Indocyanine green-hyaluronic acid-polypeptide conjugate as well as preparation method and application thereof

Abstract

The invention provides an indocyanine green-hyaluronic acid-polypeptide conjugate as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The structure of the conjugate is shown as a formula (I). The conjugate provided by the invention has good stability and biological activity, can realize the synergistic effect of tumor apoptosis and photothermal therapy, and has good practical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an indocyanine green-hyaluronic acid-polypeptide conjugate and a preparation method and application thereof. Background Art

[0002] Cancer has always been one of the killers threatening human life and is also a major challenge facing the global medical research field. Its high mortality rate, high incidence rate and trend of younger people make cancer a top priority in scientific research, so it is urgent to explore the treatment of cancer.

[0003] Hyaluronic acid can play a variety of structural functions in the extracellular matrix (ECM) through specific and non-specific interactions with a variety of functional proteins, and also plays a vital role in the communication transduction between cells and specific molecules / receptors, for example: CD44, neuroglycans, glial hyaluronan binding protein (GHAP) and lymphatic endothelial hyaluronan receptor 1 (LYVE-1) can all interact with hyaluronic acid. Current research results show that CD44 is highly expressed on the surface of various tumor cells, so hyaluronic acid is considered to play an important targeting function as a drug carrier.

[0004] Anticancer peptides (ACPs) have the potential to be developed into a new generation of anti-tumor drugs and become ideal candidate molecules for the treatment of cancer due to their broad spectrum of anticancer activity, low immunogenicity, rapid killing of cancer cells, certain selectivity, excellent tissue permeability, low resistance to drug resistance, easy synthesis, and easy modification to enhance in vivo stability and biological activity.

[0005] In recent decades, phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), has made great progress in achieving anti-tumor effects. PTT is a method that uses photothermal agents to generate local hyperthermia to destroy cancer cells. For example, gold nanoshells and indocyanine green, as common photothermal agents, have been evaluated in clinical trials for their anti-tumor therapeutic effects. PDT uses photosensitizers to generate reactive oxygen species (ROS) to damage cancer cells, such as hematoporphyrin derivatives (HpD), and has been used clinically for more than 40 years. For the photothermal dynamic therapy of cancer, the development of high-quality therapeutic drugs is crucial. Near-infrared (NIR) fluorescent dyes are popular due to their excellent properties, including stronger tissue penetration than visible light and no interference from tissue autofluorescence. Notably, indocyanine green (ICG) is the only NIR fluorescent dye approved by the US Food and Drug Administration (FDA) for clinical use. In recent years, ICG has been used in cancer phototherapy as an excellent photothermal agent and photosensitizer.

[0006] Some anti-tumor therapeutic agents containing photothermal agents have been provided in the prior art. For example, Chinese invention patent CN118267343A provides a photothermal-photodynamic synergistic antibacterial freeze-dried microneedle, which contains hyaluronic acid-encapsulated ZIF-8 nanoparticles loaded with indocyanine green; Chinese invention patent CN117731775A provides a hyaluronic acid-modified drug-loaded nanocomposite, which contains hyaluronic acid and indocyanine green; Chinese invention patent CN116712665A provides a photothermal-chemotherapeutic combined therapeutic agent, which uses microneedles to fill the drug in the cavity of the microneedle. However, the hyaluronic acid and photothermal agent in the above therapeutic agent are mixed in the form of encapsulation or compounding, and the stability is poor during application, especially after entering the human body circulation, and it is easy to fall off, which is not conducive to giving full play to the therapeutic effect of the photothermal agent. Chinese invention patent CN114349807A provides a sialic acid-linked indocyanine green Sia-ICG, in which ICG is linked to sialic acid via an amide bond. However, as a monosaccharide, sialic acid is still metabolized at a relatively fast rate in the body, and the durability of the photothermal agent's effect still needs to be improved.

[0007] At present, there is no drug or method in the prior art that has studied how to use photothermal and photodynamic methods to coordinate anticancer peptides to treat tumors. Summary of the invention

[0008] In view of the above problems, this application proposes the following technical solutions:

[0009] On the one hand, the present application provides an indocyanine green-hyaluronic acid-polypeptide conjugate, which comprises a structural formula as shown in formula (I):

[0010]

[0011] In formula (I), R1 is a polypeptide containing at least two amino acids, and R2 has a structure as shown in formula (II):

[0012]

[0013] The value range of n is 1 to 25.

