio of the nano-carrier to the DNA is 5:1-80: 1.
The nano-carrier is mixed with DNA or siRNA to prepare a gene transfection system. The N/P ratio of the nano-carrier to the DNA or siRNA is 5:1-80:1, and in the ratio range, the nano-carrier material can guide the DNA or siRNA into cells and has higher transfection efficiency.
The ability of the nano-carrier to entrap paclitaxel has better drug-loading rate and encapsulation efficiency when the cysteine proportion is 5-20%.
The nano-carrier and DNA or siRNA are mixed in a buffer solution, the pH value of the buffer solution is 5.0-7.0, the incubation is carried out for 20-30 minutes at room temperature, and the formation of a gene transfection system is ensured by reasonable pH value and incubation time.
The nano-carrier provided by the invention is suitable for therapeutic plasmid DNA or siRNA and chemotherapeutic drugs required by experiments.
The invention has the advantages that:
1. the nano-carrier provided by the invention is crosslinked by adopting a disulfide bond of lipoic acid, a formed polypeptide polymer can be rapidly degraded in cells and cannot be accumulated in the cells, amino acids in the polypeptide are all amino acids existing in vivo, and the nano-carrier has no toxic or side effect on cells and human bodies, and a CCK-8 method cell proliferation test shows that the prepared nano-carrier has lower cytotoxicity compared with bPEI-25K, and simultaneously has better gene and chemotherapeutic drug co-carrying capacity, so that the nano-carrier is very suitable for in-vivo and in-vitro chemotherapy and gene therapy research and application;
2. the nano-carrier can successfully deliver the autophagy specificity caused by siRNA inhibition chemotherapy drugs in the treatment of breast cancer, enhance the sensitivity of tumor cells to the chemotherapy drugs and promote the apoptosis of breast cancer cells, thereby forming a targeted, high-efficiency and low-toxicity nano-scale delivery system for treating the breast cancer;
3. the preparation method disclosed by the invention is simple to operate, the reaction reagent and the obtained product are non-toxic, the environment cannot be polluted, the reaction condition is mild, the nano-carrier obtained after the reaction is simple to purify, the components are low, the crosslinking degree of the nano-carrier can be controlled by controlling the ratio of the polypeptide to the cysteine, and the preparation method is beneficial to large-scale popularization in the fields of research and application.
Drawings
FIG. 1 is a NMR spectrum of L A-C L ss;
FIG. 2 is a graph showing the particle sizes of L A-C L ss/pEGFP at different degrees of crosslinking under different N/P ratios;
FIG. 3 is a potential diagram of L A-C L ss/pEGFP nanomicelle at different degrees of cross-linking;
FIG. 4 is a view of transfection effects of vectors with different degrees of cross-linking under different N/P conditions;
FIG. 5 is a transmission electron microscope image showing the particle size of the nanomicelle when the amount of cysteine is 10% and N/P is 40;
FIG. 6 is a potential diagram of nanomicelle at a cysteine content of 10% and an N/P of 40;
FIG. 7 shows the release degree investigation of DTX under different release medium conditions;
FIG. 8 shows MCF-7 cells uptake of L A-C L ss/FAM-siRNA at different N/P ratios;
FIG. 9 shows the uptake of Nile Red (Nile Red) and Nile Red loaded micelles (Nile Red PMs) by MCF-7 cells at different time points;
FIG. 10 is a study of laser confocal loading of Nile Red and FAM-siRNA carried by L A-C L ss micelles on their co-loading capability;
FIG. 11. cytotoxicity of the vector and bPEI-25K on cells at different concentrations and different times;
FIG. 12 is a graph showing 24h cytotoxicity on MCF-7 cells under different concentrations of DTX;
FIG. 13 is a graph showing the 48h cytotoxicity of MCF-7 cells under different concentrations of DTX;
FIG. 14 flow cytometry observation of the effect of different dosing groups on MCF-7 apoptosis;
FIG. 15 flow cytometry was used to observe the effect of different dosing groups on the MCF-7 cell cycle;
Detailed Description
The invention is further described below with reference to the following figures and specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Example 1: synthesis of lipoic acid modified 17 peptides
Lipoic acid (L A) modificationTrim 17 peptide amino acid sequence L ys Val Arg Val Arg Val Arg ValDProThr Arg Val Arg Gln Arg Val L ys (L ys: lysine, Val: valine, lysine, valine,DPRO D-proline, Thr threonine, Arg arginine, Gln glutamic acid) (SEQ ID NO: 1), L A-C L, synthesized by Shanghai Jier Biochemical Limited company by a polypeptide solid phase synthesis method and named as L A-C L, L A-C L purified and synthesized by preparative high performance liquid chromatography to ensure that the purity of the product reaches more than 95%, L A-is lipoic acid, and C L has the sequence KVRVRVRVRVRVRVDPTRVRERVK are provided. Wherein K is lysine; v valine; r is arginine;Dp is D-proline; t is threonine; glutamic acid, amino acids are connected by peptide bond to form 17 peptide.
