Programmable multi-modification long-acting sustained-release elastin-like polypeptide (ELP) carrier and use thereof

Abstract

Provided are a programmable multi-modification long-acting sustained-release elastin-like polypeptide (ELP) carrier and the use thereof. The carrier is an elastin-like polypeptide containing composite-feature hydroxyphenyl ring structures, composite-feature benzene ring structures, composite-feature positive charge modifications, composite-feature negative charge modifications or chimeric features composed of two or more of the above structures; can undergo an in-situ phase transition triggered by body temperature and form a drug reservoir, such that a drug is slowly released from the reservoir to the circulatory system; and is applicable to the delivery of various drugs such as proteins, polypeptides, antibodies and small-molecule drugs.

Description

A programmable, multi-modified, long-acting sustained-release elastin-like ELP vector and its applications Technical Field

[0001] This invention belongs to the fields of genetic engineering and biomedicine, specifically relating to the synthesis of a novel programmable, multimodal long-acting sustained-release elastin-like polypeptide (ELP) carrier and its application in drug delivery. Background Technology

[0002] Peptide, antibody, and protein drugs are developing rapidly in the biopharmaceutical field. Compared with traditional small molecule drugs, they have advantages such as high biological activity, strong specificity, and low toxicity, and have good affinity for receptors in the body, thus being considered highly promising biopharmaceutical drugs. To date, many peptide, antibody, and protein drugs have been marketed and have achieved significant economic benefits. However, most of these drugs suffer from poor stability, short in vivo half-life, and low bioavailability. Especially in the treatment of chronic diseases, protein and peptide drugs often require long-term and frequent administration, which not only reduces patient compliance but may also cause adverse reactions. Therefore, developing long-acting drug delivery strategies has important clinical significance. For example, glucagon-like peptide-1 (GLP-1), as a potential drug for the treatment of type 2 diabetes, is easily degraded by dipeptidyl peptidase-4 (DPP-4) and loses its biological activity. Its half-life is only about 2 minutes, requiring frequent administration to maintain therapeutic effects, which seriously affects patient adherence. Similarly, bevacizumab treatment for age-related macular degeneration requires frequent intravitreal injections, which can easily induce complications such as retinal hemorrhage and cataracts. Therefore, the development of long-acting drugs to improve patient compliance is urgently needed.

[0003] Currently, strategies for long-acting peptide, antibody, and protein drugs include techniques such as mutant construction, Fc fragment fusion, glycosylation modification, and polyethylene glycol (PEG) modification. Mutant construction extends plasma half-life by reducing unstable amino acids and enhancing protease resistance; however, finding key sites through random mutation is inefficient and yields uncertain results. While Fc fragment fusion can extend protein half-life, its complex structure leads to poor stability. Glycosylation modification is affected by factors such as sugar type, molecular size, and charge, resulting in complex processes and low yields. PEG modification can reduce renal clearance, but issues such as reduced drug activity, non-biodegradability, and immunogenicity after PEG modification limit its clinical application. These strategies still have limitations in terms of long-acting duration, safety, stability, and controllability, and are also costly to produce. Therefore, developing safer and more cost-effective long-acting drug carriers is particularly important.

[0004] Elastin-like polypeptides (ELPs) are polypeptide polymers composed of the Val-Pro-Gly-X-Gly repeating sequence, where X represents any amino acid except proline. ELPs exhibit unique temperature responsiveness, displaying a lower critical solution temperature (LCST) phase behavior. By adjusting the ELP sequence and length, its phase transition temperature can be precisely controlled, thereby achieving precise control over drug release. Because ELPs are derived from natural elastin, they possess excellent biocompatibility and extremely low immunogenicity. Different amino acids exhibit different physicochemical properties, greatly enriching the drug delivery capabilities of ELPs (including chemical conjugation, biofusion, etc.). ELPs are genetically encoded and biosynthesized, allowing for the precise preparation of desired multifunctional biomaterials capable of carrying almost any type of cargo, from small molecules to large fusion proteins, thus making them highly promising long-acting drug carriers. For chronic diseases, long-acting sustained-release drugs are one of the optimal treatment methods. By constructing ELP-drug conjugates that are recombinantly fused or chemically conjugated with drugs, and adjusting their phase transition temperature to below physiological temperature, an in-situ LCST phase transition occurs at the injection site after injection, forming an aggregated drug reservoir for slow drug release. This drug reservoir can be used for local treatment or for systemic therapy by controlling the slow release of drugs with specific kinetics and gradually entering the bloodstream. The ELP involved in this invention is a drug delivery platform based on biosynthesis, precise modification, and intelligent sustained release technologies.