[0014] In one implementation, the value of n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25.

[0015] In one embodiment, the value range of n is preferably 5-25, more preferably 8-12.

[0016] It is understandable that the polypeptide described in the present application is a compound formed by the dehydration condensation of at least two amino acid molecules connected by peptide bonds. In the present application, it can adopt small molecule peptides known to have biologically active effects, such as antibacterial, antihypertensive, hypolipidemic, thrombotic, anti-inflammatory, and anti-tumor effects.

[0017] In one embodiment, the imine bond adjacent to R1 originates from the N-terminus of the polypeptide. Specifically, the imine bond is formed by the reaction of the N-terminal amino group of the polypeptide and the aldehyde group of 4-carboxybenzaldehyde.

[0018] In one embodiment, the polypeptide is an anti-cancer peptide, such as a TAT-KLA peptide.

[0019] In one embodiment, the polypeptide is a TAT-KLA peptide.

[0020] In one embodiment, the amino acid sequence of the TAT-KLA peptide from N-terminus to C-terminus is:

[0021] YGRKKRRQRRR-GG-D(KLAKLAKKLAKLAK).

[0022] In one embodiment, the molecular weight of the TAT-KLA peptide is 3180.31 Da.

[0023] In the conjugate shown in formula (I), the R2 group is derived from indocyanine green primary amine (ICG-NH2), and the molecular formula is C 47 H 56 N4O4S, molecular weight 773.04.

[0024] In the conjugate shown in formula (I), R1 is a substituted TAT-KLA peptide, R2 is a substituted ICG-NH2, and hyaluronic acid is used as a molecular skeleton carrier to fix both of them simultaneously in the form of chemical bonds. Among them, the TAT-KLA peptide is connected to the carboxyl group of some disaccharide monomers of hyaluronic acid through a connecting bridge formed by adipic acid dihydrazide (ADH) and 4-carboxybenzaldehyde (CBA), the adipic acid dihydrazide in the connecting bridge is connected to HA through an amide bond, 4-carboxybenzaldehyde is connected to adipic acid dihydrazide through an amide bond, and the TAT-KLA peptide is connected to 4-carboxybenzaldehyde through an imine bond. The amino group of ICG-NH2 forms an amide bond with the carboxyl group of some disaccharide monomers of hyaluronic acid.

[0025] On the other hand, the present application also provides a method for preparing the indocyanine green-hyaluronic acid-polypeptide conjugate, comprising:

[0026] S1, reacting hyaluronic acid and adipic acid dihydrazide to obtain an intermediate product HA-ADH;

[0027] S2, reacting the HA-ADH obtained in step S1 with 4-carboxybenzaldehyde to obtain an intermediate product HA-ADH-CBA;

[0028] S3, reacting the polypeptide with the HA-ADH-CBA obtained in step S2 to obtain a hyaluronic acid-polypeptide conjugate;

[0029] S4, subjecting the indocyanine green primary amine to an amidation reaction with the remaining carboxyl groups in the monomer of the hyaluronic acid-polypeptide conjugate obtained in step S3 to obtain the indocyanine green-hyaluronic acid-polypeptide conjugate.

[0030] In one embodiment, the polypeptide is a TAT-KLA peptide.

[0031] In one embodiment, the molecular weight of the hyaluronic acid is 1000-20000 Da.

[0032] Preferably, the molecular weight of hyaluronic acid is 8000 Da.

[0033] In one embodiment, in S1, the molar ratio of hyaluronic acid to adipic acid dihydrazide is 1:(2-6), preferably 1:5.

[0034] In one embodiment, in S1, the molar ratio of hyaluronic acid to 4-carboxybenzaldehyde is 1:(1-3), preferably 1:1.5.

[0035] In one embodiment, in S3, the molar ratio of the polypeptide to HA-ADH-CBA is (0.5-5):(0.5-5), preferably 1:1.

[0036] In one embodiment, in S4, the molar ratio of indocyanine green primary amine to hyaluronic acid-polypeptide conjugate is (0.5-5):(0.5-5), preferably 1:1.

[0037] In one embodiment, a catalyst is added during the reaction of S1, S2 and S4, and the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS).

[0038] Preferably, the molar ratio of EDCI to NHS is 1:1.

[0039] In one embodiment, the reaction temperatures of S1-S4 are all 0-40°C, preferably 10-30°C, and the reaction times are all 1-24h.

[0040] In one embodiment, each reaction step of the above method may be followed by a purification step.