Example 2 preparation of lipoic acid modified polypeptide nanocarriers L A-C L ss
50mg of lipoic acid modified polypeptide L A-C L and different amounts of cysteine hydrochloride are dissolved in 10ml of methanol, stirred and reacted for 12 hours at the temperature of 10-30 ℃, wherein the amount of cysteine is respectively 2.5%, 5%, 10% and 20%.
As shown in Table 1, according to different proportions of L A-C L and cysteine, L A-C L ss. solutions with different molecular weights were prepared and dialyzed for 12 hours in dialysis bags with a molecular weight cut-off of 1000, the dialyzate was distilled water, and the dialyzate was replaced every 4 hours, the resulting dialyzate was freeze-dried and stored at-20 deg.C, the freeze-dried carrier was preserved for a long period of time under the condition of reconstitution, and the synthesized carrier was subjected to hydrogen nuclear magnetic resonance spectroscopy1H-NMR (600M) and molecular weight by Gel Permeation Chromatography (GPC).
TABLE 1 Synthesis of L A-C L ss of varying molecular weights
Note that:acysteine to L A-C L.bDetection by gel permeation chromatography.
As can be seen from Table 1, the molecular weight of the polymer increased significantly with the addition of cysteine, indicating that the carrier was successfully crosslinked and that the molecular weight increased with the increase in the amount of cysteine.
EXAMPLE 3 preparation of L A-C L ss in pDNA nanomicelle
Respectively dissolving a vector (L A-C L ss) and a luciferase expression plasmid pEGFP (Shanghai creative biotechnology Limited company) in water to prepare an aqueous solution, configuring a nano compound according to nitrogen-phosphorus ratios (N/P) of 5, 10, 20, 40 and 80, respectively, swirling for 10s, and standing for 30min to obtain the nano micelle, wherein the average particle size of the nano micelle is related to N/P, the optimum particle size is obtained when the N/P is 40, the particle size is between 100 and 300, specifically shown in figure 2, the Zeta potential of the nano micelle is increased to increase the N/P, and is stabilized at 0-30mV when the N/P is more than 5, and specifically shown in figure 3.
Example 4 in vitro transfection efficiency study of L AHRss/pEGFP
Cells of the human breast cancer cell line MCF-7 (purchased from cell culture center of Shanghai bioscience research institute of Chinese academy of sciences) were inoculated onto 12-well plates at 30 w/well, 1ml of DMRM medium (Gibco, USA) containing 10% FBS (Gibco, USA) was added and cultured for 24 hours to achieve 70-80% cell confluency, and the medium was replaced with a serum-free medium.
Respectively dissolving vectors (L A-C L) with different cross-linking degrees and luciferase expression plasmids pEGFP in water to prepare aqueous solution, preparing nano-composite vortex according to nitrogen-phosphorus ratios (N/P) of 2.5, 5, 10, 20, 40 and 80 respectively, standing for 30min after 10s, adding the nano-composite vortex into a pore plate, culturing for 4 hours, replacing with a fresh culture medium, and continuously culturing for 24 hours.