[0005] This invention utilizes programmable biosynthesis technology to construct a series of ELP polymer carriers with different amino acid characteristics, and screens out the preferred ELP carriers that can achieve long-term sustained release, providing an innovative solution for long-term drug delivery. Summary of the Invention

[0006] This invention aims to provide a programmable, multi-modified, long-acting sustained-release elastin (ELP) carrier and its applications. Utilizing gene-encoded biosynthesis technology, this invention develops an innovative ELP drug delivery system by arranging and combining amino acids of different structures in specific proportions. On one hand, based on isoleucine and valine, this invention combines various structures such as positively charged, negatively charged, benzene rings, and hydroxybenzene rings, or combines these structures in pairs, to biosynthesize a series of programmable ELP carriers. By detecting the temperature of in vitro critical phase behavior, candidate ELP carriers capable of undergoing in-situ phase transitions and forming ordered aggregate drug reservoirs at body temperature are screened. On the other hand, ELP carriers carrying different characteristic amino acids exhibit different sustained-release rates and metabolic efficiencies in vivo. Using Cy7 labeling of these candidate ELP carriers, long-acting novel programmable ELP carriers are screened using small animal in vivo imaging technology, thereby extending drug half-life, optimizing pharmacokinetic parameters, and improving patient compliance.

[0007] The preferred programmable sustained-release ELP obtained through screening in this invention possesses excellent temperature response characteristics, negative charge modification, suitable size, and ultra-long-acting sustained-release features. After subcutaneous injection, this programmable sustained-release ELP undergoes an in-situ phase transition triggered by body temperature, forming a drug reservoir. The drug is slowly released from the reservoir into the circulatory system, significantly prolonging the effective duration of action of a single injection, improving pharmacokinetic parameters, and enhancing patient compliance. This preferred programmable sustained-release ELP has broad application potential, suitable for the delivery of various drugs such as proteins, peptides, antibodies, and small molecule drugs, exhibiting significant advantages, especially in long-acting sustained release and drug bioavailability.

[0008] The technical solution adopted in this invention relates to a novel drug sustained-release long-acting polymer carrier system, the details of which are as follows:

[0009] In a first aspect, the present invention provides a drug-releasing, long-acting polymer carrier. The carrier is composed of an elastin-like protein (ELP) containing a positively charged or negatively charged hydroxybenzene ring, a positively charged intercalated hydroxybenzene ring, or a negatively charged intercalated hydroxybenzene ring.

[0010] Specifically, this invention utilizes a programmable biosynthesis method to construct a series of ELPs with different structures and properties based on different amino acid compositions, including a composite positive charge ELP series (IVK), a composite negative charge ELP series (IVE), a composite benzene ring ELP series (IVF), a composite hydroxybenzene ring ELP series (IVY), and a structural chimeric ELP series (IVYIVE).

[0011] In some embodiments of the present invention, the elastin-like protein can undergo an in-situ phase transition at 37 °C to form an ordered biological aggregate.

[0012] In some embodiments of the present invention, the elastin-like protein is capable of responding sensitively to external stimuli such as temperature, charge, enzymes, light, or chemicals.

[0013] In some embodiments of the present invention, the elastin-like sequence is (VPGXG)n, where n = 60 to 180, preferably n = 120 to 150, X represents any amino acid other than proline, and n is an integer.

[0014] In some embodiments of the present invention, X may be at least one of lysine, glutamic acid, tyrosine, phenylalanine, alanine, and isoleucine.

[0015] In some embodiments of the present invention, the drug delivery carrier may serve as an environmentally responsive aggregate that responds sensitively to stimuli such as temperature, pH, enzymes, charge, light, or chemicals.

[0016] In some embodiments of the present invention, the response temperature range of the drug carrier ELP polymer is 10°C to 50°C, preferably 16°C to 28°C.

[0017] In some embodiments of the present invention, the amino acid sequence of the ELP polymer is as shown in SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, or SEQ ID NO. 18. A preferred ELP amino acid sequence includes: [(VPGIG)4(VPGVG)4(VPGEG)1]2[(VPGIG)4(VPGVG)4(VPGYG)1] 14 .

[0018] In a second aspect, the present invention provides a nucleic acid molecule that encodes all of the above-mentioned elastin-like proteins.

[0019] In some embodiments of the present invention, the nucleic acid molecule is DNA or RNA, which is composed of nucleic acid molecules arranged and combined from basic units, including amino acid sequence units encoding VPGVG, or VPGIG, or VPGYG, or VPGFG, or VPGKG, or VPGGEG, or combinations of nucleic acid sequences of at least two or more units.

[0020] In some embodiments of the present invention, the amino acids encoded by the nucleic acid molecules are codon degenerate, and the nucleic acid molecule sequences of the above-mentioned elastin-like proteins are not unique. Any nucleic acid sequence that can encode the above-mentioned ELP is covered within the protection scope of the present invention.

[0021] In some preferred embodiments of the present invention, the amino acid sequence of the combinatorial unit encoding an elastin-like nucleic acid molecule is shown in SEQ ID NO.18.