[0041] It is to be understood that a person skilled in the art can select a suitable purification method for the conjugate of the present invention by using methods known in the art, such as dialysis and freeze-drying. Specifically, the dialysis bag method can be used for dialysis, the reaction product is poured into a pre-treated dialysis bag, and after dialysis with water or an ethanol / water mixture for one or more times, impurities are removed, and the mixture is pre-frozen after membrane filtration, and then placed in a freeze dryer for freeze drying.

[0042] Preferably, the dialysis uses a dialysis bag with a molecular weight cut-off of 1000-3500.

[0043] Among them, after IHTK is purified, it can form a spherical structure with good dispersion and uniform particle size in the dissolution medium through ultrasonic treatment.

[0044] Optionally, the above method for preparing the ICG-HA-TAT-KLA conjugate specifically comprises:

[0045] Preparation of HA-ADH conjugate: HA is dissolved in water, EDCI and NHS are added as catalysts to activate the carboxyl groups in HA, and a completely dissolved ADH aqueous solution is added to continue the reaction. After the reaction is completed, the reaction product is purified to obtain the HA-ADH conjugate.

[0046] Preparation of HA-ADH-CBA conjugate: CBA was dissolved in dimethyl sulfoxide, and EDCI and NHS were added as catalysts to activate the carboxyl groups in CBA; HA-ADH was dissolved in water, and the above CBA solution was slowly added to continue the reaction. After the reaction was completed, the reaction product was purified to obtain HA-ADH-CBA conjugate.

[0047] Preparation of HTK conjugate: HA-ADH-CBA was dissolved in a mixture of buffer solution (pH=7.4) and dimethyl sulfoxide, TAT-KLA was added, and the reaction was carried out at room temperature overnight. After the reaction was completed, the reaction product was purified to obtain HTK conjugate.

[0048] Preparation of IHTK conjugate: HTK is dissolved in water, and EDCI and NHS are added as catalysts to activate the carboxyl groups in HTK; ICG-Amine is dissolved in dimethyl sulfoxide and slowly added to the completely dissolved HTK solution. After the reaction is completed, the reaction product is purified to obtain the IHTK conjugate; after ultrasonic treatment, a spherical structure with good dispersion and uniform particle size can be formed in the dissolving medium.

[0049] In the process of preparing HA-ADH conjugate, the activation time is controlled to be 20 minutes; the adipic acid dihydrazide solution is added to continue the reaction, and the reaction time is controlled to be 6 hours; the purification steps of the reaction product include dialysis and freeze-drying.

[0050] In the process of preparing HA-ADH-CBA conjugate, the activation time is controlled to be 20 minutes; the above-mentioned 4-carboxybenzaldehyde solution is slowly added to continue the reaction, and the reaction time is controlled to be 6 hours; the purification steps of the reaction product include dialysis and freeze-drying.

[0051] In the process of preparing the HTK conjugate, the buffer is citric acid-disodium hydrogen phosphate buffer or disodium hydrogen phosphate-potassium dihydrogen phosphate, and after adding TAT-KLA, the reaction time is controlled to be 12 hours; the reaction product purification step includes dialysis and drying.

[0052] In the process of preparing the IHTK conjugate, the activation time is controlled to be 20 minutes; the above-mentioned ICG-Amine solution is slowly added to continue the reaction, and the reaction time is controlled to be 6 hours; the purification steps of the reaction product include dialysis and freeze-drying; the ultrasonic power is 40W, and the time is 5 minutes.

[0053] On the other hand, the present application also provides an anti-tumor pharmaceutical preparation, which comprises the indocyanine green-hyaluronic acid-polypeptide conjugate and at least one pharmaceutically acceptable excipient.

[0054] On the other hand, the present application also provides an anti-tumor pharmaceutical composition, which comprises the indocyanine green-hyaluronic acid-polypeptide conjugate.

[0055] It is to be understood that the pharmaceutical composition or pharmaceutical preparation of the present invention can be administered intravenously, by gastrointestinal administration or other pharmaceutically acceptable forms, and the dosage forms include but are not limited to injections, oral preparations, injectable implants, emulsions, liposomes, microcapsules, microspheres, nanoparticles, etc.

