Iodine complex polypeptide antibacterial hydrogel and its preparation method and application

Abstract

The present invention relates to an iodine-complexed polypeptide antibacterial hydrogel and a preparation method and application thereof. A class of iodine-complexed polypeptide derivatives is constructed through the complexation characteristics of proline and iodine, and the polypeptide self-assembly strategy is used to develop it into a new type of iodine-containing antibacterial hydrogel dressing. With polypeptide hydrogel as a carrier, iodine is complexed on the fiber structure of the hydrogel, making it more stable, sustained-release and biocompatible than iodine tincture. The present invention also applies the hydrogel to the field of infection for the first time, broadens the scope of clinical application of iodine, and provides new ideas and development directions for wound infection and repair treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an iodine complexed polypeptide derivative and a hydrogel thereof, a preparation method thereof and an application thereof. Background Art

[0002] Bacterial infection, as a public health issue that seriously threatens human health, has attracted widespread attention. Currently, antibiotics are commonly used in clinical practice to treat bacterially infected wounds. However, the widespread use and even abuse of antibiotics in recent years has led to a sharp increase in bacterial resistance, and traditional antibiotic therapy can no longer meet the treatment needs. How to treat infectious diseases without causing bacterial resistance has become an important scientific issue today.

[0003] In addition to wound infections on the skin surface, other wounds located in important physiological function parts such as the reproductive tract are more susceptible to pathogen contamination than other parts due to their complex anatomical structure and internal environment. For example, the uterine cavity has a unique anatomical structure that makes it more susceptible to pathogen contamination than other parts. In addition, the endometrium has a rich blood supply, which provides an ideal environment for the colonization and reproduction of pathogens, which can easily lead to persistent inflammation in patients, and then cause a series of female fertility disorders such as recurrent miscarriage and infertility. Such local infection wounds in the body place higher requirements on antimicrobial agents - not only requiring efficient antibacterial ability and good sustained-release performance, but also good biocompatibility and degradability.

[0004] As a well-known and highly effective antibacterial agent, iodine has broad-spectrum antibacterial properties and low drug resistance, and is widely used in many fields such as infection treatment and medical disinfection. However, the inherent low stability and strong irritation of iodine greatly limit its application scenarios as an antibacterial agent. For example, the iodine-containing antibacterial agent iodine, which is frequently used in clinical practice, is highly irritating and dehydrating. It is only suitable for the treatment of bacterial infections on surface wounds and cannot be used on wounds in the body. Polymers as carriers of iodine can enhance its stability and biocompatibility by controlling the release of elemental iodine.

[0005] With the development of moist healing theory, new hydrogel dressings that can provide a moist microenvironment have become the main development direction of iodine-containing antimicrobial agents: while ensuring that iodine continues to exert its antibacterial activity, moist dressings create a microenvironment that is conducive to wound repair and synergistically promote wound healing. Most of the iodine-containing hydrogels reported so far can only be applied to superficial open infected wounds. Few studies have explored their application in vivo wounds, and the preparation process is also relatively complicated. Therefore, we hope to design and construct new moist antimicrobial materials that retain the bactericidal advantages of iodine, have better biocompatibility, and have simple and economical synthesis methods, and expand the clinical application scenarios of iodine to make it suitable for in vivo wound treatment.

[0006] Therefore, exploring the construction of safe and effective iodine preparations that can cope with a variety of infection situations has practical and far-reaching significance for improving the treatment level of clinical wound infections. Summary of the invention

[0007] The present invention constructs a class of iodine-complexed polypeptide derivatives through the complexation characteristics of proline and iodine, and develops them into a new type of iodine-containing antibacterial hydrogel dressing using the polypeptide self-assembly strategy. With polypeptide hydrogel as a carrier, iodine is complexed on the fiber structure of the hydrogel, making it more stable, sustained-release and biocompatible than iodine tincture. The present invention also applies the hydrogel to the field of infection for the first time, broadens the scope of clinical application of iodine, and provides new ideas and development directions for wound infection and repair treatment.

[0008] The first object of the present invention is to provide an iodine-complexed polypeptide derivative or a pharmaceutically acceptable salt thereof.

[0009] The iodine complexed polypeptide derivative or a pharmaceutically acceptable salt thereof provided by the present invention has a structure as shown in formula (1):

[0010] (1)

[0011] Wherein, X in the structure of formula (1) is a capping group at the amino end of the iodine complexed polypeptide derivative, selected from the following groups:

[0012] ;

[0013] ;

[0014] ;

[0015] ;or

[0016] .