[0022] A third aspect of the present invention provides a polymer carrier as described in the first aspect of the present invention or a nucleic acid molecule-related biomaterial as described in the second aspect of the present invention, wherein the biomaterial is any one of (a1) to (d1) below:

[0023] (a1) The biomaterial comprises the ELP aggregate of the first invention or the expression cassette of the nucleic acid molecule of the second invention;

[0024] (b1) The expression cassette of (a1) includes a recombinant plasmid vector, or recombinant cells, or expression cells, or expression cells containing recombinant plasmids, such as Escherichia coli DH5α, BL21, or yeast, HEK-293T cells;

[0025] (c1) The recombinant plasmid vector of (b1) contains regulatory expression elements such as promoters, reading frames, terminators, and enhancers;

[0026] (d1) The recombinant or expression cells described in (b1) are used in prokaryotic (e.g., Escherichia coli DH5α and BL21) or eukaryotic (e.g., yeast and HEK-293T) expression systems.

[0027] A fourth aspect of the invention provides a programmable ELP polymer carrier coupled to an active molecule by a chemical or biological method, said active molecule comprising a diagnostic agent and / or a therapeutic agent and / or a molecular switch.

[0028] In some embodiments of the present invention, the diagnostic agent includes fluorescent labeling, radiolabeling, etc.

[0029] In some preferred embodiments of the present invention, the fluorescent label includes at least one of the following: Cy7, Cy5, Cy3, GFP, YFP, BFP, CFP, etc.

[0030] In some embodiments of the present invention, the therapeutic agents include peptides, antibodies, proteins, nucleic acid drugs, chemical drugs, and traditional Chinese medicines. These include monoclonal antibodies, domain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, antibody mimics, humanized antibodies and their fragments, insulin, growth factors, interleukins, interferons, energy factors, erythropoietin, neutrophil growth factor, pDNA, siRNA, miRNA, mRNA, transforming growth factor, etanercept, aflibercept, afasicept, liraglutide, pramlinide, exenatide, lantylpeptide, glucagon-like peptide-1, relaxin, octreotide, leptin, asparaginase, uricase, cyclosporine, antidiuretic hormone, oxytocin, insulin, superoxide dismutase, vaccines, Her2 receptors, VEGF receptors, etc.

[0031] In a fifth aspect, the present invention provides that the polymer carrier described in the first aspect of the present invention, or the nucleic acid molecule described in the second aspect of the present invention, or the biomaterial described in the third aspect of the present invention, or the active molecule polymer carrier conjugate described in the fourth aspect of the present invention, can be used to prepare diagnostic agents and / or therapeutic drugs, and is suitable for the treatment of chronic diseases such as cardiovascular diseases, neurodegenerative diseases, autoimmune diseases, and metabolic diseases.

[0032] A sixth aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutical polymer carrier according to the first aspect of the present invention, a nucleic acid molecule according to the second aspect of the present invention, a biomaterial according to the third aspect of the present invention, or an active molecule polymer carrier conjugate according to the fourth aspect of the present invention.

[0033] In some embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients for improving drug stability and bioavailability.

[0034] In summary, through this technical solution, the present invention provides a novel drug delivery platform with multi-stimulus responsiveness and long-acting sustained-release characteristics, possessing significant biocompatibility and application potential, capable of meeting the treatment needs of various diseases, and improving drug delivery efficiency and patient compliance.

[0035] The beneficial effects of this invention are:

[0036] This invention provides a novel long-acting, sustained-release ELP carrier, a programmable, multi-modified protein polymer (ELP) carrier. Through gene-encoded biosynthesis and rational amino acid modification techniques, this invention designs a multi-stimuli-responsive protein polymer carrier. By selecting a suitable amino acid arrangement, the ELP carrier exhibits low temperature response characteristics, undergoing a phase transition in situ after subcutaneous injection to form a drug reservoir. The drug is slowly released from this reservoir, enabling continuous in vivo delivery without relying on traditional sustained-release pumps or microsphere formulations, with a sustained-release time of up to 20 days, significantly extending the drug's half-life and improving its efficacy. Furthermore, this invention combines a low-temperature-responsive structure with charge modification, significantly improving the in-situ sustained-release effect, further optimizing pharmacokinetic parameters, extending the dosing cycle, and improving patient compliance.

[0037] The ELP polymer carrier of this invention is derived from natural elastin in vivo and has extremely low immunogenicity, bioprogrammable properties, chemical site-directed coupling modification features, good stability and biodegradability.

[0038] The drug polymer carrier ELP of the present invention uses readily available and inexpensive initial raw materials, has a simple extraction and purification process, is convenient to produce, and is easy to modify through genetic engineering. It has broad application prospects and huge market potential. Attached Figure Description

[0039] Figure 1 is a schematic diagram of ELP construction.

[0040] Figure 2a shows the SDS-PAGE electrophoresis results of IVY8, IVY12, and IVY16; Figure 2b shows the SDS-PAGE electrophoresis results of IVE8, IVE12, and IVE16; Figure 2c shows the SDS-PAGE electrophoresis results of IVK8, IVK12, and IVK16; and Figure 2d shows the SDS-PAGE electrophoresis results of IVF8, IVF12, and IVF16.