[0056] It is understood that commonly used pharmaceutically acceptable excipients are excipients, such as solvents, adhesives, fillers, wetting agents, etc., which can be selected and added when preparing a specific dosage form. If necessary, other excipients such as flavoring agents, colorants, stabilizers, lubricants, etc. can also be added. Those skilled in the art can use techniques known in the art to formulate the conjugate of the present invention into a pharmaceutical composition or a pharmaceutical preparation. For example, the preparation of pharmaceutical preparations can be carried out according to the modern pharmaceutical preparation series edited by Shenyang Pharmaceutical University. And, in addition to those mentioned in the present invention, suitable pharmaceutical excipients can also be other types known in the art, such as those recorded in the Handbook of Parmaceutical Excipients compiled by author Paul JSheskey et al., which has been revised to the eighth edition, the first edition was published in 1986, and the eighth edition was published in 2017.

[0057] It is understood that the drug carrier can be a pharmaceutically acceptable solvent, suspending agent or carrier for delivering the active pharmaceutical ingredient to an animal or human body. The carrier can be liquid or solid and is selected according to the planned mode of administration. Proteins and liposomes are also drug carriers.

[0058] On the other hand, the present application also provides the use of the indocyanine green-hyaluronic acid-polypeptide conjugate, or the indocyanine green-hyaluronic acid-polypeptide conjugate prepared by the method, or the pharmaceutical preparation, or the pharmaceutical composition in the preparation of anti-tumor drugs.

[0059] In one embodiment, the tumor comprises a melanoma, a breast tumor, a lung tumor, a colon tumor, an ovarian tumor, and a kidney tumor.

[0060] In one embodiment, when the anti-tumor drug is used, a near-infrared light source is used to irradiate the tumor site.

[0061] On the other hand, the present application also provides a system for tumor treatment, the system comprising:

[0062] a) the indocyanine green-hyaluronic acid-polypeptide conjugate, or the indocyanine green-hyaluronic acid-polypeptide conjugate prepared by the method, or the pharmaceutical preparation, or the pharmaceutical composition; and

[0063] b) An illumination device provided with a near infrared light source.

[0064] The light source emitted by the illumination device is a near-infrared light source, and specifically, the wavelength of the near-infrared light source can be 808 nm. Moreover, it has been verified by experiments that the conjugate provided by the present application effectively inhibits the growth of tumors under 808 nm laser irradiation, causing tumor ablation, and achieving good photothermal therapy and photodynamic therapy effects.

[0065] This application has at least the following technical advantages or effects:

[0066] 1. The present invention uses a derivative obtained by connecting adipic acid dihydrazide and 4-carboxybenzaldehyde as an acid-sensitive connecting bridge to connect the crude cell apoptosis peptide TAT-KLA synthesized by chemical solid phase to the hydrophilic hyaluronic acid molecular skeleton, and indocyanine green is connected to HA through an amide bond, thereby successfully preparing an ICG-HA-TAT-KLA conjugate. In this conjugate, the hyaluronic acid carrier has a good targeting effect on tumor cells and can quickly and effectively deliver anticancer peptides and ICG to the tumor cell site. The TAT in the TAT-KLA peptide is a transmembrane peptide with extremely strong cell membrane penetration ability, and KLA is a highly safe pro-apoptotic peptide. The ADH-CBA connecting bridge can release the TAT-KLA peptide under the acidic conditions in the tumor microenvironment and make it free, thereby significantly improving the therapeutic efficiency of the TAT-KLA peptide in tumor cells; at the same time, after ICG reaches the tumor site, it can exert photothermal and photodynamic effects through near-infrared light irradiation, which can promote tumor cell apoptosis on the one hand, and on the other hand, it can also help promote the release of the TAT-KLA peptide, significantly improving its release in the slightly acidic environment of the tumor, further playing a synergistic role in the treatment of tumors.

[0067] 2. The IHTK conjugate prepared in the present application fixes the anticancer peptides TAT-KLA and ICG on the molecular carrier of hyaluronic acid through chemical bonds, which has better stability after entering the human body compared with encapsulation or filling. At the same time, compared with monosaccharides or other molecular carriers, HA in the form of polysaccharides can self-assemble into nanoparticles. Nano drug carriers can increase the drug concentration in the target area, thereby improving the utilization and efficacy of the drug, and reducing the adverse reactions of the drug. In addition, the polysaccharide structure of HA is metabolized more slowly in the body, and because the polysaccharide structure is more complex, its internal chemical bonds are protected from enzymatic hydrolysis to a certain extent, thereby further improving its stability and durability in the body, and increasing the content of anticancer peptides and ICG at the tumor site.