From the above results, L A-C L ss3 has a more suitable molecular weight, smaller particle size, higher positive charge, and better transfection fluorescence intensity under these conditions, so L A-C L ss3 was taken for further examination.
EXAMPLE 5 preparation of L A-C L ss3/(DTX/siATG7) nanomicelle
1mg of docetaxel was dissolved in 1ml of dichloromethane, and 20mg of a carrier (L A-C L ss3) crosslinked with cysteine was dissolved in 1ml of dichloromethane.
Mixing the two solutions, slowly adding into 10ml of pure water containing 1% sodium cholate dropwise, stirring to volatilize the solvent, ultrafiltering to remove sodium cholate, and calculating the encapsulation efficiency and drug-loading rate. ATG7siRNA was added at N/P ═ 40, the particle size and potential were measured, and the nanocomposite morphology was observed under a transmission electron microscope, as shown in fig. 5 and 6.
As can be seen from FIGS. 5 and 6, the obtained nano-micelle has a particle size of about 160nm and a potential of more than 30mV, and can ensure the gene-loading capacity and good membrane penetration. FIG. 5 shows that the nano-micelle has a complete shape, is nearly spherical, is uniformly dispersed, and can better prevent aggregation.
Example 6: docetaxel in vitro release characteristics investigation
Examining the in vitro release of DTX by dialysis bag method, selecting a dialysis bag with molecular weight cut-off of 3500, and containing DTT (50 × 10) containing 0.5% w/v Tween-80 in PBS solution with pH of 7.4-3M) and a solution without DTT, at 37 ℃, 1mg of docetaxel is dissolved in 1ml of dichloromethane, 2mg of a carrier (L A-C L ss3) is dissolved in 1ml of dichloromethane, the two solutions are mixed and then added with 8ml of water to prepare nano micelles by an ultrasonic emulsification method, the nano micelles are slowly dripped into 10ml of pure water containing 1% of sodium cholate, the organic solvent is stirred and volatilized, the sodium cholate is removed by ultrafiltration to prepare L A-C L ss3/DTX nano micelles, 2ml of L A-C L ss3/DOX PBS solution is placed in a dialysis bag and is placed in a buffer solution containing 40ml of PBS, 100r/min, 37 ℃, 1ml of external solution is taken at time points of 2, 4, 6, 8, 10, 12, 24, 48 and 72 respectively and is added with 1ml of external solution, the concentration of the external solution is measured by high performance liquid chromatography, and an in-vitro release curve is drawn as shown in figure 7.
As can be seen from fig. 7, the release rate of docetaxel under DTT condition is significantly faster than that without DTT, and its cumulative release rate at 48 hours is nearly 85%. The result shows that the polypeptide carrier has reductive condition-sensitive behavior, and provides conditions for the release of the antitumor drug in a tumor microenvironment.
Example 7 cellular uptake of L A-C L ss3/Nile Red, L A-C L ss3/FAM-siRNA
MCF-7 cells were inoculated onto 12-well plates at 30 w/well, respectively, and 1ml of DMEM medium (Gibco, USA) containing 10% FBS (Gibco, U.S.) was added to culture for 24 hours to reach 70-80% confluency, and the medium was replaced with serum-free medium, L A-C L ss3/Nile Red, Nile Red were added to the cell wells at 0.1. mu.g/ml, and the medium was aspirated at 1, 2, and 4 hours, PBS was washed 3 times, cells were collected by digestion and centrifugation, and cell uptake was examined by flow cytometry, L A-C L ss3/FAM-siRNA was prepared as nanocomposites with different N/P and added to the cell wells for 4 hours, and the medium was aspirated, PBS was washed 3 times, collected by digestion, and flow cytometry to examine the uptake of FAM-siRNA by flow cytometry.
The results are shown in FIG. 8 (A: flow cytometry results graph; B: ratio of positive MCF-7 cell numbers of uptake of Nile Red after different administration time), the difference of uptake of Nile Red and L A-C L ss3/Nile Red by MCF-7 at different time points is more obvious, which shows that L A-C L1 ss3/Nile Red takes significantly more after formation of glia than pure Nile Red, and that carrier L A-C L ss3 can effectively mediate Nile Red into cells, and the results of uptake study of L A-C L ss3/FAM-siRNA at different N/P by MCF-7 cells show that the uptake of siRNA L A-C L ss 48/FAM-7 cells is better when N/P is equal to 40 and 80, and that the results of siRNA-7 cell uptake of siRNA into cells are more effective as seen by A-C3/FAM-7 and L.