[0041] Figure 3 shows the SDS-PAGE electrophoresis results of IVE8IVY8, IVE6IVY10, IVE4IVY12, and IVE2IVY14.

[0042] Figure 4a shows the relationship between the composite characteristic ELP and turbidity at a concentration of 10 μM, and Figure 4b shows the function of the concentration of the composite characteristic ELP and the phase transition temperature.

[0043] Figure 5 shows the optical images of the composite feature ELP at a concentration of 10 μM at 8 ℃ and 37 ℃.

[0044] Figure 6a shows the relationship between the chimeric characteristic ELP and turbidity at a concentration of 10 μM, and Figure 6b shows the function of the concentration of the chimeric characteristic ELP and the phase transition temperature.

[0045] Figure 7a shows the in vivo retention time of IVIS small animal in vivo imaging of Cy7, Cy7-IVK16, Cy7-IVE16, and Cy7-IVY16 after subcutaneous injection, and Figure 7b is a statistical graph of retention time.

[0046] Figure 8a shows the in vivo retention time of IVIS small animal in vivo imaging Cy7-IVE8IVY8, Cy7-IVE6IVY10, Cy7-IVE4IVY12, and Cy7-IVE2IVY14 after subcutaneous injection. Figure 8b is a statistical chart of retention time. Detailed Implementation

[0047] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0048] Example 1: Construction of the general monomer precursor VPGIG (VPGVG)2 and the characteristic monomer precursor (VPGIG)2VPGXG [X represents Y, E, K, F]

[0049] 1) Design and synthesize oligonucleotide gene fragments containing repeat units and restriction enzyme sites Acu I / BseRI, and then construct the target gene with sticky ends.

[0050] ;

[0051] ;

[0052] ;

[0053] ;

[0054] ;

[0055] ;

[0056] ;

[0057] ;

[0058] ;

[0059] ;

[0060] 2) Preparation of the precursor target gene. A reaction system was constructed by modifying the forward and reverse primers with 5' end phosphorylation: 5 μL forward primer gene, 5 μL reverse primer gene, 2 μL T4 DNA ligation buffer, 1 μL T4 PNK, and 7 μL ultrapure water. After incubation at 37℃ for 1 h, 1 μL of 1 M NaCl was added to terminate the reaction, followed by an annealing extension reaction. The reaction program was as follows: 95℃ for 5 min, 55℃ for 5 min, and 16℃ for 5 min, to obtain the double-stranded DNA gene fragment of the monomeric precursor.

[0061] 3) Preparation of linearized plasmid vector. The plasmid vector pET24b was digested with BseRI. The digestion system consisted of 20 μL of the plasmid, 3 μL of rCutsmart Buffer, 0.5 μL of BseRI, and 6.5 μL of ultrapure water. After incubation at 37°C for 3 h, 1 μL of CIP was added for 5′ dephosphorylation, and incubation continued at 37°C for 20 min. The linearized vector was then recovered and purified by agarose gel electrophoresis.

[0062] 4) Ligation and Transformation Reaction. Ligation system: 1 μL T4 DNA ligation buffer, 0.02 pM linearized vector, 5 μL target gene, 0.5 μL T4 DNA Ligase, and ultrapure water to a final volume of 10 μL. Incubate at 25°C for 2 h, then place on ice and let stand for 3 min. Add the ligation product to DH5α cells, incubate on ice for 20 min, followed by heat shock at 42°C for 45 s, and then on ice for another 3 min. Add 500 μL of antibiotic-free LB broth, incubate at 37°C with shaking at 200 rpm for 1 h, then spread evenly on LB agar plates containing kanamycin and incubate inverted at 37°C overnight.

[0063] 5) Single Clon and Sequencing Identification. Single-clone colony suspensions were selected for PCR identification. The reaction system consisted of: 5 μL of 2x Flash PCR MasterMix, 0.5 μL of universal primer F, 0.5 μL of universal primer R, 1 μL of bacterial suspension template, and 3 μL of ultrapure water. Positive clones were selected and sent for sequencing. Sequence alignment was used to identify the construction of the target gene.

[0064] Example 2: Constructing a composite characteristic monomer (VPGIG)2VPGXG(VPGIG(VPGVG)2)2 [X is Y, E, K, F]

[0065] 1) Preparation of (VPGIG(VPGVG)2)2. A single repeat unit was prepared using a recursive directional method for plasmid reconstruction. The plasmid pET24b-VPGIG(VPGVG)2 (hereinafter referred to as pET24b-IG2) was digested with restriction endonucleases Acu I / Bgl I and BseR I / Bgl I to obtain the gene fragment IG2 (AB digestion / BB digestion). Subsequently, IG2 (AB digestion / BB digestion) was ligated (ligation system: 1 μL T4 DNA ligation buffer, 2.5 μL IG2 (AB digestion), 1.5 μL IG2 (BB digestion), 0.5 μL T4 DNA Ligase, 4.5 μL ultrapure water) to construct the target gene and transform it. After selecting single clones, they were sent for sequencing to obtain the repeat unit (VPGIG(VPGVG)2)2.