[0017] In another embodiment, the present invention provides a preferred compound of iodine complexed polypeptide derivative NAP-FFGP, whose structure is shown in formula (2):

[0018] (2).

[0019] The second object of the present invention is to provide a hydrogel comprising the iodine-complexed polypeptide derivative and iodine-complexed.

[0020] The hydrogel provided by the present invention comprises an iodine-complexed polypeptide derivative and iodine, wherein elemental iodine is dispersed in an iodine-complexed polypeptide derivative solution to form a gel material, and the mass ratio of the iodine-complexed polypeptide derivative to iodine in the gel is in the range of 10:1-1:1.

[0021] In another embodiment, the hydrogel provided by the present invention, wherein the mass ratio of the iodine-complexed polypeptide derivative to iodine in the gel is preferably 5:1, 3:1 or 3:2, and most preferably 3:1.

[0022] In another embodiment, in the hydrogel provided by the present invention, the solution used to dissolve the iodine-complexed polypeptide derivative is selected from any buffer capable of maintaining a pH value of 5.5-8.0 in the solution state, and can be selected from phosphate buffer, disodium hydrogen phosphate-citrate buffer, TRIS, glycyl-glycine, N-di(hydroxyethyl)glycine, sodium acetate buffer, sodium carbonate buffer, lysine buffer, arginine buffer or a mixture thereof. The preferred buffer is a phosphate buffer, including a phosphate buffer, a sodium dihydrogen phosphate buffer, a disodium hydrogen phosphate buffer, a sodium phosphate buffer or a mixture thereof. The pH value range of the buffer is preferably 6.0-8.0, more preferably 7.1-7.7. The concentration of the buffer is 0.1-12 mg / mL, more preferably 0.5-6 mg / mL, and most preferably 1.0-2.0 mg / mL.

[0023] The third object of the present invention is to provide a method for preparing the iodine-complexed polypeptide derivative and the iodine-complexed hydrogel.

[0024] The present invention provides a process for preparing a hydrogel, comprising the following steps:

[0025] (1) dissolving the iodine-complexed polypeptide derivative in a buffer solution;

[0026] (2) adding iodine to the buffer solution and mixing them evenly to form a hydrogel; wherein the mass ratio of the iodine-complexed polypeptide derivative to the added iodine is in the range of 10:1-1:1.

[0027] In another embodiment, in the step (1) of the hydrogel preparation process of the present invention, the concentration of the iodine-complexed polypeptide derivative is in the range of 1-5 mg / mL, preferably 2-4 mg / mL, and more preferably 3 mg / mL.

[0028] In another embodiment, in the step (2) of the hydrogel preparation process of the present invention, the concentration of iodine added to the solution is 0.1-2 mg / mL, and the mass ratio of the iodine-complexed polypeptide derivative to the added iodine is maintained in the range of 10:1-1:1.

[0029] In another embodiment, in the step (2) of the hydrogel preparation process of the present invention, the concentration of iodine added to the solution is 0.1-2 mg / mL, and the mass ratio of the iodine-complexed polypeptide derivative to the added iodine is maintained in the range of 5:1, 3:1 or 3:2.

[0030] In another embodiment, in the hydrogel preparation process step (2) of the present invention, the concentration of iodine added to the solution is 0.3 mg / mL, 0.6 mg / mL, 1 mg / mL or 2 mg / mL, and the mass ratio of the iodine-complexed polypeptide derivative to the added iodine is maintained in the range of 10:1-1:1.

[0031] In another embodiment, in the step (1) of the hydrogel preparation process of the present invention, the concentration of the iodine-complexed polypeptide derivative is 3 mg / mL; in the step (2), the concentration of the iodine-complexed polypeptide derivative after adding the iodine element to the solution is 0.3 mg / mL, 0.6 mg / ml, 1 mg / mL or 2 mg / mL, and the mass ratio of the iodine-complexed polypeptide derivative to the added iodine element is maintained in the range of 10:1, 5:1; 3:1 or 3:2.

[0032] In another embodiment, in step (2) of the hydrogel preparation process of the present invention, the formation of the hydrogel can be promoted by heating and cooling.

[0033] In another preferred embodiment, the present invention provides a process for preparing a hydrogel, comprising the following steps:

[0034] (1) Dissolve the iodine-complexed peptide derivative in a buffer solution with a peptide concentration of 2-4 mg / mL;

[0035] (2) Adding iodine to the buffer solution to a concentration of 0.1-2 mg / mL and mixing well to form a hydrogel; wherein the mass ratio of the iodine-complexed polypeptide derivative to the added iodine is in the range of 10:1-3:2.