[0068] 3. The synthesis method of the IHTK conjugate provided in this application can successfully produce a final product with physical and chemical properties that are more suitable for practical applications. Among them, the introduction of an appropriate amount of ICG can fully exert the effects of photothermal and photodynamic therapy while avoiding the physiological discomfort of the organism and damage to normal cells caused by excessive local temperature, and even affecting the efficacy of anticancer peptides; and the hyaluronic acid carrier with an appropriate molecular weight can not only improve the product yield, but also the prepared conjugate has good water solubility, and can be made into a variety of dosage forms of pharmaceutical preparations such as intravenous injections in practical applications or production, and can be quickly and effectively absorbed after entering the human body, with better application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0070] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0071] Figure 1 The synthetic route of the IHTK conjugate in Example 1;

[0072] Figure 2 is the H NMR spectrum of the HTK conjugate in Example 1;

[0073] Figure 3 This is the near infrared absorption spectrum of the IHTK conjugate in Example 1;

[0074] Figure 4 This is the particle size morphology of the IHTK conjugate in Example 1;

[0075] Figure 5 This is the evaluation result of the environmental responsive release behavior of the IHTK conjugate in test example 1;

[0076] Figure 6 The results of the apoptotic effects of TAT-KLA, HTK conjugate and IHTK conjugate on melanoma cells in Test Example 2;

[0077] Figure 7 The HE staining results of melanoma using TAT-KLA, HTK conjugate and IHTK conjugate in Test Example 3. DETAILED DESCRIPTION

[0078] In order to more clearly explain the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided so as to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0079] The technical scheme of the present invention is further described in detail below in conjunction with several preferred embodiments and accompanying drawings. Obviously, the described embodiments are only a part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work belong to the scope of protection of the present invention. It should be noted that the experimental methods adopted in the following preparation examples and embodiments, if not otherwise specified, are conventional methods, and the reagents used in the preparation examples and embodiments, if not otherwise specified, are reagents supplied by conventional reagent manufacturers.

[0080] The experimental reagents and experimental instrument information involved in the following examples are shown in Table 1:

[0081] Table 1 Reagent and instrument information

[0082]

[0083] In the following examples, the structural formula of the HA-TAT-KLA conjugate is HA~ADH-CBA~TAT-KLA, referred to as HTK. In the embodiments of the present invention, the HA-TAT-KLA conjugate of the present invention can be represented by a structural formula or by HTK.

[0084] In the following examples, the structural formula of the ICG-HA-TAT-KLA conjugate is ICG-HA-ADH-CBA-TAT-KLA, referred to as IHTK. In the embodiments of the present invention, the ICG-HA-TAT-KLA conjugate of the present invention can be represented by a structural formula or by IHTK.

[0085] In the following examples, the amino acid sequence of the TAT-KLA peptide is:

[0086] Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Gly-Gly-Lys-Leu-Ala-Lys-Leu-Al a-Lys-Lys-Leu-Ala-Lys-Leu-Ala-Lys,

[0087] The abbreviation is: YGRKKRRQRRR-GG-D(KLAKLAKKLAKLAK),

[0088] Its molecular weight is: 3180.31Da.

[0089] Example 1: Preparation of ICG-HA-TAT-KLA conjugate (ICG-HA-ADH-CBA-TAT-KLA, IHTK)

[0090] This example provides a method for preparing an ICG-HA-TAT-KLA conjugate, and the specific preparation steps are as follows:

[0091] (1) Take 0.5mmol HA (molecular weight 8000Da, n≈10) and dissolve it in deionized water to obtain HA solution, add 1.2mmol EDCI and 1.2mmol NHS as catalysts respectively, in a ratio of 1:1, and react for 20min. In addition, weigh 2.5mmol ADH and dissolve it in deionized water, add it dropwise to the catalyzed HA, react at room temperature for 6h, then put it into a dialysis bag (molecular weight cutoff 1000), dialyze it with deionized water three times, and remove the unreacted impurities. After filtering with a 0.22μm filter membrane, pre-freeze it and put it into a freeze dryer for freeze drying to obtain HA-ADH conjugate.

[0092] (2) Dissolve 0.5 mmol HA-ADH in deionized water to obtain HA-ADH solution, weigh 0.75 mmol CBA and dissolve it in dimethyl sulfoxide, add EDCI and NHS as catalysts in a ratio of 1:1, react for 20 min, slowly add dropwise to the completely dissolved HA-ADH, react at room temperature for 6 h, then put it into a dialysis bag (molecular weight cutoff 1000), dialyze three times with ethanol / water (V:V=1:4) mixed solution to remove unreacted impurities. After filtering with a 0.22 μm filter membrane, pre-freeze and freeze dry in a freeze dryer to obtain HA-ADH-CBA conjugate.