[0066] 2) Preparation of (VPGIG)2VPGXG(VPGIG(VPGVG)2)2 [X is Y, E, K, F]. Combinatorial units were prepared using a recursive orientation method. The plasmid pET24b-(VPGIG)2VPGXG [X is Y, E, K, F] (hereinafter abbreviated as pET24b-I2X) was digested with restriction endonucleases Acu I / Bgl I to obtain gene fragment I2X (AB digestion). The plasmid pET24b-(VPGIG(VPGVG)2)2 (hereinafter abbreviated as pET24b-(IG2)2) was digested with BseR I / Bgl I to obtain gene fragment (IG2)2 (BB digestion). I2X (AB digestion) and (IG2)2 (BB digestion) were ligated to construct the combined target gene. The gene was transformed, single clones were selected, and sequencing was performed to identify the target gene with different characteristics. The repeating units pET24b-(VPGIG)2VPGYG(VPGIG(VPGVG)2)2, pET24b-(VPGIG)2VPGEG(VPGIG(VPGVG)2)2, pET24b-(VPGIG)2VPGKG(VPGIG(VPGVG)2)2, and pET24b-(VPGIG)2VPGFG(VPGIG(VPGVG)2)2 were obtained, and their amino acid sequences are as follows:

[0067] ;

[0068] ;

[0069] ;

[0070] ;

[0071] Example 3: Constructing a composite feature ELP [(VPGIG)2VPGXG(VPGIG(VPGVG)2)2] n [X represents Y, E, K, or F]

[0072] Tyrosine (Y), glutamic acid (E), lysine (K), and phenylalanine (F) were selected as the amino acids at the X position of ELPs, and composite characteristic ELPs with n of 1, 2, 4, 8, 12, and 16 were constructed.

[0073] The recursive orientation method for plasmid reconstruction in Example 2 was used to prepare repeating units, and the corresponding composite feature ELPs were obtained as follows: pET24b-[(VPGIG)2VPGYG(VPGIG(VPGVG)2)2] n n is 1, 2, 4, 8, 12, 16 (hereinafter abbreviated as pET24b-IVY1, pET24b-IVY2, pET24b-IVY4, pET24b-IVY8, pET24b-IVY12, pET24b-IVY16); pET24b-[(VPGIG)2VPGEG(VPGIG(VPGVG)2)2] n n is 1, 2, 4, 8, 12, 16 (hereinafter abbreviated as pET24b-IVE1, pET24b-IVE2, pET24b-IVE4, pET24b-IVE8, pET24b-IVE12, pET24b-IVE16); pET24b-[(VPGIG)2VPGKG(VPGIG(VPGVG)2)2] n n is 1, 2, 4, 8, 12, 16 (hereinafter abbreviated as pET24b-IVK1, pET24b-IVK2, pET24b-IVK4, pET24b-IVK8, pET24b-IVK12, pET24b-IVK16); pET24b-[(VPGIG)2VPGFG(VPGIG(VPGVG)2)2] n n is 1, 2, 4, 8, 12, 16 (hereinafter abbreviated as pET24b-IVF1, pET24b-IVF2, pET24b-IVF4, pET24b-IVF8, pET24b-IVF12, pET24b-IVF16).

[0074] Example 4: Constructing chimeric features ELP IVE8IVY8 / IVE6IVY10 / IVE4IVY12 / IVE2IVY16

[0075] Following the recursive orientation method for plasmid reconstruction in Example 2, various chimeric feature ELPs were prepared, and chimeric ELPs with different combinations of features were successfully constructed, as follows: pET24b-[(VPGIG)2VPGEG(VPGIG(VPGVG)2)2]8 [(VPGIG)2VPGYG(VPGIG(VPGVG)2)2]8 (hereinafter abbreviated as pET24b-IVE8IVY8); pET24b-[(VPGIG)2VPGEG(VPGIG(VPGVG)2)2]6 [(VPGIG)2VPGYG(VPGIG(VPGVG)2)2] 10 (Hereinafter abbreviated as pET24b-IVE6IVY10); pET24b-[(VPGIG)2VPGEG(VPGIG(VPGVG)2)2]4[(VPGIG)2VPGYG(VPGIG(VPGVG)2)2] 12 (Hereinafter abbreviated as pET24b-IVE4IVY12); pET24b-[(VPGIG)2VPGEG(VPGIG(VPGVG)2)2]2 [(VPGIG)2VPGYG(VPGIG(VPGVG)2)2] 14 (hereinafter abbreviated as pET24b-IVE2IVY14).