[0036] In another preferred embodiment, the present invention provides a process for preparing a hydrogel, comprising the following steps:

[0037] (1) Dissolve the iodine-complexed peptide derivative in a buffer solution at a peptide concentration of 3 mg / mL;

[0038] (2) Adding iodine to the buffer solution to a concentration of 0.3 mg / mL, 0.6 mg / mL, 1 mg / mL or 2 mg / mL, and mixing well to form a hydrogel; wherein the mass ratio of the iodine-complexed polypeptide derivative to the added iodine is in the range of 10:1, 5:1, 3:1 or 3:2.

[0039] The fourth object of the present invention is to provide a use of the above-mentioned iodine-complexed polypeptide derivative or the hydrogel formed by the derivative and iodine complex in the preparation of a pharmaceutical preparation for treating infectious diseases.

[0040] The present invention is to construct a new type of wet antibacterial material that retains the bactericidal advantage of iodine, has better biocompatibility, and has a simple and economical synthesis method, so as to expand the clinical application scenarios of iodine and make it suitable for in vivo wound treatment. Polypeptide hydrogels with antibacterial and anti-inflammatory properties are the focus of current research. Polypeptide hydrogels can not only deliver various inorganic antibacterial agents, antibiotics, and antimicrobial peptides, but also provide a good wet microenvironment for wound healing.

[0041] The present invention considers introducing proline into a polypeptide sequence with super strong self-assembly performance according to the molecular complexation characteristics of iodine and proline, and endows the self-assembling polypeptide with the function of combining iodine through the property that proline in the sequence can be complexed with iodine, thereby constructing a polypeptide antibacterial hydrogel that can complex iodine. The present invention can achieve the stabilization, low toxicity and multifunctionality of iodine-containing antibacterial dressings, thereby achieving the purpose of efficiently treating bacterial infections and promoting the repair of different wounds.

[0042] In another preferred embodiment, the present invention provides a use of the above-mentioned iodine-complexed polypeptide derivative or a hydrogel formed by the derivative and iodine complex in the preparation of a pharmaceutical preparation for treating infectious diseases, wherein the infection is caused by or is related to one or more infectious agents selected from the following group, the group consisting of the following items: bacteria, Demodex mites, fungi or yeasts and / or viruses.

[0043] In another preferred embodiment, the present invention provides a use of the above-mentioned iodine-complexed polypeptide derivative or a hydrogel formed by the derivative and iodine complexation in the preparation of a pharmaceutical preparation for treating infectious diseases, wherein the infection includes skin infection, endometritis, vaginitis, vulvovaginal candidiasis, cervicitis, salpingitis, oophoritis, ovarian abscess, pelvic inflammatory disease, genital tuberculosis, inflammatory pelvic mass and pelvic pain, sexually transmitted diseases, acute chorioamnionitis, puerperal infection, pregnancy complicated with GBS infection, pregnancy viral hepatitis and TORCH infection during pregnancy, etc.

[0044] The present invention provides a method for treating a disease, the method comprising administering a therapeutically effective amount of the iodine-complexed polypeptide derivative, a pharmaceutically acceptable salt thereof, and a hydrogel formed by the iodine-complexed polypeptide derivative of the present invention to a subject in need of treatment. The term "subject" includes humans and non-human mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, etc., and the preferred subject is a human patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Molecular structure of iodine-complexed polypeptide hydrogel molecules. A: structural formula of NAP-FFGP, NAP-FFGA, and NAP-FFGP; B: HPLC and mass spectrometry analysis of NAP-FFGP.

[0046] Figure 2: Images of iodine and peptide gel mixtures. A: images of NAP-FFGP and iodine ratios of 3:0.3, 3:0.6, 3:1, and 3:2, respectively; B: NAP-FFGA, NAP-FFGP mixed with iodine.

[0047] Figure 3 : TEM image of iodine-complexed polypeptide hydrogel.

[0048] Figure 4 : Antibacterial effect of iodine-complexed polypeptide hydrogel.

[0049] Figure 5 : Effect of iodine-complexed polypeptide hydrogel in the treatment of skin infections.