[0093] (3) Take 0.05 mmol of HA-ADH-CBA conjugate and dissolve it in a mixture of buffer (pH = 7.4) and DMSO (V: V = 1:1), add 0.05 mmol TAT-KLA, react at room temperature for 12 hours, then put it into a dialysis bag (molecular weight cutoff 3000), dialyze it three times with pH 7.4 buffer to remove unreacted impurities. After filtering with a 0.22 μm filter membrane, pre-freeze it and put it into a freeze dryer for freeze drying to obtain HTK conjugate.

[0094] (4) 0.05 mmol HTK was dissolved in a buffer solution (pH = 7.4) to obtain an HTK solution, to which EDCI and NHS were added as catalysts in a ratio of 1:1, respectively, and the reaction was carried out for 20 min. 0.05 mmol ICG-Amine was weighed and dissolved in dimethyl sulfoxide, and slowly added dropwise to the catalyzed HTK, and the reaction was carried out at room temperature for 6 h. Then, the solution was placed in a dialysis bag (molecular weight cutoff 1000) and dialyzed three times with a mixed solution of ethanol / water (V:V = 1:4) to remove unreacted impurities. After filtration with a 0.22 μm filter membrane, the solution was pre-frozen and freeze-dried in a freeze dryer to obtain an IHTK conjugate. The calculated yield was about 15%.

[0095] The IHTK conjugate is treated by ultrasound to form a spherical structure with good dispersion and uniform particle size in the dissolution medium.

[0096] Wherein, the synthetic route of the above-mentioned embodiment is as follows Figure 1 shown.

[0097] The chemical structure of the intermediate product HTK was identified by 1H NMR. Figure 2 As shown. Figure 2 It can be seen that the intermediate product prepared in this example is confirmed to be a HTK conjugate.

[0098] The structure of IHTK conjugate was identified by near infrared spectroscopy absorption. Figure 3 As shown. Figure 3 The final product obtained in this example is confirmed to be an IHTK conjugate.

[0099] The morphology of the IHTK conjugate was observed using a scanning electron microscope. Figure 4 As shown. Figure 4 It can be seen that the IHTK conjugate prepared in this example has a spherical structure under the microscopic level.

[0100] Optimization of the synthesis process:

[0101] The examples of the present application also optimize the synthesis method of the IHTK conjugate, wherein the IHTK conjugate is prepared by using the method described in Example 1 and hyaluronic acid with different average molecular weights, specifically 4000Da (n≈5), 8000Da (n≈10), 16000Da (n≈20), and 32000Da (n≈40).

[0102] However, it can be obtained by experiment that the yield of the above-mentioned hyaluronic acid raw material in IHTK conjugate is about 4%, 15%, 13%, and 10% respectively. It can be seen from this that the IHTK conjugate yield obtained by the method provided by the present application is about 15% or more when it is high, wherein when the hyaluronic acid with a lower molecular weight is used as the molecular skeleton, its less molecular chain makes the collision of the carboxyl group and ICG insufficient and thus leads to a lower yield, and when the hyaluronic acid with a high molecular weight is used, its structure that is easily cross-linked in space also reduces the synthesis efficiency of ICG. At the same time, the water solubility of the final product obtained by the hyaluronic acid with a molecular weight of 32000Da is reduced, so that it is difficult to characterize the spectrum, and it is not suitable for the application of dosage forms such as intravenous injections in actual production, and the absorbability after entering the human body will also deteriorate. Therefore, considering the preparation yield of the product and the physicochemical properties shown, the molecular weight of the raw material hyaluronic acid is preferably 5000~20000Da, i.e. n≤25.