[0076] Example 5: Preparation of composite characteristic ELP protein carrier

[0077] IVY8, IVY12, IVY16, IVE8, IVE12, IVE16, IVK8, IVK12, IVK16, IVF8, IVF12, IVF16 were selected for induced expression and purification.

[0078] 1) The recombinant plasmid was transformed into BL21(DE3) Escherichia coli for expression. First, positive clones were selected and inoculated into LB medium (containing kanamycin) for overnight culture. The next day, the culture was scaled up in LB liquid medium containing kanamycin for 2-3 h. When the OD600 value of the bacterial culture was 0.6-0.8, the culture was placed in a 4℃ refrigerator for 30 min, and IPTG was added to a final concentration of 0.4 mM. Expression was induced at 16℃ for 24 h, and the bacterial cells were collected by centrifugation.

[0079] 2) Reagent Preparation and Sample Preparation. LE equilibration buffer (50 mM Na₂HPO₄, 300 mM NaCl, pH 8.0), wash buffer (50 mM Na₂HPO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0), elution buffer (50 mM Na₂HPO₄, 300 mM NaCl, 300 mM imidazole, pH 8.0). The cell suspension was sonicated on ice for 2 seconds, followed by 3 seconds of cooling and 5 minutes of sonication, alternating between these cycles for a total of 5 repetitions. The supernatant was collected by centrifugation (9000 rpm, 4°C, 20 minutes). The supernatant was then filtered through a 0.22 μm filter membrane to obtain the filtrate.

[0080] 3) Column purification. The entire purification process was performed in a cold room. Ten column volumes of LE were added to equilibrate the Ni-NTA resin medium. The protein solution was loaded into the purification column, allowing it to flow out slowly. Fifteen column volumes of wash buffer were added to remove contaminating proteins. Elution buffer was then added to elute the target protein. Protein purity was assessed using SDS-PAGE gel electrophoresis. The results are shown in Figures 2a, 2b, 2c, and 2d. The purity of the purified target protein reached over 90%.

[0081] 4) Protein Quantification and Storage. The purified ELP was transferred to a dialysis bag (molecular weight cutoff of 14000 Da) and dialyzed in PBS buffer. The buffer was changed twice, every 6 hours. After dialysis, the solution was centrifuged (9000 rpm, 4℃, 15 min), and the supernatant was filtered through a 0.22 μm filter for sterilization. Protein concentration was determined using the BCA method. The protein solution was aliquoted and stored at -80℃.

[0082] Example 6: Preparation of chimeric ELP protein carriers

[0083] IVE8IVY8, IVE6IVY10, IVE4IVY12, and IVE2IVY14 were selected for induced expression and purification.

[0084] Using the protein expression and purification technique described in Example 5, a chimeric ELP protein was prepared. The protein purity was determined by SDS-PAGE gel electrophoresis, and the results are shown in Figure 3. The purity of the purified target protein reached over 95%. Its amino acid sequence is as follows:

[0085] ;

[0086] ;

[0087] ;

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[0089] Example 7: Physicochemical characterization parameters of ELP protein carriers with composite and chimeric characteristics

[0090] The phase transition temperatures (Tt) of complex and chimeric ELPs were determined by turbidimetric assay. Different ELP samples (including IVY8, IVY12, IVY16, IVE8, IVE12, IVE16, IVK8, IVK12, IVK16, IVF8, IVF12, IVF16) were diluted with PBS to different concentrations (20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM). The UV absorbance (OD350) of the ELP samples at different concentrations from 0℃ to 55℃ was detected using a multi-mode microplate reader. Tt is the temperature at which the turbidity of the ELP sample reaches half of its maximum stable value. The results are shown in Figure 4a, which displays the OD350 changes at different temperatures for 10 μM. Figure 4b shows the functional relationship between concentration and phase transition temperature, i.e., the phase transition temperature Tt at different reaction temperatures. In addition, the composite ELP was diluted to 10 μM and incubated at 8 °C and 37 °C, respectively, for optical imaging. The results are shown in Figure 5, corresponding to the data in Figure 4a. IVY16, IVE16, and IVK16 exhibited reversible phase transition behavior below 37 °C, indicating their potential to form drug reservoirs in situ in vivo.

[0091] Similarly, the phase transition temperatures Tt of IVE8IVY8, IVE6IVY10, IVE4IVY12, and IVE2IVY14 were detected using the same method. The detection results are shown in Figures 6a and 6b.

[0092] Example 8: Retention time of composite ELP protein carriers in vivo

[0093] The composite ELP was coupled with the fluorophore Cy7. Control Cy7 fluorophores, along with Cy7-IVY16, Cy7-IVE16, and Cy7-IVK16, were subcutaneously injected into the neck and back of mice. The Cy7-labeled composite ELP underwent in-situ phase transition assembly in the subcutaneous tissue, forming a drug reservoir. The retention time of Cy7-ELP in mice was observed using in vivo imaging (IVIS). The mice used in the experiment were male C57BL / 6 rats.