[0050] Figure 6 :The effect of iodine-complexed polypeptide hydrogel on the treatment of endometrial inflammation. A: macroscopic observation; B: H&E histology; C: TNF-α immunohistochemistry; D: IL-1β immunohistochemistry. DETAILED DESCRIPTION

[0051] Example 1: Preparation of iodine-complexed polypeptide derivatives

[0052] The molecular structure of the iodine complexed polypeptide derivative molecule designed by the present invention is shown in Figure 1 A, NAP stands for 2-naphthylacetic acid, which is the capping group of the amino terminus of the peptide sequence NAP-FFGP. The red part is proline; the green part is the part replaced by the control group compared with the experimental group: a self-assembling peptide without proline was designed as the control group, and proline (NAP-FFGP) was replaced with alanine (NAP-FFGA) to explore the complexation relationship between proline and iodine; proline was designed at a non-terminal position in the peptide sequence (NAP-FFPG) to explore the effect of the position of proline on the complexation between the peptide and iodine.

[0053] The peptide FFGP was synthesized by the classic and mature Fmoc-solid phase synthesis method, and then 2-naphthylacetic acid (NAP) was coupled to the amino end as a capping group through a condensation reaction according to the reaction conditions of ordinary Fmoc-amino acids to obtain the target molecule. After purification by semi-preparative high performance liquid chromatography (HPLC), the target product ( Figure 1 B).

[0054] Similarly, the peptide FFGP is synthesized by the classic and mature Fmoc-solid phase synthesis method, and then D-biotin, naproxen, ibuprofen or indomethacin is coupled to the amino terminus as a capping group through a condensation reaction according to the reaction conditions of ordinary Fmoc-amino acids to obtain other iodine-complexed peptide derivatives.

[0055] Example 2 Preparation of iodine-complexed polypeptide hydrogel

[0056] The polypeptide molecules prepared in Example 1 were dissolved in phosphate buffer (PBS, pH=7.4). After the solution was stabilized, iodine was added thereto. Ultrasound and shaking were used to complex the iodine with the polypeptide molecules to obtain a series of NAP-FFGP@I2 hydrogels (the ratios of polypeptide to iodine were 3:0.3, 3:0.6, 3:1, and 3:2, respectively). NAP-FFGA and NAP-FFPG were the control group sequences that were complexed with iodine to form hydrogels. NAP-FFGP can form hydrogels by heating and cooling at a concentration of 0.3 wt.%. At the same time, NAP-FFGP exhibited a dark blue gel state after being mixed with iodine in different ratios. Among them, when the ratio of polypeptide to iodine was 3:0.3 and 3:0.6, the gel had strong fluidity; at 3:1, the stability of the gel was better; at 3:2, particles were observed to precipitate in the gel ( Figure 2 A).

[0057] After NAP-FFGA was mixed with iodine, a brown turbid liquid appeared, indicating that it did not bind to iodine, proving the key role of proline in complexing with iodine; while after NAP-FFPG was mixed with iodine, dark blue small particles precipitated, indicating that it had a certain ability to bind to iodine, but the binding ability was poorer than that of NAP-FFGP with proline at the end of the sequence, proving that proline at the end was very critical for the binding of iodine ( Figure 2 B).

[0058] The ratio of iodine complexed by iodine-complexed polypeptide hydrogel NAP-FFGP@I2 was investigated by transmission electron microscopy (TEM). Figure 3 As shown in the figure, when the iodine concentration is less than or equal to 1 mg / mL, the microscopic morphology of the hydrogel is uniform nanofibers; however, when the iodine concentration increases to 2 mg / mL, precipitated iodine particles appear, indicating that this concentration has exceeded the maximum loading capacity of the polypeptide hydrogel for binding to iodine.

[0059] Example 3 Antibacterial evaluation of iodine-complexed polypeptide hydrogel in vitro

[0060] The NAP-FFGP@I2 hydrogel designed in the present invention retains the strong bactericidal effect of iodine because iodine is dispersed into the hydrogel system. At the same time, the hydrogel as a carrier also provides support for the stability of iodine. The drug-resistant bacterium methicillin-resistant Staphylococcus aureus (MRSA) was selected for the experiment. Figure 4As shown, NAP-FFGP alone has basically no inhibitory ability against MRSA, while NAP-FFGP@I2 exhibits strong bactericidal ability against MRSA, proving that their mixing with elemental iodine will not affect the bactericidal effect of iodine on bacteria, and the bactericidal efficiency is as high as over 99.99%.

[0061] Example 4 Evaluation of the therapeutic effect of iodine-complexed polypeptide hydrogel in a mouse skin infection model

[0062] Before the experiment, the back hair was shaved to facilitate the experimental operation, and the ICR mice were anesthetized by intraperitoneal injection. A full-thickness wound with a diameter of about 6 mm was made on the back of each mouse, and 10 μL of MRSA (10 8 CFU / mL) were dripped onto the wound surface, and treatment was performed 24 h after infection, with 20 μL of different drugs applied to the wound surface. Wound photos were taken every day and the wound size was measured ( Figure 5 The results showed that the wound growth and repair in the NAP-FFGP@I2 group were more significantly improved than those in the PBS control and iodine groups.