[0103] Test Example 1

[0104] This test example evaluates the environmental responsive release behavior of the IHTK conjugate prepared in Example 1. The experimental process is as follows:

[0105] (1) Experimental drug: IHTK conjugate prepared in Example 1

[0106] (2) Experimental method: In order to verify whether the synthesized IHTK is responsive in an acidic environment, the cumulative release amount of TAT-KLA peptide was detected by fluorescence spectrophotometry. 1 mL of IHTK with a TAT-KLA peptide concentration of 100 μg / mL was placed in 20 mL of buffer containing different pH values ​​(7.4, 6.8, 5.0) for an in vitro simulated acid-sensitive experiment. Each solution was incubated in a shaker at 37°C, and then the release medium was sampled at different time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72 h). After taking out 0.5 mL each time, an equal amount of fresh release medium was added. Using a multifunctional microplate reader, the TAT-KLA content was determined by fluorescence spectrophotometry and the time-release curve was drawn. The evaluation results of the environmental responsive release behavior of the conjugate are shown in Figure 5 and Table 2.

[0107] Table 2

[0108] Time / h Release (pH = 5.0) / % Release (pH = 6.8) / % Release (pH = 7.4) / % 0.5 8.220077 7.957529 5.027027 1 17.43243 15.66023 6.096525 2 20.52124 19.97683 7.027027 4 34.61004 30.61004 7.096525 6 42.08494 35.47104 6.927027 8 46.33205 41.31274 7.096525 10 47.6834 42.47104 7.061776 12 54.05405 45.17375 7.596525 24 67.18147 55.79151 7.696525 36 76.44788 62.54826 7.027027 48 81.27413 68.33977 7.096525 60 82.97683 69.49807 8.096525 72 86.48649 69.49807 8.016521

[0109] From Table 2 and Figure 5 It can be seen that there is almost no release of TAT-KLA peptide in a neutral environment of pH = 7.4. As the acidity increases, the cumulative release rate increases. Among them, the release after 72 hours at pH = 6.8 and pH = 5.0 is as high as 69% and 86%, respectively, which is significantly improved compared with the release of hyaluronic acid-peptide conjugates (disclosed in patent CN113648427B) in previous work (the release after 72 hours is about 45% at pH = 6.8 and the release after 72 hours is about 80% at pH = 5.0). It can be shown that the IHTK conjugate has good acid-sensitive responsiveness, can quickly release TAT-KLA peptide in an acidic tumor microenvironment, and exert a better effect. In addition, the introduction of ICG is conducive to assisting in improving the release of TAT-KLA peptide in an acidic environment, thereby further improving the therapeutic effect of the anticancer peptide TAT-KLA.

[0110] Test Example 2

[0111] This test example compares the apoptotic effects of TAT-KLA peptide, HTK conjugate, and IHTK conjugate on B16 melanoma cells. The experimental process is as follows:

[0112] (1) Experimental drugs: The blank group consisted of PBS solution and the control group consisted of TAT-KLA peptide (TK group), HTK conjugate (HTK group), IHTK conjugate without laser irradiation (IHTK-L group), and IHTK conjugate with laser irradiation (IHTK+L group).

[0113] (2) Experimental method: CCK-8 was used to measure and analyze the toxic and side effects of TAT-KLA, HTK and IHTK in different concentration ranges on B16 cells.

[0114] Specific operation process:

[0115] B16 melanoma cells 1×10 4 / well was added to a 96-well plate overnight, and different concentrations of TAT-KLA, HTK and IHTK (containing 2μg / ml, 4μg / ml, 8μg / ml, 16μg / ml, and 32μg / ml of TAT-KLA peptide, respectively) were added and incubated for 24 hours. PBS was used as the control group. IHTK was divided into a laser irradiation group (IHTK+L) and a non-laser irradiation group (IHTK-L). The IHTK+L group was irradiated with 808nm laser for 10min (1W / cm 2 After stabilization in a sterile constant temperature incubator containing 5% carbon dioxide for 6 hours, 10 μL of CCK-8 solution was added to each sample well. The 96-well plate was placed at 37°C for another 2 hours, and the absorbance of the sample at 450 nm was measured using an ELISA reader, and the viability of the B16 melanoma cells was calculated. The results of the CCK-8 cell apoptosis test are shown in Figure 6 .

[0116] Depend on Figure 6 It can be seen that TAT-KLA, HTK and IHTK can all promote tumor apoptosis. The ability of HTK and IHTK to promote tumor cell apoptosis is significantly enhanced. Moreover, after near-infrared laser irradiation, IHTK exerts photothermal and photodynamic therapy effects, and its ability to promote tumor cell apoptosis is further enhanced.