[0094] The specific steps are as follows:

[0095] 1. Preparation of Cy7-labeled samples

[0096] Remove IVY16, IVE16, and IVK16 stored at -80 ℃ and transfer them to reaction tubes. Add the fluorescent group stock solution to the reaction tubes at a fluorophore / labeled ELP molar ratio of 3:1, mix gently, and incubate the reaction tubes slowly at 4 ℃ for 12-24 h. Transfer the mixture to a dialysis bag and dialyze against PBS, changing the PBS every 4 hours for 2-3 times to obtain fluorescently labeled Cy7-IVY16, Cy7-IVE16, and Cy7-IVK16.

[0097] 2. Subcutaneous injection in the neck and back of mice

[0098] The labeled sample was subcutaneously injected into the back of the neck of mice at a dose of 0.22 μM / Kg. Negative control group: Cy7 was injected subcutaneously.

[0099] 3. In vivo retention time analysis

[0100] At 2 h, 1 day, 3 days, 7 days, 11 days, 15 days, and 17 days after subcutaneous injection, mice were photographed using a small animal in vivo imaging system. Imaging was performed using fluorescence channels with excitation at 740 nm and emission at 790 nm. The experimental results are shown in Figures 7a and 7b. In the control group, Cy7 fluorescence disappeared on day 3, while in the Cy7-IVK16 group, fluorescence disappeared on day 11. The Cy7-IVY16 and Cy7-IVE16 groups had the longest fluorescence retention time, disappearing on day 17. This indicates that the IVY structure with a lower surface Tt value and the IVE structure with a higher Tt value and negative charge modification have longer retention times and better sustained-release effects.

[0101] Example 9: Retention time of chimeric ELP protein carriers in vivo

[0102] To investigate whether chimeric ELPs could further prolong in vivo retention time, chimeric ELPs were prepared and coupled with a Cy7 fluorescent group. Cy7-IVE2IVY14, Cy7-IVE4IVY12, Cy7-IVE6IVY10, and Cy7-IVE8IVY8 were subcutaneously injected into the neck and back of mice. The Cy7-labeled chimeric ELPs underwent in-situ phase transition assembly in the subcutaneous tissue, forming a drug reservoir. Retention time was detected using IVIS imaging. Male C57BL / 6 mice were used in the experiment.

[0103] The specific steps are as follows:

[0104] 1. Preparation of Cy7-labeled samples

[0105] Remove the IVE2IVY14, IVE4IVY12, IVE6IVY10, and IVE8IVY8 samples stored at -80℃ and transfer them to reaction tubes. Add the fluorescent group stock solution to the reaction tubes at a fluoropolymer / labeled ELP molar ratio of 3:1, mix gently, and slowly agitate the reaction tubes at 4℃ for 12-24 hours. Transfer the mixed solution to a dialysis bag and dialyze it in PBS. Change the PBS every 4 hours, repeating 2-3 times to obtain the fluorescently labeled Cy7-IVE2IVY14, Cy7-IVE4IVY12, Cy7-IVE6IVY10, and Cy7-IVE8IVY8.

[0106] 2. Subcutaneous injection in the neck and back of mice

[0107] Mice were subcutaneously injected with a dose of 0.22 μM / Kg.

[0108] 3. In vivo retention time analysis

[0109] Mice were photographed using an IVIS imaging system at 2 h, 1 day, 3 days, 7 days, 11 days, 15 days, 19 days, 24 days, 30 days, 36 days, and 43 days after subcutaneous injection, with the fluorescence channel selected for excitation at 740 nm and emission at 790 nm. The results are shown in Figures 8a and 8b. The fluorescence of the Cy7-IVE4IVY12 group persisted in vivo until day 24, while the fluorescence of the Cy7-IVE6IVY10 and Cy7-IVE8IVY8 groups persisted until day 30. The Cy7-IVE2IVY14 group had the longest persistence time, persisting until approximately day 36. These results indicate that, compared to composite ELPs, chimeric ELPs have a significantly longer in vivo persistence time, suggesting that the in vivo persistence time of ELPs is closely related to the phase transition temperature / concentration function and the negative charge. According to the relationship between phase transition temperature and concentration function in Figure 6.B, the slope of IVE2IVY14 is much lower than that of the latter three, indicating that the slope may be the main factor determining the retention of chimeric ELPs in vivo, while charge affects the retention effect when the slopes are similar.