[0063] Example 5 Evaluation of the therapeutic effect of iodine-complexed polypeptide hydrogel in a mouse endometritis model

[0064] ICR mice were anesthetized by intraperitoneal injection. 60 μL MRSA (10 9 CFU / mL) bacterial solution, the injection tube was withdrawn, and no obvious fluid was found in the vagina. Treatment was carried out 24 hours after modeling, and 60 μL of samples from each group were infused into the uterine cavity. 24 hours after treatment, the uterine tissue was removed, and the infected uterus showed obvious swelling and congestion under naked eye observation ( Figure 6 A), further H&E histological evaluation showed ( Figure 6 B), after infection, the endometrial tissue was infiltrated with a large number of inflammatory cells, the integrity of the uterine epithelium was destroyed, and the interstitial edema was loose. The tissue edema in the iodine group was improved better, and the NAP-FFGP@I2 group had the most obvious improvement effect among all groups. H&E showed that the interstitial edema was improved and the epithelial damage was less. To further explore the effect of iodine complex hydrogel on inflammation, TGF-α and IL-1β immunohistochemical staining was performed ( Figure 6 C&D), the results showed that the inflammation level of NAP-FFGP@I2 group was lower than that of PBS control and iodine group. The overall preliminary experimental results showed that NAP-FFGP@I2 iodine complex hydrogel had a good therapeutic effect on the mouse endometrial bacterial infection model.

Claims

1. An iodine-complexed polypeptide derivative or a pharmaceutically acceptable salt thereof, wherein the structure of the iodine-complexed polypeptide derivative is as shown in formula (2): （2）。 2. A hydrogel comprising the iodine-complexed polypeptide derivative according to claim 1 and iodine, wherein elemental iodine is dispersed in an iodine-complexed polypeptide derivative solution to form a gel material, and the mass ratio of the iodine-complexed polypeptide derivative to iodine in the gel is in the range of 10:1-1:

1.

3. The hydrogel according to claim 2, characterized in that The mass ratio of the iodine-complexed polypeptide derivative to iodine in the gel is 5:1, 3:1 or 3:

2.

4. The hydrogel according to claim 2, characterized in that The solution used to dissolve the iodine-complexed polypeptide derivative in the gel is selected from any buffer capable of maintaining a pH value of 5.5-8.0 in the solution state, and the buffer is selected from phosphate buffer, disodium hydrogen phosphate-citrate buffer, TRIS, glycyl-glycine, N-bicine, sodium acetate buffer, sodium carbonate buffer, lysine buffer, arginine buffer or a mixture thereof; wherein the phosphate buffer includes phosphate buffer, sodium dihydrogen phosphate buffer, disodium hydrogen phosphate buffer, sodium phosphate buffer or a mixture thereof.

5. A method for preparing the hydrogel according to claim 2, comprising the steps of: (1) dissolving the iodine-complexed polypeptide derivative in a buffer solution; (2) adding iodine to the buffer solution and mixing them evenly to form a hydrogel; wherein the mass ratio of the iodine-complexed polypeptide derivative to the added iodine is in the range of 10:1-1:

1.

6. The method according to claim 5, characterized in that: In the preparation process step (1) of the method, the concentration range of the iodine-complexed polypeptide derivative is 1-5 mg / mL; in the preparation process step (2) of the method, the concentration of iodine after adding elemental iodine to the solution is 0.1-2 mg / mL, the mass ratio of the iodine-complexed polypeptide derivative to the added elemental iodine is maintained in the range of 10:1-1:1, and the formation of the hydrogel can be promoted by heating and cooling methods.

7. The method according to claim 5, characterized in that: The method comprises the following steps: (1) Dissolve the iodine-complexed peptide derivative in a buffer solution with a peptide concentration of 2-4 mg / mL; (2) Adding iodine to the buffer solution to a concentration of 0.1-2 mg / mL and mixing well to form a hydrogel; wherein the mass ratio of the iodine-complexed polypeptide derivative to the added iodine is in the range of 10:1-3:

2.

8. Use of the iodine-complexed polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 1 or the hydrogel according to claim 2 in the preparation of a pharmaceutical preparation for treating infectious diseases, wherein the infection is a skin infection or endometritis.

Images