[0117] Test Example 3

[0118] This test example tests the tumor inhibitory effect of TAT-KLA peptide, HTK conjugate, and IHTK conjugate on tumor-bearing mice. The experimental process is as follows:

[0119] (1) Experimental drugs: The blank group consisted of PBS solution and the control group consisted of TAT-KLA peptide (TK group), HTK conjugate (HTK group), IHTK conjugate without laser irradiation (IHTK-L group), and IHTK conjugate with laser irradiation (IHTK+L group).

[0120] (2) 2×10 5The prepared B16 cells were injected subcutaneously into BALB / c mice for tumor implantation. When the tumor grew to about 100 mm 3 After that, 100 μL of PBS solution was injected into the tail vein as a blank group. The control group was injected with 100 μL of TK, HTK and IHTK solution containing 150 nmol of TAT-KLA. Two hours after injection, the tumors in the IHTK+L group were irradiated with 808 nm laser for 10 minutes (1 W / cm 2 ) and treated 5 times under the same conditions. The mice were euthanized on day 24, and the tumors were dissected for photography and data analysis. The results of tumor HE staining are shown in Figure 7 .

[0121] Depend on Figure 7 It can be seen that HTK and IHTK had a significant inhibitory effect on tumors, and the tumor inhibition effect of the IHTK laser group was the most significant.

[0122] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. An indocyanine green-hyaluronic acid-polypeptide conjugate, characterized in that: It comprises the structural formula shown in formula (I): In formula (I), R1 is a polypeptide containing at least two amino acids, and R2 has a structure as shown in formula (II): The value range of n is 1 to 25.

2. The indocyanine green-hyaluronic acid-polypeptide conjugate according to claim 1, characterized in that: The polypeptide is TAT-KLA peptide.

3. The method for preparing the indocyanine green-hyaluronic acid-polypeptide conjugate according to claim 1 or 2, characterized in that: include: S1, reacting hyaluronic acid and adipic acid dihydrazide to obtain an intermediate product HA-ADH; S2, reacting the HA-ADH obtained in step S1 with 4-carboxybenzaldehyde to obtain an intermediate product HA-ADH-CBA; S3, reacting the polypeptide with the HA-ADH-CBA obtained in step S2 to obtain a hyaluronic acid-polypeptide conjugate; S4, subjecting the indocyanine green primary amine to an amidation reaction with the remaining carboxyl groups in the monomer of the hyaluronic acid-polypeptide conjugate obtained in step S3 to obtain the indocyanine green-hyaluronic acid-polypeptide conjugate.

4. The method according to claim 3, characterized in that The polypeptide is TAT-KLA peptide; And / or, the molecular weight of the hyaluronic acid is 1000-20000Da.

5. The method according to claim 3 or 4, characterized in that: In S1, the molar ratio of hyaluronic acid to adipic acid dihydrazide is 1:(2-6); and / or, in said S1, the molar ratio of hyaluronic acid to 4-carboxybenzaldehyde is 1:(1-3); and / or, in said S3, the molar ratio of the polypeptide to HA-ADH-CBA is (0.5-5):(0.5-5); and / or, in said S4, the molar ratio of indocyanine green primary amine to hyaluronic acid-polypeptide conjugate is (0.5-5):(0.5-5); and / or, a catalyst is added during the reaction of S1, S2 and S4, wherein the catalyst comprises EDCI and NHS; And / or, the reaction temperature of S1-S4 is 0-40°C, and the reaction time is 1-24h.

6. A pharmaceutical composition, characterized in that The invention comprises the indocyanine green-hyaluronic acid-polypeptide conjugate as claimed in claim 1 or 2.

7. A pharmaceutical preparation, characterized in that The invention comprises the indocyanine green-hyaluronic acid-polypeptide conjugate as claimed in claim 1 or 2 and at least one pharmaceutically acceptable excipient.

8. Use of the indocyanine green-hyaluronic acid-peptide conjugate according to claim 1 or 2, or the indocyanine green-hyaluronic acid-peptide conjugate prepared by the method according to any one of claims 3 to 5, or the pharmaceutical composition according to claim 6, or the pharmaceutical preparation according to claim 7 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: When the anti-tumor drug is used, a near-infrared light source is used to irradiate the tumor site.

10. A system for treating a tumor, characterized in that: The system comprises: a) the indocyanine green-hyaluronic acid-peptide conjugate according to claim 1 or 2, or the indocyanine green-hyaluronic acid-peptide conjugate prepared by the method according to any one of claims 3 to 5, or the pharmaceutical preparation according to claim 6, or the pharmaceutical composition according to claim 7; and b) An illumination device provided with a near infrared light source.

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