[0110] In summary, this invention innovatively proposes the synthesis and application of a novel programmable, multi-modified, long-acting sustained-release elastin ELP carrier. A single subcutaneous injection of chimeric ELP into mice demonstrated an exceptionally long duration of action exceeding one month. This carrier can not only encapsulate poorly water-soluble small molecule drugs, but also, through genetic engineering, fuse therapeutic proteins (such as antibodies and cytokines) with the ELP at the molecular level, significantly improving drug stability, prolonging drug retention time in vivo, increasing half-life, and avoiding side effects caused by excessively high drug concentrations. Different modifications to the amino acids and functional groups on the amino acids can be made through genetic engineering or chemical synthesis methods to obtain drug carriers with different environmental stimuli responses, thereby improving drug bioavailability. In conclusion, the programmable, multi-modified, long-acting sustained-release elastin ELP carrier of this invention is suitable for delivering proteins, enzymes, antibodies, small molecule drugs, and polymers, greatly improving pharmacokinetic characteristics, enhancing drug stability, bioactivity, and therapeutic efficacy, while also reducing the toxicity caused by frequent drug administration and improving drug biosafety. This invention provides new ideas for the design of future drug delivery and novel synthetic biomaterials, and is applicable to the delivery and treatment of a variety of drugs.

[0111] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A programmable, multi-modified, long-acting sustained-release elastin-like ELP carrier, characterized in that, The carrier is an elastin-like protein comprising a complex hydroxybenzene ring structure, a complex benzene ring structure, a complex positive charge modification, a complex negative charge modification, or a combination of two or more of these structural features.

2. The programmable, multi-modified, long-acting sustained-release elastin-like ELP carrier according to claim 1, characterized in that, The elastin-like sequence includes (VPGXG)n, where X is any amino acid other than proline (P); X is at least one of lysine, glutamic acid, tyrosine, phenylalanine, alanine, and isoleucine; the value of n ranges from 60 to 180; and the phase transition temperature of the carrier is 15-30℃.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes an elastin; the elastin-like structural sequence includes: [(VPGIG)4(VPGVG)4(VPGEG)1]2[(VPGIG)4(VPGVG)4(VPGYG)1] 14 .

4. A long-acting sustained-release biopolymer delivery carrier comprising the multimodified long-acting sustained-release elastin ELP carrier as described in claim 1 or 2 and an active molecule; said active molecule comprising a diagnostic agent and / or a therapeutic agent and / or a molecular switch.

5. The long-acting sustained-release biopolymer delivery carrier according to claim 4, characterized in that, The diagnostic agent includes fluorescent or radiolabeled substances; the therapeutic agent includes peptides, antibodies, proteins, nucleic acids, chemical drugs, or traditional Chinese medicines; the molecular switch includes pH responses of imines and non-covalently cross-linked phenylboronic esters, enzyme cleavage responses of matrix metalloproteinases, esterases, and histones, and photoresponsive functional groups incorporated into photosensitive functional groups.

6. The long-acting sustained-release biopolymer delivery carrier according to claim 4, characterized in that, The therapeutic agent comprises at least one of the following: monoclonal antibody, domain antibody, nanobody, multispecific antibody, chimeric antibody, antibody mimic, humanized antibody and its fragment, insulin, growth factor, interleukin, interferon, energy factor, erythropoietin, neutrophil growth factor, pDNA, siRNA, miRNA, mRNA, transforming growth factor, etanercept, aflibercept, liraglutide, exenatide, lantylpeptide, glucagon-like peptide-1, relaxin, octreotide, leptin, asparaginase, uricase, cyclosporine, antidiuretic hormone, oxytocin, pancreatin, superoxide dismutase, vaccine, and VEGF receptor.

7. The long-acting sustained-release biopolymer delivery carrier according to claim 5, characterized in that, The fluorescent label includes at least one of Cy7, Cy5, Cy3, GFP, YFP, BFP, and CFP.

8. A biomaterial, characterized in that, The biomaterial is any one of (a1) to (d1) below: (a1) The biomaterial comprises the expression cassette of the ELP polymer or nucleic acid molecule as described in claims 1 to 3; (b1) The expression cassette of (a1) comprises a recombinant plasmid vector, or a recombinant cell, or an expression cell, or an expression cell containing a recombinant plasmid; (c1) The recombinant plasmid vector described in (b1) contains transcriptional regulatory elements such as promoters, reading frames, terminators, and enhancers; (d1) The recombinant cells or expression cells in (b1) comprise prokaryotic cells and eukaryotic cells; the prokaryotic cells comprise Escherichia coli DH5α or BL21; the eukaryotic cells comprise yeast and HEK-293T.

9. The use of the programmable, multi-modified, long-acting sustained-release elastin ELP carrier of claim 1 or 2, or the nucleic acid molecule of claim 3, or the long-acting sustained-release biopolymer delivery carrier of any one of claims 4 to 7, or the biomaterial of claim 7, in the preparation of diagnostic agents and / or pharmaceuticals.

10. A method for preparing a long-acting, sustained-release biopolymer delivery carrier for in vivo, characterized in that, By combining the vector of any one of claims 1 to 2 with the active molecule of any one of claims 4 to 6 using genetic engineering technology, a sustained-release, long-acting, and controllable biopolymer drug with environmental stimulus response can be prepared.

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