Example 8 confocal laser confocal observations L A-C L ss/FAM-siATG 7/Nile Red Co-loaded micelle Intracellularly
The experiments were divided into 4 groups, Nile Red group (Nile Red), Nile Red micellar group (Nile Red-PMs), siATG7 micellar group loaded with FAM markers (FAM-siATG7-PMs), Co-loaded FAM-siATG7 and Nile Red group (Co-PMs). MCF cells were plated as 1 × 105The cells were inoculated into 24-well plates previously plated with sterilized glass disks and cultured for 24 hours. Removing the culture medium by suction, adding the above administration group, culturing for 4 hr, washing with PBS, fixing with paraformaldehyde for 30min, washing, adding sealing solution containing DAPI, and observing on laser confocal microscope.
The distribution of the co-loaded nile red and FAM-siATG7 polypeptide micelles in MCF-7 cells was observed by confocal laser microscopy, as shown in fig. 10, where blue indicates DAPI-stained nuclei, green indicates FAM-fluorescently labeled siATG7, and red indicates intracellular-distributed nile red.
EXAMPLE 9 cytotoxicity Studies of L A-C L ss3
The MCF-7 cells were treated as 8 × 103Per well, the cells were inoculated in a 96-well plate, incubated for 24 hours to reach 50% confluency, the medium was aspirated, 100. mu.l of medium containing L A-C L ss3(5, 10, 20, 40, 60, 100, 120. mu.g/ml) at various concentrations was added to each well, incubation was continued for 24, 48 hours, the cytotoxicity was measured by CCK-8 method, the cell viability was counted, and a commercial transfection reagent bPEI (Sigma-Aldrich, USA, molecular weight 25kDa) was used as a control.
As shown in FIG. 11, the control bPEI-25K was very cytotoxic, with a cell survival rate of approximately 20% at 40. mu.g/ml, whereas L A-C L ss3 was less cytotoxic, with little effect on cell survival at 100. mu.g/ml, with cell survival rates of greater than 90% and around 80% at 200. mu.g/ml.
EXAMPLE 10 pharmacodynamic study of L A-C L ss3/(DTX/TRAI L)
The experiment was divided into 3 groups, namely a pure DTX group, a DTX-loaded micelle group, a co-loaded DTX and siATG7 group, and MCF-7 cells were treated according to 8 × 103And inoculating the cells in a 96-well plate for 24 hours to ensure that the cell confluency reaches 50 percent. The culture medium is aspirated, 100 mul of culture medium containing different drug concentrations (DTX: 0.000050, 0.00050, 0.0050, 0.050, 0.50, 5.0, 50 mug/ml) is added into each well, the culture is continued for 24 and 48 hours, the cytotoxicity is detected by a CCK-8 method, and the cell survival rate is counted.
The results are shown in FIGS. 12 and 13, and for MCF-7 cells, the 24h IC was observed for the pure DTX group, the DTX-loaded group, and the DTX-and siATG-loaded 7 group50Respectively is 0.52 plus or minus 1.23 mu g/ml, 0.28 plus or minus 0.74 mu g/ml and 0.043 plus or minus 0.37 mu g/ml; 48h IC50Respectively is 0.22 +/-0.98 mu g/ml, 0.11 +/-1.43 mu g/ml and 0.031 +/-0.52 mu g/ml. The visible carrier can effectively mediate chemotherapeutic drugs and genes to enter cellsAnd play a role. The drug effect of the chemotherapy drug can be increased after DTX is wrapped into the gel, and after the siATG7 is loaded together, protective autophagy generated by tumor cells under the stimulation of the chemotherapy drug is inhibited, so that the sensitivity of the cells to the chemotherapy drug is enhanced.
Example 11L A-C L ss3/(DTX/siATG7) Proapoptosis study
Dividing the experiment into 5 groups, including control group, blank carrier group, pure DTX group, DTX-carrying micelle group, co-carrying DTX and siATG7 group, and treating MCF cells according to 4 × 105And inoculating the cells in a 12-well plate, and culturing for 24h to ensure that the cell confluency reaches 80-90%. The medium was aspirated off, 1ml of medium containing a certain drug concentration (DTX: 2.5. mu.g/ml) was added to each well, the culture was continued for 24 hours, and apoptosis was measured by flow cytometry.
The results are shown in fig. 14, the total ratio of the early apoptosis and the late apoptosis in the control group, i.e., the total proportion of apoptotic cells, was 6.95%, 5.51% in the blank vector group, 12.54% in the pure DTX group, 25.56% in the DTX-loaded micelle group, and 44.39% in the co-DTX-loaded and siATG7 group. Therefore, the medicine is wrapped by the carrier to promote apoptosis, and the co-loading effect is more obvious.
EXAMPLE 12L A-C L ss3/DTX cycle block study
Dividing the experiment into 4 groups, including control group, blank carrier group, pure DTX group, and DTX-carrying micelle group, and treating MCF cells according to 4 × 105And inoculating the cells into a 12-well plate, and culturing for 24 hours to ensure that the cell confluence reaches 80-90%. The medium was aspirated off, 1ml of medium containing a certain drug concentration (DTX: 2.5. mu.g/ml) was added to each well, the culture was continued for 24h, and the cycle block was measured by flow cytometry.
As shown in FIG. 15(A: control; B: blank vehicle; C: DTX; D: DTX-PMs; etc.), most of the cells were arrested in the G2/M phase after the same dose of DTX treatment. Compared with the control group (G2/M phase cell percentage 14.8% + -1.3%) and the blank vector group (G2/M phase cell percentage 17.4% + -1.5%), the DTX group and the DTX micelle carrying group G2/M phase cell percentage respectively reach 81.7% + -2.1% and 92.9% + -1.1%. Therefore, the medicine can obviously block cells in the G2/M period after being wrapped by the carrier, and the effect of the DTX-loaded micelle group is more obvious.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.
Claims (5)
1. A lipoic acid modified intrinsically disordered protein nano-carrier is characterized in that the chemical structure of the lipoic acid modified intrinsically disordered protein nano-carrier is shown as a formula (III):
formula (III)
Wherein K is lysine; v is valine; r is arginine;Dp is D-proline; t is threonine; and E, glutamic acid.
2. A method of preparing the lipoic acid modified inherently disordered protein nanocarrier of claim 1, said method comprising the steps of:
(A) synthesizing a lipoic acid modified polypeptide; the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 is shown in the specification; the lipoic acid modification means that the carboxyl of the lipoic acid is connected with the amino of the first lysine through an amido group;
(B) preparation of lipoic acid modified intrinsically disordered protein nanocarriers: dissolving the lipoic acid modified polypeptide synthesized in the step (A) in methanol, adding cysteine hydrochloride to ensure that the molar weight of cysteine is 10 percent of that of the lipoic acid modified polypeptide, and stirring and reacting for 12 hours in a dark place.
3. The method of claim 2, wherein the solution after the step (B) reaction is treated with N2Drying; with N2The dried sample is at-20 DEG CAnd (5) storing.
4. Use of the lipoic acid modified intrinsically disordered protein nanocarrier of claim 1 in the preparation of a combination chemotherapeutic or genetic drug.
5. The use of the lipoic acid modified intrinsically disordered protein nanocarrier of claim 4 in the preparation of a combination chemotherapeutic or genetic drug, wherein said use is as follows:
preparing nano micelles by using ultrasonic emulsification of the nano carriers and the chemotherapeutic drugs, and mixing the nano micelles with DNA or siRNA to prepare a co-loading system;
the encapsulation rate of the chemotherapeutic drug is 40% -50%, and the nitrogen-phosphorus ratio of the nano-carrier to the DNA or siRNA is 5:1-80: 1.
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